High-performance liquid chromatographic assay for diltiazem in small-volume blood specimens and application to...

A high-performance liquid chromatographic (HPLC) method was developed which involves the use of two 5-microns 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. Plasma samples (400 microliters) were extracted using the organic mobile phase [n-hexane-methanol-dichloromethane-ammonia (370:35:30:0.3)] and the extracts were monitored at 240 nm. Desipramine (30 micrograms ml-1) was the internal standard. The limit of quantification in plasma was 20 ng ml-1 with a correlation coefficient of > or = 0.999 within 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 the investigation of diltiazem absorption in a rat. Drug absorption was based on the intestinal single-pass perfusion model. The concentration of diltiazem in all test perfusion solutions was 1 mg ml-1 (2.4 mM) 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 diltiazem solution and studied in the in situ system. The pharmacokinetics of diltiazem hydrochloride were investigated in two study groups of Wistar 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 indicated 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 bioavailability of diltiazem.