Podium Presentations for Assays Leveraging MS

Evaluation of Analytical Techniques for Drug Checking John Halifax (Presenter) UCSF

To be presented in Track 3 (Steinbeck 3) on Wednesday at 14:00

Introduction:

Drug overdose mortality has risen dramatically in North America over the last two decades. The overdose crisis is now considered to be in its “Fourth Wave,” characterized by high mortality driven by synthetic opioids replacing traditional illicit drugs and augmented by increasing stimulant-involved deaths. In 2020 and 2021 in San Francisco County, cumulative deaths from overdose were almost double those from COVID-19, and an increase in fentanyl-involvement in deaths among individuals without prior evidence of opioid use in San Francisco suggests increased unintentional fentanyl exposure in the city.

Drug checking, the use of chemistry techniques to identify components present in a drug sample, has gained traction as one harm reduction intervention to mitigate overdose. Drug checking in the US has so far been limited to FTIR spectroscopy and fentanyl immunoassay test strip technologies to provide onsite service. San Francisco launched its own FTIR and test strip service in June 2022, with the service augmented with offsite research laboratory confirmation of all drug samples analysis performed by untargeted Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS). This work demonstrates the advantages of this onsite-offsite hybrid model of drug checking, which provides all the benefits of a point-of-service test for people who use drugs (PWUD), while gaining superior chemical analysis to better comprehend the composition of the illicit substance supply. Improved knowledge of the local illicit substance supply can be utilized to inform public health planning and toxicological case response.

Objectives:

To demonstrate the effectiveness of utilizing a clinically validated LC-QTOF-MS comprehensive drug screen method to analyze street drug products to evaluate, confirm, and augment point of service drug checking efforts.

Methods:

All drug samples submitted for point-of-service drug checking were delivered to the clinical lab dissolved in 1 mL MeOH at approximately 1 mg/mL to circumvent transportation of consumable drug material. All samples were diluted to approximately 10 mcg/mL in mobile phase and analyzed using a method previously validated for clinical testing. Briefly, chromatographic separation was performed using a C-18 column with a 10-minute gradient from 2%-100% organic. Data was collected on a SCIEX TripleTOF®5600 operating in positive-ion mode using a TOF-MS survey scan with IDA-triggered collection of high-resolution product ion spectra (20 dependent scans). Data was analyzed using an in-house library containing >5000 small molecules including >150 fentanyl analogs. Calibrator mixes were made for 25 compounds of interest to enable “semi-quantitation” in the drug products. Accurate quantitation is limited by weighing errors at the point-of-service and issues with drug product dissolution. Results were available to point-of-service drug checking staff within three days of sample intake for the purposes of informing delivery of service and to communicate confirmation results to returning service participants.

Results:

From June to November 2022, 188 drug products were evaluated at the point-of-service using FTIR spectroscopy and immunoassay lateral flow test strips for fentanyl and benzodiazepines followed by analysis using the LC-QTOF-MS method. The LC-QTOF-MS detected an additional 36 compounds in the samples that were missed by point-of-service technologies. Using the LC-QTOF-MS method as the “gold standard,” the sensitivity and specificity of the FTIR was 36.18% and 98.84% respectively for all analytes. Sensitivity and specificity of the FTIR for fentanyl only was 58.70% and 100.00% respectively, improving to 93.48% and 98.59% respectively when fentanyl test strip results were considered. Notably, the FTIR missed all benzodiazepine compounds detected by LC-QTOF-MS (n=4).

Confirmation by LC-QTOF-MS was originally planned for every sample only during the pilot period of the point-of-service program, but due to the overwhelming advantages provided by confirmation, confirmation for all samples has been extended indefinitely.

As determined by LC-QTOF-MS, samples expected to be methamphetamine (n=72) rarely contained other drug classes (n=2, 4.17%). Samples expected to be cocaine (n=42) contained unexpected drug classes more often, (n=10, 23.81%), but 9 of these 10 samples contained only lidocaine or levamisole, established cocaine adulterants. Samples expected to be fentanyl (n=37) were frequently identified as complex mixtures of compounds from a variety of drug classes (n=24, 64.86%), with semi-quantitation identifying widely varied and unpredictable potency of fentanyl in the samples ranging from 0.0092% to 46.37%. Samples expected to be heroin (n=10) also showed frequent contamination with fentanyl (n=3, 30.00%), conflicting with established thought that fentanyl is unlikely to be mixed with West Coast tar heroin. Detection of xylazine, deschloro-etizolam, flubromazolam, bromazolam, and butyryl fentanyl by confirmation but not by point-of-service demonstrate the added benefit of utilizing mass spectrometry to improve public health surveillance efforts to identify drug supply changes to inform overdose crisis response efforts.

Conclusions:

Confirmation of point-of-service drug checking by LC-QTOF-MS provides deeper comprehension of the illicit substance supply in a geographic region, aiding both harm reduction and traditional clinical practice. This information facilitates improved confidence in the capability and limitations of point-of-service drug checking, improves drug checking as public health surveillance intervention, and provides crucial context to interpret comprehensive drug screen results for clinical cases.

Multiplexed Quantification of Venlafaxine and Four Metabolites in Human Plasma Claire Knezevic (Presenter) Lurie Childrens Hospital

To be presented in Track 3 (Steinbeck 3) on Wednesday at 14:20

INTRODUCTION

Venlafaxine (VEN) and its O-demethylated metabolite, O-desmethylvenlafaxine (ODV), are commonly prescribed serotonin-norepinephrine reuptake inhibitors, approved for the treatment of depression and anxiety. Both VEN and ODV have similar antidepressant activity and their three metabolites formed by further demethylation (N-desmethylvenlafaxine (NDV), N,O-didesmethylvenlafaxine (NODDV), and N,N-didesmethylvenlafaxine (NNDDV)) are inactive. This metabolism is accomplished by CYP450 enzymes, namely CYP2D6, CYP3A4, and CYP2C19, all of which are polymorphic and can be inhibited or activated by foods and other drugs. At standard dosing levels, approximately 40% of patients have VEN and ODV concentrations outside the therapeutic range, demonstrating the potential clinical value of quantifying these drugs.

OBJECTIVES

The objectives of this study were to develop and validate a quantitative tandem mass spectrometry method for venlafaxine, desvenlafaxine, and their three inactive metabolites. A secondary objective was to quantify these molecules in remnant patient plasma and observe the relationship between administered drug and metabolites.

METHODS

K2EDTA plasma was spiked with VEN, ODV, NDV, NODDV, and NNDDV. Remnant whole blood from patients prescribed venlafaxine was obtained and plasma was obtained by centrifugation. Collection of remnant whole blood was approved by the IRB. Following the addition of isotopically-labeled internal standards (VEN-d6 and ODV-d6) to plasma and sample extraction via protein precipitation, samples were subjected to analysis by liquid chromatography-tandem mass spectrometry. Chromatographic separation was performed using a ZORBAX Eclipse Plus C18 column and detected on a SCIEX API 4500 mass analyzer operated in positive ionization mode.

RESULTS

The analytical measuring range for VEN and all four metabolites was 5 – 800 ng/mL. Standard curves were generated via weighted quadratic (NNDDV) or linear (VEN, ODV, NDV, NODDV) regression of calibrators. Inter-assay imprecision was less than 10% for all levels of all analytes. No carryover was observed after repeated injections of sample at upper limit of measuring range and no cross-talk was observed between any analyte or internal standard. Only minor matrix effects were observed and both recovery efficiency and process efficiency were >96% for all analytes. Twenty-one remnant plasma specimens were obtained from patients with current venlafaxine prescriptions (prescribed doses of 37.5 – 450 mg/day) and analyzed with this method. VEN and ODV were quantified in all samples, with concentrations ranging from 7-1200 ng/mL. NODDV was quantified in all but one sample and ranged from 10.2 to 430 ng/mL. NDV and NNDDV were quantified in 14 and 6 samples, respectively, at concentrations up to 1060 and 168 ng/mL, respectively. The ratio of total active to total inactive analytes ranged from 0.74 to 14.5, with a median of 6.39.

CONCLUSION

While previous studies have focused on quantifying VEN and ODV, fewer studies have evaluated the contribution of metabolism to the inactive metabolites to the overall pharmacokinetics of these antidepressants. This effort developed an efficient and accurate method for the quantification of VEN, ODV, and all three inactive metabolites in human plasma and demonstrated this capacity in remnant human samples. This method’s performance fulfills the criteria for an FDA Bioanalytical Method and may be used to study the pharmacokinetics of these drugs and metabolites. Additionally, these validation studies demonstrate that this method is suitable for analysis of patient samples. Although the time of dosing relative to the blood collection was not captured, all patients had been prescribed daily doses of venlafaxine for more than 10 days, during which time a daily dose would reach a steady state concentration. In agreement with published reports, ODV was the major metabolite present in most samples (18 of 21). Evaluation of the ratios of active analytes VEN and ODV to the inactive metabolites demonstrates wide inter-individual variation in metabolism. Importantly, this method enables further studies combining drug and metabolite quantification with pharmacogenetic evaluation of metabolizing genes. Future clinical studies can be performed to probe the impact of utilizing VEN and metabolite monitoring on therapeutic efficacy.

Rapid and Sensitive Protein Quantitation in Biofluids by Paper Spray Mass Spectrometry: Single Instrument Albumin/Creatinine Ratio Measurements Chris Gill (Presenter) Vancouver Island University

To be presented in Track 1 (Steinbeck 1) on Wednesday at 16:10

INTRODUCTION: Direct mass spectrometry approaches such as paper spray mass spectrometry (PS-MS) are presenting new alternatives as candidate methods for clinical workflows. PS-MS, in particular, offers a facile strategy for chemical measurements in complex samples such as biofluids. Small aliquots (i.e., ≤10µL) of sample are deposited on pointed paper strips with co-deposited internal standards. The strips are moistened with a suitable solvent, and upon the application of high voltage, ions are generated in a manner akin to electrospray, allowing direct analyte quantitation via tandem mass spectrometry. The strips are inexpensive and disposed for each measurement, eliminating carryover, and can be used to conduct ‘on-paper’ derivatization reactions as well as replace the extraction/preconcentration steps necessary in other analytical workflows.

OBJECTIVES: To demonstrate the use and effectiveness of chemically modified PS-MS paper substrates for the rapid, sensitive, and quantitative measurement of proteins in biofluids, including the rapid ‘one instrument’ measurement of albumin/creatinine ratios in urine.

METHODS: All measurements were performed by paper spray tandem mass spectrometry with a high-throughput paper spray ion source (Thermo Scientific™ TSQ Altis™ triple quadrupole mass spectrometer with a VeriSpray™ source). PS-MS paper substrates were modified by grafting polyamidoamine (PAMAM) dendrimers to their surface, increasing protein affinity. The extraction / preconcentration of target proteins from urine and serum was accomplished by vortexing the strips in 150 µL biofluid samples, followed by a short wash. Measurement by PS-MS was accomplished using barcode traceable VeriSpray™ PS-MS sample plates, allowing multiplexed measurements of 24 strips per plate, and the unattended measurement of up to 240 samples. Quantitative calibrations were achieved for both proteins and creatinine utilizing co-deposited internal standards.

RESULTS: The PAMAM functionalized PS-MS paper substrates were characterized for direct protein measurements in human urine and serum sample matrices. This included optimizing sample extraction times, washing protocols and evaluating the storage stability of the modified paper substrate. A 30 second protein vortex extraction followed by a 5 second acetonitrile wash was optimal. Once prepared, PAMAM functionalized PS-MS substrate exhibited no deterioration in performance when stored at ambient conditions over a one month period. As examples, the direct measurement of hemoglobin and albumin in urine and lysozyme and insulin in serum is presented, achieving up to 11 fold sensitivity improvements, dependent upon the charge state of the protein. The analytical performance of the functionalized paper substrate was evaluated for the analysis of albumin in urine, achieving linearity with R2 > 0.99, LOD of 1.1 µg mL-1, LOQ of 3.8 µg mL-1, precision <10%, and recoveries of 70-83%. The direct, simultaneous measurement of urinary albumin and creatinine ratios for anonymous patient samples by PS-MS demonstrates excellent comparison with validated clinical results obtained utilizing immunoturbimetry for albumin and the spectrophotometric Jaffe method for creatinine.

CONCLUSION: The use of PAMAM functionalized PS-MS paper substrate enhances the sensitivity of direct protein quantitation from biofluids. The ‘one instrument’ quantitative measurement of urinary albumin/creatinine ratios by PS-MS shows significant promise as a new candidate method for clinical analyses.

Redefining Serological Diagnostics with Immunoaffinity Proteomics Andrei Drabovich (Presenter) University of Alberta

To be presented in Track 3 (Steinbeck 3) on Wednesday at 15:30

INTRODUCTION: Serological diagnostics relies on identification of disease-specific antibodies in serum and is an essential tool in clinical diagnostics. Current serological tests utilize immunoassays and focus on fast and convenient assay development and high throughput measurements. Limitations of such tests include semi-quantitative measurements, lack of standardization, cross-reactivity, and inability to distinguish between human immunoglobulin subclasses. Advances in affinity proteomics and standardization of protocols, including our recent studies [1-4], facilitated development of sensitive and reproducible assays for quantification of low-abundance proteins and antibodies.

OBJECTIVES: We suggested that immunoaffinity proteomics may advance serological diagnostics of infectious diseases and cancer through the rational design and standardization of assays, and identification and quantification of disease-specific clonotypes of human antibodies, thus paving the way for precision immunology.

METHODS: Our approach for the rational design of serological assays utilizes immunoprecipitation of the antigen-binding human antibodies from blood serum, plasma or saliva followed by differential quantification of human antibody isotypes (IgG, IgA, IgM, IgE, IgD) and subclasses (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) with targeted mass spectrometry. Trypsin-cleavable and heavy isotope-labeled synthetic peptide internal standards targeting the Constant Heavy chains of human antibodies enable their absolute quantification (ng/mL). Simple assay design, targeted mass spectrometry and fast microflow separations provide high reproducibility, sensitivity and throughput (120 samples/day). Concurrent immunoaffinity-shotgun proteomics provides identification of immunoglobulin Fc interactomes and enables identification and quantification of antigen-specific immunoglobulin clonotypes. Our assays were validated in COVID-19 and prostate cancer patient samples.

RESULTS: A multiplex immunoprecipitation-selected reaction monitoring (IP-SRM) assay enabled differential quantification of anti-SARS-CoV-2 antibody isotypes and subclasses in blood serum, plasma and saliva. Likewise, an immunoprecipitation - parallel reaction monitoring (IP-PRM) assays quantified NCAP_SARS2 protein with a limit of detection of 313 pg/mL in serum. Evaluation of 36 antigen-antibody subclass combinations revealed receptor-binding domain (RBD)-IgG1 as a combination with the highest diagnostic specificity and sensitivity. Further validation revealed that anti-RBD IgG1, IgG3, IgM and IgA1 levels were significantly elevated in COVID-19- convalescent plasma and saliva, while IgG2, IgG4, IgA2 levels were not informative [1]. Anti-RBD IgG1 revealed a diagnostic cut-off of 408 ng/mL and provided 99.3% diagnostic specificity at 88% sensitivity to detect COVID-19 convalescent plasma. Evaluation of IgG1+IgA1+IgM combination in negative (N=143) and positive convalescent (N=82) plasma revealed 100% diagnostic specificity at 96.3% sensitivity. Interestingly, immunoaffinity-shotgun proteomics identified co-precipitation of immunoglobulin interactome (C1q complement complexes) and revealed IGHV3-7/IGKV3-20 as one of the most abundant clonotypes of anti-RBD antibodies circulating in plasma of COVID-19 patients. Finally, immunoaffinity proteomics platform enabled evaluation of serological response to prostate-specific proteins circulating in serum of patients with prostate cancer.

CONCLUSIONS: Immunoaffinity proteomics as a platform for serological diagnostics will facilitate standardization and improvement of the existing serological tests, enable rational design of novel tests, and offer novel tools for precision immunology, investigation of antibody subclass cooperation in immunity response and de novo sequencing of circulating high-affinity antibodies.

REFERENCES:
[1] Fu, Z.; Rais, Y.; Dara, D.; Jackson, D; Drabovich, A.P. Rational Design and Development of SARS-CoV-2 Serological Diagnostics by Immunoprecipitation-Targeted Proteomics. Analytical Chemistry 2022, 94, 12990–12999;
[2] Fu, Z.; Rais, Y.; Bismar, T.A; Hyndman, ME; Le, XC; Drabovich, AP. Mapping Isoform Abundance and Interactome of the Endogenous TMPRSS2-ERG Fusion Protein by Orthogonal Immunoprecipitation-Mass Spectrometry Assays. Molecular & Cellular Proteomics, 2021, 20, 100075;
[3] Drabovich, A.P.; Saraon, P.; Drabovich, M.; Karakosta, T.D.; Dimitromanolakis, A.; Hyndman, E.; Jarvi, K.; Diamandis, E.P. Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients. Molecular & Cellular Proteomics, 2019, 18, 1807;
[4] Schiza, C.; Korbakis, D.; Jarvi, K.; Diamandis, E.P.; Drabovich, A.P. Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974. Molecular & Cellular Proteomics, 2019, 18, 338.

Multiplexed Targeted LC-MS/MS Assay to Determine Immune Response to a Panel of Winter Viruses Dan Lane (Presenter) University of Leicester

To be presented in Track 3 (Steinbeck 3) on Wednesday at 15:50

Introduction
There is growing evidence that SARS-CoV2 exposure in some people can weaken our immune response to other seasonal winter viruses such as coronavirus, respiratory syncytial virus (RSV), and influenza (Inf). Also, cross reactivity of anti-viral immune responses from other viruses to SARS-CoV2 have been implicated in long COVID. Better methods for in depth antibody measurement are needed to further study these effects. Recent advances to clinical mass spectrometric assays have seen the development of a “spike-binding” test that determines immune response to SARS-CoV2 infections. Coupling a bait-and-capture extraction with multiplexed liquid chromatography tandem mass spectrometry (LC-MS/MS), in vitro viral antigens are used to capture specific/non-specific antibodies from plasma. Circulating complement proteins form complexes with the bound antibodies. Thus, entire immune-complexes can be captured prior to tryptic digestion and targeted LC-MS/MS analysis. These assays are currently useful for assessing response to recent infections, give insight on the maturation of the immune response and the degree of recognition antibodies may have to viral variants which conventional ELISA based tests cannot provide. They also have potential to be adapted for use in determining the need for booster vaccinations, or even used to screen suitable vectors in adeno-associated virus (AAV) gene therapy.

Objectives
This study sought to adapt a bait-and-capture LC-MS/MS assay for a panel of winter viruses and test its applicability on a small cohort of post-pandemic plasma samples (N=20).

Methods
Influenza A (InfA; hemagglutinin), influenza B (InfB; neuraminidase), human rhinovirus type 16 (HRV16; viral protein 0), human respiratory syncytial virus A (HRSVA; fusion), and 4 human coronavirus (229E, HKU1, NL63, OC43; spike) viral proteins were bound separately to 96-well plate (0.5 µg/well). Horse myoglobin (0.1 mg/mL) was used to block non-specific binding before the addition of 10 µL plasma. The samples were incubated for 1 hour. Wells were washed thrice with phosphate buffer solution (200 µL). Bound immunocomplexes were reduced and alkylated before tryptic digestion (45 oC, 1 h). Digestion was acidified and centrifuged (20 mins, 4500 rpm). The supernatant was analysed using reversed phase chromatography (ACQUITY Premier BEH C18 Column, 2.1 x 50 mm) coupled to a Waters Xevo TQ-XS triple quadrupole MS operating in multiple reaction monitoring (MRM) mode. Immunoglobulin (Ig) and complement peptides were normalised to the viral peptides.

Results
IgM was found in all samples (N=20/20) for each viral antigen (N=8). The mean highest and lowest IgM responses were seen in OC43 (response: 2.8x10^5) and HRV16 (1.5x10^5), respectively. IgG2 was detected in all samples and viruses, except in InfB (N=19/20). IgG1 was detected in all cases for OC43 and HRV16 (N=20/20). The mean highest and lowest IgG1 responses were OC43 (1.9x10^5) and HKU1 (1.6x104), respectively. One RSVA2 case saw elevated IgG1 (15.4-fold) and IgG2 (14.8-fold) responses compared to the mean, indicating recent exposure. IgG4 response was across viruses was low, detected between N=3/20 cases (229E) and N=10/20 cases (NL63). The complement protein C1qc was detected variably between viruses, ranging from N=2/20 cases (InfB) to N=7/20 cases (RSVA2). Similarly, C9 detection ranged between N=2/20 (NL63) and N=6/20 (RSVA2). No significance of IgG, IgM, or complement response was found on ANOVA testing between all viruses. On regression of C1qc against IgG1 z-scores, RSVA2 (R^2 0.99), NL63 (R^2 0.99), OC43 (R^2 0.83), and InfA (R^2 0.92) all correlated. Slopes for these viruses varied (1/slope: 1.28 - 1.92) indicating differential initiation of the complement pathways.

Conclusion
This proof-of-concept study demonstrated bait-and-capture assays may be readily adapted for the determination of immune response for different infections by LC-MS/MS. Variable IgG responses were seen across other coronaviruses, suggesting SARS-CoV2 exposure may have led to high-affinity immune response to some (OC43) but not others (HKU1). The assay also likely determined a recent exposure to RSVA2 for one case. Given larger clinical studies, thresholds could be set to determine recent or current infections. This test may also be adapted to pre-screen suitability to AAV gene therapy i.e., elevated Ig may predict rejection to viral vectors. A low IgG response to SARS-CoV-2 spike protein could also be used to determine need for a booster vaccination.

A Quantitative Assay for Measuring 1000 Metabolites in Serum, Urine and Fecal Samples David Wishart (Presenter) University of Alberta

To be presented in Track 2 (Steinbeck 2) on Thursday at 8:45

INTRODUCTION:
Metabolomics involves the comprehensive characterization of small molecule metabolites in specific biological specimens, such as blood, urine, feces and saliva. Metabolomics has traditionally been divided into two complementary but somewhat different approaches: untargeted and targeted metabolomics. Untargeted metabolomics is a discovery-based approach that aims to “qualitatively” measure all detectable spectral features over a large number of biospecimens. Targeted metabolomics studies are aimed at quantitatively measuring a small number of targeted compounds that can be readily used in clinical chemistry, medical diagnoses, and disease biomarker discovery studies. While untargeted methods can generate data on up to 1000 compounds, targeted methods usually generate data only on 100-200 compounds.

OBJECTIVE:
In an effort to allow targeted metabolomics to achieve the same coverage as untargeted metabolomics, we have developed a custom made, comprehensive, quantitative LC-MS/MS-based assay for targeted metabolomics analysis of biospeimens such as serum, urine and fecal extracts. This assay allows for the identification of up to 1000 metabolites along with another 400 metabolite sums and ratios.

METHODS:
Our method uses a combination of direct injection (DI) mass spectrometry with a reverse-phase LC-MS/MS. It combines the derivatization and extraction of analytes, and the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Two separate panels involving two different precolumn derivatization reactions were developed for this assay: Panel A - Phenylisothiocyanate (PITC) derivatization targeting amine-containing compounds and Panel B - 3-nitrophenylhydrazine (3-NPH) derivatization targeting keto- and carboxyl-containing compounds. Isotopically-labeled internal standards are used for metabolite quantification. Calibration of metabolite concentration ranges in both panels was adjusted for different biofluid types.

For panel A, a 96 deep-well plate with a filter plate attached via sealing tape, containing the required reagents and solvents, was used to prepare the plate assay. The first 14 wells of each plate are used for calibration and quality control purposes. For all metabolites, except organic acids, samples are first thawed on ice and then vortexed and centrifuged at 13,000x g. 10 µL of each sample are loaded onto the center of the filter on the upper 96-well plate and dried in a stream of nitrogen. Subsequently, PITC is added for derivatization. After incubation, the filter spots are dried again using an evaporator. Extraction of the metabolites is then achieved by adding 300 µL of extraction solvent. The extracts are obtained by centrifugation into the lower 96-deep well plate, followed by a dilution step with MS running solvent.

For organic acid analysis (panel B), 90 µL of ice-cold methanol is added to 30 µL of each sample for overnight protein precipitation. The sample is centrifuged at 13000x g for 20 min. 50 µL of supernatant was loaded into the center of wells of a 96-deep well plate, followed by the addition of NPH. After incubation for 2h, isotope-labeled internal standards, BHT stabilizer and water are added before LC-MS injection.

Mass spectrometric analysis was performed on Sciex 5500 QTrap® tandem MS instrument equipped with an Agilent 1290 series UHPLC system. The samples are delivered to the MS by a standard LC method followed by a direct injection (DI) method. Data analysis was done using Analyst 1.6.2. Calibration regression, accuracy and precision of QC standards, and spiked recovery of each targeted metabolite were used for method validation.

RESULTS:
This custom assay can be used for the targeted identification and quantification of up to 1000 metabolites across 21 chemical classes including amino acids and derivatives, biogenic amines, organic acids, nucleotides/nucleosides, ketone and keto acids, indole derivatives, vitamins and derivatives, sulfates, dipeptides and 11 different classes of lipids. In addition, 400 biomedically related metabolite sums and ratios can be calculated from concentrations of these 1000 targeted metabolites. In total, this custom assay can measure up to 1400 metabolites and ratios/sums. The accuracy of QC standards with 3 different concentration levels are in the range of 80% to 120% with satisfactory precision values of less than 20%. The recovery rates of spiked serum, urine and fecal extract samples with three different concentration levels are in the range of 80% to 120% with satisfactory precision values of less than 20%. More than 1500 biological samples such as serum, urine, and fecal extracts have been analyzed in our laboratory variations of this assay since 2021. The analysis time is approximately 1 sample/hour on a single LC-MS/MS instrument or about 100 samples over 4 days.

CONCLUSIONS:
We have developed a comprehensive, sensitive, high-throughput, low-volume, quantitative targeted LC-MS/MS assay for the analysis of up to 1400 metabolites and ratios, across 21 chemical classes for serum, urine, and fecal extracts. Only 40 µL of a given sample is required for the entire analysis. We have adapted this assay into a 96-well plate format to enable high-throughput analysis. This targeted assay will be able to deliver the same metabolite coverage with far more precise quantification, over much shorter periods of time at much lower costs than the most comprehensive untargeted assays. We believe that targeted metabolomics assays can now exceed the performance of untargeted assays and will continue to pull away from untargeted methods in terms of both performance and utility over the coming years.

PFAS Dark Matter and Slippery Cannabis: Disparate Problems with a Similar Path to a Solution Frederick Strathmann (Presenter) MOBILion Systems

To be presented in Track 1 (Steinbeck 1) on Thursday at 10:20

INTRODUCTION:
PFAS compounds have a long and diverse history of applications yet only recently has sufficient attention been focused on the environmental and toxicological impacts of their use. The number of PFAS compounds has expanded rapidly and it is estimated that 5,000 to 10,000 PFAS compounds exist. Despite the tremendous numbers of PFAS compounds in existence, a relatively small number have been studied in depth and are commercially tested routinely. The term “PFAS Dark Matter” has emerged to signify the recognized gap between Total Organic Fluorine, Total Oxidizable Precursors, and targeted methods using tandem mass spectrometry for PFAS concentration assessment. It is estimated that nearly every individual has at least one PFAS present in their blood, and the toxicological significance of PFAS and the implications of the PFAS Dark Matter are far from being fully appreciated. Similarly multifarious, cannabis has a complex chemical composition that includes terpenes, sugars, hydrocarbons, steroids, flavonoids, amino acids, and other compounds of potential interest. More than 700 natural constituents have been identified and more than 100 are classified as cannabinoids. The toxicological community has been challenged with the appearance of isomers of various cannabinoids causing numerous analytical challenges with limited solutions beyond chromatographic run time extension. Though diverse in the context of the challenges presented, trends in PFAS and cannabinoid production and laboratory-associated detection methods are quickly evolving in a manner reminiscent of Novel Psychoactive Substances with similar complexities involving identification, testing, and interpretation of toxicological data.

OBJECTIVES:
The primary objective of this study was to leverage a relatively novel analytical combination of LC, HRIM and QTOF approaches to unravel the complexity seen with existing separation challenges. A secondary objective of this study was to call attention to the need for clinical laboratories to evolve beyond existing workflows in anticipation of the challenges with emerging analytical needs associated with higher complexity biomarkers on the horizon.

METHODS:
We used the MOBILion HRIM system based on Structures for Lossless Ion Manipulation (SLIM) to assess cannabinoids and PFAS in a variety of matrices. A combination of Flow Injection Analysis or Liquid Chromatography with HRIM was used prior to detection using an Agilent QTOF. Accurate mass, isotope spacing, isotope ratios, and mobility aligned fragmentation were used in various combinations for tentative and absolute identification depending upon available standards. In several cases, CCS values were derived providing a unique, molecular identifier that was leveraged to generate 2 dimensional plots of CCS vs. m/z to elucidate trendlines and characteristic subclasses revealing distinctive relationships within and across compound classes. Lastly, previously established CCS values were used to generate reference plots of CCS vs. m/z as a tool to understand potential impact of interferences with known, endogenous compounds where applicable.

RESULTS:
Herein we report our use of the MOBIE® high-resolution ion mobility system (HRIM) from MOBILion with an Agilent LC-QTOF system to resolve fourteen different cannabinoid species reported in cannabis including positional isomers delta-8 and delta-9 THC, with an approximately 0.4% CCS difference, sufficiently resolved in matrix-free samples in the absence of chromatographic separation. Eight previously identified perfluorooctane sulfonates including the tentative identification of one additional branched form previously unseen, were found with enhancement of existing chromatographic separation by the HRIM system. Lastly, we explored an emerging link between hemp and PFAS in a subset of available extracts as a potential consequence of phytoremediation efforts with implications into as yet unknown toxicological significance.

CONCLUSION:
The LC-HRIM-QTOF system used is a powerful combination that can be implemented to enhance existing LC workflows for deeper sample characterization, reduce LC reliance to boost throughput, and add CCS values to existing compound identification and classification approaches. The examples provided here are relatively simple analytical challenges where existing separation technology has been limited in its utility for long-term, routine use. The clinical laboratory field has been eagerly watching the various “omics” fields with the anticipation of multiomics, diagnostic test availability, yet we have been largely idle in integrating truly novel analytical techniques outside of molecular testing. As the intricacies of each of the omics fields continues to be unraveled, novel technologies being applied to applications residing on the outskirts of existing and established workflows hold promise for meeting the ever-increasing complexity of novel testing expectations to come.

Time to Fly; TOF Mass Spec for Quantitation of Therapeutic Monoclonal Antibodies Paula Ladwig (Presenter) Mayo Clinic

To be presented in Track 1 (Steinbeck 1) on Thursday at 14:00

Introduction:
Therapeutic monoclonal antibodies have drastically improved the treatment of autoimmune disorders, cancers and other rare conditions. As recent as 5 years ago, clinical laboratories were struggling to develop quantitative methods for this class of proteins, based on large molecule drugs, as traditional methods for small molecules were not directly applicable.

Application of research proteomics mass spectrometry methods have increased choices for this class of therapeutics. The first mass spec methods were tryptic peptide and detection of unique peptides in the drug. The peptide methods have been very successful for chimeric therapeutic antibodies, such as infliximab for example (previously published). As therapeutics became more humanized, the peptide method loses some of its specificity due to overlapping peptides within the human proteome. Intact methods were then developed for these more humanized therapeutics such as eculizumab and vedolizumab for example (previously published). These methods use an enrichment method followed by Orbitrap detection.

Recent commercial advances in both hardware and software has allowed for TOF mass spectrometers to enter the arena as both a screening and quantitation tool for therapeutic monoclonal antibodies. The beauty of the TOF platform is the ability to screen or quantitate multiple analytes from the same injection without the loss in sensitivity found when utilizing an Orbitrap; sensitivity and resolution having an inverse relationship. This allows for more versatility in types of testing along with advantages for multiplexing panels.

Objectives:
To provide proof of concept TOF mass spectrometry methods for the quantitation of therapeutic monoclonal antibodies

Methods:
Samples were enriched utilizing Life Technologies CaptureSelect&trade; Affinity Matrix. In a 96-well PVDF plate, 100 mcL of resin was added and washed twice with 200 mcL 1xPBS utilizing positive pressure to move waste to a reservoir. 30 mcL of sample, along with 30 mcL of a stable isotope labeled or surrogate internal standard, were added and incubated shaking for 1 hour. The enrichment was washed 3 times with 150 mcL water. 200 mcL of 5% acetic acid was added and plate was incubated shaking for 15 minutes. Enriched proteins were eluted by positive pressure into a 2mL 96-deep well collection plate. Enrichment was reduced with 100 mcL 100 mM DTT in 1M ammonium bicarbonate; at 55&deg;C for at least 30 minutes.

The enriched and reduced samples were analyzed using an Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF&trade; 7600 mass spectrometer. A volume of 10 mcL was injected onto a Poroshell 300SB C3, 2.1x75 mm, 5-micron column heated at 60 &deg;C. Mobile phase A was water with 1% formic acid and mobile phase B was 10% isopropanol 90% acetonitrile with 0.1% formic acid. The therapeutics eluted during an 8 min gradient from 25%B to 33%B. The diverter valve was used to direct 6.5 minutes of the gradient into the mass spec; otherwise, the LC was diverted to waste. The mass spec using positive ESI was run using intact protein workflow; CUR 30, CAD 7 GAS1 35, GAS2 30, and temperature 500 &deg;C. TOF MS data from collected from 1000 to 2500 m/z; DP 175 and CE 10.

Sciex OS was used for instrument control, data acquisition, along with qualitative and quantitative data viewing and processing of data. Sciex OS Analytics was used for batch quantitation. The +11, +12, and +13 charge states (&plusmn;0.2 m/z) for the light chain mass of the therapeutic and internal standard were used for data extraction; combined to give the extracted ion chromatograph which is integrated and utilized for quantitation. A quadratic curve was drawn from the standards and unknowns back calculated from the curve.

Results:
Extracted trays (N=7) for the current clinical eculizumab quantitation LDT test method on our ThermoFisher Scientific QExactive Plus platform were re-injected on the Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF&trade; 7600 mass spectrometer and QC (N=12) and patient results (N=56) where compared. Inter day precision for the QE and TOF (respectively) compared; QCLow 19% and 16%, QCMed 11% and 10% and QCHigh 7% and 5%. Patient comparison gave a Passing-Bablok fit y=1.04x+(-4.476) for those within the AMR.

We also performed N=5 proof of concept runs for a test method for pembrolizumab and nivolumab from 5 to 500mcg/mL. 7 levels of standards were spiked with both pembrolizumab and nivolumab. 4 levels of QC were used to determine the accuracy and precision of the method. Precision was within 20% and accuracy within &plusmn;10% of expected.

Conclusion:
We demonstrate the ability to utilize the Agilent 1260 Infinity II HPLC System connected to an AB Sciex ZenoTOF&trade; 7600 mass spectrometer for quantitation of therapeutic monoclonal antibodies.

The Power of Mass Spectrometry in the Care of Patients with Monoclonal Gammopathies Mindy Kohlhagen (Presenter) Mayo Clinic

To be presented in Track 1 (Steinbeck 1) on Thursday at 14:40

Objective: To demonstrate the roles of MALDI-TOF and ESI-TOF mass spectrometry in monitoring patients with monoclonal gammopathies.

Introduction: Over the past few years, our lab has transitioned the majority of clinical testing for serum M-proteins from gel-based immunofixation to immunoaffinity purification with MALDI-TOF MS analysis (Mass-Fix). Mass-Fix performs comparably to immunofixation, but with several important advantages. Mass spectrometry allows for improved M-protein tracking, detection of clinically relevant post-translational modifications of M-protein light chains, and distinguishes therapeutic monoclonal antibodies (tmAbs) from endogenous M-proteins with increased analytical sensitivity and specificity. MALDI-TOF MS has rapid high throughput testing which has increased the productivity of our lab. Chromatographic separation and higher-resolution ESI-TOF mass spectrometry has been advantageous as the increased resolution affords the ability to track M-proteins in serum to levels comparable to bone marrow based minimal residual disease detection. These advantages result in better care of patients with monoclonal gammopathies.

Methods:
Immunoaffinity purification with MALDI-TOF MS: 10 mcL of serum was added to 50 mcL of a 10% v/v slurry of CaptureSelect resin targeting each IgG, IgA, IgM, kappa or lambda. After a 15 minute sample incubation, 3 50 mcL PBS washes and 3 50 mcL water washes were performed. Isolated immunoglobulins were eluted with 30 mcL of 20 mM TCEP in 0.1% TFA with a 15 minute incubation for reduction. The isolates were spotted with 10 mg/mL CHCA matrix in 50% ACN with 0.1% TFA and acquired on a MALDI TOF mass spectrometer (microflex smart LS, Bruker) in positive ion mode, with spectra collection from 6000-32000 m/z. Spectra were reviewed using in-house developed software.

Immunoaffinity purification with high resolution ESI-TOF MS: 30 mcL of serum was added to 200 mcL of a 10% v/v slurry of CaptureSelect resin targeting each IgG, IgA, IgM, kappa or lambda. After sample incubation for 15 minutes, resin was washed 3 times with 500 mcL water. Isolated immunoglobulins were eluted with 100 mcL of 5% acetic acid, followed by reduction with 50 mcL DTT in 1M ammonium bicarbonate and incubated at 55°C for 30 minutes. Isolates were analyzed via an Eksigent Ekspert 200 microLC (Dublin, CA) for separation; mobile phase A was water + 1% FA, and mobile phase B was 80% acetonitrile + 10% 2-propanol + 0.1% FA. A 7 μL injection was made onto a 1.0 × 75 mm Poroshell 300SB C3, 5 μm column flowing at 25 μL/min. A 15 min gradient from 25%B to 50% B was used for immunoglobulin elution. Spectra were collected on an Sciex TripleTOF 5600 quadrupole time-of-flight mass spectrometer (Sciex, Vaughan, ON, Canada) in ESI positive mode with a Turbo V dual-ion source with an automated calibrant delivery system (CDS). Source conditions were IS, 5500; temp, 500; CUR, 45; GS1, 35; GS2, 30; and CE, 50 ± 5. TOF MS scans were acquired from m/z 600−2500 with an acquisition time of 200 ms. The instrument was calibrated every ten injections through the CDS using calibration solution supplied by the manufacturer. Data analysis was performed using Analyst TF v1.6 and PeakView version 2.2. The mass spectra of the multiply charged light-chain ions were deconvoluted to accurate molecular mass using Bio Tool Kit version 2.2 plug-in software. Deconvoluted mass spectra were reviewed manually.

Results:
Analytical comparison between mass spectrometry methods: For an IgG kappa monoclonal protein spiked at decreasing concentrations into normal human serum, the limit of detection measured by MALDI-TOF MS was 50 mcg/mL, and by ESI-TOS MS was 3.13 mcg/mL. Current gel-based limits of detection range from 20-200 mcg/mL. In a study spiking tmAbs into samples with endogenous IgG kappa clones, MALDI-TOF MS was able to resolve 87% of endogenous M-proteins from tmAbs, while ESI-TOF MS was able to resolve 100% of clones (Kohlhagen. Clin BioChem 2021 Jun: 92:61-66).

Light chain N-glycosylation: In a study from 2020, among 414 MGUS patients, 25 (6%) displayed N-glycosylated light chains and were found to have a higher likelihood of progression to AL amyloidosis over time (hazard ratio =10.1, 95% CI 2.9,34.7) (Dispenzieri. Leukemia 2020 Oct; 34 (10): 2749-53). N-glycosylated light chains were also observed in a high number of patients with cold agglutinin disease (N=9/14, 64%) compared to other IgM related gammopathies (N=31/438, 7%) (Sidana. Am J Hematol 2020 Sep: 95(9):E222-5). In an additional cross-sectional study of 6315 patients, N-glycosylation was observed at higher rates in patients with LC amyloidosis and cold agglutinin disease than in other disease groups (Mellors. Blood Cancer J 2021 Mar; 11(3):50).

Minimal residual disease: For 251/431 patients enrolled in the STAMINA trial having MRD bone marrow testing by high sensitivity flow cytometry at 1 year post induction, serum Mass-Fix negativity and MRD negativity predicted better progression free survival and overall survival (median follow-up time was 6 years with overall survival of 76%), while serum immunofixation negativity and complete response did not (Dispenzieri. Blood Cancer J 2022 Feb; 12(2): 27).

Conclusion: Mass spectrometry has advanced the way we detect and monitor M-proteins in the clinical laboratory. Observation of post translational modifications such as N-glycosylation of light chains and more sensitive detection of M-proteins already has had a positive impact on the care of patients with monoclonal gammopathies.

Accurate Quantification of Angiotensin Peptides in Human Plasma Using a Multiple Reaction Monitoring LC-MS/MS Method William Perry (Presenter) Centers for Disease Control and Prevention

To be presented in Track 3 (Steinbeck 3) on Thursday at 14:00

Introduction:
The Renin Angiotensin Aldosterone System (RAAS) is a complex molecular pathway responsible for the regulation of vascular hemodynamics. When dysregulated, the RAAS contributes to cardiovascular pathologies, such as hypertension, which affects ~40-50% of the US population. Angiotensin (Ang) peptides are the primary effector molecules of the classical RAAS and alternative RAAS pathways. Traditional clinical assay technologies, leveraging antibody-based fluorescence or luminescence, show high levels of cross-reactivity due to the sequence homologies of the Ang peptides. Quantification of these vasoactive peptides using an accurate, high throughput method, such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), may provide a better understanding of the mechanisms underlying hypertension that standard blood pressure cuff measurements cannot capture.

Aim:
Establish unique mechanistic insights into the etiology of hypertension by developing a high through-put method to accurately quantitate angiotensin peptides from the RAAS pathway.

Methods:
A LC-MS/MS method was developed to measure four (4) Ang peptides (Ang (1-7), Ang (1-9), Ang I, and Ang II) in human serum. Protease inhibitors, RAAS enzyme-specific inhibitors, and isotope labeled standards are added to 150 ul of serum. Endogenous peptides are isolated from human serum using a two-step hydroalcoholic precipitation followed by mixed-mode solid phase extraction in a 96-well format. LC (Thermo Scientific RSLCnano) separations were performed using capillary flow and a reverse phase trap-elute methodological approach (Waters Corporation nanoEase M/Z Peptide BEH C18 Trap Column, 130Å, 5 µm, 300 µm X 50 mm and nanoEase M/Z Peptide CSH C18 Analytical Column, 130Å, 1.7 µm, 300 µm X 50 mm). MS measurements were completed using a triple quadrupole (Thermo Scientific TSQ Quantiva) set to select for specific peptide precursors and fragments, i.e. multiple reaction monitoring. Peptides were quantified relative to stable isotope labeled synthetic peptides.

Results:
Calibration curves spanning a concentration range of 1 – 1000 fmol/mL were established for all peptides. Linear regression analysis established R2 values ≥ 0.98. Estimated method limits of detection (LOD) are at or below 5 fmol/mL for all 4 peptides. For initial assessments, RAAS peptides were quantified in 25 commercial serum samples. Median values (concentration ranges) were as follows: Ang (1-7) 3.7 fmol/mL (LOD – 37.9 fmol/mL), Ang (1-9) 3.8 fmol/mL (LOD – 16.4 fmol/mL), Ang II 82.0 fmol/mL (LOD – 549.6 fmol/mL), Ang I 27.2 fmol/mL (LOD – 324.0 fmol/mL). Ang II always occurred in the highest concentration, except for one sample where Ang I was the most abundant peptide. In most samples, Ang I was about 4 times lower, and Ang (1-9) and Ang (1-7) were about 25 times lower than Ang II. In one sample, Ang I was about 30 times higher, and Ang (1-7) was about 3 times higher than Ang II.

Conclusion:
This mass spectrometry method enables accurate RAAS peptide quantification in human serum samples. Application of this method will lead to the generation of new data that may provide unique, mechanistic insights into hypertension, as well as renal diseases, arteriosclerosis, and other cardiovascular pathologies associated with the RAAS pathway.

Evaluation of Mass Spectrometry Methods for Glycosaminoglycan Biomarker Quantification in Mucopolysaccharidosis and GM1 Gangliosidosis Newborn Dried Blood Spots Zackary Herbst (Presenter) University of Washington

To be presented in Track 1 (Steinbeck 1) on Thursday at 15:35

INTRODUCTION: Measurement of enzymatic activity in newborn dried blood spots (DBS) is the preferred first-tier method in newborn screening (NBS) for the mucopolysaccharidoses (MPSs). However, false positives are observed due mainly to the presence of pseudodeficiencies. Recent research has shown that second-tier measurement of glycosaminoglycan (GAG) biomarker levels in DBS for the MPSs can dramatically reduce the false positive rate in NBS. Additionally, these methods are useful tools in monitoring progression and treatment of MPSs and GM1 gangliosidosis, another Lysosomal Storage Disorder (LSD) which warrants GAG analysis.

OBJECTIVES: Two methods for measuring GAG biomarkers in 10 MPS conditions and GM1 gangliosidosis are directly compared in the analysis of DBS from newborns with positive diagnoses.

METHODS: MPS and GM1 gangliosidosis-diagnosed newborn DBS samples are analyzed against a reference range of healthy newborn DBS via two GAG mass spectrometry methods: (1) the internal disaccharide biomarker method, and (2) the endogenous biomarker method. The internal disaccharide method is the classical method for GAG analysis wherein GAG polymers in patient DBS are cleaved by bacterial lyases and hydrolases to yield many copies of a set of disaccharides from the repeating unit of GAG polymers. These disaccharides are then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A second method, the endogenous biomarker method, analyzes the non-reducing end (NRE) of the GAG polymers without in vitro enzymatic digestion. Rather, endogenous NRE fragments generated by endohydrolases and exohydrolases in the human donor are derivatized and quantified directly by LC-MS/MS. A third NRE method, called the SensiPro method, involves enzymatic depolymerization of the GAG polymer, derivatization of the resulting glycans with aniline, and selective detection of the NRE derivative by LC-MS/MS. Due to similarities between the SensiPro method and the Internal Disaccharide method, in addition to complex sample workup requirements, the SensiPro method was only studied for MPS-I newborns.

RESULTS: The minimum differential factor (lowest GAG marker level in patient samples divided by highest level in a reference range of random newborns) was determined for both biomarker methods tested. The internal disaccharide minimum differentials for MPS-I and MPS-II were 2.5-fold and 3.0-fold, for MPS-IIIA and -IIIB were 3.7-fold and 5.4-fold, for MPS-IIIC and -VI were 9.3-fold and 2.1-fold, and for MPS-VII was 4.2-fold. The endogenous biomarker method minimum differentials for MPS-I and MPS-II were 16.0-fold and 3.8-fold, for MPS-IIIA and -IIIC were 4.6-fold and 3.2-fold, for MPS-IVA and-VI were 20.0-fold and 20.6-fold, and for MPS-VII and GM1 gangliosidosis were 10.0-fold and 3.0-fold, respectively. Non-newborn MPS-IIID DBS were tested for MPS-IIID against a reference range of healthy non-newborn DBS. The minimum differentials for these MPS-IIID DBS were 2.3-fold for the internal disaccharide method and 14.7-fold for the endogenous biomarker method.

CONCLUSION: Overall, the endogenous biomarker method outperforms the internal disaccharide method with regard to low variability in the reference range. While the internal disaccharide biomarkers give significant peak signals in healthy newborn DBS, the peak signals for endogenous biomarkers in healthy newborn DBS are either background noise or an order of magnitude lower than signals in patient DBS. Based on minimum differentials, the internal disaccharide method is the preferred method for MPS-IIIB and -IIIC but it is not able to discriminate MPS-IVA or GM1 gangliosidosis newborns from the reference range. The endogenous biomarker method is the preferred method for MPS-I, MPS-II, MPS-IIIA, -IIID, -IVA, -VI, -VII, and GM1 gangliosidosis and is generally the most useful method, with the exception that no endogenous marker was identified for MPS-IIIB DBS. MPS-IVB newborn DBS were not tested. This study supports the use of second-tier GAG analysis of newborn DBS, especially the endogenous biomarker method, as part of NBS to reduce the false positive rate.

Assessment of a 60 Biomarker Health Surveillance Panel (HSP) on Whole Blood from Remote Sampling Devices by Targeted LC/MRM-MS and DIA-MS Discovery Analysis Stephen A. Whelan (Presenter) Cedars-Sinai Precision Biomarker Laboratories (PBL)

To be presented in Track 1 (Steinbeck 1) on Thursday at 15:55

INTRODUCTION: Telehealth, accessing healthcare and wellness remotely, is an expanding necessity influenced by overcoming a global pandemic, fluidity of technology, and an increase in chronic diseases from an aging population compounded by poor nutrition and sedentary lifestyles. A diverse and ailing global population has accelerated interest in precision medicine to identify the mechanisms of an individual&rsquo;s disease state(s) so that an effective intervention or treatment can be prescribed to correct or alleviate phenotypic ailments. There has also been a rise in precision health, where individuals are focused on disease prevention, optimizing their quality of life as well as prioritizing and monitoring their health through an array of strategies involving nutrition, physical fitness, healthy microbiome, wearable technology, and mental health wellbeing. The blood proteome is an informative source of biomarkers representing an individual's physiological phenotype or biosignature. The proteome also represents an individual's genetic predisposition, continuously responding to environmental factors, as well as infectious agents, physical exercise and nutritional intervention. The convenience of having a robust remote collection device for blood tests coupled with a large multiplex protein assay representing an individual's biosignature will facilitate access to both precision medicine and precision health. Herein, we tested a 60 protein health surveillance panel (HSP), containing 35 FDA/LDT assays and covering at least 14 pathological states, on 8 healthy individuals' ability to collect their own capillary blood from a lancet finger prick onto remote collection Mitra devices (n=6) and directly compared this to the traditional phlebotomist venous blood draw also placed on Mitra devices (n=6) and plasma collection method (n=3).

METHODS: All samples from the 8 individuals were spiked with stable-isotope-labelled (SIL) HSP peptides and quantitatively analyzed by liquid chromatography-multiple reaction monitoring-mass spectrometry (LC/MRM-MS) scheduled method targeting 466 ion transitions, 114 peptides representing the 60 HSP proteins and by a data-independent acquisition mass spectrometry (DIA-MS) discovery method. Four sets of quality control (QC) groups were utilized: pooled from the digestion of capillary blood of all 8 volunteers biological replicates (n=48), pooled from the digestion of venous blood of all 8 volunteers biological replicates (n=48), pooled from the digestion of plasma of all 8 volunteers (isolated from venous blood) biological replicates (n=24), and a purchased human pooled plasma (pool4) used at the start, middle and end of each sample plate analysis. The QC samples from two sample plates were analyzed on two harmonized LC/MRM-MS platforms consisting of a Thermo Ultimate 3000 HPLC and Sciex 6500+ triple quadrupole mass spectrometer which resulted in an average %CV for all quantifier transitions of <11% for capillary blood, <6% venous blood, and <10% plasma.

RESULTS: For simplicity, we compared the 8 individuals and their biological replicates average peak area ratios (PAR) of the quantifier peptide transitions of all HSP proteins between the capillary blood (n=48), venous blood (n=48) and matched plasma (n=24) and all were below 20% CV. Heat map analysis of all 8 volunteers demonstrated that each individual had a unique biosignature and that three of the volunteers had an increase signal in inflammatory proteins including C-reactive protein (CRP). Hierarchical analysis of the 8 volunteers and their 6 biological replicates clustered in both their capillary blood and venous blood. Biological replicates from capillary blood and venous blood clustered for the same volunteer in k-means clustering analysis. Pearson statistical analysis of the three biofluids indicated that there was >90% similarity suggesting that remote collection devices may be a viable option for personal blood proteome biosignature stratification and health analysis. The same samples were also analyzed by DIA-MS on a 60 minute gradient using a Thermo Ultimate 3000 HPLC interfaced with a Thermo Orbitrap Exploris 480 mass spectrometer, using both a plasma spectral library (all protein identifications totaled 1121, with 399 in venous, 396 in capillary, and 388 in plasma, observed in >66% of samples) and a pan-human spectral library (all protein identifications totaled 3811, with 1029 in venous, 877 in capillary, and 364 in plasma, observed in >66% of samples with 1% FDR). The majority of HSP proteins were also identified in the DIA-MS method, in addition to many other proteins not typically seen in plasma alone that have biological importance in oxidative stress, longevity, energy metabolism, cardiovascular health, cancer, etc. The identified proteins with CV<30% were summed for each individual volunteer and were shown to reproducibly quantitate approximately 600-700 proteins in capillary blood, ~800 proteins in venous blood and ~300-400 proteins in plasma.

CONCLUSION: This study demonstrated that all three biofluids, but more specifically whole blood from a remote collection device, could be used for targeted HSP LC/MRM-MS and DIA-MS relative quantitative discovery analysis with high reproducibility. The DIA-MS results also demonstrates the potential for the development of a multiplexed protein biomarker assay targeting hundreds to possibly over a thousand proteins to capture a comprehensive stratification of individual biosignatures that would substantially inform precision medicine and precision health decisions. The convenience of a remote collection device to patients, the array of circulating protein tests and sample processing efficiency on a Mitra device in addition to the technological advancements in mass spectrometry and bioinformatic tools to quantitively identify thousands of physiologically relevant proteins in whole blood may shift the paradigm away from analyzing plasma alone.

Assessment of Therapeutic Humanized IgG2 Antibody Concentrations in Human Plasma Using LC/PRM-MS and Elisa: A Comparative Study Claudia Gaither (Presenter) Faculté de médecine vétérinaire, Université de Montréal

To be presented in Track 2 (Steinbeck 2) on Thursday at 15:35

INTRODUCTION:
The current trend in the pharmaceutical industry to quantify therapeutic antibodies in plasma or serum samples from clinical studies involves bioassays such as enzyme-linked immunosorbent assay (ELISA). Typically, these methods have been restricted to quantification of a single analyte and require the antibody structure and large sample volume. Several literature research findings suggest the possibility of structural changes an IgG2 antibody might undergo. If true, it can diminish or compromise the quantitation results obtained from ELISA. On the contrary, mass spectrometry (MS) allows the detection of proteotypic peptides and is independent of the antibody structure. In this study, we developed and applied liquid chromatography-parallel reaction monitoring (LC-PRM)/MS assays to quantify the concentration of a therapeutic humanized IgG2 antibody for 15 human plasma samples and compared LC-PRM/MS and ELISA results.

OBJECTIVES:
The key objectives of this project were to (1) develop quantitative LC-PRM/MS assays targeting tryptic peptides unique to the therapeutic IgG2 antibody, establishing the most efficient and reproducible digestion conditions, as well as the linear ranges, and (2) to apply these assays to the analysis of 15 human patient plasma samples covering up to 8 cycles of treatment from a phase II study, and compare the PRM/MS and ELISA concentrations.

METHODS:
Heavy and light antibody chains were in-silico digested using trypsin. Following stringent selection criteria, we ended up with two proteotypic peptides for the light and heavy chains, respectively, all absent in the human proteome.

Light/natural (NAT) and 13C/15N-stable isotope-labelled standard (SIS) peptides were provided by our supplier (SynPeptide, China) and assessed in-house for accurate peptide concentrations and purities by amino acid analysis (AAA) and capillary zone electrophoresis (CZE), respectively.

Peptide precursors were optimized by loading 100 fmol per peptide onto EvoTips, followed by analysis on an Evosep One HPLC with the 60 sample per day (SPD) method (21-minute gradient, column EV1109) and a PRM detection method measuring 2+ and 3+ precursor charge states on a Thermo Q Exactive Plus Orbitrap.

After establishing detectability of the antibody peptides at 5,000 ng/mL in human plasma by PRM/MS, a time course digestion study was performed evaluating three different denaturants (urea, sodium deoxycholate, and trifluoroethanol), seven digestion time points (0, 0.5, 1, 2, 4, 6 and 18h), and spiking the internal standard peptides before or after digestion. The samples used were plasma spiked with 20,000 ng/mL target antibody. The ideal condition was selected based on plateaued light/heavy ratios across three replicate preparations per condition, coefficients of variation (CVs) below 15%, maximal light/heavy peak area ratio, and maximal light peak intensity.

Next, the linear ranges of the PRM/MS assays were assessed with plasma sample spiked with 500,000 ng/mL of antibody, serially diluted with pooled human plasma to 200 ng/mL for a total of 10 concentration levels plus blank.

Finally, the concentrations of the target peptides in 15 experimental samples provided by a biotechnology company were quantified and compared with the antibody concentrations obtained from ELISA.

RESULTS:
All four peptides were most intense with the 2+ precursors. In the time course digestion study, urea as the denaturant with SIS spiked before digestion produced the most reproducible concentrations with good accuracy (within 80%-120% range), and CVs <15% among the replicates across all time points. Using a 2-hour digestion protocol, the linear ranges were 200 to 500,000 ng/mL for three peptides, and 200 to 50,000 ng/mL for the fourth peptide, suggesting sufficient sensitivity and linear range for expected antibody concentrations. Finally, 15 plasma samples from a phase II study at different dosing time points of up to 148 days were analyzed with the final PRM/MS approach, and results were compared to ELISA. While the ELISA results indicated no accumulation of the target antibody in the patient plasma, the PRM/MS approach confirmed the expected antibody accumulation as the dosing regimen progressed, thus providing evidence that the ELISA approach falsely underrepresented the actual antibody concentration in plasma, likely due to structural changes of the antibody in plasma.

CONCLUSION:
Using a highly reproducible and sensitive LC-PRM/MS approach, we were able to confirm the accumulation of a therapeutic antibody in patient plasma over the duration of 148 days in a phase II trial, whereas ELISA failed to demonstrate this behaviour.

Quantitative Analysis of Small Molecules in DBS with Imprinted Stable Isotope Internal Standards Tim Garrett (Presenter) University of Florida College of Medicine

To be presented in Track 2 (Steinbeck 2) on Thursday at 15:55

Introduction
The application of Phenylalanine to Tyrosine ratio for Phenylketonuria (PKU) detection using tandem mass spectrometry was first published in 1993. A simple solvent extraction followed by a derivatization method has been used to improve sensitivity and specificity for the detection of amino acids in blood or plasma by MS/MS, which has been modified and customized in labs over the years.

The development of electrospray in mass spectrometry largely improved ionization and facilitated automated analysis. Here, we introduce a Selected Reaction Monitoring method (SRM) with simple Flow Injection Analysis (FIA) via strategically adding internal standards to the clean paper discs prior to the collection of whole blood or plasma samples to achieve a better recovery and reproducibility for amino acids quantitation.

Methods
Varying concentrations of the amino acids internal standard mixture in water was pipetted onto clean paper discs either from flat precut paper or using blood sample collection devices (Capitainer) and dried for 30min. 10µl of blood or plasma sample was added and dried overnight at room temperature. The paper discs were removed and extracted with 300µl methanol in a polypropylene tube with shaking for 30min. Following centrifugation, the supernatant was transferred to a clean tube and dried down under nitrogen at 30oC. Next, 50µl of HCl in n-butanol was added, incubated at 65oC for 20min, then the excess butanol was evaporated. Samples were reconstituted using 300µl of 1:1 methanol/water and transferred to LC vial for analysis.

Results
With the addition of labelled amino acids internal standards (IS) prior to the paper matrix before the collection of whole blood or plasma samples, we achieved improved reproducibility compared to the traditional method of adding the internal standards into the extraction solvent. Because the IS was added in the paper, it reflects the extraction efficiency of amino acids from the paper matrix better than adding the IS during the extraction procedure. In addition, it could account for any potential degradation that might occur to the sample before preparing for analysis since degradation would occur equally to target analytes and the internal standards. The ratio of endogenous amino acids to its labeled counterparts were calculated. We observed lower relative standard deviation (RSD) when adding internal standards prior to the collection of samples. As expected, derivatization changed the fragmentation pattern of amino acids, largely improved the ionization efficiency for target analytes, meanwhile, it helped to eliminate background ions that would cause ion suppression in mass spectrometry. Signal intensities detected from dried plasma spots using our SRM-FIA method, based on the measurement of peak areas, were increased 6 to 39-fold for amino acids butyl ester derivatives compared to free amino acids, with reduced RSD values, especially for tyrosine. Our preliminary results show that ion suppression was a major problem which introduced higher error to data analysis because of poor peak shape. Through conducting serial of dilutions, a proper reconstitution volume was chosen for the extraction workflow that reduced overall error.

We utilized quantitative Dried blood spot cards (Capitainer) which deliver a precise volume of whole blood to a separated paper disc in which we added our IS mixture. This unique approach helped to achieve better recovery and reproducibility for the quantitation of amino acids from DXS. The SRM-FIA method was performed via Thermo Scientific TSQ Altis Mass Spectrometer coupled with Vanquish LC system, without any separation and the entire analysis process was conducted automatically with a 1 min per sample analysis rate.

Conclusions
The use of internal standards imprinted on paper for the collection of biofluids in amino acid quantitation provides improved sensitivity and reproducibility in FIA-SRM analysis.

Uncertainty Calculations for Reference Measurement Procedures Andrea Geistanger (Presenter) Roche Diagnostics GmbH

To be presented in Track 3 (Steinbeck 3) on Thursday at 15:15

INTRODUCTION:
Quantitative results based on reference measurement procedures require the calculation of the uncertainty of this value. To obtain this uncertainty the different error sources have to be taken into account and combined.

OBJECTIVE:
This talk will explain different ways for calculating the uncertainty of a liquid chromatography/mass spectrometry (LC/MS) reference measurement procedure, showing the differences between Type A and Type B uncertainties and best strategies to choose from them. In addition, combinations of results from subsequent steps to a final overall uncertainty are presented.

METHODS:
A LC/MS reference measurement procedure requires as a starting point a primary material with known purity of the analyte under consideration. Nowadays this purity assessment is often done with NMR technology. However, as NMR is a measuring method, the purity estimate carries an uncertainty. Here we use the Type A uncertainty approach, based on replicate NMR measurements. In addition, we will shortly introduce a Bayesian-approach for NMR uncertainty calculation and do a comparison of both techniques.
With this primary material a first set of primary calibrators is produced, often through multiple steps of preparation of stock solutions, weighings, dilutions etc. For these steps balances, volumetric flasks or pipettes are used, all of them coming with an error budget which has to enter the uncertainty budget of the primary calibrator preparation. We will show how this can be done through Type B uncertainty calculation.

Based on the primary calibrators the LC/MS instrument is calibrated and measurements of individual samples are carried out. We will introduce a hierarchical variability experiment, which takes into account the most important error sources and results in a Type A uncertainty for the LC/MS measurements.
Finally, the errors of the LC/MS measurements have to be combined with those of the primary calibrator preparation to obtain the overall uncertainty of an LC/MS reference measurement procedure.
RESULTS: We will show the approaches and calculation strategies based on an analyte of the Roche LC/MS Reference Measurement Procedure development project.

CONCLUSION:
Uncertainty calculation for LC/MS reference measurement procedures requires the modularisation of the different execution steps, starting from the primary reference material until the final concentration value. Different strategies have to be applied and combined to obtain the final uncertainty. We have developed a sound framework for the different steps, including the management of the different input variables up to comprehensive reports of the calculations.

Using Microprobe-Capture In-Emitter Elution and High-Resolution Mass Spectrometry for Characterization and Clinical Testing of β2-Transferrin Ruben Y. Luo (Presenter) Stanford University

To be presented in Track 3 (Steinbeck 3) on Thursday at 16:50

Introduction

Cerebrospinal fluid (CSF) leak can occur as a result of laceration, blunt trauma, or surgery. It is potentially a life-threatening condition if left untreated (1). CSF leak is typically diagnosed by detecting a protein marker beta2-transferrin (beta2-Tf) in secretion samples. beta2-Tf, a proteoform of human transferrin (Tf) (2), mainly present in CSF, is barely detectable in other body fluids (3). beta2-Tf, together with the typical Tf proteoform in serum beta1-Tf, were named after their electrophoretic mobility in gel electrophoresis. The clinical utility and diagnostic value of beta2-Tf in CSF leak have been demonstrated (4). However, the structures of beta1-Tf and beta2-Tf have not been elucidated. A novel affinity capture technique for sample preparation, called microprobe-capture in-emitter elution (MPIE), was incorporated with high-resolution mass spectrometry (HR-MS) to study and elucidate the structures of beta1-Tf and beta2-Tf (5).

Method

MPIE can directly couple a label-free optical sensing technology (next-generation biolayer interferometry, BLI) with MS, resolving the challenge of lack of process monitoring in the conventional affinity capture techniques such as bead-based immunoprecipitation. To implement MPIE, an analyte is first captured on the surface of a microprobe, and subsequently eluted from the microprobe inside an electrospray emitter. The capture process is monitored in real-time via BLI. When electrospray is established from the emitter to a mass spectrometer, the analyte is immediately ionized via electrospray ionization (ESI) for HR-MS analysis. By this means, BLI and HR-MS are directly coupled in the form of MPIE-ESI-MS, which is readily deployed to analyze the Tf glycoforms and elucidate the structures of beta1-Tf and beta2-Tf. The study can pave a way for the development of novel clinical assays for beta2-Tf. The Tf glycoforms in CSF samples, serum samples, and secretion samples from patients suspected of CSF leak were analyzed using MPIE-ESI-MS. The Tf glycoforms separated by gel electrophoresis were also analyzed.

Results

Based on the MPIE-ESI-MS results of serum, CSF, and secretion samples, the structures of beta1-Tf and beta2-Tf were elucidated. As Tf glycoforms, beta1-Tf and beta2-Tf share the amino acid sequence but have varying N-glycans. beta1-Tf, the major serum-type Tf, has two G2S2 N-glycans on Asn413 and Asn611, while beta2-Tf, the major brain-type Tf, has an M5 N-glycan on Asn413 and a G0FB N-glycan on Asn611. The analytical sensitivity of MPIE-ESI-MS for CSF samples was tested to evaluate its potential clinical use, and it was demonstrated that it was able to detect beta2-Tf in the 1:9 pooled CSF : water mixture (10-fold diluted). When testing clinical specimens, the MPIE-ESI-MS method has good performance in testing secretion samples that are not blood-contaminated, especially those with ambiguous agarose gel immunofixation electrophoresis (the conventional method) test results.

Conclusion

Through the elucidation of the structures of beta1-Tf and beta2-Tf, the resolving power of MPIE-ESI-MS was demonstrated. Besides basic research, MPIE-ESI-MS can potentially be used in clinical laboratory testing. Moreover, with the known N-glycan structures in beta1-Tf and beta2-Tf, other types of new assays can be designed to detect beta2-Tf in the future. The MPIE-ESI-HR-MS method demonstrated exceedingly good sensitivity to successfully connect a label-free technology and MS, and it has substantial value for biomedical research and clinical diagnostics.

1. McCudden CR, Senior BA, Hainsworth S, Oliveira W, Silverman LM, Bruns DE, et al. Evaluation of high resolution gel beta2-transferrin for detection of cerebrospinal fluid leak. Clinical Chemistry and Laboratory Medicine (CCLM). 2013;51:311–5.
2. Papadea C, Schlosser RJ. Rapid Method for beta2-Transferrin in Cerebrospinal Fluid Leakage Using an Automated Immunofixation Electrophoresis System. Clinical Chemistry. 2005;51:464–70.
3. Gorogh T, Rudolph P, Meyer JE, Werner JA, Lippert BM, Maune S. Separation of beta2-Transferrin by Denaturing Gel Electrophoresis to Detect Cerebrospinal Fluid in Ear and Nasal Fluids. Clinical Chemistry. 2005;51:1704–10.
4. Zaret DL, Morrison N, Gulbranson R, Keren DF. Immunofixation to Quantify beta2-Transferrin in Cerebrospinal Fluid to Detect Leakage of Cerebrospinal Fluid from Skull Injury. Clinical Chemistry. 1992;38:1909–12.
5. Luo RY, Yang S. Using Microprobe-Capture In-Emitter Elution to Directly Couple Label-Free Optical Sensing Technology with Mass Spectrometry for Top-Down Protein Analysis. Chemistry; 2022 Oct.

Poster Presentations for

Validation of an Automated Whole Blood Extraction Method for Immunosuppressant Therapeutic Drug Monitoring by LC-MS/MS Ka Keung Chan (Presenter) University of Rochester Medical Center

Poster #1b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Therapeutic drug monitoring (TDM) for immunosuppressants helps providers optimize drug dosing to prevent toxicity and rejection. Therapeutic strategies rely on accurate and precise assays, particularly at low drug concentrations. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) methods are extensively employed due to their sensitivity, specificity and ability to detect multiple analytes simultaneously. However, these assays are manually intensive and require skilled personnel, who are becoming more difficult to hire due to national staffing shortages, which appear to be getting worse each year. Automation of the sample preparation needed for LC-MS/MS analysis would greatly increase efficiency and would likely lead to improved throughput, robustness, and cost savings.

Objectives:
We aimed to develop and validate an automated sample-preparation method on a liquid-handling platform for the multiplexed quantification of sirolimus, tacrolimus, and cyclosporine in whole blood. We also validated the transfer of a LC-MS/MS method to newer systems for improved performance.

Methods:
The concentrations of sirolimus, tacrolimus, and cyclosporine A were determined in whole blood samples by Xevo TQ-MS LC-MS/MS systems with either manual or automated sample preparation. The Hamilton Microlab Starlet automated liquid handler performed barcode reading, transferring of whole blood samples, addition of internal standard solution, mixing, and liquid re-suspension in 96-well plates without manual involvement. The LC-MS/MS method utilized a ballistic LC gradient of 1.5 min for a cycle time (injection-to-injection) of 3.5 min.

Results:
The assay was linear throughout the therapeutic range for each analyte and imprecision was <7%. Method comparison between manual and automated sample preparation was excellent (r2>0.99) for sirolimus (N=58), tacrolimus (N=85), and cyclosporine A (N=56).

Conclusion
Our automated sample-preparation method with a ballistic LC gradient is suitable for routine clinical analysis. It reduces hands-on time, which also reduces errors, increases staff availability for other functions, and improves analytical throughput.

Application of Magnetic Beads in Automated Sample Preparation for Simultaneous Measurement of Catecholamines and Metanephrines in Plasma by LC-MS/MS William Wu (Presenter) Calibra Diagnostics

Poster #2a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
The accurate quantitation of catecholamines and metanephrines in plasma is essential to clinically screen pheochromocytoma and paraganglioma. Automated and high throughput LC-MS/MS method for catecholamine and metanephrine measurement is desired to achieve quicker turn-around time, lower labor cost, and consistent testing results.

Methods:
Extraction of catecholamines and metanephrines from plasma samples was accomplished by using weak cation exchange (WCX) iron oxide magnetic beads. A magnetic bead extractor for 96-well plate was utilized for automated sample preparation. Sample analysis was performed by reverse phase HPLC with a PFP column and triple quadrupole mass spectrometer in the positive electrospray mode.

Results:
The method performance was fully evaluated with CLSI guideline C62-A. The accuracy of method was conducted by patient comparison as well as spiking recovery study. The overall bias of six analytes was less than 10%. The imprecision of six analytes was also less than 10%. The linearity of the method spanned 3 orders of magnitude. The lower limit of quantitation was 10 pg/mL for metanephrine as well as 3-Methoxytyromine, 15 pg/mL for dopamine, 20 pg/mL for normetanephrine as well as epinephrine, and 50 pg/mL for norepinephrine, respectively. The automated sample preparation time was 16 minutes for a 96-sample batch and LC-MS/MS analysis time was 4 minutes. This method was used for more than 5000 clinical sample analysis over the past six month and the cancelation rate was less than 0.2%, which is better than previous sample preparation method by WCX solid phase extraction (SPE).

Conclusion:
We have successfully developed an automated sample preparation method that uses WCX magnetic beads for simultaneous measurement of catecholamines and metanephrines by LC-MS/MS. This new method has excellent throughput for large number of clinical sample testing to meet the turn-around-time requirement from ordering physicians. In addition, as this new preparation method was selective, reliable, and less interference prone to the plasma matrix compared to the conventional SPE method, minimal cancelation rate should be expected for clinically screening pheochromocytoma and paraganglioma.

Can We Transfer Insulin-like Growth Factor 1 Reference Interval between Mass Spectrometry Assays with Different Traceability? Sally Ezra (Presenter) Alberta Precision Laboratories and University of Calgary

Poster #3a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Insulin-Like Growth factor 1(IGF-1) is a key mediator of growth hormone actions. Accurate assessment of IGF-1 is crucial for diagnosis and monitoring of Growth hormone deficiency and acromegaly. Current immunoassays for IGF-1 are subject to a variety of interferences and pre-analytical sample handling issues, as well as having poor agreement between different platforms. LC-MS/MS assays provide an alternative platform not prone to most analytical interferences associated with immunoassays.

OBJECTIVES: The main objective of this study was to determine the feasibility of transferring IGF-1 reference intervals between two liquid chromatography mass spectrometry assays with traceability distinct reference materials.

METHODS: We developed and validated a quantitative IGF-1 assay traceable to NIST reference standard (2926). As clinical interpretation of IGF-1 results are highly reliant on age-specific reference intervals (RIs), a reference interval transference study was conducted against the liquid chromatography-high resolution mass spectrometry (LC-MS/HRMS) IGF-1 assay that is traceable to WHO (02/254) reference standard. The analytical agreement between the assays was evaluated using the linear model and the appropriateness of the linear model for RIs transference was assessed using Deming regression, correlation coefficients, Q-Q plot, and difference plot and studentized residues for the LC-M/MS against IGF-1 immunoassay and the LC-MS/HRMS IGF-1 assay.

RESULTS: Our study showed a strong correlation (R2>0.93) and agreement (slope=1.006, intercept =negligible) between LC-MS/MS and LC-MS/HRMS regardless of the difference in their traceability and all statistical criteria were met per CLSI guidelines. Although, the LC-MS/MS and the immunoassay showed a strong correlation (R2>0.97, slope=1.055), they failed to meet all statistical criteria for RIs transference due to the bias (-44.91) and non-normal distribution of the residues. Finally, the verification of the transferred RIs showed that 95% of reference samples fell within the transferred RIs, meeting CLSI C28-A3 guidelines.

CONCLUSION: Our method employs calibrators traceable to the recently available NIST reference standard (2926) in comparison to the reference laboratory, which was traceable to the WHO reference material (02/254). Therefore, we provide the first known example of an LC-MS method traceable to the NIST standard and demonstrate sufficient agreement between these methods to allow RI transference.

Measurement of Plasma 3-Hydroxyisovalerylcarnitine Using a Commercial LC-MS/MS Assay to Predict Marginal Biotin Deficiency in Inflammatory Bowel Disease Max Diesner (Presenter) Immundiagnostik AG

Poster #3b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Case Description:
Study cohort conducted with 100 inflammatory bowel disease (IBD) patients (20-60 years, 40 females) whom diagnosed according to standard clinical, radiological and pathological criteria and 100 healthy controls (20-60 years, 40 females). Of the patients 58/100 had Crohn`s Disease (CD), 42/100 had ulcerative colitis (UC) and 29/100 were in inflammatory state (serum high sensitivity C reactive protein (hsCRP) concentrations &gt;5mg/L). Standard serum and citrate plasma samples were withdrawn between 8:00 - 10:00 hours in the clinic after overnight fasting and stored at -20 &deg;C until the analysis. Complete blood count (CBC), high sensitivity C reactive protein (hsCRP), fecal calprotectin (fCal) were determined with standard methods in serum matrix. 3-Hydroxyisovalerylcarntine (3HIVc) levels were determined by LC-MS/MS citrate plasma matrix.

Background:
Studies on biotin's role in the immune response have emergently revealed that biotin is related to inflammation and that its deficiency leads to an increase in the level of proinflammatory cytokines. A biotin-deficient diet was recently shown to induce a colitis-like phenotype in mice, alleviated by biotin substitution. Mice with dextran sulphate sodium (DSS)-induced colitis showed biotin deficiency and significantly reduced biotin absorption. Oral biotin substitution reversed DSS colitis and induced remission. Diagnosis of biotin deficiency is rather challenging since based solely on measurement of circulating biotin levels has been shown to be insufficiently sensitive for clinical purposes. Increased urinary excretion of biotin metabolites, is suggested as a sensitive indicator of biotin deficiency. However, 24 h urine collection is time consuming, expensive, and the test relies on correct collection of samples by the patient, thus if this is not done properly the results may be inaccurate. 3HIVc increases in the case of biotin deficiency as a response to the decreased activity of biotin dependent enzyme methyl crotonyl-coenzyme. Analysis of circulating 3HIVc levels by LC-MS/MS has been found to be one of the most sensitive markers of biotin depletion.

MS Method and Results:
For measurement of 3HIVc in citrate plasma the commercial LC-MS/MS kit KM 3200 (Immundiagnostik AG, Germany) was used. This kit enables the measurement of 3HIVc after a fast precipitation reaction.
Sample preparation was achieved by adding the precipitation solution including an isotopically labelled internal standard in 96-well plates. After vortexing and centrifugation, supernatants were diluted and injected into an ACQUITY UPLC H Class (Waters) with an injection cycle of 3 mins. Transitions of 3HIVc were measured in positive mode by ESI-MS/MS on TQXS (Waters). For calibration, six levels (0.47-88.82 ng/mL) covering the complete physiological range were used. Results were verified by injections of three quality control (QC)-levels throughout the measurement. The assay was validated after FDA guidelines. Finally, the performance f the kit was evaluated was evaluated on a second LC-MS system (TSQ vantage, Thermo Scientific), providing robust application.

Patients with IBD were found to have significantly higher 3HIVc levels than controls (6.1&plusmn;2.4 ng/mL vs 5.1&plusmn;2.1 ng/mL respectively, p = 0.004). 3HIVc concentrations were compared according to different disease characteristics within the patient group: circulating 3HIVc levels were found to be similar in patients with CD and UC (5.2&plusmn;2.1 ng/mL vs. 4.9&plusmn;2.1, p = 0.451). Patients were also found to have similar circulating 3HIVc concentrations irrespective of whether or not inflammatory activity was present (5.1&plusmn;2.0 ng/mL in inflammatory vs. 5.7&plusmn;2.4 ng/mL in noninflammatory conditions, p = 0.131).

Discussion and Conclusion:
The commercial LC-MS/MS assay enabled sensitive, and accurate quantification of 3HIVc. The kit allows easy implementation into the clinical laboratory, on a wide range of mass spectrometers. The clinical findings of this study are in line with the existing preclinical evidence indicating that biotin deficiency is more common in patients with IBD in comparison to healthy controls in a clinical setting. However, surprisingly, the biotin status of patients with IBD was not found to differ according to inflammatory status. Therefore, there is still a room for more comprehensive look into the relationship between IBD related inflammation and biotin deficiency and 3HIVc measurement with LC-MS/MS provides sensitive results for clinics to define biotin repletion to achieve with this future perspective. Finally, the assay is on the way to IVDR certification in Europe.

Naturally Occurring Metabolites Can Inhibit β-Glucuronidases Hydrolysis and Result in False Negatives for Urine Drug Testing Anusha Chaparala (Presenter) Integrated Micro-Chromatography Systems Inc

Poster #5a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Urine drug testing is one of the most common practices for monitoring the use of prescribed opioid medications. Testing is typically performed by a preliminary screening assay, such as immunoassay, followed by a confirmatory assay such as liquid chromatography coupled with mass spectrometry (LC-MS/MS). Screening and confirmatory assays benefit from the use of enzymes that hydrolyze, or deconjugate, glucuronidated analytes.
New generation of beta-glucuronidases can now effectively cleave glucuronides in urine at room temperature. However, during our studies, we have identified additional challenges in urine across several patient samples. Endogenous compounds in clinical samples can inhibit these enzymes and compromise hydrolysis. This can make analyzing some patient samples more difficult than others and might lead to false-negatives.

Objective
Present data to show that endogenous metabolites in clinical samples can reduce hydrolysis, and commercially available recombinant enzymes are not inhibited equally.

Methods
Recombinant β-glucuronidases were from IMCS and Kura Biotech. Drug standards were from Cerilliant. Chemicals were purchased from MilliporeSigma and Fisher Scientific. Opioid-positive urine specimens were obtained from a national testing laboratory. Drug free human urine control was from UTAK. Drug free human urine control was fortified with glucuronidated drugs of abuse. Control and patient specimens were buffered and hydrolyzed with two commercially available β-glucuronidases for 15-minutes at room temperature. The two β-glucuronidases were compared by two methods. First, by using an equivalent amount of β-glucuronidases, measured by Bradford Assay, in patient sample hydrolysis. Second, by hydrolyzing patient samples the with a range of β-glucuronidase concentrations.
Hydrolyzed urine samples were cleaned by eluting samples through β-Gone plus plates from Phenomenex and diluted with water. 10 µL of sample was injected on a Thermo Scientific™ Vanquish™ UHPLC system coupled with a Thermo Scientific™ Endura™ Triple Quadrupole Mass Spectrometer using a Phenomenex Kinetex® 2.6 μm Biphenyl 100 Å, 50 x 4.6 mm column. Mobile phase A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively.

Results
Ninety-six patient samples that tested positive for opioids were hydrolyzed with equivalent protein amounts by two different β-glucuronidases. Samples were labeled “positive” if drug free recovery was ≥ 25 ng/mL. One β-glucuronidase consistently reported more positives than the other β-glucuronidase, indicating possible false negatives for the latter.
To determine if enzyme inhibition was involved, four patient samples and a control sample were hydrolyzed with a range of β-glucuronidase amounts (0-100 µg of one β-glucuronidase and 0-200 µg of the second). Hydrolysis was considered complete when < 20% of drug glucuronide was remaining. Several patient samples required more enzyme to complete hydrolysis than the control sample. This indicates that some patient samples may have endogenous inhibitors that can reduce hydrolysis of drug glucuronides.

Conclusion
Some patient samples may contain endogenous urine inhibitors that affect enzyme hydrolysis relative to control samples. Additionally, not all enzymes are inhibited equally by these endogenous molecules.

Rapid Translation of a SARS-CoV-2 Targeted Peptide Immunoaffinity LC-MS Test from Research into Routine Clinical Use Hans Vissers (Presenter) Waters Corporation

Poster #6a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

OBJECTIVES: During 2020, the Department of Health and Social Care (DHSC) of the United Kingdom established the Moonshot programme to fund various diagnostic approaches for the detection of SARS-CoV-2, the pathogen behind the COVID-19 pandemic. Mass spectrometry was one of the technologies proposed to increase testing capacity.

METHODS: Moonshot funded a multi-phase development programme, bringing together experts from academia, industry and the NHS to develop a state-of-the-art targeted protein assay utilising enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) to capture and detect low levels of tryptic peptides derived from SARS-CoV-2 virus. The assay relies on detection of target peptides, ADETQALPQRK and AYNVTQAFGR, derived from the nucleocapsid protein of SARS-CoV-2, measurement of which allowed the specific, sensitive, and robust detection of the virus from nasopharyngeal (NP) swabs. The diagnostic sensitivity and specificity of LC-MS/MS was compared with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) via a prospective study.

RESULTS: Analysis of NP swabs (n=361) with a median RT-qPCR quantification cycle (Cq) of 27 (range 16.7-39.1) demonstrated diagnostic sensitivity of 92.4% (87.4-95.5), specificity of 97.4% (94.0-98.9) and near total concordance with RT-qPCR (Cohen&#039;s Kappa 0.90). Excluding Cq&gt;32 samples, sensitivity was 97.9% (94.1-99.3), specificity 97.4% (94.0-98.9) and Cohen&#039;s Kappa 0.95.

CONCLUSION: This unique collaboration between academia, industry and the NHS enabled development, translation, and validation of a SARS-CoV-2 method in NP swabs to be achieved in 5 months. This pilot provides a model and pipeline for future accelerated development and implementation of LC-MS/MS protein/peptide assays into the routine clinical laboratory.

Simultaneous Detection of Respiratory Infectious Diseases Using Immunoprecipitation and LC-MSMS Yvonne Song (Presenter) Thermo Fisher Scientific

Poster #10b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:

We are now living in a new era with new normal after the COVID-19 pandemic. Recently, respiratory syncytial virus (RSV) has been another concern as it surges among children. In addition to influenza viruses, not only are their symptoms similar at early stages, but they are also both enveloped viruses with several common biological properties, often leading to challenges in accurate identification. Thus, there is a need to develop a faster and more specific analytical tool that can differentiate infectious diseases.

Among different viral components, nucleocapsid protein or nucleoprotein (NP) is highly conserved and specific for infectious disease virus types. Therefore, targeting NP could provide a more robust and faster way for disease identification.

This study describes a targeted approach for the simultaneous detection of NPs from different respiratory infectious diseases using immunoprecipitation (IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Objectives:

To monitor multiple infectious diseases in a fast and sensitive way using immunoprecipitation and selected reaction monitoring.

Methods:

Prior to IP, equal amounts of all biotinylated antibodies were pooled together as one antibody panel for this study. The biotinylated antibody panel was added to samples collected via nasopharyngeal swabs in viral transport media (VTM) followed by incubation for 15 minutes at 25 C degree with rotation. The antigen-antibody complex in VTM was directly subjected to IP using Thermo Scientific™ Pierce™ MS-Compatible IP Kit (Streptavidin).

The IP purified samples were then digested for 15 minutes at 70 C degree with vortexing at 1000 rpm using SMART Digest™ Trypsin Kits and analyzed by Thermo Scientific™ Vanquish™ MD HPLC system hyphenated to Thermo Scientific™ TSQ Altis MD mass spectrometer. Data acquisition, processing and reporting were performed using TraceFinder™ LDT software 1.0.

Results:

Multiple viruses, SARS-CoV-2, influenza virus A and B types, RSV, and human coronavirus (HCoV-229E), were selected to show that this method can distinguish different disease viruses. The protein sequences of those selected NPs are mostly unique to each disease type and remain unchanged regardless of their variants, resulting in a reliable target peptides list for selected reaction monitoring (SRM). Combining with IP which uses a specific antigen-antibody interaction, it provides a highly targeted and confident detection of each disease. Thus, this approach is much simpler and robust with minimal changes when compared to other methods targeting different components such as the spike protein.

The workflow was optimized from sample preparation to LC-MS analysis. The protein precipitation and post sample clean-up were eliminated. From IP procedure, two incubation steps for antigen-antibody complex formation and immobilization on the magnetic beads were reduced to 15 minutes each (originally 1 hour each). Trypsin digestion incubation time was optimized to 15 minutes (previously 90 minutes). Particularly, the reduction of trypsin digestion time was achieved owing to a generation of much cleaner sample matrix by IP. The entire process was finalized to less than 1 hour from 4 hours. LC-MS run time was also optimized to 5 minutes.

A total of 12 peptides were successfully monitored (2 to 3 peptides per disease) by SRM. Calibration curve was generated with stable isotope-labeled standards (Thermo Scientific HeavyPeptide AQUA Ultimate). With criteria of % RSD < 15, CV < 15, and R2 > 0.99, LOQs were determined to be between 0.05 and 1 fmol of peptides on LC column with retention time variation ± 0.01 minutes. The method significantly improved the sensitivity and turn-around time, compared to other digestion or peptide enrichment methods.

Conclusion:

• Simple and robust approach by targeting the nucleoprotein component of the enveloped virus.
• Quick sample preparation taking < 1 hour and virus detection using 5-minute LC-MS method.
• MS-compatible and clean sample matrix generation by immunoprecipitation.
• Highly targeted, sensitive, and confident detection by immunoprecipitation and selected reaction monitoring.

For Research Use Only – Not For Diagnostic Procedures.

Fast Analysis of Total Vitamin C in Plasma Using the LDTD-MS/MS Technique at 8 Seconds Per Samples Pierre Picard (Presenter) Phytronix Technologies Inc

Poster #12b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Vitamin C (Ascorbic acid, AA) is an important vitamin involved in many physiological activities in living organisms such as the synthesis of collagen, acts as antioxidant, metabolism and synthesis of adrenaline, and prevention and treatment of scurvy and cold. In the presence of moisture, air, heat, light or oxygen, ascorbic acid transforms into its oxidative form, dehydroascorbic acid (DHAA). Total Vitamin C concentration in a plasma sample is defined as the sum of AA and DHAA.

Objectives
Our goal for this presentation is to develop a method for the quantification of total vitamin C in plasma in a single operation, using the LDTD-MS/MS Technology at 8 seconds per samples, instead of HPLC method at 7 minutes per sample using reduction into the sample preparation.

Methods
To stabilize ascorbic acid, plasma samples were spiked with a H2SO4 solution (0.2 N) at a ratio of 1:1. The samples were transferred into barcoded tubes, then read, and scanned by a liquid handler where an automatic batch file was created. The liquid handler mixed the stabilized plasma, the internal standard solution, the extraction buffer, and acetonitrile. After centrifugation, 4 µL of the upper layer phase were spotted onto a LazWell96 plate and evaporated to dryness before analysis by LDTD-MS/MS.
LDTD was used with a power ramp of 6 seconds to 45% followed by a 2-second hold. Air is used as carrier gas at 3 L/min. The mass spectrometer was operated in negative MRM mode.

Result
Total vitamin C is calculated as the sum of DHAA and AA. Traditional methods use a reduction agent (DTT) for the transformation of DHAA to AA during the sample preparation before HPLC analysis. The LDTD-MS/MS technology uses the APCI ionisation mode. Under this condition, a complete transformation of ascorbic acid (AA) into its oxidative form (DHAA) is observed. Without any additional reduction or oxidation step during the sample preparation, the total vitamin C content in plasma samples was quantified using DHAA’s MRM transition (173->113).

Calibration curves ranging from 1 to 100 µg/mL and QCs were prepared in an AA-depleted plasma (plasma kept at room temperature/exposed to light for 4 days). Replicate extraction samples were deposited onto a LazWell plate and dried before analysis. The peak area against the internal standard (IS) ratio was used to normalize the signal.

The calibration curves were plotted using the peak area ratio and the nominal concentration of the standards. The inter-day correlation coefficients for vitamin C show values greater than 0.999. For the inter-run precision and accuracy experiment, each QCs are analyzed in sextuplicate, on five different days. The inter-run precision and accuracy results obtained for vitamin C ranged between 3.4% and 6.0%, and between 96.2% and 101.6%, respectively.
Following the extraction, sample extracts are kept at 4°C in closed containers to evaluate the wet stability of sample extracts. After 1 day, sample extracts were spotted onto a LazWell plate, dried and analyzed. For the dry stability, extracted samples are spotted onto a LazWell plate, dried and kept at room temperature for 1 hour before analysis. Precision and accuracy of QC samples are within the acceptance criteria (Precision ≤15% CV and accuracy 100±15% of nominal value) for 1 day at 4°C (wet stability) and 1 hour at room temperature (dry stability).

Conclusion
Total Vitamin C quantification in Plasma using LDTD-MS/MS without reduction step in sample preparation at 8 seconds per analysis.

An UPLC-MS/MS Method for Quantification of 8-OHdG in Urine Chia-Ni Lin (Presenter) Chang Gung Memorial Hospital

Poster #14a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: 8-OHdG (8-hydroxy-2-deoxyguanosine) is generated after the repair of ROS-mediated DNA damages and has been widely used as a biomarker for oxidative stress. Oxidative damage to DNA has been associated with numerous pathological conditions, including cancer, diabetes and cardiovascular disease. This study aimed to develop a method to measure 8-OHdG in urine by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

METHODS: Stable isotope-labelled 8-OHdG was used as internal standard. 8-OHdG was separated from the biological fluid using solid phase extraction (SPE). The elution solvent was injected into a LC-MS/MS (Waters Xevo TQ-XS). Samples were separated on a BEH Amide column followed by positive electrospray ionization and tandem mass spectrometric detection.

RESULTS: The linearity of 8-OHdG was between 0.2 to 20 ng/mL with a correlation coefficient of 0.997. The coefficient variation for inter- and intra-day imprecision was less than 4.8%. The accuracy was evaluated by spike recovery and the mean recovery was 106%-109%. No carryover and ion suppressant or enhancement was observed in this validated method.

CONCLUSION: In conclusion, we have developed a rapid, sensitive, and robust UPLC-MS/MS method for the quantification of 8OHdG in urine, which can provide results to assist in the assessment of oxidative stress.

Quantitation of Clinical Research Steroid Analytes from Blood Serum Utilizing Solid Phase Extraction Paired with LC-MS/MS Mackenzie Freige (Presenter) Phenomenex

Poster #15b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Quantitation of Clinical Research Steroid Analytes From Blood Serum Utilizing Solid Phase Extraction Paired With LC-MS/MS

INTRODUCTION: Steroid analysis for clinical research can require very low limits of detection which demand high recoveries from solid phase extraction (SPE) and very clean extracted samples. Labs desire high-throughput methods which consolidate analytes into one panel with fast chromatographic run times. Accurate quantitation makes it necessary to chromatographically separate steroids with the same m/z. Meeting these criteria can be challenging in a single LC-MS/MS method.

OBJECTIVES: Here, we present an effective sample cleanup method and LC-MS/MS analysis method for the quantitation of an 18-analyte steroid panel from serum.

METHODS: The (SPE) technique utilized a reversed phase Strata-X 30mg 96-well plate (Phenomenex, Torrance CA). A Core-Shell 2.6µm, 50x3 mm Kinetex C18 column (Phenomenex) was used for fast chromatographic separation. Detection employed a 7500 Triple Quadrupole LC-MS/MS equipped with an ESI source (Sciex, Framingham, MA).

RESULTS: Linearity is demonstrated for all analytes with an R squared value >0.992. The calibration curves prepared in steroid-free blood serum range from 10 pg/mL to 500 ng/mL, subject to unique cutoff requirements of the analytes of interest. LLOQ values range from 10 pg/mL to 5 ng/mL. The QC samples for 3 replicate extractions at 3 different levels showed relative standard deviation (RSD) below 15% and accuracy between 80-120%. The LC method with the use of a C18 column achieves chromatographic separation of all isomers with an 8-minute method.

CONCLUSION: The proposed sample prep method utilizing a 96-well plate SPE extraction and fast LC method resulted in a simple, rapid cleanup for identification and quantitation of the tested steroid panel analytes from blood serum. The SPE extraction method was robust, reproducible, and can be automated. This makes it a reliable method that combines accurate quantitation with high throughput.

Ultra-fast, Accurate and Simultaneous Quantification of Ritonavir and Lopinavir in Human Plasma Holly Pagnotta (Presenter) SCIEX

Poster #17a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION:
Protease inhibitors (PIs) are a class of anti-viral drugs that prevent viral replication by selectively binding to viral proteases and inhibiting their function. The development of PI-based therapies has been of enormous benefit to people infected with HIV. Unfortunately, the effectiveness of protease inhibitors can fade over time. Mutations during viral replication can result in viruses that produce new, different proteases that are not targeted by current PI therapies. The best way to avoid this drug resistance is to reduce or stop HIV replication. With less HIV replication, there is less of a chance of a new strain that is resistant to antiHIV drugs. To keep HIV levels as low as possible, PIs are typically taken in combination with at least two other anti-HIV drugs. Such combination therapies are referred to as highly active antiretroviral therapy (HAART). Lopinavir and ritonavir are two protease inhibitors that are often used as part of a fixed-dose combination, and serve as the model compounds in this study.

OBJECTIVE:
To develop a method utilizing Acoustic Ejection Mass Spectrometry (AEMS), as implemented in the Echo® MS System with a SCIEX Triple Quad™ 6500+ LCMS/MS system, which offers clear benefits for quantification of lopinavir and ritonavir in human plasma. Requiring minimal sample preparation and no chromatographic separation, it provides high sample throughput without sacrificing robustness or reproducibility.

METHODS:
Lopinavir and ritonavir were spiked into human plasma samples in the range of 0.5 ng/mL to 250 ng/mL each. Samples were processed using a liquid-liquid extraction method and 0.4 mL of supernatant liquid was collected and dried under a nitrogen stream. Samples were reconstituted in 100 µL of 25% v/v methanol in water and transferred to a 384-well plate for analysis by AEMS. Methanol with 0.1% v/v formic acid was used as carrier solvent at a flow rate of 425 µL/min in the AEMS with 50 nL sample volumes. MS/MS detection was performed using the SCIEX Triple Quad 6500+ LC-MS/MS.

RESULTS:
Calibration curves, along with quality control samples, all analyzed in six replicates, demonstrated the high reproducibility of AEMS when combined with liquid-liquid extraction. Excellent %CVs were achieved across all concentration levels with no interference in blank human plasma samples. Even at the extremely short analysis time of 3 seconds per sample, the method yielded LLOQs of 0.5 ng/mL for both lopinavir and ritonavir. The assay accuracy was 85.31–112.34% for ritonavir and 85.51–113.52% for lopinavir. The calibration curve covered approximately 3 orders of magnitude (0.5–250 ng/mL) for both analytes and displayed linearity with regression coefficients (r2) of 0.9934 for ritonavir and 0.9946 for lopinavir using a weighting of 1/x2.

CONCLUSION:
The Echo® MS System produced very sensitive, accurate and reproducible results for the simultaneous quantitative analysis of lopinavir and ritonavir in human plasma. Having a very short analysis time (3 sec/sample) enabled rapid generation of quantitative data for high numbers of samples, and with the assay showing great reproducibility even without using labeled internal standards. Future use of labeled internal standards is recommended to further improve these results.

Differentiation of Leucine and Isoleucine Using Electron Activated Dissociation (EAD) Casey Burrows (Presenter) SCIEX

Poster #17b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Here, an unambiguous differentiation of leucine (Leu) and isoleucine (Ile) in peptides derived from a monoclonal antibody (mAb) therapeutic was achieved utilizing a new fragmentation type based on electron activated dissociation (EAD). This workflow demonstrates the combination of routine characterization and elucidation of challenging residues in one single analysis, without the need for sample specific optimization.

Ensuring drug safety and efficacy is essential for biotherapeutic development, which drives the need for in-depth characterization. Confirmation of the protein sequence is a standard requirement for all protein therapeutics by regulatory agencies. Although mass spectrometry (MS) has been mostly adapted for sequence verification, differentiating Leu/Ile remains a challenge. These two isomeric amino acids have the same molecular weight and an MS/MS spectrum obtained from collision-induced dissociation (CID) cannot tell them apart. Thus, Edman degradation is still widely employed today, which is reagent expensive and time consuming.

Alternative fragmentation techniques have been reported to introduce secondary fragmentation, resulting in different losses of the side chain from Leu and Ile. The derived signature ions can be used to discriminate between these two amino acids. However, previous approaches required extensive precharacterization in order to perform MS3 experiments. The use of nano-liquid chromatography (LC), offline fractionation or infusion was also common to enhance sensitivity, but they lacked reproducibility and throughput. With more and more protein therapeutics in the market and in development, the need to distinguish Leu and Ile in an easy manner has dramatically increased. In addition, it is likely that more analytical questions will need to be answered which require technologies better able to elucidate complex structural moieties.

The data presented in this work show the streamlined identification of Leu and Ile as part of a general peptide mapping study. Data were acquired in a fast, automatic and sensitive manner using data-dependent acquisition (DDA) with Zeno EAD, with streamlined data interpretation utilizing Protein Metrics Inc. software.

Methods:
The adalimumab sample was denaturated with 7.2 M guanidine hydrochloride, 100 mM Tris buffer pH 7.2, followed by reduction with 10 mM DL-dithiothreitol and alkylation with 30 mM iodoacetamide. Digestion was performed with trypsin/Lys-C enzyme at 37 °C for 16h. 3 µl (4 µg) of the trypsin/Lys-C digest was separated with a CSH C18 column (2.1×100 mm, 1.7 μm, 130 Å, Waters) using an ExionLC system. The mobile phase A consisted of water with 0.1% formic acid, while the organic phase B was acetonitrile 0.1% formic acid. A gradient profile was used at a flow rate of 350 μL/min. The column temperature was maintained at 50°C. Data were acquired with an information dependent acquisition (IDA) method using the SCIEX ZenoTOF 7600 system. The electron energy for the alternative fragmentation in the EAD cell was set to a value of 7 eV. Data were processed using Byos software (Protein Metrics Inc.).

Results:
A peptide mapping analysis of a mAb was performed capable of answering in-depth characterization questions like Leu/Ile differentiation, while maintaining sensitivity and ease-ofuse. An analytical flow LC setup and a DDA approach with Zeno EAD on the SCIEX ZenoTOF 7600 system were used. Robustness and throughput are achieved by using analytical flow rates, while the DDA method set-up further enhance ease-of-use by avoiding compound-specific method optimization. In addition, the Zeno trap enhances the detection of fragments enabling a confident assignment.6 This breakthrough technology realizes the desire of an easy, and at the same time comprehensive, method for advanced characterization in the biopharmaceutical industry.

Conclusions:
Unambiguous differentiation between Ile and Leu in one single DDA run was achieved by using Zeno EAD on the SCIEX ZenoTOF 7600 system with automatic data interpretation by Protein Metrics Inc. software. This workflow proposes a streamlined solution for distinguishing isomers previously thought a challenge by LC-MS/MS for years.

Immunoprecipitation Top-Down High-Resolution Mass Spectrometry for the Quantification of the Protein Tumor Biomarker Neuron-Specific Enolase Sebastian van den Wildenberg (Presenter) Eindhoven University of Technology

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Poster #18a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
LC-MS methods using bottom-up based and/or middle-down have been popular methods for the quantification of proteins. However, the development of these methods often come with extensive sample preparation that require thorough assay optimization. In addition to this using bottom-up proteomics sequence coverage is often limited and information about Post Translational Modification’s (PTMs) is lost. Using top-down proteomics intact protein are analyzed, without digestion, reducing sample preparation and retaining the maximum amount of information. Top-down proteomic approaches have their own specific challenges, such as limited sensitivity and the availability of internal standards and reference material.

Objectives:
The primary objective of this study is the development of an immunoprecipitation assay, followed by intact top-down protein analysis using LC-QToF-MS for the quantification of the small cell lung cancer (SCLC) tumor biomarker Neuron Specific Enolase (NSE).

Methods:
Immunoprecipitation was performed by coupling Protein-G-labeled magnetic Dynabeads™ to monoclonal antibodies against NSE. The protein-G-antibody complex was crosslinked with BS(PEG)5. Human (NSE-spiked) serum was incubated with antibody coupled beads. After incubation, the beads were collected using a magnet and serum was removed. Collected beads were washed and the captured NSE proteins were eluted from the beads. The eluted proteins were separated using RP-LC and analyzed by QToF-MS.

Results:
The immunoprecipitation method was developed and optimized for the isolation of NSE. Protein elution from the antibody-antigen-complex was conducted using water-acetonitrile (80:20) + 1% formic acid. Using this method recombinant NSE was successfully isolated from spiked human serum. Adequate linearity and sensitivity were achieved in the clinically relevant concentration range of 0 to 100 ng/mL using both QToF-Full Scan MS + Extracted Ion Chromatogram (XIC) and QToF-Selected Reaction Monitoring.

Conclusion and Outlook:
An immunoprecipitation method couple to a LC-QoF-MS method was developed and optimized for the isolation and quantification of recombinant NSE from spiked human serum. Next steps will focus on the isolation of endogenous human NSE from serum and optimization of the chromatography by transferring the method from UPLC to nano-LC. Additionally, QToF-MS- methods and data collection modes that can be used are compared, such as mass deconvolution, single reaction monitoring (precursor > product), pseudo-single reaction monitoring (precursor > precursor), extracted ion chromatograms and combinations.

Development and Validation of a LC-MS/MS Method for Simultaneous Measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, Vitamin A, alpha-Tocopherol, and gamma-Tocopherol Yi Xiao (Presenter) Children’s Hospital Los Angeles

Poster #23b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Background:
Circulatory fat-soluble vitamin levels are commonly measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We have developed and validated a rapid and robust LC-MS/MS method that simultaneously measures 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, &alpha;-tocopherol, and &gamma;-tocopherol for clinical use.

Method:
100 uL of serum was mixed with isotope-labeled internal standard and extracted using a 96-well supported-liquid extraction plate with 1.5 mL of hexanes/isopropanol (90/10) (v/v). Dried eluate was reconstituted with 100 uL of methanol/water (90/10) (v/v) and analyzed by LC-MS/MS with a 10-minute gradient. Accuracy was assessed using NIST Standard Reference Materials SRM972a and SRM968f, patient comparison analysis at a reference lab, and spike-recovery studies using patient sera and vitamin D-depleted serum. Analytical measurement range (AMR) was determined by spiking 6 analytes into vitamin D-depleted serum to give 7 specimens at varying concentrations. The lower limit of the measuring interval (LLMI) was assessed using 6 pooled specimens with varying low concentrations of each analyte over 20 days. Precision (repeatability and reproducibility) was assessed using quality control materials. Interference studies were performed using pooled patient specimens spiked with varying concentrations of hemoglobin, bilirubin, or intralipid. Matrix effect was assessed by post-column infusion and by matrix dilution with saline.

Results:
The method was linear covering physiological concentrations with r2 > 0.99. Repeatability and reproducibility were &lt;15% CV at all QC levels. LLMI for 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, &alpha;-tocopherol, and &gamma;-tocopherol were 4 ng/mL (15% CV), 4 ng/mL (15% CV), 4 ng/mL (18% CV), 1 ug/dL (20% CV), 0.2 ug/mL (20% CV), and 0.2 ug/mL (8% CV). Recoveries for NIST Standard Reference Materials were between 92 - 111% and between 81 - 122% for spike-recovery studies. Passing-Bablok analyses for vitamin D total, vitamin A, and &alpha;-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed.

Conclusions:
We have developed and validated a comprehensive and rapid LC-MS/MS method for the simultaneous measurement of 25-OH D3, epi-25-OH D3, 25-OH D2, vitamin A, &alpha;-tocopherol, and &gamma;-tocopherol for clinical use.

Evaluation of Nitrous Oxide as a Reaction Gas for the Analysis of Selenium, Copper, and Zinc in Human Serum Using a PerkinElmer Nexion 5000 Patrick Day (Presenter) Mayo Clinic

Poster #24a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Elements including selenium, copper, and zinc are often quantified in biological samples using inductively coupled plasma mass spectrometry (ICP-MS). To accurately quantitate elements in these complex matrices, helium gas can be used to attenuate potential polyatomic interferences using cell technology. Due to ongoing helium supply chain issues, it is imperative that alternate gases are evaluated for elemental analysis of biological samples in the clinical laboratory.

OBJECTIVES: Evaluate if nitrous oxide is a suitable collision or reaction cell gas for the accurate analysis of selenium, copper, and zinc in human serum.

METHODS: Human serum samples were analyzed for selenium, copper and zinc using a PerkinElmer NexION 5000 multi-quadruple ICP-MS (PerkinElmer Inc., Waltham MA) with ESI SampleSense automation technology (ESI, Omaha NE) for high-throughput analyses. Samples were measured in dynamic reaction cell mode (DRC) using nitrous oxide as a reaction gas. Selenium was measured at mass 96 after being mass shifted from mass 80 by nitrous oxide. Copper was measured on-mass at mass 65 and zinc was measured on-mass at mass 68. Method performance was evaluated by conducting analytical sensitivity, precision, accuracy, recovery, linearity, and specificity experiments.

RESULTS: Utilizing nitrous oxide as a reaction gas, our laboratory developed method demonstrated an analytical measurement range of 0.1-5.0 mcg/mL for copper and zinc and 10-500 ng/mL for selenium, respectively. Within our analytical measurement range, the within-run precision for each element was determined to be less than 12 %CV and the between-run precision for each element was determined to be less than 13 %CV. Accuracy was determined by analyzing proficiency testing material from multiple vendors and were all within the target ranges for each element. The average recovery of each element was within 6% of the theoretical target. The method displayed an adequate linear response when numerous samples were diluted x2 and x10 throughout the analytical measurement range (Se, y=0.9907x + 0.657 R2=0.9998, Cu y=0.9897x + 0.0113 R2=0.9999, Zn, y= 1.0092x + 0.0078 R2= 0.9997). Elemental carryover studies indicated that instrument and diluter carryover were minimal with analyte carryover never exceeding the lower limit of quantitation for each element.

CONCLUSION: By using nitrous oxide rather than helium to attenuate polyatomic interferences, we were able to create an accurate and precise method for quickly analyzing selenium, copper and zinc at clinically relevant concentrations using a PerkinElmer NexION 5000. The use of nitrous oxide instead of helium, represents a more sustainable alternative for clinical trace metal elemental analysis.

Reduced Ion Suppression in an Automated Extraction of Vitamins B1 and B6 from Whole Blood for LCMS Analysis Kyle Dukes (Presenter) Biotage

Poster #24b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Concentrations of Vitamin B1 (Thiamine Diphosphate, TDP) and B6 (Pyridoxal-5-Phosphate, PLP) in whole blood are measured clinically to help measure various metabolic functions. Accurate concentrations of vitamins B1 and B6 are difficult to obtain due to ion suppression found in LCMS analysis. Phospholipids and proteins are a main cause of the ion suppression. Currently, laboratories are struggling on creating accurate, precise and robust sample preparation methods focused on removal of these interferences.

Objectives:
The goal of this study is to provide a sample preparation method providing accurate and precise concentrations of vitamin B1 and B6 through the removal of interfering phospholipids and proteins. This study further aids in accuracy and precision by use of automation.

Methods:
The Biotage ISOLUTE® PLD+ protein and phospholipid removal plate is used in conjunction with the Biotage Extrahera sample preparation workstation to automate the extraction of vitamins B1 & B6 from whole blood. Vitamins B1 & B6 are highly water soluble (logP= -5.9, -2.2 respectively) and are extremely light-sensitive. An acidic, aqueous crash solvent was used for whole blood in order to extract the vitamins. MRM of three ions each were monitored for quantitation of vitamins B1 & B6. Phospholipid and protein removal is measured by monitoring the 184-product ion in precipitated samples compared to the samples prepared with ISOLUTE® PLD+.

Results:
Five replicate extractions were performed on whole blood samples which were precipitated alone and five samples which were precipitated with the ISOLUTE® PLD+ protein and phospholipid removal plate. Recoveries for vitamins B1 & B6 were 94% and 82% respectively with a %RSD < 2. Phospholipids and proteins were reduced by over 90%. Percent recovery is based upon the calculated recovery of the vitamins B1 & B6 by precipitation alone, highlighting any analyte loss due to the use of the ISOLUTE® PLD+ sample preparation plate.

Conclusion:
Matrix phospholipids and proteins were successfully removed (>90%) from whole blood vitamin B1 & B6 analysis using the ISOLUTE® PLD+ protein and phospholipid removal plate. Recoveries for vitamins B1 & B6 were 94% and 82% respectively with a %RSD < 2. Additional studies will be performed to determine if this method is suitable for other biological matrices as well.

Ultra-Sensitive Molecular Detection of a Covid-19 Nucleotide Sequence Using an Enzyme-Linked Immuno-mass Spectrometric Assay (ELiMSA) Jaimie Dufresne (Presenter) Toronto Metropolitan University

Poster #25a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: There is an urgent need for sensitive assays for both protein and molecular analytes for the drug development of biologics, pharmacokinetics/pharmacodynamics, minimal residual disease detection, basic research, and diagnostics of low abundance biomolecules. Enzyme-linked Immuno-Mass Spectrometric Assay (ELiMSA) is a technology that combines affinity-based capture with enzyme amplification and detection by liquid chromatography and mass spectrometry to sensitively detect both proteins and oligonucleotides.

OBJECTIVES: The primary objective of this study was to design a molecular ELIMSA for a synthetic Covid-19 oligonucleotide sequence and to determine the sensitivity, range and linearity of the assay. In addition, the assay was performed on PCR products and compared to detection by agarose gel electrophoresis and Gel Red fluorescence staining.

METHODS: A complimentary capture sequence to the COVID-19 target sequence was fixed to a reactive NOS 96 well plate. A biotinylated detector probe and synthetic target in concentrations ranging from 100fM to 10nM were heated, linearized, applied to the capture probes, and allowed to cool and anneal. After washing, the conjugated reporter enzyme alkaline phosphatase - streptavidin (APSA) was applied and bound the biotinylated detector probe. After washing, the substrate adenosine monophosphate was applied and converted to adenosine that is highly ionizable and may be sensitively detected by LC–ESI–MS. In a second experiment, an ELiMSA was performed on the PCR products of the synthetic Covid-19 sequence and compared to PCR with detection by electrophoresis and Gel Red fluorescence staining.

RESULTS: The standard curve of the synthetic Covid-19 sequence by ELiMSA showed the target was safely detected at 100fM, had a linear range over four orders of magnitude from 100fM to 10nM and an R2 of 0.9942. PCR with gel electrophoresis and Gel Red fluorescence staining was able to detect the synthetic Covid-19 sequence at 100 femtograms whereas PCR followed by ELiMSA was able to detect the target at 100 attograms.

CONCLUSION: Complimentary nucleotide sequences coupled to reporter enzymes can now be used to detect and quantify nucleotide sequences using ultra sensitive liquid chromatography and mass spectrometry. Similar to previous protein ELIMSA, the use of mass spectral detection results in greater sensitivity than UV/VIS or fluorescent assays.

Assessment of the Impacts of Blood Collection Tube Types on Blood Acylcarnitine Determinations Dahai Shao (Presenter) The Cleveland Clinic Foundation

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Poster #27b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Blood acylcarnitine profile analysis is a powerful tool to diagnose numerous inherited metabolic disorders, including many mitochondrial fatty acid oxidation disorders and organic acidemias. It is used for follow-up testing of screen positive results from newborn screening programs and in the evaluation of children and adults suspected of having a fatty acid or organic acid disorder. Serum or plasma samples, the latter obtained with a variety of anticoagulants, are normally accepted for acylcarnitine profile analysis. In view of the diverse types of blood collection tubes used in acylcarnitine analyses, it is important to evaluate possible matrix effects on the measurement of the many acylcarnitines that are assessed in blood acylcarnitine assays.

METHODS: More than 50 acylcarnitines and related analytes were measured by liquid chromatography-tandem mass spectrometry using plasma obtained from sodium heparin (green top tube), lithium heparin (green top tube), and EDTA (lavender top tube) anticoagulated tubes, as well as serum (red top tube). Samples from each blood tube type were obtained at the same time from three healthy individuals.

RESULTS: Several analytes showed significant (>30%) matrix effects. Among them, butyrobetaine was ~30-75% higher in heparinized plasma than in serum or EDTA anticoagulated plasma. Propionylcarnitine (C3) was about 35% lower when measured in serum than in all plasma tube types.

CONCLUSIONS: When differences of acylcarnitine concentrations between serum and plasma tubes straddle medical decision cut-offs, diagnostic decisions can be impacted; this may be particularly relevant for mild and late-onset forms of inherited metabolic disorders. These data suggest the utility of using matrix-specific reference intervals for those analytes that differ significantly between tube types.

Mass Spectrometry for Classification of Pituitary Neuroendocrine Tumors William Phipps (Presenter) University of Washington

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Poster #30b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION
Immunohistochemistry (IHC) is the primary tool used clinically for measuring proteins in histologic tissue samples. This technique is subject to innumerable problems with respect to standardization and performance. Mass spectrometry (MS) offers an alternative with the potential to overcome these challenges. A representative application for which MS may be better suited is the classification of pituitary neuroendocrine tumors (PitNETs) based on protein hormone and transcription factor expression.

OBJECTIVES
The primary objective of this study was to develop a liquid chromatograph-tandem mass spectrometry (LC-MS/MS) assay to classify pituitary neuroendocrine tumors (PitNETs) using formalin-fixed paraffin-embedded (FFPE) tissue.

METHODS
Tumor regions from archived FFPE PitNET tissue specimens (prolactin, ACTH and growth hormone producing tumors) were microdissected and processed using heat-denaturation and overnight trypsin digestion. Mass spectrometry analyses were performed on the tryptic peptides using data-dependent acquisition (DDA) and parallel reaction monitoring (PRM) on a hybrid orbitrap mass spectrometer coupled to a nanoflow chromatograph. Untargeted DDA results were utilized to inform development of a targeted PRM assay.

RESULTS
Untargeted LC-MS/MS analyses correctly differentiated tumor hormone expression in all cases (n = 6), based on spectral counting for the key protein hormone markers and exactly matched the historical typing for these specimens using anti-pituitary hormone IHC (follicle-stimulating hormone, luteinizing hormone, prolactin, proopiomelanocortin, somatotropin, and thyrotropin). For all 3 PitNET subtypes evaluated (somatroph, lactotroph, and corticotroph tumors), at least two novel and high-quality surrogate peptides were identified for their respective hormone to enable complementary analyses by PRM.

CONCLUSION
The application of LC-MS/MS to solid tissues represents an alternative pathway to IHC that allows for more objective and highly parallel quantication protein expression in tissues to improve diagnosis. Classification of PitNET tumors by LC-MS/MS was robust and successful in cases with limited tissue. The novel surrogate peptides identified can be used to develop similar testing using other MS instrumentation, such as triple quadrupole mass spectrometers. This study provides a model for the development of more sophisticated and quantitative analyses of protein expression in a broader set of pituitary tumors and additional tumor types.

Insufficient T3 Sensitivity for DBS Testing on SCIEX 6500 MS Julian Reed (Presenter) Molecular Testing Labs

Poster #32a View Map

This poster will be presented and discussed on Thursday at 12:15 for 15 minutes in Montreal 3 (Track 2).

Free T3 is present in human serum at a level of 1.3-4.5 pg/mL. Due to the inherent sample volume limitations in dried blood spot testing, developing an assay with a limit of detection low enough for clinical relevance requires high sensitivity (0.5 pg/mL). Several methods were used to improve sensitivity: manual tuning, source parameter optimization, LC flow rate reduction, derivatization, column lipid cleanup, and use of a next-generation SCIEX 7500 instrument. Although quantifiable peaks were obtained in solvent standards, the sensitivity gains were not large enough to overcome matrix effects.

1. Problem: High sensitivity is needed to quantify free T3 in dried blood spots.

2. Method information:
a. Instrument: Shimadzu LC with MPX, SCIEX 6500 MS/MS
b. MP A: 0.1% Acetic Acid in Water
c. MP B: 0.1% Acetic Acid in ACN
d. Gradient: 2.5 minute gradient from 20% to 100% B at 0.4 mL/min
e. Column: Phenomenex Synergi 4 um Hydro RP 80 Å, 100 x 4.6 mm
f. MRM transitions: 651.7/197.0

3. Troubleshooting steps:
a.Source parameter tuning:
i. Gas Tune
ii. Temperature tune
iii. IonSpray Voltage tune
b.MRM mass tuning: Parent and daughter ion masses optimized via incremental mass adjustments of 0.1 amu.
c.MS/MS/MS: Attempted to reduce noise using QTRAP MS/MS/MS function, no gain in sensitivity.
d.Flow rate optimization: Flow rate varied to maximize signal
e.Derivatization: Butyl ester formation allows higher retention, greater sensitivity
f.Phospholipid transition: Several phospholipid transitions monitored for interference with T3 ionization.

4.Outcome:
a.Sensitivity of 0.5 pg/mL standard: S/N 13.7
b.This was slightly higher than the S/N obtained in SCIEX 7500 demo
c.Although neat solvent allowed quantitation at necessary levels, matrix effects in DBS samples swamped signal.

The Study of Three HPLC Column Chemistries for Optimal Separation of THC Isomers Haley Berkland (Presenter) Restek

Poster #36a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: The emergence of Δ9-tetrahydrocannabinol (Δ9-THC) isomers, particularly Δ8- tetrahydrocannabinol (Δ8-THC), have created analytical challenges as they are often not easily resolved by traditional chromatographic methodologies. When consumed, Δ9-THC forms the metabolite 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH). Similarly, Δ8-THC is metabolized to 11-Nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH). Traditional methods for separating Δ8-THC and Δ9-THC do not adequately resolve these metabolites, resulting in quantitation issues and the inability to determine an accurate value for one or both isomers. This issue is especially prevalent in urine samples, where these metabolites may be detected at high concentrations.

OBJECTIVES: The primary objective of this study was to evaluate the capability of different HPLC column chemistries to separate Δ8-THCCOOH and Δ9-THCCOOH.

METHODS: Multiple LC-MS/MS methods were developed and optimized to separate Δ8-THCCOOH and Δ9-THCCOOH using a Raptor Biphenyl (2.7 µm, 100 x 2.1 mm), Raptor C18 (2.7 µm, 100 x 2.1 mm), and a Raptor FluoroPhenyl (2.7 µm, 100 x 2.1 mm). Each method used water and methanol as MPA and MPB respectively, both acidified with 0.1% formic acid.

RESULTS: The Raptor Biphenyl did not provide any separation of these isomers under the conditions tested. Utilizing the Raptor C18 column, partial separation of the isomers was obtained in a 7-minute method but was unable to achieve complete resolution. By extending the method to 19 minutes, greater separation was achieved, but the isomers were still unable to be completely resolved. Using the Raptor FluoroPhenyl column, complete resolution of the isomers was achieved in a 7-minute method cycle time. Results were confirmed by analysis of spiked urine samples.

CONCLUSIONS: Of the three stationary phases tested, the Raptor FluoroPhenyl column chemistry provided optimal separation of Δ8-THCCOOH and Δ9-THCCOOH in the shortest analysis time. The separation completely resolved the isomers, preventing quantitation errors caused by the analytes interfering with each other.

Vitamin B12 Detection from Self-Collect Devices Using LC-MS/MS Frances Morris (Presenter) Molecular Testing Labs

Poster #38b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Cobalamin, commonly known as vitamin B12, in an essential vitamin used to maintain biological functions in the human body, including fatty acid and amino acid metabolism. Vitamin B12 is introduced to the body typically through the consumption of animal products including meat, eggs, and dairy but is also available and commonly consumed in supplementary forms and fortified foods. Deficiency of this essential vitamin is commonly observed and can lead to fatigue, anemia, weakness, and more.

Four common forms of cobalamins can be found in human serum including biologically active forms— adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl)— and common supplemental forms— cyanocobalamin (CNCbl) and hydroxocobalamin (OHCbl). Most current clinical diagnostics use immunoassays, which are incapable of distinguishing the different forms of cobalamin. However, these forms absorb differently in the human body and have different bioavailability; therefore, a simple concentration of cobalamins in serum may not adequately indicate vitamin b12 deficiency. LC-MS/MS can distinguish between these compounds both chromatographically and through the different m/z ratios of the forms and thus can provide more specific diagnostic information. Additionally, because of the prevalence of cobalamin deficiency, there is a high demand for accessible testing, such as at-home self-collect devices. Therefore, specific detection of cobalamins from self-collect devices is of interest.

Objectives:
The primary objective of this assay is to determine clinical levels of vitamin B12 from at-home collection devices.

Methods:
Patient samples were collected using dried serum separator cards and correlated to serum collected in serum separator tubes. These patient samples were cleaned up for analysis and injected on an HPLC-MS/MS system (SCIEX 6500). Calibration standards were prepared in stripped human serum and were observed at a linear range from 50.0- 2,000 pg/mL.

Results:
Cobalamin forms were successfully chromatographically separated. Prepared quality control samples at 250 pg/mL and were successfully detected for CNCbl, MeCbl, and AdoCbl (up to 8% CV).

Conclusion:
LC-MS/MS can be used to distinguish different forms of cobalamins at some clinically significant concentration levels. Further testing is needed to validate robustness and stability to ultimately be used to analyze commercially collected samples.

Development and Validation of a Multiplexed LC-MS/MS Ketone Body Assay for Clinical Diagnostics Robin Kemperman (Presenter) Children’s Hospital of Philadelphia

Poster #41a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
The ratio of nicotinamide adenine dinucleotide (NADH) and oxidized nicotinamide adenine dinucleotide (NAD+) may be used to evaluate the energy metabolism. NADH/NAD+ ratio can be related to the phosphorylation state of the cell, which is defined as the ratio of adenosine triphosphate (ATP) to the sum of adenosine diphosphate (ADP) and inorganic phosphate (Pi). Direct measurements are complicated, however; NADH/NAD+ ratio can be estimated using the beta-hydroxybutyrate (BHB) to acetoacetate (AcAc) ratio in the mitochondria. The BHB/AcAc ratio (ketone body ratio) mirrors the hepatic mitochondrial redox state, which may serve as an indicator of mitochondrial dysfunction or disease.
Due to the stability of AcAc it is challenging to accurately measure the ketone body ratio in a clinical environment in addition to sensitivity limitations. Here, we present a novel liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) based assay that measures BHB, AcAc, and their ratio, in addition to the BHB isomers alpha-hydroxybutyrate (AHB), gamma-hydroxybutyrate (GHB), and beta-hydroxyisobutyrate (BHIB). This highly sensitive and robust assay is thoroughly validated for both human serum and plasma; moreover, stability concerns have been investigated and addressed.

Methods
Serum or plasma samples were collected, placed on ice, and spun down within 2H, followed by storage in -80C freezer until analysis. Sample preparation includes a protein precipitation on ice, whereafter all five analytes are separated using a 6.5 min reversed phased LC method (Waters Acquity) followed by electrospray ionization and MS/MS analysis (Waters TQ-S). Data was analyzed and curated with Ascent V4 (Indigo) using validated customized rules to reduce processing times and human errors.

Results
All five analyte peaks were adequately separated chromatographically or held unique MS/MS transitions; for example, isomers AHB and GHB shared the same MS/MS transition, however, their LC peaks were baseline resolved at 1.77 and 1.67 min, respectively. A thorough validation was performed starting with the analytical measurement range, a span of three orders of magnitude for all analytes was established, creating a high sensitivity assay. Linearity was determined as following: 0.0025-1.5 mM (AcAc and AHB), 0.0050-1.5 mM (BHB and BHIB), and 0.0025-1.2 mM (GHB), in addition to a dilution up to 50x is permitted. The accuracy of the assay was surveyed by spike and recovery, results were obtained from ten random patient samples; an average of 99.9% (AcAc), 102.7% (BHB), 95.3% (AHB), 85.7% (GHB), and 87.5% (BHIB) was measured. Additionally, at least 40 samples were assayed for BHB using two independent assays, a mean bias of 0.01 mM and a Pearson’s R=0.996 were reported. Other analytes were not tested due to unavailability of matching reference tests. Moreover, a 20-day precision study was completed for each analyte at three levels determining the inter-assay (day), intra-assay (replicate), and intra-assay (injection) coefficient of variation (CV); all the %CVs were reported at or below 6.3%. The analytical specificity was determined by surveying a significant presence of 16 common therapeutic drugs, unconjugated bilirubin, conjugated bilirubin, hemoglobin, triglycerides, and high protein levels for a low- and high-level ketone body samples. No significant interferences were observed, all five analytes reported a %Bias < 10% for the individually surveyed interferences; except, high levels of triglycerides and protein reported a %Bias < 15%.

To accommodate multiple sample types, a specimen compatibility test was performed, heparinized plasma, red top serum, and gold top serum show a bias of <15% for all analytes, purple EDTA specimen were not accepted due to a larger bias for multiple analytes. To diagnose patients, new reference ranges have been established using approximately 120 reference samples.

Finally, due to the stability concerns of AcAc an extensive stability study was executed for all analytes, which showed that specimen are stable up to 2H on ice after draw, followed by spin down and storage at -80C. Post-plasma/serum separation, samples are stable up to 6H at room temperature, 1 day at 5C, 3 days at -20C, or at least 90 days at -80C. Additionally, none of the analytes showed a bias >20% after 5 freeze/thaw cycles, however, AHB showed positive trending 1 day. In the situation of a repeat injection, stability of the sample extracts were evaluated, extracts were stored at 5C or -20C, increased bias was observed at day two and immediately at day 1, respectively.

Conclusion
A rapid and robust LC-MS/MS based clinical assay was developed to quantify the ketone bodies AcAc and BHB, to three other isomers (i.e., AHB, GHB, and BHIB), as well as the establishment of clear standard operating procedures through an extensive validation and stress testing.

Development and Validation of an Ultra-Performance LC-MS/MS Method for Quantitation of Serum C-peptide Sung-Eun Cho (Presenter) GCLabs ESAC

Poster #42b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION : Mass spectrometric methods exhibit higher accuracy and lower variability than immunoassays for measuring serum C-peptide. We developed and validated an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for measuring serurm C-peptide. We analyzed C-peptide in multiple charge [M+3H]3+, because of its large molecular weight, which could not be analyzed by single charge. We also used derivatization step using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (AQC, Cayman Chemical, USA) to increase the ionization efficiency.

METHODS : For the pretreatment step, we first performed the solid phase extraction using Sep-Pak tC18 96-well plate, 100 mg solvent per well, 37-55&micro;m particle size (Waters, USA), and then performed the ion exchange solid phase extraction using Oasis MCX 96-well Plate, 30 mg solvent per well, 30&micro;m particle size (Waters, USA), finally we performed the derivatization step using 1 mg/mL AQC (Cayman Chemical, USA).
We used ExionLC UPLC (SCIEX, USA) system. We used Capcell Pak C18 ACR 2.0 mm &times; 10 mm, 3 &mu;m (OSAKA SODA, Japan) as the Guard Cartridge Column, and we used Capcell Pak C18 ACR, 2.0 mm &times; 150 mm, 3 &mu;m (OSAKA SODA, Japan) column. The total run time was 50 minutes and flow rate was 0.20 mL/min. The LC condition is as belows; 0.1% Formic acid in water for Mobile phase A and 0.1% Formic acid in Acetonitrile for Mobile phase B. The gradient extraction with 0 minute of A85/B15 to 35 minutes of A70/B30 was performed.
We used SCIEX Triple Quad 6500+ (SCIEX, USA) MS/MS in electrospray ionization and positive ion modes with multiple reaction monitoring transitions and 40 Psi of Curtain Gas, 11 Psi of Collision Gas, 5000 Ion Spray Voltage, 400&deg;C of Temperature, etc. The MS conditions of C-peptide and Internal standard are as belows; Q1 Mass 1064.262 Da, Q3 Mass 171.2 Da, 300 msec of Dwell Time, 71 volts of Deculstering Potential (DP), 10 volts of Exit Potential (EP), 169 volts of Collision Energy (CE), 12 volts of Collision Cell Exit Potential (CXP), and for Internal standard which is D8-Val7,10-C-Peptide (Bachem, Switzerland), Q1 Mass 1069.915 Da, Q3 Mass 171.2 Da, 300 msec of Dwell Time, 76 volts of DP, 10 volts of EP, 159 volts of CE, 16 volts of CXP, respectively.

We evaluated precision, accuracy using NMIJ CRM 6901-C (Wako, Japan), linearity, limit of detection (LOD), limit of quantitation (LOQ), carryover, ion suppression.

RESULTS : The intra- and inter-run precision CVs were less than 7 %, and the accuracy bias values were less than 7 %, which were all acceptable. The verified linear interval was 0.05-15 ng/mL, the LLOQ was 0.05 ng/mL. No significant carryover and ion suppression were observed.

CONCLUSION: The UPLC-MS/MS assay showed acceptable performance for measuring serum C-peptide. We will compare the data using this method with those from immunoassay. Even though this method would be difficult to be introduced as the actual clinical test method because of its long performance time and complex sample treatment method, this might be able to be used in a limited way for the purpose of accurate measurement of serum C-peptide.

Method Development and Validation of Paper Spray Mass Spectrometry Method for Quantitation of Remdesivir and Active Metabolite, GS-441524, in Plasma Lindsey Kirkpatrick (Presenter) Indiana University School of Medicine

Poster #47a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Remdesivir (GS-5734) is a nucleoside analog prodrug with antiviral activity against several single-stranded RNA viruses, including ebolavirus (EBOV), severe respiratory distress syndrome virus 1 (SARS-CoV-1), and Middle East respiratory syndrome coronavirus (MERS-CoV). In vitro studies of remdesivir and its active metabolite, GS-441524, also showed antiviral activity against the novel severe respiratory distress syndrome virus 2 (SARS-CoV-2), the causative viral pathogen of COVID-19, and it is one of the FDA-approved antiviral agents for the treatment of individuals with COVID-19. However, remdesivir pharmacokinetics and pharmacodynamics (PK/PD) data in humans is limited. It is imperative that precise analytical methods for the quantification of remdesivir and GS-441524 for use in PK/PD studies, therapeutic drug monitoring, and assessment of toxicity are developed.

Methods:
Seven-point calibration curves for remdesivir and its active metabolite, GS-441524, were created utilizing seven plasma-based calibrants of varying concentrations and two isotopic internal standards of set concentrations. Four plasma-based quality controls were prepared in a similar fashion to the calibrants and utilized for validation. No sample preparation was needed. Plasma samples were spotted on a paper substrate in pre-manufactured Verispray plastic cassettes and allowed to dry. The dried plasma spots were analyzed directly utilizing paper spray-mass spectrometry (PS-MS/MS). The method was validated, and the success of the validation was assessed by evaluating linearity, limits of detection (LOD), accuracy (% bias), precision (% CV), carryover, matrix effects, stability, and metabolic interferences. All experiments were performed on a Thermo Scientific Altis triple quadrupole mass spectrometer.

Results:
The calibration ranges were 20 – 5,000 and 100 – 25,000 ng/mL for remdesivir and GS-441524, respectively. The calibration curves for the two antiviral agents showed excellent linearity (average R2 = 0.99-1.00). The inter- and intra-day precision (%CV) across validation runs at four QC levels for both analytes was less than 11.2% and accuracy (%bias) was within ± 15%. Plasma calibrant stability was assessed and degradation for the 4 °C and room temperature samples were seen beginning at Day 7. The plasma calibrants were stable at -20 °C. No interference, matrix effects, or carryover was discovered during the validation process. The final method had an analysis time of 1.2 minutes.

Conclusion:
The development and validation of the first PS-MS/MS method quantitating remdesivir and its active metabolite, GS-441524, is reported. PS-MS/MS represents a useful methodology for rapidly quantifying remdesivir, which will be useful for clinical PK/PD, therapeutic drug monitoring, and toxicity assessment, particularly during future viral outbreaks.

A Novel LC-MS/MS Method for the Analysis of Antibiotics in Plasma for Clinical Research David Ballantyne (Presenter) Waters Corporation

Poster #48b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Background:
A reliable clinical research method for the analysis of a large number of antibiotics in a single sample may play a role in understanding the pharmacokinetic and pharmacodynamic effects of their administration. Currently such behaviour is not well understood.

An example of such a clinical research method for a large panel of antibiotic drugs in plasma was developed over several analytical ranges; azithromycin (5-500 ng/mL); ciprofloxacin and clindamycin (0.1-10 μg/mL); ampicillin, cefotaxime, chloramphenicol and linezolid (0.5-50 μg/mL); cefazolin, cefepime, ceftazidime, cefuroxime, flucloxacillin, meropenem and sulbactam (1-100 μg/mL); daptomycin and piperacillin (2-200 μg/mL).

Methods:
Matrix matched calibrators and QCs were prepared using in-house stocks and pooled plasma. Samples (50 μL) were treated with internal standard in methanol. A water/methanol/ammonia gradient was used with a Waters™ ACQUITY™ UPLC™ BEH C18 2.1 x 1.7 μm, 100mm column on a Waters ACQUITY UPLC I-Class FTN and Xevo TQD mass spectrometer utilizing polarity switching in a 5-minute run.

Results:
No system carryover was observed following analysis of plasma samples containing the highest concentration calibrators. Analytical sensitivity investigations indicated precise quantification (≤20% CV, ≤15% bias) at concentrations equal to or lower than the lowest concentration calibrator. Total precision and repeatability were assessed (3 pools, 5 replicates, 5 days; n=25) and determined to be ≤12.5% RSD. Linearity experiments determined the method provided first or second order polynomial fits over the ranges analyzed; additionally, each run met acceptance criteria (coefficient of correlation ≥ 0.995, determined concentrations of calibrators ±15% of nominal, ±20% in the case of the lowest calibrator). Post-column infusion experiments demonstrated analytes eluted in regions free of major ion suppression or enhancement. Evaluation of matrix effects at low and high concentrations indicated compensation by the internal standard. Addition of high concentrations of several endogenous and exogenous materials did not affect quantification.

Conclusions:
This quantitative method for clinical research demonstrates very good precision with minimal matrix effects and allows for the multiplexing of a large panel of antibiotics in plasma in a short run time.

For Research Use Only.Not for Use in Diagnostic Procedures.

Online SPE-HPLC-MS/MS Method for Measuring Metabolites of UV Filters in Human Urine Lei Meng (Presenter) Centers for Disease Control and Prevention

Poster #49a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Sunscreens are widely used as over-the-counter products in the United States. Avobenzone, oxybenzone, octocrylene and 2-ethylhexyl salicylate are active ingredients commonly used in sunscreen formulations and other personal care products. Previously many of these active ingredients were detected in plasma, amniotic fluid, breast milk and urine. However, little is known about safety of these active ingredients. Due to their widespread use, exposure data can inform evaluations on the safety of these chemicals.

METHODS: We have developed a method using enzyme deconjugation, isotope dilution, on-line solid phase extraction (SPE) coupled with high performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-MS/MS) to measure the metabolites of avobenzone, oxybenzone, octocrylene and 2-ethylhexyl salicylate in human urine, namely, di-hydroxy avobenzone (DHAVO), 2-ethyl-5-hydroxyhexyl 2-cyano-3,3-diphenyl acrylate (5OH-OC), 2-ethyl-5-hyroxyhexyl 2-hydroxybenozate (5OH-EHS), 2-ethyl-5-oxohexyl 2-hydroxybenozate (5oxo-EHS), and 5-(((2-hydroxybezoyl)oxy)methyl) heptanoic acid (5cx-EPS).

RESULTS: In this method, we use 100uL of urine. The limits of detection range from 0.02 to 0.1 ng/mL. The intra-day precisions range from 2.8-6.3%, inter-day precisions range from 5.4-10.6%. The accuracy using spike recoveries range from 86%-124%. Detection frequency in a convenience sampling from anonymous volunteers (N=110) range from 14.5-44.5%. This method is sensitive and rugged. In addition, with this method we can measure other phenolic compounds such as bisphenol A, bisphenol F, bisphenol S, benzophnone-3, triclosan, triclocarban and parabens.

CONCLUSION: We developed a sensitive and robust method to measure metabolites of four UV filters in sunscreen products in human urine samples. This method is suitable for large epidemiological studies and for biomonitoring studies to estimate the prevalence of human exposure to these compounds.

Extraction and Quantitation of Per and Polyfluoroalkyl Substances (PFAS) in Bioanalytical Matrices Determined Using UHPLC-MS/MS Adam Senior (Presenter) Biotage GB Ltd

Poster #49b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Per and polyfluoroalkyl substances (PFAS) comprise a large number of compounds that occur in a broad range of applications and products. PFAS are of concern because of their high persistence, bioaccumulation and slow elimination, and impacts on human and environmental health. Exposure to PFAS correlates with changes in metabolism, higher cholesterol, and increased risk of some cancers. PFAS pose particular challenges in the analytical laboratory as they are present in common consumables and hardware. We present methods to determine clinically relevant levels of PFAS in common biological matrices.

Objectives
This poster will present a consistent approach for robust high-sensitivity clean-up of PFAS including Gen X from biological matrices utilising a multifunctional sorbent bed in conjunction with a solvent crash/filtration-based procedure. Analytical column lifetime is improved by preventing matrix build-up over multiple injections, while maintaining analyte sensitivity over extended analytical runs.

Methods
A suite comprising 25 target analytes from 10 classes of PFAS was spiked and extracted from human serum, plasma, whole blood, and urine matrices. The targets varied by functionality, including carboxylic acids, sulfonic acids and telomers, sulfonamides, and ethoxy compounds. Sample extraction was investigated using polymer-based solid phase extraction and compared to sample clean-up using multifunctional sorbent strategies. Extraction solvents and crash/pre-treatment ratios to matrix volume were compared. Solvent pH modifiers, and matrix/solvent first protocols were also investigated where appropriate. Extract evaporation and reconstitution was compared to dilution and injection protocols using differing reconstitution/dilution solvents. Methods were optimized for plasma and whole blood matrices. We selected the best performing sample preparation methodology for maximum recovery and repeatability in conjunction with minimal matrix factors. Vacuum processing with a Biotage® Vacmaster™ 96 manifold was used throughout to minimize the number of potential contact surfaces for PFAS transfer. Final extraction protocols using a novel sample clean-up plate in a 96-well format were used to determine target analyte linearity and sensitivity at 100 µL and 50 µL load volumes for all matrices. LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC modified with a PFAS-free flow path and a pre-injector PFAS delay column, coupled to an AB Sciex 5500 triple quadrupole MS system operating in negative ion mode.

Results
The target suite of 25 PFAS was extracted from serum and urine using polymer-based reversed phase and corresponding mixed-mode weak anion exchange SPE chemistries in 30 mg 96-well plate formats. Reverse-phase chemistry demonstrated recovery below 50% for some long-chain PFAS with correspondingly high RSD. Modification of load, wash, and elution steps did not demonstrate improvements in method performance. Weak anion exchange (WAX) SPE chemistry demonstrated comparable method performance to reverse-phase SPE. Short-chain PFAS recoveries were typically above 95%, with low RSD and matrix factors close to 1. No improvement to WAX recovery was demonstrated from method modifications. The same suite was extracted using a novel multifunctional sorbent bed in 96-well plate format using 100 µL serum and urine matrix volumes. We found optimum extraction was obtained using a solvent first protocol in a 1:7 ratio with unmodified acetonitrile, target analyte recoveries were consistently above 80% with most RSD below 5%. Evaporation/reconstitution of extracts demonstrated no recovery of some short-chain PFAS and RSD over 10% for nearly half the extracted suite. A dilute/inject strategy with dilute ammonium acetate demonstrated consistent recoveries above 80%. We demonstrate optimized recoveries typically above 80% across our target suite for all matrices. Matrix factors between 0.8 and 1.3 were demonstrated for most analytes. Our extraction method is highly reproducible, the majority of RSD are demonstrated below 5%. We demonstrate sub ng mL-1 LOQs for most analytes in all matrices, at clinical levels similar to those demonstrated from NIST SRM 1957. Calibration curves were linear over our extracted range (0.1 to 100 ng mL-1), coefficients of determination (r²) were above 0.99 for all targets while demonstrating excellent matrix reduction, removing >99.9 % phospholipids where present. Background levels of PFAS in pooled and individual donor matrices tested were in the order of 1 ng mL-1. This novel media demonstrates PFAS background well below reportable limits. Low PFAS residues are vital to ensure results are reproducible and free from product-derived interference.

Conclusion
We demonstrate high PFAS analyte recovery and sensitivity with low matrix factors and repeatability. This methodology demonstrates more consistent performance compared to other commonly used sample preparation techniques: greater cleanliness than dilute-and-shoot methodology leading to more robust methods, and consistent recovery compared to reverse-phase or mixed-mode SPE methodology.

An End-to-end and High-Throughput Workflow for Therapeutic Drug Monitoring Using Evosep MRM Angela Mc Ardle (Presenter) Evosep Biosystems

Poster #53b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Therapeutic antibodies are increasingly used for treatment of autoimmune disease and cancer. However, it is a significant clinical problem that a substantial number of patients do not respond to these drugs e.g. 26.1 % response rate in lung cancer. Monitoring drug levels would allow for personalised dosing and account for individual metabolic clearance and early detection of drug immunogenicity and reduced efficacy. Traditionally, ELISAs have been used to monitor blood-based levels of therapeutic antibodies. However, this approach is associated with limited dynamic range and high risk of cross reactivity, especially in the context of multiplexed assays. Here we present a generic strategy and end-to-end workflow to enrich and quantify IgG based drugs using Evosep-MRM.

OBJECTIVES: To develop a fully automated high-throughput Evosep-MRM workflow for the quantification of IgG based drugs.

METHODS: 5 ul drug free plasma was spiked with infliximab (a chimeric monoclonal antibody, sold under the brand name Remicade) across a clinically relevant dynamic range (2-100 ug/ml). Additionally, samples were spiked with heavy labelled protein to support data normalisation. Magnetic beads coated with protein G were used to enrich immunoglobulins from the complex plasma matrix. Following this, proteins were denatured, reduced and alkylated using 5% SDS, 10mM TCEP and 40 mM CAA. Next, protein aggregation capture (PAC) onto magnetic microparticles was implemented, followed by on-bead trypsin digestion and loading of the resulting peptides onto Evotips. Standard curves were analysed by an MRM assay monitoring 10 peptides (heavy &amp; light pairs) on an Agilent 6495 QqQ coupled to an Evosep One.

RESULTS: We have developed an end-to-end workflow supporting sensitive, robust and high-throughput detection of the therapeutic antibody Remicade from plasma. Using the various gradients supported by the Evosep One system (100-500 samples per day) the assay was linear from 2 to 100 ug/ml for all gradients assessed. Additionally, inter and intra-day variability was characterised in samples across 3 concentrations (high, medium, and low). In all experiments it was possible to achieve data with coefficient of variance below 20 %. Although Remicade was used to showcase the workflow we suggest an approach whereby it can be adapted and applied to support analysis of other IgG based drugs. Additionally, we explored and report different approaches to data normalisation using heavy labelled proteins. Finally, the workflow was adapted and integrated into the Opteron 2 robot. The reproducibility of the automated workflow has been characterised and benchmarked compared to the manual approach.

CONCLUSIONS: A complete and automated sample preparation workflow was developed to support high-throughput Evosep-MRM monitoring of therapeutic drug antibodies in plasma.

Therapeutic Drug Monitoring of Mycophenolic Acid, Four Azole Antifungals and One Active Metabolite Using LC-MS/MS Zhicheng Jin (Presenter) University of Wisconsin - Madison

Poster #54a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Mycophenolate mofetil is an immunosuppressant preventing acute rejection after solid organ transplant. Mycophenolate mofetil and active metabolite mycophenolic acid (MPA) inhibit T and B cell proliferation by blocking inosine monophosphate dehydrogenase, which is required for guanosine monophosphate biosynthesis. However, unlike other immunosuppressants, serum and plasma are optimal samples for the quantification of MPA. Another class of drugs that requires therapeutic drug monitoring for post-transplant patients is antifungal drugs. Triazole antifungal drugs are commonly prescribed for the prevention and treatment of invasive fungal infections for immunocompromised patients or immunocompetent patients with comorbid conditions. Our laboratory offers a liquid chromatography assay for mycophenolic acid quantification. We also have an antifungal drugs panel on an LC-MS/MS platform. Because serum/plasma is the optimal sample for MPA and antifungal drugs, monitoring both mycophenolic acid and azole antifungal drugs simultaneously will increase laboratory efficiency while reducing the cost per test. We reported here the method development and validation of a new LC-MS/MS assay for the quantification of mycophenolic acid and antifungal drugs.

Methods:
The internal standards are mycophenolic acid-D3, voriconazole-D3, posaconazole-D4, itraconazole-D4, isavuconazole-D4, and hydroxyitraconazole-D4, which were purchased from Cerilliant (Round Rock, TX). Calibrators and controls were prepared in-house by spiking drug-free human serum with reference material purchased from Cerilliant (Round Rock, TX) or Toronto Research Company (Toronto, Canada). Serum or plasma samples were protein precipitated. After centrifugation, the supernatant was diluted with mobile phase A and was ready for injection. Analytes were separated on an Ultra C18, 50 x 2.1 mm, 3µm, analytical column (Restek, Bellefonte, PA) on a 1260 HPLC system (Agilent, Santa Clara, CA). Mobile phase A was ammonium formate (10 mM, with 0.1% formic acid); mobile phase B was acetonitrile with 0.1% formic acid. The mass analyzer was either API 6500 or API 4000 triple quadrupole mass spectrometer with an electrospray ionization source. Injection volume was optimized for API 6500 and API 4000 instruments individually. A six-point calibrator and three levels of quality control samples were included in every batch. Raw data were processed using MultiQuant software (SCIEX, Framingham, MA).

Results:
Our laboratory currently offers two separate tests for mycophenolic acid and antifungal drugs panel. MPA assay is on a Thermo Finnigan AS3000 liquid chromatography system and antifungals panel on an LC-MS/MS platform. We sought to combine two methods into one LC-MS/MS assay. This would increase laboratory efficiencies by reducing blood draws, decreasing sample volume, and reducing cost per test. In the meantime, after the new assay goes live, we can retire an aging HPLC system. MRM transitions of MPA and antifungals were optimized on API 6500 and API 4000 triple quadrupole mass spectrometers. The quantification transitions are: mycophenolic acid, 321.0>207.1; voriconazole, 350.1>281.0; posaconazole, 701.3>683.3; itraconazole, 705.2>392.2; isavuconazole, 428.1>215.0; and hydroxyitraconazole, 721.2>408.2. LC separation time is 5 min per sample. The intraday and interday assay precisions are within 1/3 of the total allowable error. The analyte measurable ranges (AMR) are: MPA, 0.4 - 48 mcg/mL; posaconazole, 0.1 - 12 mcg/mL; and 0.2 - 24 mcg/mL for voriconazole, isavuconazole, itraconazole, and hydroxyitraconazole. The test performance on API 6500 vs. API 4000 system will be compared and evaluated.

Conclusion:
We developed a sensitive and robust LC-MS/MS method that can quantify MPA, voriconazole, posaconazole, isavuconazole, itraconazole, and hydroxyitraconazole simultaneously. Our laboratory is in the process of validating this method on both API 6500 and API 4000 mass spectrometers.

U.S. Population Exposure to Anabasine and Anatabine from Cigarette Smoking: National Health and Nutrition Examination Survey (NHANES) 2013-2014 Patrick Bendik (Presenter) CDC

Poster #54b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Anabasine and anatabine are minor alkaloids in tobacco products and are precursors of tobacco-specific nitrosamines (TSNAs). The levels of these two compounds have been used to differentiate between exposure to different tobacco products, monitor compliance with smoking cessation programs, and for biomonitoring in TSNA-related studies. Urinary anabasine and anatabine are also useful for identifying exposure to products containing nicotine from different sources (tobacco-derived vs. synthetic). There is a lack of information on urinary anabasine and anatabine levels in a representative sample of the U.S. population.

Methods:
The concentrations of anabasine and anatabine were measured in urine collected from a representative sample of U.S. adults who currently smoked cigarettes (N = 770) during the 2013-2014 National Health and Nutrition Examination Survey (NHANES) study cycle. Smoking status was confirmed by urinary cotinine concentrations ≥20 ng/mL while smoking frequency was self-reported. Urinary anabasine and anatabine concentrations were measured using an isotope-dilution high-performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC-ESI-MS/MS) method. Weighted geometric means (GM) and geometric least squares means (LSM) with 95% confidence intervals were calculated for urinary anabasine and anatabine categorized by tobacco-use status [cigarettes per day (CPD) and smoking frequency] and demographic characteristics. All statistical analyses were performed using SAS 9.4 and JMP 13.2. We included strata and primary sampling unit (PSU) variables, and sample weights from the special sample set in all analyses to adjust for unequal probabilities of selection.

Results:
Urinary anabasine and anatabine are strongly, positively correlated with each other (R = 0.97) and are moderately, positively correlated with serum cotinine (R = 0.66 and 0.68, respectively). Smoking ≥20 CPD was associated with 3.6 times higher anabasine GM and 4.8 times higher anatabine GM compared with smoking <10 CPD. Compared with non-daily smoking, daily smoking was associated with higher GMs for urinary anabasine (1.41 ng/mL vs. 6.28 ng/mL) and anatabine (1.62 ng/mL vs. 9.24 ng/mL). Urinary anabasine and anatabine concentrations exceeded the established cut point of 2 ng/mL in 86% and 91% of urine samples from people who smoke (PWS) daily, respectively; in comparison, 100% of PWS daily had serum cotinine concentrations greater than the established 10 ng/mL cut point. We compared these minor tobacco alkaloid levels to those of serum cotinine to assess their suitability as indicators of recent tobacco use at established cut points and found that their optimal cut point values are lower than the established values.

Discussion:
This is the first time that anabasine and anatabine are reported for urine collected from a U.S. population-representative sample of NHANES study participants, providing a snapshot of exposure levels for U.S. adults who smoked during 2013-2014. The results of this study serve as an initial reference point for future analysis of NHANES cycles, where changes in the national level of urinary anabasine and anatabine can be monitored among people who smoke to show the effect of changes in tobacco policy.

Disclaimer:
The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention (CDC). Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention.

Screening a Clinical Cohort for Urinary Tract Infections Directly from Specimen via Lipidomics Linda Nartey (Presenter) University of Victoria

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Poster #56a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION:
Early detection of pathogens is essential for management of urinary tract infections (UTIs). Current tests require 2-3 days for culture and antimicrobial susceptibility tests for disease diagnosis. Protein analysis techniques have become the standard for clinical microbial identification, replacing phenotypic characterization and microscopic methods. These techniques are reliable, but require culture (24-48 hours) and are often labor-intensive, which increases the cost and burden of diagnostic laboratory support. A faster workflow that maintains accuracy of protein-based methods while reducing costs and time to identification is essential.

OBJECTIVES:
To use mass spectrometry (MS)-based phenotypic profiling of species-specific membrane lipids to accurately identify microbes directly from clinically specimen without culture.

METHODS:
Urine samples were collected from the Victoria general hospital and analyzed using our novel fast lipid analysis technique (FLAT). The FLAT method allows direct lipid extraction on a MALDI plate for pathogen identification by MALDI-TOF MS (Sorensen et al. Sci Repo 2020). Briefly, 1 µL of urine was spotted on an MFX µFocus MALDI plate. Acidified and incubated in a humidified chamber at 110°C for 30 min. The plate was rinsed with distilled water and then 1 µL of Norharmane (10mg/mL) was added to each spot. After drying, samples were analyzed using a Bruker Microflex in negative ion, linear mode with automated laser operation. Results were compared against the hospital’s standard protein biotyper identification.

RESULTS:
302 urine samples were collected for this study. We identified clinically significant pathogens such as E. coli, P. aeruginosa, Proteus sp, Klebsiella sp and even as polymicrobial directly from 1 µL of urine. Overall, FLAT data produced a sensitivity of 94% and specificity of 99% with positive and negative predictive value of 93 and 99%, respectively for Gram-negatives. Two patients were found to harbor a mobile colistin resistance (mcr) gene as noted by chemical modifications to their lipid A by phosphoenthanolamine. These specimens were confirmed positive for mcr-1 by polymerase chain reaction. Additionally, ~ 40% of the negative samples showed an ion at 1446 m/z, which is reported to be the signature ion for P. aeruginosa lipid A. However, these samples containing this 1446 ion by FLAT failed to produce viable colonies when isolated on a culture plate. Tandem MS results of the 1446 ion confirmed it was a most likely a host cardiolipin (Kim et al. Journal of Lipid Research, 2011). Furthermore, FLAT detected an ion at m/z 1230 present in all urine samples with blood. Tandem MS results of this ion compared against a plasma standard analyzed by FLAT confirmed it was a heme dimer.

CONCLUSIONS:
UTIs are common infections that produce a substantial workload for a clinical microbiology laboratory. The ability to identify pathogens without need for culture allows for faster pathogen identification, reduced time to appropriate antimicrobial therapy, and improved patient outcomes. When compared to the hospital’s standard protein-based MALDI-TOF MS assay, FLAT produced accurate microbial identifications within one hour of receipt in the analytical laboratory. In addition, FLAT identified pathogens present in both mono- and poly-microbial infected UTI samples directly from specimen without culture. FLAT is an affordable, one-hour rapid test that has the potential to rule out suspected UTIs in the general population greatly reducing healthcare costs by circumventing the need for culturing negative samples. Finally, FLAT can be used to improve antimicrobial stewardship by detecting 1) UTIs negative for Gram negative pathogens circumventing the need for culture required for protein-based identification and 2) select antibiotic resistance markers, which is a major advantage of lipidomic-based microbial identification over standard protein-based methods currently used in the clinical laboratory. Our study is ongoing to optimize FLAT detection for Gram positives that have a worse limit of detection than Gram negatives.

Mitigating Matrix Effects in LC-ESI-MS/MS Analysis: Comparison of 2H- and 13C/13N-labeled Internal Standards Brett Bowman (Presenter) Centers for Disease Control and Prevention

Poster #58b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION:
Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with stable isotope-labeled internal standards (SIL-IS) is the gold standard for quantitative analysis of drugs and metabolites in complex biological samples. Relatively significant isotopic effects associated with deuterium labeling often cause the deuterated IS to elute at a different retention time from the target analyte, diminishing its capability to compensate for matrix effects.

OBJECTIVES:
The primary objective of this study is to compare the effectiveness of 2H-labeled internal standards to 13C/15N-labeled internal standards for accounting matrix effects in urine specimens.
METHODS: The analytical performance of a dilute-and-shoot LC-ESI-MS/MS method with both 2H-labeled and 13C/15N-labeled ISs was examined for two biomarkers of xylene exposure (2MHA and 4MHA). Urinary 2MHA and 4MHA concentrations were assessed in 37 human urine specimens, and an analytical method comparison was performed. Due to significant differences in urinary results between internal standards, further validation experiments were performed. Spike accuracy across 14 urine specimens was performed to assess the accuracy of each internal standard. Post-column tee-infusion experiments were performed to identify the matrix effects.

RESULTS:
Analytical method comparison between ISs demonstrated that the 2H-labeled IS resulted in significantly lower urinary 2MHA concentrations compared to concentrations generated with the 13C-labeled IS. Accuracy through spiking experiments determined that the 2H-labeled IS exhibited a negative bias for 2MHA in urine matrix whereas the 13C-labeled IS generated accurate urinary results. Post-column infusion identified that non-equivalent matrix effects experienced by the 2H-labeled IS and the unlabeled 2MHA resulted in inaccurate urinary concentrations. Due to insignificant isotopic effects of 13C-labeling, coelution of the unlabeled analyte and the 13C-labeled IS occurred, and the internal standard experienced equivalent matrix effects. No bias was observed across ISs for 4MHA, and post-column infusion demonstrated that equivalent matrix effects were observed for 2H-labeled IS, 13C/15N-labeled IS, and unlabeled 4MHA.

CONCLUSION:
Selection and validation of a SIL-IS is a critical step in the development of an effective LC-MS/MS assay for biomonitoring applications. The relatively significant isotopic effects observed with 2H-labeling causes different retention times to the unlabeled analyte, and therefore may bias quantitation in complex biological matrices.

DISCLAIMER:
The views and opinions expressed in this report are those of the authors and do not necessarily represent the views, official policy or position of the U.S. Department of Health and Human Services or any of its affiliated institutions or agencies. The use of trade names is for identification purposes and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, or the U.S. Department of Health and Human Services.

Single-Step Sample Preparation for Quantitation of Serum Nicotine and Cotinine by LC-MS/MS Richard Giles (Presenter) Cleveland Clinic Foundation

Poster #59b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

BACKGROUND

Determination of the effectiveness of patient smoking cessation programs often requires careful monitoring of both nicotine and its primary metabolite, cotinine. Accurate measurement of blood nicotine and cotinine concentrations are important in the qualification of transplant candidates and evaluating their exposure to tobacco smoke post-transplantation, but the levels of these compounds are much lower in serum or plasma than urine. Significant opportunities for improvement in the existing method employed in our laboratory were also identified in both the sample preparation procedure as well as in mitigation of carryover. A method was thus needed that could quantitate nicotine and cotinine both at low levels (2 ng/mL) as well as much higher levels (50 ng/mL and 1000 ng/mL for nicotine and cotinine, respectively) to ascertain the degree of potential exposure. Our aim was to develop and validate a method for more efficiently quantitating nicotine and cotinine in serum over an expanded analytical measurement range with a reduced sample volume while streamlining sample preparation.

METHODS

Nicotine, cotinine, and their deuterated internal standards were extracted by protein precipitation. Briefly, 100 µl of samples, controls, or calibrators were transferred to a 96-well plate and mixed with 200 µl of protein precipitation solution (aqueous 0.2 M ZnSO4 in methanol, 3:7 v/v) containing the internal standards (nicotine-d3 and cotinine-d3 at 25 ng/mL). The plate was vortexed vigorously for one minute followed by centrifugation for five minutes, after which the supernatant was ready for injection without further manipulation. This procedure eliminated both an extended incubation step as well as the addition of caustic potassium hydroxide solution utilized in the predecessor assay. Each sample was analyzed on a Transcend II LX-2 LC coupled to a TSQ Quantis™ triple quadrupole mass spectrometer (Thermo Scientific). Online sample cleanup was performed using a turbulent flow method on a TurboFlow™ Cyclone MCX-2 mixed-cation exchange column (0.5 × 50 mm; Thermo Scientific). The compounds were separated chromatographically on a porous graphitic carbon Hypercarb™ analytical column (3.0 × 50 mm, 5 µm particle size, Thermo Scientific) at ambient temperature with an injection volume of 20 µl. Multiple reaction monitoring (MRM) was used to monitor quantifier and qualifier transitions for nicotine, nicotine-d3, cotinine, and cotinine-d3. An extended column washing step was implemented after each injection to mitigate carryover. The total analytical runtime was 6.0 minutes. Ion suppression/enhancement, matrix effects, reportable range, analytical sensitivity, carryover, intra- and inter-assay precision, and method comparison were assessed and validated.

RESULTS

The chosen alternative matrix (SeraCon™ II CD Hormone Depleted Double-Stripped Negative Diluent, SeraCare) was found to be suitable for calibrator use (maximum bias observed -6.7% for nicotine, -6.9% for cotinine when mixed with patient samples). No ion suppression or enhancement near the retention time of either analyte was observed. No interferences were found for hemolyzed, lipemic, or icteric samples, other nicotine metabolites or related compounds (nornicotine, trans-3'-hydroxycotinine, anabasine, cotinine-N-oxide), or commercial interference mixtures (Cerilliant Interference Mixtures 2-7, 40+ potential interferents). Reportable range was validated from 2 ng/mL to 50 ng/mL for nicotine, and from 1.6 ng/mL to 1000 ng/mL for cotinine. For nicotine, the mean recovery at the LLOQ was 96.5%; for cotinine, the mean recovery at the LLOQ was 103.5%. To evaluate the analytical sensitivity of the method, the limit of detection (LoD), limit of blank (LoB), and lower limit of quantitation (LLOQ) were determined for each analyte. For nicotine, the LoD was 0.34 ng/mL, the LoB was 0.26 ng/mL, and the LLOQ was 2.08 ng/mL (2.55% CV). For cotinine, the LoD was 0.41 ng/mL, the LoB was 0.32 ng/mL, and the LLOQ was 2.08 ng/mL (2.87% CV). Acceptable carryover limits (<1 ng/mL) were found for nicotine at 100 ng/mL and cotinine at 1500 ng/mL. For nicotine, the intra-day precision CV ranged from 1.3% to 1.8% and the inter-day precision CV ranged from 1.4% to 3.0%. For cotinine, the intra-day precision CV ranged from 0.8% to 1.5% and the inter-day precision CV ranged from 1.6% to 1.9%. Comparison with an independent LC-MS/MS method showed an average bias of -3.6% for nicotine and -7.4% for cotinine. Deming regression analysis of the results indicated a slope of 1.049 and intercept of -1.0 (R=0.9915) for nicotine, and a slope of 0.915 and intercept of 2.8 (R= 0.9985) for cotinine.

CONCLUSION

The described LC-MS/MS method for quantitation of nicotine and cotinine in serum was developed to minimize sample preparation time while retaining accuracy and precision. The sample preparation employs a streamlined one-step protein precipitation step intended to increase the efficiency of workflow in our clinical laboratory, and the higher carryover limits target a significant reduction in the number of patient sample analyses that must be repeated.

Determination of Free Thyroxine from Human Serum Using BioSPME Sample Preparation Prior to LC-MS/MS Analysis Olga Shimelis (Presenter) MIlliporeSigma

Poster #60a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:

Absolute free thyroxine concentrations remain relatively constant, approximately 0.02% of total thyroxine (tT4) is available in circulation in the free form. The vast majority is bound to thyroxine-binding globulin and to a lesser extent albumin and transthyretin. Direct immunoassays, the most common test performed at clinical laboratories, suffer from interferences and lack of specificity. In a 2002 study of > 5,000 patients, reported this incident occurring in ~0.5% of samples (Ismail AAA et al. 2002, Ann Clin BioChem). Liquid chromatography tandem mass spectrometry (LC/MS/MS) methods have been developed over the past 10 years to afford improved specificity and accuracy. The current standard for separation of free hormones prior to analysis has been equilibrium dialysis (ED). In general, ED is a lengthy process, on average overnight at minimum. A novel sample preparation has been utilized to alleviate this. Solid phase microextraction, SPME, has been developed into a 96-pin device termed BioSPME to prepare samples in under one-hour.

Objective:

A collaboration was established between MilliporeSigma and ARUP Laboratories to investigate the results of sample preparation by a BioSPME device or ED prior to LC/MS/MS analysis for measurement of free thyroxine in serum.

Methods:

The free thyroxine (fT4) and/or free triiodothyronine (fT3) were analyzed on Agilent 1290 LC utilizing an Ascentis Express Biphenyl (10 cm x 2.1 mm, 2.7 µm) connected to an AB Sciex 6500 QQQ. Quantifier and qualifier transitions were utilized for unlabelled and isotopically 13C6-labelled thyroxine (T4), triiodothyronine (T3), and reverse triiodothyronine (rT3). The extracted calibration standards were used to directly report the concentrations of fT4 and/or fT3.
Initial method development utilized bulk serum that was tested by an external clinical laboratory for independent determination of fT4 and fT3 by a validated ED-LC-MS/MS method. These samples were additionally used to perform reproducibility assays of the method.

The BioSPME sample preparation method consisted of using 200 µL volume samples and a C18 coated 96-pin device with a Hamilton Starlet system for automation. The method included multiple steps of transferring the device between four well plates (condition, wash, sample, and desorption). The sample plate consisted of calibration standards prepared in 7.5 mM HEPES at pH 7.5 and serum samples which were diluted with 5% (v/v) of 1.15 M HEPES to adjust the pH. Extracted samples were desorbed into 40 µL of methanol containing internal standards. The samples were then diluted with 40 µL of water by the autosampler prior to injection.

Reinjection of extracted samples was investigated using 29 serum samples and 7 calibrators.

Serum samples for robustness testing were provided in conjunction with ARUP Laboratories (Salt Lake City, UT) and were previously determined by equilibrium dialysis-LC-MS/MS using the method described by Bingfang Yue (Yue, B, Rockwood, A, et al. 2008, Clinical Chem).

Results:

Six commercial serums were tested on five (n=4 per extraction) separate occasions to test reproducibility of the method for fT4 and fT3. On average, the interday %CV for fT4/fT3 were as follows: 10.9/9.2%, 5.5/9.2%, 8.6/6.9%, 10.2/9.5,% 4.0/8.5%, and 7.8/7.2%.

A series of different sets of serum samples were investigated. The first set of 24 (n=3), a correlation of ED-LC-MS/MS to BioSPME-LC-MS/MS yielded a relationship of y = 0.738x + 0.322, R2 = 0.867. The average % CV for the fT4 concentration was 8%. On average, fT4 values for BioSPME-LC-MS/MS were 3% lower than ED-LC-MS/MS.

In a second set of 45 samples (n=1), the overall correlation was y = 0.781x + 0.464, R2 = 0.819. On average, the fT4 values using BioSPME-LC-MS/MS were 22% lower than ED-LC-MS/MS. Additional method development is on-going to increase the accuracy of the BioSPME-LC-MS/MS method.

A study involving 29 serum samples and 7 calibrators were reinjected non-sequentially twice. On average, there was a 4.1% difference in concentration.

The LLOQ of the extracted calibrators was 1 pg/mL with a 40 µL injection with an % CV of 10.9% and 19.8% (quantifier and qualifier). The peak integration ratio for the LLOQ for the quantifier/qualifier was 1.00, % CV 14.6%. The sensitivity was achieved by replacing the 20 µL standard injection loop on Agilent 1290 LC instrument with a 100 µL loop to allow for larger injection volumes.

Conclusion:

A BioSPME extraction method prior to analysis by LC-MS/MS was developed, and the evaluation results showed strong correlation against equilibrium dialysis for determination of free thyroxine (fT4) from serum samples. The BioSPME method was automated by using a Hamilton robotic system and can be adapted to other robotic liquid handlers that have gripper functionality. The time to process one 96-well plate was less than an hour. The developed LC-MS/MS detection method included a built-in preconcentrated sample (5x) without dry-down steps and the ability of get repeat injections with consistent results.

Quantitative Amino Acid Analysis by Isotope Dilution LC-MS/MS for Concentration Assignment of Protein Reference Materials Pei Liu (Presenter) MilliporeSigma

Poster #63a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Accurate protein concentration measurement is crucial for the development of protein certified reference materials (CRMs), which will be used in the calibration of a variety of procedures used in nutritional and clinical analyses. Accurate concentration assignment of a chemical analyte is typically based on gravimetry and mass purity. However, this is generally not feasible for proteins, as often there is not enough solid protein standard available. , Amino acid analysis (AAA) has become a crucial technique to assign certified, SI traceable concentration values to protein solutions, with ultraviolet or fluorescent based detection being widely used. However, these methods require pre-column or post-column derivatization steps which are inherently more prone to measurement error. Isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) is considered as the &ldquo;gold standard&rdquo; for biomolecule quantification as it can provide high accuracy and precision measurements. Here we present the development of a method for quantitative determination of protein solutions using ID-LC-MS/MS.

Methods:
Primary reference standard NIST SRM 2389a, Amino Acids in HCl, was used for calibration. NIST SRM 927e, Bovine Serum Albumin 7% Solution, was used for method optimization and validation. Proteins were hydrolyzed with vapor-phase HCl for 20-24 hours at 110°C in sealed vials. An internal standard Certified Reference Material mix of stable isotope labeled amino acids (Supelco, 96378) was added to calibrators and hydrolyzed proteins. LC-MS/MS was performed using a SeQuant ZIC-cHILIC column (3&micro;m,100&Aring; 150 x 2.1 mm) with a zwitterionic stationary phase on an Agilent 1290 Flex UPLC connected to an AB Sciex 4000 QTRAP. Data were acquired for select amino acids in MRM mode with segmenting to maximize dwell times for better quantitative measurements.

Results:
A linear regression model was applied to all calibration curves and the associated coefficient of determination (R2) were &gt;0.99 for all amino acid transitions, demonstrating a low variability and good accuracy of analyte concentration. The coefficient of variation (CV) for the injection reproducibility of the LC-MS/MS measurements across all amino acids ranged from 0.92% (threonine) to 2.1% (serine), with an average CV of 1.5%, signifying a high level of measurement precision. Using this ID-LC-MS/MS method, we observed recovery of NIST BSA SRM927e ranged from 96.9% to 101.7%, with an average recovery of 99.9%.

Conclusion:
This work demonstrates that amino acid analysis by isotope dilution LC-MS/MS can provide protein quantification with excellent accuracy and precision. Measurement accuracy and traceability was established through the use of NIST SRMs for calibration and control. This method will provide an improved approach for assignment of concentration values of accuracy-based protein reference materials used for calibration of LC-MS methods, such as those used in clinical diagnostics.

Detecting Glycosylation of Monoclonal Light Chain in MGUS Patients by MALDI-TOF MS-based-assay (MASS-FIX) Qian Wang (Presenter) Mayo Clinic

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Poster #66a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Monoclonal gammopathy of undetermined significance (MGUS) is the most common plasma cell disorder found in approximately 3% of the population over 50 years old. Patients with MGUS are usually asymptomatic and have persistent risk of progression to multiple myeloma or other plasma cell disorders of 1% per year. Since the rate of risk does not decrease over time, lifelong follow-up is required. It is important to identify risk factors to predict groups of MGUS patients with high risk of progression, which can be essential for defining frequency of monitoring, and early diagnosis of multiple myeloma or related disorders. N-glycosylation of monoclonal light chains has been identified as an important risk factor for progression to primary amyloidosis. A MALDI-TOF MS based-assay with use of isotype-specific nanobody enrichment (MASS-FIX) has been developed and validated to detect and type monoclonal light chains in plasma cell disorders. MASS-FIX is more analytically sensitive, specific, cost-effective, and efficient compared with immunofixation with gel electrophoresis, and it enables easy identification of glycosylated monoclonal immunoglobulins.

Objectives:
In this study we aimed to assess the prevalence of light chain glycosylation at the time of recognition of MGUS.

Methods:
Our study cohort consisted of 849 serum samples from unique individuals who lived in the 11 counties of southeastern Minnesota. They had samples collected within 30 days of a previously established MGUS diagnosis at Mayo Clinic, and samples were kept frozen at -80oC until testing. Samples were tested for serum protein electrophoresis using agarose gels (Helena Laboratories), immunofixation (Sebia Inc.) and free light chains (FreeLite, Binding Site) were tested by nephelometry on a Siemens BNII. In addition, MASS-FIX was used to detect glycosylation of the immunoglobin light chains. Serum samples were incubated with agarose beads coupled with antibodies targeting κ or λ light chain constant domains respectively for immuno-enrichment. Beads then washed, reduced, spotted, and analyzed separately on MALDI-TOF MS (Bruker Corporation). The spectra were analyzed using FlexAnalysis software (Bruker Corporation). All analyses were conducted using R (version 4.2.1).

Results:
Of the total 849 MGUS patients tested, median age was 72 years old (range 24 to 96), and 44.9% were female. Median serum median M-spike concentration was 0.80 g/dL (range 0.20 to 4.5) although 140 out of 849 patients did not have an M-spike available. The most common isotype identified was IgG kappa (352, 41.8%), followed by IgG lambda, (219, 26.0%) and IgM kappa (92, 10.9%). Free light chain testing results for the kappa/lambda ratio were within reference intervals (0.26 to 1.65) in 66.8% of the cohort, with the remainder being abnormal. 45 patients (5.3%) were found to have glycosylated light chain. This is aligned with prevalence of glycosylation found in previous studies. Patients with and without glycosylated light chains have similar characteristics including age, gender, hemoglobin, serum creatinine, M-spike, and prevalence of abnormal free light chain ratio results (all P≥.10). 71.1% were kappa glycosylated light chains, and 28.9% were lambda glycosylated light chains, free light chain ratio was normal in 55.6% of cases. The lowest M-spike value associated with glycosylated light chains was 0.40 g/dL.

Conclusion:
MALDI-TOF mass spectrometry-based assay (MASS-FIX) can easily detect and identify glycosylation of monoclonal immunoglobulins. The prevalence of light chain glycosylation at the initial diagnosis of MGUS is about 5%. Glycosylation further characterization can provide valuable information on disease patterns, and follow-up information from patients over time may add value in stratifying patients into groups that would be at higher risk of progression to a malignancy at early stages of MGUS.

Using LC-MS/MS LDT to Determine Fentanyl Prevalence and Evaluate the FEN2 Immunoassay’s Real-World Clinical Performance in Tertiary Care Settings Marlen Menlyadiev (Presenter) UCSD Health

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Poster #66b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
LC-MS/MS laboratory-developed tests (LDTs) are the mainstay of modern clinical toxicology testing. While widely used to support the development of FDA-cleared drug immunoassays, their significance in the clinical implementation and evaluation of such assays is less recognized. In this work we report on the use of our LC-MS/MS opiates method for determining the prevalence of fentanyl in urine drug screen (UDS) samples and assessing the real-world clinical performance of the Roche FEN2 fentanyl assay in routine clinical use. This work expands on our previous report of the FEN2 assay implementation.

Methods
Excess specimens from a total of 250 consecutive random UDS clinical samples were collected between 05/04/22 and 05/17/22 under UCSD IRB protocol 181656. These specimens were first screened using the DRI assay (Thermo Fisher Scientific) followed by the analysis by the FEN2 assay (Roche Diagnostics). Each specimen in the study was sent to the clinical toxicology laboratory for quantitative analysis by LC-MS/MS method for fentanyl and norfentanyl. Fentanyl prevalence in the tested population was calculated by examining the extracted ion chromatograms from analyzed samples for fentanyl and norfentanyl peaks (retention times, quantifier-to-qualifier ion, and signal-to-noise (S/N) ratios, etc.). For calculation of the clinical sensitivity and specificity of the FEN2 (and the DRI) immunoassay, sample were classified as true positive if they contained at least 2 ng/mL of fentanyl and/or norfentanyl. The same cutoffs were used when querying EHR to evaluate real-world clinical performance (screening and confirmation positivity rates, rates of false positives and negatives) of the FEN2 and the DRI immunoassays. False positive and negative rates in queried EHR cohorts were determined from samples that screened positive by immunoassay and did not confirm by LC/MS/MS or, conversely, from samples that screened negative, but had fentanyl and/or norfentanyl by LC-MS/MS.

Results
Thirty-eight of 250 study samples were found to contain fentanyl and 49 samples - norfentanyl at ≥ 2ng/mL concentration. Fifty-one samples contained fentanyl, norfentanyl or both analytes at ≥ 2ng/mL. The median fentanyl and norfentanyl concentrations in these 51 samples were 5 and 15.5 ng/mL, respectively, with corresponding inter-quartile ranges (IQRs) of 43 and 85 ng/mL. In addition, in 6 samples from the 250-sample study pool, fentanyl and/or norfentanyl were detected (signal-to-noise ratio >3, acceptable quantifier-to-qualify ion ratios, etc.) but not quantified. These findings corresponded to 22.8% prevalence of fentanyl in our study population.
Of the 51 LC-MS/MS true positive samples in the study, 31 and 50 were classified correctly by the DRI and the FEN2, respectively. Both assays classified 198 of the 199 LC-MS/MS-confirmed true negatives as negative. The clinical sensitivity and specificity calculated from these data were 61% and 99.5% for the DRI and 98% and 99.5% for the FEN2. The real-world screening positivity rate with the DRI and the FEN2 assays during 1 month testing period was, respectively, 13.3% and 17.3%, with the corresponding LC-MS/MS confirmation rate for immunoassay-positive samples of 88.8% and 96.8%. Higher immunoassay positivity rate for FEN2 was likely due to its ability to detect norfentanyl. The false positive rates for the DRI and the FEN2 in queried EHR cohorts (1000+ entries) were, 11.2% and 3.2%, respectively, while false-negativity rates (using smaller subset of total immunoassay screens) were 22% and 5.5% for the DRI and the FEN2 assays.

Conclusion
The use of LC-MS/MS LDT enabled estimation of the prevalence of the fentanyl in the study population (urban tertiary care hospital) and evaluation of the clinical performance of the FEN2 and DRI assay in both the study population and during 1 month of routine clinical use. Our results demonstrate the FEN2 assay has greater clinical sensitivity and is less prone to false positive results as compared with the DRI assay. These findings support the implementation of the FEN2 in a routine clinical practice and underline the broader role of mass spectrometry-based LDTs in clinical toxicology testing.

Plasma and Platelet Lipidome Changes in Treated Fabry Disease Patients Bo Burla (Presenter) Sling @ National University of Singapore

Poster #67a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION
Fabry disease (FD) is an X-linked lysosomal storage disorder characterized by reduced activity of α–galactosidase A (GLA) resulting in progressive accumulation of the globotriaosylceramide (Gb3). Clinical manifestations at later stages include chronic kidney disease, cardiomyopathies, and strokes. Estimated prevalence varies from 1:40,00 to 1:200,000 for classic FD, and down to 1:1,200 for later-onset FD. FD may also be present in initially unexplained diseases, e.g., cryptogenic stroke, cardiomyopathies, and chronic kidney disease.

In FD diagnosis, determining pathogenicity of detected variants may not always possible. In these cases, mass spectrometry (MS)–based analysis of plasma and urinary Gb3 and metabolites is recommended (PMID: 27195818). This indicates the need and utility of MS-based methods in screening and diagnosis of this under-diagnosed condition.

Platelets from a FD rat model were shown to have 7,000–fold increased Gb3 levels. In humans, a proinflammatory state and systemic inflammation have been reported for FD. However, no data is currently available on Gb3 profiles in human platelets from FD patients treated with enzyme-replacement therapy (ERT).

OBJECTIVES
The primary objective is to analyze Gb3 concentrations in platelets of ERT-treated FD patients and in healthy controls. The secondary objective was to test and validate previously reported Gb3 species (PMID: 23968398) in plasma of ERT-treated FD patients as diagnostic markers of FD. The third objective was conducting a comprehensive lipidomics analysis of both plasma and platelets from FD patients.

METHODS
Platelets were isolated using differential centrifugation in presence of anticoagulants. Plasma and platelet lipids were isolated via a simple single-step butanol/methanol liquid extraction.MS–based lipidomics analyses were performed using reversed-phase liquid chromatography and triplequadrupole MS.

RESULTS
Gb3 levels in platelets from ERT-treated FD patients were not increased compared to healthy controls. However, platelets exhibited high between-subject variability of Gb3, possibly associated with specific blood groups. Plasma Gb3 species harbouring sphingadiene (d18:2) showed an indeed better discrimination of FD patients from healthy controls compared to Gb3 species containing sphingosine (d18:1) that are commonly measured. The lipidomics profiling revealed changes in plasma GM3 gangliosides, sphingosine 1–phosphates, abundance of specific fatty acids in lipids, specific plasma ceramide ratios, and platelet acylcarnitines.

CONCLUSION
This study demonstrates that changes in plasma and platelets lipidomes in FD go beyond Gb3 and its metabolites. Furthermore, ERT seems effective in preventing Gb3 accumulation in platelets of FD patients. Lastly, this study supports the potential use of specific Gb3 species as novel makers for screening and diagnosis of FD.

Simultaneous Quantification of Monosaccharides in Human Serum: Development of a High-Throughput Derivatization-free ID-LC-MS/MS Method Chui Tse (Presenter) Centers for Disease Control and Prevention

Poster #67b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Monosaccharides play an important role in human metabolism. While glucose is the major biomarker for diabetes diagnosis and treatment, emerging evidence has suggested relationships between monosaccharides and other diseases. Elevated mannose levels in blood have shown association with insulin resistance in diabetic patients. High fructose consumption has been associated with increased de novo lipogenesis in the liver leading to non-alcoholic fatty liver disease (NAFLD). Simultaneous measurement of glucose, fructose, mannose, and other monosaccharides may be helpful to understand their roles in different diseases.

OBJECTIVES: Limited data is available about levels of different monosaccharides in the population. Although some enzymatic assay kits can measure a few monosaccharides simultaneously, the analytical variance in quantification of these monosaccharides with different concentrations can be high. GC/MS methods often require lengthy sample preparation which can significantly impact the throughput. To address the limitations, there is a need for an accurate and precise laboratory method for measuring serum monosaccharide panels. We aim to develop a high-throughput analytical LC/MS/MS method for serum monosaccharides measurement that is suitable for large scale epidemiologic studies.

METHODS: The assay was calibrated using certified primary reference material NIST SRM 917c for glucose. The 13C labeled monosaccharides were spiked into serum samples and calibrators as internal standards. Using an automated liquid handler system, acetonitrile was added to precipitate monosaccharides from serum matrix and extracts were cleaned up in a 96-well protein removal plate. Monosaccharides from the samples were quantitated using a LC/MS/MS system with acetonitrile and water LC gradient.

RESULTS: Monosaccharides, including glucose, fructose, mannose, and galactose, were fully resolved under 9 minutes demonstrating a high level of specificity. The analytical measurement range for glucose covered 10 – 380 mg/dL (R2 = 0.999). Other monosaccharides were linear between measurement range of 0.3 – 12.2 mg/dL (R2 = 0.999). The assay sensitivity allowed detection of 0.27 mg/dL for glucose and 0.025 – 0.046 mg/dL for other monosaccharides. Average measurement accuracy of 97% and imprecision of 3% for serum glucose were achieved using serum-based reference material NIST SRM 965b across 8 days measurement. Spiked recovery and inter-day imprecision of other monosaccharides were above 95% and between 3-8%, respectively. Comparison with the CDC glucose GC-MS RMP using 19 single donor serum samples (glucose concentration range from 23.11 to 376.53 mg/dL) showed good agreement from Deming regression analysis. The CV% of the IS-norm MF was between 3-12% across 6 serum samples demonstrating minimal influence of the matrix on all monosaccharides analysis. Relative ME% calculated by comparison of the CV% values of standard line slopes across four different serum lot samples is less than 2% for all analytes which served as a measure of minimum relative ME between different subjects. A preliminary study of 83 human serum samples were analyzed with detection of glucose, fructose, and mannose from all samples. A statistically significant positive correlation (r=0.58; P < 0.001) was observed between glucose and mannose levels with both monosaccharides normally distributed across the population while no correlation was observed neither between glucose and fructose, nor mannose and fructose.

CONCLUSION: The current analytical method is sufficiently accurate and precise to measure concentration levels of a panel of serum monosaccharides from human serum. Using a simple and derivatization free sample procedure with automation system has significantly improved the throughput of the assay for routine serum monosaccharides measurements in clinical laboratories and for large biomonitoring studies.

Laboratory Automation to the Rescue: Evolution of the Clam Platform for Automated LC-MS/MS Sample Preparation Scott Kuzdzal (Presenter) Shimadzu Scientific Instruments

Poster #69a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Laboratory medicine is a critical component of the complex U.S. healthcare system. Since the onset of the COVID-19 pandemic, laboratory personnel have taken on greater responsibility, working longer days, picking up extra shifts and in some cases even working across multiple labs to keep up. Laboratory automation removes a lot of repetitive work required of lab professionals, increasing laboratory efficiency by providing consistent productivity and quality. LCMSMS sample prep can be automated with excellent accuracy and reproducibility on the new Shimadzu LCMS-2040 platform. Automation reduces method variability, the chance of sample mix-ups and the risk of exposure to samples.

Methods
The analysis was performed on human plasma samples using a Shimadzu LCMS-8060 triple quadrupole mass spectrometer equipped with a clinical laboratory automation module (CLAM) for fully automated sample preparation and a Nexera X2 UHPLC with a Shim-pack Scepter C18-120 column (2.1 mm I.D. &times; 50 mm, 1.9 &mu;m) in ESI positive mode.

Preliminary Data
We herein demonstrate automation of the analysis of the antiviral remdesivir and its main plasma metabolite, GS-441524, a nucleoside analogue antiviral drug, with excellent accuracy and reproducibility. We also describe recent innovations in the CLAM automated sample preparation platform. Mass spectrometric chromatograms of Remdesivir and GS-441524 are demonstrated in plasma with good accuracy and reproducibility. The repeatability of less than 4% RSD and the accuracy between 89-106% on each control sample confirmed the robustness and the efficiency of this method (n=6). Several hardware modifications are herein presented, which result in enhanced ease of use as well as performance improvements. A forensic toxicology database for LC-MS/MS was added and the CLAM is now able to connect to a Laboratory Information System (LIS) for bidirectional communications.

Novel Aspect
Advances in full automation of LCMS sample preparation for the analysis of antiviral drugs is presented. These innovations improve CLAM automated sample preparation for the clinical research of anticoagulants, immunosuppressants, toxicology (STAT screens), methylmalonic acid (MMA), anti-epileptics, amino-glycoside antibiotics, antiarrhythmic drugs and beta-blockers.

Hydrolysis Monitoring for Accurate Clinical Measurement of Total Carnitine Artis Miller (Presenter) The Children’s Hospital of Philadelphia

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Poster #71a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Carnitine plays a crucial role in transporting fatty acids into the mitochondrial matrix, and conjugation of free carnitine to an acyl group to form an acylcarnitine is a necessary step in this process. Free and total carnitine are routinely measured in order to diagnose carnitine deficiency as well as inherent mitochondrial defects. Free carnitine is measured by tandem mass spectrometry after a simple protein crash, and it represents the fraction of carnitine unconjugated to fatty acid. Similarly, total carnitine is measured by flow injection-MS/MS after alkaline hydrolysis to liberate carnitine from associated acyl groups. Acylcarnitine is calculated as the difference between total and free carnitine. Because of the dependence on efficient alkaline hydrolysis, both the total and acylcarnitine values may be artifactually low if carnitine is not fully liberated from bound acyl groups. Importantly, the ERNDIM quality assurance program only tests free carnitine, and thus it is possible to miss systematic problems in the hydrolysis step if total carnitine is also measured.

Methods:
Our objective was to develop an automated flag to detect samples in which hydrolysis was incomplete. Our underivatized free/total carnitine method monitors free carnitine (C0) by flow injection-MS/MS (quantifier: 162.25 -> 85.17; qualifier: 162.25 -> 103.19) and a d3-carnitine IS. We added an additional MRM transition to monitor acetylcarnitine (C2) (204.25 -> 85.17), which was also normalized using the C0 IS. We monitored the decrease in normalized peak area of C2 in free vs. total carnitine samples in order to detect appropriate hydrolysis. This calculation was also added to Indigo Ascent, and a rule was constructed to flag samples with insufficient % decreases in C2.

Results:
Although many acylcarnitines are present only in low abundance in healthy individuals, acytylcarnitine (C2) is normally present at levels of approximately 4-21 umol/L, with notably high values present with ketosis. As such, measuring its decrease as a result of alkaline hydrolysis may provide a convenient way to detect the efficiency of this step. We added an MRM transition for C2 to our flow injection free/total carnitine assay, and because of co-elution during flow injection we used the C0 IS to provide overall normalization of the C2 peak (designated nC2).

During assay validation, we identified 2 outlier measurements of a high control sample. Interestingly, these outliers showed a substantially higher (total nC2) / (free nC2) ratio (0.62-0.67) compared with other samples (<0.1), consistent with suboptimal hydrolysis. This corresponded with lower measured total carnitine in these results compared with the other values in the precision study, as would also be expected for incomplete hydrolysis. Free carnitine values were comparable across the precision replicates, showing that this did not reflect a global change in the ability to measure carnitine. These data provided evidence that total / free nC2 is a useful measure to identify samples in which incomplete hydrolysis has occurred. Subsequently, this measurement has been implemented as a rule in Indigo Ascent, and we show a clinical example where a sample would have been reported with an artifactually low total carnitine if the nC2 ratio had not flagged incomplete hydrolysis.

Conclusions:
Monitoring the %decrease of normalized C2 abundance between free and total carnitine samples using a rule-based flag in our MS processing software (Indigo Ascent) provides a convenient and automated method to ensure the integrity of the hydrolysis step in free/total carnitine assays.

Improvement and Validation of a Commercial LC-MS/MS Assay for a Sensitive, Robust, Automatable and Fast Measurement of 1,25-(OH)2-Vitamin D2/D3 Markus Boesche (Presenter) Immundiagnostik AG

Poster #74b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction/Background
Vitamin D is a fat-soluble steroid vitamin, of which 25-(OH)-vitamin D, the major circulating form produced in the liver, is being further metabolized in the proximal tubule of the kidney to 1α,25-dihydroxyvitamin D (calcitriol), which is the most biologically active form of vitamin D. The influence of 1,25(OH)2-vitamin D on calcium and phosphate metabolism, as well as bone metabolism, is very well researched. The innate immune system is stimulated and the risk of upper respiratory infections is decreased by 1,25(OH)2-vitamin D2/3, however severe vitamin D deficiency is the leading cause of nutritional rickets. Poor vitamin D status is strongly linked to colon cancer and dialyses patients are monitored to appraise their kidney function. Robust routine measurements of 1,25(OH)2-vitamin D2/3 by mass spectrometry can be challenging due to other isobar vitamin D species, low circulating blood concentration of the targeted metabolites (5-100 pg/mL) as well as poor ionization. Moreover, currently no commercial solution is offered combining an easy to use sample preparation and derivatization in one assay.

Method
The 1,25-(OH)2-vitamin D2/D3 IDKenhance® LC-MS/MS kit is for the LC-MS/MS based quantification of 1,25-(OH)2-vitamin D2/D3 from 500 µl of serum and plasma. Samples were mixed with two isotopically labelled internal standards and worked up by using immunoaffinity enrichment with single filter tubes (ImmuTube®) or 96-well filter plates (ImmuPlate®). After evaporation of the eluate a fast derivatization step was applied, followed by a quick second evaporation. Thereafter, samples were reconstituted and injected into the LC-MS/MS system with a total cycle time 4.5 min. LC-MS/MS systems used for analysis were an Nexera 2 LC (Shimadzu) coupled to a QTRAP 5500 (AB Sciex) and an Acquity I-class UPLC coupled to a Xevo TQ-XS (Waters). Data analysis was performed with Skyline (22.2.1.306).

Results
The IDKenhance® derivatization showed a mean signal enhancement in peak areas of factor ~15 in comparison to underivatized 1,25(OH)2-vitamin D2/3. Results showed baseline separation of 1,25(OH)2-vitamin D2/3 from isobar metabolites (25,26-(OH)2-vitamin D3; 24,25-(OH)2-vitamin D2/3; 23,25-(OH)2-vitamin D3). The analysis of a broad calibration range covering the physiological range (5-250 pg/mL) using six calibrators as well as 3 quality controls (LQC: 17 pg/mL; MQC: 40 pg/mL; HQC: 100 pg/mL) showed robust linear regression with R² >0.99 and good precision (< 15/20%). The low Limit of Quantification (LLOQ) of 5 pg/mL was achieved (CV <20%), which supports accurate determination of extremely deficient patients. Validation of the assay was performed according to the requirements published by the FDA.

Conclusion
The sensitivity and the robustness of the assay was significantly improved, when at the same time the LC-MS/MS run time for measurement of 1,25-(OH)2-vitamin D3/D2 was reduced to ~ 4.5 min. This LC-MS/MS kit is registered under IVDD and subject for registration under IVDR. The use of a 96-well filter plates (ImmuPlate®) and the reduced LC-MS/MS cycle time of 4.5 min pushed the sample through significantly. Due to derivatization the ionization of both analytes was enhanced, peak areas were increased by a factor of 15 and signal to noise ratio was significantly improved. In conclusion, this new developed assay is the first commercially available kit which offers a complete solution for the routine analysis of 1,25(OH)2-vitamin D2/3 using LC-MS/MS methodology.

LC-MS/MS Analysis of Amyloid Beta Peptides in Artificial Cerebrospinal Fluid Using the Xevo TQ Absolute Mass Spectrometer for Clinical Research John Vukovic (Presenter) Waters Corporation

Poster #75b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Alzheimer’s disease (AD), the most common underlying cause of dementia, is a progressive neurodegenerative disorder associated with aging. Due to the extension of the average life span, the prevalence of AD is dramatically on the rise. Amyloid beta (Aβ) peptides are the most prominent biomarker compounds thought to be involved in AD pathogenesis, leading to increased interest in their quantification in clinical research studies. Historically, quantification of Aβ peptides in biological fluids has relied mainly on the use of immunoassays, such as ELISA. However, these techniques can suffer from cross-reactivity, contributing to batch-to-batch variation of the methods, which can impact confidence in results. In addition, for assessments that involve multiple biomarkers, an individual ELISA method is required for each peptide, increasing overall analysis time and cost. Therefore, a single robust method providing greater analytical selectivity would help overcome these challenges. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) could be a beneficial technique in the clinical research of Aβ peptides due to the advantages it provides over traditional ligand-binding techniques. These benefits include improvements in analytical selectivity, and the capability of multi-analyte quantitative detection in a single run. Herein, we demonstrate the suitability of the ACQUITY™ Premier UPLC™ I-Class System with Xevo™ TQ-Absolute Mass Spectrometer as a tool for analytically sensitive and selective biomarker quantitation for the accurate quantitation of multiple Aβ peptides (1-38, 1-40, 1-42) extracted from 200 µL of artificial Cerebrospinal Fluid (CSF) within the concentration range of 0.1-10 ng/mL.

Methods:
Calibration and Quality Control (QC) working solutions at each level were spiked into the blank artificial CSF with 4% (w/v) Bovine Serum Albumin (BSA). An internal standard working solution was added to 200 µL of spiked samples, which was diluted with 200 µL of 5M guanidine-HCl and 200 µL of 4% (v/v) phosphoric acid. The samples were mixed at room temperature at 900 rpm for one hour. The incubated samples were loaded onto an Oasis™ MCX SPE µElution plate, washed and then eluted with 75:15:10 (v:v:v) acetonitrile:water:ammonia into a QuanRecovery™ 700uL 96-well Collection Plates with MaxPeak™ HPS. The 100 µL of eluate was evaporated with nitrogen to dryness at 50°C and reconstituted with 50 µL of 20:80:1 (v:v:v) acetonitrile:water:ammonia. The reconstituted samples were mixed at room temperature at 800 rpm for 15 min prior to injection onto the LC-MS/MS system. Chromatographic separation was performed on an ACQUITY Premier UPLC I-Class FTN System using a ACQUITY UPLC BEH™ Peptide C18 300Å, 2.1 x 150 mm, 1.7 µm Column, using a 0.3% NH4OH/water/acetonitrile gradient. The detection was performed in positive electrospray ionization mode on a Xevo TQ Absolute Mass Spectrometer.

Results:
Analytical sensitivity of the lowest calibrator at 0.1 ng/mL was demonstrated with S/N (PtP) > 10 for all Aβ peptides (1-38, 1-40, 1-42) across the five analytical runs. We successfully demonstrated linearity from 0.1 – 10 ng/mL for the Aβ peptides, with r2>0.999 over five analytical runs. Total precision and repeatability across the Aβ peptides at the QC three concentrations (0.2, 1.0 and 7.5 ng/mL) respectively, with five replicates over five analytical runs (n = 25) was < 5% CV. The accuracy of the QCs compared to nominal concentrations ranged from 96.5 – 100.2% across the Aβ peptides.

Conclusions:
An LC-MS/MS method for the analysis of Aβ peptide biomarkers in CSF was developed for clinical research. Through the use of the ACQUITY Premier UPLC I-Class System and Xevo TQ Absolute Mass Spectrometer excellent inter-day linearity, analytical sensitivity, precision, and accuracy can be achieved, providing confidence in the results obtained for quantification of Aβ peptides in AD clinical research.

For Research Use Only. Not for Use in Diagnostic Procedures.

Comparative Quantitation of 270 Human Plasma Proteins on Agilent High Flow vs. Evosep Micro- and Nanoflow by Bottom-up MRM on Agilent 6495C Triple Quadrupole Christoph Borchers (Presenter) Jewish General Hospital, McGill University Montreal, QC, Canada

Poster #76a View Map

This poster will be attended on Wednesday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION:
Quantitation of proteins by multiple reaction monitoring (MRM) based on proteotypic peptides as protein surrogates is an increasingly popular approach over antibody-based, photometric approaches such as enzyme-linked immunosorbent assays (ELISA) due to its high multiplexicity, specificity (retention times, light/heavy peptide pair, precursor-fragment transition), large dynamic range of approximately 6 orders of magnitude, and sensitivity in the low ng/mL range. Due to its robustness, high flow, ultra-high performance liquid chromatography (UHPLC) coupled to MRM is of great interest to clinical labs. While UHPLC-MRM is commonly performed at flow rates of 0.3 to 0.5 µg/µL, loading 10-20 µg of digested protein, recent improvements in LC systems such as the highly robust Evosep One, have allowed coupling nano-to-microflow systems to triple quadrupoles, reducing the flow rate and loading amounts to <1 µL/min and <1 µg, respectively, while increasing sensitivity.
Here we compared the performance of 274 MRM assays to quantify 270 human plasma proteins utilizing a 6495C triple quadrupole (Agilent), connected to three different LC setups representing varying flow rates and loading amounts: Agilent 1290 Infinity II (reference method), Evosep One (60 samples per day, SPD), and Evosep One Whisper (40 SPD).

OBJECTIVES:
A comparison of sensitivity and quantitative performance between three different LC-MS approaches.

METHODS:
Five plasma lots were reduced and denatured (urea/dithiothreitol – DTT), alkylated (iodoacetamide – IAA), digested (Worthington trypsin), then quenched with formic acid (FA). Calibration curve standards and quality control (QC) samples were prepared by spiking digested BSA background matrix with varying levels of a 274-light peptide mix, then normalized (along with the plasma samples) with constant amounts of a 274-stable isotope-labelled standard peptide mix acting as internal standards.
All standards, samples and QCs were prepared in parallel, then split into three (one set for each system/method). Samples designated for the Agilent 1290 Infinity II – 6495C method were cleaned up by solid phase extraction using Waters HLB µElution plates, then dried down, and resuspended in aqueous 0.1% FA. 10 µg worth of material was injected onto a Zorbax Eclipse Plus RP-UHPLC column (2.1 x 150 mm, 1.8 µm particle diameter; Agilent) maintained at 50 °C. Peptide separations were achieved at 0.4 mL∕min over 60 min, via a multi-step LC gradient of 2-80% mobile phase B, and a post-gradient column re-equilibration of 4 min. Mobile phase compositions were A: 0.1% FA/H2O and B: 0.1% FA/acetonitrile.
The samples designated for Evosep One 60 SPD analysis and Evosep One Whisper analysis were loaded onto Evotips following the manufacturer’s instructions. For the 60 SPD analysis (21 min gradient), 1 µg per tip was loaded and samples were injected onto an EV-1109 column (Evosep; 8 cm x 150 µm, 1.5 µm; 1 µL/min). For the Whisper analysis, 20 ng material were loaded per tip and injected onto an EV-1112 column (Evosep; 15 cm x 75 µm,1.9 μm; 100 nL/min) with the 40 SPD method (36 min gradient).
Data were acquired on an Agilent 6495C triple quadrupole with either a Jet Stream ESI source when coupled to the 1290 Infinity II UHPLC, or with an Agilent nano source when coupled to the Evosep One HPLC, in both cases, using dynamic MRM.

PRELIMINARY RESULTS:
Preliminary results show improved Lower Limit of Quantitation values (LLOQs) for 196 of the 274 peptide targets using the Evosep 60 SPD method compared to the 1290 Infinity II reference method. The Evosep 60 SPD approach was up to 92-fold more sensitive, with a median 3-fold sensitivity improvement.

Moreover, while the reference method resulted in a median of 148 of the 274 endogenous proteotypic target peptides being within their respective linear ranges, and thus quantifiable across the five-plasma sample lots, the Evosep 60 SPD method resulted in a median of 178 endogenous peptides being quantifiable, an increase of 20% at half the gradient length, using only 1/10th of the material.
The Evosep One system with its reduced flow rates allows for significant sensitivity improvements, and although data analysis for the 20 ng/mL/Whisper method is still underway, we expect even larger sensitivity improvements.

CONCLUSION:
Moving from a robust high flow system to a robust nano-/microflow Evosep One system, coupled with a 6495C triple quadrupole, allows for significant sensitivity improvements while maintaining robust protein quantitation by highly multiplexed MRM.

Clinical Performance of Mass Spectrometry for 25-OH Vitamin D Measurement Yeonjae Lee (Presenter) Konkuk University Medical Center

Poster #77a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Background:
Accurate measurement of 25-OH vitamin D(25-OH-D) is important for vitamin D status monitoring. Although Isotope dilution liquid chromatography mass spectrometry (ID-LC-MS/MS) is confirmed as reference measurement procedure, many laboratories or researchers still use immunoassays because of the convenience and availability, regardless of standardization. In present study, authors evaluated the clinical performance of mass spectrometry and immunoassay for 25-OH-D measurement.

Method:
The accuracy of MS tests was evaluated by measuring standard reference material produced by National Institute of Standards and Technology (NIST SRM 972a). Then, using the mean values from two laboratory-developed test (LDT) mass spectrometrys (MS) for vitamin D as standard, the qualitative decision concordance and quantitative correlation with another LDT MS test and four commercialized immunoassays; Roche Elecsys Vitamin D Total II, Siemens Atellica IM Vitamin D Total assay, Abbott Alinity 25-OH Vitamin D, and Beckman Coulter Access 25-OH Vitamin D Total, were evaluated for 150 residual serum samples.

Results:
In accuracy, it was confirmed that the mean values obtained from MS tests for standard materials were within the range of assigned value. When the measured 25-OH-D values from each assay were compared to those determined by MS, ranged 5.86-67.75 ng/ml, In contrary to the equivalence between two MS tests with regression slope and intercept those containing 1 and 0 in their 95% confidence interval (CI) and r2 larger than 0.95, none of the immunoassays was equivalent to MS, by showing slope or intercept not containing 1 and 0 in their 95% CI, r2 smaller than 0.95. The regression estimates at 10, 20, and 30 ng/ml as medical decision limit (MDL) were within 5.0% the Vitamin D Standardization-Certification program (VDSCP)-claimed acceptable bias limit, in Beckman test at 30 ng/ml, only. The concordance of qualitative decision with MS ranged 90.0-94.0% in immunoassays.

Conclusion:
It was revealed that the two MS showed good qualitative decision agreement and quantitative correlation. However, despite having been achieved the certification by VDSCP, most immunoassays showed difference over acceptance criteria in the 25-OH-D measurement at MDL levels, which may affect clinical decision-making. Therefore, the superiority of MS test in measuring 25-OH-D compared to immunoassay has confirmed by this study, and attention for results interpretation and efforts for test standardization is required for immunoassays.

Plasma Arginine Metabolites in Health and Chronic Kidney Disease Kevin Mantik (Presenter) New South Wales Health Pathology, Sydney, Australia

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Poster #77b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Methylated arginines, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), cause aberrant vascular remodelling by inhibition of nitric oxide synthesis and have been shown to predict chronic kidney disease (CKD) and cardiovascular disease (CVD). The occurrence of CKD is underestimated due to the lack of sufficiently sensitive and specific markers that can be used to detect early renal dysfuction.
Objectives: The aim of this study was to validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine ADMA, SDMA, L-Arginine and L-Citrulline, in a large cohort of healthy adults. This method was then applied to a group of CKD patients, and then a high-risk group within the large cohort.

Methods:
We tested 857 men and women aged 45 from the Dunedin Multidisciplinary Health and Development Study (DHMDS) in New Zealand. Within this group we defined a healthy population (n = 376) by excluding smokers, those who use specific medications, self-reported, or tested for heart problems, high blood pressure, diabetes or CKD. We then tested a separate cohort of known CKD patients (n = 38). We also used an estimated glomerular filtration rate (eGFR) trajectory analysis based on plasma cystatin C, and defined study members at higher risk of CKD amongst the individuals in the DMHDS cohort.

Results:
Contrary to previously described methods, specificity was enhanced by complete separation (peak resolution, Rs = 1.8) between the structural isomers, ADMA and SDMA, without the use of derivatisation. We found all analytes were elevated in CKD compared to our healthy cohort (ADMA: 0.61±0.11 μmol/L vs 0.40±0.06 μmol/L, SDMA: 0.65±0.25 vs 0.42±0.06 μmol/L, and citrulline: 42.7±11.8 vs 24.0±5.4 μmol/L), however L-arginine concentration was only slightly higher in the CKD subset. The values from the healthy cohort aligned with previously published reference intervals. Regarding the kidney-function trajectory groups within the DMHD study, significant elevations for ADMA (0.40±0.07 μmol/L), SDMA (0.49±0.08 μmol/L), and L-Citrulline (28.6±7.45 μmol/L), were observed in the high-risk group.

Conclusion:
The LC-MS/MS method developed for simultaneously measuring ADMA, SDMA, Arginine and Citrulline will be used in prospective studies investigating the progression of CKD.

Validation of Bioavailable Testosterone by Mass Spectrometry Jennifer Kemp (Presenter) Mayo Clinic

Poster #78b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

OBJECTIVE: Our established bioavailable testosterone assay utilized differential precipitation of sex hormone binding globulin (SHBG) followed by scintillation counting. This method relied on antiquated equipment and necessitated the use of radioactive-labeled testosterone. Therefore, we sought to overcome these shortcomings by elimination of scintillation counting and transitioning to performing these measurements by liquid chromatography-tandem mass spectrometry.

METHODS: The method is based on the differential precipitation of SHBG by ammonium sulfate. SHBG bound testosterone is precipitated in patient sample using saturated ammonium sulfate, leaving the bioavailable testosterone in the supernatant. After addition of 13C labelled testosterone IS, the bioavailable testosterone and IS are extracted from the supernatant using isopropanol. The bioavailable testosterone and IS are then derivatized and analyzed by LC-MS/MS on a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Samples were first loaded onto a Phenomenex Cartridge Security Guard (C12, 4 x 2mm) and then separated using a Waters XBridge (C18, 2.1x50mm) analytical column.

RESULTS: To ensure assay validity the following metrics were assessed: intra and inter precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), ion suppression, and interferences. Three levels were assessed (n=20) for intra assay precision, which yielded the following: 6.8% CV at a bioavailable testosterone concentration of 0.82 ng/dL, 4.1 % CV at a concentration of 6.5 ng/dL and 3.8% CV at a concentration of 259 ng/dL. Three levels were assessed for inter assay precision, which yielded 12.5 % CV at 0.90 ng/dL, 8.9% CV at 15 ng/dL and 13.1% CV at 402 ng/dL. When using linear regression analysis to compare results with the current assay, the resulting slope was 1.04 with an r2 of 0.96. Spike and recovery of five samples yielded a mean average recovery of 110.6%. For linearity 4 samples were diluted and the mean %difference from expected was -2.6%. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. 58 common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Ion suppression was performed to ensure the IS was compensating for ion suppression or enhancement.

CONCLUSION: The LC-MS/MS measurements met or exceeded the performance of the historical scintillation counting method and the acceptance criteria for all metrics assessed. Therefore, we can eliminate the use of harmful radioactive molecules and antiquated equipment, while providing superior quality testing to our patients.

Improvement of Leukotriene E4 LC-MS/MS Using Novel Extraction and Chromatographic Conditions Kayla Moehnke (Presenter) Mayo Clinic

Poster #79a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Leukotriene E4 (LTE4) is a urinary biomarker for systemic mastocytosis and inflammatory response within the pulmonary and cardiac pathways. LTE4 guides clinical care in asthma patients as LTE4 concentrations correlate with disease severity. While LTE4 production inhibitors decrease LTE4 concentration, it is of utmost importance to measure as low as possible for an accurate clinical evaluation and diagnosis. Therefore, there is a need for an assay capable of accurate and precise measurements of LTE4 at low concentrations (≤20 pg/mL). Historically, LTE4 has been quantitatively measured by enzyme-linked immunoassays (ELISA) and LC-MS/MS; however, historical methods using both technologies have demonstrated suboptimal performance, with specificity and interferences being major challenges due to the high prevalence of molecules with similar chemical structures and characteristics. Mass spectrometry has the potential to provide highly accurate, precise, and specific results. Despite advantages of LC-MS/MS, the aforementioned challenges with interferences and specificity must still be overcome with careful development of sample preparation techniques and chromatographic conditions. Therefore, we have developed and validated a highly specific anion exchange solid-phase extraction and a chromatographic method utilizing a high-efficiency analytical column with unique selectivity.

Methods
250 mcL of patient urine was pipetted into a 96-well plate, followed by 50 mcL each of deuterium labeled internal standard (10007858, Cayman Chemicals) and 1N NaOH. Lastly, 1 mL of water was added, and the plate mixed thoroughly. The SPE plate (A3967010, Agilent) was conditioned with methanol and water, with positive pressure applied between each reagent addition. The samples were transferred into the SPE plate and ~1 psi pressure was applied for 7 minutes. The SPE plate was washed with water and methanol. The elution (methanol with 1% acetic acid and 1mcg/mL estriol) was collected and 50 mcL of water was added and mixed. The plate was injected and analyzed on the HPLC-MS/MS (Thermo Scientific TLX-4 coupled to Sciex 6500+). The LC separation was performed with a RP-MS analytical column (17626-053030, Thermo Scientific). The mobile phases were 0.02% acetic acid in both water (MPA) and methanol (MPB). The mobile phase gradient was adjusted to 55% organic for 30 seconds, with a ramping increase to 80% for 300 seconds and then to 98% for 90 seconds, at a constant flow rate of 0.6 mL/min. To confirm the validity of the assay; accuracy, precision, and sensitivity studies were performed using previously tested patient urine samples and pooled patient urine samples. The analytical measuring range (AMR) was 10 – 1280 pg/mL, by setting up a linear calibration curve using a LTE4 stock standard (20410, Cayman Chemicals) prepared in methanol and diluted with 0.1% BSA. The quality control materials comprised of pooled sample aliquots, spanning the AMR, with established means and acceptance criteria within 2 SD.

Results
When compared to methods with minimal sample preparation (“dilute and shoot”) and more traditional reverse phase analytical columns, the developed method had a tremendous effect on the chromatograms, greatly reducing the intensity of the baseline and the number of potentially interfering signals in the chromatograms. Therefore, the developed method increased the signal-to-noise ratio of the target signals, improved the reproducibility of chromatogram integration, and improved the specificity of the assay, which enabled validation of the test with a lower limit of quantitation (LLOQ) of 10 pg/mL, compared to 40 or 50 pg/mL in most cases. Assay validation studies were well within the established acceptance criteria and exceeded expectations. For intra-day precision, of four pools: 11.2, 20.1, 96, and 240 pg/mL, the %CVs are all less than 10%. For inter-day precision, of five pools, 16.4, 9.7, 58.7, 294, 652 pg/mL, the %CVs are less than 12%. Of the 59 previously tested patient samples evaluated for accuracy, the linear regression comparison gives a slope of 1.044 and a R-squared of 0.992. These strong results demonstrate that this method is a viable option for the measurement of urinary LTE4 with an improved LLOQ and reduction of interferences.

Discussion
The most significant improvement of our LTE4 assay is the exceptional lower limit of quantitation, which can be attributed to the reduction of baseline and interferences. This enhances peak picking and quantitation of low LTE4 concentrations. During evaluation of the limit of detection, we were able to measure sample concentration as low as 10 pg/mL with sufficient accuracy and precision. The novel SPE, analytical column, and LC parameter improvements of our LTE4 assay benefit patient care by providing highly sensitive and specific LTE4 measurements.

Simultaneous Quantification of Trastuzumab and Pertuzumab in Human Serum Using Accurate Mass Spectrometry Robert Haufler (Presenter) SCIEX

Poster #81a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Monoclonal antibodies (mAbs) are increasingly being used in combination with other mAbs for cancer and COVID-19 treatments. In breast cancer treatment, trastuzumab and pertuzumab are commonly co-administered to target unique human epidermal growth factor receptor 2 (HER2) epitopes. With the increasing application of mAb co-administration, simple and robust quantification methods are necessary to meet the needs of pharmacokinetic (PK) studies.

Conventional analytical methods involving ligand-binding assays, such as enzyme-linked immunosorbent assays (ELISAs), are employed during PK evaluations due to high sensitivity and sample throughput. However, multiplexing can be a significant challenge for such assays with limited selectivity. In addition to selectivity, sensitivity is also a critical factor because PK studies are often challenged by low sample volumes. Therefore, LC-MS/MS based techniques are being increasingly adopted for PK measurements of combination mAb therapeutics given their enhanced specificity, selectivity and sensitivity. In this study, an immunoaffinity purification workflow with on-bead digestion was employed to simultaneously quantify trastuzumab and pertuzumab in human serum on the ZenoTOF 7600 system.

Methods:
A 3 mg/mL stock solution of trastuzumab was prepared by dissolving 1 mg of lyophilized trastuzumab in 333 µL of phosphate buffered saline (PBS) with a pH of 7.4. A 3 mg/mL stock solution of pertuzumab was prepared by adding 300 µL of PBS to a 30 mg/mL stock solution. Subsequent dilutions were performed in normal human serum to prepare spiked serum samples ranging from 0.03 µg/mL to 300 µg/mL. A 500 µg/mL stock solution of SILuMAB was prepared by dissolving 0.1 mg of lyophilized SILuMAB in 0.2 mL of PBS. The stock was diluted 250:1 (v/v) in PBS to prepare a final solution at a concentration of 2 µg/mL. Protein A beads were diluted 8:1 with PBS and washed three times with PBS before use.

Each sample contained a 10 µL sample of normal human serum, 20 µL of 2 µg/mL SILuMAB, 200 µL of diluted protein A beads and 200 µL of PBS. After samples were shaken for 30 minutes at room temperature, the beads were washed twice with PBS. The beads were resuspended in 150 µL of digestion buffer containing 150 mM ammonium carbonate and 1 mM calcium chloride, and they were denatured at 95°C for 5 minutes. After allowing it to cool to room temperature, 2 µg of trypsin/lys-c was added to each sample and on-bead digestion was performed for 2 hours at 50°C. Digestion was stopped by adding 3 µL of formic acid. Samples were separated from the beads and placed in vials or plates for LC-MS/MS analysis.
Chromatography was performed on a Shimadzu LC-40 X3 system using a Phenomenex Biozen XBC18 (2.1 × 100 mm, 2.6 µm, 100 Å) column. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The operating flow rate was 0.5 mL/minute. Column temperature was set at 40°C. A 20 µL sample was injected for analysis.

Results:
Trastuzumab and pertuzumab were selected as model mAb therapeutics to evaluate the quantification of combination mAbs in human serum on the ZenoTOF 7600 system. The calibration curve included concentrations ranging from 0.15 µg/mL to 300 µg/mL for trastuzumab and pertuzumab. The concentration of the internal standard (IS), SILuMAB, was 0.25 µg/mL. Each calibration point was measured in three replicate injections.
At the level of the LLOQ, the %CV was required to be <20% with accuracy within ±20% of the nominal concentration. For the remaining concentrations, the %CV was required to be <15%, and the accuracy was required to be within ±15% of the nominal concentration.

An LLOQ of 0.15 µg/mL was achieved for trastuzumab and pertuzumab. Matrix interferences in the blank were more than 5x lower than the peak areas in the LLOQ. The overall %CV was <10.1% for trastuzumab and <8.12% for pertuzumab. The overall accuracy was within ±8% of the nominal concentration for trastuzumab. While for pertuzumab, the overall accuracy was within ±12% of the nominal concentration. Strong linearity was achieved with correlation coefficients (r) of >0.996 for both trastuzumab and pertuzumab.

Conclusions:
A highly sensitive method for the quantification of trastuzumab and pertuzumab in human serum using the ZenoTOF 7600 system and on-bead digestion was demonstrated. Using this approach LLOQs of 0.15 µg/mL were reached for both trastuzumab and pertuzumab in human serum due to the improved MS/MS sampling efficiency offered by the Zeno trap.

LCMS Method for the Branched Chain Amino Acids and their Keto-acids for the Detection of MSUD from Neonatal Dried Blood Spot Sudheer Moorkoth (Presenter) Manipal College of Pharmaceutical Sciences

Poster #85a View Map

This poster will be attended on Thursday at 11:00 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Maple syrup urine disease (MSUD) is an aminoacidopathy caused by defective branched-chain alpha-ketoacid dehydrogenase complex, which plays a crucial role in the metabolic pathway of branched-chain amino acid (BCAA). This leads to the accumulation of the amino acids (valine, isoleucine and leucine) along with their respective keto acids (α-ketoisovaleric acid, α-keto-β-methylvaleric acid and α-ketoisocaproic acid). The existing methods of newborn screening for MSUD is BCAA specific. There are no published literatures on the simultaneous determination of all the six biomarkers which could provide a comprehensive and reliable evaluation for the detection of MSUD. Early identification and treatment of MSUD prevents encephalopathy and allows normal intellectual development.

Objective:
To develop and validate a LCMS method for simultaneously quantify the branched chain amino acids and their keto acids from DBS sample.

Methods:
The above mentioned six biomarkers for MSUD along with the internal standard α-keto isocaproic acid sodium salt (methyl-d3) and L-leucine (5,5,5-D3) were extracted from DBS using methanol:0.1% formic acid buffer (75: 25% v/v). A ThermoFisher Scientific LCMS with Dionex Ultimate 3000 liquid chromatograph interfaced with a linear ion trap analyser having an electron spray ionization source was used. Separation of these analytes was achieved on Luna PFP 3 µ column (150 X 4.6mm) with gradient flow. Studies were carried out to determine stability of the biomarkers in matrix and in stock solution. Bland-Altman analysis was carried out to determine the correlation of results of the developed DBS method with that of the plasma method.

Results:
The method showed linearity with r2 ≥ 0.984 for all the analytes, lower limit of quantification of 0.8 ng/mL(BCAA) and 0.1ng/ml (BCKA), and recovery of 92-100.45%. Method has satisfied all the performance specifications including the effect of hematocrit as per the ICH M10 guideline. Stability study data showed that the biomarkers were stable at all the stability conditions. Comparison (cross-validation) of the developed DBS method with that of the plasma method by Bland-Altman analysis showed a good correlation.

Conclusion:
The developed LCMS method involves no derivatization, employs a simple sample preparation and has a short run time. After clinical validation, this method could be used for the comprehensive screening of MSUD in neonates.

A Path From Discovery to a Targeted Proteomics Approach for the Verification and Validation of Tissue-derived Biomarkers in Coronary Artery Diseases Chi Nguyen (Presenter) Precision Biomarker Laboratories Cedars-Sinai Medical Center Los Angeles

Poster #86b View Map

This poster will be attended on Wednesday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Over the past decades, there has been significant improvements on mass spectrometry (MS) - based technology, such that proteomics is now a gold-standard for biomarker research including discovery, analytical validation, and clinical validation. Conventionally, discovery proteomics results in a tremendous amount of information which is valuable not only in the discovery of biomarkers but also in characterizing the chemical properties of those biomarkers. In particular, the discovery MS data contains not only the relative quantification information of proteins, which facilitate the decision on biomarkers, but also about the relative elution profile of peptides, the mass peptide fingerprint spectral information, the collection of most responsive peptides and defined modified peptides, as well as the information about the background of your target matrix. This valuable information can be applied in the development of targeted proteomics approaches focusing on characterizing those biomarkers in a much larger cohort with more reliable quantification information. The biological model of this study is based on coronary artery diseases (CAD), the most common type among the heart diseases, and is one of the leading causes of death in our current time. One major challenge with current therapeutic approaches for CAD is the lack of an early diagnostic tool, which can dramatically impact treatment outcomes.

OBJECTIVES: In this study, we describe in detail the path from collecting and utilizing the comprehensive information from various sources of discovery proteomics analysis to the creation of the targeted proteomics assays for the verification and validation of biomarkers in application on coronary artery disease (CAD).

METHODS: There has been a lot of effort invested in biomarker discovery utilizing MS based approaches. From the data independent acquisition (DIA) analysis of approximately 200 tissues biopsies from CAD patients, a collection of 36 potential tissue-derived biomarkers was achieved. These proteins were then searched against the plasma spectral library information derived from over 200 DIA measurements of both na&iuml;ve and depleted plasma samples for their detection ability, spectral information, and relative elution profile. Based on this information, a targeted proteomics assay could be developed in a more effective and straightforward process. The targeted assay was evaluated in a small cohort of 42 plasma samples derived from female donors, ages from 52 to 76 years old with CAD, and 26 healthy matched controls.

RESULTS: 28 proteins out of the 36 potential biomarker proteins could be detected in plasma samples with a minimum of one unique peptide. Among them, 14 proteins show significant differences between case and control and 5 proteins have strong predictive power (AUC &gt;= 0.8).

CONCLUSION: This study illustrates a more efficient workflow to develop a targeted proteomics approach from all the possible discovery data sources relevant to the disease of interest. Furthermore, the confirmation of 5 potential tissue-derived biomarker proteins showing high predictive values in plasma samples of CAD cohort demonstrate the potential utility of using these proteins as diagnostic biomarker panel for the early detection of CAD.

Concordance of Urine and Meconium Drug Screen Results by Immunoassay and Mass Spectrometry Hannah Brown (Presenter) Hennepin Healthcare; University of Minnesota

Poster #57b View Map

This poster will be attended on Thursday at 12:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION
Newborn urine drug testing faces preanalytical and analytical challenges due to difficult sample collection protocols, low drug concentrations, and unique drug metabolites differing from those targeted by immunoassay (IA) or mass spectrometry (MS)-based methods. For these reasons, paired testing of urine and meconium is often performed . Further, the testing of meconium is also used to assess drug exposure throughout the latter half of gestation and helps with diagnosis of neonatal abstinence syndrome.

OBJECTIVE
The primary objective of this study was to evaluate concordance of drug screening results of newborn urine samples using both IA and MS-based methods with paired testing on meconium.

METHODS
The number of positive drug screen results for newborns with paired urine and meconium samples from January 2021 to October 2022 were tabulated retrospectively for two independent, de-identified datasets (n=881, dataset 1; n=2056, dataset 2). Different drug screening methods were used in the two datasets. In dataset 1, urine was screened with IA and confirmed with MS, while in dataset 2, both the screen and confirmatory testing were performed by MS. All paired meconium samples were sent to an outside reference laboratory where meconium samples were screened by IA and confirmed with MS. Common drug classes tested for in urine and meconium in both datasets include amphetamines (AMP), cocaine (COC), and cannabinoids (THC). The cutoffs for the IA urine screening method (in ng/mL) were: AMP 500, COC 150, OPI 300, THC 50. The cutoffs for the MS urine screening method (in ng/mL) were: AMP 5, COC 1, OPI 25-250 (depending on specific compound), THC 20 (as 11-nor-9-Carboxy-Δ9-tetrahydrocannabinol glucuronide). The cutoffs for meconium screening (in ng/g) were: AMP 100, COC 100, OPI 100, THC 20.

RESULTS
In dataset 1, 66 newborns screened positive for amphetamines in urine and/or meconium. Of these, 3% screened positive in urine alone, 33% were positive in meconium alone, and 64% were positive in both urine and meconium. In comparison, in dataset 2 utilizing MS, 123 newborns tested positive for amphetamines. Of these, 13% were positive in urine alone, 13% were positive in meconium alone, and 74% were positive in both urine and meconium.

For cocaine, 7 newborns screened positive in dataset 1. Of these, 0% screened positive in urine alone, 29% tested positive in meconium alone, and 71% were positive in both urine and meconium. In dataset 2, 65 newborns tested positive for cocaine and of these 15% were positive in urine alone, 14% were positive in meconium alone, and 71% were positive in both meconium and urine.
For cannabinoids, in dataset 1, 435 newborns screened positive. Of these, <1% were positive in urine alone, 66% were positive in meconium alone, and 32% screened positive in urine and meconium. The vast majority of the IA urine screens presumptively positive for cannabinoids did not confirm due to either negative confirmatory results or quantity insufficient to test. In dataset 2, 874 newborns tested positive for cannabinoids. Of these, <1% were positive in urine alone, 97% were positive in meconium alone, and 3% were positive in both meconium and urine.

CONCLUSION
Conventional urine IA screening methods may be inappropriate for urine samples from newborns. Lower cutoffs afforded by the MS approach (dataset 2) increased the sensitivity for detecting both amphetamines and cocaine, which were present in urine only for a significant number of newborns. The higher cutoffs for meconium testing may explain why the observed discordance between urine and meconium drug screen results (i.e., positive in urine and negative in meconium). Even with lower cutoffs afforded by MS, meconium was positive alone in a substantial number of newborns which may reflect drug exposure earlier in gestation or dilute urine. A large number of urine cannabinoid presumptive positive screens did not confirm. Given the poor analytical performance of screening for cannabinoids in urine, meconium only testing may be appropriate for this drug class, in combination with maternal urine drug testing.