Journal
ANALYTICAL BIOCHEMISTRY
Volume 346, Issue 2, Pages 201-209Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.07.038
Keywords
HPLC; photometric detection; fluorimetric detection; methotrexate; pteridines; creatinine
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A novel HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established. A previous oxidation process using 10(-3) M KMnO4 (pH 5.0) and 35 min of oxidation time was necessary to transform the analytes in the highly fluorescent pteridinic rings. CREA was not affected by the oxidative medium. Using Tris-HCl/NaCl buffer solution (pH 6.6) as mobile phase, MTX and the assayed pteridines were monitored by fluorescence at lambda(em) = 444 nm and lambda(ex) = 280 nm and creatinine was monitored by absorption measurements at lambda(abs) = 230 nm. All components were well resolved in approximately 7 min. Detection limits, according the criteria of Clayton and co-workers, were 10 ng ml(-1) for MTX, less than 1 ng ml(-1) for all of the pteridines, and 4 mu g ml(-1) for CREA. (c) 2005 Elsevier Inc. All rights reserved.
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