| Topic(s): Other -omics > Tox / TDM / Endocrine > Pre-Analytics |
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Poster Presentation Poster #9 Attended on Wednesday at 09:15
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 Comparison of Venous and Capillary Dried Blood Spot Sampling for Lead Measurement by ICP-MS: Implications for Pediatric Testing
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Chelsea Swartchick (Presenter) Mayo Clinic >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Lead is a toxic metal associated with irreversible neurodevelopmental damage. Children are particularly at risk due to rapid development, increased gastrointestinal absorption, and an underdeveloped blood–brain barrier, making early detection and intervention critical. Blood lead testing is recommended during early childhood, typically at 12 and 24 months of age. Venous whole blood analyzed by inductively coupled plasma mass spectrometry (ICP-MS) is the reference standard for lead measurement. However, venipuncture (VP) is not always feasible due to practical challenges in pediatric sampling. Therefore, low-volume sampling strategies are needed. Dried blood spots (DBS) obtained via capillary collection offer a minimally invasive alternative suitable for decentralized testing. ICP-MS provides the analytical sensitivity required for trace metal quantification from limited-volume specimens, enabling evaluation of capillary DBS for lead measurement. This study compares capillary DBS and venous whole blood analyzed by ICP-MS. METHODS: Whole blood from normal donors (n = 31) was collected via VP into EDTA trace metal–free tubes, and in parallel, capillary blood was obtained via fingerstick and applied to Capitainer® filter paper devices (10 µL per spot). An additional 20 residual stored whole-blood samples with previously measured lead concentrations within the analytical measuring interval (AMI: 1–200 µg/dL) were also applied to the filter paper devices, extracted, and analyzed for lead. VP samples were prepared using a 1:50 dilution with 10 µM EDTA diluent, while DBS samples were extracted with 0.01% Triton X-100, followed by 1% hydrochloric acid solution, and diluted [1:100] prior to analysis. Lead concentrations were quantified using a PerkinElmer NexION inductively coupled plasma mass spectrometer (ICP-MS). Differences between VP and DBS-derived lead concentrations were calculated as [LeadVP] − [LeadDBS], and agreement was evaluated using a pre-defined acceptance criterion of ±1.45 µg/dL. RESULTS: At the medical decision point (3.5 µg/dL), DBS specimens demonstrated within-run precision of 3.9% CV and interlaboratory precision of 5.8% CV. Venous samples from normal donors were below the lower limit of quantitation (LLOQ, <1.0 µg/dL), with corresponding capillary DBS samples also below the LLOQ. Twenty residual whole-blood specimens spanning 3.5–49.6 µg/dL were evaluated. Following application to Capitainer® devices and extraction, DBS samples showed a median lead concentration of 6.5 µg/dL (IQR: 13.7 µg/dL). Compared to the corresponding venous reference measurements, DBS demonstrated minimal bias (mean ± SD: −0.002 ± 0.445 µg/dL), with a mean percent difference of −0.3% (SD: 4.0%) and a mean absolute percent difference of 2.9%. DISCUSSION: Capillary DBS sampling demonstrated strong agreement with venous whole blood ICP-MS for lead, with minimal bias (−0.002 µg/dL; −0.3%) and low variability across a clinically relevant range. DBS demonstrated within-run and between-run precision of 3.9% and 5.8% CV, respectively. The achieved precision supports reliable classification near lead’s clinically actionable threshold, where small analytical variation could influence follow-up and intervention decisions. Although prospectively collected donor samples were below the lower limit of quantitation (<1.0 µg/dL), residual specimens (original venous lead concentrations 3.5–49.6 µg/dL) enabled evaluation across clinically relevant and elevated concentrations. Across this range, DBS results closely matched reference venipuncture measurements, demonstrating accuracy and reproducibility of the collection method. These findings support DBS microsampling coupled with ICP-MS as a reliable low-volume alternative to venipuncture for trace metal analysis and may improve pediatric screening feasibility, enabling earlier detection of lead exposure and earlier clinical intervention. |
| Topic(s): Small Molecule > Metabolomics > Emerging Technologies |
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Poster Presentation Poster #11 Attended on Wednesday at 09:15
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 On-Line 2D Mixed-Mode Anion Exchange-Reverse Phase LC-DMS-MS/MS Improves the Efficiency and Specificity of Urinary 2,3-dinor 11β-Prostaglandin F2α Measurement
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Ryan Pearce (Presenter) Mayo Clinic >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: 2,3-dinor 11β-Prostaglandin F2α (2,3-BPG) is released during mast cell activation making it a non-invasive urinary biomarker for mast cell disorders.1 While 2,3-BPG is the most abundant prostaglandin D2 metabolite, it poses an analytical challenge for LC-MS/MS analysis due to numerous structurally related interferences in urine.1 Prior research has demonstrated that differential mobility spectrometry (DMS) improves specificity for 2,3-BPG when combined with off-line anion exchange solid phase extraction.2 We sought to develop an on-line two-dimensional (2-D) LC-MS/MS method using mixed-mode anion exchange (MAX) coupled with reverse phase chromatography and SelexION DMS to reduce interferences while preserving the existing liquid-liquid sample extraction method.2 A preliminary study also assessed whether the on-line 2-D method could support dilute-and-shoot preparation to reduce hands-on time. METHODS: An on-line 2-D MAX-reverse phase LC method was developed on a Thermo Scientific Transcend TLX-4 UHPLC with TriPlus RSI autosampler, Vanquish binary and quaternary pumps, and valve interface module. The first dimension used a Waters Oasis MAX Cartridge (80Å, 30 µm, 2.1 x 20 mm) loaded with 0.05% ammonium hydroxide and eluted with an acidic organic plug into a Waters CORTECS T3 column (120Å, 1.6 µm, 2.1 x 50 mm, 60 °C) for analytical separation. 40 µL of extracted urine was injected and loaded for 1.5 min. Flow was reduced and valves switched to place the elution loop in line with the trap column while combining flow with the second dimension. After transfer over 1 min, valves were switched back to isolate each column. After a gradient ramp, 40 %B was held for 6.84 min. Flow was diverted to the MS from 8.5 to 11.5 min, followed by an organic wash for 1.67 min. Total run time was 14.08 min. During separation, the trap was washed at high flow (1-2 mL/min) followed by loop filling and re-equilibration. MS was performed using a Sciex 6500+ with SelexION in negative ion mode (IS -4500, TEM 550 °C, CUR 35, CAD 9, GS1 75, GS2 20; DP -40, EP -10, CXP -12). DMS: DT low, no modifier, SV 3875, COV 10, DMO 20, DR low. Three MRM transitions were monitored: 325.3 > 163.0 (CE -24), 325.3 > 145.1 (CE -20), and IS 334.3 > 145.1 (CE -24). The reference method was performed as previously described.2 Briefly, 1 mL of urine was combined with 25 µL of IS, acidified with 50% acetic acid, and extracted with ethyl acetate. The organic phase was evaporated and reconstituted in 135 µL mobile phase. 10 µL of sample was injected onto a legacy Transcend TLX-4 system with Shimadzu LC-20 pumps and a Sciex 5000 MS. Standards, QCs, and 41 patient samples were analyzed by both methods. Results were processed in Sciex OS using linear regression (1/x weighting) from 312.5-10,000 pg/mL. Statistical analysis was performed in Microsoft Excel and Python 3.11. RESULTS: 2,3-BPG results were compared between the reference and 2D-SelexION methods using concentrations derived from calibrating each transition separately to assess specificity. For m/z 163, comparison of 2D-SelexION to reference yielded a Passing-Bablok slope of 0.772 (95% CI 0.576-0.859, y-int=5.01, r=0.938) and Bland-Altman percent bias of -37.0%. For m/z 145, the slope was 0.805 (95% CI 0.707-0.925, y-int=38.2, r=0.969) with percent bias of -20.3%. Within-method m/z 163/145 fragment ion agreement differed substantially between the reference and 2D-SelexION methods. The reference method gave a slope of 0.896 (95% CI 0.850-0.965, y-int=-6.05, r=0.990) with percent bias of -17.1%. The 2D-SelexION method gave a slope of 0.980 (95% CI 0.936-1.012, y-int=11.49, r=0.999) with percent bias of 2.6%. The 2D-SelexION method demonstrated reduced ion ratio variability across the comparison batch compared to the reference method (CV 10.9% vs 19.2%). Applying the reference method acceptance criterion for quantifier-qualifier agreement (±20% difference) reduced the number of samples exceeding this threshold from 10 to 3. In a dilute-and-shoot pilot study (n=10), comparison of 2D-SelexION m/z 163 results to reference LLE m/z 163 results gave a slope of 0.965 (95% CI 0.682-1.243, y-int=-51.90, r=0.962) with percent bias of -11.1%. Within the dilute-and-shoot 2D-SelexION method, comparison of m/z 163 and 145 results gave a slope of 0.985 (95% CI 0.885-1.106, y-int=50.43, r=0.994) with percent bias of 3.2%. 2 of 10 samples exceeded the ±20% acceptance criterion. Despite reduced signal-to-noise compared with LLE, these findings suggest dilute-and-shoot may be feasible after further optimization. DISCUSSION: Initial experiments during method development using a hydrophilic-lipophilic balanced cartridge provided no improvement over the existing method alone. In contrast, 2,3-BPG was strongly retained on MAX under basic conditions and efficiently released under acidic organic conditions. Method optimization showed that 2% formic acid in methanol was required for narrow-band elution, low initial organic conditions combined with at-column dilution were needed to refocus analyte during transfer, and a loading flow of 0.5 mL/min was necessary to avoid breakthrough. Although the on-line 2-D MAX method alone provided only modest improvement, combining it with the SelexION improved fragment-ion agreement and reduced interference-related discordance while preserving the existing extraction workflow. Furthermore, a pilot study demonstrates the potential efficacy for on-line extraction to reduce the manual sample preparation burden. The data suggest that the remaining interferences may be structurally similar acidic compounds also retained by the anion exchange dimension. REFERENCES: 1. JH Butterfield, CR Weiler. Utility of urinary mast cell mediator metabolites in systemic mastocytosis and MCAS. J Allergy Clin Immunol Pract. 2020;8:2533–41. 2. K Moehnke, et al. Differential mobility spectrometry improves specificity of 2,3-dinor-11β-PGF2α measurement. Clin Biochem. 2024;126:110745. |
| Topic(s): Small Molecule > Emerging Technologies |
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Poster Presentation Poster #17 Attended on Wednesday at 09:15
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 From Clinical Validation to Nationwide Monitoring: Capillary Dried Blood Spots for Vitamin D Assessment in the Latest Belgian Food Consumption Survey
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Rosalie Ghesquière (Presenter)  Laboratory of Toxicology - Ghent University >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Vitamin D deficiency has been associated with a wide range of chronic diseases, underscoring the importance of generating reliable epidemiological data. However, large-scale population monitoring remains challenging because of the reliance on venous blood sampling, which is invasive and logistically demanding. Emerging microsampling technologies offer a practical alternative by enabling minimally invasive, decentralized, and participant-friendly sample collection, thereby creating new opportunities for large-scale biomarker-based studies. Within this context, capillary dried blood spot (cDBS) sampling was implemented in the most recent Belgian National Food Consumption Survey to enable nationwide assessment of vitamin D status. Although the LC-MS/MS-method for 25-hydroxyvitamin D (25-(OH)D) quantification had been previously analytically validated, clinical validation was required to assess the agreement between DBS and the reference matrix, plasma, prior to large-scale implementation. METHODS: Venous plasma and whole blood, venous DBS (vDBS), and cDBS were collected from 44 healthy volunteers to evaluate agreement between matrices. A hematocrit (Hct)-dependent conversion factor was applied to transform cDBS results to plasma-equivalent concentrations. For this, we evaluated four different Hct determination approaches: the reference Hct obtained from liquid whole blood using a hematology analyzer, two non-contact cDBS based prediction technologies relying on near-infrared (NIR) and ultraviolet-visible (UV Vis) spectroscopy, and a general gender specific population-based Hct. All samples were analysed using validated LC-MS/MS methods. Following successful clinical validation, the optimized approach was applied to 793 cDBS samples collected in the context of the most recent Belgian National Food Consumption Survey. RESULTS: The clinical validation revealed no clinically relevant methodological (vDBS vs. whole blood) or sampling-site related (cDBS vs. vDBS) differences. After Hct-dependent conversion, strong agreement between cDBS-derived plasma concentrations and measured plasma concentrations was demonstrated, with 90% of results within 20% of the plasma value, independent of the Hct approach. Weighted Cohen’s kappa values (0.83-0.85) indicated substantial to almost perfect agreement in vitamin D status classification. Application of the method to the survey samples revealed a considerable prevalence of vitamin D deficiency, with 42% of participants presenting 25-(OH)D3 concentrations below 20 ng/mL, underscoring the public health relevance of reliable nationwide monitoring. CONCLUSION: Capillary DBS, following Hct-dependent conversion, are a reliable and practical alternative to plasma for large-scale vitamin D assessment. By enabling decentralized and minimally invasive sampling, this emerging microsampling approach supports large-scale implementation in population-based studies, as evidenced by its successful integration into the latest Belgian National Food Consumption Survey. |
| Topic(s): Other -omics > Multi-omics |
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Poster Presentation Poster #18 Attended on Wednesday at 13:45
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Integrated Genomic and Metabolic Profiling of Cryptococcus neoformans Reveals Virulence‑Associated Pathways Relevant to Cryptococcal Meningitis
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Raiha Fatima (Presenter) COMSATS University Islamabad, Pakistan >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Cryptococcus neoformans is an encapsulated fungal pathogen responsible for life-threatening cryptococcal meningitis, particularly in immunocompromised individuals. Its ability to invade the central nervous system and cross the blood–brain barrier is driven by complex genetic and metabolic adaptations that remain incompletely understood. A deeper understanding of its genomic architecture and functional pathways is essential for identifying mechanisms of virulence and potential therapeutic targets. METHODS: Whole genome sequencing was performed on clinical isolates of C. neoformans using Illumina sequencing technology. High-quality genome assemblies were generated and subjected to comprehensive functional annotation. Bioinformatics analyses included antiSMASH for secondary metabolite biosynthetic gene cluster prediction, KEGG and KOBAS for pathway enrichment analysis, and InterPro along with BlastKOALA for protein function classification and metabolic reconstruction. Comparative genomic approaches were applied to evaluate structural and functional conservation. RESULTS: The assembled genome exhibited a size of approximately 19.1 Mb distributed across 14 chromosomes, consistent with reference genomes of C. neoformans. Functional annotation revealed conserved metabolic pathways alongside distinct secondary metabolite biosynthetic gene clusters, including NRPS-like and terpene-associated clusters, suggesting potential roles in virulence-associated metabolite production. Pathway analysis further identified host-interactive signaling networks, including IL-17 and VEGF-associated pathways, which may contribute to immune modulation and fungal survival under host stress conditions. CONCKUSION: This study provides an integrated genomic and in-silico metabolic framework for understanding C. neoformans pathogenicity. The identification of virulence-associated biosynthetic clusters and host-interactive pathways highlights potential targets for future antifungal therapeutic strategies and biomarker development. These findings contribute to a systems-level understanding of fungal infection mechanisms relevant to cryptococcal meningitis. |
| Topic(s): Small Molecule > Metabolomics |
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Poster Presentation Poster #20 Attended on Wednesday at 13:45
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The Human Metabolome Atlas: A Metabolomics-Based Discovery Platform
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Jeremy Chan (Presenter) University of Toronto >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Cellular atlases like the Human Protein Atlas (HPA) and the Cancer Dependency Map have established themselves as foundational resources for their respective fields. In contrast, few ‘atlas-like’ resources are available for the human metabolome. The metabolomics resources that are available are often limited by chemical coverage or have samples restricted to non-human organisms. As a result, there is a need for a resource that is equivalent to the HPA for the human metabolome. Here, we present the Human Metabolome Atlas (HMA)—a comprehensive metabolomics resource and discovery platform. Using state-of-the-art liquid chromatography-mass spectrometry methodologies, we collected the metabolomic and lipidomic profiles of 70 human cell lines across 22 tissue types. We also demonstrate how the HMA can be used as an interactive tool to understand cellular processes like sialylation and ferroptosis. METHODS: From 70 human cell lines, metabolites and lipids (n = 5) were simultaneously extracted using a methyl tert-butyl ether biphasic method and spiked with isotopically labeled internal standards (UltimateSplashTM ONE and IROA TruQuant Yeast Extract). After extraction, samples were analyzed using an Orbitrap IQ-X Tribrid MS coupled to a Vanquish Horizon UHPLC (Thermo Fisher Scientific). Three complementary LC-MS methods were used to analyze cell line extracts. Metabolites were separately analyzed using either a ZIC-pHILIC column (SeQuant; 2.1 x 150 mm, 5 µm) or a BEH amide column (WATERS; 2.1 x 100 mm, 1.7 µm) in positive or negative-ionization mode, respectively. Lipids were analyzed using a CSH C18 column (WATERS; 2.1 x 100 mm, 1.7 µm) in a polarity switching method. Metabolites and lipids were identified by matching MS/MS spectra and retention time to authentic reference standards or available databases (e.g., mzCloud, LipidBlast). Peak areas were manually integrated and curated using Skyline-daily. RESULTS: The HMA is comprised of the metabolomic and lipidomic profiles of 70 human cell lines across 22 tissue types. In total, we identified 1768 metabolites and lipids at high confidence (minimum Metabolomics Standards Initiative level 2). The metabolome coverage consisted of 200 metabolites from well-characterized essential metabolic pathways such as central carbon metabolism and nucleotide metabolism. The lipidome coverage consisted of 1568 lipids from >50 lipid classes. The HMA constitutes the most extensive human-based metabolomics resource currently available in terms of chemical coverage and identification confidence. Principal component analysis of the metabolomic and lipidomic profiles revealed two major clusters separated by cell line tissue of origin (hematological vs non-hematological). Closer examination of the specific metabolites driving these differences revealed increased levels of nucleotide intermediates in hematological cell lines. Cell lines enriched with nucleotide intermediates were found to be associated with lower levels of sialylation precursors and increased cell surface sialylation in hematological cell lines. Similarly, for the lipidome, we observed a significant enrichment of unsaturated triglycerides (TGs) in hematological cell lines. We also found that this enrichment of unsaturated TGs was associated with ferroptosis susceptibility. Lastly, we developed an interactive web portal with custom data analysis tools (hma.ccbr.utoronto.ca) to provide an accessible method of probing the HMA dataset. The HMA web portal features flexible data analysis options including volcano plots, clustered heatmaps, and fatty acid composition heatmaps (FACHs) that can be tailored to cell lines and metabolites of interest. In particular, the HMA contains many human cancer cell lines, which could be leveraged to identify disease-associated metabolomic signatures and biomarkers. Ultimately, the continuous exploration of the HMA dataset has potential to drive the development of metabolomics-based clinical diagnostics and applications. CONCLUSION: We developed a robust atlas and interactive discovery platform for the human metabolome. Our work highlights how the HMA can serve as a starting point for metabolomics-based biomedical research. |
| Topic(s): Other -omics > Metabolomics |
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Poster Presentation Poster #32 Attended on Wednesday at 13:45
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Development of Targeted Quantitative LC-MS/MS Assays for Deuterated Amino Acid Internal Standards
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Yuhang Wu (Presenter) Middlebury College >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Bacterial metabolic processes are an increasingly important area of study to better understand host-microbe interactions. To better understand these metabolic processes, we are in need of reliable approaches to identify and quantify these metabolic byproducts using untargeted and targeted metabolomics. One challenge of untargeted metabolomics, however, is the lack of normalization across samples and metabolites, which makes it difficult to draw conclusions confidently without appropriate internal standards. An additional challenge faced in this realm is the complexity of bacterial media and other biological matrices. We aim to determine optimized parameters for both targeted data analysis of untargeted metabolomics assays and targeted metabolomics using a deuterated amino acid library as a reliable internal standard set across all four instrument modes: C18 and HILIC in both positive and negative ion modes. These methods will then be optimized for complex biological matrices as well. This work will improve in-house metabolomics approaches for various applications and increase data quality and interpretability OBJECTIVE(S): The objective of this study is to develop an internal standards library for HILIC and C18 chromatography in both positive and negative ion modes, for a total of four methods, using a quadrupole time-of-flight mass spectrometer (QTOF). This library will then be applied and further optimized on a triple quadrupole mass spectrometer (QqQ). METHODS: Building the Deuterated Amino Acid Internal Standards Library Deuterated amino acids were diluted at a range of concentrations and analyzed using HILIC positive and negative ion modes, as well as C18 positive and negative ion modes. One internal standard library consisting of deuterated amino acids was constructed for each of the four modes by recording and averaging the retention times of each amino acid that displayed consistent peak quality across the tested concentrations. Spiking Deuterated Amino Acids as Internal Standards For bacterial supernatants and media samples, 200 µL of sample, including Brain Heart Infusion (BHI) media and nectar, was mixed with 1 mL methanol to precipitate proteins and reduce background signal. After a 5 min incubation at room temperature, samples were centrifuged for 10 min to pellet precipitated material. A total of 880 µL of clarified extract was transferred into two tubes, spiked with 1, 2, 5, 7.5, or 10% (v/v) deuterated amino acid standards, and dried to completion in a SpeedVac. The dried sample was resuspended in 200 µL of 100% HPLC-grade water, vortexed, centrifuged, and the final supernatant was transferred to an MS vial for analysis. In MS-Dial, these samples were run against the corresponding internal standard library constructed previously to determine efficacy. RESULTS: In HILIC mode, many of the 24 deuterated amino acids showed consistent retention times across different concentrations. Positively charged amino acids, such as histidine and arginine, ionized more effectively in positive ion mode, while negatively charged amino acids, such as glutamic acid and tyrosine, ionized more effectively in negative ion mode. More than five amino acids were consistently identified in HILIC mode in plant nectar as the matrix, which is a highly diluted sugar solution. In the more complex bacterial growth medium, BHI, approximately two deuterated amino acids were consistently identified at a minimum spike concentration of 5% in both HILIC positive and negative modes. In C18 mode, most deuterated amino acids eluted within the first one to two minutes, with only a few exceptions such as methionine. This suggests that these polar amino acids are not effective internal standards for C18 chromatography. CONCLUSION / DISCUSSION: Overall, this study shows that deuterated amino acids are promising internal standards for HILIC-based metabolomics workflows. However, in C18 chromatography, many deuterated amino acids elute close to the void volume, indicating that they have very low affinity for the nonpolar stationary phase. As a result, these amino acids are unlikely to experience the same chromatographic environment as analytes that interact more strongly with the C18 column, suggesting that they may not be effective internal standards for C18-based assays. Currently, we are testing chlorpropamide as a new candidate internal standard for constructing an internal standards library in C18 mode. We are also investigating the effect of solvent composition on chromatogram quality. Previously, we used water as the sample solvent, and we are now testing whether changing the solvent to acetonitrile:water (50:50) or 100% acetonitrile affects how samples interact with the polar HILIC column and the nonpolar C18 column. After constructing the internal standard library on the quadrupole time-of-flight mass spectrometer (QTOF), we will apply and further optimize the method on a triple quadrupole mass spectrometer (QqQ), where identified metabolites can be quantified more confidently. |
| Topic(s): Proteomics > Proteomics |
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Poster Presentation Poster #33 Attended on Wednesday at 09:15
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Assessing Urine as a Noninvasive Representation of the Plasma Proteome in Paired Samples
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Katelyn Brusach (Presenter) The Mayo Clinic >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Biofluid-based assays are critical in measuring patient status without requiring biopsy. While traditionally one marker or biofluid is targeted, the relationship between protein presence, absence, or relative quantification across biofluids may provide biological insight. Our previous research has demonstrated that urine is a noninvasive biofluid that may represent blood proteins without requiring venipuncture. This would allow for more accessible, noninvasive diagnostics without requiring blood draw. Historically, the processing of multiple biofluid proteomes is expensive and time consuming, which limits clinical applicability. In this study, we use a high-throughput method to measure the relationship between urine and plasma proteomes, specifically comparing how urine may represent blood as a surrogate biofluid. By characterizing the representation of blood markers in urine, we aim to better understand and measure disease less invasively. METHODS: We measured urine and plasma from a cohort of 15 paired urine and plasma samples. Blood and urine samples were collected within 5 minutes of each other to ensure true pairing. Samples were processed in parallel using a ThermoFisher KingFisher Flex and were run on an Orbitrap Astral mass spectrometer. RESULTS: In our combined samples, we observed a total of 887 proteins in plasma and 3500 proteins in urine. Of these proteins, 722 overlapped across biofluids. Within individual samples, up to 88.1% of plasma proteins were also observed in urine (mean: 53.1%, range: 27-88.1%). In addition to the overlapping proteins, 2778 proteins were uniquely observed in urine compared to 165 proteins unique to plasma. DISCUSSION: Our study demonstrates that urine may represent a subset of the plasma proteome. This supports that urine may reflect circulating proteins, presenting an opportunity for clinical measurements without requiring venipuncture. Proteins that overlap between biofluids should be evaluated depending on disease for biological relevance to determine if urine is a suitable surrogate measurement for current blood markers. Additionally, the proteins observed in urine should be evaluated for disease-specific protein profiles which can be noninvasively measured and may be more holistic than individual blood markers. Our rapid measurement of urine and plasma in parallel enables the opportunity for downstream clinical implementation. Ongoing work will expand this dataset and further investigate how to leverage these biofluids for more systemic clinical applications. These initial findings provide a foundation for evaluating urine as a noninvasive biofluid for proteomic measurement of circulating proteins. |
| Topic(s): Small Molecule > Lipidomics > Spatialomics |
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Poster Presentation Poster #38 Attended on Wednesday at 13:45
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Mass Spectrometry Reveals Microenvironment-Driven Lipid Signaling in Ovarian Cancer
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Carlismari Grundmann (Presenter) University of California - Santa Cruz >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Ovarian cancer is the deadliest gynecologic malignancy, with high-grade serous carcinoma (HGSC) accounting for ~70% of cases. HGSC originates in the fallopian tube epithelium and metastasizes to the ovaries and then the omentum. We have found that epinephrine enhances lipid release from omental adipocytes, creating a pro-metastatic microenvironment. Using mass spectrometry imaging (MSI) of 3D co-cultures of HGSC cells, murine omental tissues or adipocyte spheroids, alongside LC-MS(/MS) lipidomic profiling and invasion assays, we investigated this metabolic crosstalk. Our findings support that epinephrine acts as an autocrine signal in the tumor microenvironment to enhance lipid release, fostering HGSC invasion and disease progression. Understanding these interactions offers insight into tumor-microenvironment dynamics and could guide the development of targeted therapies to inhibit HGSC metastasis. METHODS: A 3D agarose-based co-culture system was used to model interactions among murine omental tissue or adipocytes, and murine oviductal epithelial (MOE) cells with different mutations, MOE PTENshRNA or MOE SCRshRNA. MALDI-MSI was performed using a Bruker timsTOF fleX at 50 µm spatial resolution in positive reflectron mode. A 50:50 CHCA matrix was applied using an HTX TM-Sprayer. Spatial features enriched in cancer–omentum/adipocyte interaction regions were prioritized for further analysis using SCiLS Lab. For lipidomic profiling, reverse-phase LC-MS/MS was performed in both positive and negative ionization mode to characterize adipocyte-conditioned media (ACM) generated under β-adrenergic receptor agonist (epinephrine), antagonist (propranolol), or vehicle control treatments. Detected lipid features were annotated by subclass and compared across treatment groups using univariate and multivariate analyses. Conditioned media were further evaluated for functional effects on tumor cell phenotypes, including proliferation, migration, and invasion assays. RESULTS: MSI revealed increased epinephrine signal intensity in 3D co-cultures containing HGSC cells, omental tissue or adipocyte spheroids. Adipocyte-conditioned media generated under epinephrine treatment modestly increased HGSC cell invasion, consistent with a pro-metastatic role of β-adrenergic signaling. In contrast, propranolol treatment reduced this invasive phenotype. Following optimization of lipid extraction from adipocyte-conditioned media, LC-MS/MS data acquired in positive and negative ionization modes were used to profile lipid subclasses across treatments. Thirteen lipid classes were detected, with sterols (ST) and fatty acids (FA) representing the most abundant classes by lipid counts. While a number of classes were detected, further analysis highlighted that there were limited differences in lipid counts among treatment groups. However, multivariate analyses, including principal component analysis (PCA) and heatmaps, revealed clear treatment-dependent shifts in lipid abundance profiles. Therefore the amount of specific lipids is differentially regulated, rather than presence or absence of a specific species or class. Application of directionality filters identifying reciprocal trends between epinephrine- or propranolol-treated samples relative to vehicle control was used to prioritize biologically relevant features for further validation. This analysis identified a putative subset of sterol-derived lipids consistently enriched in epinephrine treatment, suggesting a link between β-adrenergic activation and sterol metabolism, whereas fatty acids showed less consistent regulation. Two-dimensional lipid composition maps integrating carbon chain length, unsaturation, and oxygenation further revealed enrichment of oxidized sterol specie, including putative estrogen-related derivatives, supporting a role for β-adrenergic signaling in oxidative lipid remodeling. CONCLUSION: Our findings highlight a potential role for epinephrine in modulating lipid dynamics within the HGSC tumor microenvironment. By integrating MALDI MSI and lipidomics, we provide evidence that β-adrenergic signaling promotes lipid release from omental tissues and adipocyte cells, contributing to a pro-metastatic lipid-rich niche. HGSC is typically diagnoses after metastasis with almost all women having omental tumors, which highlights that these results support further investigation of metabolic pathways as potential therapeutic vulnerabilities. |
| Topic(s): Proteomics > Emerging Technologies > Precision Medicine |
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Poster Presentation Poster #67 Attended on Wednesday at 09:15
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Robust and High-Throughput Targeted Peptide Quantitation Using a Next-Generation Triple Quadrupole and Automated Ion Source platform
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Qin Fu (Presenter) Thermo Fisher Scientific >> NO POSTER PDF SUBMITTED
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| BACKGROUND: Targeted peptide quantitation by LC–MS/MS is widely used in proteomics, biomarker verification, and translational research due to its high sensitivity and quantitative precision. As requirements for multiplexing and sample throughput increase, maintaining reproducibility, retention time stability, and sensitivity becomes increasingly challenging. Advances in acquisition speed, ion transmission efficiency, and workflow operability are required to support large-cohort studies and routine analytical use. In this study, we evaluate the quantitative performance and robustness of a next-generation triple quadrupole mass spectrometer equipped with a novel capillary-flow electrospray ionization interface. A 57 plasma protein biomarkers panel health surveillance panel was used to assess sensitivity, linearity, reproducibility, and long-term stability under high-throughput analytical conditions. METHODS: Peptide analyses were performed using a Thermo Scientific™ Vanquish™ Neo UHPLC system coupled to a Thermo Scientific™ TSQ Certis™ triple quadrupole mass spectrometer with the plug-and-play Thermo Scientific™ OptiSpray™ ion source. Ionization was achieved using a Thermo Scientific™ Optispray™ PepMap™ Neo 150µm×15cm Cartridge which contains a 15 µm tapered emitter. Stable isotope–labeled peptides were spiked into pooled human plasma digest and analyzed using a multiplexed 57 protein Health Surveillance Panel. An 11-point calibration curve spanning 0–120 fmol on-column was evaluated. Samples were analyzed in a trap-and-elute configuration with a 14.4-minute run time, enabling a throughput of 100 samples per day. Data were acquired using scheduled selected reaction monitoring transitions and processed using two independent software workflows (Skyline and TraceFinderTM). RESULTS: An automated capillary flow emitter routine was performed with the OptiSpray ion source to position the emitter in the optimal position and sheath gas was applied to obtain more stable spray. Quantitative performance was evaluated across 1,080 scheduled SRM transitions using replicate injections and extended analytical runs. More than 96% of transitions achieved over eight data points per chromatographic peak, with a median of 16 points, supporting accurate quantitation in a highly multiplexed assay. Median limits of detection and quantification were 0.019 fmol and 0.164 fmol on-column, respectively. Greater than 99% of peptides demonstrated excellent linearity across the calibration range, with coefficients of determination (R²) exceeding 0.99. Peak area reproducibility across six replicate injections showed an average relative standard deviation (RSD) of 3.2%, with over 98% of peptides exhibiting RSDs below 10%. Long-term robustness was assessed over 71 consecutive injections of plasma digest spiked with internal standards. Approximately 89% of endogenous peptide signals showed RSDs below 10%, and 95% were below 20%, with higher variability attributed to low-abundant peptides. Retention time stability was exceptional, with RSDs below 0.6% for FDA-relevant peptides across the extended run. Independent data processing using Skyline and TraceFinder produced consistent quantitative results, confirming robustness across analysis platforms. These data demonstrate high sensitivity, reproducibility, and chromatographic stability in a high-throughput, highly multiplexed targeted peptide workflow suitable for large-cohort and translational studies. CONCLUSION: Demonstration of robust, high-throughput multiplexed peptide quantitation with exceptional reproducibility using next-generation triple quadrupole and automated ion source instrumentation. |
| Topic(s): Small Molecule > Tox / TDM / Endocrine > Tox / TDM / Endocrine |
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Poster Presentation Poster #7 Attended on Thursday at 10:00
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Bridging Bioanalysis and Clinical Safety: LC–MS/MS Quantitation of Hydroxychloroquine (HCQ) and Its Metabolites in Whole Blood and Serum
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Faraz Rashid (Presenter) University of Michigan >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Hydroxychloroquine (HCQ) is commonly prescribed for the treatment of Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), and other inflammatory rheumatic diseases. HCQ is metabolized in the liver to form desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ) metabolites. Long-term use of HCQ has been linked to irreversible retinal toxicity and cardiotoxicity by a yet unknown mechanism. Therefore, therapeutic drug monitoring (TDM) of HCQ and its metabolites is necessary to address the tissue associated toxicity. In this study, we developed a rapid and accurate LC-MS/MS method for quantification of hydroxychloroquine and its metabolites in serum and whole blood and assessed the suitability of both sample types for clinical monitoring. This method provides valuable insights into potential long-term toxicity associated with the cumulative HCQ dosage and treatment duration. OBJECTIVES: To develop a rapid, LC–MS/MS method suitable to multiple biological matrices for the precise simultaneous quantification of hydroxychloroquine and its metabolites to support clinical toxicology monitoring. METHODS: A single and rapid ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantitation of HCQ and its three metabolites in human blood and serum was developed. Method demonstrates high selectivity enabling complete separation of HCQ and metabolites within 1.5 minutes under isocratic conditions, making it suitable for high-throughput analysis. Separation enables detection of all four analytes using Kinetex (2.6 µm, 2.1 × 100mm) column at the flow rate of 0.45 mL/min with column temperature at 550C. Simple protein precipitation was successfully applied for sample preparation of both blood and serum matrices. HCQ exhibited linearity over the concentration range of 8.0–2000.0 ng/mL, while the three metabolites showed linearity from 4.0 to 2000.0 ng/mL. Regression and statistical analysis yielded a Pearson correlation of 0.995, indicating excellent correlation across the concentration range of all metabolites. RESULTS: The method was applied to the simultaneous quantification of hydroxychloroquine (HCQ) and its metabolites in clinical whole blood and serum samples (n = 48) from patients receiving long-term HCQ therapy (2–10 years). Quantitation of HCQ demonstrated that its concentration in whole blood was on average 3.8-fold higher than that in serum with only BDCQ metabolite showing correlation between the levels in whole blood and serum. In our patient cohort, 10.4% of the patients were in therapeutic ranges in serum (TR: 400-600 ng/mL) and 20.8% in whole blood (TR: 750-1200 ng/mL) with the majority being sub-therapeutic 87.5% vs. 68.8% in serum and whole blood, respectively. Seven patient samples exhibited elevated BDCQ concentrations relative to HCQ in whole blood in contrast to serum. The average BDCQ concentration in serum samples was 18.8-fold lower than HCQ, whereas the same patient samples in whole blood showed a reduced ratio of 4.3-fold suggesting a potential association with cumulative exposure and long-term toxicity. CONCLUSION: Monitoring of HCQ and its metabolites in whole blood, particularly BDCQ, may substantially improve identification of patients at risk for cumulative toxicity, which may be missed with serum based or parent-drug-only monitoring of HCQ. REFERENCES: 1.Marmor, M. F. et al. Ophthalmology 2011;118:415–422. 2.Yusuf IH, Sharma S, Luqmani R, Downes SM. Hydroxychloroquine retinopathy. Eye (Lond). 2017 Jun;31(6):828-845. 3.Yusuf IH, Charbel Issa P and Ahn SJ (2023) Hydroxychloroquine-induced Retinal Toxicity. Front. Pharmacol. 14:1196783. |
| Topic(s): Small Molecule > Pre-Analytics > Tox / TDM / Endocrine |
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Poster Presentation Poster #12 Attended on Thursday at 14:15
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Improving Pediatric Dried Blood Spot Lead Screening Through Reduced Filter Paper Punch Diameter
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Nathaniel Craun (Presenter) Case Western Reserve University >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Childhood lead exposure remains a significant public health concern, as even low-level exposure can cause irreversible neurodevelopmental harm. Blood lead level (BLL) screening is therefore essential for early identification of at-risk children. Filter paper-based dried blood spot (DBS) testing offers a practical approach for large-scale pediatric BLL screening because it requires only a small capillary blood sample, avoids venipuncture, and permits low-cost collection during routine visits with centralized laboratory processing. However, specimens must adequately saturate the filter paper for testing, and insufficient samples are reported as quantity not sufficient (QNS). Because DBS screening is performed primarily in infants and toddlers, for whom specimen collection can be challenging, QNS rates may be substantial, making repeat collection burdensome for patients and families. In regions with older housing stock, such as Northeast Ohio, where lead exposure risk remains elevated, reducing QNS rates and improving specimen adequacy are important for effective population-based screening. OBJECTIVES: This study aimed to evaluate whether reducing the required filter paper collection diameter, thus lowering the blood volume needed to fully saturate each collection spot, improved specimen adequacy and decreased QNS rates in pediatric DBS collections. A secondary objective was to assess whether this change improved agreement between positive DBS screening results and confirmatory venous blood lead testing. METHODS: Pediatric filter paper BLL tests collected over a 13-month period were retrospectively reviewed to assess the effect of reducing the filter paper punch diameter from 8 mm to 6 mm on QNS rates, specimen adequacy, and DBS screening positivity. Blood lead concentrations were measured by electrothermal atomic absorption spectroscopy (ETAAS) using a graphite furnace atomic absorption spectrometer. For children with positive DBS screening results, confirmatory venous blood collection was attempted, and, when obtained, venous blood lead concentrations were analyzed using the same instrument. RESULTS: A total of 2,365 pediatric patients (mean age, 1.86 years) underwent filter paper blood lead level (BLL) screening during the 13-month study period. Before implementation of the reduced punch diameter (June 2025–March 2026; n = 1,545), the QNS rate was 8.67%, compared with 5.24% after implementation (April 2026–June 2026; n = 820), representing a 39.56% relative reduction. Among successfully processed specimens, 4.25% screened positive for elevated BLLs before implementation versus 3.90% after implementation. Among positive screening results with confirmatory venous testing, 75.76% were confirmed to have elevated BLLs before implementation, compared with 88.89% after implementation. CONCLUSION: Reducing the filter paper punch diameter from 8 mm to 6 mm was associated with a substantially lower QNS rate, no apparent increase in presumptive positive screening results, and fewer apparent false-positive screening results based on venous confirmatory testing, suggesting improved specimen adequacy while maintaining clinical screening performance for pediatric filter paper BLL testing. This simple, low-cost modification may represent a readily scalable approach to improving specimen adequacy and laboratory efficiency in pediatric BLL screening. |
| Topic(s): Small Molecule > Tox / TDM / Endocrine > none |
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Poster Presentation Poster #19 Attended on Thursday at 10:00
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Development and Validation of a Rapid LC-MS/MS Assay for Serum Amiodarone and Desethylamiodarone
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Lori Good (Presenter) Alberta Precision Laboratories >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Amiodarone is a highly lipophilic antiarrhythmic medication associated with significant toxicity and complex pharmacokinetics, necessitating therapeutic drug monitoring (TDM) to support safe and effective patient management. Desethylamiodarone, the major active metabolite of amiodarone, also contributes to pharmacologic and toxicologic effects. Traditional high-performance liquid chromatography with diode-array detection (HPLC-DAD) methods may be limited by longer analysis times, lower specificity, reduced sensitivity, and complex sample extraction procedures. To improve analytical performance and laboratory workflow, an LC-MS/MS method for the simultaneous quantitation of serum amiodarone and desethylamiodarone was developed and validated. METHODS: Sample preparation consisted of protein precipitation of 100 µL serum with acetonitrile. Chromatographic separation was achieved using a Zorbax RRHD Eclipse Plus C18 analytical column with gradient elution consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Total chromatographic runtime is 4.5 minutes. Detection was performed using tandem mass spectrometry in multiple reaction monitoring mode for the simultaneous analysis of amiodarone, desethylamiodarone, and their corresponding deuterated internal standards. Method validation included assessment of analytical measurement range, precision, carryover, matrix effects, accuracy, and patient correlation with the previous HPLC-DAD method. RESULTS: The LC-MS/MS assay demonstrated improved analytical specificity and sensitivity compared with the previous HPLC-DAD method while significantly reducing total sample preparation and analysis time. Protein precipitation provided a simple and efficient extraction approach suitable for routine clinical laboratory implementation. Chromatographic separation of amiodarone and desethylamiodarone was achieved within a 5.5-minute runtime with excellent peak shape and separation from other lipophilic compounds. Method validation, performed in accordance with clinical laboratory guidelines, demonstrated excellent linearity (r² ≥ 0.9990), intra- and inter-assay precision with (CV ≤ 2.18%), minimal carryover (< 5% of the lowest calibrator peak area), and acceptable accuracy across the analytical measurement range. Method comparison studies demonstrated acceptable agreement with the existing HPLC-DAD assay. However, desethylamiodarone demonstrated reduced recovery with the HPLC-DAD method due to analyte loss during the sample drying step, resulting in concentrations up to 20% lower than those obtained by LC-MS/MS. The HPLC-DAD internal standard did not compensate for this loss as it is not as volatile as desethylamiodarone. Elimination of the drying step in the LC-MS/MS method improved both accuracy and precision for desethylamiodarone quantitation. CONCLUSION: A rapid and robust LC-MS/MS assay for serum amiodarone and desethylamiodarone was successfully developed for clinical therapeutic drug monitoring. The method provides improved analytical performance over the previous HPLC-DAD assay while utilizing a simple protein precipitation extraction and short chromatographic runtime. Implementation of this assay supports improved laboratory efficiency, enhanced analytical specificity, and improved quantitation of desethylamiodarone for routine monitoring of amiodarone therapy. |
| Topic(s): Small Molecule > Tox / TDM / Endocrine > Precision Medicine |
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Poster Presentation Poster #30 Attended on Thursday at 14:15
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Rapid Multiplex HILIC LC-MS/MS Assay for Simultaneous Quantification of Anti-Tuberculosis Drugs and Metabolites to Support Therapeutic Drug Monitoring
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Hannah Lusk (Presenter) University of California San Francisco >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Therapeutic drug monitoring (TDM) of anti-tuberculosis (TB) therapy is critical due to narrow therapeutic windows, treatment-associated toxicity, and significant interpatient variability in drug exposure. However, current TB-TDM practices in the United States are limited to a small number of reference laboratories, resulting in prolonged turnaround times and delayed clinical decision-making. In addition, existing assays measure parent drug concentrations and do not routinely include metabolites or degradation products, limiting assessment of drug metabolism and sample integrity. This contributes to discordance between measured concentrations and clinical outcomes. These limitations represent a significant barrier to effective TB-TDM and highlight the need for improved analytical approaches.
OBJECTIVES: The objective of this study is to develop and evaluate a multiplex LC-MS/MS assay for simultaneous quantification of anti-tuberculosis drugs, metabolites, and degradation products in serum to support TDM. By enabling more comprehensive measurement of drug exposure, we aim to improve interpretation of drug concentrations and support personalized treatment strategies. METHODS: A multiplex hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC LC-MS/MS) assay was developed for simultaneous quantification of first-line TB drugs (rifampin, isoniazid, pyrazinamide, ethambutol), key metabolites (25-desacetyl-rifampin, N-acetyl-isoniazid), and select second-line agents. Serum samples underwent protein precipitation and dilution prior to analysis on a triple quadrupole mass spectrometer operating in multiple reaction monitoring mode. Chromatographic separation was achieved using a BEH amide column with a 5-minute runtime. Method validation will be performed following CLSI and FDA bioanalytical guidelines. RESULTS: The assay achieved rapid chromatographic separation of all analytes despite wide differences in polarity. Calibration curves demonstrated strong linearity (R² > 0.99) across clinically relevant concentration ranges. During preliminary validation accuracy ranged from −0.9% to 10%, and imprecision ranged from 1–8% for most analytes. Limits of quantification and analytical measurement ranges encompassed expected therapeutic concentrations. Matrix effects (78–134%) and extraction recovery (73–111%) were acceptable for multiplex analysis. These findings support the feasibility of simultaneous quantification of TB drugs and metabolites in a single assay. CONCLUSIONS: We developed a rapid, multiplex HILIC LC-MS/MS assay for simultaneous quantification of anti-TB drugs and metabolites in serum. Preliminary data demonstrate feasibility for clinical TDM applications. Ongoing optimization and full validation will further refine assay performance and support implementation in a clinical laboratory setting. This approach has the potential to improve turnaround time, enhance interpretation of drug exposure, and support individualized TB therapy. |
| Topic(s): Proteomics > Proteomics > Tox / TDM / Endocrine |
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Poster Presentation Poster #57 Attended on Thursday at 10:00
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Feasibility of LC–MS/MS Based Therapeutic Drug Monitoring for Vedolizumab and Ustekinumab Using Commercial PK and ADA Kits
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Amol Bajaj (Presenter) ARUP >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Vedolizumab and ustekinumab are monoclonal antibody therapies widely used for inflammatory bowel disease, and therapeutic drug monitoring (TDM) has been associated with improved clinical management. Quantitation from immunoassay-based drug and anti drug antibody testing can be limited by analytical interference and inter assay variability. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) directly measures drug specific peptides and offers improved analytical specificity. This study evaluated the feasibility of implementing commercial LC–MS/MS pharmacokinetic (PK) and anti drug antibody (ADA) kits in a clinical laboratory. METHODS: Commercial LC–MS/MS PK and ADA kits were evaluated on a Sciex 6500+ triple quadrupole platform. For PK evaluation, an internal standard is added at the beginning of sample preparation, enabling normalization of biases arising from different steps of the protocol (i.e., extraction, digestion). This internal standard is a Stable-Isotopically-Labelled mAb (SIL-mAb), with a sequence highly similar to that of the targeted mAb. However, the SIL-mAb is specifically labelled with [13C6,15N2] Lys and [13C6,15N4] Arg. Aliquots of human plasma or serum samples (20 µL) were mixed with SIL-mAb. Then, the mAb of interest and its SIL form are immunocaptured, extracted, and digested with an enzyme to generate peptides. For ADA evaluation in human plasma or serum samples (100 µL), the strategy relies on the fact that ADAs are antibodies, so they have two antigen-binding sites that can be used to form a “bridge” between the t-mAbs and their corresponding SIL-mAbs. After elution and trypsin digestion, SIL-peptides are detected only if free ADAs is present in the plasma/serum. The digested sample plate is then transferred to the LC autosampler, and the samples were analyzed using a 6500+ LC-MS/MS, equipped with a 1290 series HPLC (Agilent). Chromatographic separation was performed using an Accucore Vanquish C18+ column (1.5 µm, 5 cm, 2.1 mm ID, Thermo Fisher Scientific). Quantification was performed using a six-point calibration curve (0.5–100 µg/mL for both vedolizumab PK and ustekinumab PK and 10–400 ng/mL for both vedolizumab ADA and ustekinumab ADA), monitoring four mass transitions for vedolizumab PK and three for ustekinumab PK and their respective IS and monitoring two peptides, three mass transitions for vedolizumab ADA and four for ustekinumab ADA and their respective IS. Analytical performance was assessed through studies of linearity, accuracy, precision, and sensitivity. Method comparison was performed using deidentified residual clinical specimens against a national reference laboratory using LC-MS/MS methodology for vedolizumab PK and ELISA for ustekinumab PK. The ADA evaluation included deidentified residual clinical specimens and contrived specimens. RESULTS: Vedolizumab PK demonstrated a strong correlation to a reference laboratory LC–MS/MS method across 84 de-identified patient samples with a Deming regression slope of 1.40 (95% CI 1.34–1.48) and R of 0.976. Bland–Altman analysis showed a mean positive bias of 10 µg/mL (46%). Linearity was demonstrated across 2–100 µg/mL using spike in and dilution studies. Analytical precision from pooled patient samples showed total CVs of 3% at low concentrations (4-6 µg/mL) and <4% at mid to high concentrations (30–50 µg/mL). Sensitivity assessments near the LLOQ demonstrated CVs ≤10%. Ustekinumab PK demonstrated linearity from 2.5–80 µg/mL. Comparison with a reference laboratory immunoassay method across 44 de-identified patient samples showed a Deming regression slope of 1.13 (95% CI 0.96–1.30) and an R of 0.899. Bland–Altman analysis showed a mean positive bias of 0.28 µg/mL (5%). Precision studies showed total CVs of 10% at low concentrations (3–4 µg/mL), 4% at mid concentrations (7-9 µg/mL), and 13% at higher concentrations (15-18 µg/mL). Vedolizumab LC–MS/MS ADA assays detected high titer antibodies and were linear from 11–500 ng/mL for both short and long signature peptides, with average calibrator recoveries near 100%. Ustekinumab ADA demonstrated linearity from 20–470 ng/mL. Precision across ADA concentration pools ranged from 2–10% CV at low to mid-levels and 8–16% CV at higher concentrations. Reduced sensitivity at lower ADA levels was observed. CONCLUSION: Commercial LC–MS/MS kits for vedolizumab and ustekinumab PK demonstrated adequate analytical performance and an acceptable workflow, supporting progression toward formal validation for longitudinal TDM. ADA assays were analytically feasible and capable of detecting high titer antibodies but require additional optimization and broader clinical evaluation before routine implementation. LC–MS/MS represents a robust alternative to immunoassays for biologic TDM, offering improved specificity and resistance to analytical interference. |
| Topic(s): Small Molecule > Assays Leveraging Technology > none |
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Poster Presentation Poster #77 Attended on Thursday at 10:00
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A Rapid, Low-Solvent Micro-Volume QuEChERS LC-MS/MS Method for Quantifying Emerging Psychoactive Substances in Urine
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Su-Chin Chen (Presenter) Taichung Veterans General Hospital >> NO POSTER PDF SUBMITTED
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| INTRODUCTION: Accurate quantification of New Psychoactive Substances (NPS) in urine is critical for forensic and legal determinations. Conventional Solid Phase Extraction (SPE) is labor-intensive, while simple dilution methods often suffer from ESI matrix effects in LC-MS/MS, compromising data integrity. This research establishes an efficient analytical method for NPS and psychotropic substances in urine using Micro-volume QuEChERS pretreatment coupled with LC-MS/MS to minimize matrix interference and improve throughput. METHODS: To evaluate the feasibility of the proposed analytical workflow, three sample pretreatment strategies were systematically compared: SPE, direct dilution following centrifugation, and a micro-volume QuEChERS-based extraction method. Upon confirmation of method feasibility, full validation was performed, including assessments of linearity, limit of detection (LOD), lower limit of quantification (LLOQ), selectivity, and matrix effects. For matrix effect evaluation, ten urine specimens from different sources were fortified with target analytes at a threshold concentration of 50 ng/mL. Sample preparation involved combining 200 μL of urine with 200 μL of acetonitrile, followed by micro-volume QuEChERS extraction with vortex mixing for 1 minute. Chromatographic analysis was performed using an Agilent 1290 Infinity II LC coupled to an Agilent 6470 triple quadrupole system, utilizing an InfinityLab Poroshell 120 SB-AQ column (3.0 × 100 mm, 2.7 μm). RESULTS: The experimental results demonstrated that the Limit of Quantitation (LOQ) for 7-Aminonitrazepam, 7-Aminonimetazepam, Mephedrone, and Methylephedrine reached 10 ng/mL. Within the concentration range of 10–1000 ng/mL, all targets exhibited excellent linearity (R^2 > 0.995) with high precision (1.2%–5.7%) and accuracy (-9.3%–7.7%). Evaluation of Matrix Effects (ME) and Recovery yielded consistent results across analytes: 7-Aminonitrazepam showed an average ME of 113.8% (CV 8.0%) and recovery of 103.9%; 7-Aminonimetazepam had an average ME of 109.9% (CV 7.9%) and recovery of 97.9%; while Mephedrone and Methylephedrine exhibited average ME of 101.8% and 100.3% respectively, with recoveries near 89%. The Micro-volume QuEChERS pretreatment effectively simplified complex biological matrices, reducing instrument contamination and extending equipment lifespan. Validation with authentic urine samples showed results consistent with GC-MS confirmation, while achieving a processing time 3 to 5 times faster. CONCLUSION: Micro-volume QuEChERS is a simple, rapid, and safe procedure with low organic solvent consumption. Its high reproducibility and excellent matrix cleanup capabilities make it an ideal choice for the multi-residue analysis of New Psychoactive Substances (NPS) in forensic and clinical urine testing. |