Development and Validation of Stability Indicting HPLC Method for the Separation and Simultaneous Analysis of Timolol, Dorzolamide and Latanoprost Inophthalmic formulations

 

A. Krishnamanjari Pawar1*, Chandana Mannepalli2

1Associate Professor, A. U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India.

2Research Scholar, A. U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India.

*Corresponding Author E-mail: akmpawar@andhrauniversity.edu.in, chandana.mannepalli@gmail.com

 

ABSTRACT:

The present work is intended to establish a simple, precise and sensitive stability indicating HPLC method for the separation and simultaneous quantification of timolol, dorzolamide and latanoprost in pharmaceutical formulations. The separation of analytes was achieved on Spherisorb ods2 C18 (250mm 4.6mm; 5)as stationary phase, methanol, acetonitrile and phosphate buffer (pH 5.2) in 55:45:05 (v/v) as mobile phase at 1.0 mL/min and UV detection at 239nm. In this condition, well resolved, retained peaks were identified at 3.45 min fortimolol, 2.66min for dorzolamideand 5.43min for latanoprost. The method reports 0.313g/mL, 1.25g/mL and 0.003g/mL for timolol, dorzolamide and latanoprost respectivelyas LOD that proves that the method have enough sensitivity levels for the detectionanalytes in samples. The method passes all the validation parameters as per the guidelines proved that the method was valid. The method can shows very less % degradation in various stress studies such as acidic, base, peroxide, thermal and UV light conditions and can effectively separate various stress degradation compounds and confirms the stability indicating nature of the method. The method applicability was assessed by analysing the drug content in ophthalmic drops and reports the % assay of be 98.48, 99.37 and 98.32% for timolol, dorzolamide and latanoprost respectively. Based on the results, it can be concluded that the method can adequately suitable for the separation and quantification of timolol, dorzolamide and latanoprost and hence can be applicable for the routine analysis of timolol, dorzolamide and latanoprostin single or any combined ophthalmic formulations.

 

KEYWORDS: Timolol, Dorzolamide, Latanoprost, Stability indicating HPLC method, Forced degradation, Formulation analysis.

 

 


INTRODUCTION:

Timolol (Figure 1A) is a non-selective beta adrenergic blocker prescribed in single or in combination with other drugs for the treatment of increased pressure inside eye such as in ocular hypertension and glaucoma1. The oral administrated formulation of timolol is used to treat high blood pressure, prevent heart attacks and migraine headaches. The eye drop formulation of timolol is used to treat open-angle and secondary glaucoma whereas the gel formulation was applied to skin to treat infantile hemangiomas2.

 

Cardiac arrhythmias and severe bronchospasms are the serious side effects associated with the use of timolol. Fainting, depression, confusion, congestive heart failure, impotence and worsening of Raynaud's syndrome are the less serious side effects associated with use of timolol3.

 

Dorzolamide (Figure 1B) is a second-generation topical carbonic anhydrase inhibitor drug and a non-bacteriostatic sulfonamide derivative prescribed for the treatment of treats elevated intraocular pressure (IOP) associated with open-angle glaucoma and ocular hypertension5. It was used for the treatment of glaucoma and reduces the pressure inside the eye4. It lowers the excessive intraocular pressure in open-angle glaucoma and ocular hypertension. It was able to cross the cornea, reach the ciliary body of the eye, and produce systemic effects on the carbonic anhydrase enzyme within the eye5. Itching, burning, ocular stinging and bitter taste are the possible side effects possible during the use dorzolamide. It also causes swallowing of anterior chamber and leads to transient myopia6.

 

Latanoprost (Figure 2C) is a prostaglandin analog and an isopropyl ester prodrug prescribed for the treatment of treat increased pressure inside the eye (intraocular pressure). It was the first approved topical prostaglandin F2 alpha analog used for glaucoma treatment. Latanoprost is used in the treatment of ocular hypertension and open angle glaucoma7. Darkening of the iris, redness of the eye, itchiness and blurry vision are the possible side effects associated with the use of Latanoprost8. It was available in combination with other drugs such as netarsudil and timolol.

A) Timolol B) Dorzolamide

 

C) Latanoprost

 

Figure 1: Molecular structure of analytes in the study

 

The extensive literature review was conducted for the available analytical methods for the analysis of timolol, dorzolamide and latanoprost. In literature, few analytical methods were available for the analysis of timolol and dorzolamide9,10,11. The analytical methods were also reported for the estimation of timolol and latanoprost12,13,14. Based on the available literature and best of our knowledge there is no analytical method reported for the separation and simultaneous quantification of timolol, dorzolamide and latanoprostin ophthalmic formulations. Hence the present study was intended to develop a simple and precise stability indicating HPLC method for the quantification of timolol, dorzolamide and latanoprost in ophthalmic formulations.

 

MATERIALS AND METHODS:

Chemicals and reagents:

The active pharmaceutical ingredient of timolol (98.78 %) and dorzolamide (98.53) were obtained from Intas Pharmaceuticals Ltd, Hyderabad, Telangana. The latanoprost (99.01%) pure compound was procured from Micro Labs Ltd, Secunderabad, Telangana. The pharmaceutical formulation with brand TIM-DOR-LAT PF containing 0.5% of timolol, 2% of dorzolamide and 0.005% of latanoprost was procured from ImprimisRx.The HPLC grade methanol, acetonitrile and ultra-pure (Milli-Q) were purchased from Merck chemicals, Mumbai. The analytical reagent grade chemicals like hydrogen peroxide, sodium hydroxide, hydrochloric acid and buffer chemicals were also purchased from Merck chemicals, Mumbai.

 

Instrumental conditions:

The study was conducted on Agilent (USA) 1100HPLC instrument that comprises of G1311 Aquaternary pump for delivery of solvents, 0.1 1500μLvolume injectable auto-sampler with thermostat and UV detector (G 1314 A). Various configurations of stationary phases were used for the method development studies and the column eluents were integrated using Agilent chem-station software.

 

Preparation of standard solutions:

In a separate 25mL volumetric flasks filled with 10mL of methanol (diluent), an accurately weight 25mg of timolol, dorzolamide and latanoprost were mixed separately. Then it the flasks were sonicated for 2 min to dissolve the analytes in the solvent. Then the content was filtered through 0.2 membrane filter in a separate clean and dry flask separately and the final volume was made up to the mark with the same solvent. The timolol, dorzolamide and latanoproststandard solution at a concentration of 1000g/mL was obtained separately. The combined standard solutions were prepared by accurately mixing equal volumes of individual known standard stock solutions in a separate flask and were used for method development and validation study15-18

 

Preparation of test solution:

The TIM-DOR-LAT PF (0.5% of timolol, 2% of dorzolamide and 0.005% of latanoprost) ophthalmic formulations were used for the preparation of sample solution. An accurately pipetted two mL of the formulation pipetted in a 10mL volumetric flask containing 5mL of methanol. The solution was sonicated for 2min using ultrasonic bathsonicator and filtered through 0.2micron membrane filter in to a clean and dry 10mL volumetric flask. The final volume was made up to the mark using same diluent and the formulation stock solution at 100g/mL, 400g/mL and 1g/mL of timolol, dorzolamide and latanoprostrespectively. The formulation stock solution was further diluted to required concentration using the same diluent and the selected concentration solution was used for the quantification of timolol, dorzolamide and latanoprostin formulation sample.

 

Method development:

The systematic method development strategies were applied for developing method for the simultaneous analysis of timolol, dorzolamide and latanoprost. The detector wavelength was initially evaluated using UV-visible spectrophotometer. The individual UV absorption wavelength of timolol, dorzolamide and latanoprost was measured in the range of 400-200nm and the overlay absorption spectrum of the three analytesgives the iso-absorption wavelength. The iso-absorption wavelength was finalized as suitable detector wavelength for the detection of timolol, dorzolamide and latanoprost. The analytes in the study were polar in nature and the non-polar columns were utilized as stationary phases in the development of method. The high non-polar C18 columns of various brands and configurations were studied as stationary phase in the development study. The mobile phase was optimized by varying different composition of mobile phases with various pH ranges was studied. Initially, the flow rate of the mobile phase was fixed at 1.0mL/min and after optimization of the mobile phase composition, the flow rate was optimized in the range of 0.5mL/min to 1.5 mL/min. In each optimized condition studied, the standard solution containing known and selected concentration of 20g/mL, 80g/mL and 0.20g/mL of timolol, dorzolamide and latanoprostwas injected and the chromatographic response was recorded. The peak area response, peak intensity, peak shape and the system suitability was summarized in all the studied conditions. The method conditions that produce best system suitability with high peak intensity and significantly no noise was considered as suitable conditions for the separation and analysis of timolol, dorzolamide and latanoprost. These developed method conditions were further studied for method validation study.

 

Method Validation:

The system suitability of the method was assessed by analysing the chromatographic results obtained for analysing the standard solution containing 20g/mL oftimolol, 80g/mL of dorzolamide and 0.20g/ml latanoprost in the developed method. The method was further validated for linearity, precision, accuracy, sensitivity, ruggedness and robustness as per ICH guidelines18 and methods reported in literature19-32

 

The applicability of the method for the separation and identification of various stress degradation compounds was evaluated by performing various stress degradation studies such as acidic, base, peroxide, thermal and UV light. An accurately weighed 50mg of standard timolol, dorzolamide and latanoprostwas mixed separately with 50mL of hydrochloric acid (0.1N), sodium hydroxide (0.0N) and hydrogen peroxide (3%) in acid, base and peroxide degradation studies respectively. The solutions were incubated for 24h in dark, neutralized and then bring it to standard concentration prior to the analysis. The standard timolol, dorzolamide and latanoprostwas exposed to 600C for 24h in an air oven and UV light at 254nm for 24h in thermal and UV light degradation studies respectively. Both these standard drugs after stress exposer were diluted to standard concentration prior to the analysis. All the stress exposed dilute solutions were evaluated in the established method and the chromatograms observed in each analysis were observed for confirming the acceptability of the method. Further, the method applicability was evaluated by analysing the formulation solution prepared using the marketed formulation of timolol, dorzolamide and latanoprost. The prepared formulation solution was analyzed in the developed method. The peak area response of the resultant chromatogram was compared with the standard calibration and the % assay of the each analyte in the developed method was calculated. Based on the results achieved, the method applicability was assessed.

 

RESULTS AND DISCUSSIONS:

The overlay UV absorption spectrum confirms 239nm as suitable wavelength for the simultaneous detection of timolol, dorzolamide and latanoprost. Hence 239nm was selected as detector wavelength for the detection of analytes in the study. in the development of analytical method, various configurations of columns were studied for the effective separation of analytes using various compositions of mobile phases with different pH ranges. The optimized conditions obtained after the completion of method development trails along with system suitability results were summarized in table 1. The method passes the system suitability and hence was studied for further validation.

 

Table 1: Optimized HPLC conditions for the analysis of timolol, dorzolamide and latanoprost along with system suitability results

S. No.

Method Parameter

Results achieved

1

Stationary phase

Spherisorb ods2 C18 (250mm 4.6mm; 5)

2

Mobile Phase

Methanol, acetonitrile and phosphate buffer in 55:45:05 (v/v)

3

pH

5.2

4

Flow rate

1.0mL/min

5

Elution

Isocratic

6

Wavelength

239nm

7

Run time

8min

System suitability

Parameter

Results achieved for

Timolol

Dorzolamide

Latanoprost

8

Analyte strength

20g/mL

80g/mL

0.20g/mL

9

Retention Time

3.45min

2.66min

5.43min

10

Theo plate

7492

6814

12325

11

Tail Factor

1.03

0.98

0.95

12

Resolution

6.19

--

14.28

 

In these optimized chromatographic conditions, clear separation of timolol, dorzolamide and latanoprostwas achieved with no additional detection of impurities or other co-eluting compounds. The analytes were identified at a retention time of 3.45min, 2.66min and 5.43min respectively for timolol, dorzolamide and latanoprost. The chromatogram of blank in the optimized conditions doesnt any chromatographic detection throughout the run time. This confirms that the established method was specific for the detection of timolol, dorzolamide and latanoprost. The chromatogram of blank and standard observed in the developed method condition was represented in figure 4A and 4B respectively.

 

 

Figure 4: A. Blank chromatogram

 

 

Figure 4: B. Standard chromatogram

 

Figure 4: System suitability chromatograms of timolol, dorzolamide and latanoprost in the developed method

The standard calibration curve solutions of timolol, dorzolamide and latanoprostwas prepared and analysed in the optimized method. The high correlated calibration curve was attained in the analyte range of 5 30 g/mL, 20 120 g/mL and 0.05 0.30 g/mL for timolol, dorzolamide and latanoprostrespectively. The regression equation derived as y = 12543x + 5473.8 (R = 0.9994), y = 7711x + 18973 (R = 0.9991) and y = 321451x + 1181.4 (R = 0.9992) respectively for timolol, dorzolamide and latanoprost. The peak area results identified in the linearity study were represented in Table 2.

 

The repeatability and ruggedness of the method was evaluated using standard solution at 20g/mL of timolol, 80 g/mL of dorzolamide and 0.30g/mL of latanoprost. The solution was analyzed six times in the same day, three different days and six times by change in analyst for intraday precision, interday precision and ruggedness study respectively. The % relative standard deviation of the peak area response of the each analyte in each studied condition was calculated and a %RSD of less than 2 was considered as acceptable as per the guidelines. The %RSD was calculated as 0.29, 0.11 and 0.12 in intraday precision, 1.42, 0.20 and 0.26 in interday precision and 0.37, 1.34 and 0.27 in ruggedness for timolol, dorzolamide and latanoprost respectively. The %RSD was achieved under the acceptable levels for all the analytes in each study proved that the method was precise and rugged. (Table 3)

 

The influence of the variations in the developed method conditions on the chromatographic response was assessed in robustness study. The % change in the peak area response of individual analyte was calculated and results reported under the acceptable levels. The system suitability of the individual analyte in each changed conditions was summarized (Table 3) and results reported under the acceptable levels confirms that the method was rugged.


 

 

Table 2: Linearity results

S. No

Timolol

Dorzolamide

Latanoprost

Concentration

in g/mL

Peak

Area

Concentration

in g/mL

Peak Area

Concentration

in g/mL

Peak Area

1

5

71924.8

20

185638.0

0.05

18349.1

2

10

128728.7

40

319157.1

0.10

32838.1

3

15

191690.9

60

475719.2

0.15

48437.4

4

20

255463.8

80

632528.3

0.20

65461.6

5

25

316505.7

100

785250.7

0.25

80927.1

6

30

385491.7

120

954157.3

0.30

98598.7

 

Table 3: Robustness results

S. No.

Compound

Change

Peak Area

% Change

Plate Count

Tail factor

Resolution

1

Timolol

MP 1

252219.8

1.27

7485

1.03

6.15

2

MP 2

254124.5

0.52

7499

1.01

6.13

3

pH 1

253244.5

0.87

7577

1.02

6.18

4

pH 2

254166.0

0.51

7452

1.04

6.14

5

WL 1

254757.5

0.28

7491

1.04

6.15

6

WL 2

253329.2

0.84

7405

1.03

6.19

7

Dorzolamide

MP 1

627077.3

0.86

6758

0.97

--

8

MP 2

626569.6

0.94

6751

0.98

--

9

pH 1

626656.3

0.93

6788

0.98

--

10

pH 2

631364.9

0.18

6702

0.97

--

11

WL 1

629164.4

0.53

6734

0.97

--

12

WL 2

630339.5

0.35

6792

0.96

--

13

Latanoprost

MP 1

64916.8

0.83

12301

0.94

14.21

14

MP 2

65320.6

0.22

12385

0.94

14.29

15

pH 1

64951.6

0.78

12291

0.95

14.42

16

pH 2

64728.1

1.12

12287

0.94

14.58

17

WL 1

64764.5

1.06

12376

0.96

14.23

18

WL 2

64995.5

0.71

12251

0.94

14.51

Methanol, acetonitrile and phosphate buffer in 55:45:05 in MP 1 and 55:45:05 in MP 2;mobile phase pH changed as 5.1 in pH 1 and 5.3 in pH 2; detector wavelength changed as 234 in WL 1 and 244 nm in WL 2.

 

Table 4: Recovery results

S. No.

Compound

Recovery Level

Concentration prepared in g/mL

Amount found*

Mean SD

% Recovered*

Mean SD

% RSD of Recovery

1

Dorzolamide

50 %

60

59.640.1735

99.400.289

0.29

2

100 %

80

79.230.2774

99.040.347

0.35

3

150 %

100

99.520.09

99.520.090

0.09

4

Timolol

50 %

15

14.800.0728

98.690.485

0.49

5

100 %

20

19.740.1299

98.720.650

0.66

6

150 %

25

24.840.0808

99.370.323

0.33

7

Latanoprost

50 %

0.15

0.1480.001

98.900.668

0.68

8

100 %

0.2

0.1980.0009

98.950.446

0.45

9

150 %

0.25

0.2480.002

99.210.795

0.80

* n=3

 


The spiked recovery at 50%, 100% and 150% levels was performed for the evaluation of the accuracy of the method developed for the analysis of timolol, dorzolamide and latanoprost.The standard solution in the linearity study i.e 10g/mL oftimolol, 40g/mL of dorzolamideand 0.10g/mL latanoprostwas considered as target concentration in spiked recovery study. The solutions were evaluated in the optimized method and the peak area response was compared with the standard calibration results in the same level. The %recovery for each injection and the %RSD in each spiked level was calculated. The % recovery was observed to be with in the acceptable levels of 98-102% and the %RSD was observed to be less than 2 in each spiked level (Table 4) confirms the method accuracy.

 

The sensitivity of the developed method was assessed by determining the detection and quantification limit by adopting signal to noise ratio approach. The limit of detection was obtained as 0.313g/mL, 1.25g/mL and 0.003g/mL for timolol, dorzolamide and latanoprost respectively. The quantification limit was calculated as 1.033g/mL, 4.125g/mL and 0.010g/mL respectively for timolol, dorzolamide and latanoprost. This proved that the method was enough sensitivity for the detection and analysis of timolol, dorzolamide and latanoprost in pharmaceutical formulations.

 

The method was evaluated for its applicability for the separation and analysis of various compounds generated due to stress degradation of timolol, dorzolamide and latanoprost. The standard drug was exposed to various stress conditions and then the stressed sample was evaluated in the developed method. The resultant chromatograms and its results were analysed for the evaluation of its applicability for the separation of stress degradants.Among the stress degradation conditions studies, very high %degradation was observed in thermal degradation condition. In this the %degradation was observed to be 6.25, 7.32 and 5.99% respectively for timolol, dorzolamide and latanoprost with three additional degradation products were retained at 1.7 min, 4.6 min and 6.3 min. In acid degradation study, the % degradation was calculated as 4.19 min, 6.12min and 5.02 min respectively fortimolol, dorzolamide and latanoprost. In this condition, three additional degradation compounds were identified and retained at a retention time of 1.4min, 6.2min and 6.7min. The minimum degradation of 3.24, 2.91 and 2.85% was noticed for timolol, dorzolamide and latanoprost respectively and in this condition the chromatogram shows clear detection of two degradation compounds. In all the stressed conditions performed, the peaks correspond to timolol, dorzolamide and latanoprostwere detected and the retention time of analytes were in good correlation with the standard. The %degradation was noticed to be less in all the stressed conditions for all the analytesand the method can effectively resolve the stress degradants effectively proved that the method was stable.

 

Acid Degradation

 

Base Degradation

 

Peroxide Degradation

Thermal Degradation

 

UV Light Degradation

Figure 5: Forced degradation chromatograms

 

The analytical method optimized in the study was applied for its applicability for the estimation of timolol, dorzolamide and latanoprostin ophthalmic drops. The formulation solution prepared using TIM-DOR-LAT PF was used for the formulation assay study. The resultant chromatogram show clear identification and resolution of timolol, dorzolamide and latanoprost. The % assay was observed to be 98.48, 99.37 and 98.32% for timolol, dorzolamide and latanoprostrespectively. In the chromatogram (Figure 6), there no detection of impurities and there is no detection of additional compoundsas well as the formulation excipients. This confirms that the method was significantly used for the evaluation of timolol, dorzolamide and latanoprostin ophthalmic dosage forms.

 

Figure 6: Formulation chromatogram

 

CONCLUSION:

Ocular medications should be administered in the correct dose to ensure optimal efficacy and avoid patient side effects. The novel ophthalmic formulation of timolol, dorzolamide and latanoprostwas available in the challenging dosage of 0.5, 2 and 0.005%. So, there is a need to implement a simple, accurate, precise and sensitive HPLC procedure to overcome the problem raised by that ratio and allow analysingthree medications simultaneously. The suggested approach has various advantages being the first one for concurrent separation, reproducible, wide linearity ranges and has short retention time (less than 8 min). The HPLC technique was assessed as a whole approach and regarded inexpensive thanks to the comparatively low cost of the used mobile phase and the isocratic elution mode. HPLCUV apparatus is relatively available in many laboratories. The suggested method was successfully applied in real life situations by the estimation of drugs in eye drops as well as in their single eye drops with acceptable percentage recoveries. This encourages our suggested approach to be employed as an efficient, easy and eco-friendly analytical tool for routine high-throughput analysis required in research centres and quality control laboratories to ensure that precise doses of timolol, dorzolamide and latanoprost.

 

REFERENCES:

1.      Volotinen Marjo. TurpeinenMiia. Tolonen Ari. Uusitalo Jouko. Maenpaa Jukka. Pelkonen Olavi. Timolol Metabolism in Human Liver Microsomes is Mediated Principally by CYP2D6. Drug Metabolism and Disposition. 2007; 35(7): 1135. doi: 10.1124/dmd.106.012906.

2.      Atkin T,Comai S,Gobbi G. Drugs for Insomnia beyond Benzodiazepines: Pharmacology, Clinical Applications, and Discovery. Pharmacological Reviews. 2018; 70(2): 197. doi: 10.1124/pr.117.014381.

3.      Amano S,Nakai Y,Ko A, Inoue K,Wakakura M. A case of keratoconus progression associated with the use of topical latanoprost. Japanese Journal of Ophthalmology. 2008; 52(4); 334. doi: 10.1007/s10384-008-0554-6.

4.      Hasegawa T, Hara K,Kenmochi T,Hata S. In vitro metabolism of dorzolamide, a novel potent carbonic anhydrase inhibitor, in rat liver microsomes. Drug Metabolism & Disposition. 1994; 22(6): 5.

5.      Martens-Lobenhoffer J,Banditt P. Clinical pharmacokinetics of dorzolamide. Clinical Pharmacokinetics. 2002; 41(3): 197. doi: 10.2165/00003088-200241030-00004.

6.      Martens-Lobenhoffer J,Banditt P. Clinical pharmacokinetics of dorzolamide. Clinical Pharmacokinetics. 2002; 41(3): 197. doi: 10.2165/00003088-200241030-00004.

7.      Alm A. Latanoprost in the treatment of glaucoma. Clinical Ophthalmology. 2014; 26(8): 1967. doi:10.2147/OPTH.S59162.

8.      Amano S,Nakai Y,Ko A, Inoue K,Wakakura M. A case of keratoconus progression associated with the use of topical latanoprost. Japanese Journal of Ophthalmology. 2008; 52(4); 334. doi:10.1007/s10384-008-0554-6.

9.      Erk N. Rapid and sensitive HPLC method for the simultaneous determination of dorzolamide hydrochloride and timolol maleate in eye drops with diodearray and UV detection. Pharmazie. 2003; 58(7): 491.

10.   Gajanan Darwhekar, Priya Jain, Dinesh Kumar Jain,GauravAgrawal. Development and Optimization of Dorzolamide Hydrochloride and Timolol Maleate in Situ Gel for Glaucoma Treatment. Asian Journal of Pharmaceutical Analysis. 2011; 1(4): 93.

11.   Nitish Sharma, Surendra Singh Rao, Malleswara Reddy A. A Novel and Rapid Validated Stability-Indicating UPLC Method of Related Substances for Dorzolamide Hydrochloride and Timolol Maleate in Ophthalmic Dosage Form, Journal of Chromatographic Science. 2012; 1: doi:10.1093/chromsci/bms025.

12.   Adel EhabIbrahima, Hanaa Salehb, Magda Elhenaweeb. Assessment and validation of green stability indicating RP-HPLC method for simultaneous determination of timolol and latanoprost in pharmaceutical dosage forms using eco-friendly chiral mobile phase. Microchemical Journal. 2019; 148: 21. doi:10.1016/j.microc.2019.04.059.

13.   Mohamed Walash, Rania El-Shaheny. Fast separation and quantification of three antiglaucoma drugs by high-performance liquid chromatography UV detection. Journal of Food and Drug Analysis. 2016; 15(1): doi:10.1016/j.jfda.2015.11.006

14.   Rele RV, Mhatre VV,Parab JM,Warkar CB. Simultaneous RP HPLC determination of Latanoprost and Timolol Maleate in combined pharmaceutical dosage form. Journal of Chemical and Pharmaceutical Research. 2011; 3(1): 138.

15.   Bikshal Babu Kasimala, Venkateswara Rao Anna, Useni Reddy Mallu. Stability-indicating reversed-phase HPLC method for the separation and estimation of related impurities of cilnidipine in pharmaceutical formulations. Indian Drugs. 2018; 55(12): 41. doi:10.53879/id.55.12.11185.

16.   Rajesh Varma Bhupatiraju, Srinivasa Kumar B, Pavani Peddi, Venkata Swamy Tangeti. An effective HPLC method for evaluation of process related impurities of Letermovir and LC-MS/MS characterization of forced degradation compounds. J. Chem. Metrol. 2023; 2(2): 181-198 doi:10.25135/jcm.98.2311.2975

17.   Gajanan Darwhekar, Priya Jain, Dinesh Kumar Jain, Gaurav Agrawal. Development and Optimization of Dorzolamide Hydrochloride and Timolol Maleate in Situ Gel for Glaucoma Treatment. Asian Journal of Pharmaceutical Analysis. 2011; 1(4): 93.

18.   Murali Krishnam Raju P., Shyamala, Venkata Narayana B., Dantuluri HSNR., Bhupatiraju RV. A fast, validated UPLC method coupled with PDA-QDa detectors for impurity profiling in betamethasone acetate and betamethasone phosphate injectable suspension and isolation, identification, characterization of two thermal impurities. Ann. Pharm. Fr. 2022; 80(6): 837-852 doi:10.1016/j.pharma.2022.03.003

19.   ICH Expert Working Group. ICH Harmonised Tripartite GuidelineValidation of Analytical Procedures Text and Methodology: Q2 (R1). InGeneva: International Conference on Harmonisation of Technical Requirements for Registrataion of Pharmaceuticals for Human Use. 2005;

20.   Useni Reddy Mallu,VenkateswaraRao Anna, Bikshal Babu Kasimala. Rapid Stability Indicating HPLC Method for the Analysis of Leflunomide and Its Related Impurities in Bulk Drug and Formulations. Turkish Journal of Pharmaceutical Sciences. 2019; 16(4): 457. doi: 10.4274/tjps.galenos.2018.34635.

21.   Thangabalan B, Avinash Koya, Chaitanya G, Sunitha N, Manohar Babu S. Stability Indicating RPHPLC Method for the Estimation of Acamprosate in Pure and Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis. 2013; 3(4): 141

22.   Jakaria Md, Hazrat Ali Md, Areeful Haque Md, Mohammed Abu Sayeed, Shoayeb Ahmed. In vitro Comparative Forced Degradation Study of Different Brands and Active form of Montelukast sodium using UV Spectrophotometer. Asian Journal ofPharmaceutical Analysis. 2015; 5(1): 26 doi: 10.5958/2231-5675.2015.00005.8

23.   Rucha AP, Meghna PP, Hasumati AR, Nehal S. Forced Degradation Studies of OlmesartanMedoxomil and Characterization of Its Major Degradation Products by LC-MS/MS, NMR, IR and TLC. Asian Journal of Pharmaceutical Analysis. 2015; 5(3): 119 doi: 10.5958/2231-5675.2015.00019.8

24.   Patel VD, Raj Hasumati. Ranolazine: A Review on Analytical Method and Its Determination in Synthetic Mixture. Asian Journal ofPharmaceutical Analysis. 2015; 5(4): 214 doi:10.5958/2231-5675.2015.00034.4

25.   Chandana OSS, Ravichandra Babu R. Stability Indicating HPLC Method Development and Validation for Thalidomide and its Impurity Determination. Asian Journal of Pharmaceutical Analysis. 2016; 6(2): 115 doi:10.5958/2231-5675.2016.00017.X

26.   Tentu Nageswara Rao, Imad Hussain, Prashanthi Y, Patrudu TB. Forced Degradation Study for Tolterodine by HPLC with PDA Detection. Asian Journal of Pharmaceutical Analysis. 2019; 9(2):77 doi:10.5958/2231-5675.2019.00015.2

27.   Parag AP, Vinod AB, Yogesh SA, Bhaskar OA. Development and Validation of Stability Indicating UV Spectrophotometric Method for Estimation of Teneligliptine in Bulk and Tablet Dosage Form. Asian Journal of Pharmaceutical Analysis. 2019; 9(3):128 doi: 10.5958/2231-5675.2019.00024.3

28.   Surse SN, Patil SD, Deshmukh KR, Kshirsagar SJ. Development and Validation of Analytical Method by RP-HPLC and Forced Degradation Studies of Tioconazole Drug. Asian Journal of Pharmaceutical Analysis. 2019; 9(4): 229-231. doi: 10.5958/2231-5675.2019.00039.5

29.   Khushbu KP, Arati MP, Patel CN. A new simple RP-HPLC Method development, Validation and Forced degradation studies of Bilastine.Asian Journal of Pharmaceutical Analysis. 2021; 11(3):183 doi: 10.52711/2231-5675.2021.00031.

30.   Varma BHR, Rao BS. Gas Chromatography-Head Space-Mass Spectrometry Sensor based Quality Control of Dobutamine Hydrochloride Bulk Material for a mutagenic impurity, 2-bromopropane. Research Journal of Chemistry and Environment. 2023; 27: 54-61.doi:10.25303/2702rjce054061

31.   Rajesh VB, Battula SR, Kapavarapu MVNR, Mandapati VR. A novel Rivaroxaban degradation impurity detection by RP-HPLC extraction by preparative chromatography, and characterization by LC-MS, NMR and FT-IR: Analysis of novel impurity in batch samples and tablets of Rivaroxaban. Rasayan Journal Chemistry. 2022; 15: 2373-2381. https://doi.org/10.31788/RJC.2022.1547008

32.   Rajesh VB, Sreenivasa RB, Maruthi VNRK, Varaprasad RM. Assessment of gas chromatography methodology approach for the trace evaluation of carcinogenic impurity. methyl chloride, in trimetazidine dihydrochloride. Annales Pharmaceutiques Francaises. 2023; 81: 64-73.doi:10.1016/j.pharma.2022.06.012

33.   Varma RB, Rao BS. Gas Chromatography-Head Space-Flame Ionization Sensor based assessment of four residuary solvents in rivaroxaban bulk medication. Research Journal of Pharmacy and Technology. 2022; 15: 5158-5163. doi:10.52711/0974-360X.2022.00868

 

 

 

 

 

 

Received on 04.03.2023 Modified on 18.11.2023

Accepted on 04.03.2024 RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(5):1983-1990.

DOI: 10.52711/0974-360X.2024.00314