1. No category

Liquid Chromatography Mass Spectrometry
SSI LCMS 001
LCMS Sample Evaluation Report
Roark Galloway & Miguel Gallego
Introduction
Two samples were submitted to the Shimadzu
Southern California (SCA) regional laboratory to
demonstrate chromatographic performance and
LCMS analysis for components of those samples.
The samples were prepared and provided by
representatives from CONFIDENTIAL Research
Laboratories for this evaluation. The samples were
identified as:
1) Test mix (5 components)
2) Sample ID- A001
OBJECTIVE - Attain resolution of 5 components from test mix in a total method runtime of 2
minutes or less, including MS spectral peaks.
Materials and Methods
System Hardware: The complete LCMS system is comprised of the following components:
CBM-20A:
LC-20:
LC-20 A3:
LC-20ADXR:
Prominence communications module
Reservoir tray
Prominence on-line degasser
Prominence pumps [2], (A=0.05% Formic/H2O; B = ACN)
binary gradient elution [BGE] system with 20uL mixer
SIL-20AXR:
Prominence UFLCXR autosampler
CTO-20A:
Prominence column oven
SPD-M20A:
Prominence diode array detector
LCMS-2020:
LCMS with electrospray [ESI]
LCMSsolution: LCMS system control software, version 5.10.153
Open Solution: Walk-up user interface software, version 1.0.29.0
Columns:
50mm X 2.0mm ID X 2.5u Phenomenex LUNA C18 HST
75mm X 2.0mm ID X 2.2u Shimadzu ODS, XR-II
Data Station: Dell Windows XP Professional, SP3
1.
2.
3.
4.
5.
6.
7.
8.
9.
770m Solvent / Reservoir Tray Degasser LC‐20ADXR Pumps Mixer SIL‐20AXR Column / CTO‐20A SPD‐M20A with semi‐micro flow cell LCMS‐2020 / ESI probe CBM‐20A controller 550mm
550mm Figure 1: LCMS System Hardware Configuration
Method Settings: General method settings used for data acquisition:
LCMS 2020:
Data Acquire:
Column Oven:
Flow Inlet to MS:
System Mode:
Initial Pump:
LC Time Program:
Scan 140-1000Da / Rate = 10000 Da sec-1
2 minutes / PDA = 40Hz / 200-300nm
40oC
Approx 50% passive split after PDA
Pumps = XR mode / CBM = FastLC mode
1.00mL min-1 / Pmax = 9000psi / 5%B
Refer to FIGURE 2 350mm Figure 2: LC Time Program
Results
1) CONFIDENTIAL Test mix was provided, scanned and analyzed in both (+) and (-) scan
mode.
2) 1.0 uL of sample was injected per each analysis.
3) LCMS detector gain was set to 1.0kV to accommodate response of largest MS peak.
4) Using the 50mm 2.5 micron column, all 5 peaks eluted at retention times representing
the expected masses of 152, 195, 295, 311, and 472.4 m/z in under 1.3 minutes with a
total run time of under 2 minutes. TIC chromatograms for simultaneous POS/NEG
ionization acquisition and spectra are presented in Figures 3-6. Results for
CONFIDENTIAL sample #2 are shown in Figure 7.
5) Using the 75 mm, 2.2 micron column, all 5 peaks eluted at retention times in less than 3
minutes under high-pressure conditions with increased resolution for some peaks on
the longer column (Figure 8 and Figure 9).
3 Sample data entry
windows for Direct Access
Group#1 Scan(+) EI : TIC
Int
RT=1.21
6.000e6
5.000e6
RT=1.03
4.000e6
3.000e6
2.000e6
RT=0.67
1.000e6
RT=0.53
0
0
0.2
Group#2 Scan(-) EI : TIC
0.4
0.6
0.8
1.0
Int
4.000e5
1.2
1.4
1.6
1.8
RT=1.12
3‐B 3.500e5
3.000e5
RT=1.20
2.500e5
RT=0.17
2.000e5
3‐A min.
RT=0.79
RT=1.47
1.500e5
1.000e5
0.500e4
0
0
0.2
0.4
0.6
0.8
1.0
1.2
Figure 3: TIC Chromatograms CONFIDENTIAL test Mix sample
1.4
1.6
1.8
min.
Group#1 - MS Peak: 1, RT: 0.49 to 0.58 min
Int
152.050
1.400e5
1.200e5
325.050
1.000e5
0.800e4
0.600e4
174.050
0.400e4
0
303.100
215.000
0.200e4
322.250
325.950
150.0
200.0
250.0
300.0
350.0
400.0
450.0
Group#1 - MS Peak: 2, RT: 0.63 to 0.72 min
Int
195.000
3.000e5
2.500e5
2.000e5
1.500e5
1.000e5
195.950
0.500e4
0
150.0
200.0
250.0
300.0
350.0
400.0
Group#1 - MS Peak: 3, RT: 0.99 to 1.08 min
Int
295.150
3.000e6
2.500e6
2.000e6
296.100
1.500e6
1.000e6
5.000e5
0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
4‐A 500.0
m/ z
4‐B m/ z
4‐C 900.0
m/ z
Figure 4: POSITIVE ESI spectra from peaks 1,2,3 TIC 3-A CONFIDENTIAL test Mix sample
(no significant negative ion spectra) Group#1 - MS Peak: 4, RT: 1.08 to 1.17 min
Int
311.000
1.400e6
1.200e6
1.000e6
8.000e5
6.000e5
312.050
4.000e5
643.150
2.000e5
0
333.000
200.0
300.0
400.0
500.0
600.0
700.0
800.0
900.0
Group# 2 - MS Peak: 8, RT: 1.08 to 1.16 min
Int
309.050
1.000e5
0.800e4
0.600e4
641.200
972.900
0.400e4
311.050
0.200e4
0
5‐A m/ z 5‐B 200.0
310.150
642.600
445.000
300.0
400.0
641.750
974.050
708.900
500.0
600.0
700.0
800.0
900.0
m/ z
Figure 5: POSITIVE and NEGATIVE ESI spectra, peak 1.12 min TIC 3-A & 3-B CONFIDENTIAL test Mix.
Group#1 - MS Peak: 5, RT: 1.17 to 1.28 min
Int
472.350
4.000e6
3.000e6
2.000e6
473.400
1.000e6
0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
Group#2 - MS Peak: 9, RT: 1.16 to 1.23 min
Int
506.300
20000.0
516.400
15000.0
140.600
10000.0
5000.0
0
507.850
507.350
155.400
147.750
200.0
249.250
300.0
509.500
400.0
500.0
585.200
600.0
6‐A 900.0
m/ z 6‐B 977.000
628.350
700.0
800.0
900.0
m/ z
Figure 6: POSITIVE and NEGATIVE ESI spectra, peak 1.21min TIC 3-A & 3-B CONFIDENTIAL test Mix.
1.436
TIC
7000000
6000000
1.490
5000000
4000000
1.331
3000000
2000000
1000000
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
min
Inten.(x10,000)
5.0
385
4.0
450
3.0
2.0
1.0
0.0
7‐A
196
208
484
261
200
525
987
329
300
593
400
500
661 686
600
742
700
791
800
841
935
890
900
m/z
Inten.(x1,000,000)
1.25
468
1.00
0.75
0.50
7‐B
0.25
176
215
282 319
300
0.00
200
387
529
450
400
500
628
568
600
682 726
700
798
800
955
987
933
883
900
m/z
Inten.(x100,000)
579
7.5
5.0
7‐C
2.5
176 215
257
0.0
200
309
300
359 387
400
468
504
599
500
600
642 677
737
700
783 817
800
Figure 7: TIC and SPECTRA (7A, 7B, 7C) from CONFIDENTIAL sample 167015
913
900
958 984
m/z
5000000 1:195.00
1:152.00
1:472.40
4000000 1:295.00
1:311.00
3000000
2000000
8‐A
1000000
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
min
152.00
195.00
5000000 295.00
311.00
4000000 472.40
3000000
2000000
8‐B
1000000
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
min
Figure 8: 50mm column peak separation(8A) vs. 75mm column (8B), Test mix sample.
383.00
2000000 468.00
579.00
1500000
1000000
9‐A
500000
0
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
min
383.00
468.00
1500000 579.00
1000000
9‐B
500000
0
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
Figure 9: 50mm column peak separation(9A) vs. 75mm column (9B), Test sample 167015.
min
Discussion
Open Solution: The Open Solution software utility for LCMSsolution greatly simplifies complete
system operation:
• automation – “cold” startup and equilibration automatic
• simplified sample login, in as few as 5 mouse clicks
• sample login and management by assigned sample location in autosampler for multiuser environment
• provides e-mail notification and results link when sample results are completed for an
experiment (sample group)
• simplified retrieval and review of complete LCMS chromatogram and spectra, PDA
chromatogram and spectra from any computer running Internet Explorer without “node”
fees
• summary reports can be printed from local user computer through Internet Explorer
Fast LCMS Data Acquisition: The LCMS-2020 with fast data acquisition up to 15000Da sec-1 and
positive/negative polarity switching time of 15msec is best suited for fast gradient, highresolution columns.
As demonstrated, a complex test mix containing 5 different compounds can be applied to the
Shimadzu UFLC system with a gradient program, including column wash and re-equilibration
at 2 minutes. The last eluting peak produced a retention time of 1.21 minutes, with all peaks
having complete baseline resolution.
Simplified LCMS Calibration: The tuning of the Shimadzu LCMS-2020 is a completely automated
process with a prepared tune solution. The LCMS-2020 tune program also includes a negative
tune component for actual negative ion tuning - NOT simply tuning in positive mode and
switching voltages.
All data produced in this report were achieved from a standard tune file. No sample-specific
adjustments were needed to enhance or provide system performance (exception = detector
sensitivity adjustment, probe position, drying gas flow). The LCMS was not re-tuned or
adjusted for any compound specific optimized response.
UFLC-XR High Pressure LC: For extra separation, smaller particle size columns for even faster LC
method development will require allowable pressure limits up to 9500+ psi.
For the samples analyzed in this study, the 50mm X 2.0mm ID 2.2 and 2.5 um particle size
columns produced system pressures of 2000-5000 psi at 1 ml min-1 and water/acetonitrile.
The longer 75mm X 2.0mm ID 2.2um particle size column produced system pressures up to
7800psi. The improvement in resolution is shown in the following table. The gradient was
adjusted by a factor of 1.5 to maintain the same relative gradient velocity across the column
bed.
Separation factors resulting from the analyzed samples were: Sample 1
Sample 2
Peak 1
Peak 2
Peak 3
Peak 4
Peak 5
Peak 1
Peak 2
Peak 3
Retention Time
50mm
75mm
0.53
0.64
0.67
0.85
1.03
1.3
1.12
1.5
1.21
1.59
1.32
1.82
1.43
1.99
1.49
2.12
Separation
50mm
75mm
Peak 1-2
2-3
3-4
4-5
1.264
1.537
1.087
1.080
1.328
1.529
1.154
1.060
Peak 1-2
2-3
1.083
1.042
1.093
1.065
LC Time Program
50mm X 2.0mm ID column
LC Time Program
75mm X 2.0mm ID column
Conclusion
The LCMS-2020 / UFLC-XR system combines simplified walk-up multi user capabilities and
fast reliable data acquisition. Excellent baseline separations were made using 2mm X 50mm
2.2 or 2.5 um particle size columns in a total runtime of less than 2 minutes under standard
pressure conditions. However, should the need arise for extra resolution, the Prominence
UFLCXR pressure capabilities drive system performance with the use of a higher generation of
column back pressure when using a longer column or a faster method cycle time using smaller
particle size. The Prominence UFLCXR system also achieves higher linear velocity through the
column for faster gradients and peak elution.
In order to fully utilize the benefits of any high-pressure HPLC, an LCMS system must be
capable of sufficiently rapid scans across very narrow peaks. Shimadzu’s LCMS-2020 is the
only single quadrupole mass spectrometry system capable of scan speeds up to 15000
Da/sec, 15 msec positive/negative polarity switching, and extreme sensitivity all within its
standard operating mode.
Shimadzu Scientific Instruments
7102 Riverwood Drive, Columbia, MD 21046
Phone: 800-477-1227, Fax: 410-381-1222
www.ssi.shimadzu.com
webmaster@shimadzu.com
Shimadzu Scientific Instruments
Southern California Regional Office
1817 Aston Ave., Carlsbad, CA 92008
Phone: 866-862-1677, Fax: 760-931-9854