Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Non-invasive therapeutic drug monitoring: LC-MS validation for lamotrigine quantification in dried blood spot and oral fluid/saliva","authors":"Marta Pelcová ,&nbsp;Viktória Ďurčová ,&nbsp;Pavel Šmak ,&nbsp;Ondřej Strýček ,&nbsp;Miriam Štolcová ,&nbsp;Ondřej Peš ,&nbsp;Zdeněk Glatz ,&nbsp;Pavel Šištík ,&nbsp;Jan Juřica","doi":"10.1016/j.jpba.2025.116877","DOIUrl":"10.1016/j.jpba.2025.116877","url":null,"abstract":"<div><div>Epilepsy, affecting over 50 million people globally, presents a significant neurological challenge. Effective prevention of epileptic seizures relies on proper administration and monitoring of Anti-Seizure Medication (ASMs). Therapeutic Drug Monitoring (TDM) ensures optimal dosage adjustment, minimizing adverse effects and potential drug interactions. While traditional venous blood collection for TDM may be stressful, emerging alternative sampling methods, particularly Dried Blood Spot (DBS) or oral fluid offer less invasive way of sampling. This study aimed to develop and validate an analytical method for the determination of lamotrigine in such alternative samples. The sample, either DBS or oral fluid, was subjected to extraction, evaporation, and reconstitution in 15 % acetonitrile containing 0.1 % formic acid. A Kinetex C18 Polar column was used for liquid chromatographic separation and MS in ESI+ mode was used for detection and quantitation of lamotrigine using an isotopically labelled internal standard according to EMA guidelines. The calibration range of the developed method enables the determination of lamotrigine in the concentration range of 1–30 μg/mL in DBS and 0.5–20 μg/mL in oral fluid. Oral fluid and DBS samples from patients treated with lamotrigine analysed by the developed method were compared to plasma concentrations measured by the hospital's accredited laboratory. Preliminary results indicate a promising potential for these alternative matrices in clinical TDM applications. By offering a less invasive sampling approach, this method improves the accessibility and safety of pharmacotherapy for epilepsy patients. The results of this study lay the foundation for further clinical applications by implementing alternative matrix TDM, which may significantly advance personalized care in epilepsy management.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116877"},"PeriodicalIF":3.1,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828636","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":"Development of a fluorescence-based assay for screening of urate transporter 1 inhibitors (II): Optimization of fluorescent substrates and structure-activity relationships analysis","authors":"Zhixi Dai,&nbsp;Hongming Chen,&nbsp;Xiaodan Lu,&nbsp;Haojie Cai,&nbsp;Lei Zhang,&nbsp;Jing Li","doi":"10.1016/j.jpba.2025.116900","DOIUrl":"10.1016/j.jpba.2025.116900","url":null,"abstract":"<div><div>Urate transporter 1 (URAT1) inhibitors represent a promising therapeutic approach for hyperuricemia and gout. A non-radioactive cell-based platform for screening URAT1 inhibitors using 6-carboxyfluorescein (6-CFL) was previously established by our team. In this study, aiming to optimize our testing platform, 10 potential fluorescent substrates were screened from the commercially available fluoresceins using molecular docking and pKa prediction. Among them, dibromofluorescein (DBF) and diiodofluorescein (DIF) were identified as specific transport substrates for UTAR1 with significant fluorescence changes in cellular uptake. DBF and DIF exhibited Km values similar to that of 6-CFL. Using DBF or DIF as substrates, the IC₅₀ values of the URAT1 inhibitors benzbromarone and lesinurad remained within the same order of magnitude as those obtained by 6-CFL. Notably, DBF enables visual detection, further extending the utility of this non-radioactive platform for <em>in vitro</em> screening of URAT1 inhibitors. Molecular dynamics (MD) simulations and free energy calculations were employed to compare the interactions and binding affinities of 6-CFL, DBF, and DIF with URAT1, respectively. Finally, structure-activity relationships (SARs) analysis was conducted to preliminarily identify substituents modulating the binding affinity of commercially available fluoresceins.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116900"},"PeriodicalIF":3.1,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143847820","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 novel in-depth “static- dynamic” lipidomics workflow to reveal lipids reprogramming in hepatocellular carcinoma","authors":"Jianlei Yan ,&nbsp;Yiwen Zhang ,&nbsp;Xiaoxue Zheng,&nbsp;Zhengkun Tang,&nbsp;Wei Guo,&nbsp;Saiyu Li,&nbsp;Jingjing Li,&nbsp;Huarong Xu,&nbsp;Qing Li,&nbsp;Qian Zhang","doi":"10.1016/j.jpba.2025.116880","DOIUrl":"10.1016/j.jpba.2025.116880","url":null,"abstract":"<div><div>Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths globally, and with current treatments proving less effective, there is an urgent need for specific biomarkers and therapeutic targets. Lipid metabolism reprogramming is a crucial cancer hallmark, yet comprehensive studies on lipid metabolic fluxes remain limited. In this study, combined with non-targeted lipidomics, a comprehensive workflow for stable isotope tracing lipidomics was established to analyze changes in lipid levels of HepG2 cells and LO2 cells from both static and dynamic perspectives. Through the screening of differential metabolites and the enrichment analysis of lipid metabolic pathways, the most significant differential metabolic pathways were found. Finally, the TCGA and CPTAC databases were utilized to analyze the gene expression levels and protein expression levels of pivotal enzymes in the differential metabolic pathways, and these findings were verified by Western Blotting experiments. The results demonstrated that the lipid metabolism of HCC was disordered, and the metabolic pathways that caused lipid changes in HCC were mainly glycerophospholipid metabolism and sphingolipid signaling pathway. LPCAT1 and SMPD1 played a crucial role in the reprogramming of lipid metabolism in HCC. The established \"static-dynamic\" lipidomics workflow improves the coverage and accuracy of dynamic lipid monitoring, elucidating the roles of lipids in physiological and pathological processes, providing tools for studying lipid function, and offering new perspectives on the pathogenesis of HCC as well as the identification of drug targets.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116880"},"PeriodicalIF":3.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828632","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 convenient and rapid LC-MS/MS method for determination of free and liposomal amphotericin B in human plasma by simultaneous separation using SPE","authors":"Yi Jin,&nbsp;Bowen Wu,&nbsp;Yuting Gong,&nbsp;Huanyu Wei,&nbsp;Ruihan Ma,&nbsp;Yue Wang,&nbsp;Min Yuan,&nbsp;Haiyan Xu","doi":"10.1016/j.jpba.2025.116884","DOIUrl":"10.1016/j.jpba.2025.116884","url":null,"abstract":"<div><div>Effective separation and specific detection of free and encapsulated drugs in bio-samples are critical to the pharmacokinetic investigation of liposomal medicine. In this study, a simple, convenient, reliable, and selective SPE separation method coupled with sensitive LC-MS/MS technique was developed and fully validated for the detection of liposomal amphotericin B (L-AMB) and non-liposomal amphotericin B (F-AMB) in human plasma. The simultaneous separation between L-AMB and F-AMB in plasma were realized using Oasis HLB SPE cartridge. L-AMB was collected in the aqueous eluate and F-AMB was eluted from the cartridge by methanol containing 1 % formic acid. The analyte and internal standard (natamycin) were separated on a ZORBAX Eclise XDB C18 column (2.1 × 50 mm, 3.5 μm) with gradient elution at a flow rate of 0.5 mL/min, employing a mobile phase that consisted of methanol (0.1 % formic acid) and 5 mM ammonium acetate solution (0.1 % formic acid). Mass spectrometry detection was performed in positive ion mode with electrospray ionization (ESI) interface by multiple reaction monitoring (MRM) method. The linearity range was 200–50000 ng/mL for L-AMB and 10.0–1600 ng/mL for F-AMB. A series of cross quality control samples was adopted to verify the selectivity, stability, and reproducibly of the quantification. Using our methodology, we quantified F-AMB and L-AMB without mutual interferences and obtained excellent incurred sample reanalysis (ISR) results for both analytes. The method was also successfully applied to a clinical study in healthy volunteers.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116884"},"PeriodicalIF":3.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143825981","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":"Therapeutic glycoprotein hormones: Considerations for the use of in vitro alternatives to in vivo potency assays","authors":"Ben Cowper,&nbsp;Melanie Moore,&nbsp;Chris Burns","doi":"10.1016/j.jpba.2025.116895","DOIUrl":"10.1016/j.jpba.2025.116895","url":null,"abstract":"<div><div>Therapeutic protein hormones such as Erythropoietin and Gonadotrophins have historically utilised <em>in vivo</em> bioassays for potency assignment. In line with 3 R’s principles for animal research, manufacturers and regulatory authorities alike are continually seeking to implement <em>in vitro</em> alternatives to animal assays wherever possible. However, these proteins are heavily glycosylated, giving rise to an inverse correlation of <em>in vivo</em> and <em>in vitro</em> biological activities, which complicates the transition away from <em>in vivo</em> bioassays for potency assignment. Whilst manufacturers are actively pursuing the development of alternatives to <em>in vivo</em> bioassays for product quality control, these efforts inevitably result in product-specific approaches, which are unlikely to be representative of all versions of the same product from different manufacturers. This presents a further challenge for organisations tasked with ensuring harmonisation of dosage between products from different manufacturers, through provision of documentary (pharmacopoeial monographs) and physical (assay calibrators) standards, as such resources must be suitable for all licensed versions of a product. This review will describe these challenges, and the product-specific approaches which have been implemented by manufacturers, and propose a framework aimed at mitigating the risks associated with the wider introduction of <em>in vitro</em> alternatives to <em>in vivo</em> potency assays.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116895"},"PeriodicalIF":3.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143834663","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":"Interspecies comparison on the O-methylation metabolism of LJR003, a novel immunomodulator targeting acetyl-CoA acetyltransferase 1","authors":"Yongchun Chang ,&nbsp;Weilin Zhang ,&nbsp;Xiaoyu Liu ,&nbsp;Lijun Luo ,&nbsp;Jieyi Chen ,&nbsp;Yanshi Zhao ,&nbsp;Xiaoguang Chen ,&nbsp;Ming Ji ,&nbsp;Li Sheng","doi":"10.1016/j.jpba.2025.116894","DOIUrl":"10.1016/j.jpba.2025.116894","url":null,"abstract":"<div><div>Catechol structures are essential for drug activity and can undergo meta- or para-methylation, which affects their pharmacological properties. The regioselectivity and species differences in O-methylation metabolism significantly influence drug efficacy and toxicity, requiring further study. LJR003, an immunomodulator with a catechol structure, targets acetyl-CoA acetyltransferase 1 (ACAT1), a potential target for cancer immunotherapy. This study investigated the activity, methylation regioselectivity, and species differences of LJR003 and its methylated metabolites. Pharmacokinetic studies were conducted in rats, mice, and dogs, and methylation regioselectivity was analyzed in liver, kidney, and erythrocytes from these species and humans after LJR003 incubation. Results showed that meta-methylated LJR003 had weaker ACAT1 inhibitory activity and higher systemic exposure than LJR003 in rats, mice, and dogs. Erythrocytes exhibited the lowest methylation activity in vitro, while liver catalytic efficiency in rats, mice, and dogs was at least twice that of the kidney. In humans, liver and kidney showed similar catalytic activity. LJR003 favored meta-methylation in mice, dogs, and humans in vitro, with consistent in vivo results in mice and dogs. Rats displayed a unique metabolic pattern, suggesting species-specific differences. In conclusion, LJR003 is predicted to undergo meta-methylation in humans, contributing to its pharmacological effects alongside the parent compound. These findings improve understanding of methylation metabolism and provide insights for developing catechol-based drugs, emphasizing the importance of species-specific metabolic pathways in drug development.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116894"},"PeriodicalIF":3.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143823452","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":"Integrated network pharmacology and RNA sequencing analysis to reveal the mechanisms of Qici Sanling decoction in the treatment of gemcitabine resistant bladder cancer","authors":"Zhuolun Li ,&nbsp;Jinpeng Wang ,&nbsp;Wanhui Wang ,&nbsp;Bo Geng ,&nbsp;Wei Zhang ,&nbsp;Weiyang Liu ,&nbsp;Yunfeng Nan ,&nbsp;Bosen You ,&nbsp;Enyang Zhao ,&nbsp;Xuedong Li","doi":"10.1016/j.jpba.2025.116885","DOIUrl":"10.1016/j.jpba.2025.116885","url":null,"abstract":"<div><div>Bladder cancer (BCa) is the most prevalent cancer of the urinary system in adults; the prognosis is dismal for BCa treated with gemcitabine (GEM) owing to intrinsic or acquired chemoresistance. This study investigated the potential of Qici Sanling decoction (QCSL), an herbal Chinese medicine, to augment the efficacy of GEM in treating GEM-resistant BCa via network pharmacology and RNA sequencing. We screened 103 active components of QCSL and their 226 targets from the TCMSP database and identified 3985 targets of GEM-resistant BCa via transcriptome sequencing. On the basis of the 69 common targets, a protein<img>protein interaction (PPI) network was constructed to identify the top 7 targets. Disease Ontology (DO), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were conducted to uncover key pathways. CCK-8 assays, Western blotting, flow cytometry, colony formation, and EdU assays were used to assess the apoptosis and proliferation of GEM-resistant T24 and J82 cells treated with QCSL. The BCa gene set was among the top enriched gene sets in the DO analysis; GO analysis revealed enrichment of 2020 terms linked to GEM resistance, and KEGG analysis revealed 161 enriched signalling pathways. Molecular docking indicated that PTGS2 has high affinity for targets of QCSL components. In vitro experiments demonstrated that cells treated with both QCSL and GEM had significantly reduced viability, increased levels of apoptosis, and decreased proliferative capacity. Thus, QCSL enhances the therapeutic effects of GEM in BCa by promoting cell apoptosis and inhibiting cell proliferation. These findings have significant clinical implications, highlighting a potential combined treatment strategy for GEM-resistant BCa to improve patient outcomes.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116885"},"PeriodicalIF":3.1,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828638","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":"Simultaneous determination of four PDE5 inhibitors and metabolites in rat plasma by UPLC-MS/MS","authors":"Xiaonan Xia ,&nbsp;Zhe Song ,&nbsp;Weiwen He ,&nbsp;Zhou Qiao ,&nbsp;Fanglin Wang ,&nbsp;Shaoyuan Li","doi":"10.1016/j.jpba.2025.116883","DOIUrl":"10.1016/j.jpba.2025.116883","url":null,"abstract":"<div><div>Herein, we developed a sensitive UPLC-MS/MS method for the simultaneous determination of four PDE5 inhibitor and twelve metabolites in rat plasma. A total of 16 chemicals were separated on gradient elution with mobile phase A (0.1 % formate acetonitrile) and mobile phase B (0.1 % formate and 2 mmol/L ammonium formate aqueous solution) on an ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) after protein precipitation, followed by the detection on the positive ion mode of electrospray ion source (ESI) via dynamic multi-reaction monitoring mode (MRM). The method provided good linearity over the range of 0.25–20 ng/mL for all compounds with correlation coefficients R greater than 0.99. The recoveries were higher than 75.5 %, the matrix factors were 2.1 %-20.4 %. The precision, accuracy and stability were also within acceptable criteria. A rat model was established to investigate the metabolic profile of Sildenafil, Vardenafil, Acetildenafil and Tadalafil. Blood samples from the experimental group were analyzed using ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS). Notably, desmethyltadalafil and methyl-desmethyltadalafil were not detected in the plasma. However, their glucuronide conjugates, desmethyltadalafil glucuronide and methyl-desmethyltadalafil glucuronide, were identified and characterized via tandem mass spectrometry (MS/MS). At present, cases of poisoning or even death due to overdose of phosphodiesterase type 5 (PDE5) inhibitors are often encountered in the practice of forensic science. Few studies on PDE5 inhibitors have been reported in forensic science toxicology identification, and the existing detection methods cannot meet the need for simultaneous and rapid analysis of multiple trace PDE5 inhibitors in vivo. Therefore, it is of great importance to establish and optimize the detection methods for PDE5 inhibitors drugs in biological samples. The significance of this method is to provide a reference for the determination of PDE5 inhibitors poisoning cases.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116883"},"PeriodicalIF":3.1,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143828634","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":"Stability of the antibiotic ceftaroline fosamil with focus on degradation products and decomposition pathways – Integrative analysis by LC-DAD, ESI-QTOF and in silico prediction","authors":"Marianna Coelho Mendes ,&nbsp;Lilian Fanfa Machioli ,&nbsp;Graciela Carlos ,&nbsp;Tiago Franco de Oliveira ,&nbsp;Andreas Sebastian Loureiro Mendez","doi":"10.1016/j.jpba.2025.116876","DOIUrl":"10.1016/j.jpba.2025.116876","url":null,"abstract":"<div><div>Stability studies are important tools for pharmacovigilance, enabling the investigation of new compounds made available to the population under different hospital conditions. Indicated for the treatment of complicated skin and soft tissue infections and community-acquired pneumonia, ceftaroline fosamil is a fifth-generation cephalosporin marketed as Zinforo®, a novel antibiotic given the long development time, in the form of powder for infusion. Due to the presence of a phosphate group, ceftaroline fosamil is a prodrug that is converted by hepatic enzymes to active form ceftaroline. The aim of the present study was to investigate the stability of ceftaroline fosamil through forced degradation studies (thermal stress) under exposures to 40°C and 60°C, and clinical use conditions under exposures to 4°C and 25°C, in the forms of powder, reconstituted in purified water, and diluted in 5 % glucose solution and 0.9 % sodium chloride solution (2.4 mg/mL, 5.0 mg/mL, and 8.4 mg/mL), for time periods of 10 min, 30 min, 60 min, 2 h, 24 h, 48 h, and 72 h. The preliminary analysis performed by HPLC-DAD showed that ceftaroline fosamil diluted at 2.4 mg/mL in a 5 % glucose solution was the least stable under clinical use conditions, with a decay of approximately 65 % of ceftaroline fosamil. Through mass spectrometry analysis by ESI-QTOF, the degradation products <em>m/z</em> 303, <em>m/z</em> 337, <em>m/z</em> 442, <em>m/z</em> 483, and <em>m/z</em> 543 and their degradation pathways were proposed. It was possible to partially relate the results of mass spectrometry and <em>in silico</em> experimental model for predicting degradation products.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116876"},"PeriodicalIF":3.1,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143823451","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":"Unique methotrexate polyglutamates distributions in peripheral blood mononuclear cells of rheumatoid arthritis patients: Development and validation of a UPLC-MS/MS method","authors":"Janani Sundaresan ,&nbsp;Marry Lin ,&nbsp;Gerrit Jansen ,&nbsp;Renske C.F. Hebing ,&nbsp;Maja Bulatović-Ćalasan ,&nbsp;Robert de Jonge ,&nbsp;Eduard A. Struys ,&nbsp;Maurits C.F.J. de Rotte","doi":"10.1016/j.jpba.2025.116882","DOIUrl":"10.1016/j.jpba.2025.116882","url":null,"abstract":"<div><div>Methotrexate is pivotal in treating immune-mediated inflammatory diseases. Intracellularly, methotrexate is metabolized to methotrexate-polyglutamates (MTX-PG_1–7), comprising up to six additional glutamate moieties, crucial for cellular retention and therapeutic efficacy. Hitherto, quantification of MTX-PG_1–6 in peripheral blood mononuclear cells (PBMCs) from methotrexate-treated patients was challenging due to their low abundance in blood and matrix effects. We present a robust validated UPLC-MS/MS method to quantify individual MTX-PG_1–6 in PBMCs. Stable-isotope labelled internal standard mixture of MTX-PG_1–6 was added to 5 million PBMCs, followed by deproteinization with perchloric acid, and additional sample clean-up using solid phase extraction columns. MTX-PG_1–6 were detected and quantified using UPLC-MS/MS. The method was validated for lower limit of quantification (LLOQ), linearity, carryover, recovery, matrix effects, precision and stability. We assessed MTX-PG_1–6 in PBMCs derived from five methotrexate-treated rheumatoid arthritis patients. For all MTX-PG_1–6, LLOQs were &lt; 1 fmol-MTX-PG_1–6/million cells with linearities R² &gt; 0.995. The recoveries, carryover and stability were acceptable and no matrix effects were observed. The intraday and interday precision %CVs of quality controls ranged from 2.7 % to 11.4 % and 3.5–14.9 % respectively. Interday precision using nine PBMCs aliquots from a single MTX-treated patient aligned similarly (%CV &lt;15 %). In patient-derived PBMC samples, MTX-PG₁ was the highest, with decreasing concentrations of MTX-PG₂ to MTX-PG₅. No signal for MTX-PG₆ was detected in the patient samples. We validated a new UPLC-MS/MS method to quantify MTX-PG_1–6 in PBMCs, thus facilitating PBMC-based therapeutic drug monitoring studies and understand associations between MTX-PG_1–6 concentration and therapy efficacy or adherence.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"262 ","pages":"Article 116882"},"PeriodicalIF":3.1,"publicationDate":"2025-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143820417","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}