title: A sensitive and simple ultra-high-performance-liquid chromatography-tandem mass spectrometry based method for the quantification of D-amino acids in body fluids.
abstract: D-Amino acids are increasingly being recognized as important signaling molecules in mammals, including humans. D-Serine and D-aspartate are believed to act as signaling molecules in the central nervous system. Interestingly, several other D-amino acids also occur in human plasma, but very little is currently known regarding their function and origin. Abnormal levels of D-amino acids have been implicated in the pathogenesis of different diseases, including schizophrenia and amyotrophic lateral sclerosis (ALS), indicating that D-amino acid levels hold potential as diagnostic markers. Research into the biological functions of D-amino acids is hindered, however, by the lack of sufficiently sensitive, high-throughput analytical methods. In particular, the interference of large amounts of L-amino acids in biological samples and the low concentrations of D-amino acids are challenging. In this paper, we compared 7 different chiral derivatization agents for the analysis of D-amino acids and show that the chiral reagent (S)-NIFE offers outstanding performance in terms of sensitivity and enantioselectivity. An UPLC-MS/MS based method for the quantification of D-amino acids human biological fluids was then developed using (S)-NIFE. Baseline separation (R(s)>2.45) was achieved for the isomers of all 19 chiral proteinogenic amino acids. The limit of detection was <1 nM for all amino acids except d-alanine (1.98 nM), d-methionine (1.18 nM) and d-asparagine (5.15 nM). For measurements in human plasma, cerebrospinal fluid and urine, the accuracy ranged between 85% and 107%. The intra-assay and inter-assay were both <16% RSD for these three different matrices. Importantly, the method does not suffer from spontaneous racemization during sample preparation and derivatization. Using the described method, D-amino acid levels in human cerebrospinal fluid, plasma and urine were measured.
authors: University Medical Center Utrecht/Wilhelmina Children's Hospital, Department of Metabolic and Endocrine Diseases, Utrecht, The Netherlands.
authors: Wouter F
authors: WF
authors: Visser
authors: Nanda M
authors: NM
authors: Verhoeven-Duif
authors: Roel
authors: R
authors: Ophoff
authors: Steven
authors: S
authors: Bakker
authors: Leo W
authors: LW
authors: Klomp
authors: Ruud
authors: R
authors: Berger
authors: Tom J
authors: TJ
authors: de Koning
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000097:blood / Q000134:cerebrospinal fluid / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D009574:Nitro Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids; C467080:N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester; D009574:Nitro Compounds