Abstract:
A fast and reliable high performance liquid chromatography (HPLC) method with UV diode array detection
for simultaneous quantitative analysis of the anti-retroviral drugs, nevirapine (NVP) and efavirenz
(EFV) and the anti-malarial, lumefantrine (LUM) in human plasma has been developed and validated. The
sample preparation consisted of a plasma protein precipitation with 0.5% acetic acid acetonitrile solution
containing the internal standard halofantrine (HALO) prior the LC-analysis. Chromatographic separation
was carried out on a Acclaim Polar Advantage C16, column (150 mm
×
4.6 mm, particle size, 3 m) using a
gradient of mobile phase made of 0.01% TFA in 0.1 M ammonium acetate (solvent A) and 0.1% TFA in acetonitrile
(solvent B). The separation of NVP, EFV, LUM and HALO was achieved within 17 min at a flow rate
of 1.0 mL/min and detections were initially performed at three wavelengths, 275 nm (NVP), 255 nm (EFV),
and 300 nm (LUM). The method selectivity was demonstrated in six different human plasma batches.
In addition, several concomitant drugs were analyzed under our experimental conditions and none of
them co-eluted with EFV, NVP and LUM. This demonstrated that our method is highly selective. Calibration
graphs plotted with seven concentrations in duplicate for each compound were linear between the
selected ranges with a regression coefficient (R2) greater than 0.998. Absolute extraction recovery for
NVP, EFV and LUM were 99%, 98.6 and 102%, respectively. Inter- and intra-day coefficients of variation
for LUM, EFV and NVP were
≤10%. The lower limits of quantification were 0.125 g/mL for LUM and
0.250 g/mL for both EFV and NVP. Intra- and inter-assay relative standard deviation values were found
to be less than 15% at the concentrations examined (0.125–10.0 g/mL for LUM and 0.250–15.0 g/mL
for both EFV and NVP). The present method was successfully implemented in Tanzania and only one
wavelength (255 nm) was used to measure samples of patients receiving either NVP or EFV in combination
with LUM. The concentration found in human plasma samples for all three compounds were
within the calibration range. This makes our method particularly applicable and useful to resource-limited settings.
