Page 344 - CRFTApplication Compendium_090613

The clenbuterol transitions monitored were
m/z 277.042 Am/z 202.954 at a Q1 peak width of 0.7 u
FWHM for SRM scans and m/z 277.078A m/z 202.958
at a Q1 peak width of 0.1 u FWHM for H-SRM scans.
For both, the collision energy was 18 V and the scan time
was 100 ms.
FAIMS Conditions
Dispersion voltage: –4500 V
Outer bias voltage: 35 V
Compensation voltage: –14 V
Inner electrode temperature: 50°C
Outer electrode temperature: 70°C
FAIMS gas: 50% He in N2 at 3 L/min
Implementing FAIMS requires the establishment of
conditions for the transmission of the desired analyte(s)
through the interface. Stable conditions for ion transmis-
sion can be expressed by the compensation voltage (CV).
In these experiments, the CV was ramped from –30 to 0V
in 1.5 min. The maximum response for clenbuterol
occurred at –14 V and indicated the appropriate CV for
LC–FAIMS–H-SRM analysis as shown in Figure 2.
Results and Discussion
A representative LC-SRM chromatogram for the analysis
of clenbuterol in human urine collected using unit mass
resolution (0.7 u in both Q1 and Q3) is shown in Figure
3. Although LC-MS/MS is a selective
technique, many isobaric interferences
appear in the chromatogram. These
isobaric interferences increase the
chemical background and can make
reproducible integration of the
analyte peak difficult.
An increase in selectivity is
achieved by using the FAIMS device
to improve ion separation. A repre-
sentative LC–FAIMS–SRM chromat-
ogram for the analysis of clenbuterol
in human urine is shown in Figure 4.
The selectivity offered by FAIMS
provides a cleaner chromatogram
than the corresponding trace in
Figure 3, but some contribution from
interferences remain.
By utilizing both FAIMS and
H-SRM in the LC-MS/MS analysis,
an even further increase in selectivity
is achieved. A representative
LC–FAIMS–H-SRM chromatogram
for the analysis of clenbuterol in
human urine is shown in Figure 5.
The selectivity offered by the combi-
nation of LC, FAIMS, and H-SRM
results in the further removal of
interferences.
-30
100
80
60
40
20
0
-24 -18 -12 -06 -0
Relative Intensity
Compensation Voltage Value (V)
m/z 277.1 Optimum setting = -14.16
Figure 2: Compensation voltage scan from the infusion of a clenbuterol reference standard.
The maximum response for clenbuterol occurred at a compensation voltage of –14V.
RT: 1.8
AA: 31592
2.1
2.1
100
90
80
70
60
50
40
30
20
10
0
0.0 0.5 1.0 1.5
Time (min)
2.0 2.5 3.0
Relative Abundance
Figure 3: Representative LC–SRM chromatogram for clenbuterol in human urine, obtained with unit
mass resolution and without FAIMS selectivity.