Technical Note

Online Extraction Coupled to Liquid Chromatography Analysis (OLE-
LC): Eliminating Traditional Sample Preparation Steps in the
Investigation of Solid Complex Matrices
Vinícius G. Ferreira,†,∥ Gabriel M. Leme,†,∥ Alberto J. Cavalheiro,*,† and Cristiano S. Funari‡,§

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, 18610-307 Botucatu, São Paulo, Brazil
Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences, University of Tasmania, Private Bag
75, Hobart 7001, Australia
S Supporting Information

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.

W hereas 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
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, ef fective,
this key step.1−4 According to Tobiszewisky et al.,2 sample rugged, and safe extraction (QuEChERS)8 and solid-phase
preparation generates the most pollution of any step in the microextraction (SPME) methods9 allow the elimination of
analytical process. Thus, it is not a surprise that the first of the steps iv, v, and vi outlined above, although in SPME methods a
12 principles of Green Analytical Chemistry (GAC) states that desorption step is necessary to release the analytes from the
direct analytical techniques should be applied to avoid sample
Common extraction techniques usually employed in non-
Nontargeted chemical investigations of natural samples
typically include the following sample pretreatment steps targeted investigations include ultrasonic and microwave-
before analysis in a liquid chromatography system: (i) drying assisted extraction, maceration, Soxhlet extraction, and super-
of the collected material, (ii) grinding, (iii) extraction, (iv) fluid critical fluid extraction,7 whereas the most common solvents are
extract filtration, (v) solvent elimination, (vi) solid phase
extraction, (vii) eluate drying, (viii) residue solubilization in the Received: June 21, 2016
desired concentration/solvent solution, and (ix) filtration in a Accepted: August 5, 2016
micrometer filter.3,6 In some cases, the freshly ground material

© XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b02388
Anal. Chem. XXXX, XXX, XXX−XXX

thus allowing the mobile phase to flow step and a relatively large amount of sample are required. high performance liquid To determine the method’s applicability. the metabolic profiles of plants. Set-up of the six-port. analytical procedure. reported in the literature work as follows: a pump flushes an To prevent the flow of particles through the separation system. and triethylamine a practical perspective. as shown in Figure position and the LC analysis starts. T. ethanol. two-position valve used for the online extraction (“load” and “inject” positions). and to determine the compatibility of this strategy with plant parts exhibit good resolution. and water for the extraction of polar highlight that these approaches have been applied only in compounds and chloroform for the extraction of nonpolar targeted investigations and mainly to liquid samples.1021/acs. JT Baker. phosphoric acid (85%). extractive solution though the matrix. for the assessment of plant identity or for without any type of treatment. and solvents is dramatically greening. an amount graphic column is equilibrated. chromatographic−spectrometric analysis would be useful from Acetic acid (99. USA). the overconsumption of wants to stop the plant material extraction).10 other than leaves (seeds.10 Among the separation techniques used to obtain those comprising the HPLC apparatus and the mobile phase. speeding up. XXX−XXX . such 1.11−19 Briefly. in which several typical 2 mg of ground. The through the precolumn containing the plant material prior to latter is a bottleneck in many metabolomic investigations. the six- approaches require pumps and/or valves other than those port valve remains in the “load” position. dry plant material was inserted in a sample preparation steps were integrated with liquid SecurityGuard holder (Phenomenex. are easy to operate. Solvents and Additives.13. and roots). In this sustainability5.20 Additionally. and reducing the cost of the overall increased. even after massive efforts. Once the analysis is finished (or whenever the analyst be collected. more recently.7 Considering that successive extractions with On the basis of these considerations. and solvents other than methods. efforts have been made in this direction. LC techniques are compatible with almost any type sylvestris and Cryptocaria mandioccana. XXX. Finally. XXXX. e. Chem. followed directly by a a Nylon membrane (0. and environmental (99%) were of analytical grade (Sigma. compounds. and detection steps and for used such as time. which are mainly EXPERIMENTAL SECTION performed through a comparative analysis of initial profiles obtained for different samples. valves. ultrahigh integrated strategy was (i) initially applied to the establishment performance liquid chromatography (UHPLC) are the primary of chromatographic fingerprints for the leaves of Casearia techniques.12. money.6b02388 Anal.21 in analytical chemistry. saving time. (iii) subjected to Metabolic profiles have proven to be useful in various evaluation to determine its compatibility with fresh tissues applications. During the chromatographic column equilibration. resources such as solvents and energy and resolving the Online Extraction Directly Coupled to High-Perform- solubility issues encountered with complex samples. extra apparatus.12. Once the chromato- comprising the LC instrument. which is delivered by the guard column holder containing the plant material was another pump. position) by the solvent (or solution). Some ance Liquid Chromatography (OLE-LC) Analysis. the different systems volume was completed with C18 (40 μm. USA) was placed on both sides of the guard column are eluted from the SPE cartridge to the LC valve (in the “load” recipe. Baker. Metabolic profiles are also useful in metabolomics studies. Then. the mobile phase does not flow through the guard B DOI: 10.2 μm pore diameter. this work presents an different solvents and techniques are performed to extract the alternative strategy for online coupling of the comprehensive largest part of a given metabolome. flowers. and The simplification and integration of sample preparation with acetonitrile (J. reproducibility.. The methanol.analchem. the following chromatography (HPLC) and.13 Nevertheless. the valve is solvents and energy is not in line with trends regarding automatically turned back to the “load” position. The extraction of plant tissues was After sample preparation. The chamber chromatography analysis.g. and (iv) compared in terms of quality control of plant-derived products and processes in performance with strategies employing reference procedures which a metabolite profile of a sample exhibiting the desired (off-line extraction followed by LC analyses). the valve is switched to the of solvents and energy dedicated exclusively to the extraction “inject” position. a metabolic profile of the sample is completely coupled with the HPLC analysis without the need acquired by means of one or more comprehensive analytical for pumps. St Louis.Analytical Chemistry Technical Note Figure 1. energy. the analytes Schuell. entering the chromatographic column. Finally. where the separation is especially in cases in which only a few milligrams of sample can achieved. Initially. ethanol. and selectivity. can be fully automated. sealing the sample and the C18 particles inside. Mexico). Schleicher & solid phase extraction (SPE) trap cartridge. separation. methanol. (ii) subjected to analysis of compound. ■ characteristics can be used as a reference.7%). the valve is switched to the “inject” connected to the chromatographic system. USA). Mexico) used were HPLC grade. the amount of resources sample preparation. It is important to position.

Samples: (a) 2 mg of the dry ground leaves and (b) 20 μL of a 20 mg/mL extract solution (corresponding to 3. USA) six-port.3 mM) (A). 22−14% of B and 17−27% of C (30−40 min). Figure 3. 19−22% of B and 14−17% of C (20−30 min).8 mg of the original dry grounded leaves). XXX. column (Figure 1). MeOH (B). 50% of B and 0% of C (45−60 min). sylvestris at 250 nm.0 (TEA 11. 8−19% of B and 15−14% of C (10−20 min). Flow rate: 1. HPLC−UV fingerprints of leaves of C. the solid sample cell is described in Reference Sample Preparation for Comparative positioned in the injection valve at the place of the sample loop. Mobile phase: H2O and EtOH: 2.8−90 min). 14−50% of B and 27− 0% of C (40−45 min). 5 μm. analyses (OLE-LC) and the chromatographic analyses of the a WPS-3000 SL model autosampler. 4 μm.0 mg of the original dry ground leaves). Column: Phenomenex Luna LC8 250 × 4. XXX−XXX . 83. XXXX. Mobile-phase: buffer pH = 2. C DOI: 10. 250 × 4.0 mL/min.analchem.6 mm.7−83. a TCC-3000RS model samples previously prepared using the traditional procedures column oven. mandioccana at 250 nm. Chem. Purposes were performed in a Dionex Ultimate 3000 system The sample extraction.1021/acs.7% of EtOH (0−61.8 min). In summary. and ACN (C): 6−8% of B and 11−15% of C (0−10 min).6b02388 Anal.7 mL/min. USA) equipped with a DGP-3600RS model pump. HPLC−UV fingerprints of leaves of C. followed by the chromatographic (Sunnyvale. Flow rate: 0.Analytical Chemistry Technical Note Figure 2. and a Rheodyne (Oak Harbor. Samples: (a) 2 mg of the dry ground material and (b) 20 μL of a 20 mg/mL extract solution (corresponding to 1.6 mm. Column: Synergi Hydro-RP.9 mM and H3PO4 14.7% of EtOH (61.

mandioccana. and the number of peaks was the output under consideration.22 and Bandeira et al. A representative chromatogram obtained directly could be explained by the fact that peaks acquired by OLE-LC from the dry ground leaves of C.80 (Dionex. However.22 and Bandeira and Cavalheiro. which might lead to coelution of compounds.8 mg of the original dried materials were first extracted and then analyzed by HPLC. XXXX.3 mg extractor mixture (EtOH: 2.0 mL of hexane. While similar chromatographic profiles were observed in the These species were selected to expand the variety of secondary second half of the chromatograms.2 (n = 5) for the reference procedure. XXX. Tocoyena formosa and seeds of Pterogyne nitens were analyzed.9.2 ± 2.0 mg of the original dried. it established methods of extraction and separation were can be inferred that OLE-LC was more efficient than the selected. However. yielding 21. The mixture was then centrifuged at 1200g for 10 The chromatograms were qualitatively similar. C.28 Figure S-3 in the SI shows the reference procedure. OLE-LC. sylvestris and C. please refer to the captures shown in Figures 2 procedure in terms of the outcomes of the number of peaks and 3. USA). The alkaloids. followed by 30 min of procedure with leaves of C. Sunnyvale.4 ± 8.7 to 83. the OLE-LC approach was more sylvestris and C.4 ± 548 A Varian HPLC system equipped with Prostar 210 pumps. The OLE-LC results were was 21. C. a and 19278 ± 200 au. respectively. which was 3).22 μm poly(ether sulfone).24 the leaves of C. Stems of and analysis is shown in Figure 2b. sylvestris.23 While the leaves of C. and retention times of the main corresponding compounds (Figure weighed. respectively.302. Next. it was possible to conclude that 95% of the extracted by maceration with three aliquots of 0.0 mg of The HPLC methods used were those previously described by the original dried. Chem.9 mL of EtOH material was extracted in the first extraction. corresponding chromatogram obtained by offline extraction Testing Different Plant Tissues with OLE-LC. mandioccana material. The hydroalcoholic phase was collected. mandioccana) with different chemical compositions and well.3%.9%). sylvestris leaves procedure (Figure S-1 in the SI). The combined solutions were efficiency could be related to the use of a gradient in the concentrated under reduced pressure at 35 °C to yield 33. for OLE-LC analyses and 20 μL of a 40 mg/mL extract for D DOI: 10. Reference Sample Preparation for Comparative Additionally. sylvestris leaves. The whole system was controlled by Regarding the total peak area. For a description of efficient per mass of original sample than the reference the conditions.7% in water) and to the of dried extract. mandioccana leaves were sylvestris dry leaves were subjected to three sequential analyses pretreated and analyzed by HPLC according to Funari et by OLE-LC to evaluate the efficiency of this extraction al. XXX−XXX . sylvestris leaves were analyzed by Funari et al. ensuring better solubilized to yield a concentration of 20 mg/mL. respectively. Considering that the yield of Rheodyne 7725i model six-port valve. The eluate was dried and better access to the solvent inside the matrix. fresh material. This high at 40 °C with constant stirring.23 for the analysis of C. mandioccana directly analyzed by OLE-LC (Figure 3a). In other words. mandioccana. For ground leaves of C.25 number of peaks for OLE-LC and the reference procedure were Comparison of OLE-LC and Reference Procedure for 44.23 Briefly. 20 μL of the 20 mg/mL solution injected in the compared to those of reference procedures in which plant HPLC system corresponds to 1. 250 mg was extractable material. sonication.23 those observed for the reference procedure.6b02388 Anal. Of the resulting obtained directly from the dry ground leaves of C. the observed values for the OLE- Chromeleon software version 6.22. mandioccana extract. Purposes.analchem. approximately 2 mg of dry ground leaves of C. This value this purpose. LC approach and the reference approach were 2894. sylvestris by the proposed were wider.22 Comparison between OLE-LC and Reference Proce- Leaves of C. strategy that combined the extraction and separation of plant Considering that the yield of the reference extraction procedure materials was tested in this work. sylvestris and C. whereas the resulting in a lower number of peaks. during which the compounds resulting in peaks presented UV spectra compatible with those of alkaloids (Figure S-2 in the SI).4 and 57.Analytical Chemistry Technical Note two-position valve. 4. The dried extract was subjected to SPE to high pressure in the online extraction system. it can be inferred detector controlled by the Galaxie chromatography data system that the 20 μL of 20 mg/mL extract solution injected into the software (version 1. relatively dissimilar profiles metabolites sampled in this work.9%.0 mL of 1:1 methanol/ corresponding chromatogram obtained from the reference acetic acid 10% (v/v) was added. OLE-LC approach is shown in Figure 2a. the peaks acquired by OLE-LC were broader than then solubilized to obtain a 20 mg/mL solution.27 the latter is and number of polar compounds with a retention time ≤ 35 known to have glycosylated and nonglycosylated iridoids and min was observed for the OLE-LC compared to those of the triterpene saponins. styrylpyrones. ground C.530) was also used for the analysis of HPLC system corresponded to 3. detectable compounds. sylvestris contain mainly reference procedure for the extraction of a given mass of flavonoids and clerodane diterpenes.2 chromatograms obtained using 2 mg of dried plant materials for OLE-LC and 89 ± 4. Of the resulting material. Finally. in the sample preparation procedures that often precede LC The observed values for the total peak area were 12523 ± 74 analyses and to make the investigation itself greener. On the other hand. and then areas observed for the three extractions as 100% of the ground in a knife mill. and total peak area. the extraction. two medicinal plants (C. and a Prostar 320 UV/vis the reference extraction procedure was 13. which enabled eliminate very nonpolar compounds.26 solution was filtered through a 0.1021/acs. only 2. This contrast C. a new and 793 ± 62 au for OLE-LC and the reference procedure.9 (n = 5). The total number of peaks was 108 ± 8. The former is characterized were observed for the first half (Figure 2). dried.9 mg of dried extract (21. A higher peak area by phenolic derivatives and guanidine alkaloids. This was especially ■ RESULTS AND DISCUSSION Aiming to reduce the time and resource-consumption involved important for the first half of the chromatogram. Considering the sum of the were dried at 40 °C in an oven with air circulation. corresponds to 90% of the 2 mg of dried ground leaves of C. mandioccana. Thus. whereas Figure 3b shows the aliquot of 4. 100 mg was extracted by sonication for 30 min with an by OLE-LC is shown in Figure 3a. A representative chromatogram air circulation and ground in a knife mill. as were the min. ground C. mandioccana were dried at 40 °C in an oven with dure for C. this was not the case when mandioccana generally contain flavonoids.

2−43.0 mm. sample chamber (Figure 1) and analyzed by OLE-LC.1−49. E DOI: 10.2 min).7% of B (48. 4).9−48.analchem.). C. (b) 5 mg of ground fresh leaves. 4 μm. Flow rate: 0. Samples: (a) 2 mg of dried and ground leaves. XXX. The results obtained so the profiles of the fresh and dried leaves. The chromatographic profiles and peak intensities obtained Increasing the Greenness of the Analytical Procedure from these three approaches show marked similarities between by Direct Analysis of Fresh Tissues.2−43. 5.7−100% of B (43. particularly from the far show that it is possible to achieve satisfactory analysis of analysis of dried ground leaves and fresh ground leaves (Figure dried ground tissues employing the OLE-LC approach. which might disrupt adopted: (i) fresh leaves were dried and ground and (ii) tissue cells.7% of B (0−37.9 min) and 2.0 mg of fresh material was used to show qualitatively similar profiles between the chromatograms compensate for the water content in the fresh leaves. Mobile phase: H2O (A) and EtOH (B). These fragments were added to the efficiency could be eliminated when OLE-LC is employed.1 min. HPLC-UV fingerprints of C. predisruption by milling becomes a redundant analytical mill. 100−2. The results for these analyses also chromatograms.1021/acs. contained in these matrices.1 min).9 min.6b02388 Anal. leaves. column: Synergi Hydro-RP. 83.7% of B (37. sylvestris. a fresh leaf of C.2 min).Analytical Chemistry Technical Note Figure 4. XXX−XXX .26 Thus.7−83. 150 × 4. For the (Figure 4).7−83. To obtain similar peak intensities between the procedure. 83.7% of B (49.6 mm. two different procedures were also pressure in the online extraction system. This result chromatograms obtained from these three approaches are indicates that OLE-LC is compatible with tissues other than shown in Figure 4.7−2. the drying and milling processes sylvestris was only cut with scissors to obtain fragments of adopted in traditional procedures to increase the extraction approximately 2. conventional procedures. these results still do not address whether the OLE- materials is used to compensate for the water naturally LC approach can be used to analyze tissues without the need for drying and grinding procedures. XXXX. The obtained from both approaches (Figure S-3). 2. Chem. and (c) 5 mg of fresh unground leaves.)/ 100−100% of B (43.7 mL/min. sylvestris at λ = 254 (0−25 min) and 235 nm (25−55 min). Such observations could also be related to the high sake of comparison.9−54. These findings suggest that if a higher amount of plant fresh However. enabling better access of the solvent inside the fresh leaves were only ground using liquid nitrogen and an matrix. Thus.

K. A. 2013. 1422. plant materials analyzed by OLE-LC were very similar to those D. E. 2016. 59−67. Gonçalves. Chromatographia 2009. Biomed. D. ■ (16) Zhang. F. Anal. selected fragment of fresh material by (10) Tistaert. O.. Chem. Chem. S. L. Qi. suggested experiments. K. Oliveira.. 21. Biomed. Pérez. The data. sample ∥ These authors contributed equally to this work. because the amount of solvent dedicated exclusively to sample Notes extraction was reduced to zero. J. Figure S-1 presents three chromatograms obtained Biomed. Lin. 4237−4245. Dejaegher.. Durando. Biomed. A. (15) Herviou. 690. Li..L.J. observed when traditional extraction was followed by HPLC (4) Vuckovic.2016. L.. (19) De Jager. K. 2009. Andres-Lacueva.. Similarly. Adaime. allow the detection of nonchromophoric compounds and the N. W. Prestes. M. A. Ind. C.. TrAC. 1221.. presents the UV spectra of some representative peaks in (21) Desmet... Eschalier. Table S1 in the SI provides a comparative overview (1) Tulipani. Pabst. Chem. R. Zanella. and wrote the manuscript. L. F.. M. A. D. J.. no extra apparatus was required to stop the mobile phase flow at any time during the full analytical REFERENCES procedure. Anal. J. B. J. M. G. Chromatogr.C.. Kusu. 2013. R. Pawliszyn. V. and the use of energy was minimized (principle number 9) by eliminating the drying and grinding procedures... 164−174. Chem. 1208. direct analysis of a single. Sci... 815. Cavalheiro. The use of detectors which may (13) Murakami. species. 2015. 88. chromatographic profile of different plant tissues and Cavalheiro.03.. J. Y. Table S-1 presents a comparative overview (23) Bandeira.1021/acs.. The authors declare no competing financial interest. T. an 2011. 284−291. Chem. Trends Anal. chem. ASSOCIATED CONTENT (17) Kirchner. L. Kishi. J.. 879. Figure S-3 presents the (22) Funari. the Casearia sylvestris fingerprint.. Anal. Chromatogr. 4−13. E. Brazil- been extracted by the mobile phase. Chem. 2010. J. Roche... Eng. 1999. B.6b02388 Anal. These samples might be satisfactorily packed in the sample B. soil. 84−98. chamber (Figure 1) and extracted with the mobile phase. tumors. ACS Publications website at DOI: 10. T... XXX−XXX . Authier. and A. P. F. Franzke. Appl.anal. C.. in untargeted metabolomic investigations. Z.1016/ purpose. Chim.. A... S. Alpendurada. Richard. here is being investigated.. and Brazilian Coordination for the Improvement of Personnel in Higher Education. Y.. K. N. Martins. Chromatogr. OLE-LC suggests that this approach might be applied to study (8) Ferreira.. 1084. C. M. The (9) Risticevic. 721.. B: Anal. R. Andrade. C.. L. S. A 2008. J. Sep. matrix. and efficient strategy to ■ AUTHOR INFORMATION Corresponding Author minimize or eliminate sample preparation procedures while *Tel. Acta OLE-LC serves as a “snapshot” of the plant in fluxomics. Z. Sewing... 2012. Smith.. The possibility of expanding the range of extractable (12) Tajuddin. 1523−1548. F. N.. between OLE-LC and known protocols regarding resources Anal.. Bailey. H.. J. J. Bottoli. compared with traditional off. Mechlińska.018. S. S. J. Talamine. G. C. Anal. M. Souza-Silva. TrAC. 2011. Trends Anal. J. Trends Anal.. Trends Anal. L. K.: +55 16 33019791. D. Investigations of the use of online extraction in j. Chim.. 85. it was demonstrated that OLE-LC could be used for (5) Gałuszka. Chem.1021/acs. C. Chromatogr.F. U. 85. between OLE-LC and known protocols (PDF) 1455−1460. 54. A.. Urpi-Sarda. S. R.6b02388. M. eventual difficulties related to the solubilization of complex samples were avoided due to the direct transfer of This research was supported by the São Paulo Research analytes from the solid matrices to the column once they had Foundation (grants #013/07600-3 and #010/18840-7). use of UV absorbing solvents is being evaluated for this (14) Grant.. Christians. 2005. Further investigations are under development in our group. B.Analytical Chemistry Technical Note ■ CONCLUSIONS This work describes a new. V. F DOI: 10... rative chromatography for direct isolation from the matrix are 118. J. Minimal sample size was used as compared to traditional procedures (principle Author Contributions number 2).. A 2012. Zygmunt. Chem.. performed the experiments. A 2015. 341−348. Y. Barceló. 543−556. Facco.G. DeEll. simple. A.. G. TrAC.. 2016. D. R. 37−44. M. Y... C. Life Sci. XXXX. XXX.. Because the qualitative chromatographic profiles of the TrAC. S. etc. * S Supporting Information Hallensleben. M. 2014. Pharm.. J. comprehensive multidimensional chromatography and prepa. Fukutsu. Figure S-2 (20) Pan. The Supporting Information is available free of charge on the (18) Sadagopan. J.. (11) Chang... J.F. increasingly popular subject in biology. A. An efficient extraction was ian National Council for Scientific and Technological Develop- completely coupled with the HPLC analysis without the need ment (grant #45398 2014-5). 1−15. G. C. Carneiro. ■ line extraction. 70.. animal or human tissues. F. 37. N. 28. Chromatogr..S. and steps needed for a complete analysis of a complex solid (2) Tobiszewski.5 Author Contributions When fresh unground leaves were analyzed by OLE-LC. Anal.. 222−229. 1266−1274.M. sequentially using the OLE-LC approach. Verpoorte. Hilder. Anal. 943−951. (3) Farré. 3009−3017.. R. S.trac. A. Rizzetti. Namieśnik. Heyden. ACKNOWLEDGMENTS Additionally. Anal. directly upholding the principles of green analytical chemistry. Phytochem. F. 820.. E-mail: albjcava@gmail. Takemura. Cheng. Methods 2015. Li. Acta 2014.. Namieśnik. Zhang. I. quality control of plant materials and their derived products and Chem. B: Anal. The fact that tissues (6) Ramos. Bioanal. Technol. and wrote the manuscript. 7.. J. Liu. analyzed the integrating analytical procedures (principle number 4). 2013. B. presenting different consistencies were satisfactorily analyzed by (7) Kim. C. Cohen. Vidal. R. 50. 1347−1362. Ferreira. microorganisms. 2010. 403. N. F. Eeltink. DOI: 10. J. and G. Technol.. Zhang. M. J. 2016. Chromatogr. 78−84. Llorach. analyzed the volume of analytical waste was reduced (principle number 7) data. A. also in progress. Li. S. C. Pinguet. Libert. treatment was avoided (principle number 1). Navickiene. Migaszewski. C. analysis. J. Cochran. Anal.analchem. Energy and solvent consumptions were reduced by V... J. 148−161. Chem. Res. Jacobsen.. J. X. 29. 285−294. T. Life Sci. 2642−2652. A 2005. 194− metabolites by expanding the extraction-LC analyses presented 200. ■ for pumps and valves other than those comprising the HPLC apparatus. Kawasaki.

Jamal. A. 53.analchem. (25) Cavalheiro. (28) Hamerski.. M. X. Nova 2005. G DOI: 10. da Silva Bolzani. C.. J. 34. 2010.. 2009. 72... C. Jiang. Borges. De Lima. 457−460. C. Duarte. Quim. (26) Prasad.. A. Carbonezi. 601−604. Cavalheiro. Zhao. 28.6b02388 Anal. M.. L. M. J. B.1021/acs. et al. 473−476. J. XXX. S. L.. Yang. M.. Boll Chim Farm. N. K. J. E. M. Yoshida. Young.. M. Y.. (27) Regasini. D. Castro-Gamboa. Silva. Nat.. A. XXXX. 141. Food Biochem.. 811−819. S. 2002.Analytical Chemistry Technical Note (24) Raslan.. Wei. XXX−XXX .. M.. D. I. 838−855. Prod. Sun. Chem. D. V. Phytochemistry 2000.. H.