Stable Isotope Labeled Fatty Acid Analysis in Plasma Using LC-FAIMS-SRM

Stable Isotope Labeled Fatty Acid Analysis in Plasma Using LC-FAIMS-SRM
Application
Note: 382
Stable Isotope Labeled FattyAcid Analysis
in Plasma Using LC-FAIMS-SRM
Kevin Bateman1, Sebastien Gagne1, Sheldon Crane1, Jean-Francois Levesque1, and James T. Kapron2
1
Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec, Canada; 2Thermo Fisher Scientific, Ottawa, Canada
Introduction
Key Words
• TSQ Quantum
Ultra™
• Surveyor™ HPLC
• Improved
selectivity
• Isobaric
interferences
The analysis of fatty acids is generally performed using
GC-MS methods. While these methods provide the
necessary sensitivity, sample preparation involves
derivatization, which is time consuming and labor
intensive. In comparison to GC methods, fatty acid
analysis by LC-MS is simplified because derivatization
is not required. The resulting LC-MS chromatogram,
however, often exhibits a high chemical background
that ultimately limits detection.
In this work, the gas-phase separation of high-Field
Asymmetric waveform Ion Mobility Spectrometry (FAIMS)
is used in combination with LC and zero neutral loss
tandem MS to increase the selectivity of the method.
The chemical background in the resulting LC-FAIMS-SRM
chromatograms is significantly reduced with respect to
that collected using the LC-SRM method. The result is
improved detection limits for fatty acid analyses.
Goal
To reduce the chemical background in the analysis of
stable isotope labeled stearic and oleic acids by FAIMS
with LC and tandem MS.
Experimental Conditions
Sample Preparation
Rat plasma samples containing labeled stearic and oleic
acids were prepared at a concentration level of 1000 nM.
HPLC
10 µL samples were separated on a 2.1×50 mm C8 column.
A binary gradient was formed using the Surveyor MS
Pump (Thermo Fisher Scientific, San Jose, CA) delivering
mobile phases A (0.1% ammonium hydroxide in water)
and B (0.1% ammonium hydroxide in methanol) at a
flow rate of 250 µL/min as described in Table 1.
Time (min.)
0
4
4.1
7
8
14
Table 1. Gradient profile
%B
60
74
95
95
60
60
MS
MS analysis was carried out on a TSQ Quantum Ultra
triple stage quadrupole mass spectrometer with a heated
electrospray ionization (H-ESI) probe (Thermo Fisher
Scientific, San Jose, CA). See Figures 1 and 2.
The MS and FAIMS Conditions were as Follows
MS Conditions
Ion source polarity: Negative ion mode
Spray voltage: 4000 V
Vaporizer temperature: 250°C
Sheath gas (N2): 40
Auxiliary gas (N2): 40
Ion transfer tube temperature: 300°C
Q1 peak width: 0.7 u FWHM
Collision energy: 10 eV
Scan time: 100 ms
Scan type: SRM
The SRM transitions were survivor ion scans for the
analytes of interest. Survivor ion scanning, also known as
zero neutral loss tandem MS, involves Q1 and Q3 of a
triple quadrupole MS. Both mass resolving quadrupoles are
set to the same m/z. The labeled oleic acid transition that was
monitored was m/z 288.2 → m/z 288.2. The labeled stearic
acid transition monitored was m/z 290.2 → m/z 290.2.
FAIMS Conditions
Dispersion voltage: +4500 V
Outer bias voltage: –35 V (identical to ion transfer tube
voltage offset)
Inner electrode temperature: 50°C
Outer electrode temperature: 80°C
FAIMS gas: 50% He in N2 at 4.5 L/min
Implementing FAIMS requires the establishment of
conditions for the transmission of the desired analyte(s)
through the interface. Stable conditions for ion transmission is expressed as the compensation voltage (CV). The
maximum response for the infusion of labeled oleic and
stearic acid reference standards occurred at –14 V, and
this value indicated the appropriate CV for LC-FAIMSSRM analysis. See Figure 3.
Results and Discussion
Representative LC-SRM chromatograms for the analysis
of labeled oleic acid (tR 5.6 min) and labeled stearic acid
(tR 5.8 min) in rat plasma extracts are presented in Figure 4.
Both chromatograms show that between 0.5 min. and
4.0 min. multiple species elute from the column and are
transferred to the mass spectrometer. As a result, the
baselines of the chromatograms are very high.
An increase in selectivity is achieved by using the
FAIMS device to improve ion separation. Representative
LC-FAIMS-SRM chromatograms from the injection of a
rat plasma extract of labeled oleic acid (tR 5.6 min) and
stearic acid (tR 5.8 min) are shown in Figure 5. The use of
FAIMS provides enrichment of the analytes by removing
some of the endogenous isobaric interferences and by
reducing the chromatographic baseline.
Figure 6 shows the effect of FAIMS on an absolute
scale. A representative LC-SRM chromatogram for
labeled oleic acid is compared with an LC-FAIMS-SRM
chromatogram from the injection of identical rat plasma
extract. The signal heights are approximately identical,
but the baseline for the LC-FAIMS-SRM chromatogram
is greatly reduced.
Figure 7 shows the effect of FAIMS on a relative
intensity scale for labeled stearic acid. A representative
LC-SRM chromatogram is overlaid with an LC-FAIMSSRM chromatogram from the injection of identical rat
plasma extract. The baseline for the LC-SRM trace shows
a constantly varying baseline, but the LC-FAIMS-SRM
trace shows resolved individual chromatographic peaks.
H-ESI probe
FAIMS interface
Inner electrode
Outer electrode
MS inlet
Figure 1: Drawing of the ion source (H-ESI probe), a cross-section of the
FAIMS interface, and the MS inlet. FAIMS separation involves selective
transfer of only a subset of the ions produced by the H-ESI probe.
FAIMS
waveform
generator
Figure 3: Representative LC-SRM chromatogram for labeled oleic acid
(above) and stearic acid (below) in rat plasma extracts.
Compensation
voltage
CV = –14V
H-ESI (heated
electrospray)
FAIMS
electrodes
between source
and vacuum
Figure 2: Photograph of FAIMS-enabled Quantum triple quadrupole MS
showing the asymmetric waveform generator and the heated electrospray
(H-ESI) source. FAIMS separates ions based on differences in mobility at
very high vs. low electric fields.
Figure 4: CV scan from the infusion of labeled stearic and oleic acids.
The optimum CV at which the acids emerge from FAIMS is –14V.
The LC-SRM ion chromatograms for both of the
labeled fatty acids demonstrate that a high chemical
background is present, limiting the level of quantitation.
Signal-to-noise values of 155 and 77 were obtained for
labeled oleic and labeled stearic acid, respectively. See
Table 1. The ion filtering action of FAIMS provided a
reduction in chemical background of approximately
10-fold, while the ion signal was maintained for labeled
oleic acid and reduced by 50% for labeled stearic acid.
The signal-to-noise values with FAIMS were 688 and
173 for labeled oleic and labeled stearic acids, respectively.
Therefore, FAIMS provided a 4-fold improvement in
the signal-to-noise ratio for labeled oleic acid and a
2-fold improvement in the signal-to-noise ratio for
labeled stearic acid.
Conclusion
The use of FAIMS significantly reduced the chemical
background and enriched the labeled fatty acid
chromatograms by partially removing endogenous isobaric
interferences. This reduction in chemical background
helped to define the chromatographic peaks, which
resulted in a more reliable integration of the ion signal.
The overall result was an improved assay for the detection
of tracer fatty acids in fat metabolism studies.
References
1
Kapron, J.; Jemal, M.; Duncan, G.; Kolakowski, B.; Purves, R. “Removal
of metabolite interference during liquid chromatography/tandem mass
spectrometry using high-field asymmetric waveform ion mobility
spectrometry”; Rapid Commun. Mass Spectrom. 2005, 19(14), 1979–1983.
2
Kapron, J.; Wu, J.; Mauriala, T.; Clark, P.; Purves, R.; Bateman, K.
“Simultaneous analysis of prostanoids using liquid chromatography/highfield asymmetric waveform ion mobility spectrometry/tandem mass
spectrometry”; Rapid Commun. Mass Spectrom. 2006, 20(10), 1504–1510.
5.8
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
2
Figure 5: Representative LC-FAIMS-SRM chromatogram for oleic and stearic
acid in rat plasma extract.
5.6
40000
35000
Intensity
30000
25000
LC-SRM
20000
15000
10000
500
0
5.6
30000
Intensity
25000
20000
LC–f–SRM
15000
10000
500
0
2
4
6
8
10
12
Time (min)
Figure 6: Labeled oleic acid chromatograms from LC-SRM (above) and
LC-FAIMS-SRM (below) analysis. The absolute height of these peaks
is approximately equal, but the background is reduced with FAIMS.
14
4
6
8
10
12
Figure 7: Overlayed chromatograms of labeled stearic acid with and
without FAIMS.
14
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