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Pre-Column Derivatization of Amines with 1,2-Benzo-3,4-Dihydrocarbazole-9-Isopropyl Chloroformate Followed by LC-Fluorescence and LC-APCI-MS

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Abstract

A pre-column derivatization method for the sensitive determination of amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl chloroformate (BCIC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives is carried out by high performance liquid chromatography/atmospheric pressure chemical ionization (LC-APCI-MS-MS). The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent is replaced by 1,2-benzo-3,4-dihydrocarbazole-9-isopropyl functional group, which results in a sensitive fluorescence derivatizing reagent BCIC-Cl. BCIC-Cl can easily and quickly label amines. Derivatives are stable enough to be efficiently analyzed by high-performance liquid chromatography and show an intense protonated molecular ion corresponding m/z [MH]+ under APCI in positive-ion mode. The collision-induced dissociation of protonated molecular ion formed a product at m/z 260 corresponding to the cleavage of CH2-OCO bond. Studies on derivatization demonstrate excellent derivative yields over the pH 9.0–10.0. Maximal yields close to 100% are observed with a 3 to 4-fold molar reagent excess. In addition, the detection responses for BCIC derivatives are compared with those obtained using CEOC and FMOC as derivatization reagents. The ratios of I BCIC/I CEOC and I BCIC/I FMOC are, respectively, 1.23–3.14 and 1.25–3.08 for fluorescent (FL) responses (here, I is relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C8 column. Detection limits are calculated from 1.0 pmol injection, at a signal-to-noise ratio of 3, are 10.6–37.8 fmol. The mean interday accuracy ranges from 94 to 105% for fluorescence detection with the largest mean %CV<7.5. The mean interday precision for all standards is < 6.0% of the expected concentration. Excellent linear responses are observed with coefficients of > 0.9997.

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Authors and Affiliations

  1. Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining, P.R. China

    Jinmao You, Xiangming Chen & Xianen Zhao

  2. College of Chemistry Science, Qufu Normal University, Qufu, Shandong, 273165, P. R. China

    Jinmao You, Yourui Suo, Honglun Wang, Yulin Li & Jing Sun

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  1. Jinmao You
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  2. Xiangming Chen
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  3. Xianen Zhao
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  4. Yourui Suo
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  5. Honglun Wang
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  6. Yulin Li
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  7. Jing Sun
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Correspondence to Jinmao You.

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Revised: 12 December 2005 and 13 Febrauary 2006

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You, J., Chen, X., Zhao, X. et al. Pre-Column Derivatization of Amines with 1,2-Benzo-3,4-Dihydrocarbazole-9-Isopropyl Chloroformate Followed by LC-Fluorescence and LC-APCI-MS. Chroma 63, 337–343 (2006). https://doi.org/10.1365/s10337-006-0774-1

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