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AOAC First Action Method 2016.02 (Biotin) MLT Report
Page 7 of 16
3.3.7. Sample Preparation
Reconstitution of powder samples:
Accurately weigh and transfer 25g sample (W1) to a 250mL amber
glass screw cap bottle. Dissolve using warm water (~50ºC) and make up to a total weight of 225g (W2)
using water. Mix well until the suspension is homogeneous or use a laboratory homogeniser (Polytron®) if
necessary. Weigh an aliquot of homogeneous reconstituted sample (W3) as specified in Appendix 1 of the
protocol into 100mL amber glass screw cap bottle and continue with IAC Clean-up extraction. Some
homogenous powder samples are excluded from reconstitution, please refer to Appendix 1.
Liquid samples:
Mix well to ensure homogeneity of the sample portion and weigh the specified quantity as
in Appendix 1 (W3)
into 100mL amber glass screw cap bottle and continue with IAC Clean-up extraction
(3.3.8).
3.3.8. IAC Clean-up Extraction
1. Add 0.15 M sodium phosphate buffer to an approximate volume of 50mL.
2. Swirl gently to mix well
3. Autoclave the sample preparation at 121ºC for 25 minutes.
4. Cool the sample to room temperature. Quantitatively transfer the extracts into a 100mL volumetric
flask and make up to volume with 0.15 M sodium phosphate buffer, mix well.
5. Transfer extracts into centrifuge tubes and centrifuge the samples at 4000rpm for 15min.
6. Filter the samples using the Whatman glass microfiber filter paper, collect the filtrate.
7. Set up the SPE manifold. Attach the immunoaffinity column (IAC) connected to a 10mL reservoir.
Drain off the buffer just above the gel.
8. Load the specified volume of sample filtrate onto the IAC as per Appendix 1 and initialise the flow
with the gentle suction of a vacuum pump.
9. Let the solution pass through the column by gravity at the rate of one drop per second.
10. Wash the column by passing 10mL of PBS through the column followed by 10mL of water (initialise
the flow with the help of vacuum at every step and leave it for gravity).
11. Remove any residual liquid from the column by introducing gentle vacuum.
12. Introduce a reacti-vial and elute the analyte under gravity with 2mL methanol. Elute further with
additional 1mL of methanol. Back flush at least 3 times when eluting and this can be achieved by
gentle up and down motion of the syringe plunger to maximize the elution.
13. Evaporate the eluent to dryness using a heating block set at 85±50C, under a gentle stream of
nitrogen.
14. Cool down to room temperature by keeping it outside for about 15 minutes.
15. Re-dissolve with 1mL water; cap the reacti-vials and vortex for 30 seconds.
16. Filter using syringe filter into a clean glass insert for HPLC analysis.
3.3.9. Standard Preparation
Stock Standard Biotin (100µg/mL)
Weigh 25mg of biotin reference standard into a 250mL amber volumetric flask. Add 150mL of water and
sonicate at room temperature for 90 minutes with occasional shaking. Make up to volume with water.
Stock Standard Biocytin (100µg/mL)
Weigh 10mg of biocytin reference standard into a 100mL amber volumetric flask. Add 60mL of water and
sonicate at room temperature for 90 minutes with occasional shaking. Make up to volume with water.
Mixed Intermediate Standard (100µg/100mL)
Pipette 1mL of the biotin and biocytin stock standards to a 100mL volumetric flask and dilute to volume
with water.
Calibration Standards
Standard 1 (1.0µg/100mL): Dilute 0.10mL of mixed intermediate standard to 10mL with water.
2016.02 (FEBRUARY 2017) BIO-02 MLT REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE