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.

Methyl jasmonate alleviates drought stress in young sugar beet (Beta vulgaris L.) plants

“…Betaine concentration was determined by modification of the method of Hefni, McEntyre, Lever, and Slow (). Deionized water (1.5 ml) was added to 25 mg freeze‐dried tissue, and the mixture was sonicated for 15 min at room temperature and centrifuged for 15 min at 14,000 g .…”

New approaches for extraction and determination of betaine from Beta vulgaris samples by hydrophilic interaction liquid chromatography-tandem mass spectrometry

“…The overall ASE/SPE procedure takes advantages of both the high-pressure conditions of ASE, which in turn provide faster extraction times than traditional extraction apparatus [ 9 ] (12 min against 3 h), and of the higher safety conditions of SPE purification which is based on water and methanol, rather than on more toxic organic solvents (e.g. dichloromethane [ 10 ]).…”

“…It is interesting to highlight that B. vulgaris species have higher concentrations of betaine if compared with other entities that belong to the same family. As an example, betaine concentration is typically about 0.2–0.3% (2–3 mg/g) also in sugar beets [ 23 ], while in fresh spinach, which belongs to the same family, betaine is present at concentration 5 to 10 folds lower [ 10 ].…”

“…Methanol, dichloromethane and chloroform, sometimes partially mixed with water, were used as solvents for cold extraction [ 7 , 8 ], while methanolic KOH was used together with a boiling Goldfisch apparatus [ 9 ]. High-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS) detection [ 7 ], UV (using pre- or post-derivatization) [ 10 ] and light scattering detection (LSD) [ 8 ] were used for the analytical determination. Ion chromatography in the non-suppressed conductivity mode was also employed for analysis [ 11 ].…”

Betaine in Cereal Grains and Grain-Based Products

“…Slow et al [ 3 ] have extracted betaine from various foods by using water and dichloromethane, and by derivatization of betaine with 2-naphthacyl trifluoromethanesulphonate. Hefni et al [ 17 ] developed a simple HPLC-UV method in several different food matrices such as spinach, whole grain wheat flour, wheat, and sugar beet, with the help of derivatization on the cation exchange column. More recently, the same group has used the same method to analyze 14 cereal samples, representing different genera and cultivars [ 18 ].…”