Previous meta-analyses examining the association between calcium supplementation and CVD risk have reported inconsistent findings. However, whether calcium supplementation increases the risk of adverse cardiovascular outcomes remains unclear. In randomized evidence, standard-dose calcium supplementation was not associated with a material increase in major cardiovascular outcomes; inverse associations seen in cohort studies should be interpreted cautiously because of heterogeneity and residual confounding.

Antimetabolite drugs are cornerstones in treating various cancers and autoimmune diseases; however, their clinical utility is often hampered by systemic toxicity caused by drug-induced gut microbiota dysbiosis. Predicting patient responses remains a significant challenge. Several studies have highlighted the influence of gut microbiota on antimetabolite treatment outcomes, revealing complex bidirectional interactions between the drugs and microbial communities. This review synthesizes the effects of common antimetabolites (including 5-fluorouracil, methotrexate, gemcitabine, capecitabine, 6-mercaptopurine, and thioguanine) on gut microbial communities and outlines a framework (pharmacokinetics, endogenous metabolite production, immune modulation, and apoptotic pathway modulation) for assessing chemotherapy-microbiota interactions. Additionally, potential microbial biomarkers for predicting treatment responses and strategies for manipulating the gut microbiota to enhance therapeutic efficacy are discussed. Therefore, advances in methodologies such as metagenomics and real-time microbial monitoring will be essential for unraveling these interactions and promoting the precise application of antimetabolite drugs.

Drug-induced liver injury (DILI) is one of the most common adverse reactions in clinical settings, and drug hepatotoxicity is also a primary factor contributing to the suspension and withdrawal of drug development. Currently, there are no specific biomarkers available for the early diagnosis and monitoring of DILI in clinical practice. In recent years, metabolomics has emerged as a powerful tool for identifying small molecule biomarkers in DILI, owing to its unique advantages. In this article, we comprehensively reviewed the metabolomics studies related to DILI to investigate the pathophysiological changes in DILI and to assess the potential application of metabolomics in its prediction and diagnosis. Relevant literature published before 30 April 2024 was retrieved from four online databases (PubMed, EMbase, Cochrane Library, and Web of Science). Research data were systematically collected, and the metabolites involved were analyzed. Metabolic pathway analysis related to DILI was conducted using the online software MetaboAnalyst 5.0. A total of 55 studies were included, identifying significantly altered metabolic pathways and valuable metabolic biomarkers. The most frequently reported biomarkers included glycocholic acid, taurochenodeoxycholic acid, taurocholic acid, creatine, glycochenodeoxycholic acid, taurodeoxycholic acid, glycodeoxycholic acid, and α-ketoglutaric acid. The disrupted metabolic pathways were mainly related to bile acid metabolism, lipid metabolism, and the tricarboxylic acid cycle, among which bile acid metabolism showing the most prominent alterations. Lipid metabolism-related pathways included fatty acid biosynthesis, glycerophospholipid metabolism, and sphingolipid metabolism. This article provides a systematic review of metabolomics in DILI, deepening our understanding of the metabolic disturbances caused by DILI.

Progestogens are essential for female reproductive homeostasis, and their dysregulation is implicated in disorders such as polycystic ovary syndrome (PCOS). Conventional immunoassays lack adequate selectivity and sensitivity for multi-analyte progestogen quantification, particularly at low serum concentrations, owing to antibody cross-reactivity and matrix interference. This study developed a highly sensitive and specific LC-MS/MS method by combining magnetic solid-phase extraction (MSPE) with a novel quaternary ammonium oxime (QAO) derivatization. This method enabled the simultaneous quantification of three key progestogens-pregnenolone (Preg), 17-hydroxypregnenolone (17OHPreg), and 17-hydroxyprogesterone (17OHP)-in serum from patients with PCOS. QAO derivatization significantly enhanced the ionization efficiency and improved detection sensitivity for all three targets. The validated method demonstrated excellent linearity across the tested ranges, with precision (RSD%) below 13.56%, accuracy (RE%) within +/- 15%, consistent recovery, and negligible matrix effects. The method was successfully applied to the clinical samples, enabling reliable measurement of low-abundance progestogens and providing a robust analytical tool for endocrine investigations in PCOS.

Bile acids (BAs) facilitate the digestion and absorption of fats and influence lipid and glucose homeostasis, making them potential therapeutic targets for obesity and related metabolic disorders. The liver and intestinal microbiota modify BAs structurally, generating diverse chemical forms and isomers. Comprehensive profiling of the BA pool is critical for understanding their key biological functions and as a therapeutic approach for related diseases. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is usually chosen as the preferred method for BA detection due to the complex chemical structures, the wide range of actual concentrations and the complexity of fecal sample matrices. However, free BAs are difficult to ionize, resulting in low detection signals and a lack of characteristic structural fragments to assist in structural identification. In this method, the labeling reagent (2-aminoethyl) trimethylammonium (AETMA) is employed to label the carboxyl group of BAs. Compared with underivatized BAs, the detection sensitivity of unconjugated BAs was enhanced by 25-180 fold, while that of conjugated BAs increased by 6-160 fold. It also generates unique fragment ions and enhances MS response, facilitating the discovery of potential BAs. Methodological parameters were validated using 38 BAs as representatives. Through methodological validation, it was verified that the precision, recovery, matrix effect and stability parameters of the method met acceptable criteria. We also identified 61 confirmed BAs and 55 additional candidate BAs in human pooled fecal samples. It has been successfully applied to fecal BA analysis in obese populations, providing valuable insights into potential therapeutic strategies for obesity.

BackgroundThe development of Bruton's tyrosine kinase (BTK) inhibitors has revolutionized the management of B-cell malignancies and holds potential in autoimmune diseases. However, safety concerns remain regarding treatment-associated adverse events: first-generation ibrutinib has been associated with off-target adverse event profiles, second-generation agents (acalabrutinib and zanubrutinib) require further validation of long-term safety patterns, and the real-world safety characteristics of third-generation pirtobrutinib continue to be evaluated. This study aims to characterize differences in adverse event (AE) disproportionality reporting signal patterns among four BTK inhibitors using data from the FDA Adverse Event Reporting System (FAERS).MethodsThis study analyzed AE reports for four BTK inhibitors from the FAERS database via disproportionality analysis with four distinct algorithms, complemented by a clinical prioritization scoring system. Disproportionality reporting signal patterns were characterized across System Organ Class (SOC), Preferred Term (PT), and temporal distribution dimensions. A standardized 2-year observation window was applied for sensitivity analysis, and AE-mortality associations were evaluated.ResultsA total of 77,014 AE reports were included in this study. The four BTK inhibitors showed positive disproportionality reporting signals in blood and lymphatic system disorders, cardiac disorders, and infections and infestations. However, distinct disproportionality signal patterns were observed at the PT level. Ibrutinib showed prominent cardiovascular-related disproportionality reporting signals, particularly for atrial fibrillation (ROR = 9.8). Acalabrutinib was characterized by a distinct headache signal. Zanubrutinib showed prominent signals for myelosuppression (ROR = 16.24) and haemorrhage subcutaneous (ROR = 146.81). Pirtobrutinib demonstrated the strongest signal for increased white blood cell count (ROR = 54.28). Descriptive analysis of reported time-to-onset distributions showed that pneumonia reports for ibrutinib represented a larger proportion among cases reported after longer treatment durations, whereas haemorrhage and decreased white blood cell count reports were more frequently observed within earlier reporting intervals. In terms of fatal outcomes, multiple PTs associated with ibrutinib showed statistically significant associations with reported death outcomes within the FAERS dataset.ConclusionThis descriptive pharmacovigilance study identified distinct disproportionality reporting signal patterns for four BTK inhibitors based on FAERS data, providing potential signals for post-marketing safety surveillance. These findings should be interpreted as differences in reporting patterns.

Background: Prophylaxis in children with hemophilia B (HB) lacks quantitative approaches that integrate both plasma exposure and tissue distribution. This study aimed to develop and validate a physiologically based pharmacokinetic (PBPK) model of factor IX (FIX) for pediatric HB. The model incorporated the binding of FIX to type IV collagen (Col4) to characterize its distribution in both plasma and extravascular tissues. Methods: A total of 20 children with severe HB were included, contributing 219 plasma samples. The base PBPK model was first established and verified using adult and plasma-derived FIX (pdFIX) data. It was subsequently extrapolated to children by integrating FIX-CTBB parameters and pediatric observations for model calibration. The validated model was used to characterize plasma pharmacokinetics, predict tissue distribution and target attainment, and simulate alternative prophylactic dosing regimens. Results: The model adequately described the plasma pharmacokinetics of FIX in children and predicted substantial extravascular distribution. Total extravascular exposure was approximately sixfold higher than plasma exposure. Marked heterogeneity in target attainment was identified across tissues. Lower target attainment was observed in the colon, pancreas, and brain, whereas delayed attainment occurred in bone and muscle. Simulations of prophylactic dosing regimens suggested that 75 IU/kg twice weekly may provide a favorable balance among sustained FIX exposure, tissue-level target attainment, and treatment burden. Conclusions: This PBPK model provides a mechanistic and quantitative framework for characterizing plasma and tissue exposure to FIX in children with HB and may support individualized optimization of FIX prophylactic dosing.

Accurate quantification of circulating androgens, particularly the biologically active free fraction, is analytically challenging due to ultra-low concentrations and extensive protein binding in human serum. Here, we developed and validated an integrated ultrafiltration-LC-MS/MS workflow for simultaneous quantification of nine endogenous serum free androgens. Free fractions were isolated by phosphate-buffered saline dilution followed by temperature-controlled centrifugal ultrafiltration (37 °C) using pretreated regenerated-cellulose membranes to minimize non-specific adsorption. Ultrafiltrates were purified and enriched by magnetic solid-phase extraction (MSPE), and in-situ oximation derivatization with a quaternary aminooxy reagent was applied to enhance electrospray response. The validation data demonstrated pg/mL-level sensitivity with LLOQ of 0.5-10 pg/mL, good linearity (r > 0.99), acceptable matrix effects, and high recovery. The method was applied to serum samples from 86 female patients (21-50 years) undergoing infertility evaluation, showing higher free testosterone, 11β-hydroxyandrostenedione, and androstenedione in an AMH-enriched PCOS-suspicion subgroup versus a control group. Overall, this ultrafiltration-MSPE-in-situ derivatization LC-MS/MS method provides a practical and robust platform for multiplexed measurement of serum free androgens at pg/mL levels for clinical research applications.

Inflammatory bowel disease (IBD) is characterized by destruction of the intestinal barrier, dysregulation of mucosal immunity, and imbalance of the gut microbiota homeostasis. Conventional immunosuppressants such as azathioprine (AZA), commonly used in the treatment of IBD, can partially alleviate symptoms but fail to restore normal barrier and immune functions, while exhibiting non-negligible adverse effects. Here, we report an AZA-loaded microbiota-modulating and colon-targeted nanoparticle constructed from pectin, Zein, and Eudragit®S100 (APZE). APZE could effectively encapsulate AZA and enhance its cellular and intestinal uptake, thereby improving oral bioavailability in rats compared to AZA suspension. Additionally, its colon targeting ability and mucoadhesive properties prolonged colonic retention, leading to higher colonic accumulation, as indicated by the AUC of colon fluorescence intensity in rats after oral administration of the DiR-labeled formulation. Animal studies demonstrated that APZE significantly reduced inflammation in IBD mice, repaired the intestinal barrier, modulated the gut microbiota, and upregulated short-chain fatty acid levels, exhibiting significant therapeutic efficacy with good safety. The protective effect of APZE against colitis is largely microbiota-dependent. When co-administered with commercially available Bifidobacterium, APZE attenuated colitis effectively, demonstrating excellent therapeutic effects and favorable safety. In summary, our findings indicate that APZE and Bifidobacterium improved intestinal barrier repair and colitis-related outcomes in a DSS-induced model, supporting their potential as therapeutic agents for IBD.

Oral nanomedicines provide a promising, noninvasive drug delivery platform, offering advantages in patient compliance and ease of administration. However, their clinical translation for both gastrointestinal (GI) and extra-GI diseases is hampered by multiple physiological and pharmacokinetic barriers, including the harsh GI environment, poor translocation across the mucus and epithelial layers, rapid hepatic and renal clearance, limited tissue accumulation, inefficient cellular uptake, insufficient lysosome escape, and suboptimal intracellular drug release. These challenges collectively result in low oral bioavailability and limited therapeutic outcomes. This review provides a comprehensive overview of recent advances in oral nanomedicines, delineating six key attributes for effective GI-targeted drug delivery and eight design principles for systemic applications. We also discuss the translational bottlenecks and highlight the emerging strategies to overcome these barriers, offering insights into the integration of oral nanotherapeutics into precision medicine.

Purpose:Therapeutic drug monitoring-based individualized busulfan (Bu) administration improves hematopoietic stem cell transplantation (HSCT) outcomes, but optimal Bu exposure in Chinese pediatric patients remains unclear; we aim to identify the optimal exposure level of Bu in such patients receiving Bu-based conditioning regimens of different intensities. Patients and Methods:Pediatric patients from 5 transplant centers in China who received Bu-based myeloablative conditioning regimens were included. Bu exposure, defined as the area under the concentration-time curve (AUC), was estimated using a population pharmacokinetic model, including the first-dose AUC and cumulative AUC (cAUC). The primary and secondary outcomes included event-free survival (EFS) and transplantation-related complications. Restricted cubic splines and Cox proportional hazards models were used to analyze the exposure‒response relationships between the Bu AUC and outcomes. Results:A total of 455 pediatric patients were enrolled, with 326 receiving a 3-day Bu-based (Bu3) regimen and 129 receiving a 4-day Bu-based (Bu4) regimen. Among all patients, a first-dose Bu AUC ≥ 4.5 mg×h/L was associated with a significantly higher 2-year EFS compared with AUC < 4.5 mg×h/L (84.6% vs 76.4%; P = 0.02). In the Bu3 cohort, a cAUC < 54 mg×h/L was associated with a lower 2-year EFS rate (77.7% vs 86.4%; P=0.04). In the Bu4 cohort, a cAUC of 70-90 mg×h/L was associated with a greater 2-year EFS rate (88.9%) than an exposure <70 mg×h/L (71.0%; P = 0.03) or >90 mg×h/L (66.1%; P = 0.04). Conclusion:This study demonstrated an association between Bu exposure and EFS in Chinese pediatric HSCT patients, with optimal cumulative AUC values of ≥ 54 mg×h/L and 70-90 mg×h/L identified for the Bu3 and Bu4 regimens, respectively. Additionally, a first-dose Bu AUC ≥ 4.5 mg×h/L was associated with a favorable 2-year EFS.

Acute kidney injury (AKI) is a common syndrome among critically ill patients with high incidence and high mortality, characterized by elevated serum creatinine (SCr) and uric acid (UA) levels. Given that xanthine is a key intermediate in nucleotide metabolism and the direct precursor of UA, profiling of nucleotide metabolic dysregulation in AKI remains largely unexplored. Using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), we simultaneously quantified 29 nucleotide intermediates both in plasma and matched urine samples from 58 propensity score-matched pairs of AKI and non-AKI (NAKI) critically ill patients. Compared with NAKI controls, AKI patients showed decreased levels of 7 urinary nucleotide intermediates and increased levels of 4 plasma metabolites. Urinary nucleotide levels correlated more strongly with SCr and estimated Glomerular Filtration Rate (eGFR) than their plasma counterparts. Elevated urinary xanthine and other 5 metabolites were identified as protective factors for AKI, while elevated plasma adenine, thymine and cytosine were risk factors. A combination of urinary guanine and xanthine with plasma thymine discriminated AKI risk with an area under the curve (AUC = 0.880, 95% CI = 0.800-0.934). Interestingly, alterations in nucleotide intermediates influenced AKI occurrence mediated by SCr, eGFR, UA, urea and aspartate aminotransferase (AST), with hypoxanthine and thymidine exerting specifically through AST. In summary, dysregulated nucleotide metabolism was closely associated with AKI onset and participated in kidney-liver crosstalk through modulation of liver function, providing new mechanistic insights into AKI pathogenesis.

The folate cycle is essential for regulating metabolic processes. Simultaneous measurement of folate cycle intermediates is crucial for understanding metabolic disruptions in hematopoietic, nervous, renal and cardiovascular diseases. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for folate cycle metabolites showed poor retention for highly polar compounds in reversed-phase separations with long analytical run time (30 min). Herein, we developed a novel LC-MS/MS method using hydrophilic interaction liquid chromatography (HILIC) mode for simultaneous measurement of 10 key folate cycle metabolites in human plasma, including 5 folate intermediates, 4 related amino acids, and a cofactor (VB12), with enhanced chromatographic retention and reduced analysis time (8.5 min) without derivatization. Through further method validation, all analytes demonstrated acceptable linearity (R² > 0.989), precision (intra-day precision: 1.3-11.3 %; inter-day precision: 3.4-14.6 %), recovery (89.5-113.8 %) and reasonable matrix effect (81.6-115.8 %). The results presented that all intermediates were stable for 5 h at 5°C (autosampler), 12 h at -40°C and 24 h at -80°C. Moreover, the method was successfully applied in clinical plasma from critically ill patients, revealing distinct metabolic perturbations in acute kidney injury (AKI) inpatients compared with non-AKI controls (NAKI). Levels of 5-MTHF and Gly were significantly elevated in the AKI group. Correlation analysis revealed that SCr levels were positively correlated with both 5-MTHF (r = 0.26, p = 0.04) and hCys (r = 0.27, p = 0.04) concentrations. The study is promising to evaluate folate nutritional status to mitigate the risks of folate-related diseases, such as megaloblastic anemia and neural tube defects.

Supporting information for manuscript

High-dose methotrexate (HD-MTX) is essential in treating acute lymphoblastic leukemia (ALL), but its pharmacokinetics and toxicity are influenced by transporter and metabolic enzyme gene polymorphisms. This study examines the impact of gene polymorphisms in the MTX metabolic pathway on toxicity and pharmacokinetics in Chinese patients with ALL to aid individualized therapy in order to ensure the safety of patients. We prospectively collected 273 serum MTX concentration data from 92 patients with ALL (15-71 years) undergoing HD-MTX. Differences in liver and kidney toxicity markers were analyzed across gene polymorphisms. A population pharmacokinetic model was developed using nonlinear mixed-effect modeling to explore the impact of demographic, biochemical, and genetic covariates. Patients who were homozygous for γ-glutamyl hydrolase gene rs13248452 (GG + AA genotypes) had higher urea levels. SLCO1B1 mutant allele carriers had elevated alanine aminotransferase and aspartate aminotransferase levels compared with homozygous patients. SLC19A1 rs2838957 CT genotype patients had significantly higher aspartate aminotransferase. Population pharmacokinetic analysis indicated that the glomerular filtration rate significantly affected the MTX clearance, whereas gene polymorphisms did not significantly alter the clearance rate or volume of distribution of MTX. In conclusion, gene polymorphisms in SLCO1B1, SLC19A1, and γ-glutamyl hydrolase correlate with MTX-induced alterations in liver and kidney function indicators, but the toxicity mechanisms may be independent of systemic pharmacokinetics. In clinical practice, dosage adjustments should be made based on glomerular filtration rate, with enhanced monitoring for high-risk genotypes. Genetic markers should be combined with renal function monitoring to optimize individualized MTX therapy and improve safety. SIGNIFICANCE STATEMENT: This study identifies γ-glutamyl hydrolase/SLCO1B1/SLC19A1 polymorphisms as key predictors of methotrexate toxicity in Chinese acute lymphoblastic leukemia patients, whereas population pharmacokinetic reveals glomerular filtration rate-not genetics-drives clearance. Findings advocate for genotype-toxicity monitoring and glomerular filtration rate-based dosing to improve safety.

In end-stage osteoarthritis, total joint arthroplasty (TJA) represents the definitive therapeutic intervention. Cefuroxime, a second-generation cephalosporin, exhibits a broad spectrum of activity against both Gram-negative and Gram-positive microorganisms, making it a cornerstone of surgical antimicrobial prophylaxis (SAP) to mitigate prosthetic joint infection (PJI) risk. However, the escalating demand for revision arthroplasties has paralleled rising implant-associated infections, necessitating target-site pharmacokinetic optimization to ensure effective antibiotic exposure at the bone-implant interface. Therefore, we developed a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of cefuroxime in human plasma and bone tissues. The separation was completed in 7.5 min on a BEH C18 column (2.1 × 50 mm, 3.5 μm), and the gradient elution was performed in a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water at a flow rate of 0.3 mL min-1. The correlation coefficients of calibration curves were all greater than 0.99. The detection accuracy of plasma ranged from 93.11% to 98.60% (89.15-106.2% for bone). The intra- and inter-assay precision for both plasma and bone measurements were within 15% (20% at the lower limit of quantitation, LLOQ). The matrix effects were 2.34% to 2.91% in plasma and 3.13-5.17% in bone, while extraction recoveries ranged from 99.8% to 102.0% for plasma and 105.0-107.0% for bone. Upon stability assessment under varying storage conditions, all samples exhibited a difference of less than 15.0%. The method was successfully applied to the determination of cefuroxime in plasma and bone tissues of actual patients.

ABSTRACTBackground7‐Hydroxymethotrexate (7‐OHMTX) is the main metabolite in plasma following high‐dose MTX (HD‐MTX), which may result in activity and toxicity of the MTX. Moreover, 7‐OHMTX could produce crystalline‐like deposits within the renal tubules under acidic conditions or induce renal inflammation, oxidative stress, and cell apoptosis through various signaling pathways, ultimately leading to kidney damage. The objectives of this study were thus to explore the exposure–safety relationship of two compounds and search the most reliable marker for predicting HDMTX nephrotoxicity.MethodA total of 280 plasma concentration data (140 for MTX and 140 for 7‐OHMTX) for 60 pediatric patients with non‐Hodgkin lymphoma (NHL) were prospectively collected. Plasma MTX and 7‐OHMTX concentrations were determined using a high‐performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) method. A nonlinear mixed effect model approach was used to build a joint population pharmacokinetic (PopPK) model. After validation, the model estimated the peak concentration (Cmax) and area under the curve within the initial 48 h (AUC0‐48h) of the patients after drug administration by Bayesian feedback. The receiver operating characteristic (ROC) curves were generated to identify an exposure threshold associated with nephrotoxicity.ResultsA three‐compartment chain model (central and peripheral compartments for MTX and central compartment 7‐OHMTX) with the first‐order elimination adequately characterized the in vivo process of MTX and 7‐OHMTX. The covariate analysis identified that the aspartate aminotransferase (AST) was strongly associated with the peripheral volume of distribution of MTX. Moreover, the Cmax of MTX and 7‐OHMTX showed significant differences (p < 0.0001, p = 0.0472, respectively) among patients with or without nephrotoxicity. Similarly, individuals with nephrotoxicity also exhibited substantially higher ratio of 7‐OHMTX to MTX peak concentration and the sum of MTX + 2.25 times the concentration of 7‐OHMTX (p < 0.0001, p = 0.0426, respectively). By ROC analysis, the Cmax of MTX and 7‐OHMTX had the greatest area under the curve (AUC) values (0.769 and 0.771, respectively). A Cmax threshold of 9.26 μmol/L for MTX or a Cmax threshold of 0.66 μmol/L for 7‐OHMTX was associated with the best sensitivity/specificity for toxicity events (MTX: sensitivity = 0.886; specificity = 0.70; 7‐OHMTX: sensitivity = 0.886; specificity = 0.70).ConclusionsWe demonstrated that the Cmax of MTX and 7‐OHMTX were the most reliable markers associated with nephrotoxicity and proposed a Cmax threshold of 9.26 μmol/L for MTX and 0.66 μmol/L for 7‐OHMTX as the point with a high risk of nephrotoxicity. Altogether, this study may contribute to crucial insights for ensuring the safe administration of drugs in pediatric clinical practice.

The accumulation of classic or 11-oxygenated androgens is a hallmark of polycystic ovary syndrome (PCOS). Traditional electrochemiluminescence immunoassays (ECLIA) are limited in their ability to measure a broad spectrum of androgens and may suffer from potential cross-reactivity, leading to inaccurate results. In this study, we developed a highly sensitive HPLC-MS/MS method for the simultaneous determination of nine androgens, including four classic and five 11-oxygenated androgens in human serum. Magnetic solid-phase extraction (MSPE) using magnetic graphene oxide nanoparticles followed by in-situ derivatization further improved the detection efficiency. A novel quaternary ammonium aminooxy (QAO) reagent was employed as the derivatization agent, featuring a permanently charged MS-tag and an aminooxy group that reacts with steroid ketone groups. This derivatization significantly enhanced MS sensitivity compared to traditional derivatization agents. The linear ranges for different targets were set based on clinical use and the lower limit of quantification (LLOQ) of the target androgens ranged from 0.005 to 1 ng/mL. The calibration curves exhibited excellent linear correlation (r ≥ 0.99) across the validated ranges for all the analytes. The intra- and inter-run precision was below 9.8 % and 9.7 % for the lower limit of quantification (LLOQ), and 7.5 % and 6.9 % for quality controls (QCs). Intra- and inter-day relative errors for LLOQ ranged from -8.2 % to 10.7 %, and from -7.4 % to 13.8 % for QCs. Extraction recoveries varied from 41.8 % to 80.2 %. The method was applied to 107 clinical samples, demonstrating it as a fast, sensitive and accurate method for androgens’ quantification. By offering the ability to detect and quantify a broader range of androgens, including those not measurable by ECLIA, and avoiding potential cross-reactions. This method provides a superior analytical tool for the diagnosis and management of PCOS in clinical settings.