AOACSPIFANMethods-2017Awards

106

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

(UPLC) interfaced to a triple-quadrupole mass spectrometer for analysis. The mass spectrometer is configured to monitor parent–daughter (precursor–fragment) ion pairs for each analyte and internal standard. This reaction forms the basis for method selectivity. Analytes are quantified by least-squares regression using the response ratio of the analyte to its internal standard.

containing compounds tend to tail considerably with conventional chromatographic equipment because of stainless steel plumbing and column frits (8). 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 (6). 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 (9). Method performance was demonstrated in SPIFAN II matrixes. SPIFAN II matrixes are a range of infant formula and adult nutritionals prepared by a number of the infant formula manufacturers to enable single- and multilaboratory validation of methods. SPIFAN II matrixes include partially hydrolyzed, milk-based infant formula powder; partially hydrolyzed, soy-based infant formula powder; milk-based toddler formula powder; two milk-based infant formula powders; a soy-based infant formula powder; child formula powder; elemental (extensively hydrolyzed) infant formula powder; infant formula powder with fructooligosaccharides and galactooligosaccharides; milk-based, ready-to-feed (RTF) infant formula; low fat, adult nutritional powder; high protein, RTF adult nutritional; and high fat, RTF adult nutritional. The matrixes also include the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) for infant formula, SRM 1849a; and five, unfortified, placebo matrixes matching a select subset of those previously mentioned: partially hydrolyzed, milk-based infant formula powder; partially hydrolyzed, soy-based infant formula powder; milk-based, RTF infant formula; high protein, RTF adult nutritional; and high fat, RTF adult nutritional. In these 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 all SPIFAN SMPR. AOAC Official Method 2015.14 Simultaneous Determination of Total Vitamins B 1 , B 2 , and B 6 in Infant Formula and Related Nutritionals Enzymatic Digestion and LC-MS/MS First Action 2015 [Applicable for simultaneous quantitation of total B 1 , B 2 , and B 6 in infant formula] This method facilitates simultaneous quantitation of three water-soluble vitamins in infant formula and related nutritional products, including all SPIFAN SMPR relevant forms of vitamins B 1 , B 2 , and B 6 . Samples are prepared by enzymatic digestion with papain and α-amylase to hydrolyze protein and complex carbohydrate and acid phosphatase to free phosphorylated vitamin forms. Stable-isotope labeled internal standards are incorporated into the sample preparation to correct for variability in both the sample preparation and instrument response. A series of six mixed working standard (MWS) solutions spanning two orders of magnitude in vitamin concentration are used to generate calibration curves based on the peak response ratio of the analyte to its stable-isotope labeled internal standard. Prepared samples and working standard solutions are injected onto an ultra-high pressure liquid chromatograph A. Principle

B. Apparatus and Materials

(a) Control sample. —NIST SRM 1849a, or current lot. Store at 4°C. National Institute of Standards and Technology, Gaithersburg, MD. (b) Waters Acquity BEH C18 column. —2.1 × 100, 1.7 μm. Part No. 186002352. Waters Corp., Milford, MA (c) UPLC system. —Waters Acquity Classic, or equivalent. Waters (d) Tandem quadrupole mass spectrometer with ESI probe. —Waters Xevo TQ-S, or equivalent. Waters (e) Analytical balances ( 3 ) . —( 1 ) One capable of accurately weighing 5.00 mg (for standards), six-place balance. ( 2 ) An analytical five-place balance for samples. ( 3 ) A top loading two-place balance capable of weighing to several hundred grams. (f) Water purifier. —Millipore Milli-Q Water Purification System, or equivalent. EMD Millipore, Billerica, MA. (g) Reciprocal shaking bath capable of maintaining 37°C. (h) Bottle-top dispenser. —Capable of dispensing volumes of approximately 24 mL. (i) pH meter. —Capable of measuring a pH of 4.0–5.0. (j)  Karl Fischer for moisture determination in the thiamin hydrochloride United States Pharmacopeia (USP) standard. Metrohm AG, Riverview, FL. (k) Oven. —For drying riboflavin USP standard at 105°C. (l) Vacuum chamber. —For drying pyridoxine hydrochloride USP standard. (m) Vortex mixer. (q) Room light shields. —A.L.P. Protect-A-Lamp, UV cutoff at 460 nm, or equivalent. A.L.P Lighting Components, Inc. Chicago, IL. (r) Graduated cylinders. —Various sizes, including 100, 500, 1000, and 2000 mL. (s) Beakers. —Various sizes, including 100, 200, 400, 600, 1000, and 2000 mL. (t) Volumetric flasks. —Various sizes, including 10, 25, 50, 100, 250, and 2000 mL. (u) Mobile phase bottles. —Glass, various sizes, including 250, 500, 1000, and 2000 mL. (v) Disposable, plastic Pasteur pipets. (w) Amber bottles. —Volume capacity of 50 and 100 mL (for stock standard storage). (x) Weighing vessels. —Various, including disposable weighing boats and glass weighing funnels. (y) Positive displacement pipets. —Gilson Microman: 10, 100, 250, and 1000 μL; Part Nos. F148501, F148504, F148505, and F148506. Gilson, Inc. Middleton, WI. (Z) Positive displacement pipet tips. —Gilson Capillary Piston: 10, 100, 250, and 1000 μL; Part Nos. F148312, F148314, F148014, and F148560. Gilson, Inc. (aa) Plastic syringes. —3 mL. (n) Multiposition magnetic stir plate. (o) Teflon coated, magnetic stir bars. (p) Hot plate with magnetic stirring.