Analytical
method development and validation of ambroxol hydrochloride by UV spectroscopy
and forced degradation study and detection of stability.
Mrs. M.
Sumithra*, P. Yuvanesh*, Anamika Mistry1
Department of Pharmaceutical Analysis, School of
Pharmaceutical Science, Vels University, Chennai-600 117
*Corresponding Author E-mail: sumithrapharmanalysis@gmail.com,
yuvaneshyuvi46@gmail.com, anamikachameli@gmail.com
ABSTRACT:
Aim: The
aim is to develop simple validated analytical method for analysis of
Ambroxol hydrochloride by UV
Spectroscopy and to study the forced degradation and stress conditions have
been used to detect the stability of Ambroxol hydrochloride. Method: Ambroxol hydrochloride was
estimated at 306nm. Linearity range was found to be2-10mcg/ml. The correlation
coefficient was found to be 0.99987. The molar absorptivity was found to be
3947 L mol/cm. The proposed method Sandell’s sensitivity was found to be
0.111111 μg cm−2/0.001AU. The limit of detection and limit of
quantification were found to be 3.94210and
11.94577μg /ml respectively.
The degradation behavior of Ambroxol hydrochloride was carried out as
per the standard procedures and guidelines. Forced acid hydrolytic degradation,
alkali degradation and oxidative degradation of Ambroxol hydrochloride was
performed in bulk and solid oral formulation using 1N Hydrochloric acid and
0.1M Sodium hydroxide at room temperature in different time intervals such as
0mins,30mins, 60mins and 90mins. The resulting solutions were analyzed for
content by UV spectrophotometry at the maximum absorption of 306 nm.. The assay
value ofAmbroxol hydrochloride in bulk and formulation was calculated at
different time intervals for intraday and interday experiments. Results and conclusion: The proposed
method was successfully applied for the determination of Ambroxol hydrochloride
in bulk and Pharmaceutical formulations (Tablets). The results were
demonstrated, that the procedure is accurate, precise and reproducible
(relative standard deviation < 2%), For acid degradation studies the assay
values of Ambroxol hydrochloride at the end of the 90mins and 3rd
day study for standard and sample were 58.42% and 50.53% respectively. For
alkali degradation studies the assay values of Ambroxol hydrochloride at the
end of the 90mins and 3rd day study for standard and sample were were
70.85% and 77.06% respectively. For oxidative
degradation studies the assay values of Ambroxol hydrochloride at the end of
the 90mins and 3rd day study for standard and sample were were
58.42% and 43.42% respectively. Ambroxol
hydrochloride was found to degrade extensively under acid, alkali and oxidative
conditions. Ambroxol hydrochloride has to be stored under such condition where
the possibility of acid, alkali and
oxidative hydrolysis does not arise.
KEYWORDS: Ambroxol
hydrochloride, UV –Spectroscopy, Validation, ICH guidelines, Forced degradation
studies.
INTRODUCTION:
Ambroxol hydrochloride1,2
(AMH) is chemically 4-[(2-amino-3,5-dibromophenyl)methylamino]cyclohexan-1-ol;hydrochloride3
which is a semi-synthetic derivative of vasicine
from the Indian shrub “Adhatodavasica.”
It is a mucolytic agent. Ambroxol is indicated as "secretolytic therapy in
bronchopulmonary diseases associated with abnormal mucus secretion and impaired
mucus transport. It promotes mucus clearance, facilitates expectoration and
eases productive cough, allowing patients to breathe freely and
deeply".4
Fig.1:Structure of Ambroxol
Hydrochloride
Only limited analytical methods were
reported in the literature for Ambroxol. The aim of this work was to develop
and validate a simple, fast, and reliable isocratic ultraviolet (UV)
spectroscopic method for the determination of Ambroxol hydrochloride in bulk and pharmaceutical dosage forms. Forced
degradation study like alkali, acid and oxidative degradation were performed.
Confirmation of the applicability of the developed method validated according
to the International Conference on Harmonisation (ICH) to determine the
Ambroxol hydrochloride in bulk and pharmaceutical preparations. The objective
of this study was to develop and validate an assay for the estimation of
Ambroxol using HPLC.
Chemicals reagents:
Preparation of stock solution
10 mg of Ambroxol hydrochloride raw material was
weighed and transferred in to 100 ml volumetric flask, then dissolved in
methanol and made up to the volume with the same solvent. This solution
contains 100µg/ ml concentration.
The standard stock solution was further diluted with
water to get the concentration of 10 µg/ ml and the solution was scanned
between 200 and 400 nm using the same solvent as blank. The spectrum was
observed in that range and the λ max was found to be 306 nm and was
selected as analytical wavelength.7,8,9
Preparation
of linearity studies
The standard stock solution of Ambroxol
hydrochloride was transferred into series of 10 ml volumetric flasks and made
up to the volume with water. The absorbance of 2,4,6,8,10 g/ ml solutions were
measured at 306 nm. The calibration curve was plotting between concentration
vs. absorbance. Ambroxol hydrochloride was linear within the concentration
range of 2,4,.....,10g/ ml at 306 nm. 9
Validation
of method development
Linearity
A calibration curve was plotted
between concentration and absorbance. Ambroxol hydrochloride was linear in the
concentration range of 2-10g/ ml at 306 nm. The linearity was repeated for five
times and LOD and LOQ values for calculated.,5,7
The linearity is shown in fig.3 and the concentration with the absorbance
value are given in table.1
Quantification of
formulation:
20 tablets (mucolite containing ambroxol equivalent to
30mg)were weighed accurately. The average weight of the tablets are calculated and the tablets are made into powdered form. The
powdered equivalent to 30mg of ambroxol was weighed and transferred into 100 ml
volumetric flask. Added a minimum quantity of methanol to dissolve the
substance and made upto the volume with the same(100µg/ml). The solution was
filtered with whatmann filter paper. From the clear solution, further dilutions
were made by 1ml to 10 ml volumetric flask with water to get 10µg/ml solution
theoretically. The absorbance of six replicates were measured and the amount
was calculated by using regression equation. This procedure is repeated for six
times.6,11
Precision
The repeatability of the developed method was
confirmed by the precision analysis. The intermediate precision of the method
was confirmed by intraday and interday analysis i.e. the analysis of
formulation was repeated three time in the same day and on three successive
days. For this process Ambroxol hydrochloride 30mg was used. The amount of
drugs present was determined and the percentage RSD also calculated.5,7,10,12
Accuracy
Accuracy of the method was confirmed by the recovery
studies. To the preanalysed formulation a known quantity of raw material of
Ambroxol hydrochloride was added in 6 concentration and recovery process are
followed as per the quantification process. The amount of recovery was
calculated. This procedure is repeated for 6 times and the %RSD was calculated.
The results are shown below.9,10,13
Study of acid degradation
Ambroxol Hydrochloride by UV spectroscopy method
Standard Preparation (stress) Ambroxol hydrochloride
was transferred to volumetric flask and dissolved methanol to achieve a
concentration of 1mg/mL. After 30mins, an aliquot solution was diluted with 1N
hydrochloric acid to get a final concentration of 100mcg/mL. The solution was
scanned in the UV region and the maximum absorbance was recorded at 306 nm. The
same procedure was repeated for 60mins, and 90mins time interval.14
Sample Preparation (stress) Ambroxol hydrochloride
tablets were powdered and weighed and then transferred into volumetric flask
and dissolved methanol to achieve a concentration of 1mg/mL. After 30mins, an
aliquot solution was diluted with 1N hydrochloric acid to get a final
concentration of 100mcg/mL. The solution was scanned in the UV region and the
maximum absorbance was recorded at 306 nm. repeated for 60mins, and 90mins time
interval.
Blank Preparation: A blank preparation of Hydrochloric
acid (1N) solution was prepared in a
similar manner. The procedure was repeated thrice. After the stipulated time,
the absorption of the resulting solution showed maxima 306nm (Table:8) against
reagent blank treated in the same way. Three such determinations were made and
the assay value was estimated the obtained values are concurrent.
For Inter day study Standard preparation The standard
preparation was prepared in a similar manner which was mentioned in an intraday
preparation Standard stress preparation Same method was followed, but the final
solution was scanned and absorption was recorded at the following time
intervals 1st, 2nd, and 3rd day.
Sample Preparation: Same method was followed, but the
final solution was scanned and absorption was recorded at the following time
intervals 1st, 2nd, and 3rd day. Blank preparation Similar to intraday
preparation The procedure was repeated thrice. After the stipulated time, the
absorption of the resulting solution showed maxima 306nm (Table:9) against
reagent blank treated in the same way. Three such determinations were made and
the assay values are estimated and concurrent values were obtained.
Study of alkali degradation
Ambroxol Hydrochloride by uv spectroscopy method
Stand ard Preparation (stress) Ambroxol hydrochloride
10mg was transferred to volumetric flask and dissolved methanol to achieve a
concentration of 1mg/mL. After 30mins, an aliquot solution was diluted with
distilled with 0.1M Sodium hydroxide to get a final concentration of 100mcg/mL.
The solution was scanned in the UV region and the maximum absorbance was
recorded at 306nm. The same procedure was repeated for 60mins, and 90mins time
interval. 15
Sample Preparation (stress) Ambroxol hydrochloride
granules were weighed and transferred to volumetric flask and dissolved
methanol to achieve a concentration of 1mg/mL. After 30mins, an aliquot
solution was diluted with 0.1M Sodium hydroxide to get a final concentration of
100mcg/mL. The solution was scanned in the UV region and the maximum absorbance
was recorded at 306nm. The same procedure was repeated for 60mins, and 90mins
time interval
Blank Preparation: A blank solution of Sodium
hydroxide (0.1M) solution was prepared in a similar manner. The procedure was
repeated thrice. After the stipulated time, the absorption of the resulting
solution showed maxima 306nm (Table:10) against reagent blank treated in the
same way. Three such determinations were made and the assay value was estimated
the obtained values were concurrent.
For Inter day study Standard preparation The standard
preparation was prepared in a similar manner which was mentioned in an intraday
preparation Standard stress preparation Same method was followed, but the final
solution was scanned and absorption was recorded at the following time
intervals 1st, 2nd, and 3rd day.
Sample Preparation: Same method was followed, but the
final solution was scanned and absorption was recorded at the following time
intervals 1st, 2nd, and 3rd day. Blank preparation Similar to intraday
preparation The procedure was repeated thrice. After the stipulated time, the
absorption of the resulting solution showed maxima 306nm (Table:11) against
reagent blank treated in the same way. Three such determinations were made and
the assay value was estimated and concurrent values were obtained.
Study of oxidative
degradation Ambroxol Hydrochloride by uv spectroscopy method
Standard Preparation (stress) Ambroxol hydrochloride
10mg was transferred to volumetric flask and dissolved methanol to achieve a
concentration of 1mg/mL. After 30mins, an aliquot solution was diluted with
distilled with 10% hydrogen peroxide to get a final concentration of 100mcg/mL.
The solution was scanned in the UV region and the maximum absorbance was
recorded at 306nm. The same procedure was repeated for 60mins, and 90mins time
interval.16
Sample Preparation (stress) Ambroxol hydrochloride
granules were weighed and transferred to volumetric flask and dissolved
methanol to achieve a concentration of 1mg/mL. After 30mins, an aliquot
solution was diluted with 10% hydrogen peroxide to get a final concentration of
100mcg/mL. The solution was scanned in the UV region and the maximum absorbance
was recorded at 306nm. The same procedure was repeated for 60mins, and 90mins
time interval.
Blank Preparation: A blank solution of hydrogen
peroxide(10%) solution was prepared in a similar manner. The procedure was
repeated thrice. After the stipulated time, the absorption of the resulting
solution showed maxima 306nm (Table:12) against reagent blank treated in the
same way. Three such determinations were made and the assay value was estimated
the obtained values were concurrent.
For Inter day study Standard preparation The standard
preparation was prepared in a similar manner which was mentioned in an intraday
preparation Standard stress preparation Same method was followed, but the final
solution was scanned and absorption was recorded at the following time
intervals 1st, 2nd, and 3rd day.
Sample Preparation: Same method was followed, but the
final solution was scanned and absorption was recorded at the following time
intervals 1st, 2nd, and 3rd day. Blank preparation Similar to intraday
preparation The procedure was repeated thrice. After the stipulated time, the
absorption of the resulting solution showed maxima 306nm (Table:13) against
reagent blank treated in the same way. Three such determinations were made and
the assay value was estimated and concurrent values were obtained.
RESULT
AND DISCUSSION:
The new, simple and cost
effective UV-Spectrophotometric method
was developed for the estimation of Ambroxol hydrochloride in bulk and
pharmaceutical formulations and study of acid, alkali and oxidative degradation
Ambroxol hydrochloride was
estimated at 306 nm by using water. The drug was soluble in aqueous solvent but it is not produce stable λmax and absorbance. So we tried
with water and 0.1N HCl.
Linearity range was found
to be 2−10 μg/ml. The correlation
coefficient was found to be 0.999876 and the molar absorptivity was found to be
3947 L mol−1 cm−1 in water and in 0.1M
HCl.. The proposed method Sandell's sensitivity was found to be about
0.111111 μg cm−2/ 0.001AU.
Fig.2:UV absorption spectrum of
Ambroxol hydrochloride.
Fig.3:Calibration curve of Ambroxol
hydrochloride
Table
no.1:Linearty
of Ambroxol hydrochloride
|
S. no. |
Concentration (μg /ml ) |
Absorbance |
|
1. |
2 |
0.023 |
|
2. |
4 |
0.041 |
|
3. |
6 |
0.060 |
|
4. |
8 |
0.077 |
|
5. |
10 |
0.095 |
The limit of detection and the limit
of quantification were determined by the linearity studies, theprocess was
repeated for six times and the limit of detection (3.9420) and the limit of
quantification (11.9477) were calculated. It has been shown in table no.2.
Table no:2. Optical Characteristics of Ambroxol Hydrochloride
|
PARAMETERS |
VALUES* |
|
λmax (nm) |
306 |
|
Beer’s law limit (µg/ ml) |
2-10 |
|
Sandell’s sensitivity (g/cm2/0.001 A.U) |
0.111111 |
|
Molar absorptivity (L mol–1 cm–1) |
3947 |
|
Correlation coefficient (r) |
0.999876 |
|
Regression equation (y=mx+c) |
Y = 0.009x + 0.00520 |
|
Slope(m) |
0.009 |
|
Intercept(c) |
0.0052 |
|
LOD (µg/ ml) |
3.9420 |
|
LOQ (µg/ ml) |
11.9477 |
|
Standard error |
0.0032 |
Table no.3:Quantification of
formulation.
|
|
Sample no. |
Amount added (µg/ ml) |
Amount present (µg/ ml) |
Percentage obtained |
Average% |
S.D |
%RSD |
S.E |
|
|
1. |
6 |
5.86 |
97.66 |
|
|
|
|
|
|
2. |
6 |
6.08 |
101.4 |
|
|
|
|
|
Ambroxol |
3. |
6 |
5.97 |
99.61 |
98.6 |
1.25 |
1.00 |
0.0032 |
|
|
4. |
6 |
5.86 |
97.6 |
|
|
|
|
|
|
5. |
6 |
5.64 |
94.07 |
|
|
|
|
Table
no.4: Recovery analysis of
Ambroxol hydrochloride
|
Drug |
Sample no. |
Amount present µg/ ml |
Amount added µg/ ml |
Amount found µg/ ml |
Amount recovered µg/ ml |
% recovered |
S.D |
%RSD |
S.E |
|
|
1. |
3 |
3 |
5.86 |
2.86 |
97.77 |
|
|
|
|
|
2. |
3 |
3 |
6.03 |
3.03 |
100.55 |
1.89 |
2.00 |
0.0032 |
|
Ambroxol |
3. |
3 |
3 |
6.08 |
3.08 |
101.4 |
|
|
|
|
|
4. |
3 |
3 |
5.97 |
2.97 |
99.61 |
|
|
|
|
|
5. |
3 |
3 |
6.06 |
3.06 |
101.10 |
|
|
|
From the linearity curve, the mean
concentration of 6μg/ml was selected and quantification in tablets was
performed. The 30 mg tablets was
selected for analysis. The amount present was determined by average of six
replicate analysis and the amount present were found to be 5.86,6.08,
5.97,5.86,5.64 mg respectively. The results were shown in Table no.3
The accuracy of the developed method was
carried out by standard addition method. The known amount of pure drug was
added to the previously analyzed solution containing tablets and the mixture
was analyzed by the proposed method and the recoveries were calculated. The
percentage recovery of formulation (Mucolite 30 mg) was found to be
97.77,100.55,101.4,99.61,101.10. The resultswere shown in Table no. 4.
Precision of the method has done by making repeated
analysis of the same sample and it was carried out three times in a day for 3
days. The percentage standard deviation for inter day and intraday analysis of
Ambroxol hydrochloride was found to be 1.4120 and 1.89 respectively and shown
in table .5 and 6.
Table no.5:Intraday analysis of Ambroxol hydrochloride
|
Drug |
Sample no. |
Amount Present (µg/ml) |
Amount found (µg/ml) |
Percentage obtained |
Average % |
S.D |
%RSD |
S.E |
|
|
1. |
6 |
6.08 |
101.4 |
|
|
|
|
|
Ambroxol |
2. |
6 |
5.97 |
99.62 |
98.36 |
1.1352 |
1.4120 |
0.0032 |
|
|
3. |
6 |
5.64 |
94.07 |
|
|
|
|
Table no.6: Interday analysis of Ambroxol hydrochloride
|
Drug |
Sample no. |
Amount present (µg/ml) |
Amount found (µg/ml) |
Percentage obtained |
Average % |
S.D |
%RSD |
S.E |
|
|
1. |
6 |
5.86 |
97.77 |
|
|
|
|
|
Ambroxol |
2. |
6 |
5.97 |
99.92 |
98 |
1.85 |
1.89 |
0.0032 |
|
|
3. |
6 |
5.75 |
95.62 |
|
|
|
|
Table no.7:Ruggedness study by different analysts
|
Drug |
Condition |
Sample No |
Amount present (µg/ml |
Amount found (µg/ml) |
Percentage Obtained |
Average (%) |
S.D |
%R.S.D |
S.E |
|
AMB |
Analyst 1 |
1 2 3 4 5 6 |
6 6 6 6 6 6 |
6.088 6.2 6.077 5.97 6.033 6.066 |
101.46 103.35 101.26 99.62 100.55 101.10 |
101.22 |
0.95 |
0.94 |
0.0032 |
|
AMB |
Analyst 2 |
1 2 3 4 5 6 |
6 6 6 6 6 6 |
5.86 6.08 6.06 6.05 6.2 6.08 |
97.77 101.46 101.1 100.97 103.35 101.46 |
101.01 |
0.87 |
0.86 |
0.0032 |
Ruggedness
The ruggedness of the developed method was
confirmed by using different instruments and different analysts.The % RSD was
calculated. The % RSD by using different analyst 0.94% and 0.86%. The results
are within the limit. So it indicates the developed method was more rugged. The
results were shown in Table 7.
Acid Degradation studies:
Ambroxol hydrochloride was found to be unstable under
acid condition. Table: 8shows the results of intraday degradation and how much
of standard and sample remaining in the solution after certain time intervals.
Table no. :8. Acid degradation study of AMB(Interday)assay stress
conditions
|
Stress condition |
Time in minutes |
Standard* |
Sample* |
Remarks |
|
Hydrochloric acid (1N) Acid Hydrolysis |
30 |
63.08% |
54.75% |
Degradation
observed |
|
60 |
61.31% |
51.97% |
Degradation
observed |
|
|
90 |
59.46% |
50.53% |
Degradation
observed |
The assay values of the standard and sample was found
to be 58.42% and 50.53% at the end of 90 mins.Table:9 shows the interday
degradation and how much standard and sample were remaining after the
degradation. The assay value of standard and sample was found to 58.42% and 50.03% at the end of first day of
acid hydrolysis. At the end of 3rd day the assay value of standard
and sample were found to be 52.69% and 44.24%
Table no.9: Acid degradation study of AMB(Intraday)assay
stress conditions
|
Stress condition |
Time in Hours |
Standard* |
Sample* |
Remarks |
|
Hydrochloric acid (0.1N) Acid Hydrolysis |
24 |
58.42% |
50.03% |
Degradation observed |
|
48 |
56.97% |
47.08% |
Degradation observed |
|
|
72 |
52.69% |
44.24% |
Degradation observed |
Alkali degradation studies:
Ambroxol hydrochloride was found to be unstable under
alkali condition. Table: 10 shows the results of intraday degradation and how
much of standard and sample remaining in the solution after certain time
intervals.
Table no.:10. Alkali degradation study of AMB(Intraday)assay
stress conditions
|
Stress condition |
Time in minutes |
Standard* |
Sample* |
Remarks |
|
Sodium Hydroxide (0.1M) Alkali Hydrolysis |
30 |
76.08 |
82.2 |
Degradation
observed |
|
60 |
73.86 |
77.06 |
Degradation
observed |
|
|
90 |
70.85 |
73.75 |
Degradation
observed |
The assay values of the standard and sample was found
to be 70.85% and 73.75% at the end of 90 mins.Table:11 shows the interday
degradation and how much standard and sample were remaining after the
degradation. The assay value of standard and sample was found to 66.35% and
71.62% at the end of first day of alkali hydrolysis. At the end of 3rd day the
assay value of standard and sample were
54.24% and 60.31%.
Table no.11.Alkali degradation study of AMB(Interday)assay stress
conditions.
|
Stress condition |
Time in Hours |
Standard* |
Sample* |
Remarks |
|
Sodium Hydroxide
(0.1M) Alkali hydrolysis |
24 |
66.35 |
71.62 |
Degradation
observed |
|
48 |
60.64 |
66.53 |
Degradation
observed |
|
|
72 |
54.24 |
60.31 |
Degradation
observed |
Oxidative Degradation:
Ambroxol hydrochloride was found to be unstable under
oxidative condition.Table.12shows the results of intraday degradation and how
much of standard and sample remaining in the solution after certain time
intervals.
Table no.: 12. Oxidative Intraday Degradation study of Ambroxol Hydrochloride.
|
Stress condition |
Time in minutes |
Standard* |
Sample* |
Remarks |
|
Hydrogen Peroxide (10%) Oxidative
Hydrolysis |
30 |
64.86% |
49.86% |
Degradation
observed |
|
60 |
61.62% |
46.31% |
Degradation
observed |
|
|
90 |
58.42% |
43.42% |
Degradation
observed |
The assay values of the standard and sample was found
to be 58.42% and 43.42% at the end of 90 mins.Table:13 shows the interday
degradation and how much standard and sample were remaining after the
degradation. The assay value of standard and sample was found to 57.28% and
40.09% at the end of first day of oxidative hydrolysis. At the end of 3rd day
the assay value of standard and sample were 45.22% and 33.54% respectively.
Table no. 13:Oxidative Interday Degradation study of Ambroxol Hydrochloride.
|
Stress condition |
Time in Hours |
Standard* |
Sample* |
Remarks |
|
Hydrogen Peroxide (10%) Oxidative
Hydrolysis |
24 |
57.28% |
40.09% |
Degradation observed |
|
48 |
51.31% |
37.32% |
Degradation observed |
|
|
72 |
45.22% |
33.54% |
Degradation observed |
CONCLUSION:
In this study a simple ,precise, accurate and
sensitive UV-spectroscopy methods were developed for the simultaneous
estimation of Ambroxol hydrochloride in bulk and in tablet dosage form. The
Correlation coefficient (γ) values of the proposed method was close to
1.0, it indicate that the concentration used for plotting calibration curve
were obeying Beer’s law strictly. Additives and impurities commonly present in
the dosage forms but did not show any interference in the proposed method.
Statistical validation was done it shows that the method was reproducible and
accurate. Also the various parameters were calculated such as standard
deviation and percentage relative standard deviation. The values are complies
all the limit as per ICH guidelines. The forced acid, alkali and oxidative
degradation study of Ambroxol hydrochloride
was studied by UV spectroscopy at various time interval (30mins, 60mins,
90mins; 1st, 2nd, 3rd day ;) it is observed that the drug Ambroxol hydrochloride is
degrading. Therefore the drug Ambroxol
hydrochloride has to be stored under such condition where the possibility of
acid, alkali and oxidative hydrolysis
does not arise.
ACKNOWLEDGEMENT:
We wish to thank Tablets India Pvt. Ltd., Chennai,
India for providing the gift sample of Ambroxol hydrochloride and Mrs. M. Sumithra,
Asst. Professor from School of Pharmaceutical Sciences, Vels University for
providing the technical support during the research.
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Received on 07.04.2016
Modified on 22.04.2016
Accepted on 27.04.2016 ©
RJPT All right reserved
Research J. Pharm. and Tech. 2016;
9(7):794-800
DOI: 10.5958/0974-360X.2016.00152.9