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Chromatograms and methods gallery

UHPLC-ESI-MS/MS analysis of >40 physiological amino acids and related molecules in 10 min

Ultra-sensitive detection of monoamines in the microdialysate samples


Fluorescence derivatization of serotonin (5-HT), dopamine (DA), noradrenaline (NA) and adrenaline (A). At mild alkaline conditions, 5-hydroxyindoles and catecholamines react with benzylamine, forming highly fluorescent 2-phenyl-4,5-pyrrolobenzoxazoles and 2-phenyl(4,5-dihydropyrrolo) [2,3-f]benzoxazoles, respectively. However, for derivatization of DA a higher fluorescence intensity is achieved by reaction with 1,2-diphenylethylenediamine. rather than with benzylamine, therefore for simultaneous determination of 5-HT, NA and DA in brain microdialysates, a two-step derivatization with benzylamine followed by DPE was developed.


   
Chromatograms of a standard solution containing BA-DPE-derivatized 5-HT, NA and DA at final concentrations of 0.3 fmol each in 10 µl injected on-column and a representative chromatogram of the microdialysis sample from the rat PFC at basal conditions. The basal levels of 5-HT, NA and DA in the dialysates from the PFC were 7.3, 5.3 and 8.1 fmol/10 µl, respectively.



A chromatogram showing the separation of NA, 5-HT and 5-HIAA in 10 µl microdialysis sample collected at basal conditions from the hippocampus of the awake mouse. The estimated concentration of NA was 2.99 fmol/10 µl; for 5-HT and 5-HIAA the levels were 1.15 and 426.8 fmol/10 µl, respectively.  



Ultra-sensitive detection of histamine



Intramolecular excimer-forming fluorescence derivatization of histamine with 4-(1-pyrene)butyric acid N-hydroxysuccinimide ester (PSE).

      
A typical chromatogram of a standard solution containing 50 fmol histamine derivatized with PSE in 30 µl injected on-column.
  Effects of the forced swimming (20 min) on release of histamine in the hypothalamus, prefrontal cortex, hippocampus and striatum of the rat (mean ± SEM, n = 5 rats).



Determination of monoamines and their metabolites by liquid chromatography/electrochemistry


Electrochemical detection of catecholamines and hydroxyindoles. Oxidation reaction schemes for catecholamines (DA: R1, R2 = H; NA: R1 = OH, R2 = H; A R1 = OH, R2 = CH3, 5-hydroxyindoles (5-HT) and their acidic metabolites. Each catechol or hydroxyindole molecule is oxidized to its respective quinone form while generating two protons and two electrons.

A typical chromatogram illustrating separation of DA and its main metabolites DOPAC and HVA in a standard mixture (150 fmol/15 µl) and in  15 µl of a microdialysis sample from the rat striatum at basal conditions. Chromatograms of a standard solution containing NA, DA and 5-HT at final concentrations of 5 fmol each in 20 µl injected on-column and a typical microdialysis sample from mPFC of an awake rat at basal conditions.


Determination of plasma catecholamines:

a) a chromatogram of a standard mixture containing NA, A and DHBA (internal standard) 167 fmol each












b) determination of catecholamines in 150 µl rat plasma, the sample analyzed repeatedly two times





Chromatograms illustrating separation of a standard mixture containing MHPG, NE, E, DOPAC, NM, DA, 5-HIAA, Isoprenaline (int. standard), HVA, 3-MT and 5-HT at concentrations of 0.5; 1 and 2 pmol/15 µl.


Determination of monoamines and their metabolites in the tissue extract from the rat striatum (red) without internal standard (isoprenaline) and in the standard mixture (blue).


Determination of acetylcholine by liquid chromatography/electrochemistry with immobilized enzyme reactor

 
Determination of acetylcholine by HPLC with electrochemical detection is based on enzymatic conversion of acetylcholine and choline to hydrogen peroxide in an immobilized enzyme reactor (IMER) placed between the outlet of the chromatographic column and the inlet to the electrochemical cell. Oxidation of choline via choline oxidase (ChOx) generates two molecules of hydrogen peroxide, which is then easily detected (oxidized) at a platinum electrode operating at +500 mV versus an Ag/AgCl reference electrode. Acetylcholine separated from the choline peak must be first hydrolyzed to choline and acetate via acetylcholinesterase (AChE), thereafter choline is oxidized to betaine and hydrogen peroxide.


Chromatogram illustrating determination of extracellular acetylcholine levels in the microdialysate from the ventral hippocampus of the awake rat at basal conditions and perfusion with neostigmine free artificial CSF, and a chromatogram of the ACh standard (20 fmol/15 µl injected).


Determination of amino acid neurotransmitters by liquid chromatography with fluorescence detection


Derivatization of physiological amino acids containing primary amino groups with o-phthaldialdehyde and 2-mercaptoethanol (OPA/MCE) reagent. The reaction takes place in the alkaline medium (typically a borate buffer, pH 9.5) within 1-2 min, even at low (+4 °C) temperatures. The resulting isoindole derivatives can be detected either by fluorescence detection (excitation/emission wavelengths of 330/440 nm) or by electrochemical detection at a glassy carbon electrode operating at +700 mV.

   
Chromatograms of (a) a standard mixture of amino acids and (b) a microdialysis sample from the striatum of the awake rat at basal conditions. The samples (10 µl) were derivatized with 3 µl OPA/3-mercaptopropionic acid reagent, thereafter 5 µl was injected onto the reversed-phase column. For full separation of amino acids eluting after GABA including Tyr, Met, Val, Trp, Iso, Leu and Lys, there is a need to optimize the gradient program as described elsewhere (Kehr, 1999). The concentrations of amino acids in the standard mixture were 2.5 µM each and in the microdialysate as follows (in µM): Asp 0.129; Glu 0.275; Asn 1.358; Ser 6.106; Gln 7.62; His 1.604; Gly 3.187; Thr 5.897; citrulline 0.608; Arg 0.652; Ala 5.955; Tau 2.12 and GABA 0.056.


   
Chromatograms of (a) a standard mixture of amino acids and (b) a microdialysis sample from the striatum of the awake rat at basal conditions. The samples (10 µl) were derivatized with 5 µl OPA/3-mercaptopropionic acid reagent, thereafter 6 µl was injected onto the reversed-phase microbore column. The separation was achieved by use of a binary gradient elution mode; the OPA-derivatized amino acids eluted in the order: Asp, Glu, Asn, Ser, Gln, His, Gly, Thr, Arg, Ala, Tau, GABA, Tyr, Met, Val, Phe, Iso, Leu and Lys.



Determination of GABA derivatized with OPA/ t-butylthiol reagent by HPLC with electrochemical detection: A mixture of physiological amino acid containing 500 fmol GABA and a typical microdialysis sample from the rat striatum at basal conditions.