Rapid Mass Spectrometric Study of a Supercritical CO2-extract from Woody Liana Schisandra chinensis by HPLC-SPD-ESI-MS/MS

Woody liana Schisandra chinensis contains valuable lignans, which are phenylpropanoids with valuable biological activity. Among green and selective extraction methods, supercritical carbon dioxide (SC-CO2) was shown to be the method of choice for the recovery of these naturally occurring compounds. Carbon dioxide (CO2) was the solvent with the flow rate (10−25 g/min) with 2% ethanol as co-solvent. In this piece of work operative parameters and working conditions were optimized by experimenting with different pressures (200–400 bars) and temperatures (40–60 °C). The extraction time varied from 60 to 120 min. HPLC-SPD-ESI -MS/MS techniques were applied to detect target analytes. Twenty-six different lignans were identified in the S. chinensis SC-CO2 extracts.

The lignans of S. chinensis have been typically extracted with ethanol or hazardous potentially toxic organic solvents such as methanol, chloroform and n-hexane. A valid green alternative, in which there is no need to work with a large number of organic solvents and the production does not need explosion-proof rooms, is represented by supercritical fluid extraction (SFE) with many advantages compared to common extraction methods (maceration, percolation Soxhlet extraction) [21,22].  The lignans of S. chinensis have been typically extracted with ethanol or hazardous potentially toxic organic solvents such as methanol, chloroform and n-hexane. A valid green alternative, in which there is no need to work with a large number of organic solvents and the production does not need explosion-proof rooms, is represented by supercritical fluid extraction (SFE) with many advantages compared to common extraction methods (maceration, percolation Soxhlet extraction) [19,20].
SFE is a green, mild and selective extraction process, one of the best processes to get rid of residual solvent in the extract. Among the supercritical solvents, carbon dioxide is the most common, offering several advantages, because it is non-toxic, non-flammable, cost-effective, environmentally friendly and renewable [25][26][27]. The SFE method is actively studied and applied in the processing of plant materials [28,29].
The lignans of S. chinensis were extracted by supercritical CO 2 (SC-CO 2 ) using ethanol as co-solvent [30][31][32]. Different parts of the plant were extracted by SFE, isolating 36 compounds from the leaves, 43 compounds from lignified stems and 36 compounds from rhizomes and roots. S. chinensis extracts contain a volatile fraction rich in essential oils (terpenes: monoterpenes, sesquiterpenes; terpenoids: alcohols, esters, ketones) and a non-volatile part (carboxylic acids and lignans).

Results and Discussion
Aiming to optimize the extraction of target analytes from the S. chinensis woody liana, several experimental conditions were investigated. Carbon dioxide (CO 2 ) was the solvent with the flow rate (10−25 g/min) and 2% ethanol as co-solvent in the liquid phase. Extraction was performed in the pressure range of 200-400 bar and the temperature range of 40-60 • C. The best results were obtained at 350 bar and 60 • C. Increasing the pressure from 350 to 400 bar practically gave no increase in yields. The temperature of 60 • C was chosen as the maximum allowable to avoid the decomposition of target analytes. In this work HPLC-SPD-ESI-MS/MS techniques were used with additional ionization and analysis of fragmented ions. High-accuracy mass spectrometric data were recorded on an ion trap amaZon SL BRUKER DALTONIKS equipped with an ESI source in the mode of negative ions. The three-stage ion separation mode was implemented. Under these conditions a total of 800 peaks were detected in the ion chromatogram ( Figure 2). three-stage ion separation mode was implemented. Under these conditions a total of 800 peaks were detected in the ion chromatogram ( Figure 2). Although this approach is not quantitative for evaluating each analyte, it is semiquantitative when comparing a series of extractions and allows better comparison of the yield without loss of individual analytes during fractionation and sample preparation. Only the total extraction yields were completely quantified. Table 2 summarizes all the molecular masses of the target analytes isolated from SC-CO2 of S. chinensis. Among them, 26 biologically active substances were authenticated as lignans (m/z values and fragment ions) by comparison with literature data [2,24,25,[35][36][37][38].  668 669 670 671 672 673 674 678 679 680 681 682 683 685 686 687 688 689  1213 14 15 17 18 19 20 27 28 29 30 39 40 55 56 57 58 59 60 61 62 66   Although this approach is not quantitative for evaluating each analyte, it is semiquantitative when comparing a series of extractions and allows better comparison of the yield without loss of individual analytes during fractionation and sample preparation. Only the total extraction yields were completely quantified. Table 2 summarizes all the molecular masses of the target analytes isolated from SC-CO 2 of S. chinensis. Among them, 26 biologically active substances were authenticated as lignans (m/z values and fragment ions) by comparison with literature data [2,22,23,[33][34][35][36].   Figure 3 shows examples of the decoding spectra (collision-induced dissociation (CID) spectrum) of the ion chromatogram obtained using tandem mass spectrometry. The CID spectrum in positive ion modes of schisandrin B (gomisin N, isokadsuranin) from Russian S. chinensis.  The CID spectrum in positive ion modes of benzoylgomisin Q is shown in Figure 5. Intens. The CID spectrum in positive ion modes of schisantherin A (gomisin C) from S. chinensis is shown in Figure 4. The CID spectrum in positive ion modes of schisantherin A (gomisin C) from S. chinensis is shown in Figure 4. The CID spectrum in positive ion modes of benzoylgomisin Q is shown in Figure 5. Intens.

Materials
As the objects of the study, samples of S. chinensis (woody liana) were purchased from the area of the Peschanka river near Lazovsky district (Sikhote Alin), Primorsky Krai, located at 43°32′ N and 134°33′ E, Russia. All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [39].

Chemicals and Reagents
HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from a SIEMENS ULTRA clear (SIEMENS water technologies, Germany), and all the other chemicals were analytical grade.

SC-CO2 Extraction
SC-CO2 extraction was performed using the SFE-500 system (Thar SCF Waters, Milford, USA) supercritical pressure extraction apparatus. System options included co-solvent pump (Thar Waters P-50 High Pressure Pump), for extracting polar samples. CO2 flow meter (Siemens, Germany), to measure the amount of CO2 being supplied to the system, multiple extraction vessels, to extract different sample sizes or to increase the throughput of the system. Flow rate was 50 mL/min for liquid CO2 and 1.76 mL/min for EtOH. Extraction samples of 10 g Schisandra chinensis wood were used. The extraction time was counted after reaching the working pressure and equilibrium flow, and it was 6 h for each sample.

Materials
As the objects of the study, samples of S. chinensis (woody liana) were purchased from the area of the Peschanka river near Lazovsky district (Sikhote Alin), Primorsky Krai, located at 43 • 32 N and 134 • 33 E, Russia. All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [37].

Chemicals and Reagents
HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from a SIEMENS ULTRA clear (SIEMENS water technologies, Germany), and all the other chemicals were analytical grade.

SC-CO 2 Extraction
SC-CO 2 extraction was performed using the SFE-500 system (Thar SCF Waters, Milford, USA) supercritical pressure extraction apparatus. System options included co-solvent pump (Thar Waters P-50 High Pressure Pump), for extracting polar samples. CO 2 flow meter (Siemens, Germany), to measure the amount of CO 2 being supplied to the system, multiple extraction vessels, to extract different sample sizes or to increase the throughput of the system. Flow rate was 50 mL/min for liquid CO 2 and 1.76 mL/min for EtOH. Extraction samples of 10 g Schisandra chinensis wood were used. The extraction time was counted after reaching the working pressure and equilibrium flow, and it was 6 h for each sample.

Mass Spectrometry
MS analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Germany) equipped with an ESI source in negative ion mode. The optimized parameters were obtained as follows: ionization source temperature: 70 • C, gas flow: 4 L/min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500V, fragmentary: 280 V, collision energy: 60 eV. An ion trap was used in the scan range m/z 100−1.700 for MS and MS/MS. The capture rate was one spectrum for MS and two spectra for MS/MS. Data collection was controlled by Windows software for BRUKER DALTONIKS. All experiments were repeated three times. A two-stage ion separation mode (MS/MS mode) was implemented.

Conclusions
An optimized extraction process with SC-CO 2 (and co-solvent 2% ethanol) of woody liana S. chinensis provided the samples for an accurate analytical study by HPLC-SPD-MS/MS techniques. Twenty-six different lignans typical of S. chinensis species were identified. This method allows one to get all the studied ligands in a single extract without using a series of approaches and solvents, different in polarity, which not only reduces the environmental pressure, but also simplifies the production process. These data could support future investigations on the quality of pharmaceutical preparations containing these S. chinensis extracts. This is because the biological activity is related to the presence of the identified lignans. Their excellent transcutaneous penetration may offer new therapeutic approaches with transdermal preparations based on SC-CO 2 extracts of S. chinensis.