Poster Presentations for Poster Session D : Thu 14:30 - 15:45

Molecular Hallmark Principles to Guide Protein and Metabolite Quantitation for Integrative Molecular Health Assessment Elliott James Price (Presenter) RECETOX, Masaryk University

Poster #2d View Map

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

INTRODUCTION:
Precision medicine depends on understanding the molecular basis of health and disease. However, there is limited availability of high-capacity assays for the quantitation of extended sets of clinically relevant protein and metabolite biomarkers.

METHODS:
Based on the intersection of the Hallmarks of Health [1] and Hallmarks of Environmental Insults [2] frameworks, we have outlined a suite of mass spectrometry assays to measure protein and metabolite markers and serve as a platform for integrative molecular health assessment. These assays are in various development stages, but each has been designed for application to small samples volumes (e.g. <50 µL of blood plasma and serum), relatively high-throughput (<15 minute runtime) and viable automation for routine operation. Clinical practitioners were consulted regarding analyte inclusion and disease relevance, and the designed assay panels comprise markers of inflammation, immunity, redox status, co-metabolism, and cellular signalling. Once established, standard operation procedures and associated resources shall be made openly available to facilitate adoption and replication in other laboratories for research use.

RESULTS:
To illustrate our approach, we will present a recently established LC-multiple reaction monitoring (LC-MRM) method for the absolute quantitation of 33 proteins & proteoforms [3]. We will describe its application to clinical research studies, discuss the assay development process, report the challenges encountered, and highlight the advantages of the developed LC-MRM method compared to alternative measurement techniques.

CONCLUSION:
Our ambition is to develop of a suite of quantitative mass spectrometry assays for protein and metabolite biomarkers that can be applied in a modular fashion to support study of the molecular mechanisms underlying the onset and progression of various diseases. We welcome input to help prioritise key biomarkers and guide future assay development.

REFERENCES:
[1] López-Otín, C., & Kroemer, G. (2021). Hallmarks of Health. Cell, 184(1), 33–63. https://doi.org/10.1016/j.cell.2020.11.034.
[2] Peters, A., Nawrot, T. S., & Baccarelli, A. A. (2021). Hallmarks of environmental insults. Cell, 184(6), 1455–1468. https://doi.org/10.1016/j.cell.2021.01.043.
[3] Biomarker Analytical Laboratories. (2025). SOP for Quantification of inflammatory proteins in serum/plasma using UHPLC/SRM-MS. Zenodo. https://doi.org/10.5281/zenodo.14974161.

A Simple LC-MS/MS Method for Simultaneous Analysis of 35 Anti-psychotics in Human Plasma for Clinical Research Praveen Kumar (Presenter) Waters

Poster #3d View Map

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

INTRODUCTION:
Antipsychotic drugs (APDs) are frequently used in combination with other drugs, leading to potential pharmacokinetic and pharmacodynamic drug interactions. Many of these interactions have not been adequately studied in clinical research. This study presents a simple, cost-effective and robust LC-MS method which is based on protein precipitation for the simultaneous analysis of 35 APDs in human plasma.

METHODS:
In brief, 50 µL of plasma and 100 µL of protein precipitation solvent, a 70:30 (v:v) mixture of MeOH:0.1M ZnSO4(aq) containing stable isotope labeled internal standards was added to a microfuge tube. The samples were vortex mixed briefly and centrifuged at 18,000 g for five minutes at room temperature. Subsequently, 100 µL of the supernatant was diluted with 100 µL distilled water and the samples were mixed on a shaker for three minutes at 850 rpm prior to analysis. Samples were injected onto a Waters XSelect™ HSS C18 SB XP Column using an ACQUITY™ UPLC™ I-Class PLUS FL System and eluted using a water/methanol/formic acid/ ammonium acetate gradient. The 35 APDs were quantified using a Xevo™ TQ-S micro Mass Spectrometer operated in electrospray positive mode with multiple reaction monitoring.

RESULTS:
For each analyte, the method was demonstrated to be linear across calibration range, with analytical sensitivity investigations showing precise quantification (<20% CV, ≤15% bias). Non-linearity at all levels for all compounds was less than 10% across the targeted linearity interval. Coefficients of variation (CV) for total precision and repeatability for low, mid and high QCs were ≤8.7% CV and ≤7.4%, respectively (n = 25). Recoveries for the low and high QC across all analytes ranged from 91.5-108.7% and 85.7-112.7%, respectively. Mean extraction efficiencies for the 35 APDs ranged from 52.2%-96.9%.

CONCLUSION:
A fast (less than 5 minutes per injection) and inexpensive LC-MS/MS clinical research method was developed for 35 APDs, based on protein precipitation and requiring only 50µL of plasma. The method demonstrated good recovery and extraction efficiency for each analyte, with minimal matrix effects, and total precision ≤8.7% CV.

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

ACQUITY, UPLC, XSelect and Xevo are trademarks of Waters Technologies Corporation.

Rapid Therapeutic Drug Monitoring of Immunosuppressants in Whole Blood: Achieving 8-Second Analysis with LDTD-MS/MS Serge Auger (Presenter) Phytronix

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Poster #4d View Map

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

INTRODUCTION
Everolimus, sirolimus, tacrolimus, and cyclosporine-A are commonly prescribed to prevent organ transplant rejection. However, these immunosuppressants have narrow therapeutic windows and significant variability in their pharmacokinetics, both within and between patients. Therefore, rapid and accurate blood sample analysis is essential to ensure optimal dosing. Monitoring these anti-rejection medications in blood is crucial to maintain drug concentrations within the therapeutic range, ensuring effective prevention of organ rejection while minimizing the risk of toxicity.

OBJECTIVES
The goal of this project is to develop an analytical method that uses the new Axino Ion Source, which employs the LDTD (Laser Diode Thermal Desorption) process in combination with a mass spectrometer for ultra-fast quantification. This approach allows for precise drug level measurements using a reference calibration curve, delivering accurate and timely results to improve patient management.

METHODS
For sample preparation, 100 µL of whole blood (with EDTA-K2) is combined with 100 µL of the internal standard solution in a 2 mL centrifuge tube. To precipitate the red blood cells, 40 µL of 1N ZnSO4 and 145 µL of methanol are added. The mixture is vortexed for 10 seconds and then centrifuged at 14,000 rpm for 2 minutes. For liquid-liquid extraction, 200 µL of the upper layer is transferred to a new tube and mixed with 67 µL of water and 167 µL of MTBE. After phase separation, the MTBE layer is collected and combined with the desorption solution. Finally, 5 µL of the resulting solution is spotted onto a fresh desorption plate and allowed to dry.

The size of immunosuppressant molecules implies a need for higher energy to achieve vaporization. To minimize the binding energy of these molecules, a desorption buffer is optimized. Adding a dopant to the carrier gas enhances ionization, promoting the formation of ammonium adducts rather than protonation. Ammonia (5.6% v/v at 1 µL/min) is introduced into the carrier gas (air at 3 L/min) to achieve the optimal signal. For LDTD-MS/MS analysis, the laser power is set at 85%, with a 6-second ramp. The mass spectrometer operates in positive mode, using a specific MRM transition.

RESULT
For method validation, three-point calibration curves were prepared for each immunosuppressant across specific concentration ranges: 2.25 ng/mL to 50 ng/mL for tacrolimus, everolimus, and sirolimus, and 25 ng/mL to 1000 ng/mL for cyclosporine-A. The correlation coefficients obtained ranged from 0.99511 to 0.99999. Due to the low stability of the standards, a fresh three-point calibration curve is generated for each inter-run analysis. Precision and accuracy are evaluated using a quality control sample, with cyclosporine-A at 500 ng/mL and tacrolimus, everolimus, and sirolimus at 20 ng/mL, analyzed immediately following the calibration curve. The precision (%CV) should be below 20%, and accuracy must remain within 100 ± 15% of the nominal value. Dry stability is also assessed by spotting extracted samples on a Lazwell plate and drying the sample. Quality control samples, including 125 ng/mL and 800 ng/mL for cyclosporine-A, and 6 ng/mL and 25 ng/mL for tacrolimus, everolimus, and sirolimus, are used to evaluate this stability. After one hour at room temperature, the %CV remains below 15%, and the accuracy ranges between 92.4% and 105.2% for all analytes.

CONCLUSION
In conclusion, the method validation demonstrates reliable performance with excellent calibration curve correlations and precision. Accuracy was within the required 100 ± 15% range, and %CV values were below 20%. Dry stability was confirmed with minimal deviation after one hour at room temperature. Additionally, the rapid analysis of immunosuppressant drugs in whole blood using LDTD-MS/MS provides results in just 8 seconds, making it highly suitable for therapeutic drug monitoring (TDM). These results highlight the method's robustness and efficiency for quantifying tacrolimus, everolimus, sirolimus, and cyclosporine-A.

Metabolic Characterization of Common Bacterial Growth Media Emma Guiberson (Presenter) Middlebury College

Poster #5d View Map

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

INTRODUCTION:
Over the past decade, research into the interface between a host and its microbial communities has flourished with a growing interest in the signaling molecules produced to facilitate bacterial communication. While bacteria may be grown under a variety of conditions, several common liquid culture media have been developed. Unfortunately, most media content remains poorly characterized, making metabolic studies challenging to interpret. Although there have been efforts to address this characterization gap on a study-based level, thus far these have not accounted for media batch effects or vendor differences, and offers minimal insight to the community to guide media selection for future studies. Here, we profile various bacterial media to facilitate bacterial metabolic flux research.

OBJECTIVES:
We introduce a resource to aid in media selection when studying bacterial metabolism through characterization of media using targeted and untargeted metabolomics.

METHODS:
Media samples were prepared according to vendor recommendations for bacterial growth. Aliquots of media were then prepared using published methods for LC/MS metabolomics analysis. For targeted analysis, HILIC and reverse phase were performed on a Agilent 1290 UPLC coupled to a 6495C triple quadrupole mass spectrometer in fast polarity switching mode. A known library of ~500 microbially-associated compounds was used to make the transition list. Data was analyzed in Agilent Quant and Metaboanalyst. For untargeted analyses, samples were analyzed using a Waters G2 QTOF coupled to an HPLC for both chromatographic methods and ionization modes. Data were analyzed using MS-Dial against publicly available libraries, and normalized against internal standards; statistical analyses were performed using graphpad Prism.

RESULTS:
Using the targeted method we were able to detect over 450 metabolites reliably within the media samples, which included: Brain-Heart Infusion (BHI), a rich broth for microbiome work, Phenol Red (PR) used for metabolism assays, Luria-Bertani broth (LB) used mostly for E. coli growth, and Nutrient Broth (NB) a general use media. All media included the 20 natural amino acids in comparable levels across media, as well as the four nitrogen bases, which fits with expected values as amino acids are necessary for microbial growth. Some metabolites were not observed in certain broths but present at high relative concentrations in others. For instance, maleamic acid was only detected in LB and PR broths, and was 2-fold higher in LB than PR. N-acetylgalactosamine was detected in all but PR, and was 2-fold higher in BHI and LB broth compared to NB. Caffeic acid was detected in BHI and LB, but not detected in NB or PR. Adenylosuccinic acid was not detected in BHI media, and was 2-fold higher in LB and PR compared to NB. Meanwhile some metabolites were only detected in one media, like 3-amino-5-hydroxybenzoic acid and 2-Aminoethylphosphonic acid only detected in BHI, and galacturonic acid only detected in LB.

CONCLUSIONS:
Overall, these preliminary data highlight the molecular complexity and differences between these different media, and the importance of media selection to minimize molecular interferences and matrix effects in bacterial metabolism studies.

Development of a Specific LC-MS/MS Method for the Quantification of Brain Cholesterol Metabolites in Neurodevelopmental Disorders Sophie Bouhour (Presenter) University of Sherbrooke

Poster #6d View Map

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

INTRODUCTION:
Several neurodevelopmental disorders, including Smith-Lemli-Opitz syndrome, Fragile X syndrome, and autism spectrum disorders, are associated with impairments in peripheral and cerebral cholesterol homeostasis. Oxidized derivatives of brain cholesterol, known as oxysterols, are promising biomarkers due to their ability to cross the blood-brain barrier. The quantification of these oxysterols in plasma offers a non-invasive approach to assess cerebral cholesterol homeostasis in these disorders. However, their low plasma concentrations and their structural similarities present significant analytical challenges, with only a few methods available for their simultaneous analysis.

OBJECTIVE:
This study aims to develop and validate a sensitive and specific method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the analysis of 18 compounds: 5 cholesterol precursors, 5 auto-oxidized oxysterols, and 8 enzyme-derived oxysterols.

METHODS:
The LC-MS/MS method was developed using an Acquity UPLC system coupled with a Xevo TQ-S micro mass spectrometer (Waters). The ionization source was electrospray ionization in positive mode. Stable deuterium-labeled analogs were used as internal standards for each compound.

RESULTS: The dynamic Multiple Reaction Monitoring (MRM) acquisition mode was employed. Specific transitions were designated for the 18 oxysterols and their respective internal standards. A preliminary isocratic separation of oxysterols was obtained using a C8-silica column at 62.5% acetonitrile with 0.1% formic acid (B) and 37.5% HPLC-grade water with 0.1% formic acid at a flow rate of 0.3 mL/min. This initial step was followed by a linear gradient from 62.5% to 98% acetonitrile in order to enhance the separation of the most hydrophobic compounds. This chromatography setup provided an initial resolution of the 18 oxysterols, with a total run time of 17.5 minutes. This preliminary method demonstrated linearity between 5 and 1450 nM for 17 oxysterols in pure standards solutions, with limits of quantification of 1 nM for 10 compounds and 5 nM for the remaining 7 oxysterols.

CONCLUSION:
Mass spectrometry, ionization, and chromatographic separation parameters will be further optimized. The method validation will be performed according to Clinical & Laboratory Standards Institute (CLSI) C62 guidelines. Oxysterols will then be quantified in individuals with neurodevelopmental disorders. In the long term, this method will be implemented in clinical laboratories to support potential diagnostic applications and prognostic monitoring of cholesterol-related neurodevelopmental disorders.

HistoProbe: A Dual-Mode Thermally Assisted Microfluidic Platform for Rapid, Solvent-Minimal Mass Spectrometry Analysis of FFPE Tissues Malek Hassan (Presenter) Queen's University

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Poster #7d View Map

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

INTRODUCTION:
Formalin-fixed paraffin-embedded (FFPE) tissues are essential clinical resources, yet conventional deparaffinization methods are slow, laborious, and environmentally unfriendly, relying heavily on toxic solvents. Addressing these limitations, we introduce HistoProbe, a novel microfluidic platform enabling rapid, localized, and minimally solvent-dependent analysis and imaging of FFPE tissues by mass spectrometry (MS).

METHODS:
HistoProbe integrates a modified liquid microjunction surface sampling probe (LMJ-SSP) with precise thermal regulation adapted from 3D printer hardware. The platform operates in two distinct modes: Online and Offline. Online-HistoProbe enables direct metabolite extraction and real-time analysis from FFPE tissue sections at elevated temperatures (~60 °C) without any prior sample preparation or deparaffinization. It functions as a standalone tool by interfacing directly with the electrospray ionization (ESI) source of the mass spectrometer, offering a plug-and-play solution for rapid FFPE tissue analysis. Offline-HistoProbe enables targeted thermal deparaffinization using reduced volumes of solvents (i.e., ethyl acetate, toluene, or xylene) at controlled temperatures (up to 75 °C). This mode drastically lowers solvent usage compared to standard protocols while maintaining compatibility with high-resolution imaging. Both modes were coupled with desorption electrospray ionization mass spectrometry (DESI-MS) for metabolic profiling and hyperspectral molecular imaging. Data were analyzed using principal component analysis (PCA) for dimensionality reduction and unsupervised clustering of metabolic features.

RESULTS:
Optimization studies demonstrated that Online-HistoProbe eliminated the need for sample preparation and solvent use, reducing total analysis time to approximately 17 minutes per sample and enabling high-throughput analysis exceeding 80 samples per day. The practical spatial resolution of the online mode was approximately 1 millimeter, suitable for rapid tissue screening and metabolic profiling. Offline-HistoProbe, optimized with ethyl acetate at 75 °C, achieved comparable or superior metabolite recovery to traditional xylene-based deparaffinization, while reducing solvent consumption by over 99.5%. It supported a practical spatial resolution of 50–100 micrometers, allowing fine-grained molecular imaging. Both modes reliably distinguished neoplastic from non-neoplastic regions in melanoma FFPE tissues and enabled annotation of key biomolecules, including fatty acids, phosphatidylinositols, and oxidized lipids relevant to cancer metabolism.

CONCLUSION:
HistoProbe represents a significant advancement in mass spectrometry-based histopathology, combining speed, analytical precision, automation, and sustainability. Its dual-mode functionality addresses clinical demands by offering high-throughput direct analysis (Online mode) and high-resolution molecular imaging (Offline mode). HistoProbe substantially reduces solvent consumption, sample preparation time, and environmental impact, demonstrating immense potential for clinical diagnostics, biomarker discovery, and rapid intraoperative tissue assessments.

Back to Basics When Testing for Medetomidine in a Clinical Setting Gwen McMillin (Presenter) NMS Labs

Poster #8d View Map

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

INTRODUCTION:
Stereoselectivity among drug isomers is common and can account for differences in their biological effects. Creating a chiral version of a drug is one strategy that pharmaceutical companies apply to impact dosing/potency as well as improve both safety and response profiles.(1) In the case of the alpha-2 agonist medetomidine, the purified dexmedetomidine enantiomer (Precedex®) is administered intravenously to induce sedation before and during medical procedures, and in the emergency room to manage intoxicated or combative patients. Therapeutic monitoring of dexmedetomidine concentrations is rarely needed in this setting. Levomedetomidine is not produced or used clinically. Racemic mixtures of medetomidine are available for veterinary use, and like xylazine, medetomidine has now emerged as an adulterant in the recreational drug supply, seen most commonly with illicit opioids including fentanyl and heroin. Differentiation of the medetomidine enantiomers is helpful in forensic toxicology and for surveillance of the illicit drug supply. Analytical methods that are designed to differentiate enantiomers however, are relatively complex, limited in scope, costly to perform, and may not be necessary when testing patients in a clinical setting. A chiral method to separate the medetomidine enantiomers was developed by the Center for Forensic Science Research and Education (CFSRE) and required 11 min run time per sample. This method was applied to 100 authentic human specimens obtained from emergency room visits (non-fatal, n=70) or forensic post-mortem investigations(n=30) that screened positive for medetomidine; 90% were positive for both dexmedetomidine and levomedetomidine, while only 10% were positive for dexmedetomidine alone. Concentrations ranged from 0.1 – 32 ng/mL; median for non-fatal overdose was 1.5 ng/mL.(2) These data indicate predominance of racemic medetomidine, suggesting exposure to adulterated illicit drug rather than medically administered drug. The high percentage of samples with the racemic mixture present suggests that a method for detection of medetomidine without chiral separation can have clinical utility, with faster run time and lower test costs.

OBJECTIVE:
A quantitative method for undifferentiated medetomidine by liquid chromatography tandem mass spectrometry (LC-MS/MS) is described in blood with intentions to maximize efficiency of clinical testing by avoiding chiral separation.

METHODS:
A six-point calibration curve (0.1-10.0 ng/mL) and two quality control samples (0.3 and 8.0 ng/mL) were prepared in drug-free blood with medetomidine hydrochloride (Cayman Chemical). To each calibrator, quality control and authentic blood sample medetomidine-13C-d3 was added along with borax buffer and extraction solvent (70:30 1-chlorobutane:ethyl acetate). After mixing by inversion for 15 minutes, tubes were centrifuged at 3600 rpm for 10 min and the organic layer was decanted. After evaporating to dryness, the extract was reconstituted with 80:20 0.1% formic acid and methanol. Analysis occurred with a Shimadzu LC40 configured with an inline filter (Phenomenex KrudKatcher) and Biphenyl analytical column (Restek, 2.1 x 100 mm, 2.7 µm), coupled to a Sciex 6500+ mass spectrometer operated with an ESI source and multiple reaction monitoring in positive mode. Two transitions were monitored for both the analyte (201.0>95.0 and 201.0>115.0) and internal standard (205.0>99.0 and 205.0>115.0). Total run time per sample was 5 minutes. Validation experiments included sensitivity, specificity, imprecision, bias, linearity, matrix effects, recovery, stability, robustness and carryover. Serum and plasma (EDTA) and a 10x dilution were also validated.

RESULTS:
Assay performance data for the LC-MS/MS method demonstrate that it was sufficient to support clinical needs of testing. Empirical limit of detection was 0.01 ng/mL with intra- and inter-assay imprecision throughout the analytical measurement range <4% CV. Estimated bias at the reporting limit (0.1 ng/mL) was -1%. Recovery in all matrices was 97-102% and no interfering substances were identified. Stability was acceptable for at least 30 days at ambient, refrigerated and frozen temperature, as well as after 3 freeze-thaw cycles. Carryover was <1% at 20 ng/mL. Concentrations observed in casework evaluated with this method (n=9) have ranged from 0.38 - 23.0 ng/mL (median 1.7 ng/mL, average 5.9 ng/mL).

CONCLUSIONS:
Laboratory testing designed to detect medetomidine and other adulterants is critically important to forensic toxicology, and for monitoring trends in the drug supply. However, differentiating between isomers of medetomidine may not be needed in a clinical setting. Here a robust and rapid method to detect medetomidine is described to support clinical needs and avoid the complexity and costs of a chiral separation.

REFERENCES:
1. Vallamkonda B, Sethi S, Satti PR, Das DK, Yadav S, Vashistha VK. Enantiomeric analysis of chiral drugs using mass spectrometric methods: a comprehensive review. Chirality, 2024,36:e23705.
2. Walton SE, Stang BN, Kacinko S, Papsun DM, Logan BK, Krotulski AJ. Medetomidine quantitation and enantiomer differentiation in biological specimens collected after fatal and non-fatal opioid overdoses. J Analyt Tox, 2025. Epub ahead of print.

Bringing Precision Omics to Life: Occam's Razor and Real Clinical Applications John Wilson (Presenter) ProtiFi LLC

Poster #9d View Map

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

INTRODUCTION:
The advent of omics, first of genomics and later proteomics, promised significant advancements in clinical diagnostics and personalized medicine. However, we haven’t yet realized these oft-repeated promises: static genes cannot reflect dynamic life and unstandardized, inefficient proteomics workflows yield inconsistent, unactionable results.

We present the journey, guided by Occam's razor, of forging maximum simplicity and effectiveness into real, robust and reliable solutions for every step. These solutions break down the barriers preventing practical application of omics advancements in clinical settings and build the foundation needed to fulfill unrealized long-standing promises. We explain how we maximized the minimal and illustrate with a suite of solutions for clinical application.


OBJECTIVES:
We aim to demonstrate how this concrete strategy can transform bench discoveries into bedside results.

I) To make real-world impact, problems must be distilled into solutions meeting the “Rule of Rs”. They must be:
1) Real: tangible, concrete solutions;
2) Reachable: commercially available with requisite QC, certifications, shipping, tech support…;
3) Realistic: pragmatic, meeting all real-world requirements including being
4) Reasonable (in price);
5) Robust, resilient and reliable: they just work irrespective of sample, apparatus, person, experimental error…, make no assumptions, accepts all data;
6) Rapid;
7) Routine and recognized: everyone knows it just works, so everyone uses it;
8) Reproducible, replicable, repeatable;
9) Refined: Occam commands “As little in to get all needed out”; and hopefully, they are
10) Revolutionary: transform, redefine and make real the “impossible”!

In addition, omics solutions must fundamentally be:
11) Really sensitive;
12) Ready- and really easy-to-use.

III) Finally, to make omics accessible and clinically relevant, solutions must dovetail into a complete, seamless pipeline with robustness, speed, sensitivity, and cost-effectiveness.


Methods:
1) S-Trap sample preparation system
2) S-Trap Turbo
3) S-Trap MAM kit
4) Si-Trap sample preparation system
5) BCA-no-more
6) glyPAQ for glycan structure analysis
7) HYPERsol
8) Tryp-N
9) The Shredder
10) SimpliFi


RESULTS:

1) The S-Trap sample preparation system begins with very strong lysis (fully liquidize a whole mouse heart, brain or skin, etc.), removes all contaminants, and reproducibly, robustly and rapidly prepares samples in spin columns or 96- or 384- well plates. It’s been used worldwide by thousands of researchers to process every kind of sample imaginable from serum, tissues and cells through to feces and dirt, Wharton’s jelly and sputum.
2) The S-Trap Turbo eliminates the need for concentration (speedvacing), which is highly variable, often slow and always annoying. We eliminated it to increase throughput: elute and shoot!
3) The S-Trap MAM kit allows immediate assessment of quality and contamination in biological drugs; multiple sites, instruments and people get the same 100% sequence coverage of mAbs.
4) The Si-Trap sample preparation system yields multiple sample pots for multiomics. We applied it to clear cell renal carcinoma, finding dysregulation of proteins, fatty acids, acyl carnitines and bile acids, and the corresponding enzymes with roles implicated in cancer plasticity.
5) glyPAQ enables reproducible analysis of native, reduced N-glycans
6) BCA-no-more replaces protein assays with on-plate, non-destructive and highly sensitive protein quantification (down to < 1 ug);
7) HYPERsol allows direct processing of FFPE, unlocking a truly massive resource of clinical samples. With it we found new biomarkers to distinguish histological mimics of otherwise indistinguishable tumor types.
8) Tryp-N, “N-terminal trypsin,” affords complementarity and greater sensitivity to tryptic digestions; tau from CSF of Alzheimer’s patients was quantified with it;
9) The Shredder generates thousands of overlapping peptides of all lengths from one protein, allowing full sequencing [of mAbs]; and
10) SimpliFi is a cloud-based data-to-meaning engine that accepts all kinds of omics data and outputs biomarkers, biological pathways, co- or anti-regulated genes and proteins, etc. We used it to determine biomarkers from the FFPE samples of histological mimics.


CONCLUSION:
To tangibly help people, we must bring precision omics to life in the form of products and services for every step of the omics workflow that are as rapid, robust, reproducible and as simple as possible. From solutions to transform a sample into data, then data into meaning, our work has been guided by the principles of Occam’s razor: remove everything down to the bare minimum of work and time to yield the needed output. When integrated, our workflow ensures that precision omics is a rapidly reachable reality for laboratories worldwide, expediting the transition from bench to bedside.

Note to reviewers:
This is NOT intended to be a sales job, rather it is a way of thinking and journey that resulted in a company and products. (NB we're currently installing a clean room to enable ISO13485 manufacturing for clinical settings.) Yes, we provide these products, and this talk will emphasize the why and how of seeking Occam's razor solutions; designing for simplicity is actually really hard!

MALDI MSI and Lipidomics Reveal Lipid Release in Ovarian Cancer Models Carlismari Grundmann (Presenter) University of California - Santa Cruz

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Poster #10d View Map

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

INTRODUCTION:
Ovarian cancer is the deadliest gynecologic malignancy, with high-grade serous carcinoma (HGSC) accounting for ~70% of cases. HGSC originates in the fallopian tube epithelium and metastasizes to the ovaries and omentum. We have found that epinephrine enhances lipid release from omental adipocytes, creating a pro-metastatic microenvironment. Using mass spectrometry imaging (MSI) of 3D co-cultures of HGSC cells, murine omental tissues, and adipocyte spheroids, alongside LC-MS(/MS) lipidomic profiling and invasion assays, we investigated this metabolic crosstalk. Our findings suggest that epinephrine acts as an autocrine signal in the tumor microenvironment to enhance lipid release, fostering HGSC invasion and disease progression. Understanding these interactions offers insight into tumor-microenvironment dynamics and could guide the development of targeted therapies to inhibit HGSC metastasis.

METHODS:
A 3D co-culture system in agarose was optimized to study interactions between murine omental tissues, adipocytes, and murine oviductal epithelial (MOE) cells. MALDI MSI was performed using a Bruker timsTOF flex mass spectrometer with a 50-micron spatial resolution in positive reflectron mode. A 50:50 CHCA:DHB matrix was applied using an HTX TMSprayer, enabling prioritization of lipid signals intensified in cancer-omentum/adipocyte interactions.
For lipidomic profiling, LC-MS(/MS) analysis was performed in reverse-phase using both positive and negative ionization modes to identify differentially expressed lipids in adipocyte-conditioned media treated with a β-adrenergic receptor agonist (epinephrine) or antagonist (propranolol). These conditioned media were also used to evaluate cell behaviors, including proliferation, migration, and invasion.

RESULTS:
MSI data revealed that epinephrine is produced in 3D co-cultures involving HGSC cells, omental tissues, and adipocyte spheroids. Epinephrine-treated adipocyte-conditioned media significantly enhanced HGSC cell invasion, demonstrating the pro-metastatic effects of β-adrenergic signaling. Conversely, propranolol, a β-adrenergic receptor antagonist, effectively inhibited these effects by blocking endogenous epinephrine action.

After optimizing lipid extraction strategies for adipocyte-conditioned media, LC-MS/MS data acquired in both ionization modes were analyzed to classify lipid subclasses and their relative abundance across treatments. Visualization tools such as Volcano Plots and Molecular Networks were used to interpret the data, leveraging Cytoscape for network analysis and LinexWorkflow for lipid metabolic pathway mapping.

Lipidomic profiling revealed an increased release of specific lipid classes under epinephrine treatment, particularly glycerolipids and sterols, such as MG O-6:0 and ST 26:6. These findings suggest that β-adrenergic receptor activation enhances omental lipolysis, potentially contributing to metabolic reprogramming within the tumor microenvironment. This reprogramming may create a lipid-rich niche that supports HGSC progression.

Furthermore, MALDI MSI provided spatially resolved confirmation of lipid accumulation at tumor-omentum interfaces within the 3D co-culture system. These localized lipid enrichments further support the hypothesis that metabolic crosstalk mediated by β-adrenergic signaling plays a pivotal role in facilitating tumor invasion and metastasis.

CONCLUSION:
Our findings highlight the role of epinephrine in modulating lipid dynamics within the HGSC tumor microenvironment. By integrating MALDI MSI and lipidomics, we reveal how β-adrenergic signaling enhances omental lipolysis, creating a pro-metastatic lipid-rich niche. These insights could guide the development of therapeutic strategies targeting metabolic vulnerabilities in HGSC.

Flexible Steroid Hormone Quantification: Analytical Equivalence of Two C8 Columns for LC-MS/MS John Rohloff (Presenter) Tecan, IBL International

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Poster #11d View Map

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

INTRODUCTION:
Ensuring the continuous availability of consumables is a key requirement in LC-MS analysis. Relying on a single chromatographic column can create supply chain vulnerabilities and limit laboratory flexibility. This study investigates whether the Tecan Steroid Panel LC-MS, originally developed for a specific C8 column, can also be reliably applied using the KNAUER Eurospher II C8 HPLC column, thereby providing an alternative for robust and flexible steroid hormone quantification.

METHODS:
The Steroid Panel LC-MS method, based on solid phase extraction (SPE) and subsequent LC-MS analysis, was applied to human serum samples from both male and female subjects. Fourteen steroid hormones were measured in parallel using both the original kit-provided C8 column and the KNAUER Eurospher II 100-3 C8 column. For the KNAUER column, only the flow rate was adjusted (from 0.35 to 0.50 mL/min); all other chromatographic and mass spectrometric parameters, as well as sample preparation, remained unchanged. Method comparison was performed on at least 40 samples per analyte, using Passing-Bablok regression and calculation of correlation coefficients (r). Chromatographic separation was evaluated, particularly for analytes with similar m/z transitions.

RESULTS:
The method comparison demonstrated excellent agreement between the two columns for all 14 steroid hormones, with correlation coefficients (r) ranging from 0.909 to 0.998. Passing-Bablok regression analysis confirmed the comparability of quantitative results. Baseline separation was achieved for critical analytes such as 17-OH-progesterone, 11-deoxycorticosterone, cortisone, and cortisol, which are essential for reliable quantification due to overlapping mass transitions. The workflow remained efficient, and no significant changes in sample preparation or instrument handling were required.

CONCLUSION:
The Tecan Steroid Panel LC-MS can be used interchangeably with both the original kit-provided C8 column and the KNAUER Eurospher II C8 column, without compromising analytical performance or reliability. This flexibility enhances supply chain security and supports robust implementation in laboratories. The results provide a foundation for future validations and broader adoption of alternative columns in routine steroid hormone analysis.

Screening of Classic and 11-Oxygenated Androgens in Polycystic Ovary Syndrome (PCOS): Method Development Vera Dosedelova (Presenter) MRC Laboratory of Medical Sciences

Poster #12d View Map

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

INTRODUCTION:
Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder affecting approximately 10% of women. The defining feature of PCOS is androgen excess, which contributes to several symptoms such as hirsutism, acne and irregular periods. Androgen excess has also been linked to metabolic dysfunction in women with PCOS. The international PCOS guidelines recommend the measurement of testosterone (T) and, if normal, also of androstenedione (A4) and DHEA sulfate. However, the adrenal-derived 11-oxygenated androgens were shown to significantly contribute to androgen excess in PCOS (1). 11-ketotestosterone (11KT) binds and activates the androgen receptor with similar potency as T and circulates in similar concentrations as T in healthy women. However, the clinical relevance and ranges of 11-oxygenated androgens in PCOS remain insufficiently understood.

OBJECTIVES:
To develop and optimize a UPLC-MS/MS assay for the quantification of 18 steroids in serum samples from women with PCOS.

METHODS:
The steroid panel included 18 steroids encompassing precursors and active glucocorticoids, mineralocorticoids and androgens, both classic, e.g., A4, T, DHEA, and 11-oxygenated, e.g., 11KT, 11β-hydroxyandrostenedione (11OHA4), and 11-ketoandrostenedione. Steroids were extracted from 100 µL of serum using supported liquid extraction (SLE) with ethyl acetate and analysed by UPLC-MS/MS. The assay was applied to a preliminary screen of 109 serum samples from women with PCOS.

RESULTS:
Optimization of UPLC-MS/MS focused mostly on the chromatographic separation and ionization efficiency. Comparing post-column infusion of ammonium fluoride and formic acid in the mobile phase revealed that ammonium fluoride enhanced the signal intensity of most steroids up to fourfold. However, steroids with a saturated A ring (e.g., DHEA, 5α-androsterone) ionized better with formic acid. To balance this, a timed post-column infusion of 2 mmol/L ammonium fluoride was employed during chromatographic separation. The mobile phase consisted of water and methanol with 0.01% formic acid. Different extraction solvents were tested, i.e., ethyl acetate, MTBE, dichloromethane, and MTBE : ethyl acetate (1:1, 4:1). Ethyl acetate provided the best recovery and reproducibility. Preliminary profiling results revealed DHEA, A4 and 11OHA4 as the predominant androgens in the PCOS serum samples.

CONCLUSION:
The UPLC-MS/MS assay was developed for comprehensive steroid profiling and is currently undergoing full validation. The assay will be applied to the large-scale prospective DAISy-PCOS cohort study to investigate steroid metabolome patterns and related clinical phenotypes and characterize the roles of classic vs. 11-oxygenated androgens in women with PCOS.

REFERENCES:
1. O´Reilly, M.W., et al., 11-Oxygenated C19 steroids are the predominant androgens in Polycystic Ovary Syndrome. J Clin Endocrinol Metab, 2017. 102(3): p. 840-848.

ACKNOWLEDGMENTS:
This research was supported by the Medical Research Council (MC_UP_1605/15) and the Wellcome Trust (209492/Z/17/Z).

Development and Validation of an LC-MS/MS Assay for Measuring KEAP1 Cys151 Target Engagement in Tumor Biopsies Melissa Hoffman (Presenter) Vividion Therapeutics

Poster #13d View Map

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

INTRODUCTION:
The transcription factor NRF2 plays an important role in defense against oxidative stress, and its constitutive activation in cancer contributes to therapeutic resistance and tumor progression. KEAP1 (Kelch-like ECH-associated protein 1), a substrate adaptor for the CUL3 E3 ligase, regulates NRF2. Vividion recently developed VVD-130037, a first-in-class covalent allosteric molecular glue that selectively binds KEAP1 at cysteine 151 (C151), enhancing KEAP1-CUL3 complex formation leading to NRF2 degradation. To support clinical translation of this approach, a quantitative LC-MS/MS assay was developed to measure KEAP1 target engagement in tumor biopsies from a Phase I clinical trial (NCT05954312) following treatment with VVD-130037. Pre-clinically, a KEAP1 Target Engagement (TE) assay was developed and applied to mouse xenografts and tissues by comparing vehicle and compound treated samples.1 Building on this work, a novel approach was developed to miniaturize the chemoproteomics workflow to support needle biopsy sample analysis and to calculate TE in the absence of a pre-treatment sample.

OBJECTIVES:
This study aimed to (1) optimize the KEAP1 C151 TE assay for tumor biopsy samples without a pre-treatment sample and (2) validate assay sensitivity, linearity, precision, and accuracy at a GCLP compliant lab.

METHODS:
Tumor biopsies were homogenized in IP lysis buffer by probe sonication. A single-pot, solid-phase-enhanced sample preparation (SP3) method was implemented, which used 50 µg of tumor protein (reduced from 500 µg) mixed with 50 µg of heavy isotope-labeled (SILAC) MDA-MB-231 cell lysate. Free cysteines were covalently labeled with a desthiobiotin-iodoacetamide probe, followed by tryptic digestion, and streptavidin enrichment of labeled peptides. Resulting peptides were separated on a nanoflow C18 column using a Dionex UltiMate 3000 UPLC and analyzed on an Orbitrap Exploris 480 using parallel reaction monitoring (PRM) mass spectrometry. SILAC MDA-MB-231 cell lysates were used as internal standard and quantification was performed using an external calibration curve using a full length purified KEAP1 protein spiked into a blank matrix (insect cell lysate). The assay was successfully transferred to a GCLP compliant lab (Proteome Sciences) and validation included optimization of LC-MS parameters for KEAP1 C151, C288, C297, C319, and C434, protein extraction from small biopsies, calibration curve generation to establish the linear range and lower limit of quantification (LLOQ), assessment of inter- and intra-day precision and accuracy for low, mid, and high spiked samples, and evaluation of TE in xenograft tumor models comparing TE calculated with and without pre-treatment samples. Inter-operator and inter-instrument performance was also evaluated.

RESULTS:
The assay achieved a LLOQ of 0.06 fmol/µg for the KEAP1 C151 peptide and 0.03–0.12 fmol/µg for other KEAP1 peptides. Linearity (R² ≥ 0.986) was confirmed over the 0.06–1.50 fmol/µg range. Protein extraction yielded sufficient material from 5–19 mg tumor biopsies from 10 donors with consistent peptide concentrations across replicates. KEAP1 C151 intra-day precision ranged from 9–12% across the low, mid, and high spiked samples and inter-day precision was 22% for the low spike and 15% for the mid and high spiked samples. Assay precision for the other KEAP1 peptides was also within the acceptability criteria of ≤ 20% for mid and high spiked samples and ≤ 30% for low spike samples. TE values calculated using vehicle-treated baselines were comparable to those using concentrations of C297, C319, and C434 in the same treated sample. The validated assay was successfully applied in a clinical trial (data not shown).

CONCLUSION:
The KEAP1 C151 TE assay was successfully optimized for low protein input samples and TE calculated using non-C151 KEAP1 peptides. The assay was successfully transferred to a GCLP compliant lab, validated for use with human tumor biopsies, and proof-of-concept demonstrated by a reduction in C151 signal after VVD-130037 treatment. Covalent TE from patient tumor biopsies will support clinical evaluation of VVD-130037.

REFERENCES:
1. Roy N, et al., Suppression of NRF2-dependent cancer growth by a covalent allosteric molecular glue. bioRxiv 2024.10.04.616592.

Quantification of Plasma Nicotinic Acid and Two Metabolites by LC-MS/MS Stephen Merrigan (Presenter) ARUP Laboratories

Poster #14d View Map

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

INTRODUCTION:
Niacin (nicotinic acid, NA), or vitamin B3, is a required nutrient that is metabolized to nicotinamide (NAM), a constituent of the essential coenzyme nicotinamide adenine dinucleotide (NAD). NA and NAM are absorbed from food; they are also available as nutritional supplements and are used therapeutically as cholesterol-lowering agents. NA can be metabolized by conjugation with glycine to form nicotinuric acid (NUA), which causes flushing. NA deficiency can result from inadequate dietary intake, carcinoid syndrome, prolonged use of the antibiotic isoniazid, or a rare congenital defect of niacin metabolism (Hartnup's disease); severe NA deficiency is termed pellagra. Niacin toxicity has been associated with ingestion of excessive amounts of NA supplements or niacin-containing energy drinks. Our aim was to develop an LC-MS/MS assay for NA and its metabolites, NAM and NUA, and to evaluate the methods’ performance.

METHODS:
Aliquots of human serum or plasma samples (50 µL), stable isotope-labeled internal standard mix (IS, nicotinic acid-13C6, nicotinamide-13C6, and nicotinuric acid-d4, 20 µL) and 250 µL of 2% formic acid were added to the wells of a 96-well plate; the plate was sealed and vortexed for 5 min. The adsorbent in wells of the cation exchange solid phase extraction (SPE) plate (Biotage) was preconditioned with methanol and 2% formic acid; samples were loaded and the adsorbent was washed with acidified water, followed by acidified methanol. The analytes were eluted using 10% ammonium hydroxide in acetonitrile; the extracts were dried and reconstituted. Instrumental analysis was performed on an Agilent series 6475 triple quadrupole mass spectrometer with electrospray ionization, using multiple reaction monitoring acquisition in positive ion mode. HPLC separation was accomplished using a Kinetex Biphenyl 2.6 µm, 3.0 x 50 mm column (Phenomenex) at 30 °C, flow rate 0.35 mL/min; injection volume 2µL. Injection to injection time was 7.5 min. Quantification was performed using a six-point calibration curve (40 – 4,000 nmol/L), with two mass transitions monitored for each analyte and IS. Evaluation of the method’s performance included assessment of precision, sensitivity, linearity, accuracy, specificity, matrix effects, dilution integrity, carryover, and correlation with a validated method of another laboratory. Blood collection containers and stability of the analytes were evaluated.

RESULTS:
We developed a method for quantifying NA, NAM, and NUA in plasma samples using LC-MS/MS and evaluated its analytical performance. The issues of poor analyte retention by SPE adsorbents and chromatographic columns were resolved through optimization of sample preparation and chromatographic separation. Given the highly polar nature of the analytes, cation exchange SPE was used for sample preparation. Due to the sensitivity of highly polar molecules' ionization to pH changes, which affects their separation using SPE, extraction recovery was optimized by maintaining tight pH control throughout all steps of sample preparation. The Kinetex biphenyl column provided the best chromatographic performance with adequate retention and separation of NA, NAM, and NUA peaks. The linear range of the assay for NA, NAM, and NUA was 40 to 4,000 nmol/L. Within-run imprecision for analysis of two plasma sample pools analyzed on one day in 10 replicates was <5%. Between-run imprecision for analysis of three plasma sample pools over nine runs was <10%. Comparison of 200 patient plasma specimens with a validated method of another laboratory showed 169 sample concentrations within the analytical measurement range for NAM (Deming regression y = 0.988*x – 14.5 nmol/L, R = 0.963). Because of the short half-lives of NA and NUA, the majority of the samples did not contain measurable amounts of NA or NUA. Out of the entire set, one sample contained NA at 157 nmol/L (130 nmol/L measured by the comparative method). NUA was present in two of the samples; the measured concentrations were 177 nmol/L and 507 nmol/L (233 nmol/L and 611 nmol/L, respectively, when measured by the comparative method). Evaluation of method accuracy for the targeted analytes using four human plasma sample pools spiked with 200 and 800 nmol/L, demonstrated within 10% agreement between the expected and the observed concentrations. We observed differences in NAM concentrations between serum and plasma samples collected from the same individuals, with lower concentrations measured in serum samples; no difference in the measured concentrations of NA and NUA was observed.

CONCLUSION:
An LC-MS/MS method was developed for quantifying vitamin B3 and its metabolites (NA, NAM and NUA) in plasma samples. Evaluation of the method performance demonstrated reliable analytical performance with adequate precision, accuracy, and robustness. NAM was present at measurable concentrations in ~70% of patient samples tested; NA and NUA were present in ≤1% of the analyzed specimens. We observed reasonably good agreement in quantitation of the three analytes with a validated method of another laboratory.

Optimization of Human Milk Metabolome and Proteome Coverage by LC-MS/MS Danko Brukner (Presenter) Université du Québec à Montréal (UQAM)

Poster #15d View Map

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

INTRODUCTION:
Maternal milk is a complex and dynamic biofluid whose composition varies depending on different factors, including maternal health. Variations in metabolite and protein profiles of human milk could influence infant nutrition and development. Untargeted LC-MS/MS offers a powerful approach to explore the variations in maternal milk profiles. Optimization of sample preparation protocols is essential to maximize the detection of key biomarkers, including those from xenobiotic exposure.

OBJECTIVE:
The study aims to compare different extraction methods to optimise the recovery of small molecules and proteins from maternal milk. The objective of this work will be to achieve maximal coverage for biomarker discovery in the context of different diseases or exposures.

METHODS:
Different solvent-based and solid phase extractions were employed to assess the detectability of different classes of metabolites as well as overall proteome coverage from human milk samples. Several chromatographic separations were combined with untargeted metabolomics and proteomics workflows on high-resolution quadrupole-time of flight platforms (Sciex TripleTOF 5600+ coupled to a Shimadzu Nexera UHPLC and Sciex ZenoTOF 7600 coupled to a Evosep One LC). Metabolites were putatively identified using spectral matching in Sciex OS-Q, and protein identification was performed using PEAKS 12.5 software (Bioinformatics solutions).

CONCLUSION:
The optimization of metabolite and protein extraction from human milk is a crucial step in refining untargeted LC-MS/MS workflows. The selected methods will be applied to a cohort to investigate metabolomic and proteomic differences related to gestational diabetes.

Multiomics Analysis of Mucopolysaccharidoses (Mps II) Mouse Model by LC-MS/MS Nathan Ghafari (Presenter) University of Quebec in Montreal (UQAM)

Poster #16d View Map

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

INTRODUCTION:
Hunter syndrome or mucopolysaccharidosis type 2 (MPS II) is a rare disease with a prevalence of 1/100 000, occurring mainly in boys. It is caused by a deficiency of iduronate sulfatase (IDS) enzyme, resulting in the accumulation of glycosaminoglycans (GAGs), such as dermatan and heparan sulfate, in lysosomes of cells. This accumulation leads to multiple symptoms, such as hydrocephalus, enlargement of the liver and hearing loss. In this study, a combination of targeted and untargeted LC-MS/MS methods were used to observe metabolic variations in liver and brain caused by MPS II in an IDS knock-out mouse model. The obtained results were complemented with an untargeted quantitative proteomics analysis.

OBJECTIVES:
This project aims to investigate the metabolic variations induced by MPS II in mouse liver and brain model as well as the impact of a treatment called enzymatic replacement therapy (ERT). Previous studies1,2 have shown the positive impact of ERT on GAGs levels in multiple tissues, especially in the liver where accumulated levels are very significant in MPS II. The levels of GAGs are unfortunately not reduced in the brain upon ERT due to limitations in crossing the blood-brain barrier. We have combined untargeted metabolomics and proteomics to better understand the repercussions of the disease and its treatment on in the liver and brain.

METHODS:
Livers (n=21) were obtained from three different groups of mice (WT, and MPS II model with and without ERT) and were homogenized using a Bead Ruptor with glass beads. Liver homogenates were extracted using methanol, supernatants dried and reconstituted in 25% methanol for metabolomics analysis. Dried pellets were kept for further processing bottom-up proteomics sample preparation. Metabolomics LC-MS/MS data were acquired on a quadrupole-time-of-flight system (Sciex TripleTOF 5600+). For proteomics analysis, a micro-flow LC (Evosep One) coupled to a new generation time of flight system (ZenoTOF 7600) were used for both identification and quantitation of proteins. Putative metabolites were identified using spectral database matching. Data processing was focused on the difference between each group to find statistically significant changing metabolites and proteins. Brain samples (n=10) from two groups of mice (MPS with and without ERT) were analyzed using the same workflow.

RESULTS:
These methods resulted in the identification of many significantly changing metabolites (157) and proteins (1045) between WT and MPS mice liver. Numerous metabolic pathways, including purine metabolism, associated with various liver disruptions, were identified with high confidence using both metabolites and proteins. Additionally, 35 proteins were linked to lysosomal degradation. We are in the process of analysing the brain samples and will compare our findings in this presentation.

CONCLUSION:
The multiomics analysis indicates that ERT treatment does not significantly impact most of the metabolic disturbances observed in the liver of the MPS II model, indicating that metabolic perturbation that occurs in the liver are not covered by the actual version of the enzymatic treatment. The ongoing analysis of brain samples should reveal brain-specific metabolic variations, to be compared to those found in the liver.

References:
1Menkovic I, Auray-Blais C. Diagnostics. 2019; 9(4):195.
2Menkovic I, Auray-Blais C. Bioanalysis. 2019; 11(8):727-740.

Proteome Characterization from Ocular Fluids Maggy Lepine (Presenter) UQAM

Poster #17d View Map

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

INTRODUCTION:
Tears are crucial for eye health, as they protect the surface of the eye and provide proper lubrication. The vitreous humor, a transparent gelatinous substance filling the eyeball behind the lens, plays an important role in coordinating eye growth and supporting the retina. This biofluid can be accessed through vitrectomy, a standard procedure used to treat various retinal issues, while tear proteins can be collected through non-invasive approach. Both fluids offer valuable insights for studying different eye diseases, whether related to the ocular surface or the retina. The goal of this study was to characterize the proteomic coverage from tears and vitreous humor to better understand the differences between these two ocular biofluids.

METHODS:
Tear samples were collected on Schirmer strips from healthy volunteers and vitreous humor samples were collected from patients undergoing vitrectomy procedures. Reductive alkylation and tryptic digestion was performed, followed by solid phase extraction for sample preparation prior to LC-MS/MS analysis. A EVOSEP One LC coupled to a Sciex ZenoTOF 7600+ (QqTOF) system was employed for untargeted bottom-up proteomics in low-microflow regime using data-independent acquisition (DIA-SWATH) with a 44-minute separation (SPD30 method).

RESULT:
A set of 53 biobanked vitreous humor samples including diabetic retinopathy (DR), age-related macular degeneration (AMD), retinal detachment (RD) and eye floaters were received from a biobank of ocular fluids. Tear samples were collected from a group of 16 volunteers (31 samples) with no known eye diseases affecting the ocular surface. Samples from both biofluids were analyzed and processed using Peaks 12.5 software (Bioinformatics solutions). A list of 2048 and 1144 proteins have been confidently identified (at 1% FDR) in tears and vitreous humor samples, respectively, with approximately 25% in common. Preliminary data indicates a higher proportion of extracellular matrix proteins, protease inhibitors, and apolipoproteins in the vitreous humor compared to the tears. Data analysis is ongoing to further profile these two biofluids.

CONCLUSION:
The comparative analysis of proteins in tears and vitreous humor is a novel approach for assessing eye health and potentially improve the classification of various eye diseases. Each of these ocular biofluids offers complementary utility for detecting biomarkers, depending on the specific pathology.

A Novel Ion Source with Integrated Separation Column and Replaceable Emitters Facilitates Ultra High-Throughput Multiplex PRM-MS Qin Fu (Presenter) Thermo Fisher Scientific

Poster #18d View Map

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

INTRODUCTION:
Targeted MS-based proteomics approaches, such as multiple reaction monitoring (MRM) and parallel reaction monitoring (PRM), are used to validate candidate biomarkers. MRM assays, performed with a triple quadrupole mass spectrometer, isolate and fragment a precursor, monitoring predefined fragment ions. PRM assays, using Quadrupole-Orbitrap or Quadrupole-Time of Flight (TOF) systems, accumulate and measure all fragment ions from a targeted precursor in a single event, offering sensitivity and selectivity advantages. A recent innovation combines a dual-pressure linear ion trap with a triple quadrupole, enhancing acquisition rate, ion storage, resolution, and dynamic range. Additionally, a novel ion source with integrated cartridges containing a column, liquid junction, and replaceable emitter assembly has been developed. We evaluated this new configuration with a multiplexed health surveillance panel using ultra high-throughput PRM-MS and a novel ion source with integrated separation column and replaceable emitters.

METHODS:
Parallel Reaction Monitoring (PRM-MS) method development and optimization were conducted using a pool of healthy plasma. We evaluated a modified 57-protein multiplexed Health Surveillance Panel (HSP) PRM method on a high-speed hybrid nominal mass platform (Thermo Scientific™ Stellar™ MS) combined with an Thermo Scientific™ OptiSpray™ ion source and a prototype OptiSpray cartridge with a PepMap™ Neo 150 µm x 15 cm column and a tapered emitter. The “capillary flow quick optimization” automated routine was used to determine the optimal position for the emitter and all data was acquired with the emitter in this position. This modified 57-protein multiplexed HSP PRM method was developed based on a previously published MRM assay. The current iteration of the assay includes 83 prototypic peptides, their corresponding SIL peptides, and a 14 Peptide Retention Time Calibration (PRTC) mixture (Thermo Fisher Scientific), totaling 180 peptides. To develop a large-scale and high-throughput PRM assay, we assessed four different throughputs: 100, 144, 180, and 300 samples per day (SPD). Methods for both the Thermo Scientific™ EASY-Spray™ ion source and OptiSpray ion source were developed using Skyline™ software and PRM Conductor. PRM Conductor, a software program that integrates into the Skyline external tool ecosystem, aids in the creation of PRM mass spectrometry methods.

RESULTS:
The HSP assay quantifies 57 plasma proteins, including 21 that are FDA-approved and commonly used in clinical diagnostics, such as C-reactive protein (CRP). This multiplexed PRM-MS assay demonstrates the potential to streamline the measurement of multiple clinically relevant biomarkers in a single analysis, offering advantages over traditional immunoassays in terms of throughput and specificity. Previously, we developed a multiplexed health surveillance panel PRM assay with four different throughputs: 100 SPD, 144 SPD, 180 SPD, and 300 SPD, using an EASY-Spray ion source. Now, we have evaluated the quantifiability and portability of these four different throughputs with an OptiSpray source, as well as optimized the PRM method using PRM Conductor.

The translation between the EASY-Spray ion source with an EASY-Spray HPLC column and the OptiSpray ion source with an integrated column cartridge is seamless. The implementation of the Adaptive Retention Time (ART) algorithm, which allows real-time retention time adjustments, proved advantageous in achieving robustness and enhancing reproducibility when switching between ion sources. With the aid of the Adaptive RT algorithm in the acquisition methods, out of 83 SILs and four different throughputs (run times ranging from 3.7 to 13 minutes), only a few peptides (<10) needed rescheduling. A majority of the scheduled peptides were captured using the same methods generated with the EASY-Spray source and column.

We compared the performance of the EASY-Spray system to the OptiSpray system. We observed an improvement in points per peak with the OptiSpray system, an average increase from 13 to 14 points per peak. Similarly, we observed better reproducibility, with the average CV% improving from 11% to 8%. Then, we optimized the OptiSpray ion source PRM methods acquisition parameters using the PRM conductor method's automated scheduled method generation feature. Additional improvements in points per peak and reproducibility were observed after this optimization step: the points per peak increased to 17, and the average CV% reduced to 6%. The peak areas were similar or slightly increased with the OptiSpray source, with average peak areas increasing from 4.7 X107 to 5.1 X107.
The OptiSpray ion source provides improved ionization efficiency and ensures consistent and stable spray performance, leading to reliable and reproducible results. The user-friendly design simplifies setup and operation, while its compatibility with various mass spectrometry systems enhances versatility and integration into existing workflows.

CONCLUSION:
The Stellar MS for PRM (Parallel Reaction Monitoring), equipped with a retention time adaptor algorithm, provides high sensitivity and ensures precise quantification of target peptides, making it an excellent platform for biomarker validation and quantitative proteomics. Additionally, the PRM conductor provides a seamless workflow, while the Stellar MS for PRM with the OptiSpray ion source ensures durability and long-term use, minimizing maintenance requirements and downtime.

NOVEL ASPECT:
The combination of the Stellar MS with the Adaptive RT algorithm and the PRM conductor with the automated OptiSpray ion source provides a seamless workflow and a robust platform to quantify highly multiplexed and scheduled analytes in a high-throughput fashion.

Development of Novel LC-MS/MS Psychoactive Panel (including Xylazine and Medetomidine) for Investigation of Prevalence in New Haven, CT Region Jill Kodger (Presenter) Yale University

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Poster #19d View Map

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

INTRODUCTION:
Novel psychoactive substances are synthetic compounds designed to mimic the effects of commonly misused illicit drugs. This study aims to develop a method for detecting and quantifying emerging veterinary anesthetics, kratom, synthetic opioids, synthetic cannabinoids, and synthetic benzodiazepines. The inclusion of veterinary drugs in this study is driven by the increasing prevalence of xylazine and medetomidine in patients with polydrug abuse. Kratom, used by approximately 0.7% of the U.S. population, is often consumed by individuals with opioid use disorder or polydrug abuse, and kratom-related deaths are frequently linked to polydrug use. The rise of synthetic opioids, particularly fentanyl, has contributed to what is now recognized as the "fourth wave" of the opioid epidemic, highlighting the urgent need for effective testing methods for these substances. Synthetic cannabinoids, often referred to as "bath salts," are synthetic stimulants chemically related to the khat plant and are commonly used in vapes. These compounds have been found in overdose victims and are increasingly involved in toxicological cases. There has been an uptick in novel psychoactive drug use, particularly veterinary drugs, in New Haven and surrounding areas, prompting our efforts to quantify this in our polydrug user population for surveillance purposes.

METHODS:
Urine (150uL) and internal standard (150uL at 200 ng/mL), were combined in micro centrifuge tube, vortexed, and centrifuged and poured into sample vials. 2uL of the resulting solution was injected onto an Acquity HSS T3 column (Waters; 1.8um, 2.1 x 50mm) with an Acquity UPLC H33 T3 VanGuard pre-column (Waters; 1.8um) coupled to a Xevo TQD(Waters). The mass spectrometer was operated in positive ion mode. Quantification was based on peak area ratios of xylazine (m/z 221.19>89.90) to xylazine D6 (m/z 227.18>89.94), 7-OH mitragynine (m/z 415.32>190.02), mitragynine (m/z 399.25 >174.14) , Gabapentin (m/z 172.03 >54.90) to gabapentin 13C3( m/z 175.17> 140.17), metonitazene (m/z 383.25 > 100.07) to metonitazene 13C6( m/z 175.17> 140.17), etonitazene (m/z 397.25>100.01) to etonitazene 13C6 (m/z 403.30 > 99.95) and medetomidine (m/z 201.13 > 94.94) to medetomidine 13C-D3 (205.13 > 98.93).

RESULTS:
A seven minute gradient of 50-100% mobile phase B was sufficient to separate all compounds using water +0.1% formic acid (FA) (mobile phase A) and methanol +0.1% FA (mobile phase B). The analytical measureable range (AMR) was within %CV <20% and had a recovery between 80-120%. AMR was 5 to 1000 ng/mL for all analytes except gabapentin, which was 20-1000 ng/mL. No significant carryover was observed up to a concentration of 200 ng/mL for all analytes. In a small sampling of 48 patients in our polydrug user population we found around 2.1% prevalence of medetomidine, 27.1 % prevalence of xylazine and 33.3% prevalence of gabapentin.

CONCLUSION:
In conclusion, novel psychoactive substances are detectable in our polydrug user population in the New Haven area, with xylazine being the most common, followed by medetomidine.

Comprehensive Multi-Steroid Profiling From 100 µL Sample Volumes Using An LC-MS Kit Dajana Domik (Presenter) Tecan

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Poster #20d View Map

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

INTRODUCTION:
The accurate quantification of steroid hormones is essential for the diagnosis and monitoring of a wide range of endocrine disorders. Laboratories strive to increase efficiency and adapt to the growing demand for high-throughput analysis. There is a clear trend toward obtaining more data from ever smaller sample volumes. This is especially important in situations where only small amounts of sample material are available. Mass spectrometry has become the method of choice for multi-analyte steroid profiling due to its high sensitivity, specificity, and multiplexing capabilities. In this study, we present the application and validation of the Tecan Steroid Panel LC-MS kit in combination with UHPLC-MS/MS instrumentation, enabling the simultaneous quantification of 18 relevant steroids from as little as 100 µL of serum.

METHODS:
Sample preparation was performed using a modified solid phase extraction (SPE) protocol, reducing the required serum volume from the standard 250 µL to just 100 µL. The extracted analytes were reconstituted and injected into an Agilent 1290 Infinity II UPLC System coupled to a SCIEX QTRAP®6500 LC-MS/MS System. The IonDrive™ Turbo V source was operated in polarity switching mode, allowing for the simultaneous detection of positive and negative MRM transitions within a single 10-minute run. The method was thoroughly tested by evaluating parameters such as linearity, trueness and precision, lower limit of quantification (LLOQ), recovery, and matrix effects. Calibration was achieved using lyophilized human serum standards provided in the kit, ensuring reliability and stability.

RESULTS:
The modified protocol enabled robust quantification of all 18 steroids, including glucocorticoids, mineralocorticoids, androgens, estrogens, and progestogens, from minimal sample volumes. The method demonstrated good linearity across all analytes, with correlation coefficients (R²) exceeding 0.99. Intra and interday and precision variance over six days were below 15% for all analytes, meeting expected validation criteria. LLOQs for each steroid were below the lowest calibrator, ensuring high sensitivity. Recovery rates, based on peak area, ranged from 50% to 90%, and matrix effects, also calculated from peak area, were within 80% to 120%, indicating reliable extraction and minimal interference. The reduction in sample volume did not compromise analytical performance, and the use of a standardized commercial kit ensured consistent quality and reproducibility across runs.

CONCLUSION:
The combination of the Tecan Steroid Panel LC-MS kit with advanced UHPLC-MS/MS technology enables comprehensive, high-quality steroid profiling from as little as 100 µL of serum. This reduction in sample volume expands the applicability of multi-steroid analysis to cases with limited sample availability. The validated method delivers reliable, precise, and reproducible results across a broad range of key relevant steroids, supporting improved analytical performance and data quality. Furthermore, the use of a commercial kit streamlines laboratory workflows, reduces the risk of analytical deviations, and supports cost and time savings. Keeping pace with technological advances in LC-MS/MS is essential for laboratories to remain competitive and deliver the highest standards of analytical quality in a rapidly evolving scientific landscape.

Mass Spectrometry Imaging of the Gut Microbiome Samarth Ganjoo (Presenter) University of Montreal

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Poster #21d View Map

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

INTRODUCTION:
The gastrointestinal microbiota is the largest microbial community within mammals. It is estimated that the human gut contains more than 100 trillion bacterial cells. This represents a diverse and complex array of amphiphilic membrane lipid structures spatially distributed along the gut. A multitude of these lipids have been shown to mediate host-microbe interactions, which are essential for maintaining the symbiotic relationship between the mammalian and bacterial cells. Understanding their spatial distribution in the gut can help identify the role of bacterial lipids on host biology, which remains unclear to date. This study focuses on the detection of lipid signals of bacterial origin by MALDI MSI to spatially resolve the microbiome biofilms across the intestine.

METHOD:
Fresh intestine harvested from mice was straightened and frozen on dry ice. 0.5-1 cm segments from different regions across the length of the intestine were cut and embedded in 10% CMC or NEG-50 embedding media. 10 μm thick transversal sections were cut at -16°C using a cryostat and mounted on ITO-coated glass slides. When dried, the sections were coated by sublimation (Shimadzu iMLayer) with 1,5-diaminonaphthalene (DAN) or 2,5-Dihydroxybenzoic acid (DHB) matrix. In addition to this, matrix spray deposition (HTX TM sprayer) was also employed in scenarios where MS/MS fragmentation was performed using lithium bromide (LiBr) salt with the MALDI matrices. Dual polarity MALDI MSI data was acquired from serial sections using a Shimadzu MALDI iMScope QT in the 400-1000 m/z range with 10 µm pixel size, 100 shots per pixel at a 5 kHz laser repetition rate. Lipid identification was performed by MALDI MS/MS directly from the sections.

RESULTS:
The preliminary high-resolution images of different intestinal regions produced an array of lipid signals, accounting to >500-600 unique spectral peaks per tissue section. Spatial segmentation analysis with spatially aware nearest shrunken centroid clustering and gaussian mixture models (GMM) highlighted numerous lipid signals clearly differentiating the intestine wall from the lumen. The segmentation varied across the length of the small intestine as well, portraying changes in the lipid localization in the tubular structure. A thorough library search using exact mass narrowed down our search list for lipid signals that may originate from bacterial cells around or within the gut lumen region. This is for example the case for m/z 603.5320 ([M+H]+; DG35:4), 796.5850 ([M+H]+; PC37:4), 771.5182 ([M-H]-; PG36:3), and 883.5977 ([M-H]-; PG44:3) all formally identified by MS/MS. These lipid species have been reported in literature to be preferentially expressed within bacteria. Several bacterial lipid gradients were also detected along the length of the intestine, presumably showing the localization of different bacteria. This is for example the case for PG36:3 with intensities increasing ~50% moving from jejunum to ileum in the small intestine.

In addition to bacterial lipid detection, gradients for certain mammalian lipids were also observed across the length of the small intestine, which may be a consequence of lipid transfer from bacterial cells to the mammalian host cells as a metabolic pathway for immunomodulation. This is for example the case for SM34:1; O2 observed in high abundance around the jejunum section when compared to ileum or duodenum of the intestine, accompanied by a spatial localization shift to only one side of the intestine around the ileum region. This trend is indicative not only of lipid uptake by mammalian cells, but also of bacteria distribution from which this specific lipid may originate.

Other interesting lipid trends are being investigated, with observations of phosphatidyl serine (PS) and phosphatidyl inositol (PI) lipids in the lumen regions of the tissues. These lipids are abundantly present in the gut and have been previously reported to play an important role in modulating the microbiome and its integrity. Work is also being done to assess bacterial-specific metabolite distributions within the gut to further characterize the various bacterial populations and the relationship with their host.

CONCLUSION:
Based on our findings, bacterial lipid signals as well as bacteria affected modulations in the lipids can be detected and mapped by MALDI-MSI from gut tissue sections. Additionally, a sub-population of lipids demonstrates intestine segment specific relative abundances and (or) distributions. These findings are hypothesized to be linked to certain lipid metabolic pathways that may be involved in immunomodulation, signaling, inflammation, etc. These findings and ongoing investigations will help us to better resolve the ongoing challenge of understanding the host-bacteria symbiosis.

Boronic Acid Mass Tags for the Specific Detection and Imaging of Diol Compounds Justine Gatein (Presenter) Université de Montréal

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Poster #22d View Map

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

INTRODUCTION
The advancement of on-tissue chemical derivatization techniques for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of endogenous metabolites in tissues has attracted significant interest due to their benefits in enhancing detection sensitivity and ionization efficiency for poorly ionizable or/and low-abundance metabolites. Among these, glucose was found of high interest because of its implication in numerous diseases. The boron group of boronic acids exhibits high reactivity with compounds containing a diol function. Chemical derivatization enables the selective targeting of diols while enhancing the sensitivity and specificity of the method while reducing background noise, leading to more precise and reliable analysis.

METHODS
A new boronic acid containing a positively charged amine function was developed to compensate for the low ionization efficiency of compounds and enhance their detection in MSI. The analyte to tag ratio that allows for a complete reaction in solution and detection yield was first optimized. For MALDI MS of the 4-hydroxyestrone adduct, the best matrix was 9-aminoacridine (9AA) and it was 2′,6′-dihydroxyacetophenone (DHA) for the glucose adduct. For glucose, using brain homogenates, an optimized solution of 5mg/ml of boronic acid in 50% methanol was used to determine the best on-tissue reaction. Spray deposition parameters were then optimized for the boronic acid tag and DHA matrix. MALDI MSI was subsequently performed in the positive ion mode. Since the boron group has an easily recognizable isotopic signature, it is possible to more precisely identify the adducts that have reacted with the tag, in addition to confirming their identification by exact mass. For direct MALDI MSI glucose detection, N-(1-naphthyl) ethylenediamine dinitrate (NEDC) matrix was also spray deposited on all sections. MALDI MSI was then performed in the negative ion mode.

RESULTS
Boronic acid reactivity was initially tested in solution using several diol standards including 4-hydroxyestrone, ascorbic acid and several hexoses. In particular, we were able to successfully derivatize glucose in solution. After optimizing the reaction conditions (tag quantity, solvent, etc.), several matrices including 9AA, 1,5-dihydroxybenzoic acid, 1,5-diaminonaphthalene and DHA, were tested using the dry droplet method. For MALDI MSI, the tag and the matrix spray deposition method then were optimized. Thin tissue sections from mouse brain homogenates and different mouse organs were analyzed using the optimized methods using a Shimadzu MALDI iMScopeQT system. For glucose, the results were compared with MALDI MSI methods previously reported in the literature.
Glucose was successfully detected in liver, kidney, testis and brain tissue sections (12 µm thick) by MALDI MSI at a spatial resolution of 25 µm using NEDC as matrix ([glucose + Cl]⁻, m/z = 215.032) and after on-tissue chemical derivatization with boronic acid ([glucose-boronic ac]+, m/z = 401.1868) using DHA as matrix with a mass accuracy ~1ppm. For liver and testis, glucose was homogenously detected across the sections. For brain, in all cases glucose was successfully detected predominantly in the gray matter. This distribution is consistent with that previously described in the literature, as the gray matter is the site of intense metabolic activity. For kidney, glucose was mostly detected in the medulla, which is again consistent with that previously described in the literature. However, when comparing glucose signal intensities between the ([glucose + Cl]⁻ NEDC produced ions and the ([glucose-boronic ac]+ ions, for brain, on-tissue chemical derivatization proved to be more sensitive by ~600-fold, while for liver, testis and kidney tissue, the detection improvements were ~800-fold, ~200-fold and ~70-fold, respectively.

CONCLUSION
As glucose metabolism is severely altered in many diseases, the development of MSI-based analytical methods is necessary to better understand local metabolism and potentially identify new therapeutic targets to increase treatment efficiency. By providing spatially resolved metabolic information, these methods offer valuable insights into disease-specific metabolic reprogramming and may contribute to the development of more effective, targeted therapeutic interventions.

Development and Validation of an LC-MS/MS Method for the Quantification of 5-Fluorouracil in Plasma Chandra Nesbitt (Presenter) Dynacare

Poster #23d View Map

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

INTRODUCTION:
5-Fluorouracil (5-FU) is a common chemotherapy drug used in the treatment of various types of solid tumor cancers including colorectal, stomach, breast and head and neck cancers. Dosing of 5-FU has historically been based on body surface area (BSA)1, a method resulting in significant inter-individual variability in drug levels which may lead to severe toxicities for overdosed patients or sub-optimal drug efficacy for underdosed patients. To obtain optimal dosing for patients, pharmacokinetics guided dosing has been demonstrated to reduce toxicity and improve patient outcomes. Measurement of 5-FU plasma concentrations, expressed by area under the curve (AUC), can be useful in determining optimal individual doses of 5-FU. Target levels for 5-FU AUC have been reported to be in the range of 20-30 mg*h/L2,3.

OBJECTIVE(S):
Develop and validate a selective and sensitive liquid chromatography tandem mass spectrometry method for quantifying plasma concentrations of 5-Fluorouracil in cancer patients.

METHODS:
Plasma samples containing 5-FU were extracted using a protein precipitation method with acidified acetonitrile, filtered and then concentrated using nitrogen evaporation of the supernatant. The LC-MS/MS system consisted of an ExionLC coupled to a SCIEX 4500 triple quadrupole. Chromatographic separation was performed on a Phenomenex LUNA Omega PS C18 column (150 x 4.6 mm, 3 µm). The mobile phase consisted of water with 0.1% formic acid in water (A) and methanol (B). Analyte separation was achieved using a mobile phase gradient program. Electrospray ionization in negative ion mode was used to monitor MRM transitions for each analyte. For quantification, peak area ratios of the analytes to their corresponding deuterated analogues were calculated as a function of the concentration of the analyte. Method parameters were fully validated and included the determination of sensitivity, linearity, precision and accuracy, carryover, matrix effects, and interferences.

RESULTS:
LC-MS/MS method validation studies for the quantification of 5-FU in plasma demonstrated a limit of quantification (LOQ) of 5 µg/L and linearity over the calibration range 5 to 7500 µg/L (r ≥ 0.999). Intra and inter-day precision were evaluated using QC samples at three different concentrations in replicates of 20. Inter-day studies were performed on four different days with five replicates of each QC level. The intra and inter-day imprecision ranged from 1.3 to 1.6% and 2.0 to 3.1% respectively. Method accuracy was demonstrated by recovery values of the nominal concentrations for a series of calibration and QC standards spanning the analytical range. Accuracy values were within acceptance and ranged between 87 - 110%. No carryover was observed following the analysis of plasma samples spiked with 5-FU concentrations of up to 7500 µg/L. Matrix effects were shown to be minimal and did not interfere with the accuracy and precision of the method. No interferences were observed after spiking the low QC level with high concentrations of potential interferences and determining the recovery. Recoveries of 5-FU were within the acceptable range of ±20% for each interference tested.

CONCLUSION:
A simple and sensitive LC-MS/MS method was developed and validated for determining plasma concentrations of 5-FU and can be utilized to assist in the therapeutic drug management of cancer patients undergoing chemotherapy treatment. Monitoring plasma concentrations of cytotoxic drugs, such as 5-FU, can help to achieve optimal outcomes for cancer patients, by increasing the efficacy of the drug while minimizing adverse toxic side effects experienced during treatment.

REFERENCES:
1.Beumer JH, Chu E, Allegra C, et al. Therapeutic drug monitoring in oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology recommendations for 5-fluorouracil therapy. Clin Pharmacol Ther. 2019;105(3):598-613
2.Kaldate, Haregewoin, Grier et al. Modeling the 5-Fluorouracil Area Under the Curve Versus Dose Relationship to Develop a Pharmacokinetic Dosing Algorithm for Colorectal Cancer Patients Receiving FOLFOX6. The Oncologist. 2012;17:296–302
3.Gamelin et al. Individual fluorouracil dose adjustment based on pharmacokinetic follow-up compared with conventional dosage: results of a multicenter randomized trial of patients with metastatic colorectal cancer. J Clin Oncol. 2008 May 1;26(13):2099-105.

Microbial Bile Acids Impair Memory via the Gut–Brain Axis: A Multi-Omics Perspective Dakshat Trivedi (Presenter) University of Southampton

Poster #24d View Map

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

INTRODUCTION:
Cognitive decline in ageing can occur in the absence of overt neurodegeneration, suggesting a role for systemic metabolic changes. Emerging evidence supports the gut–brain axis as a key regulator of brain health, with microbial metabolites—particularly bile acids—acting as potential neuromodulators. However, the specific bile acids and microbial pathways contributing to memory impairment during healthy ageing remain understudied.

OBJECTIVES:
To investigate whether specific plasma bile acids are associated with cognitive decline in older adults, determine the microbial contribution to their biosynthesis, and assess their mechanistic role in modulating brain function via the gut–brain axis.

METHODS:
Targeted LC-MS/MS quantified 45 bile acids in plasma from 976 older adults in the IMAGENOMICS cohort, alongside clinical, biochemical, dietary, and cognitive metadata. Metagenomic sequencing identified KEGG orthologues in bile acid biosynthesis pathways to assess microbial contributions to circulating bile acid levels.

Causality was tested in two murine models. In the first, faecal microbiota from human donors stratified by cognitive performance were transplanted into germ-free mice, followed by cognitive assessment via the Morris water maze. In the second, specific-pathogen-free mice received oral hyodeoxycholic acid (HDCA) or vehicle control by gavage. Cognition was tested using the Y-maze. Frontal cortex and hippocampus were harvested from these mice for transcriptomic profiling of bile acid receptors FXR and TGR5.

RESULTS:
Higher plasma HDCA levels were significantly associated with poorer short-term memory performance. Metagenomic pathway analysis revealed enrichment of microbial genes linked to secondary bile acid biosynthesis in individuals with elevated HDCA. KEGG orthologue abundance correlated with plasma HDCA concentrations, supporting a microbial origin. Mice receiving FMT from high-HDCA donors exhibited impaired spatial memory, with longer path lengths and reduced efficiency. Oral HDCA recapitulated these behavioural deficits, confirming a direct role for this metabolite. Transcriptomic profiling revealed HDCA-induced downregulation of FXR and TGR5 in the cortex and hippocampus, indicating disrupted bile acid signalling in brain regions involved in cognition.

CONCLUSION:
Using a targeted, hypothesis-driven multi-omics approach, we identify microbial-derived HDCA as a modifiable contributor to age-related cognitive decline. These findings establish a mechanistic link between gut microbial bile acid metabolism and brain dysfunction, highlighting bile acid signalling pathways as potential therapeutic targets for preserving cognitive health in ageing.

Development of a Quantitative Clinical Assay for Hexosylsphingosines Rachel Harris (Presenter) Children's Hospital of Philadelphia

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Poster #25d View Map

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

INTRODUCTION:
Krabbe disease and Gaucher disease are rare, autosomal recessive lysosomal storage disorders caused by impaired degradation of galactosylceramides and glucosylceramides, respectively. In Krabbe disease, undegraded galactosylceramides undergo deacylation to form psychosine (galactosylsphingosine), a cytotoxic metabolite. Similarly, in Gaucher disease, lyso-Gb1 (glucosylsphingosine) is generated through deacylation of accumulated glucosylceramides. The elevation of psychosine and lyso-Gb1 are among the biochemical hallmarks for Krabbe and Gaucher, respectively. Both disorders manifest as a clinical continuum, ranging from severe to attenuated forms. With disease-modifying therapies now available, accurate quantitation of these analytes is essential for not only for diagnosis and prognosis, but also for treatment monitoring. However, psychosine analysis has been challenging due to its low abundance in biological matrices and the need for baseline separation from its isomer, lyso-Gb1. Therefore, we aimed to develop a robust duplex assay for psychosine and lyso-Gb1, suitable for quantifying these biomarkers across a broad range of biological matrices, and to implement it in a clinical laboratory following validation.

METHODS:
The biological matrices of interest included dried blood spots (DBS), plasma, red blood cell (RBC) lysates, and cerebrospinal fluid (CSF). Hexosylsphingosines in biological specimens were extracted with methanol solution containing the IS. The samples were than acidified and purified using mixed-mode solid phase extraction (SPE) prior to the UPLC-MS/MS analysis. The UPLC-MS/MS analysis was carried out on a Waters Xevo TQ Absolute triple quad (QQQ) mass spectrometer (MS) coupled to an Acquity I-Class UPLC. A Waters UPLC Glycan BEH Amide column (2.1 x 150 mm, 1.7 µm, 130 Å) was utilized for the chromatographic separation of the two isomers. Mobile phases A and B consisted of 95:5 and 5:95 (v:v) acetonitrile mixed with water, respectively, with both containing 0.1% ammonium hydroxide and 5 mM ammonium acetate. The following gradient elution conditions were used: from 0 to 4 minutes, mobile phase A increased from 0.5% to 8%, then increased to 40% in 0.2 min, was held at 40% for 1 minute, decreased back down to 0.5% in 0.1 minute, and finally held at starting conditions for column re-equilibration for 5.2 minutes. The sphingosine backbone remaining after the loss of the sugar headgroup (m/z 282.2) was utilized as the quantifier ion, while the subsequent water loss (m/z 264.2) was utilized as the qualifier ion. The internal standards (IS) for psychosine and lysoGB1 were chosen such that the labels were localized on different parts of the molecule (sugar headgroup vs. sphingosine backbone) such that each IS gave unique transitions even though the analytes are isomeric.

RESULTS:
Mobile phase composition and the chromatographic separation gradient was optimized to achieve baseline separation of psychosine and lyso-GB1. Extended column re-equilibration times were necessary to ensure reproducible retention times. Other parameters, such as source settings, cone voltage, and collision energy were optimized to maximize the sensitivity. SPE cleanup was utilized to reduce matrix effects and prevent rapid buildup of column backpressure. The SPE protocol was further optimized to maximize analyte recovery. These efforts have enabled the accurate quantification of 0.5 nM psychosine in dried blood spots (DBS) and 0.04 nM in other biological matrices, highlighting the high sensitivity of this platform. Calibration curves were prepared in either neat solution (plasma, CSF, and RBC) or DBS, spanning 2–4 orders of magnitude for psychosine and Lyso-Gb1. Quadratic regression with 1/x² weighting was utilized for curve fitting. Preliminary validation studies have demonstrated that this protocol can measure both biomarkers in a precise, sensitive, and accurate manner in all matrices. Further analytical and clinical validation experiments will be conducted to fully validate the assay according to CLIA guidelines.

CONCLUSION:
We have developed a quantitative LC-MS/MS assay for psychosine and Lyso-Gb1. The optimized HILIC gradient and mixed-mode SPE protocol have significantly enhanced assay performance across a wide range of biological specimens. This duplex hexosylsphingosines assay will be useful for diagnosis and prognosis, but also treatment monitoring.

Nanosheet-Assisted LDI-ToF Studies for Therapeutic Drug Monitoring of Human Blood Samples Tae Geol Lee (Presenter) Korea Research Institute of Standards and Science

Poster #26d View Map

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

INTRODUCTION:
Two-dimensionally layered transition metal dichalcogenides (TMDs) have been widely used in various biomedical applications such as biosensing and cancer therapy due to their characteristics including UV absorbance, photoinduced thermal effect, tunable bandgap and photoluminescence [1]. However, their applications in therapeutic drug monitoring, which is essential for individualizing drug dosage regimens, is rarely reported.

METHODS:
A facile platform for rapid quantitative determination was fabricated via inkjet printing of TMD exfoliates and the concentrations of therapeutic drug in the patient blood sample were analyzed by using laser desorption/ionization time-of-flight (LDI-ToF) method.

RESULTS:
In this work, we report that chemically exfoliated TMD nanosheets including tungsten disulfide (WS2), tungsten ditelluride (WTe2) and molybdenum ditelluride (MoTe2) are suitable for therapeutic drug monitoring based on laser desorption/ionization mass spectrometry (LDI-MS). The WS2 nanosheets allowed for sensitive detection of commonly prescribed immunosuppressive drugs including cyclosporine A and tacrolimus [2]. Quantification of antiepileptic drugs in human blood was achieved using WTe2 and MoTe2 nanosheets, while they also facilitated the concentration determination of vitamin D3 [3]. Computational investigation using density function theory further elucidated that desorption can be facilitated by electrostatic repulsion between positively charged analytes and holes generated on the substrate surface upon laser irradiation (i.e., charge-driven desorption).

CONCLUSION:
Employing TMDs as matrices in LDI-MS enhances the sensitivity of quantitative drug and biomarker analysis, even at low concentrations. The process benefits from improved reproducibility through inkjet printing and is supported by the statistical analysis for method agreement. The compatibility of LDI-MS with LC-MS/MS techniques will facilitates the broader adoption in clinical diagnostics. The DFT calculations allowed for estimation of potential protonation sites for various drugs, where the electronic interaction between the TMD nanosheets can be critical. It is expected that the influence of the properties of TMD in LDI mechanism can be elucidated by incorporating the drugs and the TMDs into a single system so that the interactions during the ionization process can estimated.

REFERENCES:
[1] Agarwal, V., et al., Recent advances in the field of transition metal dichalcogenides for biomedical applications. Nanoscale, 2018. 10: p. 16365-16397.
[2] Joh, S., et.al., Quantitative analysis of immunosuppressive drugs using tungsten disulfide nanosheet-assisted laser desorption ionization mass spectrometry. ACS Nano, 2021. 15: p. 10141-10152.
[3] Joh, S., et al., Role of chalcogenides in sensitive therapeutic drug monitoring using laser desorption and ionization. ACS Nano, 2024. 18: p. 17681-17693.

Accurate Quantitation of 47 Therapeutic Drugs in Serum Using a Rapid and Efficient Sample Extraction Procedure Holly Pagnotta (Presenter) SCIEX

Poster #27d View Map

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

INTRODUCTION:
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely adopted technique for the quantification of therapeutic drugs in biological matrices due to its high sensitivity and specificity. However, accurate quantitation can be compromised by matrix interferences such as proteins and phospholipids. Traditional sample preparation methods like protein precipitation and solid phase extraction (SPE) have limitations—protein precipitation does not remove phospholipids, while SPE is often time-consuming. To overcome these challenges, a single-step Phree phospholipid removal (PLR) procedure was developed to simultaneously eliminate proteins and phospholipids. This method, combined with the SCIEX QTRAP 6500+ system, was applied to quantify 47 therapeutic drugs in serum, including anticonvulsants, antidepressants, antipsychotics, and tricyclic antidepressants. The method aimed to achieve high recovery, sensitivity, and precision across a concentration range of 1 to 50 ng/mL.

METHODS:
A panel of 47 drug compounds was obtained and prepared in methanol to create a 1 µg/mL stock solution. Calibrators were prepared by spiking this solution into serum to achieve final concentrations of 1, 5, and 50 ng/mL. For sample preparation, 100 µL aliquots of each calibrator were processed using the Phree PLR plate, which contains a sorbent and oleophobic membrane to selectively remove phospholipids. Acetonitrile was added to precipitate proteins, and the mixture was filtered through a 0.2 µm membrane using positive pressure. The resulting filtrate was transferred to vials for LC-MS/MS analysis.

Chromatographic separation was performed using the SCIEX ExionLC AC system with a Phenomenex Kinetex Biphenyl column at 40°C. The mobile phases included water, methanol, and ammonium formate, with a flow rate of 0.4 mL/min and a total run time of 9.5 minutes. A 2 µL injection volume was used.

Mass spectrometry was conducted on a QTRAP 6500+ system in positive electrospray ionization (ESI) mode. A scheduled MRM method with 94 transitions (2 per analyte) was used to optimize dwell times and ensure reliable quantitation. Data were processed using SCIEX OS software (v3.1.6), employing the MQ4 algorithm for peak integration and the Analytics module for calibration curve generation, concentration calculations, and statistical analysis. Recovery was assessed by comparing extracted samples to neat solvent standards.

RESULTS:
The method achieved baseline chromatographic separation of all 47 drug compounds within a 9.5-minute run. The single-step Phree PLR procedure effectively removed proteins and phospholipids, resulting in high extraction efficiency. Recovery values ranged from 70% to 130% for most analytes across all three calibrator levels, with the exception of dehydroaripiprazole and mirtazapine (below 70%) and ziprasidone (above 130%).

Calibration curves demonstrated excellent linearity, with R² values exceeding 0.991 for the majority of compounds across the 1–50 ng/mL range. At the lowest calibrator level (1 ng/mL), the method showed strong quantitative performance: precision (%CV) was below 10% and accuracy (% bias) ranged from 95% to 105% for most analytes. These results confirm the robustness and reliability of the method for quantifying trace levels of a diverse panel of therapeutic drugs in serum.

CONCLUSION:
This study demonstrates the successful development and validation of a rapid, efficient, and highly sensitive LC-MS/MS method for the quantitation of 47 therapeutic drugs in serum. The single-step Phree PLR sample preparation procedure effectively removed matrix interferences, enabling high recovery and clean extracts. The method exhibited excellent linearity, precision, and accuracy across a clinically relevant concentration range. These results support the method’s suitability for routine therapeutic drug monitoring and clinical research applications involving complex drug panels.

Integrated Analysis of Proteins and Post-translational Modifications from Dried Blood: Application to Sepsis Phenotyping and Newborn Screening Matt Foster (Presenter) Duke University

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Poster #28d View Map

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

INTRODUCTION
The term “blood proteomics” is most often associated with the analysis of plasma or serum and ignores the contribution of abundant cell-types. We have recently developed methods for mass spectrometry-based quantification of proteins, N-glycopeptides and phosphopeptides from dried blood collected by volumetric microsampling, and we applied these methods to a longitudinal study of sepsis (Foster MW et al. biorxiv 2025). Numerous acute phase and neutrophil-derived derived proteins, and related phosphosites and glycoforms, were reduced between presentation and recovery at d28-42. Here, we sought to replicate those findings in a cross-sectional sepsis cohort and to further extend these methods to dried blood spots (DBS).

METHODS
Venous EDTA-blood from adult healthy controls, patients with sepsis-3 criteria was collected on 20 µL Mitra devices. Alternatively, venous EDTA-blood was obtained from infants with or without Pompe disease (GSD type II) diagnosis based on deficient acid alpha-glucosidase (GAA) enzyme activity, and adult healthy controls, and spotted onto dried blood spot cards. Mitra tips, or 6 mm punch from DBS cards, were processed using deoxycholate-assisted in solution trypsin digestion. Approximately 1 mg of tryptic digests were enriched for N-glycopeptides using hydrophilic ion chromatography (HILIC), and the flow through was enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC). The unenriched proteome was analyzed using microflowLC and Orbitrap Astral data-independent acquisition, and the N-glycoproteome and phoshoproteomes were analyzed using an Evosep LC and stepped collision energy Orbitrap data-dependent acquisition or Orbitrap Astral DIA. Data was analyzed using Spectronaut, FragPipe and Skyline.

RESULTS
Approximate 10,000 analytes (1,600 proteins, 4,000 glycopeptides and 4,600 phosphosites) were quantified from Mitra devices loaded with venous EDTA-blood. Multi-omics factor analysis (MOFA) separated healthy control and sepsis-3 patients on a latent factors plot, with factor 1 weighting acute phase (e.g. LBP, CRP and FGL1) and immune cell proteins (e.g. MMP8, IFM3, PERM), glycoforms of AACT and phosphosites of OSTP, GOLM1 and GAS7. These data validated longitudinal changes observed in a discovery cohort of sepsis-2 patients at presentation versus recovery (day 28-42). This approach was scaled to process a 6 mm DBS punch. Comparable numbers of proteins and PTMs were quantified from DBS versus Mitra devices. A comparison of the proteomes of newborns (n=4 controls and n=3 Pompe disease) and unaffected adult controls (n=5) showed many differentially-abundant proteins, including expected higher levels of fetal hemoglobin and lower IGM complex (IGHM, CD5L and ApoJ) in newborns.

CONCLUSION
Signatures of recovery from sepsis, reflected in the blood proteome, glycoproteome and phosphoproteome, are replicated in a cross-sectional analysis of sepsis versus healthy controls. Methods for processing and “multi-proteome” analysis of dried venous blood are easily translated to DBS. Discovery of blood-based markers of GAA enzyme activity and Pomp disease-causing variants may require a larger number of cases due to heterogeneity of disease and inter-individual blood proteomes.

Simple, Large-Scale Absolute Quantitative Proteomics Through Stable Isotope-Labeled Mouse Internal Standard Timon Geib (Presenter) Lady Davis Institute

Poster #29d View Map

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

INTRODUCTION:
Large-scale proteomics faces persistent challenges, including batch effects, often extensive offline fractionation, and long LC run times, that repeatably undermine the statistical significance of data and as a result the detection of subtle yet critical changes in complex biological samples. These barriers are magnified when inter-laboratory reproducibility is required. Stable isotope-labeled (SIL) standards offer a solution, but their high cost and lack of off-the-shelf availability frequently limit widespread adoption. We have introduced “SysQuan”, leveraging SIL mouse as a versatile, system-wide internal standard, enabling the absolute quantitation of the human proteome. SysQuan simplifies proteomics workflows, reduces costs, and enhances scalability, while offering absolute proteome quantitation within a single sample.

METHODS:
C57BL/6 mice were fed 13C-lysine chow over six generations. Human and SIL mouse fresh-frozen livers were cryo-homogenized and then ultrasonicated. Homogenized livers, as well as human and SIL mouse plasma were heated in sodium dodecyl sulfate (SDS) buffer. A one-to-one mix of human and mouse liver, and one-to-one mix of human and mouse plasma were reduced with tris (2-carboxyethyl) phosphine (TCEP), alkylated with iodoacetamide and then digested overnight using trypsin and S-Trap cartridges. Up to 1 µg of digests were injected on column and analyzed by dynamic multiple reaction monitoring (MRM), performed on an Evosep One LC system, equipped with an IonOpticks Aurora Elite 15 cm x 75 μm C18 UHPLC column, coupled to an Agilent 6495 triple quadrupole. In addition, digests were offline fractionated by high-pH reversed-phase LC. Untargeted top 10 data-dependent acquisition (DDA)–parallel accumulation-serial fragmentation (PASEF) was run on an Evosep One coupled to a Bruker timsTOF HT.

RESULTS:
Through in-depth untargeted analysis of extensively fractionated liver digests (48 fractions), we identified over 48,000 SIL/light peptide pairs representing more than 6,900 unique human proteins in liver samples. In parallel, we identified over 5,700 peptides in plasma with quantifiable SIL and light features, representing more than 1,400 unique human plasma proteins. Building on this, we shifted focus to liver metabolism, targeting up to 800 proteins using targeted MRM analysis of unfractionated liver digests. From an initial set of 5,324 peptides—comprising 2,662 SIL mouse/human pairs with at least three pairs per protein—we refined the data using ion ratio matching. This resulted in the successful detection and quantitation of 541 peptides representing as many as 296 liver proteins. This was accomplished without optimizing MRM transitions, collision energies, or MRM retention time windows. Instead, a 60-min nano-flow gradient and scheduled MRM scanning were employed. Over 500 peptides were detected with dwell times as short as <1 msec.

We are actively working to further enhance detection and quantitation rate by reducing transitions to three fragments per precursor and optimizing retention times and collision energies to maximize the performance of each feature. For this, we are synthesizing and optimizing light peptide standards for the remaining ~500 hepatic protein targets. Similarly, we are implementing the same approach to the 1,400 plasma proteins.

Additionally, light peptide standards are being utilized to reverse-quantify mouse SIL liver and plasma proteins. This will allow for the direct, off-the-shelf quantitation of hundreds of proteins in a single MRM run, even with complex, unfractionated liver and plasma samples.

CONCLUSION:
SysQuan transforms absolute quantitation of the human liver and plasma proteome with a novel, single-injection workflow, making scalability and cross-laboratory comparisons accessible.

A Targeted Proteomic Approach to Monitor βT87Q-Globin Production in Transgenic Hematopoietic Cells Fan Yi (Presenter) Children's Hospital of Philadelphia

Poster #30d View Map

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

INTRODUCTION:
Beta-globinopathies are among the most prevalent inherited disorders worldwide, affecting approximately 0.27% of conceptions. They result from either reduced β-globin synthesis or structural abnormalities in the protein, caused by variants in the HBB gene (1). Curative interventions are being developed for beta-globinopathies. Since the substitution at position 87 from threonine to glutamine afford a strong antisickling effect, ex vivo gene therapies utilizing lentiviral vectors carrying the therapeutic β-globin gene (βT87Q -globin) has been developed. The safety and efficacy of this approach has been demonstrated in multiple clinical trials for β-thalassemia and sickle cell disease (2).

Measurement of βT87Q -globin has been employed as a quality control to verify the ability of transduced cells to produce the therapeutic βT87Q-globin in the vector production. A reverse phase HPLC method was utilized to distinguish βT87Q-globin chain from the normal proteoform in a semi-quantitative fashion. (2) However, the implementation of the HPLC method remains challenging in the individual lab. Herein, a UPLC-MS/MS method was developed in the Center for Diagnostic Innovation (CDI) at the Children’s Hospital of Philadelphia (CHOP) to quantitatively determine the ratio of transgenic 87Q β-globin to wild type 87T β-globin.

METHODS:
A targeted proteomic approach was adopted in this method. Tryptic peptides (87T: GTFATLSELHCDK and 87Q: GTFAQLSELHCDK) encompassing position 87 in β-globin were selected. Stable isotope labeled internal standard (IS) of these peptides were synthesized. Red blood cell (RBC) or hematopoietic cells after differentiation underwent reduction, alkylation, and tryptic digestion. IS was added to the reaction mixture before centrifugation and then the supernatant was transfer to sample analysis plate. The UPLC-MS/MS system used were Waters Acquity I class PLUS UPLC coupled with Waters Xevo TQ Absolute or Shimadzu LC 40X3 UPLC coupled with Sciex 7500+. The analytical column was Waters UPLC CSH C18 column (1.7μm, 2.1 mm x 50 mm).

Since adult human RBCs contain extremely low or undetectable levels of the 87Q peptide, and cynomolgus monkey RBCs lack the 87T peptide, mixing the two at defined ratios generates a range of 87Q/87T peptide ratios. Standard calibrators and quality controls were prepared by combining human and monkey RBC lysates in specific proportions. Theoretical peptide ratios were inferred from hemoglobin concentrations determined using the standard cyanmethemoglobin method.

RESULTS:
The 87T and 87Q peptides were chromatographically separated and specific MRM transitions were selected, ensuring accurate quantitation of both analytes. The digestion protocol was optimized to maximize the digestion efficiency. The assay validation was conducted according to the FDA M10 validation guidance. In all analytical runs, the calibration curves had coefficient of determinations above 0.998. With four level of QCs, the intra-day accuracy of all QC was between -2.9% to 7.4% bias; the intra-day precision was within 4.7% CV; the inter-day accuracy was between -0.5% to 4.6% bias; and the inter-day precision was within 3.6% CV. The 87Q/87T ratio was stable in the samples for up to 18 hours at 4°C or RT in the short-term; and up to 22 days at -80 °C in the long-term. Furthermore, the 87Q/87T ratio was consistent after 5 freeze/thaw cycles. With six lots of different human and monkey RBC combination, the assay had an accuracy between -13.0% to 14.5% bias; and a precision within 6.9% CV. There were no carryover and interference concern.

CD34⁺ cells transduced with the ALS20 vector (3) were evaluated against wild-type controls upon erythroid differentiation (>90% of cells resulted positive to benzidine staining). The transduced cells exhibited 87Q/87T ratios ranging from 0.64 to 0.93, with higher ratios correlating to increased vector copy number (VCN), whereas the control cells showed a ratio of 0.14.

DISCUSSION:
This quantitative assay described herein measures the ratio of 87T to 87Q peptides in a highly accurate, precise and specific manner. The assay’s specificity was demonstrated by its ability to identify an off-target vector used during the production process, enabled by the combination of chromatographic separation and specific MRM for each peptide. Notably, the use of real biological matrices proved critical, as initial evaluations revealed that extracted hemoglobin or recombinant β-globin yielded peptide levels several orders of magnitude lower than those recovered from hemoglobin in RBCs, for reasons not yet fully understood. Therefore, adult human and cynomolgus monkey RBCs were utilized as β-globin source. This targeted proteomic approach serves as a valuable complementary tool to existing quality control measures, supporting both vector selection and the assessment of transgenic protein expression.

REFERENCES:
(1) Modell B, Darlison, M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008;86(6):480-487.
(2) Negre O, Eggimann AV, Beuzard Y, et al. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene. Hum Gene Ther. 2016;27(2):148-165.
(3) Breda L, Ghiaccio V, Tanaka N, et al. Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies. Mol Ther. 2021;29(4):1625-1638.

A Novel Method for Urine Drug Screening Based on Desorption ElectroSpray Ionization (DESI) MS Analysis Steven Pringle (Presenter) Waters Corporation

Poster #31d View Map

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

INTRODUCTION:
Substance use disorders remain a significant public health concern in the US. Mitigating efforts are further complicated by the emergence of novel psychoactive substances (NPSs).

Urine drug testing in clinical and forensic settings, typically consists of an immunoassay-based drug screen followed by LC-MS-based confirmatory testing. Immunoassays, although fast and inexpensive, show limited sensitivity and specificity, increasing the likelihood of false-negative and -positive test results, and show limited NPS detection capability. LC-MS-based analysis enables definitive identification of drugs in urine, however is relatively lengthy. The goal of this study was to develop an ultra-fast urine drug screen based on DESI MS Analysis. The non-chromatographic method is intended to deliver increased sensitivity, specificity and required flexibility needed in the ever-changing NPS landscape.

OBJECTIVE(S):
The goal of this study was to develop an ultra-rapid mass spectrometry-based screening method capable of routine detection of drug metabolites in urine samples.

METHODS:
Drug standards from different drugs of abuse and medications were used to spike drug-free pooled urine. 1ul sample was spotted on PFTE-coated glass slides, and analyzed by a Waters XevoTM G2 XS Mass Spectrometer equipped with a prototype Desorption Electrospray Ionization (DESI) Autoloader source for high throughput plate reading. A Waters SELECT SERIESTM CyclicTM IMS equipped with a DESI XS source was used for compound validation and the separation of isobaric compounds. Spectra were acquired in positive and negative ion mode, m/z 50-600 and a targeted method using extracted ion chromatograms and MS/MS validation was developed to identify the presence of the different known drugs, while an untargeted method using principal component analysis was used to identify anomalies within the sample.

RESULTS:
A research prototype DESI Autoloader was used for automatic plate acquisition and data export. Drug standards from 44 different drugs of abuse and 4 different medications were tested in concentrations ranging from 0.2-500 ng/mL. A total of 1 ul was spotted resulting in 0.2-500 pg of material within each measured sample spot. The time of measurement was 1-2 seconds/per sample resulting in less than 3 minutes per 96-well plate, however this time can be reduced to 2 minutes per plate if necessary. The MS and DESI parameters were optimized to the mass range of m/z 50-500 in positive mode and all sprayer and flow settings were optimized for highest sensitivity.

While all drug compounds were visible in positive ion mode, Amphetamine, Ethanol-Sulfate and THC metabolites had a higher signal in negative ion mode. Using simple principal component analysis, samples in ‘fake urine’, e.g. Creatinine containing buffer could be 100% separated from urine samples. The general limit of detection for the drugs was ranging between 2-40 ng/mL after 5-10x dilution with Isopropyl-alcohol (IPA), which could be improved in some cases (e.g. Buprenorphine variants) with an extra dilution of the urine sample in IPA. For some drugs in the drug panel, the currently optimized method without sample preparation is already suitable as a screening method, however as the aimed limit of detection for most of the drugs is 50-100 ng/mL, while for medicative buprenorphine and drugs fentanyl, mitragynine and 6-acetyl-morphine is 5-10 ng/mL pre sample dilution, with an extra matrix removal or concentrating step it is possible to reach the expected LoD for all the drugs within the drug panels.

CONCLUSIONS:
In this study, we demonstrated that DESI-MS has the potential of becoming a screening method for the parallel detection of known compounds and sample anomalies in urine.

Waters, Xevo, SELECT SERIES and Cyclic are trademarks of Waters Technologies Corporation.

Development of a Phenotypic Assay for Functional Screening of Dihydropyrimidine Dehydrogenase Deficiency in Clinical Oncology Kerene Brown (Presenter) Dynacare

Poster #32d View Map

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

INTRODUCTION:
Fluoropyrimidines (FP), such as 5-fluorouracil, are a widely used class of chemotherapy drugs for treating various cancers. Despite their effectiveness, 10–40% of patients experience severe FP-related toxicities (grade 3 or higher), and 0.1–1% may suffer fatal toxicity. These adverse effects are largely attributed to a deficiency in dihydropyrimidine dehydrogenase (DPD), the key enzyme responsible for metabolizing FP drugs. To assess DPD function, genotype testing of the DPYD gene—which encodes the DPD enzyme—is recommended before initiating treatment. However, this genetic approach typically screens for only 5–6 common DPYD variants known to impair enzyme activity. In contrast, phenotypic testing evaluates actual enzyme function by measuring concentrations of uracil and its metabolite, dihydrouracil, in plasma.

OBJECTIVES:
To develop a phenotypic assay for evaluating dihydropyrimidine dehydrogenase (DPD) enzyme activity through quantification of plasma uracil and dihydrouracil levels.

METHODS:
For phenotypic analysis, whole blood was collected via standard venipuncture into EDTA (lavender-top) tubes. Samples were centrifuged, and plasma was aliquoted into transport tubes and stored frozen until analysis. Uracil and dihydrouracil were extracted from plasma using supported liquid extraction (SLE) and quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS).
For genotypic analysis, whole blood was similarly collected in EDTA tubes. DNA was extracted and analyzed using a TaqMan assay targeting five specific DPYD gene variants.

RESULTS:
To evaluate the performance of the phenotypic assay, 24 samples underwent both genotypic and phenotypic testing. Of the total samples tested, 22 samples were identified as wild type and had no identifiable genetic variants. However, genetic variants were identified in two samples. Sample B35 was heterozygous for the c.1905+1G>A mutation—a non-functional allele with an activity score of 1. Its uracil concentration exceeded 16 ng/mL, indicating reduced DPD enzyme activity.

Sample N68 carried two variants, c.1236G>A and c.1129-5923C>G, present on the same allele. These are categorized as reduced-function variants with a combined activity score of 1.5, suggesting potentially impaired enzyme function. However, phenotypic testing for N68 revealed the uracil concentration was below 16 ng/mL, consistent with normal enzyme activity.

Conversely, sample R19 exhibited a markedly elevated uracil concentration and a dihydrouracil-to-uracil ratio of 2, both suggestive of diminished DPD activity, despite the absence of any of the five DPYD variants screened by genotyping.

CONCLUSIONS:
The combined genotypic and phenotypic analysis provided complementary insights into DPD enzyme function across the 24 tested samples. While genotyping identified reduced- or non-functional DPYD variants in two cases—both supported by corresponding or partially aligned phenotypic findings—phenotyping alone revealed a case (Sample R19) with biochemical evidence of impaired enzyme activity despite the absence of known genetic variants. These results highlight the added diagnostic value of phenotypic testing in identifying patients at risk for fluoropyrimidine toxicity, particularly in cases where rare or untested DPYD variants may be involved. Together, these findings support the use of a dual testing strategy to improve the safety and personalization of fluoropyrimidine-based chemotherapy.

REFERENCES:
1. Etienne-Grimaldi M-C, Pallet N, Boige V, Ciccolini J, Chouchana L, Barin-Le Guellec C, et al. Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency. Eur J Cancer. 2023 Mar;181:3–17.
2. Henricks LM, Lunenburg CATC, de Man FM, Meulendijks D, Frederix GWJ, Kienhuis E, et al. DPYD genotype-guided dose individualisation of fluoropyrimidine therapy in patients with cancer: a prospective safety analysis. Lancet Oncol. 2018;19(11):1459–67.

Performance Evaluation of Reflex Tg-MS testing in TgAb-positive Samples: A Retrospective Concordance Analysis with Immunoassay Preejith Vachali (Presenter) Cleveland Clinic

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Poster #33d View Map

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

BACKGROUND:
Thyroglobulin (Tg) is an essential biomarker for monitoring differentiated thyroid cancer. However, the presence of Tg antibodies (TgAb) can interfere with Tg measurements obtained by immunoassays. To address this, reflex testing using a mass spectrometry-based Tg assay (Tg-MS) is employed when TgAb levels exceed a defined threshold. But recent literature findings have raised concerns regarding the validity of using a fixed TgAb threshold to trigger reflex MS testing (1). Expert recommendations now suggest that MS test may not be needed if Tg is <0.1 µg/L with no evidence of structural disease (1). This study assesses the analytical agreement and correlation between Tg-MS (LC-MS/MS) and the initial Beckman Tg immunoassay in TgAb-positive samples.

OBJECTIVE:
To determine if reflex testing with the Tg- MS assay is necessary in TgAb-positive samples by evaluating its concordance with the initial Beckman Tg immunoassay, and whether strong agreement between the two methods supports limiting or omitting reflex testing in certain clinical scenarios.

METHODS:
Consecutive tests from March 2023 to February 2025 included 484 unique patients, and 928 total of Tg measurements. Tg values were assessed using two assays: an initial Beckman Tg immunoassay for all samples, with reflex Tg-MS testing conducted only for those with elevated TgAb (>4 IU/mL). Analytical focus was placed on values ≥0.5 ng/mL due to differences in detection thresholds (Beckman: ≥0.1 ng/mL, Tg-MS: ≥0.5 ng/mL). Tg values were categorized as <1, 1–2, and >2 ng/mL based on clinical cut-offs. Descriptive statistics were applied to demographic and assay data, using medians and interquartile ranges for non-normally distributed continuous variables, and frequencies and percentages for categorical variables. Non-normality was confirmed for both Tg assays, and log transformations failed to achieve normalization. Spear-man correlation analysis was performed to assess the strength and direction of association be-tween assays. To handle repeated measures, rank-transformed values were analyzed using the rmcorr package in R to produce within-subject correlation coefficients. Concordance correlation coefficient (CCC) was calculated based on a mixed-effects Poisson regression model incorporating random intercepts for patient ID, assay type, and their interaction, as per Tsai and Lin’s method (2) for non-normal distributions. The delta method was used to estimate 95% confidence intervals (CI). All analyses were conducted using SAS 9.4 and R version 4.3.

RESULTS:
The patient cohort had a median age of 57 years (IQR: 42–69), with 80.1% female. Across all 928 observations, the median Beckman Tg was 0.1 ng/mL and Tg-MS was 0.5 ng/mL. When restricted to values ≥0.5 ng/mL, Beckman Tg also had a median of 0.5 ng/mL, indicating baseline alignment with Tg-MS. The median absolute difference between assay values was 0.0 ng/mL, confirming minimal deviation. Among 624 observations with Beckman Tg levels <0.5 ng/mL, 93.7% also had Tg-MS below this threshold. Conversely, of the 262 observations with Beckman Tg >0.5 ng/mL, 95.8% showed corresponding Tg-MS values >0.5 ng/mL. The Spearman correlation coefficient between Tg-MS and Beckman Tg was 0.60 (95% CI: 0.53–0.65, p<0.001), indicating moderate positive correlation. The CCC was 0.98 (95% CI: 0.96–1.00, p<0.001), reflecting very strong agreement. The discrepancy between correlation and concordance is attributed to the non-linear relationship between values, suggesting that although trends are only moderately aligned, individual test values are nearly identical. Among the 484 unique patients, 57.2% had a single Tg measurements, while the remainder had multiple Tg measurements over time, enabling robust within-subject repeated measures correlation analysis.

CONCLUSIONS:
This analysis demonstrates a very high level of concordance between Tg-MS and Beckman Tg assays in TgAb-positive samples. While Tg-MS is currently performed reflexively when Tg anti-bodies are elevated, the strong agreement observed raises the question of whether reflex testing is always needed. In cases where Beckman Tg and Tg-MS values align closely, it may be reasonable to reconsider the routine necessity of reflex testing, particularly in settings where assay agreement is reliably high. However, further prospective testing and validation would be required before modifying clinical protocols.

REFERENCES:
1. Giovanella L., et al., hsTg&TgAb Consensus Working Group. Thyroglobulin and thyroglobulin antibody: an updated clinical and laboratory expert consensus. Eur J Endocrinol, 2023 189(2): R11-R27.
2. Miao-yu T and Chao-Chun L., Concordance correlation coefficients estimated by variance components for longitudinal normal and Poisson data. Comput Stat and Data An, 2018. 121 p. 57-70.

Improvements in Process Efficiency and Sample Throughput with the Introduction of a Multi-Channel LC-MS/MS System for Applications in the Clinical Laboratory Dylan Flohrschutz (Presenter) Biodesix Inc.

Poster #34d View Map

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

INTRODUCTION:
Clinical laboratories encounter unique challenges as they scale. These include laboratory workflow constraints which may require technological adaptations to improve operational efficiencies. One approach to mitigating these challenges is by adapting higher throughput instruments for data acquisition to analyze patient specimens. Importantly, improvements must maintain consistency in test results. Improvements that result in increased operational efficiency can allow clinical laboratories to scale with the demands of an increasing number of patient specimens, optimize the utilization of physical space within the clinical laboratory, and deliver patient results more quickly.

METHODS:
Improvements in process efficiency and sample throughput for a clinical proteomics MRM (multiple reaction monitoring) assay utilizing LC-MS/MS were assessed by comparing single-channel LC configurations with a multi-channel configuration. The multi-channel LC-MS/MS system consists of a Thermo Fisher Scientific Transcend Multi-Channel LX-4 UHPLC system in tandem with a Thermo Fisher Scientific TSQ Quantis Plus QQQ mass spectrometer (Transcend). Two configurations of single-channel LC-MS/MS systems were included in the evaluation: a Thermo Fisher Scientific Vanquish Flex UHPLC system in tandem with a Thermo Fisher Scientific TSQ Quantis Plus QQQ mass spectrometer (Thermo single-channel), and an Agilent Infinity 1290 UHPLC system in tandem with an Agilent 6490 Series mass spectrometer (Agilent single-channel). All systems were validated for the MRM clinical diagnostic test that measures the ratio of two plasma proteins (LG3BP and C163A), combined with several clinical and radiological factors. This test is used in the clinic to help identify patients with likely benign pulmonary nodules who may benefit from further CT surveillance.

The efficiency improvements that were examined within this study included evaluating the Transcend with respect to increasing capacity to process patient specimens, optimization of physical space, and time savings all while maintaining consistency of our results. The improvement in capacity was evaluated by considering the number of samples analyzed during a typical shift in the laboratory. The optimization of physical space was assessed by measuring the total footprint occupied by each of the three systems within the laboratory. Improvements in efficiency of time were evaluated by comparing the total runtime required for each system to run a single sample using the validated MRM method.

RESULTS:
The Transcend was capable of analyzing a total of 172 samples (4 separate batches containing 43 samples each, with each batch analyzed in a staggered multi-channel configuration) in approximately 18.5 hours while the Thermo single-channel and Agilent single-channel systems were able to process 43 samples in approximately 19.1 hours and 19.0 hours, respectively. These results show that an increase in capacity of over 4x was able to be achieved within the same timeframe following the introduction of the Transcend for the MRM assay. The total estimated footprint of the Transcend was 37.3 sq. ft. while the Thermo single-channel and Agilent single-channel systems were estimated to be 28.0 sq. ft. and 33.3 sq. ft., respectively. Considering four single-channel systems would be required to achieve approximately the same throughput as a Transcend, the multi-channel system saves between 75-96 sq. ft. of bench space within the clinical laboratory. An improvement in the efficiency of time to analyze a single sample was observed with the introduction of the Transcend system by saving approximately 1 minute and 20 seconds per sample when compared to both single-channel systems, saving nearly 1 hour over the course of a batch containing 43 samples. This improvement in efficiency is attributed to the difference in autosampler design where the Transcend has a pushed-loop design while the Thermo single-channel and Agilent single-channel systems both have split-loop designs.

There were several opportunities to improve on the out-of-the-box provided Transcend that were addressed to adapt it to our plasma-based MRM assay in the clinical laboratory. The default stainless-steel lines initially provided with the Transcend led to the target peptides binding to active sites along the stainless-steel lines resulting in poor peak shape. This was resolved by installing Viper capillaries lined with biocompatible material. Additionally, due to utilizing larger mobile phase bottles to compensate for the increase in mobile phase consumption by the Transcend, it was difficult for personnel to physically work with full bottles during operations at the default height of the system. We have then installed a shorter, custom-built table that lowered the overall height of the Transcend system, making it easier and safer to operate.

CONCLUSION:
The introduction of the Transcend in our clinical laboratory allowed us to successfully scale our capacity and throughput of a clinically validated MRM assay. The multi-channel system we implemented was able to maintain consistency in our results while optimizing the physical space utilized within our laboratory. Additional process improvements are being investigated as the test continues to be adopted in the clinic.

Unlocking Latent Phenotypic Predictive Power in Unstructured Molecular Data via Self-Supervised Machine Intelligence Baljit Ubhi (Presenter) Matterworks

Poster #35d View Map

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

INTRODUCTION:
Traditional mass spectrometry (MS)-based omics workflows rely on human-curated features such as peaks and MS/MS identifications. While effective, these approaches force structure onto inherently unstructured data, discarding large volumes of raw signals that may contain valuable predictive information.

OBJECTIVES:
To overcome these limitations, we developed Large Spectral Models (LSMs)—self-supervised foundation models trained on diverse, unlabeled MS spectra. LSMs learn a semantic representation of raw mass spectral data, enabling phenotype prediction directly from instrument output without prior peak picking or molecular annotation.

METHODS:
In this study, we fine-tuned LSMs on small, labeled phenotype datasets to demonstrate predictive capabilities in real-world biological contexts. Using public LC-MS data from Sah et al. (2024) on ovarian and cervical cancer patients, we trained a predictive model using raw MS spectra alone.

RESULTS:
The LSM-based approach significantly outperformed a conventional supervised method based on manually selected lipid biomarkers. Our model achieved mean accuracy of 0.96, sensitivity of 0.98, and specificity of 0.95, with a tenfold reduction in false negative error rates. Importantly, LSM embeddings captured latent biochemical signals not detected through traditional workflows. Visualization of the reduced LSM representations revealed clear phenotypic clustering, and model benchmarking across 1,000 test/train splits confirmed reproducibility and robustness. By removing the need for feature engineering or annotation, LSMs reduce time-to-insight and expand access to predictive biology for users with limited data science expertise.

CONCLUSION:
This work illustrates a scalable, application-agnostic path for phenotype prediction from raw MS data. As a first-of-its-kind foundation model in mass spectrometry, the LSM approach unlocks unstructured molecular information previously discarded, redefining how spectral data can drive diagnostics, translational research, and precision medicine.

Analytical Accuracy Evaluation of Six Routine Lipid Assays Based on CRMLN-Certified ID-GC/MS and Abell-Kendall Reference Methods Yeo-Min Yun (Presenter) Konkuk University School of Medicine

Poster #36d View Map

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

BACKGROUND:
Lipid testing, including total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) is a cornerstone of cardiovascular risk assessment and chronic disease management. However, the clinical utility of lipid tests relies heavily on their accuracy and comparability across laboratories. Standardization using reference measurement procedures (RMPs) is essential, and isotope dilution gas chromatography–mass spectrometry (ID-GC/MS) is widely recognized as the gold standard for lipid quantification due to its superior specificity, precision, and traceability.

METHODS:
Six routine lipid assays (Roche, Beckman, Siemens, Sekisui, Wako, Kyowa) were evaluated for TC, TG, HDL-C, and LDL-C using three types of serum matrices: fresh single-donor, frozen single, and pooled serum. Reference values for TC and TG were determined by the Korean National Medical Reference Laboratory (NMRL), a CRMLN- and JCTLM-accredited laboratory, using the ID-GC/MS method in accordance with CDC and international protocols. This method provides high-order traceability and has been internationally validated for its long-term stability and analytical robustness. HDL-C and LDL-C reference values were determined using the CDC-modified Abell–Kendall and ultracentrifugation-based reference methods. The mean percentage bias (%bias) of each assay was calculated.

RESULTS:
For total cholesterol, five of six MPs exhibited acceptable mean %bias within the NCEP criterion of ±3%. Wako showed the largest negative bias (–3.87%), while Sekisui had the highest positive bias (+1.71%). Triglyceride measurements also met the NCEP allowable limit of ±5%, with mean %bias ranging from –3.68% (Wako) to +1.29% (Kyowa). These results indicate high analytical accuracy and standardization for TC and TG assays across all platforms.

In contrast, HDL-C and LDL-C results showed greater variability. HDL-C assays from Siemens (+7.41%) and Wako (–5.87%) exceeded the NCEP accuracy limit of ±5%, suggesting limitations in standardization for HDL-C. For LDL-C, Beckman showed the highest positive bias (+5.89%), while Kyowa displayed the largest negative bias (–4.14%). These deviations exceeded the NCEP recommended bias limit of ±4%, particularly in frozen pooled serum specimens with elevated TG levels. The findings underscore the impact of matrix effects and sample-dependent performance variability, especially for lipoprotein-based measurements.

DISCUSSION & CONCLUSION:
The use of ID-GC/MS in reference measurement plays a pivotal role in the standardization of lipid assays. Unlike routine enzymatic methods, ID-GC/MS offers unmatched analytical specificity and is unaffected by matrix interferences, enzymatic variability, or reagent lot differences. As a primary reference method, it enables direct linkage of patient results to certified reference materials, ensuring long-term traceability and international comparability.

This study demonstrates that routine assays for TC and TG, when anchored to ID-GC/MS-derived reference values, can achieve high accuracy and meet international quality targets. The stability and reproducibility of ID-GC/MS data also support its application in external quality assessment (EQA) programs and development of commutable reference materials. In contrast, the persistent variability in HDL-C and LDL-C assays highlights the need for further refinement of reference procedures and broader adoption of traceable calibration systems.

In conclusion, expanding the use of ID-GC/MS in reference measurement is essential to advance lipid assay standardization. Its continued application by CRMLN-certified laboratories will enhance diagnostic accuracy, support public health monitoring, and ultimately contribute to better clinical outcomes through more reliable lipid testing.

Automated Extraction and Chromatographic Separation of Δ 8, 9, 10 THC Isomers from Whole Blood Kyle Dukes (Presenter) Biotage

Poster #37d View Map

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

INTRODUCTION:
Analysis of tetrahydrocannabinol (THC) and its isomers is crucial for understanding their pharmacological effects and legal implications of cannabis use, especially in clinical testing, forensic toxicology, and sport antidoping. THC, the primary psychoactive component of cannabis, exists in several isomeric forms, including Δ9, Δ8, and Δ10-THC, each with distinct pharmacokinetic and pharmacodynamic properties. Accurate identification and quantification of these isomers are essential for evaluating impairment, exposure, or intoxication. However, their analysis presents significant challenges due to their chemical similarities, which can result in co-elution during chromatographic separation and difficulty in distinguishing between isomers using traditional analytical methods.

OBJECTIVE:
This work will demonstrate extraction strategies for cannabigerol, Δ8, Δ9, and Δ10 THC, as well as the common metabolites 11-hydroxytetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) from whole blood. In addition, two chromatographic methods of separating the isomers will be presented.

METHODS:
Δ9, Δ8, and Δ10-THC and the metabolites 11-hydroxytetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) were spiked in whole blood samples concentrations ranging from 0.15-100ng/mL and extracted using ISOLUTE® SLE+ supported liquid extraction plates. Extraction performance was investigated by comparing recovery and matrix effects using MTBE, EtOAc, EtOAc followed by Hexane, and Hexane followed by EtOAc as an elution solvent. Chromatographic separation was investigated by comparing two different analytical column chemistries, biphenyl and fluorophenyl. Protocols were transferred to the Extrahera™ Classic automated sample preparation workstation and evaluated for linearity and precision. UHPLC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC coupled to a Sciex 5500 triple quad mass spectrometer. In addition, column chemistry and oven temperature were investigated to achieve optimal separation of the isomers

RESULTS:
Extraction performance was investigated by comparing recovery and matrix effects using the follow elution solvents: MTBE (750µLx2), EtOAc (750µLx2), 750 µL EtOAc followed by 750 µL Hexane, and 750 µL Hexane followed by 750 µL EtOAc. While these four elution protocols yielded similar matrix effects, EtOAc achieved the highest recoveries of analytes, 70-90% with minimal matrix effects (0.9-1.2). The optimized extraction procedure using ISOLUTE® SLE effectively removed more than 99% of the phospholipids present compared to the protein precipitation method. Final automated method performance and calibration curves demonstrated excellent linearity with coefficients of determination, r2 > 0.99 and excellent reproducibility with RSDs below 10% for all analytes.

CONCLUSION:
THC and its metabolites are effectively extracted by supported liquid extraction (SLE) due to favorable partitioning into the organic extraction solvent layer from the aqueous whole blood matrix. The SLE workflow is comparatively simple compared to a traditional SPE approach which requires additional conditioning, equilibration, and wash steps. Baseline separation of THC isomers can be challenging. Investigation of column chemistry showed that fluorophenyl chemistry achieved better resolution of isomers than biphenyl. Moreover, investigation of column oven temperature demonstrated that a lower oven temperature, 20◦C, is more effective in separating THC isomers and its metabolites.

Click and Detect: A New Generation of Tagged Probes for Customable Targeted Multiplex MS Imaging Alexandre Goossen (Presenter) PRISM

Poster #38d View Map

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

INTRODUCTION
MS Imaging (MSI) has revolutionized biology and clinics by adding a spatial dimension to mass spectrometry (MS) while being a non-targeted approach enabling accessing the distribution of thousands of different analytes in a single acqusition. Among MSI modalities, MALDI-MSI has probably the broadest application range because of its capacity to offer analysis of peptides and proteins. However, despite all the instrumental and methodological developments made over the past twenty-five years many analytes remain undetectable due to limited abundance, poor ionization yield, or high molecular weight and high hydrophobicity in the case of proteins. This difficulty can be overcome by shifting from non-targeted mode to targeted analyses using probes which are designed for MSI detection as introduced in 2007 with the TagMass concept [1]. Advantageously, MS offer higher capabilities of multiplexing (>50) than other detection modality (e.g., fluorescence). Here we have developed a new generation of TagMass probes for highly multiplex immunohistochemistry (IHC) which are highly flexible enabling the modification of any probe including antibodies, aptamers, lectins and RNA probes thanks to bioorthogonal conjugation as well as more quantitative because of the closely related structure designed for the MS reporters which is released for detection by photocleavage.

METHODS
The probes are modified with a tag, including a linker, a UV photo-cleavable group, and a reporter for highly sensitive MS detection. The tagged probe recognizes a target in the tissue, and UV light induces a cleavage of the Tag releasing the MS reporter. The released MS reporter is detected and imaged by MSI, revealing the target distribution. Various reporters with similar mass chemical functions were synthesized and grafted onto the probe after bioorthogonal conjugation. These tagged probes are used for multiplex targeted MALDI MSI.

RESULTS
The synthesized reporters were first characterized by NMR and MS to check their purity and confirm their structure. They were then tested under UV to determine the photocleavable yields and time, and their MS response, both alone and mixed. Indeed, signal response is paramount for the quantitative capacity of the method. More than 10 different probes were synthesized starting with antibodies using bioconjugation, followed by strain-promoted azide alkyne click chemistry (SPAAC) to graft several reporters onto the probe and amplify the signal. Each synthesized reporter was grafted onto an anti-rat primary antibody for immunohistochemistry, targeting GFAP in rat brain tissue sections. MALDI MS images revealed that the different tags showed a similar distribution and detection efficiency. The reporters were then used to functionalize different primary antibodies directed against various targets in the rat brain, including GFAP (astrocytic marker), NeuN (neurons), Myelin (oligodendrocytes), and tubulin beta-3 (stem cells and neural progenitors). All these antibodies were used in a multiplex experiment, resulting in a detectable signal. Finally, a proof-of-concept was made by customizing other probes directed against markers for breast cancer, such as PDL-1, CD47, FOX-P3, Reg-Alpha, HER2, and markers of immune cells and immune check points. The same tags were also successfully used with aptamer, lectin and oligonucleotide probes demonstrating the high flexibility of their design. These results highlight the high sensitivity of the method, capable of detecting low-abundance proteins in inflammatory contexts.

The rise of bioorthogonal chemistry — particularly click chemistry — paves the way for methodological expansion of labeling strategies in mass spectrometry imaging (MSI). This approach allows methods originally developed for antibodies to be extended to other classes of biomolecules, provided that appropriate reactive functional groups can be introduced.
In this context, we present an innovative immunohistochemistry–mass spectrometry imaging (IHC-MSI) strategy based on the use of various probes including aptamers with high multiplexing capacities, sensitivity and quantification performances.

CONCLUSION
Together, these results demonstrate the robustness, versatility, and sensitivity of this photo-cleavable tag strategy for mass spectrometry imaging. The successful application of over more than ten distinct probes in multiplex immunohistochemistry experiments, targeting both neural and tumor markers, confirms the broad applicability of the method across biological contexts. Importantly, the integration of bioorthogonal click chemistry opens promising avenues for adapting this strategy to alternative probes such as aptamers or olignosnucleotides. This work not only validates a scalable and modular MSI-compatible labeling pipeline but also establishes a foundation for future clinical and translational applications in tissue-based diagnostics, with deep characterization of the tissue microenvironment for various applications including neuroinflammation or tumor microenvironment (TME)

REFERENCES
[1] Lemaire, R. et al. (2007) Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag. Journal of proteome research 6, 2057-206727

Enhancing Steroid Analysis: Automated Sample Drying for LC-MS Using the Tecan Steroid Marcel Debong (Presenter) Tecan-IBL

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Poster #39d View Map

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

INTRODUCTION
The Tecan Steroid Panel LC-MS Kit is widely used for the quantitative determination of a broad spectrum of steroids in human serum and plasma. The standard protocol, as described in the Instructions for Use (IFU), includes a validated solid phase extraction (SPE) and a drying step using an external evaporator at elevated temperature. However, the need for additional equipment and the desire for streamlined, automated workflows have prompted the development of alternative methods. Having a completely automated workflow is needed by some laboratories, whereas other investigators prefer a semi-automated solution, offering more flexibility and options. This study aimed to optimize the sample preparation process by introducing a new drying method directly on the Tecan Resolvex A200 system using a nitrogen stream using positive pressure, and to evaluate its comparability to the established protocol. Nevertheless, this would be the first step to set up a complete automated workflow.

METHODS
The new drying method involved evaporating eluates at ambient temperature (18–21°C) under a nitrogen stream at 6 bar, eliminating the need for a separate evaporator. Feasibility was assessed through six independent runs, each including calibration standards, controls, and spiked samples. In order to compare the streamlined drying method with the external evaporation, spiked serum and plasma samples were analyzed. Measurements were conducted several times, over a timespan of days and with different lots to eliminate possible variabilities. For method comparison, a parallel study was conducted: one set of samples was processed according to the IFU (drying at 40°C on an external evaporator), while the other set was dried on the Resolvex A200 using the streamlined method under ambient temperatures using an evaporation plate. Quantification was performed by LC-MS/MS according to IFU, and results were evaluated using Passing-Bablok regression and correlation analysis to assess agreement between methods according to CLSI guideline EP09-A3.

RESULTS
The new drying method achieved complete evaporation of eluates within 58–90 minutes, depending on matrix composition, at ambient temperature. The deviations in drying times are caused by room temperature variances and the sample matrix. Therefore, for setting the drying time in an automation protocol, worst-case conditions were tested and led to a drying time of 90 min in the streamlined protocol. Overall, feasibility studies demonstrated high reproducibility across six independent runs, whereby all controls met their specific ranges mentioned in the QC certificate.

In the method comparison study, results from the new and standard protocols showed excellent agreement for all 18 steroid analytes. Passing-Bablok regression coefficients (R) ranged from 0.947 to 1.00, with most analytes exceeding 0.99, indicating strong linear correlation. All regression intercepts fell within the 95% bootstrap confidence intervals. For key analytes such as androstenedione, aldosterone, and progesterone, the regression lines were nearly identical to the line of identity, confirming method equivalence. Slight discrepancies observed for DHEA and DHEAS were traced to chromatographic column aging rather than the extraction or drying method. Overall, the alternative protocol provided results fully comparable to the IFU, supporting its use as a valid alternative in research workflows.

CONCLUSION
This study demonstrates that this streamlined protocol on the Tecan Resolvex A200 is fully comparable to the standard IFU protocol for the Tecan Steroid Panel LC-MS Kit. The alternative approach eliminates the need for additional drying equipment, streamlines the workflow, and supports further automation in sample preparation. The agreement between methods across a wide range of steroid analytes confirms that users can confidently adopt these alternative procedures without compromising data quality. Any deviation from the Instruction for Use will need to be validated by the customer. These findings expand the flexibility of sample preparation and pave the way for further integration of automated solutions in steroid quantification by LC-MS.

Investigating Proteome Variations in Tears and Plasma of Patients with Liver Diseases by Untargeted and Targeted LC-MS/MS Carina Lima (Presenter) University of Quebec in Montreal

Poster #40d View Map

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

INTRODUCTION:
Acute or chronic liver damage caused by excessive alcohol consumption or a metabolic disorder (obesity, type 2 diabetes) can lead to the development of serious conditions such as non-alcoholic fatty liver disease (NAFLD), liver fibrosis, cirrhosis and liver cancer (Luo, 2025). Liver disease diagnosis relies on biopsy, an invasive procedure that carries a major risk of complications. Furthermore, currently available non-invasive biomarkers have limited accuracy in early-stage development of the disease, hampering early detection and intervention (Niu, 2022). It is therefore essential to develop highly sensitive and minimally invasive diagnostic approaches. The aim of this study was to investigate by an untargeted and targeted LC-MS/MS approach, proteome variations in tears and plasma samples from patients with liver disease.

METHODS:
Tear samples were collected on Schirmer strips from healthy volunteers and patients suffering from a variety of liver diseases. Proteins were alkylated and digested with trypsin, and peptides were extracted on solid phase extraction prior to LC-MS/MS analysis. For plasma, sample preparation included the combination of three different bottom-up proteomics methods: MagNet, PCA and PPT-IgG. First, membrane-bound proteins were enriched on SAX beads based on the MagNet approach (Wu, 2024). Unbound proteins were recovered for PCA and PPT-IgG protocols. The PCA protocol consists of the depletion of high abundant proteins by their selective precipitation with perchloric acid. For the PPT-IgG protocol, plasma proteins are first depleted of Igs, solubilized with SDS, and precipitated with acetone. For untargeted proteomics, a EVOSEP One LC coupled to a Sciex ZenoTOF 7600+ (QqTOF) system was employed in low-microflow regime, with a 44-minute separation (SPD30 method) and in data-independent acquisition mode (DIA-SWATH). Targeted analysis of S100 and annexins proteins by LC-MRM was carried out using a Sciex QTRAP 5500 (QqQ).

RESULTS:
For the untargeted proteomic results of tears samples, a total of 2908 proteins were confidently identified and quantified (1% FDR). Among those proteins, 147 were shown to change significantly in the disease group compared to the healthy controls (fold change ≥ |-1.5|, p-value < 0.01). Additionally, several ribosomal proteins (21) were found to be increased in the disease group. For targeted analysis of S100 and ANX proteins, preliminary data seems to show that several of these proteins are increased in the tears of patients with liver disease.

CONCLUSION:
The results obtained in this study are very promising, insofar as tears offer a very interesting alternative for studying these different liver diseases. Comparison of tear results with those from plasma samples should reveal specific variations in both fluids, providing complementarity for biomarker detection.

REFERENCES:
Luo et al., 2025, Genes & Diseases, 12, 101512
Niu et al., 2022, Nature medicine, 28(6), 1277-1287
Wu et al., 2024, bioRxiv, 2023-06.

Taming the Beast: Use of a pH LC Gradient and Other Tricks in a Quantitative Clinical LC-MS/MS Method for Phosphatidylethanol, a Marker of Ethanol Use Alex Lyashchenko (Presenter) Columbia University

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Poster #41d View Map

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

INTRODUCTION:
Quantification of phosphatidylethanol (PEth) homologs in whole blood for assessment of ethanol use is analytically challenging, as evidenced by the wide variability of the published LC-MS/MS methods, all of which are rather complicated. Clinical PEth tests tend to rely on a combination of extensive sample preparation and/or complicated chromatography with prolonged column washing or regeneration, which extends analysis times and lowers throughput. Some notable analytical challenges include: 1) the need for comparable extraction efficiencies of presumably soluble exogenous versus membrane-bound endogenous PEths in calibrators versus patient samples; 2) chromatographic separation of the two major clinically informative PEth homologues (PLPEth and POPEth) from each other due to potential cross-talk; 3) additional sample clean up and chromatography in order to limit cross-talk from other, far more abundant, phospholipids that produce the same fragments as the PEth analytes; 4) achieving adequate chromatographic separation and ionization efficiency for required sensitivity while maintaining short run times for high throughput. Here we demonstrate novel ways to overcome these challenges with simple sample preparation and short run times suitable for clinical analysis.

METHODS:
Extensive method development evaluating a wide breadth of mass spectrometric, liquid chromatographic, and sample preparation parameters was necessary to overcome the abovementioned challenges and develop a satisfactory clinical phosphatidylethanol method on our laboratory’s LC-MS/MS instruments.

RESULTS:
Mass spectrometric evaluation demonstrated the feasibility of using positive ionization mode (Na+ adduct) as an alternative to the previously described negative ionization mode. Retention on a C18 column and ionization of PEths were found to be exquisitely sensitive to pH and to proportion of organic solvent, respectively. Consequently, a novel LC approach using a low-to-high pH gradient in high percentage of acetonitrile allowed for initial analyte retention on a C18 column despite high proportion of organic solvent in the sample, baseline separation of the two PEth homologs, and elution of the analytes in 95% acetonitrile - resulting in excellent ionization efficiency (required for sufficient sensitivity). The pH component of this LC gradient allowed tuning the separation of PEth (pH sensitive retention) from other phospholipids (pH insensitive). Using a quaternary pump with four different mobile phases also allowed to incorporate an isopropanol column wash. An 85% isopropanol wash was found to be much superior to higher proportions of isopropanol for elution of remaining phospholipids to mitigate crosstalk. The above enabled a 4-minute analytical run time with a simple protein precipitation for sample preparation.

DISCUSSION:
None of the published PEth methods provided adequate performance on our laboratory’s LC-MS/MS instruments, necessitating extensive method development. We discovered that positive ionization using Na+ adduct resulted in similar analyte peak areas as the well-described negative ionization mode, making it a viable alternative. However, it resulted in higher background noise on our systems but may nevertheless be worthwhile exploring on other instruments. While using a low-to-high pH gradient in acetonitrile overcame chromatographic and ionization challenges, it requires tuning of the gradient to standardize retention times between columns/instruments and additional attention must be paid to preparation and storage of basic mobile phases. Using an 85% isopropanol wash versus higher percentage organic significantly reduced the time required for column washing. Ultimately, while our method requires a quaternary pump to enable pH gradient with acetonitrile elution and isopropanol wash, it allows for a simple protein precipitation sample preparation and quick 4-minute analysis time.

Enhancing Toxicology Clinical Consultation through Retrospective Analysis of Preliminary Positive Immunoassay-Based Urine Drug Screening Results Sheng-Ying Lo (Presenter) Geisinger Medical Center

Poster #42d View Map

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

INTRODUCTION:
Urine drug testing is an important tool in clinical toxicology for detecting and monitoring substance exposure. Advancements in mass spectrometry (MS) have significantly improved the sensitivity and specificity of urine drug testing by reducing false positives and false negatives. Despite these advancements, immunoassay-based urine drug testing remains widely used in clinical laboratories due to its lower cost, faster turnaround time, and adaptability for automation. Understanding the performance of immunoassay-based drug screens in a patient-specific population is valuable for aiding clinicians to make rapid, better-informed decisions.

OBJECTIVE:
This study aims to evaluate the accuracy of preliminary positive screening results to enhance toxicology consultation. This is particularly important in clinical situations where timely patient management and treatment decisions are needed before MS confirmation results are available.

METHODS:
Immunoassay and MS confirmatory results generated over four years within a rural health system from a urine drug screening panel with reflex to MS confirmation of positive screens only (N = 55,054) were queried for analysis. Positive screening results with subsequent drug/drug metabolite(s) present above the quantitation cutoffs of MS-based assays were defined as true preliminary positives. Positive predictive value (PPV) was determined for six specific screening assays that remained consistent throughout the retrospective analysis period. Data were further divided into age groups: 0 to 11 years (Group 1), 12 to 19 years (Group 2), and 20 years and older (Group 3), to determine the PPVs for pediatric and adult populations when the number of results in the group exceeded 20.

RESULTS:
The overall PPV for the amphetamines screen is 77.3% (N = 5358), the cannabinoids screen is 98.3% (N = 11946), the cocaine metabolite screen is 96% (N = 1075), the methadone metabolite screen is 96% (N = 1112), the opiates screen is 98.4% (N = 4564), and the oxycodone screen is 98.2% (N = 5845). Stratifying the amphetamines screen by age group showed a PPV of 95.6% (N = 45) for Group 1, 81.4% (N = 156) for Group 2, and 77% (N = 5157) for Group 3. The PPV of the cannabinoids screen also differed between pediatric and adult populations. Stratifying the cannabinoids screen by age group showed a PPV of 50% (N = 60) for Group 1, 98.1% (N = 930) for Group 2, and 98.6% (N = 10956) for Group 3. The remaining immunoassay-based screens showed minimal variation in PPV among the different age groups.

CONCLUSION:
Based on this retrospective analysis, five out of six immunoassay-based urine drug screens showed a high likelihood of preliminary positive results being confirmed by MS-based testing, except for the amphetamines screen. Notably, while the amphetamines screen performed well in the younger pediatric patients, its reliability declined with increasing age. In contrast, the cannabinoids screen was less reliable in the younger pediatric populations, indicating that alternative methods — such as umbilical cord tissue or meconium testing — may be more suitable for detecting in utero cannabinoid exposure. Overall, this study highlights the value of evaluating immunoassay screen performance within specific patient populations to inform clinical decision-making when MS confirmation is in progress.

NexProQ: Multiplexed Quantitation of 500+ Proteins in Whole Blood and Dried Blood Spots Using PRM-PASEF and TMTpro Laleh Ebrahimi Ghahnavieh (Presenter) McGill University / Jewish General Hospital

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Poster #43d View Map

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

INTRODUCTION:
Quantitative proteomics in clinical samples have remained constrained by limited multiplexing capacity, high reagent costs, and low sample throughput. To address these limitations, we developed NexProQ, a novel platform combining TMTpro multiplexing with PRM-PASEF acquisition on the timsTOF HT. This approach enables us simultaneous absolute quantitation of over 500 protein targets in whole blood (WB) and dried blood spot (DBS) samples in a single run.

METHODS:
Proteotypic peptides were selected from 500+ clinically relevant targets and synthesized as unlabeled NAT standards. Internal calibration curves were prepared using TMT-pro 18-plex labels and peptide dilutions (spanning 1000× to 1× of the lower limit of quantitation (LLOQ)) mixture to reach five-point internal calibration curves. Then multiplexed with plasma and DBS digests preloaded onto EvoTips. Peptides analysis has been done at 30SPD using PRM-PASEF, and assay performance was assessed in terms of linearity, limits detection, intra-assay reproducibility, and matrix effects.

RESULTS:
Assay linearity was robust across most targets (R² > 0.98), with median LLOQs of 0.1–0.5 fmol on-column. Utilizing non-adjacent TMTpro reporter ions boosts quantitation reproducibility and decreases interference due to isotopic overlap. Most targets' intra-assay CVs are below 5%, hence quantitation is reproducible across different protein concentrations and sample types.
Comparison across platforms illustrates the higher mass accuracy and consistency of reporter ion intensities on the QE+, while PRM-PASEF enables superior throughput. The strong concordance for high-abundance proteins has done by validating 274-peptide panel against reference MRM data.

CONCLUSION:
NexProQ offers significant escalating for scalability and accessibility of clinical proteomics. Its capacity to quantify hundreds of proteins across multiple samples within minutes supports its advantages in biomarker discovery, clinical validation, and large-cohort screening studies. This study paves the way for population-scale proteomic screening with performance exceeding current antibody- or aptamer-based methods. Commercial kit development is in progress to support standardization across labs.

Proteomic Analysis of Human Cells Exposed to Mono-n-butyl Phthalate (MnBP) using DIA-MS Hwangkyo Jeong (Presenter) Prometabio Research Institute

Poster #44d View Map

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

INTRODUCTION:
Exposome is a concept that explains how exposure to internal and external substances, such as chemicals, in people's daily lives affects their health. Mono-n-butyl phthalate (MnBP), one of the environment pollutants, is easily exposed in daily life because it is widely used in plastic containers, etc. Although MnBP is known to be associated with respiratory diseases, studies on biological pathways and mechanisms are lacking. Therefore, we performed proteomics analysis using DIA-MS technology to identify differentially expressed proteins (DEPs) in monocytic cell line (THP-1) treated with MnBP and to elucidate the complex interactions related to MnBP exposure.

METHODS:
The control group was not treated with MnBP, while THP-1 cells were exposed to different concentrations of MnBP. All samples were incubated for 72 hours. Following incubation, the collected cell pellets and supernatants were lysed using 5% SDS. The extracted proteins were digested with trypsin for subsequent proteomic analysis. For proteomic analysis, DIA-MS was performed using the Exploris 480. Cell (500 ng) and supernatant (250 ng) samples were injected and analyzed in triplicate using a 35-minute gradient at a flow rate of 0.4 L/min. The DIA files from each sample were processed using DIA-NN (version 1.8.1) in library-free mode, with Human SwissProt (Version 2022.04) as the reference database. Data interpretation was conducted using PLS-DA and statistical analysis.

RESULTS:
MnBP, a component of the environment pollutants, may contribute to various diseases, including respiratory and skin disorders. However, the impact of environmental factors on biological changes remains poorly understood. In this study, we investigated the effects of MnBP on THP-1 cells using a proteomics-based approach. THP-1 cells were treated with MnBP at 250, 500, and 1,000 μg/mL. Control groups were maintained without MnBP treatment. After 72 hours of incubation, cell pellets and supernatants were collected. DIA-MS was performed for both qualitative and quantitative proteomic analysis across all samples.

A total of 6,281 proteins were identified from the cell pellet samples. Comparative analysis between MnBP-treated and untreated HaCaT cells revealed 164 DEPs. For THP-1 cells, comparative analysis across different MnBP concentrations identified 51 DEPs in the 250 μg/mL-treated group, 170 DEPs in the 500 μg/mL-treated group, and 537 DEPs in the 1,000 μg/mL-treated group, compared to the untreated control. Further analysis of DEPs identified in HaCaT and THP-1 cells led to the identification of 12 significant proteins. GO analysis indicated that the enriched biological processes included protein polymerization, intermediate filament organization, and protein repair. A total of 2,433 proteins were identified from the supernatant samples. Comparative analysis between MnBP-treated and untreated HaCaT supernatants revealed 267 DEPs. For THP-1 supernatants, comparative analysis across different MnBP concentrations identified 141 DEPs in the 250 μg/mL-treated group, 224 DEPs in the 500 μg/mL-treated group, and 312 DEPs in the 1,000 μg/mL-treated group, compared to the untreated control. GO analysis using 87 overlapping proteins among the DEPs identified by comparing THP-1 supernatan showed that Staphylococcus aureus infection was enriched in the KEGG pathway. These findings provide insights into MnBP-induced proteomic alterations and their potential biological implications.

CONCLUSION:
Our findings demonstrate that MnBP induces significant, dose-dependent changes in the THP-1 proteome, affecting both intracellular and secreted protein profiles. These alterations suggest disruption in structural protein integrity and cellular repair systems.

DISCUSSION:
Using the differentially expressed proteins (DEPs) commonly identified in both the monocytic cell line (THP-1) and keratinocyte cell line (HaCaT), GO analysis suggests that MnBP induces a conserved stress-related cellular response across different cell types. Furthermore, analysis of the THP-1 supernatant indicates that MnBP may influence immune regulatory mechanisms at the secretory level. These data collectively provide mechanistic insights into cell-type-specific immune responses to MnBP exposure and may serve as a foundation for future investigations into its immunotoxic effects.

Acknowledgement:
This work was supported by Korea Environment Industry & Technology Institute(KEITI) through Core Technology Development Project for Environmental Diseases Prevention and Management, funded by Korea Ministry of Environment(MOE)(RS-2022-KE002048) and the Research Program funded Korea National Institute of Health (2008-E33030-00, 2009-E33033-00, 2011-E33021-00, 2012-E33012-00, 2013-E51003-00, 2014-E51004-00, 2014-E51004-01, 2014-E51004-02, 2017-E67002-00, 2017-E67002-01, 2017-E67002-02, 2020E670200, 2020E670201).

Development of a Liquid Chromatography Mass Spectrometry Quantification Assay for human Relaxin H2 Annabelle Fülöp (Presenter) Immundiagnostik AG

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Poster #45d View Map

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

INTRODUCTION:
Human relaxin peptides are a group of seven closely related peptide hormones that have been shown to be part of various physiological processes such as pregnancy, fertility and cardiovascular adaptations. Thus, peptides of this family are believed to be a promising diagnostic marker for several correlated clinical fields such as heart disease, fibrosis and infertility. Low expression levels in the pmol/ml range and the lack of robust proteotypic peptides make these hormones difficult peptides for identification and quantification using conventional proteomic approaches and may explain inconsistent information on their expression. To date there are only a few commercial immunoassays targeting human relaxin peptides for routine application, with varying quality. A routine LC-MS/MS method for the quantification of relaxin peptides from biological matrices does not yet exist. So far, only two approaches have been described to quantify relaxin from biological matrices, and in both cases the choice of internal standard was not ideal.1,2 Furthermore, there is only very limited mass spectrometric data that verifies immunogenic methods and results, as cross reactivity between the different members of the peptide family can occur. We addressed the aforementioned problems as well as the lack of a fast routine LC-MS/MS based method for human relaxin peptides, and decided to develop a precise method for quantification of relaxin peptides by LC-MS/MS.

METHODS:
A solid phase extraction method optimized for RLX-2 was compared to an immunoaffinity enrichment from serum or plasma by different anti-relaxin antibodies. As LC-MS/MS detection of the A-chain of RLX-2 was more sensitive than the B-chain or the intact RLX-2, the A-chain of RLX-2 was set as target and measured in positive ion mode by ESI-MS/MS or nanoLC-ESI-MS/MS. A 13C6-15N-leucine labeled endogenous RLX-2 peptide was used as an internal standard with both the A-chain and B-chains labeled, allowing the flexibility to quantify the individual RLX2 chains or fragments thereof separately. Aside from RLX-2, transitions for relaxin-1, relaxin-3 and insulin were investigated as well.

RESULTS:
The limit of quantification for RLX-2 was around 100 pg/ml (approx. 16 fM) using an ExionLC coupled with a QTRAP 5500 (AB Sciex) or 8 pg/ml (approx. 1.3 fM) using EASY-nLC 1000 coupled with an Orbitrap Velos Pro (Thermo Fisher). Thereby the sample preparation for the quantification of RLX-2 by LC-MS/MS was based on immunoaffinity enrichment. Efficient, non-denaturing dissociation of the A-chain and B-chain during the reduction step at a lower pH improved the further quantification of RLX-2. In addition, the detection of the A-chain could be enhanced by adding DMSO to the mobile phases.

CONCLUSION:
The UPLC-MS/MS and nanoLC-MS/MS allows the specific quantification of RLX-2 in the endogenous range. Proof-of-principle experiments with the first human samples from pregnant women showed a clear identification and quantification of relaxin-2. Furthermore, it was shown that CKD patients have higher RLX-2 values compared to healthy volunteers.3

REFERENCES:
1. Rais, Y. and Drabovich, A. P., Identification and quantification of human relaxin proteins by immunoaffinity-mass spectrometry. J. Proteom Res. 2024. 23(6): p. 2013-2027.
2. Fülöp, A., Armbruster, F.P., Grön H.J., Dschietzig T.B. and Diesner M., Development of a LC-MS/MS Quantification Assay for Human Relaxin H2 and Extraction of Human Relaxin H1 and Relaxin H3. Clin Lab. 2024. 1;70(2).
3. Dahlke, M., Ng, D., Yamaguchi, M., Machineni, S., Berger, S., Canadi, J., Rajman, I., Lloyd, P. and Pang, Y. Safety and tolerability of serelaxin, a recombinant human relaxin-2 in development for the treatment of acute heart failure, in healthy Japanese volunteers and a comparison of pharmacokinetics and pharmacodynamics in healthy Japanese and Caucasian populations. J. Clin. Pharmacol. 2015. 55(4): p. 415-422.

Development of a Liquid Chromatography Mass Spectrometry Quantification Assay for 5-FU, Tegafur, Capecitabin and Their Metabolites Jacqueline Fromke-Donath (Presenter) Immundiagnostik AG

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Poster #46d View Map

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

INTRODUCTION:
5-Fluorouracil (5-FU) is an antimetabolite and one of the most frequently used cytostatics in oncology for systemic tumor therapy (chemotherapy). It can be either administered directly by transfusion or as prodrug (capecitabine, tegafur). The prodrugs are metabolized into 5-fluorouracil after absorption in the liver. 5-FU is metabolized into Dihydro-5-Fluorouracil (5-DHFU) by the dihydropyrimidin-dehydrogenase (DPD), which under normal physiological conditions metabolizes uracil (U) into 5,6-dihydro-uracil (DHU), in the liver as well as mononuclear cells of the immune system. To exert a substantial anti-tumor effect, it is necessary to maintain high levels of 5-FU in the blood and tissues. 5-FU is associated with potential severe adverse events leading in some cases to substantial morbidity and mortality. DPD deficiency is thought to be the main mechanism leading to 5-FU accumulation and toxicity. Identifying patients with DPD deficiency and notably patients with complete deficiency, is then a relevant approach to limit the risk of severe adverse event (Tron, 2021). A side from PCR diagnostics of DPD mutations, LC-MS/MS has been shown great potential for initial phenotyping of DPD deficient patients by measuring U and DHU as surrogate markers for DPD activity as well as measuring the prodrugs, 5-FU and 5-DHFU as therapeutic drug monitoring (TDM) approach. We present here a prototype of a commercial diagnostic kit for the phenotyping and TDM of the aforementioned drugs and metabolites.

METHODS:
For the measurement of capecitabine, tegafur, U, DHU, 5-FU, 5-DHFU a prototype of the commercial LC-MS/MS kit (Immundiagnostik AG, Germany) was used. Analytes were extracted from 50 µl sample (serum, EDTA, citrate and lithium-heparin) or CAL/CTRL (6 CALs/ 3 CTRLs) by protein precipitation after application of 13C/15N/D labelled internal standards. Subsequently, supernatants were collected, evaporated, reconstituted and 5 µl injected into an Exion/ QTRAP 5500 system (Sciex). Total cycle time between injections was 10 mins.

RESULTS:
The chromatographic run allowed baseline separation of all analytes in all matrices in a run of 10mins. EDTA plasma gave the highest signal responses in comparison to all other matrices. The measuring interval covered the complete physiological and treatment related range (5-2500ng/mL 5-FU r²=0.998; 5-5000ng/ml 5-DHFU r²=0.996; 2-500ng/mL U r²=0.996; 50-5000ng/ml DHU r²=0.998; 2-1250ng/ml capecitabin r²=0.995; 10-5000ng/mL Tegafur r²=0.997). Precision and repeatability was <15/20% as stated by the FDA guidelines.

CONCLUSION:
The prototype of the commercial kit allowed robust quantification from all matrices for the physiological and treatment related ranges of all analytes. While the developed chromatographic run allowed baseline separation of all analytes, the final runtime has further room for improvement. Moreover, stability of real clinical samples, due to prolonged enzymatic activity in the given matrices needs to be firmly investigated, in order to achieve robust area under the curve determination for the given drugs. After the final adaptation of the chromatographic separation full validation under the IVDR and a clinical study for the TDM purpose as well as phenotyping of patients is planned.

REFERENCES:
Tron C, Lemaitre F, Boisteau E, Sourd SL, Lièvre A. When helping the minority of patients may hurt the majority: The case for DPD phenotyping and 5-fluorouracil therapeutic drug monitoring. Dig Liver Dis. 2021 Feb;53(2):258-260. doi: 10.1016/j.dld.2020.11.004. Epub 2020 Nov 20. PMID: 33229275.

Extraction of Tobacco-Specific Nitrosamines (TSNAs) from Urine Using ISOLUTE® SLE+ Prior to UPLC-MS/MS Analysis Mario Merida (Presenter) Biotage

Poster #47d View Map

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

INTRODUCTION:
TSNAs, or tobacco-specific nitrosamines, are carcinogens found in tobacco products, including e-cigarettes and smokeless tobacco. NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), and NNN (n-nitrosonornicotine) are the most analyzed TSNAs. In laboratory animal studies, NNK has been identified as a cause of lung cancer, a finding also associated with its metabolite, NNAL. Furthermore, NNN has been observed to induce esophageal cancer in these same animal models.

TSNAs can be difficult to accurately detect, as, like nicotine, there can be false positives for secondhand and third-hand contamination. Contamination due to secondhand and thirdhand smoke causes non-smokers to test positive for TSNAs if the detection limits for the method are low. Additionally, due to the ubiquitous nature of nitrosamines, contamination could originate from test tubes, extraction media, pipette tips, or any number of items used throughout the sample extraction and analysis processes.

OBJECTIVES:
This study demonstrates an optimized and streamlined extraction workflow for TSNAs in urine with high analyte recoveries, low matrix effect, and excellent reproducibility.

METHODS:
TSNAs (NNAL, NNK, and NNN) were spiked at 200pg/mL and extracted from non-hydrolyzed, non-smoker human donor urine. Sample extractions were investigated using ISOLUTE® SLE+ (Supported Liquid Extraction) plates comparing extraction performance with different elution solvents. Extraction performance was evaluated by assessing recovery, matrix effect, and reproducibility. Calibration curves were constructed using concentrations ranging from 0.1 to 200 pg/mL of synthetic urine (UriSub, pH 7.4) to avoid signal interferences. Limit of quantifications (LoQs) were determined from the calibration curves for each analyte by applying linear regression model. LC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC system coupled to a SCIEX 5500 QTrap MS system.

RESULTS:
Extraction recovery for TSNAs with ethyl acetate as elution solvent were 91.8%, 92.1%, and 80.7% for NNAL, NNK, and NNN respectively, which shows robustness of the method. Matrix effects for TSNAs were 0.82, 0.92, and 0.87 for NNAL, NNK, and NNN respectively indicating relatively lower matrix suppression for the analytes representing clean sample extract. Other elution solvents such as hexane, dichloromethane,10% isopropanol in dichloromethane showed consistent matrix effect (>0.80) but gave a wide range of extraction recovery (30-104%). Calibration curves for TSNAs from ethyl acetate elution showed good linearity with R2 values > 0.99 for each analyte. LoQs obtained from linear regression were 16.0, 9.0, and 3.0 pg/mL for NNAL, NNK, and NNN respectively.

CONCLUSIONS:
The load-wait-elute extraction procedure utilizing ISOLUTE® SLE+ offers a simple, fast, and automation compatible solution to extract TSNAs for LC-MS/MS analysis with high recovery and less matrix interference. The ability to quantify TSNAs at low pg/mL levels would allow the accurate monitoring of second-hand tobacco exposure and greatly facilitate toxicological studies.

REFERENCES:
1. Smith, C. J.; Hansch, C. The Relative Toxicology of Compounds in Mainstream Cigarette Smoke Condensate. Food Chem. Toxicol. 2000, 38, 637–646.
2. Xia, B., Xia, Y., Wong, J., Nicodemus, K.J., Xu, M., Lee, J., Guillot, T. and Li, J. (2014), Quantitative analysis of five tobacco-specific N-nitrosamines in urine by liquid chromatography–atmospheric pressure ionization tandem mass spectrometry†. Biomed. Chromatogr., 28: 375-384.

Optimisation of a LC-MS/MS Method for Quantitation of Human Serum Albumin (HSA) and Vitamin D Binding Protein (VDBP) with Identification of Three VDBP Isoforms Shauna O'Brien (Presenter) MRC-Laboratory of Medical Sciences

Poster #48d View Map

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

INTRODUCTION
Three common polymorphisms of VDBP are encoded by one of three codominant alleles of the VDBP gene. These isoforms are termed GC1f, GC1S and GC2, with each having a differing affinity of vitamin D metabolite binding, for example 25(OH)D3 and 1,25(OH)2D3, Gc1f > Gc1s > Gc2. In practice, individuals with the Gc1S or Gc2 isoforms tend to have higher circulating vitamin D levels compared to individuals with the Gc1f isoform. Vitamin D metabolites also bind to Human Serum Albumin (HSA), though with a lower affinity than VDBP. Routine clinical measurements of circulating vitamin D metabolites only measure 25(OH)D, regardless of its bioavailability which is dependent on VDBP and HSA levels. This is particularly evident in population and human health studies, such as pregnancy, ethnicity and early life stages. The concentration of both VDBP and HSA, alongside the distinction between different VDBP isoforms, could have implications on the clinical interpretation of measured VDBP levels, alongside 25OHD3 concentrations in human serum that are currently under explored.

OBJECTIVES
This work aimed to develop a “bottom-up” proteomics LC-MS/MS method for measurement of circulating VDBP and HSA levels in human serum, as well as VDBP isoform identification.

METHODS
The LC-MS/MS method has been developed and optimised using synthesised peptide standards of VDBP and HSA tryptic digest peptides; VLEPTLK and ELPEHTVK for the quantification of VDBP, LPEATPTELAK, LPDATPK and LPDATPTELAK for VDBP isoform identification, and THLPEVFLSK for quality control, while LVNEVTEFAK is used to quantify HSA. Each metabolite was measured by multiple reaction monitoring following optimization of MS parameters. LC-MS/MS method development was performed on a Waters ACQUITY UPLC coupled to a Waters Xevo TQ-XS, using a HSS T3 column with a method run time of 5 min. The denaturation, alkylation, reduction and digestion method has been optimised using 2,2,2-trifluoroethanol, dithiothreitol, iodoacetamide and TPCK-treated trypsin, respectfully.

RESULTS
The method development process focused on maximising MS sensitivity and digestion efficiency to ensure complete protein digestion, peptide recovery and accurate quantitation. Digestion factors including reagent concentrations and digestion time have been optimised using pooled human serum spiked with VDBP and HSA. The optimal trypsin concentration for peptide recovery for both 10 µL and 20 µL serum sample volumes was 4 mg/mL. Increasing digestion time beyond 15 minutes had a negative impact on peptide recovery, presumably due to over-digestion. The optimised assay achieves a linear range of 10 – 800 µg/mL VDBP and 1 – 80 mg/mL HSA using 20 µL serum.

CONCLUSIONS
A UPLC-MS/MS assay has been developed to quantify HSA and VDBP, and identify VDBP isoforms in serum samples, achieving optimal digestion and sample preparation parameters. The developed assay will be fully validated and employed alongside multi metabolite vitamin D metabolite assays across human health cohort studies in future work.

Analysis of Direct Free and Total Testosterone Using a Single LC-MS/MS Method and Supported Liquid Extraction Procedure Leslie Farris (Presenter) Cleveland Clinic Foundation

Poster #49d View Map

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

OBJECTIVE(S):
To develop and validate a single LC-MS/MS method for the analysis of direct free and total testosterone suitable for clinical use in females, children and males.

METHODS:
Free testosterone was isolated from the bound fractions through equilibrium dialysis (ED) using the Thermo Scientific RED Device and HEPES buffer. Serum and HEPES buffer were added to their respective chambers (1:1.8 ratio) and incubated at 37 °C with gentle mixing for 8 hours. Dialysates containing free testosterone and serum samples containing total testosterone were extracted along with a C-13 labeled internal standard in an identical supported liquid extraction (SLE) procedure using modified sample and dilution volumes. Dialysates (300 µL) or serum (200 µL) samples were added to a 96-well plate with 20 µL of internal standard, briefly mixed, then allowed to equilibrate/acclimate for 10 minutes. Internal standard spiked dialysates and serum were diluted with water to a final volume of 400 µL and mixed. The diluted samples were immediately loaded onto the SLE media using positive pressure and allowed to absorb for at least 5 minutes, and extracted with three elutions of (9:1, hexane:ethyl acetate) into a glass-coated collection plate, evaporated to dryness, reconstituted in 60% methanol (200 µL for total and 125 µL for free testosterone), and mixed. Samples were analyzed by injecting 50 µL of extract on a Transcend II LX-2 LC with a C18 100×3 mm, 2.6 µm analytical column (40 °C) coupled to a TSQ Altis™ triple quadrupole mass spectrometer (Thermo Scientific). The mobile phase A and B consisted of 0.2 mM ammonium fluoride in LCMS grade water and LCMS grade (7:3, methanol:acetonitrile), respectively, with a flow rate of 0.5 mL/min over a runtime of 7 minutes (5 minutes multiplexed, two channels). The performance characteristics of the methods for free and total testosterone were evaluated according to CLSI C-62, including: matrix effect, accuracy, precision, linearity, sensitivity, specificity/selectivity, carryover, and stability.

RESULTS:
The alternative matrix (Golden West Diagnostics, LLC, Mass Spect Gold Human Serum, Ultra-Low Testosterone, Lipid Free, MSG3100) was found suitable for use as calibrator matrix (bias range -0.7 to 4.4%, overall mean bias 2.6% when mixed with patient specimens). No ion suppression or enhancement near the retention time of testosterone was observed for either assay. No interferences were observed for hemolyzed, lipemic, or icteric specimens. The assay specificity was evaluated using commercial interference mixes (40+ potential interferents) and individual isobaric compounds (androstenedione, DHEA, epitestosterone, and estriol). We identified two potential interferences from the interference mixes: bupivacaine, which is isobaric with testosterone, and sertraline, an antidepressant that belongs to the class of selective serotonin reuptake inhibitors (SSRIs). However, the concentration of sertraline in the interference mix is approximately 50-300 times the efficacious dose. The methods were linear in the analytical measurement ranges of 0.10-200 ng/dL for free testosterone, and 1.0-2000 ng/dL for total testosterone. To evaluate the analytical sensitivity of the method, the limit of detection (LOD), limit of blank (LOB), and lower limit of quantitation (LLOQ) were determined for each analyte. For free testosterone, the LOD was 0.02 ng/dL, the LOB was 0.00 ng/dL, and the LLOQ was 0.12 ng/dL (12.4% CV). For total testosterone, the LOD was 0.2 ng/dL, the LOB was 0.0 ng/dL, and the LLOQ was 1.0 ng/dL (5.7% CV). The sample stability for both free and total testosterone was determined to be 3, 14, and 60 days at ambient, refrigerated, and frozen conditions respectively. Acceptable carryover were found for free testosterone at 0.026 and 0.010 ng/dL and total testosterone at 0.08 and 0.10 ng/dL, with each assay evaluated over two channels at concentrations twice the upper limit of the analytical measurement range. For free testosterone, the intra-day precision CV ranged from 3.2% to 8.8% and the inter-day precision CV ranged from 2.8% to 19.0%. For total testosterone, the intra-day precision CV ranged from 1.9% to 2.4% and the inter-day precision CV ranged from 2.7% to 3.0%. Comparison with independent reference laboratory LC-MS/MS and ED methods showed an average bias of 10.2% for free testosterone (9.8% bias in the subrange 0.1-10.0 ng/dL) and 3% for total testosterone. Deming regression analysis indicated a slope of 1.184 and intercept of -0.220 (R=0.9362) for free testosterone, and a slope of 1.025 and intercept of 0.54 (R= 0.9959) for total testosterone.

CONCLUSION:
We described an LC-MS/MS method for the quantitation of direct free and total testosterone in serum from females, children and males. Despite the analytical challenges associated with the measurement of direct free and total testosterone across the wide analytical measurement range required for a universal clinical method, our method demonstrated robust analytical performance for clinical use in diverse patient populations. Coupled with a short ED of 8 hours for free testosterone, these methods are well-suited for routine use in a clinical laboratory. The streamlined workflow supports in-house testing, decreasing test cost and turnaround time for patients undergoing biochemical workup for altered levels of total and biologically active testosterone.

A Five-Metabolite Panel Combined with Early and Prodromal Symptoms as Biomarkers for Parkinson´s Disease Yamile Lopez (Presenter) University of Alberta

Poster #50d View Map

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

INTRODUCTION:
Parkinson’s disease (PD) urgently requires blood-based markers that flag pathology before disabling motor decline. Despite advances in symptomatic treatments that focus primarily on motor impairments, there are currently no curative or disease-modifying therapies. This underscores the pressing need to deepen our understanding of PD’s underlying biochemical and physiological mechanisms.Despite the promising findings from metabolomic research, a critical limitation persists, as most studies rely on untargeted analyses or relative quantifications rather than absolute quantitative methodologies. This limitation restricts the robustness, reproducibility, and clinical applicability of findings, hindering the identification and validation of reliable biomarkers. Consequently, there is an urgent need for studies employing absolute quantitative metabolomic analyses to provide more precise and reproducible biomarkers, facilitating clinical translation and improving patient outcomes. The objective of this study is to perform an absolute quantitative analysis of the plasma metabolome in healthy controls and patients with intermediate and advanced stages of PD, aiming to identify specific metabolic alterations associated with the disease and to explore their relationship with disease progression.

METHODS:
This study measured absolute concentrations of 144 plasma metabolites (amino acids, biogenic amines, organic acids, acylcarnitines, sphingomyelins, phosphatidylcholines and lysophosphatidylcholines) in 20 neurologically healthy adults and in 40 PD patients clinically classified as intermediate (PD-I) or progressive (PD-II). A multi-targeted assay (TMIC PRIME ASSAY) consisting of a combination of LC-MS/MS and DFI was used to measure the metabolites and metabolite ratios. A multinomial logistic-regression model was built to examine how metabolite concentration changes relate to disease stage and to explore their diagnostic usefulness.

RESULTS:
Five metabolites: glutamine, butyric acid, indoleacetic acid, phosphatidylcholine aa C40:2 and acylcarnitine C12:1 emerged as the smallest biomarker set that consistently separated controls, PD-I and PD-II. When three non-motor manifestations often present in the prodromal phase (excessive salivation, sleep disturbance and depression) were added, the combined profile clearly distinguished controls from early-stage patients and improved classification of intermediate versus progressive disease. The selected metabolites play roles in gut-derived signaling, mitochondrial β-oxidation and membrane-lipid homeostasis, while the clinical variables mirror the recognized early spread of α-synuclein pathology, together offering a coherent snapshot of systemic change across PD progression.

CONCLUSIONS:
Because the panel can be quantified from a single small plasma aliquot and a brief clinical interview, it represents a practical candidate for pre-symptomatic screening and staging in PDOverall, this integrative methodological framework balances accuracy, interpretability, and clinical relevance, illustrating a promising avenue for metabolomics-based diagnostics but also emphasizing the necessity for continued methodological refinement and validation. A validated PD diagnostic panel including these five metabolites could enable earlier diagnosis, monitor disease progression, and guide therapeutic interventions by reflecting peripheral and central metabolic disturbances.

Comprehensive Quantitative Analysis of Multiclass Steroids in Serum Using the Steroid Panel LC-MS Kit and Agilent 6495 Triple Quadrupole LC-MS: Semi-Automation-Sample Preparation with 96-Well Plate Format Ayham Al Ahmad (Presenter) tecan

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Poster #51d View Map

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

INTRODUCTION
Steroids are essential endogenous metabolites that regulate numerous physiological processes, making their accurate quantification critical for clinical research. Traditional immunoassays often lack the specificity required for structurally similar steroids, leading to cross-reactivity and limited analytical performance. Liquid chromatography-mass spectrometry (LC-MS) has become the gold standard for steroid analysis due to its superior specificity and sensitivity. However, achieving high precision and accuracy across a broad panel of analytes remains challenging. This study presents a robust workflow integrating the Tecan Steroid Panel LC-MS kit with the Agilent 6495 triple quadrupole LC-MS, enabling comprehensive and efficient quantification of 17 steroids and dexamethasone in serum.

METHODS
Chromatographic separation was performed using an Agilent 1290 Infinity III LC system with a C8 column (Tecan REF: 30215928), coupled to an Agilent 6495 triple quadrupole mass spectrometer. The system operated in both positive and negative ESI modes, with MRM transitions optimized using Agilent MassHunter Optimizer software. Data acquisition and analysis were conducted with Agilent MassHunter software.
Sample preparation utilized a semi-automated solid phase extraction (SPE) protocol on a Tecan Resolvex® A200 positive pressure manifold, using 96-well SPE plates provided in the kit. All solvents were LC-MS grade, and the kit supplied all necessary reagents, calibrators, controls, and internal standards. Chromatographic conditions were optimized for the resolution of closely related analytes. Calibration curves were constructed using six points and a zero calibrator for each analyte.

RESULTS
The workflow enabled robust separation and quantification of all target analytes, including challenging pairs such as corticosterone, 11-deoxycortisol, and 21-deoxycortisol, as well as cortisone and cortisol. Precision, expressed as coefficient of variation (CV%), was typically below 10% for most analytes, with all remaining within the 20% acceptance criterion even at low and high concentration levels. Linearity was good, with high coefficients of determination (R²) for all analytes; most were fit by linear regression, while some required quadratic models and appropriate weighting. Trueness, assessed by comparison to QC samples with metrological traceability, showed deviations within ±20% for all analytes, with most well below 15%. The semi-automated SPE protocol improved throughput and reduced manual handling errors compared to traditional methods.

CONCLUSION
The optimized Steroid Panel LC-MS workflow, combining the Tecan kit and Agilent 6495 LC-MS, provides a reliable, high-throughput solution for comprehensive steroid profiling in serum. The method demonstrates high precision, excellent linearity, and robust trueness across a wide concentration range. The semi-automated sample preparation using the Resolvex A200 enhances laboratory efficiency and consistency. While some analytes showed minor variability or bias, all results met established acceptance criteria. The kit is compatible with the Agilent platform and suitable for advanced research applications, though end users should perform validation in their own laboratories. Overall, this workflow represents a robust and efficient tool for multiclass steroid quantification in clinical research.

Newborn Screening for X-linked Adrenoleukodystrophy: Method Development and Validation of LC-MS/MS assay for Lysophosphatidylcholine Quantification Kandeepan Karthigesu (Presenter) McMaster University

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Poster #52d View Map

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

BACKGROUND:
X-linked adrenoleukodystrophy (X-ALD), due to pathogenic variants of the ABCD1 gene, is the most common peroxisomal disorder and affects 1:17000 births. Among the several forms, the childhood cerebral ALD (CCALD) is the most severe and fatal at the age of 4-8 years in males. CCALD presents with adrenal insufficiency and aggressive inflammatory demyelination. Treatment by bone marrow transplantation is effective, but must be done before onset of clinical neurological symptoms. Hence, early diagnosis is crucial in a clinical setting. Inclusion of X-ALD for newborn screening (NBS) in Canada has been limited due to a lack of validated analytical methods. This study aimed to clinically validate C20:0 to C26:0 lysophosphatidylcholines (LPCs) using liquid chromatography (LC) MS/MS and to implement it as a second-tier test for CCALD screening in Ontario.

METHODS:
Screen positive samples from the first-tier method under flow injection analysis (FIA) MS/MS were subjected to C20:0-LPC, C22:0-LPC, C24:0-LPC, and C26:0-LPC measurements using LC-MS/MS. To achieve this, various mobile phases, gradients, columns, internal standards, and MS parameters were evaluated to optimize the method. Analysis was conducted using a Waters Xevo-TQS micro MS/MS instrument coupled to a Waters ACQUITY H class plus UPLC binary solvent LC system. An ACQUITY Premier BEH C8 VanGuard FIT Column, 1.7 µm, 2.1 mm x 50 mm, with a matching 5 mm guard column was employed. Underivatized analytes and stable isotope internal standards were monitored in positive electrospray ionization (ESI+) mode by multiple reaction monitoring (MRM), using the same quantification or qualification product ions for all species (M/Z 104.1 and 184.1, respectively) with the following precursor ions: 552.4 (C20:0-LPC), 580.4 (C22:0-LPC), 608.5 (C24:0- LPC), 636.5 (C26:0-LPC), 556.4 (C20:0-d4-LPC), 586.4 (C22:0-13C6-LPC), 614.5 (C24:0-13C6-LPC), and 642.5 (C26:0-13C6-LPC). Dried blood spot (DBS) samples used as calibrators, quality controls, and blank filter paper during validation were sourced in-house and from the U.S. Centers for Disease Control and Prevention (CDC).

RESULTS:
Sample elution of DBS shaking at 600 rpm and 28°C with 100% methanol showed good recovery. The mobile phases A1 and B1 comprising water/acetonitrile (50/50, v/v) and methanol/acetonitrile (50/50, v/v), respectively, with 5 mM ammonium acetate, at a flow rate of 0.4 mL/min with gradient elution starting at 85% A1, showed the best chromatographic separation. MS parameters were optimized, including cone voltage 26 V, collision energy of 30 V, and capillary voltage of 4.25 kV. Method validation demonstrated good linearity ranging from 0.1 to 8.0 μM with an R-squared value greater than 0.99 for all LPCs. The limit of detection (LOD) for C20:0 LPC, C22:0 LPC, C24:0 LPC, and C26:0 LPC was 0.035, 0.008, 0.012, and 0.018 μM, respectively. The between-day precision of C20:0, C22:0, C24:0, and C26:0-LPCs were 6.3%, 11.9%, 9.2%, and 7.0%, respectively. Two-way ANOVA followed by multiple comparisons using Tukey’s HSD (Day 0 vs. Day 5; storage temperatures: 80°C, -20°C, 4°C, 25°C, and 45°C) revealed no significant differences in the concentration of C26:0 LPC extracted from DBS.

CONCLUSION:
We developed and validated an LC-MS/MS method for quantitation of C20:0, C22:0, C24:0 and C26:0 LPCs for CCALD second-tier NBS testing, with implementation anticipated in 2025.

Cortisol and Cortisone in Scales of Atlantic Cod by LC-MS/MS - A Possible Extrapolation to Clinical Study of Chronic Stress Biomarkers Daniela Dulgheriu (Presenter) Norwegian University of Life Sciences

Poster #53d View Map

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

INTRODUCTION:
Cortisol analysis is used in research to study the relationship between stress, health and disease. High cortisol levels in blood are one of the major endocrine primary stress responses in vertebrates and are widely used as stress indicators in clinical studies - mainly for acute stress measurement. However, it does not reflect long-time stress exposure, and blood sampling is an invasive and stressful procedure, which may influence the results. Thus, alternative matrices which may reflect long-term stress exposure and cortisol accumulation over time need to be investigated. Hair of mammals, feathers of birds and fish scales have been previously used for chronic stress assessment. These analyses can be used to identify potential stress-related disorders, monitor glucocorticoid treatment, and assess the impact of stress on various health conditions. Scales are a target for endocrine stimuli and can provide insights into chronic stress caused by environmental stressors and anthropogenic disturbances. Prolonged and persistent situations such as pollution or boat noise may cause stress in wild fish.

OBJECTIVES:
The aim of this study was to measure concentrations of cortisol and cortisone in scales of Atlantic cod from different regions of Oslofjord - as chronic stress assessment, to understand any potential stress effects on wild fish populations.

METHODS:
After removal of the mucus, fish scales were washed thrice by using isopropanol, then dried on paper and stored at -80°C until subsequent analysis. A sensitive LC-MS/MS (liquid chromatography-tandem mass spectrometry) analytical method for quantification of cortisol and cortisone without derivatization, in fish scales was developed and validated, with detection levels of both cortisol and cortisone in 20 mg dry fish scale sample of 0.05 ng/g and good extraction recoveries (80% for cortisol and 90% for cortisone). Stable isotope labelled internal standards of cortisol and cortisone were used to spike each sample, which ensured a reliable quantification of steroid hormones in fish scale matrix. Due to the endogenous nature of steroid hormones, assay performance was evaluated by spiking cleaned and dried fish scales with the corresponding isotope-labelled standards analogues of each compound as a surrogate standard.

RESULTS:
This new methodology using solid phase extraction (SPE), allows simultaneous quantification of cortisol and cortisone in just 10-20mg of dried fish scales. Additionally the sample preparation time is much shorter and effective compared to other methods found in literature.

CONCLUSIONS:
This analytical method allows chronic stress quantification by measuring cortisol and cortisone levels from fish scales-as welfare indicator and could be extrapolated to other similar matrices, such as hair to investigate chronic stress in humans.