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In situ extraction and derivatization method for rapid analysis of short-chain

fatty acids in rat fecal sample by gas chromatography tandem mass
spectrometry

Article in Analytical methods · April 2017

DOI: 10.1039/C7AY00168A

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 Analytical
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An in situ extraction and derivatization method for

Cite this: Anal. Methods, 2017, 9, 2351
rapid analysis of short-chain fatty acids in rat fecal
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samples by gas chromatography tandem mass

spectrometry†
Na Hyun Park,‡a Min-Sun Kim,‡b Wonwoong Lee,a Myoung Eun Leea
and Jongki Hong *a

Received 18th January 2017
Accepted 12th March 2017
DOI: 10.1039/c7ay00168a
rsc.li/methods
Short-chain fatty acids (SCFAs) are major products of gut microbial fermentation and changes in their levels
in a biological system reflect the progress of various diseases. However, analysis of SCFAs in biological
samples is limited due to their easy loss during sample workup, their low concentrations, and the
coexistence of matrices. In this study, we developed a feasible method for the analysis of SCFAs in rat
fecal samples using gas chromatography-tandem mass spectrometry (GC-MS/MS) combined with an in
situ extraction and derivatization method. More specifically, in situ extraction and derivatization were
applied for the determination of SCFAs using tetrabutylammonium hydrogen sulfate (TBAHS) and
pentafluorobenzyl bromide (PFBBr). TBAHS concentration and pH values, as well as the temperature and
time of the derivatization reaction were effectively optimized. Moreover, the proposed process reduced
the analysis time and limited the loss of SCFAs during the sample workup. It also improved their peak
shapes and resulted in longer GC retentions and high sensitivity in MS detection. Use of GC-MS/MS
could offer high sensitivity and selectivity for SCFA-PFB derivatives in complicated matrix samples by
selecting a multiple-ion reaction monitoring (MRM) transition ion at m/z 181 that corresponds to the
[PFB]+ ion. The established method was also validated in terms of linearity, limits of detection (LODs) and
quantification (LOQs), and matrix effects. It was proved to be linear (r2 > 0.997), with lower LODs of 5–
24 ng mL
1, and lower LOQs of 0.05–0.1 mg mL
1 for all SCFAs. The proposed method will be useful for
the determination of SCFAs in rat fecal samples and for the clinical diagnosis of various diseases.

Several analytical methods such as HPLC, LC-MS, GC, and

Introduction
Short chain fatty acids (SCFAs) are the main colonic fermenta-
tion products of carbohydrates caused by intestinal bacterial
ora. There is a growing interest in intestinal SCFA levels due to
their positive physiological effects. They not only act as
substrates or signal modulators of glucose, lipid, and choles-
terol metabolism, but also produce T-helper cells and anti-
bodies (Fig. 1).1,2 The changes in SCFA levels in biological
samples are closely related to some diseases such as inam-
matory bowel diseases (IBD),3 cancer,4 obesity, and diabetes.5,6
Thus, accurate determination of SCFAs in biological samples is
essential for clinical diagnosis of various diseases.
a
College of Pharmacy, Kyung Hee University, Seoul 02447, Korea. E-mail: jhong@khu.
ac.kr
b
Integrated Metabolomics Research Group, Korea Basic Science Institute, Seoul, 02841,
Korea
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ay00168a
‡ These authors contributed equally.
GC-MS have been developed for the quantitative determination
of SCFAs in biological samples.7–12 Among them, GC-MS has
been popular for their analysis owing to its high peak capacity
and high detection sensitivity.11,12 However, the unique physi-
cochemical properties of SCFAs (low vapor pressure and boiling
point and relatively high solubility in the aqueous phase) and
their low concentrations in biological samples need to be taken
into account. In addition, their lower GC retention behavior
could be signicantly inuenced by sample matrix interference.
To overcome these problems, various derivatization and sample
preparation methods have been introduced. Recently, simple
derivatization methods using a single reagent have been sug-
gested for the protection of various types of carboxylic acids in
biological samples.13,14 These methods involve extraction of
organic acids including SCFAs with organic solvents before or
aer derivatization. Nevertheless, a consequent loss of SCFAs
during sample handling and extraction is expected. The head-
space solid-phase microextraction (HS-SPME) techniques have
also been applied for the extraction of volatile compounds from
biological uids.15–17 Despite effective purication and a low

This journal is © The Royal Society of Chemistry 2017 Anal. Methods, 2017, 9, 2351–2356 | 2351

Analytical Methods
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Fig. 1 Overall analytical procedure for the determination of SCFAs in
fecal samples by GC/MS/MS.
detection limit, SPME is sensitive to the adsorption rate and
distribution coefficient according to the kinds of bers used.
In many cases, detection of trace amounts of SCFAs can be
easily masked by high-concentrations of biological constituents.
The complex matrices of fecal samples may reduce the recovery,
accuracy, and reproducibility of the method, and lead to
instrumental contamination in repetitive analysis. Thus, selec-
tive extraction and derivatization of SCFAs from fecal samples are
required for clinical analysis of a biological sample. To this end,
we have developed an in situ method using phase-transfer
catalysis (PTC) with tetrabutylammonium hydrogen sulfate
(TBAHS) and base-catalyzed derivatization with 2,3,4,5,6-penta-
uorobenzyl bromide (PFBBr). Both TBAHS and PFBBr show
highly selective reactivity with carboxylic acids,18–20 enabling
selective extraction and derivatization of SCFAs in complex bio-
logical samples. This approach aims to eliminate the potential
loss of the desired fatty acids during the sample treatment, while
it offers several advantages such as in situ sample preparation,
effective purication, enhanced GC retention of volatile SCFAs,
and the identication of middle chain fatty acids (MCFAs) in
fecal samples. In addition, SCFA-PFB derivatives provided
specic MRM transition ions in GC-MS/MS analysis, enabling
high sensitivity, selectivity, and specicity for SCFAs in complex
sample matrices. The in situ method using PTC was the rst
application for the determination of SCFAs in fecal samples. The
method was validated in terms of linearity, limits of detection,
precision, accuracy, and matrix effects. The established method
will be useful for the reliable conrmation and quantication of
SCFAs as well as untargeted MCFAs in fecal samples and aid in
better understanding the metabolism of fatty acids in the gut.
Experimental
Chemicals and reagents
Sodium acetate, sodium propionate, sodium butyrate, and
isotopically labeled propionic acid-d3 used as an internal
2352 | Anal. Methods, 2017, 9, 2351–2356
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Technical Note
standard, and phenanthrene-d10 used as the recovery standard
for GC/MS analysis were obtained from Sigma-Aldrich (St.
Louis, MO, USA). The derivatization reagent 2,3,4,5,6-penta-
uorobenzyl bromide (PFBBr) was purchased from Sigma-
Aldrich (Steinheim, Germany). The phase transfer catalyst,
tetrabutylammonium hydrogen sulfate (TBAHS), was obtained
from J. T. Baker (Rockford, IL, USA). All organic solvents
(dichloromethane, ethyl acetate, and methanol) were
purchased from J. T. Baker (Phillipsburg, NJ, USA). Laboratory
– distilled water was obtained from Millipore (Billerica, MA,
USA). All other chemicals were of the highest analytical grade
available.
Preparation of standard solutions
Individual SCFA standards were prepared at 100 mg in 0.5 mL
water, taking sodium weight into consideration. The working
standard solution was diluted with methanol at different
concentrations and were used to determine the linearity range.
All of the stock and working solutions were stored at 4  C and
diluted prior to analysis.
Ion-pair extraction and PFB derivatization of SCFAs from fecal
samples
A volume of 40 mL of the standard mixture solution (2 mg mL
1)
was added to 500 mL of distilled water containing 0.1% formic
acid in a 4 mL glass vial prior to derivatization. Then, 500 mL of
the catalyst TBAHS (0.1 M) was added and the pH was adjusted
to 9 using sodium hydroxide (0.5 M). Aer mixing by vortexing
for about 1 min, 2.5 mL of the derivatization reagent (PFBBr)
dissolved in 500 mL of dichloromethane was added to it. The
mixture was heated at 60  C for 20 min, and centrifuged at
3000 rpm for 5 min to separate the organic and aqueous layers.
The organic layer was transferred to another tube and evapo-
rated to dryness under a stream of nitrogen gas. The dried
residue was dissolved in 80 mL of ethyl acetate and 20 mL of the
internal standard phenanthrene-d10 (1 mg mL
1) was added to
the solution.
Preparation of fecal samples
18 rats with weights ranging from 175 to 200 g were provided
by Daehan Biolink (Seoul, Korea). The rats were housed in
cages in a temperature-controlled environment (22  2  C).
The compositions of experimental diets are presented in
Table S2.† The handling of animals and the experimental
procedure were in accordance with the guidelines of Kyung
Hee University, and were approved by the ethical committee
of Kyung Hee University Seoul, Korea. The fecal samples (n ¼
18) were collected into micro-centrifuge vials and stored at

20  C until analysis. The fecal samples (0.1 g) were weighed
and homogenized in 500 mL of 0.1% formic acid spiked with
deuterated propionic acid-d3 in an ice bath. Aer ultrasonic
extraction for 10 min, the samples were centrifuged at
3500 rpm for 10 min. The supernatant was transferred to
a glass vial and the derivatization procedure described in
Section 2.3 was followed. The overall analytical ow chart is
depicted in Fig. 1.
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 View Article Online

Technical Note Analytical Methods

GC-MS/MS equipment SCFAs into SCFA-TBA organic salts, which show good solubility
in organic solvents and can rapidly react with PFBBr to form
An Agilent 7000C mass spectrometer (EI mode, 70 eV) con-
SCFA-PFB derivatives (Fig. 2).
nected to an Agilent 7890B gas chromatograph equipped with
To optimize the designed method, parameters such as the
a DB-5MS capillary column (30 m  0.25 mm i.e., 0.25 mm lm
concentration of TBAHS, pH of the aqueous phase, and deriv-
thickness, J & W Scientic, Folsom, CA, USA) was used for the
atization temperature and time were investigated to achieve
analyses. The samples were introduced via split (ratio 5 : 1)
reactions with high yields. As shown in Fig. 3, a range of TBAHS
injection with the port heated to 250  C. Helium was used as the
concentrations (0.01–0.4 M) was tested to obtain the highest
carrier gas at a ow rate of 1.0 mL min
1. The oven temperature
peak area ratios of SCFAs from fecal samples. The efficacy of the
was initially held at 80  C for 1.0 min, increased to 150  C with
reaction increased proportionally with the concentration of the
a 10  C per min rate, and then raised to 300  C with a 20  C
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catalyst, reaching a maximum yield when 0.1 M TBAHS was

per min rate, where it was held for 5 min. The mass spec-
used (Fig. 3A). However, higher concentrations of TBAHS and
trometer interface temperature was set to 260  C. For moni-
PFBBr could result in an unfavorable reaction yield because the
toring and conrmation analysis, the multiple-ion reaction
intermediate SO4H
species can easily react with PFB to form
monitoring (MRM) mode was used. The manifold temperature
PFB–SO4H during the in situ process. Therefore, concentrations
was maintained at 230  C. The temperatures for the Quad MS1
and MS2 were set at 150  C. For the MS/MS experiments, the of TBAHS and PFBBr were optimized at 0.1 M and 0.5% (2.5 mL
ow rate of nitrogen as the collision gas was set at 1.5 mL of PFBBr dissolved in 500 mL of dichloromethane), respectively.
Alkaline pH in aqueous solutions is important for the depro-
min
1. The MRM conditions were optimized experimentally
tonation of SCFAs. The pH conditions in the range of 6–11 were
and the precursor-product MRM transition ions were selected
tested. As expected, the reaction yield in acidic pH was low,
based on their mass fragmentations (Table S1†).
while the highest yield was obtained at pH 9 [Fig. 3B]. At pH
values higher than 9, the reaction yield was reduced, possibly
Method validation
because of the decomposition of TBAHS.23 Thus, the optimum
Validation of the established method was performed according conditions for an ion-pair reaction, so far, were 500 mL of TBAHS
to the FDA criteria for bioanalytical method validation.21 The (0.1 M) and pH 9. The tested reaction temperatures of PFB
calibration curves were designed in the range of 0.05–40 mg L
1 derivatization were 40, 60, 80, and 100  C. The best yield of
for acetic acid and 0.1–40 mg L
1 for propionic acid and butyric
acid (seven concentration levels, three replicates for each level),
by adding known amounts of the analytes to fecal samples with
a determined SCFA level. The lower limit of quantication
(LLOQ) was considered the lowest calibration standard
concentration. The accuracy of the method was evaluated by
analyzing the spiked working standard solution samples at
three concentration levels. The intermediate precision,
expressed as percent relative standard deviation (% RSD), was
investigated by performing ve independent replicate extrac-
tions of the spiked samples over ve consecutive days. The
matrix effect (ME) was evaluated by comparing the response of Fig. 2 Schematic illustration of the mechanism of extraction and
SCFA standard solutions and the post-extraction spiked derivatization of SCFAs with PFBBr in phase-transfer catalysis with
samples. The unspiked samples were also analysed to account TBAHS.
for the initial amount of each compound present in the sample.
The ME was calculated as follows; ME (%) ¼ (area of SCFAs in
the post-extraction spiked sample – area in the unspiked
sample)/(area of standard solution)  100, according to
a previously reported method.22

Results and discussion

Optimization of selective extraction and PFB derivatization
Generally, SCFAs show relatively high solubility in aqueous
media, while they have low pKa values and low vapor pressure
compared to long chain fatty acids and other organic acids.
Conventional extraction and derivatization could lead, as re-
ported, to loss of SCFAs during the sample workup. In this
study, the in situ extraction and derivatization method for the
analysis of SCFAs in fecal samples used TBAHS and PFBBr. Fig. 3 Optimization of ion-pair extraction and derivatization condi-
TBAHS as the phase transfer catalyst, could readily convert tions of SCFAs.

This journal is © The Royal Society of Chemistry 2017 Anal. Methods, 2017, 9, 2351–2356 | 2353
 View Article Online

Analytical Methods Technical Note

SCFAs was achieved at 60  C [Fig. 3C]. Increasing the tempera-
ture above 60  C was not suitable for dichloromethane due to its
low boiling point. Furthermore, a reaction time over 20 min did
not generate a signicant increment in the detected peak ratios,
as indicated in Fig. 3D. Consequently, the selected conditions
for the in situ extraction and derivatization were 500 mL of
TBAHS (0.1 M) at pH 9 and 60  C with a reaction time of 20 min.
GC-MS/MS analysis of SCFA-PFB derivatives
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The total ion chromatogram of the SCFA spiked water
sample obtained by the MRM mode is illustrated in Fig. S1-B.†
Three SCFA-PFB derivatives together with the internal standard
were clearly and sensitively detected in the extracted ion chro-
matogram of m/z 181. The GC-MS/MS-MRM mode combined
with the described in situ method would offer selective extrac-
tion, reasonable GC retention behavior, and superior sensitivity
and selectivity for the analysis of SCFAs in complex fecal
matrices.

EI-mass spectra of SCFA-PFB derivatives can be characterized by Analytical method validation

the molecular ions and abundant common fragments of m/z
181 [PFBc]+ and m/z 161 [PFB–HF]+ [Fig. S1-A†]. The abundant
PFB ions at m/z 181 and 161 are readily produced by the benzylic
cleavage of each SCFA-PFB derivative due to their aromatic
stability. Thus, these ions were selected in the MRM mode for
quantitative and qualitative analysis of SCFAs. The MRM tran-
sition ions of each compound are summarized in Table S1.†
A LLOQ value of 0.1 mg mL
1 was obtained for both propionic
acid and butyric acid, whereas a LLOQ of 0.05 mg mL
1 was
calculated for acetic acid. At the LLOQ level, the RSD was less
than 15% for all compounds (Table 1). The calibration equa-
tions were calculated by linear least squares regression analysis
using peak area ratios of the analytes to the internal standard.
Excellent linearity was obtained with a correlation coefficient

Table 1 Method validation data of SCFAs obtained by GC-MS/MS-MRM

Accuracy and precision% (n ¼ 5)

Inter day
Intra day (mg mL
1) (mg mL
1)
Linear range Calibration LOD
Compound (mg mL
1) curve R2 (ng mL
1) 0.5 5 20 0.5 5 20

Acetic acid 0.05–40 y ¼ 0.0004x + 0.997 5 100.5  101.6  101.4  102.7  104.9  99.0 
0.6977 10.2 3.6 6.5 3.2 2.8 2.4
Propionic acid 0.1–40 y ¼ 0.0002x + 0.998 21 113.1  95.9  92.4  108.6  102.8  95.2 
0.2523 0.7 2.4 2.9 3.6 5.9 0.7
Butyric acid 0.1–40 y ¼ 0.0002x
0.999 24 94.5  98.8  100.4  98.0  101.3  100.3 
0.0301 6.1 3.3 5.2 4.7 2.8 0.2

Fig. 4 (A) Extracted ion chromatogram and MRM chromatograms and (B) MS spectra of SCFAs extracted from rat fecal samples. Peak identities
as follows: (1) acetic acid–PFB, (2) propionic acid–PFB, (3) butyric acid–PFB, (4–6) isomers of decatrienoic acid (C10:3), (7 and 8) isomers of
undecatrienoic acid (C11:3), and (9) PFB–SO4H.

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Technical Note
(R2) of 0.99 for all analytes. Within-day and between-day coef-
cients of variation ranged from 92.4% to 113.1%, with preci-
sion ranging from 0.2% to 10.2%, indicating excellent method
accuracy and precision.
According to the aforementioned ME equation, the values of
ME were less than 8% for all SCFAs, implying that no signif-
icant ME was observed for analytes. Thus, the established
method could achieve sub-ppb levels of LLOD and highly
selective analysis of SCFAs in fecal samples, which is shown to
be equivalent to or better than other existing methods.10,11,14
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Although the developed method would require a slightly longer
analysis time due to introduction of in situ derivatization than
previous SPME methods,15–17 it could provide reliable quanti-
cation of SCFAs with high precision and accuracy by GC-MS/MS
analysis.
Application of rat fecal samples
The established method was successfully applied to the iden-
tication and quantication of SCFAs in rat fecal samples. The
extracted ion chromatogram (EIC) of m/z 181 in these samples is
illustrated in Fig. 4A. The SCFA-PFB derivatives in complicated
fecal samples were clearly detected without any interfering
peaks in both EIC and MRM chromatograms. Besides SCFAs,
suspected MCFAs (peaks 4–8) extracted from fecal samples were
also observed in the EIC of m/z 181. They were characterized as
PFB derivatives of fatty acid species, based on the presence of m/
z 181 (PFB+), 161 (PFB–HF)+ and their molecular ions in the
mass spectra (Fig. 4B). Peaks 4, 5, and 6 were assumed to be the
isomers of decatrienoic acids (C10:3) which might have double
bonds at different positions. Peaks 7 and 8 were tentatively
characterized as the isomers of undecatrienoic acids (C11:3)
from the presence of a molecular ion at m/z 360 and their
common ion at m/z 181 and 161. Another abundant peak 9 was
always detected in the TICs of fecal samples and was attributed
to PFB–SO4H, formed by ionic interaction between the inter-
mediate SO4H
and PFB+ ions. The EIC of m/z 181 will be
a useful strategy for the proling of SCFAs together with MCFAs
in various types of biological samples.
The developed method was applied to quantify fecal samples
of 18 rats from GC-MS/MS MRM chromatograms. As displayed
in the inset of Fig. 4A, the extracted SCFAs were clearly detected
without any matrix interference in their MRM chromatograms
and were accurately quantied by the peak area ratio of SCFAs
to the internal standard (propionic acid-d3). The concentrations
of SCFAs ranged from 7.7 to 62.5 mmol g
1 for acetic acid, from
0.27 to 2.16 mmol g
1 for propionic acid, and from 0.45 to 12.8
mmol g
1 for butyric acid. Thus, the in situ extraction and
derivatization method could sensitively detect and precisely
quantify SCFAs as well as MCFAs in fecal samples, providing
insight into fatty acid metabolism in biological systems.
Conclusions
The developed in situ extraction and derivatization method
using ion-pair TBAHS and PFBBr reagents offers a simple and
rapid approach for the analysis of the SCFAs in aqueous fecal
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Analytical Methods
samples. By using this method, no signicant loss of SCFAs was
observed during the sample workup. GC-MS/MS combined with
the in situ procedure allowed positive peak identication, and
high sensitivity and selectivity for SCFAs by reducing signicant
interfering peaks from complicated sample matrices. The
established method was proved suitable for reliable quanti-
cation of SCFAs in complex fecal samples by using isotopically
labelled internal standards. Additionally, it could be applied for
the SCFA target and untargeted MCFA analysis in complex
biological samples. In conclusion, this method will be useful for
better understanding the metabolism of fatty acids in various
types of biological samples.
Acknowledgements
This study was nancially supported by the National Research
Foundation of Korea (No. 2014-0012671).
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