Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Table 2. Concentrations and Ratios of VLCFAs in Plasma from Patients with ALD and from Normal Controls as
Measured by SALDI-TOFMS
Postsource Decay (PSD) of TMAE-VLCFAs. The PSD MS
spectra of the derivatized VLCFAs in SALDI-TOFMS, which
detected a neutral loss of trimethylamine (59 Da), were similar
to those in LC-MS/MS. 30 SALDI-PSD MS spectra of TMAE-
VLCFAs are recorded in Figure 2. Fragment ions were detected
at m/z 339.3 for C20:0, m/z 367.4 for C22:0, m/z 395.4 for C24:0,
and m/z 423.4 for C26:0. Those data demonstrate that derivatized
VLCFAs not only increased the ionization efficiency in SALDI but
also made the PSD easier in TOFMS. Furthermore, the neutral
loss fragmentations helped confirm the presence of VLCFAs in
the complex plasma matrix.
Quantitative Analysis of the TMAE-VLCFAs. CNT-based
SALDI has been shown to have excellent reproducibility of
spectrum signals, making it an acceptable modality for quantitative
analysis. 22,19,32 In addition, MALDI-MS with and without the stable
isotope-labeled method has been shown to be suitable for
quantifiation of small compounds in biologic samples. 38-40 In this
study, we used stable isotope-labeled VLCFAs (C26:0-d4, C22:0d3,
C20:0-d3) as the internal standards. The mass difference of
3-4 Da between VLCFAs and isotope-labeled VLCFAs provided
sufficient m/z separation space to avoid interference with
isotopologues of the ions. Figure 3 shows the SALDI-TOF mass
spectra of the four derivative VLCFAs. Three of the derivative
isotope-labeled VLCFAs had a molar ratio of 2:1 and a peak
intensity ratio of 2:1. The isotope-labeled C22:0-d3 and C26:0-d4
were both evaluated in the quantification of C24:0 as the
internal standards, with C26:0-d4 showing superior linearity (R 2
regression coefficient varied from 0.9876 to 0.9969). The
accuracy of the method was measured by determining the
mean concentration at various concentrations of analyte and
was calculated as percentage error of theoretical versus
measured concentrations. Both the intra- and interday accuracy
and precision of the method were determined by triplicate
analysis of standard samples containing VLCFAs at the concentrations
used to construct the calibration curves. Precision
was estimated as the coefficient of variance (CV) of the
analyses. The interday accuracy ranged from -5.41 to 3.23%,
and the intraday accuracy ranged from -3.95 to 4.27% (Table
C24:0/C22:0
(ion peaks
intensity ratio)
C24:0/C22:0
(conc ratio)
C20:0 (µg/mL) C22:0 (µg/mL) C24:0 (µg/mL)
1 16.23 15.21
patients with ALD
17.58 1.02 1.16
2 17.25 11.81 22.21 1.15 1.88
3 14.53 9.10 15.30 1.49 1.68
4 17.89 11.47 14.70 1.19 1.28
mean ± SD 16.47 ± 1.46 11.90 ± 2.5 17.45 ± 3.41 1.21 ± 0.20 1.50 ± 0.34
GC/MS median a
(5-90% range)
20.8 (11.8-29.5) 13.7 (7.0-21.1) 1.50 (1.18-1.74)
1 22.52 28.32
control group
19.52 0.64 0.69
2 20.28 26.30 20.95 0.76 0.80
3 18.86 22.13 18.25 0.76 0.82
4 18.16 18.45 14.96 0.74 0.81
mean ± SD 19.95 ± 1.92 23.80 ± 4.40 18.42 ± 2.55 0.72 ± 0.06 0.78 ± 0.06
GC/MS median a
(5-90% range)
25.3 (10.5-51.0) 17.4 (8.5-35.7) 0.73 (0.48-0.89)
a Value reported by Schutgens. 27
S-2 in the Supporting Information). The interassay precision
ranged from 1.05 to 7.52% and the intra-assay precision ranged
from 0.89 to 8.85% (Table S-2 in the Supporting Information). It
was found that the quantification of VLCFAs by MWCNT-based
SALDI, using isotope-labeled VLCFAs as internal standards, was
of acceptable accuracy and precision.
Patient Plasma Analysis of VLCFAs. In order to test the
clinical applicability of our assay, plasma samples collected from
four subjects with X-linked adrenoleukodystrophy (ALD) and four
normal controls were analyzed by MWCNT-based enrichment and
SALDI-TOFMS. The SALDI-TOFMS spectra of VLCFAs in ALD
patients and in normal controls are shown in Figure 4. The
intensity ratio of C24:0/C22:0 was significantly higher in the ALD
patients (1.02-1.49) than in the normal controls (0.64-0.76). The
peaks at m/z 440.4 and 452.4 were assumed to be C23:0 and
C24:1. 25,27,30,41 Table 2 summarizes the quantification data for
VLCFAs concentrations and ratios in this study and compares
them with the reference range reported by Schutgens. 27 In their
study, samples from 109 healthy subjects and 41 patients with
X-ALD were analyzed by GC-MS following derivatization by methyl
ester and purification by thin-layer chromatography. The quantification
results in our study were within the reported range.
Usually, concentration ratios of C24:0/C22:0 are used to diagnose
ALD. Stable-isotope quantification is important in the MALDI-
TOFMS method because of the suppression effect of matrix
interference peaks. In the MWCNT-based SALDI-TOFMS method
described herein, the relative quantification can also be directly
determined by measuring the ion peak intensities without the
isotope-labeled internal standards calibration. Although the sample
numbers are small, the concentration ratios of C24:0/C22:0
correlated well with the ion peak intensity ratios of C24:0/C22:0
(Pearson Correlation coefficient ) 0.88) (Figure S-8 in the
(38) Koulman, A.; Petras, D.; Narayana, V. K.; Wang, L.; Volmer, D. A. Anal.
Chem. 2009, 81, 7544–7551.
(39) Sleno, L.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2005, 19, 1928–
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(40) Hsu, W. Y.; Lo, W. Y.; Lai, C. C.; Tsai, F. J.; Tsai, C. H.; Tsai, Y.; Lin, W. D.;
Chao, M. C. Rapid Commun. Mass Spectrom. 2007, 21, 1915–1919.
(41) Aveldano, M. I.; Donnari, D. Clin. Chem. 1996, 42, 454–461.
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