Page 88 - CRFTApplication Compendium_090613

2 Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-MS/MS System
Overview
Purpose: To verify research methods to quantify a variety of chemotherapeutic drugs
on the new Thermo Scientific™ Prelude™ sample preparation-liquid chromatography
(SPLC) system, coupled to a Thermo Scientific TSQ mass spectrometer. The
LC/MS/MS platform reduces solvent consumption, requires less maintenance, and is
easier to use then traditional systems.
Methods: Prelude SPLC, Turbulent Flow Chromatography, LC/MS/MS,
chemotherapeutics
Results: Methods for the chemotherapeutic drugs Busulfan, Docetaxel, Methotrexate
and Imatinib, were validated using a Prelude SPLC system and TSQ mass
spectrometer platform.
Introduction
Bioanalysis using LC-MS/MS can be difficult due to complex sample preparation and
errors in sample handing, which can lead to variability. The use of liquid
chromatography-tandem mass spectrometry (LC-MS/MS) to quantify
chemotherapeutic drugs (i.e., busulfan, methotrexate, imatinib, and docetaxel) is
common practice. We demonstrate the application of Prelude SPLC system in
developing a faster, more reproducible and lower solvent consuming methods for
quantification of chemotherapeutic drugs.
Methods
Sample Preparation
The samples in every method were prepared in human plasma . Sample preparation
consisted of protein precipitation with organic solvents that contained internal standard,
vortexed, followed by centrifugation. The supernatant was removed and injected into
the Prelude SPLC system for analysis.
Liquid Chromatography
On-line sample clean-up was performed by 0.5x50 mm Thermo Scientific™
TurboFlow™ columns and analytical separation was performed with Thermo
Scientific™ Accucore 50x2.1mm, 2.6 µm particle size columns. For iminatib, busulfan,
and methotrexate, the TurboFlow HTLC-C18 XL column and Accucore PFP analytical
column were used. For docetaxel, the TurboFlow HTLC-C8 XL column and Accucore
C8 analytical column were used.
Mobile phases were (A) 10 mM ammonium formate, 0.05% formic acid in water;
(B) 10 mM ammonium formate, 0.05% formic acid in methanol; and (C) 45/45/10
acetonitrile/isopropanol/acetone for iminatib, busulfan, and methotrexate. Docetaxol
used acetonitrile for mobile phase B. All run times were 4 minutes or less and
multiplexed to 2 minutes per sample. These methods consumed less than 3 mL of
mobile phase per injection.
The Prelude SPLC system us
method for on-line clean-up,
1. The first step (Figure 1A – l
load the sample with aqueou
onto the TurboFlow column u
conditions. Under turbulent fl
large molecule (>15 kDa) can
the stationary phase and are
while the analytes of interest
the column. Since the majorit
interferences are from the ma
analyte of interest is removed
during step 1. Once the samp
from the matrix, the valves s
TurboFlow column is back-flu
solvent stored in the loop of t
from the previous injection),
analyte of interest from the T
to the analytical column (Figu
step). In order to focus the an
analytical column, the flow fro
column is teed to a valve with
from a second pump. The se
the analytical column provide
chromatographic peaks and f
any interferences. The elution
switches the second valve so
now eluted to the mass spect
loop can be filled with the cor
organic solvent for the next s
steps are complete, the valve
the loading position where th
cleaned and equilibrated for t
injection.
Structures of Chemotherapeutic Drugs
Docetaxel
Iminatib
Methotrexate
Busulfan
Results
Accuracy and precision experi
separate preparations of calibr
day accuracy and precision re
for busulfan, 10–2000 ng/mL f
methotrexate. The method pre
tested. Additionally, accuracy
correlation coefficient values f
linearity throughout all concent
top stability, autosampler stabil
effects, were all ~>90%. Data
standard curves for each com
of quantitation (LLOQ) for eac
The improvement in run times
system verses a conventional
phases and columns were use
duration of certain steps cann
chromatographic separation n
to how long it takes for solvent
sample elution steps are depe
steps remain the same. Howe
can be reduced. On a conventi
on the Prelude SPLC system.
from 150 to 60 seconds. The r
minutes). A shorter run time al
Mass Spectrometry
Detection of eluting analytes
stage quadrupole mass spect
(HESI II) probe in positive ion
Data Analysis
Quantitation was calculated