Development and validation of a UPLC method for quantifying trans-crocin 4 and crocetin from saffron in plasma: A pharmacokinetic study

 

Saffron is the dried stigmas of the flower Crocus sativus L. [Iridaceae]. The main saffron constituents include crocins which are mono- and bis-esters of crocetin with glucose and/or gentiobiose [1]. Several studies indicate that the main carotenoid constituent, trans-crocin-4 (TC4) has shown remarkable activity against Alzheimer's disease [2]. Therefore, a novel UPLC-UV methodology was developed and fully validated, according to FDA and EMA guidelines, for the rapid and accurate quantification of TC4 and crocetin in mice plasma after i.p administration. The separation of the analytes was performed on a C8 Hypersil Gold column with 2.5 min run time, employing the internal standard (IS) methodology. The results show adequate linearity (r² > 0.996) over a wide concentration range [0.01 – 6 µg/mL (n = 10)] with a Lower Limit of Quantification (LLOQ) value of 0.01 and 0.03 µg/mL for crocetin and for TC4 respectively, whereas the aforementioned methodology demonstrated adequate precision, accuracy, sensitivity and selectivity. The method was successfully applied for the determination of crocetin and TC4 in mouse plasma after 50 and 150 mg/kg TC4 i.p administration in mice. In details, 42 samples of the two administered groups (both male and female mice), covering a sampling period of 0 – 240 min were analyzed. The results showed that circulating TC4 levels were found to be statistically different in female and male population. Indicatively, for the female mice [50 mg/kg TC4], the experimentally determined plasma Cmax TC4 levels were 3304 ± 413 ng/mL (T_max= 60 min). Interestingly, crocetin was not detected in any plasma sample, although it has been reported that TC4 quickly hydrolyzes to crocetin after p.o administration [3]. Overall, the developed methodology offers important information about the bioavailability of TC4, as well as the differences in TC4 absorption between males and females.

Acknowledgements: Despoina Papasavva is acknowledged for technical assistance in the animal experiments.

Keywords: UPLC, Validation, trans-crocin 4, crocetin, mice plasma, pharmacokinetics.

References:

[1] Koulakiotis NS, Gikas E, Iatrou G, Lamari FN, Tsarbopoulos A. Quantitation of crocins and picrocrocin in saffron by HPLC: Application to quality control and phytochemical differentiation from other crocus taxa. Planta Med 2015; 81: 606 – 612

[2] Khazdair MR, Boskabady MH, Hosseini M, Rezaee RM, Tsatsakis A. The effects of Crocus sativus (saffron) and its constituents on nervous system: A review. Avicenna J phytomed 2015; 5: 376 – 391

[3] Asai A, Nakano T, Takahashi M, Nagao A. Orally administered crocetin and crocins are absorbed into blood plasma as crocetin and its glucuronide conjugates in mice. J Agric Food Chem 2005; 53: 7302 – 7306