VALIDATED UV SPECTROPHOTOMETRIC METHOD FOR IN-VITRO DISSOLUTION STUDIES IN PHOSPHATE BUFFER pH 7.4

VALIDATED UV SPECTROPHOTOMETRIC METHOD FOR IN-VITRO DISSOLUTION STUDIES IN PHOSPHATE BUFFER pH 7.4

Shashank Chaturvedi ^{* 1}, Sumbul Ayaz ^{2, 3} and Kamal Shah ¹

Institute of Pharmaceutical Research ¹, GLA University, Mathura - 281406, Uttar Pradesh, India.

Invertis Institute of Pharmacy ², Invertis University, Bareilly - 243123, Uttar Pradesh, India.

Steller Institute of Pharmacy ³, Bareilly - 243407, Uttar Pradesh, India.

ABSTRACT: A simple, accurate, and economical, least time-consuming method has been developed for Losartan potassium (LP) by using UV spectrophotometer. This method was developed using phosphate buffer solution pH 7.4 (PBS) for quantifying the amount of LP released from the colon-specific formulations. The wavelength maximum (λ_max) of LP in phosphate buffer pH 7.4 was found to be 228.5 nm. The developed UV spectroscopic method exhibited linearity in the range of 2-10 μg/mL with a correlation coefficient (R2) value of 0.999 and was validated with respect to linearity, specificity, accuracy (recovery), and precision. These parameters were determined according to International Conference on Harmonization (ICH) guidelines. Results of the analysis were validated statistically and by recovery studies, which proved the suitability of the developed method for the routine estimation of LP in colon-specific control release formulation. Hence, the reported method for the estimation of LP from the colon-specific formulations was simple, accurate, and least time-consuming.

Losartan potassium, Colon specific, ICH, Spectrophotometric method, Phosphate buffer pH 7.4

INTRODUCTION: Losartan potassium, is chemically 2-butyl-4-chloro-1-[[2'-(1H-tetrazol-5-yl) [1,1'-biphenyl]-4-yl]methyl]-1H imidazole -5-methanol monopotassium salt. LP is categorized as Angiotensin-II receptor antagonist. It is a colorless, crystalline powder with excellent flow properties ¹. LP is a frequently prescribed drug for the manage-ment of Hypertension (HTN). The hypotensive effect is observed due to its Angiotensin II receptor (AT1) antagonistic property ². Furthermore, LP, together with its active carboxylic acid metabolite, cumulatively accounts for the antagonistic action on Angiotensin II receptor ^{3, 4}.

LP is a Biopharmaceutics Classification System (BCS) Class III drug being freely soluble in water. It is soluble in alcohols and slightly soluble in common organic solvents ⁵. The structural formula of LP is shown underneath Fig. 1.

FIG. 1: STRUCTURE OF LP

The United States Pharmacopoeia (USP) endorses the use of HPLC for the estimation of LP in pharmaceutical samples 6. Many other analytical methods for LP determination alone or in combination have been reported like UV spectrophotometry ^{7, 8}, HPLC ^{10, 11}, HPTLC ¹², ¹³, electrochemical ^{14, 15}.

The present work describes the development of validated UV spectro-photometric method for quantifying the amount of in-vitro LP release in PBS from colon-specific formulations ¹⁶.

MATERIALS AND METHODS: Losartan potassium was obtained as a generous gift sample by ZIM Laboratories Nagpur, India. Disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride were procured from CDH, New Delhi, India. All other reagents used were of analytical grade.

Instrument: A double beam UV-visible spectro-photometer (Lab India 3200) having matched pair of 1 cm quartz cells was employed for analytical estimations.

Preparation of PBS pH 7.4: PBS was prepared by dissolving 2.38 g of disodium hydrogen phosphate, 0.19 g of potassium dihydrogen phosphate, and 8.0 g of sodium chloride in sufficient water to produce 1000 mL, pH was adjusted as per requirements ¹⁷.

Preparation of LP Standard Stock Solution: Standard stock solution of LP was prepared by accurately weighing 100 mg of drug and transferred into 100 mL volumetric flask. The drug was dissolved in PBS, and final volume was made with PBS to obtain the concentration of 1000 μg/mL ¹⁸.

Preparation of LP Working Dilutions: The working standard solution was prepared by taking 10 mL of standard stock solution in 100 mL volumetric flask. The volume was made up with PBS to obtain the concentration of 100 100 µg/mL ^{18, 19}.

Determination of Wavelength Maximum (λ_max): LP standard concentration (6 μg/mL) was prepared in PBS and UV spectrum was taken using double beam UV‐Visible Spectrophotometer (Lab India 3200) for the wavelength region of 200-400 nm and the wavelength maximum (λ_max) was found to be 228.5 nm Fig. 2.

Calibration Curve of LP in Phosphate Buffer Solution (pH 7.4): Aliquots with different volumes were withdrawn from working dilution into 10 mL volumetric flasks which were subsequently diluted with PBS (pH 7.4), in order to get standard drug concentrations between 2-10 µg/mL.

The respective absorbance of these LP dilutions was estimated at 228.5 nm ²⁰. A calibration curve was plotted between concentration (x-axis) and absorbance (y-axis) shown in Fig. 3.

Validation Procedure:

Linearity or Range: Working solution, 100 μg/ml was further diluted with PBS to get the final concentration in the range 2-10 μg/ml solutions.

The respective absorbance from the different concentrations (2-10 μg/ml) was measured at 228.5 nm. The calibration curve was constructed by plotting absorbance versus concentration, and the linear range was analyzed by regression equation ²¹.

Precision: The precision was expressed either for intraday or inter-day analysis as percent relative standard deviations.

The intraday study was conducted with a concentration of replicates of LP on the same day three times.

In the inter-day study, the concentration of the drug was analyzed on three successive days to get an insight of variable laboratory conditions on different days ²¹.

Repeatability: Repeatability study was conducted by analyzing the standard drug concentration for minimum in triplicate, and the percent relative standard deviation was calculated ²¹.

Accuracy or % Recovery: Recovery studies were performed to analyze the accuracy of the method. Different aliquots were taken to produce concentration levels at 80%, 100%, and 120% of standard test concentration.

The analysis of the sample was done in triplicate for each level. Percent recovery was then calculated as mentioned in the equation below ²⁰.

% Recovery = A-B / C × 100.................... (1)

Where, A= amount of drug estimated in totality, B= amount of drug found on a pre-analyzed basis, C= amount of pure drug added to a formulation.

Limit of Detection (LOD): The Limit of Detection (LOD) is the smallest concentration of the analyte that can be measured. LOD was estimated employing the formula mentioned underneath ²¹.

LOD = 3.3 × s / x..............................(2)

Where, X = slope of the standard curve, S = standard deviation of the response.

Limit of Quantification (LOQ): The Limit of Quantification (LOQ) is the response by a minimum quantity of the analyte that can be accurately quantified. LOQ was estimated employing the formula mentioned underneath ²¹.

LOQ = 10 × s/ x..........................................(3)

Where, X = slope of a calibration curve, X = standard deviation of the response.

% RSD:  % RSD values were determined by using the formula based on the SD of response and mean of the response.

The % R.S.D. values found to be less than 2, indicating that the proposed method is precise ^{20, 22}.

% RSD = SD of Response / Mean of Response × 100.....................(4)

RESULTS AND DISCUSSION:

Linearity or Range: The standard aliquots in the concentration range of (2‐10 μg/ml) were analyzed for the absorbance at a wavelength maximum of 228.5 nm. The LP standard dilutions exhibited absorbance in the range 0.1392‐0.6522.

The standard dilutions in this range were found to obey Beer‐Lambert’s law with regression of 0.999.

The values of Limit of detection (LOD) and limit of quantification (LOQ) for the assay have been given in Table 1.

TABLE 1: OPTICAL PARAMETERS OF LP

Accuracy or % Recovery: The concentration of recovery was found to be in the range of 10.70-13.26, and % RSD value was 0.07-0.885 respectively, shown in Table 2.

TABLE 2: RESULTS FOR DETERMINATION OF RECOVERY

Results for Determination of Intraday Precision: The % RSD for intraday precision for the concen-tration 2 µg/mL, 4 µg/mL and 6 µg/mL was determined at 10 am, 1 pm, and 4 pm the results have been shown in Table 3. Results obtained from the analytical method had a good intraday precision.

TABLE 3: RESULTS FOR DETERMINATION OF INTRADAY PRECISION

Results for Determination of Interday Precision: The % RSD for interday precision for the concen-tration 2 µg/mL, 4 µg/mL and 6 µg/mL was according to three days analysis and the results have been shown in Table 4. Results obtained from the analytical method had a good interday precision.

TABLE 4: RESULTS FOR DETERMINATION OF INTERDAY PRECISION

Results for Determination of Repeatability: The repeatability of the proposed method was assessed by analyzing LP at 6 μg/mL concentration in triplicate. Results of repeatability analysis were expressed in terms of % RSD and found to be 0.195, 0.146, and 0.144, as shown in Table 5.

TABLE 5: RESULTS FOR DETERMINATION OF REPEATABILITY

CONCLUSION: The experimental findings and the statistical parameters are in agreement that the developed UV spectrophotometric method of LP in PBS is simple, quick, specific, accurate, and precise. Furthermore, the analytical method developed has been analyzed on various parameters to confirm stability like specificity, linearity, accuracy, precision and robustness.

Hence this method can be used for the routine analysis of LP as API in pharmaceutical formulations designed for colon-specific release.

ACKNOWLEDGEMENT: The authors wish to thank the Management of Invertis University, Bareilly, U.P, for providing necessary facilities in the successful completion of this work and GLA university for the continuous encouragement in manuscript preparation. We are also thankful to ZIM Laboratories Nagpur, Maharashtra, India, for providing LP as a gift sample.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

REFERENCES:

1. Weber M and MD: Clinical safety and tolerability of Losartan, Clinical Therapeutics 1997; 19(4): 604-16.
2. Goa KL and Wagstaff AJ: Losartan potassium. Drugs, 1996; 51(5): 820-45.
3. Abraham HM, White CM and White WB: The comparative efficacy and safety of the angiotensin receptor blockers in the management of hypertension and other cardiovascular diseases. Drug Safety 2015; 38(1): 33-54.
4. Sinha AD and Agarwal R: Clinical pharmacology of antihypertensive therapy for the treatment of hypertension in CKD. Clinical Journal of the American Society of Nephrology 2019; 14 (5): 757-64.
5. Domenic AS, Todd WBG and Ghosh S: Clinical Pharmacokinetics of Losartan. Clinical Pharmacokinetics 2005; 44: 797-14.
6. United States Pharmacopeia, 32 ed. USP Convention, Rockville, MD 2008.
7. Abdel-Fattah L, Abdel- L Aziz and Gaied M: Enhanced spectrophotometric determination of Losartan potassium based on its physicochemical interaction with cationic surfactant, Spectrochimica acta. Part A, Molecular and Biomolecular Spectroscopy 2015; 136: 178-84.
8. Shah SA, Vyas RB, Vyas BA, Maniyar NR, Chauhan RS and Shah DR: A method for content uniformity determination of atenolol and losartan potassium in combined tablet dosage form. Indian Journal of Pharmaceutical Sciences 2010; 72(6): 792-94.
9. Lima MJA and Reis BF: A clean photometric method for the determination of losartan potassium in pharmaceuticals exploiting light scattering effect and employing a multi-commuted flow analysis approach, Talanta 2017; 164: 183-88.
10. Bonfilio R, Tarley CRT, Pereira GR, Salgado HRN and de Araújo MB: Multivariate optimization and validation of an analytical methodology by RP-HPLC for the determination of losartan potassium in capsules. Talanta 2009; 80 (1): 236-41.
11. Latif A, Akbar F, Khan AJ, Shafi H and Mazhar M: Development and validation of analytical method for quantification of losartan potassium in solid dosage form. Pharmaceutica Analytica Acta 2018; 9: 592.
12. Shelar K, Rao JR and Dhale C: Stability indicating HPTLC method development and validation for simulataneous estimation of Amlodipine besylate and Losartan potassium and characterisation of acid degradant product of Losartan. International Journal of Pharma Science & Research 2019; 10 (5): 2456-64.
13. Salazar C, Contreras N, Mansilla HD, Yáñez J and Salazar R: Electrochemical degradation of the antihypertensive losartan in aqueous medium by electro-oxidation with boron-doped diamond electrode. Journal of Hazardous Materials 2016; 319: 84-92.
14. Santos MCG, Tarley CRT, Dall’Antonia LH and Sartori ER: Evaluation of boron-doped diamond electrode for simultaneous voltammetric determination of hydro-chlorothiazide and losartan in pharmaceutical formulations. Sensors and Actuators B: Chemical 2013; 188: 263-70.
15. Chaturvedi S and Ayaz S: Colon specific polyelectrolyte complexation-based formulations of losartan potassium for effective management of hypertension. MOJ Bio-equivalence and Bioavailability 2018; 5(1): 00080.
16. Indian Pharmacopoeia, 5^th edition, volume 1, New Delhi, Govt. of India, Ministry of Health and Family Welfare, 248, 2007.
17. Singh M, Kaur R, Raput R, Agrawal S, Kumar S and Sharma M and Sharma A: Analysis of process and formulation variables on chitosan-based losartan potassium nanoparticles: preparation, validation and in vitro release kinetics. Recent Innovations in Chemical Engineering 2020; 13: 41-54,
18. Chaudhari SS and Phalak SD: Development and validation of UV spectrophotometric method for simultaneous equation of aspirin and omeprazole in tablet dosage form. Pharmaceutica Analytica Acta 2019, 11: 618.
19. Prasad N and Sharma A: Ultraviolet-visible spectro-photometric method for estimation of gliclazide in presence of excipients interacting in UV-visible region. Indian Journal of Pharmaceutical Education and Research 2020; 54 (2s): s337-43.
20. International Conference on Hormonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology Q2(R 1), Complementary Guideline on Methodology dated 06 November 1996, London, incorporated in November 2005.
21. Sing A, Sharma PK and Majumdar DK: Development and validation of different UV-spectrophotometric methods for the estimation of fluconazole in bulk and solid dosage form. Indian J of Chemical Technology 2011; 18: 357-62.
    

    ---

    How to cite this article:

    Chaturvedi S, Ayaz S and Shah K: Validated UV spectrophotometric method for in-vitro dissolution studies in phosphate buffer pH 7.4. Int J Pharm Sci & Res 2021; 12(4): 2417-21. doi: 10.13040/IJPSR.0975-8232.12(4).2417-21.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---