Synthesis of toluene-4-sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10- d]imidazol-1-yl)-ethyl ester and its application for sensitive determination of free fatty acids in ginkgo nut and ginkgo leaf by high performance liquid chromatography with fluorescence detection

Abstract

A novel fluorescent labeling reagent toluene-4-sulfonic acid 2-(2-thiophen-2-yl-phenanthro[9,10-d]imidazol-1-yl)-ethyl ester has been designed and synthesized. It was used to label twenty-six fatty acids (C5-C30) successfully in the presence of K₂CO₃ catalyst in N,N-dimethylformamide solvent. The reaction conditions were optimized by employing a three-factor, three-level Box-Behnken design. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography with fluorescence detection. All fatty acid derivatives were separated on a hypersil BDS-C8 column in conjunction with a gradient elution with a good baseline resolution. Good linear correlations were observed for all fatty acids with correlation coefficients > 0.993. The established method exhibited high sensitivity and excellent repeatability. The limit of detection (at a signal-to-noise ratio of 3 : 1) was 8.8-45.5 fmol. The method was used to quantify free fatty acids in ginkgo nut and ginkgo leaf samples with satisfactory results.

Fig. 1. Synthesis route of TSTPE and derivatization scheme of TSTPE with fatty acids.

Fig. 2. Excitation and emission spectra of TSTPE in DMF (A) and TSTPE-C18 in acetonitrile (B). 1: 100% acetonitrile, 2: 90% acetonitrile, 3: 80% acetonitrile, 4: 70% acetonitrile, 5: 60% acetonitrile, 6: 50% acetonitrile.

Fig. 3. The 3D response surface plots showing effects of the examined factors on the response. (A) The effect of reaction time and TSTPE amounts on the peak area; (B) the effect of derivatization temperature and TSTPE amounts on the peak area; (C) the effect of derivatization temperature and reaction time on the peak area.

Fig. 4. Chromatogram of standard fatty acids derivatives. Chromatographic conditions: Hypersil BDS-C8 column (4.6 × 150 mm, 5 μm), column temperature 30 °C; flow rate 1.0 mL min⁻¹; excitation and emission: λ_ex/λ_em = 314/398 nm. Peaks are labeled with abbreviations for all fatty acids: C5 (pentanoic acid); C6 (hexanoic acid); C7 (heptanoic acid); C8 (caprylic acid); C9 (pelargonic acid); C10 (decanoic acid); C11 (undecanoic acid); C12 (dodecanoic acid); C13 (tridecanoic acid); C14 (myristic acid); C15 (pentadecanoic acid); C16 (hexadecanoic acid); C17 (heptadecanoic acid); C18 (octadecanoic acid); C19 (nonadecanoic acid); C20 (arachidic acid); C21 (heneicosoic acid); C22 (docosanoic acid); C23 (tricosanoic acid); C24 (tetracosanoic acid); C25 (pentacosanoic acid); C26 (hexacosanoic acid); C27 (carboceric acid); C28 (montanic acid); C29 (motanic acid); C30 (triacontanoic acid).

Fig. 5. Chromatogram of fatty acid derivatives from the extracted ginkgo leaf sample (A) and ginkgo nut sample (B). Chromatographic conditions and peaks labels are the same with Fig. 4.