Abstract
Microdialysis is a powerful sampling technique used to monitor small molecules in vivo. Despite the many applications of microdialysis sampling, it is limited by the method of analyzing the resulting samples. An emerging technique for analysis of microdialysis samples is liquid chromatography-tandem mass spectrometry (LC-MS/MS). This technique is highly versatile, allowing multiplexed analysis of neurotransmitters, metabolites, and neuropeptides. Using LC-MS/MS for polar neurotransmitters is hampered by weak retention reverse phase LC columns. Several derivatization reagents have been utilized to enhance separation and resolution of neurochemicals in dialysate samples including benzoyl chloride (BzCl), dansyl chloride, formaldehyde, ethylchloroformate, and propionic anhydride. BzCl reacts with amine and phenol groups so that many neurotransmitters can be labeled. Besides improving separation on reverse phase columns, this reagent also increases sensitivity. It is available in a heavy form so that it can be used to make stable-isotope labeled internal standard for improved quantification. Using BzCl with LC-MS/MS has allowed for measuring as many as 70 neurochemicals in a single assay. With slightly different conditions, LC-MS/MS has also been used for monitoring endocannabinoids. LC-MS/MS is also useful for neuropeptide assay because it allows for highly sensitive, sequence specific measurement of most peptides. These advances have allowed for multiplexed neurotransmitter measurements in behavioral, circuit analysis, and drug effect studies.
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Ungerstedt U, Hallström Å. In vivo microdialysis—a new approach to the analysis of neurotransmitters in the brain. Life Sci. 1987;41(7):861–4.
Watson CJ, Venton BJ, Kennedy RT. In vivo measurements of neurotransmitters by microdialysis sampling. Anal Chem. 2006;78(5):1391–9.
Grosche J, Matyash V, Möller T, Verkhratsky A, Reichenbach A, Kettenmann H. Microdomains for neuron–glia interaction: parallel fiber signaling to Bergmann glial cells. Nat Neurosci. 1999;2(2):139–43.
Torres GE. The dopamine transporter proteome. J Neurochem. 2006;97(Suppl 1):3–10.
Antkiewicz-Michaluk L, Ossowska K, Romańska I, Michaluk J, Vetulani J. 3-Methoxytyramine, an extraneuronal dopamine metabolite plays a physiological role in the brain as an inhibitory regulator of catecholaminergic activity. Eur J Pharmacol. 2008;599(1):32–5.
Georgescu D, Sears RM, Hommel JD, Barrot M, Bolanos CA, Marsh DJ, et al. The hypothalamic neuropeptide melanin-concentrating hormone acts in the nucleus accumbens to modulate feeding behavior and forced-swim performance. J Neurosci. 2005;25(11):2933–40.
Mazzuferi M, Palma E, Martinello K, Maiolino F, Roseti C, Fucile S, et al. Enhancement of GABA(A)-current run-down in the hippocampus occurs at the first spontaneous seizure in a model of temporal lobe epilepsy. Proc Natl Acad Sci U S A. 2010;107(7):3180–5.
Seidel S, Singer EA, Just H, Farhan H, Scholze P, Kudlacek O, et al. Amphetamines take two to tango: an oligomer-based counter-transport model of neurotransmitter transport explores the amphetamine action. Mol Pharmacol. 2005;67(1):140–51.
Löscher W, Potschka H. Blood-brain barrier active efflux transporters: ATP-binding cassette gene family. NeuroRx. 2005;2(1):86–98.
Lada MW, Vickroy TW, Kennedy RT. High temporal resolution monitoring of glutamate and aspartate in vivo using microdialysis on-line with capillary electrophoresis with laser-induced fluorescence detection. Anal Chem. 1997;69(22):4560–5.
Slaney TR, Nie J, Hershey ND, Thwar PK, Linderman J, Burns MA, et al. Push–pull perfusion sampling with segmented flow for high temporal and spatial resolution in vivo chemical monitoring. Anal Chem. 2011;83(13):5207–13.
Justice J. Quantitative microdialysis of neurotransmitters. J Neurosci Methods. 1993;48(3):263–76.
Wong J-MT, Malec PA, Mabrouk OS, Ro J, Dus M, Kennedy RT. Benzoyl chloride derivatization with liquid chromatography–mass spectrometry for targeted metabolomics of neurochemicals in biological samples. J Chromatogr A. 2016;1446:78–90.
Nader MA, Morgan D, Gage HD, Nader SH, Calhoun TL, Buchheimer N, et al. PET imaging of dopamine D2 receptors during chronic cocaine self-administration in monkeys. Nat Neurosci. 2006;9(8):1050–6.
Okumoto S, Looger LL, Micheva KD, Reimer RJ, Smith SJ, Frommer WB. Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors. Proc Natl Acad Sci U S A. 2005;102(24):8740–5.
Westerink B. Correlation between high-performance liquid chromatography and automated fluorimetric methods for the determination of dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in nervous tissue and cerebrospinal fluid. J Chromatogr. 1982;233:69–77.
Carboni E, Imperato A, Perezzani L, Di Chiara G. Amphetamine, cocaine, phencyclidine and nomifensine increase extracellular dopamine concentrations preferentially in the nucleus accumbens of freely moving rats. Neuroscience. 1989;28(3):653–61.
Herrera-Marschitz M, Goiny M, You ZB, Meana JJ, Pettersson E, Rodriguez-Puertas R, et al. On the release of glutamate and aspartate in the basal ganglia of the rat: interactions with monoamines and neuropeptides. Neurosci Biobehav Rev. 1997;21(4):489–95.
Darvesh AS, Carroll RT, Geldenhuys WJ, Gudelsky GA, Klein J, Meshul CK, et al. In vivo brain microdialysis: advances in neuropsychopharmacology and drug discovery. Expert Opin Drug Discov. 2011;6(2):109–27.
Chirita R-I, West C, Finaru A-L, Elfakir C. Approach to hydrophilic interaction chromatography column selection: application to neurotransmitters analysis. J Chromatogr A. 2010;1217(18):3091–104.
Danaceau JP, Chambers EE, Fountain KJ. Hydrophilic interaction chromatography (HILIC) for LC–MS/MS analysis of monoamine neurotransmitters. Bioanalysis. 2012;4(7):783–94.
Zhang X, Rauch A, Lee H, Xiao H, Rainer G, Logothetis NK. Capillary hydrophilic interaction chromatography/mass spectrometry for simultaneous determination of multiple neurotransmitters in primate cerebral cortex. Rapid Commun Mass Spectrom. 2007;21(22):3621–8.
Cai H-L, Zhu R-H. Determination of dansylated monoamine and amino acid neurotransmitters and their metabolites in human plasma by liquid chromatography–electrospray ionization tandem mass spectrometry. Anal Biochem. 2010;396(1):103–11.
Nirogi R, Komarneni P, Kandikere V, Boggavarapu R, Bhyrapuneni G, Benade V, et al. A sensitive and selective quantification of catecholamine neurotransmitters in rat microdialysates by pre-column dansyl chloride derivatization using liquid chromatography–tandem mass spectrometry. J Chromatogr B. 2013;913:41–7.
Park J-Y, Myung S-W, Kim I-S, Choi D-K, Kwon S-J, Yoon S-H. Simultaneous measurement of serotonin, dopamine and their metabolites in mouse brain extracts by high-performance liquid chromatography with mass spectrometry following derivatization with ethyl chloroformate. Biol Pharm Bull. 2012;36(2):252–8.
Guo K, Ji C, Li L. Stable-isotope dimethylation labeling combined with LC−ESI MS for quantification of amine-containing metabolites in biological samples. Anal Chem. 2007;79(22):8631–8.
Greco S, Danysz W, Zivkovic A, Gross R, Stark H. Microdialysate analysis of monoamine neurotransmitters—a versatile and sensitive LC–MS/MS method. Anal Chim Acta. 2013;771:65–72.
Zhang D, Wu L, Chow DS, Tam VH, Rios DR. Quantitative determination of dopamine in human plasma by a highly sensitive LC–MS/MS assay: application in preterm neonates. J Pharm Biomed Anal. 2016;117:227–31.
Song P, Mabrouk OS, Hershey ND, Kennedy RT. In vivo neurochemical monitoring using benzoyl chloride derivatization and liquid chromatography–mass spectrometry. Anal Chem. 2011;84(1):412–9.
Anari MR, Bakhtiar R, Zhu B, Huskey S, Franklin RB, Evans DC. Derivatization of ethinylestradiol with dansyl chloride to enhance electrospray ionization: application in trace analysis of ethinylestradiol in rhesus monkey plasma. Anal Chem. 2002;74(16):4136–44.
Kang X, Xiao J, Huang X, Gu Z. Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples. Clin Chim Acta. 2006;366(1):352–6.
Yamada H, Yamahara A, Yasuda S, Abe M, Oguri K, Fukushima S, et al. Dansyl chloride derivatization of methamphetamine: a method with advantages for screening and analysis of methamphetamine in urine. J Anal Toxicol. 2002;26(1):17–22.
Inagaki S, Tano Y, Yamakata Y, Higashi T, Min JZ, Toyo'oka T. Highly sensitive and positively charged precolumn derivatization reagent for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom. 2010;24(9):1358–64.
Zestos AG, Mikelman SR, Kennedy RT, Gnegy ME. PKCβ inhibitors attenuate amphetamine-stimulated dopamine efflux. ACS Chem Neurosci. 2016;7(6):757–66.
Olson R, Justice J Jr. Quantitative microdialysis under transient conditions. Anal Chem. 1993;65(8):1017–22.
Hershey ND, Kennedy RT. In vivo calibration of microdialysis using infusion of stable-isotope labeled neurotransmitters. ACS Chem Neurosci. 2013;4(5):729–36.
Clément R, Malinovsky J-M, Dollo G, Le Corre P, Chevanne F, Le Verge R. In vitro and in vivo microdialysis calibration using retrodialysis for the study of the cerebrospinal distribution of bupivacaine. J Pharm Biomed Anal. 1998;17(4):665–70.
Bengtsson J, Boström E, Hammarlund-Udenaes M. The use of a deuterated calibrator for in vivo recovery estimations in microdialysis studies. J Pharm Sci. 2008;97(8):3433–41.
Peters JL, Michael AC. Modeling voltammetry and microdialysis of striatal extracellular dopamine: the impact of dopamine uptake on extraction and recovery ratios. J Neurochem. 1998;70(2):594–603.
Robinson TE, Jurson PA, Bennett JA, Bentgen KM. Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)-amphetamine: a microdialysis study in freely moving rats. Brain Res. 1988;462(2):211–22.
Sulzer D, Chen T, Lau Y, Kristensen H, Rayport S, Ewing A. Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci. 1995;15(5):4102–8.
Mikelman S, Mardirossian N, Gnegy ME. Tamoxifen and amphetamine abuse: are there therapeutic possibilities? J Chem Neuroanat. 2016;
Manji HK, Lenox RH. Protein kinase C signaling in the brain: molecular transduction of mood stabilization in the treatment of manic-depressive illness. Biol Psychiatry. 1999;46(10):1328–51.
Zarate CA, Singh JB, Carlson PJ, Quiroz J, Jolkovsky L, Luckenbaugh DA, et al. Efficacy of a protein kinase C inhibitor (tamoxifen) in the treatment of acute mania: a pilot study. Bipolar Disord. 2007;9(6):561–70.
Carpenter C, Sorenson RJ, Jin Y, Klossowski S, Cierpicki T, Gnegy M, et al. Design and synthesis of triarylacrylonitrile analogues of tamoxifen with improved binding selectivity to protein kinase C. Bioorg Med Chem. 2016;24(21):5495–504.
Vander Weele CM, Porter-Stransky KA, Mabrouk OS, Lovic V, Singer BF, Kennedy RT, et al. Rapid dopamine transmission within the nucleus accumbens: dramatic difference between morphine and oxycodone delivery. Eur J Neurosci. 2014;40(7):3041–54.
Robinson TE, Becker JB. Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis. Brain Res Rev. 1986;11(2):157–98.
Berridge CW, Stratford TL, Foote SL, Kelley AE. Distribution of dopamine b-hydroxylase-like immunoreactive fibers within the shell subregion of the nucleus accumbens. SYNAPSE-NEW YORK. 1997;27:230–41.
Berridge KC, Robinson TE. What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res Rev. 1998;28(3):309–69.
Kohlert JG, Meisel RL. Sexual experience sensitizes mating-related nucleus accumbens dopamine responses of female Syrian hamsters. Behav Brain Res. 1999;99(1):45–52.
Mas M, Fumero B, Fernandez-Vera JR, Gonzalez-Mora JL. Neurochemical correlates of sexual exhaustion and recovery as assessed by in vivo microdialysis. Brain Res. 1995;675(1):13–9.
Mas M, Fumero B, González-Mora J. Voltammetric and microdialysis monitoring of brain monoamine neurotransmitter release during sociosexual interactions. Behav Brain Res. 1995;71(1):69–IN5.
Pfaus J, Damsma G, Nomikos GG, Wenkstern D, Blaha C, Phillips A, et al. Sexual behavior enhances central dopamine transmission in the male rat. Brain Res. 1990;530(2):345–8.
Hamid AA, Pettibone JR, Mabrouk OS, Hetrick VL, Schmidt R, Vander Weele CM, et al. Mesolimbic dopamine signals the value of work. Nat Neurosci. 2016;19(1):117-+.
Buczynski MW, Parsons LH. Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls. Br J Pharmacol. 2010;160(3):423–42.
Béquet F, Uzabiaga F, Desbazeille M, Ludwiczak P, Maftouh M, Picard C, et al. CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus. Eur J Neurosci. 2007;26(12):3458–64.
Walker JM, Huang SM, Strangman NM, Tsou K, Sañudo-Peña MC. Pain modulation by release of the endogenous cannabinoid anandamide. Proc Natl Acad Sci. 1999;96(21):12198–203.
Zhou Y, Mabrouk OS, Kennedy RT. Rapid preconcentration for liquid chromatography–mass spectrometry assay of trace level neuropeptides. J Am Soc Mass Spectrom. 2013;24(11):1700–9.
Giuffrida A, Parsons L, Kerr T, De Fonseca FR, Navarro M, Piomelli D. Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat Neurosci. 1999;2(4):358–63.
Rimmerman N, Hughes H, Bradshaw H, Pazos M, Mackie K, Prieto A, et al. Compartmentalization of endocannabinoids into lipid rafts in a dorsal root ganglion cell line. Br J Pharmacol. 2008;153(2):380–9.
Patel S, Carrier EJ, Ho WV, Rademacher DJ, Cunningham S, Reddy DS, et al. The postmortal accumulation of brain N-arachidonylethanolamine (anandamide) is dependent upon fatty acid amide hydrolase activity. J Lipid Res. 2005;46(2):342–9.
Caillé S, Alvarez-Jaimes L, Polis I, Stouffer DG, Parsons LH. Specific alterations of extracellular endocannabinoid levels in the nucleus accumbens by ethanol, heroin, and cocaine self-administration. J Neurosci. 2007;27(14):3695–702.
Long JZ, Li W, Booker L, Burston JJ, Kinsey SG, Schlosburg JE, et al. Selective blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects. Nat Chem Biol. 2009;5(1):37–44.
Wiskerke J, Irimia C, Cravatt BF, De Vries TJ, Schoffelmeer AN, Pattij T, et al. Characterization of the effects of reuptake and hydrolysis inhibition on interstitial endocannabinoid levels in the brain: an in vivo microdialysis study. ACS Chem Neurosci. 2012;3(5):407–17.
Grouzmann E, Aubert J, Waeber B, Brunner H. A sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide Y. Peptides. 1992;13(6):1049–54.
Emmett MR, Andrén PE, Caprioli RM. Specific molecular mass detection of endogenously released neuropeptides using in vivo microdialysis/mass spectrometry. J Neurosci Methods. 1995;62(1):141–7.
Haskins WE, Wang Z, Watson CJ, Rostand RR, Witowski SR, Powell DH, et al. Capillary LC-MS2 at the attomole level for monitoring and discovering endogenous peptides in microdialysis samples collected in vivo. Anal Chem. 2001;73(21):5005–14.
Zhou Y, Wong J-MT, Mabrouk OS, Kennedy RT. Reducing adsorption to improve recovery and in vivo detection of neuropeptides by microdialysis with LC-MS. Anal Chem. 2015;87(19):9802–9.
Svensson M, Sköld K, Nilsson A, Fälth M, Nydahl K, Svenningsson P, et al. Neuropeptidomics: MS applied to the discovery of novel peptides from the brain. Anal Chem. 2007;79(1):14–21.
Haskins WE, Watson CJ, Cellar NA, Powell DH, Kennedy RT. Discovery and neurochemical screening of peptides in brain extracellular fluid by chemical analysis of in vivo microdialysis samples. Anal Chem. 2004;76(18):5523–33.
Schmerberg CM, Li L. Mass spectrometric detection of neuropeptides using affinity-enhanced microdialysis with antibody-coated magnetic nanoparticles. Anal Chem. 2013;85(2):915–22.
Li Q, Zubieta J-K, Kennedy RT. Practical aspects of in vivo detection of neuropeptides by microdialysis coupled off-line to capillary LC with multistage MS. Anal Chem. 2009;81(6):2242–50.
Maidment N, Brumbaugh D, Rudolph V, Erdelyi E, Evans C. Microdialysis of extracellular endogenous opioid peptides from rat brain in vivo. Neuroscience. 1989;33(3):549–57.
Takeda S, Sato N, Ikimura K, Nishino H, Rakugi H, Morishita R. Novel microdialysis method to assess neuropeptides and large molecules in free-moving mouse. Neuroscience. 2011;186:110–9.
Herbaugh AW, Stenken JA. Antibody-enhanced microdialysis collection of CCL2 from rat brain. J Neurosci Methods. 2011;202(2):124–7.
Pettersson A, Markides K, Bergquist J. Enhanced microdialysis of neuropeptides. ACTA BIOCHIMICA POLONICA-ENGLISH EDITION. 2001;48(4):1117–20.
Sköld K, Svensson M, Nilsson A, Zhang X, Nydahl K, Caprioli RM, et al. Decreased striatal levels of PEP-19 following MPTP lesion in the mouse. J Proteome Res. 2006;5(2):262–9.
Schultz W, Dickinson A. Neuronal coding of prediction errors. Annu Rev Neurosci. 2000;23(1):473–500.
Palacios JM, Kuhar MJ. Neurotensin receptors are located on dopamine-containing neurones in rat midbrain. 1981.
Patterson CM, Wong J-MT, Leinninger GM, Allison MB, Mabrouk OS, Kasper CL, et al. Ventral tegmental area neurotensin signaling links the lateral hypothalamus to locomotor activity and striatal dopamine efflux in male mice. Endocrinology. 2015;156(5):1692–700.
Mabrouk OS, Li Q, Song P, Kennedy RT. Microdialysis and mass spectrometric monitoring of dopamine and enkephalins in the globus pallidus reveal reciprocal interactions that regulate movement. J Neurochem. 2011;118(1):24–33.
Mabrouk OS, Falk T, Sherman SJ, Kennedy RT, Polt R. CNS penetration of the opioid glycopeptide MMP-2200: a microdialysis study. Neurosci Lett. 2012;531(2):99–103.
André PE, Caprioli RM. In vivo metabolism of substance P in rat striatum utilizing microdialysis/liquid chromatography/micro-electrospray mass spectrometry. J Mass Spectrom. 1995;30(6):817–24.
Svensson M, Sköld K, Svenningsson P, Andren PE. Peptidomics-based discovery of novel neuropeptides. J Proteome Res. 2003;2(2):213–9.
Kaplan A, Björkesten L, Åström J, Andren PE. A neuroproteomic approach to targeting neuropeptides in the brain. Proteomics. 2002;2:447–54.
Wei H, Nolkrantz K, Parkin MC, Chisolm CN, O'Callaghan JP, Kennedy RT. Identification and quantification of neuropeptides in brain tissue by capillary liquid chromatography coupled off-line to MALDI-TOF and MALDI-TOF/TOF-MS. Anal Chem. 2006;78(13):4342–51.
Dowell JA, Vander Heyden W, Li L. Rat neuropeptidomics by LC−MS/MS and MALDI−FTMS: enhanced dissection and extraction techniques coupled with 2D RP-RP HPLC. J Proteome Res. 2006;5(12):3368–75.
Behrens HL, Chen R, Li L. Combining microdialysis, NanoLC-MS, and MALDI-TOF/TOF to detect neuropeptides secreted in the crab. Cancer borealis Analytical chemistry. 2008;80(18):6949–58.
Fälth M, Sköld K, Norrman M, Svensson M, Fenyö D, Andren PE. SwePep, a database designed for endogenous peptides and mass spectrometry. Mol Cell Proteomics. 2006;5(6):998–1005.
Acknowledgments
This work was supported by NIH R37 EB003320 (Robert T. Kennedy), NIH T32 grant DA007268 (Alexander G. Zestos), and Seed Funding for Innovative Projects in Neuroscience on behalf of Michigan Brain Initiative Working Group (MiBrain Initiative).
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Guest Editors: Robert E. Stratford, Nimita Dave, and Richard F. Bergstrom
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Zestos, A.G., Kennedy, R.T. Microdialysis Coupled with LC-MS/MS for In Vivo Neurochemical Monitoring. AAPS J 19, 1284–1293 (2017). https://doi.org/10.1208/s12248-017-0114-4
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DOI: https://doi.org/10.1208/s12248-017-0114-4