Use of Coulometric Array HPLC in Metabolomics

Use of Coulometric Array HPLC in Metabolomics
Gurmil Gendeh,1 Ian Acworth,2 John Waraska,2 Paul Gamache,2 and Dennis Price2
Dionex Corporation, Sunnyvale, CA, USA; 2ESA – A Dionex Company, Chelmsford, MA, USA
1
Introduction
Experimental
Metabolomics–as investigated for drug discovery–involves the use
of nuclear magnetic resonance (NMR) and LC-MS to assess dynamic
responses of biological systems to compound exposure. Rapid analyses
can determine changes in multivariate profiles of endogenous low-molecular weight metabolites in tissue or biofluids.1 NMR is primarily applicable to the measurement of high-level metabolites; LC-MS augments
these capabilities to lower-level metabolites that are eluted and ionized
under a given set of conditions. It is widely recognized, however, that no
single analytical technique is capable of addressing the chemical diversity
and concentration range of metabolites present in a biological system.
Treatment and Sample Preparation
Described herein is the use of HPLC with electrochemical coulometric
array (EC-array) and MS detectors in parallel to facilitate the study of
changes in redox-active small molecules associated with xenobioticinduced toxicity. Biological redox reactions are highly relevant to:
Sprague-Dawley rats were fed Purina 5001 rodent laboratory chow and
water ad libitum. They were given oral doses of xenobiotic compounds,
including acetaminophen (APAP) and acetylsalicylic acid (ASA). Urine
was collected for 8 h periods and diluted 10-fold in water prior to the
analysis of 5.0 or 20 µL volumes.
Conditions
HPLC Analytical Conditions
Column: Shiseido CapCell Pak® C18 MG 3 µm, 7.5 cm ×
4.6 mm i.d.
Binary Gradient:
• Metabolism (e.g., CYP450 oxidases, reductases)
• Disease (e.g., atherosclerosis, cancer, neurodegenerative disorders)3
• Therapeutic action (e.g. antioxidants, bioreductive agents:
adriamycin, quinones)3
• Toxicity (e.g., reactive species generation, covalent binding,
oxidative stress)3–6
1.0% to 80% aqueous CH3CN (v/v) each
containing 20 mM NH4OAc in 6 min,
2 min hold, and 2 min re-equilibration
Flow Rate: 1.0 mL/min. Postcolumn flow was split (4:1)
between EC and electrospray MS detectors,
respectively (Figure 1)
This approach has the potential advantages of applicability to a mechanistically targeted subset of the metabolome.
EC-Array: CoulArray® Electrochemical Detector, 8-channel
(P/N 70-4325)
EC Conditions:
0 to 840 mV vs. Pd, 120 mV increments
MS:
Agilent 1100 MSD (Palo Alto, CA) single quadrupole ES-MS operated in +/- scan mode
(m/z 70–500)Sciex API365 (Concord, Ontario,
CAN) triple quadrupole ES-MS/MS was used for
neutral-loss scan experiments
2
Detection
Data Processing
Apparatus
800000
A. ES-MS
Control
B. EC-Array
M3
M4
M1
µA
API-MS or
MS/MS
Pump
300 mg/kg APAP
300 mg/kg APAP
M2
Solvents
M4
TIC
35
Passive Flow Split
M3
TIC
0
HPLC pump and autosampler with CoulArray 8-channel detector.
M2
EIC 152
Ion Abundance
EC-array data were transferred to Pirouette® (Infometrix, Inc., Woodinville, WA) for chemometric analysis using a CoulArray Version 2.0
Software Utility (Pattern Recognition Setup Wizard).
0
840 mV
300 mg/kg APAP
600 mV
300 mg/kg APAP
600 mV
Control
Degasser
To Waste
Autosampler
0
1
2
3
4
5
6
Minutes
Coulometric Electrode Array
27252
27251
Figure 1. System schematic.
Figure 2. A) LC-ES-MS positive TIC (70–500 m/z scan range) of urine from control and APAP-dosed animal and corresponding EIC (m/z 152) from dosed animal
and B) Overlay of EC-array channel 6 (600 mV vs. Pd) of urine from control and
APAP-dosed animals and channel 8 (840 mV vs. Pd) from dosed animal.
RESULTS AND DISCUSSION
The combined use of MS and EC-array resulted in highly complementary detection for these rapid multivariate analyses. EC-array allowed pg
sensitivity for detection of poorly ionized redox-active compounds (e.g.,
phenols, thiols, sulfides, arylamines), as previously reported.7 Two
primary xenobiotic metabolite peaks were evident from the comparison
of positive total ion chromatograms (TIC) of control (n = 20) and APAPdosed (n = 40) animals (M2, M3; Figure 2A). Extracted ion chromatograms (EIC, m/z 152, APAP + H) revealed an additional metabolite (M4,
Figure 2B). EC-array data also clearly indicated detection of these three
metabolites. An additional metabolite, not detected by MS, was also
evident (M1, Figure 2B). The lack of MS response for this metabolite in
positive ion mode may be attributed to either increased N-acidity or loss
of the nitrogen group.
Using the conditions described here, approximately 70 peak clusters
of redox-active endogenous urinary metabolites were observed using
electrochemical detection. Estimated on-column detection range was
50 pg to 1 mg for a single, 20 uL sample of urine diluted 10-fold. This
corresponds to an estimated urinary concentration of 25 ng/mL to 500
ug/Ml. High and lower-level metabolites are shown in Figures 3A and
3B, respectively.
of Coulometric
HPLC Power
in Metabolomics
2 Use
Optimization
of the Array
Separation
of 1-D Nano LC Analysis of Proteomics Samples
Full Data
Several Treatment Groups
300
15
1A-3
2A-3
3A-3
4A-3
1B-3
2B-3
3B-3
4B-3
E2
280
All 240 mV
nA
5
0
Control
20 mg/kg APAP
300 mg/kg APAP
Response
10
M1
(no MS)
S)
3.0
3.2
3.4
3.6 3.8
Minutes
4.0
4.2
4.4
M4
M
1152
AP
APAP
M3
313, 232
APAP-M+S
E1
151
E3
245
E4
484
0
Raw Data
c5-6t1.33
80 APAP, 200 mg/kg (4A-3)
c5-6t2.16
c5-6t3.00
27254
300×
Figure 4. EC data from five each of controls, low- and high-dose APAP urines.
Base peak m/z values are shown for possible endogenous marker (E1-4) and
drug metabolite peaks (M1, M3 and M4). Peak M3 is attributed to both
APAP-mercapturate (APAP-M) and APAP-sulfate (APAP-S).
µA
0
840 mV
720 mV
600 mV
480 mV
360 mV
240 mV
120 mV
0 mV
0
c5-6t3.83
Variable
1
2
3
Minutes
4
5
27253
Figure 3. A) Eight-channel chromatogram from animal receiving a single 200
mg/kg (p.o.) dose of APAP (0–8 h collection) displayed at 100 µA full scale and
B) Overlay of channel 3 from several treatment groups amplified 300-fold vs. (A)
to demonstrate sensitive detection of endogenous metabolites. Conditions as
described in Experimental section.
These initial studies have utilized EC-array data primarily for quantitative
and chemometric analyses while using the stored MS data primarily for
subsequent qualitative and confirmatory characterization of specific
interesting variables revealed. A software utility capable of retention time
normalization, binning, concatenation, and formatting of data allowed
rapid (e.g. < 2 min for 80 sample profiles) transfer of data for exploratory pattern recognition analysis.
The widely reported APAP-sulfate (M3, m/z 232) and glucuronide
metabolites (M2, m/z 328) along with unchanged APAP (M4, m/z 152)
were evident based on chromatographic, voltammetric, and MS data
(Figures 2 and 5). APAP-related hepatotoxicity is believed to be
subsequent to CYP-450 oxidative metabolic activation to form reactive
quinoneimine species with subsequent covalent binding and scavenger
glutathione (GSH) depletion.5, 6 Constant neutral-loss scan of infused
10-fold diluted urine provided little evidence of APAP-SG. However, MS
(Figure 5b) and EC-array voltammetric (Figure 5c) data demonstrated
that peak M3 consisted of two major components (m/z 232, oxidation potential Eox 840 mV) and (m/z 313, Eox 600 mV) consistent with
APAP-sulfate and APAP-mercapturate. Sulfate substitution of the phenol
is expected to increase the oxidation potential while aromatic N-acetylcysteine is expected to retain the voltammetric profile of APAP. Thus,
these voltammetric data further support the presence of these two metabolites assuggested by MS data. Furthermore, as APAP-mercapturate
is a reported urinary endproduct of APAP metabolic activation, reactive
quinoneimine species formation, and GSH conjugation, these data
provide evidence of the oxidative pathway associated with the toxicity of
this xenobiotic.5, 6
3
A
APAP
NH
APAP-M NH
OH
OH
Principal component analysis (PCA) was used as an exploratory approach to provide suggestions of sample outliers and to investigate individual and pattern associations between variables and between samples.
PCA of EC-array data resulted in consistent differentiation of high-dose
APAP (200 and 300 mg/kg, 0–8 h collection) from control, low-dose
(20 mg/kg) APAP, and high-dose (200 mg/kg) ASA. This differentiation
was observed both exclusive (Figure 6) and inclusive (not shown) of
xenobiotic metabolite variables (shaded areas in Figure 4 indicate some
excluded variables). Possible endogenous marker peaks, indicated from
eigenvector analysis, were also qualitatively characterized using the
stored mass spectral and EC voltammetric data. Base peak m/z values
for some of these peaks are indicated in Figure 4. Further structural
characterization using MS/MS of potential endogenous and xenobiotic
markers will be reported elsewhere.
O
O
O
APAP-S NH
SCH2CHOOH
NHCOCH3
B
7000
OSO3–
TIC
EIC 152 APAP
EIC 232 APAP Sulfate
EIC 313 APAP
Mercapturate
µA
0
2.25
50
2.50
2.75
3.00
3.25
Minutes
3.50
3.75
4.00
4.25
High-Dose APAP
200 mg/kg ASA
C
Low-Dose APAP
µA
Controls
10
27256
2.6
2.8
3.0
3.2
3.4
3.6
3.8
Minutes
27255
Figure 5. A) Structures, B) Overlay of total ion and extracted ion chromatograms
showing evidence of APAP sulfate and mercapturate metabolites of APAP, and C)
EC-Array response across 0–840 mV cells showing evidence of two constituents
with differing voltammetric response.
Figure 6. PCA results of EC-Array data after exclusion of xenobiotic metabolite
variables analyzed as described in Experimental. Each data point represents a
different animal where A** = 200 mg/kg NAS; C*** = controls; APAP =
acetaminophen-dosed animals with 20 (APAP20); 200 (200 APAP) or 300 (300
APAP) mg/kg APAP.
of Coulometric
HPLC Power
in Metabolomics
4 Use
Optimization
of the Array
Separation
of 1-D Nano LC Analysis of Proteomics Samples
Conclusion
References
• EC-Array augmented the metabolic profiling capabilities of LC
electrospray-MS through parallel detection of condensed phaseneutral and nonpolar redox-active metabolites at pg to µg levels.
• High-density multivariate data were rapidly acquired and transferred to pattern recognition analytical software and high-dose
APAP samples were differentiated based on endogenous and
xenobiotic redox metabolic profiles.
• Stored MS and EC-Array data were subsequently used for effective
characterization of specific endogenous and xenobiotic redox
active components and evidence of oxidative metabolic activation
associated with high-dose APAP was observed.
• As many organic chemicals are thought to exert toxicity via redox
processes, the acquired redox profiles may be particularly useful
in drug discovery and development for tissue and regiospecific
modeling, diagnostic marker identification, and mechanistic insight
to xenobiotic-induced toxicity.
1. Holmes, E.; Shockor, J. P. Current Opinion in Drug Discovery and
Development 2000, 3(1), 72–78.
2. The Pharmaceutical Basis of Therapeutics Sixth Edition; Gilman,
A.G., Goodman, L.S., Gilman, A., Eds.; Macmillan Publishing, NY,
USA, 1980; pp 12–20.
3. Free Radicals in Biology and Medicine, Third Edition; Halliwell, B.,
Gutteridge, J.M.C., Eds.; Oxford University Press, NY, USA, 1999.
4. Eyer, P. Environ. Health Perspect. 1994 102, 123–132.
5. Yamamoto, K.; Sachiko, K.; Tajima, K.; Mizutani, T. Biol. Pharm. Bull.
1997, 20, 571–573.
6. Heitmeier, S.; Blaschke, G. J. Chromatogr., B 1999, 721, 93–108.
7. Gamache, P.; Meyer, D.; Granger, M.; Acworth, I. J. Am. Soc. Mass
Spectrom. 2004, 15, 1717–1726.
CoulArray is registered trademark of Dionex Corporation.
Pak is a registered trademark of Shiseido Co. Ltd.
Pirouette is a registered trademark of Infometric, Inc.
All third party trademarks and registered trademarks are the properties of their respective owners.
Passion. Power. Productivity.
North
America
Dionex Corporation
Europe
Asia Pacific
1228 Titan Way
P.O. Box 3603
Sunnyvale, CA
94088-3603
(408) 737-0700
Austria (43) 1 616 51 25 Benelux (31) 20 683 9768; (32) 3 353 4294
Denmark (45) 36 36 90 90 France (33) 1 39 30 01 10 Germany (49) 6126 991 0
Ireland (353) 1 644 0064 Italy (39) 02 51 62 1267 Sweden (46) 8 473 3380
Switzerland (41) 62 205 9966 United Kingdom (44) 1276 691722
Australia (61) 2 9420 5233 China (852) 2428 3282 India (91) 22 2764 2735
Japan (81) 6 6885 1213 Korea (82) 2 2653 2580 Singapore (65) 6289 1190
Taiwan (886) 2 8751 6655
U.S./Canada (847) 295-7500
South America
Brazil (55) 11 3731 5140
www.dionex.com
5
LPN 2481-01 3/10
©2010 Dionex Corporation