column is attached to a10mL reservoir using aSPEmanifold. The buffer in the column is
drained off just above the immunoaffinity gel and the sample filtratewas loaded onto the
column bygravity. The loaded columnwaswashed using 10mL of PBS followed by 10mL of
water and thebiotinwas eluted under gravitywith 2mLmethanol. The columnwas back
flushedat least 3 timeswhen eluting. Theeluent was evaporated todryness using aheating
block set at 100
0
C and the samplewas extractedwith 1mLwater with a vortexmixer for 30
seconds. The extract was filtered using aPTFE syringe filter and vialed for HPLCanalysis.
Please refer toTable1 for sampleweight and loading volume for different biotin range
Chromatography
Chromatographic separationwas achieved on aPhenomenexKinetexPhenyl-Hexyl
column (150mm x 4.6mm x 2.6µm x 100A) withan isocratic partition usingmobile phase,
0.1%Phosphoric acid: Acetonitrile (85:15%v/v). For analysis, the column temperature
was kept at 25
⁰
C. The flow ratewas 0.2mL/min over 30min run timewithan injection
volume of 100µL. Detectionwas by diode array set at 200nm.
Results andDiscussion
Linearityand range
Linearity of biotinwas determined using a five point linear calibration curvewith effective
concentration of biotinat 1.0, 2.5, 5.0, 7.5 and 10µg/100mL. The five standardswere
injected in duplicate and the average peak areas of eachwere plottedagainst respective
concentration. These concentrations of the calibration standardswere suitable for the
range of samples that could be expected in commercially available nutritional formulas.
The correlation coefficient was not less than 0.999 throughout the validation study.
Precision
BIO-02
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