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MEBENDAZOLE IN API AND PHARMACEUTICAL FORMULATIONS

RP-HPLC METHOD DEVELOPMENT AND

VALIDATION FOR QUANTIFICATION OF
MEBENDAZOLE IN API AND
PHARMACEUTICAL FORMULATIONS

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ABOUT AUTHORS
Durgesh Rameshlal Parakh1*, Jayshri K. Madagul1, Harshad Rajendra Mene2, Moreshwar P.
Patil1, Sanjay J. Kshirsagar1
1
Department of Pharmaceutics,
MET’s Institute of Pharmacy, Adgaon, Nashik, Maharashtra, India.
2
Department of Pharmaceutics,
Government College of Pharmacy, Osmanpura, Aurangabad, Maharashtra, India.
* aryanparakh57@gmail.com (mailto:aryanparakh57@gmail.com)

ABSTRACT
A new simple and sensitive RP-HPLC method was developed and validated for quantification of Mebendazole in
Active Pharmaceutical Ingredient (API) and pharmaceutical formulation.The gradient RP-HPLC method was
developed on Agilent (India) C18 250 × 4.6 mm, 5 μ column using mobile phase as acetonitrile: water pH 3.0
with orthophosphoric acid (90:10 v/v) at a flow rate of 1 mL/min and detection was carried out at 234 nm

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using UV-Visible detector (UV 3000 M). The method was validated linearity, limit of detection, limit of
quantification, precision, ruggedness, robustness, accuracy and specificity were found to be satisfactory. The
method was found to be linear in the concentration range of 20-100 μ/mL with correlation coefficient of 0.999.
The method was validated according to the ICH guidelines and was proved to be specific, linear, accurate,
precise and economical for the analysis of Mebendazole.

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Meer informatie

REFERENCE ID: PHARMATUTOR-ART-2409

PharmaTutor (Print-ISSN: 2394 - 6679; e-ISSN: 2347 - 7881)

Volume 4, Issue 5

Received On: 21/12/2015; Accepted On: 04/01/2016; Published On: 01/05/2016

How to cite this article: Parakh DR, Madagul JK, Mene HR, Patil MP, Kshirsagar SJ; RP-HPLC
method development and validation for Quantification of Mebendazole in API and Pharmaceutical
Formulations; PharmaTutor; 2016; 4(5); 46-51

INTRODUCTION

Figure 1. Chemical structure of Mebendazole

Mebendazole, methyl (5-benzoyl-1H-

benzimidazol-2yl) carbamate [1 ] (Figure 1), is
broad spectrum anthelmintic agent[1 ]. It is active
against nematodes infections like whipworm
(Trichuris trichura), threadworm (Enterobius
vermicularis), roundworm (Ascaris lubricoides),
hookworm (Ancylostoma duodenale, Necator americanus), etc[1 ]. It is BCS class II drug having low aqueous
solubility (71.3 mg/L) and High permeability (Log P = 2.8) which may leads to variable absorption of
mebendazole [1 ]. It undergo extensive hepatic first pass metabolism [2 ]. It has very low bioavailability (5-10%)
and maximum amount of drug is protein bound (90-95%) [1 - 2 ].

Analysis of the drug is the most important for the formulation development. Hence there is a need to develop
a validated method for the analysis of Mebendazole from API and pharmaceutical formulations. It is white to
slightly yellow, amorphous powder, almost odourless. It is practically insoluble in water, alcohol, methylene
chloride, ether, chloroform and in dilute mineral acids whereas freely soluble in formic acids [1 - 3 ]. It irreversibly
block glucose uptake in susceptible helminthes, thereby depleting glycogen stored in parasites [1 - 3 ]. It is
official in Chin., Eur., Int., US, British, Indian, and Viet. Pharmacopoeia [4 ]. It is available as chewable tablet,
suspension and sachets in market [4 ]. It is given as 100 mg twice daily for 3 consecutive days. Repeated after
2-3 weeks if needed (to kill ova developed later) [3 ]. The estimation of Mebendazole in bulk and in tablet
dosage form was done by UV [5 - 6 ] and RP-HPLC method in alone or combination with forced degradation
studies [7 - 1 4 ]. There are many reported methods for analysis of Mebendazole either alone or in combination
with other drugs in pharmaceutical dosage forms or individually in biological fluids. The quantification of
Mebendazole in human plasma by LC-MS has been reported The estimation of Mebendazole in bulk and in
tablet dosage form was done by UV and RP-HPLC method in alone or combination with forced degradation
studies [7 - 1 4 ]. There is a need for a simple, rapid, cost effective and reproducible method for development of
Mebendazole. Therefore objective of the study was to develop a simple, accurate, precise, cost effective and
reproducible RP-HPLC method for estimation of mebendazole as per International Conference on
Harmonization (ICH) guidelines Q2(R1) [1 5 ].

MATERIALS AND METHODS

Materials
Mebendazole was gifted from Holden Pharmaceuticals (Nashik, India). Methanol was purchased from Merck
(Mumbai, India). The aceotonitrile (ACN) was obtained from Fischer scientific co. (Mumbai, India). Aerosil 200
was purchased from Research lab fine chem Industries (Mumbai, India). The HPLC grade water was produced
by double distillation method in laboratory. Membrane filters of 0.45 μ was of Millipore pvt. Ltd. (Bangalore,
India). All the chemicals and reagents used were of analytical grade.

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Equipment
It consist of RP-HPLC (Agilent, India) equipped with binary pump (P-3000 M, reciprocating), UV/Visible detector
(UV 3000 M) and Rheodyne injector port with 20 μL loop. The output signal was monitored and processed
using HPLC Workstation software. Greece C18 column (250 × 4.6 mm; 5μ particle size) was purchased from
Phenomenex (USA).

Method Methods Method New Method Volume

EXPERIMENTAL

Method Development

Solvent Selection
Various solvents like water, methanol, chloroform, acetonitrile were selected for the solubility and stability
study and it was found that Mebendazole was soluble in the following solvents; formic acid, N, N-dimethyl
formamide, 0.5 M methanolic hydrochloride, etc.In the present investigation 0.5 M methanolic hydrochloride
was selected as a solvent.

Chromatographic conditions
Chromatographic analysis was performed on HPLC column at 40 o C. The mobile phase consisted of ACN: Water
pH 3.0 with orthophosphoric acid (90:10 v/v). The mobile phase was degassed in an ultrasonic bath (PCI
analytics, India).

Preparation of standard solution

Standard stock solution was prepared by dissolving accurately weighed 10 mg of mebendazolein 50 mL of
solvent (0.5 M methanolic hydrochloride). Keep it on sonicator bath for 15 min. After 15 minutes, the volume
was made up to the mark with 100 mLvolumetric flask with same solvent to give a solution of 100 mg/mL.

Analysis of formulation
Weight equivalent to 10 mg was weighed accurately. Transferred it to 100 mL volumetric flask, dissolved in 50
mL of 0.5 M methanolic hydrochloride by ultrasonication of the flask for 15 minutes and volume was made up
to the mark with respective solvent.

Validation of the method [15]

System suitability
The system suitability was determined by calculating retention time, no. of theoretical plates (T. plate no.) and
tailing factor (asymmetry). It was resulted from HPLC method analysis of Mebendazole.

Linearity and range

The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are
directly proportional to the concentration (amount) of analyte in the sample. The range of an analytical
procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample
(including these concentrations) for which it has been demonstrated that the analytical procedure has a
suitable level of precision, accuracy and linearity.
The linearity of the analytical method was demonstrated over the concentration range of 20-100 µg/mL of the
target concentration for both the drugs. Aliquots of 2, 4, 6, 8 and 10 mL were pippeted out from working
standard solution into series of 100 mL volumetric flasks. The volume was made up to the mark with 0.5 M
Methanolic Hydrochloride. The calibration curve was obtained by plotting peak area versus concentration.

Limit of Detection (LOD)

The detection limit is the lowest amount of analyte in a sample which can be detected but not quantitated.

LOD = 3.3 × σ/S

Where,
σ = standard deviation of response and S = slope of calibration curve

Limit of Quantification (LOQ)

The quantitation limit is the lowest amount of analyte in a sample which can be quantitatively determined.

LOD = 10 × σ/S

LOD = 3.3 × σ/S

Where,
σ = standard deviation of response and S = slope of calibration curve

Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a
series of measurements obtained from multiple sampling of the same homogeneous sample under the
prescribed conditions. Precision may be considered at three levels: repeatability (intra-day), intermediate
precision (inter-day)and reproducibility. In order to determine the repeatability of method, three different
concentration solutions of 48, 60, 72 µg/mL of mebendazole solution were prepared in triplicate (n = 3). The
intermediate precision was determined by analyzing 3 samples (n = 6) of 60 μg/mL on three consecutive days.
Recovery (%), relative standard deviation i.e. RSD (%) and standard error (SE) were calculated.

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Ruggedness
The ruggedness is degree of reproducibility of test results under verify of condition like different analysts,
different instruments and different days.
To establish ruggedness of the proposed method the solutions of 60 µg/mL of Mebendazole standard solution
was prepared and analyzed with the change in the different analyst.

Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides an indication of its reliability during normal usage.
The robustness of the proposed method the solutions of 60 µg/mL of mebendazole standard solution was
prepared and analyzed by changing flow rate. The flow rate was selected as deviated to normal flow rate ±
0.1 mL/min i.e. 0.9 and 1.1 mL/min for Mebendazole standard solution.

Accuracy
The value which is accepted either as a conventional true value or an accepted reference value and the value
found. This is sometimes termed trueness. Accuracy of proposed method was ascertained on the basis of
recovery study. Recovery studies were carried out by spiking standard working solution to preanalyzed sample
solution (formulation) at three different levels 80 %, 100 % and 120%. Each sample was prepared in triplicate
(n = 3) and all the samples were analyzed by the proposed method. Recovery (%), relative standard deviation
i.e. RSD (%) and standard error (SE) were calculated.

Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be
expected to be present. Typically these might include impurities, degradants, matrix, etc.

Specificity was done by using an excipient, Aerosil 200 (adsorbent). The three different concentrations of
standard solution at three levels 80 %, 100 %, 120 % respectively was spiked on Aerosil 200 concentration
(60 µg/mL). At each levels of the amount, three determinations were performed to check effect of Aerosil 200.
Recovery (%), relative standard deviation i.e. RSD (%) and standard error (SE) were calculated.

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