Poster Presentations for Poster Session C : Thu 12:15 - 13:30

Prostate Cancer: Are Male Testosterone Assays Fit For Purpose? James Hawley (Presenter) Wythenshawe Hospital

Poster #2c View Map

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

BACKGROUND:
Androgen-deprivation therapy (ADT) is recommended to treat advanced prostate cancer. Evidence suggests that lowering testosterone concentrations below 0.7 nmol/L (20 ng/dL) improves patient survival and reduces disease progression. To date, limited work has been undertaken to assess the performance of testosterone assays in this concentration range in males receiving ADT therapy.

DESIGN:
Surplus serum from males taking ADT was obtained prior to disposal from a tertiary cancer centre. Samples were anonymised and distributed to collaborating laboratories for testosterone analysis by 5 routine assays. Simultaneously, we collaborated with UK NEQAS to arrange a small survey to assess how male prostate cancer samples are processed in the UK.

RESULTS:
The UK NEQAS survey indicated that 4/64 laboratories in the UK routinely refer samples for LC-MS/MS analysis in ADT patients. We observed considerable variation across routine immunoassay platforms compared to LC-MS/MS. All routine immunoassays displayed a mean positive bias, this ranged from 0.12 to 0.75 nmol/L (3.5 to 21.6 ng/dL).

SUMMARY:
Despite the clinical importance of suppressing testosterone in ADT, our results suggest this is a cohort of patients that is often overlooked. The performance of many routine immunoassays in this low nanomolar range is sub-optimal for these patients.

UHPLC-MS/MS Analysis of Traditional and Novel Benzodiazepines in Urine Lisa Calton (Presenter) Waters Corporation

Poster #3c View Map

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

INTRODUCTION
Benzodiazepines are important forensic toxicology drugs which are prescribed for neurological and psychiatric disorders but also have a high potential for abuse.

The aim was to develop a forensic toxicology method for confirmatory analysis of 26 traditional and designer benzodiazepines via UHPLC-MS/MS.

METHODS
Drug-free urine samples were fortified with benzodiazepines to generate calibrator and QCs. Urine samples (100µL) were added to a Waters Oasis™ MCX µElution™ Plate followed by 100 µL of a solution containing recombinant beta-glucuronidase enzyme, hydrolysis buffer and internal standards. The sample was loaded, washed and then eluted using 50:50 acetonitrile:methanol containing 5% ammonia. Samples were diluted with 2% acetonitrile: 1% formic acid prior to analysis.

Samples were injected onto an ACQUITY™ UPLC™ BEH™ C18 Column using an ACQUITY UPLC I-Class PLUS System and eluted using a water/formic acid/acetonitrile gradient with detection on a Xevo™ TQ-S micro Mass Spectrometer.

RESULTS
The panel of benzodiazepines were chromatographically separated, with baseline resolution obtained for most compounds. The extraction recovery was within 80-120% for the majority of compounds. The matrix effects were mostly minimized to ±20% after internal standard correction. Calibration curves were linear with r2≥0.99 and coefficients of variation were less than 15% for all analytes. Results for proficiency samples showed excellent agreement to existing methods.

CONCLUSION
Leveraging SPE sample preparation and UHPLC-MS/MS with the Xevo TQ-S micro Mass Spectrometer, a rapid, accurate and precise method was demonstrated for analyzing benzodiazepines in urine.

For Forensic Toxicology Use Only.
Waters, Oasis, µElution, ACQUITY, UPLC, Xevo, and BEH are trademarks of Waters Technologies Corporation.
© 2025 Waters Corporation.

Unraveling Sitosterolemia: A Case of Pediatric Hypertensive Cardiomyopathy and Lipid Disorder Xiaochun Zhang (Presenter) University Hospitals Cleveland Medical Center, Case Western Reserve University SOM

Poster #4c View Map

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

CASE DESCRIPTION:
An 8-year-old boy presented to the emergency department with a one-week history of progressive fatigue, abdominal pain, and non-bloody, non-bilious vomiting. Over the preceding few weeks, he had developed a cough, tactile fever, and failure to gain weight, along with decreased food intake and urine output. According to his parents, the boy had not acted like himself for the past three months, exhibiting increased tiredness and reduced activity levels. His past medical history was unremarkable.

On physical examination, he exhibited significant hepatomegaly and pale skin, with hard, raised, non-tender, flesh-colored nodules on his knees and legs—later identified by dermatology as tuberous xanthomas present for several years. Vital signs were: temperature 37.1°C, heart rate 118 beats per minute, respiratory rate 20 breaths per minute, blood pressure 130/91 mmHg, and oxygen saturation 100% on room air.

The patient was admitted to the pediatric intensive care unit (PICU) due to concerns of hypertensive cardiomyopathy and cardiac dysfunction.

Laboratory findings revealed slight hyponatremia, mild hyperkalemia, mildly elevated liver enzymes, microcytic anemia, and thrombocytosis. A lipid panel showed markedly abnormal results: total cholesterol 317 mg/dL, HDL 13.8 mg/dL, LDL 271 mg/dL, triglycerides 161 mg/dL, and a cholesterol/HDL ratio of 23.0 (desirable <3.4; high risk >5.0).

Imaging and cardiologic evaluation confirmed hypertensive cardiomyopathy, mid-aortic syndrome, bilateral renal and coronary artery stenosis, and hypertension. Endocrinology and medical genetics considered differential diagnoses, including familial hypercholesterolemia, sitosterolemia, or Alagille syndrome and ordered a plasma sterol panel for further investigation.

BACKGROUND:
Sitosterolemia, also known as phytosterolemia, is a rare autosomal recessive disorder caused by mutations in the ATP-binding cassette (ABC) transporter genes ABCG5 or ABCG8. These genes encode proteins that form heterodimers expressed exclusively in hepatocytes and enterocytes. The ABCG5/ABCG8 heterodimers preferentially export plant sterols from enterocytes into the intestinal lumen and from hepatocytes into bile canaliculi, limiting net absorption to less than 2% for plant sterols and approximately 55% for dietary cholesterol in healthy individuals. Mutations in ABCG5 or ABCG8 result in the accumulation of plant sterols (primarily sitosterol and campesterol) in the blood and tissues, leading to clinical manifestations such as xanthomas, premature cardiovascular disease, and early cardiac death.

MS METHOD AND RESULTS:
The plasma sterol panel, analyzed using gas chromatography-mass spectrometry (GC-MS), included plant sterols (sitosterol, campesterol, stigmasterol) and sterol intermediates of cholesterol biosynthesis (7-dehydrocholesterol, 8-dehydrocholesterol, 8(9)-cholesterol, cholestanol, desmosterol, dihydro T-MAS, lathosterol). The plasma specimen was hydrolyzed, extracted, evaporated to dryness under nitrogen, derivatized, and analyzed using selected ion-monitoring electron impact GC-MS. Results revealed dramatically elevated levels of campesterol (>100 mg/L, reference interval ≤8.0 mg/L), cholestanol (78.6 mg/L, reference interval ≤6.0 mg/L), sitosterol (>100 mg/L, reference interval ≤15.0 mg/L), and stigmasterol (10.2 mg/L, reference interval ≤0.5 mg/L), while other sterols remained within normal ranges. These results supported the diagnosis of sitosterolemia and distinguished it from other inherited lipid disorders. Later, genetic testing identified a homozygous mutation in the ABCG8 gene, further confirming the diagnosis.

DISCUSSION AND CONCLUSION:
Because of its low prevalence and heterogeneous clinical presentation, sitosterolemia is frequently underdiagnosed. A definitive diagnosis is achieved through genetic testing for the ABCG5 /8 genes. However, whole exome sequencing can be costly and time-consuming. As a result, quantitative analysis of plasma phytosterols is a valuable diagnostic tool that helps distinguish sitosterolemia from other types of hyperlipidemias. Routine enzymatic lipid assays cross-react with plant sterols and cannot distinguish them from cholesterol or other xenosterols (non-cholesterol sterols), often leading to misdiagnosis as familial hypercholesterolemia. GC-MS can accurately differentiate and quantify plant sterols, cholesterol, and other xenosterols, are essential for diagnosis and monitoring treatment responses.

Sitosterolemia is often diagnosed later in life till clinical suspicion is triggered by symptoms such as xanthomas or unexplained hypercholesterolemia. Early detection via newborn screening could enable timely interventions such as the use of medications like ezetimibe and dietary modifications to prevent premature cardiovascular disease, but implementation is hindered by high costs and limited access of confirmatory test.

Previously considered exceedingly rare and thought to require near total loss of ABCG5/8 function, recent research suggests a higher prevalence, potentially exceeding 1 in 200,000 individuals. Expanded screening in recent years has revealed a growing number of ABCG5/8 variants, with some heterozygotes exhibiting elevated sitosterol levels. In a large-scale study of over 200,000 individuals, a significant association between hypercholesterolemia and sitosterolemia was identified in 4% of patients. Additionally, plant sterols have been used as dietary supplements for decades to lower plasma cholesterol, yet the outcome remain controversial. Measuring phytosterol profiles by GC-MS can aid in diagnosing sitosterolemia, assessing plant sterol absorption in carriers, and investigating the relationship between plasma plant sterol levels and cardiovascular risk.

LC-MS/MS Analysis of Bromazolam and Its Metabolites in Clinical Specimens from a San Francisco Patient Cohort Hannah Lusk (Presenter) University of California San Francisco

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Poster #5c View Map

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

INTRODUCTION:
Benzodiazepines are central nervous system (CNS) depressants commonly used to treat anxiety, insomnia, seizures, and muscle spasms. Sedative effects induce relaxation and reduce anxiety but can also cause side effects such as drowsiness, altered mental status, dizziness, respiratory depression, and bradycardia. Due to their anxiolytic properties, benzodiazepines are frequently misused and carry a high risk of overdose, particularly when combined with other depressants. While several FDA-approved benzodiazepines, including alprazolam, lorazepam, and diazepam, are legally available by prescription in the United States, the illicit drug supply increasingly contains non-FDA-approved designer benzodiazepines. These compounds pose a significant public health concern due to their highly variable potency and poorly characterized pharmacokinetic profiles, increasing the risk of unintentional overdose. Bromazolam is one such designer benzodiazepine. Our laboratory conducts biosurveillance in San Francisco (SF) using a comprehensive mass spectrometry (MS)-based drug testing method, which has detected a recent rise in bromazolam-positive cases. In 2024, we identified 19 positive cases compared to 4 in 2023. To date, limited data exist on bromazolam’s pharmacokinetics, with only one study evaluating its metabolism using in vitro pooled human liver S9 fractions and in vivo analysis of serum and urine from two patients. To address this knowledge gap, we analyzed remnant serum and urine samples from SF patients to quantify bromazolam and characterize its metabolites. This study provides real-world data on bromazolam exposure and metabolism, offering insights into its pharmacokinetics and potential health risks.

OBJECTIVES:
The objective of this study is to characterize the metabolism of bromazolam in patient urine and serum samples. By analyzing bromazolam and its metabolites in real clinical samples, we aim to improve our understanding of its major metabolic pathways, thereby supporting toxicological and forensic investigations.

METHODS:
Ten remnant serum and 18 remnant urine samples were collected following a positive bromazolam result from our clinically validated comprehensive drug test using untargeted LC-QTOF-MS. Chromatographic separation was achieved with a C-18 column using a 10-minute gradient (2%-100% organic). Samples were analyzed on a SCIEX ZenoTOF® 7600 in positive mode with a TOF-MS survey scan and SWATH-triggered acquisition of high-resolution product ion spectra. Urine metabolites were identified through untargeted analysis. Serum concentrations of bromazolam and alpha-hydroxy bromazolam were quantified using 13 calibrators, a double blank, a blank, and four quality controls, all prepared in drug-free human serum to cover a dynamic range from 0.1 ng/mL to 1 µg/mL. Bromazolam-D5, at a concentration of 100 ng/mL, served as the internal standard. Quantification was performed using multiple reaction monitoring (MRM) on a SCIEX 4500 triple quadrupole MS. Chromatographic separation was carried out with a C-18 column using a 2-minute gradient (2%-100% organic). Sample preparation involved protein precipitation, centrifugation, evaporation under a steady stream of nitrogen gas, and reconstitution in the initial mobile phase. Data analysis was performed using PeakView software, and quantification was conducted using MultiQuant software.

RESULTS:
Bromazolam, bromazolam glucuronide, and hydroxy-bromazolam glucuronide were detected in all urine specimens analyzed using LC-QTOF-MS. Other metabolites identified in the original study that used human liver S9 fractions in vitro were not detected. Hydroxy-bromazolam glucuronide was the predominant metabolite, with an area under the curve (AUC) an average of 40% higher than the bromazolam peak. The ratios of bromazolam glucuronide and hydroxy-bromazolam glucuronide to bromazolam varied across samples, suggesting inter-individual differences in metabolic clearance. Concentrations in serum samples covered the calibration range and varied significantly between patients. IRB-approval was recently obtained to allow for medical chart review of positive bromazolam cases to evaluate if clinical symptoms and outcomes are associated with serum concentrations.

CONCLUSIONS:
The consistent detection of bromazolam glucuronide metabolites in patient urine samples underscores the significance of phase II metabolism in bromazolam clearance. In several samples, bromazolam was detected at lower levels than glucuronidated metabolites, indicating that failing to screen for these metabolites may lead to an underestimation of bromazolam-positive urine. This suggests that hydrolyzing samples before MS analysis or directly monitoring the glucuronide metabolite could extend the detection window and improve sensitivity. Variability in metabolite-to-parent drug ratios highlights inter-individual differences that may impact bromazolam’s pharmacologic effects. However, correlating bromazolam concentrations in serum with clinical symptoms is challenging due to poly-drug exposure, which complicates the attribution of symptoms to bromazolam versus other substances. Notably, no published clinical cases of bromazolam exist, with current literature primarily focusing on postmortem analyses. Our findings enhance the understanding of bromazolam pharmacokinetics, confirming its glucuronidation and metabolism to hydroxy-bromazolam.

Simultaneous Quantification of 17 Steroids Using the Tecan Kit and Shimadzu 8060 LC-MS/MS Platform Milaan Thirukumaran (Presenter) Shimadzu Scientific Instruments

Poster #7c View Map

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

BACKGROUND:
Steroid hormones are essential regulators of metabolism, immune function, stress response, and reproduction. Accurate quantification of steroid hormones is crucial for clinical diagnostics and biomedical research, especially in the context of endocrine disorders and metabolic diseases. Traditional immunoassays, while widely used, are often limited by cross-reactivity and lack of multiplexing capability, which can compromise specificity and throughput. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold standard for steroid quantification, offering superior specificity, sensitivity, and the ability to simultaneously measure multiple analytes in complex biological matrices (1,2).

OBJECTIVE:
The objective of this study was to assess the analytical performance of a research-use-only steroid quantification kit in conjunction with the Shimadzu Nexera X3 UHPLC and LCMS-8060 platform for the simultaneous determination of 17 steroids. Key evaluation criteria included intra-assay precision, and trueness across three sample levels, with the goal of establishing the suitability of this workflow for clinical research applications in the field of steroidomics.

METHODS:
Sample preparation followed the Tecan kit IFU (Cat. No. 30220266), which includes solid-phase extraction (SPE) to ensure optimal recovery and ionization efficiency for all target analytes. The workflow consists of two LC-MS/MS runs: the first run quantifies 15 steroids, while the second run quantifies two additional steroids, for a total of 17 steroids. The calibrators and quality control samples are lyophilized in serum and reconstituted with LC-MS grade water before analysis. Chromatographic separation was optimized to resolve analytes with similar structures and mass transitions, with particular attention to those known to be challenging, such as 11-deoxycortisol, 21-deoxycortisol, corticosterone, cortisol and cortisone, as well as 17-hydroxyprogesterone and 11-deoxycorticosterone(3). Proper chromatographic separation of these analytes is essential to avoid cross-talk and ensure accurate quantification. The Shimadzu LC-MS/MS system was operated in multiple reaction monitoring mode (MRM), with compound-specific transitions and collision energies. Data acquisition and quantification were performed using LabSolutions software, with internal standard correction applied to all analytes. Precision was evaluated as the relative standard deviation (%) of replicate measurements at each quality control level, with a classical acceptance criterion of less than 20%. Trueness was assessed by comparing the measured values to the corresponding target values.

RESULTS:
The Tecan kit demonstrated robust analytical performance across all tested analytes and quality control levels. Precision was assessed for all analytes at both high- and low-quality control levels using 15 replicates each. All analytes demonstrated %RSD values below 20%, indicating strong repeatability. For example, 17-OHP4 showed %RSD values of 3.9% (QC H) and 3.7% (QC L), testosterone 1.9% (QC H) and 3.2% (QC L), and cortisol 4.5% (QC H) and 8.7% (QC L) showed %RSDs well within this range at both QC levels. The method provides reliable and robust precision across the entire steroid panel.

Trueness was evaluated at three sample levels (S1, S2, S3) for each analyte. Most analytes showed trueness within the generally accepted range. For example, 17-OHP4 demonstrated trueness values of 0.2% (S1), 1.3% (S2), and 6.2% (S3); testosterone showed 13.0% (S1), 1.6% (S2), and -5.4% (S3); and cortisol showed 7.0% (S1), 7.2% (S2), and 4.5% (S3). All analytes were within ±20% of the target value across all levels, supporting the method’s suitability for clinical research applications.

Calibration curves for all analytes demonstrated good linearity, with R² values >0.99 for almost all analytes. For key clinical markers, R² values were 0.9978 for 17-OHP4, 0.9989 for testosterone, and 0.999 for cortisol. All analytes were fitted using a linear model with either 1/C or 1/C² weighting, as appropriate.

CONCLUSION:
The comprehensive evaluation of the Steroid Panel LC-MS kit in combination with the Shimadzu platform for high-throughput, multiplexed steroid analysis. The ability to reliably quantify a broad panel of steroids in just two runs offers significant advantages for research laboratories investigating complex endocrine pathways, disease mechanisms, or therapeutic interventions. The standardized workflow reduces operator variability and enhances reproducibility, while the high sensitivity and selectivity of the Shimadzu LC-MS/MS platform ensure confident detection and quantification even at low physiological concentrations. The method meets established acceptance criteria for precision across the vast majority of analytes and quality control levels. Trueness results further support the method’s reliability for clinical research applications.

References:

1. Taylor, A.E., Keevil, B.G., & Huhtaniemi, I.T. (2015). Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow. European Journal of Endocrinology, 173(2), D1–D12.
2. Vogeser, M., & Parhofer, K.G. (2007). Liquid chromatography tandem-mass spectrometry (LC-MS/MS)–technique and applications in endocrinology. Experimental and Clinical Endocrinology & Diabetes, 115(09), 559-570.
3. Zhu, Y., Li, Y., Wang, Y., Wang, Y., & Wang, Y. (2022). Simultaneous determination of 11 endogenous corticosteroids in human plasma by liquid chromatography–tandem mass spectrometry for the diagnosis of adrenal diseases. Frontiers in Endocrinology, 13(9), 940314. https://doi.org/10.3389/fendo.2022.940314.

Impact of Endogenous Insulin Antibodies on Immunoaffinity Purification of Human Insulin and Pharmaceutical Analogs in Serum Michelle R. Campbell (Presenter) Mayo Clinic

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Poster #8c View Map

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

INTRODUCTION:
Commonly utilized immunoassays for quantitating insulin exhibit variable cross-reactivity with commonly prescribed pharmaceutical insulins and their metabolites, making it difficult to differentiate endogenous insulin from insulin analogues that may be present in a patient sample. As such, factitious hypoglycemia, defined as a deliberate attempt to induce low blood glucose levels occurring secondary to the surreptitious administration of exogenous insulin, is challenging to diagnose. The development of a method using immunoaffinity purification of insulin from samples, followed by analysis by liquid chromatography high resolution accurate mass spectrometry (LC-HRAM-MS), has enabled the differentiation and quantitation of human insulin and insulin analogs. Development of insulin autoantibodies is common in patients with type 1 diabetes mellitus. Although the levels of autoantibodies typically decrease with full manifestation of type 1 diabetes, they can rise in response to therapeutic insulin. Therefore, it is imperative to demonstrate that the presence of endogenous insulin antibodies does not interfere with the purification methods in the newly developed LC-HRAM-MS assay to detect insulin analogs.

OBJECTIVES:
Evaluate whether endogenous insulin antibodies affect the recovery of human insulin and insulin analogs using immunoaffinity purification with a monoclonal anti-insulin antibody.

METHODS:
N= 11 patient serum samples with known positive levels of insulin antibody results were measured neat and after spiking with a high calibrator solution consisting of human insulin and three insulin analogs (aspart, glulisine, and lispro) in insulin-stripped serum at concentrations of 100 mcIU/mL. Immunoaffinity purification of insulin from calibrators, quality control material, and spiked patient samples was performed using magnetic beads coupled with a commercial anti-insulin monoclonal antibody. Eluate from the purification protocol was submitted for analysis using a TLX-2 multiplex LC system paired with a Thermo Scientific Q Exactive Plus mass spectrometer using a 1000 Å Diphenyl, 2.7 µm, 2.1 x 100 mm analytical column in a column heater maintained at a temperature of 80°C. Percent recoveries were calculated to determine whether the presence of insulin antibodies interfered with the capture and subsequent quantitation of human insulin or insulin analogs.

RESULTS:
Average percent recoveries (with ranges) were as follows: aspart 109% (101-113%); glulisine 110% (104-117%); human 92% (82-98%); and lispro 101% (91-113%). Insulin recovery was not correlated with the concentration of insulin antibody present in the sample.

CONCLUSION:
Immunoaffinity purification of human insulin and pharmaceutical analogs from serum using magnetic beads coupled with a monoclonal anti-insulin antibody was not affected by the presence of endogenous insulin antibodies in patient samples. Increasing concentrations of insulin antibodies did not negatively impact insulin recovery. A strength of this study was the use of 11 unique patient samples for spiking, providing variability in the endogenous antibodies evaluated compared to spiking with a single commercial antibody. These results suggest that this immunoaffinity purification method is unaffected by the presence of naturally occurring insulin antibodies in patient samples.

REFERENCES:
1. Egan AM, Galior KD, Maus AD, et al. Pitfalls in Diagnosing Hypoglycemia Due to Exogenous Insulin: Validation and Utility of an Insulin Analog Assay. Mayo Clin Proc. 2022;97(11):1994-2004.

Simultaneous Measurement of 25(OH)D and 24,25(OH)2D Among Healthy Population in Japan Using a Fully-Automated LC-MS/MS System Daisuke Kawakami (Presenter) SHIMADZU EUROPA GMBH

Poster #9c View Map

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

BACKGROUND:
Vitamin D sufficiency is currently assessed by serum 25-hydroxy vitamin D [25(OH)D] levels. However, a proportion of people show low 25(OH)D levels but high bone mineral density, which is called 'vitamin D paradox'. Among such population, measuring vitamin D metabolite ratio (VMR), the ratio of 24,25(OH)2D and 25(OH)D, is reported to be useful. As serum vitamin D levels are affected by genetic background, reference ranges of 25(OH)D and VMR may vary by ethnicities. Understanding variations of the reference ranges in different population is important for effective intervention. However, so far measuring VMR in clinical practice is challenging due to long turnaround time (TAT). We have established fully automated liquid chromatography tandem mass spectrometry (LC-MS/MS) system that simultaneously measure 24,25(OH)2D and 25(OH)D with short TAT.

METHOD:
The conditions of quantification of 25(OH)D and 24,25(OH)2D using a fully-automated sample preparation system (CLAM-2030) and a Nexera UHPLC system coupled with an ultrafast triple quadrupole mass spectrometer (LCMS-8060NX; Shimadzu Corp., Kyoto, Japan) were established. Electrospray ionization (ESI) was employed for mass spectrometric detection. Fifty serum samples of healthy population were collected measurement using the LC-MS/MS system and VMRs were calculated. Data were compared against measurements from a chemiluminescent enzyme immunoassay to establish accuracy.
Result: By optimizing LC-MS/MS conditions and analytical column, linearity was obtained at the concentration of 0.5-100 ng/mL of 24,25(OH)2D with measurement error &lt;6%. The TAT was as short as 25 minutes. Interestingly, serum 25(OH)D level and VMR as well as 24,25(OH)2D showed positive correlation, suggesting VMR itself may reflect vitamin D sufficiency status among people with lower levels of 25(OH)D.

DISCUSSION:
Although there is an accumulating data about vitamin D sufficiency/deficiency, data among Asian population is still lacking. Our data suggest 24,25(OH)2D levels as well as VMR among Japanese people might be different from European ones. Our data highlights the importance of mass-screening of vitamin D status in different populations using accurate measurement systems with high throughput.

Unravelling Metabolic Dysregulation in Head and Neck Cancer with Laser-Desorption Rapid Evaporative Ionisation Mass Spectrometry Jasmin Werner (Presenter) Imperial College London

Poster #10c View Map

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

INTRODUCTION:
Head and Neck Squamous Cell Carcinoma (HNSCC) ranks as the seventh most common cancer worldwide and presents a growing global health challenge (1). Incidence is associated with alcohol consumption, tobacco use, and certain high-risk HPV strains. Despite its prevalence, HNSCC has poor survival and morbidity outcomes. With significant intra- and inter-tumoural heterogeneity and a high mutational burden, metabolic dysregulation remains poorly characterised.
Ambient Ionisation Mass Spectrometry (AIMS) techniques, such as Laser-Desorption Rapid Evaporative Ionisation Mass Spectrometry (LD-REIMS), can be used for metabolic analysis on minimally prepared HNSCC samples (2). For this reason, this technology is also being utilised during interventions for interoperative margin surveillance.

Our aim was to demonstrate LD-REIMS as an effective technique for mapping the spatial distribution of metabolic features of ex-vivo head and neck cancer samples, thereby revealing the metabolic heterogeneity within and between tumours.

METHODS:
Paired tumour to normal samples were collected from consenting HNSCC patients during surgery (REC: 14/EE/0024). A total of 15 tissue samples from the oral cavity, larynx, and pharynx were collected, flash-frozen, and serially sectioned for LD-REIMS imaging - a 3 &micro;m wavelength Optical Parametric Oscillator (OPO) laser was used to generate an aerosol directed to a Waters Xevo G2-XS QToF instrument via a REIMS source. The data was processed using our established in-house mass spectrometry imaging pipeline, followed by unsupervised analysis. A logistic regression (LR) model was applied for pixel-by-pixel classification, enabling robust metabolic differentiation between tumour and normal tissue. Metabolic features identified from previous studies were spatially mapped and compared with H&amp;E-stained sections, annotated by a trained histopathologist.


RESULTS:
Each mass spectrometry image consisted of approximately 12,000 to 20,000 individual pixels mapping to a corresponding mass spectrum. Using this setup, a spatial resolution of 50 &micro;m was achieved. Overall, more than 4000 distinct m/z peaks were detected, with over 1000 peaks exhibiting significant dysregulation of at least two-fold.

Principal component analysis (PCA) revealed apparent separation between tumour and normal tissues, highlighting distinct metabolic differences. Comparison with histopathological annotations confirmed a strong correlation between metabolomic profiles and histological features. Key dysregulated metabolic species included glycerophospholipids, ceramides, fatty acids, and triglycerides. Lipidomic profiling further demonstrated a significant upregulation of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in tumour regions, suggesting enhanced membrane turnover and metabolic adaptation. Additionally, sphingolipid metabolism was notably altered, with a marked increase in certain ceramide species.

Further classification refinement using machine learning algorithms improved the spatial resolution of tumour delineation. Recursive feature elimination demonstrated an improved classification accuracy of 97.2% (sensitivity 98.2%, F1 score 93.6%), confirming the robustness of LD-REIMS for metabolic tissue profiling. Importantly, metabolic classifiers maintained high performance across samples from different anatomical sites (oral cavity, larynx, and pharynx), indicating the broad applicability of this technique in head and neck cancer subtypes.


CONCLUSION:
These findings show the potential of LD-REIMS as a high-resolution, preparation-free approach for metabolic tissue characterisation in HNSCC. The observed metabolic dysregulation patterns provide insights into tumour biology and may offer future avenues for biomarker development. Furthermore, the opportunity for translation into a clinical setting, using this specific AIMS technique, may set the stage for additional interoperative insights and downstream personalised medicine approaches.


REFERENCES:
(1) Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram, I., Jemal, A. and Bray, F. (2021). Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a Cancer Journal for Clinicians, [online] 71(3), pp.209&ndash;249. doi:https://doi.org/10.3322/caac.21660.
(2) Higginson, J., Manoli, E., Ford, L., Zhang, H., Dhanda, J., Takats, Z. and Paleri, V. (2022). A better way to determine tumour margins intraoperatively, using ambient mass spectrometry (interim report). British Journal of Oral and Maxillofacial Surgery, 60(1), p.e2. doi:https://doi.org/10.1016/j.bjoms.2021.12.012.

Enhancing Diagnostic and Throughput Efficiency for Total Estrone Levels Analysis in Serum Using LDTD-MS/MS Mégane Moreau (Presenter) Phytronix Technologies

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Poster #11c View Map

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

INTRODUCTION
Total estrone analysis in serum is a vital tool for assessing hormonal balance, particularly in postmenopausal women and individuals undergoing hormone-related treatments1,2. Estrone, one of the primary estrogens, plays a crucial role in various physiological processes, and its levels can provide valuable insights into reproductive health, metabolic conditions, and endocrine disorders3. By accurately measuring estrone concentrations, clinicians can better diagnose and monitor conditions such as estrogen imbalances, ovarian tumors, and hormone-dependent cancers, facilitating more effective and personalized treatment strategies3,4. This method allows for rapid and precise quantification, offering improved diagnostic accuracy and faster clinical decision-making.

OBJECTIVES
The aim of quantifying total estrone in serum is to precisely assess hormonal balance, facilitating the diagnosis and monitoring of reproductive and endocrine health conditions. This includes developing a fast extraction and analytical method to efficiently measure total estrone in serum using LDTD-MS/MS.

METHODS
The calibration curve and quality control samples are spiked into a fresh artificial matrix consisting of 20 mg/mL BSA in PBS. To each 2 mL centrifuge tube, 40 µL of the sample (or calibration curve/quality controls) are added, followed by 40 µL of hydrolysis buffer (BGS buffer from Kura) and 10 µL of internal standard solution (prepared in a methanol:water (10:90) mixture). To account for the matrix effect, the samples are diluted using the artificial matrix by adding 270 µL of 20 mg/mL BSA in PBS. The tubes are then mixed for 30 seconds at 1000 rpm. To hydrolyze estrone-sulfate, 40 µL of BGS enzyme from Kura is added, mixed, and incubated at 52°C for 30 minutes. After the incubation, the samples are extracted using 800 µL of a Hexane:MTBE (90:10) mixture. The tubes are centrifuged at 13,500 rpm for 4 minutes, and 500 µL of the upper layer is transferred into a 10x75 mm borosilicate tube. The samples are evaporated to dryness, and 125 µL of a methanol:water (1:1) mixture are added. Finally, 6 µL of the extracted sample are spotted onto a Lazwell plate, and the samples are dried at 40°C for 8 minutes before analysis. The Luxon laser power rises to 45% within 3 seconds, and the gas flow is set at 5 L/min. Mass spectrometry analysis is performed using a triple quadrupole QTrap 5500 from Sciex. MRM transitions are used to quantify estrone with transition 269.0 - 145.1, and for estrone-d4 with transition 273.0 - 147.1, utilizing a collision energy of -55 eV. The mass spectrometer operates in negative ionization mode. To normalize the signal, the ratio of the analyte's area to the internal standard's area is use.


RESULTS
To validate the analytical method, the evaluation of precision, accuracy, linearity, stability and hydrolysis efficiency was evaluated. The accuracy of quality control samples should be within ±20% of the nominal value, with a coefficient of variation (%CV) of less than 20%. For the intra-run evaluation, the precision ranged from 1.5 to 11.5 %CV, and the accuracy ranged from 91.5 to 102.0 % of the nominal value for the quality control samples (LLOQ, QCL, QCM, QCH, and ULOQ). For the inter-run evaluation, the precision ranged from 11.5 to 18.2 %CV, and the precision ranged from 96.6 to 102.9 % of the nominal value for the quality control samples (QCL, MCQ, QCH). In assessing linearity, the correlation coefficient must be greater than 0.995. All correlation coefficients obtained were greater than 0.996. When analyzing with LDTD-MS/MS, stability was assessed both in the extracted solution (wet stability) and in the sample after deposition and drying on the Lazwell plate (dry stability), which differs from the injector stability assessment used in LC-MS/MS. For wet stability, samples were kept at room temperature for 24 hours. Precision (%CV) was below 15%, and accuracy was within ±15% of the nominal value for all QCs. For dry stability, samples were stored at room temperature for 2 hours. As with wet stability, precision and accuracy met the required criteria for all QCs.

To verify the efficiency of the enzyme for estrone-sulfate hydrolysis, estrone-sulfate spiked samples (at the QCM level) were extracted according to the outlined procedure. The extraction efficiency was close to 100% for all estrone-sulfate enriched samples. Finally, cross-validation with a Shimadzu LC-MS/MS system (LC-8060) was conducted to evaluate the correlation between the two analysis methods. All matrices showed less or equal than a 15% difference in the calculated values obtained.


CONCLUSIONS
A high-throughput analysis of total estrone using the LDTD-MS/MS method was developed. The method demonstrates reliable precision and accuracy, with all quality control samples meeting the necessary criteria for both intra-run and inter-run evaluations. The high extraction efficiency and successful cross-validation with LC-MS/MS further confirm the robustness and reliability of the analysis for estrone sulfate hydrolysis.

REFERENCES

1. Davis SR, Martinez-Garcia A, Robinson PJ, Handelsman DJ, Desai R, Wolfe R, Bell RJ; ASPREE Investigator Group (2020). J Clin Endocrinol Metab.;105(9):e3348–54.
2. Harper-Harrison G, Carlson K, Shanahan MM (2025). Hormone Replacement Therapy. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing.
3. Gao WL, Wu LS, Zi JH, Wu B, Li YZ, Song YC, and Cai DZ (2015). Brazilian Journal of Medical and Biological Research, 48(2): 146-153.
4. Ankarberg-Lindgren C, Dahlgren J, Andersson MX (2018). The Journal of Steroid Biochemistry and Molecular Biology, Volume 183, Pages 116-124.

Evaluation of Prioritized Peptide Acquisition for Multiplexed Single-Cell Proteomics on an Orbitrap Astral Zoom Mass Spectrometer Tanis Correa (Presenter) Thermo Fisher Scientific

Poster #12c View Map

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

INTRODUCTION:
Single-cell proteomics by mass spectrometry (scp-MS) enables the characterization of cell states through protein abundance measurements, offering potentially more accurate phenotypic readouts than mRNA-based methods. However, current scp-MS methods face limitations in measurement throughput and proteome coverage. Sample multiplexing via isobaric tags (SCoPE-MS) offers the highest throughput in scp-MS. However, these methods have not yet been evaluated on the Thermo Scientific Orbitrap Astral MS, which promises superior sensitivity for scp-MS.

METHODS:
We implemented prioritized targeting of known precursors from the carrier channel of SCoPE-MS samples using the standard instrument method editor. Precursor target lists were assembled from a survey DIA run based on the biological question, enabling the selection of proteins involved in specific pathways or limiting the number of peptides per protein to increase coverage. To evaluate the quantitative performance, we measured a TMTpro 35-plex species-mix SCoPE-MS sample with a 30SPD method using the Thermo Scientific Vanquish Neo LC system with a trap-and-elute setup and a Thermo Scientific μPAC Neo Plus column, coupled to a Orbitrap Astral Zoom MS.

RESULTS:
This setup enabled profiling over 3000 proteins in nearly 1000 single cells per day. Using a 150pg protein load in the species-mix single-cell channels, we compared the quantitative performance between the TMT-multiplexed method and a label-free DIA species mix measured at 100SPD. The TMT data showed expected ratio-compression by isobaric interferences, absent in the DIA data. However, the absolute error distribution of fold-changes was comparable, indicating that the TMT-multiplexed method recovers a similar number of accurate ratios at 100ms Astral injection time. Importantly, the TMT-multiplexed method increases cell throughput 10-fold.

CONCLUSION:
Implementation of pSCoPE in the method editor. Evaluation of SCoPE-MS on a Orbitrap Astral Zoom MS using a TMT-labelled species-mix.

Plasma Metabolomic and Proteomic Profiling of Anxious Dogs by HPLC-MS/MS: A Case-Control Study Claudia Gaither (Presenter) Faculté de médecine vétérinaire, Université de Montréal

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Poster #13c View Map

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

INTRODUCTION:
The prevalence of anxiety disorders in dogs highlights the need for novel/better diagnostics, prediction of treatment success, and treatment progress monitoring that can be achieved using innovative MS-based analyses. Behavioral problems affect up to 85% of dogs, with many stemming from underlying anxiety disorders. Some of the most common anxiety-related behavioral problems are separation anxiety, generalized anxiety, aggression, and compulsive behaviors. Although MS has been used for some disease profiling experiments in dogs and other species, it is usually overlooked in the veterinary and animal science fields, particularly in the animal behavior and psychiatry disciplines. Thus, here we investigate plasma proteomic and metabolomic differences between a group of anxious dogs and a group of non-anxious dogs.

METHODS:
Venous blood was collected, and plasma generated at the Faculté de médecine vétérinaire, Université de Montréal. Ten control and ten patient dogs underwent behavioral evaluations by a board-certified veterinary behaviorist and either diagnosed with an anxiety disorder or confirmed lack thereof, respectively. Samples were prepared for proteomic and metabolomic analyses by in-solution enzymatic digestion and solvent extraction of metabolites, respectively, for untargeted HPLC-MS/MS. Label-free quantitative MS data were acquired using a Vanquish Flex UHPLC, and an Evosep One system, both interfaced to a Q Exactive Plus Orbitrap MS (Thermo). Data were analyzed using Proteome Discoverer 2.2 or Compound Discoverer 2.1 (Thermo). Plasma protein and metabolite differences between the groups were assessed based on fold changes and t-tests (p-value ≤ 0.05).

RESULTS:
A total of 49 proteins had statistically significantly different plasma levels between anxious and non-anxious/control dogs, as determined by two-tailed student t-tests (p-value ≤ 0.05). Among the differential proteins were Apolipoproteins A-II, C-I and C-IV, involved in the packaging, transporting, and metabolizing of lipids, including cholesterol. Cholesterol is the precursor for cortisol, a well-studied biomarker for stress that leads to various physiological effects, including the flight or fight response. Other proteins found to be at different levels between the two groups were Fibrinogen Beta and Gamma chains, Serine Protease Inhibitors and Coagulation Factor XII, which are part of the blood coagulation cascade. These higher levels could be a result from cortisol production resulting in dysfunctional coagulation and excess clot formation. Finally, Complement Factors I and D, complement components C5, C6, C8A, and Complement Component 4 Binding Protein (C4BP) were differential and are involved in the complement system cascade, part of the innate immune response. The activation of C3 and C5 convertases is regulated by C4BP, and its differential expression could result in dysfunctional regulation of such enzymes, leading to increased or continuous inflammation.

Preliminary metabolomics results also show plasma profile differences between the two groups of dogs. Some of the metabolites that appear to be higher in the anxious dogs include phenylalanine, creatine, certain bile acids and certain fatty acids. In humans, higher levels of phenylalanine, a precursor to catecholamine neurotransmitters, are linked to anxiety. Bile acids can directly bind to brain receptors through the blood-brain barrier and their signaling may induce anxiety. Long chain saturated fatty acids are linked to anxiety-like behavior in mice and other fatty acids promote inflammation. Finally, metabolites like adenosine were lower in the anxious dogs, compared to controls. Low adenosine in humans can decrease sleep quality thus resulting in anxiety and depression.

CONCLUSION:
To our knowledge, this is the first unbiased/comprehensive clinical in-depth metabolomic/proteomic profiling of plasma from dogs with anxiety disorders. On the proteomics side, there are three main pathways that appear differential between the control and anxious dogs: those involved in lipid metabolism, the coagulation cascade, and the complement system. On the metabolomics side, phenylalanine, creatine, certain bile acids and certain fatty acids were found to be differential. Targeted assays will need to be developed for the validation of our findings and bring the field a step closer to elucidating the pathophysiology of anxiety.

MassHunter Explorer 2.0: Non-Targeted Feature Identification and Statistical Analysis Software for Metabolomic Differentiation of Plasma Samples Marcus Kim (Presenter) Agilent Technologies

Poster #14c View Map

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

INTRODUCTION:
Common workflows for untargeted metabolomics by HRMS utilize multiple acquisition types. Data dependent acquisition provides rich fragment information important for identification, and iterative injections give deeper metabolite identification utilizing exclusion lists. MS1 affords sensitive and comprehensive surveys of individual samples. This combination workflow gives analysts a wealth of information for observing changes in known metabolites while also identifying metabolites or other analytes less frequently present in biologically curated metabolomic libraries. However, this heavy data load can create a steep barrier for some investigators. Herein is novel software that combines complex analysis into a streamlined workflow, untangling the interpretation of multiple data file types and gives researchers clear and confident interpretation of metabolomic alterations.

METHODS:
Metabolites were extracted from 20 µL of plasma using SPE and separated by HILIC chromatography, both using established and standardized methodology. 20 male and 20 female mouse samples were used for this study. A bioinert LC combined with a LC/Q-TOF designed for the analysis of small molecules was used for the acquisition of both MS and iterative MS/MS methods.

Following collection of two different modes of acquisition all the data files were collectively loaded into a novel software solution for HRMS untargeted analysis. Feature extraction, filtering, normalization, statistics, and identification, including comparison to method specific libraries but also direct export to SIRIUS and NIST, was achieved.

RESULTS:
The LC/Q-TOF methods were arranged into a worklist designed to identify metabolites by evaluating a library of known targets and unknown significant features. This was accomplished by including initial iterative DDA injections on a pooled sample followed by MS1 acquisition of each individual sample. The MS1 data was evaluated for the sensitive identification of molecular features. Acquiring in both polarities yielded thousands of features. Of those extracted features 1261 and 2101 were accompanied by MS/MS data for negative and positive polarities, respectively. A blank was used for first iteration to maximize the biological relevance of the MS/MS acquired from the plasma iterations. The MS/MS data was leveraged for the more confident identification of compounds assigned from spectral libraries, and also the evaluation of compounds not in the metabolite libraries by direct export to SIRIUS:CSI FingerID.

The plasma iterative and MS1 injections were batched, MS1 features extracted with the matched MS/MS data from iterative injections, normalized using LOESS normalization, and filtered for sufficient variability and frequency. Features were compared statistically for fold change variation, and significant features were compared to spectral libraries and processed with SIRIUS:CSI FingerID for identification.

The batching, feature extraction, normalization, filtering, statistical analysis, and library-based identification can all be accomplished within a single novel software platform. This streamlines the process of a historically difficult workflow extracting rich information on the differential metabolome of untargeted data files.

For negative mode, 1053 features were statistically different by 2-fold between the two sample groups. Of those features 332 were tentatively identified by spectral libraries. Of the 693 remaining features 54 had associated MS/MS data and were exported to SIRIUS:CSI Finger ID to yield 42 tentative identifications and 2 with scores >-20.

CONCLUSION:
A novel software was shown for non-targeted metabolite feature extraction, statistical preparation and identification of differential metabolites between male and female mouse plasma samples. Further, features not identified by available libraries were processed with SIRIUS CSI:FingerID for additional characterization through direct export. This easy additional characterization workflow is enabled by the new integration of MS/MS analysis into the novel software.

Prevalence of Vitamin D2 Toxicity in American Population Ravinder Singh (Presenter) Mayo Clinic

Poster #16c View Map

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

BACKGROUND:
Vitamin D supplementation by human individuals has significantly increased in the world. In US the total stored 25-OH-Vitamin D3 and 25-OH-Vitamin D2 has significantly increased for both in summers and winters. Under sunlight exposure, 7 dehydrocholesterol is converted to Vitamin D3 and is endogenous source in mammals. However, most of the exogenous source for Vitamin D used to be Vitamin D2 which used to be extracted only from mushrooms but is now manufactured by supplement industry as well. In last decade most of the supplementation has been due to chemically synthesized Vitamin D3. Food is now supplemented by both D2 and D3. It is not clear what is the prevalence of D2 supplementation/consumption in American population. Competitive immunoassays do not consistently recover 25-OH-D2 compared to 25-OH-D3 and some antibodies used have variable or no cross reactivity with 25-OH-D2. LC-MS/MS method can easily distinguish between the two and can help in providing the precise and accurate results for 25-OH-D2 and 25-OH-D3. Here we present what is the prevalence of Vitamin D2 consumption and toxicity in American population based on our LC-MS/MS methods.

METHODS:
The study measured vitamin 25-OH-D levels (total, D2, and D3) in 148,654 patients from Mayo Clinic as well as from other medical centers within the U.S. Patients age ranging from new-born babies to over 100 years, and represents Male, Female and Neutral genders. Samples were analyzed for 25-OH-Vitamin D by LC-MS/MS as published earlier. Unlike immunoassays, LC-MS/MS gave us separate results for 25-OH-D3 and 25-OH-D2 with 100 percent specificity.

RESULTS:
25-OH-Vitamin D2 levels of greater than 10 was present in at least 10,358 and ranged from 10 to 205 ng/mL. Of 10,358, 51 percent had 20-50 ng/mL, 10 percent had 51-80 ng/mL, and 1.3 percent had >80 ng/mL, ranging from 81-205 ng/mL with a median of 92 ng/mL. Of the patients found at Total Vitamin D >80 ng/mL, toxic levels of both D2 and D3 were found more in females than males. There was no significant gender difference for Vitamin D3 consumption but for D2 consumption, mean and median levels were different in males and females.

CONCLUSION:
Clinical lab testing should accurately measure both 25-OH-D3 and 25-OH-D2, otherwise the patient’s total Vitamin D values in circulation will be underestimated.

Fast and Robust Phosphoproteomics Sample Prep with AttractSPE® Disks C18 Tips for High Phosphopeptide Recovery and Identification Kaynoush Naraghi (Presenter) Affinisep

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Poster #17c View Map

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

INTRODUCTION
Phosphorylation is one of the most prevalent and important post-translational modifications proteins can undergo. Over 50% of the human proteome is phosphorylated and understanding the dynamic phosphorylation across the proteome can understand the progression of many diseases including cancers. As the stoichiometry of phosphorylation sites is generally very low, enrichment steps are required, followed by SPE clean-up before LC-MS/MS analysis to enhance identification and quantification of each site. However, recovery of phosphopeptides can be greatly affected by the choice of clean-up method, resulting in severe losses.

METHODS
Different SPE C18 options, including SPE Tips and cartridges, were compared for the desalting and purification of phosphopeptides, after automated enrichment using magnetic beads (Ti/Zr-IMAC). Effects of sample acidification prior to SPE clean-up on phosphopeptides detection were also assessed. To do so, the enrichment elution was acidified with different percentages (2, 3, 4 and 5%) of phosphoric acid or trifluoracetic acid (TFA).

RESULTS
Among all SPE options tested, AttractSPE® Disks Tips C18 provided the highest recovery of phosphopeptides (up to 2.4 times more identifications), with high reproducibility (RSD < 10%). Moreover, these SPE Tips captured hydrophilic peptides more efficiently, with smaller phosphopeptides also generally retained compared to other brands, thus leading to higher phosphopeptide identification.

Quenching the enrichment elution with 3% phosphoric acid had the best results, with 8% more phosphosites identifications compared to 5% TFA. In addition, lower acid concentration interestingly provided more singly phosphorylated peptides, while higher acid concentration recovered more hydrophilic peptides. This trend was observed for both acids tested but was more pronounced for TFA.

CONCLUSION
AttractSPE® Disks Tips C18 are shown to be the best choice for phosphopeptide purification, offering simplicity of use by centrifugation, high sample recovery, and robustness. These SPE Tips are easily scalable with their availability in different sizes and binding capacities to perfectly adapt to different sample amounts, and can be provided as 96 and 384 SPE well plates for high throughput processing and fully automated workflows.

The Fibroid Secretome Possesses Distinct Metabolomic Features Compared to Adjacent and Control Myometrium Beth Harrison (Presenter) University of Liverpool

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Poster #18c View Map

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

INTRODUCTION:
The most common gynaecological condition and indication for hysterectomy worldwide is uterine leiomyomas (fibroids). Despite this, the pathophysiology of the condition remains poorly understood. Metabolomic analysis of the fibroid secretome offers novel mechanistic insight and potential identification of a future therapeutic target to reduce the heavy, irregular menstrual bleeding, chronic pelvic pain and infertility associated with the disease.

OBJECTIVES:
Identify significant differences in secreted metabolite abundances between fibroids, control myometrium and adjacent myometrium with future promise of better mechanistic understanding of disease and non-hormonal therapeutic targets.

METHODS:
Fresh surgical fibroid or myometrial samples were placed into basal DMEM medium with primocin and incubated for 24 hours. The resultant conditioned media was rendered acellular by centrifugation (4000 rpm, 4°C, 10 min). Deproteinised media containing deuterated internal standards were analysed using the 1290 Infinity II liquid chromatography system coupled to a 6550-quadrupole time-of-flight mass spectrometry (Agilent, UK). Prior to the analysis of study samples, optimal injection and reconstitution volumes were established. Data scaling by total protein concentration (BCA assay) and total metabolite abundance were compared. Ethical approval was obtained for the collection and usage of samples (LWRTB REC: ethical approval 19/WA/0271 and INTERPRET REC: approval 19/SC/0449).

Raw data was processed using the Agilent MassHunter software suite and Metaboanalyst (Version 6.0). Features were extracted using ±10 ppm theoretical accurate mass and ±0.3 mins retention time window and were filtered based on their frequency (>70%) and variability across quality control samples (CV <30%). An accurate mass, retention time database containing 469 intermediary metabolites (Mw 72-785) was used to facilitate compound identification. Subsequent multivariant analysis using ANOVA (p<0.05 adjusted for FDR), univariant analysis using fold change and two paired t-test (FC 1.2, p<0.05 adjusted for FDR), and analysis of clinicodemographic variables was conducted following data pre-processing. Temporal profiling of the secretome was achieved by comparing time intervals collected and significant metabolite abundances. Iterative MS/MS was performed to aid in metabolite identification.

RESULTS:
129 samples from 48 patients were included in the final study, 19 samples from 2 patients were used for temporal profiling over 17, 24 and 50 h. Optimisation experiments showed that a 5 µL injection and a 50 µL reconstitution volume were superior.

Covariate metadata analysis was performed to assess the contribution of fibroid subtype (by anatomical location), BMI (normal, overweight and obese), ethnicity (Caucasian, Black, Asian), pre-operative haemoglobin (<120 g/L Hb defined as anaemic), hormonal agents, menstrual phase, menopausal status (pre and post) and parity had on metabolite abundances. No demographic data demonstrated distinct metabolic profiles in either polarity.

For targeted secretome analysis, 141 and 121 features were matched in positive and negative polarities respectively using an in-house metabolite database (level 1 identification). Of these matched features, 42(25 positive and 29 negative respectively, with duplicates accounted for), showed a significant change in metabolite abundance across univariant t-test (p<0.05) and multivariant ANOVA (p<0.05) analysis. For univariate analysis, most of the differences in metabolite abundances were shown between fibroid and control myometrium ((13 significant metabolites in positive and 20 in negative (blank contribution<5%)), where metabolite abundance was increased in fibroids relative to control (FC >1.2, no scaling). Most significant metabolites included amino acids and derivatives (including L-proline, L-ornithine, D-alanine), energy metabolism intermediates (including succinic acid, isocitrate, and malic acid), carbohydrates and derivatives (including glycerol, gluconic acid, and raffinose), and nucleotide metabolites (including hypoxanthine, inosine, and uracil).

For untargeted secretome analysis, 5000 features in positive and negative polarities were matched by RT and accurate mass. 2950 positive and 2094 negative features remained after filtering by QC frequency and sample variation. Of these, 252 positive and 335 negative features showed a significant change in metabolite abundance across univariant t-test (p<0.05) and multivariant ANOVA (p<0.05) analysis. For univariate analysis, most of the differences in metabolite abundances were shown between fibroid and control myometrium ((166 significant metabolites in positive and 31 in negative (blank contribution<5%)), where metabolite abundance was increased in fibroid relative to control (FC >1.2, no scaling). Raw data was manually interrogated to assess metabolite peak quality and abundance of the precursor ion, and of which, 35 positive and 101 negative features were confirmed. Metabolite identification is ongoing, including targeted MS/MS experiments to identify unknown significant features.

CONCLUSION:
This is the first study to demonstrate metabolomic differences in the secretome of fibroids compared to both control myometrium and adjacent myometrium using LC-QTOF-MS. We have shown that there are 48 metabolites with significant differences in abundance, that have been named and identified using RT and accurate mass from a reliable database. We have shown that metadata did not correlate with these metabolite differences. Further work is required to name and identify the significant metabolites found using the untargeted workflow, and to perform metabolite enrichment/pathway analysis to understand the biological significance in terms of pathophysiology and as a therapeutic target.

Overcoming the Dynamic Range Difficulties in CSF Samples Through an Optimized Enrichment Step for Biomarker Discovery Studies Cameron Ellis (Presenter) PreOmics

Poster #19c View Map

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

INTRODUCTION:
Cerebrospinal fluid (CSF) is a vital component of the central nervous system, especially important for investigating brain disorders. However, proteomic analysis of CSF by liquid chromatography-mass spectrometry (LC-MS) is challenging due to its low protein concentration and wide dynamic range of protein abundance. ENRICH-iST offers a powerful solution to tackle the difficulties in CSF-based proteomics by efficiently enriching low-abundance proteins onto paramagnetic beads. This enrichment step allows for a significant increase in protein identification and deeper proteome coverage. In this study, we optimized the ENRICH-iST workflow for CSF samples to provide a robust, easy-to-use, and standardized workflow for in-depth proteome analysis.

METHODS:
Rat CSF samples were prepared using the ENRICH-iST kit (PreOmics). To address the challenge of low protein concentration in CSF samples, the enrichment step was optimized by adjusting the starting volumes (20200 µL). For subsequent LC-MS sample preparation, the iST-BCT protocol (PreOmics) was employed for on-bead denaturation, reduction, alkylation, digestion, and peptide cleanup. Peptides were analyzed by nanoElute® 2 coupled to a timsTOF HT instrument (Bruker) using dia-PASEF® acquisition mode. Real-time MS data processing was carried out using ProteoScape™ (Bruker), followed by post-processing in which individual runs were assigned to sub-groups of replicates without match-between-runs.

RESULTS:
Given the low protein concentration of CSF samples (~0.1 mg/mL) compared to plasma or serum (~60–80 mg/mL), the ENRICH step was adapted to ensure effective dynamic range compression. To optimize the workflow, different CSF input volumes were tested. Notably, the ENRICH-iST workflow was successfully applied to CSF volumes ranging from 20 µL to 200 µL, yielding enhanced proteome coverage. The workflow is therefore suitable for a wide range of starting volumes and can be scaled up to the available CSF volume, providing maximum flexibility. For example, processing 300 µL of mouse CSF using ENRICH-iST led to the identification of approximately 1,800 proteins—a 1.8-fold increase over the iST-BCT workflow without enrichment. Importantly, as an antibody-free approach, ENRICH-iST is not limited to human samples and can be readily applied to other mammalian species, making it particularly valuable for preclinical research.

CONCLUSION:
The ENRICH technology offers an innovative approach to overcoming the dynamic range challenge of protein abundance in biological fluids, such as CFS, for proteomic analysis. By allowing the detection of low-abundance proteins while preserving the information about high-abundance proteins, the optimized ENRICH-iST workflow enables in-depth proteome analysis of large CSF cohorts, simplifying and improving biomarker discovery studies.

Steroid Panel LC-MS by Tecan: Exploring Some Essential Issue For Sexual Steroids Determination. Maike Arndt (Presenter) University of Florence

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Poster #20c View Map

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

INTRODUCTION:
Tecan offers the kit “Steroid Panel LC-MS” for the simultaneous determination of 17 steroids and dexamethasone. Interesting features of this kit are the limited volume of serum required (250 μL), the automated and simple sample preparation, and the high sensitivity in particular for sexual steroids testosterone and estradiol with the possibility to also determine estrone. In our laboratory at Andrology, Female Endocrinology and Gender Transition Unit of Azienda Universitaria Ospedaliera Careggi (Florence, Italy). The essential requirements for sexual hormones in which we are interested are high sensitivity and accuracy for testosterone at low concentrations (females and children) and for estradiol at low concentrations (men and postmenopausal women) together with the possibility to quantify estrone (adults). Before adopting this new method, it is imperative to ascertain that the crucial analytical concerns mentioned above can be satisfactorily addressed.

METHODS:
The Steroid Panel LC-MS kit, including the quantitative analysis of 17 steroids and dexamethasone, was used. Calibration range and retention time for all the included steroids are reported. After sample preparation via SPE, 20 μL of the extract was injected into the system. Focus was set on the comparison of estradiol and testosterone on their agreement on external standard material from UK-NEQAS.

RESULTS:
The analytical performance was in line with the internal standard material. We were able to show an accuracy between 80 and 110% for the measurement of standard material from UK-NEQAS, as well as interassay CV% below 8% for the measurement of Estradiol samples.

CONCLUSION
The performance of the Steroid Panel LC-MS met our first goals, especially regarding accuracy for the two main sexual steroids. For validation purposes, precision and accuracy will be evaluated on all the other steroids. Moreover, LoD, LoQ and linearity will be assessed and a study in order to verify the comparability with the existing LDT will be performed in the future on a large number of real samples.

Establishment of Reference Intervals for Eight Steroid Hormones in Korean Adults Using LC-MS/MS: Emphasis on Testosterone and Outlier Detection Methods Hyung-Doo Park (Presenter) Samsung Medical Center

Poster #21c View Map

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

BACKGROUND:
Liquid chromatography–tandem mass spectrometry (LC–MS/MS) is the most accurate and precise method for determining steroid hormone concentrations. Simultaneous measurement of endogenous steroid hormones using LC–MS/MS, which is less affected by cross-reactivity, may offer advantages over conventional immunoassays. However, no study has yet investigated the reference intervals and distribution of sex steroid hormones, including testosterone, in the Korean adult population using LC–MS/MS.

METHODS:
We established reference intervals for eight steroid hormones—cortisol, cortisone, corticosterone, 11-deoxycortisol, androstenedione, 17-hydroxyprogesterone, progesterone, and testosterone—in 111 Korean females and 113 males aged 18–49 years. Outlier detection was performed using both Tukey’s fence criteria and the Reed–Dixon method. Reference intervals stratified by sex and age were determined using a robust method following exclusion of outliers identified by Tukey’s fence. These intervals were compared with those derived using a nonparametric approach.

RESULTS:
Among the eight steroid hormones, testosterone and androstenedione showed significant sex differences. Testosterone levels also decreased with age in both sexes. Depending on the hormone, the proportion of outliers identified by Tukey’s fence ranged from 0% to 17.6%, whereas the Reed–Dixon method rarely detected extreme outliers. Comparison of outlier detection methods revealed a significant difference in the lower reference limit for testosterone in males when using the robust method. The reference intervals derived using Tukey’s fence were generally consistent with previously reported values in Caucasian populations. The lower limit of testosterone in Korean males aged 18–49 years was 7.69 nmol/L, which falls within the range of values reported for harmonized reference intervals in European and American male cohorts aged 19–39 years (8.02 nmol/L) and 40–49 years (7.28 nmol/L). A total of 12.0% of our subjects had testosterone concentrations below 10.4 nmol/L (300 ng/dL), which is the cut-off value for low testosterone according to the American Urological Association (AUA) guidelines. Compared to the 10th percentile values in European and American male cohorts (ranging from 11.13 to 9.91 nmol/L), 10.4 nmol/L appears to be an appropriate cut-off value for defining low testosterone in Korean males. In contrast, the nonparametric method with Reed–Dixon outlier detection yielded a lower limit of 0.80 nmol/L for testosterone, a value that would be considered pathologically low in adult males.

CONCLUSIONS:
Our study highlights the importance of careful selection of the reference population, outlier detection method, and statistical approach when establishing reference intervals for steroid hormones. Additionally, clinical decision limits should be considered to avoid generating reference intervals that are inconsistent with clinical practice or biological plausibility.

Lipid Dysregulation in an ALS Drosophila Model: Insights from Time-of-Flight Secondary Ion Mass Spectrometry Hyunkyong Shon (Presenter) Korea Research Institute of Standards and Science

Poster #22c View Map

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

INTRODUCTION:
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron loss. The expansion of GGGGCC (G4C2) hexanucleotide repeats in the C9orf72 gene is the most common genetic cause of ALS. Lipid metabolism dysfunction has been implicated in ALS pathogenesis, but its early-stage dynamics remain poorly understood. This study applies time-of-flight secondary ion mass spectrometry (ToF-SIMS) to an ALS Drosophila model to investigate lipid alterations and their role in disease progression.

OBJECTIVES: This study aims to analyze the spatiotemporal changes in lipid composition in the brains of ALS Drosophila models. Additionally, we seek to determine whether specific lipid dysregulations occur before the onset of motor dysfunction. Furthermore, we aim to identify genetic factors related to lipid metabolism that contribute to ALS pathology.

METHODS: Drosophila expressing (G4C2)36 repeats in the C9orf72 gene were used as an ALS model. ToF-SIMS was employed to map lipid distributions in Drosophila heads at different disease stages (days 1, 3, 5, and 9). Principal component analysis (PCA) was conducted to identify key lipid alterations. Genetic screening using RNAi knockdown of lipid regulatory genes was performed to assess their effects on lipid profiles and neurodegeneration.

RESULTS: ToF-SIMS analysis revealed a significant increase in fatty acids, triacylglycerols, and ceramides in ALS model flies by day 5, preceding observable motor dysfunction at day 10. These lipids accumulated primarily in the fat body regions surrounding the brain. Genetic screening identified FATP1 and ACBP as key regulators of lipid alterations, with their knockdown reducing retinal degeneration and restoring lipid homeostasis. PCA confirmed that FATP1 and ACBP knockdowns resulted in lipid profiles similar to control flies, suggesting their role in ALS pathology.

CONCLUSION: Our study demonstrates that lipid dysregulation occurs early in ALS progression, prior to clinical symptoms. ToF-SIMS provides high-resolution spatial analysis of lipid alterations, revealing potential biomarkers for early-stage ALS. FATP1 and ACBP emerge as key contributors to lipid dysregulation, offering new targets for therapeutic intervention. These findings enhance our understanding of ALS pathogenesis and highlight the importance of lipid metabolism in neurodegeneration.

Nanotrap Protein Enrichment Affinity Kit Enhance Proteome Coverage and Deplete High-Abundance Proteins Ben Lepene (Presenter) Ceres Nanosciences, Inc.

Poster #23c View Map

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

BACKGROUND:
Complex biofluids such as human plasma encompass a concentration range of over ten orders of magnitude, with albumin alone comprising approximately 50% of total protein mass, thereby masking lower-abundance biomarkers during LC-MS/MS analysis. Traditional depletion approaches often require extensive handling, large volumes, or specialized instrumentation, limiting throughput and reproducibility. The Nanotrap® Protein Enrichment Affinity Kit (PEAK) addresses these challenges by employing three proprietary magnetic hydrogel particle chemistries (Nanotrap® Protein Particles A, Nanotrap® Protein B, and Nanotrap® Protein C) to selectively capture, concentrate, and release target proteins and peptides while concurrently reducing the presence of high abundance proteins all within a single 45-minute workflow. This universal enrichment platform is compatible with multiple commercial digestion chemistries—including Promega Rapid-Digest Trypsin/Lys-C, Pierce In-Solution Tryptic Digestion and Guanidination Kit, PreOmics® iST 8X Kit, and Thermo Scientific™ SMART Digest™ Trypsin Kit—without requiring instrument-specific hardware.

METHODS:
K2EDTA human plasma samples were thawed and centrifuged briefly using low-speed centrifugation. 50 µL plasma samples were diluted four-fold in Nanotrap® Buffer 4. Samples were then incubated with Nanotrap Protein A, B, and C Particles according to the Combined-, 2-, or 3-Particle methods for 30 minutes at room temperature with gentle agitation. After magnetic separation and two washes with HPLC-grade water, bound proteins were chemically reduced (TCEP) and alkylated with iodoacetamide before proteolysis. Four digestion workflows were evaluated in parallel: Promega Rapid-Digest Trypsin/Lys-C (2 hours at 70°C), Pierce™ In-Solution Tryptic Digestion and Guanidination, PreOmics iST 8X Kit with on-column cleanup, and Thermo Scientific SMART Digest Trypsin Kit with magnetic bulk resin; each required ≤45 min of additional processing. Peptides were desalted using ZipTip™. LC-MS/MS analysis was performed on a Thermo Scientific™ Orbitrap Exploris™ 480 in data-dependent acquisition mode.

RESULTS:
Integration of Nanotrap® PEAK with each digestion chemistry yielded substantial improvements in proteome depth compared to unenriched controls. Using the Promega Rapid-Digest workflow, enrichment delivered 2.5- to 4.0-fold increases in unique protein identifications, with the 3-Particle Method consistently achieving the highest coverage. Experiments with the Pierce In-Solution kit demonstrated between 2.2- to 4.0-fold improvements across the Combined, 2-, and 3-Particle methods. The Thermo SMART Digest workflow yielded 1.1- to 2.4-fold enhancements relative to neat plasma, identifying a total of 1,058 unique proteins with a single-particle enrichment strategy. PreOmics iST workflows corroborated these trends, producing 2.0- to 4.3-fold gains across gradient lengths and digestion formats. Crucially, the Combined Particle Method depleted albumin to <3 % of total peptide signal—corresponding to >97 % removal—enabling enhanced detection of low-abundance analytes. Gel-based evaluation via SYPRO Ruby staining confirmed robust exclusion of the 65 kDa albumin band by each particle chemistry while preserving a diverse range of protein profiles. Technical reproducibility of identified protein groups was assessed across six biological replicates. Unenriched plasma exhibited a median CV of 13% across replicates, whereas manual Nanotrap PEAK protocols produced a median CV of 8.4% and semi-automated enrichment on the KingFisher™ Apex System achieved a median CV of 2.3%. Rank-abundance and dynamic range analyses further demonstrated that both manual and automated workflows extended protein detection into lower-abundance ranges and improved proteome coverage.

CONCLUSION:
Nanotrap PEAK enables highly reproducible protein enrichment from human plasma while effectively depleting high-abundance proteins such as albumin, resulting in significantly improved proteome coverage. Across multiple workflows and digestion chemistries, Nanotrap PEAK demonstrated consistent albumin depletion (>97%) and enhanced detection of low-abundance proteins, enabling up to a 4.3-fold increase in unique protein identifications. The platform showed excellent technical reproducibility, with median coefficients of variation as low as 2.3% using semi-automated protocols. Nanotrap PEAK’s compatibility with diverse LC-MS/MS platforms and digestion kits, combined with rapid processing and scalable formats – makes it a powerful tool for biomarker discovery and quantitative plasma proteomics.

Differentiation of Basal Cell Carcinoma From Benign Skin Using Reims: Towards Intraoperative Assessment of Surgical Margins During Skin Cancer Excision Martin Kaufmann (Presenter) Queen’s University

Poster #24c View Map

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

INTRODUCTION
Basal cell carcinoma (BCC) is the most common type of cancer. Typical management of BCC involves resection of the apparent tumor, followed by histopathology assessment of the surgical resection margins. Histopathology results are typically available within days following surgery. In up to 10% of cases, pathology findings reveal that the tumor is incompletely excised (known as a positive margin), which requires a second surgery to remove any remaining cancerous tissue. Positive margin cases exhibit a high recurrence rate (26%), and re-excisions increase the risk of poor cosmetic outcomes. Although the rate of positive margins for BCC surgery is relatively low, the large and increasing number of BCC cases worldwide translates into significant healthcare costs associated with re-excisions, in addition to complications arising from poor cosmetic outcomes. Tissue profiling tools such as rapid evaporative ionization mass spectrometry (REIMS) have the potential to alert the surgeon to any abnormal tissue encroached upon during surgery. The surgeon may then opt to take a wider excision that could potentially avoid a positive margin as well as the need for a second surgery.

OBJECTIVE
To assess the intraoperative potential of REIMS in BCC surgery, we studied the ability of REIMS methodology to differentiate between BCC and non-cancerous tissues typically dissected during BCC excision, using fresh ex vivo skin specimens.

METHODS
A mobile REIMS unit was placed within an out-patient clinic at Kingston Health Sciences Centre, where patient participants underwent standard-of-care surgical treatment for biopsy-confirmed BCC. Over a series of 4 clinics, 13 resection specimens were obtained from 9 patients. Specimens were cross-sectioned, and sampled by REIMS immediately after surgery. A pathologist performed REIMS point-burns using a fine-tip cautery on regions observed to contain BCC or non-cancerous epidermis, dermis or adipose. Specimens were formalin fixed and paraffin-embedded, sectioned, and stained with HPS. Mass spectra arising from REIMS point burns were assigned tissue-type labels based on histopathology annotations of the tissue surrounding each point burn. A total of 225 spectra were acquired, however 76 were excluded on the basis of poor spectral quality, the likelihood of tissue type mixtures within a point burn, and inconclusive presence of BCC. A total of 149 labeled spectra were selected for analysis, including: BCC-n=42, epidermis-n=31, dermis-n=27 and adipose-n=49. Spectra were binned using 0.01 Da bin widths, mass shift corrected, background subtracted, and normalized by total ion current; prior to creation of multivariate models based on PCA followed by LDA, using Abstract Model Builder (Waters).

RESULTS
A binary PCA/LDA model using the m/z 50-1000 range exhibited a 97% accuracy for classifying BCC and all other non-cancerous tissues treated as a single class, based on 5-fold cross-validation; or 94% when using leave-one-patient-out cross validation. Cross-validation of a 4-class model where each tissue type was treated as a separate class, also resulted in promising accuracies of 93% and 89% respectively. Slightly lower accuracies of 95% and 91% were observed using 5-fold cross-validation, when only the m/z range of 600-1000 was considered. Classification failures in binary models involved BCC being misclassified as dermis, and vice versa. Misclassification of adipose among non-cancerous tissue types contributed to reduced accuracy of multi-class models. In addition, certain spectra obtained from adipose were determined to be outliers. Features that differentiated adipose from epithelial tissue types included m/z 893.75 (TG(52:2)); and features that differentiated epithelial tissues from each other include m/z 647.45 (elevated in BCC), 699.45 and 742.55 (PE(36:2),elevated in BCC and epidermis). Variation in the relative amount of triglycerides as compared with phospholipids in adipose-labeled spectra may have contributed to the frequency of misclassifications and outliers involving the adipose-labeled spectra; presumably arising from different amounts of subcutaneous fat across patients, as well as anatomical location of the lesion.

CONCLUSIONS
Based on a limited number of BCC resection specimens, our results reveal that REIMS can be used to differentiate BCC from non-cancerous skin. Although we targeted three cell type classes that are all epithelial, REIMS was able to differentiate the various sublayers of skin on the basis of complex lipid and small molecule profiles. Importantly, our sampling methodology and detailed histopathology annotation optimized for small specimens, was critical for maximizing spectral purity and minimizing spectral labelling error. BCC has several advantages as a case-use for studying the clinical utility of REIMS: (1) There is no shortage of samples as BCC is common (2) BCC excision surgery is rapid, enabling access to a large number of specimens within a short time, for creation of robust tissue recognition models, and to eventually facilitate intraoperative testing and (3) BCC is a low risk cancer that rarely metastasizes, so pathologists are able provide access to large proportions of clinically-relevant excision specimens for REIMS sampling without compromising clinical diagnostics. We are currently expanding the number of pathology-annotated specimens for model development, and evaluating the performance of models on spectra acquired intraoperatively during BCC excision surgery.

Chemical Metabolomics – Innovative Chemical Biology Approaches to Investigate Gut Microbiome Metabolism Wawrzyniec Haberek (Presenter) Uppsala University

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Poster #25c View Map

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

INTRODUCTION:
The metabolism of the gut microbiota plays a critical role in shaping human physiology. Disruptions in this metabolic network have been associated with the onset and progression of various diseases. Despite its significance, the metabolic interactions between host and microbiome remain underexplored. Mass spectrometry (MS) is the gold standard for analyzing microbiome-derived metabolites due to its ability to resolve complex biological matrices such as plasma, feces, and urine. Profiling microbial metabolites holds promise for the discovery of novel bioactive compounds, disease biomarkers, and deeper insights into pathophysiological mechanisms.

OBJECTIVES:
A major limitation in MS-based metabolomics is the poor ionization efficiency of certain metabolite classes. This challenge underlines the need for new methodologies that enhance detection and broaden metabolite coverage. Our objective is to develop innovative chemical biology tools capable of identifying previously undetectable metabolites.

METHODS:
We have developed a suite of chemoselective probes tailored to derivatize specific functional groups of metabolites. These probes are combined with 13C/12C isotope labeling, enabling accurate quantification and comparative analysis. This platform, termed quantitative Sensitive CHEmoselective MetAbolomics (quant-SCHEMA), incorporates magnetic bead-assisted extraction for targeted isolation of metabolite classes. The method is compatible with various biological sample types and significantly enhances MS sensitivity, enabling detection of metabolites at attomole levels.

RESULTS:
Our chemoselective strategies enable the targeted analysis of several metabolite classes, including carbonyls, thiols, amines, and carboxylic acids. Using these tools, we confirmed the presence of clinically relevant, microbiota-derived metabolites in human samples. In a dietary intervention study involving 156 samples, we identified four novel food-derived biomarkers. These findings validate the robustness of our approach. We are now integrating quant-SCHEMA with standard metabolomics workflows in neuroscience, microbiome research, and biomarker discovery.

CONCLUSION:
We have successfully developed and implemented a powerful set of chemoselective tools for exploring host–microbiome metabolic interactions. To date, our work has led to the identification of over 300 previously unknown metabolites, the majority of which originate from gut microbial metabolism. Current efforts focus on characterizing the biological activity and functional roles of these newly discovered compounds

RERERENCES:
(1) Lin W et al., Angew. Chem. Int. Ed. 2021, 60, 23232.
(2) Kaur A et al., Chem. Sci. 2023, 14, 5291.
(3) Garg N et al., Angew. Chem. Int. Ed. 2018, 57, 13805.
(4) Lin W et al., Chem. Commun. 2023, 59, 5843.
(5) Conway L. P. et al., Chem. Commun. 2019, 55, 9080.

Spatial Multi-Omics Using DESI Imaging-Guided Laser Microdissection for LC-MS/MS Proteomics Brittannie Willis (Presenter) Imperial College London

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Poster #26c View Map

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

INTRODUCTION:
Glioblastoma (GBM) is a highly aggressive and treatment-resistant brain cancer with a poor prognosis, presenting significant challenges to effective treatment despite interventions including surgery, radiation, and chemotherapy. The tumour&#039;s genetic mutations and immune microenvironment contribute to treatment resistance, further complicating therapeutic strategies. To improve clinical outcomes, it is critical to understand GBM&#039;s molecular heterogeneity in response to treatment and explore innovative metabolic therapeutic strategies, such as arginine deprivation.

To understand drug responses in GBM, it is essential to consider the spatial context of proteins and metabolites within the tumour and its microenvironment. A powerful approach is the ability to capture metabolomic and proteomic data from the same tissue section. Mass spectrometry imaging (MSI) techniques, such as matrix-assisted laser desorption/ionisation (MALDI-MSI) and desorption electrospray ionisation mass spectrometry imaging (DESI-MSI), are valuable tools for metabolomic and lipidomic analyses. However, they do not provide the same depth of proteomic coverage as liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Furthermore, spatial multi-omics approaches often face challenges, such as poor protein recovery due to laser ablation or interference from embedding matrices, leading to biases in spatial representation. Additionally, many workflows often require consecutive tissue sections, which introduces variability and may cause loss of spatial context. To address these limitations, we have developed a spatially resolved multi-omics pipeline that integrates DESI-MSI for metabolomic imaging with LC-MS/MS-based quantitative proteomics from a single tissue section.

OBJECTIVE:
To develop a spatially resolved multi-omics pipeline that integrates DESI imaging-guided laser microdissection with LC-MS/MS proteomics, enabling enhanced molecular profiling from a single tissue section while preserving spatial context.

METHODS:
Fresh-frozen mouse brain tissue was sectioned at 8 &mu;m thickness and analysed using DESI-MSI on a XEVO-G2-XS-QTOF mass spectrometer (Waters) to acquire spatial metabolomic profiles. Data were acquired in both positive and negative ion modes, with a spatial resolution of 50&ndash;100 &mu;m. Data processing was performed using an in-house MATLAB pipeline for spectral preprocessing and peak picking. Regions of interest (ROIs) were identified based on high metabolomic intensities and resected using laser microdissection (Leica LMD7).

Proteins from these resected ROIs were extracted, digested using trypsin, and analysed via LC-MS/MS on a ZenoTOF 7600 mass spectrometer (SCIEX). Peptides were separated using a 2.6 &mu;m Kinetex XB-C18 100 Column, 150 &times; 0.3 mm (Phenomenex). Both DESI-MSI and LC-MS/MS conditions were optimised to maximise protein recovery while preserving spatial integrity. Database searching was performed using DIA-NN (version 1.8.1) and FragPipe (version 21.1) for protein identification and quantification.

RESULTS:
Our pipeline successfully combines spatial metabolomics and proteomics, preserving sample integrity and enhancing reproducibility. Over 1,100 proteins were identified with high sensitivity and spatial resolution within 10 &times; 10 &mu;m ROIs, enabling detailed and comprehensive molecular profiling. By eliminating the need for consecutive tissue sections, variability was minimised, allowing for direct comparisons between metabolic and proteomic data. Additionally, optimised DESI-MSI conditions ensured maximal protein recovery, supporting accurate downstream analyses and a deeper understanding of molecular processes.

CONCLUSION:
In conclusion, we demonstrate the basis for a spatially resolved multi-omics workflow that integrates DESI-MSI and LC-MS/MS, enabling high-resolution mapping of both metabolites and proteins from a single tissue section. This approach overcomes the limitations of traditional methods by maximising data extraction while maintaining the spatial resolution, making it particularly advantageous for studies involving rare or limited clinical samples.

Using mouse brain tissue as a model, we highlight the potential of our pipeline to uncover detailed differences in tissue composition through region-specific molecular profiling. This method offers a robust, quantitative approach for tissue profiling, with strong potential for advancing our understanding of therapy response and biomarker discovery. Our approach enables relative quantification by scaling spatial signals against a bulk reference channel and has demonstrated sensitivity in detecting 10 &times; 10 &micro;m ROIs, ensuring robust and reproducible results. We believe that this workflow has the potential for widespread application in both research and clinical settings, facilitating the broader adoption of spatial multi-omics across diverse tissue types and disease models.

Quantitative and Structural Characterization of Lipid Mediators by High-Resolution Mass Spectrometry Robert Di Lorenzo (Presenter) SCIEX

Poster #27c View Map

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

INTRODUCTION:
Lipid mediators are cell-derived signaling molecules that modulate numerous biological functions. Of note, they play a significant role in all stages of inflammation, including its initiation, propagation, and resolution. Consequently, this class of molecules and their biochemical pathways are attractive candidates for anti-inflammatory drug development. Lipid mediators are potent signaling molecules whose endogenous concentrations are in the pM to nM range, making them a challenge to analyze. Typically, these molecules are measured with highly sensitive triple quadrupole mass spectrometers using multiple reaction monitoring (MRM) scan modes. However, the recent advent of an ultra-sensitive high-resolution mass spectrometer (HRMS) now enables accurate and precise measurements of lipid mediators in vivo while simultaneously providing critical structural characterization.

The ZenoTOF 7600 system is an HRMS instrument capable of measuring the low endogenous concentrations of many lipid mediators with a sensitivity level rivaling that of high-end triple quadrupole instruments. In addition to traditional fragmentation by collision-induced dissociation (CID), the instrument has a complementary fragmentation mode, electron-activated dissociation (EAD), that provides diagnostic fragment ions for the structural characterization of singly charged small molecules. Lipid mediators are derived from a limited number of precursor fatty acids. They are distinguished not necessarily by mass but by the location of their hydroxyl functional group(s), double bonds, and, potentially, carbon rings. Consequently, many of these compounds are isomeric at the precursor and the CID-based product ion levels; hence, the identification and quantitation of these compounds have relied heavily on careful chromatographic separation to achieve isomeric resolution. The ZenoTOF 7600 instrument, however, has a tunable electron beam to produce EAD-based fragments that can distinguish lipid mediator isomers.

OBJECTIVES:
The objective of this study was to quantify and fully characterize lipid mediators and other small-molecule metabolites using both CID- and EAD-based fragmentation with the SCIEX ZenoTOF 7600 system. In particular, the use of EAD to achieve specific structural identification of lipid molecular species in simple and complex matrices was investigated.

METHODS:
Rats were subjected to an ischemic stroke (MCAO model) according to an institutionally approved protocol. Plasma samples were taken after 24 hours and extracted using a solid phase extraction (SPE) column. Extracts were reconstituted in 100 µL MeOH and stored at -20oC until analysis.

Samples were injected on a ExionLC system using a Phenomenex Kinetex C18 column for chromatographic separation. Mobile phases were (A) 0.1% acetic acid in water and (B) 0.1% acetic acid in 84:16 (v/v) acetonitrile/ methanol. Gradient conditions were used for a total run time of 21 min. The injection volume was 10 µL, column oven was set to 60oC and flow rate was 0.4 mL/min. Analysis was performed on the ZenoTOF 7600 system with OptiFlow Turbo V ion source using scheduled, high-resolution multiple reaction monitoring (MRMHR) scan mode. Samples were acquired using both CID and EAD fragmentation.

RESULTS:
The high sensitivity of the ZenoTOF 7600 was able to detect endogenous levels of the lipid mediators in the control and ischemic stroke sample sets. For example, the relative changes in the 12,13-EpOME concentrations resulting from ischemic stroke were observed. In addition, since full product ion spectra were simultaneously acquired, MS/MS spectral library matching was used to provide additional compound detection confirmation.

The MarkerView software was used to evaluate differences between the two sample sets. PCA analysis of the endogenous concentrations between healthy and ischemic rats showed significant differences in some lipid mediators due to ischemic stroke. The expense and difficulty in acquiring authentic standards for each lipid mediator prohibited absolute quantitation for each lipid mediator; however, using a single deuterated standard, as was done in these experiments, enabled relative quantitation between multiple sample sets. These data were sufficient to identify lipid mediators that changed in response to the treatment and can be used to direct targeted efforts with the appropriate primary reference and internal standards.

The EAD fragmentation mode of the ZenoTOF 7600 system was used to produce unique fragments that were used to distinguish between isomeric compounds. For example, the prostaglandins, PGE2 and PGD2, have the same precursor mass (m/z 397.2 Da) and coeluted during the chromatographic gradient. Using CID-formed fragments – m/z 189 Da for PGE2 and m/z 233 Da for PGD2 – the two compounds could not be fully distinguished. However, the EAD fragmentation mode produced several unique fragments, such as m/z 227.067 Da for PGE2 and m/z 215.066 Da for PGD2. The two compounds could be fully distinguished using the EAD-formed fragments.

CONCLUSIONS:
In summary, the ZenoTOF 7600 system was used to measure approximately 90 different lipid mediators in vivo with a sensitivity like that of a high-end triple quadrupole mass spectrometry system. An added benefit to high-resolution quantitative analysis is the ability to collect an accurate mass product ion spectrum that can be used for structural characterization and confirmation. In particular, the EAD fragmentation mode was used to distinguish between isomeric compounds.

Label-Free Quantitative Proteomics With High Precision And Accuracy, Driven By Ultra-High Sensitivity MS/MS David Colquhoun (Presenter) SCIEX

Poster #28c View Map

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

INTRODUCTION:
The ability to identify protein biomarkers and quantify them with high precision and accuracy is critical to proteomics research. Data-independent acquisition (DIA) workflows using high-resolution mass spectrometry (HRMS) have become highly utilized for proteomics research, providing qualitative and quantitative information on proteins and their impact on biological pathways. DIA methods are well-suited to label-free quantitation (LFQ) workflows, which have several advantages over other protein/peptide labeling techniques. However, MS sensitivity and quantitative dynamic range often limit the quantitative analysis of low-abundance proteins and peptides. This work demonstrates the impact of improved MS sensitivity on LFQ proteomics workflows using Zeno SWATH DIA on a novel QTOF system.

METHODS:
Sample analysis was performed using a Waters M-Class HPLC system coupled to a novel quadrupole time-of-flight (QTOF) system. Commercial tryptic digests (human K562, yeast and E. coli) were mixed in different ratios/dilutions and analyzed using microflow reverse-phase chromatography, with various separation gradient lengths. LFQ analysis was done using Zeno SWATH DIA and the resulting data were processed using PEAKS Studio software (BSI Inc). The precision and accuracy of the LFQ analysis at the protein and peptide levels were measured for the varied species ratios and dilutions.

RESULTS:
The MS/MS sensitivity of the novel QTOF platform was approximately 5x higher than that of its predecessor platform. The impact of this sensitivity gain is demonstrated by the number of quantitative identifications of peptides in proteins in the various LFQ mixtures. The novel QTOF system demonstrated higher precision and accuracy for the quantitation of protein ratios in the different human/yeast/E. coli mixtures, particularly for injections of low levels of sample. These results underscore the importance of sensitivity for Zeno SWATH DIA when used for quantitative proteomics research.

CONCLUSIONS:
Using IonOpticks nanoflow chromatography, Zeno SWATH DIA on the ZenoTOF 8600 system, and data processing using PEAKS Studio software, deep proteome coverage and high rates of protein group and precursor identifications were observed in the different HYE mixtures over a range of on-column loadings. A high degree of LFQ precision and accuracy was demonstrated for all HYE mixtures and across all on-column loadings.

REFERENCES:
1. Van Puyvelde, B., et al. (2022). A comprehensive LFQ benchmark dataset on modern acquisition strategies in proteomics. Sci Data 9(1):126.
2. Flexibility, speed and throughput for high proteome coverage using Zeno SWATH data-independent acquisition (DIA) coupled with the Evosep One system. SCIEX technical note, RUO-MKT-02-15461.

Monitoring UDCA and Other Bile Acids in Intrahepatic Cholestasis of Pregnancy May Improve Clinical Outcomes Kat Iacob (Presenter) ARUP Institute for Clinical and Experimental Pathology

Poster #29c View Map

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

BACKGROUND:
Intrahepatic cholestasis of pregnancy (ICP) is a condition which is characterized by elevated levels of serum bile acids (BAs) in mothers. High serum BAs in pregnancy are associated with adverse perinatal outcomes, including preterm birth and stillbirth. Ursodeoxycholic acid (UDCA), a bile acid with anticholestatic effect, is widely used to treat ICP by lowering BA concentrations and is considered safe. However, its efficacy remains controversial as several studies have shown no significant change in total BAs (TBA) and uncertain clinical benefits. Since TBA includes UDCA, the effect of UDCA supplementation on other BAs may be hidden by the assessment of TBA only. The goal of this study was to evaluate changes in concentrations of various bile acids after UDCA treatment in women with ICP.

METHODS:
Retrospective data analysis was performed on bile acid results from de-identified patients, obtained by LC-MS/MS analysis of patient serum. The analysis included 12 individual BAs, total (unconjugated, glyco (G)- and tauro (T)-conjugated) concentrations of cholic (tCA), chenodeoxycholic (tCDC), deoxycholic (tDCA), and ursodeoxycholic (tUDC) acids, TBA and non-UDC BAs (TBA - tUDC). A total of 315 samples from 196 patients (mean age 31+/-3 yo) with elevated TBA (&gt;7.0 &mu;mol/L) and ICD10 diagnostic codes indicating liver/biliary tract disorders in pregnancy were selected for the study. All patients had at least one bile acid measurement before and after UDCA treatment.

RESULTS:
High pre-treatment serum concentrations were driven mostly by glyco- and tauro-conjugated BAs, especially by GCA and TCA. A strong positive correlation (Spearman coefficient &gt;0.70) was observed between TCA and GCA, TDCA and GDCA, TCA and TCDCA. Levels of unconjugated BAs were 5-50x lower than conjugated BAs with only traces of tUDC being detected. Following UDCA treatment, normalization of pre-treatment levels of tCA, tCDC or tDCA occurred in 36%, 55% and 61% of samples, respectively. Average TBA was comparable before and on UDCA supplementation, partly due to an increase in tUDC (mean 10.7&mu;mol/L; range 1.1-75 &mu;mol/L). The biggest change in response to UDCA was observed in glyco- and tauro-conjugated BAs. Paradoxically, post-treatment reduction in non-UDC BAs was associated with lower serum concentrations of tUDC. When tUDC was &lt;10 &mu;mol/L, there was a net-decrease of 24% for glyco-conjugated BAs and of 33% for tauro-conjugated BAs. When tUDC &gt;20 &mu;mol/L, 64% of samples experienced increases in non-UDC BAs and 84% increases in TBA. The concentrations of GCA, GCDCA, GDCA and TCA exceeded their pre-treatment levels by 154%, 172%, 159% and 113% on average. Further analysis of these samples also revealed strong positive correlations between TUDCA and non-UDCA tauro-conjugated BAs as well as between GUDCA and GCDCA, suggesting a potential pro-cholestatic effect of high UDCA concentrations.

CONCLUSION:
In patients with ICP on UDCA treatment, low serum concentrations of tUDC were associated with a significant decrease in individual BA, tCA, tCDC and tDCA levels. However, the beneficial effect was less pronounced or even reversed with higher serum concentrations of tUDC. Therefore, changes in individual BAs and tUDC should be closely monitored during UDCA treatment. This would allow clinicians to finetune UDCA treatment in ICP for improved health outcomes. Clinical studies are necessary to define optimal serum levels of UDCA in order to achieve the best benefits without inducing undesirable toxicity.

VOC-Based Microbial Detection and Growth Phase Classification Using PTR-MS and Machine Learning Lauren Aufdembrink (Presenter) PureBioX

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Poster #30c View Map

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

INTRODUCTION:
Timely detection of microbial contamination is essential in healthcare, diagnostics, and industrial product monitoring. Conventional methods are labor-intensive and slow. This study demonstrates a rapid, non-invasive approach for identifying and quantifying bacterial growth by coupling real-time volatile organic compound (VOC) profiling with machine learning classification. The strategy was validated across multiple bacterial strains, media formulations, and treatment conditions.

METHODS:
Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida were cultured in TSA media with nutritional enhancements (tryptophan, betaine, vitamin B12, trace metals, vitamin mixes, glucose). Headspace VOCs were analyzed at defined timepoints (0–24 h) using proton transfer reaction mass spectrometry (PTR-MS) and ion intensity profiles were analyzed. Random Forest models were developed using ion intensity data to classify samples across incubation periods. Principal component analysis (PCA) was used to visualize temporal evolution of VOC patterns.

RESULTS:
Ion intensity profiles were unique to each bacterium. Bacterial cultures grown in nutritionally enhanced TSA media exhibited significantly amplified VOC emissions compared to standard TSA, with up to threefold increases in signal intensity. Early VOC markers, including indole (m/z 118), were detectable from E. coli cultures as early as 2–3 hours post-inoculation. These enhanced emissions enabled early differentiation of bacterial activity using mass spectrometry. Principal component analysis (PCA) of PTR-MS data revealed clear temporal separation of samples, with progressive shifts in VOC profiles corresponding to bacterial growth phases. Random Forest machine learning models trained on these data achieved 100% test accuracy at high inoculum levels (Log 8 CFU/mL) and 98% accuracy at low inoculum levels (Log 3 CFU/mL). Even closely spaced early time points (3–5–8 hours) were successfully distinguished, highlighting the model’s robustness. These findings confirm that TSA media, when supplemented with tryptophan, betaine, and vitamin B12, supports rapid bacterial metabolic activity and enhances the detectability and temporal resolution of VOC-based microbial monitoring.

CONCLUSION:
VOC-based profiling enables early differentiation of bacterial activity and response to media modifications through real-time, non-invasive monitoring. This method specifically depends on live, metabolically active bacteria to produce volatile metabolites, which are captured via direct headspace analysis without the need for sample pre-treatment. The approach offers a powerful alternative to traditional culture-based methods, with potential for rapid diagnostics, contamination monitoring, and quality control. Ongoing efforts are focused on expanding the VOC reference library across diverse microbial species and improving signal normalization to enhance reliability and applicability in complex, real-world settings.

Drugs of Abuse in Urine Analysis Using High-Speed Acoustic Ejection High-Resolution Mass Spectrometry Pierre Negri (Presenter) SCIEX

Poster #31c View Map

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

INTRODUCTION:
Drug abuse testing in urine specimens is conventionally performed by commercially available FDA-approved immunoassays. Although immunoassays provide a quick way to determine the presence of a drug, the cross-reactivity of structurally similar compounds lowers assay sensitivity and increases the rates of false positive or false negative results. Alternate chromatographic techniques, such as GC-MS or LC-MS/MS, are therefore often utilized as secondary confirmatory tests. However, both techniques are inefficient because they require long run times. Here, we show a rapid, efficient, and highly specific method to analyze drugs in urine using Acoustic Ejection Mass Spectrometry on an Echo® MS+ system and the ZenoTOF 7600 system.

OBJECTIVES:
The objective of this study was to develop a fast method for sample preparation and the rapid analysis of 15 drugs of abuse in urine using acoustic ejection high-resolution mass spectrometry.

METHODS:
The accuracy and precision of our methods are demonstrated by establishing seven calibrators for 15 drugs in synthetic urine, with concentrations ranging from 10 to 1000 ng/mL. These calibrants were prepared by serial dilutions using the Biomek i7 liquid handler. Quality control samples in human drug-free urine were prepared at three different concentrations using the liquid handler. Blank samples were also included and run between calibrators. Solid-phase extraction (SPE) was conducted on these samples using the Phenomenex plates and the liquid handler, and the samples were analyzed using the EchoMS+ system and the ZenoTOF 7600 system. Data was acquired using TOF MS and MRM HR experiments in positive and negative ionization modes. Data review and processing were performed using SCIEX OS.

RESULTS:
Linear data was obtained for the calibration curves with R>0.99, and accuracies were within 20% of the expected values. These results confirm the precision and accuracy of our methods, which are crucial for reliable drug analysis in urine. The carrier solvent, flow rate, and ejection volume were optimized to establish the appropriate system parameters. Tuning the delay interval achieved baseline resolution between peaks. Seven-point calibration curves were constructed for urine to determine the linearity and sensitivity of the developed method. Blank urine was also screened as a control. A triplicate analysis of the samples confirmed the reproducibility of the optimized method.

DISCUSSION:
The OPI and ADE interface, in conjunction with the high-resolution mass spectrometer, can analyze compounds at a rate of up to 1 sample per second, making the Echo MS+ system and the ZenoTOF 7600 system ideal for high-throughput screening. The low-volume sample size ensures no carryover and reduces matrix suppression. These benefits, therefore, relieve some of the bottlenecks common to conventional LC-MS/MS approaches.

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

Poster #32c View Map

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

INTRODUCTION:
The Multicenter AIDS Cohort Study/Women’s Interagency HIV Study Combined Cohort Study (MACS/WIHS) represents a collaboration dedicated to advancing our understanding of chronic health conditions among individuals living with HIV/AIDS. Their goal is to investigate the prevalence and impact of heart, lung, blood, and sleep disorders within this population. Particularly, the potential compounding effect of tobacco and cannabis use may further elevate cardiac and pulmonary risks in these immunocompromised individuals.

In our current analytical approach, we focus on quantifying nicotine, cotinine, 3-OH-cotinine, THC-COOH, and creatinine. The detection of nicotine in urine serves as a marker for both active and passive exposure, depending on concentration, while cotinine and 3-OH-cotinine, the primary metabolites of nicotine, are reliable indicators of secondhand smoke exposure at specific thresholds. THC-COOH, the principal metabolite of delta-9-THC, is the most definitive biomarker for cannabis use. We incorporated creatinine into our assay to enable normalization for kidney function. Although immunoassay remains the clinical standard for creatinine measurement, mass spectrometry presents a promising alternative for quantitation. To evaluate the reliability of our mass spectrometry-based creatinine measurements, we conducted a comparative analysis by also assessing creatinine levels in approximately 50 samples using an established immunoassay method. This approach allowed us to directly benchmark our LCMS data against clinical practice. By measuring these metabolites, we aim to elucidate the relationships between tobacco and cannabis exposure and cardiac and pulmonary health outcomes in people living with HIV/AIDS.

The MACS/WIHS cohort has provided our laboratory with 11,761 biospecimens, each accompanied by comprehensive data on pulmonary and cardiac function, enabling a robust assessment of these associations. Although individual methods for the quantitation of creatinine, nicotine, and THC metabolites are well established, there remains a gap in the literature for a unified approach capable of simultaneously quantifying all these analytes. Our work addresses this unmet need, offering a novel methodology to support future research in this critical area of clinical chemistry.

METHODS:
Urine samples are thawed and vortexed, then centrifuged. 200µL of urine are aliquoted to a well in a glass coated plate. 30µL of IMCSzyme RT glucuronidase mix is added to each sample for the reaction to begin. After 15 minutes, 20µL of deuterium labeled IS mix is added to each sample. As the reaction is taking place, prep of a µElution HLB SPE occurs simultaneously. 200 µL of MeOH is pushed through to waste with a positive pressure N2 manifold. Next, 200µL LCMS grade H2O is pushed through to prime and equilibrate the sorbent. Next, 200uL of the urine mixed with glucuronidase and IS is added to the sorbent and pushed through. Two washes of 10mM Ammonium Acetate (aqueous) are pushed through. The waste plate is switched out to a collection plate, a new glass coated plate. Two steps of 25µL 50:50 ACN: Ethyl Acetate are used to elute the desired analytes off of the sorbent. The elute is dried down under a stream of N2 for 5 minutes. The dried sample is then resuspended in 50µL of 80:20 MPA:MPB (MPA = 10mM Ammonium Acetate, MPB = 80:20 ACN:MeOH). The plate is sealed and ready for injection. The calibration curve is made by spiking stripped human urine from a range of 1ng/mL to 10,000 ng/mL, the only variation being creatinine which is measured from 0.4-400 mg/dL. The LC/MSMS method is 6.1 minutes beginning at 10%B and gradually reaching a point of 99%B. Chromatographic separation was achieved using a Phenomenex 100mm Kinetex EVO C18 2.6um column. The injection volume is 10µL. The detector used is a SciEx 6500+ Qtrap TripleQuadrupole Mass Spectrometer. The MS was run in MRM-ESI mode.

RESULTS:
The resulting method’s reproducibility was tested by inter-day injections of calibration curves (1-1000ng/mL)(n=20), and the injection of QCs at 10ng/mL (n=20). The R2 values for all curves are over 0.987, however most averaged over 0.996. All curves were weighted with 1/x, using a linear regression when possible, otherwise quadratic regressions. Inter-day imprecision met clinical guidelines for all curves and generally were far below the acceptable range of imprecision. The inter-day imprecision for the QCs were mostly under 6%. No carryover was observed for any of the analytes after injections at the high end of the calibration curve (10,000ng/mL). There is no crosstalk with the current set of deuterium-labeled standards (THC-COOH-d3 had some crosstalk with THC-COOH but was replaced with THC-COOH-d9 to resolve the issue). There is some matrix suppression dependent on the saturation of the urine, but the assay is sensitive enough and the concentration of the target analytes is high enough that it does not make a significant difference. The LLOQ for nicotine, cotinine, 3-OH-cotinine, and THC-COOH were below 1ng/mL (the lowest calibration concentration), and below 0.4 mg/dL for creatinine. The lowest calibrator concentration being much lower than the accepted concentration of what would be considered “positive” in a clinical setting. So far all 11,761 patient specimens from MACS/WIHS have been run with this method with several thousand more coming imminently.

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

Multimodal Molecular Imaging of Human Colorectal Cancer Biopsies Combining MALDI IMS, CODEX, and Spatial Transcriptomics Martin Dufresne (Presenter) Vanderbilt University

Poster #33c View Map

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

INTRODUCTION
Matrix-assisted laser desorption/ionization (MALDI) is a key high spatial resolution (≤ 10 µm) imaging mass spectrometry (IMS) technology, owing to its broad molecular coverage and ability to target selected molecular classes through tunable sample preparation. Combined with complimentary spatial biology platforms (e.g., CODEX, Visium, and Xenium), we can link IMS-derived molecular profiles with specific cell types and neighborhoods to discover how systems are altered in normal aging and disease. This approach is well-suited for studying cancer where characterizing tumor heterogeneity and disease progression is key to understanding cancer across stages, from pre-cancerous to advanced malignancies. Here, we present our integrated multimodal molecular imaging approach for the study of human colorectal cancer as part of the Human Tumor Atlas Network (HTAN).

METHODS
Preliminary data was acquired on 9 serial sections of a single colorectal cancer patient biopsy and two control biopsies for multi-omics analysis. All serial sections were initially scanned for autofluorescence (AF) prior to further analysis allowing for high accuracy registration to be performed using a single non-destructive modality prior to all experiments. Serial sections were then subjected to MALDI IMS, histological staining (H&E), spatial transcriptomic (Visium and Xenium), or immunofluorescence (CODEX). MALDI IMS was performed using a timsTOF flex (Bruker Daltonics) at 10 µm spatial resolution using minimal laser power after sublimation of VANDY37 matrix using the SubliMATE system (HTX Imaging). Co-registration and analysis were performed using in-house software and SCILS lab (Bruker Daltonics).

PRELIMINARY DATA
HTAN aims to build molecular atlases using integrated multi-omics to define tumor microenvironments and characterize tumor evolution at the cellular level in 2- and 3-dimensions. We have started the work on early-stage and late-onset human colorectal cancer using a multi-omics approach to achieve this goal. Because most molecular imaging modalities, including those proposed here, are inherently 2-dimensional, our strategy for generating 3-dimensional data is to co-register multiple serial sections using common, low-cost modalities that can be collected in tandem with molecular assays, e.g. autofluorescence and stained microscopy. This allows us to quickly assess the quality of each of the biopsies and histologically score the sample, allowing the selection of the best candidates for full 3-D multimodal analysis. Serial tissue sections are then guided through different spatially resolved omics and co-registered back to their respective AF pre-acquisition scan, which is also used for 3-dimensional reconstruction.

Our current workflow involves MALDI IMS in both polarities followed by CODEX of the same section, MALDI IMS of glycans, and LCM for targeted bulk metabolomics and proteomics analysis. Further sections are reserved for spatial transcriptomics using Visium and Xenium, along with traditional histological staining for the first and last sections of the 3-D stack. Initial MALDI IMS data has shown a strong correlation between certain classes of lipids such as carnitines, ether phosphatidylcholine (ether PC), and some sphingomyelin (SM) to the proliferation region of the tumor. Other signals, such as long chain PCs, ether PCs, and SMs, have intriguing distribution that do not overlap with the tumor proliferation region or healthy tissue. Further integration of these data with CODEX and spatial transcriptomics is underway, enabling mechanistic information to be layered onto molecular distributions and cellular organization.

CONCLUSION
This workflow will now be applied to a larger cohort of human colorectal cancer samples in 3D using single tissue multimodal imaging approach using lipid, glycan, and glycogen MALDI IMS which will be combined with serial section CODEX imaging and spatial transcriptomic.

Urinary Metabolites as Noninvasive Biomarkers for Neonatal Sepsis Kristaps Klavins (Presenter) Riga Technical University

Poster #34c View Map

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

INTRODUCTION:
Neonatal bacterial sepsis is a major contributor to morbidity and mortality worldwide. Early diagnosis is critical but remains challenging due to nonspecific clinical signs and limitations of current biomarkers. Blood sampling in neonates, especially those with very low birth weight, carries risks such as iatrogenic blood loss and pain. As a result, there is a strong clinical need for accurate, noninvasive diagnostic tools.

OBJECTIVE(S):
To identify urinary metabolites that serve as potential noninvasive biomarkers for neonatal sepsis and to develop a predictive model based on targeted metabolomics.

METHODS:
A prospective study was conducted involving 93 neonates, including 23 diagnosed with sepsis and 70 age-matched controls. Urine samples were collected noninvasively using standard neonatal care procedures. A targeted LC-MS-based workflow was employed to quantify 57 urinary metabolites. In the control group, confounding factors such as antibiotic exposure, chronological age, postmenstrual age, and feeding type were assessed to exclude metabolites influenced by these variables. Multivariate analysis and logistic regression were applied to identify the most discriminatory metabolites and construct a predictive model.

RESULTS:
Septic neonates showed significant decreases in normalized concentrations of adenosine and hydroxylysine. These changes were independent of potential confounding variables. A logistic regression model based on the absolute concentrations of three key metabolites yielded an area under the curve (AUC) of 0.9115, with 91% sensitivity and 90% specificity for sepsis diagnosis.

CONCLUSION:
Targeted urinary metabolomics offers a promising noninvasive approach for early diagnosis of neonatal sepsis. The identified metabolite-based model demonstrated high diagnostic accuracy and independence from common confounding factors. These findings support further validation and potential clinical integration of urinary metabolomics in neonatal care settings.

Identifying ‘DESI’red Prognostic Features of Human Ductal Carcinoma In Situ and Invasive Ductal Carcinoma using Spatialomics Natasha Iaboni (Presenter) Queen's University

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Poster #35c View Map

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

INTRODUCTION:
Breast cancer is the leading cause of new cancer cases and second leading contributor to cancer-related deaths among Canadian women. In 2024, ~30,500 Canadian women were diagnosed with breast cancer, and >5500 women died of breast cancer related complications. Between 20-25% of all breast cancer cases diagnosed were classified as ductal carcinoma in situ (DCIS), a form of breast cancer that presents as an abnormal epithelial cell population contained within the breast ducts. While DCIS is non-invasive, it may act as a precursor to invasive ductal carcinoma (IDC) by growing through the ducts and invading surrounding tissue. Women diagnosed with DCIS face challenging treatment decisions due to our current inability to predict the risk of DCIS tumour invasiveness or DCIS recurrence. Due to these knowledge gaps, many women are over treated with aggressive therapies, leading to physical and mental distress, surgical comorbidities and an overall lower quality of life.

Our lab is interested in the peroxisome proliferator-activated receptor γ (PPARγ), a key nuclear transcriptional regulator of lipid and sugar metabolism, and known to stop the growth and spread of breast tumours. Activation of PPARγ signaling via endogenous fatty acids and anti-inflammatory prostaglandins suppresses breast tumour progression via many putative signaling pathways, which are often cell and context specific. PPARγ-dependent protective pathways may include increasing expression of tumour suppressor genes such as BRCA1 or PTEN, decreasing inflammatory pathways by downregulating COX-2, and/or promoting apoptosis-related genes, all leading to decreased growth and spread of breast tumours. Notably, a report of increased PPARγ expression was associated with lower grade DCIS versus invasive breast cancer samples, suggesting PPARγ may normally act to reduce the invasive progression of DCIS. It remains unknown if loss of PPARγ signaling, common in some forms of aggressive breast tumours, plays a role in the transition from DCIS to IDC.

OBJECTIVE:
To address these clinical challenges, the primary objective of this study is to define the metabolomic profiles of human ex vivo DCIS and IDC tumours to enrich the pathological prognostic information available at time of diagnosis.

METHODS:
Formalin fixed and paraffin embedded (FFPE) human ex vivo DCIS/IDC tumour samples (n=8 DCIS, n=16 DCIS/IDC, n=10 IDC, n=34 total)) were obtained from the Kingston Health Sciences Centre. Sections of FFPE tissues were deparaffinized and then assessed using desorption electrospray ionization-mass spectrometry imaging (DESI), over a spectrum of m/z 50-1200 in negative ionization scanning mode, using a spatial resolution of 100um. The DESI-analyzed slides were then stained and annotated by a pathologist for DCIS, IDC, and non-tumour pathological regions. Of the n=34 FFPE samples, n=17 had additional corresponding tissues that were embedded in OCT and DESI assessed to serve as a direct comparison to the analytes detected via FFPE.

The pathologist-guided annotations and DESI 2D heatmaps were then meticulously overlaid with one another using the imaging analysis SlicerMSI software MassVision. Regions of Interest (ROI’s) composed of nine 100X100um pixels were plotted in each pathological zone per slide and merged to form one mean spectra per ROI. A total of n=38,757 ROI’s were selected across 5 pathological zones. Supervised machine learning was conducted along with binary statistical analysis to identify highly significant (p<0.001) ions that had a Log2 fold change >1.5 between DCIS and IDC pathological regions. To complement the metabolomic expression profiles, the samples were assessed using immunohistochemistry (IHC) to obtain an H-Score (intensity x percent positivity) of specified protein expression.

RESULTS:
Notable ions significantly elevated in FFPE IDC versus DCIS tissues included 6 different omega-6 fatty acids such as Linolenic acid (m/z 279.2303), Arachidonic acid (m/z 303.2301) and Docosatetraenoic acid (m/z 331.2600). Omega-6 fatty acids are involved in the production of pro-inflammatory molecules such as pro-tumorigenic prostaglandins (PGs) from arachidonic acid metabolism, which promote invasiveness, progression and immunosuppression. In contrast, we observed the anti-inflammatory PG 15-Deoxy-d-12,14-PGJ2 (m/z 315.11) was significantly increased in FFPE DCIS versus IDC tissue. Interestingly, 15d-PGJ2 is also a natural ligand of PPARγ, and reported to induce apoptosis and decrease oxidative stress via PPARγ activation.

The samples were then stained for PPARγ using IHC to define PPARγ expression within DCIS, IDC and surrounding benign tissues. Our data suggest a significant increase in PPARγ expression both within benign glands and ducts, and in DCIS versus IDC. PPARγ expression was not significantly different when comparing benign glands and ducts to DCIS. Within our cohort, there were no significant differences between intermediate versus high DCIS grades or moderate versus high IDC grades.

CONCLUSION:
This data suggests the significant increased expression of 15d-PGJ2 and PPARγ within DCIS versus IDC samples may aid in revealing the aggressive potential of these tumours and as well as further our understanding of those patients who are at risk for progression. Taken together, our findings also suggest a decrease in PPARγ expression correlates to progression from DCIS to IDC. Validating these expression profiles will allow us to provide pathologists with potential prognostic targets that may be routinely used to help optimize clinical treatment decision-making for DCIS patients.

Bgs Enzymatic Hydrolysis: A Recombinant Tool for Comprehensive Endocrine-Disrupting Chemicals Monitoring in Biological Models Elías Villalobos (Presenter) Kura Biotech

Poster #36c View Map

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

INTRODUCTION:
This literature review presents BGS, a recombinant beta-glucuronidase and arylsulfatase enzyme used for single-step hydrolysis in various applications. When incorporated into liquid chromatography-tandem mass spectrometry quantification methods, it enables the detection of conjugated xenobiotics and steroid hormones in human and animal models.

OBJECTIVE:
Evaluate the incorporation of enzymatic hydrolysis for different applications in the space of metabolomics, exposomics, aiming to improve the sensitivity, signal-to-noise, and reproducibility.

METHOD:
The animal model studies incorporated BGS to measure steroid hormone fluctuations and gene expression changes in zebrafish(1), revealing endocrine-disrupting effects of environmental pollutants. Similarly, research in rat plasma and liver(2) presented an optimized method for steroid hormone profiling in tissue, broadening the available matrices to look into.

The human exposome studies explore using enzymatic hydrolysis to quantify total xenobiotic exposure, focusing on plasticizers, bisphenols, PFAS, and steroid hormones. These studies (3,4) demonstrated the effectiveness of enzymatic hydrolysis in different human matrices, accompanied by lower background noise due to its recombinant nature. Hence, incorporating an effective enzymatic tool ensures a more accurate measure of EDCs in human matrices.

RESULTS:
BGS has proven to be a reliable and versatile tool for exposome-scale biomonitoring and endocrine disruption research. Used across a range of biological matrices—including human plasma, urine, serum, reproductive fluids, breast milk, as well as rat liver and zebrafish embryos—BGS consistently delivered efficient and simultaneous deconjugation of glucuronide and sulfate metabolites. Compared to traditional enzymes like Helix pomatia, BGS offered greater purity, lower background interference, and broad substrate coverage, making it particularly well-suited for both high-throughput workflows and low-volume samples. Its use significantly improved the quantification of total xenobiotic and steroid levels, providing a more accurate understanding of chemical exposure and its biological effects.

CONCLUSION:
As exposome monitoring advances, enzymatic hydrolysis can become a crucial step, facilitating cross-species comparisons, streamlining workflows, and enabling high-throughput analyses. Its integration with conjugated biomarker research can enhance method development in environmental health, toxicology, and clinical diagnostics, ensuring precise and consistent detection of chemical exposures and their effects.

REFERENCES:
1. Maria Revenikioti, 2023, An Investigation of the Link Between Endocrine Disruption and Developmental Neurotoxicity Induced by Environmental Pollutants In Zebrafish Embryos.
2. Sara Evangelista et. al, Talanta, 2024, 266, 124981.
3. Yasmin Fareed et al, Exposome, 2022, 2(1), osac008.
4. Jaye Marchiandi et al, Environ. Sci. Technol. 2024, 58, 46, 20352–20365.

Development of a Plasma Amino Acid Method using LC-MS/MS for the Biochemical Genetic Laboratory Adam Ptolemy (Presenter) Boston Childrens Hospital, Harvard Medical School

Poster #37c View Map

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

INTRODUCTION
Quantitative plasma amino acid analysis is primarily used in clinical biochemical genetics laboratories for the diagnosis and monitoring of inborn errors of metabolism (IEM). A majority of biochemical genetics laboratories measure amino acids using chromatographic methods that utilize optical detectors. These methods are limited by long analytical run times and are prone to interferences. Our laboratory uses an Acquity™ ultra-performance liquid chromatography (UPLC) system with an integrated UV detector and the MassTrak™ AAA Solution Kit (Waters Corporation). This method utilizes pre-column derivation of amino acids with 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate (AccQTag®) followed by reversed-phase UPLC on a MassTrak™ C18 column (2.1 x 150 mm, 1.7µm, Waters Corporation) followed by single wavelength UV detection at 260 nm. The challenges associated with chromatographic resolution of all plasma amino acids with this UPLC method (35 min runtime) and their potential impact on IEM patient management have been previously reported (1). Transitioning this testing to a liquid chromatography tandem mass spectrometry (LC-MS/MS) based procedure represented an opportunity to improve the efficiency, sensitivity and specificity of our existing plasma amino acid protocol.

OBJECTIVES
To develop an LC-MS/MS assay to quantify plasma amino acids and evaluate its potential use within our patient population with suspected IEMs.

METHODS
Method development and clinical feasibility studies were performed using an Acquity™ iClass UPLC coupled to Xevo TQ-S Micro tandem mass spectrometer (Waters Corporation) operated in positive electrospray ionization mode (ESI+). Sample pretreatment was performed using a Karios™ Amino Acid Kit (Waters Corporation), which utilizes protein precipitation and AccQTag® chemical derivation prior to analysis. This kit also contains calibration standards and QC for 45 different amino acids, including several isotopically labelled internal standards. Optimization of the sample preparation protocol permitted the use of 20 µL of material for testing. All amino acids and internal standards were detected as their derivatized products using their respective MS/MS transitions. Three different reverse phase C18 columns were tested to develop the final chromatographic conditions: CORTECS® C18 (2.1 x 150 mm, 1.6 µm), HHS C18 (2.1 x 150 mm, 1.8 µm), and ACCQ-TAG™ Ultra C18 (2.1 x 15 mm, 1.7 µm) columns were all obtained from Waters Corporation. Mobile phase composition, flow rate and gradient conditions were optimized for each column to provide the greatest isobaric amino acid resolution and sensitivity, within the shortest analytical runtime. Performance of each LC-MS/MS assay was preliminarily evaluated through plasma amino acid correlation studies with the predicate LC-UV method and an LC-MS/MS assay from a national reference laboratory. Method accuracy was then further assessed using archived amino acid external quality assurance (EQA) material from the European Research Network for the evaluation and improvement of screening, Diagnosis and treatment of Inherited disorders of Metabolism (ERNDiM).

RESULTS
The optimized sample preparation and chromatographic conditions of each C18 column yielded LC-MS/MS assays that were linear over a wide amino acid concentration range (2.5 to 4000 µM, R>0.99). LC-MS/MS derived plasma amino acid results were relatively higher than the UPLC method, with average plasma amino acid recoveries from the CORTECS®, HHS, and ACCQ-TAG™ Ultra C18 column methods being 130%, 119% and 117%, respectively. The blinded split sample analysis with a national reference laboratory demonstrated good agreement for the ACCQ-TAG™ Ultra C18 column method, with an average plasma amino acid accuracy of 102% relative to the reference laboratory mean. The average plasma amino acid accuracy from the HHS (114%) and CORTECS® (119%) C18 methods relative to the reference laboratory were respectively higher than the ACCQ-TAG™ Ultra C18 column method. The highest relative resolution of all isobaric amino acids included in the Karios™ Amino Acid kit (e.g., 1- and 3-methylhistidine; isoleucine, alloisoleucine and leucine; γ-, β-, and α-aminobutyric acid; sarcosine, β-alanine and alanine) was also achieved with the ACCQ-TAG™ Ultra C18 column method. Resolution of isoleucine and alloisoleucine ranged from 0.68 to 0.85 with the HHS and ACCQ-TAG™ Ultra C18 column methods, respectively. Based on these studies, the optimized plasma amino acid LC-MS/MS method chosen for validation will utilize the ACCQ-TAG™ Ultra C18 column with mobile phase compositions of 0.1% (v/v) formic acid in water (mobile phase A) and acetonitrile (mobile phase B). Under the devised gradient conditions of this method, which utilized a flow rate of 0.5 mL/min and column temperature of 55°C, all amino acids were eluted within a 17 min runtime. The accuracy of this protocol was further challenged by testing archived amino acid EQA material (N=4 samples) from ERNDiM. Good agreement relative to the ERDiM all method laboratory means was achieved, with amino acid (N=27) accuracies ranging from 87% (citrulline) to 111% (sarcosine) and an average accuracy of 98%.

CONCLUSION
We have developed an accurate LC-MS/MS method for plasma amino acids that utilizes the Kairos™ Amino Acid kit. This assay is desirable as a full amino acid panel may be achieved within a relatively short runtime (17 min), while also maintaining adequate resolution of clinically relevant isobaric analytes. The targeted metabolomics assay also requires a small sample volume (20 µL), which is desirable for potential use within our pediatric IEM patient population. The devised LC-MS/MS protocol must be validated as a laboratory developed test (LDT) within our institution.

REFERENCES
(1) Peake RWA, Law T, Hoover PN, Gaewsky L, Shkreta A, Kellogg MD. Clin Chim Acta 2013; 423:75-82.

Removing Carryover Concerns and Validation of a 6-Analyte Barbiturate Panel on GC-MS by Split Injection Alison Lightfoot (Presenter) Mayo Clinic

Poster #38c View Map

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

INTRODUCTION:
Barbiturates represent a class of drugs that were originally introduced as sedatives/sleep inducers. Amobarbital is an intermediate-acting barbiturate that has sedative/hypnotic properties used to relieve anxiety and insomnia. It also has analgesic properties. Butalbital is a short-acting barbiturate and is used to control severe headaches and pain. Pentobarbital is a short-acting barbiturate and is used in the treatment of seizures and brain injuries. Phenobarbital is frequently used to control major motor (grand mal) seizures and the treatment of epilepsy. Secobarbital and butabarbital are short-acting barbiturates and are used as a preanesthetic agent and the short-term treatment of insomnia.
Prior to this re-development and validation, an in-house assay was live clinically but had significant problems with carryover leading to inefficient workflows and frequent repeat testing of patients.

OBJECTIVE(S):
The re-development, validation, and implementation of a GC-MS method to quantify the levels of six barbiturates in patient serum samples with a focus on reducing significant carry-over observed in the previous clinical test.

METHODS:
All six barbiturates were extracted from serum using solid phase extraction techniques. The serum was buffered with a pH 6.0 sodium phosphate solution and eluted with 25% hexane in ethyl acetate. The organic phase was then dried and reconstituted in ethyl acetate. Standards and in-house controls were prepared by spiking in bovine serum. Clinical controls were prepared in human serum.

The samples were analyzed using a 1 mcL injection on an Agilent GC-MS (GC model 6890 and MS model 5975). A J&W Scientific DB-5MS, 15m, 0.25mm I.D. capillary column with a film thickness of 1 micron was installed and used for separation in the GC. The GC and MS methods were built using Agilent Chemstation. The following GC method parameters were defined: 260 °C for the inlet temperature, 280 °C for the auxiliary temperature, and an oven ramp from 85 °C to 295 °C over the course of 11 minutes. The samples were injected using split flow injections with a split of 9:1. For the MS, an inert EI (electron-impact) source was installed in the MS and Helium was used as carrier gas. Chemstation was used to build and submit each batch. Agilent Mass Hunter was used as quantification software.

RESULTS:
Carryover was negligible for all six analytes. This is a significant improvement, as the previous assay struggled with significant carryover for phenobarbital and pentobarbital, which caused potential repeat extractions of patient samples as well as requiring a blank injection between every sample on the run. Re-development of the extraction method led to a small reduction in carryover; however, the biggest factor in reducing carryover was changing the GC inlet flow from splitless to split. With the split ratio set at 9:1, less sample was injected onto the column yielding a significant reduction to carryover without sacrificing sensitivity. The split injection also improved analyte chromatography, which previously showed fronting due to overloading on the column, especially at the upper limit of quantitation (ULOQ).

The linearity across the analytical measuring interval (AMI, 0.5-10 mcg/mL) returned a linear regression with R2=1 and slopes between 0.97 and 0.99 for all six analytes (N=5 runs). The precision for all control levels (0.75, 4, and 8 mcg/mL) averaged %CV’s < 3.5% for intraday precision and %CV’s < 4.5% for interday precision (N=20 runs). Accuracy samples for pentobarbital were compared to the current clinical assay and returned a linear regression with R2=0.99 and a slope of 1 (N=19 samples). All six analytes were spiked and compared to theoretical values for accuracy and returned linear regressions with R2=0.99 or 1 and slopes between 0.97 to 1(N=30 samples).

CONCLUSIONS:
Through a full re-development and validation, we have demonstrated a new robust method for the accurate quantitation of six barbiturates in serum that has resolved carry-over concerns and has now been implemented into the clinical laboratory.

Strategies for Mitigating the Effects of High Gabapentin Concentrations in Urine Specimens Haley Berkland (Presenter) Restek

Poster #39c View Map

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

INTRODUCTION:
Gabapentin is an anti-convulsant drug that is prescribed for the treatment of neuropathic pain and seizures, as well as for many off label uses. Gabapentin is prescribed in high doses relative to other therapeutic drugs and is eliminated in urine predominantly in its unchanged form, which often results in extremely high concentrations of this compounds occurring in patient urine samples.1 When analyzed by LC-MS/MS, high concentrations of gabapentin can have significant analytical implications, particularly for the compound amphetamine. Interference between gabapentin and amphetamine has been well documented, and can result in signal suppression, poor peak shape, and shifting retention times.2 Other analytical challenges include saturation of the mass spectrometry detector and column overload. In this work, we explored several strategies to mitigate the effects of high gabapentin concentrations in urine samples.

OBJECTIVES:
The primary objective of this study was to capture the analytical challenges presented by high concentrations of gabapentin in urine samples when analyzed by LC-MS/MS and investigate different strategies to mitigate them.

METHODS:
A method developed for the analysis of 60 drugs of abuse in urine was used to test two samples: one containing 0.1 µg/mL of both gabapentin and amphetamine, and one containing 250 µg/mL of gabapentin and 0.1 µg/mL of amphetamine. After data was collected, the signal of amphetamine was compared in each sample to determine if interference from gabapentin was occurring. The method utilized a Raptor Biphenyl 50 x 2.1 mm, 2.7 µm column and a mobile phase A of water and mobile phase B of methanol, both acidified with 0.1% formic acid. The flow rate was 0.6 mL/min, the column temperature was 45°C, and the injection volume was 5 µL. Gradient elution was employed, with a total runtime of 9 minutes. Once the data had been analyzed, different strategies were tested to see if the interference between gabapentin and amphetamine could be resolved. These strategies included using alternate column lengths and diameters, decreasing injection volume, deoptimizing the analyte transition, and testing different mobile phase additives.

RESULTS:
Under the original method conditions tested, amphetamine showed a diminished signal and shifted retention time when in the presence of a high concentration of gabapentin. The method was redeveloped using several of the strategies tested that showed improved performance for both analytes. It was determined that the best approach for reducing interference was to fully chromatographically resolve gabapentin and amphetamine and ensuring that gabapentin was the first compound to elute. This was done by switching the additive in mobile phase A from 0.1% formic acid to 10 mM ammonium formate, which affected the elution order of early eluting compounds. Resolution was further improved by switching from a 50 x 2.1 mm column to a 100 x 2.1 mm column. Detector saturation and column overload were improved by deoptimizing the mass transition for gabapentin and reducing the injection volume from 5 µL to 2 µL. Significant carryover was observed due to the high analyte concentrations and was eliminated by adding a small amount of 2-propanol to mobile phase B. Performance of the other analytes in the method was not negatively affected by the change in method parameters.

DISCUSSIONS/CONCLUSIONS:
Interference between gabapentin and amphetamine, chromatographic overload, and detector saturation were all observed when analyzed under the original method conditions. Altering the mobile phase composition, using an extended column length, deoptimizing the mass transition, and reducing the injection volume were all successful in mitigating these analytical challenges. The addition of 2-propanol to mobile phase B helped to reduce carryover by washing contaminants off the analytical column more efficiently. The redeveloped method can be used to effectively analyze 60 drugs of abuse in urine without interference between gabapentin and amphetamine.

REFERENCES:
1. Heltsley, Rebecca & Depriest, Anne & Black, David & Robert, Tim & Caplan, Yale & Cone, Edward. (2011). Urine Drug Testing of Chronic Pain Patients. IV. Prevalence of Gabapentin and Pregabalin. Journal of analytical toxicology. 35. 357-9. 10.1093/anatox/35.6.357.
2. Sarah B Shugarts, Pervasive Gabapentin Interference in the LC-MS/MS Analysis of Amphetamine, The Journal of Applied Laboratory Medicine, Volume 2, Issue 4, 1 January 2018, Pages 527–534, https://doi.org/10.1373/jalm.2017.024117.

Quality And Sustainability: Enhancing Workflows Using Concentrated LC-MS Kits Dagmar Kasper (Presenter) IBL International, a Tecan company

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Poster #40c View Map

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

INTRODUCTION:
The increasing demand for sustainability and cost-efficiency in laboratories has prompted the development of innovative solutions that do not compromise analytical quality. In this context, we have tested concentrated LC-MS kits as an alternative to conventional, Ready-to-Use kits. These concentrated kits are designed to reduce shipping weight, packaging material, and storage volume, thereby supporting both environmental and economic goals. In addition to their ecological and logistical advantages, the kits provide enhanced flexibility, as all components are available individually or as part of starter and solvent kits, allowing for tailored usage and efficient inventory management. This study systematically evaluates the analytical equivalence of the new concentrated kits compared to their standard counterparts, focusing on a broad panel of relevant analytes.

METHODS:
A total of seven analytes were selected to represent a diverse range of compound classes, including water-soluble vitamins (Vitamin B1, B6), fat-soluble vitamins (Vitamin A, E), steroid hormones (cortisol, cortisone), and organic acid (methylmalonic acid).The method comparison was performed in accordance with CLSI guideline EP09-A3, using at least 40 samples to assess agreement between the two analytical methods.

Identical samples were analyzed in parallel using both the standard and the concentrated kits. For each analyte, method comparison was performed using linear regression analysis (slope, intercept, correlation coefficient R), estimation of mean bias, and assessment of precision through the standard deviation of differences. The evaluation focused on both the agreement of results and the practical interchangeability of the two kit formats. The concentrated format was also assessed for its impact on laboratory workflow, including ease of sample preparation, storage requirements, and potential for reducing shipping and packaging costs.

RESULTS:
The comparison revealed a consistently high correlation between the concentrated and standard kits for all analytes tested, with correlation coefficients (R) exceeding 0.98 and typically above 0.99. Regression slopes were close to unity, ranging from 0.98 to 1.14, indicating that the mean values obtained by both methods were highly comparable. The mean bias between the two methods was generally small relative to the respective measurement ranges, with no evidence of significant systematic deviations. For most analytes, the bias was less than 10 nmol/l (or equivalent), and the standard deviation of the differences was low, reflecting good precision and reproducibility. Notably, for cortisone, vitamin E, and MMA, the concentrated kits tended to yield slightly higher values (slope > 1.1), but these differences remained within acceptable limits and did not affect the overall analytical equivalence.

CONCLUSION:
The concentrated LC-MS kits demonstrate analytical equivalence to their standard counterparts across a diverse range of important analytes, delivering reliable, precise, and reproducible results. Beyond maintaining high analytical quality, the concentrated format offers many advantages in terms of sustainability, cost-effectiveness, and operational flexibility. These findings support the adoption of concentrated LC-MS kits as a robust, sustainable, and economical solution for modern laboratories, enabling both laboratory staff and purchasers to benefit from consistent performance, efficient processes, and a reduced environmental footprint.

Atmospheric Pressure MALDI and DESI High-Resolution MS Histochemistry for FFPE Biobanked Tissue Analysis Aligned with Pathology Practice Peter Verhaert (Presenter) ProteoFormiX

Poster #41c View Map

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

INTRODUCTION / OBJECTIVE
To better align mass spectrometry imaging workflows with the realities of clinical pathology, we develop applications that make formalin-fixed paraffin-embedded (FFPE) tissue samples accessible for direct MS-based imaging using cutting-edge high-resolution (HR) instrumentation.

METHODS
FFPE samples procured straight from the university hospital pathology biobank are sectioned at 5 μm thickness (standard microtome). An essential step prior to MS analysis is short deparaffinization of the sections to maximally avoid extra delocalization of small molecular analytes.
We work with two types of atmospheric pressure ionization HR MS: [1] OrbitrapTM (ThermoFisher Scientific) instruments interfaced with an AP/MALDI UHRTM (MassTech) source, and [2] DESI MRT (SelectTM Waters). MALDI is done on DHB coated sections, DESI is performed on matrix-free deparaffinized sections using different extraction solvents (90% acetonitrile, 0.01% formic acid and 90% methanol, 0.01% formic acid).

RESULTS
Using optimized protocols we successfully image a variety of physiologically relevant biomolecules, including endogenous neuropeptides and metabolites, directly from FFPE sections.
This capability, which we call Mass Spectrometry Histochemistry (MSHC), mirrors the workflow of immunohistochemistry (IHC), improves molecular specificity, offers a spatial resolution down to 5×5 μm² and can be applied directly to the millions of FFPE tissue blocks stored in biobanks worldwide. We illustrate this by showing MALDI and DESI HR MSHC data on different FFPE tissue samples.

DISCUSSION
The potential of MSHC is huge; we can now analyze archived samples from pathology biobanks to find small molecule biomarkers linked to the seriousness of the disease, how patients respond(ed) to treatment, or their long-term outlook. Since MSHC works directly on the same slides already used for routine pathology, it fits easily into existing lab workflows – making it much simpler to bring into clinical practice. This also facilitates integration of MSHC data with other pathology imaging modalities currently in practice on FFPE samples, including IHC, H&E. MSHC is an elegant way to generate an extra dimension of (large) data to be assimilated with the complete pathology knowledgebase archived up in well-documented biobanked disease samples. Even without molecular identification of MSHC ion signals, we predict that in the near future AI will help to link MSHC data to relevant clinical decisions, similar to microbial identifications confidently based on MALDI profiles.

The promise of MSHC is no longer theoretical. The tools exist, the protocols are feasible, and the samples are waiting. MSHC transforms existing pathology archives into living resources for discovery and diagnosis, offering the potential to revolutionize how we classify disease and tailor treatment. What’s needed now is collaborative engagement from the pathology community – to help select cases, validate findings, regulate protocols and integrate this new layer of molecular insight into everyday diagnostics.

Sample Preparation for the Determination of an Extended Panel of Per- and Polyfluoroalkyl Substances (PFAS) from Human Serum Using UHPLC-MS/MS Lee Williams (Presenter) Biotage GB Limited

Poster #42c View Map

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

INTRODUCTION
Per and polyfluoroalkyl substances (PFAS) are of concern because of their high persistence, bioaccumulation, and slow elimination with potential impacts on human and environmental health. Recently, human exposure has been linked with changes in metabolism, increased cholesterol, and high incidence of some forms of cancer. Additionally, PFAS pose particular challenges in the analytical laboratory due to their ubiquitous nature.

OBJECTIVES
This poster presents a novel method for the simultaneous determination of long and short chain PFAS in human serum at clinically relevant levels. ISOLUTE® PLD+ for PFAS, due to its optimised sorbent chemistry and functionality, allows for a simple workflow utilising solvent crash and filtration. With subtle modification to allow suitable extraction of challenging longer chain PFAS targets we have developed a robust, high sensitivity method for the clean-up of 36 PFAS compounds from human serum.

METHODS
A panel comprising 36 PFAS was spiked and extracted from human serum. Solvent crash/filtration extraction incorporating a 1:7 or 1:4 matrix/solvent ratio, utilizing ISOLUTE® PLD+ for PFAS in 96-well format was investigated. Processing was optimized using a Biotage® PRESSURE+96 positive pressure manifold. A modified procedure utilizing a 2 step sequential elution protocol was developed to optimize the extraction of longer chain 13C-18C PFAS. Extraction efficiency was compared using a range of solvents and blends of varying polarity. Final methods were developed accommodating various workflows, depending on exact application needs. Optimized methods were selected for maximum recovery and repeatability, with minimal matrix factors. LC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC modified with a PFAS-free flow path and a pre-injector PFAS delay column, coupled to an AB Sciex 5500 triple quadrupole MS system operating in negative ion mode.

RESULTS
A previously developed PFAS extraction protocol was used as the starting point for method development. 100 µL of serum was precipitated with acetonitrile (in a 1:7 ratio) and extracted using ISOLUTE® PLD+ for PFAS, followed by 1:1 dilution with 20 mM ammonium acetate for injection. Recoveries of carbon chains up to PFDoA (C12) were 78-90% with corresponding RSDs below 10%; however, PFAS > 12C demonstrated minimal recoveries. Matrix factors were typically 1.0-1.5 relative to dilute standards.

Extraction method modification to incorporate additional solvent volumes and/or aliquots was investigated. Addition of a second acetonitrile extraction aliquot increased recoveries of PFAS > 14C to between 5-16%. Evaluation of other solvent combinations demonstrated recovery between 4-80%, while PFODA (18C) remained unrecovered. Using a non-polar solvent as a second extraction aliquot increased recovery of PFAS >14C to between 2-82%, with PFODA recovery between 2-51%. MTBE as a second solvent provided the greatest potential for further optimization. MTBE was therefore modified with increasing proportions of methanol as a polar solvent (5-40%). These modifications demonstrated recoveries between 34-90% for longer chain PFAS, the optimum proportion was determined to be 90:10 MTBE/MeOH (% w/v).

Reinvestigation of evaporation and reconstitution conditions further enhanced sample repeatability and improved assay performance. Some longer chain PFAS RSD were approximately 15% when evaporating standards from pure solvent. Introduction of a keeper solvent to prevent full evaporation reduced RSD to below <5%. DMF proved to be the optimum keeper solvent for this assay. Workflow comparison between injecting fractions separately, combining and/or evaporation of one fraction and reconstitution with the other was evaluated.

Using the optimized protocol to extract 100 µL serum with 400 µL acetonitrile followed by 90:10 MTBE/MeOH demonstrated analyte recoveries typically 70 90% with RSD <5%. Excellent matrix depletion with respect to phospholipids, lyso-phospholipids and protein removal was also demonstrated. Full results, discussion, and conclusions will be presented in the final poster.

CONCLUSION
This poster demonstrates enhanced extraction efficiency of long chain serum PFAS in a single LC-MS acquisition, through optimized solvent selection using a multifunctional sorbent. Methods accommodate various workflows depending on exact application needs: dual acquisition for separation performance, evaporation/reconstitution for sensitivity. ISOLUTE® PLD+ for PFAS demonstrates enhanced cleanliness compared to dilute-and-shoot methodology leading to more robust methods.

Metabolic Changes from Pollinator Interactions and Dietary Implications Josephine Dolan (Presenter) Middlebury College

Poster #43c View Map

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

INTRODUCTION:
Understanding plant reproduction and plant-pollination interactions are of vital importance to supporting biodiversity and maintaining optimal reproduction for plant populations. Pollination, the reproductive process for plant species, carries male gametes in pollen grains, via pollinators, wind, water, or self-pollination, from an anther to the stigma of a flower. Pollination requires a complex assortment of regulatory signals and metabolic processes such as germination, seed development, and formation of the pollen tube. Nectar, which is produced by the nectary glands, is a liquid rich in sugar that attracts pollinators and functions as their source of energy. Yet, metabolic perspectives on these processes and signals are limited, leaving many unanswered questions about the pollination processes impact on the plants themselves. There is also a lack of metabolic understanding and research of how nectar metabolites vary depending on pollinator visitation. We are using untargeted metabolomics to examine squash nectar of male and female squash flowers, following different levels of pollinator visitation. The nectar reflects metabolic activity of the nectaries through its components, including altered sugar concentrations. Here, we aim to discover these quantitative metabolic changes in squash nectar as a result of pollination, giving further insight into this complex and crucial biological process.

OBJECTIVES:
To metabolically characterize changes in pollination-related metabolites between male and female squash nectar using untargeted metabolomics.

METHODS:
Female and Male flower nectar squash samples were collected from the Crook-Neck Yellow (Cucurbita pepo) squash plant in Middlebury, VT over the course of 4 days in varying environmental conditions. Aliquots of nectar samples were then diluted 1:5000 and prepared using previously published methods for LC/MS metabolomics analysis. In future investigations, time of day will be examined through the collection of samples twice daily, during high and low pollinator visitation, for the nectar of CN (Crook-Neck yellow), SN (Straight-Neck yellow), or BZ (Black Zucchini) male and female squash flowers. For the untargeted analyses, the samples were analyzed using a Waters G2 QTOF coupled to an HPLC using HILIC and both ionization modes. Data were examined using MS-Dial software against publicly available libraries, and normalized to internal standards; statistical analyses and figures were created through graphpad Prism.

RESULTS:
Using untargeted mass spectrometry, we gathered preliminary results of 62 detected and 44 identified metabolites with MS2 within the nectar squash samples of male (M) and female (F) flowers. Of those metabolites, Proline-hydroxyproline, Abscisic acid, and Monoolein, presented notable differences in quantity between female and male flowers. Proline-hydroxyproline rich glycoproteins aid in the processes of pollination and fertilization in the reproductive tissues of plants. Abscisic acid is a phytohormone that assists in plant growth regulation, and accumulates in response to abiotic stressors such as drought or salt. Monoolein is a lipid involved in the process of energy release used for plant growth and maintaining membrane stability. In positive ionization mode, on average, Monoolein, Abscisic acid, and Proline-hydroxyproline were 4 fold, 6.7 fold, and 6 fold higher in F than M nectar. On April 21st proline-hydroxyproline was 28 fold higher and on April 25th, 19 fold higher in F nectar than M. On April 30th, Abscisic acid was 38 fold greater in F to M nectar. This gives some insight into the time-specific changes in pollination, especially when paired with weather data for these time points. The ability to detect sex-dependent changes in these metabolites over time gives a unique insight into these processes in a real-time manner.

CONCLUSIONS:
These preliminary data highlighting varying quantities of metabolites, dependent on time and sex, can communicate useful information about the energetic composition of nectar, and measured metabolic changes in response to pollination, for future plant-pollinator investigations. This understanding of the metabolic components of squash nectar is vital in its application of food as medicine and to optimize vegetation reproduction.

Novel Applications of Magnetic Nanoparticle-Based Biofluid Enrichment Methods to Enable Proteomic Ovarian Cancer Biomarker Development Using Ascites Fluid Kathleen Madden (Presenter) University of Minnesota

Poster #44c View Map

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

INTRODUCTION:
Ascites, the pathological accumulation of fluid in the peritoneal cavity, is common at diagnosis and with recurrence in high-grade serous ovarian carcinoma (HGSOC). Higher volumes of ascites fluid are associated with greater likelihood of recurrent disease and poor prognosis, as indicated by shorter progression-free survival and overall survival. Due to its availability – volume and frequency – and its reflection of tumors and their microenvironments, ascites is a valuable resource for proteomic biomarker discovery. However, ascites has not been widely studied. Similar to plasma, relatively few proteins comprise the majority of the broad dynamic range of protein mass in ascites fluid, necessitating an enrichment or depletion strategy for proteomic sample preparation. Nanotrap® Protein Enrichment Affinity Kits (PEAK) developed by Ceres Nanosciences contain magnetic hydrogel Nanotrap Particles, whose complementary chemistries selectively isolate low abundance proteins while excluding high abundance proteins to enhance the depth of proteome coverage. ENRICH-iST® and ENRICHplus® kits developed by PreOmics enrich low abundance proteins using paramagnetic beads. Neither of these enrichment strategies have previously been validated for use with ascites fluid.

OBJECTIVES:
Our goal was to evaluate the performance of Nanotrap particles and the PreOmics ENRICH-iST and ENRICHplus kits using ascites fluid from ovarian cancer patients with differing extents of tumor resection during debulking surgery. The technical performance of these enrichment strategies was evaluated based on proteome depth, reproducibility of identified proteins, Gene Ontology (GO) term enrichment (cellular compartments and biological processes), and Human Plasma Proteome Project (HPPP)-based protein abundance ranking.

METHODS:
Twelve HGSOC ascites fluid specimens were obtained from an ovarian cancer biorepository at the University of Minnesota, and they were stratified by residual disease at initial debulking surgery into four groups with n=2 or 3 biological replicates: suboptimal resection vs. optimal resection with tumor implants < 1cm, < 5mm, or with no visible residual tumor. Nanotrap Protein Particles A, AC, and ABC (Ceres Nanosciences) were used to enrich 150 µL of ascites fluid from each patient sample, and ENRICH-iST and ENRICHplus (PreOmics) beads were used to enrich 20 µL and 50 µL of ascites fluid, respectively, before reduction, alkylation, and overnight digestion with trypsin at 37°C. Neat samples without enrichment were processed in parallel. Peptides (500 ng) were separated using an EvoSep One liquid chromatography system with a C18 PepSep column (8 cm x 150 µm; 10 µm emitter) and a 60 SPD method. Mass spectrometry data acquisition was conducted using a timsTOF HT mass spectrometer (Bruker) operated in DIA-PASEF mode. Direct DIA analysis was performed using Spectronaut® v. 20.

RESULTS:
Twelve samples were enriched using Nanotrap Particles A, AC, and ABC, and the ENRICHplus beads. Eight samples were enriched using the ENRICH-iST beads. The protein concentration of the individual ascites fluid samples ranged from 17.8 µg/µL – 58.0 µg/µL (median 43.9 µg/µL) as determined by BCA assay. The enrichment methods yielded a 2.3 – 3.3-fold increase in proteome depth vs. the neat samples, which had an average of 1090 ± 302 identified proteins. A total of 1106 proteins were commonly identified across all the enrichment methods. Dimensionality reduction analysis revealed the greatest proteome similarity among the samples processed using similar enrichment methods: Nanotraps or ENRICH kits. The AC Nanotrap Particle combination resulted in the highest number of proteins identified among the Nanotraps (2612 ± 897 proteins), whereas ENRICHplus out-performed the ENRICH-iST beads (3638 ± 1749 vs. 2577 ± 841 proteins). Although ENRICHplus enabled the greatest proteome depth, its reproducibility (48% CV) was the lowest among the evaluated enrichment methods. Nanotrap Particle A had the highest reproducibility of proteome coverage depth: 32% CV. The widest proteome dynamic range was achieved with ENRICHplus and ENRICH-iST: 5.4 orders of magnitude vs. 5.1 orders of magnitude for the neat samples.

DISCUSSION:
Utilizing ascites fluid to develop diagnostic, predictive, or prognostic proteomic biomarkers could have a significant impact on HGSOC patient care. We successfully evaluated the technical performance of Nanotrap Particles, ENRICH-iST and ENRICHplus protein enrichment methods based on the DIA-MS proteomic analysis of ascites fluid. Analysis of the differential abundance of the proteins enriched from ascites fluid using these enrichment strategies could greatly facilitate HGSOC biomarker development. These proteomic biomarkers could form the basis of a multiplexed targeted MS assay that is validated according to clinical laboratory guidelines.

Discovering Arginylation Substrates by ATE1-Based Arginylation Profiling with Bottom-Up and Top-Down Proteomics Richard Searfoss (Presenter) Washington University School of Medicine

Poster #45c View Map

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

INTRODUCTION:
Arginylation is a PTM installed by ATE1 to signal a protein for degradation by the N-degron pathway. Knockout of the ATE1 enzyme is embryonic lethal due to various cardiac defects including thinned myocardium and cardiac contractility deficiencies, however the exact mechanism of how this occurs is still unknown. Arginylation is also implicated as a regulator of aSyn folding and function, preventing aggregation as a potential mechanism in the prevention of synucleinopathies. Other roles for arginylation include protein secretion, notably serum albumin, and B-actin subcellular translocation.

This modification most commonly occurs on the N-terminus of substrates, but studies show it can be installed on aspartic and glutamic acid side chains as well. Study of arginylation is challenging due to the low abundance of the modification, aspecific antibodies, and the difficulty of distinguishing a PTM from a missed cleavage. initial qualitative proteomics studies have identified two small sets of candidates (43 and 19 proteins) potentially equipped with N-term and mid-chain arginylation, respectively. These results indicate that arginylation serves as a biological regulator of protein function and thus raises the question of how many proteins/sites are arginylated and what their functions are. We have recently developed an ABAP strategy for the discovery of arginylation sites with both bottom-up and top-down proteomics. This method takes advantage of isotopically labeled arginine to validate the N-terminal installation of the arginyl modification by ATE1 both in vitro and in vivo.

METHODS:
Peptides, peptide mixtures, proteins, cell proteomes, patient heart and brain tissue, and mouse tissues (lung, heart, and brain) were arginylated by ATE1 assay. The enzyme assay was successfully reconstituted including two key enzymes involved in the protein arginylation: RARS1 and ATE1. RARS1 charged the tRNA for arginine with either R0 or isotopically labeled R10 in solution, and ATE1 used this newly charged Arg-tRNA to label substrates. To incorporate isotopic Arg into proteomes, lysates from biological samples were used as ribosome-inactive conditions. Labeled proteomes were mixed, digested, and fractionated for proteomics analysis in data-dependent acquisition mode. For top-down proteomics, the platform was developed using calreticulin as a working standard as a known substrate of ATE1 arginylation. This protein was subjected to arginylation in vitro, in vivo, and on-bead during pull-down. Other individual protein standards were labeled with the same ATE1 assay. Labeled proteins were kept separately or in mixtures and analyzed in MRMHR mode to target specific charge states. Data was analyzed with a custom software “ArginylomePlot” for bottom-up and ProSight for top-down.

RESULTS:
In bottom-up proteomics, we first established the workflow using peptide (e.g.: standard peptide) and proteome-wide peptides (e.g.: HEK293T digest) as a proof-of-concept, then applied the technology to protein (e.g.: CALR) and whole proteomes (including iPSC cells, iPSC cardiac fibroblasts, iPSC cardiac myocytes, HEK293T, various cancer cells, patient heart, and patient brains) for arginylation discovery. As a result, a large catalog of unbiased arginylation sites (>200) has been established from various cell and tissue samples. Representative sites were validated and followed up for their biological pathways. In top-down proteomics analyses, analysis showed clear presence of two features with the 156 Da mass shift between WT and arginylated proteoforms visible following the application of the ATE1 assay. Calreticulin was readily arginylated and quantitatively determined to be an efficient target of this assay is various experiments. Further experiments validating substrates identified in bottom-up experiments demonstrated similar findings, with N-terminal arginylation reproducibly reconstituted.

DISCUSSION:
Arginylation is an essential PTM as demonstrated by the embryonic lethality of the knockout of ATE1. Given its wide role in diseases from cardiac disease, circulatory protein secretion, and aSyn folding and aggregation, understanding this PTM is crucial from a basic sciences and diagnostics perspective. Our work has generated the largest arginylation site library so far and established a series of arginylation assays (in vitro, in-bacteria, ex vivo, and in vivo) for functional validation. Arginylation profiling revealed new protein N-termini, thus opening new frontiers in protease cleavage. Further, we demonstrated the first ever experiment studying arginylation using top-down proteomics. This work could serve as the technological foundation for studying the functions of this essential PTM, and it will have a long-lasting impact on the arginylation field by opening new biochemical and biological frontiers. Further, given the observed arginylation across a diverse set of proteomes, including cancer cell lines and real patient samples, we believe there could be deep relevance to clinical pathologies that are not yet known where clinical mass spectrometry would excel.

A Chromatography-Free MS Method for the Ultrafast Quantification of Therapeutic Agents by DART-MS/MS for Research Purposes Zahuindanda Aventura (Presenter) Bruker Applied Mass Spectrometry

Poster #46c View Map

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

INTRODUCTION:
Many therapeutic agents require optimal concentration levels in matrix to function properly. Investigations using conventional LC-MS/MS analysis take ~5 minutes per run which means when blanks, calibrators, QCs, and patient samples need to be analyzed, the number of analyses can build up to several hours of turnaround time for a result. This study demonstrates that DART-MS/MS can provide a chromatography-free alternative for rapidly quantifying multiple classes of therapeutic agents in serum or plasma.

Direct Analysis in Real Time (DART) is a soft ionization technique coupled with tandem mass spectrometry to facilitate the rapid analysis of diverse sample types while minimizing both solvent and consumable usage. We detail the application of this approach to quantifying multiple panels of therapeutic agents including antiepileptics, antimycotics, tricyclic antidepressants, and immunosuppressants.

METHODS:
For preparation, 50 µL of matrix-matched calibrators, quality controls, and patient samples were spiked with a mixture of labeled internal standards. Liquid-liquid extraction is performed using 100 µL of solvent. For some analyses, 50 µL of a salt solution was added to assist in the phase separation. The samples were vortexed for 30 seconds and centrifuged at 10,000 rpm for 5 minutes. Aliquots of the upper layer were spotted on a QuickStrip HTS-96 DART plate and allowed to dry for 5 minutes on the bench.

For analysis, the plate was loaded onto an EVOQ DART TQ+ (Bruker Daltonics) triple quad mass spectrometer. Unique MS/MS transitions (quantitative and confirmatory) were determined for all analytes. DART source temperature, cone temp, cone gas pressure, and grid voltage as well as collision energies (CE) and scan time were optimized respectively for all panels. Spot size and scanning modes were also evaluated. Linearity and precision across the AMRs were tested for all analytes. High-throughput analysis was achieved with run times of less than 30 seconds per sample, allowing for the analysis of 96 samples in under 48 minutes.

Cross-correlation for DART-MS/MS results was tested with compounds which laboratories regularly have high volumes of patient samples. Topiramate, clozapine, levetiracetam, zonisamide, mycophenolic acid, fluconazole, itraconazole, OH-itraconazole, isavuconazole, posaconazole, and voriconazole were selected to be compared with LC-MS/MS results.

RESULTS:
In this study, linearity was evaluated by preparing five replicate calibration series in serum at 12 levels which cover the relevant concentration range for each compound. The method demonstrated excellent linearity (R2> 0.99) with all compounds showing %CV < 15% at all levels. Inter- and intra-day precision was measured in this study using matrix-matched QCs at three concentration levels. The inter-day precision study was performed over 10 days with four timepoints per day. The results for both precision studies were within acceptable limits and trueness ranged between 85% and 115% for all analytes.

A comparison with LC-MS/MS results was obtained in this study by preparing spiked serum samples as well as patient samples. These were measured on the DART-MS/MS method as well as a LC-MS/MS method. The comparison passed criteria for all tested analytes. The stability of extracted samples after spotting on the mesh was verified at room temperature over 7 h. No interference was found in this study for 144 substances including neuroleptics, antidepressants, and benzodiazepines. LOD and LOQ values obtained in this study were sufficiently sensitive to measure relevant concentrations for all therapeutic agents. Matrix effect and extraction efficiency experiments met criteria, confirming the method’s reliability in serum and plasma. No carryover was observed between low and high samples and internal standard contribution at LLOQ was < 20% for all analytes.

CONCLUSION:
The study data demonstrates the suitability of DART-MS/MS for analysis of therapeutic agents. The methods’ high throughput and simple sample preparations provide an advantageous alternative to traditional liquid chromatography-based methods which require expensive HPLC columns, buffers, and long analysis times. This allows for reduced operational costs and supports green chemistry. The research work also demonstrates early success in expansion to further compound classes such as neuroleptics, benzodiazepines, vitamins, and antibiotics.

Quantitative, Large Panel Metabolomics and Lipidomics with the MxP® Quant 1000 Kit Optimized and Validated for the Bruker EVOQ® TQ Mass Spectrometer Sebastian Gottfried (Presenter) biocrates

Poster #47c View Map

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

INTRODUCTION:
Standardized protocols and methods capable of generating reproducible results are essential for producing high quality scientific findings. The MxP® Quant 1000, a new ready-to-use, quality-controlled kit, has been developed and validated for quantitative metabolomics and lipidomics profiling. The kit targets 1,233 analytes across 49 biochemical classes including 327 small molecules and 906 lipids related to microbiome, metabolic health, and disease metabolism. The kit was built modularly; the small molecule panel and the lipid panel are available as separate kits: MxQuant and LxQuant, respectively. Instrument methods were optimized and validated for the Bruker EVOQ® TQ mass spectrometer. NIST SRM 1950, biocrates quality controls and human plasma samples were used to assess coverage, accuracy and reproducibility.

METHODS:
The kit consists of three patented 96-well filter plates, system suitability test mixtures, calibration standards, internal standards and quality controls (QCs), which were reconstituted according to protocol. Small molecules are analyzed across two plates: one designed for 3-NPH (3-nitrophenylhydrazin) and another one for PITC (phenyl isothiocyanate) derivatization. Lipids are analyzed on an individual plate without derivatization. Experimental samples, consisting of 11 human plasma samples (5 female, 6 male, age 17-65, absence of medical diagnosis), and NIST SRM 1950 were registered in the WebIDQ software and arranged together with the calibration and QC samples on a 96-well plate layout. All samples except the calibration standards were measured in replicates of three. The worklist was directly exported to the mass spectrometer software. The kit was prepared according to the user manual with a total of 40 μL of each sample pipetted per well across the three plates (20 µL for the 3-NPH plate, 10 µL for the PITC and lipid plates). Following sample preparation, extraction, and dilution, two measurement plates were prepared for small molecule analysis (3-NPH and PITC) and two for lipid analysis. Small molecules were analyzed using optimized LC-MS/MS methods, with one injection per plate: the 3-NPH plate in negative mode and the PITC plate in positive mode. Lipids were analyzed using optimized FIA-MS/MS methods, with two injections per plate: three in positive mode and one in negative mode. Data files were directly processed in WebIDQ. The quantified data was exported to R for plotting and evaluating analytical performance.

RESULTS:
The MxP® Quant 1000 kit was previously developed and validated with human plasma and urine samples and tested with human feces samples. Instrument methods were adapted and optimized on the Bruker EVOQ® TQ mass spectrometer connected to the Elute+ UHPLC system. All data collected was processed directly in WebIDQ with automated AI-driven peak picking, quantification, validation, and normalization. Data was quantified against internal standards or calibration curves. Built-in validation tools evaluated performance for accuracy and reproducibility based on included QC samples. The QCs were also used for data normalization, a crucial step in the workflow to correct for technical deviations caused by preparation, measurement, or batch effects, ensuring the consistency of the data.

Plasma-based human samples, NIST reference and pooled samples, and feces pooled samples were used to assess metabolite coverage and variability expectations in real samples. NIST SRM 1950 was used to assess accuracy based on established reference values. Samples which are part of a validation dataset were compared to show correlation with other validated instrument platforms.

CONCLUSION:
The EVOQ® TQ showed good accuracy, reproducibility, and coverage. Results highlight how standardized methods and preparation techniques combined with robust instrumentation can improve reproducibility enabling enhanced confidence in cross-laboratory and longitudinal studies.

Research use only.

Development of A Rapid Chromatography-free Method for the Quantification of 8 Antimycotic Drugs by DART-MS/MS for Research Purposes Lukas Brunner (Presenter) RECIPE Chemicals + Instruments

Poster #48c View Map

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

INTRODUCTION:
This work aimed to validate a rapid and quantitative method for measuring antifungal agents utilizing Direct Analysis in Real Time-Mass Spectrometry (DART-MS). Quick and accurate testing is crucial for effective antifungal therapy; therefore, the focus was on ensuring the method's robustness while reducing analysis time compared to LC-MS. We detail the development and validation of a chromatography-free approach to quantifying eight common antifungal agents in serum: 5-fluorocytosine, fluconazole, hydroxy-itraconazole, isavuconazole, itraconazole, ketoconazole, posaconazole, and voriconazole.

METHODS:
For preparation, 50 µL of matrix matched calibrators, quality controls, and patient samples were spiked with a mixture of labeled internal standards and liquid-liquid extraction is performed using 100 µL of solvent. The samples were vortexed for 30 seconds and centrifuged at 10,000 rpm for 5 minutes. Aliquots of the upper layer were spotted on a QuickStrip HTS-96 DART mesh and allowed to dry for 5 minutes on the bench. For analysis, the mesh was loaded onto a DART TQ-Plus (Bruker Daltonics) mass spectrometer. Mass transitions, collision energy, source temperature, spot size, cone gas, and grid voltage were optimized for the DART. Linear scanning was employed to successfully measure 96 samples in under 42 minutes, with an analysis time of 26 seconds per sample.

RESULTS:
Linearity for the DART-MS/MS method was tested by preparing five replicate calibration series in serum at 12 levels covering the relevant concentration ranges. The method demonstrated excellent linearity (R² > 0.99) with all compounds showing %CV of less than 10% at all levels. Inter and Intra-day precision was determined using matrix matched quality controls at three levels with the inter-day precision study being performed over 20 days with two timepoints per day. The results for both precision studies were within acceptable limits (<15% CV), and accuracy ranged between 85% and 115% for all analytes. Correlation with LC-MS results was tested by preparing 105 spiked serum samples as well as 20 patient samples that were measured on the DART-MS method as well as a validated LC-MS method. Correlation passed criteria for all analytes. LOD and LOQ values were sufficiently sensitive to measure therapeutic and subtherapeutic drug concentrations for all 8 antifungal agents. Matrix effect and extraction efficiency experiments met criteria, confirming the method’s reliability in serum and plasma.

CONCLUSION:
The validated DART-MS method is a robust, accurate, and efficient tool for quantification of antifungal agents. The method’s high throughput and simple sample preparation provide an advantageous alternative to traditional liquid chromatography-based methods.

Sample Cleanup Approaches for the Analysis of A Multi-Class Drugs of Abuse Panel in Breast Milk Esraa Abojasser (Presenter) Biotage

Poster #49c View Map

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

INTRODUCTION:
Breastfeeding offers significant health benefits for both infants and mothers, promoting optimal nutrition, immune protection, and developmental outcomes. However, maternal use of illicit drugs or misuse of prescription medications during lactation can result in the transfer of potentially harmful substances into breast milk, posing potentially harmful risks to the nursing infant. Consequently, the ability to accurately detect and quantify drugs of abuse in breast milk is critical for clinical toxicology, forensic investigations, and public health monitoring

OBJECTIVE:
This study demonstrates a reproducible and sensitive analytical workflow for the extraction and detection of commonly encountered drugs of abuse in human breast milk with good recoveries, low detection limits, and reliable quantification across the range of analytes.

METHODS:
A panel of 55 analytes with diverse chemical backbones and a wide range of LogP (-0.6-7.05) and pKa (0.13-10.47) was spiked into breast milk aliquots at a concentration range of 0.01-50ng/mL. Sample extraction was investigated by comparing protein precipitation, protein and phospholipid depletion (PLD), supported liquid extraction (SLE), and cSPE for QuEChERS. Extraction performance was compared by evaluating extract recovery, matrix effect, and reproducibility. The optimized protocol using cSPE for QuEChERS was transferred to the Extrahera® automated sample preparation workstation and evaluated for linearity and precision. LC-MS/MS analysis was performed using a Shimadzu Nexera X2 UHPLC system coupled to a SCIEX 5500 QTrap MS system.

RESULTS:
Breast milk can be a challenging biomatrix due to the high protein and lipid content. These matrix components can interfere with target drug analytes analysis causing ion suppression. We observed consistent extraction performance using the cSPE for QuEChERS workflow, with the majority of investigated analytes achieving 70%-90% recovery with excellent reproducibility (RSD=6-15%) with the exception of amphoteric analytes such as ritalinic acid, gabapentin, and pregabalin, with recoveries around 30-35%, and strongly and moderately polar benzodiazepines and opioids, which recoveries ranged between 55-70%. The cSPE QuEChERS extraction effectively removed over 99% of the phospholipids that co-extracted during protein precipitation, while maintaining good extraction recoveries for the target analytes. In comparison, lower recoveries and higher matrix effects were observed when analytes were extracted with the other techniques. The optimized cSPE QuEChERS extraction method was simpler and faster than traditional SPE.

CONCLUSION:
The cSPE for QuEChERS provided better recoveries for the target panel of drugs of abuse testing in breast milk while maintaining low matrix effects and RSDs below 3%. The final optimized protocol demonstrated excellent linearity and coefficients of determination, r2 > 0.99 for all analytes.

Lipidomic Analysis of the Adrenergic Response in Microvascular Endothelial Cells Óttar Rolfsson (Presenter) University of Iceland

Poster #50c View Map

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

INTRODUCTION:
Poor prognosis of patients suffering from acute critical illness (ACI) is associated with endothelial dysfunction and hyper-activation of the sympathetic nervous system as evident by toxic levels of catecholamines. This syndrome is entitled shock-induced endotheliopathy (SHINE)(1). Clinical phenotypes of SHINE show similarities to non-thyroidal illness syndrome. To investigate the molecular basis of SHINE, we developed an in vitro model using human pulmonary microvascular endothelial cells (HPMVECs). Transcriptomic analysis revealed that the cholesterol transporter GRAMD1B is differentially regulated in response to catecholamines.

OBJECTIVES:
We are working towards confirming changes to cholesterol metabolism and to understand the significance of cholesterol transport following the catecholamine hit in the context of SHINE.

METHODS:
Cells were treated with catecholamines at 0.5, 5 and 50 μM concentration and harvested at 4h and 24h. Proteins were extracted from whole cell lysates, denatured and digested using a Trypsin/Lys-C mix. The peptide mix was desalted using solid-phase extraction, dried and resuspended prior to liquid chromatography-mass spectrometry (LC-ESI-MS) analysis using a Waters Synapt G2 system. Lipids were extracted using the Bligh and Dyer method and analysed by LC-ECI-MS using a Waters Synapt G2 system operating in ion mobility mode using the method of Paglia et. al. (2) Proteomic data was analyzed within Progenesis and lipidomic raw data were analysed using Progenesis and Lipostar. Significance thresholds were set at p < 0.05 with the Benjamini-Hochberg correction applied for multiple testing. Immunostaining for markers of endothelial dysfunction and seahorse flux analysis were employed to support and verify changes to cell phenotypes following the catecholamine hit.

RESULTS:
Thus far our analysis indicates that cholesterol drops during the adrenergic response in vascular endothelium and that knockout of the GRAMD1B cholesterol transporter blocks this decrease and is accompanied by impaired oxidative phosphorylation. Changes to bile acid profiles were also observed following the hit and altered following GRAMD1B knockout.

CONCLUSION:
We hypothesize that the adrenergic response induces bile acid synthesis that regulates T4 synthesis in endothelium as has been reported previously in adipose tissue. This link may explain similarities in clinical phenotypes of patients with SHINE and those suffering from non-thyroidal illness syndrome.

REFERENCES:
1. Henriksen, Hanne, et al. Metabolic Systems Analysis of Shock-Induced Endotheliopathy (SHINE) in Trauma: A New Research Paradigm. Annals of Surgery 272(6):p 1140-1148, December 2020.
2. Paglia G. et al. Ion mobility-derived collision cross section as an additional measure for lipid fingerprinting and identification. Anal Chem. 2015 Jan 20;87(2):1137-44.

A Comprehensive LC-MS Exposomics Assay for Quantitative Analysis of Serum and Urine Rupasri Mandal (Presenter) University of Alberta

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Poster #51c View Map

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

INTRODUCTION:
Exposomics, the study of environmental exposures, life style, diet and their effects on health, and metabolomics, the analysis of small molecules (metabolites) in biological samples, are increasingly used together to understand how environmental and other factors influence human health. Metabolomics can provide insights into the impact of environmental exposures on metabolic processes, helping to identify potential biomarkers and mechanisms of disease.

OBJECTIVE:
In this quest, we have developed a custom made, comprehensive, quantitative LC-MS/MS-based assay for targeted exposomic (environmental and diet) compounds analysis of biospeimens such as serum and urine. This assay allows for the identification and quantification of up to 265 exposomic compounds.

METHODS:
Our method uses a reverse phase LC-MS/MS in both positive and negative ionization modes to separate metabolites. It combines the derivatization and extraction of analytes, and the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Two separate panels involving two different precolumn derivatization reactions were developed for this assay: Panel A - Phenylisothiocyanate (PITC) derivatization targeting amine-containing compounds and Panel B - 3-nitrophenylhydrazine (3-NPH) derivatization targeting keto- and carboxyl-containing compounds. Isotopically-labeled internal standards are used for metabolite quantification. Calibration of metabolite concentration ranges in both panels was adjusted for different biofluid types.
For panel A, a 96 deep-well plate with a filter plate attached via sealing tape, containing the required reagents and solvents, was used to prepare the plate assay. The first 14 wells of each plate are used for calibration and quality control purposes. For all metabolites, except organic acids, samples are first thawed on ice and then vortexed and centrifuged at 13,000x g. 10 µL of each sample are loaded onto the center of the filter on the upper 96-well plate and dried in a stream of nitrogen. Subsequently, PITC is added for derivatization. After incubation, the filter spots are dried again using an evaporator. Extraction of the metabolites is then achieved by adding 300 µL of extraction solvent. The extracts are obtained by centrifugation into the lower 96-deep well plate, followed by a dilution step with MS running solvent.

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

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

RESULTS:
This custom assay can be used for the targeted identification and quantification of up to 265 metabolites across 16 chemical classes including amino acids and derivatives, biogenic amines, acylcarnitines, organic acids, fatty acids, nucleotides/nucleosides, bile acids, uremic toxins, parabens, plasticizers, pharmaceutical intermediates and other exoposomic compounds. The accuracy of QC standards with 3 different concentration levels are in the range of 80% to 120% with satisfactory precision values of less than 20%. The recovery rates of spiked serum, urine and fecal extract samples with three different concentration levels are in the range of 80% to 120% with satisfactory precision values of less than 20%.

CONCLUSIONS:
We have developed a comprehensive, sensitive, high-throughput, low-volume, quantitative targeted LC-MS/MS assay for the analysis of up to 265 exposomic compounds, across 16 chemical classes for serum and urine samples. Only 40 µL of a given sample are required for the entire analysis. We have adapted this assay into a 96-well plate format to enable high-throughput analysis.

Evaluation of Two Point of Care Drug Testing Kits Against Liquid Chromatography Tandem Mass Spectrometry Lisandra Cubero Herrera (Presenter) Nova Scotia Health & Dalhousie University

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Poster #52c View Map

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

INTRODUCTION:
This study evaluates the performance of two immunoassay-based point of care testing (POCT) kits for urine drug screening in remote clinics where access to advanced technologies like liquid chromatography tandem mass spectrometry (TMS) is limited. The goal is to determine if the POCT kits can serve as a reliable first line testing approach for monitoring patient adherence to pain management programs compared to TMS.

METHODS:
Forty-six (46) medical urine samples were tested by TMS (SCIEX 6500+ QTRAP, Concord, ON, Canada), and two POCT kits, the Rapid Response™ Multi-Drug Test Panel (RR) (BTNX Inc., Markham, ON, Canada) and the SpecCheck Multi-Panel Test Cup (SC) (Spectrum Medical Diagnostics, Mississauga, ON, Canada). Both POCT kits use lateral flow immunoassay, where labeled drug-protein conjugates compete with drugs in urine for antibody binding sites on a membrane strip. Drugs detected and their respective cutoff concentrations (in ng/mL) were (TMS, RR, SC): cocaine metabolite (benzoylecgonine) (100, 150, 150), methamphetamine (250, 1000, 500), amphetamine (250, 1000, 500), benzodiazepines (100, 300, 300), methadone metabolite (EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine) (100, 100, 100), buprenorphine (40, 10, 10), morphine (300, 300, 300), hydromorphone (100, 500, 250), oxycodone (100, 100, 100) and fentanyl (10, 10, 20).

RESULTS:
TMS detected the following true positives across the 46 samples: benzoylecgonine (17), methamphetamine (7), amphetamine (19), benzodiazepines (18), EDDP (9), buprenorphine (10), morphine (9), hydromorphone (23), oxycodone (7), and fentanyl (2). Several discrepancies were noted when comparing POCT with TMS. Below, the term POCT refers to scenarios where both RR and SC yield the same result, either negative or positive.

All 17 TMS-positive samples for benzoylecgonine were detected by POCT. However, one additional sample, which was below the TMS cutoff, tested positive with POCT despite the higher POCT cutoff, indicating a false positive.

All 7 TMS-positive samples for methamphetamine were detected with RR, but two with low concentrations (<250 ng/mL and 293 ng/mL) tested negative with SC, indicating stricter SC cutoff adherence. Amphetamine, detected in 19 samples by TMS (either alone or as a metabolite of methamphetamine), was consistently identified by both POCT kits. Notably, five of these samples were below the RR cutoff, and two of these, were also below the SC cutoff. One sample without amphetamine, but with high hydroxy bupropion (metabolite of bupropion and a known interferent), tested negative by POCT, indicating low cross reactivity in this case.

POCT showed good agreement for benzodiazepines with TMS for six samples containing diazepam metabolites (nordiazepam, oxazepam, and temazepam), and one with only oxazepam. Five of six lorazepam-only samples were POCT positive, four above the POCT cutoff, and one (120 ng/mL) below. A sample with 145 ng/mL lorazepam tested positive only with RR, indicating variability near the cutoff. Four out of five samples with only 7-amino clonazepam (metabolite of clonazepam), tested negative with POCT; three were below the POCT cutoff (233, 298 and 111 ng/mL) while one was above (620 ng/mL). Another sample with 947 ng/mL tested negative with SC, suggesting a ~20% false negative rate for clonazepam detection for POCT.

Eight out of nine EDDP-positive samples by TMS were detected by both POCT assays. One at 256 ng/mL, tested negative with POCT (false negative), and one TMS-negative sample tested positive with RR (false positive).

All ten buprenorphine-positive samples with TMS were detected by POCT. Additionally, one sample negative for buprenorphine but positive for norbuprenorphine (125 ng/mL), tested positive with POCT. Another sample negative for both buprenorphine and norbuprenorphine, tested positive with RR, indicating a likely false positive.

All nine TMS-positive samples for morphine were also detected by POCT, eight of these also contained hydromorphone. Ten additional samples that were morphine-negative but had high hydromorphone levels, tested positive for morphine with POCT, indicating false positives for morphine, due to poor differentiation between morphine and hydromorphone in POCT. One sample with neither morphine nor hydromorphone, tested positive for morphine by POCT. Hydromorphone was detected in 24 samples by TMS. Of these, 16 contained hydromorphone as the primary drug with no morphine, while 8 contained hydromorphone as a metabolite of morphine. One sample with high morphine, but no hydromorphone, tested positive for hydromorphone with POCT, while another sample with high hydromorphone tested negative with POCT. Four samples with no morphine or hydromorphone tested negative with RR, but positive with SC; two of these contained oxymorphone, however, four other oxymorphone-containing samples tested negative for hydromorphone across all methods. Oxycodone was detected in 7 samples, without morphine or hydromorphone. However, 3 other samples without oxycodone, but with high hydromorphone, tested positive for oxycodone with POCT. One more sample tested positive only with SC, indicating possible false positives due to hydromorphone interference. The two fentanyl positive samples detected by TMS showed consistent results across the assays.

CONCLUSION:
Two POCT kits, Rapid Response (RR) and SpecCheck (SC), were evaluated against TMS for urine drug screening. While POCT provides rapid, cost-effective results, it is prone to both false positives (e.g., morphine when high hydromorphone is present) and false negatives (e.g., missed clonazepam detection due to lack of sensitivity to 7-amino clonazepam), requiring cautious interpretation. Both kits demonstrated limitations in reliably distinguishing between certain opiates (especially SC) and detecting specific benzodiazepines, suggesting TMS may be needed for confirmation when POCT results are inconclusive.

Time-Dependent Metabolite Stability & General Suitability of DBS and Volumetric Absorptive Microsampling VAMS® Using Mitra® Tips for Quantitative Metabolomics Traci Mizuno (Presenter) Trajan Scientific and Medical

Poster #53c View Map

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

BACKGROUND:
Remote sampling for health monitoring is gaining traction due to its convenience and potential for decentralized diagnostics. This study evaluates the stability and detectability of metabolites in dried blood samples collected via two methods: classical dried blood spots (DBS) and volumetric absorptive microsampling (VAMS®) using Mitra® tips. The goal is to determine their suitability for quantitative metabolomics, particularly under conditions mimicking home sampling and transport delays.

METHODS:
Using the Biocrates MxP® Quant 500 kit, which quantifies up to 630 metabolites, samples from three healthy individuals were analyzed after storage at room temperature for up to seven days. Stability was assessed based on detectability in ≥50% of samples, concentration retention within 85–115% of baseline, and acceptable coefficient of variation (CV) thresholds.

RESULTS:
After three days at room temperature, 273 metabolites were stable and detectable in DBS, while 249 were stable and detectable in Mitra tips. DBS showed superior lipid stability, detecting 219 stable lipids versus 180 in Mitra tips. Conversely, Mitra tips outperformed DBS in preserving polar small molecules, including amino acids, biogenic amines, bile acids, and acylcarnitines. For example, Mitra tips enabled quantification of 18 acylcarnitines compared to only five in DBS.

Extended storage revealed a decline in metabolite stability: after five days, only 194 metabolites remained stable in Mitra tips. Lipids were more prone to degradation due to oxidation, which is influenced by sample surface area. DBS, with a smaller surface area, preserved lipids better, while Mitra tips preserved small molecules more effectively due to rapid drying, limiting enzymatic degradation and reducing hemolysis to a minimum.

Extraction methods significantly affect metabolite recovery. Methanol-based extraction used in this study proved effective for both lipids and small molecules. However, inter-laboratory comparisons remain challenging due to variability in extraction protocols. Stabilizers can enhance the stability of specific metabolites, such as omega-3 and omega-6 fatty acids, though they may interfere with other analytes.

CONCLUSIONS:
From a clinical perspective, Mitra tips are preferable for monitoring health indicators like neurotransmitters, bile acids, and acylcarnitines. They also have the advantage of a reduced hematocrit bias. DBS are more suitable for assessing lipid profiles, including triacylglycerols, and trimethylamine N-oxide (TMAO), which are relevant for cardiovascular risk. Hexose stability in DBS also supports their use in blood sugar monitoring.

In conclusion, both DBS and Mitra tips are viable for remote metabolomics, with each method offering distinct advantages. Mitra tips provide better stability for small molecules and ease of use, making them ideal for personalized, predictive, preventive, and participatory (4P) medicine. DBS remain valuable for lipid-focused analyses. The choice of sampling method should align with the target metabolite class and intended clinical application.

Automated Liquid Chromatography Tandem Mass Spectrometry Analysis of Phosphatidylethanol (PEth) Spencer Seely (Presenter) University of California, San Diego

Poster #54c View Map

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

INTRODUCTION:
Alcohol Abuse Disorder (AUD) or excessive ethanol consumption is detrimental to human health. Questionnaires regarding consumption can be inaccurate for the identification of at-risk patients. Indirect biomarkers, such as ethyl glucuronide (EtG) and ethyl sulfate (EtS), have short half-lives, limiting their effectiveness in the determination of chronic abuse. Phosphatidylethanols (PEths), phospholipids conjugated to ethanol, are biomarkers of ethanol use and are detectable 2-4 weeks post-consumption. Most recent published procedures for PEth analysis require time-consuming and complex extraction protocols. We describe an LC-MS/MS laboratory developed test (LDT) which employs a simple extraction process that can be performed manually or with an automated liquid handler to reduce overall turnaround time.

METHODS:
The measurement of two PEth homologues: 16:0/18:1 (POPEth) and 16:0/18:2 (PLPEth) was performed by isotope dilution LC-MS/MS in clinical whole blood specimens. Samples were extracted using an automated liquid handler using 0.1 M zinc sulfate solution and 50:50 acetonitrile: isopropanol. Extracts were vortexed for 5 minutes, followed by centrifugation at 2000 RCF for 5 minutes. Supernatants were collected and directly injected for analysis by LC-MS/MS. A retrospective analysis of 1847 patient results was performed to identify trends in the two measured homologues.

RESSULTS:
The analytical measurement range (AMR) for both POPEth and PLPEth was determined as 10 - 1,500 ng/mL, with strong linearity observed across the measure range (R2 >0.995). Calibrator and quality control biases were found to be within 15% of correlated values from an external reference laboratory. Biases were observed for the measurement of POPEth in a subset of externally correlated patient specimens. Isotope dilution studies were performed to assign the appropriate quantifier ion transition to resolve an unknown interferant. Retrospective patient data was used to evaluate PLPEth concentrations relative to established POPEth reference ranges; revealing similar ranges. The PLPEth:POPEth ratio was evaluated and found to be statistically higher in patients with light consumption (10-20 ng/mL POPEth) compared to moderate (20-200 ng/mL POPEth, p-value <0.0001), and heavy (>200 ng/mL POPEth, p-value= 0.0014) consumption; revealing additional utility for the measurement of PLPEth.

CONCLUSION:
This development of and LC-MS/MS LDT enables the accurate measure of PEth and compares well to external reference laboratory results. Reduction in turnaround time provides more actionable information for clinicians treating alcohol abuse and preventing organ damage during transplantation. More investigation is needed to establish reference ranges for PLPEth, which can provide additional insights to monitoring alcohol consumption and abuse.

Evaluation of the Detuning Ratio as a Tool to Detect Potential Interference in LC-MS/MS Analysis Katharina Habler (Presenter) LMU University Hospital, LMU Munich

Poster #15c View Map

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

INTRODUCTION:
While MS/MS is in general a highly specific technique, the presence of unexpected substances in samples can result in interference due to shared mass transitions with target analytes (isomeric/isobaric interferences). Clinical laboratories typically address this issue by monitoring ion ratios (IR) to recognize potential interferences. To supplement this approach, differential tuning effects can be assessed. We aimed to evaluate a complementary method - the detuning ratio (DR) - for its ability to detect isomeric or isobaric interferences.

METHODS:
A DR was based on differential influences of instrument settings of MS systems on the ion yield of a respective target analyte; isomeric or isobaric interferences can lead to a shift of the DR in an affected sample. By quantifying the concentration and determining the IR and DR in samples in which known isomeric interference substances have been spiked to the target analyte, the applicability of DR detection was quantitatively investigated and compared with concentration as well as IR. This experiment was performed for two compound pairs (Cortisone/Prednisolone and O-Desmethylvenlafaxine/cis-Tramadol HCl). In both pairs matching mass transitions can be observed in a collision induced dissociation scan.

RESULTS:
The DR method correctly indicated the presence of isomeric interferences in two independent test systems: Cortisone/Prednisolone and O-Desmethylvenlafaxine/cis-Tramadol HCl. In these two spiking experiments, we observed that the DR and the IR were both suited to indicate interference in a quantitative model system of interference by known compounds sharing mass transitions with the respective target analyte. DR was found highly reproducible in analytical runs (with a CV ≤5.3%, n=20).

CONCLUSION:
The DR approach provides a valuable supplementary tool for detecting isomeric or isobaric interferences in individual samples analyzed by LC-MS/MS. When used alongside conventional IR monitoring, it can improve the analytical reliability of clinical MS-based assays. A major advantage of DR over IR is its applicability to target analytes with only a single sufficiently abundant mass transition. This is particularly important for immunosuppressants such as tacrolimus and cyclosporine.

The DR principle has a broad potential and can be used in all areas of LC-MS/MS application, even beyond biomedical analyses.

Python Scripts Generated Using a Large Language Model for Automated IGF-1 Variant Identification in Clinical Specimens Ievgen Motorykin (Presenter) Quest Diagnostics

Poster #6c View Map

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

INTRODUCTION:
The detection and identification of insulin-like growth factor-1 (IGF-1) variants by liquid chromatography, high-resolution accurate mass, mass spectrometry (LC-HRAM-MS) have become increasingly adopted in recent years. Knowledge of the presence of these variants may help clinicians understand growth disorders. Quest Diagnostics reports the presence and, if available, identity of homo- and heterozygous IGF-1 variants along with quantitative assay results. However, LC-HRAM-MS is targeted to currently search for a list of only 22 previously reported variants. Achieving untargeted searches for unreported variants requires automation of data analysis with the help of programming languages and executable algorithms.

The advent of artificial intelligence (AI) and large language models (LLM) has revolutionized the field of data analysis, and these technologies are accessible to individuals with limited prior programming knowledge. Beginners can focus on learning the underlying concepts rather than getting bogged down by syntax details. For example, these cutting-edge technologies can enable users to write Python code for complex data analysis tasks by simply describing their requirements in plain language. In this way, AI-powered tools can generate customized Python scripts tailored to the user specifications, thus eliminating the need for extensive coding knowledge. This integration between human language and machine-executable code has opened doors for individuals with limited coding experience to explore data analysis, allowing them to extract valuable insights from vast datasets without needing to learn complex programming languages.

OBJECTIVE:
Write a Python code for automated untargeted searches for IGF-1 variants in LC-HRAM-MS results from routine clinical specimens using an LLM (GitHub Co-pilot).

METHODS:
Software included Microsoft Visual Studio with built-it Git Hub Co-pilot, latest version of Python (v. 3.13), and Python language support extension. The practice data file was a de-identified patient sample that contains an IGF-1 variant. No proprietary or patient identifying data was shared with the LLM. All suspected IGF-1 variants will be confirmed with DNA sequencing.

Co-pilot was initially prompted to write a Python script to scan a specific folder, and subfolders within, containing MS data and convert any RAW file to MGF using a free open-source utility ThermoRawFileParser.exe with commands -i=[file path] and -f=0 for MGF output. Simple language prompts, such as “write a script that reads an MGF file and then parse variables SCANS, RTINSECONDS, and pairs of mass-to-charge ratio (m/z) and intensity of detected ions for each scan” were then used.

After verifying the functionality of the script, task complexity was increased to find clusters of ≥9 IGF-1 variant (MH7)7+ isotopic peaks at m/z’s within wider isotopic envelopes for each variant. Within each variant cluster, the most intense peak was specified to have an intensity of ≥60% of the most intense peak in the (MH7)7+ isotopic envelope of the wild type (WT) protein, or at least 15,000 counts. Finally, Co-pilot was prompted to list the findings as m/z clusters with corresponding cluster intensity. In addition to the text output, the script created an image file of the identified cluster and placed it in the original folder.

RESULTS:
Two scripts and executable programs were produced. The first script, containing only 30 lines of code, converts RAW files to MGF. The second script, containing 95 lines of code, does the cluster search. Dozens of peak clusters with specified characteristics were identified in data generated from the practice data file, and listed in the report as m/z’s, cluster intensity, and images for each MS file.

Below is an example of the output of the script that identifies a cluster suspected to be an IGF-1 variant, which could be either T41I, or a novel IGF-1 variant.

SCAN: 85
RTINSECONDS: 25.0328448
Clusters:
Cluster 1: [1094.67334, 1094.81482, 1094.95703, 1095.09814, 1095.24121, 1095.38464, 1095.52698, 1095.67029, 1095.81335, 1095.95667, 1096.09888, 1096.24194, 1096.3833]
Most intense peak: m/z: 1095.38464, intensity: 780548.438

Some clusters corresponded to known compounds, such as IGF-1, IGF-2, isotopically labeled internal standard, and known variants. A limitation of the method was that some clusters were redundant (e.g., isotopic peaks IP0-IP8 and IP2-IP10 being identified as different clusters but belonging to the same isotopic envelope [subscript corresponds to the increasing number of heavy atoms for each the isotopic peak]).

DISCUSSION:
We have successfully implemented LLM supported script to write a Python code for automated untargeted searches for IGF-1 variants. Further development requires a universal function that identifies unique isotopic envelopes and eliminates redundant clusters. Conversely, the function should also recognize different clusters in overlapping isotopic envelopes. In addition, the script will work on the background on all acquisition instruments. Ultimately, we hope to attain more accurate search results with just a few potential isotopic envelopes per specimen that need manual review.