Quantitative Speciation of Selenium in Human Blood Serum and Urine with AE- RP- and AF-HPLC-ICP/MS

  • Jeong, Ji-Sun (Department of Chemistry Education, Korea National University of Education) ;
  • Lee, Jonghae (Division of Metrology for Quality of Life, Korea Research Institute of Standards and Science) ;
  • Pak, Yong-Nam (Department of Chemistry Education, Korea National University of Education)
  • Received : 2013.08.16
  • Accepted : 2013.10.15
  • Published : 2013.12.20


Various separation modes in HPLC, such as anion exchange (AE), reversed-phase (RP), and affinity (AF) chromatography were examined for the separation of selenium species in human blood serum and urine. While RP- and AE-HPLC were mainly used for the separation of small molecular selenium species, double column AF-HPLC achieved the separation of selenoproteins in blood serum efficiently. Further, the effluent of AF-HPLC was enzymatically hydrolyzed and then analyzed with RP HPLC for selenoamino acid study. The versatility of the hybrid technique makes the in-depth study of selenium species possible. For quantification, post column isotope dilution (ID) with $^{78}Se$ spike was performed. ORC ICP/MS (octapole reaction cell inductively coupled plasma/mass spectrometry) was used with 4 mL $min^{-1}$ Hydrogen as reaction gas. In urine sample, inorganic selenium and SeCys were identified. In blood serum, selenoproteins GPx, SelP and SeAlb were detected and quantified. The concentration for GPx, SelP and SeAlb was $22.8{\pm}3.4\;ng\;g^{-1}$, $45.2{\pm}1.7\;ng\;g^{-1}$, and $16.1{\pm}2.2\;ng\;g^{-1}$, respectively when $^{80}Se/^{78}Se$ was used. The sum of these selenoproteins ($84.1{\pm}4.4\;ng\;g^{-1}$) agrees well with the total selenium concentration measured with the ID method of $87.0{\pm}3.0\;ng\;g^{-1}$. Enzymatic hydrolysis of each selenium proteins revealed that SeCys is the major amino acid for all three proteins and SeMet is contained in SeAlb only.


Selenium speciation;Seleno-proteins;HPLC-ICP/MS;Blood serum;Post column isotope dilution


  1. Tolu, J.; Hecho, I. L.; Bueno, M.; Thiry, Y.; Potin-Gautier, M., Anal. Chim. Acta 2011, 684, 126-133.
  2. Pyrzynska, K. Microchim. Acta 2002, 140, 55-62.
  3. Chen, Y. W.; Belzile, N. Anal. Chim. Acta 2010, 671, 9-26.
  4. Giusti, P.; Schaumloffel, D.; Preud'homme, H.; Szpunar, J.; Lobinski, R. J. Anal. At. Spectrom. 2006, 21, 26-32.
  5. Duan, J.; Hu, B. J. Mass Spectrom. 2009, 44, 605-612.
  6. B'Hymer, C.; Caruso, J. A. J. Chromatogr. A 2006, 1114, 1-20.
  7. Emteborg, H.; Bordin, G.; Rodriguez, A. R. Analyst 1998, 123, 245-253.
  8. Harwood, J. J.; Su, W. J. Chromatogr. A 1997, 788, 105-111.
  9. Mazej, D.; Falnoga, I.; Veber, M.; Stibilj, V. Talanta 2006, 68, 558-568.
  10. Dietz, C.; Landaluze, J. S.; Ximenez-Embun, P.; Madrid-Albarran, Y.; Camara, C. J. Anal. At. Spectrom. 2004, 19, 260-266.
  11. Shrivas, K.; Patel, D. K. Food Chem. 2011, 124, 1673-1677.
  12. Ordonez, Y. N.; Montes-Bayon, M.; Blanco-Gonzalez, E.; Sanz-Medel, A. Anal. Chem. 2010, 82, 2387-2394.
  13. Zoorob, G. K.; McKiernan, J. W.; Caruso, J. A. Mikrochim. Acta 1998, 128, 145-168.
  14. Montes-Bayon, M.; DeNicola, K.; Caruso, J. A. J. Chromatogr. A 2003, 1000, 457-476.
  15. Reyes, H. L.; Garcia-Ruiz, S.; Tonietto, B. G.; Godoy, J. M.; Alonso, J. I. G.; Sanz-Medel, A. J. Braz. Chem. Soc. 2009, 30, 1878-1886.
  16. Rappel, C.; Schaumloffel, D. Anal. Chem. 2009, 81, 385-393.
  17. Diaz Huerta, V.; Fernandez Sanchez, M. L.; Sanz-Medel, A. J. Anal. At. Spectrom. 2004, 19, 644-651.
  18. Pedrero, Z.; Madrid, Y. Analytica Chimica Acta 2009, 634, 135-152.
  19. Andoh, M.; Hirashima, H.; Maeda, K.; Hata, O.; Inatomi, T.; Tsujikawa, M.; Sasaki, K.; Takahashi, Y,; Fujiyama, Y. Nutrition 2005, 21, 573-581.
  20. Jitaru, P.; Goenaga-Infante, H.; Vaslin-Reimann, S.; Fisicaro, P. Anal. Chim. Acta 2010, 657, 100-107.
  21. Suzuki, Y.; Sakai, T.; Furuta, N. Anal. Sci. 2012, 28, 1-5.
  22. Jitaru, P.; Cozzi, G.; Gambaro, A.; Cescon, P.; Barbante, C. Anal. Bioanal. Chem. 2008, 391, 661-668.
  23. Jitaru, P.; Cozzi, G.; Seraglia, R.; Traldi, P.; Cescon, P.; Barbante, C. Anal. Methods 2010, 2, 1382-1387.
  24. Jitaru, P.; Prete, M.; Cozzi, G.; Turetta, C.; Cairns, W.; Seraglia, R.; Traldi, P.; Cescon, P.; Barbante, C. J. Anal. At. Spectrom. 2008, 23, 402-406.
  25. Rodriguez-Gonzalez, P.; Marchante-Gayon, J. M.; Alonso, J. I. G.; Sanz-Medel, A. Spectrochim. Acta B 2005, 60, 151-207.
  26. Wallschlager, D.; London, J. J. Anal. At. Spectrom. 2004, 19, 1119-1127.
  27. Roman, M.; Jitaru, P.; Agostini, M.; Cozzi, G.; Pucciarelli, S.; Nitti, D.; Bedin, C.; Barbante, C. Michrochem. Journal 2012, 105, 124-132.
  28. Kotrebai, M.; Tyson, J. F.; Block, E.; Uden, P. C. J. Chromatogr. A 2000, 866, 51-63.
  29. Bierla, K.; Szpunar, J.; Lobinski, R. Anal. Chim. Acta 2008, 624, 195-202.
  30. Afton, S.; Kubachka, K.; Catron, B.; Caruso, J. A. J. Chromatogr. A 2008, 1208, 156-163.
  31. Kuehnelt, D.; Kienzl, N.; Juresa, D.; Kevin, A. F. J. Anal. At. Spectrom. 2006, 21, 1264-1270.
  32. Stewart, I. I. Spectrochim. Acta B 1999, 54, 1649-1659.
  33. Rosenberg, E. J. Chromatogr. A 2003, 1000, 841-889.
  34. Yang, X.; Tian, Y.; Ha, P.; Gu, L. Wei Sheng Yan Jiu 1997, 26, 13-16.
  35. Reyes, H.; Marchante, L.; Gayon, J. M.; Garcia Alonso, J. I.; Sanz-Medel, A. J. Anal. At. Spectrom. 2003, 18, 1210-1216.
  36. Cho, H.; Pak, Y. J. Korean Chem. Soc. 2011, 55, 472-477.

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