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AsureQuality Laboratory Services
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
10
0
18.0
90
10
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)
Bio-02 w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE