LIQUID CHROMATOGRAPHIC SEPARATION AND ANALYSIS OF TELMISARTAN AND HYDROCHLOROTHIAZIDE IN PHARMACEUTICAL FORMULATIONS

LIQUID CHROMATOGRAPHIC SEPARATION AND ANALYSIS OF TELMISARTAN AND HYDROCHLOROTHIAZIDE IN PHARMACEUTICAL FORMULATIONS

Krishna Veni  P.R. ¹, Jyothi K. Kasthuri ¹, A. Vasu Babu ¹, K. Vijay ³, G.V. Raman ², I.E. Chakravarthy ⁴, B. Hari Babu*¹

Department of Chemistry, Acharya Nagarjuna University ¹, Nagarjunanagar, Guntur-522510, Andhra Pradesh, India

Department of Chemistry, Andhra Loyola College ², Vijayawada-520008, Andhra Pradesh, India

Department of Pharmaceutical Sciences, Acharya Nagarjuna University ³, Nagarjunanagar, Guntur-522510, Andhra Pradesh, India

Department of Chemistry, Rayalaseema University ⁴, Karnool, Andhra Pradesh, India

ABSTRACT: An accurate, simple and precise RP-HPLC method for the simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical formulations was developed and validated. Chromatographic separation of two drugs was achieved on a Kromasil C18, (250x4.6nm, 5µm) column by using the mobile phase consisted of methanol, tetrahydrofuran and water in the ratio 55:05:40 at a flow rate of 1mL/min and the wavelength of detection was at   234 nm. The retention time for telmisartan and hydrochlorothiazide were found to be 2.3 and 3.8 min respectively. The linearity of the developed method was tested over a concentration range of 50-100 µg/mL and the correlation coefficient (r²) of regression was 0.999 which is almost equal to 1. The limit of quantification was 0.5µg/mL for telmisartan and 0.4µg/mL for hydrochlorothiazide and the limit of detection was 0.1µg/mL for both telmisartan and hydrochlorothiazide. The mean percentage recoveries were 99.2% for telmisartan and 99.8% for hydrochlorothiazide respectively.

Telmisartan, Hydrochlorothiazide, RP-HPLC, Simultaneous estimation

INTRODUCTION:  Telmisartan (TEL), (fig1) a non-peptide molecule and its IUPAC name is 4^׳-[(1,4^׳-dimethyl-2^׳-propyl [2,6^׳-bi-1H-benzimidazol]-1^׳-yl) methyl] [1,1^׳-biphenyl]-2-carboxylic acid ¹. It is an angiotensin II, type I blocker and is used as an antihypertensive along with hydrochlorithiazide.

It is a thiazide diuretic which reduces the reabsorption of electrolytes from the renal tubules, thereby increasing the excretion of sodium and chloride ions and consequently water.

Hydrochlorothiazide (HCTZ), (fig. 1) is chemically 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-sulfonamide-1, 1-dioxide.

The combination is useful in the treatment of mild to moderate hypertension, well tolerated with a lower incidence of cough than ACE inhibitors ².

FIG.1: TELMISARTAN & HYDROCHLOROTHIAZIDE

A   literature survey revealed that there are some liquid chromatographic ^3-9 methods reported   for the determination of simultaneous estimation of telmisartan and hydrochlorothiazide in pharmaceutical dosage forms. In the present work an attempt was made to develop a simple, precise and accurate RP-HPLC method for the simultaneous estimation of telmisartan and hydrochlorothiazide in pharmaceutical dosage forms.

MATERIALS AND METHODS:

Chemicals and reagents: THF, methanol and water of HPLC grade were produced from Merck specialties Pvt. Ltd. Mumbai. A working standard of telmisartan and hydrochlorothiazide was provided by V.V.Med laboratories, Hyderabad.

Equipment: Analysis of the samples was carried out using PEAK 7000 isocratic HPLC with rheodyne manual sample injector with switch (77251) and the column used was analytical column Kromasil 100-5 C18(250X4.6nm). Electronic balance; ELB 300, DIGISUN for pHmeasurements.

Chromatographic conditions: Kromasil C18 (250X4.6mm) column was used for the separation; a mobile phase of methanol, THF and water in the ratio 55:05:40 was filtered through 0.45µm membrane filter paper and delivered at a flow rate of 1mL/min and the wavelength of detection was at 234 nm. The injection volume was 20µL, and the total run time 8min. The resulting chromatogram was shown in Fig. 2.

FIG. 2: STANDARD CHROMATOGRAM OF TELMISARTAN (t_R 2.3min) AND HYDROCHLOROTHIAZIDE (t_R 3.8min)

Preparation of standard solutions: Standard stock solutions of 1.0 mg/mL telmisartan and hydrochlorothiazide were prepared separately using methanol as solvent. In order to get the required ratio (3.2:1) of both the drugs telmisartan and hydrochlorothiazide appropriate quantities of respective solutions of each drug were mixed and diluted with the mobile phase.

Preparation of sample solution: Twenty tablets were weighed and finely powdered. A quantity of powder equivalent to 40 mg of  telmisartan and 12.5 mg of hydrochlorothiazide were weighed and transferred to a 50 mL volumetric flask; methanol was added to the same flask and shaken for some time and the volume was made up to the mark. The solution was filtered using Whatman filter paper. From the filtrate appropriate dilution was done to get a solution of 80 µg/mL of telmisartan and 50 µg/mL of hydrochlorothazide.

System suitability studies: The resolution, numbers of theoretical plates were calculated for the working standard solutions and are shown in table 1. The values obtained demonstrated the suitability of the system for the analysis of these drugs in combination.

TABLE 1: SYSTEM SUITABILITY STUDIES

Method development: Different mobile phases containing of methanol, THF and water in different proportions were tried, and finally, methanol: THF: H₂O in the ratio 55:05:40 was found to be an appropriate mobile phase, which gave acceptable peak parameters for both telmisartan and hydrochlorothiazide.

Method validation: The validation of the method was carried out in terms of parameters like linearity, precision, accuracy, limit of detection, limit of quantification, specificity and robustness.

Specificity: The Specificity of the RP-HPLC method was determined by the complete separation of telmisartan and hydrochlorothiazide as shown in fig. 2 with parameters like retention time (R_t= 2.3 min for TEL and R_t=3.8 min for HCTZ) and tailing factor (0.07 for TEL and 1.92 for HCTZ). Moreover, the clear baseline separation of the drugs and sharp peaks also supports the specificity of the method.

Linearity: The linearity of the method was tested over a concentration range of 50 to 100µg/mL of TEL and HCTZ. 20µL of each concentration was injected in duplicate into the HPLC system.

The response was read at 234nm and the corresponding chromatograms were recorded. The regression value of the plots was computed by least square regression method. Peak area (A) and concentration (c) of each drug substance was subjected to regression analysis to calculate the correlation coefficients. A correlation coefficient r²=0.999 for both TEL and HCTZ of the regression was found to be almost equal to 1. Linearity results were presented in Table 2 and graph in Fig. 3.

FIG. 3: LINEARITY OF TELMISARTAN AND HYDROCHLOROTHIAZIDE

TABLE 2: RESULTS OF LINEARITY

Precision: The precision of the method was determined by inter day and intraday studies, solutions of standard and sample were repeated thrice in a day and percentage relative standard deviation (%RSD) was calculated. Results of system precision studies were shown in Table 3 and 4. The %RSD found were less than 1%.

The intraday %RSD of telmisartan and hydrochlorothiazide were found to be 0.135% and 0.44%, respectively. The inter day %RSD of telmisartan and hydrochlorothiazide were found to be 0.9% and 0.51% respectively. The values of %RSD within a day, day to day variation <1% proves that the method is precise.

TABLE 3: RESULTS OF PRECISION STUDY (INTRA DAY)

 TABLE 4: RESULTS OF PRECISION STUDY (INTER DAY)

 Accuracy: The accuracy of the method was determined by recovery experiments. The recovery studies were performed by the regular addition method. At 50%, 75%, 100% level, the percentage recovery was calculated and presented in table 5. Recovery was found to be within the range of 99.2% to 99.8%, which indicates the accuracy of the method. For both the drugs, recovery was performed in the same way. The recovery studies were performed in triplicate. The results of recover studies were found to be satisfactory.

TABLE 5:  ACCURACY DATA OF THE DEVELOPED METHOD

 Robustness: Robustness of the method was studied by making slight changes in chromatographic conditions, such as mobile phase ratio, pH and wavelength of detection (table 6). It was observed that there were no marked changes in the chromatograms obtained. So the developed RP-HPLC method was robust.

TABLE 6:   RESULTS OF ROBUSTNESS

 Limit of Detection and Limit of Quantification: The limit of detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula:

LOD = 3.3 X Standard Deviation

Slope of calibration curve

The LOD for telmisartan and hydrochlorothiazide was found to be 0.1ppm for both the drugs (Table 1).

The limit of quantification (LOQ) is the smallest concentration of the analyte, which gives a response that can be accurately quantified. LOQ was calculated using the formula:

LOQ = 10 X Standard Deviation

Slope of calibration curve

The LOQ was 0.5ppm and 0.4ppm for telmisartan and hydrochlorothiazide, respectively (Table 1).

RESULTS AND DISCUSSION: The method was developed using the mobile phase MeOH: THF: H₂O in the ratio 55:05:40, and the standard chromatogram obtained showed the elution of telmisartan at t_R =2.3 min, tailing factor 0.07 and elution of hydrochlorothiazide at t_R =3.8 min, tailing factor 1.92. The separation of two drugs for the selected mobile phase is much prominent. Moreover, the order of elution of the two drugs is in good agreement with the earlier reported method ⁴, which was developed, with the mobile phase MeOH: CH₃CN in the ratio 70:30.

However these two methods in respect of order of elution of drugs were contradictory to other methods [3, 5, 6, 7, and 8] which were developed using 1% ortho phosphoric acid as one of the solvents in the mobile phases with either methanol or acetonitrile. This order of elution of telmisartan (at t_R =2.3 min) and hydrochlorothiazide (at t_R =3.8 min) may be because of greater affinity of telmisartan towards the mobile phase compared to hydrochlorothiazide i.e. the change is determined by the polarity of the mobile phase selected in the present study.

Moreover, the LOD and LOQ for telmisartan were found to be 0.1µg/mL and 0.5µg/mL, respectively, and for hydrochloro-thiazide the values are 0.1µg/mL and 0.4µg/mL, respectively (Table 1).A system suitability test was applied to representative chromatograms for various parameters. A graph was constructed covering a concentration range 50 to 100ppm. The calibration curve was obtained for a series of concentrations in the range of 50 to 100ppm and it was found to be linear. The data of regression analysis of the calibration curves are shown in table 2. The method was found to be accurate and precise, as indicated by the recovery studies and %RSD calculated is less than 1.

CONCLUSION: The developed RP-HPLC method for the simultaneous estimation of telmisartan and hydrochlorothiazide in combined dosage form is more precise, robust, and accurate in respect of validation and the quality of separation achieved of the two drugs is also good in terms of retention times and tailing factor.

Hence, the proposed method is applicable to the determination of telmisartan and hydrochloro-thiazide in pharmaceutical formulations.

ACKNOWLEDGEMENTS: The authors are Thankful to Acharya Nagarjuna University for constant encouragement and RV Labs, Guntur for providing instrumental support.

REFERENCES:

1. Merck index, (2001).13^th ed. Merck & Co.,Inc., white house station, NJ.
2. Sharpe M., Jarvis B. and Karen L.: Telmisartan a review of its use in hypertension. Drugs,  2001; 61(10): 1501-29.
3. Rane V.P., Sangshetti J.N. and Shinde D.B.: Simultaneous High-Performance Liquid Chromatographic determination of Telmisartan and Hydrochlorothiazide in pharmaceutical preparation. J.chromatogr.sci, 2008; 46: 887-891.
4. Bhat R. Leena., Rahul K.Godge., Asfak T. Vora and Mrinalini C.Damle: Validated RP-HPLC method for simultaneous determination of Telmisartan and    Hydrochlorothiazide in pharmaceutical formulation. J.liq.chromatogr. &related technologies, 2007; 30: 3059-3067.
5. Anand Kumar R. Tengli., Gurupadayya B.M.Neeraj Soni and Chandan R.S.: RP-HPLC method for simultaneous estimation of Hydrochlorothiazide, Ramipril and Telmisartan in tablet dosage form. J.pharmacy research, 2012; 5(3):1290-1293.
6. Wankhde S.B., Tajne M.R., Gupta K.R. and Wadodkar S.G.: RP-HPLC method for the simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablet dosage form. IndianJ.Pharm.Sci, 2007; 69(2): 298-300.
7. Sulirtho M., Kiran K., Laxman S., Dhanashree N. and Nancy P.: Simultaneous determination of related substances of Telmisartan and Hydrochlorothiazide in tablet dosage form by using reversed phase high performance liquid chromatographic method. J.Pharm Bioallied Sci, 2011; 3(3): 375-383.
8. Gopala Swamy T., Nagraju K. and Lakshmana Rao A.: RP-HPLC method for the simultaneous estimation of Telmisartan and Hydrochlorothiazide in pharmaceutical dosage form. Int.J.Drug Dev. & Res., 2011; 3(4): 362-368.
9. Shah N.J., Suhagia B.N., Shah R.R. and Shah P.B.: Development and validation of HPTLC method for the simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablet dosage form. IndianJ.Pharm.Sci, 2007; 69(2): 202-205.
   

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   How to cite this article:

   Krishna VPR, Kasthuri JK, Babu AV, Vijay K, Raman GV, Chakravarthy IE, Babu BH: Liquid chromatographic separation and analysis of Telmisartan and Hydrochlorothiazide in Pharmaceutical Formulations. Int J Pharm Sci Res 2013: 4(9); 3630-3634. doi: 10.13040/IJPSR. 0975-8232.4(9).3630-34

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