Chemical Papers

, Volume 64, Issue 4, pp 415–423 | Cite as

Use of a diazocoupling reaction for sensitive and selective spectrophotometeric determination of furosemide in spiked human urine and pharmaceuticals

  • Kalsang Tharpa
  • Kanakapura BasavaiahEmail author
  • Kanakapura Basavaiah Vinay
Original Paper


Two simple, sensitive, and selective spectrophotometric methods for the determination of 5-(aminosulfonyl)-4-chloro-2-((2-furanylmethyl)amino)benzoic acid (furosemide, FUR) are described. The methods are based on acid hydrolysis of FUR to free primary aromatic amine and diazotization followed by coupling with N-1-napthylethylene diamine (NEDA) (method A) or 4,5-dihydroxynaphthalene-2,7-disulfonic acid (chromotropic acid, CTA) (method B). The colored reaction product can be measured spectrophotometrically at 520 nm (method A) or 500 nm (method B). Beer’s law is obeyed over the ranges of 1.75–21.0 μg mL−1 and 2.5–30.0 μg mL−1, for method A and method B, respectively. Apparent molar absorptivities and Sandell’s sensitivities (in L mol−1 cm−1 and μg cm−2 per 0.001 absorbance unit, respectively) were 1.34 × 104 and 0.0253 using NEDA as the coupling agent, and 8.5 × 103 and 0.0389 using CTA for the same purpose. Analysis of solutions containing seven different concentrations of FUR gave a correlation coefficient of 0.9979 using NEDA and 0.9984 using CTA, while the slope and the correlation coefficient of the regression equation were calculated. The reaction stoichiometry in both methods was evaluated by the limiting logarithmic method and was found to be 1: 1 (diazotized FUR: NEDA or diazotized FUR: CTA). The methods were successfully applied to the determination of FUR in spiked human urine and in pharmaceutical formulations. The recovery of FUR from spiked urine was satisfactory resulting in the values of (109.4 ± 4.37) % using NEDA and (113.0 ± 4.74) % using CTA. Results of the analysis of pharmaceuticals demonstrated that the proposed procedures are at least as accurate and precise as the official method while a statistical analysis indicated that there was no significant difference between the results obtained by the proposed methods and those of the official method.


diazotization spiked human urine pharmaceuticals spectrophotometry 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abdel-Hamid, M. E. (2000). High-performance liquid chromatography-mass spectrometric analysis of furosemide in plasma and its use in pharmacokinetic studies. Il Farmaco, 55, 448–454. DOI: 10.1016/S0014-827X(00)00064-1.CrossRefGoogle Scholar
  2. Abou-Auda, H. S., Al-Yamani, M. J., Morad, A. M., Bawazir, S. A., Khan S. Z., & Al-Khamis, K. I. (1998). High-performance liquid chromatographic determination of furosemide in plasma and urine and its use in bioavailability studies. Journal of Chromatography B: Biomedical Sciences and Applications, 710, 121–128. DOI: 10.1016/S0378-4347(98)00058-9.CrossRefGoogle Scholar
  3. Basavaiah, K., Chandrashekar, U., & Nagegowda, P. (2005). Rapid titrimetric and spectrophotometric determination of frusemide (furosemide) in formulations using bromatebromide mixture and methyl orange. Indian Journal of Chemical Technology, 12, 149–155.Google Scholar
  4. Carda-Broch, S., Esteve-Romero, J., Ruiz-Angel, M. J., & García-Alvarez-Coque, M. C. (2002). Determination of furosemide in urine samples by direct injection in a micellar liquid chromatographic system. Analyst, 127, 29–34. DOI: 10.1039/b108358a.CrossRefGoogle Scholar
  5. Espinosa Bosch, M., Ruiz Sánchez, A. J., Sánchez Rojas, F., & Bosch Ojeda, C. (2008). Recent developments in analytical determination of furosemide. Journal of Pharmaceutical and Biomedical Analysis, 48, 519–532. DOI: 10.1016/j.jpba.2008.07.003.CrossRefGoogle Scholar
  6. European Directorate for the Quality of Medicines (2001). European pharmacopoeia (IV ed., pp. 1228–1229). Strasbourg, France: Council of Europe.Google Scholar
  7. European Medicines Agency (2005). Validation of analytical procedures: Text and methodology Q2(R 1). London, UK: European Medicines Agency.Google Scholar
  8. García, M. S., Sanchez-Pedreño, C., Albero M. I., & Ródenas, V. (1997). Flow-injection spectrophotometric determination of frusemide or sulphathiazole in pharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 15, 453–459. DOI: 10.1016/S0731-7085(96)01874-2.CrossRefGoogle Scholar
  9. Gomez, C. G., von Plessing, C. R., Godoy, C. G. M., Reinbach, R. H., & Godoy, R. R. (2005). Method validation for the determination of furosemide in plasma by liquid-liquid extraction and high-performance liquid chromatography with fluorescence detection. Journal of the Chilean Chemical Society, 50, 479–482. DOI:10.4067/S0717-97072005000200008.Google Scholar
  10. Gotardo, M. A., Gigante, A. C., Pezza, L., & Pezza, H. R. (2004). Determination of furosemide in pharmaceutical formulations by diffuse reflectance spectroscopy. Talanta, 64, 361–365. DOI:10.1016/j.talanta.2004.02.034.CrossRefGoogle Scholar
  11. Gölcüu, A. (2006). Spectrophotometric determination of furosemide in pharmaceutical dosage forms using complex formation with Cu(II). Journal of Analytical Chemistry, 61, 748–754. DOI: 10.1134/S1061934806080053CrossRefGoogle Scholar
  12. Guzmán, A., Agüí, L., Pedrero, M., Yáñez-Sedeño, P., & Pingarrón, J. M. (2003). Flow injection and HPLC determination of furosemide using pulsed amperometric detection at microelectrodes. Journal of Pharmaceutical and Biomedical Analysis, 33, 923–933. DOI: 10.1016/S0731-7085(03)00422-9.CrossRefGoogle Scholar
  13. Higuchi, T., & Brochmann-Hanssen, E. (1997). Pharmaceutical analysis (5th ed., pp. 142). New Delhi, India: CBS Publishers.Google Scholar
  14. Ioannou, P. C., Andrikopoulou, D. A., Glynou, K. M., Tzompanaki, G. M., & Rusakova, N. V. (1998). Spectrofluorimetric determination of anthranilic acid derivatives based on terbium sensitized fluorescence. Analyst, 123, 2839–2843. DOI: 10.1039/a806093b.CrossRefGoogle Scholar
  15. Issopoulos, P. B. (1989). Spectrophotometric determination of microquantities of frusemide using iso- and heteropolyanions of molybdenum(VI) as oxidizing agents. Fresenius’ Journal of Analytical Chemistry, 334, 554–557. DOI: 10.1007/BF00483576.CrossRefGoogle Scholar
  16. Jankowski, A., Skorek-Jankowska, A., & Lamparczyk, H. (1997). Determination and pharmacokinetics of a furosemide-amiloride drug combination. Journal of Chromatography B: Biomedical Sciences and Applications, 693, 383–391. DOI: 10.1016/S0378-4347(97)00055-8.CrossRefGoogle Scholar
  17. Llorent-Martínez, E. J., Ortega-Barrales, P., & Molina-Díaz, A. (2005). Multicommuted flow-through fluorescence optosensor for determination of furosemide and triamterene. Analytical and Bioanalytical Chemistry, 383, 797–803. DOI: 10.1007/s00216-005-0079-5.CrossRefGoogle Scholar
  18. Martindale, W. (1989). In J. E. F. Reynolds (Ed.), Martindale: The extra pharmacopoeia (29th ed., pp. 977–978, 987–991). London, UK: The Pharmaceutical Press.Google Scholar
  19. Mendham, J., Denney, R. C., Barnes, J. D., & Thomas, M. (2004). Vogel’s textbook of quantitative chemical analysis (6th ed., pp. 88). Harlow, UK: Pearson Education.Google Scholar
  20. Miller, J. N., & Miller, J. C. (2000). Statistics and chemometrics for analytical chemistry (5th ed.). Harlow, UK: Pearson Education.Google Scholar
  21. Millership, J. S., Parker, C., & Donnelly, D. (2005). Ratio spectra derivative spectrophotometry for the determination of furosemide and spironolactone in a capsule formulation. Il Farmaco, 60, 333–338. DOI:10.1016/j.farmac.2005.02.001.CrossRefGoogle Scholar
  22. Mishra, P., Katrolia, D., & Agrawal, R. K. (1990). A simple colorimetric determination of furosemide in dosage forms. Indian Journal of Pharmaceutical Sciences, 52, 155–157.Google Scholar
  23. Ptǎček, P., Vyhnálek, O., Breuel, H. P., & Macek, J. (1996). Determination of furosemide in plasma and urine by gas chromatography/mass spectrometry. Arzneimittelforschung, 46, 277–283.Google Scholar
  24. Reeuwijk, H. J. E. M., Tjaden, U. R., & van der Greef, J. (1992). Simultaneous determination of furosemide and amiloride in plasma using high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B: Biomedical Sciences and Applications, 575, 269–274. DOI: 10.1016/0378-4347(92)80155-J.CrossRefGoogle Scholar
  25. Rose, J. (1964). Advanced physico-chemical experiments (pp. 67). London, UK: Pitman.Google Scholar
  26. Sastry, C. S. P., Prasad, T. N. V., Sastry, B. S., & Rao, E. V. (1988). Spectrophotometric methods for the determination of some diuretics using 3-methyl-2-benzothiazolinone hydrazone. Analyst, 113, 255–258. DOI: 10.1039/AN9881300255.CrossRefGoogle Scholar
  27. Sastry, C. S. P., Suryanarayana, M. V., & Tipirneni, A. S. R. P. (1989). Application of p-N,N-dimethylphenylenediamine dihydrochloride for the determination of some diuretics. Talanta, 36, 491–494. DOI: 10.1016/0039-9140(89)80234-6.CrossRefGoogle Scholar
  28. Semaan, F. S., & Cavalheiro, É. T. G. (2006). Spectrophotometric determination of furosemide based on its complexation with Fe(III) in ethanolic medium using a flow injection procedure. Analytical Letters, 39, 2557–2567. DOI: 10.1080/00032710600824698.CrossRefGoogle Scholar
  29. Semaan, F. S., Neto, A. J. S., Lanças, F.M., & Cavalheiro, É. T. G. (2005a). Rapid HPLC-DAD determination of furosemide in tablets using a short home-made column. Analytical Letters, 38, 1651–1658. DOI: 10.1081/AL-200065813.CrossRefGoogle Scholar
  30. Semaan, F. S., De Sousa, R. A., & Cavalheiro, E. T. G. (2005b). Flow injection spectrophotometric determination of furosemide in pharmaceuticals by the bleaching of a permanganate carrier solution. Journal of Flow Injection Analysis, 22, 34–37.Google Scholar
  31. Semaan, F. S., Nogueira, P. A., & Cavalheiro, É. T. G. (2008). Flow-based fluorimetric determination of furosemide in pharmaceutical formulations and biological samples: use of micelar media to improve sensitivity. Analytical Letters, 41, 66–79. DOI: 10.1080/00032710701746782.CrossRefGoogle Scholar
  32. Shabir, G. A. (2003). Validation of high-performance liquid chromatography methods for pharmaceutical analysis: Understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. Journal of Chromatography A, 987, 57–66. DOI: 10.1016/S0021-9673(02)01536-4.CrossRefGoogle Scholar
  33. Shah, J., Jan, M. R., & Khan, M. A. (2005). Determination of furosemide by simple diazotization method in pharmaceutical preparations. Journal of the Chinese Chemical Society, 52, 347–352.Google Scholar
  34. Sevillano-Cabeza, A., Campíns-Falcó, P., & Serrador-García, M. C. (1997). Extractive-spectrophotometric determination of furosemide with sodium 1,2-naphthoquinone-4-sulphonate in pharmaceutical formulations. Analytical Letters, 30, 91–107. DOI: 10.1080/00032719708002293.Google Scholar
  35. Tescarollo Dias, I. L., Martins, J. L. S., & de Oliveira Neto, G. (2005). Furosemide determination by first-derivative spectrophotometric method. Analytical Letters, 38, 1159–1116. DOI: 10.1081/AL-200057227.Google Scholar
  36. The British Pharmacopoeia Commission (2002). The British pharmacopoeia (pp. 809–811, 2183–2184). London, UK: The Stationary Office.Google Scholar
  37. The United States Pharmacopoeial Convention (2000). The United States pharmacopoeia XXIV (pp. 756). Rockville, MD, USA: United States Pharmacopoeial Convention.Google Scholar
  38. Živanović, L., Agatonović, S., & Radulović, D. (1990). Spectrophotometric determination of furosemide as its Fe(III) complex in pharmaceutical preparations. Microchimica Acta, 100, 49–54. DOI: 10.1007/BF01244497.CrossRefGoogle Scholar

Copyright information

© Institute of Chemistry, Slovak Academy of Sciences 2010

Authors and Affiliations

  • Kalsang Tharpa
    • 1
  • Kanakapura Basavaiah
    • 1
    Email author
  • Kanakapura Basavaiah Vinay
    • 1
  1. 1.Department of ChemistryUniversity of MysoreManasagangothri, MysoreIndia

Personalised recommendations