Poster Presentations for Poster Session B : Wed 12:15 - 13:30

Lighting the Path From Research to Practice: LeMbA-MS for Pathology-Based Proteomic Biomarker Panel Michelle Hill (Presenter) ProSeek Bio Pty Ltd

Poster #1b View Map

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

INTRODUCTION
Liquid chromatography–mass spectrometry (LC-MS) is widely used in proteomic biomarker discovery but is often replaced by immunoassays during clinical translation. However, multiple reaction monitoring (MRM) on triple quadrupole (QQQ) MS can offer superior specificity and sensitivity in selected applications. Advantages include multiplexing without antibody cross-reactivity and avoiding the high cost and timelines of antibody development. Despite these benefits, clinical adoption of proteomic LC-MS is limited by sample complexity and labor-intensive workflows.

To address this, we developed a translational glycoprotein biomarker platform—Lectin Magnetic Bead Assay coupled with Mass Spectrometry (LeMBA-MS)—which enables single-step glycoform enrichment [1]. LeMBA-MS has shown success in cancer biomarker research using biobanked samples [2, 3].

OBJECTIVES
To support LeMBA-MS implementation in pathology laboratories, we aimed to reduce hands-on time (HoT) and turnaround time (TAT), while ensuring robustness in real-world sample conditions.

METHODS
We established in vitro diagnostic (IVD)-quality procedures for manufacturing lectin-magnetic beads, enabling simplified sample processing for clinical serum. Instructions for Use were developed for both manual and liquid handler workflows. Biomarker stability was assessed in fresh and frozen serum and plasma samples under various handling and shipping conditions.

RESULTS
Selected ovarian cancer biomarkers showed equivalence between fresh and frozen serum samples. IVD-grade LeMBA beads reduced HoT from 30 to 5 hours and TAT from 7 to 1 day without loss of assay performance. Stability studies demonstrated robustness under a range of pre-analytical conditions.

CONCLUSION
This study supports the feasibility of deploying LeMBA-MS multi-marker panels in pathology workflows, enabled by reagent manufacturing under a quality management system and performance validation in clinically relevant conditions.

REFERENCES
[1] Dutt et al. 2023 Methods Mol Biol. 2628:395-411.
[2] Dutt et al. 2023 Proteomics Clin Appl. 17(4):e2200114.
[3] Sheahan et al. 2025 Proteomes 13(2):23.

Development of LC-MS/MS Assays for Inborn Errors of Metabolism Using 3-Nitrophenylhydrazine Derivatization. Ingvi Jonsson (Presenter) University of Iceland

Poster #3b View Map

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

INTRODUCTION:
Inborn errors of metabolism are a group of rare inherited disorders that can have early onset during infancy. If not diagnosed and treated, these disorders can lead to organ damage, developmental delay and death. Newborn screening detects and quantifies acyl carnitines and amino acids, biomarkers for these disorders, with high-throughput tandem mass spectrometry. Organic acids and acyl glycines are important indicators of multiple inborn errors of metabolism, but are not detected in standard newborn screening and need additional assays for quantification. Second-tier assays such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be used to aid in diagnosis and reduce false positives. A single LC-MS/MS assay covering multiple compounds decreases system down-time by eliminating the need to change columns and mobile phases sample-to-sample, minimizes variations in sample preparations and simplifies workflow. However, the diverse physicochemical properties of these biomarkers make the development of a single multiplexed LC-MS/MS assay challenging.

OBJECTIVE:
Our goal is to develop multiplex LC-MS/MS assays for 1) full amino acid profile, 2) acyl carnitines and 3) organic acids and acyl glycines in neonatal dried blood spots, plasma and urine. All assays will use 3-nitrophenylhydrazine (3-NPH) derivatization. 3-NPH alters the chemical structure of analytes, reducing their polarity and making them amenable to reverse-phase liquid chromatography. Once developed, the assays will be validated according to the "ICH M10 on bioanalytical method validation" guideline.

METHODS:
Three LC-MS/MS methods are currently in development. Standards in neat solutions were used to determine provisional MS parameters and screen columns and mobile phases, further development used matrix samples. Upon selecting the column and mobile phases, additional variables such as flow rate, column temperature and capillary voltage were optimized for compound sensitivity.

RESULTS:
Initial screening has revealed a column and mobile phase combination that show acceptable performance for all three methods. Peak shapes for analytes are symmetric and retention of all analytes is successful. Amino acids elute at low organic percentages, thus their gradient is shallow and resolution of alloisoleucine from isoleucine and leucine is achieved with a total analysis time of 10 minutes. Monitoring of phospholipids in plasma indicates minimal ion suppression of amino acids. Acyl carnitines elute linearly in order of increasing chain length with a total analysis time of 5 minutes. Organic acids and acyl glycines are analyzed with a 10 minute method, resolving key isobaric compounds.

DISCUSSION:
While still in development, preliminary results are promising. Amino acids and acyl carnitines are easily observed in endogenous concentrations from non-spiked matrix samples. However, some organic acids need resolution from matrix interferences. Our results indicate the method has the potential to accurately quantify biomarkers for multiple inborn errors of metabolism and give a comprehensive amino acid profile. Current limitations to the method are interferences with organic acids, challenging the clinical application of the organic acid panel to accurately identify and quantify multiple markers for organic acidemias. To resolve these challenges, further optimization of the derivatization, sample cleaning and altered elution gradient may improve resolution of isobaric compounds and interferences.

Simplified Equilibrium Dialysis and SPE Method for the UHPLC-MS/MS Analysis of Free T4 and T3 for Clinical Research John Vukovic (Presenter) Waters Corporation

Poster #4b View Map

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

Background:
Measurement of free thyroxine (FT4) and free triiodothyronine (FT3) is typically performed by immunoassay; however, these can be prone to suffer from interferences. Currently, the accepted gold standard for the measurement of FT4 and FT3 is using equilibrium dialysis (ED) combined with LC-MS/MS, which can overcome the issues with immunoassay in the measurement of FT4 and FT3. However, the ED procedure can be quite time consuming and often is performed overnight.
Here, we describe a simple clinical research method to perform ED using a commercially available device followed by sample preparation using solid phase extraction (SPE), and UHPLC-MS/MS analysis using the Waters ACQUITY™ UPLC™ System and Xevo™ TQ Absolute Mass Spectrometer. This has allowed for the complete workflow including ED to be performed within a typical working day.

Methods:
Only 200µL of serum sample was added to the sample chamber of the ED device and 400µL of dialysate buffer was added to the dialysate chamber. ED was performed at 37˚C until equilibrium was reached and then samples were extracted using a simple SPE approach on an Oasis™ MAX µElution™ Plate. Calibrators were prepared directly into dialysate buffer across the ranges of 1.3 – 129pmol/L for FT4 and 1.55 – 154pmol/L for FT3 and were not subjected to the ED process, but extracted by SPE. Samples were injected onto an ACQUITY Premier HSS T3 Column with a VanGuard™ FIT Guard Column and FT4 and FT3 were chromatographically separated using a mobile phase gradient consisting of water, methanol, acetonitrile and formic acid with an injection to injection time of only 3.5 minutes. Detection was performed using the Xevo TQ Absolute Mass Spectrometer and data analysis was simplified using waters_connect™ with QUAN Review Software.

Results:
Within-run and total precision were ≤5.0%CV for the dialysate QC samples and ≤9.6%CV for the serum QC samples across all concentration levels for both FT4 and FT3. The LLoQ for FT4 and FT3 were determined to be 0.97pmol/L and 1.15pmol/L respectively. All calibration lines performed during testing were linear, having a coefficient of determination (r2) of >0.995 and % deviations of within ±15% (±20% for Cal 1) and no significant carryover was observed. Method comparison to the CDC HoSt Phase 1 Program for FT4 gave a Passing-Bablok fit of y = 0.9198x + 1.243 and a Bland Altman bias of 0.070% was obtained.

Conclusion:
A clinical research method has been developed for the UHPLC-MS/MS analysis of free thyroxine (FT4) and free triiodothyronine (FT3) from human serum using a simple equilibrium dialysis and SPE procedure which can be completed within a typical working day.

For Research Use Only. Not for use in diagnostic procedures
ACQUITY, UPLC, Xevo, Oasis, µElution, VanGuard, waters_connect, are trademarks of Waters Technologies Corporation.

Streamlined Analysis of Polar and Non-Polar Metabolites in Plasma Using Automated Micro-SPE LC-MS Guenter Boehm (Presenter) CTC Analytics

Poster #5b View Map

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

INTRODUCTION
The comprehensive profile of the metabolome and lipidome is closely associated with cellular processes and provides insight into biochemical pathway changes. Untargeted analysis of these diverse compounds in biological matrices like plasma requires robust sample preparation. Traditional liquid-liquid extraction (LLE) methods, while common, often suffer from limitations in reproducibility, manual handling, and speed. Here, we present two use cases for streamlined lipid profiling in plasma using automated Micro Solid Phase Extraction (µSPE) on a robotic sample preparation system.

METHODS
All workflows were developed on a PAL System robotic autosampler using NIST (SRM 1950) plasma spiked with an internal standard (Ultimate SPLASH ONE). Two parallel µSPE strategies were explored. The first strategy employed a Mixed-Mode Cation Exchange (MCX) cartridge to fractionate lipids using different solvent mixtures. These fractions were subsequently analyzed by Reversed-Phase LC/MS on a Kinetex EVO C18 column. The second strategy utilized a C18 cartridge to perform a high-recovery collection of the total lipidome via an automated dilution scheme, with subsequent analysis by HILIC LC/MS. For all experiments, a SCIEX ZenoTOF® 7600 mass spectrometer was used, combining Collision-Induced Dissociation (CID) for quantification and Electron Activated Dissociation (EAD) for characterization. Data was processed using SCIEX OS software.

RESULTS
The automated µSPE workflow was successfully implemented for both use cases. The MCX cartridge workflow successfully fractionated lipids into distinct groups, which were then characterized in a single run using the combined CID/EAD method. The C18 cartridge workflow demonstrated an effective automated scheme for high-recovery collection of the total lipidome, with recovery calculated for specific lipid groups like Sphingomyelins. Both approaches offered comparable or superior lipidome coverage to traditional LLE while reducing manual handling and sample consumption. The platform's successful application to NIST SRM 1950 plasma demonstrates its reliability and robustness.

CONCLUSION
This work demonstrates a flexible, automated µSPE-LC-MS/MS platform for comprehensive lipid analysis. The method simplifies sample preparation and increases throughput. The strategic use of different µSPE cartridges showcases the platform's versatility, allowing for either detailed, fraction-based analysis (MCX) or broad, high-recovery screening (C18) within a single automated system. This presents a robust solution for large-scale metabolomic and lipidomic studies.

Assessment of Utility of Cortisol-To-Cortisone Ratio in Detecting Exogenous Cortisol Contamination in Late-Night Salivary Cortisol Samples Jack Wu (Presenter) Mayo Clinic

Poster #6b View Map

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

INTRODUCTION:
Late-night salivary cortisol is routinely used to assess abnormal cortisol production and aid in the diagnosis of Cushing syndrome. While this test is non-invasive and convenient for patient self-collection, it may present with elevated cortisol results due to contamination from topical or oral hydrocortisone use, as well as oral bleeding, as cortisol concentrations are high in blood. False elevation of cortisol from such contamination might lead to unnecessary follow-up testing, increased healthcare burden, and patient anxiety.

The enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), secreted by salivary glands, converts cortisol to cortisone, resulting in lower cortisol levels in saliva when compared with blood, with a typical salivary cortisol-to-cortisone ratio < 1. Previous studies have shown that hydrocortisone and blood contamination only affect salivary cortisol but not cortisone (PMID: 36383173), and an elevated cortisol-to-cortisone level may indicate exogenous cortisol contamination of salivary samples based on findings from a small cohort of 8 patients (PMID: 22377529). However, the statistical correlation between the ratio and sample contamination has not been established.

OBJECTIVE:
We aimed to evaluate the utility of the cortisol-to-cortisone ratio in distinguishing elevated cortisol levels due to contamination from those caused by Cushing syndrome in a cohort of late-night salivary cortisol samples.

METHODS:
We conducted a cross-sectional single center study of patients (n = 5008) with late-night salivary cortisol assessed between January 7, 2014, and November 22, 2023. While only salivary cortisol was reported, all participants also had salivary cortisone measured and available. Salivary cortisol and cortisone were measured with the high-performance liquid chromatography followed by triple quadrupole mass spectrometry (LC-MS/MS). The cortisol-to-cortisone ratios were calculated, and samples with cortisol > 50 ng/dL and a cortisol-to-cortisone ratio > 1.5 were identified. Medical records were reviewed to identify the causes of elevated cortisol levels in those samples.

RESULTS:
Among the 5,008 samples analyzed, 3,441 (68.7%) had cortisol concentrations > 50 ng/dL, and 215 (4.3%) had a cortisol-to-cortisone ratio > 1.5. A total of 111 samples (2.2%) met both criteria. Of these, 82 samples (1.6%) had sufficient clinical information to allow assessment of the underlying cause of cortisol elevation.

Medical record review revealed that 42 of the 82 samples (51.2%) had confirmed contamination from topical or oral hydrocortisone use or oral bleeding. Cortisol concentrations in this group ranged from 131.0 ng/dL to 366,000.0 ng/dL, with a median value of 1,120.0 ng/dL (95% CI, 606.5 to 2,605.0 ng/dL). The cortisol-to-cortisone ratios ranged from 1.55 to 492, with a median of 6.64 (95% CI, 3.75 to 9.30).

In contrast, four of the 82 samples (4.8%) were from patients with a confirmed diagnosis of Cushing syndrome and no indication of contamination. Cortisol concentrations in these samples ranged from 538.0 ng/dL to 1,940.0 ng/dL, with a median of 1,166.5 ng/dL (95% CI, 538.0 to 1,940.0 ng/dL). The cortisol-to-cortisone ratios ranged from 2.20 to 3.75, with a median of 3.14 (95% CI, 2.20 to 3.75).

DISCUSSION:
Among all the late-night salivary cortisol samples with elevated cortisol levels, only a small proportion had elevated cortisol-to-cortisone ratios suggestive of potential exogenous cortisol contamination. Of these only 4 samples were from patients with confirmed Cushing syndrome. The median cortisol concentration did not differ significantly between the confirmed contamination group and the Cushing syndrome group (1,120.0 ng/dL vs. 1,166.5 ng/dL; p = 0.10). In contrast, the median cortisol-to-cortisone ratio was significantly higher in the contaminated samples compared to those from patients with Cushing syndrome (6.64 vs. 3.14; p < 0.05). Our results suggest that the cortisol-to-cortisone ratio may be a useful marker for identifying sample contamination in late-night salivary cortisol assessments.

Lipid Metabolism in Traumatic Brain Injury – Investigating the Effect of Xenon Treatment Elizabeth Want (Presenter) Imperial College London

Poster #7b View Map

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

INTRODUCTION:
Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. The World Health Organization (WHO) estimates that around 5.3 million individuals live with a TBI disability in the USA alone. TBI results from external mechanical forces (e.g., road traffic accidents, blast injuries) resulting in physical, cognitive and psychological impairment. With no current treatments targeting injury development, there is an urgent need for better treatments to aide with a more rapid and complete recovery. The importance of lipid metabolism associated with neuronal membrane integrity is well known, and lipid alterations during and after TBI are being explored. However, the impact of TBI on individual lipid species is yet to be fully characterised. Treatment with xenon gas shortly after TBI has been shown to reduce lesion volumes and attenuate neuronal loss, yet the impact of such xenon treatment on the brain lipid metabolism is unknown. Comprehensively profiling the plasma and brain lipidome using a sensitive, untargeted approach would a) aid biomarker identification and b) improve mechanistic understanding of the role of specific lipids in response to TBI. Here we investigate the plasma and brain lipidomes of TBI subjects using the Xevo MRT in order to maximise lipid coverage.

METHODS:
A rat model study of TBI was utilised, consisting of a naïve cohort and TBI cohort (treated with control gas or xenon gas). Treatment began 30 minutes after injury and continued over a 3 hour period to model the scenario of treatment by first responders. Samples were taken at 3 and 24 hours, in addition to a sham at the same timepoints with control gas only. Lipids were extracted from plasma using a Folch procedure and diluted appropriately for UPLC-MS analysis. Brain samples were extracted following a two-step in-house protocol producing aqueous and organic extracts. Lipids were separated using an ACQUITY™ Premier UPLC™ configured for reversed-phase (RP) chromatography, with a Phenyl-Hex column (2.1 x 100mm). The UPLC system was interfaced with a Xevo MRT mass spectrometer and data collected with both data independent (DIA) and data dependent (DDA) modes of acquisition.

RESULTS:
Lipid data were collected for all samples using the UPLC-MS methods outlined. The column was appropriately conditioned using a study QC (pool of all samples) prior to collecting data for all samples (in a randomized order) as technical triplicates, with QCs injected periodically every eighth injection. The acquired data were processed and database-searched using LipoStar software and configured with an in-house lipid database (constructed from reference standards) for lipid identification. DIA was used to collect data for all samples, whilst DDA was solely used for the study QC to provide additional lipid compound verification. Features with a mass accuracy of sub 1 ppm (precursor and fragment ions) and CV of <20% (based on study QC reproducibility) were used for further multivariate statistical analysis (MVA) to establish differences between groups. The data inherently showed that the lipidome is significantly affected after TBI. Importantly, there were clear differences in the lipidome after treatment with either xenon or control gas. A variety of lipid classes were shown to change in their levels of abundance over time (e.g., ceramides and cholesterol esters). In particular, Cer (36:2) and cholesterol ester (16:0) were decreased after xenon treatment, whilst in contrast PC (22:6) and (36:0) were increased. Ceramides are thought to be altered in TBI due to their association with neuronal bodies and are linked to oxidative stress (as are cholesterol esters) with induced abnormalities. The link with choline-based phospholipids is related to their involvement with maintaining integrity of neuronal glial cell membranes and involved in pathways, such as cholinergic neuronal transmission.

CONCLUSION:
Here we show improved lipid coverage using multi-acquisition strategies for comprehensive lipidomic profiling of a traumatic brain injury cohort, compared with conventional approaches. Specific lipid changes can be linked to TBI and subsequent treatment with xenon gas. This work is still ongoing in terms of lipid identification and extension of coverage to small molecules using a HILIC-MS assay. These efforts enhance our understanding of the brain and plasma lipidome and their alterations due to TBI, as well as providing insights into the impact of xenon gas. We hope this work will ultimately contribute to better treatment for TBI patients and improved quality of life.

Fully Automated Analysis of 165 analytes in Serum / Plasma Using RECIPE® ClinMass® TDM Kit with CLAM-LC/MS/MS System Doriane Toinon (Presenter) Shimadzu Europa

Poster #8b View Map

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

BACKGROUND
For all compounds of clinical use therapeutic drug monitoring (TDM) is recommended to monitor the variability of pharmacokinetics. The RECIPE® ClinMass® TDM Kit System opens a wide range of clinical relevant substances for a patient. Only one column, one pair of mobile phases to cover the most relevant drug classes.

LC-MS/MS has become an essential tool for monitoring drug concentrations but the manual sample preparation doesn’t suited for an effective routine work. Thus, Shimadzu CLAM (Clinical Laboratory Automated sample preparation Module for LCMS) in front of the LC-MS/MS system provides a fully automated sample preparation with a fast and high-precision analytical workflow. The combination of Shimadzu CLAM and LC-MS/MS system to the RECIPE® ClinMass® TDM Kit System achieves to a perfect suitable solution for an effective routine work. In addition, To demonstrate that this automated sample preparation provides reliable and reproducible results with minimal variation a method verification procedure was evaluated according to the CLSI Guidelines EP06-A, EP15-A3, EP17-A2.

METHODS
For the analytical verification, a Shimadzu fully automated sample preparation module for LCMS (CLAM-2040) coupled with a Nexera X3 UHPLC system and a LCMS-8060 triple quadrupole mass spectrometer was used. Human serum samples spiked with 165 widely used 8 antimycotic, 15 antibiotic, 27 antiepileptic, 36 antidepressant, 28 neuroleptic, 35 benzodiazepines, 15 tricyclic antidepressants and mycophenolic acid compounds were subjected to published verification procedures using the ClinMass® TDM Platform (order no. MS9000) in combination with the ClinMass® Add-on Set for Antimycotics in Serum /Plasma (order no. MS9600), the ClinMass® Add-on Set for Antibiotics in Serum / Plasma(order no. MS9700), the ClinMass® Add-on Set for Antiepileptic Drugs in Serum / Plasma (order no. MS9200), the ClinMass® Add-on Set for Antidepressants in Serum / Plasma (order no. MS9400), the ClinMass® Add-on Set for Neuroleptics in Serum / Plasma (order no. MS9300), ClinMass® Add-on Set for Benzodiazepiness in Serum / Plasma (order no. MS9500), ClinMass® Add-on Set for Tricyclic Antidepressants in Serum / Plasma (order no. MS9100) and ClinMass ® Add-on Set for Mycophenolic Acid in Serum /Plasma (order no. MS99100) (RECIPE®,Germany). Lyophilized, matrix-based calibrator and control samples were reconstituted, aliquoted and stored until use. Then the samples were loaded directly into the CLAM-2040. It was programmed to perform protein precipitation using Precipitant P including internal standards from the ClinMass® TDM Kit System followed by filtration and sample collection. The sample is then transported using an arm from the CLAM-2040 to the LC without human intervention, for LC-MS/MS analysis. The throughput of LC/MS/MS analysis was 5.5 minutes for antimycotics, 13 minutes for antibiotics, 6.2 minutes for antiepileptics, 5.0 minutes for antidepressants, 7.5 minutes for neuroleptics, 8 minutes for benzodiazepines, 6 minutes for tricyclic antidepressants, and 4 minutes for mycophenolic acid.

RESULTS
The trueness was determined by two different quality control (QC) samples in a single analysis sequence. The precision in CV% and deviation from the target in % Bias (N=4) of each compound met the CLSI Guidelines: for antimycotics, CV < 7.3% and Bias < 10.5%; for antibiotics, CV < 6.6% and Bias < 14.8%; for antiepileptics, CV < 7.0% and Bias < 12.7%; for antidepressants, CV < 5.5% and Bias < 11.4%; and for neuroleptics, CV < 6.1% and Bias < 14.0%. The intraassay precision (N=8) for each level were low: for antimycotics, CV < 3.6%; for antibiotics, CV < 11.4%; for antiepileptics, CV < 5.1%; for antidepressants, CV < 7.6%; for neuroleptics, CV < 5.5%; for benzodiazepines, CV < 7.6%; for tricyclic antidepressants, CV < 4.3%; and for mycophenolic acid, CV < 2.4%. Several dilutions of the Calibrator Set showed good linearity, including LLOQ, of each compound: for antimycotics, R^2 >0.998; for antibiotics, R^2>0.992; for antiepileptics, R^2>0.991; for antidepressants, R^2>0.991; for neuroleptics, R^2>0.992; for benzodiazepines, R^2>0.992; for tricyclic antidepressants, R^2>0.991; and for mycophenolic acid, R^2>0.998.

CONCLUSIONS
The ClinMass® TDM Kit System for Antimycotics, Antibiotics, Antiepileptic Drugs, Antidepressants, Neuroleptics, benzodiazepines, tricyclic antidepressants and mycophenolic acid in Serum /Plasma were successfully verified on the CLAM-2040 with the analytical system LCMS-8060 from Shimadzu. All 165 analytes passed the acceptance criteria for accuracy (trueness, precision) and linearity.

So, an automated solution for TDM analysis provided by the collaboration between Shimadzu and RECIPE®. Fast, sensitive and reliable solution adapted for routine analysis in clinical laboratories. This solution increase the throughput and reduce the human time consumption.

Metabolic Profiling of Tissues for Biomarker Discovery in Breast Cancer Valdis Gunnarsdottir Thormar (Presenter) University of Iceland

Poster #9b View Map

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

INTRODUCTION:
Breast cancer (BC) is the most prevalent cancer worldwide and the second leading cause of cancer-related deaths in women. Challenges relating to timely detection and the heterogeneous nature of BC complicate treatment strategies and influence patient outcomes. Conventional X-ray mammography screening, commonly used, exhibits limited sensitivity, particularly in young women with dense breast tissue, and lacks specificity for accurate BC diagnosis. Advanced diagnostic techniques that better characterize the disease enable more personalized treatment approaches, which improves prognosis.

OBJECTIVE(S):
This study aims to reveal associations between metabolites, lipids, and BC-related parameters, thereby facilitating the discovery of BC-specific biomarkers.

METHODS:
In this study, we use a metabolic fingerprinting method for analyzing fresh frozen (FF) tissue samples and archived formalin-fixed and paraffin-embedded (FFPE) tissue microarray (TMA) samples, on a well-defined Icelandic BC study cohort. Additionally, targeted and untargeted metabolomics analyses, covering both metabolites and lipids, have been performed on plasma samples from a subset of the same study cohort using LC-MS and NMR. The comprehensive metabolomics profiling of tissue was performed using desorption electrospray ionization mass spectrometry imagining (DESI-MSI) on TMAs from 222 BC patients and 30 normal tissues for diagnostic purposes.

Advanced machine learning techniques for predictive modeling will be applied to the data. By combining the analysis of metabolites and lipids with various BC-related parameters, we aim to explore underlying associations in the data. Both univariate and multivariate methods will be applied, as each provides distinct information about the data structure. Unsupervised algorithms like PCA, t-SNE, and UMAP will be used to visualize and elucidate data patterns, whilst supervised algorithms such as Gradient Descent, SVM, and Random Forest, including ensemble learning techniques, will be deployed to build robust classifiers. Feature selection methods will be implemented to help identify biomarkers and regulate model complexity. To ensure model reliability and generalizability, rigorous cross-validation and data partitioning into training, testing, and validation sets are conducted. Partitioning and cross-validation allow for the robust evaluation of how well the models perform on new, unseen data.

RESULTS:
DESI-MSI has shown the ability to distinguish between tumor and normal breast tissues based on their metabolomics profiles. Additionally, the results showed that despite formalin fixation and paraffin embedding on the tissue, enough information could be obtained to characterize the tissue. To confirm and expand upon these findings, the DESI-MSI data is being pre-processed again according to a new pipeline, effectively increasing the number of detectable metabolites in FFPE BC tissue and lipids in FF BC tissues.

CONCLUSION:
In this ongoing research, we investigate the correlations between BC subtypes, relevant patient characteristics, and their metabolomic profiles, aiming at a holistic view of the biomolecular changes associated with BC. Ultimately, to identify novel biomarkers.

Bioinformatics Pipeline for Targeted Glycosylation Profiling in Intact Protein Mass Spectrometry Zhewei Liang (Presenter) Mayo Clinic

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Poster #10b View Map

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

INTRODUCTION:
Clinical application of mass spectrometry (MS) and proteomics is primarily aimed at detecting and measuring targeted proteolytic peptides associated with the biomarker proteins. Intact protein and top-down proteomics approaches can capture global modifications including sequence variation and post-translational modifications (PTM). With high-resolution MS, intact protein methods can be used to profile relative abundance of various proteoforms associated with proteins of interest. Existing analysis workflows are based on exact mass matches to pre-defined protein sequences and modifications, thus have limited capability in biomarkers with multiple sequence variants or variable modifications. Here we developed a flexible bioinformatic workflow to annotate and profile glycosylation features of alpha-fetoprotein (AFP) based on intact protein measurement. The workflow can establish a proteoform database to annotate glycoforms, calculate their relative abundance, utilize machine learning to select relevant features, and detect differentiating features relevant to patient characteristics.

METHODS:
AFP was first enriched from human serum or plasma samples via immune-enrichment workflow. Chromatographic separation was performed using PLRP 300&Aring; column (Agilent), followed by intact protein analysis on an Exploris 480 Orbitrap (Thermo). Multiple charge envelopes corresponding to chromatographic peaks of AFP were deconvoluted using the ReSpec algorithm in BioPharma FinderTM software (Thermo). The deconvolution was performed with target m/z range between 500-3200 Da. Intact masses, sum peak intensities, retention time, and deconvolution scores were exported.

An initialized glycan residues database was audited based on a small training patient dataset (n=30), and non-viable glycan residues were removed based on the feasibility of the composition. To generate initial AFP proteoforms database, we consider 60 combination of mammalian glycan modification and a variable modification of oxidation (for methionine). The glycan modifications were further reduced to 43 based on structural viability. Then deconvoluted peaks were compared with AFP proteoform database by binary search. There were two rounds for the search with a mass tolerance of 7 Da: the first was based on wild type AFP; if we could not find any match, we continued to do second-round search on AFP with oxidized methionine. Furtherly, an individualized retention time (RT) window was applied to each sample to rule out deconvoluted peaks outside of the set tolerance. For each mass assigned as an AFP proteoform, we calculated minimum mass, maximum mass, mean of mass, range of mass, accuracy of mass mean, and frequency of proteoforms among all examined patient samples.

Consequently, the combinations of features were tested by K-Mean elbow method, which could determine the optimal number of clusters(k). As a result, dimensionality reduction algorithms such as PCA, t-SNE, and UMAP were used for the feature extraction. Additionally, LightGBM (Light Gradient-Boosting Machine) model was explained with SHAP (SHapley Additive exPlanations) values for the feature selection. Finaly, differentiating glycan features were associated with different disease phenotypes.

RESULTS:
Among 242 authentic patient samples, there were 2789 deconvoluted intact mass peaks. After database search, 1242 peaks were annotated, which correspond to 59 AFP proteoforms (40 corresponding to wild type AFP, and 19 to AFP with oxidized methionine). The average deconvoluted mass accuracy observed was -6.28 Da, with a standard deviation of 2.53 Da. We use AFP_5401 to represent the AFP glycoprotein with glycan composition of 5 &ldquo;H&rdquo; (Mannose or Galactose), 4 &ldquo;N&rdquo; (N-Acetylglucosamine), 0 &ldquo;F&rdquo; (Fucose), 1 &ldquo;A&rdquo; (N-Acetylneuraminic Acid, sialic acid); where &ldquo;5401&rdquo; represents the total numbers of &ldquo;HNFA&rdquo;, respectively. Four most identified glycoforms were AFP_5401, AFP_5402, AFP_5411, AFP_5412, with frequency of 55.8%, 72.7%, 68.6%, and 66.5%.

RT window was used to further rule out deconvoluted peaks outside of the set tolerance in each sample. For each annotated AFP proteoforms, we defined confidence score as 100*(1-abs(tm-em)/w), where tm denotes theoretical mass, em denotes experimental mass, and w denotes mass tolerance 7 Da. For each sample, we defined the RT window based on the proteoforms with confidence score above 60, use mean of RTs from these selected proteoforms as a baseline, and a tolerance of 0.3 was set to filter out other proteoforms. After filtering, there were 116 false positive proteoforms filtered across these 1242 peaks. As a result, the total number of remaining annotated proteoforms was 1126. The glycosylation features were extracted, to include the total intensity of peaks corresponding to the identified glycoforms, fractional abundance of AFP_5401, AFP_5402, AFP_5411, AFP_5412, AFP-L3, S1, S2 in each of the samples. Where AFP-L3 represents the glycoproteins contain a single fucose (F), S1 represents the glycoproteins contain a single sialic acid (A), S2 represents the glycoproteins contain 2 A&rsquo;s.

Among all study samples, seven consistent AFP glycoforms were identified, while their relative abundances varied.

CONCLUSION:
The developed algorithm involved establishing a proteoforms database based on training patient dataset and filtering based on RT windows. Feature extraction and feature selection were used to improve model performance by handling redundant or irrelevant features. The results demonstrate that the developed algorithm allows to facilitate data processing to identify protein glycosylation profiles and extract pertinent features for disease association. To the best of our knowledge, our algorithm is the first flexible bioinformatics pipeline to accommodate fit-for purpose proteoforms database and RT filter to annotate PTM of targeted proteins in intact protein MS.

Synthesis of Labeled Antiepileptic Drugs to Be Used as Reference Standards for LC-MS/MS Based Therapeutic Drug Monitoring (TDM) Lars Kattner (Presenter) Endotherm GmbH

Poster #11b View Map

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

INTRODUCTION:
Therapeutic drug monitoring (TDM), or measuring patient blood drug levels, is particularly important for correct dosing and avoidance of overdoses of therapeutics with undesirable side effect profiles, as it is true for antiepileptic drugs. For effective use of methods in practice, LC-MS/MS has been established as a gold standard in diagnostic routine. Reliable results are best achieved by the inclusion of stable isotope-labelled standards.

METHODS:
The synthesis of most antiepileptic drugs is well documented in the literature. However, for TDM, accordingly labelled molecules, in turn labelled multifold by deuterium or, favorably, by 13C are needed. Labelling can be achieved either by careful selection of commercially available labelled starting material, by using isotopically labelled alkylation reagents or by appropriate H/D-exchange reactions.

RESULTS:
Efficient syntheses of 11 examples of isotopically labelled antiepileptic drugs in a chemical purity of >95%, and >98% isotopic enrichment are described. Appropriate labelling is achieved either by using labelled starting material, labelled reducing agents (LiAlD4), labelled alkylation agents (D3CCI) or catalytic H/D-exchange reactions.

CONCLUSION:
Isotopically labeled drugs can by synthesized in high purity and >98% isotopic enrichment by application of established synthesis procedures found in the literature for the synthesis of the corresponding non-labelled drugs, in combination with an appropriate choice of commercially available labelled starting material, substrates, reagents, or catalytic H/D-exchange reactions.

Integrated Charged Absorbing Module Based Reactive Tag Monitoring of Steroid Distribution in Tissues Rachel Pryce (Presenter) Université De Montréal

Poster #12b View Map

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

INTRODUCTION:
Steroid abundance and distribution are critical to many clinically relevant disorders. However, steroids are not easily detected using mass spectrometry, with most methods relying on reactive mass tags to aid steroid desorption and ionization. In MALDI mass spectrometry imaging (MSI), several such mass tags have been proposed, with an attempt to increase the detection of these molecules. In this research, we have developed a platform to rapidly screen potential Integrated Charged Absorbing Modules (ICAMs) to develop mass tags that are uniquely suited for MALDI MSI.

METHODS:
To target ketone steroids, a novel modular reactive tag design was used that contains two major features, a reactive moiety and an ICAM. Adducts were formed by combining these ICAM tags with cholesterol, generating a stable facsimile of the steroid adducts formed from on-tissue reactions. This allowed potential ICAMs to be screened for detectability under both MALDI and LDI MS conditions, with each condition analyzed in triplicate. Selected ICAMs were synthesized into tags, which contain a hydrazine reactive moiety that specifically reacts with ketone functional groups. These tags were then deposited on 12 µm thick rabbit adrenal gland tissue sections using an automated sprayer system and incubated for 1 hour prior to matrix application. Samples were then analysed by MSI on a MALDI TOF mass spectrometer. All experiments were performed in triplicate to determine the best candidates. The identity of the steroids detected was confirmed using exact mass and MS/MS measurements. Preliminary results have also been obtained on human intraductal prostate cancer.

RESULTS:
The adduct screening platform identified several ICAMs with high levels of detectability by LDI and MALDI MS. Certain traits were found to be common to several of these ICAMs, leading to the development of new ICAMs combining several functional groups to enhanced detectability and ultimately the generation of high-performance reactive tags. These ICAM-based mass tags were then compared to Girard’s reagent P (GP), a commonly used mass tag to enhance keto-steroid detection by MALDI MSI. By comparing LODs and intensity measurements, several of these tags were shown to detect keto-steroids with over 10-fold improvements over GP. Optimization of MSI conditions allowed increased reproducibility of these measurements, generating a reliable and sensitive method for keto-steroid detection. The analysis of serial sections of adrenal gland showed consistent localization of specific steroids.

CONCLUSION:
The use of an adduct screening platform for the optimization of ICAMs allowed for a significant reduction in necessary time and resources to develop novel reactive tags that are well-adapted to MALDI MSI. These tags greatly outperform GP for the detection and mapping of ketone steroids in clinical samples.

Integrating Targeted Proteomics and Clinical Data for Early-Stage Breast Cancer Biomarker Discovery in Human Plasma Kristrun Yr Holm (Presenter) University of Iceland

Poster #13b View Map

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

INTRODUCTION:
Breast cancer (BC) is the most prevalent cancer in women and ranks as the second leading cause of cancer-related deaths. Fortunately, the prognosis of BC is good when detected at an early stage, however, sensitive diagnostic tools for early detection of BC are vital for improving survival rates. Blood-based biomarkers may offer an alternative minimally invasive strategy to improve BC screening, with better sensitivity than the routinely used X-ray mammography.

OBJECTIVES:
The objective of this study is to discover protein biomarkers in human plasma samples for early breast cancer diagnosis, with an emphasis on clinical variables.

METHODS:
An absolute quantification of 131 proteins was performed on 270 well-defined Icelandic biobank-based plasma samples, 135 BC patients, and 135 healthy controls by UPLC-MRM-MS assay. This Icelandic study cohort is well-defined with respect to BC subtypes, clinicopathological variables and BRCA germline mutations. Among the BC patients, 33% were from BRAC2 999del5 mutation carriers. The absolute quantification of the proteins was conducted using a PeptiQuantTM protein human kit, which contains synthetic light peptides and matching heavy peptides as an internal standard for each protein. Sample preparation prior to analysis was fully automated using the liquid handling robot coupled with a solid-phase extraction (SPE) unit, where plasma samples were proteolytically cleaved with trypsin, internal standards were added, and the samples concentrated by SPE. Peak area data was generated using Skyline Quantitative Analysis software (v22.2.0.351). Further data analysis was conducted using RStudio (v4.2.2), and SIMCA Pro 17 software was used for statistical analysis, multivariate data analysis, and machine learning.

RESULTS:
The targeted proteomics assay was successfully implemented for the absolute quantification of 131 proteins in human plasma samples with precision and accuracy for calibration standards and quality controls within 20% relative standard deviation. Out of the 131 proteins, 98 were quantifiable in the Icelandic bio-bank plasma samples, surpassing the lower limit of quantification. The samples were analyzed in eight batches, each containing matched pairs of cases and controls. Following data acquisition, the data was normalized, and minimal batch effects were corrected, ensuring accurate comparisons across all samples in downstream analysis. Considering the heterogeneous nature of BC, incorporating BC subtype, BRCA status, and clinicopathological variables such as tumor size, histological grade, and age appear to be important for assessing variations in protein concentrations. We detected several proteins that were significantly upregulated in BC cases, particularly in those with positive nodal metastasis, large tumors, and high histological grade. Other plasma proteins were found to be significantly downregulated in the Luminal B, triple-negative BC, HER-2 subtypes. Notably, two proteins were significantly downregulated in BRCA2 BC cases compared to both paired healthy controls and paired non-carrier BC patients, suggesting their potential as biomarkers for this specific mutation.

CONCLUSIONS:
Targeted proteomics using UPLC-MRM-MS demonstrates the potential for discovering and quantifying protein biomarkers in human plasma that could aid in the early detection of breast cancer, particularly in BRCA2 mutation carriers and subtypes such as Luminal B, triple-negative, and HER-2.

Quantitative LC-MS/MS Determination of Methylmalonic Acid in Human Serum Employing Strata SE SLE for Clinical Research Applications Shahana Huq (Presenter) Phenomenex

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Poster #14b View Map

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

INTRODUCTION:
Methylmalonic acid (MMA) serves as an important indicator of functional vitamin B12 status. Increased concentrations of MMA may reflect impaired intracellular processing of cobalamin and have been linked to neurological and cardiovascular complications. Accurate measurement of MMA in human serum is essential for early identification of metabolic imbalances. However, analytical challenges arise due to MMA’s low abundance, high polarity, and structural resemblance to isobaric compounds such as succinic acid. These factors demand a well-optimized extraction and detection strategy using a sensitive and selective LC-MS/MS approach for routine laboratory use.

OBJECTIVE:
To develop a streamlined LC-MS/MS method for quantifying MMA in human serum, employing a supported liquid extraction (SLE) strategy using Strata™ SE SLE, optimized for clinical research workflows.

METHOD:
A 200 µL aliquot of human serum was mixed with an equal volume of 4.5 M aqueous formic acid and applied to a Strata™ SE SLE MAX 96-well plate. A brief vacuum pulse was used to initiate flow, followed by a 5-minute equilibration period to allow complete absorption of the sample into the SLE sorbent. MMA was extracted using two sequential 900 µL aliquots of ethyl acetate/hexane (9:1, v/v), with a brief vacuum pulse applied after each addition. The combined organic eluates were evaporated to dryness under a gentle stream of nitrogen and reconstituted in a mobile phase-compatible solvent. Chromatographic separation was performed on a thermally modified, polar-embedded Luna Omega PS C18 column (3.0 × 150 mm, 3 µm particle size) exhibiting a positive charge on the surface. The mobile phase consisted of 0.5% formic acid in water (A) and acetonitrile (B), using a linear gradient from 85% to 55% B at a flow rate of 0.5 mL/min. The total analysis time, including column re-equilibration, was 5 minutes. Detection was conducted using a SCIEX 6500 triple quadrupole mass spectrometer operating in negative electrospray ionization (ESI) mode.

RESULTS:
The positively charged Luna Omega PS C18 column, when operated under hydrophilic interaction chromatography (HILIC)-like conditions, enabled baseline separation of methylmalonic acid (MMA) from succinic acid, with a retention time difference of 0.7 minutes. The distinct polar-embedded stationary phase chemistry contributed to enhanced selectivity and significantly reduced matrix interferences in extracted serum samples, resulting in a cleaner chromatographic baseline compared to conventional reversed-phase columns. A calibration curve spanning a 1000-fold dynamic range (1ng/mL – 1000 ng/mL) was established using extracted serum matrix, exhibiting excellent linearity (r² = 0.998) with 1/x weighting. At the lower limit of detection (LOD), recovery, process efficiency, and matrix effect were determined to be 103%, 79%, and 80%, respectively, supporting the method’s robustness for accurate
quantitation.

CONCLUSION:
This study describes a robust and highly selective LC-MS/MS method for the quantification of methylmalonic acid in human serum, specifically optimized for clinical research applications. The use of Strata™ SE SLE MAX 96-well plates facilitated efficient matrix cleanup while preserving analyte recovery and ensuring streamlined sample preparation. Chromatographic separation using a polar-embedded, positively charged Luna Omega PS C18 column under HILIC-like conditions enabled baseline resolution of MMA from the structurally similar succinic acid, a critical factor for analytical specificity. The method demonstrated excellent linearity across an extended dynamic range and showed strong sensitivity at low concentrations. Collectively, these results support the method’s suitability for high-throughput clinical research studies aimed at evaluating functional vitamin B12 status with precision and reliability.

Comprehensive 2D-LC-MS/MS Assay for High-Sensitivity Profiling of Classical and 11-Oxygenated Steroid Hormones in Serum Henrik Ryberg (Presenter) Sahlgrenska University Hospital

Poster #15b View Map

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

INTRODUCTION:
Recent research suggests that the adrenal glands produce significant amounts of 11-oxygenated androgens that can further be converted into bioactive 11-ketoandrostendione, 11-ketotestosterone and 11-ketodihydrotestosterone. Although these keto-androgens are typically not produced in clinically relevant concentrations when gonadal androgen production is high, they may play a significant role in women, children, and in conditions characterized by low gonadal output or during anti-androgen therapy. This underscores the need for validated, sensitive steroid assays suitable for both clinical routine and research applications.

OBJECTIVE:
To develop a sensitive and robust LC-MS/MS assay capable of quantifying a comprehensive panel of androgens (including classical and keto-variants), estrogens, and their precursors in serum/plasma down to low picomolar levels, with performance parameters suitable for clinical implementation.

METHODS:
Steroid hormones were quantified using a two-dimensional liquid chromatography system coupled to a Waters TQ-XS triple quadrupole mass spectrometer (2D-LC-MS/MS). Following initial fractionation on a reversed-phase C8 column, steroid-containing fractions were trapped and subsequently resolved on a biphenyl column under gradient elution. This high-sensitivity method enables precise steroid profiling from 50-250 µL serum samples, optimized for targeted quantification via multiple reaction monitoring. Our initial tests showed using this 2D-LC-MS/MS setup it would be possible to create an assay including 12 different steroid hormones relevant for clinical routine and research: estrone (E1), estradiol (E2), androstenedione (A), dehydroepiandrostenedione (DHEA), progesterone (P), 17-alphahydroxyprogesterone (17OHP), testosterone (T), dihydrotestosterone (DHT), 11β-hydroxyandrostenedione (11OHA,) 11-ketoandrostenedione (KA), 11-ketotestosterone (KT) and 11-keto-DHT (KDHT). Among the parameters evaluated during assay validation were analytical sensitivity, accuracy, precision, linearity, and matrix effects. The assay was benchmarked in a three-center round robin study involving laboratories in Norway and Finland.

RESULTS:
Internals standards containing 13C-labelled steroids were spiked to samples and steroid hormones were extracted on supported liquid extraction 96-well plates (Biotage) using heptane/MTBE. The exact conditions were optimized, and 11-oxygenated androgens needed more polar conditions compared to the classical sex steroids to be extracted. Recoveries were generally >80%, however recoveries for KT were around 75% for the matrices tested. Calibration curves were prepared in PBS/0.5% BSA. Chromatographic separation was performed using methanol gradient to transfer steroids from the C8 phase to the analytical column, there a methanol gradient at 40 °C containing ammonium fluoride was used. One challenge during method development was to find the optimal conditions allowing the 11-oxygenated steroids to pass from the C8 column to the analytical column, but simultaneously having good sensitivities for E2, DHT and DHEA. Lower limits of quantifications (LLOQ) for the final assay were between 0.5-25 pg/mL (2-80 pmol/L) except for DHEA which had a LLOQ at 280 pg/mL. The precisions for the assay were evaluated at two different levels and were between 1-11%. The round robin study demonstrated strong inter-laboratory agreement. KDHT was not seen in normal human serum or plasma.

DISCUSSION:
We have successfully developed and validated a high-sensitivity 2D-LC-MS/MS assay for profiling 12 steroid hormones, including four 11-oxygenated androgens. The assay’s low LLOQs and high precision render it well suited for clinical research, and it is currently being applied to large cohort studies. Based on these findings, inclusion of KT as a laboratory-developed test under the EU CE/IVDR legislation in routine clinical diagnostics is planned.

A Scalable and Accessible Workflow for Metabolic Flux Analysis in Human Cells Anna Weiser (Presenter) ETH Zurich

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Poster #16b View Map

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

INTRODUCTION:
Metabolic dysregulation is a hallmark of diseases including cancer, cardiovascular disease, and diabetes. A quantitative understanding of how metabolism is altered in these conditions is critical for therapeutic development. Metabolic flux analysis, which examines the rate at which metabolites flow through biochemical pathways, offers deep insights into metabolic activity and regulation. Stable isotope tracing experiments remain the most optimal method for dynamic measurement of metabolic processes. When combined with holistic computational models, they also offer a powerful tool for the prediction and validation of clinical interventions. In order to guide co-clinical applications robust and easy to use solutions are urgently needed.

OBJECTIVES:
Here we aim to create an easy-to-use toolkit for quantitative 13C metabolic flux analysis in human cells, for use by clinicians and researchers investigating metabolic activity, drug action, and potential resistance mechanisms. By lowering the technical barrier to flux analysis, we aim to provide a practical tool for interpreting metabolic dynamics beyond metabolite abundance. To this end, we have developed a comprehensive and user-friendly workflow that integrates liquid chromatography-mass spectrometry and stable isotope tracer analysis with machine learning-driven flux inference using Bayesian neural networks. Currently the workflow supports the design of isotope tracer experiments, exploratory data analysis, and probabilistic flux inference, with a focus on key flux ratios in human central carbon metabolism.

METHODS:
Cell Culture and Isotope Labeling:
Human cancer cell lines including adult leukemia (HAP1), pediatric osteosarcoma (HOS), and neuroblastoma (SKNBE2), were cultured under standard conditions and treated with three targeted metabolic inhibitors: (1) 2-deoxyglucose (2DG): glycolysis inhibitor; (2) 6-aminonicotinamide (6AN): oxidative pentose phosphate pathway inhibitor (PPP); (3) CB-839 (CB): glutaminase inhibitor (glutaminolysis). Cells were incubated with the stable isotope-labeled tracer [1,2-13C]-glucose to focus on glycolysis and PPP, and prepared for LC-MS analysis using a simple solvent extraction and centrifugation to obtain a final extract of approximately 250k cells in 300 µL 1:4 (v/v) water/acetonitrile.

Metabolite Profiling:
We developed a semi-targeted high-resolution HILIC-LC-MS method specifically focused on central carbon metabolites, enabling sufficient separation within a timeframe appropriate for clinical translation. Calibration curves were obtained using non-enriched QC samples, enabling quantification in the presence of sample matrix in the approximate range of 5 nM - 0.1 mM of extracted sample for most compounds. As full scan acquisitions on Orbitrap instruments often have observably biased isotope ratios, semi-targeted MS1 acquisition windows were incorporated for more accurate quantification of the relevant compounds. Data were preprocessed and analyzed in R prior to downstream analysis and modelling.

Flux Inference Framework:
Fluxes were inferred using a simulation-based approach that couples 13C-metabolic flux analysis with Bayesian neural networks in Python. The inference workflow consists of: (1) atom-resolved metabolic network modeling (EMU-based), (2) simulation of expected isotopologue patterns for candidate flux distributions, (3) training of a Bayesian neural network to map observed data to posterior flux ratios, incorporating uncertainty.
This architecture enables rapid, interpretable, and probabilistically grounded flux inference across different flux ratios.

RESULTS:
To optimize tracer selection for flux inference, Bayesian neural networks were trained on synthetic datasets simulating isotope labeling patterns under different tracer conditions. This yielded design-of-experiment maps that guide users in selecting the most informative tracers for specific regions of metabolism. For example, [1,2-13C]-glucose was optimal for resolving fluxes between glycolysis and the oxidative pentose phosphate pathway, while [U-13C]-pyruvate and [3-13C]-glutamine provided complementary information on anaplerotic versus oxidative TCA cycle activity. The selection focused on five flux ratios covering upper glycolysis, PPP, pyruvate utilization regarding TCA cycle, serine biosynthesis, and glutaminolysis, each chosen to inform on flux partitioning in central carbon metabolism.

For biological validation, our workflow was applied to the three cancer cell lines described above using [1,2-13C]-glucose to specifically probe the flux split between glycolysis and PPP. Key findings include: (1) 6AN treatment significantly increased glycolytic flux relative to PPP in HAP1 and HOS, indicating compensatory rerouting under PPP inhibition. (2) SKNBE2 displayed a non-significant trend in the same direction. (3) 2DG, unexpectedly, increased glycolytic flux relative to PPP in HOS and SKNBE2. (4) CB-839 consistently increased relative PPP flux across all cell lines, potentially reflecting enhanced NADPH production under glutaminolysis-inhibition.

CONCLUSION:
Our workflow offers a streamlined approach for integrating isotope tracing, high-resolution metabolomics, and Bayesian neural networks to infer pathway activity. The initial application to three cancer cell lines illustrates the potential of this approach to detect drug-induced metabolic rewiring and to capture context-specific metabolic adaptations. While the method shows promise for accessible and interpretable flux analysis, further validation with additional tracers and experimental systems is needed. These early results support its future use in mechanistic studies, biomarker exploration, and therapeutic profiling. With continued development, this workflow may contribute to broader adoption of metabolic flux analysis in biomedical research.

Standardization and Harmonization of Clinical Measurements: A Case Study in Phenylketonuria Monitoring Chloe Deaves (Presenter) National Measurement Laboratory at LGC

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Poster #17b View Map

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

INTRODUCTION:
Certified reference materials (CRMs) are crucial for ensuring method validation and underpinning the reliability of clinical measurements. Their analysis enables a laboratory’s methods to be standardised and their performance to be benchmarked against other measurement providers. However, in many clinical areas, commercially available matrix CRMs are limited. This is the case for the monitoring of phenylketonuria (PKU), an inherited metabolic disease that causes raised levels of phenylalanine in blood. If left untreated, this condition can lead to severe health repercussions such as irreversible brain damage. PKU patients manage their condition by severely restricting dietary phenylalanine intake, which is monitored against target decision limits through regular phenylalanine blood concentration measurements. However, the lack of a matrix-matched amino acid CRM with SI traceable assigned values, impedes the reliable comparability of hospital results obtained over a period of time. Additionally, the quality of this service is known to be inconsistent between providers, with between laboratory variability estimated to be greater than 35%, highlighting the need for measurement harmonisation and standardisation.

METHODS:
The National Measurement Laboratory (NML), hosted by LGC, collaborated with Synnovis at Guy’s and St Thomas’ Hospital to produce four traceable value assigned whole blood materials, to be used in a three-part inter-laboratory study in the absence of suitable CRMs. The aim of the study was to improve quality and comparability of phenylalanine measurements for PKU monitoring across the UK by comparison of results to traceable value assigned materials, with proven stability for the course of the project. The first part of the study assessed the variability in measurements when laboratories (n = 15) used their individual routine clinical methods. In the second part, the effect of using a harmonised method on the main source of variance in the results, as between laboratory variability (n = 5), was investigated. Furthermore, three routine calibration approaches were applied to the data and compared. Finally, the accuracy of the harmonised method and most promising calibration approach was assessed against traceable materials value assigned by the NML.

The amount of phenylalanine in each of the materials was assigned fully traceable to the International System of Units (SI) with an expanded measurement uncertainty below 2%. In order to value assign the materials for phenylalanine mass fraction with the required low associated uncertainty, a double exact matching isotope dilution gas chromatography tandem mass spectrometry (DEM-ID-GC-MS/MS) methodology was used. The use of GC-MS/MS was a key factor in achieving a lower total measurement uncertainty than typically achievable, which increased the reliability and strength of conclusions drawn through evaluation against the materials. GC-MS/MS provided enhanced analyte selectivity in comparison with other mass spectrometry systems, which decreased chromatographic interferences in the natural and labelled channels. This allowed for excellent peak area ratio precision, which is the main contributor to the total measurement uncertainty associated with traceable value assignment.

RESULTS:
The main source of variability using routine methods was the between-laboratory variation. All the laboratories used a similar analytical method, however the specifics of these varied considerably between participants. One of the key parameters that differed was how measurements were calibrated, with three general calibration approaches typically being used. For this reason, the second study aimed to assess how these three approaches affected the measurement variability when a common measurement approach was used. This demonstrated that the agreement between results improved by at least 85% when a harmonised method, including isotopically labelled internal standards, was implemented in combination with external calibration. Therefore, in the final study, this calibration strategy was used alongside the common analytical method for measuring the materials produced by the NML. This final evaluation demonstrated the accuracy of measurements, through comparison with a traceable value assigned matrix material, and showed agreement between every participant’s result and the assigned value at the 95% confidence interval, k = 2.

CONCLUSION:
A harmonised analysis method for PKU monitoring measurements was developed and evaluated using a standardised calibration approach. This significantly improved agreement between laboratories when compared to the varied methods which are routinely used. Supplying traceable value assigned whole blood materials as part of an inter-laboratory study underpinned PKU measurements to a true value. This allowed laboratories to conclusively determine the accuracy of the implemented method and benchmark their performance against other measurement providers, without the additional complexity required for CRM production.

REFERENCES:
1. Carling, R., et al., Investigation of the relationship between phenylalanine in venous plasma and capillary blood using volumetric blood collection devices. Clin Chem Lab Med, 2023. 64(6): p. 468-476.

Quantification of Cannabinoid Metabolites in Urine Samples from First-Responders Who Self-report CBD Use Samuel Krug (Presenter) Dartmouth Hitchcock Medical Center

Poster #18b View Map

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

INTRODUCTION:
Ambiguity between federal and state legislation has led to extensive commercialization of cannabinoid-related products. Cannabidiol (CBD) containing products are gaining popularity as a homeopathic remedy for stress and pain relief. Unregulated production of CBD products can be particularly precarious for individuals who have jobs requiring random drug screening. A number of studies have shown CBD containing products can have inaccurate labels depicting the concentration and presence of &Delta;9-tetrahydrocannabinol (THC). THC and CBD have documented cross-reactivity in a number of routinely used screening assays. This can result in legal disputes between the consumer and manufacturer, depending on how results are reported. There are limited studies that examine the use of CBD products, specifically by first-responders, and subsequent toxicological analysis that may result in job termination on the basis of zero-tolerance workplace policies.

OBJECTIVES:
The primary objective was to analyze urine samples by routine toxicology immunochemistry-based screening and LC-MS/MS confirmation workflows. Cross-reactivity for screening techniques and quantification of THC, THC-COOH, CBD, and CBD-COOH was reported. Secondary objectives determined whether urine samples contained any other identifiable drug metabolites present by LC-HRMS, and the impact of forced heat or acid exposure on the quantification of urine cannabinoid metabolites.

METHODS:
A cohort of ~20 firefighters who regularly use CBD products were surveyed for frequency and quantity of use. Volunteers were sent at-home urine collection kits and refrigerated at (4oC) until shipment for sample analysis. Samples were then split and sent for analysis by two independent labs for screening and confirmatory testing. Lab 1 used Bio-Rad immunochromatographic testing (Bio-Rad Cat #194-5187) followed by LC-MS/MS confirmation using a Waters I-Class LC coupled to a Waters TQ-XS. Lab 2 used ELISA (Cayman Chemical Part No. 701570) followed by LC-MS/MS confirmation using an Agilent 1290 LC coupled to an Agilent 6495 mass spectrometer. Both labs have a reportable range of 0.5-250 ng/mL for quantified urine cannabinoids. For forced degradation studies, urine samples incubated at 37oC overnight (~12 hours), acidified with 4% phosphoric acid immediately before extraction, or acidified before overnight incubation. Statistical differences were determined using a pair-wise t-tests between the original samples and the stressor conditions. Drug screening was performed using a 10mM borax buffer (pH 10.4) liquid-liquid extraction in 70:30/N-butyl chloride:ethyl acetate followed by analysis on a Waters H-Class LC coupled with a Waters G2S Synapt.

RESULTS:
BioRad immunochromatographic analysis for THC-COOH identified 11 positive samples and 11 negative samples. ELISA results of these samples identified 8 urine samples as positive and 14 as negative. LC-MS/MS results for urine cannabinoids only showed 1 sample as positive for THC-COOH (1.9 ng/mL), which also was positive during both immunochromatographic and ELISA screening. Three samples had no detectable cannabinoids that tested negative for both screening tests. CBD concentrations ranged from 0.6-86.6 ng/mL, while CBD-COOH ranged from 0.5-457 ng/mL. Degradation studies showed a significant decrease in concentration in CBD when urine was acidified under heat. No significant findings were found in the general drug screening by LC-HRMS.

CONCLUSION:
Urine samples analyzed in duplicate showed only one sample that tested positive during confirmatory testing for THC-COOH despite a large number of positives by screening. This study further demonstrates confirmatory LC-MS/MS testing is necessary for workplace drug testing. Degradation studies show there is an impact of heat and acid on the quantification of cannabinoids in urine. Further studies, such as a ring trial, would benefit the community to understand how changes in analysis parameters impact cannabinoid quantitation.

ENRICHplus Enables Enhanced Plasma Proteome Coverage While Preserving Quantitative Accuracy Jessica Wohlfahrt (Presenter) PreOmics

Poster #19b View Map

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

INTRODUCTION:
Blood is a valuable biofluid for biomarker discovery, containing a comprehensive proteome, including cytokines and tissue leakage proteins that reflect disease states. However, mass spectrometry (MS)-based plasma proteomics faces challenges due to the vast dynamic range of protein concentrations and the strong suppression effects from high-abundance proteins, masking low-abundance protein detection. Although enrichment-based strategies improve proteome coverage, they often disrupt original protein concentrations, requiring linear quantitation for accurate relative quantification. ENRICHplus, a paramagnetic bead-based enrichment method, compresses the dynamic range while maintaining quantitative accuracy. Here, a Controlled Quantitative Experiment (CQE) was conducted to assess the performance of ENRICHplus in terms of proteome coverage, reproducibility, and quantitative accuracy, compared to a neat plasma workflow.

METHODS:
To evaluate ENRICHplus, a multi-species proteome spike-in experiment was performed using five different ratios of bovine and human plasma (1:0, 1:9, 1:1, 9:1, 0:1). For ENRICHplus samples, protein enrichment, lysis, reduction, and alkylation steps were automated up to digestion on the KingFisher™ system (Thermo Scientific™). The neat plasma workflow was performed manually. All samples were analyzed using nanoElute® 2 coupled to timsTOF HT in dia-PASEF® mode. Raw data were processed with directDIA™ in Spectronaut® 19 (Biognosys).

RESULTS:
Our results demonstrate that ENRICHplus significantly compressed the dynamic range of plasma protein concentrations, resulting in a sevenfold increase in proteome coverage for human plasma compared to the neat workflow. Evaluation using Controlled Quantitative Experiment (CQE) metrics confirmed that ENRICHplus maintains robust quantitative accuracy, with observed protein fold changes closely matching those from neat plasma. Notably, the workflow exhibited high reproducibility (coefficient of variation ~13%) despite the inclusion of an enrichment step.
ENRICHplus demonstrated exceptional linearity in protein quantification, consistently outperforming the neat plasma workflow, with Pearson correlation coefficients ≥0.9. Across all spike-in ratios, the ENRICHplus maintained high linearity, underscoring its reliability for accurate relative quantification in complex plasma samples.

CONCLUSION:
These findings establish ENRICHplus as a powerful tool for plasma proteomics. It enables superior plasma proteome coverage through dynamic range compression while preserving fold-change accuracy and high linearity in protein quantification, which are essential for the discovery of clinically relevant biomarkers, where low-abundance proteins are often of great interest.

Distinct IgG Fc Glycosylation Profiles Associated with COVID-19 Vaccine Response in Patients with End-Stage Renal Disease I-Lin Tsai (Presenter) Taipei Medical University

Poster #20b View Map

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

BACKGROUND:
Patients with end-stage renal disease (ESRD) exhibit impaired immune responses due to uremia-induced dysfunction and are particularly vulnerable to severe COVID-19. Although vaccination is critical for protection, its effectiveness in this population remains variable. Immunoglobulin G (IgG) Fc glycosylation has been implicated in modulating antibody effector functions and may influence vaccine responsiveness.

METHODS:
This study analyzed Fc glycosylation patterns of anti–receptor binding domain (RBD) IgG in 46 vaccinated ESRD patients. Based on prior anti-RBD antibody measurements, participants were classified as vaccine responders (n = 23) or non-responders (n = 23). Seventeen IgG glycoforms were quantified and compared between groups. Additionally, antibody-dependent cellular cytotoxicity (ADCC) activity was assessed to evaluate functional relevance.

RESULTS:
Significant differences in glycosylation were observed between responders and non-responders. Responders showed lower IgG1 fucosylation and higher levels of bisection, monogalactosylation, digalactosylation, and sialylation. Alterations in IgG3/4 bisection were also noted. Several glycoforms correlated with ADCC activity, suggesting their role in enhancing effector responses to vaccination.

CONCLUSION:
Distinct IgG Fc glycosylation patterns are associated with differential COVID-19 vaccine responses in ESRD patients. These glycoform features may serve as biomarkers of vaccine-induced immunity and offer insights into personalized vaccination strategies for immunocompromised populations.

Quantification of Underivatized Acylcarnitines and Carnitine Intermediates Using RP Chromatography and Ion Funnel Triple Quadrupole Line D'Astous (Presenter) Agilent

Poster #21b View Map

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

INTRODUCTION:
Acylcarnitine profile analysis is a commonly requested analysis in genetic disease research laboratories due to their wide metabolic involvement. Acylcarnitines detection can be important markers for numerous disorders as they are intermediates of fatty acid oxidation and amino acids metabolism in tissues and body fluids. Currently, acylcarnitines are often tested using flow injection analysis, which compromises their quantification because various species occur as isomers and/or have very low concentrations. To address many issues inherent to such analysis, a new comprehensive LC-MS/MS method was developed to quantify 32 acylcarnitine species, with acyl-chain lengths from C0 to C20 in plasma samples without derivatization step.

METHODS:
Acylcarnitines were extracted using acidified isopropanol (0.3% formic acid) to precipitate proteins. After mixing and centrifuging, samples were diluted and analyzed on an ion funnel triple quadrupole mass spectrometer coupled to a biocompatible LC system, which is coated with an iron free alloy. Optimal source conditions and MRM transitions for all compounds were determined individually using commercial optimization software. Robust chromatography separation for the main isomers compounds was achieved without ion pairing reagents. A gradient elution was obtained using a solution of methanol:water (95:5) (B) and water (A) both phases containing 15mM of Ammonium acetate and 0.3% formic acid. Absolute quantification was obtained using commercial labeled compounds spiked into samples. Relative response calibration curves were made for quantitative analysis of each analyte.

RESULTS:
The method achieved the desired separations of the main isomers, such as: (C5) isovaleryl, valeryl and 2-methylbutyryl-L-carnitine; (C4) butytryl and isobutytryl-L-carnitine and (C6DC) 3-methylglutaryl and Adipoyl-L-carnitine without ion pairing additives, which can cause ion suppression. Method reproducibility showed RSD < 10%. Limits of quantification ranged from 0.01 to 1 ng/L for all metabolites. All the compounds showed a great linearity (R2 > 0.994) and RSD below 12%.

CONCLUSIONS:
This separation used a standardized configuration intended for discovery analysis and workflow to allow flexibility for different studies. The chromatography was reproducible, and the figures of merit evaluated for each analyte were linearity, detection and quantification limits, recovery, and precision. Simple sample preparation was obtained avoiding any derivatization. Protein precipitation is well known for its efficiency in extracting metabolites from plasma and reduces processing time. This data shows the method is capable of quantifying acylcarnitines and carnitines intermediates at levels relevant to plasma for monitoring metabolic alterations. Compared with literature and previous data, the ion funnel LC/TQ contributes to the analyte sensitivity and reproducibility. The method developed on the standardized system can be easily transferred and applied to perform different experiments from pathway discovery metabolomics to absolute quantitation.

NOVEL ASPECT:
New sensitive method developed to quantify acylcarnitines using a wide acyl-chain length without derivatization in a standardized metabolomics system.

Protein Corona Formation and Cellular Effects of Multi-Walled Carbon Nanotubes Based on Pre-Coated Bovine Serum Albumin Concentration Jin Gyeong Son (Presenter) KRISS

Poster #22b View Map

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

INTRODUCTION:
The formation of a protein corona (PC) on nanomaterials upon exposure to biological environments significantly influences their cellular interactions and biocompatibility. Multi-walled carbon nanotubes (MWCNTs) have been widely studied for biomedical applications; however, their cellular effects vary depending on surface modifications. In this study, we investigate how pre-coating MWCNTs with different concentrations of bovine serum albumin (BSA) affects PC composition and cellular responses in A549 lung epithelial cells.

OBJECTIVES:
This study aims to (i) determine how BSA pre-coating concentration influences PC composition on MWCNTs, (ii) analyze secondary structural changes in adsorbed proteins, and (iii) evaluate the impact of these modifications on cellular uptake and intracellular signaling pathways.

METHODS:
MWCNTs were pre-incubated with varying concentrations of BSA (0, 1, 5, and 10 mg/mL) to form stabilized PC layers. Spectroscopic (circular dichroism, Fourier-transform infrared) and mass spectrometric (nanoLC-ESI-MS/MS) analyses were performed to characterize protein structures and compositions. Cellular uptake was quantified using optical absorption measurements, and proteomic analysis was conducted to assess intracellular responses in A549 cells.

RESULTS:
BSA pre-coating influenced both PC composition and secondary protein structures. Higher BSA concentrations (≥5 mg/mL) led to increased α-helix content and reduced cellular uptake of MWCNTs. Proteomic analysis revealed differential regulation of ribosomal and oxidative phosphorylation pathways at low BSA concentrations, whereas higher concentrations primarily affected mRNA surveillance pathways. These findings suggest that the protein corona composition dictates cellular interactions more than the intrinsic properties of MWCNTs.

CONCLUSION:
Pre-coating MWCNTs with BSA alters protein corona formation and downstream cellular responses in a concentration-dependent manner. Our results highlight the importance of surface modifications in nanomaterial biocompatibility and provide insights for designing safer nanocarrier systems.

Targeted LC-MRM Method to Monitor Biomarkers of Liver Metabolic Activity in a Rat Model Kahina Chabi (Presenter) Université du Québec à Montréal

Poster #23b View Map

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

INTRODUCTION:
Liver disease progresses silently and is often diagnosed and monitored with clinical biomarkers including alanine aminotransferase (ALT) and aspartate aminotransferase (AST); enzymes released during hepatocyte cell death. However, these biomarkers not only lack specificity and sensitivity, but they do not reflect real-time liver metabolic capacity. A minimally invasive liver test to monitor function, progression as well as to adjust treatments remains an unmet clinical need. Acetaminophen (APAP), a widely used analgesic, is predominantly metabolized in the liver through three main pathways: glucuronidation, sulfation, and cytochrome P450–mediated oxidation into NAPQI, a toxic intermediate subsequently conjugated with glutathione and eliminated via the mercapturic acid pathway. The goal of this study is to develop a method to quantitatively measure circulating APAP metabolites following a low-dose administration in a rat model.

METHODS:
We developed a targeted LC-MRM method, on a Sciex QTRAP 5500 platform, to quantify six key APAP-derived metabolites, from glucuronidation, sulfation, oxidation, and conjugation with glutathione. Chromatographic separation was optimized using a ZORBAX Eclipse Plus C18 column with a gradient elution program. APAP was administered orally at 25 and 50 mg/kg to naïve male and female rats (~225 g) and blood samples were longitudinally collected. Serum proteins were precipitated using acetonitrile, and supernatants were dried under vacuum, reconstituted in aqueous solution, and analyzed by LC-MRM. Method performance (sensitivity, reproducibility, linearity) is being evaluated using both matrix-matched calibration curves and using stable-isotope-labeled internal standards.

PRELIMINARY RESULTS:
The developed method enables robust detection and quantification of six APAP metabolites in rat serum. The MRM transitions were optimized to ensure high selectivity and sensitivity, and the chromatographic conditions resulted in excellent peak shape and retention time reproducibility. Peak serum concentrations of metabolites were observed at approximately 30 minutes post-dose, suggesting this timepoint as optimal for capturing metabolic activity. The lower dose tested (25 mg/kg) was sufficient to produce a measurable metabolic profile, supporting its use in future low-dose longitudinal or clinical studies. Sex-based differences were also observed: female rats displayed a delayed clearance of certain conjugated metabolites, notably the glucuronide and cysteine derivatives, compared to their male counterparts. This trend, which suggests possible sex-specific differences in phase II conjugation or renal excretion, requires to be further investigated. These findings underscore the importance of including both sexes in preclinical biomarker development. The method is being applied to rats of a model of cholestatic liver injury, to assess the impact on APAP metabolism. Ultimately, metabolite ratios will be explored as composite biomarkers of liver enzymatic capacity and redox status.

CONCLUSION and PERSPECTIVES:
We present a robust and reproducible LC-MS/MS method to monitor low-dose APAP metabolism in rats as a potential tool for assessing liver health status in a rat model. Preliminary data in SHAM-operated animals confirm the method’s sensitivity and reveal metabolic differences between sexes. The use of low-dose APAP was adequate to capture relevant metabolic profiles, supporting its applicability in minimally invasive and translational contexts. This platform holds promise for the development of a functional liver biomarker strategy.

The Growing Utility of Salivary Testing in Routine Clinical Practice Brian Keevil (Presenter) University Hospital of South Manchester

Poster #24b View Map

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

BACKGROUND:
The adrenal gland secretes multiple hormones including cortisol, which is essential for survival and affects multiple organs. Abnormalities of cortisol secretion include over-secretion (hypercortisolism) or under-secretion (hypocortisolism) with a myriad of clinical presentations for both conditions.

This makes the diagnosis of adrenal disorders clinically challenging and expensive because of the need for the patient to attend hospital for testing. Cortisol dysfunction is therefore included in the differential diagnosis of many common presentations and can be assessed on its own (static testing) or as part of a stimulation or suppression test (dynamic function testing). In reality, several tests are used together to assess different aspects of the hypophyseal pituitary adrenal axis. Stimulation tests are used to assess adrenal reserve (hypocortisolism) and are designed to predict the ability of a patient to survive trauma. Historically, the main cause of adrenal insufficiency was tuberculosis or autoimmune disease; however, in the develop world use of glucocorticoid medication now predominates. Suppression tests are used to assess overt hypercortisolism (Cushing syndrome) but also mild autonomous cortisol excretion (MACE) by incidentally detected adrenal tumours. MACE is now recognised as a significant cause of cardiovascular morbidity

SUMMARY:
Serum, saliva and urine are used for static testing whilst serum samples are used for dynamic function testing. The requirement for serum samples ensures that patients must attend the hospital, which is expensive and inconvenient for the patient. Alternative strategies are therefore required to safeguard patients, improve the patient journey and also reduce costs. This presentation will focus on the strengths and weaknesses of current testing regimes and propose saliva testing as a good alternative to dynamic function testing that can be performed in the patient’s home. This approach is gaining traction in the UK and has been adopted in one major centre already.

Dolutegravir in Plasma and Dried Blood Spots (DBS) as an Adherence Monitoring Technique Amanda Schauer (Presenter) University of North Carolina-Chapel Hill

Poster #25b View Map

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

INTRODUCTION:
Dolutegravir (DTG, an integrase strand transfer inhibitor (INSTI)) is a commonly prescribed drug for HIV treatment due to its limited side effects, potency and co-formulation with other antiretrovirals. Understanding patient adherence is critical to monitoring the effectiveness of a treatment regimen due to the possibility of developing drug resistance. To assess the utility of two potential biomeasures for adherence monitoring we conducted a directly observed therapy (DOT) pharmacokinetic study of DTG.

METHODS:
We enrolled 12 healthy volunteers into our DOT of oral 50mg DTG and followed the participants across three 28-day long dosing phases with 10-14 days of washout between each phase (clinicaltrials.gov NCT03218592). All volunteers took a single dose in phase 1 followed by daily dosing in phase 2 and 0, 1, or 3 doses per week in phase 3 (n=4 participants randomized to each group). Whole blood was collected into 3ml K2EDTA vacutainers at 3, 7, 14, 21, and 28 days within each phase. The average time post last dose in hours was 24 for daily dosing, 64 for thrice weekly dosing and 146 for once weekly dosing. Fifty uL was spotted onto a Whatman 903 DBS card before the remaining blood was centrifuged to plasma. DBS was allowed to dry overnight at ambient temperature and all samples were stored at -80°C until the time of analysis. DTG was quantified in 180 3mm DBS punches and 178 plasma samples following 1) extraction by protein precipitation with the internal standard dolutegravir-13C, d5, 2) chromatographic separation on an Xterra MS C18 (50 x 2.1mm, 3.5um) column, and 3) detection using electrospray ionization on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibrated assay ranges were 10.0-10,000ng/mL for DBS and 1.00-10,000ng/mL for plasma. Values below the lower limit of quantification (LLOQ) were imputed at ½ of LLOQ. Descriptive statistics were performed using WinNonlin (Certara Inc, Cary NC) and reported as median (interquartile range) unless otherwise stated.

RESULTS:
We enrolled 12 participants (92% white, 75% female) with weight, age, and creatine clearance of 76.1(71.4-82.5)kg, 42(31-52)years, and 118.3(98.6-138.5)ml/min. In phase 1, plasma DTG persisted above LLOQ in 100% and 67% of participants at 3- and 7-days post dose with concentrations of 79.75(52.2-130.5) and 1.9(.5-3.9)ng/ml, respectively. DBS DTG was above LLOQ in 100% of participants at 3 days with concentrations of 50.4 (32.0-80.8)ng/ml but not quantifiable at 7 days. In phase 2 and 3, respective steady-state concentrations for participants taking 1, 3, and 7 doses/week were 2.38(0.69-3.57), 69.3(32.6-112), and 1240(792-1550)ng/mL for plasma and unquantifiable, 50.0(21.7-73.5), and 718.5(457.5-941.3)ng/ml for DBS. DTG concentrations were quantifiable for 10-12 days after the final DTG dose in the 4 participants randomized to take 0 doses/week in phase 3 and ranged from 1.3-2.5ng/ml.

CONCLUSIONS:
Our approach demonstrates that concentrations of DTG are distinguishable between different patterns of dosing for both plasma and DBS. Concentration profile patterns were similar between DBS and plasma, thus the two measures are likely interchangeable for adherence monitoring. We propose concentrations collected at the end of the daily dosing interval that fall between 500-1600ng/ml are a likely indicator of high adherence. Concentrations between 20-500ng/ml likely indicate inconsistent use of 2-4 doses per week and those that are quantifiable but below 20ng/ml are a likely indicator that a dose of DTG has not been taken for several days or is being taken at a very low <2 doses/week frequency. One limitation of our study is that we did not test whether our interpretation would be confounded by white coat adherence. Our PK study was designed to capture concentrations at the end of a daily dosing interval (i.e. the trough) and the typical Cmax following a single dose of DTG exceeds the concentrations described herein. Thus, this adherence monitoring technique should be intentionally employed in clinical practice to collect concentrations at the end of the dosing interval and caution is advised in interpreting concentrations exceeding this typical daily trough value.

LC-MS/MS Method Development and Validation for Quantification of Steroids in Serum Younus Mohammad (Presenter) Canterbury Health Laboratories

Poster #26b View Map

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

INTRODUCTION:
An accurate, precise and rapid measurement of steroids is crucial in diagnosis, monitoring and providing personalised treatment for many disorders, such as Cushing’s syndrome, Addison’s disease and congenital adrenal hyperplasia. Compared to traditional immunoassays LC-MS/MS provide more specificity, sensitivity and accuracy in measuring very low levels of steroids.

OBJECTIVE:
The aim of present work was to develop a simple LC-MS/MS method for the simultaneous quantification of eight steroid hormones: aldosterone, cortisol, 11-deoxycortisol, 21-deoxycortisol, androstenedione, 17-hydroxypregnenolone, testosterone, 17-hydroxyprogesterone, and dihydrotestosterone (DHT).

METHODS:
Supported liquid extraction was used for sample preparation. Chromatographic separation was achieved on a Cortecs premier C18 column, and 0.05 mM ammonium fluoride in water (+ 2% v/v methanol) and 0.05 mM ammonium fluoride in methanol (+2% v/v water) were employed as mobiles phase solvents. The analytes were detected on Water TQ-XS system, aldosterone was assessed in negative mode and all other analytes were assessed in positive mode. Chrome system calibrators and quality control samples (panel 1 and panel 2) were used. The method was validated for selectivity, sensitivity, accuracy, precision, recovery studies, effect of matrix and dilution integrity. The method also used quality assurance samples for external comparison and was compared against an external laboratory (Pathology Queensland, Australia).

RESULTS:
The analytical run was less than 10 mins. The validation results were found to be within acceptable limits, accuracy <15% compared to reference samples and % CV <10%. Passing-Bablok regression analysis for the method comparison study was within acceptable limits.

CONCLUSIONS:
The method can be a useful tool in determination of steroids in both clinical and scientific laboratory research.

Evaluation of an Ultra-High Throughput Method for the Analysis of Tacrolimus in Whole Blood using the SCIEX Echo MS+ System Michael Jarvis (Presenter) SCIEX

Poster #28b View Map

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

INTRODUCTION:
The SCIEX Echo MS+ system utilizes acoustic droplet ejection technology to provide ultra-high throughput, carryover-free analysis of samples from a variety of aqueous matrices. This technique is typically most attractive for assays that have large sample volumes, due to the speed (up to 3 Hz) of the sample analysis. In this study we have chosen to evaluate the applicability of the Echo MS+ system for the analysis of Tacrolimus. MS analysis is widely considered the gold standard for highly accurate, highly specific quantitation of immunosuppressant drugs. The attractiveness of this ultra-rapid technique for the analysis of immunosuppressant drugs such as Tacrolimus is not only its throughput, but also its ability to deliver rapid, same-day results.

METHODS:
The analysis of Tacrolimus was performed in pure solvent and in whole blood samples, after appropriate sample preparation. Whole blood calibrators and controls were obtained from Diagnotix (Appingedam, Netherlands). For the whole blood samples, calibrators, and controls, 100uL of sample was added to 200uL of concentrated ZnSO4 solution, the samples were vortex mixed, then allowed to incubate at room temperature for 5 minutes, after which the samples were again vortex mixed and finally centrifuged at 10,000 g. The clean supernatant was transferred to a 384-well microtiter plate. Sample injection (or “ejection”) using acoustic droplet ejection technology on the SCIEX EchoMS+ system uses sound energy to causes 2.5nL droplets to be ejected from the well plate for capture in the “open port” probe, which transfers the sample to the mass spectrometer source. In this study, we have evaluated sample ejections ranging from 1 droplet (2.5nL) up to 400 droplets (1uL) per sample. Regardless of the number of droplets ejected, the sample-to-sample injection time was always <5 seconds. MS/MS analysis was performed using electrospray ionization in positive mode on the SCIEX Triple Quad 6500+ system.

RESULTS:
A variety of parameters were evaluated to determine optimum method conditions, including carrier solvent composition, carrier solvent flow rate, carrier solvent modifiers, ion source conditions, and the number of droplets per injection. Our results demonstrate that although a higher percentage of methanol or acetonitrile in the carrier solvent provides enhanced ionization (raw signal, measured in counts-per-second) for Tacrolimus, there is an increase in the background that negatively impacts the S/N, therefore it is preferable to operate with predominantly aqueous carrier solvent. Increasing the ejection volume has the effect of increasing the signal, and hence the LOQ, however it also results in slightly wider peaks. Nevertheless, even with ejection volumes as large as 1000nL we were able to easily achieve sample-to-sample injection times of less than 5 seconds. A linear response was achieved over a concentration range from 1 ng/mL to 1000 ng/mL of Tacrolimus, with %CVs <15% and accuracy +/- 15% across the entire concentration range. Tacrolimus concentrations determined in whole blood samples on the Echo MS+ system were compared to measurements obtained by immunoassay on the same samples, and demonstrated a good correlation.

CONCLUSION:
In this study we have successfully demonstrated the applicability of the ultra-high throughput Echo MS+ system to the measurement of Tacrolimus in whole blood samples. The work presented here should be readily extensible to the measurement of additional immunosuppressant drugs such as Cyclosporine A, Everolimus, and Sirolimus in whole blood. The method presented here demonstrated a linear response across a concentration range from 1 ng/mL to 1000 ng/mL of tacrolimus, with acceptable %CVs and Accuracy% across the measured concentration range. A direct comparison of Tacrolimus measurements in whole blood samples using the method on the Echo MS+ system demonstrated a good correlation with measurements obtained via immunoassay on the same samples.

Highly Multiplex Targeted Proteomics Assays in Plasma using Stellar Mass Spectrometer with Adaptive RT Brian Brewster (Presenter) Thermo Fisher Scientific

Poster #31b View Map

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

INTRODUCTION:
The development of targeted assays to monitor biomedically relevant proteins is crucial for translating discovery experiments into large-scale clinical studies. However, current targeted assays struggle to scale to hundreds or thousands of targets. To overcome the challenge, Stellar mass spectrometer(MS) combined with SkylineTM were utilized to generate large-scale assays. With hyper-fast acquisition speeds, Stellar MS handles shifting retention times through real-time alignment called adaptive RT and maintains the sensitivity and speed required to manage numerous concurrent targets. We developed a multiplex targeted proteomics method with adaptive RT within 3 days using PQ500 peptides as heavy standards. This method was further applied to the quantitation of potential protein biomarkers in plasma from lung cancer, Alzheimer's disease and colorectal cancer patients.

METHODS:
Disease and healthy plasma were purchased from BioIVT and digested using AccelerOmeTM. Pooled plasma sample was used to develop large scale peptide panel. PQ500 peptides were obtained from Biognosys AG. Peptide standard was resuspended following the manufacturer’s instruction.
A VanquishTM Neo UHPLC coupled with Stellar mass spectrometer scheme was used. Mobile phase A was 0.1% FA in H2O and mobile phase B was 0.1% FA in 80% ACN. EasySpray™ PepMap™ column temperature was set at 55 °C and autosampler temperature was 7 °C. Mass spectrometer parameters such as AGC values and maximum injection time were optimized. Skyline was used to generate scheduled PRM panel. Peptides were analyzed using 24, 60 and 100 samples per day (SPD) gradients.

RESULTS & DISCUSSION:

The Biognosys PQ500 peptides were utilized as heavy standards to develop a large-scale targeted peptide panel. Initially, unscheduled methods were generated using a pure heavy peptide transition list to determine retention times. Subsequently, heavy peptides were spiked into 200 ng neat plasma digest to schedule a final method with a 0.7-minute adaptive retention time (RT) window. This workflow was completed within three days, demonstrating a highly time-efficient method development process for large-scale targeted peptide quantitation.

To optimize the separation and elution of compounds, different gradient conditions were compared. Results indicated that a 60 SPD gradient provided slightly better separation compared to a 100 SPD gradient, although the overall improvement was not significant. The developed method achieved limits of detection (LOD) below 50 amoles for more than 90% of peptides in the plasma matrix. Additionally, the peptides exhibited good linearity and reproducibility across a concentration range spanning over three orders of magnitude.

The method enabled the detection of potential protein biomarkers, such as Histidine-rich glycoprotein (HRG), which is associated with sepsis and cancer, in plasma samples. Significant changes in certain protein levels were observed in plasma from diseased patients compared to healthy donors. Notably, distinct protein alterations were detected in colorectal cancer plasma samples.

The adaptive RT function, combined with a 0.7-minute scheduled window, successfully captured shifted retention times for over 800 peptides without the need for rescheduling the retention time window. This feature significantly enhances the robustness and reliability of the method.

Overall, the results presented here demonstrate the effectiveness of large-scale, multiplexed targeted assays in increasing sample throughput. Innovative features such as Adaptive RT and PRM Conductor enhance the development and robustness of the assay, enabling routine quantification of potential biomarkers in diseased patient plasma and paving the way for more efficient and comprehensive biomarker discovery and validation.

MALDI MSI Analyses of a Tamoxifen-Induced Mouse Model of Neurodegeneration Zeinab Habbouche (Presenter) University of Montreal

Poster #32b View Map

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

INTRODUCTION:
Plasmalogens are essential glycerophospholipids, highly enriched in neuronal membranes, where they play a critical role in membrane structure, cell signaling, and redox homeostasis. Their depletion has been linked to several neurodegenerative diseases, although the underlying mechanisms remain poorly understood. To further study plasmalogen depletion in neurodegenerative diseases, an adult murine model was developed by Med-Life Discoveries, in which tamoxifen intraperitoneal injections induce targeted gene recombination, resulting in a reduction of brain plasmalogen levels (PMID 39053687). This model allows for temporal control of lipid depletion and supports longitudinal monitoring to track lipidomic dynamics and correlate them with potential neurological effects. It therefore provides a robust and reproducible experimental platform for studying lipid alterations associated with neurodegenerative pathologies and offers a valuable preclinical tool for evaluating therapeutic strategies aimed at restoring plasmalogen homeostasis.

OBJECTIVES:
The aim of this study is to further evaluate and map the extant of plasmalogen brain depletion in an tamoxifen inducible mouse model. Using high-resolution MALDI mass spectrometry imaging (MSI), we assessed changes in the distribution and spatial organization of plasmalogen-related species across brain structures, with a particular focus on the cerebellum, a region known for its high plasmalogen content and its relevance to motor coordination. This work seeks to establish a baseline lipidomic signature of inducible plasmalogen deficiency and to identify region-specific susceptibilities in the adult brain.

METHODS:
Sagittal brain sections of 12 μm thickness were prepared from adult mice, with three biological replicates per group (control and tamoxifen induced) and per sex. One brain section per condition and per sex was mounted on each slide, enabling direct intra-slide comparison across experimental groups. Tissue sections were coated with 1,5-diaminonaphthalene (1,5-DAN) matrix via sublimation using the iMLayer device (Shimadzu) following a protocol that ensured homogeneous and reproducible matrix deposition. Plasmalogen MALDI MSI was then performed using the iMScopeQT system (Shimadzu), in the negative ion mode. The spatial resolution was set at 30 μm for the cerebellum and 100 μm for all other brain regions, allowing for high-resolution mapping of lipid distribution across brain structures. On-tissue MALDI MS/MS analyses were also conducted to confirm the structural identity of the detected plasmalogens.

PRELIMINARY RESULTS:
Initial MSI results revealed significant alterations in lipid composition between the control and tamoxifen induced groups, particularly in brain regions such as the cerebellum, hippocampus, and cortex. Plasmalogen-associated ion signals were found to be consistently lower in the tamoxifen induced group, especially in the cerebellum, indicating successful induction of plasmalogen deficiency. These changes were not uniformly distributed, but rather region-specific, with the cerebellum displaying a markedly distinct lipidomic signature compared to other brain areas. This suggests that certain brain regions may be more vulnerable to plasmalogen loss. The spatial specificity of these changes highlights the utility of MALDI MSI in capturing fine scale lipid dynamics across the brain.

CONCLUSION:
This study presents a deeper characterization of a reproducible, inducible mouse model of plasmalogen deficiency in the adult brain and demonstrates the ability of MALDI MSI to detect fine scale, spatially resolved changes in lipid composition. The comparison between control and tamoxifen-induced animals highlights region-specific plasmalogen lipid depletion, with the cerebellum appearing particularly affected. These findings further support the utility of this model for studying lipid dynamics under pathological conditions and for evaluating therapeutic strategies aimed at restoring plasmalogen homeostasis.

A Comparison of Sample Preparation Techniques on Drugs of Abuse in Oral Fluids Jared Burkhart (Presenter) Restek

Poster #33b View Map

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

INTRODUCTION:
The “gold standard” biological matrices for toxicological testing are typically blood and urine, however, the collection of these two matrices is considered invasive. The analysis of drugs of abuse(DoA) in oral fluids is a solution that is gaining popularity due to its ease of collection compared to that of blood or urine. However, when performing the oral fluid analysis, it can be difficult to remove all of the buffer surfactants present in the oral fluid collection device’s buffer solution. These surfactants can cause matrix effects and poor column lifetime. Often times solid phase extraction (SPE), or lengthy extraction techniques are utilized. Finding a workflow that uses a simple sample preparation paired with accurate and robust quantitation of the analytes is important for laboratories running these tests. In this work, a dilute-and-shoot sample preparation was compared against supported liquid extraction (SLE) and salt-assisted liquid-liquid extraction (SALLE).

OBJECTIVE:
The primary objective of this work is to compare the recovery for DoA and novel psychoactive substances (NPS) in oral fluids using three sample preparation techniques: dilute-and-shoot, SLE, and SALLE.

METHODS:
An LC-MS/MS method was developed using a Raptor Biphenyl 50 x 2.1 mm, 2.7 µm analytical column equipped with a Raptor Biphenyl EXP 5 x 2.1 mm, 2.7 µm guard column. A total of 68 analytes were separated using gradient conditions, consisting of water (MPA) and methanol (MPB), both containing 0.1% formic acid, for a total cycle time of 10 minutes. Samples were prepared in oral fluid and combined with QuantisalTM buffer. Aliquots from the buffer underwent each of the sample preparation techniques, dilute-and-shoot, SALLE, and SLE. Samples were dried down and reconstituted in 90:10 MPA: MPB and moved to the instrument for analysis.

RESULTS:
The biphenyl stationary phase resolved all 68 analytes, including sets of isobaric analytes, such as methamphetamine and phentermine, isotonitazene and protonitazene, and eutylone and pentylone. All sets of isobars achieved a resolution of 1.5 or greater, providing accurate quantitation of the analytes. Performance metrics such as recovery, linearity, matrix effects, and accuracy and precision were evaluated. All analytes passed both intra and inter-day accuracy and precision requirements, without matrix interferences or effects being observed. Linearity was demonstrated using 1/x weighted linear regression and with an r2 of ≥ 0.991. An evaluation was completed, comparing analyte recovery when using dilute-and-shoot, to SLE and SALLE approaches. Dilute-and-shoot failed to remove the buffer surfactants compared to SLE and SALLE. Both SALLE and SLE returned increased sensitivity compared to dilute-and-shoot for all analytes (excluding pregabalin, gabapentin, THC, and CBD for the SLE workflow). SALLE yielded higher sensitivity for a broader range of analytes, while SLE achieved increased sensitivity for most of the opiates.

DISCUSSION:
This work demonstrates an accurate and robust solution for the analysis of these analytes. Additionally, this workflow demonstrates quick and efficient sample clean up procedures that remove buffer surfactants without the need for SPE or other tedious extraction techniques. All analytes showed an improvement in recovery when using the SALLE technique as compared to dilute-and-shoot. Most analytes showed an improvement in recovery when using SLE as compared to dilute-and-shoot aside from a few (THC, CBD, pregabalin, and gabapentin). These sample preparations can be catered based on analyte list and LOD requirements to ensure the best recovery for all analytes of interest. Utilizing quick and efficient sample preparation techniques leads to faster processing of samples in high through-put laboratories.

Development of a Ready-To-Use Evotip-Based Workflow for Plasma Protein Quantification Elodie Logerot (Presenter) Segal Cancer Proteomics Centre, Jewish General Hospital

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Poster #34b View Map

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

INTRODUCTION:
Accurate and reproducible quantification of plasma proteins poses a significant challenge to mass spectrometry-based proteomics due to its high dynamic range and inherent matrix complexity. Highly abundant proteins can mask the detection of biologically relevant, low-abundance species, and matrix effects and batch-to-batch variability further complicate quantitative analyses. As part of the SysQuan project, we use stable isotope-labeled mouse plasma (SILAC) as an internal standard for absolute quantification, which helps us address these limitations and improve consistency between workflows. Our goal is to develop an innovative, solution that will streamline plasma proteomics and enable its routine use in clinical and research environments.

OBJECTIVE:
The long-term goal is to create Evotips preloaded with SILAC mouse plasma, which will enable the direct addition and subsequent digestion of human plasma samples for quantification. This system is designed to be a robust tool for absolute protein quantification in clinical and research applications.

METHODS:
Currently, method development and optimization are based on human plasma samples. A "one-pot" digestion strategy is employed directly on Evotips to ensure compatibility with high-throughput workflows. This digestion protocol eliminates transfer steps and reduces sample handling errors by sequentially adding denaturation, reduction, alkylation, and digestion reagents directly onto the Evotip. Resulting digests were analyzed using an Evosep One LC system coupled with a timsTOF HT mass spectrometer operating in DIA-PASEF mode. Data were analyzed using a spectral library generated from fractionated, depleted and non-depleted plasma, and identification was performed using DIA-NN.

RESULTS:
On average, 450 proteins and 3,500 peptides were consistently identified in all technical replicates per sample. The workflow demonstrated high reproducibility, with coefficients of variation below 15% between replicate analyses. Notably, 91% of the proteins identified using the conventional S-Trap-based workflow were recovered during the "one-pot" digestion on Evotips. This significant overlap underscores the analytical depth of the streamlined protocol while significantly reducing processing times and preparation steps. Furthermore, around 75% of the peptides detected using the Evotip approach overlap with those included in the SysQuan panel (i.e., lysine-containing and shared between mouse and human), underlining the method's compatibility with absolute quantification pipelines.
These initial results validate the digestion strategy on Evotips and support its integration into SILAC-based quantification workflows, including MRM or PRM-PASEF methods developed on a triple quadrupole mass spectrometer such as the Agilent 6495D or timsTOF HT as part of the SysQuan project.

CONCLUSION:
We present a reproducible and simplified method for plasma proteome analysis using Evotips that yields promising results in terms of protein and peptide coverage. The next phase of this project will implement SILAC mouse plasma on the Evotips to serve as a protein-level internal standard, which will enable robust quantification. The ability to integrate an internal standard directly into the Evotip format paves the way for standardized, plasma quantification solutions, with potential applications in translational research and routine workflows.

REFERENCES:
Ye, Z., Sabatier, P., Martin-Gonzalez, J. et al. One-Tip enables comprehensive proteome coverage in minimal cells and single zygotes. Nat Commun 15, 2474 (2024). https://doi.org/10.1038/s41467-024-46777-9.

Optimized Method for LC-MS/MS Quantification of Underivatized Estrone(E1) and Estradiol (E2) in Human Serum Using Supported Liquid Extraction Stephanie Marin (Presenter) Phenomenex

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Poster #35b View Map

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

INTRODUCTION:
Precise measurement of estrone (E1) and estradiol (E2) is essential for assessing reproductive and endocrine function in clinical research. Due to their low circulating levels, poor ionization efficiency, and structural similarity, direct LC-MS quantification without derivatization is analytically challenging. Although recent improvements in sample preparation and mass spectrometry sensitivity have enhanced assay performance, derivatization remains a common approach to achieve the sensitivity required for reliable detection of E1 and E2 in serum. Here, we present a sensitive method for LC-MS/MS quantitation of estrone and estradiol without derivatization using supported liquid extraction (SLE).

OBJECTIVE:
To develop a robust extraction and LC-MS/MS method for quantification of underivatized E1 and E2 in human serum using supported-liquid extraction (SLE) and optimized chromatographic separation.

METHODS:
Four reversed-phase LC columns were screened for optimal separation: Kinetex C18, Kinetex Biphenyl, Luna Omega Polar C18, and Kinetex F5. The Kinetex Biphenyl column (50 × 3.0 mm, 2.6 µm) provided the best resolution (~1 min) and highest signal response for E1.
Sample preparation was SLE using Strata-SE SLE MAX 96-well plates. Human serum was pre-treated with ammonium acetate buffer and extracted with ethyl acetate/hexane (9:1). Extracts were dried under nitrogen and reconstituted in ammonium fluoride/methanol (60:40, v/v).
LC-MS/MS analysis was performed on an Agilent® 1290 Infinity system coupled with a SCIEX® 7500 Triple Quad mass spectrometer. The mobile phase consisted of 0.5 mM ammonium fluoride in water (A) and methanol (B), with a gradient elution over 5 minutes at 0.8 mL/min.

RESULTS:
The method demonstrated excellent linearity for E1 (1–500 pg/mL, R² = 0.998) and E2 (5–500 pg/mL, R² = 0.993). LOQs were 5 pg/mL for E1 and 10 pg/mL for E2, with signal-to-noise ratios of 12 and 8, respectively. Improved S/N for E2 was observed at 20 pg/mL (S/N = 19).

DISCUSSION AND CONCLUSIONS:
A sensitive and selective LC-MS/MS method was successfully developed for quantifying underivatized estrone and estradiol in human serum with acceptable signal to noise ratio for ensuring accurate quantitation at LOQs as low as 5 pg/mL for E1 and 10 pg/mL for E2. It is reported in the literature that for E1, 95th percentile values in a population including males and females of all categories fall within 40-200 pg/mL and for E2 95th percentile values in a population of all males and females range from 40-350 pg/mL (1). This method using Strata SE SLE and Kinetex Biphenyl column (50 × 3.0 mm, 2.6 µm) on a high sensitivity MS provides a solution for the majority of samples and avoids time consuming, labor intensive derivatization typically used in LC-MS/MS estrogen methods.

Strata SE SLE extraction coupled with a Kinetex Biphenyl column and a sensitive LC-MS/MS system provided optimal chromatographic performance suitable for clinical research applications. Where lower concentration levels are desired, various derivatization procedures can be followed to attain higher sensitivity.

References:
1. The Journal of Clinical Endocrinology & Metabolism, Volume 105, Issue 3, March 2020, Pages 754–768.

Sensitive Linear Quantification of Free Opiates in Variety Biologic Matrices by SLE-LC-MSMS with Wide Analytical Measurement Range in a Forensic Laboratory Heng Shi (Presenter) NMS Labs

Poster #36b View Map

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

INTRODUCTION:
Free Opiates test is one of the high-volume tests at the NMS laboratory. This test covers wide range of biologic samples such as in whole blood, postmortem blood, serum/plasma, vitreous fluid and urine. Due to current method that is tedious, costing and time-consuming with high repeatable rate, the organization decided to redevelopment of the current test. The challenge of the redevelop the method is to achieve a faster and cost-effective method with extended analytical measurement ranges (AMR) to reduce the sample repeatable rates. The work described here is the new development of free opiates ( Morphine, Hydromorphone, Codeine, Dihydrocodeine, 6-MAM, Oxycodone and hydrocodone) in both sample preparation and chromatographic separations with AMRs extended up to 2.5 times and the final ranges are 1-200 ng/mL for 6-MAM, oxymorphone and hydromorphone; 5-500 ng/mL for codeine and dihydrocodeine; 5-1000 ng/mL for all other analytes in the panel.

METHODS:
A Bioatge Isolute SLE+ 400 µL plate on ExtraHera was used for sample preparation. After 200 µL of calibrators, QCs and samples are aliquoted on the SLE plate, 25 µL of internal standard was added to each well followed by 255 µL 1.5% NH4OH solution. The plate was then mixed and spin down at 4000 rpm for 10 minutes before load onto the Extrahera. A “load only” SLE method was applied to the automatic sample preparation. The Extrahera method will load 300 µL on to the extraction plate and elute three times with a total volume of 1350 µL of ethyl acetate. The receiving plate was drydown on a TurboVap-96 and reconstituted with 300 µL 95:5 (v:v) of mobile phase A : mobile phase B . Mobile phase A is 0.05% Formic acid with 5 mM Ammonium Acetate in water and mobile phase B is 0.05% formic acid in methanol.

The 96-well sample plate was mixed and spun at 4000 rpm for 5 min, then load onto a Waters Acquity UPLC/Xevo TQXS Triple Quadrupole Mass Spectrometer system. 2-µL sample was injected on LC-MSMS using a Phenomenex Kinetex Biphenyl column with dimensions of 50 x 2.1 mm, part. size 5 µm. The column temperature is maintained at 55 oC. The LC gradient starts at 2 % mobile phase B ( 0.1% formic acid in methanol) after hold for 0.6 min, mobile phase B was linearly gradient to 15% at 1.1 min; 20% at 1.9 min and 35% at 2.4 min respectively. From time 2.5-3.0 min, mobile phase B at 98% to wash the column and ready for the next injection.

RESULTS:
Under the chromatographic conditions, all the free opiates on the panel yield excellent peak shape and isobaric separations with a total analysis time of 3 minutes which is about 50% faster than previous method. The isobaric separations of morphine vs hydromorphone and codeine vs hydrocodone were achieved through the slow gradient at beginning and the oven temperature at 55 oC . The five intra-days and inter-days CVs are < 10% for all analytes in blood, serum and urine matrices. Interferences check with around 90 analytes that includes from broad OTCs, designer’s/synthetic opiates, cocaine panel, fentanyl panel, benzodiazepines, amphetamines and cannabinoids were performed during the new method validation. There were no interferences found under the method LC-MSMS conditions.

CONCLUSIONS:
A new SLE-LCMSMS method was developed, validated and now in live at the NMS laboratory. The method is accurate, sensitive and rugged. Compared to previous method, it is simper, faster and more cost effective. Not only the lower limits of quantitation were met, but the extended AMRs up to 2.5 times) were achieved with linear correlation coefficient > 0.997 for each analyte compared previous method. The extended AMRs reduced the sample repeatable rate significantly, as well as turnaround time. The method is adaptable for accurate and reproducible quantification of free opiates in variety of biologic matrices with high throughput at any laboratory.

Increasing Diagnostic Accuracy of High-Throughput Analysis of Testosterone, Androstenedione and Dhea in Human Serum With Ldtd-MS/MS Pierre Picard (Presenter) Phytronix Technologies Inc

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Poster #37b View Map

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

INTRODUCTION
High-throughput analysis of serum concentrations of testosterone, androstenedione (A4), and dehydroepiandrosterone (DHEA) plays a crucial role in the diagnosis and management of various endocrine disorders. Although testosterone is the primary androgen, other androgenic hormones are important when evaluating conditions such as polycystic ovarian syndrome (PCOS), adrenal insufficiency, and disorders involving hyperandrogenism or hypoandrogenism1,2. These hormones, mainly produced by the gonads and adrenal glands, are essential for the regulation of sexual development, reproductive functions and metabolic processes1,2. Accurate and efficient quantification of these steroids in serum is essential for detecting hormonal imbalances. High-throughput techniques, particularly steroid immunoassays, have become the standard for measuring these hormones due to their sensitivity and ability to process large sample volumes. However, these methods are often limited by the specificity of the antibodies for epitopes, which can lead to cross-reactions with structurally similar steroids, thereby affecting the accuracy of the measurements. Overcoming these limitations through advanced analytical techniques, such as laser diode thermal desorption coupled to mass spectrometry (LDTD-MS/MS), is essential to ensure accurate diagnostics and effective treatment strategies.

OBJECTIVES
To facilitate screening and diagnosis of endocrine disorders, a rapid analytical method to separate testosterone and DHEA, isobaric compound, a specific APCI ionisation, extraction method and a LDTD-MS/MS analytical method was developed. This approach aims to improve clinical decision-making by providing timely results while maintaining high sensitivity and precision across a broad range of samples.

METHODS
A calibration curve and quality controls are spiked into an artificial matrix consisting of 20 mg/mL BSA in PBS. To prepare the samples, 12.5 µL of internal standards and 250 µL of samples are mixed at 1000 rpm for 5 seconds. Then, 1000 µL of a Hexane/MTBE (90:10) mixture is added, followed by mixing at 1000 rpm for 30 seconds and centrifugation at 14,000 rpm for 4 minutes. The upper layer (500 µL) is transferred to a borosilicate tube (10x75mm) and evaporated to dryness. The samples are reconstituted with 100 µL of an Acetonitrile:Water (1:1) mixture, and 4 µL are spotted onto a LazWell96 plate for LDTD-MS/MS analysis.

Testosterone and DHEA are isobaric compounds, and to enhance selectivity between them, an ammonia solution is infused into the carrier gas to form an adduct. The infusion of ammonia into the LDTD carrier gas is optimized to maximize the signal of both adduct and protonated molecules. Ammonium hydroxide (1% in water) is introduced into the Luxon at a rate of 2 µL/min, with a carrier gas flow of 4.5 L/min. This process selectively generates ammonium adducts on DHEA. Under these ionization conditions, specific primary masses are targeted for all analytes, and the mass spectrometer operates in positive ionization mode, using a designated MRM transition for detection.

RESULTS
Specificity is confirmed by running separate calibration curves for each analyte, monitoring all MS/MS transitions. Data indicates no detectable interferences between the analytes. The solvents used in the liquid-liquid extraction (LLE) process are optimized to minimize unwanted noise in MS/MS transitions. Calibration curves range from 100 to 10,000 pg/mL for testosterone, and from 200 to 10,000 pg/mL for DHEA and androstenedione. A set of fortified quality controls (QCs) is prepared in serum matrix with low levels of testosterone, androstenedione, and DHEA. The peak area to internal standard (IS) ratio is used to normalize the signal. For linearity evaluation, the coefficient of correlation must exceed 0.995, with obtained values ranging from 0.99390 to 0.99876 for testosterone, androstenedione, and DHEA across six runs. Precision is evaluated by ensuring that the %CV for quality control samples (QCL, QCM, QCH) remains below 15%. For inter-assay precision, the %CV was below 11.4% for all QCs. Accuracy for testosterone ranged from 101.5% to 105.6%, while A4 ranged from xx% to yy%, and DHEA ranged from 99.7% to 104.9%. Using LDTD-MS/MS technology, sample stability, both in the extracted (wet stability) and dry state on the Lazwell plate (dry stability), is evaluated rather than injector stability (as in LC-MS/MS). Precision values for these stability assessments ranged from 0.9%CV to 10.9%, and accuracy ranged from 98.2% to 106% for both dry and wet stability. Finally, cross-validation with LC-MS/MS was performed, and the percentage differences between the two methods were all below 20% for the evaluated matrices.

CONCLUSION
The selective high-throughput shotgun analysis of steroids, utilizing specific APCI adduct formation and optimized liquid-liquid extraction (LLE) with LDTD-MS/MS, provides excellent specificity, precision, and accuracy for the quantification of testosterone, androstenedione, and DHEA in serum. Cross-validation with LC-MS/MS further confirms the robustness and comparability of the two techniques, ensuring consistent and reliable hormone analysis across different platforms.

REFERENCES
1. Frank Z. Stanczyk, Diagnosis of hyperandrogenism: Biochemical criteria, Best Practice & Research Clinical Endocrinology & Metabolism, Volume 20, Issue 2, 2006, Pages 177-191, ISSN 1521-690X, https://doi.org/10.1016/j.beem.2006.03.007.
2. Nassar GN, Leslie SW. Physiology, Testosterone. In: StatPearls. StatPearls Publishing, Treasure Island (FL); 2023. PMID: 30252384.

Evaluation of the New Strata™ SE SLE for Sustainable and Reproducible Steroid Extraction in Clinical Research Applications Rajashree Chakravarti (Presenter) Phenomenex

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Poster #38b View Map

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

INTRODUCTION:
Accurate quantitation of low-level steroids in clinical research necessitates sample preparation methods that are both efficient and highly reproducible. Supported Liquid Extraction (SLE) offers a streamlined alternative to Solid Phase Extraction (SPE), particularly in high-throughput laboratories, due to its simplified workflow and reduced need for labor-intensive method development. This study evaluates the performance of the new synthetic Strata™ SE SLE product in two critical areas: batch-to-batch reproducibility and comparative extraction efficiency against other commercial SLE products.
A rigorous evaluation of batch-to-batch reproducibility was conducted prior to product release, recognizing that variability between production lots can lead to inconsistent analyte recovery, matrix effects, or background contamination—factors that can compromise data integrity in clinical research. Additionally, a comparative analysis was performed using a panel of eleven clinically relevant steroids with low limit of detection (LOD) requirements, reflecting their importance in disease-state research. A key focus of this study was to develop a sustainable extraction method by avoiding halogenated solvents such as dichloromethane, which are traditionally used in SLE but pose environmental and regulatory concerns.

METHODS:
Eleven steroid analytes were extracted from human serum at LOD, 2×LOD, and 5×LOD concentrations using Strata SE SLE. Ethyl acetate, a non-halogenated solvent, was used as the primary eluent and compared with dichloromethane to assess performance. Batch-to-batch reproducibility was evaluated across three independently manufactured lots of Strata SE. Comparative performance was assessed against Novum Pro and a leading competitor SLE product using both eluents. LC-MS/MS analysis was conducted using an Agilent® 1290 Infinity LC system coupled with a SCIEX® 7500 Triple Quad MS.

RESULTS:
Strata SE demonstrated excellent batch-to-batch reproducibility, with recovery values ranging from 76–112% and inter-batch %CVs consistently below 20%. In comparative studies, Strata SE using ethyl acetate achieved superior recovery (73–118% at LOD) and cleaner extracts than both Novum Pro and the competitor SLE. Matrix effects and process efficiency were also favorable. Importantly, Strata SE eliminated the need for dichloromethane, aligning with recent U.S. EPA restrictions and supporting laboratories' sustainability initiatives.

CONCLUSION:
The new Strata SE SLE platform delivers high-quality, reproducible extraction of low-level steroids from complex biological matrices using a sustainable, non-halogenated solvent. Its performance not only meets but exceeds that of established commercial SLE products, offering superior recovery, reduced matrix interference, and minimal background contamination. The demonstrated batch-to-batch consistency further reinforces its reliability for routine clinical research applications. By enabling laboratories to transition away from chlorinated solvents without compromising analytical performance, Strata SE SLE represents a forward-looking solution that supports both scientific excellence and environmental responsibility.

Evaluation of an Assay for Seven Nontuberculosis Mycobacterial Pulmonary Disease Drugs Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Kyunghoon Lee (Presenter) Seoul National University Bundang Hospital

Poster #39b View Map

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

BACKGROUND:
Nontuberculous mycobacteria (NTM), with over 200 identified species, are ubiquitous in natural environments such as soil and water. Recently, the global prevalence of nontuberculous mycobacterial pulmonary disease (NTM-PD), caused by lung tissue damage and inflammation, has risen rapidly, with a reported rate of 56.7 cases per 100,000 population in Korea as of 2021. Despite the growing burden of NTM-PD, treatment remains empirical, typically involving two or three antibiotics administered over 18 to 24 months. Therefore, there is am increasing need for therapeutic drug monitoring (TDM) to enable optimized treatment. The aim of this study was to develop and evaluate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the simultaneous quantification of seven antibiotics commonly used in NTM-PD treatment.

METHODS:
Seven antibiotics – azithromycin, clarithromycin, clofazimine, ethambutol, linezolid, moxifloxacin, and rifampicin – were selected for assay development. Each serum sample was analyzed using a UPLC-MS/MS system. Analytical performance was evaluated in terms of precision, linearity, lower limit of quantification (LLOQ), carryover, and method comparison.

RESULTS:
All analytes and internal standards were clearly separated on the UPLC-MS/MS system without ion suppression. Within-run and between-day precision at three concentration levels for each drug ranged from 2.2% to 9.2% and from 5.8% to 16.7%, respectively. Calibration curves were linear over the following range: 0 – 10 μg/mL for azithromycin, clarithromycin, ethambutol, moxifloxacin, and rifampicin; 0 – 5 μg/mL for clofazimine; and 0 – 20 μg/mL for linezolid. The LLOQ was 0.0625 μg/mL for clofazimine; 0.125 μg/mL for azithromycin, clarithromycin, and rifampicin; and 0.25 μg/mL for ethambutol, linezolid, and moxifloxacin. No carryover was observed. Method comparison using four serum samples showed a difference ranging from -0.5% to 12.3% between the new and existing methods for ethambutol and rifampicin.

CONCLUSIONS:
The newly developed antibiotics assay demonstrated acceptable analytical performance including precision, linearity, LLOQ, carryover and method comparability. This method may provide a valuable foundation for implementing TDM and supporting personalized therapy for NTM-PD in clinical practice.

Enhancement Of Traceability, Throughput And Ecologic Footprint Of An Immunosuppressant LC-MS Workflow Via Fluent Automation Magali Wolff (Presenter) Tecan IBL International GmbH

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Poster #40b View Map

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

BACKGROUND:
Immunosuppressants are critical agents used to modulate the immune system, primarily to prevent organ transplant rejection and to manage autoimmune diseases. Precise quantification of immunosuppressant drugs is essential for optimizing therapeutic efficacy and minimizing toxicity, as these agents often have narrow therapeutic windows and significant inter-individual variability in pharmacokinetics. Accurate measurement of immunosuppressant levels typically requires analysis of whole blood samples, which presents unique challenges due to the complexity and variability of the matrix[1]. Automated sample preparation and analysis workflows have become increasingly important [2], enabling high-throughput, standardized, and reproducible processing of whole blood specimens. These advances help ensure reliable monitoring of immunosuppressant concentrations in clinical practice and research settings.

OBJECTIVE:
This study aimed to evaluate the sample preparation and analysis of four commonly used immunosuppressants, namely Tacrolimus, Everolimus, Sirolimus and Cyclosporine A, by automation compared to a manual sample preparation. The focus was on evaluating improvements in sample tracking, processing efficiency, and resource consumption compared to conventional methods, and determining the suitability of the automated workflow for routine laboratory applications in immunosuppressant monitoring.

METHODS:
Sample preparation followed the Tecan LC-MS Kit Ref # 30261557 / 30261558. Hereby, whole blood samples were being homogenized as well as extracted with a zinc sulphate and a deproteinization solution containing isotopically labeled internal standards of each of the analytes. Six calibrators and quality control samples at three concentration levels, within the expected physiological range, were reconstituted from a lyophilized whole blood matrix and treated as samples thereafter. Subsequently, the supernatant of these extracts was injected into an LC-MS/MS system consisting of a Thermo Scientific Vanquish UHPLC and a TSQ Quantis mass spectrometer operated by Chromeleon. For the automated procedure a Tecan Fluent 780 liquid handler equipped with a robotic gripper arm as well as an 8-channel pipetting arm with piercing tips was utilized. The liquid handler furthermore was equipped with a 4-slot plate centrifuge, four orbital shakers as well as two tube rotators with integrated barcode scanners. Total durations of manual as well as automated sample preparation were taken. Furthermore, the analytical performance of both procedures and their respective environmental impact was evaluated.

RESULTS:
The average total duration per plate with the automated workflow in a five-plate-scenario was approximately 34 minutes. The approximation included a sample scanning duration of 3 minutes, a sample rotation duration of 2 minutes, reagent distribution durations of 5 minutes, sample distribution times of 15 minutes, centrifugation of 4 minutes and a supernatant transfer of 5 minutes. The average total duration per plate with the manual workflow in a five-plate-scenario was 62 minutes. This included a manual sample scanning duration of 3 minutes, manual tube handling duration of 19 minutes (including de- and recapping etc.), sample distribution times of 20 minutes, reagent distribution durations of 3 minutes, agitation of 2 minutes, centrifugation of 5 minutes, as well as a supernatant transfer of 10 minutes. Due to the piercing capability of the sample tubes and the pipette tips, the de- and recapping step was omitted within the automated workflow. Calibration curves for both, the manual as well as the automated workflow were linear with an R² > 0.99 and all controls fell within their target range.

CONCLUSION:
Automation proved to be a robust and valuable enhancement of immunosuppressant LC-MS worklflows. Sample prep time and most importantly staff occupation has been shown to be reduced significantly compared to a manual workflow. While pipetting steps themselves did not necessarily show significant differences in duration, especially the automated tube rotation in combination with cap piercing and automated barcode scanning had an immense impact on the overall workflow. With its analytical performance and its low environmental impact the automated workflow should be the preferred fit for a sustainable and state of the art laboratory environment.

Enhanced Sensitivity of a Modified Orbitrap Astral Mass Spectrometer for Deeper Proteome Coverage in Single-cell Proteomics Applications Scott Peterman (Presenter) Thermo Fisher Scientific

Poster #41b View Map

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

INTRODUCTION:
Single cell proteomics using liquid chromatography-mass spectrometry is transforming our understanding of cellular heterogeneity and function by enabling detailed analysis of protein expression in individual cells. However, to properly characterize these functions and heterogeneity, many single cells need to be analyzed. This raises challenges related to sample throughput and depth of proteome coverage. While throughput can be increased by multiplexing, e.g using TMT, achieving depth of coverage requires high sensitivity. Methods that provide the highest proteome coverage and throughput would be of great interest to single cell proteomics applications. Here we evaluated the depth of proteome coverage and throughput that can be achieved for low-input and single cell samples using a modified Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer.

METHODS:
For the dilution series, Pierce™ HeLa digest (20 µg) was reconstituted by adding 200 µL of 0.015% DDM/0.1% TFA, sonicated for 5 minutes, then diluted in 0.015% DDM/0.1% TFA to 5 ng/µL by adding the stock solution to 95 µL of 0.015% DDM solution in a low-binding sample well plate and vortexed for a few seconds. Diluted bulk HeLa digest (at different concentrations) and pre-digested single cells were separated using different gradients on the Thermo Scientific™ Vanquish™ Neo UHPLC system. Eluting peptides were measured on a modified Orbitrap Astral MS equipped with a Thermo Scientific™ FAIMS Pro™ interface. Data was processed with Thermo Scientific™ Proteome Discoverer™ software using Chimerys™ search algorithm and Spectronaut™ 19 software.

RESULTS:
The modified Orbitrap Astral MS was operated in DIA mode using the low input application mode. To evaluate its performance, 250 pg from the bulk HeLa digest was separated and analyzed. Triplicate raw data files were processed together without using a library, in Spectronaut 19. Compared to the standard Orbitrap Astral MS, we observed an increase in protein groups identifications of over 5% from 5,986 to 6,308 and over 6% in peptides identifications from 50,935 to 54,181. Next, we performed a dilution series from 50 pg to 10 ng by injecting the appropriate amounts from the 5 ng/µL stock solution.

DISCUSSION:
For mass spectrometers utilizing ion accumulation, the efficiency and time devoted to accumulation becomes the most critical element for achieving ultimate sensitivity. By introducing parallel ion packet management, trapping instruments can increase data acquisition speed while spending longer accumulation times. The original Oribtrap Astral MS parallelized five different ion packets and dual mass analysis of precursors and product ions to deliver high protein sample coverage while maximizing data quality. The new instrument enhancements applied to the Orbitrap Astral MS added an additional accumulation stage in the source region to initiate the accumulation step for an extra 1-3 milliseconds. While this seems minor, we observed an increase in identified proteins and peptides across all concentrations, with an average gain of 5% in protein groups and 10% in peptides. Among the identified protein groups, more than 80% had less than 20% CVs.Furthermore, we also analyzed a small number of single cells and observed an average increase in protein groups identifications of 5.5 % (6,041 compared to 5,724) and 4% for peptide groups (36,156 to 34,993) when using the modified Orbitrap Astral MS and the low input application mode compared to the standard Orbitrap Astral MS.

Evaluating Trends in Methodology for Urine Drug Testing Christine Snozek (Presenter) Mayo Clinic Arizona

Poster #42b View Map

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

INTRODUCTION:
With increasing use of mass spectrometry (MS) in clinical and toxicology laboratories, there is much discussion about labs employing MS for testing, either in addition to or instead of immunoassays (IAs). Due to the rapidly-changing nature of therapeutic and non-therapeutic drug use, toxicology testing requires labs to examine their test methodologies more frequently than other areas of the clinical lab. However, despite numerous anecdotal reports of labs implementing MS, there are no studies systematically evaluating patterns of IA versus MS utilization for drug testing on a large scale. College of American Pathologists (CAP) proficiency testing surveys offer a unique opportunity to evaluate these patterns in a large group of laboratories.

OBJECTIVE:
To quantify patterns of MS utilization in laboratories performing drug testing and using CAP surveys.

METHODS:
Data were extracted for the CAP Drug Monitoring in Pain Management (DMPM), Urine Drug Screening (UDS) and Urine Toxicology (UT) Surveys. UDS is screen-only, with nearly all respondents reporting automated or point-of-care IA results. DMPM and UT include both screening and confirmation results, including MS. Methodology data were retrieved from odd-numbered years 2015-2023 for the cocaine metabolite benzoylecgonine (BZE), and 2017-2023 for morphine (MOR) and fentanyl (FEN). A single challenge for each drug was identified from each Survey per year. Only one methodology was included for labs responding to >1 Survey, prioritizing DMPM and UT responses over UDS to incorporate confirmation testing.

RESULTS:
IA screen-only results consistently comprised >80% of responses for BZE and MOR, but increased from 13.5% in 2017 to 40.4% in 2023 for FEN, reflecting greater availability of IA for that drug. For labs reporting methods other than IA-only, the traditional IA to MS reflex pattern predominated for both BZE (58.1%) and MOR (47.1%) in the first year, but declined by 2023 to 36.5% and 30.8%, respectively. At the same time, the percentage of labs reporting MS-only increased steadily for both BZE (32.5% to 58.7%) and MOR (45.2% to 63.2%). In contrast, FEN showed a stable pattern throughout the study period, with >75% using MS only compared to approximately 20% reporting IA to MS reflex. The choice of MS platform also shifted over time, with fewer labs using gas chromatography (GC-MS) and more implementing time-of-flight (TOF) compared to relatively stable fractions of respondents using liquid chromatography with single-stage (LC-MS) or tandem (LC-MS/MS) detectors. However, LC-MS/MS remained the predominant MS methodology throughout the study.

CONCLUSION:
CAP proficiency testing data allowed evaluation of trends in drug testing methodology over nearly a decade. IA tests are readily available for BZE and MOR; BZE has minimal therapeutic use whereas MOR has both therapeutic & recreational uses. FEN testing is comparatively newer, due to its implications in the North American opioid epidemic. Despite stable percentages of IA screen-only responses for BZE and MOR, laboratories using MS reported decreasing reliance on traditional IA to MS reflex testing and increasing MS-only strategies. In contrast, IA-only responses increased for FEN, reflecting greater availability of commercial assays developed in response to emerging FEN usage in the opioid epidemic. However, MS utilization for FEN remained stable, with the vast majority of labs relying on MS-only testing rather than IA to MS reflex. These results provide some quantitation of the growth of MS within clinical and toxicology laboratories, including shifts from older techniques such as GC-MS to newer platforms such as TOF.

Exploring Tryptophan Metabolism in Schistosomiasis: Potential Biomarkers for Chronic Inflammation Rachel Alfaro Leone (Presenter) UCSF

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Poster #43b View Map

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

INTRODUCTION:
Schistosomiasis is a common helminth infection that affects an estimated 250 million people worldwide. Adult schistosomes reside in the vasculature and extrude eggs through the liver and gut or bladder and cervix, leading to chronic inflammation in these tissues. While acute infection may manifest with minimal symptoms, inflammation generated by chronic infection can cause long term complications such as liver fibrosis, genitourinary damage, and cancer.

Products of tryptophan metabolism have been utilized as tools in understanding inflammatory response triggered by various diseases. Inflammatory signaling induces downstream metabolism of tryptophan along different pathways. Enzyme activity may be shunted towards kynurenine or serotonin production, due in part to the cytokine response.

We hypothesize that key metabolites of tryptophan metabolism may correlate to chronic inflammatory state in schistosomiasis. To study this, we evaluated metabolite levels in serum from pediatric patients with S. haematobium infections pre- and post-anthelmintic (praziquantel) treatment compared to uninfected controls.

METHODS:
This study utilized remnant serum samples from 20 S. haematobium-infected and 18 non- infected patients collected during a previous prospective longitudinal school-based study of 5-15 year old children in Eswatini. Pre- and 12-month post-treatment samples were analyzed for 17 of the 20 infected patients. The BCM IRB and the Eswatini Health and Human Research Review Board approved use of these specimens (IRB #H-34210).

We established a panel of 7 target analytes – Tryptophan (Trp), Kynurenine (Kyn), Kynurenic Acid (KynA), 5-hydroxyindole acetic acid (5-HIAA), Serotonin/5-Hydroxytryptamine (Ser), 3-Hydroxykynurenine (3-HK), and 3-hydroxyanthranilic acid (3-HAA). These analytes were chosen due to their relationship with pro-inflammatory cytokines and their ability to indicate key shifts in tryptophan degradation based on published studies. A 10-point calibration curve was developed across a range of concentrations (16.4–50,000 ng/mL for tryptophan, 1.1-25,000 ng/mL for serotonin, 6.6– 25,000 ng/mL for all else) based on published studies. L-tryptophan-d5 (250 ng/mL) was used as an internal standard for all analytes.

The samples were run through a Phenomenex Kinetex 5 uM EVO C18 100A (100x2.1mm) column and analyzed on a Sciex 4500. Mobile Phase A and B were 0.05% Formic Acid in Water and 0.05% Formic Acid in Acetonitrile respectively. The time gradient was as follows: 0-0.1 min, 5% B; 0.1-0.5 min, 5% B; 0.5-3.5 min 5-40% B; 3.5-4.5 min, 40-80% B; 4.5-7.5 min, 80-5% B.

The two best performing transitions were chosen for each analyte and were run together on the same assay. Small molecule extraction was performed by protein precipitation of 25 uL of serum. The supernatant was dried under nitrogen to concentrate the yield and reconstituted using Mobile Phase A for LC-MS analysis.

RESULTS:

Method Validation
The standard calibration curve exhibited an average r value of >0.98 and %error for each calibration point had an average of 12.21% across all analytes. Two analytes (3-HAA and 5-HIAA) were below the limit of quantitation were not analyzed.

Analysis of Patient Samples
Serum samples were split into three groups for analysis: infected pre-treatment, infected post-treatment, and uninfected controls. The Kyn/Trp ratio was elevated in infected pre-treatment serum compared to uninfected controls (median, 0.023 vs. 0.015; P < 0.002) and treated versus uninfected (median, 0.021 vs. 0.015; P = 0.01) pairings. The Ser/Trp ratio was lower in pre-treatment serum versus uninfected controls (median, 0.015 vs. 0.030; P = 0.01), but there was no difference between treated and uninfected samples (median, 0.024 vs. 0.030; P = 0.43). The tryptophan concentrations were not significantly different between pre-, post-treatment, or uninfected serum, reflecting that the ratio differences were driven by kynurenine and serotonin concentrations alone. The Kyn/Trp ratio was not significantly different between pre- and post-treatment in serum (median, 0.023 vs. 0.021; P = 0.52). However, the Ser/Trp ratio was increased (median, 0.015 vs. 0.024; P = 0.03) between these groups.

DISCUSSION:
Studies of chronic inflammation via tryptophan metabolism predominantly utilize the Kyn/Trp ratio as the main indicator for inflammatory state. This ratio is affected by cytokines, which induce the enzyme, indoleamine 2,3-dioxygenase (IDO), to metabolize tryptophan to kynurenine. Our assay goes beyond the Kyn/Trp axis to assess an alternate metabolic pathway from tryptophan to serotonin, which may also be induced by different patterns of inflammatory signals. Our study demonstrates disturbances in both the Kyn/Trp and Ser/Trp ratio depending on Schistosoma infection status and treatment. The Ser/Trp ratio is significantly lower in pre-treatment samples when compared to post-treatment and uninfected controls, while the Kyn/Trp ratio was elevated in both pre- and post-treatment groups compared to uninfected controls. This suggests that Kyn/Trp ratio does not revert to the levels prior to Schistosoma infection, while the Ser/Trp ratio does, indicating that different metabolite ratios may serve as helpful biomarkers of inflammatory status at different stages of infection. Further investigation is needed to assess whether the pattern of systemic cytokines in these samples can be related to the tryptophan metabolite levels.

Our findings strengthen the role of tryptophan-derived metabolites as biomarkers of systemic inflammation. Many studies only evaluate the Kyn/Trp ratio as it has classically served as the main marker of chronic inflammatory conditions. This study suggests that other ratios of tryptophan metabolism may be valuable tools in understanding alternative states of chronic inflammation within different diseases or conditions.

Transferrin Analysis versus Glycomics in Congenital Disorders of Glycosylation Testing Matthew Schultz (Presenter) Mayo Clinic

Poster #44b View Map

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

INTRODUCTION:
Congenital disorders of glycosylation (CDG) are a heterogeneous group of rare genetic conditions that present with a broad clinical spectrum. For CDG affecting N-linked glycosylation, transferrin glycosylation analysis performed by a variety of methods has been the cornerstone of biochemical testing. More recently, glycomics approaches have become available in clinical laboratories. In this study, we retrospectively evaluated the incremental diagnostic utility of glycomics testing compared to transferrin analysis alone across a range of CDG conditions in our laboratory.

METHODS:
Combined transferrin and ApoCIII glycoform analysis is performed by intact protein analysis using online immunoaffinity enrichment (via an in-house produced column), followed by electrospray ionization mass spectrometry (ESI-MS) on a SCIEX X500B QTOF high-resolution instrument. Spectra are deconvoluted to a single charge state profile and major glycoform ratios are reported along with qualitative profile assessment of any abnormal glycoforms present. Glycomics analysis entails overnight PNGase F–mediated glycan release, graphitized carbon enrichment, permethylation, and analysis using a SCIEX 5800 MALDI-TOF instrument. Glycomic profiles are reviewed and assessed utilizing Collaborative Integrative Laboratory Reports (CLIR) multivariate pattern recognition software. Results were reviewed from January 2022 to April 2025 and includes 10,484 transferrin/ApoCIII tests with 421 having combined glycomic analysis also performed.

RESULTS:
Of cases analyzed by intact protein analysis, 552 samples demonstrated elevated mono-glycosylated transferrin ratio (type I pattern) and 211 samples demonstrated an elevated ratio of a glycan lacking sialic acid (trisialo; type II pattern). Among cases with both transferrin and glycomics data available, both tests were abnormal in 48 samples. Notably, for known individuals with PMM2-CDG, the most prevalent subtype (representing 60–70% of CDG cases in the literature), transferrin and glycomics were both abnormal in all cases (n=18). Three diagnostic samples were identified by glycomics alone (SLC35C1-CDG (2) and MOGS-CDG(1)) while glycomic analysis suggested abnormalities in four additional individuals in which outcomes could not be confirmed.

CONCLUSION:
Transferrin glycoform analysis remains a valuable first-tier screening method for N-linked CDG. Transferrin and glycomic analysis provide complementary information for many conditions and glycomic analysis enhances detection of several CDG including SLC35C1-CDG and MOGS-CDG. Further studies are warranted to outline the role of glycomics in specific diagnostic contexts.

Leveraging MALDI-MSI for Studying Spatial Molecular Mechanisms Involved in Vibrio cholerae Biofilm Formation. Ethan Older (Presenter) University of California Santa Cruz

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Poster #45b View Map

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

INTRODUCTION:
Cholera is an infection of the small intestine caused by pathogenic strains of the bacterium Vibrio cholerae and is characterized by severe diarrhea and rapid dehydration, left untreated, the mortality rates are 50-60% (1,2). In 2025, the World Health Organization (WHO) recorded 37,500 cholera cases and 2,400 deaths across 26 endemic countries. However, WHO estimates that the number of officially reported cases represents only 5-10% of the actual number of cases (3). The V. cholerae infection cycle relies on biofilm formation and dispersal. V. cholerae biofilm formation is controlled by QS molecules including cholerae autoinducer-1 (CAI-1), autoinducer-2 (AI-2) and 3,5-dimethyl-pyrazin-ol (DPO) which suppress biofilm formation through the repression of the critical transcriptional activator vpsT (4). The second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) has also been found to bind to VpsT, activating multiple genes involved in biofilm formation (4,5). Interestingly, certain members of the human gut microbiome have been found to modulate V. cholerae infection through microbial metabolites (6,7). However, the identities of these metabolites, and the mechanisms by which they regulate V. cholerae biofilms remain unknown. We hypothesize that some of these unknown metabolites may be leveraged as a mechanism to modulate cholera infection, providing a potential treatment or preventative factor.

OBJECTIVE(S):
Probe the spatial distribution of excreted and intracellular V. cholerae metabolites during biofilm formation using Expansion Mass Spectrometry (ExMS).

METHODS:
V. cholerae microcolonies will be grown on solid media alone and imaged. Microcolonies will also be grown on functionalized hydrogels and physically expanded for analysis by Expansion Mass Spectrometry (ExMS). We have recently developed ExMS for use with human high grade serous ovarian cancer cell lines by applying an ionically crosslinked alginate and covalently crosslinked polyacrylamide to create double-crosslinked hydrogels capable of 20x stretch. These chemically inert materials require surface functionalization for 2D microcolony attachment. We have systematically optimized a procedure for attachment using Sulfo-SANPAH, a water-soluble heterobifunctional crosslinker. Surface adhesion is essential for cells to endure the physical stress of stretching and prevents premature detachment. A mechanically engineered platform will be used to equibiaxially stretch hydrogels with attached cells, enhancing expansion factors before MALDI-MSI, while maintaining cell viability and structural integrity. A MALDI matrix will be applied using an HTX-TMSprayer and MALDI-MSI will be performed using a Bruker timsTOF flex mass spectrometer with a 50 µm spatial resolution in positive and negative mode. The detection of microbial metabolites will be optimized by tuning experimental conditions including matrix composition, sample preparation, and MALDI parameters.

RESULTS:
Previous work in the Sanchez lab has enabled the label-free detection of c-di-GMP, the second messenger involved in regulating biofilm formation, in microbial colony biofilms using MALDI-MSI (8). We expect that MALDI-MSI of V. cholerae microcolony growth will reveal longitudinal metabolite production patterns that can be used to delineate stages of biofilm development. For example, increased c-di-GMP production during early microcolony growth facilitates expansion of V. cholerae cell populations and extracellular matrix production leading to biofilm formation. In addition to c-di-GMP, the production of QS molecules (i.e., CAI-1, AI-2, and DPO) also control V. cholerae biofilm formation. We will correlate these signals with stages in the V. cholerae life cycle to develop a baseline model for V. cholerae biofilm formation. We will also develop and optimize an LC-based method for rapid and quantitative validation of V. cholerae metabolites. Using this method, we will analyze a cohort of intestinal samples from V. cholerae-infected and uninfected mice to prioritize additional infection-relevant signals.

DISCUSSION:
This work is envisioned to spatially map V. cholerae metabolite production during the natural progression from microcolony to biofilm. While these experiments will focus on the detection of c-di-GMP and the known QS molecules, the data generated provide the unique ability for the untargeted discovery of additional exogenous microbial metabolites that affect V. cholerae biofilm regulation. Investigation of these metabolites may then reveal a factor that can be leveraged to prevent cholera disease through control of V. cholerae biofilms.

REFERENCES:
1. Charles, R. C. & Ryan, E. T. Cholera in the 21st century. Curr. Opin. Infect. Dis. 24, 472 (2011).
2. Sack, D. A., Sack, R. B., Nair, G. B. & Siddique, A. Cholera. The Lancet 363, 223–233 (2004).
3. Ali, M. et al. The global burden of cholera. Bull. World Health Organ. 90, 209–218 (2012).
4. Papenfort, K. et al. A Vibrio cholerae autoinducer-receptor pair that controls biofilm formation. Nat. Chem. Biol. 13, 551–557 (2017).
5. Krasteva, P. V. et al. Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP. Science 327, 866–868 (2010).
6. You, J. S. et al. Commensal-derived metabolites govern Vibrio cholerae pathogenesis in host intestine. Microbiome 7, 132 (2019).
7. Pauer, H. et al. Bioactive small molecules produced by the human gut microbiome modulate Vibrio cholerae sessile and planktonic lifestyles. Gut Microbes 13, 1918993 (2021).
8. McCaughey, C. S. et al. A label-free approach for relative spatial quantitation of c-di-GMP in microbial biofilms. Anal. Chem. 96, 8308–8316 (2024).

Development of Rapid Methicillin-Resistant Staphylococcus aureus Screening Software Based on MALDI-TOF MS and Machine Learning Jong-Min Park (Presenter) Hallym University

Poster #46b View Map

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

INTRODUCTION:
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections such as bacteremia, pneumonia, and surgical wound infection, and it is challenging to deal with as it has resistance against many other antibiotics. Rapid detection of MRSA is essential to prevent such deadly infections, and prompt treatment of antimicrobials against MRSA improved treatment outcomes. However, traditional MRSA screening/confirmatory tests based on molecular diagnostics involve PCR, sequencing, and DNA chips plus bacterial culture with antimicrobial susceptibility tests, which are time-consuming (> 2 days), labor-intensive, and costly. In this study, AMRQuest software was developed to screen for MRSA and identify bacterial species simultaneously. The AMRQuest software provides a score that represents the likelihood that the bacterial isolate is MRSA by comparing the MALDI-TOF spectra of S. aureus with the database using a machine learning technique. We embedded the AMRQuest software into the MALDI-TOF MS with a bacterial identification system and used it to identify S. aureus isolates from patients and perform methicillin-resistance testing simultaneously, enabling faster treatment of patients with severe MRSA infections and preventing unnecessary antimicrobial overuse.

METHODS:
The AMRQuest software was trained using mass spectra of 927 S. aureus isolates as the training set and logistic regression machine learning algorithm. Single-blinded and randomized MRSA screening by AMRQuest was performed at the three tertiary-care hospital. The cefoxitin disk diffusion test was conducted to use the test results as the reference values. To evaluate the clinical performance of AMRQuest, diagnostic performance parameters, including PPV, NPV, sensitivity, specificity, and Cohen’s kappa, were calculated from the results of AMRQuest and compared to those of conventional tests and the cefoxitin disk test.to determine the accuracy of the AMRQuest software. The SCCmecA gene was detected to compare the discrepancy between the cefoxitin disk diffusion test and the results of AMRQuest MRSA screening.

RESULTS:
MRSA screening was performed using 537 of the S. aureus isolates including 231 of MRSA and 306 of methicillin-susceptible S. aureus (MSSA) from three tertiary-care hospitals. The AMRQuest scores of MRSA isolates had a higher median value (0.99995) than those of MSSA isolates (1.82498 × 10−4). The Mann−Whitney unpaired test confirmed that MRSA and MSSA were clearly categorized using the AMRQuest score (P < 0.0001), with only seven isolates being classified differently using the cefoxitin disk diffusion test. when the cutoff value for screening MRSA using AMRQuest was set to >0.5, the sensitivity, specificity, the area under the ROC curve of AMRQuest for the testing set were 98.7%, 97.7%, and 0.979, respectively.

CONCLUSION:
In this study, we presented the AMRQuest software based on MALDI-TOF MS and logistic regression as a rapid MRSA screening method. The AMRQuest results were similar to those of the cefoxitin disk diffusion test, which was used as the reference method. In conclusion, it is suggested that the AMRQuest software can be used as a rapid MRSA screening method in a clinical laboratory, and further studies are needed to determine the causes of the discrepancy from the reference method as well as to identify unknown features.

Increasing the Accessibility of Creatinine Screening Through At-Home Collected Dry Blood Spot Samples Using an LC-MS/MS Assay Carlos Torres (Presenter) LetsGetChecked

Poster #47b View Map

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

INTRODUCTION:
Health disparities in diagnosis and treatment of chronic kidney disease (CKD) are apparent. Improving access to CKD screening may decrease some of these outcomes. Blood creatinine concentrations are a mainstay in CKD screening, as it is commonly used to estimate glomerular filtration rate. Creatinine is classically measured in serum/plasma using high-throughput chemistry analyzers. In order to make screening more accessible, a non-traditional approach was taken and the sample type finger prick Dried blood spots (DBS) was chosen. This approach allows for at-home collection as an option rather than a more invasive venipuncture collection from a clinic. With the sensitivity that LCMSMS can provide, creatinine is accurately measured from dried blood spots even with minimal sample volume extract. We aim to demonstrate how dried blood spot creatinine screening via LCMS/MS has contributed to and impacted patient accessibility for CKD screening.

METHODS:
Since DBS Creatinine has become available to the public, LetsGetChecked has been able to provide a CKD screening program in collaboration with various health plans to screen patients nationwide where medically necessary. The test is CLIA certified with licensure approval from New York Department of Health; the following results were procured using data from real patients in a live clinical setting.

RESULTS:
Since March 2024, 85,667 individuals have been screened for CKD via dried blood spot creatinine with LCMSMS for analysis. The normal rate of these individuals is 85% to the abnormal rate of 15%. Out of the 85,677 patients, 3155 individuals were below the age of 50 and 82,472 were above the age of 50;46,203 were women and 39,464 were men. The total data men and women tend to increase in creatinine levels with increased in age.

CONCLUSION:
Creatinine via dried blood spot analysis has been able to contribute to CKD screening through collaboration with health plans, as 85,667 individuals have been reached. By improving awareness and participation of DBS creatinine testing, patient screening numbers could be comparable to blood creatinine screening. Future directions would be increasing contributions from individuals <50 years old that are at-risk. In addition, analysis of cohort data with collaborations with health plans to generate statistical trends that could be used to enhance the effectiveness of such screening programs.

LC-MS/MS Separation of 11-Nor-9-Carboxy-Δ9-THC and 11-Nor-9-Carboxy-Δ8-THC in Urine Using the SCIEX Citrine System Mahesheema Ali (Presenter) The MetroHealth Medical Center

Poster #48b View Map

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

INTRODUCTION
11-Nor-9-carboxy-Δ8-tetrahydrocannabinol (Carboxy-Δ8-THC) is a metabolite of Delta-8-tetrahydrocannabinol (Δ8-THC), a cannabinoid found in cannabis, while 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (Carboxy-Δ9-THC) is the primary metabolite of Delta-9-tetrahydrocannabinol (Δ9-THC). Due to their structural similarities with other cannabinoids and metabolites, their separation and analysis present significant analytical challenges. Accurate quantification and differentiation of Carboxy-Δ8-THC and Carboxy-Δ9-THC are critical for regulatory compliance, forensic toxicology, and clinical research.

OBJECTIVE
To develop and optimize an LC-MS/MS method for the separation, quantification, and reporting of Carboxy-Δ8-THC and Carboxy-Δ9-THC in urine.

LC-MS/MS METHODOLOGY and MATERIALS
Standards:
(-)-11-nor-9-Carboxy-Δ8-THC (Item No. T-010)
(±)-11-nor-9-Carboxy-Δ9-THC (Racemic mixture; not for immunoassay, Item No. T-170)
Deuterated internal standards from Cerilliant
Preparation of Internal Standard
Add 100 µL of Δ8-THC-D3 and 100 µL of Δ9-THC-D3 to a test tube.
Add 12.3 mL of methanol.
Mix thoroughly and vortex.
Preparation of Top Stock Solution
Transfer 15 µL of Δ8-THC and 15 µL of Δ9-THC into a 10 mL volumetric flask.
Adjust the volume to 10 mL using methanol.
Mix thoroughly.
Follow the Standard Operating Procedure (SOP) for further steps.
Instrumentation and Conditions
System: SCIEX® Citrine/6500 Triple Quadrupole
Column: Phenyl-hexyl (Kinetex®)
Mobile Phase:
(A) 0.1% formic acid in water
(B) 0.1% formic acid in methanol
Gradient: 2–75% over 9 minutes
Mass Spectrometry (MS/MS) Conditions:
Ionization Mode: Negative
Optimized Parameters:
Ion source: Turbo Spray Ion Drive
Polarity: Negative
Curtain gas (CUR): 30
Collision gas (CAD): 8
Ion spray voltage (IS): -4500V
Temperature: 650°C
Ion source gas 1 (GS1): 60
Ion source gas 2 (GS2): 50
Declustering potential (DP): -110V
Entrance potential (EP): -10V
Collision energy (CE): -28V
Collision cell exit potential (CXP): -11V
Liquid Chromatography (LC) Conditions
Column Oven Temperature: 50°C
Injection Volume: 2 µL
Flow Rate: 0.8 mL/min
Detection: MS/MS (SCIEX® 6500 Triple Quad)

RESULTS
The method demonstrated robust analytical performance, ensuring optimized separation, increased throughput, and reproducible quantification across relevant analyte concentration ranges. It exhibited excellent repeatability, with calibration curves showing good linearity. The complete resolution of Carboxy-Δ8-THC and Carboxy-Δ9-THC stereoisomers was achieved, allowing for both qualitative and quantitative analysis. The respective retention times for Carboxy-Δ8-THC and Carboxy-Δ9-THC were determined to be 7.45 and 7.70 minutes.

CONCLUSION
The developed LC-MS/MS method effectively differentiates Carboxy-Δ8-THC and Carboxy-Δ9-THC metabolites. The separation of these compounds was accomplished within approximately 9 minutes using a phenyl-hexyl column and 0.1% formic acid in methanol as the mobile phase under optimized conditions on the SCIEX® Triple Quad system. This method provides a reliable approach for regulatory, forensic, and clinical applications.

Automated Microelution Plate Solid Phase Extraction for Quantitative LC-MS/MS Analysis of Fentanyl Analogs, Nitazenes, and Xylazine in Human Urine Qi Huang (Presenter) Tecan

Poster #49b View Map

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

INTRODUCTION:
The continued prevalence of fentanyl and its analogues, as well as nitazenes and xylazine, underscores the urgent need for reliable analytical tools in clinical and forensic toxicology, as these substances continue to drive overdose fatalities. Accurate detection of these substances in biological matrices is critical in clinical and forensic toxicology. This presentation evaluates the use of a 96-well microplate based SPE format offering microelution capabilities for the extraction of fentanyl analogs, nitazenes, and xylazine from human urine, using automated and semi-automated liquid handling and positive pressure instruments.

OBJECTIVES:
This study demonstrates the extraction of fentanyl analogs, nitazenes, and xylazine from human urine using a strong cation exchange SPE sorbent, packed into a microelution plate. The adoption of such plate enables streamlined automation, improved sample throughput, and significantly lower elution volumes compared to traditional SPE columns. This study explores a fully automated and a semi-automated workflow.


METHODS:
The sample panel consisting of 30 analytes was spiked into drug-free human urine (Utak) yielding a concentration of 1 ng/mL. Samples were loaded onto Tecan HPSCX 96-well microelution SPE plates. For each of the SPE workflows, both the Resolvex® A200 and i300 instruments were used for positive pressure loading and solvent dispensing, while the Resolvex® i300, integrated on a Fluent automated workstation, also provided drying of purified samples. After wash steps, a low elution volume totaling 50uL, delivered as two 25uL dispense and elute steps were used to elute the analytes. LC-MS/MS quantitation was performed on the eluates, which were either injected directly or dried and reconstituted, before injection. The Sciex 7600 ZenoTOF in HR-MRM mode with internal standards, was used for quantitation.

RESULTS:
The microelution plate SPE workflow was evaluated for extraction efficiency, reproducibility, and operational robustness. LC-MS/MS analysis on the Sciex 7600 of the extracted samples provided ample sensitivity for the majority of the compounds, at a concentration of 1ng/mL in urine. To assess the efficiency of the SPE extraction, the recovery of the samples was calculated using a post-extraction spike in. The sample recovery showed high extraction efficiency, with 90% of analytes achieving recoveries ≥80%. Technical replicate samples were processed to evaluate the reproducibility, demonstrating low CVs and high quantitative stability of the workflow. Two thirds of the samples analyzed resulted in CVs being lower than 10%.

CONCLUSION:
The SPE methods have been proven to effectively extract a panel of 30 compounds consisting of fentanyl and its analogues, as well as nitazenes and xylazines from human urine. By utilizing liquid handling and positive pressure instruments and the microelution plate based SPE workflow, the method was automated enabling low-volume elution, reduced hands-on time, and a simple, efficient protocol ideal for high-throughput toxicology assays and testing.

Lipid Makeover: How Aging Transforms Adipose and Liver Tissues Yoann Rodriguez (Presenter) University of Illinois Chicago

Poster #50b View Map

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

INTRODUCTION:
Aging is associated with widespread metabolic changes that impair energy homeostasis and increase disease risk. Adipose tissue and liver play central roles in lipid storage, thermogenesis, and metabolic regulation, making them particularly susceptible to age-related dysfunction. While previous studies have described shifts in lipid composition with aging, comprehensive molecular profiling across multiple tissues remains limited. In this study, we employed untargeted lipidomics using high-resolution LC/Q-TOF mass spectrometry to systematically investigate lipid remodeling in white adipose tissue (WAT), brown adipose tissue (BAT), and liver across the lifespan of mice. By obtaining broad lipid coverage, including low-abundant and isomeric lipids, we aimed to uncover age-dependent signatures and pathways underlying metabolic inflexibility.

METHODS:
Liver, WAT, and BAT were collected from female C57BL/6 mice across three ages groups (young, mature, and old; n = 8 per group). Lipids were extracted from 10 – 15 g of tissue using a modified methanol/MTBE water protocol, incorporating the EquiSPLASH internal standard mix for normalization. Samples were homogenized, phase-separated, and dried under nitrogen before reconstitution in 9:1 methanol/chloroform for LC-MS/MS analysis. Untargeted lipidomics was performed using a 16-minute reversed-phase method on a ZORBAX Eclipse Plus C18 column coupled to the Revident LC/Q-TOF system operating in both positive and negative ion modes. Pooled QC samples were used for system suitability and Iterative MS/MS acquisition to enhance lipid annotation. Lipid species were identified using MS-DIAL 5, Lipid Annotator, and MassHunter Explorer. Data was normalized to the corresponding internal standard and tissue weight and downstream visualization was conducted using the lipidR package.

RESULTS:
Lipidomic profiling across liver, WAT, and BAT revealed distinct tissue-specific alterations associated with aging. Principal component analysis (PCA) showed clear clustering by tissue type, underscoring unique lipidomic signatures. Differential abundance analysis demonstrated a dramatic increase in the number of significantly altered lipid species in old versus young mice, with the liver and BAT showing the largest shifts. Triacylglycerols, EtherTG and TG esters were consistently upregulated in aged tissues, while phospholipids (PC, PE) and sphingolipids (SM, Cer) were commonly downregulated. Chain length analysis indicated enrichment of long-chain lipids in older mice, particularly in BAT and liver, suggesting age-related shift in lipid biosynthesis or degradation pathways. Notably mature age groups displayed intermediate profiles, reinforcing a progressive pattern of lipid remodeling. These findings highlight a systemic loss of lipid homeostasis with age, marked by increased lipid storage and reduced membrane and mitochondrial lipid species.

CONCLUSION:
Untargeted lipidomics using the Revident LC/Q-TOF platform revealed that aging induces profound, tissue-specific remodeling in metabolically active organs such as liver, white adipose tissue, and brown adipose tissue. With over 650 lipids confidently identified, we observed consistent age-related accumulation of long-chain and neutral lipids, including triacylglycerols and ether triacylglycerols. These changes were most pronounced in older mice and were associated with disrupted lipid homeostasis, mitochondrial dysfunction, and membrane composition shifts. Collectively, our findings highlight how aging drives metabolic inflexibility across key energy-regulating tissues, offering insight into lipid-based mechanisms that may contribute to age-associated disease risk.

LC-MS Determination of NO2-Modified Fatty Acids in Human Plasma Jesse Mclaughlin (Presenter) Concordia University

Poster #51b View Map

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

INTRODUCTION:
Nitro-modified fatty acids (NO2-FAs), ethanolamides, and oxylipins are bioactive lipids with roles in inflammatory response, oxidative stress, cell proliferation, and lipid metabolism. They are being investigated for therapeutic potential. Numerous LC-MS assays exist for oxylipins. However, NO2-FAs remain comparatively understudied.

OBJECTIVE:
To develop an LC-HRMS method for the simultaneous analysis of NO2-FAs, oxylipins, and ethanolamides in murine and human plasma.

METHODS:
Sample preparation included C18 solid-phase extraction (SPE), solvent evaporation, and reconstitution, followed by reversed-phase C18 LC-HRMS (Q-TOF). Extraction recoveries and matrix effects were evaluated, and sources of recovery losses were investigated. Analyte stability in the autosampler at 4 ˚C over 72 hours was assessed with and without the inclusion of acetic acid in the SPE elution volume.

RESULTS:
Oxylipin extraction recoveries ranged from 71-83%, and matrix effects ranged from 87-98%. NO2-FA and ethanolamide recoveries ranged 50-94%, indicating significant recovery losses for select analytes. Matrix effects ranged from 68-150%, indicating significant ionization effects. Troubleshooting recovery issues showed no significant losses due to incomplete elution from the SPE cartridges. No significant difference was found in recoveries or autosampler stability of analytes based on the inclusion of acetic acid in the SPE elution volume.

CONCLUSION:
Recoveries remain low for nitrooleate isomers, and further investigation of sources of incomplete recovery is ongoing. Significant matrix effects, particularly for the nitro-conjugated linoleic acid isomers and 10-nitrooleate, present challenges for reliable quantification. Acetic acid in the SPE elution does not have a significant impact on the results, including recovery efficiency and stability within the autosampler.

Additional optimization is required to support robust, simultaneous analysis of these lipid classes in a single LC-HRMS assay.

Unrealized Gains: Advancing Toxicology Methods with Multi-Factorial HPLC-MS/MS Optimization in Oral Fluid and Urine Matrices Phillip Gibbs (Presenter) Clinical Lab Consulting, LLC

Poster #52b View Map

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

INTRODUCTION:
Toxicology laboratories routinely employ HPLC-MS/MS methods with manufacturer default settings on a subset of available parameter settings, potentially limiting analytical performance and sensitivity. In addition, many automated optimization programs do not account for two-factor interactions. A truly global parameter optimization accounting for interactions with these less common settings remains a critical yet underexplored aspect of analytical chemistry that significantly affects detection limits, quantitative accuracy, and method reliability. Traditional approaches rely on standard operating conditions and lack systematic evaluation of key instrumental parameters. This study addresses the gap between common chromatographic assumptions, default instrument settings, and optimized analytical performance in toxicology applications for oral fluid and urine matrices across multiple mass spectrometry platforms.

OBJECTIVES:
The primary objective was to evaluate analytical gains achievable through systematic HPLC-MS/MS parameter optimization compared to manufacturer default conditions on Sciex and Shimadzu systems. Specific aims included: (1) assessing effects of mobile phase additives on ionization efficiency and chromatographic performance; (2) optimizing electrospray ionization source parameters including gas flows, temperatures, and voltages; and (3) conducting compound-specific optimization of multiple reaction monitoring (MRM) transitions and collision energies using non-standard protocols versus automated optimization. Secondary objectives were to quantify sensitivity improvements, evaluate method robustness, and develop optimization protocols applicable to various forensic analyte classes and sample matrices.

METHODS:
A systematic multi-factorial approach employed oral fluid and urine matrices with representative panels including stimulants/illicit drugs, opioids/opiates, benzodiazepines, non-benzodiazepine hypnotics, antidepressants, barbiturates, antipsychotics, anticonvulsants, anesthetics/dissociatives, muscle relaxants, and ethanol biomarkers. Mobile phase optimization evaluated common volatile organic acids, associated ammonium salts, and ammonium fluoride using definitive screening design (DSD). Source parameter optimization on Shimadzu platforms used response surface methodology (RSM) to evaluate nebulizing gas flows, heating gas flows, interface temperature, DL temperature, heat block temperature, and drying gas flows. Equivalent parameters were examined on the 4500 Sciex platform for ethanol biomarkers. Interface voltage optimization employed a custom experimental design for the 8060NX and single factor experiments for the 8050, which lacks the ion focus voltage parameter available in the NX source configuration. Compound-specific parameters, including collision energies and MRM transitions, were optimized using manufacturer-automated fragment selection or stepwise manual methodology for both platforms. Performance metrics included integrated area counts, signal-to-noise ratios, limits of detection and quantification (LOD/LOQ), linearity, precision, accuracy, and assessment of ion suppression or enhancement.

RESULTS:
Optimized conditions showed significant improvements over default parameters. Analyte area counts increased by approximately 1.3 to more than 10-fold across target analytes, with source settings and interface/ion focus voltages providing the most significant gains. Mobile phase optimization confirmed acetic acid as the preferred phase modifier when negative mode analysis was included, while ammonium fluoride improved positive mode analysis but suppressed negative mode signals. Source parameter optimization identified nebulizing gas and heating gas flow as most critical, with optimal drying gas flow differing significantly between the 8060NX and 8050 platforms. Interface voltage showed significant improvements compared to default tune file settings on both Shimadzu platforms, and optimal ion focus parameter setting diverged significantly from the default setting for the 8060NX. Compound-specific MRM transition and filter settings resulted in average signal enhancement of 5-30% compared to default values.

DISCUSSION:
The multi-factorial optimization approach yielded substantial analytical improvements over manufacturer defaults, revealing unrealized potential in routine toxicology methods. Sensitivity improvements exceeding 10-fold for some analytes have direct implications for detecting lower analyte concentrations in forensic samples, potentially expanding detection windows A systematic multi-factorial approach using employed oral fluid and urine matrices for investigative purposes. This systematic approach provides a framework for forensic laboratories to enhance existing methods without requiring new instrumentation, underscoring the importance of moving beyond default conditions toward evidence-based method optimization in forensic toxicology.

Spatial Post-Translational Modifications as Precision Indicators of Breast Cancer Risk and Progression: Are these the NextGen Biomarkers? Peggi Angel (Presenter) MUSC Proteomics Center

Poster #53b View Map

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

INTRODUCTION:
It is a dichotomy that collagen breast stroma is clinically imaged for breast cancer risk and progression, yet molecular regulation of the breast collagen proteome remains poorly defined. Collagen proteins represent the majority of the extracellular microenvironment in any tissue; emergence of collagen stroma is significantly associated with risk of breast cancer (BC). Post-translational modifications (PTMs), particularly those on collagen proteins, play a significant role in cell function within the tissue microenvironment leading to disease. In current studies, we test if the breast microenvironment shows specific PTMs linked to histopathological and clinical characteristics over the continuum of breast health.

METHODS:
Formalin-fixed, paraffin-embedded sections of breast tissue were prepared for multiplexed glycomic and proteomic imaging experiments with appropriate institutional review board permissions. Tissues were dewaxed, sprayed with enzymes (HTX Imaging), digested, and imaged using published protocols. Multiplexed, sequential glycomics used EndoF3 (N-zyme Scientifics) to target core fucosylated N-glycans and PNGaseF (N-zyme Scientifics) to target all other N-glycans. After glycomics, collagenase was used to target collagens and other extracellular proteins. Serial sections were used to define select cell markers (PhotoCleavable Mass-Tags (PCMT); AmberGen). Glycomic, proteomic, and PCMT imaging were done by MALDI FT-ICR or MALDI QTOF (timsTOF fleX, Bruker). Recently reported proteomic workflows were used to derive peptide identifications. Images were visualized by commercial software (SCiLS; Bruker or Weave; Aspect Analytics). Exported data were statistically evaluated (Prism v10).

RESULTS:
This report summarizes data from several independent mass spectrometry imaging studies across the continuum of breast health. Collagen proteins increase with breast density and contain PTMs that facilitate localized cell recruitment, and signaling. Very few studies investigate spatial distribution and regulation of the collagen proteome across the breast continuum. Tissue sections and tissue microarrays were used to investigate post-translational modifications in healthy breast from the Susan G. Komen tissue bank (n=86 samples), ductal carcinoma in situ (DCIS) (n>210) normal adjacent and matched invasive breast carcinomas (IBC; n=79) and triple-negative breast cancer (TNBC; n=80). A main comparator was differences in populations at high risk versus low risk for breast cancer mortality. Area under the receiver operating curve (AUROC) ≥70%, p-value≤0.05 was used to distinguish between groups; nonparametric testing p-value <0.05, FDR controlled, was used to evaluate staging.

ECM-targeted proteomic imaging reported collagen peptides post translationally modified by hydroxylation of proline (HYP) are spatially regulated within the breast microenvironment. In normal breast, single collagen peptides from stroma could differentiate by clinical imaging category (47 peptides, AUROC≥70%, p-value 0.01). In DCIS, specific collagen peptide intensities decreased with progression to invasive breast cancer and associated with an 19.5% decrease in disease free survival probability (hazard ratio 2.8, p-value 0.041). In a cohort of 27 patients with matched primary tissue and later tissue collected with invasive breast cancer, collagen peptides with hydroxylated prolines showed a significant decrease (Mann-Whitney test with multiple comparison correction performed q-value 0.002). Exploratory machine learning suggested a 6-peptide signature could predict between primary DCIS and all later recurrence (AUROC 99.6%, Accuracy 98.5%, positive predictive value 97.1%, negative predictive value 100%. TNBC showed differences in staging with increases in specific peptides from stage I-III, followed by decreases at metastasis. A total of 9 peptides associated with survival based on HYP status, including discoidin domains binding sites, known for immune exclusion. HYP variants tested against normal and TNBC breast epithelial cells demonstrated changes in transcriptional signaling dependent on the site localization of proline hydroxylation.

CONCLUSION:
The collective studies suggest that post-translational modification on collagen peptides contribute to profound pathological changes within the breast microenvironment. Larger studies are warranted to leverage PTM variation as breast cancer targets for improved clinical management, new therapeutic avenues, and as predictors of outcome.

Development of an MS1 Peptide Profiling Analytical Workflow and Feasibility Evaluation for Lung Cancer Detection Honglei Huang (Presenter) Oxford Cancer Analytics

Poster #29b View Map

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

INTRODUCTION:
Early detection of lung cancer is crucial for improving patient outcomes. This study aims to enhance lung cancer detection through a liquid chromatography-mass spectrometry (LC-MS)-based peptide profiling approach. Conventional proteomics relies on precursor (MS1) and fragment ion (MS2) quantification, but complexity of plasma samples and MS2 duty cycles hinder the accuracy and throughput in a clinical setting. Given the limitations this presents to the clinical adoption of LC-MS based proteomics, MS1-only profiling methods have been proposed as an alternative, improving quantification sensitivity and throughput. To assess the ability of such an approach to detect lung cancer we have conducted a pilot study evaluating the robustness, quantification accuracy, and feasibility of MS1-profiling in distinguishing lung cancer cases from controls using machine learning.

METHODS:
A gold reference standard (GRS) was created by pooling plasma from eight healthy individuals. Neat plasma preparation was compared with affinity-based depletion and perchloric acid precipitation. Protein digestion was performed using a single-port solid-phase sample preparation (SP3) method. Peptide samples were analyzed using an Evosep One–Exploris 480 system in MS1-only mode at 120,000 resolution (22 min per sample).

This protocol was applied to a pilot study including 50 non-small cell lung cancer (NSCLC) cases and 50 age- and sex-matched controls. GRS samples were used to define stable features and included for quality control. Features were quantified using Dinosaur software, with in-house tools used for batch correction and retention time alignment. A balanced random forest model evaluated the ability of stable features (CV < 50%) to differentiate cancer from controls using out-of-bag estimates of ROC AUC.

RESULTS:
Neat plasma preparation was selected for MS1 profiling due to simplicity, minimal sample loss, and the lowest coefficient of variation (CV). Our approach detected 30,000 - 40,000 features in GRS samples, with 4,695 consistently identified across 20 replicates (median CV ~21%). Peptides from over 300 proteins were quantified with high reproducibility. Using a balanced random forest machine learning model, we achieved an out-of-bag ROC AUC of 84% for lung cancer discrimination.

CONCLUSIONS:
We developed a sensitive, high-throughput, quality-controlled MS1-based analytical approach for proteomic feature detection in a pilot lung cancer study. Machine learning demonstrated reasonable performance in distinguishing lung cancer from controls. These results support the feasibility of MS1-based analytics for lung cancer detection, but clinical validation is required before application in high-risk population screening.

Volumetric Blood Microsampling and LC-MS/MS Quantitation: A Novel Approach for the Determination of an Anti-Tuberculosis Drug Panel Deema Qasrawi (Presenter) Altasciences

Poster #30b View Map

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

INTRODUCTION:
The collection of low volume capillary blood processed as a dried blood spot (DBS) is a widely used microsampling technique, offering a minimally invasive alternative to conventional venipuncture whilst facilitating simplified sample storage and transport. Moreover, DBS represents an attractive specimen of choice supporting patient-centric blood sampling and enabling the conduct of decentralized clinical trials. Its simplicity and suitability for enabling patient self-sampling is particularly advantageous in remote or resource-limited geographic locations.

The last decade has resulted in innovative designs focused on volumetric blood collection, overcoming previous challenges related to hematocrit-induced sampling volume bias. Consequently, the accuracy and precision with which blood microsamples can now be collected aligns with the statistical requirements for assay performance, particularly in regulated bioanalysis.

To date, there have been no reports for the application of microsampling in support of the current anti-tuberculosis (TB) drug panel, despite TB being the world’s leading infectious disease killer according to a 2024 WHO report. Since treatment for TB involves the co-administration of several anti-TB drugs, the availability and advantages of convenient blood sampling combined with advanced analytics enables reaching rural populations globally.

The current research therefore reports the coupling of modern microsampling technology with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify, in a single run, the following panel of anti-TB drugs and relevant metabolites: sutezolid (SZD), sutezolid sulfoxide (SUS), pretomanid (PA), quabodepistat (QBS), delamanid (DLM), bedaquiline (BDQ) and N-desmethyl bedaquiline (NDB).
 
METHODS:
The Ahlstrom 222 pre-cut cellulose discs in the Capitainer®B microsampling device were pretreated with 1.0 M ascorbic acid (aq) and dried prior to blood collection. Extraction from dried blood involved pre-soaking discs in acetonitrile:methanol:water (10:9:1) for 30 minutes followed by sonication for 60 minutes in acetonitrile (containing stable-label internal standards).
Detection was performed using a SCIEX TripleQuad 6500+ operated in positive-ion electrospray mode with multiple-reaction monitoring (MRM). Chromatographic gradient separation was achieved using a Phenomenex Synergi 2.5 µm Polar-RP column (100 x 2 mm i.d.) operated at 0.50 mL/min and 50°C with mobile phase components consisting of ammonium acetate, citric acid, acetic acid, methanol and acetonitrile. Blood samples and the anti-TB drugs and relevant metabolites were sourced commercially.

RESULTS:
A significant challenge in assay development for such a physicochemically disparate panel of analytes involved establishing chromatographic conditions suitable for retention of the most polar analyte (SUS, clogP = -0.1), whilst keeping run time reasonable by minimizing retention for the most hydrophobic analyte (BDQ, clogP = 7.2). To this end, optimized gradient separation conditions on a Synergi Polar-RP column resulted in a K´of 4.5 for SUS, with BDQ eluted under five minutes. The overall analyte elution order of SUS, SZD, PA, QBS, DLM, NDB, BDQ correlated with increasing clogP, and retention time differences allowed for the application of MRM in discrete periods. The approach of “period-splitting” to optimize MS duty cycle resulted in analyte peaks defined, minimally, from fifteen data points, ensuring reproducible quantitation.

Extractability from dried blood collected using the Capitainer®B device was optimal when using a two-stage process involving pre-soaking and sonication, with recoveries between 85-96%. However, as microsamples aged, reduced yields were noted for DLM, BDQ and NDB. In accelerated stability evaluations with microsamples stored for six days at reduced temperature (-80°C, 4°C), room temperature and elevated temperature (40°C, low and high humidity), recovery increased with decreasing temperature and humidity. Such noted stability trends indicate analyte transformation as opposed to actual decreases in extractability, the latter most often related to changes in dried blood morphology over time.

Since literature reports indicate DLM degradation in plasma (catalyzed by albumin) can be mitigated at pH ≤ 6, and as both NDB and BDQ oxidation products were detected in abundance from aged microsamples, the Ahlstrom 222 pre-cut cellulose discs used as collection substrate in the Capitainer®B device were pretreated with 1.0 M ascorbic acid (aq). With this novel substrate pre-treatment approach, the presence of antioxidant and reduced pH conferred 21-days of stability for all analytes with the oxidative products of NDB and BDQ notably absent.

Analytes extracted from a 10 μL dried blood volumetric sample were evaluated for accuracy, precision, linearity, selectivity and matrix effect (including HCT bias from 25% -55%), successfully meeting all acceptance criteria based upon FDA guidelines for bioanalytical method validation. The analytical range spanned from 10 – 10,000 ng/mL for all analytes. The average coefficient of determination derived from linear regression weighted 1/x2 were ≥ 0.99, with inter-run precision (% CV) and accuracy (% nominal) between 2.2% – 7.5% and 94% – 104%, respectively, for all analytes.

CONCLUSIONS:
The research herein represents the first application of microsampling for determination of the reported anti-TB drug panel, leveraging a novel substrate pre-treatment approach to confer stability for those labile analytes. Combined with LC-MS/MS detection incorporating chromatography which overcomes challenges associated with retention, separation and throughput, method performance data indicates the assay is fit to support future drug development efforts leveraging patient-centric blood sampling.