, Volume 75, Issue 1–2, pp 17–23 | Cite as

Sample Treatment Approaches for Trace Level Determination of Cesium in Hepatitis B Vaccine by Suppressed Ion Chromatography

  • Kulamani DashEmail author
  • Shanmugam Thangavel
  • Lori Rastogi
  • Shrinivas M. Dhavile
  • Jayaraman Arunachalam


The hepatitis B surface antigen manufactured by recombinant DNA technology is extracted from the culture media by density gradient centrifugation using cesium salts. Cesium is considered to be toxic, because it affects active ion transport by blocking potassium channels. The residual trace levels of cesium in hepatitis B vaccine samples are determined by suppressed ion chromatography. Hepatitis B vaccines contain various buffer salts, aluminum-containing adjuvants, proteins and traces of iron. The polyvalent cations (Al3+, Fe3+) and proteins degrade the chromatographic performance in terms of decreased retention time and poor reproducibility. Different sample preparation approaches were evaluated with the aim of eliminating these foulants: (1) filtration, (2) digestion and (3) digestion-protein precipitation. Quantitative elimination of these foulants was achieved in the digestion-protein precipitation sample clean-up approach. Cesium was separated on the IonPac CS17 column with suppressed conductivity detection. The results of the ion chromatography (IC) method were compared with ICP-MS analysis. The precision of determination was better than 6.5% (relative standard deviation) with a method detection limit of 45 ng mL−1. The expanded uncertainty in the measurement at 95% confidence level (coverage factor 2) is better than 16.3%.


Hepatitis B vaccine Cesium Polyvalent cations Proteins Suppressed ion chromatography 



Grateful acknowledgement is made to Dr. T. Mukherjee, Director, Chemistry Group, BARC, for his keen interest and encouragement throughout this work. We express our sincere thanks to Director, C-MET, Hyderabad for giving permission to carry out ICP-MS measurements at their facility.


  1. 1.
    Hepatitis B (2008) Fact sheet no. 204, World Health OrganizationGoogle Scholar
  2. 2.
    Kaplan NP, Colowick NP, Wu R (1980) Recombinant DNA (part F; Methods in enzymology), vol 68, Academic PressGoogle Scholar
  3. 3.
    Zhang H, Chen HT, Glisin V (2003) Isolation of DNA-free RNA, DNA, and proteins by cesium trifluoroacetate centrifugation. Biochem Biophys Res Commun 312:131–137CrossRefGoogle Scholar
  4. 4.
    Karlovsky P, de Cock AWAM (1991) Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs. Anal Biochem 194:192–197CrossRefGoogle Scholar
  5. 5.
    Seiler HG, Sigel A, Sigel H (1994) In: Seiler HG (ed) Handbook on metals in clinical and analytical chemistry Marcel Dekker pp 627–630Google Scholar
  6. 6.
    Pinsky C, Bose R, Taylor JR, McKee JSC, Lapointe C, Birchall J (1981) Cesium in mammals: acute toxicity, organ changes and tissue accumulation. J Environ Sci Health A 16:549–567CrossRefGoogle Scholar
  7. 7.
    El-Yazigi A, Martin C, Siqueira E (1988) Concentrations of chromium, cesium, and tin in cerebrospinal fluid of patients with brain neoplasms, leukemia or other non-cerebral malignancies, and neurological diseases. Clin Chem 34:1084–1086Google Scholar
  8. 8.
    Anderson P, Davidson CM, Littlejohn D, Ure AM, Shand CA, Cheshire MV (1996) The determination of cesium and silver in soil and fungal fruiting bodies by electrothermal atomic absorption spectrometry. Anal Chim Acta 327:53–60CrossRefGoogle Scholar
  9. 9.
    Robb P, Owen LMW, Crews HM (1995) Stable isotope approach to fission product element studies of soil-to-plant transfer and in vitro modelling of ruminant digestion using inductively coupled plasma mass spectrometry. J Anal At Spectrom 10:625–629CrossRefGoogle Scholar
  10. 10.
    Oughton D, Day J (1993) Determination of cesium, rubidium and scandium in biological and environmental materials by neutron activation analysis. Radioanal Nucl Chem 174:177–185CrossRefGoogle Scholar
  11. 11.
    Butcher DJ, Sneddon JA (1998) Practical guide to graphite furnace atomic absorption spectrometry, chemical analysis: a series of monographs on analytical chemistry and its applications (vol 149). Wiley, New YorkGoogle Scholar
  12. 12.
    Sarzanini C (1999) Liquid chromatography: a tool for the analysis of metal species. J Chromatogr A 850:213–228CrossRefGoogle Scholar
  13. 13.
    Sarzanini C, Bruzzoniti MC (2001) Metal species determination by ion chromatography. Trends Anal Chem 20:304–310CrossRefGoogle Scholar
  14. 14.
    Buldini PL, Cavalli S, Sharma JL (2002) Matrix removal for the ion chromatographic determination of some trace elements in milk. Microchem J 72:277–284CrossRefGoogle Scholar
  15. 15.
    Vanatta LE, Coleman DE (2002) Reproducible digestion method for ion-chromatographic analysis of anions in 30% hydrogen peroxide. J Chromatogr A 956:23–33CrossRefGoogle Scholar
  16. 16.
  17. 17.
    Dash K, Thangavel S, Chaurasia SC, Arunachalam J (2009) Determination of mercury in hepatitis-B vaccine by electrothermal atomic absorption spectrometry (ETAAS) using colloidal palladium modifier. At Spectrosc 30:26–31Google Scholar
  18. 18.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  19. 19.
    Iyer S, Robinett RSR, HogenEsch H, Hem SL (2004) Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant. Vaccine 22:1475–1479CrossRefGoogle Scholar
  20. 20.
    Garfin DE (2000) In: Ahuja S (ed) Handbook of bioseparations. Academic Press, SandiegoGoogle Scholar
  21. 21.
    Mikes O (1988) High performance liquid chromatography of biopolymers and bio-oligomers, separation of individual compound classes (part B). J Chromatogr Lib 41B:B1–B56Google Scholar
  22. 22.
    Matsushita S (1985) Determination of protein-free and protein-bound calcium and magnesium in biological samples by use of ultrafiltration and ion chromatography. Anal Chim Acta 172:249–255CrossRefGoogle Scholar
  23. 23.
    Haddad PR, Jackson PE (1990) Ion chromatography principles and applications. J Chromatogr Lib 46:409–453Google Scholar
  24. 24.
    Ding Y, Mou S (2002) Effects of common metal ions on the determination of anions by suppressed ion chromatography. J Chromatogr A 956:65–70CrossRefGoogle Scholar
  25. 25.
    Christian GD (2003) Analytical Chemistry, 6th edn. Wiley, SingaporeGoogle Scholar
  26. 26.
    Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143CrossRefGoogle Scholar
  27. 27.
    Material safety data sheet, Thimerosal, Science Lab.comGoogle Scholar
  28. 28.
    Gustavo González A, Ángeles Herrador M (2007) A practical guide to analytical method validation, including measurement uncertainty and accuracy profiles. Trends Anal Chem 26:227–238CrossRefGoogle Scholar
  29. 29.
    Montaser A (1998) Inductively coupled plasma mass spectrometry. Wiley-VCH, New YorkGoogle Scholar
  30. 30.
    EURACHEM/CITAC guide CG 4: Accessed 4 Jul 2011

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Kulamani Dash
    • 1
    Email author
  • Shanmugam Thangavel
    • 1
  • Lori Rastogi
    • 1
  • Shrinivas M. Dhavile
    • 1
  • Jayaraman Arunachalam
    • 1
  1. 1.National Centre for Compositional Characterization of Materials (CCCM)Bhabha Atomic Research CentreHyderabadIndia

Personalised recommendations