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

minutes with magnetic stir plate and stir bar. Adjust to pH 4.0-4.5 with formic acid (~ 100 μL). 200 mL is adequate for 6 working standards and 32 samples. Scale as needed. Prepare fresh daily. 5. Stable-Isotope Labeled Compounds – Individual, Internal Standard Stock Solutions a) 2 H 4 -Nicotinamide Stock Solution (Approximate Concentration: 560 µ g/mL) - Weigh 14.0 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 mL volumetric flask 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. b) 2 H 4 -Nicotinic Acid Stock Solution (Approximate Concentration: 500 µ g/mL) - Weigh 12.5 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 mL volumetric flask 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. c) 13 C 4 -Pyridoxine Stock Solution (Approximate Concentration: 70 µ g/mL) - Weigh 7.0 ± 0.1 mg into a tarred 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) 2 H 3 -Pyridoxal Stock Solution (Approximate Concentration: 40 µ g/mL) - Weigh 4.0 ± 0.1 mg into a tarred 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. e) 2 H 3 -Pyridoxamine Stock Solution (Approximate Concentration: 40 µ g/mL) - Weigh 4.0 ± 0.1 mg into a tarred 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. e) 13 C 4 -Thiamine Chloride Stock Solution (Approximate Concentration: 100 µ g/mL) i) 0.12 M HCl - Add approximately 300 mL water to a 500 mL graduated cylinder. Add 5.0 ± 0.1 mL conc. HCl and swirl to mix. Bring to 500 mL with laboratory water and mix well. ii) Weigh 5.0 ± 0.1 mg 13 C 4 -thiamine into a tarred weighing vessel. Quantitatively transfer to a 50 mL volumetric flask with 0.12 N HCl and QS with 0.12 N HCl. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. f) 13 C 4 , 15 N 2 -Riboflavin Stock Solution (Approximate Concentration: 73 µ g/mL) i) 1.0% Acetic acid in water - Add approximately 30 mL water to a 500 mL graduate cylinder. Add 5.0 ± 0.1 mL glacial acetic acid and swirl to mix. Bring to 500 mL with laboratory water and mix well. ii) Weigh 7.3 ± 0.1 mg 13 C 4 , 15 N 2 -riboflavin into a tarred weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with 1.0% acetic acid and QS with 1.0% acetic acid. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. 6. Internal Standard Stock Mixture (ISSM) – Combine 2500 μL of 50 mM ammonium formate with 250 μL 2 H 4 -nicotinamide stock, 250 μL 2 H 4 -nicotinic acid stock, 250 μL 13 C 4 -pyridoxine stock, 200 μL 2 H 3 - pyridoxal stock, 50 μL 2 H 3 -pyridoxamine stock, 250 μL 13 C 4 -thiamine stock, and 250 μL 13 C 4 , 15 N 2 -