Highly sensitive coupled-column high-performance liquid chromatographic method for the separation and quantitation of the diastereomers of leucovorin and 5-methyltetrahydrofolate in serum and urine.
A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-microns Microbore C18 column in (6R,S)-LV and (6R,S)-METHF. (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-microns bovine serum albumin column through a Rheodyne valve system and are separated into their diastereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4-12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.
Schleyer
E
E
Department of Hematology and Oncology, University of Göttingen, Germany.
Reinhardt
J
J
Unterhalt
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Hiddemann
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D000970:Antineoplastic Agents / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D002955:Leucovorin / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics / Q000652:urine; D012016:Reference Values; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism; D013763:Tetrahydrofolates / Q000032:analysis* / Q000097:blood / Q000493:pharmacokinetics / Q000652:urine
D000970:Antineoplastic Agents; D013763:Tetrahydrofolates; D002955:Leucovorin; C005984:5-methyltetrahydrofolate