High-performance liquid chromatographic assay for catecholamines and metanephrines using fluorimetric detection with pre-column 9-fluorenylmethyloxycarbonyl chloride derivatization

被引:74
作者
Chan, ECY [1 ]
Wee, PY [1 ]
Ho, PY [1 ]
Ho, PC [1 ]
机构
[1] Natl Univ Singapore, Dept Pharm, Singapore 119260, Singapore
来源
JOURNAL OF CHROMATOGRAPHY B | 2000年 / 749卷 / 02期
关键词
derivatization; LC; catecholamines; metanephrines; 9-fluorenylmethyloxycarbonyl chloride;
D O I
10.1016/S0378-4347(00)00423-0
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A convenient HPLC-fluorescent assay of norepinephrine (NE), epinephrine (E), dopamine (DA) and their 3-O-methylated metabolites, normetanephrine (NM) and metanephrine (MN) was developed. These analytes were coupled to 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before assays. Results showed that using a linear gradient elution, peaks of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM, FMOC-MN and FMOC-DHBA (3,4-dihydroxybenzylamine, internal standard) were simultaneously resolved within 40 min. Optimization of the chromatographic and derivatization conditions, and validation of the assay were further discussed in the paper. The structures of these derivatives were confirmed by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). The molecular ions [M+H](+) of FMOC-NE, FMOC-E, FMOC-DA, FMOC-NM and FMOC-MN were m/z 836, 850, 820, 628 and 642, respectively. Based on these findings, the FMOC-derivatives of metanephrines and catecholamines were confirmed to be hi-substituted and tri-subutituted respectively at the amino and catechol functional groups. Finally, the assay was successfully applied to the measurement of urinary E, DA, NM and MN after direct derivatization and simple cleaning. (C) 2000 Elsevier Science B.V. All rights reserved.
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页码:179 / 189
页数:11
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