Determination of Methyldopa in Pharmaceutical Formulations by Combined Spot Test-Diffuse Reflectance Spectroscopy

Este trabalho descreve um método simples e rápido por “spot test” quantitativo, utilizando a espectroscopia de reflectância difusa para determinação de metildopa em formulações farmacêuticas. O método proposto baseia-se na reação de complexação entre metildopa e íons molibdato, formando um complexo amarelo na superfície do papel de filtro. As medidas de reflectância foram realizadas a 410 nm. A curva analítica obtida a partir do gráfico de densidade óptica do sinal de reflectância (A R ) vs. log da concentração de metildopa apresentou linearidade na faixa de 6,30 × 10 a 1,89 × 10 mol L, com um coeficiente de correlação de 0,998. O limite de detecção foi de 2,74 × 10 mol L (R.S.D. = 1,02%). As substâncias comumente utilizadas como excipientes nas formulações farmacêuticas não interferem no método proposto. O método foi aplicado para a determinação de metildopa em formulações farmacêuticas comerciais. Os resultados obtidos por este método foram comparados favoravelmente com aqueles obtidos pelo método oficial, com 95% de nível de confiança.

In recent years more and more strict regulations related to the quality control of pharmaceuticals led to increasing demands on the simplicity and rapidity of analytical assay. 34,35Considering the simplicity of the spot test method, the use of small quantities of chemicals, the confidence and the rapidity of such kind of procedure, the quantitative spot-test analysis by diffuse reflectance spectroscopy could be interesting to develop analytical procedures for routine analysis of drugs in pharmaceuticals formulations.
For many years, the use of reflectance spectroscopy as an analytical technique was limited to paints, pigments, paper, ceramic and textile areas.However, with the development of optical devices including optical fibers 36 and reflectance spheres, [37][38][39][40] the situation has changed and acceptable results were obtained.
Several quantitative reflectometric methods have been reported in the literature.The exact value for the absolute reflectance of KBr powder was utilized to detect a low concentration of dioctyl phthalate. 41The properties of redox media equilibrium of some quinonoid indicators were studied when they are immobilized by adsorption on XAD-2, synthetic copolymer, by reflectometry measurement. 42he determination of Ni(II), Fe(III) and Cr(VI), 37 hydrogen chloride, 43 cations, 44 and free active chlorine, 45 in water, ammonium, phosphate and Cu(II), 46 Fe(III), 47 Cr(VI), 48 acetylsalicylic acid 49 and furosemide in pharmaceutical formulations 50 by reflectance spectroscopy were related.Moreover, a multiplexed colorimetric solid-phase extraction for determination of Ag(I), Ni(II), and sample pH by diffuse reflection spectroscopy was developed 51 and the interaction of aqueous iodine solutions with immobilized poly(vinylpyrrolidone) (PVP) by diffuse reflection spectroscopy was investigated. 52To the best of our knowledge, there are no reports on the use of quantitative spot test by diffuse reflectance for the determination of methyldopa in pharmaceutical formulations.
It has long been known that molybdate can react with catechol to form colored complexes. 53,54The cathecolate functionalities on the MTD ligand (Figure 1) suggest that it is capable of binding at available coordination sites on a cisdioxo Mo(VI) center to produce species analogous to the well known bis(catecholate)complex, MoO 2 (cat) 2 2-(H 2 cat=catechol). 54In previous studies, a spectrophotometric method 55 and a flow-injection analysis procedure 56 by using this reaction was developed in this laboratory.The combined spot test-diffuse reflectance spectroscopy offers advantages over the mentioned spectrophotometric method, such as simplicity and extremely low consumption of reagents.The flow injection procedure can not be performed in locus.In this aspect, quantitative spot test procedures can be a very good alternative when combined with diffuse reflectance spectroscopy.The reflectance measurements can be performed by using a very simple homemade reflectometer or a portable diffuse reflectance spectrophotometer, 57 which are small, lightweight, inexpensive and battery operated, characteristics highly attractive for many applications in any location by nearly everyone.
In the present work, a quantitative spot test procedure carried out by measuring the reflectance of the color developed in complexation reaction of MTD with molybdate ions is described.This method is simple, rapid, inexpensive and does not involve any pre-treatment procedure or heating steps.The results obtained by the proposed method were in excellent agreement with those given by the official method, 33 proving that the method is a reliable alternative for the analysis of methyldopa in pharmaceutical formulations.

Reagents and solutions
Analytical reagent or pharmaceutical grade chemicals were used.For the preparation of the solutions and samples, deionised water and grade A glassware were used throughout.Whatman 41 filter paper was used as solid support.
Methyldopa standard was purchased from Purifarma, Brazil, (purity > 99.99%).A 2.1 × 10 -2 mol L -1 stock solution of MTD was freshly prepared.This solution was standardized according the literature. 33Working standard solutions were obtained by appropriate dilution of this stock solution in water.
Four commercial samples (A -D) of MTD containing 250 or 500 mg MTD per unit were purchased from local drugstores in Araraquara, Brazil.

Recommended procedure for the calibration curve
For the spot reaction, 20 μL of the analyte solution was spotted onto 1 cm 2 Whatman 41 filter paper followed by addition of 20 μL of the reagent solution.The solutions were dropped onto the paper with a micropipette fixed in a holder according to procedure described by Tubino et al. 37 and the reflectance measurements were carried out at 410 nm after 15 min.The blank solution is prepared in a similar way, but omitting MTD.Calibration graphs are prepared by plotting the optical density of the reflectance signal (A R ) vs. the log of the mol L -1 drug concentration.These graphs or the corresponding linear least squares equations are used to convert reflectance into MTD concentration, for any analysed sample.

Procedure for the assay of MTD in pharmaceutical samples
Twenty tablets of each commercial brand of MTD were weighed and finely powdered.A portion of this powder, equivalent to approximately 125.0 mg of MTD was accurately weighed and dissolved with 35 mL of water by shaking for 15 min in a mechanical shaker.The resulting mixture was transferred into 50 mL standard flask and the volume completed with deionised water.An aliquot of this solution was taken for the spot test reflectance analysis according to the recommended procedure.

Study of interferences
Since the aim of this study was to determine MTD in pharmaceuticals, the effects of the most commonly used excipients were carefully examined.The excipients studied were sucrose, glucose, talc, fructose, lactose, poly(ethylene glycol), microcrystalline cellulose, croscarmellose sodium, starch, polyvinylpyrrolidone and magnesium stearate.For this study, solutions containing MTD and each of the excipients taken separately in concentrations equal or 10 times greater than that of MTD were shaken with water in a magnetic mixer for 15 minutes, diluted and analyzed under the same conditions described in recommended procedure.

Results and Discussion
The method involves the complexation reaction of MTD with molybdate ions to produce yellow coloured product, in aqueous media.The reflectance spectrum of the reaction product shows that the best analytical wavelength is located at 410 nm.
Investigations were carried out to establish the optimum conditions for complex formation.Thus, the influence of the molybdate concentration on the reaction was studied in order to achieve maximum absorbance, repeatability, stability, sensitivity and linearity.The solutions of this reagent were evaluated in the following concentrations: 5.0 × 10 -2 , 2.5 × 10 -1 , 5.0 × 10 -1 , 1.0, 2.0, 4.0, 8.0 and 10.0% (m/v).The 1.0% ammonium molybdate solution was found to be sufficient for providing maximum and reproducible color intensity.

Analytical curves and stability
Under optimized experimental conditions, a series of standard solutions was analyzed to test the linearity.The calibration curve (Figure 2) was found to be linear in the 6.30 × 10 -3 -1.89 × 10 -2 mol L -1 concentration range (A R = -0.6674+ 0.4644 × C; r = 0.998), where A R is the reflectance measurement to 410 nm and C = log (10 4 [MTD]/mol L -1 ).The factor 10 4 was used to adjust the calibration graph to log values higher than zero.The limit of detection was estimated to be 2.74 × 10 -3 mol L -1 (R.S.D. = 1.02%), according to the analytical curve data and using the criteria of the mathematical model given by Miller and Miller. 58 The data given in Table 1 show that the color development is immediate at room temperature (25±1 °C), but the stability of color was obtained after 15 min.The resulting chromogen is stable for at least 60 min at room temperature.In this manner, all intensity reflectance values were taken after 15 minutes, the drying time of filter paper.Alternatively, using a hair-dryer this time is reduced for 25 seconds, without any alteration in the intensity of the reflectance measurement.

Effect of interferences, repeatability and recovery studies
The influence of excipients that commonly accompany MTD in pharmaceutical formulations was studied.No interference in the proposed method was observed up to ca. 10-fold excess of sucrose, glucose, talc, fructose, lactose, poly(ethylene glycol), microcrystalline cellulose, croscarmellose sodium, starch, polyvinylpyrrolidone and magnesium stearate.
The area of solid support used for spot test is very important.In this work we observed that good reflectance readings could be assured if 1 cm 2 sized pieces of good quality filter paper are used as support for the spot test reactions.The most important thing here is the uniformity of the spot color test over the entire surface.In the repeatability study, the R.S.D. was 3% for solutions containing equivalent to 1.68 × 10 -2 mol L -1 of MTD (n = 10).This is good evidence of repeatability of the proposed method.
To study the recovery of the MTD from pharmaceuticals formulations, four commercial samples were used.The recovery of MTD was examined by adding MTD reference solutions at four levels (4.20 × 10 -3 , 6.30 × 10 -3 , 8.40 × 10 -3 and 10.5 × 10 -3 mol L -1 ) to the samples containing equivalent to 8.40 × 10 -3 mol L -1 MTD.The results obtained (Table 2) were compared with the added concentrations.The average recoveries obtained ranged from 100.1 to 101.0%, evidencing the absence of matrix effect on the proposed method.

Analytical applications
In order to assess the utility of the presently developed method it was applied to the determination of methyldopa in pharmaceutical formulations.The results, presented in Table 3, compare favorably with the official method of the Brazilian Pharmacopoeia. 33Statistical analysis of the results obtained by the proposed and official methods using t-test and F-test 58 showed no significant difference between the performances of these methods, for 95% confidence level.

Conclusions
In the present study, we have demonstrated the potential of diffuse reflectance spectroscopy for the analysis of methyldopa in pharmaceutical preparations.The results obtained from the present study showed the good performance of this technique, suggesting its use as an advantageous alternative for quantitative analytical purposes.The proposed method results a simple, fast, inexpensive, precise and accurate analytical technique to determine MTD in commercial pharmaceutical preparations with satisfactory recoveries.Moreover, it does not require the removal of usual excipients present in pharmaceutical formulations since they were found not to interfere with the determination of MTD.Label content for tablets: mg unit -1 ; b average value ± standard deviation (SD) of four determinations; c relative standard deviation (RSD) of four determinations; d the figures between parentheses are the theoretical values of t and F at P = 0.05.

Figure 2 .
Figure 2. Calibration curve for methyldopa.Formation of yellow coloured product on filter paper square (1 cm 2 ).A R values were taken at 410 nm.The concentration of methyldopa, [MTD], is given in mol L -1 .

Table 1 .
Reflectance measurements at room temperature (25±1 °C) as related to the time of reaction a

Table 2 .
Results of the addition-recovery experiments a Average ± relative standard deviation (RSD) for the four determinations.

Table 3 .
Determination of MTD in commercial pharmaceutical preparations