Development and validation of a RP-UHPLC-ESI-MS/MS method for the chiral separation and determination of flavanone, naringenin and hesperetin enantiomers

• Simultaneous chiral separation of flavanone, naringenin and hesperetin enantiomers.
• Comparison of acid and enzymatic hydrolysis of flavanone glycosides.
• Application to the real samples: liquorice, sweet, hot and chilli pepper.

A quick and sensitive RP-UHPLC-ESI-MS/MS method for the separation of flavanone, naringenin and hesperetin enantiomers was developed. The separation of analytes was performed using a Chiralpak AD-3R column, and methanol was used as the mobile phase. Detection was carried out using a triple quadrupole tandem mass spectrometer with an electrospray ionisation source. Positive ionisation and multiple reaction monitoring (MRM) were used. The developed method showed satisfactory linearity with determination coefficients greater than 0.996 in the concentration ranges of 2.5–100.0 ng mL−1 for naringenin and flavanone enantiomers and 0.5–100.0 ng mL−1 for hesperetin enantiomers. The limits of quantification varied from 0.1 to 2.0 ng mL−1. The intra-day and inter-day precisions were below 15%, and the accuracy varied from −13.6% to 13.5%. The described method was successfully applied for the chiral separation and determination of flavonoid enantiomers in real samples of spices and herbal root. Due to the occurrence of the natural compounds in the forms of free aglycones and their glycosides, these samples were subjected to hydrolysis in order to obtain free aglycones from the glycosylated forms. Acid and enzymatic hydrolysis techniques were also compared. In the course of this study, the enzymatic hydrolysis technique was selected.

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