Wongchang Thamrong, Winterberg Markus, Tarning Joel, Sriboonvorakul Natthida, Muangnoicharoen Sant, Blessborn Daniel
Mahidol-Oxford Tropical Medicine Research Unit (MORU), Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Wellcome Open Res. 2022 May 31;4:47. doi: 10.12688/wellcomeopenres.15141.3. eCollection 2019.
Ceftriaxone is a cephalosporin antibiotic drug used as first-line treatment for a number of bacterial diseases. Ceftriaxone belongs to the third generation of cephalosporin and is available as an intramuscular or intravenous injection. Previously published pharmacokinetic studies have used high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) for the quantification of ceftriaxone. This study aimed to develop and validate a bioanalytical method for the quantification of ceftriaxone in human plasma using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Sample preparation was performed by protein precipitation of 100 µl plasma sample in combination with phospholipid-removal techniques to minimize matrix interferences. The chromatographic separation was performed on an Agilent Zorbax Eclipse Plus C18 column with 10 mM ammonium formate containing 2% formic acid: acetonitrile as mobile phase at a flow rate of 0.4 ml/min with a total run time of 10 minutes. Both the analyte and cefotaxime (internal standard) were detected using the positive electrospray ionization (ESI) mode and selected reaction monitoring (SRM) for the precursor-product ion transitions 555.0→396.1 for ceftriaxone and 456.0→324.0 for cefotaxime. The method was validated over the concentration range of 1.01-200 μg/ml. Calibration response showed good linearity (correlation coefficient > 0.99) and matrix effects were within the ±15% limit in 6 different lots of sodium heparin plasma tested. However, citrate phosphate dextrose plasma resulted in a clear matrix enhancement of 24% at the low concentration level, which was not compensated for by the internal standard. Different anticoagulants (EDTA, heparin and citrate phosphate dextrose) also showed differences in recovery. Thus, it is important to use the same anticoagulant in calibration curves and clinical samples for analysis. The intra-assay and inter-assay precision were less than 5% and 10%, respectively, and therefore well within standard regulatory acceptance criterion of ±15%.
头孢曲松是一种头孢菌素类抗生素药物，用作多种细菌性疾病的一线治疗药物。头孢曲松属于第三代头孢菌素，有肌内注射或静脉注射剂型。先前发表的药代动力学研究使用高效液相色谱结合紫外检测（HPLC-UV）来定量头孢曲松。本研究旨在开发并验证一种使用液相色谱串联质谱（LC-MS/MS）定量人血浆中头孢曲松的生物分析方法。通过对100 μl血浆样品进行蛋白沉淀并结合去除磷脂技术来进行样品制备，以尽量减少基质干扰。色谱分离在安捷伦Zorbax Eclipse Plus C18柱上进行，流动相为含2%甲酸的10 mM甲酸铵：乙腈，流速为0.4 ml/min，总运行时间为10分钟。使用正电喷雾电离（ESI）模式和选择反应监测（SRM）检测分析物和头孢噻肟（内标），头孢曲松的前体-产物离子跃迁为555.0→396.1，头孢噻肟为456.0→324.0。该方法在1.01 - 200 μg/ml的浓度范围内进行了验证。校准响应显示出良好的线性（相关系数>0.99），在测试的6个不同批次的肝素钠血浆中，基质效应在±15%的限度内。然而，在低浓度水平下，枸橼酸盐磷酸盐葡萄糖血浆导致明显的基质增强24%，内标无法对此进行补偿。不同的抗凝剂（乙二胺四乙酸、肝素和枸橼酸盐磷酸盐葡萄糖）在回收率方面也表现出差异。因此，在校准曲线和临床样品分析中使用相同的抗凝剂很重要。批内和批间精密度分别小于5%和10%，因此完全符合标准监管接受标准±15%。
