August 8-11, 2005 Bristol, Rhode Island Characterization and Performance of MALDI on a Triple Quadrupole Mass Spectrometer for Analysis and Quantification of Small Molecules Jason S. Gobey, Mark J. Cole, John S. Janiszewski Q0 Q1 Q2 Q3
Dec 30, 2015
August 8-11, 2005 Bristol, Rhode Island
Characterization and Performance of MALDI on a Triple Quadrupole Mass Spectrometer for Analysis and Quantification of Small Molecules
Jason S. Gobey, Mark J. Cole, John S. Janiszewski
Q0 Q1 Q2 Q3
August 8-11, 2005 Bristol, Rhode Island
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
# S
am
ple
s A
naly
zed
2000 2001 2002 2003
Year
HT ADME Sample History 2000-2003
0
10000
20000
30000
40000
50000
60000
70000
80000
# S
am
ple
s/w
eek
August 8-11, 2005 Bristol, Rhode Island
MALDI Potential For HT Quantitation
1. Speed: potential for <1sec/sample
2. Simple analyses: no pumps, solvents, etc
3. Capacity: ~2mm sample size; no format constraints
4. Universality: ???
August 8-11, 2005 Bristol, Rhode Island
Small Molecule MALDI Quantitation Has 4 Requirements
1. MS/MS
2. High repetition (firing) rate laser
3. Sample Cleanup
4. Internal Standard
August 8-11, 2005 Bristol, Rhode Island
MS/MS Is Critical For Distinguishing Signal
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z
0.0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
6.0e6
7.0e6
8.0e6
9.0e6
1.0e7
1.1e7
1.2e7
1.3e7
1.4e7
1.5e7
Inte
nsity, cp
s
378.8288.1189.8
172.9143.6
335.2171.5
294.2
156.1
316.3122.0
90.9107.1
360.8188.0130.9 278.1
524.1 568.0270.1238.0
115.9 304.2362.9
202.1 323.3220.9 438.8423.077.2 522.1 643.8 665.9141.1 512.1309.2 855.1757.1 887.0 962.9 1054.1
Q1 MS scan of -cyano matrix (“blank”)
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z
0.0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
6.0e6
7.0e6
8.0e6
9.0e6
1.0e7
1.1e7
1.2e7
1.3e7
1.4e7
1.5e7
Inte
nsity, cp
s
378.8288.1189.8
172.9143.6
335.2171.5
294.2
156.1
316.3122.0
90.9107.1
360.8188.0130.9 278.1
524.1 568.0270.1238.0
115.9 304.2362.9
202.1 323.3220.9 438.8423.077.2 522.1 643.8 665.9141.1 512.1309.2 855.1757.1 887.0 962.9 1054.1
Q1 MS scan of -cyano matrix (“blank”)
August 8-11, 2005 Bristol, Rhode Island
Q1 MS 4ng Carbamazepine
40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500m/z
2.9e7
Inten
sity, cps
316.0287.9
172.2115.9
270.4145.7 190.0
147.1 378.9317.4
304.4106.1 335.2244.289.1 122.0 298.4191.0 237.1 250.2
290.3 416.4264.1162.0 256.388.2 194.1 272.3117.9 313.2234.0223.0102.0 336.2156.1 251.1 278.2245.2 361.3 376.9207.069.9 299.4305.3266.2130.976.8 175.1 413.1 441.0 477.2197.0 363.3 400.994.8 328.264.8 232.2 394.9282.292.0 459.2114.7 185.0 465.1449.0384.1351.2181.0150.1
MH+ 237
40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260m/z
1.7e6
Inten
sity, cps
194.2
192.1
237.3
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
Intensity, cps
25pg Carbamazepine SRM 237/194Carbamazepine MS/MS 237
August 8-11, 2005 Bristol, Rhode Island
0.502 0.504 0.506 0.508 0.510 0.512 0.514 0.516 0.518 0.520
Time, min
~280Laser Shots
~200msec
“Peaks” Produced By Laser Drilling Through Sample
Laser:• JDSU Nanolaser• Solid state diode-
pumped• 355nm wavelength• 500psec pulse width• ~16uJ/pulse• 1,400Hz pulse rate
August 8-11, 2005 Bristol, Rhode Island
Energy Per Pulse vs. Laser Firing Rate At 355 nm
0
2
4
6
8
10
12
14
16
18
0 500 1000 1500 2000
laser firing rate (Hz)
energ
y p
er
puls
e a
t 3
55
nm
(uJ)
August 8-11, 2005 Bristol, Rhode Island
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
2.4042.4062.4082.4102.412 2.4142.4162.418 2.420 2.4222.424Time, min
0.0
2000
4000
6000
8000
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
Laser rate = 10 Hz Laser rate = 1400 Hz
180 msec252 shots
24 sec240 shots
Desorption Time Is Linear With Laser Rate“band focusing” effect
Area = 1597 Area = 1625
August 8-11, 2005 Bristol, Rhode Island
“Good” crystals from clean samples
“Bad” crystals from raw samples
Raster Laser
Raster Laser
Direct Sampling Of Biological Matrices Does Not Produce Useful Crystals
August 8-11, 2005 Bristol, Rhode Island
Sample Cleanup By Simple SPE With MALDI Matrix In Eluent
Elute with 25uL MALDI matrix soln.
Cleanup biological samples with SPE
Pipette directly onto MALDI target
• SPE removes suppressing interferences• Eluent contains matrix and int. std.• High co-crystallization uniformity• Rapid and easy to automate
August 8-11, 2005 Bristol, Rhode Island
Aqueous MP
Organic MP
Waste
MassSpectrometer
Current HT Column-Switching LC/MS 3M/Tomtec SPExpress Card System
SPE-Type Sample Cleanup Is ComparableTo Contemporary HT Methodology
August 8-11, 2005 Bristol, Rhode Island
0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84
Time, min
3.4e4
~1.5 mm~1.5 mm
August 8-11, 2005 Bristol, Rhode Island
“Peak” Integration for Quantitation
Each “peak” consists of ~10 separate measurementsEach measurement is an average of ~280 laser shots
0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84
Time, min
3.4e4
August 8-11, 2005 Bristol, Rhode Island
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
0.0
4.4e4
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
0.0
8.9e5Internal Standard
Analyte
Example of Microsomal Incubate Timecourse
Blank
T=45 min
T=30 minT=15 min
T=5 min
T=0 min
0.25uM Std.
August 8-11, 2005 Bristol, Rhode Island
0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42
Time, min
3.3e4
August 8-11, 2005 Bristol, Rhode Island
0.70 0.71 0.72 0.73 0.74 0.75 0.76 0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84
Time, min
3.4e4
~1.5 mm~1.5 mm
August 8-11, 2005 Bristol, Rhode Island
Internal Standard is Necessary for Quantitation
0 5 10 15 20 25 30 35 40 45 50
time (min)
AnalyteInt. Std.Ratio
Compound 1 Microsomal Timecourse
0 5 10 15 20 25 30 35 40 45 50
time (min)
AnalyteInt. Std.Ratio
Compound 2 Microsomal Timecourse
August 8-11, 2005 Bristol, Rhode Island
Verapamil Std. Curve
y = 0.0033x - 0.0106R2 = 0.9788
0
2
4
6
8
10
12
14
16
18
20
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
Concentration (ng/ml)
Analy
te/I
S
Buspirone Std. Curve
y = 0.2461x - 0.0429R 2 = 0.9986
0
1
2
3
4
5
6
7
8
0 5 10 15 20 25 30
Concentration (uM)
Analy
te/I
S
August 8-11, 2005 Bristol, Rhode Island
Human Microsome T1/2 ESI vs. MALDI
y = 1.0228x + 1.8996
R2 = 0.9372
0.2
20.2
40.2
60.2
80.2
100.2
120.2
140.2
0.2 20.2 40.2 60.2 80.2 100.2 120.2 140.2
MALDI T1/2 (min)
ESI T
1/2
(min
)
August 8-11, 2005 Bristol, Rhode Island
Other Biological Samples Analyzed
1. Hepatocyte Metabolic Stability
2. Caco-2 Absorption
3. MDR Pgp Transport
4. Serum - standard curves
August 8-11, 2005 Bristol, Rhode Island
The Inevitable Comparison:
MALDI vs. ESI For Small Molecule Analysis
• Universality
• Coverage
• Sensitivity
• Speed
August 8-11, 2005 Bristol, Rhode Island
MALDI vs ESI conditions
Generic system for high throughput LC/ESI/MS/MS• Standard API3000• Standardized mobile phases• Standardized columns• Use only a few template MS/MS conditions; define polarity
MRM and choose a collision energy (little optimization)
Generic system for high throughput MALDI/MS/MS• Modified API3000 with MALDI source -CHC used exclusively as MALDI matrix• Simple generic SPE cleanup used• Template MS/MS method taken from the ESI studies
August 8-11, 2005 Bristol, Rhode Island
Universality: Ionization Success
Experiment:
•208 compounds from compound collection•Representative of total chemical space•25 Std. Curves used to correlate 50nM signals with detection limits•MALDI success: S/N 5 and estimated detection limit <50nM
Failed by MALDI: 33/208 16%
Failed by ESI: 14/208 6.7%
Failed ESI, Good MALDI: 4
Weekly Percent Compound Failure By ESI
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Week # (2002)
% F
aile
d C
om
pound
s
August 8-11, 2005 Bristol, Rhode Island
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9Time, min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
MALDI MS
ESI LC/MS
Sensitivity: Sample Consumption
Blank
T=45 min
T=30 minT=15 min
T=5 min
T=0 min
0.25uM Std.
August 8-11, 2005 Bristol, Rhode Island
MALDI MS
ESI LC/MS
Ionization/Ion Transfer Efficiency
25 uL injectedArea = 217,100
25 nL consumedArea = 26,970
2.2nL
MALDI: 100-fold increase in efficiency over ESI
August 8-11, 2005 Bristol, Rhode Island
0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84Time, min
0.77 0.78 0.79 0.80 0.81 0.82 0.83 0.84Time, min
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9Time, min
Speed: Effect On Precision
Multiple Measurementsvs.
Single Measurement
August 8-11, 2005 Bristol, Rhode Island
Averaged vs. Single(n=8) Measurements
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0 5 10 15 20 25 30 35 40 45 50
time (min)
are
a r
ati
o (
x1
00
00
)
4% CV
6% CV
8% CV
6% CV
10% CV
T1/2 = 30.7 min
C.V. = 7%
August 8-11, 2005 Bristol, Rhode Island
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6
Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e4
8ng Quinidine
10ng Clozapine
Capillary LC method and data collection by Dr. John Soglia
Inte
nsity,
cps
August 8-11, 2005 Bristol, Rhode Island
MALDI / MS/ MS LC/ ESI / MS/ MS
I on-suppression YES YES
Sample prep SPE ACN precipitation
Suffi cient Sensitivity f or HT ADME YES YES
Coverage GOOD EXCELLENT
Simple plumbing YES NO
Space constraints NO YES
Solvent use <50µL/ sample >1 mL/ sample
Current speed ~8 samples/ minute 3-4 samples/ minute
Potential speed 6-8 samples/ minute or
360 samples/ hr
1 sample/second or
3600 samples/hr
August 8-11, 2005 Bristol, Rhode Island
Potential Uses For MALDI Analyses(small molecule measurements)
1. Large numbers of samples (ADME screening)
2. Rapid just-in-time or real-time analyses (spot checking, quick analyses, designing larger studies)
3. Asynchronous LC-MS “write once, read many times”
August 8-11, 2005 Bristol, Rhode Island
Summary
• The instrument was capable of generating calibration curves for a variety of compounds. Suitable linearity and dynamic range was obtained to support typical ADME assays
• ADME data obtained using this prototype instrument was compared with data obtained using ESI/MS/MS, revealing a good correlation
• A calibration curve for verapamil in human serum was linear from 5-1000 ng/ml. Lower detection limits were not possible, due to severe ion-suppression
• Overall, the instrument shows promise as a tool for ADME screening, with possibilities for increased speed, perhaps plate-reader like speed
August 8-11, 2005 Bristol, Rhode Island
The future…
• “Autosampler” – not trivial, 3600 samples/hr• Faster motors• Software • Cheaper sample cleanup or more tolerant MALDI
methodology• Disposable plates/plates with sorbent• Negative ionization not addressed• Integration strategy to match analysis speed
with complementary use of ESI