Download Capillary Electrophoresis/Mass Spectrometry: From One Meter

EXTENED ASMS ABSTRACT
Capillary Electrophoresis/Mass Spectrometry: From One Meter Capillaries to Chip-Based
Devices
Jack Henion, Katja Heinig, and Timothy Wachs, Analytical Toxicology, New York State College of
Veterinary Medicine, Cornell University, 927 Warren Dr., Ithaca, NY 14850, and Gary Schultz and
Tom Corso, Advanced BioAnalytical Services, Inc., 15 Catherwood Rd., Ithaca, NY 14850.
INTRODUCTION
Capillary electrophoresis coupled with mass spectrometry (CE/MS) was first reported by R. Smith
et al. in the last decade. Although one can argue that CE/MS should be a powerful complement
to LC/MS techniques, this promise has not been fulfilled. As a so-called nanoscale technique
most researchers have not been particularly successful with practical applications which
convincingly demonstrate either qualitative or quantitative capabilities. Some of the problems are
due to the rather low levels of sample that are typically injected (loaded) onto the capillary as well
as the somewhat limited detection limits afforded by current MS techniques. In spite of the
relatively good sensitivity that we typically have from MS, the demands of CE capillary loadings,
etc. place stringent detection limit demands upon the CE/MS combination.
This paper presents a brief review of the historical developments in CE/MS using atmospheric
pressure ionization (API) techniques. It will review the successful strategies for interfacing CE to
API MS followed by recent examples showing the CE/MS and CE/MS/MS determination of
carnitine and its acyl analogs in biological samples including human plasma and urine. Finally,
recent developments will be presented which focus on miniaturizing CE/MS techniques to chipbased devices. It is the authors’ belief that nanofabricated devices which can provide rapid,
routine, serial and/or parallel separations on a chip-based substrate may contribute in the future to
expanded use of CE/MS techniques in the pharmaceutical, agrochemical, and chemical
industries.
EXPERIMENTAL
A Crystal CE model 310 (ATI Unicam, Madison, WI) was used for the electrophoretic separations.
Uncoated fused-silica capillaries of 75 m i.d., 360 m o.d. and 80 cm length were filled with CE
electrolyte solutions containing ammonium acetate (10-30 mmol/l) with acetic or formic acid in
aqueous and organic (methanol, acetonitrile) solution. The separation voltage was 30 kV. A
pressure between 0 and 50 mbar was applied at the injection (inlet) end during the electrophoretic
separation. Injection was performed hydrodynamically or electrokinetically. The PE Sciex API 365
tandem mass spectrometer was coupled to the CE apparatus via an in-house built coaxial sheathflow interface, placed into the commercial ion spray interface housing. A 2 M resistor was used
to decouple the electrospray voltage from the CE voltage. Pneumatically assisted electrospray
(ion spray) with 2-propanol/water or methanol/water containing 0.1% acetic acid as sheath liquid
at 4-10 l/min and nitrogen as nebulizer gas was used for all CE/MS experiments. The sheath
liquid was delivered by a Harvard Apparatus syringe pump. All experiments were carried out in
the positive ion mode. Full-scan, selected ion monitoring (SIM) or selected reaction monitoring
(SRM) modes were used. The Q1 and Q3 quadrupoles were operated at unit mass resolution (0.7
Da at half-height.
RESULTS AND DISCUSSION
This report introduces the development and application of capillary electrophoretic methods with
electrospray mass spectrometric detection for the determination of carnitine and acylcarnitine
homologues in human blood, urine and plasma. The selected reaction monitoring mode provides
high selectivity and sensitivity, although SIM and full-scan analyses are also possible for the
detection of the targeted analytes in biological samples. Performing a CE/MS analysis provides
the possibility to separate isomers that are not distinguishable by MS detection without prior
separation. This work shows the feasibility for determining carnitine and acylcarnitines in human
urine and plasma samples with acceptable selectivity, sensitivity, precision and accuracy. The
simultaneous determination of all analytes with one sample preparation and separation method
was not practical in this work. Nonaqueous CE electrolytes (methanol/acetonitrile as solvent
mixture) provided good homologous peak resolution of carnitine and all acylcarnitines inclusive of
C2 to C18 in a relatively short separation time, but could only accommodate relatively “clean”
biological samples prepared by liquid-liquid extraction. Therefore, this approach can be applied if
the determination of long-chain homologues is required. A 100% aqueous electrolyte was wellsuited for each sample matrix studied and showed good isomeric acylcarnitine separation
efficiency (Figure 1). Although the C16 and C18 homologues could not be detected, this method is
applicable to characterize important metabolic disorders, such as propionyl and isovaleryl
acidemia and MCAD. Therefore, CE/MS might be used as an alternative for acylcarnitine
determinations in clinical samples.
1
400000
110000
4b
A
4a
300000
C
7
90000
8
4
200000
1
70000
8
100000
8.5
10
6
5
9
3
9
2
50000
intensity, cps
10
0
12
11
30000
4
370000
6
8
10
12
14
12
650000
B
320000
14
16
18
D
550000
270000
450000
220000
350000
170000
12
1
120000
12
250000
1
150000
70000
20000
50000
4
5
6
time, min
7
8
2
3
4
5
6
7
8
time, min
Figure 1. Separation of synthetic carnitine/acylcarnitine standard mixtures in different
electrolytes by SIM CE/MS. (A) aqueous buffer containing 0.8% formic acid, (B) buffer
containing 3.2% acetic acid and 50% acetonitrile, (C) buffer containing 3.2% acetic acid and 50%
methanol, (D) nonaqueous buffer (6.4% formic acid in methanol/acetonitrile 1:1); buffer: 10 mmol/l
ammonium acetate; voltage: 30 kV; injection: 0.1 min 50 mbar standard mixture 1.5 g/ml; for
peak assignment see Table 1, 4a) iso-butyrylcarnitine, 4b) n-butyrylcarnitine
These developments provide a useful test platform to evaluate the analytical potential of chipbased CE/MS techniques. We have initiated studies to evaluate the potential to accomplish
competitive results to those described above using chip-based substrates. Preliminary results
from nanofabricated capillary channels in silicon substrates will be shown. A novel monolithic
nanosprayer nozzle fabricated in a silicon chip in a radial array will be described with a focus on
new strategies for high-throughput CE/MS analyses in the future.
ACKNOWLEDGMENTS
The authors wish to thank SmithKline Beecham for financial support of our research and PE Sciex
for the loan of the API 365 mass spectrometer and financial support. We also thank ATI Unicam
for the loan of the Crystal CE apparatus. Furthermore, we thank Dr. Carla Vogt for supplying the
acylcarnitine standards and helpful information as well as Dr. Donald Chace of Neo Gen
Screening for generously providing blood spot samples and internal standards.