Journal of Chromatography B最新文献

{"title":"Development of a validated and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to simultaneous determination of crizotinib, alectinib and lorlatinib in human plasma","authors":"Haohua Chi ,&nbsp;Shu Li ,&nbsp;Fang Liu ,&nbsp;Bo Li ,&nbsp;Wenqian Chen ,&nbsp;Cheng Zhang ,&nbsp;Wei Zhou ,&nbsp;Meng Yang ,&nbsp;Pengmei Li","doi":"10.1016/j.jchromb.2025.124770","DOIUrl":"10.1016/j.jchromb.2025.124770","url":null,"abstract":"<div><div>Crizotinib, alectinib, and lorlatinib are tyrosine kinase inhibitors used sequentially in non-small cell lung cancer (NSCLC) therapy. Therapeutic drug monitoring (TDM) is valuable during sequential treatment, enabling simultaneous quantification. Consequently, an analytical method capable of multiplexing these three compounds into a single assay is highly applicable for routine TDM. A multiplexed UHPLC-MS/MS method quantifying all three drugs was validated for selectivity, matrix effects, linearity (20–1000 ng/mL), accuracy, precision, carryover, and stability. Matrix effects were negligible in both plasma from healthy volunteers and plasma from patients with hyperlipidemia. No significant interfering responses were observed for crizotinib, alectinib, lorlatinib, or their corresponding isotope-labeled internal standards. Sample stability was confirmed under ambient conditions (25 °C) for at least 24 h. Furthermore, the pretreated supernatant remained stable in the auto sampler (15 °C) for 24 h. This validated method is suitable for application in pharmacokinetic studies and exposure-response assessments.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1267 ","pages":"Article 124770"},"PeriodicalIF":2.8,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144933189","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":"LC-MS/MS method for quantifying the HIV-1 broadly neutralizing antibody PGT 121.414.LS in human serum","authors":"Connor E. Gould ,&nbsp;Qing Ma ,&nbsp;Raymond Cha ,&nbsp;Kevin J. Zemaitis ,&nbsp;Robin DiFrancesco ,&nbsp;Gene D. Morse ,&nbsp;Troy D. Wood","doi":"10.1016/j.jchromb.2025.124774","DOIUrl":"10.1016/j.jchromb.2025.124774","url":null,"abstract":"<div><div>Quantitation of human immunodeficiency virus-1 (HIV-1) broadly neutralizing antibodies (bNAbs) in human serum is required for clinical trials investigating the pharmacokinetics, pharmacodynamics, and drug interactions of these treatments. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is gaining interest as an alternative to ligand binding for therapeutic antibody quantitation in serum. We report the validation of a method using nonspecific purification and targeted LC-MS/MS to quantify PGT 121.414.LS (a bNAb in development for HIV-1 prevention and treatment) in human serum. High-resolution spectra of tryptic peptides derived from the variable region were obtained on an Orbitrap for surrogate peptide selection, followed by multiple reaction monitoring using triple quadrupole mass spectrometry. Surrogate peptides were evaluated for linearity and reproducibility across the therapeutic concentration range using immunopurification or ammonium sulfate precipitation. Using ammonium sulfate precipitation, linear calibration curves were validated over 10–500 μg/mL (LLOQ at 10 μg/mL) using stable isotope labeled peptide internal standards. Method accuracy and reproducibility were evaluated using quality control samples (QCs) at four concentrations in the linear range. The average concentrations of all QCs fell within ICH M10 acceptance criteria. Matrix effects were investigated at the low and high QC concentrations across six lots of human serum. Dilutional integrity, stability, and effects of hemolysis were also assessed. The method exhibits minimal carryover and negligible crosstalk. The assay provides accurate quantification of PGT 121.414.LS in serum over the range of concentrations anticipated in specimens from treated persons living with HIV (PLWH) after initial dosing and prior to subsequent dosing of PGT 121.414.LS.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1266 ","pages":"Article 124774"},"PeriodicalIF":2.8,"publicationDate":"2025-08-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144916975","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 and validation of a LC–MS/MS method for the detection of 38 benzodiazepines and 2 Z-drugs in blood","authors":"Yu Liu ,&nbsp;Xin-ze Liu ,&nbsp;Zhen-shuo Guo ,&nbsp;Ping Xiang ,&nbsp;Hui Yan","doi":"10.1016/j.jchromb.2025.124772","DOIUrl":"10.1016/j.jchromb.2025.124772","url":null,"abstract":"<div><div>Benzodiazepines and <em>Z</em>-drugs are commonly used as prescription medications to treat anxiety, epilepsy, insomnia, and alcohol withdrawal syndrome, but their use can lead to tolerance, dependence, and withdrawal reactions if taken against official guidelines. Furthermore, designer benzodiazepines, most of which lack clinical and toxicological data, have entered the illicit drug market as new psychoactive substances and are used for recreational purposes. Their abuse can cause confusion, memory loss, respiratory depression, and even death, especially when combined with other sedative-hypnotics or alcohol. Therefore, new qualitative and quantitative methods for benzodiazepines and <em>Z</em>-drugs are needed for use in forensic and clinical toxicology. This study explored the sample preparation and liquid-liquid extraction using 0.5 mL blood samples and 2 mL of extraction solvent for analysis of these drugs by liquid chromatography-tandem mass spectrometry and multiple reaction monitoring. The benzodiazepines were separated on a pentafluorophenylpropyl (PFPP) column using a mobile phase gradient consisting of A (water, 0.1 % formic acid, 5 % acetonitrile, and 20 mmol/L ammonium acetate) and B (acetonitrile) for 9 min. Validation steps confirmed that the method demonstrated good selectivity, sensitivity (limit of detection: 0.2 ng/mL, lower limit of quantification: 0.5 ng/mL), linearity (R² ≥ 0.99), accuracy, and precision (&lt;20 %). Matrix effects ranged from 35 to 126 %, and recoveries ranged from 17 to 99 %, with 35 compounds having recoveries of more than 50 %. The method was successfully applied for the identification and quantification of benzodiazepines and nonbenzodiazepines in blood samples from 15 authentic poisoning cases. Ten analytes were detected: alprazolam (130.2–575.3 ng/mL), hydroxyalprazolam (2.3–37.3 ng/mL), clonazepam (11.7–773.2 ng/mL), lorazepam (63.4–166.9 ng/mL), 7-aminoclonazepam (17.4–385.3 ng/mL), oxazepam (2.6–964.1 ng/mL), diazepam (227.2 ng/mL), nordazepam(22.4 ng/mL), zolpidem (11.8–64.0 ng/mL), midazolam (70.2 ng/mL), and hydroxymidazolam (162.8 ng/mL).</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1266 ","pages":"Article 124772"},"PeriodicalIF":2.8,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144922508","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 automated Immunoaffinity liquid chromatography-tandem mass spectrometry assay for quantification of aldosterone in human plasma","authors":"Liang-Xing Li ,&nbsp;Jian-You Xue ,&nbsp;Wan-Ying Lin ,&nbsp;Xiao-Feng Yin ,&nbsp;Ting-Ting Luo ,&nbsp;Zhi-Liang Cai ,&nbsp;Chao-Chao Wu ,&nbsp;Qiang Gao ,&nbsp;Xin Li ,&nbsp;Ru-Yi Zhang","doi":"10.1016/j.jchromb.2025.124773","DOIUrl":"10.1016/j.jchromb.2025.124773","url":null,"abstract":"<div><div>The diagnosis of primary aldosteronism (PA) relies on the accurate determination of aldosterone. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has long been considered the gold standard for aldosterone quantification but it is hindered by labor-intensive sample preparation. To address this, we developed an immunoaffinity-mass spectrometry (iMS) assay on a fully automated device combining anti-aldosterone antibodies with stable isotope-labeled internal standards (IS). This method completes sample preparation within 15 min for at least six parallel samples in parallel with minimal manual intervention.</div><div>The key performance metrics include a lower limit of quantitation (LOQ) of 50 pg/mL, recovery rates between 105.1 and 113.9 %, and linearity in the range of 50–2000 pg/mL (R² = 0.9993). Inter-assay coefficient of variation (CV) ranged from 2.33 % to 3.91 %. In addition, plasma aldosterone concentrations by iMS and immunoassay had a high correlation coefficient (<em>R</em> = 0.947).</div><div>Overall, this automated high-throughput platform delivers clinical-grade sensitivity, precision, and scalability, making it suitable for routine testing and adaptable for other clinical analytes.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1266 ","pages":"Article 124773"},"PeriodicalIF":2.8,"publicationDate":"2025-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144912164","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 pseudotargeted metabolomics approach for relative quantification of free fatty acid double-bond isomers in beagle plasma","authors":"Wen Xiao ,&nbsp;Haihui Ren ,&nbsp;Yueyi Zhang ,&nbsp;Jiameng Han ,&nbsp;Qiaorong Liu ,&nbsp;Zunjian Zhang ,&nbsp;Yuan Tian","doi":"10.1016/j.jchromb.2025.124765","DOIUrl":"10.1016/j.jchromb.2025.124765","url":null,"abstract":"<div><div>Free fatty acids (FFAs) play a key role in living organisms and participate in metabolic processes, mainly as a source of energy for cells. The C<img>C position isomer of FFAs is strongly associated with many diseases, but quantification of its double bond position isomer remains a challenge in lipid analysis. In this study, based on the requirements of the pseudotargeted metabolomics, an analytical method based on a two-step derivatization LC-MS/MS method and a multiple reaction monitoring (MRM) mode was established and fully validated in terms of linearity, precision, and stability. The results demonstrated that the method relatively quantified 30 FFAs in plasma samples. These included 8 saturated fatty acids (SFAs), 12 monounsaturated fatty acids (MUFAs), and 10 polyunsaturated fatty acids (PUFAs). The developed method was applied to analyze the increase or down-regulation of the relative content of FFAs before and after the administration of the drug in beagles which may help to explain the therapeutic effect of sacubitril valsartan. The establishment of this method allows the study of FFAs in beagles plasma to be no longer limited to analysis at the subclass level but can be focused at the isomer level.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1266 ","pages":"Article 124765"},"PeriodicalIF":2.8,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144912163","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 separation on a bisphenylureido β-cyclodextrin column and determination of eight antihistamine enantiomers in human plasma by HPLC","authors":"Jing Lin,&nbsp;Dan Li,&nbsp;Peiyuan Luo,&nbsp;Laisheng Li","doi":"10.1016/j.jchromb.2025.124771","DOIUrl":"10.1016/j.jchromb.2025.124771","url":null,"abstract":"<div><div>A <em>bis</em>(3,5-dichlorophenylureido)-β-cyclodextrin stationary phase (CUCDP) was prepared and characterized. It had strong chiral separation abilities for antihistamines (<em>R</em>s = 1.61–2.58) in isocratic elution, including chlorpheniramine maleate, bromopheniramine maleate, promethazine hydrochloride and trimeprazine tartrate. Based on CUCDP, a new HPLC method for simultaneous separation and determination of the above eight antihistamine enantiomers (<em>R</em>s = 1.58–1.92) in human plasma by gradient elution was established. The optimized conditions were as follows: a simple sample pretreatment with acetonitrile (ACN) to precipitate protein, a gradient elution of 0.5 % triethylammonium acetate (TEAA, pH = 4.0)-ACN as the mobile phase at a flow rate of 0.5 mL/min, column temperature at 20 °C, a photo-diode array detector (PDA) at 260 nm, and the sampling volume of 10 μL. The good linear relationships for all enantiomers were observed in the concetration range of 0.25–10.0 μg/mL (<em>R</em>² = 0.9986–0.9993). The average recoveries were 84.80 %–103.30 % with the RSDs of 1.24 %–2.26 % (<em>n</em> = 5). The limits of detection (LODs) and limits of quantification (LOQs) were 0.015–0.05 μg/mL and 0.05–0.15 μg/mL, respectively. At present, the reported cyclodextrin-based CSPs had not such separation ability for antihistamines by HPLC. However, the new CUCDP exhibited high chiral selectivity for antihistamines, which was mainly related to the introduction of <em>bis</em>phenylureido group into the rim of cyclodextrin, and the strengthening of hydrogen bonding, π–π stacking and inclusion between CUCDP and analytes. This method could save analysis time, reduce solvent consumption, and improve efficiency at low cost.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1266 ","pages":"Article 124771"},"PeriodicalIF":2.8,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144892279","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 ionic liquid 3-(2-hydrazinyl-2-oxoethyl)-1-methyl-1H-imidazol-3-ium chloride as a derivatization reagent for HPLC-HRMS determination of steroid hormones in urine","authors":"M. Zorina ,&nbsp;Y.-Q. Feng ,&nbsp;Sanka N. Atapattu ,&nbsp;S. Girel ,&nbsp;Dzh. Konshina ,&nbsp;V.V. Konshin ,&nbsp;A. Temerdashev","doi":"10.1016/j.jchromb.2025.124760","DOIUrl":"10.1016/j.jchromb.2025.124760","url":null,"abstract":"<div><div>This work demonstrates for the first time the possibility of using a novel ionic liquid 3-(2-hydrazinyl-2-oxoethyl)-1-methyl-1H-imidazol-3-ium chloride ([HOMI]Cl) as a derivatization reagent for steroid hormones and their subsequent analysis using ultra-high performance liquid chromatography–quadrupole time-of-flight mass spectrometry in human urine. Derivatization conditions, such as pH, reagent concentration, reaction time and temperature were evaluated and optimized. A comparative study of [HOMI]Cl and hydroxylamine effectiveness revealed a formation of both <em>syn</em>- and anti-forms of the steroid derivatives for the novel [HOMI]Cl reagent similarly to a derivatization with hydroxylamine. Hence obtained limits of quantitation were comparable to the literature values obtained with Girard P and hydroxylamine reagents (0.15 to 7.5 ng/mL). Finally, in-depth optimization of [HOMI]Cl hydrophobicity and ionization source conditions were found necessary to further improve the electrospray response of hence derivatized steroids.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1265 ","pages":"Article 124760"},"PeriodicalIF":2.8,"publicationDate":"2025-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144885559","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":"Screening of compounds in ginseng that activate CYP19A1 enzyme promoters for the treatment of postmenopausal osteoporosis using molecular docking and affinity ultrafiltration technology","authors":"Yiying Tan ,&nbsp;Yajing Li ,&nbsp;Haiqing Luo ,&nbsp;Yishan Li ,&nbsp;Lu Han ,&nbsp;Jiaming Shen ,&nbsp;Xuesheng Hu ,&nbsp;Xiaochen Gao ,&nbsp;Chunnan Li ,&nbsp;Jiaming Sun","doi":"10.1016/j.jchromb.2025.124767","DOIUrl":"10.1016/j.jchromb.2025.124767","url":null,"abstract":"<div><div>The primary etiology of postmenopausal osteoporosis is estrogen deficiency. The enzyme aromatase (CYP19A1) serves as the key rate-limiting enzyme in the conversion of androgens to estrogens. In this study, ginseng was selected as the subject of investigation. The study identified that the 50 % ethanol extract of ginseng exhibited the most potent activity in the determination of CYP19A1 enzyme activity. This extract was subsequently analyzed using UHPLC-QE Orbitrap-MS in conjunction with mass spectrometry molecular network technology. To identify the active components effective against postmenopausal osteoporosis, enzyme ultrafiltration affinity, molecular docking, and kinetic simulation techniques were employed. Cross-analysis of binding energy and affinity rate results revealed that ginsenoside Re and ginsenoside Rf possessed the highest absolute binding energy and affinity values, establishing them as the most effective active components. The mechanisms involving ALP, OPG, apoptosis, and qPCR were validated in vitro to confirm the anti-PMOP effects of these active ingredients. This study offers an efficient and rapid method for screening natural products to identify active components for the treatment of postmenopausal osteoporosis.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1265 ","pages":"Article 124767"},"PeriodicalIF":2.8,"publicationDate":"2025-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144864251","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":"UHPLC-MS/MS analysis of PFAS in the serum of patients with Alzheimer's disease, mild cognitive impairment and controls: A preliminary study","authors":"O. Deda ,&nbsp;S. Adams ,&nbsp;M. Tsolaki ,&nbsp;A. Lioupi ,&nbsp;A.C. Tsolaki ,&nbsp;G. Theodoridis ,&nbsp;I.D. Wilson ,&nbsp;R.S. Plumb ,&nbsp;H. Gika","doi":"10.1016/j.jchromb.2025.124738","DOIUrl":"10.1016/j.jchromb.2025.124738","url":null,"abstract":"<div><div>Complimentary UHPLC-MS methods for 6 ultra short chain (polar) and 30 long chain (and relatively non-polar) PFAS compounds have been developed and used to profile these compounds in human serum. The methods were developed to be rapid, with both having analysis times of ca. 5 min, to enable the rapid screening of samples. Both methods were sensitive enough to detect the target PFAS over the range 0.5–50 ng/mL. These methods were then applied to the screening of the serum of 137 subjects comprising healthy controls (HC), subjects with mild cognitive impairment (MCI) and those with Alzheimer's disease (AD). These methods demonstrated the presence of the 33 of targeted PFAS in these serum samples, presenting over a wide concentrations range with PFPrA found at the highest maximum concentration of 707 ng/mL. Interestingly the PFAS profiles found for the HC and MCI subjects differed from those of the AD patients. The reason(s) for differences in the PFAS profiles obtained for serum from AD patients compared to HC/MCI subjects is unclear and may be unrelated to disease but clearly warrants further investigation to clarify the underlying reasons for this observation.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1266 ","pages":"Article 124738"},"PeriodicalIF":2.8,"publicationDate":"2025-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144903277","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":"UHPLC-based C-phycocyanin quantification method to support the validation of remote sensing models for harmful cyanobacterial blooms","authors":"Elisabetta Canuti","doi":"10.1016/j.jchromb.2025.124759","DOIUrl":"10.1016/j.jchromb.2025.124759","url":null,"abstract":"<div><div>The deployment of hyperspectral satellite missions has opened new opportunities for integrated approaches to address the escalating issue of cyanobacterial algal blooms in marine and inland waters. Despite these advancements, the validation of satellite data concerning C-phycocyanin (C-PC) content requires robust analytical methods. Currently, the available techniques, predominantly spectrophotometric or fluorometric, exhibit low reproducibility due to the interference of chlorophyll <em>a</em> and perform optimally with water samples. The samples collected during ship-based campaigns and commonly used for satellite data validation are concentrated samples on filters, which limiting the application of these methods. In this study, we aim to establish an Ultra High Pressure Liquid Chromatography (UHPLC) -based method for C-PC quantification in water samples concentrated on filters to validate satellite derived algorithm for monitoring cyanobacteria blooms. This involves the extraction of C-PC from pure algal cultures (<em>Synechococcus</em> spp. and <em>Anabaena</em> spp.) and natural samples fixed on filters and the quantification employing UHPLC analysis—a recognized gold standard for phytoplankton pigment analysis. Based on a Design of Experiment (DoE) full factorial design approach, we compared different extraction techniques. Subsequently, we compared different methods and developed and validated a rapid, simple, and sensitive reverse phase UHPLC method. The optimized chromatographic parameters for C5 phase, were acetonitrile with trifluoroacetic acid (0.1 % <em>v</em>/v) in a 5′ linear gradient (from 20 % to 100 %), flowrate 0.8 mL min⁻¹, with an accuracy of 10 %, a reproducibility of 9.8 % and a limit detection of 0.025 mg L⁻¹.</div></div>","PeriodicalId":348,"journal":{"name":"Journal of Chromatography B","volume":"1265 ","pages":"Article 124759"},"PeriodicalIF":2.8,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144878290","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}