Quantitative analysis of piperine and the derivatives in long pepper extract by HPLC using relative molar sensitivity

Abstract

A novel method was developed for quantification of five major piperine derivatives（piperanine, piperine, chavicine, isopiperine, and isochavicine）in a hot water extract of long pepper fruit（LPE）using the relative molar sensitivity（RMS）based on the combination of HPLC/UV and 1H- quantitative NMR（1H-qNMR）. The RMSs of piperanine, chavicine, isopiperine, and isochavicine to piperine of which the absolute purity was determined by 1H-qNMR were calculated to be 0.3693, 1.138, 0.9164, and 1.277, respectively. The total amount of piperine derivatives in LPE was quantified by both 1H-qNMR and HPLC/UV based on the RMS using piperine as a single-reference material （RMS method）. The relative difference in quantitation values of 1H-qNMR and calibration curve method from the RMS method was 2.01％ or less. The relative difference of the total cis-trans piperine isomers content between before and after photoirradiation in piperine solution was quantified to be 2.84％ by the RMS method. In addition, the interlaboratory difference of the RMS method was confirmed in the range of 0.600 to 4.00 μg/g when analysis was performed on piperine derivatives in LPE containing tablets, while the total amount of piperine derivatives in the tablets was quantified at 606 μg/g. Our proposed method is a reliable tool for determining the contents of piperine and the derivatives in LPE and processed foods containing LPE.