AI-64310-CRFT

Thermo Scientific Poster Note

•

PN ASMS13_TP29_JDiBussolo_e 06/13S

od performance utilizing an

uid chromatography (SPLC)

whole blood when measuring

urposes by tandem mass

ion (ESI).

ed clean up of whole blood

tion by aqueous zinc sulfate and

by high-resolution liquid

µm HPLC column. Reversed-

were done in a way that avoided

ids, which would have suppressed

f four ISDs was achieved by

ards (IS).

stently reproduced within systems

d levels were reliably measured:

limus and Tacrolimus; and

. A throughput of 21 samples per

th channels, which generated only

er between samples was detected.

lyzed in whole-blood using LC-MS

nterference by phospholipids.

ble to compensate, minimizing

The Thermo Scientific™ Prelude™

t automates sample preparation

ed to the ESI of a tandem mass

he Prelude SPLC system

chnology and high-efficiency LC

vatives D

-Cyclosporin-A and

rds in the whole-blood sample

zed to reliably minimize

ta quality. The method was also

FIGURE 4. Optimized HPLC Condit

Elution from Accucore C8, 2.6 µ

FIGURE 3. Non-Optimized HPLC

Elution from Accucore PFP, 2.6 µ

Mass Spectrometry

The Thermo Scientific™ TSQ Vantage™ triple-stage quadrupole system with

heated electro-spray interface (HESI-II) was used to measure the transitions from

ammonium-adduct precursor ions to product ions:

Everolimus: 975.7 > 908.4

Sirolimus: 931.6 > 864.6

Tacrolimus: 821.5 > 824.4

Tacrolimus IS: 824.4 > 771.0

Cyclosporin A: 1202.8 > 425.3

Cyclosporin A IS: 1214.9

> 437.2

During method development, the elution of phospholipids and dioctylphthalate

were tracked by adding the following transitions:

Dioctylphthalate: 391 >149

Lyso-Phosphotidylcholine;16:0: 496 > 184

Lyso-Phosphotidylcholine;18:0: 524 > 184

Phosphotidylcholine;38:6: 806 > 184

Data Analysis

Thermo Scientific™ TraceFinder™ software with Aria MX was used for instrument

control, data acquisition and data processing. The internal standards (IS) shown

above were used for quantitation by stable-isotope dilution technique.

Results

Identifying the HPLC Column and

Because ISDs are as hydrophobic as

extracted and transferred to the HP

Therefore, the HPLC conditions mu

detector in a reasonable timeframe

as well as buildup of interfering co

processing many samples. Figure 3

optimized conditions, which resulte

peak areas for internal standards in

from optimized conditions, which r

reproducibility (RSDs < 10%).

Methods

Off-Line Sample Preparation

ChromSystems 6PLUS1® ISD multilevel calibrator set and MassCheck® whole-

blood controls as well as in-house test samples were mixed with aqueous zinc

sulfate solution and then with methanol containing internal standards:

Tacrolimus-

(Toronto Research Chemicals, Canada) and D

-Cyclosporin A

(Alsachim, France). After centrifugation, supernatants were harvested into glass

autosampler vials.

On-Line Sample Preparation & Liquid Chromatography (SPLC)

In each channel, 20 µL injections of supernatants were extracted with a Thermo

Scientific™ TurboFlow™ Cyclone-P™ TurboFlow column (0.5 x 50mm) using a

mobile phase mixture of 7:3

water:methanol

containing 10 mM ammonium

formate and 0.05% formic acid at 1.5 mL/min. A slow flow of methanol eluted

extracted ISDs, which merged with a higher flow of a 7:3 water: methanol

mixture, to transfer and focus the ISDs to an Accucore C8, 2.6 um, 3.0 x 30 mm

HPLC column, which was maintained at 70 °C by the built-in heater. The ISDs

were separated from matrix interferences and eluted to the heated electrospray

ionization (HESI) source by a gradient of increasing methanol. Figure 2 shows this

focus method.

ysed

MW:

Sirolimus

MW: 914.17

FIGURE 2. Summary of SPLC Focus Method.

Solvents:

Water + 10mM NH

OOCH +

0.05% HOOCH

Methanol + 10mM NH

OOCH

+ 0.05% HOOCH

45% Acetonitrile + 45%

Isopropanol + 10% Acetone

Total solvent consumption is

3.37 mL A, 3.25 mL B, 1.5 mL C

for each injection.

Dioctylphthalate:

Lyso-Phosphotidylcholine;16:0:

Lyso-Phosphotidylcholine;18:0:

Phosphotidylcholine;38:6:

.Tacro:

Siro:

Evero: