WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

Volume 3, Issue 2, 2090-2097. Research Article ISSN 2278 – 4357

SPECTROPHOTOMETRIC ESTIMATION OF AMBROXOL

HYDROCHLORIDE IN BULK AND TABLET FORMULATION

Suma.B.V.*1, Kannan.K2, Madhavan. V3, Chandini R Nayar4

*
Research scholar, SASTRA University, Thanjavur-613402, Tamil Nadu, India,
1
Department of Pharmaceutical Chemistry, M.S.Ramaiah College of Pharmacy, Bangalore -
560054, Karnataka India,
2
Department of pharmacy, Annamalai University, Annamalai Nagar-608002, Chidambaram,
Tamil Nadu, India.
3
Department of PharmaCognosy, M.S.Ramaiah College of Pharmacy, Bangalore - 560054,
Karnataka India.
4
School of Chemical and Biotechnology, SASTRA University, Thanjavur-613402, Tamil
Nadu, India.

ABSTRACT
Article Received on
19 November 2013, A simple and sensitive spectrophotometric method in visible region
Revised on 23 December
2013, has been developed for the estimation of ambroxol hydrochloride in
Accepted on 27 January
2014 bulk drug and in tables. In this method the free amino group of
ambroxol hydrochloride reacts with ninhydrin reagent in alkaline

*Correspondence for
medium to form a violet color chromogen which is determined
Author: spectrophotometrically at 567 nm. The optimum experimental
Suma.B.V. condition for effect of heating, concentration of ninhydrin reagent and
Assistant Professor,
buffers were determined. It obeyed the Beer’s law range 2-10µg/ml.
Department of Pharmaceutical
Percentage purity of drug in formulation by this proposed method was
Chemistry, M.S.Ramaiah
College of Pharmacy, found to be 99.60%. The proposed method was found to be simple,
Bangalore, Karnataka, India. accurate and precise method for routine estimation of ambroxol
hydrochloride in bulk and from tablet dosage form.
Key words: Ambroxol hydrochloride, ninhydrin, spectrophotometric.

INTRODUCTION
Ambroxol Hydrochloride is trans-4-[(2Amino-3, 5-dibromobenzyl) amino] cyclohexanol
(Figure 1). It shows molecular formula as C13H18Br2N2O.HCl with molecular weight
414.57. Ambroxol is a metabolite of bromhexine. It is an expectoration improver and
mucolytic agent used in the treatment of acute and chronic disorders characterized by the

www.wjpps.com Vol 3, Issue 2, 2014. 2090

Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

production of excess or thick mucus. It is administered as a hydrochloride salt in daily doses

of 30-120 mg using mostly oral formulations like tablets and syrups. Similar doses have been
given by inhalation, injection or rectally1, 2. A literature survey reveals a spectrophotometric3-
10
, UV- HPLC11-17, LC- MS/MS18-20, HPTLC21 and miscellaneous methods 22-26
. However
many of these methods are limited in applications or time consuming or involves tedious
extraction procedures or involves costlier instruments therefore there is a need for a simple
spectrophotometric method for the assay of AMB.

This paper describes a rapid, simple and sensitive spectrophotometric method for
determination of AMB. The determination is based on the reaction of the primary amino
group of AMB with ninhydrin in methanol medium.

Figure 1 Chemical structure of AMB.

The aim of the present work is to determine AMB spectrophotometrically with ninhydrin
reagent in its pure and tablet dosage form.

MATERIAL AND METHODS

A SHIMADZU -UV1700 double beam UV-visible recording spectrophotometer with pair of
1cm matched quartz cell was used to measure absorbance of solutions.

Chemical and reagents used: Ambroxol hydrochloride (AMB) was procured from yarrow
chemicals private limited, Mumbai and ninhydrin, methanol, sodium hydroxide was procured
from Qualigens Fine Chemicals (Glaxo Ltd). The drug was purchased from the local market
(Mucolite containing AMB of 30 mg as per the label claim, marketed by Dr. Reddy‘s
laboratories, India).

Preparation of standard solution and reagents: - A stock solution of 1000µg/ml solution

was prepared by dissolving in methanol. Working standard solution concentration of
100µg/ml was used for experiment prepared in methanol. A 3% solution of ninhydrin was

www.wjpps.com Vol 3, Issue 2, 2014. 2091

Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

prepared and always a fresh solution was used. A 3 N concentration of sodium hydroxide was
prepared in distill water and used.

EXPERIMENTAL
Method
Aliquots of the working standard solution of ambroxol hydrochloride (2-10 µg/ml) were
transferred in a series of 10 ml volumetric flask. Then 1 ml of 3% freshly prepared ninhydrin
reagent and 1 ml 3 N concentration of sodium hydroxide were successively added to each
flask and volume was made upto the mark with methanol. The solutions were allowed to
stand for 5 minutes to complete the reaction. The absorbance was measured at 567 nm against
reagent blank prepared in similar manner.

Estimation from tablets

Twenty tablets of labeled claim 30 mg of ambroxol hydrochloride were weighed accurately.
Average weight of each tablet was determined. Tablets were crushed into fine powder using
motor and pestle. An accurately weighed quantity of powder equivalent to 100 mg of
ambroxol hydrochloride was transferred into a volumetric flask and it was shaken with 10 ml
of methanol and sonicated for 30 minutes, filtered through Whatman no.42 filter paper and
washed with methanol. The filtrate and the washing were collected in a 100 ml volumetric
flask. This filtrate and the washing were diluted up to the mark with methanol to obtain
concentration of 1000µg /ml. A 10 ml of 1000 µg /ml solution was further diluted to 100 ml
with methanol to give final concentration of 100 µg/ml. Appropriate aliquots of drug solution
were taken. The values of optical and regression terms of analysis are given in table no 1.
The individual assay procedure was carried out for the estimation of drug contents in tablets.
The concentration of the drug in the tablets was calculated using calibration curve as shown
in the table no 2. The recovery experiment was carried out by standard addition method and
results are given in the table no 3.

RESULTS AND DISCUSSION

The ninhydrin has been known as a reagent for the detection of amino acids and amines since
ages and therefore a number of theories have been put forward to explain the mechanism of
its reaction, It was suggested that the reactions of ninhydrin with amine, amino acid and
imino acids all proceed by the same mechanism to give di ketohydrindylidene-
27
diketohydrindamine or the Ruhemann’s purple . This compound further reacts with amino
group to give the product which absorbed maximally at 567 nm.

www.wjpps.com Vol 3, Issue 2, 2014. 2092

Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

The optimum conditions for determination of AMB were established through a number of
preliminary experiments.

Effect of heating
A 6 µg /ml solution of AMB of 0.6 ml was mixed with 1 ml of 3% freshly prepared ninhydrin
reagent and 1 ml 3 N concentration of sodium hydroxide in a boiling tube. The reaction
mixture was heated on a water bath at 100±1o C, upon heating the color of the solution used
to disappear within few minutes, but when kept at room temperature without heating,
remind stable for 30 minutes, hence no heating was performed which is the advantage of this
method.

Effect of ninhydrin concentration and sodium hydroxide concentration

To 0.6 ml of AMB, different volumes of (0.1-1 ml) of 3% ninhydrin were added with (1 ml)
of 3 N sodium hydroxide. The reaction mixture was allowed to stand for 5 minutes. The
coloured product was diluted with methanol and the absorbance was measured against a
reagent blank at 567 nm. The results obtained showed highest absorbance at 0.8 ml which
remains unaffected with higher volume of ninhydrin as shown in the figure no 2. Therefore
1ml of 3% ninhydrin and 1 ml of 3N sodium hydroxide solution were added for the
determination.

Hence for the determination of AMB in bulk and in tablet dosage form 1 ml of 3 % fresh
ninhydrin reagent and 1 ml of 3 N sodium hydroxide solution was used.

Figure 2 Effect of ninhydrin concentration

The method was validated as per the ICH guidelines. The proposed method for determination
of AMB showed Beer’s law range from 2-10 µg/ ml, molar absorptivity of 35984.85. Linear

www.wjpps.com Vol 3, Issue 2, 2014. 2093

 Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

regression of absorbance on the concentration gave the equation Y= 0.089x + 0.076 with a
correlation coefficient of r2 0.999. The percentage purity was found to be 99.60% with
average of three trials. Recovery studies were carried out at three different levels of 80%.
100%, 120% based on standard addition method and was found to be in the range of 100.48%
for 80%, 101.27% for 100% and 100.44 for 120% respectively. The percentage recovery
indicates that there is no interference of the excipients present in the formulations.

Figure 3: Linearity curve for AMB

Table No. 1 Statistical analysis and Optical characteristics of AMB

Parameters Values
Detection wave length 567 nm
Beer range 2-10 µg/ ml
Correlation coefficient 0.9996
Slope 0.08930
Y intercept 0.07620
Absorptivity for 10 µg/ ml 95
Molar absorptivity 35984.85
Specific absorptivity 951.703
Sand ell’s Sensitivity 1.0252x10-2 µg/ cm2

Table no 2 Analysis of tablet formulation: Mucolite containing AMB of 30 mg

Sl.No. Parameters AMB

1 Label claim(mg/tab) 30 mg
2 Drug content % 99.60
3 % RSD 0.3331

www.wjpps.com Vol 3, Issue 2, 2014. 2094

 Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

Table No. 3 Recovery studies of AMB

Amt of Total amt Amt of Recovery

Amt of %
Standard of AMB from of
Sl.No Sample Recovery*
(µg/ml) AMB standard standard*
(µg/ml) ±RSD
(µg/ml) graph*
1 1.6 2 3.6 3.607 1.607 100.48±0.3984
2 2 2 4 4.025 2.025 101.27±0.3175
3 2.4 2 4.4 4.410 2.410 100.44±0.051

*Average of three readings

CONCLUSION
The developed method was found to be accurate, precise, repeatable and reproducible and
can be used for the routine analysis of AMB in bulk and drug formulations.

ACKNOWLEDGEMENT
The authors are thankful to Gokula Education Foundation for providing necessary facilities to
carry out the research work. The authors are also thankful to Prof. R.Sethuraman,Vice
chancellor of SASTRA university, Dr. K.Thiagarajan research dean, Dr. R. Chandramouli,
associate research dean of SASTRA University Thanjavur, Tamil Nadu for their support and
encourage ment.

REFERENCES
1. The Merck Index. , 13th Ed. Merck & Co. Inc.: New Jersey, 2001.
2. Gillissen, A.; Nowak, D. Characterization of N-acetylcysteine and ambroxol in anti-
oxidant therapy. Respir. Med., 1998, 92, 609-623.
3. Tomas Perez-Ruiz, Carmen Martínez-Lozano,Antonio Sanz,M. Teresa San Miguel,
Automatic extraction-spectrophotometric method for the determination of ambroxol in
pharmaceutical preparations, Talanta July 1996;(43), (7):1029–1034
4. Naryana Reddy M; Kanna Rao KV; Swapna M; Sarkar DG; Reddy NM; Rao KKV,
Spectrophotometric determination of ambroxol, Indian Journal of Pharmaceutical
Sciences. July 1998; 60(4): 249-251
5. Kuchekar BS, Shinde GS, Naikawadi IT,Todkar KJ, Kharade SV, Spectrophotometric
Estimation Of Ambroxol Hydrochloride In Tablets Indian journal of pharmaceutical
sciences, 2003;(65),(2):193-195

www.wjpps.com Vol 3, Issue 2, 2014. 2095

Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

6. PNS Pai, N Lalitha, B Balakrishna, GK Rao Determination of ambroxol hydrochloride

using dithiocarbamic acid colorimetric method Indian journal of pharmaceutical
sciences, 2006 : (68),(4): 501-502
7. Raju A. I., Kiran Babu; Simple, sensitive and rapid spectrophotometric method for
determination method ambroxol hydrochloride; The Indian Pharmacist (New Delhi)
A.2006, 05(54), 71-72.
8. Asha Immanuel Raju Chaduvula, Kiran Babu Uppuluri, New Spectrophotometric Method
for the Determination of Ambroxol Hydrochloride Using Artificial Neural Networks. The
Preliminary Program for 2007 Annual Meeting
9. Pritam S Jain A new, simple, sensitive spectrophotometric method in uv region for the
determination of ambroxol hydrochloride in bulk and in pharmaceutical formulations
journal of pharmacy Researc,. 2009, 02 (8) 1131-1132
10. Rele Rajan V Gurav Pankaj J, Simple spectrophotometric methods for the determination
of Ambroxol hydrochloride from pharmaceutical formulation, International Journal of
Pharm Tech Research, July Sept 2012; (3):994-998
11. Milan Nobilis,Jiří Pastera, Dalibor Svoboda, Jaroslav Květina, Journal of High-
performance liquid chromatographic determination of ambroxol in human plasma
Chromatography B: Biomedical Sciences and Applications, 23 October 1992,(581), (2),
251–255
12. Gunawan Indrayanto and Ratna Handajani, Quantitative Determination of Ambroxol
Hydrochloride in Syrups by RP-HPLC and UV Spectroscopy, Drug development and
industrial pharmacy, 1994;(20),(9):1639-1647
13. Kiss, BD; Nemes, KB; Urmos, I; Szunyog, J; Klebovich, I, Determination of ambroxol in
dog plasma by high-performance liquid chromatography and UV detection,
Chromatographia, 2000, (51):217 - 220
14. John E. Koundourellis, Eleftheria T. Malliou,Theodora A. Broussali, High performance
liquid chromatographic determination of ambroxol in the presence of different
preservatives in pharmaceutical formulations. Journal of Pharmaceutical and Biomedical
Analysis , 15 August 2000(23) :(2–3): 469–475
15. Zarzuero Aranzazu, Sayaere Ma Lusia, Lopez Franciso G determination of ambroxol
hydrochloride in solution, Journal of liquid Chromatography and related technologies A,
2001, 24(7) 1007-1014

www.wjpps.com Vol 3, Issue 2, 2014. 2096

Suma.B.V. et al. World Journal of Pharmacy and Pharmaceutical Sciences

16. Dinçer Z, Basan H, Göger NG, Quantitative determination of ambroxol in tablets by

derivative UV spectrophotometric method and HPLC,J Pharm Biomed Anal. 2003 Apr 1;
(31), (5):867-872
17. Satana E, Basan H and Goğer NG. Determination of ambroxol hydrochloride in tablets
using flow-injection UV spectrophotometry and HPLC, Journal of Analytical Chemistry ,
2008 ;( 63), (5): 451-454.
18. Kim H, Yoo JY, Han SB, Lee HJ, Lee KR. Determination of ambroxol in human plasma
using LC-MS/MS, J Pharm Biomed Anal. 2003 Jun 1; 32(2):209-216.
19. Su F, Wang F, Gao W, Li H, Determination of ambroxol in human plasma by high
performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-
MS/ESI),Journal of Chromatography. B, Analytical Technologies in the Biomedical and
Life Sciences , 2007; 853(1-2):364-368
20. Hu W, Xu Y, Liu F, Liu A, Guo Q, Rapid and sensitive liquid chromatography tandem
mass spectrometry method for the quantification of ambroxol in human plasma, Biomed
Chromatogr. 2008 Oct; 22(10):1108-1114
21. P.S. Jain, Stability-Indicating HPTLC Determination of Ambroxol Hydrochloride in Bulk
Drug and Pharmaceutical Dosage Form, Journal of Chromatographic Science, January
2010; (48), 45-48.
22. Schmid J,Assay of ambroxol in biological fluids by capillary gas-liquid chromatography
Journal of Chromatography B: Biomedical Sciences and Applications, 1987; 414: 65–75
23. Tomas Perez-Ruiz, Carmen Mariı́ nez-Lozano, Antonio Sanz, Eva Bravo, Sensitive
method for the determination of ambroxol in body fluids by capillary electrophoresis and
fluorescence detection Journal of Chromatography B: Biomedical Sciences and
Applications, 26 May 2000,(742); (1): 205–210
24. Szostak Roman Mazurek Sylwester, FT-Raman quantitative determination of ambroxol in
tablets, Journal of molecular structure 2004; (704), (1-3): 229-233
25. Hwang MS Cho S Chong H, Woo IA Non destructive determination of the ambroxol
content in tablets by Raman spectroscopy Journal of pharma Biomed Anal. 2005, 38(2)
210-215
26. Levent A, Sentürk Z,Colorimetric and atomic absorption spectrometric determination of
mucolytic drug ambroxol through ion-pair formation with iron and thiocyanate. Comb
Chem High Throughput Screen. 2010 Sep; 13(8):675-682.
27. D.J.Mc Caldin. The chemistry of ninhydrin.Chem Rev. 1960 (60) :39-51.

www.wjpps.com Vol 3, Issue 2, 2014. 2097