Karthi

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 (AT1) 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 1 Chemical structure of Telmisartan and its related impurities Figure 2 Chemical structure of Hydrochlorthiazide and its related impurities The chemical names for all components are listed in Table 1. Table 1 Chemical names of all related impurities of Hydrochlorthiazide and Telmisartan Potassium dihydrogen phosphate was obtained from Merck Limited, Mumbai, India; Sodium 1- Hexane 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 min – 1 [Table 2]. Table 2 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 5 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 5 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 5 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 5 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 3 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. 5 ml of mobile phase A was added and sonicated for 5 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 3 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 5 N (Normal) HCl (Hydrochloric acid) and 5 N NaOH (Sodium Hydroxide) respectively. These were subjected to the condition mentioned in Table 3. The solutions were neutralized as needed by (5 N NaOH or 5 N HCl). Table 3 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 3. 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 5 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 4 hours, cooled and volume was made up the with mobile phase A. Further 3 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 5 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 3 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 5 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 3 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 5 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 3 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 5 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 3 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; The feature of stationary phase (RP-C8 and RP-C18). 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 3 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 > 2 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 4 Typical HPLC chromatograms of stressed samples treated with Acid. (a) For HCTZ (b) For TE Figure 7 Typical HPLC chromatogram of sample exposed to light Figure 5 Typical HPLC chromatograms of stressed samples treated with Alkali (a) For HCTZ (b) For TE Figure 6 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, 1 and 0.994 respectively. The Y-Intercept for Impurity B of HCTZ was -502.40 and the slope of linearity graph was found to be 38643.69. In case of HCTZ, the Y-Intercept was -1066.00 40 and the slope of linearity graph was found to be 52848.28. Determination of limit of quantification and detection (LOQ and LOD) The linearity performed above, was used for the determination of limit of quantification and detection. The results are tabulated in Table 4. Table 4 Limit of quantification, detection Determination of relative response factor (RRF) with linear calibration curve The RRF was determined as the ratio of slope of the regression line of the linearity graph, of the impurity to that of corresponding main drug component. From the linearity curves the slope of the regression line of the HCTZ impurity B was 38643.69 and that of HCTZ main drug peak was 52848.28. Hence the relative response factor calculated for HCTZ impurity B was 0.74. Accuracy Accuracy was evaluated by the simultaneous determination of analytes in solution prepared by standard addition method. Placebo preparation of dosage form were spiked with Telmisartan, Hydrochlorothiazide and Hydrochlorothiazide impurity-B at four different levels of LOQ, 50%, 100% and 200% of the specification level in sample solution each level in triplicate. The quantification of added analyte (%weight/weight) was carried out by using an external standard of corresponding main drug prepared at the analytical concentration. Relative response factors of the related impurities were used to calculate the weight percentage of related impurities in drug product. (Note: In routine analysis, the RRF of the related impurities that were either not tested in the method validation with unknown identities were used as 1). The accuracy limits were kept at 75 to 125% at LOQ levels and 80 to 120% for other levels. The experimental results revealed that approximately 97–107% recoveries were obtained for all the investigated related compounds. Therefore, based on the recovery data [Table 5] the estimation of related compounds that are prescribed in this report has been demonstrated to be accurate for intended purpose and is adequate for routine analysis. Table 5 Recovery data Method precision and ruggedness ICH (International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human Use) considers ruggedness as the method reproducibility and intermediate precision. During method precision six independent sample preparations were injected. During intermediate precision the same exercise was repeated using a fresh set of samples on a separate day, on a separate instrument, using a different HPLC column serial number by a different analyst. The results of the precision for the tablet strength of 80/12.5 are revealed in the data given in Table 6. Table 6 Method precision (80/12.5 mg tablets) Acceptance criteria Relative standard deviation (RSD) of six determinations should not be more than 10.0%. Relative standard deviation (RSD) of 12 determinations of two analysts should not be more than 10.0%. Robustness In order to demonstrate the robustness of the method, system suitability parameters were verified by making deliberate change in chromatographic conditions, i.e. change in flow rate by ±0.1 ml min–1, change in pH of the buffer by ±0.2 units, change in column oven temperature by ±5°C. The standard and sample was injected and the system suitability conditions and final result was monitored. The method was demonstrated to be robust over an acceptable working range of its HPLC operational conditions. At higher wavelength, i.e. at 275 nm the response of HCTZ Imp B increases sharply; thus the RRF is not valid for this wavelength. In robustness study the higher wavelength used is limited to 272nm. Hence it was concluded that method is Robust. Solution stability The standard and sample solution was kept at sample temperature for 24 hours were injected on to the HPLC. The data obtained are summarized in Table ​Table77–9. Table 7 Robustness Table 9 Stability in analytical solution (Hydrochlorothiazide) Table 8 Stability in analytical solution for Telmisartan For telmisartan The data shows that RSD of Standard solution up to 24 hours is less than 10%. The data also shows that % difference up to 24 hours is ? 0.05. Thus the standard solution found to be stable for at least upto 24 hours at 20°C. For hydrochlorothiazide The data shows that RSD of Standard solution up to 24 hours is less than 10%. Thus the standard solution for Hydrochlorothiazide was found to be stable for at least upto 24 hours at 20°C. The % difference up to 6 hours is ? 0.05 for Hydrochlorothiazide Impurity-B. Thus the sample was found to be stable for at least upto 6 hours at 20°C and has to be injected within 6 hours after preparation. Filter compatibility Spiked sample solution filtered through different types of membrane syringe filters (Centrifuged, Glass, Nylon, PVDF and Teflon) were injected on HPLC. The % difference was calculated against centrifuged sample solution. The data obtained is summarized in Table 10. Table 10 Data for filter compatibility The data shows that % Difference against centrifuged is within the limit ± 0.05. Filter used: 0.45μ Nylon membrane filter (supplied by MDI, India). Go to: Conclusion A stability study was carried out and an efficient HPLC method for the quantification of related substances of HCTZ and TE in drug product was developed and validated. The results of the stress testing of the drug, undertaken according to the ICH guidelines, revealed that the degradation products were formed in hydrolytic and oxidative conditions. Validation experiments provided proof that the HPLC analytical method is linear in the proposed working range as well as accurate, precise (repeatability and intermediate precision levels) and specific, being able to separate the main drug from its degradation products. The proposed method was also found to be robust with respect to flow rate, column oven temperature, pH of mobile phase. Due to these characteristics, the method has stability indicating properties being fit for its intended purpose; it may find application for the routine analysis of the related substances of HCTZ and TE in Telmisartan and Hydrochlorothiazide tablets. Go to: Footnotes Source of Support: Nil Conflict of Interest: None declared. Go to: References 1. Wienen W, Entzeroth M, Meel JC, Stangier J, Busch U, Ebner T, et al. A review on telmisartan: A novel, longacting angiotensin II-receptor antagonist. Cardiovasc Drug Rev. 2000;18:127–56. 2. Unger T. Significance of angiotensin type 1 receptor blockade: why are angiotensin II receptor blockers different. Am J Cardiol. 1999;84:9–15. [PubMed] 3. Dollery C. 2nd ed. United Kingdom: Churchill Livingstone; 1999. Therapeutic Drugs; pp. 52–7. 4. Wellington K, Faulds DM. Valsartan/hydrochlorothiazide: A review of its pharmacology, therapeutic efficacy and place in the management of hypertension. Drugs. 2002;62:1983–2005. [PubMed] 5. 6th ed. Strasbourg: 2006. European Pharmacopoeia, Telmisartan; p. 3851. 6. The Directorate for the Quality of Medicines of the Council of Europe. 4th ed. 2002. European Pharmacopoeia; pp. 1352–4. 7. Palled MS, Chatter M, Rajesh PM, Bhat AR. Difference spectrophotometric determination of telmisartan in tablet dosage forms. Indian J Pharm Sci. 2006;68:685. 8. Bhat L, Godge R, Vora A, Damle M. Validated RP-HPLC Method for Simultaneous Determination of Telmisartan and Hydrochlorothiazide in Pharmaceutical Formulation. J Liq Chromatogr Related Techno. 2007;30:3059–67. 9. Tamai S, Yamamura N, Sarashina A, Yong CL, Igarashi T, Tanigawa Y. Pharmacokinetic Comparison of an Angiotensin II Receptor Antagonist, Telmisartan, in Japanese and Western Hypertensive Patients Using Population Pharmacokinetic Method. Drug Metab Pharmacokinet. 2004;19:15–23. [PubMed] 10. Muthu AK, Sankhla R, Gupta S, Smith AA, Manavalan R. Development and Validation of a Reversed Phase HPLC Method for Simultaneous Determination of Amlodipine and Telmisartan in Pharmaceutical Dosage Form. J Applied Chem Res. 2010;12:43–52. 11. Xu M, Song J, Liang Y. Rapid determination of telmisartan in pharmaceutical preparations and serum by linear sweep polarography. J Pharm Biomed Anal. 2004;34:681–7. [PubMed] 12. Yan T, Li H, Deng L, Guo Y, Yu W, Fawcett JP, et al. Liquid chromatographic-tandem mass spectrometric method for the simultaneous quantitation of telmisartan and hydrochlorothiazide in human plasma. J Pharm Biomed Anal. 2008;48:1225–9. [PubMed] 13. Joshi S, Karbhari PA, Bhoir SI, Bindu KS, Das C. RP-HPLC method for simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet formulation. J Pharm Biomed Anal. 2010;52:362–71. [PubMed] 14. Zecevic M, Zivanovic LJ, Agatonovic-Kustrin S, Ivanovic D, Maksimovic M. Statistical optimization of a reversed-phase liquid chromatographic method for the analysis of amiloride and hydrochlorothiazide in tablets. J Pharm Biomed Anal. 2000;22:1–6. [PubMed] 15. Rockville, USA: Centre for Biologics Evaluation and Research (CBER); 2000. FDA, Guidance for Industry, Analytical Procedures and Method Validation (Chemistry, Manufacturing and Controls Documentation), Center for Drug Evaluation and Research (CDER) 16. Bakshi M, Singh S. Development of validated stability-indicating assay methods -critical review. J Pharm Biomed Anal. 2002;28:1011–40. [PubMed] 17. USA: ICH; 2003. International Conference on Harmonization (ICH) Q1A (R2): Stability Testing of New Drug Substances and Products. 18. USA: ICH; 1996. International Conference on Harmonization (ICH) Q1B, Stability Testing; Photostability Testing of New Drug Substances and Products. 19. Geneva, Switzerland: ICH; 2005. International Conference on Harmonization (ICH) Q2 (R1): Validation of Analytical Procedures-Test and Methodology. 20. United State Pharmacopeia Convention, System Suitability Testing. 30th ed. Rockville, USA: 2007. The United State Pharmacopeia. 21. Washington, USA: Center for Drug Evaluation Research (CDER); 1994. FDA, Reviewer Guidance: Validation of Chromatographic Methods. 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 (AT1) 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 1 Chemical structure of Telmisartan and its related impurities Figure 2 Chemical structure of Hydrochlorthiazide and its related impurities The chemical names for all components are listed in Table 1. Table 1 Chemical names of all related impurities of Hydrochlorthiazide and Telmisartan Potassium dihydrogen phosphate was obtained from Merck Limited, Mumbai, India; Sodium 1- Hexane 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 min – 1 [Table 2]. Table 2 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 5 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 5 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 5 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 5 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 3 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. 5 ml of mobile phase A was added and sonicated for 5 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 3 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 5 N (Normal) HCl (Hydrochloric acid) and 5 N NaOH (Sodium Hydroxide) respectively. These were subjected to the condition mentioned in Table 3. The solutions were neutralized as needed by (5 N NaOH or 5 N HCl). Table 3 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 3. 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 5 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 4 hours, cooled and volume was made up the with mobile phase A. Further 3 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 5 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 3 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 5 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 3 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 5 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 3 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 5 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 3 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; The feature of stationary phase (RP-C8 and RP-C18). 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 3 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 > 2 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 4 Typical HPLC chromatograms of stressed samples treated with Acid. (a) For HCTZ (b) For TE Figure 7 Typical HPLC chromatogram of sample exposed to light Figure 5 Typical HPLC chromatograms of stressed samples treated with Alkali (a) For HCTZ (b) For TE Figure 6 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, 1 and 0.994 respectively. The Y-Intercept for Impurity B of HCTZ was -502.40 and the slope of linearity graph was found to be 38643.69. In case of HCTZ, the Y-Intercept was -1066.00 40 and the slope of linearity graph was found to be 52848.28. Determination of limit of quantification and detection (LOQ and LOD) The linearity performed above, was used for the determination of limit of quantification and detection. The results are tabulated in Table 4. Table 4 Limit of quantification, detection Determination of relative response factor (RRF) with linear calibration curve The RRF was determined as the ratio of slope of the regression line of the linearity graph, of the impurity to that of corresponding main drug component. From the linearity curves the slope of the regression line of the HCTZ impurity B was 38643.69 and that of HCTZ main drug peak was 52848.28. Hence the relative response factor calculated for HCTZ impurity B was 0.74. Accuracy Accuracy was evaluated by the simultaneous determination of analytes in solution prepared by standard addition method. Placebo preparation of dosage form were spiked with Telmisartan, Hydrochlorothiazide and Hydrochlorothiazide impurity-B at four different levels of LOQ, 50%, 100% and 200% of the specification level in sample solution each level in triplicate. The quantification of added analyte (%weight/weight) was carried out by using an external standard of corresponding main drug prepared at the analytical concentration. Relative response factors of the related impurities were used to calculate the weight percentage of related impurities in drug product. (Note: In routine analysis, the RRF of the related impurities that were either not tested in the method validation with unknown identities were used as 1). The accuracy limits were kept at 75 to 125% at LOQ levels and 80 to 120% for other levels. The experimental results revealed that approximately 97–107% recoveries were obtained for all the investigated related compounds. Therefore, based on the recovery data [Table 5] the estimation of related compounds that are prescribed in this report has been demonstrated to be accurate for intended purpose and is adequate for routine analysis. Table 5 Recovery data Method precision and ruggedness ICH (International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human Use) considers ruggedness as the method reproducibility and intermediate precision. During method precision six independent sample preparations were injected. During intermediate precision the same exercise was repeated using a fresh set of samples on a separate day, on a separate instrument, using a different HPLC column serial number by a different analyst. The results of the precision for the tablet strength of 80/12.5 are revealed in the data given in Table 6. Table 6 Method precision (80/12.5 mg tablets) Acceptance criteria Relative standard deviation (RSD) of six determinations should not be more than 10.0%. Relative standard deviation (RSD) of 12 determinations of two analysts should not be more than 10.0%. Robustness In order to demonstrate the robustness of the method, system suitability parameters were verified by making deliberate change in chromatographic conditions, i.e. change in flow rate by ±0.1 ml min–1, change in pH of the buffer by ±0.2 units, change in column oven temperature by ±5°C. The standard and sample was injected and the system suitability conditions and final result was monitored. The method was demonstrated to be robust over an acceptable working range of its HPLC operational conditions. At higher wavelength, i.e. at 275 nm the response of HCTZ Imp B increases sharply; thus the RRF is not valid for this wavelength. In robustness study the higher wavelength used is limited to 272nm. Hence it was concluded that method is Robust. Solution stability The standard and sample solution was kept at sample temperature for 24 hours were injected on to the HPLC. The data obtained are summarized in Table ​Table77–9. Table 7 Robustness Table 9 Stability in analytical solution (Hydrochlorothiazide) Table 8 Stability in analytical solution for Telmisartan For telmisartan The data shows that RSD of Standard solution up to 24 hours is less than 10%. The data also shows that % difference up to 24 hours is ? 0.05. Thus the standard solution found to be stable for at least upto 24 hours at 20°C. For hydrochlorothiazide The data shows that RSD of Standard solution up to 24 hours is less than 10%. Thus the standard solution for Hydrochlorothiazide was found to be stable for at least upto 24 hours at 20°C. The % difference up to 6 hours is ? 0.05 for Hydrochlorothiazide Impurity-B. Thus the sample was found to be stable for at least upto 6 hours at 20°C and has to be injected within 6 hours after preparation. Filter compatibility Spiked sample solution filtered through different types of membrane syringe filters (Centrifuged, Glass, Nylon, PVDF and Teflon) were injected on HPLC. The % difference was calculated against centrifuged sample solution. The data obtained is summarized in Table 10. Table 10 Data for filter compatibility The data shows that % Difference against centrifuged is within the limit ± 0.05. Filter used: 0.45μ Nylon membrane filter (supplied by MDI, India). Go to: Conclusion A stability study was carried out and an efficient HPLC method for the quantification of related substances of HCTZ and TE in drug product was developed and validated. The results of the stress testing of the drug, undertaken according to the ICH guidelines, revealed that the degradation products were formed in hydrolytic and oxidative conditions. Validation experiments provided proof that the HPLC analytical method is linear in the proposed working range as well as accurate, precise (repeatability and intermediate precision levels) and specific, being able to separate the main drug from its degradation products. The proposed method was also found to be robust with respect to flow rate, column oven temperature, pH of mobile phase. Due to these characteristics, the method has stability indicating properties being fit for its intended purpose; it may find application for the routine analysis of the related substances of HCTZ and TE in Telmisartan and Hydrochlorothiazide tablets. Go to: Footnotes Source of Support: Nil Conflict of Interest: None declared. Go to: References 1. Wienen W, Entzeroth M, Meel JC, Stangier J, Busch U, Ebner T, et al. A review on telmisartan: A novel, longacting angiotensin II-receptor antagonist. Cardiovasc Drug Rev. 2000;18:127–56. 2. Unger T. Significance of angiotensin type 1 receptor blockade: why are angiotensin II receptor blockers different. Am J Cardiol. 1999;84:9–15. [PubMed] 3. Dollery C. 2nd ed. United Kingdom: Churchill Livingstone; 1999. Therapeutic Drugs; pp. 52–7. 4. Wellington K, Faulds DM. Valsartan/hydrochlorothiazide: A review of its pharmacology, therapeutic efficacy and place in the management of hypertension. Drugs. 2002;62:1983–2005. [PubMed] 5. 6th ed. Strasbourg: 2006. European Pharmacopoeia, Telmisartan; p. 3851. 6. The Directorate for the Quality of Medicines of the Council of Europe. 4th ed. 2002. European Pharmacopoeia; pp. 1352–4. 7. Palled MS, Chatter M, Rajesh PM, Bhat AR. Difference spectrophotometric determination of telmisartan in tablet dosage forms. Indian J Pharm Sci. 2006;68:685. 8. Bhat L, Godge R, Vora A, Damle M. Validated RP-HPLC Method for Simultaneous Determination of Telmisartan and Hydrochlorothiazide in Pharmaceutical Formulation. J Liq Chromatogr Related Techno. 2007;30:3059–67. 9. Tamai S, Yamamura N, Sarashina A, Yong CL, Igarashi T, Tanigawa Y. Pharmacokinetic Comparison of an Angiotensin II Receptor Antagonist, Telmisartan, in Japanese and Western Hypertensive Patients Using Population Pharmacokinetic Method. Drug Metab Pharmacokinet. 2004;19:15–23. [PubMed] 10. Muthu AK, Sankhla R, Gupta S, Smith AA, Manavalan R. Development and Validation of a Reversed Phase HPLC Method for Simultaneous Determination of Amlodipine and Telmisartan in Pharmaceutical Dosage Form. J Applied Chem Res. 2010;12:43–52. 11. Xu M, Song J, Liang Y. Rapid determination of telmisartan in pharmaceutical preparations and serum by linear sweep polarography. J Pharm Biomed Anal. 2004;34:681–7. [PubMed] 12. Yan T, Li H, Deng L, Guo Y, Yu W, Fawcett JP, et al. Liquid chromatographic-tandem mass spectrometric method for the simultaneous quantitation of telmisartan and hydrochlorothiazide in human plasma. J Pharm Biomed Anal. 2008;48:1225–9. [PubMed] 13. Joshi S, Karbhari PA, Bhoir SI, Bindu KS, Das C. RP-HPLC method for simultaneous estimation of bisoprolol fumarate and hydrochlorothiazide in tablet formulation. J Pharm Biomed Anal. 2010;52:362–71. [PubMed] 14. Zecevic M, Zivanovic LJ, Agatonovic-Kustrin S, Ivanovic D, Maksimovic M. Statistical optimization of a reversed-phase liquid chromatographic method for the analysis of amiloride and hydrochlorothiazide in tablets. J Pharm Biomed Anal. 2000;22:1–6. [PubMed] 15. Rockville, USA: Centre for Biologics Evaluation and Research (CBER); 2000. FDA, Guidance for Industry, Analytical Procedures and Method Validation (Chemistry, Manufacturing and Controls Documentation), Center for Drug Evaluation and Research (CDER) 16. Bakshi M, Singh S. Development of validated stability-indicating assay methods -critical review. J Pharm Biomed Anal. 2002;28:1011–40. [PubMed] 17. USA: ICH; 2003. International Conference on Harmonization (ICH) Q1A (R2): Stability Testing of New Drug Substances and Products. 18. USA: ICH; 1996. International Conference on Harmonization (ICH) Q1B, Stability Testing; Photostability Testing of New Drug Substances and Products. 19. Geneva, Switzerland: ICH; 2005. International Conference on Harmonization (ICH) Q2 (R1): Validation of Analytical Procedures-Test and Methodology. 20. United State Pharmacopeia Convention, System Suitability Testing. 30th ed. Rockville, USA: 2007. The United State Pharmacopeia. 21. Washington, USA: Center for Drug Evaluation Research (CDER); 1994. FDA, Reviewer Guidance: Validation of Chromatographic Methods. RP-HPLC Estimation of Ramipril and Telmisartan in Tablets V. P. Kurade,* M. G. Pai, and R. Gude Author information ► Article notes ► Copyright and License information ► This article has been cited by other articles in PMC. Go to: Abstract A rapid high performance liquid chromatographic method has been developed and validated for the estimation of ramipril and telmisartan simultaneously in combined dosage form. A Genesis C18 column having dimensions of 4.6×250 mm and particle size of 5 μm in isocratic mode, with mobile phase containing a mixture of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid): methanol:acetonitrile (15:15:70 v/v/v) was used. The mobile phase was pumped at a flow rate of 1.0 ml/min and the eluents were monitored at 210 nm. The selected chromatographic conditions were found to effectively separate ramipril (Rt: 3.68 min) and telmisartan (Rt: 4.98 min) having a resolution of 3.84. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation. Linearity for ramipril and telmisartan were found in the range of 3.5-6.5 μg/ml and 28.0-52.0 μg/ml, respectively. The percentage recoveries for ramipril and telmisartan ranged from 99.09-101.64% and 99.45-100.99%, respectively. The limit of detection and the limit of quantitation for ramipril was found to be 0.5 μg/ml and 1.5 μg/ml respectively and for telmisartan was found to be 1.5 μg/ml and 3.0 μg/ml, respectively. The method was found to be robust and can be successfully used to determine the drug content of marketed formulations. Keywords: Simultaneous estimation, RP-HPLC, ramipril, telmisartan, validation Telmisartan is a new angiotensin II receptor antagonist for the treatment of essential hypertension usually given in combination with ramipril. Telmisartan (TEL) is chemically 4'-((1,4'-dimethyl-2'-propyl(2,6'-bi-1H-benzimidazol)-1'-yl)methyl)-(1,1'-biphenyl)-2-carboxylic acid. Ramipril (RAM) is chemically (1S,5S,7S)-8-((2S)-2-(((1S)-1-ethoxycarbonyl-3-phenyl-propyl)amino)propanoyl)-8-azabicyclo(3.3.0)octane-7-carboxylic acid. RAM is a highly lipophilic, long acting ACE inhibitor. Literature survey revealed that telmisartan is not yet official in any of the pharmacopoeia. RAM is official in USP and BP where HPLC and potentiometric titration is the official method of analysis[1,2]. There are numerous methods reported for estimation of these drugs alone as well as in combination with other drugs in pharmaceutical dosage forms[3–8] and/or in biological fluids. However, no method has been reported so far for the estimation of these two drugs simultaneously in combined dosage forms. Hence, in the present study, a new reversed-phase high performance liquid chromatography method was developed and validated for the simultaneous estimation of RAM and TEL in tablets. The liquid chromatographic system consisted of the following components: Knauer, Advanced Scientific Instruments containing Smartline Pump 1000, PDA detector and Rheodyne injector with 20 μl fixed loop. Chromatographic analysis was performed using Eurochrome software on a Genesis C18 column with 250×4.6 mm i.d. and 5 μm particle size. Analytically pure RAM and TEL were obtained as gift samples from M/s Cipla Ltd., Verna, Goa, India). Acetonitrile, methanol, water (E. Merck, Mumbai, India) were of HPLC grade, while orthophosphoric acid and potassium dihydrogenphosphate (KH2PO4) (S. D. Fine Chemicals, Mumbai, India) were of analytical grade used for the preparation of mobile phase. Tablet formulation containing labeled amount of 5 mg RAM and 40 mg TEL was procured from the local Pharmacy. Potassium dihydrogenphosphate buffer (0.01 M) was prepared in water and pH was adjusted to 3.4 by using ortho phosphoric acid solution. The mobile phase components, buffer (pH 3.4): methanol:acetonitrile (15:15:70 v/v/v) were then mixed and finally filtered through a nylon membrane filters of 0.45 μ and sonicated. Around 50 mg of RAM and 50 mg of TEL were accurately weighed and transferred to a standard 100 ml and 50 ml volumetric flasks, respectively. To this 30 ml of methanol was added, shaken for 20 min and sonicated to dissolve the solids. After complete dissolution of the drugs, volume was made up to the mark with methanol to give stock solutions of 500 μg/ml of RAM and 1000 μg/ml of TEL separately. A reverse phase C18 column equilibrated with the optimum composition of the mobile phase containing 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 with orthophosphoric acid):methanol:acetonitrile (15:15:70 v/v/v) was used to resolve the peaks of RAM and TEL. The mobile phase flow rate was maintained at 1 ml/min and effluents were monitored at 210 nm. The sample was injected using a 20 μl fixed loop, and the total run time was 10 min. Appropriate aliquots of standard stock solutions of RAM and TEL were diluted with acetonitrile to obtain final concentrations in the range of 3.5-6.5 μg/ml of RAM and 28.0-52.0 μg/ml of TEL. The solutions were injected in triplicates for each concentration using a 20 μl fixed loop system and chromatograms were recorded. Calibration curves were constructed by plotting average content of the drug versus respective concentrations and regression equations were computed for RAM and TEL. The plots of average content Vs respective concentration of RAM and TEL were found to be linear in the range of 3.5-6.5 μg/ml and 28.0-52.0 μg/ml with coefficient of correlation (r2) 0.9963 and 0.9957 for RAM and TEL, respectively. Ten tablets were weighed and finely powdered. Tablet powder equivalent to 5 mg of RAM and 40 mg of TEL was accurately weighed and transferred to a 100 ml volumetric flask. To this was added about 50 ml of methanol and flask was sonicated for 15 min. The flask was shaken, and the volume was made up to the mark with methanol. The above solution was then filtered through 0.45 μ Whatman filter paper. One millilitre of the above filtrate was further diluted up to 10 ml with acetonitrile to obtain final concentrations of 5 μg/ml and 40 μg/ml of RAM and TEL, respectively. Sample solution was then filtered using sample filtration assembly through nylon membrane filter of 0.45 μ. The solution was injected under above chromatographic conditions and peak areas were measured. The quantification was carried out by keeping these values to the straight line equation of calibration curve. The method was validated for accuracy, precision, specificity, detection limit, quantitation limit and robustness as per ‘ICH’ guidelines[9]. Preanalyzed sample solution was spiked with ramipril and telmisartan in the same proportion as that present in the tablet formulation. Spiking was done at three different concentrations 70%, 100%, and 130% of the label claim. Accuracy of the method was studied by calculating the recovery of the spiked samples. The average recoveries are 99.09 to 101.64 and 99.45 to 100.99 for RAM and TEL, respectively. Repeatability of the method was validated by performing six replicate assays of the homogeneous sample. Results were calculated in terms of %RSD of the content of RAM and TEL. Method was also validated for intermediate precision by comparing the performance of the method on different day by different chemist. Six replicate assays of homogeneous sample were performed using the same procedure and chromatographic conditions, % RSD of the contents of RAM and TEL were calculated. This results and repeatability (performed on previous day, by different chemist) results were compared. The specificity of the RP-HPLC method was determined by the complete separation of RAM and TEL as shown in (fig. 1) with parameters like retention time (tR), resolution (Rs) and tailing factor (T). The peaks obtained for RAM and TEL were sharp and have clear baseline separation. Sample matrix did not show any interference with the analyte peaks. Retention times for RAM and TEL were 3.68 and 4.98 min, respectively. Different standard solutions were prepared in the concentration range of 0.1-2 μg/ml for RAM and 0.5-5 μg/ml for TEL and the limit of detection (LOD) and quantitation (LOQ) for ramipril and telmisartan was determined. Robustness of the method was studied by changing the flow rate of the mobile phase by ± 2% and also by observing the stability of the drugs for 24 h at room temperature in the dilution solvent. Fig 1 Typical sample chromatogram of RAM and TEL. Chromatogram of sample showing well resolved peaks of ramipril (RAM) at Rt-3.68 min and telmisartan (TEL) with Rt of 4.98 min. Optimization of mobile phase was performed based on resolution, tailing factor and peak area obtained for both ramipril and telmisartan. The mobile phase, 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid):methanol:acetonitrile (15:15:70 v/v/v) was found to be satisfactory and gave two symmetric and well-resolved peaks for RAM and TEL. The resolution between RAM and TEL was found to be 3.84, which indicates good separation of both the compounds. The retention time for ramipril and telmisartan were 3.68 min and 4.98 minute, respectively (fig. 1). The asymmetry factors for RAM and TEL were 1.01 and 1.19, respectively. The 3D dimension spectra of both RAM and TEL showed that both the drugs absorb appreciably at 210 nm so, 210 nm was selected as the detection wavelength in liquid chromatography. The calibration curve for RAM was obtained by plotting the peak area of RAM versus the respective concentration of RAM over the range of 3.5-6.5 μg/ml, and it was found to be linear with r2 = 0.9963. Similarly, the calibration curve for TEL was obtained over the range of 28.0-52.0 μg/ml and was found to be linear with r2 = 0.9957. The detection limit for RAM and TEL were 0.5 μg/ml and 1.5 μg/ml, respectively. The quantitation limit for RAM and TEL were 1.5 μg/ml and 3.0 μg/ml, respectively, which suggest that a nanogram quantity of both the compounds can be estimated accurately. The validation parameters are summarized in (Table 1). TABLE 1 SUMMARY OF VALIDATION PARAMETERS The recoveries of ramipril and telmisartan were found to be in the range of 99.09-101.64% and 99.45-100.99 %, respectively. The system suitability test parameters are shown in (Table 2). The liquid chromatographic method was applied to the determination of RAM and TEL in their combined dosage forms (tablet formulation) and the average assay values were 98.94% and 99.84% of the labeled claim of RAM and TEL, respectively. TABLE 2 SYSTEM SUITABILITY TEST PARAMETERS FOR RAMIPRIL AND TELMISARTAN BY THE PROPOSED METHOD In the present study the attempt has been undertaken to develop most simple, economical, sensitive and accurate analytical HPLC method for the simultaneous estimation of these drugs without their prior separation. The method gives good resolution between both the compounds with a short analysis time (< 10 min). The method was validated and found to be simple, sensitive, accurate and precise. Percentage of recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of ramipril and telmisartan in their combined dosage form. Go to: Acknowledgments The authors are grateful to Goa College of Pharmacy for providing necessary facilities for the research work and M/s Cipla Ltd., Verna, Goa, India for providing gift samples of ramipril and telmisartan. Go to: Footnotes Kurade, et al.: RP-HPLC Estimation of Ramipril and Telmisartan Go to: REFERENCES 1. The British Pharmacopoeia. Vol. 2. London: British Pharmacopoeial Commission; 2003. p. 1609. 2. The United States Pharmacopoeia 29, National Formulary 24. Asian Edition. Rockville, MD: United States Pharmacopoeial Convention, Inc; 2006. p. 1890. 3. Baing MM, Vaidya VV, Sane RT, Menon SN, Dalvi K. Simultaneous RP-LC determination of losartan potassium, ramipril, and hydrochlorothiazide in pharmaceutical preparations. Chromatographia. 2006;64:293–6. 4. Rao KV, Vijaya Kumari K, Bhanuprakash I, Prabhakar G, Begum J. Determination of ramipril in pharmaceutical dosage forms by reversed-phase liquid chromatography. Asian J Chem. 2006;18:788–92. 5. Belal F, Al-Zaagi IA, Gadkariem EA, Abounassif MA. A stability-indicating LC method for the simultaneous determination of ramipril and hydrochlorothiazide in dosage forms. J Pharm Biomed Anal. 2001;24:335–42. [PubMed] 6. Rao RN, Sen S, Nagaraju P, Reddy VS, Radha Krishnamurthy P, Udaybhaskar S, et al. HPLC determination of Telmisartan in bulk and pharmaceutical formulations. Asian J Chem. 2006;18:775–82. 7. Palled MS, Rajesh PM, Chatter M, Bhat AR. RP-HPLC determination of Telmisartan in tablet dosage forms. Indian J Pharm Sci. 2005;67:108–10. 8. Kumar MV, Muley PR. Stability indicating RP-HPLC method for determination of Telmisartan in solid dosage forms. Indian Pharmacist. 2005;4:69–72. 9. ICH, Q2B- Validation of Analytical Procedures: Methodology, International Conference on Harmonization. 1996. Nov, Indian J Pharm Sci. 2009 Mar-Apr; 71(2): 148–151. doi:  10.4103/0250-474X.54283 PMCID: PMC2839403 RP-HPLC Estimation of Ramipril and Telmisartan in Tablets V. P. Kurade,* M. G. Pai, and R. Gude Author information ► Article notes ► Copyright and License information ► This article has been cited by other articles in PMC. Go to: Abstract A rapid high performance liquid chromatographic method has been developed and validated for the estimation of ramipril and telmisartan simultaneously in combined dosage form. A Genesis C18 column having dimensions of 4.6×250 mm and particle size of 5 μm in isocratic mode, with mobile phase containing a mixture of 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid): methanol:acetonitrile (15:15:70 v/v/v) was used. The mobile phase was pumped at a flow rate of 1.0 ml/min and the eluents were monitored at 210 nm. The selected chromatographic conditions were found to effectively separate ramipril (Rt: 3.68 min) and telmisartan (Rt: 4.98 min) having a resolution of 3.84. The method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation. Linearity for ramipril and telmisartan were found in the range of 3.5-6.5 μg/ml and 28.0-52.0 μg/ml, respectively. The percentage recoveries for ramipril and telmisartan ranged from 99.09-101.64% and 99.45-100.99%, respectively. The limit of detection and the limit of quantitation for ramipril was found to be 0.5 μg/ml and 1.5 μg/ml respectively and for telmisartan was found to be 1.5 μg/ml and 3.0 μg/ml, respectively. The method was found to be robust and can be successfully used to determine the drug content of marketed formulations. Keywords: Simultaneous estimation, RP-HPLC, ramipril, telmisartan, validation Telmisartan is a new angiotensin II receptor antagonist for the treatment of essential hypertension usually given in combination with ramipril. Telmisartan (TEL) is chemically 4'-((1,4'-dimethyl-2'-propyl(2,6'-bi-1H-benzimidazol)-1'-yl)methyl)-(1,1'-biphenyl)-2-carboxylic acid. Ramipril (RAM) is chemically (1S,5S,7S)-8-((2S)-2-(((1S)-1-ethoxycarbonyl-3-phenyl-propyl)amino)propanoyl)-8-azabicyclo(3.3.0)octane-7-carboxylic acid. RAM is a highly lipophilic, long acting ACE inhibitor. Literature survey revealed that telmisartan is not yet official in any of the pharmacopoeia. RAM is official in USP and BP where HPLC and potentiometric titration is the official method of analysis[1,2]. There are numerous methods reported for estimation of these drugs alone as well as in combination with other drugs in pharmaceutical dosage forms[3–8] and/or in biological fluids. However, no method has been reported so far for the estimation of these two drugs simultaneously in combined dosage forms. Hence, in the present study, a new reversed-phase high performance liquid chromatography method was developed and validated for the simultaneous estimation of RAM and TEL in tablets. The liquid chromatographic system consisted of the following components: Knauer, Advanced Scientific Instruments containing Smartline Pump 1000, PDA detector and Rheodyne injector with 20 μl fixed loop. Chromatographic analysis was performed using Eurochrome software on a Genesis C18 column with 250×4.6 mm i.d. and 5 μm particle size. Analytically pure RAM and TEL were obtained as gift samples from M/s Cipla Ltd., Verna, Goa, India). Acetonitrile, methanol, water (E. Merck, Mumbai, India) were of HPLC grade, while orthophosphoric acid and potassium dihydrogenphosphate (KH2PO4) (S. D. Fine Chemicals, Mumbai, India) were of analytical grade used for the preparation of mobile phase. Tablet formulation containing labeled amount of 5 mg RAM and 40 mg TEL was procured from the local Pharmacy. Potassium dihydrogenphosphate buffer (0.01 M) was prepared in water and pH was adjusted to 3.4 by using ortho phosphoric acid solution. The mobile phase components, buffer (pH 3.4): methanol:acetonitrile (15:15:70 v/v/v) were then mixed and finally filtered through a nylon membrane filters of 0.45 μ and sonicated. Around 50 mg of RAM and 50 mg of TEL were accurately weighed and transferred to a standard 100 ml and 50 ml volumetric flasks, respectively. To this 30 ml of methanol was added, shaken for 20 min and sonicated to dissolve the solids. After complete dissolution of the drugs, volume was made up to the mark with methanol to give stock solutions of 500 μg/ml of RAM and 1000 μg/ml of TEL separately. A reverse phase C18 column equilibrated with the optimum composition of the mobile phase containing 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 with orthophosphoric acid):methanol:acetonitrile (15:15:70 v/v/v) was used to resolve the peaks of RAM and TEL. The mobile phase flow rate was maintained at 1 ml/min and effluents were monitored at 210 nm. The sample was injected using a 20 μl fixed loop, and the total run time was 10 min. Appropriate aliquots of standard stock solutions of RAM and TEL were diluted with acetonitrile to obtain final concentrations in the range of 3.5-6.5 μg/ml of RAM and 28.0-52.0 μg/ml of TEL. The solutions were injected in triplicates for each concentration using a 20 μl fixed loop system and chromatograms were recorded. Calibration curves were constructed by plotting average content of the drug versus respective concentrations and regression equations were computed for RAM and TEL. The plots of average content Vs respective concentration of RAM and TEL were found to be linear in the range of 3.5-6.5 μg/ml and 28.0-52.0 μg/ml with coefficient of correlation (r2) 0.9963 and 0.9957 for RAM and TEL, respectively. Ten tablets were weighed and finely powdered. Tablet powder equivalent to 5 mg of RAM and 40 mg of TEL was accurately weighed and transferred to a 100 ml volumetric flask. To this was added about 50 ml of methanol and flask was sonicated for 15 min. The flask was shaken, and the volume was made up to the mark with methanol. The above solution was then filtered through 0.45 μ Whatman filter paper. One millilitre of the above filtrate was further diluted up to 10 ml with acetonitrile to obtain final concentrations of 5 μg/ml and 40 μg/ml of RAM and TEL, respectively. Sample solution was then filtered using sample filtration assembly through nylon membrane filter of 0.45 μ. The solution was injected under above chromatographic conditions and peak areas were measured. The quantification was carried out by keeping these values to the straight line equation of calibration curve. The method was validated for accuracy, precision, specificity, detection limit, quantitation limit and robustness as per ‘ICH’ guidelines[9]. Preanalyzed sample solution was spiked with ramipril and telmisartan in the same proportion as that present in the tablet formulation. Spiking was done at three different concentrations 70%, 100%, and 130% of the label claim. Accuracy of the method was studied by calculating the recovery of the spiked samples. The average recoveries are 99.09 to 101.64 and 99.45 to 100.99 for RAM and TEL, respectively. Repeatability of the method was validated by performing six replicate assays of the homogeneous sample. Results were calculated in terms of %RSD of the content of RAM and TEL. Method was also validated for intermediate precision by comparing the performance of the method on different day by different chemist. Six replicate assays of homogeneous sample were performed using the same procedure and chromatographic conditions, % RSD of the contents of RAM and TEL were calculated. This results and repeatability (performed on previous day, by different chemist) results were compared. The specificity of the RP-HPLC method was determined by the complete separation of RAM and TEL as shown in (fig. 1) with parameters like retention time (tR), resolution (Rs) and tailing factor (T). The peaks obtained for RAM and TEL were sharp and have clear baseline separation. Sample matrix did not show any interference with the analyte peaks. Retention times for RAM and TEL were 3.68 and 4.98 min, respectively. Different standard solutions were prepared in the concentration range of 0.1-2 μg/ml for RAM and 0.5-5 μg/ml for TEL and the limit of detection (LOD) and quantitation (LOQ) for ramipril and telmisartan was determined. Robustness of the method was studied by changing the flow rate of the mobile phase by ± 2% and also by observing the stability of the drugs for 24 h at room temperature in the dilution solvent. Fig 1 Typical sample chromatogram of RAM and TEL. Chromatogram of sample showing well resolved peaks of ramipril (RAM) at Rt-3.68 min and telmisartan (TEL) with Rt of 4.98 min. Optimization of mobile phase was performed based on resolution, tailing factor and peak area obtained for both ramipril and telmisartan. The mobile phase, 0.01 M potassium dihydrogen phosphate buffer (adjusted to pH 3.4 using orthophosphoric acid):methanol:acetonitrile (15:15:70 v/v/v) was found to be satisfactory and gave two symmetric and well-resolved peaks for RAM and TEL. The resolution between RAM and TEL was found to be 3.84, which indicates good separation of both the compounds. The retention time for ramipril and telmisartan were 3.68 min and 4.98 minute, respectively (fig. 1). The asymmetry factors for RAM and TEL were 1.01 and 1.19, respectively. The 3D dimension spectra of both RAM and TEL showed that both the drugs absorb appreciably at 210 nm so, 210 nm was selected as the detection wavelength in liquid chromatography. The calibration curve for RAM was obtained by plotting the peak area of RAM versus the respective concentration of RAM over the range of 3.5-6.5 μg/ml, and it was found to be linear with r2 = 0.9963. Similarly, the calibration curve for TEL was obtained over the range of 28.0-52.0 μg/ml and was found to be linear with r2 = 0.9957. The detection limit for RAM and TEL were 0.5 μg/ml and 1.5 μg/ml, respectively. The quantitation limit for RAM and TEL were 1.5 μg/ml and 3.0 μg/ml, respectively, which suggest that a nanogram quantity of both the compounds can be estimated accurately. The validation parameters are summarized in (Table 1). TABLE 1 SUMMARY OF VALIDATION PARAMETERS The recoveries of ramipril and telmisartan were found to be in the range of 99.09-101.64% and 99.45-100.99 %, respectively. The system suitability test parameters are shown in (Table 2). The liquid chromatographic method was applied to the determination of RAM and TEL in their combined dosage forms (tablet formulation) and the average assay values were 98.94% and 99.84% of the labeled claim of RAM and TEL, respectively. TABLE 2 SYSTEM SUITABILITY TEST PARAMETERS FOR RAMIPRIL AND TELMISARTAN BY THE PROPOSED METHOD In the present study the attempt has been undertaken to develop most simple, economical, sensitive and accurate analytical HPLC method for the simultaneous estimation of these drugs without their prior separation. The method gives good resolution between both the compounds with a short analysis time (< 10 min). The method was validated and found to be simple, sensitive, accurate and precise. Percentage of recovery shows that the method is free from interference of the excipients used in the formulation. Therefore, the proposed method can be used for routine analysis of ramipril and telmisartan in their combined dosage form. Go to: Acknowledgments The authors are grateful to Goa College of Pharmacy for providing necessary facilities for the research work and M/s Cipla Ltd., Verna, Goa, India for providing gift samples of ramipril and telmisartan. Go to: Footnotes Kurade, et al.: RP-HPLC Estimation of Ramipril and Telmisartan Go to: REFERENCES 1. The British Pharmacopoeia. Vol. 2. London: British Pharmacopoeial Commission; 2003. p. 1609. 2. The United States Pharmacopoeia 29, National Formulary 24. Asian Edition. Rockville, MD: United States Pharmacopoeial Convention, Inc; 2006. p. 1890. 3. Baing MM, Vaidya VV, Sane RT, Menon SN, Dalvi K. Simultaneous RP-LC determination of losartan potassium, ramipril, and hydrochlorothiazide in pharmaceutical preparations. Chromatographia. 2006;64:293–6. 4. Rao KV, Vijaya Kumari K, Bhanuprakash I, Prabhakar G, Begum J. Determination of ramipril in pharmaceutical dosage forms by reversed-phase liquid chromatography. Asian J Chem. 2006;18:788–92. 5. Belal F, Al-Zaagi IA, Gadkariem EA, Abounassif MA. A stability-indicating LC method for the simultaneous determination of ramipril and hydrochlorothiazide in dosage forms. J Pharm Biomed Anal. 2001;24:335–42. [PubMed] 6. Rao RN, Sen S, Nagaraju P, Reddy VS, Radha Krishnamurthy P, Udaybhaskar S, et al. HPLC determination of Telmisartan in bulk and pharmaceutical formulations. Asian J Chem. 2006;18:775–82. 7. Palled MS, Rajesh PM, Chatter M, Bhat AR. RP-HPLC determination of Telmisartan in tablet dosage forms. Indian J Pharm Sci. 2005;67:108–10. 8. Kumar MV, Muley PR. Stability indicating RP-HPLC method for determination of Telmisartan in solid dosage forms. Indian Pharmacist. 2005;4:69–72. 9. ICH, Q2B- Validation of Analytical Procedures: Methodology, International Conference on Harmonization. 1996. Nov, Indian J Pharm Sci. 2013 Jan-Feb; 75(1): 31–35. doi:  10.4103/0250-474X.113538 PMCID: PMC3719147 Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cefpodoxime Proxetil and Dicloxacillin Sodium in Tablets D. R. Acharya* and Dipti B. Patel Author information ► Article notes ► Copyright and License information ► Go to: Abstract 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. Keywords: Cefpodoxime proxetil, dicloxacillin sodium, reversed-phase high-performance liquid chromatographic method, tablet, validation Cefpodoxime proxetil (CEF) is chemically 1-(isopropoxy carbonyloxy) ethyl (6R,7R)-7-[2-(2-amino-4-thiazolyl)-(z)-2-(methoxyimino) acetamido]-3-methoxymethyl-3-cephem-4-carboxylate,[1] is a third generation cephalosporin anti-biotic. It is used for infections of the respiratory tract, urinary tract and skin and soft tissues. It has greater activity against Staphylococcus aureus.[2] CEF is official in IP[3] and USP,[4] which describes liquid 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 9 (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, 5 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 min. 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 5 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 1. Fig. 1 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 1 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 2. Table 2 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%, which indicates that the proposed method is repeatable. The low % RSD values of interday (0.70-1.45 and 0.63-1.42%) and intraday (0.12-0.48 and 0.11-0.59%) for CEF and DCX, respectively, reveal that the proposed method is precise (Table 2). limit of detection (LOD) values for CEF and DCX were found to be 0.072 and 0.368 μg/ml, respectively, and limit of quantification (LOQ) values for CEF and DCX were found to be 0.22 and 1.11 μg/ml, respectively (Table 2). These data show that the proposed method is sensitive for the determination of CEF and DCX. Specificity of the method was confirmed from the resolution factor and peak purity data of the analyte. The resolution factor obtained for the CEF and DCX from the nearest resolving peak was >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 3. Fig. 2 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 3 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 4. Table 4 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. 2. Table 5 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., 5 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 0.11-0.59% 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 1. 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. 2. Sweetman SC. The Martindale: The Complete Drug Reference. 35th ed. London: Pharmaceutical Press; 2007. p. 207. 3. Indian Pharmacopeia. Vol. 3. New Delhi: The Controller Publication, Govt. of India; 2010. p. 1018. 4. USP28-NF23. Rockville, MD: United State Pharmacopeial Convention, Inc; 2005. The United State Pharmacopeia; p. 397. 5. 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. 6. 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. 7. Gandhi SV, Patil UP, Patil NG. Simultaneous spectrophotometric determination of cefpodoxime proxetil and potassium clavulanate. Hindustan Antibiot Bull. 2009;51:24–8. [PubMed] 8. 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. 9. Sweetman SC, editor. The Martindale: The Complete Drug Reference. 35th ed. London: Pharmaceutical Press; 2007. p. 237. 10. Indian Pharmacopeia. Vol. 3. 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 Reversed-phase 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; 5 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 min. 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. 1 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; 5 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 min and diluted to 100 ml with methanol. The resulting solution was centrifuged at 3000 rpm for 5 min. 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 3 consecutive days. Three different concentrations of ASP, ATO and CLO were analyzed in six independent series in the same day (intra-day precision) and 3 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 5 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 3 and 4. 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 min. 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<k<10) for eluted peaks, assay sensitivity, solvent noise were also considered. Finally a mobile phase consisting of a mixture of acetonitrile: phosphate buffer pH 3.0 adjusted with o-phosphoric acid in ratio 50:50 (v/v), was selected as mobile phase to achieve maximum separation and sensitivity. Flow rates between 0.8 to 1.4 ml/min were studied. A flow rate of 1.2 ml/min gave an optimal signal to noise ratio with a reasonable separation time. Using a reversed phase C18 column, the retention times for ASP, ATO and CLO were observed to be 1.89, 6.6, 19.8 min, respectively. Total time of analysis was less than 20 min. The chromatogram at 235 nm showed a complete resolution of all peaks (fig. 2). Fig. 2 Representative chromatograms of standard solutions (a) Standard solution of ASP (75 μg/ml); (b) standard solution of ATO (10 μg/ml); (c) standard solution of CLO (75 μg/ml) and (d) a standard solution containing 75 μg/ml ... Validity of the analytical procedure as well as the resolution between different peaks of interest is ensured by the system suitability test. All critical parameters tested met the acceptance criteria on all days. As shown in the chromatogram, all three analytes are eluted by forming symmetrical single peaks well separated from the solvent front (fig. 2). Excellent linearity was obtained for all the three drugs in the range of 30-105 μg/ml for ASP and ATO and 5-30 μg/ml CLO. The correlation coefficients (r2) were found to be greater than 0.999 (n=6) in all instances. The results of calibration studies are summarized in Table 1. The proposed method afforded high recoveries for ASP, ATO and CLO capsules. Results obtained from recovery studies presented in Table 2, indicate that this assay procedure can be used for routine quality control analysis of this ternary mixture in capsules. Precision of the analytical method was found to be reliable based on % RSD (< 2%) corresponding to the peak areas and retention times. As can be seen in Table 3, the % RSD values were less than 2, for intra-day and inter-day precision. Hence, the method was found to be precise for all the three drugs. TABLE 1 LINEARITY PARAMETERS FOR THE SIMULTANEOUS ESTIMATION OF ASP, ATO AND CLO (N=6) TABLE 2 RESULTS OF ACCURACY STUDIES OF ASP, ATO AND CLO TABLE 3 RESULTS OF INTRA-DAY PRECISION AND INTER-DAY PRECISION FOR SIMULTANEOUS DETERMINATION OF ASP, ATO AND CLO STANDARDS The chromatograms were checked for the appearance of any extra peaks. It was observed that single peak for ASP (Rt±SD, 1.89±0.01), ATO (Rt±SD, 6.6±0.01) and CLO (Rt±SD, 19.8±0.01) were obtained under optimized conditions, showing no interference from common capsule excipients and impurities. Also the peak areas were compared with the standard and % purity calculated was found to be within the limits. These results demonstrate the specificity of the method (fig. 3). Fig. 3 Representative chromatogram obtained for marketic formulations. Representative chromatogram obtained for Ecosprin Gold-10 and Deplatt CV capsule formulations LOD and LOQ were found to be 1.8 μg/ml and 5.5 μg/ml for ASP, 0.0959 μg/ml and 0.290 μg/ml for ATO and 0.66 μg/ml and 1.89 μg/ml for CLO. In all deliberately varied conditions, the SD of retention times of ASP, ATO and CLO were found to be well within the acceptable limit. The tailing factor for all the three peaks was found to be < 1.5 (Table 4). The validated method was used in the analysis of marketed conventional capsules Ecosprin Gold-10 and Deplatt-CV with a label claim: 75 mg ASP, 10 mg ATO and 75 mg CLO per capsule. Representative chromatogram is shown in (fig. 3). The results for the drugs assay show a good agreement with the label claims (Table 5). TABLE 4 RESULTS OF ROBUSTNESS STUDY TABLE 5 RESULTS OF ASSAY IN COMMERCIAL SAMPLES The developed HPLC method is simple, specific, accurate and precise for the simultaneous determination of ASP, ATO and CLO from capsules. The developed method provides good resolution between ASP, ATO and CLO. It was successfully validated in terms of system suitability, linearity, range, precision, accuracy, specificity, LOD, LOQ and robustness in accordance with ICH guidelines. Thus, the described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs either as such or in combination. Go to: ACKNOWLEDGEMENTS The authors are thankful to Torrent Pharmaceuticals, Gujarat, for providing aspirin, atorvastatin calcium and clopidogrel bisulphate as gift samples for this work; Prof. M. N. Navale, Founder President and Dr. (Mrs.) S. M. Navale, Founder Secretary, Sinhgad Technical Education Society, Vadgaon (Bk), Pune, for providing required facilities for research work. Go to: Footnotes Londhe, et al.: RP-HPLC Method for Aspirin, Atorvastatin Calcium and Clopidogrel Bisulphate Go to: REFERENCES 1. 24th ed. Rockville, MD: Pharmacopeial Convention; 2000. United States Pharmacopoeia/National Formulary; p. 161. 2. Vol. 1. London: HMSO Publication; 2007. British Pharmacopoeia; p. 184. 3. Vol. 1. Ghaziabad: The Indian Pharmacopoeia Commission; 2007. The Indian Pharmacopoeia; p. 745. 4. Saraf S, Saraf S, Garg G. Spectrophotometric determination of the aspirin and atenolol in combined dosage forms. Indian J Pharm Educ Res. 2008;42:74–7. 5. Luo G, Lan Q, Wang Z, Zhou G. 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Manoj K, Shanmugapandiyan P, Anbazhagan S. RP-HPLC method for simultaneous estimation of atorvastatin and aspirin from capsule formulations. Indian Drugs. 2004;41:284–9. 16. Rajeswari K, Sankar G, Seshagirirao J. RP-HPLC method for the simultaneous determination of Atorvastatin and Amlodipine in tablet dosage form. Indian J Pharm Sci. 2006;68:275–6. 17. Havaldar F, Chaudhari V. Simultaneous determination of atorvastatin calcium and amlodipine besylate from pharmaceutical formulation by reversed phase HPLC. Asian J Chem. 2006;17:2502–8. 18. Yadav S, Mhaske D, Kakad A, Dhaneshwar S. Simple and sensitive HPTLC method for the determination of content uniformity of atorvastatin calcium tablets. Indian J Pharm Sci. 2005;67:182–7. 19. Gomez Y, Adams E. Analysis of purity in 19 drug product tablets containing clopidogrel: 18 copies versus the original brand. J Pharm Biomed Anal. 2004;34:341–8. [PubMed] 20. Anandakumar K, Ayyappan T, Raman V, Vetrichelvan T, Sankar A, Nagavalli D. 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