6. AOACSPIFANMethods-2018Awards

105

S alvati et al .: J ournal of AOAC I nternational V ol . 99, N o . 3, 2016  1

INFANT FORMULA AND ADULT NUTRITIONALS

Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 , and B 6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS: Single-Laboratory Validation, First Action 2015.14 L ouis M. S alvati , S ean C. M c C lure , T odime M. R eddy , and N icholas A. C ellar 1 Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219

This method provides simultaneous determination of total vitamins B 1 , B 2 , B 3 , and B 6 in infant formula and related nutritionals (adult and infant). The method was given First Action for vitamins B 1 , B 2 , and B 6 , but not B 3 , during the AOAC Annual Meeting in September 2015. The method uses acid phosphatase to dephosphorylate the phosphorylated vitamin forms. It then measures thiamine (vitamin B 1 ); riboflavin (vitamin B 2 ); nicotinamide and nicotinic acid (vitamin B 3 ); and pyridoxine, pyridoxal, and pyridoxamine (vitamin B 6 ) from digested sample extract by liquid chromatography-tandem mass spectrometry. A single-laboratory validation was performed on 14 matrixes provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) to demonstrate method effectiveness. The method met requirements of the AOAC SPIFAN Standard Method Performance Requirement for each of the three vitamins, including average over-spike recovery of 99.6 ± 3.5%, average repeatability of 1.5 ± 0.8% relative standard deviation, and average intermediate precision of 3.9 ± 1.3% relative standard deviation. A OAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) released a call for methods for total vitamins B 1 , B 2 , B 3 , and B 6 in infant formula and related nutritionals. In the European Union and other countries, label claim is regulated based upon total vitamin content and not just the fortified form. Historically, microbiological methods were used to estimate total vitamin. However, these methods are challenged with newer, more diverse nutritional products and are no longer considered the gold standard. Newer, chromatographic methods, especially with mass spectral detection, are quickly becoming the new standard because their specificity enables accurate quantitation across more complex and diverse matrixes. However, Submitted for publication February 18, 2016. The method was approved by the AOAC Expert Review Panel for SPIFAN Nutrient Methods as First Action. The expert review panel invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. 1 Corresponding author’s e-mail: nick.cellar@abbott.com DOI: 10.5740/jaoacint.15-0315

that specificity then requires explicit definition of the vitamin forms necessary for a “total” vitamin determination. SPIFAN gathered experts in industry, government, and academia to provide these definitions. Total vitamin B 1 is defined as the sum of thiamine, thiamine monophosphate, thiamine pyrophosphate, and thiamine triphosphate in the Standard Method Performance Requirement (SMPR ® ; 1). Total B 2 is defined as riboflavin, riboflavin-5′- phosphate, and flavin adenine dinucleotide (2). Total B 3 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). SPIFAN defined forms of vitamins B 1 , B 2 , B 3 , and B 6 that represent the major contributors to total vitamin concentration in formulas and enter the formulation through both fortification and from ingredient sources. For example, depending on the protein fraction of milk and the degree of processing, the contribution of inherent (unfortified) vitamin can be virtually 0 to >45% of the total value (5). But, regardless of source, the concentration of each vitamin must be verified to meet label claim. Remarkably, the necessary sample treatments and separation as described later are similar for the intended vitamins and thus lend themselves to simultaneous determination, saving both time and cost. Further, the availability of modern mass spectrometry (MS) instrumentation with electrospray ionization (ESI) facilitates simultaneous determination by removing remaining hurdles associated with detection. Suppression of ionization is problematic for quantitation with ESI, but is overcome with the use of stable-isotope labeled internal standards. The associated cost of isotopically labeled standards, although perceived as great, only adds a few cents to the cost of a sample because of the small amount necessary. By contrast, the syringe filter required to prevent clogging the liquid chromatography (LC) column adds about $1 (USD) to the cost of a sample. 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. Thiamine is not well retained in reverse phase at low pH without an ion pairing agent (6). However, ion pairing agents bring additional challenges to LC-tandem MS (MS/MS) determination. Improved retention of thiamine has been previously demonstrated by increasing the mobile phase pH (6). In fact, there is a striking improvement in retention for many water-soluble vitamin under reverse-phase conditions at moderate pH (5–7). This improvement in retention was harnessed to achieve good method performance for a subset of the targeted vitamin forms; however, elution of phosphorylated compounds is notoriously difficult (8). The phosphate moiety complexes with Fe 3+ and thus phosphate