Simultaneous analysis of antiretroviral drugs abacavir and tenofovir in humanhair by liquid chromatography tandem mass spectrometry

A sensitive and reproducible method has been developed and validated for the simultaneous quantification of the key antiretroviral drugs abacavir and tenofovir in hair using LC-MS/MS. The method was validated according to the US Food and Drug Administration (FDA) guidelines for the parameters: specificity, stability, limits of detection (LOD), limits of quantification (LOQ), linearity, accuracy, precision and recovery. Hair samples (50mg) were decontaminated and subjected to methanolic extraction, where 1ml methanol was added along with the internal standard abacavir-d4 at a final concentration of 0.15ng/mg hair. After 16h, the drugs were recovered by liquid-liquid extraction using ammonium acetate buffer and a mixture of methyl tert-butyl ether:ethyl acetate (1:1). The samples were reconstituted with 200μl acetonitrile:water (1:1) prior to injection for LC-MS/MS. The LOD and LOQ values were 0.06 and 0.12ng/mg (drug/hair) for both drugs. Calibration curves were linear in the concentration range of 0.12-4.0ng/mg of drug/hair with regression coefficient (r(2)) value of 0.999 for both drugs. The data for accuracy, precision and recovery were within the FDA limits. The concentrations of the drugs in the hair samples ranged from 0.12ng/mg to 4.48ng/mg and 0.32ng/mg to 1.67ng/mg for tenofovir and abacavir, respectively. This is the first full report of a method for the simultaneous determination of these two key antiretroviral drugs in hair. The newly developed method is useful for future routine analysis of tenofovir and abacavir in human hair and could be used in therapeutic drug monitoring and adherence to medicines studies, which would be helpful in decision making regarding treatment change in combination anti-retroviral therapies.