Abstract:
ABSTRACT
A reverse phase-high performance liquid chromatographic method was developed and validated
for the simultaneous determination of lamivudine, tenofovir disoproxil fumarate and efavirenz in
lamivudine, tenofovir and efavirenz finished formulation product. The method was developed by
altering various organic solvents such as acetonitrile and methanol, column, detection, flow rate
and temperature. An isocratic elution mode with a mixture of acetonitrile and water in the ratio
of (55:45 % v/v) was selected for the mobile phase with a nucleosil (Macherey Nagel-Germany)
C18 (4.6 mm x 250 mm x 5m) column as stationary phase for simultaneous separation of
lamivudine, tenofovir disoproxil fumarate and efavirenz. The separation was achieved at a flow
rate of 1 mL/min and detection wavelength of 252 nm at room temperature. Further, different
dissolution media were investigated for optimal release of lamivudine, tenofovir disoproxil
fumarate and efavirenz from lamivudine, tenofovir and efavirenz tablets. The optimization of
dissolution medium was preceded by establishment of the sink concentration for efavirenz which
was found at 0.5% sodium dodecyl sulphate in water with a release of more than 75% of each of
the three active pharmaceutical ingredients at 37°C with a paddle method, 75 rpm at 45 min. The
analytical method was validated and the linear range was found in the concentration range of
0.05 to 0.12 mg/mL of lamivudine, tenofovir disoproxil fumarate and efavirenz with regression
coefficient (r2) of 0.9984 which met the acceptance criteria of r2 equal or greater than 0.98. The
% rsd for the intra-day precision were 1.08%, 1.23% and 1.46% for lamivudine, tenofovir
disoproxil fumarate and efavirenz respectively. The % rsd for the inter-day precision were
1.95%, 1.99% and 1.67% for lamivudine, tenofovir disoproxil fumarate and efavirenz
respectively. The test method had an acceptable level of accuracy for the assay of lamivudine,
tenofovir disoproxil fumarate and efavirenz in lamivudine, tenofovir and efavirenz tablets from
50 % to 120 % of test concentration with % rsd less than 2% for all three active pharmaceutical
ingredients. The test solution remained stable when stored at 4°C for 72 hours. The method was
robust as it remained largely unaffected by small variations in temperature and mobile phase. All
of these assessed parameters complied with the acceptance criteria hence indicated the usefulness
of the reverse phase-high performance liquid chromatographic method for determination of assay
and dissolution release testing for finished formulation product which contain lamivudine,
tenofovir disoproxil fumarate and efavirenz active substances.
