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AssigneesRijksuniversiteit Groningen, Academisch Ziekenhuis Groningen
InventorsIdo Peter KEMA, Claude Pascal VAN DER LEY, Hermannus Johannes Roelof VAN FAASSEN
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The invention relates to analytical chemistry, in particular to means and methods for the quantitative determination of derivatized biogenic amines, precursors or metabolites thereof. Provided is a method for the in situ derivatization of at least one biogenic amine, precursor or metabolite thereof in an isolated aqueous sample, the derivatization comprising the steps of: (i) contacting said sample with a propionic anhydride/acetonitril solution in the presence of a phosphate buffer having a pH in the range of 7.0 to 9.0 and allowing the conversion of amine and/or hydroxyl moieties of said biogenic amine or metabolite thereof to form a propionyl derivative of said biogenic amine; followed by (ii) adding to the reaction mixture obtained in step (i) a carbodiimide compound and an electrophilic amine-containing compound, and allowing the carbodiimide-mediated derivatization of carboxylic acid moieties of a biogenic amine, precursor or metabolite thereof.
A method for the in situ derivatization of at least one biogenic amine, precursor or metabolite thereof in an aqueous sample, the derivatization comprising the steps of:
Method according to any one of the preceding claims, wherein the pH of said phosphate buffer is in the range of 8.0 to 8.5.
Method according to any one of the preceding claims, wherein an internal standard is added to the sample.
Method according to any one of the preceding claims, wherein step (i) comprises mixing 1 volume of (biological) sample, 1 volume of internal standard and 5 volumes of phosphate buffer, and contacting the mixture with 1 volume of propionic anhydride in acetonitrile.
Method according to any one of the preceding claims, wherein step (ii) comprises adding 2 volumes of the carbodiimide and 2 volumes of the primary amine-containing compound to 1 volume of the reaction mixture obtained in step (i).
Method according to any one of the preceding claims, wherein step (ii) comprises adding 2,2,2-trifluroethylamine (TFEA) and N-ethyl-N′-(3-dimethylaminopropyl) carbothimide (EDC), preferably 0.4 M TFEA and 0.4 M EDC.
Method according to any one of the preceding claims, further comprising, following step (ii), a centrifugation step.
Method according to any one of the preceding claims, wherein said biogenic amine is selected from the group consisting of L-DOPA, epinephrine, norepinephrine, dopamine, 5-hydroxytryptophan, serotonin, 5-hydroxyindolacetic acid, metanephrine, normetanephrine and 3-methoxytyramine.
Method according to any one of the preceding claims, further comprising detecting at least one derivatized biogenic amine by subjecting the derivatized sample to (in-line) SPE-LC-MS/MS.
Dependencies are inferred from the claim text.