Separation of a New Antidiabetic Agent,N-(Trans-4-Isopropylcyclohexylcarbonyl)-D-Phenylalanine,and its Isomers by Chiral High-Performance Liquid Chromatography

Abstract

N-(trans-4-Isopropylcyclohexylcarbonyl)-D-phenylalanine (A4166) is a new oral antidiabetic agent. In order to determine the purity of chemical samples of A4166, a high-performance liquid chromatographic method for the separation of A4166 and synthetic by-products (an L-enantiomer and a cis-iosmer of A4166) has been developed. A chiral stationary phase column packed with 5 μm N-(tert-butylaminocarbonyl)-L-valylaminopropyl silica gel was used for the direct separation of A4166 and its isomers after derivatization with a non-chiral reagent.     

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.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

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.     

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract

A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.     

HPLC determination of valproic acid in human plasma by derivatization with O-p-nitrobenzyl-N,N′-diisopropylisourea

A sensitive and specific method for the determination of valproic acid in plasma has been developed. After the proteins in the plasma have been precipitated with a saturated solution of ammonium sulfate in 1N HCl, the valproic acid, together with the internal standard, is extracted from the plasma with dichloromethane. An aliquot of the organic solution is taken for derivatization of the valproic acid and the internal standard with O-p-nitrobenzyl-N,N′-diisopropylisourea. Separation is carried out by HPLC using two chromatographic systems: an adsorption system with a μ Porasil column, hexane-chloroform (94:6) as mobile phase, and caproic acid as internal standard and a partition reverse phase system comprising a μ Bondapak T_M/C₁₈ column, acetonitrile/methanol/0.0035 M phosphate buffer (60:10:30), and caprylic acid as internal standard. UV detection is at 254 nm. This method, developed in both systems, permits the determination of plasma levels of valproic acid in the reported range of 50-100 μg/mL. With adequate sensitivity, specificity, precision, and accuracy. The plasma levels of valproic acid may be determined by this method without interference from the commonest antiepileptic drugs. Good correlation is obtained with the enzymatic immune analytic method: EMIT.     

An HPLC Method for Determination of Atropine in Human Plasma

Abstract

A development of a high performance liquid chromatographic method for the determination of atropine in human plasma is presented. Atropine is extracted from plasma basified with 0. 1N sodium hydroxide using chloroform, subsequently subjected to base hydrolysis, followed by derivatization of the generated tropic acid with 4-bromomethyl-7-methoxycoumarin (Br-Mmc). The derivative produced has a strong blue fluorescence at excitation wavelength of 328 nm and emission cutoff filter of 389 nm. d1-Mandelic acid as internal standard (I. S.) was added after hydrolysis. The chromatographic separation was achieved on a reversed phase ODS column with a mobile phase of 33% acetonitrile in 0. 01M ammonium phosphate buffer (pH 5). The minimum quantitative limit was 125 ng/ml of plasma.     

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).     

An Ion-Pairing High-Pressure Liquid Chromatography Assay for the Determination of Cefoperazone in Plasma and Urine

Abstract

A high-performance liquid chromatographic assay for cefoperazone (cfp) in plasma and urine is described. For analysis, the internal standard ticarcillin (ticar) is solvated in acetonitrile, which is then added to plasma or urine. The supernatant is drawn off of the resulting protein precipitate and injected directly onto the reverse-phase C₁₈ column, with detection at 254 nm. The mobile phase is composed of phosphate-acetonitrile-tetramethylammonium chloride (TMA). Coefficients of variation for reproducibility were less than 9% for extra-low, low, medium, and high controls. Limits of detection were 0.5 μG/mL for plasma and 1 μG/mL for urine. No interference from other cephalosporins or other antibiotics was found. This high-pressure liquid chromatographic ion-pairing assay is simple, accurate, inexpensive, and requires no extraction.     

Liquid Chromatographic Analysis of Pentobarbital

Abstract

A method is described for a one step acetonitrile precipitation of serum or plasma and subsequent analysis of pentobarbital by reverse phase HPLC. The results of using two internal standards, N,N-Diethyl-m-toluamide and 5-(p-Methylphenyl)-5-phenylhydantoin are compared. Internal standard is added to serum (as little as 25 μL) and vortex-mixed with acetonitrile followed by centrifugation. An aliquote of the supernatant is analyzed on a C18 reverse phase column eluted with metanol/0.05 M (NH₄)₂HPO₄, pH 8/water (55/20/25, v/v/v). The effluent is monitored at 220 nm.     

Determination of Ketoconazole in Plasma and Dosage Forms by High-Performance Liquid Chromatography and a Microbiological Method

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of ketoconazole in plasma and in tablets was developed. the method employs benzafibrate as internal standard and is sufficiently rapid and sensitive for use in pharmacokinetic studies. Separation of the drug from plasma was achieved by extraction with acetonitrile followed by a reversed phase chromatography on a μ Bondapak column using the isocratic mobile phase of methanol-water-glacial acetic acid (67.5:32:0.5). With this eluting solvent ketoconazole and the internal standard. were well separated from the components of plasma. A linear relationship was obtained between the ratio of the area under the peak of drug to that of the internal standard versus the concentration of the drug. Data comparing the microbiological assay with the HPLC procedure, which was developed, are shown. In the microbiological assay, Candida albicans, was the test organism, using the agar diffusion technique. Both methods were applied to the assay of ketoconazole in plasma and in tablets. Excellent agreement was observed between the results from the two methods.     

High Performance Liquid Chromatographic Determination of Pentamidine in Plasma

Abstract

A high performance liquid chromatographic method for quantitating pentamidine in plasma has been developed. Sample clean-up involved precipitating plasma with acetonitrile containing the internal standard, hexamidine. The supernatant was passed through a C₈ Bond Elut column and eluted with a methanolic solution of sodium 1-heptanesulfonate. The eluate was then analyzed on an Altex C₈ column with a mobile phase consisting of 45% CH₈CN, 0.02% detramethylammonium chloride and 0.1% H₃PO₄. Using fluorescence detection (EX: 275 nm and EM: 340 nm), the detection limit was 1.25 ng/ml for 0.5 ml of plasma. The coefficients of variation for interday and intraday were around 10%.     

Determination of Chlorzoxazone in Rat Plasma by LC-ESI-MS/MS and Its Application to a Pharmacokinetic Study

A sensitive LC-ESI-MS/MS method for determination of chlorzoxazone in rat plasma has been developed. Chromatographic separation was achieved on a Zorbax SB-C₁₈ column, with 45:55 (v/v) acetonitrile–water as the mobile phase. A LC-ESI-MS/MS was performed in a multiple reactions monitoring (MRM) mode using target ions m/z 167.5→131.6 for chlorzoxazone and m/z 230.7→185.6 for phenobarbital (internal standard). The calibration plots were linear over the range of 10.0–2,000 ng/mL. Intra-day and inter-day precisions were better than 5.1% and 6.8%, respectively. The validated method was successfully used to analyze the drug in samples of rat plasma for pharmacokinetic study.     

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.     

An HPLC Method for the Determination of Diltiazem and Desacetyldiltiazem in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of diltiazem and its metabolite desacetyldiltiazem in human plasma. Diltiazem and desacetyldiltiazem are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 7.4) using 1% 2-propanol in n-hexane containing diazepam as an internal standard. A reversed phase cyanopropylsilane column was used with a mobile phase of 45% acetonitrile and 55% 0.05M acetate buffer (pH 4.0). The minimum detectable limit was 2ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.     

Simultaneous Determination of Hydrochlorothiazide and Valsartan in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

Abstract

A rapid and specific liquid chromatography/tandem mass spectrometry method was described for the simultaneous determination of hydrochlorothiazide and valsartan in human plasma. After extracted from plasma using methanol, hydrochlorothiazide, valsartan and hydroflumethiazide, irbesartan, used as the internal standard, respectively, were chromatographically analyzed on a Phenomenex Kromasil C₈ column with water and methanol (27:73, v/v) as the mobile phase. Selected reaction monitoring was specific for mass detection employing negative electrospray ionization. The calibration standards were linear over the concentration range (3.13–800 ng/ml for hydrochlorothiazide and 11.72–3000 ng/ml for valsartan). The method was found to be suitable for application to a pharmacokinetic study after oral administration of dispersible tablet containing 12.5 mg hydrochlorothiazide and 80 mg valsartan to 20 healthy volunteers.     

Bioanalytical method development,validation and quantification of dorsomorphin in rat plasma by LC‐MS/MS

The present investigation describes the development and validation of a sensitive liquid chromatography–mass spectrometry/mass spectrometry (LC‐MS/MS) method for the estimation of dorsomorphin in rat plasma. A sensitive LC‐MS/MS method was developed using multiple reaction monitoring mode, with the transition of m/z (Q1/Q3) 400.2/289.3 for dorsomorphin and m/z (Q1/Q3) 306.2/236.3 for zaleplon. Chromatographic separation was achieved on a reverse phase Agilent XDB C₁₈ column (100 × 4.6 mm, 5 µm). The mobile phase consisted of acetonitrile and 5 mm ammonium acetate buffer (pH 6.0) 90:10 v/v, at a flow rate of 0.8 mL/min. The effluence was ionized in positive ion mode by electrospray ionization (ESI) and quantitated by mass spectrometry. The retention times of dorsomorphin and internal standard were found to be 2.13 and 1.13 min, respectively. Mean extraction recovery of dorsomorphin and internal standard in rat plasma was above 80%. Dorsomorphin calibration curve in rat plasma was linear (r² ≥ 0.99) ranging from 0.005 to 10 µg/mL. Inter‐day and intra‐day precision and accuracy were found to be within 85–115% (coefficient of variation). This method was successfully applied for evaluation of the oral pharmacokinetic profile of dorsomorphin in male Wistar rats. Copyright © 2013 John Wiley & Sons, Ltd.     

High Performance Liquid Chromatographic Determination of (-)-N-Formylnorephedrine in Plasma

Abstract

An HPLC procedure for the detection and quantitative estimation of (-)-N-formylnorephedrine in rabbit plasma had been developed. The procedure involved the extraction of (-)-N-formylnorephedrine from plasma spiked with the internal standard (phenacetin), using ethyl acetate. The ethyl acetate extract is evaporated under nitrogen and the residue is reconstituted in water and injected onto the column. A u-Bondapak-C18 column 30 cm × 3.9 mm ID was used. The mobile phase is 20% acetonitrile in water; at a flow rate of 1.5 ml/min and uv detection at 256 nm. A linear relationship between concentration and peak height ratio (I/internal standard) was obtained (r = 1.00). The reported procedure allows the measurement of (-)-formylnorephedrine in concentrations as low as 150 ng/ml of plasma with total procedure time of about 10 min. The applicability of the procedure to pharmacokinetic studies is illustrated and metabolites are shown not to interfere with the assay procedure.     

An HPLC Method for the Determination of Verapamil and Norverapamil in Human Plasma

Abstract

A high performance liquid chromatographic method is presented for the determination of verapamil and its metabolite norverapamil in human plasma. Verapamil and norverapamil are extracted from plasma basified with 0.5M dibasic sodium phosphate (pH 9.5) using ethyl acetate containing trimipramine as an internal standard. A reverse-phase cyanopropylsilane column was used with a mobile phase of 65% acetonitrile and 35% 0.02M acetate buffer (pH 7.0). The minimum detectable limit was 2 ng/ml of plasma. The effect of the pH, molarity, and percent acetonitrile of the mobile phase on the capacity factor was studied. Possible interferences from other drugs administered concurrently are presented.