Review Team (February 2016)

BVit-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Determination of B-Vitamins in Infant Formula and Adult/Pediatric Formulas ESI LC-MSMS

c) Standards Preparation: 1) Preparation of Stock Standards: Table-1

Standard Weight (mg)

Standard Makeup volume (mL)

Standard Name

Diluent

Thiamine Hydrochloride

10.0 10.0 10.0 10.0 10.0 10.0 10.0

10.0

0.1M Hydrochloric acid

Riboflavin

100.0

0.1 M Acetic acid

Niacinamide Nicotinic acid

10.0 10.0 10.0 10.0 50.0

Milli-Q water Milli-Q water Milli-Q water Milli-Q water Milli-Q water

Calcium Pantothenate Pyridoxine hydrochloride

Biotin

Folic acid 0.02 M Potassium Hydrogen Carbonate Note 1: Individual stock standards can be used for 30 days when stored in dark under refrigerated conditions. Note 2: Apply appropriate salt and purity corrections to the prepared stock standards using the formula. Conc. after salt correction (mg/L) = Molecular weight of compound x Wt of Reference Materials x % Purity X 10 ----------------------------------------------------------------------------------------------------------------- Formula weight of CRM X Makeup volume (ml) 2) Preparation of Intermediate standards: Prepare 10 mg/L of mix standard by transferring required volume of each stock solution into a 100 mL low actinic volumetric flask and makeup with laboratory water. 3) Preparation of working standards: Prepare 1.0 mg/L and 0.1 mg/L working standards from 10 mg/L standard using laboratory water as diluent. 4) Preparation of calibration standards: Table-2: 10.0 10.0

Working Standard Conc. (mg/L)

Volume of Working Std (mL)

Volume of Diluent (mL)

Final Volume (mL)

Final Conc.(μg/L)

10.0 10.0 10.0

0.500 0.200 0.100 0.500 0.200 0.100 0.500 0.200

9.500 9.800 9.900 9.500 9.800 9.900 9.500 9.800

10.000 10.000 10.000 10.000 10.000 10.000 10.000 10.000

500.00 200.00 100.00

1.0 1.0 1.0

50.00 20.00 10.00

0.10 0.10

5.00 2.00

Diluent: Laboratory water .

2