Online Extraction Coupled to Liquid Chromatography Analysis (OLE-LC)

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Online Extraction Coupled to Liquid Chromatography Analysis (OLE-LC): Eliminating Traditional Sample Preparation Steps in the Investigation of Solid Complex Matrices Vinícius Guimarães Ferreira, Gabriel Mazzi Leme, Alberto José Cavalheiro, and Cristiano Soleo Funari Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02388 • Publication Date (Web): 05 Aug 2016 Downloaded from http://pubs.acs.org on August 6, 2016

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Analytical Chemistry

Online Extraction Coupled to Liquid Chromatography Analysis (OLE-LC): Eliminating Traditional Sample Preparation Steps in the Investigation of Solid Complex Matrices Vinícius G. Ferreira1 ‡; Gabriel M. Leme1 ‡; Alberto J. Cavalheiro1*; Cristiano S. Funari2,3 1

Chemistry Institute, São Paulo State University (UNESP), 14800-900 Araraquara, São Paulo, Brazil. College of Agricultural Sciences, São Paulo State University (UNESP), Private Bag 237, Botucatu 18610-307, São Paulo, Brazil. 3 Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences, University of Tasmania, Private Bag 75, Hobart 7001, Australia. ‡ These authors contributed equally to this work. 2

Tel.: +55 16 33019791, E-mail: [email protected]

ABSTRACT: Current methods employed for the analysis of the chemical composition of solid matrices (such as plant, animal or human tissues, soil, etc.) often require many sample treatment steps, including an extraction step with exclusively dedicated solvents. This work describes an optimized analytical setup in which the extraction of a solid sample is directly coupled to its analysis by high-performance liquid chromatography. This approach avoids (i) the use of pumps and valves other than those comprising the HPLC instrument, (ii) the use of solvents other than those of the mobile phase and (iii) the need to stop the mobile phase flow at any time during the full analytical procedure. The compatibility of this approach with the direct analysis of fresh tissues (leaves, stems and seeds of four plant species with dissimilar chemical compositions) was successfully demonstrated, leading to the elimination of sample preparation steps such as drying, grinding, concentration, dilution, and filtration, among others. This work describes a new, simple, and efficient green approach to minimize or eliminate sample treatment procedures. It could be easily applied for quality control of plant materials and their derived products through chromatographic fingerprints and for untargeted metabolomic investigations of solid matrices, among other applications.

Whereas the success of the analysis relies on the quality of the sample inserted in the analytical system, the time and the greenness of the overall process is strongly impacted by this key step.1–4 According to Tobiszewisky,2 sample preparation generates the most pollution of any step in the analytical process. Thus, it is not a surprise that the 1st of the 12 principles of Green Analytical Chemistry (GAC) states that direct analytical techniques should be applied to avoid sample treatment.5 Non-targeted chemical investigations of natural samples typically include the following sample pre-treatment steps before analysis in a liquid chromatography system: (i) drying of the collected material, (ii) grinding, (iii) extraction, (iv) fluid extract filtration, (v) solvent elimination, (vi) solid phase extraction, (vii) eluate drying, (viii) residue solubilization in the desired concentration/solvent solution and (ix) filtration in a µm filter.3,6 In some cases, the freshly ground material is directly extracted, thus eliminating step (i).7 On the other hand, other sample preparation steps may be eliminated in targeted investigations. For example, the quick, easy, cheap, effective, rugged and safe extraction (QuEChERS)8 and solid-phase

microextraction (SPME) methods9 allow the elimination of steps (iv), (v) and (vi) outlined above, although in SPME methods a desorption step is necessary to release the analytes from the fiber. Common extraction techniques usually employed in nontargeted investigations include ultrasonic and microwaveassisted extraction, maceration, Soxhlet extraction, and supercritical fluid extraction,7 whereas the most common solvents are methanol, ethanol and water for the extraction of polar compounds and chloroform for the extraction of non-polar compounds.7 Considering that successive extractions with different solvents and techniques are performed to extract the largest part of a given metabolome, the amount of resources used such as time, energy and solvents, is dramatically increased. After sample preparation, a metabolic profile of the sample is acquired by means of one or more comprehensive analytical methods.10 Among the separation techniques used to obtain the metabolic profiles of plants, high performance liquid chromatography (HPLC) and, more recently, ultra-high performance

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liquid chromatography (UHPLC), are the primary techniques. LC techniques are compatible with almost any type of compound, are easy to operate, can be fully automated, and exhibit good resolution, reproducibility, and selectivity.10 Metabolic profiles have proven to be useful in various applications, e.g., for the assessment of plant identity or for quality control of plant-derived products and processes in which a metabolite profile of a sample exhibiting the desired characteristics can be used as a reference. Metabolic profiles are also useful in metabolomics studies, which are mainly performed through a comparative analysis of initial profiles obtained for different samples. The simplification and integration of sample preparation with chromatographic-spectrometric analysis would be useful from a practical perspective, saving time, money and environmental resources such as solvents and energy and resolving the solubility issues encountered with complex samples. Some efforts have been made in this direction, in which several typical sample preparation steps were integrated with liquid chromatography analysis.11–19 Briefly, the different systems reported in literature work as follows: a pump flushes an extractive solution though the matrix, followed directly by a solid phase extraction (SPE) trap cartridge. Then, the analytes are eluted from the SPE cartridge to the LC valve (in the “load” position) by the solvent (or solution), which is delivered by another pump. Finally, the valve is switched to the “inject” position and the LC analysis starts.12,13 Nevertheless, such approaches require pumps and/or valves other than those comprising the LC instrument.12,13,20 Additionally, an amount of solvents and energy dedicated exclusively to the extraction step and a relatively large amount of sample are required. The latter is a bottleneck in many metabolomic investigations, especially in cases in which only a few milligrams of sample can be collected, even after massive efforts; the overconsumption of solvents and energy is not in line with trends regarding sustainability5,21 in analytical chemistry. It is important to highlight that these approaches have been applied only in targeted investigations and mainly to liquid samples. Based on these considerations, this work presents an alternative strategy for online coupling of the comprehensive sample preparation, separation, and detection steps and for greening, speeding up and reducing the cost of the overall analytical procedure. The extraction of plant tissues was completely coupled with the HPLC analysis without the need for pumps, valves, extra apparatus and solvents other than those comprising the HPLC apparatus and the mobile phase. To determine the method’s applicability, the following integrated strategy was (i) initially applied to the establishment of chromatographic fingerprints for the leaves of Casearia sylvestris and Cryptocaria mandioccana; (ii) subjected to analysis to determine the compatibility of this strategy with plant parts other than leaves (seeds, flowers and roots); (iii) subjected to evaluation to determine its compatibility with fresh tissues without any type of treatment; and (iv) compared in terms of performance with strategies employing reference procedures (offline extraction followed by LC analyses). EXPERIMENTAL SECTION Solvents and additives. The methanol, ethanol and acetonitrile (J. T. Baker, Mexico) used were HPLC grade. Acetic acid (99.7%), phosphoric acid (85%) and triethylamine (99%) were of analytical grade (Sigma, St Louis, USA).

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Online extraction directly coupled to high-performance liquid chromatography (OLE-LC) analysis. Initially, 2 mg of ground, dry plant material was inserted in a SecurityGuard holder (Phenomenex, USA). The chamber volume was completed with C18 (40 µm, JT Baker, Mexico). To prevent the flow of particles through the separation system, a Nylon membrane (0.2 µm pore diameter, Schleicher & Schuell, USA) was placed on both sides of the guard column recipe, sealing the sample and the C18 particles inside. Finally, the guard column holder containing the plant material was connected to the chromatographic system, as shown in Figure 1. During the chromatographic column equilibration, the 6-port valve remains in the “load” position. Once the chromatographic column is equilibrated, the valve is switched to the “inject” position, thus allowing the mobile phase to flow through the precolumn containing the plant material prior to entering the chromatographic column, where the separation is achieved. Once the analysis is finished (or whenever the analyst wants to stop the plant material extraction), the valve is automatically turned back to the “load” position. In this position, the mobile phase does not flow through the guard column (Figure 1). In summary, the solid sample cell is positioned in the injection valve at the place of the sample loop.

Figure 1. Set-up of the 6-port, 2-position valve used for the online extraction (“load” and “inject” positions).

The sample extraction, followed by the chromatographic analyses (OLE-LC) and the chromatographic analyses of the samples previously prepared using the traditional procedures described in section 2.4 were performed in a Dionex® Ultimate 3000 system (Sunnyvale, USA) equipped with a DGP3600RS model pump, a WPS-3000 SL model autosampler, a TCC-3000RS model column oven, and a Rheodyne® (Oak Harbor, USA) 6-port, 2-position valve. The whole system controlled by Chromeleon software version 6.80 (Dionex, Sunnyvale, USA). A Varian® HPLC system equipped with Prostar 210 pumps, a Rheodyne 7725i model ® 6-port valve, and a Prostar 320 UV/Vis detector and controlled by the Galaxie chromatography data system software (version 1.9.302.530) was also used for the analysis of fresh material. The HPLC methods used were those previously described by Funari et al.22 and Bandeira et al.23 for the analysis of C. sylvestris and C. mandioccana, respectively. For a description of the conditions, please refer to the captures shown in Figures 2 and 3. Reference sample preparation for comparative purposes. C. sylvestris and C. mandioccana leaves were pre-treated and analyzed by HPLC according to Funari22 and Bandeira.23 Briefly, C. sylvestris leaves were dried at 40°C in an oven with air circulation, and then ground in a knife mill. Of the resulting material, 250 mg was extracted by maceration with three aliquots of 0.9 mL of EtOH at 40°C with constant stirring. The


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combined solutions were concentrated under reduced pressure at 35°C to yield 33.3 mg of dried extract. The dried extract was subjected to SPE to eliminate very non-polar compounds. The eluate was dried and solubilized to yield a concentration of 20 mg/mL. Finally, the solution was filtered through a 0.22 µm polyethersulfone.22 Leaves of C. mandioccana were dried at 40°C in an oven with air circulation and ground in a knife mill. Of the resulting material, 100 mg was extracted by sonication for 30 min with an aliquot of 4.0 mL of hexane. Next, 4.0 mL of methanol:acetic acid 10% 1:1 (v/v) was added, followed by 30 min of sonication. The mixture was then centrifuged at 1200 g for 10 min. The hydroalcoholic phase was collected, dried and weighed, yielding 21.9 mg of dried extract (21.9%), which was then solubilized to obtain a 20 mg/mL solution.23 RESULTS AND DISCUSSION Aiming to reduce the time and resource-consumption involved in the sample preparation procedures that often precede LC analyses and to make the investigation itself greener, a new strategy that combined the extraction and separation of plant materials was tested in this work. The OLE-LC results were compared to those of reference procedures in which plant materials were first extracted and then analyzed by HPLC. For this purpose, two medicinal plants (C. sylvestris and C. mandioccana) with different chemical compositions and wellestablished methods of extraction and separation were selected.22,23 While the leaves of C. sylvestris contain mainly flavonoids and clerodane diterpenes,24 the leaves of C. mandioccana generally contain flavonoids, styrylpyrones, and alkaloids.25 Comparison of OLE-LC and reference procedure for C. sylvestris. A representative chromatogram obtained directly from the dry ground leaves of C. sylvestris by the proposed OLE-LC approach is shown in Figure 2a, whereas the corresponding chromatogram obtained by offline extraction and analysis is shown in Figure 2b. While similar chromatographic profiles were observed in the second half of the chromatograms, relatively dissimilar profiles were observed for the first half (Figure 2). A higher peak area and number of polar compounds with retention time ≤ 35 min was observed for the OLE-LC compared to those of the reference procedure. The total number of peaks was 108 ± 8.2 for OLE-LC and 89 ± 4.2 (n=5) for the reference procedure. Regarding the total peak area, the observed values for OLELC approach and the reference approach were 2894.4 ± 548 and 19278 ± 200 au, respectively. Considering that the yield of the reference extraction procedure was 13.3%, it can be inferred that the 20 µL of 20 mg/mL extract solution injected into the HPLC system corresponded to 3.0 mg of the original dried, ground C. sylvestris leaves. On the other hand, only 2.0 mg of the original dried, ground C. sylvestris leaves were analyzed by OLE-LC. In other words, the OLE-LC approach was more efficient per mass of original sample than the reference procedure in terms of the outcomes of the number of peaks and total peak area. Additionally, approximately 2 mg of dry ground leaves of C. sylvestris dry leaves were subjected to three sequential analyses by OLE-LC to evaluate the efficiency of this extraction procedure (Figure S-1 in the SI).Considering the sum of the areas observed for the three extractions as 100% of the extractable material, it was possible to conclude that 95% of the material was extracted in the first extraction. This high effi-

ciency could be related to the use of a gradient in the extractor mixture (EtOH: 2.7 to 83.7% in water) and to the high pressure in the online extraction system, which enabled better access to the solvent inside the matrix, ensuring better extraction.26

Figure 2. HPLC–UV fingerprints of leaves of C. sylvestris at 250 nm. Column: Synergi Hydro-RP, 250×4.6 mm; 4 µm. Mobile phase: H2O and EtOH: 2.7–83.7% of EtOH (0-61.8 min); 83.7% of EtOH (61.8-90 min). Flow rate: 0.7 mL/min. Samples: a) 2 mg of the dry ground leaves and b) 20 µL of a 20 mg/mL extract solution (corresponding to 3.0 mg of the original dry ground leaves).

Comparison between OLE-LC and reference procedure for C. mandioccana. A representative chromatogram obtained directly from the dry ground leaves of C. mandioccana by OLE-LC is shown in Figure 3a, whereas Figure 3b shows the corresponding chromatogram obtained from the reference procedure with leaves of C. mandioccana. The chromatograms were qualitatively similar, as were the retention times of the main corresponding compounds (Figure 3). However, the peaks acquired by OLE-LC were broader than those observed for the reference procedure. This was especially important for the first half of the chromatogram, during which the compounds resulting in peaks presented UV spectra compatible with those of alkaloids (Figure S-2 in the SI). The observed values for the total peak area were 12523 ± 74 and 793 ± 62 au for OLE-LC and the reference procedure. Considering that the yield of the reference extraction procedure was 21.9%, 20 µL of the 20 mg/mL solution injected in the HPLC system corresponds to 1.8 mg of the original dried ground leaves of C. mandioccana extract. This value corresponds to 90% of the 2 mg of dried ground leaves of C. mandioccana directly analyzed by OLE-LC (Figure 3a). Thus, it can be inferred that OLE-LC was more efficient than the reference procedure for the extraction of a given mass of detectable compounds. However, this was not the case when the number of peaks was the output under consideration. The number of peaks for OLE-LC and the reference procedure were 44.4 ± 8.4 and 57.2 ± 2.9 (n=5), respectively. This contrast could be explained by the fact that peaks acquired by



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OLE-LC were wider, which might lead to coelution of compounds, resulting in a lower number of peaks.

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content in the fresh leaves. The chromatograms obtained from these three approaches are shown in Figure 4. The chromatographic profiles and peak intensities obtained from these three approaches show marked similarities between the profiles of the fresh and dried leaves, particularly from the analysis of dried ground leaves and fresh ground leaves (Figure 4). These findings suggest that if a higher amount of plant fresh materials is used to compensate for the water naturally contained in these matrices, the drying and milling processes adopted in traditional procedures to increase the extraction efficiency could be eliminated when OLE-LC is employed. (Figure 4). Such observations could also be related to the high pressure in the online extraction system, which might disrupt tissue cells, enabling better access of the solvent inside the matrix.26 Thus, pre-disruption by milling becomes a redundant procedure.

Figure 3. HPLC–UV fingerprints of leaves of C. mandioccana at 250 nm. Column: Phenomenex Luna LC8 250×4.6 mm; 5 µm. Mobile-phase: Buffer pH = 2,0 (TEA 11,9 mM and H3PO4 14,3 mM) (A), MeOH (B) and ACN (C): 6–8% of B and 11-15% of C (0-10 min); 8-19% of B and 15-14% of C (10-20 min); 19-22% of B and 14-17% of C (20-30 min); 22-14% of B and 17-27% of C (30-40 min); 14-50% of B and 27-0% of C (40-45 min); 50% of B and 0% of C (45-60 min). Flow rate: 1.0 mL/min. Samples: a) 2 mg of the dry ground material and b) 20 µL of a 20 mg/mL extract solution (corresponding to 1.8 mg of the original dry grounded leaves).

Testing different plant tissues with OLE-LC. Stems of Tocoyena formosa and seeds of Pterogyne nitens were analyzed. These species were selected to expand the variety of secondary metabolites sampled in this work. The former is characterized by phenolic derivatives and guanidine alkaloids27; the latter is known to have glycosylated and nonglycosylated iridoids and triterpene saponins.28 Figure S-3 in the SI shows the chromatograms obtained using 2 mg of dried plant materials for OLE-LC analyses and 20 µL of a 40 mg/mL extract for conventional procedures. The results for these analyses also show qualitatively similar profiles between the chromatograms obtained from both approaches (Figure S3). This result indicates that OLE-LC is compatible with tissues other than leaves. Increasing the greenness of the analytical procedure by direct analysis of fresh tissues. The results obtained so far show that it is possible to achieve satisfactory analysis of dried ground tissues employing the OLE-LC approach. However, these results still do not address whether the OLE-LC approach can be used to analyze tissues without the need for drying and grinding procedures. Thus, a fresh leaf of C. sylvestris was only cut with scissors to obtain fragments of approximately 2.0 mm. These fragments were added to the sample chamber (Figure 1) and analyzed by OLE-LC. For the sake of comparison, two different procedures were also adopted: i) fresh leaves were dried and ground and ii) fresh leaves were only ground using liquid nitrogen and an analytical mill. To obtain similar peak intensities between the chromatograms, 5.0 mg of fresh material was used to compensate for the water

Figure 4. HPLC-UV fingerprints of C. sylvestris at λ=254 (0-25 min.) and 235 nm (25-55 min.) (C. sylvestris. Column: Synergi Hydro-RP, 150 x 4.6 mm; 4 µm. Mobile phase: H2O (A) and EtOH (B): 2.7-83.7% of B (0-37.2 min.); 83.7-83.7% of B (37.243.2 min.); 83.7-100% of B (43.2-43.9 min.)/ 100-100% of B (43.9-48.1 min.); 100-2.7% of B (48.1-49.9 min.) and 2.7-2.7% of B (49.9-54.1 min.). Flow rate: 0.7 mL/min. Samples: (a) 2 mg of dried and ground leaves, (b) 5 mg of ground fresh leaves and (c) 5 mg of fresh unground leaves.

CONCLUSIONS This work describes a new, simple and efficient strategy to minimize or eliminate sample preparation procedures while directly upholding the principles of Green Analytical Chemistry.5 When fresh unground leaves were analyzed by OLE-LC, sample treatment was avoided (principle number 1); minimal sample size was used as compared to traditional procedures (principle number 2); energy and solvent consumptions were reduced by integrating analytical procedures (principle number 4); the volume of analytical waste was reduced (principle number 7) because the amount of solvent dedicated exclusively to sample extraction was reduced to zero, compared with traditional off-line extraction; and the use of energy was min-


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imized (principle number 9) by eliminating the drying and grinding procedures. Additionally, eventual difficulties related to the solubilization of complex samples were avoided due to the direct transfer of analytes from the solid matrices to the column once they had been extracted by the mobile phase. An efficient extraction was completely coupled with the HPLC analysis without the need for pumps and valves other than those comprising the HPLC apparatus. Similarly, no extra apparatus was required to stop the mobile phase flow at any time during the full analytical procedure. Table S1 in the SI provides a comparative overview between OLE-LC and known protocols regarding resources and steps needed for a complete analysis of a complex solid matrix. Because the qualitative chromatographic profiles of the plant materials analyzed by OLE-LC were very similar to those observed when traditional extraction was followed by HPLC analysis, it was demonstrated that OLE-LC could be used for quality control of plant materials and their derived products and in untargeted metabolomic investigations. The fact that tissues presenting different consistencies were satisfactorily analyzed by OLE-LC suggests that this approach might be applied to study animal or human tissues, soil, tumors, microorganisms, etc. These samples might be satisfactorily packed in the sample chamber (Figure 1) and extracted with the mobile phase. The direct analysis of a single, selected fragment of fresh material by OLE-LC serves as a “snapshot” of the plant in fluxomics, an increasingly popular subject in biology. Further investigations are under development in our group. The possibility of expanding the range of extractable metabolites by expanding the extraction-LC analyses presented here is being investigated. The use of detectors which may allow the detection of non-chromophoric compounds and the use of UV absorbing solvents is being evaluated for this purpose. Investigations of the use of online extraction in comprehensive multidimensional chromatography and preparative chromatography for direct isolation from the matrix are also in progress.

ASSOCIATED CONTENT Supporting Information Figure S-1 presents three chromatograms obtained sequentially using the OLE-LC approach. Figure S-2 presents the UV spectra of some representative peaks in Casearia sylvestris fingerprint. Figure S-3 presents the chromatographic profile of different plant tissues and species. Table S-1 presents a comparative overview between OLE-LC and known protocols. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION * Corresponding author:

e-mail address: [email protected] Tel.: +55 16 33019791 Author Contributions V.G.F. and G.M.L. performed the experiments, analyzed the data and wrote the manuscript.

C.S.F. and A.J.C. analyzed the data, suggested experiments and wrote the manuscript.

ACKNOWLEDGMENT This research was supported by the São Paulo Research Foundation (grants #013/07600-3 and #010/18840-7), Brazilian National Council for Scientific and Technological Development (grant #45398 2014-5) and Brazilian Coordination for the Improvement of Personnel in Higher Education.

The authors have declared no conflicts of interest.

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For TOC only


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Figure 1. Set-up of the 6-port, 2-position valve used for the online extraction (“load” and “inject” positions). 503x225mm (96 x 96 DPI)



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Figure 2. HPLC–UV fingerprints of leaves of C. sylvestris at 250 nm. Column: Synergi Hydro-RP, 250×4.6 mm; 4 µm. Mobile phase: H2O and EtOH: 2.7–83.7% of B (0-61.8 min); 83.7% of B (61.8-90 min). Flow rate: 0.7 mL/min. Samples: a) 2 mg of the dry ground leaves and b) 20 µL of a 20 mg/mL extract solution (corresponding to 3.0 mg of the original dry ground leaves). 334x235mm (96 x 96 DPI)


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Figure 3. HPLC–UV fingerprints of leaves of C. mandioccana at 250 nm. Column: Phenomenex Luna LC8 250×4.6 mm; 5 µm. Mobile-phase: Buffer pH = 2,0 (TEA 11,9 mM and H3PO4 14,3 mM) (A), MeOH (B) and ACN (C): 6–8% of B and 11-15% of C (0-10 min); 8-19% of B and 15-14% of C (10-20 min); 19-22% of B and 14-17% of C (20-30 min); 22-14% of B and 17-27% of C (30-40 min); 14-50% of B and 27-0% of C (40-45 min); 50% of B and 0% of C (45-60 min). Flow rate: 1.0 mL/min. Samples: a) 2 mg of the dry ground material and b) 20 µL of a 20 mg/mL extract solution (corresponding to 1.8 mg of the original dry grounded leaves). 333x228mm (96 x 96 DPI)



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Figure 4. HPLC-UV fingerprints of C. sylvestris at λ=254 (0-25 min.) and 235 nm (25-55 min.) (C. sylvestris. Column: Synergi Hydro-RP, 150 x 4.6 mm; 4 µm. Mobile phase: H2O (A) and EtOH (B): 2.7-83.7% of B (037.2 min.); 83.7-83.7% of B (37.2-43.2 min.); 83.7-100% of B (43.2-43.9 min.)/ 100-100% of B (43.948.1 min.); 100-2.7% of B (48.1-49.9 min.) and 2.7-2.7% of B (49.9-54.1 min.). Flow rate: 0.7 mL/min. Samples: (a) 2 mg of dried and ground leaves, (b) 5 mg of ground fresh leaves and (c) 5 mg of fresh unground leaves. 333x329mm (96 x 96 DPI)


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Figure S-1. HPLC–UV fingerprints of leaves of C. sylvestris at 250 nm. Column: Synergi Hydro-RP, 250×4.6 mm; 4 µm. Mobile-phase: H2O and EtOH: 2.7–83.7% of EtOH for 61.8 min, followed by 10 min in isocratic mode, posteriorly 10 min in isocratic mode of 100% of EtOH was used to ensure the cleaning of the system. Flow rate: 0.7 mL/min. Samples: a) First extraction of 2 mg of dry ground material; b) second sequential extraction of the same material and c) third sequential extraction. 333x342mm (96 x 96 DPI)



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Figure S-2. HPLC–UV fingerprints of leaves of C. sylvestris at 250 nm showing UV spectra of the peaks. Column: Synergi Hydro-RP, 250×4.6 mm; 4 µm. Mobile phase: H2O and EtOH: 2.7–83.7% of B (0-61.8 min); 83.7% of B (61.8-90 min). Flow rate: 0.7 mL/min. Samples: a) 2 mg of the dry ground leaves and b) 20 µL of a 20 mg/mL extract solution (corresponding to 3.0 mg of the original dry ground leaves). 333x228mm (96 x 96 DPI)


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Figure S-3. HPLC–UV fingerprints of different species and tissues at 250 nm. Column: Phenomenex Luna C18, 250×4.6 mm; 5 µm. Mobile-phase: H2O and MeOH: 5/5/100/100% B in 0/5/30/40 min. Flow rate: 0.7 mL/min. Samples: a) steams of Tocoyena formosa and b) seeds of Pterogyne nitens. Left chromatograms: 20 µL (corresponding to 0.08 mg) of a 40 mg/mL extract and right chromatograms: 2 mg of the dry ground material. 583x235mm (96 x 96 DPI)


Online Extraction Coupled to Liquid Chromatography Analysis (OLE-LC)

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