Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic
Sample Clean-up Method
Jessalynn P. Wheaton, Erin E. Chambers, John Martin and Kenneth J. Fountain
A P P LIC AT ION BEN EFIT S
n
Simple, universal sample prep protocol
n
Speeds up workflow through direct
injection of eluates
n
n
n
INT RO DUC T ION
Drug discovery is a vital segment of pharmaceutical research where vast numbers
of compounds are screened to determine therapeutic efficacy, activity, and ADME
properties. This process helps identify the handful of drug candidates that will
progress further. Many closely related drug compounds must be rapidly analyzed
Eliminates the vast majority (>99%)
and quick decisions must be made as to which drugs will continue into development
of plasma phospholipids
and eventually clinical trials. During the drug discovery stage, speed, time, ease of
Reduces sample variability, eliminating
use, and high throughput are key aspects of everyday work. There is little time for
a major source of suppression
method development, making simple and universal sample prep methods such as
Facilitates use of shorter runtimes,
improving throughput
protein precipitation (PPT) an attractive choice. Crude techniques such as PPT are
often quite efficient in terms of generating high analyte recovery but result in
relatively dirty samples. In particular, PPT does little to eliminate phospholipids
(PLs), a major source of concern in bioanalysis. PLs build up in LC/MS/MS systems
and are one of the major sources of matrix effects in plasma-based assays. Amongst
Wate rs solutions
n
ACQUITY UltraPerformance LC® technology
nXevo ®
TQ MS system
nOstro™
sample preparation products
nIntelliStart
™
software
other problems, matrix effects also alter mass spectrometry response in an unpredictable manner, decrease method robustness, and add to method variability. In this
publication, Ostro™ 96-well sample preparation plates are used to eliminate both
proteins and the vast majority of PLs while maintaining high analyte recovery, all
with a simple single step method. A screening method for a group of 26 structural
analogs and metabolites (see Figure 1 for representative structures) in plasma was
developed using this technique.
O
N
N
N
k e y wo rds
Cl
Phospholipid removal, LC/MS/MS, Ostro, matrix
OH
O
HN
N
nordazepam
OH
Cl
temazepam
Cl
O
N
Cl
HN
N
N
diazepam
effects, drug discovery, screening
O
Cl
lormetazepam
O
HN
O
OH
N
N
Cl
Cl
oxazepam
Cl
lorazepam
Figure 1. Representative structures of 6 of the 26 analogs utilized in this study.
1
Table 1. MRM transitions and MS conditions for the 26 structural analogs in
plasma, generated using IntelliStart1
E X P E RIM ENTA L
ACQUITY UPLC Conditions
Column: ACQUITY UPLC® BEH C18,
2.1 x 50 mm, 1.7 µm
Mobile Phase A: 0.1% HCOOH in H2O
Mobile Phase B: Methanol
Flow Rate: 0.6 mL/min
Gradient:
Time
Profile Curve
(min) %A %B
0.0
98
2
6
2.0
1
99
6
2.5
1
99
6
2.6
98
2
6
3.0
98
2
6
Compound Name
Precursor
Ion
Product
Ion
Cone
Collision
Voltage (V) Energy (eV)
Triazolam
343.1
238.9
42
40
Alpha-hydroxymidazolam
342.0
203.0
38
28
2-hydroxyethylflurazepam 333.1
108.9
36
26
Clozapine
327.2
269.9
50
24
Midazolam
326.1
290.9
42
26
Prazepam
325.2
270.9
32
22
Alpha-hydroxyalprazolam
325.1
296.9
40
24
Bromazepam
318.1
181.9
38
32
Clonazepam
316.1
269.9
38
24
Flunitrazepam
314.1
268.0
42
24
Alprazolam
309.1
280.9
42
26
Injection Volume:
18.0 µL
Temazepam
301.1
254.9
26
24
Injection Mode: Partial Loop
Clobazam
301.1
223.9
32
30
Column Temperature:
35 °C
n-Desmethylflunitrazepam 300.2
253.9
38
22
Chlordiazepoxide
300.2
226.9
28
22
Estazolam
295.1
266.9
34
22
Desalkylflurazepam
289.1
139.9
42
26
Sample Temperature: 15 °C
Strong Needle Wash: 60:40 ACN:IPA + 0.2% conc. HCOOH (600 µL)
Weak Needle Wash: 80/20 H2O/MeOH (200 µL)
Waters Xevo™ TQ MS Conditions, ESI +
Oxazepam
287.1
241.0
28
32
7-aminoflunitrazepam
284.2
135.0
46
26
Nitrazepam
282.2
207.0
38
34
Nordiazepam
271.1
139.9
32
30
Capillary Voltage:
1.0 V
7-aminonitrazepam
252.1
121.0
40
26
Desolvation Temp:
400 °C
7-aminoclonazepam
286.1
120.9
38
28
Cone Gas Flow:
Not used
Desolvation Gas Flow: 1000 L/Hr
Collision Cell Pressure: 2.6 x 10 (-3) mbar
MRM transition monitored, ESI+: See Table 1
2
Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method
Basic Protocol
SAMPLE PREPARATION PROTOCOL
Ostro 96-well sample preparation plates
Add plasma
Sample preparation was performed using the standard protocol
included with the Ostro 96-well plate (see Figure 2). Sample
volume was 100 µL of plasma. This was extracted in the wells
of the Ostro plate using 300 µL of 1% HCOOH in acetonitrile.
Add organic
Samples were mixed by aspiration. Vacuum was then applied
to collect the eluates. Eluates were diluted 1:1 with water to
ensure compatibility with the LC mobile phase and directly
injected onto the LC/MS/MS system.
Aspirate to mix
RESULTS AND DISCUSSION
Apply vacuum
Separation of the 26 structural analogs (Figure 3) was achieved
using a two-minute gradient at low pH with methanol. MS was
performed in positive ion mode. Precursor and product ions
Analyze
were automatically optimized and an MS method automatically
generated using IntelliStart software. A previous application
note (720002569EN) by Rainville et al describes the
Figure 2. Ostro 96-well plate protocol.
capabilities of IntelliStart in detail.
14, 15, 16, 17, 18, 19, 20, 21
100
1
0.80
10, 11, 12, 13
%
26
9
22, 23, 24
25
3
46
2
5
7
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
7-aminonitrazepam
7-aminoclonazepam
7-aminoflunitrazepam
Clozapine
Midazolam
Chlordiazepoxide
Alpha -Hydroxymidazolam
Bromazepam
n-Desmethylflunitrazepam
Nitrazepam
Clonazepam d4
Clonazepam
Flunitrazepam
Triazolam
2-Hydroxyethylflurazepam
Hydroxyalprazolam d5
Alpha -Hydroxyalprazolam
Alprazolam
Alprazolam d5
Oxazepam
Clobazam
Estazolam
Desalkylflurazepam
Temazepam
Nordiazepam
Prazepam
8
0
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
2.20
2.40
2.60
2.80 min
Figure 3. Representative ESI+ MRM chromatogram of a mixture of 26 structural analogs and metabolites.
Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method
3
The Ostro 96-well plate was used to remove residual
PLs prior to LC/MS/MS analysis. Utilizing the generic,
simple protocol provided by the manufacturer (Figure
2), a group of analogous compounds were extracted.
100
80
The resulting analysis demonstrated an average recovery
of 86% for the group of structural analogs in plasma
(Figure 4), which is equal to or better than traditional
PPT. To assess PL removal, eight individual PLs were
60
40
20
summed. Results showed that Ostro plates remove
>99% of the 8 PLs relative to traditional PPT (Figure
5). In addition, the MRM transition, 184>184, was
monitored to visually demonstrate the significant
decrease in residual PLs using Ostro plates compared to
0
)
)
)
)
)
)
)
)
)
)
)
0) 0 2 01 0 4 9) 0 8 6) 0 5 5 ) 3) 7) 7) 0 2 0 3 7 ) 9) 1 9 2 ) 1) 5 ) 2) 1 5 1 2 6 ) 5) 14
91 H-9 (F-9 (A-9 -05 M-9 -9 0 (A-9 -90 -9 0 -90 F-90 (A-9 A-9 -90 -9 0 D-9 -02 -90 D-91 -9 0 A-9 (A-9 -90 -90 A-9
N
( m 5
(
C
( m
( (C (E ( (O
C ( (P
(
T (C
( (B (C ( 5 m
(N m (
(
(
m
m
m
e
4
d e
a
d la m m am id am am m a pa am a m pa
a p
lam la p m in l am a m la d a m a m pam
p e
a
p a
zo zo ze la ap o p zo m ep zep aze o lam razo zep b az aze pox azol a ze zep aze raz a zep azep raze
i a d a ra o z az ze ra pa z
Tr ymi lf lu praz Clo Mid Pra yalp aze oma lon a nit r p raz Alp e ma Clo ni tr ia ze Est flur Oxa clon uni t Nitr ordi oni t
y
l
u
l
d
u
r
l
l
x
T
o
n
x
f
C Fl A
N in
lfl o r
ro Clo B
in n o
ro eth ya
lky
hy Chl
am
sa
yd
yd xy ox
a m mi
et
7-H
-H ro d r
De
7- 7-a
sm
ha
ha -h yd Hy
e
p
p
l
D
a
al 2
n(T-
traditional PPT (Figure 6). One of the primary reasons
to eliminate PLs is to improve method robustness.
Overnight runs of both Ostro plates and traditional PPT
samples were carried out using the generic gradient
Figure 4. Average analyte recovery data for 26 structural analogs and metabolites in
plasma using an Ostro 96-well plate. Overall average recovery was 86%.
and PLs were monitored continuously. Figure 7 shows
the LC/MS trace for a representative PL at the beginning
9000000
and end of the runs. When Ostro plates are used, the
PL levels are negligible and no build-up occurs. When
PPT is used, a significant amount of PLs are present
sum of 8 phospholipids
8000000
7000000
and accumulate throughout the run. The result of this
undesirable build-up is a continuous downward trend in
area counts throughout the duration of the run (Figure
6000000
5000000
8). This in turn results in high signal variability relative
to samples prepared with Ostro plates, 33% using
PPT vs. 9% for Ostro samples. In addition, area counts
4000000
3000000
decrease by 57% from the first injection to the final
injection when PPT is used. In discovery bioanalysis,
high throughput is of utmost importance. If one tries
to increase throughput by shortening gradient time,
the impact of residual PLs is further magnified. To
2000000
1000000
0
Ostro
PPT
demonstrate the negative effect PLs have on analytical
throughput, the gradient time was decreased by half.
Flow rate was increased and organic content was
Figure 5. Comparison of phospholipids remaining after traditional PPT and PPT using
Ostro 96-well plate.
ramped from 50 to 98% in 0.5 minutes. 200 Ostro
samples and 200 PPT samples were injected using the
shorter gradient. The MRM transition 184>184 was
injection. These resultant chromatograms demonstrate the inability to shorten
monitored to reveal overall PL build-up and elution in
gradient time with PPT due to PLs which continue to elute significantly after the
the shortened gradient window (Figure 9). Using the
gradient ends at 0.5 minutes. Overall, the Ostro plate allows for increased method
2 minute gradient, PLs elute within 0.2 minutes of
robustness and reduced variability. Additionally both improved instrument uptime
the end of the gradient. Under the truncated gradient
and the ability to significantly shorten run times are realized through the elimination
conditions PLs continue to elute for more than 1 minute
of PLs, all of which are highly desirable in a discovery setting. Calibration curves from
after the end of the gradient and well into the re-
1-500 ng/mL for each of the 26 structural analogs had a resulting average r2 value
equilibration phase and beginning of the next
of 0.925, sufficient for discovery screens.
4
Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method
100
7.50
Traditional PPT
7.69
184.4 > 184.4 (Lipid 184)
1.31e8
7.43
%
7.76
9.18
8.80
7.31
8.31
9.88
10.08
0
2
4
6
8
10
12 min
100
Ostro Sample Preparation Products
%
184.4 > 184.4 (Lipid 184)
1.31e8
8.30
0
2
4
6
8
10
12 min
Figure 6. Representative chromatograms of MRM transition 184>184 to demonstrate total remaining residual
phospholipids from traditional PPT and Ostro eluates.
gradient
end
%
100
0
%
100
0
%
100
0
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.13
2.00
2.28
758.4 > 184.4 (lipid 758)
3.19e6
2.25
2.50
PPT 1st
injection
0.25
758.4 > 184.4 (lipid 758)
3.19e6
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
Ostro last
injection
0.25
2.75 min
2.75 min
758.4 > 184.4 (lipid 758)
3.19e6
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
Ostro 1st
injection
2.75 min
758.4 > 184.4 (lipid 758)
3.19e6
%
100
1.99
PPT last
injection
end
organic hold
0
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75 min
Figure 7. Representative chromatograms of MRM transition 758>184 to demonstrate build-up of an individual
PL over subsequent injections using traditional PPT. A gradient from 2-98% B in 2 minutes was used followed by
a 0.5 minute hold at high organic.
Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method
5
70000
Ostro
9% RSD
60000
50000
PPT
Ostro
40000
30000
20000
PPT
33% RSD
10000
0
time
Figure 8. Variability of area counts over subsequent injections using Ostro 96-well plate vs. traditional PPT.
%
100
0.65
0.73
0.55
184.4 > 184.4 (Lipid 184)
1.34e8
0.87
0.02
0
0.2
0.4
0.6
0.8
1.0
PPT 1
injection
1.2
1.4 min
184.4 > 184.4 (Lipid 184)
1.34e8
%
100
end
gradient
PPT 200
injections
0.73
0
%
100
0.2
0.6
0.8
1.0
1.2
1.4 min
184.4 > 184.4 (Lipid 184)
1.34e8
Ostro 200
injections
0.33
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 min
184.4 > 184.4 (Lipid 184)
1.34e8
Ostro 1st
injection
%
100
0.4
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 min
Figure 9. Representative chromatograms of the MRM transition 184>184 to demonstrate the overall build-up of PLs
over subsequent injections using traditional PPT vs. Ostro 96-well plate. A fast gradient from 50-98% in 0.5 minutes
was used. When PPT is used, PLs continue to elute well after the end of the gradient and when the Ostro plate is used, the
short gradient can readily be implemented without concern over PL build-up.
6
Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method
CONCLUSIONS
n
Simple, universal sample preparation protocol
n
Direct injection of the eluate streamlines workflow
n
Removes >99% of phospholipids from plasma resulting in improved
instrument uptime and more robust methods
n
High recovery for analogs and metabolites with no method
development
n
Reduces sample variability
n
Facilitates use of shorter runtimes, improving throughput
REFERENCES
1
Rainville, P.D., Mather J, Waters Application Note; 2008, 720002569EN
7
Eliminating Phospholipids in Drug Discovery Extractions Using a Fast, Generic Sample Clean-up Method
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