Journal of the Iranian Chemical Society

, Volume 15, Issue 7, pp 1603–1612 | Cite as

Simultaneous spectrophotometric determination of thiamine and pyridoxine in multivitamin dosage forms using H-point standard addition and Vierodt᾿s methods

  • O. H. Rebwar
  • Y. M. Hunar
  • S. J. Hijran
Original Paper


Two simple, reliable and accurate spectrophotometric methods were described for the simultaneous determination of thiamine hydrochloride (THC) and pyridoxine hydrochloride (PYH) in pharmaceutical formulation without any pre-separation procedures. These methods are based on the difference in the color observed from diazotization coupling methods of THC and PYH. When the diazonium salts formed from the reaction of HNO2 with p-nitroaniline (PNA), they were coupled with THC and PYH in sodium hydroxide medium and the formed dyes had maximum absorption bands at 452 nm for THC and 482 nm for PYH. All experimental parameters were optimized to reduce experimental error. The first method was based on the Vierodt᾿s method, in which the two wavelengths 452 and 482 nm λmax of THC and PYH, respectively, were selected for the formation of simultaneous equation. In the second method the H-point standard addition method (HPSAM) was applied, when absorbances at wavelengths pair, 452 and 500 nm, were monitored with the addition of a standard solution of THC to a solution containing a fixed concentration of THC and PYH. The results from applying Vierodt᾿s and HPSA methods showed that THC and PYH can be determined simultaneously in the concentration range 1.5–11.0 μg mL−1 (LOD = 0.48 μg mL−1) and 0.4–5.0 μg mL−1 (LOD = 0.23 μg mL−1), respectively. The methods were successfully applied for simultaneous determination of THC and PYH in different synthetic and pharmaceutical products. To assess the obtained vitamins (THC and PYH) analysis by both methods (Vierodt᾿s and HPSAM) in the several pharmaceutical formulations, the results are compared with that obtained by HPLC method as a reference, and good agreements between the results indicate the reliability of the proposed methods.


Thiamine hydrochloride Pyridoxine hydrochloride Vierodt᾿s method H-point standard addition method Simultaneous determination 



The authors would like very much to thank respectful head and staff of the Chemistry Department, College of Science University of Salahaddin, Erbil, Iraq.


  1. 1.
    A. Afkhami, M. Abbasi-Tarighat, M. Bahram, H. Abdollahi, A new strategy for solving matrix effect in multivariate calibration standard addition data using combination of H-point curve isolation and H-point standard addition methods. Anal. Chim. Acta 613, 144–151 (2008). CrossRefGoogle Scholar
  2. 2.
    T.N. Al-Sabha, A.A. Bunaciu, H.Y. Aboul-Enein, H-point standard addition method (HPSAM) in simultaneous spectrophotometric determination of binary mixtures: an overview. Appl. Spectrosc. Rev. 46, 607–623 (2011). CrossRefGoogle Scholar
  3. 3.
    M. Amin, J. Reusch, High-performance liquid chromatography of water-soluble vitamins. J. Chromatogr. A 390, 448–453 (1987). CrossRefGoogle Scholar
  4. 4.
    R.D. Amrutkar, V.D. Rakibe, M. Amin Prakruti, Simultaneous estimation of thiamine hydrochloride and pyridoxine hydrochloride in multivitamin injection by UV-spectrophotometric method. Int. J. Res. Pharm. Biomed. Sci. 4, 1229–1232 (2013)Google Scholar
  5. 5.
    J.V. Andres, F.B. Reig, P.C. Falco, H-point standard additions method for analyte determination in ternary mixtures. Analyst 120, 299–304 (1995). CrossRefGoogle Scholar
  6. 6.
    J.S. Annino, R.W. Giese, Clinical Chemistry principle and Procedure, 4th edn. (Little Brown and Company, Boston, 1976)Google Scholar
  7. 7.
    P. Campíns-Falcó, F. Bosch-Reig, J. Verdú-Andrés, Evaluation and elimination of the “blank bias error” using the H-point standard addition method. Anal. Chim. Acta 270, 253–265 (1992). CrossRefGoogle Scholar
  8. 8.
    P. Campíns-Falcó, F. Blasco Gómez, F. Bosch-Reig, The H-point and generalized H-point standard additions methods for flow injection procedures. Talanta 47, 193–202 (1998). CrossRefGoogle Scholar
  9. 9.
    E. Dinç, G. Kökdil, F. Onur, A comparison of matrix resolution method, ratio spectra derivative spectrophotometry and HPLC method for the determination of thiamine HCl and pyridoxine HCl in pharmaceutical preparation. J. Pharm. Biomed. Anal. 22, 915–923 (2000). CrossRefGoogle Scholar
  10. 10.
    M.A. Elsayed, S.F. Belal, A.M. Elwalily, H. Abdine, Spectrophotometric determination of tolbutamide, thiamine hydrochloride, and pyridoxine hydrochloride in combination products. J. Pharm. Sci. 68, 739–741 (1979)CrossRefGoogle Scholar
  11. 11.
    F. Feng, K. Wang, Z. Chen, Q. Chen, J. Lin, S. Huang, Flow injection renewable drops spectrofluorimetry for sequential determinations of Vitamins B1, B2 and B6. Anal. Chim. Acta 527, 187–193 (2004). CrossRefGoogle Scholar
  12. 12.
    L. Garcıa, S. Blázquez, M.P. San Andrés, S. Vera, Determination of thiamine, riboflavin and pyridoxine in pharmaceuticals by synchronous fluorescence spectrometry in organized media. Anal. Chim. Acta 434, 193–199 (2001). CrossRefGoogle Scholar
  13. 13.
    H.-Y. Gu, A.-M. Yu, H.-Y. Chen, Electrochemical behavior and simultaneous determination of vitamin B2, B6, and C at electrochemically pretreated glassy carbon electrode. Anal. Lett. 34, 2361–2374 (2001). CrossRefGoogle Scholar
  14. 14.
    S.A. Halvatzis, M. Timotheou-Potamia, Kinetic-Potentiometric determination of ascorbic acid, biotin, pyridoxine hydrochloride and thiamine hydrochloride with N-bromosuccinimide. Anal. Chim. Acta 227, 405–419 (1989). CrossRefGoogle Scholar
  15. 15.
    B. Hemmateenejad, Chemometrics in Iran. Chemom. Intell. Lab. Syst. 81, 202–208 (2006). CrossRefGoogle Scholar
  16. 16.
    I.I. Hewala, Determination of two binary vitamin mixtures using difference and derivative spectrophotometry. Anal. Lett. 26, 2217–2237 (1993). CrossRefGoogle Scholar
  17. 17.
    E. Hund, D.L. Massart, J. Smeyers-Verbeke, Evaluation of the H-point standard additions method (HPSAM) and the generalized H-point standard additions method (GHPSAM) for the UV-analysis of two-component mixtures. J. Pharm. Biomed. Anal. 21, 23–42 (1999). CrossRefGoogle Scholar
  18. 18.
    A.H. Kamal, S.F. El-Malla, S.F. Hammad, A review on UV spectrophotometric methods for simultaneous multicomponent analysis. Eur. J. Pharm. Med. Res. 3, 348–360 (2016)Google Scholar
  19. 19.
    R.L. Kirchmeier, R.P. Upton, Simultaneous determination of niacin, niacinamide, pyridoxine, thiamine, and riboflavin in multivitamin blends by ion-pair high-pressure liquid chromatography. J. Pharm. Sci. 67, 1444–1446 (1978). CrossRefGoogle Scholar
  20. 20.
    A. Lebiedzińska, M.L. Marszałł, J. Kuta, P. Szefer, Reversed-phase high-performance liquid chromatography method with coulometric electrochemical and ultraviolet detection for the quantification of vitamins B1 (thiamine), B6 (pyridoxamine, pyridoxal and pyridoxine) and B12 in animal and plant foods. J. Chromatogr. A 1173, 71–80 (2007). CrossRefGoogle Scholar
  21. 21.
    K. Li, Simultaneous determination of nicotinamide, pyridoxine hydrochloride, thiamine mononitrate and riboflavin in multivitamin with minerals tablets by reversed-phase ion-pair high performance liquid chromatography. Biomed. Chromatogr. 16, 504–507 (2002). CrossRefGoogle Scholar
  22. 22.
    J.F.C. Lima, M.C.B.S.M. Montenegro, A.M.R. Silva, Vitamins B1 and B6 tubular electrodes as FIA detectors; their use in the analysis of pharmaceutical products. J. Pharm. Biomed. Anal. 9, 1041–1046 (1991). CrossRefGoogle Scholar
  23. 23.
    C.K. Markopoulou, K.A. Kagkadis, J.E. Koundourellis, An optimized method for the simultaneous determination of vitamins B1, B6, B12, in multivitamin tablets by high performance liquid chromatography. J. Pharm. Biomed. Anal. 30, 1403–1410 (2002). CrossRefGoogle Scholar
  24. 24.
    M.L. Marszałł, A. Lebiedzińska, W. Czarnowski, P. Szefer, High-performance liquid chromatography method for the simultaneous determination of thiamine hydrochloride, pyridoxine hydrochloride and cyanocobalamin in pharmaceutical formulations using coulometric electrochemical and ultraviolet detection. J. Chromatogr. A 1094, 91–98 (2005). CrossRefGoogle Scholar
  25. 25.
    R.C. Martínez, F.B. Domínguez, I.M.S. García, J.H. Méndez, R.C. Orellana, R.S. Guzmán, Electrochemical response of a polypyrrole-dodecylsulphate electrode with multicharged cations and vitamins B1 and B6. Application as a microsensor in flow-injection analysis. Anal. Chim. Acta 336, 47–56 (1996). CrossRefGoogle Scholar
  26. 26.
    P. Ortega Barrales, A. Domínguez Vidal, M.L. Fernández de Córdova, A. Molina Díaz, Simultaneous determination of thiamine and pyridoxine in pharmaceuticals by using a single flow-through biparameter sensor. J. Pharm. Biomed. Anal. 25, 619–630 (2001). CrossRefGoogle Scholar
  27. 27.
    D. Ozdemir, E. Dinc, Determination of thiamine HCl and pyridoxine HCl in pharmaceutical preparations using UV-visible spectrophotometry and genetic algorithm based multivariate calibration methods. Chem. Pharm. Bull. (Tokyo) 52, 810–817 (2004)CrossRefGoogle Scholar
  28. 28.
    A.R. Pires, A.N. Araújo, J.A. Lopes, M.C.B.S.M. Montenegro, Simultaneous potentiometric determination of thiamine and pyridoxine in multivitamins using a single cyclodextrin-based thiamine-selective electrode. Anal. Lett. 42, 1923–1939 (2009). CrossRefGoogle Scholar
  29. 29.
    F.B. Reig, P.C. Falco, H-point standard additions method. Part 1. Fundamentals and application to analytical spectroscopy. Analyst 113, 1011–1016 (1988). CrossRefGoogle Scholar
  30. 30.
    M.C. Walker, B.E. Carpenter, E.L. Cooper, Simultaneous determination of niacinamide, pyridoxine, riboflavin, and thiamine in multivitamin products by high-pressure liquid chromatography. J. Pharm. Sci. 70, 99–101 (1981)CrossRefGoogle Scholar
  31. 31.
    Z.-R. Zhang, Y.X. Li, D.Y. Mao, V.V. Cosofret, Sensitive membrane electrodes for the determination of vitamin B1 and vitamin B6. J. Pharm. Biomed. Anal. 8, 385–388 (1990). CrossRefGoogle Scholar

Copyright information

© Iranian Chemical Society 2018

Authors and Affiliations

  1. 1.Department of Chemistry, College of ScienceUniversity of SalahaddinErbīlIraq

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