June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Research Paper

Analyses of the fatty acid separation principle
using liquid chromatography-mass spectrometry
Shigeo Takashima*, Kayoko Toyoshi, Nobuyuki Shimozawa
Division of Genomics Research, Life Science Research Center, Gifu University
Abstract

Fatty acids vary in their hydrocarbon chain length and the number and position of the double bonds. In thisstudy, we investigated the principle of the separation of fatty acids by reverse-phase liquid chromatography coupled withnegative electrospray ionization mass spectrometry (LC-negative ESI-MS). In particular, we aimed to detect very-longchain fatty acids (VLCFAs) and to distinguish between isomeric unsaturated fatty acids differing in their double bond positions. We found that both saturated and unsaturated fatty acids are not separated by regular chromatography but instead
separated by sequential detachment from the column s stationary phase, according to the strength of the organic mobile
phase. The interaction of the fatty acids with the stationary phase during chromatographic migration had a minor effecton their separation. Unsaturated fatty acid isomers with different double bond positions were found to be separable, andtheir elution order was determined by the position of the double bonds relative to the omega carbon. We also found thatﬁbroblasts from the patients with peroxisomal disease show characteristic distribution patterns for some fatty acid isomers
in the proposed separation method. Our method is, thus, useful for analyzing a wide range of fatty acid species, including
VLCFAs and unsaturated fatty acid isomers, to understand the physiological changes in fatty acid metabolism from clinicalsamples.
Key words: fatty acid, very-long-chain fatty acid, separation principle, liquid chromatography-mass spectrometry, peroxisomal
diseasevery-long-chain fatty acids (VLCFAs; fatty acids with 22 orIntroduction

more carbon atom) with carbon numbers of up to 32 andFatty acids are essential components of living organisms.

3‒5)

They form the structure of the cellular membrane, act as
signaling molecules (for example, eicosanoids and lysolip-

longer existids), and serve as cellular energy sources. Fatty acids arefunctional metabolic organelles, the peroxisomes, accumu-

classiﬁed by multiple chemical characteristics, such as thelate VLCFAs in their tissues

hydrocarbon chain length, the number of double bonds, andin saturated fatty acids (SFAs), one or two in monounsatu-

the position of the double bonds. In humans, fatty acids

rated fatty acids (MUFAs) or diunsaturated fatty acids

with a chain length of 16 to 22 carbon atoms are the most

(DUFAs), respectively, and more in polyunsaturated fatty

1,2)

common. Moreover, patients with speciﬁc types ofperoxisomal diseases, a group of diseases caused by dys6,7). In some organs such as the brain and the retina,. There are no double bonds

acids (PUFAs). Unsaturated fatty acids are further classiﬁedby the ﬁrst double bond position counted from the terminal
methyl group (omega carbon), for example, ω-3, ω-6, and*Corresponding author
Shigeo Takashima

ω-9. Distinguished by such chemical features, fatty acids

Division of Genomics Research, Life Science Research Center,
Gifu University, 1‒1 Yanagido, Gifu, Gifu 501‒1193, Japan

diverge into a number of species, and their physiological
functions vary with their chemical characteristics.

Tel: 058‒293‒3174, Fax: 058‒293‒3172
E-mail: staka@gifu-u.ac.jp

To identify fatty acid species in biological samples, gas
chromatography-mass spectrometry (GC-MS) and liquid

Received November 27, 2017. Accepted January 18, 2018.

chromatography-mass spectrometry (LC-MS) are commonly used. In a GC-MS assay, fatty acids in biological

Epub April 20, 2018.
DOI: 10.24508/mms.2018.06.002
21

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Medical Mass Spectrometry Vol. 2 No. 1

samples are extracted from lipids by hydrolysis and meth8)
yl-esteriﬁed for better detection . GC-MS requires theacid; Sigma-Aldrich, #85679), nonadecanoic acid (C19:0,
nonadecylic acid; Sigma-Aldrich, #72332), eicosanoic acidvaporization of analytical samples, so it is reliable andapplicable for volatile short- to long-chain fatty acids; how-

(C20:0, arachidic acid; Sigma-Aldrich, #A-3631), docosanoic acid (C22:0, behenic acid; Tokyo Chemical Industry/
TCI, Tokyo, Japan, #B1747), tetracosanoic acid (C24:0, lig-

ever, the application of GC-MS to VLCFAs, especially saturated ones, is limited by their poor vaporization capacity
9,10)
and detectability . The double bond position is also iden-noceric acid; TCI, #T0076), pentacosanoic acid (C25:0,

tiﬁable with GC-MS. For this purpose, unsaturated fatty(C26:0, cerotic acid; TCI, #C0829), octacosanoic acid
(C28:0, montanic acid; Sigma, #284432), cis-2-hexadece-

pentacosylic acid; TCI, #P0882), hexacosanoic acidacids are derivatized with dimethyl disulﬁde or pyrrolidine,

noic acid (C16:1 ω-14; TCI, #H0428), cis-6-hexadecenoic
acid (C16:1 ω-10, sapienic acid; Abcam, Cambridge,

ionized, and fragmented by electron ionization; then, thedouble bond position is determined from the characteristic
MS spectra. LC-MS complements GC-MS for fatty acid

United Kingdom, #ab143911), cis-9-hexadecenoic acid
(C16:1 ω-7, palmitoleic acid; Cayman Chemical, Annanalysis. The extracted fatty acids are analyzed without
11‒13). For
modiﬁcation or often derivatized in LC-MS assay

Arbor, MI, USA, #10009871), cis-6-octadecenoic acid
(C18:1 ω-12, petroselinic acid; Sigma-Aldrich, #P8750),

lipidomic analysis, lipids are directly examined, and theconstituent fatty acids are identiﬁed by the collision-in-

cis-9-octadecenoic acid (C18:1 ω-9, oleic acid; Cayman
Chemical, # 90260), cis-11-octadecenoic acid (C18:1 ω-7,

2,14)
duced dissociation (CID)-mediated lipid fragmentation .
Although LC-MS can distinguish different fatty acid spe-

cis-vaccenic acid; Matreya LLC, State College, PA, USA,#1266), cis,cis,cis-9,12,15-octadecatrienoic acid (C18:3

cies according to the chain length and the double bond

ω-3, α -linolenic acid; Cayman Chemical, #90210), cis,cis,cis-6,9,12-octadecatrienoic acid (C18:3 ω-6, γ -linolenic

number, the determination of the double bond position isstill challenging. A method that is applicable to a wideacid; Cayman Chemical, #90220), cis,cis,cis-5,11,14-eicosatrienoic acid (20:3 ω-6, sciadonic acid; Cayman Chemi-

range of fatty acid species and distinguishes the chemical
speciﬁcities would be useful to understand the entire range

cal, #10009999), cis,cis,cis-8,11,14-eicosatrienoic acid
(20:3 ω-6, dihomo- γ -linolenic acid; Cayman Chemical,

of fatty acids in biological samples.
Previously, we reported a method for analyzing a series#90230), high-performance liquid chromatography (HPLC)
grade tert-butyl methyl ether (t-BME, Sigma-Aldrich,of fatty acids and related molecules in human ﬁbroblast and
10)serum samples . In the previous report, a reverse phase
LC conjugated with a negative electrospray ionization-MS#34875), HPLC grade acetonitrile (Wako, Osaka, Japan,#019-08631), HPLC grade acetone (Wako, #014-08681),(LC-negative ESI-MS) was used, and fatty acids with chain

HPLC grade 1 mM ammonium acetate (Wako, #01821041), and 10% ammonia solution (Wako, #013-17505).

length ranging from 14 (C14) to 44 carbons (C44) with adouble bond number of up to 13 (e.g., C44:13) were
because the ionization efﬁciency is higher for VLCFAs

The fatty acid compounds were dissolved with two volumes of chloroform and one volume of methanol contain-

than for shorter fatty acids. In this study, we investigated
the separation principle for the fatty acids on reverse-phaseing 0.05% 2,6-di-t-butyl-p-cresol (butylated hydroxytoluene,
BHT; Nacalai Tesque, Kyoto, Japan, #11421-92) and stored

LC by comparing several types of LC columns andat −20°C until use.

detected. This method is ideal for analyzing VLCFAs

expanded the application of the technique to distinguish the

Liquid chromatography-mass spectrometry

double bond positions in the unsaturated fatty acids.

For all analyses, we used a Waters Acquity ultra-perfor-

Materials and Methods

mance liquid chromatography (UPLC) system, which was

Reagents

equipped with an autosampler/injector and XevoQTof mass

The following fatty acid standards and other chemical

spectrometer. For LC, aqueous mobile phase A was puriﬁedreagents were used in this study: tetradecanoic acid (C14:0,
common name: myristic acid; Sigma-Aldrich, St. Louis,

water containing 1 mM (0.0077% w/v) ammonium acetate

MO; #70079), hexadecanoic acid (C16:0, palmitic acid;
Sigma-Aldrich, #76119), octadecanoic acid (C18:0, stearic

phase B was acetonitrile. Linear gradient conditions withascending mobile phase B were applied, as shown in Sup-and 5.78 mM (0.01% w/v) ammonia. The organic mobile

22June 2018

Medical Mass Spectrometry Vol. 2 No. 1

plementary Fig. S1. The ﬂow rate was set to 0.4 mL/min or

L of 5 M hydrochloric acid in a glass tube and lysed by vig-

0.2 mL/min, and the column temperature was maintained at
40°
C. The injection volume was 5 μL. After LC, the ana-

orous vortexing for 1 min. The cell lysate was incubated at
100°
C in an oil bath for 1 h. After bringing the lysate toroom temperature, 800 μL of t-butyl methyl ether (t-BME),

lytes were ionized by negative-ion electrospray ionization(negative ESI) and mass spectra were obtained by the time-

100 μL of methanol containing C19:0 (as an internal stan-of-ﬂight MS. The scan range was set to 100‒1000 m/z. The
setting for the MS system was as follows: capillary voltage,

dard; 1 μg/mL), and 400 μL of puriﬁed water were added,
and the sample was vortexed again for 1 min. After centrifu-

1.0 kV; sampling cone, 56 (arbitrary value); extraction

gation at 200×g for 5 min, the upper phase was collected,cone, 4.0 (arbitrary value); source temperature, 125°C;

800 μL of water was added, and the sample was vortexed

desolvation temperature, 350°
C; cone gas ﬂow, 60 L/h; andagain. After phase separation by 5 min centrifugation, theupper organic phase (t-BME) containing fatty acids wasdesolvation gas ﬂow, 1000 L/h. The data were analyzed by
prepared by Microsoft Excel. ACD ChemSketch was usedcollected in a glass sample vial with a polytetraﬂuoroethylene (PTFE)-lined cap (GL Sciences, Tokyo, Japan,

to draw chemical structural formula.

#1030‒51023 and #1030-45260). The organic solvent was

Masslynx and Quanlynx software (Waters) and graphs were

evaporated under a stream of nitrogen gas. The fatty acids

LC columns

were reconstituted in 100 μL of acetone and used for
LC-MS analysis.

The following columns (Waters Acquity UPLC columns)
were used: BEH (Ethylene Bridged Hybrid) C18 (2.1×100 mm,particlesize

1.7 μm,

pore

size

Double bond nomenclature of fatty acids

130 Å,#186002352), BEH C8 (2.1×50 mm, particle size 1.7 μm,

In this paper, the position of the double bond from thepore size 130 Å, #186002877), Protein BEH C4 (2.1×
50 mm, particle size 1.7 μm, pore size 300 Å, #186004495,

terminal methyl group (omega carbon) is denoted by omega(ω) numbers so that ω-3 or ω-9 indicates the position of theand 2.1×100 mm, particle size 1.7 μm, pore size 300 Å,#186004496), BEH C18 VanGuard Pre-Column (2.1×5 mm,ﬁrst double bond between the 3 and 4 carbons (ω-3) orthth
9 and 10 carbons (ω-9), respectively. In some speciﬁc

particle size 1.7 μm, pore size 130 Å, #186003975), BEH
C8 VanGuard Pre-Column (2.1×5 mm, particle size 1.7 μm,

cases, we use the cis-x nomenclature, such as cis-12 or cis-rdth15, in which the double bond position is counted from theanti-terminal carboxyl group to clarify that the double bondpore size 130 Å, #186003978), and Protein BEH C4 VanGuard Pre-Column (2.1×5 mm, particle size 1.7 μm, poreis in the cis conformation rather than trans conformation as
appears in the chemical formula.

size 300 Å, #186004623).

Fatty acid puriﬁcation from ﬁbroblasts

This study was approved by the Ethical Committee of the

A neonatal human skin ﬁbroblast line (NB1RGB,

Graduate School of Medicine, Gifu University (permission
number: 29-286).

#RCB0222) was obtained from RIKEN BRC through the
National Bio-Resource Project (NBRP) of the Ministry of
Education, Culture, Sports, Science, and Technology

Results(MEXT), Japan. An adult skin ﬁbroblast line (contY) was

Mobile phase strength-dependent detachment behaviorof fatty acids from the LC column: The fatty acidseparation principle

15)

established by us from a healthy adult male . Written
informed consent was obtained from the donner. A neonatalskin ﬁbroblast line (F-12) was also established from a

We previously reported a method for the analysis of abiopsy sample obtained from a peroxisome biogenesis dis-range of fatty acids found in biological samples . The totalorder (PBD) patient carrying PEX2 mutation, who was

fatty acid species including saturated and unsaturated fatty
acids, as well as very-long-chain fatty acids (VLCFAs)

10)

10)

diagnosed with Zellweger syndrome . Written informed
consent was obtained from the patient s parents. The puriﬁ-

found in peroxisomal disease patients, were analyzed in acation of total fatty acids from ﬁbroblasts was performed as

single assay. In the present study, we modiﬁed our method,

10)

described previously . Brieﬂy, a pellet of approximately

aiming for improved detection of VLCFAs and the precise
separation of unsaturated fatty acids according to the num-6

1×10 cells was mixed with 400 μL of acetonitrile and 50 μ23June 2018

Fig. 1.

Medical Mass Spectrometry Vol. 2 No. 1

Comparison of LC columns with different stationary phases upon fatty acid analysis.

(A‒D) A fatty acid standard mixture (C14:0, C16:0, C18:0, C20:0, C22:0, C24:0, C25:0, C26:0, and C28:0) was separated with C18(A and B), C8 (C), or C4 (D) columns. A 15-min linear gradient was used except for (B), where a 50-min gradient was used. Mass
chromatograms of the fatty acid species are shown. The x-axis indicates the retention time and the y-axis indicates the relative intensity. (E, F) Comparison between LC columns. The x-axes indicate the number of carbons, and the y-axes indicate the retention timein (E) or % B in (F). Trend lines are shown as black lines in (F). (G) Comparison of the column length. Five-centimeter (cm) and10-cm C4 columns are compared. The retention time for each fatty acid species is normalized by the retention time of C14:0, andthe time difference against C14:0 ( Δ RTC14:0) is shown.

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not suitable for analyzing saturated VLCFAs.
For the C8 column, the fatty acids eluted earlier than with
the C18 column (Fig. 1C), and all standard fatty acid molecules including VLCFAs were successfully eluted. For theC4 column, all fatty acids eluted much earlier (Fig. 1D). We

assessed the correlation between fatty acid length and retention time (Fig. 1E). The C4 and C8 columns had a high correlation coefﬁcient (r＝0.9985 for C4, and r＝0.9982 for C8)
with a linear trend line, whereas the C18 columns had a low

correlation coefﬁcient (r＝0.9864; calculated from the dataof C14:0 to C24:0). This indicates that C4 and C8 columns
are suitable for separating a long-range fatty acid mixture,including VLCFAs.
To understand the relationship between the fatty acids,
stationary phases, and the organic mobile phase in moredetail, we plotted the detachment time of the fatty acids(calculated from the column hold-up time and the fatty acidretention time) and the strength of the organic mobile phase(% B, acetonitrile) at the detachment time. With C18 columns, the best-ﬁt trend line on the plot was exponential(Fig. 1F), and the function of the trend line is given by%B＝10.106 e
Fig. 1.

0.0904 lenFA,

where lenFA indicates the carbon chain length of the saturated fatty acid. We estimated the longest fatty acid detect-

Continued.

able with the C18 column using this function when the maxber and position of the double bond(s). We evaluated threeimum mobile phase B was 95%. The estimated longest

types of LC columns with different stationary phases: C18,

fatty acid was C24:0, which is consistent with the above

C8, and C4 (see Materials and Methods for details), where
C18 has the highest hydrophobicity and C4 has the lowest. A

observation that C25:0 and longer fatty acids failed to elutefrom the C18 column. The best-ﬁt trend line became linear

saturated fatty acid standard mixtures ranging from C14 towhen the data up to C22:0 was used (data not shown).

C28 (1 μg/mL of C14:0, C16:0, C18:0, C20:0, and C22:0,
and 0.1 μg/mL of C24:0, C25:0, C26:0, and C28:0) wereThese data conﬁrmed that the C18 column does not ﬁt the
VLCFA analysis. For the C8 column, the trend line was lin-

analyzed using the three columns with a gradient LC (LCear (Fig. 1F):

condition 1: 0.4 mL/min ﬂow rate, 15 min linear gradient%B＝2.7925 lenFA−7.483 ,from 20% B to 95% B followed by 2.5 min static 95% B;
see Supplementary Fig. S1). With the C18 column, the fatty

and C36:0 is estimated to be the longest detectable fatty acidacids eluted from shorter ones at constant time intervals,

if this trend continues. We previously reported the detection

but, VLCFAs such as C25:0 and longer failed to eluteof C34:0 in human plasma and ﬁbroblast samples using a C8

within the assay time (Fig. 1A). When the gradient timecolumn, although C36:0 has not been detected . With the C4

was extended to 50 min (LC condition 2; Fig. 1B) or thecolumn, the trend line was also linear (Fig. 1F):

10)

organic phase was kept at 95% for 2 h after the LC gradient%B＝2.0341 lenFA−1.7692 ,(LC condition 3; data not shown), the VLCFAs failed toelute or eluted with broad peaks, respectively, indicating

and the estimated longest fatty acid was C47:0, although

the intense adhesion of the VLCFAs to the C18 stationary

the existence of C47:0 is unlikely in biological samples.

phase. Thus, we conclude that the C18 stationary phase is

Overall, the above observations indicate that each column
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Medical Mass Spectrometry Vol. 2 No. 1

has a speciﬁc limit in the length of detectable saturated

C14:0 eluted at 2.81 min and C28:0 at 8.18 min. The timefatty acids.

difference between C14:0 and C28:0 is 5.37 min, which iscomparable to the case of the 5-cm column. To compensate

Next, we examined the effect of the column length.
Five-centimeter (cm) and 10-cm columns with the C4 sta-for the difference in the hold-up time between the two col-tionary phase were compared. With the 5-cm C4 column,
C14:0 eluted at 1.61 min with a 0.4-mL/min ﬂow rate andumns, the retention time of C14:0 was used as a reference.
The adjusted retention times of the standard fatty acids
became almost identical between the 5-cm and 10-cm col-C28:0 eluted at 6.94 min. Thus, the time difference between
C14:0 and C28:0 is 5.33 min. With the 10-cm C4 column,

Fig. 2.

umns (Fig. 1G), indicating that lengthening the column

The static phase in the LC gradient affects the migration of speciﬁc fatty acid species.

Fatty acid standards were separated by the LC conditions with a 5-min static phase, as shown in Supplementary Fig. S1. The x-axis
indicates the retention time and the y-axis indicates the relative intensity. The migration of some fatty acid species is affected byeach LC condition.

26

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Medical Mass Spectrometry Vol. 2 No. 1

does not improve the separation of fatty acids. From theabove results, the retention of the fatty acids seemed to belargely dependent on the concentration of the organic
mobile phase (i.e., acetonitrile), and the interactions between
the fatty acids and the column-ﬁlled stationary phase during
chromatographic migration has a minor effect.
As described, the mobile phase strength seemed to determine the timing of the detachment of fatty acids from thestationary phase. Utilizing this rule, we divided the fatty
acid standard mixture into portions by inserting a 5-minstatic period in the LC gradient at several time points, as
indicated in Fig. 2 (LC conditions 3 to 8; C4 column wasused). For the ﬁrst condition, the gradient paused after
1 min of analytical time, when the mobile phase B was
25%, until 6 min; then, the gradient was resumed. Under
this condition, C14:0 eluted normally, whereas C16:0 and
C18:0 were trapped in the static phase; thus, their migration
slowed. C20:0 and longer fatty acids eluted with a 5-mindelay but in the same proportion as under the continuous
gradient condition. When the gradient was paused at 3 min(35%B; LC condition 5), C14:0 to C18:0 had eluted
already, the migration of C20:0 to C24:0 slowed, and C26:0
and C28:0 were proportionally delayed by 5 min. All of thestandard fatty acids eluted by 7 min (55%B), indicating thatfatty acids up to C28:0 had detached from the C4 stationary
phase before the concentration of mobile phase B reachedFig. 3.

55%, which is also predicted by the above formula. This
simple separation principle for fatty acids enables the frac-(A) A 2-D map analysis of the fatty acids. Fatty acids
from control human ﬁbroblasts (NB1RGB) analyzed
with the C8 column. The x-axis represents the retention
time and the y-axis represents m/z. The fatty acid species
are shown on the map as black spots. The carbon number
and the double bond number in the fatty acids are annotated in the panel (the same panel without annotations isshown in Supplementary Fig. S2). (B) Retention timesof MUFAs (C14:1 to C28:1), DUFAs (C16:2 to C28:2),
and PUFAs with three double bonds (tri-unsaturated fatty
acids; C18:3 to C28:3) and four double bonds (tetra-unsaturated fatty acids; C20:4 to C28:4), as well as SFAs
(C14:0 to C28:0), are plotted on the graph. All show linear correlations.

tioning of fatty acid mixtures, which is also shown below.

Separation of unsaturated fatty acids with doublebond numbers
Next, we examined unsaturated fatty acids to see if they
behave in the same manner as the SFAs in biological samples. Total fatty acids were extracted from human ﬁbroblasts (NB1RGB) and analyzed with the C8 column. As in
our previous report, unsaturated fatty acids showed a regu10)

lar elution pattern

Correlation between the retention time and the hydrocarbon chain length of unsaturated fatty acids.

(Fig. 3A; Supplementary Fig. S2).

Among fatty acids with the same hydrocarbon chain length,with less double bonds. For example, among the C20 fatty

tively) and found a linear correlation (Fig. 3B). The unsaturated fatty acids show constant and predictable elution pat-acids, the elution order is C20:5, C20:4, C20:3, C20:2, andtern, as in the case of SFAs.

the one with more double bonds eluted earlier than the ones

C20:1, and C20:0 elutes last. We examined the relationshipTo separate the fatty acid species from the biologicalof hydrocarbon chain length and retention time for MUFAs,

samples, we improved the separation method by modifying
the LC gradient. We inserted a 5-min decrease in the

DUFAs, and PUFAs with three and four double bonds(tri-unsaturated and tetra-unsaturated fatty acids, respec-

organic phase (LC condition 9) rather than inserting a static
27

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Medical Mass Spectrometry Vol. 2 No. 1

including MUFAs, DUFAs, and PUFAs, are detectable inhuman tissue samples. Moreover, very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are elevated in peroxi2,10,14)

somal diseases. All can be separated by LC according

to the chain length and double bond numbers. Because theunsaturated fatty acids are also distinguished by their double bond positions, we further separated unsaturated fatty
acids according to their double bond positions.
To determine the basic rule concerning double-bond-position-dependent fatty acid separation, we compared standard
unsaturated fatty acids on LC with the 50-min gradient (LCcondition 2). First, we used three octadecenoic acids(C18:1) with different double bond positions such as at ω-9
position (oleic acid), ω-7 (cis-vaccenic acid), and ω-12
(petroselinic acid) (Fig. 5A). Five microliters of 10 μM
solutions were injected and analyzed with the C8 column.
Among the three octadecenoic acids, cis-vaccenic acid(ω-7) eluted ﬁrst, oleic acid (ω-9) eluted second, andpetroselinic acid (ω-12) eluted last. Three peaks separating
a mixture of the octadecenoic acids were also detectable.
The double bond position, which is closest to the omega
carbon in cis-vaccenic acid, farthest in petroselinic acid,and in between in oleic acid, is a possible determinant ofFig. 4.

the elution order. It seems that as the continuous saturatedhydrocarbon chain terminated with a methyl group (con-

Separation of biological fatty acids into fractions.
(A) Fatty acids from control ﬁbroblasts (contY) wereanalyzed with a modiﬁed gradient LC (LC condition
9; upper graph) with a C4 column and are shown in a
2-D map (lower panel). Fatty acids are divided into two
portions, one with elution index (EI) 18 and below andthe other with EI 19 and above. Saturated fatty acids andselected unsaturated fatty acids at the separation border
are labeled. (B) An EI chart of fatty acids. The EI valuesof even-numbered fatty acids detected in control humanﬁbroblasts (C14 to C28) are shown.

taining the omega carbon) becomes longer, the unsaturated
fatty acid becomes more hydrophobic, thus prolonging theelution time.
We also analyzed three hexadecenoic acids (C16:1) withdouble bond positions at ω-7 (palmitoleic acid), ω-10 (sapienic acid), and at ω-14 (cis-2-hexadecenoic acid) (Fig. 5B).
As indicated by the results for the 18:1 fatty acids, the double bond position relative to the omega carbon correlated tothe elution order (thus the elution order was palmitoleic
acid, sapienic acid, and cis-2-hexadecenoic acid). This con-phase as described above. Using this modiﬁcation, we wereable to clearly separate the biological fatty acids into frac-ﬁrmed the idea that a double bond closer to the omega car-tions (Fig. 4A). The separation was very clear according tolonger, saturated hydrocarbon chains ending with thethe elution index, which shows total hydrophobicity of theomega carbon on the terminal methyl group makes MUFAs

given fatty acid species (Fig. 4B; see Discussions for moredetail). Using this method, fatty acid mixtures can be sepa-

more hydrophobic.

rated into fractions divided by their hydrophobicity.

linolenic acid (octadecatrienoic acid, C18:3), α-linolenicacid and γ-linolenic acid. Alpha-linolenic acid possesses

Separation of unsaturated fatty acids according to theposition of the double bonds

three double bonds at the ω-3, 6, and 9 positions, andγ-linolenic acid has double bonds at the ω-6, 9, and 12 positions

As shown above, a number of unsaturated fatty acids(Fig. 5C). The peaks of these two fatty acids were separated

bon makes the MUFAs less hydrophobic; in other words,

To expand this idea to PUFAs, we compared two types of28

June 2018

Fig. 5.

Medical Mass Spectrometry Vol. 2 No. 1

Elution order of unsaturated fatty acids.

(A) Mass chromatogram of three octadecenoic acids (C18:1), cis-vaccenic acid (ω-7), oleic acid (ω-9), petroselinic acid (ω-12), andtheir mixture are shown. (B) Mass chromatogram of three hexadecenoic acids (C16:1), palmitoleic acid (ω-7), sapienic acid (ω-10),
cis-2 hexadecenoic acid (ω-14), and their mixture. (C) Mass chromatogram of two octadecatrienoic acids (18:3), α -linolenic acid(ω-3, 6, 9), γ -linolenic acid (ω-6, 9, 12), and their mixture. (D) Mass chromatogram of two eicosatrienoic acids (C20:3), dihomo- γ-linolenic acid (ω-6, 9, 12), sciadonic acid (ω-6, 9, 15), and their mixture.

clearly, and α-linolenic acid, which has the ﬁrst doublebond closer to the omega carbon, again, eluted beforeγ-lin-

identical proximal and second-proximal double bonds, but
the positions of the distal double bond are different.
Dihomo- γ -linolenic acid eluted before sciadonic acid, thus

olenic acid, which has a more distant double bond. Concerning the C18:1 and C16:1 MUFAs, the elution order of
the C18:3 PUFAs was found to be determined by the posi-

indicating the non-proximal double bond also affects thetion of the ﬁrst double bond.

double bonds to the omega carbon is the determining factor

elution order. Taken together, the total proximity of the all

We also examined PUFAs with the same proximal dou-in the elution order of PUFAs.

ble bond but different distal double bond. Two C20:3
PUFAs (eicosatrienoic acids), dihomo- γ -linolenic acid

Detailed fatty acid proﬁle of human ﬁbroblasts(ω-6, 9, and 12) and sciadonic acid (ω-6, 9, and 15), were

Finally, we compared the fatty acids of human ﬁbroblasts
retrieved from a control subject and a patient with peroxi-

examined (Fig. 5D). These two eicosatrienoic acids have29June 2018

Fig. 6.

Medical Mass Spectrometry Vol. 2 No. 1

Quantitatively changed unsaturated fatty acid isomers found in ﬁbroblasts of peroxisome biogenesis disorder.

(A) Multiple peaks representing different fatty acid isomers in the C20 lineage (C20:2 and C20:3) are shown. The x-axis indicatesthe retention time and the y-axis indicates relative intensity. In the PBD ﬁbroblasts, the proportion of each two peaks in C20:2 andC20:3 are altered. The numbers in parentheses indicate the relative amount with respect to C20:0. The arrows indicate an increaseor a decrease of each fatty acid isomer. (B) Pie charts representing the proportion of C20:2 isomers or C20:3 isomers in the control
and PBD ﬁbroblasts are shown.

some biogenesis disorder (PBD), a type of peroxisomal dis6,7)

ease involving defective peroxisomal biosynthesis(Supplementary Fig. S3). The amount of C20:2a andC20:2b fatty acids were comparable in the control ﬁbro-. Fatty

acids were extracted from the total lipids by acid hydrolysis
and assayed with the C8 column with a 50-min gradient (LCblasts, whereas the amount of C20:2a was double that ofcondition 2). We searched for unsaturated fatty acids giving
multiple peaks representing double-bond position differ-

two peaks, C20:3a and C20:3b (Fig. 6A, Supplementaryences and found several examples (Fig. 6A). Interestingly

ﬁbroblasts compared to C20:3b (Fig. 6B). The relativesome of the peaks differ in intensity between the control
and PBD ﬁbroblasts. C20:2 had two peaks, and we tenta-

quantiﬁcation against internal C20:0 revealed the nearly
two-fold increase or decrease of C20:2a or C20:3b, respec-tively identify that which eluted earlier as C20:2a and thattively, in the PBD ﬁbroblasts (Fig. 6A). The separation of
fatty acid isomers was, thus, found to be useful to under-

C20:2b in the PBD ﬁbroblasts (Fig. 6B). C20:3 also had
Fig. S3). C20:3a was clearly more abundant in the PBD

which eluted later as C20:2b. We conﬁrmed that they both13

have the same carbon number by comparing their C ratios

stand the metabolic changes in cellular fatty acids.
30

June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Discussion

determinant of the elution order among the isomers. A dou-

Fatty acids separation principle: Organic phasestrength-dependent detachment from LC column

ble bond closer to the omega carbon makes the unsaturated
fatty acid less hydrophobic; thus, it elutes earlier than iso-

We investigated the separation principle for fatty acids

mers having the ﬁrst double bond further from the omega

and found that the separation depends on their detachment

carbon. Moreover, we found that the proximity of otherbehavior from the stationary phase of the LC column,

double bonds also affects the elution order. By and large,

unlike regular chromatography. The fatty acids sequentiallywhen the hydrocarbon chain length is identical, fatty acids

detached from the stationary phase along with the organic

with more double bonds elute earlier than those with fewer

mobile phase once an appropriate concentration to retrieve

double bonds, and, if the number of the double bonds is theeach fatty acid species from the stationary phase wassame, those with double bond(s) closer to the omega carbon

achieved. Among the SFAs, longer SFAs elute after shorter

elute earlier than others. Following these criteria, we found

SFAs because of their higher hydrophobicity, whichthat many isomeric unsaturated fatty acids exist in humanincreases the adhesion strength to the stationary phase (e.g.,ﬁbroblasts and the concentrations of some of these fatty

C22:0 elutes after C20:0). When SFAs and unsaturated

acids change in PBD patients.

fatty acids are compared, SFAs elute after the MUFAs with

We could not identify the exact double bond position of

the same or longer hydrocarbon chain by one carbon, butthese fatty acids because locating the double bond by
ESI-MS is still challenging. Collision-induced dissociation

elute before MUFAs longer by two hydrocarbons (forexample, C22:0 elutes after C22:1 and C23:1 but before
C24:1). MUFAs elute after the SFAs shorter by two hydro-(CID) is often used with ESI-MS to fragment target ionscarbons, MUFAs shorter by one hydrocarbon, and DUFAs

position of the double bond(s) of unsaturated fatty

with the same hydrocarbon length (C22:1 elutes afteracids

C20:0, C21:1, and C22:2) but elute before MUFAs longer
by one hydrocarbon and DUFAs longer by two hydrocar-. The derivatization of
the terminal carboxyl group with N-(4-aminomethylphenyl)bons (C22:1 elutes before C23:1 and 24:2). These featuresindicate that acquiring one double bond can compensate forpyridinium (AMPP) enables fatty acids to be detected withmore sensitive positive ESI (ESI＋), and the CID fragmen-

the increased hydrophobicity provided by one hydrocarbon.

tation generates product ions for double bond location

but does not give deﬁnitive product ions to determine the16,17). To overcome this problem, several derivatization
18,19)

methods have been developed

18,20)

A simple method to estimate the elution order (or retention.
Zhao et al. recently reported that the epoxidation of unsatu-time) of given fatty acids is to calculate and compare therated fatty acids using low temperature plasma followed byelution index (EI). The EI is calculated by deducting the

CID fragmentation gives product ions generated at the site

number of double bonds from the number of carbon atomsof epoxidation, and, thus, the double bond position can beof a given fatty acid. For example, in the case of C22:3,

determined . A combination of our method with thesederivatization methods will further improve our understand-19)[total carbon number] 22−[double bond number] 3＝[EI]
19. With the C4 column, a fatty acid elutes before the fatty
acids with larger EI, and fatty acids sharing the same EI

ing of the entire fatty acid species in biological samples,allowing the hydrocarbon chain length, double bond num-

elute around the same time, aside from the SFAs that elutebers, and double bond positions to be distinguished.

slightly later than the others. With the C8 column, longer

VLCFAs increase in some human peroxisomal disease

fatty acids elute slightly earlier than the shorter fatty acids
when their EI is the same. The EIs of selected human ﬁbro-

patients and are considered pathogenic, although their
6,7)

blast fatty acids are shown in Fig. 4B.

. Relatively
few VLCFA species, such as C26:0 and C24:0, are com-

Double bond position-speciﬁc fatty acid separation

monly used as diagnostic markers for peroxisomal dis6,21)
eases , but signiﬁcantly more VLCFA species accumu-

rated fatty acids that differ from each other by the position

late in patients, as we and others have previously
2,10,14). Our current analysis of the patient ﬁbroreportedof the double bond(s). By examining C16:1 and C18:1

blasts with distinguishing double bond positions revealed

MUFAs, as well as C18:3 and C20:3 PUFAs, we found thatclear ﬂuctuations in additional fatty acid species such as

the ﬁrst double bond from the omega carbon is the major

C20:2a and C20:3b, for which we do not know the exact

pathogenicity should be conﬁrmed clinicallyWe also investigated the elution order of isomeric unsatu-

31June 2018

Medical Mass Spectrometry Vol. 2 No. 1

double bond position. Proﬁling the entire fatty acid range,

Moser AB, Moser HW: Peroxisome biogenesis disorders.

distinguishing the double bond position, and identifying thechanges in additional fatty acid species in peroxisomal dis-

Biochim Biophys Acta 1763: 1733‒1748, 2006.
7) Shimozawa N: Molecular and clinical ﬁndings and diag-ease patients will give us a new insight of the metabolic

nostic ﬂowchart of peroxisomal diseases. Brain Dev 33:

changes in patients and reveal the pathology of peroxisomal

770‒776, 2011.

diseases.

8) Takemoto Y, Suzuki Y, Horibe R, Shimozawa N, Wanders
RJ, et al: Gas chromatography/mass spectrometry analy-

Acknowledgements

sis of very long chain fatty acids, docosahexaenoic acid,phytanic acid and plasmalogen for the screening of per-We thank Dr. Tsuneo Imanaka at Hiroshima International

oxisomal disorders. Brain Dev 25: 481‒487, 2003.

University, Dr. Masashi Morita and Dr. Kosuke Kawaguchiat the University of Toyama, and Dr. Kazuaki Yokoyama,
Dr. Kotaro Hama, and Dr. Yuko Fujiwara at Teikyo Univer-

9) Liu A, Chang J, Lin Y, Shen Z, Bernstein PS: Long-chainsity for their valuable comments. This work was supported

aging and age-related macular degeneration. J Lipid Res

by JSPS KAKENHI grant numbers JP25860855 for ST,

51: 3217‒3229, 2010.

and very long-chain polyunsaturated fatty acids in ocularand 15H048750001, 15K153890001 for NS from the Japan

10) Takashima S, Toyoshi K, Itoh T, Kajiwara N, Honda A, etSociety for the Promotion of Science, Japan. This work was

al: Detection of unusual very-long-chain fatty acid andalso supported by research aid funds from the Koshiyama

ether lipid derivatives in the ﬁbroblasts and plasma of pa-

Foundation for the Promotion of Science and Technology,
and the Takahashi Industrial and Economic Research Foun-tients with peroxisomal diseases using liquid chromatog-

dation for ST.

268, 2017.

raphy-mass spectrometry. Mol Genet Metab 120: 255‒
11) Johnson DW: A rapid screening procedure for the diagno-

Conﬂict of Interest

sis of peroxisomal disorders: quantiﬁcation of very long-

The authors declare no conﬂicts of interest.

chain fatty acids, as dimethylaminoethyl esters, in plasma
and blood spots, by electrospray tandem mass spectrome-

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