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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.
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 2231
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
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 2232
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].
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 2233
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
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 2234
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].
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 2235
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
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 2236
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].
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 2237
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
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 2238
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
colour change precipitation crystals formation
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
CB - colour changed to brown colour
NC – No change
Colour change of Epinephirine due to Oxygen
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 2239
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
04 75°C No colour change No precipitation No crystal formation
05 95°C No colour change No precipitation No crystal formation
Observations
colour change precipitation crystals formation
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
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 2240
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
1 Atropine sulphate USP 93% to 107% 96.43%w/v
2 Ephinephrine bi tartararte BP 87.5% to 112.5% 95.57%w/v
3 Lignocaine hydrochloride USP 95% to 105 % 103.42%w/v
Batch No.3
1 Atropine sulphate USP 93% to 107% 100.55%w/v
2 Ephinephrine bi tartararte BP 87.5% to 112.5% 104.60%w/v
3 Lignocaine hydrochloride USP 95% to 105 % 104.06%w/v
B.No 1 - Atropine Standard B.No 1 - Atropine Injection B.No 2 - Atropine Standard
Observations
colour change precipitation crystals formation
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
CB - colour changed to brown colour
NC – No change
Colour change of Epinephirine due to Oxygen
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 2241
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
RESEARCH ARTICLE S.Abraham jebaraj et.al / IJIPSR / 2 (10), 2014, 2230-2249
Department of Pharmaceutics ISSN (online) 2347-2154
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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
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 2243
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.
S.NO Drugs Pharmacopeia
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
RESEARCH ARTICLE S.Abraham jebaraj et.al / IJIPSR / 2 (10), 2014, 2230-2249
Department of Pharmaceutics ISSN (online) 2347-2154
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Table 12: Stress testing – Photolysis
S.NO Drugs Pharmacopeia
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
Atropine sulphate Ephinephrine bi tartararte Lignocaine hydrochloride
Table 13: Stress testing – Hydrolysis
S.NO Drugs Pharmacopeia
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
Atropine sulphate Ephinephrine bi tartararte Lignocaine hydrochloride
Acid
degradation
Base
degradation
Acid
degradation
Base
degradation
Acid
degradation Base degradation
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Table 14: Stress testing – Thermal degradation
S.NO Drugs Pharmacopeia
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
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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
RESEARCH ARTICLE S.Abraham jebaraj et.al / IJIPSR / 2 (10), 2014, 2230-2249
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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.
RESEARCH ARTICLE S.Abraham jebaraj et.al / IJIPSR / 2 (10), 2014, 2230-2249
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