Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 5. Analysis of urine sample using a running buffer consisting
of 50 mM H3PO4-Tris, pH 4. Separation voltage: -10 kV with an
injection time of 5sat-10 kV. Detection: BDD at +1.0 V vs Ag/
AgCl, 3 M NaCl. The urine samples were filtered using a 0.22 µm
Millipore filter and diluted in the injection buffer with different dilution.
(A) 10-fold dilution, pristine and spiked urine samples were presented
as the lower and upper curve, respectively. Peak identification: (1)
IXS; (2) VMA; (3) AA; (4) HVA; (5) UA; and (6) TRP. The diluted
urine sample was spiked with 20 µM IXS, 20 µM VMA, 100 µM AA,
20 µM HVA, 100 µM UA, and 20 µM TRP. (B) 15-fold dilution, pristine
and spiked urine samples were presented as the lower and upper
curve, respectively. Peak identification: (1) IXS; (2) VMA; (3) AA; (4)
HVA; (5) UA; and (6) TRP. The diluted urine sample was spiked with
20 µM IXS, 20 µM VMA, 100 µM AA, 20 µM HVA, 100 µM UA, and
20 µM TRP.
For further LOD improvement, a series of experiments based
on field-amplified sample stacking (FASS) 30 was performed by
exploiting the conductivity difference between the sample zone
and the running buffer to effect preconcentration. A standard
solution of 6 analytes including ascorbic and uric acids, two
endogenous urinary compounds, was prepared in 10 mM H3PO4
pH 2 and injected at -10 kVfor 10 s with the separation
performed at -10 kV using 50 mM H3PO4 pH 4 as the running
buffer. It should be noted that at pH 3, ascorbic acid migrated
very closely with HVA, whereas uric acid was not baseline
separated from TRP. In principle, the amount of stacking is
proportional to the conductivity difference between the running
(30) Weng, Q.-F.; Xu, G.-W.; Yuan, K.-L; Tang, P J. Chromatogr. B 2006, 835,
55–61.
6902 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 6. Analysis of an urine sample using a running buffer
consisting of 50 mM H3PO4-Tris, pH 4. Separation voltage: -10 kV
with an injection time of 5sat-10 kV. Detection: BDD at +1.0Vvs
Ag/AgCl, 3 M NaCl. The urine sample was filtered using a 0.22 µm
Millipore filter and diluted 8-fold in the injection buffer. (A) with sample
stacking and (B) without sample stacking.
buffer and the sample solution. A preconcentration factor of 4
was observed for IXS, AA, and HVA compared to 3 for VMA
and UA and only 1.25 for TRP (Figure 4, curve b). With sample
stacking, the detection limit of IXS, AA, and HVA was 75 nM
compared with nonstacking (300 nM, Figure 4, curve a), significantly
lower than the physiological levels of these biomarkers in
urine as discussed later. Notice that peak-width was broadened
when the injection time was greater than 10 s (data not shown).
In another attempt, the capillary was first filled with 50 mM H3PO4
pH 4 followed by a sample injection as described above. The
cathodic running buffer was changed to 10 mM H3PO4, pH2
instead of 50 mM H3PO4, pH 4. In this case, the separation
window was narrower, resulting in the comigration of three
pairs: IXS/VMA, AA/HVA, and UA/TRP (Figure 4, curve c).
No further improvement in separation resolution was attained by
adding several organic modifiers including 10% methanol, 10%
acetonitrile, 5 mM methyl �-cyclodextrin, and 5 mM cetyl
trimethylammonium bromide in the running buffer.
Analysis of Biomarkers in Urine. The electropherogram of
an authentic urine sample obtained from a healthy female shows
5 low peaks and one very high peak. All biomarkers were detected
even if the urine sample was diluted to 10- and 15-fold, respectively
in the injection buffer (Figure 5). Standard IXS, HVA, VMA,