DETERMINATION OF DROLOXIFENE AND 2 METABOLITES IN SERUM BY HIGH-PRESSURE LIQUID-CHROMATOGRAPHY

In this assay of the nonsteroidal antiestrogen droloxifene and two met abolites in human serum, the serum samples were deproteinized with an equal volume of acetonitrile and then injected into an analytical octa decylsilane column. The analytical column was equilibrated with aceton itrile/water (1/1, vol/vol) containing acetic acid and diethyl amine a nd eluted isocratically with 66% acetonitrile in the same buffer. Drol oxifene, N-desmethyldroloxifene, and 4-methoxydroloxifene were post-co lumn converted to fluorophors by ultraviolet illumination while passin g through a 10-m transparent knitted polytetrafluorethylene reaction c oil. Analytical recovery was close to 100%. Within- and between-day pr ecision corresponded to a coefficient of variation (CV) of 2-5% at ser um concentrations of greater than or equal to 25 ng/ml, except for 4-m ethoxydroloxifene (CV 7-10% at a concentration of 25 ng/ml). By increa sing the injection volume from 50 to 250 mu l, the detection limits co uld be decreased from similar to 5 to 1 ng/ml. Conjugated droloxifene could be estimated in a second run after treatment of sample with an e nzyme preparation containing beta-glucuronidase plus sulphatase. The r ecovery of droloxifene glucuronide was close to 100%. Sulphate conjuga tes have not been identified and were not accounted for.