Development and validation of reversed phase-high-performance liquid chromatography method for simultaneous quantification of temozolomide and alpha-lipoic acid in bulk and lipidic nanovesicles.

A new and precise single-isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous analysis of Temozolomide (TMZ) and Alpha-lipoic acid (ALA) in bulk and lipidic nanovesicles. The chromatographic separation of both drugs was carried out on an Eclipse Plus (C18) column (250 ×4.6 mm, 5 μm). An acetonitrile: acetate buffer (pH 4.5) mixture (60:40% v/v) was used as the mobile phase at a flow rate of 0.7 mL/min in isocratic mode. The eluted compounds were detected with a diode array detector at 328 nm (TMZ) and 332 nm (ALA). The retention times for TMZ and ALA were recorded as 3.63 min and 5.59 min, respectively. The developed method was subsequently validated for various parameters in accordance with ICH Q2(R1). The method showed linearity within the concentration range of 2-60 μg/mL for TMZ (r2 = 0.9998) and 15-750 μg/mL for ALA (r2 = 0.9999). The % recovery for both drugs was within acceptable limits with minimal variability. Moreover, the limit of detection and limit of quantitation were 1.0 µg/mL and 2.9 µg/mL for TMZ, and 5.0 µg/mL and 15.1 µg/mL for ALA, respectively. The validated method was found to be adequately sensitive and specific in simultaneously quantifying TMZ and ALA entrapped in liposomes [73.01 ± 3.96% entrapment efficiency for TMZ and 79.63 ± 0.24% for ALA (average ± SD, n = 3)]. Therefore, the proposed RP-HPLC method was found to be quick, precise, and specific for the simultaneous analysis of TMZ and ALA in bulk and in lipidic nanovesicles.