Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography

Hesperetin (HT) is a flavanone abundantly found in citrus fruits. It has been reported that HT possesses significant antioxidant, anticancer, anti-inflammatory and analgesic activities. This explains the necessity of developing new methods more powerful and sensitive for analyzing HT in biological fluids. Taking into account the chiral nature of HT, the study of the stereospecific kinetics of in vitro and in vivo metabolism and tissue distribution could be a useful tool for further understanding stereoselective biotransformations in human body. A simple nano-liquid chromatographic method for the determination of the enantiomeric composition of hesperetin in human urine was developed. Chiral separation was achieved using a 100 μm I.D. capillary, packed with phenyl-carbamate-propyl-β-cyclodextrin stationary phase, employing a mobile phase composed by a mixture of triethylammonium acetate buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v) at room temperature. The detection was done by using on-column UV detector at 205 nm. Calibration curves were linear in the studied concentration range from 0.25 to 25 μg/mL (r2 > 0.999). Precision assay was <4.5% and was within 3% at the limit of quantification (0.5 μg/mL). The recovery of 7-ethoxycoumarin (IS), R- and S-hesperetin was greater than 82.48%, utilizing a liquid–liquid extraction procedure. The developed method was successfully applied to the determination of hesperetin enantiomers in urine samples obtained from a male volunteer, after the ingestion of 1 L of a commercial blood orange juice.