Sundar_1.pptx opthalmic injectable dosage form

opthalmic dosage form containing phenylepherine. lidocaine and tropicamide

1. GENERIC FORMULATION OF PHENYLEPHRINE HCL, LIDOCAINE, TROPICAMIDE AND EVALUATION OF ANALYTICAL METHOD VALIDATION OF ASSAY BY HPLC Presented by, SUNDHARA MOORTHY V M. Pharm Department of Pharmaceutics, Nandha College of Pharmacy, Erode-52. Under the guidance of, Dr. K.P. MOHANRAJ , M.Pharm ., Ph.D ., Department of Pharmaceutics, Nandha College of Pharmacy, Erode-52.

2. CONTENTS Introduction Literature review Aim and Objective Plan of work Drug profile Methodology Evaluation Result and discussion References

3. INTRODUCTION An essential part of medical care is the administration of drugs to the eye. Pharmaceutical scientists face several fascinating and difficult tasks, one of which is ophthalmic medication delivery. Ophthalmic preparations are specific dosage forms intended to be applied topically (topical), intraocular, periocular (near the eye), or in combination with any specialized instrument. The medication is administered intracamerally (IC) to the anterior chamber of the eye. Ophthalmic preparations must meet the same sterility standards as parentral dosage forms, in addition to taking into account preservation, tissue compatibility, osmotic pressure (tonicity), that can be avoidance of the pyrogens and their particle matter, and their appropriate packaging. Solutions and suspensions are common topical ophthalmic therapeutic dose formulations. The majority of the time, ophthalmic solutions are multidose products with appropriate preservatives that satisfy the standards of the compendial Preservative Efficacy Tests (USP, BP, EU, and JP). Medication is used topically to treat local conditions such mydriasis, miosis, bacterial infections, and intraocular pressure reduction.

4. Ideal characteristics Good penetration of the cornea. Optimizing the absorption of ocular drugs by extending the duration of interaction with corneal tissue. Less harmful effects and side effects. Reducing precorneal medication loss. Comfortable and non-irritating form (a viscous fluid shouldn't cause reflex blinking or lachrymal secretion). Advantages production and affordability in comparison to alternative dosage designs. It's possible that ophthalmic solutions contain more consistent doses. Increased bioavailability for eyes.

5. LITERATURE REVIEW Emma D. Deeks et al., (2019) Tropicamide 0.02% and phenylephrine 0.31% and lidocaine 1% injectable solution is the first fixed-dose mydriatic /anaesthetic combination approved for intracameral use in adults undergoing cataract surgery. The intracameral preparation, which is administered via a single injection, provides rapid and sustained mydriasis through to the end of surgery, with recipients spending around half as long in preoperative/surgery rooms as standard topical regimen recipients in the phase 3 study. Overall, the preparation was generally well tolerated, with no serious adverse events leading to hospitalization or permanent vision loss. Thus, tropicamide and phenylephrine and lidocaine injectable solution is an emerging option for mydriasis/ anaesthesia in adults undergoing cataract surgery. Dhruvil Nayak et al., (2023) The aim of the study is to assess the safety and efficacy of topical and intracameral application of a combination of tropicamide , phenylephrine and lidocaine during phacoemulsification surgery. A total of 50 patients were recruited patients who were operated with phacoemulsification surgery. During the intraoperative period, pupil size was studied and eventual adverse events have been monitored. Also, comfort reported by patients and surgeons has been investigated. It was observed that the intracameral drug combination did not have any impact on blood pressure, pulse rate as well as did not affect the Intra Ocular Pressure (IOP) dynamics and was successful in maintaining pupil size after its application during surgery. The combination also proved effective as an anaesthetic agent, which was proven by pain score findings, as the patients were comfortable and compliant enough to tolerate cataract surgery.

6. Yung-Chi Lee a et al., (1999) A Gelfoam ® based ocular device containing 1.7 mg of phenylephrine and 0.6 mg of tropicamide was formulated and evaluated for pupillary dilation in rabbits. The manufacturing procedure is fairly simple and the required excipients are inexpensive. The in vivo results show that the mydriatic response produced by the proposed device is larger and longer lasting than that produced by eyedrops with an equivalent amount of phenylephrine and tropicamide . The results reported in this study, along with those of previous studies, imply that Gelfoam ® is a versatile drug carrier for either local or systemic drug delivery via the ophthalmic route. M. J. Galmier et al., (1999) A high-performance liquid chromatographic method for the simultaneous determination of phenylephrine and tropicamide in human aqueous humor was developed. After centrifugation, an aliquot of the supernatant was injected onto the column and the eluent was monitored at 280 nm then 254 nm after 5 min. Separation was performed on a CN column with 0.01 M Pic B8(octane sulfonic acid)–acetonitrile (65:35, v/v) as mobile phase. The standard curves were linear in the detection range. The precision of the method (expressed by relative standard deviation) and the accuracy (mean error in per cent) were <5% for both intra- and inter assays.

7. AIM OF WORK Aim: Our aim of the project to develop generic formulation of phenylephrine HCL, lidocaine, tropicamide injectable solution for the treatment of cataract surgery. Objective To assess the efficacy and safety of intracameral mydriatic and anesthetic combination for pupillary dilation in pediatric cataract surgery by preparing the combination of Tropicamide 0.02%, phenylephrine 0.31%, lidocaine 1% for intracameral injectable solution.

8. DRUG PROFILE Lidocaine Solubility: Soluble in ethanol (50 mg/ml), water (9 mg/ml at 25° C), methanol, DMSO (47 mg/ml at 25° C), and chloroform. Very soluble in ethanol and in chloroform, freely soluble in benzene and in ether practically insoluble in water. Melting Point: Melts at about 62°C- 69°C with decomposition. Molecular Formula: C14H22N2O Molecular Weight: 234.3373 g/mol PH: 6.09

9. TROPICAMIDE Molecular Formula: C17H20N2O2 Molecular Weight: 284.353 g/mol Physiochemical Properties: Tropicamide is melts at about 96.5°C with decomposition Solubility: I. Tropicamide is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide. The solubility of tropicamide in these solvents is approximately 30 mg/ml. Tropicamide is sparingly soluble in aqueous buffers. II. Freely soluble in chloroform in ethanol (95 percent) and in solutions of strong acid; slightly soluble in water. PH: 5

10. PHENYLEPHERINE HCL Molecular Formula: C9H14ClNO2 Molecular Weight: 203.66 g/mol Physiochemical Properties: Phenylephrine hydrochloride is an odorless white microcrystalline powder. Bitter taste. Solubility: Freely soluble in water and in ethanol (95%) practically insoluble in chloroform. Melting Point: 143-145° C. PH: 5.8

11. Plan of work STAGE 1 Pre-formulation studies Solubility studies STAGE 2 Preparation of Tropicamide, phenylephrine hydrochloride and lidocaine ophthalmic injection STAGE 3 Evaluation Physical characterization description P H Conductivity Particulate matter 2. Analytical studies Identification test Assay potency Endotoxin test Sterility test Assay validation by HPLC method STAGE 4 Stability studies

12. METHODS OF PREPARATION Preparation of Tropicamide, Phenylephrine HCL, lidocaine ophthalmic injection Weigh accurately Tropicamide 0.2g, Phenylephrine hydrochloride 3.1g and Lidocaine 10g transferred into a beaker and dissolved in a beaker by using 700ml of water for injection and make up to 1000ml. Ingredients (mg) FORMULATION CODE F1 F2 F3 Tropicamide 0.16 0.2 0.05 Phenylephrine 2.58 3.1 0.02 Lidocaine 9.43 10 0.12 Phosphate buffer QS - - Water for injection - QS - Sterile water - - QS

13. EVALUATION 1. Description - A clear and colourless solution 2. pH - Calibrate the probe and meter according to the manufacturer’s directions. Use buffers with pH 4.0 and 7.0 to calibrate. Check the pH of the injection solution. The pH should be within the range of 6.0– 8.0 3. Standard calibration curve - A stock solution of (1mg/ml) of standard drug was prepared, later required dilutions were made with a water for injection pH 6.09. To a series of 10ml volumetric flasks aliquots standard solutions were taken and the volume was made up using a water for injection pH 6.09. The absorbance of these solutions was measured at respective wave length of maximum absorbance, using 1cm quartz cuvette in UV-Visible spectrophotometer. Absorbance values were plotted against respective concentration to obtain standard calibration curve.

14. Method of analysis by HPLC Preparation of buffer Weigh and transfer 5.7g of sodium acetate into a 1000ml beaker add 700ml of water sonicate to dissolve the content then volume make up to 1000ml after adding 1.636ml of acetic acid adjust pH4.4 with acetic acid and filter through 0.2µ membrane filter . Tropicamide stock solution Weigh and transfer 10mg of tropicamide into 10ml vol. flask add diluent to dissolve and sonicate for 5 minutes then make up to 10ml with diluent. Instrument Shimadzu HPLC equipped with UV Detector Mode Isocratic Column C8 Column Dimension 250 mm x 4.6 mm, 5 µm ( agilentzorbox )particle size Column Temperature 40°C Sampler temperature 25°C Flow rate 1.0 mL /minute Wavelength 234nm Detector UV Visible Injection Volume 20 µL Run time 15 minutes  

15. Phenylephrine hydrochloride stock solution Weigh and transfer 31mg of phenylephrine hydrochloride into 10ml volumetric flask add diluents to dissolve and sonicate for 5 minutes then make up to 10ml with diluent Standard Preparation Weigh and transfer 10mg of lidocaine in 10ml volumetric flask, add 5ml diluent sonicate to dissolve the content, add 0.2ml of tropicamide stock solution into it, then add 1ml phenylephrine stock solution and make up to 10ml using diluent (lidocaine HCl 1000µg/ml, tropicamide 20µg/ml, phenylephrine HCl 310µl/ml) Sample preparation From the sample solution pipette 1.0ml into 10ml volumetric flask volume make up with diluent Procedure (injection sequence) S. No Name Injections 1 Blank 1 2 Standard solution 6 4 Sample preparation 2 5 Bracketing standard solution 1

16. System suitability The RSD for area counts of replicate injections of standard preparation must be not more than 2.0 %. Theoretical plate counts for tropicamide , phenylephrine hydrochloride and lidocaine peak should be not less than 2000. Tailing factor for tropicamide , phenylephrine hydrochloride and lidocaine peak must be not more than 2.0. % RSD for bracketing standard should be within 2.0% with standard injection. Specification limits Test Specifications Limits mg/ml PLT Ophthalmic Injection   Tropicamide-99% to 101%w/v Phenylephrine Hydrochloride-95% to 105%w/v Lidocaine-95% to 105%w/v  

17. Bacterial endotoxin The endotoxin limit for a given test material or preparation is calculated from the expression K/m Endotoxin limit =K/M where K=maximum number of units of endotoxin 5.0 EU/kg of body weight (70kg) of intravenous administration. Maximum dose administrated to the patient of the test product per kg per hour. The test should be carried out in a manner that avoid any contamination. The container which is used in the test must be de- pyrogenated at 250°C for 2hrs Control Standard Endotoxin The control standard endotoxin (CSE) is freeze dried endotoxin which can be stored in refrigerator for not more than 28 days. Mixing vigorously not more than 3min before use should make subsequent dilution of the concentrate. Each dilution should be swirled for NLT 30 sec before processing to make the next dilution. Calculation of maximum valid dilution (MVD )

18. Sterility Membrane Filtration Method 1. Collect the samples to be tested for sterility as per SOP, comprising about 2% of the total batch material. Wipe the sample article individually with 70% IPA solution and keep in a clean S.S (Stainless steel) trays marked with Product Name, Batch No and Lot No, and then transfer the samples to the sterility room. 2. Prepare the media (FTM and SCDM) as per the SOP for preparation of culture media dispense 100 ml quantity in conical flasks & plug them. Sterilize both the media at 1210C and 15 psi pressure for 20 minutes as per SOP for Media Sterilization by Autoclaving. 3. After autoclaving label the tubes with Name of Media, Media Batch No. and pre-incubate the media tubes at appropriate temperature i.e. SCDM tubes at 20 to 250C whereas FTGM tubes at 30 to 35ºC for 24 - 48hrs before subjecting them for sterility operations. 4. Autoclave Dress, S.S. cups, receptacle unit, scissors and forceps in a S.S Container at 1210C temperature and 15psi pressure for 30 minutes as per SOP. 5. After sterilization cool the contents and aseptically transfer in a S.S. container to sterile working bench/ LAF. Start the LAF as per SOP for operating Instruction for LAF.

19. 6. Connect the Filtration manifold holder assembly with the S.S. reservoir properly with pipe and place sterilized S.S. cups in the sterile receptacle under laminar airflow unit. Place 0.45µ sterile membrane filters between filtration cup and receptacle with the help of sterilized forceps. 7. Wet the membrane filter by adding approximately 15 ml of sterilized Fluid A (0.1% peptone water) to filter holder, and filter the fluid by employing vacuum. 8. Cut the tip of bottle/vial with sterile SS blade in front of the gas burner and immediately transfer the contents to membrane 9. Immediately filter the solution with the aid of vacuum and wash the membrane three times with 100 ml of sterile water. 10. After complete filtration, stop the vacuum of manifold with the help of manifold vacuum control key. 11. Lift the membrane carefully with the help of sterile forceps, aseptically cut the membrane filter into two halves with sterile SS scissor and transfer one half to FTM and one half to SCDM tubes by unplugging in front of gas burner only . 12. Label both the tubes with product name, B.No , lot No., Date of testing, Completion date & Tested by.

20. 13. Simultaneously prepare a negative control by filtering 100 ml of 0.1% peptone water instead of product sample, cut the membrane into two halves with sterile SS scissor and transfer one half to FTM and one half to SCDM and label both the tubes as Negative control. 14. Incubate the FTM tubes at 300C – 350C and SCDM tubes at 200C – 250C for 14 days. 15. Prepare four positive Control tubes by inoculating aseptically 10 to 100cfu in FTM tubes with S. aureus, P. aeruginosa, B. subtilis. Similarly prepare three SCDM positive control by inoculating approximately 10 to 100cfu separately with C. albicans , A. niger . Incubate FTM positive control tubes at 30 – 35ºC for 3 days & SCDM positive control tubes at 20 – 25ºC for 5 days. 16. Visually examine the media tubes daily to its conclusion for macroscopic Evidence of microbial growth . 17. If no evidence of microbial growth is found in the repeat test the product examined complies with the test for sterility. If microbial growth is found in the repeat test the product examined does not comply with the test for sterility. 18. The test is not valid unless the Negative control shows negative till at end of incubation, and positive control shows growth within specified incubation period.

21. VALIDATION PARAMETERS 1. SPECIFICITY - To determine the ability of the method in differentiating the target analyte from other interfering substances from sample matrix . S. No Solution No. of Injections 1 Blank 1 2 Standard solution 6 3 Blank 1 4 Plain placebo 1 5 Sample 1 6 Bracketing Standard solution 1 2. PRECISION - To establish the closeness of agreement between a series of measurements from a multiple sampling of the same homogeneous sample for determination of tropicamide , phenylephrine hydrochloride and lidocaine content in tropicamide , phenylephrine hydrochloride and lidocaine ophthalmic injection . System precision Solution No. of Injections Purpose Blank 1 Blank Standard solution 6 Method validation

22. Method precision To determine the precision of the method, standard solutions, six-test solutions from sample solution will be prepared according to the procedure given in the methodology section 6.0 and standard deviation and % RSD for the same will be reported. S. No Name Injections 1 Blank 1 2 Standard Solution 6   4 Sample preparation 2 5 Bracketing standard solution 1 Intermediate precision Intermediate precision shall be evaluated by different analyst using a different HPLC system, different column and on a different day by using same sample solution used for method precision S. No Name Injections 1 Blank 1 2 Standard solution 6   4 Sample preparation 2 5 Bracketing standard solution 1

23. 3. ACCURACY - It is the closeness of the measured value to the true value . S. No Solution No. of Injections   Blank 1   Standard solution 6   Blank 1   Accuracy solution-1 50% level 2   Accuracy solution-2 50% level 2   Accuracy solution-3 50% level 2   Accuracy solution-1 100% level 2   Accuracy solution-2 100% level 2   Accuracy solution-3 100% level 2   Accuracy solution-1 150% level 2   Accuracy solution-2 150% level 2   Accuracy solution-3 150% level 2   Bracketing standard solution 1 4. Linearity To establish the linearity of the relationship of concentration and measurement results for determination of tropicamide , phenylephrine hydrochloride and lidocaine. Solution No. of Injections Purpose Blank 1 Blank Standard solution 6 System suitability/Quantification Blank 1 Blank Linearity solution-1 3 Method validation Linearity solution-2 3 Method validation Linearity solution-3 3 Method validation Linearity solution-4 3 Method validation Linearity solution-5 3 Method validation Bracketing standard solution 1 System suitability/Quantification

24. 5. Range The range of analytical method is the interval between the upper and lower levels of analyte that has been demonstrated to be determined with a suitable linearity, accuracy and precision. Robustness (mobile phase PH4.2 ) S. No Name Injections 1 Blank 1 2 Standard solution 6   4 Sample preparation 2 5 Bracketing standard solution 1 Robustness (mobile phase PH4.6) S. No Name Injections 1 Blank 1 2 Standard solution 6   4 Sample preparation 2 5 Bracketing standard solution 1

25. STABILITY OF SOLUTIONS ANALYTICAL SOLUTIONS STABILITY To establish the stability of standard and sample solutions by injecting them into various stipulated intervals up to 24 hours at bench top . Solution No. of Injections Purpose Blank 1 Blank Standard solution 6 System suitability/Quantification Standard solution (12 Hours) 1 Method validation Sample solution (24Hours) 1 Method validation Filter compatibility To determine the filter compatibility by using different types of 0.2µm membrane filters. Solution No. of Injections Purpose Blank 1 Blank Standard solution 6 System suitability Standard solution (Centrifuged) 1 Method validation Standard solution (0.2µ nylon filter) 1 Method validation Standard solution (0.2µm PTFE) 1 Method validation Standard solution (0.2µm PVDF filter) 1 Method validation Bracketing standard solution 1 Bracketing standard

26. RESULT AND DISCUSSION SOLUBILITY TEST Tropicamide - Freely soluble in chloroform, ethanol (95%) and strong acids. Slightly soluble in water phenylephrine hydrochloride - Freely soluble in water and 95% ethanol. lidocaine - Very soluble in ethanol and chloroform, freely soluble in benzene and ether . Lambda max

27. Calibration curve Lidocaine S. no Concentration (µg/ml) Absorbance (263nm) 1 2 20 0.215 3 40 0.402 4 60 0.615 5 80 0.802 6 100 0.982 Phenyl ephreine S. no Concentration (µg/ml) Absorbance (270nm) 1 2 20 0.015 3 40 0.026 4 60 0.037 5 80 0.048 6 100 0.057

28. Tropicamide S. no concentration (µg/ml) Absorbance (423nm) 1 2 20 0.127 3 40 0.246 4 60 0.367 5 80 0.478 6 100 0.592 pH 5.0

29. Particulate matter Light obscuration particle count test : ≥ 10 µm particle size ≥25 µm particle size 3000 per container 300 per container

30. Assay validation by HPLC method

31. Precision I. System precision Tropicamide Name of sample Area Mean %RSD Standard solution-1/1 219210     219805.5         0.14   Standard solution-1/2 220111 Standard solution-1/3 219939 Standard solution-1/4 219924 Standard solution-1/5 219872 Standard solution-1/6 219777 Phenylephrine Name of sample Area Mean %RSD Standard solution-1/1 794063     793709         0.07   Standard solution-1/2 794515 Standard solution-1/3 793624 Standard solution-1/4 793837 Standard solution-1/5 793358 Standard solution-1/6 792857 Lidocaine Name of sample Area Mean %RSD Standard solution-1/1 10019606     10015703         0.065   Standard solution-1/2 10024189 Standard solution-1/3 10017330 Standard solution-1/4 10016074 Standard solution-1/5 10011990 Standard solution-1/6 10005030

32. Method precision Tropicamide Phenylephrine

33. Lidocaine

34. Intermediate precision Tropicamide Name of the sample Area Mean %RSD % assay Standard-1/1 219724       218565.5             0.26         101% Standard-1/2 218446 Standard-1/3 218335 Standard-1/4 218317 Standard-1/5 218412 Standard-1/6 218159 Sample solution-1/1 220999     220960     0.02   Sample solution-1/2 220921 Phenylephrine Name of the sample Area Mean %RSD %assay Standard-1/1 781604       782208.8         0.06     Standard-1/2 781652 Standard-1/3 782763 Standard-1/4 782334 Standard-1/5 782621 Standard-1/6 782279 Sample solution-1/1 784261   784769     0.09   100.3 Sample solution-1/2 785277

35. Lidocaine Name of the sample Area Mean %RSD %assay Standard-1/1 9995999       9998153         0.015     Standard-1/2 9997066 Standard-1/3 9997872 Standard-1/4 9998145 Standard-1/5 10000313 Standard-1/6 9999525 Sample solution-1/1 10150057   10155471     0.075   101.5 Sample solution-1/2 10160884

36. ACCURACY Tropicamide Phenylephrine Lidocaine Name of the sample Area Mean %RSD Standard solution-1/1 219164   218713.2         0.12       Standard solution-1/2 218858 Standard solution-1/3 218433 Standard solution-1/4 218656 Standard solution-1/5 218601 Standard solution-1/6 218567 Name of the sample Area Mean %RSD Standard solution-1/1 785103       785098.8             0.02       Standard solution-1/2 785262 Standard solution-1/3 785207 Standard solution-1/4 785280 Standard solution-1/5 784938 Standard solution-1/6 784803 Name of the sample Area Mean %RSD Standard solution-1/1 10019162     10016982         0.01       Standard solution-1/2 10018545 Standard solution-1/3 10016126 Standard solution-1/4 10017652 Standard solution-1/5 10015348 Standard solution-1/6 10015058

37. Linearity Tropicamide Phenylephrine Lidocaine Name of the sample Area Mean %RSD Standard solution-1/1 227594     229076.8         0.38   Standard solution-1/2 228363 Standard solution-1/3 229557 Standard solution-1/4 229657 Standard solution-1/5 229785 Standard solution-1/6 229505 Name of the sample Area Mean %RSD Standard solution-1/1 688611     688611.7         0.03     Standard solution-1/2 688589 Standard solution-1/3 688378 Standard solution-1/4 688409 Standard solution-1/5 689032 Standard solution-1/6 688651 Name of the sample Area Mean %RSD Standard solution-1/1 10011201     10004425         0.03     Standard solution-1/2 10002246 Standard solution-1/3 10002839 Standard solution-1/4 10003980 Standard solution-1/5 10003011 Standard solution-1/6 10003275

38. Robustness (Mobile phase pH 4.6) Tropicamide Phenylephrine

39. Lidocaine

40. Stability of solutions Tropicamide Phenylephrine No of the sample Area Mean %RSD %ASSAY % Difference Standard solution-1/1 225844 226804.5   0.26         97.5%   Standard solution-1/2 226400 Standard solution-1/3 226809 Standard solution-1/4 227019 Standard solution-1/5 227393 Standard solution-1/6 227362 Standard solution24Hr-1/1 227479 221159.5 4.04   Standard solution 24Hr-1/2 214840 No of the sample Area Mean %RSD ASSAY % Difference Standard solution-1/1 618815 618437.5   0.038 99.5%   Standard solution-1/2 618589 Standard solution-1/3 618427 Standard solution-1/4 618360 Standard solution-1/5 618313 Standard solution-1/6 618121 Sample 24Hr-1/1 622152 615436.5   1.54   Sample 24Hr-1/2 608721

41. Lidocaine No of the sample Area Mean %RSD ASSAY % Difference Standard solution-1/1 9684117 9691374   0.04 97.5               Standard solution-1/2 9694619 Standard solution-1/3 9692961 Standard solution-1/4 9694264 Standard solution-1/5 9692977 Standard solution-1/6 9689304 Sample 24Hr-1/1 9723361 9451225   4.07   Sample 24Hr-1/2 9179088

42. Robustness pH 4.2 Tropicamide Phenylephrine

43. Lidocaine

44. Filter compatibility Tropicamide Phenylephrine No of the sample Area Mean %RSD ASSAY Standard solution-1/1 228271 229836.8   0.36   Standard solution-1/2 229596 Standard solution-1/3 230187 Standard solution-1/4 230609 Standard solution-1/5 230028 Standard solution-1/6 230330 Sample 0.2µ PTFE-1/1 215715 NA NA 93.85% Sample 0.2µ PVDF -1/1 215824 NA NA 93.9% Sample0.2µ nylon -1/1 215609 NA NA 93.8% No of the sample Area Mean %RSD ASSAY Standard solution-1/1 689971   689844.2   0.03   Standard solution-1/2 690065 Standard solution-1/3 689482 Standard solution-1/4 689947 Standard solution-1/5 689707 Standard solution-1/6 689893 Sample0.2µ PTFE-1/1 609971 NA NA 88.4% Sample 0.2µ PVDF -1/1 611326 NA NA 88.6% Sample0.2µ nylon -1/1 609844 NA NA 88.4%

45. Lidocaine No of the sample Area Mean %RSD ASSAY Standard solution-1/1 10021837 10018159     0.02   Standard solution-1/2 10018176 Standard solution-1/3 10016225 Standard solution-1/4 10018061 Standard solution-1/5 10016487 Standard solution-1/6 10018168 Sample0.2µ PTFE-1/1 9195065 NA NA 91.78% Sample 0.2µ PVDF -1/1 9189137 NA NA 91.72% Sample0.2µ nylon -1/1 9192072 NA NA 91.75%

46. DISCUSSION The formulation containing Phenylephrine HCl, Lidocaine, and Tropicamide serves various therapeutic purposes, including ophthalmic applications and local anesthesia. Developing a robust analytical method for the quantitative determination of these active pharmaceutical ingredients (APIs) is crucial to ensure the safety, efficacy, and quality of the formulated product. High-Performance Liquid Chromatography (HPLC) is a widely used technique for pharmaceutical analysis due to its sensitivity, specificity, and ability to separate complex mixtures . During the method development phase, parameters such as mobile phase composition, column selection, and detection wavelength were optimized to achieve adequate separation and resolution of the target compounds. The method validation process involved assessing various parameters, including specificity, linearity, accuracy, precision, and robustness, to ensure the reliability and reproducibility of the analytical results.

47. Specificity studies confirmed that the developed method could effectively separate Phenylephrine HCl, Lidocaine, and Tropicamide from potential interfering substances present in the formulation matrix. Linearity studies demonstrated a linear relationship between the concentration of the analytes and their respective peak areas over the specified range, indicating the method's suitability for quantitative analysis. Accuracy and precision studies revealed that the method provided accurate and precise results, with low relative standard deviations (RSDs) for both intra-day and inter-day analyses. Robustness studies further confirmed the method's ability to produce consistent results under different experimental conditions, such as variations in mobile phase composition and flow rate. Overall, the analytical method validation results indicate that the developed HPLC method is suitable for the quantitative determination of Phenylephrine HCl, Lidocaine, and Tropicamide in the generic formulation. This method can be employed for routine quality control testing to ensure the potency and uniformity of the formulated product.

48. CONCLUSION The development and validation of an HPLC method for the assay of Phenylephrine HCl, Lidocaine, and Tropicamide in the generic formulation have been successfully achieved. The method demonstrated good specificity, linearity, accuracy, precision, and robustness, meeting the regulatory requirements for pharmaceutical analysis. These results indicate that the developed method is suitable for routine analysis of the mentioned compounds in their combined formulation. Further studies could focus on the application of this method in stability studies, impurity profiling, and pharmacokinetic investigations to ensure the quality and efficacy of the formulated product.

49. REFERENCES