SIMULTANEOUS DETERMINATION OF TACRINE AND 1-HYDROXYTACRINE, 2-HYDROXYTACRINE AND 4-HYDROXYTACRINE IN HUMAN PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

An analytical method was developed for the simultaneous reversed-phase (cyano) high-performance liquid chromatographic determination with fl uorescence detection of tacrine and 1-hydroxy-, 2-hydroxy-, and 4-hydr oxytacrine in human plasma. Alkalinized human plasma samples were extr acted with a mixture of chloroform/l-propanol (90:10, v/v). Extraction recoveries generally ranged from 68% to 83% for all four compounds an d did not appear to be concentration dependent over the range examined . Calibration curves were constructed over the following ranges: 0.5-3 0.0 ng/mL for tacrine (THA) and 4-hydroxytacrine (4-OH-THA), 1.0-30.0 ng/mL for 2-hydroxytacrine (2-OH-THA), and 0.925-46.2 ng/mL for 1-hydr oxytacrine (1-OH-THA). The intra- and interassay precision (RSD) for t he quality control specimens analyzed during validation were less than or equal to 11.8% and less than or equal to 9.28%, respectively. The intra- and interassay accuracy (RE) for the quality control specimens analyzed during validation ranged from 14.1% to -7.5% and 12.1% to -3. 33%, respectively, for all four compounds. The limit of quantitation w as estimated as the level of the lowest concentration in the calibrati on curve for each compound based on acceptable interassay precision an d accuracy statistics generated at this concentration. The sensitivity of the method was adequate for the determination of THA, 1-OH-THA, 2- OH-THA, and 4-OH-THA following oral administration of Cognex (40 mg si ngle dose) to normal volunteers.