Transcript and dehydro-aripiprazole

Use of High Resolution Mass Spectrometry (HRMS) to Solve Severe Issues
Due to Isotopic Distribution in Regulated Bioanalysis
Richard Lavallée, Nicolaos Soilis, Jean-Nicholas Mess and Fabio Garofolo*
Algorithme Pharma Inc., Laval (Montreal), QC, Canada
Figure 1: Aripiprazole and Dehydro Aripiprazole
Aripiprazole
C23H27Cl2N3O2
m/z 448.1559
Dehydro Aripiprazole
C23H25Cl2N3O2
m/z 446.1402
CHROMATOGRAPHY
• Agilent Technologies Series 1100
• Ascentis Express C18, (30 X 2.10) mm, 2.7µm
• 10mM HCOONH4 pH 4.0 : MeOH 45:55% v/v
• 1.50 minutes run time
DETECTION
• AB SCIEX API5000TM operated in MRM mode
• TripleTOFTM5600 operated in full scan TOFMS mode
at 30K resolution
• Electrospray positive ionization for both systems
Drug name
Drug MRM
Similarly, the isotopic contribution from lamotrigine to
lamotrigine-13CD3 was determined to be 13.21% on a
triple quadrupole while it decreased by 13 times (1.00%)
in HRMS (10mDa XIC). Narrowing the mass extraction
window to 5 mDa further decreased even further the
contribution to 0.31%.
riluzole-13C15N2,
1.4e6
Contribution
322.07/212.1
Clopidogrel-D3
325.07/215.1
3.57
Lamotrigine
256.0/211.0
Lamotrigine-13CD3
260.0/215.0
13.21
Lamotrigine
256.0/211.0
Lamotrigine-13C3
259.0/214.0
7.54
Riluzole
235.01/138.0
Riluzole-13C15N2
238.01/141.1
0.41
M
a
x. 1
.4
e6 cps.
0.71
%
1.2e6
1.0e6
Blank
8.0e5
6.0e5
4.0e5
2.0e5
Table 2: Isotopic contribution at 1:1
TripleTOFTM5600 high resolution MS
Compounds
Clopidogrel vs
Clopidogrel-D3
Lamotrigine vs
Lamotrigine-13CD3
Lamotrigine vs
Lamotrigine-13C3
Riluzole vs
Riluzole-13C15N2
using a
Extraction window
% Contribution
2mDa
0.40%
5mDa
0.49%
10mDa
0.65%
25mDa
1.98%
50mDa
7.49%
2mDa
0.24%
5mDa
0.31%
10mDa
1.00%
25mDa
1.64%
50mDa
7.24%
2mDa
1.98%
5mDa
2.22%
10mDa
3.70%
25mDa
9.88%
50mDa
12.21%
2mDa
0.48%
5mDa
0.66%
10mDa
0.50%
25mDa
0.71%
50mDa
0.73%
0.0
A calibration curve of aripiprazole and quality control (QC)
samples (fortified with and without dehydro-aripiprazole at
equimolar concentration) were extracted and injected on
an API5000TM. The chromatographic conditions were
adjusted in order to have co-elution of aripiprazole and
dehydro-aripiprazole (Figure 2). The data was acceptable
in term of linearity (linear weighted 1/x2 regression) and
showed precision and accuracy for the QC samples
without dehydro-aripiprazole; however, the low and high
QC spiked with dehydroaripiprazole had nominal values of
148.3% and 145.8%, respectively, due to isotopic the
contribution of dehydro-aripiprzole (Table 3).
0.4
0.6
0.8
Tim
e, min
1.0
1.2
1.4
LLOQ
Concentration (ng/mL)
Curve
Code
Line #
IOO724.02
18
10
13
22
Mean
S.D.
N
% C.V.
% Nominal
QC1(Aripiprazole)
QC2(Aripiprazole)
QC3(Aripiprazole)
Line #
Line #
Line #
1.500
10.000
120.000
1.418
1.453
1.558
1.488
1.479
0.060
4
4.0
98.6
16
14
19
21
9.967
9.745
10.196
10.525
10.108
0.333
4
3.3
101.1
23
27
28
---
118.137
115.015
117.524
SLP
116.892
1.654
3
1.4
97.4
QC4(Aripiprazole+Dehydro
QC5(Aripiprazole+Dehydro
Aripiprazole)
Line #
Aripiprazole)
1.500
120.000
9*
29*
32*
35*
2.170
2.138
2.289
2.304
2.225
0.084
4
3.8
148.3
15
30
33
---
173.043
177.128
174.665
SLP
174.946
2.057
3
1.2
145.8
Figure 5: Calibration curve of aripiprazole using a
TripleTOFTM5600 high resolution MS
18.00
Calibrant
16.00
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
14.00
On the HRMS system, the resolution of 30K was still too low
to allow full resolution of aripiprazole from dehydroaripiprazole 37Cl isotope (Figure 3). Nevertheless, using a
narrow extraction window of 10mDa, the isotopic contribution
was kept below 5%.
12.00
10.00
Nominal
(ng/mL)
0.50
1.00
2.50
6.00
15.00
30.00
60.00
110.00
130.00
150.00
Back-Calculated
(ng/mL)
0.51
1.02
2.17
5.29
15.53
31.41
61.57
114.52
134.08
154.31
Deviation
(%)
2.6
1.6
-13.4
-11.8
3.5
4.7
2.6
4.1
3.1
2.9
8.00
6.00
Linear (weighted 1/x2)
r2 = 0.9945
4.00
2.00
The same samples previously injected on the API5000TM
were re-analyzed by HRMS on the TripleTOFTM5600. The
data presented in Table 4 demonstrate that HRMS was able
to solve a bioanalytical issue where the triple quadrupole
failed. As per quantification data, the system was proven to
be sensitive and linear (Figures 4 and 5)
RESULTS: ISOTOPIC CONTRIBUTION OF DRUG AND
METABOLITE
Using LC-MS/MS on a triple quadrupole, chromatographic
separation is usually needed when metabolites can
isotopically contribute to the parent drug. This is the case
for aripiprazole and dehydro-aripiprazole (Figure 1) where
dehydro-aripiprazole
theoretically
contributes
to
aripiprazole at 68.5% (unit resolution MS).
0.2
Table 3: Precision and accuracy of aripiprazole in
presence of dehydro-aripiprazole using a API5000TM triple
quadrupole
0.0
+TO
FM
S: 1.9831 to 2.0819 m
in fromSam
ple 17 (R
EF-1 (Drug) 1µL) of TO
FM
S ESI+.w
iff different calibrations (DuoSpray ())
40.0
60.0
80.0
100.0
120.0
140.0
160.0
Table 4: Precision and accuracy of aripiprazole in
presence of dehydro-aripiprazole using a TripleTOFTM5600
high resolution MS
Concentration (ng/mL)
Curve
Code
Line #
M
ax. 4.3e5 cps.
QC1(Aripiprazole)
QC2(Aripiprazole)
QC3(Aripiprazole)
Line #
Line #
Line #
1.500
10.000
120.000
QC4(Aripiprazole+Dehydro
QC5(Aripiprazole+Dehydro
Aripiprazole)
Line #
Aripiprazole)
1.500
120.000
448.16
1.4e4
1.2e4
20.0
Concentration (ng/mL)
Figure 3: Overlay of HRMS scans of aripiprzole (blue) and
dehydro-aripiprazole (red)
Aripiprazole
Dehydro-aripiprazole
IOO724.03
1.0e4
Mean
S.D.
N
% C.V.
% Nominal
450.15
8000.0
6000.0
17
9
12
21
1.324
1.289
1.384
1.386
1.346
0.047
4
3.5
89.7
15
13
18
20
9.164
9.403
9.613
9.740
9.480
0.253
4
2.7
94.8
22
16
26
27
122.444
126.797
122.842
125.241
124.331
2.057
4
1.7
103.6
8
28
31
34
1.416
1.410
1.416
1.393
1.409
0.011
4
0.8
93.9
10
14
29
32
129.025
127.922
127.100
126.670
127.679
1.036
4
0.8
106.4
4000.0
2000.0
0.0
446
448
450
m/z, Da
452
454
+TO
FM
S: 1.9831 to 2.0819 m
in fromSam
ple 17 (R
EF-1 (Drug) 1µL) of TO
FM
S ESI+.w
iff different calibrations (DuoSpray ())
M
ax. 4.3e5 cps.
448.16
1.4e4
1.2e4
Aripiprazole
1.0e4
Intensity, cps
However, for riluzole and
where isotopic
contribution was already low on a triple quadrupole
(0.41%), HRMS did not succeed to lower this value
(0.50% at 10 mDa XIC). Data are from isotopic
distribution experiments are presented in Table 1 and
Table 2.
MRM (IS or MET)
Clopidogrel
RESULTS: ISOTOPIC CONTRIBUTION OF DRUG
AND INTERNAL STANDARD
Three different drugs and their corresponding stable
labeled internal standards were used to evaluate if HRMS
can solve typical interference from isotopic distribution
observed on triple quadrupole mass spectrometer. The
isotopic contribution from clopidogrel to clopidogrel-D3
was determined to be 3.57% when using a triple
quadrupole. This can be explained since unit resolution
cannot distinguish between clopidogrel-13C37Cl isotope
(m/z 325.0673) and clopidogrel-D3 (m/z 325.0857).
However, when analyzed under HRMS on the
TripleTOFTM5600 (30K resolution, 10mDa XIC), this
contribution dropped by a factor of 5.5 times (0.65%).
Internal Standard (IS)
or metabolite (MET)
Intensity, cps
Interference due to isotopic distribution is a common issue
bioanalytical scientists must deal with during LC-MS/MS
method development. As an example, clopidogrel
contributes to its internal standard clopidogrel-D3 at 7%
(theoretical value, unit resolution MS). Therefore, the ratio
of drug and IS must be carefully adjusted to avoid any
bias in quantification. Furthermore, some dehydrogenated
metabolites may interfere with the parent drug
quantification due to isotopic distribution. This is the case
for dehydro-aripiprazole which contributes to aripiprazole
at 68.5% (theoretical value, unit resolution MS). In most
cases, specific action must be taken to circumvent these
issues, which is time consuming. In this study, HRMS will
be evaluated as a powerful tool to easily solve
bioanalytical issues caused by isotopic distribution.
SAMPLE EXTRACTION
• Aripiprazole, dehydroaripiprzole and aripiprazole-D8
were extracted by LLE from human plasma samples.
• Analytical range: 0.500 to 150.00 ng/mL
XIC of +T
OF M
S: 4
48.1 to 448
.1
5 Da fromSam
ple 1 (Q
C0
3-52
) o
f Re
-inj-02
9.wiff (D
uoSp
ray ())
Figure 4: Representative chromatograms of extracted
blank and LLOQ (0.500 ng/mL) of aripiprazole using a
TripleTOFTM5600 high resolution MS
Peak Area Ratio
INTRODUCTION
SOLUTION PREPARATION
For isotopic contribution determination, all drugs and
stable labeled internal standards solutions were
prepared separately at a concentration of 100ng/mL.
Figure 2: Representative chromatogram of aripiprazole
(blue), aripiprzole-D8 (red) and dehydro-aripiprzole (green)
Table 1: Isotopic contribution at 1:1 using a
API5000TM triple quadrupole mass spectrometer
Intensity, cps
• Purpose
– To demonstrate the advantages of using HRMS over
triple quadrupole mass spectrometers to solve
bioanalytical issues related to isotopic distribution in
a regulated environment.
• Method
– Isotopic contribution of drugs on their stable labeled
internal standards was evaluated in solution
prepared at equimolar concentrations.
– Aripiprazole precision and accuracy was evaluated
in plasma extracts spiked with and without dehydroaripiprazole.
• Results
– HRMS was successful in reducing the isotopic
contribution of drugs to their stable labeled internal
standards.
– Quantification of aripiprazole on a triple quadrupole
mass spectrometer showed inaccuracy for samples
containing dehydro-aripiprazole.
– Accuracy of these samples was maintained on the
Q-TOF due to its high resolving power.
METHODS
Intensity (cps)
OVERVIEW
8000.0
Dehydro-aripiprazole
37Cl isotope
6000.0
CONCLUSION
In conclusion, we have shown that HRMS can simply and
efficiently solve typical bioanalytical issues caused by isotopic
contribution. Moreover, the HRMS was able to deliver
acceptable quantification performance in regards to sensitivity,
linearity and precision and accuracy.
4000.0
2000.0
0.0
448.05
448.10
448.15
448.20
m/z, Da
448.25
448.30
* CORRESPONDING AUTHOR