INTRODUCTION:

Glutathione (GSH) is chemically known as (2S)-2-amino-4-[[(1R)-[(carboxymethyl) carbamoyl]-2-sulfanylethyl] carbamoyl] butanoic acid. Glutathione exists in reduced and oxidized states. Its molecular formula and molecular weights are C10H17N3O6S and 307.32 g/mol. It is used as anti-aging, skin whitening, and anti-acne, protecting the liver. L-Glutathione also plays a role in the hepatic biotransformation and detoxification process. It acts as a hydrophilic molecule that is added to other lipophilic wastes before entering biliary excretion. A tripeptide with many roles in cells. It conjugates to drugs to make them more soluble for excretion. It is a cofactor for some enzymes.

It is involved in protein disulfide bond rearrangement and reduces peroxides. L-Glutathione is freely soluble in water, diluted alcohol, liquid ammonia, and dimethylformamide. Glutathione is an endogenous peptide with antioxidant and other metabolic functions. Glutathione and glutathione sodium is used to prevent neurotoxicity associated with cisplatin.

Congo red is an organic compound. The sodium salt of 3,3′-([1,1′-biphenyl]-4,4′-dial)bis(4-amino naphthalene-1-sulfonic acid). It is an azo dye. Congo red is water-soluble showing a red colloidal solution. Its solubility is greater in organic solvents.

In the literature review, various methods are available for the determination of L-Glutathione by spectrofluorimetric methods chemiluminescence methods, chromatographic methods, spectrophotometric methods. The present work is concentrated to achieve the optimum chromatographic conditions for the determination of L-Glutathione by UV-Spectroscopic method.

Figure 1. L-glutathione

Figure 2. Congo red

MATERIALS AND METHODS:

Materials:

The drug was purchased from Morril Fox (Panaji, Goa, India). All chemicals and reagents used were of analytical grade. The formulation of L-Glutathione was purchased from West-Coast Pharmaceutical Works Ltd (Ahmedabad, Gujrat, India). Congo red was purchased from Research – Lab (Mumbai, India).

Instrumentation:

Analytical balance (Shimadzu AT220),

UV-Visible Spectrophotometer (Shimadzu 1800).

Method Development:

Preparation of standard stock solution:

Accurately weighed 10mg of L-Glutathione then transferred to a 10ml calibrated volumetric flask. The volume was made up the mark with the distilled water to make up the volume (solutionA).

Accurately weighed 10mg congo red then transferred to 10ml calibrated volumetric flask. The volume was made up the mark with the distilled water to make up the volume (solution B).

Procedure for plotting a calibration curve:

Five different concentration 10,30,50,70,90µg/ml was prepared respectively. 0.1to 0.9 ml of standard solution was pipetted out separately from solution A and 0.2ml from solution B in a series of 10ml volumetric flask. The volume was made up to the mark with distilled water. The absorbance was measured at lambda max 568nm against the blank solution.

Estimation of L-Glutathione in Formulation:

10mg equivalent weight of L-Glutathione capsule was weighed and transferred to a 10ml calibrated volumetric flask. The volume was made up to the mark with distilled water (solution C). 0.5ml solution C and 0.2ml solution B were pipetted out and transferred to the 10ml calibrated volumetric flask. The volume was made up to the mark with distilled water and it was analyzed at 568nm then % purity of L-glutathione formulation was calculated.

RESULT:

The absorption spectrum obtained from L-glutathione and congo red mixture showed lambda max at 568nm. The developed method for L-glutathione was validated as per ICH guidelines for validation of analytical procedure.

Figure 3. UV Spectrum of Congo red

Figure 4. UV Spectrum of L-glutathione with congo red

Linearity:

The linearity of an analytical procedure is its ability to obtain test results directly proportional to the analyte concentration in the sample. Five different concentrations of L-glutathione and congo red were prepared and analyzed at 568nm wavelength. The correlation coefficient was found to be 0.9983.

Figure 5. Calibration curve for L-glutathione with congo red

Table 1. Linearity Result

Sr. No	Concentration (µg/ml)	Abs
1	10	0.159
2	30	0.264
3	50	0.352
4	70	0.441
5	90	0.525

Table 2. Optimization parameters

Parameters	Results
Lambda max	568nm
Range	10-90(µg/ml)
Regression equation	y = 0.0045x + 0.121
Correlation coefficient	0.9983
Intercept	0.121
Slope	0.0045
LOD	5.05(µg/ml)
LOQ	15.30(µg/ml)

Range:

The range of an analytical procedure is the interval between the upper and lower analyte concentration in the sample. It has been demonstrated that the analytical procedure has a suitable level of precision, accuracy, and linearity. The range was derived from the linearity studied. The range is an interval between the analyte's highest and lowest concentration limit. The range was found to be 10-90µg/ml.

The detection limit (LOD):

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The limit of detection was found to be 5.05µg/ml as per ICH guidelines.

Quantification limit (LOQ):

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The quantitation limit is a parameter of quantitative assays for low levels of compounds in sample matrices. The limit of quantification was found to be 15.30µg/ml as per ICH(Q2R1) guidelines.

Precision:

The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision, and reproducibility. The precision was performed at 30 µg/ml. six readings of the same concentration were prepared and %RSD was calculated. The %RSD was found to be less than 2% i.e.it should be found within the limit.

Table 3. Precision Results

Sr. No	Concentration (µg/ml)	Intraday	Intraday	Interday
1	30	0.247	0.243	0.243
2	30	0.245	0.242	0.243
3	30	0.244	0.245	0.242
4	30	0.245	0.245	0.239
5	30	0.246	0.244	0.237
6	30	0.244	0.241	0.239
Mean	-	0.245	0.243	0.240
Standard deviation	-	0.0011	0.0016	0.0025
%RSD	-	0.4768	0.6710	1.0436

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Accuracy should be established across a specified range of analytical procedures. Once linearity, precision has been established accuracy may be determined. Accuracy was determined at 3 levels 80,100,120% of label claim.

Table 4. Accuracy results

Level	Spiked Conc. (µg/ml)	Absorbance	Amount Recovered	%Recovery	Average	Standard Deviation	%RSD
	40	0.464	39.93	99.82
	40	0.461	39.85	99.62	99.65	0.1527	0.1532
80%	40	0.460	39.81	99.52
	50	0.571	49.91	99.82
	50	0.569	49.89	99.78	99.80	0.02	0.0200
100%	50	0.570	49.90	99.80
	60	0.695	59.93	99.88
120%	60	0.693	59.91	99.85	99.82	0.0793	0.0795
	60	0.689	59.84	99.73

System suitability:

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations, and samples are to be analyzed. System suitability is an integral part of analytical procedures. Six replicate readings of (30µg/ml) standard preparation were taken and %RSD was calculated and acceptance criteria should be less than 2%.

Table 5. System suitability study

Sr. No	Absorbance
1	0.237
2	0.239
3	0.239
4	0.242
5	0.242
6	0.243
Mean	0.240
Standard deviation	0.0025
%RSD	1.0436

Robustness

The robustness evaluation should be considered during the development phase and depends on the type of study under study. It should show the reliability of analysis concerning deliberate variations in method parameters. The developed analytical method was found to be robust. The evaluation of robustness should be considered during the development phase. Minor variation in wavelength never varies in the result. The result was found satisfactory as shown in table 6.

Table 6. Robustness

Sr. No	567nm	568nm	569nm
1	0.184	0.182	0.181
2	0.186	0.185	0.183
3	0.182	0.187	0.185
4	0.181	0.181	0.183
5	0.188	0.184	0.186
6	0.179	0.183	0.182
Mean	0.183	0.183	0.183
Standard deviation	0.0033	0.0021	0.0016
%RSD	1.1845	1.1761	1.0155

CONCLUSION:

A novel UV-Spectroscopy method was developed and validated for the determination of L-Glutathione in bulk and formulation with congo red. The developed method was accurate, precise, economical, linear, robust. The developed method was used for the analysis of L-glutathione and its formulation.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful to the Principal, D.S.T.S Mandal’s College of Pharmacy Solapur, for providing the necessary facilities.

REFERENCES:

1. British Pharmacopoeia, vol. 1, Her Majesty’s Stationary Office, London, United Kingdom, 2005, pp. 924–926.

2. L-glutathione [internet]. Drug Bank [cited Oct 24, 2021]. Available from: https://go.drugbank.com/drugs/DB00143.

3. L-glutathione image [internet]. wikipidia [cited Oct 24 2021]. Available from: https://en.wikipedia.org/wiki/File:Glutathione-skeletal.svg

4. A.P. Senft, T.P. Dalton, H.G. Shertzer, Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde, Anal. Biochem. 280 (2000) 80–86.

5. H. Wang, W.S. Wang, H.S. Zhang, A Spectrofluorimetric method for cysteine and glutathione using the fluorescence system of Zn(II)-8-hydroxyquinoline-5-sulfonic acid complex, Spectrochim. Acta A 57A (2001) 2403–2407.

6. S.C. Liang, H. Wang, Z.M. Zhang, X. Zhang, H.S. Zhang, Direct spectrofluorimetric determination of glutathione in biological samples using 5-maleimidyl-2-(m- methyl phenyl) benzoxazole, Anal. Chim. Acta 451 (2002) 211–219.

7. ICH Q2(R1). Validation of Analytical Procedures: Text and Methodology. ICH Harmonised Tripartite Guidelines, 1994.

8. British Pharmacopoeia, vol. 1, Her Majety’s Stationary Office, London, United Kingdom, 2005, pp. 924–926.

9. D.W. Lamson, M.S. Brignall, The use of nebulized glutathione in the treatment of emphysema: a case report, Altern. Med. Rev. 5 (2000) 429–431.

10. Zainab A. Bagalkote, Ganesh Gajeli. UV Spectrophotometric Method Development and Validation of Carbimazole in Bulk and Tablet Dosage form. Asian Journal of Pharmaceutical Research. 2021; 11(3):163-166.

11. Suryawanshi Anuja, Ansari Afaque, Kalshetti Mallinath. UV Spectrophotometric Method Development and Validation of Luliconazole in Bulk and Formulation. Research Journal of Pharmacy and Technology; Raipur Vol. 14, Iss. 7, (Jul 2021): 3826-3828.

12. PA Jadhav, CS Raut, JP Bidada, BB Buwa, PN Dhabale, SC Dhawale. Development and Statistical Validation of UV Spectrophotometric Method for Estimation of Griseofulvin in Tablet Dosage Form. Asian J. Research Chem. 3(2): April- June 2010; Page 404-406.

13. K. Vijaya Sri, S. Sravani, M. Shiva Kumar. Development and Validation of UV Spectrophotometric Method for Estimation of Lurasidone in Bulk and Pharmaceutical Formulations. Asian J. Pharm. Res. 5(2): April-June 2015; Page 102-107.

14. DVS Roopa Sirisha Doppa, Sathish Kumar Konidala, Sheik Khanabhi. Development and Validation of UV Spectroscopic Method for the Determination of Ranolazine in Bulk and Formulation. Research J. Pharm. and Tech. 2019; 12(10):5007-5010.

15. A.J.C. Hartfield, C. Bachelor-McAuley, R.C. Compton, Electrochemical determina- tion of glutathione: a review, Analyst 137 (2012) 2285–2296.

16. Vaishnavi Dulange and G.B. Gajeli Development and Validation of UV Spectroscopy Method for the Estimation of Dolutegravir in Bulk and Pharmaceutical Dosage Form. Asian Journal of Pharmaceutical Analysis,2021; 11(3): July-sep.

17. E. Bolonska-Sikora, J. Ozczuldlowski, Z. Witkiewicz, D. Widel, Glutathione: method of sample preparation for chromatography and capillary electrophoresis, Chemik 66 (2012) 929–942.

18. P. Monostori, G. Wittmann, E. Karg, S. Turi, Determination of glutathione and glu- tathione disulfide in biological samples: an in-depth review, J. Chromatogr. B 877 (2009) 3331–3346.

19. L. Manna, L. Valvo, P. Betto, Determination of oxidized and reduced glutathione in pharmaceuticals by reversed-phase high-performance liquid chromatography with dual electrochemical detection, J. Chromatogr. A 846 (1999) 59–64.

20. C.S. Yang, S.C. Chang, P.J. Tsai, W.Y. Chen, J.S. Kuo, Simultaneous measurement of ascorbic acid and glutathione: application of microdialysis and online HPLC with Au/Hg electrode in anaesthetized rat liver, J. Liq. Chromatogr. Relat. Technol. 21 (1998) 3139–3148.

21. Sushil D. Patil, Sayali K. Chaure, Sanjay Kshirsagar. Development and validation of UV spectrophotometric method for Simultaneous estimation of Empagliflozin and Metformin hydrochloride in bulk drugs. Asian J. Pharm. Ana. 2017; 7(2): 117-123.

22. X.M. Guan, B. Hoffman, C. Dwivedi, D.P. Matthees, A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples, J. Pharm. Biomed. Anal. 31 (2003) 251–261.

23. A.F. Loughlin, G.L. Skiles, D.W. Alberts, W.H. Schaefer, An ion exchange liquid chromatography-mass spectrometry method for the determination of reduced and oxi- dized glutathione and glutathione conjugates in hepatocytes, J. Pharm. Biomed. Anal. 26 (2001) 131–142.

24. Jyoti Mittha, Bhavana Habib. UV Spectrophotometric Method Development and Validation of Dasatinib in Bulk and Formulation. Asian Journal of Pharmaceutical Analysis. 2021; 11(3):203-206.

25. Y. Ma, F. Xiang, W. Jin, L. Yu, Determination of total glutathione in yeasts by high- performance liquid chromatography with dansylation, Z. Naturforsch. C 65 (2010) 391–394.

26. R. Kand’a´r, P. Za´kova´, H. Lotkova´, O. Kucera, Z. Cervinkova´, Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection, J. Pharm. Biomed. Anal. 43 (2007) 1382–1387.

27. S.I. Yakubu, I.A. Yakasai, A. Musa, Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane, J. Basic Clin. Pharmcol. 2 (2011) 151–158.

28. Z. Shen, H. Wang, S.C. Liang, Z.M. Zhang, H.S. Zhang, Spectrofluorimetric deter- mination of reduced glutathione in human blood using N-[p-(2-benzothiazolyl) phe- nyl] maleimide, Anal. Lett. 35 (2002) 2269–2278.

29. Y. Avi-Dor, R. Lipkin, A spectrophotometric method for the determination of reduced glutathione, J. Biol. Chem. 233 (1958) 69–72.

30. M.A. Raggi, L. Nobile, A.G. Giovannini, Spectrophotometric determination of glu-tathione and of its oxidation product in pharmaceutical dosage forms, J. Pharm. Biomed. Anal. 9 (1991) 1037–1040.

Received on 25.01.2022 Modified on 27.02.2022

Asian J. Pharm. Ana. 2022; 12(2):78-82.

DOI: 10.52711/2231-5675.2022.00014