Cloud point extraction for analysis of antiretrovirals in human plasma by UFLC-ESI-MS/MS

An analytical methodology based on cloud point extraction (CPE) coupled to Ultra-Fast Liquid Chromatography and electrospray tandem mass spectrometry (UFLC-MS/MS) was developed for analysis of Abacavir (ABC), Efavirenz (EFV), Lamivudine (3 TC) and Nelfinavir (NFV) in human plasma. It is the first time that CPE was used for extraction of antiretrovirals (ARV) from plasma. The effects of relevant physic-chemical variables on analytical response of each ARV, including pH, surfactant concentration, equilibration time and temperature, were study and optimized; as well as its coupling to UFLC-ESI-MS/MS. Under optimized conditions, the resulting methodology was as follows: a 500 μL aliquot of human plasma was diluted with 2 mL deionized water in a 10 mL centrifuge tube. A 500 μL aliquot Triton X-114 5% w/v was added and homogenized using a vortex stirrer. The resulting cloudy solution was kept at 65 °C for 20 min for promoting the condensation of surfactant micelles. Then it was centrifuged at 3000 × g for 5 min for separation of the surfactant-rich phase. After discarding the aqueous supernatant, 400 μL ACN were added to the remaining surfactant rich phase and centrifuged in order to precipitate proteins and separate them. A 150 μL aliquot of the supernatant was transferred to 2 mL vial and further diluted with 400 μL deionized water. A 30 μL aliquot of the so-prepared solution was injected and analyzed into the UFLC-MS/MS. The method detection limits for ABC, EFV, 3 TC and NFV under optimized conditions were 31, 77, 57 and 21 ng mL⁻¹, respectively. The RSD% for the studied analytes were <15%, except at the LOQ, which were <19%. Recovery values ranged from 81 to 107%. The proposed methodology was successfully applied for the analysis of ABC, EFV, 3 TC and NFV in human plasma within the concentration range of 43–6816, 125–4992, 81–3248 and 49–7904 ng mL⁻¹, respectively. Under optimized working conditions the proposed analytical methodology meets standard requirements of international guidelines, which makes it suitable for pharmacokinetic studies of the four ARV, as well as for therapeutic monitoring of HIV patients.