Utility of Schiff’s base Formation Reactions for the Spectrophotometric Determination of Pemetrexed Disodium in bulk and in its Pharmaceutical Formulations

P. Saifulla Khan*, P. Raveendra Reddy and V. Krishna Reddy

Department of Chemistry, SriKrishnadevaraya University, Anantapuramu - 515003.

*Corresponding Author E-mail: pskhan002@gmail.com

ABSTRACT:

Two simple, accurate, sensitive and extraction-free Spectrophotometric methods (A and B) have been developed for the determination of pemetrexed disodium in bulk and in pharmaceutical dosage forms. The methods A and B are based on the reaction of pemetrexed disodium with p-Dimethyl aminocinnamaldehyde (PDAC) and 4-Hydroxy-3-methoxybenzaldehyde (Vanillin) in the presence of dilute Sulphuric acid gives red colored chromogenes, which show maximum absorbance at 515nm and 470nm respectively. Beer’s law is obeyed in the concentration range 2.5-40µg mL-1 for method A and 2.5-35 µg mL-1 for method B.The molar absorptivity and sandell’s sensitivity values are 1.3 X104 Lit mo1-1cm-1 and 0.046 μg cm-2for method A and 1.5 x104 Lit mo1-1cm-1and 0.039 μg cm-2 for method B. The results of the two methods are validated statistically and by recovery studies. The proposed methods are economical, sensitive and can be used for the determination of pemetrexed disodium in bulk and pharmaceutical formulations.

KEYWORDS: UV-Visible Spectrophotometry;Pemetrexed disodium; p-Dimethylamino cinnamaldehyde (PDAC); 4-Hydroxy-3-methoxybenzaldehyde (Vanillin); Sulphuric acid; Methanol.

INTRODUCTION:

Pemetrexed disodium heptahydrate has the chemical name L- Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-disodiumsalt heptahydrate¹_,with a molecular formula of C₂₀H₁₉N₅O₆.7H₂O and a molecular weight of 597.49 gmol^-1. The basic structure of the drug is shown in Fig-1.

Fig 1 Structure of Pemetrexed disodium

Pemetrexed (Alimta; Eli Lilly, Indianapolis, IN) is a folate anti metabolite that primarily inhibits thymidylate synthase.^2-3 pemetrexed shows activity against a variety of solid tumor in clinical trails, that is non-small-cell lung^4-5and brest^6-7cancers. It also inhibits both dihydrofolate reductase and glycinamide ribonucleotide formyl transferase.⁸ Literature survey indicated that few analytical methods have been reported for the determination of pemetrexed disodium by HPLC⁹_, RP-HPLC¹⁰and LC^11-12_.Few Spectrophotometric methods were also reported for its validation in drug samples^9,13. Aromatic aldehydes (Vanillin, PDAC and PDAB etc) were employed as chromogenic reagents for developing spectrophotometric methods for the determination of different drugs containing amino group (both primary and secondary amines) producing Schiff’s bases or enamines.^14-18 In the present study, we are reporting simple, sensitive, accurate and economical Spectrophotometric methods A and B for the determination of pemetrexed disodium using p-Dimethylamino cinnamaldehyde (PDAC) and 4-Hydroxy-3-methoxybenzaldehyde (Vanillin).

MATERIALS AND METHODS:

Instrument: Shimadzu UV-1700 Pharmaspec with 1cm matched quartz cell was used for spectrophotometric measurements.

Reagents: All the chemicals used were of analytical grade.

P-Dimethyl amino cinnamaldehyde (PDAC), 4-Hydroxy-3-methoxybenzaldehyde (Vanillin):

5% solutions of these reagents were prepared by dissolving 5gm of each aldehyde in 100mL of methanol.

Experimental

A standard stock solution of the pemetrexed disodium was prepared by dissolving 10mg of the compound in 10mL of distilled water to get a concentration of 1000 μg mL^-1. This was further diluted with distilled water to get the working standard solution of 100 μg mL^-1_.

Assay procedure

Methods A and B:

Different aliquots of standard solution of pemetrexed disodium (0.5-5.5mL, 100µgmL^-1) were transferred into a series of 10mL volumetric flasks. To each flask, 1mL of p-Dimethyl amino cinnamaldehyde (PDAC) or 4-Hydroxy-3-methoxybenzaldehyde (Vanillin) (5%w/v) and 1mL of con H₂S0₄were added. After thoroughly shaking, the flasks were set aside for 10 minutes for the reaction to complete. The volumes in each flask were made up to the mark with methanol. The absorbances of the red colored solutions were measured at 515 nm for PDAC and at470nm for vanillin against reagent blank. The colored species of PDAC and Vanillin were stable for 60 and 90 minutes respectively. The amount of pemetrexed disodium present in the sample solutions were determined from the respective calibration curves.

The methods were extended for the determination of pemetrexed disodium in parental formulations (pemzem® 100mg, Dr.Reddy,s Laboratories, Hyderabad, India). It is supplied as a sterile lyophilized powder for intravenous available in single-dose vials. Each 100mg vial of pemzem® contains pemetrexed disodium equivalent to 100mg pemetrexed and 106mg mannitol. The total contents of vial were weighed and sterile powder equivalent to 10mg was dissolved in distilled water in a10mL volumetric flask. The above solution was further diluted and analyzed as described under the assay procedure for bulk samples. The analysis procedure was repeated five times to evaluate the precision.

RESULTS AND DISCUSSIONS:

Both PDAC and Vanillin form red colored chromo gens with pemetrexed disodium with λ_maxat 515nm and at 470nm respectively. They obey Beer’s law in the concentration range 2.5-40µg mL^-1and 2.5-35 µg mL^-1respectively. The colored Schiff’s bases formed between pemetrexed disodium and PDAC as well as Vanillin are shown in figures 2 and 3 respectively (Methods A and B).

Table 1 Optical characteristics and precision data

Parameters	Method A	Method B
λ_max(nm)	515	470
Beer’s law limits (μgmL^-1)	2.5-40	2.5-35
Molar Absorptivity (Lt mole^-1cm^-1)	1.2966x10⁴	1.5117x10⁴
Sandell’s Sensitivity (µg cm^-2/0.001 absorbance unit)	0.046	0.0395
Regression Equation* (Y= mx +c) Slope(m) Intercept(c)	0.023 -0.031	0.027 -0.019
Correlation Coefficient (r)	0.998	0.999
Precision(% RSD)	0.450	0.280

In both the methods, systematic and detailed study on the various parameters such as reaction time, effect of reagent concentration and solvent’s effects on reaction were carried out and the optimum conditions were established. The optical characteristics such as absorption maxima, Beer’s law limits, molar absorptivity, Sandell’s sensitivity, the regression analysis using the method of leastsqure and the correlation coefficient of the regression plots were evaluated and summarized in Table-1.

The reproducibility and precision of the methods are very good as shown by the corresponding RSD values. To assess the applicability of the proposed methods, they were employed for the determination of pemetrexed disodium in pemzem® 100mg commercially available tablets from local pharmacy. The results obtained in the studies are presented in the Table-2.

The recovery percentage of the drug was evaluated by spiking different known amounts of pemetrexed disodium in to the drug sample and determining their amounts by the proposed methods. In all the methods 99.0 to 100.0% recoveries were achieved which indicates the selectivity of the proposed methods.

Table 2 Assay of pemetrexed disodium in pemzem® 100mg formulations

S.No	Labeled Amount(mg)	*Amount obtained by proposed method (mg)		** % Recovery by the proposed method
S.No	Labeled Amount(mg)	Method A	Method B	Method A	Method B
1	100	98.77	99.74	99.81	100.14
2	100	99.65	99.12	98.94	99.84
3	100	98.62	99.58	99.18	99.25

* Average five determinations. ** After spiking the sample.

CONCLUSION:

The proposed methods are simple, extraction-free, sensitive, accurate and economical for routine analysis of pemetrexed disodium in bulk and in its pharmaceutical formulation. Based on molar absorptivity data and Beer’s law range, it may be concluded that among the proposed methods, method B is more sensitive than method A.

ACKNOWLEDGEMENT:

The authors acknowledge M/s Vishnu Chemicals Limited, Hyderabad for the supply of pemetrexed disodium as gift sample.

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Received on 22.11.2013 Modified on 07.12.2013

Asian J. Research Chem. 7(3): March 2014; Page 323-326