Journal of pharmaceutical and biomedical analysis最新文献

{"title":"Development and validation of a label-free HPLC-CAD method to determine total sialic acid in therapeutic proteins","authors":"Yifei Wang ,&nbsp;Yuechuan (Alex) Xu ,&nbsp;Björn-Hendrik Peters ,&nbsp;Michael Peddicord ,&nbsp;Difei Qiu ,&nbsp;Julie Cheng ,&nbsp;Ming Zeng ,&nbsp;Zhi Chen","doi":"10.1016/j.jpba.2025.116937","DOIUrl":"10.1016/j.jpba.2025.116937","url":null,"abstract":"<div><div>Sialylation plays a crucial role in influencing the pharmacokinetic, chemical, physical, and immunogenic properties of therapeutic proteins and is commonly tracked through total sialic acid analysis. In this study, we developed a novel label-free HPLC method to monitor total sialic acid levels in protein drug products. The method utilizes a replaceable, pre-column C18 cartridge that removes protein in-line, preventing protein interference with sialic acid and contamination of the analytical column and enabling direct sample injection. The optimized mixed-mode hydrophilic interaction-ion exchange liquid chromatography (HILIC-IEX) separation condition includes a volatile mobile phase for charged aerosol detection (CAD) of sialic acids. The method was validated to demonstrate satisfactory specificity, linearity (R &gt; 0.999), precision (RSD between 0.4 % and 2.1 %), sensitivity (QL &lt; 25 ng), and accuracy (recovery between 93 % and 102 %). It was successfully applied to determine total sialic acids in a model protein drug product, demonstrating promising potential as an accurate, robust, efficient, and cost-effective approach for sialylation evaluation.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116937"},"PeriodicalIF":3.1,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143898557","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":"Study on the mechanism of action of Penehyclidine hydrochloride on LPS-induced acute lung injury by regulating autophagy through the mTOR/Keap1/Nrf2 signaling pathway","authors":"Junting Weng ,&nbsp;Zhicheng Chen ,&nbsp;Shuoyun Weng ,&nbsp;Rongjie Guo ,&nbsp;Bingbing Shi ,&nbsp;Danjuan Liu ,&nbsp;Shanjiao Huang","doi":"10.1016/j.jpba.2025.116938","DOIUrl":"10.1016/j.jpba.2025.116938","url":null,"abstract":"<div><div>Acute lung injury (ALI) is a clinical syndrome characterized by pulmonary inflammation and edema, leading to impaired oxygenation and respiratory failure. Penehyclidine hydrochloride (PHC) has anticholinergic, anti-inflammatory, and antioxidant properties. In this paper, we investigated the protective role of PHC in ALI and explored its mechanism of action. Both <em>in vivo</em> and <em>in vitro</em> experiments were performed using LPS induction to establish an ALI model. Following PHC intervention, the assessment of lung injury was conducted using pathological section examination, mouse lung injury scoring, and ELISA to measure oxidative stress markers including myeloperoxidase (MPO), malondialdehyde (MDA), Super Oxide Dismutase (SOD), and Glutathione Peroxidase (GSH-Px), as well as inflammatory cytokine levels of TNF-α, IL-1β, and IL-18. Immunoblotting and immunofluorescence assays were employed to detect autophagy markers and the mTOR/Keap1/Nrf2 signaling pathway. To confirm the role of autophagy in the protective effects of PHC against ALI, we administered PHC in combination with Rapamycin (RAPA) or 3-Methyladenine (3-MA) to the model groups and evaluated the aforementioned parameters. Our findings revealed that in the LPS-induced ALI model, there was significant pulmonary histopathological damage and increased levels of MPO, MDA, TNF-α, IL-1β, and IL-18, along with decreased levels of SOD and GSH-Px in lung tissue or serum. These alterations were all reversed following PHC treatment. Additionally, compared to the ALI group, PHC administration reversed the expression of mTOR/Keap1/Nrf2 and autophagy proteins LC3, Beclin-1 and p62 induced by LPS. Treatment with the mTOR inhibitor (autophagy inducer RAPA) blocked the protective effects of PHC on lung injury, the mTOR/Keap1/Nrf2 signaling pathway, and autophagy, while co-treatment with the autophagy inhibitor 3-MA showed a significant protective effect on ALI. The results suggest that PHC has a notable protective effect on ALI, which may be achieved by modulating the mTOR/Keap1/Nrf2 signaling pathway to inhibit autophagy.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116938"},"PeriodicalIF":3.1,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143883075","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 3D-printed integrated maneuverable device for sensitive colorimetry of Pseudomonas aeruginosa","authors":"Jizhou Li,&nbsp;Shiruoyu Zhou,&nbsp;Ruining Bai,&nbsp;Zhifeng Fu","doi":"10.1016/j.jpba.2025.116934","DOIUrl":"10.1016/j.jpba.2025.116934","url":null,"abstract":"<div><div>Timely and sensitive monitoring of pathogens in clinical specimen is highly demanded for efficient control and precise treatment of infectious diseases. Herein, a 3D-printed maneuverable device integrating incubation, washing, and detection functions was manufactured for colorimetry of <em>Pseudomonas aeruginosa</em> (<em>P. aeruginosa</em>) using transparent resin. The device combined with magnetic beads (MBs) can achieve specific separation and efficient enrichment of <em>P. aeruginosa</em>. Furthermore, it can be directly fixed onto a 96-well plate holder for colorimetry. Specifically, a <em>P. aeruginosa</em> bacteriophage termed as JZ1 acquired from river water was applied as a recognition reagent to functionalize the separation vectors MBs. Then, a nanoconfinement MOFs material termed as PCN-222(Pt) with remarkable peroxidase-like activity was conjugated with polymyxin B to act as a signal tracer. With the formation of target bacterial complexes, the bound PCN-222(Pt) catalyzed the color reaction of 3,3′,5,5′-tetramethylbenzidine to enable quantitative colorimetry of <em>P. aeruginosa</em> by the maneuverable device. With this device, <em>P. aeruginosa</em> can be quantified within 40 min, with a dynamic range of 1.9 × 10² ∼ 1.9 × 10⁶ cfu mL⁻¹. The results for colorimetry of <em>P. aeruginosa</em> in diverse sample matrixes demonstrated its satisfactory practicability. This work provides a facile, timely, and cost-effective technique for point-of-care testing of pathogens.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116934"},"PeriodicalIF":3.1,"publicationDate":"2025-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891607","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":"Impurity profiling and stability analysis of enzalutamide: Identification, genotoxicity assessment, and development of UHPLC methods for critical impurities","authors":"Burcu Oktar Uzun ,&nbsp;Pelin Kaygu ,&nbsp;Elif Keskin ,&nbsp;Gamze Bostancıoğlu ,&nbsp;Okşan Soyer Can ,&nbsp;Melike Ceren Miser ,&nbsp;Çağan Ağtaş ,&nbsp;Esen Bellur Atici ,&nbsp;Sibel A. Özkan","doi":"10.1016/j.jpba.2025.116926","DOIUrl":"10.1016/j.jpba.2025.116926","url":null,"abstract":"<div><div>This study aims to comprehensively evaluate and control the impurity profile of enzalutamide, an androgen receptor signaling inhibitor used to treat metastatic castration-resistant prostate cancer. Using liquid chromatography-mass spectrometry (LC-MS), twenty impurities were identified based on their mass-to-charge ratios (<em>m/z</em>) during synthetic method development and stress testing studies. Classification of these compounds according to ICH M7 guidelines revealed two potentially genotoxic impurities: Enzal-2 (4-isothiocyanato-2-(trifluoromethyl)benzonitrile), classified as a Class 3 impurity due to its isothiocyanate group, and Enzal-2A (4-amino-2-(trifluoromethyl)benzonitrile), classified as a Class 2 impurity due to a positive Ames test result. Based on the threshold of toxicological concern (TTC) of 1.5 µg/day and the maximum daily dose of enzalutamide of 160 mg, a control limit of 9.4 ppm was established for Enzal-2 and Enzal-2A to mitigate safety risks. Both Enzal-2 and Enzal-2A were identified as process-related impurities, with Enzal-2A also recognized as a hydrolysis degradation product. Specific ultra-high-performance liquid chromatography (UHPLC) methods were developed and validated for the precise, accurate, and robust quantification of enzalutamide, Enzal-2, Enzal-2A, and related impurities. These methods were applied to enzalutamide samples subjected to various stress conditions, including elevated temperature, daylight, UV radiation, and exposure to oxidative, neutral, alkaline, and acidic environments, as well as under accelerated and long-term stability testing. The findings underscore the importance of comprehensive impurity profiling and validated analytical methods to ensure the safe and effective manufacture, quality control, and use of enzalutamide.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116926"},"PeriodicalIF":3.1,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143886868","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":"From bedside to bench: Evaluating the effect of different storage and extraction techniques on protease activity analysis in inflammatory bowel disease patients","authors":"Robert W. Perry ,&nbsp;Frederic Buemi ,&nbsp;Xinyu Guo ,&nbsp;Shiva T. Radhakrishnan ,&nbsp;Sharmili Balarajah ,&nbsp;Lucy C. Hicks ,&nbsp;Julian R. Marchesi ,&nbsp;Horace R.T. Williams ,&nbsp;Rachael Barry","doi":"10.1016/j.jpba.2025.116924","DOIUrl":"10.1016/j.jpba.2025.116924","url":null,"abstract":"<div><div>Proteolytic enzymes (proteases) are integral to multiple physiological processes, and increased protease activity is observed in inflammatory bowel disease (IBD) and other gastrointestinal diseases. Therefore, protease activity has been explored for potential diagnostic biomarkers and therapeutic targets. Faecal samples are commonly used to measure protease activities associated with gastrointestinal conditions as they allow non-invasive access to the diseased environment. However, faecal sample storage and protein extraction methods are not standardised for the measurement of faecal protease (FP) activity. In addition to being of scientific importance, the evaluation of the viability of FP activity as a potential biomarker for IBD management is also of clinical interest. In this study, the effect of different sample storage conditions and extraction techniques on FP activity were measured, on samples from IBD patients, using fluorescent substrates highly selective for serine proteases. FP activity was found to be stable for up to 72 hours at both 4°C and room temperature. Activity was found to decline more rapidly when samples were stored in extraction buffer rather than as crude samples. Regarding extraction techniques, FP activity was highest when extracted in Tris based buffers at pH 7.5–8; bead-beating of samples during extraction was found to have no significant effect on activity; and processing the extracted sample on ice was shown to minimise loss of FP activity. These results provide a framework for standardised evaluation of FP activity and suggest that protease activities have the potential to be a viable future biomarker.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116924"},"PeriodicalIF":3.1,"publicationDate":"2025-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902582","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":"Comprehensive quality evaluation of Huricha Liuwei Pills using multidimensional fingerprinting and multicomponent quantification","authors":"Lingkui Chen ,&nbsp;Zhenwei Zhang ,&nbsp;Mengfan Bao ,&nbsp;Sha Yang ,&nbsp;Ming Cai ,&nbsp;Lili Lan ,&nbsp;Wanyang Sun ,&nbsp;Guoxiang Sun","doi":"10.1016/j.jpba.2025.116925","DOIUrl":"10.1016/j.jpba.2025.116925","url":null,"abstract":"<div><div>In recent years, the application of traditional Chinese medicine (TCM) has gradually increased in the world, the study of quality control has become more and more important. As a branch of TCM, Mongolian medicine has complex compositions and lacks research on the overall quality. Therefore, in this paper, three fingerprints, high performance liquid chromatography (HPLC), differential scanning calorimetry (DSC) and electrochemical (EC), were established using Huricha Liuwei pills (HLP) as an example, and the quality of the three fingerprints was evaluated by the comprehensive ratio quantitative fingerprint method (CRQFM). To ensure the accuracy of the quantitative analysis, the purity of the fingerprint peaks was verified using the dual-wavelength absorption coefficient ratio (DWACR). And four components were quantified by the multi-markers assay by monolinear method (MAML): gallic acid (GA), chlorogenic acid (CA), hydroxysafflor yellow A (HSYA) and ellagic acid (EA). Based on the EC redox characteristics, the fingerprint efficacy relationship between two fingerprints (HPLC and DSC) and EC activity was established. The results showed that after CRQFM evaluation, 16 batches of samples were categorized into different quality grades. Subsequently, the evaluation results were integrated using the equal weighing method, and the 16 batches of samples had quality grades ranging from 1 to 3. The fingerprint efficacy relationship indicated that more than half of the fingerprint peaks showed good antioxidant activity, including GA, CA and EA. This paper established an effective quality control method for HLP and provided new ideas for the quality evaluation of other TCM.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116925"},"PeriodicalIF":3.1,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143883068","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":"Rapid diagnosis of diquat and paraquat poisoning: A dual approach using colloidal gold immunochromatographic assay and hydrophilic interaction liquid chromatography","authors":"Gongwei Han ,&nbsp;Jiahui He ,&nbsp;Teng Zhang ,&nbsp;Ge Li ,&nbsp;Xinxin Huang ,&nbsp;Yin Liu ,&nbsp;Dongmei Tian ,&nbsp;Shuai Song ,&nbsp;Quan Xia","doi":"10.1016/j.jpba.2025.116923","DOIUrl":"10.1016/j.jpba.2025.116923","url":null,"abstract":"<div><div>Accurate diagnosis and treatment in poisoning cases necessitate the monitoring of diquat and paraquat concentrations. This study presents a competitive colloidal gold immunochromatographic assay (GICA) strip for qualitative detection, alongside hydrophilic interaction liquid chromatography with ultraviolet detection (HILIC-UV) for quantitative assessment. Both methods underwent rigorous validation. The GICA strip achieved a detection limit of 20 ng/mL and demonstrated no cross-reactivity with glyphosate, deltamethrin, or dichlorvos in spiked serum samples. A compliance rate of 100 % confirmed its repeatability, validated by ten quality control samples at a concentration of 200 ng/mL. HILIC-UV exhibited a detection limit of 0.2 μg/mL and excellent linearity for paraquat and diquat in serum and urine across a range of 0.2–6.4 μg/mL (r² &gt; 0.99). Accuracy ranged from 86.4 % to 111.4 %, with relative standard deviation (RSD) below 10.8 %. Sigma metrics for quality control samples varied from 4.47 to 6.09, establishing a 1–3 s/2/3–2 s/R-4s internal quality control scheme (n = 3, r = 1). A total of 24 patient specimens were subjected to dual testing with HILIC-UV and GICA strips. Among these, 21 samples from 17 patients tested positive, while three patients with confirmed glyphosate, deltamethrin, and dichlorvos poisoning tested negative, indicating complete consistency. Of the positive results, 11 patients were diagnosed with paraquat poisoning, and six with diquat poisoning, both of which significantly impair liver, kidney, and coagulation functions. Integrating GICA for rapid qualitative assessment with HILIC-UV for quantitative analysis enhances the identification of diquat and paraquat poisoning, making it particularly suitable for emergency diagnostics.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116923"},"PeriodicalIF":3.1,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891605","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":"Discovery of bioactive compounds targeting endothelin A receptor and angiotensin II type 1 receptor in Gegen Qinlian decoction by co-immobilized receptor chromatography","authors":"Chan Li ,&nbsp;Yanbing Zhu ,&nbsp;Yu Fang","doi":"10.1016/j.jpba.2025.116918","DOIUrl":"10.1016/j.jpba.2025.116918","url":null,"abstract":"<div><div>An increased interest in multi-target compounds screening from complex matrices has revolutionized drug development paradigms, as these compounds often exhibit superior therapeutic efficacy and reduced off-target liabilities compared to the single-target compound. However, conventional discovery methods have been constrained by their singular focus on single-target screening methodologies. To address this limitation, we introduce a co-immobilization strategy tailored for G protein-coupled receptors(GPCRs) implicated in cardiovascular pathologies-specifically the endothelin A receptor (ET_AR) and angiotensin II type 1 receptor(AT₁R)-to facilitate the identification of multi-target compound within traditional herbal formulations. This innovative approach involves the oriented co-immobilization of ET_AR and AT₁R onto silica gel surfaces via histidine-tag anchoring, ensuring precise spatial orientation and functional integrity. Rigorous chromatographic characterization using receptor-specific ligands validated the dual-receptor column’s functionality. Subsequent screening of Gegen Qinlian Decoction (GQD) identified puerarin as a novel multi-target compound capable of engaging both the two receptors. Zonal elution revealed that puerarin competes with native ligands for overlapping binding sites on ET_AR and AT₁R. Injection-amount-dependent method, demonstrated that puerarin binds ET_AR and AT₁R with association constants of 2.5 × 10⁵ M⁻¹ and 2.0 × 10⁵ M⁻¹, respectively. These findings validate our co-immobilization platform as a powerful tool for dissecting multi-target interactions in traditional Chinese medicines (TCMs), offering a transformative strategy to unlock the therapeutic potential of complex herbal mixtures.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116918"},"PeriodicalIF":3.1,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143883074","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":"Automated derivatization with 6-aminoquinolyl-N-hydroxysccinimidyl carbamate for the enantioselective amino acid analysis of neurotensin synthesized by liquid phase peptide synthesis","authors":"Ryan Karongo ,&nbsp;Feiyang Li ,&nbsp;Franz Fiessinger ,&nbsp;Adrian Sievers-Engler ,&nbsp;Isabell Kroth ,&nbsp;Sarah Resch ,&nbsp;Lars Baumann ,&nbsp;Alexander Novak ,&nbsp;Mimi Gao ,&nbsp;Terence Hetzel ,&nbsp;Wiebke Holkenjans ,&nbsp;Werner Hoheisel ,&nbsp;Reinhard Pell ,&nbsp;Michael Gottfried ,&nbsp;Michael Lämmerhofer","doi":"10.1016/j.jpba.2025.116916","DOIUrl":"10.1016/j.jpba.2025.116916","url":null,"abstract":"<div><div>This study presents an automated derivatization protocol utilizing 6-aminoquinolyl-<em>N</em>-hydroxysuccinimidyl carbamate (AQC) for enantioselective amino acid analysis of peptides synthesized via liquid phase peptide synthesis, as exemplified by neurotensin. The primary aim was to enhance operational efficiency to manage the derivatization of large sample sets and reduce human error in routine enantioselective amino acid analysis of peptide therapeutics. The chromatographic method based on Chiralpak QN-AX demonstrated enantio- and chemoselectivity for all proteinogenic amino acids (except D-Leu/D-Ile and Glu/pGlu), with quantitative analysis achieved by HPLC-ESI-MS/MS with MRM acquisition through external calibration using stable isotope-labeled internal standards. The goal was to test for racemization of amino acids during peptide synthesis and process optimization, respectively. The results confirmed varying susceptibility to racemization among amino acids during peptide synthesis and cleavage of protection groups, with specific amino acids exhibiting higher levels of D-enantiomer formation. The developed protocol effectively assessed the amino acid composition and stereointegrity of the liquid phase synthesized neurotensin. This research and application highlights the critical role of automation in optimizing peptide analysis workflows and sets the foundation for future improvements in peptide synthesis and chromatographic conditions to enhance specificity, particularly for challenging amino acid pairs. Ultimately, the findings contribute to advancing laboratory practices in peptide chemistry, ensuring the quality and efficacy of peptide-based therapeutics.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116916"},"PeriodicalIF":3.1,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143876560","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":"Purification of testosterone and its metabolites in urine using two-dimensional high-performance liquid chromatography for 13C/12C ratios analysis by gas chromatography/combustion/isotope ratio mass spectrometry","authors":"Mengmeng Yan,&nbsp;Tianshuo Zhu,&nbsp;Chao Wen","doi":"10.1016/j.jpba.2025.116921","DOIUrl":"10.1016/j.jpba.2025.116921","url":null,"abstract":"<div><div>Two-dimensional high-performance liquid chromatography (2D-HPLC) is employed as a sample purification method for the isolation and enrichment of testosterone and its main metabolites in urine samples, thereby facilitating subsequent determination of ¹³C/¹²C ratios (<em>δ</em>¹³C) using Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry (GC/C/IRMS). The orthogonality of the HPLC columns was optimized by leveraging the distinct differences in polarity and steric hindrance effects between the stationary phases, enabling effective separation of endogenous interferents from testosterone in urine. Urine samples purified with 2D-HPLC exhibit significantly reduced matrix interferences in the fractions of testosterone (T), 5α-androstane-3<em>α</em>,17<em>β</em>-diol (5<em>α</em>-diol), and 5β-androstane-3<em>α</em>,17<em>β</em>-diol (5<em>β</em>-diol), thereby enhancing the accuracy and reliability of GC/C/IRMS analysis. The method achieves limit of quantification as low as 2 ng/mL for testosterone, and 3 ng/mL for both 5β-diol and 5α-diol. Furthermore, the simple instrument configuration enables direct transfer of 1D-purified fractions to 2D-HPLC for further isolation and enrichment, providing a flexible and efficient workflow for method development, particularly in handling complex biological matrices.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"263 ","pages":"Article 116921"},"PeriodicalIF":3.1,"publicationDate":"2025-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143879448","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}