Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

Abstract

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)(2) and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[alpha]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC-Cl. The new reagent BCEC-Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC-amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)(2) and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I (BCEC)/I (BCEOC) = 1.17-3.57, I (BCEC)/I (FMOC) = 1.13-8.21, and UV(BCEC)/UV(BCEOC) = 1.67-4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C(18) column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)(2). Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)(2) and Try from bee-collected pollen (bee pollen) samples.

Optimization of Extraction Condition of Bee Pollen Using Response Surface Methodology: Correlation between Anti-Melanogenesis, Antioxidant Activity, and Phenolic Content

“…Bee pollen is flower pollen with nectar and salivary substances of bees and considered as perfect foods due to its nutritional value. It is rich in essential components, including amino acids, proteins, hormones, minerals, and vitamins, and exerts diverse beneficial effect on humans [11,12,13,26,27,28].…”

Advances in Capillary Electrophoretically Mediated Microanalysis for On‐line Enzymatic and Derivatization Reactions

“…The authors reported the use of (‐)‐1‐(9‐Fluorenyl)ethyl chloroformate (FLEC) reagent for amino acid derivatization. Previously, FLEC has been used for chiral separation of various molecules, including amino acids by HPLC . It has rarely been implemented in CE until now.…”

Analysis and Evaluation of Fatty Acid in Rattan Tea by Highly Selective and Sensitive HPLC- FLD-MS Method Coupled with Pre-Column Fluorescent Labeling

“…With APCI/MS in positive ion detection mode, intense ion current signals for fatty acid derivatives should be attributed to the introduction of the weak basic nitrogen in corresponding TSNPE molecular core structure resulting in high ionization efficiency 16 .…”