LC-MS/MS Assay of Flavanones in Citrus Juices and Beverages

Flavanones are a class of flavonoid compounds, an important phytonutrient found in fruit and vegetables. Considered one of the most important phytonutrient groups, flavonoids may promote beneficial health effects as antioxidants, anti-inflammatory modulators, and as ‘statin-like’ hypocholesterolemic agents, among other reactions. Flavonoids are also useful as markers for identifying the origin of food ingredients. Di Donna and co-authors¹ report a new method using ultra high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) that is faster as well as more sensitive and selective than traditional methods. Beverage and fruit juice samples were centrifuged and degassed prior to chemical derivatization with methoxamine. This step converts the flavanones into stable alkyloxymes for assay. Internal flavanone standards were also derivatized, but using methoxyamine labeled with a stable isotope. Samples and standards were then analysed using UHPLC coupled to a Thermo Scientific TSQ Vantage triple-stage quadrupole mass spectrometer. The total run time per sample was 16 minutes. Calibration curves generated for dilutions of each each flavanone standard were compared with an internal standard of 100µg/L in each assay. Linearity was good, as shown by the correlation coefficient, R², generated for all flavanone standard curves. Calibration was further tested by assaying two known concentrations each of commercial flavanones dissolved in a suitable blank beverage solution. The researchers report accuracy rates between 95% and 111% for the spiked samples, showing that the derivatization process resulted in complete analyte recovery. Other assay parameters tested showed an assay working range between 20 and 250 parts per billion for detecting flavanone concentrations. Validation on real juice and beverage samples confirmed the suitability of the method for assaying flavanones in foodstuffs. Results from orange beverages containing varying concentrations of orange juices showed dose-dependant levels of flavanones, hesperetin and hesperidin, for example. The results were consistent with previous reports and correctly reported flavanone specificity in the fruits tested. The assay correctly detected brutieridin and melitidin only in grapefruit-containing drinks. The authors conclude that their method presents a faster and more sensitive assay and is suitable for assaying flavanones in beverages. Furthermore, they suggest that with the lower detection limits, this method will be useful for detecting adulteration or contamination in foodstuffs. References

1. Di Donna et al. (2013) “Comprehensive assay of flavanones in citrus juices and beverages by UHPLC–ESI-MS/MS and derivatization chemistry,” Food Chemistry 141 (pp. 2328–2333)

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