Standardization of Crude Extract of Neem Seed Kernels (Azadirachta indica A. Juss) and Commercial Neem Based Formulations Using HPTLC and Extended Length Packed-Columns SFC Method

Two chromatographic techniques are described for the separation and quantitative determination of azadirachtin A and B, salannin, and nimbin present in the crude extract of neem seed kernels and commercial neem based formulations. The high performance thin-layer chromatography (HPTLC) separation of markers was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using ethylacetate-benzene (7.0: 3.0, v/v) as mobile phase. The other technique was based on extended length packed column supercritical fluid chromatographic (PC-SFC) separation of the markers using Cyano column (250 mm × 4.6 mm I.D, S-5.0 μ) and Kromasil 100 NH₂ column (250 mm × 4.6 mm I.D, 5.0 μ) connected in series. The detection was carried out using photodiode array detector at 338 K using methanol-modified carbon dioxide (10%) as the mobile phase at flow rate of 2.0 mL min⁻¹. The current study assesses the effect of extending column length during PC-SFC experiment to obtain maximum resolution between a number of unknown components and known markers present in neem seed extracts. Both the chromatographic methods were validated in terms of precision, robustness, recovery, limits of detection and quantitation. The analysis of variance (ANOVA) and Student’s t-test were applied to correlate the results of quantitative determination of markers by means of HPTLC and PC-SFC method.