Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Table 2. Concentrations and Ratios of VLCFAs in Plasma from Patients with ALD and from Normal Controls as<br />
Measured by SALDI-TOFMS<br />
Postsource Decay (PSD) of TMAE-VLCFAs. The PSD MS<br />
spectra of the derivatized VLCFAs in SALDI-TOFMS, which<br />
detected a neutral loss of trimethylamine (59 Da), were similar<br />
to those in LC-MS/MS. 30 SALDI-PSD MS spectra of TMAE-<br />
VLCFAs are recorded in Figure 2. Fragment ions were detected<br />
at m/z 339.3 for C20:0, m/z 367.4 for C22:0, m/z 395.4 for C24:0,<br />
and m/z 423.4 for C26:0. Those data demonstrate that derivatized<br />
VLCFAs not only increased the ionization efficiency in SALDI but<br />
also made the PSD easier in TOFMS. Furthermore, the neutral<br />
loss fragmentations helped confirm the presence of VLCFAs in<br />
the complex plasma matrix.<br />
Quantitative Analysis of the TMAE-VLCFAs. CNT-based<br />
SALDI has been shown to have excellent reproducibility of<br />
spectrum signals, making it an acceptable modality for quantitative<br />
analysis. 22,19,32 In addition, MALDI-MS with and without the stable<br />
isotope-labeled method has been shown to be suitable for<br />
quantifiation of small compounds in biologic samples. 38-40 In this<br />
study, we used stable isotope-labeled VLCFAs (C26:0-d4, C22:0d3,<br />
C20:0-d3) as the internal standards. The mass difference of<br />
3-4 Da between VLCFAs and isotope-labeled VLCFAs provided<br />
sufficient m/z separation space to avoid interference with<br />
isotopologues of the ions. Figure 3 shows the SALDI-TOF mass<br />
spectra of the four derivative VLCFAs. Three of the derivative<br />
isotope-labeled VLCFAs had a molar ratio of 2:1 and a peak<br />
intensity ratio of 2:1. The isotope-labeled C22:0-d3 and C26:0-d4<br />
were both evaluated in the quantification of C24:0 as the<br />
internal standards, with C26:0-d4 showing superior linearity (R 2<br />
regression coefficient varied from 0.9876 to 0.9969). The<br />
accuracy of the method was measured by determining the<br />
mean concentration at various concentrations of analyte and<br />
was calculated as percentage error of theoretical versus<br />
measured concentrations. Both the intra- and interday accuracy<br />
and precision of the method were determined by triplicate<br />
analysis of standard samples containing VLCFAs at the concentrations<br />
used to construct the calibration curves. Precision<br />
was estimated as the coefficient of variance (CV) of the<br />
analyses. The interday accuracy ranged from -5.41 to 3.23%,<br />
and the intraday accuracy ranged from -3.95 to 4.27% (Table<br />
C24:0/C22:0<br />
(ion peaks<br />
intensity ratio)<br />
C24:0/C22:0<br />
(conc ratio)<br />
C20:0 (µg/mL) C22:0 (µg/mL) C24:0 (µg/mL)<br />
1 16.23 15.21<br />
patients with ALD<br />
17.58 1.02 1.16<br />
2 17.25 11.81 22.21 1.15 1.88<br />
3 14.53 9.10 15.30 1.49 1.68<br />
4 17.89 11.47 14.70 1.19 1.28<br />
mean ± SD 16.47 ± 1.46 11.90 ± 2.5 17.45 ± 3.41 1.21 ± 0.20 1.50 ± 0.34<br />
GC/MS median a<br />
(5-90% range)<br />
20.8 (11.8-29.5) 13.7 (7.0-21.1) 1.50 (1.18-1.74)<br />
1 22.52 28.32<br />
control group<br />
19.52 0.64 0.69<br />
2 20.28 26.30 20.95 0.76 0.80<br />
3 18.86 22.13 18.25 0.76 0.82<br />
4 18.16 18.45 14.96 0.74 0.81<br />
mean ± SD 19.95 ± 1.92 23.80 ± 4.40 18.42 ± 2.55 0.72 ± 0.06 0.78 ± 0.06<br />
GC/MS median a<br />
(5-90% range)<br />
25.3 (10.5-51.0) 17.4 (8.5-35.7) 0.73 (0.48-0.89)<br />
a Value reported by Schutgens. 27<br />
S-2 in the Supporting Information). The interassay precision<br />
ranged from 1.05 to 7.52% and the intra-assay precision ranged<br />
from 0.89 to 8.85% (Table S-2 in the Supporting Information). It<br />
was found that the quantification of VLCFAs by MWCNT-based<br />
SALDI, using isotope-labeled VLCFAs as internal standards, was<br />
of acceptable accuracy and precision.<br />
Patient Plasma Analysis of VLCFAs. In order to test the<br />
clinical applicability of our assay, plasma samples collected from<br />
four subjects with X-linked adrenoleukodystrophy (ALD) and four<br />
normal controls were analyzed by MWCNT-based enrichment and<br />
SALDI-TOFMS. The SALDI-TOFMS spectra of VLCFAs in ALD<br />
patients and in normal controls are shown in Figure 4. The<br />
intensity ratio of C24:0/C22:0 was significantly higher in the ALD<br />
patients (1.02-1.49) than in the normal controls (0.64-0.76). The<br />
peaks at m/z 440.4 and 452.4 were assumed to be C23:0 and<br />
C24:1. 25,27,30,41 Table 2 summarizes the quantification data for<br />
VLCFAs concentrations and ratios in this study and compares<br />
them with the reference range reported by Schutgens. 27 In their<br />
study, samples from 109 healthy subjects and 41 patients with<br />
X-ALD were analyzed by GC-MS following derivatization by methyl<br />
ester and purification by thin-layer chromatography. The quantification<br />
results in our study were within the reported range.<br />
Usually, concentration ratios of C24:0/C22:0 are used to diagnose<br />
ALD. Stable-isotope quantification is important in the MALDI-<br />
TOFMS method because of the suppression effect of matrix<br />
interference peaks. In the MWCNT-based SALDI-TOFMS method<br />
described herein, the relative quantification can also be directly<br />
determined by measuring the ion peak intensities without the<br />
isotope-labeled internal standards calibration. Although the sample<br />
numbers are small, the concentration ratios of C24:0/C22:0<br />
correlated well with the ion peak intensity ratios of C24:0/C22:0<br />
(Pearson Correlation coefficient ) 0.88) (Figure S-8 in the<br />
(38) Koulman, A.; Petras, D.; Narayana, V. K.; Wang, L.; Volmer, D. A. Anal.<br />
Chem. 2009, 81, 7544–7551.<br />
(39) Sleno, L.; Volmer, D. A. Rapid Commun. Mass Spectrom. 2005, 19, 1928–<br />
1936.<br />
(40) Hsu, W. Y.; Lo, W. Y.; Lai, C. C.; Tsai, F. J.; Tsai, C. H.; Tsai, Y.; Lin, W. D.;<br />
Chao, M. C. Rapid Commun. Mass Spectrom. 2007, 21, 1915–1919.<br />
(41) Aveldano, M. I.; Donnari, D. Clin. Chem. 1996, 42, 454–461.<br />
<strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
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