Y. Tsuruta, S. Kitai, S. Watanabe
Apr 10, 2000
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15
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Journal
Analytical biochemistry
Abstract
Highly sensitive fluorescent labeling reagents, 2-(alkyloxy)-4-(2-phthalimidinyl)phenylsulfonyl chlorides (alkyloxy = methoxy, ethoxy, and propoxy; MPS-Cl, EPS-Cl, and PPS-Cl, respectively), for determination of phenols by high-performance liquid chromatography (HPLC) have been developed. These reagents react with phenols in basic medium to produce the corresponding fluorescent sulfonyl esters. The maximum fluorescence wavelengths of the derivatives were 308 nm for excitation and 410 nm for emission. The peaks due to phenol labeled with MPS-Cl, EPS-Cl, and PPS-Cl eluted at 6.3, 8.8, and 12.8 min, respectively, on a reversed-phase column with isocratic elution of methanol/water (2:1, v/v), and the detection limits (signal-to-noise = 3) of the derivatives were 10, 12, and 17 fmol per injection, respectively. Among these reagents, MPS-Cl was advantageous because its derivatives had shorter retention times and higher sensitivities in HPLC. The efficiency of converting phenol to the fluorescent derivative by MPS-Cl was about 100%. When MPS-Cl was applied to the determination of urinary phenol and p-cresol by HPLC using p-ethylphenol as an internal standard, the derivatives were separated at retention times of 6.3, 8.7, and 12.3 min, respectively, under the HPLC conditions described above. The concentrations of phenol and p-cresol in normal human urine were 11.9-293.5 and 8.2-346.1 nmol/mg creatinine, respectively.