Analytical Chemistry Chemical Cytometry Quantitates Superoxide

solution in an equal volume, and 1 µL of the mixture was
pipetted onto the MALDI-MS sample plate without the enrichment
procedure.
Adsorption Efficiency of VLCFAs by MWCNTs in the
Enrichment Procedure. Adsorption efficiency was determined
using standard mixtures of four VLCFAs (40 µg/mL each).
Standard mixtures were derivatized first and then subjected to
the enrichment procedure with MWCNTs as mentioned above.
After the standard mixture had adsorbed to the surface of the
MWCNTs, the supernatants were analyzed by HPLC/ESI-MS.
HPLC analysis was performed on a 3 µm C18 column (AtlantisdC18,
2.1 mm i.d × 50 mm, Waters) with an isocratic program
(mobile phases of acetonitrile/methanol/formic acid ) 80/20/
0.1). The autosampler was a Finnigan Surveyor autosampler fitted
with a 10 µL loop. The HPLC and autosampler systems were all
synchronized via Xcalibur software (Xcalibur, Finnigan Corp.). A
Finnigan LCQ DECA XP PLUS quadrupole ion trap mass spectrometer
(Finnigan Corp., San Jose, CA) equipped with a
pneumatically assisted electrospray ionization source was used.
The mass spectrometer was operated in positive ion mode by
applying a voltage of 4.5 kV to the ESI needle. The temperature
of the heated capillary in the ESI source was set at 260 °C.
The flow rate of the sheath gas (nitrogen) was set at 25
(arbitrary units). Helium was used as the damping gas at a
pressure of 10 -3 Torr. Voltages across the capillary and the
octapole lenses were tuned by an automated procedure to
maximize the signal of the ion of interest. We used the
following SIM (selected ion monitoring) transitions for the
analysis: C20:0 (center mass 398.2), C22:0 (center mass 426.2),
C24:0 (center mass 454.2), and C26:0 (center mass 482.2). The
isolation width was set as 1.5 Da. The experimental programs
and data analyses were performed with the software package
Xcalibur (Xcalibur, Finnigan Corp.).
Preparation of Standard Solutions and Calibration Curves.
Stock solutions of internal standard mixtures of three stable
isotope-labeled VLCFAs (C20:0-d3, C22:0-d3, and C26:0-d4) and
standard mixtures of four VLCFAs (C20:0, C22:0, C24:0, C26:
0) were prepared at a concentration of 50 µg/mL in methanol
and kept in the dark at -20 °C when not in use. For the
calibration curve, the concentrations of the calibration solutions
of standard mixture were 5, 10, 15, and 20 µg/mL, and the
concentration of the calibration solution of the internal standard
mixture was 10 µg/mL. The calibration solutions (50 µL) were
evaporated in a stream of nitrogen prior to the derivatization
procedure and reconstituted in 10 µL of 50% (v/v) methanol
for the enrichment procedure.
Hydrolysis of Lipids and Extraction of Total Fatty Acids
from Plasma. For hydrolysis of lipids, a solution of acetonitrile
(720 µL) and 5N hydrochloric acid (80 µL) was added to 50 µL of
plasma in a1mLglass tube and heated at 80 °C for 1 h. After
that, 50 µL of internal standard mixture (C20:0-d3, C22:0-d3, and
C26:0-d4) at a concentration of 10 µg/mL was added before
extraction of fatty acids with 2 mL of hexane. The hexane
extraction step was repeated three times. The hexane extracts
were combined and evaporated in a stream of nitrogen prior
to the following derivatization procedure and reconstituted in
10 µL of 50% (v/v) methanol for the enrichment procedure.
Figure 1. Enrichment and SALDI-MS analysis of VLCFAs (1 µg/
mL) derivatized to quaternary ammonium salt (TMAE-VLCFAs)
(A-D); and MALDI-MS analysis of VLCFAs with CHCA as matrix (E).
(* indicated the background peaks).
RESULTS AND DISCUSSION
Preparation of Quaternary Ammonium Salt Derivatives
of VLCFAs. We found that VLCFAs (stock concentration 50 µg/
mL) could not been detected in MALDI-MS or SALDI-MS ether
in the positive or negative ion mode. Using ESI-MS, Johnson et
al. 30 reported that trimethyaminoethyl-VLCFAs (TMAE-VLCFAs)
afforded 8- to 12-fold greater signal intensity than the corresponding
dimethylaminoethyl-VLCFAs (DMAE-VLCFAs). In order to
enhance the detection in SALDI-MS, the same derivatization
strategy was used in this study. The carboxylic acid groups of
VLCFAs were first derivatized to DMAE-VLCFAs. The limit of
detection (LOD) in MALDI-MS with CHCA as the matrix ranged
from 10 to 50 µg/mL (Figure S-1B in the Supporting Information).
Subsequently, DMAE-VLCFAs were reactived with methyl iodide
to form TMAE-VLCFAs. The LOD of TMAE-VLCFAs in MALDI-
MS (15 µg/mL) were about 10 times greater than the LOD of
DMAE-VLCFAs (Figure S-1A in the Supporting Information). The
DMAE-VLCFAs were not detected in the SALDI analysis. TMAE-
VLCFAs are quaternary ammonium salts with permanent positive
charges. The mode of ionization of quaternary ammonium salts
under SALDI conditions is by dissociation of the salts in the ion
source, leading to the detection of the positively charged moieties.
So, the quaternary TMAE-VLCFAs iodide is expected to lead to
higher SALDI-TOFMS sensitivity due to the dissociation of the
iodide ion. Besides, in the process of SALDI, no additional acids
were added. This ensured that the permanent positively charged
VLCFAs have high priority in ionization over the uncharged or
neutral compounds.
Detection of Derivatized VLCFAs with Various MWCNTs
for Enrichment and as SALDI Substrates (Scheme 1).
MWCNTs come in a variety of diameters and lengths, depending
on the growth process. In this study, MWCNTs of different
diameters and lengths were chosen from commercial products
with the same producing method (CVD method), carbon content,
melting point, and density. The sizes of the four MWCNTs listed
in Table 1 were provided by the manufacturer. The structural
information was confirmed by SEM and TEM and is provided in
Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
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