A New Method for the HPLC Analysis of Pt(II) in Urine

Abstract

An analytical method has been developed to measure Pt(II) in urine via derivatization and UV or HPLC analysis. A measured quantity of urine is heated briefly with diethyl ammonium diethyl-dithiocarbamate, and the resulting Pt(Et₂NCS₂)₂ is extracted into a measured volume of chloroform. Concentrations of Pt(II) are determined by UV absorption at 346 nm or by reverse phase HPLC analysis. The detection limit for Pt(II) as its dithiocarbamate is ～ 1 ng by HPLC; the concentration limit for HPLC analysis by direct extraction was ～ 25 ng/ml. Chromatographic response was linearly related to Pt(II) concentration over the range 100-4, 000 ng/ml; dilution of more concentrated samples has extended this range to at least 30, 000 ng/ml. This method has been applied to the analysis of Pt(II) in the urine of patients who have received cis-dichlorodiamniineplatinum(II) (CDDP) chemotherapy.     

Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

High Performance Liquid Chromatographic Assay for Basic Amine Drugs in Plasma and Urine Using a Silica Gel Column and an Aqueous Mobile Phase. II. Chlorpheniramine

Abstract

A high performance liquid chromatographic [HPLC] method that involves the use of a silica gel column and an aqueous mobile phase for quantitation of chlorpheniramine in plasma and urine is presented. Alkalinized samples are cleaned by extraction with pentane [containing 1% CH₃CN], and the extraction is followed by evaporating the solvent and reconstituting the residue in a small amount of mobile phase. An aliquot of this solution is analyzed by an HPLC system with an Ultrasphere Si Column, an aqueous mobile phase at pH 7 containing 60% CH₃CN and 7.5 mM [NH₄]₂HPO₄, and UV detection at 200 nm. Although the average recovery of extraction is 58% ± SD 10%, the detection limit for the method is 0.7 ng/ml in plasma and 100 ng/ml in urine [s/n = 3] for 0.5 ml samples. The coefficients of variation [CV] on the results of samples run to measure interday and intraday precision and the bias on control samples were all 10% or less. We have used the method in a bioavailability study of a controlled release formulation involving over 1000 samples.     

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.     

A Simple High Performance Liquid Chromatography Procedure for the Determination of N1-Methylnicotinamide in Urine

Abstract

A simple high performance liquid chromatography procedure for the determination of N¹-methylnicotinamide in urine samples is described. The procedure eliminates the need for extraction or ion exchange clean-up of urine samples prior to their analysis. Human and rat urine samples can be analyzed for N¹-methylnicotinamide directly following a simple pH adjustment. The metabolite was separated and quantitated on a 5μ Ultrasphere ODS (C18) reverse-phase column. The mobile phase contained 10 mM K₂HPO₄ and 10 mM sodium 1-octanesulfonate in 8% acetonitrile at pH 7.0. The system has been used to conduct over 1000 determinations during a period of three months without reduction in performance or efficiency.     

High-Performance Liquid Chromatographic Determination of SAZ-VII-23, A Novel Antiarrhythmic Agent,in Dog Plasma and Urine

Abstract

A HPLC method has been developed to determine the concentrations of SAZ-VII-23 (3-benzoyl-7-isopropyl-3,7-diazabicyclo[3.3.1]nonane HClO₄), a novel antiarrhythmic agent, in dog plasma and urine. Plasma treated with acetonitrile and alkalinized urine were extracted with chloroform- propanol (9:1). An aliquot was injected on to HPLC system using a C₆ reversed-phase column and acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8 (28.5:28.5:43 v/v) containing 4.0 mM triethylamine as mobile phase. Detection wavelength was 255 nm. The linear range were 0.04–8 μg/ml, and the lower limit of quantitation was 0.04 μg/ml in plasma and urine, respectively. The method was applied to determine plasma and urine concentrations and preliminary pharmacokinetic profiles of SAZ-VII-23 in a dog.     

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.     

Quantitation of R-836, a New Oral Bronchodilator,by High Performance Liquid Chromatography using Manual or Robotic Sample Preparation

Abstract

A simple and selective high performance liquid chromatography (HPLC) method has been developed for the quantitation of R-836 (an investigational oral bronchodilator) in human plasma and urine, dog plasma and urine, and rat plasma. The method consists of reversed-phase HPLC with ultraviolet detection. Sample preparation involved a one-step protein precipitation procedure and was performed both manually and by a Zymate robotic system. Precision and accuracy results showed excellent reproducibility; results using the robotic procedure were slightly better than the manual procedure. The robotic procedure was capable of preparing the samples with minimal operator handling.     

Determination of Belotecan in the Plasma,Bile, and Urine of Rats by High-Performance Liquid Chromatography with Fluorescence Detection and Its Application to a Pharmacokinetic Study

Abstract

A simple and reliable high-performance liquid chromatographic (HPLC) method was developed for the determination of belotecan in the plasma, urine, and bile samples of rats. Belotecan was analyzed with HPLC using a C18 column with fluorescence detector. A mixture of acetonitrile–0.1 M potassium phosphate buffer at pH 2.4 (25:75, v/v) and 0.2% trifluoroacetic acid was used as the mobile phase. The lower limits of quantitation (LOQ) were 5 ng mL^?1 for the plasma and 5 µg mL^?1 for the urine and bile samples. The method has been readily applied for the routine pharmacokinetic study of belotecan in small laboratory animals.     

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.     

Use of Alkaline Degradation for the Analysis of Dihydrotriazines via High Pressure Liquid Chromatography

Abstract

An HPLC method is described for the determination of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3′,4′-dichlorobenzyloxy)-1,3,5-triazine hydrochloride (WR 38839). The procedure required the isomerization of the drug sample by alkaline treatment with sodium hydroxide, as the parent compound was retained by the column. The reaction product of the drug was analyzed by HPLC using a strong cation exchange resin as the stationary phase and glycine buffer, pH 10.4 as the mobile phase. The product was isolated and identified by TLC, UV, IR, mass spectroscopy and elemental analysis. The postulated mechanism indicates that this would be a general analytical method for dihydrotriazine compounds. This technique, developed for the assay of the dihydrotriazine in an aqueous system, was successfully applied to rat urine samples spiked with the drug.     

Separation and Quantitation of Urinary Phthalates by HPLC

Abstract

A method was developed for the separation and quantitation of plasticizers and their metabolites from human urine using HPLC, Urine was diluted with an equal volume of water and extracted at pH 2.0 with diethyl ether, The extract was dried, the solvent vacuum stripped, and the residue dissolved in methanol for injection into the chromatograph. A C₁₈ reverse phase column containing 10 μ particles was used for the analysis. Ionic suppression, 0.5% acetic acid in water, at pH 3.0 was used to resolve the acidic components. A step gradient of acetonitri1e:water (containing acetic acid) was used to elute the polar metabolites as well as the non-polar plasticizers. Mass spectrometry was used t o identify the compounds in the HPLC fractions. From the HPLC fractions of the urine extract collected, phthalic acid, MEHP, DEHP and normal urinary constituents (e.g., hippuric and benzoic acid derivatives) were identified     

HPLC Assay for Trimethoprim in Plasma and Urine

Abstract

A sensitive HPLC method for the quantitation of trimethoprim in plasma and urine was developed using fluorescence detection. Plasma (or urine) samples were made basic by the addition of 3.8N sodium hydroxide and extracted with chloroform:2-propanol (95:5). After evaporation of the organic layer, a portion of the residue was analyzed by HPLC with fluorescence detection. The minimum detectable quantity is 0.1 μg/ml for this method. This method has been applied to the analysis of plasma and urine obtained from subjects after a single 160 mg dose of trimethoprim.     

Headspace liquid-phase microextraction of methamphetamine and amphetamine in urine by an aqueous drop

This study developed a headspace liquid-phase microextraction (LPME) method by using a single aqueous drop in combination with high performance liquid chromatography (HPLC)-UV detection for the determination of methamphetamine (MAP) and amphetamine (AP) in urine samples. The analytes, volatile and basic, were released from sample matrix into the headspace first, and then protonated and dissolved in an aqueous H₃PO₄ drop hanging in the headspace by a HPLC syringe. After extraction, this drop was directly injected into HPLC. Parameters affecting extraction efficiency were investigated and optimized. This method showed good linearity in the investigated concentration range of 1.0-1500 μg L⁻¹, repeatability of the extraction (R.S.D. < 5%, n = 6), and low detection limits (0.3 μg L⁻¹ for both analytes). Enrichment factors of about 400-fold and 220-fold were achieved for MAP and AP, respectively, at optimum conditions. The feasibility of the method was demonstrated by analyzing human urine samples.     

Thiazides XII: A Simple HPLC Method for Determination of Thiazides in Urine

Abstract

A simple HPLC procedure is described for the determination of nine thiazides in the urine samples.     

Rapid High Performance Liquid Chromatographic Determination of Ampicillin in Human Urine

Abstract

A rapid, sensitive and specific HPLC assay for the determination of ampicillin in human urine is developed.

Ampicillin was directly measured in human urine at 225 nm using a reversed phase column (Synchropack RP-P) and a mobile phase composed of (1:9 methanol-sodium acetate solution, 0.01 M, pH 4). The analysis required no longer than 10 min. Linear correlation between the peak height ratio of ampicillin to cefoxitin sodium (internal standard) and ampicillin concentration in urine over the range 10–100 μg ml^?1 was obtained. The developed method proved to be advantageous as it monitors ampicillin level in urine. Moreover, the urinary excretion of ampicillin in human subjects after an oral administration of 500 mg ampicillin capsules was established using the proposed method.     

Synthesis of (+)- and (−)-Tetrabenazine from the Resolution of α-Dihydrotetrabenazine

Abstract

Tetrabenazine (1) was reduced with NaBH₄ to α-dihydrotetrabenazine (2) and then resolved with di-p-toluoyl-L-tartrate and di-p-toluoyl-D-tartrate to subsequently give (+)- and (?)-α-dihydrotetrabenazine. The enantiomers were oxidized under Swern conditions to prepare samples of (+)-tetrabenazine and (?)-tetrabenazine. The samples were optically pure by chiral HPLC analysis.     

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.     

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.     

The Determination of Sodium Monofluoroacetate (Compound 1080) in Formulation and Technical Samples by High Pressure Liquid Chromatography

Abstract

A high pressure liquid chromatographic (HPLC) procedure was developed for the determination of sodium monofluoroacetate (Compound 1080). The procedure utilized an amine (NH₂) bonded column for the reverse phase determination of sodium monofluoroacetate in formulation and technical samples.