Differential Measurement of Cortisol and Cortisone in Human Saliva by HPLC with Uv Detection

Abstract

Both cortisol and its dehydro metabolite cortisone are present in normal human saliva. A method for differential Measurement of both compounds in 1 ml samples of saliva by HPLC/UV is described. the method uses an extraction column having a cyclodextrin bonded phase to retain the compounds of interest while allowing elution of interfering compounds. A steroid-bearing fraction is eluted from the cyclodextrin column, dried, reconstituted in a weak mobile phase, and injected on a reversed phase HPLC/UV system provided with an injector-mounted reversed phase extraction column. Samples containing corticosteroid concentrations as low as 0.5 ng/ml can be effectively analyzed by this method.     

Coprecipitative Preconcentration and Differential Pulse Cathodic Stripping Voltammetric Determination of Selenium (IV)

Abstract

A DPCSV procedure for the determination of selenium (IV) with a prior preconcentrative coprecipitation on iron (III) hydroxide has been developed. The experimental conditions for coprecipitation of selenium (IV) onto iron (III) hydroxide, viz. pH, iron (III) concentration, volume of aqueous phase and selenium concentration, were optimized. The coprecipitated selenium (IV) is dissolved in 10 ml of 0.1 M HCl and analysed using DPCSV in the presence of copper (II). Selenium concentrations as low as 10–100 ng present in 500 ml of the aqueous phase could be determined. The method is precise and has been applied to the analysis of sea water and reference material samples.     

A High-Performance Liquid Chromatographic Approach for Monitoring Ethofumesate in Plant,Soil and Water

Abstract

A simple quantitative HPLC-UV method for the analysis of ethofumesate in fodder beet, soil and water samples is described. This method is based on a simple extraction step, gel-chromatographic and/or minisilica gel column clean up step and a separation step on a reversed phase column with uv-detection. The method is sensitive down to 10 μg/L in water and 50 μg/kg in plant and soil.

This approach shows recoveries between 90% and 110% in almost all matrices. Preliminary tests show that this method is also applicable for the analysis of ethofumesate in other plant materials.     

High-Performance Liquid Chromatographic Determination of Atenolol in Tablets

Abstract

A reversed phase high-performance liquid chromatographic method was developed for the determination of atenolol in four oral 100 mg atenolol preparations.

An aliquot of the sample is dissolved in a mobile phase consisting of 0.0612 M potassium hydrogen phosphate - isopropanol-tetrahydrofuran (84:10:6) v/v). The pH was adjusted to 6.7 with phosphate buffer. Nicotinamide was used as internal standard and chromatographed on a Pinkerton column ISRP (GFF-S5–80) 5 μm, 150 × 4.6 mm i.d. The applied column is convenient for the assay at least 90 samples of atenolol without degrading column performance. The detection was performed at 272 nm. The retention time for atenolol was 5.07 min.

The proposed HPLC method was found to be suitable for the rapid and precise routine analysis of atenolol in tablets.     

Selective Separation of Polar Compounds Using an Electric Field Coupled Normal Phase HPLC Column

Abstract

An electric field coupled method for separation of polar compounds using normal phase high performance liquid chromatography is described. The method, which is not based on electrophoresis or electrochromatography, selectively changes the column capacity for polar analytes. The use of different columns showed improved column efficiency and up to a 62% increase in the number of theoretical plates. The theory for the improvement is presented. The significance and utility of the method for selective analysis of polar fractions in environmental samples is discussed.     

Quantitative Analysts of 5-Hydroxytryptamtne in Biological Material by High Perfornance Liquid Chromatography and Electrochmical Detection

Abstract

A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.     

Quantitation of Serotonin in Human Plasma,Serum and Cerebrospinal Fluid Samples by HPLC-EC Using 6-Hydroxytryptamine as an Internal Standard

Abstract

A simple method for sample clean up and concentration of serotonin (5-HT) in biological samples, such as human cerebro-spinal fluid and serum, is described. To the sample 6-hydroxy-tryptamine (6-HT) is added as an internal standard and it is then absorbed either on C₁₈ SEP-PAK cartridge or Biorex-70 short column. 5-HT and 6-HT are then eluted from the column with methanolic formic acid. After evaporation, the residue is dissolved in the mobile phase and an aliquot is used for LC-EC quantitation.     

Corticosteroid Analysis by HPLC-UV Facilitated byv Use of an Injector-Mounted Extraction Column

Abstract

A method for integrating solid phase extraction of corticosteroids from biological samples with normal operation of reversed-phase HPLC is described. This method uses an extraction column mounted in place of the sample loop of a conventional injection valve to separate corticosteroids from some interfering compounds, and to effectively concentrate steroids from dilute samples. Both manual and fully automated chromatographs using this principle are described. A specific application to measurement of corticosterone in a rabbit serum preparation allows routine measurement of as little as 300 pg corticosterone per sample.     

High Performance Liquid Chromatographic Method for Quantitation of a New Antidiabetic Agent in Plasma

Abstract

An HPLC procedure for the detection and quantitation of a new antidiabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166), in dog plasma was developed. The drug and internal standard were extracted from plasma using a reversed phase C₁₈ extraction column (Sep-pak). Separation was accomplished on a ERC-ODS-1161 reversed-phase column with a mobile phase of acetonitrile/0.1M phosphate buffer, pH 6.6 (30/70). Quantitation was achieved by monitoring the ultraviolet absorbance at 210 nm. A linear relationship between concentration and peak height ratio (A4166/internal standard) was obtained. The method has been successfully used for analysis of plasma samples from beagle dogs following oral administration of A4166.     

High Performance Liquid Chromatographic Determination of Triazolam in Human Serum

Abstract

A high performance liquid chromatographic method which utilizes UV-detection has been developed for the sensitive and specific determination of triazolam in human serum. Using 8-chloro-6-phenyl-l-ethoxymethyl-4H-s-triazolo[4, 3-a][1, 4]benzodiazepine as an internal standard, serum samples were buffered with 2 ml of 4M NaOH and extracted twice with 5 ml aliquots of toluene. The combined toluene extracts were evaporated to dryness and the residue dissolved in the chromatographic mobile phase. The samples were chromatography on a microparticulate reverse-phase column using a 0.06M acetic acid:acetonitrile (61:39) mobile phase. Known metabolites of triazolam did not interfere in the analysis. A linear relationship between peak height ratios and concentrations was observed, with the lower limit of detection being approximately 1 ng of triazolam. The utility of the method was demonstrated by administering therapeutic doses of the drug to human volunteers and monitoring serum triazolam concentrations as a function of time.     

A Simple HPLC Method for the Determination of Trazodone Human Serum

Abstract

A simple HPLC method with minimal sample preparation and good reproducibility for the determination of trazodone in serum is described. Basified serum samples were extracted using ethyl acetate containing diazepam as the internal standard (IS). Chromatography was performed on a cyanopropylsilane column with 15 μL sample injection. The mobile phase consisted of 0.02 M ammonium phosphate, pH 7.5 : acetonitrile (70:30 v/v). The eluent was monitored at 220 nm. The serum standard curve was linear from 10.0 to 8000.0 ng/mL serum. The overall within-run quality control CV was 6.3% for five concentrations (20.0, 40.0, 100.0, 250.0 and 1000.0 ng/mL) and the overall recovery from serum was 85.4%. This method has been applied to the analysis of human serum samples.     

Determination of Free Fatty Acids in Natural Oils and Alkyd Resins by High Performance Liquid Chromatography

Abstract

Mixtures of free fatty acids in natural oil and alkyd resin samples have been analyzed using a μBondapak Free Fatty Acid column in conjunction with a ternary mobile phase. Variation of the mobile phase composition allows ‘‘fingerprinting'’ as well as quantitation of the fatty acid components. Samples can be analyzed in ten minutes by this method. The results of the application of this technique to the identification of oil sources of fatty acids as well as the production of fatty acids during alkyd resin synthesis are given. Good agreement is observed for fatty acid compositions determined via HPLC with those obtained by gas chromatographic methyl ester analysis.     

HPLC Separation of Theophylline,Paraxanthine, Theobromine,Caffeine and Other Caffeine Metabolites in Biological Fluids

Abstract

An HPLC method is described for rapid analysis of caffeine and seven of its metabolites in plasma, urine, milk and saliva in a single operation using a 5 μ C₁₈ reverse phase column. The metabolites are extracted with chloroform - iso-propanol (85:15) from 100 μL samples added to NH₄HCO₃. No interference from normal blood, urine, milk or saliva constituents was observed. The method is accurate and precise and separates 1,7-dimethylxanthine (paraxanthine) from 1,3-dimethylxanthine (theophylline). Sensitivity for most metabolites is in the range of 0.1 to 0.3 μg/mL and the detectability is at the nanogram level.     

Determination of Copper by Faas After Preconcentration with Lead-4-Methylpiperidinedithiocarbamate on Microcrystalline Naphthalene By Solid Phase Extraction

ABSTRACT

By using the Pb-4-methylpiperidinedithiocarbamate complex (Pb(4-MPDC)₂) on microcrystalline naphthalene in a column a method was developed for the preconcentration of copper in water samples prior to its determination by FAAS. In this method, copper in liquid phase quantitatively replaces lead on the Pb(4-MPDC)₂-naphthalene solid phase in the column, forming solid Cu(4-MPDC)₂ complex.

Afterwards, copper on Cu(4-MPDC)₂-naphthalene can be easily eluted by potassium cyanide into the aqueous phase, and the Cu is measured by FAAS. The optimum experimental parameters such as pH, flow rate, sample volume, Pb(4-MPDC)₂-naphthalene ratio, concentration of the potassium cyanide solution and effect of matrix ions for the preconcentration of copper were investigated. The obtained recovery was nearly 100 %, when the enrichment factor was 100 for standard solutions and spiked water samples. The proposed method has been employed for the determination of copper in various standard metal alloys and natural water samples.     

The Identification of Drugs of Abuse in Urine Using Reverse Phase High Pressure Liquid Chromatography

Abstract

A rapid, isocratic HPLC procedure for the identification of drugs of abuse in urine is described. The procedure utilizes a reverse phase μC₁₈ column, a methanol/water mobile phase, buffered to pH 7.5, and a UV detector operating at 254 nm. Using indole as an external standard, 13 common drugs of abuse can be differentiated in less than 15 minutes. Nine different drugs of abuse were identified in actual drug screen urine samples, confirming TLC results. Reproducibility and quantitative capabilities of this method were also demonstrated. Due to apparent interferences from drug metabolites this method must at present be used in conjunction with another established method such as TLC or GC for positive drug identification.     

Microbore Liquid Chromatography For Analysis of Plasma Pindolol Concentrations

Abstract

Isocratic high-performance liquid chromatographic assay of pindolol with use of a microbore column was developed. the sample preparation involves extraction of alkalized plasma into ether and back extraction into 0.05 N H₂SO₄. Metoprolol was used as the internal standard. Chromatographic separation is performed on a microbore C18 (5 μm) column using acetonitrile-disodium hydrogenphosphate buffer (37:63) containing 20 mM sodium dodecyl sulfate as the mobile phase. the detection is achieved by using a fluorescence detector operated at the excitation and emission wavelengths of 260 and 310 nm, respectively. Acceptable reproducibility and accuracy data are presented over the concentration range normally encountered in human plasma samples. the lower detection limit is 2.5 ng/ml. This sensitivity has been found to be adequate for routine analysis of pindolol in human plasma samples, making the method applicable to pharmacokinetic studies and clinical trials.     

Determination of 2′,3′-Dideoxyinosine in Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatography analysis method has been developed for the quantitation of 2′,3′-dideoxyinosine (DDI) in plasma. Proteins were precipitated from plasma samples with acetonitrile containing the internal standard, 6-methylaminopurine riboside. The treated samples were evaporated to dryness and reconstituted in mobile phase for the analysis. Separation of the components was achieved on a 5 μm octadecylsilane column with ultraviolet detection at 254 nm. The method was validated at nine concentrations between 0.015 and 150 μg/mL. Using 500 μL of human plasma, the limit of quantitation was 120 ng/mL and the limit of detection was 60 ng/mL. The mean intra-day precision of the method was 1.6%. The mean accuracy of the method was within 2% of the actual values. This method is currently being used for pharmacokinetic studies in the rat.     

Iron speciation by solid phase extraction and flame atomic absorption spectrometry using N,N′-bis-(2-hydroxy-5-bromobenzyl)-2-hydroxy-1,3-diiminopropane

A new Schiff base, N,N′-bis-(2-hydroxy-5-bromobenzyl)-2-hydroxy-1,3-diiminopropane, has been synthesized for the very sensitive determination of iron(III) and iron(II) in natural water samples. It enabled a very selective and rapid method for iron determination to be developed. The method has also been applied to total iron determination in sediment samples. In the preconcentration system, the Schiff base reagent is mixed with the samples and chelates containing iron(III). The complexes are then adsorbed on silica gel within a column system. Elution of the adsorbed chelate from the silica gel was performed with a small volume of acetone containing 2.5% nitric acid. The iron is measured off-line by flame atomic absorption spectrometry. The method can be applied to the preconcentration, separation and speciation of iron. The effects of parameters such as pH, sample flow rate, eluent flow rate, foreign ions and ligand concentration have been investigated. The effect of humic acid that can produce complexes with metal ions in natural systems has also been investigated. The results obtained indicate that the method is not affected by the presence of up to 10 ppm humic acid, which would be a very high concentration to be present in natural systems. The solid phase extraction method developed has been applied to the determination of iron in both natural water samples and sediment samples. The LOD was found to be 0.17 mg L⁻¹ when no preconcentration was used, although preconcentration factors of 100 could be achieved. The recovery values for spiked samples were between 100 and 104%. The results were compared statistically with those from the standard 1,10 phenanthroline method used for iron speciation in water systems. A Student’s t-test indicated no significant difference between the two methods. In addition, this method was applied to the analysis of a certified sediment sample, LGC 6156. Generally, a 10-fold preconcentration factor was required for the analysis of natural water samples.     

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.     

Rapid Determination of Propranolol and 4-Hydroxypropranolol in Plasma by High Pressure Liquid Chromatography

Abstract

The resolving power of high pressure liquid chromatography has been combined with the sensitivity of fluorescence detection to develop a method for the determination of propranolol and 4-hydroxypropranolol in plasma. A single selective extraction step precedes chromatography on a high efficiency bonded phase column. The limits of quantitation are approximately 2 ng/ml of plasma for each drug. The analysis of a number of clinical samples has demonstrated the application of the method in pharmacokinetic studies, but the possible interference of other drugs and their metabolites is still under investigation.