International Journal of Innovative Pharmaceutical ...ijipsr.com/sites/default/files/articles/IJIPSRMNR-102.pdfA.2.Preformulation Studies a) Effect Of Temperature 1% solution of each

RESEARCH ARTICLE S.Abraham jebaraj et.al / IJIPSR / 2 (10), 2014, 2230-2249

Department of Pharmaceutics ISSN (online) 2347-2154

Available online: www.ijipsr.com October Issue 2230

FORMULATION AND EVALUATION OF FIXED DOSE

COMBINATION OF ATROPINE SULPHATE, LIDOCAINE

HYDROCHLORIDE AND EPINEPHRINE BITARTATE

INJECTION

1S.Abraham jebaraj*,

2 T.Sivakumar,

3R.Prasana

1Department of pharmaceutics, Karpagam university.Coimbatore, INDIA

2Prinicipal, Nandha college of pharmacy.Erode, INDIA

3Adcock ingram limited Bangalore, INDIA

Corresponding Author:

S.Abraham jebaraj

Department of pharmaceutics

Karpagam university, Coimbatore, Tamilnadu, INDIA

Email: [email protected]

Phone: +91 8220008811

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

The aim of the project is combining three drugs which is already present in the market and formulate

the fixed dose combination injectable formulation. This will be used for intracameral injection. This

ophthalmic product contains the active ingredients Atropine sulfate – 3 microgram, Lignocaine

hydrochloride – 5.175 mg, Epinephrine bitartarate – 4.5 microgram per 0.5ml with this required

quantity of sodium meta bisulphate, sodium chloride, Disodium EDTA, Acetate buffer and the

sufficient quantity of water for injection are added to make the stable formulation. This product can be

used any type of intraocular surgery and having advantage of less maneuvers during ophthalmic

surgery, No pain, Longer duration of mydriatic ensuring sufficient time for surgery and post operative

analgesia, Cost effective and no preservative present in the formulation to avoid toxic effects to eye.

The materials and methods used in this experiment proves that drugs does not have any physical and

chemical incompatibilities and found good stability during stability studies. The assay results are found

within pharmacopoeia limits.

Keywords: Atropine,Lignocaine Hydrochloride, Epinephrine bitartarate, ophthalmic, Mydriatic.




INTRODUCTION

The eye is one of the most important organ in the human body. Due to the change in the food

habit and lifestyle, the defects of an eye are more common around the world. Ophthalmology is

the study of the eye and its diseases.Ophthalmist is the person one who treats an eye disease either

by drug therapy or by surgery. The common eye diseases are glaucoma, conjuctival congestion,

ocular hyper tension, conjunctivitis, reduction in the power of the lens in an eye, cataract etc.

Now a day’s eye surgery is common in cataract and other diseases. The drugs used in the surgery

and treatment of the diseases are Local anesthetics, Mydriatis, Miotics, cycloplgics, Antibiotics,

Stains, Lubricants and Anti inflammatory agents. Ocular surgery is necessary for cataract removal

and intra ocular implantations, prior to operation local anaesthesia is must for patients. At early

stage opthalmogist considered topical anaesthesia is the best for this sugery. But anxious patients

cannot maintain the fixation, more over in topical method cannot remove the sensation of pressure

and discomfort during intraocular maneuvers like phacoemulsification and lens insertion.Intra

cameral injection is best approach for to eliminate the complications arises during surgery [1].

Intracameral lignocaine is a potent local anaesthetic and produces faster onset of action.The

duration of anaesthic effect also sufficient for surgery and it has low toxicity. More over

interacameral injection method is highly specific, produces efficacious results and patients are

very comfortable. Surgens were found effective pupil dilation and increased duration local

anaesthetic effect when used intracameral Ephinephrine (1:10000) injection along with lignocaine

injection [2]. Because of steps involved in the ocular surgery like capsulorhexis,

phacoemulsification and intra ocular lens implantation are more difficult in ocular surgery

maneuvers. Topical anesthesia with or without intracameral injection is not contraindicated if

pupillary enlargement required intraoperatively. Combining Atropine with Lingnocaine and

Ephinephrine combination will be very convenient for the patient to get faster medriasis that lasts

for the entire duration of intra ocular surgery during cataract removal, Intra ocular implantations

and Retinal detachment.More over Atropine is the first choice mydriatic drug used by

ophthalmologists.Atropine produces full mydriasis for the entire duration of the surgery and time

for mydriasis takes less than any other drugs. In the market atropine sulphate injection as single

dose injection available in market. Combination of Lignocaine hydrochloride with Epinephrine

bitartrate injection are available in market.Aim of this research combine these two formulation

into a single formulation for intra Ocular surgery. In these three combinations, Lignocaine is a




local anesthetic agent. Local anesthetics are chemical agents that reversibly block the

transmission of nerve impulses along sensory fibres. They will also block motor nerves but in

higher concentrations than are normally obtained by topical instillation. Different sensations are

lost according to the size of axons serving them. Pain, which is carried by the smallest fibres, is

lost first, followed by touch and temperature sensitivity; pressure, which is carried by the largest

nerve fibres, is lost last. Not all local anesthetics are suitable for topical ophthalmic use. This is

because of the poor absorption characteristics of some agents. For example, procaine is an

excellent injectable anesthetic but, because of its highly polar nature, has a poor lipid solubility

and therefore crosses mucous membranes very slowly [3]. Atropine induces mydriasis by

blocking contraction of the circular pupillary sphincter muscle, which is normally stimulated by

acetylcholine release, thereby allowing the radial pupillary dilator muscle to contract and dilate

the pupil. Atropine induces cycloplegia by paralyzing the ciliary muscles, whose action inhibits

accommodation to allow accurate refraction in children, helps to relieve pain associated with

iridocyclitis, and treats ciliary block (malignant) glaucoma. Atropine is contraindicated in patients

pre-disposed to narrow angle glaucoma. Atropine can be given to patients who have direct globe

trauma [3,4,5]. Epinephrine (also known as adrenaline) is a hormone and a neurotransmitter.

Epinephrine has many functions in the body, regulating heart rate, blood vessel and air passage

diameters, and metabolic shifts; epinephrine release is a crucial component of the fight-or-flight

response of the sympathetic nervous system. Generally an ophthalmic product contains the active

ingredient, Buffer, Antioxidant if needed, preservatives if needed, isotonic agent and WFI. As per

guideline provided for fixed dose combination, the concentration of active ingredients was fixed,

as per that concentration of Atropine sulfate – 3 microgram, Lignocaine hydrochloride – 5.175

mg, Epinephrine bitartrate – 4.5 microgram per 0.5ml with this required quantity of sodium meta

bisulphate, sodium chloride,Disodium EDTA,Acetate buffer and the sufficient quantity of WFI.

The excipients selected in this formulation are already exist in the present market formulations.

Formula was derived from the existing market products.

1.Hand book of Pharmaceutical manufacturing formulations,sterile pharmaceutical

formulation,Part 2 of Volume 6,Page no 33,179 by Dr.Sarfarz Niazi mentioned that both the

formulations are having similar excipents.

2.Hand book on speciality parentral drugs by R.M.Gupta also mentioned similar kind of formula

for both the formulations. So the existing market formulations of atropine sulphate injection and

ephinephrine bitartarate, Lignocaine injection contains the following excipients [13,15,16,17].




Table 1: Existing Marketed Formulations Compositions of Atropine Sulphate

Injection and Epinephrine bitartrate, Lignocaine Injection

Atropine and Lignocaine is unstable in alkaline pH, but Epinephrine bitartarate are stable in

alkaline and acidic pH. So the pH is adjusted to acidic pH in between 3.0 to 4.0 by glacial acetic

acid. Excipent selection and criteria for injectable dosage forms page no 281, says that for PH 3

to 6, acetate buffer can be used. Sodium chloride is used as to maintain the osmololity.The

osmololity will be inbetween 280 to 350 mOsm which is more compatable with the

tissue.Chelating agent Di sodium EDTA 0.01% to 0.075% is included in this formulation in order

prevent the oxidation process induced by the free radicals from the molecular oxygen. [8,10,11]

MATERIALS

Atropine sulphate, Epinephrine bitartrate, Lignocaine Hydrochloride, Manufacturing tank,

Autoclave, Filling machine, Stability chamber, pH meter, Osmometer

METHODS

A.1.Preformulation:

a) Solubility Studies

The three drugs were dissolved in WFI, in 10 ml Stoppard three different volumetric

flasks. The volumetric flasks were then placed at room temperature for 8 hours. The

solution was filtered through membrane and appropriate dilutions were made to measure

the absorbance at UV visible spectrophotometer, and water as blank. [7]

S.No

Atropine

Sulphate

injection

Epinephrine bitartrate +

Lignocaine

hydrochloride injection

Strength Uses

01 Sodium chloride Sodium chloride 1.5% Osmololity

O2 Sodium

metabisulphate Sodium metabisulphate 0.1% Anti oxidant

03 Sodium acetate Sodium acetate 1%to 2% Buffering agent

04 Glacial acetic

acid Glacial acetic acid 1%to2% Buffering agent

05 Water For

Injection Water For Injection QS Vehicle




b) Effect of Temperature on Stability of Drugs

1 gram of each drugs were dissolved together in WFI in 100 ml volumetric and filtered

through 0.22µ membrane filter and filled in to a vials. The filled vials are kept in

refrigerator, room temperature, 50°C, 75°C and 95°C for 1 week. Vials kept in

refrigerator and room temperature are considered as controls. The above samples are

inspected for colour change formation of precipitation and crystals formation [7].

c) Light Stability of Drug

1gram of each drugs were dissolved separately in WFI in 100 ml volumetric flask and

filtered through 0.22 μm micron membrane filter. The filtered solution was poured into a

vial to expose to the open atmosphere, remaining solution was filled into 20 ml amber

colored Vials and 20 ml flint vial and packed into the cardboard box as controls.The

above vials were checked for 5 weeks for visible colour change formation of precipitation

and crystals formation [7].

d) Effect of Oxygen

1 gram of each drugs were dissolved separately in WFI in 100 ml volumetric flask and

filtered through 0.22 micron membrane filter and filled into 20 ml vial and place at room

temperature. One group of vials were purged with nitrogen gas another group was purged

with compressed air.The above vials were checked for 5 weeks for visible colour change

formation of precipitation and crystals formation [7].

e) Physical Compatibility Study

Compatibility studies were carried out for Atropine sulfate, Lidocaine Hydrochloride,

Epinephrine Bitartarate and with excipients by FTIR. Eqivalent parts of drugs were mixed

and checked physical incompatibility. [5, 7]

A.2.Preformulation Studies

a) Effect Of Temperature

1% solution of each drugs (1:1:1) were dissolved together in WFI with all excipients, and filtered

through 0.22 micron membrane filter and filled in to a vials. The filled vials are kept in

refrigerator, room temperature, 50°C, 75°C and 95°C for 1 week. Vials kept in refrigerator and

room temperature are considered as controls. The above samples are inspected for colour change,

formation of precipitation and crystals formation [7].




b) Light stability of drugs:

1% solution of each drugs(1:1:1) were dissolved together in WFIwith all excipients and filtered

through 0.22 μm micron membrane filter. The filtered solution was poured into a vial to expose to

the open atmosphere, remaining solution was filled into 20 ml amber colored Vials and 20 ml flint

vial and packed into the cardboard box as controls.The above vials were checked for 6 weeks for

visible colour change formation of precipitation and crystals formation [19, 7].

c) Effect of oxygen:

1% of each drugs (1:1:1) were dissolved together in WFI with all excipients and filtered through

0.22 micron membrane filter and filled into 20 ml vial and place at room temperature. One group

of vials were purged with nitrogen gas another group was purged with compressed air.The above

vials were checked for 5 weeks for visible colour change formation of precipitation and crystals

formation [7].

Table 2: Dispensing of Active materials and Excipients was carried out in a separate

dispensing booth under laminar air flow

d) Compounding procedure for Formulation

All the process are carried out class C area

Step 1 - Checked the cleanliness of 2 L manufacturing vessel from inside and outside.

Step 2 - 2.0 L of fresh WFI collected in a manufacturing vessel and cooled to room

temperature.0.22 micron filtered nitrogen was purged for about 30 minutes.

S.No Ingredients

Wt/2lts

Batch no. 1

Ampoule

Batch no. 2

Vial

Batch no. 3

Vial

1 Atropine Sulphate 6.235 mg 6.235 mg 6.235 mg

2 Epinephrine bitartrate 16.78 mg 16.78 mg 16.78 mg

3 Lidocaine HCl 11.43 gms 11.43 gms 11.43 gms

4 sodium meta bisulphite 1 gm 1 gm 1 gm

5 Disodium Edetate 0.50gm 0.50gm 0.50gm

6 Sodium acetate 1.2gm 1.2gm 1.2gm

7 Sodium chloride 6gm 6gm 6gm

8 Glacial acetic acid 0.3ml 0.3ml 0.3ml

9 Water for Injection QS QS QS




Step 3 - 1gm of sodium Metabisulfate added and dissolved in step 2. Ensured complete

dissolution.

Step 4 - 1.2gms of Sodium acetate added and dissolved in Step 2. Ensured complete dissolution.

Step 5 - 0.3ml of Glacial acetic acid IP added and dissolved in Step 2. Ensured complete

dissolution.

Step 6 - 6.0gms of Sodium chloride added and dissolved in Step 2. Ensured complete

dissolution.

Step 7 - 0.50grams of Disodium Edetate IP added and dissolved in Step 2. Ensured complete

dissolution.

Step 8 - Lidocaine hydrochloride 11.43 grams followed by Epinephrine bitartate 16.78mg and

followed by Atropine sulfate 6.235 mg added to Step 2 and dissolved completly.

Step 9 - The pH was adjusted to 3.7 by using glacial acetic acid solution. (Limit 3.0 to 4.0)

Step 10 - The total volume was made upto 2 liters and mixed for about 30 minutes.R16,

k. Sterilization by filtration:

The solution was filtered through sterile 2 micron glass fiber pre filter and followed by 0.22

micron PVDF membrane filter (make: millipore).the sterile solution was collected in a sterile

vessel by using silicon tubing sets and membrane filters. [12]

l. Filling and sealing

The filling was carried out Ampoule filling machine in sterile area under class A ,surrounded by

class B area .All the solution contact machine parts were sterilized by autoclaving at 121 degree

celcious.All ampoules are washed with water for injection by using ampooles washing machine

and dehydrogenated at 300 degree celcious for 3 minutes. Three consecutive batches were

manufactured.The above solution was filled automatic vial filling machine under aseptic

conditions and nitrogen purging done on the head space of the vial. [12]

- 100 vials were filled with compressed air purging on the head space of the product in order to

find the oxygen sensitivity of the drug product.

- 100 vials were filled flint vials,in order to find the light sensitivity. [12]

m. Visual inspection:

Visual inspection was carried out for all the filled ampoules for particulate matters,Glass

pieces,Precipiation,and sealing defects.Rejects were cursed by the crusher [12].




n. Stress testing

The part quantity of filled ampoules were subjected to stress testing in order to find stability

related problems which includes.

1.Oxidation –The product filled and purged with compressed air ampoules are analyzed for

assay content in order identify the potential loss of drug product by the atmospheric oxygen. The

datas are given at table 13.

2.Photolysis-The product filled in flint ampoules are subjected to sunlight for 4 hrs and analyzed

for assay content. The datas are given at table 14.

3.Hydrolysis-the product PH are gradually adjusted to lower PH and Higher PH in order to

identify the stabilization of product. The datas are given at table 15.

4.Thermal degradation:The product is subjected different temperatures in order to break the

bonds that is called as pyrolysis. The datas are given at table 16. [18]

o. Accelerated stability studies

First batch was charged for accelerated stability studies. Stability studies conduct to identify the

shelf life of the product or to identify any degradation. As per ICH guidelines Real Time Studies

Conducted for 30˚C ± 2, 65 ±5%, Accelerated studies conducted for 40˚ C ± 2, 75 ±5%.Analysis

carried out for 0,3.6,9,12,18,24,36 month for Real time, For accelerated studies 0,3,6 month.

[5,20]

p. Analytical procedure

Assay of individual drugs are carried out at HPLC with Photodiode array detector(PDA).Atropine

sulphate and Lignocaine hydrochloride was done by USP method.Ephinephrine hydrochloride

done by BP method.The assay results are given at table 11.

RESULT

A.1.preformulation:

Table 3: Solubility Profile: Atropine sulphate, Ephineprine Bitartarate and Lignocaine hydrochloride are

found to be freely soluble in water for injection

Table 4: Temperature Stability Profile: The vials kept in refrigerator, room temperature, 50°C, 75°C and

95°C for 20 minutes were inspected for colour change and formation of precipitation and crystals

formation and there is no such change found in the preparation

S.No Name of the

Drugs

Solvent Wave

length

Absorbanc

e

Concentration

mg/ml

Standard

deviation

01 Atropine WFI 225nm 3.08 750 microgram/ml 0.10328

02 Epinephrine WFI 233nm 3.8 800microgram/ml 0.10467

03 Lidocaine WFI 272nm 2.83 700 microgram/ml 0.10238




Table 5: Light Stability profile:Colour change was observed in flint vials as well as in amber

vials due to exposure to the light

Table 6: Effect of Oxygen :Vials purged with compressed air changed to brown colour due oxidation of

epinephrine bitartarate.

Fig. 1: FTIR: Atropine + Epinephrine + Lidocaine + Disodium EDTATE+ Sodium met

bisulphate+ Sodium Chloride+ Sodium Acetate

e. FTIR report -Physical compatablity studies

Compatibility studies were carried out for Atropine sulfate, Lidocaine Hydrochloride,

Epinephrine Bitartarate and with excipients by FTIR. Eqivalent parts of drugs were mixed and

checked physical incompatablity for the following functional groups and found no change.

S.No Temperature (°C) Duration colour change Precipitation crystals formation

01 Refrigerator Control No change No change No change

02 Room temperature Control No change No change No change

03 50°C 20min No change No change No change

04 75°C 20 min No change No change No change

05 95°C 20 min No change No change No change

Observations

colour change precipitation crystals formation

No of

week

1st

week

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

we

ek

1st

we

ek

2nd

we

ek

3rd

we

ek

4th

we

ek

5th

we

ek

1st

we

ek

2nd

we

ek

3rd

we

ek

4th

we

ek

5th

we

ek

Flint vial CB CB CB CB CB NC NC NC NC NC NC NC NC NC NC

Amber

vial CB CB CB CB CB NC NC NC NC NC NC NC NC NC NC

CB - colour changed to brown colour

NC – No change

Colour change of Epinephrine due

to light

Observations


1st

wee

k

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

wee

k

1st

wee

k

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

wee

k

1st

wee

k

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

wee

k

Vial -

Purged

with

nitrogen

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

Vial -

Purged

with

compres

sed air

CB CB CB CB CB NC NC NC NC NC NC NC NC NC NC


NC – No change

Colour change of Epinephirine due to Oxygen




60070080090010001100120013001400150016001700180019002000

1/cm

40

45

50

55

60

65

70

75

80

85

90

95

100

%T

1613.52

1594.23

1577.84

1558.55

1523.83

1473.68

1407.13

1387.84 1378

.20 1365.66

1339.62

1319.37

1280.79

1275.97

1246.07

1201.70

1167.95 1143

.84

1112.01

1064.75

1021.35

969.27 702.

12 639.43

608.57

All sample 11-01-14 11.52

Details of Functional group:

Atropine

1) Presence of Ketone group,

2) Presence of Aromatic ring.

Epinephrine

1) Presence of primary amine group,

2) Presence of alcholol.

Lignocaine

1) Presence of primary amine group,

2) Presence of Carboxylic acid.

A.2.preformulation

Table 7: Effect of temperature

Table 8: Light Stability Study

S.No Temperature (°C) Colour change precipitation Crystal formation

01 Refrigerator No colour change No precipitation No crystal formation

02 Room temperature No colour change No precipitation No crystal formation

03 50°C No colour change No precipitation No crystal formation



Observations


1st

we

ek

2nd

we

ek

3rd

we

ek

4th

we

ek

5th

we

ek

1st

we

ek

2nd

we

ek

3rd

we

ek

4th

we

ek

5th

we

ek

1st

we

ek

2nd

we

ek

3rd

we

ek

4th

we

ek

5th

we

ek

Flint

vial

N

C NC

N

C NC NC NC NC

N

C NC NC NC NC

N

C NC

N

C

Amber

vial

N

C NC

N

C NC NC NC NC

N

C NC NC NC NC

N

C NC

N

C

NC – No change




Table: 9 Oxygen sensitivity

Table 9: Assay Results

S.NO Drugs Pharmacopeia

standards Pharmacopeial limits Obtained results

Batch No. 1

1 Atropine sulphate USP 93% to 107% 95.55%w/v

2 Ephinephrine bi tartararte BP 87.5% to 112.5% 97.63%w/v

3 Lignocaine hydrochloride USP 95% to 105 % 104.96%w/v

Batch No. 2




Batch No.3




B.No 1 - Atropine Standard B.No 1 - Atropine Injection B.No 2 - Atropine Standard

Observations


1st

wee

k

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

wee

k

1st

wee

k

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

wee

k

1st

wee

k

2nd

wee

k

3rd

wee

k

4th

wee

k

5th

wee

k

Vial -

Purged

with

nitrogen

NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC

Vial -

Purged

with

compres

sed air

CB CB CB CB CB NC NC NC NC NC NC NC NC NC NC


NC – No change

Colour change of Epinephirine due to Oxygen




Assay HPLC graphs

B.No 2 - Atropine Injection B.No 3- Atropine Standard B.No 3 - Atropine Injection

Atropine Placebo B.No 1 - Epinephrine Standard B.No 1 - Epinephrine Injection

B.No 2 - Epinephrine Standard B.No 2 - Epinephrine Injection B.No3 - Epinephrine Standard

B.No 3 - Epinephrine Injection Epinephrine Placebo B.No 1- Lignocaine standard




B.No 1- Lignocaine Injection B.No 2- Lignocaine standard B.No 2- Lignocaine Injection

B.No 3- Lignocaine standard B.No 3- Lignocaine Injection Lignocaine Placebo

Table 10: PH and Osmolality: Were checked for consistently for 5 weeks and found the results were

within the specified limits

S.N

o

Product

Name

Prod

uct

PH

limit

Observed PH

Osmololity

limit

(mOsmol/k

g)

Observed Osmololity

(mOsmol/kg)

1st

we

ek

2nd

we

ek

3rd

we

ek

4th

wee

k

5th

we

ek

1st

we

ek

2nd

we

ek

3rd

we

ek

4th

we

ek

5th

we

ek

1 Batch

no. 1 3-4 3.7 3.4 3.5 3.6 3.5 280- 350 330 327 332 318 329

2 Batch

no. 2 3-4 3.84 3.7 3.6 3.73 3.84 280- 350 323 318 333 320 322

3 Batch

no. 3 3-4 3.6 3.7 3.6 3.66 3.74 280- 350 328 332 318 325 318




RESULTS OF STRESS TESTING

Table 11: a.Stress testing –Oxidation:During stress study by oxidation all the three drugs were

found to be degraded by oxidation.The assay values are found to be lesser than the pharmacopeial

limits.The osmolality was found to be higher than the limit.


standards

Pharmacopeial

limits Obtained results

1 Atropine sulphate USP 93% to 107% 40.24

2 Ephinephrine bi tartararte BP 87.5% to 112.5% 51.64

3 Lignocaine hydrochloride USP 95% to 105% 56.72

4 Osmolality 280 to

350mOsmol/kg 954mOsm

Atropine sulphate Ephinephrine bi tartararte Lignocaine hydrochloride

b. Stress testing – Photolysis

During stress study by photolysis all the three drugs were found to be degraded by photolysis.The

assay values are found to be lesser than the pharmacopeial limits.There is no elution found in

Atropine and Epinephrine and assay content of lignocaine was drastically reduced. The product

were filled in both Flint and Amber coloured vials for Photolysis test




Table 12: Stress testing – Photolysis


standards

Pharmacopeial

limits

Obtained results

1 Atropine sulphate USP 93% to 107% No elution

2 Ephinephrine bi tartararte BP 87.5% to 112.5% No elution

3 Lignocaine hydrochloride USP 95% to 105% 40.32


Table 13: Stress testing – Hydrolysis


standards

Pharmacopeial

limits Obtained results

1 Atropine sulphate USP 93% to 107% No elution

2 Ephinephrine bi tartararte BP 87.5% to 112.5% No elution

3 Lignocaine hydrochloride USP 95% to 105%

60.32


Acid

degradation

Base

degradation

Acid

degradation

Base

degradation

Acid

degradation Base degradation




Table 14: Stress testing – Thermal degradation


standards

Pharmaco

peial limits Obtained results

at 50 deg C at 75 deg C at 95 deg C

1 Atropine

sulphate USP

93% to

107% Peak splitting Peak splitting

Peak

splitting

2 Ephinephrine

bi tartararte BP

87.5% to

112.5% 40.32%w/v Peak splitting No elution

3 Lignocaine

Hydrochloride USP

90% to

110% 101.61%w/v 126.54%w/v 119.99%w/v

4 Osmolality

280 to

350mOsmo

l/kg

557 mOsm 902 mOsm 802 mOsm

Atropine sulphate 50C Atropine sulphate 75C Atropine sulphate 95C

Ephinephrine bi tartararte 50C Ephinephrine bi tartararte 75C Ephinephrine bi tartararte 95C

Lignocaine hydrochloride 50C Lignocaine hydrochloride 75C Lignocaine hydrochloride

95C




Accelerated Stability study report

The pH and Osmololity of the product found to be stable during six months accelerated stability

studies

Table 15: Osmolarity and pH Limits

Date Osmolality limit 280 to 350 mOsmol/kg PH limit (3.0 to 4.0)

13/08/2013 315 3.70

03/09/2013 330 3.82

17/10/2013 328 3.84

27/11/2013 323 3.81

13/12/2013 332 3.80

21/01/2013 332 3.78

Table 16: Accelerated stablity studies report

S.No Name of the drug Assay for 3rd

month Assay for 6rd

month

1 Atropine sulphate USP 96.58% w/v 96.55% w/v

2 Epinephrine bitartarate BP 98.65% w/v98.65 97.45% w/v

3 Lignocaine hydrochloride

USP 102.46% w/v 101.95% w/v

DISCUSSION

From the above experiment, it is clear that there is no incompatibility in between the three

combinational drugs and with excipients. The FTIR studies show that there is no change in the

functional groups of individual drugs. So the three drugs are combined and formulated in Fixed

dose parenteral formulation for intra Occular injection. Prevention form Photolysis: The Amber

colour ampoule was selected and it is suitable for this injectable formulation due to Ephinephrine

is more sensible to light and atropine is also slowly degrades on light which is filled in the flint

vials. Prevention from Oxidation: Nitrogen purging is must throughout the manufacturing process

since the product very much sensitive to oxygen. All the three drugs are loses its potency

drastically due to oxygen sensitivity. Simultaneously the osmolality also increased from above

from the limit. In order to prevent the oxidation of the product, while manufacturing 0.22 micron

filtered nitrogen was bubbled in the water for injection collected for compounding, this is to




eliminate the dissolved oxygen in the water for injection. The head space of the filled ampoule is

purged with filtered nitrogen. Purging of Pre gassing and post gassing of nitrogen gas was done at

filling stage in vials. Grade 2 nitrogen should be used for manufacturing and purging purpose.

Because this grade contains less than 2ppm of oxygen content in nitrogen gas. Prevention from

thermal degradation: The product was stored in room temperature that is 23C because

ephineprine and atropine is degrading during change of temperature .The formulation should not

be freezed during storage. The assay of all the three drugs were within the limit when stored in

the room temperature. In higher temperature the assay of atropine and epinephrine are drastically

decreased.The osmolality was increased above the limit. Prevention form Hydrolysis: In the acid

hydrolysis and base hydrolysis all the three drugs loses its potency. At above pH 7.2 product was

got precipitated. At 3to 4 PH there is no change in osmolality, pH and clarity of the product.

There is no preservative added in this formulation. Since preservative produces toxic effects in the

eye. Because of no preservative in the formulation it is preferred to fill in the single dose

container. The Accelerated stability studies carried out as per ICH guidelines also shows that the

formulation was stable for 6 months in accelerated condition.

Advantage of this preparation

1. Single dose administration

2. This product can be used any type of intraocular surgery

3. Less maneuvers during opthalmic surgery

4. No pain

5. Longer duration of mydriatic and post operative analgesia

6. No preservative

7. Cost effective

CONCLUSION

The fixed dose combination of sterile injectable formulation of Atropine sulphate,Epinephrine

bitartarate and Lignocaine hydrochloride is very stable 6 months of accelerated stability with the

excipients selected in this formulation.This product is packed in amber ampoules and should be

stored in room temperature.Further this product can be checked for animal studies for

toxicology,Eye irritation test ,ocular tissue damages and clinical trials so that it will be helpful to

glaucoma and cataract patients and easy handling of surgery by ophthalomogist.




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Documents

International Journal of Innovative Pharmaceutical ...ijipsr.com/sites/default/files/articles/IJIPSRMNR-102.pdfA.2.Preformulation Studies a) Effect Of Temperature 1% solution of each