High-performance liquid chromatographic assay for the determination of 5-methyltetrahydrofolate in human plasma.
An isocratic high-performance liquid chromatographic method for the determination of 5-methyltetrahydrofolate (5-MTHF) in human plasma is described. The method involves solid-phase extraction of 5-MTHF and p-aminoacetophenon (an internal standard) using Sep-Pak C18 cartridges. Separation was achieved 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). The method was validated in the range of 5-MTHF concentrations from 0.0625 micromol/l to 4.0 micromol/l. Within-day and inter-day precision expressed by the relative standard deviation was less than 8.1% and inaccuracy did not exceed 8.7%. The method is specific, accurate and sensitive enough to be used in pharmacokinetic studies for the assessment of the systemic availability of 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 physiological concentrations in plasma (5-20 nmol/l).
Chládek
J
J
Department of Pharmacology, Faculty of Medicine, Charles University, Hradec Králové, Czech Republic. chladekj@lfhk.cuni.cz
Sispera
L
L
Martínková
J
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013763:Tetrahydrofolates / Q000097:blood* / Q000493:pharmacokinetics
D013763:Tetrahydrofolates; C005984:5-methyltetrahydrofolate