Triggered MRM: Simultaneous

Quantitation and Confirmation

Using Agilent Triple Quadrupole
LC/MS Systems

Technical Overview

Triggered MRM (tMRM) acquisition is an analytical method which is available for

all Agilent Triple Quadrupole LC/MS systems. tMRM acquisition combines MRM
with the generation of a product ion spectrum which can then be used for library
identification and confirmation. As a result, tMRM analysis decreases analysis time,
increases throughput, and allows for fast, sensitive, quantitative and qualitative
analysis on a single instrument, in a single analytical run.

How tMRM Acquisition Works

In tMRM analysis, up to 10 MRM transitions (primary and secondary) are defined
for each target analyte in the method. The primary transitions are acquired for
all analytes, but when the signal of one of the primary transitions exceeds a
user-defined threshold, the secondary transitions are triggered and acquired for
a specified number of scans (Figure 1).

Secondary MRM Triggered cycle (above threshold)

×104
transitions are triggered Compound Precursor Product
2 Analyte 1 575.3111 937.463
Analyte 1 575.3111 694.235
Analyte 1 575.3111 1036.532
Counts

Analyte 1 575.3111 595.309

1 Analyte 1 575.3111 469.235
Analyte 1 575.3111 823.4
Primary cycle (below threshold) Threshold
0 Compound Precursor Product
Analyte
2 1 575.31112.5 937.463 3 3.5 4 4.5 5
Analyte 1 575.3111 694.235
Analyte 1 575.3111 1036.532 Acquisition time (min)
Analyte 1 575.3111 595.309
Analyte 1 575.3111 469.235
Analyte 1 575.3111 823.4

Figure 1. A tMRM experiment with two primary transitions for each analyte. Secondary MRM transitions
are triggered when the primary MRM signals cross a user-defined threshold.
tMRM Library Searching tMRM product ion spectrum. This ion spectra, rather than conventional
product ion spectrum is then used for product ion scanning techniques, is
Using tMRM, the secondary MRM library search and identification of the that acquiring all 10 MRM transitions
spectra acquired during tMRM compound of interest. takes less than 50 ms, while a typical
experiments are combined with the product ion scan on an ion trap, or
primary MRM transition spectra for With up to 10 product ions present triple quadrupole instrument takes
a particular analyte to generate a in each tMRM product ion spectrum approximately 200 ms. This fast cycle
product ion spectrum. Figure 2 shows for each target analyte, users can time means that more data points are
nine MRM transitions (two primary confidently confirm target analytes with acquired across each peak, and that
and seven secondary transitions) a library search. The main advantage of the quantitative data acquired is more
which are combined to make one using tMRM to generate these product sensitive, robust, and accurate.

A x10 3 334 > 76 x10 3 334 > 91 x10 3 334 > 105 x10 3 x10 3 334 > 119
334 > 117
117.0

5 5 5 5 5
76.0 91.1 105.1 119.0

0 0 0 0 0
80 80 100 80 100 120 80 100 120 80 100 120

x10 3 334 > 132 x10 3 334 > 145 145.0 x10 3 334 > 147 x10 3 334 > 171

5 5 5 147.0 5
132.1 171.1
0 0 0 0
100 140 100 140 180 100 140 180 80 100 120 140 160 180

B
×10 3 145.0 tMRM Product Ion Spectrum
117.0
Counts

76.0
91.1 105.1 132.1
171.1 334.2
0
80 100 120 140 160 180 200 220 240 260 280 300 320 340
Acquisition time (min)

Figure 2. tMRM product ion spectra are generated by combining the primary (●) and secondary MRM spectra for a target analyte A. The
product ion spectrum B may be used for library search and analyte confirmation.

2
New Enhanced Triggering
Functions
The Agilent Mass Hunter B.06
Acquisition for Agilent 6400 Series
Triple Quadrupole LC/MS systems
adds triggering functions that increase
specificity and tMRM data quality:
• Trigger delay
Once the triggering threshold is
met, the trigger delay defines the
number of cycles to skip between
triggers, spreading the acquisition of
secondary MRM transitions across a
peak. Trigger delay can be combined
with the entrance delay function.
Figure 3 shows data acquired using
the entrance delay triggering function.
Panel A shows an entrance delay of
0 scan cycles and Panel B shows an
entrance delay of three scan cycles. In
this example, the entrance delay moves
the acquisition of secondary spectra
closer to the apex of the peak.

• Entrance delay • Trigger window

If the signal for the designated
primary MRM transitions cross the
triggering threshold, the entrance
delay postpones triggering for a user-
defined number of cycles, moving
the acquisition of secondary MRM
transitions closer to the apex of the
peak.
The trigger window confines the
activation of all triggering functions
to a user-defined window around
the expected retention time for
a particular peak. This function
increases triggering specificity based
on the target compounds and known
retention times for a particular tMRM
method.

A ×106 Cpd 3: Acephate: +ESI tMRM Frag=380.0V B ×106 Cpd 3: Acephate: +ESI MRM Frag=380.0V
3.4 3.4
2.971
3.2 3.2
3.0 3.0
2.8 2.8
2.6 2.6
2.4 2.4
2.2 2.2
5
×10 2.0 ×105 2
6.4 2.971
1.8 9 1.8
5.6 1.6 8 1.6
4.8 1.4 7 1.4
4.0 1.2 6 1.2
Counts

Counts

1 5 1
3.2
0.8 0.8
4
2.4 0.6
3 0.6
1.6 0.4 0.4
2
0.8 0.2 0.2
1
0 0
0 2.5 2.6 2.7 0 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3
2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.4
2.80 2.90 3.00 3.10 Acquisition time (min) 2.85 2.95 3.05 3.15 Acquisition time (min)
Acquisition time (min) Acquisition time (min)

Figure 3. Data acquired for acephate using the entrance delay triggering function.

3
Avoiding False Positives with
tMRM Acquisition
Tebufenpyrad is a toxic insecticide
which can be present in ginger.
However, there is an endogenous
compound found in ginger which
co-elutes with tebufenpyrad and shares
the same two primary MRM transitions
with a similar qualifier ion ratio. In
a typical triple quadrupole analysis,
without tMRM analysis, a ginger
extract could easily give a false positive
result for tebufenpyrad. However,
the product ion spectra generated by
tMRM analysis allow differentiation
of the endogenous ginger compound
from the tebufenpyrad contaminant. In
order to acquire the same quantitative
Rapid Quantitative Analysis
with tMRM Acquisition
The tMRM product ion spectrum for
tebufenpyrad was generated while
quantitation was performed, without
sacrificing the quality of either result.
Triggering additional MRM transitions
with a primary MRM threshold means
that the tMRM cycle time is always
as short as possible, maximizing the
number of data points acquired across
each peak. When the number of data
×103
91.1
8 117.0
6
Counts
points acquired across an analytical
peak is maximized, the sensitivity and
accuracy of the quantitative analysis
is optimal. Quantitation using tMRM
is also compatible with dynamic
MRM1 (Stone, 2009). This is ideal
for confirmation and identification
of large numbers of analytes, such
as those encountered in pesticide
residue screening. In addition, the
tMRM method allows each MRM to be
acquired at optimal collision energy and
hence maximizes sensitivity.
Acquired tMRM spectrum
145.0 native ginger compound

and qualitative data without tMRM

acquisition, one would have to first 4
analyze the extract using a fast
scanning triple quadrupole, and then 2

reanalyze the sample using an ion trap 334.2

or a time-of-flight (TOF) instrument for 0
qualitative analysis. ×103
91.1
1 117.0 145.0 Comparison acquired versus
tMRM library spectrum
Figure 4 shows a comparison of the
tebufenpyrad tMRM library spectrum
and the endogenous compound
Counts

334.2
spectrum found in the ginger extract. 0
171.1
The peaks circled in the tebufenpyrad 76 91.1 105.1 132.1
spectrum differ significantly or are
absent from the endogenous ginger
-1 145
compound. In this case, a false 117
positive result was avoided due to
×103 117
the qualitative data generated by 1 tMRM library spectrum
145 tebufenpyrad
tMRM acquisition.
H 3C
CH3
H3C Cl
Counts

0.5
H 3C N
NH
76 91.1 N
105.1 132.1
171.1 O CH3
334.2
0
80 100 120 140 160 180 200 220 240 260 280 300 320 340
Acquisition time (min)

Figure 4. The comparison of an acquired tMRM spectrum for an endogenous ginger compound compared
with the authentic library tMRM spectrum for tebufenpyrad.

4
Conclusions
tMRM is a data dependent scan
function which increases throughput,
provides both quantitative and
qualitative information, and minimizes
the cost of analyses. Standard product
ion scanning techniques require
approximately 200 ms per transition, at
least four times longer than a tMRM
cycle. The longer cycle time for a
typical product ion scan means that
fewer data points are acquired across
each analytical peak, compromising
sensitivity and robustness for
qualitative and quantitative results.
In addition, the LC methods for
instruments with longer product ion
scan cycle times are often lengthened
to minimize analyte co-elution. With
tMRM, high throughput LC methods
with narrow peak widths can be easily
adopted on a triple quadrupole LC/MS
instrument, enabling quantitative
analysis and qualitative confirmation
on a single instrument, in a single
injection.

SCREEN
NTIFY

CONF
UA

RM
Q

5
Reference
1. New Dynamic MRM Mode
Improves Data Quality and Triple Quad
Quantification in Complex Analyses.
Agilent publication 5990-3595EN.

www.agilent.com/chem/QQQ
For Research Use Only. Not for use in diagnostic
procedures. This information is subject to change
without notice.
© Agilent Technologies, Inc. 2013
Published in the U.S.A., January 18, 2013
5990-8461EN