Development and Validation of an LC/MS/MS Method for the Determination of Tenofovir in Monkey Plasma
Development and Validation of an LC/MS/MS Method for the Determination of Tenofovir in Monkey Plasma
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用于测定猴血浆中替诺福韦的 LC/MS/MS 方法的开发和验证
DOI：
10.1248/bpb.34.877
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发表时间：
2011-06-01
影响因子：
2
通讯作者：
Zhao, Yimin
中科院分区：
文献类型：
--
作者：
Guo, Jifen;Meng, Fanhua;Zhao, Yimin
建立了一种简便、特异、灵敏的LC/MS/MS定量猴子血浆中替诺福韦（ttfv）的方法。加入阿德福韦作为内标（IS）后，用甲醇制备无蛋白提取物。采用Discovery C-18反相色谱柱（4.6 x 250 mm, 5 μ m）进行等温色谱分离。流动相为甲醇-水-甲酸（20:80:0.5,v/v/v）。采用电喷雾电离(ESI)-MS/MS在正离子模式下分别采用288/176和274/162跃迁实现TFV和IS的检测。使用200 μ l血浆样品，分析范围为0.005-1.250 μ g/ml。日内、日间精密度值均小于11.4%，准确度在0.4 ~ 2.9%之间。对该方法的灵敏度、选择性、准确度和精密度、基质效应、回收率和稳定性进行了充分验证。由于TFV的高极性，在使用LC/MS/MS方法定量TFV的血浆浓度时，主要的挑战是避免离子抑制。为了避免离子抑制，充分的色谱分离是目前最有效的方法。此外，发现干燥残留物的重构溶剂对LC峰形状有显著影响。经验证的方法成功应用于6只猴子口服两种酯类TIN前药（相当于TFV 20 mg/kg）后的生物等效性研究。该方法使实验室科学家能够使用适当的仪器进行快速的TFV测定。
A simple, specific, sensitive LC/MS/MS method for the quantitation of tenofovir (TFV) in monkey plasma was developed and validated. After the addition of adefovir as an internal standard (IS), methanol was used to produce a protein-free extract. Isocratic chromatographic separation was performed on a reverse-phase Discovery C-18 column (4.6 x 250 mm, 5 mu m). The mobile phase consisted of methanol-water-formic acid (20 : 80 : 0.5, v/v/v). Detection of TFV and the IS was achieved with electrospray ionization (ESI)-MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The analytical range was set at 0.005-1.250 mu g/ml using a 200 mu l plasma sample. The intra- and inter-day precision values were less than 11.4%, and accuracy ranged from 0.4 to 2.9% in all quality control samples. The method was fully validated for its sensitivity, selectivity, accuracy and precision, matrix effect, recovery, and stability. Due to the high polarity of TFV, the major challenge was to circumvent ion suppression when quantitating the plasma concentration of TFV using the LC/MS/MS method. To avoid ion suppression, sufficient chromatographic separation was the most effective means for the present purposes. Moreover, it was found that the reconstitution solvents of the dried residue had a significant impact on LC peak shapes. The validated method was successfully applied to a bioequivalence study in 6 monkeys after the oral administration of two ester prodrugs of TIN (equivalent to TFV 20 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid TFV determination.