Development and experimental application of an HPLC procedure for the determination of capsaicin and dihydrocapsaicin in serum samples from human subjects

Abstract

Total capsaicins are extracted from 2 mL aliquots of serum or plasma using methyl-isobutyl ketone, evaporation of the extract to dryness and reconstitution with 200 μL of acetonitrile. The HPLC mobile phase is 40:60 water:acetonitrile. The absorbance of the eluent is monitored at 205 nm. Standardisation uses a known mixture of pure capsaicin and dihydrocapsaicin. Accuracies are 98.9 and 100.6 % for capsaicin and dihydrocapsaicin respectively. Inter batch reproducibility for both is 15 %. The limits of detection are 2.6 and 3.8 ng/mL for capsaicin and dihydrocapsaicin respectively. Analyses of sera obtained previously from human subjects who had eaten chilli containing meals showed that in those that absorbed capsaicins (N = 30) then the median, mean and SD of their serum capsaicin were: 13.4, 18.9 and 16.3 ng/mL. The corresponding data for those sera (N = 13) that had measurable levels of dihydrocapsaicin were: 6.9, 7.5 and 3.6 ng/mL. This procedure is suitable for use in prospective studies of the metabolism of orally ingested chilli.

Keywords: Capsaicin; Dihydrocapsaicin; HPLC; Post oral dose; Serum levels.

Figures

Fig. 1

Typical chromatogram obtained for the mixed standard (capsaicin = 200 ng/mL, dihydrocapsaicin = 250 ng/mL) when extracted with MIBK and run on the Agilent 1100 instrument with 60:40 acetonitrile:water as the eluant

Fig. 2

Typical chromatogram obtained for a post chilli dose serum sample when extracted with MIBK and run on the Agilent 1100 instrument with 60:40 acetonitrile:water as the eluent

Fig. 3

Box and whisker plots and parametric statistics of the results obtained from the serum samples collected post ingestion of a meal containing 20 g of chilli sauce