A High-Methionine Diet for One-Week Induces a High Accumulation of Methionine in the Cerebrospinal Fluid and Confers Bipolar Disorder-like Behavior in Mice.

Methionine (Met) is considered the most toxic amino acid in mammals. Here, we investigated biochemical and behavioral impacts of ad libitum one-week feeding of high-Met diets on mice. Adult male mice were fed the standard rodent diet that contained 0.44% Met (1×) or a diet containing 16 graded Met doses (1.2×-13×). High-Met diets for one-week induced a dose-dependent decrease in body weight and an increase in serum Met levels with a 2.55 mM peak (versus basal 53 µM) on the 12×Met diet. Total homocysteine (Hcy) levels were also upregulated while concentrations of other amino acids were almost maintained in serum. Similarly, levels of Met and Hcy (but not the other amino acids) were highly elevated in the cerebrospinal fluids of mice on the 10×Met diet; the Met levels were much higher than Hcy and the others. In a series of behavioral tests, mice on the 10×Met diet displayed increased anxiety and decreased traveled distances in an open-field test, increased activity to escape from water soaking and tail hanging, and normal learning/memory activity in a Y-maze test, which were reflections of negative/positive symptoms and normal cognitive function, respectively. These results indicate that high-Met ad libitum feeding even for a week can induce bipolar disorder-like disease models in mice.

Diet Effects on Cerebrospinal Fluid Amino Acids Levels in Adults with Normal Cognition and Mild Cognitive Impairment.

BACKGROUND: Exploration of cerebrospinal fluid (CSF) amino acids and the impact of dietary intake on central levels may provide a comprehensive understanding of the metabolic component of Alzheimer's disease. OBJECTIVE: The objective of this exploratory study was to investigate the effects of two diets with varied nutrient compositions on change in CSF amino acids levels in adults with mild cognitive impairment (MCI) and normal cognition (NC). Secondary objectives were to assess the correlations between the change in CSF amino acids and change in Alzheimer's disease biomarkers. METHODS: In a randomized, parallel, controlled feeding trial, adults (NC, n = 20; MCI, n = 29) consumed a high saturated fat (SFA)/glycemic index (GI) diet [HIGH] or a low SFA/GI diet [LOW] for 4 weeks. Lumbar punctures were performed at baseline and 4 weeks. RESULTS: CSF valine increased and arginine decreased after the HIGH compared to the LOW diet in MCI (ps = 0.03 and 0.04). This pattern was more prominent in MCI versus NC (diet by diagnosis interaction ps = 0.05 and 0.09), as was an increase in isoleucine after the HIGH diet (p = 0.05). Changes in CSF amino acids were correlated with changes in Alzheimer's disease CSF biomarkers Aß42, total tau, and p-Tau 181, with distinct patterns in the relationships by diet intervention and cognitive status. CONCLUSION: Dietary intake affects CSF amino acid levels and the response to diet is differentially affected by cognitive status.

Effects of time-of-day on the concentration of defined excitatory and inhibitory amino acids in the cerebrospinal fluid of rats: a microdialysis study.

Amino acid neurotransmitters are responsible for many physiological and pathological processes, and their cerebral concentrations respond to external influences such as the light-dark cycle and to the synthesis, release, and recapture rhythms and form part of the biochemical relationships derived from excitatory-inhibitory (E/I), glutamine-glutamate sum (GLX), glutamatergic processing (glutamine-glutamate ratio) and excitotoxic indexes. The changes in these variables during a 24-h period (1 day) are important because they allow organisms to adapt to external stimuli and form part of physiological processes. Under pathological conditions, the damage produced by acute events may depend on diurnal variations. Therefore, it is important to analyze the extracellular levels of amino acids as well as the above-mentioned indexes over a 24-h period. We focused on determining the cerebrospinal fluid levels of different amino acid neurotransmitters, and the E/I, GLX, glutamatergic processing and excitotoxic indexes, determined by microdialysis over a 24-h cycle. Our results showed significant changes during the 24-h light/dark cycle. Specifically, we found increments in the levels of glutamate (325%), GABA (550%), glutamine (300%), glycine (194%), alanine (304%) and the GLX index (263%) throughout the day, and the maximum levels of glutamate, glutamine, glycine, and alanine were obtained during the last period of the light period. In conclusion, the concentration of some amino acid neurotransmitters and the GLX index show variations depending on the light-dark cycle.

Some CSF Kynurenine Pathway Intermediates Associated with Disease Evolution in Amyotrophic Lateral Sclerosis.

The aim of this study was to evaluate the kynurenine pathway (KP) and amino acids profile, using mass spectrometry, in the cerebrospinal fluid (CSF) of 42 amyotrophic lateral sclerosis (ALS) patients at the diagnosis and 40 controls to detect early disorders of these pathways. Diagnostic and predictive ability (based on weight loss, forced vital capacity, ALS Functional Rating Scale-Revised evolution over 12 months, and survival time) of these metabolites were evaluated using univariate followed by supervised multivariate analysis. The multivariate model between ALS and controls was not significant but highlighted some KP metabolites (kynurenine (KYN), kynurenic acid (KYNA), 3-Hydroxynurenine (3-HK)/KYNA ratio), and amino acids (Lysine, asparagine) as involved in the discrimination between groups (accuracy 62%). It revealed a probable KP impairment toward neurotoxicity in ALS patients and in bulbar forms. Regarding the prognostic effect of metabolites, 12 were commonly discriminant for at least 3 of 4 disease evolution criteria. This investigation was crucial as it did not show significant changes in CSF concentrations of amino acids and KP intermediates in early ALS evolution. However, trends of KP modifications suggest further exploration. The unclear kinetics of neuroinflammation linked to KP support the interest in exploring these pathways during disease evolution through a longitudinal strategy.

Untargeted GC/TOFMS unravel metabolic profiles in cerebrospinal fluid of Chinese people living with HIV.

BACKGROUND: Metabolic syndrome becomes a focus of clinical cares to people living with HIV (PLHIV) globally. This study aimed to explore the metabolic profiles in cerebrospinal fluid (CSF) of Chinese people living with HIV (PLHIV). METHODS: Cerebrospinal fluid samples from PLHIV and healthy controls were collected from our hospital. Then, the metabolic profiles of CSFs were analyzed PLHIV with healthy individual as the normal controls using the untargeted GC/TOFMS. Following this, kyoto encyclopedia of genes and genomes annotation and pathway analysis were performed to further explore the underlying mechanism of these metabolic alterations in cognitive impairment of PLHIV. RESULTS: Both PCA analysis and OPLS-DA had presented that most samples were localized in 95% CI and the gap between control and HIV could significantly separate from each other. Upon this quality control, a total of 82 known metabolites were identified in CSF between PLHIV and healthy controls. Clustering of these metabolites presented that these differentially expressed metabolites could markedly distinguish HIV from healthy controls. Further pathway analyses showed that TCA cycle (citric acid, fumaric acid, lactate, et al.), amino acid (arginine, proline, alanine, aspartate, glutamine, et al.), lipid (cholesterol, butyrate, et al.) metabolisms were significantly changed in CSF of PLHIV, which might affect the cognitive status of PLHIV via affecting neuron energy support, signaling transduction, and neuroinflammation. CONCLUSION: Metabolic profiles were significantly altered in CSF and might play key roles in the etiology of cognitive impairment of PHLIV. Further explore the exact mechanism for these metabolic changes might be useful for cognitive impairment management of PHLIV.

[Recommendations for aminoacids chromatography analysis]. / Recommandations concernant l'analyse de la chromatographie des acides aminés.

Biochemical diagnosis of hereditary metabolic diseases requires the detection and simultaneous identification of a large number of compounds, hence the interest in metabolic profiles. Amino acid chromatography allows the identification and quantification of more than forty compounds. As part of the accreditation process for medical biology examinations according to standard NF EN ISO 15189, the group from SFEIM recommends an approach to accredit amino acid chromatography. Validation parameters and recommendations are discussed in this specific framework.

Choroid plexus LAT2 and SNAT3 as partners in CSF amino acid homeostasis maintenance.

BACKGROUND: Cerebrospinal fluid (CSF) is mainly produced by the choroid plexus (CP) located in brain ventricles. Although derived from blood plasma, it is nearly protein-free (~ 250-fold less) and contains about 2-20-fold less free amino acids, with the exception of glutamine (Gln) which is nearly equal. The aim of this study was to determine which amino acid transporters are expressed in mouse CP epithelium in order to gain understanding about how this barrier maintains the observed amino acid concentration gradient. METHODS: Expression of amino acid transporters was assessed in isolated choroid plexuses (CPs) by qRT-PCR followed by localization studies using immunofluorescence with specific antibodies. The impact of LAT2 (Slc7a8) antiporter deletion on CSF amino acids was determined. RESULTS: The purity of isolated choroid plexuses was tested on the mRNA level using specific markers, in particular transthyretin (Ttr) that was enriched 330-fold in CP compared to cerebral tissue. In a first experimental round, 14 out of 32 Slc amino acid transporters tested on the mRNA level by qPCR were selected for further investigation. Out of these, five were considered highly expressed, SNAT1 (Slc38a1), SNAT3 (Slc38a3), LAT2 (Slc7a8), ASC1 (Slc7a10) and SIT1 (Slc6a20b). Three of them were visualized by immunofluorescence: SNAT1 (Slc38a1), a neutral amino acid-Na+ symporter, found at the blood side basolateral membrane of CP epithelium, while SNAT3 (Slc38a3), an amino acid-Na+ symporter and H+ antiporter, as well as LAT2 (Slc7a8), a neutral amino acid antiporter, were localized at the CSF-facing luminal membrane. In a LAT2 knock-out mouse model, CSF Gln was unchanged, whereas other amino acids normally 2-20-fold lower than in plasma, were increased, in particular the LAT2 uptake substrates leucine (Leu), valine (Val) and tryptophan (Trp) and some other amino acids such as glutamate (Glu), glycine (Gly) and proline (Pro). CONCLUSION: These results suggest that Gln is actively transported by SNAT1 from the blood into CP epithelial cells and then released luminally into CSF via SNAT3 and LAT2. Its efflux via LAT2 may drive the reuptake from the CSF of essential amino acid substrates of this antiporter and thereby participates to maintaining the amino acid gradient between plasma and CSF.

Abnormal Amino Acid Profiles of Blood and Cerebrospinal Fluid from Cystathionine ß-Synthase-Deficient Mice, an Animal Model of Homocystinuria.

Mental retardation is the most common feature among inborn errors of amino acid metabolism. Patients with homocystinuria/homocysteinemia caused by cystathionine ß-synthase (CBS) deficiency suffer from thromboembolism and mental retardation from early ages; therefore, detection by newborn screening is performed. Furthermore, elevated levels of serum homocysteine during pregnancy are associated with the occurrence of neural tube defects (NTDs) in newborns. However, the causes of such central nervous system (CNS) defects are unknown. We found previously impaired learning abilities in Cbs-deficient (Cbs-/-) mice (but not NTD births). Here, we investigated the amino acid profiles of serum and cerebrospinal fluid (CSF) from Cbs-/- mice. Mice deficient in cystathionine γ-lyase (Cth), a downstream enzyme of CBS in transsulfuration, as well as wild-type mice, were analyzed as controls. Cbs-/- and Cth-/- mice were smaller than wild-type mice, and CSF yields in Cbs-/- mice were lower than the others. CSF amino acid levels were generally lower than those in serum, and compared with the dramatic amino acid level alterations in Cbs-/- mouse serum, alterations in CSF were less apparent. However, marked upregulation (versus wild-type) of aspartic acid/asparagine (Asp/Asn), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), methionine (Met), total homocysteine, and citrulline, and downregulation of lysine (Lys) were found in Cbs-/- mouse CSF. Because similar regulation of total homocysteine/citrulline/Lys was observed in the CSF of Cth-/- mice, which are free of CNS dysfunction, the reduced CSF volumes and the level changes of other amino acids could be relevant to Cbs-/--specific CNS defects.

Altered Cerebrospinal Fluid Concentrations of Hydrophobic and Hydrophilic Compounds in Early Stages of Multiple Sclerosis-Metabolic Profile Analyses.

The lack of a single predictive or diagnostic test in multiple sclerosis (MS) remains a major obstacle in the patient's care. The aim of this study was to investigate metabolic profiles, especially lipids in cerebrospinal fluid (CSF) using 1H-NMR spectroscopy and metabolomics analysis to discriminate MS patient group from the control ones. In this study, 19 MS patients and 19 controls, without neurological problems, patients were enrolled. To obtain the CSF metabolic profiles, NMR spectroscopy was used. Hydrophilic and hydrophobic compounds were analyzed using univariate and multivariate supervised analysis orthogonal partial least square discriminant analysis (OPLS-DA). Targeted OPLS-DA analysis of 32 hydrophilic and 17 hydrophobic compounds obtained 9 hydrophilic metabolites and 8 lipid functional groups which had the highest contribution to patient's group separation. Lower concentrations of CSF hydrophilic and hydrophobic compounds were observed in MS patients as compared to control group. Acetone, choline, urea, 1,3-dimethylurate, creatinine, isoleucine, myo-inositol, leucine, and 3-OH butyrate; saturated and monounsaturated acyl groups of ω-9, ω-7, ω-6, ω-3, and fatty acid, triglycerides, 1,3-DG, 1-MG, and unassigned component signal at 3.33 ppm were the most important signal compounds in group separation. Analysis of metabolic profile of raw CSF and their lipid extract shows decreased levels of many compounds and led to the conclusion that MS patients could have a disturbance in many metabolic pathways perhaps leading to the decreased level of acetyl-CoA and/or inflammation. CSF metabolic profile analyses could be used as a fingerprint for early MS diagnosis.

A Metabonomics Investigation into the Therapeutic Effects of BuChang NaoXinTong Capsules on Reversing the Amino Acid-Protein Interaction Network of Cerebral Ischemia.

BACKGROUND: Amino acids (AAs) in cerebrospinal fluid (CSF) play a pivotal role in cerebral ischemia (CI). BuChang NaoXinTong Capsules (BNC) are widely prescribed in Chinese medicine for the treatment of cerebrovascular and cardiovascular diseases. METHODS: In order to investigate the therapeutic effects and pharmacological mechanisms of BNC on reversing CI from a system level, an amino acid-protein interaction imbalanced network of CI containing metabolites of AAs, key regulatory enzymes, and proteins was constructed for the first time. Furthermore, a novel method for detecting the ten AAs in CSF was developed by UPLC-QQQ-MS in an effort to validate the imbalanced networks and the therapeutic effects of BNC via analysis of metabolites. RESULTS: Based on a middle cerebral artery occlusion (MCAO) rat model, the dynamic levels of amino acids in CSF 3, 6, 12, and 24 h after MCAO were analyzed. Up to 24 h, the accumulated nine AA biomarkers were found to significantly change in the MCAO group compared to the sham group and exhibited an obvious tendency for returning to baseline values after BNC treatment. In addition, based on the imbalanced network of CI, four key enzymes that regulate the generation of BNC-mediated AA biomarkers were selected and validated using an enzyme-linked immunosorbent assay and western blotting. Finally, aromatic-L-amino-acid decarboxylase (AADC) was found to be one of the putative targets for BNC-mediated protection against CI. CONCLUSION: This study provides new strategies to explore the mechanism of cerebral ischemia and help discover the potential mechanism of BNC.

Elevated inflammatory cytokine expression in CSF from patients with symptomatic thoracic disc herniation correlates with increased pain scores.

BACKGROUND: The pathophysiology of pain in patients with symptomatic thoracic disc herniation (TDH) remains poorly understood. Mere mechanical compression of the spinal cord and/or the exiting nerve root by a prolapsed disc cannot explain the pathogenesis of pain in all cases. Previous studies report a direct correlation between the levels of proinflammatory cytokines in disc biopsies and the severity of leg pain in patients with lumbar disc herniation. A similar correlation in patients with TDH has not been investigated. PURPOSE: To correlate the cerebrospinal fluid (CSF) expression of cytokines and pain-related amino acids with preoperative pain scores in patients with symptomatic TDH. STUDY DESIGN: A prospective human study of CSF samples and clinical outcome scores. METHODS: Using enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), we determined inflammatory cytokine levels (TNF-α, IL-1ß, and IL-10) and amino acid levels (glutamate, aspartate, gamma-aminobutyric acid, glycine, and arginine) in CSF samples from 10 patients with TDH and 10 control subjects who did not suffer an inflammatory disease nor pain related to spinal cord compression and subsequently correlated these levels with preoperative pain scores. Differences between both groups were evaluated by a Mann-Whitney U test. In order to estimate the correlation between cytokine or amino acid expression and pain scores, data were analyzed using a linear regression analysis. RESULTS: No inflammatory cytokines were found in CSF samples from control subjects, whereas TNF-α, IL-1ß, and IL-10 were detectable by ELISA in all CSF samples from patients with TDH. TNF-α and IL-10 but not IL-1ß levels moderately correlated with preoperative pain scores. Elevated TNF-αlevels positively correlated with high pain scores; elevated IL-10 levels negatively correlated with high pain scores. Amino acids were detectable in all samples from both groups. There were no significant differences between the groups in any of the amino acids measured with HPLC. CONCLUSION: Increased proinflammatory cytokine expression is associated with elevated pain scores in patients with symptomatic TDH. On the other hand, there is no conclusive correlation between the intensity of pain and the local or systemic presence of amino acids associated with pain transmission.

Chiral capillary electrophoresis with UV-excited fluorescence detection for the enantioselective analysis of 9-fluorenylmethoxycarbonyl-derivatized amino acids.

The potential of capillary electrophoresis (CE) with ultraviolet (UV)-excited fluorescence detection for sensitive chiral analysis of amino acids (AAs) was investigated. DL-AAs were derivatized with 9-fluorenylmethoxycarbonyl chloride (FMOC)-Cl to allow their fluorescence detection and enhance enantioseparation. Fluorescence detection was achieved employing optical fibers, leading UV excitation light (< 300 nm) from a Xe-Hg lamp to the capillary window, and fluorescence emission to a spectrograph equipped with a charge-coupled device (CCD). Signal averaging over time and emission wavelength intervals was carried out to improve the signal-to-noise ratio of the FMOC-AAs. A background electrolyte (BGE) of 40 mM sodium tetraborate (pH 9.5), containing 15% isopropanol (v/v), 30 mM sodium dodecyl sulfate (SDS), and 30 mM ß-cyclodextrin (ß-CD), was found optimal for AA chemo- and enantioseparation. Enantioresolutions of 1.0 or higher were achieved for 16 proteinogenic DL-AAs. Limits of detection (LODs) were in the 10-100-nM range (injected concentration) for the D-AA enantiomers, except for FMOC-D-tryptophan (536 nM) which showed intramolecular fluorescence quenching. Linearity (R2 > 0.997) and repeatability for peak height (relative standard deviations (RSDs) < 7.0%; n = 5) and electrophoretic mobility (RSDs < 0.6%; n = 5) of individual AA enantiomers were established for chiral analysis of DL-AA mixtures. The applicability of the method was investigated by the analysis of cerebrospinal fluid (CSF). Next to L-AAs, endogenous levels of D-glutamine and D-aspartic acid could be measured in CSF revealing enantiomeric ratios of 0.35 and 19.6%, respectively. This indicates the method's potential for the analysis of low concentrations of D-AAs in presence of abundant L-AAs.

Diurnal Variation of Melatonin Concentration in the Cerebrospinal Fluid of Unanesthetized Microminipig.

BACKGROUND/AIM: The aim of this study was to develop a method for sequentially collecting cerebrospinal fluid (CSF) from an unanesthetized microminipig, which shares many physiological and anatomical similarities with humans, such as diurnality, and investigate the diurnal variation of melatonin concentration in the CSF. MATERIALS AND METHODS: A catheter was placed percutaneously into the subarachnoid space of an anesthetized animal, and the tip of the catheter was placed into the cisterna magna under X-ray. We then sequentially collected CSF at light-on and -off times from the unanesthetized animal for several weeks. After catheter placement, a period of one week or more was necessary to relieve the contamination of RBCs in the CSF. RESULTS: A higher melatonin level in the CSF was noted during lights-off time, and the level was higher than that in the serum. CONCLUSION: This model of sequential collection of CSF will contribute to research in brain functions.

Species fractionation in a case-control study concerning Parkinson's disease: Cu-amino acids discriminate CSF of PD from controls.

BACKGROUND: Parkinson's disease is affecting about 1% of the population above 65 years. Improvements in medicine support prolonged lifetime which increases the total concentration of humans affected by the disease. It is suggested that occupational and environmental exposure to metals like iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn) can influence the risk for Parkinson's disease. These metals play a key role as cofactors in many enzymes and proteins. METHODS: In this case-control study, we investigated the Mn-, Fe-, Cu- and Zn-species in cerebrospinal fluid (CSF) by size-exclusion chromatography hyphenated to inductively coupled plasma mass spectrometry (SEC-ICP-MS) and the total concentration of these metals by inductively coupled plasma sector field mass spectrometry (ICP-sf-MS). RESULTS: The investigation of total metal concentration and speciation provided only minor changes, but it produced strong significance for a number of ratios. The analysis revealed a strong change in the ratio between total concentration of Fe and the amino acid-fraction of Cu. This could be observed when analyzing both the respective element concentrations of the fraction (which also depends on individual variation of the total element concentration) as well as when being expressed as percentage of total concentration (normalization) which more clearly shows changes of distribution pattern independent of individual variation of total element concentrations. CONCLUSION: Speciation analysis, therefore, is a powerful technique to investigate changes in a case-control study where ratios of different species play an important role.

Effects of L-theanine on anxiety-like behavior, cerebrospinal fluid amino acid profile, and hippocampal activity in Wistar Kyoto rats.

RATIONALE AND OBJECTIVES: The amino acid L-theanine (N-ethyl-L-glutamine) has historically been considered a relaxing agent. In the present study, we examined the effects of repeated L-theanine administration on behavior, levels of amino acids in the cerebrospinal fluid (CSF), and hippocampal activity in Wistar Kyoto (WKY) rats, an animal model of anxiety and depressive disorders. METHODS: Behavioral tests were performed after 7-10 days of L-theanine (0.4 mg kg-1 day-1) or saline administration, followed by CSF sampling for high-performance liquid chromatography (HPLC) analysis. An independent set of animals was subjected to [18F]fluorodeoxyglucose positron emission tomography (PET) scanning after the same dose of L-theanine or saline administration for 7 days. RESULTS: In the elevated plus maze test, the time spent in the open arms was significantly longer in the L-theanine group than in the saline group (P = 0.035). In addition, significantly lower CSF glutamate (P = 0.039) and higher methionine (P = 0.024) concentrations were observed in the L-theanine group than in the saline group. A significant increase in the standard uptake value ratio was observed in the hippocampus/cerebellum of the L-theanine group (P < 0.001). CONCLUSIONS: These results suggest that L-theanine enhances hippocampal activity and exerts anxiolytic effects, which may be mediated by changes in glutamate and methionine levels in the brain. Further study is required to more fully elucidate the mechanisms underlying the effects of L-theanine.

Hollow fiber-stir bar sorptive extraction and microwave assisted derivatization of amino acids in biological matrices.

A kind of solid phase microextraction configuration combining the principles of hollow fiber solid phase microextraction (HF-SPME) and stir bar sorptive extraction (SBSE) is presented. The main feature of HF-SBSE is the use of microporous hollow fiber acting as the carrier and filter, while a thin stainless steel wire and silica microspheres in the lumen of hollow fiber respectively acting as the magnetic stirrer and the dispersed sorbents for the collection and extraction of the target analytes, thus affording extraction process like SBSE. Moreover, the prepared hollow fiber stir bar was applied to direct microextraction and microwave assisted derivatization with N,O-Bis(trimethylsilyl)trifluroacetamide (BSTFA) of four amino acids in rats' urine and cerebrospinal fluid followed by gas chromatography mass spectrometric analysis. The limits of detection for four amino acids were found to be in the range of 0.0003-0.017µgmL-1, and all the analytes did not exhibit any lack of fit. The extraction recoveries using HF-SBSE techniques ranged from 71.8% to 102.3%. The results indicated that hollow fiber stir bar sorptive extraction was a promising technique for the enrichment and direct derivatization of analytes extracted from biological matrices without sample clean-up.

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.

A micellar electrokinetic chromatography-mass spectrometry approach using in-capillary diastereomeric derivatization for fully automatized chiral analysis of amino acids.

In the context of bioanalytical method development, process automatization is nowadays a necessity in order to save time, improve method reliability and reduce costs. For the first time, a fully automatized micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) method with in-capillary derivatization was developed for the chiral analysis of d- and l-amino acids using (-)-1-(9-fluorenyl) ethyl chloroformate (FLEC) as labeling reagent. The derivatization procedure was optimized using an experimental design approach leading to the following conditions: sample and FLEC plugs in a 2:1 ratio (15s, 30mbar: 7.5s, 30mbar) followed by 15min of mixing using a voltage of 0.1kV. The formed diastereomers were then separated using a background electrolyte (BGE) consisting of 150mM ammonium perfluorooctanoate (APFO) (pH=9.5) and detected by mass spectrometry (MS). Complete chiral resolution was obtained for 8 amino acids, while partial separation was achieved for 6 other amino acid pairs. The method showed good reproducibility and linearity in the low micromolar concentration range. The applicability of the method to biological samples was tested by analyzing artificial cerebrospinal fluid (aCSF) samples.

Enantioselective analysis of proteinogenic amino acids in cerebrospinal fluid by capillary electrophoresis-mass spectrometry.

d-Amino acids (AAs) are increasingly being recognized as essential molecules in biological systems. Enantioselective analysis of proteinogenic AAs in biological samples was accomplished by CE-MS employing ß-CD as chiral selector and ESI via sheath-liquid (SL) interfacing. Prior to analysis, AAs were fully derivatized with FMOC, improving AA-enantiomer separation and ESI efficiency. In order to optimize the separation and MS detection of FMOC-AAs, the effects of type and concentration of CD in the BGE, the composition of the SL, and MS-interfacing parameters were evaluated. Using a BGE of 10 mM ß-CD in 50 mM ammonium bicarbonate (pH 8) containing 15% v/v isopropanol, a SL of isopropanol-water-1 M ammonium bicarbonate (50:50:1, v/v/v) at a flow rate of 3 µL/min, and a nebulizer gas pressure of 2 psi, 15 proteinogenic AAs could be detected with enantioresolutions up to 3.5 and detection limits down to 0.9 µM (equivalent to less than 3 pg AA injected). The selectivity of the method was demonstrated by the analysis of spiked cerebrospinal fluid, allowing specific detection of d-AAs. Repeatability and linearity obtained for cerebrospinal fluid were similar to standard solutions, with peak area and migration-time RSDs (n = 5) below 16.2 and 1.6%, respectively, and a linear response (R(2) ≥ 0.977) in the 3-90 µM range.