Analysis of neolignans compounds of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck by HPLC

A high performance liquid chromatographic (HPLC) method was developed and validated for quantitative determination of neolignans in extracts of Piper regnellii var. pallescens. The analysis were carried out on a Metasil ODS column (150 mm x 4.6 mm, 5 microm) at 30 degrees C, using as mobile phase acetonitrile-water (60:40, v/v) containing 2% acetic acid. The flow rate was 1.0 ml/min and the detection was at 280 nm. The validation using conocarpan as standard demonstrated that the method presents linearity (linear correlation coefficient=0.9991), precision (relative standard deviation <5%) and accuracy (mean recovery=104.55%) in the concentration range 31.25-500 microg/ml. The limit of detection (LOD) was 1.68 microg/ml and the limit of quantitation was 5.60 microg/ml. This method allowed the identification and quantification of conocarpan, eupomatenoid-5 and eupomatenoid-6 in the hydroethanolic extracts obtained from the leaves, stems and roots by maceration process. All the extracts showed the same chromatographic profile, being that the extract of the roots presented the highest concentration of neolignans.     

A stability-indicating HPLC method for the determination of glucosamine in pharmaceutical formulations

A stability-indicating high performance liquid chromatographic (HPLC) method was developed for the assay of glucosamine in bulk forms and solid dosage formulations. The HPLC separation was achieved on a Phenomenex Luna amino column (150 mm x 4.6 mm, i.d., 5 microm particle size) using a mobile phase of acetonitrile-phosphate buffer (75:25, v/v, pH 7.50) at a flow rate of 1.5 ml min(-1) and UV detection at 195 nm. The method was validated for specificity, linearity, solution stability, accuracy, precision, limit of detection, and limit of quantitation. The detector response for glucosamine hydrochloride was linear over the selected concentration range from 1.88 to 5.62 mg ml(-1) with a correlation coefficient 0.9998. The accuracy was between 98.9 and 100.5%. The precision (R.S.D.) amongst six sample preparations was 1.1%. The limit of detection and the limit of quantitation are 0.037 and 0.149 mg ml(-1), respectively. The sample and standard solutions were stable for 1 week. The method was successfully used for analysis of active-excipient compatibility samples used for development of a solid dosage formulation in our laboratory and subsequent stability studies. The method was also used for the analysis of glucosamine in several commercially available solid dosage forms.     

LC method for the analysis of Oxiconazole in pharmaceutical formulations

A LC method has been developed for the quantitative determination of Oxiconazole (Ox) in bulk form, lotion and cream pharmaceutical formulations. The method validation yielded good results including the range, linearity, precision, accuracy, recovery, specificity, robustness, limit of quantitation and limit of detection. The LC separation was carried by reversed phase chromatography using a LiChrocart C(8) column (125 mm x 4.0 mm i.d., 5 microm particle size). The mobile phase was composed of methanol-0.02 M ammonium acetate buffer (85:15 v/v), pumped isocratically at flow rate 1 ml min(-1). The detection was carried out on UV detector at 254 nm. The calibration curve for Ox was linear from 40.0-140.0 microg ml(-1) range. The precision of this method, calculated as the relative standard deviation (R.S.D.) was 1.57% for lotion and 0.71% for cream. The R.S.D. values for intra- and inter-day precision studies were 0.57 and 1.34%, respectively. The recovery of the drug ranged between 98.84-102.2% (lotion) and 100.54-101.59% (cream). The stability indicating capability of the assays was proved using forced degradation. Chromatograms showed Ox well resolved from the degradation product.     

Validated enantiospecific LC method for determination of (R)-enantiomer impurity in (S)-efavirenz

A high-performance liquid chromatographic method was developed for separation of the enantiomers of efavirenz. The developed method was applied for the determination of (R)-enantiomer in (S)-efavirenz and satisfactory results were achieved. The base line separation with a resolution of more than 4.0 was achieved on Chiralcel OD (250 mm x 4.6 mm, 10 microm) column containing tris-(3,5-dimethylphenylcarbomate) as stationary phase. The mobile phase consists of n-hexane: isopropyl alcohol (80:20 v/v) with 0.1% (v/v) of formic acid as additive. The flow rate was kept at 1.0 ml/min and the UV detection was monitored at 254 nm. The (R)-enantiomer was found linear over the range of 0.1 microg/ml--6 microg/ml. The limit of detection (LOD) was 0.03 microg/ml and the limit of quantification (LOQ) was 0.1 microg/ml (n=3. The precision of (R)-enantiomer at LOQ level was evaluated through six replicate injections and the RSD of the peak response was achieved as 1.34%. The results demonstrated that the developed LC method was simple, precise, robust and applicable for the purity determination of efavirenz.     

Rapid determination of amoxicillin in premixes by HPLC

A rapid analytical procedure for routine identification and quantification of amoxicillin in premixes by high performance liquid chromatography was developed and tested. The ground premix samples were extracted for 10 min using 100ml extraction mixture water-methanol (800:200, v/v). The extract was analyzed by reversed-phase on Agilent Zorbax SB-C18 column (4.6 mm x 150 mm, i.d., 5 microm particle size) with water-methanol-phosphoric acid-triethylamine (842:150:4:4) containing 10 mM hexane-1-sulfonic acid sodium salt (pH 3.5) as mobile phase. UV detection was carried out at 230 nm. The method was validated for specificity, linearity, solution stability, accuracy, precision, limit of detection, and limit of determination. The detector response for amoxicillin was linear over the selected concentration range from 2.0 to 40.0 mg ml(-1) with a correlation coefficient 0.9999. The mean accuracy was 100.1% with a standard deviation of 0.6%. The limit of detection and the limit of determination are 0.1 and 0.3 mg ml(-1), respectively, which corresponds to 10 and 30 mg kg(-1), respectively, in real premix sample. The sample and standard solutions were stable for 4 h. The method is selective and can be used in routine analysis.     

Development and validation of bioanalytical method for the determination of asulacrine in plasma by liquid chromatography

Asulacrine (9-[(2-methoxy-4-methylsulphonylamino)phenylamino]-N,5-dimethyl-4-acridinecarboxamide), an analogue of the antileukaemia drug amsacrine, has high antitumour activity in mice and has also shown clinical activity. A simple method is described for the quantitation of asulacrine in plasma by liquid chromatography. Chromatographic separation was achieved on a reversed phase C 18 column (250 mm x 4.6mm, particle size 5 microm, Gemini) using isocratic elution (acetonitrile and 0.01 M sodium acetate buffer pH 4.0, 45/55, v/v) at a flow rate of 1 ml/min. Asulacrine and internal standard (the ethylsulphonanilide analogue) were measured using UV detection at 254 nm. The total chromatographic run-time was 8 min with asulacrine and internal standard eluting at approximately 4.7 and approximately 6.5 min, respectively. Limit of quantification was 0.1microg/ml. The linearity range of the method was 0.1-10 microg/ml (r2=0.9995). Mean recoveries from plasma were 100-105%. Intra-batch and inter-batch precision was 7.1 and 7.8%, respectively, and intra-batch and inter-batch accuracy (relative error) was 4.9 and 8.4%, respectively (n=8 in all cases). The bench top, freeze thaw, short-term storage and stock solution stability evaluation indicated no evidence of degradation of asulacrine. The validated method is simple, selective and rapid and can be used for pharmacokinetic studies in mice.     

Development and validation of an ultra performance liquid chromatography method for venlafaxine hydrochloride in bulk and capsule dosage form

A simple, specific, accurate, and precise ultra performance liquid chromatographic method was developed and validated for the estimation of venlafaxine hydrochloride in tablet dosage forms. A acquity TM BEH column having C18, 100×2.1 mm i.d. in isocratic mode, with mobile phase containing dipotassium hydrogen phosphate: Acetonitrile (30:70 v/v; pH 7.00 with dilute o-phosphoric acid) was used. The flow rate was 0.75 ml/min and effluents were monitored at 227 nm. The retention time of venlafaxine hydrochloride was 0.548 min. The method was validated for specificity, linearity, accuracy, precision, limit of quantification, limit of detection, robustness and solution stability. Limit of detection and limit of quantification for estimation of venlafaxine hydrochloride were found to be 6.11 μg/ml and 20.33 μg/ml, respectively. Recoveries of venlafaxine hydrochloride in tablet formulations were found to be in the range of 99.3-99.5%. Proposed method was successfully applied for the quantitative determination of venlafaxine hydrochloride in tablet dosage forms.     

Sensitive LC determination of ciprofloxacin in pharmaceutical preparations and biological fluids with fluorescence detection

A simple and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of ciprofloxacin. Separation of ciprofloxacin and anthranilic acid (internal standard) was achieved on a Kromasil 100, C(18), 5 microm (250 x 4.6 mm i.d.) reversed-phase column, using fluorescence detection with lambda(exc)=300 nm and lambda(emi)=458 nm. The mobile phase consisted of acetonitrile-methanol-acetate buffer (pH 3.60; 0.05 M) (10:30:60 v/v/v) containing 1% v/v acetic acid. The analysis was performed in less than 9 min, with a flow rate of 0.8 ml min(-1). A rectilinear relationship was observed for concentrations between 0.005 and 1.0 microg ml(-1) of ciprofloxacin in aqueous standard solutions and serum and the detection limit was 20 pg injected on-column. The intra- and inter-day relative standard deviation (n=8) ranged from 1.6 to 2.6% and from 1.9 to 4.8%, respectively, calculated at three concentration levels of standard solutions. Direct measurements of ciprofloxacin in pharmaceutical preparations and in serum, after precipitation of proteins, were performed with high precision and accuracy. The application of the method to urine samples involved a solid-phase extraction treatment of the samples using C(18) cartridges. The linear working range in urine extended from 0.05 to 2.0 microg ml(-1) and the detection limit was 0.2 ng injected on-column.     

Stability indicating ultraviolet spectroscopic method for the estimation of ezetimibe and carvedilol

In this study, new and rapid stability indicating ultraviolet spectroscopic methods were developed and validated for the estimation of ezetimibe and carvedilol in pure form and in their respective formulations. Since both the drugs are poorly water soluble, 20% v/v acetonitrile in triple distilled water was selected as the solvent system for both the drugs. This ensured adequate drug solubility and maximum assay sensitivity. The linearity range for ezetimibe and carvedilol at their respective wavelength of detection of 232 nm and 238 nm was obtained as 2-50 microg/ml and 2-20 microg/ml respectively. The linear regression equations obtained by least square regression method, were Y = 0.0443 x (X) + 0.0106 for ezetimibe and Y = 0.1080 x (X) + 0.034 for carvedilol, where Y is the absorbance and X is the concentration (in microg/ml) of pure drug solution. The detection and quantitation limit as per the error propagation theory were found to be 0.4 microg/ml and 1.3 microg/ml respectively for ezetimibe and 0.7 microg/ml and 2.1 microg/ml respectively for carvedilol. The methods were employed with high degree of precision and accuracy for the estimation of total drug content in two commercial tablet formulations of each of the two drugs. It was concluded that both the developed methods are accurate, sensitive, precise, and reproducible. They can be applied directly for the estimation of drug content in pharmaceutical formulations.     

An improved HPLC method with fluorescence detection for the determination of pyrene in rat plasma and its pharmacokinetics

A high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) for the determination of pyrene in rat plasma was developed and validated. The method used fluorene as internal standard (IS), following a single-step protein precipitation, the analyte and internal standard were separated on a C18 column with a mobile phase containing methanol-water (90:10, v/v) at a flow rate of 1 ml/min. The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 265 and 394 nm, respectively. Two calibration curves were constructed in the range of 2-100 ng/ml and 0.1-5 microg/ml for pyrene with a lower limit of quantitation (LLOQ) of 2 ng/ml. Both intra-day and inter-day precision were less than 6% except at LLOQ, for which the precision was 10.6 and 9.8, respectively. Accuracy ranged from 98.3 to 103.6%, except at LLOQ, for which the accuracy was about 85%. The recovery ranged from 84.7 to 95.0% at the low, medium and high concentrations. The present HPLC-FLD method was rapid, sensitive, and reliable. The method described herein had been successfully applied for the pharmacokinetic studies in female Wistar rats after administration of 10mg equivalent pyrene/kg dose of solution of pyrene and 1mg equivalent pyrene/kg dose of pyrene-loaded nanoparticle.     

Development and Validation of a Reversed-Phase High-performance Liquid Chromatography Method for Determination of Desmopressin in Chitosan Nanoparticles

A simple isocratic reversed-phase high performance liquid chromatographic method was developed for determination of released desmopressin from chitosan nanoparticles in the in vitro media. The chromatographic separation was achieved with acetonitrile/water (25:75, v/v), in which water contained 0.1% v/v trifluoroacetic acid with pH=2.5 as mobile phase, a Chromolith^® Performance RP-18e column (150×4.6 mm; 5 μm) kept at 40° and ultraviolet detection at 220 nm. The compound was eluted isocritically at a constant flow rate of 1.6 ml/min. The method was validated according to the International Conference on Harmonisation guidelines. The validation characteristics included accuracy, precision, linearity rang, selectivity, limit of detection, limit of quantitation and robustness. The calibration curve was linear (r>0.9999) over the concentration rang 0.5-100 μg/ml. The limit of detection and limit of quantitation in the release media were 0.05 and 0.5 μg/ml, respectively. The proposed method had an accuracy of and intra- and inter-day precision <4.2. Furthermore, to evaluate the performance of the proposed method, it was used in the analysis of desmopressin level in real samples containing chitosan nanoparticles in the in vitro media.     

A validated LC method for the determination of vesamicol enantiomers in human plasma using vancomycin chiral stationary phase and solid phase extraction

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.     

Determination of cetylpyridinium chloride and tetracaine hydrochloride in buccal tablets by RP-HPLC

The HPLC method for simultaneous determination of cetylpyridinium chloride (CPC), tetracaine hydrochloride (TTC) in Xipiluan buccal tablets was developed and validated. The HPLC method was performed on a CN column (150 x 4.6 mm i.d., 5 microm particle size); the mobile phase was methanol-tetramethylammonium hydroxide (20 mM)-potassium dihydrogen phosphate (3 mM) (90:10:3, v/v/v) (pH* 5.0), pumped at a flow rate 1.5 ml min(-1). The UV detector was set at 230 nm. The retention time for CPC and TTC was 3.52 and 3.10 min, respectively. Calibration curves were linear (r=0.9999, n=6) in the range of 5-2000 microg ml(-1) for CPC and 1-500 microg ml(-1) for TTC. Limit of detection and quantitation for CPC was 0.033 and 0.11 microg ml(-1), for TTC were 0.0056 and 0.019 microg ml(-1). The R.S.D. of repeatability and intermediate precision for CPC and TTC were less than 2.0%.     

A Validated RP-HPLC Method for Simultaneous Estimation of Nebivolol and Hydrochlorothiazide in Tablets

A simple, selective, rapid, precise and economical reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of nebivolol and hydrochlorthiazide from pharmaceutical formulation. Phenomenex Gemini C(18) (25 cm×4.6 mm i.d., 5 μ) column with a mobile phase consisting of acetonitrile: 50mM ammonium acetate (adjusted to pH 3.5 using orthophosphoric acid) (70:30 v/v) at a flow rate of 1.0 ml/min was used. Detection was carried out at 254 nm. Probenecid was used as an internal standard. The retention times of probenecid, nebivolol and hydrochlorthiazide were 13.05, 3.32 and 4.25 min, respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for the estimation of these drugs in combined dosage forms.     

Development and validation of a liquid chromatographic method for determination of enantiomeric purity of citalopram in bulk drugs and pharmaceuticals

A simple, rapid and robust LC method for enantiospecific separation and determination of citalopram in drugs and pharmaceuticals was developed using UV and polarimetric detectors connected in series. Baseline separation with resolution > or = 3.0 was achieved within 20 min on Chiralcel OD-H (250 mm x 4.6 mm) 5 microm column using a mobile phase containing of n-hexane:2-propanol:triethylamine (TEA) (95:05:0.1 v/v/v) at a flow rate of 1.0 ml/min at 25 degrees C. Effects of 2-propanol, triethylamine and temperature on enantioselectivity and resolution of the enantiomers were evaluated. Clopidogrel hydrogen sulphate was used as an internal standard (IS) for quantitative determinations using UV detector at 240 nm. Polarimetric detector was used for identification of enantiomers. The limits of detection (LOD) and quantification (LOQ) were 0.5 and 1.3 microg/ml respectively for both the enantiomers. The linearity of the method was in the range of 50-600 microg/ml with r2 > 0.9999. The inter- and intra-day assay precision was less than 0.63% (%R.S.D.) and recoveries were in the range 99.38-100.41%. The method was validated and found to be suitable for determination enantiomeric purity of citalopram in bulk drugs and pharmaceutical formulations.     

LC determination of rosiglitazone in bulk and pharmaceutical formulation

An isocratic reversed phase liquid chromatographic (RP-LC) method has been developed and subsequently validated for the determination of rosiglitazone and its related impurities. Separation was achieved with a Symmetry C18 column and sodium phosphate buffer (pH adjusted to 6.2):acetonitrile (50:50, v/v) as eluent, at a flow rate of 1.0 ml/min. UV detection was performed at 245 nm. The method is simple, rapid, selective and stability indicating. Indole was used as internal standard for the purpose of quantification of rosiglitazone. The described method is linear over a range of 0.45-10 microg/ml for related impurities and 180-910 microg/ml for assay of rosiglitazone. The method precision for the determination of assay and related compounds was below 1.0 and 3.6% RSD, respectively. The mean recoveries of impurities were found to be in the range of 95-102%. The percentage recoveries of Active Pharmaceutical Ingredient (API) from dosage forms ranged from 99.02 to 101.30. The method is useful in the quality control of bulk manufacturing and also in pharmaceutical formulations.     

Simultaneous RP-HPLC Estimation of Cefpodoxime Proxetil and Clavulanic Acid in Tablets

A new, simple, precise, rapid and accurate RP-HPLC method has been developed for the simultaneous estimation of cefpodoxime proxetil and clavulanic acid from pharmaceutical dosage forms. The method was carried out on a Zorbax Eclipse XDB 5 μ C 18 (150×4.6 mm) column with a mobile phase consisting of acetonitrile:50 mM potassium dihydrogen phosphate buffer (pH 3.0, 70:30 v/v) at a flow rate of 1.0 ml/min. Detection was carried out at 228 nm. Aspirin was used as an internal standard. The retention time of clavulanic acid, cefpodoxime proxetil and aspirin was 4.43, 6.44 and 5.6 min, respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification and solution stability. The proposed method can be used for the estimation of these drugs in combined dosage forms.     

Development and validation of an accurate HPLC method for the quantitative determination of picroside II in tablets

A simple, sensitive and accurate high performance liquid chromatographic method (HPLC) with UV detection was developed and validated to determine picroside II in a new tablet formulation with paeoniflorin as internal standard. Chromatographic separation was achieved on an Agilent XDB C18 column (250 x 4.6 mm I.D., 5 microm) using a mobile phase consisting of acetonitrile-water-acetic acid (18:82:0.4, v/v/v) at a flow rate of 1.0 ml/min. The UV detection wavelength was set at 265 nm. Linear calibration curves were obtained in the concentration range of 0.10-100 microg/ml with the limit of quantification (LOQ) of 0.10 microg/ml. The within- and between-run precisions in terms of % relative standard deviation (RSD) were lower than 5.7% and 6.3%, respectively. The accuracy in terms of % relative error (RE) ranged from -2.3% to 5.0%. This validated method was successfully applied to the determination of the content of picroside II in a new tablet formulation.     

An improved and validated LC method for resolution of bicalutamide enantiomers using amylose tris-(3,5-dimethylphenylcarbamate) as a chiral stationary phase

An improved HPLC method for determination of enantiomeric purity of bicalutamide in drugs and pharmaceuticals was developed and validated. Baseline separation with resolution >/=6.0 was achieved within 10 min on Chiralpak AD-H (250 mm x 4.6 mm; particle size 5 microm) column using n-hexane:2-propanol (65:35 v/v) as mobile phase at a flow rate of 1.0 ml/min at 25 degrees C. The detection was made at 270 nm using UV detector while a polarimetric detector connected in series was used for identification of enantiomers. The effects of 2-propanol, ethanol and temperature on enantioselectivity and resolution of enantiomers were evaluated. The method was validated in terms of accuracy, precision and linearity in the range of 10-250 microg/ml and the r(2) was >0.9999. The recoveries were 99.68-100.25% with <1% R.S.D. The limits of detection (LOD) and quantification (LOQ) of enantiomers were (2.4, 3.0 and 7.6, 9.3) x 10(-8)g/ml for (S)-(+)-BCT and (R)-(-)-BCT enantiomers, respectively. The method was found to be suitable for rapid determination of enantiomeric purity of bicalutamide in bulk drugs and pharmaceutical formulations.     

An enantiospecific HPLC method for the determination of (S)-enantiomer impurities in (R)-tolterodine tartarate

A high-performance liquid chromatographic method was developed for the separation of the enantiomers of tolterodine tartarate. The proposed method was applied to the determination of (S)-isomer in (R)-tolterodine tartarate, and satisfactory results were obtained. The enantiomers of tolterodine tartarate were separated on a Chiralpak AD-H (250 mm x 4.6 mm) column containing amylose tris-(3,5-dimethylphenyl-carbamate) at room temperature. The mobile phase consisted of n-hexane and isopropyl alcohol in the ratio of 85:15 (v/v) with 0.075% triethylamine (TEA) and 0.05% trifluoroacetic acid (TFA) as the additive. The flow rate was kept at 0.5 ml/min, and UV detection wavelength was set at 283 nm. The calibration curves of (S)-enantiomer in the concentration range from 0.05 microg/ml to 1 microg/ml range were linear. The relative standard deviations of within-day and between-day were less than 2% (n = 3). The limit of detection (LOD) was 0.75 ng (S/N = 3) and the limit of quantification (LOQ) was 0.05 microg/ml (RSD < 4.1%, n = 3). The determination recoveries of the (S)-enantiomer were in the range of 98.2-104.8%. The results demonstrated that the developed HPLC method was a reliable, simple technique and was applicable to the purity determination of (R)- tolterodine tartarate.