Analysis of Arsenical Metabolites in Biological Samples

Araceli Hernandez‐Zavala1, Zuzana Drobna2, Miroslav Styblo2, David J. Thomas3

1 Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 2 Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 3 Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.33
DOI:  10.1002/0471140856.tx0433s42
Online Posting Date:  November, 2009
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Quantitation of iAs and its methylated metabolites in biological samples provides dosimetric information needed to understand dose‐response relations. Here, methods are described for separation of inorganic and mono‐, di‐, and trimethylated arsenicals by thin‐layer chromatography. This method has been extensively used to track the metabolism of the radionuclide [73As] in a variety of in vitro assay systems. In addition, a hydride generation‐cryotrapping‐gas chromatography‐atomic absorption spectrometric method is described for the quantitation of arsenicals in biological samples. This method uses pH‐selective hydride generation to differentiate among arsenicals containing trivalent or pentavalent arsenic. Curr. Protoc. Toxicol. 42:4.33.1‐4.33.17. © 2009 by John Wiley & Sons, Inc.

Keywords: arsenic; methylated arsenicals; [73As]; thin‐layer chromatography; hydride generation‐cryotrapping‐gas chromatography‐atomic absorption spectrometry; pH‐selective hydride generation; arsenic oxidation state

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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Arsenical Metabolites by Thin‐Layer Chromatography
  • Alternate Protocol 1: Analysis of Arsenical Metabolites by Hydride Generation‐Atomic Absorption Spectrophotometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Analysis of Arsenical Metabolites by Thin‐Layer Chromatography

  • Samples (see unit 4.32)
  • 0.2 M CuCl/0.2 N HCl
  • 30% (v/v) H 2O 2
  • Baker‐flex PEI‐F cellulose TLC plates (20 × 20–cm) with fluorescent indicator (J.T. Baker)
  • Isopropanol, HPLC grade or equivalent
  • Acetic acid, glacial, analytical grade or equivalent
  • Acetone, HPLC grade or equivalent
  • 0.5‐ml heat‐resistant microcap tubes with lid locks or clips
  • 100°C incubator
  • Benchtop centrifuge
  • Ice bath
  • Microcon concentrators with a nominal MWCO of 10 kDa (Microcon YM‐10 Centrifugal Filter Unit; Millipore)
  • 18‐G needles
  • Soft lead pencil
  • Micropipettor with a narrow tip or a capillary tube
  • Glass TLC tank (25 × 29 × 9–cm)
  • Chemical fume hood
  • Phosphorimager or Radio‐TLC scanner
NOTE: High‐grade chemicals should be used for preparation of all reagents used in TLC separations described here. This is especially important if one intends to use the TLC methods to separate stable (i.e., nonradiolabeled) arsenicals. A 0.2 M CuCl solution in 0.2 N HCl is used for extraction of arsenicals from biological samples. Hydrogen peroxide (30%) is needed for oxidation of extracted samples.NOTE: Additional materials, chemicals, or equipment will be needed for TLC analysis of nonradioactive arsenicals.

Alternate Protocol 1: Analysis of Arsenical Metabolites by Hydride Generation‐Atomic Absorption Spectrophotometry

  • Chromosorb WAW‐dimethyldichlorosilane 46/60 with 15% OV‐3 (Supelco)
  • Rejuv‐8 silylating reagent (Sigma)
  • Liquid nitrogen
  • Deionized water, 18 MΩ cm−1 or better
  • 4% (w/v) NaBH 4 (minimum 98%; EMD Chemicals) in 0.02 M NaOH
  • 6 N HCl [prepared from reagent‐grade concentrated (38%) HCl]
  • Antifoam B silicone emulsion (J.T. Baker)
  • Samples (see unit 4.32)
  • 2.5 M Tris⋅Cl, pH 6 ( appendix 2A)
  • As calibration standards: sodium arsenate (96% pure, Sigma); sodium arsenite (99% pure, Sigma); sodium methylarsonate (monosodium acid methane arsonate, 98% pure, Chem Service); dimethylarsinic acid (98% pure, Strem Chemicals)
  • Ethanol
  • 30‐cm long borosilicate glass U‐tube (4‐mm i.d. and 6‐mm o.d.)
  • 1.6 Ω ft−1 Ni80/Cr20 wire (Omega Engineering)
  • Plugs of silanized glass wool
  • Helium, high‐purity gas in cylinder
  • Borosilicate tubing (6‐mm i.d.)
  • 4‐way Teflon valve
  • 3‐way connector for carrier gas lines
  • Computerized atomic absorption spectrometer equipped with an air/hydrogen flame unit (e.g., Perkin Elmer Model 5100 atomic absorption spectrometer or equivalent)
  • As electrodeless discharge lamp (System II, Perkin Elmer), operated per manufacturer's specifications in conjunction with a Perkin Elmer Model 5100 atomic absorption spectrometer)
  • Hydrogen, high‐purity gas in cylinders or from a hydrogen generator
  • Manual hydride generation (HG) system including:
    • Power source with a 30‐ to 60‐V output for heating the U‐tube
    • Gas flow controllers (e.g., FMA‐2400 or 2600 Series, Omega Engineering)
    • Timer
    • Miscellaneous stoppers, valves, and tubing for carrier gas lines
  • 1‐liter Dewar flask
NOTE: Methylated trivalent arsenicals and TMAO are not commercially available. Previous studies have used oxomethylarsine (CH 3AsIIIO), iododimethylarsine [(CH 3) 2AsIIII], and trimethylarsine oxide synthesized by Professor William R. Cullen (University of British Columbia, Vancouver, Canada).
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Literature Cited

Literature Cited
   Braman, R.S. and Foreback, C.C. 1973. Methylated forms of arsenic in the environment. Science 182:1247‐1249.
   Buchet, J.P. and Lauwerys, R. 1985. Study of inorganic arsenic methylation by rat liver in vitro: Relevance for the interpretation of observations in man. Arch. Toxicol. 57:125‐129.
   Crecelius, E.A. 1977. Changes in the chemical speciation of arsenic following ingestion by man. Environ. Health Perspect. 19:147‐150.
   Del Razo, L.M., Styblo, M., Cullen, W.R., and Thomas, D.J. 2001. Determination of trivalent methylated arsenicals in biological matrices. Toxicol. Appl. Pharmacol. 174:282‐293.
   Devesa, V., Del Razo, L.M., Adair, B., Drobna, Z., Waters, S.B., Hughes, M.F, Styblo, M., and Thomas, D.J. 2004. Comprehensive analysis of arsenic metabolites by pH‐specific hydride generation atomic absorption spectrometry. J. Anal. At. Spectrom. 19:1460‐1467.
   Hernandez‐Zavala, A., Matousek, T., Drobna, Z., Walton, F., Adair, B.M., Dedina, J., Thomas, D.J., and Styblo, M. 2008. Speciation of arsenic in biological matrices by automated hydride generation‐cryotrapping‐atomic absorption spectrometry with multiple microflame quartz tube atomizer. J. Anal. At. Spectrom. 23:342‐351.
   Hirata, M., Mohri, T., Hisanaga, A., and Ishinishi, N. 1989. Conversion of arsenite and arsenate to methylarsenic and dimethylarsenic compounds by homogenates prepared from livers and kidneys of rats and mice. Appl. Organomet. Chem. 3:335‐341.
   Katano, S., Matsuo, Y., and Hanaoka, K. 2003. Arsenic compounds accumulated in pearl oyster Pinctada fucata. Chemosphere 53:245‐251.
   Matoušek, T., Hernández‐Zavala, A., Svoboda, M., Langerová, L., Adair, B.M., Drobná, Z., Thomas, D.J., Stýblo, M., and Dedina, J. 2008. Oxidation state specific generation of arsines from methylated arsenicals based on L‐cysteine treatment in buffered media for speciation analysis by hydride generation‐automated cryotrapping‐gas chromatography‐atomic absorption spectrometry with the multiatomizer. Spectrochim. Acta Part B: Atomic Spectroscopy 63:396‐406.
   Reay, P.F. and Asher, C.J. 1977. Preparation and purification of 74As‐labeled arsenate and arsenite for use in biological experiments. Anal. Biochem. 78:557‐560.
   Resano, M., Garcia Ruiz, E., Mihucz, V.G., Moricz, A.M., Zaray, G., and VanHaecke, F. 2007. Rapid screening method for arsenic speciation by combining thin layer chromatography and laser ablation‐inductively coupled plasma‐dynamic reaction cell‐mass spectrometry. J. Analyt. Atom. Spect. 22:1158‐1162.
   Styblo, M., Delnomdedieu, M., Hughes, M.F., and Thomas, D.J. 1995. Identification of methylated metabolites of inorganic arsenic by thin‐layer chromatography. J. Chromat. B 668:21‐29.
   Styblo, M., Hughes, M.F., and Thomas, D.J. 1996. Liberation and analysis of protein‐bound arsenicals. J. Chromat. B 677:161‐166.
   Waters, S.B., Devesa, V., Fricke, M., Creed, J., Styblo, M., and Thomas, D.J. 2004. Glutathione modulates recombinant rat arsenic (+3 oxidation state) methyltransferase‐catalyzed formation of trimethylarsine oxide and trimethylarsine. Chem. Res. Toxicol. 17:1621‐1629.
   Yoshinaga, J., Chatterjee, A., Shibata, Y., Morita, M., and Edmonds, J.S. 2000. Human urine certified reference material for arsenic speciation. Clin. Chem. 46:1781‐1786.
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