Two Mass-Spectrometric Techniques for Quantifying Serine Enantiomers and Glycine in Cerebrospinal Fluid: Potential Confounders and Age-Dependent Ranges

Abstract

Abstract Background: The recent discovery and specific functions of d-amino acids in humans are bound to lead to the revelation of d-amino acid abnormalities in human disorders. Therefore, high-throughput analysis techniques are warranted to determine d-amino acids in biological fluids in a routine laboratory setting. Methods: We developed 2 chromatographic techniques, a nonchiral derivatization with chiral (chirasil-l-val column) separation in a GC-MS system and a chiral derivatization with Marfey’s reagent and LC- MS analysis. We validated the techniques for d-serine, l-serine, and glycine determination in cerebrospinal fluid (CSF), evaluated several confounders, and determined age-dependent human concentration ranges. Results: Quantification limits for d-serine, l-serine, and glycine in cerebrospinal fluid were 0.14, 0.44, and 0.14 μmol/L, respectively, for GC-MS and 0.20, 0.41, and 0.14 μmol/L for LC-MS. Within-run imprecision was <3% for both methods, and between-run imprecision was <13%. Comparison of both techniques with Deming regression yielded coefficients of 0.90 (d-serine), 0.92 (l-serine), and 0.96 (glycine). Sample collection, handling, and transport is uncomplicated—there is no rostrocaudal CSF gradient, no effect of storage at 4 °C for 1 week before storage at −80 °C, and no effect of up to 3 freeze/thaw cycles. Conversely, contamination with erythrocytes increased d-serine, l-serine, and glycine concentrations. CSF concentrations for 145 apparently healthy controls demonstrated markedly and specifically increased (5 to 9 times) d-serine concentrations during early central nervous system development. Conclusions: These 2 clinically applicable analysis techniques will help to unravel pathophysiologic, diagnostic, and therapeutic issues for disorders associated with central nervous system abnormalities, NMDA-receptor dysfunction, and other pathology associated with d-amino acids.