title: A validated solid-phase extraction HPLC method for the simultaneous determination of the citrus flavanone aglycones hesperetin and naringenin in urine.
abstract: A simple, specific, precise, accurate, and robust HPLC assay for the simultaneous analysis of hesperetin and naringenin in human urine was developed and validated. Urine samples were incubated with beta-glucuronidase/sulphatase and the analytes were isolated by solid-phase extraction using C18 cartridges and separated on a C8 reversed phase column using a mixture of methanol/water/acetic acid (40:58:2, v/v/v) at 45 degrees C. The method was found to be linear in the 50-1200 ng/ml concentration range for both hesperetin and naringenin (r > 0.999). The accuracy of the method was greater than 94.8%, while the intra- and inter-day precision for hesperetin was better than 4.9 and 8.2%, respectively and for naringenin was better than 5.3 and 7.8%, respectively. Recovery for hesperetin, naringenin and internal standard 7-ethoxycoumarin was greater than 70.9%. The method has been applied for the determination of hesperetin and naringenin in urine samples obtained from a male volunteer following a single 300 mg oral dose of each of the corresponding flavanone glycosides hesperidin and naringin. The intra- and inter-day reproducibility through enzyme hydrolysis was less than 3.9% for both total (free + conjugated) hesperetin and naringenin. Stability studies showed urine quality control samples to be stable for both hesperetin and naringenin through three freeze-thaw cycles and at room temperature for 24 h (error < or = 3.6%).
authors.affiliation: Department of Pharmacy, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece.
authors.forename: Feras Imad
authors.initials: FI
authors.lastname: Kanaze
authors.forename: Eugene
authors.initials: E
authors.lastname: Kokkalou
authors.forename: Manolis
authors.initials: M
authors.lastname: Georgarakis
authors.forename: Ioannis
authors.initials: I
authors.lastname: Niopas
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002957:Citrus / Q000737:chemistry*; D044950:Flavanones / Q000302:isolation & purification / Q000652:urine*; D006569:Hesperidin / Q000302:isolation & purification / Q000652:urine; D006801:Humans; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling
chemical_list: D044950:Flavanones; D006569:Hesperidin; C005274:naringin