Reverse Phase HPLC Determination OF 5,6-Dihydro-5-azacytidine in Biological Fluids

Abstract

A sensitive and specific reverse phase HPLC method which allows measurement of the new antitumor agent 5,6-dihydro-5-azacytidine (DHAC) in biological fluids at concentrations as low as 50 ng/ml (2 × 10^?7 M) has been developed. After addition of 5′-chloro-5′-deoxy-5,6-dihydro-5-azacytidine as an internal standard, sequential ultrafiltration, boronate gel affinity chromatography and cation exchange chromatography are employed to isolate DHAC from plasma or urine. DHAC is then reacted with N,N-dimethylformamide diethylacetal to form a dimethylaminomethylene derivative with enhanced UV detectability (λmax = 264 nm, log ε = 4.3) and better retention on a reverse phase column. Isocratic separation is then accomplished on a fully loaded and end-capped ODS column with 0.050 M formic acid in 20% acetonitrile/water. This assay has been used to determine the plasma pharmacokinetics of DHAC in rats given a single i.v. bolus dose of 50 mg/kg. Analysis of the drug in human plasma indicates that this method is suitable for determining DHAC disposition and pharmacokinetics in human subjects.     

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.     

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.     

Combined High-Performance Liquid Chromatography and Radioimmunoassay for A Novel Retinobenzoic Acid Derivative, 4-[(5,6,7,8-Tetrahydro-5,5,8,8-Tetramethyl-2-Naphthyl)-Carbamoyl] Benzoic Acid (AM-80), in Human Plasma

Abstract

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid (Am-80) is a novel retinobenzoic acid derivative possessing retinoid activity. Plasma concentration of this drug in clinical use is very low, less than 1 ng/ml, and could not be measured with direct immunoassays. A combination of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) was developed for this drug in human plasma. Am-80 in 0.5 ml of human plasma was extracted by solid-phase extraction, and the extract was purified by reversed-phase HPLC. The immunoreactivity in the fraction of the eluate was measured by competitive RIA. The within- and between-assay variances were 4.1 to 15.5% and 4.1 to 7.0%, respectively. The limit of quantification was 0.04 ng/ml in human plasma, which was much lower than that of direct RIA, 0.6 ng/ml, previously reported. This assay system could be used to observe the pharmacokinetics of Am-80 in human even after dermal application at very low dose.     

A High Performance Liquid Chromatography Method for the Determination of Ranitidine in Plasma

Abstract

Ranitidine hydrochloride is a highly potent H₂ receptor antagonist of value in the treatment of peptic ulceration. A method is described for the determination of ranitidine in biological fluids. The drug is extracted from plasma prior to determination by reverse phase high performance liquid chromatography. The method is sensitive enough to determine 10ng ranitidine per ml in plasma. The h.p.l.c. method has been automated so that analysis can be carried out without attention over a 24 hour period. The automated h.p.l.c. method has been used for studying the pharmacokinetics of oral and intravenous doses of ranitidine hydrochloride administered to man.     

Reverse Phase HPLC Determination and Murine Pharmacokinetics of Arabinosyl-5-azacytosine

Abstract

A sensitive and specific reverse phase HPLC assay has been developed to measure the new antitumor agent arabinosyl-5-aza-cytosine (ara-AC) in biological fluids at concentrations as low as 50 ng/ml (0.2 μM). This assay also detects arabinosyl-N-formyl-guanylurea (AGU-CHO), the initial hydrolytic metabolite of ara-AC. 2′-Deoxy-5-azacytidine, an analogue with similar chemical stability, is used as an internal standard. Chromatographically interfering plasma ribosides are removed by solid phase extraction on a phenyl boronic acid cartridge. Separation of ara-AC, AGU-CHO and internal standard is then accomplished isocratically (1% CH₃CN in 10 mM pH 6.8 phosphate buffer) on fully carbon loaded and end-capped C₈ and C₁₈ columns connected in tandem. The compounds of interest are detected by UV absorption at 240 nm and total analysis time is 20 min. This assay has been used to determine bolus dose plasma kinetics in male BDF₁ mice given 200 mg/kg ara-AC as a tail vein injection. Plasma elimination of the ara-AC is triphasic with a terminal phase half-life of 52 min and the elimination of the AGU-CHO metabolite parallels that of the parent drug. Analysis of ara-AC in human plasma indicates that this method is suitable for determining drug disposition and pharmacokinetics in human subjects.     

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.     

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%.     

Determination of Fleroxacin in Plasma by Direct Injection High Performance Liquid Chromatography with Column Switching

ABSTRACT

A high-performance liquid chromatography (HPLC) assay was developed for the determination of fleroxacin in plasma. The plasma samples were directly introduced onto a HPLC column after filtering through a MolcutII® membrane filter, which removes high molecular weight proteins. The fleroxacin in filtrate was separated from interfering substances and retained on a pre-column using an ODS stationary phase and then was introduced to an analytical column with an ODS stationary phase by column switching. Fleroxacin and lomefloxacin, as an internal standard, were detected by ultraviolet absorbance at 295 nm. Determination of fleroxacin was possible over the concentration range 50-4000 ng/ml; the limit of detection was 20 ng/ml. The recovery of fleroxacin added to plasma was 97.3-100.4% with a coefficient of variation of less than 2.2%. This method is applicable to drug level monitoring in the plasma of patients being treated with fleroxacin and of healthy volunteers participating in pharmacokinetic studies.     

Sensitive HPLC Assay for Ketoprofen in Human Plasma and its Application to Pharmacokinetic Study

Abstract

A selective and sensitive HPLC method was developed for the analysis of ketoprofen in human plasma. The assay involves an extraction of the drug and the internal standard (piroxicam) into diethyl ether from acidified plasma and then back-extracted into a small volume of alkaline aqueous solution before injection onto the HPLC column. A microbore column (2 mm I.D. × 10 cm) packed with a C18 reversed-phase material (5 pm ODS Hypersil) was used. The chromatographic separation was accomplished with a mobile phase comprising a mixture of acetonitrile-methanol-water (15 :20 : 65, v/v) containing 10 mM Na2HP04 buffer, pH 4. The mobile phase was pumped at a flow rate of 0.5 dmin. The eluant was monitored at 258 nm. With this procedure coefficients of variation were less than 10%. The detectionlimit was 0.05 μg/ml (i.e., 50 ng/ml) of plasma. The highly sensitive nature of this method was applied successfully to the dewmination of ketoprofen in human plasma for phmacokinetic studies.     

Determination of LY217332, A New 3'-Quaternary Ammonium Cephalosporin,In Plasma by Solid Phase Column Extraction and HPLC

Abstract

A sensitive and rapid assay has been developed for the determination of LY217332, a 3'-imidazolo[4,5-c]pyridinium cephalosporin, in plasma. The method utilizes cyano solid phase column extraction and HPLC with ultraviolet detection. The lower limit of detection is 5 ng/ml plasma and the relative standard deviation for precision and accuracy was 5% or less from 50–500 ng/ml. The method is applicable to the assay of ceftazidime, cephaloridine, cefpirome and BMY-28142 with minor modification of the mobile phase and the detection wavelength.     

A Sensitive Method for the Determination of Baclofen in Human CSF by High Performance Liquid Chromatography

Abstract

Baclofen (4-amino-3p-chlorophenylbutyric acid) is used clinically for the treatment of multiple sclerosis and other spastic conditions. The intrathecal route of administration is now prefered to the oral route. To optimize efficacy, the levels of the drug in CSF need to be monitored after intrathecal administration. In this paper, the authors describe a sensitive reverse phase high performance liquid chromatographic method for the determination of baclofen in human CSF. This assay employed cation exchange extraction, pre-column derivatization with PITC, and ultra-violet detection (254 nm). The method was shown to be highly sensitive (5 to 10 ng/ml). This method was compared to a method using direct spectrophotometric detection of baclofen at 220 nm after direct application of CSF samples onto the chromatograph without prior extraction.     

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.     

Quantitation of Meclizine Dihydrochloride in Serum by Reversed Phase Ion Pair High Performance Liquid Chromatography

Abstract

A specific and sensitive HPLC method has been developed for the assay of meclizine dihydrochloride in dog serum using an internal standard technique with a single step extraction. The extracts are injected into a reversed phase ion pair HPLC system using a solvent containing camphorsulfonate as paring anion. The detection limit is 5 ng/ml and the range of linearity is 5–250 ng/ml. The method has been used to quantitate meclizine dihydrochloride levels in bioavailability and pharmacokinetic studies in dogs.     

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.     

Determination of Dirithromycin,LY281389 and Other Macrolide Antibiotics by HPLC with Electrochemical Detection

Abstract

Sensitive and rapid assays have been developed for the determination of the macrolide antibotics dirithromycin, erythromycylamine, and LY281389 in plasma. The methods utilize dichloromethane extraction of alkalinized plasma and isocratic reverse phase HPLC with electrochemical detection. The lower limit of detection is 10 ng/ml. Calibration curves are linear and highly reproducible over the range of 20–500 ng/ml. Precision of the calibration curves is very good having relative standard deviations of 5% or less over the dynamic range. These methods can be used for other macrolide antibiotics with minor modifications to the mobile phase. The electrochemical response of various macrolides was found to be dependent upon the functionality at C-9 of the macrolide ring.     

Single Column HPLC Method for Drug Level Monitoring by Direct Plasma Sample Injection. Application to 6-Mercaptopurine,Theophylline and Chlorpromazine

Abstract

An HPLC method with direct plasma sample injection onto a reverse phase column and stepwise elution was applied to the drug level monitoring of 6-mercaptopurine, theophylline and chlorpromazine by using spectrophotometric or electrochemical detection. The analysis of the drug spiked in human plasma was quantitative, and 100% of the drug was recovered regardless of the entity free or bound to plasma protein. Owing to a preliminary procedure of protein coating on the ODS silica gel the column characteristics were somewhat deteriorated, but accurate analyses could be achieved by a single column method including the simultaneous determination of some metabolites of the drug.     

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.     

A Sensitive High Performance Liquid Chromatographic Method for U-80, 278A,A Substituted Aminotetralin,in Rat Plasma,Whole Blood and Brain Tissue

Abstract

A sensitive analytical method for U-80, 278A, a substituted aminotetralin analogue in rat plasma, whole blood and brain tissue has been developed. The method involves solid phase extraction, efficient reversed phase HPLC and fluorescence detection, and can measure 1 ng/ml from 50 μl samples. During method development, many analogues were investigated and a wide range of extraction and HPLC conditions were explored. These experiments enabled rapid modification and revalidation of the method to support animal experiments with novel analogues.     

Liquid Chromatographic Analysis of a New Antihypertensive Agent,PD 78,799, in Plasma on Silica Gel with Reversed-Phase Eluent

Abstract

A sensitive and selective HPLC assay has been developed for the analysis of a new antihypertensive agent in human plasma. The drug and internal standard were isolated from plasma by liquid-liquid extraction. Separation was accomplished on unmodified silica gel with a mobile phase of 60:40 acetonitrile:10 mM dibasic ammonium phosphate. Detection was by UV absorbance at 291 nm. The method is linear over a range of 20 to 4000 ng/ml. Relative error of calibration and control standards ranged from ?1.5 to 5.0% and precision, as indicated by relative standard deviation, ranged from 0.8 to 5.2%. The method has been successfully used for analysis of plasma samples from human volunteers following oral administration of PD 78,799.