Multiple Reaction Monitoring Mass Spectrometry Research Articles

Integrated proteomic and genomic analysis to identify predictive biomarkers for valproate response in bipolar disorder: a 6-month follow-up study

BackgroundSeveral genetic studies have been undertaken to elucidate the intricate interplay between genetics and drug responses in bipolar disorder (BD). However, there has been notably limited research on biomarkers specifically linked to valproate, with only a few studies investigating integrated proteomic and genomic factors in response to valproate treatment. Therefore, this study aimed to identify biological markers for the therapeutic response to valproate treatment in BD. Patients with BD in remission were assessed only at baseline, whereas those experiencing acute mood episodes were evaluated at three points (baseline, 8 ± 2 weeks, and 6 ± 1 months). The response to valproate treatment was measured using the Alda scale, with individuals scoring an Alda A score ≥ 5 categorized into the acute-valproate responder (acute-VPAR) group. We analyzed 158 peptides (92 proteins) from peripheral blood samples using multiple reaction monitoring mass spectrometry, and proteomic result-guided candidate gene association analyses, with 1,627 single nucleotide variants (SNVs), were performed using the Korean chip.ResultsThe markers of 37 peptides (27 protein) showed temporal upregulation, indicating possible association with response to valproate treatment. A total of 58 SNVs in 22 genes and 37 SNVs in 16 genes showed nominally significant associations with the Alda A continuous score and the acute-VPAR group, respectively. No SNVs reached the genome-wide significance threshold; however, three SNVs (rs115788299, rs11563197, and rs117669164) in the secreted phosphoprotein 2 gene reached a gene-based false discovery rate-corrected significance threshold with response to valproate treatment. Significant markers were associated with the pathophysiological processes of bipolar disorders, including the immune response, acute phase reaction, and coagulation cascade. These results suggest that valproate effectively suppresses mechanisms associated with disease progression.ConclusionsThe markers identified in this study could be valuable indicators of the underlying mechanisms associated with response to valproate treatment.

Exploration of the Amino Acid Metabolic Profiling and Pathway in Clonorchis sinensis-Infected Rats Revealed by the Targeted Metabolomic Analysis.

Background: Clonorchiasis remains a serious public health problem. However, the molecular mechanism underlying clonorchiasis remains largely unknown. Amino acid (AA) metabolism plays key roles in protein synthesis and energy sources, and improves immunity in pathological conditions. Therefore, this study aimed to explore the AA profiles of spleen in clonorchiasis and speculate the interaction between the host and parasite. Methods: Here targeted ultrahigh performance liquid chromatography multiple reaction monitoring mass spectrometry was applied to discover the AA profiles in spleen of rats infected with Clonorchis sinensis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis (KEGG) was performed to characterize the dysregulated metabolic pathways. Results: Pathway analysis revealed that phenylalanine, tyrosine, and tryptophan biosynthesis and β-alanine metabolism were significantly altered in clonorchiasis. There were no significant correlations between 14 significant differential AAs and interleukin (IL)-1β. Although arginine, asparagine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine were positively correlated with IL-6, IL-10, tumor necrosis factor (TNF)-α as well as aspartate aminotransferase and alanine aminotransferase; β-alanine and 4-hydroxyproline were negatively correlated with IL-6, IL-10, and TNF-α. Conclusion: This study reveals the dysregulation of AA metabolism in clonorchiasis and provides a useful insight of metabolic mechanisms at the molecular level.

Abstract 5925: Safety, efficacy, pharmacokinetics, and pharmacodynamics of a second generation Irofulven analog CAP-0121 in preclinical cancer models

Abstract Background: Irofulven is a semisynthetic sesquiterpene anticancer agent initially investigated for its unique mechanism of action and potential to overcome resistance observed in other chemotherapeutics. This study investigates the pharmacokinetics and pharmacodynamics of a second generation irofulven analog, CAP-121 (Califia Pharma), and its efficacy and safety in preclinical cancer models relative to irofulven. Methods: 24-hour comprehensive PK was performed at two different intravenous (IV) doses in canines for irofulven (0.2 mg/kg and 1 mg/kg; MTD) and CAP-121 (1 mg/kg and 3 mg/kg). Plasma concentrations were measured by ultra-high performance liquid chromatography multiple reaction monitoring mass spectrometry. PK parameters were determined for both compounds using noncompartmental methods (Phoenix WinNonlin, version 8.4). PK parameters included area under the concentration vs time curve (AUC), maximum observed concentration (Cmax), clearance (CL), volume of distribution (Vd), and elimination half-life (t1/2). Canine platelet and neutrophil counts were measured across 28 days to ascertain the maximum tolerated dose (MTD); platelet count reversibility was also assessed. Mice xenograft models implanted with a drug resistant human ovarian cancer (A2870), or a drug-resistant human metastatic lung cancer (MV522), were dosed with irofulven (9 mg/kg; MTD) and CAP-121 (15, 20, and 25 mg/kg) and resulting tumor mass, % change in body weight, and platelet counts were compared relative to vehicle only. Results: Systemic exposure, CL, Vd, and t1/2 were similar between irofulven and CAP-121 at a 1 mg/kg IV dose. For irofulven, exposure increased approximately proportionally at doses of 0.2 mg/kg and 1 mg/kg. For CAP-121, exposure increased less than proportionally at doses of 1 mg/kg and 3 mg/kg. MTD of CAP-121 was found to be 3-fold higher in canines compared to irofulven as 3-fold more CAP-121 was required to match the platelet reduction with irofulven; platelet nadir was rapidly reversible for CAP-121. Canine nadir platelets were 3-fold higher for CAP-121 vs irofulven at the same dose (1 mg/kg). CAP-121 was superior to irofulven (p < 0.02) with regards to final tumor weights in mice xenograft models, even though MTD was not reached for CAP-121; a change in body weight and platelet loss was not observed for CAP-121 at up 25 mg/kg in mice. Conclusion: The canine PK/PD study of CAP-121 demonstrates that CAP-121 has favorable PK parameters. With regards to in vivo anticancer cytotoxic activity, the two drug-resistant xenograft models confirm the superiority of CAP-121 over irofulven. Together, these findings indicate a wider therapeutic window in humans is expected for CAP-121 compared to irofulven, emphasizing the potential anticancer utility of CAP-121 for solid cancers without prolonged thrombocytopenia. Further investigation in clinical trials is warranted Citation Format: Raymond T. Suhandynata, T K. Nguyen, Venkata R. Kotamraju, Jeremiah D. Momper, Michael J. Kelner. Safety, efficacy, pharmacokinetics, and pharmacodynamics of a second generation Irofulven analog CAP-0121 in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5925.

Associations between inflammatory and angiogenic proteomic biomarkers, and cardiovascular events and mortality in relation to kidney function

ABSTRACT Background The links between chronic kidney disease (CKD) and the high burden of cardiovascular disease remain unclear. We aimed to explore the association between selected inflammatory and angiogenic biomarkers, kidney function and long-term outcome in patients with an acute coronary syndrome (ACS) and to test the hypothesis that CKD status modifies this association. Methods A total of 1293 ACS patients hospitalized between 2008 and 2015 were followed until 31 December 2017. Plasma was collected on days 1–3 after admission. A total of 13 biomarkers were a priori identified and analysed with two proteomic methods, proximity extension assay or multiple reaction monitoring mass spectrometry. Boxplots and multiple linear regression models were used to study associations between biomarkers and kidney function and adjusted standardized Cox regression with an interaction term for CKD was used to assess whether CKD modified the association between biomarkers and major adverse cardiovascular events and death (MACE+). Results The concentrations of nine biomarkers—endothelial cell-specific molecule-1 (ESM-1), fibroblast growth factor 23 (FGF-23), fractalkine (CX3CL1), interleukin-1 receptor antagonist (IL-1RA), interleukin-18 (IL-18), monocyte chemotactic protein-1 (MCP-1), placenta growth factor (PlGF), transmembrane immunoglobulin 1 (TIM-1) and vascular endothelial growth factor A (VEGFA)—were inversely associated with kidney function. ESM-1, FGF-23 and TIM-1 showed associations with MACE+. Only FGF23 remained independently associated after adjustment for the other biomarkers (hazard ratio per standard deviation increase 1.34; 95% Bonferroni corrected confidence interval 1.19–1.50). None of the biomarkers showed an interaction with CKD. Conclusions The concentrations of 9 of the 13 prespecified inflammatory and angiogenic proteomic biomarkers increased when kidney function declined. Only FGF-23 demonstrated an independent association with MACE+, and this association was not modified by CKD status. These findings further support FGF-23 as an independent prognostic marker in ACS patients with and without CKD.

GC-MS/MS and LC-MS/MS analysis followed by risk ranking of mepiquat and pyrethroids in coffee

Pesticides used in coffee cultivation may remain behind and pose a risk to humans even after various processing stages of coffee beans (washing, drying, roasting, and grinding). In addition to pesticides applied to battle pests and diseases, mepiquat formation occurs during roasting. A comprehensive LC-MS/MS and GC-MS/MS multi-residue screening approach to determine pesticides in coffee was used in this study. Residues were extracted using the QuEChERS method and analyzed using multiple reaction monitoring (MRM) mass spectrometry after chromatographic separations. Good linearity (r2˃0.9919), recovery (80.2–118%), and RSD (≤19%) were obtained for the procedure including sample preparation and both instrumental techniques. Nine residues were identified as commonly present in coffee samples collected from supermarkets in Serbia. The human intake risk of pesticides was categorized and prioritized using the matrix ranking scheme. Also, health risk assessment was conducted by calculating acute and chronic hazard indices. Multi-residues were present in 79.2% of samples, whereas mepiquat chloride (88.9%) and permethrin (81.9%) were the most detected residues. Risk ranking classified mepiquat chloride, permethrin, and methiocarb sulfone in a high-risk group. On the other hand, the human health risk assessment indicated no risks in the short- and long-term and no cumulative chronic risk.

Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. Sex- and strain-specific differences in protein abundances are identified and described, and the measured values are freely accessible via our MouseQuaPro database: http://mousequapro.proteincentre.com . Together, this large library of quantitative MRM-MS assays established in mice and the measured baseline protein abundances represent an important resource for research involving mouse models.

Rapid detection of heat stress biomarkers in Atlantic salmon (Salmo salar) liver using targeted proteomics

AbstractMost fish are ectothermic; therefore, their physiology is significantly affected by temperature. Aquaculture fish have limited ability to avoid elevated water temperatures, with impacts increasing as a result of climate change. To date, quantifying gene expression has been proposed to monitor heat stress in salmon liver. This study aimed to establish a faster multiplexed proteomics method to measure the abundance of thermal stress biomarkers in liver of salmon reared at 15°C or 20°C. Moreover, this study aimed to determine the effects that sample pooling, and data normalisation using housekeeping (HK) protein peptides would exert over the statistical significance of these thermal stress markers. A multiple reaction monitoring (MRM) mass spectrometry method, comprised 45 peptides derived from thermal stress markers and 10 peptides from HK proteins, was applied to measure these markers in liver of salmon reared at 15°C or 20°C. When samples were processed individually, 34 peptides were significant between salmon livers at 15°C or 20°C. In pooled samples, this decreased to five significant peptides. Peptides hprt1_HYADDLDR (hypoxanthine phosphoribosyl transferase) and gapdh_VPTPNVSVVDLTVR (glyceraldehyde‐3‐phosphate dehydrogenase) were the most stable and unstable HK protein peptides, respectively. When data was normalised with hprt1_HYADDLDR, 16 peptides were significant in individual samples and 13 in pooled samples. Significant peptides serpinh1a_ADLSNISGK, SerpinH1_TNSILFIGR, ela2_VVGGEDVR and gapdh_VPTPNVSVVDLTVR were common regardless of data strategy. A fast and reliable MRM method was established to validate thermal stress markers in salmon liver, where individual samples yielded better results than pooled samples. Sample pooling was only better when combined with normalisation as it validated twice the number of markers than sample pooling alone. This method could be applied to monitoring stress response in experiments involving feeding additives designed to mitigate thermal stress or in selective breeding programs to help understanding family variance in thermal tolerance.

A pilot study of S100A4, S100A8/A9, and S100A12 in dilated cardiomyopathy: novel biomarkers for diagnosis or prognosis?

Circulating biomarkers can provide important information for the diagnosis and prognosis of dilated cardiomyopathy (DCM). We explored novel biomarkers for the diagnosis and prognosis of DCM to improve clinical decision-making. A total of 238 DCM patients and 65 control were consecutively enrolled at Zhongshan Hospital between January 2017 and January 2019. In the screening set, four DCM patients and four controls underwent measurements of serum proteomic analysis. Seventy-six differentially expressed circulating proteins were screened by data-independent acquisition proteomics, and three of these proteins (S100A4, S100A8/A9, and S100A12) were validated by multiple-reaction monitoring-mass spectrometry. In the validation set, subsequently, a total of 234 DCM patients and 61 control subjects were evaluated by enzyme-linked immunosorbent assay. Circulating S100A4, S100A8/A9, and S100A12 were significantly increased in DCM patients (P<0.001). These three proteins were significant positively correlated with other parameters, such as Lg (NT-proBNP), IL-1β, TGF-β, CRP, left ventricular end-diastolic diameter, and left ventricular end-systolic diameter, whereas they were negatively correlated with left ventricular ejection fraction, respectively (P<0.05). The receiver operator characteristic curve showed the combination of S100A4, S100A8/A9, and S100A12 [area under curve (AUC) 0.88, 95% confidence interval (CI) 0.84-0.93] was better than single S100A4 (AUC 0.74, 95% CI 0.68-0.81), S100A8/A9 (AUC 0.82, 95% CI 0.77-0.88), or S100A12 (AUC 0.80, 95% CI 0.72-0.88) in the diagnosis of DCM (P<0.01). After a median follow-up period of 33.5months, 110 patients (47.01%) experienced major adverse cardiac events (MACEs), including 46 who had cardiac deaths and 64 who had heart failure rehospitalizations. Kaplan-Meier analysis indicated that the DCM patients with ≥75th percentile level of S100A4 had a significantly higher incidence of MACEs than those with <75th percentile level of S100A4 (61.40% vs. 42.37%, P<0.05). There were no significant differences of MACE rate among DCM patients with different concentrations of S100A8/A9 and S100A12 (P>0.05). Cox proportional hazards regression analysis revealed that S100A4 [≥75th percentile vs. <75th percentile: hazard ratio (HR) 1.65; 95% CI 1.11-2.45] remained significant independent predictors for MACEs (P<0.05); however, S100A8/A9 and S100A12 were not independent factors for predicting MACE (P≥0.05). S100A4, S100A8/A9, and S100A12 may be additional diagnostic tools for human DCM recognition, and the combination of these three indicators helped to improve the accuracy of a single index to diagnose DCM. Additionally, S100A4 was identified as a significant predictor of prognosis in patients with DCM.

Evaluation of a Nondepleted Plasma Multiprotein-Based Model for Discriminating Psychiatric Disorders Using Multiple Reaction Monitoring-Mass Spectrometry: Proof-of-Concept Study.

Psychiatric evaluation relies on subjective symptoms and behavioral observation, which sometimes leads to misdiagnosis. Despite previous efforts to utilize plasma proteins as objective markers, the depletion method is time-consuming. Therefore, this study aimed to enhance previous quantification methods and construct objective discriminative models for major psychiatric disorders using nondepleted plasma. Multiple reaction monitoring-mass spectrometry (MRM-MS) assays for quantifying 453 peptides in nondepleted plasma from 132 individuals [35 major depressive disorder (MDD), 47 bipolar disorder (BD), 23 schizophrenia (SCZ) patients, and 27 healthy controls (HC)] were developed. Pairwise discriminative models for MDD, BD, and SCZ, and a discriminative model between patients and HC were constructed by machine learning approaches. In addition, the proteins from nondepleted plasma-based discriminative models were compared with previously developed depleted plasma-based discriminative models. Discriminative models for MDD versus BD, BD versus SCZ, MDD versus SCZ, and patients versus HC were constructed with 11 to 13 proteins and showed reasonable performances (AUROC = 0.890-0.955). Most of the shared proteins between nondepleted and depleted plasma models had consistent directions of expression levels and were associated with neural signaling, inflammatory, and lipid metabolism pathways. These results suggest that multiprotein markers from nondepleted plasma have a potential role in psychiatric evaluation.

The synaptic biomarker contactin5 differentiates between neurodegenerative dementias

AbstractBackgroundSynapses are crucial for memory formation, and their disruption is among the earliest events in Alzheimer’s disease (AD) pathogenesis. Less is known about synaptic alterations in other types of dementia. Contactin5 (CNTN‐5) is a synaptic protein and one of the neural adhesion molecules. It contributes to synaptic formation, plasticity and is associated with dendritic morphogenesis. A GWAS identified a risk variant (rs 141684 G) in CNTN‐5 gene, which is associated with increased risk of AD development. We aimed to assess whether CNTN‐5 could be a differential diagnostic cerebrospinal fluid (CSF) biomarker for AD from other types of dementia.MethodCNTN‐5 levels in CSF samples were measured with two methods: an in‐house developed immunoassay on the automated ELLA platform, and a multiple reaction monitoring mass spectrometry (MRM) assay. The clinical cohort included controls (n = 27), patients with AD (n = 19), patients with behavioral variant frontotemporal dementia (bvFTD) (n = 18), and patients with dementia with Lewy bodies and Parkinson’s disease dementia (PDD/DLB) (n = 18; table 1). CNTN‐5 results obtained with both methods were correlated using spearman’s correlation. Differences in CNTN‐5 levels between the diagnostic groups were assessed with Kruskal‐Wallis test and corrected with Dunn´s factor for multiple comparison.ResultsAs shown in Figure 1, CNTN‐5 measurements between methods were strongly and positively correlated (rho = 0.58, p&lt;0.001). In AD patients, CNTN‐5 levels were significantly elevated in comparison to bvFTD patients with both assays (both: p&lt;0.05), as well as, compared to patients with DLB/PDD (MRM: p&lt;0.05 and ELLA: p&lt;0.01). There was no significant difference in CNTN‐5 levels between AD and controls, or between bvFTD and PDD/DLB, as shown in figure 2.ConclusionOur results showed that elevated CNTN‐5 is a promising synaptic biomarker, with specificity for AD. As a next step, we would look further into a larger clinical cohort, where associations between CNTN‐5 levels and cognitive stage could be thoroughly studied.

Triazole Antifungal Quantification for Therapeutic Drug Monitoring in Serum by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Posaconazole, Voriconazole, Itraconazole, and Hydroxyitraconazole.

Antifungal therapy with triazole drugs including posaconazole, voriconazole, itraconazole, and its active metabolite hydroxyitraconazole is routinely accompanied by therapeutic drug monitoring to ensure optimal dosing. The method presented here simultaneously quantitates these compounds in serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specimen preparation includes protein precipitation with a methanol and acetonitrile mixture, centrifugation, and filtration. Analyte separation is achieved by reverse-phase chromatography using a dC18 column and a linear gradient of methanol in water. Analytes are detected by multiple reaction monitoring mass spectrometry and quantitated by comparison to a standard curve.

Quantitative Analysis of Buprenorphine, Norbuprenorphine, and Their Glucuronide Metabolites in Human Urine.

We present a quantitative clinical LC-MS/MS assay for the simultaneous analysis of buprenorphine, norbuprenorphine, and their glucuronide metabolites in human urine. The assay is based on the direct and hydrolysis-free sample preparation protocol, i.e., dilute and shoot, whereby clarified urine specimens are diluted in internal standard reagent and injected into the LC-MS/MS instrument. The analytical platform employs reversed-phase liquid chromatography for upfront separation and electrospray ionization multiple reaction monitoring MS detection via the triple-quadrupole (TSQ Quantiva) instrument. The assay has a quantitative analytical range of 5ng/mL-1000ng/mL represented at seven levels for each of the four analytes. A unique stable isotopically labeled analogue is used as internal standard for each analyte. For high-throughput performance, the assay can be multiplexed between two LC channels.

Proteomic profiling in the progression of psychosis: Analysis of clinical high-risk, first episode psychosis, and healthy controls

Background and hypothesisRecent evidence has highlighted the benefits of early detection and treatment for better clinical outcomes in patients with psychosis. Biological markers of the disease have become a focal point of research. This study aimed to identify protein markers detectable in the early stages of psychosis and indicators of progression by comparing them with those of healthy controls (HC) and first episode psychosis (FEP). Study designThe participants comprised 28 patients in the clinical high-risk (CHR) group, 49 patients with FEP, and 61 HCs aged 15–35 years. Blood samples were collected and analyzed using multiple reaction monitoring-mass spectrometry to measure the expression of 158 peptide targets. Data were adjusted for age, sex, and use of psychotropic drugs. Study resultsA total of 18 peptides (17 proteins) differed significantly among the groups. The protein PRDX2 was higher in the FEP group than in the CHR and HC groups and showed increased expression according to disease progression. The levels of six proteins were significantly higher in the FEP group than in the CHR group. Nine proteins differed significantly in the CHR group compared to the other groups. Sixteen proteins were significantly correlated with symptom severity. These proteins are primarily related to the coagulation cascade, inflammatory response, brain structure, and synaptic plasticity. ConclusionsOur findings suggested that peripheral protein markers reflect disease progression in patients with psychosis. Further longitudinal research is needed to confirm these findings and to identify the specific roles of these markers in the pathogenesis of schizophrenia.

Optimization of a ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry method for monitoring haloacetic acids as chlorinated disinfection by-products in the fresh-cut industry

Haloacetic acids (HAAs) are one of the most important chlorinated disinfection by-products generated during water disinfection in the fresh-cut industry, and they can remain in the product, resulting in a consumer health risk. In this study, ultra-high-pressure liquid chromatography-tandem multiple reaction monitoring mass spectrometry (UHPLC-MRM) analysis used for drinking water was optimized and applied for the quantification of nine HAAs (HAA9) in fresh-cut lettuce and process water samples, with the complex matrix interferences for separation, and quantification problems. The method showed good selectivity, specificity and linearity, satisfactory values for trueness (recoveries of 80–116 %), precision (<22 %), and uncertainty (<55 %). Quantification limits varied from 1 to 5 µg L−1 or µg kg−1. The matrix effect for tribromoacetic, bromochloroacetic and chlorodibromoacetic acid was corrected by matrix-matched calibration and standard addition. After storage at -20 °C, only monobromoacetic acid was the HAA which loss happened after 7 days. The application of the methodology in lettuce and process water samples from the industry was successfully implemented. Therefore, this method could be employed for the quality control and regulatory analysis of HAAs in fresh products and process water from the fruit and vegetable industry.

B-325 Retrospective Study Comparing Immunoassay Screen and Mass Spectrometry Confirmation Results in an Opioid Dependant Population

Abstract Background Retrospective analysis of clinical laboratory data is employed to monitor the performance of instruments, evaluate test utilization and support operational changes. Urine drug testing in the context of opioid use disorder is used to assist in the management of patients. This testing is typically performed by an immunoassay screen followed by mass spectrometry confirmation. Immunoassay screens identify drug class and can cross-react with off-target substances to produce false-positive or false-negative results. Mass spectrometry confirmations provide more sensitive and specific results. The objective of this study was to utilize historical data to determine the concordance between immunoassay and mass spectrometry results from patients in an opioid dependency treatment program. Methods Urine drug screening results spanning a 3-year period (2020–2023) were mined to identify samples that screened positive for amphetamines, benzodiazepines, opiates, fentanyl/carfentanil, oxycodone/oxymorphone, and cocaine. These results were compared to the mass spectrometry confirmation results. The immunoassay results were collected on an Olympus AU480 instrument and the mass spectrometry results were collected using an in-house developed dynamic multiple reaction monitoring method on both Agilent 6470 and 6460 triple quadrupole instruments. Results Of the 15 850 urine drug records reviewed, 28.8% screened positive for amphetamines, 23.5% screened positive for benzodiazepines, 14.2% screened positive for opiates, 23.8% screened positive for fentanyl/carfentanil, 4.2% screened positive for oxycodone/oxymorphone, and 11.2% screened positive for cocaine. Of the amphetamine class samples that screened positive, 95.3% confirmed positive and 4.7% confirmed negative. Of the benzodiazepine class samples that screened positive, 50.8% confirmed positive and 49.2% confirmed negative. Of the opiate class samples that screened positive, 79.8% confirmed positive and 20.2% confirmed negative. Of the fentanyl/carfentanil samples that screened positive, 97.2% confirmed positive and 2.8% confirmed negative. Of the oxycodone/oxymorphone samples that screened positive, 58.5% were confirmed positive and 41.5% confirmed negative. Of the cocaine samples that screened positive, 99.8% confirmed positive and 0.2% confirmed negative. Conclusion Our findings determined there were discrepancies among all classes when comparing immunoassay vs mass spectrometry results. There was good agreement between immunoassay and mass spectrometry results in the detection of fentanyl/carfentanil, cocaine and amphetamines. The differences between methods were most significant in the benzodiazepine, opiate and semi-synthetic opioid classes. This may be due to different cut-off levels between methods, specificity limitations of immunoassays and/or changing drug use patterns resulting in mass spectrometry multiple reaction monitoring false negative results.

Serum proteomic biomarkers diagnostic of knee osteoarthritis

To better understand the pathogenesis of knee osteoarthritis (OA) through identification of serum diagnostics. We conducted multiple reaction monitoring mass spectrometry analysis of 107 peptides in baseline sera of two cohorts: the Foundation for National Institutes of Health (NIH) (n=596 Kellgren-Lawrence (KL) grade 1-3 knee OA participants); and the Johnston County Osteoarthritis Project (n=127 multi-joint controls free of radiographic OA of the hands, hips, knees (bilateral KL=0), and spine). Data were split into (70%) training and (30%) testing sets. Diagnostic peptide and clinical data predictors were selected by random forest (RF); selection was based on association (p<0.05) with OA status in multivariable logistic regression models. Model performance was based on area under the curve (AUC) of receiver operating characteristic (ROC) and precision-recall (PR) curves. RF selected 23 peptides (19 proteins) and body mass index (BMI) as diagnostic of OA. BMI weakly diagnosed OA (ROC-AUC 0.57, PR-AUC 0.812) and only symptomatic OA cases. ACTG was the strongest univariable predictor (ROC-AUC 0.705, PR-AUC 0.897). The final model (8 serum peptides) was highly diagnostic (ROC-AUC 0.833, 95% confidence interval [CI] 0.751, 0.905; PR-AUC 0.929, 95% CI 0.876, 0.973) in the testing set and equally diagnostic of non-symptomatic and symptomatic cases (AUCs 0.830-0.835), and not significantly improved with addition of BMI. The STRING database predicted multiple high confidence interactions of the 19 diagnostic OA proteins. No more than 8 serum protein biomarkers were required to discriminate knee OA from non-OA. These biomarkers lend strong support to the involvement and cross-talk of complement and coagulation pathways in the development of OA.

Metabolomic Reconfiguration in Primed Barley (Hordeum vulgare) Plants in Response to Pyrenophora teres f. teres Infection.

Necrotrophic fungi affect a wide range of plants and cause significant crop losses. For the activation of multi-layered innate immune defences, plants can be primed or pre-conditioned to rapidly and more efficiently counteract this pathogen. Untargeted and targeted metabolomics analyses were applied to elucidate the biochemical processes involved in the response of 3,5-dichloroanthranilic acid (3,5-DCAA) primed barley plants to Pyrenophora teres f. teres (Ptt). A susceptible barley cultivar ('Hessekwa') at the third leaf growth stage was treated with 3,5-DCAA 24 h prior to infection using a Ptt conidia suspension. The infection was monitored over 2, 4, and 6 days post-inoculation. For untargeted studies, ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-MS) was used to analyse methanolic plant extracts. Acquired data were processed to generate the data matrices utilised in chemometric modelling and multi-dimensional data mining. For targeted studies, selected metabolites from the amino acids, phenolic acids, and alkaloids classes were quantified using multiple reaction monitoring (MRM) mass spectrometry. 3,5-DCAA was effective as a priming agent in delaying the onset and intensity of symptoms but could not prevent the progression of the disease. Unsupervised learning methods revealed clear differences between the sample extracts from the control plants and the infected plants. Both orthogonal projection to latent structure-discriminant analysis (OPLS-DA) and 'shared and unique structures' (SUS) plots allowed for the extraction of potential markers of the primed and naïve plant responses to Ptt. These include classes of organic acids, fatty acids, amino acids, phenolic acids, and derivatives and flavonoids. Among these, 5-oxo-proline and citric acid were notable as priming response-related metabolites. Metabolites from the tricarboxylic acid pathway were only discriminant in the primed plant infected with Ptt. Furthermore, the quantification of targeted metabolites revealed that hydroxycinnamic acids were significantly more prominent in the primed infected plants, especially at 2 d.p.i. Our research advances efforts to better understand regulated and reprogrammed metabolic responses that constitute defence priming in barley against Ptt.

Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets.

Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. Platelets from 12-LOX+/+ (wild-type [WT]) and 12-LOX-/- mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry-multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX-/- mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. Stored platelets from 12-LOX-/- mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX-/- than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX-/- platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1-dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX-inhibited platelets transfused to humans.

Scout triggered multiple reaction monitoring mass spectrometry for the rapid transfer of large multiplexed targeted methods in metabolomics

The field of metabolomics based on mass spectrometry has grown considerably in recent years due to the need to detect and, above all, quantify a very large number of metabolites, simultaneously. Up to now, targeted multiplexed analysis on complex samples by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) has relied almost exclusively on compound detection based on absolute retention times, as in the Scheduled-MRM (sMRM) approach. Those methods turn out to be poorly transferable from one instrument to another and result in a time-consuming and tedious method development involving a significant number of critical parameters that need specific re-optimisation. To address this challenge, we introduce a novel acquisition mode called scout-triggered MRM (stMRM). In stMRM, a marker transition is used to trigger MS analysis for a group of dependent target analytes. These marker transitions are strategically distributed throughout the chromatographic run, and the dependent analytes are associated based on their retention times. The result is a targeted assay that remains robust even in the presence of retention time shifts. A 3 to 5-fold increase in the number of detected transitions associated to plasma metabolites was obtained when transferring from a direct application of a published sMRM to a stMRM method. This significant improvement highlights the universal applicability of the stMRM method, as it can be implemented on any LC system without the need for extensive method development.We subsequently illustrate the robustness of stMRM in modified chromatographic elution conditions. Despite a large change in metabolite’s selectivity, the multiplexed assay successfully recovered 70% of the monitored transitions when consequently modifying the gradient method. These findings demonstrate the versatility and adaptability of stMRM, opening new avenues for the development of highly multiplexed LC-MS/MS methods in metabolomics. These methods are characterized by their analytical transparency and straightforward implementation using existing literature data.

Quantification of caffeoylquinic acids and triterpenes as targeted bioactive compounds of Centella Asiatica in extracts and formulations by liquid chromatography mass spectrometry

Centella asiatica (CA) is a culinary vegetable and well-known functional food that is widely used as a medicinal herb and dietary supplement. CA is rich in pentacyclic triterpenes (TTs), including asiaticoside (AS), madecassoside (MS) and the related aglycones asiatic acid (AA), madecassic acid (MA). Traditionally, TTs have been associated with the bioactivity and health promoting effect of CA. Recently, mono-caffeoylquinic acids (MonoCQAs) and di-caffeoylquinic acids (DiCQAs) have been found to contribute to the bioactivity of CA as well. This work reports an analytical strategy based on liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM–MS) for the simultaneous rapid and accurate quantification of 12 bioactive compounds in CA, namely AS, MS, AA, MA, 5-CQA, 4-CQA, 3-CQA, 1,3-DiCQA, 3,4-DiCQA, 1,5-DiCQA, 3,5-DiCQA, 4,5-DiCQA. Method selectivity, accuracy, precision, repeatability, robustness, linearity range, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The validated LC-MRM-MS method has been successfully applied to quantify the 12 bioactive compounds in CA aqueous extracts and two related formulations: a standardized CA product (CAP) used in a phase I clinical trial and formulated CA rodent diets used in preclinical studies. The validated method allows us to support the standardization of CA products used for clinical trials and conduct routine LC-MRM-MS analyses of formulated preclinical diets to confirm correct levels of CA phytochemical markers.