HPLC method for determination of diclofenac in human plasma and its application to a pharmacokinetic study in Turkey

A simple high-performance liquid chromatography (HPLC) method has been developed for determination of diclofenac in human plasma. The method was validated on Ace C(18) column using UV detection. The mobile phase consisted of 20 mM phosphate buffer (pH 7) containing 0.1% trifluoroacetic acid-acetonitrile (65:35, v/v). Calibration curve was linear between the concentration range of 75-4000 ng/mL. Intra- and inter-day precision values for diclofenac in plasma were less than 3.6, and accuracy (relative error) was better than 5.3%. The limits of detection and quantification of diclofenac were 25 and 75 ng/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of diclofenac in healthy Turkish volunteers who had been given 50 mg diclofenac.     

An HPLC method for the determination of bisoprolol in human plasma and its application to a pharmacokinetic study

A new high-performance liquid chromatographic method is described for the determination of bisoprolol in human plasma. The proposed method was based on the derivatization of bisoprolol with 4-chloro-7-nitro-2,1,3-benzoxadiazole in borate buffer at pH 9.5 to yield a fluorescent product. Chromatographic separation of bisoprolol was achieved by using isocratic elution at a flow rate of 1.2 mL/min on a C18 reversed-phase column (Inertsil, 4 μm, 150 4.6 mm) at 40°C. The mobile phase used for the analysis was methanol-water (70:30, % v/v). Fluorescence detector was used at the excitation and emission wavelengths of 458 and 525 nm, respectively. The method was validated for linearity, limit of detection, limit of quantification, precision, accuracy, recovery and system suitability. The assay was linear over the concentration range of 10-2000 ng/mL. This method was applied in pharmacokinetic studies of bisoprolol preparations in healthy volunteers.     

HPLC method for determination of moxifloxacin in plasma and its application in pharmacokinetic analysis

Moxifloxacin is a representative of the fourth generation of fluoroquinolones. It possesses bacteriostatic activity against Gram-positive and Gram-negative bacteria. A simple and fast high performance liquid chromatography-ultraviolet detection (HPLC-UV) method was developed for moxifloxacin analysis. The separation was performed on C18 column. The mobile phase was a mixture of acetonitrile and 0.4% triethylamine solution. The regression curve was linear over the range 0.2–10.0?µg/mL. The validation parameters obey the European Medicine Agency limits. The in vivo study confirmed the practical application of the method.     

Validation of a simple HPLC method for assay of haplamine and its metabolites in plasma suitable for pharmacokinetic application in rats

A simple HPLC method with ultraviolet detection has been developed and validated for the simultaneous determination of haplamine and its metabolites (trans/cis-3,4-dihydroxyhaplamine) in rat. A liquid-liquid extraction was used to extract the compounds from rat plasma. The analysis was performed on a C(18) Nucleosil Nautilus column. The mobile phase consisted of water (A) and a mixture of methanol and acetonitrile (85:15; v/v) (B) used in gradient mode (38-40% B for 10 min, 40-58% B for 49 min, 58-38% B for 1 min, and 38% for 5 min) pumped at 1 mL/min. The calibration curves showed good linearity with correlation coefficients greater than 0.999 for the analytes in the investigated concentration range. The lower limit of detection was 0.007, 0.008 and 0.009 microg/mL and the lower limit of quantification was 0.014, 0.017 and 0.018 microg/mL for haplamine, and trans/cis-3,4-dihydroxyhaplamine, respectively. The method was applied to a preliminary pharmacokinetic study in rats. This method proved to meet fully the standards required of experimental pharmacokinetic studies and should be used in further preclinical investigation.     

A simple RP‐HPLC method for quantification of columbianadin in rat plasma and its application to pharmacokinetic study

A rapid and sensitive reversed‐phase high‐performance liquid chromatographic (RP‐HPLC) method was developed to investigate pharmacokinetics of columbianadin, one of the main bioactive constituents in the roots of Angelica pubescens f. biserrata, in rat plasma after intravenous administration to rats at two doses of 10 and 20 mg/kg. The method involves a plasma clean‐up step using liquid–liquid extraction by diethyl ether, followed by RP‐HPLC separation and detection. Separation of columbianadin was performed on an analytical Diamonsil? ODS C₁₈ column, with a mobile phase of MeOH–H₂O (85 : 15, v/v) at a flow‐rate of 1.0 mL/min, and UV detection was set at 325 nm. The retention time of columbianadin and scoparone (internal standard) was 6.7 and 3.5 min, respectively. The calibration curve was linear over the range of 0.2–20.0 μg/mL (r² = 0.9986) in rat plasma. The lower limits of detection and quantification were 0.05 and 0.1 μg/mL, respectively. The extraction recovery from plasma was in the range of 81.61–89.93%. The intra‐ and inter‐day precisions (relative standard deviation) were between 1.01 and 9.33%, with accuracies ranging from 89.76 to 109.22%. The results indicated that the method established was suitable for the determination and pharmacokinetic study of columbianadin in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study

A new, simple, and reproducible method for determination of carboxylic acid metabolite of clopidogrel in human plasma has been developed. After liquid-liquid extraction in acidic medium with chloroform, samples were quantified on a Nova-pak C(8), 5 microm column using a mixture of 30 mM K(2)HPO(4)-THF-acetonitrile (pH = 3, 79:2:19, v/v/v) as mobile phase with UV detection at 220 nm. The flow rate was set at 0.9 mL/min. Ticlopidine was used as internal standard and the total run time of analysis was about 12 min. The method was linear over the range of 0.2-10 microg/mL of clopidogrel metabolite in plasma (r(2) > 0.999). The within-day and between-day precision values were in the range 1.0-4.8%. The limit of quantification of the method was 0.2 microg/mL. The method was successfully used to study the pharmacokinetics of clopidogrel in healthy volunteers.     

Validated HPLC method for the quantitative determination of CoQ(10) in dog plasma and its application to a pharmacokinetic study

Coenzyme Q₁₀ (CoQ₁₀) is a naturally occurring compound located in all membranes throughout the cell. A rapid and sensitive HPLC method was developed to determine the concentration of CoQ₁₀ in dog plasma using a surrogate matrix. Chromatographic separation was carried out on a Diamonsil C₁₈ column with the UV detector set at 275 nm. Methanol–2‐propanol (40:60, v/v) was used as a mobile phase delivered at a flow rate of 1.0 mL/min. Calibrators were prepared using blank plasma–K₂HPO₄ buffer (50 mm , pH 8.0)–saline (1:3:6, v/v/v) as surrogate matrix. It was shown that the surrogate matrix had similar properties to dog plasma for CoQ₁₀ in extraction, freeze–thaw and stability. The assay was linear over the concentration range of 0.10–100 µg/mL. The intra‐ and inter‐day precisions were within 13.3% in terms of relative standard deviation (RSD%) and the accuracy was within ±7.5% in terms of relative error. This simple and reproducible HPLC method with less plasma volume (0.4 mL) and adequate sensitivity was successfully applied to pharmacokinetic studies of CoQ₁₀ in dogs and an investigation of the effect of CoQ₁₀ formulation on CoQ₁₀ baseline levels. Copyright © 2010 John Wiley & Sons, Ltd.     

Development of a method for the determination of ibafloxacin in plasma by HPLC with flourescence detection and its application to a pharmacokinetic study

A simple, rapid, and sensitive high-performance liquid chromatographic method is developed for the determination of ibafloxacin in rabbit plasma. Plasma proteins are precipitated with acetonitrile, and after extraction with methylene chloride followed by desecation, ibafloxacin is determined by reversed-phase chromatography with fluorescence detection exciting at 330 nm and emission at 368 nm. Peaks corresponding to ibafloxacin and the internal standard (salycilic acid) are obtained at 9.8 and 5.2 min, respectively. The method is validated for a limit of quantitation of 10 ng/mL. The intraday relative standard deviation ranges from 4.78-7.15%, and the interday precision ranges from 1.32-4.03%. The method shows linearity for the two calibration curves used (10-100 ng/mL and 100-2000 ng/mL). The procedure described is applied successfully to a pharmacokinetics study of ibafloxacin in rabbits.     

Liquid chromatographic/mass spectrometry assay of bromotetrandrine in rat plasma and its application to pharmacokinetic study

A rapid and sensitive liquid chromatography–tandem mass spectrometric method (LC‐MS/MS) for the determination of bromotetrandrine in rat plasma has been developed and applied to pharmacokinetic study in Sprague–Dawley (SD) rats after a single oral administration. Sample preparation involves a liquid–liquid extraction with n‐hexane–dichlormethane (65:35, containing 1% 2‐propanol isopropyl alcohol, v/v). Bromotetrandrine and brodimoprim (internal standard, IS) were well separated by LC with a Dikma C₁₈ column using methanol–ammonium formate aqueous solution (20 mm ) containing 0.5% formic acid (60:40, v/v) as mobile phase. Detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode. The ionization was optimized using ESI(+) and selectivity was achieved using MS/MS analysis, m/z 703.0 → 461.0 and m/z 339.0 → 281.0 for bromotetrandrine and IS, respectively. The present method exhibited good linearity over the concentration range of 20–5000 ng/mL for bromotetrandrine in rat plasma with a lower limit of quantification of 20 ng/mL. The intra‐ and inter‐day precisions were 2.8–7.5% and 3.2–8.1%, and the intra‐ and inter‐day accuracy ranged from ?4.8 to 8.2% and ?5.6 to 6.2%, respectively. The method was successfully applied to a pharmacokinetic study after a single oral administration to SD rats with bromotetrandrine of 50 mg/kg. Copyright © 2009 John Wiley & Sons, Ltd.     

HPLC determination of irbesartan in human plasma: its application to pharmacokinetic studies

A simple and rapid HPLC method using fluorescence detection was developed for determination of irbesartan in human plasma. Sample preparation was accomplished through a simple deproteinization procedure with 0.4 mL of acetonitrile containing 800 ng/mL of losartan (internal standard), and to a 0.1 mL plasma sample. Chromatographic separation was performed on a Zorbax Xclipse XDB C₁₈ column (150 × 4.6 mm, i.d., 5 µm) at 40°C. An isocratic mobile phase, acetonitrile:0.1% formic acid (37:63, v/v), was run at a flow‐rate of 1.0 mL/min, and the column eluent was monitored using a fluorescence detector set at excitation and emission wavelengths of 250 and 370 nm, respectively. The retention times of irbesartan and losartan were 4.4 and 5.9 min, respectively. This assay was linear over a concentration range of 10–5000 ng/mL with a lower limit of quantification of 10 ng/mL. The coefficient of variation for this assay precision was less than 8.48%, and the accuracy exceeded 94.4%. The mean relative recoveries of irbesartan and losartan were 98.4 and 99.1%, respectively. This method was successfully applied for pharmacokinetic study after oral administration of irbesartan (300 mg) to 23 Korean healthy male volunteers. Copyright © 2009 John Wiley & Sons, Ltd.     

Pre-column derivatization method for determining phenylephrine in human plasma and its application in a pharmacokinetic study

In the present study, a rapid derivatization liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated to evaluate phenylephrine in human plasma. The plasma samples were processed to precipitate the proteins, followed by derivatization of the phenylephrine in the plasma with dansyl-chloride solution and extraction with methyl tert-butyl ether–n-hexane (2:1, v/v). The treated samples were analyzed on a Gemini C₁₈ column with 3 min gradient elution, and sensitive detection was achieved with a Waters TQ-s. The method gave linear results over a concentration range from 0.020 to 10.0 ng/ml. The lower limit of quantification was 0.020 ng/ml. Intra- and inter-day precision was <15%, and accuracy was 95.0–105.3%. The validated LC–MS/MS method was successfully applied in the pharmacokinetic analysis of phenylephrine in Chinese subjects with common cold after a single-dose administration of 5, 10 or 20 mg phenylephrine. This pre-column derivatization method may also be applied for the analysis of endogenous hormones such as norepinephrine and adrenaline in a biological matrix.     

A rapid and simple RP‐HPLC method for quantification of kirenol in rat plasma after oral administration and its application to pharmacokinetic study

A rapid and simple reverse‐phase high‐performance liquid chromatography (RP‐HPLC) was developed and validated for the quantification of kirenol in rat plasma after oral administration. Kirenol and darutoside (internal standard, IS) were extracted from rat plasma using Cleanert™ C₁₈ solid‐phase extraction (SPE) cartridge. Analysis of the extraction was performed on a Thermo ODS‐2 Hypersil C₁₈ reversed‐phase column with a gradient eluent composed of acetonitrile and 0.1% phosphoric acid. The flow rate was 1.0 mL/min and the detection wavelength was set at 215 nm. The calibration curve was linear over the range of 9.756–133.333 µg/mL (r² = 0.9991) in rat plasma. The lower limits of detection and quantification were 2.857 and 9.756 µg/mL, respectively. The intra‐ and inter‐day precisions (relative standard deviation, RSD) were between 2.24 and 4.46%, with accuracies ranging from 91.80 to 102.74%. The extraction recovery ranged from 98.16 to 107.62% with RSD less than 4.81%. Stability studies showed that kirenol was stable in preparation and analytical process. The present method was successfully applied to the pharmacokinetic study of kirenol in male Sprague–Dawley rats after oral administration at a dose of 50 mg/kg. Copyright © 2010 John Wiley & Sons, Ltd.     

Validated RP‐HPLC/UV method for the quantitation of abiraterone in rat plasma and its application to a pharmacokinetic study in rats

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of abiraterone (ART) in rat plasma. The analytical procedure involves extraction of ART and diclofenac (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system with a Betasil C₁₈ column maintained at ambient room temperature and an isocratic mobile phase [acetonitrile–water–10 mm potassium dihydrogen phosphate (pH 3.0), 55:5:40, v/v/v] at a flow rate of 1.00 mL/min with a total run time of 10 min. The eluate was monitored using an UV detector set at 255 nm. Method validation was performed as per FDA guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 93.4–3251 ng/mL (r² = 0.997). The intra‐ and inter‐day precisions were 0.56–4.98 and 3.03–7.18, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of ART in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Development and validation of a RP‐HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid–liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C₁₈ column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182–5035 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the range of 1.41–11.2 and 3.66–8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Simple and sensitive HPLC method for determination of amrubicin and amrubicinol in human plasma: application to a clinical pharmacokinetic study

A simple and sensitive high‐performance liquid chromatographic (HPLC) method was developed for determination of amrubicin and its metabolite amrubicinol in human plasma. After protein precipitation with methanol without evaporation procedure, large volume samples were injected and separated by two monolithic columns with a guard column. The mobile phase consisted of tetrahydrofuran–dioxane–water (containing 2.3 mM acetic acid and 4 mM sodium 1‐octanesulfonate; 2:6:15, v/v/v). Wavelengths of fluorescence detection were set at 480 nm for excitation and 550 nm for detection. Under these conditions, linearity was confirmed in the 2.5–5000 ng/mL concentration range of both compounds. The intra‐ and inter‐day precision and intra‐ and inter‐day accuracy for both compounds were less than 10%. The method was successfully applied to a clinical pharmacokinetic study of amrubicin and amrubicinol in cancer patients. Copyright © 2009 John Wiley & Sons, Ltd.     

A rapid and simple HPLC method for the determination of curcumin in rat plasma: assay development,validation and application to a pharmacokinetic study of curcumin liposome

This paper describes a sensitive, specific and rapid high‐performance liquid chromatography (HPLC) method for the determination of curcumin in rat plasma. After a simple step of protein precipitation in 96‐well format using acetonitrile containing the internal standard (IS), emodin, plasma samples were analyzed by reverse‐phase HPLC. Curcumin and the IS emodin were separated on a Diamonsil C₁₈ analytical column (4.6 × 100 mm, 5 µm) using acetonitrile–5% acetic acid (75:25, v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was sensitive with a lower limit of quantitation of 1 ng/mL, with good linearity (r² ≥ 0.999) over the linear range 1–500 ng/mL. All the validation data, such as accuracy and precision, were within the required limits. A run time of 3.0 min for each sample made high‐throughput bioanalysis possible. The assay method was successfully applied to the study of the pharmacokinetics of curcumin liposome in rats. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated LC-MS/MS assay for the quantitative determination of nalbuphine in human plasma and its application to a pharmacokinetic study

A solid‐phase extraction–liquid chromatographic–tandem mass spectrometry method for the determination of nalbuphine concentrations in human plasma has been developed. Samples (1 mL) were extracted using a Strata™‐X solid phase extraction cartridges. Chromatographic separation of nalbuphine and naloxone (internal standard) was achieved on a Phenomenex Kinetex PFP (2.6 μm, 100 A, 100 × 2.1 mm) column using a mobile phase consisting of 0.1% formic acid, 15 mM ammonium acetate in deionized water and acetonitrile (60:40, v/v). The flow rate was 0.3 mL/min and the total run time was 2 min. Detection of the analytes was achieved using positive ion electrospray ionization via multiple reactions monitoring mode. The mass transitions were m/z 358 → 340 for nalbuphine and m/z 328 → 310 for naloxone. The assay was linear over the concentration range 0.50–500.00 ng/mL, with correlation coefficients ≥0.995. The lower limit of quantitation was set at 0.5 ng/mL plasma based on an average signal‐to‐noise ratio of 44.79. The intra‐ and inter‐day precision was less than 8.07% in terms of relative standard deviation and accuracy ranged from 94.97 to 106.29% at all quality control levels. The method was applied successfully to determine nalbuphine concentrations in human plasma samples obtained from subjects receiving intravenous administration of nalbuphine. The method is rapid, sensitive, selective and directly applicable to human pharmacokinetic studies involving nalbuphine. Copyright © 2011 John Wiley & Sons, Ltd.     

An HPLC method for the determination and pharmacokinetic study of lehmannine in rat plasma

A high-performance liquid chromatographic (HPLC) method for determining lehmannine (LMN) in rat plasma was developed for application in the pharmacokinetics study. The plasma was deproteinized with acetonitrile that contained an internal standard and was separated from the aqueous layer by adding sodium chloride. The HPLC assay was carried out using a VP-ODS column at 40 degrees C. The mobile phase was acetonitrile-0.02 mol/L ammonium acetate buffer-triethylamine (35:65:0.04, v/v/v). The flow rate was 1.0 mL/min. The detection wavelength was set at 220 nm. The method was used to determine the concentration-time profiles of LMN in the plasma following oral administration or bolus injection of LMN aqueous solution. The pharmacokinetic parameters of LMN were calculated for the first time by Drug and Statistics 1.0 program.     

A sensitive column-switching HPLC method for aripiprazole and dehydroaripiprazole and its application to human pharmacokinetic studies

A simple and sensitive column‐switching HPLC‐UV method was developed for the simultaneous determination of aripiprazole, a novel atypical antipsychotic drug, and its active metabolite, dehydroaripiprazole in human plasma. Aripiprazole, its active metabolite and 7‐[5‐[4‐(3‐chloro‐2‐methylphenyl)‐1‐piperazinyl]pentyloxy]‐3,4‐dihydro‐2(1H)‐quinolinone (OPC‐14558) as an internal standard were extracted from 1 mL of plasma using a mixture of chloroform/n‐heptane (3:7, v/v), and the extract was injected into a column I (TSK BSA‐ODS/S precolumn, 5 μm) for cleanup and column II (C₁₈ STR ODS‐II analytical column, 5 μm) for separation. Peaks were detected with an UV detector set at a wavelength of 254 nm, and the total time for chromatographic separation was ～20 min. Mean absolute recoveries were 74.0 and 74.7% for aripiprazole and dehydroaripiprazole, respectively. Intra‐ and inter‐day CVs were less than 7.5 and 7.1% for aripiprazole concentrations ranging from 2 to 600 ng/mL, and 9.2 and 4.5% for dehydroaripiprazole concentrations ranging from 2 to 160 ng/mL. The validated concentration ranges for this method were 1–500 ng/mL and the limits of detection were 0.5 ng/mL for both aripiprazole and dehydroaripiprazole. This method was applied to pharmacokinetic study in human volunteers and patients taking aripiprazole.     

A simple and sensitive HPLC method for quantitation of low phenoprolamine hydrochloride concentrations in human plasma and its pharmacokinetic application

Phenoprolamine hydrochloride is a novel compound that works against a variety of types of hypertension. The purpose of this study was to develop a simple and sensitive high-performance liquid chromatographic method for quantitation of low phenoprolamine hydrochloride concentrations in human plasma and to apply it to pharmacokinetic study. The procedure involved extraction of the drug and clonidine (internal standard) from the plasma using diethyl ether. Chromatographic separations were carried out on a 4.6 x 200 mm Hypersil silica column with UV detection at 230 nm. The isocratic mobile phase, 1% ammonium acetate (pH 5.4) and methanol (0.3:99.7, v/v), was run at 1 mL/min. Extraction recovery was 84% for phenoprolamine hydrochloride at a concentration level of 200 ng/mL, and 76% for clonidine at 200 ng/mL. The method was linear in the concentration range 5-4000 ng/mL with a lower limit of quantitation of 5 ng/mL for phenoprolamine hydrochloride. Inter- and intra-day coefficients of variation were less than 10%. The validated method was successfully applied to a pharmacokinetic study in human after an oral administration of the drug, and the pharmacokinetic parameters are presented.