Chromatographia

, 74:541 | Cite as

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

Original

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-μm capillary and a 380-μm 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.

Keywords

Column liquid chromatography Amino acids FITC In-capillary optical fiber laser-induced fluorescence 

Notes

Acknowledgments

Financial supports from National Natural Science foundation of China (20927007, 20575042 and 20775050) and Education Ministry of China (105141 and KJ091207) are gratefully acknowledged.

References

  1. 1.
    Graser TA, Godel HG, Albers S, Foldi P, Furst P (1985) An ultra rapid and sensitive high-performance liquid chromatographic method for determination of tissue and plasma free amino acids. Anal Biochem 151(1):142–152. doi:0003-2697(85)90064-8[pii] CrossRefGoogle Scholar
  2. 2.
    Hanczkó R, Molnár-Perl I (2003) Derivatization, stability and chromatographic behavior of o-phthaldialdehyde amino acid and amine derivatives: o-Phthaldialdehyde/ 2-mercaptoethanol reagent. Chromatographia 57:S103–S113. doi:10.1007/bf02492091 CrossRefGoogle Scholar
  3. 3.
    Roach MC, Harmony MD (1987) Determination of amino acids at subfemtomole levels by high-performance liquid chromatography with laser-induced fluorescence detection. Anal Chem 59(3):411–415. doi:10.1021/ac00130a007 CrossRefGoogle Scholar
  4. 4.
    Ahnoff M, Grundevik I, Arfwidsson A, Fonselius J, Persson B-A (1981) Derivatization with 4-chloro-7-nitrobenzofurazan for liquid chromatographic determination of hydroxyproline in collagen hydrolysate. Anal Chem 53(3):485–489. doi:10.1021/ac00226a024 CrossRefGoogle Scholar
  5. 5.
    Haynes PA, Sheumack D, Kibby J, Redmond JW (1991) Amino acid analysis using derivatisation with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography. J Chromatogr A 540:177–185CrossRefGoogle Scholar
  6. 6.
    Liu H, Sañuda-Peña MC, Harvey-White JD, Kalra S, Cohen SA (1998) Determination of submicromolar concentrations of neurotransmitter amino acids by fluorescence detection using a modification of the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid analysis. J Chromatogr A 828(1–2):383–395CrossRefGoogle Scholar
  7. 7.
    Zhao X, Suo Y (2008) LC Determination of Amino Acids in Rat Plasma with Fluorescence Detection: aapplication to Exercise Physiology. Chromatographia 67(5):375–382. doi:10.1365/s10337-008-0522-9 CrossRefGoogle Scholar
  8. 8.
    Muramoto K, Kawauchi H, Yamamoto Y, Tuzimura K (1976) Analysis of fluorescein–thiohydantoin amino acids by high speed liquid chromatography. Agricic Biol Chem 40(4):815–817CrossRefGoogle Scholar
  9. 9.
    Muramoto K, Kamiya H, Kawauchi H (1984) The application of fluorescein isothiocyanate and high-performance liquid chromatography for the microsequencing of proteins and peptides. Anal Biochem 141(2):446–450CrossRefGoogle Scholar
  10. 10.
    Lu XN, Chen Y (2002) Chiral separation of amino acids derivatized with fluoresceine-5-isothiocyanate by capillary electrophoresis and laser-induced fluorescence detection using mixed selectors of β-cyclodextrin and sodium taurocholate. J Chromatogr A 955(1):133–140CrossRefGoogle Scholar
  11. 11.
    Lalljie SPD, Sandra P (1995) Mekc analysis of FITC and DTAF amino-acid derivatives with LIF Detection. Chromatographia 40(9–10):513–518CrossRefGoogle Scholar
  12. 12.
    Takizawa K, Nakamura H (1998) Separation and determination of fluorescein isothiocyanate-labeled amino acids by capillary electrophoresis with laser-induced fluorescence detection. Anal Sci 14:925–928CrossRefGoogle Scholar
  13. 13.
    Mank AJG, Velthorst NH, Brinkman UAT, Gooijer C (1995) Near-infrared laser-induced fluorescence detection in column liquid-chromatography—a comparison of various lasers and detection systems. I. continuous-wave lasers. J Chromatogr A 695(2):165–174CrossRefGoogle Scholar
  14. 14.
    Mank AJG, Velthorst NH, Brinkman UAT, Gooijer C (1995) Near-infrared laser-induced fluorescence detection in column liquid-chromatography - a comparison of various lasers and detection systems. II. Pulsed lasers. J Chromatogr A 695(2):175–183CrossRefGoogle Scholar
  15. 15.
    Sepaniak MJ, Yeung ES (1980) Determination of adriamycin and daunorubicin in urine by high-performance liquid chromatography with laser fluorometric detection. J Chromatogr 190(2):377–383CrossRefGoogle Scholar
  16. 16.
    Zhao SL, Yuan HY, Xiao D (2005) Detection of dd-serine in rat brain by capillary electrophoresis with laser induced fluorescence detection. J Chromatogr B 822(1–2):334–338. doi:10.1016/j.jchromb.2005.06.027 CrossRefGoogle Scholar
  17. 17.
    Wang CL, Zhao SL, Yuan HY, Xiao D (2006) Determination of excitatory amino acids in biological fluids by capillary electrophoresis with optical fiber light-emitting diode induced fluorescence detection. J Chromatogr B 833(2):129–134. doi:10.1016/j.jchromb.2006.01.013 CrossRefGoogle Scholar
  18. 18.
    Zhao SL, Wang B, Yuan HY, Xiao D (2006) Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection. J Chromatogr A 1123(1):138–141. doi:10.1016/j.chroma.2006.05.038 CrossRefGoogle Scholar
  19. 19.
    Zhao SL, Yuan HY, Xiao D (2006) Optical fiber light-emitting diode-induced fluorescence detection for capillary electrophoresis. Electrophoresis 27(2):461–467. doi:10.1002/elps.200500300 CrossRefGoogle Scholar
  20. 20.
    Hu L, Yang X, Wang C, Yuan H, Xiao D (2007) Determination of riboflavin in urine and beverages by capillary electrophoresis with in-column optical fiber laser-induced fluorescence detection. J Chromatogr B 856(1–2):245–251. doi:10.1016/j.jchromb.2007.06.011 CrossRefGoogle Scholar
  21. 21.
    Xiao D, Zhao SL, Yuan HY, Yang XP (2007) CE detector based on light-emitting diodes. Electrophoresis 28(1–2):233–242. doi:10.1002/elps.200600473 CrossRefGoogle Scholar
  22. 22.
    Yang XP, Yuan HY, Wang CL, Su XD, Hu L, Xiao D (2007) Determination of penicillamine in pharmaceuticals and human plasma by capillary electrophoresis with in-column fiber optics light-emitting diode induced fluorescence detection. J Pharmaceut Biomed 45(2):362–366. doi:10.1016/j.jpba.2007.05.017 CrossRefGoogle Scholar
  23. 23.
    Yang XP, Yuan HY, Wang CL, Zha SL, Xiao D, Choi MMF (2007) In-column fiber-optic laser-induced fluorescence detection for CE. Electrophoresis 28(17):3105–3114. doi:10.1002/elps.200600815 CrossRefGoogle Scholar
  24. 24.
    Xiao D, Yan L, Yuan HY, Zhao SL, Yang XP, Choi MMF (2009) CE with LED-based detection: an update. Electrophoresis 30(1):189–202. doi:10.1002/elps.200800415 CrossRefGoogle Scholar
  25. 25.
    Huo F, Yuan H, Breadmore MC, Xiao D (2010) Multi-wavelength light emitting diode array as an excitation source for light emitting diode-induced fluorescence detection in capillary electrophoresis. Electrophoresis 31(15):2589–2595. doi:10.1002/elps.201000071 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.College of Chemistry and College of Chemical Engineering, Sichuan UniversityChengduChina
  2. 2.Test Center for Feed Quality Supervision and Inspection (Chengdu) of the Ministry of AgricultureChengduChina
  3. 3.College of Chemistry and Environmental Engineering, Chongqing University of Arts and SciencesChongqingChina
  4. 4.Department of ChemistryHong Kong Baptist UniversityHong Kong SARChina

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