Considerations when using LC-MS/MS Systems with Fast
and High Resolution Liquid Chromatography
Mark Kuracina and André Schreiber
AB SCIEX Concord, Ontario (Canada)
Overview
XIC of +MRM (32 pairs): Period 4, 293.2/237.0 amu from Sample 8 (All Pesticides) of RT...
Max. 4.1e5 cps.
2.26
This application note describes the benefits of using Fast and
High resolution HPLC systems with small particle size columns
and mass spectrometric detection. Requirements for HPLC and
MS/MS systems are discussed to enable quantitation with
highest throughput, sensitivity and/or resolution.
4.0e5
3.8e5
3.6e5
3.4e5
3.2e5
3.0e5
2.8e5
2.6e5
2.4e5
Introduction
2.2e5
2.0e5
1.8e5
The advent of small particle size High Performance Liquid
Chromatography (HPLC) columns has caused a renewed focus
on the front end separation technology leading into Mass
Spectrometry (MS) systems. This is due to both the development
in small particle manufacturing processes as well as the
development of commercially available HPLC systems that can
operate at high backpressures in order to provide the flows
required for efficient separation. The benefit of such technology
is that it can be used to either increase resolution or maintain
resolution at faster run times or a combination of both depending
on the column length and particle size chosen.
The practical benefits to LC-MS/MS include the ability to
increase sample throughput by reducing run times, the ability to
increase sensitivity 2-3 times by generating more concentrated
sharper peaks, and the ability to utilize enhanced resolution to
better separate targeted analytes and matrix ions in complex
matrices that may lead to ion suppression effects.
The downside being that high pressure HPLC systems are both
more expensive to purchase as well as maintain. Also, that the
use of small particle size columns may also require more
extensive sample preparation and filtering to avoid column
clogging.
1.6e5
1.4e5
1.2e5
1.0e5
8.0e4
6.0e4
4.0e4
2.0e4
0.0
0.5
1.0
1.5
2.0
Time, min
2.5
3.0
3.5
4.0
Figure 1. Detection of 16 priority pesticides using a fast gradient and a
1.8 μm particle size column – dwell times of all 32 MRM transitions were
adjusted to give a minimum of 15 data points per peak
The following example in Figure 2 depicts the advantages of
Fast HPLC utilizing small particles size columns, namely the
ability to use the combination of faster gradients and higher flow
rates to provide sharper HPLC peaks, thus providing better
sensitivity, while reducing run time.
A mixture of 17 pesticides was analyzed by LC-MS/MS using a
3200 QTRAP® LC/MS/MS System with Turbo V™ Source and
Electrospray Ionization (ESI) probe. All analytes were detected
using two Multiple Reaction Monitoring (MRM) transitions.
Separation was performed on a 2.5 μm column with a mobile
phase of water/methanol + 5 mM ammonium formate. The
chromatograms in Figure 2 show clearly the sensitivity
enhancements and reduced run times using higher flow rates
and fast gradients.
p1
XIC of +MRM (34 pairs): 253.2/171.2 amu from Sample 1 (traditional gradient) of Data Pesticides...
Max. 3.2e4 cps.
A
6.9
3.0e4
Intensity, cps
2.0e4
~12sec
1.0e4
0.0
1
2
3
4
5
6
7
8
9
10
Time, min
XIC of +MRM (34 pairs): 253.2/171.2 amu from Sample 10 (fast LC 10) of Data Pesticides...
11
12
13
14
Max. 4.1e4 cps.
3.3
4.0e4
15
B
Intensity, cps
3.0e4
2.0e4
~8sec
1.0e4
0.0
1
2
3
4
5
6
7
8
9
10
Time, min
XIC of +MRM (34 pairs): 253.2/171.2 amu from Sample 4 (faster LC 10) of Data Pesticides...
11
12
13
14
Max. 4.9e4 cps.
C
2.1
4.9e4
15
Intensity, cps
4.0e4
3.0e4
2.0e4
~6sec
1.0e4
0.0
1
2
3
4
5
6
7
8
Time, min
9
10
11
12
13
14
15
Figure 2. Use of small particle size column with fast gradients to increase sensitivity and throughput (LC-MS/MS detection of 17 pesticides in Multiple
Reaction Monitoring with A) 8 min gradient at 250 μL/min B) 5 min gradient at 500 μL/min C) 2 min gradient at 500 μL/min on a Phenomenex Synergi
Fusion-RP 50x2 mm column with 2.5 μm particles)
HPLC Requirements
In order to fully utilize small particle size columns, the HPLC
system used requires the following attributes:
• Accurate and reproducible HPLC gradient pumps that operate
from 0.1 to 1.5mL/min at high back pressure with a minimum
delay volume
• An autosampler with a fast cycle time to reduce the overall run
time and also, with low carry-over in order to avoid
contamination between injections
• A column oven to accurately control the column temperature
and to reproducibly elevate the column temperature in order
to reduce solvent viscosity and backpressure
It should be noted that the Waters Acquity UPLC system, the
Agilent 1200 Series Rapid Resolution LC system, the Dionex
Ultimate 3000 LC system, and the Shimadzu Prominence
UFLCXR system are all designed to be used with regular as well
as small particle size columns to provide fast chromatography.
Furthermore, that all of these systems are fully controlled by
®
Analyst Software to provide fast chromatography when used in
conjunction with AB SCIEX Mass Spectrometers. However, it
should be noted that when choosing a fast HPLC system, the
performance as well as cost of purchase and maintenance
should all be considered prior to purchase.
p2
Filter precursor ion
Fragmentation
Filter product ion
3.9
7500
7000
6500
216/174
6000
5500
5000
4500
4000
3500
216/104
3000
2500
2000
1500
1000
500
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, min
4.0
4.5
5.0
5.5
6.0
6.5
7.0
Figure 3. Schematics of a triple quadrupole mass spectrometer operating in Multiple Reaction Monitoring (MRM) with a resulting chromatogram
MS/MS Requirements
• An ionization source with excellent ionization efficiency at flow
rates between 0.1 and 1.5 mL/min
In order for a triple quadrupole LC-MS/MS operating in MRM
mode (Figure 3) to be compatible with Fast HPLC, it requires the
following:
XIC of +MRM (60 pairs): 308.2/235.1 amu from Sample 2 (Benzomix f ast LC) of Data Benzodiazepines...
Max. 1.4e6 cps.
A
2.6
1.4e6
Intensity, cps
1.0e6
5.0e5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Time, min
XIC of +MRM (60 pairs): 308.2/235.1 amu from Sample 7 (Benzomix f aster LC) of Data Benzodiazepines...
4.0
4.5
5.0
Max. 1.5e6 cps.
B
1.6
1.5e6
5.5
Intensity, cps
1.0e6
5.0e5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Time, min
XIC of +MRM (60 pairs): 308.2/235.1 amu from Sample 9 (Benzomix much faster LC) of Data Benzodiazepin...
4.5
5.0
Max. 1.7e6 cps.
C
1.2
1.7e6
1.5e6
5.5
Intensity, cps
1.0e6
5.0e5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Time, min
3.5
4.0
4.5
5.0
5.5
Figure 4. Use of Turbo V™ Source at high flow rates with small particle size column (LC-MS/MS detection of 30 benzodiazepines in Multiple Reaction
Monitoring with A) 0.6 mL/min and 500°C B) 1 mL/min and 600°C C) 1500 mL/min and 700°C on a Zorbax SB-C18 50x4.6 mm column with 1.8 μm
particles)
p3
• A fast and efficient collision cell that can operate at low dwell
and pause times to enable the acquisition of enough data
points across narrow LC peaks with a minimal loss in
sensitivity and no cross-talk
Scan Speed and Dwell Time
Given the general confusion about scan speed versus dwell
time, it should be noted that scan speed is only applicable to full
scan qualitative experiments like a Q1, Product Ion, Precursor
Ion, and Neutral Loss scan where the quadrupole is set to scan
over a range of masses in a set period of time in order to
determine what compounds may be present in the unknown
sample.
The AB SCIEX industry standard Turbo V™ Source with
orthogonal spray and optimized flow dynamics provides excellent
performance across a broad flow rate range without splitting or a
roll off in sensitivity, thus providing the ability to utilize both
standard Fast LC flow rates as well as to go up to 1.5 mL/min, to
maximize throughput without sacrificing sensitivity.
However, when performing a MRM experiment for targeted
quantitation where Q1 and Q3 are set to filter specific ions as
they pass through the ion optics, it is the dwell and the pause
times and the number of MRM transitions monitored that
determine the cycle time and thus the number of data points
obtained across a given HPLC peak.
The example chromatograms shown in Figure 4 illustrates the
advantages of Turbo V™ Source operating using ESI in
combination with Fast HPLC at higher flow rates without loss in
sensitivity due to optimized ion source temperature.
A mixture of 30 benzodiazepines was analyzed by LC-MS/MS
using a 3200 QTRAP ® LC/MS/MS System with Turbo V™
Source and ESI probe. All compounds were detected in MRM
mode. Separation was performed on a 1.8 μm column with a
mobile phase of water/acetonitrile + 0.2% formic acid and 2 mM
ammonium formate at different flow rates. The ion source
temperature was optimized for complete evaporation of solvents
depending on the flow rate.
In terms of quantitation, the amount of MRM transitions and thus
compounds that can be monitored in a given run is directly
dependent on the ability of the collision cell to use short dwell
and pause times to provide enough, typically 10-15, data points
across a peak for good quantitative reproducibility of less than
10% while still maintaining sensitivity and avoiding cross-talk.
Figure 6. Illustration of Dwell and Pause times during an MRM
experiment. The MRM experiment contains dwell time to detect ion of
interest and pause time to avoid cross talk: Total cycle time = number of
MRM transitions multiplied with (dwell + pause); Total cycle time must
give 10-15 data points over HPLC peak to allow accurate quantitation.
The industry standard AB SCIEX Linear Accelerator (LINAC®)
Collision Cell provides the ability to analyze hundreds of
compounds in a set period of time, with a single time window
while utilizing dwell and pause times on the order of a few
milliseconds as shown in Figure 5.
1ms
2ms
Sample Name: "1msec" Sample ID: "01" File: "Data d...
Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"
Comment: "" Annotation: ""
7.9
1.8e5
3ms
Sample Name: "2msec" Sample ID: "02" File: "Data d...
Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"
Comment: "" Annotation: ""
1.8e5
4ms
Sample Name: "3msec" Sample ID: "03" Fil e: "Data d...
Peak Name: "Atrazine" Mass(es): "216.1/174.0amu"
Comment: "" Annotation: ""
8.1
1.8e5
5ms
Sample Name: "4msec" Sample ID: "04" File: "Data d...
Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"
Comment: "" Annotation: ""
10ms
SampleName: "5msec" Sample ID: "05" File: "Data d...
Peak Name: "Atrazi ne" Mass(es) : "216.1/174.0 amu"
Comment: "" Annotation: ""
15ms
Sample Name: "10msec" Sample ID: "10" File: "Data...
PeakName: "Atrazine" Mass(es): "216.1/174.0 amu"
Comment:"" Annotation: ""
20ms
Sample Name: "15msec" Sample ID:"15" File: "Data ...
Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"
Comment: "" Annotation: ""
25ms
Sample Name: "20msec" Sample ID: "20" File: "Data ...
PeakName: "Atrazine" Mass(es): "216.1/174.0amu"
Comment: "" Annotation: ""
50ms
Sample Name: "25msec" Sample ID: "25" File: "Data ...
Peak Name: "Atrazine" Mass(es): "216.1/174.0 amu"
Comment: "" Annotation: ""
SampleName: "50msec" Sample ID: "50" File: "Data ...
Peak Name: "Atrazine" Mass(es):"216.1/174.0 amu"
Comment: "" Annotation: ""
1.8e5
1.8e5
1.7e5
1.7e5
1.7e5
1.7e5
1.7e5
1.7e5
1.7e5
1.6e5
1.6e5
1.6e5
1.6e5
1.6e5
1.6e5
1.6e5
1.6e5
1.6e5
1.6e5
1.5e5
1.5e5
1.5e5
1.5e5
1.5e5
1.5e5
1.5e5
1.5e5
1.5e5
1.5e5
1.4e5
1.4e5
1.4e5
1.4e5
1.4e5
1.4e5
1.4e5
1.4e5
1.4e5
1.4e5
1.3e5
1.3e5
1.3e5
1.3e5
1.3e5
1.3e5
1.3e5
1.3e5
1.3e5
1.3e5
1.2e5
1.2e5
1.2e5
1.2e5
1.2e5
1.2e5
1.2e5
1.2e5
1.2e5
1.2e5
1.1e5
1.1e5
1.1e5
1.1e5
1.1e5
1.1e5
1.1e5
1.1e5
1.1e5
1.1e5
1.0e5
1.0e5
1.0e5
1.0e5
1.0e5
1.0e5
1.0e5
1.0e5
1.0e5
1.0e5
9.0e4
9.0e4
8.1
1.8e5
1.8e5
8.1
1.8e5
9.0e4
Intensity, cps
Intensity, cps
9.0e4
9.0e4
9.0e4
8.0e4
8.0e4
8.0e4
8.0e4
8.0e4
8.0e4
8.0e4
8.0e4
8.0e4
8.0e4
7.0e4
7.0e4
7.0e4
7.0e4
7.0e4
7.0e4
7.0e4
7.0e4
7.0e4
7.0e4
6.0e4
6.0e4
6.0e4
6.0e4
6.0e4
6.0e4
6.0e4
6.0e4
6.0e4
6.0e4
5.0e4
5.0e4
5.0e4
5.0e4
5.0e4
5.0e4
5.0e4
5.0e4
5.0e4
5.0e4
4.0e4
4.0e4
4.0e4
4.0e4
4.0e4
4.0e4
4.0e4
4.0e4
4.0e4
4.0e4
3.0e4
3.0e4
3.0e4
3.0e4
3.0e4
3.0e4
3.0e4
3.0e4
3.0e4
2.0e4
2.0e4
2.0e4
2.0e4
2.0e4
2.0e4
2.0e4
2.0e4
2.0e4
1.0e4
0.0
1.0e4
7.0
7.5
8.0
1.31
8.5
9.0
9.5
0.0
1.0e4
7.0
7.5
8.0
8.5
1.13
9.0
9.5
0.0
1.0e4
7.0
7.5
8.0
8.5
1.14
9.0
9.5
0.0
1.0e4
7.0
7.5
8.0
8.5
1.08
9.0
9.5
0.0
1.0e4
7.0
7.5
8.0
8.5
9.0
9.5
0.0
1.0e4
7.0
7.5
8.0
8.1
1.7e5
7.9
Intensity, cps
9.0e4
1.8e5
8.1
1.7e5
8.1
Intensit y,cps
9.0e4
Intensity, cps
9.0e4
Intensity, cps
Intensity, cps
Intensit y,cps
9.0e4
8.1
1.8e5
1.7e5
8.1
8.5
9.0
9.5
1.13
1.10
peak area x10e6 counts
0.0
1.0e4
7.0
7.5
8.0
8.5
1.13
9.0
9.5
0.0
3.0e4
2.0e4
1.0e4
7.0
7.5
8.0
8.5
1.11
9.0
9.5
0.0
1.0e4
7.0
7.5
8.0
8.5
1.12
9.0
9.5
0.0
7.0
7.5
8.0
8.5
9.0
9.5
1.15
Figure 5. Sensitivity in Multiple Reaction Monitoring depending on dwell time in milliseconds
p4
The amount of compounds that can be analyzed in a set period
of time is determined by the cycle time and the HPLC peak
width. As an example, the monitoring of 100 MRM transitions
with a dwell time and pause time of 5ms each would have a total
cycle time of 1 second, thus providing 15 data points across a 15
second wide HPLC peak. If a larger number of analytes has to
be monitored and/or the peak width is narrower because of the
use of fast HPLC the MRM dwell time has to be adjusted
accordingly.
An example of detecting 16 priority pesticides using 2 MRM
transitions and a fast HPLC gradient over 3 min is presented in
Figure 1 on page one. The use of a 1.8 μm particle size column
and a flow rate of 2 mL/min resulted in a typical peak width of 2.5
s. Dwell times of all MRM transitions were adjusted to allow the
collection of sufficient data points over each peak.
Summary
In summary, small particle size columns can be used in
conjunction with triple quadrupole LC-MS/MS systems to
increase throughput, sensitivity and/or resolution for targeted
quantitative applications.
AB SCIEX LC-MS/MS systems, with the patented Turbo V™
®
Source and LINAC Collision Cell can fully utilize Fast and High
Resolution HPLC systems, such as the Waters Acquity UPLC
system, the Agilent 1200 Series Rapid Resolution LC system,
the Dionex Ultimate 3000 LC system, and the Shimadzu
Prominence UFLC XR system.
However, when doing so, peak width, dwell and pause time all
need to be considered in order to provide the best LC-MS/MS
performance while providing enough data points across a peak
for accurate and reproducible quantitation.
While Fast HPLC can provide the enhancements discussed, the
results obtained are still dependent on the same factors that
govern traditional HPLC separation, namely, stationary phases,
particle size, solvent systems, and column temperature.
Because AB SCIEX mass spectrometers and a range of HPLC
systems can be fully controlled with Analyst® Software, the best
combination of HPLC system and MS/MS detector can be
chosen to meet specific needs.
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: 1282110-01
353 Hatch Drive Foster City CA
Headquarters
94404 USA
Phone 650-638-5800
www.absciex.com
International Sales
For our office locations please call the division
headquarters or refer to our website at
www.absciex.com/offices