Enantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant.
The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R(2) > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%-8.0% and 0.34%-0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.
Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
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A
Prior
Laboratory for the Analysis of Medicines, Department of Pharmacy, Faculty of Medicine, CIRM, University of Liège, Avenue Hippocrate 15, 4000 Liège, Belgium; Department of Analytical Chemistry and Instrumental Analysis, Faculty of Pharmacy, University of Medicine and Pharmacy "luliu Haţieganu" Cluj-Napoca, 4 Louis Pasteur Street, 400349 Cluj-Napoca, Romania.
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RC
Moldovan
Laboratory for the Analysis of Medicines, Department of Pharmacy, Faculty of Medicine, CIRM, University of Liège, Avenue Hippocrate 15, 4000 Liège, Belgium.
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J
Crommen
Laboratory for the Analysis of Medicines, Department of Pharmacy, Faculty of Medicine, CIRM, University of Liège, Avenue Hippocrate 15, 4000 Liège, Belgium.
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Servais
Laboratory for the Analysis of Medicines, Department of Pharmacy, Faculty of Medicine, CIRM, University of Liège, Avenue Hippocrate 15, 4000 Liège, Belgium.
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M
Fillet
Biomolecular Analysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
G J
GJ
de Jong
Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
G W
GW
Somsen
D000596:Amino Acids / Q000134:cerebrospinal fluid*; D019075:Electrophoresis, Capillary / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D013237:Stereoisomerism
D000596:Amino Acids
Amino acids; Capillary electrophoresis-mass spectrometry; Cerebrospinal fluid; Chiral separation