Tandem Mass Spectrometry Research Articles

A miniaturized QuEChERS and UPLC–MS/MS method for the determination of mycotoxins in cashew nuts

This study aimed to develop and validate a multi-mycotoxin analysis method applied to cashew nuts by employing a miniaturized QuEChERS method followed by determination by ultra-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). Satisfactory recoveries for the concentrations 1, 10 and 30 ng g−1, ranging from 66% (fumonisin B1) to 110% (ochratoxin A) and relative standard deviations lower than 9% (fumonisin B2) were obtained for the target compounds. Limits of quantification ranged from 0.004 ng g−1 (sterigmatocystin) to 0.59 ng g−1 (alternariol). The applicability of the analytical method was verified by analyzing 30 cashew nut samples from the city of Rio de Janeiro, RJ, southeastern Brazil. Aflatoxins M1, G2, G1, B2, B1, ochratoxin A and sterigmatocystin were detected, respectively, in 27%, 10%, 17%, 30%, 30%, 30% and 50% of the analyzed samples, at maximum concentrations of 0.56, 0.67, 1.43, 2.02, 4.93, 4.81, and 0.35 ng g−1. The maximum limit established by Brazilian legislation for aflatoxins was not exceeded by any of the analyzed samples.

Urinary complement factor D is increased in primary malignant hypertension: a single-center, cross-sectional study

Kidney injury is one of the detrimental consequences of primary malignant hypertension (pMHTN). There is a paucity of non-invasive biomarkers to enhance diagnosis and elucidate the underlying mechanisms. This study aims to explore urine protein biomarkers for pMHTN associated renal damage. In the discovery phase, urine samples were collected from 8 pMHTN, 19 disease controls (DCs), and 5 healthy controls (HCs). In-gel digestion combined with liquid chromatography–tandem mass spectrometry (LC–MS/MS) approach was used for identification of proteins associated with pMHTN. In the validation phase, the differentially expressed proteins were validated by ELISA assay in cohort with 10 pMHTN patients, 37 DCs, and 30 HCs. Compared to DCs and HCs, a specific band between 15 and 25 kDa was found in 7 out of 8 patients with pMHTN. Further LC–MS/MS analysis revealed 5 differentially expressed proteins. ELISA validation demonstrated that urinary complement factor D (CFD) was significantly up regulated in pMHTN. By receiver operating characteristic curve analysis, urinary CFD/Cr showed moderate potential in discriminating pMHTN from DCs (the area under curve: 0.822, 95% CI 0.618–0.962). Urinary CFD may be a potential biomarker for pMHTN with its elevation indicative of the activation of the alternative complement pathway in pMHTN.

The construction of a novel supplementary diagnostic model for patients with indeterminate HIV infection: a proteomics study.

The window period, defined as HIV nucleic acid test (NAT) reactivity but Western blot (WB) test inconclusive, is garnering more attention. Improving the detection efficiency of HIV high-risk populations in the window period is critical to reducing the risk of unanticipated transmission. The purpose of this study was to create an additional strategy for distinguishing indeterminate HIV infection cases. Based on WB follow-up results, the individuals in this study were divided into persons in the HIV window period and persons without HIV. Plasma was analyzed using quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) to detect differentially expressed proteins (DEPs). The biological implications of these DEPs were investigated using enrichment analysis. Protein-protein interaction (PPI) analysis and LASSO regression were used to identify key proteins. The calibration curve, decision curve, and nomogram were utilized to create the model. Fifty-seven DEPs were screened out, with 33 up-regulated and 24 down-regulated in persons with HIV at window period. The most important Gene Ontology (GO) enrichment items are oxidoreductase activity and heme binding. Oxidoreductases account for half of the 10 main proteins identified from various DEPs. An auxiliary diagnostic model comprised of Peroxiredoxin-2 (P32119), Band 3 anion transport protein (P02730), and Histone H2A type 1 (P0C0S8) was developed. The results of the confusion matrix parameters revealed that this diagnostic approach had strong practicability in distinguishing indeterminate HIV infection cases. The three DEPs identified and predicted by proteomics are useful for the supplemental identification of persons in the HIV window period.

Cannabinoids Detected in Exhaled Breath Condensate after Cannabis Use.

Cannabinoids can be detected in breath after cannabis use, but different breath matrices need to be explored as studies to date with filter-based devices that collect breath aerosols have not demonstrated that breath-based measurements can reliably identify recent cannabis use. Exhaled breath condensate (EBC) is an unexplored aqueous breath matrix that contains condensed volatile compounds and water vapor in addition to aerosols. EBC was collected from participants both before and at two time points (0.7 h ± 0.2 h and 1.7 h ± 0.3 h) after observed cannabis use. Eleven different cannabinoids were monitored with liquid chromatography tandem mass spectrometry (LC-MS/MS). Five different cannabinoids, including Δ9-tetrahydrocannabinol (THC), were detected in EBC collected from cannabis users. THC was detected in some EBC samples before cannabis use, despite the requested abstinence period. THC was detected in all EBC samples collected at 0.7 h post use and decreased for all participants at 1.7 h. Non-THC cannabinoids were only detected after cannabis use. THC concentrations in EBC samples collected at 0.7 h showed no trend with sample metrics like mass or number of breaths. EBC sampling devices deserve further investigation with respect to modes of cannabis use (e.g., edibles), post use time points, and optimization of cannabinoid recovery.

Circulating adrenal and gonadal steroid hormones heterogeneity in active young males and the contribution of 11-oxy androgens

The classical androgens, testosterone and dihydrotestosterone, together with dehydroepiandrosterone, the precusrsor to all androgens, are generally included in diagnostic steroid evaluations of androgen excess and deficiency disorders and monitored in androgen replacement and androgen suppressive therapies. The C11-oxy androgens also contribute to androgen excess disorders and are still often excluded from clinical and research-based steroids analysis. The contribution of the C11-oxy androgens to the androgen pool has not been considered in androgen deficiency. An exploratory investigation into circulating adrenal and gonadal steroid hormones in men was undertaken as neither the classical androgens nor the C11-oxy androgens have been evaluated in the context of concurrent measurement of all adrenal steroid hormones. Serum androgens, mineralocorticoids, glucocorticoids, progesterones and androgens were assessed in 70 healthy young men using ultra high performance supercritical fluid chromatography and tandem mass spectrometry. Testosterone, 24.5 nmol/L was the most prominent androgen detected in all participants while dihydrotestosterone, 1.23 nmol/L, was only detected in 25% of the participants. The 11-oxy androgens were present in most of the participants with 11-hydroxyandrostenedione, 3.37 nmol, in 98.5%, 11-ketoandrostenedione 0.764 in 77%, 11-hydroxytestosterone, 0.567 in 96% and 11-ketotestosterone: 0.440 in 63%. A third of the participants with normal testosterone and comparable 11-ketotestosterone, had significantly lower dehydroepiandrosterone (p < 0.001). In these males 11-hydroxyandrostenedione (p < 0.001), 11-ketoandrostenedione (p < 0.01) and 11-hydroxytestosterone (p < 0.006) were decreased. Glucocorticoids were also lower: cortisol (p < 0.001), corticosterone (p < 0.001), cortisone (p < 0.006) 11-dehydrocorticosterone (p < 0.001) as well as cortisol:cortisone (p < 0.001). The presence of dehydroepiandrosterone was associated with 16-hydroxyprogesterone (p < 0.001), which was also significantly lower. Adrenal and gonadal steroid analysis showed unexpected steroid heterogeneity in normal young men. Testosterone constitutes 78% of the circulating free androgens with the 11-oxy androgens abundantly present in all participants significantly contributing 22%. In addition, a subset of men were identified with low circulating dehydroepiandrosterone who showed altered adrenal steroids with decreased glucocorticoids and decreased C11-oxy androgens. Analysis of the classical and 11-oxy androgens with the additional measurement of dehydroepiandrosterone and 16-hydroxyprogesterone may allow better diagnostic accuracy in androgen excess or deficiency.

Master equation modeling of water dissociation in small ionic water clusters: Ag+(H2O) n , n = 4-6.

We model temperature-dependent blackbody infrared radiative dissociation (BIRD) rate coefficients of Ag+(H2O) n , n = 4-6, a system with loosely bound water molecules. We employ a master equation modeling (MEM) approach with consideration of absorption and emission of blackbody radiation, comparing single and multiple-well descriptions. The unimolecular dissociation rate coefficients are obtained using the Rice-Ramsperger-Kassel-Marcus (RRKM) theory, employing two approaches to model the sum of states in the transition state, the rigid activated complex (RAC) and the phase space limit (PSL) approach. A genetic algorithm is used to find structures of low-lying isomers for the kinetic modeling. We show that the multiple-well MEM approach with PSL RRKM in the All Wells and Transition States Are Relevant (AWATAR) variant provides a reliable description of Ag+(H2O) n BIRD, in agreement with previously published experimental data. Higher-lying isomers contribute significantly to the overall dissociation rate coefficient, underlying the importance of the multiple-well ansatz in which all isomers are treated on the same footing.

Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial for understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy human donors and sequenced BCRs at bulk and single-cell levels, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.

UPLC-LTQ-Orbitrap Study on Rat Urinary Metabolites of 5-Methoxy-Alpha-Methyltryptamine.

5-Methoxy-α-Methyltryptamine (5-MeO-AMT) is a new psychoactive substance which is abused due to its hallucinogenic and euphoric effects. This study aimed to study the metabolic characteristics of 5-MeO-AMT. Five rats were given intraperitoneal injection at a dose of 50 mg/kg of 5-MeO-AMT, and their urine was subsequently collected at different times within 7 days. Ultra-high performance liquid chromatography-- tandem high-resolution mass spectrometry (UPLC-LTQ-Orbitrap) was used to detect the precise molecular weight and fragment ions of 5-MeO-AMT and its possible metabolites in the urine sample extracted with benzene-ethyl acetate. Three metabolites, including OH-5-MeO-AMT, α-Me-5-HT, and N-Acetyl-5-MeO-AMT were identified in rats' urine. The major metabolic pathways involved O-demethylation, hydroxylation of indole ring, and Acetylation on aliphatic amines. The results of this study are an important reference for the identification and screening of toxicants of 5-MeO-AMT.

Quantitative Label-Free Proteomic Analysis of Mouse Ovarian Antral Follicles Following Oral Exposure to a Human Relevant Mixture of Three Phthalates.

Dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), and benzyl butyl phthalate (BBP) are used in personal and medical care products. In the ovary, antral follicles are essential for steroidogenesis and ovulation. DBP, BBP, and DEHP are known to inhibit mouse antral follicle growth and ovulation in vitro, and associate with decreased antral follicle counts in women. Given that the in vivo effects of a three-phthalate mixture on antral follicles are unknown, we evaluated the effects of a human relevant mixture of DBP, BBP, and DEHP on ovarian follicles through proteome profiling analysis. Adult CD-1 female mice were fed corn oil (vehicle), or two dose levels of a phthalate mixture based on estimated exposures in general (32 µg/kg/day; PHT 32) and occupationally exposed (500 µg/kg/day; PHT 500) populations for 10 days. Antral follicles (>250 µm) were isolated and subjected to proteome profiling via label-free tandem mass spectrometry. A total of 5,417 antral follicle proteins were detected, of which 194 were differentially abundant between vehicle and PHT 32, and 136 between vehicle and PHT 500. Bioinformatic analysis revealed significantly different responses between the two phthalate doses. Protein abundance differences in the PHT 32 exposure mapped to cytoplasm, mitochondria, and lipid metabolism; while those in the PHT 500 exposure mapped to cytoplasm, nucleus, and phosphorylation. When both doses altered proteins mapped to common processes, the associated predicted transcription factors were different. These findings provide novel mechanistic insight into phthalate-associated, ovary-driven reproductive outcomes in women.

Chondroitin sulfate glycan sulfation patterns influence histochemical labeling of perineuronal nets: a comparative study of interregional distribution in human and mouse brain.

Perineuronal nets (PNNs) are a condensed subtype of extracellular matrix that form a net-like coverings around certain neurons in the brain. PNNs are primarily composed of chondroitin sulfate (CS) proteoglycans from the lectican family that consist of CS-glycosaminoglycan (CS-GAG) side chains attached to a core protein. CS disaccharides can exist in various isoforms with different sulfation patterns. Literature suggests that CS disaccharide sulfation patterns can influence the function of PNNs as well as their labeling. This study was conducted to characterize such interregional CS disaccharide sulfation pattern differences in adult human (n=81) and mouse (n=19) brains. Liquid chromatography tandem mass spectrometry was used to quantify five different CS disaccharide sulfation patterns, which were then compared to immunolabeling of PNNs using Wisteria Floribunda Lectin (WFL) to identify CS-GAGs and anti-aggrecan to identify CS proteoglycans. In healthy brains, significant regional and species-specific differences in CS disaccharide sulfation and single versus double-labeling pattern were identified. A secondary analysis to investigate how early-life stress (ELS) impacts these PNN features discovered that although ELS increases WFL+ PNN density, the CS-GAG sulfation code and single versus double PNN-labeling distributions remained unaffected in both species. These results underscore PNN complexity in traditional research, emphasizing the need to consider their heterogeneity in future experiments.

A carrier-based quantitative proteomics method applied to biomarker discovery in pericardial fluid

Data-dependent liquid chromatography tandem mass spectrometry (LC-MS/MS) is challenged by the large concentration range of proteins in plasma and related fluids. We adapted the SCoPE method from single-cell proteomics to pericardial fluid, where a myocardial tissue carrier was used to aid protein quantification. The carrier proteome and patient samples were labeled with distinct isobaric labels, which allowed separate quantification. Undepleted pericardial fluid from patients with type 2 diabetes mellitus and/or heart failure undergoing heart surgery was analyzed with either a traditional LC-MS/MS method or with the carrier proteome. In total, 1398 proteins were quantified with a carrier, compared to 265 without, and a higher proportion of these proteins were of myocardial origin. The number of differentially expressed proteins also increased nearly four-fold. For patients with both heart failure and type 2 diabetes mellitus, pathway analysis of upregulated proteins demonstrated enrichment of immune activation, blood coagulation, and stress pathways. Overall, our work demonstrates the applicability of a carrier for enhanced protein quantification in challenging biological matrices such as pericardial fluid, with potential applications for biomarker discovery. Mass spectrometry data are available via ProteomeXchange with identifier PXD053450.

NAT10 Overexpression Promotes Tumorigenesis and Epithelial-Mesenchymal Transition Through AKT Pathway in Gastric Cancer.

N-acetyltransferase 10 (NAT10), the only RNA cytosine acetyltransferase known in humans, contributes to cancer tumorigenesis and progression. This study aims to investigate the effect of NAT10 on the malignant biological properties of gastric cancer (GC) and its underlying mechanism. The expression and prognostic significance of NAT10 in GC were analyzed using The Cancer Genome Atlas (TCGA) and Sun Yat-sen University (SYSU) cohorts. The influence of NAT10 on the malignant biological behaviors of GC was detected by Cell Counting Kit-8 (CCK-8) assay, plate colony formation assay, 5-ethynyl-2'-deoxyuridine (EdU), Transwell migration and invasion assays, scratch wound assay, flow cytometric analysis, and animal studies. The overall level of N4 acetylcytidine (ac4C) in GC was detected by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The downstream signal pathways of NAT10 were analyzed by Gene Set Enrichment Analysis (GSEA) and verified by Western blot (WB) and immunofluorescence (IF). The significant upregulation of NAT10 expression in GC was associated with a poor prognosis. The knockdown of NAT10 markedly suppressed GC cell proliferation, migration, invasion, and cell cycle progression. Downregulating NAT10 reduced ac4C levels and inhibited AKT phosphorylation and epithelial-mesenchymal transition (EMT) in GC. NAT10 functions as an oncogene and may provide a new therapeutic target in GC.

Biological Evaluation of Lysionotin: a Novel Inhibitor of 5-Lipoxygenase for Anti-glioma.

To explore the potential mechanism of lysionotin in treating glioma. First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively. Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling. Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.

Enhancing anthocyanin extraction efficiency in vegetables and fruits: a high-speed shear homogenization technology.

To extract anthocyanins with high efficiency, a hypothesis for high-speed shear homogenization extraction (HSHE) method was established through a combination of solvent and ultrasonic-assisted extractions. The efficacy of this hypothesis was demonstrated by performing qualitative and quantitative analyses of 16 anthocyanins extracted from five northern vegetables, and five berry fruits using ultra-high-performance Q-Exactive Orbitrap tandem mass spectrometry. Single-factor experiments were conducted by varying ethanol concentration, temperature, pH and extraction cycles to determine the optimal conditions for this method. Optimal extraction conditions (ethanol 70-80%, 40-50 °C, pH 3-4, performed twice) were determined using an HSHE (5 min, 10 000 rpm, 25 °C) assisted shaker (60 min) and ultrasonication (40 kHz, 160 W cm-2, 30 min, 25 °C) procedure. Compared to the traditional non-HSHE method, the total anthocyanin content obtained through HSHE extraction showed a significant increase, ranging from 1.0 to 3.9 times higher, with purple cabbage exhibiting the most pronounced enhancement in content. More types of anthocyanins were detected in blueberry (9), black bean (7) and raspberry (5), of which malvidin was the major anthocyanin (0.426 g kg-1) in blueberry, having an amount five times than previously obtained. The established HSHE method has been proven to be a superior technique for anthocyanin extraction, with higher extraction efficiency and concentrations. This technique also provides a new avenue for extracting bioactive compounds from diverse food sources, with potential applications in improving the functional properties of food products. © 2024 Society of Chemical Industry.

Effects of the antitumor drugs adagrasib and asciminib on apixaban metabolism in vitro and in vivo

Apixaban is an oral anticoagulant that directly inhibits the target Factor Xa (FXa). In this study, we focused on the in vivo and in vitro effects of adagrasib and asciminib on apixaban metabolism, to discover potential drug-drug interactions (DDI) and explore their inhibitory mechanisms. The levels of apixaban and its metabolite, O-desmethyl-apixaban (M2), were determined by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In vitro evaluation, the maximum half inhibitory concentration (IC50) of adagrasib in rat liver microsomes (RLM) and human liver microsomes (HLM) against apixaban was 7.99 μM and 117.40 μM, respectively. The IC50 value of asciminib against apixaban in RLM and HLM was 4.28 μM and 18.42 μM, respectively. The results of the the analysis on inhibition mechanisms showed that adagrasib inhibited the metabolism of apixaban through a non-competitive mechanism, while asciminib inhibited the metabolism of apixaban through a mixed mechanism. Moreover, the interaction of apixaban with adagrasib and asciminib in Sprague-Dawley (SD) rats was also investigated. It was found that the pharmacokinetic characteristics of apixaban were significantly changed when combined with these two antitumor drugs, where AUC(0-t), AUC(0-∞), t1/2, Tmax, and Cmax were increased, while CLz/F was significantly decreased. But both drugs did not appear to affect the metabolism of M2 in a significant way. Consistent results from in vitro and in vivo demonstrated that both adagrasib and asciminib inhibited the metabolism of apixaban. It provided reference data for the future clinical individualization of apixaban.

Improved Characterization of Soil Organic Matter by Integrating FT-ICR MS, Liquid Chromatography Tandem Mass Spectrometry, and Molecular Networking: A Case Study of Root Litter Decay under Drought Conditions.

Understanding of how soil organic matter (SOM) chemistry is altered in a changing climate has advanced considerably; however, most SOM components remain unidentified, impeding the ability to characterize a major fraction of organic matter and predict what types of molecules, and from which sources, will persist in soil. We present a novel approach to better characterize SOM extracts by integrating information from three types of analyses, and we deploy this method to characterize decaying root-detritus soil microcosms subjected to either drought or normal conditions. To observe broad differences in composition, we employed direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR MS). We complemented this with liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify components by library matching. Since libraries contain only a small fraction of SOM components, we also used fragment spectral cosine similarity scores to relate unknowns and library matches through molecular networks. This integrated approach allowed us to corroborate DI-FT-ICR MS molecular formulas using library matches, which included fungal metabolites and related polyphenolic compounds. We also inferred structures of unknowns from molecular networks and improved LC-MS/MS annotation rates from ∼5 to 35% by considering DI-FT-ICR MS molecular formula assignments. Under drought conditions, we found greater relative amounts of lignin-like vs condensed aromatic polyphenol formulas and lower average nominal oxidation state of carbon, suggesting reduced decomposition of SOM and/or microbes under stress. Our integrated approach provides a framework for enhanced annotation of SOM components that is more comprehensive than performing individual data analyses in parallel.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) β-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG β-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Simultaneous detection of three hypoxia-inducible factor stabilizers-molidustat, roxadustat, and vadadustat-in multiple keratinized matrices and its application in a doping context.

In a doping case, a top athlete challenged an anti-doping rule violation, involving molidustat. Molidustat is a stabilizing agent of the hypoxia-inducible factor (HIF) recently developed. It is currently undergoing clinical trials for anemia associated with chronic kidney disease. HIF stabilizers are banned at all times by the World Anti-Doping Agency (class S2). Because of their pharmacological proprieties, these new drugs can enhance athletic performance. The athlete's defense wanted to analyze multiple keratinized matrices as they allow long-term investigations. Requests concerning HIF stabilizers are constantly growing. We have therefore developed a liquid chromatography coupled with tandem mass spectrometry method to identify and quantify three molecules of this class: molidustat, vadadustat, and roxadustat. Thirty milligrams of keratinized matrices were incubated in 1mL of pH8.4 diammonium hydrogen phosphate buffer for 16 h at 40°C with 1ng of testosterone-D3, used as internal standard. After extraction with ethyl acetate/diethyl ether (80/20), the organic phase was evaporated, and the dry residue was reconstituted in 30 μL of initial phase. The method was linear from 5 to 1000 pg/mg for the three analytes. Limits of quantification were 2, 0.5, and 5pg/mg for molidustat, roxadustat, and vadadustat, respectively. The analysis of the athlete's head hair (collected 1 month after the urine test) showed a concentration of molidustat of 135 pg/mg, and his beard hair and his fingernails clippings contained 55 and 40 pg/mg, respectively.

Determination of warfarin in volumetric absorptive microsampling by liquid chromatography-tandem mass spectrometry

ObjectiveThis study aims to develop and validate bioanalytical method for quantifying warfarin in VAMS samples using liquid chromatography tandem mass spectrometry (LC-MS/MS), directly implementing the method to patients receiving warfarin therapy. MethodsThe UPLC-MS/MS method was developed and optimized, with quercetin as the internal standard. Sample preparation was carried out using protein precipitation with methanol-acetonitrile (1:3 v/v). ResultsChromatographic separation was achieved using Acquity® UPLC BEH C18 column with 0.1 % formic acid-acetonitrile-methanol (30:69:1 v/v) as mobile phase, in isocratic elution. Multiple Reaction Monitoring (MRM) detection was done using m/z values of 307.10 → 161.06 for warfarin and 301.03 → 150.98 for quercetin as internal standard, using Electrospray Ionization (ESI) negative ion source. The clinical application of the bioanalytical method was carried out on 25 patients receiving warfarin therapy at Universitas Indonesia Hospital and warfarin levels were well within the calibration range from 6.05 to 431.39 ng/mL. ConclusionA novel method has been developed to analyze warfarin in VAMS samples. This method has been fully validated according to guideline from FDA 2022 and is linear in the range of 5–500 ng/mL and the value of r ≥ 0.9977, and successfully applied for the analysis of warfarin in VAMS samples of clinical patients.

The usefulness of determining plasma and tissue concentrations of phosphorodiamidate morpholino oligonucleotides to estimate their efficacy in Duchenne muscular dystrophy patients.

Currently four kinds of phosphorodiamidate morpholino oligomers (PMOs) such as viltolarsen have been approved for the treatment of Duchenne muscular dystrophy (DMD); however, it is unclear whether human efficacy can be estimated using plasma concentrations. This study summarizes the tissue distribution of viltolarsen in mice and cynomolgus monkeys and evaluates the relationship between exposure and efficacy based on exon skipping. In the tissue distribution studies, all muscles in DMD model mice showed higher concentrations of viltolarsen than those in wild-type mice and cynomolgus monkeys, and the concentrations in skeletal muscle were correlated with the exon-skipping efficiency in mice and cynomolgus monkeys. In addition, a highly sensitive bioanalytical method using liquid chromatography with tandem mass spectrometry shows promise for determining plasma concentrations up to a later time point, and the tissue (muscle)/plasma concentration ratio (Kp) in DMD model mice was shown to be useful for predicting changes in pharmacodynamic (PD) markers in humans. Our results suggest that pharmacokinetic (PK)/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD model mice. This information will be useful for the efficient and effective development of PMOs as therapeutic agents. Significance Statement We compare that plasma and tissue concentrations with the efficiency of exon skipping for viltolarsen as an example phosphorodiamidate morpholino oligomers in skeletal and cardiac muscle of mice and cynomolgus monkeys for pharmacokinetics/pharmacodynamics (PK/PD) analysis. Our results suggest that PK/PD analysis can be conducted by using the human PK profile or Kp values and skipping efficiency in DMD model mice.