LC-UV-Solid-Phase Extraction-NMR-MS Combined with a Cryogenic Flow Probe and Its Application to the Identification of Compounds Present in Greek Oregano

Abstract

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.

Characterization of Flavonoid Subgroups and Hydroxy Substitution by HPLC-MS/MS

“…In dihydroflavonols the C-ring fission fragment with the higher relative abundance is the Peak C presented a retention time and spectrum similar to that of eriodictyol ([M+H] + with m/z=289, and a fragmentation pattern identical to eriodictyol). The addition of an internal standard confirmed the identity of component C as eriodictyol [25][26][27][28][29][30]. Peak D eluted at 43.5 min and had the typical spectrum of flavanones or dihydroflavonols.…”

“…Compound D thus presented a naringenin-type structure i.e. an A-dihydroxy-B-monohydroxyflavanone [28,29]. The identity of compound D was confirmed to be naringenin by the use of internal standard.…”

“…Peak A presented a retention time and spectrum similar to taxifolin, while the MS/MS analysis revealed a molecular ion peak and fragmentation identical to that of this compound (Table 3). Taxifolin was then co-injected with the diethyl ether extract and the HPLC-DAD analysis showed an increase in peak A [25][26][27][28][29]. + ion (m/z=289) indicated that it could be a trihydroxy-monomethoxy flavanol, a tetrahydroxy flavanone, or a trihydroxy dihydroflavonol.…”

Phenolic Molecules in Virgin Olive Oils: a Survey of Their Sensory Properties, Health Effects, Antioxidant Activity and Analytical Methods. An Overview of the Last Decade Alessandra

“…GC-MS [192] [174] reported the first application of the hyphenated LC-SPE-NMR technique using postcolumn solid-phase extraction to direct identification of new phenolic compounds in the polar fraction of VOO. The addition of a post-column SPE system to replace of the loop system of the LC-NMR, resulted in higher sensitivity (significant increase of the signal to noise [S/N] ratio); in fact, S/N improvements by up to a factor of 4 could be demonstrated with this new technology [16]. The spectra recorded were one dimensional (1D) 1 H-NMR and two dimensional (2D)…”

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

“…Developments in NMR continue to take place. Hyphenation with HPLC via an SPE trap and cooling of the measuring coils to ultra low temperatures (so called ''cryo probes'') leads to cleaner samples and an increase of the concentration sensitivity with a factor 10 to 100 depending on the HPLC and LC/NMR probe parameters (Exarchou et al 2003). Additionally the SPE step allows the use of normal nondeuterated HPLC eluents and the recording of NMR spectra in 100% deuterated solvents.…”