The Characterization of Ibuprofen Metabolites Isolated from Urine by Tandem Quadrupole (MS/MS) Directed Purification
Paul M. Lefebvre1, Ian Wilson2, Paul Rainville1, Chris Stumpf1, Robert S. Plumb1, Ronan Cleary1, Warren B Potts III1
1
Waters Corporation, Milford, MA; 2Astra-Zeneca, UK
POSITIVE ION COLLECTION
UPLC QTOF RESULTS
To demonstrate the benefit of exact mass for metabolite fraction
characterization, a peak with the mass equivalent to the ibuprofen sulfate
metabolite (m/z = 302) was detected and isolated by the positive ion
MS data.
Each collected fraction was injected to determine the exact mass of
collected peak and also to evaluate the purity. The data below shows the
analysis of a fraction for each metabolite.
100
100
1.14
Positive TIC
Separation
Water w/10 mM ammonium formate: Acetonitrile, 20 ml/min total flow
gradient. 0 – 5 minutes: 5%, 5 – 35 minutes: 5 – 60% B, 35 – 35.5
minutes: 60 – 95% B, 35.5 – 39.5 minutes: 95% B, 39.5 – 40 minutes:
95 – 5% B, 45 minutes end
24.04
100
IBUPROFEN FRAGMENTATION
The fragmentation of the ibuprofen gluceronide metabolite reported by
Kearney et al. [4], is shown below. The major ions generated include
205, 175, 193, and 161.
6.25
Negative BPI
CONCLUSIONS
1.32
1.72
Isolation
100
2.15
24.68
26.19
12.42
25.09
18.07
0
22.25
22.50
22.75
23.00
23.25
23.50
23.75
24.00
24.25
24.50
24.75
25.00
25.25
25.50
25.75
26.00
26.25
26.50
26.75
27.00
27.25
27.50
27.75
28.00
28.25
28.50
MS Detection
Electrospray positive and negative, 3 kV capillary voltage, 30 V cone
voltage.
8.58
•
Collection by nominal mass does present the possibility to collect
non-metabolite related isobaric interferences. However, UPLC
exact mass fraction analysis provide rapid way to eliminate the
undesired fractions.
•
Another option for improving the collection selectivity would be to
trigger fractionation from MS/MS functions (MRM, Constant
Neutral Loss, and Precursor Ion) with the Quattro micro tandem
quadrupole mass spectrometer.
%
%
14.36
16.73
10.22
10.81
27.82
16.17
18.44
3.93
36.57 36.94
22.76
Ibuprofen gluceronide metabolite fragmentation.
28.49
11.43
37.25
6.53
8.96
5.99
19.43
8.21
0
100
21.14
24.03
22.15
40.52
NEGATIVE ION COLLECTION
150
200
250
300
350
400
m/z
MASS ACCURACY
29.56
6.33
0
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
42.00
44.00
Time
0.50
The positive ion data shows that a fraction was collected for the target
mass. Again, the collection threshold could have been reduced to collect
the smaller peaks.
15.36
26 .1 2
100
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
Time
9.50
97
FRACTION CHARACTERIZATION
2 8.47
Gluceronide Parent
382.1550
382.1544
-1.6
C19H26O8
Gluceronide Fragment 1
175.0245
175.0243
-1.1
C6H8O6
Gluceronide Fragment 2
113.0237
113.0239
1.8
C5H6O3
OH Gluceronde Parent
397.1487
397.1499
3.0
C19H26O9
OH Gluceronde Fragment 1 175.0238
175.0243
2.8
C6H8O6
100
19 .42
18.97
OH Gluceronde Fragment 2 113.0239
113.0239
0
C5H6O3
2 4.86
12.50
13.50
14.00
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
18.50
19.00
19.50
20.00
20.50
21.00
21.50
22.00
Time
0
24.00
24.50
25.00
25.50
26.00
26.50
27.00
27.50
28.00
28.50
29.00
29.50
30.00
30.50
31.00
31.50
32.00
32.50
33.00
Tim e
Negative TIC
1.06
2.42
26.12
System
Waters ACQUITY UPLC™
System with a 2.1 x100,
1.7 μm ACQUITY UPLC
BEH C18 column @ 40°
C. The total flow is
directed into the Q-TOF
Premier Mass
Spectrometer
0
100
150
200
250
300
350
400
5.01
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
This table illustrates that the observed mass for both the parent and
fragment ions correspond to the theoretical values . Having this accuracy
allows for increased confidence in the elemental composition calculation
to track metabolic transformation of the target drug.
9.75
9.26
0.50
%
m/z
0.39
-3
12.34
The improved speed and resolution of the UPLC analysis provides
rapid, high quality fraction analysis results. The observed purity
for the collected fractions are greater than 90% pure.
•
The potential ibuprofen sulfate metabolite was not found and the
collected fraction is an isobaric interference.
•
Using high / low collision energy switching allows for simultaneous
collection of both LC/MS and LC/MS/MS of data.
9.50
39.58
4.30
16.20
16.74
37.22
18.43
9.00
6.04
25.57
19.42
8.18
Waters ACQUITY UltraPerformance LC™ System with a Q-Tof Premier™.
27.27
24.60
7.54
11.27
13.7014.15
21.03
23.04
30.40
35.62
0
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
30.00
32.00
34.00
36.00
38.00
40.00
IBUPROFEN SULFATE
Analysis of the first hydroxy—gluceronide metabolite collected (preparative retention
time = 15.36 minutes)
28.43
42.00
44.00
The negative ion data shows that 3 fractions were collected for both
metabolites. Note that for each extracted ion trace there are additional
peaks observed but were not collected. The collection threshold can
be reduced to collect these peaks. Since these ions were collected as
a “positive control”, 3 fractions are sufficient to demonstrate the
functionality.
Time
Separation
Water w/10 mM ammonium formate: Acetonitrile, 0.6 ml/min total flow
gradient. 0 – 4 minutes: 0 – 20% B, 4 – 9 minutes: 20 – 95% B, 9-10
minutes: 95% B, 10-12 minutes: 95 – 5% B, 45 minutes end,
MS Detection
Electrosray positive or negative, 2800V, capillary voltage, 80V cone
voltage, 5 or 25 eV collision cell voltage.
The chromatographic results show that the collected fractions are greater
than 90% pure. The data was acquired using high / low collision energy
switching to simultaneously collect both LC/MS and LC/MS/MS data.
This allows for exact mass MS/MS experiments to be performed on the
fly and eliminates the need for additional injections for separate MS and
MS/MS experiments.
REFERENCES
1. Ismail IM. Dear GJ.. Xenobiotica. 29(9):957-967, 1999
2. Dear GJ, Mallett DN and Plumb RS. LC-GC Europe 14(10) 616-624, 2001
3. Lefebvre et al. “The Application of MS/MS—Directed Purification to the
Identification of Drug Metabolites in Biological Fluids” Waters Application Note
# 720001129EN)
Time
15.32
2.00
•
4. Kearney et al. “Exact Mass MS/MS of Ibuprofen Metabolites using Hybrid
Quadrupole-Orthogonal Time-of-Flight Mass Spectrometry Equipped with a
Lockspray Source”, Waters Corporation Application Note # 720000706EN.
To determine if the collected fraction for m/z 302 is actually the
ibuprofen sulfate metabolite, exact mass of the target was obtained.
356.2434
302.2334
100
%
1.24
13.00
%
17.71
14.83
0
%
27.29
18 .1 0
100
Characterization
%
%
Formula
4.72
Negative BPI
28.11
16.75
Obs. Mass Calc. Mass
Mass
(M–H)
(M-H)
Error (ppm)
Name
15 .48
16.17
The table below shows the calculated mass accuracy for the parent and
fragment ions for both negative ion metabolites.
Analysis of the first gluceronide metabolite collected (preparative retention time =
26.12 minutes)
m/z = 382
m/z = 398
100
2.00
-0
The known gluceronide and hydroxy-gluceronide metabolites are
detected in negative ion mode [4]. The respective molecular ions
monitored are 398 and 382. Fractionation will occur each time the
intensity of one of these masses rises above a set threshold.
28 .6 2
Waters® Mass—Directed AutoPurification™ System *Quattro micro™ Mass
Spectrometer used in place of the ZQ™ as shown.
Isolation of metabolites using nominal mass fraction collection
provide a useful way to collect LC fraction for further use and
analysis, compared to the previous non-specific collection trigger.
4.39
18.42
METABOLITE ISOLATION
•
Time
13.38
24.67
In this poster we will show the characterization of
the ibuprofen metabolites isolated by MS/MS
directed purification. We will subject the isolated
fractions to the same the analytical tests typically
performed, including exact mass MS and MS/MS.
The characterization will help determine if the
ibuprofen sulfate metabolite is present and isolated.
Another indication that this fraction is not related to ibuprofen is the
lack of the common fragment ions observed in the other metabolites
m/z = 302
15.27
9.31
A more recent approach was to use MS/MS directed
purification of the LC stream. [3] In demonstrating
this approach, the metabolites of ibuprofen were
isolated from human urine. Fractionation was
triggered based on peaks detected in the precursor
ion trace of common ibuprofen fragment. One
fraction detected has a corresponding mass,
equivalent to the ibuprofen sulfate metabolite. This
sulfated-ibuprofen metabolite has not previously
been detected or isolated.
The exact mass for the fraction is 302.2334. The theoretical mass for
the ibuprofen sulfate metabolite is 302.0824 (C13H17O6S). The
calculated mass accuracy is 500 ppm. Therefore, it is not possible for
the collected fraction to be a sulfate metabolite.
%
An essential part of the drug discovery and
development process is the identification and
characterization of drug metabolites. This has
traditionally been achieved using liquid or gas
chromatography coupled with mass spectrometry.
[1-2] Often times, however, the metabolites require
additional analysis and need to be isolated from
biological fluids. The traditional approach has been
to fractionate the LC eluent by time and then
analyze each fraction to find the metabolites. The
problem with this approach is that it is time
consuming and requires the chromatographic
retention time to be reproducible with experimental
variations, i.e. column life, temperature and system
to system variation.
System
Waters® 2525 Binary Gradient Module, 2767 Sample Manager,
Column Fluidics Organizer with a Sunfire™ C18 5μm 19 x 100 mm
column. Flow is split 1:1000 with a LC Packings Splitter. 99.9% of the
flow passes to the fraction collector on the 2767. The remaining 0.1% is
combined with a 1ml/minute makeup of 50:50 H2O w/10mM
ammonium formate: ACN using a 515 makeup pump. This flow is then
split 80% to a 2996 PDA detector and 20 % to the Quattro micro Mass
Spectrometer equipped with an ESCi™ Multi-Mode Ionisation Source.
%
INTRODUCTION
357.2484
243.1572
0
50
100
150
200
250
378.2280
300
350
400
450
m/z
MS/MS of the m/z = 302 triggered fraction
720001537EN
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