Electromembrane extraction of verapamil and riluzole from urine and wastewater samples using a mixture of organic solvents as a supported liquid membrane: Study on electric current variations.

In this study, the application of a mixture of organic solvents as a supported liquid membrane for improving the efficiency of the electromembrane extraction procedure was investigated. The extraction process was followed by high-performance liquid chromatography analysis of two model drugs (verapamil and riluzole). In this research, four organic solvents, including 1-heptanol, 1-octanol, 2-nitrophenyl octyl ether, and 2-ethyl hexanol, were selected as model solvents and different binary mixtures (v/v 2:1, 1:1 and 1:2) were used as the supported liquid membrane. The mixture of 2-ethyl hexanol and 1-otanol (v/v, 2:1) improved the extraction efficiency of model drugs by 1.5 to 12 times. It was found that extraction efficiency is greatly influenced by the level of electric current. In this study, for various mixtures of organic solvents, the electric current fluctuated between 50 and 2500 µA, and the highest extraction efficiencies were obtained with low and stable electric currents. Finally, the optimized extraction condition was validated and applied for the determination of model drugs in urine and wastewater samples.

Electromembrane extraction through a virtually rotating supported liquid membrane.

Electromembrane extraction (EME) of model analytes was carried out using a virtually rotating supported liquid membrane (SLM). The virtual (nonmechanical) rotating of the SLM was achieved using a novel electrode assembly including a central electrode immersed inside the lumen of the SLM and five counter electrodes surrounding the SLM. A particular electronic circuit was designed to distribute the potential among five counter electrodes in a rotating pattern. The effect of the experimental parameters on the recovery of the extraction was investigated for verapamil (VPL), trimipramine (TRP), and clomipramine (CLP) as the model analytes and 2-ethyl hexanol as the SLM solvent. The results showed that the recovery of the extraction is a function of the angular velocity of the virtual rotation. The best results were obtained at an angular velocity of 1.83 RadS(-1) (or a rotation frequency of 0.29 Hz).The optimization of the parameters gave higher recoveries up to 50% greater than those of a conventional EME method. The rotating also allowed the extraction to be carried out at shorter time (15 min) and lower voltage (200 V) with respect to the conventional extraction. The model analytes were successfully extracted from wastewater and human urine samples with recoveries ranging from 38 to 85%. The RSD of the determinations was in the range of 12.6 to 14.8%.

Simultaneous analysis of six cardiovascular drugs by capillary electrophoresis coupled with electrochemical and electrochemiluminescence detection, using a chemometrical optimization approach.

CE coupled with dual electrochemical (EC) and electrochemiluminescence (ECL) detection was optimized for simultaneous analysis of six cardiovascular drugs (alprenolol, propafenone, acebutolol, verapamil, atenolol and metoprolol) via central composite design. Following this study, three critical electrophoretic factors governing the CE separation were investigated: Tris-H(3)PO(4) buffer concentration, buffer pH value and separation voltage. A modified chromatographic response was adopted for evaluating CE separation quality. Optimum conditions were achieved using Tris-H(3)PO(4) buffer 35.6 mM (pH 2.3) separated at 13.9 kV, which was employed experimentally and led to the successful simultaneous separation of the above six drugs. The good agreement of the chromatographic response was observed between predicted data and actual experimental results using these optimized conditions (RSD=3.75%). The proposed method was validated for linearity, repeatability and sensitivity, and subsequently successfully applied to determine six basic drugs in urine samples.

Dietary salt does not influence the disposition of verapamil enantiomers in relation to efflux transporter ABCB1 genetic polymorphism in healthy Korean subjects.

To evaluate the effects of dietary salt on the stereoselective disposition of verapamil enantiomers in relation to the transporter ABCB1 2677GG/3435CC and 2677TT/3435TT haplotypes, ten healthy subjects were asked to take diets of three different salt levels for 7 days in a randomized, three-way crossover manner. The plasma concentrations of verapamil and norverapamil enantiomers were determined after a single oral dose of 240 mg verapamil on the last day of each phase. Pharmacokinetic parameters were calculated by non-compartmental analysis techniques and compared among the three different dietary salt phases. Compared with the medium salt diet, the high and low salt diets had no significant effect on the disposition of verapamil enantiomers. Moreover, the ABCB1 haplotypes did not alter the impact of dietary salt, although ABCB1 2677TT/3435TT subjects had slightly, but not significantly, higher C(max) and area under the curve (AUC) and lower T(max) for the verapamil enantiomers than did 2677GG/3435CC subjects in each salt phase.

Direct determination of verapamil in urine and serum samples by micellar liquid chromatography and fluorescence detection.

Verapamil, a calcium channel antagonist, is one of the most commonly prescribed drugs in the treatment of hypertension. In this work, it was determined in serum and urine samples by a sensitive and precise chromatographic procedure without any pre-treatment step in a C18 column using a micellar mobile phase of 0.15M sodium dodecyl sulfate and 5% pentanol at pH 7. Fluorescence detection set at 230 nm (excitation) and 312 nm (emission) was used. Verapamil is eluted at 12.5 min with no interference by the protein band or endogenous compounds. Linearities (r > 0.998), as well as intra- and inter-day precision, were studied in the validation of the method. LODs were also calculated to be 11.0, 18.5 and 20.2 ng/mL in micellar solution, serum and urine, respectively. Recoveries in the biological matrices were in the 97-99% range. Drug excretion in urine was studied in a volunteer receiving treatment for hypertension, and verapamil, as an unchanged drug, was separated from other metabolites. The procedure developed can be useful in the field of toxicology and clinical analysis.

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry.

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.

Verapamil: identification of novel metabolites in cultures of primary human hepatocytes and human urine by LC-MS(n) and LC-NMR.

1. Verapamil is a well-known and world-wide prescribed calcium antagonist, but it suffers from extensive first-pass metabolism. Although it has been marketed for many years, a complete understanding of its biotransformation in humans is still lacking. 2. The metabolism of verapamil was therefore investigated in cultures of primary human hepatocytes and in extracts of human urine after oral dosing. Identification of metabolites was done with LC-MS(n) and LC-NMR (600 MHz) to obtain in-depth information on its biotransformation products and definitive proof of the proposed chemical structures of metabolites. 3. Hyphenation of LC-MS(n) and LC-NMR was shown to be a powerful and effective platform for the identification of metabolites. Indeed, 21 Phase I and 16 Phase II metabolites were identified. Basically, all the Phase II metabolites (glucuronides) and 11 of the Phase I (oxidative) metabolites were not reported previously. 4. New insight into verapamil's biotransformation pathway is provided as well as evidence about its true complexity of metabolic disposal.

Determination of verapamil by adsorptive stripping voltammetry in urine and pharmaceutical formulations.

A sensitive reduction peak of verapamil is obtained by adsorptive stripping voltammetry in 0.01 M phosphate (pH 7.4) at an accumulation time of 30 s. The peak potential is -1.81 V (vs. Ag/AgCl). The peak current is directly proportional to the concentration of verapamil (1x10(-8)-1x10(-6) M), with a 3sigma detection limit of 5x10(-10) M (0.246 ng/ml). The R.S.D. at the 1x10(-7) M level is 1.8%. The interference of some metal ions, and some amino acids, and the application of the method to analysis of urine, and pharmaceutical formulations are described. The method is simple (no extraction), rapid (30 s accumulation time), sensitive (the detection limit of verapamil is 0.491 ng/ml), reproducible(within day R.S.D. of 1.28-1.8%), and suitable for routine analysis of verapamil, urine, and pharmaceutical formulation.

Effects of grapefruit juice and smoking on verapamil concentrations in steady state.

OBJECTIVE: Human gut wall cytochrome P(450) (CYP)3A4 is inhibited by grapefruit juice (G), whereas smoking increases CYP1A2 activity. Both enzymes contribute to verapamil biotransformation. This study was performed to quantitatively assess the effect of these factors on verapamil pharmacokinetics in steady state. METHODS: Twenty-four young healthy volunteers of both sexes (12 smokers, 12 non-smokers) participated in this randomised crossover study. Prolonged release verapamil (120 mg, Isoptin KHK) was given bid for 7 days in two periods. During days 5-7, 1 l of either G or water was coadministered daily. On day 7, concentrations of verapamil and norverapamil enantiomers were determined during one dosing interval, and model independent pharmacokinetic parameters were estimated. PR intervals were monitored for pharmacodynamics. Statistical evaluation was done essentially using bioequivalence methods. RESULTS: G significantly increased ( R, S)-verapamil the area under the concentration-time curve at steady state (AUC(tau,ss)) by a mean of 1.45-fold [90% confidence interval (CI) 1.29, 1.63] and peak plasma concentration at steady state (C(max,ss)) by 1.63-fold (90% CI 1.38, 1.91). The increase in concentrations present for ( R)- and ( S)-enantiomers was slightly greater for verapamil than for norverapamil. Smokers had significantly lower AUC(tau,ss) and C(max,ss) values than non-smokers by (means) 0.61-fold to 0.85-fold for verapamil and norverapamil enantiomers, respectively. G effects were unrelated to naringenin pharmacokinetics. Prolongation of PR intervals by G coadministration was borderline significant; an increase above 350 ms occurred in two individuals during the G period. Significantly increased urinary 6-beta-hydroxycortisol excretion by G suggests induction of hepatic CYP3A. CONCLUSIONS: Patients on verapamil treatment should abstain from grapefruit juice. Smoking habits should be considered for verapamil dosing.

Pharmacokinetic interaction between verapamil and almotriptan in healthy volunteers.

OBJECTIVE: To assess the interaction between almotriptan, a 5-HT1B/1D-receptor agonist used to treat migraine, and verapamil, an agent for migraine prophylaxis. METHODS: Twelve healthy volunteers received the following treatments in a crossover design: (1) 120-mg sustained-release verapamil tablet twice daily for 7 days and one 12.5-mg almotriptan tablet on day 7 and (2) one 12.5-mg almotriptan tablet alone on day 7. Serial plasma and urine samples were obtained on day 7. Almotriptan plasma concentrations were determined by liquid chromatography-tandem mass spectrometry; urine samples were analyzed by ultraviolet HPLC. Safety measures included blood pressure and pulse measurements, electrocardiography, and adverse event monitoring. Statistical comparisons of pharmacokinetic parameters and vital sign data were made by ANOVA. RESULTS: Mean almotriptan peak concentration and area under the plasma concentration-time curve were significantly higher and volume of distribution and oral clearance were significantly lower after coadministration of almotriptan and verapamil compared with administration of almotriptan alone. The magnitudes of these differences were approximately 20%. Renal clearance was unaffected by verapamil coadministration. No significant effects of treatment on blood pressure or pulse were detected, with the exception of sitting systolic blood pressure at 2 hours after administration. However, the difference in mean change from baseline at this time point was only 8 mm Hg. CONCLUSIONS: Verapamil modestly inhibited almotriptan clearance to a degree consistent with the modest contribution of CYP3A4 to almotriptan metabolism. This observation and the lack of effect of verapamil on the tolerability to almotriptan administration suggest that no reduction of the almotriptan dose is warranted.

Solid-phase extraction-high-performance liquid chromatography determination of verapamil and norverapamil enantiomers in urine.

A simple, rapid, sensitive and selective method has been developed for the stereospecific determination of verapamil and its metabolite, norverapamil in urine. For sample preparation we utilized a membrane-based solid-phase extraction (SPE) disk consisting of a thin, particle-loaded membrane inserted in a plastic syringe-like barrel. The particles, which may be C8 or C18 bonded phase (C8 in this work), are embedded within a matrix of PTFE (Teflon) fibrils. Overall analyte recoveries were above 85%, even at low concentration of 3.0 ng/ml with reproducibilities (C.V. values) below 13.1%. This method of extraction has the advantage of speed and considerable reduction in solvent volumes compared to conventional SPE and solvent extraction. The separation of all the enantiomers was achieved using a single chiral stationary phase column, the cellulose-based reversed-phase, Chiralcel OD-R. Analyte concentrations of less than 3.0 ng/ml could be quantitated with C.V. values below 14%. Calibration curves were linear in the range 2.5-300 ng/ml. Intra-day and inter-day reproducibilities were 10.5-14.2% at 3 ng/ml, 4.8-9.3% at 138.5 ng/ml and 7.8-10.1% at 280 ng/ml level, respectively, for all the enantiomers.

Determination of the stereochemical composition of the major metabolites of verapamil in dog urine with enantioselective liquid chromatographic techniques.

The stereochemical composition of verapamil and seven of its basic-extractable metabolites, isolated from the urine of dogs administered oral racemic verapamil, was determined by HPLC, using an Ultron OVM (ovomucoid) column. One dog was given oral (R)-verapamil alone in order to discriminate the (R)- and (S)-enantiomers of the metabolites. Structure identification of the isolated verapamil metabolites was accomplished using a combination of HPLC-MS and FAB-MS-MS techniques. Six of the urinary verapamil metabolites, including verapamil, were predominantly of the (R)-configuration, whereas one of the metabolites was predominantly in the (S)-form. The remaining isolated metabolite was comprised of approximately equal amounts of the two forms.

Two tricyclic antidepressant poisonings: levels of amitriptyline, nortriptyline and desipramine in post-mortem biological samples.

Two deaths due to amitriptyline and desipramine overdoses are reported. The first case deals with a 20-year-old Caucasian male who was found dead at his residence. Toxicological analysis of the blood, urine, liver and kidney revealed the presence of amitriptyline (1.7 mg/l, 0.13 mg/l, 36.0 mg/kg and 98.0 mg/kg) and nortriptyline (0.66 mg/l, 0.74 mg/l, 12.0 mg/kg and 37.0 mg/kg). The gastric content contained only 220 mg of amitriptyline. The urine also contained norverapamil, which was consistent with previous verapamil therapy. The second case involved a 19-year-old Caucasian male who attempted suicide earlier and was on desipramine medication. The blood, urine, liver and gastric content disclosed the presence of desipramine in the concentrations of 14.2 mg/l, 33.7 mg/l, 112.5 mg/kg and 180 mg, respectively. The levels of these tricyclics analyzed by high pressure liquid chromatography were in agreement with the levels reported in the literature. Though with the amitriptyline poisoning no significant anatomic changes were noted, the desipramine-caused death was further supported by the multisystem vascular congestion and ischemic changes consistent with cardiopulmonary failure.

Lack of interaction between verapamil and cimetidine.

Nine healthy normal subjects received verapamil, 10 mg iv, before (control) and during cimetidine dosing (300 mg every 6 hours), and verapamil, 120 mg po, twice in the same manner. After intravenous doses, the t1/2 (means +/- SE: control, 3.60 +/- 0.40 hours; cimetidine trial, 4.30 +/- 0.60 hours), volume of distribution (5.8 +/- 0.6 vs. 6.6 +/- 0.9 L/kg), and total clearance (19.2 +/- 1.5 vs. 18.4 +/- 1.6 ml/min/kg) did not change during cimetidine dosing. After oral doses, the t1/2 (4.25 +/- 0.57 vs. 4.60 +/- 0.70 hours), plasma AUC (585 +/- 113 vs. 506 +/- 82 ng/ml X hr) and absolute bioavailability (35% +/- 7% vs. 30% +/- 5%) did not differ between control and cimetidine trials, respectively. Five of the subjects also received lidocaine, 25 mg iv, once in the control state and once during the cimetidine regimen described above. Lidocaine clearance fell (665 +/- 216 vs. 527 +/- 134 ml/min; P less than 0.05) during cimetidine therapy, resulting in a trend toward a longer t1/2 (1.81 +/- 0.41 vs. 2.44 +/- 0.42 hours; 0.1 greater than P greater than 0.05) with no change in volume of distribution (1.77 +/- 0.66 vs. 1.99 +/- 0.81 L/kg). Verapamil pharmacodynamics (ECG PR interval, blood pressure, and heart rate) were evaluated after intravenous doses. A decrease in mean arterial pressure (8 +/- 1 vs. 9 +/- 2 mm Hg) and a reflex increase in heart rate (14 +/- 3 vs. 17 +/- 2 bpm) were no different in the control and cimetidine trials.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma levels and urinary excretion of verapamil, norverapamil, N-dealkylverapamil (D617), N-dealkylnorverapamil (D620) following oral administration of a slow-release preparation.

The kinetics of verapamil and of its N-dealkylated metabolites (norverapamil, D617, D620) were studied in six cardiac patients with normal cardiac indexes after 120 mg oral administration of the drug both as conventional preparation and as slow-release preparation. Following a dose of the slow-release preparation, the drug concentration curves were smoother and the mean bioavailability was lower in comparison with the conventional preparation. A patient taking inducing agents (phenobarbital and phenytoin) exhibited a strikingly low bioavailability. Following administration of the conventional preparation, the mean plasma half-lives of verapamil, norverapamil, D617 and D620 were 4.4, 6.6, 8.5, and 15.8 h respectively and the drug concentrations showed a triexponential decay. Urinary excretion data indicate that a saturation phenomenon may occur at level of renal tubular transport and that a competition may be suspected between D620 and the other compounds. It is concluded that various mechanisms, i.e. changes in hepatic and renal clearances, occurrence of a deep compartment, and the properties of the pharmaceutical preparation may affect verapamil kinetics during long-term treatment.

Rapid high-performance liquid chromatographic method for the measurement of verapamil and norverapamil in blood plasma or serum.

A simple high-performance liquid chromatographic method for the simultaneous measurement of plasma verapamil and norverapamil concentrations has been developed. The sample (100 microliters) is vortex-mixed for 30 sec with 4 M sodium hydroxide solution, pH 13 (50 microliters), internal standard solution (aqueous 5,6-benzoquinoline, 0.20 mg/l) (50 microliters) and methyl tert.-butyl ether (200 microliters). After centrifugation at 9950 x g for 2 min, a portion (100 microliters) of the resulting extract is analysed on a microparticulate (5 microns) silica column using a methanolic solution of potassium bromide (3.0 mM) and perchloric acid (0.37 mM) as the mobile phase, and the column effluent is monitored by fluorescence detection using an excitation wavelength of 203 nm. A specimen, together with a quality control sample, can be analysed, in duplicate, within 30 min. The limit of accurate measurement of the assay is 2 micrograms/l, and no potential sources of interference have been identified. The method has advantages of speed, small sample requirement and complete resolution of the three major metabolites of verapamil.