Derivatization of amines for electrochemical detection
AssigneeThe United States of America as represented by the Department of Health and Human Services
InventorsKenneth A. Jacobson, Kenneth L. Kirk, Markku I. Linnoila, Thomas Miller, Kazunori Mine
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Abstract
Primary and secondary amines in biological fluids are selectively derivitized by reaction with esters of N-hydroxysuccinimide or N-hydroxysulfosuccinimide to form an N-acylated derivative. Those N-acylated derivatives may then be extracted into a polar organic phase and extracted. Non-electroactive amines may be made suitable for coulometric analysis by selecting the ester used so as to attach an electroactive group to the amine. For example, histamine may be reacted with the Bolton-Hunter reagent to form a suitable derivative in high yield.
Claims25
A process for the quantitative detection of amines in a fluid sample, comprising:
- reacting a fluid sample, under a non-reactive atmosphere, with a reactive O-acyl N-hydroxysulfosuccinamide to form an N-acylated derivative of the amine to be detected, wherein said acyl group is selected so as to enhance the separability of said N-acylated amine derivative from polar substances in the remainder of the fluid which may interfere with electrochemical measuring;
- separating the N-acylated amine derivative from polar substances in the remainder of the fluid sample which may interfere with electrochemical measuring;
- and
- subjecting the separating N-acylated amine derivative to electrochemical detection coupled to high pressure liquid chromatography.
In the process for the quantitative detection of electroactive amines in an aqueous fluid sample containing other polar substances, comprising electrochemical detection coupled to high pressure liquid chromatography, the improvement whereby the sensitivity of such process can be substantially increased, comprising the steps of, prior to said detection step:
- reacting the fluid sample, under a non-reactive atmosphere, with a reactive O-acyl hydroxlamine derivative to form an N-acylated derivative of the electroactive amine to be detected, wherein said reactive O-acyl hydroxylamine derivative is selected to have the property that it is not reactive toward the portion of the electroactive amine to be detected which renders the amine electroactive, and wherein said acyl group is selected so as to render said N-acylated amine derivative soluble in an organic solvent and thereby separable from polar substances in the remainder of the fluid which may interfere with electrochemical measuring;
- and
- separating the N-acylated amine derivative from polar substances in the remainder of the fluid sample which may interfere with electrochemical measuring by extracting the N-acylated amine derivative with a suitable organic solvent.
In the process for the quantitative detection of non-electroactive amines in an aqueous fluid sample containing other polar substances, comprising derivatizing said amines with an electroactive group and detecting said derivatized amine by electrochemical detection coupled to high pressure liquid chromatography, the improvement whereby such non-electroactive amines can be quantitatively detected in the fluid sample at substantial sensitivity, comprising the steps of, prior to said detecting step:
- reacting the fluid sample, under a non-reactive atmosphere, with a reactive O-acyl hydroxylamine derivative to form an N-acylated derivative of the non-electroactive amine to be detected, wherein said reactive O-acyl hydroxlamine derivative is selected to have the property that it is not reactive toward hydroxyl groups, and wherein said acyl group is selected so as to render said N-acylated amine derivative soluble in an organic solvent and thereby separable from polar substances in the remainder of the fluid sample which may interfere with electrochemical measuring and to render said N-acylated amine derivative electroactive;
- and
- separating the N-acylated amine derivative from polar substances in the remainder of the fluid sample which may interfere with electrochemical measuring by extracting the N-acylated amine derivative with a suitable organic solvent.
Dependencies are inferred from the claim text.