Development and validation of RP-HPLC method for simultaneous determination of lamivudine, stavudine, and zidovudine in perfusate samples: Application to the Single-Pass Intestinal Perfusion (SPIP) studies

A reversed-phase high performance liquid chromatography (RP-HPLC) method with ultraviolet detection was developed and validated for the simultaneous quantification of antiretroviral drugs lamivudine (3TC), stavudine (d4T), and zidovudine (AZT) in perfusate samples obtained from the Single-Pass Intestinal Perfusion studies. The chromatographic analysis was performed using a Gemini C18 column and didanosine as internal standard (IS). The following parameters were considered for the validation procedure: system suitability, accuracy, precision, linearity and selectivity. The limits of detection were 0.32 µg/mL for 3TC, 0.11 µg/mL for d4T and 0.45 µg/mL for AZT and the limits of quantification were 1.06 µg/mL for 3TC, 0.38 µg/mL for d4T and 1.51 µg/mL for AZT. Repeatability and intermediate precision ranged from 1.05 to 1.31 and 1.50 to 1.87, respectively, and are expressed as percent of relative standard deviation (RSD). Based on these results, the developed and validated RP-HPLC method can be used for simultaneous determination of 3TC, d4T, and AZT in perfusate samples. Furthermore, this method is simple and adequate for measurements of the antiretroviral drugs in the same sample, since those compounds are mostly co-administered. Besides, this work can be used as an initial base for the development of similar methods in the same conditions presented in our study.

本研究建立并验证了一种采用紫外检测的反相高效液相色谱（Reversed-Phase High Performance Liquid Chromatography, RP-HPLC）方法，用于同时定量测定单次灌流肠道实验（Single-Pass Intestinal Perfusion studies）所得灌流液样品中的抗逆转录病毒药物拉米夫定（lamivudine, 3TC）、司他夫定（stavudine, d4T）与齐多夫定（zidovudine, AZT）。色谱分析采用Gemini C18色谱柱，并以去羟肌苷（didanosine）作为内标（internal standard, IS）。验证过程考察了系统适用性、准确度、精密度、线性范围与选择性等指标。3TC、d4T与AZT的检出限分别为0.32 μg/mL、0.11 μg/mL与0.45 μg/mL，定量限分别为1.06 μg/mL、0.38 μg/mL与1.51 μg/mL。重复性与中间精密度分别介于1.05~1.31与1.50~1.87之间，以相对标准偏差（relative standard deviation, RSD）百分比形式表征。基于上述结果，本研究建立并验证的RP-HPLC方法可用于同时测定灌流液样品中的3TC、d4T与AZT。此外，由于上述三种药物多为联合给药，该方法简便易行且可满足同一样品中抗逆转录病毒药物的含量测定需求。本研究可为在本研究采用的相同实验条件下开发同类分析方法提供初始参考依据。