AI-64310-CRFT

Thermo Scientific Poster Note

•

PN-64152-ASMS-EN-0614S

t of using active flow

y for LC/MS research and

i ll

Methods

Columns were connected in the manner shown in Figure 2. For the conventional

2.1 mm i.d. column, a 1 mL/min direct flow to source was used with a 2 µL injection

FIGURE 4. A) Comparison of pea

with PCS, conventional 2

1 mm i.

%RSD of peak area for six replica

%RSD peak height. Individual co

espec a y w en comp ex

reproducibility, a model

ventional 50 x 4.6 mm i.d.

g a series of standard steroids.

FIGURE 2 Illustrations of column installation The length and inner diameter of

volume to maintain concentration consistency. The conventional 4.6 mm i.d. column

included a 70% post-column split to waste for consistent comparison of sample

introduction to the MS source. All columns were packed with Thermo Scientific™

Accucore™ C18 2.6 µm stationary phase.

, %RSD, and %Diff (precision

se flow rates were adjusted to

ere also determined using

ed plasma. Column robustness

a 5 fg/µL alprazolam solution in

the connective tubing was adjusted to provide similar backpressures (linear

velocities and split flow ratios). A) Conventional 50 x 2.1 mm i.d. column

connected directly to injector and MS source. B) Conventional 50 x 4.6 mm i.d.

column connected directly to injector but including a 70% split to waste post

column C) Curtain flow 50 x 4 6 mm i d column installation Flow split from

he Thermo Scientific™ TSQ

mm i.d. column with a post-

50 x 4.6 mm i.d. column

the CF column over both the

Pump

Injector

HESI

Pump

Injector

HESI

m x 70mm

. .

injector to peripheral inlet. Peripheral outlet flow to waste. Central flow zone

from injector to MS source.

e) and conventional 2.1 mm i.d.

precision and accuracy of the

showed increased sensitivity,

2 fg on column. Further

ion following more than 220

NanoViper10

Waste

HESI

Peripheral

flowtowaste

Injector

Pump

CurtainFlow

Central flow

urine.

e for LC/MS separations,

The LC system was a Thermo Scientific™ UltiMate™ 3000 RS system. For curtain

flow column installation, the flow was split immediately prior to the injection valve in the

autosampler. Mobile phase composition for the column performance study was

A: 0.1% formic acid (aq), B: methanol (MeOH) + 0.1% formic acid in a 65:35 ratio

For all test compounds, the curtain f

performance in peak area and peak

performance measurements

lectrospray and atmospheric

e of small-bore HPLC columns

phic efficiency. A study by

formance in a 2.1 mm i.d.

on the reduced plate height,

)

isocratically. Gradient elution was used for all other studies with mobile phase A:

2 mM ammonium acetate (NH

OAc) (aq) and B: acetonitrile (ACN) (sensitivity and

linearity) and 2 mM NH

OAc (aq) and B: MeOH + 2 mM NH

OAc (urine study). Flow

rates were measured volumetrically to determine the actual flow entering the MS

source (with the exception of the 2

1 mm i.d. column). Samples were analyzed by

Calibration curves were generated

from 1 pg/µL to 100 pg/µL. RSD%

Figure 5 shows the CF 4.6 mm i.d.

conventional 4 6 mm i d column wit

ters of 1.7 to 2.7 µm was

mn length, and manufacturer.

ty in LC/MS research and

bility of the column, which is

us. One approach to remove

t d h

t i

Results

selected-reaction monitoring (SRM) with a TSQ Quantiva MS in heated-electrospray

ionization (HESI) mode

. .

Table 1 lists RSD% on the lowest co

column provides better reproducibili

the conventional 4.6 mm i.d. or the

s presen e ere uses cur a n

e phase is managed at both the

inside the analytical column

iameter; the dimensions are

through the central zone

n active flow management

Column Performance

Column performance was measured using a mixture of steroids – ketotestosterone,

nortestosterone, testosterone and epitestosterone – in concentrations from 1 to

100 pg/µL with six replicate injections per sample. Figure 3 shows the TIC

chromatograms of the test compounds (1 pg/µL) The mean of the performance

FIGURE 5. Calibration curves for

A) conventional 4.6 mm i.d. colu

column, and C) CF 4.6 mm i.d. col

eter (4.6 mm) adds an

ng larger injection volumes)

e phase is split prior to the

parameters of peak area, height, S/N, and RSD% (area and height) were graphed and

compared. Figure 4 shows peak area comparison (A) and area RSD% (B), peak

height comparison (C), and %RSD for peak height

FIGURE 3. TIC Chromatograms (SRM) of ketotestosterone, nortestosterone,

t t t

d it t t

A) C ti

l 4 6 i d l

ith

11-ketotestosterone

Y= -559.255+3.12757e+006*X R^2=0.9988 W:1/X

150000

200000

250000

300000

Area

w region. Mobile phase flow

olumn. Virtual column

entral flow region and the

es os erone an ep es os erone on onven ona . mm . . co umn w

PCS, B) Conventional 2

1 mm i.d. column and C) CF 4.6 mm i.d. column.

Intensity was normalized to most intense base peak.

RT:0.38

AA 4275

NL:

6.00E3

TIC MS

Genesis

S1-6

0.00

0.02

0.04

0.06

0.08

50000

100000

Central flow

RT:0.31

AA:2484

RT:0.52

AA:1345

RT:0.18

AA:801

NL:

6.00E3

TIC MS

Genesis s1-6

Y= -1

400000

500000

600000

700000

800000

Area

to source

RT:0.34

AA:1835

RT:0.28

AA:999

RT:0.46

AA:802

RT:0.16

AA:429

RT:0.39

AA:9012

NL:

6.00E3

TIC MS

Genesis s1 6

100000

200000

300000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

RT:0.33

AA:4876

RT:0.52

AA:4760

RT:0.20

AA:2358

Time (min)