A rapid and sensitive profiling of free fatty acids using liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) after chemical derivatization

Article abstract of DOI:10.1039/c6ra01344a

Free fatty acids (FFAs) have diverse roles in cellular energy and signaling and they are critical molecules in various biological states. Due to the poor ionization efficiency of FFAs under electrospray ionization mass spectrometry (ESI-MS) conditions, it is a challenging aspect to construct a robust platform for profiling of various FFAs in biological samples using liquid chromatography ESI-MS. In the present study, we applied trimethylsilyldiazomethane (TMSD) derivatization to improve ionization efficiencies in the profiling of FFAs. Multiple reaction monitoring (MRM) was used for the selective quantification of methylated FFAs. The optimal TMSD methylation was validated for a reliable FFA profiling. Furthermore, the high-throughput analysis of FFAs was successfully performed in short analysis and derivatization times. To verify the utility and effectiveness of the developed method, we compared both methylation and nonmethylation (intact FFA) data in the profiling of FFAs in mice liver and plasma. It is noteworthy that the methylation derivatization provided better results in FFA profiling. Further, we performed statistical data analysis where HBV and mock mice tissues were discriminated when the methylated FFAs data were used. In the lipidomics field, the present method can also be applied for the profiling of FFAs in biological samples for biomarker discovery. The present validated LC/ESI-MS/MS assay method may also be used for FFA profiling modeling studies in other biomedical samples.

Full text of DOI:10.1039/c6ra01344a

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A rapid and sensitive profiling of free fatty acids
using liquid chromatography electrospray
ionization tandem mass spectrometry (LC/ESI-MS/
MS) after chemical derivatization†
Cite this: RSC Adv., 2016, 6, 32130
Hyuck Jun Mok,‡a Jae Won Lee,‡a Raju Bandu,a Hong Seok Kang,b Kyun-Hwan Kimb
a
*
and Kwang Pyo Kim
Free fatty acids (FFAs) have diverse roles in cellular energy and signaling and they are critical molecules in
various biological states. Due to the poor ionization efficiency of FFAs under electrospray ionization mass
spectrometry (ESI-MS) conditions, it is a challenging aspect to construct a robust platform for profiling of
various FFAs in biological samples using liquid chromatography ESI-MS. In the present study, we applied
trimethylsilyldiazomethane (TMSD) derivatization to improve ionization efficiencies in the profiling of
FFAs. Multiple reaction monitoring (MRM) was used for the selective quantification of methylated FFAs.
The optimal TMSD methylation was validated for a reliable FFA profiling. Furthermore, the high-
throughput analysis of FFAs was successfully performed in short analysis and derivatization times. To
verify the utility and effectiveness of the developed method, we compared both methylation and
nonmethylation (intact FFA) data in the profiling of FFAs in mice liver and plasma. It is noteworthy that
the methylation derivatization provided better results in FFA profiling. Further, we performed statistical
data analysis where HBV and mock mice tissues were discriminated when the methylated FFAs data
were used. In the lipidomics field, the present method can also be applied for the profiling of FFAs in
biological samples for biomarker discovery. The present validated LC/ESI-MS/MS assay method may also
be used for FFA profiling modeling studies in other biomedical samples.
Received 16th January 2016
Accepted 20th March 2016
DOI: 10.1039/c6ra01344a
www.rsc.org/advances
(GC) coupled to electron ionization (EI) MS was usually applied
to analyse fatty acid methyl ester (FAME) which is one of the
Introduction
most common fatty acid derivatives.12–14 By using this tech-
nique, the separation of positional and geometrical (cis/trans)
isomers was also achieved in approximately 20 min.12 In addi-
tion to GC-MS, liquid chromatography (LC)-MS has also been
established as an effective analytical technique in the proling
of FFAs with short run times.15 The use of electrospray ioniza-
tion (ESI) MS alone, which is a so ionization technique, only
provides the information of molecular ions. Thus, tandem MS
(MS/MS) is generally applied for the sensitive and selective
analysis of FFAs.16,17 FFAs have been analysed in the negative ion
mode and there is a limitation to the ionization efficiency. In
the previous study, barium acetate was used as the cationization
agent for the sensitive proling of FFAs in the positive ion
multiple reaction monitoring (MRM) mode using triple quad-
rupole mass spectrometer.18 Some researchers, Lee et al., and
Pettinella et al., have demonstrated that trimethylaminoethyl
ester iodide derivatization provided good sensitivity in the
analysis of FFAs.19,20 Zhou et al. has also demonstrated that
isotope-labelling derivatization by using 2,4-dimethoxy-6-
piperazin-1-yl pyrimidine (DMPP) could improve ionization
efficiency for analysing of FFAs.21 Yang et al. derivatized
Free fatty acids (FFAs), categorized as a class of lipids,1 provide
an important energy source as nutrients,2 and they also function
as signalling molecules in various cellular processes including
insulin secretion.3 FFAs are biomedical indicators of the
abnormal lipid metabolism in various metabolic diseases
including diabetes mellitus.4 Several researchers have applied
FFA proling to nd biomarkers for the diagnosis and charac-
terization of various metabolic diseases.5–7 In some studies,
FFAs were also proled to nd the potential biomarkers
of Alzheimer's disease,8 coronary heart disease9 and other
diseases.
Mass spectrometry (MS) has been widely used for the
proling of FFAs in biological samples.10,11 Gas chromatography
aDepartment of Applied Chemistry, The Institute of Natural Science, College of Applied
Science, Kyung Hee University, Yongin, 446-701, Republic of Korea. E-mail: kimkp@
khu.ac.kr
bDepartment of Pharmacology, School of Medicine, Konkuk University, Seoul 143-701,
Republic of Korea
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra01344a
‡ Author contribution: Hyuck Jun Mok and Jae Won Lee contributed equally.
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FFAs with 2-bromo-1-methylpyridinium iodide and 3-carbinol- protocols and fed normal laboratory chow and tap RO water. All
1-methylpyridinium iodide, forming 3-acyloxymethyl-1- mice related experiments were carried out in accordance with
methylpyridinium iodide (AMMP). They observed that the Konkuk University IACUC (Institutional Animal Care and Use
detection sensitivity was approximately 2500-folds higher in Committees). The four week aged female mice (C57BL/6) were
positive-ion mode ionization than negative-ion mode.22 Simi- purchased from Nara biotech co., ltd (Seoul, Korea). Animals
larly, Bollinger et al. have reported a method on conversion of were housed at temperature 23 ꢀ 2 ꢁC, with relative humidity of
FFAs carboxylic acid group in to an amide bearing permanent 50 ꢀ 10% and light controlled environment. To obtain plasma
positive charge, N-(4-aminomethylphenyl)pyridinium (AMPP) samples, mice were anesthetized by intraperitoneal injection of
and that method showed approximately 60 000-folds increased 200 mL of zoletil–xylazine (40 mg mLꢂ1)–DPBS solution
detection sensitivity in positive ion mode ionization when (8 : 2 : 90; v/v) and plasma was collected by cardiac puncture
compared to negative ion mode.23 However, those methods are and stored at ꢂ80 ꢁC. In addition, livers particularly right lateral
complex and time-consuming processes to derivatize FFAs.
lobe were also excised from the same set of mice and washed
In particular, a rapid and sensitive proling method is with PBS to remove the plasma and connective tissue. Aer that
required for the analysis of FFAs in clinical samples. The they blotted dry with lter paper wiper and then stored at ꢂ80
chemical derivatization can signicantly increase the sensitivity ꢁC until analysis.
and specicity of GC-MS and LC/ESI-MS method to analyse
For applying the developed FFAs proling method to
highly acidic compounds such as phosphopeptides, phospho- diseased animal models, we made acute hepatitis B infected
lipids and FFAs.24–29 Particularly, trimethylsilyldiazomethane mice using male BALB/c mice (six weeks old, 18–20 g). The
(TMSD) derivatization method is extremely simple and it was hepatitis B virus infection model of mice and its mock were
applied for analysis of phosphopeptides26 and phospholipids29 used. In this model, HBV (hepatitis B virus) plasmid DNA (pHBV
to improve detection sensitivity. Recently, Lee et al. also 1.2) and control vector (pGEM-4z) were used. A total of 25 mg of
demonstrated TMSD derivatization method that could improve plasmid DNA diluted in PBS which is equivalent to 10% of mice
peak shape and detection limits of many lipid classes.29 In the body were hydrodynamically injected into mice tails. To make
present study, LC/ESI-MS/MS method with TMSD methylation the injection easier, the mice were placed in cylinder-shaped
was used for the proling of FFAs. The utility and effectiveness restraining device to prevent their moving and vasodilate
of the developed method was veried by the proling of FFAs in veins of their tails by using its light-bulb device. The injection
plasma and liver samples.
was performed with high pressure within 5 seconds by using
Liver is a core organ which plays a key role in lipid metab- a 26 G needle with 3 mL syringe as described by Park E. S. et al.35
olism and has various enzymes related to lipid synthesis such as The mice were sacriced 3 days aer hydrodynamic injection
fatty acid synthase (FAS), a key enzyme for FFA.30 Hepatitis B and liver were extracted as described above.
virus can cause various liver diseases such as cirrhosis and
hepatocellular carcinoma.31 It is known that HBV virus disrupts
various lipid synthesis-related enzymes in liver.32–34 Therefore,
Sample preparation
we have applied our developed method to HBV and mock mice Each lipid standard was dissolved in methanol and stored at
tissues to identify FFA changes.
ꢂ20 ꢁC. We compared three common lipid extraction methods
to select the better one for analysing FFAs.
First, we applied Bligh and Dyer method which is the most
common lipid extraction method.36 In this method, a 750 mL of
chloroform/methanol (1 : 2; v/v) was added to 50 mL of plasma
samples and 50 mg of liver tissues which were taken in different
Eppendorf tubes (1.5 mL). Next, a 10 mL of arachidonic acid-d8
(internal standard (IS)) solution was added to above plasma and
tissue samples at a concentration of 10 ng mLꢂ1. In the case of
liver tissues, an additional step of homogenization was per-
formed aer the addition of chloroform and methanol. Aer
vortexing for 1 min, samples were incubated in ice for 10 min.
Aer that a 250 mL of chloroform and 450 mL of water were
added to both plasma and tissue sample tubes. Aer the
Experimental
Materials
HPLC-grade acetonitrile, methanol, water and isopropanol were
purchased from J.T. Baker (Avantor Performance Material, Inc,
PA, USA). Trimethylsilyldiazomethane (TMSD) reagent was
purchased from Sigma-Aldrich, St. Louis, MO, USA. The FFAs,
palmitic acid (16:0), oleic acid (18:1) and arachidonic acid (20:4)
were purchased from Sigma-Aldrich St. Louis, MO, USA. The
FFA standard, arachidonic acid-d8 (20:4-d8) was purchased
from Cayman chemical, Ann Arbor, MI. The 26 G needle with 3
mL syringes were purchased from KOVAX syringe, Korea
vaccine CO, Seoul, Korea. Zoletil and xylazine were purchased
from Virbac S.A, France and Sigma-Aldrich Co. LLC., USA,
respectively. Dulbecco's phosphate-buffered saline (DPBS) was
purchased from Thermo Fisher Scientic Inc., USA.
ꢁ
centrifugation (13 800 ꢃ g, 2 min at 4 C), the organic phases
were collected into different Eppendorf tubes.
Secondly, we performed extremely simple lipid extraction
method where we used only methanol as extraction solvent. For
about 50 mL of plasma sample, 10 mL of IS followed by 1 mL of
ice cold methanol was added. Aer that, the sample mixture was
vortexed for 30 s and incubated for 10 min in ice. Aer centri-
Animal samples
All mice used for our study are maintained in Konkuk University fugation (10 000 ꢃ g, 5 min at 4 ꢁC), about 1 mL of supernatant
of Seoul, Republic of Korea according to standard animal care was collected into another Eppendorf tube. In the case of liver
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Table 1 Optimized MRM transitions for profiling of free fatty acids
Table 1 (Contd.)
MRM
transitions
MRM
transitions
Species
Ion mode Q1 m/z Q3 m/z CE (eV)
Species
Ion mode Q1 m/z Q3 m/z CE (eV)
Intact FFAs
C8:0
C10:0
C12:0
C14:0
C14:1D9
C16:0
C16:1D9
C18:0
C18:1n9
C18:2n6
C18:3n3
C18:3n6
C18:4
Methylated-C22:6n3
Methylated-C24:0
Methylated-C24:1D15 or n9
Positive
Positive
Positive
343
383
381
311
351
349
10
10
10
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
143
171
199
227
225
255
253
283
281
279
277
277
275
311
309
307
305
303
311
301
339
337
335
333
331
329
327
367
365
99
127
155
183
181
211
209
239
237
235
233
233
231
267
265
263
261
259
267
257
295
293
291
289
287
285
283
323
321
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
ꢂ8
a
Internal standard (IS).
tissue, approximately 50 mg of tissue was homogenized in 1 mL
of ice cold methanol and 10 mL of IS solution was added to it.
Further, tissue samples were mixed for 30 s and incubated for
10 min in ice. The centrifugation was performed at 10 000 ꢃ g
ꢁ
for 5 min at 4 C and the supernatants were collected in to 1.5
C20:0
mL collection tubes.37
C20:1D11
C20:2D11,14
C20:3n3 or n6
C20:4n6
(d8) C20:4n6 Isa
C20:5n3
C22:0
C22:1n9
C22:2D13,16 or n6
C22:3
Lastly, Dole's extraction method was used for FFA analysis in
plasma and liver tissues.38 Plasma and liver tissues were mixed
separately with 500 mL of the Dole's mixture (methanol, n-
hexane, phosphoric acid (2 mol Lꢂ1), 20 : 10 : 1 (v/v)) and IS
solution. An additional step of homogenization was performed
in case of liver tissues, as described above in the context of Bligh
& Dyer and methanol extraction methods. Aer vortexing,
samples were incubated for 10 min at room temperature. Then
300 mL of water and 200 mL of n-hexane were added and
centrifuged for 5 min at 13 000 rpm. The organic supernatants
were collected into 1.5 mL tubes. All extracted FFA samples were
divided into two; one of them was used for negative ion mode
MS analysis and another one was used for TMSD methylation.
All samples were stored at ꢂ80 ꢁC before analysis. For TMSD
methylation, the samples were dried in SpeedVac concentrator
and reconstituted with 100 mL methanol.
C22:4n6
C22:5n3
C22:6n3
C24:0
C24:1D15 or n9
Methylated FFAs
Methylated-C8:0
Methylated-C10:0
Methylated-C12:0
Methylated-C14:0
Methylated-C14:1D9
Methylated-C16:0
Methylated-C16:1D9
Methylated-C18:0
Methylated-C18:1n9
Methylated-C18:2n6
Methylated-C18:3n3 or n6
Methylated-C18:4
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
159
187
215
243
241
271
269
299
297
295
293
291
327
325
323
321
321
319
327
317
355
353
351
349
347
345
127
155
183
211
209
239
237
267
265
263
261
259
295
293
291
289
289
287
295
285
323
321
319
317
315
313
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
TMSD methylation
A simple and rapid TMSD methylation derivatization method
was used for the analysis of FFAs in the plasma and liver
samples. A solution of TMSD (2 mol Lꢂ1) in hexane (50 mL) was
mixed in equal proportions with sample and standard (v/v).
ꢁ
Aer vortexing for 30 s, mixture was incubated at 30 C for 10
min under optimized conditions which has been described in
the succeeding text. For quenching the derivatization reaction,
glacial acetic acid (6 mL) was added as described previously.29
Methylated-C20:0
Methylated-C20:1D11
Methylated-C20:2D11,14
Methylated-C20:3n3
Methylated-C20:3n6
Methylated-C20:4n6
Methylated-(d8) C20:4n6 Isa
Methylated-C20:5n3
Methylated-C22:0
Methylated-C22:1n9
Methylated-C22:2D13,16 or n6 Positive
Methylated-C22:3
Methylated-C22:4n6
Methylated-C22:5n3
LC-ESI/MS/MS equipment and conditions
The HPLC analysis was performed on an Agilent 1290 innity
series HPLC instrument (Agilent Technologies, USA) equipped
with binary pump (G4220A, USA), an autosampler (G4226A,
USA), a column compartment (G1316C, USA) and a thermostat
(G1330B, USA). The temperaturꢁe of column oven and auto-
Positive
Positive
Positive
ꢁ
sampler was set at 40 C and 4 C, respectively. For the sepa-
ration of FFAs, Hypersil GOLD column (2.1 ꢃ 100 mm ID; 1.9
mm Thermo scientic) was used. The mobile phase solvent A
consisted of a acetonitrile/methanol/water mixture (19 : 19 : 2)
with 0.1% (v/v) formic acid and 20 mmol Lꢂ1 ammonium
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formate; and the mobile phase solvent B consisted of iso- of 15 Da to their intact molecular weights which corresponds to
propanol with 0.1% (v/v) formic acid and 20 mmol Lꢂ1 ammo- the addition of methyl group (Fig. 1). The fragmentation pattern
nium formate. The ow rate of the mobile phase was 0.25 mL of methylated and intact FFAs were conrmed by using tandem
minꢂ1 and the injection volume was 3 mL. A 20 min lipid elution MS analysis. In the negative ion mode, intact FFAs showed
gradient was performed as follows: rst 10 min, solvent similar fragmentation behaviour that was reported earlier by
composition was set at 95% A and 5% B to elute FFAs; followed Nagy et al., and other researchers.15,39–42 The diagnostic product
by a linear gradient to solvent 90% A and 10% B for 2 min and ion observed in the negative ion mode MS spectra of FFAs is [M
kept for 3 min for elution of other lipids. Finally, the column ꢂ H-44]ꢂ which corresponds to the neutral loss of CO2. On the
was equilibrated at 5% solvent B for 5 min before reuse.
other hand, the positive ion mode analysis of methylated FFAs
LC-MS analysis was performed on a triple quadrupole mass showed [M + H-32]+ ion as the base peak which corresponds to
spectrometer (QQQ LC-MS 6490 series, Agilent Technologies, the loss of CH3OH. The peak corresponds to the loss CH3OH
USA) equipped with an ESI source which provides high sensi- was selected as Q3 transition in the MRM analysis of all FFAs
tivity by iFunnel technology that consists of three components: (Fig. 1). For the proling of various FFAs, we xed the MRM
a hexabore capillary, Agilent Jet Stream technology, and a dual transitions of each species based on their abundant character-
ion funnel. The typical operating source conditions for MS istic product ions of [M ꢂ H ꢂ CO2]ꢂ and [M + H ꢂ CH3OH]+ for
scan in the positive and negative ion ESI mode were optimized intact and methylated FFAs, respectively.
as follows: capillary voltage 4000 V, nozzle voltage 500 V. The
Further, we also optimized the tested time (10, 15, 20, 25 and
nebulizer was set at 40 psig and the nitrogen drying gas was set 30 min) and temperatures (10, 20, 30, 40, and 50 ꢁC) for TMSD
at a ow rate of 13 L minꢂ1 and the temperature was main- methylation of FFAs. In these experiments, we analysed FFA
tained at 250 ꢁC. For collision-induced dissociation (CID) standards, C16:0 (palmitic acid-saturated FFA), C18:1 (oleic
experiments, the precursor ion of each FFA species was acid-monounsaturated FFA) and C20:4 (arachidonic acid-
selected using the quadrupole analyser and the product ions polyunsaturated FFA) with different derivatization time and
were analysed using another quadrupole analyser. Ultra-pure temperatures. The arachidonic acid-d8 standard was used as an
nitrogen was used as collision gas. The collision energies for IS for this analysis. According to different derivatization time
methylated and nonmethylated FFAs (intact FFA) were also and temperature, the efficiency of methylation was estimated by
optimized by using FFA standards (8 eV for intact FFAs and 10 the peak area of FFA standards normalized by IS. 10 min of
eV for methylated FFAs). All the spectra of FFAs were recorded derivatization time was enough for methylation of FFAs and 30
under optimized experimental conditions and the quantitative ꢁC of reaction temperature showed the best efficiency for the
analysis was performed in multiple reaction monitoring derivatization (Table 2). Thus, 10 min and 30 ꢁC were selected as
(MRM) mode using computed transitions for methylated and the optimized conditions for methylation of FFAs.
intact FFAs (Table 1).
The derivatization efficiency of TMSD methylation for FFA
proling is given in Table 3. The methylated and intact FFAs
showed difference in ionization efficiency which may presum-
ably be due to their structural differences. To evaluate the effi-
ciency of TMSD methylation, we compared the peak areas of
intact FFAs in both methylated and nonmethylated mixtures.
The TMSD methylation efficiency of each FFA was described as
follows: the percentage of methylated FFAs ((peak area of
compound in nonmethylated FFAs ꢂ peak area of non-
methylated compound in methylated FFAs)/peak area of
a compound in a nonmethylated FFAs ꢃ 100 (%)) (Table 3). All
FFA standards showed high TMSD derivatization efficiency as
shown in Table 3 (the lowest efficiency was 82.9%).
Result and discussion
FFA proling by LC/MS/MS with TMSD methylation
In this study, TMSD methylation was applied to increase the
sensitivity of FFA analysis. To the best of our knowledge, this is
the rst attempt to analyse FFAs in plasma and liver samples
with TMSD methylation by LC/MS/MS. Thus, we established the
MRM transition (m/z value of precursor ion (Q1) > m/z value of
product ion (Q3)) for the proling of methylated FFAs. The ESI-
MS conditions were also optimized in the analysis of methyl-
ated FFA standards (palmitic acid-saturated FFA, oleic acid-
monounsaturated FFA and arachidonic acid-polyunsaturated
FFA). The methylated FFAs were analysed as [M + H]+ ions in
Validation study
positive ion mode whereas intact FFAs were predominately The performance of TMSD methylation was validated to
analysed as [M ꢂ H]ꢂ ions in negative ion mode. The m/z value conrm whether the methylation is applicable to FFA proling
of the molecular ions of methylated FFAs showed the mass shi in biological samples (Table 4). First, the reproducibility of
Fig. 1 Formation of TMSD derivative of arachidonic acid (C20:4) and its proposed fragmentation pattern.
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Table 3 Methylation efficiency of fatty acid standards
The percentage of
Species
methylated compoundsa (%)
Palmitic acid
Oleic acid
Arachidonic acid
96.15 ꢀ 5.93b
82.90 ꢀ 5.69
99.95 ꢀ 0.01
a
((Peak area of compound in nonmethylated FFAs ꢂ peak area of
nonmethylated compound in methylated FFAs)/peak area of
b
a compound in a nonmethylated FFAs) ꢃ 100 (%). The values of
percentages are in mean ꢀ SD (n ¼ 6).
methylation was estimated by calculating the relative standard
deviation (% RSD) of 1 ng of FFA standards in intra- and inter-
day experiments. In the analysis of four FFA standards, the %
RSDs of the retention time and peak areas showed less than
15.1% and 1.6% RSD in both intra- and inter-day studies,
respectively (Table 4). Second, the sensitivity of the FFA
proling with and without methylation were also compared.
The limitation of detection (LOD) and coefficient (R2) were
calculated in terms of methylated and nonmethylated condi-
tions. As a result, both methylated and intact FFA standards
showed good coefficient (higher than 0.975). However, in the
case of LOD, methylated standards showed better sensitivity
than nonmethylated standards. It can be that noted that the
intact oleic acid showed 25 fold higher LOD than the corre-
sponding methylated standard (Table 4). Furthermore, sensi-
tivity was signicantly increased in case of palmitic acid and
oleic acid standards (Fig. 2). Even though the intact arachidonic
acid showed same LOD and sensitivity as similar to its meth-
ylated form, its average peak area was smaller than TMSD
(Fig. 2). The results of lower LOD, good coefficient and low %
RSD of inter and intraday variation supported the reproduc-
ibility and reliability of our developed method. The matrix effect
has been evaluated, where the matrix factor was ranged from
0.86–1.05. These data indicate that there was no signicant
endogenous interference. The stabilities of samples were within
the limits and the mean percentage changes were less than
ꢀ10% from their nominal concentrations. Our results indicate
that the samples were stable at 4 ꢁC for 24 h in autosampler at
ꢁ
ꢂ80 C for 30 days. The samples were stable at room tempera-
ture for 6 h.
Comparison of extraction methods of FFA
For the optimization of extraction method, we compared three
common lipid extraction methods (Method 1: Bligh and dyer;
Method 2: extraction with methanol; Method 3: Dole's proce-
dure) with liver sample from C57BL/6 mice. A 50 mg each of
liver tissue was used for each experiment to compare the
extraction efficiency. As shown in Fig. 3, the overall area of
FFAs extracted from methods 1 and 2 showed higher value
than method 3. When we compared the extraction methods 1
and 2, they showed similar results in terms of the number of
identied FFAs (14 FFAs in both methods) and relative area
(area of FFA/area of IS) of FFAs. As these extraction methods
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Table 4 Data of validation of methylated fatty acids and LODs
Intraday
Interday
variationc
variationb
Coefficient (R2)
LOD (ng)
Intact
Linearity
range (ng)
Species
RTa (min)
RT
Peak area
RT
Peak area
Intact
Methylated
Methylated
Palmitic acid
Oleic acid
Arachidonic acid
(d8) arachidonic acid-IS
1.59
2.8
2.15
2.09
1.6
0.2
0.4
0.9
5.6
5
4.6
1.7
1.1
0.4
0.4
0.6
15.1
7.1
4.8
15
0.9931
0.9819
0.9909
0.991
0.9869
0.9927
0.9943
0.975
1600
1000
100
800
40
100
100
800–25 000
40–25 000
100–100 000
100–100 000
100
a
Average of retention time (n ¼ 9). b % RSD (n ¼ 9) of intraday variation. c % RSD (n ¼ 27) of interday variation (3 days).
Fig. 2 Improved sensitivity of FFA standards by TMSD methylation. Panel (A) MRM chromatogram of intact FFA standards (C16:0 (palmitic acid-
saturated FFA), C18:1 (oleic acid-monounsaturated FFA) and C20:4 (arachidonic acid-polyunsaturated FFA)); Panel (B) MRM chromatograms of
methylated FFAs where they showed different RT due to methylation (addition of methyl group to compound).
Fig. 3 Comparison among three common FFA extraction methods (Method 1: Bligh & dyer; Method 2: extraction using methanol; Method 3:
Dole's mixture).
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are very complex and tedious time consuming processes which FFAs in mice liver and plasma were detected (Table 5). It is
may cause the experimental handling error, we moved to the noteworthy that ve methylated FFAs (C14:1, 18:1, 18:2, 18:3,
simple extraction method which involves methanol as extrac- and 18:4) were additionally analysed in mice plasma when
tion solvent. Finally, methanol extraction method was selected compared to intact FFAs. Furthermore, ten FFAs (C16:1, 18:0,
due to its simplicity and good performance (Fig. 3).
18:1, 18:2, 18:3, 18:4, 20:1, 20:2, 22:2, and 22:3) were only
detected by TMSD methylation in mice liver. The average
relative areas of methylated FFAs are much higher than the
intact FFAs (Fig. 4 and Table 5). All the detected FFAs were also
validated by tandem mass spectrometric experiments (Fig. 5
and S1†).
Application of TMSD methylation for proling of FFAs in mice
liver and plasma
In order to validate the utility of TMSD methylation for the
proling of FFAs in biological samples, we applied our vali-
dated method for analysing mice liver and plasma (C57BL/6
mice). The arachidonic acid-d8 (20:4) was used as the IS for
the normalization of each FFA species. The relative area (area
Application of TMSD methylation in the FFA proling in HBV
infected mice
of FFA/area of IS) of FFA species was characterized for the We also applied the developed method for proling of FFAs in
detailed phenotype of mice samples. Each sample (plasma and three samples (HBV infected samples and its mock mice liver
tissues) was analysed four times (n ¼ 4). Furthermore, the tissue). In these experiments, each tissue was analysed four
results of FFA proling with and without methylation were times (n ¼ 4). The peak area of individual FFA species was
compared. Compared to intact FFAs, the RT of methylated divided by internal standard for normalization and relative
FFAs was slightly delayed due to the formation of methyl ester quantication.29,43 The normalized FFA data of mice samples
structure (Fig. 2 and 4). It can be noted that the methylated were subjected to principal component analysis (PCA) and
FFAs were eluted within 5 min even though their RTs were hierarchical clustering analysis (HCA) (heat map, dendro gram)
delayed. Thus, it is appropriate to perform the high- which were performed through the web-based data analysis
throughput proling of FFAs. Upon methylation, 11 FFAs in soware, MetaboAnalyst 3.0. In addition, the uses of FFA
mice plasma and 16 FFAs in mice liver were successfully proling with and without methylation were also compared
analysed. On the other hand, in the case of intact FFAs, six in this study. When the data used that was obtained by
Fig. 4 Comparison between intact and methylated FFAs in liver tissues. Panel (A) MRM chromatograms of intact FFAs, C18:1, C18:2, C18:3 and
C18:4 in liver (no FFAs were detected). Panel (B) The MRM chromatograms of methylated FFAs (the data indicated that the sensitivity was
significantly increased after methylation derivatization).
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Table 5 Fatty acid species analysed in mice liver and plasma by QqQ with and without methylation
RTa (min)
Intact
Liverb
Intact
Plasmab
Intact
Species
Methylated
Methylated
Methylated
C8:0
C10:0
C12:0
C14:0
C14:1D9
C16:0
C16:1D9
C18:0
C18:1n9
C18:2n6
C18:3n3 or n6
C18:4
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
2.2
1.9
1.7
—
—
—
—
2.2
1.9
1.7
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1.6
3
2.3
3.7
3
2.5
2.1
1.4
—
3.9
3.1
2.6
2.3
1.8
—
0.48 ꢀ 0.05c
—
—
—
1.09 ꢀ 0.12
0.09 ꢀ 0.10
17.7 ꢀ 1.65
8.44 ꢀ 0.82
0.68 ꢀ 0.10
0.91 ꢀ 0.11
—
1.13 ꢀ 0.09
1.54 ꢀ 0.21
10.5 ꢀ 0.94
21.5 ꢀ 2.39
2.26 ꢀ 0.18
—
1.47 ꢀ 0.22
0.72 ꢀ 0.13
0.23 ꢀ 0.04
1.10 ꢀ 0.15
C20:0
—
C20:1D11
C20:2D11,14
C20:3n3 or n6
C20:4n6
C20:5n3
C22:0
C22:1n9
C22:2D13,16 or n6
C22:3
C22:4n6
C22:5n3
C22:6n3
C24:0
—
—
0.26 ꢀ 0.01
0.01 ꢀ 0.00
0.19 ꢀ 0.04
22.2 ꢀ 1.42
1.03 ꢀ 0.01
0.15 ꢀ 0.04
5.43 ꢀ 0.25
0.16 ꢀ 0.01
23.0 ꢀ 3.07
—
—
—
—
4
3.3
2.7
2.4
2
—
—
—
—
—
—
—
—
—
—
0.14 ꢀ 0.03
0.41 ꢀ 0.05
1.54 ꢀ 0.20
4.28 ꢀ 0.44
21.7 ꢀ 2.32
—
0.19 ꢀ 0.02
3.40 ꢀ 0.22
52.7 ꢀ 1.98
—
0.02 ꢀ 0.00
0.30 ꢀ 0.01
2.59 ꢀ 0.11
—
0.20 ꢀ 0.00
6.26 ꢀ 1.10
1.55 ꢀ 0.24
—
—
—
C24:1D15 or n9
—
—
—
—
a
Average of retention time. b Average of relative peak area (compound/internal standard) ꢀ S.D (n ¼ 4). c The values of percentages are in mean ꢀ
SD (n ¼ 4).
nonmethylation, HBV and mock mice tissues were not well signicantly changed FFAs (C16, C16:1, C20:1, C20:2, C20:3,
separated in the PCA score plot and HCA (Fig. 6A). On the other C20:4, C20:5, C22:1, C22:3, C22:4 and C22:5; P-value < 0.05) in
hand, HBV and mock mice tissues were clearly distinguished methylated conditions. These results indicated that the use of
when the data of methylated FFAs was used in the statistical methylation can provide the comprehensive information of FFA
analysis (Fig. 6B). As it can be seen from Fig. 6B, many FFAs proles with higher intensity and more number of identica-
were decreased in HBV induced mice and nally we found some tions than the nonmethylation.
Fig. 5 Tandem mass spectra of representative methylated C18:1 FFA (the m/z 297 ([M + H]+) ion corresponds to methylated C18:1 FFA (oleic
acid) in positive ion mode).
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Fig. 6 Principal component analysis (PCA) and hierarchical clustering analysis (heat map, dendro gram) of (A) intact and (B) methylated FFAs data
in mock and HBV mice (triplicate MS run).
supported by Proteogenomic Research Program through the
National Research Foundation of Korea funded by the
Conclusions
Korean Ministry of Education, Science and Technology (to K.
P. K.), Korea and supported by Proteogenomic Research
Program.
In this study, the LC/MS/MS with simple and rapid methylation
method was used for the proling of FFAs. The proposed method
is simple for proling of methylated and various other FFA
species within a short analysis time (5 min) and derivatization
time (10 min). We observed that our method has high derivati-
zation efficiency aer the optimization of TMSD methylation by
using FFA standards (palmitic acid-saturated FFA, oleic acid-
monounsaturated FFA, arachidonic acid-polyunsaturated FFA).
The results also revealed that methylated FFAs showed higher
detection sensitivity, reproducibility and reliability in the positive
ion mode than intact FFAs in the negative ion mode. The data
showed that our methylation method provided in detail infor-
mation of FFAs with higher intensity and more number of iden-
tications than the nonmethylation. Besides, we also applied the
developed method for proling of FFAs in animal models. The
utility of FFA proling with methylation was also proved by PCA
and HCA of HCV infected and control liver tissues. In the lip-
idomics, this method can be used for analysing even low abun-
dant FFA species qualitatively and quantitatively. This method
can also be applied to many biological and clinical samples to
discover FFA biomarker. Furthermore, this study also explained
the utility and effectiveness of methylation derivatization in
proling of FFAs in biological samples (mice liver and plasma).
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