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Volume 27.2
Sample Handling
Troubleshooting Analyte Recovery when
Using HybridSPE-Precipitation Technology
Craig Aurand, Charles Mi, Xioaning Lu,
An Trinh, and Michael Ye
Introduction
HybridSPE™-Precipitation (HybridSPE-PPT) is a new
sample prep platform for pharmaceutical bioanalysis. The
technology merges the simplicity of protein precipitation
with the selectivity of SPE for the selective removal of
proteins and phospholipids from biological plasma. Removal
of these two key interferences greatly reduces the risk of
ion-suppression during LC-MS-MS analysis for improved
assay sensitivity and reproducibility. In Reporter issues 26.3
and 26.5, we provided an overview of HybridSPE-PPT and
offered application examples that illustrate the benefits of
the technology. In this article, we discuss non-phospholipid
specific interactions between the HybridSPE phase and the
sample that could potentially lead to low recovery of certain
basic and acidic chelator compounds. We conclude our
discussions with strategies for improving the recovery of
such problematic compounds.
How does HybridSPE-PPT work?
When using 96-well HybridSPE-PPT, 100 μL of plasma is
added to the individual wells followed by 300 μL precipita-
tion agent (1% formic acid in acetonitrile). After a brief
mixing step to adequately precipitate endogenous proteins,
vacuum is applied to the plate. As the sample flows
through the packed-bed/filter-frit assembly, both proteins
and phospholipids are concurrently removed. Proteins are
physically removed by low porosity filters whereas phos-
pholipids are chromatographically removed by the station-
ary phase. The resulting eluent is free of both phospholipids
and proteins, and can be directly analyzed via LC-MS-MS.
The HybridSPE stationary phase is a patent pending
zirconia coated silica that is highly selective towards
phospholipids. Retention is based on a Lewis acid-base
interaction between the empty zirconia d-orbitals (Lewis
acid) and the electron pair of the phosphate moiety (Lewis
base) inherent of all phospholipids. Phosphate is a very
strong Lewis base and will preferentially interact with
zirconia over other Lewis bases (Figure 1).
The Importance of Formic Acid
Most acidic pharmaceutical compounds contain carboxyl
(-COOH) groups. When processing acidic compounds using
HybridSPE-PPT, the HybridSPE Zr-Si stationary phase will
likely co-retain acidic compounds along with phospholipids,
resulting in low absolute recovery. To rectify the situation,
formic acid is added to the precipitation agent and becomes
part of the sample during HybridSPE processing. Formic acid
is a stronger Lewis base than most -COOH groups found in
acidic pharmaceutical compounds. As a result, formate ions
will tie up the phase’s zirconia ions, minimizing retention of
acidic analytes of interest. Because formate is not a strong
enough Lewis base to displace the phosphates, phospholip-
ids preferentially retain on the HybridSPE-PPT phase.
In this application, we
process three acidic
compounds (ketoprofen,
naproxen, and flunixin) and
two neutral compounds
using HybridSPE-Precipita-
tion. The analytes were
spiked into plasma at the
level of 20 ng/mL. 100 μL
of plasma was precipitated
with 300 μL of one of two
reagents prior to HybridSPE-
PPT: 1) 1% formic acid in
acetonitrile or 2) neat
acetonitrile. The resulting
HybridSPE-PPT eluent was
analyzed by LC-MS-MS
(MRM) using an Ascentis
(continued on page 18)
Figure 1. HybridSPE-PPT 96-well Schematic and Phospholipid Retention Mechanism
The phosphate moiety of phospholipids is a strong Lewis base (electron
donor) that interacts with Zr atoms coated on the silica surface.
Proprietary HybridSPE
Zirconia Coated Silica
The Zr atom acts as a
Lewis acid (electron
acceptor) because it
has empty d-orbitals.
:
Si-OH
O
O
O
O
Zr
Zr
Si-O–
O
Zr+-OH
Upper 5 μm PTFE Frit
Lower 0.2 μm
Hydrophobic Filter
G004255
G004389
sigma-aldrich.com/hybridspe-ppt
Excerpt from Reporter, Volume 27.2.
18
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Volume 27.2
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Sample Handling
RP-Amide column. Figure 2 compares the results between
the two precipitation agents. From the results described in
Figure 2, complete loss in recovery was observed for the
three acidic compounds (ketoprofen, naproxen, and
flunixin) when formic acid was not added to the precipita-
tion agent. When formic acid is added to the precipitation
agent, greater than 88% absolute recovery was observed
for each of the acidic compounds (data not shown). In
contrast, the two neutral compounds, phenylbutazone and
oxyphenylbutazone were unaffected by the presence of
formic acid resulting in high recovery under both conditions.
Troubleshooting Recovery of Chelator and
Acidic Chelator Compounds
In our research thus far, we have found that certain
chelator and acidic chelator compounds retain exceptionally
strong on the Zr-Si stationary phase used in HybridSPE-PPT
resulting in low
absolute
recovery
(< 40%) when
using the
recommended
primary
HybridSPE-PPT
method (100
μL plasma +
300 μL formic
acid in
acetonitrile). Such chelating compounds can be identified
as having functional groups with oxygen atoms in the alpha
and beta positions. To improve recovery, a Lewis base
stronger than formate is required as a modifier in the
(continued from page 17)
Figure 2. Comparison of Precipitation Agents (with and
without formic acid) using HybridSPE-PPT
Precipitation Agent: 1% formic acid in acetonitrile
Precipitation Agent: neat acetonitrile
precipitation agent. Table 1 lists various Lewis bases and
their relative retention strength on zirconia. From our
experience, replacing formic acid with citric acid and adding
a simple conditioning step can significantly increase the
recovery of certain chelator and acidic chelator compounds.
Figure 3 describes specific chelation functional groups and
lists example compounds with such functional groups.
Details for the secondary procedure we recommend are
described in Table 3. By using the secondary procedure
described in Table 3, recovery for chelator compounds can
improve from <40% to 65-95%. Mechanistically, citric acid
is a stronger Lewis base than formic acid, inhibiting
retention of chelator compounds. However, citric acid is not
a strong enough Lewis base to displace retention of
phosphates (i.e., phospholipids).
Troubleshooting Recovery of Basic and
Neutral Compounds
Although the primary retention mechanism for HybridSPE
is based on Lewis acid-base interactions between Zr ions on
the stationary phase and negatively charged functional
groups in the sample (e.g., phosphate moiety of phospho-
lipids), secondary interactions derived from the silica surface
can retain basic and neutral compounds resulting in poor
recovery. These secondary interactions with silica surface
include: 1) weak cation exchange and 2) HILIC interactions.
To disrupt any weak-cation exchange interactions between
the silica silanol groups (Si-O-) and basic compounds (e.g.,
contains amine functional groups), formic acid should be
substituted with ammonium formate. The primary procedure
we recommend uses formic acid as part of the precipitation
agent. As a result, H+ is the resulting counter-ion used to
Table 1. Relative Retention Strength
of Lewis Bases to Zirconia
Relative Retention
Lewis Base
Strength on Zirconia
Hydroxide
Strongest
Phosphate
Fluoride
Citrate
Sulfate
Acetate
Formate
Chloride
Weakest
Figure 3. Low Recovery Chelation Functional Groups
with Example Compounds
Chelation functional groups that can lead to low HybridSPE-PPT
recovery and may require citric acid secondary procedure:
Ketoprofen
Naproxen
Flunixin
0 1 2 3 4 5
Min
0 1 2 3 4 5
Min
Oxyphenylbutazone
Phenylbutazone
Beta-hydroxy
caroboxylic
acids
1,3-Diketone
type
compounds
Beta-
hydroxy
ketones
1,3-Diol
type
compounds
G004760
G004761
G004762
G004764
G004763
G004765
G002553
G003164
Pravastatin
G002555
Atorvastatin
G002554
Fluvastatin
Apigenin
Example compounds with chelation functional groups:
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Sample Handling
neutralize exposed silanol groups on the Hy-
bridSPE-PPT phase (Si-O- => Si-OH). For some
basic compounds, H+ is not a strong enough
counter-ion to inhibit cation-exchange reten-
tion between silanol groups and basic com-
pounds. This results in poor recovery. By re-
placing formic acid with ammonium formate,
a stronger ammonium counter-ion (NH
4
+) is
employed. Ammonium ions are sufficient in counter-ion
strength to inhibit most (if not all) basic compounds from
interacting with silanol groups.
For more polar compounds, secondary HILIC interactions
(e.g., hydrogen bonding) may occur between basic/neutral
analytes of interest and the silica surface. These secondary
HILIC interactions can be disrupted by substituting ac-
etonitrile with methanol as the precipitation agent. To
further minimize potential secondary HILIC interactions, the
sample needs to be 25% aqueous prior to HybridSPE-PPT
processing. Therefore, combining 100 μL plasma with 300
μL organic precipitation agent is recommended for
HybridSPE-PPT. For smaller plasma volumes (e.g., 20-50 μL),
the sample should be diluted with DI water to maintain a
Table 2. Improvement of Absolute Recovery when Incorporating Ammonium
Formate in the Precipitation Agent
Standard
Standard
Analyte
(no matrix)
Standard
(no matrix)
Plasma + 1%
(% Absolute
+ 1% formic
(no matrix)
+ 1% NH
4HCO2
NH
4HCO2
Recovery)
acid in MeCN
+ MeOH
in MeOH
in MeOH
Mirtazapine
(266/195)
0.0%
13.2%
96.0%
104.0%
Risperidone
(411/191)
0.0%
10.4%
99.1%
123.3%
Olanzapine
(313/256)
0.0%
13.6%
89.4%
56.4%
final sample volume of 100 μL prior to addition of 300 μL
precipitation agent. If greater sensitivity is required after
sample dilution, an evaporation and reconstitution step can
be added prior to LC-MS-MS analysis.
In this study, 3 basic compounds experienced low
recovery when using the primary method (100 μL plasma +
300 μL 1% formic acid in acetonitrile). 100 μL of spiked
(20 ng/mL) plasma samples or standard samples (no matrix)
were precipitated with 300 μL neat methanol, 1% formic
acid in acetonitrile, or 1% ammonium formate in methanol
prior to 96-well HybridSPE-PPT processing using the
“In-well” precipitation method. Absolute recovery was
assessed by reversed-phase LC-MS-MS (Table 2). From
these results, significant improvements in HybridSPE-PPT
recovery of the basic compounds observed when substitut-
ing 1% formic acid in acetonitrile with 1% ammonium
formate in methanol as the precipitation agent.
Table 3 summarizes the primary and secondary procedures
recommended when optimizing conditions for HybridSPE-PPT.
Conclusion
HybridSPE-PPT technology is a new sample prep platform
designed for pharmaceutical bioanalysis. The technology
combines the simplicity of protein precipitation and the
selectivity SPE by specifically targeting the removal of
precipitated proteins and phospholipids. The phospholipid
removal mechanism is based on a Lewis acid-base interac-
tion between the phosphate moiety inherent with all
phospholipids and the Zr-Si stationary phase. Although a
primary method that is suitable for most applications is
available, low recovery can occur. In this report, we
described the secondary interactions that can take place
resulting in low recovery and strategies for how to trouble-
shoot these recovery issues.
For more information and to download the latest
HybridSPE-PPT instruction sheet, please visit our website
sigma-aldrich.com/hybridspe-ppt
Table 3. Summary of Recommended Primary and
Secondary Procedures for 96-well HybridSPE-PPT
Primary Procedure (suitable for 80% of applications):
Recommended for most applications (basic, neutral, acidic analytes)
1. To each well, add 100 μL plasma followed by 300 μL
1% formic acid in acetonitrile. Mix the sample well
(e.g., vortex).
2. Apply vacuum and collect the resulting eluent for
LC-MS-MS analysis.
3. If low recovery is observed, proceed to Secondary
Procedures.
Secondary Procedure (acidic & chelator compounds):
Recommended for low recovery chelator and acidic chelator compounds
1. Condition each well with 400 μL 0.5% citric acid in
acetonitrile (until flow has ceased).
2. To each well, 100 μL plasma followed by 300 μL 0.5%
citric acid in acetonitrile. Mix the sample well (e.g., vortex).
3. Apply vacuum and collect the resulting eluent for
LC-MS-MS analysis.
Note:
Recovery of chelator compounds can improve from < 40% to 65-95%
Citric acid is a stronger Lewis base than formic acid inhibiting the retention of chelator
compounds.
Citric acid is not a strong enough Lewis base to inhibit phosphates (phospholipids)
from retaining on the HybridSPE phase.
Secondary Procedure (basic & neutral compounds):
Recommended for low recovery basic and neutral compounds
1. To each well, add 100 μL plasma followed by 300 μL
1% ammonium formate in methanol. Mix the sample well
(e.g., vortex).
2. Apply vacuum and collect the resulting eluent for
LC-MS-MS analysis.
Note:
Recovery of basic and neutral compounds can improve from < 40% to > 89%
NH
4
+ (ammonium formate) is a stronger counter-ion than H+ (formic acid) inhibiting
most basic compounds from interacting with HybridSPE silanol groups (Si-O-).
Methanol is a more polar solvent than acetonitrile further inhibiting any potential
secondary HILIC interactions between the analyte and HybridSPE silica surface.
Featured Products
+
Description Cat.
No.
HybridSPE-Precipitation
96-well Plate, 50 mg/well, pk. 1
575656-U
Cartridge, 30 mg/1 mL, pk. 100
55261-U
