Intelligent Use of Retention Time during Multiple Reaction

Monitoring for Faster and Extended Compound Screening

with Higher Sensitivity and Better Reproducibility
André Schreiber and Nadia Pace
AB SCIEX Concord, Ontario (Canada)

Key Features of Scheduled MRM™ Algorithm

• Intelligent use of retention times to maximize dwell times and
optimize cycle time of MRM methods
• Increased number of monitored MRM transitions to screen
and quantify more analytes per analysis
1.7e6

Scheduled MRM™ Algorithm

• Better Signal-to-Noise due to higher dwell times
1.5e6

1.0e6

• Greatly improved reproducibility and accuracy by detecting 5.0e5

0.58

more data points across chromatographic peaks

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4 .0 4.5 5.0 5.5 6.0 6.5 7.0

• Faster sample analysis by applying UHPLC without

Time, min

compromising data quality

Introduction
LC-MS/MS instruments operating in Multiple Reaction Monitoring
(MRM) are widely used for targeted quantitation and screening
on triple quadrupole and hybrid triple quadrupole linear ion trap
®
(QTRAP ) systems because of their well known selectivity and
sensitivity. Extensive panels with a few hundred MRM transitions
are used routinely in many laboratories, for example to screen
for food contaminants and environmental pollutants or to identify
drugs in intoxication cases in forensic laboratories.
However, the current limit of a few hundred transitions per
chromatographic run limits the number of analytes that can be
monitored per injection. This is further complicated by the
demand for faster analysis through Ultra High Pressure Liquid
Chromatography (UHPLC) without reducing the number of
monitored analytes and without compromising reproducibility and
accuracy.
or to speed up the analysis by the use of UHPLC or to combine
both concepts without compromising data quality.
Key Principles of MRM and Scheduled
MRM™ Algorithm
Dwell time is the time spent acquiring the targeted MRM
transition during each cycle. While very short dwell times can be
used (5-10 ms) for extended compound screening, higher dwell
times are desirable for better Signal-to-Noise (S/N).
Duty cycle is effectively the amount of time spent monitoring an
analyte, therefore the higher the duty cycle the better the data
quality. Duty cycle is inversely proportional to the number of,
concurrent MRM transitions monitored.

With the new Scheduled MRM™ Algorithm offered in the
Analyst® software version 1.5, MRM transitions of the targeted
analytes are monitored only around the expected retention time.
Thus, automated MRM scheduling decreases the number of
concurrent MRM transitions, allowing both the cycle time and the
dwell time to be optimized for highest sensitivity, accuracy, and
reproducibility. In addition Scheduled MRM™ allows the
monitoring of many more MRM transitions in a single acquisition
Therefore, an increase in multiplexing resulting in more
concurrent MRM transitions can decrease the analytical
reproducibility.
The ideal cycle time for an MRM method is a chromatographic
consideration. A cycle time which provides 10-15 data points
across the LC peak is optimal for accurate quantitation and
reproducibility, especially for low abundant analytes. The
relationship between number of MRM transitions, dwell time,
duty cycle, and cycle time is illustrated in Figure 1.

p1
 3.1
1.7e6 1.11e5
1.5e6 1.00e5 cycle
A 8.00e4 time
1.0e6
6.00e4

4.00e4
5.0e5
0.58
2.00e4

0.0 0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 2.9 3.0 3.1 3.2 3.3
Time, min Time, min

cycle
time
3.1
1.7e6 9.1e4
1.5e6
B 8.0e4

6.0e4
1.0e6
cycle
4.0e4
time
5.0e5
0.58
2.0e4

0.0 0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 2.9 3.0 3.1 3.2 3.3
Time, m in Time, min

cycle
time

3.1
1.7e6 1.11e5
1.5e6 1.00e5 cycle
C 8.00e4 time
1.0e6
6.00e4

4.00e4
5.0e5
0.58
2.00e4

0.0 0.00
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 2.9 3.0 3.1 3.2 3.3
Time, min Time, min

cycle
time

Figure 1. Considerations for Multiple Reaction Monitoring

(A) Traditionally, few MRM transitions are detected to quantify targeted analytes with high dwell times for best S/N and cycle times to collect enough
data points across the LC peak for accurate and reproducible data (the width of the bars indicate the dwell time and the space between bars indicate the
cycle time).
(B) Increasing the number of MRM transitions by maintaining the dwell time extends the cycle time resulting in very poor quantitative results because of
an insufficient number of data points across the LC peak.
(C) Increasing the number of MRM transitions by decreasing the dwell time results in lower duty cycle and, thus, in lower S/N and higher limits of
detection.

The Scheduled MRM™ Algorithm is illustrated in Figure 2. Prior significantly reduced resulting in much higher duty cycles for
knowledge of the retention of each analyte allows the MRM each analyte. The software computes maximum dwell times for
transition to be monitored only in a short time window. At any the co-eluting compounds while still maintaining the desired
one point in time, the number of concurrent MRM transitions are cycle time.

p2
 1.7e6
1.5e6

1.0e6

5.0e5
0.58

0.0
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0
Time, min

lower number of MRM monitored

higher number of MRM monitored

Figure 2. The Scheduled MRM™ Algorithm uses the knowledge of the elution of each analyte to monitor MRM transitions only during a short retention
time window. This allows many more MRM transitions to be monitored in a single LC run, while maintaining maximized dwell times and optimized cycle
time.

Good Chromatogra-
phy is the Key for the
Best LC-MS/MS Data
The key to the highest order
multiplexing and optimal
MS/MS performance is high
quality and highly reproducible
LC separation.

One of the user inputs to the

software to automatically
create the Scheduled MRM™
methods is the MRM Detection
Window. This is an estimate of
the LC peak width and
chromatographic
reproducibility expected, and
should therefore reflect the
time window around the Figure 3. Acquisition method interface for Scheduled MRM™, in addition to traditional MRM parameters, the user
provides retention times of all analytes, an MRM detection window, and a Target scan time. The software then
supplied retention time which
automatically designs and optimizes the Scheduled MRM™ acquisition method.
will contain the entire LC peak
plus any shifts in monitored. Reduced concurrency also means that higher dwell
chromatography. The narrower the peak widths and the more times will be used for each MRM, improving the data quality.
reproducible the elution, the tighter this MRM detection window
can be and, thus, less concurrent MRM transitions are

p3
Easy Method Creation characteristic and high sensitivity spectra can be searched
against a mass spectral library for compound identification.
Another key advantage in Scheduled MRM™ is the ease at
XIC of +MRM (761 pairs): Exp 1, 303.2/109.1 amu Expected RT: 4.7 ID: 17-alpha-Methyltestosterone from S... Max. 2.9e4 cps.
which powerful quantitative MRM acquisition methods can be
2.4e6
Screening with Scheduled MRM™
created. The user is required to specify a few key parameters 2.0e6

(Figure 3):1
1.5e6

• MRM transition: (Q1, Q3) and any compound dependent 1.0e6

parameters 5.0e5

• Expected retention time for each MRM transition 0.0

1.0 2.0 3.0 4.0
4.7 5.1
5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
Time, min

• MRM detection window must be wide enough to allow the +EPI (376.10) Charge (+0) CE (35) CES (15) FT (50): Exp 3, 7.413 min from Sample 1 (mix 757 100ngmL) o... Max. 4.9e6 cps.

165.1

MRM peak to stay entirely within the window across all 4.5e6
Dependent EPI spectrum
376.0
for confirmation
4.0e6 123.0
Haloperidol: Fit = 88.0%
injections – consider the width of the LC peak at the base and 3.5e6

the retention time stability 3.0e6

2.5e6

• Target scan time is effectively the cycle time – how often the 2.0e6

1.5e6
358.0
chromatographic peak should be sampled. This is determined 1.0e6

5.0e5 206.2
135.1
from the peak width at the base. The best accuracy and 60 80
109.1
100 120 140 160
200.0
180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z, Da
reproducibility is between 10-15 points across the peak
Figure 4. Using Scheduled MRM™ to increase the number of monitored
• Additionally, MRM ID, like compound name, for easier data MRM transitions for screening applications. The example shows an
processing and reporting injection of more than 750 compounds relevant in forensic toxicology.
The Scheduled MRM™ survey was used to automatically acquire EPI
The software algorithm then automatically builds an acquisition spectra for identification by library searching.
method that schedules the appropriate MRM transitions to be
screened over the chromatographic analysis at the appropriate Better Sensitivity and Reproducibility
times. Instead of monitoring all transitions all of the time, it will
Figure 5 shows a comparison of using traditional MRM and
only look for those transitions within the targeted time window.
Scheduled MRM™ detection for the screening of pesticides in
®
Results of Using the Scheduled MRM™ fruit and vegetable samples. A 4000 QTRAP LC/MS/MS system
was used to detect 150 MRM transitions.
Algorithm
XIC of +MRM (150 pairs): 254.2/198.1 amu Expected RT: 4.9 ID: Irgarol 1 from Sample 4 (fast LC sMRM 20) of Test_fast sMRM.wiff (Turbo ... Max. 1.7e6 cps.

Increased Number of MRM Transitions 1.7e6

Pesticide screening using 150 MRM transitions
4.91

1.5e6

1.0e6
The number of MRM transitions which can be monitored in a
5.0e5
single analysis depends on chromatographic peak width and
0.0
required S/N (dwell time). Several publications show that AB 0.5 1.0 1.5

XIC of +MRM (150 pairs): 203.2/175.1 Da ID: Metamitron ...

2.0 2.5 3.0

Max. 1.7e5 cps.

3.5
Time, min
4.0 4.5 5.0 5.5

XIC of +MRM (150 pairs): 254.2/198.1 Da ID: Irgarol 1 fro...

6.0 6.5 7.0

Max. 1.6e6 cps.

®
SCIEX systems equipped with Linear Accelerator collision cell 1.7e5
Metamitron
1.48
CV = 8.8% 1.6e6
Irgarol
4.91
CV = 5.6%
1.5e5 1.4e6
I n te n s it y , c p s

can be used to detect several hundred transitions using MRM CV = 6.6% 1.2e6
1.0e6
MRM CV = 8.4%
1.0e5
2-4
traditional LC configurations. 8.0e5
6.0e5
5.0e4
4.0e5
2.0e5

The automated MRM scheduling decreases the number of 0.0

1.20 1.25 1.30 1.35 1.401.45 1.50 1.55
Time, min
1.60 1.65 1.70 1.75
0.0
4.65 4.70 4.75 4.80 4.85 4.90 4.95 5.00
Time, min
5.05 5.10 5.15

XIC of +MRM (150 pairs): 203.2/175.1 amu Expected RT: ... Max. 2.3e5 cps. XIC of +MRM (150 pairs): 254.2/198.1 amu Expected RT: ... Max. 1.7e6 cps.

concurrent MRM transitions. Thus Scheduled MRM™ allows the 1.50 4.91
2.3e5
Scheduled CV = 2.4% 1.7e6
Scheduled CV = 2.3%
monitoring of many more MRM transitions per cycle without the 1.5e6
I n t e n s it y , c p s

2.0e5
MRM™ CV = 2.2% MRM™ CV = 2.9%
1.5e5

need to sacrifice data quality. 1.0e5

1.0e6

5.0e5
5.0e4

The example in Figure 4 shows an injection of more than 750 0.0

1.20 1.25 1.30 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75
0.0
4.65 4.70 4.75 4.80 4.85 4.90 4.95 5.00 5.05 5.10 5.15
Time, min Time, min

compounds typically analyzed in forensic laboratories to screen

Figure 5. Using Scheduled MRM™ to optimize dwell times and number
for toxic substances, such as drugs of abuse, pharmaceuticals of data points across the LC peak in a pesticide screening method with
and their metabolites. 150 MRM transitions. The Scheduled MRM™ method shows significantly
better sensitivity and reproducibility.
Such screening methods are used frequently to screen for a
large number of targeted compounds. The Scheduled MRM™
survey was used to automatically acquire Enhanced Product Ion
(EPI) spectra on a 3200 QTRAP ® LC/MS/MS system. The

p4
 XIC of +MRM (150 pairs): 254.2/198.1 Da ID: Irgarol 1 from Sample 2 (traditional LC) of Test_trad.wiff (Turbo Spray) Max. 1.6e6 cps.

The Scheduled MRM™ Algorithm automatically optimizes dwell 9.24

1.5e6 Traditional LC
times enabling detection with higher sensitivity and better 150 MRM
1.0e6

reproducibility by collecting more data points across the LC 5.0e5

peak. The improvement in sensitivity and reproducibility depends 0.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0
Time, min
8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

XIC of +MRM (150 pairs): 254.2/198.1 amu Expected RT: 4.9 ID: Irgarol 1 from Sample 4 (fast LC sMRM 20) of Test_fast sMRM.wiff (Turbo ... Max. 1.7e6 cps.

on the number of concurrent MRM transitions. Narrow LC peaks 4.91

1.7e6
Fast LC
and highly stable retention times allow setting a smaller MRM 1.5e6

150 scheduled MRM

detection window for best Scheduled MRM™ performance. 1.0e6

5.0e5

Faster analysis using UHPLC without compromising data 0.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
Time, min

quality XIC of +MRM (150 pairs): 254.2/198.1 amu Expected RT: 2.9 ID: Irgarol 1 from Sample 3 (faster LC sMRM 15) of Test_faster sMRM.wiff (T...

2.89
Max. 2.5e6 cps.

2.5e6
Ultra Fast LC
2.0e6

The use of small particle size columns and faster gradients 1.5e6
150 scheduled MRM
results in narrower LC peaks. Traditional MRM would require 1.0e6

5.0e5

decreasing the number of transitions or compromising quality to 0.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0
Time, min
maintain the number of transitions.
Figure 6. Scheduled MRM™ allows fast and ultra fast LC separation
The chromatograms in Figure 6 show examples of traditional, using small particle column while maintaining the number of monitored
MRM transitions without compromising data quality.
fast and ultra fast LC to monitor 150 MRM transitions. Scheduled
MRM™ allows accelerated analysis without the need to
compromise the number of monitored compounds and/or data
quality. The data were acquired using a 4000 QTRAP®
LC/MS/MS system. A Phenomenex Synergi 2.5u Fusion-RP
50x2 mm column with different gradients of water/methanol and
5 mM ammonium formate was used. The gradient conditions are
shown in Table 1.

®
Table 1. Traditional, fast and ultra fast LC gradients to detect 150 MRM transitions of pesticides on a 4000 QTRAP LC/MS/MS system

Traditional LC (2150 psi) Fast LC (4330 psi) Ultra Fast LC (4570 psi)

Step Time (min) Flow (µL/min) A%/B% Time (min) Flow (µL/min) A%/B% Time (min) Flow (µL/min) A%/B%

0 0 250 80/20 0 500 70/30 0 500 60/40

1 8 250 10/90 5 500 10/90 2 500 10/90

2 14 250 10/90 6 500 10/90 4 500 10/90

3 15 250 80/20 7 500 70/30 5 500 60/40

4 20 250 80/20 10 500 70/30 8 500 60/40

Figure 7 shows results of the analysis of fruit extracts analyzed
with a traditional LC and MRM method in comparison to a fast
LC and Scheduled MRM™ method. The samples were extracted
using a QuEChERS procedure before analysis.
Several pesticides were detected, quantified and identified using
MRM ratio calculation, including Imazalil at 42 μg/kg and
Thiabendazole at 3.4 μg/kg in grapefruit, Metazachlor at
8.9 μg/kg in apricot, and Methomyl at 4.7 μg/kg in grapes. The
use of Scheduled MRM™ for this analysis allowed faster sample
analysis with better sensitivity and reproducibility. In addition,
data exploration was easier because of a more selective
acquisition.

p5
 XIC of +MRM (150 pairs): 297.1/159.2 Da I... Max. 3.1e4 cps. XIC of +MRM (150 pairs): 297.1/159.2 am... Max. 1.1e5 cps.
References
3.0e4 Imazalil + Thiabendazole
9.3
Imazalil + Thiabendazole
5.1

I n t e n s it y , c p s
1.00e5
in Grapefruit in Grapefruit 1
2.0e4
8.00e4
Scheduled MRM™ tutorial
6.00e4

1.0e4 4.00e4
2
2.00e4
C. A. Mueller et al.: ‘Development of a Multi-Target
0.0 0.00
2 4 6 8 10 12 14 1.0 2.0 3.0 4.0 5.0 6.0 7.0
Time, min
XIC of +MRM (150 pairs): 278.2/210.2 Da... Max. 8200.0 cps.
Time, min
XIC of +MRM (150 pairs): 278.2/210.2 am... Max. 1.5e4 cps.
Screening Analysis for 301 Drugs Using a QTRAP Liquid
8000
7.9
Metazachlor 1.5e4
3.9
Metazachlor Chromatography/Tandem Mass Spectrometry System and
In te n s ity , c p s

6000 in Apricot 1.0e4

in Apricot Automated Library Searching’ Rapid Commun. Mass
4000
5000.0
Spectrom. 19 (2005) 1332-1338
2000

0 0.0 3
2 4 6 8
Time, min
10 12 14 1.0 2.0 3.0 4.0
Time, min
5.0 6.0 7.0
A . Schreiber et al.: ‘Simultaneous Quantitative Screening
XIC of +MRM (150 pairs): 163.1/88.1 Da I... Max. 1400.0 cps. XIC of +MRM (150 pairs): 163.1/88.1 am... Max. 2989.7 cps.

7800 Methomyl Methomyl

and Qualitative Confirmation of 300 Pesticides Using the
5000
I n t e n s it y , c p s

®
6000 in Grapes 4000 in Grapes New 3200 QTRAP LC/MS/MS System’ Application Note AB
0.8
3000
4000
2000
SCIEX (2005)
2000 2.8 1000
4
0
2 4 6 8
Time, min
10 12 14
0
1.0 2.0 3.0 4.0
Time, min
5.0 6.0 7.0 C. Borton et al.: ‘Analysis of Endocrine Disruptors,
Pharmaceuticals, and Personal Care Products in River
Figure 7. Comparison of traditional LC and MRM with fast LC and
Scheduled MRM™ for the analysis of pesticides in fruit extracts, the new Water’ Application Note AB SCIEX (2007)
method allowed faster analysis with better sensitivity and reproducibility.
Also cleaner data display made data exploration easier.

Summary
®
The new Scheduled MRM™ Algorithm offered in Analyst
software version 1.5 automatically monitors MRM transitions of
the targeted analytes only around the expected retention time.
The scheduling decreases the number of concurrent MRM
transitions, allowing both the cycle time and the dwell time to be
optimized for highest sensitivity, accuracy, and reproducibility. In
addition, Scheduled MRM™ allows the monitoring of many more
MRM transitions in a single acquisition and/or accelerating the
analysis by the use of UHPLC maintaining highest data quality.

For Research Use Only. Not for use in diagnostic procedures.

© 2010 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.

Publication number: 1282310-01

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