ESTIMATION OF TIMOLOL MALEATE AND DORZOLAMIDE

HYDROCHLORIDE IN OPHTHALMIC SOLUTION DOSAGE

FROM BY HPLC – METHOD
Thesis submitted to St. Joseph’s College of Arts and Science, (Autonomous)

In partial fulfillment of the requirements for the award of the degree of

BACHELOR OF SCIENCE IN CHEMISTRY

Submitted By

SANGAVI. A

(Reg. No: A15CHE37)

Under the Guidance of

Dr. S. DAVID AMAL RAJ, M.Sc., M.phil., Ph.D.,

Asst.Prof.in chemistry

St. Joseph’s College of Arts and Science (Autonomous)

Cuddalore – 607 001.

POST GRADUATE AND RESEARCH DEPARTMENT OF CHEMISTRY

ST. JOSEPH’S COLLEGE OF ARTS AND SCIENCE (AUTONOMOUS)

CUDDALORE – 607 001

APRIL – 2018
 CERTIFICATE

This is to certify that this thesis entitled “ESTIMATION OF TIMOLOL

MALEATE AND DORZOLAMIDE HYDROCHLORIDE IN
OPHTHALMIC SOLUTION DOSAGE FROM BY HPLC-METHOD”
Submitted to St. Joseph’s College of Arts and Science (Autonomous) in partial
fulfillment for the award of the Degree of Bachelor of Science in Chemistry is a
bonafide record of work carried out by SANGAVI. A (A15CHE37) under my
guidance for the Academic period 2015 – 2018

Signature of the Guide

Dr. S. DAVID AMAL RAJ

Forwarded by

(Head of the Department) (Principal)

Viva – voce examination held on ----------------

Examiners

1. 2.
DECLARATION
 DECLARATION

I hereby declare that this project entitled (ESTIMATION OF TIMOLOL

MALEATE AND DORZOLAMIDE HYDROCHLORIDE IN
OPHTHALMIC SOLUTION DOSAGE FROM BY HPLC METHOD)
submitted to the St. Joseph’s College of Arts and Science ( Autonomous ),
Cuddalore – 607 001, in partial fulfillment of the requirement for the award of the
degree of Bachelor of Science in Chemistry is a record of original project work
done by me under the supervision and guidance of Dr. S. DAVID AMALRAJ,
M.Sc., M.phil., Ph.D., Asst. Professor in Chemistry, St. Joseph’s College of Arts
and Science (Autonomous), Cuddalore – 607 001 and the project has not formed
the basis for the award of any degree / diploma / associate ship / fellowship / other
similar titled to any candidate of any other University.

Signature of Candidate

SANGAVI. A
(Reg. No: A15CHE37)
Place : Cuddalore - 1
Date :
ACKNOWLEDGEMENT
 ACKNOWLEDGEMENT

First and foremost my gratitude goes to the “ALMIGHTY” for keeping me

healthy and fit mentally to carry out project work to completion.

I extremely indebted to my dad and mom Mr. V. ARUMUGAM and

Mrs. A. VALARMATHI for having elevated me to this status and made me
successful in my life so far.

I wish to express my sense of gratitude to our Rev. Fr. G. PETER

RAJENDIRAM , M.A.,M.Sc.,M.Ed.,M.phil St. Joseph’s College of Arts and
Science (Autonomous), Cuddalore for granting me permission undertake the
project work.

I am extremely grateful and thankful to Mr. A. AMALORPAVADOSS,

M.Sc.,B.Ed.,M.phil., Head of the department of chemistry, St. Joseph’s College of
Arts and Science (Autonomous), Cuddalore for his constant help and
encouragement in executing the project work successfully.

I have immense pleasure in expressing my gratitude to my guide

Dr. S. DAVID AMALRAJ, M.Sc., M.phil., Ph.D., Asst. professor in chemistry,
St. Joseph’s College of Arts and Science (Autonomous), Cuddalore, for her
dynamic guidance valuable suggestion and motivation throughout the course of
this dissertation work.

Finally I thank my fellow friend. They were always supporting and

encouraging me with their best wishes.

SANGAVI. A
(Reg. No: A15CHE37)
 CONTENT

CHAPTER DESCRIPTION

1 INTRODUCTION

2 SCOPE OF THE WORK

3 EXPERIMENTAL METHODS

4 RESULTS AND DISCUSSION

5 CONCLUSIN

6 REFERENCES
 CHAPTER – 1
INTRODUCTION
 SHASUN PROFILE

SHASUN HISTORY

 From the modest beginning in 1976 Shasun has acquired a worldwide and
their intermediates.
 The company product s are exported to customers in countries across
Europe, North America and Latin Americe & Asia.
 SHASUN is derived from the name of the founder, late Shrishankarlal Jain
and his wife Smt. Sundarbai.
MILESTONES
 1976 – 1990
 Incorporated as a private limited company, Shasun chemicals in Chennai.
 First production facility established at Velanchery, Chennai for manufacture
of analgin (anti pyretic).
 Second production facility was established at Puducherry for manufacture of
Ibuprofen (anti – inflammatory).
 1990 – 2000.
 Third manufacturing unit setup at Cuddalore to manufacture anti – ulcerative
ranitidine HCL.
 Shasun converted into a public limited company, incorporating its present
name.
 US subsidiary, Shasun inc., is established the company shares were listed on
Mumbai, Ahmedabad and Chennai Stock Exchanges. Board approves for
hiving of its manufacturing unit manufacturing Ibuprofen into separate joint
venture company with a foreign drug major.
 From an alliance with eastiman chemical Co., Ltd of US to license the letters
technology for the manufacture of a acrylic emulsion.
 Fire accidents in one of the packing sections of Cuddalore factory.
 Obtains US FDA approval of its 3 bulk active pharmaceuticals products.
 Enters into alliances with suven pharmaceutical Ltd and innovasynth
technologies Ltd for drug discovery.
 Enters into a pact with Eli Lilly and Co., for manufacture of APL
tuberculosis.
 Brings down the average effective cost of its loans to 5% from the level 10%
in last year.
 Board approved for the issue and allotment of 9,40,000 shares on a
preferential basis.
 2007 – 2009.
 Shasun chemicals sings technology licensing agreement with lund beck.
 Shasun chemicals technology has announced that it has entered into a non
exclusive licensing agreement with merck & Co., Inc.
 Shasun chemical board recommends divided of 5 % equity share of 2/- each.
 2014.
 Shasun pharma successful completion of US FDA and Mexican COFEPRIS
inspection. Shasun pharma updates joint venture agreement with sequent
Ltd, Shasun pharma acquisition of global rights of Ibuprofen 12hr extened
release OTC & nuprin brand. Shasun & strides acrolab Ltd, have approved a
scheme of amalgamation between the two compound.
 ABSTRACT

Dorzolamide Hydrochloride is used to lower the increased intraocular

pressure in open – angle glaucoma and ocular hypertension. Timolol maleate is
indicated in the treatment of elevated intraocular pressure in patients with ocular
hypertension or open – angle glaucoma. In this article, a high performance liquid
chromatography (HPLC) method with UV – visible detector wavelength (254 nm
& 295 nm) for Dorzolamide Hydrochloride & Timolol maleate was developed and
validated. The mobile phase consisted of methanol : buffer (60:40), the pH was
adjusted to 3 with glacial acetic acid, was run through a Column C-18, (EC 150/4.6
NUCLEOSIL 100-5), reverse phase analytical column. The experiment was
carried out at room temperature for Dorzolamide Hydrochloride & Timolol
maleate. Analytical run time was less than 10 min. The assay exhibited good
linear relationship. Accuracy & Precision were over the concentration range of 10
to 30 µ g/ml & 40 to 120 µ g/ml for Dorzolamide Hydrochloride and Timolol
Maleate respectively. This method was found to be applicable for determination of
the Dorzolamide Hydrochloride & Timolol Maleate in active pharmaceutical
ingredient (API) and pharmaceutical product also.
 INTRODUCTION

Dorzolamide is an ophthalmic solution ( a liquid that is placed in the eyes)

that is for treating glaucoma. It is in a class of drugs called carbonic anhydrase
inhibitors which includes brinzolamide. Many parts of the body, including the eye,
contain the enzyme carbonic anhydrase which plays a key role in controlling the
pressure within the eye. Carbonic anhydrase controls secretion of fluid within the
eye and thereby determines the pressure within the eye (intraocular pressure): the
greater the amount of fluid that is secreted, the higher the pressure. Patients with
glaucoma have increased intraocular pressure. Dorzolamide blocks carbonic
anhydrase thereby decreasing intraocular pressure. This reduces the risk of nerve
damage and loss of vision that is caused by increased intraocular pressure in
patients with glaucoma.

Timolol is a beta – adrenergic blocking drug that is used to treat high blood
pressure, angina (heart pain), and heart attacks and to prevent migraine headaches
Timolol is a first generation beta blocker in a class that includes propranolol
(Inderal, InnoPran), nadolol (Corgard), penbutolol sulfate (Levatol), Sotalol
hydrochloride (Betapace), and pindolol (Visken). They differ from other beta
blockers because they are non – selective in nature, meaning that they block both
beta – 1 and beta – 2 receptors on nerves and, therefore, will affect not only the
heart but also the kidneys, lungs, gastrointestinal tract, liver, uterus, muscles
surrounding blood vessels, and skeletal muscle. As a result, they could cause such
effects as reduced pumping of blood by the heart and reduced kidney function
among other actions. Timolol specifically works by blocking the stimulating
actions of the sympathetic nervous system thereby allowing the heart to relax and
beat more slowly. This reduces the amount of blood that the heart must pump.
Timolol was approved by the FDA in November 1981. The aim of the present
study is to develop a sensitive and rapid RP-HPLC method with UV detection for
the quantitative determination of Dorzolamide Hydrochloride and Timolol Maleate
in active pharmaceutical ingredient (API). This method offers the advantage of
simplicity with adequate sensitivity, selectivity, precision and accuracy.
Comparative to other methods this method shows less run time and good
sensitivity. This method has been satisfactorily applied to the determination of
estimation of Dorzolamide Hydrochloride and Timolol Maleate as API and in
ophthalmic solution dosage form. The review of literature reveals that methods
including LC/MS, UV, HPTLC, HPLC, Volta metric methods etc. have been
reported for the estimation of drugs individually / simultaneously mixture in
biological specimen but no method has been reported so far in literature for
estimation of Dorzolamide Hydrochloride and Timolol Maleate as API and in
ophthalmic solution dosage form by RP-HPLC method with UV detector.

1.1 DORZOLAMIDE

This drug, developed by Merck, was the first drug in human therapy (market
introduction 1995) which resulted from structure – based drug design. It was
developed to circumvent the systemic side effects of acetazolamide which has to
be taken orally. Carbonic anhydrase inhibitors are a class of pharmaceuticals that
suppress the activity of carbonic anhydrase. Their clinical use has been established
as, anti glaucoma agents diuretics, antiepileptics, in the management of mountain
sickness, gastic and duodenal ulcers, neurological disorders, or osteoporosis.

Carbonic anhydrase inhibitors are primarily used for the treatment of

glaucoma. They may also be used to treat seizure disorder and acute mountain
sickness. Because they encourage solubilization and excretion of uric acid, they
can be used in the treatment of gout.

1.1.1 MOLECULAR STRUCTURE

Dorzolamide Hydrochloride Ophthalmic solution is a carbonic anhydrase

inhibitor formulated for topical ophthalmic use.
Dorzolamide hydrochloride is described chemically as: (4S-trans) – 4-
(ethylamino) – 5, 6 – dihydro – 6 –n methyl – 4 H – thieno (2,3-b) thiopyran – 2-
sulfonamide 7,7 – dioxide monohydrochloride. Dorzolamide hydrochloride is
optically active.

Fig : 1 Dorzolamide hydrochloride.

 Dorzolamide hydrochloride has a molecular weight of 360.9 and a melting
point of about 264oC. It is a white to off-white, crystalline powder, which is
soluble in water and slightly soluble in methanol and ethanol.

Dorzolamide Hydrochloride Ophthalmic solution is supplied as a sterile,

isotonic, buffered, slightly viscous, aqueous solution of dorzolamide
hydrochloride. The pH of the solution is approximately 5.6, and the osmolarity is
260-330 mOsM. Each mL of Dorzolamide Hydrochloride Ophthalmic solution,
2% contains 20 mg dorzolamide (22.3 mg of dorzolamide hydrochloride). Inactive
ingredients are hydroxyethyl cellulose, mannitol, sodium citrate dehydrate, sodium
hydroxide (to adjust pH) and water for injection. Benzalkonium chloride 0.0075%
is added as a preservative.

1.1.2 MECHANISM OF ACTION

Carboin anhydrase (CA) is an enzyme found in many tissues of the body

including the eye. It catalyzes the reversible reaction involving the hydration of
carbon dioxide and the dehydration of carbonic acid. In humans, carbonic
anhydrase exists as a number of isoenzymes, most active being carbonic anhydrase
II (CA – II), found primarily in red blood cells (RBCs), but also in other tissues.
Inhibition of carbonic anhydrase in the ciliary processes of the eye decrease
aqueous humor secretion, presumably by slowing the formation of bicarbonate ions
with subsequent reduction in sodium and fluid transport. The result is a reduction
in intraocular pressure (IOP).

Dorzolamide Hydrochloride Ophthalmic solution contains dorzolamide

hydrochloride, an inhibitor of human carbonic anhydrase II. Following topical
ocular administration, Dorzolamide Hydrochloride Ophthalmic solution reduces
elevated intraocular pressure. Elevated intraocular pressure is a major risk factor
in the pathogenesis of optic nerve damage and glaucomatous visual field loss.

1.1.3 PHARMACODYNAMICS

When topically applied, dorzolamide reaches the systemic circulation. To

assess the potential for systemic carbonic anhydrase inhibition following topical
administration, drug and metabolite concentrations in RBCs and plasma and
carbonic anhydrase inhibition in RBCs were measured. Dorzolamide
accumulateds in RBCs during chronic dosing as a result of binding to CA – II. The
parent drug forms a single N-desethyl metabolite, which inhibits CA-II less
potently than the parent drug but also inhibits CA-I. The metabolite also
accumulates in RBCs where it binds primarily to CA – I. Plasma concentrations of
dorzolamide and metabolite are generally below the assay limit of quantization (15
nM). Dorzolamide binds moderately to plasma proteins (approximately 33%).
Dorzolamide is primarily excreted unchanged in the urine; the metabolite also is
excreted in urine. After dosing is stopped, dorzolamide washes out of RBCs
nonlinearly, resulting in a rapid decline of drug concentration initially, followed by
a slower elimination phase with a half – life of about four months.

To simulate the systemic exposure after long-term topical ocular

administration, dorzolamide was given orally to eight healthy subjects for up to 20
weeks. The oral dose of 2 mg b.i.d closely approximates the amount of drug
delivered by topical ocular administration of Dorzolamide Hydrochloride
Ophthalmic solution 2% t.i.d steady state was reached within 8 weeks. The
inhibition of CA – II and total carbonic anhydrase activities was below the degree
of inhibition anticipated to be necessary for a pharmacological effect on renal
function and respiration in healthy individuals.

1.1.4 CLINICAL STUDIES

The efficacy of Dorzolamide hydrochloride Ophthalmic solution was

demonstrated in clinical studies in the treatment of elevated intraocular pressure in
patients with glaucoma or ocular hypertension (baseline IOP=23mmHg). The IOP
– lowering effect of Dorzolamide Hydrochloride Ophthalmic solution was
approximately 3 to 5 mmHg throughout the day and this was consistent in clinical
studies of up to one year duration.

The efficacy of Dorzolamide Hydrochloride Ophthalmic solution when

dosed less frequently than three times a day (alone or in combination with other
products) has not been established.

In one year clinical study, the effect of Dorzolamide Hydrochloride

Ophthalmic solution 2% t.i.d on the corneal endothelium was compared to that of
betaxolol ophthalmic solution b.i.d and timolol maleate ophthalmic solution 0.5%
b.i.d. There were no statistically significant differences between groups in corneal
endothelial cell counts or in corneal thickness measurements. There was a mean
loss of approximately 4% in the endothelial cell counts for each group over the one
year period.
1.1.5 DRUG INTERACTION

Although acid – base and electrolyte disturbances were not reported in the
clinical trials with Dorzolamide Hydrochloride Ophthalimc solution, these
disturbances have been reported with oral carbonic anhydrase inhibitors and have,
in some instances, resulted in drug interactions (e.g., toxicity associated with high-
dose salicylate therapy). Therefore, the potential for such drug interactions should
be considered in patients receiving Dorzolamide Hydrochloride Ophthalmic
solution.

1.1.6 DORZOLAMIDE DROPS ARE USED FOR:

Treating increased pressure in the eye in patients with ocular hypertension

and open – angle glaucoma. Dorzolamide drops are a carbonic anhydrase
inhibitor. It works by decreasing fluid production and pressure inside the eye.

1.1.7 DO NOT USE DORZOLAMIDE DROPS IF:

 You are allergic to any ingredient in dorzolamide drops
 You have severe kidney problems
 You are taking another carbonic anhydrase inhibitor (e.g
acetazolamide, methazolamide)
 Contact your doctor or health care provider right away if any of these
apply to you.

1.1.8 BEFORE USING DORZOLAMIDE DROPS:

Some medical conditions may interact with dorzolamide drops. Tell your
doctor or pharmacist if you have any medical conditions, especially if any of the
following apply to you:
 If you are pregnant, planning to become pregnant, or are breast –
feeding
 If you are taking any prescription or nonprescription medicine, herbal
preparation, or dietary supplement
 If you have allergies to medicines, foods, or other substances
 If you have had a severe allergic reactioin (e.g severe rash, hives,
difficulty breathing, dizziness) to any other sulfonamide (sulfa)
medicine (e.g sulfamethoxazole)
 If you have kidney or liver problems
  If you have an eye injury or infection, or if you will be having eye
surgery

Some MEDICINES INTERACT with dorzolamide drops. Tell your health

care provider if you are taking any other medicines, especially any of the
following:

 Salicylates (e.g aspirin) because the risk of their side effects may be
increased by dorzolamide drops

 Other carbonic anhydrase inhibitors (e.g acetazolamide,

methazolamide) because the risk of their side effects may be increased
by dorzolamide drops

This may not be a complete list of all interactions that may occur. Ask your
health care provider if dorzolamide drops may interact with other medicines that
you take. Check with your health care provider before you start, stop or change the
dose of any medicine.

1.1.9 HOW TO USE DORZOLAMIDE DROPS:

Use dorzolamide drops as directed by your doctor. Check the label on the
medicine for exact dosing instructions.
 An extra patient leaflet is available with dorzolamide drops. Talk to
your pharmacist if you have questions about this information.
 Remove contact lenses before you use dorzolamide drops; lenses may
be placed back in eyes 15 minutes after use of dorzolamide drops.
 Dorzolamide drops are only for the eye. Do not get it in your nose or
mouth.
 To use dorzolamide drops in the eye, first, wash your hands. Tilt your
head back. Using your index finger, pull the lower eyelid away from
the eye to form a pouch. Drop the medicine into the pouch by lightly
pressing the “finger push area” of the bottle with your thumbs or
index finger, then gently close your eyes. Immediately use your
finger to apply pressure to the inside corner of the eye for 1 to 2
minutes. Do not blink. Remove excess medicine around your eye.
Wash your hands to remove any medicine that may be on them.
 If you use other medicines in your eyes, wait at least 5 minutes
between using dorzolamide drops and your other eye medicines.
  To prevent germs from contaminating your medicine, do not touch the
applicator tip to any surface, including the eye. Keep the container
tightly closed.
 If you miss a dose of dorzolamide drops, use it as soon as possible. If
it it almost time for your next dose, skip the missed dose and go back
to your regular dosing schedule. Do not use 2 doses at once.

Ask your health care provider any questions you may have about how to use
dorzolamide drops.

1.1.10 IMPORTANT SAFETY INFORMATION :

 Dorzolamide drops may cause blurred vision. This effect may be

worse if you take it with alcohol or certain medicines. Use
dorzolamide drops with caution. Do not drive or perform other
possibly unsafe tasks until you know how you react to it.

 Dorzolamide drops are a sulfonamide (sulfa) medicine. Some patients

who are allergic to sulfonamides have experienced severe and rarely
fatal allergic reactions to sulfonamide medicines. Contact your doctor
right away if you develop fever, chills, or sore throat; red, swollen,
blistered, or peeling skin (with or without fever); red or irritated eyes;
sores in the mouth, throat, nose, or eyes; symptoms of liver damage
(e.g yellowing of the eyes or skin, pale stools, dark urine, severe
stomach pain, loss of appetite); unusual tiredness or weakness; or
unusually pale skin.

 Tell your doctor or dentist that you take dorzolamide drops before you
receive any medical or dental care, emergency, care or surgery.

 PREGNANCY and BREAST – FEEDING : If you become pregnant,

contact your doctor. You will need to discuss the benefits and risks of
using dorzolamide drops while you are pregnant. It is not known if
this medicine is found in breast milk. Do not breast – feed while takin
dorzolamide drops.
1.1.11 POSSIBLE SIDE EFFECTS OF DORZOLAMIDE DROPS:

All medicines may cause side effects, but many people have no, or minor,
side effects. Check with your doctor if any of these most COMMON side effects
persist or become bothersome:

Bitter taste in the mouth; blurred vision; dry eyes; eye sensitivity to light;
increased tear production; mild burning, redness, or stinging of the eyes.

Seek medical attention right away if any of these SEVERE side effects
occur: Severe allergic reactions (rash; hives; itching; difficulty breathing or
swallowing; tightness in the chest; swelling of the mouth, face, lips, throat, or
tongue; unusual hoarseness); eyes or eyelid irritation, swelling, pain or discharge;
vision changes.

This is not a complete list of all side effects that may occur. If you have
questions about side effects, contact your health care provider. Call your doctor
for medical advice about side effects. To report side effects to the appropriate
agency, please read the Guide to Reporting Problems to FDA.

1.1.12 PROPER STORAGE DORZOLAMIDE DROPS:

Store dorzolamide drops at room temperature, between 59 and 86 degrees F (15

and 30 degree C). Store away from heat, moisture, and light. Do not store in the
bathroom. Keep dorzolamide drops out of the reach of children and away from
pets.

1.2 TIMOLOL

Timolol is a medication used either by mouth or as eye drops. As eye drops

it is used to treat increased pressure inside the eye such as in ocular hypertension
and glaucoma. By mouth it is used for high blood pressure, chest pain due to
insufficient blood flow to the heart, to prevent further complications after a heart
attack, and to prevent migraines.

Common side effects with the drops is irritation of the eye. Common side
effects by mouth include tiredness, slow heart beat, itchiness, and shortness of
breath. Other side effects include masking the symptoms of low blood sugar in
those with diabetes. Use is not recommended in those with asthma, heart failure,
or COPD. It is unclear if use during pregnancy is safe for the baby. Timolol is in
the non-selective Beta blocker family of medication.

Timolol was patented in 1968 and came into medical use in 1978. It is on
the World Health Organization’s List of Essential Medicines, the most effective
and safe medicines needed in a health system. Timolol is available as a generic
medication. The wholesale coast in the developing world is about 0.86 to 2.29
USD per 5 ml bottle. In the United States it costs 25 to 50 USD per month.

1.2.1 MOLECULAR STRUCTURE

Its molecular formula is C13H24N4O3S-C4H4O4 and its structural formula

is:

Fig – 2: Timolol Maleate

Timolol maleate has a molecular weight of 432.50. It is a white, odorless,

crystalline powder which is soluble in water, methanol, and alcohol. Timolol
maleate ophthalmic solution is stable at room temperature. Timolol Maleate
Ophthalmic solution is supplied as a sterile, isotonic, buffered, aqueous solution of
timolol maleate in two dosage strengths:

1.2.2 MECHANISM OF ACTION

Timolol maleate is a beta 1 and beta 2 (non – selective) adrenergic receptor

blocking agent that does not have significant intrinsic sympathomimetic, direct
myocardial depressant, or local anesthetic (membrane – stabilizing) activity.
 Beta – adrenergic receptor blockade reduces cardiac output in both healthy
subjects and patients with heart disease. In patients with severe impairment of
myocardial function, beta-adrenergic receptor blockade may inhibit the stimulatory
effect of the sympathetic nervous system necessary to maintain adequate cardiac
function.

Beta – adrenergic receptor blockade in the bronchi and brochioles results in

increased airway resistance from unopposed parasympathetic activity. Such an
effect in patients with asthma or other bronchospastic conditions is potentially
dangerous.

The onset of reduction in intraocular pressure following administration of

Timolol maleate ophthalmic solution can usually be detected within one-half hour
after a single dose. The maximum effect usually occurs in one two hours and
significant lowering of intraocular pressure can be maintained for periods as long
as 24 hours with a single dose. Repeated action may be related to reduced aqueous
formation. However, in some studies a slight increase in outflow facility was also
observed.

1.2.3 PHARMACOKINETICS

In a study of plasma drug concentration in six subjects, the systemic

exposure to timolol was determined following twice daily administration of timolol
maleate ophthalmic solution 0.5%. The mean peak plasma concentration
following morning dosing was 0.46 ng/ml and following afternoon dosing was
0.35 ng/ml.

1.2.4 CLINICAL STUDIES

In controlled multiclinic studies in patients with untreated intraocular

pressures of 22mgHg or greater, timolol 0.25% or 0.5% administered twice a day
produced a greater reduction in intraocular pressure than 1,2,3 or 4 % pilocarpine
solution administered four times a day or 0.5, 1, or 2 % epinephrine hydrochloride
solution administered twice a day.

In these studies, timolol was generally well tolerated and produced fewer
and less severe side effects than either pilocarpine or epinephrine. A slight
reduction of resting heart rate in some patients receiving timolol ( mean reduction
2.9 beats / minute standard deviation 10.2) was observed.

1.2.5 DRUG INTERATION

Beta – adrenergic blocking agents:

Patients who are receiving a beta – adrenergic blocking agent orally and
timolol should be observed for potential additive effects of beta – blockade, both
systemic and on intraocular pressure. The concomitant use of two topical beta-
adrenergic blocking agents is not recommended.

Calcium antagonists:

Caution should be used in the co administration of beta – adrenergic

blocking agents, such as timolol, and oral or intravenous calcium antagonists
because of possible atrioventricular conduction disturbances, left ventricular
failure, and hypotension. In patients with impaired cardiac function, co
administration should be avoided.

Catecholamine – depleting drugs:

Close observation of the patient is recommended when a beta blocker is

administered to patients receiving catecholamine – depleting drugs such as
reserving, because of possible additive affects and the production of hypotension
and / or marked brady cardia, which may result in vertigo, syncope, or postural
hypotension.

Digitalis and Calcium antagonists:

The Concomitant use of beta – adrenergic blocking agents with digitalis and
calcium antagonists may have additive affects in prolonging atrioventricular
conduction time.

CYP2D6 inhibitors :

Potentiated systemic beta-blockade (e.g decreased heart rate, depression) has

been reported during combined treatment with CYP2D6 inhibitors (e.g., quinidine
SSRIs) and timolol.
Clonidine:

Oral beta-adrenergic blocking agents may exacerbate the rebound

hypertension which can follow the withdrawal of clonidine. There have been no
reports of exacerbation of rebound hypertension with ophthalmic timolol maleate.

1.2.6 TIMOLOL DROPS ARE USED FOR

Treating increased pressure in the eye (ocular hypertension) and open –

angle glaucoma. It may also be used for other conditions as determined by your
doctor.

Timolol drops are a beta – blocker. It works to decrease fluid production

and pressure inside the eye.

1.2.7 DO NOT USE FOR TIMOLOL DROPS

 You are allergic to any ingredient in timolol drops

 You have severe chronic obstructive pulmonary disease (COPD) or a
history of asthma
 You have heart block, heart failure, or an unusually slow heart beat
 You are in shock caused by severe heart problems
 You are using another beta-blocker eye drop (e.g betaxolol)
Contact your doctor or health care provider right away if any of these
apply to you.

1.2.8 BEFORE USING TIMOLOL DROPS

Some medical conditions may interact with timolol drops. Tell your doctor
or pharmacist if you have any medical conditions, especially if any of the
following apply to you:
 If you are pregnant, planning to become pregnant, or are breast –
feeding
 If you are taking any prescription or nonprescription medicine, herbal
preparation, or dietary supplement
 If you have allergies to medicines, foods, or other substances
  If you have a history of lung or breathing problems (e.g bronchitis,
COPD, emphysema), diabetes, low blood sugar, heart problems,
certain muscle problems (e.g myasthenia, gravis, muscle weakness),
blood vessel problems, or an overactive thyroid
 If you have narrow – angle glaucoma, double vision, a drooping
eyelid, or an eye infection or injury

Some MEDICINES MAY INTERACT with timolol drops. Tell your health care
provider if you are taking any other medicines, especially any of the following:

 Bupivacaine, calcium channel blockers (e.g verapamil), certain

antiarrhythmics (e.g disopyramide, flecainide, quinidine), cimetidine,
digoxin, ketanserin, reserpine, or selective serotonin reuptake
inhibitors (SSRIs) (e.g fluoxetine) because serious heart problems (e.g
conduction problems, heart failure, slow heartbeat) or low blood
pressure may occur
 Clonidine because increased blood pressure may occur
 Insulin or oral antidiabetics (e.g glyburide, repaglinide) because the
risk of low blood sugar (e.g dizziness, headache, hunger, shakiness or
weakness, sweating) or slow heart rate may be increased. Timolol
drops may also hide certain signs of low blood sugar
 Alpha – blockers (e.g alfuzosin, prazosin), oral beta-blockers (e.g
propranolol), or other beta-blocker eye drops (e.g betaxolol) because
the risk of their side effects may be increased by timolol drops
 Certain sympathomimetics (e.g albyterol, salmeterol), epoinephrine,
or theophylline because their effectiveness may be decreased by
timolol drops
 This may not be a complete list of all interactions that may occur.
Ask your health care provider if timolol drops may interact with other
medicines that you take. Check with your health care provider before
you start, stop, or change the dose of any medicine.

1.2.9 HOW TO USE TIMOLOL DROPS

 Use timolol drops as directed by your doctor. Check the label

on the medicine for exact dosing instructions.
 An extra patient leaflet is available with timolol drops. Talk to
your pharmacist if you have questions about this information.
  Timolol drops are only for the eye. Do not get it in your nose
or mouth.
 Soft contact lenses may absorb a chemical contained in some
brands of timolol drops. Remove contact lenses before you use
timolol drops; lenses may be placed back in the eyes 15 minutes
after use of timolol drops.
 To use timolol drops in the eye, first, wash your hands. Tilt
your head back. Using your index finger, pull the lower eyelid
away from the eye to form a pouch. Drop the medicine into the
pouch and gently close your of the eye for 1 to 2 minutes. Do
not blink. Remove excess medicine around your eye with a
clean, dry tissue, being careful not to touch your eye. Wash
your hands to remove any medicine that may be on them.
 To prevent germs from contaminating your medicine, do not
touch the applicator tip to any surface, including the eye. Keep
the container tightly closed.
 Use timolol drops at least 10 minutes before or after any other
medicine that you put in your eye.
 Using timolol drops at the same time each day will help you
remember to use it.
 Continue to use timolol drops even if you feel well. Do not
miss any doses.
 If you miss a dose of timolol drops, use it as soon as possible.
If it is almost time for your next dose, skip the missed dose and
go back to your regular dosing schedule. Do not use 2 doses at
once.
Ask your health care provider any questions you may have about how to use
timolol drops.

1.2.10 IMPORATANT SAFETY INFORMATION

 Timolol drops may cause drowsiness, dizziness, or blurred vision.

These effects may be worse if you take it with alcohol or certain
medicines. Use timolol drops with caution. Do not drive or perform
other possibly unsafe tasks until you know how you react to it.
 Tell your doctor or dentist that you take timolol drops before you
receive any medical or dental care, emergency care, or surgery.
  Contact your doctor if you have an eye injury or infection, or if you
will be having eye surgery.
 Diabetes patients – Timolol drops may hide signs of low blood sugar,
such as a rapid heartbeat. Be sure to watch for other signs of low
blood sugar. Low blood sugar may make you anxious, sweaty,
weak, dizzy, drowsy, or faint. It may also make your vision change;
give you a headache, chills, or tremors; or make you more hungry.
Check blood sugar levels closely. Ask your doctor before you change
the dose of your diabetes medicine.
 If you have a history of any severe allergic reaction, talk with your
doctor. You may be at risk for an even more severe allergic reaction
if you come into contact with the substance that caused your allergy.
Some medicines used to treat severe allergies may also not work as
well while you are using timolol drops.
 Timolol drops may cause harm if it is swallowed. If you may have
taken it by mouth, contact your poison control centre or emergency
room right away.
 Lab tests, including eye pressure, may be performed while you use
timolol drops. These tests may be used to monitor your condition or
check for side effects. Be sure to keep all doctor and lab
appointments.
 Timolol drops should be used with extreme caution in CHILDREN;
safety and effectiveness in children have not been confirmed.
 PREGNANCY and BREAST – FEEDING; If you become pregnant,
contact your doctor. You will need to discuss the benefits and risks of
using timolol drops while you are pregnant. Timolol drops are found
in breast milk. Do not breast – feed while taking timolol drops.

1.2.11 POSSIBLE SIDE EFFECTS OF TIMOLOL DROPS

All medicines may cause side effects, but many people have no, or minor
side effects. Check with your doctor if any of these most COMMON side effects
persist or become bothersome:

Blurred vision; dizziness; dry eyes, feeling that something is in your eye;
headache; increased tear production; minor burning, itching, or stinging of the eye;
nausea seek medical attention right way if any of these SEVERE side effects
occur:
 Severe allergic reactions (rash; hives; itching; difficulty breathing; tightness
in the chest; swelling of the mouth, face, lips, or tongue); chest pain or discomfort;
confusion; eye irritation, swelling, pain, or discharge; eyelid pain, redness, scaling,
drooping, or swelling; fainting; pain, numbness, weakness, or tingling of an arm or
leg; severe or persistent headache or dizziness; shortness of breath; slow or
irregular heartbeat; swelling of the hands, ankles or feet; vision changes.

This is not a complete list of all side effects that may occur. If you have
questions about side effects, contact your health care provider. Call your doctor
for medical advice about side effects. To report side effects to the appropriate
agency, please read the Guide to Reporting Problems to FDA.

1.2.12 PROPER STORAGE OF TIMOLOL DROPS

Stored timolol drops at room temperature, between 59 and 86 degrees F (15

and 30 C). Protect from freezing. Store away from heat, moisture, and light.
Keep timolol drops out of the reach of children and away from pets.

1.2.13 GENERAL INFORMATION

If you have any questions about timolol drops, please talk with your doctor,
pharmacist or other health care provide.
Timolol drops are to be used only by the patient for whom it is prescribed.
Do not share it with other people.
If your symptoms do not improve or if they become worse, check with your
doctor.
Check with your pharmacist about how to dispose of unused medicine.

1.3 MEDICATION PRESCRIBED

The combination of dorzolamide and timolol and is used to treat eye

conditions, including glaucoma and ocular hypertension, in which increased
pressure can lead to a gradual loss of vision. Dorzolamide and timolol is used for
patients whose eye condition has not responded to another medication.
Dorzolamide is in a class of medications called topical carbonic anhydrase
inhibitors. Timolol is in a class of medications called topical beta blockers.
Dorzolamide and timolol lowers pressure in the eye by decreasing the production
of natural fluids in the eye.

1.3.1 HIGH PRESSURE IN THE EYE

Glaucoma or ocular hypertension have pressures in one or both of their

eye(s) that are too high for them. High pressure in the eye may damage the optic
nerve. This may lead to loss of vision and possible blindness. There generally are
few symptoms that you can feel to tell you whether you have high pressure within
your eyes. Your doctor needs to examine your eyes to determine this. If you have
high pressure in your eye, you will need your pressure checked and your eyes
examined regularly

1.3.2 DORZOLAMIDE HYDROCHLORIDE AND TIMOLOL MALEATE

OPHTHALMIC SOLUTION

Dorzolamide Hydrochloride and Timolol Maleate Ophthalmic solution is an

eyedrop. It contains dorzolamide hydrochloride, which is an ophthalmic carbonic
anhydrase inhibiting drug. It also contains timolol maleated, which is a beta –
blocking drug. Both drugs work to lower pressure in the eye, but in different ways.

Dorzolamide hydrochloride and Timolol Maleate ophthalmic solution is a

medicine for lowering pressure in the eye in people with open – angle glaucoma or
ocular hypertension. It is used when a beta – blocker eye drop alone is not
adequate to control eye pressure.

1.3.3 DRUG INTERACTION

Carbonic anhydrase inhibitors: There is a potential for an additive effect on

the known systemic effects of carbonic anhydrase inhibition in patients receiving
an oral carbonic anhydrase inhibitor and Dorzolamide Hydrochloride and Timolol
maleate ophthalmic solution. The concomitant administration of Dorzolamide
hydrochloride and timolol maleate ophthalmic solution and oral carbonic
anhydrase inhibitors is not recommended.

Acid – base disturbances: Although acid – base and electrolyte disturbances

were not reported in the clinical trials with dorzolamide hydrochloride ophthalmic
solution, these disturbances have been reported with oral carbonic anhydrase
inhibitors and have, in some instances, resulted in drug intractions (e.g toxicity
associated with high – dose salicylate therapy). Therefore, the potential for such
drug interaction should be considered in patients receiving Dorzolamide
Hydrochloride and timolol maleate ophthalmic solution. Beta – adrenergic
blocking agents: Patients who are receiving a beta – adrenergic blocking agent
orally and Dorzolamide hydrochloride and Timolol Maleate ophthalmic solution,
and oral or intravenous calcium antagonists because of possible atrioventricular
conduction disturbances, left ventricular failure, and hypotension. In patients with
impaired cardiac function, coadministration should be avoided.

Catecholmine – depleting drugs: Close observation of the patient is

recommended when a beta – blocker is administered to patients receiving
catecholamine – depleting drugs such as reserpine, because of possible additive
effects and the production of hypotension and / or marked bradycardia, which may
result in vertigo, syncope, or postural hypotension.

Digitalis and calcium antagonists : The concomitant use of beta-adrenergic

blocking agents with digitalis and calcium antagonists may have additive effects in
prolonging atrioventricular conduction time.

CYP2D6 inhibitors : Potentiated systemic beta-blockade (e.g decreased heart

rate, depression) has been reported during combined treatment with CYP2D6
inhibitors (e.g quinidine, SSRIs) and timolol.

Clonidine : Oral beta – adrenergic blocking agents may exacerbate the

rebound hypertension which can follow the withdrawal of clonidine. There have
been no reports of exacerbation of rebound hypertension with ophthalmic timolol
maleate.

1.3.4 NONCLINICAL TOXICOLOGY

Carcinogenesis, Mutagenesis, Impairment of Fertility

In a two – year study of dorzolamide hydrochloride administered orally to

male and female Sprague – Dawley rats, urinary bladder papillomas were seen in
male rats in the highest dosage group of 20 mg/kg/day (250 times the
recommended human ophthalmic dose). Papillomas were not seen in rats given
oral doses equivalent to approximately 12 times the recommended human
ophthalmic dose. No treatment – related tumors were seen in a 21 – month study
in female and male mice given oral doses up to 75 mg/kg/day ( ̴ 900 times the
recommended human ophthalmic dose).

The increased incidence of urinary bladder papillomas seen in the high –

dose male rats is a class – effect of carbonic anhydrase inhibitors in rats. Rasts are
particularly prone to developing papillomas in response to foreign bodies,
compounds causing crystalluria, and diverse sodium salts.

No changes in bladder urothelium were seen in dogs given oral dorzolamide

hydrochloride for one year at 2 mg/kg/day (25 times the recommended human
ophthalmic dose) or monkeys dosed topically to the eye at 0.4 mg/kg/day ( ̴ 5
times the recommended human ophthalmic dose) for one year.

In a two – year study of timolol maleate administered orally to rats, there

was a statistically significant increase in the incidence of adrenal
pheochromocytomas in male rats administered 300 mg/kg/day (approximately
42,000 times the systemic exposure following the maximum recommended human
ophthalmic dose). Similar differences were not observed in rats administered oral
doses equivalent to approximately 14,000 times the maximum recommended
human ophthalmic dose.

In a lifetime oral study of timolol maleate in mice, there were statistically

significant increases in the incidence of benign and malignant pulmonary tumors,
benign uterine polyps and mammary adenocarcinomas in female mice at 500
mg/kg/day, (approximately 71,000 times the systemic exposure following the
maximum recommended human ophthalmic dose), but not at 5 or 50 mg/kg/day
(approximately 700 or 7,000 respectively, times the systemic exposure following
the maximum recommended human ophthalmic dose). In a subsequent study in
female mice, in which post-mortem examinations were limited to the uterus and
the lungs, a statistically significant increase in the incidence of pulmonary tumors
was again observed at 500 mg/kg/day.

The increased occurrence of mammary adenocarcinomas was associate with

elevations in serum prolactin which occurred in female mice administered oral
timolol at 500 mg/kg/day, but not at doses of 5 or 50 mg/kg/day. An increased
incidence of mammary adenocarcinomas in rodents has been associated with
administration of several other therapeutic agents that elevate serum proclatin, but
no correlation between serum proclatin levels and mammary tumors has been
established in humans. Furthermore, in adult human female subjects who received
oral dosages of up to 60 mg of timolol maleate (the maximum recommended
human oral dosage), there were no clinically meaningful changes in serum
prolactin.

Timolol maleate was devoid of mutagenic potential when tested in

vivo(mouse) in the micronucleus test and cytogenetic assay (doses up to 800
mg/kg) and in vitro in a neoplastic cell transformation assay (up to 100 mcg/ml).
In Ames tests the highest concentrations of timolol employed, 5,000 or 10,000
mcg/plate, were associated with statistically significant elevations of revertants
observed with tester strain TA100 (in seven replicate assays), but not in the
remaining three strains. In the assays with tester strain TA100, no consistent dose
response relationship was observed, and the ratio of test to control revertants did
not reach 2. A ratio of 2 is usually considered the criterion for a positive Ames test.

Reproduction and fertility studies in rats with either timolol maleate or

dorzolamide hydrochloride demonstrated no adverse affect on male or female
fertility at doses up to approximately 100 times the systemic exposure following
the maximum recommended human ophthalmic dose.

1.3.5 WARNING AND PRECAUTIONS

Potentiation or Respiratory Reaction Including Asthma

Dorzolamide hydrochloride – timolol maleate contains timolol maleate, a

beta – adrenergic blocking agent; and although administered topically, is absorbed
systemically. Therefore, the same types of adverse reactions that are attributable to
systemic administration of beta-adrenergic blocking agents may occur with topical
administration. For example, severe respiratory reactions, including death due to
bronchospasm in patients with asthma, and rarely death in association with cardiac
failure, have been reported following systemic or ophthalmic administration of
timolol maleate.

Cardiac Failure

Sympathetic stimulation may be essential for support of the circulation in

individuals with diminished myocardial contractility, and its inhibition by beta –
adrenergic receptor blockade may precipitate more severe failure. In patients
without a history of cardiac failure continued depression of the myocardium with
beta – blocking agents over a period of time can, in some cases, lead to cardiac
failure. At the first sign or symptom of cardiac failure, dorzolamide hydrochloride
– timolol maleate should be discontinued.

Sulfonamide Hypersensitivity

Dorzolamide hydrochloride – timolol maleate contains dorzolamide, a

sulfonamide; and although administered topically, it is absorbed systemically.
Therefore, the same types of adverse reactions that are attributable to sulfonamides
maleate. Fatalities have occurred, although rarely, due to severe reactions to
fulminant hepatic necrosis, agranulocytosis, aplastic anemia, and other blood
dyscrasias. Sensitization may recur when a sulfonamide is readministered
irrespective of the route of administration. If signs of serious reactions or
hypersensitivity occur, discontinue the use of this preparation.

Obstructive Pulmonary Disease

Patients with chronic obstructive pulmonary disease (e.g chronic

bronchitis,emphysema) of mild or moderate severity, bronchospastic disease, or a
history of bronchospastic disease (other than bronchial asthma or a history of
bronchial asthma, in which dorzolamide hydrochloride – timolol maleate is
contraindicated) should, in general, not receive beta-blocking agents, including
dorzolamide hydrochloride – timolol maleate.

Increased Reactivity to Allergens

While taking beta – blockers, patients with a history of atopy or a history of

severe anaphylactic reactions to a variety of allergens may be more reactive to
repeated accidental, diagnostic, or therapeutic challenge with such allergens. Such
patients may be unresponsive to the usual doses of epinephrine used to treat
anaphylactic reactions.

Potentiation of Muscle Weakness

Beta-adrenergic blockade has been reported to potentiate muscle weakness

consistent with certain myathenic symptoms (e.g diplopia, ptosis, and generalized
weakness). Timolol has been reported rarely to increase muscle weakness in some
patients with myasthenia gravis or myasthenic symptoms.

Masking of Hypoglycemic Symptoms in Patients with Diabetes Mellitus

Beta – adrenergic blocking agents should be administered with caution in

patients (especially those with labile diabetes) who are receiving insulin or oral
hypoglycemic agents. Beta - adrenergic receptor blocking agents may mask the
signs and symptoms of acute hypoglycemia.

Masking of Thyrotoxicosis

Beta – adrenergic blocking agents may mask certain clinical signs (e.g
tachycardia) of hyperthyroidism. Patients suspected of developing thyrotoxicosis
should be managed carefully to avoid abrupt withdrawal of beta-adrenergic
blocking agents that might precipitate a thyroid storm.

Renal and Hepatic Impairment

Dorzolamide has not been studied in patients with severe renal impairment
(CrCl <30 mL/min). Because dorzolamide and its metabolite are excreted
predominantly by the kidney, dorzolamide hydrochloride – timolol maleate is not
recommended in such patients.

Dorzolamide has not been studied in patients with hepatic impairment and
should therefore be used with caution in such patients.

Impairment of Beta – Adrenergically Mediated Reflexes During Surgery

The necessity or desirability of withdrawal of beta – adrenergic blocking

agents prior to major surgery is controversial. Beta – adrenergic receptor blockade
impairs the ability of the heart to respond to beta – adrenergically mediated reflex
stimuli. This may augment the risk of general anesthesia in surgical procedures.
Some patients receiving beta – adrenergic receptor blocking agents have
experienced protracted severe hypotension during anesthesia. Difficulty in
restarting and maintaining the heartbeat has also been reported. For these reasons,
in patients undergoing elective surgery, some authorities recommend gradual
withdrawal of beta – adrenergic receptor blocking agents. If necessary during
surgery, the effects of beta – adrenergic blocking agents may be reversed by
sufficient doses of adrenergic agonists.

Corneal Endothelium

Carbonic anhydrase activity has been observed in both the cytoplasm and
around the plasma membranes of the corneal endothelium. There is an increased
potential for developing corneal edema in patients with low endothelial cell counts.
Caution should be used when prescribing dorzolamide hydrochloride – timolol
maleate to this group of patients.

Bacterial Keratitis

There have been reports of bacterial keratitis associated with the use of
multiple – dose containers of topical ophthalmic products. These containers had
been inadvertently by patients who, in most cases, had a concurrent corneal disease
or a disruption of the ocular epithelial surface.
 CHAPTER – II
SCOPE OF THE WORK
 SCOPE OF THE WORK

It is evident from the study that the developed methods are simple, specific,
precise, accurate and cost effective. The newly developed method can be used for
routine analysis as method for the estimation of Timolol Maleate and
Dorzolmalide Hydrochloride present in pure drug and in the pharmaceutical
preparation. So the developed method could be suitable for the quantitative
estimation of dorzolamide hcl in pharmaceutical preparation from the current work
it was finally concluded that the developed RP-HPLC method is found to very
simple, reliable and selective providing satisfactory accuracy and precision.
 CHAPTER – III
EXPERIMENTAL METHODS
 EXPERIMENTAL METHODS

Materials
Dorzolamide Hydrochloride and Timolol Maleate Jawa Pharmaceuticals (P)
Ltd were available from Oasis Laboratories Jaipur, India. HPLC grade
acetonitrile, methanol, triethylamine were purchased from Merck. Double distilled
water for analytical purpose was obtained from Milli – Q- R- O system.

Chromatographic conditions
The HPLC system consisted of a Shimadzu LC–10Advp liquid
chromatographic pump, Rheodyne injection port with a 20 µl sample loop and UV
– visible detector (Shimadzu). Data collection, integration and calibration were
accomplished using LC solutions chromatography Data system. The
chromatographic separation of Dorzolamide Hydrochloride and Timolol Maleate
was accomplished using EC 150 / 4.6 NUCLEOSIL 100 – 5C 18, 5 µm reverse
phase analytical column. The mobile phase consisted of methanol: buffer (0.02 M
Octane – 1 – sufonic acid buffer) (60:40), the pH was adjusted to 3 with glacial
acetic acid. Before use, the mobile phase was filtered by passing it through a 0.45
µm filter and the filtrate is degassed by using bath sonicator. The mobile phase
was pumped at 1 ml/min at room temperature. All separations were performed at
ambient temperature.
Preparation of stock and standard solutions
Stock solution of Dorzolamide Hydrochloride and Timolol Maleare was
prepared in methanol at (1000 + 250 µg/ml). This stock solution was diluted with
methanol to obtain the concentrations required for preparation of standard working
solutions. Dorzolamide Hydrochloride and Timolol Maleate working solutions
were in the range of 6.43 to 120 µg/ml & 2.28 to 30 µg/ml. Sample for the
determination of recovery, precision and accuracy were prepared by spiking
quality control (QC) standard Dorzolamide Hydrochloride (40,60,80,100,120
µg/ml) and Timolol Maleate & (10,15,20,25,30 µg/ml) concentrations.
Validation parameters and procedures
The RP – HPLC assay validation was done as per ICH Q2 (R1) guidelines.
These tests included determination of accuracy, precision, linearity, sensitivity and
limit of detection and quantification.
Linearity, limit of detection (LOD) and limit of quantification (LOQ)
Standard calibration samples were prepared by making serial dilutions from
the stock solution of Dorzolamide Hydrochloride and Timolol Maleate.
Calibration curve of concentration versus peak areas was plotted at concentration
range of Dorzolamide Hydrochloride and Timolol Maleate working solutions 6.43
to 120 µg/ml & 2.28 to 30 µg/ml 0.5 – 50 µg/ml simultaneously. The limit of
detection (LOD) and the lower limit of quantification (LLOQ) were measured
according to the FDA’s guidance for bioanalytical method validation in 2001. The
limit of detection was defined as the lowest concentration of Dorzolamide
Hydrochloride and Timolol Maleate resultin in a peak height greater or equal to
three times from background noise (S/N=3). The LOQ was investigated in
extracted samples from five different days. For the determination of LOQ, the
percentage deviation and % RSD are to be less than 20%.
Precision and Accuracy
The precision and accuracy were determined by analyzing spiked standard
and extracted samples of Dorzolamide Hydrochloride at (40,60,80,100,120 µg/ml)
and Timolol Maleate at (10,15,20,25,30 µg/ml) different concentrations ranging.
The precision of an HPLC method was determined as the coefficient of variation
(%RSD) of intra – and inter – day. The intra – day precision was determined by
analyzing the spiked standard and extracted samples prepared within a day. The
inter – day precision was determined by analyzing the spiked standard and
extracted samples analyzed on five different days. After concentrations were
calculated by re – fitting peak areas obtained with different standard solutions into
a derived regression equation from the set of these standard solutions, % R.S.D
was determined at each concentration of the standard solutions from their average
value and S.D. The accuracy of the HPLC method was demonstrated by
percentage deviation. The calculated concentrations (or conc. Found) were
obtained by re-fitting peak areas from standard solutions of known concentrations
(or conc. Added) into a derived regression equation. The conc. Found and conc.
added was then used to determine the absolute percentage deviation at each
concentration of the standard solutions.
Recovery
The absolute recovery was calculated by comparing the peak areas of
compounds after liquid extraction with those obtained on direct injection onto the
column of the same amount of Dorzolamide Hydrochloride and Timolol Maleate
dissolved in mobile phase. Each measurement was made in triplicates.
Peak area of extracted standard
Recovery (%) = -----------------------------------------------------
Peak area of un extracted standard
System suitability
The purpose of system suitability to define a set of parameters that are
measured prior to each experiment that will tell the analyst if the system is
performing adequately or not. The suitability parameters that are evaluated for
HPLC method includes peak area reproducibility and retention time.
 CHAPTER – IV
RESULTS AND DISCUSSION
 RESULTS AND DISCUSSION
Chromatography
To facilitate equality control study of Dorzolamide Hydrochloride and
Timolol Maleate, a sensitive, specific and reproducible HPLC method has been
developed and validated for quantitative determination of Dorzolamide
Hydrochloride and Timolol Maleate by reverse phase HPLC with UV detection at
254 nm & 295 nm.
The representative chromatograms of Dorzolamide Hydrochloride and
Timolol Maleate spiked in unextracted standard concentration and blank. The
retention time of Dorzolamide Hydrochloride and Timolol Maleate was 6.08 min
& 3.46 min respectively, and the peaks were sharp. There was good baseline
separation of Dorzolamide Hydrochloride and Timolol Maleate.
Linearity, limit of detection (LOD) and limit of quantitation (LOQ)
Peak areas of Dorzolamide Hydrochloride and Timolol Maleate were
measured. A representative calibration graph of peak area versus concentration in
the range Dorzolamide Hydrochloride at (40,60,80,100,120 µg/ml) and Timolol
Maleate at (10,15,20,25,30 µg/ml) resulted in regression equation of the calibration
curve was calculated as y = 7330.x + 3699for Dorzolamide Hydrochloride was
calculated from calibration curve, where y is peak area and x is the value of
various concentrations of standard solutions and correlation coefficient was found
to be r2 = 0.999.
 The linear regression equation the linear regression equation y = 16109 x –
48294 for Timolol maleate was calculated from calibration curve, where y is peak
area and x is the value of various concentrations of standard solutions and
correlation coefficient was found to be r2 = 0.999. These results demonstrated a
good linearity between the peak areas versus concentrations. The LOD was found
to be 0.755 µg/ml and LOQ was found to be 2.289 µg/ml and 6.432 µg/ml for
Timolol Maleate and Dorzolamide Hydrochloride respectively.
Precision
The precision of the assay method was validated by the determination of the
intra – and inter – day coefficient of variation (% R.S.D) and percentage deviation.
The intra – day precision data over the concentration range of 40,60,80,100,120
µg/ml for Dorzolamide Hydrochloride and 10,15,20,25,30 µg/ml for Timolol
Maleate and all mean RSD (%) was found to be within acceptance limit (<2%), for
Dorzolamide Hydrochloride and Timolol Maleate.
Repeatability
The repeatability study which was conducted on the solution having the
concentration of about 20 µg/ml for Timolol Maleate and 80 µg/ml for
Dorzolamide Hydrochloride showed a RSD of 0.573 % for Timolol Maleate and
0.429% for Dorzolamide Hydrochloride. Thus it was concluded that the analytical
technique showed a good repeatability.
Accuracy
The accuracy of the method was verified by comparing the concentrations
measured from market sample with actual added concentrations. The recovery of
Dorzolamide Hydrochloride and Timolol Maleate was evaluated at concentrations
of 80, 100 and 120 µg/ml prepared by using standard & spiked solutions of
Dorzolamide Hydrochloride and Timolol Maleate. % recovery was calculated and
was found to be 99.31 % for Timolol Maleate and 99.89 % for Dorzalamide
Hydrochloride. The limit for mean % recovery is 98 – 102 % and as both the
values are within the limit, hence it can be concluded that the proposed method is
accurate.
Tablet : 1 Recovery of Dorzolamide Hydrochloride and Timolol Maleate

S. No Conc. Before Reference Std. Conc. After % recovery

spiking C1 (µ added C2 (µ spiking C3 (µ
µµ µg/ml) µµ µg/ml)* µµ µg/ml)*
1 40 40 81.34 101.67
10 10 20.03 100.15
2 40 60 98.87 98.87
10 15 24.80 99.21
3 40 80 118.95 99.13
10 20 29.56 98.53

Mean = SD DORZOLAMIDE = 99.8 + 1.55

TIMOLOL MALEATE = 99.31 + 0.83
System suitability
System suitability testing is an integral part of many analytical procedures.
The tests are based on the concept that the equipment, electronics, analytical
operations and samples to be analyzed constitute an integral system that can be
evaluated as such. Following system suitability test parameters were established.
The data are shown in Tab. 2.
 Table 2: Data of system Suitability Parameter
S.No Parameter
1 Resolution
2 Theoretical plate
3 Retention time
4 Tailing factor
5 Asymmetric Factor
Result
3.09
Timolol Maleate 2100 Dorzolamide
Hydrochloride 2300
Timolol Maleate 6.08 min Dorzolamide
Hydrochloride 3.7 min
Timolol Maleate 1.09 Dorzolamide
Hydrochloride 1.15
Timolol Maleate 1.11 Dorzolamide
Hydrochloride 1.17

The present method for the determination of Dorzolamide Hydrochloride

and Timolol Maleate is sensitive, specific accurate, and reproducible. The
excellent separation is demonstrated in the chromatograms and no interfering
peaks were observed. The calibration curve was liner and the method was suitable
for the analysis of samples. The accuracy of the method was in compliance with
the proposed limits and the precision of the method was satisfactory. The system
suitability of the method shows that the performance of the chromatographic
system is not significantly influenced by variations of the operational parameters
inside an accepted domain. This method shows the system suitability parameters
are within the limits only.
The validation parameters according to I.C.H Q2 (R1) guidelines were
studied. The accuracy of the methods was proved by performing recovery studies
in the available formulations. Values obtained were found to be greater than 98 %,
which indicated that the proposed method is accurate for the analysis of drug.
Summary of various validation parameters performed for RPHPLC method are
shown in Tab. 3.

Table 3: Summary of Validation Parameters by RP-HPLC Method

DORZOLAMIDE TIMOLOL
Validation Parameters
HYDROCHLORIDE MALEATE

Specificity % interference < 0.5 %

Linear range 40-102 10-30

Working range 30-140 2.28-40

Range
40 Target range 64,80,100 16,20,24

Target 80 20
concentration
Repeatability 0.429 0.573

Inter day 0.16 0.366

Precision (% RSD)
Inter day 0.422 0.788

Accuracy (% recovery) 99.89 99.31

LOD 2.122 0.751

LOQ 6.432 2.289

CHAPTER – V
CONCLUSION
 CONCLUSION

It is evident from the study that the developed methods are simple, specific,
precise, accurate and cost effective. The newly developed method can be used for
routine analysis as method for the estimation of Timolol Maleate and Dorzolamide
Hydrochloride present in pure drug and in the pharmaceutical preparation. So the
developed method could be suitable for the quantitative estimation of dorzolamide
HCl in pharmaceutical preparation from the current work it was finally concluded
that the RP – HPLC method is found to very simple, reliable and selective
providing satisfactory accuracy and precision. The method is suitable for routine
quantitative analysis in pharmaceutical dosage forms. The recoveries achieved
were highly signification the developed method. Hence, the chromatographic
method developed for Timolol Maleate and Dorzolamide Hydrochloride can be
effectively applied for routine analysis in research institutions, quality control
department in industries, approved testing laboratories and in pharmacokinetic
studies.
CHAPTER – VI
REFERENCES
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