AOAC Final Action Methods in 2019

AOAC Official Method 2015.14 Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 , and B 6 in Infant Formula and Related Nutritionals Enzymatic Digestion and LC-MS/MS First Action 2015 Revised First Action 2018 Final Action 2019

Table 2015.14B. Repeatability (RSD r ) summary statistics

No. fortified products Passed SMPR Failed SMPR

Avg. RSD r , %

SMPR for RSD r , %

Total vitamin B 1 (thiamine) B 2 (riboflavin)

2.3 4.2 2.7 2.2

14 11 14 14

0 3 0 0

≤5

B 3 (niacinamide) B 6 (pyridoxine)

[Applicable to determination of total vitamins B 1 , B 2 , B 3 , and B 6 in fortified milk powders, infant formulas, and adult/pediatric nutritional formulas with concentrations. Total B 1 , B 2 , B 3 , and B 6 can be quantified in ready-to-feed products with concentrations (mg/100 g) as low as 0.0062, 0.0043, 0.044, and 0.0056, respectively.] See Table 2015.14A for method performance requirements. See Tables 2015.14B and C for method performance information supporting acceptance of the method. A. Principle The method facilitates simultaneous quantitation of four water- soluble vitamins in infant formula and related nutritional products, including all AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Standard Method Performance Requirements (SMPRs ® ) relevant forms of vitamins B 1 , B 2 , B 3 , and B 6 . Samples are prepared by enzymatic digestion with papain, α-amylase, and phosphatase to hydrolyze protein and complex carbohydrates and 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 2 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 (UPLC) interfaced to a triple-quadrupole mass spectrometer (MS/MS) for analysis. The MS/MS is configured to monitor molecular–fragment (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. B. Apparatus and Materials (a) Control sample. —NIST SRM 1849a (U.S. National Institute of Standards and Technology, Gaithersburg, MD, USA), or current lot. Store at 4°C. (b) UPLC column. —Waters Acquity BEH C18, 2.1 × 100 mm, 1.7 μm (Part No. 186002352; Waters Corp., Milford, MA, USA). (c) UPLC system. —Waters Acquity Classic, or equivalent.

(d) Tandem quadrupole mass spectrometer with ESI probe. — Waters Xevo TQ-S, or equivalent. (e) Analytical balances (3). —( 1 ) 6-Place balance, capable of accurately weighing 5.00 mg (for standards). ( 2 ) 5-Place balance (for samples). ( 3 ) Top-loading 2-place balance, capable of weighing to several hundred grams. (f) Water purifier. —Millipore Milli-Q Water Purification System, or equivalent (EMD Millipore, Billerica, MA, USA). (g) Water bath shaker.— Capable of maintaining 37°C (Lab- Line Orbit, or equivalent). (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) Vortex mixer. (k) Multiposition magnetic stir plate. (l) Room light shields. —A.L.P. Protect-A-Lamp, UV cutoff at 460 nm, or equivalent (A.L.P Lighting Components, Inc., Chicago, IL, USA). (m) Graduated cylinders. —Various sizes, including 10, 100, 500, and 1000 mL. (n) Beakers. —Various sizes, including 100, 200, 400, 600, 1000, and 2000 mL. (o) Volumetric flasks. —Various sizes, including 10, 25, 50, 100, 250, and 2000 mL. (p) Mobile phase bottles. —Glass, various sizes, including 250, 500, 1000, and 2000 mL. (q) Pasteur pipets.— Disposable, plastic. (r) Amber bottles. —Volume capacity of 50 and 100 mL (for stock standard storage). (s) Weighing vessels. —Various, including disposable weighing boats and glass weighing funnels. (t) Positive displacement pipets. —Gilson Microman 10, 100, 250, and 1000 μL (Part Nos. F148501, F148504, F148505, and F148506, respectively; Gilson, Inc., Middleton, WI, USA).

Table 2015.14C. Reproducibility (RSD R ) summary statistics

Table 2015.14A. Method performance requirements

No. fortified products Passed SMPR Failed SMPR

Analytical range (μg/100 g) 20–2000 30–4000

Avg. RSD R , %

SMPR for RSD R , %

LOQ (μg/100 g) Rec. % RSD r , % RSD R , %

Total vitamin B 1 (thiamine) B 2 (riboflavin)

Total vitamin B 1 (thiamine) B 2 (riboflavin)

2.3 6.9 6.7 5.8

10 13 14 13

4 1 0 1

≤20 90–110 ≤5

≤10

≤30

≤10

B 3 (niacinamide) B 6 (pyridoxine)

B 3 (niacinamide) B 6 (pyridoxine)

200–10000 ≤200

10–2000

≤10

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