HPLC with In-Capillary Optical Fiber Laser-Induced Fluorescence Detection of Picomolar Amounts of Amino Acids by Precolumn Fluorescence Derivatization with Fluorescein Isothiocyanate

Abstract

In this paper, a fluorescein isothiocyanate (FITC) precolumn derivatization technique in conjunction with an HPLC-in-capillary optical fiber laser-induced fluorescence (HPLC-ICOF-LIF) detection method has been developed for determination of amino acids. The HPLC separation of FITC-labeled amino acids and the ICOF-LIF detection system are studied and optimized. Optimum separation conditions were obtained with a gradient elution program of acetonitrile and phosphate buffer (10 mM, pH 6.8). The ICOF-LIF detection system comprises a 530-lm capillary and a 380-lm optical fiber. The analyses of amino acids display excellent linear relationship between peak area and concentration with correlation coefficients greater than 0.999 and the method also provides good repeatability with RSD \ 3%. The detection limits for FITC-tagged amino acids are very low and the lowest LOD for tyrosine is 51 pM. The proposed method has been successfully applied to determination of amino acids in human serum. Our developed HPLC-ICOF-LIF system is cheap, simple, stable, and sensitive which is potentially useful for the formulation analysis and bioanalysis.

Design and synthesis of a novel pre-column derivatization reagent with a 6-methoxy-4-quinolone moiety for fluorescence and tandem mass spectrometric detection and its application to chiral amino acid analysis

“…Until now, several fluorophores, such as fluorescein, coumarin, and benzofurazan, have been reported [30][31][32][33]. They are widely used for HPLC analysis, and the sensitivity using these fluorophores is ca.…”

Rapid Determination of Vitamin B2 in Foods by HPLC with in Capillary Optical Fiber Laser-Induced Fluorescence Detection Technique

“…All solutions were stored in amber glass bottles in a refrigerator at 4 °C. HPLC-capillary in optical fiber laser induced fluorescence detector was used 13 . The excitation of blue laser was entried directly into the detection window in 530 µm capillary by a 14.0 cm × 380 µm solid bare fiber, which excitating analyses and eject fluorescence.…”