Determinationof BiotinbyHighPerformanceLiquidChromatography coupledwith
EASI-EXTRACTBiotin Immunoaffinitycolumncleanup extraction
George Joseph, Ranjani Devi andRaj Naganaboyina
AsureQuality Ltd, POBox 41, ShortlandStreet, Auckland1140, NewZealand
ElaineC. Marley andDavidLeeman
R-BiopharmRhoneLtd,West of ScotlandSciencePark, Glasgow, ScotlandG200XA
Corresponding author’semail:
Abstract
A new simple cost-effectiveHPLCmethod for the routine analysis of biotin in infant, adult
and / or pediatric formula is described. Themethod fullymeets theAOACSMPR finalised on
March 2014 for the vitamin. The samplewas dispersed in sodium phosphate buffer and
autoclaved at 121
⁰
C for 25minutes, cooled to room temperature anddiluted to100mLusing
a volumetric flask. The extract is centrifugedand filtered usingWhatmanglassmicrofiber
filter paper. The clear filtrate is collected for cleanup using biotin immunoaffinity column. The
buffer in the affinity columnwas drained and the sample filtrate is loaded andallowed to flow
throughby gravity. The column iswashedwith phosphate buffered saline followed bywater.
Biotin from the columnwas elutedwithmethanol, evaporated todryness and is re-extracted
inwater for HPLCanalysis. Chromatographic analyseswere performedusingC18 phenyl
hexyl solid-core columnwith isocratic elutionandUV detection at 200nm. A single laboratory
validationwas performedwith recoveries of 93
–
104% and repeatabilityRSDs of notmore
than 3% andwithin laboratory reproducibility of notmore than6%. Themethod is optimised
for routine analysis of samples ranging from0.1 to 250µg/100g of biotin.
Introduction
Biotin is the fusionof an imidazolidone ringwith a tetrahydrothiophen group linked toa
valeric acid side chain. Themolecule contains three asymetric carbon atoms so eight
stereoisomers arepossible. All eight biotin isomers have been synthesized, but only d-
biotin has been found tobe biologically active and present in nature (1, 2).
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