Page 90 - CRFTApplication Compendium_090613

4 Development and Validation of Methods for Chemotherapy Drugs on the New Prelude SPLC LC-MS/MS System
e
Compound name Method Range (ng/mL) Linearity (r2 ) Recovery
Busulfan 20-2000 0.995-0.998 89.4-93.5
Docetaxel 5-1000
Imatinib 10-2000 0.991-0.998 92.0-110.2
Methotrexate 10-750 0.992-0.998 102.0-110.2
TABLE 1. Method Range, Linearity, and Recovery.
TABLE 2. Intraday Accuracy and Precision.
TABLE 3. Interday Accuracy and Precision.
Low QC Mid QC High QC Low QC Mid QC High QC
Busulfan 0.56-16.5 0.17-8.17 0.22-5.83 1.1-10.9 1.8-3.3 1.6-4.2
Docetaxel
Imatinib 1.0-9.5 0.3-9.8 0.0-11.7 1.0-1.9 1.1-7.4 1.3-6.2
Methotrexate 0.13-18.5 0.12-9.74 0.10-10.5 3.3-7.5 0.6-5.9 2.8-7.8
Intraday Accuracy Intraday Precision (%RSD)
Compound name (%Difference from Theoretical)
Low QC Mid QC High QC Low QC Mid QC High QC
Busulfan 4.76 0.35 3.85 5.6 5.4 3.9
Docetaxel
Imatinib 11.00 1.33 3.74 4.0 2.0 5.9
Methotrexate 2.33 2.80 0.48 5.5 2.8 7.5
Interday Accuracy Interday Precision (%RSD)
Compound name (%Difference from Theoretical)
0 100 200 300 400 500 600 700 0.0
0.5
1.0
1.5
2.0
2.5
3.0 Methotrexate
Concentration (ng/mL)
Intensity (mv)
0.5 1.0 1.5 2. 0
5000
10000
15000
20000
Busu
Time (min)
Intensity (mv)
0.5 1.0 1.5 2. 0
2000
4000
6000
8000
10000
12000
14000
Time (min)
Intensity (mv)
FIGURE 2. Standard Curves for Each Compound Tested Using a Prelude SPLC
System with TSQ MS
FIGURE 3. Representative
Tested Using a Prelude S
Compound name Bench Top Stability Autosampler Stability Selectivity (% of LOQ)
Busulfan 104.2-121.5 104.1-111.3 0.01-7.08
Docetaxel
Imatinib 98.0-105.1 88.2-96.5 N/A
Methotrexate 102.4-102.9 101.4-102.5 2.14-5.50
TABLE 4. Bench Top Stability, Autosampler Stability, and Selectivity.
Discussion
LC/MS methods for these
liquid extraction and longer
liquid-liquid extraction are t
disposal point of view. On-li
and variability of the metho
Prelude SPLC system redu
allows for higher sample th
capabilities of the Prelude
the solvent consumption, l
The Prelude SPLC system
down time, and operating c
sample clean-up prior to H
designed to deliver the vol
single push of the piston. T
seals and check valves be
several hundred if not thou
required on traditional HPL
therefore, syringe pumps a
Prelude SPLC system syri
the Prelude SPLC system
mobile phases possible. Th
reduced, resulting in shorte
0 500 1000 1500 2000 0
5
10
15
20
25
30 Busulfan
Concentration (ng/mL)
Peak Area Ratio (Sample/IS)
0 200 400 600 800 0
5
10
15
20
25
Imatinib
Concentration (ng/mL)
Peak Area Ratio (Sample/IS)
FIGURE 4. Comparison of
SPLC System with TSQ M
0
Prelude SPLC
Conventional HPLC Sample
Clean-up
Sample
Clean-up
HPLC
pump 1
MS
waste
HPLC
pump 2
TFC column
Analyticalcolumn
plug
200 µ L loop
plug
HPLC
pump 1
MS
waste
HPLC
pump 2
TFC column
Analyticalcolumn
plug
200 µ L loop
plug
HPLC
pump 1
MS
waste
HPLC
pump 2
TFC column
Analyticalcolumn
plug
200 µ L loop
plug
(A)
(B)
(C)
med for system verification from three
on three different days. Interday and intra-
d at concentration ranges of 20–2000 ng/mL
ng/mL for docetaxel, and 1–750 ng/mL for
lues less than 15% for all compounds
eoretical value for all the methods. The
s ranged from 0.991 to 0.998, showing
es. All the analytes passed carryover, bench
criterion. Recoveries, including matrix
Tables 1–4. Figure 2 depicts representative
resentative chromatograms at the lower limit
own in Figure 3.
lower void volumes of the Prelude SPLC
in Figure 4 for Docetaxel. The same mobile
on. When using on-line clean-up, the
ause they are dependent on the
n of other steps in the process are related
the column. The sample clean-up and
atography, and therefore, the time for those
lumn cleaning and re-equilibration steps
nsfer step was 75 seconds vs. 60 seconds
up and equilibration steps were reduced
in run time of 29% (5:15 minutes to 3:45
t consumption by 33%.
FIGURE 1. Valve positions for On-line
Sample Clean up and Analytical
Separation.
hermo Scientific™ TSQ Vantage™ triple-
with a heated electrospray ionization
ted reaction monitoring (SRM).
tific™ LCQUAN™ software.