Simultaneous determination of verapamil and norverapamil in biological samples by high-performance liquid chromatography using ultraviolet detection

In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane–isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1–1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.     

Achiral and chiral high-performance liquid chromatography of verapamil and its metabolites in serum samples

Rapid and simple achiral and chiral HPLC assays have been developed for the determination of verapamil and its metabolites in serum samples. Two achiral reversed-phase columns, Hisep C₁₈ (150×4.6 mm) and NovaPak C₁₈ (150×3.9 mm) were used for the simultaneous separation of all analyzed compounds. An α₁-AGP column (100×4.0 mm) was recommended for successful chiral separations of verapamil and its seven metabolites. All analyses were realised with fluorescence detection at λ_ex=276 nm and λ_em=310 nm. Limits of quantitation were in the range 1.0 to 5 ng/ml for all compounds. Both off-line SPE (SepPak C₁₈ cartridges) and the on-line SPE with a semipermeable surface SDS C₈ pre-column, (10×4.6 mm) were used for the clean-up and sample preconcentration. Extraction recoveries for all analyzed compounds were 87.7±5.8 to 92.7±4.0% for off-line SPE and 94.3±4.2 to 98.2±5.1% for on-line SPE. The complete assay could be applied for achiral and chiral monitoring verapamil and all its metabolites in serum samples.     

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.     

Simultaneous analysis of verapamil and norverapamil enantiomers in human plasma by high-performance liquid chromatography

An improved HPLC method for the simultaneous determination of the enantiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N-acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexane). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an α₁-acid glycoprotein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision and robustness of the method. The assay is sensitive and specific. Baseline separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precision and accuracy better than 15% at those levels. Detector response is linear in the range tested (3–200 ng/ml for V and 2–100 ng/ml for NV, single enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers.     

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.     

Determination of the enantiomers of chlorpheniramine and its main monodesmethyl metabolite in urine using achiral–chiral liquid chromatography

The enantiomers of chlorpheniramine and its monodesmethyl metabolite were determined separately in urine by using a coupled achiral–chiral chromatographic system. The two enantiomers of the studied compound and the internal standard were separated from the biological matrix on a cyanopropyl column and reinjected into a chiral amylose AD column where the two enantiomers were separated and quantified by UV detection. The method was validated for chlorpheniramine and for the metabolite within the range 0–1000 ng/ml. It was also applied in a pilot pharmacokinetic study to samples from a volunteer given 8 mg of racemic chlorpheniramine by mouth.     

Determination of pindolol enantiomers in human plasma and urine by simple liquid–liquid extraction and high-performance liquid chromatography

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(−)-α-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C₁₈ silica column and detected using fluorescence (excitation λ: 215 nm, emission λ: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r²≥0.998 over the concentration range 8–100 ng ml⁻¹ for plasma and 0.1–2.5 μg ml⁻¹ for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently <10%. Assay parameters are similar to those of previously published assays for pindolol enantiomers, however this assay is significantly easier and cheaper to run. Clinically relevant concentrations of each pindolol enantiomer can readily be measured.     

A novel solid‐phase extraction–spectrofluorimetric method for the direct determination of atenolol in human urine

A novel, simple, sensitive and selective solid‐phase extraction (SPE)–spectrofluorimetric method has been developed for the determination of atenolol (ATE) in human urine. Because an extraction procedure is required to isolate ATE or eliminate the interfering molecules present in complex human urine for the direct spectrofluorimetric determination, a pH‐sensitive poly(acrylic acid‐ethylene glycol dimethacrylate) [poly(AA‐EGDMA)] hydrogel was developed and used as a SPE adsorbent. Some factors affecting the ATE extraction efficiency, such as washing solvent type and volume, and the volume of elution solvent were optimized. Eluates from SPE cartridges were analyzed using a spectrofluorimeter (λ_ex = 277 nm and λ_em = 300 nm). The calibration graph was linear over the concentration range 0.15–4.0 µg/mL. Limit of detection (LOD) and limit of quantification (LOQ) values were found to be 0.03 and 0.10 µg/mL, respectively. Relatively high intraday [2.06%, mean relative standard deviation (RSD)] and interday (2.6%, mean RSD) precisions were achieved. High mean recovery (95.4%) and low RSD values (3.8%) were obtained for spiked ATE in human urine. The spectrofluorimetric method presented here can be easily applied to assay trace amounts of ATE in pharmaceuticals and biological samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of salbutamol enantiomers in human plasma and urine by chiral high-performance liquid chromatography

Enantiomers of salbutamol were directly separated (R_s=1.16) and quantitated at therapeutic concentrations after solid-phase extraction from human plasma and urine by normal-phase high-performance liquid chromatography on a chiral column with fluorescence detection. The assay was linear for each enantiomer between 1.25 and 500 ng ml⁻¹ and had a minimum limit of detection of 250 pg ml⁻¹. A 3-ml plasma or 1-ml urine sample was required for quantitation at therapeutic doses. Inter-day variation was 50% for S-(+)- and 6.5% for R-(−)-salbutamol. The assay was used to compare enantioselective disposition after single doses of racemate by the intravenous, oral and rectal routes.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Identification and determination of the enantiomers of moprolol and their metabolites in human urine by high-performance liquid chromatography and gas chromatography—mass spectrometry

A simple and sensitive high-performance liquid chromatographic (HPLC) method using chiral derivatization was developed to screen and determine the enantiomers of moprolol and their metabolites in human urine. The recovery of (+)- and (−)-moprolol from urine was 70.8–81.1% at different concentrations. The coefficients of variation (C.V.) were less than 3.2 and 6.5% for intra- and inter-assays, respectively. Moprolol could be detected in urine up to 24 h after oral administration of a 50-mg dose of moprolol. Unconjugated and conjugated enantiomers of moprolol and their metabolites were analyzed by gas chromatography (GC). A gas chromatographic—mass spectrometric (GC—MS) confirmatory method was established to identify the metabolites of moprolol. The double derivatization procedure for moprolol and their metabolites with S-(−)-menthyl chloroformate [(−)-MCF] and N-methyl(trimethylsilyl)trifluoroacetamide (MSTFA) gave very good GC—MS properties of the derivatized compounds and provided reliable structural information for their confirmation analysis. This is the first published report on the use of a GC—MS method for the detection of the enantiomers of moprolol and their metabolites in human urine.     

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane—isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound α₁-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.     

Tryptophan and kynurenine determination in untreated urine by reversed-phase high-pressure liquid chromatography

A fast and sensitive method for the analysis of tryptophan and some of its metabolites is discussed. A reversed-phase chromatographic system with water mobile phase can separate tryptophan, N-formalkynurenine, kynurenine and 3-hydroxykynurenine in less than 15 min at a flow-rate of 1 ml/min. The application of the method to the analysis of tryptophan and kynurenine in untreated urine of a patient loaded with tryptophan is described. The ease and speed of analysis makes the method very attractive for clinical purposes. Among other things, it was found that tryptophan in untreated urine degrades with time, even if the sample is frozen at ?11°.     

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.     

Enantioselective assay for the determination of perhexiline enantiomers in human plasma by liquid chromatography

Effective use of the antianginal agent perhexiline is difficult because saturable metabolism by the polymorphic cytochrome P450 2D6 (CYP2D6) isoform produces elevated plasma perhexiline concentrations that have been associated with serious hepatic and neurological toxicity. Perhexiline is marketed for therapeutic use as a racemate and there is evidence for differences in the disposition of its enantiomers. The current study describes an enantioselective HPLC-fluorescent method utilising pre-column derivatization with (R)-(-)-1-(1-napthyl)ethyl isocyanate. Following derivatization, the enantiomers are resolved on a C18 column with gradient elution using a mobile phase composed of methanol and water. The method described is suitable for the quantification of (+)- and (-)-perhexiline in human plasma following clinical doses and demonstrates sufficient sensitivity, accuracy and precision between 0.01 and 2.00 mg/l for each enantiomer, with intra-assay coefficients of variation and bias <20% at 0.01 mg/l and <10% at 2.00 mg/l, and inter-assay coefficients of variation and biases <15% at 0.03 mg/l and <10% at 0.40 and 0.75 mg/l. The application of this method to plasma samples collected from a patient treated with perhexiline revealed that (+)-perhexiline concentrations were higher than (-)-perhexiline concentrations, confirming the stereoselective disposition of perhexiline. The current study describes an enantioselective method that utilises pre-column formation of fluorescent diastereomers that are resolved on a C18 HPLC column using a gradient of methanol and water.