Technical Note

pubs.acs.org/ac

Online Extraction Coupled to Liquid Chromatography Analysis (OLE-

LC): Eliminating Traditional Sample Preparation Steps in the
Investigation of Solid Complex Matrices
Vincius G. Ferreira,, Gabriel M. Leme,, Alberto J. Cavalheiro,*, and Cristiano S. Funari,

Chemistry Institute, Sao Paulo State University (UNESP), 14800-900 Araraquara, Sao Paulo, Brazil

College of Agricultural Sciences, Sao Paulo State University (UNESP), Private Bag 237, 18610-307 Botucatu, Sao 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 ow 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 ltration, among
others. This work describes a new, simple, and ecient 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 ngerprints 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
this key step.14 According to Tobiszewisky et al.,2 sample
preparation generates the most pollution of any step in the
analytical process. Thus, it is not a surprise that the rst of the
12 principles of Green Analytical Chemistry (GAC) states that
direct analytical techniques should be applied to avoid sample
treatment.5
Nontargeted chemical investigations of natural samples
typically include the following sample pretreatment steps
before analysis in a liquid chromatography system: (i) drying
of the collected material, (ii) grinding, (iii) extraction, (iv) uid
extract ltration, (v) solvent elimination, (vi) solid phase
extraction, (vii) eluate drying, (viii) residue solubilization in the
desired concentration/solvent solution, and (ix) ltration in a
micrometer lter.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, ef fective,
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
ber.
Common extraction techniques usually employed in non-
targeted investigations include ultrasonic and microwave-
assisted extraction, maceration, Soxhlet extraction, and super-
critical uid extraction,7 whereas the most common solvents are
Received: June 21, 2016
Accepted: August 5, 2016

XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b02388

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Analytical Chemistry
Figure 1. Set-up of the six-port, two-position valve used for the online extraction (load and inject positions).
methanol, ethanol, and water for the extraction of polar
compounds and chloroform for the extraction of nonpolar
compounds.7 Considering that successive extractions with
dierent 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 prole of the sample is
acquired by means of one or more comprehensive analytical
methods.10 Among the separation techniques used to obtain
the metabolic proles of plants, high performance liquid
chromatography (HPLC) and, more recently, ultrahigh
performance 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 proles 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 prole of a sample exhibiting the desired
characteristics can be used as a reference. Metabolic proles are
also useful in metabolomics studies, which are mainly
performed through a comparative analysis of initial proles
obtained for dierent samples.
The simplication and integration of sample preparation with
chromatographicspectrometric 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
eorts have been made in this direction, in which several typical
sample preparation steps were integrated with liquid
chromatography analysis.1119 Briey, the dierent systems
reported in the literature work as follows: a pump ushes 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 eorts; the overconsumption of
solvents and energy is not in line with trends regarding
sustainability5,21 in analytical chemistry. It is important to
B DOI: 10.1021/acs.analchem.6b02388
Technical Note
highlight that these approaches have been applied only in
targeted investigations and mainly to liquid samples.
On the basis of 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 methods applicability, the following
integrated strategy was (i) initially applied to the establishment
of chromatographic ngerprints 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, owers, 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
(o-line 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).
Online Extraction Directly Coupled to High-Perform-
ance 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 m, JT Baker, Mexico).
To prevent the ow of particles through the separation system,
a Nylon membrane (0.2 m 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 six-
port valve remains in the load position. Once the chromato-
graphic column is equilibrated, the valve is switched to the
inject position, thus allowing the mobile phase to ow
through the precolumn containing the plant material prior to
entering the chromatographic column, where the separation is
achieved. Once the analysis is nished (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 ow through the guard
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Analytical Chemistry Technical Note

Figure 2. HPLCUV ngerprints of leaves of C. sylvestris at 250 nm. Column: Synergi Hydro-RP, 250 4.6 mm; 4 m. Mobile phase: H2O and
EtOH: 2.783.7% of EtOH (061.8 min); 83.7% of EtOH (61.890 min). Flow rate: 0.7 mL/min. Samples: (a) 2 mg of the dry ground leaves and
(b) 20 L of a 20 mg/mL extract solution (corresponding to 3.0 mg of the original dry ground leaves).

Figure 3. HPLCUV ngerprints of leaves of C. mandioccana at 250 nm. Column: Phenomenex Luna LC8 250 4.6 mm, 5 m. Mobile-phase:
buer pH = 2.0 (TEA 11.9 mM and H3PO4 14.3 mM) (A), MeOH (B), and ACN (C): 68% of B and 1115% of C (010 min); 819% of B and
1514% of C (1020 min); 1922% of B and 1417% of C (2030 min); 2214% of B and 1727% of C (3040 min); 1450% of B and 27
0% of C (4045 min); 50% of B and 0% of C (4560 min). Flow rate: 1.0 mL/min. Samples: (a) 2 mg of the dry ground material and (b) 20 L of
a 20 mg/mL extract solution (corresponding to 1.8 mg of the original dry grounded leaves).

column (Figure 1). In summary, the solid sample cell is described in Reference Sample Preparation for Comparative
positioned in the injection valve at the place of the sample loop. Purposes were performed in a Dionex Ultimate 3000 system
The sample extraction, followed by the chromatographic (Sunnyvale, USA) equipped with a DGP-3600RS model pump,
analyses (OLE-LC) and the chromatographic analyses of the a WPS-3000 SL model autosampler, a TCC-3000RS model
samples previously prepared using the traditional procedures column oven, and a Rheodyne (Oak Harbor, USA) six-port,
C DOI: 10.1021/acs.analchem.6b02388
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Analytical Chemistry
two-position valve. The whole system was controlled by
Chromeleon software version 6.80 (Dionex, Sunnyvale, USA).
A Varian HPLC system equipped with Prostar 210 pumps, a
Rheodyne 7725i model six-port valve, and a Prostar 320 UV/vis
detector 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
pretreated and analyzed by HPLC according to Funari et
al.22 and Bandeira and Cavalheiro.23 Briey, 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 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 nonpolar compounds. The eluate was dried and
solubilized to yield a concentration of 20 mg/mL. Finally, the
solution was ltered through a 0.22 m poly(ether sulfone).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 1:1 methanol/
acetic acid 10% (v/v) was added, followed by 30 min of
sonication. The mixture was then centrifuged at 1200g 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 rst extracted and then analyzed by HPLC. For
this purpose, two medicinal plants (C. sylvestris and C.
mandioccana) with dierent chemical compositions and well-
established methods of extraction and separation were
selected.22,23 While the leaves of C. sylvestris contain mainly
avonoids and clerodane diterpenes,24 the leaves of C.
mandioccana generally contain avonoids, 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 oine extraction
and analysis is shown in Figure 2b.
While similar chromatographic proles were observed in the
second half of the chromatograms, relatively dissimilar proles
were observed for the rst half (Figure 2). A higher peak area
and number of polar compounds with a 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.
D DOI: 10.1021/acs.analchem.6b02388
Technical Note
Regarding the total peak area, the observed values for the OLE-
LC 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 L 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
ecient 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 eciency 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 rst extraction. This high
eciency 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
Comparison between OLE-LC and Reference Proce-
dure 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 rst 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 L 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 ecient 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 OLE-LC
were wider, which might lead to coelution of compounds,
resulting in a lower number of peaks.
Testing Dierent 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 alkaloids;27 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 L of a 40 mg/mL extract for
Anal. Chem. XXXX, XXX, XXXXXX
Analytical Chemistry Technical Note

Figure 4. HPLC-UV ngerprints of C. sylvestris at = 254 (025 min) and 235 nm (2555 min). C. sylvestris, column: Synergi Hydro-RP, 150 4.6
mm, 4 m. Mobile phase: H2O (A) and EtOH (B); 2.783.7% of B (037.2 min); 83.783.7% of B (37.243.2 min); 83.7100% of B (43.243.9
min.)/ 100100% of B (43.948.1 min); 1002.7% of B (48.149.9 min) and 2.72.7% of B (49.954.1 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.

conventional procedures. The results for these analyses also
show qualitatively similar proles between the chromatograms
obtained from both approaches (Figure S-3). 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 dierent 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
E DOI: 10.1021/acs.analchem.6b02388
chromatograms, 5.0 mg of fresh material was used to
compensate for the water content in the fresh leaves. The
chromatograms obtained from these three approaches are
shown in Figure 4.
The chromatographic proles and peak intensities obtained
from these three approaches show marked similarities between
the proles of the fresh and dried leaves, particularly from the
analysis of dried ground leaves and fresh ground leaves (Figure
4). These ndings 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
eciency 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, predisruption by milling becomes a redundant
procedure.
Anal. Chem. XXXX, XXX, XXXXXX
Analytical Chemistry
CONCLUSIONS
This work describes a new, simple, and ecient 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 o-
line extraction, and the use of energy was minimized (principle
number 9) by eliminating the drying and grinding procedures.
Additionally, eventual diculties 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 ecient 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 ow 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 proles 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 dierent 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 uxomics, 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 nonchromophoric 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 prepa-
rative chromatography for direct isolation from the matrix are
also in progress.
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.anal-
chem.6b02388.
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
the Casearia sylvestris ngerprint; Figure S-3 presents the
chromatographic prole of dierent plant tissues and
species; Table S-1 presents a comparative overview
between OLE-LC and known protocols (PDF)
F DOI: 10.1021/acs.analchem.6b02388
Technical Note
AUTHOR INFORMATION
Corresponding Author
*Tel.: +55 16 33019791. E-mail: albjcava@gmail.com.
Author Contributions
These authors contributed equally to this work.
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.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
This research was supported by the Sao Paulo Research
Foundation (grants #013/07600-3 and #010/18840-7), Brazil-
ian National Council for Scientic and Technological Develop-
ment (grant #45398 2014-5), and Brazilian Coordination for
the Improvement of Personnel in Higher Education.
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G DOI: 10.1021/acs.analchem.6b02388
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