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4
Versatile Solutions to Current Demands for Automated Sample Cleanup, High Throughput, and High-Resolution Chromatography on a Single LC-MS Platform
RVs (saquinavir, nevirapine,
Mass Spectrometry
All data were collected on the Thermo Scientific™ Q Exactive™ hybrid quadrupole-
Orbitrap MS (HRAM, high-resolution accurate mass) with heated electrospray
ionization source (HESI) and full scan exact mass extraction used to quantitate results.
FIGURE 4. Upper trace: 384
data file, 9 s data window. L
E:\Q-ExactiveData\...\Sample1537
11
N/C
RT:
0.00-107.25
SM:
5G
earch [hospital] lab: acetonitrile-
-crashed whole blood, and an
r each ARV was from 5 to
ernal standard for use with either the
Case Study #1: The HRAM MS was used in full scan positive (250-700
m/z
) and
negative (250-350
m/z
) ionization modes at resolution 70,000. A 30 s MS data window,
necessary for simultaneous positive/negative switching, was used.
Case Study #2: The Q Exactive HRAM MS was used in full scan negative ionization
100
nce
0
50
100
RelativeAbundance
RT:0.68
AA:102558
SN:319
RT:9.64
AA:96896
SN:314
RT:23.56
AA:91786
SN:306
RT:16.34
AA: 95527
SN: 302
RT:26.92
AA: 91004
SN:302
RT:0.68
AA:665874
SN:1756
RT:9.64
AA:622674
SN:1686
RT:16.35
AA:634708
SN:1674
RT:26.35
AA:583436
SN:1661
RT: 31.4
AA:592
SN:161
e) standard in neat solution, C8
e supplied by a local pharmaceutical
mode across a mass range of 804-895
m/z
at resolution 36,000. Two acquisition styles
of high-throughput data collection workflows were examined. The first style of
acquisition (collection of 1000 individual data files with 21 s data windows) involved a
high-throughput collection with complete baseline chromatographic separation of the
analytes in order to avoid potential matrix interferences (data not shown). A two-tiered
d i i i
l i
l d fi
384 i j
i
ll
d i
i
l d fil
0
5
10
15
20
25
30
0
50
RelativeAbunda
RT:
53.13 - 54.13
SM:
5G
100
ce
RT:53.21
AA:96067
SN:311
as a Thermo Scientific™
Scientific™ Dionex™
mps, a dual-valve VIM (valve
utosampler (CTC Analytics AG,
ump flow to the MS was diverted to
secon acqu s t on sty e nvo ve , rst,
n ect ons co ecte nto a s ng e ata e
using the 21 s data window (Figure 3). The second high-throughput tier demonstrated
the use of the Q Exactive MS to theoretically separate compounds based solely on
their accurate mass using a 9 s data window (Figure 4).
Data Analysis
50
100
tiveAbundance
0
50
RelativeAbundan
RT:53.16
AA:9375
SN: 31
RT:
AA
SN:
RT:53.21
AA:605017
SN:1626
f the method, to allow for faster LC
ration time.
figuration, two LC channels (each
s) were multiplexed to one at-source
tlined in Table 1 Use of the VIM’s
Results
C St d #1
Case Study #1: Post-acquisition data analysis was performed using Thermo
Scientific™ Xcalibur™ Quan Browser software version 3.0.
Case Study #2: Post-acquisition data processing of single, individually collected data
files was performed using Xcalibur Quan Browser software version 3.0. Post-
53.2
53.3
53.4
0
Rela
.
s (up to 5 mL/min) as necessary for
L were injected in staggered fashion
riven by Thermo Scientific™Aria™
ultant chromatographic comparison
s
ase u y :
Using a 100 s LC/MS research
resolution, antiretrovirals were
multiplexed (two-channel) LC s
standard curve matrices gave l
acquisition data processing of the multiple data files collected into a single file was
performed using Generic Chromatographic Viewer of Thermo Scientific™ QuickCalc
TM
software version 8.3.24.
FIGURE 2 Comparison of ARV standard (250 ng/mL) across four matrix preparations
.
re outlined in Table 2. Multiple
ed onto a UHPLC column (C8, 2.1 x
C channel (four) and a cross-
ftware version 1.6.
%RSD across two channels w
Case Study #2:
The first workflow (1000 separ
acquisition of an average of 1.
Nevirapine [M+H]+
Saquinavir [M+H]+
RT:
0.00 -0.53
SM:
7G
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
RT: 0.25
AA: 84262314
RT: 0.28
AA: 276689068
RT: 0.24
NL: 1.54E7
m/z=
267.12326-267.12594
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL: 4.90E7
m/z=
671.38819-671.39491
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL: 8.93E3
RT:
0.00 -0.52
SM:
7G
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
RT: 0.29
AA: 26676271
RT: 0.29
AA: 131704845
RT: 0.24
NL: 5.59E6
m/z=
267.12326-267.12594
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_003
NL: 2.82E7
m/z=
671.38819-671.39491
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_003
NL: 1.97E4
RT:
0.00 -0.52
SM:
7G
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
RT: 0.25
AA: 22978793
RT: 0.29
AA: 83570949
RT: 0.03
AA: 45509
RT: 0.24
NL: 4.78E6
m/z=
267.12326-267.12594
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL: 1.74E7
m/z=
671.38819-671.39491
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL: 3.82E3
RT:
0.00 - 0.52
SM:
7G
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
RT: 0.25
AA: 19856397
RT: 0.29
AA: 48388013
RT: 0.26
NL: 4.15E6
m/z=
267.12326-267.12594
F: FTMS + pESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL: 9.82E6
m/z=
671.38819-671.39491
F: FTMS + pESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL: 3.29E3
.
resolution of three analytes. Th
earmarked as the internal stan
The second workflow (three se
using 21 s data windows) resul
minute with peaks chromatogr
Zidovudine [M-H]-
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
AA: 41758
RT: 0.37
AA: 6864606
RT: 0.34
AA: 147554194
RT: 0.05
AA: 658433
RT 035
m/z=
266.08815-266.09081
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
NL: 1.37E6
m/z=
314.01854-314.02168
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
NL: 2.87E7
m/z=
309.11059-309.11369
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL:224E7
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
MA: 96299
RT: 0.36
AA: 3693436
RT: 0.33
AA: 14888169
RT: 0.18
AA: 33359
RT 034
m/z=
266.08815-266.09081
F: FTMS - pESI Full
ms [250.00-350.00]
MS StdA_250_003
NL: 7.60E5
m/z=
314.01854-314.02168
F: FTMS - pESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_003
NL: 3.49E6
m/z=
309.11059-309.11369
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_003
NL:351E7
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
AA: 16730
RT: 0.36
AA: 3235427
RT: 0.31
AA: 51351138
RT 034
m/z=
266.08815-266.09081
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
NL: 6.82E5
m/z=
314.01854-314.02168
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
NL: 1.19E7
m/z=
309.11059-309.11369
F: FTMS + p ESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL:316E7
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
AA: 15227
RT: 0.36
AA: 3068603
RT: 0.31
AA: 64432405
RT 034
m/z=
266.08815-266.09081
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
NL: 6.49E5
m/z=
314.01854-314.02168
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
NL: 1.46E7
m/z=
309.11059-309.11369
F: FTMS + pESI Full
ms [250.00-700.00]
MS ICIS
StdA_250_004
NL:386E7
Efavirenz [M-H]-
IS [M+H]+
The final workflow (one set of
resulted in the acquisition of 4.
in an ultra-high-throughput wor
Robustness of the assay was
Cyclone™ column, 0.5 x 50 mm
Diluted
ACN
ACN-Crashed
Plasma
Diluted Plasma
ACN-Crashed
Whole Blood
0.0
0.2
0.4
Time (min)
0
20
40
60
80
100
RelativeAbundance
: .
AA: 132197193
.
m/z=
307.09604-307.09912
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
0.0
0.2
0.4
Time (min)
0
20
40
60
80
100
RelativeAbundance
: .
AA: 193817363
RT: 0.13
AA: 67616
.
m/z=
307.09604-307.09912
F: FTMS - pESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_003
0.0
0.2
0.4
Time (min)
0
20
40
60
80
100
RelativeAbundance
: .
AA: 175642230
.
m/z=
307.09604-307.09912
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
0.0
0.2
0.4
Time (min)
0
20
40
60
80
100
RelativeAbundance
: .
AA: 215104504
RT: 0.13
AA: 98765
.
m/z=
307.09604-307.09912
F: FTMS -p ESI Full
ms [250.00-350.00]
MS ICIS
StdA_250_004
IS [M-H]-
TABLE 3. Overview of acqui
i
th CTC D l A DL
injections (1152# multiplexed) f
%RSDs of multiple injections (
≤2.8% .
e, 10 x 4.6 mm
formate + 0.05% formic acid (aq)
acetonitrile
ile/isopropanol/acetone
FIGURE 3. Upper trace: View of 384 sulfatide sample injections collected into a
single data file, 21 s data window. Lower trace: Scaling to 1.75 min (four injections).
us ng e
ua - rm
running cross-channel stag
D
Wi
C:\Xcalibur\...\Sample001
11/14/1311:30:06
Sample001
N/C
RT:
0.00 -176.59
SM:
5G
0
100
0
100
RT:46.05
AA:26337
SN:310
RT:0.50
AA:35391
SN:309
RT:62.61
AA:29960
SN:298
RT:64.44
AA:27658
SN:298
RT:40.53
AA:28248
SN:292
RT:25.81
AA:29567
SN:290
RT:120.10
AA:28013
SN:267
RT:96.18
AA:27267
SN:259
RT:90.66
AA:22590
SN:258
RT:155.08
AA:25839
SN:254
RT:13.39
AA:26323
SN:254
RT:162.44
AA:25516
SN:253
RT:107.24
AA:24086
SN:253
RT:132.99
AA:24979
SN:248
RT:147.72
AA:26397
SN:247
RT:42.51
AA:99948
SN:440
RT:57.24
AA:105250
SN:431
RT:62.76
AA:99856
SN:422
RT:27.80
AA:96168
SN:406
RT:143.23
AA:80268
SN:384
RT:163.47
AA:79306
SN:382
RT:0.65
AA:113228
SN:377
RT:150.59
AA:77699
SN:372
RT:95.38
AA:76662
SN:366
RT:121.14
AA:75790
SN:365
RT:86.19
AA:78012
SN:363
RT:71.46
AA:76807
SN:360
RT:115.62
AA:75443
SN:359
RT:137.72
AA:75846
SN:357
RT:18.11
AA:78468
SN:355
RT 4628
NL: 3.54E4
m/z=
806.54173-
806.54979 MS
ICISSample001
NL: 9.64E4
m/z=
862.60405-
862.61267 MS
ICISSample001
NL:239E5
AcquisitionStyle
Le
(
StandardLC-MS
w/384datafiles
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
Time (min)
0
100
0
100
: .
AA:291648
SN:408
RT:62.85
AA:295440
SN:405
RT:38.93
AA:286329
SN:395
RT:0.30
AA:311812
SN:374
RT:26.05
AA:280182
SN:362
RT:69.71
AA:237171
SN:361
RT:143.31
AA:222421
SN:356
RT:119.39
AA:221149
SN:354
RT:95.47
AA:221198
SN:354
RT:167.23
AA:219008
SN:341
RT:158.04
AA:220406
SN:341
RT:88.12
AA:221427
SN:340
RT:112.04
AA:216178
SN:339
RT:130.44
AA:217567
SN:337
RT:16.36
AA:224530
SN:321
RT:46.19
AA:671315
SN:1931
RT:62.76
AA:648226
SN:1865
RT:38.84
AA:655611
SN:1846
RT:27.80
AA:645725
SN:1760
RT:69.63
AA:539813
SN:1723
RT:0.65
AA:716499
SN:1667
RT:143.23
AA:515626
SN:1635
RT:176.36
AA:499585
SN:1620
RT:163.47
AA:503520
SN:1603
RT:119.30
AA:495420
SN:1602
RT:91.71
AA:502366
SN:1591
RT:97.23
AA:496196
SN:1572
RT:137.72
AA:492410
SN:1567
.
m/z=
890.63521-
890.64411 MS
ICISSample001
NL: 6.05E5
m/z=
888.61957-
888.62845 MS
ICISSample001
RT:
10306 10481
SM:
5G
7 μm heated to 60 °C
rmate + 0.2% formic acid (aq)
TranscendIILX-4system
with384datafiles
TranscendIILX-4system
with1datafile
(DT Submit)
. -
.
100
0
100
0
100
RT:103.10
AA:24554
SN:239
RT:104.45
AA:19941
SN:239
RT:103.56
AA:27204
SN:234
RT:104.02
AA:24515
SN:220
RT:104.59
AA:75250
SN:335
RT:103.24
AA:75603
SN:321
RT:103.70
AA:86764
SN:304
RT:104.16
AA:77207
SN:293
RT:104.54
AA:8014
SN:40
RT:103.65
AA:11077
SN:39
RT:103.19
AA:7985
SN:34
RT:104.68
AA:217095
SN:310
RT:103.34
AA:219431
SN:293
RT:104.25
AA:219845
SN:261
RT:103.80
AA:241909
SN:258
RT:104.62
AA:96459
SN 151
RT:103.27
AA:93813
RT:104.19
AA:99221
RT:103.73
AA:104726
NL: 2.67E4
m/z=
806.54173-
806.54979 MS
ICISSample001
NL: 7.35E4
m/z=
862.60405-
862.61267 MS
ICISSample001
NL: 1.82E5
m/z=
890.63521-
890.64411 MS
rmate + 0.2% formic acid in 1:1
l
_
TranscendIILX-4system
with1datafile
(DT_Submit)
103.1
103.2
103.3
103.4
103.5
103.6
103.7
103.8
103.9
104.0
104.1
104.2
104.3
104.4
104.5
104.6
104.7
104.8
Time (min)
0
100
0
:
SN:145
SN:136
SN:135
RT:104.59
AA:493233
SN:1473
RT:103.25
AA:497242
SN:1417
RT:103.70
AA:552731
SN:1311
RT:104.16
AA:485637
SN:1253
ICISSample001
NL: 4.62E5
m/z=
888.61957-
888.62845 MS
ICISSample001