Development and Validation of UV Spectrophotometric Methods for Quantitative Estimation of Antihypertensive Drugs in Fixed Dose Combinations: A Green Analytical Approach

Shweta S. Borkar¹*, Suwarna S. Bobde²

¹Department of Pharmaceutical Analysis, Goa College of Pharmacy,

Goa University, 18^th June Road, Panaji 403001, Goa, India.

²Department of Pharmaceutics, P.E.S’s Rajaram and Tarabai Bandekar College of Pharmacy,

Farmagudi, Ponda 403401, Goa, India.

*Corresponding Author E-mail: swe_bha26@rediffmail.com

ABSTRACT:

The aim of the current research work was to develop simple, precise and accurate UV spectrophotometric methods for quantitative estimation of amlodipine besylate (AMLO) and nebivolol hydrochloride (NEBI) in binary mixtures and their fixed dose combination. The principle employed was based on isobestic point, area under curve (AUC) and dual wavelength approach. In isobestic point method, wavelength for AMLO and NEBI was found to be 268nm. Concentration of NEBI was determined at isobestic wavelength 268nm while AMLO was determined at 360nm and 268nm as there was no interference of NEBI. In AUC, wavelength of maximum absorption for AMLO and NEBI was found to be 360nm and 280nm. The measurements of AUC were carried out at wavelength range of 348-372nm for AMLO and 268-292 nm for NEBI. In case of dual wavelength, two wavelengths were selected for each drug so that the difference in absorbance would be zero for another drug. The wavelengths selected for determination of AMLO were 272.42nm and 292.42nm, whereas, the wavelengths selected for determination of NEBI were 234.37nm and 239.97nm. Validation of methods were carried out as per ICH Q2(R2) guidelines. Linearity was established over concentration range of 14-30µg/mL for AMLO (r² = 0.999 at 268nm and r² = 0.999 at 360nm) and NEBI (r² = 0.999 at 268nm) in isobestic point method, whereas in case of AUC, it was established at concentration range of 5-25µg/mL for AMLO (r² = 0.999) and NEBI (r² = 0.980). However, in dual wavelength method, linearity was determined at concentration range of 10-26µg/mL for AMLO (r² = 0.999 at 272.42 – 292.42nm) and NEBI (r² = 0.993 at 234.37 – 239.97nm). Percentage relative standard deviation (%RSD) value of less than 2% for intra-day and inter-day precision confirmed the precision of the methods. Accuracy of the methods was found to be in range of 98-102%. The assay results pertaining to the methods were within the range of pharmacopoeial limits. The greenness of the method was assessed using AGREE and ComplexGAPI tools. The routine analysis of fixed dose combination in terms of time and cost renders the method suitable and valid as compared to other expensive techniques using sophisticated instruments. The developed analytical methods stand out not only for its accuracy and efficiency but also for its profound commitment to sustainability and environmental safekeeping.

KEYWORDS: Amlodipine Besylate, Nebivolol Hydrochloride, UV Spectrophotometry, Green Chemistry.

INTRODUCTION:

Nebi-AM is a fixed dose combination bilayer tablet containing the drugs, amlodipine besylate (AMLO) and nebivolol hydrochloride (NEBI) both belonging to the class of anti hypertensive agents are prescribed to treat hypertension and manage conditions such as angina and heart failure.

Chemically, AMLO is dialkyl 1,4-dihydropyridine-3,5-dicarboxylate derivative, which is calcium channel blocker used for the treatment of hypertension, chronic stable angina and confirmed or suspected vasospastic angina^1,2. Chemically, NEBI is 2,2’-iminobis[1-(6-fluoro-3,4-dihydro-2H-chromen-2-yl)ethanol]. NEBI serves as a beta blocker which is used to treat high blood pressure and in the treatment of angina, to decrease the heart rate and contractile force^3,4. The chemical structures of AMLO and NEBI are depicted in Figure 1.

(a) (b)

Figure 1. The chemical structures of amlodipine besylate (AMLO) (a) and nebivolol hydrochloride (NEBI) (b)

Literature survey was carried out to gather credible references pertaining to reported analytical approaches used for simultaneous quantification of AMLO and NEBI. The reported analytical methods based on estimation of AMLO and NEBI in binary mixture were based on spectrophotometry^5,6, RP-HPLC^7,8, RP-LC⁹, HPTLC¹⁰, HPLC¹¹and Spectrofluorimetry¹². The reported spectrophotometric methods were based on absorbance ratio, Q Analysis, Absorbance correction, partial simultaneous equation method and first order derivative spectrophotometric approach. With the presence of current technology, UV-Visible spectrophotometer acts as a boon for analysis. It is an instrument with advantages of being simple, less time consuming and an economical method for analysis^{13,14, 15}. Throughout the literature survey it was seen that there were no methods on isobestic point, AUC and dual wavelength approach. Hence an attempt was made to work on these approaches for effective determination of AMLO and NEBI with simple, easy and less time consuming concept. These approaches were very useful, resource efficientt, cost effective and time saving, as it leads to determination of the above drugs concurrently in combination by eliminating interference with each other and without the need of prior separation. In present scenario, these proposed methods have tilted analytical advantages on the side of spectrophotometric analysis as compared to chromatographic approach in terms of cost, simplicity, time, expertise, accuracy and impact on environment. These methods will align with environmentally sustainable practices which can exhibit characteristic indicative of “Green methods”.

MATERIALS AND METHODS:

Materials:

Instrument and Softwares:

The samples were analyzed using UV-Visible double beam spectrophotometer of make Shimadzu (Model UV-2700). The spectra were recorded and processed using UV Probe 2.51 software. Greenness of method were assessed using AGREE version 0.5 (beta) and complexGAPI version 0.2 (beta).

Chemicals and Standard Solutions:

Drug samples of amlodipine besylate IP (AMLO) and nebivolol hydrochloride IP (NEBI) having percent purity of 99.74 % and 99.9 % were received from Pure and Cure Health Care Pvt. Ltd., Uttarakhand, India and Aristo Pharmaceutical Pvt. Ltd., Mumbai as gratis. The samples were used as such without any further purification. The tablets having brand name Nebi-AM manufactured by Otsira Genetica were purchased from local pharmacy store. Each uncoated bi-layered tablet contains amlodipine besylate IP equivalent to amlodipine (5.0 mg) and nebivolol hydrochloride IP equivalent to amlodipine (5.0 mg). Methanol HPLC grade was purchased from Thermo Fisher Scientific Pvt. Ltd., Mumbai.

Methods:^16,17

Preliminary analysis of AMLO and NEBI:

Monographs of AMLO and NEBI were referred for solvent selection and stability studies. NEBI and AMLO are official in IP. AMLO is also official in USP, BP and EP. For the selection of solvent for NEBI and AMLO, stability studies were performed using distilled water and methanol. The spectra’s for both NEBI and AMLO having concentration of 10µg/mL were recorded. The stability of the NEBI and AMLO were determined by recording the spectra’s of same sample solutions at 0.0, 2.0, 4.0, 6.0, 8.0 and 24hrs.

Preparation of solutions and measurements:

The stock solutions of AMLO (1000µg/mL) and NEBI (1000µg/mL) were prepared separately by accurately transferring 139mg and 109mg of the drug sample respectively into 100mL of volumetric flask and diluting with methanol upto the mark. Working standards of AMLO (100µg/mL) and NEBI (100µg/mL) were prepared separately by appropriately diluting individual stock solution using methanol. Accurate dilutions were made from working standard solutions to get incremental concentrations of AMLO and NEBI in the range of 14-30µg/mL, 5.0-25µg/mL and 10-26µg/mL respectively. The samples were diluted using distilled water. The zero order spectra of AMLO and NEBI were recorded for each concentration in triplicate and stored in computer.

Isobestic Point Method:

The isobestic wavelength for AMLO and NEBI was determined by overlapping UV spectra’s of both drugs having concentration of 10µg/mL. To prepare 10µg/mL of AMLO and NEBI, 1ml was withdrawn from working standard solution of AMLO (100µg/mL) and NEBI (100µg/mL) in a 10mL volumetric flask separately and diluted upto the mark with distilled water. Spectra’s of these solutions were obtained after scanning them over the UV range of 200-400nm.

Area Under Curve Method:

The wavelength region for simultaneous determination of AMLO and NEBI was carried out by overlapping UV spectra’s of both the drugs having concentration of 10µg/mL. To prepare 10µg/mL of AMLO and NEBI, 1ml was withdrawn from working standard solution of AMLO (100µg/mL) and NEBI (100µg/mL) respectively in 10ml volumetric flask separately and diluted upto the mark with distilled water.Spectra’s of these solutions were obtained after scanning them over the UV range of 200-400nm.

Dual Wavelength Method:

In case of dual wavelength method, two wavelength were selected for each drug so that the difference in their absorbance would be zero for another drug. This was done by overlapping UV spectra’s of both the drugs having concentration of 10µg/mL. To prepare 10µg/mL of AMLO and NEBI, 1ml was withdrawn from working standard solution of AMLO (100µg/mL) and NEBI (100µg/mL) respectively in 10ml volumetric flask separately and diluted upto the mark with distilled water. Spectra’s of these solutions were obtained after scanning them over the UV range of 200-400nm.

Application of Isobestic point, AUC and Dual wavelength methods for marketed formulation of NEBI-AM:^®^18,19

Twenty tablets were weighed (8.003g) and the average weight of the tablet (0.400g) was determined. The powder content equivalent to one tablet (0.400g) was transferred into 100mL volumetric flask. To it 20mL of methanol was added and the solution was sonicated for 20mins. The solution was again sonicated for 20min by adding more 20mL of methanol and the solution was diluted upto the mark using methanol as a solvent to get a concentration of 50μg/mL of AMLO and 50μg/mL of NEBI. The resultant solution was centrifuged for 45 min at the speed of 4000rpm to obtain clear supernatant solution. The supernatant solution (10mL) was withdrawn and diluted to 100mL using methanol to obtain concentration of AMLO (5.0μg/mL) and NEBI (5.0μg/mL). This resulted in a mixture of AMLO and NEBI which were then analyzed in a UV-visible spectrophotometer at the wavelength range of 200 - 400 nm by isobestic point, AUC and dual wavelength method.

Analytical method validation:

The proposed methods were validated to ensure their reliability. Method validation was performed using the following parameters: Linearity and range, Specificity, Precision, Accuracy and Assay as per ICH Q2 (R2) guidelines^20,21,22.

Assessment of greenness of analytical method:^23,24

Evaluating the established analytical methods greenness is crucial. AGREE metric tool and ComplexGAPI softwares were used. For AGREE, input criteria was referred to the 12 significance principles of green chemistry wherein each of the 12 input variables was transformed into common scale in 0-1 range. The final output was a circular diagram with overall score and colour representation in the centre. The performance of the procedure in each of assessment was reflected with intuitive red-yellow-green colour scale and weight of each principle was reflected with the width of its corresponding segment. ComplexGAPI was presented to assess whole analytical procedures’ green character. Comprehensive data about analytical process to be analyzed was gathered. Final output was represented by a three colour scale; green for low ecological impact, yellow for moderate environmental effects and red for high ecological impact. The analytical process utilized E-factor input wherein closer the E-factor towards zero, the greener and more sustainable will be the process.

RESULT:

Preliminary analysis of AMLO and NEBI:

The solvent was selected in such a way that both AMLO and NEBI were soluble in the chosen solvent. AMLO and NEBI were found to be soluble in methanol. Hence, it was decided to use methanol as a solvent to prepare stock solution (1000 µg/mL) and working standard (100 µg/mL) solution. However in order to make analysis cost effective and eco-friendly; distilled water was used as diluents to prepare further solutions. Overlain spectra depicting the stability of AMLO and NEBI in distilled water are as shown in Figure 2 and Figure 3.

Figure 2. Stability spectra of AMLO in distilled water (10µg/mL) recorded at 0.0, 2.0, 4.0, 6.0, 8.0 and 24hrs

Figure 3. Stability spectra of NEBI in distilled water (10µg/mL) recorded at0.0, 2.0, 4.0, 6.0, 8.0 and 24hrs

Selection of wavelength for isobestic point, AUC and dual wavelength method:

By overlapping the UV spectra of AMLO and NEBI having same concentration, wavelength was determined for isobestic point method. Isobestic wavelength was found to be 268nm.Concentration of NEBI was determined at isobestic wavelength 268nm and AMLO at 268nm and 360nm as at this wavelength absorbance of NEBI was found to be zero and did not interfere with AMLO concentration. An overlain spectrum of AMLO, NEBI and mixture is as shown in Figure 4.

Figure 4. Overlain Spectra of AMLO (10µg/ml) and NEBI (10µg/ml) and its mixture (10+10µg/ml)

Selection of wavelength region is one of the most important criteria during the development of the area under curve method.^The^λ_max^{for AMLO and NEBI was found to be 360nm and 280nm. It
was decided to select}wavelength range of ±12 from 360nm and ±12 from 280nm which were wavelength maxima. The measurement of AUC was carried out at wavelength range of 348-372nm for AMLO and 268-292nm for NEBI. An overlain spectra of AMLO, NEBI and mixture is as shown in Figure 5.

Figure 5. Overlain Spectra of AMLO (10µg/ml), NEBI (10µg/ml) and its mixture (10+10µg/ml)

In case of dual wavelength method, AMLO showed equal absorbance at 234.37nm and 239.97nm where the difference in absorbance were measured for the determination of NEBI. Similarly differences in absorbance at 272.42nm and 292.42nm were measured for determination for AMLO. An overlain spectra of AMLO, NEBI and mixture is as shown in Figure 6.

Figure 6. Overlain Spectra of AMLO (10µg/ml), NEBI (10µg/ml) and its mixture (10+10µg/ml)

Analytical method validation:

Linearity and Range:

Linearity for AMLO and NEBI was established using five incremental concentrations in the concentration range of 14 - 30µg/mL for both AMLO and NEBI in isobestic point method, 5.0 - 25µg/mL for both AMLO and NEBI in AUC and 10 - 26µg/mL for both AMLO and NEBI in dual wavelength method. In isobestic point method, the absorbances were measured at 268nm and 360 nm for AMLO and 268nm for NEBI. The AUC was determined for AMLO at wavelength range 348-372nm and NEBI at 268-292nm. However in case of dual wavelength method, difference in absorbance was measured at wavelength range of 272.42-292.42nm for AMLO and 234.37 - 239.97nm for NEBI. Calibration curves were constructed by plotting absorbance verses concentration in isobestic point and dual wavelength method whereas AUC versus concentration in area under curve method. The data was subjected for regression analysis to determine regression equation. The results are summarized in Table 1.0.

Table 2. Results of the precision study carried out for AMLO and NEBI by using Isobestic point, AUC and dual wavelength approach (n=6).

Sr. No.	Isobestic point				AUC				Dual wavelength
	Amount calculated (μg/mL)				Amount calculated (μg/mL)				Amount calculated (μg/mL)
	Intra-day		Inter-day		Intra-day		Inter-day		Intra-day		Inter-day
	AMLO	NEBI	AMLO	NEBI	AMLO	NEBI	AMLO	NEBI	AMLO	NEBI	AMLO	NEBI
1.0	21.81	21.99	21.94	22.05	15.21	14.89	15.24	15.22	18.27	18.23	17.93	18.23
2.0	21.83	22.17	22.09	22.45	15.98	15.04	15.70	15.14	17.93	18.23	17.93	18.23
3.0	21.91	22.47	22.05	22.68	15.26	15.19	15.42	15.15	18.27	17.49	17.93	17.82
4.0	21.83	22.17	22.30	22.79	15.38	15.49	15.49	15.02	18.27	18.23	18.27	18.23
5.0	21.87	22.68	22.29	22.62	15.68	15.19	15.84	15.36	17.93	18.23	18.60	18.23
6.0	21.98	22.56	22.09	22.45	15.33	14.92	15.30	14.91	18.62	18.23	18.60	17.46
Average	21.87	22.33	22.12	22.50	15.47	15.12	15.50	15.13	18.21	18.10	18.21	18.03
SD	0.064	0.267	0.142	0.260	0.296	0.221	0.232	0.157	0.259	0.302	0.329	0.325
% RSD	0.292	1.195	0.639	1.155	1.914	1.462	1.498	1.040	1.422	1.668	1.809	1.803

Table 3. Results of accuracy studies of AMLO and NEBI by isobestic point, AUC and dual wavelength method (n=3).

Sr. No

Level of tablet concentration

Isobestic point

AUC

Dual wavelength

Recovery (%)

Mean±SD,

% RSD

Recovery (%)

Mean±SD,

% RSD

Recovery (%)

Mean±SD,

% RSD

Recovery (%)

Mean±SD,

% RSD

Recovery (%)

Mean±SD,

% RSD

Recovery (%)

Mean±SD,

% RSD

AMLO

NEBI

AMLO

NEBI

AMLO

NEBI

1.0

80 %

100±0.866, 0.861

99.58±0.721,

0.724

98.1±1.040,

1.048

98.6±0.503,

0.510

100.5±0.787,

0.786

101±1.412

1.399

2.0

100 %

100.29±1.09,

1.09

100.29±0.796,

0.794

102.11±0.151,

0.148

98.88±0.546,

0.553

98.76±0.522,

0.529

100.87±0.436,

0.434

3.0

120 %

100.19±0.996,

0.994

100.30±0.725,

0.723

99.8±0.404,

0.406

100.92 ± 0.460,

0.458

101.55 ±0.785,

_0.779

98.35±0.704,

_0.720

Table 4. Result of the assay carried out for AMLO and NEBI by isobestic point, AUC and dual wavelength method (n=3).

Weight taken (tablet powder)	Isobestic point		AUC		Dual wavelength
	Amount found (%)		Amount found (%)		Amount found (%)
	AMLO	NEBI	AMLO	NEBI	AMLO	NEBI
396.1 mg	99.45	99.18	101.4	99.20	101.5	101.2
	99.68	100.0	101.71	100.26	101.5	101.2
	99.68	99.18	102.0	101.32	99.6	101.28
Average	99.60	99.45	101.73	100.26	100.23	101.22
SD	0.133	0.473	0.304	1.060	1.096	0.047
% RSD	0.134	0.476	0.299	1.057	1.094	0.046

Assessment of greenness of analytical method:

It was found from the results of assessment that by using AGREE software; greenness score of 0.63 was obtained. Whereas in ComplexGAPI, a close to zero E-factor value of 0.02 was obtained, indicating that the three UV spectroscopic methods were within the threshold and accepted as green for AMLO and NEBI following principles of green analytical chemistry. For AGREE, input was provided in software in terms of 12.0 green analytical principles and the output was obtained in terms of circular diagram as shown in figure 8. The result of complexGAPI assessment, as conveyed through pictorial representation is as shown in figure 9.

Figure 8. Green matrix output for 12 principles of green chemistry reporting overall score at the center (AGREE).

Figure 9. Pictogram depicting results of greenness assessment (ComplexGAPI).

DISCUSSION:

Preliminary studies conducted revealed that there were no significant changes in spectral characteristics of AMLO and NEBI at the end of 24hrs when distilled water was used as a solvent, thus AMLO and NEBI were found to be stable in distilled water, and hence distilled water was used as a solvent to carry out development of method and the further analytical studies. Correlation coefficient values obtained for all the three methods were close to 1 which indicated the method to be linear. In specificity studies, the spectra obtained for standard mixture coincided with the tablet mixture which exhibited no interference from tablet (NEBI-AM) excipients. Precision studies showed that calculated %RSD value for AMLO and NEBI was less than 2%, which indicated the method was precise. The mean accuracy at each level was in the range of 98 to 102% for both AMLO and NEBI which concludes the method to be accurate. The obtained assay values of AMLO and NEBI were matching with the labelled claim and %RSD for each drug was less than 2.AGREE diagram displayed a central score representing overall greenness and an outer circle with a clockwise motion from 1.0 to 12.0 that represented 12.0 green analytical chemistry ideas in the range of 0.0 to 1.0 with red, yellow, and green color coding. Dark green color denotes a value that is near to 1.0, red indicates a number that is close to 0.0, and yellow shows a value that is in the middle of dark green and red. The developed analytical approach received a 0.63 central score and the calculated scores for reported HPTLC and HPLC methods were 0.39 and 0.46. The complex GAPI software uses the environmental impact and procedural aspects for assessing greenness. The pentagonal chart generated by the software for proposed UV methods consists of 5 divided parts each representing an aspect in analytical method. The more greener are the pentagonal charts more eco-friendly is the method.

CONCLUSION:

Simple UV Visible spectrophotometric methods were developed for quantification of AMLO and NEBI in fixed dose combination, NEBI-AM. These methods were found to be advantageous over other methods used for simultaneous quantification of binary mixture without prior separation. The produced results were validated according to ICH Q2(R2) guidelines which were found to comply within the label claim and provide alternate method for accurate determination of these drugs in fixed dose combination. The applied method was found to be sensitive, accurate and precise. These methods were also subjected for assessment of greenness using AGREE and ComplexGAPI that were found greener than the reported chromatographic methods. By embracing Green Chemistry principles, optimizing resource use, minimizing waste, and embracing environmental responsibility, we emphasize on minimizing the use of hazardous chemicals and solvents, use of more energy-efficient instruments, minimizing exposure to harmful substances to reduce health risks and enhance workplace safety, conserving energy, decreasing waste output, support sustainable development goals and contribute positively to corporate sustainability strategies. Hence aligning the developed and validated UV spectroscopic methods more favorably with green and sustainable analytical practices and can be successfully applied for routine analysis of marketed formulations in terms of cost and time. These methods were found to be suitable and valid as compared to other techniques which may be expensive using sophisticated instruments.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Aristo Pharmaceutical Pvt. Ltd.,Mumbai and Pure and Cure Healthcare Pvt. Ltd. for gifting the sample of Nebivolol hydrochloride and amlodipine besylate.

REFERENCES:

1. Amlodipine besylate. In: The United States pharmacopeia 41: The National Formulary 36: Official from May 1, 2018. Rockville, MD: United States Pharmacopeial Convention. 2018: 262–63.

2. Amlodipine besylate. In: Indian Pharmacopoeia, 2014. 7th ed. Ghaziabad, New Delhi: Indian Pharmacopoeia Commission. 2014: 1045–46.

3. Tripathi KD. Essentials of Medical Pharmacology. 8th ed. New Delhi, Delhi: Jaypee Brothers Medical Publishers. 2019.

4. Nebivolol Hydrochloride. In: Indian Pharmacopoeia, 2014. 7th ed. Ghaziabad, New Delhi: Indian Pharmacopoeia Commission. 2014: 2310–11.

5. Mishra P, Shah K, Gupta A. Spectrophotometric methods for simultaneous estimation of nebivolol hydrochloride and amlodipine besylate in tablets.Int. J. Pharm. Pharm. Sci. 2009; 1(2): 55-61.

6. Giri CK, Kondawar MS, Chougule DD. Simultaneous estimation of nebivolol hydrochloride and amlodipine besylate in combined tablet dosage form by Q analysis method. Int. J. Pharm. Res. Dev. 2010; 2(5): 13.

7. Deepak S, Anurekha J, Alankar S, Simultaneous estimation of amolodipine besylate and nebivolol hydrochloride in tablet dosage forms by reverse high performance liquid chromatographic using ultraviolet detection. Pharm. Methods. 2011; 2(1): 9-14. doi:10.4103/2229-4708.81083

8. Al Bratty M, Manoharan G, Alhazmi HA. Development and validation of RP-HPLC method for the simultaneous estimation of Amlodipine and Nebivolol in raw and tablet formulation. Int. J. Pharm. Res. Allied Sci. 2016; 5(4): 37-44.

9. Joshi SJ, Karbhari PA, Bhoir SI. RP-LC simultaneous determination of nebivolol hydrochloride and amlodipine besilate in uncoated bi-layer tablets. Chromatographia. 2009; 70(3): 557-561.

10. Dhandapani B, Anjaneyulu N, Venkateshwarlu Y, Rasheed SH. HPTLC method development and validation for the simultaneous estimation of amlodipine besylate and nebivolol hydrochloride in tablet dosage form. J. Pharm. Res. 2010; 3(2): 332-334.

11. Hinge MA, Patel JA, Mahida RJ. Spectrophotometric and High Performance Liquid Chromatographic Determination of Amlodipine Besylate and Nebivolol Hydrochloride in Tablets Dosage Form. Pharm. Methods. 2016; 7(1): 1-7.

12. Sunitha G, Gayatri T, Manaswini Y, Mounika G, Anumolu P.D. Synchronise derivative spectroflourimetric simultaneous quantification of nebivolol hydrochloride and amlodipine besylate.Der. Pharm. Lett., Sch. Res. Libr. 2016; 8(2): 447-451.

13. Priyanka MS, Mankar SD, Dighe SB. An Overview on Analytical Method Development and Validation for Ertugliflozin in Bulk and Pharmaceutical Dosage form. Asian J. Pharm. Anal. 2023; 13(3): 222-228. doi:10.52711/2231-5675.2023.00036

14. Narender M, Anka RA, Sreehasa A, Mounika P, Sonekar S, Vijaya DC, Navya SN. UV-Spectroscopic Method development and Validation for simultaneous estimation of Doxylamine succinate and Pyridoxine hydrochloride in bulk and Pharmaceutical dosage form. RJPT. 2020; 13(10): 4613-4620,doi: 10.5958/0974-360X.2020.00812.4

15. Lakpa DS, Bibhas P, Bhupendra S. A Review of various Analytical Methods Developed for Estimation of Paracetamol, Diclofenac sodium, and Chlorzoxazone in Single-component and Multi-component Dosage Form. RJPT. 2023; 16(8): 3977-3982. doi:10.52711/0974-360X.2023.00653

16. Vijaykumar TP, Mrunalle DM. Development of Visible Spectrophotometric Method and its Validation for Dolutegravir in Tablet dosage form. Asian J. Pharm. Anal. 2023; 13(4): 249 – 254. doi:10.52711/2231-5675.2023.00041

17. Shivprasad P, Ajay K, Kartik A, Akash B, Madhav B, ashish B, Mahesh C. Development and Validation of UV Spectroscopy Method for determination of Posaconazole in Bulk and Formulation. Asian J. Pharm. Anal. 2023; 13(3): 171-174, doi:10.52711/2231-5675.2023.00027

18. Rajan VR. UV Spectrophotometric estimation of Drotaverine hydrochloride by zero order and area under curve methods in bulk and Pharmaceutical Dosage Form. RJPT. 2018; 11(12): 5576-5580, doi: 10.5958/0974-360X.2018.01014.4

19. Santosh K, Sushma A, Shripad P.Quantification of Vortioxetine in Pharmaceutical preparations by validated area under curve UV Spectrophotometric Analytical method. RJPT. 2023; 16(12): 5986-5989, doi:10.52711/0974-360X.2023.000971

20. ICH. Validation of analytical procedures: text and methodology, Q2 (R1), International Conference on Harmonization, Geneva, Switzerland, 2005. 6–13.

21. Shweta J, Karuna M, Rohit J, Dyade GK.AQBD Approach in Chemo metric assisted Method Development for the Estimation of Ciprofloxacin and Metronidazole by UV-VIS Spectrophotometry. AJPRes. 2022; 12 (3): 183-191. doi: 10.52711/2231-5691.2022.00030

22. Pavan C, Sandip B, Santosh G, Sagar P, Shitalkumar P.Development and Validation of UV Spectrophotometric method for Estimation of Itraconazole in Bulk Drug and Solid Dosage Form. AJPRes. 2021; 11 (1): 13-16, doi: 10.5958/2231-5691.2021.00004.6

23. Francisco PP, Pereira, Wojnowski W, Tobiszewski M, AGREE-Analytical GREEnness metric approach and software, Anal. Chem. 2020; 92(14): 10076-10082.doi.org/10.1021/acs.analchem.0c01887.

24. Kiran BA, Girija BB, Madhav RS, Sadikali FS.Environmentally Friendly Analytical Methods for Quantitative Measurement of Sofosbuvir in Pharmaceutical Formulations: Comparison of HPTLC and UV Spectrophotometric Approaches. RJPT. 2023; 16(8): 3830-3836. doi: 10.52711/0974-360X.2023.00632

Received on 06.09.2024 Revised on 09.01.2025

Accepted on 15.04.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5702-5708.

DOI: 10.52711/0974-360X.2025.00823

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Parameters	Isobestic point method			AUC		Dual wavelength method
Parameters	AMLO	AMLO	NEBI	AMLO	NEBI	AMLO	NEBI
Range (µg/mL)	14-30	14-30	14-30	5-25	5-25	10-26	10-26
Wavelength (nm)	268	360	268	348-372	268-292	272.42-292.42	234.37-239.97
Correlation coefficient	0.999	0.999	0.999	0.999	0.980	0.999	0.993
Slope	0.0062	0.0173	0.0062	0.4014	0.237	0.0029	0.0013
_Intercept	0.0003	0.0004	0.0008	0.1527	0.9171	0.001	0.0013