Review Team (February 2016)

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

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Determination of Biotin in Infant, Pediatric and Adult Nutritionals by HPLC and Fluorescence Detection

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Example: Level 7 (WS7) = 5 mL x 0.9962 P g/mL y 50 mL x 1000ng/ P g = 99.62 ng/mL d) Working Standards (WS) Concentration Level 1-6 = (volume of WS7 used in mL) x (concentration of WS7 in ng/mL) y (WS volume in mL) Example:

Level 1 (WS1) = 0.5 mL x 99.62 ng/mL y 10 mL = 4.981 ng/mL Level 2 (WS2) = 1 mL x 99.62 ng/mL y 10 mL = 9.962 ng/mL Level 3 (WS3) = 2 mL x 99.62 ng/mL y 10 mL = 19.92 ng/mL Level 4 (WS4) = 4 mL x 99.62 ng/mL y 10 mL = 39.85 ng/mL Level 5 (WS5) = 6 mL x 99.62 ng/mL y 10 mL = 59.76 ng/mL Level 6 (WS6) = 8 mL x 99.62 ng/mL y 10 mL = 79.70 ng/mL

Standard Concentration (ng/mL)

Level

HPLC peak area

1 2 3 4 5 6 7

4.981 9.962 19.92 39.85 59.76 79.70 99.62

0.2019 0.6600 2.2932 6.1743 9.2305 11.353 12.863

Polynomial regression: y = ax 3 + bx 2 + cx + d

y = peak area, x = concentration a -0.0000094 b 0.0007713 c 0.1482 d -0.7451 R 2 =0.9994

2. Calculation of biotin concentration in samples a) The biotin concentration in the injected product sample, C i and the standard curve generated from the standards. b) The original product concentration, C p

, is calculated from its biotin peak area

, ( in P g/100g) is then calculated based on the dilution scheme

employed for the sample C p = C i x 50 y ss x D 1 x D 2 where C p:

Original product concentration ( P g/100g) C i : Injected sample’s biotin concentration, from standard curve (ng/mL) 50: Dilution of original product prior to filtration in mL