Design of Experiment Utilization to Develop a Simple and Robust RP-UPLC Method for Stability Indicating Method of anti glaucoma Ophthalmic Drops
More details
Hide details
Quality Control Department, EIPICO, Zagazig University, Zagazig, Egypt
Medicinal Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
Publish date: 2017-10-05
Eurasian J Anal Chem 2015;10(1):46–67
Brimonidine Tartrate and Timolol Maleate are used in treatment of glaucoma by decreasing intra ocular pressure. It is necessary to establish a Validated UPLC method for the assay of Brimonidine Tartrate and Timolol Maleate as well as Benzalkonium Chloride in eye drops. Using of DOE techniques provides more information about different factors affects system suitability of new method. DOE led to highly robust methods through creating design space. The method was performed on Phenomenex Kinetex C18 (50x4.6 mm, 2.6 µm) and the mobile phase consisted of Methanol and 1- Decane sulphonic acid sodium (pH 3.0, 0.01M) which pumped at a flow rate 0.6 mLmin-1 at 50 °C in gradient manner. 10 µL of drugs sample solutions were monitored at wavelengths 320 nm.Brimonidine Tartrate, Timolol Maleate and Combigan ED were stressed under different conditions in forced degradation studies. Degradation products had a good resolution with main drugs and Benzalkonium Chloride. The proposed method was validated in terms of linearity, accuracy, precision and limits of detection and quantitation according to ICH.
Hani M. Hafez   
Quality Control Department, EIPICO, Zagazig University, Zagazig, Egypt
1. Jushuf IHA et al (2009) British national formulary 57, BMJ Group, London.
2. British Pharmacopoeia (2014), London with the monographs of the sixth edition of the European pharmacopoeia monograph Accessed 03/10/2014
3. Health wise Knowledgebase, US Pharmacopoeia (2014) Rockville,, Accessed 03/10/ 2014
4. Merianos JJ (1991) Quaternary Ammonium Antimicrobial Compounds in: Block SS (Ed.), Disinfection, Sterilization, and Preservation, 4th Ed. Lea & Febiger, Malvern, PA, 225–255.
5. Elzanfaly ES, Saad AS, AbdElaleem AB (2012) A smart simple Spectrophotometric method for simultaneous determination of binary mixtures. J Pharm Anal 2(5):382-385
6. Jain PS, Khatal RN, Jivani HN, Surana SJ (2011) Development and Validation of TLC-densitometry Method for Simultaneous Estimation of Brimonidine tartrate and Timolol maleate in Bulk and Pharmaceutical Dosage Form. J Chromatograph Separat Techniq 2: 113-117
7. Phogat A, Kumar MS, Mahadevan N (2011) Simultaneous Estimation of Brimonidine Tartrate and Timolol Maleate in Nanoparticles Formulation by RP-HPLC. Int J Rec Adv Pharm Res 3: 31-36
8. Nagaraju P, Kiran KS, Poornima CH (2014) Development and Validation of RPHPLC Method for the Simultaneous Estimation of Brimonidine tartrate and Timolol Maleate in Combined Dosage form, AJCPR, 2: 58-64
9. Huynh-Ba K (2009) Handbook of Stability Testing in Pharmaceutical Development, Springer Science & Business Media, LLC New York, USA, 1st ed.
10. Baertschi SW (2005) Pharmaceutical Stress Testing, Predicting Drug Degradation, Taylor & Francis Group, New York, 1st ed.
11. Preparing Buffered Mobile Phases for Reversed Phase HPLC, Accessed 01/10/2014
12. Schmidta AH, Molnar I (2013) Using an innovative Quality-by-Design approach for development of a stability indicating UHPLC method for ebastine in the API and pharmaceutical formulations. Journal of Pharmaceutical and Biomedical Analysis 78-79: 65– 74
13. Snyder LR, Kirkland JJ, Glajch JL (1997) Practical HPLC Method Development, 2nd ed, Wiley Interscience, New York
14. Nickerson B (2011) Sample Preparation of Pharmaceutical Dosage Forms, Springer, Heidelberg.
15. Neue UD, O’Gara JE, Mendez A (2006) Selectivity in reversed-phase separations influence of the stationary phase. J Chromatogr A, 1127: 161–174
16. Monks K, Molnar I, Rieger HJ, Bogati B, Szabo E (2012), Quality by design: multidimensional exploration of the design space in high performance liquid chromatography method development for better robustness before validation. J Chromatogr A, 1232: 218–230.
17. ICH Harmonized Tripartite Guideline (1997) Validation of Analytical Procedures: Text and Methodology Q2 (R1).
18. Mehta J, Patel V, Kshatri N, Vyas N (2010) A versatile LC method for the simultaneous quantification of latanoprost, timolol and benzalkonium chloride and related substances in the presence of their degradation products in ophthalmic solution. Anal Methods 2: 1737-1744.