Determination of Saccharin in Plasma and Urine by High Performance Liquid Chromatography

Abstract

A sensitive and specific high performance liquid chromatographic (HPLC) assay for the determination of saccharin in plasma and urine was developed. Saccharin is extracted into diethyl ether at acid pH, evaporated, and reconstituted prior to instrumental analysis. Overall recovery of saccharin is 86.9 + 8.6% and the sensitivity limits of detection is 0.15 μg per ml of plasma or urine using a fluorescence detector. The sensitivity limit in plasma can be extended to 20 ng per ml by use of a 2 ml assay volume and detector attenuation. The assay was used for the determination of saccharin in plasma and urine of rats following oral doses of 5 mg/kg.     

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.     

Gas Chromatographic Analysis of Cyclophosphamide in Plasma and Tissues Using Nitrogen-Phosphorus Detection

Abstract

A gas chromatographic method for the analysis of cyclophosphamide in plasma, blood, and organ tissues is described. This method involves extraction of aliquots of plasma or tissue homo-genate in alkaline condition with ether. The extracted drug is derivatized with heptafluorobutyric anhydride followed by gas chromatographic separation via a glass column of 183 cm × 2 mm i. d. packed with 3% SE-30 on chromosorb W-HP. The derivatized cyclophosphamide and isophosphamide, an added internal standard, are detected by a nitrogen-phosphorus detector. The sensitivity limit of this method is 10 ng per gm or ml of sample and gives linearity over 100-fold of concentration range.     

Rapid and Sensitive Determination of Furosemide in Human Plasma by High Pressure Liquid Chromatography

Abstract

A rapid and sensitive determination of furosemide in human plasma by high pressure liquid chromatography is described. The drug is extracted from plasma after addition of the internal standard (2,4-dinitrophenol), the extract is concentrated by means of evaporation and injected on to the liquid chromatograph. Separation is achieved on a reversed-phase column with 50 % methanol in water, containing 0.5 % glacial acetic acid; detection is carried out at 340 nm. The method is linear up to 5 μml and can determine concentrations down to 0.1 μg/ml in a 1ml plasma sample. It is much faster than existing methods and is at least as good with respect to accuracy and sensitivity.     

Determination of Plasma Norepinephrine and Epinephrine by High Performance Liquid Chromatography Using a Two-Column System and an Electrochemical Detector

Abstract

A method for the simultaneous plasma norepinephrine (NE) and epinephrine (E) determination by reversed-phase ion-pair liquid chromatography with electrochemical detection has been developed. Catecholamines were extracted from a 4 ml plasma sample using an alumina adsorption procedure. A two-pump, two-injection valve, two-column system allowed both to detect plasma NE and E with a good sensitivity due to large injected volumes of extract without any electrochemical detector disturbance and to eliminate uric acid and dopa the low k' of which would prevent the NE detection. Using this method, NE and E would be detected in respective injected amounts down to 30 and 50 picograms. Plasma NE and E determinations were found to be linear in the range of 288 to 788 pg/ml and 24 to 274 pg/ml respectively. The reproducibility, expressed as the coefficients of variation, varied from 2.1% for NE to 10.8% for E.     

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.     

Determination of Flecainide in Human Plasma by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, selective, and sensitive high-performance liquid chromatographic (HPLC) method for the monitoring of plasma flecainide levels in a therapeutic or research environment is described. The drug is first separated from plasma by a single-step extraction with hexane and then quantitated by HPLC with fluorescence detection. Two linear ranges have been established; 100–2000 ng/ml for drug monitoring in clinical management of patients and 3–300 ng/ml for pharmacokinetic studies. The intra-day variation is less than 6%.     

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.     

Reverse Phase HPLC Determination of Azq in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method employing a simple sample preparation procedure and utilizing an internal standard was developed to measure the new antitumor agent AZQ in biological fluids. A single chloroform extraction gave drug recoveries of greater than 88% from plasma, urine and CSF in the range of expected physiological concentrations (20–800 ng/ml). Isocratic reverse phase HPLC with UV detection at 340 nm resulted in a limit of quantisation of 5 ng/ml although smaller amounts of the drug could be detected. This assay was successfully applied to determine the single dose plasma pharmacokinetics of AZQ in rats. The potential of this method for determining AZQ disposition and pharmacokinetics in human subjects was demonstrated by analysis of patient CSF.     

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 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.     

High-Performance Liquid Chromatography of MDL-035 in the Plasma of Rats,Dogs and Humans

Abstract

A sensitive and reproducible high performance liquid chromatographic method was set up for the assay of MDL-035, a new non-steroidal, nonacidic analgesic antiinflammatory agent, in the plasma of rats, dogs and humans. Plasma samples (0.5ml) containing flurazepam as the internal standard, were diluted and extracted with ethyl ether. After centrifugation, the organic phase was taken to dryness, the residue was redissolved and injected into an RP-2 column. The elution was made in isocratic conditions using a CH.CN/phosphate buffer solution as mobile phase. The UV detection was made at 320 nm. The method was validated for the concentration range from 0.05 to 10 ug/ml, and applied to pharmacokinetic studies. A typical plasma concentration-time profile in the rat after an oral administration is here presented.     

High Performance Liquid Chromatographic analysis of the Biological Response Modifier Synthetic Double-Stranded RNA (dsRNA,Ampligen)

Abstract

A high performance liquid chromatographic method was developed for analysis of the antitumor agent synthetic, double-stranded RNA (dsRNA, Ampligen) in plasma. In this procedure the agent was extracted from plasma by ion-exchange chromatography and then degraded to its primary components, inosine and cytidine, by treatment with nuclease and alkaline phosphatase. Inosine and cytidine derived from degradation of ampligen were quantified by reversed phase HPLC. Standard curves for quantitation of inosine derived from ampligen were generated by addition of various amounts of ampligen to plasma. Utilizing this method, extraction of drug from the plasma was approximately 70–80%. Standard curves were linear over the concentration range of 0.25 to 100 ug/ml plasma. There were no peaks from plasma which interfere with quantitation of inosine. The approximate lower limit of detection of drug by this method was 0.25 ug/ml plasma. Interassay and intra-assay mean variability of standards was 9.6 and 3.2% respectively. Analysis of plasma samples obtained from one patient after infusion of ampligen (640 mg/m²) show that this method is sensitive enough to monitor plasma for clinical pharmacology studies of ampligen.     

Determination of Reserpine in Plasma Using High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

A high-performance liquid chromatographic method for determining reserpine in plasma has been developed. The procedure involves extraction of reserpine from buffered plasma into benzene, oxidation of reserpine to a fluorophor by treatment with vanadium pentoxide in phosphoric acid, and chromatographic separation of the reserpine fluorophor on an octadecylsilane column by ion-pairing with heptanesulfonate ions. Fluorescence monitoring of the column effluent provides high sensitivity of detection and increases the specificity of the procedure. A detection limit of approximately 100 pg of reserpine per ml of plasma was obtained following analysis of 2 ml samples. Analysis of a number of samples demonstrated the applicability of this method in confirming the presence of reserpine in equine plasma specimens collected at various horse shows and in evaluating the pharmacokinetic behavior of reserpine following intramuscular administration to horses.     

Determination of Almitrine in Human Plasma by a Gas Chronatographic Method

Abstract

A sensitive and specific method for the quantitative determination of the new drug, almitrine bismesylate, was developed based on the procedure reported by Baune et al. The method depends on initial extraction of drug from plasma with cyclohexane followed by separation from acidic constituents by a base wash. Final separation and quantitation of almitrine was achieved by GLC with NPD detection using the dtmethyl derivative (S-2082) as internal standard. The method was shown to be specific for almitrfne and for internal standard by a parallel run using GC/MS and anaiysis of the M.S. species. The procedure was linear from 25 to 175 ng/ml. Assays were routinely performed with 1 ml of plasma though the analysis could be performed with 0.5 ml. The method is rugged, having been run by at least three analysts over a span of more than 5000 samples, and efficient, allowing 60 samples per day throughput on a routine basis.     

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.     

GLC Determination of Chlorpheniramine in Human Plasma Using a Nitrogen Sensitive Thermionic Detector

Abstract

A highly sensitive procedure for the determination of plasma levels of the antihistamine, chlorpheniramine (CPA) is presented. Following the administration of therapeutic doses (4–8 mg) of CPA, plasma levels of the drug can be expected to be low. To reliably and accurately measure such levels, an ultrasensitive means of determining the concentration is required. A preliminary extraction with ether, a back-wash into acid, a final basic extraction into hexane with subsequent measurement by a gas chromatograph equipped with a nitrogen sensitive detector, provides the required selectivity, sensitivity and speed of analysis. In an expenditure of less than 10 minutes, 1 ng of CPA per ml of plasma can be reliably determined.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

Determination of Chlordiazepoxide and Its Metabolites in Plasma by High Pressure Liquid Chromatography

Abstract

A rapid, sensitive and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of chlordiazepoxide and its metabolites from plasma. The assay involves extraction of chlordiazepoxide and its metabolites into diethyl ether from plasma buffered to pH 9. The overall recovery of chlordiazepoxide is 80 ± 5.0% (S.D.) and the sensitivity limit of detection is 50 to 100 ng/ml of plasma, using a 1 ml specimen. The assay was used in the determination of plasma levels of chlordiazepoxide and its metabolites in man following oral administration of chlordiazepoxide. HCl.

The chromatographic behavior of other clinically important benzodiazepines and their major metabolites is also reported.     

Solid-Phase Extraction of Carbamazepine and Two Major Metabolites from Plasma for Analysis by HPLC

Abstract

A rapid, sensitive and simple to operate HPLC method for the simultaneous determination of carbamazepine, carbamazepine 10,11-epoxide and 10,11-dihydro-10,11-trans-dihydroxycarbamazepine in plasma is described. The drug and its metabolites are extracted from plasma using commercially available reversed-phase octadecylsilane bonded-silica columns (Bond Elut C₁₈, 2.8 ml capacity). Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of acetonitrile - methanol - water (19:37:44) at a flow-rate of 1.8 ml/min in conjunction with a Waters Assoc. Nova-Pak C₁₈ column. The analytical column, in Radial-Pak cartridge form, was used in combination with a Waters Assoc. Z-module RCSS and protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak μBondapak C₁₈ insert. Using ultraviolet detection at 214 nm, levels in the region of 50–100 ng/ml for CBZ and its metabolites can be measured with only 250 μl of plasma. The method has been used to determine steady-state concentrations of the drug and its metabolites in paediatric patients.