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 C۱۸ reversed-phase column and a mobile phase composed of methanol/water/acetic acid (۸۵:۱۴.۵:۰.۵ v/v/v) at the flow rate of ۰.۸ ml/min. The UV detector was set at ۴۲۳ nm and ۱۳-cis retinoic acid was used as the internal standard. Serum samples were pretreated with solid-phase extraction using Bond Elut C۱۸ (۲۰۰mg) cartridges or with direct precipitation using acetonitrile. Results:The calibration curves were linear over the range of ۰.۰۵-۱.۲۵ µg/ml for direct precipitation method and ۰.۵-۵ µg/ml for solid-phase extraction. The mean recoveries of crocetin over a concentration range of ۰.۰۵-۵ µg/ml serum for direct precipitation method and ۰.۵-۵ µg/ml for solid-phase extraction were above ۷۰ % and ۶۰ %, respectively. The intraday coefficients of variation were ۰.۳۷- ۲.۶% for direct precipitation method and ۰.۶۴ - ۵.۴۳% for solid-phase extraction. The inter day coefficients of variation were ۱.۶۹ – ۶.۰۳% for direct precipitation method and ۵.۱۳-۱۲.۷۴% for solid-phase extraction, respectively. The lower limit of quantification for crocetin was ۰.۰۵ µg/ml for direct precipitation method and ۰.۵ 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

Keywords:

Crocetin , Crocus sativus , Direct precipitation , High Performance Liquid Chromatography , Human serum samples , Saffron , Solid phase extraction