HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR DILTIAZEM IN SMALL-VOLUME BLOOD SPECIMENS AND APPLICATION TO PHARMACOKINETIC STUDIES IN RATS

A high-performance liquid chromatographic (HPLC) method was developed which involves the use of two 5-mu m BDS silica gel columns (15 cm x 4 .6 mm I.D.) in series for increased resolution and sensitivity, and an organic mobile phase for both extraction and elution of diltiazem. Pl asma samples (400 mu l) were extracted using the organic mobile phase [n-hexane-methanol-dichloromethane-ammonia (370:35:30:0.3)] and the ex tracts were monitored at 240 nm. Desipramine (30 mu g ml(-1)) was the internal standard. The limit of quantification in plasma was 20 ng ml( -1) with a correlation coefficient of greater than or equal to 0.999 w ithin the 20-800 ng ml(-1) standard window. The inter- and intra-assay R.S.D.s were within 5%. The recovery of diltiazem varied from 101.1% at 20 ng ml(-1) to 93.7% at 400 ng ml(-1). The method was applied to t he investigation of dilitiazem absorption in a rat. Drug absorption wa s based on the intestinal single-pass perfusion model. The concentrati on of diltiazem in all test perfusion solutions was 1 mg ml(-1) (2.4 m M) and the flow-rate through the system was 3.33 . 10(-3) ml s(-1). A non-specific mucolytic absorption enhancer was also added to a diltiaz em solution and studied in the in situ system. The pharmacokinetics of diltiazem hydrochloride were investigated in two study groups of Wist ar rats (n = 4), A two-sample Student's t-test was employed to compare values of the area under the curve (AUC). The pharmacokinetic data in dicated that the AUC in the group which received the enhancer [18.12 /- 5.43 ng ml(-1) h(-1) (+/-S.D.)] was higher than that in the control group (11.49 +/- 3.67 ng h(-1) ml(-1)), t-test; p = 0.0483. Hence it was shown that administration of an enhancer could increase the oral b ioavailability of diltiazem.