Determination of trichlormethiazide in human plasma and urine by high-performance liquid chromatography

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

Simultaneous Determination of Salicylic Acid and Salicyluric Acid in Plasma and Urine by High Pressure Liquid Chromatography

Abstract

A sensitive, rapid, and specific high pressure liquid chromatographic (HPLC) assay was developed for the determination of salicylic (SA) and salicyluric (SU) acids in plasma and urine. The compounds are extracted into ethyl ether at acid pH, evaporated, and reconstituted prior to instrumental separation. Overall recovery of both compounds is 90 ± 5%, and the sensitivity limits are 150 ng of SU and 300 ng SA per ml of biological fluid. The assay was used for the determination of both compounds in plasma and urine of man following oral doses of 40 mg/kg of sodium salicylate.     

Bioanalysis of Naproxen by High Performance Reversed Phase Liquid Chromatography with Photometric and Fluorimetric Detection

Abstract

Methods for the quantitative determination of NAPROXEN and its main metabolite in plasma and urine are described. The separation is based on reversed phase liquid chromatography with LiChrosorb RP 8 (5 μm) as the support and methanol/phosphate buffer pH 7 as mobile phase, in some cases with addition of tetrabutyl ammonium ion as ion-pairing agent to improve the chromatographic selectivity. With UV-detector and a simple filter fluorometer an extraction-evaporation procedure is used for both plasma and urine determinations, while the high selectivity and sensitivity of a sophisticated fluorescence detector permits the direct injection of diluted samples on to the column. Use of an internal standard improves the within-run precision (s_rel%), which for plasma determinations of NAPROXEN are - with UV-detection, 0.2 – 1.7% (range 10 – 40 μg/ml), with filter fluorometer, 2.4 – 5.9% (range 12 – 58 μg/ml), and with fluorescence detector, 0.8 – 4.1% (range 5 – 20 μg/ml).     

A Validated Ion-Pairing High Performance Liquid Chromatographic Method for the Determination of Enoxacin and its Metabolite Oxo-Enoxacin in Plasma and Urine

Abstract

A rapid, sensitive, and specific determination of enoxacin and its principal metabolite, oxo-enoxacin, in plasma and urine is described. the method, which employs the structurally related compound ciprof loxac in as internal standard, involves a protein precipitation step for plasma and solid-phase extraction for urine. Liquid chromatographic analysis is carried out on a C-18 bonded silica column; the mobile phase consists of 0.1 M citric-acid/acetonitrile employing ammonium perchlorate and tetrabutyl-ammonium hydroxide as ion-pairing agents. Quantitation is performed by UV-detection at 340 nm.

The analytical method was validated by examining the performance characteristics specificity, linearity, precision, accuracy, sensitivity, and recovery. Enoxacin calibration curves were linear between 0.02 and 3.2 μg/ml of plasma and from 0.5 to 125 μg/ml of urine. Limits of quantitation in plasma and urine were 0.01 and 0.5 μg/ml, respectively. For oxo-enoxacin, linear of calibration curves were obtained i n the range 0.05 to 1.6 μg/ml (plasma) and 1 to 50 μg/ml (urine); the respective quantitation limits were approximately 0.02 and 1 μg/ml.

The present assay procedure has been applied to monitoring plasma and urine concentrations in several pharmacokinetic studies in humans and different animal species.     

Determination of Meperidine in Human Plasma and Urine by Gas-Liquid Chromatography

Abstract

A highly sensitive method for the determination of meperidine and normeperidine in biological fluids is presented. Concentrations as low as 5 ng meperidine and 25 ng normeperidine per ml of sample can be determined. The recovery of meperidine from control plasma and urine was 91.3 to 102.5% and the recovery of normeperidine was 64.7 to 82.8%. The method has been successfully used for the determination of meperidine in human plasma and urine and of normeperidine in human urine.     

High Performance Liquid Chromatographic Separation of Estradiol-17 α and -17/β in Biological Fluids; Application to Plasma,Milk and Urine of Cows

Abstract

A rapid HPLC technique was developed to separate estradiol epimers. In order to improve the sensitivity of the detection, a radioitmmunoassay was used.

Estrone, estradiol-17α and estradiol-17β were separated within 20 min using 10 ml of chloroform: acetone (90:10), as the mobile phase. The efficiency of the technique was assessed with ₃ steroids and the assay of collected fractions with antlsera specific to each estrogen. Using a non-specific radioimmunoassay, profiles of endogenous estrogens in different biological fluids (blood plasma, milk, urine) were obtained.

The efficiency of HPLC as a separation method and the high sensitivity of radioimmunoassay as a detector allows us to obtain profiles of estrogens from biological samples where steroid concentration is below lOOpg/ml.     

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.     

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.     

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.     

Determination of the New Ace-Inhibitor Quinapril and Its Active Metabolite Quinaprilate in Plasma and Urine by High-Performance Liquid Chromatography and Pre-Column Labelling for Fluorescent-Detection

Abstract

A sensitive and selective method for the determination of quinapril and its active metabolite quinaprilate in human plasma and urine is described. The method is based on isolation using C18 Bond Elut cartridges, pre-column derivatization with 9-anthryldiazo-methane and purification of the reaction mixture on CBA columns followed by reversed-phase high performance liquid chromatography with fluorometric detection. Calibration curves were linear between 20 ng and 1000 ng/ml of plasma (100-2000 ng for urine) for both substances, the lower limit of detection being 5-10 ng/ml.

The present assay procedure has been applied to monotoring plasma and urine concentrations in several pharmacokinetic studies in humans.     

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.     

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.     

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

Abstract

A reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for quantifying chlormezanone in plasma and urine. an extraction step was needed to avoid endogenous interferences especially in urines and led to an average recovery of 90%. the sensitivity limit was 20 ng/ml in both plasma and urine. the method was reproducible with intra- and inter-assay coefficients of variations below 5%. This method was applied to the determination of plasma and urine levels during a pharmacokinetic study in the elderly. It was found suitable to follow the concentrations as long as 120 h after a single oral administration of 400 mg chlormezanone.     

A Specific and Highly Sensitive Method for the Determination of Protriptyline in Body Fluids and Tissues

Abstract

Protriptyline, a tricyclic antidepressant, was converted to its heptafluorobutyramide using heptafluorobutyrylimidazole as the acylating agent. Conditions for the gas chromatography of the derivative and its quantitation by an electron capture detector were established. The smallest detectable amount of the protriptyline derivative was 0.05 nanogram. Isolation and purification procedures were devised which permit the determination of the drug in biological materials without interference from reagents, drug metabolites or naturally occurring substances. Protriptyline extracted from plasma, erythrocytes, urine and tissues was determined in concentrations as low as 10 nanograms/ml.     

High Pressure Liquid Chromatography Assay for Determination of 18-β-Glycyrrhetinic Acid in Plasma

Abstract

This report presents a useful high pressure chromatography assay for determination of the proposed chemopreventive agent 18-β-glycerrhetinic acid (GA) in murine and human plasma. Drug was released from plasma proteins through precipitation with a mixture of sodium bisulfate and sodium chloride after which it was extracted with acetonitrile. Standard calibration curves of GA covered the concentration range of 2.5 to 120 μg/ml. The lower limit of detection was 0.5 μg/ml. No endogenous plasma constituents from plasma were found to interfere with the determination of GA. Recover of GA from plasma was greater than 95% over a concentration range of 20 to 100 μg/ml. Linearity of the assay was excellent; within-run precision showed a C.V. of 4.2% at 25 μg/ml, 5.6% at 20 μg/ml and 3.4% at 120 μg/ml. Between-run assay precision and accuracy was also considered to be excellent. The specificity, sensitivity and reproducibility of the procedure are adequate for proposed clinical pharmacology studies of this agent.     

Determination of Bendroflumethiazide in Plasma by High Performance Liquid Chromatography and Fluorescence Detection

Abstract

An accurate, precise, and specific assay is described for the determination of bendroflumethiazide (BFTZ) in plasma. The procedure employs a C18 column, a mobile phase consisting of 35% acetonitrile in 0.015M phosphoric acid, and a fluorescence detector with a 254nm excitation filter and a 400nm emission filter. Furosemide is used as the internal standard. Using lml of plasma, this method can detect 10ng/ml of BFTZ.     

Determination of Ritodrine in Plasma Using HPLC

Abstract

An HPLC instrument coupled with an electrochemical detector was used to determine ritodrine (erythro-p-hydroxy-α-[1-[(p-hydroxyphenethyl)-amino]ethyl] benzyl alcohol hydrochloride) at nanogram levels in serum. Extraction of ritodrine was accomplished using a multistep ethyl acetate procedure, and the mobile phase consisted of acetonitrile, ammonium acetate, glacial acetic acid, and a counterion. The stationary phase was a Biophase ODS 5 μm column at ambient temperature. Nalbuphine hydrochloride (Nubain®) was used as an internal standard to quantitate the ritodrine levels of pregnant patients receiving ritodrine. The procedure's linearity for both ritodrine standards and spiked plasma samples was demonstrated. The precision of the assay was found to be 3.4% at 20 ng/ml ritodrine. The minimum detectable concentration, with a signal-to-noise ratio of 6, was determined to be 0.31 ng per 50 μl injected, corresponding to a concentration of 0.6 ng/ml plasma. The sensitivity, precision, and reproducibility of the assay were all found to be acceptable for determining ritodrine in patient serum.     

A simplified assay of furosemide in plasma and urine by high-pressure liquid chromatography.

A simplified high-pressure liquid chromatograhic method for determination of furosemide in plasma and urine has been developed using a fluorometric detector directly coupled to the column effluent. The method includes an ether extraction from acidified biologic samples. The mobile phase used for chromatography on a reversed-phase column (C15 hydrocarbon permanently bonded to silica particles) is sufficiently acidic to induce fluorescence of furosemide. The methylester of furosemide is employed as an internal standard. The sensitivity is 0.1 and 0.25 microgram per ml plasma and urine, respectively. The applicability to pharmacokinetic studies of furosemide is shown.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

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.