Spectrophotometric determination of Methyldopa in pure form and in the pharmaceutical preparations

A simple, rapid and sensitive spectrophotometric method for determination of methyldopa (MD) using 4-chloro-7-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) as reagent in an alkaline medium (pH 12.3). Absorbance of the resulting brown-colored product is measured at 470 nm. Beer’s Law is obeyed in a concentration range of (1.6-17.6 µg/mL) with molar absorptivity (1.9337×10 4 L/mol.cm), correlation coefficient 0.9988, and the limit of detection (5.536×10 -3 µg/mL). The method has been successfully applied to the determination of Methyldopa in pharmaceutical preparations.


Material and Methods
Apparatus UV-VIS spectrophotometer single beam from A&E Lab (UK) -S60-Series with 1 cm quartz cells, pH meter from (Senz pH tester, China), Balance from Mettler AB 104-S (Switzerland).

Procedure for stoichiometric ratio
The reaction stoichiometry between the studied drug and NBD-Cl has been determined spectrophotometrically by applying molar ratio and continuous variation methods.In the former method, equimolar solutions of (MD) and NBD-Cl (2×10 -3 M) were used.Different aliquots of NBD-Cl were added to fixed aliquots of drug solution -total volume (25 ml) and the absorbance were measured at 470 nm against the reagent blank treated similarly.While in the latter method, a series of MD-NBD-Cl solutions were kept at (5 ml) (0:5, 0.5:4.5, 1:4, 1.5:3.5, 2:3, …… 5:0).The absorbance of the resulting solutions were measured at 470 nm against the reagent blank treated similarly.

Results and Discussion
Absorption spectra of MD-NBD-Cl system against reagent blank in an alkaline medium at room temperature (25 o C) were produced brown colored product which absorbs maximally at 470 nm, the result shown in Figure (3), and reagent blank against ethanol, the result shown in Figure (4).

Optimization of reaction variables
In order to establish optimum experimental conditions, necessary for rapid and quantitative formation of colored product with maximum stability and sensitivity, the effect of various parameters such as volumes of NBD-Cl, in addition of alkaline medium, the reaction time and the stability of colored product were studied at room temperature (25 0 C).

Effect of NBD-Cl concentration
The effect of NBD-Cl concentration on the reaction were studied at room temperature (25 ± 5°C).The reaction of (MD) with NBD-Cl were dependent on the concentration of NBD-Cl reagent.So, the reagent concentration in solution were studied by varying the NBD-Cl volume of (8×10 -3 M) NBD-Cl, while the (MD) concentration were maintained constant at 8 μg/mL.The study revealed that the reaction were dependent on concentration of NBD-Cl reagent.The highest absorption intensity were attained when the volume of NBD-Cl were (1.5 ml, 4.8×10 -4 M) of (8×10 -3 M) NBD-Cl, and decrease in the absorbance at volume large than 1.5 ml of NBD-Cl, the result shown in Figure (5).

Effect of pH
An alkaline medium was necessary, since the results revealed that (MD) does not react with NBD-Cl in acidic media, the results revealed that the absorbances at pH < 8 were close to 0, indicating that under acidity, (MD) has difficulty to react with NBD-Cl.Different concentrations from NaOH were tested, Best results were obtained in the case of higher concentrations of NaOH (1M), the result shown in Figure (7).

Effect of Time
Under the above described optimum conditions, the absorbance-time curve for the reaction of (MD) with NBD-Cl in alkaline medium were constructed, and the product remained stable for (3h.), the result shown in Figure (8).

Stoichiometry of the reaction
Under the optimum conditions, (cons. of NBD-Cl, pH, temperature, time) the stoichiometry of the reaction between (MD) and NBD-Cl were investigated by mole-ratio and continuous variation methods (23) .The stoichiometric ratio between NBD-Cl and (MD) was found to be 1:1, the results shown in Figures (9, 10).

Calibration curve
The calibration curves for (MD) pure form through complexation with NBD-Cl showed excellent linearity at concentration ranges of (1.6-17.6 μg/mL).The result shown in Figure (11).

Construction of calibration curves
Calibration curves were constructed according to the optimum conditions in

Application of the method
Twenty tablets from each one of the pharmaceutical preparations were weighed and average weight were calculated.Tablets were crushed into fine powder.An accurately weighed quantity of powder equivalent to 250 mg of (MD) were transferred into a beaker and it were shaken with 50 ml of distilled water and filtered.The filtrate and the washing were collected in a 100 ml volumetric flask.The proposed method was successfully applied for the determination of (MD) in various commercial tablets , the results obtained are shown in Table (2).

Conclusion
The method described is simple, rapid, convenient and do not require special working conditions unlike many other reported methods.The procedure were showed shorter reaction time, stable colored species with inexpensive reagents.The determination can be performed at room temperature and do not require heating step.The proposed method can be applied to assay of (MD) in pharmaceutical preparations.

Fig. ( 5 )
Fig. (5):-Effect of volume of NBD-Cl 8×10 -3 MEffect of temperatureThe effect of temperature on the reaction of (MD) with NBD-Cl in alkaline medium was studied at different values (20-80°C) by continuous monitoring of the Fig. (8):-Absorbance-time curve for the reaction of (MD) with NBD-Cl in alkaline medium