Radioenzymatic determination of phenylpropanolamine in plasma

We modified a norepinephrine radioenzymatic method for determination of plasma phenylpropanolamine (PPA) concentrations. PPA is converted to N[methyl-3H]ephedrine ([3H]EPD) by the enzyme phenylethanolamine N-methyltransferase (PNMT) and S-[methyl-3H]adenosyl-L-methionine ([3H]AdoMet). The product, [3H]EPD, is isolated from unreacted [3H]AdoMet and labeled side products by an organic extraction and a TLC procedure. In addition, a preincubation organic extraction procedure is included to remove inhibitors of PNMT from plasma and to concentrate the sample for enhanced enzymatic conversion. In order to accurately quantitate PPA across the wide range of possible concentrations, the assay is conducted at two plasma volumes. PPA concentrations between 0.3 and 50 micrograms/liter can be detected with 1 ml of plasma, while concentrations between 4 and 1500 micrograms/liter can be detected with 0.1 ml of plasma. The intra-assay coefficients of variation (CVs) are 9.3 and 5.7% at 0.5 and 1500 micrograms/liter, respectively, while the mean interassay CV is 13.8%