Related Experiment Video
Updated: May 11, 2026
HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis
Published on: November 11, 2022
Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum
Amir Hooshang Mohammadpour1, Mohammad Ramezani, Nasim Tavakoli Anaraki
1Pharmaceutical Research Center, Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran.
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.
