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.     

Analysis of Bunaprolast,An Esterase Unstable Drug,with An Active Metabolite Subject to Oxidative Degradation,in Blood Plasma

Abstract

This report describes a sensitive, selective and robust assay for bunaprolast and an active metabolite in canine and human plasma. Method development was complicated in that bunaprolast is quickly hydrolysed by esterases even in vitro, and the metabolite is rapidly oxidised in dilute aqueous solutions. Effective measures to stabilize analytes in biological matrices and during sample extraction are described.

The method involves the selective solid phase extraction of analytes with a close analogue as internal standard followed by reversed phase HPLC and fluorescence detection. The limit of quantification was typically less than 1 ng/ml, and the assay was linear over the range 1 - 1,000 ng/ml.     

An HPLC Assay for a Prostacyclin Analogue,Ciprostene Calcium,in Human Plasma

Abstract

A sensitive assay has been developed for the quantification of the prostacyclin analogue, ciprostene calcium, in human plasma. The method involves solid phase extraction of ciprostene calcium and internal standard, carbacyclin, from a small volume of human plasma. The extract is derivatized with 4-bramamethyl-7-acetoxycoumarin, and the derivatized product extracted with a polar solid phase cartridge and concentrated by evaporation. The final extract is separated by reversed phase HPIC and measured by a fluorimetric detector following post-column alkaline hydrolysis. The overall extraction efficiency is better than 75%, and the assay is linear over the concentration range studied (5–20 ng/ml). The limit of quantification was approximately 5 ng/ml. Ultimate sensitivity was limited by interfering peaks endogenous to the biological matrix. Coefficients of variation at mid-range concentrations are less than 10%.     

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 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.     

High Performance Liquid Chromatoraphic Determination of R-831, A New Antiallergic Agent,in Dogs and Humans

Abstract

A simple, selective and sensitive HPLC method has been developed to measure R-831 levels in dogs and humans. It is an internal standard technique with a single step extraction and one wash. Samples are chromatographed on a reversed-phase system with ultraviolet detection. The lowest detectable concentration for plasma is 25 ng R-831/ml with a 1 ml sample and the linear range is 25–8000 ng R-831/ml. The lowest detectable concentration for urine is 250 ng R-831/ml with a 0.1 ml sample and the linear range is 250–8000 ng R-831/ml. This method has been used to quantitate levels of R-831 in bioavailability and toxicity studies in dogs, and in pharmacokinetic and efficacy studies in humans.     

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.     

Routine Determination of a New 1–4 Dihydropyridine,Oxodipine, in Human Plasma by High Performance Liquid Chromatography with Electrochemical Detection

Abstract

A rapid, specific and reproducible high-performance liquid chromatographic routine assay with electrochemical detection was developed for the determination of Oxodipine in human plasma.

After extraction at alkaline pH by cyclohexane, Oxodipine and its internal standard were chromatographied on a reversed-phase column.

Calibration curves were linear over a concentration range of 1–50 ng/ml with relative errors within-day or between-day not exceeding 8% at any level.

The limit of detection was 30 pg injected based on a signal-to- noise ratio of 7. However, the reliable limit of quantification was 1 ng/ml using 1 ml of human plasma.

A dual-electrode coulometric detector was operated in a screening mode of oxidation, providing a greater specificity and reducing background noise.

This method allowed the complete follow-up of clinical pharmacokinetic studies and drug monitoring in patients.     

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.     

Assay of Yohimbine in Human Plasma Using High Performance Liquid Chromatography with Electrochemical Detection

Abstract

Yohimbine is a selective α₂ adrenoreceptor antagonist used in the study of α₂ adrenoreceptors in man. In order to better improve administration regimens for the study of yohimbine in man, we have developed an assay for the determination of yohimbine in plasma utilizing reverse phase high performance liquid chromatography with electrochemical detection. Using a C₁₈ column and a methanol:acetate (60:40) mobile phase, we detected yohimbine in plasma following a simple chloroform extraction. Reserpiline was used as an internal standard. The assay was linear over a concentration range of 50–250 ng/ml in spiked plasma and had a lower limit of sensitivity of 10 ng/ml. It was used to detect yohimbine in plasma sampled from 4 volunteers during an infusion of the alkaloid.     

Determination of Almitrine in Human Plasma by a Gas Chronatographic Method

Abstract

A sensitive and specific method for the quantitative determination of the new drug, almitrine bismesylate, was developed based on the procedure reported by Baune et al. The method depends on initial extraction of drug from plasma with cyclohexane followed by separation from acidic constituents by a base wash. Final separation and quantitation of almitrine was achieved by GLC with NPD detection using the dtmethyl derivative (S-2082) as internal standard. The method was shown to be specific for almitrfne and for internal standard by a parallel run using GC/MS and anaiysis of the M.S. species. The procedure was linear from 25 to 175 ng/ml. Assays were routinely performed with 1 ml of plasma though the analysis could be performed with 0.5 ml. The method is rugged, having been run by at least three analysts over a span of more than 5000 samples, and efficient, allowing 60 samples per day throughput on a routine basis.     

A Liquid Chromatographic Method for the Determination of Atenolol in Human Plasma

Abstract

A reliable, highly reproducible, accurate and time-efficient high performance liquid chromatographic (HPLC) method to measure atenolol concentration in human plasma was developed and validated. Sample clean-up consists of simple and efficient liquid-liquid extraction (mean recovery 103%) which allows a high sample throughput. Chromatography on a CN-propyl column yields symmetrical and well resolved peaks for atenolol and for the internal standard (metoprolol) without any interference from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection of 12.6 ng/ml (calculated at a 99.9% confidence level) with %CV (precision) ≥ 8.8% and bias (accuracy) ≥ 3.8% for concentrations in the range of 10 – 1000 ng/ml. We now routinely use this method in human pharmacokinetic studies of atenolol dosage forms.     

Simultaneous Analysis of Cimetidine and Ranitidine in Human Plasma by HPLC

Abstract

A rapid method for the simultaneous quantitation of the H₂-receptor antagonist drugs cimetidine and ranitidine in human plasma by isocratic ion-pair reverse-phase HPLC is described. The method involves a simple organic extraction step of the alkalinized plasma containing added internal standard followed by back extraction of the extract with dilute acetic acid and subsequent analysis of the aqueous acidic phase on a reverse-phase (C18) column. The eluting solvent was acetonitrile-water (20:80 v/v) containing 0.005 mole/litre octanesulphonic acid and was monitored at 229 nm. The run time for the assay was 12.5 minutes, with a detection limit for cimetidine of 50 ng/m1/(0.2 μmole/1) and that for ranitidine was 20 ng/ml (0.06 umole/1).     

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.     

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.     

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 Separation of Tropatepine in Biological Fluids. Application to a Healthy Volunteer

Abstract

A high performance liquid chromatographic method for the determination of tropatepine in human plasma and urines is described here. After addition of an internal standard (2 chloro-11-(4-methyl piprazine 1-yl) dibenzo (b-f)(1–4) thiazepine) to the biological fluid and extraction at pH 12.0 in hexane, the analysis was performed on a reversed phase column (C18 microBondapak) with UV detection at 231 nm. The compound was eluted by a perchlorate buffer-acetonitrile mixture with a flow rate of 1.7 ml/min. The detection limit was about 25 ng/ml; reproducibility was around 7.5% for plasma concentrations below 50 ng/ml. Mass spectrometry by direct insertion probe had validated the chromatographic results. The method was successfully applied to plasma specimen collected from a healthy human volunteer following a single intravenous administration of 20 mg of tropatepine.     

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.     

HPLC Determination of Methylphenidate in Human Plasma

Abstract

A simple, rapid and sensitive method for measuring methylphenidate in human plasma by HPLC has been developed. After the addition of the internal standard, ethylphenidate, the two compounds are extracted under basic conditions. The residue obtained is resuspended in acetonitrile and analysed on an ODS reversed phase column with detection by UV absorbance at 192 nm. The limit of sensitivity is 5 ng/ml and the procedure is linear over the 5–50 ng/ml concentration range.     

Gas Chromatographic Analysis of Cyclophosphamide in Plasma and Tissues Using Nitrogen-Phosphorus Detection

Abstract

A gas chromatographic method for the analysis of cyclophosphamide in plasma, blood, and organ tissues is described. This method involves extraction of aliquots of plasma or tissue homo-genate in alkaline condition with ether. The extracted drug is derivatized with heptafluorobutyric anhydride followed by gas chromatographic separation via a glass column of 183 cm × 2 mm i. d. packed with 3% SE-30 on chromosorb W-HP. The derivatized cyclophosphamide and isophosphamide, an added internal standard, are detected by a nitrogen-phosphorus detector. The sensitivity limit of this method is 10 ng per gm or ml of sample and gives linearity over 100-fold of concentration range.