Sundar_1.pptx opthalmic injectable dosage form

GENERIC FORMULATION OF PHENYLEPHRINE HCL, LIDOCAINE, TROPICAMIDE AND
EVALUATION OF ANALYTICAL METHOD VALIDATION OF ASSAY BY HPLC
Presented by,
SUNDHARAMOORTHY 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.

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

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.

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.

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.

• 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.

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.

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

TROPICAMIDE
• Molecular Formula: C17H20N2O2
• 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

PHENYLEPHERINE HCL
• Molecular Formula: C9H14ClNO2
• 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

Plan of work
• STAGE 1
1. Pre-formulation studies
a) Solubility studies
• STAGE 2
• Preparation of Tropicamide,
phenylephrine hydrochloride
and lidocaine ophthalmic
injection
• STAGE 3
• Evaluation
1. Physical characterization
a. description
b. PH
c. Conductivity
d. Particulate matter
2. Analytical studies
a) Identification test
b) Assay potency
c) Endotoxin test
d) Sterility test
e) Assay validation by HPLC
method
• STAGE 4
1. Stability studies

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

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.

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

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
Injecti
ons
1 Blank 1
2 Standard solution 6
4 Sample preparation 2
5
Bracketing standard
solution
1

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

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)

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.

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.

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.

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

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
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
1 Blank 1
2 Standard solution
6
5 Bracketing standard solution 1

3. ACCURACY - It is the closeness of the
measured value to the true value.
S. No Solution No. of Injections
1. Blank 1
1. Standard solution 6
1. Blank 1
1. Accuracy solution-1 50% level 2
1. 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/Quantificatio
n
Blank 1 Blank
Linearity solution-1 3 Method validation
Bracketing standard
solution
1
System
suitability/Quantificatio
n

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)
1 Blank 1
2 Standard solution
6
Robustness (mobile phase PH4.6)
1 Blank 1
2 Standard solution
6

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

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

Calibration curve
Lidocaine S. no Concentratio
n (µg/ml)
Absorbance
(263nm)
1 0 0
2 20 0.215
3 40 0.402
4 60 0.615
5 80 0.802
6 100 0.982
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60 80 100 120
Absorbance
at
263nm
Concentration (µg/ml)
Calibration curve Lidocaine
Phenyl ephreine
S. no Concentration(
µg/ml)
Absorbance
(270nm)
1 0 0
2 20 0.015
3 40 0.026
4 60 0.037
5 80 0.048
6 100 0.057
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60 80 100 120
Absorbance
at
270nm
Calibration curve for Phenylephrine HCL

Tropicamide
S. no concentration(
µg/ml)
Absorbance
(423nm)
1 0 0
2 20 0.127
3 40 0.246
4 60 0.367
5 80 0.478
6 100 0.592
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60 80 100 120
Absorbance
at
423nm
Calibration curve for Tropicamide
pH 5.0

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

Assay validation by HPLC method

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

Method precision
Tropicamide
Phenylephrine

Intermediate precision
Tropicamide
Name of the
sample
Area Mean %RSD % assay
Standard-1/1
2197
24
218565.5
0.26 101%
Standard-1/2
2184
46
Standard-1/3
2183
35
Standard-1/4
2183
17
Standard-1/5
2184
12
Standard-1/6
2181
59
Sample
solution-1/1
2209
99
220960
0.02
Sample
solution-1/2
2209
21
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

Lidocaine
Name of the sample Area Mean %RSD %assay
Standard-1/1 9995999
9998153 0.015
Standard-1/5 10000313
Sample solution-1/1
10150057
10155471
0.075 101.5
Sample solution-1/2
10160884

ACCURACY
Tropicamide
Phenylephrine
Lidocaine
Name of the sample Area Mean %RSD
Standard solution-1/1 219164
218713.2
0.12
785098.8 0.02
10016982 0.01

Linearity
Tropicamide
Phenylephrine
Lidocaine
229076.8 0.38
688611.7 0.03
10004425 0.03

Robustness (Mobile phase pH 4.6)
Tropicamide
Phenylephrine

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 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%
Sample 24Hr-1/1 622152 615436.5 1.54
Sample 24Hr-1/2 608721

Lidocaine
No of the sample Area Mean %RSD ASSAY % Difference
Standard solution-1/1 9684117 9691374 0.04 97.5
Sample 24Hr-1/1 9723361 9451225 4.07
Sample 24Hr-1/2 9179088

Robustness pH 4.2
Tropicamide
Phenylephrine

Filter compatibility
Tropicamide
Phenylephrine
No of the sample Area Mean %RSD ASSAY
Standard solution-1/1 228271 229836.8 0.36
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%
689844.2
0.03
Sample0.2µ PTFE-1/1 609971 NA NA 88.4%

Lidocaine
Standard solution-1/1 10021837 10018159 0.02
Sample0.2µ PTFE-1/1 9195065 NA NA 91.78%

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.

• 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.

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.

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