Method Development and Validation for the Simultaneous Estimation of Aflatoxins in Botanical Origin Samples Using High-Performance Thin-Layer Chromatography

Background and Objectives: Aflatoxins are toxic secondary metabolites produced by certain Aspergillus species and are frequently found in contaminated food and herbal products. Their presence poses serious health risks, necessitating effective analytical methods for detection and control. In this study, a simple, sensitive, and cost-effective High-Performance Thin-Layer Chromatography (HPTLC) method was developed and validated for the simultaneous estimation of aflatoxins B₁, B₂, G₁, and G₂ in a polyherbal formulation, Haridrakhand. Materials and Methods: The method employed silica gel 60 F₂₅₄ plates and a mobile phase of chloroform: acetone: water (140:20:0.3, v/v/v), with detection at 366 nm under UV light. The method was validated as per ICH Q2(R2) guidelines for linearity, accuracy, precision, specificity, robustness, Limit of Detection (LOD), and Limit of Quantification (LOQ). Results: Calibration curves showed excellent linearity with Correlation Coefficients (R²) greater than 0.996. Recovery studies at 80%, 100%, and 120% levels yielded recovery rates within 98-102%. The LOD and LOQ values confirmed the method’s sensitivity, and all validation parameters met the required acceptance criteria. Conclusion: This HPTLC method is suitable for routine screening and quality control of aflatoxins in herbal formulations.