Determination of [(13)C]galactose enrichment in human plasma by gas chromatography/positive chemical ionization tandem mass spectrometry.
Galactosemia is a potentially fatal disease resulting from a deficiency of galactose-1-phosphate uridyl transferase. In order to perform mechanistic studies designed to elucidate further the etiology of the disease, we required a method to monitor (13)C enrichment in plasma galactose following a single oral dose or intravenous infusion of [1-(13)C]galactose. Determinations of plasma [(13)C]galactose enrichment requires methodology with extremely high specificity because of potential interference from other low molecular mass plasma constituents and from glucose, an isomer which is present in much higher concentrations. We have developed a method based on gas chromatography/positive chemical ionization tandem mass spectrometry (GC/PCI-MS/MS) for the precise and accurate determination of plasma [(13)C]galactose enrichment. The method employed a pentaacetylaldononitrile derivative of galactose in order to improve its GC and MS characteristics. Peak areas resulting from the transitions m/z 328 --> 106 and m/z 329 --> 107 were used to quantify the relative abundance of labeled and unlabeled galactose. Validation of the method was performed by determination of the precision and accuracy over a wide range of galactose concentrations and (13)C enrichments. The GC/PCI-MS/MS method was able to determine accurately enrichments at galactose concentrations down to 0.8 microM in the presence of 4 mM glucose, making it both highly selective and the most sensitive method currently available.
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Center for Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania, 421 Curie Boulevard, Philadelphia, Pennsylvania, 19104-6160, USA.
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D002138:Calibration; D005690:Galactose / Q000031:analogs & derivatives / Q000032:analysis* / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007202:Indicators and Reagents; D009570:Nitriles; D015203:Reproducibility of Results
D007202:Indicators and Reagents; D009570:Nitriles; C405610:galactose aldonitrile pentaacetate; D005690:Galactose