IJRPC 2015, 5(4), 641-644 Patel Faijal et al.

ISSN: 22312781

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND CHEMISTRY

Available online at www.ijrpc.com Research Article

AUV-SPECTROPHOTOMETRIC DETERMINATION OF
METHOTREXATE IN TABLET DOSAGE FORM
Patel Faijal*, Shelke Maroti and Suryawanshi Samrat
Padm.Dr. D. Y. Patil College of Pharmacy, Akurdi, Pune-411044, Maharashtra, India.

ABSTRACT
Two simple, precise and economical UV methods have been developed for estimation of Methotrexate
in bulk formulation. Method A involves measurement of UV absorbance in zero order derivativeat
303nm. Method B deals with area under curve measurement (AUC method), which involves the
calculation of integrated value of absorbance with respect to wavelength between 294-308nm. The
drug follows Beer-Lambert’s law in the concentration range of 3-10μg/ml in both the methods. Results
of analysis were validated statistically and were found to be satisfactory. Thus proposed methods can
be successfully applied for estimation of Methotrexate in routine analytical work.

Keywords: Methotrexate, Zero order derivative, Area Under Curve method (AUC).

INTRODUCTION Stock solution

Methotrexate is described chemically L- Accurately about 5 mg of Methotrexate was
Glutamic acid, N-{4-[[(2,4-Diamino-6- weighed and transferred to 50 ml volumetric
pteridinyl)methyl]methylamine]benzoyl}-, flask; 10 ml of 0.1N NaOHwas added to
Folex: Methotrexate; Mexate.It is a class of dissolve the drug completely with vigorous
anticancer drug. It is abbreviated MTX and as shaking. Then the volume was made up with
amethopterin is antimetabolite and antifolate the distilled water up to the mark to give the
[1-3]
drug. The drug is official in Indian drug stock solution of concentration 100μg/ml.
[4] [5] [6]
pharmacopoeia ,USP and BP. Literature
survey reveals that there are few UV Method A
[7-9] [10]
Spectroscopic methods and one HPLC The zero order derivative spectra at
method is reported for the determination of n=0showed a sharp peak at 303 nm (Figure
methotrexate in plasma and urine of humans, 1). The absorbance difference at n=0
rats and dogs. So an attempt was made to (dA/dλ)was calculated by the inbuilt software
develop two simple, accurate, rapid and of the instrument which was directly
precise spectrophotometric methods for the proportional to the concentration of the
determination of Methotrexate in tablet and standard solution.The standard drug solutions
formulation. were scanned in the zero order derivative
spectra.A calibration curve was plotted taking
EXPERIMENTAL the absorbance difference (dA/dλ) against the
MATERIALS concentration of Methotrexate. The coefficient
Methotrexate was obtained as gift sample from of correlation (r2), slope and intercept values of
Matrix Ltd. Methanol AR grade and 0.1N this method are given in table 1.
NaOHwere used as a solvent in the study.
Method B
INSTRUMENT The AUC (area under curve) method involves
A shimadzu UV-1700 UV/VIS the calculation of integrated value of
Spectrophotometer was used with 1cm absorbance with respect to the wavelength
matched quartz cells were used for spectral between two selected wavelengths λ1 and λ2.
measurements. Area calculation processing item calculates
the area bound by the curve and the horizontal
axis. This wavelength range is selected on the

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IJRPC 2015, 5(4), 641-644 Patel Faijal et al. ISSN: 22312781

basis of repeated observations so as to get the calculated as shown in table 2. All the
linearity between area under curve and methods were validated for linearity, accuracy
concentration. Suitable dilutions of standard and specificity.
stock solution (100μg/ml) of Methotrexate
were prepared and scanned in the spectrum RESULT AND DISCUSSION
mode from the wavelength range 400nm to The methods A and B for the estimation of
200nm (figure 2) and the calibration curve was Methotrexate in tablet form were found to be
plotted as AUC against concentration of simple, precise, accurate, rapid &
Methotrexate. The method was checked by reproducible. Beer- Lambert’s law was obeyed
analyzing the samples with known in the concentration range of 3-10μg/ml in both
concentration. As the results obtained were the methods. The values of standard deviation
satisfactory low, the method was applied for were satisfactory low and the recovery studies
pharmaceutical formulations. were close to 100%.The derivative
spectroscopic method applied has the
Analysis of tablet formulation advantage that it locates the hidden peaks in
For the estimation of Methotrexate in tablet the normal spectrum when the spectrum is not
formulation by the two methods, twenty sharp and it also eliminates the interference
tabletswere weighed and ground into a fine caused by the excipients present in the
powder. Tablet powder equivalent to 2.5 mg of formulation. The AUC method has advantage
Methotrexate weighed and transferred to 25 ml that it is applicable to be drug which shows the
volumetric flask and dissolve in 10 ml 0.1N broad spectra without a sharp peak. Hence the
NaOH. It was kept for ultra sonification for 45 two methods can be employed for routine
min, finally the volume was made up to the analysis of the drugs in quality control, R&D
mark with 0.1N NaOH, this was then filtered laboratories.
through Whatman filter paper to get tablet
stock solution of concentration to 100μg/ml. CONCLUSION
Various dilutions of the tablet solution were An accurate and precise zero order derivative
prepared and analyzed for six times and and AUC method have been developed and
concentration was calculated by using evaluated for the analysis of methotrexate
calibration curve for the two methods. Both the using (0.1N) NaOH as solvent. The
methods were validated according to percentage recovery and obtained
[11]
ICHguidelines . Recovery studies were concentrations of active ingredient where
carried out atthree different levels i.e. 80%, within the acceptable limits. These methods
100% and 120% by adding the pure drug (4, 5 can be used for the estimation of methotrexate
and 6mg respectively) to previously analyzed in bulk and formulation for quality control
tablet powdered sample (2.5mg) as per ICH studies.
guidelines[12] and percentage recovery was

Table 1: Optical characteristics and parameters

Parameters Method A Method B
Wavelength(nm)(λ Max) 303 294-308
Beer’s – Lambert’s range (µg/ml) 3-10 3-10
2
Coefficient of correlation (r 0.9971 0.981
Regression equation : Y = mx + c
a – Slope (m) 0.052 0.635
b – Intercept (c) 0.015 0.457
LOD 8.730 8.5011
LOQ 28.57 28.3370
Molar absorptivity 0.04990 0.7203

Table 2: Assay of the Tablet

Tablet Label Amount found
Method % mean S.D. R.S.D. S.E.
Formulation claim(mg) (mg)*
A T1 2.5 2.45 98.0 1.2296 1.2546 0.5020
B T1 2.5 2.32 92.8 3.2936 3.5491 1.3446

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IJRPC 2015, 5(4), 641-644 Patel Faijal et al. ISSN: 22312781

Table 3: Recovery Studies

Level of S.E.*
Tablet Mean* S.D.* R.S.D.*
S. No. recovery
Sample
% A B A B A B A B
01 80 98.3 99.2 0.5 2.000 0.5086 2.016 2.886 1.154
02 T1 100 97.6 98.2 1.3 1.1150 1.3319 1.1354 0.7505 0.6437
03 120 96.7 97.66 1.8 0.5507 1.8650 0.5638 1.0400 0.3179
When *n=3 at each level of recovery

Fig. 1: Spectrum by Zero order derivative method

Fig. 2: Spectrum by AUC method

IJRPC 2015, 5(4), 641-644 Patel Faijal et al. ISSN: 22312781

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