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Volume-5, Issue-4, Oct-2014
1108
Available Online at www.ijppronline.in
International Journal Of Pharma Professional’s
Research
Research Article
DEVELOPMENT AND VALIDATION OF RP-HPLC
METHOD FOR SIMULTANEOUS ESTIMATION OF
LEVOCETIRIZINE DIHYDROCHLORIDE AND
AMBROXOL HYDROCHLORIDE IN ORAL SYRUP
FORMULATION
Tholla haseena banu*, C.gopinath, vineet kumar,
dipankar karmakar, Achhrish goel
ISSN NO:0976-6723
Annamacharya college of pharmacy
Arvind Remedies Limited, Chennai
Abstract The present work describes a simple, precise and accurate RP-HPLC method for estimation of
Levocetirizine Di-Hydrochloride and Ambroxol Hydrochloride in Oral syrup dosage form. The separation
was achieved by using Sun fire column (C18) and mixture of acetonitrile and buffer in the ratio of 50:50
(ammonium dihydrogen phosphate dissolved in HPLC grade water pH 6.0 ±0.05 with Triethylamine as
mobile phase, at a flow rate of 1 ml/min. Detection was carried out at 230 nm. The retention time of
Levocetirizine Di-Hydrochloride & Ambroxol Hcl were found to be 4.239 &2.631min. The accuracy and
reliability of the proposed method was ascertained by evaluating various validation parameters like
linearity, precision, accuracy and specificity. The proposed method provides an accurate and precise
quality control tool for routine analysis of Levocetirizine Di-Hydrochloride & Ambroxol Hcl in Oral syrup
dosage form.
Keywords: - : Levocetirizine Di-Hydrochloride , Ambroxol HCL, Validation
Introduction High Performance Liquid Chromatography, or HPLC,
is the most common analytical separation tool and is
used in many aspects of drug manufacture and
research. HPLC is used for the Qualitative and
quantitative analysis of unknown mixtures –
determining what is there, and how much.Separation
of mixtures for later analysis.In this stationary phase
is less polar than the mobile phase and isusually
comprised of spherical silica particles (typically, 3–
5µm in diameter). Typicalmobile phases used in RP-
HPLC consist of mixtures of aqueous buffers
mixedwith water-miscible organic solvents, such as
methanol and acetonitrile. Inaddition to modified
silica stationary phases, other new developments
inRP-HPLC are now available, e.g. porous polymeric,
carbon and mixed mobile phases [1].
Incidence of allergic diseases such as allergic rhinitis
and asthma is increasing to epidemic proportions
(allergic rhinitis: 10-50%; and asthma: 5-15%), both
in the developed and the developing world, with a
histamine receptors [4].
reduced quality of life of the patients, lower
productivity and increasing medical costs. Rhinitis
frequently precedes asthma, and treating allergic
rhinitis has beneficial effects on asthma, suggesting
that upper airway disease is a risk factor for asthma.
[2]. Histamine plays a vital role in the allergic
immediate reaction. The biological effects of
histamine in the allergic reaction are mediated
through H1 receptors. H1 antihistamines work as
inverse agonists and suppress the effects of
histamine.levocetirizine is used to treat allergic
reactions and chronic idiopathic urticaria having
molecular formula C21H25ClN2O3.2HCl [3].
Levocetirizine di-hydrochloride is chemically 2-(2-
{4-[(R)-(4 chlorophenyl) (phenyl)methyl]piperazin-
1-yl}ethoxy)acetic acid is a third-generation non-
sedative antihistamine, developed from the second-
generation antihistamine cetirizine.Levocetirizine is
the active enantiomer of cetirizine. Levocetirizine is
non-sedative anti-histaminic and competitive
inhibitors of H1 receptors and acts by blocking
Volume-5, Issue-4, Oct-2014
1109
Ambroxol is mainly used to reduce bronchial hyper-
reactivity and acts as a mucolytic and cough
suppressant having molecular formula C13H18Br2N2O.
Ambroxol is chemically trans-4-(2-Amino-3,5-
dibrombenzylamino)-cyclohexanol [5]. Ambroxol is
one of the most popular medicines used to relieve the
symptoms of cough asthma and colds [6]. Ambroxol
is an active N-desmethyl metabolite of the mucolytic
bromhexine possesses mucokinetic (improvement in
mucus transport) and secretolytic (liquifies secretions)
properties. It promotes the removal of tenacious
secretions in the respiratory tract and reduces
mucusstasis (arresting the secretion of mucus) [7].
Ambroxol may stimulate the synthesis and secretion
of pulmonary surfactant; the drug has been referred to
as a “surfactant activator” [8]. It is administered as
the hydrochloride in daily doses of 30 –120 mg and is
available commercially as syrups, tablets and granules
similarly doses have been given by inhalation,
injection [9].
Syrup contains Levocetirizine di hydrochloride LCT
(antihistamines), Ambroxol hydrochloride (AMB)
(bronchosecretolytic and expectorants) and
Montelukast sodium. Combinations of these are not
available in market as only two each of the
combinations are available. Therefore the
simultaneous determination of these analyte’s
becomes motivating and significant. Literature
survey revealed that various analytical Methods like
HPLC, UV and HPTLC have been reported for the
determination of Ambroxol and Levocetirizine in
different formulation either individually or in
combination with some other drugs but for the syrup
it is not available. So our aim is to develop an
accurate, selective and precise simultaneous Method
for the estimation of Ambroxol Hydrochloride and
Levocetirizine in syrup formulation.
MATERIALS AND METHODS:
Chemicals and reagents:
Working standards of Levocetirizine and Ambroxol
were obtained from Shilpa Medicare lmt., Rayachur,
India. Water used was double distilled and filtered
through 0.45 µm filter. HPLC grade methanol,
acetonitrile were obtained from merck, Mumbai,
India. Ammonium dihydrogen phosphate and
Triethylamine are of analytical grade, obtained from
Merck, Mumbai, India.
Instruments:
Analysis was performed on Water alliance 2695
HPLC model (e series) containing Separator
module, Photodiode array detector waters 2996 and
variable wave length programmable UV/visible
detector waters 2489 and rheodyne injector (7725i)
with 100µl fixed loop. Chromatographic analysis was
performed using Sun fire C-18 column with 250mm
x 4.6mm internal diameter and 5µm particle size.
Sartorius electronic balance (CPA224S) was used for
weighing purpose. Empower 2 software was used for
handling the data.
Chromatographic conditions:
Isocratic mobile phase of acetonitrile and buffer in
the ratio of 50:50 (ammonium dihydrogen phosphate
dissolved in water pH 6.0 ±0.05 with
Triethylamine).Flow rate of mobile phase was set as
1 ml/min with detection wavelength as 230 nm.
Injection volume was maintained constant as 20 µl
mobile phases was filtered through 0.45µm
membrane filter and degassed using ultrasonicator
(pci), Mumbai.
Volume-5, Issue-4, Oct-2014
1110
Preparation of standard solution:
Standard Solution A:
Weighed accurately about 25 mg of Levocetirizine in
to 100 ml standard volumetric flask, Add 10ml of
methanol and sonicated for 5 minutes and then made
up to the volume with mobile phase.
Standard Solution B:
Weighed accurately about 30 mg of Ambroxol and
transferred in to 200 ml standard volumetric flask. To
it add 30 ml of methanol & 20 ml of solution A and
sonicated for 5 minutes. Then made up to volume
with mobile phase and mixed well.
Preparation of sample solution:
About 5 g of syrup was taken in a 100 ml volumetric
flask. Add 10 ml of methanol and sonicate to dissolve
for 5 minutes. Allowed to cool at room temperature
then made up to volume with mobile phase.
.
OPTIMIZATION OF CHROMATOGRAPHIC
METHOD:
All the compounds are subjected to analysis for
mobile phase of different pH under different
chromatographic conditions. The changes in
retention time of drugs were noted as a function of
changing in mobile phase, pH, strength and
selectivity .After completion of trials it was found
that ammonium dihydrogen phosphate buffer ( pH
6.0 adjusted with Triethylamine):acetonitrile in the
ratio of (50:50) with a flow rate of 1ml/min gives
good resolution and retention time.
Volume-5, Issue-4, Oct-2014
1111
METHOD VALIDATION
SPECIFICITY
The evaluation of the specificity of the method was
determined against placebo. The interference of the
excipients of the claimed placebo present in
pharmaceutical dosage form was derived from
placebo solution. Further the specificity of the
method toward the drug was established by means of
checking the interference of the degradation products
in the drug quantification for assay during the forced
degradation study. Prepare sample solution in
triplicate, sample solution spiked with known
impurities in triplicate and analyse as per testing
procedure above mentioned. Calculate the percentage
difference between the mean assay of spiked and
unspiked results with respect to the unspiked result.
LINEARITY
Linearity test solutions for the assay method were
prepared at five concentration levels from 80 to 120
% of assay analyte concentration (80, 90, 100, 110,
and 120 μg/ml. The peak areas versus concentration
data were evaluated by linear regression analysis.
PRECISION
The precision of the assay method was evaluated in
terms of repeatability by carrying out six independent
assays of Ambroxol & Levocetirizine test sample
preparation and calculated the % RSD of assay
(intraday). Intermediate precision of the method was
checked by performing same procedure on the
different day (interday) by different person under the
same experimental condition.
ACCURACY
An accuracy study was performed by adding known
amounts of Ambroxol & Levocitrizine to the placebo
preparation. The actual and measured concentrations
were compared. Prepare sample solution by spiking
the respective standard with placebo at 75%, 100%
and 125% of standard working concentration in
triplicate alternatively 90%, 100% and 110% is also
acceptable.Analyse as per testing procedure.
Calculate the percentage recovery and percentage
relative standard deviation (% RSD) at each level.
ROBUSTNESS OF METHOD
The robustness of study was carried out to evaluate
the influence of small but deliberate Variations in the
chromatographic conditions. The factors chosen for
this study were the flow rate (1.5 ml/min), mobile
phase composition [ammonium dihydrogen phosphate
buffer (pH 6.0 adjusted with Triethylamine):
acetonitrile in the ratio of (50:50) and using different
lot of Liquid chromatographic column.
RESULT AND DISCUSSION: -
SYSTEM SUITABILITY
System suitability test was carried out to verify that
the analytical system is working properly and can give
accurate and precise results. System suitability results
were tabulated in table no: 2-3.
The System suitability parameters are tailing
factor, theoretical plates, resolution, and % RSD
of number of injections are within the limits. So,
the system is suitable for all sample sequence and
conditions outlined in the method.
BLANK INTERFERENCE
Blank solution was prepared and injected. It was
observed that no blank peaks were eluting at the
retention time of Levocetirizine Di-hydrochloride and
Ambroxol Hydrochloride peaks.
PLACEBO INTERFERENCES
Placebo is spiked at their specification level with
known concentration of standard and sample solution
and unspiked sample solution and standard solution
was analyzed. It was observed that no placebo peaks
were eluting at the retention time of Levocetirizine
Di-hydrochloride and Ambroxol Hydrochloride in the
spiked standard and spiked sample solution and was
found to be within the acceptable limit as shown in
Fig no: 4-5. Results were tabulated in table no: 4.
LINEARITY
A series of Levocetirizine Di-hydrochloride and
Ambroxol Hydrochloride solutions were prepared in
the range of about 80 to 120% and injected into the
HPLC system. Linearity was established by plotting
graph of concentration versus response of
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride. Results are tabulated in table no: 5-6
and figure no. 8-9.
Volume-5, Issue-4, Oct-2014
1115
The correlation coefficients are 0.9999 and 0.9997 for
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride respectively. Therefore the HPLC
method for the simultaneous estimation of
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride in syrup formulation was found to be
linear.
System precision:
To evaluate system precision for Levocetirizine Di-
hydrochloride and Ambroxol Hydrochloride method,
five replicate injections were injected and analyzed.
System precision results were tabulated in table no: 7.
Chromatogram for system precision is shown in the
Fig no: 10.
Method precision
To evaluate the method precision for Levocetirizine
Di-hydrochloride and Ambroxol Hydrochloride
method six samples solutions were prepared as per
test procedure and analyzed. % recovery and % RSD
of six samples were calculated and found to be within
the acceptable limits. Method precision results were
tabulated in table no: 8-9. Chromatogram is shown in
Fig no: 11.
INTERMEDIATE PRECISION /RUGGEDNESS
The ruggedness of the method was performed by
changing the analyst, instrument, and column. The
retention time by analyst 1 was found to be 2.647 &
4.439 for Levocetirizine Di-hydrochloride and
Ambroxol Hydrochloride respectively and the same
was found to be 2.675 & 4.422 for Levocetirizine Di-
hydrochloride and Ambroxol Hydrochloride
respectively when performed by analyst 2 and the
results were found to be comparable. The % RSD for
six replicate injections was found to be within the
acceptable limits. Hence the developed method was
found to be sufficiently rugged. Results of
ruggedness are tabulated in table no-10
The % RSD was found to be 0.39 & 0.28 for
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride for analyst1 and 0.20 & 0.23 for
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride for analyst2. Therefore, The HPLC
system for the simultaneous estimation of
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride is precise.
RANGE
Based on the method precision, linearity and accuracy
data it can be concluded that the Levocetirizine Di-
hydrochloride and Ambroxol Hydrochloride method is
precise, linear and accurate in the range of and 80 to
120% of Levocetirizine Di-hydrochloride (120-180
µg/ml) and Ambroxol Hydrochloride (32-48 µg/ml).
LIMIT OF DETECTION & LIMIT OF
QUANTIFICATION:-
To establish the LOD and LOQ for Levocetirizine Di-
hydrochloride and Ambroxol Hydrochloride,
appropriate concentrations were injected. The signal
to noise ratio was found to be 3 and 10 respectively.
Chromatograms for LOD and LOQ were tabulated in
table no: 11.
ROBUSTNESS
Robustness indicates reliability of the procedure
during the normal usage. Robustness chromatograms
are shown in Fig no: 12-14. Results are tabulated in
table no: 12-17.
Change in flow rate:-
When robustness was carried out by changing the
flow rate, the retention time for Levocetirizine Di-
hydrochloride and Ambroxol Hydrochloride was
found to be 2.835 &4.321 for Levocetirizine Di-
hydrochloride and Ambroxol Hydrochloride
respectively with an acceptable tailing factor i.e. < 2
when analysis was performed at low flow rate
(1.3ml/min) and the same was shifted to 2.504 &
3.803 for Levocetirizine Di-hydrochloride and
Ambroxol Hydrochloride respectively with an
acceptable tailing factor when performed at high flow
rate (1.7ml/min).
Change in wavelength:
When the robustness studies were carried out by
changing the wavelength the retention time of
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride was shifted to 2.659 and 4.055 min
respectively with an acceptable tailing factor i.e. < 2
when the analysis was carried out at a wavelength of
about 228 nm instead of 230 nm and the same was
shifted to 2.660 and 4.066 respectively Levocetirizine
Di-hydrochloride and Ambroxol Hydrochloride with
an acceptable tailing factor at 232 nm. The results
were comparable with the results of optimized
Volume-5, Issue-4, Oct-2014
1116
chromatographic conditions.
Change in temperature:
When the robustness studies were carried out by
changing the temperature the retention time of
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride was shifted to 2.606 and 4.023 min
respectively with an acceptable tailing factor i.e. < 2
when the analysis was carried out at a temperature of
about 200C instead of ambient temperature and the
same was shifted to 2.833 and 4.463 min respectively
Levocetirizine Di-hydrochloride and Ambroxol
Hydrochloride with an acceptable tailing factor at
300C. The results were comparable with the results of
optimized chromatographic conditions
Volume-5, Issue-4, Oct-2014
1120
The % RSD was found to be within the acceptable
limits under modified parameters that are, by
changing the flow rate, changing the wavelength as
well as by changing the temperature. Thus the
method was concluded to be robust and not effected
by deliberate variations in method parameters.
REFERENCE:-
1. David c. lee et al., “Pharmaceutical Analysis”
Blackwell publication . Page no 44-45.
2. Ramalingam et al., “HPLC method for the
simultaneous determination of Levocetirizine,
Ambroxol and Montelukast in human Plasma
employing response Surface Methodology”
International Journal of Drug Development &
Research, ,Vol. 4 Issue 3,173-185.
3. Patel et al. “spectrophotometric determination of
Montelukast sodium and Levocetirizine di-
hydrochloride in tablet dosage form by AUC curve
method” Scholars Research Library, 3 (5): 135-140.
4.Prabhu et al., “simultaneous estimation of
gatifloxacin and ambroxol hydrochloride by uv-
spectrophotometry” International Journal of
Pharmaceutical Sciences Review and Research,
Volume 3, Issue 2, 123-126.
5. Reddy et al., “uv estimation of ambroxol
hydrochloride in bulk and pharmaceutical
formulations by simple visible spectrophotometry”
International Journal of Pharmaceutical Sciences
Review and Research, Article No. 04, 32-36.
6. Kimbhahune et al., “spectrophotometric
simultaneous analysis of ambroxol hydrochloride,
guaifenesin and terbutaline sulphate in liquid dosage
form (syrup)” international journal of pharmaceutical
sciences review and research, Article-004, 24-28.
7. Nagappan et al, Research J. Pharm. and Tech. 1(4),
ISSN 0974-3618, 366-369.
8. Prabhu et al., “ Simultaneous UV
Spectrophotometric Estimation of Ambroxol
Hydrochloride and Levocetirizine Dihydrochloride”
Indian J Pharm Sci, 70(2), 2008, 236-238.
9. Falgun et al., “simultaneous estimation of ambroxol
hydrochloride and doxofylline in pharmaceutical
formulation by hptlc-desitometric
method”Chromatography Separation Techniques,
volume 4,issue 3.
Correspondence Address:
Tholla haseena banu
Annamacharya college of pharmacy
E-mail- [email protected]
Phone: +919640757713