Iranian Journal of Basic Medical Sciences

Iranian Journal of Basic Medical Sciences

Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples

Document Type : Original Article

Authors
Amir Hooshang Mohammadpour 1
Mohammad Ramezani 2
Nasim Tavakoli Anaraki 3
Bizhan Malaekeh-Nikouei 4
Sara Amel Farzad2 2
Hossein Hosseinzadeh 3
1 Pharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
2 2Pharmaceutical Research Centre, Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
3 1Pharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
4 3Nanotechnology Research Centre, Department of Pharmaceutics, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
10.22038/ijbms.2013.248
Abstract
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
Keywords
Crocetin
Crocus sativus
Direct precipitation
High Performance Liquid Chromatography
Human serum samples
Saffron
Solid phase extraction
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Iranian Journal of Basic Medical Sciences
Volume 16, Issue 1 - Serial Number 1
SAFFRON SPECIAL ISSUE
January 2013
Pages 47-56