ACTA CHROMATOGRAPHICA, NO.

19, 2007

HIGH-PERFORMANCE
THIN-LAYER CHROMATOGRAPHIC METHOD
FOR DETERMINATION OF ERDOSTEINE
IN PHARMACEUTICAL DOSAGE FORMS

D. V. Mhaske and S. R. Dhaneshwar*

Department of Quality Assurance Techniques and Pharmaceutical Chemistry, Bharati
Vidyapeeth University, Poona College of Pharmacy, Centre for Advanced Pharmaceutical
Research, Erandwane, Pune 411038, Maharashtra State, India

SUMMARY
A sensitive, selective, precise, and stability-indicating method for
quantitative analysis of erdosteine, in the presence of its degradation pro-
ducts, both as the bulk drug and in a formulation has been established and
validated. High-performance thin layer chromatography (HPTLC) on alu-
minium-backed silica gel 60 F254 plates with toluene–methanol–acetone–
ammonia 3.5:3.5:2.5:0.05 (v/v) as mobile phase was followed by densito-
metric measurement at 254 nm. This system was found to give compact
bands for erdosteine (RF 0.45 ± 0.02). The method was validated in accor-
dance with ICH guidelines There was no chromatographic interference
from capsule excipients. Erdosteine was subjected to acid and alkaline hy-
drolysis, oxidation, dry heat, wet heat, and UV degradation. The drug is
degraded by acid and alkaline hydrolysis, oxidation, and UV irradiation.
The drug was found to be stable under wet and dry heat conditions. Because
the method could effectively separate the drug from its degradation products
it can be regarded as stability-indicating.

INTRODUCTION
Erdosteine (erdotin; (+)-1S-(2-[N-3-(2-oxotetrahydrothienyl)aceta-
mido)thioglycolic acid; Fig. 1 [1]) is a thiol derivative developed for treat-
ment of chronic obstructive bronchitis, including acute infective exacerba-
tion of chronic bronchitis.
Erdosteine contains two blocked sulfhydryl groups which are relea-
sed after first-pass metabolism. The three active metabolites have mucolytic
and free radical-scavenging activity. Erdosteine modulates mucus produc-

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 O
H
N
S S COOH
O
Fig. 1
The chemical structure of erdosteine

tion and viscosity and increases mucociliary transport, thereby improving

expectoration [2]. It is registered as expectorating and for the treatment of
acute exacerbations of chronic bronchitis in adults, and for the treatment
of lower respiratory infections in children above the age of 2 years in 7 to
10 days [3]. It also suppresses the chemical stimulation-induced cough re-
flex and plasma leakage into the airways. Erdosteine is used for treatment
of acute and chronic bronchitis on the basis of its unique mechanism of
action – muco-modulatory activity, antibacterial activity, anti-inflammatory
activity, and antioxidant activity [4].
Erdosteine and its optical active metabolite has been analysed by
high-performance liquid chromatography using a fluorescent chiral tagging
reagent [5]. HPLC with on-line mass spectrometric detection has been used
in a preliminary study to elucidate the metabolism of erdosteine [6]. Sen-
sitive determination of erdosteine in human plasma has been achieved by
automated 96-well solid-phase extraction and LC–MS–MS [7]. As far as
we are aware no HPTLC methods for stability-indicating chromatographic
determination of erdosteine in pharmaceutical dosage forms have been re-
ported in literature.
The International Conference on Harmonization (ICH) guideline
entitled “Stability Testing of New Drug Substances and Products” requi-
res stress testing to be conducted to elucidate the inherent stability charac-
teristics of the active substance [7]. Susceptibility to oxidation is one of the
tests required, as are hydrolytic and photolytic stability. An ideal stability-
indicating method is one that quantifies the standard drug alone and also
resolves its degradation products [8].
The objective of this work was to develop stability-indicating HPTLC
method for determination of erdosteine in presence of its degradation pro-
ducts for assessment of the purity of the bulk drug and the stability of its
dosage forms. The proposed method is simple, accurate, specific, repeatable,
and stability-indicating, reduces the duration of analysis, and is suitable
for routine determination of erdosteine. The method was validated in accor-
dance with ICH guidelines [9,10] and their recent updates [11,12].

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EXPERIMENTAL
Chemicals and Reagents
Pharmaceutical grade erdosteine was kindly supplied as a gift by
Glenmark Pharmaceuticals, Nasik, India; it was certified to contain 99.85
% (w/w) on dry basis and was used without further purification. All other
chemicals and reagents used were analytical grade and were purchased
from Merck Chemicals, India.
A stock solution (1000 µg mL−1) of erdosteine was prepared by dis-
solving 100 mg in 100 mL methanol. Standard solutions for calibration we-
re prepared by dilution of the stock solution with methanol; the concentra-
tions were such that amounts of erdosteine between 30 and 1000 ng were
applied to the plates.
Chromatography
HPTLC was performed on 20 cm × 10 cm aluminium plates coated
with 250-µm layers of silica gel 60 F254 (E. Merck, Darmstadt, Germany;
supplied by Anchrom Technologists, Mumbai). Before chromatography the
plates were pre-washed by development with methanol and activated at
60°C for 5 min. Samples were applied to the plates as bands 6 mm wide
and 6 mm apart by use of a Camag (Muttenz, Switzerland) Linomat IV appli-
cator fitted with a 100 microlitre syringe (Hamilton, Switzerland). The rate
of sample application was constant at 0.1 µL s−1.
Linear ascending development with toluene–methanol–acetone–am-
monia 3.5:3.5:2.5:0.05 (v/v) as mobile phase was performed, in the dark,
in a Camag twin-trough glass chamber previously saturated with mobile
phase vapour for 10 min at room temperature (25°C) and relative humidity
of 60 ± 5% by lining the two largest sides with filter paper that had been
soaked thoroughly in the mobile phase. The development distance was 9 cm
and the development time approximately 30 min. The volume of mobile
phase used for chromatography was 10 mL.
After development the plates were dried in a current of air by means
of an air dryer in wooden chamber with adequate ventilation. The flow of
air in the laboratory was maintained unidirectional (laminar flow, towards
exhaust). Densitometric scanning at 254 nm in reflectance–absorbance mode
was then performed with a Camag TLC Scanner III operated by CATS soft-
ware (V 3.15; Camag). The slit dimensions were 5 mm × 0.45 mm and the
scanning speed was 10 mm s−1. The monochromator bandwidth was 20
nm. Each track was scanned three times and baseline correction was used.

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The source of radiation was a deuterium lamp emitting a continuous UV
spectrum between 190 and 400 nm.
Calibration of the Method
Each calibration solution was applied to the plate six times and the
plate was developed as described above. Peak areas were plotted against
the corresponding concentrations to obtain the calibration graph.
Method Validation
Precision
The precision of the method was determined with the product, sam-
ples of which were accurately weighed and assayed. System repeatability
was determined by performing six replicate analyses of each of three dif-
ferent amounts (300, 500, and 900 ng band−1), measurement of peak area
for the active compound, and calculation of relative standard deviation
(RSD, %) and standard error (SE). Method repeatability was measured as
the RSD obtained by repeating each assay six times on the same day
(intra-day precision). Intermediate precision was assessed by sixfold assay
of the three samples on each of two different days (inter-day precision).
Robustness
To measure robustness the experimental conditions were delibera-
tely changed by small amounts and the effects on the results were exami-
ned. Chromatograms were obtained with mobile phases of composition to-
luene–methanol–acetone–ammonia 3.5:2.5:3.5:0.05, 3.5:3.0:2.5:0.05, and
3.5:3.5:2.5:0.05 (v/v). The amount of mobile phase, the temperature, and the
relative humidity were also varied by ±5%. After prewashing of the plates
with methanol they were activated at 60 ± 5°C for 2, 5, or 7 min before
chromatography. The time from application to chromatography and from
chromatography to scanning were also varied (0, 20, 40 and 60 min). The
robustness of the method was measured for three different amounts of er-
dosteine – 300, 500, and 900 ng band−1.
Limits of Detection and Quantitation
To estimate the limit of detection (LOD) and the lower limit of quan-
titation (LLOQ), blank methanol was applied six times and the band was
chromatographed by following the same method. The signal to noise ratio
(S/N) was determined. LOD was considered as 3 × S/N and LLOQ as 10 × S/N.

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Specificity
The specificity of the method was ascertained by analysing drug
standard and sample. The identity of the band for erdosteine in the sample
was confirmed by comparison of the RF and spectrum of the sample band
with those from the standard. The peak purity of erdosteine was assessed
by comparing spectra acquired at the peak start (S), peak apex (A), and
peak end (E) positions of the band by use of the densitometer.
Recovery
Recovery was determined by applying the method to drug sample
to which known amounts of erdosteine corresponding to 50, 100, and 150%
of the label claim had been added (standard-addition method). Six analy-
ses were performed for each amount added and the results obtained were
compared with those expected.
Analysis of the Marketed Formulation
To determine the erdosteine content in conventional capsules (la-
bel claim 300 mg per capsule) the capsule contents were weighed. Powder
equivalent to 300 mg erdosteine was extracted with methanol, the sample
being sonicated for 30 min to ensure complete extraction of the drug. The
volume of the extract was diluted to 100 mL and 1 µL of the resulting solu-
tion was diluted to 10 mL with methanol. The resulting solution was cen-
trifuged at 3000 rpm for 5 min and the supernatant was analysed for drug
content by applying 3 µL of the solution to a plate (600 ng band−1) and
analysis as described above. The analysis was repeated in triplicate. The
possibility of excipient interference in the analysis was studied.
Accelerated Degradation of Erdosteine [13,14]
A stock solution containing 100 mg erdosteine in 100 mL methanol
was prepared. This solution was used for forced degradation to provide an
indication of the stability-indicating nature and specificity of the method.
In all degradation studies the average peak area of erdosteine after appli-
cation (500 ng band−1) for HPTLC of seven replicates was obtained. To
study the degradation of erdosteine by use of the HPTLC method most of
the study was conducted by single development of the plate to prevent the
movement of non-polar degradation products to the extreme end of the plate.
Acid and Base-Induced Degradation
HCl (0.1 M, 10 mL) or NaOH (0.1 M, 10 mL) were added separate-

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ly to 10 mL methanolic stock solution and the mixtures were heated under
reflux for 4.0 h at 50°C in the dark (to exclude the possible degradative
effect of light). The resulting solutions (10 µL; 500 ng band−1) were then
analysed as described above.
Hydrogen Peroxide-Induced Oxidation
Hydrogen peroxide (10.0% or 30% (w/v), 10 mL) was added to 10
mL methanolic stock solution and the mixtures were heated in a boiling
water bath for 10 min to completely remove excess hydrogen peroxide.
Then the same mixtures were heated under reflux for 4.0 h at 50°C. The
resulting solutions (10 µL; 500 ng band−1) were then analysed as descri-
bed above.
Dry and Wet Heat Treatment
The drug was stored in oven at 100°C for 5.0 h to study dry thermal
degradation and the stock solution was heated under reflux for 5.0 h on a
boiling water bath to study wet thermal degradation.
UV Degradation
The UV stability of the drug was studied by exposing the stock so-
lution to direct outdoor sunlight for 15 days. After suitable dilution the re-
sulting solution (10 µL; 500 ng band−1) was analysed as described above.
Neutral Hydrolysis
Double-distilled water (10 mL) was added to 10 mL methanolic stock
solution and the mixture was heated under reflux for 5.0 h at 50°C to study
the degradation occurring under neutral conditions.

RESULTS AND DISCUSSION

Optimization of the Procedure
The TLC procedure was optimized with the objective of developing
a stability-indicating method. Both pure drug and the degraded products
were applied to the TLC plates and chromatographed with different mobile
phases. Initially toluene–methanol–carbon tetrachloride–acetone 2.0:3.0:3.0:2
(v/v), 3.0:3.0:3.5 (v/v), and 3.5:4.5:2.5: (v/v) were tried. Addition of 0.05
mL ammonia to these mobile phases improved the characteristics of the
bands. Finally, the mobile phase toluene–methanol–acetone–ammonia

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3.5:3.5:2.5:0.05 (v/v) was found to enable good resolution with a sharp
and symmetrical peak of RF 0.45. Well defined bands were obtained when
the chamber was saturated with mobile phase for 30 min at room tempe-
rature (Fig. 2).

Fig. 2
Densitogram obtained from erdosteine standard (peak 1, RF 0.45 ± 0.02, 500 ng band−1)

Linearity
The calibration graph was linear, i.e. the system adhered to Beer’s
law, over the range 30–1000 ng band−1 (r2 ± SD = 0.998 ± 0.002). Linearity
was evaluated by duplicate analysis of six standard working solutions equi-
valent to 30–1000 ng band−1 erdosteine. The relationship between peak area
and concentration was subjected to least-squares linear-regression analysis
to calculate the calibration equation and correlation coefficients. The reg-
ression data showed linearity was good over the concentration range inve-
stigated; this was apparent from the high value of the correlation coefficient

Table I
Linear regression data for the calibration plotsa

Regression data Value

Linear range 300–1000 ng band−1
r2 ± SD 0.998 ± 0.002
Slope ± SD 0.073 ± 0.004
Intercept ± SD 36.78 ± 1.50
Confidence limitb, slope 0.059–0.066
Confidence limitb, intercept 38.74–40.48
a
n=6
b
95% confidence limit

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and the SD for the intercept of <2%. There was no significant difference be-
tween the slopes plots from 30–100 ng band−1 or from 300–1000 ng band−1
(ANOVA; P < 0.05). Typical linearity data are given in Table I.

Validation
Precision
Repeatability of sample application and measurement of peak area,
as RSD (%), was 1.12 and 1.21, respectively. RSD for inter-day and intra-
day analysis was <2%. The values are shown in Table II.

Table II
Results of measurement of intra and inter day precisiona

Intra-day precision Inter-day precision

SD of area RSD (%) SE SD of area RSD (%) SE
1.42 1.12 0.51 1.86 1.21 0.76
a
n=6
Results are averages from analysis of three concentrations – 300, 500, and 900 ng band−1

Robustness
The standard deviation of peak area was calculated for each change
of conditions parameter and RSD was found to be <2% (Table III).

Table III
Results from robustness testinga

Condition changed SD of peak area RSD (%)

Mobile phase composition 1.74 1.50
Amount of mobile phase 1.48 1.28
Temperature 1.23 1.12
Relative humidity 1.18 0.98
Plate pretreatment 1.02 0.86
Time from application to chromatography 0.82 0.59
Time from chromatography to scanning 0.78 0.37
a
(n = 6)
Results are averages from analysis of three concentrations – 300, 500, and 900 ng band−1

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LOD and LOQ
The LOD and LLOQ, for signal-to-noise ratios were 3:1 and 10:1,
respectively, were 5 and 10 ng band−1.
Specificity
When the peak purity of erdosteine standard was assessed by com-
paring spectra acquired at the peak-start, peak-apex, and peak-end positions
of a band, r(start, middle) = 0.996 and r(middle, end) = 0.9994. Good correlation
(r = 0.9998) was also obtained between spectra of erdosteine obtained from
standards and samples.
Recovery
When the method was used for extraction and subsequent analysis
of erdosteine in pharmaceutical dosage form spiked with 50, 100, and 150
% extra drug, recovery was 98–102% of erdosteine as bulk and in dosage
form results are listed in Table IV.

Table IV
Results from recovery studiesa

Amount of standard Amount

Recovery (%) RSD (%) SE
added to matrixb (mg) recovered (mg)
0 198.8 99.44 0.82 0.25
100 300.15 100.05 0.47 0.12
200 403.24 100.81 0.58 0.29
300 505.2 101.41 1.71 0.85
a
n=6
b
The matrix contained 200 mg drug

Stability in Sample Solution

Solutions of erdosteine of two different concentrations, equivalent
to 400 and 800 ng band−1, were prepared and stored at room temperature
for 3 days. The solutions were then chromatographed on the same TLC pla-
te and the densitograms obtained were evaluated for additional bands, if
any. There was no instability in the sample solution. The results obtained are
listed in Table V.

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Table V
Stability of erdosteine in sample solutions (n = 6)

Amount (ng band−1) Mean area Area range RSD (%) SE

400 7724.56 7720.12–7730.58 0.58 0.36
800 15449.12 1544.36–15457.75 1.58 0.72

Band Stability
If the sample is not stable on the layer, the time the sample is left
on the plate before chromatographic development can affect the results
from analysis of the separated bands. This must be investigated during vali-
dation [15]. Two dimensional chromatography using same mobile phase
was used to discover decomposition occurring during development. If decom-
position occurs during development, peak(s) of decomposition product(s)
should be obtained from the analyte in both the first and second develop-
ments. No decomposition was observed after application or during deve-
lopment.
Analysis of the Marketed Formulation
A single band at RF 0.45 was observed in the densitogram of drug
samples extracted from capsules. There was no interference from excipients
commonly present in the capsules. The drug content was found to be
100.28%, RSD 0.57%. Statistical evaluation of the accuracy and precision
of the results was performed by use of Student’s t-test and the F-ratio at
95% confidence level. It was found that degradation of erdosteine had not
occurred in the marketed formulations analysed. The low RSD value indi-

Table VI
Applicability of the proposed method for analysis of commercial capsulesa

Property Value
Label claim (mg) 300
Drug content (%) ± SD 100.28 ± 0.88
RSD (%) 0.57
SE 0.36
t-valueb 2.44
F-valueb 9.27
a
n=6
b
Theoretical values for t and F (P = 0.05)

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cated the suitability of this method for routine analysis of erdosteine in phar-
maceutical dosage forms (Table VI).
Stability-Indicating Property of the Method (Accelerated Degradation)
Acid and Base-Induced Degradation
The densitogram obtained from the acid-degraded sample of erdo-
steine contained two peaks of degradation products at RF 0.10 and 0.65
(Fig. 3). The densitogram obtained from the base-degraded sample for er-
dosteine contained two peaks of degradation products at RF 0.28 and 0.55

Fig. 3
Densitogram obtained from acid-degraded erdosteine. Peaks 1 and 3 (RF 0.10 and 0.65, res-
pectively) are degradation products

Fig. 4
Densitogram obtained from base-degraded erdosteine. Peaks 1 and 3 (RF 0.28 and 0.55, res-
pectively) are degradation products

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(Fig. 4). The concentration of the drug was found to have changed from the
initial concentration indicating that erdosteine undergoes degradation under
acidic and basic conditions.
Hydrogen Peroxide-Induced Degradation
The densitogram obtained from the sample degraded with 10% hy-
drogen peroxide contained two peaks of degradation products at RF 0.16 and
0.55 (Fig. 5).

Fig. 5
Densitogram obtained from hydrogen peroxide-degraded erdosteine. Peaks 1 and 3 (RF 0.16
and 0.55, respectively) are degradation products

Dry and Wet Heat Degradation

No peaks of degradation products were observed in densitograms ob-
tained from samples subjected to wet heat and dry heat conditions (Fig. 6).

Fig. 6
Densitogram obtained from sample subjected to wet heat

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UV Degradation
The densitogram obtained from the sample for 15 days subjected
to UV irradiation contained a degradation product peak at RF 0.51 (Fig. 7).

Fig. 7
Densitogram obtained from sample subjected to UV irradiation. Peak 2 (RF 0.51) is that
of a degradation product

Neutral Degradation
No peaks of degradation products were observed in densitograms
obtained from samples degraded under neutral conditions.
Results obtained from study of erdosteine degradation are
summarized in Table VII.

Table VII
Results from study of erdosteine degradation

Recovery RF values of degradation

Condition
(%) products
Acid, 0.1 M HCl, reflux, 4.0 h, 50°C 66.13 0.10, 0.65
Base, 0.1 M NaOH, reflux, 4.0 h, 50°C 82.65 0.28, 0.55
H2O2 10% (w/v), reflux, 4.0 h, 50°C 87.20 0.16, 0.55
Neutral hydrolysis, 50°C, 5 h 98.02 –
Dry heat, 100°C, 5 h 98.00 –
Wet heat, 100°C, 5 h 99.05 –
UV, sunlight, 15 days 72.01 0.51

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CONCLUSION
This HPTLC method for quantitative analysis of erdosteine in phar-
maceutical formulations is precise, specific, accurate, reproducible, and
stability-indicating, without interference from the excipients or from degra-
dation products resulting from treatment with acid, alkali, oxidizing agent,
or from UV irradiation. The method was validated in accordance with ICH
guidelines. The method reduces analysis time compared with other methods
and seems to be suitable for routine analysis of pharmaceutical formulations
in quality-control laboratories, where economy and speed are essential.
This drug is separated from its degradation products, and hence can be re-
garded as stability-indicating.

ACKNOWLEDGEMENT
The authors thank Glenmark Pharmaceuticals, Nasik, India, for pro-
viding a sample of erdosteine as a gift.

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