An Improved Method for the Determination of Reserpine in Plasma Using Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method coupled with fluorescence detection was developed to measure plasma reserpine concentrations. After extraction from 3 ml of plasma, the reserpine and internal standard (methyl-18-triethoxy benzoyl reserpate) residues were oxidized to their respective fluorophors by vanadium pentoxide and chromatographed on a reversed phase trimethylsilyl column. These compounds were detected at excitation wave length 460 nm and analyzed at 570 nm. The minimum quantifiable level was ca 0.3 ng/ml and the absolute recovery was determined to be between 78–83%. The coefficient of variation was less than 9% for day-to-day and within run analyses. This method is suitable for pharmacokinetic studies of reserpine in man.     

Simultaneous Determination of Chloroquine and Desethylchloroquine in Blood,Plasma and Urine by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method is described for the determination of chloroquine and its major metabolite desethylchloroquine in blood, plasma and urine. The procedure employs reversed-phase chromatography, with ultraviolet detection, and chlorpheniramine as an internal standard. One milliliter samples of biologic fluid are extracted in a single step with ether. The method has a sensitivity limit of 5 ng/ml for chloroquine and its metabolite. The applicability of the method is demonstrated by the analysis of blood and plasma samples obtained from rabbits following intravenous administration of chloroquine.     

Analysis of Norethindrone In Plasma by High-Performance Liquid Chromatography

Abstract

A sensitive specific high-performance liquid chromatographic procedure for the determination of norethindrone in plasma is described. The organic solvent extract from plasma is chromatographed on a reversed phase column using a high-performance liquid chromatograph fitted with an ultraviolet detector (254 nm); quantitation from plasma samples containing 2 ng/ml norethindrone is reported. Metabolites and endogenous substances do not interfere with the assay. The determination of norethindrone concentrations in plasma following administration of single oral dose to a mini-pig is described.     

Improved High Pressure Liquid Chromatographic Method for Phenylpropanolamine in Human Plasma

Abstract

A high-pressure liquid chromatographic analysis of phenylpropanolamine in plasma following extraction, back extraction and pre-column derivitization with O-phthalaldehyde is presented. The method is improved by the use of phenylethanolamine as internal standard. Using fluorescence detection, the method is sufficiently sensitive to quantitate 5 ng/ml in 0.5 ml plasma with a standard error of estimate of 2.7 ng/ml when calibrated over the O to 240 ng/ml range. Analysis of over 2000 clinical samples have shown the method to be highly specific and reliable.     

High Performance Liquid Chromatographic Determination of Phenacemide in Biological Fluids

Abstract

A sensitive and reliable high performance liquid chromatographic procedure has been developed for the quantitation of phenacemide in plasma or urine. After simple extraction of the drug with ethylacetate from alkalinized samples and evaporation to dryness, the reconstituted extract was chromatographed using a C₈ reversed phase analytical column with UV detection at 254 nm. Regression analyses for the calibration plots obtained on 3 different days for the drug concentrations ranging 1–15 mcg/ml indicated excellent linearity (r >0.999) and reproducibility (CV< 4%, p >0.01). The mean recovery of spiked phenacemide in plasma and urine from the lower limit of quantitation (1 mcg/ml) to 15 mcg/ml was 97.9 and 96.3%, respectively and their respective CV was 3.53 and 2.58%. The method was applied to monitor the plasma vs. time profile of the drug following a single bolus IV dose of 12 mg/kg in a dog.     

Quantitation of Cyproheptadine in Plasma and Urine by HPLC

Abstract

A sensitive, reliable and specific high performance liquid chromatographic procedure has been developed for the quantitation of cyproheptadine in plasma or urine. After extraction of the drug with ethyl acetate from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with acetonitrile and chromatographed using a C₈ reversed-phase analytical column with UV detection at 254 nm. The average recoveries of cyproheptadine from spiked plasma and urine samples in the concentrations ranging from 0.2 – 3 mcg/ml were 95.7 and 100.3%, respectively and their respective CV was 4.1 and 3.9%. Regression analyses for the calibration plots for plasma and urine standards obtained on three different days for the drug concentrations between 0.2 – 3 mcg/ml indicated excellent linearity (r > 0.999) and reproducibility (CV < 2.0%, p > 0.01). The limit of sensitivity was 50 ng/ml for both plasma and urine samples. The method was applied to monitor the plasma concentration versus time profile of cyproheptadine following a single bolus IV dose of 1 mg/kg in a dog.

Urine samples taken from a human subject for the duration of 24 hours following a single oral dose of 8 mg showed that the cumulative amount excreted in urine as cyproheptadine was approximately 1% of the dose.     

Determination of Ciprofloxacin (Bay o 9867) in Biological Fluids by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for the analyses of ciprofloxacin (BAY o 9867) (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid hydrochloride) in human serum, plasma and urine samples is described. Diluted serum, plasma, and urine samples are injected onto a RP-18 column without prior extraction or clean-up procedure. Ciprofloxacin is separated from the ballast by an eluent consisting of an 0.025M H₃PO₄ solution adjusted to pH=3 with tetrabutylammonium hydroxide and acetonitrile.

Ciprofloxacin is detected fluorimetrically giving a detection limit of 8ng/ml in plasma and serum and of 50ng/ml in urine. A statistical evaluation of the assay showed acceptable accuracy and precision for 10 to 500ng of BAY o 9867 per ml in serum and plasma and for 50ng to 600ng of BAY o 9867 per ml of diluted urine specimens. This method was used to monitor the concentrations of BAY o 9867 in serum, plasma and urine of volunteers after oral administration of ciprofloxacin.     

High Performance Liquid Chromatographic Separation of Tropatepine in Biological Fluids. Application to a Healthy Volunteer

Abstract

A high performance liquid chromatographic method for the determination of tropatepine in human plasma and urines is described here. After addition of an internal standard (2 chloro-11-(4-methyl piprazine 1-yl) dibenzo (b-f)(1–4) thiazepine) to the biological fluid and extraction at pH 12.0 in hexane, the analysis was performed on a reversed phase column (C18 microBondapak) with UV detection at 231 nm. The compound was eluted by a perchlorate buffer-acetonitrile mixture with a flow rate of 1.7 ml/min. The detection limit was about 25 ng/ml; reproducibility was around 7.5% for plasma concentrations below 50 ng/ml. Mass spectrometry by direct insertion probe had validated the chromatographic results. The method was successfully applied to plasma specimen collected from a healthy human volunteer following a single intravenous administration of 20 mg of tropatepine.     

Quantitative Analysis of Terbutaline (Bricanyl®) in Human Plasma with Liquid Chromatography and Electrochemical Detection Using On-Line Enrichment

Abstract

A liquid chromatographic method with electrochemical detection (LC-EC) has been developed for the quantitative analysis of terbuta-line in the range 5–50 pmole ml^?1 of human plasma. Terbutaline is isolated from 2 ml of plasma on an ion-exchange column and the eluate is concentrated on a hydrophobic precolumn on-line in the chromatographic system. The precolumn is then back-flushed for further separation onto a hydrophobic analytical column. The mobile phase is a methanol-aqueous buffer to which sodium perchlorate is added to achieve resolution from interfering peaks. A glassy carbon electrode is used for detection. Comparison has been made with gas chromatography-mass spectrometry (GC-MS) to examine the accuracy of the method.     

High Performance Liquid Chromatographic Analysis of Desipramine in Human Plasma Using a Microbore Column Technique

Abstract

A reversed-phase, isocratic HPLC method has been developed for the quantitation of desipramine in human plasma. the method involved the use of cloimpramine as an internal standard. the chromatographic separation was accomplished with a mobile phase comprising acetonitrile-aqueous solution (60:40. v/v) containing 10 mM disodium hydrogenphosphate and 80 mM sodium dodecyl sulfate adjusted to pH 2. the mobile phase was pumped at a flow rate of 0.5 ml/min. the column used was a microbore column (2 mm I.D. × 100 mm) packed with a C18 reversed-phase material (5μm ODS Hypersil). Plasma samples were extracted at basic pH with diethyl ether followed by back-extraction into 0.1 N sulfuric acid. Using UV detection at 250 nm, the lower limit of sensitivity was 10 ng/ml. the inter- and intra-assay coefficients of variation were found to be less than 10%. the assay procedure was applied to a long term oral dosing study in patients to monitor the plasma concentration of desipramine.     

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.     

Ion-Pair Liquid Chromatographic Analysis of Phenylpropanolamine in Plasma and Urine by Post-Column Derivatization with O-Phthalaldehyde

Abstract

A high pressure Liquid chromatographic analysis of phenylpropano Lamine in plasma and urine by post-column derivatzaition with o-phthalaldehyde is described. Plasma samples are extracted with methylene chloride under alkaline conditions. Urine is diluted with mobile phase without extraction. Using fluorescence detection, the method is sufficiently sensitive (2 ng/ml in 0.5 ml of plasma and 0.5 mcg/ml in 0.2 ml of urine) so that phenylpropano lamine concentrations in plasma or urine may be measured for up to 24 hours following a 75 mg oral dose. Coefficients of variation for inter-day and intra-day precision are less than 10%.     

Determination of Doxorubicin,Daunorubicin and some of their Metabolites in Mouse Plasma by High-Performance Reversed-Phase Liquid Chromatography with Amperometric Detection

Abstract

A selective and sensitive reversed-phase liquid chromatographic method with electrochemical detection for the analysis of doxorubicin, daunorubicin and some of their metabolites in plasma is reported. A mobile phase consisting of acetonitrile-phosphate buffer solution-tetrahydrofuran (25–71,5–3,5) flowing at 1 ml/min through a Lichrocart RP 18 column was employed. The influence of various parameters on the separation (solvent composition, pH, tetrahydrofuran content) has been examined. An extraction of anthracyclines from plasma was performed using chloroform-ethanol mixture (4: 1) with high extraction efficiency; reproducible results were attained by working with a 1 M phosphate buffer which ensured a real buffering of the plasma samples. The sensitivity of amperometric detection makes this method suitable for analyzing small amounts of the parent drugs and their metabolites. The precision was better than 4% in the range 0.2 to 5 μg/ml plasma.     

High Performance Liquid Chromatographic Assay of Methoclopramide in Plasma Using a Silica Gel Column and An Aqueous Meobile Phase

Abstract

A high performance liquid chromatographic method for quantitating matoclopramide in plasma is presented. Proteins ara precipitated from the plasma sample with acetonitri la containing the internal standard, procainamida. The treated samples ara than analyzad using an Ultrasphere Si column, an aqueous solution at pH 7 of 65% CH³CN and 5.0 mM (NH₄)₂HPO₄ as a mobile phase, and a fluorescence detector. The retention times for drug and intarna1 standard ara 11.2 and 13.2 min, respectively. The caibration curve is Linear from 0.89 to 44.5 ng/ml. The detection limit is 0.89 ng/ml [signaL/hoisa = 31] for 0.2 ml plasma samples Pracision is measured by intraday and intarday coefficients o f variation, which are less than 10%. This method is currently being used for pharmacokinetic studies of methoclopramide.     

Determination of Ascorbic Acid and Dehydroascorbic Acid in Blood Plasma Samples

Abstract

Following the stabilization of the plasma samples with HClO₄ and EDTA, the samples could be directly analyzed by HPLC using electrochemical detection and reversed-phase columns. The accuracy and precision of the method was evaluated using plasma samples spiked with ascorbic acid (10 μg/ml) and the results were also compared to the classical colorimetric procedure. Dehydroascorbic (5 μg/ml) was determined in plasma samples using UV detection following derivatization at room temperature for 45 minutes with o-phenylenediamine.     

A New Sensitive Reversed‐phase High‐performance Liquid Chromatography Method for the Quantitative Determination of Metoclopramide in Canine Plasma

Abstract

A specific and sensitive high‐performance liquid chromatographic method was developed for the determination of metoclopramide in canine plasma. The procedure involves fast liquid–liquid extraction and analysis on an octadecyl silane (ODS) column. A preliminary pharmacokinetic study was performed by applying the developed method to a single oral administration of metoclopramide (MCP) to a dog. The validation method yielded good results regarding linearity, precision, accuracy, and specificity. The procedure is suitable for separation and quantification of MCP in canine plasma, enough to quantify 0.2 ng/ml when 0.5 ml of plasma is used. This assay procedure might be useful for the pharmacokinetic study of MCP in dogs.     

A Liquid Chromatographic Method for the Determination of Atenolol in Human Plasma

Abstract

A reliable, highly reproducible, accurate and time-efficient high performance liquid chromatographic (HPLC) method to measure atenolol concentration in human plasma was developed and validated. Sample clean-up consists of simple and efficient liquid-liquid extraction (mean recovery 103%) which allows a high sample throughput. Chromatography on a CN-propyl column yields symmetrical and well resolved peaks for atenolol and for the internal standard (metoprolol) without any interference from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection of 12.6 ng/ml (calculated at a 99.9% confidence level) with %CV (precision) ≥ 8.8% and bias (accuracy) ≥ 3.8% for concentrations in the range of 10 – 1000 ng/ml. We now routinely use this method in human pharmacokinetic studies of atenolol dosage forms.     

Detection of Sulfamethazine Residues in Milk by High Performance Liquid Chromatography

Abstract

A simple high performance liquid chromatographic (HPLC) procedure for the detection of sulfamethazine residues in milk is described. Milk is extracted with chloroform, the extract evaporated to dryness and then redissolved in potassium phosphate buffer (pH 5.0). The chloroform extract, in buffer, is passed through a cyclobond I solid phase extraction (SPE) column. The SPE column is washed with 10 ml potassium phosphate buffer and then sulfamethazine is eluted with 2 ml aqueous (50%) methanol. The eluent is directly analyzed by HPLC with uv detection at 265 nm. The recoveries ranged from 83.2% to 88.2% in samples fortified between 5 to 40 ppb levels.     

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.     

High Performance Liquid Chromatographic Determination of Oxamniquine in Plasma Samples

Abstract

A sensitive and reliable liquid chromatographic assay procedure for the quantitation of oxamniquine in plasma or urine was developed. Chromatographic separation was achieved on a reversed-phase phenyl colum using U.V. Detection at 254 nm. The eluting solvent was the mixture of 0.05 M acetate buffer pH 5 and acetonitrile (3:7). With this mobile phase the drug and its external standard were well separated from the interference of the blank samples. The average recovery of oxamniquine from 3 or more replicate dog plasma samples of different concentration (0.125 ? 4.00 μg/ml) was 95.5% and its coefficient of variation was 4.17%. The reproducibility of the assay was confirmed by the analysis of variance test for the slopes of the three standard plots obtained from plasma samples at three different occasions (F=4.2, p > 0.01). The detection limit for plasma samples was approximately 20 ng/ml. The method was applied to measure the plasma level vs, time profile of this drug following a single bolus intravenous dose of 16 mg/kg to a dog.