title: A novel liquid chromatography/tandem mass spectrometry method for the quantification of glycine as biomarker in brain microdialysis and cerebrospinal fluid samples within 5min.
abstract: Glycine is an important amino acid neurotransmitter in the central nervous system (CNS) and a useful biomarker to indicate biological activity of drugs such as glycine reuptake inhibitors (GRI) in the brain. Here, we report how a liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the fast and reliable analysis of glycine in brain microdialysates and cerebrospinal fluid (CSF) samples has been established. Additionally, we compare this method with the conventional approach of high performance liquid chromatography (HPLC) coupled to fluorescence detection (FD). The present LC-MS/MS method did not require any derivatisation step. Fifteen microliters of sample were injected for analysis. Glycine was detected by a triple quadrupole mass spectrometer in the positive electrospray ionisation (ESI) mode. The total running time was 5min. The limit of quantitation (LOQ) was determined as 100nM, while linearity was given in the range from 100nM to 100μM. In order to demonstrate the feasibility of the LC-MS/MS method, we measured glycine levels in striatal in vivo microdialysates and CSF of rats after administration of the commercially available glycine transporter 1 (GlyT1) inhibitor LY 2365109 (10mg/kg, p.o.). LY 2365109 produced 2-fold and 3-fold elevated glycine concentrations from 1.52μM to 3.6μM in striatal microdialysates and from 10.38μM to 36μM in CSF, respectively. In conclusion, we established a fast and reliable LC-MS/MS method, which can be used for the quantification of glycine in brain microdialysis and CSF samples in biomarker studies.
authors.affiliation: CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397 Biberach, Germany.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Voehringer
authors.forename: René
authors.initials: R
authors.lastname: Fuertig
authors.forename: Boris
authors.initials: B
authors.lastname: Ferger
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000134:cerebrospinal fluid; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D005998:Glycine / Q000134:cerebrospinal fluid*; D008297:Male; D017551:Microdialysis / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D005998:Glycine
keywords: 2,4-dinitrofluorobenzene; ANOVA; Biomarker; CE; CE–MS; CNS; CSF; CXP; DNFB; DP; ESI; FD; GABA; GC–MS; GRI; GlyT; GlyT1; Glycine; HILIC; HPLC; In vivo microdialysis; LC–MS; LC–MS/MS; LOD; LOQ; MCE; MRM; N-methyl-d-aspartate; NMDA; OPA; UV; aCSF; analysis of variance; artificial cerebrospinal fluid; capillary electrophoresis/mass spectrometry; cell exit potential; central nervous system; cerebrospinal fluid; collision energy; declustering potential; electrospray ionisation; fluorescence detection; gas chromatography/mass spectrometry; glycine reuptake inhibitors; glycine transporter; high-performance liquid chromatography; hydrophilic interaction chromatography; limit of detection; limit of quantitation; liquid chromatography/mass spectrometry; liquid chromatography/tandem mass spectrometry; mercaptoethanol; multiple reaction monitoring; o-phthaldialdehyde; ultraviolet; γ-aminobutyric acid