Optimization and Validation of RP-HPLC Method for Quantification of Timolol Maleate in Bulk Drug and Ophthalmic Formulation

Analytical chemistry is an area of chemistry that deals with the analysis of the composition of matter by breaking down chemical substances in most cases into their individual components and secondly identifying the sub components and lastly detecting impurities that may be present. It is used on nearly all sectors in the modern industry. It is majorly concerned with particular methods to establish and record the chemical composition of natural and artificially-produced materials. [1] Analytical methods are classified into Instrumental method and chemical method. Instrumental method is based on the measurement of light absorption or emission of fluorescence via conductivity to electrode potential. Chemical method involves mass of the analyte estimated by gravimetric and volumetric titration. Quality control techniques, including analysis are very important for the up keep and guarantee of material and are important parts of quality assurance and quality control. Several instrumental methods are used in pharmaceutical analysis, amongst these some important methods are separation techniques, spectrometric techniques and other analytical techniques.  Pharmaceutical analysis is the integral part of the pharmaceutical sciences. In pharmaceutical analysis section, the research analyst is responsible for three important functions

viz: a) Raw material, active compound and product analytical method development for feeling performer oil and feeling well breaker.

b) Method development for selective estimation of drug and constituents components, impurities and degradation products, as well as detection of a degradation product, a degradation pathway and degree of degradation during kept at ambient and accelerated conditions.

c) Development of analytical method for micro and semi micro quantities of drugs and its metabolites in biological system. [2,3]

Analytical chemistry has been providing tools and techniques since the early days of chemistry. Founding scientists like Justus von Liebig contributed to the development of elemental analysis, while Wittgenstein built upon their work. The earliest instrumental method for determining trace elements was flame emission spectroscopy, developed in 1860 by Robert Bunsen and Gustav Kirchhoff. The central themes of analytical chemistry developed in the 20th century after 1930, with the 76th IUPAC council starting a new style that would continue into the 21st century. Instrumental methods are now the dominant tools in chemical analysis, with researchers developing new techniques and those applying them. Analytical chemistry plays a critical role in the pharmaceutical industry, used for drug candidate screening, in-process monitoring of product quality, and clinical monitoring of drug-patient interaction. [4] Method validation is the process of ensuring the analytical procedure used in tests is suitable for its purpose. It assesses the quality, reliability, and consistency of testing results and is crucial for sound data analysis. It involves defining an analysis requirement and verifying the proposed method's expected performance. The use of suitable equipment and competent investigators are essential. Methods must be validated or re-validated before routine use, when conditions change, or when a method change is beyond its scope. [6,7]

RP-HPLC Method Design and Validation:

Reversed-phase HPLC method development is a routine yet essential exercise for many analytical chemists. For complex mixtures, the procedure is both systematic and empirical. Choice of suitable column, mobile phase composition and detection conditions is critical in effective separation. The most widely successful (and used) Tech-schedule system in LC is that of Reversed-Phase systems using bonded silica such as the C18, due its overall compatibility to a large number of compounds. [8,9]

Solubility Consideration in Method Development:

Solubility is the active ingredient should be screened for solubility in different solvents, both aqueous (buffer, water, saline) and organic (methanol, THF, chloroform, hexane). This also aids in the assessment of the API as acidic or basic: 2 The pH of the aqueous-based solution is determined to keep in control the titrant addition. The API would ideally be completely dissolvable at 1 mg/mL for the selected solvent. UV scan readings from 200 to 400 nm can be used to target absorbance peaks for additional method development.

Diluent selection:

The ideal diluent should:

• Dissolve both the API and its related impurities.

• Aid in generating well-defined peaks.

• Maintain analyte chemical stability.

• Prevent interaction with the sample container.

• Avoid degradation of the drug substance.

Wavelength optimization:

Selection of the detection wavelength is important. Nanoparticles made of such potential drugs are prepared and scanned in a UV spectrophotometer for best absorbance. The product is verified by HPLC equipped with a diode array detector. The wavelength with maximum, highest signal intensity and minimum variability is chosen for routine use.

Optimization of Chromatographic Parameters:

Normal Phase Vs Reversed Phase: [10]

It is not an absolute although RP chromatography is the workhorse because it can be so widely applied; NP chromatography is used in preference when:

• The sample dissolves with non-polar solvents (hexane, chloroform) exclusively.

• The analyte is too strongly retained on the RP column and the elution is too slow.

• The compound is not kept in RP.

• The resolution of isomer compounds is not very good.

• The analyte is not stable in solvent with base water.

The terms "normal phase" and "reverse phase" refer to having the mobile phase more polar or less polar than the stationary phase, respectively. A common initiating condition would be 100% isopropanol on a cyano-bonded column and attenuate using less aggressive solvents such as hexane.

Developing RP-HPLC Method:

In RP-HPLC, the mobile phase is less polar than the stationary phase. It generally stained with solvent blends (like water/methanol or acetonitrile) with our without other additives.

• Let's pick appropriate column to start.

• Retention and selectivity can be fine-tuned by changing solvent ratios.

• Begin by trying isocratic runs and, when necessary, use gradients.

• Make certain that the solvent is suitable for the column and detector.

• If possible, use HPLC quality solvents; the less crap in your solutions, the better.

Buffer Selection:

Buffer choice depends on:

• UV transparency.

• Chemical stability.

• Work with column materials.

• Preferred pH (30 is generally from 2.0 to 8.0).

Second, the buffer has to minimize the ionization of the analyte close to its pKa to prevent peak distortion. Keep a buffer strength of at least 2% to prevent separation.

pH consideration:

If the pH of the mobile phase is close to the pKa of the analyte, only partial 47 ionization occurs in the mobile phase and it leads to tailing or broad peak shapes of the analyte. A pH at least ±1 from the pKa is typically recommended to ensure the stability of the analyte form.

Mobile Phase Optimization:

Typical organic solvents include methanol and acetonitrile, and occasionally THF. The mobile phase must:

• Effectively remove and separate all impurities.

• Tolerate ±5% composition variation.

• There are degradants in such method, and it requires a long time to elute the API.

• The use of THF should be done carefully as it is prone to oxidation.

Column Selection:

Important factors include:

• Particle size and shape.

• Column length and diameter.

• The pore sizes, bonding (C8, C18, CN, NH2), and end-capping.

The greater retention is observed with C18 columns than with C8. Performance of column is manufacturer dependent. screen several columns during method scouting and select one that efficiently separates the API from its impurities.

Column temperature:

It is also preferred that the column temperature be maintained at 20-?C. If symmetry or peak efficiency is not good, column heating to 80degreesC will give good results. Retention decreases on average 1–2% per every 1°C increase.

Detector Selection:

Detector selection also depends on drug and impurity or degradation product characteristics. Popular detectors are UV, PDA (photodiode array), fluorescence and mass spectrometer detector.

Validation Parameters Of Hplc Method:[11,12]

1. Precision:

There are also two conceptually different measures that address precision in different ways; the first considers how similar individual test results are when using a method on a homogenous sample. It is commonly reported as standard deviation or relative standard deviation (coefficient of variation).

ii. Accuracy:

Accuracy is a measure of how close test results are to the true value. It needs to be inspected in the use of the method. In evaluating drug assays, recovery in the range of 99%-101%, as determined by the recovery studies, is obtained.

iii. Limit of Detection (LOD):

Limit of Detection (LOD) (Hill, 1998, p.1) LOD is the lowest amount of analyte that can reliably be observed, but not necessarily quantitated under particular testing conditions. En3 It is given in ratio or ppm and depends on the type of instrument and procedure.

iv. LOQ (Limit of Quantitation):

LOQ is the lowest concentration of the analyte that can be measured accurately and precisely. Its value is reported in % or ppb with an RSD < 3%, moreover, the signal-to-noise ratio must be higher than 10.

v. Specificity:

Specificity The ability of the method to determine the analyte unequivocally without interference from other components such as impurities, degradation products and excipients. It is demonstrated through spiked and stressed samples.

vi. Linearity and Range

Linearity indicates that the test result is directly proportional to the analyte concentration. This can be seen in the correlation coefficients (r² ≥ 0.999). The concentration span between which accuracy, precision and linearity fall is the working range.

vii. Ruggedness:

Method ruggedness is a measure of method consistency under modified conditions such as operators, instruments, laboratories, or days. It tests whether the results are influenced by exogenous causes.

viii. Robustness:

Robustness gauges the extent to which the method is tolerant to minor deliberate variabilities in parameters for example the temperature, pH, or buffer strength highlights the stability of the method.

Timolol Maleate: [13,14,15]

Timolol is a beta-adrenergic receptor blocking agent and non-selective of the beta-blocker class of drugs. It is largely used in the form of timolol maleate, a salt which makes the drug more soluble in water for ophthalmic and oral formulations. Timolol is distinguished by a morpholine ring and a thiadiazole group, which are responsible for its high affinity at β? and β? adrenergic receptors. Its molecular structure is C13H24N4O. Timolol is a non-selective beta -adrenergic receptor blocker that inhibits the action of epinephrine and norepinephrine. When used in an ocular form to treat glaucoma, particularly between refractory variousthis medication is thought to act by decreasing the production of aqueous humour in the ciliary body, thereby decreasing the intra-ocular pressure (IOP); the same applies to ocular hypertension. Timolol is also given orally for systemic use in conditions including hypertension, stable chronic angina, post-myocardial infarct inhibition, and migraine prophylaxis. In its systemically administered form, it decreases heart rate, cardiac contractility, and blood pressure by blocking β?-receptors in the heart. Because of the non-selective nature it could also cause bronchoconstriction through effect on β?-receptors on bronchial smooth muscle, making it less desirable for those with asthma or chronic obstructive pulmonary disease (COPD). Timolol taken orally has good bioavailability (about 60–80%) and reaches peak plasma levels 1–2 hours after taking. It is metabolized by the liver and mainly excreted in the urine. Ophthalmic onset can be found at 25-5% and is dosed two times a day, though higher concentrations with long-acting gel formulations are dosed once a day. Systemic absorption from the eye may take place resulting in, for example, cardiovascular side-effects such as bradycardia or hypotension - in particular in the elderly or in patients with pre-existing cardiac conditions. Adverse effects include stinging or burning upon instillation, blurred vision, and, rarely, beta-blockade side effects. Due to its systemic absorption and potential therapeutic effects, timolol should be administered with caution in those with asthma, bradycardia, heart block, or hypotension, and should not be discontinued suddenly following long-term use to prevent rebound effects.

Ophthalmic Preparations: [16,17,18,19]

Ophthalmic preparations are dosage forms that are specifically formulated for drug delivery to the eye or the structures surrounding the eye to manage ocular disorders and other eye diseases including to improve patient acceptability and drug effectiveness. These can be roughly divided into liquid, semi-solid and solid forms with different characteristics, advantages and limitations. Ophthalmic dosage forms Eye Drops are the most prevalent ocular dosage form, which refers to solutions, suspensions, emulsions and microemulsions. Generally, the solutions are sterile, isotonic, and buffered to pH near that of tears (~7.4) to avoid irritation. Topical eye drops are the easiest to use, have quick onset of action and are convenient to patients. Nonetheless, these agents have poor bioavailability due to rapid turnover of tears and nasolacrimal drainage, thereby requiring frequent dosing. Microemulsions, a relatively new type of eye drops, are able to increase bioavailability by producing nanodroplets and forming long drug reservoirs on the corneal surface which lead to increased drug retention and permeation, but at the same time they are stable and easy to be sterilized. Ophthalmic Ointments are semi-solid preparations intended for conjunctival administration. Upon instillation, ointments split into tiny droplets that are retained in the conjunctival sac for a longer time, enhancing the contact time, and drug availability. This renders them very suitable for night patient or when continuous drug release required. They also result in blurry vision and occasionally irritation, thus restricting their use during the day and by patients. In Situ Gels are novel delivery systems which are instilled in the form of liquids which further form gels in situ upon contact with the physical factors, such as their temperature, pH and ionic strength as s tear fluid. This gelling facilitates longer contact time of the drug with the ocular mucosa, which leads to increased bioavailability and decreased dosing frequency. Polymers such as gel gum, for example, and poloxamer may also be employed. The primary benefit is extended drug release and increased patient compliance with the drug and some challenges are development complexity and potential in variability. Ophthalmic Inserts and Devices including soluble ophthalmic drug inserts (SODI) and minidiscs provide controlled and prolonged drug delivery by spanning the conjunctival sac. Resorbable or drug-releasing intravaginal inserts can be prepared from polymers with sustained drug release or slow dissolution, with the goal of decreasing the dosing schedule and enhancing therapeutic effect. They are best suited for longstanding conditions that need ongoing treatment. Nevertheless, patient discomfort, inserter difficulty and foreign body sensation are possible limiting factors of usage. Factors including drug bioavailability, patient compliance, delivery, and stability are considered by each ophthalmic formulation types. Eye drops are more convenient, though less retentive; ointments increase contact time but blur the vision; in situ gels show improved retention along with better patient compliance; inserts give prolonged release but are discomforting. The selection of formulation depends on the physicochemical properties of the drug, desired therapeutic effects, and patient convenience with continued research aimed at maximizing these formulations to bypass ocular barriers and increase treatment efficiency.

Advantages:

1. Provide direct drug delivery to the eye for targeted action.

2. Ensure rapid onset of action for ocular conditions.

3. Are non-invasive and easy for patients to administer.

4. Bypass first-pass metabolism, enhancing local bioavailability.

5. Improve patient compliance with convenient dosing forms.

Disadvantages:

1. Have short contact time due to tear drainage and blinking.

2. Show low ocular bioavailability (<5%) due to absorption barriers.

3. Often require frequent dosing to maintain effect.

4. May cause systemic side effects via nasolacrimal absorption.

5. Risk contamination if handled improperly.

6. Can temporarily blur vision, especially with ointments.

drug profile

Timolol Maleate is a non-selective β-adrenergic blocker widely used for the treatment of glaucoma and ocular hypertension. It acts by reducing the production of aqueous humor in the ciliary body, thereby lowering intraocular pressure (IOP). Commonly administered as a 0.25% or 0.5% ophthalmic solution, the typical dosage is one drop in the affected eye(s) twice daily. Timolol exhibits an onset of action within 20–30 minutes and has a duration of effect lasting up to 24 hours. Though primarily used topically, it has about 60% systemic bioavailability, and systemic absorption can occur through the conjunctiva, potentially leading to side effects such as bradycardia or hypotension. The drug is primarily excreted via the kidneys and requires caution in patients with respiratory or cardiac conditions.

MATERIALS AND METHODS

The RP-HPLC method was developed using a Waters C18 column (250 mm × 4.6 mm, 5 µm) with a mobile phase consisting of Methanol and 0.05% Formic Acid in a 75:25 v/v ratio. The flow rate was maintained at 0.7 mL/min, and detection was carried out at 294 nm using a UV detector. Timolol Maleate (API) was procured from Indoco Remedies, while the ophthalmic formulation (IOTIM® 0.5%) was obtained from FDC Ltd. HPLC-grade solvents and reagents were sourced from Merck Ltd. Standard and sample solutions were prepared in methanol, filtered, and injected in volumes of 20 µL. The method was optimized for retention time, peak shape, and resolution, and validated according to ICH guidelines for linearity, accuracy, precision, specificity, robustness, LOD, and LOQ.

RESULTS AND DISCUSSION

Validation of Optimised RP-HPLC method as per ICH guidelines

Linearity:

The linearity of the method was indicated the ability of the method to generate a degree of response that is directly proportional to the concentration of the analyte within the anticipated range, was done. A set of standard solutions in the concentration range of 10–50 µg mL-1 were prepared and injected, and the peak areas were plotted against concentrations to obtained calibration curve. At a concentration of 10 μg/mL, the mean area was 308.31 mAU·s, at 30 μg/mL it was 902.85 mAU·s and at 50 μg/mL it was 1485.07 mAU·s and linearity response, consistent retention times of 3.196–3.224 min and good peak symmetry (0.48–0.50), and an indication of the suitability of the method for quantification.

Figure 1: Chromatogram of linearity-10 µg-01

Figure 2: Chromatogram of linearity-10 µg-02

Figure 3: Chromatogram of linearity-20 µg 01

Figure 4: Chromatogram of linearity-20 µg-02

Figure 5: Chromatogram of linearity-30 µg-01

Figure 6 Chromatogram of linearity-30 µg-02

Figure 7: Chromatogram of linearity-40 µg-01

Figure 8: Chromatogram of linearity-40 µg-02

Figure 9: Chromatogram of linearity-50 µg-01

Figure 10  Chromatogram of linearity-50 µg-02

Table 1 : Chromatogram Parameters for Linearity

Accuracy:

The accuracy of the method was determined through recovery studies at three different levels of 80%, 100% , and 120%. Different fixed amounts of the API were supplemented to a constant amount of the marketed formulation and analysed. The percentage of recovery (at the 80% reference value) was 100.54% (mean value between 542.81 and 544.39 mAU ·s), at 100% level 99.80% (mean area of 602.30 and 605.27 mAU·s), and at 120% level 100.62% (mean area of 665.37 and 665.11 mAU·s). All mean values were within the acceptable recovery range (98–102%), showing that there was no interferences in the method, which means that this method was precise and free for the determination of matrix interferences.         

Figure 11: Chromatogram of Accuracy 80%-01

Figure 12: Chromatogram of Accuracy 80%-02

Figure 13: Chromatogram of Accuracy 100%-01

Figure 14: Chromatogram of Accuracy 100%-02

Figure 15: Chromatogram of Accuracy 120%-01

Figure 16: Chromatogram of Accuracy 120%-02

Table 2: Chromatographic Results of Accuracy Trials (80%, 100%, 120%)

Table 3 : Results of Accuracy

Precision (repeatability):

Repeatability was calculated to study the same method under the same experimental conditions in a short space of time. Two injections were injected at a concentration of 30 × g/mL, and the retention times were 3.190 and 3.191 minutes. The resulting peak areas were 904.14 and 904.14 mAU·s, respectively: the mean area was 904.14 mAU·s, and the %RSD value was 0.00%. The repeatability was satisfactory with the retention time relatively consistent, peaks symmetric (peak symmetry=0.49), and peak area changes slightly with little variation. Low theoretical plate counts (3755 and 3773, respectively) indicated good column performance and method reproducibility at one concentration level.

Figure 17: Chromatogram of System suitability -1(15 µg)

Figure 18: Chromatogram of System suitability No-2

Table 4 : Repeatability

Table 5 : System Suitability Parameters

Table 6 : Results of Repeatability

Intraday:

Intraday precision was measured by carrying out analyses of samples at three concentration levels (10, 30 and 50 µg/mL) in duplicate on the same day. mean peak area was 306.19 mAU·s and % RSD 0.17%, for 30 µg/mL mean was 902.65 mAU·s and % RSD 0.17%, and for 50 µg/mL, the average area was 1486.05 mAU·s (0.15 % RSD). The retention times was constant (between 3.190 and 3.196 min) and the peaks symmetry were 0.48–0.49. The results from three synthetic samples proved the precision and applicability of the method in a single day and in routine practice.

Figure 18: Chromatogram of Intraday Precision (10 µg -01)

Figure 19: Chromatogram of Intraday Precision (10 µg -02)

Figure 20: Chromatogram of Intraday Precision (30 µg -01)

Figure 21: Chromatogram of Intraday Precision (30 µg -02)

Figure 22: Chromatogram of Intraday Precision (50 µg -01)

Figure 23: Chromatogram of Intraday Precision (50 µg -02)

Table 7: Chromatogram of Precision (Intraday)

Table 7 : Precision (Intraday)

Precision (Interday)

Interday precision was determined by repeating the analysis of 10, 30, and 50 µg/mL on three successive days. The corresponding area of 10 µg/mL was 305.50 mAU·s with a % RSD of 0.14%; 30 µg/mL, 899.21 mAU·s with a % RSD of 0.02%; and 50 µg/mL, with a % RSD of 0.01%, and they were 1490.52 mAU·s, respectively. The retention times were reproducible throughout all days, varying only slightly from 3.179 to 3.184 minutes. The low % RSD values and constant chromatographic conditions showed that the method was highly precision interday and the robustness of the method was established over a period of time.

Figure 24: Chromatogram of interday Precision (10 µg -01)

Figure 25: Chromatogram of interday Precision (10 µg -02)   

Figure 26: Chromatogram of interday Precision (30 µg -01)

Figure 27: Chromatogram of interday Precision (30 µg -02)

Figure 28: Chromatogram of interday Precision (50 µg -01)

Figure 29: Chromatogram of interday Precision (50 µg -02)

Table 8 Chromatogram of Precision (Interday)

Table 9 Precision (Interday)

Ruggedness:

Ruggedness of the HPLC method was determined using two replicate injections of 30µg/mL timolol maleate samples under the same chromatographic conditions. The retention time in both injections was the same 3.192 min demonstrating reproducibility of the method. The peak areas were 906.40240 mAU·s (Run 01) and 904.51880 mAU·s (Run 02) with minimal %RSD (0.10%) indicating the reproducibility. The symmetry factor (0.49) and theoretical plate number (~3790) were within the acceptable range to indicate the peak shape and column efficiency, respectively.

Figure 30: Chromatogram of Ruggedness 30 µg/ml 01

Figure 31: Chromatogram of Ruggedness 30 µg/ml 01

Table 10 : Chromatogram of Ruggedness

Robustness:

Robustness was measured by changing some of the analytical parameters, e.g. mobile phase compositions (74:26 and 76:24 MeOH: Buffer) and detection wavelength (293 nm and 295 nm). The retention time and peak area effects of these variations were investigated to check the robustness of the method. The mean area and retention time were 589.31 mAU·s and 3.224 min (RSD = 0.17%), respectively, with mobile phase change to 74:26. And the mean area is 593.65 mAU·s and retention time is 3.166 min at 76:24 (RSD = 0.23%), respectively. In terms of the wavelength, the mean area was 580.76 mAU·s at 293 nm and 602.83 mAU·s at 295 nm, with the RSDs of 0.13% and 0.11%, respectively. These findings indicated that the method was robust and could tolerate minor deviations in the analysis.                    

Figure 32: Chromatogram of Mobile phase composition (74+26) 01

Figure 33: Chromatogram of Mobile phase composition (74+26) 02

Figure 34: Chromatogram of Mobile phase composition (76+24) 01

Figure 35: Chromatogram of Mobile phase composition (76+24) 02

Figure 36: Chromatogram of wavelength change 293 nm +295 nm 01

Figure 37: Chromatogram of wavelength change 293 nm +295 nm 02

Table 11 : Chromatogram of Robustness

Table 12 : Robustness

LOD AND LOQ:

Limit of Detection:

The sensitivity of the HPLC method was determined in terms of LOD and LOQ based on the standard deviation of the response and the slope of the calibration curve. LOD was estimated to be LOD = 3.3 × SD / Slope with a standard deviation (SD) of 1.35 and a slope of the curve of 29.74. According to the computation, the LOD was 0.15 µg/ml. The detection limit (LOD) of the method was determined by the following equation:

LOD = 3.3 × Avg. SD / Slope

LOD = 1.35 × 3.3 / 29.74

LOD = 0.15 µg/ml

Where,

SD = Standard Deviation = 1.35,

S = Slope of the curve = 29.74.

The Limit of Detection (LOD) was found to be 0.15 µg/ml.

Limit of Quantification (LOQ)

The LOQ was calculated by LOQ = 10 × SD / Slope and was 0.45 µg/mL. These results demonstrate that the method is sensitive enough for the determination of Timolol Maleate, and the low LOD and LOQ values verify the reliability of the method for routine analysis even at trace concentrations.

The quantification limit (LOQ) of the method was determined by the following equation:

LOQ = 10 × Avg. SD / Slope

LOQ = 1.35 × 10 / 29.74

LOQ = 0.45 µg/ml

Where,

SD = Standard Deviation = 1.35,

S = Slope of the curve = 29.74.

The Limit of Quantification (LOQ) was found to be 0.45 µg/ml.

Table 13 : Result of LOD & LOQ

Table 14 : X Optimized Method

The final chromatographic conditions selected were as follows:

• Analytical column: Waters C18 Column 250 mm × 4.6 mm, 5 μm particle size
• Injection volume : 20 μL
• Flow rate: 0.7 mL/min
• Mobile phase: Methanol: 0.5% formic acid (75:25% v/v)
• Detection: 294 nm
• Run Time: 7 min

Figure 38 Chromatogram of Final Trial 8

Assay:

The validated HPLC method was used to analysed the drug in a ophthalmic formulation (claimed content of the analyte was 40 µg/mL). Two replicates were performed, and the areas were 1211.14 and 1209.63 mAU·s, which represent % label claim of 100.14% and 99.86%, respectively. The average drug content was found to be 100.00% with an RSD of 0.09%, which proved that the method was accurate, precise and could be used for the quality control of the pharmaceutical products.

Figure 39: Chromatogram for Marketed Formulation (40 µg) 01

Figure 40: Chromatogram for Marketed Formulation (40 µg) 02

Table 15 : Chromatogram of Assay of Marketed Formulation of Timolol Maleate

Table 16 Result of Assay of Marketed Formulation of Timolol Maleate