Ananthula Suryatheja, Janagam Dileep R, Jamalapuram Seshulatha, Johnson James R, Mandrell Timothy D, Lowe Tao L
Department of Pharmaceutical Sciences, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA.
Department of Comparative medicine, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Oct 15;1003:47-53. doi: 10.1016/j.jchromb.2015.09.006. Epub 2015 Sep 11.
Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3μM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies.
建立了快速、灵敏、选择性好且准确的液相色谱/串联质谱（LC/MS/MS）方法，用于定量测定大鼠血浆中的左炔诺孕酮（LNG），并进一步对其特异性、线性、准确性、精密度、灵敏度、基质效应、回收率和稳定性进行了验证。采用正己烷：乙酸乙酯体积比为80:20的液-液萃取程序，从大鼠血浆中高效萃取LNG。在AB SCIEX Triple Quad™ 4500 LC/MS/MS系统上安装反相Luna C18(2)柱（50×2.0mm内径，3μm）进行色谱分离。LNG在2分钟内被特异性地鉴定出来。在0.5-50ng·mL(-1)浓度范围内绘制线性校准曲线。所建立的方法经日内和日间准确性及精密度验证，其值均在可接受范围内。发现基质效应最小。在0.5（低）、5（中）和50（高）ng·mL(-1)三个质量控制（QC）浓度下的回收率>90%。使用QC样品评估了LNG在包括储存、萃取和分析在内的各个实验阶段的稳定性，结果表明LNG在所有条件下均稳定。该验证方法成功用于研究皮下注射后大鼠体内LNG的药代动力学，证明了其在相关临床前研究中的适用性。
