Focusing Review

CHROMATOGRAPHY, Vol.32 No.2 (2011)

Focusing Review

Development and application of chromatographic methods
for glycerolipid analysis
Yutaka Itabashi
Faculty of Fisheries Sciences, Hokkaido University
3−3−1 Minato−cho, Hakodate 041−8611, Japan

Abstract
In the past 30 years, chiral-phase high-performance liquid chromatography (HPLC) has progressed from an application practiced by just a few
chromatographers to a popular method widely used in many areas of pharmaceutical and biological sciences, including lipid chemistry. Since
we demonstrated HPLC resolution of mono- and diacylglycerol enantiomers on chiral stationary phases in the 1980s, the methodology has
been extended to the resolution of various synthetic and naturally occurring chiral glycerolipids. Using reversed-phase HPLC, we also demonstrated the successful resolution of 1,2-diacylglycerol regioisomers (reverse isomers), which had remained a major unsolved problem in glycerolipid chromatography. These developments of the methodology permit further expansion of lipidomics and better understanding of lipid
metabolism.
Keywords: glycerolipid; enantiomer; diastereomer; reverse isomer; derivatization; high-performance liquid chromatography; mass spectrometry

cur in cells and tissues requires complementary profiling of lipid-

1. Introduction
There is no uniformly adopted definition for lipids and it isderived metabolic products (lipidomics). This has led to the devel-

widely accepted that lipids are fatty acids, their derivatives, andopment of mass spectrometry (MS) -based lipidomics methodology

substances related biosynthetically or functionally to these com-

[2,3]. MS, however, is unable to differentiate between enantiomers,

pounds [1]. Glycerolipids –lipids based on glycerol (acylglycerols,

and in many instances among regioisomers. This shortcoming is

phospholipids, glycolipids, etc.) –in living organisms are complex

especially obvious in glycerolipids due to the prochiral nature of

mixtures of different types of molecules that contain stereoisomers

glycerol [4].

and regioisomers. Although the technology of chromatography has

There were two unsolved problems in glycerolipid chromatog-

advanced rapidly in recent years, there still exist many lipid mole-raphy in the 1980s, one of which was enantiomer separation andcules whose separations are difficult to achieve and physiologicalthe other regioisomer (reverse isomer) separation. We have solved

significance and biological activities are unknown. Accurate andimportant aspects of these problems by using chiral-phase HPLC

concise methods for the analysis of lipids are therefore essential for[5] and reversed-phase HPLC [6], respectively. This review de-many research studies.

scribes the chromatographic methods developed for both stereoi-

In the present “post-genomic era” the focus of investigation issomeric and regioisomeric glycerolipids and their applications tomoving away from gene sequences and interest is gradually return-lipid research. Readers are referred to earlier comprehensive re-ing to the gene products themselves (proteomics). It is well recog-

views for background covering chiral analysis of glycerolipids [4,nized that an understanding of pathophysiological changes that oc-

7–14].

Correspondence: Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-8611, Japan
Phone & Fax: +81-138-40-8802
E−mail: yutaka@fish.hokudai.ac.jp―５９―

CHROMATOGRAPHY, Vol.32 No.2 (2011)of n forms. For example, one set of enantiomers can exist for 1,2-

2. Nomenclature of glycerolipids
Glycerol and common fatty acids are achiral compounds that

diacylglycerol and 1 (and 2)-lysophosphatidylcholine (lyso-Ptd

have no chiral centers in their molecules, while glycerolipids be-Cho), in which only the C-2 of the glycerol skeleton is a chiral cen-come chiral molecules when the C-1 and C-3 positions of the glyc-ter. Four stereoisomers (enantiomers of 2 sets) can exist for phos-

erol backbone have different fatty acids, or when the C-2 position

phatidylglycerol (PtdGro) and phosphatidylserine (PtdSer), whichmakes a chiral center. Almost all of the naturally occurring glycer-

have two chiral centers in their molecules.

olipids are chiral and can exist as both enantiomers and

The steric configuration of glycerol-based lipids is usuallydiastereomers. The structures of some glycerolipids adopted in thisrepresented by an sn (stereospecific numbering) notation rather

review are shown in Figure 1. Generally, stereoisomers of 2n forms

than R/S in the Cahn-Ingold-Prelog system for designating abso-(enantiomers of 2n-1 forms) exist in molecules having chiral centerslute configuration. When the secondary hydroxyl group of an (S )glycerol derivative is put in the left side of C-2 in a Fisher projection, the top and bottom carbons are made to be C-1 and C-3, re-

O

A
H2C O

O
R2 C

O

C

H

C

R1 (sn-1)

C

C
H2C O

R2 C OCS

HO

R3 (sn-3)

C

R1

O
R2 C

O

C

R

H

H2C O

R1 C

O

HO

C

R

H

H2C O

C

and its enantiomer, 2,3-diacyl-sn-glycerol, corresponds to R. Simi-

R2

larly, 1,2-diacyl-sn-glycero-3-phospho-1’-sn-glycerol fits with R,S(Figure 1 E), and one of the diastereomers, 1,2-diacyl-sn-glycero-3-phospho-3’-sn-glycerol, corresponds to R,R. It should be notedthat although the steric configuration at C-2 of 1,2-diacyl-sn-

O

R2 C

O

C

R

C

R1

O

3,5-dinitrophenylurethane (3,5-DNPU) and 1(2)-anthrylurethane

P O CH2OSC
HNH2derivatives of 1,2-diacylglycerol, 1-alkylglycerol, etc., the R/S notation is opposite from that of the original compounds. In addition

COOH

OH (sn-3')O
R2 C O+P O (CH2)2N(CH3)3O-

to enantiomers, natural glycerolipids containing 1,2-diacyl-snglycerol moiety can also exist as regioisomers (reverse isomers),

O

H

R

glycerol is the same as that of 1,2-diacyl-sn-glycero-3-phospho-1’sn-glycerol, the R/S notation reverses each other. In the case of the

H

H2C O

( sn-1')P O CH2OS
H (sn-2')
HOCO

ing to this notation, 1,2-diacyl-sn-glycerol is S (Figure 1 C, 1 D)

H

H2C O

O

O

C

C

S

F
R1

O
H2C O

glycerol backbone, similar to the manner described above. Accord-

H2C OH

H2C

G

bones, such as PtdGro, C-1’ and C-3’ are replaced for the second

H2C O

O

O
C

the compound [15]. When compounds have two glycerol back-HO

H2C OH

H2C O

S

spectively, and the prefix “sn” is placed before the stem-name ofR

H2C OH

H

E

C

C

D

O
O

H2C O

(sn-2)

O

H2C O

O

B

H2C O CC

R

R1such as 1-palmitoyl-2-linoleoyl-sn-glycerol (16:0-18:2) and 1linoleoyl-2-palmitoyl-sn-glycerol (18:2-16:0) (see Figure 1 C, 1

H

H2C O
HO

D).

CH2OH

O
OH

OH

3. Chiral-phase HPLC of stereoisomeric glycerolipids
I

H2C O
HO

C

S

R

O

J

H2C O

H

H2C OHH

C

C

3.1 Chiral stationary phases (CSP) for glycerolipid analysis

R

There are a number of different materials employed as CSP.

R

According to Taylor and Maher [16], these materials are classifiedO
H2C

into six types based on separation principles: ligand exchange

Figure 1. Structures of some chiral glycerolipids. (A) 1,2,3-triacylsn-glycerol (triacylglycerol); (B) 1-acyl-sn-glycerol(monoacylglycerol); (C, D) 1,2-diacyl-sn-glycerol (diacylglycerol reverse isomer, R1≠R2); (E) 1,2-diacyl-snglycero-3-phospho-1’-sn-glycerol (phosphatidylglycerol,
PtdGro); (F) 1,2-diacyl-sn-glycero-3-phospho-L-serine(phosphatidylserine, PtdSer); (G) 1-acyl-2-hydroxy-snglycero-3-phosphocholine( lysophosphatidylcholine ,lyso-PtdCho); (H) 1,2-diacyl-[ß-D-galactopyranosyl-(1’→ 3 ) ] -sn-glycerol( monogalactosyldiacylglycerol ,MGDG); (I) 1-alkyl-sn-glycerol (glyceryl ether); (J) (R )1-acyl-2,3-epoxypropane (glycidol fatty acid ester).

phases, charge transfer packings, asymmetric strand packings(brush-type CSP or Pirkle-type CSP), chiral cavity packings, CSP
based on polymers, and silica-bound protein CSP. Of these six, theasymmetric strand packings (low molecular weight CSP) and CSP
based on polymers (synthetic and natural macromolecules, such as
polymethacrylate, polymethacrylamide, cellulose, amylose andtheir derivatives) are effective for enantiomer separation of glycerolipids. Generally, polycyclic aromatic compounds containing
naphthyl or anthryl groups as π donors form charge-transfer complexes with aromatic rings having electron-withdrawing substitu-―６０―CHROMATOGRAPHY, Vol.32 No.2 (2010)

Yutaka Itabashi

H
O2N

N
O

H
N
O

C
C
O
N

C
NO2H

Generally, compounds having hydroxyl or amino groups canreact with isocyanates to form the corresponding urethane or urea
CC

derivatives, respectively.

O
N

H

C

R1−OH＋R2−N＝C＝O → R2−NHCOO−R1 (urethane)H

R1−NH2＋R2−N＝C＝O → R2−NHCONH−R1 (urea)

H

CH3

CH3

The 3,5-dinitrophenyl isocyanate and 1- and 2-anthryl isocyanates react readily with glycerolipids having hydroxyl or amino

Figure 2. Possible interactions between (R )-1-(1-naphthyl)ethylamine CSP and glycerolipid derivatives. (A) 3,5dinitrophenylurethane, (B) 2-anthrylurethane. From
[13].

groups, such as PtdGro and PtdSer, at room temperature in toluenein the presence of a base (pyridine or triethylamine). The derivatization not only enables separation of enantiomers but also facilitates detection. For example, the bis(3,5-DNPU) derivatives ofents such as nitro or cyano groups. We showed that this interaction

monoacylglycerols have a strong absorption at 226 nm (λmax), as

could also be formed between a naphthyl group and an anthryl

indicated by the ε value of ca. 60,000 in ethanol, and have a goodgroup, which would serve as an electron accepter and a donor, re-absorption at 254 nm (ε 31,000) [5]. Isocyanates are moisture sen-spectively [17,18]. In this case, the formations of additional

sitive and therefore, long-term storage is not desirable. Althoughhydrogen-bonding and dipole-dipole stacking between solutes and3,5-dinitrophenyl isocyanate is commercially available, it can be

CSP would better separate the enantiomers. Therefore, if these in-easily synthesized in the laboratory by thermal decomposition ofteractions do not occur between enantiomers and CSP, it might be

3,5-dinitrobenzoyl

possible to make them occur by converting them into the appropri-

dinitrobenzoyl chloride and sodium azide [20]. The 3,5-

ate derivatives using achiral reagents. We have found that thisdinitrobenzoyl azide is stable for storage and loses nitrogen at ap-

derivatization method is very effective for enantiomer separation ofproximately 120℃ to yield 3,5-dinitrophenyl isocyanate [20]. Wechiral glycerolipids containing hydroxyl or amino groups (seehave found that 3,5-dinitrophenyl isocyanate and 1(2)-anthryl iso-Figure 1). Figure 2 illustrates the possible interactions between the

cyanate can be easily synthesized by the reactions of 3,5-1-(1-naphthyl)ethylamine

dinitroaniline

moiety

in

CSP

and

the

3,5-

and

azide,

whichisprepared1(2)-aminoanthracene,fromrespectively,

3,5-

with

dinitrophenylurethane (3,5-DNPU) and 2-anthrylurethane moieties

triphosgene [bis(trichloromethyl) carbonate] as a phosgene substi-

of the glycerolipid derivatives. Enantiomer resolution on Pirkle-

tute [13,17,18]. Triphosgene is a stable solid, which is safer andtype CSPs, which consist of relatively small molecular weight

more convenient to transport and store than phosgene and diphos-chiral substances bonded to silica, is due mainly to attractive inter-gene [21].

actions between solutes and CSP, i.e., hydrogen-bonding, π-π com3.2 Enantiomeric mono-, di- and triacylglycerols

plexing, and dipole stacking [19]. The 1-(1-naphthyl)ethylamine
CSP has the ability to serve as either a donor or an acceptor in

Enantiomeric 1- and 3-monoacyl-sn-glycerols were the firsthydrogen-bonding and π-π complexing and to participate in dipole

chiral glycerolipids resolved by chiral-phase HPLC [5]. Synthetic

stacking. Thus, the diastereomeric hydrogen-bonding association, π

racemates with C12-C18 saturated acyl groups in the molecules-π donor-acceptor interaction, and dipole-dipole interaction be-

were converted into the bis(3,5-DNPU) derivatives and then sepa-

tween CSP and solutes are probably the major factors contributing

rated into enantiomers on CSP prepared by Oi and Kitahara [23]to the separation of the derivatives of enantiomeric glycerolipids.

from acylated amino acid and chiral 2-(4-chlorophenyl)isovalericIn this case, better enantiomer separation is obtained for 3,5-DNPU

acid bonded chemically to silica through ionic linkage (Sumipax

than for 2-anthrylurethane [13]. This suggests that the charge-

OA-2100). To obtain clear enantiomer resolution, a ternary solvent

transfer complex formation between the 3,5-dinitrophenyl groupsystem, hexane/1,2-dichloroethane/ethanol (40:12:3, by vol), wasand the anthracene ring is stronger than that between the naphthyl

used as the mobile phase. Sumichiral OA-4100 [24,25] and YMCgroup and the anthracene ring. On the other hand, polymer type

A-K03 [26], both of which contained chiral (R )-1-(1-naphthyl)

CSPs, such as amylose tris(3,5-dimethylphenylcarbamate), have aethylamide groups as a chiral selector, were also used for this pur-large advantage in that enantiomer separation can be obtained with-pose. The best resolution was obtained on OA-4100 within 10 min

out converting chiral lipids into derivatives or into simple deriva-

on a 25 cm-long column. This OA-4100 column showed an earlier

tives, such as acetates and methyl esters. Thus, the packing materi-elution of the sn-1-enantiomer than the sn-3-enantiomer using theals containing polymer type CSP are useful when derivatization issame solvent system as that used for OA-2100. However, the sn-2-

undesirable or difficult to obtain or when the recovery of eachregioisomer almost overlapped with the sn-1-enantiomer on theenantiomer is needed.

column. Therefore, if the sn-2-regioisomer exists in samples, a pre-―６
１―

CHROMATOGRAPHY, Vol.32 No.2 (2011)

A

liminary separation of the sn-2-isomer from the sn-1(3)-sn-1,2enantiomer on thin-layer chromatography (TLC) or achiral-phase
HPLC is necessary before enantiomer separation on OA-4100. Although the enantiomer resolutions on OA-2100 (α = 1.18–1.19)

sn-2,3and A-K 03 (α = 1.12) are poorer than that on OA-4100 (α = 1.31–
1.35), the sn-2-isomer can be completely resolved with a faster elution than the sn-1-enantiomer on OA-2100 (α = 1.28). The elution
orders of the sn-1- and sn-3-enantiomers on OA-4100 and A-K03
are reversed on OA-4100 R and A-L03, respectively, which have
opposite configurations. Chiral-phase HPLC of enantiomericmonoacylglycerols has been utilized for the determination of fatty
acid positional distribution in triacylglycerol molecules (called
10

stereospecific analysis of triacylglycerols) [7,9,10].

15

20

25

30 minDeng et al. reported direct HPLC separation of enantiomericandregioisomericmonoacylglycerolsoncellulosetris(4-

chlorophenylcarbamate) (Chiralcel OF) and by using hexane/2-

B

propanol eluent mixtures [27]. Without previous derivatization of
the samples, García et al. also reported a similar separation of enantiomeric and regioisomeric monoacylglycerols on other polymer
type CSPs, namely amylose tris(3,5-dimethylphenylcarbamate)(Chiralpak IA) and cellulose tris(3,5-dichlorolphenylcarbamate)(Chiralpak IC), using hexane/2-propanol eluent mixtures [28]. We
have observed that much better separation of both enantiomers andmolecular species can be obtained by chiral reversed-phase HPLCon Chiralpak IA and Chiralpak IC by using acetonitrile containing
10-30% methanol as the mobile phase (Figure 3) [29].
A chiral-phase HPLC resolution of enantiomeric 1,2- and 2,3diacyl-sn-glycerols is also based on the conversion of diacylglycer-15

ols into the 3,5-DNPU derivatives by reaction with 3,5dinitrophenyl isocyanate [22,30] and the best separation has beenmethacrylamide polymeric phase (YMC A-K03) [26]. Figure 4
B

sn-2

25

30

35

40

min

Figure 4. Chiral-phase HPLC profiles of the 3,5-dinitrophenylurethane derivatives of 1,2- and 2,3-diacyl-sn-glycerols
derived from linseed oil triacylglycerols on a column
containing (R)-1-(1-naphthyl)ethylmethacrylamide
polymer (A-K03, 250 x 4.6 mm ID). Column temperature: (A) 10℃; (B) 28℃. Mobile phase: hexane/CH2Cl2/
EtOH (40:10:1, by vol). Flow rate: (A) 1 mL/min; (B)
0.5 mL/min. Detection: 254 nm. From [13].

achieved on a column containing bonded (R )-1-(1-naphthyl)ethyl-

A

20

sn-2

sn-3 sn-1
sn-3

sn-1

shows the HPLC chromatograms of the 3,5-DNPU derivatives of
the 1,2- and 2,3-diacyl-sn-glycerol moieties of linseed oil triacyl7

8

9

10

11 min

22

24

26

28

30

min

glycerols at different column temperatures. The derivatives were

Figure 3. Direct resolution of enantiomeric (sn-1 and sn-3) and regioisomeric (sn-2) monoarachidonoylglycerols (20:4) by
chiral-phase HPLC on amylose tris(3,5-dimethylphenylcarbamate). Column: Chiralpak IA (250 x 4.6 mm ID, 5µm particles). Mobile phase: (A) CH3CN/MeOH (90:10,
by vol); (B) hexane/MeOH (96:4, by vol); Flow rate: 0.5
mL/min. Column temperature: (A) 20℃; (B) 25℃. Detection: ELSD (50℃).

resolved by an isocratic elution with hexane/1,2-dichloroethane/
ethanol (40:10:1, by vol) as the mobile phase, with the sn-1,2enantiomers emerging ahead of the sn-2,3-enantiomers. Typically,
for such CSP, the magnitude of the separation factors for enantiomeric pairs increases as the column temperature of the CSP is reduced [19]. In fact, better separation of enantiomeric diacylglycerols was achieved at lower temperatures (Figure 4A). Thus, analysis―６２―CHROMATOGRAPHY, Vol.32 No.2 (2010)

Yutaka Itabashi

at lower temperatures is especially effective for the separation of an

rectly separated without any derivatization. The method is based on

enantiomer mixture consisting of many molecular species witha cellulose tris(3,5-dimethylphenylcarbamate) column (Chiralcel

saturated and unsaturated acyl groups, which are partially resolved

OD), which allows for separation of underivatized 1,2- and 2,3-

on CSP according to their equivalent carbon numbers (ECN = total

diacyl-sn-glycerols, provided a conventional silica gel column is

number of carbon atoms in the two constituent fatty acids−2 x to-used in tandem to separate 1,3-diacylglycerols from 1,2(2,3)-

tal number of their double bonds) [26]. Thermodynamic considera-

diacylglycerols before they enter the CSP column. There is no needtion for enantiomer separation of diacylglycerols on CSP has been

to isolate the fraction of 1,2(2,3)-diacylglycerols in advance. For

described elsewhere [13,31]. The chiral-phase HPLC method de-this purpose, a silica gel column (Ultrasphere, 5µm column, 250 x

scribed here has been extensively utilized in stereospecific analyses

4.6 mm ID) was connected in series with the CSP column (250 xof diacylglycerol moieties derived from natural triacylglycerols by

4.6 mm ID). The mobile phase was hexane/2-propanol (300:7, byrandom degradation with Grignard reagent [32–36].

vol) at a flow rate of 1.0 mL/min at 25℃. An authentic mixture of

We utilized chiral-phase HPLC for the determination of the

1,3-dicapryloyl- and racemic 1,2-dicapryloylglycerols subjected tostereochemical configuration of glycerol moieties in glycoglycerol-

tandem column HPLC yielded sn-1,3-, sn-2,3-, and sn-1,2-isomers

ipids [37]. For this purpose di- and monoacylglycerols were re-at 26, 32, and 34 min, respectively. Comparable results were ob-leased from glycosyldi- and glycosylmonoacylglycerols (see Figure

tained for isomeric mixtures of dioleoylglycerol. The method was1H), respectively, by periodate oxidation followed by hydrazinoly-

used for the evaluation of the stereospecificity of action of immobi-sis. The released di- and monoacylglycerols were converted into

lized Candida antarctica lipase B and Rhizomucor miehei lipase,

3,5-DNPU and bis(3,5-DNPU) derivatives, respectively, as de-which possessed opposite stereospecificity. Although enantiomericscribed above. The derivatives were separated on two CSP of

separation without derivatization is convenient, it does not exclude

opposite configuration, (R )- and (S )-1-(1-naphthyl)ethylmeth-isomerization of diacylglycerols in the free form on silica column

acrylamide polymers (A-K03 and A-L03) for diacylglycerols and

HPLC. Free diacylglycerols are known to undergo isomerization

N -(R )-(1-naphthyl)ethylaminocarbonyl-(S )-valine (OA-4100) andeven in a solid state [39]. Recently, we have observed that much

N -(S )-1-(1-naphthyl)ethylaminocarbonyl-(R )-valine (OA-4100 R)

better resolution of enantiomeric 1,2- and 2,3-diacyl-sn-glycerols

for monoacylglycerols. Using the above method, we determined

and their molecular species as free forms is obtained by chiralthereversed-phaseglycerolconfiguration

in

the

glycosyldiacylglycerols

HPLC

on

amylosetris(3,5-dimethylphenyl-(monogalactosyl-, digalactosyl-, and sulfoquinovosyldiacylglycer-

carbamate) (Chiralpak IA) using acetonitrile/methanol eluent mix-ols) and glycosylmonoacylglycerols (monogalactosyl-, digalactosyl

tures (Figure 5).

-, and sulfoquinovosylmonoacylglycerols) isolated from spinach

Some asymmetric triacylglycerols with very different acyl

leaves and the coralline red alga Corallina pilulifera. The resultsgroups have been successfully resolved without derivatization by

clearly showed that the glycerol moieties in all the glycoglycerolip-chiral-phase HPLC on polysaccharide-based CSP [40]. Enanti-

ids examined have S -configuration (1,2-diacyl-sn-glycerols and 1-

omeric 1-eicosapentaenoyl-2,3-dicapryroyl-sn-glycerol (20:5-8:0-

monoacyl-sn-glycerols).

8:0) and 1,2-dicapryroyl-3-eicosapentaenoyl-sn-glycerol (8:0-8:0-

We have also developed a highly sensitive method for the20:5) were almost completely resolved with a peak resolution (Rs)separation and detection of enantiomeric and regioisomeric diacyl-of 1.07 on a cellulose tris(4-chlorophenylcarbamate) column

glycerols as 1- and 2-anthrylurethanes by chiral-phase HPLC with

(Chiralcel OF, 250 x 4.6 mm ID) using hexane/2-propanol (100:1,

fluorescence detection [17,18]. The derivatives were obtained by

by vol) as the mobile phase. Excellent resolution with an Rs value

reaction with 1(2)-aminoanthracene and triphosgene in acetonitrile

greater than 11 was achieved for enantiomeric 1-docosahexaenoyl-

at 100℃. Again, satisfactory resolution of the enantiomers and re-

2,3-dicapryroyl-sn-glycerol (22:6-8:0-8:0) and 1,2-dicapryroyl-3-

gioisomers was achieved on A-K03, using a mixture of hexane/di-

docosahexaenoyl-sn-glycerol (8:0-8:0-22:6) on a Chiralcel OD col-chloromethane/ethanol (150:10:1, by vol) as the mobile phase (seeumn (250 x 4.6 mm ID) using hexane/2-propanol (200:1, by vol) as

Figure 2). Under working conditions, the sn-1,2-enantiomers

the mobile phase at 1 mL/min and 25℃. Recently, Nagai et al. em-

eluted ahead of the sn-2,3-enantiomers and both were preceded byployed recycle HPLC for the separation of enantiomeric triacyl-

the X-1,3-regioisomers. Like the 3,5-DNPU, the anthrylurethanes

glycerols possessing minor differences in chain lengths and degrees

eluted from the columns according to their ECN values. The detec-of unsaturation [41]. Effective enantiomer separation was obtainedtion limit of 2-anthrylurethanes was 1 fmol when the signal-to-in 150 min for 1,2-dipamitoyl-3-oleoyl-rac-glycerol (16:0-16:0-18:

noise ratio was 3:1.

1), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (18:1-18:1-16:0), and 1,2

Piyatheerawong et al. [38] have reported an analytical method-dipalmitoyl-3-linoleoyl-rac-glycerol (16:0-16:0-18:2) on a Chiral-in which enantiomeric 1,2- and 2,3-diacyl-sn-glycerols can be di-cel OD-RH column (150 x 4.6 mm ID, 5µm particles) using―６３―

CHROMATOGRAPHY, Vol.32 No.2 (2011)

be expected since it is effective in HPLC in conjunction with mass

sn-2,3spectrometry (HPLC/MS) and does not need expensive chiral ma-

Aterials and toxic mobile phases. Hexane and dichloromethane (or1,2-dichloroethane), which have frequently been used in chiralphase HPLC, are now considered neurotoxic and carcinogenic, respectively.

sn-1,2Natural glycerolipids consist of various molecular species dif-sn-1,3fering in polarity, which may be exploited by both normal- andreversed-phase chromatography. The glycerolipids with shorter
10

14

18

22

chain lengths and higher degrees of unsaturation are more polarminthan those with longer and more saturated chains. In normal-phase
chromatography, the less polar compounds are eluted ahead of thesn-2,3B

sn-1,2more polar ones, while during reversed-phase chromatography themore polar ones are eluted ahead of the less polar ones. During2chiral-phase HPLC, we found that the glycerolipids were subject to
1.
2.
3.
4.
5.

3
4

both polar and non-polar interactions with CSP, in addition to

18:3-18:2
18:2-18:2
18:1-18:3
18:1-18:2
18:1-18:1

chiral interaction [5]. Thus, chiral-phase HPLC gives complex
chromatograms for natural glycerolipids (see Figure 4). Complete
separation of individual molecular species in both enantiomers canbe obtained when polar capillary gas chromatography [45] or2
51

reversed-phase HPLC [13] is used jointly with chiral-phase HPLC.
3
1

4

Reversed-phase HPLC/ESI-MS provides both sensitive detection

5and identification of the solutes [33,46].
10

20

30

40

50

60

min

3.3 Diastereomeric glycerophospholipids

Figure 5. Direct resolution of isomeric diacylglycerols by chiralreversed-phase HPLC on amylose tris(3,5-dimethylphenylcarbamate). (A) a mixture of enantiomeric (sn1,2 and sn-2,3) and regioisomeric (sn-1,3) dilinoleoylglycerols (18:2-18:2) ; (B) a mixture of enantiomeric 1,2- and 2,3-diacyl-sn-glycerols having various pairs ofacyl groups. Column: Chiralpak IA (250 x 4.6 mm ID, 5µm particles). Mobile phase: CH3CN/MeOH (90:10, byvol); Flow rate: 0.5 mL/min. Column temperature: (A)25℃; (B) 15℃. Detection: ELSD (50℃).

Glycerophospholipids are similar to triacylglycerols in that itis difficult to achieve clear HPLC resolution of stereoisomers in appropriate elution times. One of the reasons is that phospholipids do
not have the suitable functional groups for derivatization used instereoisomer separation by chiral-phase HPLC. However, some
phospholipids with hydroxyl or amino groups, such as lysoPtdCho, PtdGro, and PtdSer, react easily with 3,5-dinitrophenyl
isocyanate to form the corresponding urethane or urea derivatives,
respectively, which are separable on chiral-phase HPLC. For themethanol as the mobile phase (0.5 mL/min at 25℃), but neither

first time, we used chiral-phase HPLC for the resolution of enanti-

1,2-dioleoyl-3-linoleoyl-rac-glycerol (18:1-18:1-18:2) nor 1,2-

omeric and diastereomeric glycerophospholipids during a reassess-

dipalmitoyl-3-stearoyl-rac-glycerol (16:0-16:0-18:1) was resolved.

ment of the stereochemical configuration of natural PtdGro mole-

These techniques used are simple and useful for the determination

cules [47]. For this purpose, synthetic and natural PtdGro samples

of enantiomeric purity of synthetically structured lipids and somewere converted to their bis(3,5-DNPU) derivatives, which were

natural triacylglycerols.

confirmed by electrospray ionizarion (ESI)-MS and then separated

For enantiomer separation of glycerolipids, columns of 150–

by HPLC using two columns having chiral phases of opposite con-

250 mm length and 4–4.6 mm inside diameter are usually used.

figuration, (R )- and (S )-1-(1-naphthyl)ethylmethacrylamide poly-

Separation can be further improved by using the same two or threemers (YMC A-K03 and A-L03). Figure 6 shows chiral-phasecolumns connected in series [22,25]. Capillary columns packed

HPLC profiles of some synthetic and natural PtdGro as bis(3,5-with OA-2100 (300 x 0.32 mm ID) have also been used for enanti-

DNPU) derivatives. Absolute configurations of the resolved peaks

omer resolution of racemic 1-alkylglycerols (glyceryl ethers), α-

were assigned by comparison with the elution order of the corre-hydroxy acids and 1,2-alkanediols [42–44]. We believe that future

sponding 1- and 3-monoacyl-sn-glycerol enantiomers as bis(3,5-

popularization of chiral-phase HPLC using capillary columns can

DNPU) derivatives on the same column. We employed this method―６４―CHROMATOGRAPHY, Vol.32 No.2 (2010)

A

Yutaka Itabashi

sn-3,sn-3’
(R,R)

B

sn-3,sn-3’(R,R)

sn-3,sn-1’
(R,S)

5

10

15

20

sn-3,sn-1’(R,S)

25 min5sn-3,sn-1’
(R,S)

C

10

15

20

25

30 minsn-3,sn-1’
(R,S)

D

sn-3,sn-3’(R,R)

sn-3,sn-3’
(R,R)

5

10

15

BMP

20

25

30 min

5

10

15

20

25

30 minFigure 6. Chiral-phase HPLC profiles of the bis(3,5-dinitrophenylurethane) derivatives of synthetic and natural phosphatidylglycerols(PtdGro). Upper panel: Synthetic PtdGro generated from 1,2-dioleoyl-sn-glycero-3-phosphocholine (A) or salmon roe phosphatidylcholines (B) and glycerol by transphosphatidylation with phospholipase D from Streptomyces septatus TH-2; Lower panel:
PtdGro from Escherichia coli (C) and rat lung (D). sn-3, sn-1’: 1,2-diacyl-sn-glycero-3-phospho-1’-sn-glycerol (R,S configuration) ; sn-3, sn-3’: 1,2-diacyl-sn-glycero-3-phospho-3’-sn-glycerol (R,R configuration); BMP: bis(monoacylglycerol) phosphate.
Column: YMC A-K03 (250 x 4.6 mm ID, 5µm particles). Mobile phase: hexane/CH2Cl2/MeOH/TFA (60:20:20:0.2, by vol, 1 mL/
min). Column temperature: 10℃. Detection: 254 nm UV.

for the determination of the stereochemical configuration of PtdGro

50% R,R and 50% R,S at 50–60℃ to 70% R,R and 30% R,S at 0

prepared by phospholipase D-catalyzed transphosphatidylation℃. Based on these studies, we developed a simple method for syn-from PtdCho and glycerol [48]. All chromatograms obtained forthesizing diastereomerically pure R,R and R,S PtdGro molecules

bacterial phospholipase D products showed two clearly resolved[50]. For this purpose, 1,2-O -isopropylidene PtdGro diastereomers

peaks of different proportions of diastereomers ranging from 30–

were

40% 1,2-diacyl-sn-glycero-3-phospho-1’-sn-glycerol (R,S configu-(PtdCho) and enantiomeric 1,2-O - and 2,3-O -isopropylidene-sn-

ration) and 60–70% 1,2-diacyl-sn-glycer-3-phospho-3’-sn-glycerol

glycerols [51] by transphosphatidylation with phospholipase D

preparedfrom

1,2-diacyl-sn-glycero-3-phosphocholine(R,R configuration). (Figure 6 A, 6 B). The asymmetric propor-

from Actinomadura in acetate buffered diethyl ether at 30℃. The

tions were absent from the diastereomers prepared with cabbage or

isopropylidene protective group was removed by heating the iso-peanut phospholipase D, which gave equimolar mixtures of R,Spropylidene PtdGro diastereomers at 100℃ in trimethylborate in

and R,R diastereomers. It was concluded that bacterial phospholi-

the presence of boric acid [49]. Using the method, we have foundpase D catalyzed transphosphatidylation proceeds to a large extent

that some bacterial [47,52,53] and mammalian PtdGro molecules

stereospecifically, showing a preference for the sn-3’-position ofcontain a significant amount of R,R isomer, of which existence had

the glycerol molecule. We used this method to demonstrate a

not been previously reported (Figure 6 C, 6 D). We have also

marked effect of temperature on the stereoselectivity of phospholi-

found that the R,R isomer in bacteria increased gradually with in-pase D toward the two primary hydroxyl groups of glycerol in thecreasing growth temperature, suggesting adaptation toward envi-

transphosphatidylation reaction of PtdCho to PtdGro [49]. The pro-ronmental change [54]. These studies demonstrated that chiral-

portion of the R,R isomer produced with phospholipase D fromphase HPLC and ESI-MS provided direct and unambiguous infor-

Streptomyces TH-2 and Actinomadura sp. changed gradually frommation about the configuration, identity, and quantity of molecular―６５―CHROMATOGRAPHY, Vol.32 No.2 (2011)

A

though approximately 1% of total PtdSer was detected in rat cere-

L-PtdSerB

D-PtdSerbrum by reversed-phase HPLC of the fluorescent derivatives ofserine liberated from PtdSer by hydrolysis [56].
L-PtdSer

3.4 Enantiomeric ether lipids
Glycerolipids with ether linkages are widely distributed in nature from microorganisms to animals, especially in the liver oil of

10

15

20 minL-PtdSer

C

10

15

20 mindeep-sea sharks. It is known that the ether linkage of the glycerolipids from animals is located at the sn-1 position in the glycerol

L-PtdSerD

skeleton, but is at the sn-3 position in the phospholipids of archaebacterium [57]. In platelet activating factor (PAF, 1-alkyl-2-acetylsn-glycero-3-phosphocholine), the ether linkage at the sn-1 position is essential for expression of activity [58]. The position of an
ether linkage in glycerolipids can be determined by chiral-phase
HPLC. The acyl and/or phosphate groups of ether lipids are hydro-

10

15

20 min10

15

L-PtdSer

E

20 minlyzed to generate monoalkylglycerols (glyceryl ethers). These arethen converted into the bis(3,5-DNPU) derivatives for chiral-phase

L-PtdSerF

HPLC and on-line ESI-MS. Each isomer of 1-, 2-, and 3-alkyl-snglycerols is clearly separated on Sumichiral OA columns [59–61].
The HPLC chromatograms and HPLC/ESI-MS spectra of themonoalkylglycerols derived from diacylalkylglycerols in the liver
oil of the megamouth shark Megachasma pelagios [60] and in the

1015

20 min10

15

20 minmuscle lipids from a deep-sea teleost fish Stromateus stellatus [61]showed no peaks that correspond to sn-2- and sn-3-isomers, dem-

Figure 7. Chiral-phase HPLC profiles of synthetic and natural
phosphatidylserines (PtdSer) as 3,5-dinitrophenylurea
derivatives on polymeric (R )-1-(1-naphthyl)ethylmethacrylamide. (A) a mixture of synthetic L- and D-PtdSer;(B) synthetic L-PtdSer; (C) rat brain; (D) mackerel eye;(E) rat liver; (F) mackerel liver. Column: YMC A-K03(250 x 4.6 mm ID, 5µm particles). Mobile phase: hexane/CH2Cl2/MeOH/TFA (70:20:10:0.2, by vol, 1 mL/
min). Column temperature: 10℃. Detection: 254 nm
UV.

onstrating that the ether linkage occurs only at the sn-1 position of
the glycerol backbone. The enantiomers of alkylacylglycerols anddialkylglycerols have also been well separated on OA-4100 [62]after conversion into their 3,5-DNPU derivatives.
3.5 Enantiomeric lysophosphatidylcholines (lyso-PtdCho)
We have investigated the chiral-phase HPLC resolution of intact lyso-PtdCho (Figure 8) [63]. Lyso-PtdCho is an abundant com-

species in natural and synthetic PtdGro.

B

A

sn-3

sn-3

We have also reported the chiral-phase resolution of

sn-1
sn-1

diastereomeric phosphatidyl-D-serine and L-serine following derivatization with 3,5-dinitrophenyl isocyanate and purification bynormal-phase HPLC on a polyvinyl alcohol bonded silica column(YMC PVA-SIL, 100 x 3 mm ID) [14,55]. Complete resolution ofdiastereomeric L-PtdSer and D-PtdSer was achieved by HPLC on
two CSP of opposite configuration, (R )- and (S )-1-(1-naphthy)60

ethylmethacrylamide polymers (YMC A-K03 and A-L03 columns,

70

80

90 min

70

80

90

100

110 minFigure 8. Chiral-phase HPLC resolution of the 3,5-dinitrophenylurethane derivatives of (A) 1-palmitoyl-rac-glycero-3phosphocholine (2-lyso-PtdCho) and (B) 1-hexadecylrac-glycero-3-phosphocholine (lyso-PAF). Column: two
Chiralcel OD-RH columns (each 150 x 4.6 mm ID, 5µm
particles) in series; Mobile phase: MeOH/H2O (90:10,
by vol) at 0.25 mL/min; Column temperature: 5℃; Detection: 254 nm UV.

each 250 x 4.6 mm ID), using hexane/dichloromethane/methanol/
trifluoroacetic acid (70:20:10:0.2, by vol) as the mobile phase. The
diastereomers eluted with a reversal in the elution order on the twocolumns. Using this method, we have demonstrated that the serinemoiety of the PtdSer isolated from rat brain, rat liver, mackerel eye,and mackerel liver only had the L-configuration (Figure 7), al-―６
６―

CHROMATOGRAPHY, Vol.32 No.2 (2010)

Yutaka Itabashi

ponent of plasma and oxidized low-density lipoprotein (LDL) thatoils and have a high health risk potential because of the epoxy ring

displays several biological activities, some of which may occur

structure. Free glycidol, which may be released from GE during thethrough the PAF receptor [64]. Near baseline resolution of enanti-

digestive process by lipase-catalyzed reactions, is classified as pos-omeric 2-lyso-PtdCho (1-acyl-2-hydroxy-sn-glycero-3-phospho-

sibly carcinogenic [65]. The detection and quantification of GE

choline and 3-acyl-2-hydroxy-sn-1-glycero-1-phosphocholine) as

molecules in refined edible oils have been carried out using HPLC-

3,5-DNPU has been achieved on a CSP column of the reversed-

MS or GC-MS [66]. Although GE has enantiomers (see Figure 1

phase type (Chiralcel OD-RH) (Figure 8A). A good separation wasJ), no studies have been carried out for their enantiomer resolution

also achieved for its alkyl homolog (lyso-PAF) (Figure 8B). The

and composition in refined edible oils. Using chiral normal-phaseanalysis was conducted at 5℃ on two columns connected in series

or chiral reversed-phase HPLC in conjunction with atmospheric(each 150 mm x 4.6 mm ID) using a simple solvent system withpressure chemical ionization (APCI)-MS on Chiralpak IA, we havemethanol/water (90:10, v/v) as the mobile phase. Under the samedemonstrated that the GE molecules in some refined edible oils areconditions, partial separation can be obtained for enantiomeric 1(3)composed of approximately equal amounts of R and S enantiomers-lyso-PtdCho (1-hydroxy-2-acyl-sn-glycero-3-phosphocholine and(Figure 9). A column-switching HPLC/APCI-MS method was de-

3-hydroxy-2-acyl-sn-glycero-1-phosphocholine) as 3,5-DNPU

veloped for this purpose [67]. In the case of chiral normal-phase(chromatograms not shown).

HPLC, an edible diacylglycerol-rich oil (0.6 mg) dissolved in hexane was injected into a silica column (250 x 4.6 mm ID) and GE
was fractionated as a mixture of palmitate (16:0-GE), stearate (18:0

3.6 Enantiomeric glycidol fatty acid esters (GE)Finally, we have investigated the determination of 2,3-epoxy--GE), oleate (18:1-GE), linoleate (18:2-GE), and linolenate (18:3-

1-propanol (glycidol) fatty acid esters, which are undesirable con-

GE). The GE mixture was introduced to the analytical column

taminants formed mainly during the refining processes of edible(Chiralpak IC, 250 x 4 mm ID, 5 µm particles) and individual GE
molecules were separated and detected by on-line APCI-MS. The
HPLC analyses on silica and chiral columns were done underR

isocratic elutions with hexane/2-propanol (99.5:0.5, by vol). Post-

16ᾉ0-GES

column addition of ethanol was used to assure APCI in the positive
40

R

50

60 m inmode [61]. Quantification was successfully performed in multiple
reaction monitoring (MRM) mode using a precursor ion [M + H]+.

S

18ᾉ0-GE

The contents of 16:0-GE, 18:0-GE, 18:1-GE, 18:2-GE, and 18:3GE were 6.6, 2.6, 74, 118, and 49 µg/g, respectively. The total

40

50

Intensity ( cps )R

60 m inS

amount of GE (250 µg/g) was less than that reported previously by
Masukawa et al. (295 µg/g), where a combination of double SPE

18ᾉ1-GEand reversed-phase HPLC/MS was used [66]. The columnswitching HPLC/MS method is simple and rapid and can be util40

50
R

60 m inS

ized for the identification and quantification of GE mixtures invarious refined edible oils. Synthetic racemates of fatty acid mono-18ᾉ2-GEand diesters of 3-chloro-1,2-propanediol, which is a glycidol re40

50

lated substance, can also be clearly resolved into enantiomers by

60 m inR

18ᾉ3-GE

both chiral normal-phase HPLC and chiral reversed-phase HPLCS

on Chiralpak IA columns (250 x 4.6 mm ID, 5 µm particles) (chromatograms not shown).

40

50

60 m in

4. Reversed-phase HPLC of regioisomeric glycerolipids

Figure 9. A column-switching HPLC/APCI-MS profiles of theglycidol fatty acid esters in a diacylglycerol-rich oil. 1
st column: silica (250 x 4.6 mm ID, 5µm particles); 2nd column: Chiralpak IC (250 x 4.6 mm ID); Mobile
phase: isocratic elution with hexane/2-propanol, 99.5:
0.5, by vol) at 5℃ (1 st column) and 20℃ (2nd column). MS: API 2000 Q Trap MS/MS. Scan mode:
MRM (precursor ion, [M + H]+). Injection volume: 20µL (30 mg/mL hexane).

4.1 Diacylglycerol reverse isomers
The 3,5-DNPU derivatives are suitable for the resolution ofregioisomers (reverse isomers) within an enantiomeric class of diacylglycerols (sn-1,2- or sn-2,3-), as well as for the resolution ofenantiomers. The UV absorbing derivatives are readily detected bya spectrophotometer or by a mass spectrometer. The reverse isomers of diacylglycerols (see Figure 1 C, 1 D) having various pairs―６７―

CHROMATOGRAPHY, Vol.32 No.2 (2011)

16:0

A

B

18:3

22:6
16:0

16:0

18:3

C

18:1

22:6

18:1

18:3
18:3

16:0

105

D

115

18:1
16:0

125 min

16:0
18:1

100

110

min

18:1

E

100

16:0

F

18:0

110

14:0

min

14:0
16:0

18:0
18:1

25

35 min50

60 min40

50

60 minFigure 10. Reversed-phase HPLC resolution of the reverse isomers of 1,2-diacyl-rac-glycerols as 3,5-dinitrophenylurethanes. Peak identification: (A) 1-linolenoyl-2-palmitoyl-rac-glycerol and 1-palmitoyl-2-linolenoyl-rac-glycerol; (B) 1-docosahexaenoyl-2-palmitoylrac-glycerol and 1-palmitoyl-2-docosahexaenoyl-rac-glycerol; (C) 1-linolenoyl-2-oleoyl-rac-glycerol and 1-oleoyl-2-linolenoylrac-glycerol; (D) 1-oleoyl-2-palmitoyl-rac-glycerol and 1-palmitoyl-2-oleoyl-rac-glycerol; (E) 1-oleoyl-2-stearoyl-rac-glyceroland 1-stearoyl-2-oleoyl-rac-glycerol; (F) 1-palmitoyl-2-myristoyl-rac-glycerol and 1-myristoyl-2-palmitoyl-rac-glycerol. Column: (upper panel) a fully endcapped ODS, Superspher RP-18e (250 x 4 mm ID, 4µm particles) and isocratic elution with CH3
CN at 18℃; (lower panel) a polymeric non-endcapped ODS, Zorbax Eclipse PAH C18 (150 x 4.6 mm ID, 3.5µm particles) andisocratic elution with MeOH at 30℃. Detection: 254 nm UV.

of acyl groups, including saturated and highly unsaturated chains,verse isomers of very different pairs of acyl groups are clearly re-when chromatographed as the 3,5-DNPU derivatives, give excel-

solved on both endcapped and non-endcapped monomeric ODS

lent separations on highly efficient and fully endcapped ODS col-columns, which retain less polar isomers longer with lower degrees

umns (Figure 10B) and on silver loaded cation-exchange columnsof unsaturation or shorter chain lengths at the sn-1(3) position. The[6]. No satisfactory resolution by any of the HPLC methods, how-

non-endcapped columns, however, markedly shorten the elution

ever, was obtained for the reverse isomers possessing only minor

times of the derivatives compared to the endcapped columns [68].

differences in chain lengths and degrees of unsaturation, such as

Most commercially available ODS columns are fully endcapped topalmitate/linolenate (16:0/18:3) and oleate/linolenate (18:1/18:3)

decrease undesirable influence of residual silanol. Our study, how-

(Figure 10A, 10C). Recently, we have found that these criticalever, demonstrates that non-endcapped ODS phases are more suit-pairs of reverse isomers can be resolved clearly on non-endcappedable for the separation of diacylglycerol reverse isomers than fully

polymeric ODS columns, as shown in Figure 10D–10F. The

endcapped ones.

highly dense packing of octadecyl groups in polymeric ODS and

We have utilized the reversed-phase HPLC method of resolu-

the planar structure of the 3,5-DNPU moiety in the derivatives

tion of reverse isomers of diacylglycerols in a chromatographic

would contribute to such reverse isomer separation. The derivatives

search for reverse isomers in natural diacylglycerophospholipids

with various pairs of acyl groups could deeply penetrate the narrow(Figure 11). For this purpose the glycerophospho- and glycolipids

space between polymeric ODS groups and interact more strongly

were dephosphorylated by phospholipase C and by periodate oxi-with polymeric ODS than with monomeric ODS. Thus, the reverse

dation/hydrazinolysis, respectively. The released diacylglycerols

isomers having minor differences in chain lengths and degrees ofwere converted into the 3,5-DNPU derivatives, which were then re-unsaturation would be clearly resolved on polymeric ODS. The re-solved on the fully endcapped ODS columns (Superspher RP-18e,―６８―

CHROMATOGRAPHY, Vol.32 No.2 (2010)

Yutaka Itabashi

35

A
Relative Abundance (%)

30
25
18:1
20:4

20

18:1
22:6
20:4

15

22:6

18:1

18:1
10
5
0
10

20

30

40

50

60

70

80

90

100

Relative Abundance (%)

100
90

110

120

130

140

150

min

20:5

B

16:0
16:0

80

20:5

70
60
50
40
30
20
10
0

20

30

40

50

60

70

80

90

100 minFigure 11. Reversed-phase HPLC/ESI-Ion Trap MS profiles (TIC chromatograms) of the 3,5-dinitrophenylurethane derivatives of 1,2-diacylsn-glycerols derived from PtdEtn of human red cell (A) and monogalactosyldiacylglycerols of the red alga Porphyra yezoensis(B). Column: Superspher RP-18e (250 x 4 mm ID, 4µm particles). Other HPLC conditions as given in Figure 10.

250 x 4 mm ID, 4 µm particles). Excellent reverse isomer separa-

4.2 Regioisomeric glycerophospholipids

tions were obtained (when present) for 18:0-22:6, 18:1-22:6, 18:0-

We have reported the direct chromatographic resolution of re-

20:5, 18:1-20:4, 18:1-20:5, 18:0-20:4, 16:0-22:6, 16:0-20:4, andgioisomers of intact diacylglycerophospholipids [71] on the same

16:0-20:5 species [4,69,70]. The results confirmed the preferential

reversed-phase column as that used for the resolution of the 3,5-

location of saturated fatty acids in the sn-1- and the polyunsatu-

DNPU derivatives of diacylglycerols (Superspher RP-18e, 250 x 4

rated fatty acids in the sn-2-position as demonstrated by phospholi-

mm ID). Complete resolution was realized for the pairs of 1-

pase A 2 digestion. However, notable exceptions were also seen inpalmitoyl-2-docosahexaenoyl-sn- (16:0-22:6) and 1-docosahexa-

mammalian samples, representing that several % of the total phos-

enoyl-2-palmitoyl-sn- (22:6-16:0), as well as for 1-palmitoyl-

pholipid class (phosphatidylethanolamine, PtdEtn) was in the re-2-eicosapentaenoyl-sn- (16:0-20:5) and 1-eicosapentaenoyl-2-

verse isomer form (Figure 11A). In contrast, the results also con-palmitoyl-sn- (20:5-16:0), and 1-palmitoyl-2-arachidonoyl-sn- (16:firmed the preferential location of polyunsaturated fatty acids in the

0-20:4) and 1-arachidonoyl-2-palmitoyl-sn-glycero-3-phosphocho-

sn-1-position in seaweed glycolipids, representing that the relativelines (20:4-16:0). The separations were effected using acetonitrile/

content of 20:5-16:0 and 16:0-20:5 in total monogalactosyldiacyl-

methanol/water/triethylamine (65:30:5:0.1, by vol) as the mobile

glycerols (MGDG) were 7.4% and 2.4%, respectively (Figure 11

phase.

B). This method provided the first direct demonstration of the posi4.3 Regioisomeric triacylglycerols

tional distribution of fatty acids in individual molecular species of

The determination of triacylglycerol regioisomers and enanti-natural glycerophospho- and glycolipids.

omers has always been a challenging analytical task. It plays an important part in the understanding of the role of triacylglycerol struc-―６９―CHROMATOGRAPHY, Vol.32 No.2 (2011)

5. Conclusion

ture in lipid metabolism and is essential for full characterization of
triacylglycerols in lipid-containing foods and food additives. Reso-

This review describes that chiral-phase and reversed-phaselution of regioisomeric triacylglycerols has been obtained only re-

HPLC separations of synthetic mono- and diacylglycerol enanti-

cently using highly efficient reversed-phase HPLC columns. We

omers and synthetic diacylglycerol reverse isomers, which wereported the resolution of some regioisomeric disaturated anddemonstrated in the 1980s and 2000s, respectively, can be readily

monounsaturated triacylglycerols by reversed-phase HPLC [72]. A

extended to the resolution of naturally occurring enantiomericliquid chromatograph equipped with an evaporative light scatteringmono- and diacylglycerols and regioisomeric acylglycerols derived

detector (operating temperature 50℃, 2.4 bar) and a fully end-

from complex lipids. A combination of chiral-phase HPLC andcapped Superspher 100 RP-18e column (250 x 4 mm ID, 4 µm par-reversed-phase HPLC with on-line MS permits the identification of

ticles) was used. Three to six µg mixtures of the corresponding tri-

the molecular species of enantiomers, diastereomers, and reverse

acylglycerol isomers were injected using a 10 µL injection loop.

isomers. A practical consequence of this development has been an

The analyses were performed isocratically at 18℃ using mobile

accurate determination of the stereospecific positional placement ofphases of acetonitrile modified with various solvents and at various

fatty acids in all glycerolipids. The development of improved CSP

flow rates as required. Regioisomeric pairs with saturated and un-

and reversed-phase columns along with optimized chromatographic

saturated acyl groups were prepared by esterification of corre-

elution conditions has permitted the further extension of chiral-sponding diacylglycerols and free fatty acids in the presence of N,Nphase and reversed-phase HPLC resolution of enantiomers,-dicyclohexylcarbodiimide. Specifically separated triacylglycerol

diastereomers, and reverse isomers of intact glycerophospholipids.

species were the following pairs: 1,3-dipamitoyl-2-oleoylglycerol

However, there still exist some compounds for which enantiomer(16:0-18:1-16:0)/1,2(2,3)-dipalmitoyloleoylglycerol (16:0-16:0-18:

resolution and regioisomer resolution are difficult to achieve, such1), 1,3-dipamitoyl-2-linoleoylglycerol (16:0-18:2-16:0)/1,2(2,3)-

as PtdCho and PtdEtn. With the development of new derivatization

dipalmitoyllinoleoylglycerol (16:0-16:0-18:2), 1,3-dipalmitoyl-2-

reagents and chromatographic techniques, these difficult com-eicosapentaenoyglycerol(16:0-20:5-16:0)/1,2(2,3)-dipalmitoyle-pounds should not be hard to resolve in the near future. Organisms

icosapentaenoylglycerol (16:0-16:0-20:5) and 1,3-dipalmitoyl-2-

usually maintain the chirality of their constituents, but some excep-

docosahexaenoylglycerol(16:0-22:6-16:0)/1,2(2,3)-dipalmitoyl-

tions have been recently found in amino acids and phospholipids.

docosahexaenoylglycerol (16:0-16:0-22:6). The resolution im-

We believe that chiral-phase and reversed-phase HPLC will be-

proved with increasing number of double bonds in the acyl resi-

come much more important in the near future as a valuable tool fordues. While 16:0-18:1-16:0 and 16:0-16:0-22:6 were only partiallystudying

resolved, 16:0-22:6-16:0 and 16:0-16:0-22:6 were fully separated

diastereomers, and regioisomers of glycerolipids.

thephysiologicalsignificanceofenantiomers ,with all tested mobile phases except those containing methanol. We
Acknowledgements

also reported the separation of the regioisomeric 16:0-18:3-16:0/
16:0-16:0-18:3 and 16:0-20:4-16:0/16:0-16:0-20:4 pairs of triacyl-

The author wishes to thank the late Professor Toru Takagi

glycerols on the Superspher RP-18e columns (250 x 4 mm ID, 4(Hokkaido University) and Professor Arnis Kuksis (University ofµm particles) [73]. Separated species also included the four triacyl-

Toronto) for their help and advice regarding the subject of this re-glycerols with ECN = 42: 16:0-20:5-16:0, 16:0-16:0-20:5, 16:0-22:

view. The author also thanks Professor Takao Tsuda (Nagoya Insti-

6-16:0, and 16:0-16:0-22:6, on a single reversed-phase HPLC col-tute of Technology), Professor Kanji Miyabe (Toyama University)

umn with mobile phase acetonitrile/2-propanol (70:30, by vol) atand Dr. Naobumi Oi (Chiralchromatography Laboratory) for their

0.8 mL/min. In all cases the isomer with the unsaturated acyl resi-

continued interest and encouragement. Thanks are due to all col-due in either the 1- or 3-position was retained more strongly thanlaborators, co-workers, and colleagues of his studies.

the corresponding 2-isomer. This may be due to a residual polarity

Financial supports from the Ministry of Education, Science,of the reversed-phase column, which would interact more strongly

Sports and Culture of Japan and the Research Fellowships of thewith the more polar unsaturated fatty acids located in the more ac-

Japan Society for the Promotion of Science, as well as from com-cessible primary positions of the triacylglycerol molecule. Re-

panies are gratefully acknowledged. The work in Canada was sup-cently, Nagai et al. reported a rapid separation of regioisomeric tri-

ported by the Heart and Stroke Foundation of Ontario, Toronto,

acylglycerols with two saturated fatty acids in the molecules using

Canada and the Medical Research Council of Canada, Ottawa.

an octacosyl (C 28) column [74]. Good resolution has been

Finally, the author is grateful to the Society for Chroma-achieved for 16:0-18:1-16:0/16:0-16:0-18:1, 16:0-22:6-16:0/16:0-

tographic Science for selecting him as a recipient of the 2010

16:0-22:6, and 16:0-18:1-18:0/18:1-18:0-16:0 within 30 min afterAward of the Society for Chromatographic Science.

injection using acetone as the mobile phase at 10–15℃.

―７０―

CHROMATOGRAPHY, Vol.32 No.2 (2010)

Yutaka Itabashi

1198-1199, 67-72.

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