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AOAC First Action Method 2016.02 (Biotin) MLT Report
Page 6 of 16
washed with phosphate buffered saline followed by water. Air is passed through the column to remove
residual liquid.
Biotin from the column is eluted with methanol and collected in a reacti-vial. The eluent is evaporated to
dryness using a heating block set at 85±5°C under a gentle stream of nitrogen and the sample is re-
constituted in 1mL of water. The biotin in the reconstituted sample is quantified by HPLC using a UV
detector set at 200nm. Identification of biotin peak is based on absolute retention time. Spectrum scan can
be used for peak purity confirmation if required.
3.3.4. Chemicals
1. Laboratory Reagent Grade Water
2. Sodium Dihydrogen Phosphate Dihydrate
3. Disodium Hydrogen Phosphate Dihydrate
4. Sodium Hydroxide
5. Methanol, HPLC grade
6. Acetonitrile, HPLC grade
7. Ortho-phosphoric acid, 85%
8. Phosphate buffered saline (PBS) pH 7.4 (Life Technologies 100100.31 or equivalent)
9. Biotin, purity
≥
99% (Sigma B4501 or equivalent)
10. Biocytin, purity
≥
98% (Sigma B4261 or equivalent)
3.3.5. Reagents
1.
2 M Sodium Hydroxide
: Weigh 80g of sodium hydroxide into a 1L volumetric flask, dissolve in
water and make up to mark.
2.
0.15 M Sodium Phosphate Buffer
: Weigh 9.15g of
sodium dihydrogen phosphate dihydrate and
16.31g of disodium hydrogen phosphate dihydrate into a 1L volumetric flask. Dissolve in water and
make up to mark. Adjust pH to 7 with 2M sodium hydroxide.
3. 0.1% Phosphoric acid:
In a 1L volumetric flask, add 500 mL water. Add 1.2 mL of ortho-phosphoric
acid. Mix and make up to mark with water.
3.3.6. Apparatus
1. Whatman Glass Microfiber Filters, CAT No. 1820-125
2. R-Biopharm Rhone Easy Extract Biotin Immunoaffinity Column P82/P82B or equivalent
3. SPE Manifold with accessories
4. Autoclave set at 121±3°C
5. Centrifuge (variable speed)
6. Analytical Balance 4 dp
7. Amber glass screw cap bottle, 250mL and 100mL
8. Horizontal shaker
9. Volumetric flasks, 1L, 250mL, 100mL, 10mL
10. Pipettors, calibrated, 10.0mL, 5.0mL, 1.0mL, 200µL, 100µL and 50µL.
11. Measuring cylinder, 100mL, 50mL
12. Reacti-vials
13. Reacti-therm heating block with nitrogen blow down (Thermo Scientific)
14. Ultrasonic bath set at 50ºC
15. Centrifuge tubes, 50mL
16. Vortex mixer
17. Syringe filter, PTFE 0.45µm (Advantec Cat No HP045AN)
18. Disposable syringes, 10mL and 1mL
19. HPLC vials, 2mL with glass inserts (200µL)
2016.02 (FEBRUARY 2017) BIO-02 MLT REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE