Development and validation of an HPLC-UV method for the simultaneous determination of Efavirenz and Dolutegravir in human plasma

Abstract

Background: Although HIV/AIDS possess a challenge, global efforts have been mounted to address the epidemic. These include the massive role out of anti-retroviral therapy (ART) globally. To date either efavirenz or dolutegravir in combination with a nucleotide and nucleoside constitute the recommended first line HIV drug regimens. Measuring adherence to ART and monitoring the potential toxicities of ARVs including EFV and DTG is a requisite for improvement of ART outcomes. A simultaneous method for the determination of efavirenz and dolutegravir in plasma was developed and validated. Methods: Stock solution of efavirenz was prepared by weighing approximately five milligrams of efavirenz powder and dissolving it in an appropriate amount of HPLC grade methanol to achieve a concentration of 1000mg/L. Stock solution of dolutegravir was prepared by weighing approximately zero point five milligrams of dolutegravir powder and dissolving in appropriate amount of dimethyl sulfoxide to achieve a concentration of 100mg/L. The stock solutions were diluted and spiked in plasma appropriately to make calibrators and quality controls ranging from 0.5-10mg/L and 0.05-1mg/L for efavirenz and dolutegravir respectively. Developing and optimizing the method involved identifying parameters that affect the chromatographic separation including pH, mobile phase, column, flow rate, wavelength, injection volume and temperature. The method was applied on stored samples of participants on efavirenz and dolutegravir ARV based regimens, samples were aliquoted and run in triplicates. Results: Sample processing involved a combination of protein precipitation from plasma, evaporation to dryness and reconstitution followed by high performance liquid chromatography with a C18 column, detection at 260nm, flow rate 0.8ml/min, mobile phase 70% methanol and 30% phosphate buffer and temperature 400c. The method was accurate (85-119%) with a coefficient of variation of less than 15% and a limit of quantification of 0.5mg/L for efavirenz and 0.05mg/L for dolutegravir. All the samples analyzed to test the method had a coefficient of variation of 2.10 to 8.15% for efavirenz and 0.79 to 8.98% for dolutegravir. Conclusion: An HPLC-UV method suitable for the simultaneous determination of efavirenz and dolutegravir in plasma was developed and validated.