development and validation of hplc method for determination of crocetin, a constituent of saffron, in human serum samples

objective(s):the present study reports the development and validation of a sensitive and rapid extraction method beside high performance liquid chromatographic method for the determination of crocetin in human serum. materials and methods:the hplc method was carried out by using a c18 reversed-phase column and a mobile phase composed of methanol/water/acetic acid (85:14.5:0.5 v/v/v) at the flow rate of 0.8 ml/min. the uv detector was set at 423 nm and 13-cis retinoic acid was used as the internal standard. serum samples were pretreated with solid-phase extraction using bond elut c18 (200mg) cartridges or with direct precipitation using acetonitrile. results:the calibration curves were linear over the range of 0.05-1.25 µg/ml for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction. the mean recoveries of crocetin over a concentration range of 0.05-5 µg/ml serum for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction were above 70 % and 60 %, respectively. the intraday coefficients of variation were 0.37- 2.6% for direct precipitation method and 0.64 - 5.43% for solid-phase extraction. the inter day coefficients of variation were 1.69 – 6.03% for direct precipitation method and 5.13-12.74% for solid-phase extraction, respectively. the lower limit of quantification for crocetin was 0.05 µg/ml for direct precipitation method and 0.5 µg/ml for solid-phase extraction. conclusion: the validated direct precipitation method for hplc satisfied all of the criteria that were necessary for a bioanalytical method and could reliably quantitate crocetin in human serum for future clinical pharmacokinetic study