Development and validation of HPLC-MS/MS method for simultaneous analysis of B vitamins present naturally or after fortification in fruit juices

Abstract

In this study, a multivitamin analysis method with a run time of 20 min was developed for nine major B vitamins in fruit juices using HPLC coupled with a triple quadrupole mass spectrometer. A reverse phase column with ammonium formate buffers in methanol and water as mobile phases under gradient elution mode were used to accomplish chromatographic separation. A simple dilute and shoot method was used for sample preparation. A calibration and accuracy study for method validation was conducted. The accuracy profiles were created using measurement uncertainty estimated from the validation data according to the ISO/DTS 21748. The proportional bias due to the matrix effect were ranging between 80% and 115% in the matrices investigated in this study. The measurement uncertainty and the corresponding coefficient of variation were within the acceptable limits defined in the literature and international regulatory standards i.e. EU regulation. The limits of quantitation were low enough to prove fitness for purpose for all vitamins except cyanocobalamin. The study concludes with a method with the potential to be used in quality control laboratories for high throughput routine vitamin analysis.