Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

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.     

Liquid Chromatography–Electrospray Ionization–Mass Spectrometric Method for the Determination of Hydrochlorothiazide in Human Plasma: Application to a Pharmacokinetic Study

Abstract

A rapid, convenient, and sensitive liquid chromatography–electrospray ionization–mass spectrometry method was developed and validated for the quantification of hydrochlorothiazide in human plasma. The samples were first spiked with the internal standard, and the analyte was then extracted with ethyl acetate. The chromatographic separation was achieved on a C₁₈ column by using water–acetonitrile (68:32, v/v) as mobile phase. The method was linear within the range of 2.5–200 ng/ml. The lower limit of quantification was 1.0 ng/ml. Finally, the validated method was successfully applied for the evaluation of the pharmacokinetic profiles of hydrochlorothiazide in healthy male Chinese volunteers.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and 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).     

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.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were 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.     

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.     

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.     

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.     

RP-HPLC Method for Determination of Oseltamivir Phosphate in Capsules and Spiked Plasma

A new, sensitive RP-HPLC method was developed for the determination of oseltamivir phosphate in capsules and plasma. The method was based on the reaction of the drug with 4-chloro-7-nitrobenzofurazan in borate buffer solution of pH 8.50. Isocratic chromatography was performed on a C₁₈ column with acetonitrile–10 mM nitric acid (pH 3, 60 + 40, v/v) as the mobile phase with fluorescence detection (λex: 470 nm, λem: 541 nm). Mexiletine hydrochloride was used as an internal standard. Analytical parameters were evaluated. The calibration range was linear from 50.0–750.0 ng/ml. The mean percentage recovery in capsules and plasma were 99.95% and 95.42%, respectively.     

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.     

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%.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

Development and validation of a highly sensitive and robust LC‐ESI‐MS/MS method for simultaneous quantitation of simvastatin acid,amlodipine and valsartan in human plasma: application to a clinical pharmacokinetic study

A high‐throughput, simple, highly sensitive and specific LC‐MS/MS method has been developed for simultaneous estimation of simvastatin acid (SA), amlodipine (AD) and valsartan (VS) with 500 µL of human plasma using deuterated simvastatin acid as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode (MRM) using electrospray ionization. The assay procedure involved precipitation of SA, AD, VS and IS from plasma with acetonitrile. The total run time was 2.8 min and the elution of SA, AD, VS and IS occurred at 1.81, 1.12, 1.14 and 1.81 min, respectively; this was achieved with a mobile phase consisting of 0.02 m ammonium formate (pH 4.5):acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on an X‐Terra C₁₈ column. A linear response function was established for the range of concentrations 0.5–50 ng/mL (r > 0.994) for VS and 0.2–50 ng/mL (r > 0.996) for SA and AD. The method validation parameters for all three analytes met the acceptance as per FDA guidelines. This novel method has been applied to human pharmacokinetic study. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Captopril in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry

Abstract

In this study, a fast and sensitive liquid chromatography/tandem mass spectrometry method for the determination of captopril in human plasma was developed and validated. The analyte and enalaprilat, used as the internal standard, were extracted from plasma using methanol directly precipitate protein. Analysis was performed on a Lichrospher CN column with water (containing 0.1% formic acid) and methanol (60∶40, v/v) as the mobile phase. Linearity was assessed from 6.25 to 800 ng/ml in plasma. The analytical method proved to be applicable in a pharmacokinetic study of captopril after oral administration of 20 mg captopril tablet to 20 healthy volunteers.     

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.