Use of An Improved Version of C8+SCX Mixed-Mode
Solid Phase Extraction Material for Clean Extraction
and Recovery of Basic, Zwitterionic, Neutral and
Acidic Compounds from Biological Fluids
Yuhui Yang, An Trinh, Michael Ye, and Tom Henderson
595 North Harrison Road, Bellefonte, PA 16823
T404109
HKB
Abstract
C8+SCX mixed-mode bonded silica is the most commonly used
sample prep tool for systematic toxicological analysis. However, both
zwitterionic compounds and polar basic compounds cannot be
effectively extracted and recovered from most commercially available
mixed-mode phases. This is primarily due to the low ion-exchange
capacity inherent with phases commercially available. In this
presentation, we describe the use of a mixed-mode phase in which
ion-exchange capacity has been optimized for extraction of polar
basic and zwitterionic compounds. Three generic extraction
protocols have been determined. The first protocol offers superior
selectivity when recovering non-polar to moderately polar basic and
zwitterionic compounds. The second protocol is used to recover very
polar basic and zwitterionic compounds. The third protocol is aimed
to systematically fractionate basic and zwitterionic compounds from
neutral and acidic compounds for further analysis. The novel mixedmode SPE phase offers improved performance over other
commercially available silica- and polymer- based mixed-mode
products.
Introduction
1. Reliable and reproducible sample preparation methodology is
critical for analyzing pharmaceutical drug candidates, drugs of
abuse, and their metabolites from biological matrices.
2. In systematic toxicological analysis (STA), the undirected drug
screening must be a generic procedure where the substances of
interest are isolated at a yield as high as possible and the
biological matrixes should be removed. Only one SPE tube should
be used.
3. However, most commercially available mixed-mode phases have
inadequate ion-exchange capacity to retain more polar basic and
zwitterionic compounds. As a result, the compounds are lost
during load and wash steps during the extraction procedure.
4. In contrast, polymer-based mixed-cation phases have inadequate
hydrophobic capacity and thus many substances of interest are
frequently lost in the methanol wash step.
Experimentation/Results
General Mixed-Mode SPE Methodology
Condition with methanol
& equilibrate with
pH 3-6 buffer
Load
Sample @
low pH 3-6
Sample must be aqueous to support reversed-phase
component of interaction.
Low pH environment ionizes basic compounds for ionexchange & neutralizes acidic compounds to promote
reversed-phase interactions
Wash 1 further reinforces Ion-Exchange interaction with
basic compounds, neutralizes acidic compounds for
Reversed-Phase retention, and removes all non-basic
hydrophilic interferences
Wash 1: low pH 3-6 buffer
Wash 2: 100% methanol
Elute with basified
(high pH)methanol
Wash 2 is a powerful wash step used to remove all
hydrophobic interferences. If fractionation of acidic and
neutral components are necessary, both compound
groups will be eluted in this step
Methanol alone will not elute basic compounds due to the
Ion-Exchange component of interaction. By basifying
methanol, basic compounds are neutralized disrupting
both electrostatic and hydrophobic interaction
Generic Extraction Protocol 1
For Non-Polar Basic and Zwitterionic Compounds
(100mg/1mL or 100mg/3mL SPE tube)
1. Sample Pre-Treatment: Dilute urine (plasma/serum) sample (1.0mL)
with 50mM ammonium acetate buffer or 10mM potassium phosphate
buffer (pH6.0) (1.0mL).
2. SPE Tube Conditioning: Solvate tube with 1mL methanol
3. SPE Tube Equilibrium: Equilibrate tube with 50mM ammonium
acetate buffer or 10mM potassium phosphate buffer (1mL)
4. Sample Loading: Load pre-treated urine or plasma (1mL) at a flow
rate of 1mL/min
5. Interference Elution: Elute interference with 50mM ammonium
acetate buffer (or 10mM phosphate buffer) (1mL), 1M acetic acid
(1mL) and methanol (1mL).
6. Analyte Elution: Elute analytes with 5% ammonium hydroxide in
methanol
The generic protocol is only suitable for compounds that are ionized at pH 6 load
conditions to facilitate ion-exchange interaction, and/or of sufficient non-polar
character to interact with bonded phase C8 functional groups.
Case 1: Extraction of Tricyclic
Antidepressants Using DSC-MCAX SPE
mAU
2
4
Spiked Serum
Sample Prior to
SPE cleanup
0
Compound
(10ng/mL)
0
2
4
6
-0.2 0.0 0.2 0.4 0.6 0.8
mAU
Time (min)
1
2
0
2
Spiked Serum
Sample After
DSC-MCAX
cleanup
3
4
4
% Abs Recovery ±
RSD (n=3)
1. Nordoxepin
99.4 ± 4.3%
2. Doxepin
102.1 ± 1.8%
3. Nortriptyline
95.6 ± 2.7%
4. Amitryptyline
101.3 ± 3.2%
6
Time (min)
SPE: Discovery DSC-MCAX, 100mg/3mL, HPLC: Discovery C8, 15cm x 2.1mm ID, 5µm, Mobile
Phase: 10mM ammonium acetate, pH 4.5:MeCN (45:55), Flow Rate: 0.42mL/mi,; Temp.: 40ºC, Det.:
210nm, UV, Inj. Vol.: 10µL
Case 2: Extraction of 2-Aminopyridine
& Piroxicam from Human Urine
Spiked Urine Sample
Without SPE Cleanup
1. 2-aminopyridine
(4.0µg/mL spike)
0
2
Time (min)
1
0
Spiked Urine Sample
After DSC-MCAX
2
2
Time (min)
2. Piroxicam (10µg/mL spike)
4
% Abs Recovery
+ RSD (n=4)
2-aminopyridine
Piroxicam
Discovery DSCMCAX SPE
102 ± 3.5%
101 ± 1.2%
Vendor A MixedCation SPE
30 ± 52.5%
98 ± 3.2%
Vendor B MixedCation SPE
36 ± 24.2%
83 ± 4.3%
4
SPE: Discovery DSC-MCAX, 100mg/3mL, Vendor A & B Mixed-Cation SPE, 100mg/3mL, HPLC:
Discovery HS F5, 15cm x 4.6mm ID, 5µm, Mobile Phase: 10mM potassium phosphate, pH
6:MeCN (85:15), Flow Rate: 2mL/min, Temp.: 25°C, Det.: 220nm, UV, Inj. Vol.: 10µL
Case 3: Importance of Ion-Exchange
Capacity on Recovery
Drug
DSC-MCAX
Vendor A
Vendor B
Buprenorphine
98.4±1.2
91.3±8.2
85.7±6.4
Codeine-6-glucuronide
89.3±1.1
88.4±10.8
72.3±14.2
Ecgonine Methyl Ester
89.4±2.8
84.2±13.3
76.8±15.5
Isoxsuprine
97.4±1.8
92.3±6.9
84.7±7.7
Levallorpham
98.3±1.9
98.2±2.8
92.6±6.7
6-Monoacetyl Morphine
98.4±1.2
91.3±8.2
85.7±6.4
Morphine
94.5±3.9
84.3±11.2
68.7±15.4
3-Pyridylacetontrile
94.6±4.1
51.4±21.2
33.7±28.5
Vendor A has an ion-exchange capacity of 2.4mg atrazine per gram
and the value for Vendor B is 1.2mg/g. Lower recoveries of these
compounds are assigned to the partial losses in the previous washing
step. The present study evidences that an ion-exchange capacity over
3.6mg atrazine per gram is essential. DSC-MCAX has the value
between 3.6mg/g and 4.8mg/g.
Generic Extraction Protocol 2
Weakly Basic, Polar Basic, Weak Zwitterionic,
or Polar Zwitterionic Drug Generic Extraction Protocol
(for 100mg/1mL or 100mg/3mL SPE tube)
When a target may have (even a minor but detectable) leakage
during loading at neutral pH, the second protocol should be used:
1. Sample Pre-Treatment: Dilute urine (plasma) sample (1mL) with
10mM potassium phosphate buffer (pH3.0) or 10mM acetic acid
buffer (pH3.0) (1mL).
2. SPE Tube Conditioning: Solvate the tube with 1mL methanol
3. SPE Tube Equilibrium: Equilibrium the tube with 10mM pH3.0
potassium phosphate buffer or 10mM acetic acid buffer (1mL)
4. Sample Loading: Load pre-treated urine or plasma (1mL) at a flow
rate of 1mL/min
5. Interference Elution: Elute interference with 1mL 10mM pH3.0
phosphate buffer (or acetic acid buffer) and methanol (1mL).
6. Analyte Elution: Elute analytes with 5% ammonium hydroxide
in methanol
Case 4: Vendor Comparison - Extraction of
Cytosine Using Mixed-Mode Exchange SPE
Discovery DSC-MCAX SPE
Absolute Recovery: 99.2 ± 4.8% (n=3)
Vendor A Mixed-Cation SPE
Average Recovery: 1.8%
0
2
Time (min)
4
Vendor B Mixed-Cation SPE
Average Recovery: 4.2-44.0%
SPE: Discovery DSC-MCAX, 100mg/3mL, Vendor A & B Mixed-Cation SPE: 100mg/3mL, HPLC:
Discovery HS F5, 15cm x 4.6mm ID, 5µm, Mobile Phase: 10mM potassium phosphate, pH 3, Flow
Rate: 1mL/min, Temp.: Ambient, Det.: 280nm, UV, Inj. Vol.: 10µL
Absolute Recoveries of Polar Zwitterions
Compounds by DSC-MCAX
Drug
Alprazolam
Brombuterol
Buprenorphine
Clenbuterol
Diazepam
Ecgonine
Estazolam
Mabuterol
Mapenterol
Methaqualone
Nordazepam
Oxazepam
Oxytetracycline
S(+)-Salbutamol
Terbutaline
DSC-MCAX
98.7 ± 1.9
99.7±1.1
102.4±4.6
104.8±3.3
96.3±2.6
92.9±3.8
101.8±2.8
89.3±4.2
94.5±2.7
99.5±2.2
94.8±3.2
92.8±3.8
87.9±3.3
91.4±2.5
90.4±4.2
Comments
Zwitterions compounds such as
benzodiazepines have been
considered as neutral compounds,
and thus are frequently distributed
into two fractions, i.e. the methanol
fraction and the final fraction when
the mixed-mode SPE cartridges
from the current vendors are used.
However, at pH3.0, the compounds
are positively charged and when
DSC-MCAX is used, they are fully
eluted into the final fraction with
high recoveries.
Generic Extraction Protocol 3
Systematic Fractionation of Neutral and Acidic
Compounds from Basic and Zwitterionic Compounds
The protocol was developed by Takeda et al. (J. Chromatogr. B. 2001,
758, 235) 1g/6mL SPE tube was used via the protocol described below:
1.
2.
3.
4.
5.
6.
7.
Equilibrate the SPE tube by 6mL of methanol
Condition the SPE tube by 6mL of 10mM acetic acid (pH3)
The pH of the biological fluid sample should be adjusted to pH 3.5 or
below in order to keep acidic compounds by the SPE tube via reversedphase mechanism. Under the low pH condition, both basic and
zwitterionic compounds were kept mainly by ion-exchange mechanism.
1mL or more of the sample was loaded by the SPE tube.
Wash the SPE by 6mL of 10mM acetic acid (pH3) and this process should
last at least ten minutes
Dry the SPE tube by 6mL chloroform. The moisture in the SPE tube is
removed.
Recover acid and neutral compounds by 6mL acetone:chloroform (20:80).
Recover the basic and zwitterionic fraction by 6mL 5% ammonium
hydroxide in methanol.
Case 5: Systematic Fractionation and Analysis
of Acidic and Basic Drugs by DSC-MCAX
Sample Prior to DSC-MCAX SPE
DSC-MCAX Fraction 2: Basic Compounds
3
3 5
1
5
0
mAU
mAU
2 4
2
4
Time (min)
6
0
0
0
DSC-MCAX Fraction 1: Acidic Compounds
1
4
Time (min)
0
2
4
Time (min)
6
6
Compound
% Abs Recovery ±
RSD (n=3)
1. Secobarbital
105.8±2.1
2. Keprofen
101.7±1.3
3. Nortriptyline
100.3 ±0.5
4. Naproxen
101.5±0.8
5. Amitryptyline
103.3±1.7
0
mAU
10
24
2
Case 6: Fractionation of Benzoic and
p-Nitrobenzoic Acid Using DSC-MCAX
Sample Prior to
DSC-MCAX SPE
3
DSC-MCAX Fraction 1:
Acidic Compounds
3
1
2
2
0
2
4
Time (min)
1
0
0
2
4
Time (min)
1. o-aminobenzoic acid
2. Benzoic acid
3. p-nitrobenzoic acid
DSC-MCAX Fraction 2:
Only o-aminobenzoic acid
2
Time (min)
4
SPE: Discovery DSC-MCAX, 300mg/3mL, HPLC: Discovery C18, 15cm x 4.6mm ID, 5µm, Mobile
Phase: 0.1% TFA:MeOH (60:40), Flow Rate: 2mL/min, Temp.: Ambient, Det.: 254nm, UV, Inj. Vol.:
10µL
Case 6. Vendor Comparison
Acidic fraction by Vendor B: 92%
o-aminobenzoic acid eluted into the
fraction.
1
mAU
mAU
20
20
Acidic fraction by Vendor A: 22%
o-aminobenzoic acid eluted into the
fraction.
2
Time (min)
4
0
mAU
0 2 4 6 8 10
Basic fraction: 77% o-aminobenzoic acid
1
0
2
Time (min)
4
2
Time (min)
4
Basic fraction: only 8% o-aminobenzoic acid
in the fraction
1
mAU
0.0 0.2 0.4 0.6
0
0
0
1
0
2
Time (min)
4
Comparison Between Silica- and
Polymer-Based Mixed-Mode SPE
Silica-Based Mixed-Mode:
Polymer-Based Mixed-Mode:
1. Biological samples loaded at
pH 3-6. At higher pH, the final
basic and zwitterions fraction
has cleanest background.
Reducing ion-suppression for
LC-MS quantitation is critical.
1. Biological samples loaded at pH 1.
A centrifuge step may be required
to remove sediments. Extract
background is frequently not as
clean as that from silica-based
mixed-mode SPE.
2. A number of successful
applications for systematic
fractionation of acid, neutral
and basic compounds.
2. Few applications on file for
systematical fractionation of acid,
neutral and basic compounds.
3. Adequate hydrophobicity for
non-polar compounds.
3. Many basic compounds such as
morphine were lost during the
methanol wash step due to the
inadequate hydrophobicity.
C8+SCX Mixed-Mode SPE:
Vendor Comparison
Current C8+SCX
Mixed-Mode SPE:
1. The application range is
severely limited to non-polar
basic compounds.
2. Greater risk to lose polar
compounds and/or basic
compounds with lower pKa.
3. Zwitterions compounds may be
partially lost during sample load
and wash steps resulting in
elution distribution across
multiple fractions.
Discovery DSC-MCAX:
1. Optimized ion-exchange
capacity to extract and
recover a wide range of basic
compounds (from weak to
strong, polar to hydrophobic).
2. Reduced risk of prematurely
eluting polar basic, weak
basic and polar zwitterions
compounds.
3. Zwitterionic compounds will
be retained at pH 3.0, and can
be recovered with basified
methanol during final elution.
Conclusion
• Three generic extraction protocols were described in this report.
The first two methods discussed the extraction of basic compounds
at two pH levels. By loading samples at the appropriate pH, strong
ionic and hydrophobic interactions can occur during the extraction
process. As a result, strong wash solvents (e.g., methanol) can be
used to elute co-retained interferences thereby drastically
improving selectivity final elution with basified methanol. The third
method illustrated the use of mixed-cation phases to fractionate
basic and zwitterionic compounds from neutral and acidic
compounds.
• Each application describes the importance of ion-exchange
capacity on the recovery of polar basic and polar zwitterionic
compounds. Unlike most commercially available mixed-cation
phases, Discovery DSC-MCAX offers the ion-exchange capacity
necessary to retain and adsorb all basic and zwitterionic
compounds until final elution. For fractionation applications, poor
ion-exchange capacity can result in partial distribution of polar
basic and zwitterionic compounds across multiple fractions.