| Summary: | In the present study, the determination of histidine (HIS) by an on-line flow method based on the concept of zone fluidics is reported. HIS reacts fast with <i>o</i>-phthalaldehyde at a mildly basic medium (pH 7.5) and in the absence of additional nucleophilic compounds to yield a highly fluorescent derivative (<i>λ</i><sub>ex</sub>/<i>λ</i><sub>em</sub> = 360/440 nm). The flow procedure was optimized and validated, paying special attention to its selectivity and sensitivity. The LOD was 31 nmol·L<sup>−1</sup>, while the within-day and day-to-day precisions were better than 1.0% and 5.0%, respectively (<i>n</i> = 6). Random urine samples from adult volunteers (<i>n</i> = 7) were successfully analyzed without matrix effect (<1%). Endogenous HIS content ranged between 116 and 1527 μmol·L<sup>−1</sup> with percentage recoveries in the range of 87.6%–95.4%.
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