Method validation for determination of piperine in black pepper by high performance liquid chromatography

Abstract

The determination of piperine in black pepper samples using High performance liquid chromatography (HPLC) was successfully performed and validated. The method demonstrated specificity and good linearity in the concentration range of 1 - 6 mg/L with a coefficient of determination (R²) of 0.9998. Accuracy was evaluated through %recovery studies and obtaining results between 100.25 - 101.65%. The precision expressed as a relative standard deviation (%RSD), showed good repeatability for both intra-day and inter-day analyses, with %RSD values less than 5%. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.99 and 1.09 mg/L, respectively. The linearity, percent recovery, precision, LOD and LOQ values were acceptable and complied with AOAC guidelines for single laboratory, validation of chemical methods for dietary supplements and botanicals. Measurement uncertainty was calculated as 3.49% at a 95% confidence interval (k=2). All results presented that the method was reliable and suitable for the determination of piperine in black pepper sample. Subsequently, 28 black pepper samples from various sources were analyzed using a validated method. The results showed that piperine content ranged from 3.15% to 6.41% w/w, with 20 samples (71.4%) meeting the Thai Industrial Standard (TIS. 297-2556) requirement of piperine content ≥4%. The results demonstrate that the proposed HPLC technique for analyzing piperine content in black pepper samples is effective and can be applied to samples from various sources. Furthermore, the data from this study can serve as a guideline for the quality control of Thai herbal raw materials and extracts, ensuring compliance with standard requirements and enhancing global competitiveness.