LC-HRMS/MS analysis of fenugreek saponins

Foreword

Fenugreek (Trigonella foenum graecum L.) is a herb cultivated in Southern Europe, Northern Africa, and some places in Asia. The seeds of Fenugreek are known to have hypocholesterolemic activity. A number of saponins and flavonoids were found in the seeds of fenugreek. The hypocholesterolemic effect of fenugreek is attributed to its saponins. Identification of fenugreek saponins using HPLC-UV analysis is not easy due to the absence of strong chromophores. The lack of appropriate pure standards turns the identification of saponins into a very ambiguous task using conventional HPLC techniques. Analysis of crude extracts is even more complicated because most fenugreek saponins are eluted in a very narrow time range. The peak-to-peak separation of all fenugreek saponins was not achieved after extensive HPLC studies.

On the other side, mass spectrometry, and in particular, high-resolution MS/MS, is a reliable technique for the analysis of multi-component mixtures even if chromatographic separation is not complete. The atomic composition of a compound may be obtained from the accurate mass data while MS/MS spectra can be used for structure elucidations. 

Most known and a number of new (unreported) fenugreek saponins were detected in this study.

Experimental

The HPLC method development and analyses of soybean extracts were performed using the Accela high-speed LC system (which consisted 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. 

The compounds were separated on Zorbax HPLC column (150×2.1 mm, 3µm, Agilent) employing a linear gradient of acetonitrile/water with 0.1% acetic acid.

Sample Preparation

Fenugreek was purchased from a local distributor. Whole fenugreek grains were ground and the powdered sample was defatted with hexane in a Soxhlet apparatus for 6 hours. A defatted grounded fenugreek sample was 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 the LC-MS analysis.

References for structures identified in this study

* - Masayuki Yoshikawa, Toshiuki Murakami, Hajime Komatsu, Nobotushi Murakami, Johji Yamahara, Hisashi Matsuda, “Medicinal Foodstuffs. IV. Fenugreek Seed. (1): Structures of trigoneosides Ia, Ib, IIa, IIb, IIIa and IIIb, new furastanol saponins from the seeds of Indian Trigonella foenum-graecum L., Chem. Pharm. Bull. 45, 1, 1997, 81-87.

** - Masayuki Yoshikawa, Toshiuki Murakami, Hajime Komatsu, Johji Yamahara, Hisashi Matsuda, “Medicinal Foodstuffs. VII. Fenugreek Seed. (2): Structures of six new furastanol saponins, trigoneosides Iva, Va, Vb, VI, VIIb and VIIIb, from the seeds of Indian Trigonella foenum-graecum L.”, Heterocycles, 47, 1, 1998, 397-405.

*** - Toshiuki Murakami, Akinobu Kishi, Hisashi Matsuda, Masayuki Yoshikawa, “Medicinal Foodstuffs. XVII. Fenugreek Seed. (3): Structures of new furastanol-type steroid saponins, trigoneosides Xa, Xb, XIb, XIIa, XIIb, and XIIIa, from the seeds of Egyptian Trigonella foenum-graecum L., Chem. Pharm. Bull. 48, 7, 2000, 994-1000.