Liquid chromatographic analysis of atenolol enantiomers in human plasma and urine.

Abstract

A sensitive, stereospecific high-performance liquid chromatographic assay of atenolol (AT) enantiomers in human plasma and urine was developed. After addition of internal standard (IS, methoxamine) and alkalinization of the plasma, the drug and IS were extracted with ethyl acetate. The organic layer was evaporated and, after addition of saturated sodium carbonate, the residue was derivatized with (-)-menthyl chloroformate at ambient temperature; the reaction was complete within 30 s, with an efficiency of 97.2 +/- 2.6%. The diastereomeric derivatives of AT and IS were then extracted into chloroform and analyzed on a C18 column with a mobile phase consisting of water: acetonitrile: methanol. The samples were detected utilizing a fluorescent detector. Water-diluted urine samples were derivatized directly and then subjected to the same procedure as plasma. Under these conditions, AT diastereomers were separated with a resolution factor of 1.94, free of any interfering peak. An excellent linear relationship (r greater than or equal to 0.998) was obtained between the peak area ratios and the corresponding concentrations in the ranges 12.5-250 and 250-2500 ng/mL for plasma and urine, respectively. The applicability of the method is demonstrated by analysis of plasma and urine concentrations of individual enantiomers of AT after oral administration of a single 100-mg dose to a healthy subject.