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Investigation of tropicamide and benzalkonium chloride stability

using liquid chromatography

Article  in  Journal of Liquid Chromatography & Related Technologies · January 2012

DOI: 10.1080/10826076.2011.597072

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 Journal of Liquid Chromatography & Related Technologies, 35:231–239, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2011.597072

INVESTIGATION OF TROPICAMIDE AND BENZALKONIUM

CHLORIDE STABILITY USING LIQUID CHROMATOGRAPHY
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M. Jovović,1 N. Kostić,2 B. Jančić-Stojanović,2 and A. Malenović2

1
Clinical Center of Serbia, Pharmacy and Medical Supplies, Belgrade, Serbia
2
Department of Drug Analysis, Faculty of Pharmacy, University of Belgrade,
Belgrade, Serbia

& In this study, a development of stability indicating method (SIM) that could be used to detect a
decrease in the amount of tropicamide and benzalkonium chloride due to their degradation is
described. In the method development, stress samples should be used. Benzalkonium chloride proved
to be very stable under all stress conditions. Acid stress with 0.1 M HCl at 70
C led to 10% degra-
dation of tropicamide after 30 min. The alkaline stress with 1 M NaOH at 70
C induced faster
degradation and more than 20% of tropicamide degraded after 30 min. On the other hand, tropi-
camide proved to be exceptionally stable to oxidative stress and no degradation was observed with
30% H2O2 even after heating at 70
C for 90 min. The mobile phase consisted of acetonitrile and
water phase 70:30 v=v (water phase: 5 mM sodium heptan sulphonate and 1% triethylamine –
TEA) with pH of the mobile phase adjusted to 3.5 with ortophosphoric acid. The flow rate was
2 mL min1. In order to confirm the method adequacy for intended purpose, the proposed method
was validated. Finally, the proper validation of developed SIM performed according to the official
guidelines proved that the method accomplishes its intended purpose.

Keywords benzalkonium chloride, eye drops, forced degradation studies, liquid

chromatography, tropicamide, validation

INTRODUCTION
Forced degradation or stress testing is undertaken to demonstrate
selectivity when developing stability-indicating methods (SIM). These
studies also provide information about the degradation pathways and
degradation products that could form during storage. They may facilitate
pharmaceutical development where knowledge of chemical behavior can
be used to improve a drug product. Information such as stereochemical
stability, identification thresholds of degradation products, polymorphism

Address correspondence to A. Malenović, Department of Drug Analysis, University of Belgrade,

Vojvode Stepe 450, Belgrade, Serbia. E-mail: andja@pharmacy.bg.ac.rs
 232 M. Jovovic´ et al.

and crystal forms, stability of (parenteral) combination products, and mass

balance are very useful and can be obtained with stress tests.[1]
The International Conference on Harmonization (ICH)[2] guidance
provides very little information about strategies and principles for conduct-
ing forced degradation studies, including problems of poorly soluble drugs
and exceptionally stable compounds. In particular, the issue of adequate
stress level in stress testing is not addressed specifically. Overstressing a mol-
ecule can lead to degradation profiles that are not representative of the real
storage conditions and perhaps not relevant to the method development.
Therefore, stress-testing conditions should be realistic and not excessive.
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In this regard, it is the amount of stress that is important and not necessar-
ily the extent of degradation. Indeed, some compounds may not degrade
significantly after considerable exposure to stress conditions.
In general, the number of investigation focused on the stability of
extemporaneous preparations is limited. The eye drops intended only for
hospital use were the subject of this study. Eye drops contained 1% w=v
of tropicamide and 0.01% w=v of benzalkonium chloride as preservative,
so in this paper forced degradation studies of tropicamide using high
performance liquid chromatography (HPLC) is described. Tropicamide
((R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(4-pyridylmethyl)propanamide) is a
medical substance with parasympatholytic activity similar to atropine. How-
ever, it acts more rapidly (after ca. 15 min) and for a shorter time (1.5–3 h
max.). It causes dilatation of the pupils, cycloplegia, and increases intraocu-
lar pressure. For this reason, 0.5% and 1% aqueous solutions of tropica-
mide are used in ophthalmology locally during medical examination of
eyes and in short operations.[3]
In the literature, for the determination of tropicamide, different meth-
ods were described: high performance capillary electrophoresis with square
wave amperometric detection,[4] an isocratic, reversed-phase liquid chro-
matography using atropine as an internal standard,[5] and reaction with
ammonium meta-vanadate reagent in acidic medium and then re-titration
of unconsumed reagent.[6] For determination of tropicamide and phenyl-
ephrine in human aqueous humor, HPLC method was applied.[7] Degra-
dation products of tropicamide were separated and determined applying
TLC, HPLC-DAD, and HPLC-FT-IR.[3] On the other hand, benzalkonium
chloride, mostly used as preservative in pharmaceutical preparations, was
determined by RP-HPLC method using a butyl (C4) column.[8] A simple
and rapid RP-LC method using C8 column was developed for determi-
nation of benzalkonium chloride and separation from its potential
impurities.[9] There are also several described methods for the benzalko-
nium chloride determination in ophthalmic formulations.[10–13]
As there is no SIM method for tropicamide and benzalkonium chloride
described, the objective of this study was to establish inherent stability
 Tropicamide and Benzalkonium Chloride 233

through stress studies under a variety of ICH recommended test conditions

and to develop a validated stability-indicating assay.

EXPERIMENTAL
Chemicals and Reagents
Working substances of tropicamide and benzalkonium chloride were
donated by Clinical Center of Serbia, Pharmacy and Medical Supplies.
All reagents used were of an analytical grade. Acetonitrile – HPLC
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gradient grade was obtained from Sigma (St. Louis, MO, USA). Water –
HPLC grade, sodium 1-heptane-sulfonate (Sigma, St. Louis, MO, USA),
triethylamine – TEA (Acros Organics, New Jersey, USA) and orto-phos-
phoric acid (J.T. Baker, Holland) were used to prepare a water phase.
For stress-samples preparation 0.1 M HCl, 1 M NaOH, 30% H2O2 were used.
The analyzed eye drops were produced by Laboratory of Clinical Center
of Serbia, Belgrade, only for hospital use. Eye drops contain 1% w=v of
tropicamide and 0.01% w=v of benzalkonium chloride as preservative.

Chromatographic Conditions
The chromatographic system Waters Breeze consisted of Waters 1525
Binary HPLC Pump, Waters 2487 UV=VIS detector, and Breeze Software,
Windows XP, for data collection. Separations were performed on the
SupelcoSil LC-C8 4.6 mm  250 mm, 5 mm particle size column with UV
detection at 210 nm. The flow rate was 2 mL min1. The samples were intro-
duced through a Rheodyne injector valve with a 20 mL sample loop. Mobile
phase was prepared by mixing acetonitrile and water phase, 70%:30% v=v.
Water phase consisted of 5 mM sodium 1-heptane-sulfonate and 1% v=v of
TEA. The pH was adjusted at 3.5 with orto-phosphoric acid. After treating
in an ultrasonic bath for 30 min, mobile phase was filtered through a
0.45 mm membrane filter Alltech (Loceren, Belgium).

Forced Degradation Studies

All stress decomposition studies were performed at an initial drug
concentration of 1 mg mL1 prepared in the mobile phase. Solutions for
use in forced degradation studies were prepared by diluting the stock sol-
ution with NaOH or HCl or H2O2 to achieve the final concentration of
100 mg mL1 of tropicamide. Acid decomposition studies were carried out
using 0.1 M HCl; alkaline degradation studies were performed in 1 M
NaOH and oxidative in 30% H2O2. The samples were analyzed after
 234 M. Jovovic´ et al.

stressing at room temperature and also after combining acid, alkaline, or

oxidative stress with heating at 70
C. Samples were injected into HPLC sys-
tem after 0, 15, 30, 45, 60, 75, and 90 min.

Standard Solutions for Linearity Testing

Solutions of tropicamide working standard were prepared in mobile
phase to provide concentration from 0.3 to 1.3 mg mL1, while the solu-
tions of benzalkonium chloride were prepared in concentration range
from 20 to 60 mg mL1.
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Standard Solutions for Accuracy Testing

For accuracy testing, laboratory mixture of tropicamide and benzalko-
nium chloride was prepared in placebo solution. Furthermore, three series
of dilutions calculated as 80%, 100%, and 120% were prepared in the
mobile phase, with three solutions for each concentration level:

1. Mixture 1 (80%) contained 0.4 mg mL1 of tropicamide and 24 mg mL1

of benzalkonium chloride;
2. Mixture 2 (100%) contained 0.5 mg mL1 of tropicamide and 30 mg mL1
of benzalkonium chloride; and
3. Mixture 3 (120%) contained 0.6 mg mL1 of tropicamide and 36 mg mL1
of benzalkonium chloride.

Eye Drops Sample Solution for Precision Testing

Precision was tested as repeatability, and the solutions for this testing
were prepared from analyzed eye drops separately for tropicamide and ben-
zalkonium chloride. For tropicamide three series of dilutions were pre-
pared in the mobile phase to obtain 0.4 mg mL1 (80%), 0.5 mg mL1
(100%), and 0.6 mg mL1 (120%). Three different solutions were prepared
for each concentration level. As the concentration of benzalkonium chlor-
ide in eye drops is 100 times lower than the concentration of tropicamide,
solutions for precision-repeatability testing for benzalkonium chloride
were prepared separately to obtain concentrations of 24 mg mL1 (80%),
30 mg mL1 (100%), and 36 mg mL1 (120%). For each concentration level
three different solutions were prepared.

RESULTS AND DISCUSSION

Forced degradation or a stress testing study is part of the development
strategy and also an integral component of validating analytical methods
 Tropicamide and Benzalkonium Chloride 235

that indicate stability and detect impurities.[2] The forced degradation

studies are expected to elucidate possible degradation pathways and to
identify the degradation products that may be spontaneously generated
during drug storage and use, as well as to facilitate improvements in the
manufacturing process and formulations parallel with the accelerated
stability studies.
The choice of stress conditions should be consistent with the product’s
decomposition under normal manufacturing, storage, and usage condi-
tions, which are specific in each case.[14] Also, choice of forced degradation
conditions should be based on sound scientific understanding of the pro-
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duct’s decomposition mechanism under typical use conditions and usually

to get a decomposition level of 10 to 15% which is considered adequate for
validation of a chromatographic purity assay.
Although it is expected that the analytical method used in such analysis
have to be able to detect, separate, and quantify all the observed degradati-
on products, if it is not necessary to fully identify all products of forced
degradation studies, we have enough information on retention times to
enable their monitoring in stability studies. If they start to appear at levels
greater than thresholds of 0.1%, they will require identification. For this
reason, degradation of tropicamide is observed as a percent of decrease
in the peak area of tropicamide.
Based on its chemical structure (Figure 1) and present amide func-
tional group, degradation of tropicamide was expectable under different
stress conditions. As with 0.5 M HCl at room temperature, total degradation
of tropicamide was observed; the next step was to choose a weaker acid for

FIGURE 1 Chemical structures of tropicamide and benzalkonium chloride.

 236 M. Jovovic´ et al.

stability examination. This proved to be not so simple, as the effect of

lowering the acid concentration had an abrupt change on tropicamide
stability. Namely, all the concentrations from 0.5 M to 0.1 M degraded the
tropicamide totally at room temperature. However, at room temperature,
tropicamide proved to be stable in 0.1 M HCl and the next logical step
was to apply heating simultaneously. It was shown that heating at 70
C
for 30 min led to 10% degradation of tropicamide.
In alkaline medium, total degradation was observed with 2 M NaOH at
room temperature. However, in 1 M NaOH at room temperature it proved
to be stable, so simultaneous heating was applied. After alkaline stress with
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1 M NaOH and heating at 70
C for 30 min, more than 20% of tropicamide
degraded. As tropicamide proved to be stable in 3% and 10% H2O2, for oxi-
dative stress studies, 30% H2O2 was chosen. Surprisingly, no degradation
products were observed after oxidative stress with 30% H2O2 even after
heating at 70
C for 90 min.
Chemically, bezalkonium chloride is a mixture of alkylbelzyldimethy-
lammonium chlorides that usually contains C-10, C-12, C-14, and C-16
homologues. According to this chemical structure (Figure 2) and literature
data,[15,16] it could be concluded that benzalkonium chloride will be stable
under various stress agents’ effects. Despite the observed degradation of
tropicamide, no interfering peaks at the retention time for the benzalko-
nium chloride were observed in any of the stressed samples.

FIGURE 2 The chromatograms of the analyzed eye drops containing: A. 0.5 mg mL1 of tropicamide
and B. 30 mg mL1 of benzalkonium chloride.
 Tropicamide and Benzalkonium Chloride 237

TABLE 1 The Important Parameters for the Calibration Curves

Compound Concentration range y ¼ ax þ b r Sb

Tropicamide (mg mL1) 0.3–1.3 22927.1x þ 13135.9 0.9988 549.3

Benzalkonium chloride (mg mL1) 20–60 13.01x  164.64 0.9989 15.36

r – correlation coefficient; Sb – standard deviation of the intercept.

TABLE 2 Accuracy of the Assay

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Compound Injected Found R (%)

Tropicamide (mg mL1) 0.4 0.394  0.002 98.5

0.5 0.493  0.003 98.6
0.6 0.59  0.01 98.3
Benzalkonium chloride (mg mL1) 24 24.64  0.08 98.5
30 30.3  0.04 101.0
36 36.2  0.1 100.5

S (n ¼ 3).

Stability indicated method was then validated in order to confirm the

adequacy for intended purpose. The important data from method vali-
dation, necessary for the current analysis, are given in Table 1.
Response for the drug was linear in the concentration range between
0.3–1.3 mg ml1 for tropicamide (coefficient of correlation 0.9988) and
20–60 mg mL1 for benzalkonium chloride (coefficient of correlation
0.9989). The average recovery for tropicamide ranged from 98.3 to
98.6% and from 98.5 to 101.0% for benzalkonium chloride. The method
proved to be precise and accurate. Results for the accuracy are given in
Table 2 and for the precision in Table 3.
The chromatograms of the analyzed eye drops containing 0.5 mg mL1
of tropicamide and 30 mg mL1 of benzalkonium chloride are given in
Figure 2A and 2B, respectively.

TABLE 3 Precision of the Assay

Compound Injected Found RSD (%)

Tropicamide (mg mL1) 0.4 0.364  0.004 1.10

0.5 0.470  0.005 1.00
0.6 0.56  0.01 1.80
Benzalkonium chloride (mg mL1) 24 22.6  0.2 0.84
30 28.0  0.3 1.03
36 33.8  0.1 0.38

S (n ¼ 3).
 238 M. Jovovic´ et al.

CONCLUSION
Developed stability indicated method is used to detect the decrease in
amount of tropicamide due to its degradation under different stress con-
ditions. As tropicamide possesses an amide functional group, hydrolytic
degradation in acid and alkaline medium is expected. Stress was first
initiated at room temperature with 0.1 M HCl and 1 M NaOH, but since
no degradation occurred, an elevated temperature was applied simul-
taneously. After heating at 70
C, acid induced degradation led to a decrease
in the amount of tropicamide to approximately 10%; whereas, in alkaline
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medium, degradation of tropicamide was around 20%. On the other hand,

tropicamide proved to be exceptionally stable to oxidative stress even after
heating for 90 min in 30% H2O2. As the observed changes can significantly
influence drug efficacy, its contact with acids and bases must be prevented.
It means that the special attention has to be paid to the type and quality of
bottles for packing of eye drops prepared as extemporaneous preparations.

ACKNOWLEDGMENTS
The authors thank to the Ministry of Science of the Republic of Serbia
for supporting these investigations through the Project 172052.

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