Experimental design approach for the optimisation of a HPLC-fluorimetric method for the quantitation of the angiotensin II receptor antagonist telmisartan in urine.

A high performance liquid chromatographic method with fluorimetric detection has been developed for the quantitation of the angiotensin II receptor antagonist (ARA II) 4-((2-n-propyl-4-methyl-6-(1-methylbenzimidazol-2-yl)-benzimidazol-1-yl)methyl)biphenyl-2-carboxylic acid (telmisartan) in urine, using a Novapak C18 column 3.9 x 150 mm, 4 microm. The mobile phase consisted of a mixture acetonitrile-phosphate buffer (pH 6.0, 5 mM) (45:55, v/v) pumped at a flow rate of 0.5 ml min(-1). Effluent was monitored at excitation and emission wavelengths of 305 and 365 nm, respectively. Separation was carried out at room temperature. Chromatographic variables were optimised by means of experimental design. A clean-up step was used for urine samples consisting of a solid-phase extraction procedure with C8 cartridges and methanol as eluent. This method proved to be accurate (RE from -12 to 6%), precise (intra- and inter-day coefficients of variation (CV) were lower than 8%) and sensitive enough (limit of quantitation (LOQ), ca. 1 microg l(-1)) to be applied to the determination of the active drug in urine samples obtained from hypertensive patients. Concentration levels of telmisartan at different time intervals (from 0 up to 36 h after oral intake) were monitored.

Evaluation of the cytogenetic damage induced by the antihypertensive drug nimodipine in human lymphocytes.

The aim of this work was a study of the genotoxic potential of chronic long-term therapy with the antihypertensive drug nimodipine by measures of sister chromatid exchanges (SCE) and micronuclei (MN) in peripheral human lymphocytes of patients with long-term exposure to this drug. Peripheral human lymphocytes of control individuals exposed in vitro to nimodipine were also studied to assess the effect of the drug itself. Fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out on five patients under antihypertensive treatment with nimodipine. The in vitro study was performed on five control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma (controls/medium). The in vivo study showed no genotoxic effects of long-term therapy with nimodipine because the frequencies of SCE and MN in exposed patients did not show significant differences as compared with control individuals. A statistically significant increase in the frequency of MN was detected in controls/medium as compared with control individuals without the drug. FISH analysis revealed statistically significant differences with respect to the frequency of centromeric signals in nimodipine-induced MN in vitro. With regard to the in vivo results, chronic long-term therapy with nimodipine is not associated with increased genotoxicity. The differing results in vivo and in vitro could be due to extensive metabolism of nimodipine, indicating that the cytogenetic effect observed was due to the drug itself rather than its metabolites or to an adaptive response to nimodipine in vivo.

Determination of the beta-blocker atenolol in plasma by capillary zone electrophoresis.

A capillary zone electrophoretic method was optimised for the determination of the beta-blocker atenolol in plasma. Separation was performed in an uncoated silica capillary of 58.5 cm (effective length 50 cm) x 75 microm I.D., and detection was at 194 nm. The effects of the buffer (concentration and pH), the injection time, the voltage applied and the plasma clean-up procedure were studied. The determination of atenolol was achieved in less than 3 min, using an electrolyte of 50 mM H3BO3-50 mM Na2B4O7 (50:50, v/v) pH 9, injected hydrodynamically for 4 s at 50 mbar and applying a voltage of +25 kV. This method was applied to the determination of atenolol in plasma of nine hypertensive patients (male and female, aged from 39 to 73 years). Atenolol concentrations found vary from 30 to 585 ng/ml.

In vitro and in vivo evaluation of the antihypertensive drug atenolol in cultured human lymphocytes: effects of long-term therapy.

The genotoxicity of atenolol, a beta-blocker antihypertensive drug, both in vitro and in vivo, was cytogenetically tested for its ability to induce sister chromatid exchange (SCE) and micronuclei (MN) in cultured peripheral lymphocytes. Also, fluorescence in situ hybridization (FISH) with a centromeric probe was performed to determine the origin of the induced MN. The in vivo study was carried out, on the one hand, on four patients under antihypertensive treatment with atenolol and, on the other hand, on four matched control individuals taking an oral dose of atenolol. The in vitro study was performed on the control individuals by adding the drug to the culture medium at a final concentration similar to the levels found in plasma. When a comparison was made, the frequency of SCE did not show significant differences in any case. A statistically significant increase in the frequency of MN was detected in patients but not in control individuals either in vitro or in vivo. FISH analysis revealed statistically significant differences between patients and control individuals without the drug with respect to the frequency of centromeric signals in MN. Taking all these observations together, our data suggest that chronic exposure to atenolol resulted mainly in the induction of chromosome loss, so an aneugenic activity could be predicted. Different sensitivity to the compound was observed among control individuals. Nevertheless, all of them responded to the presence of atenolol in the same way in both assays. Interindividual variability was also reported. The intervariability seen in patients suggested an adaptive response to the chemical after long-term therapy.