Determination of droloxifene and two metabolites in serum by high-pressure liquid chromatography

In this assay of the nonsteroidal antiestrogen droloxifene and two metabolites in human serum, the serum samples were deproteinized with an equal volume of acetonitrile and then injected into an analytical octadecylsilane column. The analytical column was equilibrated with acetonitrile/water (1/1, vol/vol) containing acetic acid and diethyl amine and eluted isocratically with 66% acetonitrile in the same buffer. Droloxifene, N-desmethyldroloxifene, and 4-methoxdroloxifene were post-column converted to fluorophors by ultraviolet illumination while passing through a 10-m transparent knitted polytetrafluorethylene reaction coil. Analytical recovery was close to 100%. Within- and between-day precision corresponded to a coefficient of variation (CV) of 2-5% at serum concentrations of > or 25 ng/ml, except for 4-methoxydroloxifene (CV 7-10% at a concentration of 25 ng/ml). By increasing the injection volume from 50 to 250 microliters, the detection limits could be decreased from approximately 5 to 1 ng/ml. Conjugated droloxifene could be estimated in a second run after treatment of sample with an enzyme preparation containing beta-glucuronidase plus sulphatase. The recovery of droloxifene glucuronide was close to 100%. Sulphate conjugates have not been identified and were not accounted for.