Simultaneous determination of relatedsubstances of telmisartan andhydrochlorothiazide in tablet dosage form byusing reversed phase high performanceliquid chromatographic method

J Pharm Bioallied Sci 2011 Jul-Sep; 3(3): 375–383 doi: 10.4103/0975-7406.84441 PMCID: PMC3178944 Simultaneous determination of related substances of telmisartan and hydrochlorothiazide in tablet dosage form by using reversed phase high performance liquid chromatographic method Sutirtho Mukhopadhyay, Kiran Kadam, Laxman Sawant,1 Dhanashree Nachane,1 and Nancy Pandita1 Author information ► Article notes ► Copyright and License information ► Go to: Abstract Objective: Telmisartan is a potent, long-lasting, nonpeptide antagonist of the angiotensin II type-1 (AT 1) receptor that is indicated for the treatment of essential hypertension Hydrochlorothiazide is a widely prescribed diuretic and it is indicated for the treatment of edema, control of essential hypertension and management of diabetes insipidus In the current article a new, accurate, sensitive, precise, rapid, reversed phase high performance liquid chromatography (RP-HPLC) method was developed for determination of related substances of Telmisartan and Hydrochlorthiazide in tablet dosage form Materials and Methods: Simultaneous determination of related substances was performed on Kromasil C18 analytical column (250 × 4.6 mm; 5μm pertical size) column at 40°C employing a gradient elution Mobile phase consisting of solvent A (solution containing 2.0 g of potassium dihydrogen phosphate anhydrous and 1.04 g of Sodium 1- Hexane sulphonic acid monohydrate per liter of water, adjusted to pH 3.0 with orthophosphoric acid) and solvent B (mixture of Acetonitrile: Methanol in the ratio 80:20 v/v) was used at a flow rate of 1.0 ml min–1 UV detection was performed at 270 nm Results: During method validation parameter such as precision, linearity, accuracy, specificity, limit of detection and quantification were evaluated, which remained within acceptable limits Conclusions: HPLC analytical method is linear, accurate, precise, robust and specific, being able to separate the main drug from its degradation products It may find application for the routine analysis of the related substances of both Telmisartan and Hydrochlorthiazide in this combination tablets Keywords: Hydrochlorothiazide, related substances, RP-HPLC, telmisartan, validation Telmisartan (TE), 4_-[(1,4_-dimethyl-2_-propyl[2,6_- bi-1H-benzimidazol]-1_-yl) methyl-[1,1_biphenyl]-2-carboxylic acid, is a potent, long-lasting, nonpeptide antagonist of the angiotensin II type-1 (AT1) receptor that is indicated for the treatment of essential hypertension It selectively and insurmountably inhibits stimulation of the AT1 receptor by angiotensin II without affecting other receptor systems involved in cardiovascular regulation In clinical studies, TE shows comparable antihypertensive activity to members of other major antihypertensive classes, such as angiotensin-converting enzyme (ACE) inhibitors, beta-blockers and calcium antagonists Experiments have confirmed the placebo like safety and tolerability of TE in hypertensive patients.[1] Telmisartan (TE) is widely used in the treatment of hypertension and heart failure.[2] Hydrochlorothiazide (HCTZ) (6-chloro-3, 4-dihydro-2H-1, 2, 4-benzo-thiadiazine-7-sulfonamide 1,1-dioxide) is a widely prescribed diuretic It is indicated for the treatment of edema, control of essential hypertension and management of diabetes insipidus.[3] Hydrochlorothiazide, a thiazide diuretic, is also used to treat mild to moderate hypertension, usually in combination with other antihypertensive agents with different mechanisms of action [4] This is not only because blood pressure control is often inadequate using monotherapy but also because combination therapy can simplify dosing regimens, improve compliance, decrease side effects and reduce cost The literature survey reveals that, TE and HCTZ are reported in British Pharmacopoeia.[5,6] There have been several publications describing analytical methods for the determination of HCTZ and TE individually or with other drugs as combination Although there are a few papers published on simultaneous determination of TE and HCTZ in formulation most of them deal with the assay of each constituent Several methods are reported for the determination of TE like Spectrophotometric[7] and HPLC.[8–10] The other methods available in the literature are based on Linear Sweep polarography,[11] LC–MS.[12] Articles on the determination of HCTZ in combination with other drugs by HPLC are also reported in literature.[13,14] However the exhaustive literature survey revealed that none of the most recognized pharmacopoeias or any journals includes these drugs in combination for the simultaneous determination of related substances of TE and HCTZ and the information regarding the stability of the drugs is not available So the aim of this work was to develop a liquid chromatographic procedure which will serve a reliable, accurate, sensitive and stability indicating HPLC method for the simultaneous determination of related substances of TE and HCTZ in TE + HCTZ tablets The Regulatory agencies recommend the use of stability indicating methods (SIMs)[15] for the analysis of stability samples.[16] This requires stress studies in order to generate the potential related impurities under stressed conditions, method development and validation With the evident of the International Conference on Harmonization (ICH) guidelines,[17] requirements for the establishment of SIMs have become more clearly mandated The production of the potential impurities in a drug product generally take place under various environmental conditions like exposure to light, heat, hydrolysis or oxidation Hence Stress testing can help identifying degradation products and provide important information about intrinsic stability of the drug product Several methods have been studied earlier for simultaneous determination of Telmisartan and Hydrochlorothiazide, but there is no report on method for related substances of these drugs in combination So the aim of our study is to develop simple, fast, accurate and specific reversed phase high performance liquid chromatographic method for simultaneous determination of related substances of Telmisartan and Hydrochlorothiazide in tablet dosage form Go to: Experimental Reagents and materials Hydrochlorthiazide and Telmisartan active pharmaceutical ingredient (API) and test sample (Each tablet containing 80mg telmisartan and 12.5mg HCTZ or 40mg telmisartan and 12.5mg HCTZ) were kindly supplied by Getz Pharma Research, Ambarnath, India Individual reference standards for Telmisartan impurities [Figure 1] were not available The EP CRS (European Pharmacopoeial Commission of Reference Substances) for system suitability, consisting of a mixture of all the impurities (Impurity-A, B, C, E and Impurity-F) of telmisartan was procured from (LGC Promochem, India) The related substances of Hydrochlorthiazide [Figure 2] were supplied by the API (active pharmaceutical ingredient) vendor (Unichem, Mumbai, India) Figure Chemical structure of Telmisartan and its related impurities Figure Chemical structure of Hydrochlorthiazide and its related impurities The chemical names for all components are listed in Table Table Chemical names of all related impurities of Hydrochlorthiazide and Telmisartan Potassium dihydrogen phosphate was obtained from Merck Limited, Mumbai, India; Sodium 1Hexane sulphonic acid monohydrate was obtained from Alfa Aesar Mumbai, India; Methanol was procured from Merck Mumbai, India; Acetonitrile was obtained from Rankem Mumbai, India; Monobasic sodium phosphate, Ortho-Phosphoric acid, Sodium Hydroxide, Hydrochloric acid, 50% Hydrogen peroxide were obtained from Merck Limited, Mumbai, India High purity deionised water was obtained from [Millipore, Milli-Q (Bedford, MA, USA)] purification system Instrumentation HPLC system (Waters 2695 Alliance Separation Module) (eg Waters Milford, USA) equipped with inbuilt autosampler and quaternary gradient pump with an on-line degasser was used The column compartment having temperature control and Photodiode Array/ Ultraviolet (PDA/UV) Detector (2996/2487) was employed throughout the analysis Chromatographic data was acquired using Empower software The Analytical Balance used for weighing was of the make – Mettler Toledo, Model- XS205DU The pH meter used was of the make -Thermo Electron Corp., Model-Orion-4star 1117000 Chromatographic conditions Kromasil C-18, 250 × 4.6 mm, 5μm (AKZO NOBEL) column was used as stationary phase maintained at 40°C The mobile phase involved a variable composition of solvent A (2.0 gm of Potassium dihydrogen phosphate anhydrous and 1.04 gm of Sodium 1- Hexane sulphonic acid monohydrate dissolved in 1000 ml of water, adjusted to pH 3.0 with orthophosphoric acid) and solvent B (A mixture of Acetonitrile: Methanol in the ratio 80:20 v/v) The mobile phase was pumped through the column with at a flow rate of 1ml – [Table 2] Table Mobile phase program for gradient elution The optimum wavelength selected was 270 nm which represents the wavelength where all impurities has suitable responses in order to permit simultaneous determination of related impurities of Telmisartan and HCTZ in Telmisartan + HCTZ tablets The stressed samples were analyzed using a Photodiode Array (PDA) detector covering the range of 200–400 nm Go to: Solution Preparation Standard solution Preparation of standard stock solution – Telmisartan 40.0 mg of Telmisartan working standard was weighed accurately and transferred into a 200 ml volumetric flask About 70 ml of methanol was added and the solution was sonicated to dissolve the standard The volume was made up to the mark with methanol Further ml of this solution was diluted to 50 ml with mobile phase A Preparation of standard stock solution – Hydrochlorothiazide 60.0 mg of Hydrochlorothiazide working standard was weighed accurately and transferred into a 200 ml volumetric flask About 70 ml of methanol was added and the solution was sonicated to dissolve the standard The volume was made up to the mark with methanol Further ml of this solution was diluted to 50 ml with mobile phase A Preparation of standard solution 15 ml of standard stock solution of Telmisartan and ml of standard stock solution of Hydrochlorothiazide were in taken in 100 ml volumetric flask, and the volume was made up with mobile phase A System suitability solution The standard solution prepared was used for system suitability evaluation Sample solution For 80 – 12.5 mg 10 tablets accurately weighed were transferred in 100 ml volumetric flask 10 ml of mobile phase A was added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled to room temperature and the volume was made up with mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A and the solution was filtered through 0.45 μ Nylon filter For 40 – 12.5 mg 10 tablets accurately weighed were transferred in 50 ml volumetric flask ml of mobile phase A was added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled to room temperature and the volume was made up with mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A and the solution was filtered through 0.45 μ Nylon filter Forced degradation sample solution for specificity study Multiple stressed samples were prepared as indicated below They were carried out on the higher strength tablets (80mg_12.5mg) and chromatographed along with a non-stressed sample (control) Hydrolytic conditions: Acid- base-induced degradation Solution containing 0.150mgml–1 of Hydrochlorthiazide and 0.960mgml–1 of Telmisartan was treated with N (Normal) HCl (Hydrochloric acid) and N NaOH (Sodium Hydroxide) respectively These were subjected to the condition mentioned in Table The solutions were neutralized as needed by (5 N NaOH or N HCl) Table Hydrolytic, oxidizing thermal, and photolytic stress conditions Oxidative condition: Hydrogen peroxide-induced degradation Solution containing 0.150mgml–1 of Hydrochlorothiazide and 0.960mgml–1 of Telmisartan was treated with 50% v/v H2O2 under the condition shown in Table Thermal degradation study 10 tablets of Telmisartan + Hydrochlorothiazide were weighed and transferred into 100 ml volumetric flask 10 ml of Mobile phase A was added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled at room temperature The solution was heated in the oven at 70°C for hours, cooled and volume was made up the with mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A Photolytic degradation study As per guidelines for photostability testing of new drug substances and products, samples should be exposed to light providing an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy of not less than 200Wh m-2 to allow direct comparisons to be made between the drug substance and drug product.[19] For photo stability testing 10 tablets of Telmisartan + Hydrochlorothiazide were weighed and transferred into each of 100 ml clear glass, 100 ml flask covered with aluminum foil and 100 ml amber colored volumetric flask 10 ml of Mobile phase A was added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled at room temperature These flasks were kept under UV and white light for 1.2 million lux hours in photo stability chamber/ 200Wh m-2 After study the sample was cooled and diluted upto the mark with Mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A and filtered through 0.45μ Nylon filter Go to: Placebos were Treated Similarly Preparation of placebo solution For telmisartan Telmisartan Placebo equivalent to 10 tablets was weighed and transfered in 100 ml volumetric flask 10 ml of Mobile phase A added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled at room temperature, and the volume made up with mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A and filtered through 0.45 μ Nylon filter For hydrochlorothiazide Hydrochlorothiazide Placebo equivalent to 10 tablets was weighed and transfered in 100 ml volumetric flask 10 ml of Mobile phase A added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled at room temperature and the volume made up with Mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A and filtered through 0.45 μ Nylon filter For placebo without telmisartan + hydrochlorothiazide Placebo without TE and HCTZ equivalent to 10 tablets was weighed and transfered in 100 ml volumetric flask 10 ml of Mobile phase A was added and sonicated for to 10 minutes with intermittent shaking till the tablets disintegrate The solution was cooled at room temperature and the volume made up with Mobile phase A Further ml of this solution was diluted to 25 ml with mobile phase A and filtered through 0.45 μ Nylon filter Go to: Results and Discussion Optimization of chromatographic conditions The maximum absorption wavelength of the reference drug solution and of the forcefully degraded drug solution was found to be 270 nm This was observed from the UV absorption spectra and was selected as detection wavelength for LC analysis The main objective of this chromatographic method was separation of degraded impurities from both the drugs Forced degradation study revealed a critical separation of closely eluting impurities of Hydrochlorthiazide, formed from the HCTZ peak The possible impurities of TE and HCTZ are very similar to respective drug substances To obtain a good resolution among the impurities and main drug substances different stationary phases were tested considering; a The feature of stationary phase (RP-C8 and RP-C18) b The particle size of the column (3μm and 5μm) The detector response for all the components found suitable at 270 nm; hence the typical chromatogram was recorded at this wavelength The typical HPLC chromatograms [Figure 3a] represent the satisfactory separation of all components among each other Figure a) Typical HPLC Chromatogram of mixture of all components b) Typical HPLC chromatograms of unstressed control sample in forced degradation studies Selection of stationary phase Kromasil C18 was chosen due to its proven robust nature and more importantly for the fact that the API monograph for Telmisartan as published in European Pharmacopoeia, uses the same column (although different dimensions) Since the impurity standards for telmisartan were available only in the form of mixture, it was easier to track these impurities (during development) based on their elution order in the new method developed for this combination formulation Influence of addition of ion-pair reagent in the mobile phase HCTZ and Telmisartan lie in opposite spectrum in terms of retention in reverse phase chemistry Thus ion Pair reverse phase had to be incorporated to retain HCTZ and gradient elution was used to elute Telmisartan and its impurities The robustness of separation depends on the quality/ purity of ion pair reagent used Influence of pH of mobile phase buffer A pH change of ±0.2 units did not have any adverse effect on the separation After optimizing various parameters, the method was finalized on Kromasil C18 250 × 4.6mm; 5μ HPLC column using variable composition of solvent A: KH2PO4 (2.0 g L–1), hexane sulfonic acid sodium salt (1.04 g L–1) pH 3.0 with orthophosphoric acid and solvent B: A mixture of Acetonitrile: Methanol in the ratio 80:20 v/v as mobile phase [Table 2] The mobile phase pumped through the column at a flow rate of 1.0 ml min–1 and column compartment temperature kept at 40°C Method validation The optimized RP-HPLC method was validated according to ICH guidelines.[19] The various validation parameters that were performed are as follows: Specificity, Accuracy, Precision (Repeatability And Intermediate Precision), Linearity, Range And Robustness System suitability features were also assessed Solution stability and filter compatibility were also studied System suitability test The system suitability test performed according to USP 30.[20] The Standard solution was injected six times into the chromatograph and the chromatograms were recorded The relative standard deviation of the area for individual peaks, for six replicate injections of standard solution should not be more than 5.0 % The USP theoretical plates for HCTZ should not be less than 10000 and for Telmisartan should not be less than 500000 The relative standard deviation for six replicate injections of standard solution was found to be less than 5.0 % The results obtained for Theoretical plates, USP tailing factor (Tf) were also all within acceptable limits Specificity The peak purity indices for the analytes in stressed solutions determined with PDA detector under optimized chromatographic conditions were found to be better (purity angle < purity threshold) indicating that no additional peaks were co-eluting with the analytes and evidencing the ability of the method to assess unequivocally the analyte of interest in the presence of potential interference Baseline resolution was achieved for all investigated compounds The FDA guidelines indicated that well separated peaks, with resolution, Rs > between the peak of interest and the closest eluting peak, are reliable for the quantification.[21] All the peaks meet this specification, visibly confirmed in Figures Figures44–7 Figure Typical HPLC chromatograms of stressed samples treated with Acid (a) For HCTZ (b) For TE Figure Typical HPLC chromatogram of sample exposed to light Figure Typical HPLC chromatograms of stressed samples treated with Alkali (a) For HCTZ (b) For TE Figure Typical HPLC chromatograms of thermal-stressed samples (a) For HCTZ (b) For TE Linearity and range The nominal concentration of test solutions for HCTZ and TE were 0.150 mgml–1 and 0.960 mgml–1, respectively The limit of any impurity related to telmisartan was kept at not more than 0.1% for unknown and 1% for total and for HCTZ it was not more than 1% for Impurity B, not more than 1% for any other impurity and not more than 2.5% for total impurity The relative response function was determined by preparing standard solution of each component at different concentration levels ranging from lower limit of quantification to at least 200% of impurity tolerance level and that identification of impurities below lower level of quantification were not considered to be necessary unless the potential impurities are expected to be unusually potent or toxic The plots of area under the curve (AUC) of the peak responses of the analytes against their corresponding concentrations fitted straight lines responding to equations The y-intercepts were close to zero with their confidence intervals containing the origin The correlation co-efficient (r) for Impurity B of HCTZ, HCTZ and Telmisartan were found to be 0.9998, and 0.994 respectively chromatography method for its estimation Literature survey reveals the high-performance thin layer chromatography (HPTLC)[5] method for the determination of CEF individually Literature survey also reveals the reversed-phase high-performance liquid chromatographic (RP-HPLC)[6] and spectrophotometric[7] methods for determination of CEF with other drugs Dicloxacillin (DCX) is chemically (2S,5R,6R)-6-[3-(2,6-dichlorophenyl)-5-methyl-1,2-oxazole-4-amido]3,3-dimethyl-7-oxo-4-thia-1azabicyclo[3.2.0] heptane-2-carboxylic acid,[8] and is a penicillinase resistant penicillin, used in the treatment of bacterial infections such as pneumonia and bone, ear, skin and urinary tract infection.[9] It is official in IP[10] and USP,[11] and describe RP-HPLC method for its estimation Literature survey reveals HPLC[12] method for determination of DCX in pharmaceutical dosage forms as well as in biological fluids Literature survey also reveals spectrofluorimetric[13] and RP-HPLC[14–16] methods for determination of DCX with other drugs The combined dosage form of CEF and DCX are available in the market for the treatment of infections caused by susceptible microorganisms like urinary tract infections and gonococcal urethritis The combination of these two drugs is not official in any pharmacopoeia, hence, no official method is available for the simultaneous estimation of CEF and DCX in their combined dosage forms Literature survey does not reveal any simple spectrophotometric or other method for simultaneous estimation of CEF and DCX in combined dosage form The present communication describes simple, sensitive, rapid, and accurate RP-HPLC method for simultaneous estimation of both drugs in their combined tablet dosage forms Go to: MATERIALS AND METHODS Shimadzu, LC-2010CHT, Japan instrument provided with a kromasil C18 column (250×4.6 mm, μ) and LC 20 AD Pump and Prominence SPD 20A UV-deuterium detector was employed in the study Data acquisition was performed by using LC solution software HPLC grade methanol (MeOH) and acetonitrile (ACN) were purchased from Finar Chemicals Ltd., Ahmedabad, India and water from Sisco Research Laboratories Pvt Ltd., Mumbai, India Trifloroacetic acid (TFA) AR grade were purchased from SD Fine Chem., Mumbai, India Preparation of solutions and reagents: A solution containing acetonitrile, methanol and water in the proportion of 20:50:30 v/v/v was used as diluent for the proposed method A mixed standard stock solution of CEF (1.0 mg/ml) and DCX (1.0 mg/ml) was prepared by accurately weighing CEF (25 mg) and DCX (25 mg) and dissolving in diluent and make up to 25 ml with diluent in the 25 ml volumetric flask An aliquot of 1.0 ml of stock solution was transferred in 10 ml volumetric flask and adjusted up to the mark with diluent having concentration (100 μg/ml) Preparation of sample solution: Twenty tablets were weighed and powdered The powder equivalent to 200 mg CEF and 500 mg DCX was transferred to 100 ml volumetric flask Diluent (50 ml) was added to it and sonicated for 20 The volume was adjusted with diluent after the filtration of sonicated solution An aliquot of 1.0 ml of this solution was transferred in 100 ml volumetric flask and adjusted up to the mark with diluent CEF (20 μg/ml) and DCX (50 μg/ml) From this solution, 2.5 ml was transferred to 10 ml volumetric flask and adjusted up to the mark with diluent to achieve the concentration of CEF μg/ml and DCX 12.5 μg/ml Methodology: To optimize the RP-HPLC parameters, several mobile phase compositions were tried A satisfactory separation and good peak symmetry for CEF and DCX was obtained with a mobile phase ACN:MeOH:TFA (0.001%) with pH 6.5 (30:50:20 v/v/v) at a flow rate of 1.0 ml/min to get better reproducibility and repeatability Quantification was carried out at 235 nm based on peak area Complete resolution of the peaks with clear baseline was obtained (fig 1) System suitability test parameters for CEF and DCX for the proposed method are reported in Table Fig Chromatogram of standard solution of DCX and CEF DCX (30 μg/ml) is dicloxacillin concentration and CEF (10 μg/ml) is cefpodoxime concentration in chromatogram of standard solution at UV radiation 235 nm Table SYSTEM SUITABILITY PARAMETERS Validation developed method: The proposed method has been validated for the simultaneous determination of CEF and DCX in tablet dosage form.[17] Calibration curves were constructed by plotting peak areas versus concentrations of CEF and DCX, and the regression equations were calculated The calibration curves were plotted over the concentration range 0.5-20 μg/ml for CEF and 5-50 μg/ml for DCX Accurately measured working standard solutions of CEF (0.05, 0.1, 0.5, 1.0, 1.5 and 2.0 ml) and measured working standard solution of DCX (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 ml) were transferred to a series of 10 ml of volumetric flasks and diluted to the mark with diluent Aliquots (20 μl) of each solution were injected and analysed under the operating chromatographic conditions described as above Regression parameters are mentioned in Table Table REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION PARAMETERS For the repeatability, the relative standard deviation (RSD) values for CEF and DCX were found to be 0.21 and 0.38%, respectively The RSD values were found to be 2 in all samples The standard chromatogram of CEF and DCX display good resolute peak (fig 1) and no interference from excipients present in the formulation indicate specific nature of the method (fig 2) The peak purity data of the CEF and DCX show that no other excipient is coeluted with the drug and the peak of the drug is pure in nature The recovery experiment was performed by the standard addition method The mean recoveries obtained were 99.77±0.89 and 99.94±0.45% for CEF and DCX, respectively (Table 2) The low value of standard deviation indicates that the proposed method is accurate Results of recovery studies are shown in Table Fig Chromatogram of sample solution of DCX and CEF DCX (12.5 μg/ml) is dicloxacillin concentration and CEF (5 μg/ml) is cefpodoxime concentration in chromatogram of marketed formulation at UV radiation 235 nm Table RECOVERY DATA The robustness was studied by analysing the same samples of CEF and DCX by small but deliberate variation in the method parameters and the change in the responses of CEF and DCX were noted Robustness of the method was studied by changing the composition of mobile phase by ±1.0 ml of organic solvent (0.001% trifloroaceticacid:methanol:acetonitrile, composition (a) 19:51:30, (b) 21:49:30, (c) 19:50:31, (d) 21:50:29), flow rate by ±0.1 ml/min (flow rate 1.1 and 0.9 ml/min), column oven temperature by ±5° (25 and 35°) and mobile phase pH by ±0.05 unit (pH 6.45 and 6.55) None of the alterations caused a significant change in resolution between CEF and DCX, peak area % RSD, retention time % RSD, tailing factor and theoretical plates and the results are reported in Table Table ROBUSTNESS DATA Solution stability: The difference in the initial value of percentage assay and the values obtained at 0, 12, 24 and 48 h of percentage assay should not be more than 2.0% The assay obtained at different time intervals were compared with the initial assay values Solution stability period for sample solution and standard solution was determined Standard and sample solutions were stable till 48 h Solution stability period for standard preparation and sample preparation were found to be within the acceptance criteria Analysis of marketed formulation: The proposed validated method was successfully applied to determine CEF and DCX in their tablet dosage form The amount found to be 201.1±2.62 and 501.45±2.61 mg/tablet for CEF and DCX, respectively The result obtained for CEF and DCX was comparable with the corresponding labelled amounts (Table 5) The RP-HPLC chromatogram for CEF and DCX of sample was recorded and shown in fig Table ANALYSIS OF FORMULATION OF CEFPODOXIME PROXETIL AND DICLOXACILLIN BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD Go to: RESULTS AND DISCUSSION A RP-HPLC method was developed and validated for the determination of CEF and DCX in tablet dosage forms on a column (C18, 250×4.6 mm i.d., μm) with variable wavelength detection at 235 nm The retention times of DCX and CEF was 1.92 and 3.35 min, respectively Linear correlation was obtained between area and concentrations of CEF and DCX in the concentration ranges of 0.5-20 and 5-50 μg/ml, respectively The low RSD values of interday (0.70-1.45% for CEF and 0.63-1.42% for DCX) and intraday (0.12-0.48% for CEF and 0.110.59% for DCX) at 235 nm, reveal that the proposed method is precise The LOD and the LOQ for CEF and DCX were found to be 0.07 and 0.36 μg/ml and 0.22 and 0.38 μg/ml, respectively These data show that method is sensitive for the determination of CEF and DCX The recovery experiment was performed by the standard addition method The mean recoveries were 99.77±0.89 and 99.94±0.45 for CEF and DCX, respectively (Table 2) The results of recovery studies indicate that the proposed method is highly accurate The proposed validated method was successfully applied to determine CEF and DCX in their tablet dosage form The results obtained for CEF and DCX were comparable with the corresponding labelled amounts (Table 3) No interference of the excipients with the absorbance of interest appeared; hence, the proposed method is applicable for the routine simultaneous estimation of CEF and DCX in pharmaceutical dosage forms A simple, linear, accurate, specific and selective RP-HPLC method was developed and validated for estimation of CEF and DCX in their combined dosage form In this proposed method the linearity is observed in the concentration range of 0.5-20 μg/ml for CEF and 5-50 μg/ml for DCX with coefficient of correlation, (r2)=0.9996 and (r2)=0.9987 for CEF and DCX, respectively at 235 nm The result of the analysis of pharmaceutical formulation by the proposed method is highly reproducible and reliable and it is in good agreement with the label claim of the drug The method can be used for the routine analysis of the CEF and DCX in combined dosage form without any interference of excipients Go to: ACKNOWLEDGEMENTS The authors thank the Indica Laboratories Ltd., Ahmedabad, Gujarat, India for providing gift sample of CEF and DCX for research The authors also thank the Center for Health Science Studies, Ganpat University, Ganpat Vidyanagar - 384 012, Mehsana, Gujarat, India for providing all the facilities to carry out the work Go to: Footnotes Acharya and Patel: RP-HPLC Method for Cefpodoxime Proxetil and Dicloxacillin Sodium Go to: REFERENCES O’Neill MJ, editor An Encyclopedia of Chemicals, Drugs and Biologicals 14th ed Whitehouse Station, New Jersey: Merck Research Laboratories, Division of Merck and Co., Inc; 2006 The Merck Index; p 319 Sweetman SC The Martindale: The Complete Drug Reference 35th ed London: Pharmaceutical Press; 2007 p 207 Indian Pharmacopeia Vol New Delhi: The Controller Publication, Govt of India; 2010 p 1018 USP28-NF23 Rockville, MD: United State Pharmacopeial Convention, Inc; 2005 The United State Pharmacopeia; p 397 Darji BH, Shah NJ, Patel AT, Patel NM Development and validation of a HPTLC method for the estimation of cefpodoxime proxetil Indian J Pharm Sci 2007;69:331–3 Singh S, Dubey N, Jain D, Tyagi L, Singh M Spectrophotometric and RP-HPLC methods for simultaneous determination of cefpodoxime proxetil and clavulanate potassium in combined tablet dosage form Am Eurasian J Sci Res 2010;5:88–93 Gandhi SV, Patil UP, Patil NG Simultaneous spectrophotometric determination of cefpodoxime proxetil and potassium clavulanate Hindustan Antibiot Bull 2009;51:24–8 [PubMed] O’Neill MJ, editor An Encyclopedia of Chemicals, Drugs and Biologicals 14th ed Whitehouse Station, New Jersey: Merck Research Laboratories, Division of Merck and Co., Inc; 2006 The Merck Index; p 523 Sweetman SC, editor The Martindale: The Complete Drug Reference 35th ed London: Pharmaceutical Press; 2007 p 237 10 Indian Pharmacopeia Vol New Delhi: The Controller Publication, Govt of India; 2010 pp 1201–2 11 USP28-NF23 Rockville, MD: United States Pharmacopeial Convention, Inc; 2005 The United State Pharmacopeia; pp 451–2 12 Alderete O, González-Esquivel DF, Del Rivero LM, Castro Torres N Liquid chromatographic assay for dicloxacillin in plasma J Chromatogr B Analyt Technol Biomed Life Sci 2004;805:353–6 [PubMed] 13 Morelli B Second-derivative spectrophotometric assay of mixtures of dicloxacillin sodium and ampicillin sodium in pharmaceuticals J Pharm Sci 1988;77:1042–6 [PubMed] 14 Barot T, Patidar K, Kshartri N, Vyas N Development and validation of LC method for the determination of ampicillin and dicloxacillin in pharmaceutical formulation using an experimental design Eur J Chem 2009;6:955–64 15 Kathiresan K, Murugan R, Hameed SM, Inimai GK, Kanyadhara T Analytical method development and validation of cefixime and dicloxacillin tablets by RP-HPLC Rasayana J Chem 2009;2:588–92 16 Zhang MJ, Zhang HJ, Guan X, Xiao ZH HPLC determination of dicloxacillin and amoxicillin in human plasma Yaowu Fenxi Zazhi 2006;26:228–31 17 The International Conference on Harmonization Q2 (R1) Validation of Analytical Procedure: Text and Methodology 2005 [Last Accessed on 2012 Dec 28] Available from: http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/ Q2_R1 Guideline.pdf Indian J Pharm Sci 2011 Jan-Feb; 73(1): 23–29 doi: 10.4103/0250-474X.89753 PMCID: PMC3224406 Development and Validation of a Reversedphase HPLC Method for Simultaneous Determination of Aspirin, Atorvastatin Calcium and Clopidogrel Bisulphate in Capsules S V Londhe, R S Deshmukh, S V Mulgund, and K S Jain* Author information ► Article notes ► Copyright and License information ► Go to: Abstract A simple, accurate, rapid and precise isocratic reversed-phase high-performance liquid chromatographic method has been developed and validated for simultaneous determination of aspirin, atorvastatin calcium and clopidogrel bisulphate in capsules The chromatographic separation was carried out on an Inertsil ODS analytical column (150×4.6 mm; μm) with a mixture of acetonitrile:phosphate buffer pH 3.0 adjusted with o-phosphoric acid (50:50, v/v) as mobile phase; at a flow rate of 1.2 ml/min UV detection was performed at 235 nm The retention times were 1.89, 6.6 and 19.8 for aspirin, atorvastatin calcium and clopidogrel bisulphate, respectively Calibration plots were linear (r2>0.998) over the concentration range 5-30 μg/ml for atorvastatin calcium and 30-105 μg/ml for aspirin and clopidogrel bisulphate The method was validated for accuracy, precision, specificity, linearity, and sensitivity The proposed method was successfully used for quantitative analysis of capsules No interference from any component of pharmaceutical dosage form was observed Validation studies revealed that method is specific, rapid, reliable, and reproducible The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of aspirin, atorvastatin calcium and clopidogrel bisulphate in bulk drug and capsule dosage form Keywords: Aspirin, atorvastatin calcium, clopidogrel bisulphate, HPLC Aspirin (ASP) (fig 1a) is a well-known antithrombotic, antipyretic, analgesic agent Chemically 2-(acetyloxy)benzoic acid, it is official in USP-NF[1], BP[2] and IP[3] It is an antiplatelet agent approved by the Food and Drug Administration, USA, for use in secondary prevention of heart attacks and stroke[4,5] Besides it is mainly used as an analgesic, antipyretic, antiinflammatory and antithrombic agent Atorvastatin calcium (ATO) (3R,5R)-7-[2-(4-fluorophenyl)-3-phenyl-4(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid, calcium salt, (2:1) trihydrate) (fig 1b), a synthetic lipid-lowering agent is official in IP[3] It is a selective competitive inhibitor of the enzyme HMG-CoA reductase, which catalyses the conversion of HMG-CoA to mevalonate, an important rate limiting step in cholesterol biosynthesis[6] Fig Chemical structures of the analytes Structures of (a) aspirin, (b) atorvastatin calcium and (c) clopidogrel bisulphate (c) Clopidogrel bisulphate (CLO) (+)-(S)-methyl2-(2-chlorophenyl)-2-(6,7-dihydrothieno[3,2-c] pyridin-5(4H)-yl)acetate (fig 1c), sulfate is a new thienopyridine derivative It is an antiplatelet agent, which selectively inhibits the binding of adenosine diphosphate (ADP) to its platelet receptor and blocks the subsequent ADP-mediated activation of the glycoprotein GPIIb/IIIa complex, thereby inhibiting platelet aggregation[7] It is official in USP-NF[8] It has been shown to prevent ischemic stroke, myocardial infraction and vascular disease and has demonstrated its clinical efficacy superior to that of aspirin Thus, clopidogrel is indicated for the patients with atherosclerosis documented by recent stroke, recent myocardial infraction or cardiovascular disease[9] The ternary combination of ASP, ATO and CLO is used for atherosclerotic patients suffering from various heart diseases Literature survey reveals many reported methods for the analysis of ASP by high-performance liquid chromatography (HPLC)[10–12] and high-performance thin-layer chromatography (HPTLC)[13] Chromatographic methods have been reported for determination of ATO, in combination with other drugs, in bulk and pharmaceutical dosage forms[14–18] Estimation of CLO by HPLC[19–21] and HPTLC, either individually or in combination with other drugs is reported[22,23] To the best of our knowledge hitherto there is no analytical method reported for simultaneous determination of ternary mixture containing ASP, ATO and CLO Therefore, an attempt has been made to develop a simple, accurate, rapid and reproducible reverse phase HPLC method for simultaneous determination of ASP, ATO and CLO in capsule dosage form and validate it, in accordance with ICH guidelines[24] Go to: MATERIALS AND METHODS Pharmaceutical grade of ASP, ATO and CLO were kindly supplied as gift samples by Torrent Pharmaceuticals, Gujarat, India, certified to contain > 99% (w/w) on dried basis Commercially available Ecosprin Gold-10 (Tristar Formulation Pvt Ltd., Pondicherry, India) and Deplatt-CV (Surien Pharmaceuticals, Pondicherry, India) capsules claimed to contain 75 mg aspirin; 10 mg atorvastatin calcium and 75 mg clopidogrel bisulphate have been utilized in the present work All chemicals and reagents used were of HPLC grade and were purchased from Merck Chemicals, India Chromatographic system and conditions: The HPLC system consisted of a Perkin Elmer (USA) Series 200 instrument equipped with a binary pump, rheodyne injector with 20 μl capacity loop and UV detector The software used was Total Chrom Navigator version® 6.3 Separation was achieved on a reverse phase Inertsil® ODS analytical column (150×4.6 mm; μm) The mobile phase consisted of acetonitrile and 10 mmol/l potassium phosphate buffer adjusted to pH 3.0 with o-phosphoric acid in the ratio 50:50 (v/v) Before analysis the mobile phase was filtered through a 0.2 μm membrane and degassed by ultrasonification The flow rate was 1.2 ml/min Detection was monitored at 235 nm and injection volume was 20 μl All the experiments were performed at ambient temperature Standard solutions and calibration graphs for chromatographic measurement: Stock standard solutions were prepared by dissolving separately 100 mg of ASP, ATO and CLO in 100 ml methanol (1000 μg/ml) The standard calibration solutions were prepared by appropriate dilution of the stock solution with methanol to reach a concentration range of 30-105 μg/ml for ASP and CLO and 5-30 μg/ml for ATO Triplicate 20 μl injections were made for each concentration and chromatographed under the optimized conditions described above The peak area were plotted against the corresponding concentrations to obtain the calibration graphs Sample preparation: Twenty capsule contents were accurately weighed, their mean weight was determined and they were mixed and finely powdered A portion equivalent to about one capsule was accurately weighed and transferred into a 100 ml volumetric flask containing 50 ml methanol, sonicated for 30 and diluted to 100 ml with methanol The resulting solution was centrifuged at 3000 rpm for Supernatant was taken and after suitable dilution the sample solution was then filtered using 0.45 μ filter (Millipore, Milford, MA) The original stock solution was further diluted to get sample solution of drug concentration of 75 μg/ml ASP, 10 μg/ml ATO and 75 μg/ml CLO A 20 μl volume of sample solution was injected into HPLC, six times The peak areas for the drugs were measured at 235 nm and amounts of ASP, ATO and CLO were determined using the related linear regression equations Method validation: The developed method was validated according to the ICH guidelines[24] The system suitability was evaluated by six replicate analyses of ASP, ATO and CLO mixture at a concentration of 75 μg/ml ASP, 10 μg/ml ATO and 75 μg/ml CLO The acceptance criteria were a R.S.D of peak areas and retention times less than 2%, Theoretical plate numbers (N) at least 2500 for each peak and tailing factors (T) less than 1% for ASP, ATO and CLO Standard calibration curves were prepared in the mobile phase with six concentrations ranging from 30-105 μg/ml for ASP and CLO and 5-30 μg/ml for ATO in triplicate into the HPLC system keeping the injection volume constant The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs To study the reliability and suitability of the developed method, recovery experiments were carried out at three levels 80, 100 and 120% Known concentrations of commercial capsules were spiked with known amounts of ASP, ATO and CLO At each level of the amount six determinations were performed and the results obtained were compared with expected results Recovery for pharmaceutical formulations should be within the range 100±5% The percent R.S.D of individual measurements was also determined Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day) for consecutive days Three different concentrations of ASP, ATO and CLO were analyzed in six independent series in the same day (intra-day precision) and consecutive days (inter-day precision) Every sample was injected in triplicate The repeatability of sample application and measurement of peak area for active compounds were expressed in terms of percent RSD All chromatograms were examined to determine if compounds of interest co-eluted with each other or with any additional excipients peaks Marketed formulations were analyzed to determine the specificity of the optimized method in the presence of common capsule excipients Limit of detection (LOD) and limit of quantitation (LOQ) were estimated from the signal-to-noise ratio LOD and LOQ were calculated using 3.3σ/s and 10σ/s formulae, respectively, where, σ is the standard deviation of the peak areas and s is the slope of the corresponding calibration curve To evaluate robustness of HPLC method a few parameters were deliberately varied The parameters included variation of flow rate, percentage of buffer in the mobile phase, and pH of mobile phase Go to: RESULTS AND DISCUSSION During the optimization of HPLC method, two columns (HI-Q Sil C18 5μm; 250 mm × 4.6 mm and Inertsil ODS C18 μm; 150×4.6 mm), two organic solvents (acetonitrile and methanol), two buffers (acetate and phosphate) at two different pH values (3 and 4) were tested Initially methanol:water, acetonitrile:water, acetonitrile:acetate buffer, methanol:acetate buffer were tried in different ratios at pH and ASP and ATO eluted with the tried mobile phases, but CLO was retained Then, with acetonitrile: phosphate buffer all the three drugs eluted, but the analysis time was more than 30 In order to decrease the analysis time, column length was reduced from 250 to 150 mm The mobile phase conditions were optimized so the peak from the first-eluting compound did not interfere with those from the solvent, excipients Other criteria, viz time required for analysis, appropriate k range (1