2015.14 Vitamin B (July 2018)

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

FOR ERP USE ONLY DO NOT DISTRIBUTE

10. 13 C 4

-Pyridoxine: pyridoxine:HCl (4,5-bis(hydroxymethyl)- 13 C 4

); Cambridge Isotope Laboratory;

Catalog # CLM-7563.

11. 2 H 3 12. 2 H 3 13. 13 C 4 14. 13 C 4

-Pyridoxal: IsoSciences; Catalog # 7098. -Pyridoxamine: IsoSciences; Catalog # 7099. -Thiamine chloride; IsoSciences; Catalog # 9209.

, 15 N 2 -Riboflavin: IsoSciences, Catalog# 7072. 15.Acid phosphatase, type II from potato, 0.5 – 3.0 U/mg: Sigma, catalog# P3752. 16.Papain from Carica papaya, ≥ 3 U/mg: Sigma, catalog# 76220. 17.α–amylase from aspergillus oryzae, 150 U/mg: Sigma, catalog# A9857. 18.Hydrochloric acid concentrated (12M); ACS grade, or equivalent. 19.Ammonium formate: for mass spectrometry (≥99.0%), Fluka 70221 or equivalent.

20.Glacial acetic acid: Sigma ACS Reagent Grade, or equivalent. 21.Formic acid: Sigma ACS Reagent Grade, or equivalent. 22.Laboratory Water: 18.0 MΩ, < 10 ppb TOC, or equivalent. 23.Methanol: Fisher LC-MS/MS Optima grade or EMD Omni-Solve LC-MS grade. 24.Ethylenediaminetetracetic acid, disodium salt dihydrate (EDTA): ACS grade (99-101%), or equivalent.

25.Potassium phosphate dibasic: ACS grade (>98%), or equivalent. 26.meta-Phosphoric acid: ACS grade (33.5-36.5%), or equivalent. 27.pH 4.0, 7.0, and 10.0 buffer solutions for pH meter calibration. 28.Phosphoric acid, 85%, ACS grade, or equivalent. 29.Potassium hydroxide, 40%, ACS grade, or equivalent.

D.

Standard and Solution Preparation 1. 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. 2. Mobile Phase B: Methanol. 3. Weak needle wash: 10% methanol in water, expiration 3 mo. 4. Strong needle wash: Methanol. 5. 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 6 working standards and 32 samples. Scale as needed. Expiration: 3 days. 6. Enzyme Cocktail - 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 ten