Fast separation and quantification of three antiglaucoma drugs by high-performance liquid chromatography UV detection

In this study, a simple and accurate high-performance liquid chromatography method was developed and validated for fast separation of three anti-glaucoma drugs: timolol maleate (TM), brimonidine tartrate (BM), and latanoprost (LP). Separation of the three drugs was achieved in < 6 minutes using a BDS Hypersil phenyl column and a mobile phase consisting of acetonitrile: 25mM phosphate buffer, pH 4.0 (50: 50, v/v) at 1.2 mL/min with UV detection at 210 nm. The method was linear over the concentration ranges of 5.0–200.0 μg/mL, 2.0–80.0 μg/mL and 1.0–25.0 μg/mL with lower detection limits of 0.21 μg/mL, 0.10 μg/mL and 0.11 μg/mL for TM, BM and LP, respectively. The method was applied for the determination of two fixed-dose combination eye drops for the treatment of glaucoma, containing TM together with either BM or LP. Commercial samples of single-ingredient ophthalmic solutions containing the studied drugs were also successfully analyzed. The results obtained by the proposed method were favorably compared with those obtained by the comparison methods using Student’s t test and the variance ratio F test.     

Development and Validation of Different Ultraviolet-Spectrophotometric Methods for the Estimation of Besifloxacin in Different Simulated Body Fluids

In the present study a simple, accurate, precise, economical and specific UV-spectrophotometric method for estimation of besifloxacin in bulk and in different pharmaceutical formulation has been developed. The drug shows maximum λ_max289 nm in distilled water, simulated tears and phosphate buffer saline. The linearity range of developed methods were in the range of 3-30 μg/ml of drug with a correlation coefficient (r²) 0.9992, 0.9989 and 0.9984 with respect to distilled water, simulated tears and phosphate buffer saline, respectively. Reproducibility by repeating methods as %RSD were found to be less than 2%. The limit of detection in different media was found to be 0.62, 0.72 and 0.88 μg/ml, respectively. The limit of quantification was found to be 1.88, 2.10, 2.60 μg/ml, respectively. The proposed method was validated statically according to International Conference on Harmonization guidelines with respect to specificity, linearity, range, accuracy, precision and robustness. The proposed methods of validation were found to be accurate and highly specific for the estimation of besifloxacin in different pharmaceutical formulations.     

Preclinical study of the α<Subscript>2</Subscript>-adrenostimulant Brimozolin (brimonidine)

A preclinical study of the α₂ -adrenostimulant Brimozolin (brimonidine tartrate), synthesized by an original method at OAO Center for the Chemistry of Therapeutic Agents_All-Russian Chemico-Pharmaceutical Research Institute (TsKhLS-VNIKhFI), was performed using 29 rabbits. Brimozolin eye drops decreased intraocular pressure in the intact eyes of experimental animals by 3 – 5 mmHg for one day by producing significant increases in the outflow of aqueous humor and suppressing its production. Histological studies of the outer and inner layers of the eye showed that there were no changes. This agent is recommended for clinical trials.     

A new spectrophotometric method for the determination of methyldopa

A new, simple and low cost spectrophotometric method for the determination of methyldopa in pharmaceutical preparations was developed. The method was based on the coupling of methyldopa with 2,6-dichloroquinone-4-chlorimide (DCQ). The absorbance maximum (λ_max) of the resulted colored product was at 400 nm. Different buffers were used to determine the optimal pH for the reaction. About 1% w/v acetate buffer with pH 8.0 gave the optimal pH required for the reaction. Of the different solvents tried, water and ethanol were found to be the most suitable solvents. Beer’s law was obeyed in concentration range of 4–20 μg/ml methyldopa. The correlation coefficient was found to be (r = 0.9975). The limit of detection and limit of quantification were 1.1 μg/ml and 3.21 μg/ml, respectively. The reaction ratio between methyldopa and DCQ was studied and found to be 1:3. The work included the study of the possible interference of hydrochlorothiazide found in combination with methyldopa tablets. The method was validated and results obtained for the assay of two different brands of methyldopa tablets were compared with the BP method (colorimetric). The repeatability and reproducibility of the developed method were evaluated and the obtained results quoted. The derivative formed as a result of the reaction of methyldopa with DCQ was isolated and its possible mechanistic pathway was suggested.     

Development and Validation of New HPLC Method for Simultaneous Estimation of L-Lysine Hydrochloride and L-Carnitine-L-Tartrate in Pharmaceutical Dosage Form

A new analytical method is developed and validates for simultaneous estimation of L-lysine hydrochloride and L-carnitine-L-tartrate in single pharmaceutical dosage form by means of high resolution HPLC. The chromatographic procedure was carried out using C₁₈-5 μm (4.6 mm × 250 mm), column. Optimally suited mobile phase was made to run in an isocratic elution mode whose composition was 10 mM of potassium dihydrogen phosphate adjusted with triethylamine to a pH of 7.5 and pumped at a flow rate of 0.50 ml/min through chromatographic system. The detector''s wavelength was 214 nm. The accuracy of the analytical method was determined keeping into account the recovery of analytes, which in this case remains well within the accepted standards that is 95-105%. Detection limit for L-lysine hydrochloride and L-carnitine-L-tartrate are 1.47 μg/ml and 0.85 μg/ml while quantitation limit for L-lysine hydrochloride and L-carnitine-L-tartrate is 4.41 and 2.55 μg/ml, respectively. All the statistical results were validated performing precision, accuracy, linearity, specificity studies for analytes concentration from 70-130%. The outcome of results confirmed that the method was in consonance with the acceptance criteria.     

Development and Validation of Stability-indicating HPLC Method for Simultaneous Estimation of Cefixime and Linezolid

A stability-indicating reverse phase high performance liquid chromatography method was developed and validated for cefixime and linezolid. The wavelength selected for quantitation was 276 nm. The method has been validated for linearity, accuracy, precision, robustness, limit of detection and limit of quantitation. Linearity was observed in the concentration range of 2-12 μg/ml for cefixime and 6-36 μg/ml for linezolid. For RP-HPLC, the separation was achieved by Phenomenex Luna C₁₈ (250×4.6 mm) 5 μm column using phosphate buffer (pH 7):methanol (60:40 v/v) as mobile phase with flow rate 1 ml/min. The retention time of cefixime and linezolid were found to be 3.127 min and 11.986 min, respectively. During force degradation, drug product was exposed to hydrolysis (acid and base hydrolysis), H₂O₂, thermal degradation and photo degradation. The % degradation was found to be 10 to 20% for both cefixime and linezolid in the given condition. The method specifically estimates both the drugs in presence of all the degradants generated during forced degradation study. The developed methods were simple, specific and economic, which can be used for simultaneous estimation of cefixime and linezolid in tablet dosage form.     

Development and Validation of Stability-indicating High Performance Liquid Chromatographic Method for the Estimation of Everolimus in Tablets

The present study depicts the development of a validated reversed-phase high performance liquid chromatographic method for the determination of the everolimus in presence of degradation products or pharmaceutical excipients. Stress study was performed on everolimus and it was found that it degrade sufficiently in oxidizing and acidic conditions but less degradation was found in alkaline, neutral, thermal and photolytic conditions. The separation was carried out on Hypersil BDS C18 column (100×4.6 mm, 5 μ) column having particle size 5 μ using acetate buffer:acetonitrile (50:50 v/v) with pH 6.5 adjusted with orthophosphoric acid as mobile phase at flow rate of 1 ml/min. The wavelength of the detection was 280 nm. A retention time (R_t) nearly 3.110 min was observed. The calibration curve for everolimus was linear (r²=0.999) from range of 25-150 μg/ml with limit of detection and limit of quantification of 0.036 μg/ml and 0.109 μg/ml, respectively. Analytical validation parameters such as selectivity, specificity, linearity, accuracy and precision were evaluated and relative standard deviation value for all the key parameters were less than 2.0%. The recovery of the drug after standard addition was found to be 100.55%. Thus, the developed RP-HPLC method was found to be suitable for the determination of everolimus in tablets containing various excipients.     

pH对酒石酸溴莫尼定滴眼液稳定性的影响

目的:考察pH对酒石酸溴莫尼定滴眼液稳定性的影响,确定本品最佳的pH范围及贮存条件.方法:采用HPLC法测定酒石酸溴莫尼定滴眼液的含量及有关物质,考察本品在强光、高温条件下的稳定性.结果:酒石酸溴莫尼定滴眼液对强光、高温较敏感,且随着pH值的升高,稳定性下降.结论:本品最适宜的pH范围为偏酸性,最适宜的贮存条件为低温、避光.     

Development and Validation of a HPLC Method for the Determination of Cyclosporine A in New Bioadhesive Nanoparticles for Oral Administration

A simple and reliable high performance liquid chromatography method was developed and validated for the rapid determination of cyclosporine A in new pharmaceutical dosage forms based on the use of poly (methylvinylether-co-maleic anhydride) nanoparticles. The chromatographic separation was achieved using Ultrabase C₁₈ column (250×4.6 mm, 5 μm), which was kept at 75°. The gradient mobile phase consisted of acetonitrile and water with a flow rate of 1 ml/min. The effluent was monitored at 205 nm using diode array detector. The method exhibited linearity over the assayed concentration range (22-250 μg/ml) and demonstrated good intraday and interday precision and accuracy (relative standard deviations were less than 6.5% and the deviation from theoretical values is below 5.5%). The detection limit was 1.36 μg/ml. This method was also applied for quantitative analysis of cyclosporine A released from poly (methylvinylether-co-maleic anhydride) nanoparticles.     

Estimation Based on Emission Wavelength of Dabigatran Etexilate Mesylate in Bulk and Capsule Dosage Form

A simple, rapid, specific and highly sensitive spectrofluorimetric method has been developed for the quantification of dabigatran etexilate mesylate in bulk and capsule dosage form. A linear relationship was found between fluorescence intensity and concentration in the range of 0.01-1.0 μg/ml in dimethyl sulphoxide as solvent at an emission wavelength of 391 nm after excitation at 334 nm, with a good correlation coefficient (0.989). The detection and quantification limits were found to be 0.005 and 0.015 μg/ml, respectively. The proposed method was applied for dabigatran etexilate mesylate capsules, results reveal with percentage recovery of 102% and percentage relative standard deviation values were found to be less than 2 for accuracy and precision studies. The proposed method was validated for linearity, range, accuracy, precision, limit of detection and quantification according to International Conference on Harmonization guidelines. Statistical analysis of the results revealed high accuracy and good precision. The suggested procedures could be used for the determination of dabigatran etexilate mesylate in bulk and capsule dosage form in quality control laboratories of industries as well as in academic institutions.     

Spectrophotometric Estimation of Raltegravir Potassium in Tablets

Ultra violet spectrophotometric estimation of the raltegravir potassium, an integrase inhibitor antiretroviral agent was estimated by Ultra violet absorption maxima method at λ_max of 328 nm and UV area under curve method in the wave length range of 323-333 nm. The Beer''s law obeyed in the concentration range of 3-55 μg/ml and correlation coefficients were found to be more than 0.996 for both methods. The results of the analysis were 100.58±0.99 and 99.69±0.59 by absorption maxima and area under curve method respectively. Both the methods were validated as per ICH guidelines.     

Development and Validation of Stability-indicating RP-HPLC Method for Estimation of Pamabrom in Tablets

The present study depicts the development of a validated RP-HPLC method for the determination of the pamabrom in presence of degradation products or other pharmaceutical excipients. Stress study was performed on pamabrom and it was found that it degrade sufficiently in acidic, alkali and oxidative condition but less degradation was found in thermal and photolytic condition. The separation was carried out on Enable G 120 A⁰ (250×4.6 mm, 5 μ) column having particle size 5 μ using methanol: water (75:25 v/v) with pH 4.0 adjusted with ortho phosphoric acid as mobile phase at flow rate of 1 ml/min. The wavelength of the detection was 280nm. A retention time (R_t) nearly 3.9 min was observed. The calibration curve for pamabrom was linear (r² = 0.9997) from range of 10-60 μg/ml with limit of detection and limit of quantification of 1.41 μg/ml and 4.28 μg/ml, respectively. Analytical validation parameter such as selectivity, specificity, linearity, accuracy and precision were evaluated and relative standard deviation value for all the key parameters were less than 2.0%. The recovery of the drug after standard addition was found to be 101.35%. Thus, the developed RP-HPLC method was found to be suitable for the determination of pamabrom in bulk as well as stability samples of tablets containing various excipients.     

Determination of Cephalexin Monohydrate in Pharmaceutical Dosage Form by Stability-Indicating RP-UFLC and UV Spectroscopic Methods

An ultra-fast liquid chromatographic method and two UV spectroscopic methods were developed for the determination of cephalexin monohydrate in pharmaceutical dosage forms. Isocratic separation was performed on an Enable C₁₈G column (250 mm × 4.6 mm i.d., 5 μm) using methanol:0.01 M TBAHS (50:50, v/v) as the mobile phase at a flow rate of 1.0 ml/min. The PDA detection wavelength was set at 254 nm. The UV spectroscopic method was performed at 261 nm and at 256–266 nm for the AUC method using a phosphate buffer (pH=5.5). The linearity was observed over a concentration range of 1.0–120 μg/ml for UFLC and both of the UV spectroscopic methods (correlation coefficient=0.999). The developed methods were validated according to ICH guidelines. The relative standard deviation values for the intraday and interday precision studies were < 2%, and the accuracy was > 99% for all of the three methods. The developed methods were used successfully for the determination of cephalexin in dry syrup formulation.     

Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium in Tablets

A simple, accurate, rapid and precise reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of cefpodoxime proxetil and dicloxacillin sodium in tablet. The chromatographic separation was carried out on kromasil C₁₈ analytical column (250×4.6 mm; 5 μm) with a mixture of acetonitrile:methanol:trifloroacetic acid (0.001%) with pH 6.5 (30:50:20, v/v/v) as mobile phase; at a flow rate of 1.0 ml/min. UV detection was performed at 235 nm. The dicloxacillin sodium and cefpodoxime proxetil were eluted at 1.92 and 3.35 min, respectively. The peaks were eluted with better resolution. Calibration plots were linear over the concentration range 0.5-20 μg/ml for cefpodoxime proxetil (r²=0.9996) and 5-50 μg/ml for dicloxacillin sodium (r²=0.9987). The method was validated for accuracy, precision, linearity and specificity. The method was very sensitive with limit of detection 0.0726, 0.3685 μg/ml and limit of quantification 0.220, 1.116 μg/ml for cefpodoxime proxetil and dicloxacillin sodium, respectively. The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of cefpodoxime proxetil and dicloxacillin sodium in bulk drug and tablet dosage form.     

双波长法测定乳酸环丙沙星滴眼液的含量

目的 :建立乳酸环丙沙星滴眼液含量的测定方法。方法 :采用双波长分光光度法 ,以0.1mol/L盐酸为稀释液 ,检测波长为237nm和279nm。结果 :乳酸环丙沙星浓度在3～9μg/ml范围内有良好的线性关系 ,r=0.9998 ;平均回收率为101 6 %,RSD为2 16 %。结论 :本法简便快捷、定量准确 ,可作为乳酸环丙沙星滴眼液的含量测定方法。     

Stability-indicating High-performance Liquid Chromatography Method for Simultaneous Determination of Aminophylline and Chlorpheniramine Maleate in Pharmaceutical Formulations

The present work deals with the development and validation of method for simultaneous determination of antihistaminic drugs in pharmaceutical formulations. A precise, specific and accurate reverse phase-high-performance liquid chromatography method for the simultaneous measurement of aminophylline and chlorpheniramine maleate was developed. The separation of drugs was achieved on C-18 (5 μm, 250×4.6 mm) high-performance liquid chromatography column. The runtime for analysis was 10 min. Mobile phase is mixture containing dilute H₂SO₄:methanol (60:40% v/v) with flow rate adjusted at 1.5 ml/min. The detection of components was performed at a wavelength of 264 nm. Retention times of aminophylline and chlorphinramine maleate were found to be 2.00 and 3.25 min, respectively. Linearity was found in the range of 16-24 μg/ml for chlorpheniramine maleate and 102.4-153.6 μg/ml for aminophylline with a correlation coefficient of 0.9998 and 0.9996, respectively. High peak purity index of 99.99% indicated the complete separation of analytes in the presence of degradation products is justification of method stability. Linearity, accuracy, specificity, precision and robustness studies were performed for method validation.     

Quality by Design Approach for Simultaneous Estimation of Doxycycline Hyclate and Curcumin by RP-HPLC Method

A simple, rapid, reliable, robust and optimized reversed phase high performance liquid chromatographic method for simultaneous estimation of doxycycline hyclate and curcumin was successfully developed and validated as per International Conference on Harmonization guidelines. The objective was achieved in terms of well separated peaks within 10 min on a Waters Sunfire C8 column with dimensions of 250×4.6 mm, particle size 5.0 μm using mobile phase consisting of 30 volumes of potassium dihydrogen phosphate buffer (50 mM) adjusted to pH 6.5±0.1 with triethylamine and 70 volumes of methanol at flow rate of 0.85 ml/min. The column effluents were monitored at 400 nm maintained at ambient column temperature (28^o). The developed method was found linear over the concentration range of 200-700 μg/ml for doxycycline hyclate and 8-28 μg/ml for curcumin, the detection and quantitation limit was found to be 26.063 and 78.97 μg/ml for doxycycline hyclate; 0.795 and 2.13 μg/ml for curcumin, respectively. The developed method was optimized using Minitab software version 16 to meet the current quality by design requirements. The method validation was done for linearity, range, detection and quantitation limit, accuracy, precision, specificity, system suitability testing, and robustness.     

Development and Validation of a HPTLC Method for Simultaneous Estimation of L-Glutamic Acid and γ-Aminobutyric Acid in Mice Brain

A new robust, simple and economic high performance thin layer chromatographic method was developed for simultaneous estimation of L-glutamic acid and γ-amino butyric acid in brain homogenate. The high performance thin layer chromatographic separation of these amino acid was achieved using n-butanol:glacial acetic acid:water (22:3:5 v/v/v) as mobile phase and ninhydrin as a derivatising agent. Quantitation of the method was achieved by densitometric method at 550 nm over the concentration range of 10-100 ng/spot. This method showed good separation of amino acids in the brain homogenate with R_f value of L-glutamic acid and γ-amino butyric acid as 21.67±0.58 and 33.67±0.58, respectively. The limit of detection and limit of quantification for L-glutamic acid was found to be 10 and 20 ng and for γ-amino butyric acid it was 4 and 10 ng, respectively. The method was also validated in terms of accuracy, precision and repeatability. The developed method was found to be precise and accurate with good reproducibility and shows promising applicability for studying pathological status of disease and therapeutic significance of drug treatment.     

A Validated RP-HPLC Method for the Estimation of Lapatinib in Tablet Dosage form using Gemcitabine Hydrochloride as an Internal Standard

A simple, selective, rapid, precise and economical reverse-phase high-performance liquid chromatography method has been developed for the determination of lapatinib in tablet using gemcitabine hydrochloride as an internal standard. Chromatography was carried out on an ODS C-18 RP column (4.6 mm i.d. ×250 mm) using a mixture of acetonitrile and water (50:50 v/v) as the mobile phase at a flow rate of 1.0 ml/min. The drug was monitored at 232 nm. The retention times for lapatinib and gemcitabine hydrochloride were found to be 4.25±0.05 and 6.10±0.05 min, respectively. The method produced linear responses in the concentration range of 2-60 μg/ml of lapatinib. The limit of detection and limit of quantitation were 0.265 and 0.884 μg/ml, respectively.     

Development and Validation of a Liquid Chromatographic Method for Estimation of Dicyclomine Hydrochloride,Mefenamic Acid and Paracetamol in Tablets

Liquid chromatographic method was developed for simultaneous quantitative determination of dicyclomine hydrochloride, mefenamic acid and paracetamol in their combined dosage form. The separation was achieved using a C₁₈ column (250×4.6 mm id, 5 μm) using acetonitrile:20 mM potassium dihydrogen phosphate 70:30 (v/v) adjusted to pH 4 using orthophosphoric acid as mobile phase at a flow rate of 1 ml/min and detection at 220 nm. Separation was completed within 12 min. The retention times of dicyclomine hydrochloride, mefenamic acid and paracetamol were 3.8, 9.3 and 2.5 minutes respectively. The proposed method was found to have linearity in concentration range of 10–100 μg/ml for dicyclomine hydrochloride, 0.05-10 μg/ml for mefenamic acid and 0.1−20 μg/ml for paracetamol. The developed method has been statistically validated and was found to be simple, precise, reproducible and accurate. The developed and validated method was successfully used for the quantitative analysis of commercially available dosage form.