6. AOACSPIFANMethods-2018Awards

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L in et al .: J ournal of AOAC I nternational V ol . 100, N o . 1, 2017  147

Figure 2016.11A. HPLC system configuration with a column switch.

(f)  50% Ethanol .—Use a graduated cylinder to transfer 250 mL ethanol to a 500 mL volumetric flask. Dilute to volume with laboratory water. Expiration: 1 month at room temperature. (g)  50% Methanol .—Use a graduated cylinder to transfer 500 mL of methanol into a 1 L volumetric flask. Dilute to volume with laboratory water. Expiration: 1 month at room temperature. (h)  Postcolumn reagent .—Use a volumetric pipet to transfer 0.8 mL streptavidin fluorescein into a 1 L volumetric flask and dilute to volume with biotin mobile phase. Expiration: 1 week at room temperature. (i)  Biotin stock standard solution (100 μg/mL) .— Weigh 0.05000 (±0.0050) g USP biotin reference standard. Quantitatively transfer the standard to a 500 mL volumetric flask using 50% ethanol. Mix well. Transfer to a 500 mL amber bottle. Store refrigerated. Expiration: 3 months. (j)  Biotin intermediate solution (1000 ng/mL) .—Use a volumetric pipet to transfer 1 mL biotin stock standard solution into a 100 mL volumetric flask. Dilute to volume with laboratory water. Mix well. Expiration: make fresh on day of use. (k)  Biotin working standards (5, 10, 20, 40, 60, 80, and 100 ng/mL) .—For the 100 ng/mL working standard, use a volumetric pipet to transfer 5mLbiotin intermediate solution into a 50 mL volumetric flask. Dilute to volume with biotin mobile phase and mix well. For the 5, 10, 20, 40, 60, and 80 ng/mL working standards, use volumetric pipets to respectively transfer 0.5, 1, 2, 4, 6, and 8 mL of the 100 ng/mL working standard into 10 mL volumetric flasks. Dilute the volumetric flasks to volume with biotin mobile phase and mix well. Expiration: make fresh on day of use.

E. Sample Preparation

Note: Because biotin is light-sensitive, all samples must be prepared, handled, and stored in the dark or under yellow- shielded lighting ( see B ), unless otherwise stated. (a)  Accurately weigh up to 4 g powder product or up to 20 g liquid product in a 50 mL volumetric flask. The final concentration of biotin in the 50 mL solution should be in the range of 8–60 ng/mL. Add 20 mL water to reconstitute powder samples or add an appropriate amount of water to dilute to a total volume of 24 mL for liquid products. Swirl to mix well. (b)  Add 10 mL 50% methanol to each sample. Swirl to mix well. (c)  Add 7.5 mL 2% metaphosphoric acid to each sample. Immediately swirl to mix well. (d)  Dilute the volumetric flasks to volume with laboratory water. Invert the flask to mix well. (e)  Transfer approximately 2 mL of each sample into a microcentrifuge tube. Centrifuge at 10000 × g for 10 min. (f)  Filter the supernatant of each sample through syringe filter into an HPLC autosampler vial. (a)  Instrumental operating conditions .— ( 1 )  FD wavelength parameters .— λ ex = 495 nm, λ em = 518 nm. ( 2 )  Run time .—15 min. ( 3 )  Injection volume .—20 μL. ( 4 )  Column temperature .—20°C. ( 5 )  Mobile phase flow rate .—For each injection run, the mobile phase flow rate starts at 0.4 mL/min and the column F. Instrumentation