Li Wenkui, Li Ying-He, Li Austin C, Zhou Shaolian, Naidong Weng
Department of Bioanalytical Chemistry, Covance Laboratories Inc., 3301 Kinsman Blvd. Madison, WI 53704, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Oct 25;825(2):223-32. doi: 10.1016/j.jchromb.2005.01.012.
In the present work, for the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous analysis of norethindrone, and ethinyl estradiol, was developed and validated over the concentration range of 50-10000pg/ml and 2.5-500pg/ml, respectively, using 0.5 ml of plasma sample. Norethindrone, ethinyl estradiol, and their internal standards norethindrone-(13)C2, and ethinyl estradiol-d4, were extracted from human plasma matrix with n-butyl chloride. After evaporation of the organic solvent, the extract was derivatized with dansyl chloride and the mixture was injected onto the LC-MS/MS system. The gradient chromatographic elution was achieved on a Genesis RP-18 (50 mm x 4.6 mm, 3 microm) column with mobile phase consisted of acetonitrile, water and formic acid. The flow rate was 1.0 ml/min and the total run time was 5.0 min. Important parameters such as sensitivity, linearity, matrix effect, reproducibility, stability, carry-over and recovery were investigated during the validation. The inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels were <6.8% relative standard deviation (RSD) and 4.4% relative error (RE) for norethindrone, and 4.2% RSD and 5.9% RE for ethinyl estradiol, respectively. Chromatographic conditions were optimized to separate analytes of interest from the potential interference peaks, arising from the derivatization. This method could be used for pharmacokinetic and drug-drug interaction studies in human subjects.
在本研究中，首次开发并验证了一种采用串联质谱检测的液相色谱法（LC-MS/MS），用于同时分析炔诺酮和炔雌醇。分别使用0.5 ml血浆样本，在50 - 10000 pg/ml和2.5 - 500 pg/ml的浓度范围内进行分析。炔诺酮、炔雌醇及其内标物炔诺酮-(13)C2和炔雌醇-d4，用正丁基氯从人血浆基质中萃取。有机溶剂蒸发后，提取物用丹磺酰氯衍生化，然后将混合物注入LC-MS/MS系统。在Genesis RP-18（50 mm×4.6 mm，3 µm）柱上实现梯度色谱洗脱，流动相由乙腈、水和甲酸组成。流速为1.0 ml/min，总运行时间为5.0 min。在验证过程中研究了灵敏度、线性、基质效应、重现性、稳定性、残留和回收率等重要参数。低、中、高浓度水平的质量控制样品的日间精密度和准确度，炔诺酮的相对标准偏差（RSD）<6.8%，相对误差（RE）为4.4%；炔雌醇的RSD为4.2%，RE为5.9%。优化了色谱条件，以将目标分析物与衍生化产生的潜在干扰峰分离。该方法可用于人体受试者的药代动力学和药物相互作用研究。
