Determination of catecholamines as their N-hydroxy-succinimidyl-3-indolylacetate derivatives by pre-column derivatization HPLC separation and fluorescent detection
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N-Hydroxysuccinimidyl-3-indolylacetate (SIIA) is a new fluorescent derivatizing reagent with an indole ring and ¶an N-hydroxysuccinimide ester functionality. It can react with catecholamines under mild conditions to form corresponding amides, which have strong fluorescence at λex/λem = 301 nm/¶365 nm. This paper covers the RP-HPLC separation and fluorescent determination of derivatized catecholamines with SIIA. In a mobile phase of methanol-water (36/64, v/v) containing H3cit-Na2HPO4 buffer (pH = 4.00, 10 mmol/L), the derivatives of norepinephrine (NE), epinephrine (E) and dopamine (DA) were eluted within 15 min on a C18 column. The detection limits were 0.043, 0.13 and 0.18 pmol, respectively, when the ratio of signal to noise (S/N) was 3. The excessive reagent is rapidly hydrolyzed to 3-indolylacetic acid (IA) that can be easily separated from derivatives.
KeywordsDopamine Epinephrine Catecholamine Indole Strong Fluorescence
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