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%.     

Determination of the calcium entry blocker verapamil in plasma by capillary gas chromatography with on-column injection

A specific, sensitive, and accurate assay for quantitation of verapamil has been developed. The method involves a single extraction step with n-heptane, followed by evaporation at room temperature under nitrogen. 0.1 μl of the extract was injected into a capillary column coated with crosslinked 5% phenylmethylsilicone. The column separated verapamil, norverapamil, and its internal standard within 15 minutes using temperature programming from 90 to 290°C. The lower limit of detection was 1 ng/ml for verapamil. The calibration curve was linear in the concentration range (5–300 ng/ml). Plasma concentration data from dogs receiving intravenous verapamil infusion are presented.     

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.     

Rapid analysis of ranitidine in biological fluids and determination of its erythrocyte partitioning

A reversed-phase ion-pair high-performance liquid chromatographic assay is described for the rapid and sensitive quantitation of the H2-receptor antagonist ranitidine in human plasma and urine. The method involves a single-step extraction of the alkalinized sample with methylene chloride and analysis of the evaporated extract on a cyano column. Detection was performed by ultraviolet absorbance monitored at 318 nm. The overall run time of the assay was 5 min at a flow-rate of 2.0 ml/min. The limit of sensitivity was 1 ng/ml ranitidine in human plasma. Urine and plasma samples collected from a subject after administration of an oral dose of 150 mg of ranitidine were analyzed by this method. Furthermore, the procedure was applied to determine the red blood cell partition coefficient of ranitidine in a concentration range up to 10 micrograms/ml.     

Simple and rapid high-performance liquid chromatographic assay for esmolol.

A procedure for determining esmolol concentrations in blood is described. Dichloromethane was used to extract esmolol from the blood and to inhibit the activity of blood esterases. Blood esmolol concentrations were determined by high-performance liquid chromatography using 3-methoxy-O-demethylencainide as the internal standard. The limit of detection of this assay was 5 ng/ml. The relationship between the peak-height ratio of esmolol and the internal standard was linear in the concentration ranges 10-30,000 ng/ml. The mean absolute and relative recoveries of esmolol from blood were 84 and 89%, with coefficients of variation less than 3%. This method has been used in our laboratory for pharmacokinetic and pharmacodynamic studies.     

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.     

Quantitative determination of tramadol in human serum by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the quantitative determination of tramadol in human serum, plasma or whole blood samples is described. The method involves the use of [2H2, 15N]tramadol hydrochloride as an internal standard and chemical ionization with isobutane, employing single-ion monitoring for quantification. It is specific, sensitive and precise, and has high accuracy. The within-run coefficient of variation is about 1% between 25 and 200 ng/ml and 1.8-2.9% at the lowest concentrations tested (6.25 and 12.5 ng/ml). The between-run coefficient of variation increases from 1.6% to 5.2% with decreasing concentration from 200 to 12.5 ng/ml. The calibration graphs were linear in the tested concentration range, and the accuracy of the assay was not dependent on the sample volume used. The detection limit was about 4 ng/ml for serum samples of 1 ml. The method proved suitable for pharmacokinetic studies. Its high sensitivity allows measurements of serum concentrations for at least 30 h after the single administration of therapeutic doses of tramadol hydrochloride.     

A Direct and Sensitive Method for the Determination of Salicylamide in Microplasma Samples by High-Performance Liquid Chromatography Using Fluorescence Detection

Abstract

A quantitative analysis of salicylamide in microplasma volumes by high-performance 1iquid chromatography using fluorescence detection is reported. The procedure is extremely simple and very rapid, involving the direct introduction of the plasma sample on the HPLC column. The assay procedure is linear over the concentration range studied, 0–100 ng/ml with correlation coefficient for the linear regression, r = 0.998. This assay procedure enables the detection of salicylamide as low as 5.0 ng/ml in plasma, using sample volume of 100 μl.     

HPLC Measurement of Cyclosporine in Blood Plasma and Urine and Simultaneous Measurement of its Four Metabolites in Blood

Abstract

A high performance liquid chromatographic assay has been developed for the estimation of cyclosporine and its four major metabolites in blood and for cyclosporine alone in plasma and urine samples. This assay employs a rapid and very reproducible solid-liquid extraction system. Isocratic chromatographic conditions allow the simultaneous measurement of cyclosporine and its four major metabolites in blood. The method is linear up to 2500 ng/ml and the minimum quantifiable limit for cyclosporine is 30 ng/ml, when 1 ml of sample is analyzed.     

Automated determination of verapamil and norverapamil in human plasma with on-line coupling of dialysis to high-performance liquid chromatography and fluorometric detection

A fully automated method for the simultaneous determination of verapamil and its main metabolite norverapamil in human plasma is described. This method is based on on-line sample preparation using dialysis followed by clean-up and enrichment of the dialysate on a precolumn and subsequent HPLC analysis with fluorometric detection. All sample handling operations were performed automatically by a sample processor equipped with a robotic arm (ASTED system). The plasma samples were dialysed on a cellulose acetate membrane (cut-off: 15 kD) and the dialysate was purified and enriched on a short pre-column filled with cyanopropyl silica. Before starting dialysis, this trace enrichment column (TEC) was first conditioned with the HPLC mobile phase and then with pH 3.0 acetate buffer. 370 μl of plasma sample spiked with the internal standard (gallopamil) were dialysed in the static-pulsed mode. The solution at the donor side was pH 3.0 acetate buffer containing Triton X-100 while the acceptor solution was made of the same acetate buffer. When dialysis was discontinued, the analytes were desorbed from the TEC by the HPLC mobile phase and transferred to the C₁₈ analytical column by means of a switching valve. This mobile phase consisted of a mixture of acetonitrile, pH 3.0 acetate buffer and 2-aminoheptane. The influence of different parameters of the dialysis process on the recovery of verapamil and norverapamil has been studied. The effect of the volume, the aspirating and dispensing flow-rates of the dialysis solution has been investigated. The recoveries of verapamil and norverapamil in plasma were close to 75% and the limits of quantification were 5 ng/ml for both analytes. The method was found to be linear in the concentration range from 5 to 500 ng/ml (r²: 0.9996 for both analytes). The intra-day and inter-day reproducibilities at a concentration of 100 ng/ml were 2.3% and 5.6% for verapamil and 1.7% and 5.1% for norverapamil, respectively.     

Determination of cyclosporin A in whole blood by high-performance liquid chromatography using automated column switching

A fully automated high-performance liquid chromatographic column-switching system is presented for the determination of cyclosporin A in whole blood. After blood proteins were precipitated with acetonitrile, the supernatant was automatically loaded on to a cyanopropyl column for initial separation, and then the fraction containing cyclosporin A was loaded on to a trimethylsilica column for final separation and quantitation. Cyclosporin A was detected by ultraviolet absorption at 205 nm. The minimum detectable concentration of cyclosporin A was 5 ng/ml in 100 microliter of blood. The coefficient of variation of the method was 1.755, 1.748 and 0.655% in whole blood when spiked at the 170, 425 and 850 ng/ml levels, respectively. One assay was completed in 15 min.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Gas chromatographic assay for the new antitumor agent pyrazine-2-diazohydroxide (diazohydroxide) and its stability in buffer, blood and plasma

Diazohydroxide is a new antitumor agent being considered for clinical trial. A sensitive and specific assay for diazohydroxide in physiological media, plasma and blood has been developed based on conversion of diazohydroxide to 2-chloropyrazine in the presence of strong hydrochloric acid. The 2-chloropyrazine is extracted into the ethyl acetate and separated by capillary gas chromatography with nitrogen-phosphorus detection. Using 0.2 ml plasma the assay was linear up to 100 micrograms/ml diazohydroxide and had a lower limit of detectability for diazohydroxide of 50 ng/ml. The coefficient of variation of the assay at 1 micrograms/ml was 6.7%. Breakdown of diazohydroxide was rapid under mild acid conditions but slower under alkaline conditions,. The half-life of diazohydroxide in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature was 5 min and at pH 8.0, 480 min. Breakdown of diazohydroxide in plasma was biphasic. In fresh mouse plasma diazohydroxide had a terminal half-life at 37 degrees C of 72 min while in fresh human plasma the terminal half-life was 23 min and in fresh blood 21 min. Diazohydroxide accumulated in red blood cells at 37 degrees C to a concentration 68% above the concentration in plasma. Diazohydroxide was 49% bound to human plasma proteins at room temperature.     

Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection.

An improved method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (alendronate) in human urine and an assay in human plasma are described. The methods are based on co-precipitation of the bisphosphonate with calcium phosphates, automated pre-column derivatization of the primary amino group of the bisphosphonic acid with 2,3-naphthalene dicarboxyaldehyde (NDA)-N-acetyl-D-penicillamine (NAP) or cyanide (CN-) reagents, and high-performance liquid chromatography (HPLC) with electrochemical (ED) or fluorescence detection (FD). The feasibility of ED of the NDA-CN- derivative of aldendronate has been demonstrated, and a HPLC-ED assay in human urine has been validated in the concentration range 2.5-50.0 ng/ml. In order to eliminate the cyanide ion from the assay procedure, several other nucleophiles in the NDA derivatization reaction were evaluated. An NDA-NAP reagent was found to produce highly fluorescent derivatives of alendronate. The assay in urine based on NDA-NAP derivatization and HPLC-FD has been developed and fully validated in the concentration range 1-25 ng/ml. Based on the same NDA-NAP derivatization, an assay in human plasma with a limit of quantification of 5 ng/ml has also been developed. Both HPLC-FD assays were utilized to support various human pharmacokinetic studies with alendronate.     

Ion-Paired Liquid Chromatographic Method for the Analysis of Pyridostigmine in Plasma

Abstract

A high performance Liquid chromatographic (HPLC) procedure for the analysis of pyridostigmine in plasma has been developed. Only 0.5 ml of plasma is required for the analysis. The clean-up procedure involves a protein precipitation step and a column elution step prior to separation by HPLC. The assay is quite sensitive with a detection limit of 1.37 ng/ml for pyridostigmine in plasma. Variability of results ranged from 3 to 14% on evaluations of assay precision. Accuracy of results, evaluated using blind samples in the range of 0–50 ng/ml, differed between 6 and 12% from blind sample values. Stability was also determined for pyridostigmine in plasma at ?20°C and ?80°C. The results showed no degradation for pyridostigmine at ?80°C for up to four months. In a preliminary study with one human volunteer, the drug could be detected up to 12 hours following oral syrup solution doses of between 0.4 and 0.9 mg/kg. This assay is suitable for pharmacokinetic studies involving pyridostigmine in human subjects.     

Gas chromatographic assay for the new antitumor agent sulfamic acid diester (NSC 329680) and its stability in buffer, blood and plasma

A sensitive gas chromatographic assay with electron-capture detection has been developed for sulfamic acid diester (sulfamic acid 1,7-heptanediyl ester, NSC 329680) based on its conversion to 1,7-diiodoheptane in the presence of excess sodium iodide. The assay is linear up to 1 microgram/ml sulfamic acid diester and has a lower limit of detection of 25 ng/ml from 0.5 ml plasma. The coefficient of variation of the assay is 6.4% at 1 microgram/ml and 8.0% at 100 ng/ml. Sulfamic acid diester is relatively stable in 0.9% sodium chloride and 0.1 M sodium phosphate buffers, pH 7.0-9.0, with half-lives greater than 38 h. The major breakdown product of sulfamic acid diester is sulfamic acid 1,7-heptane-monoyl ester. When added to whole blood sulfamic acid diester shows concentration-dependent breakdown. At 50 and 100 micrograms/ml sulfamic acid diester, the half-time in whole blood is 6.9 h and 65% of the drug is sequestered by the blood cells. At 10 micrograms/ml sulfamic acid diester in blood, there is no detectable breakdown of the drug over 24 h and all of the drug is sequestered by the blood cells. Protein binding of sulfamic acid diester in human plasma is 82% at 10 micrograms/ml and 68% at 100 micrograms/ml.     

Determining pirimiphos-methyl in durum wheat samples using an acetylcholinesterase inhibition assay

An electrochemical assay used for detecting acetylcholinesterase (AChE) inhibitors has been optimised to detect pirimiphos-methyl in durum wheat. Pirimiphos-methyl is a phosphothionate insecticide and so it needs to be transformed into the corresponding oxo form to act as an effective AChE inhibitor. The inhibition assay was based on the electrochemical detection of the product of AChE, choline, via choline oxidase biosensors obtained with Prussian-Blue modified screen printed electrodes. The procedure for the oxidation of pirimiphos methyl via N-bromosuccinimide (NBS) and AChE inhibition was optimised for reagent concentrations and inhibition time in a buffer solution. A calibration of the pirimiphos-methyl (25–1,000 ng/ml) was obtained in the buffer. The intra-electrode CV ranged between 1.6 and 15.0, whereas the inter-electrode CV ranged between 4.6 and 16.0. The detection limit (LOD) was 38 ng/ml, and the I_50% was 360 ng/ml. The assay conditions were then re-optimised to work with durum wheat extracts, and the calibrations were obtained under different experimental conditions, such as sample pretreatment (milled or whole grains) and extract concentration. The calibrations were slightly affected by the sample matrix, resulting in an increased LOD (65–133 ng/ml) and I_50% (640–1,650 ng/ml). The LOD found for the sample, determined under optimal conditions, was 3 mg/kg. Spiked samples were prepared at the EU regulated level (5 mg/kg) and analysed with the optimised protocol, resulting in an average recovery of 70.3%.     

High-performance liquid chromatographic method for determination of ciprofloxacin in biological fluids

A simple and precise high-performance liquid chromatographic procedure has been developed for the determination in biological fluids of ciprofloxacin, a new, with extended antibacterial spectrum, quinoline carboxylic acid. The work-up procedure involves a chemical extraction step followed by isocratic chromatography on a reversed-phase analytical column, with ultraviolet detection. The detection limit for blood levels is 10 ng/ml. The calibration curve is linear from this detection limit to 10 microgram/ml. The statistical analysis of the correlation made between this assay and an agar diffusion procedure during a pharmacokinetic study suggests the existence of one or more active metabolites which could be mainly excreted in the bile.     

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.     

A Disposable Carbon Nanotubes‐screen Printed Electrode (CNTs‐SPE) for Determination of the Antifungal Agent Posaconazole in Biological Samples

For the first time, the use of carbon nanotubes was exploited for the development of a sensitive electrochemical method for determination of the newly antifungal posaconazole (POS). The electrochemical activity of POS was investigated at the surface of multi‐walled nanotubes (MWNTs) modified electrode. The cyclic voltammograms showed a sharp oxidation peak at potential around 671 mV vs. Ag/AgCl. To reach the assay optimization, factors affecting the method sensitivity have been investigated, such as types of carbon nanotubes and its concentration in the electrode matrix, type of supporting electrolyte, pH, accumulation time and scan rate. A good linear relationship was obtained within the concentration range from 32–1280 ng/ml with the limit of detection and quantification of 11 ng/ml and 33 ng/ml, respectively. The proposed method was successfully applied for the determination of POS in its commercial dosage form, spiked human plasma samples, and dried blood spots. The in vivo results obtained were also used to study the pharmacokinetics of POS in human plasma. The results obtained were validated and found to be in accordance with those obtained by the reference methods.