Pyrimethamine analysis by enzyme inhibition and HPLC assays

Pyrimethamine is an antiparasitic agent currently used for therapy of central nervous system toxoplasmosis, a disease seen with increasing frequency in association with the AIDS epidemic. Monitoring of pyrimethamine levels may be particularly important because patients may be treated with high doses of the drug for extended periods of time. The authors have developed and validated both a new enzyme inhibition assay that can be run on an automated analyzer and an improved high performance liquid chromatography (HPLC) method. The calibration range of both methods is 100 to 3,000 micrograms/L. Both demonstrate good linearity, specificity, and precision, and correlate well with one another (r = 0.99). The CVs of the enzyme inhibition assay were < or = 8.6% and those of the HPLC method were < or = 5.4%. No interference was noted for a variety of drugs likely to be used concomitantly with or in lieu of pyrimethamine with the exception of a minor interference from trimethoprim in the enzyme inhibition assay. The major advantage of the enzyme inhibition assay is its ease of automation. The major advantages of the HPLC assay are its precision and relative simplicity. These methods should facilitate therapeutic monitoring of pyrimethamine.