Liquid chromatography-tandem mass spectrometry determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers via promotion of sodium adduct formation

Article abstract of DOI:10.1016/j.ab.2014.10.017

Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in blood plasma has been observed in various pathological conditions, including atherosclerosis. In this study, we investigated the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method for accurate quantification of PCOOH (1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC), focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH ([M+Na]⁺, m/z 812) provided not only a known product ion (m/z 147) but also characteristic product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Thus, three multiple reaction monitorings (MRMs) could be performed. MRM (812/147) enabled determination of 16:0/HpODE PC, and MRM (812/541) and MRM (812/388) allowed specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this method, we could determine plasma PCOOH concentrations in healthy subjects and patients with angiographically significant stenosis. In healthy subject and patient plasma, the concentration of 16:0/HpODE PC was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC. This finding shows that radical and/or enzymatic oxidation, rather than singlet oxygen oxidation, is recognized to cause peroxidation of PC. The newly developed LC-MS/MS method appears to be a powerful tool for developing a better understanding of in vivo lipid peroxidation and its involvement in human diseases.

Full text of DOI:10.1016/j.ab.2014.10.017

Accepted Manuscript
LC-MS/MS determination of human plasma 1-palmitoyl-2-hydroperoxyocta-
decadienoyl-phosphatidylcholine isomers via promotion of sodium adduct for-
mation
Shunji Kato, Kiyotaka Nakagawa, Yuuri Suzuki, Akira Asai, Mototsugu Nagao,
Kazuyuki Nagashima, Shinichi Oikawa, Teruo Miyazawa
PII:
S0003-2697(14)00507-7
DOI:
http://dx.doi.org/10.1016/j.ab.2014.10.017
YABIO 11896
Reference:
Analytical Biochemistry
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Revised Date:
Accepted Date:
4 September 2014
27 October 2014
28 October 2014
Please cite this article as: S. Kato, K. Nakagawa, Y. Suzuki, A. Asai, M. Nagao, K. Nagashima, S. Oikawa, T.
Miyazawa, LC-MS/MS determination of human plasma 1-palmitoyl-2-hydroperoxyoctadecadienoyl-
phosphatidylcholine isomers via promotion of sodium adduct formation, Analytical Biochemistry (2014), doi: http://
dx.doi.org/10.1016/j.ab.2014.10.017
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Analytical Biochemistry
LC-MS/MS determination of human plasma
1-palmitoyl-2-hydroperoxyoctadecadienoyl-phosphatidylcholine isomers
via promotion of sodium adduct formation
Shunji Kato1, Kiyotaka Nakagawa1, Yuuri Suzuki1, Akira Asai2, Mototsugu Nagao2,
Kazuyuki Nagashima3, Shinichi Oikawa2, Teruo Miyazawa1,*
1Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science,
Tohoku University, Sendai 981-8555, Japan
2Division of Endocrinology and Metabolism, Department of Medicine, Nippon Medical
School, Tokyo 113-8603, Japan
3The Cardiovascular Institute, Tokyo 106-0031, Japan
Short title: LC-MS/MS analysis of PCOOH
Subject category: Lipids, Lipoproteins and Prostaglandins
*Corresponding author: Food and Biodynamic Chemistry Laboratory, Graduate
School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi,
Aoba-ku, Sendai 981-8555, Japan. Fax: 81-22-717-8905.
E-mail address: miyazawa@biochem.tohoku.ac.jp (T. Miyazawa)
1

A B S T R A C T
Accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product
of phosphatidylcholine, in blood plasma has been observed in various pathological conditions,
including atherosclerosis. In this study, we investigated the use of LC-MS/MS to develop a
method for accurate quantification of PCOOH
(1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC),
focusing on isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC. Sodiated PCOOH
([M+Na]+, m/z 812) provided not only a known product ion (m/z 147), but also characteristic
product ions (m/z 541 for 16:0/13-HpODE PC and m/z 388 for 16:0/9-HpODE PC). Three
multiple reaction monitorings (MRMs) could thus be performed. MRM (812/147) enabled
determination of 16:0/HpODE PC, and MRM (812/541 and 812/388) allowed specific
measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC, respectively. By using this
method, we could determine plasma PCOOH concentrations in healthy and patients with
angiographically significant stenosis. In both plasma, the concentration of 16:0/HpODE PC
was close to the sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC.
This finding shows that radical- and/or enzymatic-oxidation, rather than singlet
oxygen-oxidation, are recognized to cause peroxidation of PC. The newly developed
LC-MS/MS method appears to be a powerful tool for developing a better understanding of in
vivo lipid peroxidation and its involvement in human diseases.
Keywords: Phosphatidylcholine hydroperoxide; Hydroperoxide positional isomer; Tandem
mass spectrometry; Human blood plasma; Atherosclerosis; Stenosis
2

Abbreviations: MxP, 2-methoxypropene; BHT, butylated hydroxytoluene; CID,
collision-induced dissociation; ESI, electrospray ionization; HpODE,
hydroperoxyoctadecadienoyl; ICAM-1, intracellular adhesion molecule-1; MRM, multiple
reaction monitoring; PC, phosphatidylcholine; PCOOH, phosphatidylcholine hydroperoxide
3

Oxidative lipid modifications play important roles in various pathological conditions,
including atherosclerosis [1–4]. Among oxidatively modified lipids, phosphatidylcholine
hydroperoxide (PCOOH) (Fig. 1), a primary oxidation product of phosphatidylcholine (PC),
is known to accumulate in arterial walls and blood plasma in atherosclerotic rabbits [5].
PCOOH was also identified in human atherosclerotic lesions [6, 7]. Furthermore, PCOOH
accumulation in the plasma has been found in human subjects with pathological conditions,
such as hyperlipidemia [8, 9], diabetes [10], and Alzheimer’s disease [11]. Because
hyperlipidemia and diabetes are strongly associated with increased incidence of
atherosclerosis [12–14], higher plasma PCOOH in these patients is recognized to be involved
in atherogenesis. As for the possible mechanism, our previous study showed that PCOOH
induced THP-1 monocytic cell adhesion to intracellular adhesion molecule-1 (ICAM-1) via
actin cytoskeletal polymerization and subsequent localization of lymphocyte
function-associated antigen-1 [15, 16]. Further, we reported the involvement of Rac GTPase
activation in the PCOOH-induced cell adhesion to ICAM-1 via actin polymerization. The
Rho-family GTPase-dependent actin cytoskeleton organization is involved in various cellular
processes such as adhesion. Via these mechanisms, phospholipid peroxidation may play a
crucial role in atherogenic progress. Therefore, accurate PCOOH determination is key to
understanding the pathophysiology of oxidative stress-related diseases, such as
atherosclerosis.
PCOOH can be quantified by liquid chromatography (LC)-chemiluminescence
detection (hydroperoxide base-specific method) [5, 8–11, 17–20], LC-mass spectrometry
(LC-MS) [7, 20], and LC-tandem mass spectrometry (LC-MS/MS) [21, 22]. The
concentrations of PCOOH have been shown to range from 55 to 227 pmol/ml in the plasma
of healthy subjects [17, 20], though some reports have failed to detect PCOOH in human
plasma [23, 24]. These conflicting results suggest the need for further investigation of
analytical concerns, such as detection capability and extraction recovery, to develop
4

additional, more reliable methods.
In recent years, plasma PCOOH concentrations have been measured at the molecular
species level using LC-MS/MS. Hui et al. reported that PCOOH
(1-palmitoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 16:0/HpODE PC)
and (1-stearoyl-2-hydroperoxyoctadecadienoyl-sn-glycero-3-phosphocholine, 18:0/HpODE
PC) concentrations in plasma samples were 89 and 32 pmol/ml, respectively [21], although
these authors did not mention the position of the hydroperoxide group in the fatty acid. As the
determination of hydroperoxide position can be helpful in the elucidation of the in vivo lipid
peroxidation mechanisms (i.e. radical-, enzyme-, and/or singlet oxygen-oxidation) [25],
analysis of these PCOOH isomers analysis has been hoped for the investigation of oxidative
stress-related pathophysiology.
In the present study, we used LC-MS/MS to investigate the above analytical
concerns (e.g., detection capability and extraction recovery), focusing on a representative
PCOOH molecular species (16:0/HpODE PC) in plasma [21, 22]. Moreover, we investigated
various analytical conditions in order to quantify major 16:0/HpODE PC isomers such as
1-palmitoyl-2-(13-hydroperoxyoctadecadienoyl)-sn-glycero-3-phosphocholine
(16:0/13-HpODE PC) and
1-palmitoyl-2-(9-hydroperoxyoctadecadienoyl)-sn-glycero-3-phosphocholine
(16:0/9-HpODE PC) in plasma from healthy subjects and patients with angiographically
significant stenosis.
Materials and methods
Materials
1-Palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0/18:2 PC) and lysoPC
5

(16:0) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Methyl linoleate was
obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of the highest
grade available.
Preparation of 16:0/13-HpODE PC and 16:0/9-HpODE PC
16:0/13-HpODE PC (Fig. 1) was prepared from 16:0/18:2 PC using soybean
lipoxygenase-1 [26, 27]. 16:0/9-HpODE PC was synthetically prepared as follows: linoleic
acid methyl ester was oxidized for 24 hours at 40°C. Among the oxidation products,
9-hydroperoxyoctadecadienoic acid methyl ester was chromatographically fractionated [28]
and its hydroperoxide group was protected by 2-methoxypropene (MxP) [26, 27]. The
protected methyl ester was purified and saponified in base solution to yield
9-hydroperoxyoctadecadienoic acid, in which the hydroperoxide group was protected by MxP.
The protected fatty acid hydroperoxide was purified and esterified with lysoPC (16:0) in
chloroform containing N, N'-dicyclohexylcarbodiimide and dimethylaminopyridine under N2
atmosphere for 24 hours [29]. Synthesized diacylphosphocholine was finally deprotected, and
the resultant 16:0/9-HpODE PC was chromatographically purified [26, 27].
MS/MS and LC-MS/MS
A 4000 QTRAP quadrupole/linear ion-trap tandem mass spectrometer (AB SCIEX,
Tokyo, Japan) was used for MS/MS analysis. Initially, we analyzed the prepared reference
PCOOH (16:0/13-HpODE PC or 16:0/9-HpODE PC). To evaluate MS/MS fragmentation,
product ion scanning was performed via direct infusion of the PCOOH solution (1 nmol/ml
methanol) into the MS/MS system (10 µl/min). Electrospray ionization (ESI) was used as an
ion source with a collision energy of 61 eV, a turbo gas temperature of 700°C, and a spray
6

voltage of 5500 V. Nitrogen pressure values for turbo, nebulizer, and curtain gases were set at
80, 70, and 20 psi, respectively. Positive ion spectra were collected in the m/z range of
100–1000.
By using the observed fragments, MRM of PCOOH (16:0/13-HpODE PC or
16:0/9-HpODE PC) was performed on an LC-MS/MS system. For LC-MS/MS analysis, a
Shimadzu liquid chromatography system, including a vacuum degasser, a quaternary pump,
and an autosampler (Shimadzu, Kyoto, Japan), was equipped with a 4000 QTRAP mass
spectrometer. PCOOH was analyzed using an ODS column (Atlantis T3 column, 3.5 µm, 2.1
× 100 mm; Waters, Tokyo, Japan) with a binary gradient consisting of solvent A (methanol
containing 5 mM ammonium acetate or 0.1 mM sodium acetate) and solvent B (water
containing 5 mM ammonium acetate or 0.1 mM sodium acetate). The gradient profile was as
follows: 0–4 min, 70%–90% B linear; 4–10 min, 90% B; 10–17 min, 90%–100% B linear;
17–30 min, 100% B; 30–30.1 min, 100%–70% B linear; 30.1–35.0 min, 70% B. The flow
rate was 0.2 ml/min, and the column temperature was 40°C. PCOOH isomers were detected
using MRM for the transition of parent ions to product ions (Table 1), and external standard
curves were individually constructed.
Extraction of PCOOH from plasma
Blood (10 ml) was collected from 3 healthy subjects (mean age, 24 2 years) into a
tube containing heparin as an anticoagulant. Plasma was prepared from the blood by
centrifugation at 1,000 × g for 10 min at 4°C. Plasma samples from 3 subjects were pooled
and used in the following studies.
To effectively extract PCOOH from plasma samples, different extraction conditions
were examined (Scheme 1) as follows:
Step 1: plasma (0.3 ml) was diluted with 0.3 ml of 0%–5.0% KCl aqueous solution.
7

Step 2: total lipids were extracted from the diluted plasma using 2.4 ml
chloroform:methanol (1:1 or 2:1, v/v) with 0.002% butylated hydroxytoluene (BHT).
The extract was partitioned by centrifugation at 1,000 × g for 20 min at 25, 4, or -20°C
into 2 layers: the chloroform layer (lower organic layer) and the methanol-water layer
(upper layer). The lower chloroform layer (lipid fraction) was collected. The remaining
aqueous layer containing a semisolid interface was re-extracted with Folch’s theoretical
lower phase [30] and subjected to centrifugation at 1,000 × g for 20 min at 25, 4, or
-20°C. The combined lipid fraction was either rinsed with Folch’s theoretical upper
phase or left without rinsing. Similarly, lipids were extracted by mixing the diluted
plasma with 2.4 ml chloroform:methanol (1:2, v/v) or 100% methanol containing
0.002% BHT. The mixture was centrifuged at 1,000 × g for 20 min and the supernatant
was collected. The extraction was repeated, and the supernatants containing lipid
moieties were combined.
Step 3: the lipid fraction was evaporated and dried under nitrogen gas.
Step 4: the dried extract was redissolved in 0.24 ml chloroform:isopropanol (2:1, v/v),
and 0.04 ml of this mixture was loaded onto an aminopropyl Sep-Pak cartridge (Waters)
equilibrated in chloroform:isopropanol (2:1, v/v). The cartridge was rinsed with 0.75 ml
chloroform:isopropanol (2:1, v/v). PCOOH was eluted with 1.5 ml methanol. The
eluent was evaporated, and the residue was dissolved in 0.2 ml methanol.
Step 5: a 10-µl final aliquot was injected into the LC-MS/MS system.
To evaluate the extraction efficiency, we spiked standard PCOOH (120 pmol
16:0/13-HpODE PC/10 µl methanol) into the plasma (Step 1), lipid fraction (Step 2), total
lipids (Step 3), dried extract (Step 4), or final aliquot (Step 5; see Scheme 1). PCOOH was
then extracted and quantified by LC-MS/MS, and extraction recoveries were calculated using
equation corresponding to the external standard curves.
8

Matrix effect
The ion suppression effect was evaluated by injecting either the plasma extract or
mobile phase (10 µl) into the LC-MS/MS system in parallel with continuous post-column
infusion of the PCOOH standard solution (0.1 pmol 16:0/13-HpODE PC or 16:0/9-HpODE
PC/ml methanol) at 10 µl/min.
Plasma PCOOH in healthy subjects and patients with stenosis
After optimization of the method, we measured the concentration of plasma PCOOH
in healthy subjects and patients with angiographically significant stenosis (≥ 50% in at least
one major coronary vessel). Blood samples were collected from 8 healthy subjects (2 women
and 6 men, mean age 25 2 years) and 12 patients (3 women and 9 men, mean age 69 11
years). Plasma samples were prepared from heparinized blood via centrifugation at 1,000 × g
for 10 min at 4°C, and plasma PCOOH was extracted and quantified under optimized
conditions. Plasma PCOOH concentrations were calculated using equation corresponding to
the external standard curves. This study was approved by the institutional review board of the
Cardiovascular Institute (Tokyo, Japan). Informed consent was obtained from all participants.
Statistics
Data are expressed as means SDs. For comparisons between 2 groups, statistically
significant differences were determined by Student’s t-tests. To compare an overall difference
among 3 or more groups, one-wayANOVA was used. If a statistically significant difference
was detected, the Turkey-Kramer test was performed for comparison between individual
groups. Differences were considered significant at P < 0.05.
9

Results
Preparation of authentic 16:0/13-HpODE PC and 16:0/9-HpODE PC
PCOOH (16:0/13-HpODE PC) was enzymatically synthesized from PC (16:0/18:2
PC) using soybean lipoxygenase-1 and chromatographically purified to yield essentially pure
PCOOH (Fig. 1) [26, 27]. The structure of PCOOH was confirmed by NMR (data not shown).
Another PCOOH isomer (16:0/9-HpODE PC) was prepared by esterifying the
9-hydroperoxyoctadecadienoyl group into lysoPC (16:0).
New MRM programs for PCOOH detection
MS analysis was carried out using the synthesized PCOOH (16:0/13-HpODE PC or
16:0/9-HpODE PC) as a reference compound. [M+H]+ and [M+Na]+ ions were detected at
m/z 790 and 812, respectively, by positive ESI (Figs. 2A, B). Collision-induced dissociation
(CID) of [M+H]+ produced fragment ions including [M+H-H2O]+ (m/z 772), [M+H-H2O-O]+
(m/z 756), and choline phosphate ion (m/z 184) (Figs. 2C, D). It was found that CID of
[M+Na]+ yielded unique neutral losses (i.e., 88 Da for 16:0/13-HpODE PC and 169 Da for
16:0/9-HpODE PC). For example, product ion (m/z 541) was generated from
16:0/13-HpODE PC through the neutral loss of 88 Da and phosphatidylcholine (Fig. 2E).
Whereas CID of 16:0/9-HpODE PC produced a product ion (m/z 388) through the neutral
loss of 169 Da and 255 Da (corresponding to the palmitic acid) (Fig. 2F). Sodiated
cyclophosphane (m/z 147) was observed in both spectra [31-33].
Based on these results, the authentic PCOOH was analyzed with LC-MS/MS under
various MRM programs (e.g., 790/184, 812/541, 812/388, and 812/147). While MRM
enabled detection of PCOOH, the peak intensity was found to be affected by Na+ in the
10

mobile phase (Figs. 3A, B). Thus, we added extra Na+ (i.e., 0.1 mM sodium acetate) to the
mobile phase, and achieved stable and quantitative detection of sodiated PCOOH by MRM
(812/541, 812/388, and 812/147) (Figs. 3C, D). MRM (812/541 and 812/388) could detect
PCOOH only if there was a hydroperoxide group at the 13- or 9-position, respectively, of
linoleic acid in the PCOOH molecule. MRM (812/147) enabled detection of PCOOH,
regardless of the position of the hydroperoxide group in linoleic acid. Under optimized
conditions, calibration curves showed good linearity within the range of 0.01 to 10
pmol/injection (Fig. 4).
Extraction of PCOOH from plasma
Next, we investigated PCOOH extraction from plasma samples. PCOOH
(16:0/13-HpODE PC) spiked into plasma (Step 1 of Scheme 1) was extracted under different
extraction conditions to check PCOOH recovery. Higher PCOOH recovery was obtained
when chloroform:methanol (1:1 and 2:1, v/v) was used as an extraction solvent (Fig. 5A).
When plasma was diluted with 0-1% KCl followed by extraction with chloroform:methanol
(2:1, v/v), higher PCOOH recovery was observed (Fig. 5B). A somewhat lower PCOOH
recovery was found when the combined lipid fraction was rinsed with Folch’s theoretical
upper phase (Fig. 5C). PCOOH recovery was not affected by extraction temperature (Fig.
5D).
In this study, the optimal extraction conditions were as follows: plasma (300 µl) was
diluted with water (0% KCl, 300 µl) and mixed with 2.4 ml chloroform:methanol (2:1, v/v) at
4°C. The lipid fraction was collected, and the remaining layer was re-extracted with Folch’s
theoretical lower phase. The combined lipid fraction was evaporated and applied to
solid-phase extraction. As shown by the recovery of PCOOH spiked into each step of the
extraction (Scheme 1, Fig. 5E), our method enabled efficient and quantitative extraction of
11

PCOOH from plasma. Higher recovery was also observed when 16:0/9-HpODE PC spiked in
plasma was extracted by the optimal extraction conditions (data not shown). Consistent with
the quantitative results, the matrix effect was not observed following injection of plasma
extracts into the LC-MS/MS system in parallel with continuous post-column infusion of
reference PCOOH (16:0/13-HpODE PC or 16:0/9-HpODE PC) (Fig. 6).
Quantification of PCOOH in plasma samples from healthy subjects and patients
Under optimized quantification procedures (e.g., detection and extraction conditions),
we measured PCOOH in plasma samples of healthy subjects. A very clear PCOOH peak was
detected at 17 min, especially for MRM 812/541 and 812/388 (Fig. 7). Plasma PCOOH
concentrations in healthy subjects were calculated to be 36.1 11.5 pmol/ml (means SDs, n
= 8) for 16:0/13-HpODE PC (MRM 812/541) and 33.1 10.2 pmol/ml for 16:0/9-HpODE
PC (MRM 812/388), while a higher concentration (72.3 23.5 pmol/ml) was observed for
MRM (812/147) (Table 2 and supplementary Table I). In patients with angiographically
significant stenosis, the PCOOH concentrations were 52.4 24.6 (812/541), 45.2 18.1
(812/388), and 97.3 39.5 (812/147) pmol/ml (means SDs, n = 12); these concentrations
tended to be higher than those of healthy subjects (P = 0.06-0.13). In healthy and patient
plasma, the concentration of 16:0/HpODE PC was found to be close to the sum of the
concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC.
Discussion
As mentioned in the Introduction, several studies have investigated the physiological
concentrations of PCOOH, particularly in human plasma, due to its possible contribution to
pathological conditions. Using synthesized authentic 16:0/13-HpODE PC and 16:0/9-HpODE
PC, we developed an LC-MS/MS method to analyze PCOOH to obtain quantitative data and
12

information on the position of the hydroperoxide group.
Analysis of authentic 16:0/13-HpODE PC and 16:0/9-HpODE PC by QTRAP
MS/MS yielded the same product ion mass spectra, and generated a well-known product ion
m/z 184 (phosphocholine) (Figs. 2C, D). This fragment ion (m/z 184) is commonly used for
LC-MS/MS MRM (790/184) analysis of PCOOH [21, 22]; however, MRM (790/184) does
not give the structural information of the fatty acid moiety. On the other hand, sodiated
PCOOH (m/z 812) generated unique product ions (i.e., m/z 541 for 16:0/13-HpODE PC and
m/z 388 for 16:0/9-HpODE PC). Therefore, three MRM of sodiated PCOOH could be
performed via LC-MS/MS analysis (Figs. 3A, B). MRM (812/541 and 812/388) allowed
specific measurement of 16:0/13-HpODE PC and 16:0/9-HpODE PC only if there was a
hydroperoxide group at the 13- and 9-position of the fatty acid, respectively. MRM (812/147)
enabled detection of 16:0/HpODE PC, regardless of the position of the hydroperoxide group
in the fatty acid of the PCOOH molecule. Nevertheless, PCOOH peak intensities were easily
affected by instrumental conditions in LC-MS/MS MRM (812/541, 812/388, 812/147),
especially metals (Na+) in the flow path (and/or ion source). In the present study, stable and
quantitative detection of PCOOH was achieved by addition of Na+ to the mobile phase (Figs.
3C, D). Generally, nonvolatile salts containing Na+ put a burden on the MS instrument [34].
In the present study, PCOOH and other matrix compounds were separated as much as
possible, and the amount of Na+ could be reduced to only 0.1 mM in the mobile phase.
Despite the use of a nonvolatile salt (sodium acetate), our method could provide hundreds of
hours of trouble-free operation and allow stable continuous analysis over 400 samples. Under
optimized conditions, the external standard curves showed good linearity. It is worthy to say
that when 16:0/13-HpODE PC and 16:0/9-HpODE PC were analyzed with MRM (812/147),
there was no difference in their standard curves (Figs. 4A, B). This means that MRM
(812/147) actually enables determination of all 16:0/HpODE PC isomers. With optimized
methods, the detection limit of PCOOH was either superior to, or in the same range of,
13

previous LC-MS/MS methods [21, 22]. Based on these results (Figs. 2–4), we expect that this
newly developed LC-MS/MS MRM could become a powerful tool for providing accurate
quantitative data, as well as structural information of PCOOH.
Our group previously developed an LC-chemiluminescence method and reported a
mean plasma PCOOH concentration of 227 pmol/ml [17], while Yamamoto et al. detected no
PCOOH in human plasma [23, 24]. The reason for the differences observed in the reported
PCOOH concentrations may be rooted in the extraction conditions, particularly considering
the fact that we failed to detect PCOOH in plasma extracts prepared using 100% MeOH as an
extraction solvent in previous experiments (Fig. 5) [23, 24]. PCOOH has been reported to be
easily decomposed by several factors, such as acids [35], metal ions [36], and temperature
[37]. Thus, we spiked standard PCOOH (16:0/13-HpODE PC) into plasma and checked
PCOOH recovery under different extraction conditions (Scheme 1). We found that PCOOH
recovery was notably affected by the extraction solvent (Fig. 5A). In addition, recovery was
low when 5% KCl was used for plasma dilution before extraction with chloroform:methanol,
although such concentrations of KCl have been frequently used for total lipid extraction from
biological samples [30]. Unexpectedly, recovery was not affected by extraction temperature.
These results suggest that PCOOH can be stably extracted by avoiding contact of PCOOH
with water-soluble materials (e.g., metals) in the plasma during extraction procedures.
Moreover, these results suggest that the present extraction procedures and LC-MS/MS MRM
assays not only allowed accurate quantification, but also provided useful structural
information of the analyte, even in the presence of background contaminants from plasma.
Our method exhibited the ability to detect 16:0/13-HpODE PC and 16:0/9-HpODE
PC in human plasma (Fig. 7). MRM (812/147) detected some peaks close to the retention
time of PCOOH; these peaks may be attributed to other PCOOH molecular species, other PC
oxidation products (e.g., phosphatidylcholine hydroxide), and nonoxidized PC, because this
product ion (147) originated from a PC backbone, not from a hydroperoxide group on a fatty
14

acid chain. Nevertheless, PCOOH detection using PC backbone-derived product ions has
generally been performed [21, 22]. In contrast, very clear chromatograms were obtained for
MRM (812/541 and 812/388). It should be noted that the plasma PCOOH (16:0/13-HpODE
PC PC) peak (17.6 min) would be consisted of two cis-trans isomers
(13-hydroperxyoctadeca-9Z, 11E-dienoyl PC and 13-hydroperxyoctadeca-9E, 11E-dienoyl
PC), whereas two peaks of PCOOH (16:0/9-HpODE PC) would represent
9-hydroperxyoctadeca-10E, 12Z-dienoyl PC (17.4 min) and 9-hydroperxyoctadeca-10E,
12E-dienoyl PC (17.6 min). Quantification of PCOOH (16:0/9-HpODE PC) was carried out
based on the total peak area of both peaks.
In patients with angiographically significant stenosis, the PCOOH concentration
tended to be higher than those of healthy subjects (Table 2 and supplementary Table I),
suggesting the involvement of phospholipid peroxidation in the development of
atherosclerosis. Accumulation of PCOOH such as 16:0/13-HpODE PC in plasma may cause
the development of atherosclerosis, as our in vitro cell culture studies showed that
16:0/13-HpODE PC induced atherogenic responses (i.e., monocyte adhesion and
angiogenesis induction). Future trials are needed to further investigate these possibilities (e.g.,
age-matched control studies), as the patients participating in this study were 69 11 years of
age, and age is thought to correlate with increased phospholipid peroxidation [8]. While these
data are still preliminary, the concentration of 16:0/HpODE PC was found to be close to the
sum of the concentrations of 16:0/13-HpODE PC and 16:0/9-HpODE PC in healthy and
patient plasma (Table 2 and supplementary Table I). Considering these concentrations, other
isomers (e.g., 16:0/10- and 12-HpODE PC) would scarcely exist in human plasma. Lack of
16:0/10- and 12-HpODE PC might provide insight into biochemical processes that initiate
phospholipid peroxidation in the plasma lipoprotein membrane. It is possible that radical-
and/or enzymatic-oxidation, rather than singlet oxygen-oxidation, are apt to cause
peroxidation of unsaturated fatty acid residues of PC. Thus, new method is required to
15

distinguish radical- and enzymatic-oxidation in future.
As mentioned above, there are published reports about LC-MS/MS determination
method of PCOOH [21, 22]. But, by using the published method, it is impossible to analyze
the position of hydroperoxide group in the fatty acid of PCOOH molecule. In the present
study, we could differentially determine 16:0/13-HpODE PC and 16:0/9-HpODE PC, and
found that there was no difference in these levels of healthy and patient plasma. Meanwhile,
it is likely that different LOXs (15-LOX and 5-LOX) generate different 16:0/HpODE PC
species (13-HpODE PC and 9-HpODE PC) in inflammatory state [38, 39]. Hence, difference
of 16:0/13-HpODE PC and 16:0/9-HpODE PC may appear in inflammatory atherosclerotic
lesions. This will provide better understanding of the pathophysiology of atherosclerosis.
These possibilities need to be addressed in future studies.
In summary, we developed a method for accurate quantification of PCOOH,
specifically isomers such as 16:0/13-HpODE PC and 16:0/9-HpODE PC in human plasma by
LC-MS/MS. MRM (812/147) enabled determination of plasma PCOOH (16:0/HpODE PC),
and MRM (812/541 and 812/388) allowed specific measurement of 16:0/13-HpODE PC and
16:0/9-HpODE PC, respectively. Using this method, we determined plasma PCOOH
concentrations in healthy subjects and in patients with angiographically significant stenosis.
The LC-MS/MS MRM system is useful for studying biological samples, including human
plasma, and will be useful for developing a better understanding of the pathophysiology of
oxidative stress-related diseases, such as atherosclerosis.
16

Acknowledgements and notice of grant support
We thank Dr. Ohki Higuchi (Biodynamic Plant Institute Co., Ltd., Sapporo, Japan) for
excellent technical assistance. This work was supported in part by KAKENHI (20228002 to
TM) of JSPS, Japan.
17

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22

Figure legends
Fig. 1. Chemical structures of PC (1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine,
16:0/18:2 PC) (A),
(1-palmitoyl-2-(13-hydroperoxyoctadecadienoyl)-sn-glycero-3-phosphocholine,
16:0/13-HpODE PC) (B), and
(1-palmitoyl-2-(9-hydroperoxyoctadecadienoyl)-sn-glycero-3-phosphocholine,
16:0/9-HpODE PC) (C).
Fig. 2. Q1 mass spectra of 16:0/13-HpODE PC and 16:0/9-HpODE PC in positive ESI mode
(A and B, respectively). The product ion mass spectra of protonated PCOOH (m/z 790
[M+H]+) for 16:0/13-HpODE PC (C) and 16:0/9-HpODE PC (D), and sodiated PCOOH (m/z
812 [M+Na]+) for 16:0/13-HpODE PC (E) and 16:0/9-HpODE PC (F). Standard PCOOH (1
nmol16:0/13-HpODE PC or 16:0/9-HpODE PC/ml methanol) was infused directly into the
MS/MS system at a flow rate of 10 µl/min. Detailed analytical conditions are described in
Materials and Methods.
Fig. 3. MRM chromatograms of standard PCOOH (16:0/13-HpODE PC or 16:0/9-HpODE
PC). 16:0/13-HpODE PC (1 pmol) was analyzed with MRM (m/z 790/184, 812/541, and
812/147) (A, C), whereas 16:0/9-HpODE PC (1 pmol) was analyzed with MRM (m/z
790/184, 812/388, and 812/147) (B, D). Mobile phases included either 5 mM ammonium
acetate (A, B) or 0.1 mM sodium acetate (C, D). Insets show Q1 mass spectra of PCOOH
detected at 17.0 (A), 16.9 (B), 17.5 (C), and 17.4 (D) min. Detailed analytical conditions are
described in Materials and Methods.
Fig. 4. Calibration curves of reference PCOOH (16:0/13-HpODE PC (A) or 16:0/9-HpODE
23

PC (B)). Different amounts of PCOOH (0.01–10 pmol) were analyzed by optimized
LC-MS/MS MRM (m/z 812/541 812/388, and 812/147) with mobile phase containing sodium
acetate. Detailed analytical procedures are described in Materials and Methods.
Fig. 5. PCOOH recovery rates from plasma spiked with standard PCOOH (16:0/13-HpODE
PC). PCOOH-spiked plasma (300 µl) was diluted with 300 µl of water and mixed with 2.4 ml
of chloroform:methanol (1:2 or 2:1, v/v). Lipid fractions were collected, and the remaining
phase was re-extracted with Folch’s theoretical lower phase at 4°C. Combined lipid fractions
were evaporated and applied to solid phase extraction followed by LC-MS/MS determination
of PCOOH. PCOOH-spiked plasma (300 µl) was diluted with 300 µl of water and mixed with
2.4 ml of chloroform:methanol (1:2, v/v) or 100% methanol. The supernatants were collected
and applied to solid phase extraction followed by LC-MS/MS. (A) Recovery of PCOOH
extracted with different solvent ratios (chloroform:methanol (1:2, 1:1, or 2:1, v/v) or 100%
methanol). (B) Recovery of PCOOH extracted using 300 µl of different concentrations of
KCl (instead of water) and 2.4 ml of solvent (CHCl3-MeOH, 2:1). (C) Recovery of PCOOH
in the case of washing (rinsing) the combined lipid fraction (obtained from extraction
conditions using CHCl3-MeOH 2:1 and 0% KCl) with Folch’s theoretical upper phase. (D)
Recovery of PCOOH extracted with CHCl3-MeOH 2:1, 0% KCl, and no rinsing at different
temperatures (25, 4, or -20°C). (E) Recovery of PCOOH after addition of PCOOH standards
to different extraction steps (Scheme 1). Extracts were analyzed by optimized LC-MS/MS
condition with mobile phase containing sodium acetate. Detailed analytical conditions and
procedures are described in Materials and Methods. Means without a common letter differ
significantly at P < 0.05.
Fig. 6. Experimental setup for evaluating LC-MS/MS ion suppression (A) and MRM
chromatograms of PCOOH when the plasma extract (10 µl) was injected into the LC-MS/MS
24

system with mobile phase containing sodium acetate in parallel with continuous post-column
infusion of a PCOOH standard solution (0.1 pmol 16:0/13-HpODE PC (B, D) or
16:0/9-HpODE PC (C, E)/ml methanol) at 10 µl/min. MRM conditions were 812/541 (B),
812/388 (C), and 812/147 (D, E). Detailed analytical conditions are described in Materials
and Methods.
Fig. 7. MRM chromatograms of PCOOH in the plasma of healthy subjects. Plasma extracts
(10 µl) were analyzed by optimized LC-MS/MS MRM (812/541 (A), 812/388 (B), and
812/147 (C)) with mobile phase containing sodium acetate. Detailed analytical conditions are
described in Materials and Methods.
25

TABLE 1. Optimal MS/MS parameters for PCOOH detection
790.4 [M+H]+
Precursor ion (m/z)
812.4 [M+Na]+
Product ion (m/z)
184.3
126
10
541.4
126
10
388.3
126
10
147.0
126
10
Declustering potential (V)
Entrance potential (V)
Collision energy (V)
Collision cell exit potential (V)
Curtain gas (psi)
47
59
41
69
10
30
16
6
20
Collision gas (psi)
7
Ion spray voltage (V)
Temperature (°C)
5000
500
40
Ion source gas 1 (psi)
Ion source gas 2 (psi)
40
26

TABLE 2. PCOOH (16:0/HpODE PC) concentrations in the plasma of healthy subjects
and patients with angiographically significant stenosis
MRM
812/541
812/388
pmol/ml
812/147*
Healthy subjects
n = 8
72.3 23.5
(69.3 23.5)
97.3 39.5
(97.6 42.2)
36.1 11.5
33.1 10.2
Patients
52.4 24.6
0.063
45.2 18.1
0.105
n = 12
P value
0.127
*Contents in parentheses ( ) represent the sum of 16:0/13-HpODE PC (812/541) and
16:0/9-HpODE PC (812/388). Values are means SD.
27

Fig. 1.
A
B
C
13
O
O
O
9
O
H3C
H3C
N
P
O
O
OH
O
O
CH3
O
28

Fig. 2.
100
A 100
B
m/z 790.4 [M+H]+
m/z 790.4 [M+H]+
O
O
O
O
H3C
N
P
O
O OH
H3C
O
O
CH3
O
m/z 813.0 [M+Na]+
m/z 812.9 [M+Na]+
m/z 772.4 [M+H-H2O]+
m/z 772.4 [M+H-H2O]+
m/z 756.4 [M+H-H2O-O]+
m/z 756.4 [M+H-H2O-O]+
0
0
100
200
300
400
500
600
700
800
900
1000
100
200
300
400
500
600
700
800
900
1000
m/z (Da)
m/z (Da)
C 100
D 100
m/z 184.4
m/z 184.2
m/z 790.8 [M+H]+
m/z 790.4 [M+H]+
m/z 86.0
m/z 772.6 [M+H-H2O]+
m/z 86.0
m/z 772.4 [M+H-H2O]+
m/z 756.6[M+H-H2O-O]+
m/z 756.4 [M+H-H2O-O]+
0
0
100
200
300
400
m/z (Da)
500
600
700
100
200
300
400
500
600
700
m/z (Da)
m/z 812.2 [M+Na]+
100
100
E
F
255 Da
m/z 812.2 [M+Na]+
O
O
m/z 541.2
O
O
P
H3C
N
O
O OH
H3C
O
O
CH
O
88 Da
169 Da
3
59 Da
124 Da
183 Da
59 Da
124 Da
183 Da
147.2
Na+
OH
O
P
724.2
541.2
O
Na+
OH
-183
-88
O
P
O
O
86.0
86.0
O
665.2
-59
147.0
-255
-169
329.4 388.2
-59
643.6
-124
753.2
-59
753.2
-88
0
0
100
200
300
400
m/z (Da)
500
600
700
800
100
200
300
400
m/z (Da)
500
600
700
800
29