Analytical Chemistry Chemical Cytometry Quantitates Superoxide

HVA and VMA in infant urine has been analyzed by capillary
electrophoresis (CE) with UV detection with a detection limit
(LOD) of 3.5 × 10 -4 M for HVA and 1.8 × 10 -4 M for VMA. 8a A
simultaneous determination of VMA, HVA, creatinine, and uric
acid uses capillary electrophoresis and a 30 mM phosphate
buffer (pH 7.0) containing 150 mM sodium dodecyl sulfate
(SDS). The detection is by UV absorbance at 245 nm and the
run is rather lengthy (15 min). 9 Although the authors claimed
lower LODs for both HVA and VMA (5.5 × 10 -5 M and 5.0 ×
10 -5 M), such LODs are still significantly above the normal
levels found in healthy subjects (8.2 to 41 µM for HVA and
11.6 to 28.7 µM for VMA). 10 Besides high LOD, UV detection
is also problematic as biological samples often consist of several
compounds with strong absorption at low wavelengths. The
neurotransmitters can be conjugated with a strong fluorophore
and analyzed by MEKC with fluorescence detection to achieve
high sensitivity. 11a This approach might be feasible for routine
analysis of a few compounds but becomes impractical and timeconsuming
for multiple analytes. The use of CE-MS for analysis
of VMA, HVA, and other biomarkers for metabolic disorders
in newborns is available elsewhere. 11b CE equipped electrochemical
detection (ECD) using a bare silica capillary can be
used to detect HVA and VMA after electrophoretic separation
at pH 5. 12a,b However, HVA and VMA are not baseline separated
and other catecholamines including IXS are not included. 12a HVA
and VMA also become less electroactive at pH > 5, the minimal
required pH for electrophoretic separation of HVA and VMA. In
general, bare fused silica capillaries at high pH are used in such
studies to resolve IXS, HVA, and VMA. The separation is lengthy
since negatively charged IXS, HVA, and VMA migrate to the
anode, i.e., opposite flow direction to the electroosmotic flow
(EOF). In addition, HPLC with isocratic elution and spectrophotometric
detection are often used for analysis of tryptophan
metabolites including IXS. 12c HPLC is also coupled with mass
spectrometry to detect compounds associated with purple urinary
bag syndrome (PUBS) and IXS is one of these toxic
compounds. 12d Micellar electrokinetic chromatography (MEKC)
using a bare fused silica capillary and laser induced fluorescence
detection (a KrF excimer laser, λ ) 248 nm) has been used to
detect HVA, VMA, and IXS. Under the best condition with this
expensive instrumentation, the detection limit for HVA and VMA
is 170 nM and 150 nM, respectively. 12e
This work describes a novel scheme for the analysis of IXS,
HVA, and VMA in the presence of tryptophan and other important
catecholamines and indoleamines (Table 1). The fused silica
capillary is coated with a thin layer of poly(diallyl dimethylammonium)
chloride (PDDA) or gold nanoparticles (AuNPs) embed-
(9) Shirao, M. K.; Suzuki, S.; Kobayashi, J.; Nakazawa, H.; Mochizuki, E.
J. Chromatogr. B 1997, 693, 463–467.
(10) Garcia, A.; Heinanen, M.; Jimenez, L. M.; Barbas, C. J. Chromatogr. A 2000,
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(12) (a) Li, X. J.; Jin, W. R. Chin. Chem. Lett. 2002, 13 (9), 874–876. (b) Li,
X. J.; Jin, W. R.; Weng, Q. F. Anal. Chim. Acta 2002, 461 (1), 123–130. (c)
Marklova, E.; Makovickova, I.; Krakorova, I. J. Chromatogr. A 2000, 870,
289–293. (d) Bar-Or, D.; Rael, L. T.; Bar-Or, R.; Craun, M. L.; Statz, J.;
Garrett, R. E. Clin. Chim. Acta 2007, 378, 216–218. (e) Paquette, D. M.;
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6896 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
ded in PDDA to reverse the EOF, allowing fast migration of IXS,
VMA, HVA, and tryptophan. In contrast, catecholamines and
indoleamines migrate against the EOF and emerge very late in
the electropherogram, i.e., they do not interfere with the analysis
of IXS, VMA, HVA, and tryptophan. The presence of AuNPs plays
an important role in baseline separation of several compounds
and a mechanism is given to decipher the interaction between
AuNPs and the analytes. Although the composite consisting of
AuNPs embedded in PDDA has been reported by Chen et al., 13
this is the first systematic application of an AuNP-PDDA coated
capillary for simultaneous analysis of IXS, HMA, and VMA in urine
samples. Together with sample stacking, the applicability of this
approach for analysis of such important biomarkers in urine
samples with improved detection sensitivity is also presented and
discussed in detail.
EXPERIMENTAL SECTION
Chemicals. Poly(diallyl dimethylammonium) chloride (PDDA,
MW ) 200 000-350 000, 20 wt % in water), hydrogen tetrachloroaurate
tetrahydrate (HAuCl4 · 4H2O), Tris (hydroxymethyl)aminomethane,
phosphoric acid (H3PO4), and other chemicals
were purchased from Sigma (Dublin, Ireland). Unless otherwise
stated, a 50 mM H3PO4 solution was adjusted to pH 3.0
with 0.5 M Tris buffer and used as the separation buffer. The
standard stock solutions (5.0 mM) of the analytes were
prepared daily in deionized water. All solutions were prepared
in Milli-Q ultrapure water and filtered through a 0.22 µm pore
size membrane followed by sonication for 5 min prior to use.
Synthesis of PDDA-Gold Nanoparticle (AuNPs) Composite.
The PDDA-AuNP composite was prepared as described by
Chen et al. 13 PDDA (250 µL, 4% wt. in H2O), 40 mL of H2O, 200
µL of 0.5 M NaOH, and 300 µL of HAuCl4 (10 mg/mL) were
thoroughly mixed in a beaker, covered with an inverted culture
dish for 2 min. The mixture was then maintained at 100 °C for
30 min, resulting in a ruby red solution. The UV-visible
spectrum of the AuNP colloid was recorded on a HP 8453
UV-visible spectrophotometer in a1cmoptical path quartz
cuvette. The size distribution of AuNPs in PDDA was measured
using a zetasizer Nano ZS system (Malvern Instruments, MA)
which is based on a dynamic light scattering (DLS) technique.
TEM micrographs were obtained by a Delong LVEM (Soquelec,
Montreal, QC, Canada) low-voltage TEM at 5 kV. A
small amount of PDDA-AuNPs was sonicated to disperse the
material. A 20 µL sample of well dispersed suspension was then
dried on a Formvar-carbon coated grid and analyzed.
Preparation of Coated Capillaries. A fused-silica capillary
(50 µm id and 365 µm od) purchased from Polymicro Technologies
(Phoenix, AZ, USA) was cut to 45 cm as the effective capillary
length. In order to expose the maximum number of silanol groups
on the silica surface, the fused-silica capillary was rinsed with 1.0
M NaOH and deionized water for 15 min each. The preconditioned
capillary was then rinsed with the PDDA or the PDDA-AuNP
(13) Chen, H.-J.; Wang, Y.-L.; Wang, Y.-H.; Dong, S.-J.; Wang, E. Polymer 2006,
47 (2), 763–766.
(14) (a) Luong, J. H. T.; Male, K. B.; Glennon, J. D. Analyst 2009, 134 (10),
1965–1979. (b) Kraft, A. Int. J. Electrochem. Sci. 2007, 2, 355–385. (c) Swain,
G. M.; Ramesham, R. Anal. Chem. 1993, 65, 345–351. (d) Xi, J.; Granger,
M.; Chen, Q.; Strojek, K.; Lister, T.; Swain, G. Anal. Chem. 1997, 69, 591A–
597A. (e) Tenne, R.; Patek, K.; Hashimoto, K.; Fujishima, A. J. Electroanal.
Chem. 1993, 347, 409–415.