Timolol is a potent β-adrenergic blocker, useful in treatment of ocular hypertension or open-angle glaucoma.

Many chromatographic analysis methods have been applied for the determination of pharmaceutical compounds containing heterocyclic rings (as timolol maleate), but the most commonly applied chromatographic technique is HPLC.

Understanding the stability characteristics for both the active pharmaceutical ingredient (API) and for the drug product (DP) is crucial for the development of a safe and effective pharmaceutical agent. For this purpose, samples from API and DP during product development are disposed under strictly controlled storage conditions to assess stability testing. Forced degradation studies are being conducted to identify the degradation products that are likely to occur during long term storage as the worst- case scenario

Darkovska-Serafimovska, Marija

Serafimovska, Tijana

Velevska Stojanovska, Aleksandra

Veleska Stojanovska, Aleksandra

UGD Academic Repository

  Macedonian pharmaceutical bulletin, 68 (Suppl 1) 199 - 200 (2022)  Online ISSN 1857 - 8969   DOI:10.33320/maced.pharm.bull.2022.68.03.095 Short communication     *v.sani79@gmail.com                                                                                                                   S1 PP 80 Forced degradation of timolol maleate on high temperature for verification of HPLC method for related substances in                Timolol eye drop 0.5%  Aleksandra Veleska Stojanovska1*, Tijana Serafimovska2,  Marija Darkovska Serafimovska1   1Faculty of Medical sciences, University “Goce Delcev”, Krste Misirkov No.10-A, 2000 Stip, North Macedonia 2Faculty of Pharmacy, University “Ss. Cyril and Methodius”, Mother Tereza 47, 1000 Skopje, North Macedonia   Introduction  Timolol is a potent β-adrenergic blocker, useful in treatment of ocular hypertension or open-angle glaucoma. Many chromatographic analysis methods have been applied for the determination of pharmaceutical compounds containing heterocyclic rings (as timolol maleate), but the most commonly applied chromatographic technique is HPLC (Mandour et al., 2020).   Understanding the stability characteristics for both the active pharmaceutical ingredient (API) and for the medicinal product (MP) is crucial for  the development of a safe and effective pharmaceutical agent. For this purpose, samples from API and MP during product development are disposed under strictly controlled storage conditions to assess stability testing. Forced degradation studies are being conducted to identify the degradation products that are likely to occur during long term storage as the worst-case scenario (Sengupta et al., 2018).  Materials and methods   The forced degradation studies were performed on API Timolol maleate (batch 005009102019) and MP Timolol eye drops 0.5% (batch 2112, expiry date: 12.2023) manufactured by Profarma Sh.a, Albania. Forced degradation was done only by exposing the API and MP on high temperature at 80°C for 48 hours to obtain degradation products quickly and to verify the method for determination of related substances listed in the British pharmacopoeia (BP, 2016). Identification and quantification of degradation products was performed at room temperature, using an isocratic method and reverse phase HPLC stainless steel column (30 cm x 4.6 mm) packed with end-capped octadecyl silyl silica gel for chromatography (10 µm) (µBondapakTM C18 was used) on HPLC system (Agilent Technologies Infinity II 1260, DAD HS 1260: Serial No. DEAEK06706). Mobile phase was consisting of mixture of 0.02 M sodium octane-sulfonate and methanol in ratio 42,5:57,5; adjusted to pH 3.0 using glacial acetic acid. An isocratic method elution was used with flow rate of 1.5 mL per minute on ambient column temperature. UV detection was on wavelength of 295 nm. Injection volume was 20 µL of each solution and run time was 4 times of the retention time of the principal peak of timolol maleate.  Results and discussion  Verification of the HPLC method was done according to ICH guidelines Q2 (R1) Validation of Analytical Procedures (ICH Q2 (R1), 2022). For forced degradation studies the chosen stress condition (temperature of 80°C for 48 hours) leads us to only one secondary peak at RRT 1,6 due to exposition of the product at higher temperature.  The parameter specificity, demonstrate good separation of all peaks derived from active substance 200  Pharmaceutical Analysis/Quality Assurance/Regulatory Affairs  Maced. pharm. bull., 68 (Suppl 1) 199 - 200 (2022) (timolol maleate, secondary peak because of force degradation and maleic acid) and excipients (placebo).  The precision and reproducibility of the proposed method were evaluated by performing replicate analysis of the standard solution timolol maleate in concentration of 0.02 mg/mL (reference limit for impurities), to determine intraday and inter-day variability [within day (n=6) and between days (n=6)]. Relative standard deviations were calculated to obtain the precision of the method. Relative standard deviations were calculated to obtain the precision of the method. The results of precision, and reproducibility of the method demonstrate a good precision (RSD =1.05%). Linearity was performed using timolol maleate standard solutions in concentration range from 0.005 mg/mL (limit of quantification-LOQ) to 0.03 mg/mL or 25%-150% from the reference limit for impurities. The response was linear over the range (R2=0.9999). Limit of Detection LOD and LOQ were determined by evaluation peak areas of timolol maleate at lower concentrations. Acceptance criteria were signal to noise ratio about 3:1 for LOD and about 10:1 for LOQ. According to results LOD for timolol was 0.05 µg/mL, and LOQ for timolol was 0.15 µg/mL.    Conclusion  From a regulatory perspective, the combined use of stress testing and accelerated degradation studies with an optimized separation technique and validation procedures is a useful approach to achieve a comprehensive understanding of API and MP stability, with regards to the nature of the relevant degradation products, within a reasonable timeframe.  The forced degradation studies and its application are mainly used for the determination of stability of molecule under accelerated conditions and for the development of stability indicating methods. In our case, forced degradation was done only by exposing the API and MP on high temperature at 80°C for 48 hours to obtain degradation products quickly and to verify the method for determination of related substances listed in the British   pharmacopoeia. Testing of specificity of the method demonstrate good separation of all peaks derived from active substance and excipients in the formulation. Verification of the HPLC method was done according to the ICH guideline Q2 (R1) to prove that monograph in BP for timolol eye drops is suitable for analyzing of degradation products in our formulation.    Acknowledgement   The authors are grateful to the management of Profarma,Sh.a,, Albania for enabling this research done in their laboratory for quality control to be presented on this Congress. Authors want to thank to all the employers in Profarma Sh.a Albania that have contributed to this research.  References  British Pharmacopoeia, 2016. Available at: https://www.pharmacopoeia.com, (last access: 29th May, 2022) ICH Q2R1: Validation of Analytical Procedures: Text and Methodology. Proceeding of the International Conference on Harmonization of Technical Requirements for the Registration of Drugs for Human Use, Geneva, Switzerland, 2022, (EMA/CHMP/ICH/82072/2006). Available at: https://www.ema.europa.eu/en/documents/ scientific-guideline/ich-guideline-q2r2-validation-analytical-procedures-step-2b_en.pdf, last access: 29th May, 2022 Mandour, A.A., Nabil, N., Zaazaa, H.E., 2020 Review on analytical studies of some pharmaceutical compounds containing heterocyclic rings: brinzolamide, timolol maleate, flumethasone pivalate, and clioquinol. Futur. J Pharm. Sci. 6, 52. https://doi.org/10.1186/s43094-020-00068-4   Sengupta, P., Chatterjee, B., Tekade, R.K., 2018. Current regulatory requirements and practical approaches for stability analysis of pharmaceutical products: A comprehensive review. International Journal of Pharmaceutics, 543(1-2), 328-344.     https://doi.org/10.1016/j.ijpharm.2018.04.007               

Forced degradation of timolol maleate on high temperature for

verification of HPLC method for related substances in

Timolol eye drop 0.5%

               Forced degradation of  timolol maleate on high temperature for verification of  HPLC method for related substances in Timolol eye drop 0.5% Aleksandra Veleska Stojanovska1; Tijana Serafimovska2; Marija Darkovska Serafimovska1   1Faculty of  Medical sciences, University “Goce Delcev”, Krste Misirkov No.10-A, 2000 Stip, North Macedonia 2Faculty of  Pharmacy, University “Ss. Cyril and Methodius”, Mother Tereza 47, 1000 Skopje, North Macedonia INTRODUCTION  Timolol is a potent β-adrenergic blocker, useful in treatment of  ocular hypertension or open-angle glaucoma. Many chromatographic analysis methods have been applied for the determination of  pharmaceutical compounds containing heterocyclic rings (as timolol maleate), but the most commonly applied chromatographic technique is HPLC. MATERIAL AND METHODS:       Chemicals and Regents Mobile phase was consisting of  mixture of  0.02M sodium octane-sulfonate and methanol in ratio 42,5:57,5; adjusted to pH 3.0 using glacial acetic acid.   Apparatus: Liquid chromatography was performed with stainless steel column (30cm x 4.6mm) packed with end-capped octadecyl silyl silica gel for chromatography (10 µm) (µBondapakTM C18 was used) on HPLC system (Agilent Technologies Infinity II 1260, DAD HS 1260: Serial No. DEAEK06706).      Chromatographic Conditions: An isocratic method elution was used with flow rate of  1.5 mL per minute on ambient column temperature. UV detection was on wavelength of  295 nm. Injection volume was 20 µL of  each solution and run time was 4 times of  the retention time of  the principal peak of  timolol maleate. RESULTS:      Verification of the method Linearity was performed using timolol maleate standard solutions in concentration range from 0.005 mg/ml (limit of  quantification-LOQ) to 0.03 mg/ml or 25%-150% from the reference limit for impurities. The response was linear and the coefficient of correlation was greater than 0.999.  The range (R2=0.9999) is shown on Figure 1. Linearity of  the method is shown in Table 1. Limit of  Detection (LOD) and (LOQ) were determined by evaluation peak areas of  timolol maleate at lower concentrations. Acceptance criteria were signal to noise ratio about 3:1 for LOD and about 10:1 for LOQ. According to results LOD for timolol was 0,05 µg/ml, and LOQ for timolol was 0,15 µg/ml.  CONCLUSION  Forced degradation was done only by exposing the API and DP on high temperature at 80°C for 48 hours to obtain degradation products quickly and to verify the method for determination of  related substances listed in the British   pharmacopoeia.   For forced degradation studies the chosen stress condition (temperature of  80°C for 48 hours) leads us to only one secondary peak at RRT 1,6 due to exposition of  the product at higher temperature.  Range (%) Concentration levels (mg/ml) Peak Area (mAU*s) Mean Peak Area (n=3) LOQ* 0.0001                                           0.0001                                         0.0001                                            0.0001                                             0.0001                                3.58993.591183.769283.898493.708013.71137 25% 0.005                                               0.005                                           0.005                                                   0.005                                             0.005 93.1           92.48            91.68   91.63           92.34 92.2550% 0.01001                                                     0.01001                                                       0.01001                                                   0.01001                                           0.01001 181.38               180.92181.31181.06         180.6  181.0575% 0.01501                                            0.01501                                                0.01501                                               0.01501                                                   0.01501  276.95          277.1    276.71  276.74276.96 276.89 100% 0.02002                                        0.02002                                  0.02002                                       0.02002                                  0.02002 363.06         363.78                  362.97            363.09                363.04 363.19 150% 0.03003                                                         0.03003                                                0.03003                                            0.03003                                           0.03003 546.71547.19   545.92      546.13       546.65 546.52 Table 1. Linearity of the method y = 18145x + 1.4852 R² = 0.9999 0 100 200 300 400 500 600 0.000 0.010 0.020 0.030 0.040 Peak Area (mAU*s) Concentration (mg/ml) Linearity - Timolol Figure 1. Linearity and the response R2=0.9999. The parameter specificity, demonstrate good separation of all peaks derived from active substance (timolol maleate, secondary peak because of force degradation and maleic acid) and excipients (placebo).  The parameter specificity, demonstrate good separation of  all peaks derived from active substance (timolol maleate, secondary peak because of  force degradation and maleic acid).  Specificity is shown in Figure 2.  Figure 2. Specificity of the method and secondary peak. The precision and reproducibility of  the proposed method were evaluated by performing replicate analysis of  the standard solution timolol maleate in concentration of  0,02mg/ml (reference limit for impurities), to determine intraday and inter-day variability [within day          (n = 6) and between days (n = 6)].   Relative standard deviations were calculated to obtain the precision of  the method. The results of  precision, and reproducibility of  the method demonstrate a good precision             (RSD =1,05%). 

Forced degradation of timolol maleate on high temperature for verification of HPLC method for related substances in Timolol eye drop 0.5%

https://eprints.ugd.edu.mk/30293/1/Forced%20degradation%20of%20timolol%20maleate%20on%20high%20temperature%20for%20verification%20of%20HPLC%20method%20for%20related%20substances%20in%20timolol%20eye%20drop%200.5%25.pdf

Forced degradation of timolol maleate on high temperature for verification of HPLC method for related substances in Timolol eye drop 0.5%

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