Chemically enhanced liquid chromatography/tandem mass spectrometry determination of glutamic acid in the diffusion medium of retinal cells.
A rapid, reproducible and highly sensitive method, based on liquid chromatography mass spectrometry, was developed for the determination of the excitatory amino acid glutamic acid released in the diffusion medium of control, ischemic and mutant cells from retinas. Signal intensity of glutamic acid was enhanced by dansyl chloride derivatization giving rise to a detection limit in the order of pmol/mL. Further, in HPLC-ESI-MS detection an MS-friendly dansyl group to glutamic acid enhanced both ionization efficiency in the ESI source and collision-activated dissociation in the collision cell. The sample processing procedure included liquid-liquid extraction, derivatization with dansyl chloride and a final cation-exchange extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (20-300 ng/mL), accurate and precise for the differential quantification of glutamic acid in the diffusion medium of retina cells. This is the first report of using chemical derivatization to enhance MS/MS detection of the glutamic acid released in the diffusion medium of wild-type and mutant retina cells, under ischemic conditions.
Timperio
Anna Maria
AM
Department of Environmental Sciences, University La Tuscia L. go dell'Università snc 01100 Viterbo, Italy. timperio@unitus.it
Fagioni
Marco
M
Grandinetti
Felice
F
Zolla
Lello
L
D000818:Animals; D002138:Calibration; D002478:Cells, Cultured / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry*; D003619:Dansyl Compounds / Q000737:chemistry / Q000592:standards; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism* / Q000592:standards; D007511:Ischemia / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012160:Retina / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry
D003470:Culture Media; D003619:Dansyl Compounds; D018698:Glutamic Acid; C029038:dansyl chloride