AI-64310-CRFT

Therapeutic Drug Monitoring of 9 New Anticancer Agents by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Analyte

Retention

time

Precur

Ion

Bortezomib

3.11

367.

Dasatinib

3.01

488.

Erlotinib

3.12

394

Imatinib

2.96

494.

D8-Imatinib

2.96

502.

Lapatinib

3.28

581.

Nilotinib

3.26

530.

Sorafenib

3.59

465.

Sunitinib

3.06

399.

Vandetanib

2.99

475.

Introduction

The treatment of some cancers has shifted from conventional chemotherapy drugs

to chronic treatment with molecular targeted therapies. Targeted therapies include

drugs such as Tyrosine kinase inhibitors (eg: Imatinib, Dasatinib, Nilotinib, Sunitinib,

Sorafenib, Vandetanib, Lapatinib, Vatalanib and Erlotinib) that present better

efficiency and lower side effects than conventional anti cancer drugs.

Goal

The goal was to develop and validate a fast, specific and sensitive method for the

quantitation of Tyrosine kinase inhibitors (eg: Imatinib, Dasatinib, Nilotinib, Sunitinib,

Sorafenib, Vandetanib, Lapatinib, Vatalanib and Erlotinib) in plasma samples using

liquid chromatography coupled to mass spectrometry.

Method

Equipment

The liquid chromatography consisted of a Thermo Scientific (Courtaboeuf, France)

Accela

autosampler and a quaternary pump. Separation was performed on an

Hypersil Gold

PFP (2.1x100 mm; pore size 1.9 µm) analytical column placed in a

thermostated column heater at 50

C. The chromatographic system was coupled to

a triple quadrupole (TSQ) Quantum Ultra mass spectrometer (MS) from Thermo

Fisher Scientific, Inc. equipped with an Ion Max electrospray ionization (ESI)

interface and operated with XCalibur 2.07 software (Thermo Fisher Scientific,

Courtaboeuf France).

LC conditions

he mobile phase used for chromatography was 10 mM ammonium formate buffer

containing 0.1% (v/v) formic acid (solution A), and acetonitrile with 0.1 % (v/v)

formic acid (solution B). The mobile phase was delivered using the following

stepwise gradient elution program: initial conditions of 95:5 (A:B) maintained for 0.5

minutes, run from 95:5 (A:B) at 0.5 minutes to obtain 5:95 (A:B) at 2 minutes,

conditions 5:95 (A:B) maintained from 2 to 4 minutes, wash using 100% of phase C

from 4 to 7 minutes, run from 5:95 (A:B) at 7.01 minutes to 95:5 (A:B) at 7.5

minutes, conditions 95:5 (A:B) maintained to 10 minutes for equilibration. The flow

was 300 µl/min. The thermostated column heater was set at 50

C and the

autosampler was maintained at 4

MS conditions

The MS conditions were as follows: ESI in positive mode, capillary temperature:

325

C: 10V, tube lens voltages range: reported in Table 1; spray voltage: 3500 V;

sheath and auxiliary gas (nitrogen) flow-rate: 45 and 25 (arbitrary units),

respectively. The Q2 collision gas (argon) pressure was 1.5 mTorr. Data are

acquired in selected reaction monitoring (SRM) mode.

The SRM transitions, the collision energy and ions ratio for each analyte are

reported in Table 1.

Sample preparation

Calibrators and QCs preparation

For each drug, two primary stock solutions were prepared at 1 mg/ml by dissolving

10-mg base equivalent aliquots of each drug in 10 mL of methanol. Stock solutions

were mixed together in order to get 2 methanolic working solutions containing all

drugs at 100 µg/mL, 10 µg/mL and 1 µg/mL.

The first set was used for the preparation of the calibration standards ranging from

2 to 250 ng/mL for BORT, DASA and SUNI and from 50 to 3 500 ng/mL for the

others drugs. The second set was used for the preparation of the 5 quality controls

(QCs): 7, 75, 150, 750 and 1 500 ng/mL for each drug.

Only QCs at 7, 75 and 150 ng/m

at 75, 150, 750 and 1 500 ng/m

imatinib, internal standard (IS) sto

chemical in 2 ml of methanol. Pl

control (QC) samples were pre

working solution to blank plasma.

Table 1:

Retention time, precur

precursor molecular ion/product i

lens voltage (TL)/collision energy(

Plasma sample extraction pro

Aliquots of 50

l of the plasma unk

placed in appropriate labeled 1.5

acetonitrile containing 20 ng/mL I

sample was centrifuged at 6 000

supernatant were diluted two-fold

ratio. After capping and vortexing,

tray that was maintained at +4

injected into the HPLC system.

Results

Chromatograms

The proposed method enables th

TKIs in 50

L-plasma aliquots by l

Typical chromatographic profiles o

shown in Fig. 2.

Internal standard, calib

quantification

Imatinib-D8 was used as IS wit

negligible memory effect. Calibrati

were best described by 1/x weight

TKI to IS

versus

the concentrations