HPLC Determination of Xylazine in Equine Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) method is described for the determination of xylazine in equine plasma. The drug and internal standard (pindolol) were separated on a 5 μm cyanopropyl-modified column (250 × 4.6 mm i.d.) using a buffer-acetonitrile mixture containing an ion pairing reagent. The drug and internal standard were isolated from plasma by liquid extraction into ethyl acetate. The method was validated over the concentration range 50–2000 ng/ml in plasma; the reproducibility, expressed as the mean co-efficient of variation was less than 5.0% for both between-day and within-day replicate determinations. The method was linear over the concentration range studied. No interferences were observed from endogenous plasma components and the limit of detection was 20 ng/ml. The method was successfully applied to the determination of xylazine in equine plasma in a crossover study design for pharmacokinetic measurements.     

A Simple High-performance Liquid Chromatographic Assay for Loracarbef in Human Plasma

Abstract

A simple, rapid and reproducible high-performance liquid chromatographic (HPLC) method for the determination of loracarbef in human plasma has been developed and evaluated. Plasma protein was precipitated with ammonium sulfate. The drug and the internal standard (Cefetamet) were eluted from a μ-bondapak C-18 column with a mobile phase consisting of acetonitrile:methanol:water:glacial acetic acid (2.5:17.5:79.2:0.8%, v/v). The column eluent was monitored at 265 nm. Quantification was achieved by the measurement of the peak-height ratio of the analyte to the internal standard and the limit of quantification for loracarbef in plasma is 0.5 ug/ml. The within-day coefficient of variation (CV) ranged from 2.28% to 3.67%, and between-day CV from 2.38% to 5.59% at three different concentrations. The absolute recoveries ranged from 91.1% to 93.88%, and the relative recoveries from 93.4% to 108% at three different concentrations. Preliminary stability tests showed that loracarbef is stable for at least 5-weeks in human plasma after freezing. The method is applied for the determination of the pharmacokinetic parameters of loracarbef after oral administration to 2 beagle dogs.     

High Performance Liquid Chromatographic Determination of Amiodarone in Plasma and Tissues

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of amiodarone (AD) in plasma and tissues was developed. The method involved deproteinization of plasma or homogenized tissue with acetonitrile containing an internal standard (N-Cetylpyridinium chloride) followed by reversed phase chromatography using μ bondapack C₁₈ column (10μm) with a mobile phase consisting of acetonitrile - methanol - sodium dihydrogen phosphate buffer (70:10:20%, v/v), the pH adjusted to 4.0 and pumped at flow rate of 1.0 ml/min. The column effluent was monitored at 242 nm. A linear relationship was obtained between peak height ratios (drug to internal standard) versus drug levels over the concentration range of 50–750 ng/ml. The detection limit of AD in plasma and tissues by this method was 20 ng/ml.     

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

High Pressure Liquid Chromatographic Determination of Mecillinam in Human Plasma and Urine

Abstract

A simple, specific, rapid and sensitive method for the analysis of mecillinam in plasma and urine using high pressure liquid chromatography is described. The assay is performed by direct injection of a plasma protein free supernatant or a dilution of urine. A μBondapak phenyl column with an eluting solvent of 16% CH₃CN-0.2% H₃PO₄ was used, with UV detection of the effluent at 220 nm. Desacetyl-cephalothin was used as the internal standard and quantitation was based on peak height ratio of mecillinam to that of the internal standard. The lowest concentration detectable without extraction was 0.25 μg/ml for plasma and 8.9 μg/ml for urine. No interference from plasma and urine was noted.     

A Sensitive High Performance Liquid Chromatographic Determination of Clopamide in Human Plasma

Abstract

A simple and sensitive high-performance liquid chromatographic method for quantitation of clopamide in human plasma has been developed. the assay uses a reversed-phase C18 microbore column (2 mm I.D. × 100 mm) packed with 5 μm ODS Hypersil. the chromatographic separation was achieved by using an isocratic mobile phase comprising acetonitrile-10 mM phosphate buffer pH 4 (17:83, v/v) at a flow rate of 0.5 ml/min. the eluant was monitored by a UV detector operating at 241 nm. the assay was based on an organic extraction before chromatographic separation. to 1 ml plasma sample, 100 μl of the internal standard, methylparaben (300 ng/ml), and 8 ml of diethyl ether were added. the samples were shaken and centrifuged, the organic layer was then transferred to a tapered centrifuge tube and evaporated to dryness. the residue was reconstituted and injected onto the HPLC column. the inter-and intra-assay coefficients of variation were found to be less than 10%. the lowest limit of detection for clopamide in plasma was 5 ng/ml. the method is sensitive, specific and allows for routine analysis in the pharmacokinetic studies.     

Routine Determination of a New 1–4 Dihydropyridine,Oxodipine, in Human Plasma by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A rapid, specific and reproducible high-performance liquid chromatographic routine assay with electrochemical detection was developed for the determination of Oxodipine in human plasma.

After extraction at alkaline pH by cyclohexane, Oxodipine and its internal standard were chromatographied on a reversed-phase column.

Calibration curves were linear over a concentration range of 1–50 ng/ml with relative errors within-day or between-day not exceeding 8% at any level.

The limit of detection was 30 pg injected based on a signal-to- noise ratio of 7. However, the reliable limit of quantification was 1 ng/ml using 1 ml of human plasma.

A dual-electrode coulometric detector was operated in a screening mode of oxidation, providing a greater specificity and reducing background noise.

This method allowed the complete follow-up of clinical pharmacokinetic studies and drug monitoring in patients.     

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 Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Determination of Metoclopramide in Serum by HPLC Assay and Its Application to Pharmacokinetic Study in Rat

Abstract

A simple, sensitive, and reproducible HPLC method has been developed for the determination of metoclopramide employing reversed phase high performance liquid chromatography with UV detection at 270 nm. The separation was performed on a Novapak C₁₈, 4 μm (3.9 × 150 mm) column. Acetonitrile (18%) in 0.02 M ammonium acetate containing 0.1% triethylamine was used as the mobile phase and the run time was 7 min. Tramadol was used as the internal standard. The mean retention times of metoclopramide and tramadol were 3.4 and 4.6 min, respectively. Linear response (r > 0.997) was observed over the range of 0.025–5 μg/ml of metoclopramide. There was no significant difference (p < 0.05) between inter- and intra-day studies for metoclopramide. The mean relative standard deviations (RSD%) of the results of within-day precision and accuracy of the drug were < 10%. The applicability of the assay was demonstrated in measuring metoclopramide pharmacokinetics in rats. The elimination half-life was 2.09 ± 0.39 h with an apparent clearance of 2.45 ± 0.70 (L/h)/kg in rat.     

A Stability-Indicating Hplc Analysis of Famotidine and Its Application to Kinetic Studies

Abstract

A stability-indicating HPLC analytical method has been developed for the determination of the H₂-receptor antagonist, famotidine in the presence of its degradation products. the method utilizes reversed phase chromatography with UV detection and internal calibration techniques. the mobile phase was comprised of 84% ammonium acetate buffer (pH 2.9) and 16% acetonitrile and pumped at a flow rate of 1.5 ml/min. Quantitation was performed by measuring the peak height ratio of drug to internal standard (salicylic acid). the limit of famotidine detection was determined to be 10 ng (0.4 ug/ml) with a signal to noise ratio of 3:1. Within day coefficient of variation of the method was 2.22% (2.5 μg/ml) and 0.82% (10 μg/ml). Between day coefficient of variation based on the slopes of daily prepared standard curves was 4.70%. the developed method was used to determine the drug content of famotidine tablets. Further, it was used to investigate the kinetics of degradation of the drug in an acidic solution.     

Reverse Phase HPLC Determination OF 5,6-Dihydro-5-azacytidine in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method which allows measurement of the new antitumor agent 5,6-dihydro-5-azacytidine (DHAC) in biological fluids at concentrations as low as 50 ng/ml (2 × 10^?7 M) has been developed. After addition of 5′-chloro-5′-deoxy-5,6-dihydro-5-azacytidine as an internal standard, sequential ultrafiltration, boronate gel affinity chromatography and cation exchange chromatography are employed to isolate DHAC from plasma or urine. DHAC is then reacted with N,N-dimethylformamide diethylacetal to form a dimethylaminomethylene derivative with enhanced UV detectability (λmax = 264 nm, log ε = 4.3) and better retention on a reverse phase column. Isocratic separation is then accomplished on a fully loaded and end-capped ODS column with 0.050 M formic acid in 20% acetonitrile/water. This assay has been used to determine the plasma pharmacokinetics of DHAC in rats given a single i.v. bolus dose of 50 mg/kg. Analysis of the drug in human plasma indicates that this method is suitable for determining DHAC disposition and pharmacokinetics in human subjects.     

High Performance Liquid Chromatongraphy of ABBOTT-53385 in Dog,Rat, and Human Plasma

Abstract

A simple, sensitive and reproducible high-performance liquid chromatographic (HPLC) method was developed to monitor plasma ABBOTT-53385 (I) levels in dogs, rats and humans. The samples were first supplemented with the internal standard, then extracted on Bond Elut® extraction columns. They were analyzed by reverse-phase HPLC with UV detection at 240 nm. The calibration curve was rectilinear over the range of 0.05–2.0 μg/ml and the interday variance was less than 4%. The limit of detection for this method was about 0.03 μg/ml.     

Rapid Determination of Bupropion in Human Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) technique has been developed for the determination of bupropion hydrocloride (Bup) in human plasma, using a reversed-phase method, with UV detection at 250 nm.

The internal standard 5-(P-methylphenyl)-5-phenylhydantoin (MPPH), was used as an aid to quantitation. The plasma was deprotemized with acetonitrile and the clear supernatant was directly injected in the chromatographic system. The lower limit of quantitation was 5.0 ng/ml using only 100 μl of plasma sample.

Linear regression analysis for the calibration plots obtained on five different days over a two-week period for the the two ranges used (10–250 ng/ml and 250–2000 ng/ml) in plasma indicated excellent linearity and reproducibility. The mean recovery of spiked Bup in plasma samples over the concentrations studied was found 96.5 ± 3.14%.

The method revealed that more than 30% of Bup was lost when the supernatant was stored at room temperature for 24 hrs.     

Sensitive HPLC Assay for Ketoprofen in Human Plasma and its Application to Pharmacokinetic Study

Abstract

A selective and sensitive HPLC method was developed for the analysis of ketoprofen in human plasma. The assay involves an extraction of the drug and the internal standard (piroxicam) into diethyl ether from acidified plasma and then back-extracted into a small volume of alkaline aqueous solution before injection onto the HPLC column. A microbore column (2 mm I.D. × 10 cm) packed with a C18 reversed-phase material (5 pm ODS Hypersil) was used. The chromatographic separation was accomplished with a mobile phase comprising a mixture of acetonitrile-methanol-water (15 :20 : 65, v/v) containing 10 mM Na2HP04 buffer, pH 4. The mobile phase was pumped at a flow rate of 0.5 dmin. The eluant was monitored at 258 nm. With this procedure coefficients of variation were less than 10%. The detectionlimit was 0.05 μg/ml (i.e., 50 ng/ml) of plasma. The highly sensitive nature of this method was applied successfully to the dewmination of ketoprofen in human plasma for phmacokinetic studies.     

A Reversed-Phase HPLC Method For Determining Camptothecin In Plasma With Specificity For the Intact Lactone Form of the Drug

Abstract

Camptothecin is a pentacyclic indole alkaloid with a terminal α-hydroxy-δ-lactone ring, which in aqueous media at physiological pH, exists in equilibrium with the dissociated open-lactone carboxylate. the rate of equilibration between the two components is slow enough to permit their separation by reversed-phase HPLC. Selective determination of the intact lactone form of the drug was achieved by direct analysis of plasma samples immediately upon deproteinization with a solution of the internal standard in methanol chilled to ?70°C. Acidification of the sample to pH 2 with perchloric acid prior to protein precipitation effected complete lactonization of the carboxylate and, therefore, provided a measure of total drug levels. Plasma concentrations of the carboxylate may be calculated from the difference between total drug and intact lactone determinations. Chromatography was performed on a 5 μm Ultrasphere ODS column (4.6 mm × 25 cm) preceded by a 1.5 cm RP-18 Brownlee Guard column with an eluent composed of acetonitrile-0.1 M ammonium acetate buffer, pH 5.5 (28:72, v/v) with 1 mM sodium dodecyl sulfate at a flow rate of 1.0 ml/min. the drug was monitored by fluorescence detection with excitation at 347 nm and a 418 nm emission cutoff filter. Approximately 3.0 hr was required to assay an 8 point standard curve and a drug-free plasma sample. Employing 50 μl of plasma, the lowest concentration on the camptothecin lactone and total drug standard curves, 2.82 nM (0.49 ng/ml), was quantified with 3.79 and 5.58% coefficients of variation, respectively. the method has been shown to be specific and reproducible.     

Rapid Determination of Teicoplanin in Human Plasma by High Performance Liquid Chromatography

Abstract

A rapid isocratic reversed-phase HPLC method for the determination of the major component of teicoplanin in plasma is described.

By using solid phase extraction C18 and UV detection at 240 nm, this method is specific, and sufficiently sensitive.

We found a good linearity over the range: 5 – 40 mg/1 of teicoplanin plasma concentrations. The coefficients of variation did not exceed 5.4% both within-day and between-day assays.

With the small plasma sample required for the analysis (250 μl) and the good accuracy, this rapid procedure appears to be suitable for the therapeutic drug monitoring.     

A Rapid High Pressure Liquid Chromatographic Assay of Benoxaprofen in Plasma

Abstract

A rapid high-performance liquid chromatographic assay for the determination of the anti-inflammatory drug benoxaprofen in human plasma, is described. Plasma samples of 1.0 ml, to which benoxaprofen, and warfarin as an internal standard, had been added, were extracted with ether under acidic conditions. The samples were analyzed on a MicroPak CN-10 column using 25% acetonitrile in water (pH 2.5 with phosphoric acid). Detection was made on a variable wavelength UV absorbance detector at 309 nm.

Samples containing 0.5–10 μg benoxaprofen gave a mean extraction recovery from control plasma of 90.6 ± 6.8% (n=18). Stability tests have shown that benoxaprofen in plasma is stable for at least two weeks after freezing.     

HPLC Assay of a Leukotriene D4 Antagonist,in Pharmaceutical Preparations and Biological Samples with UV and Fluorescence Detection

Abstract

Analytical methodology has been developed to assay L-660, 711, 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thio)propionic acid to assess drug stability in pharmaceutical preparations and drug concentration in plasma samples. Reversed-phase HPLC chromatography with UV detection at 232 nm and 300 nm was used to analyze drug content and assess stability in pharmaceutical formulations. In addition to UV, fluorescence detection with 300 nm and 400 nm as the excitation-emission wavelength pair was used to enhance sensitivity for biological samples with low drug concentrations. The latter system had a detection limit of 1–5 ng/ml for a 25 μl injection. It was used to monitor drug concentration in plasma following intrapulmonary, intravenous and oral administration.     

A Simple and Sensitive HPLC Method for Antipyrine in Plasma

Abstract

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of antipyrine in small volume (50 μl) of plasma samples. Aminopyrine was employed as the internal standard. The sample preparation is a direct plasma protein precipitation procedure so is less tedious and rapid. The assay employs a column packed with a C₁₈ reversed-phase material (5 μm Nucleosil) with an isocratic mixture of acetonitrile and water (25:75, v/v) as the mobile phase. The eluant was detected at 254 nm. The assay achieves the level of sensitivity (0.5 μg/ml) and accuracy required to obtain meaningful data about the single-dose pharmacokinetics of antipyrine in guinea pig and rat. The method gave high reproducibility with coefficients of variation less than 5%.