Simultaneous Determination of Total Vitamins B1, B2, B3, and B6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS-A Multi-Laboratory Testing Study Final Action: AOAC Method 2015.14.

A multi-laboratory study was completed for AOAC First Action Method 2015.14. Ten laboratories from eight countries participated. Each laboratory analyzed (in duplicate) a placebo and 14 fortified nutritionals. Product matrices analyzed included milk, soy, partially hydrolyzed milk, partially hydrolyzed soy, and elemental-based infant formula powders, milk based infant formula ready-to-feed liquids, adult low-fat powders, and adult high fat and high protein ready-to-drink nutritionals. Data was then compared to standard method performance requirements (SMPR). Samples were prepared by enzymatic digestion with papain, α-amylase, and phosphatase to hydrolyze protein and complex carbohydrate and to free phosphorylated vitamin forms respectively. Stable-isotope labeled internal standards were incorporated into the sample preparation to correct for variability in both the sample preparation and instrument response. Prepared samples and working standard solutions were injected onto an ultra-high-pressure liquid chromatograph, interfaced to a triple-quadrupole mass spectrometer (MS/MS). Reverse phase gradient chromatography, using mobile phases of methanol and 20 mM ammonium formate in water on a C-18 column, were used for the analysis. The MS/MS was configured to monitor precursor-fragment ion pairs for each analyte and internal standard. Vitamins B1 (thiamine), B2 (riboflavin), B3 (niacin), and B6 (pyridoxine) were quantified by least squares regression using the response ratio of the analyte to its internal standard. Total vitamins B1, B2, B3, and B6 had average repeatability of 2.3%, 3.9%, 2.7%, and 2.2% RSD, and reproducibility of 8.2%, 6.9%, 6.7%, and 5.8% RSD, respectively. Repeatability and reproducibility SMPR were met for 53/56 and 50/56 fortified-product/analyte pairs analyzed, respectively.