High-performance liquid chromatographic assay for the determination of 5-methyltetrahydrofolate in human plasma

An isocratic high-performance liquid chromatographic method for the determi nation of 5-methyltetrahydrofolate (5-MTHF) in human plasma is described. T he method involves solid-phase extraction of 5-MTHF and p-aminoacetophenon (an internal standard) using Sep-Pak C-18 cartridges. Separation was achiev ed with an ODS column using acetonitrile and phosphate buffer supplemented with octanesulfonic acid (an ion-pairing agent). The pH of the mobile phase (2.5) was optimal with respect to the mode of detection (fluorescence). Th e method was validated in the range of 5-MTHF concentrations from 0.0625 mu mol/l to 4.0 mu mol/l. Within-day and inter-day precision expressed by the relative standard deviation was less than 8.1% and inaccuracy did not exce ed 8.7%. The method is specific, accurate and sensitive enough to be used i n pharmacokinetic studies for the assessment of the systemic availability o f 5-MTHF after leucovorin administration to patients as a rescue after high -dose therapy with methotrexate. The limit of detection was 0.17 pmol which corresponds to a plasma concentration of 1.7 nmol/l. Thus, the assay could potentially be used for the measurement of 5-MTHF in the range of physiolo gical concentrations in plasma (5-20 nmol/l). (C) 2000 Elsevier Science B.V . All rights reserved.