Beijing Institute of Pharmacology and Toxicology, Beijing, P. R. China.
Biol Pharm Bull. 2011;34(6):877-82. doi: 10.1248/bpb.34.877.
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 (4.6×250 mm, 5 µm). 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 µg/ml using a 200 µl 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 TFV (equivalent to TFV 20 mg/kg). The method permits laboratory scientists with access to the appropriate instrumentation to perform rapid TFV determination.
建立并验证了一种用于猴血浆中替诺福韦（TFV）定量的简单、特异、灵敏的 LC/MS/MS 方法。加入阿德福韦作为内标（IS）后，用甲醇进行蛋白沉淀提取。采用 Discovery C(18)（4.6×250mm，5µm）反相色谱柱进行等度洗脱分离。流动相由甲醇-水-甲酸（20:80:0.5，v/v/v）组成。TFV 和 IS 的检测采用电喷雾电离（ESI）-MS/MS 在正离子模式下，分别用 288/176 和 274/162 跃迁进行。采用 200µl 血浆样品，分析范围设定为 0.005-1.250µg/ml。所有质控样品的日内和日间精密度值均小于 11.4%，准确度在 0.4-2.9%之间。该方法的灵敏度、选择性、准确度和精密度、基质效应、回收率和稳定性均得到了全面验证。由于 TFV 的高极性，使用 LC/MS/MS 方法定量测定 TFV 血浆浓度时，主要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，还发现干燥残渣的复溶溶剂对 LC 峰形有显著影响。该方法成功应用于 6 只猴子口服两种 TFV 酯前药（相当于 TFV 20mg/kg）后的生物等效性研究。该方法允许有适当仪器的实验室科学家进行快速 TFV 测定。
