ADVANCES IN SOLID PHASE EXTRACTION—
REMOVAL OF PHOSPHOLIPIDS USING A
NOVEL REVERSED-PHASE SORBENT
RESULTS
Jonathan P. Danaceau, Xin Zhang, and Erin Chambers
Waters Corporation, Milford, MA, USA
Plasma Corticosteroids—Recovery
INTRODUCTION
Strata
X RP 10mg
5 step
Competitor
2
Competitor
3 RP 10mg 5 step
Bond
Elut Plexa
20
60%
0
-20
-40
40%
-60
-80
-100
20%
0%
-20%
Figure 4. Matrix Effects obtained from Oasis PRiME HLB
and competitors’ SPE devices. Bars and error bars
represent means and standard deviations (N=5).
Recovery
Matrix Effects
-40%
Cortisol
Adione
17-OHP
Figure 1. Recovery and matrix effects for plasma
steroids (N=4).
Phospholipid Removal
1.41 E^8
A
4.00
5.00
Lyso-PL (524.4 > 184.4)
2.52 2.81
B
0
A
100
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Time
8.00
Quantitative Results
5.80
6.00
6.20
Competitor 1
0
5.60
5.80
6.00
6.20
%CV
3.0
92.3%
5.4%
30
94.8%
3.0%
300
94.9%
6.0%
Competitor 2
5.80
6.00
6.20
Competitor 3
5.80
Acc.
%CV
Acc
%CV
94.4%
5.7%
93.7%
6.5%
0
1.50
95.0%
3.6%
92.3%
5.6%
-10
15
95.4%
5.5%
93.7%
6.5%
-20
Synthetic Cannabinoids in whole blood
Recovery
Time
6.20
6.00
ME = -83%
0
5.60
5.80
6.00
ME = -88%
0
5.60
5.80
6.00
Bond
Elut Plexa
Competitor
3 RP 10mg 5 step
5.80
Time
6.20
6.00
PRiME
-30
y = -1.1605x - 11.645
R² = 0.9868
-40
-50
-60
-70
Competitor 1
-80
Competitor 2
Competitor 3
0
120
6.20
ME = -75%
0
5.60
-100
Evolute
ABN 10mg
5 step
Competitor
1
Strata
X RP 10mg
Competitor
2 5 step
6.20
Phospholipids vs. ME
-90
Oasis PRiME HLB 10mg 3 step
6.20
Figure 5. A. Traces of lysophosphatidylcholine 18:0
(Lyso-PL). B. Traces of JWH-203 post spiked into
extracted plasma.
QC Spike
Conc.
0.15
Table 1. Quality Control results for plasma steroids
extracted from plasma using Oasis PRiME HLB (N=6).
6.00
100
0
5.60
17α-OH progesterone
5.80
100
100
%
Accuracy
ME = -11%
0
5.60
100
100
100
QC Spike Conc.
2.06e6
Oasis PRiME HLB
0
5.60
0
5.60
Cortisol
B
100
2.51e7
%
Figure 2. Residual phospholipid traces for plasma
samples prepared with Oasis PRiME HLB (A) and by
protein precipitation (B). > 97% of phospholipids
were removed compared to PPT. A 20x magnification
of the SPE extract is shown at upper right.
JWH-203 (340.2 > 125)
%
1.41 E^8
%
3.00
%
2.00
Time
%
1.00
2.00
8.00
6.00 4.00
7.006.00 8.00
Examination of phospholipid traces revealed that JWH203 co-eluted with residual phospholipid
lysophosphatidycholine 18:0 (m/z 524.4) causing the
severe matrix effect with all other SPE products.
Removing the phospholipids virtually eliminated this ion
suppression.
%
%
2.34 2.75
0
Phospholipid removal—
Impact on ion suppression
%
0
100
%
100
Blk_Human_100_PL_150424_001 F1
TIC
100
20X 7.05e6
Mag
Androstenedione
Sample Preparation—Synthetic cannabinoids: 0.1 mL
of a solution of 0.1 M zinc sulfate/ammonium acetate
was added to 0.1 mL whole blood and vortexed for 5
seconds to lyse the cells. All samples were then precipitated by adding 400 µL ACN. The entire sample
was vortexed for 10 seconds and centrifuged for 5
min at 7000 rcf. The supernatant was diluted with 1.2
mL water prior to loading to all SPE devices. The SPE
protocols are shown in the figure below. All SPE devices consisted of 10 mg 96-well plates. Following
elution, all samples were evaporated to dryness and
reconstituted with 100 μL of 30% ACN.
Evolute
ABN 1
10mg 5 step
Competitor
60
Matrix Effects
Sample Preparation—Plasma corticosteroids: 150 μL
plasma samples were precipitated with 300 μL of a
solution of 4:1 MeOH and 89 g/L ZnSO4 (V:V). After
centrifugation, 300 μL of supernatant was diluted with
900 μL 4% H3PO4 and directly loaded onto Waters’
Oasis PRiME μElution SPE plates. All wells were then
washed with 25% MeOH and eluted with 2 x 25 μL of
90:10 ACN:MeOH. The sample eluates were diluted
with 25 μL water and analyzed by UPLC/MS/MS. The
SPE procedure is shown in the Figure below.
Oasis PRiME HLB 10mg 3 step
80
40
METHODS
Calibration standards were purchased from SigmaAldrich (St. Louis, MO, USA) and Cerilliant. All
deuterated internal standards were purchased from
Cerilliant (Round Rock, TX).
100
80%
%
Bioanalysis of drugs and endogenous molecules from
plasma samples often involves using solid phase
extraction (SPE) to clean up the matrix and
concentrate the analytes of interest. Despite the
advances in SPE sorbents and formats, residual
phospholipids often remain in extracts and can
interfere with analyses by causing ion suppression
and by prematurely fouling analytical columns and MS
sources. A novel reversed-phase SPE sorbent has
been developed that is designed to additionally
remove phospholipids from biological samples. This
sorbent is also water wettable, enabling extraction
without the usual requisite preconditioning and
equilibration steps. Application examples of plasma
corticosteroids and synthetic cannabinoids in whole
blood are highlighted. The resulting methods
demonstrate consistent recovery, reduced matrix
effects, and the elimination of >95% of phospholipids
compared to protein precipitation or other reversedphase sorbents.
Matrix Effects (ME)
20
40
60
80
Relative PL Abundance
100
Figure 6. Relationship between Phospholipid 524
abundance and ion suppression for JWH-203, showing
the direct correlation between this coeluting PL and the
ion suppression for JWH-203.
80
60
40
20
0
CONCLUSIONS
Figure 3. Extraction recoveries for Oasis PRiME HLB
vs. all other SPE devices (n=5). For Oasis PRiME HLB,
21/22 compounds had recoveries between 90-110%.
Table 2 Recovery and standard deviation ranges
N=5
% Rec Range
AVG
% REC
% RSD
Range
AVG
% RSD
Oasis PRiME
HLB
90-110
(1 @ 71%)
100
3-7
4
Alternate 1
60-97
85
1-17
7
Alternate 2
59-92
80
2-27
11
Alternate 3
46-84
73
3-41
11
 Simplified Load-Wash-Elute extraction protocols
 Efficient and highly consistent recovery of a variety of
compounds
 Improved recovery, consistency and reduced matrix
effects compared to other reversed-phase sorbents.
 Excellent accuracy and precision for plasma steroids
 Near elimination of phospholipids from plasma and
whole blood samples
 Removal of co-eluting PL virtually eliminated ion
suppression for JWH-203
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