Department of Chemistry, Cleveland State University, Cleveland, OH, 44115, USA.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, 210023, PR China.
J Pharm Biomed Anal. 2018 Sep 5;158:386-394. doi: 10.1016/j.jpba.2018.06.035. Epub 2018 Jun 20.
A rapid and sensitive LC-MS/MS method has been developed for quantitative analysis of cardiolipin (18:2) or CL (18:2) in human leukocytes and mouse mitochondria. The structural analog CL (14:0) was used as the internal standard. Both CL (18:2) and the IS were extracted using a modified Folch method, and separated on a Waters XBridge® BEH C XP column using a mobile phase of 0.1% ammonium hydroxide in acetonitrile/water (90:10, v/v) pumped at a flow rate of 0.4 mL/min. Quantitation was achieved by negative ESI-MS/MS in MRM mode. The total run time was 2.00 min with retention times of 0.74 min for the IS and 0.84 min for CL (18:2), respectively. The method was validated according to the US-FDA guidance for bioanalytical method validation using human leukemia and lymphoma cell lines, which had a calibration range of 0.120-60.2 nM with a correlation coefficient >0.999. The intra- and inter-assay accuracy and precision were ≤±5% and ≤8%. The IS normalized matrix factors of CL (18:2) and the IS normalized recoveries of CL (18:2) ranged 0.92-1.04, and 95-101%, respectively. The stability studies showed that CL (18:2) was stable under various test conditions. The developed method was successfully applied to the measurement of CL (18:2) in various biological samples including K562 and HL-60 human leukemia cell lines, U937 human lymphoma cell line, white blood cells from patients of Alzheimer's disease and normal cognitive controls (NCCs), and mitochondria from mouse skeletal muscles. It may be useful for preclinical and clinical studies of this compound.
已开发出一种灵敏、快速的 LC-MS/MS 方法，用于定量分析人白细胞和鼠线粒体中的心磷脂（18:2）或 CL（18:2）。结构类似物 CL（14:0）被用作内标。CL（18:2）和 IS 均采用改良的 Folch 法提取，在 Waters XBridge® BEH C XP 柱上以 0.1%氨化乙腈/水（90:10，v/v）为流动相分离，流速为 0.4 mL/min。通过负电喷雾质谱/质谱（ESI-MS/MS）在 MRM 模式下定量。总运行时间为 2.00 分钟，IS 的保留时间为 0.74 分钟，CL（18:2）的保留时间为 0.84 分钟。该方法根据美国食品和药物管理局（FDA）生物分析方法验证指南进行验证，采用人白血病和淋巴瘤细胞系，校准范围为 0.120-60.2 nM，相关系数>0.999。日内和日间准确度和精密度均≤±5%和≤8%。CL（18:2）的 IS 归一化基质因子和 IS 归一化回收率范围分别为 0.92-1.04 和 95-101%。稳定性研究表明，CL（18:2）在各种测试条件下均稳定。该方法成功应用于各种生物样本中 CL（18:2）的测定，包括 K562 和 HL-60 人白血病细胞系、U937 人淋巴瘤细胞系、阿尔茨海默病患者和正常认知对照（NCC）的白细胞以及鼠骨骼肌线粒体。它可能对该化合物的临床前和临床研究有用。
