Development of a multiple reaction monitoring liquid chromatography tandem mass spectrometry method for the simultaneous determination of five antiretroviral drugs in human blood plasma-a contrast of extraction efficiencies using selected sample preparation techniques

Abstract

A multiple reaction monitoring liquid chromatography - tandem mass spectrometry (MRM LC-MS/MS) method for the simultaneous determination of antiretroviral drugs (Emtricitabine, Tenofovir, Efavirenz, Lopinavir and Retonavir) in sterilized human blood plasma was developed for therapeutic drug monitoring (TDM) purposes and validated using United States food and drug administration (US FDA) guidelines. A comparison between seven sample extraction techniques was made. These are QuEChERS (a portmanteau for Quick, Easy, Cheap, Efficient and Rugged), solid phase extraction (SPE), liquid-liquid extraction (LLE), protein precipitation (PPT) and mixed modes i.e. QuEChERS- PPT (Q-PPT), QuEChERS-LLE (Q-LLE) and LLE-PPT. Data acquisition was done in MS fullscan and MRM and the two compared. Chromatographic and mass spectrometric parameters were optimised using peak area’s as variables. The analytical performance characteristics that were investigated for method validation were instrument detection limits (IDLs), method detection limits (MDLs), % mean recoveries, precision (% Relative standard deviation) and accuracy. Optimised LC-MS/MS parameters were employed for this purpose. The percent mean recoveries were between 68.8 – 85.6 % for single modes and 52.4 – 70.5 % for mixed mode techniques. Precision of all the techniques investigated was within acceptable range of < 15 % at all concentration levels for all analytes. Accuracy was calculated as a percentage of deviation of the mean value to the true value and the values were between 5.87 – 65.94 % for single mode techniques and between 21.73 – 51.59 % for mixed mode techniques. SPE proved to be more superior than the other techniques as it recorded the highest percentage recoveries and it satisfied all US FDA guidelines. MDLs for the other six sample preparation techniques fell below the clinically relevant therapeutic range (3 – 8 ppm) therefore all techniques can be employed for routine TDM studies.