Patel Bhargav, Jangid Arvind G, Suhagia B N, Desai Nirmal
Department of Chemistry, St. Xavier's College, Navrangpura, Ahmedabad, India.
Accutest Research Lab, Satellite, Ahmedabad, India.
J Chromatogr Sci. 2018 Nov 1;56(10):867-878. doi: 10.1093/chromsci/bmy055.
An isotope dilution selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of hydrochlorothiazide (HCTZ) and ramipril in human plasma through a new concept of periodical polarity switching. Extraction of HCTZ, ramipril and their deuterated analogs as internal standards (ISs) was carried out from 150 μL of human plasma by solid-phase extraction method. Chromatographic separation of analytes was performed on Hypurity C18 (150 mm × 4.6 mm, 5 μ) column under gradient conditions with methanol:0.2% (v/v) formic acid in water as the mobile phase. The method was validated over a concentration range of 0.750-300 ng/mL for HCTZ and 0.125-80.0 ng/mL for ramipril. The mean extraction recovery for analytes and ISs were >(86.0%), consistent across all four QC levels. The challenges to evaluate matrix effect and continuous reproducibility of method during long analytical run was studied and resolved. Processed samples, freeze-thaw, long-term and whole blood stability were evaluated for both the analytes. The method was applied to support a bioequivalence study of 25 mg of HCTZ and 5 mg of ramipril tablet formulation in nine healthy Indian subjects. Assay reproducibility was demonstrated by reanalysis of 42 incurred samples.
建立了一种同位素稀释选择性灵敏高效液相色谱-串联质谱（LC-ESI-MS/MS）法，通过周期性极性切换的新概念同时测定人血浆中的氢氯噻嗪（HCTZ）和雷米普利。采用固相萃取法从150μL人血浆中提取HCTZ、雷米普利及其作为内标（ISs）的氘代类似物。在Hypurity C18（150 mm×4.6 mm，5μm）柱上，以甲醇:0.2%（v/v）甲酸水溶液为流动相，在梯度条件下对分析物进行色谱分离。该方法在HCTZ浓度范围为0.750 - 300 ng/mL、雷米普利浓度范围为0.125 - 80.0 ng/mL内进行了验证。分析物和内标的平均提取回收率均>（86.0%），在所有四个质量控制水平上均一致。研究并解决了在长时间分析运行过程中评估基质效应和方法持续重现性的挑战。对两种分析物均评估了处理后样品、冻融、长期和全血稳定性。该方法应用于支持对9名健康印度受试者进行的25 mg HCTZ和5 mg雷米普利片剂配方的生物等效性研究。通过对42个实际样品的重新分析证明了测定的重现性。
