Cyclodextrin Micellar LC for Direct Selective Analysis of Combined Dosage Drugs in Urine.

Two cyclodextrin micellar liquid chromatographic methods were developed and applied to the simultaneous determination of bisoprolol/hydrochlorothiazide and atenolol/chlorthalidone combinations in urine matrices without sample pretreatment. These combined ß-blockers and diuretics chemotherapies are commonly used in the treatment of hypertension and cardiovascular diseases. Hybrid isocratic mobile phases containing hydroxypropyl-ß-cyclodextrin, sodium dodecyl sulfate, phosphate buffer and methanol on a Luna C18 column with 0.5 mL min(-1) flow rate and 25.0°C column temperature were used. The methods were sensitive enough for the determination of analytes at the therapeutic urine levels with limits of detections down to 1.0 µg mL(-1); relative standard deviations and recoveries were ranged between 1.5-4.4% and 98.00-109.52%, respectively. Urinary excretion studies showed that the detection of drugs is possible up to 24 h after their ingestion. The selective proposed separations with less consumption of organic solvents over the hitherto ones could be attributed to the four point competitive interactions among analysts, pseudostationary phases and a real stationary phase.

Extraction and preconcentration of beta-blockers in human urine for analysis with high performance liquid chromatography by means of carrier-mediated liquid phase microextraction.

A novel method was developed for the analysis of four beta-blockers, namely sotalol, carteolol, bisoprolol, and propranolol, in human urine by coupling carrier-mediated liquid phase microextraction (CM-LPME) to high performance liquid chromatography (HPLC). By adding an appropriate carrier in organic phase, simultaneous extraction and enrichment of hydrophilic (sotalol, carteolol, and bisoprolol) and hydrophobic (propranolol) drugs were achieved. High enrichment factors were obtained by optimizing the compositions of the organic phase, the acceptor solution, the donor solution, the stirring rate, and the extraction time. The linear ranges were from 0.05 to 10.0 mg L(-1) for sotalol and carteolol, and from 0.05 to 8.0 mg L(-1) for bisoprolol and propranolol. The limits of detection (S/N=3) were 0.01 mg L(-1) for sotalol, carteolol, and bisoprolol, and 0.005 mg L(-1) for propranolol. The relative standard deviations were lower than 6%. The developed method exhibited high analyte preconcentration and excellent sample clean-up effects with little solvent consumption and was found to be sensitive and suitable for simultaneous determination of the above four drugs spiked in human urine. Furthermore, the successful analysis of propranolol in real urine specimens revealed that the determination of beta-blockers in human urine is feasible using the present method.

Isotachophoretic determination of bisoprolol, clonidine, disopyramide and tolazoline in human fluids.

Separation and determination of bisoprolol, clonidine, disopyramide and tolazoline in control serum and in human urine was investigated by capillary isotachophoresis. The drugs were separated by using the cationic electrolyte system. viz., sodium acetate buffer (pH 4.64) (c1 = 10 mM)-beta-alanine. The compounds were almost totally isolated from serum by solid-phase extraction using a Sep-Pak C18 cartridge. The recovery of compounds varied from 87 to 99%. The linear calibration range was studied to apply the method to real human fluids. The limit of determination of the drugs was 40.0 micrograms/ml serum. The limit of determination by direct sampling for bisoprolol is 3 micrograms/ml urine.

Pharmacokinetics and metabolism of bisoprolol enantiomers in humans.

The plasma concentrations and urinary excretions of bisoprolol enantiomers in four Japanese male healthy volunteers after a single oral administration of 20 mg of racemic bisoprolol were evaluated. The AUC(infinity) and elimination half-life of (S)-(-)-bisoprolol were slightly larger than those of (R)-(+)-bisoprolol in all subjects. The metabolic clearance of (R)-(+)-bisoprolol was significantly (P < 0.05) larger than that of (S)-(-)-bisoprolol (S/R ratio: 0.79+/-0.03), although the difference was small. In contrast, no stereoselective in vitro protein binding of bisoprolol in human plasma was found. An in vitro metabolic study using recombinant human cytochrome P450 (CYP) isoforms indicated that oxidation of both bisoprolol enantiomers was catalyzed by the two isoforms, CYP2D6 and CYP3A4. CYP2D6 metabolized bisoprolol stereoselectively (R > S), whereas the metabolism of bisoprolol by CYP3A4 was not stereoselective. The S/R ratio of the mean clearance due to renal tubular secretion was 0.68, indicating a moderate degree of stereoselective renal tubular secretion. These findings taken together suggest that the small differences in the pharmacokinetics between (S)-(-)- and (R)-(+)-bisoprolol are mainly due to the stereoselectivity in the intrinsic metabolic clearance by CYP2D6 and renal tubular secretion.

Dose proportionality of bisoprolol enantiomers in humans after oral administration of the racemate.

Dose proportionality of racemic bisoprolol and the stereoselectivity of its enantiomers were studied after single oral dosing of 5 to 40 mg of bisoprolol hemifumarate in eight healthy male volunteers in an open-label, randomized, four-way cross-over trial. There were dose-proportional increases in mean peak plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) values for the racemate and the individual enantiomers. No statistically significant differences were detected between the mean half life (t 1/2), Cmax, and time to reach Cmax (tmax) of the R- and S-isomers at each of the four dose levels studied. These findings support dose proportionality and absence of stereoselective pharmacokinetics for bisoprolol in the dose range studied.

Vasoconstrictors and renal protection induced by beta 1-selective adrenoceptor antagonist bisoprolol.

We investigated the role of the vasoconstrictors endothelin-1 (ET-1) and thromboxane in renal protection by the beta 1-selective adrenoceptor antagonist, bisoprolol, in Dahl salt-sensitive rats (Dahl S) and salt-resistant rats (Dahl R). Six-week bisoprolol treatment (20 mg/kg chow) reduced systolic blood pressure (SBP) by 14% in Dahl S rats fed a high-salt (4% NaCl) diet. This BP reduction was accompanied by a decrease in aortic wall thickness. ET-1 and thromboxane released from renal cortex was significantly decreased by 17 and 30% with bisoprolol, respectively. Other prostaglandin synthesis was unaffected. Renal function such as proteinuria, N-acetyl-beta-D-glucosaminidase (NAG) excretion, and glomerular filtration rate (GFR) was not influenced by bisoprolol. Morphologic investigation showed that bisoprolol significantly improved glomerular sclerosis by 29% and attenuated arterial damage by 71%, although tubular injury was not affected. The more severe the glomerulosclerotic lesions, the greater the generation of thromboxane and ET. The arterial lesions were positively correlated to thromboxane generation. These data indicate that long-term bisoprolol treatment reduces vasoconstrictive ET-1 and thromboxane generation and that these alterations may be partly responsible for the amelioration of glomerular and arterial injury in Dahl S rats.

Sensitive determination of bisoprolol enantiomers in plasma and urine by high-performance liquid chromatography using fluorescence detection, and application to preliminary study in humans.

A sensitive, stereoselective high-performance liquid chromatographic method with fluorescence detection for the measurement of bisoprolol enantiomers in human plasma and urine has been developed. Bisoprolol was extracted at alkaline pH with chloroform, followed by solid-phase extraction. The effluent was evaporated, and the reconstituted residue was chromatographed on a Chiralcel OD column with a mobile phase of hexane-2-propanol (10:0.9, v/v) containing 0.01% (v/v) diethylamine. Within the plasma and urine enantiomeric concentration ranges of 5-100 ng/ml and 25-1250 ng/ml, respectively, a linear relationship was obtained between the peak-height ratios and the corresponding concentrations. The limit of quantitation, defined as three times the baseline noise, was 2 ng/ml for each enantiomer in plasma. A preliminary pharmacokinetic study was undertaken in three healthy male volunteers following an oral dose of 5 mg of racemic bisoprolol. The results confirm that this assay is suitable for pharmacokinetic studies of bisoprolol enantiomers in humans following oral administration of the therapeutic dose.