Page 7 - CRFTApplication Compendium_090613

3 Thermo Scienti c Poster Note • PN ASMS13_M003_TOlney_E 07/13S
The top panel on Figure 5 shows the
at 40 pg on column (289.4  97.2 +
SRMs. The lower panel shows the fu
standard in the Q1 MS scan (290.9 –
95.7 – 298.7 Da).
Figure 7 again shown the chromatog
Q1 MS scan and the product ion sca
averaged Q1 Full Scan mass spectru
shown.
The response for 6 replicate injection
Table 2. Highlighted in yellow are the
tolerances. In most cases, where int
and are likely a result of lower injecti
outside the limit for mass or resolutio
indicate a change in calibration or fai
performance while
method is used for verification of
he Internal Standard Verification
would be used. Results of the
ine the confidence that can be
used to demonstrate generation
alysis. Next, conditions are
fault conditions (mimicking
entify data that is suspicious or
to simultaneously monitor the
taining the ability to quantitate
release of results, this can
ectroscopist. Usually an
view and the recourse is usually
peaks for the various analytes.
tors the operation of the mass
smission, during the analytical
This method utilizes the internal
ample preparation. The resulting
eliminate the need for manual
nitor one or two ions to evaluate
these monitor ions must be
y should not require additional
fects on the measurement of any
al standard is present as a
d. A new mode of operating a
t uses the internal standard to
ensities, in real time, during
e parameters are out of tolerance.
de up in 50/50 MeOH/Water. For
tration in all samples was 0.8
ns, a simple 3 min
peak, which is representative of
dura™ triple-stage quadrupole
arting with a standard
estosterone: 289.4 97.2, 109.2)
292.497.2).
e IS is first identified for ISV. The
om the IS SRM. The resolution
are automatically populated. The
oduct and SRM transitions.
quired. The user also has control
transition to perform the ISV
SQ Endura MS method editor.
the ISV parameters.
FIGURE 3. Response curve for
Testosterone using the ISV met When the ISV method is generated, in addition to the expected SRM transition
specified, the method UI adds two small diagnostic scans: a) Q1 MS full scan of 3 Da
about the precursor ion and b) product ion mode over a small 3 Da mass range about
the product ion mass. The scan times for the analyte ions are unaffected but time is
taken from the internal standard scan to perform the two ISV scans.
This method shown here has a chromatographic peakwidth of 3 seconds. Ten scans
are specified across the peak. Each cycle has ~300 ms and thus each of the three
transitions will have ~100 ms dwell time. The two testosterone transitions are
unaffected and have ~100 ms dwell times. The internal standard will provide the dwell
time perform the ISV scans. It is assumed that the internal standard is a well behaved,
well understood, strong transition. This allows the user to reliably take anywhere from
20 to 80% of the IS dwell time for the ISV measurements.
In the present case 80% of the IS time is given to the ISV. The IS will have a ~20 ms
dwell time and both the precursor ion Q1 MS full scan (290.9 – 293.9 Da) and the
product ion mode (292.4 95.7 – 98.7 Da) scan will each have ~40 ms dwell times.
Data Analysis
Data was collected using Xcalibur 3.0 and processed using Qual Browser. Data was
worked up using standard statistical tools in MS Excel.
In addition, as shown in Table 1, the
approximately 2 pg/mL for both the
TABLE 1. Testosterone response,
same 2 pg/mL level for both ISV
FIGURE 1. TSQ Endura MS method. Source parameters and scan parameters for
Testosterone analysis using ISV.
FIGURE 2. TSQ Endura MS method summary.
Results
Quantitation and Monitoring
Routine sample analysis typically consists of monitoring one or two transitions for each
analyte ion and monitoring a single transition for the associated internal standard.
These analyses can also have multiple analytes and may have multiple internal
standards. To reduce complexity and to demonstrate the effectiveness of the new
method, a simple testosterone assay with one analyte (two transitions) and one
internal standard was used to show the ability to monitor the performance of the mass
spectrometer through a series of injections over time. The analyte response was
monitored across the useful analytical range while the internal standard transition is
used to both normalize the system response (the traditional function) and to evaluate
the calibration of the mass spectrometer.
Figure 3 shows the calibration curve using the ISV method. Figure 4 is the curve run
using the same method without performing the ISV measurements. This demonstrates
that including the ISV portion in the method does not affect the linearity of the assay.
Test
pg/mL
2
4
20
200
2000
Testo
pg/mL
2
4
20
200
2000
FIGURE 5. Testosterone 40 pg on
column. Chromatograms shown fr
top to bottom: Testosterone SRM;
Testosterone d3 SRM, Q1 Full Sca
Product Ion Scan.