AOAC SPIFAN Review Team Meeting Book-July 22, 2015

BVIT-02 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Abbott Nutrition Division Simultaneous Determination of Total Vitamin B 6, B 2, B 3 in Infant Formula Products by LC-MS/MS Using Enzymatic Digestion and B 1

B.

THEORY 1. Method

a) This method via an enzymatic digestion facilitates the simultaneous quantitation of the total content of three vitamins, B6, B2, and B1 from Abbott Nutrition Milk Based Pediatric products. b) The B6 is quantitated as a sum of three vitamers Pyridoxine, Pyridoxal, and Pyridoxamine. c) Samples are prepared by the addition of an enzyme mixture consisting of Papain, Alpha-Amylase, and Acid Phosphotase. The mixture breaks down the matrix freeing any bound vitamins, and dephosphorylates the vitamin phosphate forms to the free forms d) Stable-isotope labeled internal standards are incorporated into the sample preparation and are used to correct for variability in both the sample preparation and instrument operation. e) A series of 6 mixed working standard solutions spanning two orders of magnitude in concentration are used to generate calibration curves. A response ratio (ratio of analyte to stable-isotope internal standard peak responses) is calculated for all analytes and used together with known analyte concentration to generate calibration curves used for quantitation. f) Prepared samples and working standard solutions are injected onto an Acquity UPLC (Waters, Corp) interfaced to a Triple-Quadrupole Mass Spectrometer (MS/MS) for analysis using a 2.1mm x 50mm x 1.7 m m BEH C-18 (Waters, Corp) and the analytes of interest are ionized via Electrospray Ionization (ESI). Analytes elute in less than 5 minutes with an additional 2 minutes required for column re-equilibration. g) The analytes are detected by tandem mass spectrometry. The MS/MS is configured to monitor parent-daughter (precursor-fragment) ion pairs for each analyte and internal standard and form the basis for analyte detection and quantitation and form the basis for method selectivity. h) Analytes are quantified by interpolation of the analyte-stable isotope internal standard response ratio for the sample against a calibration curve. i) Analysis of the seven analytes is accomplished by a single injection of the prepared sample on the Quattro Premier XE instrument platforms in most matrices. 2. Stable Isotope Internal Standards in Mass Spectrometry The addition of an appropriate internal standard to a quantitative assay has several significant advantages. An internal standard can correct for variations in the extraction efficiency during sample preparation, correct for errors or losses in volumes during sample handling and in the case of mass spectrometry compensate for competitive ionization in the source caused by co-eluting matrix components. An appropriate internal standard must be a compound that has similar chemical and physical properties to the analyte of interest. The closer the match between the internal standard and the compound of interest the better the performance of the internal standard. In the case of mass spectrometry the ideal internal standard can be utilized, the compound itself. This can be accomplished by synthesizing the compound with a stable isotope, most commonly of hydrogen ( 2 H), carbon ( 13 C) or nitrogen ( 15 N). The resulting compound will behave almost identically to the native 20