volume-5, issue-4, oct-2014 available online at www...

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

http://en.wikipedia.org/wiki/Antihistamine

http://en.wikipedia.org/wiki/Enantiomer


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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.

http://en.wikipedia.org/wiki/Carbon

http://en.wikipedia.org/wiki/Hydrogen

http://en.wikipedia.org/wiki/Bromine

http://en.wikipedia.org/wiki/Nitrogen

http://en.wikipedia.org/wiki/Oxygen


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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.


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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.


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The correlation coefficients are 0.9999 and 0.9997 for


Hydrochloride respectively. Therefore the HPLC

method for the simultaneous estimation of


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


Hydrochloride for analyst1 and 0.20 & 0.23 for


Hydrochloride for analyst2. Therefore, The HPLC

system for the simultaneous estimation of


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


Hydrochloride was shifted to 2.659 and 4.055 min


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


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chromatographic conditions.

Change in temperature:

When the robustness studies were carried out by

changing the temperature the retention time of


Hydrochloride was shifted to 2.606 and 4.023 min


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


Hydrochloride with an acceptable tailing factor at

300C. The results were comparable with the results of

optimized chromatographic conditions


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1118


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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

volume-5, issue-4, oct-2014 available online at www...

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