1. business and industrial
2. chemicals industry
3. plastics and polymers

Oasis Sample Extraction Products
Purity by SPE
THE MOST WIDELY USED SPE
PRODUCTS IN BIOANALYTICAL
LABORATORIES
Oasis Product Family Provides:
n Highest SPE Recovery
n Cleanest Extracts
n Lowest Matrix Effects
n Lowest Method Variability
* U.S. patents 5,882,521 (1996), 5,976,376 (1998), 6,106,721 (1999), 6,254,780 (2001), 6,322,695 (2001),
6,468,422 (2002), 6,726,842 (2004), 6,773,583 (2004), 6,723,236 (2004), additional patents pending.
2
The novel design of the Oasis® family of solid-phase extraction products
is intended to simplify and improve sample preparation. By combining the
appropriate sorbent, device format and methodology, bioanalytical scientists
are routinely able to achieve robust, reproducible and sensitive SPE methods.
Oasis SPE sorbents covered by nine US patents* are unique in their purity,
stability and retention characteristics. The innovative, patented Oasis µElution
plate, for the first time, enables elution volumes of clean extracts to be as low
as 25 µL while maintaining high analyte(s) SPE recovery.
[ 3 ]
Why Solid-Phase Extraction (SPE)?
For high sensitivity analyses, such as those employing
UPLC®/MS/MS, proper sample preparation can be critical
for minimizing matrix effects and concentrating analytes of
interest. Oasis sample preparation can be used with UPLC/
MS/MS systems to provide the cleanest extracts.
The system shown here integrates the
Xevo™ TQ MS, a highly advanced mass
spectrometer, combining MassLynx™
informatics with innovative ScanWave™
and IntelliStart technologies, and the
Waters ACQUITY UPLC® system.
Sample preparation is a key component of every analytical method (such as LC/MS/MS). By some estimates, 60-80% of the work activity and
operating cost in the analytical laboratory is spent preparing samples for introduction into an instrument.
The importance of sample preparation, particularly SPE, stems from four major concerns—eliminating matrix effects including reducing ion
suppression, lowering method variability, concentrating analyte(s) of interest, and improving analytical system performance.
S P E Off ers Solut ions to T hese Major Samp l e P repa rat ion Conce rns
Eliminat ing Mat rix Eff ec t s
Concent rat ing Analyt e(s) of Int e rest
Solid-phase extraction (SPE) has proven to be an effective tool for
removing interferences, enabling sensitive, selective, and robust
LC/MS/MS analysis.
Frequently, compounds of interest are present at levels too low for
accurate and precise quantitation. SPE enables the enrichment of
selected analytes without concentrating the interferences.
Reducing Ion Su pp ression
Imp rov ing Analyt ical Syst em P e rformance
Oasis SPE products successfully remove phospholipids and lysophospholipids, key contributors to ion suppression in LC/MS/MS
analysis. Removal of these interferences results in lowering the
method variability and increasing the MS response, hence reducing
the LOQ.
Advances in SPE technology, combined with robotic automation,
make SPE not only a cost-effective, but also a time-efficient,
sample-preparation technique. This improves analytical system
performance by:
[ 4 ]
n
Introducing the analyte(s) in an MS compatible solvent
n
Extending analytical column lifetime, reducing
system downtime/maintenance
n
Minimizing ion suppression to improve signal response
CHEMISTRY
Wat ers Innovat ions in Solid- Phase E x t rac t ion
In 1996, Waters revolutionized SPE technology with the introduction of Oasis HLB, the
first water-wettable—yet hydrophobic—polymeric sorbent, permanently changing SPE
practice. The Oasis family includes five patented SPE chemistries for all your sample
preparation needs:
n
Oasis HLB:
Hydrophilic-Lipophilic-Balanced reversed-phase
sorbent for acids, bases and neutrals
n
Oasis MCX:
Mixed-mode Cation-eXchange
reversed-phase sorbent for bases
n
Oasis MAX:
Mixed-mode Anion-eXchange
reversed-phase sorbent for acids
n
Oasis WCX:
Mixed-mode Weak Cation-eXchange reversed-phase
sorbent for strong bases and quaternary amines
n
Oasis WAX:
Mixed-mode Weak Anion-eXchange reversed-phase
sorbent for strong acids (e.g., sulfonates)
OASIS MAX
OASIS MCX
OASIS HLB
pKa <1
1 meq/g
Hydrophilic
Retention of Polars
OASIS WCX
pKa >18
0.25 meq/g
Lipophilic
RP Retention
OASIS WAX
Water-wettable
Stable from pH 0–14
No silanol interactions
pKa ~6
0.6 meq/g
pKa ~5
0.75 meq/g
[ 5 ]
Chemistry
Oasis HLB Chemist ry
Unique Water-Wettable Oasis HLB Copolymer
Universal Sorbent for Acidic, Neutral,
and Basic Compounds
Oasis HLB is a Hydrophilic-Lipophilic-Balanced, water-wettable,
reversed-phase sorbent for all your SPE needs. It is made from a
specific ratio of two monomers, hydrophilic N-vinylpyrrolidone and
lipophilic divinylbenzene. It provides superior reversed-phase capacity
with a neutral polar ‘hook’ for enhanced retention of polar analytes.
Waters has built a family of SPE sorbents which inherit some key
features of this unique substrate: stability at pH extremes and in a
wide range of solvents, extraordinary retention of polar compounds,
and a relative hydrophobic retention capacity 3x higher than that of
traditional silica-based SPE sorbents like C18.
N-VINYLPYRROLIDONE
DIVINYLBENZENE
HYDROPHILIC-LIPOPHILIC BALANCE
Water-wettable Oasis sorbents exhibit excellent retention capacity for
a wider polarity spectrum of analytes, even if the sorbent bed runs
dry during conditioning or sample loading. This means that your SPE
methods will be more rugged and robust, precluding the need for
repeat sample preparation.
Specific Surface Area: 810 m2/g, Average Pore Diameter: 80 Å
Total Pore Volume: 1.3 cm3/g, Average Particle Diameter: 5 µm, 15 µm, 25 µm, 30 µm or 60 µm
Effect of Drying on Recovery
No impact of sorbent drying on HLB
High, consistent recovery
The advantage of having higher retention capacity [k] is that more
analytes are retained with less breakthrough, improving the recovery
and overall reproducibility of your SPE method.
Oasis HLB Cartridge (30 mg)
C18 Cartridge (100 mg)
100
100
Available in five particle sizes [60 µm, 30 µm, 25 µm, 15 µm, and
5 µm], Oasis HLB sorbent, in cartridge, plate, or column format,
allows you to select the appropriate product based on the volume,
viscosity, and turbidity of your sample.
80
Procainamide
Acetaminophen
Ranitidine
Propranolol
Doxepin
60
40
20
Exceptional Batch-to-Batch Reproducibility
0
Long-term, batch-to-batch reproducibility of traditional silica-based
mixed-mode sorbents may be compromised by hydrolytic instability at
pH extremes, relatively low ionic capacity, and difficulties in bonding.
To assure performance consistency for large projects, careful analysts
test for suitability, and then reserve successful, specific lots of sorbent.
0
% Recovery
% Recovery
80
5
10
Drying Time (minutes)
60
40
20
0
0
4
Drying Time (minutes)
8
Batch-to-Batch Reproducibility of Oasis HLB Sorbent —
A Decade of Dramatic Consistency
No Batch Reservations Needed: Consistently high recoveries over more than 10 years of production.
% Recovery
105
95
85
Recovery for:
Procanamide – 1.90% RSD
Oasis sorbents have demonstrated excellent long-term, batch-tobatch reproducibility for over ten years. Our careful process design
and stringent quality controls have set a new standard in batch-tobatch and lot-to-lot reproducibility for SPE sorbents. Our entire Oasis
family of sorbents and devices is manufactured in Waters ISO 9000
Ranitidine – 1.68% RSD
Acetaminophen – 1.65% RSD
registered facilities in compliance with cGMP guidelines of the U.S.
Food and Drug Administration for Class 1 medical devices.
Multiple batches of Oasis HLB, MCX, MAX, WCX, and WAX have
each been used successfully on validated bioanalytical assays in
a regulated laboratory environment.
[ 6 ]
Chemistry
Oasis MC X Chemist ry
Mixed-Mode Cation-eXchange and
Reversed-Phase Sorbent
Drug–Sorbent Interactions on Oasis MCX Sorbent
Highly selective retention enables much stronger washes,
resulting in very clean extracts.
High Selectivity and Sensitivity for Basic Compounds
Oasis MCX is designed to overcome the limitations of traditional
silica-based mixed-mode SPE sorbents. It is a strong mixed-mode
cation exchange, water-wettable, polymeric sorbent made reproducibly by a novel, Waters-patented process. Oasis MCX provides
dual modes of retention—ion exchange, and reversed phase—on
a single, clean, stable, high-surface-area, organic co-polymer that
is stable from pH 0 to 14.
O
SO–3
SO–3
Strong cationexchange mode
Best retention at least
2 pH units below pKa
In addition to the pH stability typical of DVB polymers, Oasis MCX
has far greater binding capacity than that of silica-based mixedmode SPE sorbents. The ability to fully manipulate pH [0–14]
during the development, optimization, and use of SPE methods
on mixed-mode sorbents enables not only fast, straightforward,
method development, but also ensures very rugged and robust
procedures. There is no analyte breakthrough or loss of recovery
due to dissolving silica particles at high pH or cleaving bonded
phase at low pH.
Effective use of mixed-mode SPE sorbents for extraction of basic
compounds from biological matrices (such as plasma, urine, bile,
and tissue homogenates) requires high capacity (both reversedphase for the retention of interferences and ion-exchange for
selective retention of analytes). Typical silica-based mixed-mode
sorbents are synthesized in a complex process that is hard to
N
MCX sorbent
Mixed-mode Cation eXchange
of the analyte
Reversed-phase
interaction
+
CH 3
N
H H
O
CH 3
OH
Sulfonic-acid-cationexchange capacity:
1.0 meq/g
Propranolol
(basic drug)
control, and they have relatively low exchange capacities, ranging
from 0.06–0.2 meq/g.
The novel, well-controlled Waters production process reproducibly
delivers Oasis MCX with 1.0 meq/g of sulfonic-acid-ion-exchange
capacity. Two particle sizes [60 µm and 30 µm] are available.
Oasis MAX Chemist ry
Mixed-Mode Anion-eXchange and
Reversed-Phase Sorbent
Drug–Sorbent Interactions on Oasis MAX Sorbent
Highly selective retention enables much stronger washes,
resulting in very clean extracts.
High Selectivity and Sensitivity for Acidic Compounds
Oasis MAX is designed to overcome the limitations of traditional
silica-based mixed-mode SPE sorbents. It is a strong mixed-mode
anion-exchange, water-wettable, polymeric sorbent stable from pH
0 to 14. Now you can employ reliable SPE in methods to detect,
confirm, or quantify acidic compounds and/or acidic metabolites in
biological fluids. SPE procedures using the selectivity and ruggedness of Oasis MAX enable separation of analytes from complex
samples into two fractions: acidic compounds and basic/neutral
compounds. Fractionated extracts can be analyzed by multiple
analysis methods or even multiple techniques [LC/MS and GC/MS].
O
MAX sorbent
Mixed-mode Anion eXchange
N
CH 3
+
CH 2 N C H
4 9
R+
CH 3
Strong anionexchange mode
Best retention at least
Reversed-phase
retention
The novel, well-controlled Waters production process reproducibly
delivers Oasis MAX with 0.25 meq/g of quaternary-amine-ion-exchange capacity. Two particle sizes [60 µm and 30 µm] are available.
[ 7 ]
S
COO
Quaternary-amineanion-exchange capacity:
0.25 meq/g
O
CH 3
–
2 pH units above pKa
of the analyte
Suprofen
(acidic drug)
Chemistry
Oasis WC X Chemist ry
Mixed-Mode Weak Cation-eXchange
and Reversed-Phase Sorbent
Drug-Sorbent Interactions on Oasis WCX Sorbent
Highly selective retention enables much stronger washes,
resulting in very clean extracts.
High Selectivity and Sensitivity for
Strongly Basic Compounds
Oasis WCX is designed to provide superior sample preparation for
strong bases and quaternary amines. It is a weak-cation-exchange,
mixed-mode, water-wettable, polymeric sorbent, stable from pH
0 to 14. Oasis WCX has all the advantages of Oasis HLB. Rugged
and highly selective SPE methods using Oasis WCX detect, confirm
and quantify strongly basic compounds and quaternary amines in
biological fluids.
O
N
WCX sorbent
Weak Cation eXchange
COO Weak cation-exchange
interaction
CH 3 O
The novel, well-controlled Waters production process reproducibly
delivers Oasis WCX with 0.75 meq/g of carboxylic-acid-ion-exchange
capacity. Three particle sizes [60 µm, 30 µm, and 5 µm] are available.
Reversed-phase
interaction
H3 C
CH 3
O
N
+
CH 3
CH 3
Carboxylic-acid-cationexchange capacity: 0.75 meq/g
Oasis WAX Chemist ry
Mixed-Mode Weak Anion-eXchange
and Reversed-Phase Sorbent
Drug-Sorbent Interactions on Oasis WAX Sorbent
High Selectivity and Sensitivity for
Strongly Acidic Compounds
Highly selective retention enables much stronger washes,
resulting in very clean extracts.
Oasis WAX is designed to provide superior sample preparation for
strong acids. It is a weak-anion-exchange, mixed-mode, waterwettable, polymeric sorbent, stable from pH 0 to 14. Oasis WAX
has all the advantages of Oasis HLB. Rugged and highly selective
SPE methods using Oasis WAX detect, confirm, and quantify strongly
acidic compounds in biological fluids.
O
N
WAX sorbent
Weak Anion eXchange
+
NH
SO–3
The novel, well-controlled Waters production process reproducibly
delivers Oasis WAX with 0.6 meq/g of piperazine-ion-exchange
capacity. Three particle sizes [60 µm, 30 µm, and 5 µm] are available.
Reversed-phase
interaction
Piperazine-anion-exchange
capacity: 0.6 meq/g
[ 8 ]
+
NH2
Weak anion-exchange
interaction
FORMAT
Oasis Family of S P E P roduc t s
The entire Oasis family of sorbents and devices is manufactured in Waters ISO 9000
registered facilities in compliance with cGMP guidelines of the U.S. Food and Drug
Administration for Class 1 medical devices.
n
Oasis µElution Plates
n
Oasis 96-Well Extraction Plates
n
Oasis Syringe-Barrel Cartridges
n
Oasis Glass Cartridges
n
Oasis On-Line Columns
[ 9 ]
FORMAT
Oasis Family of SP E P roduc t s
Oasis μElut ion P lat es
n Patented µElution plate design*
n Enabling technology facilitates elution
volumes as low as 25 µL
n No evaporation and reconstitution necessary;
just elute and shoot
n Ideal for small sample volumes
n Up to a 15x increase in concentration
n Compatible with most liquid-handling
robotic systems for automated, reliable
high-throughput SPE (HT-SPE)
Oasis 96-W ell E x t rac t ion P lat es
n Innovative two-stage well design
(1999 R&D 100 Award winner)
n High throughput and high recovery
n Available in 5 mg, 10 mg, 30 mg,
and 60 mg per well
n Compatible with most liquid-handling
robotic systems for automated, reliable
high-throughput SPE (HT-SPE)
* U.S. patent 6,723,236
[ 10 ]
FORMAT
Oasis Syringe-Ba rrel Ca rt ridges
n Ultra-clean syringe barrel and frits
n Available in cartridges ranging from 1 cc to 35 cc
n Available from 10 mg to 5 g of sorbent per cartridge
n Flangeless syringe-barrel cartridges available:
1 cc, 3 cc, 6 cc
n Also available: Plus cartridges with Luer inlet
hub and outlet tip, 225 mg
Oasis Glass Ca rt ridges
n Ultra-clean glass syringe with Teflon ® frit
n Endocrine disruptors analysis at part-per-trillion levels
n Available in 5 cc (200 mg) configuration
Oasis On-Line Columns
n Rugged, reproducible, ultra-fast, on-line analysis
n Compatible with all on-line analysis systems
n Wide choice of configurations, particle sizes,
and sorbent chemistries
[ 11 ]
FORMAT
Oasis �Elut ion Plat es
Minimizing SPE elution volume is critical for SPE performance,
recovery, and precision. Normal elution volume range for an Oasis
µElution plate is only 25–50 µL, far lower than the 75–300 µL
typical for SPE disk technology.
Wat ers Lat est Innovat ion in S P E Technology
n Elute in as little as 25 µL
Reproducible elution in only 25 µL enhances and streamlines SPE
methods. SPE can be performed on very small sample volumes.
Sample enrichment increases as much as 15-fold. A time-consuming
step for eluate concentration by evaporation is no longer needed—
neither is evaporation/reconstitution if the eluting solvent is properly
chosen to be compatible with the LC/MS mobile phase. The innovative
features of Oasis µElution plates enable sensitive, robust, reproducible results without evaporation and reconstitution. Analytical
performance, in terms of sensitivity, selectivity, and ruggedness,
is superior.
n No evaporation and reconstitution
n Ideal for small sample volumes
n Up to a 15x increase in concentration
Now you can confidently perform SPE cleanup and analyte enrichment of very small sample volumes (10-25 µL) up to a maximum of
375 µL. The Waters Oasis µElution plate combines patented plate
design, proven Oasis chemistries, and straightforward protocols that
deliver high analyte recovery and clean extracts in elution volumes
as low as 25 μL.
Comparison of µElution vs. Disk Technology
Elution Technology
Disk Technology
Wide and Short
Sorbent Bed
Narrow and Tall
Sorbent Bed
Oasis µElution Technology vs. Disk Technology
Ratio Bed H/D = 1.15
Ratio Bed H/D = 0.13
Vacuum
Vacuum
There are two predominant low-elution SPE technologies in the
marketplace today: SPE Disk and Oasis µElution. These technologies
differ greatly in performance primarily because of three features:
aspect ratio, holdup volume and elution volume.
Recovery Comparison of µElution vs. Disk Technology
A small holdup volume is necessary to minimize elution volume.
SPE disks have holdup volumes in the 35–65 µL range, compared
to that for an Oasis µElution bed plus frits which measures as
little as 15 µL. Having 2–4x less holdup volume minimizes elution
volume and reduces analyte loss during elution, improving both
recovery and precision.
T he innovative features of the Oasis µElution plates enable sensitive,
robust, reproducible results without evaporation and reconstitution.
Oasis µElution Technology
Disk Technology
>85% recovery in 25 µL
25 µL
Minimum of 100 µL needed for best result
75 µL
75 µL
75 µL
100
Elution Volume
80
% Recovery
There is a correlation between a sorbent bed’s aspect ratio [height to
diameter, H/D] and its performance in an SPE device. An H/D ratio
less than 1.0 often compromises extraction efficiency. In SPE disk
design (e.g. membrane/glass-fiber disk), a small mass of sorbent is
embedded in a thin support structure having good flow properties
but a small aspect ratio, H/D=0.13. In contrast, the Oasis µElution
technology uses an internally tapered well, packed with high capacity
Oasis sorbent, having an aspect ratio, H/D=1.15, nearly 9x that
of SPE disk technology. An Oasis µElution plate well functions
more like a chromatography column, minimizing loss of analyte
during critical steps in the SPE procedure and enhancing overall
extraction efficiency.
60
40
20
0
Oasis HLB
Brand E C18
Brand E UR
Brand S C18
µElution plate
100 µL Spiked Saline
acetaminophen
practolol
[ 12 ]
N-acetylprocainamide
betamethasone
caffeine
naproxen
amitriptyline
propranolol
FORMAT
µElution Plate Loading Capacity
SPE Protocol for
Oasis MCX µElution 96-Well Plate
SPE device capacity is defined as the total mass of analytes and
Condition:
200 µL CH3OH
endogenous sample components retained by the sorbent bed under
Equilibrate:
loading conditions. Breakthrough will occur when the capacity of
200 µL H2O
the sorbent bed is exceeded. The physicochemical properties of
Load:
Various volumes of:
- Plasma diluted 1:1 with 4% H3PO4 in H2O
- Urine diluted 1:1 with H2O
Oasis sorbents are designed to provide exceptionally high loading
capacity, even though each well in a Waters Oasis µElution plate
Wash 1:
contains only 2 mg of Oasis sorbent.
200 µL 2% CHOOH in H2O
Wash 2:
To determine the Oasis μElution plate capacity, increasing volumes
200 µL CH3OH
of plasma and urine samples (from 50 µL to 350 µL in 50 µL
Elute:
2 x 25 µL 5% NH4OH in 60:40
CH3CN:CH3OH
increments) were spiked with 200 ng/mL imipramine (non polar
base) and 200 ng/mL atenolol (polar base). The plasma aliquots
Dilute:
50 µL H2O
were diluted 1:1 with 4% aqueous H 3 PO 4 and the urine aliquots
Inject:
5 µL
were diluted 1:1 with H2O and then loaded onto the µElution plate.
SPE recovery was calculated and plotted for each loading level.
SPE Recovery for 200 ng/mL Imipramine and 200 ng/mL Atenolol on Oasis MCX µElution Plate
100
80
80
60
60
n=8
% Recovery
Imipramine
(non-polar)
100
% Recovery
Atenolol (polar)
SPE Recovery for Polar and
Non-Polar Analytes in Urine Example
40
40
20
20
0
0
50
100 150
200 250 300
350 µL
Matrix Effects for Polar and
Non-Polar Analytes in Plasma
Ion Suppression Ion Enhancement
SPE Recovery for Polar and
Non-Polar Analytes in Plasma Example
50
Loaded Plasma Volumes (Undiluted)
100 150
200 250 300
Loaded Urine Volumes (Undiluted)
350 µL
40
30
20
10
0
-10
-20
-30
-40
50
100 150 200 250 300
350 µL
Loaded Plasma Volumes (Undiluted)
Oasis 96-W ell Plat es
Ve rsat il e, High-T h rough put Oasis 96-W ell E x t rac t ion P lat es
Waters award-winning plate design, with five chemistry and four
sorbent-mass options, provides flexible high-throughput SPE in a
single device. The Oasis 96-well plates are designed to be used
on many manifold configurations and most robotic liquid handling
systems. Oasis sorbents’ unique balance of hydrophobicity and water-
wettability means you will never have to worry about poor results
if individual wells of the 96-well plate dry out. As always, you can
expect Oasis SPE products to perform reliably, delivering high and
reproducible recoveries for a wide range of analytes, including polar
and basic compounds, with RSDs less than 5% [n=96].
Waters 96-Well Plate Design
1999 R&D100
Award Two-Stage
Well-Design
5 mg
10 mg
30 mg
60 mg
By varying frit size and/or placement, the same plate may be filled with various
quantities of sorbent per well. Our design permits optimal recoveries, even with
low sorbent weights for smaller elution volumes.
[ 13 ]
FORMAT
Oasis On-Line Columns
On-Line SP E Columns for LC /MS/MS
2
There are three HPLC Oasis On-Line column configurations designed
to fit all your on-line-analysis needs.
1
n The Oasis Cartridge Column fits into a Sentry™ holder
that features a finger-tight fitting for fast, convenient
replacement [1]
n The Oasis Direct-Connect Column can be screwed directly
into a switching valve or connected to fittings like those
for a conventional HPLC column [2].
n The Oasis Column features traditional HPLC column
3
fittings and hardware [3].
All of these formats are available with the five Oasis patented sorbents (HLB, MCX, MAX, WCX, WAX) in a wide choice of particle sizes and
dimensions. The Oasis On-Line columns make it possible to analyze a specific analyte in a sample matrix when combined with appropriate
Waters narrow-bore analytical columns (such as XBridge™, SunFire™, Atlantis ®, XTerra ®, or Symmetry ® columns).
On-Line System Configuration
Oasis
On-Line Column
Isocratic
HPLC
Waste
10 Port Valve
2 positions
Gradient
HPLC
Mass
Spectrometer
Narrow Bore
Analytical Column
10
1
2
8
3
7
4
5
515
System
9
10
9
2
Oasis
1
Waste
INJECTION
POSITION
515
System
Waste
6
8
3
7
4
Alliance HT
Alliance HT
Mass
Spectrometer
Mass
Spectrometer
Analytical
Column
5
6
Analytical
Column
[ 14 ]
Oasis
LOAD
POSITION
METHODOLOGY
OASIS Met hodology
SPE method development starts with knowledge of the sample matrix—the nature and
relative concentration of the analytes of interest as well as of potentially interfering
endogenous compounds. Then, judicious choices of sorbent and solvents for load, wash,
and elute steps create rugged, reliable, selective protocols.
Waters application chemists have devised several approaches to streamline this process.
n
Oasis 2x4 Method
n
Oasis 2x4 Optimized Methodology
n
Oasis 2x4 Method Optimized
for µElution Plate
n
Oasis HLB Generic Method
n
Oasis HLB Optimized Methodology
n
Oasis Advanced Methodology
[ 15 ]
Methodology
3A: Oasis 2x4 Met hod
Oasis 2x4 Method
Only 2 protocols and 4 sorbents to
analyze all types of compounds:
acids, bases, and neutrals
The Oasis 2x4 Method is a simple, logical approach
to the selection of an SPE sorbent and protocol. Two
protocols and four sorbents provide the flexibility
to extract acids, bases, and neutrals with high SPE
recoveries while removing matrix components that
may interfere with analysis.
For Bases
pKa 2-10:
Use Oasis MCX
For Strong Acids
pKa <1:
Use Oasis WAX
For Acids
pKa 2-8:
Use Oasis MAX
For Strong Bases
pKa >10:
Use Oasis WCX
Follow the simple steps outlined in this flow chart to
achieve high recoveries and the cleanest extracts:
Apply Protocol 1
Apply Protocol 2
Prepare Sample
Prepare Sample
n Select one of the four Oasis sorbents.
Condition/Equilibrate
Load Sample
Condition/Equilibrate
Load Sample
n Apply the indicated Protocol [1 or 2].
Wash:
2% HCOOH
Wash:
5% NH4OH
n Characterize your analyte [Neutral,
Acid or Base, pKa].
n Determine SPE recoveries by LC analysis.
Elute 1:
100% CH3OH
Weaker
Acids
Elute 2:
5% NH4OH in CH3OH
Base
Note that neutral analytes can be isolated from any of the four
sorbents in the Elute 1 step of either protocol. Choose the particular ion-exchange sorbent that is best at removing specific matrix
interferences. A good example of this is shown below.
Choosing Optimum Sorbent and Protocol for Neutral
Compounds Example: Prednisone in Plasma
SPE Recovery
0
%
50
100%
3.08
Oasis MCX — Protocol 1
%
100
Protein
Precipitation
3.26
3.16
2.52
2.60
2.50
Oasis MAX — Protocol 2
%
100
2.522.60
Oasis WAX — Protocol 1
2.50
2.60
%
100
Strong Acid
Oasis WCX — Protocol 2
1: MRM of 5 Channels ES+
TIC
2.55e7
%
0
1.75
2.00
2.25
2.50
2.75
3.00
3.25
2% Phospholipids
1: MRM of 5 Channels ES+
TIC
2.55e7
9% Phospholipids
1: MRM of 5 Channels ES+
TIC
2.55e7
11% Phospholipids
Recovery is high on all four Oasis sorbents,
but removal of phospholipids, a primary cause
of matrix ion suppression, is best on Oasis MCX.
Protein precipitation is a poor choice for sample
preparation prior to LC/MS/MS analysis.
3.50
Strong Base
The Oasis Sorbent Selection Plates and Cartridge Kit enable rapid
development of SPE methods for LC/MS analysis. Having all four
Oasis ion-exchange sorbents [MCX, MAX, WAX and WCX] in a
single plate is convenient for scouting the best ways to accomplish
efficient isolation of unknown, zwitterionic compounds, or mixtures
of analytes with different retention/elution properties.
100% Phospholipids
1: MRM of 5 Channels ES+
TIC
2.55e7
1: MRM of 5 Channels ES+
TIC
2.55e7
2.52
2.60
Acid
Oasis Sorbent Selection Plates and Cartridge Kit
Evaluating Oasis 2x4 Method for Cephalexin
0
100
Elute 2:
2% HCOOH in CH3OH
Monitoring 5 Phospholipid
MRM Transitions
Cleanliness of 100% MeOH Elute 1
100
Weaker
Bases
Elute 1:
100% CH3OH
Neutrals
3.75
12% Phospholipids
4.00 Min
O
H
Oasis WAX
OH
NH
pKa = 7.3
2
H H
N
H
O
Oasis MAX
Protocol 2 – Elute 2
H
O
Oasis WCX
Prednisone
S
N
HO
Protocol 1 – Elute 1
OH
H
O
100%
Oasis MCX
Protocol 1 – Elute 2
O
SPE Recovery
50
O
pKa = 2.6
Cephalexin
[Cephalosporin antibiotic]
A difficult amphoteric analyte
Protocol 2 – Elute 1
Aliquots of prepared sample processed using Oasis 2x4 Method protocol designated for each
of 4 sorbents. Eluates from Elute 1 and Elute 2 steps analyzed by LC/MS/MS.
Clearly, Oasis MCX is the sorbent of choice.
[ 16 ]
Methodology
Oasis 2x4 Method—Proof of Concept
To demonstrate the logic, simplicity, and effectiveness of the
Oasis 2x4 Method, five rat plasma samples were prepared, each
containing one of these characterized test analytes:
n Imipramine, a base – pKa of conjugate acid = 9.4
n Ibuprofen, an acid – pKa = 5.2
n Decanesulfonic acid, a strong acid – pKa < 0.5
The neutral analyte was processed on all four sorbents, as shown
on the previous page. Of the four method options, Oasis MCX with
Protocol 1 proved superior at removing nearly all the phospholipids, eliminating this major source of matrix effects, a known cause
of ion suppression, loss of sensitivity, and inaccurate quantitation
in LC/MS analysis.
Essentially quantitative recovery and excellent cleanup efficiency
were achieved for each of the ionic or ionizable test analytes when
the recommended Oasis 2x4 Method sorbent/protocol combination
was used. These results are shown in the four figures below.
n Valethamate, a quaternary amine [strong base]
– pKa of conjugate acid > 12
n Prednisone, a neutral compound
Each plasma sample was diluted [1/1, v/v] and acidified with phosphoric acid [4% in water]. Respective aliquots were then processed
using the protocol and the Oasis ion-mixed-mode sorbent designated
by the Oasis 2x4 Method for the corresponding analyte type. LC/MS/
MS analysis was used to determine SPE recoveries.
Oasis 2x4 Method Test on MCX: Base Isolation
Oasis 2x4 Method Test on WAX: Strong Acid Isolation
For Bases
pKa 2–10:
Use Oasis MCX
Oasis MCX
For Strong Acids
pKa < 1:
Use Oasis WAX
Oasis WAX
Protocol 1
Protocol 1
Imipramine
Decanesulfonic
acid
SPE Recovery: 108%
n=4, 2–18% RSD
SPE Recovery: 94%
n=8, 3–6% RSD
pKa <1
3.19
2.71
pKa ~6
MRM ES+
281.2 > 85.95
4.16e6
N
N
O
Imipramine [pKa = 9.4]
0
1
3
pKa ~18
0
1
3
5 min
Oasis 2x4 Method Test on WCX: Strong Base Isolation
For Acids
pKa 2–8:
Use Oasis MAX
Oasis MAX
O– Na+
Decanesulfonic acid [pKa <0.5]
5 Min
Oasis 2x4 Method Test on MAX: Acid Isolation
S
MRM ES+
221.1 > 79.8
2.85e3
O
For Strong Bases
pKa > 10:
Use Oasis WCX
Oasis WCX
Protocol 2
Protocol 2
Ibuprofen
Valethamate
SPE Recovery: 100%
n=6, 6–13% RSD
SPE Recovery: 106%
n=4, 9–13% RSD
pKa ~5
2.63
2.71
O
MRM ES+
205 > 161
1.34e4
OH
O
N+
Br –
MRM ES+
306.2 > 163
2.10e6
O
Ibuprofen [pKa = 5.2]
0
1
3
Valethamate [pKa >12]
5 min
[ 17 ]
0
1
3
5 min
Methodology
3B: Oasis 2x4 Opt imized Met hodology
Optimization of the Oasis 2x4 Method on mixed-mode sorbents
[MCX, MAX, WCX, WAX] takes full advantage of two different
chromatographic separation mechanisms: reversed phase and ion
exchange. Changes in pH are used to manipulate the respective
ionization states of the exchange sites on the sorbent and of the
acidic or basic moieties in analytes and interferences. In this
way, retention can be directed selectively to occur through either
hydrophobic or ionic interactions. Simultaneously, organic solvent
concentration is modified to fine-tune elution based upon rela-
tive hydrophobicity. The result is cleaner extracts that reduce
matrix effects, enhance the sensitivity, and lower the viability
of the analytical method.
In the examples that follow, we will demonstrate how straightforward
wash-elute studies, in which the percentage of organic solvent is
systematically varied, are used to determine the optimal composition of SPE mobile phases for selective wash and elution steps.
Similar experiments may be designed for another variable such
as pH or ionic strength.
3B–1: Oasis MCX 2x4 Method — Optimized for Bases
When the greatest selectivity and sensitivity for basic compounds
[pKa 2–10] is required to achieve the lowest limits of detection
[LOD] and quantitation [LOQ], optimization of Protocol 1 is recommended. As an example, a wash-elute study was performed to
determine the optimal retention and elution parameters for imipramine on Oasis MCX [below left]. These experimental data were
then used to optimize a selective Oasis MCX SPE method [below
right] to remove neutral, acidic, and basic interferences.
Optimized Oasis MCX Method for Imipramine
Wash-Elute Study
Imipramine
N
[% CH3OH in H2O] : [conc. NH4OH] 95:5 v/v
N
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Condition/Equilibrate:
1 mL CH3OH/H2O
Basic drug is loaded in
ionized [or neutral] form
if sample was pretreated
with acid [or base]
Load sample:
1 mL plasma or urine
Locks ionized drug on
strong cation-exchange
sites; removes proteins
and salts
Indicates Peak Area
of Analyte in LC Analysis
of Eluate
Maximum
% Organic Wash
20
30
40
50
60
70
80
90
Removes interfering
acids [unionized] and
neutral compounds and
acidic interferences
Wash 2:
Minimum
% Organic Elute
1 mL 100% CH3OH
Neutralizes bases;
removes more polar
basic interferences
0
10
Wash 1:
1 mL 2% HCOOH
100
Wash 3:
1 mL 5% NH4OH in CH3OH/H2O 55/45
% CH3OH in H2O
Basic analyte is
eluted; hydrophobic
basic interferences
are retained
Elute:
Experiment for Oasis MCX 96-Well Plate 30 mg
1 mL 5% NH4OH in CH3OH/H2O 95/5
A second example demonstrates the dramatic benefits of an optimized
Oasis MCX method for determining methamphetamine in urine. A
wash-elute study was used to find the best concentration of triethylamine [TEA] for Wash 3 solution [below left]. In this way, tertiary
and aromatic amine interferences were removed selectively. As shown
in the LC/UV data [below right], impressive RSDs were achieved for
quantitation of the primary and secondary amine analytes.
Oasis MCX Urine Application:
Amphetamine and Methamphetamine
Optimized Oasis MCX Method for Quantitation
of Methamphetamine in Urine
Load:
10 mL spiked urine
(acidified with 100 µL 5 N HCI)
Locks ionized drug on
strong cation-exchange
sites; removes proteins
and salts
Wash 1:
Wash 2:
2 mL 100% CH3OH
Weak base removes
tertiary and
aromatic amines
Blank without
T EA Wash
0.01
2 mL 5% CH3OH in 0.1 N HCI
Removes interfering
acids [unionized] and
neutral compounds and
acidic interferences
AU
1
0.00
Wash 3:
1.5 mL 2.5% TEA in CH3OH
0
Elute:
4 µg/mL n=6
1. Amphetamine
2. Methamphetamine
3. Phentermine (I.S.)*
* Internal Standard
2 mL 5% NH4OH in CH3OH
[ 18 ]
2
Recovery
RSD
96.8% 1.35%
90.4% 4.01%
—
—
4
3
2
Blank with
2.5% T EA Wash
Spiked Sample
with 2.5% T EA Wash
6
NH
Amphetamine
2
H
N
Methamphetamine
NH
Phentermine
2
Methodology
3B–2: Oasis WAX 2x4 Method — Optimized for Strong Acids
When the greatest selectivity and sensitivity for strong acids
[pKa <1] is required to achieve the lowest limits of detection [LOD]
and quantitation [LOQ], optimization of Protocol 1 is recommended.
As an example, a wash-elute study was performed to determine the
optimal retention and elution parameters for decanesulfonic acid
on Oasis WAX [below left]. These experimental data were then
used to optimize a selective Oasis WAX method [below right] to
remove neutral, basic, and acidic interferences.
Wash-Elute Study
Optimized Oasis WAX Method for Decanesulfonic Acid
[% CH3OH in H2O] : [conc. NH4OH] 95/5 v/v
O
S
O
O– Na+
Decanesulfonic Acid
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Condition/Equilibrate:
1 mL CH3OH/H2O
Load sample:
1 mL plasma or urine
Wash 1:
Indicates Peak Area
of Analyte in LC Analysis
of Eluate
1 mL 2% HCOOH
Maximum
% Organic Wash
Minimum
% Organic Elute
Removes interfering
bases [ionized] and neutral
compounds [including
unionized acids]
0
10
20
30
40
50
60
70
80
90
100
Wash 2:
1 mL 100% CH3OH
Wash 3:
1 mL 5% NH4OH in CH3OH/H2O 25/75
% CH3OH in H2O
Strongly acidic analyte is
eluted; more hydrophobic
acidic interferences
are retained
Experiment for Oasis WAX 96-Well Plate 30 mg
Ionizes weak anionexchange sites to
lock on analytes; removes
proteins and salts
Neutralizes anionexchange sites; removes
more polar strongly
acidic interferences
Elute:
1 mL 5% NH4OH in CH3OH/H2O 80/20
3B–3: Oasis MAX 2x4 Method — Optimized for Acids
When the greatest selectivity and sensitivity for acidic compounds
[pKa 2–8] is required to achieve the lowest limits of detection [LOD]
and quantitation [LOQ], optimization of Protocol 2 is recommended.
As an example, a wash-elute study was performed to determine the
optimal retention and elution parameters for ibuprofen on Oasis MAX
[below left]. These experimental data were then used to optimize a
selective Oasis MAX SPE method [below right] to remove neutral,
basic, and acidic interferences.
Wash-Elute Study
Optimized Oasis MAX Method for Ibuprofen
OH
[% CH3OH in H2O] : [conc. HCOOH] 98/2 v/v
10%
20%
30%
40%
50%
60%
70%
O
80%
90%
Ibuprofen
100%
Condition/Equilibrate:
1 mL CH3OH/H2O
Acidic drug is loaded in
ionized [or neutral] form
if sample was pretreated
with base [or acid]
Indicates Peak Area
of Analyte in LC Analysis
of Eluate
Wash 1:
Maximum
% Organic Wash
1 mL 5% NH4OH
Minimum
% Organic Elute
Removes interfering
bases [unionized] and
neutral compounds
0
10
20
Load sample:
1 mL plasma or urine
30
40
50
60
70
80
90
1 mL 100% CH3OH
Wash 3:
100
1 mL 2% HCOOH in CH3OH/H2O 45/55
% CH3OH in H20
Experiment for Oasis MAX 96-Well Plate 30 mg
Wash 2:
Acidic analyte is eluted;
more hydrophobic acidic
interferences are retained
[ 19 ]
Locks ionized analyte
on strong anion-exchange
sites; removes
proteins and salts
Elute:
1 mL 2% HCOOH in CH3OH/H2O 90/10
Neutralizes acids;
removes more polar
acidic interferences
Methodology
3B–4: Oasis WCX 2x4 Method—Optimized for Strong Bases
When the greatest selectivity and sensitivity for strong bases such
as quaternary amines [pKa >10] is required to achieve the lowest
limits of detection [LOD] and quantitation [LOQ], optimization of
Protocol 2 is recommended. As an example, a wash-elute study was
performed to determine the optimal retention and elution parameters for valethamate on Oasis WCX [below left]. These experimental
data were then used to optimize a selective Oasis WCX SPE method
[below right] to remove neutral, acidic, and basic interferences.
Optimized Oasis WCX Method for Valethamate
Wash-Elute Study
O
O
[% CH3OH in H2O] : [conc. HCOOH] 98/2 v/v
10%
20%
30%
40%
50%
60%
70%
80%
90%
N+
Br–
100%
Valethamate
Condition/Equilibrate:
1 mL CH3OH/H2O
Load sample:
1 mL plasma or urine
Indicates Peak Area
of Analyte in LC Analysis
of Eluate
Maximum
% Organic Wash
Wash 1:
Minimum
% Organic Elute
1 mL 5% NH4OH
0
10
20
30
40
50
60
70
80
90
100
% CH3OH in H2O
Removes interfering
acids [ionized] and neutral
compounds [including
unionized bases]
Wash 2:
1 mL 100% CH3OH
Wash 3:
1 mL 2% HCOOH in CH3OH/H2O 25/75
Experiment for Oasis WCX 96-Well Plate 30 mg
Strongly basic analyte is
eluted; more hydrophobic
basic interferences
are retained
Ionizes weak cationexchange sites to lock on
analytes; removes
proteins and salts
Neutralizes cationexchange sites; removes
more polar strongly
basic interferences
Elute:
1 mL 2% HCOOH in CH3OH/H2O 75/25
3C: Oasis 2x4 Met hod Opt imized for µElution Plat e
For Bases
pKa 2-10:
Use Oasis MCX
For Strong Acids
pKa <1:
Use Oasis WAX
For Acids
pKa 2-8:
Use Oasis MAX
Apply Protocol 1
For Strong Bases
pKa >10:
Use Oasis WCX
Apply Protocol 2
Prepare Sample
Prepare Sample
Condition/Equilibrate
Load Sample
Condition/Equilibrate
Load Sample
Wash:
2% HCOOH
Wash:
5% NH4OH
Elute 1:
100% CH3OH
Elute 2: 5% NH4OH in
60:40 CH3CN:CH3OH
Base
Strong Acid
Elute 1:
100% CH3OH
Neutrals
Elute 2: 2% HCOOH in
60:40 CH3CN:CH3OH
Acid
Strong Base
The proven Oasis 2x4 method elution solvent is optimized to accommodate the elutropic requirement of the small elution volume.
Methanol is good as a generic elution solvent, but is often not
strong enough for 25 µL elution volumes. The elution solvent
recommended to be used with the μElution plate must possess
a high enough elutropic strength to fully elute analytes in small
volumes, and be appropriate for a diverse set of analytes.
The recommended elution solvent for the Oasis 2x4 method
optimized for the µElution plate format is 60% CH3 CN:40% CH3OH
with a modifier. This was chosen as a starting point as it meets all
of the above criteria.
Follow the simple steps outlined in this flow chart to achieve high
recoveries and the cleanest extracts:
n Characterize your analyte [Neutral, Acid or Base, pKa].
n Select one of the four Oasis sorbents.
n Apply the indicated Protocol [1 or 2].
n Determine SPE recoveries by LC analysis.
[ 20 ]
Methodology
Oasis 2x4 Method Proof of concept:
Recovery Study:
Analytes Spiked into Rat Plasma
To demonstrate the logic, simplicity, and effectiveness of the Oasis
2x4 method, five samples of rat plasma were prepared, each spiked
with one of the previously characterized test analytes shown below:
O
O
N
Br–
N+
O
O
— Imipramine: pKa = 9.4 (Base)
H
Imipramine (B)
pKa = 9.4, 2 ng/mL
— Ibuprofen: pKa = 5.2 (Acid)
O F
— Valethamate: pKa >12 (Quaternary Amine)
HO
F F F F
— Prednisone: Neutral
Prednisone (N)
10 µg/mL
OH
F
F
O
Nonafluoropentanoic Acid
(SA) pKa <0.5, 200 ng/mL
— Nonafluoropentanoic Acid: pKa <0.5 (Strong Acid)
Ibuprofen (A)
pKa = 5.2, 200 ng/mL
Protocol 1
Oasis WAX
Oasis MAX
Oasis WCX
Pre
Oasis MCX
Protocol 2
Pre
100
80
% SPE Recovery
Each plasma sample was diluted [1:1, v:v] and acidified with phosphoric acid [4% in water]. Respective aliquots were then processed
using the protocol and the Oasis ion-mixed-mode sorbent designated by the Oasis 2x4 Method for the corresponding sample type. LC/
MS/MS analysis was used to determine SPE recoveries. The neutral
analyte was processed on all four sorbents used.
H
O
Valethamate (QA)
pKa >12, 2 ng/mL
F
F
OH
H
N
OH
60
40
20
0
(n)
ic
no
nta
pe
)
(sb
ate
ham
let
(n)
Va
ne
iso
dn
(a)
fen
pro
(n)
Ibu
ne
iso
dn
oro
flu
ne
iso
dn
na
No
Pre
(b)
ine
am
i pr
(n)
Im
ne
iso
dn
Pre
id
Ac
Elute 1: CH3OH
)
(sa
Elute 2: 60:40 CH3CN/CH3OH + modifier
3D: Oasis HLB Generic Met hod
In 1996 Waters introduced Oasis HLB, the first hydrophiliclipophilic-balanced polymeric SPE sorbent. By careful design of
its chemical structure and particle morphology [see page 6], we
dramatically elevated the performance of SPE.
n Its higher retention power [3–5x] reduces breakthrough, increas-
es enrichment factors, and permits finer tuning of gradient steps
for more selective washing and elution sequences. Especially
hydrophobic analytes may require stronger elution solvents.
Polar compounds like phenols [> 9x higher k] that interact via
hydrogen bonding with the amide moieties in the HLB backbone
may require a protic solvent [e.g., methanol] rather than an
aprotic solvent [e.g., acetonitrile] for efficient elution.
When isolating a range of acidic, basic, and neutral compounds,
a generic method using Oasis HLB is recommended to remove
unretained matrix constituents [e.g., salts, sugars, polar lipids,
proteins) while reproducibly retaining, and subsequently eluting,
a broad chromatographic polarity range of acidic, basic, and
neutral analytes.
n Its excellent stability over a pH range of 0–14 and compat-
When transferring methods from C18 -bonded-silica phases, consider
these three unique advantages of Oasis HLB:
ibility with a broad range of organic solvents facilitates
development of rugged, reliable methods.
Oasis HLB Generic Method for Acids/Neutrals/Bases
n Its higher capacity [2–3x more surface area] means
correspondingly less sorbent weight is required. Bed
and elution volumes can be reduced. Smaller-scaledevice formats are more effective.
Prepare Sample
(typically acidify)
Condition/Equilibrate:
CH3OH/H2O
Load sample solution
Wash:
CH3OH/H2O 5/95
Elute:
CH3OH
[Optional; e.g.,
often not required in
µElution plate format]
[ 21 ]
Evaporate and Reconstitute
(optional)
[e.g., acidify
to disrupt drug–
protein binding]
Methodology
3E: Oasis HLB Opt imized Met hodology
Chemical and chromatographic principles may be applied to optimize methods on Oasis HLB. Selectivity is dramatically enhanced
by tuning pH, as well as the ratio of organic solvent to water, in
the mobile phase to manipulate retention.
If analytes or interferences are ionizable, then, as highly polar
entities in their charged states, they may be eluted in weak mobile
phases. If, by changing pH, they are converted to neutral form, they
are retained primarily by the strength of their hydrophobic interaction
with the sorbent surface. Stronger mobile phases, with higher organic
solvent concentrations, will then be required for successful elution.
Published work by Waters chemists clearly demonstrates the benefits
of such a 2-D [two-dimensional] process on Oasis HLB. The theory of
retention, wash-elute studies for verapamil and two of its metabolites, and successive selectivity improvements made by refining the
Oasis HLB method are summarized in the figures below.
Theory: Retention Factor [k] vs. pH for
Acids, Bases, and Neutrals
40
Wash-Elute Study for Verapamil and Metabolites in Plasma
Acid Retained
2% CH3COOH Modified Wash
2% NH4OH Modified Wash
Base Retained
3
Acid [HA]
Retention Factor k
32
pKa = 9.0
pKa = 4.8
16
8
0
2
4
pH 8
Recommended pH range for silica
CN
80
H
N
H 3 CO
12
10
40
60
% MeOH
H 3 CO
Low Retention for Acid
6
1
2
Maximum
% Methanol Wash
0
100
OCH
3
H 3 CO
OCH
3
H 3 CO
20
1. Norverapamil
14
40
60
% MeOH
CN
N
80
OCH
3
OCH
3
100
2. Verapamil
CN
H 3 CO
N
H 3 CO
0
Minimum
% Methanol Elution
0
20
Acid [A–]
Base [BH+]
1
0
0
Low Retention for Base
0
2
Maximum
% Methanol Wash
Neutrals
24
3
indicates
peak area of analyte
in LC analysis of eluate
Base [B]
OCH
Recommended pH range for Oasis® Sorbents
3
OCH
3
OCH
3
3. Methoxyverapamil
Effect of Method Development Optimization on Chromatography: Verapamil in Plasma
Oasis HLB Generic Method
Modified Oasis HLB Generic Method
Optimized Oasis HLB Method
Prepare sample solution
[acidified to disrupt protein binding]
Prepare sample solution
[acidified to disrupt protein binding]
Prepare sample solution
[acidified to disrupt protein binding]
Condition/Equilibrate:
Condition/Equilibrate:
Condition/Equilibrate:
1 mL CH3OH/H2O
1 mL CH3OH/H2O
1 mL CH3OH/H2O
Load sample:
Load sample:
Load sample:
1 mL plasma
1 mL plasma
1 mL plasma
Wash 1:
Wash 1:
Wash 1:
1 mL 5% CH3OH
1 mL 5% CH3OH with 2% CH3COOH
1 mL 5% CH3OH
Elute:
Wash 2:
Wash 2:
1 mL 100% CH3OH
1 mL 5% CH3OH with 2% NH4OH
1 mL 5% CH3OH with 2% NH4OH
Wash 3:
Elute:
1 mL 65% CH3OH with 2% NH4OH
1 mL 65% CH3OH with 2% CH3COOH
Elute:
Peak Identification:
Peak 1: Norverapamil
Peak 2: Verapamil
Peak 3: Methoxyverapamil [I.S.]
0.004 AU
1 mL 65% CH3OH with 2% CH3COOH
0.004 AU
1
1
2
3
2
4
6
8
2
1
3
3
Sample
Blank
0
0.004 AU
2
10 min
Sample
Blank
0
2
4
6
[ 22 ]
8
10 min
Sample
Blank
0
2
4
6
8
10 min
Methodology
3F: Oasis Advanced Met hodology
Waters Oasis product family of unique, patented sorbents in flexible
device formats and our array of method development strategies
simplify and streamline traditional SPE applications. They also
enable you to meet new sample preparation challenges, no
matter whether they have been recently ripped from the headlines
or previously relegated to the unsolvable SPE problem files.
Orthogonal SPE Using Two Cartridges—Most Powerful Methodology
For very complex matrices, the ultimate way to optimize cleanup
and isolation is to use orthogonal separation modes [e.g., reversed
phase and ion exchange] in tandem. Individual cartridges can be
connected with an adapter. By careful planning of mobile phase
compatibility and retention mode sequences, the final eluate from
the first cartridge can be loaded directly into the second cartridge.
Then, after discarding the first cartridge, the second can be washed
and eluted with appropriately scaled volumes of solvent. A larger
first cartridge removes the bulk of matrix interferences. A smaller
second cartridge further concentrates the analyte(s) and maximizes
the enrichment factor.
Tandem Oasis MAX – MCX Results for Fluoroquinolone
Antibiotics in Beef Kidney – spiked at 1 µg/kg
Chromatogram 1: Result from a single Oasis MAX Cartridge
Ciprofloxacin
Enrofloxacin
0
Animal tissue homogenates are complex matrices containing a host
of endogenous interferences that may overload the ion-exchange
and reversed-phase capacities of an SPE sorbent. Difficulty is further
compounded when analytes, such as fluoroquinolone antibiotics,
are amphoteric. Compared to a single-cartridge cleanup, the ability
of a well-designed tandem Oasis MAX–MCX method to conquer
such a challenge successfully is dramatic. Chromatograms at right
demonstrate the selective isolation and confident determination of
ciprofloxacin and enrofloxacin at sub-ppb levels.
8
16
24 min
Chromatogram 2: Result from Tandem Oasis MAX + MCX
N
HN
N
N
N
OH
F
pKa of acid ~ 5
pKa of base ~ 8–9
0
O
24 min
16
Tandem Oasis MAX–MCX Method for Amphoteric Fluoroquinoline Antibiotics
Stage 1:
Condition, Load, and Wash
Oasis MAX
6 cc 150 mg Cartridge
Stage 2:
Condition
Oasis MCX
1 cc 30 mg Cartridge
Condition/Equilibrate:
1 mL CH3OH/1 mL 5 N NaOH;
1 mL H2O
Condition:
Elute & Wash:
1 mL CH3OH
2 mL 0.2 N HCl in CH3OH
MAX Cartridge
Wash 1:
MCX Cartridge
Load:
5 mL of prepared sample
Stage 3:
Attach MCX Cartridge
to outlet of MAX Cartridge;
Elute from MAX into MCX
Stage 4:
Discard MAX Cartridge;
Wash & Elute
MCX Cartridge
Wash:
2 mL of CH3OH
Elute:
500µL of 10% NH4OH in CH3OH
MCX Cartridge
1 mL 5% NH4OH in H2O
Wash 2:
MCX Cartridge
MAX Cartridge
1 mL CH3OH
After NaOH treatment,
MAX retains amphoteric
analytes as anions.
Wash steps remove basic
and neutral matrix interferences.
HCl converts
amphoteric analytes
to cations.
They elute from MAX
into lower MCX bed where
they are retained. Acidic interferences
are neutralized by HCl and washed
out of both cartridges by CH3OH.
[ 23 ]
OH
O
Enrofloxacin
O
Ciprofloxacin
8
N
F
Neutralize eluate with HCOOH
and bring to 1 mL with
buffered mobile phase.
Smaller bed in 1-cc MCX
Cartridge enables elution
in a smaller volume,
increasing enrichment factor.
O
ORDERING INFORMATION
Oasis HLB Sample Extraction Products
Description
Oasis HLB cartridge
1 cc/10 mg
Oasis HLB cartridge
1 cc/30 mg
1 cc/30 mg
NEW Oasis HLB cartridge
Oasis HLB flangeless cartridge
1 cc/30 mg
Oasis HLB cartridge with
1 cc/10 mg
Gilson ASPC adapter
Oasis HLB cartridge with
1 cc/30 mg
Gilson ASPC adapter
Oasis HLB cartridge
3 cc/60 mg
Oasis HLB flangeless cartridge
3 cc/60 mg
Oasis HLB cartridge with
3 cc/60 mg
Gilson ASPC adapter
Oasis HLB cartridge
6 cc/200 mg
Oasis HLB
3 cc/400 mg
3 cc/540 mg
NEW Oasis HLB cartridge
Oasis HLB flangeless cartridge
3 cc/540 mg
Oasis HLB cartridge
6 cc/150 mg
Oasis HLB cartridge
6 cc/150 mg
Oasis HLB cartridge
6 cc/500 mg
Oasis HLB cartridge
12 cc/500 mg
Oasis HLB cartridge
20 cc/1 g
Oasis HLB cartridge
35 cc/6 g
Oasis HLB Plus cartridge
225 mg
Oasis HLB Vac RC cartridge
20 cc/30 mg
Oasis HLB Vac RC cartridge
20 cc/60 mg
Oasis HLB glass cartridge
5 cc/200 mg
Oasis HLB Prospekt 2/Symbiosis cartridge* 1.0 x 10 mm
Oasis HLB Prospekt 2/Symbiosis cartridge* 2.0 x 10 mm
Reservoir 30 cc for Oasis cartridges
Reservoir 60 cc for Oasis cartridges
Reservoir adapter for 1 cc, 3 cc, 6 cc cartridges
Reservoir adapter for 12 cc, 20 cc, 35 cc cartridges
Reservoir adapter for 5 cc cartridges, Teflon
Particle
Size
30 µm
30 µm
30 µm
30 µm
Qty.
Part No.
100/box
100/box
1000/box
100/box
186000383
WAT094225
186003908
186001879
30 µm
500/box
186000988
30 µm
500/box
WAT058882
30 µm
30 µm
100/box
100/box
WAT094226
186001880
30 µm
500/box
WAT058883
30 µm
60 µm
60 µm
60 µm
30 µm
60 µm
60 µm
60 µm
60 µm
60 µm
60 µm
30 µm
30 µm
60 µm
30 µm
30 µm
30/box
100/box
100/box
100/box
30/box
30/box
30/box
20/box
20/box
10/box
50/box
50/box
50/box
30/box
96/box
96/box
48/box
12/box
10/box
10/box
10/pkg
WAT106202
186003849
186004134
186003852
186003365
186003379
186000115
186000116
186000117
186000118
186000132
186000382
186000381
186000683
186001196
186003925
WAT011390
WAT024659
WAT054260
WAT048160
405000934
Oasis MCX Sample Extraction Products
(Cation Exchange)
Oasis MCX cartridge
Oasis MCX flangeless cartridge
Oasis MCX cartridge
Oasis MCX cartridge
Oasis MCX flangeless cartridge
Oasis MCX cartridge
Oasis MCX cartridge
Oasis MCX cartridge
Oasis MCX cartridge
Oasis MCX cartridge
Oasis MCX cartridge
Oasis MCX Plus cartridge
Oasis MCX Vac RC cartridge
Oasis MCX Vac RC cartridge
Oasis MCX Prospekt 2/Symbiosis cartridge*
1 cc/30 mg
1 cc/30 mg
1 cc/60 mg
3 cc/60 mg
3 cc/60 mg
3 cc/60 mg
6 cc/150 mg
6 cc/150 mg
6 cc/500 mg
20 cc/1 g
35 cc/6 g
225 mg
20 cc/60 mg
20 cc/60 mg
10 x 1 mm
Particle
Size
30 µm
30 µm
60 µm
30 µm
30 µm
60 µm
30 µm
60 µm
60 µm
60 µm
60 µm
60 µm
30 µm
60 µm
30 µm
Oasis MCX direct connect column
Oasis MCX column
Oasis MCX cartridge column
Oasis MCX column
Oasis MCX column
Oasis MCX column
2.1 x 15 mm
2.1 x 20 mm
2.1 x 20 mm
3.0 x 20 mm
3.9 x 20 mm
4.6 x 20 mm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
186002050
186002046
186002051
186002047
186002048
186002049
Oasis MCX µElution plate
Oasis MCX plate
Oasis MCX plate
Oasis MCX plate
Oasis MCX plate
96-well
10 mg/96-well
30 mg/96-well
30 mg/96-well
60 mg/96-well
30 µm
30 µm
30 µm
60 µm
60 µm
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
186001830BA
186000259
186000248
186000250
186000678
Description
Oasis HLB column
Oasis HLB column
Oasis HLB column
Oasis HLB cartridge column
Oasis HLB column
Oasis HLB column
Oasis HLB column
Oasis HLB column
Oasis HLB cartridge column
Oasis HLB column
Oasis HLB column
Oasis HLB column
Oasis HLB cartridge column
Oasis HLB column
Oasis HLB column
Oasis HLB column
Holder kit for 2.1 x 20 mm cartridge column
Holder kit for 3.9 x 20 mm cartridge column
Extraction column connector
Inline precolumn filter kit
Replacement filters
Replacement steel gaskets
2.1 x 20 mm
3.0 x 20 mm
3.9 x 20 mm
3.9 x 20 mm
4.6 x 20 mm
2.1 x 20 mm
3.0 x 20 mm
3.9 x 20 mm
3.9 x 20 mm
4.6 x 20 mm
2.0 x 15 mm
2.1 x 20 mm
2.1 x 20 mm
3.0 x 20 mm
3.9 x 20 mm
4.6 x 20 mm
5 µm
5 µm
5 µm
5 µm
5 µm
15 µm
15 µm
15 µm
15 µm
15 µm
25 µm
25 µm
25 µm
25 µm
25 µm
25 µm
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
5/pkg
1/pkg
186002034
186002037
186002040
186001413
186002043
186002035
186002038
186002041
186001414
186002044
186001792
186002036
186000706
186002039
186002042
186002045
186000262
WAT046910
WAT082745
WAT084560
WAT005139
WAT084567
Oasis HLB µElution plate
Oasis HLB plate
Oasis HLB plate
Oasis HLB plate
Oasis HLB plate
2 mg/96-well
5 mg/96-well
10 mg/96-well
30 mg/96-well
60 mg/96-well
30 µm
30 µm
30 µm
30 µm
60 µm
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
186001828BA
186000309
186000128
WAT058951
186000679
* For use with Spark Holland Prospekt 2 and Symbiosis systems
[ 24 ]
Qty.
Part No.
100/box
100/box
100/box
100/box
100/box
100/box
30/box
30/box
30/box
20/box
10/box
50/box
50/box
50/box
96/box
186000252
186001881
186000782
186000254
186001882
186000253
186000256
186000255
186000776
186000777
186000778
186003516
186000261
186000380
186002098
ORDERING INFORMATION
Oasis MAX Sample Extraction Products
(Anion Exchange)
Oasis WCX Sample Extraction Products
(Weak Cation Exchange)
Oasis MAX cartridge
Oasis MAX flangeless cartridge
Oasis MAX cartridge
Oasis MAX cartridge
Oasis MAX flangeless cartridge
Oasis MAX cartridge
Oasis MAX cartridge
Oasis MAX cartridge
Oasis MAX Plus cartridge
Oasis MAX Vac RC cartridge
Oasis MAX Vac RC cartridge
Oasis MAX Vac RC cartridge
Oasis MAX Prospekt 2/Symbiosis cartridge*
1 cc/30 mg
1 cc/30 mg
3 cc/60 mg
3 cc/60 mg
3 cc/60 mg
6 cc/150 mg
6 cc/150 mg
6 cc/500 mg
225 mg
20 cc/30 mg
20 cc/60 mg
20 cc/60 mg
10 x 1 mm
Particle
Size
30 µm
30 µm
30 µm
60 µm
30 µm
30 µm
60 µm
60 µm
60 µm
30 µm
30 µm
60 µm
30 µm
Oasis MAX direct connect column
Oasis MAX column
Oasis MAX cartridge column
Oasis MAX column
Oasis MAX column
Oasis MAX column
2.1 x 15 mm
2.1 x 20 mm
2.1 x 20 mm
3.0 x 20 mm
3.9 x 20 mm
4.6 x 20 mm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
186002056
186002052
186002057
186002053
186002054
186002055
Oasis MAX µElution plate
Oasis MAX plate
Oasis MAX plate
Oasis MAX plate
Oasis MAX plate
2 mg/96-well
10 mg/96-well
30 mg/96-well
60 mg/96-well
60 mg/96-well
30 µm
30 µm
30 µm
60 µm
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
186001829
186000375
186000373
186001256
186001205
Description
Qty.
Part No.
100/box
100/box
100/box
100/box
100/box
30/box
30/box
30/box
50/box
50/box
50/box
50/box
96/box
186000366
186001883
186000367
186000368
186001884
186000369
186000370
186000865
186003517
186000372
186000371
186000378
186002099
Description
Oasis WCX 1 cc cartridge
Oasis WCX 3 cc cartridge
NEW Oasis WCX 6 cc cartridge
Oasis WCX 1 cc cartridge
Oasis WCX 3 cc cartridge
Oasis WCX Plus cartridge
Oasis WCX µElution plate
Oasis WCX 96-well plate
Oasis WCX 96-well plate
Oasis WCX Prospekt 2/Symbiosis cartridge*
Oasis WCX 2.1 x 20 mm column
Oasis WCX 3.9 x 20 mm column
Oasis WCX 2.1 x 20 mm column
Oasis WCX 3.9 x 20 mm column
Oasis WAX 1 cc cartridge
Oasis WAX 3 cc cartridge
Oasis WAX 6 cc cartridge
Oasis WAX 1 cc cartridge
Oasis WAX 3 cc cartridge
Oasis WAX Plus cartridge
Oasis WAX µElution plate
Oasis WAX 96-well plate
Oasis WAX 96-well plate
NEW Oasis WAX 96-well plate
Oasis WAX Prospekt 2/Symbiosis cartridge*
Oasis Method Development Kits
Oasis sorbent selection plate
3 rows each:
MCX, MAX, WCX, WAX
NEW Oasis µElution Sorbent
Selection Plate, 3 rows each:
MCX, MAX, WCX, WAX
Oasis sorbent selection
cartridge kit, 10 each:
MCX, MAX, WCX, WAX
Part No.
96-well
30 µm
186003249
96-well
30 µm
186004475
1 cc/30 mg
30 µm
186003463
Part No.
186002494
186002495
186002498
186002496
186002497
186003518
186002499
186002501
186002503
186002892
30 µm
30 µm
5 µm
5 µm
Description
Oasis WAX 2.1 x 20 mm column
Oasis WAX 3.9 x 20 mm column
Oasis WAX 2.1 x 20 mm column
Oasis WAX 3.9 x 20 mm column
Particle Size
Qty.
100/box
100/box
30/box
100/box
100/box
50/box
1/pkg
1/pkg
1/pkg
96/box
186002505
186002507
186002510
186002512
Oasis WAX Sample Extraction Products
(Weak Anion Exchange)
* For use with Spark Holland Prospekt 2 and Symbiosis systems
Description
30 mg
60 mg
150 mg
30 mg
60 mg
225 mg
2 mg/96-well
10 mg/96-well
30 mg/96-well
1.0 x 10 mm
Particle
Size
30 µm
30 µm
30 µm
60 µm
60 µm
60 µm
30 µm
30 µm
30 µm
30 µm
[ 25 ]
30 mg
60 mg
150 mg
30 mg
60 mg
225 mg
96-well
10 mg/96-well
30 mg/96-well
60 mg
1.0 x 10 mm
Particle
Size
30 µm
30 µm
30 µm
60 µm
60 µm
60 µm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
30 µm
5 µm
5 µm
Qty.
Part No.
100/box
100/box
30/box
100/box
100/box
50/box
1/pkg
1/pkg
1/pkg
1/pkg
96/box
186002489
186002490
186002493
186002491
186002492
186003519
186002500
186002502
186002504
186003915
186002893
186002508
186002509
186002511
186002513
ORDERING INFORMATION
Manifold for Extraction Plate
Manifold for Extraction Cartridges
Description
Extraction plate manifold for Oasis 96-well plates
Qty
Part No.
1/box
Description
Part No.
186001831
Extraction plate manifold kit A
(includes extraction plate manifold, reservoir tray,
sealing cap and 350 µL sample collection plate)
Waters extraction manifold, 20-position without rack
(includes 20 needle tips, 25 plugs, and ejector tool)
WAT200677
WAT097944
Waters extraction manifold, 20-position
(complete with rack for 13 x 75 mm tubes)
WAT200606
Extraction plate manifold kit B
(as kit A, with 1 mL sample collection plate)
WAT097945
Waters extraction manifold, 20-position
(complete with rack for 13 x 100 mm tubes)
WAT200607
Extraction plate manifold kit C
(as kit A, with 2 mL sample collection plate)
WAT097946
Waters extraction manifold, 20-position
(complete with rack for 16 x 75 mm tubes)
WAT200608
Waters extraction manifold, 20-position
(complete with rack for 16 x 100 mm tubes)
WAT200609
Oasis 96-well µElution Plate
(Requires Manifold Spacer)
Oasis 96-well Plate
(No Spacer Required)
Spacer for µElution
Plate (Included
with Manifold Kit)
Extraction Plate
Manifold
186001831
Collection
Plate
Waters Extraction Manifold
Accessories for Extraction Plate Manifold
Description
Disposable reservoir tray
Sample collection plate, 350 µL
Sample collection plate, 2 mL
Sealing cap for 96-well collection plate
SPE vacuum pump 115 V 60 Hz
SPE vacuum pump 240 V 50 Hz
Accessories for Extraction Columns and Cartridges
Qty
Part No.
25/box
50/box
50/box
50/pkg
WAT058942
WAT058943
WAT058958
WAT058959
725000417
725000418
Vacuum box gasket kit
Kit includes: 2 foam top gaskets 2 orange O-rings
Description
Qty
Part No.
1/pkg
1/pkg
1/pkg
1/pkg
5/pkg
1/pkg
48/pkg
12/pkg
100/pkg
186000262
WAT046910
WAT082745
WAT084560
WAT005139
WAT084567
725000417
725000418
WAT011390
WAT024659
WAT024310
Adapter (to attach reservoir to 1, 3 & 6 cc
Oasis Vac cartridges)
12/pkg
WAT054260
Adapter (to attach reservoir to 12, 20 & 35 cc
Oasis Vac cartridges)
10/pkg
WAT048160
Holder kit for 2.1 x 20 mm cartridge column
Holder kit for 3.9 x 20 mm cartridge column
Extraction column connector
Inline precolumn filter kit
Replacement filters
Replacement steel gaskets
SPE vacuum pump 115 V 60 Hz
SPE vacuum pump 240 V 50 Hz
Reservoir, 30 cc (for Oasis Plus, Light, Vac & Classic cartridges)
Reservoir, 60 cc (for Oasis Plus, Light & Vac cartridges)
Adapter, male-male Luer (for Oasis Classic cartridges)
186003522
Vacuum Box Gasket Kit
SPE Vacuum Pump
(Includes two gauges and pressure regulator)
[ 26 ]
[ 27 ]
Sales Offices
Austria and European Export
(Central South Eastern Europe, CIS
and Middle East) 43 1 877 18 07
Australia 61 2 9933 1777
Belgium 32 2 726 1000
Brazil 55 11 5094-3788
Canada 1 800 252 4752 x2205
China 86 21 6879 5888
CIS/Russia +7 495 3367000
Czech Republic 420 2 617 1 1384
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Finland 09 5659 6288
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The Netherlands 31 76 508 7200
Norway 47 63 84 60 50
Poland 48 22 833 4400
Puerto Rico 1 787 747 8445
Singapore 65 6273 1221
Spain 34 93 600 9300
Sweden 46 8 555 11 500
Switzerland 41 56 676 70 00
Taiwan 886 2 2543 1898
United Kingdom 44 208 238 6100
All other countries:
Waters Corporation U.S.A.
1 508 478 2000
1 800 252 4752
www.waters.com
Hungary 36 1 350 5086
India and India Subcontinent
91 80 2837 1900
Ireland 353 1 448 1500
Italy 02 265 0983
Japan 81 3 3471 7191
Korea 82 2 820 2700
Mexico 52 55 5200 1860
T he quality management system of Waters’ manufacturing facilities
in Taunton, Massachusetts and Wexford, Ireland complies with the International
Standard ISO 9001:2000 Quality Management and Quality Assurance Standards.
Waters’ quality management system is periodically audited by the registering
body to ensure compliance.
©2008 Waters Corporation. Waters, ACQUITY UPLC, Atlantis, IntelliStart,
MassLynx, Oasis, Quattro Premier, ScanWave, Sentry, Sirocco, SunFire,
Symmetry, T he Science of W hat’s Possible, UPLC, XBridge, Xevo and XTerra
are trademarks of Waters Corporation. Teflon is a trademark of DuPont.
All trademarks used are acknowledged. All rights reserved.
July 2008,
720001692EN 2008 IH-WA