1. INTRODUCTION:

Losartan is an angiotensin II receptor antagonist with antihypertensive activity due mainly to selective blockade of AT₁ receptors and the consequent reducedpressor effect of angiotensin II. It is used in the management of hypertension¹. Hydrochlorothiazide (HCT) is a thiazide diuretic used in the treatment of hypertension, its fixed dosage combination with losartanis indicated in the treatment and management of edema and hypertension¹.

Several methods have been reported for the determination of LSP either alone or in combination with other drugs including: Spectrophotometry^2-6, high performance liquid chromatography (HPLC)^5,7-10

HCT alone or combination with other drugs was determined by capillary electrophoresis¹¹and electrochemical method¹², spectrophotometry^13-22, HPLC^23-29 and ultra-pressure liquid chromatography¹⁰. Only few spectrophotometric^31-35 and chromatographic methods^36-40are available for the simultaneous estimation of LSP and HCT in pharmaceutical dosage forms. There is always great demand for simple and affordable methods of analysis in poor countries where the cost of analysis is prohibitive, accordingly this method was proposed.

2. EXPERIMENTAL:

2.1 Chemicals and Instruments:

Losartan potassium and hydrochlorothiazide working standards were supplied by Amipharma Laboratories – Sudan and Methanol (Carlo Erba-Italy), UV-Visible spectrophotometer UV 1800 (Shimadzu-Japan), Loscar-H tablets (Cadila Healthcare Limited, India), labeled to contain 50mg Losartan potassium and 12.5mg Hydrochlorothiazide per tablet, was purchased from the local market. Aqueous methanolic solution 50% v/v was used as a diluent.

2.2 Preparation of Solutions:

2.2.1 Losartan potassium standard solution:

Accurately weighedabout 7.5mg losartan potassium working standard were weighed and transferred into 100 mL volumetric flask; dissolved and the volume was completed to the mark using methanol (75µg mL^-1).

2.2.2 Hydrochlorothiazide standard stock solution:

Accurately weighed about 22.5mg hydrochlorothiazide working standard were weighed and transferred into transferred into 100mL volumetric flask; dissolved and the volume was completed to the mark using methanol (225µg mL^-1).

2.2.3 Synthetic mixtures:

Nine laboratory synthetic mixtures having different concentrations of losartan potassium and hydrochlorothiazide covering the concentrations likely to be present in the sample, were prepared according to multilevel multifactor design⁴¹; by mixing different volumes from the two analytes stock solutions in nine separate 50 mL volumetric flask and making final volume to the mark with the diluent.

2.2.4 Analytical wavelengths determination:

Two independent solutions were prepared from the standard stock solution of the two analytes by diluting 5 mL of each to 50 mL using the diluent. The resulting solutions were scanned over the wavelength range of 200-400nm against the diluent as a blank. The spectra were recorded in the overlay mode.

2.2.5 Construction of the calibration curves:

Aliquot volumes (1-5 mL) from stock solutions of each analyte were transferred into a separate set of five 50 ml volumetric flasks and made volume with the diluent; to obtain losartan potassium concentration in the range of 1.5-7.5µg mL^-1 and hydrochlorothiazide concentration in the range of 4.5-22.5µg mL^-1. The absorbance for these solutions were measured at the selected wavelengths and the calibration curves were constructed by plotting the absorbance values obtained against their corresponding concentrations.

2.2.6 Sample preparation:

Twenty tablets were accurately weighed and crushed to fine powder, from the powder weight equivalent to one tablet was taken and transferred into 100 mL volumetric flask; 1 mL distilled water was added and the mixture was shaken to disperse the powder then 20 mL methanol were added and the mixture was sonicated for 15 minutes; cooled and made to volume with methanol. The resulting solution was filtered using 0.45 μm filter and 5 mL of the clear filtrate were diluted to 50 mL with the diluent.

2.3 Validation procedure:

The developed method was validated according to the ICH guidelines requirements⁴²; for linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ).

2.3.1 Linearity:

The calibration data was subjected to linear regression analysis using the method of least squares. The slope, intercept, the standard deviation of the slope and intercept and confidence interval at 95% confidence level were obtained.

2.3.2 Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification of the two analytes were calculated using the following equations⁴².

LOD = 3 × SD/slope of the calibration curve. (3)

LOQ = 10 × SD/slope of the calibration curve. (4)

Where S = average standard deviation of the response obtained in the linearity determination.

2.3.3 Precision:

The repeatability precision (intra-day) of the method was tested using six independent sample solutions containing analytes at 100% concentrations. The intermediate precision (inter-day) was performed by repeating the same process on a different day using fresh reagents and samples. The results were expressed as percentage content and relative standard deviation (%RSD).

2.3.4 Accuracy:

The accuracy of the method was tested by analyzing the nine synthetic mixtures, the results were expressed as percentage content and relative standard deviation (%RSD).

2.4 Theoretical background:

The absorbance subtraction method⁴³is based on the fact that; for a mixture of two drugs X and Y having overlapping spectra intersecting at isoabsorptive point (λ_iso) and Y is extended more than X where X does not show any absorbance at another wavelength (λ₂). The isoabsorptive point (λ_iso) can be exploited for separate quantitative estimation of each X and Y in their mixture (X+Y) using absorbance factor (A_iso / A₂) which is a constant for pure compound Y representing it’s average absorbance ratio at the two wavelengths λ_iso and λ₂. The absorbance values corresponding to X and Y at λ_iso in the mixture are calculated according to the following equation

Absorbance of Y in the mixture at

abs_iso

λ _iso = -------- X abs λ₂ (X + Y) ……………(1)

abs2

Absorbance of X in the mixture at

abs_iso

λ _iso = abs_iso(X+Y) - --------X abs λ₂ (X + Y) ..(2)

abs2

abs_iso

Where: (--------)

abs2

the absorbance ratio which is a constant for pure Y at λ _iso and λ₂.

abs λ_iso (X + Y) and abs λ₂ (X + Y): absorbance of the mixture at λ_iso and λ₂, respectively.

The concentration of each analyte is then calculated from its corresponding regression equation at the isoabsorptive point.

3. RESULTS AND DISCUSSION:

3.1 Selection of the analytical wavelengths:

Fig.1 shows that the overlay spectrum of LSP and HCT are intersecting 266.9 nm (isoabsorptive point) and HCT is having an absorption maximum at 325 nm of where LSP is not having any interference. These two wavelengths were selected as analytical wavelengths since they are satisfying the conditions for the application of the proposed method.

Figure 1: Absorption spectra of (a) LSP (75µg/ml) and (b) HCT (22.5µg/ml) in diluents

3.2 Method validation:

3.2.1Linearity:

Linear regression analysis of the calibration data indicated excellent linearity with correlation coefficients of 0.9996 and 0.9998; over the range of 1.5-7.5µg mL^-1and 4.5-22.5µgmL^-1for LSP and HCT, respectively at the isoabsorptive point. The linear regression analysis results are shown in Table 1. Data analysis also indicated that the residuals were normally distributed around the mean with uniform variance across all concentrations. The average absorbance factor (A_iso/ A₂) calculated from the linearity data of HCT standard solutions at the two wavelengths 266.9 and 325nm, respectively was calculated as 6.252.

3.2.2 Limit of detection and limit of quantification:

The LOD for LSP and HCT were 1.85 and 0.46μgmL^-1, respectively, whereas the LOQ was 6.51μgmL^-1 for LSP and 1.40μg mL^-1 for HCT, at the isoabsorptive point. The obtained LOQ values indicate that the determination of the two analytes with high precision and accuracy is possible at concentrations above these values as shown in Table 1.

Table 1: Regression Analysis Results.

Wavelength	266.9 nm		325 nm
Parameter	LSP	HCT	HCT
Concentration range (µg/mL)	15-75	4.5-22.5
Linearity – Regression equation
Slope (b)	0.0173	0.057	0.010
Intercept (a)	0.013	0.021	0.0005
Correlation coefficient (r)	0.9996	0.9998	0.9996
Standard deviation of the slope (s_b)	0.0002	0.0006	0.0001
Standard deviation of the intercept (s_a)	0.010	0.008	0.0017
Limit of detection (µg/mL)	1.85	0.46	0.06
Limit of quantitation (µg/mL)	6.51	1.40	1.70

3.2.3 Precision:

The absorbance values of the samples measured at 266.9 and 325 nm, were corrected using the calculated absorbance factor (6.252) to obtain the interference free absorbance values of the two analytes at 266.9 nm (Tables 2 and 3).

Table 2:Absorbance data of repeatability determination.

Sample	266.9 nm	266.9 nm	^*LSP_{266.9 nm}	^*HCT_{266.9 nm}
1	1.648	0.117	0.917	0.731
2	1.654	0.119	0.910	0.744
3	1.639	0.120	0.889	0.750
4	1.658	0.118	0.920	0.738
5	1.635	0.120	0.885	0.750
6	1.625	0.115	0.906	0.719

* at 266.9 nm, calculated using the absorption factor

Table 3:Absorbance data of intermediate precision determination.

Sample	266.9 nm	266.9 nm	^*LSP_{266.9 nm}	^*HCT_{266.9 nm}
1	1.655	0.119	0.744	0.911
2	1.655	0.118	0.738	0.917
3	1.653	0.118	0.738	0.915
4	1.651	0.117	0.731	0.920
5	1.643	0.118	0.738	0.905
6	1.651	0.116	0.725	0.926

* at 266.9 nm, calculated using the absorption factor

The concentrations of the two analytes in the samples were calculated from their unified calibration curves at this wavelength. The low percent relative standard deviations (%RSD,< 2%) values obtained for the two analytes in repeatability and intermediate precision; together with overall %RSD of the assays calculated using the data from the two-day analysis of <2%, give strong evidence that the outcomes of the determinations were statistically similar regardless the day of the assay and reagent preparation. The results of precision determination are summarized in Tables 4 and 5.

3.2.4 Accuracy:

The absorbance values of the mixtures measured at 266.9 and 325 nm, were corrected using the calculated absorbance factor (6.252) to obtain the interference free absorbance values of the two analytes at 266.9 nm (Tables 6).

Good agreement between the theoretical and the actual concentrations of the analytes in the synthetic mixtures was obtained with small relative standard deviation values (RSD %,< 2%), confirming satisfactory accuracy of the proposed method as shown in Table 7.

Table 4: Assay results of repeatability determination.

Sample	LSP(µg ml^-1)		% Content	HCT (µg ml^-1)		% Content
Sample	Theoretical	Actual	% Content	Theoretical	Actual	% Content
1	50.0	52.0	104.00	12.5	12.36	98.88
2	50.0	51.6	103.26	12.5	12.58	100.64
3	50.0	50.4	100.80	12.5	12.68	101.44
4	50.0	52.2	104.44	12.5	12.47	99.76
5	50.0	50.2	100.34	12.5	12.68	101.44
6	50.0	51.4	102.80	12.5	12.14	97.12
Average			102.60			99.88
SD			1.68			1.68
RSD%			1.64			1.67

Table 5: Assay results of intermediate precision determination.

Sample	LSP(µg ml^-1)		%Content	HCT (µg ml^-1)		%Content
Sample	Theoretical	Actual	%Content	Theoretical	Actual	%Content
1	50.0	51.68	103.36	12.5	12.58	100.64
2	50.0	52.04	104.08	12.5	12.47	99.76
3	50.0	51.93	103.86	12.5	12.47	99.76
4	50.0	52.17	104.34	12.5	12.36	98.88
5	50.0	51.35	102.70	12.5	12.47	99.76
6	50.0	52.53	105.06	12.5	12.25	98.00
Average			103.90			99.46
SD			0.812			0.908
RSD%			0.78			0.91

Table 6: Absorbance data of accuracy determination.

Mixture	Abs _{266.9 nm}	Abs _{325 nm}	Abs _LSP*	Abs _HCT*
1	1.581	0.125	0.7995	0.7814
2	1.572	0.163	0.5529	1.019
3	2.100	0.163	1.0809	1.019
4	1.850	0.125	1.0685	0.7814
5	1.807	0.163	0.7879	1.019
6	1.570	0.084	1.0448	0.5251
7	1.048	0.083	0.529	0.5189
8	1.315	0.125	0.5335	0.7814
9	1.316	0.083	0.797	0.5189

* at 266.9nm, calculated using the absorption factor

Table 7: Assay results of accuracy determination

Mixture	LSP (µg ml^-1)		% Content	HCT (µg ml^-1)		%Content
Mixture	Theoretical	Actual	% Content	Theoretical	Actual	%Content
1	45.0	45.27	100.60	13.3	13.23	99.47
2	30.0	31.07	103.57	17.5	17.36	99.20
3	60.0	61.47	102.45	17.5	17.36	99.20
4	60.0	60.75	101.25	13.3	13.23	99.47
5	45.0	44.6	99.11	17.5	17.36	99.20
6	60.0	59.39	98.98	8.8	8.773	99.69
7	30.0	29.7	99.00	8.8	8.664	98.45
8	30.0	29.96	99.87	13.3	13.23	99.47
9	45.0	45.13	100.29	8.8	8.66	98.41
Average			100.57			99.18
SD			1.61			0.45
RSD%			1.60			0.46

3.3 Commercial Product Assay:

The good results of determination of LSP and HCT in tablets by the proposed method confirmed the accuracy and precision of the method. The assay results were 102.60% and 99.88%with %RSD 1.68 and 1.68for LSP and HCT respectively.

4. CONCLUSION:

A Simple, accurate, precise and cost effective spectrophotometric methods has been developed and validated for the determination of losartan potassium and hydrochlorothiazide in tablets without prior separation. The proposed method is beneficial for poor countries where acquisition of sophisticated analytical equipment required for the analysis of multicomponent formulation is not affordable. The simplicity of the method makes suitable for quick in process check as well.

5. CONFLICTS OF INTEREST:

The authors declare no conflicts of interest.

6. REFERENCES:

1. Sweetman SC. Martindale: The Complete Drug Reference 36thedition, UK: The Pharmaceutical Press. 2009.

2. Lastra, OC, Lemus IG, Sanchez HJ and Perez RF. Development and validation of an UV derivative spectrophotometric determination of losartan potassium in tablets. J Pharm Biomed Anal. 2003; 33:175-80. DOI:10.1016/s0731-7085(03)00347-9.

3. Kolsure AK, Ingale SS, Abnawe SA and Chabukswar AR, ChoudhariVP, KuchekarBS. Spectrophotometric simultaneous determination of atorvastatin and losartan potassium in combined tablet dosage form by ratio derivative method. J Pharm Res. 2010; 3: 2262-2264.DOI

4. Bonfilio IR, Favoretto LB, Pereira GR and Azevedo RC, Araujo MB. Comparative study of analytical methods by direct and first-derivative UV spectrophotometry for evaluation of losartan potassium in capsules. Braz J Pharm Sci.2010; 46: 1-17. DOI:10.1590/S1984-82502010000100017.

5. Ansari M, Kazemipour M, Khosravi F and Badaran M. A comparative study of first-derivative spectrophotometry and high-performance liquid chromatography applied to the determination of losartan potassium in tablets. Chem Pharm Bull (Tokyo). 2004; 52: 1166-70.DOI:10.1248/cpb.52.1166.

6. Mohamed M. El-Sutohy, Salwa R. El-Shaboury, Samiha A. Hussein, Niveen A. Mohamed. Validated Non-Extractive Spectrophotometric Methods for Determination of Some Angiotensin II Receptor Antagonists. Asian Journal of Pharmaceutical Analysis. 2013; 3(1): 03-08.

7. Maio VM, Dos P, Dias CL and Bergold AM. Validation of an isocratic HPLC assay of losartan potassium in pharmaceutical formulations and stress test for stability evaluation of drug substance. Acta Farm Bonaer. 2005; 24:250-5.

8. Bonfilio R, Tarley CR, Pereira GR and Salgado HR, Araujo MB. Multivariate optimization and validation of an analytical methodology by RP-HPLC for the determination of losartan potassium in capsules. Talanta. 2009; 80: 236-41.DOI: 10.1016/j.talanta.2009.06.060.

9. Mccarthy KE, Wang Q, Tsai EW, Dominic PI, Brooks MA. Determination of losartan and its degradates in COZAAR® tablets by reversed-phase high-performance thin-layer chromatography. J Pharm Biomed Anal. 1998; 17:671-7.DOI: 10.1016/S0731-7085(97)00251-3.

10. Williams RC, Alasandro MS, Fasone VL, Boucher RJ, Edwards JF. Comparison of liquid chromatography, capillary electrophoresis and super-critical fluid chromatography in the determination of Losartan potassium drug substance in Cozaar® tablets. J Pharm Biomed Anal. 1996; 14: 1539-46.DOI:10.1016/0731-7085(96)01740-2.

11. Hillaert S, Bossche WV. Simultaneous determination of hydrochlorothiazide and several angiotensin-II-receptor antagonists by capillary electrophoresis. J Pharm Biomed Anal. 2003; 31:329-39.DOI:10.1016/s0731-7085(02)00643-x.

12. Razak AO. Electrochemical study of hydrochlorothiazide and its determination in urine and tablets. J Pharm Biomed Anal. 2004; 34: 433-40.DOI:10.1016/s0731-7085(03)00497-7

13. Atana ES, Altmay SA, Goger NG, Ozkanab SA, Senturk Z. Simultaneous determination of valsartan and hydrochlorothiazide in tablets by first derivative UV spectrophotometry and LC. J Pharm Biomed Anal. 2001; 25:1009-13. DOI:10.1016/S0731-7085(01)00394-6.

14. Bhadresh V. Savaj, Ashutosh Kumar Patidar, Hashumati A. Raj.Simultaneous determination of Propranolol hydrochloride and Hydrochlorothiazide in Tablets formulation using spectrophotometric technique (Simultaneous Equation Method). Asian Journal of Pharmaceutical Analysis. 2015, 5(1): 31-36 DOI:10.5958/2231-5675.2015.00007.1.

15. Audumbar Digambar Mali. Simultaneous Determination of Carvedilol and Hydrochlorothiazide in Pharmaceutical Dosage Form by Second Order Derivative UV Spectrophotometry. Asian Journal of Pharmaceutical Analysis. 2015; 5(3):133-138. DOI: 10.5958/2231-5675.2015.00021.6

16. Daharwal SJ, Veena D. Singh. Estimation of Binary Mixtures of Captopril and Hydrochlorthiazide by using Chemometric Assisted Method. Asian Journal of Pharmacy and Technology. 2015; 5(2): 79-82. DOI: 10.5958/2231-5713.2015.00018.5.

17. Daharwal SJ, Veena D. Singh. Development of classical least square method for the determination of Candesartan and Hydrochlorthiazide in tablet dosage form. Asian Journal of Pharmaceutical Research. 2015; 5(2): 90-95. DOI: 10.5958/2231-5691.2015.00013.1

18. Bhusari KP, Khedekar PB, Dhole S, Banode VS. Derivative and Q-analysis spectrophotometric methods for estimation of hydrochlorothiazide and olmesartan medoxomil in tablets. Indian J Pharm Sci. 2009; 71: 505-8.DOI:10.4103%2F0250-474X.58176.

19. Erturk S, Cetin SM, Atmaca S. Simultaneous determination of moexiprilhydrochloride and hydrochlorothiazide in tablets by derivative spectrophotometric and high-performance liquid chromatographic methods. J Pharm Biomed Anal. 2003; 33: 505-11. DOI:10.1016/s0731-7085(03)00252-8

20. Prasad CV, Parihar C, Sunil K, Parimoo P. Simultaneous determination of amilorideHCl, hydrochlorothiazide and atenolol in combined formulations by derivative spectroscopy. J Pharm Biomed Anal. 1998; 17: 877-84. DOI:10.1016/s0731-7085(97)00241-0.

21. Dinc E, Ustunda O. Spectophotometric quantitative resolution of hydrochlorothiazide and spironolactone in tablets by chemometric analysis methods. Farmaco. 2003; 58: 1151-61. DOI: 10.1016/j.farmac.2003.07.005.

22. Joshi SJ, Pradnya A, Suvarna K, Bhoir I, 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. DOI: 10.1016/j.jpba.2009.10.021

23. Huang T, He Z, Yang B, Shao L, Zheng X, Duan G. Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution. J Pharm Biomed Anal. 2006; 41: 644-8. DOI:10.1016/j.jpba.2005.12.007.

24. Erk N. Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma by liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci. 2003; 784:195-201. DOI: 10.1016/s1570-0232(02)00759-6.

25. Gandla Kumaraswamy, Gandla Lalitha, N. Ravindra, K.B. Pradeesha. Simultaneous Estimation of Amlodipine, Atenolol and Hydrochlorothiazide in Bulk and Tablet Dosage Form by RP-HPLC Method. Asian Journal of Pharmaceutical Analysis. 2014; 4(4):131-136.

26. Sridevi Ranjitha Karanam, V. Reena Jyothi Swarupa. Stability Indicating Analytical Method Development and Validation for Simultaneous Estimation of Valsartan and Hydrochlorothiazide in Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis. 2016; 6(1) 7-14.DOI: 10.5958/2231-5675.2016.00002.8.

27. Beula S. Janet, Suthakaran R, Prem Kumar Bichala, Shankar CH, Syed Ghouse, Suneetha K.A Prospective Validation of Hydrocholorothiazide and Bisoprolol Fumerate in its Pure and Bulk Dosage Forms by using RP- HPLC Technique. Asian Journal of Pharmaceutical Analysis. 2020; 10(1): 1-6DOI: 10.5958/2231-5675.2020.00001.0

28. Kumar R. Anantha, Babu G. Raveendra, Sowjanya M, Ramayyappa M. Validated RP-HPLC method for the estimation of Amiloride and hydrochlorothiazide in combined tablet dosage form. Asian Journal of Pharmaceutical Analysis. 2021; 11(3):207-211. DOI: 10.52711/2231-5675.2021.00037

29. Kalleshvar P. Jatte, Chakole RD, Charde MS. Degradation Profiling of Lisinopril and Hydrochlorothiazide by RP- HPLC method with QbD Approach. Asian Journal of Pharmaceutical Analysis. 2021; 11(4): 270-274.DOI: 10.52711/2231-5675.2021.00046.

30. Hamid Khan, Mushir Ali, Alka Ahuja, Javed Ali. Method for Simultaneous Determination of Telmisartan and Hydrochlorothiazide in Human Plasma. Asian Journal of Research in Pharmaceutical Science. 2016; 9(9): 1441-1446. DOI: 10.5958/0974-360X.2016. 00278.X

31. ErkN. Analysis of binary mixtures of losartan potassium and hydrochlorothiazide by using high performance liquid chromatography, ratio derivative spectrophotometric and compensation technique. Journal of Pharmaceutical and Biomedical Analysis. 2001; 24(4):603-611.DOI: 10.1016/s0731-7085(00)00434-9

32. ThubeC, DhagudeJ, PawarPY. Simultaneous estimation and validation of losartan potassium and hydrochlorothiazide in bulk and tablet dosage form by using different spectrophotometric method. Der Pharma Chemica. 2014; 6(2):24-30.

33. RaoKS, PandaM, KesharNK. Spectrophotometric methods for the simultaneous estimation of losartan potassium and hydrochlorothiazide in tablet dosage forms. Chronicles of Young Scientists. 2011; 2(3):155-160. DOI:10.4103/2229-5186.90893

34. Shah SA, Rathod IS, Suhagia BN and Savale SS, Patel JB. Simultaneous determination of losartan and hydrochlorothiazide in combined dosage forms by first-derivative spectroscopy and high-performance thin-layer chromatography. Journal of AOAC International. 2001; 84(6): 1715-1723. DOI:10.1093/jaoac/84.6.1715.

35. Maggio RM, Castellano PM, Kaufman TS. A multivariate approach for the simultaneous determination of losartan potassium and hydrochlorothiazide in a combined pharmaceutical tablet formulation. Analytical Bioanalytical Chemistry. 2008; 391(8): 2949-2955. DOI 10.1007/s00216-008-2180-z

36. United States Pharmacopoeia/ National Formulary INC. 24th edition. The United States Pharmacopoeia Convention. Rockville, MD.2010.

37. Jagadeeswararao V, Vasanth PM, Ramesh T and Ramesh M. Analytical method development and validation of losartan potassium and hydrochlorothiazide in combined dosage form by RP-HPLC. International Journal of Chemical Technology Research. 2013; 5(6): 3007-3014.

38. Shakya AK. HPLC-PDA determination of losartan potassium and hydrochlorothiazide using design of experiments. Jordan Journal of Pharmaceutical Sciences. 2015; 8(3): 153-171.

39. Md Arif Hossen, Md Haque, Irin Dewan and A.N.M. Hamidul Kabir, Md Khalid Hossain, Ashraf Islam. Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Hydrochlorothiazide and Losartan Potassium in Tablet Dosage Form. Dhaka University Journal of Pharmaceutical Sciences. 2011; 10(1): 35-42. DOI:10.3329/dujps. v10i1.10013

40. Mohammed NMS, Abdo HR, Hassan H.M. Method development and validation of simultaneous determination of hydrochlorothiazide and losartan in tablet dosage form by RP-HPLC. International Journal of Pharmaceutical Sciences and Research. 2019; 10(1): 227-231. DOI: 10.13040/IJPSR.09758232.10(1).227-31

41. Brereton RG. Multilevel multifactor designs for multivariate calibration. The Analyst. 1997; 122: 1521-1529. DOI:10.1039/A703654J

42. ICH Harmonized Tripartite Guideline: Validation of Analytical Procedures.Q2A.1996.

43. Lotfy HM. Absorbance subtraction and amplitude modulation as novel spectrophotometric methods for the analysis of binary mixtures. International Journal of Pharmacy and Pharmaceutical Sciences.2014; 6(1): 735-741.

Received on 08.08.2022 Modified on 22.03.2023

Asian J. Pharm. Ana. 2023; 13(4):243-248.

DOI: 10.52711/2231-5675.2023.00040