fast uhplc methods for analysis of amino acids uhplc methods for analysis of amino acids qi zhang,...

Fast UHPLC Methods for Analysis of Amino AcidsQi Zhang, Bruce Bailey, Marc Plante, David Thomas, and Ian Acworth Thermo Fisher Scientific, Chelmsford, MA, USA

2 Fast UHPLC Methods for Analysis of Amino Acids

Fast UHPLC Methods for Analysis of Amino AcidsQi Zhang, Bruce Bailey, Marc Plante, David Thomas, and Ian AcworthThermo Fisher Scientific, 22 Alpha Road, Chelmsford, MA, USA

OverviewPurpose: Methods that offer both rapid and sensitive measurement of amino acids are essential to neurochemical research. The ability to decrease analysis time enables an improvement in temporal resolution when studying amino acid neurotransmission. Methods: Two methods using pre-column derivatization were developed: first, a fast isocratic method with electrochemical detection for the analysis of several neurochemically important amino acids, and secondly, a fast and sensitive gradient UHPLC method with fluorescence detection that measures both free D-aspartic acid and D-serine (as well as their L-enantiomers). Results: The UHPLC methods enabled the rapid separation of various neuroactiveamino acids within half of the analysis time when compared to traditional HPLC methods, while maintaining resolution and excellent sensitivity. Data from rat brain region homogenates are presented.

IntroductionPrecolumn derivatization of amino acids with o-phthaldialdehyde (OPA) reagent prior to detection by electrochemical or fluorescence detection is a very sensitive and selective way to measure neuroactive amino acids at low levels in microdialysis perfusates or brain tissues samples. Applying UHPLC technology, the run time for these analyses can be greatly reduced, thereby further enhancing sensitivity and improving sample throughput. Together with the new UHPLC-compatible Thermo Scientific™ Dionex™ UltiMate™ 3000 electrochemical detector, a fast isocratic method using OPA/βME derivatization for the analysis of neuroactive amino acids in brain tissue homogenates and microdialysis samples was developed. Although D-enatiomers of amino acids are common in lower organisms such as bacteria and were not thought to occur in mammalian tissues, it became apparent that some D-amino acids do occur in higher organisms and that they have biochemical importance. Recent publications indicating an abundance of D-amino acids in neuroendocrine tissues have prompted the development of simple analytical methods for the measurement of these amino acid enantiomers.1D-Serine (D-Ser) occurs in glial cells, is particularly abundant in brain regionsenriched in N-methyl-D-aspartate (NMDA) receptors and is the endogenousco-agonist of the glutamatergic NMDA receptor (not glycine).D-Aspartic acid (D-Asp) is found in some specific neuronal pathways, but is more abundant in epinephrine-containing glandular tissue (e.g., adrenal medulla) where it appears to regulate hormone synthesis and release. We developed a rapid UHPLC method using chiral N-acetyl-L-cysteine (NAC)-OPA derivatization and fluorescence detection for the analysis of D- and L-Asp and D- and L-Ser with improvement over typical HPLC methods.2,3

The application of these two methods is demonstrated with the measurement of neuroactive amino acids and D- and L-Asp and Ser in different brain tissuesamples.

Methods

Results and Discussion

Analysis of Neuroactive Amino Acids with EC DetectionPump: Thermo Scientific™ Dionex™ UltiMate™ 3000 DPG-3600RS Autosampler: Thermo Scientific™ Dionex™ UltiMate™ 3000 WPS-3000TRSEC Detector: Thermo Scientific™ Dionex™ UltiMate™ 3000 ECD-3000RS

6011RS cell, E1 = 150 mV, E2 = 550 mVColumn Thermostat: Thermo Scientific™ Dionex™ UltiMate™ 3000 TCC-3000RSColumn: Thermo Scientific™ Accucore™ C18, 2.6 µm, 100 × 3 mmMobile Phase: 76.5% 75 mM Phosphate buffer, pH 6.3, 20% Methanol,

3.5% AcetonitrileTemperature: 45 ºCFlow Rate: 0.8 mL/minInjection Volume: 2 µL

Analysis of free D- and L-Asp and D- and L-Ser with Fluorescence DetectionPump: Thermo Scientific™ Dionex™ UltiMate™ 3000 HPG-3400RS Autosampler: Thermo Scientific™ Dionex™ UltiMate™ 3000 WPS-3000TRS Detector: Thermo Scientific™ Dionex™ UltiMate™ 3000 FLD-3400RS Dual PMT, Excitation: 340 nm, Emission: 450 nmColumn Thermostat: Thermo Scientific™ Dionex™ UltiMate™ 3000 TCC-3000RSColumn: Thermo Scientific™ Hypersil GOLD™, 1.9 µm, 200 × 2.1 mmTemperature: 35 ºCMobile Phase A: 50 mM Dibasic sodium phosphate, pH 6.5Mobile Phase B: MethanolGradient: 0–6 min: 3%B; 6.5–10 min: 20%B; 11–14 min: 80% B; then

equilibrate at 3%B for 7 minFlow Rate: 0.25 mL/minInjection Volume: 2 µL

Data AnalysisAll HPLC chromatograms were obtained and compiled using Thermo Scientific™Dionex™ Chromeleon™ Chromatography Data System software, 6.8 SR 11.

.

Analysis of Neuroactive Amino Acids with EC DetectionA rapid method for neuroactive amino acids in brain tissue and microdialysissamples was developed using the new UltiMate 3000 electrochemical detector(ECD-3000RS) and ultra coulometric analytical cell (model 6011RS). With ultra-low internal volume, this new cell design minimizes dispersion to improve resolution for UHPLC separation, and still maintains near 100% efficiency for highsensitivity. Separation of 11 amino acids was completed within 10 min using an Accucore column, which reduces the analysis time in half compared to most HPLCmethods that typically require >20 min. Sensitivity for these amino acids typicallyvaried from 10 to 20 pg on column, with the exception of aspartic acid (Asp) which had a detection limit at of 40 pg on column. Although we are only presenting brain tissue homogenate data in this poster, the excellent sensitivity of this method will enable the measurement of trace amino acid levels in other biological samplessuch as microdialysis perfusates.

FIGURE 1: Overlay of chromatogram of prefrontal cortex control sample and standard showing the separation of 11 amino acids.

PC PT% change over control SC ST

% change over control

Asp 758.4 694.9 -8.4 766.2 704.8 -8.0Glu 1055.1 1275.0 20.8 1373.5 1198.5 -12.7GABA 478.2 392.9 -17.8 583.3 652.9 11.9

BC BT HC HTAsp 1476.0 1873.2 26.9 1493.1 581.4 -61.1Glu 572.1 1980.6 246.2 657.0 493.6 -24.9GABA 302.7 928.9 206.9 500.0 348.9 -30.2

TABLE 1. Levels (ng/mg tissue wet weight) of measured neuroactive aminoacids in regional brain tissues

FIGURE 2: Comparison of Asp, Glu and GABA concentration levelsbetween treated tissue sample and control samples.

Figure 3 shows the separation of D- and L-Asp, D- and L- Ser and some other amino acids, for standards and brain stem tissue homogenates. Unfortunately,an interference peak was found. Figure 4 illustrates separation of D-Asp from anunknown interference peak ”1” in the presence of large amount of L-Asp. The L/Denantiomer ratio was 360 for sample BT and 1600 for sample BC. Unknown interference peak “2” was separated from D-Asp but co-eluted with L-Asp. It has been observed during method development that its amount was insignificant relativeto the endogenous amount of L-Asp, so it shouldnʼt be a concern if quantitation ofL-Asp is required. It was observed that the amount of unknown “2” increased significantly if the supernatant was not separated from the precipitant right after tissue extraction. Figure 5 shows the zoom-in view of the separation of D-Ser fromasparagine (Asn) and L-Ser in standard and a tissue sample for a better view ofseparation resolution.

FIGURE 3. Overlay of chromatograms from brain stem tissue sampletreated and control, overlaid with a 10 ng sample chromatogram

0.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.2-3,075,435

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114,719,536

1 - DL-AA FLD-2 #142 BT 1:10 Emission_12 - DL-AA FLD-2 #143 BC 1:10 Emission_13 - DL-AA FLD-2 #163 500ng/mL Emission_1counts

min

3

2

1 D-A

sp

L-A

sp

Glu

Asn D

-Ser

L-S

er

340 nm /450 nm

FIGURE 4. Chromatogram showing separation of D-Asp from interferencepeak

4.65 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.34-78,735

0

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1 - DL-AA FLD-2 #142 BT 1:10 Emission_12 - DL-AA FLD-2 #143 BC 1:10 Emission_13 - DL-AA FLD-2 #147 [modified by lab122] 10ng/mL Emission_1counts

min

321

D-A

sp

unkn

own

1

L-A

sp +

unk

now

n 2 340 nm /450 nm

FIGURE 5. Chromatogram showing separation of D-Ser from Asn and L-Ser

10.96 11.13 11.25 11.38 11.50 11.63 11.75 11.88 12.00 12.13 12.25 12.38 12.50 12.63 12.75 12.88 13.00 13.13 13.29-7,236,239

0

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15,000,000

20,000,000

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30,000,000

36,687,339


min

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Asn

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er

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er

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CC CT% change over control SC ST


D-Asp 1.8 1.2 -32.3 2.4 3.2 31.5D-Ser 30.5 28.8 -5.7 28.1 42.2 50.1

BC BT HC HTD-Asp 0.6 3.4 442.4 0.9 1.6 86.9D-Ser 0.5 41.3 8498.8 1.5 15.5 958.7

Table 2 and Figure 6 shows concentration of D- and L-Asp and D- and L-Ser in eight brain tissue samples. Amphetamine treatment was associated with region specific differences. The brain stem region was markedly stimulated with a 442% increase of D-Asp and 8498% increase in D-Ser. The hippocampus was also affected, with an increase of 875% and 958% for D-Asp and D-Ser, respectively. The frontal cortexregion was least affected.

TABLE 2. D-Asp and D-Ser concentration (ng/mg tissue wet weight) in regional brain tissues.

FIGURE 6: Comparison of D-Asp and D-Ser concentration levels between treated tissue samples and control samples.

Conclusions• A fast UHPLC method using OPA/βME and EC detection was developed for

neuroactive amino acid analysis. Separation of 11 amino acids wascompleted within 10 min. Sensitivity was 10–40 pg on column for variousamino acids.

• A UHPLC method for the simultaneous determination of D- and L-Asp and Ser enantiomers within 20 min was also developed showing improvedseparation of D-Asp from the interference peak. Sensitivity was 200–400 fgon column.

• Application of these two methods was demonstrated with analysis of regional brain tissue sample for the effects of amphetamine on Asp, Glu, GABA and D-Asp and D-Ser. Region specific differences was observed for the effect ofamphetamine treatment, with brain stem most markedly stimulated,hippocampus was also affected, prefrontal cortex was least affected.

References1. DʼAniello, A. D-Aspartic acid: An endogenous amino acid with an important

neuroendocrine role. Brain Res. Reviews 2007, 53, 216-234.2. Bruckner, H.; Haasmann, S., et al. Liquid chromatographic determination of

D- and L-amino acids by derivatization with o-phthaldialdehyde and chiral thiols. J. Chromatogr. A,1994, 611, 259-273.

3. Bruckner, H.; Wittner, R. Automated enantioseparation of amino acidsby derivatization with o-phthaldialdehyde and N-acetylated cysteines.J. Chromatogr. 1989, 476, 73-82.

Biological ExperimentsMale Sprague Dawley rats weighing 175–200 grams were administered 2.0 mg/kg d-amphetamine or vehicle (saline) via i.p. injection. One hour later animals were sacrificed by carbon dioxide asphyxiation and the brains rapidly removed, dissected and frozen at -70 oC.

Sample Preparation10 to 20 mg of brain tissue was homogenized in 1 mL 0.3N perchloric acid, then diluted either 10 or 20 times with water before analysis.

Derivatization ProcedureStock OPA/NAC: 20 mg OPA was dissolved in 1 mL methanol, 10 mg NAC and 9 mL OPA diluent was added. Stock derivatizing reagent was stable for 5 days at4 ºC. Working reagent was prepared fresh daily by diluting 1 mL of stock reagent into 4 mL OPA diluent. Automated precolumn derivatization was performed by mixing 25 µL working reagent with 25 µL tissue sample. For enhanced column life, 8 µL 1M acetic acid was mixed into sample before injection to neutralize sample pH. © 2013 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher

Scientific Inc. and its subsidiaries. This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. PO70537_E 03/13S

Eight brain tissue samples from four different brain regions including prefrontal cortex (P), striatum (S), brain stem (B) and hippocampus (H), from either saline control (C) or amphetamine treated (T) rats were analyzed. Figure 1 shows anoverlay of chromatograms of a tissue sample and separation of 11 amino acidsstandards. The effect of amphetamine treatment was most notable for Asp,glutamate (Glu) and γ-aminobutyric acid (GABA) (Table 1 and Figure 2-4).Amphetamine treatment resulted in over 200% increase in Glu and GABA, and a 26% increase in Asp in brain stem, but an overall decrease in their levels in the hippocampus. No significant changes were found in the prefrontal cortex or the striatum.

Analysis of Free D- and L-Asp acid and D- and L-Ser with FluorescenceDetectionA variety of methods, including enzymatic assay, GC and HPLC methods, have been developed to separate and quantitatively determine amino acids enantiomersin biological samples. Unfortunately, most published methods either separate theenantiomers of just one amino acid e.g., D- and L-Asp, or when separating the enantiomers of more amino acids requires an analysis time of over one hour.Presented here is a UHPLC method with precolumn derivatization using OPA and NAC for simultaneous determination of D- and L-Asp and D- and L-Ser enantiomers in only 20 min, with detection limit of 200 to 400 fg on column.

0

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ng/m

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et w

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0.00

0.50

1.00

1.50

1.99

0.72 Asp

0.95 Glu 1.93 Ser

2.09 Gln2.70 Arg

3.36 Gly

3.79 Thr 5.83 Tau 6.77 Ala 7.76 GABA

min

µA

1 - ECDRS AA #89 P contr sample 1:20 dilution ECDRS_22 - ECDRS AA #90 1ug/mL 11AA ECDRS_2

3Thermo Scientific Poster Note • PN70537_E 03/13S


OverviewPurpose: Methods that offer both rapid and sensitive measurement of amino acidsare essential to neurochemical research. The ability to decrease analysis time enables an improvement in temporal resolution when studying amino acidneurotransmission.Methods: Two methods using pre-column derivatization were developed: first, afast isocratic method with electrochemical detection for the analysis of several neurochemically important amino acids, and secondly, a fast and sensitive gradientUHPLC method with fluorescence detection that measures both free D-aspartic acid and D-serine (as well as their L-enantiomers). Results: The UHPLC methods enabled the rapid separation of various neuroactiveamino acids within half of the analysis time when compared to traditional HPLCmethods, while maintaining resolution and excellent sensitivity. Data from rat brainregion homogenates are presented.

IntroductionPrecolumn derivatization of amino acids with o-phthaldialdehyde (OPA) reagentprior to detection by electrochemical or fluorescence detection is a very sensitive and selective way to measure neuroactive amino acids at low levels in microdialysisperfusates or brain tissues samples. Applying UHPLC technology, the run time for these analyses can be greatly reduced, thereby further enhancing sensitivity and improving sample throughput. Together with the new UHPLC-compatible ThermoScientific™ Dionex™ UltiMate™ 3000 electrochemical detector, a fast isocraticmethod using OPA/βME derivatization for the analysis of neuroactive amino acidsin brain tissue homogenates and microdialysis samples was developed.Although D-enatiomers of amino acids are common in lower organisms such asbacteria and were not thought to occur in mammalian tissues, it became apparentthat some D-amino acids do occur in higher organisms and that they havebiochemical importance. Recent publications indicating an abundance of D-aminoacids in neuroendocrine tissues have prompted the development of simple analytical methods for the measurement of these amino acid enantiomers.1D-Serine (D-Ser) occurs in glial cells, is particularly abundant in brain regionsenriched in N-methyl-D-aspartate (NMDA) receptors and is the endogenousco-agonist of the glutamatergic NMDA receptor (not glycine).D-Aspartic acid (D-Asp) is found in some specific neuronal pathways, but is more abundant in epinephrine-containing glandular tissue (e.g., adrenal medulla) where itappears to regulate hormone synthesis and release. We developed a rapid UHPLCmethod using chiral N-acetyl-L-cysteine (NAC)-OPA derivatization and fluorescencedetection for the analysis of D- and L-Asp and D- and L-Ser with improvement over typical HPLC methods.2,3

The application of these two methods is demonstrated with the measurement ofneuroactive amino acids and D- and L-Asp and Ser in different brain tissuesamples.

Methods







Data AnalysisAll HPLC chromatograms were obtained and compiled using Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System software, 6.8 SR 11.

.

Analysis of Neuroactive Amino Acids with EC DetectionA rapid method for neuroactive amino acids in brain tissue and microdialysissamples was developed using the new UltiMate 3000 electrochemical detector (ECD-3000RS) and ultra coulometric analytical cell (model 6011RS). With ultra-low internal volume, this new cell design minimizes dispersion to improve resolution for UHPLC separation, and still maintains near 100% efficiency for high sensitivity. Separation of 11 amino acids was completed within 10 min using an Accucore column, which reduces the analysis time in half compared to most HPLC methods that typically require >20 min. Sensitivity for these amino acids typically varied from 10 to 20 pg on column, with the exception of aspartic acid (Asp) which had a detection limit at of 40 pg on column. Although we are only presenting brain tissue homogenate data in this poster, the excellent sensitivity of this method will enable the measurement of trace amino acid levels in other biological samples such as microdialysis perfusates.




Asp 758.4 694.9 -8.4 766.2 704.8 -8.0Glu 1055.1 1275.0 20.8 1373.5 1198.5 -12.7GABA 478.2 392.9 -17.8 583.3 652.9 11.9






0.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.2-3,075,435

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4.65 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.34-78,735

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10.96 11.13 11.25 11.38 11.50 11.63 11.75 11.88 12.00 12.13 12.25 12.38 12.50 12.63 12.75 12.88 13.00 13.13 13.29-7,236,239

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D-Asp 1.8 1.2 -32.3 2.4 3.2 31.5D-Ser 30.5 28.8 -5.7 28.1 42.2 50.1













Biological ExperimentsMale Sprague Dawley rats weighing 175–200 grams were administered 2.0 mg/kgd-amphetamine or vehicle (saline) via i.p. injection. One hour later animals weresacrificed by carbon dioxide asphyxiation and the brains rapidly removed, dissected and frozen at -70 oC.


Derivatization ProcedureStock OPA/NAC: 20 mg OPA was dissolved in 1 mL methanol, 10 mg NAC and 9 mL OPA diluent was added. Stock derivatizing reagent was stable for 5 days at4 ºC. Working reagent was prepared fresh daily by diluting 1 mL of stock reagent into 4 mL OPA diluent. Automated precolumn derivatization was performed bymixing 25 µL working reagent with 25 µL tissue sample. For enhanced column life,8 µL 1M acetic acid was mixed into sample before injection to neutralize sample pH. © 2013 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher




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0.00

0.50

1.00

1.50

1.99

0.72 Asp

0.95 Glu 1.93 Ser

2.09 Gln2.70 Arg

3.36 Gly

3.79 Thr 5.83 Tau 6.77 Ala 7.76 GABA

min

µA

1 - ECDRS AA #89 P contr sample 1:20 dilution ECDRS_2 2 - ECDRS AA #90 1ug/mL 11AA ECDRS_2






Methods








.





Asp 758.4 694.9 -8.4 766.2 704.8 -8.0Glu 1055.1 1275.0 20.8 1373.5 1198.5 -12.7GABA 478.2 392.9 -17.8 583.3 652.9 11.9


TABLE 1. Levels (ng/mg tissue wet weight) of measured neuroactive amino acids in regional brain tissues

FIGURE 2: Comparison of Asp, Glu and GABA concentration levels between treated tissue sample and control samples.



0.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.2-3,075,435

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4.65 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.34-78,735

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10.96 11.13 11.25 11.38 11.50 11.63 11.75 11.88 12.00 12.13 12.25 12.38 12.50 12.63 12.75 12.88 13.00 13.13 13.29-7,236,239

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D-Asp 1.8 1.2 -32.3 2.4 3.2 31.5D-Ser 30.5 28.8 -5.7 28.1 42.2 50.1

















Eight brain tissue samples from four different brain regions including prefrontal cortex (P), striatum (S), brain stem (B) and hippocampus (H), from either saline control (C) or amphetamine treated (T) rats were analyzed. Figure 1 shows an overlay of chromatograms of a tissue sample and separation of 11 amino acids standards. The effect of amphetamine treatment was most notable for Asp, glutamate (Glu) and γ-aminobutyric acid (GABA) (Table 1 and Figure 2-4). Amphetamine treatment resulted in over 200% increase in Glu and GABA, and a 26% increase in Asp in brain stem, but an overall decrease in their levels in the hippocampus. No significant changes were found in the prefrontal cortex or the striatum.

Analysis of Free D- and L-Asp acid and D- and L-Ser with Fluorescence DetectionA variety of methods, including enzymatic assay, GC and HPLC methods, have been developed to separate and quantitatively determine amino acids enantiomersin biological samples. Unfortunately, most published methods either separate the enantiomers of just one amino acid e.g., D- and L-Asp, or when separating the enantiomers of more amino acids requires an analysis time of over one hour. Presented here is a UHPLC method with precolumn derivatization using OPA and NAC for simultaneous determination of D- and L-Asp and D- and L-Ser enantiomers in only 20 min, with detection limit of 200 to 400 fg on column.

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0.13 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.06-0.47

0.00

0.50

1.00

1.50

1.99

0.72 Asp

0.95 Glu 1.93 Ser

2.09 Gln2.70 Arg

3.36 Gly

3.79 Thr 5.83 Tau 6.77 Ala 7.76 GABA

min

µA


5Thermo Scientific Poster Note • PN70537_E 03/13S





Methods








.





Asp 758.4 694.9 -8.4 766.2 704.8 -8.0Glu 1055.1 1275.0 20.8 1373.5 1198.5 -12.7GABA 478.2 392.9 -17.8 583.3 652.9 11.9




Figure 3 shows the separation of D- and L-Asp, D- and L- Ser and some other amino acids, for standards and brain stem tissue homogenates. Unfortunately, an interference peak was found. Figure 4 illustrates separation of D-Asp from an unknown interference peak ”1” in the presence of large amount of L-Asp. The L/D enantiomer ratio was 360 for sample BT and 1600 for sample BC. Unknown interference peak “2” was separated from D-Asp but co-eluted with L-Asp. It has been observed during method development that its amount was insignificant relative to the endogenous amount of L-Asp, so it shouldnʼt be a concern if quantitation of L-Asp is required. It was observed that the amount of unknown “2” increasedsignificantly if the supernatant was not separated from the precipitant right aftertissue extraction. Figure 5 shows the zoom-in view of the separation of D-Ser fromasparagine (Asn) and L-Ser in standard and a tissue sample for a better view of separation resolution.

FIGURE 3. Overlay of chromatograms from brain stem tissue sample treated and control, overlaid with a 10 ng sample chromatogram

0.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.2-3,075,435

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2

1 D-A

sp

L-A

sp

Glu

Asn D

-Ser

L-S

er

340 nm /450 nm

FIGURE 4. Chromatogram showing separation of D-Asp from interference peak

4.65 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.34-78,735

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

873,817


min

321

D-A

sp

unk

now

n 1

L-A

sp +

unk

now

n 2 340 nm /450 nm


10.96 11.13 11.25 11.38 11.50 11.63 11.75 11.88 12.00 12.13 12.25 12.38 12.50 12.63 12.75 12.88 13.00 13.13 13.29-7,236,239

0

5,000,000

10,000,000

15,000,000

20,000,000

25,000,000

30,000,000

36,687,339


min

321

Asn

D-S

er

L-S

er

340 nm /450 nm



D-Asp 1.8 1.2 -32.3 2.4 3.2 31.5D-Ser 30.5 28.8 -5.7 28.1 42.2 50.1



















0

500

1000

1500

2000


ng/m

gw

et w

eigh

t Asp

ControlTreated

0

500

1000

1500

2000

2500


ng/m

g w

et w

eigh

t Glu

ControlTreated

0

200

400

600

800

1000


ng/m

g w

et w

eigh

t GABA

Control

Treated

00.5

11.5

22.5

33.5

4


ng/m

g w

et w

eigh

t D-Asp

ControlTreated

0

10

20

30

40

50


ng/m

g w

et w

eigh

t

D-Ser

ControlTreated

0.13 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.06-0.47

0.00

0.50

1.00

1.50

1.99

0.72 Asp

0.95 Glu 1.93 Ser

2.09 Gln2.70 Arg

3.36 Gly

3.79 Thr 5.83 Tau 6.77 Ala 7.76 GABA

min

µA







Methods








.





Asp 758.4 694.9 -8.4 766.2 704.8 -8.0Glu 1055.1 1275.0 20.8 1373.5 1198.5 -12.7GABA 478.2 392.9 -17.8 583.3 652.9 11.9






0.1 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.2-3,075,435

10,000,000

20,000,000

30,000,000

40,000,000

50,000,000

60,000,000

70,000,000

80,000,000

90,000,000

100,000,000

114,719,536


min

3

2

1 D-A

sp

L-A

sp

Glu

Asn D

-Ser

L-S

er

340 nm /450 nm


4.65 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.34-78,735

0

100,000

200,000

300,000

400,000

500,000

600,000

700,000

800,000

873,817


min

321

D-A

sp

unkn

own

1

L-A

sp +

unk

now

n 2 340 nm /450 nm


10.96 11.13 11.25 11.38 11.50 11.63 11.75 11.88 12.00 12.13 12.25 12.38 12.50 12.63 12.75 12.88 13.00 13.13 13.29-7,236,239

0

5,000,000

10,000,000

15,000,000

20,000,000

25,000,000

30,000,000

36,687,339


min

321

Asn

D-S

er

L-S

er

340 nm /450 nm



D-Asp 1.8 1.2 -32.3 2.4 3.2 31.5D-Ser 30.5 28.8 -5.7 28.1 42.2 50.1


Table 2 and Figure 6 shows concentration of D- and L-Asp and D- and L-Ser in eight brain tissue samples. Amphetamine treatment was associated with region specific differences. The brain stem region was markedly stimulated with a 442% increase of D-Asp and 8498% increase in D-Ser. The hippocampus was also affected, with anincrease of 875% and 958% for D-Asp and D-Ser, respectively. The frontal cortex region was least affected.






• Application of these two methods was demonstrated with analysis of regionalbrain tissue sample for the effects of amphetamine on Asp, Glu, GABA and D-Asp and D-Ser. Region specific differences was observed for the effect ofamphetamine treatment, with brain stem most markedly stimulated,hippocampus was also affected, prefrontal cortex was least affected.











0

500

1000

1500

2000


ng/m

gw

et w

eigh

t Asp

ControlTreated

0

500

1000

1500

2000

2500


ng/m

g w

et w

eigh

t Glu

ControlTreated

0

200

400

600

800

1000


ng/m

g w

et w

eigh

t GABA

Control

Treated

00.5

11.5

22.5

33.5

4


ng/m

g w

et w

eigh

t D-Asp

ControlTreated

0

10

20

30

40

50


ng/m

g w

et w

eigh

t

D-Ser

ControlTreated

0.13 1.25 2.50 3.75 5.00 6.25 7.50 8.75 10.06-0.47

0.00

0.50

1.00

1.50

1.99

0.72 Asp

0.95 Glu 1.93 Ser

2.09 Gln2.70 Arg

3.36 Gly

3.79 Thr 5.83 Tau 6.77 Ala 7.76 GABA

min

µA


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