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.     

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.     

Simultaneous Determination of Paracetamol and Orphenadrine Citrate in Dosage Formulations and in Human Serum by RP‐HPLC

Rapid liquid chromatographic procedures are proposed for analysis of paracetamol and orphenadrine citrate in pharmaceutical preparations and human serum using acetonitrile: water (50:50) as a mobile phase, adjusting pH to 2.6, UV detection at 215 nm and propylparaben sodium as internal standard. The advantages of this method include good and rapid separation, well resolved peaks, and only a small amount of sample is required for assay and adequate precision. The method showed good linearity in the range of 6 to 10000 ng/mL for paracetamol serum concentrations with a correlation coefficient 0.9999 (inter and intra day CV < 3.15) and in the range 3–10000 ng/mL for orphenadrine citrate serum concentrations with a correlation coefficient of 0.9999 (inter and intra day CV < 3.58). The recovery of paracetamol and orphenadrine citrate was > 96.9% and > 96.7%, respectively. The proposed method may be used for the quantitative analysis of paracetamol and orphenadrine citrate alone or in combination from raw materials, in bulk drugs, dosage formulations and in serum.     

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.     

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 rapid RP‐HPLC method for analysis of (−)‐copalic acid in copaíba oleoresin

The Copaifera species (Leguminoseae) are popularly known as ‘copaíba’ or ‘copaíva’ and are grown in the states of Amazonas, Pará and Ceará in northern Brazil. The oleoresins obtained from these species have been extensively used owing to their pharmacological potential and their application in cosmetic and pharmaceutical preparations. In the present study, the development and validation of a novel, rapid and efficient RP‐HPLC methodology for the analysis of the diterpene (?)‐copalic acid (CA), pointed out as the only chemical marker of the Copaifera genus, are described. The regression equation (Y = 26,707x ? 29,498) was obtained with good linearity (r² = 0.9993) and the limits of quantification and detection were 9.182 and 3.032 µg/mL, respectively. The precision and the accuracy of the method were adequate (lower than 4%). Finally, the validation parameters evaluated were satisfactorily met, so the developed method represents a suitable tool for application in the quality control of such natural products. Further studies aiming to develop analytical methodologies for each Copaifera species using a more representative number of chemical markers should be performed. Copyright © 2012 John Wiley & Sons, Ltd.     

A simple validated RP‐HPLC bioanalytical method for the quantitative determination of a novel valproic acid arylamide derivative in rat hepatic microsomes

A simple and specific bioanalytical method based on reversed‐phase high‐performance liquid chromatography (RP‐HPLC) coupled with ultraviolet detection was developed and validated for the determination of a novel valproic acid arylamide, N‐(2‐hydroxyphenyl)‐2‐propylpentanamide (HO‐AAVPA) in rat hepatic microsomes (a subcellular fraction containing phase I enzymes, especially cytochrome P450). The chromatographic separation was achieved using a reversed‐phase Zorbax SB‐C₁₈ column and a mobile phase of acetic acid in water (0.2% v/v) and acetonitrile (40:60 v/v) with a flow rate of 0.5 mL/min. The calibration curve was linear over the range of 882–7060 ng/mL (r² = 0.9987), and the lower limit of quantification and the lower limit of determination were found to be 882 and 127.99 ng/mL, respectively. The method was validated with excellent sensitivity, and intra‐day accuracy and precision varied from 93.79 to 93.12%, and from 2.12 to 4.36%, respectively. The inter‐day accuracy and precision ranged from 93.29 to 97.30% and from 0.68 to 3.60%, respectively. The recovery of HO‐AAVPA was measured between 91.36 and 97.98%. The assay was successfully applied to the analysis of kinetic metabolism and pharmacokinetic parameters in vitro by a substrate depletion approach. Copyright © 2014 John Wiley & Sons, Ltd.     

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

A simple, specific, sensitive and reproducible high‐performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of odanacatib in rat and human plasma. The bioanalytical procedure involves extraction of odanacatib and itraconazole (internal standard, IS) from a 200 μL plasma aliquot with simple liquid–liquid extraction process. Chromatographic separation was achieved on a Symmetry Shield RP₁₈ using an isocratic mobile phase at a flow rate of 0.7 mL/min. The UV detection wave length was 268 nm. Odanacatib and IS eluted at 5.5 and 8.6 min, respectively with a total run time of 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 50.9–2037 ng/mL (r² = 0.994). The intra‐ and inter‐day precisions were in the range of 2.06–5.11 and 5.84–13.1%, respectively, in rat plasma and 2.38–7.90 and 6.39–10.2%, respectively, in human plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.     

Development and validation of RP‐HPLC‐fluorescence method for quantitative determination of quinidine,a probe substrate for P‐glycoprotein inhibition assay using Caco‐2 cell monolayer

A simple, sensitive and specific reverse‐phase high‐performance liquid chromatographic (RP‐HPLC) method with fluorescence detection was developed for quantitation of quinidine from HBSS buffer. The method was applicable in the bi‐directional transport assay for evaluation of the inhibitory effect of test compounds on P‐glycoprotein‐mediated quinidine transport; quinidine was used as a probe P‐glycoprotein substrate. The calibration curve was linear (correlation coefficient ≥99) in the range 0.30–100.00 nm. The method was validated and is specific and sensitive with limit of quantitation of 300 pm for quinidine. The method was found to be accurate and precise in the working calibration range. Stability studies were carried out at different storage conditions where the analyte was found to be stable. The applicability and reliability of the analytical method was evaluated by successful demonstration of efflux ratio (P_appB → A/P_appA → B) in the Caco‐2 cell monolayer efflux assay. The efflux ratio for quinidine (100 nm) alone was 10.8, which reduced to less than 2 in the presence of the classical P‐gp inhibitors verapamil and ketoconazole (100 μm each). Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of a generic RP‐HPLC PDA method for the simultaneous separation and quantification of active ingredients in cold and cough medicines

A novel generic reverse phase high performance liquid chromatography (RP‐HPLC) method is developed and validated for simultaneous determination of seven pharmaceutically active ingredients, namely, acetaminophen, dextromethorphan, doxylamine, phenylephrine, guaifenesin, caffeine and aspirin. All seven ingredients were quantified in soft gel, syrup and tablet formulations of the over‐the‐counter US‐marketed products, as per the guidelines of the International Conference on Harmonization. The separation was achieved in a 16 min run time on an Agilent Zorbax Phenyl column using a gradient method with two mobile phases. Mobile phase A was 0.15% trifluoro acetic acid in purified water and while mobile phase B was a mixture of acetonitrile and methanol (750:250 v/v) with 0.02% trifluoro acetic acid. The flow rate was 1.0 mL min^?1 and injection volume was 10 μL. Detection was performed at 280 nm using a photodiode array detector. As part of the method validation, specificity, linearity, precision and recovery parameters were verified. The concentration and area relationships were linear (R² > 0.999), over the concentration ranges 20–120 μg mL^?1 for acetaminophen, 75–450 μg mL^?1 for dextromethorphan, 31.25–187.5 μg mL^?1 for doxylamine, 25–150 μg mL^?1 for phenylephrine, 25–150 μg mL^?1 for aspirin, 6.5–39 μg mL^?1 for caffeine and 12–72 μg mL^?1 for guaifenesin. The relative standard deviations for precision and intermediate precision were <1.5%. The proposed RP‐HPLC generic method is applicable for routine analysis of cold and cough over‐the‐counter products.     

Lipophilicity determination of N-( benzothiazol-2-yl )-α-amino alkyl phosphonic diesters by RP-HPLC and RP-HPTLC

Using methanol-water mixtures as the mobile phase,the chro-matographic retention parameters k' and Rf were determined by reversed-phase high-performance liquid chromatography(RP-HPLC)and reversed-phase high-performance thin-layer chromatography(RP-HPTLC)for N-(benzothiazol-2-yl)-o) amino alkyl phosphonic diesters and the correlation with lipophilicity parameter(ClogP)was established.Logkw values obtained from RP-HPLC and R values obtained from RP-HPTLC can be used to evaluate the lipophilicity of this kind of compounds.Chromatographic method is a good alternative for lipophilicity measurement.     

Simultaneous Quantitation of Captopril and NSAID's in API,Dosage Formulations and Human Serum by RP‐HPLC

An accurate, sensitive and least time consuming reverse phase high performance liquid chromatographic (RP‐HPLC) method for the estimation of captopril in the presence of non steroidal anti‐inflammatory drugs in formulation and human serum has been developed and validated. Chromatographic separation was conducted on prepacked Purospher star C18 (5 μm, 25 × 0.46 cm) column at room temperature using methanol:water (80:20 v/v) as a mobile phase, pH adjusted at 2.8 with o‐phosphoric acid and at a flow rate of 1.0 mL min⁻¹, while UV detection was performed at 227 nm. The limit of detection and quantification for captopril were 1 and 0.35 ng mL⁻¹, while that for (NSAID's) i.e. flurbiprofen, ibuprofen, diclofenac sodium and mefenamic acid LOD were 0.2, 1, 2 and 0.4 ng mL⁻¹ respectively and LOQ were 0.9, 2.9, 8 and 1 ng mL⁻¹ Analytical recovery was > 98.1%. The method used for the quantitative analysis of commonly administered non steroidal anti‐inflammatory drugs (NSAID's) i.e. ibuprofen, flurbiprofen, diclofenac sodium and mefenamic acid alone or in combination with captopril from API (active pharmaceutical ingredients), dosage formulations and in human serum. The established method is rapid (RT < 12 min), accurate (recovery > 98.1%), selective (no interference of excepients and other commonly used drugs and food) and sensitive (LOQ 3.5 ng mL^;‐1) and reproducible (SD ± 0.003).     

Study on Determination of Six Transition Metal Ions in Biological Samples by SPE and RP‐HPLC

This paper reports the utilization of solid phase extraction and the reversed‐phase high‐performance liquid chromatography (RP‐HPLC) for the determination of six transition metal ions (iron, cobalt, nickel, copper, zinc and manganese) in biological samples. The samples were digested by microwave digestion. The iron, cobalt, nickel, copper, zinc and manganese ions in the digested samples can react with 2‐(2‐quinolinylazo)‐5‐diethylaminophenol (QADEAP) to form colored chelates in pH 4.0 acetic acid‐sodium acetic buffer solutions and cetyl trimethylammonium bromide (CTMAB) medium. These chelates were enriched by solid phase extraction with C₁₈ cartridge. Then the chelates were separated on a Waters Nova‐Pak‐C₁₈ column (3.9 × 150 mm, 5 μm) by gradient elution with methanol (containing 0.5% of acetic acid and 0.1% of CTMAB) and 0.05 mol/L pH 4.0 acetic acid‐sodium acetic buffer solution (containing 0.1% of CTMAB) as mobile phase at a flow rate of 0.5 mL/min. The detection limits of iron, cobalt, nickel, copper, zinc and manganese are 3 ng/L, 4 ng/L, 2 ng/L, 4 ng/L, 8 ng/L, 10 ng/L, respectively. This method was applied to the determination of iron, cobalt, nickel, copper, zinc and manganese in biological samples with good results.     

Determination of 3-n-butylphthalide in rabbit plasma by HPLC with fluorescence detection and its application in pharmacokinetic study

A rapid, sensitive and specific reversed-phase high-performance liquid chromatographic method was developed for the determination of 3-n-butylphthalide, a drug currently being developed for treatment of stroke, in rabbit plasma. Fluorescence detection at an excitation wavelength of 280 nm and an emission wavelength of 304 nm was used for quantification of 3-n-butylphthalide. Ibuprofen was used as internal standard. Plasma samples were extracted with diethyl ether under acidic conditions. After evaporation of the organic phase, the extract was dissolved in mobile phase and injected into the chromatograph with C(18) column and a mobile phase of 0.05 mol/L sodium acetate buffer (pH 4.5)-acetonitrile (400:600). The peak area ratio vs concentration in plasma was linear over the range of 0.0212-4.24 microg/mL (correlation coefficient r = 0.9984) and the limit of quantification was 0.0212 microg/mL. Mean recovery was determined as 101.0% by analysis of plasma standard samples containing 0.0424, 0.424, 2.12 and 4.24 microg/mL of 3-n-butylphthalide. The intra-day relative standard deviations (RSDs) ranged from 3.6 to 8.9% and inter-day RSDs were within 8.0%. Pharmacokinetics of a single intravenous dose of 3-n-butylphthalide to the rabbits was presented to illustrate the applicability of this method. 3-n-Butylphthalide exhibited linear pharmacokinetics after intravenous administration to rabbits over the dose range 1-10 mg/kg.     

Validation of a high‐performance liquid chromatography method for the determination of (−)‐α‐bisabolol from particulate systems

A reversed‐phase high performance liquid chromatography method has been developed and validated for determination and quantitation of the natural sesquiterpene (−)‐α‐bisabolol. Furthermore the application of the method was done by characterization of chitosan milispheres and liposomes entrapping Zanthoxylum tingoassuiba essential oil, which contains appreciable amount of (−)‐α‐bisabolol. A reversed‐phase C₁₈ column and gradient elution was used with the mobile phase composed of (A) acetonitrile–water–phosphoric acid (19:80:1) and (B) acetonitrile. The eluent was pumped at a flow rate of 0.8 mL/min with UV detection at 200 nm. In the range 0.02–0.64 mg/mL the assay showed good linearity (R² = 0.9999) and specificity for successful identification and quantitation of (−)‐α‐bisabolol in the essential oil without interfering peaks. The method also showed good reproducibility, demonstrating inter‐day and intra‐day precision based on relative standard deviation values (up to 3.03%), accuracy (mean recovery of 100.69% ± 1.05%) and low values of detection and quantitation limits (0.0005 and 0.0016 mg/mL, respectively). The method was also robust for showing a recovery of 98.81% under a change of solvent in standard solutions. The suitability of the method was demonstrated by the successful determination of association efficiency of the (−)‐α‐bisabolol in chitosan milispheres and liposomes. Copyright © 2009 John Wiley & Sons, Ltd.     

Structure‐guided RP‐HPLC chromatography of diastereomeric α‐helical peptide analogs substituted with single amino acid stereoisomers

An α‐helical model peptide (Ac‐EAEKAAKE‐X‐EKAAKEAEK‐amide) was used as a template to examine the efficacy of conventional reversed‐phase high‐performance liquid chromatography (RP‐HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo‐Ile, d ‐Ile and d ‐allo‐Ile. We compared differences in peptide retention behavior on a C₈ column and a C₁₈ column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP‐HPLC to separate Ile‐ and allo‐Ile‐substituted analogs of a 26‐residue α‐helical antimicrobial peptide (AMP), with the substitution site towards the C‐terminus of the α‐helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP‐HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo‐designed AMP. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous Determination of Prazosin,Atorvastatin, Rosuvastatin and Simvastatin in API,Dosage Formulations and Human Serum by RP‐HPLC

Prazosin hydrochloride is the first developed selective antagonist for α₁ — adrenoceptors, which is used as antihypertensive agent. Statins are used in the treatment of various types of hypercholesterolemia. In the present paper a simple, specific an accurate RP‐HPLC method was developed and validated for the simultaneous determination of prazosin, atorvastatin, rosuvastatin and simvastatin in active and in dosage formulations. A nucleosil 100‐10, C‐18, 10μ column having 250 × 4.6 mm i.d. in isocratic mode, with mobile phase containing methanol:water:acetonitrile (70:20:10) adjusted to pH 2.5 ± 0.02 using orthophosphoric acid. The flow rate was 1 mLmin^?1 and effluents were monitored at 240 nm. The % recovery for all the drugs in formulations was found to be 94‐105%. The parameters such as accuracy (%RSD less than 2), precision (%RSD less than 2), linearity (>0.999) were found to be satisfactory. The presented method was applied without any interference of excepients for the determination of tablets. The proposed method due to its low LOQ, excellent accuracy, precision and selectivity could be used for routine quality control.     

Development and validation of an RP‐HPLC method for the quantitation of Orteronel (TAK‐700), a CYP17A1 enzyme inhibitor,in rat plasma and its application to a pharmacokinetic study

A novel, simple, specific, sensitive and reproducible high‐performance liquid chromatography assay method has been developed and validated for the estimation of Orteronel in rat plasma. The bioanalytical procedure involves extraction of Orteronel and phenacetin (internal standard) 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 mL/min and a C₁₈ column maintained at ambient room temperature. The eluate was monitored using a photodiode array detector set at 242. Orteronel and internal standard eluted at 4.8 and 6.2 min, respectively and the total run time was 9 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 100–3149 ng/mL (r² = 0.995). The intra‐ and inter‐day precisions were in the ranges of 0.31–7.87 and 3.97–6.35, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study of Orteronel in rats. Copyright © 2013 John Wiley & Sons, Ltd.     

RP‐HPLC enantioseparation of β‐adrenolytics using micellar mobile phase without organic solvents

Enantioseparation of a few commonly administered racemic β‐adrenolytics (namely, carvedilol, betaxolol, salbutamol and bisoprolol) has been achieved using a water micellar mobile phase containing surfactants (sodium dodecyl sulphate and Brij‐35) without organic solvents as a new approach in RP‐HPLC. Two chiral derivatizing reagents based on enantiomerically pure (S )‐(−)‐levofloxacin were synthesized using N ‐hydroxysuccinimide and N ‐hydroxybenzotriazole as the activation auxiliaries. Diastereomeric derivatives of the chosen β‐adrenolytics were synthesized under microwave irradiation in a very short reaction time. The (S )‐(−)‐levofloxacin moiety enhanced molar absorbance of the diastereomeric derivatives resulting in very low limit of detection (1.618 and 4.902 ng/mL, respectively, for diastereomeric derivatives of (RS )‐betaxolol and better resolution with lower retention times (for all the analytes), in comparison to literature reports. There was 15–20 times less consumption of mobile phase because of lower retention time.