Optimization of HPLC method using central composite design for estimation of Torsemide and Eplerenone in tablet dosage form

Abstract A simple, precise, accurate and robust high performance liquid chromatographic method has been developed for simultaneous estimation of Torsemide and Eplerenone in tablet dosage form. Design of experiment was applied for multivariate optimization of the experimental conditions of RP-HPLC method. A Central composite design was used to study the response surface methodology and to analyse in detail the effects of these independent factors on responses. Total eleven experiments along with 3 center points were performed. Two factors were selected to design the matrix, one factor is variation in ratio of Acetonitrile and the second factor is flow rate (mL/min). Optimization in chromatographic conditions was achieved by applying Central composite design. The optimized and predicted data from contour diagram comprised mobile phase (acetonitrile, water and methanol in the ratio of 50: 30: 20 v/v/v respectively), at a flow rate of 1.0 ml/min and at ambient column temperature. Using these optimum conditions baseline separation of both drugs with good resolution and run time of less than 5 minutes were achieved. The optimized assay conditions were validated as per the ICH guidelines (2005). Hence, the results showed that the Quality by design approach could successfully optimize RP-HPLC method for simultaneous estimation of Torsemide and Eplerenone.

RP-HPLC method for determination of norethindrone in dissolution media and application to study release from a controlled release nanoparticulate liquid medicated formulation

Several methods are available for the determination of norethindrone. These methods are either complicated or needvalidation. The objective of this work was to develop and validate a simple reversed phase-high performance liquidchromatographic method for the determination of norethindrone in dissolution media. A Thermo Scientific C18column (250 mm × 4.6 mm ID, 5 µm pore size) was used. A mobile phase consisting of deionized water: acetonitrile(50:50, v/v) and 5 ml/l acetic acid was used. The flow rate was 1.3 ml/minute and the wavelength of the detectionwas 245 nm. Validation of linearity, accuracy and precision, limit of detection, limit of quantification, specificity, andstability (degradation) was carried out according to the International Conference on Harmonization guidelines. Thedeveloped and validated method was used to study norethindrone release from a nanoparticulate liquid medicatedformulation (LMF). The results indicated that the method was simple, accurate and precise, and met the acceptancecriteria. The drug exhibited higher stability in basic media when compared to acidic media. Drug release from a LMF(nanoemulsion) followed zero order kinetics. In conclusion, a simple method was developed, validated, and usedsuccessfully in evaluating in vitro drug release from a sustained release/controlled release nanoparticulate LMF.

Simultaneous estimation of Cefpodoxime proxetil and Ofloxacin In tablet dosage form using RP-HPLC.

A simple, rapid and precise reverse phase liquid chromatographic (RP-HPLC) method was developed and subsequently validated for simultaneous estimation of Cefpodoxime proxetil and Ofloxacin in combined fixed dose oral formulation. The analysis was carried out using X-terra C8 (4.6 x 250mm, 5μm, Make: ACE), prepacked column. The separation was carried out using a mobile phase containing a 0.25%v/v triethyl amine buffer of pH 3.5 and acetonitrile (30:70 v/v), was pumped at a flow rate of 1.2 ml/min with UV-detector and PDA detection at 227 nm. Both the drugs were well resolved on the stationary phase and the retention times were around 2.747 minute for Cefpodoxime proxetil and 2.076 minute for Ofloxacin. The method was validated and shown to be linear for Cefpodoxime proxetil and Ofloxacin. The correlation coefficients for Cefpodoxime proxetil and Ofloxacin are 0.998 and 0.999 respectively. The relative standard deviations for five replicate measurements in two sets of each drug in the tablets is always less than 2% and mean % error of active recovery not more than ±1.5%. The method was validated for precision and accuracy. The developed method could be applied for routine analysis of Cefpodoxime proxetil and Ofloxacin in tablet dosage form without any interference of excipients.

Selective separation, detection of zotepine and mass spectral characterization of degradants by LC-MS/MS/QTOF / 药物分析学报

A simple, precise, accurate stability-indicating gradient reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed for the quantitative determination of zotepine (ZTP) in bulk and pharmaceutical dosage forms in the presence of its degradation products (DPs). The method was developed using Phenomenex C18 column (250 mm ~ 4.6 mm i.d., 5 mm) with a mobile phase containing a gradient mixture of solvents, A (0.05%trifluoroacetic acid (TFA), pH ? 3.0) and B (acetonitrile). The eluted compounds were monitored at 254 nm;the run time was within 20.0 min, in which ZTP and its DPs were well separated, with a resolution of 41.5. The stress testing of ZTP was carried out under acidic, alkaline, neutral hydrolysis, oxidative, photolytic and thermal stress conditions. ZTP was found to degrade significantly in acidic, photolytic, thermal and oxidative stress conditions and remain stable in basic and neutral conditions. The developed method was validated with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness as per ICH guidelines. This method was also suitable for the assay determination of ZTP in pharmaceutical dosage forms. The DPs were characterized by LC-MS/MS and their fragmentation pathways were proposed.

RP-HPLC analysis of manool-rich Salvia officinalis extract and its antimicrobial activity against bacteria associated with dental caries

In this paper we screened the dichloromethane extract from the aerial parts of Salvia officinalis L., Lamiaceae, against a representative panel of microorganisms that cause caries, conducted a bioassay-guided fractionation to establish themselves the most active metabolite (manool) and determined the Salvia officinalis fraction with the manool highest concentration to be used to activate an ingredient in oral care products such as toothpastes and mouthwashes. Both manool and S. officinalis extract showed very promising minimal inhibitory concentration values (between 6.24 and 31.36 µg.ml-1) and time kill curves against the primary causative agents of dental caries (Streptococcus mutans) revealed that, at twice its minimal bactericidal concentration (12.48 µg.ml-1), manool required 6 h to completely kill the bacteria. Salvia officinalis extract at twice its minimal bactericidal concentration (31.36 µg.ml-1 ) needed 12 h. The results achieved with Salvia officinalis extract motivated us to develop and validate an analytical RP-HPLC method to detect and determine manool in this extract. The validation parameters were satisfactorily met and evaluated allows us to consider the developed method suitable for use in different labs. In conclusion, our results evidenced that the manool-rich S. officinalis extract can be considered an analytically validated alternative to develop novel and effective antimicrobial agents against the main bacteria responsible for dental caries.

Simultaneous estimation of drotaverine hydrochloride and paracetamol in bulk and tablet dosage form by RP-HPLC method.

A simple, rapid, reproducible, accurate and precise Reverse Phase HPLC method was developed for the quantitative simultaneous estimation of Drotaverine hydrochloride and Paracetamol in combined tablet dosage form. Drotaverine hydrochloride is an analog of papaver and is used mainly as an antispasmodic, smooth muscle relaxant. Paracetamol has analgesic and antipyretic activity. The chromatographic separation of both drugs was achieved with 250 x 4.6 mm, i.d 5 μm C-18 column using Methanol: water pH adjusted to 4.0 with O- Phosphoric acid. (60:40 v/v) at the flow rate of 1ml/min. The measurements were made at 243.0 nm using UV detector. The linearity range was found to be 5-80 μg/ml for Drotaverine hydrochloride and 5-70 μg/ml for Paracetamol. The coefficient of correlation for Drotaverine hydrochloride and Paracetamol was found to be 0.9994 and 0.9990 respectively. The retention time for Drotaverine hydrochloride and Paracetamol were 4.562 min and 8.146 min, respectively. The tailing factor for Drotaverine hydrochloride and Paracetamol was found to be 1.12 and 1.18 respectively. The percent recoveries obtained for Drotaverine hydrochloride and Paracetamol were found to be 99.85 and 99.92 respectively. The relative standard deviation for intraday and interday precision in tablet was always less than 2%. The method was validated for linearity, range, precision, accuracy, specificity, selectivity, intermediate precision, ruggedness, robustness, stability and suitability.

RP HPLC Method for the determination of Tamsulosin in bulk and Pharmaceutical Formulations.

A simple, sensitive, precise and specific reverse phase high performance liquid chromatographic method was developed and validated for the determination of Tamsulosin in bulk and tablet dosage forms. It was found that the excipient in the tablet dosage forms does not interfere in the quantification of active drug by proposed method. The HPLC separation was carried out by reverse phase chromatography on Shimadzu HPLC, 10-At detector with hypersil ODS C18 Column 250 X 4.6 mm (particle size of 5μ) and constant flow pump. Rheodyne injector with 20 μl loop with a mobile phase composed in the ratio acetonitrile: (0.05M) KH2PO4 buffer (45:55) at flow rate 1.8 ml /min. The detection was monitored at 240nm. The calibration curve for Tamsulosin was linear from 10-50g/ml and internal standard (Bromhexine) 10g/ml were prepared by suitable dilutions of the stock solution with appropriate mobile phase. The interday and intraday precision was found to be within limits. The proposed method has adequate sensitivity, reproducibility and specificity for the determination of Tamsulosin in bulk and its tablet dosage forms. LOD and LOQ for Tamsulosin were found to be 0.495 and 0.461.Accuracy (recoveries: 98.5-98.55%) and reproducibility were found to satisfactory.

Validation and stability indicating RP-HPLC method for the determination of sildenafil citrate in pharmaceutical formulations.

A simple, selective, accurate reverse phase-high Performance Liquid Chromatographic (RP-HPLC) method was developed and validated for the analysis of Sildenafil Citrate in pharmaceutical formulations. Chromatographic separation achieved isocratically on a C18 column [Use Inertsil C18, 5m , 150 mm x 4.6 mm] utilizing a mobile phase of acetonitrile/phosphate buffer (70:30, v/v, pH 7.0) at a flow rate of 0.8 ml/m with UV detection at 228 nm. The retention time was 4.087. The method is accurate (99.15-101.85%), precise (intraday variation 0.13-1.56% and inter-day variation 0.30-1.60%) and linear within range 0.1- 30μg/ml (R2=0.999) concentration and was successfully used in monitoring left over drug. The detection limit of sildenafil citrate at a signal-to-noise ratio of 3 was 1.70ng/ml in formulations while quantification limit in drug was 5.40 ng/ml. The proposed method is applicable to stability studies and routine analysis of sildenafil citrate in pharmaceutical formulations.

Determination of Three Kinds of Alkaloids in Yiqi Zhixue Granules by Ion Pair RP-HPLC Method / 中药新药与临床药理

Objective To develop a method for the determination of berberine hydrochloride,palmatine hydrochloride and jatrorrhizine hydrochloride in Yiqi Zhixue Granules. Methods The samples were extracted by ultrasonication with methanol for 30 min,and an ion pair RP-HPLC method was applied to determine three kinds of alkaloids.The column of C18 with temperature at 30 ℃ was used to separate the target components,the mobile phase consisted of the mixed solution of 0.01 mol/L sodium-heptanesulfonate solution and equal volume of 0.01 mol/L potassium dihydrogen phosphate solution(pH being adjusted at 3.1 with phosphoric acid) combined with acetonitrile(70 ∶ 30),the flow rate was 1.0 mL/min,and the detection wavelength was at 345 nm. Results Three kinds of alkaloids were separated perfectly,the average recoveries(n=6)were 100.95 %(RSD=2.10 % ) for berberine chloride,102.14 %(RSD=2.29 % ) for palmatine chloride,and 100.71 %(RSD=2.65 % ) for jatrorrhizine chloride. Conclusion The developed method is demonstrated to be simple,specific and accurate,which can be used to determine the contents of berberine chloride,palmatine chloride and jatrorrhizine chloride in Yiqi Zhixue Granula,and to control the quality of Folium Mahoniae in Yiqi Zhixue Granules.

DETERMINATION OF THE COUMATETRALYL IN BIOLOGICAL MATERIALS BY RPHPLC METHOD USING INTERNAL STANDARD / 中国法医学杂志

A simple,reliable RPHPLC method on ODS column with UV detector for deter-mination of the anticoagulant rodenticide coumatetralyl in animal tissue was developed.1,1′-Bi-(2-Naphthol)was used as an internal standard to check the process ofexperiments.The mean recoveries from the spiked rabbit liver were around 90% atthe levels of 0.4-16mg/kg.The coeffecint of variation of 5 determination was 3.2%.The results showed that this method has a satisfactory reproducibility.The methodis practical for examining poisons in poisoning cases.