Measuring multiple neurochemicals and related metabolites in blood and brain of the rhesus monkey by using dual microdialysis sampling and capillary hydrophilic interaction chromatography–mass spectrometry
- 400 Downloads
In vivo measurement of multiple functionally related neurochemicals and metabolites (NMs) is highly interesting but remains challenging in the field of basic neuroscience and clinical research. We present here an analytical method for determining five functionally and metabolically related polar substances, including acetylcholine (quaternary ammonium), lactate and pyruvate (organic acids), as well as glutamine and glutamate (amino acids). These NMs are acquired from samples of the brain and the blood of non-human primates in parallel by dual microdialysis, and subsequently analyzed by a direct capillary hydrophilic interaction chromatography (HILIC)–mass spectrometry (MS) based method. To obtain high sensitivity in electrospray ionization (ESI)–MS, lactate and pyruvate were detected in negative ionization mode whereas the other NMs were detected in positive ionization mode during each HILIC-MS run. The method was validated for linearity, the limits of detection and quantification, precision, accuracy, stability and matrix effect. The detection limit of acetylcholine, lactate, pyruvate, glutamine, and glutamate was 150 pM, 3 μM, 2 μM, 5 nM, and 50 nM, respectively. This allowed us to quantitatively and simultaneously measure the concentrations of all the substances from the acquired dialysates. The concentration ratios of both lactate/pyruvate and glutamine/glutamate were found to be higher in the brain compared to blood (p < 0.05). The reliable and simultaneous quantification of these five NMs from brain and blood samples allows us to investigate their relative distribution in the brain and blood, and most importantly paves the way for future non-invasive studies of the functional and metabolic relation of these substances to each other.
KeywordsNeurochemicals HILIC-MS Microdialysis Rhesus monkey Brain Blood
This work was supported by the Max Planck Society and BMBF Grant, Nr. 01EV0701. We thank Nadine Serr and Ulrike Passlack-Memaj for assistance in chemical preparations. We thank Mark Augath for assistance with the monkey anesthesia.
- 7.Andersson K, Arner P (1995) Cholinoceptor-mediated effects on glycerol output from human adipose tissue using in situ microdialysis. Br J Pharmacol 115:1155–1162Google Scholar
- 15.Zhu Y, Wong PSH, Cregor M, Gitzen JF, Coury LA, Kissinger PT (2000) In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain. Rapid Commun Mass Spectrom 14:1695–1700CrossRefGoogle Scholar
- 27.Viklund C, Ponten E, Glad B, Irgum K, Horstedt P, Svec F (1997) “Molded” Macroporous Poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) Materials with Fine Controlled Porous Properties: Preparation of Monoliths Using Photoinitiated Polymerization. Chem Mater 463–471Google Scholar
- 30.Food and Drug Administration (2001) Guidance for industry: bioanalytical method validation. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research. http://www.fda.gov/cder/guidance/index.htm.
- 42.Samuelsson C, Hillered L, Zetterling M, Enblad P, Hesselager G, Ryttlefors M, Kumlien E, Lewen A, Marklund N, Nilsson P, Salci K, Ronne-Engstrom E (2007) Cerebral glutamine and glutamate levels in relation to compromised energy metabolism: a microdialysis study in subarachnoid hemorrhage patients. J Cereb Blood Flow Metab 27:1309–1317CrossRefGoogle Scholar
- 43.Richards DA, Tolias CM, Sgouros S, Bowery NG (2003) Extracellular glutamine to glutamate ratio may predict outcome in the injured brain: a clinical microdialysis study in children. Pharmacol Res 48:101–109Google Scholar