Exploiting the diazotization reaction of 4-minoacetophenone for Methyldopa determination.

: Based on the diazotization reaction of 4-aminoacetophenone with sodium nitrite in acid medium to form diazonium salt, which was coupled with Methyldopa to form a violet reddish soluble azo dye with maximum absorbance at 560 nm,a batch procedure had been developed for the estamination of Methyldopa. Under optimum experimental parameters affecting on the development and stability of the colored product, Beer´s law obeyed in the range (0.5-45) μg.ml -1 with a correlation coefficient (0.9979).The proposed method was successfully applied to the determination of Methyldopa in either pure form and in commercial brands of pharmaceuticals, no interference was observed from common excipients in the formulations. The analytical results obtained by applying this method were in good agreement with labeled values.


Introduction:
Due to the magnitude use of Methyldopa (MTD), chemically known as α-methyl-3,4-dihydroxy phenyl alanine which is a catecholamine derivative widely used as an antihypertensive agent.The Methyldopa is a centrally acting α-2adrenoreceptor agonist, which reduces sympathetic tone and produces a fall in blood pressure [1].Besides, it is necessary for routine quality control in the analysis of produced medicines, thus there is an important demand for rapid and simple methods for the determination of methyldopa in pharmaceutical preparations.The official method reported in USP [2]describes a non aqueous titration for the assay of MTD,although this later one is kind of simple and being used in routine analysis laboratories, it is time consuming and very tedious.Several researches have been devoted to the development of new high performance alternative procedures for determinationof Methyldopa in medicines and/or biological specimens using different techniques such as Electrochemical [3][4][5][6], Chromatography [7][8][9][10], Nanotechnology [11].How-ever these methods either requires sophisticated equitement;or involves procedures with rigorous control of the experimental conditions.Molecular absorption spectrophotometry is by far the instrumental technique of choice in industrial laboratories.Owning mainly to its simplicity, often demanding low cost equipment and lending itself to easy automation of trace analysis procedures, therefore a various number of UV-Visible spectrophotophotometric methods for Methyldopa determination have been reported which involves the *Department of Chemistry/College of Science/University of Al-Mustansiriyah.Baghdad-Iraq use of diverse chromogenic reagents such as: O-chloranil(O-CIN), Chloranilic acid (CIA) and Dichlorodicyanobenzoqu-inone (DDQ) [12], Vanadium [13],2,6-dichloroquinone -4chlorimide(DCQ [14],p-Chloranil [15] tris (1,10phenanthroline[16],Molybdate [17,18],Salicylic acid [19],4-amino benzoic acid [20] and 3-amino pyridine [21].Nevertheless, most of the mentioned methods present some disadvantage and drawbacks such as long time for the color development [13],complex procedure [17] and limited Beer´s law range [14,18].
A vast number of aromatic amines medicines such as: Sulfamethoxazole [22],Pramipexole Dihyochloride[23],Metronidazole [24], Ceftazidime [25] and Metoclopramide Hydrochloride and Dapsone [26] have been determined by the diazotization reaction, which is based on the conversion of free primary aryl amine into a diazonium salt by the reaction with nitrous acid; the salt then rapidly form an azo dye with a chromogenic reagent.The procedure requires the removal of excess nitrous acid by sulfamic acid, the stabilization of intermediary diazonium salt at low temperature and the expulsion of nitrogen bubbles [22].
The present paper describes the application of 4-amino acetophenone as an inexpensive new diazotization agent for the determination of MTD in medication.The method is based on the diazotization reaction of 4-amino acetophenone with sodium nitrite in hydrochloric acid medium;the formed diazonium salt is then coupled with MTD in sodium hydroxide medium to form a water soluble mono azo dye.This method does not need to get rid of excess sodium nitrite (by addition sulfamic acid or ammonium sulfamate) because of the low concentration of sodium nitrite used by adding equimolar solution of 4-amino acetophenone and sodium nitrite.The reaction product has been spectrophotometrically measured at 560 nm.

Material and Methods:
Equipment: Shimadzu UV-VIS double beam spectrophotometer (VARIAN UV-Visible) with 1cm matched quartz cells was used for all spectral measurements of the resulting solutions.

Reagents and Chemicals:
All chemicals were of analytical reagents grade.1-Methyl dopa (MTD) stock standard solution (1000 μg.ml -1 ) was prepared by dissolving 0.0500 g of pure MTD (SDI) and made up to 50 ml volumetric flask with distilled water.Working standard solutions were prepared by suitable dilution of the stock standard solution.2-Hydrochloric acid solution (0.8 M) was prepared by diluting 6.68 ml of 11.64 M of concentrated hydrochloric acid (BDH) with distilled water in 100 ml volumetric flask.3-Sodium hydroxide solution (0.5 M) was prepared by dissolving 1.0000 g of sodium hydroxide (BDH) in distilled water and diluting to the mark in 50 ml volumetric flask.4-4-amino acetophenone(3mM) solution was prepared by dissolving 0.0405 g of 4-amino acetophenon(BDH) in 5 ml ethanol ,adding 20 ml distilled water and finally the acidity was adjusted with 1 ml of 0.8M hydrochloric acid.This solution was frozen to zero degree using ice bath for 5 min.To this solution equimolar of sodium nitrite was added with shaking for 10 min., after that the azo solution was transferred to 100 ml volumetric flask, diluted to the mark with distilled water and kept in the refrigerator.

Procedures: 1-Assay of Pure Methyl dopa (MTD):
Into a series of 25 ml volumetric falsk,transfer increasing volumes of standard stock solution(1000 μg.ml -1 ) containing (0.01-0.9) ml of Methyldopa to cover the range of calibration curve(0.5-45)μg.ml - in a final volume of 25 ml,to this solution add 4ml of azo 4amino acetophenon,the solution was shacked thoroughly and 2ml of 0.5M NaOH was added.The contents were diluted to the mark with distilled water, after 15 min.the absorbance of the azo dye was measured at 560 nm against the corresponding reagent blank.

2-Assay of MTD in tablets dosage form:
Ten tablets were finely weight, ground and powdered.A quality corresponding to the weight of one tablet (250 mg) was carefully weighted and made up to 100 ml with distilled water.The resultant solution was filtered and diluted to 3 different concentrations which were analyzed in five replicate as described under the assay of pure Methyldopa (MTD0.

Result and Discussion: Preliminary Studies
The proposed method involves diazotization of the 4-amino acetophenone with sodium nitrite in hydrochloric acid medium to form diazonium salt, which on coupling with Methyldopa in sodium hydroxide medium yielding a water-soluble azo dye.The visible spectrum (Fig. 1) of the yielded reaction product demonstrates that the best analytical wavelength is located at 560 nm, which has a negligible absorbance at reagent blank at the corresponding λ max .

Optimization conditions:
The optimum reaction conditions have been established by varying the factors one at a time and keeping the other parameters fixed and observing the effects of the product on the adsorbance.These factors include the NaOH, 4-amino acetophenone, HCl volumes, time and addition sequence.

Study of the dye:
The composition of the formed complex had been established using Mole ratio method which was based on the measurement of series of solution in which increased volumes(0.5-4)ml of (3 mM) diazonium reagent were added to a fixed volume(1) ml of(3 mM) Methyldopa, under optimum conditions mentioned in the analytical procedure, the results obtained in Fig( 5) indicate that 1:1 azo dye was formed between Methyldopa(D) and diazonium azo reagent(R).The probable reaction path might be written as follow: Fig ( 5): mole ratio plot.

Figures of merits:
For proposed method, the calibration graph was obtained by the procedure described previous and a series of standard solutions was analyzed to test the linearity.The molar absorptivity, the sandell´s sensitivity, the slope, the intercept and correction coefficient were evaluated by a least squares regression analysis and were included in Table (

Pharmaceutical Applications:
In order to demonstrate the applicability of the proposed method for the determination of Methyldopa, the method was successfully applied to the analysis of different pharmaceutical preparation containing MTD and results are summarized in Table (3).For all preparations examined, the assay results of proposed method were in good agreement with the labeled content.

Conclusion:
The developed methodology is very adequate for the determination of methyldopa in aqueous solution and in pharmaceutical preparation samples at a concentration level of traces(ppm) and without requiring any previous separation step nor a temperature or pH control.Morever the proposed methods are very economical when compared to other methods such as those based on the use of HPLC .