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

HUNZICKER GA; HEIN GJ; HERNANDEZ SR; ALTAMIRANO JC

ANALYTICAL CHEMISTRY RESEARCH

ELSEVIER

Lugar: Amsterdam; Año: 2015 vol. 6 p. 1 - 8

2214-1812

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 physicchemical variables on analytical response of each ARV, including pH, surfactant concentration, equilibrationtime 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 mL aliquot of human plasma was diluted with 2 mL deionized water in a 10 mL centrifuge tube. A 500 mL 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 mL ACN were added to the remaining surfactant rich phase and centrifuged in order to precipitate proteins and separate them. A 150 mL aliquot of the supernatant was transferred to 2 mL vial and furtherdiluted with 400 mL deionized water. A 30 mL 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 mL1, 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 theconcentration range of 43-6816, 125-4992, 81-3248 and 49e7904 ng mL1, 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.