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AOAC First Action Method 2016.02 (Biotin) MLT Report
Page 8 of 16
Standard 2 (2.5µg/100mL): Dilute 0.25mL of mixed intermediate standard to 10mL with water.
Standard 3 (5.0µg/100mL): Dilute 0.50mL of mixed intermediate standard to 10mL with water.
Standard 4 (7.5µg/100mL): Dilute 0.75mL of mixed intermediate standard to 10mL with water.
Standard 5 (10µg/100mL): Dilute 1.0mL of mixed intermediate standard to 10mL with water.
Standard 6 (20µg/100mL): Dilute 2.0mL of mixed intermediate standard to 10mL with water.
Note:
The concentrations given above are indicative only; calculate the actual concentrations of biotin and
biocytin in each calibration standards using the following formula.
Biotin / Biocytin (µg/100mL) = (W1 x P x 10 x Vis) ÷ (V x 10)
W1= Weight of biotin or biocytin (mg)
P = Percentage purity from the certificate of analysis
Vis = Volume of mixed intermediate standard used for the calibration standard (mL)
V = Volume of stock standard (250mL for biotin and 100mL for biocytin)
3.3.10. Chromatographic Conditions
1. Mobile Phase A : 0.1% Phosphoric acid
2. Mobile Phase B : 100% Acetonitrile
3. Mobile Phase C : 80% Acetonitrile
4. Column: Phenomenex Kinetex Phenyl-Hexyl, 00F-4495-E0 (150 x 4.6mm x 2.6µm x 100Å)
5. Column Temperature: 25±2ºC
6. Retention times: Biocytin 4.5 to 5.5 minutes and biotin 16 to 17 minutes
7. Run time: 27 minutes
8. Detector: PDA operating at 200nm (PDA scan 200 to 350nm)
9. Injection volume: 100µL
Table 3: HPLC Gradient Program
Time (min)
Flow Rate (mL/min) Mobile Phase A (%)
Mobile Phase B (%)
Mobile Phase C (%)
0.0
0.6
90
10
0
18.0
0.6
90
10
0
18.5
0.8
0
0
100
24.0
0.8
0
0
100
24.5
0.6
90
10
0
27.0
0.6
90
10
0
3.3.11. System Suitability
1. Inject the calibration
standard 4
six times at the beginning of the analytical sequence
2. Run the full set of calibration standards at the start and the end of the sequence
3. The six point calibration should give a correlation coefficient NLT 0.997.
4. Inject one of the calibration standards after every 5 sample injections.
3.3.12. Calculation and Reporting
The chromatography software will automatically calculate the concentration of the sample (as is basis) in
µg/100g, provided the concentration of the standards in (µg/100mL), sample weight (g) and dilution are
entered correctly.
Manual calculation can also be performed by using the following equation:
Biotin or Biocytin (µg/100g) =
(Sample Area x Dilution)
(Slope x Sample weight)
2016.02 (FEBRUARY 2017) BIO-02 MLT REPORT
FOR ERP USE ONLY
DO NOT DISTRIBUTE