AOAC SPIFAN ERP Meeting Book-March 16, 2016

Bio-02 w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

7. STANDARD PREPARATION Stock Standard (100µg/mL): Weigh 25mg of biotin reference material into a 250mL amber volumetric flask. Add 150mL of water and shake on an orbital shaker for 24 hours.

Intermediate Standard (100µg/100mL): Dilute 1mL of the stock standard to 100mL with water.

Standard 1 (1.0µg/100mL): Dilute 100µL of intermediate standard to 10mL with water. Standard 2 (2.5µg/100mL): Dilute 250µL of intermediate standard to 10mL with water. Standard 3 (5.0µg/100mL): Dilute 500µL of intermediate standard to 10mL with water. Standard 4 (7.5µg/100mL): Dilute 750µL of intermediate standard to 10mL with water. Standard 5 (10µg/100mL): Dilute 1mL of intermediate standard to 10mL with water.

8. CHROMATOGRAPHIC CONDITIONS 1. Mobile Phase A : 0.1% Phosphoric acid

2. Mobile Pahse B : 100% Acetonitrile 3. Mobile Phase C : 80% Acetonitrile 4. Column: Phenomenex Kinetex Phenyl-Hexyl (150mm x 4.6mm x 2.6µm x 100A) 5. Flow rate: 0.6 mL/min 6. Column Temperature: 25 0 C 7. Run time: 27 minutes 8. Detector: UV diode-array operating at 200nm 9. Injection volume: 100µL

Table 2: Gradient Program

Time (minutes)

Mobile Phase A (%)

Mobile Phase B (%)

Mobile Phase C (%)

0.0

90 90

10 10

0 0

18.0

18.5

0

0

100

24.0

0

0

100

24.5

90

10

0

27.0

90

10

0

9. SENSITIVITY Limit of Quantitation (LOQ) was calculated based on the lowest working standard and the dilution factor. LOQ = (1 x 100) / (20 x 50) = 0.1µg/100g (1ppb) 1 = 1µg/100mL lowest standard 100 = Volume (mL) 20 = 20g sample 50 = Volume (mL) loaded on immunoaffinity column 1= Final volume (mL)

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