AQbD-based development and validation of an HPLC-PDA method for simultaneous determination of sibutramine, desmethylsibutramine, and didesmethylsibutramine in herbal weight-loss supplements.

Illegal adulteration of herbal weight-loss supplements with sibutramine (SIB) and its structurally related analogues remains a significant public health concern, highlighting the need for reliable, accessible, and cost-effective analytical methods for routine regulatory surveillance. In this study, a high-performance liquid chromatography method coupled with photodiode-array detection was developed, chemometrically optimized using Box-Behnken response surface methodology, and validated for the simultaneous determination of SIB, desmethylsibutramine (DMS), and didesmethylsibutramine (DDMS). The effects of mobile-phase pH, flow rate, and column temperature on chromatographic resolution were systematically evaluated to establish optimum separation conditions. Under the optimized conditions, comprising an InertSustain C18 column with gradient elution using acetonitrile and phosphate buffer (pH 3.9), a flow rate of 1.0 mL min-1, a column temperature of 42.5 °C, and UV detection at 223 nm, complete baseline separation of the three analytes was achieved with chromatographic resolutions of 2.269 (DDMS/DMS) and 4.865 (DMS/SIB). The optimized chromatographic system satisfied all system suitability criteria, exhibiting retention-time RSDs below 0.05%, peak-area RSDs below 1.0%, theoretical plate numbers exceeding 37 000, and tailing factors below 1.45. The method was validated in accordance with ICH Q2(R2) and AOAC guidelines, demonstrating satisfactory linearity (R 2 > 0.99), precision, accuracy, and adequate sensitivity for routine analysis. Application of the validated method to commercial herbal weight-loss supplements successfully identified products adulterated with sibutramine and desmethylsibutramine, and all positive findings were independently confirmed by high-resolution mass spectrometry. The proposed analytical workflow provides a robust, chemometrically optimized, and experimentally validated strategy for the routine regulatory screening and quality control of illegal pharmaceutical adulterants in herbal weight-loss supplements.