Determination of albendazole and its main metabolites in ovine plasma by liquid chromatography with dialysis as an integrated sample preparation technique

Albendazole is a benzimidazole derivative with a broad-spectrum activity against human and animal helminth parasites. In order to determine the main pharmacokinetic parameters in sheep after oral and intravenous administration of a new formulation of albendazole (an aqueous solution), a fully automated method was developed for the determination of this drug and its main metabolites, albendazole sulfoxide (active metabolite) and sulfone in ovine plasma. This method involves dialysis as purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatography (LC). All sample handling operations were executed automatically by means of an ASTED XL system. After conditioning of the trace enrichment column (TEC) packed with octadecyl silica with pH 6.0 phosphate buffer containing sodium azide, the plasma sample, in which a protein releasing reagent (1 M HCl) containing Triton X-100 was automatically added, was loaded in the donor channel and dialysed on a cellulose acetate membrane in the static-pulsed mode. The dialysis liquid consisted of pH 2.5 phosphate buffer. By rotation of a switching valve, the analytes were eluted from the TEC in the back-flush mode by the LC mobile phase and transferred to the analytical column, packed with octyl silica. The chromatographic separation was performed at 35°C and the analytes were monitored photometrically at 295 nm. Due to the differences in hydrophobic character between albendazole and its metabolites, a gradient elution was applied. The mobile phase consisted of a mixture of acetonitrile and pH 6.0 phosphate buffer. The proportion of organic modifier was increased from 10.0 to 50.1% in 12.30 min, then from 50.1 to 66.9% in 1.70 min. First, the gradient conditions and the temperature were optimised for the LC separation using the DryLab software. Then, the influence of some parameters of the dialysis process on analyte recovery was investigated. Finally, the method developed was validated. The mean recoveries for albendazole and its metabolites were about 70 and 65%, respectively. The limits of quantification for albendazole and its metabolites were 10 and 7.5 ng/ml, respectively.     

An HPLC Method for the Determination of Verapamil and Norverapamil in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of verapamil and its metabolite norverapamil in human plasma. Verapamil and norverapamil are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 9.5) using ethyl acetate containing trimipramine as an internal standard. A reverse-phase cyanopropylsilane column was used with a mobile phase of 65% acetonitrile and 35% 0.02M acetate buffer (pH 7.0). The minimum detectable limit was 2 ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Specific HPLC Method for the Separation of Verapamil and Four Major Metabolites After Oral Dosing

Abstract

We have developed a high performance liquid chromatographic (HPLC) method which resolves verapamil, norverapamil, D620, D617 and what we believe to be another verapamil metabolite which has been previously unreported. An alkyl-phenyl column is used with a mobile phase of 0.005% sulfuric acid in methanol. The extraction recoveries of verapamil, norverapamil and the internal standard (imipramine) from plasma ranged between 98% and 104%. The day-to-day, and within-day coefficients of variations for verapamil and norverapamil at plasma concentrations of 7.3 and 233 ng/ml ranged between 1.7 and 6.1%. The limit of sensitivity was slightly less than 1 ng for both verapamil and norverapamil. Chromatograms of extracts of serum and urine obtained from five normal subjects who took single oral verapamil doses, indicated the presence of verapamil, norverapamil, and two other known metabolites. Chromatograms of serum extracts also indicated an additional peak which is probably another verapamil metabolite.     

Solid-phase extraction with liquid chromatography and ultraviolet detection for the assay of antidepressant drugs in human plasma

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase high-performance liquid chromatography (HPLC) procedure for the assay of five antidepressant drugs (trazodone, doxepin, desipramine, maprotiline and imipramine) is reported. The drugs were recovered from plasma buffered at a suitable pH using C18 Bond-Elut cartridges and mixtures of methanol-aqueous buffer as washing and elution solvents. The recoveries of the drugs using other sorbent materials (C8, C2, cyclohexyl, cyanopropyl and phenyl Bond Elut and copolymer HLB waters cartridges) were also examined. The selectivity of SPE was examined by using spiked plasma samples and the CH cartridge gave rise to the cleanest extracts. Cyclohexyl cartridges were conditioned successively with 2 ml of methanol and 1 ml of acetic acid-sodium acetate buffer (0.1 M, pH 4.0). Plasma sample was buffered at pH 4.0 and then applied to the sorbent. The washing step was performed subsequently with 1.5 ml of acetate buffer (0.1 M, pH 4.0), 100 microl of acetonitrile and 1 ml of methanol-acetate buffer (30:70, v/v). Finally, the analytes were eluted with 0.5 ml of methanol-acetate buffer (70:30, v/v). The extract was evaporated to dryness, reconstituted in mobile phase, and chromatographed on a reversed-phase C18 column with ultraviolet detection at 215 nm. The recoveries of trazodone, doxepin, desipramine, maprotiline and imipramine from spiked plasma samples using the CH cartridge were 58 2, 84 3, 83 3, 83 3 and 82 2%, respectively. The within-day and between-day repeatabilities were lower than 6% and 9%, respectively. The linearity of calibrations for the five antidepressants was between 0.005 and 2 microg/ml. The limits of detection were 1 ng/ml for trazodone, doxepin and desipramine and 2 ng/ml for maprotiline and imipramine.     

Quantitation of Verapamil and Norverapamil in Small Blood Samples From the Rat by High Performance Liquid Chromatography

Abstract

A simple, sensitive HPLC assay using flurescence detection was developed for quantitation of verapamil and its active metabolite, norverapamil in 100-200 μl blood samples from the rat. Baseline separation of verapamil, normverapamil and internal standard, propranolol, was attained within 14 minutes. Standard curves for verapamil and norverapamil were linear from 7 ng/ml to 1000 ng/ml with limit of detection of 4 ng/ml for both Compounds. the intraday and interday coefficients of variation in verapamil and norverapamil concentrations, determined from spiked whole blood samples, were less than 10%.     

Rapid Quantitation of Verapamil in Plasma by High-Performance Liquid Chromatography

Abstract

A simple and sensitive liquid chromatographic assay procedure using a fluorescence detector for the quantitative determination of verapamil in plasma without extraction was developed. After precipitating the protein with acetonitrile, the resulting supernatant liquid was injected onto the column for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the eluting solvent was the isocratic mixture of methanol, acetonitrile and pH 3.0 glycine buffer (1:4:5). With this mobile phase the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of verapamil from 3 replicate samples of different concentration (100–600 ng/mL) were 95.5 ± 5.68%. The minimum amount of verapamil detectable by this method was 40 ng/mL of sample. The elimination half-life (β-phase) of this drug in rabbits was found to be 3.7 hours.     

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre‐treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.     

Determination of thiolcarbamate herbicides in natural water by HPLC with spectrophotometric detection

Summary Thiolcarbamates used for killing weed seeds were determined in natural waters by HPLC with spectrophotometric detector. Methanol-water (80:20) containing 0.01 mol/l ammonium acetate buffer pH 5.0 was used as mobile phase. The column was thermostated at 35°C. Seppak C18 cartridges were used for sample preparation and preconcentration. The minimum detectable concentration for samples of 100 ml was 0.5 ng/ml.     

Quantitation of nifedipine in human plasma by on-line solid-phase extraction and high-performance liquid chromatography

An analytical methodology for nifedipine quantitation in plasma by on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contain C₂ and the analytes nifedipine and nitrendipine (internal standard) are separated on a C₁₈ column with a mobile phase consisting of acetonitrile–13 mM phosphate buffer pH 7 (65:35, v/v) followed by UV detection at 338 nm. Validation of the method demonstrated good recoveries (>90%), sensitivity (limit of quantification, 2 ng/ml), based on a 500 μl sample volume, accuracy and precision (<5.5% in concentrations greater than the limit of quantitation). This methodology has been used for bioequivalence studies.     

Plasma level monitoring of D,L-verapamil and three of its metabolites by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay was developed for determination of verapamil, norverapamil (M1) and its N-dealkylated metabolites (M2 and M3) in plasma. Plasma samples were vortex-mixed, deproteinized and centrifuged. The analysis was performed on a C18 reversed-phase column with fluorimetric detection. Since the polarity of verapamil and norverapamil differs considerably from that of M2 and M3, two different eluents were used for rapid high-performance liquid chromatographic separation. The eluent for the separation of verapamil and norverapamil was acetonitrile-0.07% orthophosphoric acid (33:67, v/v), and for M2 and M3 acetonitrile-0.07% orthophosphoric acid (25:75, v/v). The high-performance liquid chromatographic assay allowed rapid, sensitive and reliable quantitation of verapamil and three of its metabolites in plasma without an extraction procedure. The limit of detection was less than 5 ng/ml (plasma) for all compounds. No interferences with other commonly co-administered drugs was observed. Plasma concentrations of verapamil and its metabolites were determined in 21 patients receiving a continuous infusion of verapamil for tachyarrhythmia of acute onset. The steady-state plasma concentration data of verapamil and its three main metabolites in these patients gave evidence that the plasma concentration of verapamil and its active metabolite norverapamil was primarily determined by the extent of the formation of M2.     

Automated analysis of oxolinic acid and flumequine in salmon whole blood and plasma using dialysis combined with trace enrichment as on-line sample preparation for high-performance liquid chromatography

The use of dialysis as sample clean-up for high-performance liquid chromatography makes fully automated determination of drugs in whole blood and plasma possible. High recoveries of the analytes oxolinic acid and flumequine and the internal standard nalidixic acid are obtained after a short time of dialysis (7.3 min). The dilute dialysates are enriched on a small column packed with polystyrene. When dialysis is discontinued, the analytes are eluted by mobile phase to the analytical column. With UV detection the limit of detection was 50 ng/ml for both oxolinic acid and flumequine. Validation showed good precision and accuracy and good correlation between determinations in plasma and whole blood.     

Separation and HPLC analysis of 15 benzodiazepines in human plasma

Benzodiazepines (BZDs) are often prescribed to schizophrenic or depressed patients, as a part of polypharmacy regimens. An HPLC method has been developed for the simultaneous determination of 15 BZDs in human plasma. Separation was obtained by using a C8 RP column and a mobile phase composed of 65% aqueous phosphate buffer at pH 3.0 and 35% ACN. The UV detector was set at 220 nm and clomipramine was used as the internal standard. A careful pretreatment procedure of plasma samples was developed, using SPE with C1 cartridges, which gives high extraction yields (> 97%). The LOQs were always lower than 7.6 ng/mL and the LODs always lower than 2.6 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed and schizophrenic patients undergoing polypharmacy with one or more BZDs. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence, the method seems to be suitable for the therapeutic drug monitoring (TDM) of patients undergoing therapy with one or more BZDs.     

Determination of cibenzoline in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (CipralanTM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20). A 10-microns ion-exchange (sulfonate) column was used with acetonitrile-phosphate buffer (0.015 mol/l, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard. The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10-1000 ng/ml and 50-5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Rapid determination of sulphonamides in milk using liquid chromatographic separation and fluorescamine derivatization.

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the filtrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1-4% at 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.     

Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection

A selective, accurate and precise high-performance liquid chromatographic assay coupled to fluorescence detection was developed for the detection of some angiotensin II receptor antagonists (ARA II): Losartan, Irbesartan, Valsartan, Candesartan cilexetil and its metabolite Candesartan MI. The analytes and the internal standard (bumetanide, a high-ceiling diuretic) were extracted from plasma under acidic conditions by means of solid-phase extraction using C8 cartridges. This procedure allowed recoveries close to 80% for all these drugs excluding Candesartan cilexetil (70%) which presented adsorption processes on glass and plastic walls. The analytes and potential interferences were separated on a reversed-phase column, muBondapak C18, at room temperature. A gradient elution mode was used to carry out the separation, the optimal mobile phase being composed of acetonitrile-5 mM acetate buffer, pH 4, at variable flow-rates (from 1.0 to 1.2 ml/min). Fluorescence detector was set at an excitation wavelength of 250 nm and an emission wavelength of 375 nm. Intra- and inter-day relative standard deviations for all the compounds were lower than 8% except for Losartan (12%) and the method assesses a quite good accuracy (percentage of relative error approximately 6% in most of the cases). The limit of quantitation for these compounds was 3 ng/ml for Candesartan cilexetil and M1, 16 ng/ml for Losartan and 50 ng/ml for Irbesartan and Valsartan, which allows their determination at expected plasma concentration levels. This assay method has been successfully applied to plasma samples obtained from hypertensive patients under clinical studies after oral administration of a therapeutic dose of some of these ARA II compounds.     

Piroxicam quantitation in human plasma by high-performance liquid chromatography with on- and off-line solid-phase extraction.

A comparative study of two analytical methodologies for piroxicam quantitation in plasma by off-line and on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contained C8 for both extraction methods. The analytes piroxicam and tenoxican (internal standard) were separated on a C18 column with a mobile phase consisting of acetonitrile:20 mM phosphate buffer pH 3.1 (50:50, v/v) followed by UV detection at 360 nm. The validation of the methods demonstrated good recoveries (over 90%), sensitivity (limits of quantification of 0.05 microgram/ml with on-line SPE and 0.1 microgram/ml with off-line SPE, based on a 100 microliters and 200 microliters sample volume, respectively), accuracy and precision (better than 9.5%). Both methodologies have been used for bioequivalence studies.     

Determination of clomipramine and its hydroxylated and demethylated metabolites in plasma and urine by liquid chromatography with electrochemical detection

A procedure for the determination of clomipramine and its 8-hydroxy, demethyl, 8-hydroxydemethyl and didemethyl metabolites in plasma and urine by high-performance liquid chromatography with electrochemical detection is described. A 1-ml plasma or urine sample is made alkaline with a carbonate buffer (pH 9.8) and extracted with 20% ethyl acetate in n-heptane. After back-extraction into an acid phosphate buffer (pH 2.4), an aliquot is injected into a 5-microns ion-paired reversed-phase column and eluted with a mobile phase containing a phosphate buffer with tetramethylammonium chloride-acetonitrile (57:43). The detection is coulometric with a first cell at +0.40 V, a second at +0.73 V and a guard cell set at 0.75 V for oxidation of the mobile phase. The method provides recoveries in the general range of 80-110% and a day-to-day precision of 3.7-8.8%, depending on the compound. The minimum quantifiable level for all compounds was 0.2 ng/ml with a 20-microliters injection. Steady-state plasma concentration data and urinary levels are reported for 24 depressed patients receiving daily either 75-150 mg orally or 50-75 mg by infusion.     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

A common method for the determination of several calcium channel blockers using an HPLC system with ultraviolet detection

We report a common HPLC method for the single or simultaneous determination of four calcium channel blockers (CCB), namely diltiazem (DTZ), verapamil (VER), nifedipine (NIF) and nitrendipine (NIT) and their active metabolites demetildiltiazem and deacetildiltiazem (MA and M1), norverapamil (NOR), and dehydronifedipine (DHN). DHN was first synthesised in our laboratory and different pH values of the mobil phase were subsequently prepared and tested for chromatographic separation. The detection system and the environmental light conditions were optimised. The best separations of all analytes were obtained using a C₁₈ column and a mobile phase of methanol, 0.04 M ammonium acetate, acetonitrile and triethylamine (2:2:1:0.04 v/v). Quantitation was performed using imipramine (IMI) as the internal standard. For DTZ and its metabolites (M1 and MA), the wavelength chosen was 237 nm; for VER and its metabolite NOR, it was 210 nm; and, finally for NIF and its metabolite DHN and NIT it was 216 nm. When a simultaneous analysis was carried out the wavelength was of 230 nm. The optimum pH were 7.90 and 7.10 when the separation of NIT and DTZ or VER and NIF were carried out, respectively, and 7.90 when a simultaneous separation was carried out. The detection limit of the assay was less than 8 ng ml⁻¹ for all compounds, with coefficients of variation less than 7% (for inter- and intra-day) over the concentration range of 1–1000 ng ml⁻¹. The retention times were less than 11 min. When NIF or NIT were studied, it was necessary to use a sodium vapour lamp in order to avoid the photodegradation which takes place under daylight conditions.     

High Performance Liquid Chromatographic Determinations of Sulfonamides by Ionic Suppression

Abstract

A high pressure liquid chromatography procedure is reported for extraction and quantitation of 8 sulfonamides in stock solutions and in vitro plasma samples. This assay consists of a single, one-step extraction of sulfonamides from plasma and is sensitive to 10.0 ng/ml at 254 nm without additional concentration of the sample. Four sulfonamides (sulfamerazine, sulfamethazine, sulfapyridine and sulfathiazole) were separated from the plasma matrix by either mobile phase regardless of pH. The sulfonamides with the highest pKa, sulfanilamide (10.5) and sulfaguanidine (11.3), were only separable from plasma in a 50% water/50% methanol mobile phase at pH 7.45. The sulfonamide with the lowest pKa, sulfisoxazole (4.9), and its metabolite, acetylsulfisoxazole (N⁴), were separated from plasma by either mobile phase, 50/50 or 60/40 water/methanol, when acetate buffer reduced the pH to 4.00. Standard concentration curves of peak height were the most sensitive at 254 nm when a 60% water/40% methanol mobile phase at pH 4.00 was used. Sulfanilamide and sulfaguanidine were the most responsive to ultraviolet quantitation at 254 nm regardless of ionic suppression or polarity of the mobile phase.