Quantitative Determination of Sphingolipids by HPLC-ESI/MSn

Jin XIE Rong SONG Li ZHOU Siwen PENG Yanning HUANG Xiaoqi ZHU

Abstract The most predominant sphingoilipids bases are phytosphingosine (phyto-sph) and phytosphingosine-1-phosphate (phyto-S1P), followed by dihydrosphingosine (dh-sph) and dihydroshingosine-1-phosphate (dh-S1P). In this study, a fast, sensitive and selective method for determining those four of sphingolipids by high performance liquid chromatography coupled to electrospray ionization ion trap mass spectrometry was introduced. Comparing two methods, method B’s recovery was better than method A. And the limit of detection (LOD) was between 0.003 to 0.013 μM for 20 μl injection.

Key words Sphingoilipids; Liquid chromatography; Mass spectrometry

Received: June 9, 2022  Accepted: August 10, 2022

Supported by Science and Technology Innovation Project of Hunan Academy of Agricultural Sciences (2021CX32).

Jin XIE (1984-), female, P. R. China, assistant research fellow, devoted to research about biochemistry and molecular biology.

*Corresponding author.

Sphingolipids are a major component of membrane lipids［1］. It have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD)［2］. However, the mechanisms underpinning these processes are not well understood.

Sphingolipids are a diverse group of lipids that contain a relatively large hydrophobic moiety, known as ceramides that include a sphingoid or long-chain base (LCB) amidelinked to a fatty acid［3］. Sphingolipids are not only essential components of cellular membranes, but also act as second messengers to regulate stress responses, cell proliferation and apoptosis［4］. The structures and abbreviations of those sphingolipids analyzed in this report are shown in Table 1. And the relationships［2］ of those sphingolipids are shown in Fig. 1［2］.

So, the qualitative and quantitative determination of sphingolipids is very meaningful.

Materials and Methods

Materials

Reagents

Dihydrosphingosine, phytospingosine, dihydrosphingosine-1-phosphate and phytosphingosine-1-phosphate (Sigma, USA); LC-MS (finngan LCQ Deca XP MAX, Thermo); methanol; chloroform; ammonia water; hydrochloric acid; tetramethylene oxide; ammonium acetate;  formic acid; DMSO.

Instruments

An HPLC-ESI-MSn (Themo-Finnigan Scientific, Waltham, MA, USA) system equipped with a C8 column (Agilent, USA; 3.5μm, 2.5 mm×150 mm) was used to analysis target compound, including a Surveyor Autosampler, Surveyor LC pump and an ion-trap mass spectrometer LCQ Deca MAX (LCQ, Thermo-Finnigan, USA).

Methods

Extraction and purification of plant material

Method A［5］: First, 30 m g of freeze-dried leaf from Populus was ground to a powder with liquid nitrogen in a mortar using a pestle. Then, the powder was added with 3 ml of extraction solvent ［isopropanol: n-hexane∶water(v∶v)55∶20∶25］, and incubated at 60 ℃ for 15 min. After centrifugation at 500 g for 10 min, the supernatant was decanted to a tube and dried in vacuum. The powder, crude extract was dissolved with 2 ml of 33% methylamine solution in ethanol/water (7∶3 v/v) and incubated at 50 ℃ for 1 h. In order to desterilize the sample, it was dried in vacuum. The powder was dissolved in 1 ml of THF/methanol/water (2∶1∶2 v/v/v) containing 0.1% formic acid. The sample was centrifuged at 500 g for 10 min. The supernatant was stored at -30 ℃ until analysis.

Method B［6］: Methanol was added to 30 mg of sample which was added into a tube, and the tube was vortexed by Fastprep. Then, 5 ml of MTBE was added and the mixure was incubated for 1 h at room temperature in a shaker. Phase separation was induced by adding 1.25 ml of water. Upon 10 min of incubation at room temperature, the sample was centrifuged at 1 000 g for 10 min. The upper (organic) phase was collected, and vacuum-dried in a centrifuge. The precipitate was dried in vacuum. The sample was dissolved in CHCl3/methanol/water (60∶30∶4.5) for storage.

High-performance liquid chromatography conditions

The elution gradient was performed at constant flow rate of 0.2 ml/min: 0-3 min, 20% A (0.1% formic acid and 0.5 mM ammonium acetate in water) and 80% B (methanol); 3-14 min, 14.5% A and 85.5% B; 14-15 min,14.5% A and 85.5% B;15-22 min, 10% A and 90% B; 22-23 min 6% A and 96% B; 23-34 min 100% B.

Mass spectrometry parameters

An LCQ DECA XP MAX ion trap mass spectrometer system (Thermo-Finnigan) coupled with ESI source was applied with the following parameters: positive ionization mode, sheath gas: nitrogen, 40 abi units, Aux gas: nitrogen, 10 abi units(ca. 3.33 L/min), capillary voltage+4.5 kV, Tube Lens Offset 30 V, Multipole RF Amplifier 400Vp-p, Multipole 1 Offset -6.80 V, Multipole 2 Offset -9.50 V, Intermultipole Lens Voltage -16.00 V, Entrance Lens -50 V, Capillary temperature 280 ℃, collision energy 38.

Results and Discussion

Specific ions of parent (precursor) and fragment (product) ions

The four kinds of sphingolipids, parent ion mass-to-charge ratio, and ion trap to give certain energy, the characteristic fragment of two stage fragmentation of mass spectrometry (ms2), three stage fragmentation of mass spectrometry (ms3) are shown in Table 2.

Effect of mobile phase

In order to improve the sphingolipids signal, various chromatographic reagents and ionization reagents for sphingolipids were compared. The four sphingolipids in different reagents were consistent in the amount of signal change tendency, as shown inFig. 2. It could be seen that 1% formic acid+0.5 mM ammonium acetate+methanol was the optimal mobile phase.

Standard calibration curve and limit of detect

Discussions

During the establishment of the extraction and detection method for four sphingolipids standard samples, it was found that the recovery rate of two standard samples was low, but the detection limit was very low. No relevant samples were detected in the sphingolipids extraction and detection of poplar  tomentosa leaves, indicating that the four sphingolipids were not contained in poplar  tomentosa leaves.This method can still be used to detect and analyze sphingolipids of other plants.

References

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