Podium Presentations for Tox / TDM / Endocrine

Is the Fentanyl Result Real? Tackling Interfering Substances Amid the Opioid Epidemic Hsuan-Chieh (Joyce) Liao (Presenter) University of Washington

To be presented in Track 4 (Colton) on Wednesday at 13:30

INTRODUCTION: The surge in illicit fentanyl use has significantly escalated opioid-related overdose fatalities. In response to the ongoing opioid epidemic, clinical laboratories are increasingly incorporating fentanyl-specific testing into their urine drug screening panels, utilizing both immunoassays and LC-MS/MS assays.

OBJECTIVES: This practical training aims to outline the strengths and limitations of LC-MS/MS in detecting fentanyl, offering insights into best practices through case studies and discussions.

METHODS: While immunoassays are cost-effective and rapid, making them ideal for initial screenings, LC-MS/MS is predominantly employed as a confirmatory test due to its superior sensitivity and specificity. However, both immunoassays and LC-MS/MS assays can yield "false positive results" due to interfering substances. High-resolution mass spectrometry may be helpful in identifying these interferences.

RESULTS: Structurally similar compounds, such as fluoroquinolone antibiotics (e.g., ciprofloxacin, ofloxacin, and their metabolites), can trigger cross-reactivity in fentanyl immunoassays. While "false positive results" in LC-MS/MS are uncommon, they can occur due to interfering substances from system reagents, instrument fluidics, or the sample itself. Detecting and resolving these issues requires systematic and logical troubleshooting approaches.

CONCLUSION: The use of LC-MS/MS as confirmatory testing, along with high-resolution mass spectrometry, can effectively identify interfering substances in immunoassays. To ensure accurate drug testing LC-MS/MS results, it is essential to continuously monitor quality metrics and employ strategic troubleshooting to effectively manage and mitigate potential interferences.

Using Ion Mobility to Help Make Mass Spec Mainstream: A Case Study in Urine Toxicology Frederick Strathmann (Presenter) MOBILion Systems

To be presented in Track 1 (Steinbeck 1) on Wednesday at 15:45

INTRODUCTION:
The barrier to adopting mass spectrometry for the majority of laboratories has been well documented, yet the desire to generate higher quality results remains. High-Resolution Ion Mobility (HRIM) offers a glimpse into another dimension that can make ‘mass spectrometry for all’ a reality, where knowledge of how it works is not necessary for the everyday user.

Urine toxicology is an example of a well-established offering of most laboratories. It is a vital component to numerous patient management scenarios from emergency room visits to recovery programs and even drug screening for transplant recipients. The variability in menu design and widespread use of immunoassays in many labs has represented a challenge for physicians, laboratorians, and patients in obtaining reliable results in a timely manner. Further, the ‘screen with reflex to confirmation’ workflow is a mainstay of urine toxicology testing due to ease of use and automated workflows available with immunoassays; however, this two stage workflow is unnecessarily costly, often lacks sufficient specificity for the screening results to be reliably actionable, and relies on laboratories to adopt relatively complex mass spectrometry workflows or requires a costly sendout testing process with a delay in results based on reference laboratory performance. Using urine toxicology as one of numerous examples, we aim to demonstrate how high-resolution ion mobility is poised to put a mass spectrometer in every laboratory without anyone realizing it is there.

OBJECTIVES:
The primary objective of this study was to illustrate the various benefits of using a simplified urine toxicology, mass spectrometry-based workflow relying on a combination of Collision Cross Section and High-Resolution Mass Spectrometry in the absence of chromatographic retention time for identification. A secondary objective is to illustrate the potential power of embedding untargeted analyses into routine testing workflows in the hopes of turning the 'unused' data into gold.

METHODS:
A qualitative, urine toxicology workflow was developed using a modular, automated tip-based extraction for removal of matrix. A High-Resolution Ion Mobility-High Resolution Mass Spectrometry prototype system was used with a less than 2.5-minute injection-to-injection cycle. 62 targeted drugs were identified using an automated detection algorithm based on CCS, with helium as the drift gas, in combination with m/z. A CLIA validation protocol was followed to assess precision, accuracy, carryover, analytical sensitivity, and analytical specificity. An internally developed liquid chromatography-tandem mass spectrometry method was used to confirm all results in addition to external comparisons for the majority of included drug targets.

RESULTS:
Instrument Detection Limits were calculated to assess overall sensitivity of the prototype system, with sufficient sensitivity achieved for all included drug targets. Imprecision and accuracy were within acceptable limits and analytical sensitivity and specificity were greater than 90% when compared to internally and externally derived results using orthogonal technology. Patient comparison results (n > 120) demonstrated acceptable agreement with the prototype system detecting additional compounds not included in all externally derived results due to known variability with drug panel inclusion and established cutoffs. Additional detected features were extracted from authentic sample data files highlighting the epidemiological value and potential biomarker discovery at scale with targeted and untargeted workflows in use for routine clinical testing.

CONCLUSION:
Eliminating the dependence on chromatography for compound identification is critical to reducing barriers for mass spectrometry adoption in the field. Moreover, laboratories that have integrated mass spectrometry into their workflows need a strategy to delegate routine assays. This shift will enable them to concentrate on developing next-generation biomarkers without compromising the established performance or becoming constrained by rigid systems. Additionally, to prevent the stagnation of promising biomarker translation due to inefficient affinity-based methods, it's imperative to develop a platform that is capable of performing these novel assays without the complexity of existing mass spectrometry workflows. This will maximize the potential of innovation in health equity for precision medicine. Ultimately, enhancing patient outcomes begins with a paradigm shift from a technology-centric approach to one that prioritizes addressing patient needs.

The Old Assay in the New Era: Re-Evaluation of Mass Spectrometric Immunosuppressant Assay Junyan Shi (Presenter) Vancouver Coastal Health, University of British Columbia

To be presented in Track 1 (Steinbeck 1) on Thursday at 10:30

Introduction: The number of solid organ and stem cell transplantation continues to increase worldwide, Organ transplantation is a multidisciplinary field, requiring a diverse community of professionals to collaborate to support various transplant programs. Clinical laboratories play pivotal roles encompassing all phases of transplantation including pre-transplant, transplant, and post-transplant stages. While non-specific tests such as serum creatinine/eGFR, liver enzymes, complete blood counts are useful for routine investigation, transplant-specific tests guide more important clinical decision-making and ultimately impact clinical outcomes in transplant recipients. Therapeutic Drug Monitoring (TDM) for immunosuppressants (cyclosporine, tacrolimus, and sirolimus) stands at the intersection of precision medicine and efficient healthcare delivery, ensuring optimal drug dosages and minimizing adverse effects. In this rapidly evolving era, laboratories encounter significant challenges, from method selection concerns to operational optimization and the need for data-driven insights.

Objectives: The study aims to explore the challenges in immunosuppressant testing using mass spectrometry, provide solutions to optimize the entire workflow, and demonstrate how data analytics can greatly facilitate decision-making to maximize efficiency and quality in clinical laboratories.

Methods: Clinical-driven and data analytics-based approaches were applied to examine the bottlenecks of the laboratory-developed mass spectrometric immunosuppressant assay that has been in routine clinical use for over two decades. We conducted a retrospective data analysis from the laboratory information system, utilizing open-source R software, to identify trends in test volume, test order patterns for both inpatient and outpatient settings, turn-around time, and variations in efficiency among technologists. To address the identified barriers throughout the entire workflow, we introduced solutions at various phases. In the pre-analytical phase, we developed new sample batching and processing strategies. In the analytical phase, we re-evaluated the existing assay to improve run time and throughput. In the post-analytical phase, we reinforced procedures aimed at improving the quality of results.

Results: Over the past two decades, the total test volume of immunosuppressant testing has substantially increased by 4 folds in our laboratory, rising from 30 samples to 120 samples per day. Inpatients and outpatients from transplant clinics of our hospital contributed to 58% of the total volume. The majority of inpatient samples were received before 10am, while the majority of outpatient samples were received before 12pm. Our analysis revealed that the delay occurring in the analytical phase, as opposed to the delay from collection to receipt in the pre-analytical phase. Furthermore, we observed substantial variation and inconsistency in turn-around times among different technologists. The new workflow and the modified mass spectrometric method resulted in significant reduction in the turn-around time, an increase in testing throughput, improvement in assay precision by minimizing variations among technologists, as well to help alleviate staffing shortages to some extent from the operational perspective.

Conclusion: This study highlights the significance of re-evaluation of laboratory-developed immunosuppressant assay to meet the growing demands from transplant patients. By leveraging data analytics, we were able to objectively identify workflow bottlenecks and facilitate informed decision-making in clinical laboratories. The implementation of improvements aimed at enhancing efficiency and quality provided a model in addressing the evolving clinical needs and staffing shortages.

High Sensitivity Measurement of Free T4 in Serum by Equilibrium Dialysis-LC-MS/MS Julie Ray (Presenter) ARUP Laboratories

To be presented in Track 3 (Steinbeck 3) on Thursday at 10:50

INTRODUCTION:
According to the free hormone hypothesis, only the unbound or free fraction of the hormone exerts biological effects. Diagnosis and monitoring of thyroid disorders often requires the measurement of free T4 (fT4) and free T3 (fT3). Most clinical laboratories measure these hormones with immunoassays. Significant biases observed in immunoassays have been avoided by utilizing the sensitivity and specificity of liquid chromatography tandem mass spectrometry (LC-MS/MS). While ultrafiltration and size exclusion chromatography can be valid methodologies to extract free hormones, they are subject to variability due to protein leakage, temperature control and nonequilibrium. Equilibrium dialysis (ED) is considered to be the gold standard method for separation of free hormones from biological samples. Accuracy and specificity of detection are also of prime importance, considering very low endogenous concentrations of free hormones.

OBJECTIVES:
Our primary objective was to reduce the complexity and enhance sensitivity of an existing LC-MS/MS fT4 method that utilizes direct injection of the serum dialysates with 2-dimensional (2D) chromatographic separation (1st dimension separation being online SPE), and detection on a Triple QuadTM 5500 (Sciex, MA).

METHODS:
200 µL aliquots of serum samples and controls were subjected to equilibrium dialysis for 20 ± 1 hours. 150 µL of calibrators, and dialysates were mixed with 150 µL of internal standard 13C6 T4. Desalting of the dialysates was carried out on a Strata-X-AW 33 µm polymeric weak anion exchange, 10 mg (Phenomenex, CA) SPE plate. The sorbent was conditioned with methanol, equilibrated with water, followed by loading of sample. Two 200 µL aliquots of 10 % NH4OH in methanol were used to elute the analyte of interest. After evaporation under a flow of nitrogen at 60oC, the samples were reconstituted with 125 µL of 30% methanol in water and 50 µL was injected for analysis. Separation was performed on a 2.1 X 50 mm, 2.6 µm Kinetex C18 column (Phenomenex, CA). Mobile phase A was 0.05% formic acid in water and mobile phase B was a mixture of 1:1 Methanol: Acetonitrile. LC gradient consisted of 30% mobile phase B for 2.5 min, followed by 1 min conditioning with 95% mobile phase B and re-equilibration to the initial conditions. T4 peak eluted at 2.7 min with a total run time of 5.5 min. Sample analysis was performed on a Triple QuadTM 7500 (Sciex, MA). Data acquisition was performed using electrospray ionization in positive mode, using mass transitions of m/z 777.7>731.7 (T4 quant), 777.7>604.7 (T4 qual) and 783.7>737.7 (13C6-IS T4 quant) and 783.7>610.7 (13C6-IS T4 qual). Data analysis and quantitation were performed on OS software (SCIEX, OS 2.2.0).

RESULTS:
Lower and upper limits of quantitation (LLOQ and ULOQ) were 0.1 and 10 ng/dL respectively. Intra- and inter-day precision evaluated using three matrix-matched quality control samples (0.14, 2.05, 10.0 ng/dL) showed < 10% imprecision. Post-dialysis spiked samples spiked with T4, showed recoveries ranging between 85% and 115%. The method showed no carryover in a sequence of low-high-low T4 injected samples. Good agreement was observed with the existing LC-MS/MS method utilizing 2D LC separation for fT4 on the Triple QuadTM 5500 (slope = 0.997, r = 0.963, n = 53). Chromatograms of calibrators and patient samples (fT4 concentration ~0.11 ng/dL) demonstrated 7-10x improvement in peak area and signal-to-noise ratio, as compared to the existing method.

CONCLUSION:
We reduced the complexity of chromatographic separation by transitioning the method from 2D to 1D HPLC separation. Incorporating SPE based desalting of the dialysates and the improved detector sensitivity of the SCIEX Triple QuadTM 7500, allowed to reduce the lower limit of quantitation. Reducing injection volume from 250 µL to 50 µL permitted sufficient volume for additional injections if necessary for the reanalysis of samples (which is useful for pediatric sample testing). The new method provides significant enhancement in the signal intensity, and signal-to-noise ratio.

Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urines by LC-MS/MS Yanchun Lin (Presenter) Washington University School of Medicine in St louis

To be presented in Track 1 (Steinbeck 1) on Thursday at 13:30

Introduction: The most common cause of drug overdose fatality in the United State involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl adulterated or associated with xylazine as an emerging threat to the United States, and a national response plan will include xylazine testing. Xylazine, is a clonidine analog and is not FDA-approved for use in humans, but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers an effective way to detect xylazine in patient samples with high sensitivity and specificity.

Objectives: The objectives of this study are to develop a LC-MS/MS method to detect xylazine metabolites in urine, and to investigate the presence and abundance of xylazine metabolites in remnant urine samples submitted for clinical testing.

Methods: Remnant urine samples were used for this study under IRB approval with waiver of informed consent. Samples were identified when they tested positive for xylazine during routine clinical testing by LC-MS/MS. Remnant urine samples were diluted with xylazine-D6 internal standard (Cayman Chemical), and hydrolyzed using B-One β-glucuronidase (KURA biotech). Hydrolysis efficiency was quantified using codeine-6-glucuronide standard (Cerilliant) prepared in negative urine at 5,000 ng/mL. Liquid chromatographic separation utilized a 100 mm x 2.1 mm, 2.6 μm Kinetex C18 column (Phenomenex) with a 4 minute gradient ramping from 2% to 98% mobile phase B (A: 0.1% formic acid and 2mM ammonium acetate in water; B: 0.1% formic acid and 2mM ammonium acetate in methanol) with a flow rate of 400 μl/min. The column temperature was held at 50°C. Xylazine and 4-OH xylazine were detected in positive mode using multiple reaction monitoring, with transitions optimized from standards obtained from Sigma Adrich, and/or Cayman Chemical (xylazine: 221>90, 221>164; 4-OH xylazine: 237>90, 237>137). Xylazine and 4-OH xylazine were quantified using calibration curves prepared at 5, 50, 500, and 5000 ng/mL in negative urine. Product ion scanning was used to detect four additional xylazine metabolites, based on spectral matching to previously published MS/MS spectra, using remnant urines with high concentrations of xylazine.1 Based on the collected product ion scanning experiments, the highest abundance fragment ions were used to perform multiple reaction monitoring for the proposed sulfone xylazine (253>181, 253>147), oxo xylazine (235>122, 235>114), OH-sulfone xylazine (269>197, 269>163), and OH-oxo xylazine (251>197, 251>148) metabolites in all remnant urines using chromatography and retention times consistent with the product ion scanning experiments. All experiments were performed using an Aquity HPLC coupled to a TQS-micro triple quadrupole mass spectrometer (Waters).

Results: During the study period, 138 urines screened positive for fentanyl by immunoassay which were reflexed to confirmatory testing by LC-MS/MS. Xylazine was detected at >=1 ng/mL in 38% of these urines. A subset (n= 362) of urines that screened positive for opiates, amphetamines, or cocaine metabolite, were also tested for xylazine. Xylazine was detected in only 1 urine which did not screen positive for fentanyl but contained methamphetamine and amphetamine.
Xylazine concentrations quantified in remnant urines ranged from <5 ng/mL to 10,729 ng/mL. 4-OH xylazine concentrations ranged from <5 ng/mL to 219 ng/mL. We observed five previously reported xylazine metabolites in urines containing xylazine: 4-OH xylazine, oxo xylazine, sulfone xylazine, OH-oxo xylazine, OH-sulfone xylazine Hydrolysis of urines with glucuronidase increased the peak areas of xylazine metabolites containing hydroxyl function groups: 4-OH xylazine, OH-oxo xylazine, OH-sulfone xylazine. The mean concentration ratio of 4-OH xylazine-to-xylazine was 0.2. The mean peak area ratios of metabolite-to-xylazine for sulfone xylazine, OH-oxo xylazine, OH-sulfone xylazine, and oxo xylazine were 3.1, 1.4, 0.5, and <0.1, respectively. The proposed sulfone xylazine metabolite was observed to have the most abundant signal of all detected metabolites in the majority of urines, although there is currently no standard available to enable quantification or confirmation of this metabolite structure.

Conclusions: Xylazine and 4-OH xylazine were quantitated in patient urines. Four additional xylazine metabolites were observed and relative abundance to xylazine was estimated using peak areas. Future studies include quantitation of xylazine and xylazine metabolites in urine and serum and association of xylazine with clinical and demographic variables.

References:
1.Meyer GMJ, Maurer HH. Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC–MS, LC–MSn, and LC–HR-MSn. Anal Bioanal Chem. 2013;405(30):9779-9789. doi:10.1007/s00216-013-7419-7.

Monitoring the San Francisco Drug Supply: Results from a Bio-Surveillance Project of Opiate Treatment Patients Using High Resolution Mass Spectrometry John Halifax (Presenter) UCSF

To be presented in Track 1 (Steinbeck 1) on Thursday at 13:50

Introduction:

The overdose crisis in the United States continues to accelerate. The most recently available national data shows the age adjusted risk of overdose rose by 14% from 2020 to 2021, a continued rise driven in part by a changing drug supply. The potent synthetic opioid fentanyl emerged a decade ago with devastating consequences for overdose mortality, and recent reports from eastern United States identify further troubling changes. Xylazine, a sedative associated with severe soft tissue damage, has been increasingly detected in overdose deaths involving illicitly manufactured fentanyl (IMF). Provisional national data from 2019-2022 indicate regional heterogeneity for xylazine prevalence; xylazine was detected in 49.9% of IMF-involved deaths in the Northeast U.S. Census Bureau Region, but only 1.1% of IMF-involved deaths in the West Region. Re-analysis of all accidental overdoses in San Francisco from 2022 found xylazine in 2.4% of cases. Additionally, nitazenes, a family of synthetic opioids structurally unrelated to fentanyl but of similar or greater potency, have been identified in eastern United States in limited cases. Nitazenes have not been identified on the West Coast, but in spring 2023 patients at opiate treatment programs reported drugs marketed as &ldquo;Iso,&rdquo; assumed to refer to the nitazene isotonitazene. The historical precedent of IMF&rsquo;s westward spread after its emergence in the Northeast raises concerns that this lower prevalence of xylazine and nitazenes in Western US may be a preamble to a delayed but significant emergence in western states. Within this national context, local concerns about new changes in the drug supply prompted the initiation of a real-time surveillance of the drug supply utilizing urine from patients at a hospital-based opioid treatment program in San Francisco.

Initiated March1st, 2023, this bio-surveillance program utilized untargeted Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS) to provide toxicological analysis of de-identified urine aliquots from an at-risk patient population. Notable findings from the program include detecting a widespread but unsustained emergence of xylazine in San Francisco and refuting suggestions that drugs marketed locally as &ldquo;Iso&rdquo; consisted of isotonitazene. This real-time information on the state of the local substance supply informs clinicians, patients, and the San Francisco Department of Public Health. An interactive dashboard and a one-page document are updated weekly to communicate aggregated program results.

Objectives:

To demonstrate the effectiveness of utilizing a clinically validated LC-QTOF-MS comprehensive drug screen method to analyze de-identified urine samples from a population exposed to the local drug supply as a proxy for direct drug supply surveillance.

Methods:

Each patient presenting for treatment at San Francisco General Hospital&rsquo;s (SFGH) Opiate Treatment Outpatient Program (OTOP) provides urine for toxicology testing. Since March 1, 2023, a de-identified aliquot of each sample is analyzed on a clinically validated comprehensive drug test by untargeted high-resolution LC-QTOF-MS. Briefly, chromatographic separation is performed using a C-18 column with a 10-minute gradient from 2%-100% organic. Data is collected on a SCIEX TripleTOF&reg;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). Parent ion masses of analytes of particular interest were added to an inclusion list for the TOF-MS scan to ensure collection of high-resolution product ion spectra even when the analyte&rsquo;s abundance was below the IDA threshold. Data is analyzed using an in-house library containing &gt;5000 small molecules including &gt;150 fentanyl analogs and 12 nitazene-family compounds. Results are communicated weekly to OTOP clinicians in weekly aggregate in the form of an interactive dashboard. A one-page factsheet presenting weekly aggregated results for xylazine prevalence is updated weekly for use as a patient education material for SFGH clinics.


Results:

From March to November 2023, n = 383 urine samples were analyzed using the LC-QTOF-MS method. No samples were positive for any of the 12 nitazene compounds present in available libraries. In all samples, n =52 were positive for xylazine (13.58%), with xylazine prevalence varying temporally. The first xylazine positive sample was detected in late March 2023, but xylazine detections remained sporadic until late May, when the proportion of samples with xylazine began to steadily rise. From June 1st - July 31st, 31.9% of all samples (n=91) tested positive for xylazine compared to only 6.6% of all samples prior to June 1st (n=136). Since the end of July, xylazine prevalence has again fallen, with only 8.33% of samples (n=156) positive for xylazine. Detection of fentanyl analogs and their metabolites, predominantly fluorofentanyl (n=46) but also methoxy-furanyl fentanyl (n=33), methoxy-tetrahydrofuran-fentanyl (n=15, and norcarfentanil (n=1) provide nuanced insights into the local opioid supply, now dominated by fentanyl. All xylazine and fentanyl analog positive samples were also positive for fentanyl or its main metabolite, norfentanyl, suggesting that xylazine and fentanyl analogs are present in the drug supply as adulterants of fentanyl rather than stand alone drugs themselves.

Conclusions:

Analysis of de-identified urine samples from a patient population at-risk of overdose by LC-QTOF-MS provides deeper comprehension of the illicit substance supply in a geographic region, aiding clinical practice and patient education. The LC-QTOF-MS untargeted method adapted to ensure spectra collection for analytes of interest facilitates a hybrid untargeted-targeted approach to flexibly assess the drug supply while reliably establishing target analyte prevalence in the sample population.

Combining Toxicology Testing with Field Sobriety Test Results to Improve Impairment Classification for Cannabis Robert Fitzgerald (Presenter) University of California San Diego

To be presented in Track 1 (Steinbeck 1) on Thursday at 14:10

Introduction: The relationship between cannabis use and driving impairment is complex because of the unique pharmacokinetic and pharmacodynamic properties of delta-9-tetrahydrocannabinol (THC). With ethanol there is a clear relationship between amount of alcohol consumed, blood concentrations, and effects on driving performance. With cannabis these relationships are much more complex. The relationship between blood THC concentrations and crash risk has not been established. While it is clear that THC can impair driving, there are still uncertainties regarding the universality of such impairment and its time course. A key question remains: how to best identify drivers who are impaired by cannabis?

This presentation focuses on the toxicology results from the University of California-San Diego’s Center for Medicinal Cannabis Research recently competed randomized placebo controlled trial evaluating the effect of smoked cannabis on driving performance.

Objectives: The first objective of this study was to determine the relationship between concentrations of THC (and related cannabinoids) and performance on a driving simulator. The second objective was to evaluate how various toxicology cutoff concentrations in both blood and oral fluid affected the classification of participants deemed impaired by the field sobriety tests (FSTs) examinations.

Methods: 191 regular cannabis users were randomized to smoke 700 mg of placebo, 5.9% or 13.4% THC cannabis in a double blind manner. Blood, oral fluid and breath samples were collected serially up until 5 hours after smoking. During the study period participants drove a driving simulator and were administered FSTs by trained drug recognition experts (N=11). FSTs consisted of a walk and turn, modified Romberg, lack of convergence, one leg stand, and finger to nose tests. OF was collected using the Quantisal device. Concentrations of THC and related cannabinoids were quantified using isotope dilution liquid chromatography with tandem mass spectrometry.

Correlation between driving performance (swerving and coherence) and THC concentrations in blood, OF, and breath were determined using Spearman’s rho. P-values were adjusted for multiple testing using the False Discovery Rate (FDR) method. P-values < 0.05 were considered significant.

Results: No correlation (p > 0.05 in all cases) was observed between blood, oral fluid or breath THC concentrations and standard deviation of lateral position (swerving) or car following (coherence) on the driving simulator at any of the time points studied. The rate of impaired FST performance was significantly higher in the THC group compared to the placebo group up to 188 minutes after smoking. Seventy-one minutes after smoking, FSTs classified 81% of the participants who received active drug as being impaired. However, 49% of participants who smoked placebo (controls) were also deemed impaired. OF showed less of an impact than blood for reclassifying the active drug cohort, while reclassifying a higher percentage of the placebo group as not impaired.

Conclusion: The complete lack of a relationship between the concentration of the centrally active component of cannabis in blood, OF, and breath is strong evidence against the use of per se laws for cannabis. In the largest randomized double-blinded placebo controlled trial to date, our data confirm that THC concentrations (and/or metabolites/related cannabinoids) in blood, OF, or breath cannot be used as a sole indicator of impairment.

Adding a requirement of a positive toxicology test in OF to the FST exam slightly decreased the percentage of participants who smoked active drug that were classified as possibly being under the influence of cannabis but dramatically decreased the percentage of placebo group subjects that were classified as such.

Clinical Utility of High-Resolution Mass Spectrometry in a Pediatric Case of Altered Mental Status Adina Badea (Presenter) Lifespan/Rhode Island Hospital & the Warren Alpert Medical School of Brown University

To be presented in Track 1 (Steinbeck 1) on Thursday at 15:25

Case Description:

A 16-year-old male with a medical history of anxiety, ADHD, and multiple prior episodes of altered mental status (AMS) with no resolved etiology presents to the emergency department (ED) with AMS. He was in his baseline state at home, then went to school and was noted to exhibit extreme somnolence. He was unable to be woken up, and EMS was called. Of note, the patient had had two similar presentations to the ED over the previous two weeks. He received Narcan with no improvement. He was admitted to the neurology service and had an EEG that did not show epileptiform activity, similarly to his previous episodes when he was discharged home without any antiepileptic drugs. Other labs were unremarkable. A comprehensive drug screen was ordered and was unexpectedly positive for clozapine.

Background:

Extreme somnolence can be caused by an overdose or as a side effect of a variety of medications: antihistamines, antiemetics, antipsychotics, anticonvulsants, barbiturates, benzodiazepines and opioids to name a few. Adding to that sedatives like xylazine, a known contaminant of the illicit drug supply, along with non-toxic etiologies such as seizures and other neurological conditions, and the diagnosis of an AMS episode involving somnolence becomes more complex than what typical immunoassay drug screens can handle. Unintentional clozapine ingestions are rare. Clozapine is a second generation antipsychotic which has been used as an effective treatment alternative to traditional antipsychotics and has been deemed relatively safe in overdose. However, toxic effects in pediatric patients have been recorded and include hypersalivation, tachycardia, hypotension, sedation, delirium, leading to severe effects such as coma, respiratory depression, and even respiratory failure.

MS Method and Results:

A clinically validated comprehensive drug screen test using LC-QTOF-MS was used. The urine specimen was centrifuged to remove particulates, diluted with solvent and injected onto a SCIEX X500R platform. The untargeted data collection was performed using a positive-ion mode TOF-MS survey scan with IDA-triggered collection of high-resolution product ion spectra (20 dependent scans). The data was analyzed against an in-house validated library of 318 drugs and metabolites spanning multiple drug classes. The specimen was found positive for amphetamine, trazodone, citalopram (prescribed medications), naloxone, lorazepam (administered in the field and trauma room), and clozapine, an unexpected finding.

Discussion and Conclusion:

The patient's presentation was consistent with previously reported clozapine toxicity symptoms. However, the clozapine toxidrome includes many symptoms that are not all present concomitantly in exposed individuals. AMS in the form of extreme sedation can be attributed to other toxic agents as well as neurologic etiologies. This patient had alternative workups for this presentation in two other AMS episodes. Immunoassay drug screens were positive for amphetamine, known prescribed medication. Clozapine was not on the home medications list. Physicians and parents were notified of the comprehensive drug test result. The patient's older brother had been prescribed clozapine some months back, with discontinuation due to severe somnolence as a side effect. Patient denied taking clozapine intentionally. His symptoms improved with supportive care and the patient was discharged. Upon further investigation, the parents realized they mixed up medications and had given the patient the doses intended for his brother. Post correction, the patient's symptoms resolved permanently. Comprehensive drug testing by LC-QTOF-MS was the key to resolving this case. Had this test been employed in earlier episodes, rehospitalization of patient could have been prevented.

Liquid Chromatography or Paper Spray? Quantifying Tyrosine Kinase Inhibitors with Tandem Mass Spectrometry Rich Lahr (Presenter) Mayo Clinic

To be presented in Track 2 (Steinbeck 2) on Thursday at 15:25

Introduction: Dabrafenib and trametinib are targeted drugs used for the treatment of metastatic melanoma and other un-resectable solid tumors with BRAF V600 mutations. The drugs inhibit two protein kinases: the mutated B-Raf kinase (dabrafenib) and its downstream MEK1/2 kinase (trametinib). Although the targeted therapies are highly effective and administered orally, they can also exert significant toxicity, resulting in dose reduction and discontinuation. To maximize treatment efficacy while minimizing adverse events, clinicians should make informed treatment decisions based on the therapeutic drug levels of dabrafenib, its major metabolite, and trametinib.

Objectives: The primary objective of the study was to develop and evaluate mass spectrometry-based methods to quantify dabrafenib, hydroxy-dabrafenib (OH-dabrafenib), and trametinib in plasma to monitor drug concentrations in metastatic melanoma patients receiving targeted therapy of dabrafenib and trametinib.

Methods: We developed liquid chromatography (LC) and paper spray methods coupled to tandem mass spectrometry (MS/MS) for the quantification of dabrafenib, OH-dabrafenib, and trametinib. In the LC method, 100 µL of plasma was aliquoted into an amber plastic sample vial and 300 µL of methanol (containing Dabrafenib-D9 and Trametinib-[13C]6 internal standards [IS]) was added. Next, the samples were mixed at 2000 rpm (at 5°C) for 5 minutes, followed by 5-minute centrifugation at 10,000 x g (at 5°C). 100 µL of the supernatant were transferred into an amber 300 µL glass autosampler vial and mixed with 80 µL of water. The resulting samples (10 µL) were analyzed by LC-MS/MS using Thermo Scientific TSQ Altis triple quadrupole MS with heated electrospray ionization in positive mode. A Thermo Scientific Hypersil Gold aQ (2.1x50mm, 3um) column, connected to a Thermo Scientific Vanquish Horizon SII autosampler and LC pumps, was used to achieve chromatographic separation utilizing a linear gradient with a total run time of 5.2 minutes. Mobile phase A and B contained 0.1% formic acid in water and methanol, respectively. Compounds were identified by retention time, relative retention time to an isotopically labeled IS, and Q1/Q3 ion pair ratios.
For the paper spray ionization (via Thermo Scientific VeriSpray ion source) method, 100 µL of plasma was aliquoted into an amber plastic sample vial and 200 µL of methanol IS was added. After the same vortex and centrifugation steps, 6 µL of the supernatant was transferred onto a VeriSpray sample plate. The sample plates were air dried under dark conditions for 30 minutes. The resulting samples were analyzed with a VeriSpray ion source connected to the TSQ Altis MS. Samples were ionized directly into the MS system by first rewetting the samples with 20 µL of 0.1% formic acid in 1:1 acetonitrile: isopropanol and then adding 100 µL of the spray solvent (0.01% formic acid in 9:1 methanol: water). Both rewetting and spray solvents were added in 10 µL increments with time delays between each respective solvent addition. Chronographs were generated by applying 3800 volts to the spray paper from time point 0.1 to 1.1 minutes. Compounds were identified by Q1/Q3 ion pair ratios and relative abundance to the IS response.

Results: The LC method development proved challenging due to the lack of an isotopically labeled IS for OH-dabrafenib. When using Dabrafenib-D9 as it's IS, we observed inconsistency in the OH-dabrafenib analyte response relative to Dabrafenib-D9 at higher concentrations. When evaluating the matrix effects of patients’ EDTA plasma, we visually observed variable ion signal (via t-infusion signal suppression) at the retention time of dabrafenib-D9 that was not present at the OH-dabrafenib retention time. To mitigate these signal variations, we analyzed the extracted samples in triplicate injections and the final calculated result was averaged. The replicate injections significantly improved imprecision. Across the analytic measuring range, the total imprecision for dabrafenib and trametinib was 1.4-6.8% and 1.4-3.4%, respectively. However, for OH-dabrafenib the total imprecision was 4.0-13.7%. The final LC method resulted in a linear analytic measurement range of 10-3500, 10-2500, and 0.5-50 ng/mL for dabrafenib, OH-dabrafenib, and trametinib, respectively.
Conversely, the paper spray method only utilizes a single “injection” per sample and did not display differential suppression for the three analytes and two ISs. In addition, the approximately 2.5-minute cycle time (including the needed pauses during solvent addition) of the paper spray ionization method yielded a faster turnaround time for the entire set of samples. While the paper spray method resolved the suppression observed for OH-dabrafenib, the overall signal intensity for trametinib at the lower limit of quantitation (LLOQ, 0.5 ng/mL) was not enough to reproducibly distinguish it from a matrix blank’s signal. Therefore, the paper spray method resulted in a linear analytic measurement range of 10-3500, 10-2500, and 2.0-50 ng/mL for dabrafenib, OH-dabrafenib, and trametinib, respectively.

Conclusions: We developed both LC and paper-spray ionization methods for the detection and quantification of dabrafenib, OH-dabrafenib, and trametinib in EDTA plasma. While the LC method provides a more sensitive detection limit for trametinib, the paper spray method provides more reproducible results for OH-dabrafenib, without an isotopically labeled IS. Overall, the paper spray provided a faster analysis for all of the analytes. Based on the laboratory’s needs, the adoption of either analytical method could provide accurate results.

Poster Presentations for

Positive Bias in Fractionated Vitamin D2-D3 Method as Determined via LC-MS/MS Elizabeth Mast (Presenter) IU Health

Poster #1a View Map

This poster will be presented and discussed on Wednesday at 17:45 for 15 minutes in Montreal 3 (Track 2).

1. Problem
Method comparison of 25-hydroxyvitamin D3 showed significant number of samples with >20% positive bias while compared to reference method during validation of new instrumentation in the laboratory while 25-hydroxyvitamin D2 was within acceptable limits.

2. Method Information
200 uL serum sample prepared by acetonitrile protein precipitation.
TSQ Altis with Transcend TLX-LC; APCI
Turbo column: C18 XPL 0.5x50 mm
Turbo Mobile Phases: A- 0.1% Formic acid/ Water, B- 0.1% Formic acid/ Methanol; C- 40/40/20 ACN/IPA/Acetone
Analytical column: C18 Accucore 2.6 um 50 x 3mm
Analytical Mobile Phases: A- 0.1% Formic acid/Water, B- 0.1% Formic acid/Methanol

3. Troubleshooting Steps
Analysis of NIST standards, particularly the NIST standard with >20% epimer, showed the method was unable to separate 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3, thus showing >20% positive bias in the patients samples. We developed a new LC gradient using fluorophenyl 2.7um 100x3 mm column that was able to obtain baseline resolution of 25-hydroxyvitamin D3 from 3-epi-25-Hydroxyvitamin D3. Further verified accuracy using CAP PT samples.

4. Outcome
The new flurophenyl method was able to obtain baseline resolution of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 while maintaining the extra online extraction provided by the turbo column on the TLX Transcend HPLC. While this method is longer, performance of the new method resolved the positive bias in 25-hydroxyvitamin D3 as compared to the reference method. Additionally, the number of samples with detectable 3-epi-25-Hydroxyvitamin D3 corresponded with the number of samples with &gt;20% bias seen during previous validation studies. While 3-epi-25-Hydroxyvitamin D3 was not quantitated, Tracefinder, data analysis software, allows for an estimation of the amount present, which was in agreement with NIST standards with known 3-epi-25-hydroxyvitamin D3 values. Separation of 25-hydroxyvitamin D2 from 3-epi-25-hydroxyvitamin D2 was also achieved. No significant amount of 3-epi-25-hydroxyvitamin D2 was seen in patient samples.

Ordeals of Developing a Method to Measure Low-Level Concentrations of Serum Testosterone and my Troubleshooting Journey Leslie Farris (Presenter) Cleveland Clinic Foundation

Poster #1b View Map

This poster will be presented and discussed on Wednesday at 18:00 for 15 minutes in Montreal 3 (Track 2).

1. Problem

We needed to improve sensitivity to measure low concentrations (down to ~1 ng/dL) of total testosterone in serum by LC-MS/MS. Several issues affecting sensitivity and accuracy (to a lesser extent) were encountered throughout method development, as listed below (a-d). Through systematic examination of each potential variable, multiple modifications were implemented during method development, particularly to improve sensitivity.

a. Contamination: Initial injections of neat standards prepared in 50% acetonitrile were linear from 0.5 - 1000 ng/dL, but the low end of the curve was intermittently visible due to high background. Additionally, the baseline of neat injections was increasing over time. We suspected that either testosterone or another analyte with a similar ionization pattern was accumulating on the column or system.
b. Calibration preparation: The calibrators prepared in the candidate matrix generated biased results (>30%) relative to an outside reference lab. The sample comparison showed no bias (≤-5%) with a set of commercial calibrators.
c. Mobile phase modifier: Testosterone areas were higher with more labor-intensive sample preparations such as SPE and LLE, compared to protein precipitation methods. To glean the benefits of LLE without the partitioning time and supernatant transfer we opted for a SLE sample preparation. The cleanliness of the extracts improved the signal to noise ratio of our LLOQ (1 ng/dL), but the absolute area of the quantification peak was still lower than desired (<10,000).
d. Signal loss after sample extraction in plates: The SLE extraction procedure was optimized in cartridges but was switched to plates to improve throughput. A significant loss (3-fold) in signal was observed when the SLE extraction was switched from cartridges to plates, and the collection from glass vials to plastic plates.

2. Method Information (Optimized)
&bull; Sample Extraction
o Sample volume 200uL
o Supported liquid extraction (SLE)
o Elution solvent: 9:1 Hexane:Ethyl acetate
o Concentrate and reconstitute in 200 uL of 60% methanol
&bull; Instrument Information
o Binary Pumping system
 Multiplex over 2-channels
o MS Triple Quadrupole
&bull; Mobile Phase A: 0.2 mM Ammonium Fluoride in LCMS grade water
&bull; Mobile Phase B: 0.2 mM Ammonium Fluoride in LCMS grade (7:3)(Methanol:Acetonitrile)
&bull; 7-minute gradient, 0.5 mL/min flow rate
o Multiplexing run time ~ 5 minutes
&bull; Column: C18 100x3 mm, 2.6 um with guard cartridge (40°C)
&bull; Injection volume 50 uL
&bull; Quantitative MRM acquisition
&bull; Calibration range 1.0-2000 ng/dL in ultra-low testosterone stripped serum

3. Troubleshooting Steps
a. LC Cleaning/flushing and MS Bakeout
b. Evaluation of mobile phase reagents (i.e., water) and equilibration step
c. Assessment of candidate matrix for calibrators and preparation protocol
d. Comparing different sample preparation and mobile phase additives
e. Evaluation of plastic collection plate vs glass vials

4. Outcomes
a. Contamination:
The LC system was thoroughly cleaned/flushed and the MS was bakeout (Step 3a.) to remove any residual contaminants. To investigate further, we injected blank using a double gradient after a prolonged equilibration period at initial conditions (90% aqueous) in mobile phase A prepared using CLRW. The resulting chromatography showed a noticeable increase in the baseline signal at the analyte retention time. We repeated this step using mobile phase A prepared using bottled DI or LCMS grade water (Step 3b). LCMS grade water effectively reduced the background and risk of contamination but not the DI water.

b. Calibration preparation:
We investigated if the candidate matrix was contributing to the bias. Not surprisingly, several commercial serums and prepared alternatives using BSA and PBS screened were found to contain testosterone. We identified a double stripped ultra-low testosterone serum as a suitable candidate matrix for preparing calibrators (Step 3c.). The bias was corrected only after implementing the following steps to prepare in-house calibrators in double stripped ultra-low testosterone: sonicate the CRM in its ampule, dilute CRM in the same carrier solvent it was shipped in, sonicate and rock the high-level calibrator in matrix before preparing additional calibrators levels.

c. Mobile phase modifier:
Replacing the formic acid mobile phase modifier for a low concentration of ammonium fluoride (Step 3d.) resulted in a 3-fold increase in signal.

d. Signal loss after sample extraction in plates:
Through a series of investigations alternating the sample pre-treatment vessel (plastic vs glass), the SLE format (cartridge vs plate) and collection vessel (plastic vs glass), the root cause of the signal loss was attributed to the plastic collection plate. This was rectified by collecting the samples in glass-coated plates (Step 3e.).

Quantitative and Qualitative Analysis of Steroids by High-Resolution Mass Spectrometry Holly Pagnotta (Presenter) SCIEX

Poster #4a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Hormonal steroids regulate most body functions and the dysregulation of these molecules can play a role in the pathophysiology of human disease. Early techniques to measure endogenous steroids include immunoassay and gas chromatography-mass spectrometry (GC-MS). Immunoassays are problematic because they lack specificity for low-level steroids due to interference from endogenous steroids present at higher levels. In contrast, GC-MS offers higher specificity and is sensitive to low-level steroids, however, it requires extensive sample preparation via derivatization. More recently, steroid analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a sensitive and specific technique with a simplified sample preparation.

Here, the analysis of hormonal steroids by LC-MS/MS is explored using the ZenoTOF 7600 system. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were calculated for steroids and steroid levels were measured in plasma samples. EAD-derived fragment ions were used to structurally characterize the steroids of interest. EAD generated structure-specific fragment ions that were sufficient to distinguish steroid isomers and isobars during analysis without requiring extensive chromatographic development.

Methods:
Stock solutions of analytes and internal standards were diluted in PBS to generate standard curves using technical replicates to determine the LOD and LLOQ of steroids. PBS was used as a matrix surrogate because plasma contains significant amounts of endogenous steroids. Deuterated internal standards were added to NIST 1950 plasma samples at a final concentration of 1 ng/mL to quantify endogenous steroids. For plasma samples and quality control (QC) samples, 5 µL of the internal standard mixture was added to 200 µL of sample with and 300 µL DI water. Samples were shaken @ 700 rpm for 5 minutes, and the protein was precipitated by adding 250 µL of 0.1M zinc sulfate and shaking @ 600 rpm for 5 minutes after which 500 µL cold methanol was added. Samples were again shaken at 500 rpm for 5 minutes, centrifuged at 2637 rcf for 10 minutes at room temperature, and the supernatant was collected and loaded onto a preconditioned HLB SPE 30mg (30 µm) plate. The plate was washed with DI water and steroids were eluted with acetonitrile. The eluant was dried with N¬2, and samples were reconstituted in 50 µL 50:50 methanol:water.
Prepared samples were separated by high-performance liquid chromatography (HPLC) using a Kinetex biphenyl column from Phenomenex (2.6 µm particle size, 100 x 2.1 mm). Analytes were eluted from the column using a biphasic gradient. Mobile phase A was water with 0.2mM ammonium fluoride and mobile phase B was methanol. The flow rate was 400 µL/min. The column temperature was held constant at 50°C and the injection volume was 15 µL.
Data were acquired with a ZenoTOF 7600 system using 2 methods. The LLOQ and LOD of various steroids were determined using the scheduled high-resolution multiple reaction monitoring scan mode (sMRMHR). For DHEA-sulfate and estrone, pseudo MRM transitions were used (precursor ion to precursor ion) due to inefficient fragmentation. Experiments to structurally characterize steroids via EAD-based fragmentation were performed using a data-dependent acquisition (DDA) scan mode.

Results:
Standard curves analyzed using the sMRMHR scan mode run with CID-based fragmentation yielded LLOQ values ranging from 0.01 to 25 ng/ml and LODs ranging from 0.01 to 25 ng/mL to values for all compounds tested. However, distinguishing structural isomers is a significant challenge in bioanalysis. Traditional LC-MS/MS methodologies use CID to generate fragments for quantification. If isomers and/or isobars interfere with the analysis of CID-based fragments, then time-consuming chromatographic methods must be used to resolve these compounds. EAD-generated fragments have been shown to enable high compound specificity without the need for extensive chromatographic method development. CID product ion spectra for the isomers 11-deoxycorticosterone and 17-hydroxyprogesterone, both have masses of m/z 331.227. CID based MRM transitions are not sufficient to distinguish these molecules. In contrast, EAD-based fragmentation generated rich MS/MS data for the same pair of isomers. Three unique EAD-based fragments derived from 11-deoxycorticosterone that were not present in the spectrum for 17-hydroxyprogesterone. Similarly, 2 unique fragments were found for 17-hydroxyprogesterone. These product ions can be used in sMRMHR experiments using EAD-based fragmentation to increase the specificity of the assay by distinguishing between these 2 isomers.

Conclusions:
In this study, the ZenoTOF 7600 system enabled rapid and sensitive analysis of hormonal steroids in human plasma. The use of EAD enhanced structural details, surpassing the limitations of CID. Simultaneous CID- and EAD-based fragmentation supported high-throughput analysis while improving analyte specificity, marking a significant advancement in hormonal steroid analysis.

A Fast, Automated, and Reproducible Sensitive Method for Immunosuppressant Analysis in Whole Blood Nicholas Chestara (Presenter) DPX Technologies

Poster #5b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Immunosuppressant drug monitoring is commonly utilized in organ donation rejection prevention and in organ cancers such as kidney, pancreatic, and epithelial cell cancers. The continuous monitoring of these drugs saves lives, therefore a quick and easy sample preparation method prior to analysis is imperative in the hospital setting. Unfortunately, immunosuppressants are highly protein bound, and recoveries with traditional methods can be scarce and lack sensitivity. The accurate and robust filtration of four immunosuppressants (Cyclosporin A, Everolimus, Sirolimus, and Tacrolimus) are demonstrated in this poster.

Objectives
This method is quick, easy, and can be performed by anyone looking to analyze immunosuppressants in whole blood; this method provides clean extracts and therefore less instrument maintenance.

Methods
A MICROLAB® NIMBUS96 automated liquid handler (referred to as ALH) is utilized for sample preparation, however this can be performed manually or with other automated pipetting platforms. First, 50 µL of 0.2 M ZnSO4 in water is added to 50 µL of whole blood in a well plate for the initial step of protein precipitation. The solution is mixed thoroughly 10 times and allowed to settle for several seconds. Next, a 150 µL aliquot of acetonitrile (ACN) is added and the solution is mixed 20 times thoroughly for a fully precipitated sample. The ALH takes a fresh set of tips, aspirates the sample solution, goes Tip-on-Tip with the filtration tips, moves to a new well plate location, and dispenses the sample through the filtration tip into a well plate. The ALH disposes of the tips and picks up another set of tips. The tips transfer 100 µL of acetonitrile and 25 µL of HPLC water to the final well plate for injection. The tips then transfer a 125 µL aliquot of the filtrate to the injection well plate, mixing the solution to ensure homogenous composition for injection. The entire method from start to finish was 10 minutes.

Analysis was performed on a Shimadzu LC40 paired with a SCIEX 6500+ tandem mass spectrometer (LC-MS/MS). The analytical LC column is a Restek Force Biphenyl, 3 µm, 50 x 2.1 mm LC Column (PN 9629352) paired with a Restek Force Biphenyl, 5 x 2.1 mm EXP Guard Cartridge (PN 962950252). The choice of a biphenyl column allows for optimal separation and more flexibility in sample composition prior to injection; a 5 µL injection volume is used and the LC method is 8 minutes. The column is heated to 70°C, and the mobile phases are [A] 0.1% formic acid in HPLC water and [B] 100% methanol. The internal standards used are Everolimus D4 (Cerilliant, TX) and Cyclosporin A 11N15 (Cerilliant, TX).

Results
The immunosuppressants LOQs (level of quantitation) are determined to be 1 ng/mL for Tacrolimus, Sirolimus, and Everolimus and 10 ng/mL for Cyclosporin A. Reference ranges, based on available literature, for these analytes are above the determined LOQs. During a linearity study conducted in correlation with 2 UTAK Control QC’s, accuracy values were found between 88.5% and 106.6% for all compounds. The recovery is found to be 93% to 100% for all analytes from DPX filtration and a total recovery from filtration and protein precipitation is found to be 72% to 83%, where most of the recovery loss is from the protein precipitation.

Conclusion
For highly protein bound analytes such as immunosuppressants in whole blood, an effective sample preparation protocol is necessary to ensure accuracy of patient results. A fast and reproducible method for the analysis of immunosuppressants in whole blood is needed to determine how well a patient has acclimated to a new organ transplant.

LC-MS/MS Analysis of MAbs Using a Monoclonal Antibodies Quantification Kit – Spotlight on Infliximab John Vukovic (Presenter) Waters Corporation

Poster #6a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background: Therapeutic monoclonal antibodies (t-mAbs) have been a revolution in the therapeutic tools available to clinicians for treating a variety of conditions. In the era of personalized medicine, there is increasing awareness of the need to measure mAbs for the purposes of dose optimization and cost management. The use of Ligand Binding Assay (LBA) based techniques for measuring mAbs is well established but has some limitations, including poor performance, lack of standardization, a high cost when processing a limited number of samples, limited dynamic range, and the potential for cross-reactivity. Moreover, commercial kits are only available for a limited number of mAbs. Mass spectrometry, a technology widely used in clinical laboratories for monitoring small molecules, is an interesting alternative to overcome these limitations. Here we demonstrate Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) associated with the ready-to-use commercial mAbXmise™ kit is a simple way to implement mAbs measurement for the anti-TNFα mAb, infliximab, providing high analytical performance, ease of use, and high flexibility for laboratory personnel.

Methods: The Promise Proteomics Inflammation mAbXmise Kit contains all the calibrators, QCs, internal standard and sample preparation consumables to run the LC-MS/MS method for infliximab. The QC material was used to evaluate method precision. Anonymized plasma samples (Synnovis Analytics, UK) were extracted using the mAbXmise Kit and analyzed using an in-house developed LC-MS/MS method.
Briefly, 20µL plasma samples, including calibrator, and quality controls, are dispensed into the mAbXmise plates containing lyophilized stable labelled infliximab. Samples are transferred to the PuriXmise plate to perform the immunocapture of infliximab and allows washing of the sample to reduce matrix interferences. Samples are eluted into a collection plate and evaporated to dryness. Samples are resuspended, and the protease (CutXmise) is added to the sample to digest infliximab overnight. The digestion is quenched, with the samples immediately ready for analysis. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters XSelect™ Premier HSS T3, 2.5µm, 2.1 x 50 mm Column using a water/acetonitrile/formic acid gradient elution profile and the infliximab signature peptides SINSATHYAESVK (SIN), ASQFVGSSIHWYQQR (ASQ), GLEWVAEIR (GLE), SAVYLQMTDLR (SAV) and DILLTQSPAILSVSPGER (DIL) were quantified with both a Waters Xevo™ TQ-XS Mass Spectrometer (5µL injection) and Xevo TQ-S micro Mass Spectrometer (15µL injection), with a run time of 4.5 minutes.

Results: The method was shown to be linear from 2 - 100 µg/mL. Analytical sensitivity at lowest calibrator of 2µg/mL provided S/N (PtP) ≥15:1 for all tryptic peptides on both mass spectrometers. Coefficients of variation (CV) at 4 and 25µg/mL QC concentrations were all ≤ 10.0% (n = 5). The relative concentrations of the tryptic peptides were compared across 29 anonymized samples and good agreement was observed for GLE, SAV, DIL and ASA peptides. However, the SIN peptide demonstrated significant negative method bias, which could be attributed to its susceptibility to deamidation.

Conclusions: We have demonstrated a method for quantifying Infliximab in plasma using a commercially available kit for sample preparation followed by LC-MS/MS analysis. The use of the Promise Proteomics mAbXmise Kit makes the analysis accessible and easy to implement, while also being amenable to automation. The extended measuring range, between 2 µg/mL and 100 µg/mL, avoids the requirement for dilution sometimes observed with immunoassays, improving turnaround times for higher concentration samples. In addition, reduction in dilution and re-analysis improves utilization of the kit, which aids cost management. The Kit aids in the day-to-day reproducibility of the immunocapture and tryptic digestion of IFX for targeted LC-MS/MS analysis with the correct peptide selection. Compared to a direct digest surrogate peptide workflow, the process used in the kit reduces matrix interference, improves analytical sensitivity, and enables the use of lower sample volumes.

It has been demonstrated that the method can be run on both the Xevo TQ-XS and Xevo TQ-S micro Mass Spectrometers which provide the dynamic range to quantify IFX across the expected range, and the selectivity and analytical sensitivity to obtain low-level quantification of the IFX surrogate peptide in plasma samples.

The data presented combines the use of a kit dedicated to the preparation of samples and the use of liquid chromatography and mass spectrometry instrumentation to perform the quantitative analyses. The mAbXmise Kit described has not been cleared by any regulatory entity for diagnostic purposes outside of Europe. Proteomics mAbXmise Kits are not available for sale in all countries.

Rapid Quantification of Amiodarone and its Metabolite Using LC-MS/MS Andrea Bozovic (Presenter) University Health Network & University of Toronto

Poster #7a View Map

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

INTRODUCTION
Amiodarone (AMI) is an antiarrhythmic medication used to treat various types of irregular heartbeats (arrhythmias). It is primarily used for the treatment of ventricular arrhythmias, especially in cases where other antiarrhythmic medications have not been effective. Amiodarone has a long half-life, and this can influence the timing of dosage adjustments and potential drug interactions. Clinical testing of serum amiodarone levels is performed to monitor and optimize the therapeutic concentration of amiodarone in a patient's blood. Indication of testing include: a) monitoring therapy to ensure that the patient is receiving an effective dose for managing their arrhythmia; b) avoiding toxicity: amiodarone has a narrow therapeutic range, and levels that are too high can lead to adverse effects.

OBJECTIVES
To develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of amiodarone and its metabolite (desethylamiodarone (DEA)) and implement the test in the routine clinical diagnostic service.

METHODS
Instrument used in setting up this clinical test was a SCIEX ExionLC AD liquid chromatography system coupled to a TQ6500+ triple quadrupole mass spectrometer. ACE C18-PFP (100 x 2.1 mm, 3 um) column equipped with an in-line filter was chosen for chromatographic separation. Column oven was kept at 30 ± 1°C, while the autosampler had a thermostatted sample compartment that was maintained at 15 ± 1°C. Separation of the measurands from the matrix component was achieved using gradient elution, with solvent A: 0.1% Formic Acid in Water, and solvent B: 0.1% Formic Acid in Acetonitrile. Run time was 2.50 min. Mass spectrometer operated in the multiple reaction monitoring (MRM) mode, and positive ion polarity. Two transitions were monitored for AMI and DEA, and one for each isotopically labeled analogue used as internal standards (d4-AMI, and d4-DEA). Before LC-MS/MS analysis, proteins were precipitated from serum using acetonitrile, and diluted ten-fold using LC solvent A. Calibration standards and quality control material were made in-house from blank serum pools by adding certified reference material.

RESULTS
Method was linear between 0.1 and 20.0 mg/l for both measurands, with the correlation coefficient (r2) greater than 0.99. Within-run precision of ten replicates of 3 patient sample specimens was well below 10%, for both AMI and DEA. Between-day precision study that was monitored for two samples at mean concentrations: 1.3 mg/l and 2.0 mg/L over eleven days, generated %CVs of 5.7% and 6.3% for AMI, and 7.3% and 7.1% for DEA, respectively. Carryover was assessed for three pairs of high and low samples in triplicates, and no significant carryover was observed (<0.3% overall). Limit of quantification of of both AMI and DEA was much lower than 0.1 mg/l (Functional sensitivity, defined here as the concentration that results in a CV=20% and S/N>10) but was set to 0.1mg/l as this was medically relevant concentration. Dilution recovery was assessed by analyzing dilutions (0-, 5-, 10-, and 50-fold) of three samples within AMR (2.5, 5, 10 mg/l), and three samples above AMR (20, 35, 50mg/L) in duplicate. Assessments passed the ±15% acceptability criteria. Method comparison with the in-house HPLC method showed ~20% positive bias, while a comparison with another LC-MS/MS method a 8-10% bias was observed, with no significant interpretation changes to the test results. Evaluation of matrix effect was performed by adding the drug at a known concentration to 12 samples post-extraction (6 were non-treated and 6 were AMI-treated patient samples). Matrix effect was within total allowable error or 20%. Assessment of the sample stability showed that the concentration of both AMI and DEA is unaffected up to five freeze-thaw cycles if the serum specimen is kept at -20°C. Extract is stable in the fridge and freezer for at least 14 days.

CONCLUSION
We have established a new in-house LC-MS/MS method for rapid, simultaneous detection of amiodarone and desethylamiodarone. The improvement over the in-house HPLC method included increased specificity, shorter retention time, improved accuracy and sensitivity. The method has so far been robust and free of interferences.

Development and Validation of a TDM Assay for Nimodipine Quantitation in CSF and Plasma Jonathan Hoyne (Presenter) Mayo Clinic

Poster #11a View Map

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

INTRODUCTION:
Aneurysmal subarachnoid hemorrhage (aSAH) is a debilitating threat to life and neurological function. Amongst patients that reach the hospital, delayed cerebral ischemia (DCI) is a frequent negative sequalae of aSAH. Standard preventative treatment of delayed cerebral ischemia involves oral administration of 60 mg Nimodipine every 4 hours. Nimodipine is an L-type calcium antagonist that acts as a vasodilator. Equilibrium concentrations of nimodipine in CSF and serum on standard dosing are highly variable. Generally, higher concentrations of CSF nimodipine within the therapeutic range improve outcome but the correlation isn’t linear or universal. A subset of patients fails to achieve quantifiable nimodipine concentrations in CSF. Other patients experience systemic hypotension with equilibrium concentrations consistent with those that in other patients decrease risk of DCI. Systemic hypotension is a contraindication for full nimodipine dose, but lower doses are associated with an increased risk of DCI. The nature of the interpatient variability of equilibrium nimodipine concentrations is ill-understood. Previous studies suggest sex, age, body surface area, metabolic heterogeneity, location, or magnitude of initial insult play roles but these have not been interrogated in combination. Currently nimodipine dose is standardized across patients without accounting for these sources of individual variation. We developed a high sensitivity nimodipine assay which will allow us to evaluate CSF and plasma concentrations of nimodipine. We will utilize this assay to investigate the within- and between- patient variability in the relationship between dose and equilibrium plasma and CSF concentrations.

METHODS:
We developed an LC-MSMS assay for nimodipine in CSF and Plasma. We spiked nimodipine into negative patient pools to create calibrators and control materials as well as mock patient samples. After mixing of the spiked samples, we aliquoted 250 mcL of manufactured CSF or plasma samples and extracted with 500 mcL of acetonitrile + interanal standard. Nitrendipine at 1.25 ng /mL. Incubate 5 mins at RT. Vortex for 30 seconds and centrifuged at 3000g for 10 mins. 500 mcL of each extract are transferred to a 96 microwell plate and 40 mcL of sample are injected into the LC-MS/MS.

Liquid chromatography was performed with an LX-2 LC system and an HPLC autosampler at a flow rate of 0.4 mL / min.
Mobile phase A: 20 mM NH4Acetate in 50/50 MeOH:H2O + 0.1% formic acid.
Mobile phase B: 20 mM Ammonium Acetate + 0.1% formic acid in 100% MeOH.
Time
0 60 80% A 20% B
60 180 10% A 90% B Ramp
180 210 10% A 90% B
210 270 80% A 20% B Ramp
270 300 80% A 20% B

Tandem Mass Spectrometry was performed using an ABSciEx API 4500. The mass spectrometer was operated in ESI positive mode.
Nimodipine was monitored at 419.1/343.2 for the quantifier ion and 419.1/301.0
Nitrendipine was monitored at 361.2/315.0 for the quantifier ion and 361.2/329.1
LOQ is 10 pg/mL in plasma and 2 ng/mL in plasma. Plasma LOQ represents the lowest likely level in patient samples rather than an analytical limit. AMR is 10 pg - 750 pg/mL in CSF and 2 ng – 75 ng/mL in plasma. The assay is linear within these values. Intraassay and interassay CV is well below 20% at levels consistent with patient values expected from the literature.

CONCLUSION:
Sensitivity of the assay in CSF was sufficient to expect quantifiable levels in patient samples for all patients based on previously published data. Performance of the assay in plasma was consistent with published methods. Other parameters of assay performance were well within analytical expectations for a TDM assay. We expect to be able to use this assay to interrogate the variability between plasma and CSF nimodipine concentrations within patients. We will combine TDM data with pharmacogenetic, demographic, and physiologic data to attempt to derive a model of nimodipine pharmacokinetics which will allow clinically useful estimation of CSF nimodipine concentration based on measured plasma nimodipine concentrations. Alternately, we will measure CSF nimodipine concentrations for clinical evaluation of therapeutic dose within patients.

An Examination of Potential Interference in a Clinical Assay for Botulinum Neurotoxins through Mass Spectrometric Detection Kaitlin Hoyt (Presenter) Centers for Disease Control and Prevention

Poster #12a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Botulism is a paralytic disease caused by botulinum neurotoxin-mediated cleavage of proteins in the neuromuscular junction that facilitate acetylcholine exocytosis. The current approved confirmatory assay for BoNTs in clinical specimens is the mouse bioassay. An in vitro assay to detect and differentiate BoNT, the Endopep-MS method, has been developed and validated for clinical samples using a benchtop mass spectrometric detection. Validation included potential interfering substances of the Endopep-MS method in clinical specimens with the intent of method distribution in public health laboratories while seeking U.S. Food and Drug Administration (FDA) approval.

Methods:
The Endopep-MS method detects BoNT based on the cleavage products of a peptide substrate, which mimics its native target. Serum and stool samples spiked with potential interfering substances (such as acetaminophen and bilirubin) at therapeutic levels and with BoNTs at 5 times the limit of detection for each serotype, were tested by Endopep-MS, Toxin was extracted from the sample with high-affinity monoclonal antibodies and the cleavage of a serotype specific peptide substrate determined the presence of BoNT utilizing MALDI-TOF mass spectrometry detection.

Results:
The panel of potential interfering substances was used to assess selectivity for the validation of the Endopep-MS method. The interfering substances panel was designed to test exogeneous and endogenous substances that are commonly present in clinical specimens such as serum and stool extract that could affect the possible outcome of results for the Endopep-MS method. The exogeneous and endogenous substances presented no interference in testing spiked serum and stool extract with the BoNT Endopep-MS assay, with the one exception of sodium phosphate. Stool samples spiked with sodium phosphate at a concentration of 2.5 g/L resulted negative for BoNT.

Discussion &amp; Conclusions:
The list for interfering substances was determined from the Clinical Laboratory Standards Institute (CLSI) EP07-A2 guideline, with additions made by the FDA and the National Center for Emerging and Zoonotic Infectious Diseases epidemiologists at the Centers for Disease Control and Prevention. Sodium phosphate was the only substance to show interference with the assay. It was included as an exogenous substance because it is present in most enema solutions at ~1 g; however, patients with suspected botulism only receive sterile non-bacteriostatic water enemas for stool collection (with no sodium phosphate). Although the Endopep-MS method uses sodium phosphate in gelatin phosphate-buffered collecting fluid and 1X PBST, the limits are below the interference threshold at 6 mg/mL, 0.20 mg/mL, and 1.6 mg/mL. Therefore, although the large amounts of sodium phosphate had interference with the detection of BoNT, it would not be a substance present in stool specimens collected for botulism testing. Testing for potential interfering substances is a critical step of methods validation studies and our results demonstrate that the Endopep-MS assay for BoNT detection is not affected by 72 substances.



The findings and conclusions in this presentation have not been formally disseminated by the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry and should not be construed to represent any agency determination or policy.

Validation of a Simple Dilute-and-Shoot LCMSMS Method for Urinary Metanephrines: Time to Break Free? Jason Robinson (Presenter) Health PEI Provincial Laboratory Services

Poster #13a View Map

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

Introduction: Pheochromocytoma is a tumor of the adrenomedullary chromaffin cells. These tumors commonly produce excessive amounts of the catecholamine metabolites metanephrine, normetanephrine, and 3-methoxytyramine, which are collectively referred to as metanephrines and are generally specific for pheochromocytomas. Indeed, quantitation of fractionated urine or plasma free metanephrines are the first line biochemical test for the investigation of pheochromocytoma. Often, the fractionated urine metanephrines are measured in an acidified 24-hour collection, followed by in vitro acid hydrolysis to deconjugate urine metanephrines prior to measurement by LCMSMS or HPLC. However, the amount of free, non-conjugated urine metanephrines are more clinically relevant versus the total deconjugated amount; and moreover, urine free metanephrines are reported to be highly stable in refrigerated urine without acid preservative. We hypothesized that by measuring the free fraction of metanephrines in urine we could establish a streamlined and concise laboratory workflow in addition to more convenient collection requirements for patients.

Objectives: To develop an accurate and robust LCMSMS method to detect urinary metanephrines using a UHPLC coupled with a Thermo TSQ Quantis tandem mass spectrometer.

Methods: The mass spectrometer was tuned using pure calibrator standards for metanephrine, normetanephrine and 3-methoxytyramine. Deuterated internal standards were used for each compound. Final sample preparation involved diluting urine samples with 4-volumes of mobile phase A (10 mM ammonium formate; pH 2.8), and 2-volumes of internal standard in mobile phase B (100% methanol). Samples were injected with UHPLC through a pentafluorophenyl column (150 mm x 2.1 mm; 1.9 &micro;m) and a 13-minute gradient method eluted all three compounds of interest with column reconditioning. The concentration of each target was determined using commercial calibrators, and within-run precision and repeatability studies were performed using commercial QC material with CLSI based protocols. The method was extensively compared to an accredited referral laboratory using non-linear regression analysis and via an ISO15189 proficiency testing program for urine free metanephrines by LCMSMS. We also evaluated sample stability and various preanalytical preparations such as solid-phase extraction.

Results: Within-run precision of all targets was 0.9 to 2.5%; and repeatability was 2.7 to 3.1%. The limit of quantitation was between 1.3 to 1.5 nmol/L and the method was linear to ~10000 nmol/L with R^2&gt;0.99. Our method was compared to a referral lab using 105 clinical samples and we report an acceptable negative bias (-1.2 to -14 nmol/L) and comparison across the 3 metanephrine targets (R2 = 0.82 to 0.92). Importantly, samples above the clinical cut-offs were 100% concordant with the referral laboratory. Method accuracy was considered acceptable based on the performance of 3 proficiency testing events with an LCMS peer group of 11. Solid-phase extraction did not measurably improve our analytical sensitivity based on signal-to-noise and the instrument performed excellent with only the minimum amount of vendor recommended weekly maintenance. We established reference intervals in nmol/d using non-acidified 24-hour urines submitted to our laboratory, and transferred intervals from a referral lab as well as the literature. The samples were stable refrigerated or frozen for 4 weeks.

Conclusions: We validated a simple and robust methodology for detecting urine free metanephrines, which allowed us to repatriate a large volume send-out test. The collection requirement allows for more convenient patient collection protocols and the ability to perform more tests on 24-hour collections.

Development of a Rapid and Sensitive LC-MS/MS Assay for Dolutegravir Quantitation in Breast Milk Ashley Rackow (Presenter) Johns Hopkins University

Poster #15a View Map

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

Introduction: The use of antiretroviral therapy (ART) in pregnant people living with HIV can prevent peri- and post-natal transmission of the virus. Dolutegravir (DTG) is an integrase strand transfer inhibitor that is utilized in several combinatorial ART regimens. DTG is a lipophilic drug, and may deposit in breast milk during the postpartum period. Previous work has shown transplacental and breastmilk transfer of DTG, resulting in neonatal and infant drug exposure. However, DTG pharmacokinetics (PK) in breast milk are incompletely understood. Bioanalytical tools are required to understand the multicompartment pharmacology and efficacy of DTG in preventing mother-to-child HIV transmission.

Objectives: To develop and validate a liquid chromatographic-mass spectrometric (LC-MS/MS) assay in breast milk to support clinical trials research.

Methods: Breast milk was purchased acquired from BioIVT (Hicksville, NY). DTG was acquired from Toronto Research Chemicals (Toronto, ON), and its isotopically labeled internal standard, 13C, 2H5-DTG, was acquired from Alsachim (Illkirch, FR). DTG was spiked into breast milk to prepare calibration standards and quality controls. Drug was isolated via protein precipitation, and reconstituted material was analyzed using an API 5500 mass spectrometer (SCIEX, Redwood City, CA) interfaced with a Shimadzu LC-40. Analytical separation occurred using a Waters Acquity C8 column, and the instrument was operated in selective reaction monitoring and positive ionization modes. Monitored transitions were 420.20/277.10 for DTG and 426.20/133.10 for 13C, 2H6-DTG. The assay was validated in accordance with FDA bioanalytical method validation recommendations.

Results: The analytical measuring range of the assay was 0.5-1000 ng/mL, with an analytical run time of 2.2 minutes. Inter-assay precision and accuracy ranged from 4.6% to 15.1% and -2.4% to 9.6%, respectively, for materials prepared at the lower limit of quantification (0.5 ng/mL), low (1.5 ng/mL), mid (75 ng/mL) and high (800 ng/mL) quality control levels. Matrix effects assessments showed 12% ion suppression for DTG and 13C, 2H5-DTG, and recovery efficiencies of 99%; relative matrix effects were negligible. DTG also demonstrated stability under freeze-thaw, re-injection, and room temperature conditions.

Conclusion: An LC-MS/MS method for DTG quantification in breast milk has been developed and validated. The assay met acceptable performance criteria and may be used in downstream applications to understand the multicompartment pharmacology and distribution of DTG into breast milk.

Evaluating Analytical Performance of Tacrolimus LC-MS/MS Assay Using Ascomycin Versus Tacrolimus-C13D2 Internal Standards. Kwaku Twum (Presenter) University of North Carolina - Chapel Hill

>> POSTER (PDF)

Poster #16a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Objective: To investigate and compare imprecision, accuracy, linearity, and potential matrix interference in tacrolimus LC-MS/MS assay using ascomycin or tacrolimus-C13D2 as internal standards.

Introduction: Therapeutic monitoring of whole blood tacrolimus concentration via liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains essential to reduce the risk of rejection of a transplant organ. Conventional LC-MS/MS approaches for tacrolimus measurement have utilized the internal standard ascomycin, a structural analog. Recently, deuterated isotopically labeled tacrolimus internal standards have become increasingly commercially available, which may provide improved performance over ascomycin. This study evaluated the method performance for measurement of tacrolimus in whole blood using ascomycin versus deuterated tacrolimus (tacrolimus-C13D2) internal standards.

Methods: Whole blood tacrolimus concentrations are measured by LC-MS/MS as part of routine patient care at UNC Health McLendon Clinical Laboratories (Chapel Hill, NC). In brief, EDTA whole blood tacrolimus is extracted using an automated TECAN auto-pipetting system, which involves pipetting (in the following order) 100 µL of zinc sulfate, 100 µL of calibrator/control/sample, 250 µL lysing solution containing acetonitrile and internal standard (ascomycin or tacrolimus-C13D2) into microcentrifuge tubes. Samples are vortexed and centrifuged at 13000g for 3 minutes. A 100 µL aliquot is transferred to a 96-well microtiter plate and analyzed using a Waters Xevo TQD Mass Spectrometer equipped with an Acquity UPLC BEH C18 Column (2.1x50 mm) with an Acquity UPLC BEH C18 guard column (2.1 x 5mm). An electrospray ionization interface is operated in positive ion [ES(+)] mode and MS/MS detection in multiple reaction monitoring mode. The performance in the linearity of calibrators, precision, accuracy, and interference of the matrix using the two internal standards were investigated. To assess imprecision, three levels of UTAK quality control materials [L1(3.65 ng/mL), L2(13.26 ng/mL), and L3(20.32 ng/mL)] spanning the analytical measuring range (AMR) were tested in over 20 analytical runs spanning ten days to calculate percent coefficient of variation (CV). Accuracy was assessed by comparing a) measured results in residual external quality assessment materials with peer results and calculating standard deviation index (SDI) and b) patient result comparisons using tacrolimus-C13D2 to a validated assay that uses ascomycin. Linearity and % recovery of six-point calibration results prepared using tacrolimus-C13D2 or ascomycin for each analytical run were compared. Statistical analysis of agreement between results was all performed using Microsoft Excel.

Results: Between-day imprecision was measured at the CV of 8.40% (L1), 2.66% (L2), 3.67% (L3), and 3.63% (L1), 3.87% (L2), 9.27% (L3) for QC measured with tacrolimus-C13D2 and ascomycin respectively. Using previously reported patient samples (n=17) with ascomycin as the internal standard, tacrolimus-C13D2 showed a mean bias of +0.55 ng/ml or 3.00%. Testing of residual CAP survey samples showed a better CAP survey challenge closer to the peer group mean for tacrolimus-C13D2 (SDI -1.48, -1.17, and -1.35) compared to ascomycin (SDI -2.1, -2.2, -2.1). Six calibrator levels measured each day of testing demonstrated linear response and showed a slightly better recovery of manufacturer-assigned concentrations using tacrolimus-c13d2 (mean bias -0.02%, R2 of 0.9997) compared to ascomycin (mean bias -0.58%, R2 of 0.9984).

Conclusion: Whole blood tacrolimus measurement via LC-MS/MS demonstrated comparable analytical performance using tacrolimus-C13D2 or ascomycin as an internal standard. Linearity, imprecision, and accuracy in tacrolimus measurements showed acceptable performance for both internal standards, though a negative bias in measured tacrolimus compared with peers was observed. In evaluating the choice of internal standard, other factors such as cost and ease of obtaining pure isotopically labeled analog must also be considered.

Fentanyl Urine Drug Screen Comparison of the Ark II and Lin Zhi FEN2 Immunoassays Brian Lam (Presenter) University of California Los Angeles

Poster #18a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background: Given the opioid epidemic, fentanyl screening in urine has become increasingly important. Immunoassays remain the most common screening methodology due to the high throughput and ease of integration into automated chemistry systems. The ARK II is a widely used immunoassay, while a novel assay, FEN2 by Lin Zhi, has become available on the Roche platform; here, we evaluate and compare their performance.

Methods: 457 urine samples were analyzed for fentanyl across the FEN2 and ARK II assay on the Cobas c502 platform. Samples were analyzed immediately upon request for drug of abuse screening or frozen for subsequent analysis. For confirmation testing, a liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) method with a limit of detection of 1 ng/mL for fentanyl/norfentanyl was used. Any sample with either fentanyl or norfentanyl above the LC-MS/MS cut-off was deemed positive.

Results: Both the ARK II and FEN2 immunoassays adequately detected fentanyl. The ARK II had a sensitivity and specificity of 86.0 and 96.6, respectively, and FEN2, with a sensitivity and specificity of 87.5 and 98.4, respectively.

Conclusion: The current data set has some discrepant results between the ARK II and Lin Zhi immunoassays. Most of the discrepant results have concentrations of fentanyl and norfentanyl near the cut-off for immunoassay detection.

A Low-Level Analytical Method for the Quantification of Serum Free Testosterone using LC-MS/MS for Clinical Research Lisa Calton (Presenter) Waters Corporation

Poster #19b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Clinical research of free testosterone in human serum has long been considered challenging, partly due to the inherent low concentrations (&lt;3% of total testosterone is free). Furthermore, issues relating to variability in results obtained from equilibrium dialysis, the gold standard, have been noted due to technical challenges in addition to lack of control over temperature, pH and shifts in equilibrium. A calculation developed by Vermeulen et al has also been widely used, taking into account total testosterone serum albumin and sex hormone-binding globulin (SHBG) concentrations.

OBJECTIVES: The aim of this project was to develop a novel clinical research, method for the analysis of serum free testosterone using a short, reproducible and accurate equilibrium dialysis protocol and an analytically sensitive mass spectrometer.

METHODS: Testosterone certified reference material (Merck, UK) was used to create calibrators in 52.75 mM HEPES buffer adjusted to pH 7.4. In-house QC materials prepared in serum using individual, pooled and stripped serum matrices (Golden West Biologicals, USA and BioIVT, UK), were used to evaluate method precision. Serum samples (200 &micro;L) were incubated at 37&deg;C with 400 &micro;L pH-adjusted 52.75 mM HEPES buffer for 2 hours, mixing at 800 r.p.m. with Rapid Equilibrium Analysis (RED) devices and plates (Thermo Fisher, UK). Diasylate was spiked with testosterone-13C3 (Merck, UK), then processed with a liquid-liquid extraction (LLE) procedure. Using an ACQUITY&trade; UPLC&trade; I-Class System, samples were injected onto an ACQUITY UPLC BEH&trade; C18, 1.7&micro;m, 2.1 x 100 mm Column using a water/methanol/ammonium fluoride gradient elution profile and quantified with a Xevo&trade; TQ Absolute Mass Spectrometer. The standard procedure for achieving equilibrium in the incubation step is to incubate for 4 hours at 37&deg;C, mixing at approximately 250 r.p.m. However, we have demonstrated that a relatively quick incubation step with a fast mix can achieve equilibrium as well. Additionally, it is essential to control the pH of the dialysis buffer and, particularly, the temperature of the chamber during incubation.

RESULTS: The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.5&ndash;650 pg/mL. Addition of high concentrations of endogenous substances (albumin, bilirubin, creatinine, cholesterol, triglycerides and uric acid) did not affect quantification. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (&le;20%CV and &le;15% bias) at 0.50 pg/mL. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs at approximately 2.79, 8.80 and 134 pg/mL respectively were all &le; 8.4% (n=25). Accuracy was assessed by analyzing 45 male external quality assurance (EQA) samples (NEQAS, UK) ranging in concentration from 50.1-268.6 pg/mL and the resulting Deming equation y=-2.869+0.947x demonstrated good agreement. When LC-MS/MS determined concentrations were processed against Vermeulen equation determined concentrations (total testosterone and SHBG concentrations were provided by the scheme, a concentration of 45 g/L albumin was assumed), again good agreement was shown with a Deming equation y=-0.2008+0.9325x.

CONCLUSIONS: We have successfully quantified free testosterone using a fast equilibrium dialysis procedure with LLE and LC-MS/MS analysis, for use in clinical research. The method demonstrates sub pg/mL analytical sensitivity, excellent linearity and precision, with minimal matrix effects and a strong agreement with an EQA scheme.

For Research Use Only, Not for use in diagnostic procedures.

Unmasking Designer Benzodiazepines: Comprehensive Analysis Reveals Evasion from Targeted Detection Methods in Substance Abuse Users Raymond Suhandynata (Presenter) University of California, San Diego

Poster #21b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Designer benzodiazepines belong to the synthetic depressant class designer drugs and pose a significant health risk to substance users. Standard practice for drugs of abuse testing in clinical laboratories typically rely on targeted LC-MS/MS analysis of urine specimens, predominantly focusing on commonly prescribed benzodiazepines and their metabolites, omitting designer benzodiazepines. These limitations have prompted some laboratories to incorporate broad-spectrum high-resolution LC-MS/MS analysis into their testing protocols to facilitate the identification of designer benzodiazepines and other designer drugs.

Methods:
A total of 23,747 urine specimens, originating from pain clinics and drug rehabilitation centers, underwent testing for benzodiazepines using a clinically validated benzodiazepine immunoassay and targeted LC-MS/MS benzodiazepine panel. Out of these, 945 were positive by immunoassay (Thermo Fisher DRI Benzodiazepine), while 886 were confirmed positive by LC-MS/MS. Specimens with discordant results (positive by immunoassay but negative by targeted LC-MS/MS) underwent further investigation using a broad-spectrum LC-QTOF-MS approach. MS spectra were acquired in positive MSE mode and matched to a Waters UNIFI toxicology spectral library containing over 1,500 compounds and significant library matches were confirmed by purchasing standard or certified reference material of the suspected analyte when available Additionally, immunoassay cross-reactivity of four designer benzodiazepines was evaluated using three separate immunoassays.

Results:
Among the 945 benzodiazepine immunoassay positive urine specimens tested, 6.2% (n=59) were not positive for benzodiazepines when tested by the targeted LC-MS/MS panel. Further analysis of 51 of these specimens (8 specimens could not be sequestered) by LC-QTOF-MS revealed designer benzodiazepines in 66.7% (n=34) of them. The most prevalent designer benzodiazepines detected were flualprazolam, etizolam, and flubromazolam.

Conclusion:
This study underscores the limitations of traditional targeted LC-MS/MS analysis in detecting designer benzodiazepines among substance abuse users. The adoption of a comprehensive approach involving immunoassay screening followed by both targeted and broad-spectrum LC-MS/MS methods is crucial for developing an accurate understanding of designer benzodiazepines usage in the population.

Rapid Quantitative Screening of 18 Synthetic Cannabinoids in Urine Using DART-MS Analysis Zahuindanda Aventura (Presenter) Bruker Applied Mass Spectrometry

Poster #22a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction: Immunoassay-based (IA) detection for drugs of abuse is commonly used as an initial screening step in urine-based drugs testing due to rapid generation of results and ease of automation. However, IAs suffer from significant issues with cross-reactivity leading to false positives, thus requiring costly and time-consuming chromatography-based confirmatory testing. As a cost-effective alternative, DART-MS provides quantitative and highly selective results greatly reducing or eliminating false positives compared to conventional IA-based drug screening. In this work, we report on the development of a rapid, chromatography-free screening approach for eighteen synthetic cannabinoids in urine: (4-cyano-CUMYL-BUTINACA (1), 4-fluoro ABUTINACA N-(4-hydroxybutyl) metabolite (2), 4-fluoro MDMB-BUTICA (3), 4-fluoro BUTICA butanoic acid metabolite (4), 4-fluoro MDMB-BUTINACA (5), 4-fluoro MDMB-BUTINACA N-butanoic acid metabolite (6), 5-fluro ADB metabolite (7), 5-fluoro MDMB-PICA (8), 5-fluoro MDMB-PICA metabolite (9), ADB-4en-PINACA (10), ADB-BINACA (11), ADB-BUTINACA (12), ADB-BUTINACA N-(4-hydroxylbutyl) METABOLITE (13), ADB-HEXINACA (14), AMP-4en-PINACA (15), JWH 018 N-pentanoic acid METABOLITE (16), MDMB 4en-PINACA-butanoic acid metabolite (17), MDMB-CHMICA METABOLITE (18)). This DART-MS screening method successfully measures the targeted synthetic cannabinoids in 96 samples at a rapid throughput of 23 seconds per sample.

Methods: For method development, triplicate calibration series were prepared by spiking certified drug-free urine with standards 1-18 (0.1-2500 ng/mL) using deuterated AB-PINACA as an internal standard. Hydrolysis was performed by adding 50 µL Kura enzyme to 500 µL pre-spiked certified aliquots of drug-free urine and incubated at room temperature for 20 minutes. After hydrolysis, 500 µL 0.1 M Borax buffer (pH=10.4) and 2.5 mL 30:70 (ethyl acetate:n-chlorobutane) were added to each sample followed by a 30 second agitation. Samples were centrifuged at 4000 RPM for 10 minutes and the organic layer was transferred to glass vials and evaporated to dryness under N2 at 40°C followed by reconstitution in 100 µL MeOH. Reconstituted samples were vortexed for 30 seconds, and 2 µL aliquots of each sample were transferred onto a Bruker DART QuickStrip HTS-96 screen and allowed to dry under N2 gas at 40°C for 15 minutes. For analysis, the prepared QuickStrip-HTS 96 screen was loaded onto the automated XY transmission stage of a TQ-Plus (Bruker Daltonics) triple quadrupole mass spectrometer for DART-MS-MS analysis. Accuracy was determined in triplicate using certified drug-free urine without detectable levels of 1-18 at 2 levels for each analyte within the linear range of each calibration series. Results were validated against LC-MS that was performed using 20 urine samples confirmed as positive for one or more analytes.

Results and Discussion: DART and TQ-plus parameters were optimized for high sensitivity, precision, and fast analysis time. With DART gas temperature and grid voltage optimized at 350°C and 100 V, respectively, unique MS/MS transitions, collision energies, and MS scan times were successfully identified for 1-18. DART-MS analysis of the synthetic cannabinoid panel resulted in a good linear correlation of R2 > 0.95 and an accuracy between 87 and 107% for all 18 analytes across the defined calibration ranges. The lower level of quantitation (LLOQ) of between 0.1 to 5 ng/mL and cross-validation of the samples showed good correlation with LC-MS data, indicating that this rapid chromatography-free workflow is sufficient in detecting all 18 analytes at or below the common cutoff values without the high rate of false positives associated with IA based screening approaches.

The results presented herein demonstrate the suitability of the DART-MS workflow as a rapid, quantitative, and selective alternative to conventional IA-based urine screening by offering a quantitative method with the benefits of minimizing false positives typically associated with IA based screening, avoiding costly and unnecessary chromatography-based confirmatory testing.

Ultra-fast Online Solid Phase Extraction LC/MS/MS Method for the Simultaneous Analysis of Steroid Hormones Using the Multiplexed 4-channel System Eishi Imoto (Presenter) Shimadzu Scientific Instruments

Poster #23a View Map

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

Introduction:
　The Multiplexed 4-channel LC/MS/MS system uses up to 4 multiple, alternating sample introduction streams to keep a single mass spectrometer working continuously. Online solid phase extraction (SPE) automatically reduces matrix effects in LC/MS/MS and enriches the analyte to increase reproducibility and sensitivity without complicated sample pretreatment. Combining these technologies, multiplexed 4-channel online SPE LC/MS/MS system is expected to maximize ruggedness and robustness while increasing sample throughput. However, high-throughput online SPE LC/MS/MS requires complex method construction and optimization for target compounds.

Objectives:
　The primary objective of this study is to establishment of rapid online SPE LC/MS/MS method coupled to high-throughput multiplexed 4-channel system.

Methods:
　The multiplexed 4-channel online SPE LC/MS/MS system (Nexera QX) was composed of an autosampler, a column oven, 4 binary pumps for extraction and the other 4 binary pumps for separation, an up to 4 solvent selection pump for the needle inner rinse and a triple quadrupole mass spectrometer (LCMS-8060NX). Almost 20 steroid hormones were spiked into human serum which was commercially available and had ultra-low hormones and steroids. Spiked serum samples were treated with liquid-liquid extraction with methyl tert-butyl ether. Online SPE was conducted using newly developed C18 SPE column, and analysis was performed with a biphenyl column. Multiple reaction monitoring (MRM) acquisition was operated in positive and negative ionization mode using ultra-fast polarity switching with triple quadrupole mass spectrometer.

Results:
　Prior to the analysis of spiked serum samples, steroid hormones in methanol were analyzed to optimize LC conditions in order to maximize the efficiency of extraction from trap column. For this purpose, the parameters, such as initial solvent concentration, flow rate, loading time and elution solvent on online SPE step were optimized. Additionally, it is necessary to assess whether compounds are properly transferred effectively from SPE column to separation column. Analytical initial solvent concentration and confluent flow rate were adjusted based on peak shapes and areas. Finally, the chromatographic selectivity was demonstrated through the baseline resolution of isobaric steroids species; Deoxycorticosterone and 17-Hydroxyprogesterone; 21-Deoxycortisol, Corticosterone and 11-Deoxycortisol. Resulting from the chromatographic optimization, newly designed online SPE column demonstrated excellent recoveries which is grater than 76% compared with only analytical column and biphenyl column achieves good chromatographic separation.
　Needle inner rinse was performed through 4 solvent selection pump with low to high concentration of organic solvent in the process of gradient analysis. Additionally, the carryover in Online SPE column was eliminated by flushing during its idle time, because each flow path was independent and there was enough time to wash after its analysis. Those 2 washing techniques minimize system carryover, effectively.
　Based on the chromatographic optimization and wash techniques, the measurement time in single stream was 10 minutes including automated sample clean-up using online SPE. The multiplexed 4-channel online SPE LC/MS/MS system delivered a higher sample throughput, overlapping sample injections. Compared with single and multiplexing system, throughput increased 49%. It showed that the multiplexing system maximize the productivity.
　All the compounds showed excellent quantitative results with great accuracy. For example, 11-Deoxycorticosterone and Estriol were spiked in biological samples at 1 ng/mL. The measured concentration were 0.983 ng/mL and 1.055 ng/mL, respectively. As a result of this study, the established method for an ultra-fast online SPE accomplished adequate performance to achieve quantitative analysis using the high-throughput 4-channel online SPE LC/MS/MS.

Conclusion:
　Transferring a highly optimized LC/MS/MS method for steroid hormones from a conventional single stream system to a multiplexed 4-channel online SPE LC/MS/MS platform significantly accelerated total run time maintaining its data quality. This approach has several advantages as it increases sample throughput, maximize data acquisition times on the single MS and is simplified to use as the software is designed for multiplexed stream analysis.

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

>> POSTER (PDF)

Poster #24b View Map

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

INTRODUCTION
The interplay between maternal health and neonatal outcomes informs current guidelines that recommend universal screening for substance use disorders in obstetric patients. As part of this screening process, maternal and neonatal drug testing is frequently performed to detect in utero drug exposure. While maternal urine drug testing exhibits high sensitivity and specificity, neonatal urine drug testing faces preanalytical and analytical challenges due to difficult sample collection protocols, low drug concentrations, and drug metabolites differing from those targeted by immunoassay (IA) or mass spectrometry (MS)-based methods. Consequently, meconium is often the preferred specimen type to detect neonatal drug exposure due to a longer window of detection compared to urine. Most laboratories do not have the ability to analyze meconium and, therefore, send out meconium testing to specialized reference laboratories, which can delay results for several days or more. In response highly sensitive MS-based methods for rapid, in-house neonatal urine drug testing have been developed, although the concordance of these methods with gold-standard meconium testing is not well documented.

OBJECTIVE
The primary objective of this study was to evaluate concordance of drug screening results of paired newborn urine and meconium samples with maternal urine samples using IA and liquid chromatography mass spectrometry (LC-MS/MS) methods, aiming to investigate maternal-fetal drug transfer.

METHODS
The number of positive drug screen results for newborns born at Barnes Jewish Medical Center with paired urine and meconium samples from January 2021 to October 2022 were tabulated retrospectively for the de-identified dataset (n=1,424). Of the 1,424 newborns, 831 newborns had mothers with drug screens performed within three days of birth (+/- 3 days). Urine drug testing was performed on freshly collected urine by in-house LC-MS/MS methods. All paired meconium samples were sent to an outside reference laboratory where they were screened by IA and confirmed with MS. The menu of available meconium testing performed in our patient population allowed for a paired urine-meconium comparison for the following drugs: amphetamines, cocaine, cannabinoids, opiates, and cannabinoids. The cutoffs for the MS urine screening method (in ng/mL) were: amphetamines 5, cocaine 1, opiates 25-250 (depending on specific compound), cannabinoids 20 (as 11-nor-9-Carboxy-Δ9-tetrahydrocannabinol glucuronide). The cutoffs for meconium screening (in ng/g) were: amphetamines 100, cocaine 100, opiates 100, cannabinoids 20.

RESULTS
Of the 1,424 newborns, 120 screened positive for amphetamines, 65 screened positive for cocaine, and 2 screened positive for PCP in urine and/or meconium. Positivity rates were equivalent for amphetamines (7.4%), cocaine (3.9%), and PCP (0.1%). McNemar’s test p-values indicate there is no significant difference between urine and meconium for the detection of amphetamines and cocaine (p < 0.001). There was insufficient data to analyze concordance for PCP due to low positivity rate. For opiates, 57 newborns screened positive. The drug class with the highest positivity rate is cannabinoids, with 866 newborns screening positive. There was a significant difference in positivity rate (percent positive agreement (PPA)) for opiates and cannabinoids (20% and 2.5%, respectively). A cohort of 831 newborns with mothers having a urine drug screens performed within three days of birth were identified. Positivity rates were higher in maternal urine for all drug classes except cannabinoids (69.4% positivity rate in maternal urine; 96.9% positivity rate in meconium). Notably, in the case of amphetamines, cocaine, and opiates, an additional 7, 24, and 32 instances of in utero drug exposure were identified in maternal urine alone, accounting for 9%, 34%, and 42% of all positive screens, respectively. Specifically in the case of cannabinoid positivity, 99.8% of all newborns with in utero cannabinoid exposure screened positive in meconium and/or maternal urine.

CONCLUSION
In this retrospective analysis including paired data from meconium and urine collected from 1,424 neonates, we observed that a sensitive and rapid LC-MS/MS method for neonatal urine tests detected equivalent numbers of neonates exposed to cocaine and/or amphetamines in utero compared to common meconium drug testing methods. Further, 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. Higher positivity rates in maternal urine highlight the importance of incorporating maternal drug screening results into the interpretation of newborn urine drug screens, as it provides a comprehensive understanding of fetal exposure, especially in instances in which meconium is unavailable. This study highlights the potential value of sensitive and rapid LC-MS/MS methods for neonatal urine drug testing.

An Extraordinarily High Bromazolam Concentration in a Clinical Case Brittany Casey (Presenter) NMS Labs

>> POSTER (PDF)

Poster #26a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Case Description:
A 42-year-old male presented at a medical facility following possible drug overdose. Toxicology testing was performed and targeted commonly encountered drugs and metabolites such as amphetamines, anticonvulsants, antidepressants, antihistamines, antipsychotics, benzodiazepines, central nervous system stimulants, hallucinogens, hypnosedatives, muscle relaxants, and non-steroidal anti-inflammatory agents, in addition to a number of novel psychoactive substances (NPS). Bromazolam was identified and quantitated as part of the confirmation testing in this case.

Background:
Bromazolam was first synthesized in 1976 but was never approved for use. Today it is frequently found in conjunction with opioids such as such as fentanyl and heroin, often in the form of clandestine or counterfeit tablets. It is an increasingly prevalent NPS, as evidenced by an increase in identifications in both seized drugs and toxicology samples. Bromazolam use is generally characterized by central nervous system depression and can be life-threatening; therefore, accurate identification and quantitation of bromazolam is critical for patient care. Toxicology screening via immunoassay is common and would likely yield a presumptive positive result for benzodiazepines as a drug class but would not allow for identification of the analyte of interest. The use of powerful mass spectral techniques like TOF coupled with a comprehensive and relevant scope of analysis make it possible to efficiently and effectively test for NPS in clinical samples.

MS Method and Results:
Routine screening for THC, barbiturates, salicylate, and gabapentin was performed via immunoassay. For expanded screening, 0.5 mL of serum was buffered, extracted with organic solvent, and analyzed via LC-TOF-MS. Bromazolam confirmation testing utilized 0.5 mL of buffered serum in a liquid/liquid extraction. The reconstituted extract was separated using UPLC and analyzed with positive-ion electrospray tandem mass spectrometry for detection and quantitation. The patient’s serum bromazolam concentration was 1400 ng/mL.

Discussion and Conclusion:
Seizures, hyperthermia, and myocardial injury associated with bromazolam use were recently reported, and deaths attributed to bromazolam use are increasing. Bromazolam use can have serious and life-threating impacts on patient health, and its inclusion in clandestine or counterfeit tablets complicates patient care, as patients are often unaware of the true composition of these items. Previous reports of bromazolam-positive samples, including fatalities, were one to two orders of magnitude lower in concentration than the case reported herein.

Intelligent Reflex Automatic Worklist Intervention for Toxicological Drug Screening by High-Resolution LC/Q-TOF Cate Simmermaker (Presenter) Agilent Technologies

Poster #27a View Map

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

Introduction
Data-independent acquisition (DIA) is advantageous in large library screening methods for drug analysis. Using LC/Q-TOF, information can be gathered on targets and suspects simultaneously and further, older sample data files can easily be re-investigated for the existence of emerging analytes of interest. Screening for a large range of analytes at many different concentration levels can, however, result in the need for manual evaluation and possible sample reinjection. Intelligent reflex workflows with MassHunter Acquisition have been developed to increase instrument up time and reduce the need for manual worklist setup. Outlined herein is a demonstration of two intelligent reflex workflows performed using the new Revident LC/Q-TOF. Evaluation of drug analytes over a range of concentrations in solvent, plasma and urine is demonstrated using workflows to automatically manage carryover and detection of values above the calibration curve with adjusted sample reinjections during real-time analyses.

Objective
To demonstrate the capabilities of new intelligent reflex workflows for drug screening using a new software tool for fast reflexive worklist management for carry over and above calibration curve detection through automated worklist appending and injection volume reduction.

Method
Urine and plasma samples prepared by dilution and EMR-Lipid extraction, respectively, were spiked at 8 calibration levels from 1 to 100 ng/mL with 32 scheduled drugs and 16 heavy labeled analytes at 50 ng/mL. An 11-minute reverse phase LC gradient method was optimized on a 100 mm Poroshell 120 EC-C18 column. Non-targeted acquisition was carried out using All Ions methodology at collision energies 0, 20 and 40 eV. MassHunter Quantitation methods were built using library MS/MS spectra for comparison of molecular ion and fragment ions and curated further with retention times specific to the chromatography gradient. Intelligent reflex was enabled in the worklist during data acquisition to address carryover and samples detected above highest calibration levels in real time.

Results
All Ions acquisition was used for non-targeted analysis of spiked calibration curves between 1 and 100 ppb (n=4), and results will be shown for plasma and urine matrix. Initial analysis showed, for the 32 compounds of interest the screening method designated all compounds as identified at the 5 ng/mL level with most identified < 5 ng/mL. The screening method utilizes mass accuracy, retention time, mass match score, and number of verified ions. Most calibration curves showed good linearity over the entire concentration range, with correlations R2 > 0.99. Precision of 95% of the response RSDs for each calibration level were below 10%, and mass accuracies were within ±2 ppm for most compounds. The above calibration curve reflexive logic was tested by the worklist integration of a sample over-spiked with 6 compounds at 500 ng/mL. Upon detection of signal over the calibration levels in the associated MassHunter Quantitation method, the sample was reinjected, and injection volume was reduced from 4 uL to 0.5 uL, placing the new data within the calibration range. Carryover management was engaged by setting a blank concentration threshold of 1 ng/mL, and upon detection of carryover in the blank by the instrument, an additional blank injection was inserted into the worklist and replicated until a workflow limit or no carryover detection.

Conclusion
Intelligent reflex methodology can be utilized for non-targeted drug screening workflows, increasing instrument up time and reducing the requirement for manual interpretation between initial injection and subsequent reinjections. Shown herein, this can be beneficial for automatically handling samples with results above calibration curve levels and those showing carryover in integrated blanks. In combination with succinct LC/Q-TOF data analysis supported by the LC Screener tool, complex analysis can be simplified yielding clear and accurate results.

RA45296.487974537.

Development of Simple LC-FAIMS-MS/MS Method for the Quantification of Nicotine and Its Metabolites in Urine Danting Liu (Presenter) Mayo Clinic

>> POSTER (PDF)

Poster #28a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
The use of tobacco products, particularly smoking, is the main preventable cause of lung cancer in the U.S. Nicotine is the active component in tobacco, responsible for addiction. The clinical test for nicotine and its metabolites in urine is a widely accepted method to evaluate nicotine exposure. In addition, anabasine, an analog of nicotine present in trace amounts in tobacco products, is also used as an indicator for monitoring compliance in tobacco cessation and effectiveness in nicotine replacement therapy. Currently, the conventional methods used to detect these molecules in the clinical laboratory are direct dilution or solid phase extraction (SPE), followed by LC-MS/MS analysis. However, direct dilution and injection methods may introduce bias in quantification and are prone to interferences, especially with low concentration samples. SPE purification is effective at overcoming these limitations, but this technique adds cost and labor to the analysis. Here, we present a novel LC-FAIMS-MS/MS method capable of detecting nicotine and its metabolites in urine with better sensitivity and specificity, without adding labor and cost.

Method:
Residual urine samples from the nicotine positive and negative patients were used in this study. In the test, 50 μL of calibrator, QC, blank, or patient urine samples were mixed with 50 μL of internal standard working solution (200 ng/mL Nicotine-D4, Nonicotine-D4, Anabasine-D4 and Cotinine-D3) and 800 μL of Mobile phase A (5 mM Ammonium Bicarbonate in 0.1% Formic Acid) in a 96 well-plate at room temperature. The sample was then separated by the LC column (C18, 3.0 x 50 mm, 2.6 um PS C-18 Kinetex) using a 10 min gradient (mobile phase B, 0.1% Formic Acid in ACN) and analyzed by TSQ Altis Plus Triple Quadrupole MS with or without FAIMS installed. The solid phase extraction (SPE) method was performed to compare with the dilution method. Nicotine and its metabolites in 50 μL of the urine sample were extracted with a strong cation Bond Elut PlexaPCX 30 mg plate (Agilent), followed by the same LC-MS/MS method. The four analytes were quantitated using internal standards and calibration curves.

Result and Conclusion:
The results obtained from the LC-FAIMS-MS/MS method showed that, compared with the regular dilute-and-shoot method, using the FAIMS cleaned up the background noise up to 80% and largely increased the signal-to-noise ratio (S/N). The chemical noise signals that interfere with the quantification of anabasine and cotinine, especially at low concentrations, were completely eliminated. The Compensation Voltage (CV) values used in FAIMS for each analyte were optimized to produce the highest quantitative accuracy and precision. The agreement between the quantifier and qualifier ions was excellent, with linear regression analysis yielding R2 greater than 0.99 for all four analytes. The total imprecision of the assay was less than 10% across the analytical measuring range. Overall, our new method is simple and utilizes low cost sample preparation, requires minimal instrument maintenance, while providing accurate, precise, and specific measurements, which makes this method an ideal, high throughput analysis technique for the clinical laboratory.

Evaluation of the Roche Benzodiazepines II Immunoassay for Urine Drug Testing in Clinical Specimens Mengyuan Ge (Presenter) UC San Diego Health

>> POSTER (PDF)

Poster #29a View Map

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

Background: Benzodiazepines are one of the most commonly prescribed medications in the United States and are frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated metabolites excreted in urine. False negative results are especially prevalent with lorazepam which is almost exclusively excreted at lorazepam-glucuronide. Some clinical laboratories have addressed this problem with the addition of beta-glucuronidase to enhance assay sensitivity as a laboratory developed test (LDT). Roche Diagnostics recently received FDA clearance to offer a benzodiazepine immunoassay that includes beta-glucuronidase.

Methods: Performance characteristics of two FDA-cleared benzodiazepine urine immunoassays (Benzodiazepine Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a benzodiazepine immunoassay LDT (HS-BENZ, with glucuronidase) were evaluated using 258 urine specimens. These immunoassays were directly compared to an LC-MS/MS benzodiazepine LDT to determine clinical sensitivity and specificity. Cross-reactivity of all three immunoassays were compared and evaluated based on the measured benzodiazepine concentrations determined by the LC-MS/MS LDT. Cross-reactivity for 7-aminoclonazepam and lorazepam was assessed using drug-free urine spiked with reference materials (Cerilliant) at concentrations ranging from 100 to 1000 ng/mL.

Results: The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all three assays. Cross-reactivity of the Benzodiazepines II assay was greater across the range of benzodiazepine concentrations tested in comparison to two other immunoassays, and in particular, the 7-amino-clonazepam metabolite as it was the only immunoassay out of the three that was sensitive enough to detect the presence of specimens containing this metabolite alone. Cross-reactivity analysis shows that the Benzodiazepine II assay detected 7-aminoclonazepam or lorazepam at 300 ng/mL, while the other two assays required 800 ng/mL and 600 ng/mL for a positive result.

Conclusions: A comprehensive evaluation of these three immunoassays demonstrates that the Benzodiazepines II immunoassay has increased clinical and analytical sensitivity compared to the Benzodiazepines Plus and HS-BENZ immunoassays. The inclusion of a beta-glucuronidase greatly improved the sensitivity of the Benzodiazepines II and HS-BENZ immunoassays for lorazepam, which is primarily excreted as a glucuronide metabolite in urine. As one of the first commercially available FDA-cleared benzodiazepine urine immunoassays which incorporates a beta-glucuronidase, clinical laboratories should consider implementing this assay to detect benzodiazepines more robustly in their patient populations.

Analysis of Δ-8-THC, Δ-9-THC, and Isomer Metabolites in Whole Blood by LC-MS/MS Haley Berkland (Presenter) Restek

Poster #29b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: The testing of whole blood samples for tetrahydrocannabinol (Δ-9-THC) consumption is routine and has been around for many decades. Δ-9-THC is metabolized into 11-Hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ-9-THC) and further into 11-nor-9-carboxy-Δ-9-THC (Δ-9-THC-COOH). It is important to test for the parent and both metabolites to properly monitor for THC usage. As more isomers of Δ-9-THC become available on the market, testing becomes more complicated and novel methods are needed to achieve isomeric resolution. One such isomer, Δ-8-THC, is federally unregulated in the United States and readily available for purchase in many stores. This compound forms its own hydroxylated and carboxylated metabolites, (11-OH-Δ-8-THC and Δ-8-THC-COOH), that must be chromatographically resolved from their isomeric metabolites. The resolution of isomeric metabolites is key in reporting accurate specimen findings and poor resolution, especially when one isomer is in much greater abundance than the other, can result in quantitation issues and invalid data. In this study, an LC-MS/MS method was developed to adequately resolve all three isomeric pairs of compounds in whole blood.

OBJECTIVES: The primary objective of this study was to develop an LC-MS/MS method to adequately resolve isomeric pairs Δ-8-THC and Δ-9-THC, 11-OH-Δ-8-THC and 11-OH-Δ-9-THC, and Δ-8-THC-COOH and Δ-9-THC-COOH in a whole blood matrix.

METHODS: An LC-MS/MS method was developed on a Raptor FluoroPhenyl 100 x 3.0 mm, 2.7 µm column using a mobile phase A of water and a mobile phase B of methanol, both acidified with 0.1% formic acid. The flow rate was 0.8 mL/min and the column temperature was 40°C. The gradient started at 64% B and held for 6.50 minutes, ramped up to 68% B at 6.60 minutes, and held at 68% B until 13.00 minutes. At 13.10 minutes the gradient ramped up to 100% B until 14.00 minutes, before returning to start conditions at 14.10 minutes and held until 16.00 minutes for ample re-equilibration. Whole blood samples were prepared using a liquid-liquid extraction. Extracts were dried down and reconstituted in 50:50 MPA:MPB. Δ-8/9-THC and 11-OH-Δ-8/9-THC were collected in ESI+ mode, while Δ-8/9-THC-COOH was collected in ESI- mode.

RESULTS: All three isomer pairs were adequately resolved using the developed method. Calibration curves were tested ranging from 0.5-100 ng/mL for 11-OH-Δ-8/9-THC and Δ-8/9-THC, and 2.5-500 ng/mL for Δ-8/9-THC-COOH. Linearity was demonstrated using a 1/x2 weighted linear regression, and all analytes showed acceptable R2 values. The method showed acceptable inter-day and intra-day precision and accuracy. No matrix or cross-analyte interferences were observed.

CONCLUSION: An LC-MS/MS method was successfully developed for reliable and accurate testing of Δ-8/9-THC isomers and isomer metabolites in whole blood. The method was determined to be quick, rugged, and sensitive enough to meet reporting guidelines for clinical and forensic toxicology laboratories.

Development and Validation of a Risankizumab LDT for Therapeutic Drug Monitoring Alex Barbeln (Presenter) Mayo Clinic

Poster #30a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background: Risankizumab (RISA) is a humanized IgG1 kappa therapeutic monoclonal antibody (tmAb) that targets interleukin 23 (IL23). RISA is used to treat patients with moderate to severe plaque psoriasis or those with Crohn’s disease. Therapeutic drug monitoring is used to identify loss of response to therapy. A novel lab developed test (LDT) was developed and validated utilizing IL23 coupled to magnetic beads to enrich RISA from patients’ serum. Samples were analyzed using an Agilent Infinity II LC in front of a Sciex 7600 ZenoTOF mass spectrometer.

Methods:
IL23 bead coupling: Approximately 25 mL of 10 mg/mL DynabeadsTM M-280 Streptavidin (ThermoFisher Scientific; Waltham MA) were added to a 50 mL conical tube. Beads were washed with 12.5 mL 1x PBS + 5 mg/mL BSA and mixed on an Eppendorf Thermomixer for 1 minute at room temperature (RT) at 1000 RPM. A DynaMagTM-50 Magnet was used to collect the magnetic beads against the tube wall and allow for the removal of the supernatant. The coupling mixture consisting of 25 mL 1x PBS + 5 mg/mL BSA and 500 µg IL23 protein (Sino Biological; Wayne, PA) was added to the beads and mixed for 30 minutes at RT at 1000 RPM. The beads were washed three times with 25 mL 1x PBS. The beads were then resuspended in 25 mL 1x PBS to give a final concentration of 20 µg/mL coupled protein.

Sample enrichment: Samples were prepared by adding 100 µL IL23 coupled bead slurry, 150 µL 1x PBS, 20 µL unknown/standard/QC, and 30 µL of 2.5 µg/mL stable isotopically labeled (SIL)-RISA (Promise Proteomics; Grenoble, France) to a 96-well filter plate (Sigma-Aldrich; St. Louis, MO). Samples were mixed at RT for two hours at 1250 RPM. The sample wells were then washed twice with 150 µL 1x PBS and twice with 150 µL HPLC water, filtering via positive pressure after each wash. Elution was performed by adding 50 µL 5% acetic acid and mixing at RT for 15 minutes at 1250 RPM. The eluent was then pushed to a 96-well PCR plate after attaching the PCR plate underneath the filter plate and centrifuging for 3 minutes at 3000 RPM. The eluent was reduced with 25 µL 100 mM DTT in 1 M ammonium bicarbonate. The plate was mixed at 55°C for 45 minutes at 800 RPM.

LC-MS: The enriched and reduced samples were analyzed using an Agilent 1290 Infinity II LC connected to an AB Sciex 7600 ZenoTOF mass spectrometer. A volume of 20 µL was injected onto an Agilent Poroshell 300SB C3, 2.1x75mm column with 5 µm particles heated to 60°C, running a 7-minute gradient from 27 to 40% B at a flow rate of 400 µL/min. Mobile phases consisted of (A) 1% formic acid in water and (B) 90% acetonitrile, 10% isopropyl alcohol, and 0.1% formic acid. Sciex OS was used for data analysis. The +11, +12, and +13 charge states for the RISA light chain were combined to give the XIC used for quantitation. A calibration curve from 1 to 100 µg/mL using the Hill Equation and 1/x2 weighting was obtained from standards prepared by spiking RISA in normal human serum.

Development: During development, optimization experiments were performed to minimize the amount of IL23 coupled beads that would be needed per sample well. This was done to try and make the assay cost effective while also ensuring there was sufficient IL23 coupled beads to avoid saturation when enriching the RISA in patient serum samples and the spiked internal standard. The final coupled bead slurry of 100 µL consists of 1 mg beads coupled to 2 µg IL23. Using 100 µL was proven to be able to enrich the quantity of RISA in the highest standard (2 µg) and the internal standard (0.075 µg) while having the RISA area counts be linear across the analytical measuring range.

Results: Intra-assay and inter-assay precision was analyzed at four levels; 1, 5, 30, and 70 µg/mL. Intra-assay precision, N = 20 replicates, had coefficients of variation of 5.7%, 4.9%, 4.7%, and 4.7% respectively. Inter-assay precision was measured over twenty analytical runs and had coefficients of variation of 8.5%, 7.8%, 9.6%, and 5.2% respectively. The assay was proven to be linear from 1 to 100 µg/mL by re-extracting standards of 1, 5, 10, 25, 50, 75, and 100 µg/mL as unknowns. Carryover was assessed over twenty analytical runs by dividing the area counts of a matrix blank extracted after the highest standard (100 µg/mL) and the area counts of the low standard (1 µg/mL) to get the %LLOQ (lower limit of quantitation) area. The average %LLOQ of the carry over blank across twenty analytical runs was 9% with a range of 1-30%, which was below our acceptance criteria of <50% of the LLOQ. The LOD (limit of detection) was determined to be approximately 0.17 µg/mL, which is 17% of the LLOQ of 1 µg/mL. Residual samples, N=37, for patients being treated with RISA were analyzed during validation. Using patient chart review it was determined that N=4 patients that had a RISA quantitation below the LLOQ (limit of quantitation) had not received a RISA dose before their blood was drawn. Patients being treated for Crohn’s Disease, N=21, had an average RISA concentration of 25.1 µg/mL, while patients treated for Psoriasis, N=12, had an average RISA concentration of 6.4 µg/mL. These values coincided with the RISA drug package insert.

Conclusion: We have successfully developed and validated a new LDT for RISA therapeutic drug monitoring which can be used to monitor potential loss of response to therapy. This quantitative assay will soon be paired with an ADA (anti-drug antibody) assay and will be offered as a panel to add more value to patients receiving RISA therapy.

Analytical Validation of an Automated Dried Blood Spot Desorption LC-MS/MS Method for Levetiracetam and Lamotrigine Joshua Miller (Presenter) Mayo Clinic

>> POSTER (PDF)

Poster #30b View Map

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

Introduction: Levetiracetam and lamotrigine are antiepileptic drugs used in the treatment of tonic-clonic seizures but require monitoring due to their narrow therapeutic ranges. Traditional monitoring methods utilize serum samples, requiring frequent venipunctures for patients. Dried blood spots (DBS) are an alternative specimen type which can easily be collected at home. However, DBS require laborious sample preparation before measurements using LC-MS/MS, limiting their use in high test volume laboratories. Herein we report a novel LC-MS/MS method that allows for the coincidental quantification of levetiracetam and lamotrigine from DBS utilizing direct inline flow through desorption technology which requires no sample preparation.

Objective: The goal of this study was to validate an LC-MS/MS method utilizing inline flow-through desorption technology to quantify levetiracetam and lamotrigine from DBS samples.

Methods: DBS were prepared by spiking bovine whole blood with levetiracetam and lamotrigine and depositing these mixtures onto dried matrix cards. Direct inline matrix desorption and chromatographic separation were achieved using a Thermo Scientific DSX-1 equipped with a Vanquish HPLC system. Samples were purified online using a Cyclone-P (1 x 50 mm) TurboFlow column and then eluted onto an Accucore™ aQ (3 x 150 mm) analytical column for further chemical separation. The HPLC was coupled to a Thermo Scientific TSQ Altis Plus triple quadrupole mass spectrometer for ion selection and detection. Sample quantification was performed using isotopically labeled internal standards and by comparison to an external calibration curve. Each batch contained five calibrators with concentrations across the linear range. Twenty runs over the course of ten days were used to determine accuracy and precision.

Results: Calibration curves for both anticonvulsants maintained a linear response (R2>0.99) which encompassed their respective therapeutic windows. Within-run precision varied with %CVs of 3.6-7.8% and between-run precision varied with %CVs of 4.5-9.7% for both drugs. Passing-Bablok regression analysis of accuracy studies revealed excellent correlations (R2=0.99) for both drugs. Our method yielded a 14-day mean difference in concentration <±20% and <±10% compared to their originally measured concentration for levetiracetam and lamotrigine, respectively, indicating that the DBS are stable for at least 14 days.

Conclusions: Automatic flow through desorption technology in tandem with LC-MS/MS can accurately and precisely quantify levetiracetam and lamotrigine from DBS samples. This investigation bolsters the number of analytes that are amenable to DBS desorption LC-MS/MS analysis, highlighting the versatility of this technology for high throughput laboratories. Furthermore, the utilization of DBS versus serum has the potential to benefit patients undergoing long-term therapeutic drug monitoring.

Optimizing an Immunosuppressant Testing Workflow: Lessons Learned from Revisiting a Long-Established Assay Meng Wang (Presenter) Vancouver General Hospital

Poster #31b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction: Immunosuppressants, such as tacrolimus (TAC), sirolimus (SRO), and cyclosporine A (CSA), are medications that suppress the immune system. Immunosuppressant is commonly used in transplant patients to prevent organ rejection. Due to the narrow therapeutic window of immunosuppressant and the variable pharmacokinetics among patients, routine drug monitoring is a standard clinical practice. In British Columbia, Canada, there has been a 29% increase in transplant recipients receiving follow-up care over the past five years. In our facility, the daily test volume for TAC increased from an average of 29 in 2005 to an average of 120 per day in 2021. Same-day result is ideal to provide information for dose adjustment, especially for new transplant patients and inpatients. With the manual sample preparation, the technologists spend approximately 5-7 hours hands on time on sample preparation and there have been 5.4% of results filed the following day, which are considered as delayed results. Given the increasing test volume, extended bench time, and result delays, revisiting and optimizing the workflow of immunosuppressant testing is essential to enhance efficiency.

Objectives: The primary goal of this quality improvement study was to revisit and enhance the efficiency of our existing immunosuppressant test, which has been in use for over a decade. Our focus was on optimizing the workflow to better align with the demands of our current test volume. This included an analysis of the pre-analytical processes, leading to adjustment in the batch volume and cut-off times. In addition, we assessed several analytical parameters in our workflow, including Hamilton programming, liquid handling robotics versus manual sample preparation, sample mixing, reagent selection, and instrument parameters.

Methods: TAC, SRO, and CSA concentrations were quantified in whole blood samples. With the new method, whole blood specimens were processed by the liquid handler. Samples were mixed, cleaned up through protein precipitation by zinc sulfate and acetonitrile incubation, and filtered, prior to analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chromatographic separation was achieved using an Agilent Zorbax Eclipse Plus C18 UPLC column integrated into the Agilent 1290 Infinity LC system, coupled with an Agilent Ultivo Triple Quadrupole mass spectrometer. For pre-analytical workflow improvement, retrospective data analysis was done using the R open-source software.

Results: Pre-analytically, we set our cut-off batch time at 9AM and 12PM each day to facilitate same-day reporting based on the peak sample receiving time. Compared to our existing manual method, this optimized workflow demonstrated notable improvements, including (1) a double batch volume from 42 to 84 samples; (2) a streamlined robotic sample preparation, decreasing the hands on time from 5-7 hours to 3 hours per day; (3) a 23.7% reduction in instrument time per patient sample and a total reduction of 2 hours per batch; and 4) improved precision. The new workflow maintained comparable linearity, and the method comparison revealed R2 values of 0.99, 0.93, and 0.99 for TAC, SRO, and CSA, respectively.

Conclusion: This refined workflow not only enhances the precision of immunosuppressant measurements by eliminating user variations, but also contributes to streamlined laboratory processes, improved quality, and shorter turnaround time, ultimately benefiting patient care. Our experience also highlights the importance of incorporating prospective considerations when introducing laboratory developed tests to avoid extensive future re-validation.

Quantification of Corticosteroids and Androgens in Serum, Utilizing Waters MassTrak™ Steroid Serum Sets 2 & Sets 3 for Clinical Research Niall Tobin (Presenter) Waters

Poster #32a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background: Steroid hormones encompass a large class of small molecules that play a central role in metabolic processes, such as the regulation of sexual characteristics, blood pressure, and inflammation. Enzymes that form part of the steroid biosynthetic pathway are pivotal in these metabolic processes, and their dysfunction can be examined through the correct measurement of steroid hormones in a clinical research setting. The availability of lyophilized calibrators and QCs reduces sample preparation time, aids in method harmonization, and assists with metrological traceability in accordance with ISO15189:2022, when used alongside an analytically selective chromatographic method.

Clinical Research Method: A quantitative clinical research method utilizing Waters MassTrak Steroid Serum Sets 2 and 3, Cals & QCs and Waters Oasis™ PRiME HLB µElution plate technology for the extraction of testosterone, androstenedione, 17-hydroxyprogesterone (17- OHP), dehydroepiandrosterone sulfate (DHEAS), Cortisol, 11-deoxycortisol and 21-deoxycortisol from human serum samples. Chromatographic separation was performed on an ACQUITY™ UPLC™ I Class Plus (FL-I) System, using an ACQUITY UPLC HSS T3 Column, accompanied by a Xevo™ TQ-S micro Mass Spectrometer.

Results: Accuracy (±6%) and precision (±10%) have been confirmed through comparison to External Quality Assurance (EQA) LC-MS/MS schemes, panels and QC material for all seven steroid hormones. All analytes were assessed at the low, medium and high concentrations for each MassTrak Steroid Serum Set, which yielded excellent results across the range (±10%). Deming and Linear regression analysis were performed. No statistically significant bias was observed for each compound, with a mean method bias of ±1.3% for Set 2 and ±1.0% for Set 3. The clinical research method was shown to be linear for all analytes over the calibration ranges specified, furthermore, calibration lines created using Set 2 and Set 3 were analysed over a five-day period and were linear with a co-efficient of determination of (r2) > 0.999 for all analytes. Analytical sensitivity using Signal:Noise (S/N) of the low calibrator (Calibrator 1) of each set, was >10:1 for each analyte across several analytical runs.

Conclusion: This evaluation has demonstrated that the MassTrak Steroid Serum Calibrator and Quality Control Sets 2 and 3 can provide precise and accurate quantification of steroid hormones in serum. An analytically sensitive and selective clinical research method has been developed for the analysis of testosterone, androstenedione, 17- OHP, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol in serum using Waters I-Class Xevo TQ-S micro system.

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

Failed Proficiency Result for 11-Nor-9-Carboxy-THC Rene Garay (Presenter) Duke University Health System

>> POSTER (PDF)

Poster #34b View Map

This poster will be presented and discussed on Wednesday at 19:00 for 15 minutes in Montreal 3 (Track 2).

Title: Failed proficiency result for 11-nor-9-carboxy-THC
1. Problem
Laboratory failed a proficiency sample result for THC-COOH. Investigation was launched to determine root cause of failure.
2. Method Information
• Waters LC-MS system
o Acquity Binary Solvent Manager
o Acquity Sample Manager
o Acquity Column Manager
o Xevo TQ-S micro
• Mobile Phase A: 0.005% Formic Acid in Water
• Mobile Phase B: 0.01% Formic Acid in Acetonitrile
• 7 minute gradient method, 0.4 mL/min
• Column: 2.1 x 100 mm, 1.7 µm C18
• Column Temp: 50°C
• Injection volume: 7.5 µL
• Quantitative MRM acquisition
• UTAK drug free urine spiked with KF
• Cerilliant Certified Reference Material
o Calibrator analyte: (±)-11-nor-9-Carboxy-Δ9-THC (Item # T-006)
o Internal Standard: (±)-11-nor-9-Carboxy-Δ9-THC-d3 (Item # T-004)

3. Troubleshooting Steps
THC-COOH slope drift was observed in calibration as the 5-day work week passed. Protocol at the time was to pull new calibrator set from -80°C freezer on Monday, use throughout the week (interim storage at 4°C refrigerated temperature), and discard on Friday. Slope drift appeared to coincide with days of the week. A downward trend in slope value was determined. Stability of THC-COOH in calibrator solution was questioned. Outlier sample was recalculated using calibration slope from different days of the week. The recalculated results would pass using calibrations from earlier days of the week.

4. Outcome
Calibrator set protocol was changed so that sets would be discarded after use on Wednesday, and a new set would be pulled on Thursday. This set would then be discarded on Friday. Ideally, a new set would be pulled every day, but the current composition of the calibrator set would make it too laborious a task to make daily aliquots. The slope of the calibration curve is monitored daily, and any variation exceeding the cutoff is promptly addressed.

Implementation and Validation of an Ultra-High Performance LC-MS/MS Method for Nicotine Metabolites and Anabasine Quantification in Urine Specimens Ahram Yi (Presenter) GC Labs

Poster #35b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Background: Reliable quantification of urine nicotine metabolites is essential for identifying smoke exposure. Further, detection of minor tobacco alkaloids, such as anabasine, may aid in determining smoking status or compliance with nicotine-replacement therapy. We implemented and validated an ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for quantifying 3-OH cotinine, nornicotine, cotinine, anabasine, and nicotine in urine specimens.

Methods: The UHPLC–MS/MS method used a Triple Quad 5500+ QTRAP Ready (SCIEX, USA) with 1 μg/mL 3-OH cotinine-d3 (3-OH cotinine-IS), 1 μg/mL nornicotine-d4 (norcotinine-IS), 1 μg/mL cotinine-d3 (cotinine-IS), 1 μg/mL nicotine-d4 (nicotine-IS), and 20 μg/mL anabasine-d4 (anabasine-IS) as internal standards. The method consists of precipitation extraction with methanol and dilution with distilled water followed by the analysis of the prepared sample by UHPLC-MS/MS in multiple reaction monitoring (MRM) using electrospray ionization positive (ESI+) mode. The mass spectrometer was operated through transitions from the precursor to the product ions (m/z 193.1 to 134.0 for 3-OH cotinine, m/z 196.2 to 134.0 for 3-OH cotinine-IS, m/z 149.1 to 130.1 for norcotinine, m/z 153.2 to 134.0 for norcotinine-IS, m/z 177.1 to 98.0 for cotinine, m/z 180.2 to 101.1 for cotinine-IS, m/z 163.1 to 94.0 for anabasine, m/z 167.2 to 150.2 for anabasine-IS, m/z 163.1 to 84.1 for nicotine, and m/z 167.2 to 134.1 for nicotine-IS). The total run time was 6 min. We conducted a gradient of mobile phase A of 30 mM ammonium bicarbonate and mobile phase B of 100% MeOH at a flow rate of 0.40 mL/min on a Kinetex EVO C18 column (2.1 mm × 150 mm, 5 μm; Phenomenex, USA) in an ExionLC™ system (Sciex, USA). Linearity, recovery, precision, carry-over, and matrix effect were evaluated to validate the method.

Results: Linear dynamic ranges were 4.98–23406.90 ng/mL for 3-OH cotinine, 1.02–9381.70 ng/mL for nornicotine, 1.04–8592.06 ng/mL for cotinine, 1.00–9106.58 ng/mL for anabasine, and 0.99–9252.99 ng/mL for nicotine (R² ≥ 0.9988). The lower limits of quantification were 4.98 ng/mL for 3-OH cotinine, 1.02 ng/mL for nornicotine, 1.04 ng/mL for cotinine, 1.00 ng/mL for anabasine, and 0.99 ng/mL for nicotine. The recovery of 3-OH cotinine, nornicotine, cotinine, anabasine, and nicotine UHPLC–MS/MS measurements were within ±9% of the targeted values. The intra- and inter-day coefficients of variation were all acceptable (≤5% for all tests). Carry-over was not found for all compounds. Ion suppression or enhancement was not observed in the blank and six patient samples for all compounds.

Conclusions: The UHPLC–MS/MS nicotine metabolite and anabasine assay showed an adequate recovery, precision, sensitivity, and AMR, making it suitable for measuring urine nicotine metabolite and anabasine concentrations.

A Simple and Sensitive LC-MS/MS Method for Simultaneous Measurement of Metanephrine, Normetanephrine, and 3-Methoxytyramine Sangmi Kim (Presenter) Chosun University Hospital

>> POSTER (PDF)

Poster #36a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background: Plasma 3-methoxytyramine (3-MT), the O-methylated dopamine metabolite, is useful for detecting dopamine-producing pheochromocytomas and paragangliomas (PPGLs). Due to the difficulty in measuring the very low concentrations in plasma, its measurements have not been widely offered as part of routine workup for PPGLs. In this study, we developed a highly sensitive and specific mass spectrometric method for simultaneous measurement of plasma free metanephrine (MN), normetanephrine (NMN), and 3-MT by incorporating 3-MT measurement into previously established MN and NMN assay, and applied it to clinical practice.

Methods: All plasma samples were extracted with the solid-phase extraction (SPE) method. SPE was performed using Strata CW-X extraction cartridges (Phenomenex, Torrance, CA, USA), pretreated with 1 mL methanol, 1 mL deionized water, and 1 mL of 0.5 M ammonium acetate. Finally, 3 μL of the reconstituted eluate was injected into the liquid chromatography tandem mass spectrometry (LC-MS/MS) system. Analyses were performed on an Agilent 6490 tandem mass spectrometer equipped with an Agilent 1260 high-performance liquid chromatography (HPLC) system (Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation of MN and NMN was conducted on a Unison UK C18 column (Imtakt, Portland, OR, USA; 2.0×100 mm, 3.0 μm). The analytes were detected in multiple-reaction monitoring mode by using positive electrospray ionization: MN, transition m/z 180.1 → 165.1; NMN, m/z 166.1 → 134.1; 3-MT, m/z 150.9 → 118.9). The method was validated for linearity, precision, accuracy, lower limits of quantification (LLOQ) and detection (LOD), extraction recovery, matrix effect, and carry-over according to CLSI C62-A, C64 guidelines. From August 2022 to August 2023, 3-MT was measured in clinical samples requested for MN and NMN measurement.

Results: Analytical run time was 6 min per sample and total sample preparation time was 1.5 hour per batch. Assay range and LLOQ were as follows; 0.03–50.0 nM (R²>0.99) and 0.03 nM for 3-MT; 0.04–22.0 nM (R²>0.99) and 0.04 nM for MN; 0.08–60.0 nM (R²>0.99) and 0.08 nM for NMN. The intra- and inter-day imprecisions were CV 1.1–6.9% and 2.1–9.8%, respectively. For 3-MT, accuracy was 91.5–104.0%. No interference or carryover was observed. The matrix effects ranged from 92.8% to 98.8%, and extraction recovery ranged from 96.3% to 107.2%. Among the 2,947 clinical samples, 1.4% (42/2,947) showed elevated plasma 3-MT.

Conclusions: A highly sensitive and specific LC-MS/MS method for 3-MT quantitation in plasma that could be incorporated in an established LC-MS/MS assay for metanephrines was developed and successfully applied to clinical practice. By allowing simultaneous measurement of three catecholamine metabolites, this method would thus contribute to increasing accessibility to 3-MT measurements in clinical laboratories, and provide valuable information to clinicians when diagnosing and monitoring patients with PPGL.

Quantification of Thyroglobulin in Serum for Clinical Research using SISCAPA Workflow Combined with LC-MS/MS Dominic Foley (Presenter) Waters Corporation

Poster #39a View Map

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

Background: Thyroglobulin (Tg) is a 660 kDa homodimer synthesized by the follicular cells of the thyroid gland, acting as a substrate in the production of the hormones triiodothyronine (T3) and thyroxine (T4). Accurate measurement of Tg using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA) workflow combined with liquid chromatography - tandem mass spectrometry (LC-MS/MS) has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead. Given the general complexity of this workflow it has been predominantly implemented in laboratories experienced in LC-MS/MS protein analysis. Here we report the development of a SISCAPA™ workflow for accurate measurement of thyroglobulin on an Andrew+™ Pipetting Robot, enabling easier adoption of this workflow.

Methods: Thyroglobulin certified reference material (T-113) (Merck, UK) was used to create calibrators in surrogate serum (chicken serum, Merck, UK). In-house QC material prepared in pooled serum (BioIVT, UK) and surrogate serum, were used to evaluate method precision. External Quality Assurance (EQA) serum samples (NEQAS, UK) were analyzed using the newly developed method and the quantified results were compared to the All Laboratory Trimmed Mean (ALTM) of the scheme. The Waters™ Andrew+™ Pipetting Robot with OneLab™ Software was used to fully automate the workflow from denaturant/reduction reagent addition to the serum samples through to elution of the Tg FSP tryptic peptide off the magnetic beads, without the need for user intervention.

Briefly, 250µL serum samples were added to a 96-well plate, treated with denaturation and reduction buffer (Deoxycholate, TCEP, Trizma™ Base) and mixed for 40 minutes at 37°C. Internal standard and trypsin were added and mixed for 30 minutes at 37°C. The samples were quenched with a protease inhibitor cocktail and mixed for 10 minutes. Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes at room temperature. The plate was transferred to a magnetic array plate and the beads were left to pull down for 2 minutes. The samples were discarded, and the wells were washed with PBS/CHAPS wash buffer for 1 minute. The plate was transferred to a magnetic array plate and the beads were left to pull down, the wash buffer was discarded, and the wash step was repeated again. Tg was eluted from beads through the addition of 2% acetonitrile and 0.5% formic acid and mixed for 10 minutes at room temperature. The plate was transferred to a magnetic array plate and the beads were left to pull down and the eluate was transferred to a fresh 96-well plate. The samples were centrifuged for 5 minutes, followed by fitting of the autosampler magnetic plate. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters XSelect™ HSS T3, 2.5µm, 2.1 x 50 mm Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer with a run time of 2.6 minute.

Results: The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (<20%) at 0.1 ng/mL with S/N (PtP) >10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all < 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements.

Conclusions: A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.

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

Parallel Column Regeneration: Increasing Throughput in Clinical Research without Succumbing to Matrix Contamination Daniel Kenny (Presenter) Waters Corporation

Poster #43a View Map

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

INTRODUCTION: Typical gradient LC methods include two segments: the gradient in which the separation occurs, and the regeneration step where the column is washed with strong solvent and re-equilibrated to initial conditions, ready for subsequent sample injections. Washing with 3-5 column volumes of a suitable strong solvent is usually recommended, as this prevents accumulation of phospholipids and potentially extends the useful life of the LC column. Allowing sufficient re-equilibration time is important for method robustness. Reducing the time allowed for regeneration may produce acceptably performing high-throughput methods in some cases, but it comes with the risk of poor-quality data and limiting column longevity.

There is an alternative hardware configuration, parallel column regeneration, which splits the elution gradient and regeneration phases over two consecutive injections, allowing sufficient washing and re-equilibration to be performed on the ‘passive’ column, while the gradient separation occurs on the ‘active’ column. This has the potential to dramatically shorten the analytical run, increase sample throughput, without compromising UPLC best practices.

OBJECTIVES: To demonstrate time savings and reduction in residual sample matrix of the parallel column regeneration system using various analytes in biological matrices (whole blood, serum, and dried blood spots).

METHODS: An ACQUITY UPLC system was configured with two Binary Solvent Manager pumps, a single Sample Manager, a two-position, heated, Column Manager with fluidics valves, and a tandem mass spectrometer. The configured hardware was controlled with MassLynx instrument control software, making use of the dual-pump configuration capability of the instrument driver software. Serum-based steroid hormone calibrator and control samples were precipitated and subjected to solid phase extraction; lysophosphatidylcholine species were eluted from dried blood spot punches, and immunosuppressant drugs were analyzed in whole blood calibrator and control sample supernatant. All test matrices were extracted using stable isotope-labelled internal standard, allowing a cursory estimation of instrument-associated repeatability, by replicate injections of pooled, extracted sample (n=10 on each column, by each injection mode). The extracted samples were separated by gradient elution UPLC and analyzed by multiple reaction monitoring (MRM) with electrospray ionization, with and without parallel column regeneration. Chromatographic retention time and analyte: internal standard peak area response ratios were monitored.

The effect of changing the regeneration conditions on phospholipid accumulation was monitored in the UPLC column eluate of columns by detection of precursors of the m/z 184 fragment ion, representing the phosphocholine head group of phospholipids.

RESULTS:
Retention times and response ratios were equivalent for all analyzed samples (p<0.05), with or without parallel column regeneration. The analyte: internal standard response ratio showed <9%, <4.5% and <15.5% within-batch imprecision for immunosuppressant drugs, steroid hormones, and lysophosphatidylcholines, respectively, in either serial or parallel regeneration mode of analysis.
Using parallel regeneration for the analysis of lysophosphatidylcholines, the number of samples analyzed per hour increased from 10.3 to 18.1, representing a 75% improvement in throughput. For the measurement of steroid hormones, the throughput was improved by 14% (from 8.3 with serial, to 9.5 samples/hr with parallel regeneration). For the analysis of immunosuppressant drugs, which is already a rapid 2.1 min method by conventional serial analytical methods, the number of samples analyzed per hour increased from 28.4 to 32.1, representing a 13% improvement in throughput for this clinical research method.

For the simplest extracted sample (immunosuppressants in whole blood) the average phospholipid ion intensity in the wash/hold eluate of columns using parallel column regeneration was 71% less than with serial regeneration (n=10 injections of a pooled, extracted matrix sample). The volumes of washing and re-equilibration were more than doubled with parallel regeneration, whilst still achieving a time saving of 24 minutes per 96 samples. An even greater impact on phospholipid ion intensity was seen in the steroid method, with a 99% reduction compared with serial regeneration. In this instance, the column volumes of washing and equilibration were increased more than 5-fold using parallel regeneration, whilst also saving 1 hour 33 minutes per 96 samples analyzed. Phospholipid removal in the serial analysis of lysophosphatidylcholines was already extensive, due to a generous 2.3 and 4.4 column volumes already allowed for washing and re-equilibration, respectively. Using parallel column regeneration and dividing the method almost equally between gradient separation and reconditioning phases, a notable shortening of the analytical method was possible (4 hours saved per 96 samples), and a modest increase to 3.2 wash volumes was allowed. This was accompanied by a 99% reduction in phospholipid ion intensity compared with serial regeneration.

CONCLUSION: Parallel column regeneration can be easily configured by adding an additional pump and valves to any conventional LC-MS/MS system commonly encountered in clinical research. The greatest relative improvements in throughput will be found for analytical methods where the wash and equilibration periods are similar in length to the elution period. Even when the methods to be transferred to parallel column regeneration do not meet this ideal gradient to regeneration ratio, it is still possible to make modest gains in throughput, whilst harnessing the opportunity to improve phospholipid removal by optionally increasing the extent of column regeneration.

For Research Use Only, not for use in diagnostic procedures.

Copper State Opioid Crisis: Developing a Fentanyl and Fentanyl Analog in Blood Method for Fatal and Non-Fatal Overdoses Austin Echelmeier (Presenter) Arizona Department of Health Services

Poster #47a View Map

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

INTRODUCTION: According to the Centers for Disease Control and Prevention (CDC), nearly 300 lives are lost daily in the United States due to drug overdoses. To reduce overdoses and health disparities, the CDC launched the Overdose Data to Action (OD2A) program in 2019. ASPHL was awarded the OD2A grant in 2019 to assist with fatal overdose testing for county Medical Examiners. ASPHL has continued this work and is currently working to expand to non-fatal overdose testing. As part of this OD2A grant work, ASPHL continues to improve toxicology testing. One recent improvement was to develop and validate a method for fentanyl and fentanyl analogs in blood.

OBJECTIVES: The primary objective was to develop and validate a quantitative method on liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify and quantify fentanyl and fentanyl analogs in human blood at clinically relevant concentrations.

METHODS: Human whole blood was prepared by agitation, centrifugation, supported liquid extraction (SLE) on a Biotage Extrahera liquid handler, dry down on a Biotage Turbovap, and reconstitution on a Biotage Extrahera. The samples were analyzed on an Agilent 1260 Infinity II LC Stack with a Sciex 7500 MS. The LC method used an Agilent Poroshell 120 EC-C18, 2.1 x 100 mm, 1.9 µm column with a 16-minute gradient elution of Mobile Phases A (10 mM Ammonium Formate with 0.05% Formic Acid in Water) and B (0.05% Formic Acid in Acetonitrile). The MS method used Multiple Reaction Monitor (MRM) transitions and retention times for a total of 26 fentanyl and fentanyl analog analytes. Data was processed and evaluated using Sciex OS software. The validation data was evaluated by the ANSI/ASB Standard 036, First Edition 2019, Standard Practices for Method Validation in Forensic Toxicology.

RESULTS: The method was evaluated for the following criteria: reportable range, calibration model, carryover, precision, accuracy, ionization suppression or enhancement, interference, stability, and uncertainty.

CONCLUSION: ASPHL developed and validated a quantitative LC-MS/MS method to detect fentanyl and fentanyl analogs in blood. The method will be used for fatal overdose patient samples that fall under the OD2A Strategy 3 grant work and non-fatal overdose patient samples that fall under the OD2A Strategy 4 grant work.

Tacrolimus Concentrations in Envarsus vs Prograf Patients: An Evaluation of the Clinical Impact of LC-MS/MS and Immunoassay Methods on Tacrolimus Measurement Adekunle Alabi (Presenter) University of Colorado Anschutz Medical Campus

Poster #50b View Map

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

INTRODUCTION: Tacrolimus is a calcineurin inhibitor used as part of an immunosuppressive regimen in kidney and liver transplant patients to prevent graft-versus-host disease (GvHD) and requires therapeutic drug monitoring due to its narrow therapeutic index. Tacrolimus levels are commonly measured using two different methodologies – Immunoassay and liquid chromatography tandem mass spectrometry (LC-MS/MS). A global proficiency study to assess and compare the measurement of trough levels by these two methodologies noted a positive bias with the ARCHITECT immunoassay which is likely due to the presence of tacrolimus metabolites.

OBJECTIVES: The aim of our study is to assess the impact of two different formulations of tacrolimus on the accuracy of its analytical measurement. While differences between immunoassay and LC-MS/MS results have been evaluated for immediate-release tacrolimus (Prograf), it has yet to be characterized for extended-release formulations such as Envarsus. The discrepancy between immunoassay and LC-MS/MS has clinical implications, as dosage modifications are based on measured concentrations, analytical variation may cause intervals of subtherapeutic or supratherapeutic exposure, increasing the risk of graft rejection or toxicity.

METHODS: Development of an LC-MS/MS method for the simultaneous quantification of tacrolimus and its major metabolite, desmethyl tacrolimus, was performed in whole blood using traceable calibrators and quality control material for tacrolimus and standard material for desmethyl tacrolimus. Tacrolimus concentrations were measured by LC-MS/MS and ARCHITECT immunoassay methods in excess whole blood specimens from patients treated with either Prograf or Envarsus undergoing standard clinical monitoring at UCSD Health. Measurement biases between LC-MS/MS and immunoassay methodologies were determined for both formulations of tacrolimus.

RESULTS: External calibration curves for both tacrolimus and desmethyl tacrolimus were linear (R2>0.995), and the analytical measurement range (AMR) for tacrolimus spanned from 1.1 - 31.6 ng/ml. Calibrator and Quality control (QC) biases were within 15% of their target values throughout the AMR and within-run imprecision was less than 10% (n=25) for all calibrators and QCs. Between-run imprecision for Low, Mid, and High QC levels over a period of 2 weeks (n=5 days) was less than 10%. Comparative bias of tacrolimus concentrations between immunoassay and LC-MS/MS was significantly lower (P value=0.0342) for Envarsus (n=11 specimens) relative to Prograf (n=17 specimens).

CONCLUSION: The differential bias between immunoassay and LC-MS/MS in the measurement of tacrolimus in patients dosed with immediate-release versus extended-release formulations suggests that their distinct pharmacokinetic profile may impact the accuracy of the measurement. Therefore, applying observed measurement biases from different assays derived from Prograf patients to Envarsus patients and vice versa may result in erroneous estimation of tacrolimus concentrations.

A High Throughput Method for the Quantification of Estradiol at Male and Post-menopausal Levels in Dried Blood Spot Samples Joshua Johnson (Presenter) Molecular Testing Labs

Poster #51b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Estradiol and estrone are the two main biologically active estrogens. Of the two, estradiol is more biologically potent, and found at higher concentration in premenopausal women. Because of this, estradiol measurements are an important part of monitoring reproductive health and assessing menopausal status. Immunoassays of moderate sensitivity are adequate for measuring estradiol in premenopausal women but require large sample volume and still may not adequately detect male or postmenopausal female serum concentrations which can be as low as 20 pg/mL. An estradiol measurement may be required in diagnosing males with estrogen producing neoplasms, or for monitoring estrogen levels in a post-menopausal woman; in these cases, an LC-MS/MS technique would be employed.
Highly sensitive LC-MS/MS assays can quantify estradiol at sub pg/mL levels by extracting it from a serum sample, which requires that the patient have blood drawn by a phlebotomist, usually in a clinical setting. This requisite in-person sampling can be a barrier to care for many who live in underserved regions, so an at home self-collect sampling option would be a great benefit for ease of access to diagnostic testing.

Dried blood spots have long been used in such testing as the diagnosis of phenylketonuria in newborns, infectious disease testing, vitamin D3 testing, and increasingly other hormones and biomarkers. As a sample type, dried blood spots (DBS) have key advantages over other types, including reduced risk of bacterial contamination, lower levels of hemolysis, increased sample stability, and most importantly: the capacity for the patient to self-collect. Despite these advantages, dried blood spots assays are limited by sample volume requiring a highly sensitive method to quantify low levels of estradiol in dried blood spots.

Objective
To achieve the sensitivity required to quantify estradiol at male and post-menopausal levels in dried blood spot, while maintaining a high-throughput 96 well plate format, a combination of sample cleanup techniques, analyte derivatization and optimized instrumentation will be leveraged.

Methods
Dried blood spot patient samples were extracted using solid phase extraction and derivatized to enhance ionization prior to injection. Samples were injected on an HPLC (Schimadzu SIL-40 series) coupled with a triple quadrupole mass spectrometer (Sciex 6500+).

Results
Contrived quality control samples were successfully measured at 20 pg/mL.

Conclusions
Using high throughput sample cleanup technologies combined with the latest industry standards in instrumentation, A sensitive, high throughput, and automatable diagnostic test can be achieved for self-collected dried blood spot samples.

Development and Validation of a Novel LC/MS-MS Method for the Simultaneous Quantification of 16 Drugs for Therapeutic Drug Monitoring Jessica Miller (Presenter) University Health Network

Poster #53b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Objectives: Therapeutic drug monitoring (TDM) involves the measurement of a drug in a patient’s blood in order to tailor dosages and maintain therapeutic efficacy. Drugs that benefit from TDM have narrow therapeutic ranges, exhibit pharmacokinetic variability, and/or are used for an extended period of time. Anti-epileptic and anti-psychotic drugs are both examples wherein the drug classes are broad and often prescribed in combination, and may require lifelong treatment for symptom management. Adverse effects of anti-epileptic and anti-psychotic drugs are well known, therefore TDM is important for patient safety. However, due to the large size of these drug classes, polypharmaceutical nature of treatment, and disparate concentrations of the drugs in serum, monitoring of anti-epileptic and anti-psychotic drugs is challenging. This study developed and validated a method for the simultaneous quantification of 16 anti-epileptic and anti-psychotic drugs in human serum using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Methods: We developed and validated a rapid LC-MS/MS method for the simultaneous quantification of anti-epileptic and anti-psychotic drugs including 9-OH-risperidone/Risperidone, Clobazam/Norclobazam, Clozapine/Norclozapine, Ethosuximide, Gabapentin, Lamotragine, Levetiracetam, MHC, Phenobarbital, Pregabalin, Primidone, Quietiapine, and Topiramate in serum on the SCIEX Triple Quad 4500. Sample preparation includes a protein precipitation protocol. The calibration and quality control ranges were chosen to cover the entire therapeutic range for the respective drugs. The analytical evaluation included linearity, imprecision, accuracy, method comparison, limit of quantitation (LoQ), ion suppression, and matrix effects.

Results: The total run time was ≤ 5 minutes. Intra- and inter-day imprecision ranged from 2.7%-8.0% and 3.3%-13.3% respectively. All of the criteria set according to relevant guidelines for lab developed tests passed for linearity, accuracy, and method comparison. LoQ ranged from 4.48 µmol/L (Phenobarbital) to 3.10 nmol/L (9-OH-risperidone). No significant ion suppression or matrix effects were observed.

Conclusion: This method is suitable for therapeutic drug monitoring of indicated anti-epileptic and anti-psychotic drugs in patients undergoing mono- or polytherapy.

Incorporating an Ion-Pairing Reagent and Solid Phase Extraction to Improve Nicotine and Its Metabolites in Urine by LC-MS/MS Method Yubo Chai (Presenter) Mayo Clinic

Poster #54a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION
Cigarette smoking is the leading preventable cause of death in the United States. Nicotine, present in tobacco products, is an addicting substance that causes individuals to continue use of tobacco despite concerted efforts to quit. Nicotine is metabolized to its major metabolites of cotinine, and nornicotine. Nornicotine is pharmacologically active and may accumulate to a greater degree in the brain. The presence of anabasine is indicative of active use of a tobacco product. As a result, tobacco users in abstinence programs are routinely monitored by laboratory tests that measure nicotine, its metabolites, and anabasine.

Measurements of nicotine and metabolites in urine are prone to quantifier/qualifier mismatch, poor chromatography of anabasine and nornicotine, and increasing interferences with nicotine. Therefore, we present an improved method to monitor nicotine, cotinine, nornicotine, and anabasine in urine.

METHODS
Solid phase extraction was applied to clean up the urine samples. 50 mcL of calibrator, QC, blank and patient samples were pipetted into 96-well plate, followed by 50 mcL of deuterium labeled internal standard mixtures (200 ng/mL each), diluted with 500 mcL of 0.1% formic acid in water, followed by vortexing the plate. The strong cation Bond Elut PlexaPCX 30mg plate (Agilent) was conditioned with 0.1N HCL in methanol followed by 0.1N HCL in water. Samples were transferred to the SPE plate, pushed through with positive pressure, washed with 0.1N HCL in water and 0.1N HCL in methanol, and eluted twice with 5% of ammonium hydroxide in methanol. The final eluent was diluted with the ion-pair reagent, 1-octanesulfonic acid, at a concentration of 1.25g/L. The analytes were injected and analyzed on the HPLC-MS/MS (Thermo Scientific TLX-4 coupled to Sciex triple quadrupole 6500), separated over 10 minutes using a 3 x 50 mm, 2.6 um PS C-18 column (Kinetex) with the mobile phase A (0.1% formic acid in water and 2mM of ammonium formate), mobile phase B (0.1% formic acid in ACN). The method was validated with patient samples over 20 runs, with a maximum of two runs per day. All necessary studies, including accuracy, precision, linearity, specificity, sensitivity, LOD, interferences and ion suppression studies were performed.

RESULTS
Compared to our current dilute and shoot method, the new SPE method provided significantly reduced quantifier/qualifier discrepancies and eliminated interferences. The analytical measuring range was 5-1,200 ng/mL for nicotine/cotinine, and 2-120 ng/mL for nornicotine/anabasine No significant carryover was seen following a sample twice the upper limit of quantitation. To assess the improvements from the new method, 119 samples that previously exhibited interferences or quantifier/qualifier discrepancies were tested. With the new method, the quantifier and qualifier match were within 15% for nicotine, 7% for cotinine, 11% for nornicotine. Anabasine samples results matched within 20% with the exception of 4 samples which matched <35%. For intra-day and inter-day imprecision, the %CVs were less than 6.8% and 7.1% for all analytes. Accuracy included a minimum of 63 patient samples of each analyte that was cross validated with our current LC-MS/MS method. The results for each analyte met the CSLI validation criteria. No interferences were observed among 92 common prescriptive drugs, as well as vitamins and hormones. No hemolysis, bilirubin/icterus, and bilirubin/icterus conjugate interferences were present. Minimal ion suppression/enhancement was seen for anabasine. Some degree of ion suppression/enhancement was observed in the analytes of nicotine, cotinine and nornicotine. However, the internal standard was able to compensate for the ion suppression/enhancement.

DISCUSSION
An accurate and robust method was established for nicotine and its metabolites in urine. Our new SPE extraction, polar analytical column, and ion-pairing reagent achieved the goal of reducing quantifier/qualifier mismatches and interferences. The specificity, precision, accuracy, and quantifier-qualifier match were all improved through enhanced chromatographic performance and eliminating the potential interferences. As a result, the improved method significantly reduces the need to repeat samples; therefore, improving the turn-around time, increasing throughput, and dramatically reducing the workload for the laboratory.

Mitotane Quantification Method in Plasma Using GC-MS Chris Thompson (Presenter) Mayo Clinic

Poster #56a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction:
Mitotane, 2,4’-Dichlorodiphenyldichloroethane (2,4’-DDD), is a chemotherapy drug used to treat adrenal cortical carcinoma. Plasma concentrations of mitotane need to be monitored continuously in the treatment of this disease to maintain the narrow established therapeutic range of 14-20 mcg/mL. Additionally, one of the main metabolites of mitotane, 2,4’-Dichlorodiphenyldichloroethylene (2,4’-DDE), could be monitored as it has the potential to provide additional value for the treatment of patients. However, the clinical utility of this metabolite requires further investigation.

Methods:
Standard stocks of 2,4’-DDD (Cat#: 32098, Restek) and 2,4’-DDE (Cat#: 32099, Restek) were purchased in methanolic solution. Standards were prepared by spiking varying levels of stock standards into Na-Heparin pooled human plasma (CA# IPLANAH100ML, Innovative Research). 200 µL of standards, controls, blanks, and patient samples were added to a 2 mL 96-well plate (CA# 96-6009, Chromtech). Next, 400 µL of internal standard (CA#’s CLM-6999-1.2, CLM-4693-1.2, Cambridge Isotope Labs) in acetonitrile was added to each well. The plate was then placed on a vortex mixer for 1 minute and centrifuged at 3500 RPM for 10 minutes. After centrifugation, the resulting supernatant is transferred to a GC-MS vial, which is sealed with a crimp cap.

The extracted samples were then analyzed using a 1 µL injection by GC-MS (Agilent GC model 6890 and MS model 5975). A J&W Scientific DB-5MS, 15m, 0.25mm I.D. capillary column with a film thickness of 1 micron was installed and used for analysis in the GC. The GC and MS methods were built using Agilent Chemstation. The GC method parameters were set at 260 °C for the inlet, 280 °C for the Aux temperature with an oven ramp from 60 °C to 320 °C over the course of 9.5 minutes. For the MS an inert electron ionization source was installed in the MS. The carrier gas used for the study was helium. Agilent Chemstation was used to build and submit each batch. Agilent Mass Hunter was used as the quantification software.

Results:
The linearity for samples (N=5) spanning the AMR (0.25-40 mcg/mL) yielded a linear regression with an R2=0.998 and slope of 0.9578 and 0.9765 for both mitotane and DDE, respectively. The intraday precision of the control levels (0.5, 5, and 30 mcg/mL) averaged a CV of less than 10%, with an N=20. The carryover for mitotane is negligible, however the carryover for the metabolite is about 200% of the LLOQ area and drops off significantly after the first 2 blanks. Patient samples were run using this method and compared to an external laboratory which used a GC-FID for their analysis. This comparison yielded a slope of 1.023 and an R2 of 0.9820 using linear regression analysis.

Conclusion:
Through development and validation of a quantitative GC-MS method for mitotane and its metabolite, we demonstrate that this method can provide accurate and precise quantitative results for therapeutic monitoring of mitotane and its metabolite DDE in patient samples. A Hamilton STAR liquid handling instrument method is being developed to automate a portion of the extraction process for implementation into the clinical lab setting.

Comparison Between DRI Fentanyl II and LZI Fentanyl Enzyme Immunoassays Rejwi Dahal (Presenter) Indiana University School of Medicine

Poster #58a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction: Urine drugs of abuse panel often does not include Fentanyl at many institutions because there are not many options for FDA-cleared assays. DRI Fentanyl II has a cutoff of 1.0 ng/mL with only 7% cross reactivity towards norfentanyl at the cutoff while LZI fentanyl immunoassay is targeted towards norfentanyl with the cutoff of 5 ng/mL.

Objective: The purpose of this study was to compare clinical utility of DRI Fentanyl II and LZI Fentanyl enzyme immunoassays.

Methods: 806 urine samples were screened by DRI Fentanyl II and LZI Fentanyl immunoassays. 183 random fentanyl positive and negative samples were further analyzed by LC-MS/MS for presence of absence of fentanyl, norfentanyl, and 4ANPP. Furthermore, 4ANPP positive samples were sent to a reference laboratory for further analyses to determine if any other fentanyl analogs are present in the samples.

Results: Positive agreement and negative agreement between the two methods were 96.9% and 97.6%, respectively. Cohen’s Kappa value was 89% (84.2 %to 93.7%). There were 3 samples that were screened positive by DRI II and negative by LZI, while 17 samples were screened positive by LZI and negative by DRI II.

Conclusion: Both assays provide similar clinical utility thus, either can be adopted and incorporated into urine drugs of abuse panel.

Quantitation of Endogenous Steroids in Serum Using Dried Blood Spot Plasma Separator Card and Triple Quadruple Mass Spectrometry Vikki Johnson (Presenter) Shimadzu Scientific Instruments

Poster #58b View Map

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

INTRODUCTION: Endogenous steroids are essential to the regulation of several metabolic pathways including energy metabolism, stress, and fertility. Dried blood spots (DBS) offer an alternative to conventional venipuncture blood collection by allowing less invasive sample collection at home. A challenge for the lab is the small sample size collected from the dried blood spot resulting in low concentrations of endogenous analytes. To assist with accurate quantitation of select steroids in this biological matrix, dispersive solid-phase (dSPE) extraction was utilized for increased sensitivity and selectivity by reducing matrix interference. Triple quadrupole mass spectrometry with its sensitive quantitation capability was used to analyze endogenous steroids. Optimized source conditions and MRM transitions on the mass spectrometer further improved limit of detection in matrix.

OBJECTIVES: The primary objective of this study is to compare quantitative results from serum and plasma separator cards with immunoassay methods.

METHODS: Four steroids (cortisol, testosterone, DHEA-S, and progesterone) were evaluated. Calibration curves and quality control samples were prepared by spiking certified reference standards into charcoal striped serum. Correlation samples were prepared by spiking analytes into serum and adding two drops to the collection card and drying for 15min. The collection disk was removed and reconstituted with isotope-labeled internal standard solution, incubated, and digested. The samples were then taken through an automated dSPE extraction protocol. A triple quadrupole mass spectrometer (LCMS-8060NX) coupled to a HPLC (Nexera X2) was used. Separation occurred over 3 minutes using a Velox SP-C18 column (100mm x 3.0mm I.D., 2.7µm). The spiked serum samples were additionally analyzed on immunoassay and the results were compared.

RESULTS: A triple quadrupole mass spectrometer with polarity switching was utilized to allow for positive and negative ionization in a single method (quantitation MRM: cortisol (ESI+): 363.05>105.10, testosterone (ESI+): 289.20>109.10, progesterone (ESI+): 315.20>109.05, and DHEA-S (ESI-): 367.05>97.05). Accuracy and precision for quality control inter-day (n=6) and intra-day (n=20, 3days) were less than 10% for all analytes. Calibrations were linear with correlation coefficients (r2) greater than 0.99 over the concentration range 5-1,250 ng/mL for cortisol, 80-10,000 ng/mL for DHEA-S, 0.1-25 ng/mL for testosterone, and 0.1-25 pg/mL for progesterone. Correlation between serum aliquots and reconstituted serum collection cards ranged between -4.0% -30% differences and correlation between plasma collection cards are currently in evaluation. dSPE is completed using Hydrophilic-Lipophilic Balanced (HLB) absorbent, developed to cleanup both polar and non-polar analytes from aqueous samples to achieve these low reference ranges. The steps for the sample extraction are automated using hands-free automated electronic pipettes. Chromatography for all analytes have been evaluated on a SP-C18 (2.7um 2.1x100mm) column with an injection-to-injection time of 6 min (Cortisol, 2.1min, DHEAs: 2.4min, testosterone: 3.1min, and progesterone: 3.8min).

CONCLUSION: A highly sensitive and accurate LC-MS/MS method was developed for the quantification of four steroids on DBS cards. The approach to utilizing DBS rather than traditional immunoassay has several advantages such as non-invasive collection and smaller sample volume. Automated sample extraction increases sample throughput by analyzing all analytes on a single LC-MS/MS method.

Using a Virtual Chromatography Tool to Develop Methods for Novel Psychoactive Substances Jared Burkhart (Presenter) Restek

Poster #60a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Novel psychoactive substances (NPS) have created a challenge for toxicology laboratories. New NPS are constantly disappearing as fast as they emerge, making it difficult to stay on top of which compounds are necessary to add to laboratory testing scopes. The development and optimization of liquid chromatography (LC) separations is time consuming and costly, often requiring several steps including literature research, column selection, method scouting, method development, and method optimization. To alleviate the burden of sacrificing instrument-uptime, labor and materials, an instrument-free software modeling tool was developed to include a comprehensive drugs of abuse (DoA) library. This online tool allows users to obtain optimized separations while maintaining critical pair resolution by adjusting parameters such as column dimension, mobile phase, gradient programs, and more for almost 300 compounds including the 38 newly added NPS drugs.

OBJECTIVES: The primary objective of this study is to use a chromatographic modeling tool to develop effective LC-MS/MS methods for various NPS compounds including synthetic opioids, designer benzodiazepines, synthetic cathinones, synthetic cannabinoids, and toxic adulterants.

METHODS: The NPS library utilized the same design space as the existing DoA library. Retention times were collected using method conditions consisting of a fast (5 minute) and slow (15 minute) gradient, 30°C/60°C temperature points, and ACN/MeOH mobile phases on Raptor Biphenyl and Raptor C18 columns in a 50 x 2.1, 2.7 µm dimension. The 38 NPS compounds were divided into three small groups to account for the separation of isobars and to generate the optimal points per peak for instrument analysis. A set of 8 compounds, referred to as “meld compounds”, were then added to each group. These meld compounds spanned the chromatographic space and were used to verify instrument performance from injection to injection. Data was collected and input into the platform. Results of retention times between experimental and modeled data were compared. To verify the ability of the modeler to develop methods for NPS, three methods were developed and optimized using the chromatogram modeler for the following NPS subclasses: 1) synthetic opioids and toxic adulterants 2) designer benzodiazepines 3) stimulants and synthetic cannabinoids. All methods utilized a Raptor Biphenyl 100 x 2.1, 2.7 µm column with a MPA of water and MPB of methanol, both acidified with 0.1% formic acid. The flow rate was 0.6 mL/min and the column temperature was 40°C. The developed methods were transferred to an LC-MS/MS system and the experimental results were compared with the modeler.

RESULTS: The online chromatogram modeling tool successfully developed methods for NPS compounds. Developing the methods using the virtual chromatography tool was completed in under ten minutes per method. The acceptance criteria for retention time agreement between experimental and modeled values was set at +/- 15 seconds, chosen to represent a typical MRM window. All analytes in all three methods fell within this window, as well as maintaining elution order and resolution. For example, Isotonitazene had a predicted retention time of 2.86 minuntes and an experimental retention time 2.75 minutes, for a difference of 6.6 seconds. Eutylone had a predicted retention time of 4.42 minutes and an experimental retention time of 4.18 minutes, for a difference of 14.4 seconds. Based on the acceptance criteria as defined, each NPS method was successfully transferred from the virtual model to an LC-MS/MS instrument.

CONCLUSION: As NPS continue to proliferate the illicit drug market, the burden of adding these compounds to laboratory testing scopes becomes the obligation of LC method developers. Utilizing tools such as a virtual chromatography modeler can help method developers deal with the challenges these emerging compounds present.

Best Practices: Enzymatic Hydrolysis Protocols Are Not a "One Size Fits All" for Each Drug Class When Using B-One Elías Villalobos (Presenter) Kura Biotech

Poster #60b View Map

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

INTRODUCTION: Glucuronidation plays a crucial role in the elimination of many drugs from the body. This metabolic process involves the conjugation of drugs with glucuronic acid, resulting in the formation of more polar compounds that can be easily excreted. Consequently, these conjugated drugs can complicate drug analysis by mass spectrometry for multiple reasons and to help resolve this issue, many laboratories utilize beta-glucuronidase enzymes to cleave the glucuronide conjugates to convert them back to their original, parent form. This approach allows for more accurate detection and quantification of these drugs and metabolites while streamlining the sample preparation workflow. However, it’s important to take into consideration when hydrolyzing different drug classes such as opiates, benzodiazepines, cannabinoids, and antidepressants, utilizing a universal approach may not be the most effective option. The reason for this is that each analyte has a unique interaction with beta-glucuronidase, leading to a customized hydrolysis method that is tailored to the specific class of drugs.

OBJECTIVE: Our main objective is to emphasize that a one-size-fits-all approach is not always ideal when hydrolyzing different drug classes and analytes, and to raise awareness about the importance of a customized hydrolysis protocol. This study also shows how to apply best practices while evaluating the hydrolysis performance using B-One® (Finden® by Kura Biotech®) recombinant beta-glucuronidase for the various drug classes for use in forensic and clinical toxicology laboratories.

MATERIALS AND METHODS: Common drug classes and analytes were quantitatively analyzed in different panels for hydrolysis efficiency using the following glucuronide standards fortified in drug-free urine: Opiates, Benzodiazepines, Naloxone/Buprenorphine/Norbuprenorphine, Carboxy-THC, and Amitriptyline. Quality control standards were prepared at low, medium, and high concentrations. The hydrolysis method was performed using B-One®, an “all-in-one” recombinant beta-glucuronidase stabilized in its reaction buffer for fast room-temperature hydrolysis. The hydrolysis was followed by a clean-up protocol using XTR™ tips 5 mg HLB (DPX Technologies) and then diluted with DI water for analysis by LC-MS/MS.

RESULTS: A quantitative method was used to determine the concentrations of free drugs for each analyte in quadruplicates and then the recoveries were calculated using a hydrolysis efficiency formula. Results demonstrated good recovery and precision with an optimized hydrolysis method for each drug class.

DISCUSSION AND CONCLUSION: The hydrolysis experiments conducted on various drug classes demonstrate the need for customized hydrolysis parameters for B-One,® including different hydrolysis times and enzyme amounts, by following the best practices suggested here. The addition of DPX XTR™ tips and NGX custom standard mixtures provides a streamlined protocol for ease of use as well.

Development of LC-MS/MS Xylazine Assay for Investigation of Xylazine Prevalence in New Haven, CT Region Leah Militello (Presenter) Yale University

>> POSTER (PDF)

Poster #63a View Map

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

Introduction: Xylazine is a veterinary tranquilizer not approved for human use that has been implicated in an increasing number of overdose deaths nationwide. It is used as an adulterant in recreational drugs of abuse, including heroin, cocaine and fentanyl, and many users are exposed unknowingly. As an α2-adrenergic agonist, its physiologic effects include sedation, muscle relaxation and decreased perception of pain. Taken in combination with other drugs, especially other central nervous system depressants like alcohol or benzodiazepines, the effects can be lethal. Xylazine was involved in 10% of all drug overdose deaths in Connecticut in 2020. Typical drug abuse screening panels do not include an assay capable of xylazine detection, as no immunoassay designed to run on an automated analyzer has been FDA-approved thus far. There is increasing demand from providers in the emergency department for an assay to determine the presence of xylazine in intoxicated patients with unclear clinical presentation. Here we describe a laboratory-developed xylazine assay and investigate the presence of xylazine in patient samples that screened positive for other drugs of abuse within the geographic region served by our hospital system. This method also examined the impact of measuring the xylazine metabolite, 2,6-dimethylaniline, on assay sensitivity.

Objectives: Develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for detection of xylazine and use that assay to examine the prevalence of xylazine in the New Haven, CT region.

Methods: A total of 37 patient urine samples that screened positive by immunoassay for cocaine, opiates and/or fentanyl between October and November 2023 were selected for the study and subject to retrospective analysis. Sample preparation consisted of a centrifugation step, addition of the internal standard, methadone-d9, followed by vortexing and another centrifugation step. Mobile phase A was 0.1 % formic acid in water and mobile phase B was 0.1 % formic acid in acetonitrile. Liquid chromatography (LC) was performed using an Acquity UPLC HSS column (T31.8 µm, 2.1 x 100 mm) at 50°C. MS/MS used positive electrospray ionization and monitored m/z transitions of 221 > 164 for xylazine (primary), 221 > 90 for xylazine (qualifier), and 319 > 105 for methadone-d9 (IS). The measurement of the xylazine metabolite 2,6-dimethylaniline had m/z transitions of 122 > 105 (primary) and 122 > 107 (qualifier). Retention time was 1.94 minutes for xylazine, 2.26 minutes for 2,6-dimethylaniline, and 2.16 minutes for the methadone-d9 internal standard. Linearity was established between 1-1000 ng/mL.

Results: Xylazine was detected a total of 9 out of 37 patient samples, or 24%. Of these samples, one was positive exclusively for opiates and three were positive exclusively for cocaine. Two samples were positive for both cocaine and fentanyl, two were positive for both cocaine and opiates, and one was positive for both fentanyl and opiates. All but one of the opiate positive specimens confirmed positive for 6-MAM, indicating heroin use. The quantity of xylazine ranged from 6 ng/mL to greater than 1000 ng/mL, with a mean value of 215 ng/mL and a median of 24 ng/mL. The results of 2,6-dimethylaniline were all significantly lower than xylazine and it was not found to add value in addition to xylazine.

Conclusions: Analysis by LC-MS/MS showed a high prevalence (24%) of xylazine in patient urine samples that screened positive for cocaine, opiates, fentanyl or a combination of these drugs. This is consistent with the Center for Disease Control and Prevention (CDC) data reporting that around 30% of fentanyl overdoses have also been found to contain xylazine. This may indicate a high level of adulteration by xylazine in the illegal drug supply in New Haven. Measurement of the metabolite 2,6-dimethylaniline did not add value, so laboratories looking to develop assays for xylazine do not need to measure this metabolite.

Development and Analytical Performance of 43 Drug of Abuse Panel in Urine using the SCIEX Triple Quad™ Citrine LC-MS/MS Mahesheema Ali (Presenter) The MetroHealth Medical Center

Poster #64a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Background: This study reports the analytical performance of the SCIEX Citrine Triple Quad MS/MS system to analyze 43 drugs of abuse in human urine matrix monitoring over 125 MRM transitions (including internal standards).

Method: The evaluation of performance involved verifying identity, selectivity, linearity, the limit of detection (LOD), lowest limits of quantification (LLOQ), accuracy, precision, extraction recovery, matrix effect, and stability. The method uses the SCIEX Exion HPLC system and the SCIEX Triple Quad™ Citrine LC-MS/MS system to ensure fast, sensitive, and reproducible results were utilized, and data was processed using Multiquant MD software 3.0.3 version. To evaluate the drug compounds, commercially available urine spiked with Cerilliant was used and processed under certain conditions with an optimized scheduled MRM.

Sample Prep Conditions
To analyze the 50 μL urine sample, it needs to be hydrolyzed. Use 15 µL of IMCS Rapid Hydrolysis Buffer and 5 µL of IMCSzyme for this purpose, and let it sit for 15 minutes at room temperature. After hydrolysis, add 10 µL of internal standard with 135 µL of water. Next, add 300 µL of sample diluent, which should be a mix of 3 parts mobile phase A and 1 part mobile phase B. Centrifuge the mixture at 15,000 rpm for 15 minutes and the sample is now ready for analysis.

Liquid Chromatography Conditions
Column: Phenomenex Kinetex Phenyl-Hexyl and Security Guard: ULTRA Phenyl cartridge are used in this method. The mobile phase A is made up of water and 0.1% formic acid, whereas mobile phase B contains methanol and 0.1% formic acid. The flow rate is set at 0.8 mL/min, and the injection volume is 2 uL. A linear gradient is used from 2% to 98% B over 6.4 minutes, with a retention time of 1 to 6.5 minutes.

Mass Spectrometry Conditions
Duration of the Method: 7.5 minutes. Polarity can be Positive or Negative. Electrospray Scheduled MRM detection window is 50 seconds. Transitions are compound dependent. Source Conditions are flow rate optimized.

Results: The regression coefficients (r2) for the calibration curves in the study were ≥0.99. Intra and intraassay imprecision were found to be <10%. Comparison with a reference laboratory revealed <20% bias for all 42 analytes, deeming correlation coefficients >0.990. Linearity ranges were established from the lowest to upper limit calibrator concentrations with 10- to 100-fold maximum dilution.

Conclusion: An analytical method was developed to quantify over 43 drug components in human urine. This method provides an effective strategy for improving TAT in drug testing and confirmation of screen results.

An Automated Sample Preparation Approach for the Determination of Per and Polyfluoroalkyl Substances (PFAS) from Human Biological Fluids using UHPLC-MS/MS Adam Senior (Presenter) Biotage GB Ltd

Poster #65a View Map

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

INTRODUCTION: Exposure to Per and polyfluoroalkyl substances (PFAS) has been linked with changes in metabolism, increased cholesterol, and high incidence of some forms of cancer. PFAS pose particular challenges in the analytical laboratory due to their ubiquitous nature. Environmentally, PFAS are of concern because of their high persistence (forever chemicals), bioaccumulation and slow elimination, and potential impacts on human and environmental health. We present automated methods to determine clinically relevant levels of PFAS in common biological matrices.

OBJECTIVES: This poster presents an automated and robust, high-sensitivity method for the clean-up of 31 PFAS compounds from various biological fluids. ISOLUTE® PLD+ for PFAS, due to its optimised sorbent chemistry and functionality, allows a simple solvent crash/filtration-based approach.

METHODS: A suite comprising 31 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. Simple solvent crash/filtration extraction methodologies incorporating a 1:7 matrix/solvent ratio, utilizing ISOLUTE® PLD+ for PFAS in 96-well format were investigated. Processing parameters were optimized for the Biotage® Vacmaster™-96 vacuum manifold and the Biotage® Extrahera™ automated sample preparation platform at 100 µL and 50 µL sample volumes. Solvent crash performance was compared using solvent/matrix first approaches. Extrahera™ automation parameters were optimised for each matrix to maximise sample/solvent crash and mixing efficiency, and included system consumables, pipetting parameters (aspirate/dispense frequency, height and speed), and extraction conditions (applied pressure and duration). The optimized Vacmaster™ and Extrahera™ methods for each matrix were selected on the basis of maximum recovery and repeatability in combination with minimal matrix factors. Final ISOLUTE® PLD+ for PFAS protocols for manual and automated platforms were used to assess method performance at 100 µL and 50 µL load volumes for all matrices. Target analyte linearity was determined from 0.1 to 100 ng mL-1 over 8 levels. Sensitivity (limit of quantitation, LOQ) was estimated where signal/noise of the lowest calibration standard was >10:1, with accuracy of 80-120% and RSD <15%. 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: Optimized extraction protocols utilizing ISOLUTE® PLD+ for PFAS sample preparation demonstrate highly consistent recoveries and high repeatability for all PFAS in our target suite. Using the Extrahera™ automated sample extraction platform and 100 µL sample volumes, serum recoveries were 80-94%, typical RSD were <5%, with matrix factors typically1.0-1.5; sample extraction using Vacmaster™ demonstrated comparable performance. Results obtained using 50 µL sample volumes were similar, automated recovery RSD were lower than demonstrated from manually extracted samples. Plasma recoveries from 100 µL matrix using automation were 77-80%, with RSD <5%, matrix factors were 1.0-1.5; manual extraction recoveries demonstrated greater variability (58-89%), with correspondingly higher RSD (< 10%), matrix factors were comparable. Automation recoveries from whole blood were 79-90%, typical RSD were <5%, matrix factors were slightly higher than from blood products (1.2-1.5), probably due to increased matrix complexity. Manual recoveries were slightly higher than automated (82-94%), as were matrix factors (1.3-1.5), RSD were comparable to automated extraction. Similar results for whole blood were demonstrated using 50 µL sample volumes, automated RSD were lower than manual RSD. Urine recoveries using our automated protocol were 70-82%, RSD were <6%, and MF 1.0-1.5; manual recoveries were comparable (75-90%) with RSD <6%, matrix factors 1.0-1.5 were typically observed. Results obtained using 50 µL urine demonstrated less consistent results with greater variability. The optimized ISOLUTE™ PLD+ for PFAS protocol was used to determine extracted linearity and calibration ranges. Method performance data were similar using 100 µL matrix volumes with Extrahera™ automation or Vacmaster™ manual extraction. Most analytes in our target suite demonstrate LOQ at 0.1 ng mL-1. All analytes demonstrate good linearity, r2 > 0.995. Most analytes demonstrate repeatability <10% (<20% at LOQ). Typical analyte accuracy was 90-110% (80-120% at LOQ). Method performance using 50 µL matrix volumes is similar to 100 µL with slightly increase variation, probably due to increased variation in liquid transfer. ISOLUTE™ PLD+ for PFAS demonstrates excellent matrix depletion compared to centrifugation and dilute/shoot. Our optimized extraction protocol results in 99.9% depletion of phospholipid and lysophospholipid, almost all protein is removed. Analytical column lifetime is improved by preventing matrix build-up over multiple injections, maintaining analyte sensitivity over extended analytical runs.

CONCLUSION: Optimized extraction protocols utilizing ISOLUTE® PLD+ for PFAS demonstrate consistent PFAS recovery and sensitivity, with low matrix factors, for 31 PFAS residues extracted from a variety of biological fluids. Use of the Biotage® Extrahera™ automated sample preparation platform demonstrates comparable data to a manual vacuum-based method, helping to improve sample throughput. ISOLUTE® PLD+ for PFAS demonstrates good accuracy and precision across the calibrated range of the assay, with LOQ at clinically relevant levels. Due to its optimised sorbent chemistry and functionality, ISOLUTE® PLD+ for PFAS demonstrates enhanced cleanliness compared to diMlute-and-shoot methodology leading to more robust methods.

Optimizated Sample Preparation for Low Level Determination of Alcohol Marker EtG from Hair Using UPLC-MS/MS Analysis. Lee Williams (Presenter) Biotage GB Limited

Poster #65b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

Introduction
Hair analysis is growing in popularity due to the non-invasive nature of sample collection. Hair testing can provide advantages over other more widely used matrices, such as prolonged exposure and potential timelines. However, as a solid matrix sample preparation can often be lengthy and labour intensive. Minor ethanol metabolites such as ethyl glucuronide (EtG) and ethyl palmitate (EtPa) can be measured in hair as direct markers of alcohol consumption. As suggested by the Society of Hair Testing (SoHT) in 2019, EtG determination from hair is the preferred marker for the assessment of abstinence.

Objectives
This poster aims to demonstrate improved workflow and clean-up of hair matrix to allow low level EtG determination required for alcohol abstinence testing.

Methods
Hair samples (10 mg) were subjected to micro-pulverized extraction using the Biotage® Lysera bead mill homogenizer. Once in liquid form, sample extracts were further cleaned-up using polymer-based solid phase extraction (SPE) in 96-well plate format. Subsequent analysis was performed using a Waters ACQUITY Premier UPLC coupled to a Xevo TQ Absolute triple quadrupole mass spectrometer. Negative ions were acquired using electrospray ionization operated in the MRM mode.

Results
Initial method development focussed on investigation of LC mobile phases and column chemistry to achieve good chromatographic performance while providing adequate negative ion sensitivity. An HSS T3 column provided good reversed-phase chromatography albeit with acidic mobile phase composition, resulting in reduced negative ion sensitivity. HILIC chromatography yielded good sensitivity generally at the expense of compromised chromatographic peak shape.

Evaporative effects were investigated using a range of traditional SPE elution solvents. The results demonstrated between 15-25% analyte signal reduction depending on exact solvent combination.
Matrix homogenization was investigated after standard washing protocols. Both dry hair and pulverisation in solvent was evaluated. 10 mg of hair was extracted with up to 1 mL of H2O to effect efficient analyte solubilisation from the matrix.

Sample extraction and clean-up was performed using polymer-based mixed-mode strong and weak anion exchange SPE chemistries due to the carboxylic acid moiety on the analyte. Wash and elution optimization resulted in analyte recoveries greater than 80% for both chemistries. Suppression profiles were reduced resulting in matrix factors between 0.7-0.9, depending on exact protocol. Final workflow streamlining evaluated SPE bed reduction, minimum elution volumes and direct injection compared to evaporation/reconstitution protocols. Both approaches presented different advantages with respect to simplicity and time vs absolute sensitivity but ultimately achieved the required LOQs. Calibration curves were constructed using hair spiked between 1-100 pg/mg to cover the range of chronic excessive consumption and abstinence. Results demonstrated good linearity and coefficients of determination (r2) values greater than 0.99. LoQs were determined to be below the required SoHT guidelines for abstinence cases of equal to or below 5 pg/mg of hair.

Conclusion
This poster demonstrates optimized extraction and cleanup of hair matrix allowing low level alcohol marker determination in abstinence cases.

A Novel Method for Simultaneous Targeted LC/MSMS Quantification of THC and Nicotine Metabolites in Human Urine Emilio Mejia (Presenter) UCSF

Poster #66a View Map

This poster will be attended on Thursday at 09:15 for 1 hour 15 minutes in the Exhibit Hall.

Introduction: The Multicenter AIDS Cohort Study/ Women’s Interagency HIV Study Combined Cohort Study (MACS/WIHS) is a collaboration researching the effects of chronic health conditions of people living with HIV/AIDS. The main focus of the collaboration is to research heart, lung, blood, and sleep disorders. Tobacco and cannabis use potentially can add additional risk to cardiologic and pulmonary health in these immunocompromised individuals. The analytes targeted in this method are nicotine, cotinine, 3-OH-cotinine, THC-COOH, and creatinine. Nicotine presence in the urine is an indicator of active or passive use dependent on the concentration. Cotinine and 3-OH-cotinine are the primary metabolites of nicotine and are good indicators of secondhand smoke exposure (SHSE) at certain concentrations. THC-COOH is the primary metabolite of delta-9-THC and is the best indicator of cannabis use. Quantitation of the metabolites of nicotine and THC, respectively, can help to provide a clearer understanding of the effects of their use on lung and heart health. MACS/WIHS has a biorepository of over 8000 specimens ready to be analyzed with this method. Although various methods exist for the quantitation of nicotine and THC metabolites respectively, there are none yet published that quantify both simultaneously.

Objectives: To facilitate the quantitation of THC and nicotine metabolites in urine by providing a targeted method for the selected analytes.

Methods: Urine samples are thawed and vortexed, then centrifuged. 200µL of urine are aliquoted to a well in a glass coated plate. 30µL of IMCSzyme glucuronidase mix is added to each sample for the reaction to begin. After 15 minutes, 20µL of deuterium labeled IS mix is added to each sample. As the reaction is taking place, prep of a µElution HLB SPE takes place. 200 µL of MeOH is pushed through to waste with a positive pressure N2 manifold. Next, 200µL LCMS grade H2O is pushed through to prime and equilibrate the sorbent. Next, 200uL of the urine mixed with glucuronidase and IS is added to the sorbent and pushed through. Two washes of 10mM Ammonium Acetate (aqueous) are pushed through. The waste plate is switched out to a collection plate, a new glass coated plate. Two steps of 25µL 50:50 ACN: Ethyl Acetate are used to elute the desired analytes off of the sorbent. The elute is dried down under a stream of N2 for 5 minutes. The dried sample is then resuspended in 50µL of 80:20 MPA:MPB (MPA = 10mM Ammonium Acetate, MPB = 80:20 ACN:MeOH). The plate is sealed and ready for injection. The calibration curve is made by spiking stripped human urine from a range of 1ng/mL to 10,000 ng/mL, the only variation being creatinine which is measured from 0.4-400 mg/dL. The LC/MSMS method is 6.1 minutes beginning at 10%B and gradually reaching a point of 99%B. Chromatographic separation was achieved using a Phenomenex 100mm Kinetex EVO C18 2.6um column. The injection volume is 10µL The detector used is a SciEx 6500+ Qtrap TripleQuadrupole Mass Spectrometer. The MS was run in MRM-ESI mode. The transitions for the chosen analytes and their internal standard counterparts are as follows. The following are done in positive mode, Creatinine 113.9/44.1, Creatinine-d3 116.9/47.1, Nicotine 163.1/130.0, Nicotine-d3 166.0/130.0, Cotinine 177.1/80.0, Cotinine-d3 180.1/80.0, 3-OH-Cotinine 193.0/80.1. The following are done in negative mode, THC-COOH 343.2/299.2, THC-COOH-d9 352.1/308.2. Each analyte also has a qualifier transition used solely for confirmation of the analyte's presence in the sample.

Results: The resulting method’s reproducibility was tested by inter-day injections of calibration curves (1-10000ng/mL)(n=5), and the injection of QCs at 10ng/mL (n=5). The R2 values for all curves are over 0.987, however most averaged over 0.996. All curves were weighted with 1/x, and linear when possible, otherwise quadratic regressions. Inter-day imprecision was less than 0.035% for all curves. The inter-day imprecision for the QCs were 2.0% or less. No carryover was observed for any of the analytes after injections at the high end of the calibration curve (10,000ng/mL). There is no crosstalk with the current set of deuterium-labeled standards (THC-COOH-d3 had some crosstalk with THC-COOH but was replaced with THC-COOH-d9 to resolve the issue). There is some matrix suppression dependent on the saturation of the urine, but the assay is sensitive enough and the concentration of the target analytes is high enough that it does not make a significant difference. A test to determine recovery has not yet been performed but will be done. The LLOQ for nicotine, cotinine, 3-OH-cotinine, and THC-COOH were below 1ng/mL (the lowest calibration concentration), as well as below 0.4 mg/dL for creatinine. The lowest calibrator concentration being much lower than the accepted concentration of what would be considered “positive” in a clinical setting. So far 1702 patient specimens from MACS/WIHS have been run with this method. Within these specimens 527 (31%) have tested positive for THC-COOH (>15ng/mL), 635 (37%) tested positive for nicotine (>15ng/mL), 251 (15%) were positive for 3-OH-Cotinine (>50ng/mL), 280 (16%) were positive for cotinine, with 110 (6%) of the cotinine positives being within the SHSE range.

Conclusion: Although many studies have focused on individual metabolites of nicotine or THC, there has not been a published method that encompasses various metabolites of both compounds. It seems pertinent to run one method that quantifies all the aforementioned metabolites within one method. The developed method is very sensitive with respect to what is considered a positive value within human urine. The method is efficient and more cost-effective and faster than running multiple methods to analyze the various metabolites.

Validation of the Simultaneous Quantification of Two Long Acting Injectable Antiretroviral Drugs Cabotegravir and Rilpivirine in Plasma Using LC-MS/MS Amanda Schauer (Presenter) University of North Carolina-Chapel Hill

Poster #71a View Map

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

Introduction
Cabotegravir (CAB), an integrase inhibitor, and rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor, are used in combination as the first long acting injectable therapy for the treatment of HIV infection. Recent data suggests that variable absorption and drug distribution may warrant drug monitoring in patients with body habitus changes (eg obesity, pregnancy). Because these drugs are a co-formulated, we developed a fully validated LC-MS/MS method for simultaneous measurement of CAB and RPV in human plasma over a clinically relevant 1000-fold range for each analyte.

Methods
Calibration standards were prepared at eight concentrations in human plasma to cover the calibration range of 25.0 to 25000 ng/mL (CAB) and 5.00 to 5000 ng/mL (RPV). Quality controls (QCs) were prepared at four concentrations consisting of a lower limit of quantification (LLOQ), low, mid, and high concentrations (25.0, 75.0, 1000, and 20000 ng/mL for CAB; 5.00, 15.0, 200, and 4000 ng/mL for RPV). A QC three times higher than the upper limit of quantitation was used to evaluate the ability to dilute samples for accurate and precise concentration measures.
Human plasma (40µL) samples were extracted by protein precipitation by mixing with 200 µL of methanol containing stable, isotopically labeled internal standards (13C 2H2 15N-CAB and RPV-d6,). The samples were vortexed for approximately 2 minutes, centrifuged at room temperature for 5 minutes at 16,100 rcf, and mixed 1:5 with water before being transferred to a 96-well plate for LC-MS/MS analysis.

Chromatographic separation was achieved by reverse phase chromatography on a Waters Atlantis T3 (50x2.1 mm, 3 µm particle size) analytical column with 0.1% acetic acid in water (mobile phase A) and 0.1% acetic acid in methanol (mobile phase B) under gradient conditions. Detection of the analytes and internal standards was achieved on an AB Sciex API-5000 triple quadruple mass spectrometer under positive ion electrospray conditions with a total run time of 3.5 minutes.

Results
The method was fully validated to meet the acceptance criteria of the US Food and Drug Administration guidelines and met all acceptance criteria for precision and accuracy. Inter-assay precision (%CV) and accuracy (%Bias) was within ±15% for all four concentrations of QC samples and determined by replicates of 6 QCs in each of 3 separate runs.

Six different blank plasma lots were free of interferences in all monitored transitions. Those same six lots also were spiked at the LLOQ level and the precision and accuracy were within 20% criteria demonstrating that this assay performance is not affected by different plasma sources. Lastly, these six lots were spiked at the low, mid, and high QC concentrations and the average peak area ratios (analyte:internal standard) from the analyses (n=3) were plotted against QC concentration. The CV from the 6 slope values was <5% indicating lack of matrix effect. Analyte stability was established for 3 freeze-thaw cycles, at room temperature in human plasma for up to 24 hours, and in extracts for up to 3 days. Long-term stability in human plasma was also established for up to 691 days when stored at -80C.

Conclusions
A simple and fast LC-MS/MS assay has been validated for the quantitative analysis of cabotegravir and rilpivirine in human plasma.

The Potential of Paper Spray-Mass Spectrometry for the Analysis of NPS in the Emergency Department Ed Goucher (Presenter) Thermo Fisher Scientific

Poster #71b View Map

This poster will be attended on Wednesday at 14:30 for 1 hour 15 minutes in the Exhibit Hall.

INTRODUCTION: Ambient ionization has recently become an attractive addition to toxicology workflows, as it provides rapid analysis of drugs of abuse compared with traditional chromatographic techniques such as LC-MS. Based on electrospray ionization mechanisms, paper spray ionization (PSI) generates ions directly from a sample spotted onto a paper substrate, minimizing the cost and time of analysis. PSI uses dried matrix spots (DMS), a well-established but evolving trend in toxicology providing an alternative to collection of venous blood samples. Using DMS and PSI combined introduces the possibility of less invasive sample collection for the patient, no sample extraction or chromatography required facilitating lower cost per analysis, and a quicker turnaround of results.

Emergency department (ED) presentations due to acute drug toxicity can be life-threatening; currently clinicians rely on self-report and clinical patterns of toxicity to determine the drug(s) likely to be involved. The use of a VeriSpray™ Paper Spray Ion Source coupled to a mass spectrometer for identification and quantitation of drugs of abuse can provide results on drugs present to inform patient care early in the patients’ ED presentation. As such, there is the potential for a wide-reaching benefit to ED care, ultimately enabling clinicians to tailor patient care based on analytically confirmed drug use.

METHODS: Initial investigations have focused on the development and validation of synthetic cathinones in human urine. A synthetic cathinone working solution containing 4-CEC, 4-Cl-α-PPP, 4-Cl-α-PVP, MDPV, 4-EMC, N-ethylhexedrone, and MDPV-d8 was spiked into urine at an appropriate concentration and vortexed. The sample was spotted onto a VeriSpray™ sample plate containing Whatman™ ET 31 chromatography paper in a triangular shape and allowed to dry at room temperature. Results were acquired in less than 2.5 minutes per sample and validated according to ANSI/ASB 036 guidelines.

RESULTS: Method development focused on improving the signal to noise ratios and ion ratios per analyte to offset any interference from the paper substrate and increase the selectivity of subsequent quantitative results. By targeting less abundant ion transitions, up to a 30-fold increase in the signal to noise was seen, complemented by a significant decrease in the interference from the paper substrate and urine itself. Subsequent preliminary method validation demonstrated R2 > 0.98 and no carryover. Accuracy and precision values were between -3 % and 4 % and 16 % and 17 % respectively, despite high matrix effects and no sample extraction being carried out.

CONCLUSION: The increased signal to noise ratios and initial method validation provides an encouraging starting point for future standard operating procedures when using the VeriSpray™ Paper Spray Ion Source in a research and clinical setting. By demonstrating quantifiable results without the need for extensive sample preparation, the continued optimization for a wider panel of drugs of abuse as the study progresses will inform current and future applications to achieve the long-term aim of using a VeriSpray™ Paper Spray Ion Source in the ED.

Evaluation of a Fast and Novel Chromatography-Free Workflow for Urine Toxicology Screening Using DART-MS/MS Matthew Clabaugh (Presenter) Bruker

Poster #74b View Map

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

INTRODUCTION:
In conventional forensics testing, toxicology urine screening typically employs Immunoassay-based (IA) tests which are prone to several limitations including false negatives and cross-talking, to name a few. Further, the frequent addition of new test targets results in slow test improvements and the IA-based assay industry cannot adequately respond to this fast-moving introduction of new substances. Herein, we report on a rapid, simple, and mass spectrometry-based urine screening method using DART-MS/MS for six common forensic compound groups, which vastly improves the acquisition of positive results without false-positive consequences, and one which is amenable to quick response for new drug targets.

OBJECTIVES:
Direct Analysis in Real Time-MS/MS (DART-MS/MS) is a fast and simple chromatography-free solution for wide-audience urine screening workflows. The primary goal of this method is to provide enhanced data quality by leveraging high resolution mass spectrometry with analysis times that are similar to immunoassays. The secondary goal is to simplify and improve the conventional urine-screening workflow by introducing high quality commercial kits to lower the threshold of entry into mass spectrometry-based screening.

METHODS:
The screening workflow includes the following compound groups: 1 Stimulants (amphetamine, methamphetamine, MDA), 2 Opiates (codeine, oxycodone, oxymorphone, tapentadol), 3 Benzodiazepines (alprazolam, diazepam, oxazepam, temazepam), 4 Benzodiazepine metabolites (7-aminoclonazepam, alpha-hydroxyalprazolam), 5 Illegals (benzoylecgonine, 6-MAM, THC-COOH), and 6 Opioids (fentanyl, acetylfentanyl, nor-fentanyl).

Development:
Analysis was performed using the newly introduced Bruker EVOQ DART-TQ+ platform. This platform utilizes a fully integrated DART ion source, an improved EVOQ triple quadrupole mass spectrometer, and single point of entry software to enable automatic acquisition and processing of a standard 96 sample set.

DART parameters, including temperature, scan type, and scan time, were optimized to maximize both sensitivity and precision. All drugs were tuned prior to analysis.

Calibration series and QCs were prepared in pooled human urine with deuterated internal standards. Liquid-liquid extraction was performed by concentrating the organic layer across a 2h preparation time. Aliquots of 2 µL were loaded onto a Bruker HTS-96 plate and ionized using the integrated DART-MS source for analysis amounting to 20-30 seconds per sample and a total run time < 1.5h.

Validation:
Regression curves were comprised of 5 calibrators, blanks, matrix blanks, and 3 QCs run in triplicate. Each set of samples was run multiple times.

RESULTS:
The drug groups 1-5 were analyzed by DART-MS/MS and the DART parameters were optimized for each group. Helium stream temperature was determined to be as follows for the six groups analyzed: (1) 200 C; (2) 350 C; (3) 350 C; (4) 350 C; (5) 350 C; (6) 350 C. All samples were run in scan mode with a 5-8 second duration. Data were processed against the internal standards using tqControl and the integrated MS quantitation features. All drugs investigated were analyzed via quadratic regression curves in the range tested, with R2 values of 0.985 or better for screening. Quadratic regression was selected to extend the screen range. Most drugs passed the validation protocol following traditional FDA requirements.

CONCLUSION:
This DART-MS/MS method describes a suitable chromatography-free approach for screening urine toxicology samples using mass spectrometry. The rapid analysis times, simplicity, and data quality were deemed to exceed current immunoassay times, complexity, and rate of false positives. Furthermore, this solvent-free approach reduces costs and environmental impact. NOTE: Analysis of a correlation set against a validated assay is pending.

Enzymatic Hydrolysis of Recalcitrant Steroids with Engineered Arylsulfatases Anusha Chaparala (Presenter) Integrated Micro-Chromatography Systems Inc

Poster #25a View Map

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

INTRODUCTION
Over the last decade, detecting sulfated anabolic androgenic steroids (AAS) has been described as preferred analytical methods. However, its direct detection can be limited due to poor cleavage of the sulfate, especially in testosterone and its derivative, boldenone. Poor recovery of dehydroepiandrosterone sulfate (DHEAS) has also been a major challenge in steroid testing community.

OBJECTIVE
There are no reported sulfatases that can cleave sulfated metabolites of testosterone, boldenone or dehydroepiandrosterone. These are the first reported, genetically engineered variants of sulfatases completely hydrolyze testosterone- boldenone- 17beta sulfates and DHEA-3beta sulfates.

METHODS
Steroid standards, internal standards, and sulfated metabolites were from Cerilliant, Steraloids, TRC and Fisher. Chemicals were purchased from MilliporeSigma and Fisher Scientific. Drug free human urine controls were from UTAK. Urine controls were fortified with sulfated metabolites and hydrolyzed up to 4 hours at 37°C. After hydrolysis, samples were diluted to 50% methanol and eluted through a β-Gone Plus plate from Phenomenex. 10 uL of diluted sample was injected on a Thermo Scientific® Vanquish® UHPLC system coupled to Thermo Scientific® Endura® Triple Quadrupole Mass Spectrometer using a Phenomenex Kinetex® 2.6 um Biphenyl 100 Å, 50 x 4.6 mm column. Mobile phases A and B were 0.1% formic acid in water and methanol, respectively. Calibration curves had r2 ≥ 0.99 and quality controls were within ± 20%.

RESULTS
Engineered arylsulfatases from P. aeruginosa can hydrolyze difficult substrates such as boldenone sulfate and dehydroepiandrosterone sulfate within 2 hours at 37°C, whereas all prior purified sulfatases have little or no observed activity towards these analytes.

CONCLUSION
Difference in reactivities were observed for sulfatases towards 3beta and 17beta sulfates. Milder reaction conditions were established for liberating sulfated steroids in urine. Larger, comparative studies need to be performed in a broader range of biological matrices before enzymes are recommended for routine use in steroid testing.