AI-64310-CRFT

Thermo Scientific Poster Note

•

PN-64103-ASMS-EN-0614S

atility of an LC-MS platform for

ilities in a multi-channel

(HRAM) h b id

Methods

Sample Preparation

Case Study #1: Four standard curve matrices of ARVs (saquinavir, nevirapine,

Mass Spectrometry

All data were collected on the Ther

Orbitrap MS (HRAM, high-resolutio

ionization source (HESI) and full sc

ass

y r

efavirenz, and zidovudine) were prepared by a research [hospital] lab: acetonitrile-

crashed plasma, water-diluted plasma, acetonitrile-crashed whole blood, and an

acetonitrile-water neat mix. The dynamic range for each ARV was from 5 to

1000 ng/mL. Warfarin was added post-prep as internal standard for use with either the

positive- or negative-ionizing ARVs.

Case Study #1: The HRAM MS wa

negative (250-350

m/z

) ionization

necessary for simultaneous positiv

Case Study #2: The Q Exactive HR

) in biological matrix for

a high-throughput workflow

re injected across two LC

in a cross-sequential manner

Case Study #2: A 100 ng/mL sulfatide (from bovine) standard in neat solution, C8

UHPLC columns, and mobile phase reagents were supplied by a local pharmaceutical

lab.

Liquid Chromatography

mode across a mass range of 804-

of high-throughput data collection

acquisition (collection of 1000 indiv

high-throughput collection with co

analytes in order to avoid potential

d i i i

l i

l d fi

ulfatide molecular species in

two discrete high-throughput

inct HPLC columns, were used

ti l

The multi-channel LC used in these experiments was a Thermo Scientific™

Transcend™ II LX-4 configured with four Thermo Scientific™ Dionex™

UltiMate™ 3000 Binary Rapid Separation HPG Pumps, a dual-valve VIM (valve

interface module), and a CTC

Dual-Arm DLW Autosampler (CTC Analytics AG,

Zwingen, Switzerland). In both case studies

LC pump flow to the MS was diverted to

secon acqu s t on sty e nvo ve ,

using the 21 s data window (Figure

the use of the Q Exactive MS to th

their accurate mass using a 9 s dat

Data Analysis

cross-sequen a manner o

waste, except during the data collection window of the method, to allow for faster LC

flow rates and, therefore, faster column re-equilibration time.

Case Study #1: In a high-throughput workflow configuration, two LC channels (each

with distinct TurboFlow sample extraction columns) were multiplexed to one at-source

C18 guard column The LC method details are outlined in Table 1 Use of the VIM’s

Case Study #1: Post-acquisition da

Scientific™ Xcalibur™ Quan Brow

Case Study #2: Post-acquisition da

files was performed using Xcalibur

trices with online sample

ithin a data window of 30 s

atrices were investigated. All

detector bypass position allowed for high flow rates (up to 5 mL/min) as necessary for

TurboFlow technology.

Multiple injections of 30 µL were injected in staggered fashion

across two channels of the LC system and were driven by Thermo Scientific™Aria™

MX software version 2.1. Figure 2 illustrates a resultant chromatographic comparison

of a neat standard with three in biological matrices

acquisition data processing of the

performed using Generic Chromat

software version 8.3.24.

FIGURE 2 Comparison of ARV sta

les examined

the percent

ganic standard were

Case Study #2: The sulfatide LC method details are outlined in Table 2. Multiple

injections (10 µL) of the neat standard were injected onto a UHPLC column (C8, 2.1 x

50 mm, 1.7 µm) heated to 60 °C in both a cross-LC channel (four) and a cross-

sequential manner and were driven by Aria OS software version 1.6.

Nevirapine [M+H]+

Saquinavir [M+H]+

RT:

0.00 -0.53

SM:

100

RelativeAbundance

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:

RelativeAbundance

maceutical laboratories

ogies. Market response to

ies that can perform online

h t

ARVs LC Method Details

Zidovudine [M-H]-

100

RelativeAbundance

100

RelativeAbundance

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

RelativeAbundance

Efavirenz [M-H]-

IS [M+H]+

TABLE 1. LC method details for Case Study #1

se c roma ograp c

onsistent.

ility of a single multichannel

l production tool. A multi-

ions through multiple LC

LC Method Length

100 s

Data Window

30 s

TurboFlow Columns

Thermo Scientific™ Cyclone™ column, 0.5 x 50 mm

Diluted

ACN

0.0

0.2

0.4

Time (min)

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

RelativeAbundance

IS [M-H]-

ple throughput (Figure 1). This

cleanup utilizing Thermo

seline-resolved UHPLC

ass, all in a rugged high-

Analytical Column

C18 guard cartridge, 10 x 4.6 mm

Loading Mobile Phase

10 mM ammonium formate + 0.05% formic acid (aq)

Eluting Mobile Phase

0.1% formic acid in acetonitrile

Extraction Column Wash

45:45:10 acetonitrile/isopropanol/acetone

FIGURE 3. Upper trace: View of 38

single data file, 21 s data window.

Injection Volume

30 µL

TABLE 2. LC Method Details for Case Study #2

C:\Xcalibur\...\Sample001

11/14/131

N/C

RT:

0.00 -176.59

SM:

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:13.39

AA:26323

SN:254

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:0.65

AA:113228

SN:377

RT:18.11

AA:78468

SN:355

RT 4628

Sulfatide LC Method Details

LC Method Length

96 s

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.

AA:23

SN:36

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.

AA:539

SN:17

RT:0.65

AA:716499

SN:1667

RT:

10306 10481

SM:

Data Window

21 s (or 9 s)

Analytical Columns

C8, 2.1 x 50 mm, 1.7 μm heated to 60 °C

Loading Mobile Phase

5 mM ammonium formate + 0.2% formic acid (aq)

. -

100

RT:103.10

AA:24554

SN:239

RT:103.56

AA:27204

SN:234

RT:103.24

AA:75603

SN:321

RT:103.70

AA:86764

SN:304

RT:103.65

AA:11077

SN:39

RT:103.19

AA:7985

SN:34

RT:103.34

AA:219431

SN:293

RT:1

AA:2

SN:

RT:103.27

AA:93813

RT:103.73

AA:104726

Eluting Mobile Phase

5 mM ammonium formate + 0.2% formic acid in 1:1

acetonitrile/methanol

Injection Volume

10 µL

103.1

103.2

103.3

103.4

103.5

103.6

103.7

100

SN:145

SN:135

RT:103.25

AA:497242

SN:1417

RT:103.70

AA:552731

SN:1311