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Technical Note 125
SAMPLE PREPARATION BY MIXED-MODE SPE USING
ISOLUTE ® HCX
This technical note describes the extraction of basic drugs from biological fluids using ISOLUTE
HCX mixed-mode SPE sorbents.
Sample preparation techniques such as protein precipitation, supported liquid extraction or non-polar
SPE may not be selective enough to give extracts of sufficient purity for low level analysis. In these cases,
the selective mixed-mode approach to the extraction of basic drugs is a suitable alternative, giving very
high purity extracts with minimal levels of co-extracted material.
The ISOLUTE HCX series of mixed-mode SPE sorbents (HCX, HCX-3 and HCX-5) is based on a combination of strong cation exchange and non-polar (C8, C18 and C4 respectively) chemistries. Basic
drugs are retained by two primary retention mechanisms (see Figure 1). This allows a rigorous interference elution regime to be used to elute interferences retained by either non-polar or cation exchange
interactions alone. Only analytes with both non-polar and basic characteristics are extracted using the
ISOLUTE HCX series of sorbents, providing an extremely pure final extract.
R
NH 3+
NH 3+
R
Ionic
interaction
Hydrophobic
interaction
C8
SO 3-
SO 3SO 3-
HO
Si
O
O
O
Si
Si
HO
O
S
Si
O
OH
O
Si
Si
O
OH
OH
O
Si
O
Si
O
S
Si
HO
O
S
Si
O
OH
O
S
Si
O
S
Si
Figure 1. Multiple interactions on ISOLUTE HCX mixed-mode columns
O
The mixed-mode approach for extraction of ionizable drugs from biological fluids is extremely robust.
The initial retention mechanism for the analytes is non-polar (hydrophobic), and is unaffected by the
high or variable ionic strength of the matrix. The initial hydrophobic interaction is a function of the
sorbent chain length, with shorter chains (e.g. C4) being less retentive than longer chains (e.g. C18). If
retention of non-ionizable compounds is minimized, a cleaner extract will result.
For further information on the use of ISOLUTE HCX-3 and HCX-5, please see Technical Note TN113.
EXTRACTION PROTOCOL
Evaluate ISOLUTE HCX, 25 mg/1 mL in the ISOLUTE Array® format using the procedure detailed
below. Populate the plate with individual wells as required. Process using a VacMaster®-96 sample
processing station or automated liquid handling system.
Vacuum settings
At all stages, use a short pulse (approx 1 second) of low vacuum (< -5" Hg), unless otherwise stated.
Sample volume
This procedure is optimized for a biological fluid sample volume of 100 µL. Sample should be diluted
1:1 (v/v) with appropriate buffer before applying to the column (total volume of buffered sample
applied is 200 µL).
Note: Work in our R&D laboratory has shown that 25 mg ISOLUTE SPE columns have sufficient
capacity for extraction of up to 1 mL plasma sample without analyte breakthrough. Test conditions:
1 mL plasma spiked at 0.1 mg/mL analyte concentration and diluted 1:1 with buffer before applying to
the column (total volume of buffered sample applied is 2 mL).
Sample pre-treatment
Dilute the sample (100 µL of plasma or urine) with ammonium acetate buffer (0.05 M, pH 6.0,
100 µL) to give a 200 µL total sample volume at 1:1 dilution. Mix thoroughly.
Column conditioning
Place extraction plate on vacuum manifold. No collection plate should be used at this stage.
Condition each well with methanol (1 mL). Use gravity or a short pulse of vacuum to initiate flow.
This will ensure efficient wetting of the hydrophobic frits, promoting even flow of sample through the
wells.
Column equilibration
Rinse wells with ammonium acetate buffer (0.05 M, pH 6.0, 250 µL). Load all wells prior to applying a short pulse of vacuum to initiate flow.
Sample loading
Apply 200 µL buffered sample. Load all wells prior to applying a short pulse of vacuum to initiate
flow.
Interference elution
Elute acidic and neutral interferences with:
■ Ammonium acetate buffer (0.05 M, pH 6.0, 250 µL)
■
Acetic acid (1 M, 250 µL)
■
Apply vacuum for 30 seconds to dry sorbent bed
■
Methanol (250 µL)
For each solvent, load all wells and allow to soak for 1 minute prior to applying a short pulse of vacuum.
Analyte elution
Place collection plate in base of manifold. Ensure correct alignment (position A1 of collection plate
directly underneath position A1 of extraction plate), and that extraction plate outlet Luer tips extend
into the top of the collection plate. This will prevent sample cross contamination. Spacers are
available to ensure optimum penetration.
Elute basic analytes with methanol/NH4OH (95:5, v/v, 2 x 100 µL). This will suppress ionization of
the drug, overcoming both cationic and non-polar retention mechanisms, allowing elution of the
analytes.
Apply the first 100 µL aliquot and allow to soak for 2–4 mins. If the aliquot has not reached the
top frit at the end of the soak time, apply a short vacuum pulse.
Apply the second 100 µL aliquot and allow to soak for a further 2–4 mins. Apply low vacuum for
1 minute to complete elution.
Evaporate this elution solvent and re-constitute the sample in a solvent compatible with the analytical technique. For LC-MS the mobile phase is suggested.
Care should be taken to avoid losses of thermally labile or volatile analytes at this stage.
REAGENTS
1. Methanol
2. 0.05 M ammonium acetate buffer pH 6
Ammonium acetate 97+% reagent, FW 77.08. Dissolve 3.854 g in 1 L of water and adjust pH using 1 M
acetic acid (0.9635 g in 250 mL of water).
3. 1 M acetic acid
Acetic acid glacial 99.99+%, FW 60.05. Add 6 mL of acid to 50 mL of HPLC grade water in a 100 mL
volumetric flask, make up to volume with water.
4. Methanol/ammonium hydroxide (95:5, v/v)
Add 5 mL of ammonium hydroxide, FW 35.05, to 50 mL of HPLC grade methanol in 100 mL volumetric flask, make up to volume with methanol.
O R D E R I N G I N F O R M AT I O N
Description
Part Number
Pack size
ISOLUTE Array format
ISOLUTE Array HCX 25 mg/1 mL wells*
100
902-0025-R
ISOLUTE Array HCX 25 mg/1 mL plate
1
902-0025-RP
* As with other Array products, loose wells can be processed on a standard VacMaster-10 or -20
Sample Processing Station equipped with Array Luer adaptors (p/n 120-1201). In order to process
loose wells using a VacMaster-96 Sample Processing Station, a base plate (part number 120-1000-P01)
and base plate sealing strips (part number 120-1200 for sealing unused positions) are required.
Description
Pack size
Part Number
1
902-0025-P01
ISOLUTE-96 format
ISOLUTE-96 HCX 25 mg plate
ISOLUTE column format
ISOLUTE HCX 25 mg/1 mL
100
902-0002-A
100
902-0002-AG
Tab-less ISOLUTE column format
ISOLUTE HCX 25 mg/1 mL (tab-less)
Other configurations are available, please contact Biotage for details.
VacMaster-96 Sample Processing Station
Description
Pack size
Part Number
VacMaster-96 manifold only*
1
121-9600
VacMaster-96 Vacuum Control Unit
1
121-9601
VacMaster-96 Vacuum Control Unit with integral vacuum source
1
121-9602
VacMaster-96 with Vacuum Control Unit (121-9601)
1
121-9603
VacMaster-96 with Vacuum Control Unit (121-9602)
1
121-9604
* Option does not include a vacuum control unit.
© 2003 Biotage. All rights reserved. ISOLUTE, ISOLUTE Array, and VacMaster are registered trademarks of Biotage
057-702
www.biotage.com
UNITED STATES AND CANADA
Main Office: +1 434 979 2319
Toll Free: +1 800 446 4752
Fax: +1 434 979 4743
Order Tel: +1 434 220 2687
Order Fax: +1 434 296 8217
[email protected]
UNITED STATES AND CANADA
Main Office: +1 434 979 2319
Toll Free: +1 800 446 4752
Fax: +1 434 979 4743
Order Tel: +1 434 220 2687
Order Fax: +1 434 296 8217
[email protected]
JAPAN
Tel: +81 422 28 1233
Fax: +81 422 28 1236
[email protected]
Technical Note 127
SAMPLE PREPARATION BY MIXED-MODE SPE USING
ISOLUTE ® HAX
This technical note describes the extraction of acidic drugs from biological fluids using ISOLUTE
HAX mixed-mode SPE sorbents.
Sample preparation techniques such as protein precipitation, supported liquid extraction or non-polar
SPE may not be selective enough to give extracts of sufficient purity for low level analysis. In these cases,
the selective mixed-mode approach to the extraction of acidic drugs is a suitable alternative, giving very
high purity extracts with minimal levels of co-extracted material.
ISOLUTE HAX mixed-mode SPE sorbent is based on a combination of strong anion exchange and
non-polar chemistries. Acidic drugs are retained by two primary retention mechanisms (see Figure 1).
This allows a rigorous interference elution regime to be used to elute interferences retained by either
non-polar or anion exchange interactions alone. Only analytes with both non-polar and acidic characteristics are extracted using the ISOLUTE HAX sorbent, providing an extremely pure final extract.
R
– OOC
COO –
R
Ionic
interaction
Hydrophobic
interaction
C8
NR 3+
NR 3+
NR 3+
HO
Si
O
O
O
Si
S
Si
O
Si
OH
HO
O
Si
O
OH
O
Si
OH
O
S
Si
O
O
HO
Si
O
Si
Si
O
OH
O
Si
O
S
Si
Figure 1. Multiple interactions on ISOLUTE HAX mixed-mode columns
O
The mixed-mode approach for extraction of ionizable drugs from biological fluids is extremely robust.
The initial retention mechanism for the analytes is non-polar (hydrophobic), and is unaffected by the
high or variable ionic strength of the matrix. Loading sample at acidic pH will therefore minimize retention of small organic acid endogenous compounds, as they will not retain by hydrophobic interaction
alone. This ensures additional extract cleanliness, particularly when extracting urine samples.
EXTRACTION PROTOCOL
Evaluate ISOLUTE HAX, 25 mg/1 mL in the ISOLUTE Array® format using the procedure detailed
below. Populate the plate with individual wells as required. Process using a VacMaster®-96 sample processing station or automated liquid handling system.
Vacuum settings
At all stages, use a short pulse (approximately 1 second) of low vacuum (< -5" Hg), unless otherwise
stated.
Sample volume
This procedure is optimized for a biological fluid sample volume of 100 µL. Sample should be diluted
1:1 (v/v) with appropriate buffer before applying to the column (total volume of buffered sample
applied is 200 µL).
Note: Work in our R&D laboratory has shown that ISOLUTE 25 mg SPE columns have sufficient
capacity for extraction of up to 1 mL plasma sample without analyte breakthrough. Test conditions:
1 mL plasma spiked at 0.1 µg/mL analyte concentration and diluted 1:1 with buffer before applying to
the column (total volume of buffered sample applied is 2 mL).
Sample pre-treatment
Dilute the sample (100 µL of plasma or urine) with formic acid (2%, pH 2, 100 µL) to give a 200 µL total
sample volume at 1:1 dilution. Mix thoroughly.
Column conditioning
Place extraction plate on vacuum manifold. No collection plate should be used at this stage.
Condition each well with methanol (1 mL). Use gravity or a short pulse of vacuum to initiate flow.
This will ensure efficient wetting of the hydrophobic frits, promoting even flow of sample through the
wells.
Column equilibration
Rinse wells with formic acid (2%, pH 2, 250 µL). Load all wells prior to applying a short pulse of vacuum to initiate flow.
Sample loading
Apply 200 µL acidified sample. Load all wells prior to applying a short pulse of vacuum to initiate flow.
Interference elution
Elute basic and neutral interferences with:
■ Ammonium acetate buffer (0.1M, pH 7, 250 µL)
■
Apply vacuum for 30 seconds to dry sorbent bed
■
Methanol / water (50/50, v/v, 250 µL)*
■
Apply vacuum for 30 seconds to dry sorbent bed
*Evaluate increasing the % methanol at this stage to improve extract cleanliness. Check for analyte
breakthrough.
For each solvent, load all wells and allow to soak for 1 minute prior to applying a short pulse of vacuum.
Analyte elution
Place collection plate in base of manifold.
Ensure correct alignment (position A1 of collection plate directly underneath position A1 of extraction
plate), and that extraction plate outlet Luer tips extend below the rim of the collection plate. This will
prevent sample cross contamination. Spacers are available to ensure optimum penetration.
Elute acidic analytes with methanol/acetic acid (98:2, v/v, 2 x 100 µL). This will suppress ionization of
the drug, breaking both anionic and non-polar retention mechanisms, allowing elution of the analytes.
Apply the first 100 µL aliquot and allow to soak for 2–4 mins. If the aliquot has not reached the top frit
at the end of the soak time, apply a short vacuum pulse.
Apply the second 100 µL aliquot and allow to soak for a further 2–4 mins. Apply low vacuum for
1 minute to complete elution.
Evaporate this elution solvent and re-constitute the sample in a solvent compatible with the analytical
technique. For LC-MS the mobile phase is suggested.
Care should be taken to avoid losses of thermally labile or volatile analytes at this stage.
REAGENTS
1. Methanol
2. 2% Formic acid, pH 2
Add 2.083 mL formic acid (96%) to 100 mL volumetric flask, and make up to volume with HPLC grade
water.
3. 0.1 M Ammonium acetate pH 7
Ammonium acetate 97+% reagent, FW 77.08. Dissolve 7.708 g in 1 L of HPLC grade water.
4. Methanol / water (50:50, v/v)
Add 50 mL methanol to 100 mL measuring cylinder, make up to volume with HPLC grade water.
5. Methanol / acetic acid (98:2, v/v)
Add 2 mL acetic acid, glacial 99.99+% to 50 mL of methanol in 100 mL volumetric flask, make up to
volume with methanol.
O R D E R I N G I N F O R M AT I O N
Description
Part Number
Pack size
ISOLUTE Array format
ISOLUTE Array HAX 25 mg/1 mL wells*
100
903-0025-R
ISOLUTE Array HAX 25 mg/1 mL plate
1
903-0025-RP
* As with other Array products, loose wells can be processed on a standard VacMaster-10 or -20
Sample Processing Station equipped with Array Luer adaptors (p/n 120-1201). In order to process
loose wells using a VacMaster-96 Sample Processing Station, a base plate (part number 120-1000-P01)
and base plate sealing strips (part number 120-1200 for sealing unused positions) are required.
Description
Pack size
Part Number
1
903-0025-P01
ISOLUTE-96 format
ISOLUTE-96 HAX 25 mg plate
ISOLUTE column format
ISOLUTE HAX 25 mg/1 mL
100
903-0002-A
100
903-0002-AG
Tab-less ISOLUTE column format
ISOLUTE HAX 25 mg/1 mL (tab-less)
Other configurations are available, please contact Biotage for details.
VacMaster-96 Sample Processing Station
Pack size
Part Number
VacMaster-96 manifold only*
1
121-9600
VacMaster-96 Vacuum Control Unit
1
121-9601
VacMaster-96 Vacuum Control Unit with integral vacuum source
1
121-9602
VacMaster-96 with Vacuum Control Unit (121-9601)
1
121-9603
VacMaster-96 with Vacuum Control Unit (121-9602)
1
121-9604
Description
* Option does not include a vacuum control unit.
© 2003 Argonaut Technologies, now a Biotage company All rights reserved. ISOLUTE, ISOLUTE Array, and VacMaster are registered trademarks of Biotage.
063-702
United States and Canada
T: + 1 434 979 2319
Toll-Free: +1 800 446 4752
[email protected]
Sweden
Biotage
T: + 46 18 56 59 00
[email protected]
United Kingdom, EIRE
Biotage
T: + 44 1443 811811
[email protected]
Japan
Biotage
T: + 81 422 281233
[email protected]