Spot-test identification and rapid quantitative sequential analysis of dipyrone

ARTICLE

Spot-test identification and rapid quantitative sequential analysis of dipyrone

M. Tubino^I,*; A. C. Biondo^I; M. M. D. C. Vila^II; L. Pezza^III; H. R. Pezza^III

^IState University of Campinas - UNICAMP, Institute of Chemistry, P.O. Box 6154, 13083-970 Campinas, SP, Brazil

^IISorocaba University - UNISO, Sorocaba, SP, Brazil

^IIISão Paulo State University- UNESP, Chemistry Institute of Araraquara, Araraquara, SP, Brazil

ABSTRACT

A qualitative spot-test and tandem quantitative analysis of dipyrone in the bulk drug and in pharmaceutical preparations is proposed. The formation of a reddish-violet color indicates a positive result. In sequence a quantitative procedure can be performed in the same flask. The quantitative results obtained were statistically compared with those obtained with the method indicated by the Brazilian Pharmacopoeia, using the Student's t and the F tests. Considering the concentration in a 100 µL aliquot, the qualitative visual limit of detection is about 5×10^-6 g; instrumental LOD ≅ 1.4×10^-4 mol L^-1 ; LOQ ≅ 4.5×10^-4 mol L^-1.

Keywords: dipyrone, spot-test, analysis, qualitative, quantitative

RESUMO

Um método "spot-test" qualitativo e seqüencialmente quantitativo é proposto para análise de dipirona em fármaco "puro" e em preparações farmacêuticas. A formação de coloração vermelho-violeta indica um resultado qualitativo positivo. Na seqüência, um procedimento quantitativo pode ser realizado no mesmo frasco. Os resultados quantitativos obtidos foram comparados estatisticamente com os resultados obtidos pelo método indicado pela Farmacopéia Brasileira, utilizando o teste t de Student e o teste F. Considerando a concentração em uma alíquota de 100 µL, o limite qualitativo visual de detecção foi de cerca 5×10^-6 g; instrumentalmente o limite de detecção foi de LOD ≅ 1.4×10^-4 mol L^-1 e o limite de quantificação de LOQ ≅ 4.5×10^-4 mol L^-1.

Palavras-chave: dipirona, spot-test, análise, qualitativa, quantitativa

Introduction

Dipyrone (sodium salt of 1-phenyl-2,3-dimethyl-4-methylaminomethane-sulfonate-5-pyrazolone) (Fig. 1) is a water-soluble pyrazolone derivative widely used in therapeutics as a analgesic, antipyretic and antispasmodic drug [1]. Dipyrone was developed in Germany and was introduced into clinical practice in 1922. It is still in use in many countries for adults and children, where it is sold as an over-the counter (OTC) painkiller [2,3]. Due to its strong analgesic effect, available parenteral formulation, and low cost, dipyrone is widely used, generating a consumption of more than 10 thousand tons/year. Dipyrone is very popular in Brazil and is marketed in the sodium salt form or as the magnesium salt, as well as in association with other drugs [4]. However, the use of dipyrone was proscribed more than twenty years ago in the U.S.A. due to its putative role in depressing bone marrow, causing aplastic anemia and agranulocytosis. However, this has been criticized by many authors [2,4,5].

The metabolism of dipyrone has been recently reviewed. It was demonstrated that it inhibits cyclooxygenase (COX). However, in contrast to classical COX-inhibitors, such as aspirin-like drugs, dipyrone has no anti-inflammatory effect and a low gastrointestinal toxicity, indicating a different mode of action. The authors suggested that the pharmacologically active metabolites of dipyrone inhibit COX activity by sequestering radicals that initiate the catalytic activity of this enzyme or through the reduction of the oxidized states of the COX protein [6]. After oral intake, it is spontaneously hydrolyzed in the gastric fluid to its main metabolite, 4-methylaminoantipyrine (4-MAA), which is rapidly and nearly completely absorbed. 4-MAA is then converted to variety of metabolites by various enzymatic reactions. The effect of dipyrone occurs approximately fifteen minutes after oral administration. The biotransformation takes place at the hepatic level, the duration of this effect is approximately 4-6 hours, and its elimination occurs at the renal level [7].

Recently, additional beneficial effects of dipyrone, such as vascular smooth muscle relaxation, and as antiapoptotics and anticolvulsants, have been reported and have increased the interest in dipyrone [3].

A great problem related with pharmaceutical products around the world is falsification. These incidents probably occur more frequently in developing and poor countries but certainly Europe and United States are not completely free of them. In Brazil the problem was denounced some years ago [8,9] and involved antibiotics, contraceptives, cancer medicines and also common analgesics and antipyretics like dipyrone and aspirin. There is suspicion that such anomalies still remain. From these considerations, it is clearly apparent that it is very important to develop quick, simple, reliable and low cost analytical procedures that could be used routinely for screening examinations to detect possible falsifications.

Some methods have been developed for dipyrone determination, such as titrimetry [1], HPLC [10], spectrophotometry [11-15], potentiometry [16], amperometry [17,18], turbidimetry [19], voltammetry [20], and reflectometry [21]. The iodometric titration of dipyrone is recommended by the Pharmacopoeia [1] but this procedure is very slow and laborious, thus less applicable to large-scale analysis.

The aim of the present work is to develop a simple and reliable spectrophotometric method for the qualitative spot-test [22] associated with a rapid quantitative sequential analysis of dipyrone in pharmaceutical preparations. A similar qualitative procedure has been reported [23] but in the present work the sequential quantitative analysis using iron III as catalyst was developed. The method is based on the selective oxidation of dipyrone, a characteristic reaction of a pyrazolone, in the presence of concentrated sulfuric acid, splitting off formaldehyde which reacts with chromotropic acid producing a reddish-violet compound [11]. The sulfuric acid addition promotes the oxidation of dipyrone and guarantees the temperature necessary for the reaction, to occur, as it interacts with the small quantity of water intentionally added in order to take advantage of the highly exothermic process of the hydration of this acid. The formation of the reddish-violet compound identifies the dipyrone. In case of positive qualitative result the quantitative analysis can be sequentialy performed.

Experimental

Reagents

All chemicals were of analytical grade and were used without further purification. Concentrated sulfuric acid (96%) was obtained from Synth^®. Chromotropic acid (disodium salt, dihydrate, C₁₀H₆O₈S₂Na₂.2H₂O) and dipyrone (C₁₃H₁₆N₃NaO₄S) were obtained from Sigma-Aldrich®. Distilled water was obtained from a glass distillation device.

Pharmaceutical dosage forms of dipyrone were purchased from reliable drugstores. The commercial tablets comprising dipyrone tested were: - Novalgina^® (500 mg); Conmell^® (320 mg); Anador^® (500 mg); Lisador^® (500 mg); Buscopan^® (250 mg) and Generic Medley^® (500 mg).

Solutions

Chromotropic acid solution: prepared daily by dissolving 0.2002 g of chromotropic acid in 5 mL of distilled water, in order to obtain a 0.1 mol L¹ concentration.

Iron (III) chloride 0.1 mol L^-1 solution: to prepare 25 mL of a 0.1 mol L^-1 solution about 0.67 g of analytical grade (Vetec®) FeCl₃.6H₂O were dissolved in a volumetric flask.

Standard dipyrone solution: prepared by dissolving an adequate mass of dipyrone in 50.0 mL of distilled water, in order to obtain a 0.1 mol L¹ concentration. From this solution, by appropriate dilutions, the other solutions of the dipyrone were prepared.

Sample preparation: Twenty tablets were powdered in a mortar after weighing. An accurately weighed portion of one tablet, equivalent to about 15 mg of anhydrous dipyrone, was dissolved in 10.0 mL of water, in a volumetric flask, to obtain a solution with a concentration of approximately 4.5 × 10 ^-3 mol L^-1.

Procedure

Add, with a micro spatula, to the tube (straight walled volumetric flask of 5.0 mL) about 2 to 3 mg of solid chromotropic acid. Transfer carefully 100 µL of dipyrone solution into this tube. Gently dissolve the chromotropic acid. Add a drop of the 0.1 mol L^-1 iron (III) chloride solution and homogenize. Then add 500 µL of good quality concentrated sulfuric acid. The acid should be rapidly added, all at once. Shake the tube carefully in order to homogenize. If a reddish-violet color appears the test is positive for dipyrone. To sequentially perform the quantitative analysis the volume is completed to 5.0 mL with water. The absorbance is measured at 576 nm using the volumetric flask as the spectrophotometric cell. Alternatively spectrophotometric cuvettes can be used.

Apparatus

A Femto 600 single beam spectrophotometer was used for all absorbance measurements. The absorbance measurements were performed using a straight walled volumetric flask also used for the reaction. Spectrophotometric glass cuvettes 1.00 cm optical path were also used.

The 5.00 mL straight walled volumetric flask used is essentially an adequately calibrated tube with 11 cm total height, 1.0 cm ID, 1.4 cm OD and fitted with a stopper to allow shaking without losing solution. It is similar to the usual 1.0 and 2.0 volumetric flasks. This format was used in order to promote the most intimate contact of the analyte and of the reagents due to the format and to the dimensions of the bottom of the flask.

As the volumetric tubes were not of optical quality their transmittance (filled with water) at 576 nm was compared. No significant difference was observed and, therefore, they were used without necessity of any correction in the measured absorbances.

Volume measurements of the aliquots and of the sulfuric acid were done using Eppendorf^® micropipettes.

Reference method

To analyze the samples by the classic iodometric method, the preparation procedure recommended by the Brazilian Pharmacopoeia [1] was used.

Statistical analysis

The statistical t-Student test and the F tests [24] were applied to compare the recovery results obtained by the two analytical methods, the proposed and the titrimetric ones [1].

Results and Discussion

The reaction used in the method developed in this work is based on the reaction of dipyrone analogue, 1-phenyl-2,3-dimethyl-4-dimethylamino-5-pyrazolone [22], which makes use of a very selective oxidation of that compound in the presence of concentrated sulfuric acid, splitting out formaldehyde. The reducing action of pyramidone is related to its tendency to tautomerise [25]; the oxidation probably begins with the isomeric methoxy form of the pyrazolone [22]. The formaldehyde formed is identified by reacting it with warm chromotropic acid, yielding a red-violet color [22]. The nature of this chromogen has never been unambiguously determined but some experimental evidence suggests the hypothesis that it has a mono-cationic dibenzoxanthylium structure [26]. In this work, we use the highly exothermic hydration process of the sulfuric acid and its very low heat capacity to provoke the necessary temperature increase for the reaction to occur [23]. In order to accelerate the reaction iron III was used as catalyst. A drop of a 0.1 mol L^-1 iron (III) chloride solution was added directly in the solution containing the dipyrone with the chromotropic acid. When the sulfuric acid is added, the increase of the reaction rate can be visually observed when compared to the reaction without the addition of iron III.

The absorption spectrum of the reaction product was obtained using the final solution in the volumetric flask. With the defined working conditions the maximum absorption wavelength was observed at 576 nm.

The stability of the product formed was studied over time. Measurements performed 15 days later showed that the reddish-violet compound formed is completely stable in this time interval when stored at ambient temperature (ca. 25 ºC).

The molar ratio of the analyte to the analytical reagent and the volume ratio of the concentrated sulfuric acid were investigated. The best conditions were: 1:10 ratio of dipyrone to chromotropic acid using 500 µL of concentrated sulfuric acid.

The absorbance was measured at 576 nm using the volumetric flask as spectrophotometric cell. These results were compared with those obtained with spectrophotometric cuvettes.

The calibration curves were constructed in the range from 1.0 × 10^-3 mol L^-1 to 6.0 × 10³ mol L^-1 considering the concentration in the aliquot. They can be described by the equations: a) A = 0.15 + 110 C (r=0.999) where A is the absorbance at 576 nm and C is the concentration in mol L^-1 in the aliquot; when using the spectrophotometer with the straight walled volumetric flask and b) A = 0.16 + 114 C (r=0.998) with the cuvettes.

For both procedures, using the volumetric tubes for spectrophotometric measurements or using the cuvettes, the instrumental limits of detection, LOD, and of quantitation, LOQ, are: LOD ≅ 1.4 × 10^-4 mol L^-1 (LOD ≅ 3.3 SD / slope); limit of quantitation LOQ ≅ 4.5 × 10^-4 mol L^-1 (LOQ ≅ 10 SD / slope). The visual qualitative limit of detection is about 5 × 10^-6 g of dipyrone in the aliquot.

Alternatively, in order to obtain results more rapidly, a simple proportional calculation can be done using as standard a solution prepared with the expected concentration of dipyrone in the sample. If the obtained result for the sample is within the established limits, the pharmaceutical preparation can be considered in conformity with the pharmacopoeia recommendations.

In order to test the proposed method six commercial pharmaceutical preparations, purchased in local pharmacies, were analyzed. The results were compared with those obtained with the titrimetric procedure recommended by the Brazilian Pharmacopoeia [1], using the paired Student's statistical t test and the F test [24]. As it can be observed in Table 1, for a confidence coefficient of 0.05, in four cases values of t, slightly higher than the theoretical value, were obtained. However, the F test shows complete agreement in all cases. For the confidence coefficient of 0.01 complete agreement was achieved in the Student's t and in the F tests.

ª Other components present in the pharmaceutical preparation besides dipyrone:

A- magnesium stearate; macrogol;

B - starch; lactose; talc; magnesium stearate;

C - hypromelose; polyvidone; silicon dioxide; sucrose; quinoline yelow; starch; talc; magnesium stearate;

D - prometazin hydrochloride, adiphenine hydrochloride, magnesium stearate; silicon dioxide; talc; starch; sucrose;

E - silicon dioxide; magnesium stearate; polyvidone, magnesium silicate;

F - hyoscine butylbromide, calcium phosphate dibasic; starch; silicon dioxide; tartaric acid; stearic acid; polyvidone; sucrose; talc; gum arabic; titanium dioxide; macrogol;carnauba wax; white wax.

^b Reference titrimetric method [1].

^c Proposed method I, using straight volumetric tubes.

^d Proposed method II, using spectrophotometric cuvettes.

^e Paired calculated Student's t values, comparing the proposed method I with the reference titrimetric method.

^f Paired calculated Student's t values, comparing the proposed method II with the reference titrimetric method.

^gF values comparing method I with reference titrimetric method.

^hF values comparing method II with reference titrimetric method

ⁱF values comparing method I with method II.

Theoretical: Students's t = 2.78 (confidence coefficient, α=0.05) and 4.60 (confidence coefficient, α=0.01) for the degree of freedom ν= 4 (ν= n₁ + n₂ -2); n₁ = n₂ = 3; F = 19.00 (α=0.05).

Conclusion

The analytical method described in this work, despite the complexity of the analyte, presents extreme chemical and instrumental simplicity, very low cost, quite good accuracy and precision, rapidity and reliability. The quantitative determination can be performed in less than five minutes. Therefore, considering these characteristics, it can be suggested for the determination of dipyrone in the bulk drug and in pharmaceutical preparations.

Acknowledgements

The authors are grateful to CNPq (Conselho 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 English revision of the manuscript.

• [1] Brazilian Pharmacopoeia, 3rd ed., Organização Andrei Editora, São Paulo, Brazil, 1977, p 406-8.
• [2]I.M.Benseñor, São Paulo Med. J., 119 (2001) 190.
• [3] H. Ergün, D. A. C. Frattarelli and J. Aranda, J. Pharmaceut. Biomed., 35 (2004) 479.
• [4] P. Danielli, and M. B Leal, Rev. Bras. Farm., 84 (2003) 17.
• [5] Y. Bentur, and O. Cohen, J. Toxicol.-Clin. Toxic., 42 (2004) 261.
• [6] S. C. Pierre, R. Schmidt, C. Brenneis, M. Michaelis, G. Geisslinger, and K. Scholich, Br. J. Pharmacol., 151 (2007) 494.
• [7] G. Suarez-Kurtz , F.M. Ribeiro, R.C.E. Estrela, F.L. Vicente, and C J. Struchiner, Braz. J. Med. Biol. Res., 34 (2001) 1475.
• [8]Brasil, Anvisa, Agência Nacional de Vigilância Sanitária, Rev. Saúde Pública, 38 (2004) 748.
• [9] F.G.D. Vieira, J. M. Crubellate, I. G. Silva, and W. R. Silva, RAE eletron., 1 (2002) 1.
• [10]H.Senyuva, I. Aksahin, S. Ozcan, and B.V. Kabasakal, Anal. Chim. Acta, 547 (2005)73-7.
• [11] K.A. Sakiara, L. Pezza, C. B. Melios, H. R. Pezza, and M. Moraes, Farmaco, 54 (1999) 629.
• [12] J. L. F. C. Lima, S. M. O. Sá, J. L. Santos, and E. A. G. Zagatto, J. Pharmaceut. Biomed., 32 (2003)1011.
• [13] L. H. Marcolino Jr, R. A. Souza, O. Fatibello Filho, and F.C. Moraes, Anal. Lett., 38 (2005) 2315.
• [14] A.V. Pereira, L. Penckowski, M. Vosgerau, and M. F. Sassá, Quím. Nova 25 (2002)553.
• [15] A. P. Nascimento, M. G. Trevisan, E. R. M. Kedor-Hackmann, and R. J. Poppi, Anal. Lett., 40 (2007) 975.
• [16] J. S. Albuquerque, V. L. Silva, F. Lima, A. Araújo, and M. C. B. S. M. Montenegro, Anal. Sci., 19 (2003) 692
• [17] T. R. L. C. Paixão, C. R. Matos, and M. Bertotti, Talanta, 61 (2003) 725.
• [18] E. P. Medeiros, S. L. Castro, F. M. Formiga, S. R. B. Santos, M. C. U. Araújo, and V. B. Nascimento, Microchem. J., 78 (2004) 91.
• [19] L. H. Marcolino Jr, V. G. Bonifácio, and O. Fatibello Filho, Quím. Nova, 28 (2005) 783.
• [20] M. F. S Teixeira, L. H. Marcolino Jr., O. Fatibello Filho, E. R. Dockal, and E. T. G. Cavalheiro, J. Braz. Chem. Soc., 15 (2004) 803.
• [21] P. L.Weinert, L. Pezza, and H.R. Pezza, J. Braz. Chem. Soc., 18 (2007) 846
• [22] F. Feigl, Spot Tests in Organic Analysis, 7th ed., Elsevier, Amsterdan, 1966, p. 434-36 and 635.
• [23] L. Pezza, M. Tubino, C. B. Melios, and H. R. Pezza, Anal. Sci., 16 (2000) 313.
• [24] K. Eckschlager, Errors, Measurement and Results in Chemical Analysis, Van Nostrand Reinhold Company, London, 1972, p. 109-120.
• [25] P. Karrer, Organic Chemistry, 4th ed.; Wiley, New York, 1950, p. 798.
• [26] P. E. Georghiou, and C. K. Ho, Can. J. Chem., 67 (1989) 871

Publication Dates

• Publication in this collection
  09 June 2010
• Date of issue
  2010