Enzymatic assay of medroxyprogesterone acetate in plasma

Abstract

A method is described for the enzymatic assay of medroxyprogesterone acetate (MPA) in plasma. After extraction and chromatographic purification of the steroid it is desacetylated and rechromatographed on the same Lipidex column. The primary enzymatic reaction is carried out with 3α,20β-hydroxy-steroid dehydrogenase and the NAD + formed is enzymatically cycled with alcohol dehydrogenase and malate dehydrogenase, and the product formed (malate) is measured in a third enzymatic step with malate dehydrogenase in a medium containing glutamate oxaloacetic transaminase. NADH is measured fluorometrically. The sensitivity of the final fluorometric assay is 25–30 fmol, calculated from the minimum amount differing from zero. If 250 μl plasma samples are used the method can detect MPA at a level of 0.6–1 nmol/1 of plasma. The specificity of the method seems good because the plasma and water blanks give the same results and the values are comparable to those obtained by mass fragmentography. However, they are frequently considerably lower than those obtained by radioimmunoassay. The advantages and disadvantages of the procedure are discussed.