The LC-HRMS/MS analysis of soybean saponins and isoflavones

Soy is an important food product used worldwide. Apart from valuable nutritional properties soybean products are considered to possess various positive effects on human health, including. Some of the beneficial biological effects are attributed to soybean isoflavones and saponins. The soyasaponins consist of a triterpenoid soyasapogenol and one or more carbohydrate moieties attached to it. Up to now, 29 soyasaponins have been isolated from soybeans. All known soyasaponins are divided into three groups depending on their aglycone structure (soyasapogenols A, B, and E).

All known soyasaponins contain glucuronopyranosyl moiety linked to the C-3 oxygen of the aglycone. Negative ionization mode is preferred for organic compounds containing a carboxylic group and no significant proton acceptor. Carbohydrates can form sodium adducts in the positive ionization mode however higher CID energy is required to cause fragmentation of sodiated ions which may result in decreased abundance of fragment ions and loss of some information. Acids or volatile salts are common additives to LC-MS solvents as they improve the ionization of compounds upon ESI. 

Experimental

The HPLC method development and analyses of soybean extracts were performed using the Accela high-speed LC system (which consists of ultra-high pressure quaternary pump, autosampler, and PDA detector) coupled with the LTQ Orbitrap Discovery hybrid FT mass spectrometer (Thermo Fisher Scientific Inc.). The mass spectrometer was equipped with the Ion Max electrospray ionization ion source. The mass spectrometer was operated in the negative ionization mode. Ion source parameters were as follows: spray voltage 3.5 kV, capillary temperature 250⁰C, source fragmentation voltage 35V, sheath gas rate 30 (arbitrary), and auxiliary gas rate 10 (arbitrary). Mass spectra were acquired in the m/z 300-2000 Da range. For the data-dependent experiment, a list of masses of potential saponins was prepared after preliminary analysis of the scan chromatogram. 

Sample Preparation

Imported from the USA soybeans were purchased from a local distributor. Whole soybeans were ground and the powdered soy sample was defatted with hexane in a Soxhlet apparatus for 6 hours. Powdered and defatted soy samples (about 0.5g) were extracted in a sonicator with 80% ethanol (5 ml) for 30 min at 35-40⁰C. The ethanol extract was filtered through a 0.2 µm nylon filter before LC-MS analysis.

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