Validated RP-HPLC Method for the Estimation of Amiloride and Hydrochlorothiazide in Combined Tablet Dosage Form

R. Anantha Kumar¹*, G. Raveendra Babu², M. Sowjanya³, M. Ramayyappa²

¹Nova College of Pharmacy, Jangareddy Gudem, Andhra Pradesh, India.

²A.K.R.G. College of Pharmacy, Nallajerla, Andhra Pradesh, India.

³Vijaya Teja Degree College, Addanki, Andhra Pradesh, India.

*Corresponding Author E-mail: ananth2.pharma@gmail.com

ABSTRACT:

The aim of this work is to build up a rapid, exact, precise and accurate reverse phase liquid chromatographic method for the simultaneous analysis of amiloride and hydrochlorothiazide in tablet dose structure. The chromatographic strategy was normalized utilizing Hypersil ODS coulmn (250×4.6mm, 5μm molecule size) with UV detection at 210nm and flow rate of 1ml/min. The mobile phase includes phosphate buffer (pH acclimated to 2.5 with dilute Ortho Phosphoric acid) and acetonitrile in the proportion of 60:40 v/v. The linearity of proposed technique was found in the range of 5-30μg/ml (R²=0.999) for amiloride and 50-300μg/ml (R²=0.999) for Hydrochlorothiazide appropriately. The limit of detection (LOD) was discovered to be 0.10μg/ml and 0.40μg/ml for Amiloride and Hydrochlorothiazide appropriately. The limit of quantitation (LOQ) was discovered to be 0.30μg/ml and 1.20μg/ml for Amiloride and Hydrochlorothiazide separately. The retention times of Amiloride and Hydrochlorothiazide were found to be 3.258min and 2.383min separately. The technique was truly recommended and %RSD was found to be under 2 demonstrating high degree of exactness and accuracy. Subsequently proposed strategy can be effectively evaluated for the simultaneous estimation of Amiloride and Hydrochlorothiazide in promoted formulations.

KEYWORDS: Amiloride, Hydrochlorothiazide, RP-HPLC, ICH guidelines, Validation.

INTRODUCTION:

Amlodipine is synthetically 3-O-ethyl 5-O-methyl 2-(2-aminoethoxymethyl)- 4-(2-chlorophenyl)- 6-methyl-1,4-dihydropyridine-3,5-dicarboxylate and is a well known antihypertensive medication having a place with the gathering of medications called dihydropyridine calcium channel blockers¹.

Because of their selectivity for the fringe veins, dihydropyridine calcium channel blockers are related with a lower occurrence of myocardial sadness and cardiovascular conduction variations from the norm than other calcium channel blockers. It is ordinarily utilized in the treatment of hypertension and angina. Amlodipine has cancer prevention agent properties and a capacity to improve the creation of nitric oxide (NO), a significant vasodilator that diminishes blood pressure^2,3.

Hydrochlorothiazide is a benzothiadiazine that is 3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide subbed by a chloro bunch at position 6 and a sulfonamide at 7. It is diuretic utilized for the treatment of hypertension and congestive heart failure⁴. It has a part as a xenobiotic, an ecological toxin, a diuretic and an antihypertensive specialist. It is a benzothiadiazine, a sulfonamide and an organochlorine compound. Hydrochlorothiazide is the most usually recommended thiazide diuretic. It is shown to treat edema and hypertension. Hydrochlorothiazide use is normal yet declining for angiotensin changing over catalyst inhibitors. Numerous mix items are accessible containing hydrochlorothiazide and angiotensin changing over protein inhibitors or angiotensin II receptor blockers⁵.

The blend of Amlodipine and hydrochlorothiazide goes under the calcium-channel blocker and potassium-losing diuretic, is shown for the treatment and the executives of hypertension. The medications improve blood stream by dilatation of blood vessels⁵.

Fig. 1: Chemical structure of Amiloride

Fig. 2: Chemical structure of Hydrochlorothiazide

Literature survey reveals few analytical methods for the determination of Amlodipine alone and in combination with other drugs in pharmaceutical preparations and biological fluids, viz. Spectrophotometry^6-9, HPTLC^10-14 and HPLC^15-18. There are few analytical methods for the determination of hydrochlorothiazide alone or in its combination with other drugs in pharmaceutical preparations including spectrophotometry^19,20, TLC²¹, and HPLC^22-34. Hence, the developed method aims in separating the selected drugs simultaneously. The developed method was validated as per the ICH guideleines^35-37.

MATERIALS AND METHODS:

Chemicals and reagents:

APIs of Amiloride and Hydrochlorothiazide were obtained as a gift sample from Pharmatrain labs (Hyderabad, Andhra Pradesh). Acetonitrile and HPLC grade water were collected from Rankem lab ltd. Ortho Phosphoric acid AR grade has been purchased from E. Merck Chemicals Mumbai, India. All other reagents were AR grade. The industrial sample of BIDURET containing Amiloride 5mg and Hydrochlorothiazide 50mg is obtained from the local market.

Instrumentation:

The LC system comprises of Waters model 2695, UV-Visible detector. The output signals were monitored and integrated using Empower 2 software. Melter Electronic Balance together with Solomon pH meter were used.

Chromatographic conditions:

Chromatographic separation was performed on the hypersil ODS column (250×4.6mm, 5μm) using the mobile step consisting of ACN: buffer at a ratio of 40:60v/v. The pH buffer (0.02N potassium dihydrogen orthophosphate) is modified to 2.5 with dilute orthophosphoric acid and screened through a 0.45μm membrane filter. The column was held at room temperature (30^oC) and the flow rate was 1ml/min. Until the solution is injected, the column is balanced for at least 30 minutes, with the mobile process running through the device. The amount of the sample injected was 20μl. The UV-Visible detector was placed at a wavelength of 210nm. Under described experimental conditions, all the peaks were well defined and free from tailing. A typical chromatogram of Amiloride and Hydrochlorothiazide sample is shown in figure 3. The parameters are tabulated in table 1.

Table 1. Chromatographic Conditions

Mobile phase	ACN: Buffer (40:60%, v/v)
Diluent	Mobile phase
Column	Hypersil C18 (250 mm x 4.6 mm, 5 µm)
Column temp.	Ambient
Wave length	210 nm
Injection vol.	20 µl
Flow rate	1 ml/min.
Run time	10 min.
Retention times	2.383 min for HCT; 3.258 min for AML

Fig. 3: Chromatogram of Amiloride and Hydrochlorothiazide sample

Preparation of Standard solution:

5mg of Amiloride working standard and 50mg of Hydrochlorothiazide working standard were weighed and transferred to a 100ml volumetric flask to which 50 ml of diluent (mobile phase) was applied and dissolved. Then the solution was rendered up to the diluent point. The solution was further diluted to produce a concentration of 50μg/ml of Amiloride and 500μg/ml of Hydrochlorothiazide. Concentration solutions of 5-30μg/ml of Amiloride and 50-300μg/ml of Hydrochlorothiazide were prepared and linearity was calculated.

Preparation of Sample solution:

Approximately 20 tablets have been taken and their average weight is calculated. These pre-weighed 20 tablets are finely ground. Of the ground powder, a quantitatively comparable assay of 5mg of amiloride and 50mg of hydrochlorothiazide is moved to a 100ml volumetric flask and 50ml of diluent is added to dissolve for 10 minutes. Then the solution is rendered up to the diluent point. The solution is further routed into a 0.45μm membrane filter.10ml of the above-mentioned filtrate and diluted to 100ml with a diluent to produce a concentration of 20μg/ml of Amiloride and 200μg/ml of Hydrochlorothiazide.

Validation of the method:

The analytical method was validated as per ICH guidelines^7-9 with respect to parameters such as linearity, accuracy, precision, assay, ruggedness, robustness, limit of detection and limit of quantification as follows.

Linearity:

Linearity of this procedure was evaluated by linear regression analysis, calculated by least square method and considered by preparing standard solutions of Amiloride and Hydrochlorothiazide at different concentrations. The peak areas of Amiloride or Hydrochlorothiazide were plotted versus their respective concentrations. The response was found to be linear over the concentration range of 5-30μg/ml for Amiloride and 50-300μg/ml for Hydrochlorothiazide. The correlation coefficient (R²) for both Amiloride and Hydrochlorothiazide were estimated to be 0.999. The data is given in table2 and depicted in fig. 4 and 5.

Fig. 3: Linearity of Amiloride

Fig. 4: Linearity of Hydrochlorothiazide

Table 2: Linearity of amiloride and hydrochlorothiazide by RP-HPLC

S. No	Conc. Taken in mg/ml (AML)	Conc. Taken in mg/ml (HCT)	Peak area of AML	Peak area of HCT
1	5	50	239155	740511
2	10	100	476322	1484656
3	15	150	696810	2172994
4	20	200	952543	3005981
5	25	250	1186415	3704212
6	30	300	1409342	4406377

Accuracy:

Accuracy was conducted in triplicate with different amounts of Amiloride and Hydrochlorothiazide equal to 50 percent, 100 percent and 150 percent of the normal volume of amiloride and hydrochlorothiazide injected into the HPLC device as per test protocol. The overall percent recovery of Amiloride and Hydrochlorothiazide has been measured. The data was given in Table 3.

Table 3: Accuracy and % recovery of each analyte

Accuracy Level   %	Mean recovery of Amiloride (%)	Mean recovery of Hydrochlorothiazide (%)
50	99.74	99.62
100	100.10	100.15
150	99.34	100.80

Precision:

Method repeatability:

Six sample solutions of the same concentration were prepared and injected into the HPLC system as per test procedure.

Intermediate Precision:

Two analysts as per test procedure conducted the study. For Analyst-1 Method Repeatability and for Analyst-2 six sample solutions of same concentration were prepared and injected into the HPLC system as per test procedure. The results were given in table 4.

Table 4. Precision data for AMLandHCT

	AML			HCT
	Mean	Std. Dev.	%RSD	Mean	Std. Dev.	%RSD
Method precision	938379	12080.9	1.30	3015624	24832.0	0.80
Intermediate precision	945621	123512	1.21	312.521	25362.1	0.84

Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were performed on samples containing concentration of analytes, based on calibration curve method. Standard solution of Amiloride and Hydrochlorothiazide were injected in six replicates. Average peak area of six analytes was plotted against concentration.LOD and LOQ were calculated using the formula LOD=3.3 σ/S and LOQ=10 σ/S. The LOD and LOQ were ground in to be 0.05μg/ml, 0.81μg/ml and 0.15μg/ml, 2.44μg/ml for Amiloride and Hydrochlorothiazide respectively.

Table 5. LOD and LOQ of AML and HCT

Drug	LOD (µg/mL)	LOQ (µg/mL)
Amiloride	0.10	0.30
Hydrochlorothiazide	0.40	1.20

Assay:

The validated method was put in for the determination of Amiloride and Hydrochlorothiazide in commercially obtainable BIDURET tablets. The results of assay (n=3) undertaken yielded 98.99% (%RSD=0.07) of Amiloride and 100.79 % (%RSD= 0.18%) of Hydrochlorothiazide.

Robustness:

Robustness was done by small deliberate changes in chromatographic conditions and retention time of Amiloride and Hydrochlorothiazide was marked. The factors selected were flow rate and mobile phase. The results remained unaffected by small variation in these parameters as shown in table 6.

System Suitability:

System suitability and chromatographic measures were validated such as number of theoretical plates, asymmetry factor and tailing factor. The results are given in the table 7.

Table 6. Robustness Parameters of AML and HCT

Parameter	Amiloride			Hydrochlorothiazide
	Mean area	STDV	%RSD	Mean area	STDV	%RSD
Initial sample	936102	1837.3	0.88	3009472	9071.0	0.64
Flow (+0.1mL/min)	982188	1291.2	0.52	3015472	6644.1	0.32
Flow (-0.1mL/min)	934001	4347.9	0.91	3004642	1193.3	0.32
Org. Phase 10 % more	928735	6397.19	0.53	3017092	6356.2	0.98
Org. Phase 10 % less	921776	4357.19	0.33	3064022	2356.8	0.79

Table 7. System suitability parameters of AMLandHCT

Drug	Theoretical plates	Tailing factor	Retention time	Resolution
Amiloride	5768	1.20	3.258	---  8.32
Hydrochlorothiazide	7780	0.90	2.383

CONCLUSION:

The proposed method was found to be linear over the concentration range of 5-30µg/ml and 50-300µg/ml for Amiloride and Hydrochlorothiazide respectively. System suitability parameters indicate good resolution for both the peaks >2. The technique was found to be precise and exactnesst as shown by the consequences of ruggedness studies and accuracy considers whose %RSD isn't over 2%. There were no checked changes in the chromatograms which submitted the robustness of the technique. The standard deviation of %assay for test was determined, for every boundary in power considers the general standard deviation was ground in under 2%. The low %RSD esteem affirms the strength of technique. The recommended strategy was ground in to be quick, exact, precise and sensitive. It utilizes lesser measures of solvents and has more limited maintenance times than enduring techniques. Numerous examples can be appropriately examined by this technique. Hence developed method can be used for normal analysis of Amiloride and Hydrochlorothiazide in tablet dosage form.

Table 8. Summary of system suitability and validation parameters of AML and HCT

Parameter	Results
	AML	HCT and PIP
Linearity range (μg/mL)	5-30	50-300
Correlation coefficient	0.999	0.999
Theoretical plates (N)	5768	7780
Tailing factor	1.09	1.29
LOD (μg/mL)	0.10	0.40
LOQ (μg/mL)	0.30	1.20

ACKNOWLEGEMENT:

The authors are thankful to Pharmatrain (Hyderabad, Andhra Pradesh) for providing API of Amiloride and Hydrochlorothiazide.

REFERENCES:

1. The Merck Index, 23^rd ed. Whitehouse Station, New Jersey; 2008. p. 516, 6235.

2. Martindale, Sweetman SC. The Complete Drug Reference. 32^nd ed. Pharmaceutical Press; 1999. p. 822, 907.

3. Oneil MJ, Smith A, Heckelman PE, Budawari S. The Merck Index, an Encyclopedia of Chemicals, Drugs and Biologicals, 13^rd ed., Merck and Co Inc., White House Station, New jersey; 2001. p. 488, 865.

4. British Pharmacopoeia, Vol. 1, The Stationery Office, London, UK, 2007.

5. P. Pickkers, R. S. Garcha, M. Schachter, P. Smits, and A. D. Hughes. Inhibition of carbonic anhydrase accounts for the direct vascular effects of hydrochlorothiazide.  Hypertension. vol. 33, no. 4, pp. 1043–1048, 1999.

6. Chandan Kumar Giri, Kondawar MS, Chougule DD. Simultaneous estimation of Nebivolol hydrochloride and Amlodipine besylate in combined tablet dosage form by Q-analysis method. IJPRD/PUB/ARTI/VOV-2/ISSUE-5/JULY/013.

7. Bhatia NM, Mohite AS, Bhatia MS. Simultaneous spectrophotometric estimation of atenolol and amlodipine besylate by multiwavelength method, The Indian Pharmacist 2007; 6: 59-62.

8. Chandrashekhar TG, Rao PSN, Smrita K. Analysis of amlodipine besylate by HPTLC with fluorimetric detection: a sensitive method for assay of tablets. J. Planar. Chromatogr. Mod. TLC 1994; 7: 458.

9. Pandya KK, Satia M, Gandhi TP. Detection and determination of total amlodipine by high performance thin layer chromatography: A useful technique for pharmacokinetic studies. J Chromatogr B Biomed Appl 1995; 667(2): 315-20.

10. Alaa El Gindy, Ahmed Ashour, Laila Abdel Fattah. Application of LC and HPTLC Densitometry for the Simultaneous determination of Benazepril Hydrochloride and Hydrochlorothiazide, J Pharm Biomed Anal 2001; 25(2): 171-9.

11. Ilango K, Kumar PB, Prasad VRV. Simple and rapid high performance thin layer chromatographic determination of amlodipine in pharmaceutical dosage forms. Indian J. Pharm. Sci 1997; 59: 336.

12. Shah NJ, Suhagia BN, Shah RR, Shah PB. Development and validation of HPTLC method for Simultaneous estimation of Telmisartan and Hydrochlorothiazide in tablet dosage form, Indian J of Pharm Sci 2007; 69: 202-5.

13. Carlucci G, Palumbo G, Mazzeo P, Quaglia MG. Simultaneous determination of losartan and hydrochlorothiazide in tablets by high performance liquid chromatography. J Pharm Biomed Anal 2000; 23(1): 185-9.

14. Patel Gaurav, Patel Sanjay et al. RP-HPLC Method for Simultaneous Estimation of Amlodipine Besylate and Hydrochlorothiazide in Combined Dosage Forms. S. J. Pharm. Sci 2010; 3(1): 49-53.

15. Kartal M, Erk N. Simultaneous determination of Hydrochlorothiazide and Amiloride Hydrochloride by Ratio Spectra Derivative Spectrophotometry and High-performance Liquid Chromatography. J Pharm Biomed Anal 1999; 19(3-4): 477-85.

16. Safeer K, Anbarasi B, Senthil Kumar N. Analytical Method Development and Validation of Amlodipine and Hydrochlorothiazide in combined dosage form by RP-HPLC. Int. J. Chem Tech Res 2010; 2(1): 21-25.

17. M. A. Gotardo, L. Pezza, and H. R. Pezza. Determination of hydrochlorothiazide in pharmaceutical formulations by diffuse reflectance spectroscopy. Ecletica Qu ´ ´ımica, vol. 30, no. 2, pp. 17–24, 2005.

18. S. A. Hapse, V. S. Wagh, P. T. Kadaskar, M. D. Dokhe, and A. S. Shirsath. Spectrophotometric estimation and validation of hydrochlorothiazide in tablet dosage forms by using different solvents. Der Pharma Chemica. vol. 4, no. 1, pp. 10–14, 2012.

19. A. M. Idris and R. E. E. Elgorashe. Sequential injection chromatography with a miniaturized multi-channel fiber optic detector for separation and quantification of propranolol and hydrochlorothiazide.  Chemistry Central Journal. vol. 5, article 28, pp. 1–8, 2011.

20. P. N. Bhoya, E. M. Patelia, and Gautambhai. Development and validation of TLC-densitometry method for simultaneous estimation of Bisoprolol fumarate and hydrochlorothiazide in bulk and tablets. Journal of Chromatography and Separation Techniques. vol. 4, no. 1, article 163, 4 pages, 2013.

21. S. Gayathri, D. Sireesha, M. A. Haque, S. Harshini, V. Bhakshi, and S. K. Reddy. Method development and validation of RP-HPLC method for simultaneous estimation of Olmesartan medoxomil and Hydrochlorothiazide in bulk and pharmaceutical dosage form. International Journal of Pharma Research and Health Sciences. vol. 2, no. 6, pp. 457–462, 2014.

22. M. Mahesh, R. Kumanan, and K. N. Jayaveera. Isocratic RPHPLC UV Method development and validation for the simultaneous estimation of hydrochlorothiazide and Ramipril in tablet dosage form and bulk durg. International Journal of Current Pharmaceutical Research. vol. 3, no. 2, pp. 119–123, 2011.

23. Z. Vujic, N. Mulavdi ´ C, M. Smaji ´ C, J. Brbori ´ C, and P. Stankovic. Simultaneous analysis of irbesartan and hydrochlorothiazide: an improved HPLC method with the aid of a chemometric protocol.  Molecules. vol. 17, no. 3, pp. 3461–3474, 2012.

24. B. V. Savaj, H. A. Raj, S. Rajanit, and S. Harshita. Analytical techniques for determination of hydrochlorothiazide and its combinations: a review. International Journal of Advances in Scientific Research. vol. 1, no. 3, pp. 114–128, 2015.

25. Analytical Method Development and Method Validation for the Simultaneous Estimation of Metformin hydrochloride and Pioglitazone hydrochloride in Tablet Dosage Form by RP-HPLC. Asian J. Pharm. Ana. 2(3): July-Sept. 2012; Page 85-89.

26. K. Vijaya Sri, M. Anusha, S. Ravinder Reddy. A Rapid RP-HPLC Method development and Validation for the Analysis of Linagliptinin Bulk and Pharmaceutical Dosage Form. Asian J. Pharm. Ana. 5(1): Jan.- March 2015; Page 16-20.

27. B. Mohan Gandhi, A. Lakshmana Rao, J. Venkateswara Rao. A New Stability-Indicating and Validated RP-HPLC Method for the Estimation of Tolvaptan in Bulk and Pharmaceutical Dosage Forms. Asian J. Research Chem. 7(7): July 2014; Page 628-633.

28. Vatchavai Bhaskara Raju, Bonthu Mohan Gandhi, Kamatham Srinivas Sumanth, Kolli Srinivas, Tupakula N Venkata Lakshmi Neeraja. RP-HPLC Method Development and Validation for Simultaneous Estimation of Telmisartan and Ramipril in pure and Pharmaceutical Dosage forms. Asian J. Research Chem. 2017; 10(2):179-185.

29. Ashwini Parmar, Sandeep Sonawane, Santosh Chhajed, Sanjay Kshirsagar. Development and Validation of RP-HPLC Method for simultaneous estimation of Telmisartan, Amlodipine Besylate and Hydrochlorthiazide in their tablet dosage form. Asian J. Pharm. Ana. 2017; 7(3): 189-195.

30. S. J. Daharwal, Veena D. Singh. Development of classical least square method for the determination of Candesartan and Hydrochlorthiazide in tablet dosage form. Asian J. Pharm. Res. 5(2): April-June 2015; Page 90-95.

31. S. J. Daharwal, Veena D. Singh. Estimation of Binary Mixtures of Captopril and Hydrochlorthiazide by using Chemometric Assisted Method. Asian J. Pharm. Tech. 2015; Vol. 5: Issue 2, Pg 79-82.

32. T Venkatachalam, V Kishor Kumar, P Kalai Selvi, R Srinivasan, R Mariammal, KG Lalitha. New Spectrophotometric Method Applied to the Simultaneous Determination of Metoprolol Succinate and Hydrochlorthiazide. Asian J. Research Chem. 3(2): April- June 2010; Page 464-467.

33. G. Saravanan, SK. Bajidbhee, I. Sri Krishnanjaneyulu. Development and validation of RP-HPLC method for simultaneous estimation of Hydrochlorthiazide and Olmesartan medoxomil in bulk and pharmaceutical dosage form. Asian J. Research Chem 8(2): February 2015; Page 147-152.

34. Devika G.S., M. Sudhakar, J. Venkateshwara Rao. Isocratic RP-HPLC Method for Simultaneous Estimation of Spirinolactone and Hydrochlorthiazide in Oral Solid Dosage Form. Research J. Pharm. and Tech. 5(8): August 2012; Page 1050-1053.

35. ICH, Q2A, Text on validation of analytical Procedures International Conference on Harmonization, Geneva 1994:1-5.

36. ICH, Q2B, Validation of analytical Procedure: Methodology, International conference on Harmonization, Geneva 1996: 1-8.

37. ICH, Q2R1, Text on validation of analytical Procedures International Conference on Harmonization, Geneva 1994: 1-13.

Received on 12.03.2021                    Modified on 14.04.2021

Accepted on 09.05.2021                   ©AJRC All right reserved

Asian J. Research Chem. 2021; 14(5):316-320.

DOI: 10.52711/0974-4150.2021.00053