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(x) 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)  pH 4.0, 7.0, and 10.0 buffer solutions for pH meter calibration. (ab) Phosphoric acid. —85%, ACS grade, or equivalent. (ac) Potassium hydroxide. —40%, ACS grade, or equivalent. (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 of 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 of 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. —Using a graduated cylinder, transfer 200 mL 50 mM ammonium formate buffer to an appropriate reservoir.Add 200 ± 10mg acid phosphatase, 80 ± 5mg 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. ( 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 quantum satis (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. ( 2 )  2 H 4 -nicotinic acid stock solution (approximate concentration: 500 μg/mL) .—Weigh 12.5 ± 0.1 mg into a tared weighingvessel.Quantitatively transfer to a 25mLvolumetricflask with laboratory water and 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. ( 3 )  13 C 4 -pyridoxine stock solution. —Approximate concentration: 70 μg/mL.Weigh 7.0 ± 0.1mg into a tared weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. ( 4 )  2 H 3 -pyridoxal stock solution. —Approximate concentration: 40 μg/mL. Weigh 4.0 ± 0.1 mg into a tared weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and store refrigerated (2–8°C). Expiration: Until exhausted or evidence of contamination. D. Standard and Solution Preparation

(ab) Syringe filters, PTFE 0.45 μm. —Acrodisc 25 mm, or equivalent. Pall Corporation, Port Washington, NY. (ac) 50 mL self-standing, plastic centrifuge tubes. (ad) Autosampler vials. —9 mm amber with screw top 12 × 32 mm presplit PTFE-silicon septa (Waters, Part No. 186000847C, or equivalent). Waters (ae) Teflon coated magnetic stir bars. (a) Nicotinamide. —USP (U.S. Pharmacopeial Convention, Inc. Rockville, MD) Official Lot; Cat. No. 1462006. Store as indicated on label. (b) Niacin (nicotinic acid). —USP Reference Standard Official Lot; Cat. No. 1461003. Stored 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 from white light. Measure the moisture content of the powder before use. (f) Pyridoxamine dihydrochloride. —Fluka Analytical Standard, Cat. No. P9380. Sigma-Aldrich, St. Louis, MO. (g) Pyridoxal hydrochloride. —Sigma, Cat. No. P9130. Sigma-Aldrich (h) 2 H 4 -nicotinamide. —CDN Isotopes, Pointe-Claire, Quebec, Canada; Cat. No. D-3457. (i) 2 H 4 -nicotinic acid. —CDN Isotopes; Cat. No. D-4368. ( j ) 1 3 C 4 - p y r i d o x i n e . —P y r i d o x i n e : H C l (4,5-bis(hydroxymethyl)- 13 C 4 ); Cambridge Isotope Laboratory, Tewksbury, MA; Cat. No. CLM-7563. (k) 2 H 3 -pyridoxal. —IsoSciences, King of Prussia, PA; Cat. No.7098. (l) 2 H 3 -pyridoxamine. —IsoSciences; Cat. No. 7099. (m) 13 C 4 -thiamine chloride. —IsoSciences, King of Prussia, PA; Cat. No. 9209. (n) 13 C 4 , 15 N 2 -riboflavin. —IsoSciences, King of Prussia, PA., Cat. No. 7072. (o) Acid phosphatase, type II from potato, 0.5–3.0 U/mg. — Sigma, Cat. No. P3752. Sigma-Aldrich (p) Papain from Carica papaya, ≥3 U/mg. —Sigma, Cat. No. 76220. Sigma-Aldrich (q) α - Amylase from Aspergillus oryzae, 150 U/mg. —Sigma, 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, St. Louis, MO. (u) Formic acid. —Sigma ACS reagent grade, or equivalent. Sigma-Aldrich, St. Louis, MO. (v) Laboratory water. —18.0 MΩ, <10 ppb total organic carbon (TOC), or equivalent. (w) Methanol. —Fisher Scientific (Franklin, MA) LC-MS/ MS Optima grade or EMD (EMD Millipore) Omni-Solve LC-MS grade. Reference Standard C. Reagents