Simultaneous Estimation of Amlodipine and Chlorthalidone in Combined Dosage Form: Method Development and Validation

The problem of hypertension is among the most important public health issues and the main factor contributing to the development of cardiovascular diseases. High blood pressure can lead to such dangerous pathologies as stroke, myocardial infarction, heart failure, renal disease, and other problems associated with the vascular system. Since hypertension involves various factors, monotherapy is sometimes ineffective in obtaining therapeutic results. Thus, using combination therapy that uses antihypertensive drugs with complementary mechanisms of action has proved to be an efficient approach. [1]

Amlodipine (AML) is a long-lasting calcium channel antagonist belonging to the dihydropyridines. This drug interferes with the movement of Ca2+ ions across the membrane into the cell, specifically L-type calcium channels in the vascular smooth muscle and. Chlorthalidone (CHL) belongs to the thiazides and acts upon the distal convoluted tubules of the nephron. This increases excretion of sodium and chloride in urine. Water excretion increases secondary to sodium loss. It reduces plasma volume and extracellular fluid volume. Long-term use decreases peripheral vascular resistance, thereby lowering blood pressure. Their combination provides a synergistic antihypertensive effect and widely used in the treatment of hypertension. [2,3]

Various techniques like UV spectrophotometric analysis and RP-HPLC analysis have been documented for quantitation of these medicines either individually or along with other medicines. But some of these methodologies are found lacking in certain aspects such as sensitivity, specificity, separation of degradation products, complex composition of mobile phase, long running time, large consumption of solvents, and incomplete validation process. Moreover, some methodologies were devised for multiple drugs rather than a binary drug mixture of Amlodipine (AML) and Chlorthalidone (CHL). [4,5,6]

Fig.1. Structure of Amlodipine [7]

Fig.2. Structure of Chlorthalidone [7]

MATERIAL AND METHOD

The analysis was carried out on a Shimadzu LC-2010CHT HPLC system with LC Solution software for data acquisition and processing. All sample solutions were produced using a sonicator (Life Care Instruments Pvt. Ltd.) to guarantee full analyte dissolution. The WebMan analytical balance was used to accurately weigh standards and samples. To obtain efficient separation and consistent results, the entire system was run under chromatographic settings that were optimized. For selection of wavelength UV-VIS Spectrophotometry of LABMAN LMSP-UV 100B system was used.

Chemicals and reagents

The Amlodipine and Chlorthalidone’s working standard were provided by Arni Analytical, Nashik. The marketed formulation i.e. Amlodac 17.5 mg was procured from local medical store. The reagents and standards used in the study included potassium dihydrogen orthophosphate (AR grade), methanol, acetonitrile and water (HPLC grade), all procured from Fisher Scientific.

Selection of analytical wavelength:

Individual stock solutions for Amlodipine and Chlorthalidone were prepared separately. 10 mg of Amlodipine and Chlorthalidone were individually measured and dissolved in 100 mL volumetric flasks using methanol. Methanol up to 70 mL was added to the flasks containing the drugs and sonicated for 5 to 10 minutes until the substances were completely dissolved. The volumetric flasks were filled up with methanol, creating 100 µg/mL individual stock solutions for both Amlodipine and Chlorthalidone. By using methanol as blank the baseline was stabilized. The solution run in between 200- 400 nm and λmax found at 242 nm. Therefor, 242 nm is the selected wavelength for further analysis.

Chromatographic conditions:

 

 

Fig.3. UV Spectra of Amlodipine and Chlorthalidone

 

 

Fig.4. Optimized method chromatogram

Table.2. System suitability parameters of specificity

Sr. No.	Parameters	AML	CHL
1	Retention Time	3.000 min	5.883 min
2	Theoretical plates	5109.848	5705.414
3	Tailing factor	1.552	1.553

 

 

 

Fig.5. Chromatogram of specificity Standard

 

 

Fig.6. Linearity of Amlodipine

 

 

Fig.7. Linearity of Chlorthalidone

CONCLUSION

All the above parameters are achieved, analysed, and then compared with the acceptance criteria for each one of them. Based on the above data and analysis, it is confirmed that the above validation technique is ideal for use in estimating the amounts of Amlodipine and Chlorthalidone in tablets. The method has been successfully validated using the RPHPLC method according to the ICH guidelines. The technique showed strong specificity and no influence from blank solutions. Precision studies showed %RSD values within acceptable limits, indicating high reproducibility. The accuracy values were between 98 and 102%, which confirmed the validity of the technique. The robustness and solution stability tests provided additional evidence that the technique was consistent and stable.

CONFLICT OF INTEREST

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS

The authors express their sincere gratitude to Sandip Institute of Pharmaceutical Sciences, for providing the necessary facilities and a supportive environment to carry out this research work on the development and validation of an RP-HPLC method for the simultaneous estimation of Amlodipine and Chlorthalidone. The authors are thankful to Arni Analytical Laboratory for providing the required laboratory facilities and technical support during the experimental studies.

REFERENCES

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