Chemical Department, Saint Petersburg State University, 199034 Saint Petersburg7-9, Universitetskaya nab., Russia,
Anal Bioanal Chem. 2014 Jan;406(3):923-7. doi: 10.1007/s00216-013-7521-x. Epub 2013 Dec 13.
A reliable and high throughput high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for determining levels of the antitubercular drug-D -cycloserine in human plasma. Plasma samples analyte with an internal standard (IS) (niacin) were prepared by solid-phase extraction using Waters Oasis MCX cartridges. The chromatographic separation was performed using the HILIC mode on a YMC-Pack SIL-06 column (150 × 4.6 mm; 3 μm) under isocratic conditions. The run time of analysis was 5 min. The mobile phase consisted of methanol, propanol-2 and 0.075 % trifluoroacetic acid (66.5:28.5:5, v/v/v). Protonated ions formed by turbo ion spray in positive mode were used to detect the analyte and the IS. MS/MS detection was used to monitor the fragmentation of 103-75 m/z for cycloserine and 124 to 80 m/z for niacin (IS) on an API 4000 (AB Sciex) triple quadrupole mass spectrometer. A linear dynamic range of 0.3-30 μg/mL was established for cycloserine using 0.2 mL human plasma and a 1 μL injection volume. The mean relative recovery of cycloserine and niacin were 77.2 and 82.4 %, respectively. The procedure of sample preparation was consistent and reproducible (precision, 0.8-3.4 %; accuracy, 93.8-104.9 %). The method was validated in accordance with requirements of the European Medicines Agency and successfully applied to a bioequivalence study of 250 mg tablet formulations in 23 healthy human subjects.
建立并验证了一种可靠且高通量的高效液相色谱-串联质谱（HPLC-MS/MS）法，用于测定人血浆中抗结核药物-D-环丝氨酸的浓度。采用沃特世 Oasis MCX 萃取小柱对含内标（烟酸）的血浆样品进行固相萃取预处理。采用亲水作用色谱模式，在 YMC-Pack SIL-06 柱（150×4.6mm，3μm）上以等度洗脱方式进行色谱分离。分析运行时间为 5min。流动相由甲醇、异丙醇-2 和 0.075%三氟乙酸（66.5:28.5:5，v/v/v）组成。采用正离子模式的涡轮离子喷雾使分析物和内标形成质子化离子。采用 API 4000（AB Sciex）三重四极杆质谱仪，监测 103-75m/z 处环丝氨酸和 124-80m/z 处烟酸（内标）的离子碎片，进行 MS/MS 检测。采用人血浆 0.2mL 和进样量 1μL，建立了 0.3-30μg/mL 的线性动态范围。环丝氨酸和烟酸的平均相对回收率分别为 77.2%和 82.4%。样品制备过程稳定且重现（精密度为 0.8-3.4%；准确度为 93.8-104.9%）。该方法符合欧洲药品管理局的要求，并成功应用于 23 名健康人体的 250mg 片剂制剂的生物等效性研究。
