Improved sensitivity mass spectrometric detection of eicosanoids by charge reversal derivatization

Article abstract of DOI:10.1021/ac100720p

Combined liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is a powerful method for the analysis of oxygenated metabolites of polyunsaturated fatty acids including eicosanoids. Here we describe the synthesis of a new derivatization reagent N-(4-aminomethylphenyl) pyridinium (AMPP) that can be coupled to eicosanoids via an amide linkage in quantitative yield. Conversion of the carboxylic acid of eicosanoids to a cationic AMPP amide improves sensitivity of detection by 10- to 20-fold compared to negative mode electrospray ionization detection of underivatized analytes. This charge reversal derivatization allows detection of cations rather than anions in the electrospray ionization mass spectrometer, which enhances sensitivity. Another factor is that AMPP amides undergo considerable collision-induced dissociation in the analyte portion rather than exclusively in the cationic tag portion, which allows isobaric derivatives to be distinguished by tandem mass spectrometry, and this further enhances sensitivity and specificity. This simple derivatization method allows prostaglandins, thromboxane B₂, leukotriene B₄, hydroxyeicosatetraenoic acid isomers, and arachidonic acid to be quantified in complex biological samples with limits of quantification in the 200-900 fg range. One can anticipate that the AMPP derivatization method can be extended to other carboxylic acid analytes for enhanced sensitivity detection.

Full text of DOI:10.1021/ac100720p

Anal. Chem. 2010, 82, 6790–6796
Improved Sensitivity Mass Spectrometric
Detection of Eicosanoids by Charge Reversal
Derivatization
James G. Bollinger,† Wallace Thompson,† Ying Lai,‡ Rob C. Oslund,† Teal S. Hallstrand,‡
Martin Sadilek,† Frantisek Turecek,† and Michael H. Gelb*,†,§
Departments of Chemistry, Medicine, and Biochemistry, University of Washington, Seattle, Washington 98195
Combined liquid chromatography-electrospray ioniza-
tion-tandem mass spectrometry (LC-ESI-MS/MS) is a
powerful method for the analysis of oxygenated metabo-
lites of polyunsaturated fatty acids including eicosanoids.
Here we describe the synthesis of a new derivatization
reagent N-(4-aminomethylphenyl)pyridinium (AMPP) that
can be coupled to eicosanoids via an amide linkage in
quantitative yield. Conversion of the carboxylic acid of
eicosanoids to a cationic AMPP amide improves sensitiv-
ity of detection by 10- to 20-fold compared to negative
mode electrospray ionization detection of underivatized
analytes. This charge reversal derivatization allows detec-
tion of cations rather than anions in the electrospray
ionization mass spectrometer, which enhances sensitivity.
Another factor is that AMPP amides undergo considerable
collision-induced dissociation in the analyte portion rather
than exclusively in the cationic tag portion, which allows
isobaric derivatives to be distinguished by tandem mass
spectrometry, and this further enhances sensitivity and
specificity. This simple derivatization method allows pros-
taglandins, thromboxane B2, leukotriene B4, hydroxye-
icosatetraenoic acid isomers, and arachidonic acid to
be quantified in complex biological samples with limits
of quantification in the 200-900 fg range. One can
anticipate that the AMPP derivatization method can be
extended to other carboxylic acid analytes for enhanced
sensitivity detection.
reaction monitoring (SRM) in which precursor ions are isolated
in the first stage of the mass spectrometer followed by collision-
induced dissociation to give fragment ions, which are detected
after an additional stage of mass spectrometer isolation. The
current limit of quantification for these analytes is in the ∼10-20
pg range. This sensitivity level is appropriate for studies with
cultured cells in vitro or with relatively large tissue samples, but
it is not sufficient for studies with smaller volume samples such
as joint synovial fluid or bronchoalveolar lavage fluid from
experimental rodents. Given the importance of oxygenated fatty
acid derivatives in numerous medically important processes such
as inflammation and resolution of inflammation, we sought to
improve the LC-ESI-MS/MS sensitivity of detection of these lipid
mediators using a widely available analytical platform.
For reasons that are not well understood, cations generally
form gaseous ions better than anions in the electrospray ionization
source of the mass spectrometer. Additionally, for underivatized
carboxylic acids it is required to add a weak organic acid to the
chromatographic mobile phase, i.e., formic acid, so that the
carboxylic acid is kept in its protonated state, which allows it to
be retained on the reverse-phase column to ensure chromato-
graphic separation. However, the presence of the weak acid offsets
the formation of carboxylate anions in the electrospray source
because the weak acid carries most of the anionic charge in the
electrospray droplets, and thus formation of analyte anions is
suppressed. We reasoned that conversion of the carboxylic acid
to a fixed-charge cationic derivative would lead to improved
detection sensitivity by ESI-MS/MS. Charge-reversal derivatization
of carboxylic acids with quaternary amines has been explored in
previous work (for example, refs 4-6). However, these reagents
utilize organic cations that tend to fragment by collision-induced
dissociation near the cationic site. Fragmentation in the deriva-
tization tag is not desirable because analytes that form isobaric
precursor ions and that comigrate on the LC column will not be
distinguished in the mass spectrometer if they give rise to the
same detected fragment ion. This loss of analytical specificity is
a serious problem when analyzing complex biological samples.
Fragmentation in the analyte portion rather than in the tag portion
Liquid chromatography coupled to electrospray ionization
tandem mass spectrometry (LC-ESI-MS/MS) has emerged as a
powerful method to detect oxygenated derivatives of fatty acids
including eicosanoids (for example, refs 1-3). With these methods
it is possible to analyze a large collection of eicosanoids in a single
LC-ESI-MS/MS run. These lipid mediators are detected by single
* To whom correspondence should be addressed. Michael H. Gelb, Depts.
of Chemistry and Biochemistry, Campus Box 35100, University of Washington,
Seattle, WA 98195. Phone: 206 525-8405. Fax: 206 685-8665. E-mail:
gelb@chem.washington.edu.
† Department of Chemistry.
‡ Department of Medicine.
§ Department of Biochemistry.
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(2) Kita, Y.; Takahashi, T.; Uozumi, N.; Nallan, L.; Gelb, M. H.; Shimizu, T.
Biochem. Biophys. Res. Commun. 2005, 330, 898
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Deems, R. A.; Dennis, E. A. J. Biol. Chem. 2007, 282, 22834
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.
6790 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
10.1021/ac100720p  2010 American Chemical Society
Published on Web 07/22/2010

also reduces chemical noise, which also enhances sensitivity of
detection.
tion analysis. Internal standards were diluted to a working stock
of 5 pg/µL in absolute ethanol.
In this study we report the design and synthesis of a new
cationic tag and show that it can be quantitatively attached via an
amide linkage to the carboxyl group of eicosanoids by a simple
derivatization procedure. We then show that the derivatized
eicosanoids can be analyzed by LC-ESI-MS/MS with limits of
quantification that are well below those reported for underivatized
eicosanoids.
Preparation of Samples Prior to Derivatization with
AMPP. Standard Curves. Each sample contained 50 pg of each
internal standard and various amounts of nonisotopic eicosanoids
(added from the stock solutions described above) transferred to
a glass autosampler vial insert (Agilent catalog no. 5183-2085).
Solvent was removed with a stream of nitrogen, and the residue
was derivatized with AMPP as described below.
Mouse Serum. Analysis of endogenous eicosanoids in serum
was carried out with commercial mouse serum (Atlantic Biologi-
cals catalog no. S18110). A volume of 1, 5, or 10 µL of serum was
placed in a glass autosampler vial insert. Two volumes of methanol
(LC/MS, JT Baker catalog no. 9863-01) containing 50 pg of each
internal standard were added. The vial insert was mixed on a
vortex mixer for ∼10 s. The concentration of methanol was
lowered to 10% (v/v) by addition of purified water (Milli-Q,
Millipore Corp.), and the samples were loaded via a glass Pasteur
pipet onto a solid phase extraction cartridge (10 mg Oasis-HLB,
Waters catalog no. 186000383). The cartridges were previously
washed with 1 mL of methanol and then 2 × 0.75 mL of 95:5
water-methanol. After sample loading, the sample tube was rinsed
with 200 µL of purified water-methanol (95:5, v/v), and this was
added to the cartridge. The cartridge was washed with 2 × 1 mL
of water-methanol (95:5, v/v). Additional solvent was forced out
of the cartridge solid phase by applying medium pressure N2
(house N2 passed through a 0.2 µm cartridge filter) for a few
seconds. Column eluant receiver vials (Waters Total Recovery
autosampler vials, Waters catalog no. 186002805) were placed
under the cartridges. The cartridges were then eluted with
methanol (1 mL). All cartridge steps were carried out using a
vacuum manifold (Waters catalog no. WAT200606) attached
to a water aspirator. Solvent was removed by placing the
receiver vials in a centrifugal evaporator (Speed-Vac). These
processed samples were derivatized with AMPP (see below)
without storage.
Lung Epithelial Cells. The University of Washington Institu-
tional Review Board approved the studies involving human
subjects, and written informed consent was obtained from all
participants. Primary bronchial epithelial cells were isolated from
a volunteer with asthma during a bronchoscopy using a nylon
cytology brush of cells from subsegmental airways. To establish
primary culture, the epithelial cells were seeded into a culture
vessel coated with type 1 collagen in bronchial epithelial basal
media (BEBM, Lonza, Allendale, NJ) supplemented with bovine
pituitary extract, insulin, hydrocortisone, gentamicin, amphotericin
B, fluconazole, retinoic acid, transferrin, triiodothyronine, epi-
nephrine, and human recombinant epidermal growth factor
(serum-free BEGM) and maintained at 37 °C in a humidified
incubator. After expansion in vitro, passage 2 epithelial cells were
grown to >90% confluence on a 12-well plate. The medium was
changed to 200 µL of Hanks balanced salt solution, and the cells
were treated with either calcium ionophore (A23187, 10 µM in
DMSO) or a DMSO-containing control solution for 20 min at 37
°C. The synthesis of eicosanoids was stopped by the addition of
4 volumes of ice-cold methanol with 0.2% formic acid, and samples
were stored at -80 °C until processed. The number of epithelial
EXPERIMENTAL METHODS
Synthesis of AMPP. Pyridine (40 mmol, 3.2 mL) was
dissolved in 46 mL of absolute ethanol followed by the addition
of 1-chloro-2,4-dinitrobenzene (40 mmol, 8.2 g, Aldrich). The
mixture was heated with a reflux condenser at 98 °C for 16 h
under nitrogen. After cooling, ethanol was removed by rotary
evaporation, and the crude product was recrystallized by dissolving
in a minimal amount of hot ethanol and allowing the solution to
slowly cool. The product N-2,4 dinitrophenyl pyridinium chloride
was isolated as a yellow solid in 62% yield, and its identity was
confirmed by melting point analysis (189-191 °C observed,
189-190 °C reported7). N-2,4-Dinitrophenyl pyridinium chloride
(16.8 mmol, 4.76 g) was dissolved in 70 mL of ethanol-pyridine
(3:1). 4-[(N-Boc) -amino-methyl] aniline (33.6 mmol, 7.56 g,
Aldrich) was added, and the reaction mixture was heated under
a reflux condenser at 98 °C under nitrogen for 3 h. After cooling,
700 mL of water was added to precipitate 2,4-dinitroaniline. After
filtration, the filtrate was concentrated to dryness by rotary
evaporation, and the product was isolated as a brown oil. This oil
was treated with 112 mL of 25% (v/v) trifluoroacetic acid in
dichloromethane for 30 min at room temperature. The mixture
was concentrated by rotary evaporation, and the solid was
triturated twice with benzene to remove excess trifluoroacetic acid.
The mixture was again concentrated by rotary evaporation. The
residue was dissolved in a minimal amount of heated ethanol, the
solution was allowed to cool for ∼5 min, and then diethyl ether
was added with swirling until the solution started to cloud up.
The mixture was transferred to the freezer (-20 °C) and left
overnight. The mixture was allowed to warm to room temperature
and then decanted. The obtained mother liquor was treated with
additional diethyl ether as above to give additional AMPP solid.
The solids were combined and triturated with diethyl ether. The
solid was dried under vacuum to give 3.30 g of AMPP as a brown
solid, 59% yield. 1H NMR (300 MHz, D6-DMSO) 9.34 (d, 2H),
8.81 (t, 1H), 8.53 (broad, 3H), 8.33 (t, 2H), 7.95 (d, 2H), 7.80
(d,2H), 4.22 (s, 2H) (1H NMR spectra are shown in the
Supporting Information, estimated purity of AMPP is >95%).
Preparation of Eicosanoid Stock Solutions. The following
eicosanoid standards from Cayman Chemicals were used (PGE2
,
PGD2, PGF2R, 6-keto-PGF1R, TxB2, 5(S)-HETE, 8(S)-HETE,
11(S)-HETE, 12(S)-HETE, 15(S)-HETE, LTB4, arachidonic acid,
D4-PGE2, D4-PGD2, D4-PGF2R, D4-6-keto-PGF1R, D4-TXB2, D8-
5(S)-HETE, D4-LTB4, D8-arachidonic acid). Stock solutions of
eicosanoids were prepared at a concentration of 100 pg/µL in
absolute ethanol and stored at -80 °C under Ar in Teflon
septum, screw cap vials. Serial dilutions of the stock solutions
were made in absolute ethanol for standard curve and extrac-
(7) Park, K. K.; Lee, J.; Han, D. Bull. Korean Chem. Soc. 1985, 6, 141
.
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6791

cells was 5.0 × 105 cells per well. The studies were performed
in duplicate.
Frozen samples were thawed on ice, 50 pg of each internal
standard was added, and Milli-Q water was added to bring the
methanol to 10% (v/v). The samples were processed by solid-
phase extraction (Oasis-HLB) as described for mouse serum
samples.
(Pierce catalog no. 24460)-15 mM AMPP in acetonitrile. The vials
were mixed briefly on a vortex mixer, capped, and placed in a 60
°C incubator for 30 min. The cap was replaced with a split-septum
screw cap (Agilent catalog no. 5185-5824) for autoinjection onto
the LC-ESI-MS/MS. Samples were analyzed on the same day.
Samples were kept in the autosampler rack at 4 °C while queued
for injection.
Rat 3Y1 Cells. 3Y1 cells were a gift from Dr. H. Kuwata
(Department of Health Chemistry, School of Pharmaceutical
Sciences, Showa University). 3Y1 cells were maintained in
complete medium consisting of Dulbecco’s Modified Eagle
Medium (DMEM) with low glucose (Invitrogen catalog no. 11885-
084) containing 10% heat inactivated, fetal bovine serum (FBS)
(Invitrogen catalog no. 16140), 100 units/mL penicillin, and 100
µg/mL streptomycin (Invitrogen catalog no. 15140) in plastic
tissue culture dishes (Nunc catalog no. 172958) in a humidified
atmosphere of 5% CO2 at 37 °C. Passage of cells was performed
using 0.25% trypsin/EDTA (Invitrogen catalog no. 25200-056).
For PGE2 analysis, 3Y1 cells were plated at 5 × 104 cells/
well in a 24-well plate (Nunc catalog no. 142475) in 1 mL of
complete medium and incubated overnight. The medium was
then replaced with DMEM containing 2% FBS. After 24 h
incubation, the medium was removed from each well and
replaced with 1 mL DMEM containing 2% FBS and the
cytosolic phospholipase A2-R inhibitor Wyeth-2 (compound 10
from ref 9) or DMSO vehicle control (final concentration in
each well did not exceed 0.1% (v/v)). Cells were incubated for
20 min at room temperature. Mouse interleukin-1ꢀ (1 ng/well)
and human tumor necrosis factor-R (1 ng/well) (R & D Systems
catalog no. 401-ML and 210-TA) were both added to each well
in 10 µL of DMEM containing 2% FBS (negative control wells
received only 10 µL of DMEM containing 2% FBS). Cells were
incubated for 48 h at 37 °C in a humidified atmosphere of 5%
CO2. The supernatant was then carefully removed from each
well and transferred into a glass test tube and placed on ice
for immediate analysis. PGE2 levels were measured by an
enzyme immunoassay kit (Cayman Chemical catalog no.
500141) according to the manufacturer’s instructions using 50
µL of medium above the cells. A sample of medium (50 µL)
was also analyzed by LC-ESI-MS/MS as follows. To the
medium was added 2 volumes of methanol containing 50 pg
of each internal standard, methanol was reduced to 10% v/v
by addition of Milli-Q water, and samples were submitted to
solid-phase extraction using Oasis-HLB cartridges as described
for mouse serum samples.
LC-ESI-MS/MS Analysis. Some studies were carried out
with a Waters Quattro Micro triple quadrupole mass spectrometer,
a 2795 Alliance HT LC/autosampler system, and the QuanLynx
software package. Chromatography was carried out with a C18
reverse-phase column (Ascentis Express C18, 2.1 mm × 150 mm,
2.7 µm, Supelco catalog no. 53825-U). Solvent A is 95% H2O/5%
CH3CN/1% acetic acid, and solvent B is CH3CN/1% acetic acid.
The solvent program is (linear gradients) 0-1.0 min, 95-78%
A; 1.0-7.0 min, 78-74% A; 7.0-7.1 min, 74-55% A; 7.1-12.1
min, 55-40% A; 12.1-13.0 min, 40-0% A; 13.0-15.0 min, 0%
A; 15.0-15.1 min, 0-95% A; 15.1-20.1 min, 95% A. The flow
rate is 0.25 mL/min.
Similarly, some studies were carried out with a Waters Quattro
Premier triple quadrupole mass spectrometer interfaced to an
Acquity UPLC. Solvent A is 100% water (Fisher Optima grade
catalog no. L-13780)-0.1% formic acid (Fluka catalog no. 94318),
and solvent B is CH3CN (Fisher Optima grade catalog no.
L-14338)-0.1% formic acid. The same LC column was used but
with a modified solvent program (linear gradients): 0-1.0 min,
95% A; 1.0-2.0 min, 95%-85% A; 2.0-2.1 min, 85%-74% A;
2.1-6.0 min, 74%-71% A; 6.0-6.1 min, 71%-56% A; 6.1-10.0
min, 56% A; 10.0-14.0 min, 56%-0% A; 14.0-14.1 min, 0%-95%
A; 14.1-18.0 min, 95% A. Supplemental Material Tables 1 and
2 in the Supporting Information give the autosampler and ESI-
MS/MS data collection parameters for the Waters Quattro Micro
triple quadrupole and the Waters Quattro Premier triple quadru-
pole mass spectrometers, respectively.
For comparison purposes, we also carried out LC-ESI-MS/
MS analysis of underivatized eicosanoids in the negative ion mode.
The same LC column, solvents A and B, and flow rate were used
as for the AMPP amides. The solvent program was slightly
modified as follows: For the Waters Quattro Micro, we used 0-1.0
min, 95-63% A; 1.0-7.0 min, 63%A; 7.0-7.1 min, 63-38% A;
7.1-12.1 min, 38-23% A; 12.1-13.0 min, 23-0% A; 13.0-15.0 min,
0% A; 15.0-15.1 min, 0-95% A; 15.1-20.1 min, 95% A. For the
Waters Quattro Premier, we used 0-1.0 min, 95% A; 1.0-2.0 min,
95%-85% A; 2.0-2.1 min, 85%-59% A; 2.1-6.0 min, 59% A; 6.0-6.1
min, 59%-38% A; 6.1-11 min, 38%-23% A; 11-11.1 min, 23%-0%
A; 11.1-14 min, 0% A; 14.0-14.1 min, 0%-95% A; 14.1-19.0 min,
95% A. Values of m/z for precursor and fragment ions were as
published,8 and cone voltages and collision energies were opti-
mized for each instrument and for each analyte in the usual way
(values not shown but similar to those published8).
Eicosanoid Recovery Studies. We measured the recovery
of eicosanoids following the sample workup procedure given
above. Recovery studies were done using either 30 mg Strata-X
(Phenomenex Cat. 8B-S100-TAK-S) or 10 mg Oasis-HLB (Waters
Cat. 186000383) cartridges with 50 or 5 pg of each eicosanoid in
phosphate buffered saline. For these recovery studies, 50 pg of
each internal standard was added to samples just prior to
derivatization with AMPP (i.e., post-solid phase extraction).
Derivatization with AMPP. To the residue in the Waters
Total Recovery autosampler vial was added 10 µL of ice-cold
acetonitrile (JT Baker catalog no. 9017-03)-N,N-dimethylform-
amide (Sigma catalog no. 227056) (4:1, v:v). Then 10 µL of ice-
cold 640 mM (3-(dimethylamino)propyl)ethyl carbodiimide hy-
drochloride (TCI America catalog no. D1601) in purified water
was added. The vial was briefly mixed on a vortex mixer and
placed on ice while other samples were processed as above. To
each vial was added 20 µL of 5 mM N-hydroxybenzotriazole
(8) Kita, Y.; Takahashi, T.; Uozumi, N.; Shimizu, T. Anal. Biochem. 2005, 342,
134
.
(9) McKew, J. C.; Lee, K. L.; Chen, L.; Vargas, R.; Clark, J. D.; Williams, C.;
Clerin, V.; Marusic, S; Pong, K. Inhibitors of Cytosolic Phospholipase A2.
U.S. Patent 7,557,135 B2, July 7, 2009.
6792 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

Table 1. Extraction Yields, Liquid Chromatography Retention Times, and Tandem Mass Spectrometry Parameters
for Eicosanoid AMPP Amide Molecular Species
limit of
quantitation in
limit of
extraction LC retention positive mode quantitation in negative
fragment
cone
collision
eicosanoid
yield (%)a time (min)b
(pg) (CV)c
mode (pg)d
precursorione(m/z) ione (m/z) voltagef (V) energyf (eV)
6-keto-PGF1R
PGF2R
102/87
64/100
67/74
63/67
86/86
42/63
42/65
40/75
31/66
69/62
35/53
73/113
5.4/4.1
6.7/5.2
6.8/5.3
7.2/5.7
6.2/4.6
9.8/7.7
10.4/9.7
10.3/9.1
10.2/8.8
10.2/8.9
10.1/8.5
11.7/12.4
5.4/4.1
6.7/5.2
6.8/5.3
7.1/5.6
6.2/4.6
9.8/7.7
10.4/9.6
11.7/12.4
5/0.3 (6.8)
5/0.5 (9.1)
5/0.3 (5.5)
5/0.6 (7.5)
5/0.2 (2.3)
5/0.5 (10.3)
5/0.4 (10.4)
5/0.2 (6.7)
5/0.3 (8.9)
10/0.9 (6.6)
5/0.4 (11.1)
10/1 (5.6)
110/10
110/7
100/7
120/7
90/4
537
521
519
519
537
503
487
487
487
487
487
471
541
525
523
523
541
507
495
479
239
239
239
307
337
323
283
295
335
347
387
239
241
241
241
311
341
325
284
239
65/80
60/75
60/75
60/75
65/70
50/60
55/65
55/65
55/65
55/65
55/65
55/65
65/80
60/75
60/75
60/75
65/70
50/60
55/65
55/65
55/55
43/52
40/45
40/48
43/42
35/35
37/34
37/40
32/30
35/35
33/34
40/50
55/55
43/52
40/45
40/48
43/42
35/35
37/34
40/50
PGE2
PGD2
TxB2
LTB4
80/7
5(S)-HETE
8(S)-HETE
11(S)-HETE
12(S)-HETE
15(S)-HETE
arachidonic acid
D4-6-keto-PGF1R
D4-PGF2R
120/10
120/10
80/5
140/10
200/10
no data
D4-PGE2
D4-PGD2
D4-TxB2
D4-LTB4
D8-5(S)-HETE
D8-arachidonic acid
a The first number is for the Waters Oasis HLB cartridge, and the second number is for the Phenomenex Strata-X cartridge. b The first number
is for the Waters Quattro Micro, and the second number is for the Waters Quattro Premier. c LOQ values are for eicosanoid AMPP amides in
positive mode. The first number is for the Waters Quattro Micro, and the second number is for the Waters Quattro Premier. The values in
parentheses are the CVs based on 6 independent 15 µL injections of 0.075 pg/µL on the Waters Quattro Premier (see the main text). d LOQ values
are for underivatized eicosanoids analyzed by LC-ESI-MS/MS in negative mode. The first number is for the Waters Quattro Micro, and the
second number is for the Waters Quattro Premier. e m/z values listed are calculated monoisotopic values. The actual values used are derived
from instrument tuning, which is instrument dependent. f Cone voltages and collision energies were optimized for each analyte. These numbers
are instrument dependent. The first number is for the Waters Quattro Micro instrument, and the second number is for the Waters Quattro Premier
instrument.
Figure 1. Structure of AMPP and an AMPP amide along with the reagents used for derivatization.
Recoveries were obtained by comparing the LC-ESI-MS/MS peak
integrals to those obtained from a sample of eicosanoids that were
derivatized with AMPP and injected directly onto the LC column
without sample processing. Recovery yields are given in Table 1.
The peak areas for the internal standards were similar in all
samples studied (not shown) showing that the eicosanoid recover-
ies were similar regardless of sample matrix.
phenyl ring also serves to enhance the derivative’s interaction with
the LC reverse phase column, which is necessarily used to
minimize any matrix effects during ESI-MS/MS analysis. Density
functional theory calculations (B3LYP/6-31G*) indicated that
homolytic cleavage of the benzylic CH2-N bond in the charge
tag was a high-energy process that required >350 kJ mol-1 of
dissociation energy and was deemed not to out compete
fragmentations in the fatty acyl chain (Supplementary Material
Figure 1 in the Supporting Information). A side reaction that
could not be evaluated with the model system shown in Supple-
mentary Material Figure 1 in the Supporting Information is
elimination of 4-(N-pyridyl)benzylamine, which gives rise to the
m/z 183 marker fragment ion. An amide linkage of the charge
tag to the analytic carboxylic acid was preferred over an ester
since the former are generally more resistant to fragmentation in
the mass spectrometer.11 The final feature of the design of AMPP
is its ease of synthesis in pure form.
RESULTS AND DISCUSSION
Design and Preparation of AMPP Amides. Our goal was
to develop a simple derivatization procedure to convert the
carboxylic acid terminus of eicosanoids to a cationic group so that
mass spectrometry could be carried out in positive mode. The
tag should be designed so that fragmentation occurs in the analyte
portion rather than in the tag portion so that the power of tandem
mass spectrometry can be used for analytical specificity and
sensitivity. Our computational analysis of substituted N-pyridinium
methylamino derivatives10 indicated that these had only moderate
dissociation energies for loss of pyridine and other single bond
cleavages in the linker and thus might not be suitable for our
purposes, which require fragmentation in the fatty acyl chain.
Therefore, the charge tag was redesigned to strengthen the
pyridinium N-C bond by inserting a phenyl ring as a linker. The
We developed a simple synthesis of highly pure N-(4-amino-
methylphenyl)-pyridinium (AMPP, Figure 1) and used it as a new
derivatization reagent. We developed a simple method to convert
(10) Chung, T. W.; Turecek, F. Int. J. Mass Spectrom. 2008, 276, 127
(11) McLafferty, F. W.; Turecek, F. Interpretation of Mass Spectra, 4th ed.;
.
University Science Books: Mill Valley, CA, 1993.
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6793

Figure 2. LC-ESI-MS/MS traces of eicosanoids after workup from a solution in phosphate buffered saline: (A) early eluting eicosanoids and
(B) latter eluting eicosanoids.
the carboxyl group of lipid mediators to the AMPP amide using
a well-known carbodiimide coupling reagent. Coupling conditions
were optimized to give near quantitative formation of the AMPP
amide. The was shown by converting the fluorescent fatty acid
10-pyrene-decanoic acid into its AMPP amide and examining the
reaction mixture by fluorimetric HPLC and ESI-MS. No remaining
10-pyrenedecanoic acid was detected, and the AMPP amide was
formed in >95% yield with <1% N-acyl urea formation (a common
side product in carbodiimide-promoted couplings) (data not
shown). Derivatization is carried out using a single reagent
cocktail in a small vial heated at 60 °C for 30 min without the
need for anhydrous or anaerobic conditions.
LC-ESI-MS/MS Analysis of Eicosanoid AMPP Amides.
In this study we focused on representative eicosanoids that do
not contain peptides such as cysteinyl leukotrienes. The analytes
studied are PGD2, PGE2 and PGF2R, 6-keto-PGF1R (the spontane-
ous breakdown product from prostacyclin, PGI2), TxB2 (the
spontaneous breakdown product from TxA2), LTB4, 5(S)-
HETE), 8(S)-HETE, 11(S)-HETE, 12(S)-HETE, and 15(S)-
HETE, and AA. ESI-MS/MS analysis with fragment ion
6794 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010

scanning of AMPP amides of the analytes are shown in the
Supporting Information Supplementary Material Figure 6A-D.
In all the spectra, some cleavage of the AMPP tag occurs giving
rise to the peaks at m/z ) 169 and 183. To best ensure analytical
specificity, we quantified the eicosanoids using a fragment in the
analyte portion rather than in the AMPP tag. For PGD2, we used
the m/z ) 307 fragment, which is likely due to cross-ring
cleavage of the cyclopentanone ring and represents the most
abundant non-tag fragment ion. For PGE2 and PGF2R, we used
m/z ) 239 due to cleavage between C3 and C4. For AA and
6-keto-PGF1R, we used m/z ) 239, again due to C3-C4
cleavage. The major nontag fragment for TxB2 is m/z ) 337
due to cross-ring cleavage. For all HETE species, we could
identify a unique fragment ion (see Table 1) to avoid cross
contamination of MS/MS signals due to partially unresolved LC
peaks. Finally, for LTB4, we chose m/z ) 323, which is the most
abundant non-tag fragment ion but has a nonobvious origin.
Next we studied a mixture of eicosanoids in phosphate buffered
saline and optimized pre-ESI-MS/MS sample workup and LC-ESI-
MS/MS conditions. Critical for a successful method with ultra-
sensitive analyte detection is high yield recovery of analytes from
the sample prior to LC-ESI-MS/MS analysis. We found that solid
phase extraction with a rapidly wettable matrix (Oasis HLB
cartridges, Waters Inc. or Strata-X cartridges, Phenomenex)
combined with analyte elution with methanol gave high yield
recovery of all eicosanoids analyzed (Table 1). The data in Table
1 is based on 50 pg of each analyte submitted to recovery studies.
When the same recovery studies were carried out with 5 pg of
eicosanoid, results were essentially identical (data not shown).
Inferior recoveries were obtained using elution of the solid phase
cartridges with acidified methanol or if the sample was subjected
to liquid-liquid extraction or protein precipitation using various
solvents (Supporting Information). Figure 2A,B shows a typical
LC-ESI-MS/MS run of an eicosanoid mixture. All of the analytes
examined elute from the LC column in less than 11 min. All
HETEs except 11(S)-HETE and 12(S)-HETE are baseline resolved.
The partial overlap of the 11(S)- and 12(S)-HETEs is not a concern
because completely selective fragment ions are being monitored.
The LC elution profile and peak shape for both the derivatized
and underivatized analytes (not shown) are similar. This shows
that the presence of the quaternary ammonium group of the
AMPP tag has no negative effect on the LC. TxB2 gives rise to
the typical broad peak shape due to interconversion between
the two hemiacetal isomers. Also similar to underivatized
analysis, the fragments used to monitor PGD2 and PGE2
(isobars of each other) are not completely unique to each
species, but baseline LC resolution of these two eicosanoids
resolves the issue. The same is true for the isobaric pair TxB2
and 6-keto-PGF1R.
Table 2. Coefficient of Variation (%) for LC-ESI-MS/MS
Analysis of Eicosanoid AMPP Amides
intersample
intersample
intrasamplea (10 µL PBS)b (10 µL serum)c
6-keto-PGF1〈
TxB2
4.9
2.4
8.1
8.0
4.5
7.3
3.3
6.7
5.5
5.2
4.0
12.7
16.9
3.0
not detected
3.7
PGF2 〈
6.5
6.4
PGE2
9.2
8.3
17.2
2.8
not detected
PGD2
not detected
LTB4
7.3
6.4
5(S)-HETE
8(S)-HETE
11(S)-HETE
12(S)-HETE
15(S)-HETE
arachidonic acid
20.3
12.2
10.7
5.8
13.7
8.8
6.3
12.4
7.9
28.2
a Coefficient of variation for the analysis of the same sample of 1.25
pg/µLofeicosanoidstandardsinjectedthreetimesontotheLC-ESI-MS/MS.
b Coefficient of variation for the analysis of three independent samples
of 1.25 pg/µL of eicosanoid standards (50 pg of standards spiked into
phosphate buffered saline and worked up as described in the
Experimental Methods section for LC-ESI-MS/MS.). c Coefficient of
variation for three separate serum samples spiked with internal
standards only.
all cases, the mass spectrometry response was linear from the
limit of quantification up to the highest amount analyzed (19 pg
on-column for the Waters Quattro Micro and 5-10 pg on-column
fortheWatersQuattroPremier)(SupplementalMaterialFigure2A,B
in the Supporting Information). We define limit of quantification
as the amount of eicosanoid AMPP amide needed to give an ESI-
MS/MS signal that is 10-fold the noise (as determined using the
Waters MassLynx software). The limits of quantification for all
eicosanoid AMPP amides are listed in Table 1. The values using
the Waters Quattro Micro are 5 pg for all eicosanoids except 12(S)-
HETE (10 pg) and AA (10 pg). With the Waters Quattro Premier,
the values are 0.2-0.5 pg for all eicosanoids except 12(S)-HETE
(0.9 pg) and AA (1 pg). We also determined limit of quantification
of underivatized eicosanoids analyzed by LC-ESI-MS/MS in
negative ion mode. The values are shown in Table 1 for the Waters
Quattro Micro and the Waters Quattro Premier. The new method
using AMPP amides is found to be 10- to 20-fold more sensitive
on both instruments. This constitutes a significant improvement
in detection sensitivity of eicosanoids.
To validate the above stated estimates of the limit of quantifica-
tion, we injected eicosanoids standard mixtures six times on the
Waters Quattro Premier, where each analyte was injected at
roughly twice the estimated limit of quantification. Values of CV
for analyte to internal standard peak ratios for each analyte are
listed in Table 1. These CVs validate our estimates for the limits
of quantification by demonstrating that eicosanoid analysis can
be performed with reasonable reproducibility proximal to these
stated values.
To ensure high yield conversion of eicosanoids to AMPP
amides with minimal formation of N-acyl-ureas, we used a large
excess of derivatization reagents. The AMPP tag and the EDCI
coupling reagent elute in the void volume (not shown), and the
HOBt elutes in the large time window between PGD2 and LTB4
(not shown). Thus removal of excess derivatization reagents
is not necessary prior to LC-ESI-MS/MS.
Next, we more thoroughly investigated the reproducibility of
the AMPP derivatization method for eicosanoid analysis. The
results are summarized in Table 2. Three repetitive LC-ESI-MS/
MS analyses of an identical sample (19 pg on-column levels of
each eicosanoid) gave coefficients of variation in the range of the
2.4-12.7% range. We also prepared three independent mixtures
of eicosanoids (50 pg each) in phosphate buffered saline, submit-
ted each to sample workup, and analyzed each by LC-ESI-MS/
Standard curve analysis was performed starting below the limit
of quantification up to 2 orders of magnitude above this limit. In
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010 6795

Table 3. Eicosanoid Levels (pg/µL) in Mouse Serum and Bronchial Epithelial Cellsa
Arachi-donic
Acid
serum (µL) TxB2
PGE2
PGD2
PGF2〈
15
LTB4 5(S)-HETE 8(S)-HETE 11(S)-HETE 12(S)-HETE 15(S)-HETE
1
46
49
43
43
51
39
198
340
174
169
255
248
748
1075
792
393
545
490
469
586
593
437
11 345
17 840
14 116
16 512
15 108
14 487
240
703
617
672
749
732
28 623
23 246
14 900
16 631
12 200
15 476
880 (1210)
1
11
13
11
9
646
5
350
5
986
350
10
971
958
536
10
13
416
bronchial
epithelial
cellsb
4 (7) 105 (195) 25 (22) 170 (300)
5 (19)
a Eicosanoids not listed in the table were not detected. b The first number is for unstimulated cells, and the number in parentheses is for
stimulated cells. Values are for 0.47 million cells per sample stimulated in 200 µL of Hank’s balanced salt solution.
MS (19 pg on-column) (Table 2). Coefficients of variation ranged
from 2.8 to 28.2%. We also analyzed three independent serum
aliquots (10 µL), and the coefficient of variation ranged from 3.7
to 13.7%.
have a limit of quantification around 1 pg, and the method requires
that each analyte be quantified in a single assay well. Thus, if 12
eicosanoids are to be analyzed, one requires a sample containing
∼12 pg of each species. The AMPP amide method disclosed in
the current study can detect all 12 eicosanoid species in a single
sample containing ∼0.3-1 pg of each eicosanoid and is thus more
than an order of magnitude more sensitive than antibody based
detection. Previously reported LC-ESI-MS/MS detection of
underivatized eicosanoids in negative ion mode provide a limit of
quantification in the 10-20 pg range1–3 (see also our data in Table
1), and thus our method is more than an order of magnitude more
sensitive than these methods. We are currently studying the use
of AMPP to improve the detection sensitivity for other oxygenated
fatty acids including cysteinyl-leukotrienes, lipoxins, resolvins, and
protectans. Analysis of the full spectrum of fatty acids should be
feasible based on the results with AA reported in this study. This
new method based on AMPP amides should find widespread use
in the quantification of lipid mediator levels where sample supply
is limited.
We then analyzed the set of endogenous eicosanoids present
in mouse serum in order to test the AMPP derivatization method
on a complex biological sample. Representative LC-ESI-MS/MS
traces are shown in the Supporting Information Supplementary
Material Figure 7 for a relatively high abundant serum eicosanoid
(TxB2) and a relatively low abundant eicosanoid (PGF2R).
Traces for the full set of eicosanoids and internal standards
are shown in the Supporting Information Supplementary Ma-
terial Figure 3. Eicosanoids levels are shown in Table 3 for multiple
runs and with different volumes of serum analyzed.
As another example of low-level eicosanoid analysis in a
complex biological matrix, we analyzed eicosanoid formation in
calcium ionophore stimulated primary bronchial epithelial cells.
Results are summarized in Table 3, and selected ion traces for
the full set of eicosanoids are shown as Supplementary Material
Figure 4 in the Supporting Information.
Finally, we cross-validated the AMPP method with a com-
mercially available antibody-based assay kit. We analyzed PGE2
production by Rat 3Y1 cells and compared the results obtained
via LC-ESI-MS/MS analysis of AMPP amides with those
obtained from the EIA kit. Results are summarized in the
Supporting Information Supplementary Material Figure 8.
There is strong agreement between the two methods.
ACKNOWLEDGMENT
This work was supported by grants from the National Institutes
of Health (Grant HL50040 to M.H.G. and Grant HL089215 to
T.S.H.) and by grants from the National Science Foundation
(Grants CHE-0750048 and CHE-0349595 to F.T.).
SUPPORTING INFORMATION AVAILABLE
Additional information as noted in text. This material is
available free of charge via the Internet at http://pubs.acs.org.
CONCLUSIONS
We developed a simple derivatization procedure to convert
carboxylic acids to AMPP amides and showed that this signifi-
cantly improves the sensitivity for detection of eicosanoids by
LC-ESI-MS/MS. Antibody-based quantifications of eicosanoids
Received for review March 19, 2010. Accepted July 8,
2010.
AC100720P
6796 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010