Overview
A rapid, robust, sensitive and specific LC-MS/MS assay using the AB SCIEX QTRAP® 6500 system has been developed for the simultaneous detection of all major forms of vitamin B complex. The method detects all currently used forms of vitamin B6 and vitamin B3 in infant formula and includes vitamin B12. The sample preparation allows the same extract to be used for Vitamin C detection and the LC-MS/MS conditions have been tuned so that the response for each vitamin is linear over the various required detection ranges.
Introduction
Vitamin B is a group of water-soluble vitamins that play important roles in cell metabolism. The absence of individual B vitamins in a diet can lead to several conditions including depression and high blood pressure so they are often added to foods, especially infant formula. Human daily nutritional recommendations for the members of the vitamin B complex vary considerably, for example from 6 μg of vitamin B12 to 20 mg of vitamin B3 (Table 1). The US Food and Drug Administration regulates food labels in the United States and food labeling is required for most prepared foods such as breads, cereals, canned foods, snacks, drinks, and especially for infant formula, which is highly regulated.1
Table 1. Daily required values (DV) of different B vitamins for a human adult as obtained from the FDA1Analysis of food samples can be challenging, as the matrices are complex and sensitive methods typically require highly selective sample clean up procedures. Vitamin B is a complex mixture of highly polar compounds (Figure 1) whose pKa range from 0.5 to 10.2, making their analysis challenging. Several methodologies exist to look at these analytes in separate classes, but relatively few analytical methods exist that examine the vitamin B complex as a whole, with high throughput capabilities, minimal sample preparation, and which have high sensitivity and specificity.Here we present new data acquired by Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) from a quantitative method that contains vitamins B1, B2, B3 (two forms), B5, B6 (three forms), B7, B12 and folic acid (vitamin B9).. Instrument detection levels for these vitamins using this method have been shown to be less than a ng/mL for the neat compounds using positive mode Electrospray Ionization (ESI) and the Scheduled MRM™ algorithm. The required limits of detection vary greatly between each vitamin, but all the B vitamins can be detected in infant formula, by adjusting the MS/MS voltages accordingly, even with detection limits having a 10,000-fold range. The LC-MS/MS method utilizes a small particle size polar endcapped reversed phase (RP) column and an 11 min gradient. In this new iteration of the method very little sample preparation has been used to enable a high throughput suitable for routine food testing.
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