The current procedure is based onmonoclonal antibodywhichmakes the test highly
specific, sensitive, rapidand simple to perform. The total extraction and cleanup time
takes approximately 2hours for a set of 10 samples. The result is improved cleanup and
concentration of the vitamingiving amuch cleaner chromatogramand therefore providing
moreaccurateand sensitive detection.
Experimental
Samples
Twodairy based nutritional formula sampleswere selected for the validation study. These
sampleswere representative of the type of materials commercially available and
therefore reflect the range of samples that a laboratory performing biotin analysis could
likely encounter.
Apparatus
(a)
HPLCSystem: Dionex (ThermoScientific) Ultimate3000 LCHPLCSystem consisting
binary pumpwith a vacuum degasser, a thermostatted column compartment, an
autosampler anddiode array detector. The systemswere controlled anddata collected
and processed byChromeleon software.
(b)
Analytical Column: PhenomenexKinetexPhenyl-Hexyl (150mm x 4.6mm x 2.6µm x
100A).
(c)
Balance: Readability 0.1mg (SartoriusAGGermany)
(d)
VortexMixer (VWR)
(e)
Centrifuge (HeraeusGermany)
(f)
pHmeter and probe
(g)
WhatmanGlassMicrofiber Filters, CATNo. 1820-125 or equivalent
(h)
Biotin ImmunoaffinityColumn: R-BiopharmBiotinEasy-Extract Pack P82/ P82B or
equivalent monoclonal biotin antibody
(i)
SPEManifold (Phenomenex Inc)
BIO-02
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