Journal of Chromatography B最新文献

{"title":"Development and application of SPE-UHPLC-MS/MS methods for quantification of 29 steroids in rat serum","authors":"Haiyang Cui ,&nbsp;Dongliang Yang ,&nbsp;Yonggang Zheng ,&nbsp;Jing Li ,&nbsp;Hairui Yan ,&nbsp;Xiaoli Gao","doi":"10.1016/j.jchromb.2026.124924","DOIUrl":"10.1016/j.jchromb.2026.124924","url":null,"abstract":"<div><div>Steroids and their metabolites play crucial roles in reproductive health, endocrine diseases, and tumor development. Comprehensive analysis of these compounds is essential for both mechanistic investigations and clinical evaluations. Although comprehensive analytical methods have been established for steroids in human clinical samples, their direct application to rat specimens remains limited, particularly with the absence of validated methods for detecting certain hydroxylated steroid metabolites in vivo. This study developed highly sensitive solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry methods for the quantitative analysis of 29 steroid metabolites. Through optimized derivatization of steroids and integration of two-dimensional liquid chromatography techniques, the method effectively reduces interference from non-volatile salts and overcomes trace-level detection challenges. Method validation demonstrated a wide linear range (0.002–250 ng/mL), low limits of quantification (2–100 pg/mL), high accuracy (88–114%), and precision (intra- and inter-batch CV ≤14%). The method exhibited acceptable extraction recovery, matrix effects, and stability, confirming compliance with regulatory standards. The validated method was successfully applied to characterize serum steroid metabolic profiles in both <em>N</em>-methyl-N-nitrosourea-induced breast cancer and ovariectomized rat models, revealing alterations in estrogen 2-hydroxylation and pregnenolone 21-hydroxylation pathways mediated by key steroidogenic enzymes, including CYP1A1/CYP1B1 and potentially CYP21A2-independent mechanisms, providing robust support for preclinical research on steroid-related diseases.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124924"},"PeriodicalIF":2.8,"publicationDate":"2026-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146004851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Portable mass spectrometry systems for point-of-care testing: technologies, applications, and clinical implementation","authors":"Brooks B. Pond,&nbsp;Madison Hoskins,&nbsp;Haylie Hollis,&nbsp;Stacy Brown","doi":"10.1016/j.jchromb.2026.124923","DOIUrl":"10.1016/j.jchromb.2026.124923","url":null,"abstract":"<div><div>Portable mass spectrometry systems have shown great potential in point-of-care testing and field-based diagnostics, as they fill important gaps in decentralized clinical analysis. Conventional centralized laboratory testing may delay clinical decision making due to transport and analysis times. For conditions where transportation is limited, these delays disproportionately affect resource-poor settings. Advances in technology allow the design and assembly of lightweight, portable MS systems. Fundamental issues such as size reduction, power consumption, and vacuum requirements have been addressed as the MS miniaturization field has developed. These advances have generated commercially available devices and home-built instruments capable of rapid analysis, often without extensive sample preparation. Studies on applicability of portable MS have already shown utility in multiple fronts, such as therapeutic drug monitoring for HIV, psychiatric drugs, and cardiac interventions; drug profiling in forensic medicine; disease diagnosis including malaria detection; and cancer tissue analysis. Nevertheless, the tradeoff between miniaturization and sensitivity, small mass resolution relative to benchtop instruments, and challenges in reproducibility represent ongoing issues with miniaturized mass spectrometry. Despite these disadvantages, significant clinical potential can be concluded from current published evidence. With the development of complementary sample preparation methods and miniaturization technology, portable MS systems could see implementation in emergency departments, mobile clinics, and resource scarce clinic environments.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124923"},"PeriodicalIF":2.8,"publicationDate":"2026-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Simple ion-pairing reversed phase LC-UV method for the relative quantification of capping species in mRNA-based modalities","authors":"Jonathan Maurer ,&nbsp;Denis Bouchard ,&nbsp;Agathe Bousquier ,&nbsp;Camille Malburet ,&nbsp;Jean-François Cotte ,&nbsp;Davy Guillarme","doi":"10.1016/j.jchromb.2026.124922","DOIUrl":"10.1016/j.jchromb.2026.124922","url":null,"abstract":"<div><div>Messenger RNA (mRNA)-based therapeutics and vaccines rely on proper 5′ capping to ensure translational efficiency, stability, and reduced reactogenicity. Current analytical approaches for capping evaluation often rely on mass spectrometry (MS) and fluorinated solvents, which are costly, technically demanding, and not always suitable for quality control (QC) laboratories. Here, we describe the development of a simple, robust, and QC-compatible ion-pairing reversed-phase liquid chromatography method coupled with UV detection (IP-RPLC-UV) for the relative quantification of key 5′ capping species in mRNA modalities, including uncapped, Cap 0, Cap 1, and Cap G transcripts, as well as non-templated additions (+G). The method employs targeted cleavage of the 5′ end using an oligo hybridization and RNase H digestion strategy, enabling baseline resolution of capping species within 20 min. A systematic screening of various ion-pairing reagents demonstrated that butylammonium acetate offered the optimal balance between retention and resolution, while a design of experiments approach identified critical parameters influencing performance and ensured method robustness. Method sensitivity was confirmed with a lower limit of quantification at 0.01 mg/mL for uncapped species, and repeatability testing showed consistent results across samples. This study introduces the first IP-RPLC-UV method tailored for routine relative quantification of cap structures, providing a practical alternative to MS-based workflows and facilitating the implementation of accessible, reliable analytical control strategies for mRNA drug development.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124922"},"PeriodicalIF":2.8,"publicationDate":"2026-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Impacts of methionine oxidation on product quality and anti-tumor activity of a therapeutic monoclonal antibody","authors":"Qiangzu Sun ,&nbsp;Zhenhua Miao ,&nbsp;Liqing Bian ,&nbsp;Fei Teng ,&nbsp;Huanhuan Guo ,&nbsp;Xu-Alan Lin ,&nbsp;Xichen Zhang ,&nbsp;Fan Zhang ,&nbsp;Dong-Qiang Lin","doi":"10.1016/j.jchromb.2026.124920","DOIUrl":"10.1016/j.jchromb.2026.124920","url":null,"abstract":"<div><div>Methionine oxidation in monoclonal antibodies (mAbs) could critically compromise drug stability and efficacy. This investigation comprehensively evaluated the structural and functional impacts of oxidation on a mAb An under long-term storage (1.5 years at 5 °C) and forced oxidation conditions. Orthogonal analytical approaches including high-performance liquid chromatography and mass spectrometry revealed preferential oxidation at M253 and M429 residues, concomitant with increased charge heterogeneity (basic peaks) and hydrophobicity. Notably, while the oxidation attenuated Complement-Dependent Cytotoxicity (CDC) activity by ∼70%, it did not alter Antibody-Dependent Cellular Cytotoxicity (ADCC), binding affinity (FcγRIIIa), or <em>in-vivo</em> anti-tumor efficacy in murine models. The pharmacokinetics was similarly unaffected. These findings demonstrate that methionine oxidation, despite altering physicochemical properties, does not impact mAb A's essential biological functions. Crucially, forced oxidation samples effectively simulated the oxidation progression of mAb An under subsequent storage conditions, supporting its stability during long-term storage.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124920"},"PeriodicalIF":2.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Removal of aspirin and tetracycline hydrochloride using UiO66/Polydopamine/bacterial cellulose composite: Phenomenological modeling and theoretical study","authors":"Nadia Bouaziz ,&nbsp;Fatma Aouaini ,&nbsp;Abdelmottaleb Ben Lamine","doi":"10.1016/j.jchromb.2025.124910","DOIUrl":"10.1016/j.jchromb.2025.124910","url":null,"abstract":"<div><div>The effective removal of antibiotics and non-steroidal anti-inflammatory drugs and their combined pollutants is critical for both the aquatic environment and human health. This study evaluates the removal of pharmaceutical waste through the adsorption of aspirin and tetracycline hydrochloride (TC) onto the surface of a UiO-66/Polydopamine/Bacterial Cellulose composite (UiO-66/PDA/BC). Both theoretical and experimental approaches were considered to investigate the removal of aspirin and TC. The theoretical study, based on statistical physics theory, employs three analytical models to describe the adsorption phenomena of these drugs at the molecular level. According to the fitting results, the adsorption of both drugs onto the UiO-66/PDA/BC composite is an endothermic process and follows a monolayer adsorption mechanism with one type of site. Regarding the number of adsorbed molecules per site (n), the analysis indicates that both multi-docking (<em>n</em> &lt; 1) and multi-molecular (<em>n</em> &gt; 1) adsorption mechanisms are possible for aspirin and TC on UiO-66/PDA/BC. The adsorption capacities at saturation of the UiO-66/PDA/BC adsorbent deduced by the monolayer model are found to be 147.34–215.05 mg/g for aspirin and 193.72–246.66 mg/g for TC. Furthermore, adsorption energy calculations suggest that the removal of these pharmaceutical pollutants occurs primarily through physical interactions. The thermodynamic analysis confirms the spontaneous and feasible nature of the adsorption of both drugs onto the UiO-66/PDA/BC composite.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124910"},"PeriodicalIF":2.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145974241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Applying a vacuum ultraviolet detector for liquid chromatography: Simultaneous analysis of four organic acids and four carbohydrates in a fermentation process","authors":"Mayur Bansal ,&nbsp;Annika Dombrowski ,&nbsp;Chinoso Nwanebi ,&nbsp;Dale Harrison ,&nbsp;Hal S. Alper","doi":"10.1016/j.jchromb.2026.124921","DOIUrl":"10.1016/j.jchromb.2026.124921","url":null,"abstract":"<div><div>The initial application of a prototype vacuum ultraviolet (VUV) absorption detector system for high performance liquid chromatography (HPLC) was validated and characterized. This detector enabled simultaneous detection and quantification of four organic acids—lactic, acetic, itaconic, and citric along with four carbohydrates—glucose, fructose, galactose, and lactose. This collection of analytes was selected based on their prevalence in fermentations, metabolic production interest, and occurrence in food processes/chemistry. Full spectrum VUV data, collected across 7 discrete wavelength bands centered at 177.3, 190.3, 203.1, 215.8, 228.3, 240.7, and 252.9 nm, yielded unique fingerprints that allowed for separation and quantification of strongly co-eluting peaks, most notably the hexose sugars. Separations were performed on an Aminex HPX-87H ion exclusion column using 5 mM H₂SO₄ as the isocratic mobile phase at 0.6 mL/min, with samples preheated to 40 °C. Across the eight analytes, Hydra limits of detection spanned 0.0228–3.59 g/L, with calibration linearity of R² = 0.941–0.998. The VUV prototype maintained linear response up to 100 g/L for all analytes (60 g/L for itaconic acid), whereas refractive index detection saturated between ∼10–60 g/L depending on the analyte. Spectral deconvolution recovered co-eluting organic acids with ∼1–4% error and the co-eluting hexose cluster within ∼11–19% error, demonstrating practical spectral selectivity for overlapped peaks. This extended dynamic range can eliminate the need for sample dilution thus greatly streamlining workflow processes and minimizing the dilution of trace components. The combination of quantitative accuracy and spectral selectivity was demonstrated though the monitoring of glucose utilization and lactic acid production during a seven-day <em>Saccharomyces cerevisiae</em> fermentation with minimal sample preparation. These results support the potential of LC-VUV detection as a robust, high-throughput method for metabolite analysis in high titer bioprocessing applications. However, a current limitation of the prototype is reduced sensitivity at low concentrations relative to conventional detectors, indicating that present performance is best suited to higher titer samples.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124921"},"PeriodicalIF":2.8,"publicationDate":"2026-01-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145923080","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Library-assisted UHPLC-Q-TOF-MS/MS bioanalytical profiling of Qiling Hushen granules: In-vivo metabolic mapping and kidney-biased distribution","authors":"Pei Sheng ,&nbsp;Mingjin Zhang ,&nbsp;Shuqi Wang ,&nbsp;Shuyi Peng ,&nbsp;Jiakun Li ,&nbsp;Yi Liu ,&nbsp;Kaixuan Xu ,&nbsp;Xiaofei An","doi":"10.1016/j.jchromb.2026.124913","DOIUrl":"10.1016/j.jchromb.2026.124913","url":null,"abstract":"<div><div>Qiling Hushen Granules (QLHSG), a multi-herb prescription widely used for diabetic kidney disease (DKD), lacks system-level evidence linking chemical features with <em>in-vivo</em> disposition. A library-assisted UHPLC–Q-TOF-MS/MS workflow was developed to integrate <em>in-vitro</em> phytochemical profiling with <em>in-vivo</em> prototype/metabolite elucidation and organ-level biodistribution. An in-house spectral library was constructed using reference standards, open databases and diagnostic-ion rules, enabling high-confidence annotation based on retention time, accurate mass and MS/MS fragmentation. In total, 166 <em>in-vitro</em> constituents were cataloged. <em>In vivo</em>, 78 prototype constituents were characterized. Metabolites were assigned by matrix (bile 46, feces 44, plasma 27, urine 156), delineating phase I/II biotransformation and elimination routes. Tissue mapping demonstrated kidney-biased accumulation of multiple prototypes and metabolites, indicating that renal targeting is consistent with the therapeutic context. Dominant exposure classes included iridoids and flavonoids. Representative bioactives were astragaloside IV, calycosin, paeoniflorin, secologanoside and sweroside. Network analysis combined with molecular docking connected absorbed constituents to DKD-relevant targets (<em>e.g.</em>, TP53, EGFR, AKT1, STAT3, HIF1α), providing plausible mechanistic insights. Overall, this workflow establishes a reproducible pathway, from chemical profiling to systemic exposure and organ targeting in complex herbal formulations. For the first time, it provides system-level chemical evidence supporting the renoprotective effects of QLHSG.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124913"},"PeriodicalIF":2.8,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145923079","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A sensitive method for confirming the clearance of caffeine and its three primary metabolites in sheep tissues destined for human consumption","authors":"Gregory S. Doran ,&nbsp;Susan M. Robertson ,&nbsp;Michael A. Friend ,&nbsp;Scott H. Edwards","doi":"10.1016/j.jchromb.2026.124912","DOIUrl":"10.1016/j.jchromb.2026.124912","url":null,"abstract":"<div><div>Caffeine is a naturally occurring stimulant that is consumed in coffee, tea, and energy and soft drinks, but may also be used therapeutically in humans and animals. Caffeine initially degrades rapidly via demethylation to theobromine, paraxanthine and theophylline, prior to further degradation to other metabolites. A method was developed to separate and quantify the four chemicals, which is generally complicated by co-elution of paraxanthine and theophylline, which are the two bioactive metabolites. Processes to extract and purify the four analytes from liver, kidney, muscle, brain, perirenal and abdominal fats, plasma and milk were also developed and provided limits of quantitation as low as 1 ng/g in solid tissues and 1 ng/mL in milk and plasma using a combination of solvent and solid phase extractions. Sample homogenisation considerations and recovery at different stages was investigated to identify the areas of greatest analyte loss. A study was conducted involving the daily oral caffeine dosing of pregnant merino ewes at a rate of 25 mg/kg for 52 days until the birth of lambs. Tissue samples and milk were collected from ewes and lambs up to 28 days after birth and analysed using the new methodologies. Caffeine and the three primary metabolites appeared in all tissues analysed at high concentrations at day 0 (birthing), but declined by 99.9 % in all tissues in ewes by day 7, and all tissues except plasma (98.5 %) in lambs by day 7. All analytes were only detected in milk from ewes at day 0, but provided a source of caffeine and its metabolites to suckling newborn lambs, which may be responsible for the slower clearance from lamb plasma. Theophylline was the dominant metabolite in both ewes and lambs, followed by paraxanthine, while theobromine was only detected at much lower concentrations. The results derived from this animal study using this new method indicate that accumulation of caffeine and its three primary metabolites does not occur in the milk or tissues of ewes or lambs.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1271 ","pages":"Article 124912"},"PeriodicalIF":2.8,"publicationDate":"2026-01-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145895827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative GC–MS/MS in the clinical analysis of eicosanoids: A critical review and discussion, a 40-years historical retrospect, and possible implications for LC-MS/MS","authors":"Dimitrios Tsikas","doi":"10.1016/j.jchromb.2025.124911","DOIUrl":"10.1016/j.jchromb.2025.124911","url":null,"abstract":"<div><div>Mass spectrometry was a protagonist in the discovery of prostaglandins, thromboxane, leukotrienes and other arachidonic acid-derived molecules, collectively known as the eicosanoids. Mass spectrometry has played a significant role in exploration of their metabolic pathways in humans and animals, in health and disease, and in pharmacotherapy. Clinical researchers in the United States of America and in Europe, in close cooperation with chemist analysts, were the pioneers in the application of gas chromatography–mass spectrometry (GC–MS) and gas chromatography-tandem mass spectrometry (GC–MS/MS) to quantitate eicosanoids and index metabolites in plasma, serum and urine samples from clinical trials by using stable-isotope labeled analogs as internal standards. In the present article, the application of the stable-isotope dilution GC–MS/MS methodology in the quantitative clinical analysis is reviewed. The focus is on prostaglandins, thromboxane, leukotrienes, and their so-called index metabolites for renal and whole-body synthesis of certain eicosanoids such as PGE₂ and its major urinary metabolite (PGE-MUM), respectively. Nowadays, LC-MS/MS, which evolved later than GC–MS/MS, is increasingly used in numerous areas of research, including the eicosanoids in clinical studies. The present work critically discusses the current practice of LC-MS/MS users in the quantitative analysis of eicosanoids in biological samples. While the LC-MS/MS technology offers rapidity and high-throughput analysis, especially due to the renunciation of time-consuming analytical derivatization steps that are required in GC–MS/MS, LC-MS/MS seems to lack sufficient analytical sensitivity, i.e., lower limit of quantitation, for many eicosanoids such as thromboxane B₂ and leukotriene B₄. Reported data on basal concentrations of certain eicosanoids in plasma and urine samples from healthy humans as determined by LC-MS/MS are several orders of magnitude higher than originally reported by pioneering eicosanoid researchers, who developed, validated and used sophisticated, tailored GC–MS- and GC–MS/MS-based analytical methods for individual eicosanoids. Modern eicosanoids researchers would greatly benefit from the milestones and signposts set previously eicosanoids researchers from the very start. A key milestone and signpost is the concentration of primary eicosanoids and their metabolites in plasma, serum and urine samples of healthy humans. Issues for consideration in the GC–MS/MS and LC-MS/MS analysis of eicosanoids are discussed.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124911"},"PeriodicalIF":2.8,"publicationDate":"2025-12-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145880737","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An ID-HPLC-MS/MS based candidate reference measurement procedure for the quantification of imipenem, meropenem and ertapenem in human plasma","authors":"Jing Lin ,&nbsp;Qingqing Pan ,&nbsp;Weihua Wang ,&nbsp;Shuangshuang Chen ,&nbsp;Guangliang Wu ,&nbsp;Min Shen ,&nbsp;Huoyan Ji","doi":"10.1016/j.jchromb.2025.124908","DOIUrl":"10.1016/j.jchromb.2025.124908","url":null,"abstract":"<div><h3>Background</h3><div>Carbapenems require precise plasma levels for optimal therapy, yet no LC-MS/MS method offered SI-traceability or reference-grade performance. In this study, a candidate reference measurement procedure (RMP) using isotope dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) was developed and validated for accurate quantification of imipenem (IPM), meropenem (MEM) and ertapenem (ETP) in human plasma.</div></div><div><h3>Methods</h3><div>To ensure traceability to SI units, the absolute content of the IPM, MEM and ETP primary reference materials (PRMs) were determined by quantitative nuclear magnetic resonance (qNMR) spectroscopy. Samples were prepared by protein precipitation followed by dilution. Method validation was performed according to guidances from the Clinical and Laboratory Standards Institute (CLSI), including selectivity/specificity, matrix effect, carryover, linearity, limit of quantification (LOQ) and limit of detection (LOD), trueness and precision, and stability. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). Moreover, the candidate RMP was compared between two independent laboratories.</div></div><div><h3>Results</h3><div>The method showed high selectivity and stability. Linear ranges were 0.208 to 52.087 μg/mL (IPM, <em>r</em> = 0.9997), 0.412 to 103.427 μg/mL (MEM, <em>r</em> = 0.9999) and 0.083 to 20.791 μg/mL (ETP, <em>r</em> = 0.9999). Mean recoveries were 100.05 to 104.80 %, 92.34 to 94.77 % and 95.55 to 100.04 %, respectively. The method demonstrated high sensitivity, with limits of LOQ of 0.103, 0.103 and 0.021 μg/mL and corresponding LOD of 0.0077, 0.0018 and 0.0006 μg/mL for IPM, MEM and ETP, respectively. No obvious matrix effect and carryover were observed. The expanded uncertainties (<em>k</em> = 2) were ≤ 6.12 %.</div></div><div><h3>Conclusion</h3><div>The qNMR-based candidate RMP provides SI-traceable, accurate simultaneous quantification of IPM, MEM and ETP, enabling standardization of clinical assays and reliable patient-sample measurement.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1270 ","pages":"Article 124908"},"PeriodicalIF":2.8,"publicationDate":"2025-12-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145867136","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}