Skip to main content
Log in

A novel fluorescent labeling reagent, 2-(9-acridone)-ethyl chloroformate, and its application to the analysis of free amino acids in honey samples by HPLC with fluorescence detection and identification with online ESI-MS

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

In this study, a novel fluorescent labeling reagent 2-(9-acridone)-ethyl chloroformate (AEC-Cl) was designed, synthesized and applied for the determination of free amino acids by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The free amino acids were rapidly and efficiently labeled by AEC-Cl in the presence of basic catalyst (pH 9.0) within 5 min at room temperature (25 °C). The derivatives exhibited excellent stability and fluorescence properties, with maximum excitation and emission wavelengths at 268 nm and 438 nm, respectively. Derivatives of 22 kinds of natural amino acids were completely separated by gradient elution on a Hypersil ODS C18 column. Under the optimal conditions, the calibration curves exhibited excellent linear responses, with correlation coefficients of R2 > 0.9994. The detection and quantification limits were in the range of 0.61–2.67 μg kg−1 and 2.07–8.35 μg kg−1, respectively. Therefore, AEC-Cl was successfully applied for the detection of trace levels of free amino acids in honey samples.

A novel fluorescent labeling reagent was applied for the determination of free amino acids in honey by high-performance liquid chromatography with a fluorescence detector.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Nasuti C, Gabbianelli R, Falcioni G, Cantalamess F. Antioxidative and gastroprotective activities of anti-inflammatory formulations derived from chestnut honey in rats. Nutr Res. 2006;26:130–7.

    Article  CAS  Google Scholar 

  2. Liu JR, Ye YL, Li TY, Wang YW, Peng CC. Effect of floral sources on the antioxidant, antimicrobial and anti-inflammatory activities of honeys in Taiwan. Food Chem. 2013;139:938–43.

    Article  CAS  Google Scholar 

  3. Li SZ. Ben cao gang mu: compendium of materia medica. Beijing: Foreign Languages Press; 2003.

    Google Scholar 

  4. Williams S, King J, Revell M, Manley-Harris M, Balks M, Janusch F, et al. Regional, annual, and individual variations in the dihydroxyacetone content of the nectar of manuka (Leptospermum scoparium) in New Zealand. J Agric Food Chem. 2014;62:10332–40.

    Article  CAS  Google Scholar 

  5. Alissandrakis E, Tarantilis PA, Harizanis PC, Polissiou M. Comparison of the volatile composition in thyme honeys from several origins in Greece. J Agric Food Chem. 2007;55:8152–7.

    Article  CAS  Google Scholar 

  6. Azevedo MS, Seraglio SKT, Rocha G, Balderas CB, Piovezan M, Gonzaga LV, et al. Free amino acid determination by GC-MS combined with a chemometric approach for geographical classification of bracatinga honeydew honey (Mimosa scabrella Bentham). Food Control. 2017;78:383–92.

    Article  CAS  Google Scholar 

  7. Sun Z, Zhao L, Cheng N, Xue X, Wu L, Zheng J, et al. Identification of botanical origin of Chinese unifloral honeys by free amino acid profiles and chemometric methods. J Pharm Anal. 2017;7:317–23.

    Article  Google Scholar 

  8. Grunfeld E, Vincent C, Bagnara D. High-performance liquid chromatography analysis of nectar and pollen of strawberry flowers. J Agric Food Chem. 1989;37:290–4.

    Article  CAS  Google Scholar 

  9. Hanczkó R, Jámbor A, Perl A, Molnár-Perl I. Advances in the o-phthalaldehyde derivatizations comeback to the o-phthalaldehyde-ethanethiol reagent. J Chromatogr A. 2007;1163:25–42.

    Article  Google Scholar 

  10. Jámbor A, Molnár-Perl I. Amino acid analysis by high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride: Literature overview and further study. J Chromatogr A. 2009;1216:3064–77.

    Article  Google Scholar 

  11. Nozal MJ, Bernal JL, Toribio ML, Diego JC, Ruiz A. Rapid and sensitive method for determining free amino acids in honey by gas chromatography with flame ionization or mass spectrometric detection. J Chromatogr A. 2004;1047:137–46.

    Article  CAS  Google Scholar 

  12. López-Cervantes J, Sánchez-Machado D, Rosas-Rodríguez J. Analysis of free amino acids in fermented shrimp waste by high-performance liquid chromatography. J Chromatogr A. 2006;1105:106–10.

    Article  Google Scholar 

  13. Pereira V, Pontes M, Camara JS, Marques JC. Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure. J Chromatogr A. 2008;1189:435–43.

    Article  CAS  Google Scholar 

  14. Kovács A, Simon-Sarkadi L, Ganzler K. Determination of biogenic amines by capillary electrophoresis. J Chromatogr A. 1999;836:305–13.

    Article  Google Scholar 

  15. Dalluge JJ, Smith S, Sanchez-Riera F, McGuire C, Hobson R. Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography-tandem mass spectrometry. J Chromatog A. 2004;1043:3–7.

    Article  CAS  Google Scholar 

  16. Fiehn O. Extending the breadth of metabolite proiling by gas chromatography coupled to mass spectrometry. TrAC Trend Anal Chem. 2008;27:261–9.

    Article  CAS  Google Scholar 

  17. Kelly MT, Blaise A, Larroque M. Rapid automated high performance liquid chromatography method for simultaneous determination of amino acids and biogenic amines in wine, fruit and honey. J Chromatogr A. 2010;1217:7385–92.

    Article  CAS  Google Scholar 

  18. Rebane R, Herodes KA. Sensitive method for free amino acids analysis by liquid chromatography with ultraviolet and mass spectrometric detection using precolumn derivatization with diethyl ethoxymethylenemalonate: application to the honey analysis. Anal Chim Acta. 2010;672:79–84.

    Article  CAS  Google Scholar 

  19. Bauza T, Kelly MT, Blaise A. Study of polyamines and their precursor amino acids in Grenache noir and Syrah grapes and wine of the Rhone Valley. Food Chem. 2007;105:405–13.

    Article  CAS  Google Scholar 

  20. Cometto PM, Faye PF, Di Paula Naranjo R, Rubio MA, Aldao MA. Comparison of free amino acids profile in honey from three argentinian regions. J Agric Food Chem. 2003;51:5079–87.

    Article  CAS  Google Scholar 

  21. Bosch L, Alegría A, Faé R. Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods. J Chromatogr B. 2006;831:176–83.

    Article  CAS  Google Scholar 

  22. Tuberoso C, Congiu F, Serreli G, Mameli S. Determination of dansylated amino acids and biogenic amines in cannonau and vermentino wines by HPLC-FLD. Food Chem. 2015;175:29–35.

    Article  CAS  Google Scholar 

  23. Fleury M, Ashley D. High-performance liquid chromatographic analysis of amino acids in physiological fluids: on-line precolumn derivatization with o-phthaldialdehyde. Anal Biochem. 1983;133:330–5.

    Article  CAS  Google Scholar 

  24. Shang SF, Wang H. Sensitive determination of amino acids in kelp by reversed phase high performance liquid chromatography with precolumn derivatization using phenylisothiocyanate. J Chromatogr. 1996;43:309–12.

    Article  CAS  Google Scholar 

  25. Liu HJ. Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high-performance liquid chromatography with ultraviolet detection. J Chromatogr A. 1994;670:59–66.

    Article  CAS  Google Scholar 

  26. You JM, Ming YF, Shi YW, Zhao XE. Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) and its application for determination of amino acids from seeds and bryophyte plants using high-performance liquid chromatography with fluorescence detection and identification with electrospray ionization mass spectrometry. Talanta. 2005;68:448–58.

    Article  CAS  Google Scholar 

  27. You J, Shan Y, Zhen L, Zhang L, Zhang Y. Determination of peptides and amino acids from wool and beer with sensitive fluorescent reagent 2-(9-carbazole)-ethyl chloroformate by reverse phase high-performance liquid chromotography and liquid chromotography mass spectrometry. Anal Biochem. 2003;313:17–27.

    Article  CAS  Google Scholar 

  28. You JM, Zhao HX, Sun ZW, Xia L, Yan T, Suo YR. Application of 10-ethylacridine-3-sulfonyl chloride for HPLC determination of aliphatic amines in environmental water using fluorescence and APCI–MS. J Sep Sci. 2009;32:1351–62.

    Article  CAS  Google Scholar 

  29. Sun YN, Lv ZX, Sun ZW, Wu CX, Ji ZY, You JM. Determination of thiophenols with a novel fluorescence labelling reagent: analysis of industrial waste water samples with SPE extraction coupled with HPLC. Anal Bioanal Chem. 2016;408:3527–36.

    Article  CAS  Google Scholar 

  30. Ali R, Ali HRH, Batakoushy HA, Derayea SM, Elsutohy MM. A reductant colorimetric method for the rapid detection of certain cephalosporins via the production of gold and silver nanoparticles. Microchem J. 2019;146:864–71.

    Article  CAS  Google Scholar 

  31. You J, Zhang W, Zhang Q, Zhang L, Yan C, Zhang Y. Development of a precolumn derivatization method for the determination of free amines in wastewater by high-performance liquid chromatography via fluorescent detection with 9-(2-hydroxyethyl) acridone. Anal Chem. 2002;74:261–70.

    Article  CAS  Google Scholar 

  32. You J, Lao W, You J, Wang G. Characterization and application of −9-N-acetyl-N-hydroxysuccinimide as a pre-column derivatization agent for fluorescence detection of amino acids in liquid chromatography. Analyst. 1999;124:1755–60.

    Article  CAS  Google Scholar 

  33. Fan X, You J, Kang J, Ou Q, Zhu Q. New reagents for determination of amino acids by liquid chromatography with pre-column fluorescence derivatization. Anal Chim Acta. 1998;367:81–91.

    Article  Google Scholar 

  34. You J, Liu L, Zhao W, Zhao X, Suo Y, Wang H, et al. Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen. Anal Bioanal Chem. 2007;387:2705–18.

    Article  CAS  Google Scholar 

  35. Rebane R, Herodes K. Evaluation of the botanical origin of Estonian uni- and polyfloral honeys by amino acid content. J Agric Food Chem. 2008;56:10716–20.

    Article  CAS  Google Scholar 

  36. Bernal J, Nozal MJ, Toribio L, Diego JC, Ruiz A. A comparative study of several HPLC methods for determining free amino acid profiles in honey. J Sep Sci. 2005;28:1039–47.

    Article  CAS  Google Scholar 

  37. Spano N, Piras I, Ciulu M. Reversed-phase liquid chromatographic profile of free amino acids in strawberry-tree (Arbutus unedo L.) honey. J AOAC Int. 2009;92:S73–84.

    Article  Google Scholar 

  38. Francisco WBA, Lisangela MB, Daniel RC, Douglas WF. Amino acids profile of sugar cane spirit (cachaca), rum, and whisky. Food Chem. 2008;108:784–93.

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Science Foundation under Grant 21976105.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Zan Li or Jinmao You.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All experiments were performed and approved according to the guidelines issued by the Ethical Committee of Qufu Normal University (no. QF2019-040 L).

Informed consent

All individual participants who took part in the study gave their written informed consent before participation.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 219 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gao, Y., Tan, J., Lu, J. et al. A novel fluorescent labeling reagent, 2-(9-acridone)-ethyl chloroformate, and its application to the analysis of free amino acids in honey samples by HPLC with fluorescence detection and identification with online ESI-MS. Anal Bioanal Chem 412, 8339–8350 (2020). https://doi.org/10.1007/s00216-020-02969-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-02969-y

Keywords

Navigation