A rapid, sensitive and selective HPLC-UV method was developed for quantification of azadirachtin (1) and 3-tigloylazadirachtol (2) in Neem kernels and oil, with a good linearity over a range of 5.0–60.0 and 2.5–50.0 μg mL−1 respectively, with r2 > 0.999 for all curves. The limits of detection (LOD) for both limonoids were 25 ng mL−1 for kernels and 0.10 μg mL−1 for oil, and quantification (LOQ) for 1 and 2 were 1.5 μg mL−1 for both matrices. The quantification method was applied to Neem seeds and oil from different areas of Brazil. The low quality of Brazilian oil (concentrations of 1 = 228.5 to 630 mg kg−1 and 2 = 117.1 to 582.9 mg kg−1) could be attributed to mechanical extraction, since the content of azadirachtin and 3-tigloylazadirachtol in seeds was (1 = 2048.8 to 5117.1 mg kg−1, 2 = 224.7 to 1116.5 mg kg−1) similar to values obtained from Indian seeds (1 = 556.9 to 3030.8 mg kg−1, 2 = 43.1 to 590.6 mg kg−1).
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