2015.14 Vitamin B (July 2018)

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

FOR ERP USE ONLY DO NOT DISTRIBUTE

Appendix A:

Method

Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 by Enzymatic Digestion and LC-MS/MS. Louis M. Salvati, Sean C. McClure, and Nicholas A. Cellar. Abbott Nutrition, 3300 Stelzer Rd. Columbus, OH 43219

, and B 6

in Infant Formula and Related Nutritionals

Introduction AOAC SPIFAN released a call for methods for total vitamins B 1 , B 2 , B 3 pyrophosphate, and thiamine triphosphate in the SMPR. 1 Total B 2 phosphate, and flavin adenine dinucleotide. 2 Total B 3 nutritionals. Total vitamin B 1

, and B 6

in infant formula and related

is described as the sum of thiamine, thiamine monophosphate, thiamine

is defined as riboflavin, riboflavin-5’-

is defined as the sum of nicotinic acid and

nicotinamide. 3 Finally, total B 6 includes five forms: pyridoxine, pyridoxal, pyridoxal-5’-phosphate, pyridoxamine, and pyridoxamine-5’-phosphate. 4 A single method for all four total vitamins was developed to meet the need.

The combined method was developed to measure thiamine, riboflavin, pyridoxamine, pyridoxal, pyridoxamine, nicotinic acid, and nicotinamide directly. Separation was achieved with 20 mM ammonium formate mobile phase without ion pairing agent. Improved retention of thiamine has been previously demonstrated by increasing the mobile phase pH. 5 In fact, there is a striking improvement in water soluble vitamin retention under reverse phase conditions at moderate pH (5 -7). 6 Despite this improvement in retention, elution of phosphorylated compounds is notoriously difficult. 7 The phosphate moiety complexes with Fe 3+ and thus phosphate containing compounds tend to tail considerably with conventional chromatographic equipment due to stainless steel plumbing and column frits. 7 Further, the phosphorylated forms of these vitamins tend to be a small fraction of the free-form thus putting pressure on accuracy of quantitation. Instead of trying to overcome the challenges of direct analysis of the phosphorylated forms, this method includes an overnight, enzymatic digestion with acid phosphatase to hydrolyze the phosphate yielding the free vitamin forms. 5 Papain and α-amylase are also included in the enzymatic cocktail to digest protein and complex carbohydrate, respectively, thus aiding reduction in sample extract complexity. 8 Method performance was demonstrated in 14 SPIFAN II matrixes. Precision averaged 3.9%, and over-spike recovery was generally 95-105%. Both free and phosphorylated forms were over-spiked as part of the recovery experiment. The method was demonstrated to meet both SPIFAN SMPR requirements and Abbott Nutrition requirements.