AOAC Final Action Methods in 2019

(u) Positive displacement pipet tips. —Gilson Capillary Piston 10, 100, 250, and 1000 μL (Part Nos. F148312, F148314, F148014, and F148560, respectively; Gilson, Inc.). (v) Plastic syringes. —3 mL. (w) Syringe filters. —Polytetrafluoroethylene (PTFE) 0.45 μm, Acrodisc 25 mm, or equivalent (Pall Corp., Port Washington, NY, USA). (x) Plastic centrifuge tubes.— 50 mL, self-standing (Superior Scientific, Ltd., or equivalent). (y) Autosampler vials. —9 mm amber with screw top 12 × 32 mm presplit PTFE-silicon septa (Waters; Part No. 186000847C, or equivalent). ( z )  Stir bars .—Teflon-coated, magnetic. C. Reagents (a) Nicotinamide. —USP Reference Standard Official Lot (Cat. No. 1462006; U.S. Pharmacopeial Convention, Inc., Rockville, MD, USA). Store as indicated on label. (b) Niacin (nicotinic acid). —USP Reference Standard Official Lot (Cat. No. 1461003). Store as indicated on label. (c) Pyridoxine hydrochloride. —USP Reference Standard Official Lot (Cat. No. 1587001). Store in desiccator protected from white light. Dry according to manufacturer’s instructions before use. (d) Riboflavin. —USP Reference Standard Official Lot (Cat. No. 1603006). Store in desiccator protected from white light. Dry according to manufacturer’s instructions before use. (e) Thiamine hydrochloride. —USP Reference Standard Official Lot (Cat. No. 1656002). Store in desiccator protected fromwhite light. Measure moisture content of the powder before use. (f) Pyridoxamine dihydrochloride. —Fluka Analytical Standard (Cat. No. P9380; Sigma-Aldrich, St. Louis, MO, USA). (g) Pyridoxal hydrochloride. —Sigma, Cat. No. P9130 (Sigma- Aldrich). (h) 2 H 4 -nicotinamide. —Cat. No. D-3457 (CDN Isotopes, Pointe- Claire, Quebec, Canada). (i) 2 H 4 -nicotinic acid. —Cat. No. D-4368 (CDN Isotopes). (j) 13 C 4 -pyridoxine:pyricoxine.— HCl (4,5-bis(hydroxymethyl)- 13 C 4 ) (Cat. No. CLM-7563; Cambridge Isotope Laboratory, Tewksbury, MA, USA). (k) 2 H 3 -pyridoxal. —Cat. No. 7098 (IsoSciences, King of Prussia, PA, USA). (l) 2 H 3 -pyridoxamine. —Cat. No. 7099 (IsoSciences). (m) 13 C 4 -thiamine chloride. —Cat. No. 9209 (IsoSciences). (n) 13 C 4 , 15 N 2 -riboflavin. —Cat. No. 7072 (IsoSciences). (o) Acid phosphatase, type II from potato, 0.5–3.0 U/mg. —Cat. No. P3752 (Sigma-Aldrich). (p) Papain from Carica papaya, ≥3 U/mg. —Cat. No. 76220 (Sigma-Aldrich). (q) α - Amylase from Aspergillus oryzae, 150 U/mg. —Cat. No. A9857 (Sigma-Aldrich). (r) Hydrochloric acid, concentrated (12 M). —American Chemical Society (ACS) grade, or equivalent. (s) Ammonium formate.— For mass spectrometry (≥99.0%); Fluka 70221 or equivalent (Sigma-Aldrich). (t) Glacial acetic acid. —Sigma ACS reagent grade, or equivalent (Sigma-Aldrich). (u) Formic acid. —Sigma ACS reagent grade, or equivalent (Sigma-Aldrich). (v) Laboratory water. —18.0 MΩ, <10 ppb total organic carbon (TOC), or equivalent.

(w) Methanol. —Fisher Scientific (Franklin, MA, USA) LC- MS/MS optima grade or EMD (EMD Millipore) Omni-Solve LC- MS grade. (x) Ethylenediaminetetracetic acid (EDTA), disodium salt dihydrate. —ACS grade (99–101%), or equivalent. (y) Potassium phosphate dibasic. —ACS grade (>98%), or equivalent. (z) Metaphosphoric acid. —ACS grade (33.5–36.5%), or equivalent. (aa) Buffer solutions.— pH 4.0, 7.0, and 10.0; for pH meter calibration. (bb) Phosphoric acid. —85%, ACS grade, or equivalent. (cc) Potassium hydroxide. —40%, ACS grade, or equivalent. D. Standard and Solution Preparation (a) Mobile phase A. —20 mM ammonium formate in water. Using a graduated cylinder, transfer 500 mL laboratory water to a mobile phase reservoir. Add 0.631 g ammonium formate and mix well. Scale as needed. Expiration: 3 days. (b) Mobile phase B. —Methanol. (c) Weak needle wash. —10% methanol in water. Expiration: 3 months. (d) Strong needle wash. —Methanol. (e) 50 mM ammonium formate. —Using a graduated cylinder, transfer 1400 mL laboratory water to an appropriate reservoir. Add 4.41 g ammonium formate and mix well. 1400 mL is adequate for six working standards and 32 samples. Scale as needed. Expiration: 3 days. (f) Enzyme cocktail. —( 1 ) Using a graduated cylinder, transfer 200 mL 50 mM ammonium formate buffer to an appropriate reservoir. Add 200 ± 10 mg acid phosphatase and 200 ± 10 mg papain. Mix for 10 min with magnetic stir plate and stir bar. Adjust to pH 4.0–4.5 with formic acid (approximately 100 μL). 200mL is adequate for sixworking standards and 32 samples. Scale as needed. Prepare fresh daily. ( 2 ) If a product contains extensive protein, or alpha-linked polysaccharides such as starch, use the following preparation: Using a graduated cylinder, transfer 200 mL 50 mM ammonium formate buffer to an appropriate reservoir. Add 200 ± 10 mg acid phosphatase, 80 ± 5 mg α-amylase, and 400 ± 10 mg papain. Mix for 10 min with magnetic stir plate and stir bar. Adjust to pH 4.0–4.5 with formic acid (approximately 100 μL). 200 mL is adequate for six working standards and 32 samples. Scale as needed. Prepare fresh daily. (g) Stable-isotope-labeled compounds. —Individual, internal standard stock solutions. Note : Internal standard stock solutions have an expiration of 6 months, however the following guidelines can be used to troubleshoot internal standards and extend the expiration dates indefinitely. The channel of the nonlabeled analyte of interest must be monitored to ensure the stable isotope-labeled internal standard does not contribute more than 20% of the area count of the lowest-level calibration standard. No response should be generated in any other channels being monitored in the method, as this is a sign of contamination, in which case fresh solution should be prepared or a fresh lot of material should be ordered. The area count of the internal standard should be at least 3× the area count of the analyte in the lowest-level calibration standard and the lowest level matrix-based QC sample. ( 1 )  2 H 4 -nicotinamide stock solution (approximate concentration: 560 μg/mL). —Weigh 14.0 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 25 mL volumetric flask with laboratory water and use quantity sufficient (QS) with laboratory water. Mix well and transfer to a 50 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination.

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