Get documents about "LCMS Sample Evaluation Report - PDF
Document Sample
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: Prominence communications module
LC-20: Reservoir tray
LC-20 A3: Prominence on-line degasser
LC-20ADXR: 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. Solvent / Reservoir Tray
2. Degasser
770m
3. LC‐20ADXR Pumps
4. Mixer
5. SIL‐20AXR
6. Column / CTO‐20A
7. SPD‐M20A with semi‐micro flow cell
8. LCMS‐2020 / ESI probe
9. CBM‐20A controller
550mm
550mm 350mm
Figure 1: LCMS System Hardware Configuration
Method Settings: General method settings used for data acquisition:
LCMS 2020: Scan 140-1000Da / Rate = 10000 Da sec-1
Data Acquire: 2 minutes / PDA = 40Hz / 200-300nm
Column Oven: 40oC
Flow Inlet to MS: Approx 50% passive split after PDA
System Mode: Pumps = XR mode / CBM = FastLC mode
Initial Pump: 1.00mL min-1 / Pmax = 9000psi / 5%B
LC Time Program: Refer to FIGURE 2
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 3‐A
1.000e6 RT=0.53
0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min.
Group#2 Scan(-) EI : TIC
Int RT=1.12
4.000e5
3.500e5
3.000e5
RT=1.20
2.500e5
2.000e5 RT=0.17 RT=0.79
3‐B
RT=1.47
1.500e5
1.000e5
0.500e4
0
0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 min.
Figure 3: TIC Chromatograms CONFIDENTIAL test Mix sample
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
215.000 303.100 322.250
0.200e4
325.950 4‐A
0
150.0 200.0 250.0 300.0 350.0 400.0 450.0 500.0
m/ z
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 4‐B
0.500e4
0
150.0 200.0 250.0 300.0 350.0 400.0
m/ z
Group#1 - MS Peak: 3, RT: 0.99 to 1.08 min
Int 295.150
3.000e6
2.500e6
2.000e6
1.500e6 296.100
1.000e6
5.000e5 4‐C
0
200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0 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
5‐A
4.000e5
2.000e5 333.000
643.150
0
200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0
m/ z
Group# 2 - MS Peak: 8, RT: 1.08 to 1.16 min
Int 309.050
1.000e5
0.800e4
0.600e4
641.200
5‐B
0.400e4
310.150 642.600 641.750
972.900
311.050 974.050
0.200e4 445.000 708.900
0
200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0 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
6‐A
1.000e6
0
200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0
m/ z
Group#2 - MS Peak: 9, RT: 1.16 to 1.23 min
20000.0
Int 506.300
15000.0
516.400
10000.0
140.600 507.850 6‐B
507.350
5000.0
155.400
509.500
977.000
147.750 249.250 628.350
585.200
0
200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0 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
5000000
1.490
4000000
3000000
1.331
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
7‐A
1.0
196 484 525 987
261
208 329 593 661 686 742 791 841 890 935
0.0
200 300 400 500 600 700 800 900 m/z
Inten.(x1,000,000)
1.25
468
1.00
0.75
0.50
7‐B
0.25
176 215 955
282 319 387 450 529 568 628 682 726 798 883 933 987
0.00
200 300 400 500 600 700 800 900 m/z
Inten.(x100,000)
579
7.5
5.0
2.5 7‐C
176 215
309 468
0.0 257 359 387 504 599 642 677 737 783 817 913 958 984
200 300 400 500 600 700 800 900 m/z
Figure 7: TIC and SPECTRA (7A, 7B, 7C) from CONFIDENTIAL sample 167015
5000000 1:195.00
1:152.00
1:472.40
4000000 1:295.00
1:311.00
3000000
2000000
1000000 8‐A
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
1000000 8‐B
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
500000 9‐A
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
500000
9‐B
0
0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 min
Figure 9: 50mm column peak separation(9A) vs. 75mm column (9B), Test sample 167015.
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 multi-
user 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, high-
resolution 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:
Retention Time Separation
50mm 75mm 50mm 75mm
Sample 1 Peak 1 0.53 0.64
Peak 2 0.67 0.85 Peak 1-2 1.264 1.328
Peak 3 1.03 1.3 2-3 1.537 1.529
Peak 4 1.12 1.5 3-4 1.087 1.154
Peak 5 1.21 1.59 4-5 1.080 1.060
Sample 2 Peak 1 1.32 1.82
Peak 2 1.43 1.99 Peak 1-2 1.083 1.093
Peak 3 1.49 2.12 2-3 1.042 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 Shimadzu Scientific Instruments
7102 Riverwood Drive, Columbia, MD 21046 Southern California Regional Office
Phone: 800-477-1227, Fax: 410-381-1222 1817 Aston Ave., Carlsbad, CA 92008
www.ssi.shimadzu.com Phone: 866-862-1677, Fax: 760-931-9854
webmaster@shimadzu.com
Related docs
Other docs by aaw90594
