[ Care and Use Manual ]

Oasis HLB Cartridges and 96-well Plates

I. Introduction Contents

Oasis HLB I. INTRODUCTION

Universal Sorbent for Acidic, Neutral, and Basic Compounds
II. Sample Pre-Treatment
Oasis HLB is a Hydrophilic-Lipophilic-Balanced, water-wettable,
reversed-phase sorbent for all your SPE needs. It is made from a III. Solid Phase Extraction for Acidic, Neutral, and
specific ratio of two monomers, the hydrophilic N-vinylpyrrolidone Basic Compounds using Oasis HLB
and the lipophilic divinylbenzene. It provides superior reversed-phase
capacity with a neutral polar hook for enhanced retention of polar
iv. Oasis HLB Protocol Charts
analytes.

Waters has built a family of SPE sorbents which inherit some key
v. Oasis HLB 11 bottle Optimization Approach
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 vi. Troubleshooting
traditional silica-based SPE sorbents like C18.

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, obviating the need for
repeat preparation.

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.

Available in five particle sizes [60 m, 30 m, 25 m, 15 m, and

5 m], 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.

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II. Sample Pre-Treatment c. Aqueous Samples: Water, Beverages

Adjust pH to maximize analyte retention on the SPE sorbent. Buffer salts
a. Biological Samples
and dispersive agents may be used to increase partitioning onto the SPE
This section contains recommendations for preparing your biological
sorbent. Pretreatment to remove suspended matter prior to SPE treatment
samples (plasma, serum, urine, etc.) prior to solid-phase extraction.
may include filtration or centrifugation.
1. Prepare acidified or basified water diluents: Add 40 L of
d. Non-Aqueous Liquid
concentrated phosphoric acid, or 40 L of concentrated
When appropriate the sample may be diluted with aqueous buffers and
ammonium hydroxide, to 1 mL of water.
organic co-solvents for reversed-phased or mix-mode SPE. If sufficient
2. Dilute plasma or urine, 1:1 with acidified or basified dilution has occurred, the sample may be treated in a manner similar to an
water. Add 10 to 50 L of internal standard. aqueous sample.
Note: final concentration should be no more than 10% organic
otherwise protein precipitation will occur. III. Solid Phase Extraction for Acidic, Neutral, and
3. If necessary, clarify samples by centrifugation at 8,000 x g Basic Compounds using Oasis HLB
for 10-30 minutes. (Note: refer to Table 1 for the recommended volume to be used in each step)
4. If necessary, filter samples for suspended solids. Remove cover 1. Place Oasis HLB cartridge or plate on the vacuum manifold.
from vacuum manifold and place appropriate collection or waste
tray inside. Replace manifold cover. 2. Condition: Draw through methanol.

i. Environmental 3. Equilibrate: Draw through water.

ii. Water Analysis 4. Load: Draw through diluted sample.

iii. Food Safety 5. Wash: Draw through 5% methanol in water (v/v). (Release
vacuum and discard waste fluids. Insert collection device,
b. Solid Samples: Soil, whole foods, tissue replace the cover, and turn on vacuum).
1. Homogenize the sample with an appropriate solvent to obtain
an aqueous based or an organic solvent based extract of the 6. Elute: Draw through methanol (or other elution solvents),

sample. Initial extraction conditions are chosen to maximize collecting eluates in a suitable device.

analyte recovery, while minimizing matrix interference. In 7. Evaporate/Reconstitute or Dilute (optional).

many cases, it may be beneficial to add buffers, dispersive salts,
8. Inject: Cover collection plate and inject onto column.
or co-solvents to improve extraction efficiency.
Note: For the Load and Elute steps, it is recommended that flow rates should
2. Adjust the initial extract to optimize analyte retention onto
not exceed 1.0 mL/min In all other steps, flow rates up to 5 mL/min are
the SPE sorbent. This can include pH adjustment, solvent
acceptable.
adjustment, or solvent exchange through evaporation and
reconstitution (refer to Sections IV and V). It may be necessary
to centrifuge or filter the sample prior to loading.

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iv. Oasis HLB Protocol Charts ii. Strategy for Optimizing the Generic SPE Method
i. Generic Method Oasis HLB SPE
Prepare sample solution
Prepare sample either in acid or base
(typically acidfy)

Condition
Methanol
4a: Condition
Methanol

Equilibrate
4b: Equilibrate Water
Water

Load
4c: Load sample solution
sample solution

Wash 1
5% methanol in water
4d: Wash
5% methanol in water
Acidic Compounds Basic Compounds

4e: Elute
Wash 2 Wash 2
Methanol 2% formic acid in methanol/water 5% NH4OH in methanol/water

Evaporate and reconstitute Elute Elute

(optional) 5% NH4OH in methanol/water 2% formic acid in methanol/water

Avoid using methods developed for C18 or other silica-based cartidges

(see note below) The strategic procedure to obtain cleaner extracts start with the generic
The fast Oasis HLB generic method is ideal for LC/MS/MS method through the first wash.
analysis
By adjusting the pH of the additional washes to increase analyte
The generic method is an excellent starting point since it retention, higher concentrations of organic solvent may be applied to
works for a wide range of compounds, yielding high recoveries remove interferences.
(> 85%) and consistent results (< 5% RSD)
The pH is decreased for acidic analytes (below the pKa of the
When cleaner backgrounds are required for higher sensitivity
compound) to increase retention.
or selectivity, the generic protocol can be optimized using a
straighforward method development strategy. The pH is raised for basic compounds (above the pKa of the
The strategy uses the full pH range (pH 1 to pH 14) and varies the organic compound) to increase retention.
solvent level. The pH is then changed to elute the analyte. The % solvent
in the Wash 2 and Elute steps is determined by varying the
Note: Wash and Elute steps developed for C18 or other silica-based sorbents
% methanol in 10% increments at each pH.
may not be appropriate for the polymeric Oasis HLB sorbent.

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Analyze the Wash 2 and Elute samples to determine optimum Wash/Elution Steps: 20 Bottle Optimization for Oasis HLB
% methanol. The 20 Bottle Optimization method for Oasis HLB is set up first by spiking
the analyte into saline and loaded it into the wells of the 96-well plate
Select the highest % methanol in Wash 2 that does not remove
or 20 cartridges of Oasis HLB. We prepare the 20 bottle of solvents as
any analytes.
described bellow. First, we start with 5% MeOH with base wash step to
Select the lowest % methanol in the Elute step that elutes the remove proteins, to prevent the wells from clogging, as well as putting
analytes. bases in a neutral state for more retention by reverse phase.
Figure 1: Retention Factor Versus pH For Acids, Bases, and Neutrals
The 5% MeOH wash step (removes salts and proteins) in the generic Oasis
HLB method is weak enough that the analyte should not wash off. You pass
through the Oasis HLB devices these 20 solutions of acid and base in MeOH.

You plot the response against the organic solvent ratio, and determine what
percent organic to use for the wash and elution step After running the 2-D
optimization (20 Bottle Optimization), it is important to select a wash
step that is not too strong, or you may lose your analyte, and is the most
effective in the removal of unwanted components.

Also, to select the elution organic solvent ratio that is just strong enough to
elute the analyte and retain the most hydrophobic interferences on
the sorbent.

Summary
With the generic SPE method, one method yields good results for a wide
range of compounds. With an optimized method, cleaner extracts can be
obtained.

V. Oasis HLB 11 bottle Optimization Approach 20 Bottle Optimization Oasis HLB: Example
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,
Wash 1: Base with 5% MeOH (remove proteins to prevent
by changing pH, they are converted to neutral form, they are retained
clogging of wells; bases are in neutral state for more retention)
primarily by the strength of their hydrophobic interaction with the sorbent
surface. Stronger mobile phases, with higher organic solvent concentrations, Wash 2: Base with 40% MeOH (removes hydrophilic bases and
will then be required for successful elution. neutrals and all acids)

Wash 3: 100% Water (removes residual ammonium hydroxide)

Published work by Waters chemists clearly demonstrates the benefits of
such a 2-D [two-dimensional] process on Oasis HLB. The theory of retention, Elute: Acid with 70% MeOH (101% recovery from rat plasma)
wash-elute studies for alprenolol, and successive selectivity improvements
Note: Depending on matrix and sensitivity requirements, additional washes
made by refining the Oasis HLB method are summarized in the figures
may be required.
below.
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vi. Troubleshooting

a. Adjustment to Optimize Recoveries

Spike an appropriate volume of reagent water with all analytes and internal/surrogate standards. Follow steps 4a-4e in Section II, but use a rack to collect the
eluates in the Load (4c), Wash (4d), and Elute (4e) steps in separate collection vessels. In addition, repeat step 4e with a second portion of methanol and col-
lect the eluate. Analyze all four collected fractions. Use the provided table to determine adjustments, if necessary, to optimize sample recovery.

If the fraction from this step contains the analyte: Make this adjustment for optimum analyte recovery:
Load (4c) The Oasis HLB sorbent has been found to retain ionized analytes more strongly than silica-based reversed-phase
sorbents. However, recoveries may be enhanced when analyte ionization is suppressed. For acidic analytes, adjust
the sample pH to at least two pH units below the pKa of the acid. For basic analytes, adjust the pH to at least two pH
units above the pKa of the conjugate acid.
Wash (4d) Recoveries of very polar analytes can be increased by using only water (not 5% methanol in water) as the wash
solution.
First Elution (4e) If an acceptable recovery of analyte(s) is obtained in this fraction (usually > 90%), no adjustments are necessary.
Second Elution For very non-polar analytes, methanol may not have adequate elution strength. Stronger solvents such as acetonitrile
or ethyl acetate may be substituted, or used in sequence. In addition, for ionizable analytes, methanol may need to be
(4e repeated) modified with the addition of 2% acid or 2% base, as appropriate. If solvents stronger than methanol or acetonitrile
are used for the elution, then a preliminary conditioning step (see step 4a) should be performed prior to the methanol
conditioning step. For example, if ethyl acetate is to be used as an eluent, condition the cartridge with ethyl acetate,
followed by methanol (4a) and then water (4b).

Table 1: Recommended Volume for Generic Methods (assuming 1:1 dilution)

Cartridges 96-well plate Elution Plate
Cartridge size/sorbent mass 1 cc 3 cc 6 cc 12 cc 20 cc 35 cc 5 mg 10 mg 30 mg 60 mg 2 mg
Condition/ Equilibration (mL) 1mL 2 mL 3 mL 5 mL 10 mL 50 mL 0.2 mL 0.5 mL 0.5-1mL 0.5-2 mL 0.2 mL
Load (mL urine or plasma)
1mL 2 mL 5 mL 15 mL 30mL 100mL 1 mL 1-2 mL 1-2 mL 1-2 mL 0.025-0.75 mL
(total of matrix and dilution)
Wash (mL) 1mL 2 mL 4 mL 5 mL 10 mL 40 mL 0.2 mL 0.5 mL 0.5-1 mL 1-2 mL 0.2 mL
Elute (mL) 1mL 2 mL 4 mL 5 mL 10 mL 60 mL 0.05-0.2 mL 0.15-0.3 mL 0.4-1.0 mL 0.8-2 mL 0.025-0.10 mL

Note: Above listed sample volumes are for biological samples; for certain types of samples (i.e. drinking water) up to 20 times of sample solution is possible.
Note: Solution should be made fresh daily.

Table 2: Load Volumes for Large Volume WATER Analysis

Cartridge size/sorbent mass 6 cc 6 cc
1 cc 3 cc 12 cc 20 cc 35 cc
(200 mg) (200 mg)
Load (mL water)
50 mL 200 mL 500 mL 1000 mL 1000 mL 2000 mL 5000 mL
(total of matrix and dilution)

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[ Care and Use Manual ]

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Possible, and Oasis are trademarks of Waters Corporation. Milford, MA 01757 U.S.A.
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