Chromatographic Analysis of Amino and Organic Acids in Physiological Fluids to Detect Inborn Errors of Metabolism

Michael Woontner1, Stephen I. Goodman1

1 University of Colorado Health Sciences Center, Aurora, Colorado
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.2
DOI:  10.1002/0471142905.hg1702s51
Online Posting Date:  November, 2006
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Abstract

This unit describes methods for the preparation of samples for analysis of physiological amino acids and organic acids. Amino acids are analyzed by ion‐exchange chromatography using an automated system. Organic acids are analyzed by gas‐chromatography/mass spectrometry (GC‐MS). Analysis of amino and organic acids is necessary to detect and monitor the treatment of many inborn errors of metabolism.

Keywords: amino acids; organic acids; LC; GC‐MS

     
 
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Table of Contents

  • Basic Protocol 1: Quantitative Determination of Amino Acids in Physiological Fluids
  • Basic Protocol 2: Analysis of Organic Acids in Urine
  • Alternate Protocol 1: Analysis of Organic Acids in Plasma or Serum
  • Basic Protocol 3: Quantitative Determination of Orotic Acid in Urine
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Quantitative Determination of Amino Acids in Physiological Fluids

  Materials
  • Patient specimen: serum, plasma, urine, or cerebrospinal fluid
  • 9.33% (w/v) sulfosalicylic acid (SSA)
  • Diluent buffer: Li‐D high‐performance amino acid analysis buffer, pH 3.3 (purchased as liquid from Beckman)
  • Internal standard: 0.5 mM D‐glucosaminic acid in Li‐D
  • Column buffers: Li‐A (pH 2.83), Li‐B (pH 3.60), and Li‐C (pH 3.75) high‐performance amino acid analysis buffers, and Li‐R column regeneration buffer (purchased as liquids from Beckman)
  • Ninhydrin reagent (Nin‐RX plus additive, Beckman)
  • Centrifugal filter device (0.22‐µm filter/tube unit to fit microcentrifuge, e.g., Costar Spin‐X LC)
  • Amino acid analyzer (Beckman 6300 instrument)
  • Dedicated computer and analytical software (amino acid analyzer software or any graphical software for analysis of chromatography)

Basic Protocol 2: Analysis of Organic Acids in Urine

  Materials
  • Patient specimen: random urine (i.e., collected at any time), 2 ml
  • External standard: 0.25 mg/ml n‐tetracosane in ethyl acetate
  • Internal standard: 0.5 mg/ml dimethylmalonic acid, in water
  • 6 N hydrochloric acid (HCl)
  • Diethyl ether (highest purity available)
  • Ethyl acetate (highest purity available)
  • Sodium sulfate, anhydrous
  • Nitrogen source
  • N,Obis[trimethylsilyl]trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS), e.g., Pierce
  • 98:1:1 (v/v/v) cyclohexane/pyridine/hexamethyldisilazane
  • Creatinine assay instrument (e.g., Beckman Coulter Creatinine 2 Analyzer), optional
  • 10‐ml round‐bottom extraction tubes (Kimble/Kontes with flathead TS 13 glass stoppers)
  • 13‐ml conical‐bottom concentration tubes (Kimble/Kontes)
  • Tabletop centrifuge: e.g., IEC Clinical Model with six‐place swinging‐bucket rotor
  • Dry block heater for conical‐bottom tubes (18‐mm holes for the tubes specified above)
  • Gas chromatograph (GC) vials
  • Gas chromatograph with a mass spectrometer as the detector (e.g., Agilent 6890N gas chromatograph with an Agilent 5973 mass detector)

Alternate Protocol 1: Analysis of Organic Acids in Plasma or Serum

  Materials
  • Patient specimen: random urine (i.e., collected at any time)
  • Internal standard: 15N 2‐orotic acid (Cambridge Isotope Laboratories), 50 µg/ml in water
  • 6 N hydrochloric acid (HCl)
  • Diethyl ether (highest purity available)
  • Ethyl acetate (highest purity available)
  • Sodium sulfate, anhydrous
  • Nitrogen source
  • N,Obis[trimethylsilyl]trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS), e.g., Pierce
  • 10‐ml round‐bottom extraction tubes (Kimble/Kontes; with flathead TS 13 glass stoppers)
  • 13‐ml conical‐bottom concentration tubes (Kimble/Kontes)
  • Tabletop centrifuge: e.g., IEC Clinical Model with six‐place swinging‐bucket rotor
  • Dry block heater for conical‐bottom tubes (18‐mm holes for the tubes specified above)
  • Gas chromatograph (GC) vials
  • Gas chromatograph with a mass spectrometer as the detector (e.g., Agilent 6890N gas chromatograph with an Agilent 5973 mass detector)
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Figures

Videos

Literature Cited

   Bremer, H.J., Duran, M., Kamerling, J.P., Przyrembel, H., and Wadman, S. 1981. Disturbances of Amino Acid Metabolism: Clinical Chemistry and Diagnosis. Urban and Schwarzenberg, Baltimore.
   Deyl, Z., Hyanek, J., and Horakova, M. 1986. Profiling of amino acids in body fluids and tissues by means of liquid chromatography. J. Chromatogr. 379:177‐250.
   Gibson, K.M., Aramaki, S., Sweetman, L., Nyhan, W.L., DeVivo, D.C., Hodson, A.K., and Jakobs, C. 1990. Stable isotope dilution analysis of 4‐hydroxybutyric acid: An accurate method for quantification in physiological fluids and the prenatal diagnosis of 4‐hydroxybutyric aciduria. Biomed. Environ. Mass Spectrom. 19:89‐93.
   Hoffmann, G., Aramaki, S., Blum‐Hoffmann, E., Nyhan, W.L., and Sweetman, L. 1989. Quantitative analysis for organic acids in biological samples: Batch isolation followed by gas chromatographic‐mass spectrometric analysis. Clin. Chem. 35:587‐595.
   Hoffmann, G.F., Sweetman, L., Bremer, H.J., Hunnemen, D.H., Hyanek, J., Kozich, V., Lehnert, W., Nyhan, W.L., Speidel, I., Trefz, F.K., and Gibson, K.M. 1991. Facts and artefacts in mevalonic aciduria: Development of a stable isotope dilution GC‐MS assay for mevalonic acid and its application to physiological fluids, tissue samples, prenatal diagnosis and carrier detection. Clin. Chim. Acta 198:209‐227.
   Markey, S.P., Urban, W.G., and Levine, S.P. 1974. Mass spectra of compounds of biological interest. Vol 1: Indices. Vol 2: Spectra. National Technical Information Service, Springfield, Va.
   Moore, S. and Stein, W.H. 1963. Chromatographic determination of amino acids by the use of automated recording equipment. Methods Enzymol. 6:819‐831.
   Scriver, C.R. and Rosenberg, L.E. 1973. Amino Acid Metabolism and Its Disorders. W.B. Saunders Company, Philadelphia.
   Shapira, E., Blitzer, M.G., Miller, J.B., and Affrick, D.K. 1989. Biochemical Genetics: A Laboratory Manual. Oxford University Press. New York.
   Thompson, J.A. and Markey, S.P. 1975. Quantitative metabolic profiling of urinary organic acids by gas chromatography‐mass spectrometry: Comparison of isolation methods. Anal. Chem. 47:1313‐1321.
   Tuchman, M., Whitley, C.B., Ramnaraine, M.L., Bowers, L.D., Fregien, K.D., and Krivit, W. 1984. Determination of urinary succinylacetone by capillary gas chromatography. J. Chromatogr. Sci. 22:211‐215.
Key References
   Goodman, S.I. and Markey, S.P. 1981. Diagnosis of Organic Acidemias by Gas Chromatography‐Mass Spectrometry. Alan R. Liss, Inc. New York.
  Contains discussion of GC‐MS as well as lists of common ions and examples of common spectra.
   Hommes,  F.A. (ed.) 1991. Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual. Wiley‐Liss, New York.
  Contains detailed chapters on amino acid and organic acid protocols.
   Sweetman, L. 1984. Qualitative and Quantitative Analysis of Organic Acids in Physiologic Fluids for Diagnosis of the Organic Acidurias. In Abnormalities in Amino Acid Metabolism in Clinical Medicine (W. L. Nyhan). Appleton‐Century‐Crofts. Norwalk, Conn.
  An outstanding discussion of issues related to methodology.
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