manual of methods for determining micronutrients in fortified foods

8. Pipette exactly 4.0 mL of the filtrate obtained into a centrifuge tube which contains 4.0 mL
methanol. Mix and use the centrifuge to separate the precipitate from the supernatant liquid.
9. Pipette 4.0 mL of the clear supernatant into a test tube, dilute with 2.0 mL water and mix on a
Vortex mixer. This is the final extract of the sample for HPLC. Filter this solution through a
0.45 μm membrane.
B. Chromatography
10. Start HPLC system and allow to warm up and equilibrate the column for at least 1 hour with
mobile phase flowing. Flow rate should be 1.0 mL/min. Use the chromatographic conditions
indicated in Table 1.
11. Adjust flow to 1.5 mL/min and inject the working standard solution in duplicate, in the following
order 0.04 mg/L, 0.08 mg/L and 0.12 mg/L.
12. Inject the samples under the same conditions as the riboflavin standard and intersperse with
standard solution injections after every nine samples to ensure accurate quantification.
Table 1. Chromatographic conditions to determine riboflavin
Parameter
Condition
Column
C18. Waters. 150 mm x 4.1mm ID
Flow
1.5 mL/min
Detector
Fluorescence:
Excitation wavelength: 423 nm
Emmision wavelength: 525 nm
Injection volume 50 µL
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