Simultaneous determination and validation of emtricitabine, rilpivirine and tenofovir from biological samples using LC and CE methods

Abstract

A combination of antiretroviral agents is frequently used in effective treatment of the human immunodeficiency virus infection. In this study, two different separation methods are presented for the simultaneous determination of emtricitabine, rilpivirine and tenofovir from raw materials and urine samples. Developed liquid chromatography and capillary electrophoresis methods were thoroughly optimized for high analytical performances. Optimization of multiple variables at the same time by performing a minimum number of experiments was achieved by the Box-Behnken design, which is an experimental design in response surface methodology, in capillary electrophoresis. The results of the experimental design ensure minimum analysis time with well-separated analytes. Separation conditions, such as different stationary phases, pH level, organic modifiers and temperatures in liquid chromatography method, were also optimized. In particular, among stationary phases, the core-shell column especially enhanced the effectiveness of separation in liquid chromatography. Both methods were fully validated and applied to real samples. The main advantage of the developed methods is the separation of the drug combination in a short time with high efficiency and without any time-consuming steps.

Detection and quantification of Covid-19 antiviral drugs in biological fluids and tissues

“…One of the major challenges when developing a method for the quantification of pharmaceutical compounds in bio-fluids and tissues is the practical issues related to acquiring large volumes of sample for the preparation of calibration standards and quality controls [ 42 ]. Although the calibration standards should be prepared in the same biological matrix as the one that is going to be analysed, by adding an internal standard as well [ 28 ], many methods for antiviral drugs’ quantification have been validated in aqueous solutions [ 20 , [43] , [44] , [45] , [46] , [47] , [48] , [49] ].…”

“…Moreover, in some cases, acetonitrile [ 45 , 63 , 64 ] acidified with 0.01% HCl [ 65 ] or water adjusted with 8% (v/v) trichloroacetic acid (TCA) [ 66 ] and methanol alone [ 67 ] have been used too. In addition to plasma samples, PP has been applied to other matrices, such as seminal plasma [ 42 ], cell lysates [ 68 ], cerebrospinal fluid [ 67 ], mice vaginal lavage [ 69 ], saliva [ 64 ], urine [ 46 ] and breast milk [ 70 ]. Yamada et al [ 64 ] used protein precipitation for extracting darunavir, tenofovir and other antiviral drugs from saliva samples, through a three-step protocol, including the addition of ACN, drying of the supernatant and its regeneration with the mobile phase.…”

“…Yamada et al [ 64 ] used protein precipitation for extracting darunavir, tenofovir and other antiviral drugs from saliva samples, through a three-step protocol, including the addition of ACN, drying of the supernatant and its regeneration with the mobile phase. Moreover, ACN was used also for the extraction from urine samples assisted by sonication in an ultrasonic bath, in a method developed by Gumustas et al [ 46 ] for the quantification of emtricitabine, rilpivirine and tenofovir. For mice vaginal lavage, the addition of ACN is used to precipitate mucins and dilute salts [ 69 ], while in the case of breast milk, PP is conducted after lipid removal through hexane treatment [ 70 ].…”

“…Some methods based on potentiometry [ 44 ], spectrofluorimetry [ 45 ] or matrix-assisted laser desorption/ionization (MALDI) [ [106] , [107] , [108] ] were employed. However, as expected, the most widely used methods were based on hyphenated techniques, typically liquid chromatography but also capillary electrophoresis [ 46 ] (CE), coupled to UV-Vis detection or electrospray ionization (ESI) with mass spectrometry (MS). Indeed, besides their higher sensitivity, they offer the possibility to carry out multiresidue analyzes, i.e.…”

“…However, the main disadvantage of CE is its low concentration sensitivity. In this regard, Gumustas et al [ 46 ] compared LC-UV and CE-UV methods for the simultaneous determination of emtricitabine, rilpivirine and tenofovir. LC-UV sensitivity, calculated as the limit of detection (LOD) and limit of quantification (LOQ), was better than CE.…”