Technical Note
Multiple Techniques for Simultaneous
Quantitative & Qualitative Data
Acquisition Using a Hybrid QuadrupoleLinear Ion Trap Mass Spectrometer
Gary Impey, Applied Biosystems/MDS Sciex, Concord, ON Canada
Overview
The goal of this work was to evaluate the multiple options available for simultaneous collection of
quantitative data with confirmatory information using a hybrid quadrupole-linear ion trap mass
spectrometer.
- Selectivity and sensitivity of Multiple Reaction Monitoring (MRM) scan capabilities combined with
linear ion trap functionality in an automated experiment
- Dedicated full scan MS/MS
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- Ultimate specificity using MS
Introduction
Multiple reaction monitoring (MRM) is the standard technique for quantitative LC/MS/MS
experiments. The LOD’s/LOQ’s, precision, and accuracy achieved with this technique are difficult
to match. However, it is often desirable to obtain confirmatory or qualitative information in addition
to quantitative data. This can help determine possible interferences or troubleshoot an analysis.
The ability to acquire both types of data in a single experiment would save significant amounts of
both time and money, with the advantages of obtaining additional information. A hybrid triple
quadrupole/linear ion trap mass spectrometer has the capabilities to perform such an experiment in
a variety of ways. These include, single MRM transitions per analyte, multiple MRM transitions per
analyte (one for quantitation and the others for confirmation), MRM transitions used as survey
scans to trigger the collection of MS/MS in an automated fashion, dedicated full scan MS/MS per
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analyte, and dedicated MS for improved selectivity.
As with any technique, the possibility of interferences can cause significant problems with
reproducibility as well as inaccurate results, even in MS/MS mode. To improve specificity, several
solutions have been proposed; 1) improve chromatographic separation, or 2) increase resolution to
improve parent mass selection. Improving chromatographic separation generally leads to
increases in analysis time, which is undesirable for large sample sets. Using higher resolution can
improve parent mass selection, but a compromise between resolution and sensitivity must be
realized. Taking advantage of improved specificity as opposed to resolution can be achieved using
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MS in a quantitative application, without the need for lengthy chromatography.
Each one of these techniques was evaluated for a quantitative application. The quantitative data
obtained from a typical MRM experiment was used as a benchmark in terms of LOD’s/LOQ’s,
precision and accuracy. Statistical analysis was performed on all sets of data and the results
compared.
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Technical Note
Experimental Conditions
The test mixture consisted of eight benzodiazepines and a deuterated internal standard (see Table
1 for names and corresponding transitions monitored). Nine standards were prepared covering the
concentration range from 50 fg/µL to 1 ng/µL as standard solutions as well as in urine matrix. Five
replicate injections (5 µL) were made for each concentration level. The LC/MS system consisted of
®
an Agilent 1100 autosampler, Agilent 1100 LC pump, and an AB/MDS Sciex 4000 Q TRAP (see
Figure 1). Separation was achieved on a monolithic column (Chromolith SpeedROD 4.6 x 50 mm)
with 50:50 ACN:H2O with 0.1% formic acid at a flow rate of 1.0 mL/min and total run time of 3.0
minutes. Five different combinations of MRM and full scan MS/MS experiments were performed
(see Table 2 for details).
Table 1. Test Mixture of Benzodiazepines and their
Corresponding MRM Transitions
Analyte
Alprazolam
Bromazepam
Clonazepam
Diazepam
Oxazepam
Flunitrazepam
Prazepam
Temazepam
Transition
IST
D
309/281 Alprazolam-d5
309/205
316/209
316/270
285/193
287/241
314/268
325/271
301/255
Figure 1. Schematic of 4000 Q TRAP ® System
Transition
Q0
314/286
Q1
q2
Q3
LIT
Table 2. Multiple Options for Simultaneous Quantitative and Qualitative Data Acquisition
Scan Type
Q1
q2
Q3/LIT
Experiment(s)
Use
MRM
resolving
fragment
resolving
EPI
MS3
resolving
resolving
fragment
fragment
trap/scan
isolate/fragment/trap/scan
Single MRM transitions/analyte
Multiple MRM transitions/analyte
Automated collection of MS/MS
Full Scan quantification
pathway specific quantification
quantification
quantification with confirmation (ion ratios)
quantification with confirmation (full Scan)
quantification with confirmation (full Scan)
ultimate specificity for quantification
The specific comparisons made for quantitation were (see Table 2): 1) using a single MRM
st
transition per analyte, 2) MRM with multiple transitions per analyte (1 transition for quantitation and
nd
the 2 for confirmation, 3) MRM transitions triggering the collection of linear ion trap MS/MS in an
nd
automated experiment (full scan MS/MS collected, no need for the 2 transition), 4) full scan linear
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ion trap MS/MS (extracted around the ion of interest) and 5) using MS .
Results
MRM For Quantitation: Multiple reaction monitoring (MRM) is the standard mode used to perform
quantitative LC/MS experiments. It is also often desirable to obtain confirmatory or qualitative
information in addition to the quantitative data. Many areas use multiple MRM transitions per
analyte in order to confirm the presence of the desired compound (typically 2, but in some cases 3).
Based on ion ratios and comparisons between standard and sample area ratios of the chosen
transitions, it is possible to determine interferences that may cause erroneous results.
The number of analytes to be monitored, and the number of transitions per analyte needed for
confirmation will add to the overall cycle time of the method. A triple quadrupole can monitor a
large number of transitions simultaneously and still achieve the necessary number of points per
chromatographic peak for precise quantitation. Typically, 10 points per chromatographic peak is
the minimum one would need to achieve reasonable %CV’s. Figure 2 shows 8 analytes with 2
transitions each (one for quantitation and the other for confirmation) and a deuterated internal
standard. 17 transitions were monitored simultaneously in less than 3 minutes.
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Technical Note
A
Figure 2. A) 17 MRM transitions were used to
monitor and confirm (with secondary transitions)
the presence of 8 benzodiazepines (+ 1 ISTD) in a
high throughput chromatographic method in under
3 minutes. The total cycle time is 510 msec. This
equates to 18 points per peak (peaks at the base
are ~9 sec). The average LOQ’s are 250 to 500 fg
on column with CV’s under 10% for 5 replicate
injections. B) Example of 500 fg on column for
Prazepam
B
MRM Triggered MS/MS for Quantitation: Instead of using 2 or 3 transitions per analyte and
calculating specific ion ratios or area ratios around the compound of interest, it is possible to collect
full scan MS/MS simultaneously. This provides the normal quantitative precision and accuracy of
the MRM transitions, while at the same time collecting MS/MS for confirmation. This is
accomplished through an automated experiment (IDA). When the corresponding transition goes
above a preset threshold, a linear ion trap MS/MS scan (EPI) is triggered.
Figure 3. A) 8 analytes (+ ISTD, 9 MRM transitions) in an IDA
method were used to trigger the collection of confirmatory
MS/MS. The total cycle time for this method is 583 msec (270
ms for the 9 MRM’s and 313 for the EPI scans @ 4000
amu/sec). This is comparable to the multiple MRM example in
Figure 2 (cycle time of 510 ms). B) Calibration curve for
Prazepam from 100 fg/uL to 250 pg/uL. C) 500 fg of Prazepam
on column, with 5 replicate injections giving a CV of 10%.
A
B
C
Dedicated MS/MS (EPI Scans) for Quantitation: Full scan data can also be collected as
dedicated experiments for each analyte. From this data set, it is possible to quantitate around any
of the ions present for a given analyte, or even sum them together.
Figure 4. This data contains 5 dedicated EPI scans in a
looped experiment. A mass range 220 amu covered the
needed region for confirmatory ions of the chosen
benzodiazepines. For quantitation, the dominant ion pair
(same for the MRM experiments) were used to calculate
the calibration curve, LOD/LOQ’s and the %CV for 5
replicate injections. It should also be noted that it is
possible to sum multiple ions together for quantitation
with the full scan data.
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MS for Quantitation: MS will offer much better selectivity than any resolution optimization in Q1
for an MRM experiment. It can filter out potential interferences based on fragmentation pathway
discriminations and the data can be collected on the same time scale as a typical MRM transition.
The LOD and LOQ’s are also comparable. Figure 6 shows a data set for Alprazolam, monitoring
309-281-205 with a total cycle time of 180 msec.
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Technical Note
Figure 5. MS3 is really the most selective
scan function to quantitate with, without
worrying about any potential interferences.
The data represents Alprazolam
(309/281/205) from 250 fg/uL to 100 pg/uL
on column. The LOQ for Alprazolam on
column is 1.25 pg. Comparing this to the
traditional MRM experiment (500 fg on
column for the 309/205 transition), the LOQ
for the MS3 data is only 2.5 times higher, but
with only a 0.25% chance of potential
interferences.
Table 3. Comparison LOQ’s for the Multiple Quantitation Techniques
Scan Type
MRM
EPI
MS3
Experiment(s)
Single MRM Transition per analyte
Multiple MRM Transitions per analyte
Automated collection of MS/MS (IDA)
Full Scan Quantitation
Pathway Specific Quantitation
# of species monitored
LOQ*
8 + ISTD
16 + ISTD
8 + ISTD
1 + ISTD
1 + ISTD
250-500 fg
250-500 fg
250-500 fg
500 fg
1.25 pg
* On column; based on Fast Chromatography (1.0 mL/min) with 9 second wide peak s
Also compound dependent
Table 3. Each of the techniques shown above were evaluated for precision and accuracy in a quantitative application. MRM is the
traditional benchmark for quantitative results. The various techniques available for quantitation also provide qualitative information as
well. The LOQ’s presented are for Alprazolam, but on average are consistent over the set of benzodiazepines tested. Duty cycle can
play a role in the detection limits observed as demonstrated by the full scan (EPI) experiment. For 5 analytes, a 1.2 second cycle
time is observed which corresponds to about 8 points per chromatographic peak. The same experiment with only 2 analytes has the
same duty cycle as one of the MRM experiments (~500 msec), and so leads to a much better LOQ of 500 fg on column, which is
similar to what was observed in the MRM experiments.
Summary
ü
The hybrid quadrupole-linear ion trap mass spectrometer offers flexibility in designing and
running a multitude of quantitative applications that can provide confirmatory information at
the same time.
ü
Ultimately, more information can be collected while maintaining precision and accuracy of
the results.
ü
Fast chromatography can be employed for high throughput applications without sacrificing
performance. Scan speeds of 4000 amu/sec in linear ion trap mode allow the collection of
full scan data on the time scale of an MRM transition.
ü
MS offers the ultimate in selectivity for quantitative applications with detection limits
approaching those of traditional MRM experiments, without potential interferences.
Fragmentation pathways can be used to eliminate possible interferences that would
normally be dealt with through lengthy chromatography or an increase in resolution, which
can have a negative effect on sensitivity.
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Acknowledgements
The author wishes to thank Yves LeBlanc and Nic Bloomfield.
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For Research Use Only. Not for use in diagnostic procedures. Applera, is a trademarks and Applied Biosystems is a registered
trademark of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. MDS and Sciex are trademarks
of MDS Inc., 4000 Q TRAP is a registered trademark of Applied Biosystems/MDS Sciex.
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