62Eclética Química, 36 (4), 62-67, 2011. furantoin is absorbed after oral intake, however it does not reach therapeutic concentrations in the plasma and in the tissues since it is rapidly eliminated through both renal and biliary routes after rapid absorption in the gas- tro-intestinal tract.2 Since macrocrystalline nitrofuranto- in is more slowly absorbed and presents less collateral effects, it is usually commercialized in this form [2,6]. Some analytical methods are employed for the determination of nitrofurantoin in pharmaceutical preparations. For example: spectrophotometric [7-10]; spectrofluorimetric [11,12]; voltametric [13,14]; chro- matographic [15,16], methods have been described. The development of simple and rapid analyt- ical methods is always interesting. Diffuse reflectance spectroscopy in the ultraviolet and visible region of the spectrum frequently allows the direct quantitative analysis of analytes with a minimum of treatment, with consequent decreases in manipulation and in the use of organic solvents and chemical reagents. The diffuse re- flectance spectroscopy using spot tests has been used with success in quantitative analysis of many drugs [17- 19, 21-24]. In aim of the present work was the develop- ment of a quantitative diffuse reflectance method, in the ultraviolet-visible range of the spectrum, for the deter- Introduction Nitrofurantoin, [N-(5-nitro-2-furyld- ine)-1-aminohydantoin], C8H6N4O5, whose structural formula is shown in Figure 1, is a nitro compound very active against gram-positive and gram-negative bacte- ria [1]. It is also effective against Proteus and Pseudo- monas aeruginosa, particularly in the acidic medium of the urinary treat. This drug is used as antibacterial agent in urinary infections with doses from 5 to 10 mg per day[2,3]. Figure 1. Structural formula of nitrofurantoin [1] The mechanism of action of the 5-nitrofuranic derivatives is not yet completely elucidated [4,5]. Nitro- Two quantitative analytical methods for the determination of nitrofurantoin in pharmaceutical preparations in the visible-ultraviolet range of the spectrum are reported. One of them uses the diffuse reflectance technique and the other transmittance measurements in aqueous solution. Both procedures are very simple to be performed and also present green characteristics as only polyethylene glycol and water are used as solvents. Commercial pharmaceu- tical preparations of the drug were analyzed using the proposed methods. The results were compared with those obtained when the HPLC procedure recommended by the USP was applied. Statistical comparison using the paired Student’s t test and the Snedecor’s F test showed good agreement between all the methods. Keywords: nitrofurantoin ; UV-visible diffuse reflectance ; transmittance, pharmaceutical preparations GREEN AND SIMPLE UV-VISIBLE DIFFUSE REFLECTANCE AND TRANSMITTANCE METHODS FOR THE DETERMINATION OF NITROFURANTOIN IN PHARMACEUTICAL PREPARATIONS M. Tubino1*, L. F. Bianchessi1, M. Palumbo1 and M. M.D.C. Vila2 1Institute of Chemistry, State University of Campinas, CP 6154, CEP 13083-970, Campinas, SP, Brazil 2University of Sorocaba, Pharmacy Course, Sorocaba, SP, Brazil * tubino@iqm.unicamp.br 63Eclética Química, 36 (4), 62-67. 2011. the excipients starch, talc and lactose (1:1:1 w/w) in or- der to obtain a content of about 274 mg g-1, which is the nominal concentration of the analyte in the pharmaceu- tical preparation. Exact masses of that mixture, weighed to 0.1 mg, from ca. 0.015 g to ca. 0.045 g, were dissolved in ca. 3.0 g of polyethylene glycol, also weighed to 0.1 mg. The analytical curve from ca. 1.4 mg g-1 to ca. 4.0 mg g-1 of nitrofurantoin in polyethylene glycol was con- structed in this way. The spectra were registered from 300 nm to 500 nm but the absorbance values at the maximum absorp- tivity (385 nm) were considered for analytical purposes. The nitrofurantoin solutions were put on the support (waxed paper) without the necessity of weigh- ing this aliquot: a quantity was placed on a folded pa- per (Fig. 2A). The two parts were closed, followed by heating for 5 minutes at 60 oC in an oven. In sequence the paper was opened, the excess of the “ointment” was removed manually with absorbent paper, for example, a paper handkerchief. In this part of the procedure, the insoluble excipients are all mechanically removed leav- ing the sample clean. A foil of black PVC (polyvinyl- chloride) was put between the two parts of the paper (Fig. 2B) and the absorbance of the diffuse reflectance process was determined at 385 nm in the Shimadzu spectrophotometer or in the portable device constructed in our laboratory using a blue light emitter diode [20]. In the manual reflectometer the measurements are made by placing it on the paper containing the analyte. For the blank, paper imbedded with the polyethylene glycol was used. Treatment of the samples The content of 20 capsules of the pharmaceu- tical preparation was carefully crushed in a mortar. A quantity of this powder large enough to obtain a con- centration of ca. 3.5 mg g-1 in polyethylene glycol was weighed. The procedure followed was the same de- scribed above for the analytical curve. Transmission method using the UV-VIS spectro- photometer The solubility of the nitrofurantoin in water is very low. However in polyethylene glycol it is very high. Solutions of nitrofurantoin in polyethylene glycol are missible in water, forming clear solutions stable for mination of nitrofurantoin in the bulk drug and in phar- maceutical preparations. A priori the intention was to obtain a procedure with two fundamental characteris- tics, i.e., it must be green and it must be easy to perform. Experimental Chemicals All the reagents and solvents used were of ana- lytical grade. Polyethylene glycol (40 k) and bidistilled water were used as solvents. Reflectance support For the reflectance measurements waxed paper was used as support for the analyte solution in polyeth- ylene glycol. Apparatus HPLC: Shimadzu Prominence with SPP- M20A diode array; column (Waters) - m-Bonbapak C18 (300 mm ´ 3.9 mm I.D.), particle size, 10 mm; pore size, 125 angstrons; injection volume, 20 mm. Spectrophotometer: Shimadzu UV-2450 ultraviolet visible spectrophotometer equipped with a reflectance accessory. Lab-made reflectometer: The home made reflectometer has already been described in detail [20]. In the present case a blue E1L31-3G0A2 Toyoda Gosei Co. Ltd. LED was used: Æ 3 mm E.D.; view angle 15 degrees. Analytical balance: Mettler Toledo AX-205, 0.1/0.01 mg. Procedures Diffuse reflectance method using Shimadzu 2405 device or the lab-made reflectometer Analytical curve The analytical curve was constructed by preparing solutions of nitrofurantoin in 40 k polyeth- ylene glycol. In order to adequately dissolve the ana- lyte these solutions were heated to 60 oC, a temperature where the solvent is liquid, allowing perfect homogene- ization. The analytical grade nitrofurantoin used to construct the calibration curve was initially mixed with 64Eclética Química, 36 (4), 62-67. 2011. The final solution was ready to be injected in the chro- matograph, whose detector was adjusted to 254 nm. The flow rate was 1.0 mL min-1. Results and Discussion Diffuse reflectance procedure From the beginning of this work the aim was to develop a reliable green analytical method that could allow the analysis of nitrofurantoin in the bulk drug and in pharmaceutical preparations with a minimum of ma- nipulation. As is well known for a reflectance procedure a homogeneous reflecting surface is necessary. In the present case, waxed paper was used with the purpose to homogeneously fix the analyte on its surface. In this case the intensity of light reflected by the analyte was expected to be proportional to its concentration. Nitro- furantoin was dissolved in 40 k polyethylene glycol. The paste formed was applied to the paper with a small laboratory spatula. It is not necessary to weigh this ali- quot as the surface and the thickness of the paper delim- it the quantity of the analyte. The solution of the analyte must be applied on the rough side of the paper because the absorption is more effective on this surface. The commercial form of nitrofurantoin is pre- sented as a solid mixed with starch, talc and lactose, all encapsulated into a brownish material that is not speci- fied by the manufacturer. After crushing, this brown ma- terial remains in the powder impeding the direct reflec- tance measurement of the drug in the solid preparation. Due to this, dissolution in polyethylene glycol was nec- essary. The brown material is insoluble and remains in suspension whereas nitrofurantoin is easily dissolved. When the waxed paper is soaked with the solution, the non soluble material is automatically separated and re- mains on the outer surface of the paper, being removed by simply passing a soft tissue such as, for example, a paper handkerchief. The analytical curve obtained with the Shi- madzu UV-2450 spectrophotometer is described by the equation A = 0.02154+ 0.06994 C; (r=0.998); where A is the measured absorbance of the light at 385 nm in the reflection process and C is the concentration of the drug in mg g-1giving LD @ 0.23 mg g-1 and LQ @ 0.69 mg g-1. With the portable reflectometer built in our lab- at least 48 hours, allowing transmission measurements that were done at 368 nm. Figure 2. A- Spreading of the nitrofurantoin solution in polyethylene glycol on the rough side of the waxed paper; B- Black foil of PVC (polyvinylchloride) introduced between the two sides of the folded waxed paper. a – waxed paper (clean); b – laboratory spatula; c – nitrofurantoin solution in 40 k polyethylene glycol; d - black foil of PVC (polyvinylchloride). Analytical curve To construct the calibration curve 272.92 mg g-1 of a mixture of nitrofurantoin with starch, talc and lactose (1:1:1 w/w) was used. This mixture was used to prepare a polyethylene glycol nitrofurantoin solution containing 2.1025 mg g-1 of the analyte. This last solu- tion was used to prepare all the aqueous solutions used to construct the analytical curve from ca. 1.6 ´ 10-3 mg g-1 to ca. 9.0 ´ 10-3 mg g-1. The absorbance was measured in the Shimadzu UV-2450 spectrophotometer at 368 nm, using certified 1.00 cm path length quartz cells. HPLC method The HPLC method recommended by USP [15] was employed. In this procedure acetanilide, 1.00 mg mL-1 in water, is used as internal standard. The mobile phase was a solution containing aqueous phos- phate buffer (pH = 7.0) and acetonitrile in the proportion 88:12 v/v. Nitrofurantoin (0.04996 g) was dissolved in 40.0 mL of dimethylformamide. To this solution 50.0 mL of the internal standard solution was added. This last solution was filtered through a 0.45 mm porosity nylon filter, rejecting the first milliliters of the filtrate. 65Eclética Química, 36 (4), 62-67. 2011. Transmission procedure From a solution of nitrofurantoin in polyeth- ylene glycol containing 2.10 mg g-1 a series of solutions diluted in water, from ca. 1.7×10-3 mg g-1 to 1.0×10-2 mg g-1 were prepared. The absorbances of these solutions were measured at 368 nm, constituting the analytical curve described by the equation A = -0.01228 + 73.32 C; (r = 0.998); where A absorbance and C the concentra- tion of nitrofurantoin in mg g-1, having LD @ 0.63 ´10-3 mg g 1 and LQ @ 1.9´10 3 mg g 1. The results are shown in Table 1 with the title “Transmission”. HPLC procedure The HPLC procedure was performed accord- ing to the USP [15], using acetanilide as internal stan- dard. The results are shown in Table 1 under the title “HPLC”. oratory [20-22] the following analytical curve was ob- tained: r = 67.080 + 47.724 x C; (r =0.997); where r is the resistance in ohms of the LDR used as sensor in the device and C is the concentration of the drug in mg g-1, with LD @ 0.27 mg g-1 and LQ @ 0.81 mg g-1. Several samples of pharmaceutical prepara- tions obtained in the local market were analyzed. The results are shown in Table 1 where the title “Reflectance I” are those obtained with the Shimadzu spectropho- tometer using the diffuse reflectance sphere accessory and the results with the title “Reflectance II” were ob- tained with the portable reflectometer constructed in our laboratory. These same samples were also used for the transmission and for the HPLC methods. Table 1. Determination of nitrofurantoin in pharmaceutical preparations by the visible- ultraviolet diffuse reflectance, ultra- violet transmission and HPLC methods. Table 2. Determination of nitrofurantoin in pharmaceutical preparations. Statistical comparison among the results obtained using the diffuse reflectance, transmission and HPLC methods, using the paired Student’s t test and the Snedecor’s F test. The degrees of freedom () is 4 as = n1 + n2 - 2 and n1 = n2 = 3 in the present case; t critical is 2.78 (= 0.05) and 4.60 (= 0.01); F tabled is 19.0 (=0.05) and 99.0 (=0.01)[20]. 66Eclética Química, 36 (4), 62-67. 2011. References A. Kleemann, J. Engel, B. Kutschera, D. Reichest, Pharmaceutical Substances: Syntheses, Patentes, Appli- cations, N-Z, fourth ed. Thieme, Berlin, 2001, p 1453- 1454. W. Tavares, Manual de Antibióticos e Quimioterápicos Antiinfecciosos, Atheneu, São Paulo, 2001. V. Lorian, Antibiotic in Laboratory Medicine, fifth ed., Lippincott Williams & Wilkins, Philadelphia, 5rd edn, 2005. A. Masunari, L. C. Tavares, Rev. Bras. Cienc. Farm., 42(2) (2006) 203. F. R. Paula, S. H. P. Serrano, L.C. Tavares, Quim. Nova, 32(4) (2009) 1013. L. L. Bruton, J. S. Lazo, K. L. Parker, in: J. G. Hardam, L.E. Limbird, A.G. Gilman (eds), Goodman & Gilman: as Bases Farmacológicas da Terapêutica, eleventh ed., McGraw Hill, Rio de Janeiro, 2006. M. I. Walash. A. M. Elbrashy, M. A. Sultan, Anal. Lett., 26(3) (1993)499. J. J. B. Nevado, J. R. Flores, M. L. D. Pardo, Analusis, 21(1) (1993) 33. M. I. Walash, A. M. Elbrashy, M. S. Eldin, M.A., Abuir- jeie M. A. E. Sultan, Pharmazie, 49(9) (1994) 698. M.C. Mahedero, G.T. Díaz, S. G. Pascua, J. Pharm. Biomed Anal., 29 (2002) 477. T. S. Belal, J. Fluoresc., 18 (2008) 771. W. Zhang, C. R. Wilson, N.D. Danielson, Talanta, 74 (2008) 1400. E. Hammam, J. Pharm. Biomed. Anal., 30 (2002) 651. A. Guzman, L. Agui, M. Pedrero, P. Yanez-Sedeno, J. M. Pingarron, Eletroanalysis, 16 (21) (2004) 1763. The United States Pharmacopeia. The National Formu- lary, twenty fifty ed. Pharmacopeial Convention, Rock- ville, 2005, p 1380-1383 F. Belal, Chromatographia, 25 (1) (1988) 61. M. A. Gotardo, R. Sequinel, L. Pezza, H. R. Pezza. Eclet. Quim 33 (4) (2008) 7 M. A. Gotardo, L. Pezza, H.R. Pezza, Eclet. Quím. 30 (2) (2005) 17 M. A. Gotardo, J. O. Tognolli, L. Pezza, H. R. Pezza, Spectrochim. Acta, Part A 69 (4) (2008) 1103 F. A. A. Matias, M. M. D. C. Vila, M. Tubino, Sens. Actuators B Chem., 88 (2003) 60. F. A. A. Matias, M. M. D. C. Vila, M. Tubino, J. Braz. Chem. Soc.,15 (2004), 327. M. Tubino, R.L. Souza, Talanta, 68 (2006) 776. M. Tubino, L. F. Bianchessi, Marta M. D. C. Vila, Anal. Sci. 26 (2010) 121. Comparison of the methods Table 1 shows the results obtained by the three procedures proposed in this work and by the HPLC method. In Table 2 the statistical comparison between all the procedures using the paired Student’s t test and the Snedecor’s F test [25] is shown. Observing the values of Table 1, it can be noted that all the analyzed pharmaceutical preparations are in agreement with the declared content of the active prin- ciple. In the statistical comparison between the meth- ods, whose results are shown in Table 2, it can be ob- served that all procedures offered equivalent results in terms of accuracy at a 95 % confidence level (a=0.05), as the calculated values of t are all below the critical t values. With respect to the precision, statistical equiv- alence was observed except in two cases (Reflec II vs. HPLC; samples A and B). However at the 99% confi- dence level (a=0.01) complete agreement among the precision of the methods is observed. Comparing all the methods used from the point of view of laboratory manipulation diffuse reflectance and transmission are doubtless the simplest to be per- formed and the least expensive. They are also more en- vironmentally friendly as they use only polyethylene glycol and water as solvents in small quantities. In the particular case of the transmission procedure it presents the particular advantage that most common laboratories have at least a small spectrophotometer, which is quite enough for carrying out this analysis. Conclusion It can thus be concluded that the diffuse reflec- tance and the transmission methods reported in the pres- ent article can be proposed for the green quantitative analysis of nitrofurantoin in pharmaceutical prepara- tions and in the bulk drug. Acknowledgements The authors are grateful to CNPq (Consel- ho Nacional de Pesquisa Científica e Tecnológica), to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for financial support and to Prof. Dr. Carol H. Collins for an English revision of the manuscript. 67Eclética Química, 36 (4), 62-67. 2011. M. Tubino, M.M.D.C. Vila, J.Braz.Chem.Soc. 20 (2009) 1901. K. Eckschlager, Errors, Measurement and Results in Chemical Analysis, Van Nostrand: Reinhold Company, London, 1972.