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A Novel Sample Preparation Device for Improved Phospholipid Removal in
Bioanalytical Assays
Jessalynn P. Wheaton, Erin E. Chambers, and Diane M. Diehl
Waters Corporation, 34 Maple St., Milford, MA 01757 USA
A P P LIC ATION BENEFITS
INTRODUC TION
Simple and universal phospholipid removal
Matrix effects are residual components of biological matrix that alter mass spectrometry
Performance superior to competitive devices
and techniques
Cleaner samples in less time
response, decrease method robustness, add to method variability, and lead to poor
limits of detection/quantification. A major source of matrix effects in plasma-based
bioanalytical assays is residual phospholipids. If not removed from the sample,
phospholipids can build up on analytical columns, elute at unpredictable times
in subsequent injections, and negatively impact assay robustness in a number of
different ways. Over the past few years, it has become commonplace to monitor
phospholipids during method development and to implement strategies to remove
them from plasma samples. Sample preparation strategies for phospholipid (PL)
removal include the use of specific PL removal plates, liquid-liquid extraction (LLE),
and solid-phase extraction (SPE.) During the method development process, decisions
are made regarding the choice of a “fit-for-purpose” sample prep technique. Ostro™
Sample Preparation Products are designed to provide a novel solution for the rapid
and simple cleanup of phospholipids in biological samples prior to LC/MS/MS analysis.
Using a convenient 96-well format, in-well protein precipitation is performed with a
single pass through method which provides consistent, high quality results, including
significant removal of phospholipids for cleaner extracts, optimal recovery for diverse
analytes, and increased reproducibility for consistent, robust methods.
Basic Protocol
Wat e rs solut ions
Add plasma
ACQUITY UPLC
Xevo TQ
Add organic
Ostro Sample Preparation Products
ACQUITY UPLC BEH C18 Column
MassLynx 4.1
k e y wo rds
Phospholipid removal, Ostro,
Aspirate to mix
Apply vacuum
Analyze
LC/MS/MS
Figure 1: Basic Protocol.
1
E X P ERIM ENTAL
RESULTS AND DISCUSSION
The Ostro 96-well plate was directly compared to protein precipitation (PPT),
LC system:
Waters ACQUITY UPLC® System with a Waters Xevo™ TQ MS triple quadrupole mass spectrometer operated in positive ion MRM mode
Column: ACQUITY UPLC BEH C18,
2.1 x 50 mm, 1.7 µm
Injection volume:
8 µL
Flow rate: 0.6 mL/min
2 competitive phospholipid removal plates, LLE and solid-supported liquid-liquid
extraction (SSLE.) Analyte recovery, phospholipid removal, and reproducibility of
phospholipid removal were all evaluated.
Ostro
PPT
Competitor 1
Competitor 2
120
100
80
% Recovery
LC conditions
60
40
20
iol
str
ad
iny
lE
Eth
Py
ren
ac
N-
(n)
(a)
Ni
flu
mi
cA
cid
(z)
en
Ke
top
rof
T6
69
2
ic
on
es
ulf
Pro
(qa
)
)
(sa
Ac
no
pra
am
ain
oc
ety
lpr
id
lol
ide
(b)
(b)
0
Figure 2a: Average analyte recovery of a diverse analyte mix using PPT, Ostro 96-well plate,
2 competitive phospholipid removal 96-well plates.
b)
100
Ostro
LLE
SSLE
90
80
% Recovery
70
60
50
40
30
20
10
rof
top
Ni
flu
mi
cA
cid
(z)
Eth
iny
lE
str
ad
iol
(n)
(a)
en
(qa
)
Ke
id
Ac
ic
on
es
ulf
T6
69
2
)
(sa
b)
l(
olo
Pro
p
ran
Py
ren
Nac
ety
lpr
oc
ain
am
ide
(b)
0
Figure 2b: Average analyte recovery of a diverse analyte mix using Ostro, LLE, and SSLE.
2
A Novel Sample Preparation Device for Improved Phospholipid Removal in Bioanalytical Assays
Level of Phospholipids Remaining
5000000
Phospholipids Remaining (Total Area Count)
100
90
4000000
80
70
60
3000000
Dramatic decrease
in phospholipids
50
40
2000000
30
20
10
1000000
0
1
Ostro
2
3
Competitor 1
4
5
6
Competitor 2
0
Ostro
Competitor 1
Competitor 2
PPT
Figure 3a: Comparison of phospholipids remaining in extracted samples using Ostro,
two competitive phospholipid removal products, and PPT.
Samples were processed on the Ostro 96-well plate using the
400000
Phospholipids Remaining (Total Area Count)
Figure 4: % phospholipid removal in 6 lots of human plasma.
generic, simple protocol (Figure 1) provided by the manufacturer
350000
and a set of diverse analytes. Ostro sample preparation products
provided overall significantly better recovery for the analytes
300000
tested (Figures 2a and b) than PPT, LLE, SSLE and competitive PL
250000
removal plates. The Ostro 96-well plate also removed significantly
200000
more phospholipids than the current phospholipid removal products
150000
on the market, PPT, LLE, and SSLE (Figures 3a and 3b.) In addition,
not only was the level of PL removal better, but PLs were removed
100000
in a significantly more reproducible manner when Ostro Sample
50000
Preparation Products were used versus competitive PL removal
0
devices (Figure 4). The average RSD for lipid removal across six
Ostro
LLE
SSLE
Figure 3b: Comparison of phospholipids remaining in extracted samples prepared
using Ostro 96-well plate, LLE, and SSLE.
sources of human plasma using Ostro was <1%, while competitive
plates exhibited RSD’s between 24% and 41% for lipid removal.
A Novel Sample Preparation Device for Improved Phospholipid Removal in Bioanalytical Assays
3
CONC LUSION
Providing cleaner, more reproducible extracts than competitive phospholipid removal
devices or techniques, Ostro allows for more robust analyses and increased sample
throughput. Requiring minimal to no method development, this technology can be
rapidly implemented in order to improve laboratory workflow. Method simplicity,
reproducibility, and highly efficient phospholipid removal make this technique an
attractive alternative to liquid-liquid extraction or protein precipitation for obtaining
higher quality data from large numbers of samples. In addition, the generic nature of
this device and the significant clean-up provided facilitate productivity increases by
reducing system downtime.
© 2010 Waters Corporation. Waters, T he Science of W hat’s
Possible, Ostro, ACQUITY UPLC, and Xevo are trademarks of
Waters Corporation.
September 2010 720003730EN KK-PDF
Waters Corporation
34 Maple Street
Milford, MA 01757 U.S.A.
T: 1 508 478 2000
F: 1 508 872 1990
www.waters.com