Kuklenyik Zsuzsanna, Martin Amy, Pau Chou-Pong, Garcia-Lerma J Gerardo, Heneine Walid, Pirkle James L, Barr John R
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA.
J Chromatogr Sci. 2009 May-Jun;47(5):365-72. doi: 10.1093/chromsci/47.5.365.
The HIV-1 reverse transcriptase inhibitors tenofovir (TFV), emtricitabine (FTC), and lamivudine (3TC) are widely used in the treatment of HIV-1-infected persons and are now being considered as chemoprophylactic drugs for the prevention of sexual HIV transmission. Assays that measure these drugs after either oral or topical application are critical to the understanding of the pharmacokinetic profiles of the drugs and allow a rational design of chemoprophylaxis modalities for evaluation in macaque models and human trials. We developed a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method for sensitive measurement of FTC, 3TC, and TFV in plasma from macaques. To achieve detection limits of 10 pg on column, the plasma analytes were measured using acidic mobile phase and positive electrospray ionization MS-MS detection. However, this caused various chromatographic peak distortions, which were minimized by using mobile phase additives that induced ion-pairing interactions. Chromatographic peak tailing was minimized by adjusting the organic mobile phase concentration while considering the simultaneous effect of organic content on buffer and analyte pKa. Injection solution interferences were corrected by chromatographic peak focusing using column switching. The final method provides simultaneous measurement of all three analytes with a wide linear range of 1-3000 ng/mL using 0.1 mL plasma (10 pg on column) and coefficients of variation from 5% to 15% in the high ng/mL concentration range and from 16% to 20% in the low ng/mL concentration range.
人类免疫缺陷病毒1型（HIV-1）逆转录酶抑制剂替诺福韦（TFV）、恩曲他滨（FTC）和拉米夫定（3TC）被广泛用于治疗HIV-1感染者，目前正被考虑用作预防性传播HIV的化学预防药物。在口服或局部应用后测量这些药物的分析方法对于了解药物的药代动力学特征至关重要，并且有助于合理设计化学预防方案，以便在猕猴模型和人体试验中进行评估。我们开发了一种高效液相色谱-串联质谱（HPLC-MS-MS）方法，用于灵敏测量猕猴血浆中的FTC、3TC和TFV。为了实现柱上检测限为10 pg，使用酸性流动相和正电喷雾电离MS-MS检测来测量血浆分析物。然而，这导致了各种色谱峰变形，通过使用诱导离子对相互作用的流动相添加剂将其最小化。通过调整有机流动相浓度并同时考虑有机成分对缓冲液和分析物pKa的影响，将色谱峰拖尾最小化。通过使用柱切换进行色谱峰聚焦来校正进样溶液干扰。最终方法使用0.1 mL血浆（柱上10 pg）可同时测量所有三种分析物，线性范围宽达1 - 3000 ng/mL，在高ng/mL浓度范围内变异系数为5%至15%，在低ng/mL浓度范围内为16%至20%。
