Online hyphenation of HPTLC-Fluorescence Detection by Intensity Changes (FDIC)-Mass Spectrometry for sphingolipid analysis
Researcher:
Jarne Lardiés, Carmen
Congress:
1st International Caparica Conference on Chromagenic and Emissive Materials, IC3EM 2014
Participation type:
Póster
Year:
2014
Location:
Costa de Caparica (Portugal)
Sphingolipids have been qualitatively visualized from a long time on silica gel plates using unspecific fluorescent revealing agents like primuline. They showed an increase in fluorescence emission without any apparent chemical reaction [1]. We demonstrate here that primuline behaves as a FDIC fluorophore, and that can be incorporated in a hyphenated system based on High-Performance Thin-Layer Chromatography (HPTLC) for detecting and quantitatively determining target neutral sphingolipids of medical interest in human plasma samples at low concentrations. Hyphenation makes here reference to the combination of a gradient-based chromatographic system (Automated Multiple Development-HPTLC) and detectors (primuline-FDIC by densitometry, MS) in an automated, sequentially coupled mode to get further information about the sample from one silica gel HPTLC plate.
After gradient separation of sample, the HPTLC plate is impregnated in a methanolic solution of primuline (200 mg L-1). The use of primuline as FDIC fluorophore:
- enables a universal detection of all components of a mixture. All non-absorbing sphingolipids can also be detected by FDIC, e.g. sphinganine
- allows quantification of separated sphingolipid peaks to be carried out, using standards
- makes possible to precisely locate peak coordinates by densitometry and transfer the desired peaks to a mass spectrometer using an elution-based interface for further sphingolipid identification
- is compatible with atmospheric pressure ionization techniques as electrospray (ESI) and atmospheric pressure chemical ionization (APCI).
FDIC responses on primuline depend on sphingolipid structure, which contains long hydrocarbon chains but also highly polar groups [2]. Increases in emission are produced by weak electrostatic interactions between primuline and hydrocarbon chains in sphingolipid's ceramidic backbones. Therefore, responses increase in function of chain length, via polarizability [3]. On the other hand, polar groups establishes specific donor-acceptor interactions which produce decreases in emission, and even fluorescence quenching [4].
Sphingolipids have been qualitatively visualized from a long time on silica gel plates using unspecific fluorescent revealing agents like primuline. They showed an increase in fluorescence emission without any apparent chemical reaction [1]. We demonstrate here that primuline behaves as a FDIC fluorophore, and that can be incorporated in a hyphenated system based on High-Performance Thin-Layer Chromatography (HPTLC) for detecting and quantitatively determining target neutral sphingolipids of medical interest in human plasma samples at low concentrations. Hyphenation makes here reference to the combination of a gradient-based chromatographic system (Automated Multiple Development-HPTLC) and detectors (primuline-FDIC by densitometry, MS) in an automated, sequentially coupled mode to get further information about the sample from one silica gel HPTLC plate.
After gradient separation of sample, the HPTLC plate is impregnated in a methanolic solution of primuline (200 mg L-1). The use of primuline as FDIC fluorophore:
- enables a universal detection of all components of a mixture. All non-absorbing sphingolipids can also be detected by FDIC, e.g. sphinganine
- allows quantification of separated sphingolipid peaks to be carried out, using standards
- makes possible to precisely locate peak coordinates by densitometry and transfer the desired peaks to a mass spectrometer using an elution-based interface for further sphingolipid identification
- is compatible with atmospheric pressure ionization techniques as electrospray (ESI) and atmospheric pressure chemical ionization (APCI).
FDIC responses on primuline depend on sphingolipid structure, which contains long hydrocarbon chains but also highly polar groups [2]. Increases in emission are produced by weak electrostatic interactions between primuline and hydrocarbon chains in sphingolipid's ceramidic backbones. Therefore, responses increase in function of chain length, via polarizability [3]. On the other hand, polar groups establishes specific donor-acceptor interactions which produce decreases in emission, and even fluorescence quenching [4].