Detection and Quantification of Neurotransmitters in Dialysates

Agustin Zapata1, Vladimir I. Chefer1, Sandrine Parrot2, Luc Denoroy2

1 National Institute on Drug Abuse Intramural Research Program, National Institutes of Health, Baltimore, Maryland, 2 AniRa‐Neurochem and BioRaN, Centre de Recherche en Neurosciences de Lyon, Lyon, France
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.4
DOI:  10.1002/0471142301.ns0704s63
Online Posting Date:  April, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Sensitive analytical methods are needed for the separation and quantification of neurotransmitters obtained in microdialysate studies. This unit describes methods that permit quantification of nanomolar concentrations of monoamines and their metabolites (high‐performance liquid chromatography [HPLC] electrochemical detection), acetylcholine (HPLC‐coupled to an enzyme reactor), and amino acids (HPLC‐fluorescence detection, capillary electrophoresis with laser‐induced fluorescence detection). Curr. Protoc. Neurosci. 63:7.4.1‐7.4.35. © 2013 by John Wiley & Sons, Inc.

Keywords: high‐performance liquid chromatography (HPLC); electrochemical detection; capillary electrophoresis; laser fluorescence

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Quantification of Dopamine and Serotonin by HPLC/EC
  • Alternate Protocol 1: Quantification of Dopamine and Serotonin by UHPLC/EC
  • Basic Protocol 2: Quantification of Acetylcholine by HPLC/EC
  • Alternate Protocol 2: Quantification of Acetylcholine by HPLC/EC Using a Peroxidase‐Wired Electrode
  • Basic Protocol 3: Quantification of OPA‐Derivatized Amino Acids in Microdialysates by HPLC with Fluorescence Detection
  • Alternate Protocol 3: Quantification of OPA‐Derivatized Glutamate, Aspartate, or GABA in Microdialysates by HPLC with Fluorescence Detection
  • Basic Protocol 4: Quantification of Excitatory Amino Acids in Microdialysates by Capillary Electrophoresis with Laser‐Induced Fluorescence
  • Alternate Protocol 4: Quantification of Amino Acids by Micellar Electrokinetic Chromatography with Laser‐Induced Fluorescence
  • Support Protocol 1: Manual Derivatization of Microdialysates with NDA/NaCN
  • Support Protocol 2: Online Derivatization of Microdialysates with NDA/NaCN
  • Support Protocol 3: In‐Machine Automatic Derivatization of Microdialysates with NDA/NaCN
  • Support Protocol 4: In‐Capillary Automatic Derivatization of Microdialysates with NDA/NaCN
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Quantification of Dopamine and Serotonin by HPLC/EC

  Materials
  • Mobile phase I or II (see reciperecipes)
  • HPLC‐grade water
  • Monoamine standards (see recipe)
  • Microdialysis samples (units 7.2& 7.3), frozen
  • 10 µM 3,4‐dihydroxybenzylamine (DHBA, internal standard, optional)
  • Filtration apparatus:
    • Borosilicate glass vacuum flask
    • Fritted filter support and spring clamp
    • 0.22‐µm filter discs (e.g., Millipore)
    • Vacuum source
  • Ultrapure helium with regulator and Teflon tubing
  • HPLC/EC system consisting of:
    • HPLC dual‐piston pump equipped with pulse dampener
    • C18 column: 5‐µm particle size, 100‐mm length × 1‐mm i.d. (Bioanalytical Systems, for mobile phase I) or 3‐µm particle size, 100‐mm length × 2‐mm i.d. (UniJet LC column, Bioanalytical Systems, for mobile phase II)
    • Manual injector (20‐µl sample loop/gas‐tight HPLC glass microsyringe) or autoinjector
    • BAS LC‐4C amperometric electrochemical detector (or equivalent) equipped with a radial flow cell with a glassy carbon working electrode (Bioanalytical Systems)
    • Pen recorder and integrator (or equivalent)
    • Computer software program

Alternate Protocol 1: Quantification of Dopamine and Serotonin by UHPLC/EC

  • Mobile phase VII (see recipe)
  • UHPLC/EC system consisting of:
    • C18 Kinetex column, 1.7‐µm particle size, 100‐mm length × 2‐mm i.d. (Phenomenex)
    • SIL‐30AC autoinjector and LC‐30AD pump from Shimadzu (Nexera series)
    • Decade 2 amperometric electrochemical detector equipped with a VT03 cell made with a 2‐mm glassy carbon working electrode, an Ag/AgCl electrode, and a 25‐µm spacer (Antec‐Leyden)
    • Computer software program

Basic Protocol 2: Quantification of Acetylcholine by HPLC/EC

  Materials
  • Mobile phase III (see recipe)
  • HPLC‐grade water
  • 20% (v/v) nitric acid
  • 30% (v/v) acetic acid
  • Acetylcholine and choline standards (see recipe)
  • Microdialysis samples (units 7.2& 7.3), frozen
  • Filtration apparatus:
    • Borosilicate glass vacuum flask
    • Fritted filter support and spring clamp
    • 0.22‐µm filter discs (e.g., Millipore)
    • Vacuum source
  • Ultrapure helium with regulator and Teflon tubing
  • HPLC/EC system consisting of:
    • HPLC dual‐piston pump equipped with pulse dampener
    • Analytical column (UniJet, Bioanalytical Systems, cat. no. MF‐8904)
    • Guard column (UniJet; Bioanalytical Systems, cat. no. MF‐8935)
    • Immobilized enzyme reactor (IMER, Bioanalytical Systems, cat. no. MF‐8903)
    • Amperometric electrochemical detector equipped with platinum electrode and Ag/AgCl reference electrode (Bioanalytical Systems)
    • 20‐µl injection loop
    • Pen recorder and integrator (or equivalent)
    • Computer software program

Alternate Protocol 2: Quantification of Acetylcholine by HPLC/EC Using a Peroxidase‐Wired Electrode

  • Peroxidase/redox polymer coating kit (Bioanalytical Systems, cat. no. MF‐2095)
  • Glassy carbon electrode (Bioanalytical Systems)

Basic Protocol 3: Quantification of OPA‐Derivatized Amino Acids in Microdialysates by HPLC with Fluorescence Detection

  Materials
  • Mobile phase IV (see recipe)
  • Borax buffer (see recipe)
  • 1 mg/ml OPA stock solution (see recipe)
  • 10% (v/v) β‐mercaptoethanol (see recipe)
  • Microdialysis samples (units 7.2& 7.3), frozen
  • Amino acid standards (see recipe)
  • Filtration apparatus:
    • Borosilicate glass vacuum flask
    • Fritted filter support and spring clamp
    • 0.22‐µm filter disks (e.g., Millipore)
    • Vacuum source
  • HPLC system consisting of:
    • 10‐ml gas‐tight HPLC priming syringe (e.g., Kloehn or Waters)
    • Chromatography pump (e.g., Waters 510) capable of delivering 1.0 ml/min at ≤4000 psi
    • Injector, automated (e.g., Waters 717 autosampler equipped with glass low‐volume inserts) or manual
    • C 18 column (Alltech Adsorbosphere, cat. no. 287095)
    • Fluorescence detector (e.g., Waters 420) equipped with 338‐ and 450‐nm excitation filters and 450‐nm emission filter
    • Data acquisition software (e.g., Waters Millennium)

Alternate Protocol 3: Quantification of OPA‐Derivatized Glutamate, Aspartate, or GABA in Microdialysates by HPLC with Fluorescence Detection

  • 40% and 100% (v/v) acetonitrile
  • Mobile phase V or VI (see reciperecipes)

Basic Protocol 4: Quantification of Excitatory Amino Acids in Microdialysates by Capillary Electrophoresis with Laser‐Induced Fluorescence

  Materials
  • HPLC‐grade water, autoclaved and filtered
  • 1% (w/v) NaOH
  • Background electrolyte I (see recipe)
  • NDA‐derivatized microdialysates, excitatory amino acid standards, and artificial cerebrospinal fluid (aCSF) (see Support Protocols protocol 91 protocol 124)
  • 1‐ml glass vials
  • CE‐LIF system consisting of:
    • CE instrument with 1 to 30 kV capability and refrigerated sampler (Beckman‐MDQ, Agilent 3D)
    • Fused silica capillary (50‐µm i.d., 340‐µm o.d., Polymicro Technology)
    • LIF detector (Picometrics Zetalif ) equipped with 410‐nm diode laser (Melles Griot) or 442‐nm He‐Cd laser (Melles Griot)
  • 500‐µl polyethylene PCR tubes or plastic or glass microtubes

Alternate Protocol 4: Quantification of Amino Acids by Micellar Electrokinetic Chromatography with Laser‐Induced Fluorescence

  Materials
  • 87 mM aqueous NaCN (4.264 mg/ml; store up to 1 week at 4°C in a capped glass vial)
  • Derivatization buffer (see recipe)
  • Microdialysis samples (units 7.2& 7.3), amino acid standards (see 7.4), and aCSF to be derivatized
  • Internal standard: 10 µM α‐aminoadipic acid (see recipe)
  • 2.925 mM NDA stock solution (see recipe)
  • 1‐ml glass vial with cap

Support Protocol 1: Manual Derivatization of Microdialysates with NDA/NaCN

  Materials
  • Epoxy glue
  • Microdialysis samples (units 7.2& 7.3), amino acid standards (see 7.4), and aCSF to be derivatized
  • Internal standard: 10 µM α‐aminoadipic acid or 50 µM cysteic acid (see reciperecipes)
  • NaCN/borate solution: 14.66 mM NaCN in 0.5 M borate buffer, pH 8.7
  • 2.925 mM NDA stock solution (see recipe)
  • Fused‐silica capillary tubing (Polymicro Technology):
    • One with 40‐µm i.d., 105‐µm o.d.
    • Four with 75‐µm i.d., 150‐µm o.d.
  • Microdialysis instrument
  • Polyethylene tubing, 2.5‐cm long with 300‐µm i.d.
  • 50‐, 100‐, and 500‐µl microsyringes
  • Microsyringe pump (Harvard Apparatus, Instech, CMA), equipped with a multiple‐syringe holder

Support Protocol 2: Online Derivatization of Microdialysates with NDA/NaCN

  Materials
  • Microdialysis samples (units 7.2& 7.3), amino acid standards (see 7.4), and aCSF to be derivatized
  • HPLC‐grade water
  • 300 mM NaCN in 0.5 M sodium borate buffer, pH 10.5
  • 15 mM NDA in 75% (v/v) dimethyl sulfoxide
  • Running buffer: background electrolyte II (see recipe) with 5% (v/v) methanol
  • 1‐ml glass vials
  • 200‐µl polyethylene PCR tube
  • CE instrument: P/ACE MDQ (Beckman) with 62‐cm‐long (46 cm from injection to detection window), 50‐µm‐i.d. fused‐silica separation capillary and Expert software

Support Protocol 3: In‐Machine Automatic Derivatization of Microdialysates with NDA/NaCN

  Materials
  • 87 mM aqueous NaCN (4.264 mg/ml; store up to 1 week at 4°C in a capped glass vial)
  • Derivatization buffer (see recipe)
  • 30 mM NDA in 1:1 (v/v) acetonitrile/water
  • HPLC‐grade water
  • Microdialysis samples (units 7.2& 7.3), amino acid standards (see 7.4), and aCSF to be derivatized
  • Running buffer: 75 mM sodium borate buffer, pH 9.2
  • 1‐ml glass vial with cap
  • 200‐µl polyethylene PCR tubes
  • CE instrument: P/ACE MDQ (Beckman) with 70‐cm‐long (56 cm from injection to detection window), 75‐µm‐i.d. fused‐silica separation capillary
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Abercrombie, E.D., Keller, R.W. Jr., and Zigmond, M.J. 1988. Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: Pharmacological and behavioral studies. Neuroscience 27:897‐904.
   Anton, A.H. and Sayre, D.F. 1962. A study of the factors affecting the aluminum oxidetrihydroxyindole procedure for the analysis of catecholamines. J. Pharmacol. Exp. Therap. 138:360‐375.
   Babu, C.V., Chung, B.C., Lho, D.S., and Yoo, Y.S. 2006. Capillary electrophoretic competitive immunoassay with laser‐induced fluorescence detection for methionine‐enkephalin. J. Chromatogr. A 1111:133‐138.
   Baseski, H.M., Watson, C.J., Cellar, N.A., Shackman, J.G., and Kennedy, R.T. 2005. Capillary liquid chromatography with MS3 for the determination of enkephalins in microdialysis samples from the striatum of anesthetized and freely‐moving rats. J. Mass Spectrom. 40:146‐153.
   Bowser, M.T. and Kennedy, R.T. 2001. In vivo monitoring of amine neurotransmitters using microdialysis with on‐line capillary electrophoresis. Electrophoresis 22:3668‐3676.
   Chefer, V.I., Denoroy, L., Zapata, A., and Shippenberg, T.S. 2009. Mu opioid receptor modulation of somatodendritic dopamine overflow: GABAergic and glutamatergic mechanisms. Eur. J. Neurosci. 30:272‐278.
   Church, W.H. and Justice, J.B. 1989. On‐line smallbore chromatography for neurochemical analysis in the brain. In Advances in Chromatography (J.C. Giddings, J.C. Grushka, and R.P. Brown, eds.) pp. 165‐194. Marcel Dekker, New York.
   Church, W.H., Justice, J.B. Jr., and Neill, D.B. 1987. Detecting behaviorally relevant changes in extracellular dopamine with microdialysis. Brain Res. 412:397‐399.
   Damsma, G., Westerink, B.H.C., Imperato, A., Rollema, H., de Vries, J.B., and Horn, A.S. 1987. Automated brain dialysis of acetylcholine in freely moving rats: Detection of basal acetylcholine. Life Sci. 41:873‐876.
   Denoroy, L., Parrot, S., Renaud, L., Renaud, B., and Zimmer, L. 2008. In‐capillary derivatization and capillary electrophoresis separation of amino acid neurotransmitters from brain microdialysis samples. J. Chromatogr. A 1205:144‐149.
   Griffin, W.C., Middaugh, L.D., and Becker, H.C. 2007. Voluntary ethanol drinking in mice and ethanol concentrations in the nucleus accumbens. Brain Res. 1138:208‐213.
   Guillarme, D., Ruta, J., Rudaz, S. and Veuthey, J.L. 2010. New trends in fast and high‐resolution liquid chromatography: A critical comparison of existing approaches. Anal. Bioanal. Chem. 397:1069‐1082.
   Kehr, J. 1998a. Determination of glutamate and aspartate in microdialysis samples by reversed‐phase column liquid chromatography with fluorescence and electrochemical detection. J. Chromatogr. B 708:27‐38.
   Kehr, J. 1998b. Determination of γ‐aminobutyric acid in microdialysis samples by microbore column liquid chromatography and fluorescence detection. J. Chromatogr. B 708:49‐54.
   Kehr, J., Dechent, P., Kato, T., and Ogren, S.O. 1998. Simultaneous determination of acetylcholine, choline and physostigmine in microdialysis samples from rat hippocampus by microbore liquid chromatography/electrochemistry on peroxidase redox polymer coated electrodes. J. Neurosci. Methods 83:143‐150.
   Kennedy, R.T., Watson, C.J., Haskins, W.E., Powell, D.H., and Strecker, R.E. 2002. In vivo neurochemical monitoring by microdialysis and capillary separations. Curr. Opin. Chem. Biol. 6:659‐665.
   Kissinger, P.T., Bruntlett, C.S., Davis, G.L., Felice, L.J., Riggin, R.M., and Shoup, R.E. 1977. Recent developments in the clinical assessment of the metabolism of aromatics by high‐performance, reversed‐phase chromatography with amperometric detection. Clin. Chem. 23:1449‐1455.
   Koslow, S.H., Racagni, G., and Costa, E. 1974. Mass fragmentographic measurement of norepinephrine, dopamine, serotonin and acetylcholine in seven discrete nuclei or rat diencephalon. Neuropharmacology 13:1123‐1130.
   Lanckmans, K., Van Eeckhaut, A., Sarre, S., Smolders, I., and Michotte, Y. 2006. Capillary and nano‐liquid chromatography‐tandem mass spectrometry for the quantification of small molecules in microdialysis samples: Comparison with microbore dimensions. J. Chromatogr. A 1131:166‐175.
   Lindroth, P. and Mopper, K. 1979. High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with o‐phthaldialdehyde. Anal. Chem. 51:1667‐1674.
   Maidment, N.T., Brumbaugh, D.R., Rudolph, V.D., Erdelyi, E., and Evans, C.J. 1989. Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo. Neuroscience 33:549‐557.
   Manica, D.P., Lapos, J.A., Jones, A.D., and Ewing, A.G. 2003. Analysis of the stability of amino acids derivatized with naphthalene‐2,3‐dicarboxaldehyde using high‐performance liquid chromatography and mass spectrometry. Anal. Biochem. 322:68‐78.
   Massieu, L., Morales‐Villagrán, A., and Tapia, R. 1995. Accumulation of extracellular glutamate by inhibition of its uptake is not sufficient for inducing neuronal damage: An in vivo microdialysis study. J. Neurochem. 64:2262‐2272.
   Parrot, S., Sauvinet, V., Riban, V., Depaulis, A., Renaud, B., and Denoroy, L. 2004. High temporal resolution for in vivo monitoring of neurotransmitters in awake epileptic rats using brain microdialysis and capillary electrophoresis with laser‐induced fluorescence detection. J. Neurosci. Methods 140:29‐38.
   Pettit, H.O. and Justice, J.B. 1991. Procedures for microdialysis with smallbore HPLC. In Microdialysis in the Neurosciences (T.E. Robinson and J.B. Justice, eds.) pp. 117‐154. Elsevier Science Publishers, New York.
   Potter, P.E., Meek, J.J., and Neff, N.H. 1983. Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. J. Neurochem. 41:188‐194.
   Powell, P.R. and Ewing, A.G. 2005. Recent advances in the application of capillary electrophoresis to neuroscience. Anal. Bioanal. Chem. 382:581‐591.
   Prata, C., Bonnafous, P., Fraysse, N., Treilhou, M., Poinsot, V., and Couderc, F. 2001. Recent advances in amino acids analysis by capillary electrophoresis. Electrophoresis 22:4129‐4138.
   Robert, F., Bert, L., Parrot, S., Denoroy, L., Stoppini, L., and Renaud, B. 1998. Coupling on‐line brain microdialysis, precolumn derivatization and capillary electrophoresis for routine minute sampling of O‐phosphoethanolamine and excitatory amino acids. J. Chromatogr. A 817:195‐203.
   Roth, M. 1971. Fluorescence reaction for amino acids. Anal. Chem. 43:880‐882.
   Sauvinet, V., Parrot, S., Benturquia, N., Bravo‐Moraton, E., Renaud, B., and Denoroy, L. 2003. In vivo simultaneous monitoring of γ‐aminobutyric acid, glutamate, and L‐aspartate using brain microdialysis and capillary electrophoresis with laser‐induced fluorescence detection: Analytical developments and in vitro/in vivo validations. Electrophoresis 24:3187‐3196.
   Tossman, U., Jonsson, G., and Ungerstedt, U. 1986. Regional distribution and extracellular levels of amino acids in rat central nervous system. Acta Physiol. Scand. 127:533‐545.
   Umagat, H., Kucera, P., and Wen, L.‐F. 1982. Total amino acid analysis using pre‐column fluorescence derivatization. J. Chromatogr. 239:463‐474.
   Welch, A.S. and Welch, B.L. 1969. Solvent extraction method for simultaneous determination of norepinephrine, dopamine, serotonin, and 5‐hydroxyindoleacetic acid in a single mouse brain. Anal. Biochem. 30:161‐179.
   Winters, R.A., Zukowski, J., Ercal, N., Matthews, R.H., and Spitz, D.R. 1995. Analysis of glutathione, glutathione disulfide, cysteine, homocysteine, and other biological thiols by high‐performance liquid chromatography following derivatization by N‐(1‐pyrenyl)maleimide. Anal. Biochem. 227:14‐21.
   Wu, N., and Clausen, A.M. 2007. Fundamental and practical aspects of ultrahigh pressure liquid chromatography for fast separations. J. Sep. Sci. 30:1167‐1182.
   Zetterstrom, T., Sharp, T., Marsden, C.A., and Ungerstedt, U. 1983. In vivo measurement of dopamine and its metabolites by intracerebral dialysis: Changes after D‐amphetamine. J. Neurochem. 41:1769‐1773.
   Zhang, L.Y. and Sun, M.X. 2004. Determination of histamine and histidine by capillary zone electrophoresis with pre‐column napthtalene‐2,3‐dicarboxaldehyde derivatization and fluorescence detection. J. Chromatogr. A 1040:133‐140.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library