Validation of HPLC-UV Methods for the Quantification of Betaine in Foods by Comparison with LC-MS

Abstract

The importance of dietary betaine is increasingly recognized. The aim of this study was to develop a simple high-performance liquid chromatography with standard ultraviolet spectrometric detection (HPLC-UV) method for betaine (N,N,N-trimethylglycine) determination in foods after derivatization. Two methods were used for betaine derivatization. Thereafter, derivatized betaine was quantified using HPLC-UV, and the results were compared with liquid chromatography mass spectrometry (LC-MS). The established derivatizing agent 2′-naphthacyl triflate and a novel derivatizing agent 2-bromo-2′-acetonaphthone produced the same cationic derivative when they react with betaine. The calibration curves were linear up to 1000 μmol/L (R ² = 0.9974 for 2′-naphthacyl triflate and 0.9995 for 2-bromo-2′-acetonaphthone). The limit of detection was 1 μmol/L for both methods (2′-naphthacyl triflate and 2-bromo-2′-acetonaphthone), confirming sufficient sensitivity for betaine quantification in foods. The average recovery from different food matrices (wheat flour and spinach) (n = 12) was 99 ± 9 %, 95 ± 10 %, and 101 ± 8 % for LC-MS, 2′-naphthacyl triflate, and 2-bromo-2′-acetonaphthone, respectively. Inter- and intra-assay coefficients of variation (CVs) in the control samples (whole wheat flour) were below 10 %. Quantitative results for foods analyzed using 2′-naphthacyl triflate and 2-bromo-2′-acetonaphthone were comparable to LC-MS (R ² = 0.992 and 0.990), respectively. The highest betaine content (~160 mg/100 g) was found in spinach followed by faba bean, wheat flour, and beetroot. These methods can be widely used for betaine quantification because of the simplicity of the derivatization procedures, and the commercial availability of the derivatizing reagent (2-bromo-2′-acetonaphthone) or through the relatively easy synthesis of 2-naphthacyl triflate.