Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Scheme 1. (A) Procedure of Derivatizating VLCFAs to Quaternary Ammonium Salt and a Neutral Loss in the
Postsource Decay (PSD) and (B) Multiple Functions of MWCNTs for Analyte Enrichment and as the SALDI Substrate
was added to dissolve the residue at room temperature for 10
min. The residue was removed in a stream of nitrogen at 40 °C
and was reconstituted in 50% (v/v) methanol immediately before
analysis.
Enrichment of VLCFAs and Analysis with SALDI-TOFMS
by MWCNTs. The procedure was performed according to the
procedure reported by Pan et al. with minor modifications 32
(Scheme 1B). MWCNTs (10 mg) were first suspended in 1 mL
of 50% (v/v) methanol and then sonicated for 3 min.
Then, 10 µL of the suspension was pipetted immediately into
a centrifuge tube containing the analyte solution. The tube was
sonicated for less than 5 s, after which the MWCNTs with analytes
solution were homogeneously spread in the solution and the
analytes were adsorbed from the liquid phase to the surface of
MWCNTs within 10 min. The analyte-adsorbed MWCNTs were
precipitated by centrifugation at 10 000 rpm for 10 min. Then, the
supernatant was removed, and 4 µL of dispersant solution of 50%
methanol (v/v) with 5% glycerol (v/v) and 1% sucrose (w/w) was
added to the centrifuge tube to resuspend the MWCNTs. Finally,
about 2 µL of the MWCNT solution was pipetted onto the SALDI-
TOFMS sample plate. The sample plate was left at room temperature
for 10-15 min to allow for the evaporation of the solvent
before analysis by SALDI-TOFMS.
(32) Pan, C.; Xu, S.; Zou, H.; Guo, Z.; Zhang, Y.; Guo, B. J. Am. Soc. Mass
Spectrom. 2005, 16, 263–270.
6816 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
SALDI-TOFMS analyses were performed on an AXIMA-CFR
plus (Shimazu/KRATOS, Manchester, UK) instrument equipped
with a nitrogen laser (wavelength at 337 nm) and delayed
extraction. Ions were accelerated by energy of 20 kV before
entering the TOF mass spectrometer. All spectra were acquired
in the linear mode. In the SALDI-TOF/PSD MS experiments,
spectra were acquired in the reflectron mode. The mass spectra
of 50 different profiles of a sample spot were averaged. Each
profile included five laser shots. The mass calibration was
achieved using low-mass standards (MassPREP Calibration Mix-
DIOS, Waters) in the mass range 20-1500 Da. Quantitative
analysis of VLCFAs was carried out by measuring the ion peak
intensity of the individual mass peaks.
Limit of Detection (LOD) of Derivatized VLCFAs with
MWCNT-Based SALDI-MS and MALDI-MS. Serial concentrations
of the standard solutions of four VLCFAs (60 µL) were
prepared: 50, 25, 10, 5, 1, and 0.5 µg/mL. Following the derivatization
of VLCFAs to quaternary ammonium salt derivatives, the
products were reconstituted in 60 µL of 50% (v/v) methanol. The
derivatized products (10 µL) were enriched by MWCNTs according
to the procedure mentioned above. The amounts of VLCFAs
analyzed per spot were 250, 125, 50, 25, 5, and 2.5 ng.
The sensitivity of MALDI-TOFMS to detect VLCFAs was
tested using CHCA (10 mg/mL in 80% acetone) as the matrix.
The reconstituted solution (10 µL) was mixed with CHCA