High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol comp onents of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside a nd isoquercitrin, in rat plasma. Following extraction from the plasma sampl es with ethyl acetate-methanol (2:1, v/v), these four compounds were succes sfully separated using a C-18 column with a gradient elution of 5 and 25% a cetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm f or epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 mug) was used as the internal standard and was detected at 278 nm. Th e method is linear over the studied range of 0.16-40, 0.63-160, 0.13-32 and 0.13-30 mug/ml for chlorogenic acid, epicatechin, hyperoside and isoquerci trin, respectively. The correlation coefficient for each analyte was greate r than 0.995. The intra-day and inter-day precision of the analysis was bet ter than 4 and 7%, respectively. The extraction recoveries at low to high c oncentration were greater than 85% for both epicatechin and chlorogenic aci d, and greater than 94% for both hyperoside and isoquercitrin. The detectio n limits were 0.04, 0.20, 0.03 and 0.03 mug/ml for chlorogenic acid, epicat echin, hyperoside and isoquercitrin. The developed method was used to analy ze the plasma concentrations of the four analytes after the intravenous adm inistration of hawthorn polyphenol extract to rats. (C) 2001 Elsevier Scien ce BV All rights reserved.