Journal of Chromatography B最新文献_第6页

Bioanalysis of protein-unbound prednisolone in serum using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry 用平衡透析-液相色谱-串联质谱法分析血清中蛋白不结合的强的松龙。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2024.124440

J.E. Möhlmann , M. van Luin , E.G.W.M. Lentjes , A.D.R. Huitema , A.M. Punt

Introduction

High-dose systemic prednisolone is the cornerstone treatment of many autoimmune- and inflammatory diseases. Since prednisolone shows non-linear protein binding at higher serum concentrations, quantification of the unbound prednisolone concentration is important to understand prednisolone pharmacokinetics. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify protein-unbound prednisolone in serum.

Methods

Protein-unbound prednisolone was obtained using an equilibrium dialysis technique. Prednisolone was extracted from the dialysate using methyl tert-butyl ether. After evaporation to dryness, the organic phase residue was reconstituted and ready for injection onto the LC-MS/MS. Prednisolone was analysed by selected reaction monitoring with MS/MS operating in positive ion mode.

Results and discussion

The equilibrium between bound and unbound prednisolone was stable after 24 h. The calibration model for prednisolone in serum ranged from 0.25 to 811 µg/L and had an average linearity of 0.998. The coefficient of variation (CV) for precision at the lower limit of quantification was ≤ 4.3 % and for the other quality control samples ≤ 7.8 %. Prednisolone protein binding showed no significant degradation after 30 months of storage at −80 °C and was not influenced by multiple cycles of freezing and thawing. The recovery for the tested matrix effects in serum ranged from 85 % to 115 % (CV 10.3 %) and throughout the validation, no carry-over was observed.

Conclusion

An LC-MS/MS assay for prednisolone in serum was developed and validated, with a successful equilibrium dialysis technique to obtain protein-unbound prednisolone prior to quantification. This assay is considered suitable for pharmacokinetic studies.

大剂量全身性强的松龙是许多自身免疫性疾病和炎症性疾病的基础治疗。由于强的松龙在较高的血清浓度下显示非线性蛋白结合，因此对未结合的强的松龙浓度进行定量分析对于了解强的松龙的药代动力学非常重要。我们建立了一种液相色谱-串联质谱（LC-MS/MS）测定血清中非蛋白结合强的松龙的方法。方法：采用平衡透析法提取非蛋白强的松龙。用甲基叔丁基醚从透析液中提取强的松龙。蒸发至干燥后，有机相残留物重组，准备注射到LC-MS/MS上。采用正离子模式的质谱联用（MS/MS）对强的松龙进行选择性反应监测。结果与讨论：强的松龙与非强的松龙在24 h后平衡稳定。强的松龙血清中强的松龙的校准模型在0.25 ~ 811µg/L范围内，平均线性为0.998。定量下限的精密度变异系数(CV)≤4.3%，其余质控样品≤7.8%。强的松龙蛋白结合在-80°C下储存30个月后没有明显降解，也不受多次冷冻和解冻的影响。血清中基质效应的回收率从85%到115%不等（CV 10.3%），在整个验证过程中，没有观察到携带效应。结论：建立并验证了血清中强的松龙的LC-MS/MS检测方法，并成功地利用平衡透析技术在定量前获得非蛋白结合的强的松龙。该试验被认为适用于药代动力学研究。

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引用次数: 0

Development and validation of a UPLC–MS/MS method for simultaneous quantification of polymyxins and caspofungin in human plasma for therapeutic drug monitoring 用于治疗药物监测的人血浆中多粘菌素和卡泊霉素同时定量的UPLC-MS/MS方法的建立和验证。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2025.124465

Tong Wu , Libin Pu , Wenqing Liu , Yinliang Bai , Jingjing Ma , Xia Song , Aijia Cao , Shunli Pan , Jiahui Yang , Chang Wang , Wen Qiu

Objective

To develop a rapid, convenient, accurate, and low-residual-effect ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for the determination of polymyxin B sulfate and colistin sulfate in the blood of patients with multidrug-resistant bacterial infections, as well as caspofungin acetate in the blood of patients with fungal infections, thus facilitating the rational use of antibiotics in clinical applications.

Methods

All analytes were diluted with 0.2 % aqueous formic acid, and plasma proteins were precipitated using acetonitrile. The selected reaction monitoring (SRM) mode was used for measurement. Separation of all analytes was completed on a Hypersil GOLD C18 column (100 × 2.1 mm, 3.0 µm). They were quantitatively analyzed using electrospray ionization on a triple quadrupole mass spectrometer in the positive ion mode. The mobile phase consisted of water (containing 0.1 % formic acid) and acetonitrile, which was delivered by gradient elution at a flow rate of 0.3 ml/min. The internal standard was bacitracin zinc (BcZn), and the column temperature was maintained at 25 °C. The runtime for each analysis was 3.5 min.

Results

The procedure was validated following the recommendations of the U.S. Food and Drug Administration, which included measurements of accuracy (ranging from 83.27 % to 105.86 % for within-run and between-run accuracy), precision (with coefficients of variation from 2.50 % to 16.51 % for within-run precision and between-run precision), and matrix effects (ranging from 88.65 % to 103.94 %). The extraction recoveries ranged from 38.01 % to 42.76 for polymyxin B1 (PMB1), polymyxin B2 (PMB2), polymyxin E1 (PME1), polymyxin E2 (PME2), and 88.65 % to 89.84 % for caspofungin (CPF). Plasma samples were stable under various storage conditions, including three freeze–thaw cycles at −80 °C, 24-hour periods at room temperature and 4 °C, and 30 days of freezing at both −20 °C and −80 °C, with relative standard deviations (RSD) of less than 15 %.

Conclusion

In this study, a UPLC–MS/MS method was developed to simultaneously quantify PMB1, PMB2, PME1, PME2, and CPF in human plasma. The method was validated in blood samples from patients with multidrug-resistant bacteria combined with fungal infections and is suitable for therapeutic drug monitoring.

目的：建立一种快速、方便、准确、低残留效应的超高效液相色谱-串联质谱（UPLC-MS/MS）测定多药耐药细菌感染患者血液中硫酸多粘菌素B和硫酸粘菌素，以及真菌感染患者血液中醋酸卡泊芬净的方法，为临床合理使用抗生素提供依据。方法：所有分析物均用0.2%甲酸水溶液稀释，血浆蛋白用乙腈沉淀。选择反应监测（SRM）模式进行测量。在Hypersil GOLD C18色谱柱（100 × 2.1 mm, 3.0µm）上完成所有分析物的分离。在正离子模式下，在三重四极质谱仪上使用电喷雾电离对它们进行定量分析。流动相为水（含0.1%甲酸）和乙腈，梯度洗脱，流速为0.3 ml/min。内标品为杆菌肽锌（BcZn），柱温保持在25℃。每次分析的运行时间为3.5分钟。结果：该程序按照美国食品和药物管理局的建议进行验证，包括准确度（运行内和运行间精度范围从83.27%到105.86%），精度（运行内精度和运行间精度的变异系数从2.50%到16.51%）和矩阵效应（范围从88.65%到103.94%）的测量。多粘菌素B1 （PMB1）、多粘菌素B2 （PMB2）、多粘菌素E1 （PME1）、多粘菌素E2 （PME2）的提取回收率为38.01% ~ 42.76%,caspofungin （CPF）的提取回收率为88.65% ~ 89.84%。血浆样品在-80℃冻融3次、室温和4℃冻融24小时、-20℃和-80℃冻融30天等多种条件下均保持稳定，相对标准偏差（RSD）均小于15%。结论：本研究建立了同时定量人血浆中PMB1、PMB2、PME1、PME2和CPF的UPLC-MS/MS方法。该方法在多药耐药菌合并真菌感染患者血液样本中得到验证，适用于治疗药物监测。

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引用次数: 0

Unraveling the in vivo pharmacokinetic behavior of mPEG5-NH2 polymer in rats by UHPLC-MS/MS assay UHPLC-MS/MS法研究mPEG5-NH2聚合物在大鼠体内的药动学行为。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2025.124460

Yingxia Guo , Meichen Liu , Jiye Tian , Chunpeng Feng , Qingbin Wang , Xuan Zhao , Lei Yin

As an important chemical reagent, methoxy polyethylene glycol amine (mPEG-NH₂) is widely used in biomedical field. Unraveling the pharmacokinetic behavior of mPEG-NH₂ polymers is essential for revealing the toxicity and efficiency of mPEG-NH₂ related drug delivery systems. In this study, a simple analytical assay based on mass spectrometry (MS) was first established and validated for quantification of mPEG₅-NH₂ in biological matrix. The multiple reaction monitoring (MRM) transitions at m/z 252.2 (precursor ions) → 87.7 (fragment ions) and m/z 371.2 (precursor ions) → 89.2 (fragment ions) were chosen to determine mPEG₅-NH₂ and OH-PEG₈-OH, respectively. The UHPLC-MS/MS assay showed excellent linearity over the range of 0.01–10 μg/mL. Intra-day and inter-day accuracies and precisions of the assay were all within ± 6.44 %. The analytical assay was successfully applied to reveal the in vivo pharmacokinetic behavior of mPEG₅-NH₂ in rats.

甲氧基聚乙二醇胺（mPEG-NH2）作为一种重要的化学试剂，在生物医学领域有着广泛的应用。阐明mPEG-NH2聚合物的药代动力学行为对于揭示mPEG-NH2相关给药系统的毒性和效率至关重要。本研究首次建立并验证了一种基于质谱（MS）的简单分析方法，用于定量生物基质中的mPEG5-NH2。分别选择m/z 252.2（前体离子）→87.7（片段离子）和m/z 371.2（前体离子）→89.2（片段离子）的多重反应监测（MRM）跃迁来测定mPEG5-NH2和OH-PEG8-OH。UHPLC-MS/MS法在0.01 ~ 10 μg/mL范围内线性良好。日内、日间准确度和精密度均在±6.44%以内。该分析方法成功地揭示了mPEG5-NH2在大鼠体内的药动学行为。

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引用次数: 0

LC-MS/MS analysis of surface and lysate N-glycans of CHO-K1 cells: Structure, relative quantity, and absolute quantity CHO-K1细胞表面和裂解物n -聚糖的LC-MS/MS分析：结构、相对数量和绝对数量。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2024.124441

Chulmin Moon, Chi Soo Park, Chang Myeong Jeong, Han Seul Lee, Kyuran Kim, Haeun Byeon, Daeun Eom, Ha Hyung Kim

Chinese hamster ovary (CHO)-K1 cells are widely used in biomedical research relevant to cancer, toxicity screening, and viruses, as well as in the production of recombinant proteins for biopharmaceuticals. In this study, liquid chromatography (LC)-electrospray ionization (ESI)-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS) was used to characterize the surface and lysate N-glycans of CHO-K1 cells and analyze their structures. The relative quantity (%) of each N-glycan and absolute quantity (pmol) of total N-glycans were also obtained. In total, 37 surface and 30 lysate N-glycans were identified. Each of these two fractions contained eight high-mannose type (required for protection against proteolysis and N-glycosylation of recombinant proteins) at 28.8 % (the sum of the relative quantities of each N-glycan) and 66.5 %, respectively. Additionally, the surface and lysate N-glycans differed in their levels of sialyation (affect cell–cell interactions; 48.1 % and 13.5 %), fucosylation (affect cell signaling; 37.9 % and 25.5 %), and terminal-galactosylation (prerequisite for subsequent sialylation; 36.6 % and 20.9 %). These results indicate that the lysate of CHO-K1 cells contained more mannosylated (2.3-fold) N-glycans compared to the surface, which contained relatively more sialylated (3.6-fold), slightly more highly fucosylated (1.5-fold), and more terminal-galactosylated (1.8-fold) N-glycans. The sum of the absolute quantity of each N-glycan was obtained as a ratio of 1 (1,778.7 pmol; surface):2.2 (3,887.3 pmol; lysate) from approximately 5 × 10⁶ CHO-K1 cells. This study is the first to compare the surface and lysate N-glycans of CHO-K1 cells using LC-ESI-HCD-MS/MS. The results can be used to control and optimize biotechnology and biomedical research using CHO-K1 cells.

中国仓鼠卵巢(CHO)-K1细胞广泛应用于与癌症、毒性筛选、病毒相关的生物医学研究以及生物制药重组蛋白的生产。本研究采用液相色谱法(LC)-电喷雾电离法(ESI)-高能碰撞解离法(HCD)-串联质谱法（MS/MS）对CHO-K1细胞的表面和裂解n -聚糖进行表征，并对其结构进行分析。得到了每种n -聚糖的相对数量（%）和总n -聚糖的绝对数量（pmol）。共鉴定出37个表面n -聚糖和30个裂解n -聚糖。这两个部分分别含有8种高甘露糖类型（用于防止重组蛋白的蛋白质水解和n -糖基化），分别为28.8%（每种n -聚糖的相对数量的总和）和66.5%。此外，表面和裂解物n -聚糖的分泌水平不同(影响细胞-细胞相互作用；48.1%和13.5%)，聚焦化(影响细胞信号传导；37.9%和25.5%)和末端半乳糖基化(后续唾液酰化的先决条件；36.6%和20.9%)。这些结果表明，CHO-K1细胞的裂解液含有更多的甘露糖基化（2.3倍）n -聚糖，而表面含有相对较多的唾液化（3.6倍），高度聚焦化（1.5倍）和末端半乳糖化（1.8倍）n -聚糖。各n -聚糖的绝对数量之和为1 (1,778.7 pmol；表面):2.2 (3,887.3 pmol；裂解液)从大约5 × 106 CHO-K1细胞中提取。本研究首次采用LC-ESI-HCD-MS/MS比较CHO-K1细胞的表面和裂解n -聚糖。该结果可用于控制和优化使用CHO-K1细胞的生物技术和生物医学研究。

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引用次数: 0

Chitosan functionalized two-dimensional covalent organic framework nanosheets with high hydrophilicity for efficient glycopeptide enrichment 壳聚糖功能化具有高亲水性的二维共价有机框架纳米片，用于糖肽的高效富集。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2025.124461

Zhuo Mi, Wenkang Zhang, Han Wang, Xingyi Qi, Shuo Wang, Jiayi Song, Ping Su, Yi Yang

Glycopeptides are an important biomarker, which play a crucial role in various biological processes. Due to their low abundance and the presence of interfering macromolecular proteins, enrichment of glycopeptides is necessary before testing. However, most materials for enriching glycopeptides have high site resistance, relatively low surface area, and limited recognition sites. Herein, a highly hydrophilic two-dimensional (2-D) covalent organic framework (NUS-10) loaded with chitosan (CS) (denoted as NUS-10@CS) had been synthesized. After enrichment with NUS-10@CS, a total of 34 glycopeptides from horseradish peroxidase (HRP) tryptic digests were detected, demonstrating a high enrichment efficiency for glycopeptides from model glycoprotein digestion. Meanwhile, the material exhibited ultra-high adsorption capacity (1 fmol/μL HRP), excellent selectivity (HRP tryptic digest/bovine serum albumin (BSA) tryptic digest = 1:2000), macromolecular protein anti-interference ability (HRP tryptic digest/BSA = 1:2000) and good binding capacity (200 mg/g). Additionally, 712 glycopeptides corresponding to 200 glycoproteins were identified from 3 µL human serum. NUS-10@CS was promising for glycopeptide analysis, helping to identify potential disease biomarkers more efficiently, and leading to easier and more accurate diagnosis of diseases, which was essential for early intervention and treatment.

糖肽是一种重要的生物标志物，在多种生物过程中起着至关重要的作用。由于其低丰度和干扰大分子蛋白的存在，糖肽在检测前必须富集。然而，大多数糖肽富集材料具有较高的位点抗性、相对较小的表面积和有限的识别位点。本文合成了一种负载壳聚糖（CS）（表示为NUS-10@CS）的高亲水性二维（2-D）共价有机骨架（NUS-10）。用NUS-10@CS富集后，从辣根过氧化物酶（HRP）胰蛋白酶中共检测到34个糖肽，表明对模型糖蛋白消化的糖肽具有很高的富集效率。同时，该材料具有超高的吸附量（1 fmol/μL HRP）、极好的选择性（HRP胰蛋白酶/牛血清白蛋白（BSA）胰蛋白酶= 1:2000）、大分子蛋白的抗干扰能力（HRP胰蛋白酶/牛血清白蛋白= 1:2000）和良好的结合能力（200 mg/g）。此外，从3µL人血清中鉴定出200个糖蛋白对应的712个糖肽。NUS-10@CS有望用于糖肽分析，有助于更有效地识别潜在的疾病生物标志物，并导致更容易和更准确的疾病诊断，这对早期干预和治疗至关重要。

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引用次数: 0

Method development and validation on RP-HPLC method for estimation of xanthohumol in nanostructured lipid carriers drug delivery systems 纳米脂质载体给药体系中黄腐酚含量的RP-HPLC测定方法的建立与验证。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2024.124437

Shubham Singh , Himani Sharma , Vijay Kumar , Gaurav Gupta , Samir Patel , Archita Patel , Kamal Dua , Sachin Kumar Singh

Xanthohumol(Xn) is isolated from female inflorescences of Humulus lupulus. It has been discovered that Xn and its formulation are useful in the treatment of cancer. As this bioactive compound has medicinal importance, hence, a novel, precise, and sensitive HPLC method should be developed. In the present study, an RP-HPLC method has been developed and validated as per ICH Q2(R1) guidelines using column C18 having particle size 5 µm and dimension 250 × 4.6 mm. The detection wavelength (λ) used was 370 nm. The mobile phase consisted of a combination of HPLC grade Methanol and HPLC grade water buffer at a ratio of 95:5 v/v, with a flow rate of 1.0 mL/min. The total run time was 10 min with Xn retention time (Rt) at 4.5 min. The calibration plot was found linear in the range 2–10 μg/mL. The recovery of 97.65 % indicated good accuracy of method. The method’s precision is within 2 % of acceptable limits. LOD and LOQ values of 0.85 and 2.6 μg/mL indicated good sensitivity of method. The uniqueness of the research work is relying on achieving peak of Xn at lesser retention time as compared to existing methods. Further the results of specificity studies revealed absence of any interference of Xn peak with the excipients used in the nanostructured lipid carriers (NLCs). Overall, the study provided an accurate, precise, sensitive and specific method to quantify Xn in bulk and NLCs.

黄腐酚（Xn）是从葎草（Humulus lupulus）的雌花花序中分离出来的。已经发现Xn及其配方对治疗癌症有用。由于该生物活性化合物具有重要的药用价值，因此需要开发一种新颖、精确、灵敏的高效液相色谱方法。在本研究中，根据ICH Q2（R1）指南，使用粒径为5µm，尺寸为250 × 4.6 mm的柱C18，开发并验证了RP-HPLC方法。检测波长（λ）为370 nm。流动相为HPLC级甲醇与HPLC级水缓冲液，比例为95:5 v/v，流速为1.0 mL/min。总运行时间为10 min， Xn保留时间（Rt）为4.5 min。校准曲线在2 ~ 10 μg/mL范围内呈线性。回收率为97.65%，表明该方法具有良好的准确度。该方法的精密度在可接受范围的2%以内。定量限和定量限分别为0.85和2.6 μg/mL，灵敏度较高。与现有方法相比，该研究工作的独特之处在于在更短的保留时间内获得Xn的峰值。进一步的特异性研究结果表明，Xn峰与纳米结构脂质载体（nlc）中使用的赋形剂没有任何干扰。总的来说，本研究提供了一种准确、精确、敏感和特异的方法来定量散装和NLCs中的Xn。

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引用次数: 0

Development and validation of a RP-HPLC method for simultaneous determination of cimetidine, metoprolol tartrate and phenol red for intestinal perfusion studies 肠道灌注研究中同时测定西咪替丁、酒石酸美托洛尔和酚红的反相高效液相色谱方法的建立与验证。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2025.124449

Meryem Ermis , Fatma Kir , Selma Sahin

A new reversed phase high-performance liquid chromatography (RP-HPLC) method, with a short analysis time and easy to apply, was developed for the simultaneous detection of cimetidine (CIM), metoprolol tartrate (MT) and phenol red (PR) for use in intestinal perfusion studies. The analysis was performed with phosphate buffer (pH 5.0, 12.5 mM)-acetonitrile mixture as mobile phase and C₁₈ column (Inertsil ODS-3; 5 µm, 4.6 × 250 mm) as stationary phase. Gradient analysis conditions were used and the acetonitrile ratio in the mobile phase varied from 10 to 50% in 10 min. Total run time for analysis was 10 min and the injection volume was 20 µL. Detection of compounds was performed at 207 nm. Under optimum HPLC conditions, retention times were 4.03 min for CIM, 6.99 min for MT and 8.49 min for PR. The method was validated according to ICH Q2 (R1) guideline for specificity, linearity, sensitivity, precision, accuracy, stability and robustness. Developed method was linear and determination coefficients of the calibration curves were 0.9993, 0.9991 and 1.0 for CIM, MT and PR, respectively. The limits of quantification were 6.20, 2.78 and 0.45 μg/mL for CIM, MT and PR, respectively. The precision and accuracy values of the developed analytical method met the ICH Q2 (R1) limits. The applicability of the method was demonstrated by preliminary in-situ intestinal perfusion studies. In conclusion, samples obtained from in-situ intestinal perfusion studies performed to examine the absorption/permeability of CIM, MT, and PR can be analyzed with the developed HPLC method.

建立了一种分析时间短、易于应用的反相高效液相色谱（RP-HPLC）方法，用于肠道灌注研究中同时检测西咪替丁（CIM）、酒石酸美托洛尔（MT）和酚红（PR）。以磷酸缓冲液(pH 5.0, 12.5 mM)-乙腈混合物为流动相，C18色谱柱(Inertsil ODS-3；5µm, 4.6 × 250 mm)作为固定相。采用梯度分析条件，流动相乙腈比为10 ~ 50%，分析时间为10 min，总运行时间为10 min，进样量为20µL。在207 nm处检测化合物。在最佳HPLC条件下，CIM的保留时间为4.03 min， MT的保留时间为6.99 min， PR的保留时间为8.49 min。方法的特异性、线性度、灵敏度、精密度、准确度、稳定性和鲁棒性按照ICH Q2 （R1）指南进行验证。所建立的方法是线性的，CIM、MT和PR的标定曲线决定系数分别为0.9993、0.9991和1.0。CIM、MT和PR的定量限分别为6.20、2.78和0.45 μg/mL。所建立的分析方法精密度和准确度符合ICH Q2 （R1）的要求。初步的原位肠道灌注研究证实了该方法的适用性。综上所述，通过原位肠道灌注研究来检测CIM、MT和PR的吸收/渗透性，所获得的样品可以用所开发的HPLC方法进行分析。

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引用次数: 0

Development and validation of an LC-MS/MS multiplex assay for the quantification of bedaquiline, n-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine in dried blood spots 液相色谱-质谱联用法测定干血斑中贝达喹啉、n-去甲基贝达喹啉、利奈唑胺、左氧氟沙星和氯法齐明含量的建立和验证

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2025.124470

Gerard Aime Kenfack Teponnou , Anton Joubert , Saskia Spaltman , Marthinus van der Merwe , Edda Zangenberg , Sharon Sawe , Paolo Denti , Sandra Castel , Francesca Conradie , Richard Court , Gary Maartens , Lubbe Wiesner

Dried blood spot (DBS) assays to quantify novel and repurposed drugs for the treatment of rifampicin-resistant tuberculosis (RR-TB) would facilitate pharmacokinetic studies and therapeutic drug monitoring in low-middle income settings, considering their ease of application and simple sample storage requirements. We describe a DBS method for the simultaneous quantification of bedaquiline and metabolite N-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine. The analytes were extracted from the matrix and isolated by solid-phase extraction. Two LC-MS/MS systems were used, optimized for the separate analysis of the more polar compounds (linezolid and levofloxacin), and less polar compounds (bedaquiline, N-desmethyl bedaquiline, and clofazimine), employing gradient elution. Electrospray ionization and multiple reaction monitoring were used to quantify the analytes on a Sciex API3200 and an API5500 triple quadrupole mass spectrometer, for the more polar and less polar analytes, respectively. Isotopically labelled internal standards were used to compensate for variability in the quantification of each analyte. The method was validated according to international guidelines and applied to samples from a clinical trial. We performed correlation and agreement analysis of the DBS assay and in-house plasma methods using Deming regressions and Bland–Altman plots. Coefficients of correlation between measured plasma and DBS concentrations ranged from 0.866 (95% CI: 0.817–0.902) to 0.989 (95% CI: 0.985–0.992). More than 67% of the samples showed a difference between the observed and estimated plasma concentrations within 20% of their means, meeting EMA requirements for method reproducibility and demonstrating the interchangeability of our DBS and plasma LC-MS/MS methods.

考虑到干血点（DBS）检测易于应用和简单的样品储存要求，用于定量治疗利福平耐药结核病（RR-TB）的新药和再用途药物，将促进中低收入环境下的药代动力学研究和治疗药物监测。我们描述了一种同时定量贝达喹啉及其代谢物n -去甲基贝达喹啉、利奈唑胺、左氧氟沙星和氯法齐明的DBS方法。分析物从基质中提取，用固相萃取法分离。采用两套LC-MS/MS系统，采用梯度洗脱对极性较高的化合物（利奈唑胺和左氧氟沙星）和极性较低的化合物（贝达喹啉、n -去甲基贝达喹啉和氯法齐明）进行分离分析。在Sciex API3200和API5500三重四极质谱仪上使用电喷雾电离和多重反应监测分别对极性较高和极性较低的分析物进行定量。使用同位素标记的内部标准来补偿每种分析物定量的可变性。该方法根据国际准则进行了验证，并应用于临床试验样本。我们使用Deming回归和Bland-Altman图对DBS测定法和内部血浆方法进行了相关性和一致性分析。血浆与DBS浓度的相关系数范围为0.866 （95% CI: 0.817-0.902）至0.989 （95% CI: 0.985-0.992）。超过67%的样品显示观察到的和估计的血浆浓度之间的差异在其平均值的20%以内，满足EMA对方法重复性的要求，并证明了我们的DBS和血浆LC-MS/MS方法的互换性。

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引用次数: 0

A simple HPLC-UV method for monitoring therapeutic adherence in pulmonary arterial hypertension 一种简单的HPLC-UV方法监测肺动脉高压患者的治疗依从性。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2024.124443

Paweł K. Kunicki , Maciej T. Grymm , Tomasz Pawiński , Daniel Szulczyk , Marcin Waligóra , Grzegorz Kopeć

A considerable percentage of ineffective treatment in pulmonary arterial hypertension (PAH) may be related to subtherapeutic dosage or non-adherence. The aim of the study was to develop a simple analytical method suitable for plasma determination of selected drugs: riociguat (RIO), bosentan (BOS) and macitentan (MAC) administered to PAH patients. An isocratic HPLC-UV system (Spectra Physics – Shimadzu) with a manual injector (50 μL loop) was applied. Chromatographic analysis was performed using a Suplecosil LC-CN column (150 × 4.6 mm, 5 μm) protected with a Supelguard precolumn at room temperature. The separation was carried out using the mobile phase: CH₃CN:H₂O:0.5 M KH₂PO₄:85 % H₃PO₄ (172:324.2:3.7:0.1, v/v) at a flow rate of 1.8 mL/min. Ethyl acetate (4 mL) was used for 10-min liquid–liquid extraction from 0.4 mL alkalized plasma sample. Detection was performed at λ = 245 nm chosen as a compromise between signal intensity and matrix interference. The analytes were eluted at retention times of 4.4 min (RIO), 5.4 min (BOS), 8.9 min (MAC) and 7.8 min for gallopamil (internal standard, GAL). The method was found linear and calibrated in the ranges: 5–1000 ng/mL for RIO, 10–2000 ng/mL for BOS and 20–2000 ng/mL for MAC, with r² of 0.9991 for RIO, 0.9983 for BOS, and 0.9949 for MAC, respectively. Within the given ranges, the method ensured reliable results with the required precision and accuracy: ≤15 % (≤20 % for LLOQ). There was no significant carryover effect. The method has been successfully used in pilot study on adherence in patients treated for PAH, enabling monitoring of RIO, BOS and MAC. Drug concentrations were assessed in samples taken before (C0) and 3 h after drug administration (C3). For RIO, BOS and MAC, the developed method was suitable for both C0 and C3 samples, allowing steady-state drug determination if used. The presented method can be recommended to laboratories equipped with basic HPLC apparatus as an attractive analytical tool for both TDM and adherence studies.

相当大比例的肺动脉高压（PAH）无效治疗可能与亚治疗剂量或不依从性有关。本研究的目的是建立一种简单的分析方法，适用于PAH患者血浆中所选药物的测定：瑞西奎特（里约热内卢）、波生坦（BOS）和马西坦（MAC）。采用手动进样（50 μL回路）等密度HPLC-UV体系（Spectra Physics - Shimadzu）。色谱分析采用Supelguard预柱保护的Suplecosil LC-CN柱（150 × 4.6 mm, 5 μm），室温下进行。采用流动相CH3CN:H2O:0.5 M kh2po4: 85% H3PO4 （172:324.2:3.7:0.1, v/v）进行分离，流速为1.8 mL/min。0.4 mL碱化血浆样品，用乙酸乙酯（4ml）液液萃取10min。在λ = 245 nm处进行检测，选择信号强度和矩阵干扰之间的折衷。洗脱时间为4.4 min（里约热内卢）、5.4 min （BOS）、8.9 min （MAC）和7.8 min （GAL）。该方法在里约热内卢的5-1000 ng/mL、BOS的10-2000 ng/mL和MAC的20-2000 ng/mL范围内呈线性关系，里约热内卢、BOS和MAC的r2分别为0.9991、0.9983和0.9949。在给定的范围内，该方法保证了可靠的结果，所需的精密度和准确度：≤15% （LLOQ≤20%）。没有明显的结转效应。该方法已成功用于PAH治疗患者依从性的试点研究，可以监测里约热内卢，BOS和MAC。在给药前（C0）和给药后3小时（C3）的样品中评估药物浓度。对于里约热内卢、BOS和MAC，该方法适用于C0和C3样品，使用该方法可以进行稳态药物测定。该方法可推荐给配备基本HPLC设备的实验室，作为TDM和粘附性研究的有吸引力的分析工具。

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引用次数: 0

Determination of eldecalcitol in human plasma by SIL-IS UPLC-APCI-MS/MS method for pharmacokinetics study SIL-IS UPLC-APCI-MS/MS法测定人血浆中艾尔地骨糖醇的药代动力学研究。

IF 2.8 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Chromatography B

Pub Date : 2025-02-01 DOI: 10.1016/j.jchromb.2025.124468

Wanyu Wang , Zhichao Yin , Pengfei Du , Jianbang Wu , Minhui Wang , Yaqin Wang , Ping Wu , Xiaocui Xia , Lin Zhang , Yamin Liu , Zhenyue Gao , Jie Shen , Yuanwei Jia

Eldecalcitol is a novel analog of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] for the treatment of patients with osteoporosis. A highly sensitive and specific ultra-performance liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (UPLC-APCI-MS/MS) technique featuring a lower limit of quantitation (LLOQ) as low as 5 pg/ml, has been established and validated for the rapid and accurate quantification of eldecalcitol in human plasma samples. Plasma samples were extracted by solid phase extraction (SPE). Stable isotope-labeled compound eldecalcitol-d6 was used as an internal standard (SIL-IS). The detection process was carried out utilizing Multi-Reaction Monitoring (MRM) mode, employing an atmospheric pressure chemical ionization (APCI) source operating in the positive ion modality. The target fragment ion pairs for eldecalcitol and SIL-IS were identified as m/z 508.6 transitioning to 397.4, and m/z 514.6 transitioning to 403.3, respectively. This method was validated regarding selectivity, LLOQ, linearity, accuracy and precision, recovery, matrix effects, dilution reliability, stability, carryover test, and incurred sample reanalysis (ISR). This methodology had been successfully employed to assess the pharmacokinetics (PK) profile of eldecalcitol in a clinical investigation.

Eldecalcitol是一种新的类似于1α，25-二羟基维生素D3 [1,25(OH)2D3]的药物，用于治疗骨质疏松症。建立了一种高灵敏度、高特异性的超高效液相色谱-常压化学电离串联质谱（UPLC-APCI-MS/MS）技术，该技术的定量下限（LLOQ）低至5 pg/ml，可快速、准确地定量测定人血浆样品中的艾尔地钙醇。血浆样品采用固相萃取法（SPE）提取。采用稳定同位素标记化合物钙醇-d6作为内标（SIL-IS）。检测过程采用多反应监测（MRM）模式，采用正离子模式的常压化学电离（APCI）源。eldecalcitol和SIL-IS的目标片段离子对分别为m/z 508.6向397.4过渡，m/z 514.6向403.3过渡。对该方法进行了选择性、定量限、线性度、准确度和精密度、回收率、基质效应、稀释度可靠性、稳定性、遗留试验和引起的样品再分析（ISR）等方面的验证。该方法已在临床研究中成功地用于评估依地骨糖醇的药代动力学（PK）特征。

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引用次数: 0