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Research J. Pharm. and Tech. 7(10): October 2014
1208
ISSN 0974-3618 www.rjptonline.org
REVIEW ARTICLE
Analytical Methods for Quantitative Estimation of Ambroxol HCl in
Pharmaceutical Preparation: A Review
Veena Devi Singh1*, Sanjay J. Daharwal2
1Research Scholar, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur CG 492010
2Assistant Professor, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur CG 492010
*Corresponding Author E-mail: veena1806@gmail.com
ABSTRACT:
Context: Ambroxol Hcl is an active metabolite of bromohexine. It is an expectoration improver and a mucolytic agent
used in the treatment of bronchial asthma and chronic bronchitis.The available formulation of Ambroxol was
authorized first in market since 1978.
Objective:This article highlights on published analytical methods reported in the literature for the determination of
Ambroxol Hcl in biological samples and pharmaceutical formulations.
Material and Methods: Various techniques like electrochemical, spectrophotometry, high-performance liquid
chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), Gas chromatography, Ultra
performance liquid chromatography (UPLC) and high-performance thin layer chromatography (HPTLC) were used
for the qualitative and quantitative estimation of Ambroxol Hcl.
Result and Discussion: Literature reveals that most widely used diluents are methanol and distilled water in HPLC
methods, which prolonged the run times with greater tailing factor. For spectrometric determination, the presence of
multiple entities and excipients includes complexity with multi- component dosage forms, which could produce
significant challenge to the analytical chemist during the development of assay procedure. For such instances, chemo-
metric methods can be preferred to routine spectrophotometric methods.
Conclusion: Amongst various analytical techniques available for the quantification of single and multicomponent
dosage form. HPLC methods are most extensively used for analysis of Ambroxol Hcl.
KEYWORDS: Ambroxol HCl, Analytical methods, biological fluid, Mucolytic agent and pharmaceutical
formulation.
INTRODUCTION:
Ambroxol hydrochloride is chemically 4-{((2-amino-3,5-
dibromophenyl)-methyl)-amino)}-cyclohexanol or N-
{((trans-phydroxycyclohexyl),(2-amino-3,5-dibromo
benzyl))}-amine,(Figure 1.) which is a semi synthetic
derivative of vasicine obtained from the Indian herbal shrub
“Adhatoda vasica”. Ambroxol HCl is an active metabolite
of bromohexin, and it is a mucolytic agent, used as an
expectoration improver as well as in the treatment of
chronic bronchitis and bronchial asthma (Pai et al.; 2005). It
was authorized first in market since 1978. Here, many
formulations are available such as tablet, syrup, pastilles,
sachet and dry powder, drops, ampoules and inhalation
solution, as well as effervescent tablet in market in
individual component and in combination with other drugs.
Received on 20.07.2014 Modified on 10.08.2014
Accepted on 18.08.2014 © RJPT All right reserved
Research J. Pharm. and Tech. 7(10): Oct. 2014 Page 1208-1219
The drug is official in Indian Pharmacopoeia (Indian
Pharmacopoeia 2007), British Pharmacopoeia (British
Pharmacopoeia 2005), and European Pharmacopoeia
(European Pharmacopoeia 2005).It is sparingly soluble in
water and ethanol, soluble in dimethyl formamide (DMF),
methanol and insoluble in chloroform and benzene. It is a
white yellowish crystalline powder; melting point 240°C;
administered orally. The dose of Ambroxol hydrochloride is
30-60 mg per day. The molecular weight of Ambroxol
Hydrochloride is 414.6, and their Pharmacological
properties namely surfactant stimulatory, anti-
inflammatory, and anti-oxidant and local anesthetic effects
in addition to the muco-kinetic and mucociliary effects of
the parent compound. Recognition of the surfactant
stimulatory and anti-inflammatory properties of the drug
has led to the resurgence of interest in the molecule in the
management of difficult to treat obstructive airway
disorders. (Gupta 2006)
Research J. Pharm. and Tech. 7(10): October 2014
1209
Figure-1. Structure of Ambroxol HCl.
The present review of literature stated that till date various
methods have been reported for the determination of
Ambroxol in biological samples and pharmaceutical
formulations. Techniques such as Electrochemical,
Spectrophotometry, High performance liquid
chromatography (HPLC), Ultrathin layer chromatography,
Gas chromatography, High-performance thin layer
chromatography (HPTLC) and Liquid chromatography–
mass spectrometry (LC–MS), from which HPLC methods
are used most extensively. Overview of the methods for
determination of Ambroxol shown in (Figure. 2).
2. SAMPLE PREPARATION:
2.1. Solubility
With reference to Biopharmaceutics Classification System
(BCS), classification of Ambroxol comes under BCS class-
I, it means Ambroxol has high solubility and high
permeability (Yazdanian et al. 2005; Polli et al. 2004). To
control the release of drug, various strategies like
hydrophilic matrix system (Shaikh et al. 2011) and
Microsphere (Kumar 2014) have been proposed . The
solubility of the drug was tested in solvents routinely used
for analytical methodology.
2.2. Sample preparation strategies
Sample preparation is an important part of analytical
methodology, and about approximately 30% error generated
in sample analysis was reported due to sample preparation
(Hendriks et al. 1996).(Figure. 3) shows various diluents
used for the analysis of Ambroxol. In major cases methanol
was used as a diluent. The sample preparation techniques
for the extraction of Ambroxol from biological matrices
(plasma, serum and urine) (Hu et al. 2008) include protein
precipitation, Liquid-Liquid Extraction had been chosen
among various techniques. The solvents like acetonitrile,
ethanol and n-Hexane etc. were tried alone as well as in
combination (Tompe et al. 2013).
3. ANALYTICAL METHODS
3.1. Electrochemical methods
Yuanzhe et al. constructed a new electrochemical method
for determination of Ambroxol HCl, the method was based
on MWCNT/ Nafion modified glassy carbon electrodes. In
cyclic voltammetry, the compound showed an irreversible
oxidation peak. It was compared with the results on bare
glassy carbon electrode and Nafion modified glassy carbon
electrode, and obtained significant improvements in the
sensitivity on MWCNT/Nafion modified glassy carbon
electrode. The differential pulse voltametric detection limits
were determined to be 3×10- 8 M, 4×10-9 M and 1×10-9 M on
bare glassy carbon electrode, Nafion modified glassy
carbon electrode and MWCNT/Nafion modified glassy
carbon electrode, respectively. At the optimized condition, a
sensitive linear differential pulse voltametric response range
for the Ambroxol was between 1×10-8 and 1.8×10-6 M on
MWCNT/Nafion modified glassy carbon electrode.
(Piao 2012)
Habib et al. constructed an electrochemical procedure for
the determination of Ambroxol in mucolytic. The method
was based on adsorptive accumulation of the species at the
hanging mercury drop electrode (HMDE), The behaviour of
adsorptive stripping response was studied by using various
experimental conditions, such as type of supporting
electrolyte, pH, accumulation time, pulse amplitude, scan
rate and mode of sweep. In Britton-Robinson buffer
solution, an irreversible reduction process involving transfer
of one electron and one proton was took place. The
response was linear over the 0.2-6 µ/ml concentration
range. The average of determinations obtained by the
square wave adsorptive voltametric method with its relative
standard deviation was 99.8 +/- 2.40%. (Habib et al. 2005)
Figure-2. Overview of analytical methods for estimation of Ambroxol HCl in biological and pharmaceutical samples.
Research J. Pharm. and Tech. 7(10): October 2014
1210
Figure-3. Various diluents used for the analysis of Ambroxol HCl.
3.2. Spectrophotometry
In the literature about 17 methods were reported for the
estimation of Ambroxol using Spectrophotometry (Akhtar
et al. 2013; Ponnilavarasan et al. 2011; Prasanthi et al. 2010
) of which 1method are for determining Ambroxol alone,
while the others are for quantifying Ambroxol in
combination with other drug substances. Table 1. shows
the summary of the reported spectrophotometric methods
indicating the basic principle, λ max, solvent and limit of
detection (LOD).
3.3. Capillary Electrophoresis (CE)
CE methods have excellent performance for separation of
pharmaceuticals, which makes it the first-choice technique
for separation of stereoisomer’s. For Ambroxol HCl
analysis few authors have used CE as a separation and
determination technique.
Tomas et al. constructed a sensitive capillary
electrophoretic method combined with laser-induced
fluorescence detection for the determination of Ambroxol
HCl. Samples were derivatized with 5·10−4Mfluorescein
isothiocyanate. A linear relationship between concentration
and peak area was in the concentration range 0.008–42 μg
ml−1 with a correlation coefficient of 0.9999. The method
was demonstrated their applicability to serum and urine
samples. (Tomas et al. 2000)
3.4. Chromatography
3.4.1. HPLC
3.4.1.1. Biological samples.
Various methods for the determination of Ambroxol in
biological samples like plasma, serum and urine(Suresh et
al. 2012; Dharuman et al. 2011 ) are listed in Table 2.
3.4.1.2. Pharmaceutical samples.
Analytical methods for the determination of Ambroxol HCl
in pharmaceutical dosage forms using HPLC (Mallapur et
al. 2011; Kotkar et al. 2012 )are shown in Table 3.
3.5. HPTLC
In the literature about 6 methods were reported for the
determining Ambroxol HCl alone, and in combination with
other drug substancesin pharmaceutical formulation and in
biological samples by using high performance thin layer
chromatography (Kasture et al. 2010; Bagada et 2013).The
HPTLC methods are shown in Table 4.
Research J. Pharm. and Tech. 7(10): October 2014
1211
Table 1. Representative spectrophotometric method for the analysis of Ambroxol Hydrochloride.
Compound Method λmax Solvent LOD(µg/ml) Ref.
Doxofylline(DOX) and
Ambroxol Hcl (AMB
HCl)
Simultaneous equation
Q-Absorption ratio method
274nm (DOX)
247nm(AMB)
Methanol 0.353(DOX)
0.157(AMB)
( Akhtar et al.
2013)
Ambroxol HCl and
Loratadine(LOR)
Simultaneous equation method
308nm(AMB)
245nm(LOR)
Methanol 2.31 (AMB)
0.482 (LOR)
(Ponnilavarasan
et al. 2011 )
Ambroxol HCl and
Guiaphensin(GUA)
Simultaneous equation method 242 (AMB)
272(GUA)
Methanol -------- (Prasanthi et al.
2010 )
Salbutamol (SAL) and
Ambroxol (AMB)
Simultaneous equation (I)
Area Under Curve method
223 nm (SAL)
244 nm(AMB)
Wavelength range
232
‐
217nm(SAL)
252-237nm(AMB)
0.1N HCL -------- (Patel et al.
2011)
Gatifloxacin(GFC)
andAmbroxol HCl
(AMB)
Simultaneous equations 286nm(GFC)
242 nm(AMB)
Methanol 0.65 (GFC)
1.37 (AMB)
(Prabu et al.
2010)
Ambroxol HCl,
Guaifenesin(GUA) and
Terbutaline
sulphate(TER)
Simultaneous equation,
Second order derivative,
Colorimetry
244.5nm(AMB)
279.4nm (GUA)
307.5nm(TER)
0.1NNaOH,
0.1N Hcl
Distilled
water
0.35 (AMB)
1.77 (GUA)
0.2(TER)
(Kimbahune
et al. 2011 )
Levocetirizine2. HCl
(LEVC) and Ambroxol
HCl (AMB)
Simultaneous equation,
Absorbance ratio analysis
Area under curvemethods
231 nm (LEVC)
245 nm (AMB)
228-234nm(LEVC)
242-248nm(AMB)
0.1M HCl 5.49(LEVC)
4.9(AMB)
(Patil et al. 2013)
Levofloxacin (LVF) and
Ambroxol (AMB)
Simultaneous equation method
(vierodt’smethod)
288 nm (LVF)
244 nm(AMB)
Distilledwate
r
0.6(AMB) (Chanda et al.
2013)
Salbutamol sulphate
(SAL) and Ambroxol
Hcl (AMB)
Simultaneous equation method
Area Under Curve method
224.5 nm (SAL) 244.5
nm (AMB)
229.5-219.5nm(SAL)
249.5-239.5nm(AMB)
Distilled
water, 0.1N
Hcl and
phosphate
buffer(pH
6.8)
--------- (Chitlange et al.
2011)
Ambroxol Diffuse reflectance
spectroscopy
520 nm(AMB) Methanol 145 μg/ml (Rufino et al.
2009)
Guaifenesin(GUA)
and Ambroxol HCl
(AMB)
Absorption ratio,
First order derivative
spectroscopy
238 nm (GUA)
255 nm (AMB)
223nm (λ1)
273nm(λ2)
Double
distill water
0.6 (GUA)
0.5 (AMB)
0.50 (GUA)
0.40(AMB)
(Deshpande et al.
2012)
Ambroxol (AMB)
Guaiphenesin (GUA)
and Levo Salbutamol
sulphate (L-SAL)
Four rapid derivative
spectrophotometric methods
252.2 nm(AMB)
282.6nm(GUA)
285nm(L-SAL)
Double
distill water
--------- (Thakkar et al.
2012)
Ambroxol HCl,(AMB)
Guaiphensin (GUA)
Cetirizine HCl (CTZ)
and Phenylephirine HCl
(PHE)
Multivariate analysis 246nm (AMB)
273 nm(GUA)
231 nm (CTZ)
274 nm(PHE)
0.1NHCl:
Methanol
(1:9)
1.01(AMB)
1.07 (GUA)
0.48 (CTZ)
2.11 (PHE)
(Arora et al.
2013)
Ambroxol HCl(AMB)
Guaifenesin(GUA) and
Loratadine(LOR)
Simultaneous equations 310 nm(AMB)
274 nm(GUA)
248 nm(LOR)
Methanol ------------ (Nalluri et al.
2013)
Paracetamol(PAR)
Ambroxol HCl (AMB)
Levocetirizine.2 HCl
(LCT)Phenylephrine
HCl (PHEP)
first-order derivative
spectrosphotometry
305.5 nm (PAR)
321 nm(AMB),
244 nm (LCTZ)
280 nm (PHEP)
Distilled
water
0.0352
(PAR)
0.0373(AM)
0.0645(LCT)
0.0557 PHEP)
(Kumar et al.
2014)
Ambroxol HCl(AMB)
Cetirizine Hcl(CTZ)
Absorbance ratio methods 243 nm (AMB)
229 nm (CTZ)
Methanol 0.0328(AMB)
0.0648 (CTZ)
(Bhatia et al.
2008)
Ambroxol HCl (AMB)
and Guaifenesin (GUA)
in presence of the
oxidative degradate
(AD) of AMB
andguaicol (GD), the
impurity of GUA
MultivariateSpectro-
photometric principal
component regression (PCR)
,Partial least squares (PLS)
246nm (AMB)
273 nm(GUA)
Methanol ----------- (Abdelkawy et
al. 2011)
Research J. Pharm. and Tech. 7(10): October 2014
1212
Table 2. Summary of HPLC methods to determine Ambroxol hydrochloride in biological sample.
Matrix Internal
Standard
Sample
preparation
Mobile phase Column Detectio
n
λ
max
(nm)
Flow rate
(ml/min)
Rt and
LOD
Ref.
Human
Plasma
Probenecid
liquid–liquid
extraction
MeCN: 30-
40%, buffer
conc.
Phenomene
xRC18
(150mm×4
.6mm,
5μm)
SPD
10A UV
detector
230 0.8-1.2 8.665 min (Suresh
et al.
2012)
Human
plasma
Nebivolol liquid–liquid
extraction
Acetonitrile–
phosphate
buffer 20 mM)
(20:80, %v/v)
(pH 3.5)
Acetonitrile:tri
ethylamin
(0.5%) in
phosphate
buffer 20 mM)
(55:45, v/v)
(pH 3.5)
Supelcosil
TM
LCABZ
(250 mm ×
4.6 mm, 5
μm)
Kromasil
C18
column
(250 mm ×
2.1 mm, 5
μm)
PDA
detector
230 0.5 7. 5min
11.5min
(Dharu
man et
al.
2011)
Human
plasma
Furosemide protein
precipitation
with methanol
0.15 M SDS,
8% npropanol,
0.3% TEA, in
0.02 M ortho-
phosphoric
acid (pH 4.0)
Spherisorb-
ODS 2 C
18
(150 mm ×
4.6 mm, 5
μm)
SPD
10A UV
detector
220 1.0 3.4 min and
0.26μg/mL
(LEV)
5.2 and
0.07
μg/mL
(AMB)
(Belal
et al.
2013 )
Rat
plasma
------ liquid–liquid
extraction
50mM pho-
sphate buffer
and methanol
(3:7 v/v)
Capcell
Pac C18
MG-
Shiseido
(150 mm ×
4.6 mm, 5
μm)
elector
chemical
detection
850
mv
0.2 ----- (Rupali
et al.
2012 )
Human
Serum
----- Protein
precipitation
Methanol:
distilled water
(containing
1% triethyl-
amine (50:50
%v/v)(pH 3.5)
protein-
coated µ-
Bondapak
CN silica
pre-column
(150 mm ×
4.6 mm, 5
μm)
SPD
10A UV
detector
254
nm
0.5 2.6min (Emara
et al.
2012)
Human
Plasma
------- liquid- liquid
extraction
Acetonitrile
and phosphate
buffer (0.1M)
in the ratio of
20:80 (v/v)
µbondapak
C18column
(300 x 3.9
mm, 10μ)).
SPD
10A UV
detector
254
nm
1.0 ------ (Singh
et al.
2014)
.
Table 3. Reported analytical HPLC methods for determination of Ambroxol HCl either alone or in combination with other drugs in
pharmaceutical dosage form.
Study aim Mobile phase Column Detection λmax
(nm)
Flow rate
(ml/ min)
Rt
(in min)
LOD/
LLOD
µg/ml
Ref.
Simultaneous
estimation of
Cefixime(CEF) and
Ambroxol HCl
(AMB)in tablet
Acetonitrile:methan
ol:0.5% ammonium
acetate buffer(pH
5.54)
(44: 16: 40 %v/v/v)
Phenomenex
-Gemini, RP-
18 column
(250 X 4.6
mm, 5µm)
SPD-M
20A PDA
220 1.0 2.18
(CEF)
4.32
(AMB)
3 (CEF)
0.8
(AMB)
(Mallapur
et al.
2011)
Cefpodoxime -
Proxetil(CFP)
andAmbroxol HCl
(AMB) in Bulk and
in Tablets
Acetonitrile: 0.025
M potassium di-
hydrogen-phosphate
buffer (70:30 v/v)
(pH 4.0with ortho-
phosphoric Acid).
IS- Diclofenac
sodium
Qualisil RP
C-8
(250 x 4.6
mm, 5 μm)
SPD-20A
UV
detector
248 1.0 3.89
(CFP)
2.69
(PRO)
IS- 5.52
0.18
(CFP)
0.09
(PRO)
(Kotkar et
al. 2012 )
Research J. Pharm. and Tech. 7(10): October 2014
1213
Simultaneous
Estimation of
Cefixime(CEF)and
Ambroxol HCl
(AMB) in Tablets
Acetonitrile:
Phosphate
Buffer(60:40 %v/v)
PH-7.0
Luna
Phenomenex
RP-C18
(250x 4.6
mm, 5 μm)
SPD-M
20A PDA
detector
230 1.0 2.75
(CEF)
5.01
(AMB)
0.1(CEF
) 0.15
(AMB)
(Kamarap
u et al.
2010)
Simultaneous
Estimation of (AMB)
Ambroxol HCl And
Cetirizine Hcl
(CTZ)In Tablet
Dosage Form
Methanol–Pota-
ssiumdihydrogen
phosphate buffer
80:20 (%v/v) (10
mM, pH 3.5 ± 0.02,
adjusted with ortho-
phosphoric acid)
Princeton C-
8
(4.6 ×
250mm, 5
µm)
UV 276 1.0 2.7
(AMB)
4.2 (CTZ)
0.60
(AMB)
1.5(CTZ
)
(Maithani
et al.
2010)
Simultaneous
estimation of
Ambroxol(AMB)
Chlorpheniramine
maleate(CHL) and
Phenylephrine Hcl
(PHE)inbulk and
liquid dosage form
Methanol :
Acetonitrile
(50%:50%) and
phosphate buffer 5
pH containing
0.75% TEA set by
formicacid(40%:60
%v/v)
C-18
(250 ×
4.6mm,
5μm)
UV 261 0.95 11.1
(AMB)
13.58
(CHL)
2.8
(PHE)
11.58
(AMB)
0.66
(CHL)
7.04
(PHE)
(Bagada
et al. 2014
)
Estimation of
Guaifenesin (GUA)
and Ambroxol HCl
(AMB) in Tablet
Acetonitrile: 50mM
potassium di-
hydrogen phosphate
buffer(22:78 v/v)
(pH -3.2 using
orthophosphoric
acid)
HiQSil C18
(250 X 4.6
mm, 5μm)
UV 262 1.0 7.5
(GUA)
9.5
(AMB)
-------- (Deshpan
de et al.
2012)
Simultaneous
Estimation of
Ambroxol
HCl(AMB) and
Loratadine(LOR) in
Tablet
0.02M Potassium
dihydrogenOrtho-
phosphate:
Acetonitrile (30:70
%v/v) ( pH-5 using
Ortho phosphoric
acid
Symmetry
C18
( 4.6 x
250mm,
5µm )
UV 245 1.0 2.25
(AMB)
7.58
(LOR)
3.18
(AMB)
2.87
(LOR)
(Sateesh
et al.
2013)
Simultaneous
estimation of
salbutamol sulphate
(SAL) and ambroxol
hydrochloride(AMB)
in pharmaceutical
dosage forms
15mM Ammonium
acetate:acetonitrile
(16:84%v/v) in
isocratic mode
Phenomenex
C18 RP
(250 x
4.6mm; 5
μm)
SPD-
M20A
PDA
detector
227 1.0 3.1(SAL)
4.5
(AMB)
0.021
(SAL)
0.034
(AMB)
(Nalluri et
al. 2013)
Simultaneous
Determination of
Gatifloxacin(GAT)
and Ambroxol
Hydrochloride
(AMB) in Tablet
0.1 M phosphate
buffer : Acetonitrile
(55:45 % v/v)
( pH 5.5)
Phenomenex
RP-C18
(250×4.6
mm, 5μm).
UV 254 1.0 2.2 (GAT)
4.5
(AMB)
----------- (Prabu et
al. 2010)
Simultaneous
analysis of
Levocetirizine2Hcl(L
CTZ),
AmbroxolHcl(AMB),
and montelukast
sodium(MONT)
15 mm of Ammo-
nium acetate:
Acetonitrile
(40:60%,v/v)in
isocratic mode
Phenomenex
RP-C18
(250×4.6
mm, 5μm).
UV 215 1.0 2.21
(LCTZ)
4.46
(AMB)
13.35
(MONT)
-----------
---
(Srividya
et al.
2013)
Simultaneous
Determination
ofCefiximeTrihydrate
(CEF) and Ambroxol
HCl(AMB) in
Pharmaceutical
Dosage Form
Acetonitrile:Methan
ol: Triethylamine
(50:50:0.1, %v/v/v)
pH 3.0
Phenomenex
RP-C18
(250×4.6
mm, 5μm).
UV 254 1.0 1.6(CEF)
3.7
(AMB)
0.615
(CEF)
1.86
(AMB)
(Kasture
et al.
2010)
Simultaneous
estimation of
Ambroxol
HCl(AMB)
Dextromethorphan
Hbr(DEX) and
Guaifenesin(GUA) in
pharmaceutical
cough cold
preparation
Acetonitrile
:Methanol :10mM
Phosphate Buffer in
0.3% Triethyl
Amine (25:15:60
%v/v) ( pH – 3 )
Hibar RP-
C18
(250 ×
4.6mm.,
5μm)
PDA 205 1.0 5.87
(AMB)
5.87
(AMB)
7 (DEX)
12.33
(GUA)
0.14
(AMB)
1.36
(GUA)
0.068
(DEX)
0.147
(AMB)
(Bagada
et al.
2013)
Research J. Pharm. and Tech. 7(10): October 2014
1214
Determination of
Bromhexine (BRO)
Guaifenesin (GUA),
Ambroxol (AMB)
Salbutamol (SAL)
/Terbutaline(TER)
Pseudoephedrine(PS
E) Triprolidine
(TRP)and
Chlorpheniramine
Maleate (CPM) in
Cough-cold syrups
Acetonitrile:0.25
MM sodium hexane
sulphonate:0.2 M
ammonium acetate
(35:4:10:51, %
v/v/v/v)
(pH 3.0-water)
RP-
Octadecylsil
ane
(250
X4.6mm,
5µm )
UV 254 1.0 3.0
(BROM)
3.5
(GUA)
4.3(SAL)
5.9(TER)
7.8(PSE)
9.4(TRP)
18.3
(CPM)
0.0013
(BRO)
0.0013
(GUA)
0.008
(SAL)
0.0014
(TER)
0.003
(PSE)
0.0017
(TRP)
0.011
(CPM)
(Njaria
2013)
Simultaneous
analysis of
Phenylephrine Hcl
(PHE), Guaiphenesin
(GUA), Ambroxol
HCl (AMB) and
Salbutamol(SAL)
Methanol-
Acetonitrile (1:1
%v/v)
(pH 3.0 phosphate
buffer)
Phenomenex
C8 column
250 × 4.6
mm, 5µm )
UV 273 1.0 ----------- 0.22
(PHE),
0.50
(GUA),
0.70
(AMB)
0.55
(SAL)
(JOshi et
al. 2011)
Simultaneous
Estimation of
Salbutamol(SAL)
Ambroxol(AMB) and
Guaifenesin (GUA)
in Tablet Dosage
Forms
Acetonitrile: pota-
ssium di-hydrogen
phosphate
(70:30 % v/v)(pH-
4)
SHISEIDO
C18 column
(250 x
4.6mm ,
5μm)
UV 215 1.2 2.57
(SAL)
7.1
(AMB)
5.85
(GUA)
4.28
(SAL)
1.23
(AMB)
3.56
(GUA)
(Kamatha
m et al.
2013)
Stability indicating
method for the
determination of
Terbutalinesulphate
(TER),
Guaifenesin(GUA)
Ambroxol
Hcl (AMB) and
preservatives(MP.PP)
content in liquid
formulations
Phosphate buffer :
Acetonitril(0.01M
Potassium
dihydrogen
orthophosphate
buffer pH (6.0± 0.1)
Sunfire C18
column,
(250
x4.6mm,
5Μm).
UV 214 1.2 3.68
(TER)
15.17
(GUA)
18.71
(AMB)
23.27
(MP)
24.38
(PP)
----------- (Bapatu et
al. 2011)
Ambroxol HCl
(AMB) Potassium
clavulanate (PC) and
Amoxicillin
trihydrate (AMX) in
bulk drugs and
laboratory prepared
synthetic mixture
Methanol: 0.01 M
phosphate buffer
(14:86%V/V) (pH
3.0)
Phenomenex
Luna C18
(250 mm×4
mm i.d.,
5μm)
UV 220 1.2
4.26
(AMB)
5.15 (PC)
6.25
(AMX)
0.264
( AMB)
0.504
( PC)
0.269
(AMX)
(Rajput et
al. 2014)
Simultaneous
estimation of
Ambroxol (AMB)
and
Desloratadine (DES)
in its pharmaceutical
dosage form
Acetonitrile: Ammo
nium acetate buffer
( 40: 60 %v/v)( pH -
3.5,adjusted with
0.01M glacial acetic
acid)
Inertsil
C18column
(4.6× 250
mm, 5 µm)
UV 252 1.0 4.35
(AMB)
6.132
(DES)
0.609
(AMB)
0.580
(DES)
(MOses et
al. 2013)
Determination of
Ambroxol
HCl(AMB)
in pharmaceutical
preparations
Acetonitril–Water
(60:40, %v/v)
(pH 3.5adjusted
orthophosphoric
acid
Princeton
SPHERC
(250 × 4.6
mm, 5µm)
UV 250 1.0 ---------- 0.001 (Muralidh
aran et al.
2013 )
Determination of
Ambroxol (AMB)
and
Gemifloxacin(GMF)
in tablet dosage
forms.
Sodium Phosphate
buffer: Acetonitrile,
(40:60%V/V)
HypersilC18
(4.6X250mm
, 5 μm)
UV 246 1.2 3.4
(AMB)
2.3
(GMF)
2.93
(AMB)
3.0
(GMF)
(Begum et
al. 2013)
Research J. Pharm. and Tech. 7(10): October 2014
1215
Estimation of (AMB)
Ambroxol HCl,
CetirizineHcl
(CTZ)in Combined
Dosage Form
Methanol:
Acetonitrile : water
(40:40:20 %v/v/v)
HIQ SIL-C
column
(2504X4.6
mm ,10 μm)
UV 229 1.0 10.495
(AMB)
4.155
(CTZ)
0.033
(AMB)
0.065
(CTZ)
(Bhatia et
al. 2008)
Determination of
ternary mixtures of
Ambroxol
HCl,Guaifenesin, and
Theophylline
0.05 M phosphate
buffer:methanol:ace
tonitrile:triethyl-
amine (63.5 : 27.5
:9 :0.25, v/v/v/v)
pH 5.5 adjusted
with
orthophosphoric
acid)
RP-C18
analytical
column
(4.6X250mm
, 5 μm)
220 1.2 -------- ----- (Abdelwa
hab 2012)
Determination of a
mixtureofAmbroxol
HCl (AMB) and
Guaifenesin (GUA)
in presence of the
oxidative degradate
(AD) of AMB and
guaicol (GD), the
impurity of GUA
Water: Methanol
containing 1% tri-
ethylamine(80:20,v/
v)
(pH 2.9)
Waters
Bondapack
C18 column
(4.6X250mm
,5 μm)
220 1.5 3.93
(AD)
8.44
(GD)
10.3±0.1(
GUA)
16.3± 0.1
(AMB)
----- (Abdelka
wyet al.
2011)
Table 4. Representative HPTLC methodsfortheanalysisof Ambroxol Hydrochloride.
Study aim Mobile Phase Detector λmax
(nm)
Retention
factor
(Rf)
LLOQ in
(µg/band)
Ref.
Bioanalytical method for
determination of (AMB)
Ambroxol HCl and (CEF)
Cefadroxil in human
plasma
Toluene : Methanol
(6:4% v/v)
UV
254
0.69(AMB)
0.18(CEF)
0.2(AMB)
(Suresh et al 2012)
Simultaneous estimation
of Ambroxol HCl(AMB)
Dextromethorphan
Hbr(DEX) and
Guaifenesin(GUA) in
cough cold preparation
Toluene: Methanol:
Chloroform:
Glacial Acetic Acid
(6.5:1.5:1.5:0.5
%v/v/v/v).
UV 275 0.056(AMB)
0.056(DEX)
0.612(GUA)
0.171(AMB)
0.171(DEX)
1.853(GUA)
(Bagada et al.2013 )
Simultaneous estimation
of Amoxicillin trihydrate
(AMX)and Ambroxol HCl
(AMB) in Pharmaceutical
Dosage Form
n-Butanol: 1.0 M
Ammonium
acetate: Methanol
(7.5:2.0:1.5)
(v/v/v)
UV 222
0.45 ± 0.02
(AMX)
0.68±0.02
(AMB)
(Dhaneshwar et al.
2011)
Stability-Indicating
HPTLC Determination of
Ambroxol HCl(AMB) in
Bulk Drug and
Pharmaceutical Dosage
Form
Methanol–
Triethylamine
(4:6 v/v).
UV 254 0.53 ±
0.02(AMB)
0.01(AMB)
(Jain 2010)
Simultaneous
determination of
Roxythromycin (ROX)
and Ambroxol
Hydrochloride (AMB) in
tablets
Benzene: Diethyl
ether:Triethylamine
( 4 :5: 1 v/v/v)
UV 365 (ROX)
255
(AMB)
0.95(ROX)
0.36(AMB)
0.48 (ROX)
0.45(AMB)
(Mulla et al. 2012)
Determination of
Ambroxol HCl and
Guaifenesin by TLC-
in Cough
Cold Formulations
Chloroform:
Methanol: Ethyl
acetate: acetic acid
(70: 8: 2:10v/v/v/v)
UV 270
0.14(AMB)
0.66(GUA)
0.79(AD)
0.88 (GD)
--- (Abdelkawy et al.
2011)
Research J. Pharm. and Tech. 7(10): October 2014
1216
3.6. Ultra Performance Liquid Chromatography
(UPLC) Method
Trivedi et al. (2011) constructed stability indicating
reversed phase ultra-performance liquid chromatography
(RP-UPLC) method for simultaneous determination of
Ambroxol hydrochloride (AMB), Cetirizine hydrochloride
(CTZ), Methylparaben (MP) and Propylparaben (PP) in
liquid pharmaceutical formulation. Chromatographic
separation was carried outby using Agilent Eclipse plus
C18, 1.8 μm (50 x 2.1 mm) column, at 237 nm detector
wavelength. The optimized mobile phase containing
mixture of 0.01 M phosphate buffer and 0.1 %
triethylamine as a solvent-A and acetonitrile as a solvent-B.
The developed method separates AMB, CTZ, MP and PP in
presence of twelve known impurities/degradation products
and one unknown degradation product within 3.5 min. At a
flow rate of 0.5 mL/min at 50°C (column oven)
temperature. Under the backpressure in the system was
about 6,000 psi. The LLOQ (µg/ml) were found to be 0.12,
0.18, 0.13 and 0.16 of MP, CTZ, PP, and AMB
respectively, and %RSD were found to be 3.5, 4.3, 5.7 and
4.8 of methyl paraben (MP), Cetirizine hydrochloride
(CTZ), Propylparaben (PP),and Ambroxol hydrochloride
(AMB) respectively.( Trivedi et al. 2011)
3.7. Gas Liquid Chromatography
Marucci et al. studied Ambroxol HCl in biological material
by using gas chromatography with electron-capture
detection. Pinazepam, was used as internal standard, at a
concentration of 5 ng/ml in methanol. The % Recovery was
found to be 100 ±2.3 % R .S.D. The limit of detection was 4
ng/ml of Ambroxol HCl for all the biological samples.
(Colombo et al. 1990)
3.8. LC–MS
Xin et al. developed a novel, rapid and sensitive LC-
MS/MS method operated in segmental and multiple
reactions monitoring for the simultaneous determination of
amoxicillin and Ambroxol HCl in human plasma.
Amoxicillin degrades in plasma at room temperature and
readily undergoes hydrolysis by the plasma amidase. The
degradation of amoxicillin in plasma was well prevented by
immediate addition of 20 μL glacial acetic acid to 200 μL
aliquot of freshly collected plasma samples before storage
at −80°C. The lower limits of quantitation of Ambroxol and
Amoxicillin were 0.5ng/mL and 5 ng/mL respectively.
Furthermore, the mass response saturation problem with
amoxicillin was avoided by diluting the deproteinized
plasma samples with water before injection into the LC-
MS/MS system. (Dong et al. 2013)
Kim et al. constructed a sensitive and selective liquid
chromatographic method coupled with tandem mass
spectrometry (LC-MS/MS) for the quantification of
Ambroxol HCl in human plasma. Domperidone was used as
internal standard, with plasma samples extracted using
diethyl ether under basic condition. A centrifuged upper
layer was then evaporated and reconstituted with 200 µl
methanol. The reconstituted samples were injected into a C-
18 XTerra MS column (2.1 x 30 mm) with 3.5 µm particle
size. The analytical column lasted for at least 600
injections. The mobile phase was composed of 20 mM
ammonium acetate in 90% acetonitrile (pH 8.8), with flow
rate at 250 µl/min. The mass spectrometer was operated in
positive ion mode using turbo electron spray ionization.
Nitrogen was used as the nebulizer, curtain, collision, and
auxiliary gases. Using MS/MS with multiple reactions
monitoring (MRM) mode, Ambroxol HCl was detected
without severe interferences from plasma matrix. Ambroxol
HCl produced a protonated precursor ion ((M+H)(+)) at m/z
379 and a corresponding product ion at m/z 264. And
internal standard (domperidone) produced a protonated
precursor ion ((M+H)at m/z 426 and a corresponding
product ion at m/z 174. Detection of Ambroxol HCl in
human plasma was accurate and precise, with quantification
limit at0.2 ng/ml. (Kim et al. 2003)
Wen et al. developed LC-MS/MS method for simultaneous
determination of amoxicillin and Ambroxol in human
plasma using Clenbuterol as internal standard (IS). The
plasma samples were subjected to a simple protein
precipitation with methanol. Separation was achieved on a
Lichrospher C(18) column (150 mm x 4.6mm ID, dp 5
microm) using methanol (containing 0.2% of formic acid)
and water (containing 0.2% of formic acid) as a mobile
phase by gradient elution at a flow rate of 1.0 mL/min.
Detection was performed using electron spray ionization in
positive ion multiple reaction monitoring (MRM) mode by
monitoring the ion transitions from m/z 365.9-->348.9
(amoxicillin), m/z 378.9-->263.6 (Ambroxol HCl) and m/z
277.0-->203.0 (IS). Calibration curves were linear in the
concentration range of 5-20,000 ng/mL for amoxicillin, and
1-200 ng/mL for Ambroxol HCl, with the intra- and inter-
run precisions of <9% and the accuracies of 100+/-
7%.(Wen et al. 2008)
Thummala.et al. studied the isolation, identification, and
characterization of Ambroxol HCl’s unknown impurity.
One unknown impurity of Ambroxol was formed in the
formulated drug under stress conditions (40°C /75% relative
humidity (RH) for 6 months) with the relative retention
time (RRT) 0.68 in RP-HPLC. The impurity was enriched
by exposing it to heat and it was isolated by using
preparative HPLC. The enriched impurity was purified and
characterized using the following sophisticated techniques:
2D NMR (gDQ-COSY, gHSQC, and gHMBC), FTIR, and
LC-MS/MS. On the basis of the spectral data, the impurity
was characterized as trans-4-(6,8-dibromoquinazolin-
3(4H)-yl)cyclohexanol.( Thummala.et al. 2014). The
comparative sensitive methods of various techniques are
shown in Figure. 4.
Research J. Pharm. and Tech. 7(10): October 2014
1217
Figure-4. Comparative sensitive methods of various techniques.
4. CHALLENGES:
Ambroxol HCl is used in preventing bronchial hyper-
reactivity, as well as decreased airway hyper-reactivity by
either increasing the lyso-phosphatidyl-choline turnover, or
modifying epithelial secretion. As discussed earlier,
Ambroxol HCl belongs to BCS class-I, it means Ambroxol
has high solubility and high permeability. However, for
successful treatment a constant and uniform supply of drug
is needed. Hence, it represents significant formulation
challenges. To overcome these problems and challenges an
important strategy is considered, hydrophilic matrices are
commonly used which achieves slow release of drug over
an extended period of time. The onset of its pharmacologic
action is often delayed and the duration of its therapeutic
effect is sustained. Hence, in development of hydrophilic
matrix, there is having chances to interference of excipients
to drug. Furthermore, it becomes necessary to analysis of
drugs, and selection of solvent is greater challenges for the
analysis. The review of literature reveals that most widely
used diluents are methanol and distilled water in HPLC
methods, which prolonged the run times with greater tailing
factor. It was observed that the drug gets slowly degraded in
acidic conditions over a period of time. For spectrometric
determination, the presence of multiple entities and
excipients includes complexity with multi- component
dosage forms, which could produce significant challenge to
the analytical chemist during the development of assay
procedure. Estimation of the individual drugs in these
multicomponent dosage forms becomes difficult. For such
instances like multicomponent dosage forms, chemo-metric
methods can be preferred to routine spectrophotometric
methods.
5. CONCLUSION:
The review is concluded that various methods such as
spectroscopy, chromatography, electrophoresis and
hyphenated technique are available for the quantification of
single and multicomponent dosage form of Ambroxol HCl
in biological fluids and pharmaceutical formulations. HPLC
was extensively used for the determination of Ambroxol
HCl in various matrices like plasma, serum and urine. For
determination of Ambroxol HCl in biological samples, were
commend the LC–MS/MS method, since this method
combines the LC separation ability with MS sensitivity and
selectivity, allowing the unambiguous identification of
Ambroxol HCl and its metabolites. For analysis of
Ambroxol HCl in pharmaceuticals, HPLC with UV
detection is applicable because this method provides
accurate results and low cost compared to more advanced
detection techniques. This review revealed an overview of
the recent state-of-art analytical methods for the
determination of Ambroxol HCl.
6. DECLARATION OF INTEREST:
There is no conflict of interest with any financial
organization regarding the material discussed in the
manuscript. The authors alone are responsible for the
content and writing of the manuscript.
Research J. Pharm. and Tech. 7(10): October 2014
1218
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