NOVEL REAGENT FOR THE SENSITIVE DETERMINATION OF FREE FATTY ACIDS BY HPLC WITH FLUORESCENCE DETECTION AND IDENTIFICATION WITH MASS SPECTROMETRY AND APPLICATION TO SEVERAL MEDICINAL HERBS

Abstract

A new fluorescence reagent 2-(2-(4-(dimethylamino)-phenyl)-1H-phenanthro-[9,10-d]- imidazol-1-yl)-ethyl-4-methylbenzenesulfonate (PPMB) has been designed for fatty acids labeling. Fifteen fatty acids containing six saturated and nine unsaturated fatty acids were used to evaluate the analytical potential of this reagent. The derivatization reaction of PPMB with fatty acids was carried out in the presence of K₂CO₃ catalyst at 95°C for 40 min in N, N-dimethylformamide solvent. The separation of fatty acids was achieved on a reversed-phase Hypersil BDS C₈ column (4.6 × 200 mm, 5 µm) with a good baseline resolution. The fluorescence excitation and emission wavelengths were set at λ_ex 260 nm and λ_em 430 nm, respectively. The identification was carried out by the online APCI-MS in positive-ion detection mode. Results showed the fatty acid derivatives not only enabled sensitive fluorescence detection but also had high MS ionizability. Excellent linear responses were observed using fluorescent detection with coefficients of >0.9993. Detection limits, at a signal-to-noise ratio of 3:1, were in the range of 29.88–69.37 fmol. Fatty acid extracts from Lycium chinese mill, Long pepper, and Evodiae fructus were successfully analyzed with satisfactory results.

Keywords:

• derivatization
• Evodiae fructus
• fatty acids
• HPLC
• Long pepper
• Lycium chinese mill

Notes

ND: not detectable or with values below LODs.