Page 8 - CRFTApplication Compendium_090613
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4
Development of a Method for Evaluation of Mass Spectrometer Performance in Real Time
The top panel on Figure 5 shows the SRM chromatographic response for testosterone
at 40 pg on column (289.4
97.2 + 101.2) and internal standard (292.4
97.2)
SRMs. The lower panel shows the full scan chromatographic response for the internal
standard in the Q1 MS scan (290.9 – 293.9 Da) and the product ion mode (292.4
95.7 – 298.7 Da).
Figure 7 again shown the chromatographic response for the full scan internal standard
Q1 MS scan and the product ion scan in the upper panel. In the lower panels the
averaged Q1 Full Scan mass spectrum and the Product Ion Mode mass spectrum are
shown.
The response for 6 replicate injections over the 2 to 2000 pg/mL range are shown in
Table 2. Highlighted in yellow are the values that fall outside of the predetermined
tolerances. In most cases, where intensities are low, they track the internal standard
and are likely a result of lower injection volume. In only two cases are the values
outside the limit for mass or resolution. These deviations were not large enough to
indicate a change in calibration or failure in the system.
FIGURE 3. Response curve for
Testosterone using the ISV method
FIGURE 4. Response curve for
Testosterone using standard method
(No ISV)
e expected SRM transition
scans: a) Q1 MS full scan of 3 Da
er a small 3 Da mass range about
e ions are unaffected but time is
e two ISV scans.
akwidth of 3 seconds. Ten scans
ms and thus each of the three
stosterone transitions are
rnal standard will provide the dwell
internal standard is a well behaved,
ser to reliably take anywhere from
ents.
e ISV. The IS will have a ~20 ms
an (290.9 – 293.9 Da) and the
ll each have ~40 ms dwell times.
d using Qual Browser. Data was
cel.
In addition, as shown in Table 1, the LLOQ, as defined by <15% RDS, is unchanged at
approximately 2 pg/mL for both the ISV and non-ISV methods.
TABLE 1. Testosterone response, corrected for IS. LOD is approximately at the
same 2 pg/mL level for both ISV and non-ISV methods.
Error Conditions
To demonstrate the utility of the
spectrometer to show the ability
shows a series of injections rep
drift represents the Q1 mass po
meters and scan parameters for
ring one or two transitions for each
associated internal standard.
d may have multiple internal
e the effectiveness of the new
yte (two transitions) and one
onitor the performance of the mass
e. The analyte response was
the internal standard transition is
aditional function) and to evaluate
method. Figure 4 is the curve run
measurements. This demonstrates
ot affect the linearity of the assay.
Testosterone with ISV Measurements
pg/mL
fg on Col
Response
% St Dev
2
40
2113
12.8%
4
80
3994
5.8%
20
400
19329
1.7%
200
4000
197403
1.2%
2000
40000
2117342
1.2%
Testosterone without ISV Measurements
pg/mL
fg on Col
Response
% St Dev
2
40
1922
9.7%
4
80
3824
5.6%
20
400
19301
1.1%
200
4000
188281
1.3%
2000
40000
2029272
0.8%
FIGURE 5. Testosterone 40 pg on
column. Chromatograms shown from
top to bottom: Testosterone SRM;
Testosterone d3 SRM, Q1 Full Scan,
Product Ion Scan.
FIGURE 6. Testosterone 40 pg on
column. Chromatograms shown from
top to bottom: Testosterone d3 Q1
Full Scan, Product Ion Scan; Mass
Spectra: Testosterone d3 Q1 Full
Scan, Product Ion Scan
TABLE 3. Effect on Testoster
drift.
TABLE 2. Testosterone respon
tolerance limits are shown at t
Conc
fg/uL
Analyte Internal Std Resp/I
40
2948 732910
2
40
2492 703486
2
40
2775 721359
2
40
2432 637542
2
40
1731 617986
1
40
2425 647537
2
80
4480 737877
3
80
4513 785476
3
80
4246 649547
4
80
4036 660396
3
80
4896 748874
4
80
4401 682479
4
400
21176 749495
19
400
20982 767396
19
400
15863 571678
19
400
16293 587573
19
400
13233 491762
18
400
13344 489467
19
4000 197336 702203 201
4000 200435 731429 196
4000 162271 597776 194
4000 159666 579981 197
4000 186138 677821 197
4000 169513 621454 195
40000 1628083 544745 2151
40000 1484947 500983 2133
40000 1522016 515818 2123
40000 1226096 416741 2117
40000 1279791 443751 2076
40000 1113194 381350 2101
Expect
600000
Error
150000
Min
450000
Max
750000
Response
Figure 7. Simulated Q1 mas
Conc
fg/uL Analyte Internal Std
400 ND ND
ND
400 3201 101823
400 15765 568519
400 18019 568924
400 21541 669797
400 7010 221336
400
502 15619
ISV Table
Response
