The Determination By Means of Capillary Gas Chromatography of the Benzodiazepines-Clobazam,Clonazepam, Diazepam,and Nitrazepam-and Their Principal Metabolites Currently Used In the Treatment of Epilepsy

Abstract

The authors developed a gas chromatographic method allowing the quantification of those benzodiazepines in current use in the treatment of epilepsy, name ly clobazam, clonazepam, diazepam and nitrazepam. This method involved a butyl acetate extraction from plasma, and an analysis on a CP-Sil 5 WSCOT capillary column with electron-capture detection. Intra-day precision and accuracy were better than 10% for each of the compounds and their main metabolites, N-desmethyldiazepam, oxazepam, N-desraethylclobazam. the quantification limit was about 0.5 to 1 ng/ml for each compound. Linearity proved satisfactory between 1 and 4000 ng/ml. Adequate han previously. Besides, its sensitivity is sufficient for kinetic studies following a single dose administration.     

Simultaneous Determination of CGS 10787B and Its Major Metabolite (CGS 12094) in Plasma by High Performance Liquid Chromatography

Abstract

A method for the simultaneous determination of CGS 10787B and its major, metabolite (CGS 12094) in plasma is described. The two compounds, and the internal standard (dichlorinated analog), are extracted from acidified plasma with ethyl acetate, taken to dryness, and reconstituted in chromatographic mobile phase. The analytes are determined automatically by high performance liquid chromatography in the reversed-phase mode as paired ions, using [N(Bu)₄]⁺ as the counterion. The separation of the compounds is achieved on a 3u C-8 column, with detection at 254 nm.

Recovery and reproducibility assessments indicate good accuracy and precision over the range of 1.0 to 250 ug/ml for CGS 10787B and 1.0 to 100 ug/ml for CGS 12094.

The method has a limit of detection of 0.2 ug/ml for both compounds, and has been shown to be adequate for studying the disposition kinetics of CGS 10787B.     

Determination of Pyridostigmine in Human Plasma by High-Performance Liquid Chromatography

Abstract

An easy to perform, specific, reproducible and sensitive high performance liquid chromatographic (HPLC) method to measure pyridostigmine concentration in human plasma was developed and validated. Sample clean-up consists of ion-pair extraction into dichloromethane in the presence of neostigmine as internal standard, followed by back extraction into an aqueous phase. Mean recovery of 110% (with a standard deviation of 10%) was determined for concentrations of 5 – 100 ng/ml. Chromatography on a 125·4 mm CN-propyl column using a mobile phase composed of 10% acetonitrile in 3.5×10^?4M NaH₂PO₄ and UV detection at 270 nm, yields clean chromatograms without any interferences from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection (LD) of 3 ng/ml, with %CV (precision) and bias (accuracy) ≥ 10% for concentrations in the range of 0–100 ng/ml. The method is being used in human pharmacokinetic studies of oral dosage forms of pyridostigmine.     

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.     

High Performance Liquid Chromatography Determination of Pcmx In Blood Plasma Using Electrochemical Detection

Abstract

A rapid and specific high performance liquid chromatographic (HPLC) procedure has been developed for the determination of p-chloro-mxylenol (PCMX) in blood plasma. the method is based on the extraction of PCMX from plasma with benzene in the presence of a known amount of dichloro-m-xylenol (internal standard). the benzene extract is evaporated to dryness and the residue dissolved in 200 μl of mobil phase. the HPLC system consisted of a reversed-phase column and a 65% methanol:35% ammonium carbonate 0.05% solution as a mobile phase. an electrochemical (EC) detector/glassy carbon electrode set a potential of +0.9V versus Ag/AgCl/3M NaCl is used to monitor the drug. the recovery of PCMX is approximately 98%, the limit of quantitation is 2 ng/ml of plasma for the HPLC-EC procedure. the coefficient of variation is 5.1% over the range of 10-1000 ng/ml of plasma. Data are presented to illustrate the practicality of this method for evaluation of PCMX plasma levels after a single intravenous administration of 500 mg dose of PCMX to five mongrel dogs.     

Conductometric Titration of Chlorhexidine and Proguanil

Abstract

Conductometric titrations of chlorhexidine and proguanil are reported. The procedure is based on the copper-bi-guanide reaction which gives a pink solid. Studies at several pH values, and presence of NH₃ and ethanol are carried out. 3 ml of 0.2 M NH3, 9ml of 0.01 M NaOH diluted to 60 mL with 15% ethanol are added to 5ml of biguenide aqueous solution and titrated awith cooper acetate. Concentrations of Chlorhexidine in 5.9x10^?5 - 3.4x10^?4 M range are determined. Fareignspecies presence is studied too.

Biguanides are found in several pharmaceutical formulations and industrial samples.

Estimetion of chlorhexidine and proguanil salts with the standard method lacks selectivity since it is based on perchloric acid titration in acetic acid medium¹. Some recent papers about determination of chlorhexidine and proguanil have been publisghed: potentiometric titration of chlorhexidine² polarographic determinetion of praguanil and chlorhexidine^3,4 spectrophotometric assay of chlorhexidine in contact lens solutions⁵ and suppositories⁶, HPLC of proguanil in serum sam-ples⁷ and chlorhexidine in several samples ^8,9,10,10,11 or G C ^12,13 and mass fragmentography^14,15.     

The Simultaneous Determination of Δ9-Tetrahydrocanna-Binol and 11-Hydroxy -Δ9 -Tetrahydrocannabinol in Plasma

Abstract

A technique capable of determining both Δ⁹-tetrahydrocannabinol and 11-hydroxy-Δ⁹-tetrahydrocannabinol is described. The method is based on the reactivity of the phenolic functionality common to both compounds, and is sensitive to 5 ng of Δ⁹-tetrahydrocannabinol and 1 ng of 11-hydroxy-Δ⁹-tetrahydrocannabinol per ml of plasma.     

Revised HPLC Determination of Atenolol in Plasma and Urine

Abstract

An HPLC method for analysis of atenolol in human plasma and urine is presented. Based on alkaline extraction, acid backextraction and reverse phase ion-pair chromatography this method is quite specific for atenolol. For a 0.5 ml plasma sample the sensitivity ranges from 20 ng/ml in fasted healthy volunteers to 50 ng/ml in various groups of patients. A sensitivity in urine of 1.0 mcg/ml was sufficient for all samples studied. As presented this method has been used in several clinical pharmacokinetic studies involving hundreds of samples.     

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.     

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 Simple Isocratic HPLC Method for the Determination of Metoclopramide in Plasma and Urine

Abstract

Metoclopramide concentrations in plasma and urine were determined by high performance liquid chromatography using a cyanopropylsilane column and UV detection. The mobile phase consisted of 0.03M sodium acetate (pH 7.4) and acetonitrile. The plasma samples were extracted with dichloromethane after pH adjustment. Urine proteins were precipitated with acetonitrile. The reproducibility and precision of the methods were demonstrated by the analysis of samples containing 5 – 200 ng/ml plasma and 0.25 – 200 ug/ml urine.

The glucuronide and sulfate conjugates of metoclopramide were also quantitated after differential acid hydrolysis of urine samples. The conditions for acid hydrolysis were studied. The methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

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.     

An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH₂Cl₂ containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH₃CN containing IS.

Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH₂PO₄, pH 3.5 - CH₃OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

An HPLC Method for Measuring 5-Fluorouracil in Plasma

Abstract

5-Fluorouracil in plasma was determined by extraction with methyl isobutyl ketone, evaporation of the ketone, and reverse phase high performance liquid chromatography of the evaporation residue. With UV detection at 280 nm the lower limit of detection is 10.0 ng/ml and interfering peaks eliminated. The method is highly reproducible.     

Determination of Arsenic in Organic Compounds and Other Matrices by Flame Emission Spectrometry

Abstract

A direct and simple method for arsenic in organic compounds involves merely dissolution of the sample in an organic solvent, such as benzene, cyclohexane, or methyl isobutyl ketone, and direct aspiration into a fuel-rich acetylene-oxygen flame. The arsenic line at 2350 Å is used; the detection limit is 2.2 ü/ml. From iron and copper matrices arsenic is isolated by extraction from a solution 9 M in HCI and 0.25 M in KI with benzene.     

High-Performance Liquid Chromatographic Determination in Human Plasma of a Anticonvulsant Benzodiazepine: Clonazepam

Abstract

A rapid and specific high-pressure liquid chromatography method for determination of clonazepam in human plasma is described.

The analysis is linear for concentrations ranging from 5 to 100 ng. ml^-1 plasma for clonazepam.

The method is applicable to quantitation of clonazepam in human plasma of subjects receiving 0.05 at 0.20 mg.kg^-1 orally, with satisfactory accuracy and precision.     

Metformin and Moroxidine Determination With Cu (II)

Abstract

Conductometric titrations of metformin and moroxidine are reported. The procedure is based on the copper-biguanide reaction which gives a pink soluble complex. Studies at several pH values and presence of NH₃ are carried out. NaOH-NH₃ (1:15) are added to 5 ml of biguanide aqueous solution and diluted to 60 ml and titrated with copper sulphate. Concentrations of metformin in 205x10^?4-1,7x10^?3 M range are determined. Foreign species presence is studied too.     

Assay of underivatized intrazepam and clonazepam in plasma by capillary gas chromatography applied to pharmacokinetic and bioavailability studies in humans

The assay procedure of underivatized, intact nitrazepam and clonazepam in human plasma is described, using gas chromatography with a support-coated open tubular column (OV-17), a solid injection system and electron-capture detection. Clonazepam is used as a internal standard in the assay of nitrazepam and vice versa. Linear calibration curves after a single extraction step were obtained in the concentration range 10--100 ng/ml plasma, with standard deviations less than 4.9%. The sensitivity limit of the method is about 1 ng/ml plasma for both drugs. The method was applied to pharmacokinetic and bioavailability studies of nitrazepam in humans. Seven healthy volunteers received two nitrazepam-containing tablet preparations (5 mg) and plasma concentrations were determined regularly from 15 min to 80 h following drug administration. The mean elimination half-life of nitrazepam was 27 h (range 13-34 h). Considerable intra-individual differences in peak level times between the two preparations were observed, whereas the extent of bioavailability was rather similar.     

Densities and Viscosities of the Ternary Mixture (Benzene + 1-Propanol + Ethyl Acetate) at 298.15 K

Abstract

Densities and viscosities of the ternary mixture (benzene + 1-propanol + ethyl acetate) and the corresponding binary mixtures (benzene + 1-propanol, benzene + ethyl acetate and 1-propanol + ethyl acetate) have been measured at the temperature 298.15 K. From these measurements excess volumes, V^E , excess viscosities, η^E, and excess Gibbs energies of activation for viscous flow, G*^E , have been determined. The equation of Redlich-Kister has been used for fitting the excess properties of binary mixtures. The excess properties of the ternary system were fitted to Cibulka's equation.     

Size Exclusion Chromatographic Determination of PEG 3350 in Human Plasma and Urine

Abstract

A size exclusion chromatographic method is reported that quantitated PEG 3350 in human plasma and urine to a concentration of 10 μg/ml in plasma, or urine. The chromatographic system consisted of a 500 A gel permeation analytical column, a differential refractometer detector, and chloroform as the eluting solvent. Organic extraction was used as an initial separation technique. PEG 400 was used as the internal standard for PEG 3350, and showed similar extraction properties. Spiked plasma standards yielded standard calibration curves with correlation coefficients of greater than 0.99, and relative standard deviations (n=3) of 2.19% (500 μg/ml) and 6.97% (20 μg/ml). The analytical technique was used to estimate the elimination rate constant of PEG 3350 from 5 normal human subjects after oral administration of 240 grams of PEG 3350. K_E was found to be 0.1079 hr^?1 0.03781^?1 hr (mean s.d.) using the Sigma-minus data analysis method on total urines collected from the 5 subjects at varying intervals.