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Original Article
SMJ
Jureeporn Sri-in, M.Sc.*, Waraphorn Sisan, M.Sc.*, Phonphailin Kingkhangphloo, B.Sc.*, Pinpilai Jutasompakorn,
M.D.*,
Weerawadee Chandranipapongse, M.D.*, Somruedee Chatsiricharoenkul, M.D.*, Onchira Buranakan,
MD.**,
Arpha Pornseth, M.D.**, ammanoon Surachatkumtonekul, M.D.**
*Department of Pharmacology, ** Department of Ophthalmology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, ailand.
Stability and Sterility of Extemporaneously Prepared
0.01% Atropine Ophthalmic Solution in Articial
Tears and Balanced Salt Solution
ABSTRACT
Objective: e aim of this study was to investigate the physicochemical and microbiological stability of extemporaneously
prepared 0.01% atropine ophthalmic solution in unopened eyedropper and in simulated use condition.
Materials and Methods: Two formulations of 0.01% atropine solutions, atropine in articial tear and atropine in
balanced salt solution (BSS), were prepared using 0.5 mL insulin syringes. In unopened conditions, 0.01% atropine
solutions were stored for six months at refrigerated temperature (2-8°C) or room temperature (25±2°C). Visual
inspection, atropine quantication, pH measurements, and sterility assay were analyzed at baseline, and every month
for six months. In simulated use condition, 0.01% atropine solutions stored at refrigerated and room temperature
were analyzed at 0, 15 and 30 days.
Results: In unopened conditions, both of 0.01% atropine formulations stored at refrigerated temperature showed
satisfactory stability. Atropine remained within 90% to 110% of the initial concentration up to six months. Under
room temperature, both formulations of atropine were less than 90% of their initial value aer 4 months storage. In
simulated use condition, atropine concentration was within 90% to 110% of initial value aer 30 days at refrigerated
and room temperature. All atropine solutions prepared in articial tear and BSS were free from bacterial contamination
throughout the study. No alteration of physical appearance (i.e., precipitation, discoloration) was observed, and
pH values also remained nearly unchanged.
Conclusion: Both formulations of 0.01% atropine are physicochemically stable for up to 6 months when kept
unopened in refrigerator, and for 1 month at refrigerated and room temperatures in simulated use condition.
Keywords: Myopia; atropine; stability; sterility; articial tear; balanced salt solution (Siriraj Med J 2022; 74: 91-99)
Corresponding author: ammanoon Surachatkumtonekul
E-mail: si95thim@gmail.com
Received 15 September 2021 Revised 28 October 2021 Accepted 17 November 2021
ORCID ID: https://orcid.org/0000-0002-0037-6863
http://dx.doi.org/10.33192/Smj.2022.12
INTRODUCTION
Myopia is an eye disorder and is the principal type of
refractive error. Previous population-based studies have
reported that the prevalence rates of myopia are highest
in East Asian populations.
1,2
eir ndings showed that
80% of schoolchildren in Taiwan, Hong Kong, and China,
as well as up to 96% of schoolchildren in South Korea,
suered from myopia.
3
It is estimated that myopia will
aect nearly 5 billion people by 2050.
2,4,5
Currently, there
are many methods for controlling myopia progression,
such as spectacles, contact lens, and pharmaceutical
strategies. Most of the studies in this eld use atropine
All material is licensed under terms of
the Creative Commons Attribution 4.0
International (CC-BY-NC-ND 4.0)
license unless otherwise stated.
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eye drops to reduce the rate of myopia progression in
children.
6
Atropine is a nonselective muscarinic antagonist, it
binds to and inhibit muscarinic acetylcholine receptors,
producing a wide range of anticholinergic eects. e
precise mechanisms underlying the ecacy of atropine
in slowing myopia progression are remains unclear.
Various hypotheses have been postulated, including the
action via muscarinic receptor pathways in the retina,
choroid and sclera. ese resulting in the prevention of
axial elongation, inhibition of scleral proliferation and
matrix synthesis. Moreover, atropine may be exerting
its eect via other receptors present in the eye.
7-9
Most “Atropine for the Treatment of Myopia (ATOM)”
studies focus on the ecacy and safety of 1%, 0.5%, 0.1%,
and 0.01% atropine in myopic Asian children aged 6-12
years old. eir ndings illustrated that 0.01% atropine is
eective for retarding myopia with minimal side eects,
compared with higher doses of atropine.
10-13
Recently,
the studies of Low-concentration Atropine for Myopia
Progression (LAMP) have demonstrated the ecacy
and safety of atropine concentrations of 0.05%, 0.025%,
and 0.01% in China in children aged 4-12 years old with
myopia. All these concentrations drastically reduced
the rate of myopia progression without any vision-
threatening side eects.
14
Generally, 0.01% atropine
is the most common strategy for managing childhood
myopia and is widely used all over the world, including
in Asian countries, such as Singapore, Taiwan, China,
and ailand.
2
e treatment period usually lasts for at
least 2 years, and may take longer if myopia progression
persists.
15
Since 0.01% atropine ophthalmic solution is not
commercially available in Thailand, eye drops are
prepared by ophthalmologists or hospital pharmacists.
e 1% commercial atropine is diluted with 0.9% sodium
chloride solution, balanced salt solution, or various
brands of articial tears depending on the discretion of
the ophthalmologist. Long-term treatment with atropine
is required for myopia control, and hence a longer shelf-
life is necessary to extend the follow-up intervals for
patients. However, there is little data concerning the
long-term stability of low-dose atropine eye drops. Only
two studies have been published demonstrating that
0.01% atropine in 0.9% sodium chloride solution with
or without preservatives is stable for six months in an
unopened container, both at room temperature and
refrigerated temperature.
16,17
However, there are no studies
on the stability of 0.01% atropine eye drops prepared
in articial tears (with preservatives) or balanced salt
solution (without preservatives). e lack of long-term
stability and sterility data limits the conservation period
of these preparations. Consequently, the aim of this study
was to determine the long-term chemical, physical, and
microbiological stability of extemporaneously prepared
atropine in artificial tears containing preservatives and in
balanced salt solution at refrigerated and room temperature.
e chemical, physical, and microbiological stability
of both formulations were also tested in simulated use
conditions.
MATERIALS AND METHODS
Reagents and materials
Atropine sulfate monohydrate, the reference
standard of atropine, and scopolamine hydrobromide,
the internal standard for atropine, were obtained from
e United States Pharmacopeial Convention, Inc., USA.
1% Atropine sulfate solution was obtained from Alcon-
Couvreur, Belgium. Balanced salt solution (BSS) was
obtained from Alcon Research LLC, USA. Hydroxypropyl
methylcellulose (HPMC), an articial tears solution
with sodium perborate as a preservative, was obtained
from Silom Medical Co., Ltd., ailand. LC/MS grade
acetonitrile and formic acid were obtained from Scharlau,
Barcelona, Spain. HPLC-grade methanol was obtained
from Fisher Scientic UK, the United Kingdom. Type I
water was produced using a Milli-Q water purication
system from Millipore Corporation, USA.
0.01% Atropine eye drops preparation
e preparation processes were undertaken by
scientists in a clean room of class 1.0×10
5
(air cleanliness
level of a maximum of 2.93×10
4
particles (≥0.5 µm)
per cubic meter). Two formulations of 0.01% atropine
ophthalmic solutions were prepared aseptically using a
0.5 mL insulin syringe:
- Atropine in preserved articial tears (HPMC),
prepared by dissolving 0.1 mL of 1% atropine
sulfate solution into 10 mL articial tears.
- Atropine in balanced salt solution, prepared by
dissolving 0.15 mL of 1% atropine sulfate solution
into 15 mL balanced salt solution (BSS).
Clear low-density polyethylene commercial eyedroppers
of HPMC and BSS were used as the containers in this
study.
Study design
e stabilities of the 0.01% atropine ophthalmic
solutions were studied at refrigerated temperature (2-8
°C) or room temperature (25±2 °C). e durations of the
study for the unopened eyedroppers and in simulated use
conditions were 6 months and 1 months, respectively.
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Original Article
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In short, there were 4 subgroups in each study (for the
unopened eyedroppers and simulated use conditions)
as shown below:
(i) 0.01% atropine in HPMC at refrigerated temperature,
(ii) 0.01% atropine in HPMC at room temperature,
(iii) 0.01% atropine in BSS at refrigerated temperature,
(iv) 0.01% atropine in BSS at room temperature.
e eyedroppers stored at room temperature were
kept on the shelf, protected from light in their commercial
packages at 50%±10% residual humidity (RH).
Physicochemical and microbiological stability of
the 0.01% atropine ophthalmic solutions in simulated
use conditions
At day 0, 60 eyedroppers with two formulations
of 0.01% atropine solutions were prepared, with 15
eyedroppers for each subgroup (i–iv). For illustration
purposes, subgroup (i) with a total number of 15 eyedroppers
was used as an investigation process example. Each of the
15 eyedroppers was emitted daily (1 drop of the 0.01%
atropine solutions), that is, a drop was squeezed out
of the eyedropper and collected for analysis instead of
being dropped into the eye. Out of the 15 eyedroppers,
10 eyedroppers were obtained for visual inspection and
sterility assay. Next, 5 eyedroppers were tested at day 0,
15, and discarded. Another 5 eyedroppers were tested
at day 0 and 30. It is important to note the reason why
the eyedroppers were discarded aer the sterility assay
on day 15. Namely, subgroups (i) and (ii) both had an
approximate volume of 10 mL, while the sterility assay
required at least 4 mL. Hence, aer the daily emission and
two sterility assays, there would be an insucient amount
of solution remaining for another sterility assay and so
these were discarded. e remaining 5 eyedroppers from
the 15 totals were used for the atropine quantication
and pH measurements at days 0, 15, and 30.
Aer completing the 1-month study under simulated
use conditions, further investigations were planned, with the
aim to extend the experimental period of both formulations
at refrigerated temperature to 2 months. ere were 2
subgroups of eyedroppers here: (i) 6 eyedroppers of 0.01%
atropine in HPMC at refrigerated temperature, and (ii)
6 eyedroppers of 0.01% atropine in BSS at refrigerated
temperature. ese two subgroups were investigated
in the exact same manner as in the 1-month study. Out
of the 6 eyedroppers in each subgroup, 4 eyedroppers
were obtained for visual inspection and sterility assay at
day 0, one at another time point (days 15, 30, 45, or 60),
and one discarded (n = 1 for each time point/subgroup).
e remaining 2 from the 6 eyedroppers were used for
atropine quantication and pH measurements at days 0,
15, 30, 45, and 60 (n = 2 for each time point/subgroup).
Physicochemical and microbiological stability of
0.01% atropine ophthalmic solutions in the unopened
eyedroppers
In total, 120 eyedroppers of 0.01% atropine solutions
were prepared, comprising 30 eyedroppers for each
subgroup: atropine in HPMC at refrigerated temperature,
atropine in HPMC at room temperature, atropine in BSS
at refrigerated temperature, and atropine in BSS at room
temperature. In each subgroup, 5 unopened eyedroppers
were used for the analysis at days 30, 60, 90, 120, 150, and
180 (n = 5 for each time point/subgroup). Each eyedropper
was subjected to the following analyses: visual inspection,
atropine quantication, pH measurement, and sterility
assay. e baseline values for atropine quantication, pH
measurement, and the sterility assay were obtained from
the studies of the 0.01% atropine ophthalmic solutions
under the simulated use conditions.
Analyses
Quantication of atropine
The liquid chromatography with tandem mass
spectrometry (LC-MS/MS) method was applied for
quantitative analysis of the extemporaneously prepared
atropine solution. LC-MS/MS analysis was performed
using an Acquity Ultra Performance LC
TM
(Waters,
Co., Ltd. USA) coupled to a Quattro Premier XE Mass
Spectrometer (Micromass Technologies, UK) equipped
with an electrospray interface. For data acquisition and
processing, a MassLynx 4.1 SCN627 system (Micromass
Technologies, UK) was used.
Scopolamine hydrobromide was used as an internal
standard (IS). e chromatographic separation of atropine
and the internal standard was performed using a Kinetex
C18 column (50×2.10 mm, 1.7 µm; Phenomenex Ltd.,
USA). e mobile phase was an 85:15 (v/v) mixture of
0.1% (v/v) formic acid and acetonitrile in an isocratic
elution mode over a 2 min total run time. e ow rate
was 0.3 mL/min and the column temperature were set
at 30±5 °C. e injection volume was 1 μL. MS analyses
were carried out using the multiple reaction monitoring
(MRM) mode with positive electrospray ionization (ESI+).
e mass transition ion-pair was selected as m/z 290.1
to 124.1 for atropine and m/z 304.1 to 138.1 for the IS.
Validation of this method was performed according
to the International Conference on Harmonisation (ICH)
guidelines.
12
Linearity was determined by preparing
one calibration curve daily using six concentrations
of atropine (50, 100, 150, 200, 300, and 400 ng/mL),
obtained from atropine standard solution diluted in
diluent solutions (methanol and Milli-Q water at a ratio
of 1:1, v/v). e inuence of dierent weighting factors
(1/x and 1/x
2
) on the sum of the percentage relative error
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was evaluated and the results were compared with an
unweighted calibration curve. Accuracy was tested by
spiking the atropine reference standard with the atropine
test sample (at a concentration of 200 ng/mL) to obtain
three concentration levels, namely 80%, 100% and 120%, of
the test sample concentration. e accuracy was evaluated
on the basis of the calculated recovery values, and the
results should be found within the range of 95%-105%.
e precision of the methods was determined in terms of
the intra-day precision (repeatability) and intermediate
precision (within-laboratory reproducibility). e intra-
day precision was assessed by injecting six replicates
of three dierent concentrations of atropine standard
solutions (100, 200, and 300 ng/mL) on the same day. e
intermediate precision was determined by injecting the
same solutions for three consecutive days. e intra-day
and intermediate precisions are expressed as the relative
standard deviation (RSD, %). A value of less than 5%
was acceptable for both RSDs.
For sample preparation, 0.01% atropine solution from
each eyedropper was diluted with the diluent solution to
obtain a theoretical concentration of 200 ng/mL. A 100
µL aliquot of diluted atropine was transferred into a 1.5
mL micro tube and mixed with 20 µL internal standard
solution at a concentration of 1,000 ng/mL. e micro
tubes were thoroughly mixed by vortex mixing for 10
seconds. en, 1 µL of the mixed solution was collected
and transferred into an autosampler vial and submitted
to LC-MS/MS analysis.
In the chemical stability assessment, the baseline
concentration (day 0) was dened as 100% and the
subsequent concentrations of each time point were
calculated as percentages of the initial concentration.
Acceptance criteria for the stability were dened as
90%-110% of the baseline concentration (including the
limit of a 95% condence interval of the measures).
13,14
Visual inspection and pH measurements
During the study period, the physical appearance
of the solutions was examined when the samples were
taken from each eyedropper for the sterility assay. An
approximately 4 mL sample was dispensed from each
eyedropper into a 5 mL sterilized tube. Before sending the
sample for the sterility assay, the atropine solutions were
visually inspected under white light. e transparency,
color, and presence of visible particles or haziness were
noted.
For pH measurement, a 0.5 mL aliquot of 0.01%
atropine from each sample was transferred into a 2.0
mL micro tube. Hand-held pH testing was performed
on a SevenCompact S220 pH/ion meter with an InLab
Micro Pro-ISM electrode (Mettler Toledo, Switzerland),
which was calibrated at 25 °C in pH 4.01, 7.00, and 9.21
buer solutions (Mettler Toledo, Switzerland). e pH
change was considered acceptable if it did not vary by
more than one pH unit from the initial value.
14
Sterility assay
e sterility assay was carried out by the Department
of Microbiology, Faculty of Medicine Siriraj Hospital,
Mahidol University, in line with the United States
Pharmacopeia (USP) for pharmaceutical microbiology
testing.
15
First, 4 mL of 0.01% atropine solution from
each eyedropper was aseptically taken and sent to the
Department of Microbiology in a 5 mL sterilized tube for
the sterility assay, using a direct inoculation method. Each
sample was transferred directly to a uid thioglycolate
medium and soybean casein digest medium, and then
incubated at 30-35 °C and 20-25 °C, respectively, for 14
days. e culture medium was then carefully examined
for microbial growth.
RESULTS
Quantication of atropine
e retention times were 1.02 min for atropine
and 0.70 min for scopolamine. e method was shown
to be selective, as no interferences were observed at the
retention times corresponding to 0.01% atropine in the
articial tears or in the BSS (Figs 1A-E). e calibration
curve was linear for the concentrations ranging from
50-400 ng/mL and the determination coefficient R
2
was greater than 0.999 (Fig 1F). e weighting factor of
1/x was selected, since it was the one that reproduced
the least sum of percentage relative errors (%RE). is
method showed acceptable accuracy as the percentage
recovery ranged from 99.42%-102.18% in the three
dierent concentrations of atropine standard solutions. e
precision was satisfactory, with the RSD of the intra-day
and intermediate precision ranging from 1.05%-2.99%
and 1.66%-2.94%, respectively.
Chemical stability
In the simulated use study, both formulations
demonstrated chemical stability (concentration range
between 90%-110% of the initial concentration) for up to
30 days at room temperature and 60 days at refrigerated
temperature. e concentrations of the 0.01% atropine
solutions stored at room temperature were between
97.60%-99.44% of the initial concentrations in HPMC
and 102.26%-106.93% in BSS, and the 95% condence
interval was a maximum of +4.34%. For the 0.01% atropine
solutions stored in refrigerator, the concentrations were
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Original Article
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Fig 1. Chromatograms of: (A) BSS solution, (B) HPMC solution, (C) atropine reference standard spiked at 200 ng/mL, (D) atropine test
sample in BSS at 200 ng/mL, and (E) atropine test sample in HPMC at 200 ng/mL. (F) Calibration standard of atropine.
TABLE 1. Percentage of atropine concentration remaining (mean ± 95% CI) of 0.01% atropine for each formulation
and conservation condition in the simulated use study.
Storage
Solutions
Percentageofatropineconcentrationremaining(mean±95%CI)
conditions Day0 Day15 Day30 Day45 Day60
At room
HPMC 100 97.60±4.10 99.44±4.34
ND ND
temperature
(n=5) (n=5) (n=5) (n=5)
(25±2°C)
BSS 100 106.93±1.15 102.26±2.72
(n=5) (n=5) (n=5) (n=5)
ND ND
HPMC 100 101.44±2.20 104.86±2.47 96.57±1.53 105.80±3.53
Inrefrigerator (n=7)* (n=7) (n=7) (n=7) (n=2) (n=2)
(2–8°C) BSS 100 104.50±2.29 103.73±2.31 102.75±3.70 97.40±1.87
(n=7)* (n=7) (n=7) (n=7) (n=2) (n=2)
* n = 5 in the 1-month study and n = 2 in the 2-month study.
ND = not determined.
between 96.57%-105.80% of the initial concentration
in HPMC and 97.40%-104.50% in BSS, and the 95%
condence interval was a maximum of ±3.70%. e
chemical stability results for the simulated use conditions
are presented in Table 1.
In the unopened study, the 0.01% atropine in HPMC
and BSS stored at refrigerated temperature remained stable
up until 180 days of storage. e concentrations were
between 93.61%-102.99% of the initial concentrations in
HPMC and 92.66%-105.11% in BSS, with the maximal
and the 95% condence interval at a maximum of +4.61%.
At room temperature, the 0.01% atropine solutions were
still within an acceptable range for 60 days in HPMC and
for 90 days in BSS. e chemical stability results for the
unopened study are presented in Table 2. e chemical
stability trend for all the conditions are presented in
Fig 2.
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TABLE 2. Percentage of atropine concentration remaining (mean ± 95% CI) of 0.01% atropine for each formulation
and conservation condition in the unopened eyedroppers.
Storage
Solutions
Percentageofatropineconcentrationremaining(mean±95%CI)
conditions Day0 Day30 Day60 Day90 Day120 Day150 Day180
At room
HPMC 100 96.51±1.46 94.07±1.36 83.67±2.94 78.71±0.92 73.09±3.21 67.74±3.15
temperature
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
(25±2°C)
BSS 100 98.32±2.36 93.32±2.38 92.54±2.92 80.90±1.61 78.53±1.84 74.57±1.25
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
In
HPMC 100 100.71±1.69 102.99±3.19 100.30±1.62 93.61±1.53 95.17±1.95 99.62±1.38
refrigerator
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
(2–8°C)
BSS 100 100.57±2.04 105.11±3.93 104.27±3.32 92.66±1.63 99.34±3.78 101.58±4.61
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
* Baseline values of the atropine concentration were obtained from the studies of 0.01% atropine ophthalmic solutions in 1-month simulated
use conditions.
Fig 2. (A) Percentage of atropine concentration remaining (mean ± 95% CI) for each formulation and conservation condition in the simulated
use study. (B) Percentage of atropine concentration remaining (mean ± 95% CI) for each formulation and conservation condition in the
unopened eyedroppers.
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Visual inspection and pH measurements
All the samples that were sent for the sterility assay
(from 168 eyedroppers) remained clear and colorless,
with no precipitation or visible particles observed during
the study period in all the study conditions. e pH of all
the samples showed insignicant changes throughout the
study. For both formulations, when stored at refrigerated
temperature and room temperature, the pH did not vary
by more than 0.20 and 0.23 pH units from the initial
value for the simulated use conditions and unopened
conditions, respectively (Table 3 and 4).
Sterility assay
e results indicated that the sterility was preserved
in all the samples, i.e., for every subgroup in the simulated
use and unopened conditions. No microbiological growth
was observed when incubated for 14 days at 30-35 °C in
uid thioglycolate medium and at 20–25 °C in soybean
casein digest medium.
TABLE 3. e pH value of 0.01% atropine sulfate for each formulation and conservation condition in the simulated
use study.
TABLE 4. e pH value of 0.01% atropine sulfate for each formulation and conservation condition in the unopened
eyedroppers.
Storage
Solutions
pH value (mean ± SD)
conditions Day0 Day15 Day30 Day45 Day60
At room
HPMC 6.93±0.02 6.90±0.04 6.92±0.01
ND ND
temperature
(n=5) (n=5) (n=5) (n=5)
(25±2°C)
BSS 7.02±0.04 6.82±0.08 6.88±0.04
ND ND
(n=5) (n=5) (n=5) (n=5)
In
HPMC 6.95±0.05 6.86±0.05 6.93±0.02 6.89±0.02 6.90±0.03
refrigerator
(n=7)* (n=7) (n=7) (n=7) (n=2) (n=2)
(2–8°C)
BSS 6.98±0.08 6.92±0.06 6.90±0.06 6.81±0.05 6.78±0.07
(n=7)* (n=7) (n=7) (n=7) (n=2) (n=2)
* n = 5 in the 1-month study and n = 2 in the 2-month study.
ND = not determined.
Storage
Solutions
pH value (Mean ± SD)
conditions Day0 Day30 Day60 Day90 Day120 Day150 Day180
At room
HPMC 6.93±0.02 6.91±0.01 6.92±0.01 6.91±0.01 6.83±0.00 6.85±0.01 6.90±0.02
temperature
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
(25±2°C)
BSS 7.02±0.04 6.86±0.05 6.89±0.02 6.81±0.07 6.79±0.10 6.81±0.06 6.83±0.05
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
In
HPMC 6.97±0.03 6.90±0.01 6.88±0.03 6.92±0.01 6.87±0.01 6.91±0.03 6.92±0.03
refrigerator
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
(2–8°C)
BSS 7.02±0.04 6.88±0.04 6.85±0.06 6.85±0.04 6.85±0.02 6.86±0.05 6.88±0.05
(n=30) (n=5)* (n=5) (n=5) (n=5) (n=5) (n=5) (n=5)
* Baseline values of the pH measurement were obtained from the studies of 0.01% atropine ophthalmic solutions in the 1-month simulated
use conditions.
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DISCUSSION
To assess the accuracy of the extemporaneous
prepared ophthalmic solutions in clinical practice, this
study investigated the accuracy of using 0.5 mL and
1 mL insulin syringes compared to an auto pipette for
the extemporaneous preparation. e 0.01% atropine
solutions were prepared using an auto pipette, and 0.5
mL and 1 mL insulin syringes. Here, 0.1 mL of 1%
atropine sulfate was mixed with 9.9 mL of HPMC and
0.15 mL of 1% atropine sulfate was mixed with 14.85 mL
of BSS (n =5 for each apparatus in each formulation).
e preparation using the 1 mL insulin syringe was the
same as for the preparation using the 0.5 mL insulin
syringe. e mean concentration of atropine in HPMC
compared to the expected concentration ranged from
98.24%-104.37%, 100.25%-102.91%, and 154.29%-
157.05% for the auto pipette, and the 0.5 mL and 1 mL
insulin syringes, respectively. e mean concentration of
atropine in BSS ranged from 98.00%-103.03%, 100.01%-
104.79%, and 140.63%-145.89% for the auto pipette, and
the 0.5 mL and 1 mL insulin syringes, respectively. In
this study, a 0.5 mL insulin syringe was then used in the
preparation process, since it was more accurate than the
1 mL insulin syringe.
In the stability assessment of the ophthalmic solutions,
the physicochemical and microbiological stability should
be evaluated. Previous recent studies have also focused on
the long-term stability of ophthalmic atropine solutions.
Saito et al.
16
demonstrated that the physical, chemical,
and microbiological stability of 0.01%, 0.10%, 0.25%, and
0.5% atropine in 0.9% sodium chloride solution were
maintained for at least 6 months when stored unopened
in polyethylene bottles at 25 °C or 5 °C. Berton et al.
17
showed that 0.01% atropine in 0.9% sodium chloride
solutions with and without antimicrobial preservative
were physicochemically stable for 6 months when stored
unopened in low-density polyethylene bottles at 25 °C.
e aim of our study was to investigate the long-term
stability of 0.01% atropine in HPMC and BSS when
stored unopened at room temperature (25 °C) or at
refrigerated temperature (5 °C), and the stability of the
0.01% atropine solutions in a simulated use condition
for up to 2 months.
In the simulated use study, 0.01% atropine in
HPMC and BSS demonstrated physicochemical and
microbiological stability for up to 30 days at room and
refrigerated temperature. For the 2-month extension
study at refrigerated temperature, 0.01% atropine in
HPMC and BSS also maintained its physicochemical and
microbiological stability throughout the study period.
In the unopened conditions, 0.01% atropine in HPMC
and BSS stored at refrigerated and room temperature
showed both physical and microbiological stability over 6
months. e pH values remained nearly constant, and no
visual changes or microbial contamination were observed
over the study period. Regarding the chemical stability,
the mean atropine concentrations in HPMC and BSS
remained well within 90%-110% of the initial concentration
for 6 months at refrigerated temperature. However at
room temperature, the mean atropine concentrations in
HPMC and BSS were considered to be at an acceptable
level of stability for only 2 and 3 months, respectively.
ese results supported the eect of temperature on the
chemical stability. e dierences in chemical stability
at room temperature between our study and previous
studies
16-18
are particularly related to the formulation.
In previous studies, atropine was mostly prepared in
0.9% sodium chloride solution with a pH value of 5.3-
6.2, compared to the pH value ranging from 6.8-7.0 for
atropine in HPMC and BSS. e stability of atropine
sulfate solution is enhanced in acidic conditions, as it has
a lower degree of hydrolysis. Atropine sulfate solution is
most stable at a pH between 3–6, and the ideal storage pH
ranges between 3-4.
19-20
However, ophthalmic solution
should better fall within the ocular comfort range (pH
6.6–7.8) to avoid eye discomfort and irritation.
21
From the results from the unopened study, the
conservation period of 0.01% atropine in HPMC and
BSS could be ensured for 6 months when stored at 5 °C
and for 2 months when stored at 25 °C.
Hence, the follow-up intervals for patients receiving
these formulations could be extended to up to 6 months
when a refrigerator is available.
ere are some limitations of this study to note. First,
the number of samples in the 2-month extension simulated
use study was limited. Second, the room temperature in
this study was 25±2 °C, which is actually lower than the
average indoor temperature in most parts of ailand.
Since the storage temperature significantly aects the
chemical stability, the conservation period for 0.01%
atropine in HPMC and BSS outside the refrigerator
might be, consequently, shorter than in our study.
CONCLUSION
is study demonstrated that 0.01% atropine solution
both in HPMC and BSS retained good physicochemical
and microbiological stability for 6 months both when
le unopened and when stored at 5±3 °C; whereas, the
atropine concentration in unopened eyedroppers stored
at 25±2 °C generally declined over time. is study also
confirmed the physicochemical and microbiological
stability of both formulations at 5±3 °C or 25±2 °C for 30
Sri-in et al.
Volume 74, No.2: 2022 Siriraj Medical Journal
https://he02.tci-thaijo.org/index.php/sirirajmedj/index
99
Original Article
SMJ
days aer opening. In conclusion, the extemporaneously
prepared 0.01% atropine ophthalmic solution both in
HPMC and BSS could be kept for up to 6 months in the
refrigerator at a temperature of 2-8 °C until the bottle is
opened.
ACKNOWLEDGEMENTS
The authors wish to thank the Department of
Microbiology Faculty of Medicine Siriraj Hospital,
Mahidol University for their analysis of the sterility tests.
Potential conicts of interest
e authors have no conicts of interest with the
manufacturers or suppliers of any of the products or
materials in this study. It is to be noted though that
the authors were supported by a Chalermprakiat grant
from the Faculty of Medicine Siriraj Hospital, Mahidol
University.
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