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WA20252
Environmental
& Agrochemical
Applications
Notebook
www.waters.com
Waters Corporation, 34 Maple Street, Milford,
Massachussetts 01757-3696 U.S.A.
Tel: 508 478-2000
Fax: 508 872-1990
© 2002 Waters Corporation.
Oasis® Sample Extraction Products
Agrochemical and Environmental Applications Notebook
Oasis® Sample Extraction Products – General Information
Oasis® HLB.......................................................................................ii-iv
Oasis® MCX .....................................................................................v-vi
Oasis® MAX....................................................................................vii-ix
Oasis® Methods ..............................................................................x-xv
Applications ................................................................................................1-54
Product Information ................................................................................55-57
Appendix A – Oasis® Glass Cartridge Instructions ..........................................58-59
Appendix B – Oasis® for EPA (SW-846) ...................................................... 60-61
Bibliography .............................................................................................62-63
Click anywhere on list; type page #; click OK. Compound Index
Page
Acenaphthene.........................................1
Acenaphthylene.......................................1
Acephate ...............................................5
Acetaminophen ................................51-53
Acifluorfen ..........................................4, 7
Alachlor .................................................6
Aldicarb ..........................................31-32
Aldicarb Sulfone ...............................31-32
Aldicarb Sulfoxide.............................31-32
AMPA..................................................25
Anthracene .............................................1
Atrazine....................................2-3, 33-35
Azinphos Methyl......................................9
Benomyl ..........................................33-35
Bentazon ................................4, 7, 42-45
Benzo(a)anthracene .................................1
Benzo(a)pyrene .......................................1
Benzo(b)fluoranthene................................1
Benzo(k)fluoranthene ................................1
Benzoperylene ........................................1
Benzylbutyl phthalate...................29-30, 38
Bis(ethylhexyl)phthalate ................29-30, 38
Bisphenol A.........................27, 33-35, 39
Bolstar....................................................9
Cafteric acid....................................48-49
Carbaryl .........................................31-35
Carbendazim...................................17-20
Carbofuran ......................................31-32
Chloramben ............................4, 7, 42-45
2-Chloro-2,6-diethylacetanilide...................6
4-Chloro-3-methylphenol .........................12
Chlorogenic acid..............................48-49
2-Chlorophenol .....................................12
Chlorpyrifos ............................................9
Chlortetracycline ...............................13-14
Chrysene................................................1
Cichoric acid ...................................48-49
Clofibric acid...................................51-53
Clopyralid .......................................10-11
Coumaphos............................................9
Cyanazine .............................................2
2,4-D...................................4, 7-8, 42-45
2,4-DB............................................4, 7-8
Demeton ................................................9
Desethylatrazine....................................2-3
Desethyldesisopropylatrazine .....................3
© 2002 Waters Corporation. Rev 3, 01/02
Page
Desisopropylatrazine .............................2-3
Diazinon ................................................9
Dibenzo(g,h,I)perylene .............................1
Dibutyl phthalate.........................29-30, 38
3,5-Dichlorobenzoic ................................4
2,6-Diethylaniline.....................................6
4,4-Difluorobiphenyl...............................28
Dicamba.................................4, 8, 42-45
Dichlorobenzoic ......................................7
2,4-Dichlorophenol ................................12
Dichlorprop......................................4, 7-8
Dichlorvos ..............................................9
Diethyl phthalate.........................29-30, 38
Diethylstilbestrol ...............................36, 39
2,4-Dimethylphenol................................12
2,4-Dinitrophenol...................................12
4,6-Dinitro-2-methylphenol.......................12
Dimethyl phthalate ......................29-30, 38
Dinoseb ..........................................4, 7-8
Dioctyl phthalate.........................29-30, 38
Diphenhydramine..............................51-53
Diquat..................................................24
Disulfoton ...............................................9
Endothall ..............................................16
Estradiol ...............................................39
Estrogen...............................................40
Estrone.................................................39
17b-estradiol ..................................36, 40
Ethoprop ................................................9
17a-ethynylestradiol ...............................36
Ethynylestradiol...........................39, 51-53
Fensulfothion ...........................................9
Fenthion .................................................9
Fluoranthene ...........................................1
Fluorene .................................................1
Fusidic acid ..........................................54
Glyphosate...........................................25
Hydroxyatrazine ...................................2-3
3-Hydroxycarbofuran.........................31-32
Hydroxydesethylatrazine ...........................3
Hydroxydesisopropylatrazine.....................3
Ibuprofen...................................41, 51-53
Indenopyrene..........................................1
Isorhamnetin..........................................47
Kaempferol...........................................47
Ketoprofen .....................................41, 54
Page
Malvidin-3-glucoside ..............................42
MCPA..................................4, 7-8, 42-45
MCPP .............................................4, 7-8
Meclofamic acid ...................................41
Merphos ................................................9
Methiocarb......................................31-32
Methomyl ........................................31-32
Metolachlor ............................................6
Mevinphos .............................................9
Minocycline..........................................15
Naled....................................................9
Naphthalene ..........................................1
1-Naphthylamine...................................21
Naproxen ............................................41
Naptalam........................................21-23
2-Nitrophenol........................................12
4-Nitrophenol ...............................4, 7, 12
Nonylphenol..............................27-28, 38
Oxamyl...........................................31-32
Oxytetracycline.................................13-14
Paraquat ..............................................24
Parathion Methyl......................................9
Pentachlorophenol (PCP).....................7, 12
Phenanthrene ..........................................1
Phenol .................................................12
Phenylbutazone .....................................41
Phenylpropanolamine ........................51-53
Phorate ..................................................9
Phthalic Acid.........................................21
Picloram ...............................4, 7-8, 42-45
Propoxur..........................................31-32
Pyrene....................................................1
Quercetin .............................................47
Ronnel ...................................................9
Salicylic acid ...................................51-53
Simazine ................................................2
Tamoxifen ........................................51-53
2,4,5-T ...........................................4, 7-8
2,4,5-TP..........................................4, 7-8
Tetrachlorovinphos....................................9
Tetracycline ......................................13-15
Thiabendazole .................................17-20
Tokuthion ................................................9
2,4,6-Trichlorophenol .............................12
Trichloronate ...........................................9
Triclopyr...........................................10-11
i
Oasis® HLB General Information
I.
Figure 1: Structure of Oasis® HLB solid-phase
extraction sorbent [poly(divinylbenzeneco-N-vinylpyrrolidone)] and physical characteristics.
Introduction
Considerable time and effort is consumed in choosing an appropriate
solid-phase extraction (SPE) sorbent and extraction protocol. The limitations of today’s sorbents require the analyst to watch carefully and control
closely the extraction procedure. Even then it is difficult and time-consuming to achieve high, reproducible recoveries for analysis of important
polar drugs and metabolites. A new polymeric SPE sorbent has been
developed that allows more samples to be processed in one batch,
resulting in a significant increase in throughput. The bottleneck at the
sample preparation step can now be significantly reduced or eliminated.
O
N
Traditional Methods for Solid-Phase Extraction
The most commonly used sorbents are porous silica particles surfacebonded with C18 or other hydrophobic alkyl groups. Prior to use, the sorbent must first be conditioned with a water-miscible organic solvent to solvate the alkyl chains, and then equilibrated with water or buffer solution.
Because these sorbents are not hydrophilic, or water-wettable, care must
be taken to ensure that the sorbent stays wet before loading the aqueous
sample. Failure to do so prevents proper sample-sorbent-contact and is
the major cause of low analyte recoveries and poor assay-to-assay reproducibility.
N
Average Pore Diameter (Å)
Specific Surface Area (m2/g)
Total Pore Volume (cc/g)
Mean Particle Diameter (µm)
Percent Fines < 10 µm
II.
Waters has designed Oasis® HLB sample extraction products to overcome these limitations of reversed-phase SPE and to streamline the
sample preparation process. The key to this advancement has been the
development of a novel patented* polymeric reversed-phase sorbent.
This macroporous copolymer [poly(divinylbenzene-co-N-vinylpyrrolidone)]
exhibits both hydrophilic and lipophilic retention characteristics. HLB is an
acronym for hydrophilic-lipophilic balance which describes two major
features of this sorbent: the unique abilities to (1) remain wetted with
water, and (2) to retain a wide spectrum of both polar and nonpolar
compounds. Oasis® HLB extraction plates and cartridges deliver higher,
more reproducible recoveries for a wide range of analytes using rapid,
straightforward extraction protocols. These results can be achieved without having to worry about watching your samples as they are processed,
because the sorbent can be allowed to dry out during the extraction
Oasis® HLB sample extraction products are available in various cartridge,
plates, and column configurations. A Certificate of Analysis (COA) in
each product box reports recoveries, with RSDs, for three polar
pharmaceutical compounds. The Certificate of Analysis displays results
from stringent quality control tests on the batch of polymer sorbent and
the lot of packed cartridges.
Current Oasis® Patents:
Patent No. 5,882,521 (1996), Patent No. 5,976,376 (1998)
Patent No. 6,106,721 (1999), Patent No. 6,254,780 (2001)
Patent No. 6,322,695 (2001), Additional Patents Pending
ExpertEase, Oasis, Symmetry, SymmetryShield and Waters are trademarks of Waters Corporation.
Bond Elut is a trademark of Varian.
© 2002 Waters Corporation
ii
O
82
831
1.4
31.4
0.1%
Effect of Drying Time on Recovery
The effect of recovery on cartridge drying time for pharmaceutical compounds in porcine serum is shown for Oasis® HLB extraction cartridges
and for Bond Elut® C18 cartridges. The Waters HPLC system used in this
experiment consists of a 600 Multi-Solvent Delivery System, a 486
Tunable Absorbance Detector and a 717plus Autosampler. Data
acquisition was performed using 860 ExpertEase™ software installed
on an 845 workstation. A 20-position vacuum manifold (MSE,
Torrance, CA) with a vacuum pump was used to process solid-phase
extraction cartridges.
Freshly thawed porcine serum was spiked with either a polar solution
containing 10 µg/mL each of procainamide, acetaminophen and ranitidine (Solution 1) or a non-polar solution containing 10 µg/mL each of
doxepin and propranolol (Solution 2). All analyses were performed in
triplicate. 1 cc, 100 mg Bond Elut® C18 and 1 cc, 30 mg Oasis® HLB
extraction cartridges were conditioned with 1 mL of methanol. When the
methanol level reached the top frit of the cartridge, the vacuum was maintained for 0, 10, 30, 60, 120, 240, or 480 seconds to vary the cartridge drying time. I mL of water was then applied to the cartridge followed by 1.0 mL of porcine serum spiked with Solution 1 or Solution 2.
Cartridges were washed with either 1 mL deionized water for the C18
Bond Elut® cartridges, or 1 mL of 5% methanol in water for Oasis® HLB
extraction cartridges. The analyte was eluted with 1 mL of methanol. Ten
µL of 1000 µg/mL internal standard (sulfanilamide for Solution 1, and
butyl paraben for Solution 2) was added. Samples were vortexed and
analyzed by HPLC.
Figure 2 shows the percent recovery versus cartridge drying time for pharmaceutical compounds in porcine serum on Bond Elut® cartridges. The
results show that recovery drops markedly for the most polar compounds
even before the first minute of cartridge drying. The results are consistent
with hydrophobic collapse of the C18–alkyl chains on the silica surface
when the conditioning solvent is lost. All reversed-phase silica sorbents
behave similarly, regardless of the manufacturer.
The results in Figure 2 are most compelling when multiple samples are
processed simultaneously on vacuum manifolds. If some of the cartridges
are inadvertently dried out, poor recovery and irreproducibility result. For
polar compounds such as drug metabolites, this drying has tremendous
impact. When samples are processed using traditional hydrophobic (like
C18 or PS-DVB) sorbents, every cartridge must be watched to keep it wet
with the conditioning solvent until the sample is loaded. This becomes difficult when many solid-phase extraction cartridges are being processed
simultaneously and virtually impossible when plates are processed. The
results in Figure 2 emphasize that the Oasis® HLB sorbent is essential to
the reliable use of the 96-well plate format.
The percent recovery for several drying times for Oasis® HLB extraction
cartridges demonstrates the ability of the sorbent to maintain surface conditioning even if the cartridge is allowed to run dry. Reproducibility and
recoveries are not affected and sample processing becomes easier and
more efficient. In addition, samples can be processed faster since the
vacuum can be run continuously instead of stopping and starting the
vacuum or manipulating stopcocks to stop the flow of solvent before it
reaches the sorbent bed.
Figure 2: Percent recovery versus cartridge drying time
for pharmaceutical compounds in porcine serum using
Bond Elut C18 and Oasis® HLB extraction cartridges.
This section suggests SPE methods for Oasis® HLB extraction cartridges
and plates. Slight differences in the procedure are noted for specific
applications. Section A describes a Quick Start SPE Procedure for acids,
bases and neutral analytes. Section B outlines adjustments that may
be needed to optimize your recoveries. Section C describes solution
preparation.
Section A: Quick Start SPE Procedure for Oasis® HLB
1.
Add mix 10 to 50 µL of internal standard to 1 mL of
sample. For analytes, that are highly bound to serum
protein, add / mix 20 µL of concentrated phosphoric
acid with the sample.
2.
If necessary, clarify samples by centrifugation at
8,000 x g for 20 minutes.
3.
Place Oasis® HLB extraction cartridges or plates on
vacuum manifold and set vacuum to 5" Hg. No
individual stopcocks are necessary for cartridges.
4.
Solid-Phase Extraction Procedure: The following
simple protocol should be used in preparing and
using the cartridges or plates for the isolation of a
wide spectrum of acidic, basic, and netural analytes.
Procedure optimization is discussed in Section B.
Note 1: Once the HLB sorbent has been conditioned and
equilibrated there is no need to keep the cartridges or plate
wells wet prior to sample loading. Maintain a continuous vacuum
on all cartridges throughout steps 4a–4d except to empty waste.
This convenience will save you time. For the load (4c) and Elute
(4e) steps, flow rates should not exceed 2 mL/min. In all other
steps, flow rates up to 10 mL/min are acceptable. You may
need to momentarily increase the vacuum to start the flow of
aqueous solutions.
Note 2: The solution volume in each step can be adjusted to meet
the specific application. For example, the Oasis® HLB extraction
plate, 96-well with 10 mg of sorbent per well, may use ≤ 200 µL
volumes at each step. For more details, see specific product
instruction booklet.
% Recovery on Oasis® HLB
% Recovery on C18
Drying Time (mins)
III. Methods for Oasis® HLB Extraction
Cartridges and Plates
4a. Condition:
Add to and draw through each
cartridge 1 mL methanol.
4b. Equilibrate:
Add to and draw through each
cartridge 1 mL water.
Drying Time (mins)
ExpertEase, Oasis, Symmetry, SymmetryShield, Sentry and Waters are trademarks of Waters Corporation.
Bond Elut is a trademark of Varian.
© 2002 Waters Corporation
iii
4c. Load:
Add to and draw through each
cartridge 1 mL of sample.
4d. Wash:
Add to and draw through each
cartridge 1 mL of 5% methanol in
water (v/v). Release vacuum,
remove manifold cover, and
discard waste fluids. Insert rack
containing collection vessels,
replace cover and turn on vacuum.
4e. Elute:
Table 1
If the fraction from this
step contains the analyte
Load (4c)
Add to and draw through each
cartridge 1 mL methanol, collecting
eluates in suitable vessels.
5.
If desired, evaporate eluates to dryness and reconstitute in mobile phase or another suitable solvent.
6.
Analyze.
Wash (4d)
Section B: Adjustments to Optimize Recoveries
Spike 1 mL of PBS solution (for preparation, see Section C) with analytes
and internal standard. Follow steps 4a–4e in Section A, 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 1
mL portion of methanol and collect the eluate. Analyze all four collected
fractions. Use Table 1 to determine adjustments, if
necessary, to optimize sample recovery.
Section C: Preparation of Phosphate-Buffered
Saline (PBS)
To make isotonic saline solution:
1.
To a 1 liter flask, add the following anhydrous salts:
a. 200 mg KCl
b. 8000 mg NaCl
c. 200 mg KH2PO4
d. 1150 mg Na2HPO4
2.
Add 1 liter deionized water. Stir to dissolve.
3.
Adjust pH to 7.0 with10% phosphoric acid.
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
iv
First Elution (4e)
Second Elution
(4e repeated)
Make this adjustment for
optimum sample recovery
The Oasis® HLB sorbent has been
found to retain ionized analytes more
strongly than silica-based reversedphased 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.
Recoveries of very polar analytes can
be increased by using only 1 mL of
water (not 5% methanol in water) as
the wash solution.
If an acceptable recovery of analyte(s)
is obtained in this fraction (usually
> 90%), no adjustments are
necessary
For very nonpolar analytes, methanol
may not have adequate elution
strength. Stronger solvents such as
acetonitrile and ethyl acetate may be
substituted or used in sequence. In
addition, for ionizable analytes,
methanol may need to be modified
with the addition of 2% acid or
2% base, as appropriate.
Oasis® MCX General Information
I.
Section A: Quick Start SPE Procedure
for Oasis® MCX Sorbent
Introduction
Oasis® MCX extraction products contain a mixed-mode polymeric
patented sorbent in 30 µm and 60 µm (LP) particle sizes with reversedphase and cation-exchange functionalities The strong cation-exchange
sulfonic acid groups are on the surface of the Oasis® HLB sorbent, a poly
(divinylbenzene-co-N-vinylpyrrolidone) copolymer. The major difference
between the Oasis® HLB and MCX sorbents is the high selectivity of the
MCX sorbent for basic compounds.
The cation-exchange groups impart high selectivity for basic drugs
allowing you to obtain clean extracts from urine, whole blood, serum,,
plasma, soils, and water for analysis by HPLC, GC, GC-MS, or LC/MS.
The mixed-mode Oasis® MCX sorbent gives high and reproducible
recoveries for acidic, basic, and neutral compounds—even if the cartridge runs dry. Therefore, the Oasis® MCX cartridges can be used for
all stages of drug monitoring: screening, identification/confirmation, and
quantification. The two available particle sizes allow you to select the
appropriate product based on the viscosity and turbidity of your sample.
For viscous samples, excellent flow can be achieved using the 60 µm
large particle (LP) size sorbent in either cartridges or plates. Oasis® MCX
extraction products are available in cartridges and 96-well plates as listed in the ordering information
The Certificate of Analysis (COA) also contained in this package reports
recoveries with RSDs for the weak acid barbital and neutral drug acetaminophen and the basic compounds amphetamine, toluidine, and procainamide isolated according to the method in Section A. The COA
displays results from stringent quality control tests on the batch of polymer
sorbent and the lots of packed cartridges.
The Quick Start SPE Procedure for Oasis® MCX Sorbent (Table 2) is
an excellent starting point for any method. Further optimizations are
also discussed. Methods developed on the 1 cc/30 mg cartridges
are directly transferable to the 30 mg per well extraction plates. The
procedure also applies to the 10 mg per well plates except, lower
volumes (>300 mL) can be used for each step.
Table 2
Steps for 1 cc/30 mg
Cartridges
1 mL methanol/
1 mL water
Load
1 mL spiked and acidified
plasma or urine
N
SO3 H
SO3 H
HO3S
N
O
Prepares sorbent for use.
Optional step in extracting
basic drugs from human
urine (see Wash 3) and
plasma.
At low pH, all bases are in the
ionized form for retention by
cation-exchange. Neutral and
acidic analytes are retained by
reversed-phase mechanism.
Wash 1
1 mL 0.1N HCl
Removes proteins and locks basic
drugs to sorbent by ion-exchange
mechanism.
Wash 2
1 mL 100% methanol
(MeOH)
Removes interferences retained by
hydrophobic interaction. Can be
used as an elution step for neutral
and acidic compounds, if desired.
Figure 3: Structure of Oasis® MCX sorbent
O
Purpose
Wash 3 (optional)
1 mL <60% methanol with
5% ammonium hydroxide
(NH4OH)
Removes basic compounds that
are more polar than analyte
of interest.
Elute 1 mL 5%
NH4OH in methanol
Elutes basic drug(s) of interest.
Evaporate and Reconstitute
in 100 µL of an appropriate
solvent or solution
Concentrates sample and/or
changes solvent for analysis.
Sulfonation of poly(divinylbenzene-co-N-vinylpyrrolidone) is done at a
tightly controlled level of 1.0 meq/gram, producing a unique, strong,
cation-exchange sorbent.
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
v
Section B: Troubleshooting/Method
Optimization (Table 3)
1.
2.
3.
Protein-binding: the pH can be lowered to disrupt drug-protein
interaction. Add either 20 µL concentrated H3PO4 per mL plasma
or 10 mL of 5N HCL per mL urine.
Cartridges run dry during processing: if the cartridges run dry there
is no impact on performance, whether analyzing a mixture of
acidic, basic, and neutral drugs or extracting basic drugs.
Reuse: Do not reuse. The cartridges are designed for single
use only.
4.
Adjustments to optimize recovery: Repeating the Quick Start SPE
Procedure in Section A using spiked PBS instead of plasma or urine
allows method optimization to improve recovery. Spike an
appropriate volume of PBS with all analytes and internal standard.
Follow the steps in the SPE procedure and collect the eluates from
each step. A second final elution step should also be performed.
Analyze all fractions for the presence of the analyte. Use the
following suggestions, if necessary, to optimize the analyte recovery.
Table 3
If the fraction from this
step contains the analyte
Load
Wash 1 (0.1 N HCl)
The Oasis® MCX sorbent can be used from pH 0 to 14 enabling you to optimize pH for analyte retention,
hence achieve higher recoveries. Recoveries for acidic compounds 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. Basic compounds should be in the ionized form for best retention. For basic analytes,
adjust the pH to below 5.
Use a cartridge or plate containing more sorbent mass.
Wash 2
(100% methanol)
Neutral and acidic compounds elute in this step and can be collected for analysis. If acceptable recoveries
of these analytes are obtained in this fraction (usually > 90%), no adjustments are necessary. If recoveries
are not acceptable and analytes were not found in previous fractions, use either a larger volume of
methanol or a stronger elution solvent such as acetonitrile or ethyl acetate. Note: If water immiscible
solutions are used in this step, dry cartridges or plates under vacuum for 5 minutes to remove residual
water prior to the Wash 2 step.
Wash 3 (optional)
(<60% methanol
with 5% NH4OH)
Reduce the percent methanol if basic analytes are present. If neutral and acidic analytes are found, use
either a larger volume of methanol or a stronger elution solvent in Wash 2 as described above.
Elution (5% NH4OH/
95% MeOH)
Second Elution
(5% NH4OH/
95% MeOH)
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
vi
Make this adjustment for optimum sample recovery
If an acceptable recovery of analyte(s) is obtained in this fraction (usually > 90%), no adjustments
are necessary.
The volume of elution solution can be increased to improve recoveries. For very nonpolar basic analytes,
methanol may not have adequate elution strength. Stronger solvents such as acetonitrile or ethyl acetate
(EA) with 5% NH4OH may be substituted, or used in sequence. If water immiscible solutions are used
for elution and an aqueous Wash 3 has been performed, dry the cartridges or plates under vacuum
for 5 minutes to remove residual water prior to the Elution step.
Oasis® MAX General Information
I.
Preparation of Reagents
Introduction
Oasis® MAX extraction products are available in cartridges and 96-well
plates as listed in the ordering information. These products contain a
mixed-mode polymeric (patented) sorbent in 30 µm or 60 µm (LP) particle size with reversed-phase and anion-exchange functionalities. Strong
anion-exchange quaternary amine groups (0.25 meq/g dimethylbutylamine) are on the surface of a poly (divinylbenzene-co-N-vinylpyrrolidone) copolymer. The major difference between the Oasis® HLB and
MAX sorbents is the presence of the anion-exchange groups that provide
high selectivity for acidic compounds.
The anion-exchange groups impart high selectivity for acidic drugs allowing you to obtain clean extracts from urine, whole blood, serum, plasma,
soils, and water for analysis by HPLC, GC, GC-MS, or LC/MS. The
mixed-mode Oasis® MAX sorbent gives high and reproducible recoveries
for acidic, basic, and neutral compounds—even if the cartridge runs dry.
Therefore, the Oasis® MAX cartridges can be used to extract drugs for
monitoring, screening, confirmation, and quantitation. The two available
particle sizes allow you to select the appropriate product based on the
viscosity and turbidity of your sample. For viscous samples, excellent flow
can be achieved using the 60 µm large particle (LP) size sorbent in either
cartridges or plates.
The Certificate of Analysis (COA) also contained in this package reports
recoveries with RSDs for the weakly acidic drug secobarbital, the basic
drug nortriptyline, and three acidic drugs salicylic acid, ketoprofen and
naproxen isolated according to the method in Section A. The COA
displays results from stringent quality control tests on the batch of polymer
sorbent and the lots of packed cartridges.
Figure 4: Structure of Oasis® MAX sorbent
O
50 mM Sodium Acetate pH 7/ Methanol (95/5) Wash Solution—1 L
Prepare the 50mM Sodium Acetate pH 7 buffer by adding 6.80 grams
of sodium acetate trihydrate to 1 liter of deionized water. Mix to dissolve.
Adjust to pH 7.0 using a 10% glacial acetic acid aqueous solution.
Add 53 mL (41.6 grams) of methanol and mix. Transfer to a suitable
container. Cap and store at 4 °C and discard after six months.
2% Formic Acid (CHOOH) in Methanol—100 mL
Prepare the 2% formic acid (CHOOH) in methanol elution solution by
pipetting 2 mL of concentrated formic acid (90%) into a 100 mL volumetric flask containing ~60 mL of methanol. (Note: After pipetting concentrated formic acid be sure to displace the vapors from the pipette body by
repeatedly drawing up and dispensing air.) Fill to the mark with
methanol. Cap and mix by inversion. Make fresh daily.
Section A: Quick Start SPE Procedure
for Oasis® MAX Sorbent
The Quick Start SPE Procedure for Oasis® MAX sorbent (Table 4) is
an excellent starting point for any method. Further optimizations are
also discussed in Section 5. Methods developed on the 1 cc/30 mg
cartridges are directly transferable to the 30 mg/well 96-well extraction
plates. The procedure also applies to the 10 mg per well plates except
lower volumes (>300 mL) can be used for each step.
N
+
CH2 NR 3
+
CH 2NR3
+
R3NCH2
Phosphate-Buffered Saline (PBS)—250 mL
Prepare the PBS by adding 250 mL deionized water, 0.050 g KCl,
2.000 g NaCl, 0.050 g KH2PO4, and 0.288 g Na2HPO4 to a 250
mL beaker. Stir to dissolve. Adjust the pH to 7.0 ±0.05 using
concentrated H3PO4. Store in a capped bottle under refrigeration and
discard after three months.
N
O
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
vii
Table 4
Note: If necessary, clarify the samples by centrifugation at 8000 x g for
20 minutes prior to loading on the cartridge.
Steps for 1 cc/30 mg
Condition and Equilibrate
1 mL methanol/1 mL water
Purpose
Prepares sorbent for use. Optional
step in extracting acidic drugs from
human urine (see Section A)
and plasma.
Load
1 mL spiked and acidified
plasma or urine
At low pH, bases are ionized and
acidic and neutral compounds are
neutral. All analytes are retained by
reversed-phase interactions. Note:
Acidic analytes typically bind
strongly to matrix proteins. To break
these interactions, acidify (<2.5)
samples prior to loading.
Wash 1
1 mL pH 7, NaOAc 50mM/
MeOH (95/5)
Removes proteins and locks acidic
drugs to sorbent by ion-exchange
mechanism.
Elute 1 or Wash 2
1 mL 100% methanol
(MeOH)
Removes interferences retained by
hydrophobic interaction. Can be
used as an elution step for neutral
and basic compounds, if desired.
Elute 2
1 – 2 mL 2% formic
acid in methanol
Evaporate and Reconstitute
In 100 µL of an appropriate
solvent or solution
1 mL elutes most acidic analyte
of interest. 2 mL needed for
salicylic acid.
Concentrates sample and/or
changes solvent for analysis.
Note 1: The volume of solvent or solution used in each step depends on
the sorbent mass in the cartridges or plates: >300 mL for 10 mg/well
plates, >1 mL for 1 cc cartridges or 30 mg/well plates, >2 mL for 3 cc
cartridges, and >5 mL for 6 cc cartridges.
Note 2: For the load and elute steps, respective flow rates should not
exceed 1 mL/min for 1 cc cartridges, 2 mL per minute for 3 cc
cartridges, or 5 mL /min for 6 cc cartridges. To avoid exceeding the
recommended elution flow rates on cartridges containing Oasis® MAX
60 µm (LP) sorbent, use gravity feed or a low vacuum setting to elute
analytes. In all other steps flow rates up to 10 mL/min are acceptable.
You may need to increase the vacuum momentarily to start the flow of
aqueous solutions.
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© 2002 Waters Corporation
viii
Note 3: There is no need to keep the cartridges wet prior to sample
loading. Maintain a continuous vacuum on all cartridges throughout all
steps. Individual stopcocks are not required. This convenience will save
you time.
Note 4: The addition of 100 µL of a low volatility solvent such as
dimethylformamide (DMF) to samples prior to evaporation reduces evaporative loss of volatile analytes. Alternatively, acidifying a solution will
reduce evaporative loss of volatile basic analytes. Basifying a solution
will reduce evaporative loss of volatile acidic analytes.
Note 5: The elution solvent can be modified in order to be compatible
with the method of analysis, such as GC or GC/MS. The Oasis® MAX
sorbent is compatible with a wide range of organic solvents.
Note 6: Recommendations for using Oasis® MAX 96-well
extraction plates:
a. The capacity of the vacuum manifold tray is 200 mL; the tray
should be emptied after each step.
b. The 96-well collection trays should be used at the Elute 1 and/or
Elute 2 steps, depending on the application.
c. Momentarily increase vacuum to maximum after each step for
optimal precision.
d. If the entire plate is not being used, place a sheet of 3M Scotch™
clean label protection sheet (3M part no. 822-p) or other suitable
cover over the unused portion of the plate to avoid contamination
and maintain a consistent vacuum.
Section C: Troubleshooting Method Optimization
Table 5
If the fraction from this
step contains the analyte
Load
Wash 1 (pH 7 50mM
NaOAc/methanol)
Elute 1 or
Wash 2
(100% methanol)
Wash 3 (optional) (<60% methanol
with 2% formic acid)
Make this adjustment for optimum sample recovery
The Oasis® MAX sorbent can be used from pH 0 to 14 enabling you to optimize pH for analyte retention,
hence achieve higher recoveries. Recoveries for acidic compounds may be enhanced when the analyte
is ionized. For acidic analytes, adjust the sample pH to at least two pH units above the pKa of the acid.
The best retention for basic compounds is achieved when ionization is suppressed. For basic analytes,
adjust the pH to two pH units above its pKa.
Use a cartridge or plate containing more sorbent mass.
Neutral and basic compounds elute in this step and can be collected for analysis. If acceptable recoveries
of these analytes are obtained in this fraction (usually > 90%), no adjustments are necessary. If recoveries
are not acceptable and analytes were not found in previous fractions, use either a larger volume of
methanol or a stronger elution solvent such as acetonitrile or ethyl acetate. Note: If water immiscible
solutions are used in this step, dry cartridges or plates under vacuum for 5 minutes to remove residual
water. If acidic compounds are found in this fraction, use 2% NH4OH in methanol as the wash 1 solution.
Reduce the percent methanol if acidic analytes are present. If neutral and basic analytes are found,
use either a larger volume of methanol or a stronger elution solvent in Elute 1 as described above.
Elute 2 (2% formic
acid in MeOH)
If an acceptable recovery of analyte(s) is obtained in this fraction (usually > 90%), no adjustments
are necessary.
Second Elution 2
(2% formic acid in
MeOH)
The volume of elution solution can be increased to improve recoveries. For very nonpolar acidic analytes,
methanol may not have adequate elution strength. Stronger solvents such as acetonitrile or ethyl acetate (EA)
with 2% CHOOH may be substituted, or used in sequence. If water immiscible solutions are used for
elution and an aqueous solution was used in the previous step, dry the cartridges or plates under vacuum
for 5 minutes to remove residual water prior to the Elution step.
Elution Step
SPE Method Examples
I. Protein-binding: The pH can be lowered to disrupt drug-protein
interaction. Add either 20 µL concentrated H3PO4 per mL plasma or
urine or 10 - 30 mL of 5N HCl per mL urine.
A method development strategy can be tailored to specific applications.
At least three different approaches to method development can be used.
2. Cartridges run dry during processing: If the cartridges run
dry there is no impact on performance whether analyzing a mixture of
acidic, basic, and neutral drugs or extracting acidic drugs.
3. Reuse: The cartridges are designed for single use only.
Do not reuse.
4. Adjustments to Optimize Recovery: Repeating the Quick
Start SPE Procedure using spiked PBS instead of plasma or urine allows
method optimization to improve recovery. Spike an appropriate volume
of PBS with all analytes and internal standard. Follow the steps in the SPE
procedure and collect the eluates from each step. A second final elution
step should also be performed. Analyze all fractions for the presence of
the analyte. Use the suggestions, if necessary, to optimize the analyte
recovery (Table 5).
1. Collecting only Elute 2: The 100% methanol step can be used
to remove neutral and basic impurities prior to eluting a clean extract of
acidic drugs with 2% CHOOH in methanol.
2. Collecting Elute 1 and Elute 2: The sample can be divided
into two fractions, a basic and neutral analyte fraction and an acidic
analyte fraction.
3. Collecting only Elute 1: Clean extracts of basic and/or neutral
compounds can be obtained by eluting with 100% methanol and leaving all acidic endogenous interferences on the sorbent.
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
ix
Oasis® HLB Generic SPE Method for GC Analysis
Conditions for Oasis® HLB Cartridge, 5 cc, 200 mg glass cartridge
Prepare Sample
Condition/Equilibrate: 3 mL solvent*/
3 mL methanol/3 mL water
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in water
Elute:
6 mL solvent*
Dry, Adjust to Final Volume
*typical solvents:
ethyl acetate, MTBE,
methylene chloride.
For high polarity
analytes use 5% MeOH
in the solvent of choice
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
x
Oasis® HLB Generic SPE Method for HPLC Analysis
Conditions for 3 cc, 60 mg Oasis® HLB cartridge
Prepare Sample
Condition/Equilibrate:
1 mL methanol/1 mL water
Load:
up to 200 mL sample
Wash:
1 mL 5% methanol in water
Elute:
1 mL methanol
Evaporate and Reconstitute
This general protocol works for most
analytes in many matrices.
Use this generic method first.
Use optimized method to
reduce interferences
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
xi
Oasis® MAX Generic SPE Method
Typical Analysis: Acidic Analytes
Conditions for 6 cc cartridges
Prepare Sample
(neutral pH for most samples)
Condition:
3 mL methanol/ 3 mL water
Load:
up to 300 mL sample
Wash #1:
3 mL 50mM NaOAc (pH 7)
Elute 1 (Wash #2):
4 mL methanol
Elute 2*:
4 mL methanol (2 % formic acid)
Evaporate and Reconstitute
*Analytes with pKa < 3 require stronger acid
(i.e trifluoroacetic) at this step
Retention on Oasis® MAX sorbent is based on both
hydrophobicity and anion exchange.
Wash #2 (methanol) will contain bases and neutrals retained by reversed-phase
interaction. This fraction may be analyzed for those compounds if desired.
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
xii
Oasis® MCX Generic SPE Method
Conditions for 3 cc cartridges
Prepare Sample:
acidify, 0.05 N HCl
Condition:
1mL methanol/ 1 mL water
Load
up to 200 mL sample
Wash #1:
2 mL 0.1 N HCl
Wash #2:
2 mL methanol
Elute
2 mL methanol(4% NH4OH)
Evaporate and Reconstitute
This method allows for high retention of bases in acidified samples.
Wash #2 (methanol) will contain acids and neutrals retained by reversed-phase
interaction. This fraction may be analyzed for those compounds if desired.
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
xiii
Solid-Phase Extraction Method for Oasis® MCX
(Tissue Homogenate)
Procedure for extracting Basic drugs from Tissue homogenate
using 6 cc/150 mg LP Oasis® MCX cartridges.
1. Prepare Sample
6. Wash 2
•
•
Prepare Tissue homogenate (5.0 g) by diluting with
(50-60 mLl) 0.1M KH2PO4 (or other buffer), vortex
and centrifuge @ 3K RPM.
•
Collect supernatant (Filter*) and transfer to conditioned
Oasis® MCX Cartridge with 60 mL reservoir attached
* Filter if necessary, 0.45 micron filter
4mL- 0.1N HCl
(Removes proteins and non-retained aqueous
materials Portonates Bases)
7. Wash 3
•
4 mL - 5% MEOH in H2O
2. Condition Oasis® cartridge
8. Wash 4
•
•
2 mL MEOH
3. Equilibrate
•
9. Elute 2
2 mL Phosphate Buffer
•
4. Load
•
1. 2 mL -5% NH4OH in 70:30 ACN/MEOH)
(Elutes the basic drug(s)
Supernatant at 4-5ml/min.
10. Evaporate
5. Wash 1
•
4 mL -70:30 ACN/MEOH)
(Removes acidic and neutral drugs)
•
@ 40° C and reconstitute in appropriate solvent
4mL- H2O (Try 2 x 2 mL)
Note: No stopcock needed, turn vacuum
on add MEOH then add buffer!
Oasis® MCX Extraction Cartridge 6 cc 150 mg LP
Reservoir 60 cc for Oasis® cartridges
Reservoir Adapter for 1, 3, 6 cc cartridges
30/box
12/box
12/box
186000255
WAT024659
WAT054260
Notes: The Oasis® MCX sorbent is a Mixed-mode polymeric sorbent with Cation- eXchange and reversed-phase functional
groups. The strong Sulfonic acid cation exchange groups impart high selectivity for basic drugs, producing very clean extracts
from urine, whole blood, serum or plasma for GC, GC/MS, LC/MS or HPLC
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
xiv
Oasis® HLB method for fractionation of
natural product crude extracts
Selecting elution solvents by relative strength
for the generic SPE method (1-D)*
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Dilute sample to 95%
aqueous
Low—adjust pH—High
Prepare Sample
Condition:
3 mL methanol/3 mL water
Load:
up to 500 mL of sample
Wash 1:
3 mL 5% methanol in water
Remove weakly retained
polar compounds
Low—adjust pH—High
Wash 2:
3 mL methanol
Remove moderately-polar
and weakly retained nonpolar compounds
Low—adjust pH—High
Solvent Type
Relative
Elution Strength**
Methanol
Acetonitrile
Tetrahydrofuran
Acetone
Ethyl Acetate
proton donor
dipole-dipole
dipole-dipole
dipole-dipole
dipole-dipole
1.0
3.1
3.7
8.8
high
Methylene Chloride
dipole-dipole
high
Comments
disrupts H-bonding
medium polarity drugs
medium polarity drugs
medium polarity drugs
nonpolar drugs and
GC compatible
nonpolar drugs and
GC compatible
* When using solvents other than methanol, add 10-30% methanol to disrupt H-bonding
on the Oasis® HLB sorbent.
** High-Purity Solvent Guide. Burdick & Jackson Laboratories, Inc. Solvent Properties of
Common Liquids, L.R. Snyder, J. Chromatogr., 92, 223 (1974); J. Chromatogr.
Sci. 16, 223 (1978)
Remove moderately retained
non-polar compounds
Low—adjust pH—High
Wash 3:
3 mL 70/30 acetonitrile/MeOH
Solvent
Remove strongly retained
non-polar compounds
Low—adjust pH—High
Wash 4:
3 mL 90/10 MeOH/DCM
Selecting pH for optimal retention and elution
Low pH retain
acidic compounds
High pH retain
basic compounds
20
Theory
18
Acids (HA)
16
B
Retention Factor
14
(pKa = 9.0 )
(pKa = 4.8 )
Neutrals
12
10
8
6
4
Low pH elute
basic compounds
A-
Bases (BH+)
2
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
High pH elute
acidic compounds
pH
Recommended pH range of Silica
0
Recommended pH range of Oasis®
14
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
xv
Polycyclic Aromatic Hydrocarbons
in Drinking Water
Compounds
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
% Recovery
Tap Water
Tap Water
1.0 µg/L
200 ng/L
4 replicates
4 replicates
66.5
55*
99.2
78*
98.4
77*
105
82.4
114
94.4
103
83.0
115
91.9
117
98.9
102
85.9
105
90.3
104
80.1
90.9
77.4
94.3
69.8
92.1
59.5
92.0
65.9
92.8
62.3
RSDs < 5 %
RSDs < 10 %
*result @ 220 nm
Naphthalene
Acenaphthylene
Acenaphthene
Fluorene
Phenanthrene
Anthracene
Fluoranthene
Pyrene
Benzo(a)anthracene
Chrysene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Dibenzo(g,h,I)perylene
Benzoperylene
Indenopyrene
Oasis® HLB Extraction Method
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Condition:
5 mL methylene chloride
Rinse: 5 mL methanol
Rinse: 5 mL reagent H2O
Load:
500 mL sample @ 15 mL/min
Wash:
5 mL reagent H2O
Elute: 8 mL methylene chloride
(5 mL bottle wash + 3 mL)
Evaporate and Reconstitute:
with 1 mL acetonitrile
6
10
5
11
Phenanthrene
HPLC Method
9
Column:
Mobile Phase:
12
4
78
13
16
Gradient:
14 15
1 2
spiked tap water (1 µg/L)
3
nonspiked sample
0
10
Minutes
20
30
Flow Rate:
Detection:
Injection:
SepServe PAH, 125 x 4.6 mm
A: Water
B: Acetonitrile
60% A for 1 min, then linear
gradient to 100% B in 15 min
1.2 mL/min
UV @ 254 nm (0.02 AUFS)
20 µL
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
1
Triazine Herbicides in Drinking Water
Compounds
1.
2.
3.
4.
5.
6.
% Recovery
Tap Water
Tap Water
Spike Level
Spike Level
500 µg/L
200 ng/L
5 replicates
7 replicates
98.4 (5.0)
95.6 (5.8)
132 (1.3)
109 (11)
106 (5.1)
104 (4.0)
not determined
97.7 (3.9)
not determined
93.1 (3.7)
101 (5.0)
101 (4.4)
Desisopropylatrazine
Hydroxyatrazine
Desethylatrazine
Simazine
Cyanazine
Atrazine
Oasis® HLB Extraction Method
Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Condition: 3 mL methanol
Rinse: 3 mL H2O
Load:
75 mL sample @ 4 mL/min
Wash:
1 mL H2O
Elute:
1.2 mL methanol
Evaporate to 0.2 mL
Cl
Reconstitute to exactly 1.0 mL with H2O
N
N
NHCH(CH3) 2
N
C2H5NH
Atrazine
spiked well water
3
4
6
5
HPLC Method
2
1
nonspiked sample
Column:
Mobile Phase:
blank cartridge
Gradient:
0
5
Minutes
10
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
2
15
Flow Rate:
Detection:
Injection:
SymmetryShield™ RP8
3.9 mm x 150 mm
A: 15% Acetonitrile in
phosphate buffer (5 mM, pH 6.7)
B: Acetonitrile
100% A for 2 min, then
linear gradient to 70% B in 25 min
1.0 mL/min
UV @ 214 nm (0.02 AUFS)
75 µL
Atrazine and Metabolites in Drinking Water
Compounds
1.
2.
3.
4.
5.
6.
7.
(% Recovery
0.2 µg/L
94 (3)
75 (8)
89 (6)
79 (4)
107 (7)
79 (5)
89 (5)
Hydroxydesisopropylatrazine
Desethyldesisopropylatrazine
Hydroxydesethylatrazine
Desisopropylatrazine
Hydroxyatrazine
Desethylatrazine
Atrazine
Oasis® MCX SPE Method
- % RSD, n=5)
1.0 µg/L
85 (3)
76 (5)
76 (7)
83 (2)
101 (2)
83 (3)
77 (3)
Conditions for Oasis® MCX Cartridge, 6 cc, 150 mg
Part Number 186000256
Prepare Sample:
adjust to pH 1.5 with HCI
Condition: 2 mL methanol
Rinse with 2 mL H2O
Load Sample
Wash #1:
2 mL 0.1 N HCI
Cl
N
H2N
Wash #2:
2 mL methanol
N
N
Elute:
3 mL methanol (4% NH4OH)
NH2
Evaporate and Reconstitute
Desethyldesisopropylatrazine
PDA (0.08 AUFS)
Drinking water samples (100 mL) were spiked
with the herbicides and adjusted to pH 1.5. The
samples were then analyzed using 6 cc
Oasis® MCX cartridges using the protocol for
basic compounds
HPLC Method
4
1
Column:
Mobile Phase:
5
6
2
3
Gradient:
7
Spiked Sample
Blank Sample
0
10
20
Flow Rate:
Detection:
Injection:
SymmetryShield™ RP18, 3.9 x 150 mm
A: phosphate buffer (20 mM, pH 6.8)
B: acetonitrile
95% A for 2 min
then linear to 25% A in 20 min
0.8 mL/min
PDA (215nm)
80 µL
Minutes
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
3
Acidic Herbicides in Drinking Water
Compounds
Oasis® HLB Extraction Method
% Recovery
Tap Water
Well Water
400 ng/L
2.0 µg/L
5 replicates
5 replicates
126 (5.3)
97.5 (3.8)
98.5 (3.8)a
115 (4.4)a
99.2 (6.9)a
95.1 (10)
Well Water
400 ng/L
5 replicates
106 (2.3)
96.3 (8.3)
90.6 (5.6)
1.
2.
3.
Picloram
Dicamba
Chloramben
Tap Water
2.0 µg/L
5 replicates
90.9 (7.0)
85.1 (7.2)
86.7 (7.3)
4.
5.
6.
4-Nitrophenol
Bentazon
2,4-D
83.3 (6.1)
89.3 (6.0)
92.3 (7.1)
113 (6.0)a
114 (5.6)a
107 (3.1)
90.4 (1.7)a
91.2 (3.0)
86.5 (1.8)
112 (13)
104 (8,8)
122 (12)
7.
8.
9.
MCPA
Dichlorprop
2,4,5-T
97.6 (8.2)
96.4 (11)
106 (6.2)
104 (4.5)
107 (9.0)
116 (8.8)
80.8 (3.6)
87.4 (3.0)
95.1 (5.0)
96.7 (5.5)
103 (6.0)9
96.6 (12)
10. MCPP
100 (7.7)
11. 3,5-Dichlorobenzoic 93.3 (6.3)
12. 2,4-DB
95.4 (5.1)
116 (6.6)
119 (9.7)
110 (8.4)
93.8 (3.0)
84.3 (2.7)a
83.7 (5.6)
94.7 (2.9)
96.9 (5.9)
83.3 (5.2)
13. 2,4,5-TP
14. Acifluorfen
15. Dinoseb
92.5 (6.7)
102 (8.5)
73.8 (6.8)
87.7 (5.3)
70.0 (17)
54.7 (5.2)
82.7 (10)
81.3 (8.2)
88.1 (1.9)b
89.3 (7.9)
94.8 (8.3)
71.7 (7.1)
Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Prepare Sample: 75 mL sample,
adjust to pH 2 with H3PO4
Condition:
3 mL 10:90 methanol/MTBE*
Risnse: 2 mL methanol
Rinse: 2 mL H2O
Load:
75 mL sample @ 4 mL/min
Wash:
1 mL H2O
Elute:
2 mL 10:90 methanol/MTBE*
Evaporate to 0.2 mL
Reconstitute to 0.5 mL with H2O
subtracted blank value was greater than 10% of spike level
b
result measured at 268 nm
a
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
1
OCH2COOH
Cl
2
3
HPLC Method
spiked tap water
(400 ng/L)
Cl
45
6
8
7
13
12
910
14 15
11
nonspiked sample
0
10
20
Gradient:
Minutes
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
4
2,4-D
Column:
Mobile Phase:
30
Flow Rate:
Detection:
Injection:
SymmetryShield™ RP8
A: pH 3.4 phosphate
buffer (13 mM)
B: Acetonitrile
85% A linear to 70% A in 8 min,
hold until 15 min, then linear
to 40% A in 30 min, then linear
to 10% A in 35 min.
1.0 mL/min
UV @ 230 nm (0.015 AUFS)
75 µL
Acephate in Drinking Water
Oasis® HLB Extraction Method
% Recovery
Spike Level
Spike Level
10 µg/L
5 µg/L
5 replicates
7 replicates
91.0% (16.3% RSD)
83.6% (15.0% RSD)
(Source water: a chlorinated municipal tap water)
Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Prepare Sample:
To 20 mL sample add 5 g NaCl
Adjust to pH 3 with H3PO4
Condition:
3 mL methylene chloride
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Acephate
H 3 CS
H CO
3
O
O
P
C
N
H
Load:
20 mL sample @ 4 mL/min
CH 3
Elute:
2 mL methylene chloride
- contact and systematic
organophosphorous insecticide
- highly polar, water soluble
Evaporate and Reconstitute:
with 250 µL mobile phase
HPLC Method
acephate
spiked tap water
nonspiked sample
blank cartridge
0
5
Minutes
10
Column:
Mobile Phase:
Flow Rate:
Detection:
Injection:
SymmetryShield™ RP8
3.9 mm x 150 mm
4% acetonitrile in water
1.0 mL/min
UV @ 200 nm (0.02 AUFS)
75 µL
15
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
5
Acetamide Herbicides and Metabolites
in Drinking Water
Compounds
1.
2.
3.
4.
5.
Tap Water
2.0 µg/L
5 replicates
99.1 (10)
98.5 (3.8)
93.3 (11)
93.9 (3.2)
97.1 (6.6)
Metolachlor-metabolite*
2-Chloro-2,6-diethylacetanilide
2,6-Diethylaniline
Alachlor
Metolachlor
% Recovery
Tap Water
200 ng/L
5 replicates
78.9 (22)
109 (34)
80.3 (23)
90.1 (21)
93.0 (16)
Oasis® HLB Extraction Method
Well Water
200 ng/L
5 replicates
88.3 (3.3)
105 (24)
89.2 (7.8)
89.9 (1.2)
112 (3.3)
Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Condition:
3 mL methanol
Rinse:
2 mL H2O
Load:
150 mL sample @ 4 mL/min
* 2-[(2-ethyl-6-methylphenyl)amino]1-propanol
Wash:
1 mL H2O
Elute:
1.2 mL methanol
CH2CH3
Evaporate to 0.2 mL
Reconstitute to 0.5 ml with reagent H2O
CH2OCH3
N
COCH2Cl
CH2CH3
Alachlor
spiked well water
(200 ng/L)
HPLC Method
1
2
3
4 5
Column:
Mobile Phase:
nonspiked sample
Gradient:
blank cartridge
0
10
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
6
Minutes
20
Flow Rate:
Detection:
Injection:
Symmetry® C8
A: 30% acetonitrile in pH 6.8
phosphate buffer (10 mM)
B: acetonitrile
100% A initial, then linear
to 60% B in 20 min
1.2 mL/min
UV @ 214 nm (0.02 AUFS)
80 µL
Determination of Acidic Herbicides
in Drinking Water
Compound
picloram
chloramben
4-nitrophenol
(non-linear above 500)
bentazon
(non-linear above 300)
RF (slope)
3.7
1.8
r2
0.999
0.989
LOQ
300
200
RSD*
16 %
9.3 %
474
0.990
<100
5.6 %
181
--------
<100
6.1 %
2,4-D
MCPA
2,4,5-T
dichlorprop
MCPP
dichlorobenzoic
51
43
105
105
136
64
0.999
0.980
0.999
0.999
0.992
0.988
100
200
100
100
100
100
7.2 %
9.2 %
6.3 %
5.0 %
7.0 %
5.6 %
50
91
77
-------0.997
0.999
100
100
100
11 %
6.6 %
6.3 %
acifluorfen
(non-linear above 300)
2,4,5-TP
2,4-DB
dinoseb
(non-linear above 200)
pentachlorophenol
>500
67
-------0.998
<100
100
10.7 %
11.7 %
Oasis® MAX SPE Method
for Acidic Herbicides
Conditions for Oasis® MAX Cartridge, 6 cc, 150 mg
Part Number 186000369
Sample is first hydrollized at
pH 12 for 60 min. Then, pH
is adjusted to approx. neutral
with HCl before SPE.
Prepare Sample
Condition:
3 mL methanol/ 3 mL water
Load:
300 mL sample
Wash #2 (methanol) will
contain bases and neutrals
retained by reversed-phase
interaction. This fraction
may be analyzed for those
compounds if desired.
Wash #1:
3 mL 50 mM NaOAc (pH 7.5)
Analytes with pKa < 3
require strong acid
(i.e trifluoroacetic) at this step.
Elute 1 (Wash #2):
4 mL methanol
Elute 2*:
4 mL methanol(2 % TFA)
Evaporate and Reconstitute
*average of RSD from 4 levels
4
LC/MS (ESI-)
river water
1 µg/L spike level
16
5
8,9
7
1
10
15
13
14
11
2
HPLC Method
(low MS response for dicamba)
Instrument:
Column:
Injection Volume:
Mobile Phase:
6
3
12
Gradient:
Flow Rate:
Mass Spec:
Waters 2690 Separations Module
Waters XTerra® MS C18, 2.1 x 100 mm
20 µL
A: 15mM ammonium formate,
B: acetonitrile
25% B to 60% B in 9 min, hold
5 min, to 90% B in 16 min
200 µL/min
Waters/Micromass ZMD™, Positive
and Negative Electrospray (ESI+, ESI-)
Multiple Selected-Ion Recording (SIR)
Oasis, XTerra and Waters are trademarks of Waters Corporation.
ZMD is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
7
GC/MS Analysis of Acidic Herbicides
in Drinking Water
Compound
1. dicamba
2. MCPP
3. MCPA
4. dichloroprop
5. 2,4-D
6. 2,4,5-T
7. 2,4,5-TP
8. 2,4-DB
9. dinoseb
10. picloram
Oasis® MAX SPE Method
for Acidic Herbicides
% Recovery
< 95%
< 95%
< 95%
< 95%
< 95%
< 95%
< 95%
< 95%
< 95%
< 60%
Conditions for Oasis® MAX Cartridge, 6 cc, 150 mg
Part Number 186000369
Sample is first hydrollized at
pH 12 for 60 min. Then, pH
is adjusted to approx. neutral
with HCl before SPE.
Prepare Sample
Condition:
3 mL methanol/ 3 mL water
Load:
250 mL sample
40 µg/L spike level
Wash #2 (methanol) will
contain bases and neutrals
retained by reversed-phase
interaction. This fraction
may be analyzed for those
compounds if desired.
Analytes with pKa < 3
require strong acid
(i.e trifluoroacetic) at this step.
8,9
2
Wash #1:
3 mL 50 mM NaOAc (pH 7.5)
Wash #2:
4 mL methanol
Elute*:
4 mL MeOH/MTBE/TFA 89:10:1
Evaporate and Reconstitute
to 0.5 mL in MTBE
3
* methylated with diazomethane
1
4
5
6
7
HPLC Method
10
Column:
Carrier Gas:
Temp Program:
spiked sample
blank sample
5
Minutes
Oasis and Waters are trademarks of Waters Corporation.
RTX 5 is a trademark of Restek Corporation.
© 2002 Waters Corporation
8
10
15
Detection:
Injection:
RTX-5 capillary, 30 meters, 0.32 mm ID,
0.25 µm film thickness
Helium @ 20 cm/sec
50 °C 1 min initial hold, 25 oC/min
to 100 °C, then 10 °/min to 290 °C
FID
2 µL
Organophosphorous Pesticides
in Drinking Water and Fruit
Cl
Oasis® HLB Extraction Method
O-
N
O
Cl
Cl
OC2H5
S
P
O
N+
O
OC2H5
Chlorpyrifos
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
S
P
O
O
Condition:
6 mL 10:90 methanol/MBTE*
Parathion methyl
Rinse: 6 mL methanol
Rinse: 6 mL H2O
Fruit Extraction:
5-10 g fruit extracted with 20-40 mL acetonitrile, diluted
with H2O 1:10, and loaded onto the Oasis® cartridge.
Load:
500 mL sample @ 15 mL/min
Wash:
5 mL H2O
GC Method
Column:
SGE BPX5 capillary, 30 meters,
0.32 mm ID, 0.25 µm film thickness
Helium @ 30 cm/sec
50 °C initial, 30 °C/min
to 150 °C, then 6°/min to 300 °
NPD
2 µL
Carrier Gas:
Temp Program:
Detection:
Injection:
Elute:
8 mL 10:90 methanol/MBTE
Dry over sodium sulfate
Evaporate to 1.0 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
Compounds
17
4
1
19
2
9
6
20
7
12
14
16
18
10
15
11
ISTD
5
8
13
3
tap water (500 ng/L)
tap water blank
0
20
Oasis and Waters are trademarks of Waters Corporation.
BPX5 is a trademark of SGE Corporation.
© 2002 Waters Corporation
Minutes
40
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Dichlorvos
Mevinphos
Naled
Ethoprop
Phorate
Demeton
Diazinon
Disulfoton
Parathion Methyl
Ronnel
Chlorpyrifos
Fenthion
Trichloronate
Tetrachlorovinphos
Tokuthion
Merphos
Fensulfothion
Bolstar
Azinphos Methyl
Coumaphos
% Recovery
Tap Water
Apple
Spike Level
Spike Level
250 µg/L
400 ng/L
4 replicates
3 replicates
86.7 (7.9)
104 (8.8)
99.6 (7.8)
51.0 (3.5
112 (8.0)
95.6 (9.6)
110 (8.9)
121 (5.8)
88.2 (14)
84.0 (10)
110 (9.9)
104 (10)
113 (11)
108 (11)
115 (12)
102 (12)
112 (9.8)
101 (12)
111 (11)
89.5 (13)
120 (11)
84.7 (12)
112 (8.5)
88.4 (12)
118 (7.9)
81.3 (11)
111 (9.8)
87.0 (12)
112 (10)
73.8 (8.3)
107 (9.9)
139 (5.2)
108 (9.4)
101 (10)
111 (7.8)
72.3 (9.8)
113 (9.3)
82.3 (2.7)
113 (9.0)
77.0 (9.6)
9
LC/UV Determination of Clopyralid
and Triclopyr in River Water
Compounds
1.
2.
% Recovery (% Recovery ± % RSD, n=5)
Drinking Water
River Water
0.4 µg/L
2.0µg/L
0.4 µg/L
2.0 µg/L
100 (8)
110 (4)
94 (5)
110 (2)
85 (3)
87 (2)
82 (11)
81 (8)
Clopyralid
Triclopyr
Oasis® MAX Extraction Method
Conditions for Oasis® MAX Cartridge, 6 cc, 500 mg
Part Number 186000865
MTBE is employed as
elution solvent (elute 2) to
minimize humic interference
from surface water. Therefore
precondition with this solvent.
Prepare Sample:
pH 5 to 8
Condition:
3 mL each: MTBE*/MeOH/H20
Load:
300 mL sample
Wash 1:
3 mL 50 mM NaOAc (pH 7)
Cl
N
COOH
Cl
Cl
Cl
PDA (290 nm, 0.05 AUFS)
Clopyralid
0
N
COOH
Triclopyr
1.
2.
Clopyralid
Triclopyr
LC Conditions
Instrument:
10
Minutes
Elute:
4 mL MeOH/MTBE/TFA** (89:10:1)
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
** TFA - trifluoroacetic acid
1
0.4 µg/L spike level
Wash 2:
4 mL methanol
Evaporate and Reconstitute
Cl
Oasis, Alliance, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
10
O
Clopyralid is a stronger acid
than formic acid. Therefore,
formic acid cannot be utilized
to elute this compound
from Oasis® MAX sorbent.
TFA was employed for
elution of clopyralid.
2
20
Waters Alliance® Separations Module
with 996 PDA
Column:
Waters XTerra® RP18, 4.6 x 100 mm,
(3.5 µm dP)
Injection Volume: 50 µL
Mobile Phase:
20% acetonitrile/80% 10mM TFA
(pH 2.1) to 80% acetonitrilein 20 minutes
Flow Rate:
1.0 mL/min
LC/MS Determination of Clopyralid
and Triclopyr in River Water
Cl
N
COOH
Cl
Cl
Cl
Clopyralid
N
O
Optimized Oasis® MAX method
for clopyralid and triclopyr
COOH
Conditions for Oasis® MAX Cartridge, 6 cc, 500 mg
Part Number 186000865
Cl
Triclopyr
Prepare Sample:
pH 5 to 8
MTBE is employed as
elution solvent (elute 2) to
minimize humic interference
from surface water. Therefore
precondition with this solvent.
Condition:
3 mL each: MTBE*/MeOH/H20
Load:
300 mL sample
Wash 1:
3 mL 50 mM NaOAc (pH 7)
Clopyralid is a stronger acid
than formic acid. Therefore,
formic acid cannot be utilized
to elute this compound
from Oasis® MAX sorbent.
TFA was employed for
elution of clopyralid.
Wash 2:
4 mL methanol
Elute:
4 mL MeOH/MTBE/TFA** (89:10:1)
Evaporate and Reconstitute
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
** TFA - trifluoroacetic acid
0.4 µg/L spike level - river water
100
triclopyr
m/z = 258
% abundance
LC/MS Conditions
Instrument:
100
clopyralid
m/z = 192
0
5
10
Waters Alliance® Separations Module with
Waters/Micromass ZMD™
Interface:
Positive Electrospray (ESI+)
Multiple Selected-Ion Recording (SIR)
Column:
Waters XTerra® MS C18, 2.1 x 100 mm
Injection Volume: 20 µL
Mobile Phase
Gradient:
25% acetonitrile/75% 10mM TFA
(pH 2.1) to 90% acetonitrile in 6 minutes
Flow Rate:
200 µL/min
Oasis, XTerra, Alliance and Waters are trademarks of Waters Corporation.
ZMD is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
11
Phenols in Drinking Water
Compounds
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
% Recovery
Tap Water
Tap Water
500 ng/L
200 µg/L
5 replicates
5 replicates
98.8 (5.6)
99.7 (2.8)
98.4 (1.2)
95.6 (3.6)
89.2 (4.6)
93.2 (3.8)
90.8 (6.1)
86.2 (4.4)
101 (3.7)
95.3 (2.0)
99.8 (3.9)
95.6 (3.4)
101 (5.7)
97.4 (3.6)
85.0 (5.5)
84.1 (10)
91.5 (4.4)
94.2 (2.1)
93.0 (3.4)
87.8 (11)
90.5 (4.2)
81.0 (19)
Phenol
4-Nitrophenol
2-Chlorophenol
2-Nitrophenol
2,4-Dinitrophenol
2,4-Dimethylphenol
4-Chloro-3-methylphenol
2,4-Dichlorophenol
4,6-Dinitro-2-methylphenol
2,4,6-Trichlorophenol
Pentachlorophenol (PCP)
Oasis® HLB Extraction Method
Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Prepare Sample: 75 mL sample,
adjust to pH 2 with H3PO4
Condition:
3 mL 10:90 methanol/MTBE*
Risnse: 2 mL methanol
Rinse: 2 mL H2O
Load:
75 mL sample @ 4 mL/min
Wash:
1 mL H2O
Elute:
2 mL 10:90 methanol/MTBE*
OH
Evaporate to 0.2 mL
Reconstitute to 0.5 mL with H2O
2
280 nm
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
45
1
Phenol
9
3
1
6
7
273 nm
HPLC Method
8
Column:
Mobile Phase:
10
11
spiked tap water
(500 ng/L)
Gradient:
11
Flow Rate:
Detection:
Injection:
300 nm
5
10
Minutes
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
12
15
20
Symmetry® C8
A: 1% acetic acid in water
B: 1 % acetic acid in acetonitrile
30% B initial, linear gradient
to 100% B in 30 minutes
1.2 mL/min
UV (0.02 AUFS)
70 µL
Tetracyclines in Meat
Oasis® HLB Extraction Method
The homogenized meat sample is extracted with 2 x 20 mL of
McIlvaine (mixed citrate/phosphate, pH 4.1) buffer with added
EDTA. The combined extracts are filtered before proceeding to the
Oasis® HLB solid-phase extraction method.
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Condition:
3 mL methanol
For details of the meat extraction procedure see
J.AOAC Int., 79 (2), p 405 (1996).
Compounds
1.
2.
3.
Rinse:
2 mL H2O
% Recovery
Beef
Pork
Spike Level
Spike Level
0.5 µg/g
0.5 µg/g
4 replicates
4 replicates
96.2 (5.7)
103 (5.0)
91.4 (5.5)
99.8 (6.1)
80.6 (6.8)
83.4 (6.1)
Oxytetracycline
Tetracycline
Chlortetracycline
Load:
40 mL sample @ 4 mL/min
Wash:
2 mL of 5% methanol in H2O
Elute:
3 mL methanol
Evaporate to 0.2 mL
HO
H
OH
N
N
1
Absorbance @365nm
0.004 AUFS
Reconstitute to 1.0 mL with
50 mM oxalic acid
OH
NH 2
OH
2
OH
O
OH
O
O
Oxytetracycline
HPLC Method
3
spiked pork
nonspiked sample
0
5
Minutes
Column:
Mobile Phase:
Flow Rate:
Detection:
Injection:
Nova-Pak® C8
20% acetonitrile in
50 mM oxalic acid/water
0.8 mL/min
UV @ 365 nm
60 µL
10
Nova-Pak, Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
13
Tetracyclines in Milk
Oasis® HLB Extraction Method
The milk sample (15 mL) is diluted with 25 mL of McIlvaine (mixed citrate/
phosphate, pH 4.1) buffer with added EDTA. The diluted sample is
centrifuged at 8000 x g for 10 minutes at 5° C. Any floating lipid layer is
removed and the remaining supernatant is processed using Oasis® HLB
solid-phase extraction cartridges.
Conditions for Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Condition:
3 mL methanol
For details of the buffer preparation procedure see
J.AOAC Int., 79 (2), p 405 (1996).
Compounds
1.
2.
3.
Rinse:
2 mL H2O
% Recovery
Tap Water
Apple
Spike Level
Spike Level
25 µg/L
50 µg/L
4 replicates
4 replicates
70.7 (3.5)
67.7 (5.8)
73.7 (7.3)
68.5 (5.1)
76.7 (2.9)
67.3 (1.8)
Oxytetracycline
Tetracycline
Chlortetracycline
Load:
15 mL sample @ 5 mL/min
Wash:
1.5 mL of 5% methanol in H2O
Elute:
2 mL methanol
Evaporate to 0.2 mL
N
HO
1
OH
NH 2
2
OH
Reconstitute to 1.0 mL with
50 mM oxalic acid
OH
O
OH
O
O
Absorbance @365nm
0.004 AUFS
Tetracycline
HPLC Method
Column:
Mobile Phase:
3
spiked milk
0
5
Minutes
Nova-Pak, Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
14
10
Flow Rate:
Detection:
Injection:
Nova-Pak® C8
13% acetonitrile, 13 % methanol
in 50mM oxalic acid/water
0.8 mL/min
UV @ 365 nm
60 µL
Tetracyclines in Serum
Oasis® HLB Extraction Method
Results from analysis of 6 replicates, spike level 2.5 µg/mL
Compounds
1. Minocycline
2. Tetracycline
% Recovery % RSD
94.8%
1.4%
104%
0.55%
Conditions for Oasis® HLB Cartridge, 1 cc, 30 mg
Part Number WAT094225
Prepare Sample:
Mix 20 µL (H2PO4) to 1 mL serum
Compare with results obtained using C18 cartridges:
Compounds
1. Minocycline
2. Tetracycline
Condition:
1 mL methanol
% Recovery % RSD
40.7%
0.82%
67.4%
0.44%
Load:
1 mL sample
N
HO
OH
OH
OH
Rinse:
1 mL H2O
O
OH
Wash:
1 mL of 5% methanol in H2O
NH 2
O
Elute:
1 mL methanol
O
Tetracycline
Evaporate to 0.2 mL
1. Minocycline
2. Tetracycline
3. Demeclocycline (IS)
1
2
Reconstitute with mobile phase
3
HPLC Method
Sample:
sample
blank
10.0
20.0
Minutes
30.0
Column:
Mobile Phase:
Flow Rate:
Detection:
Injection:
1 mL spiked porcine serum
2% phosphoric acid
(demecycline as IS)
SymmetryShield™ RP8
0.1% TFA in water:
acetonitrile:methanol (91:7:2)
0.9 mL/min
UV @ 279 nm
20 µL
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
15
Endothall in Drinking Water or Soil
A) LC/MS Method
Instrument:
Oasis® HLB Extraction Method
Conditions for Oasis® HLB Cartridge, 6 cc, 500 mg LP
Part Number 186000115
Waters Alliance LC/MS with
Micromass Platform LC™ Mass Detector
SymmetryShield™ RP8
(2.1 mm x 100 mm, 3.5 µm)
5% Acetonitrile in 1% formic acid/water
200 µL/min
Electrospray (neg. ion)
(SIR mode, m/z = 185)
75 µL
®
Column:
Mobile Phase:
Flow Rate:
Detection:
Injection:
Prepare Sample:
100 mL, adjust to pH 2 with H3PO4
Condition:
6 mL methanol
Rinse:
6 mL H2O
Load:
100 mL @ 15 mL/min
B) GC/MS or GC/FID Method
Column:
Carrier Gas:
Temp Program:
Detection:
Injection:
RTX 5 capillary,
30 meters, 0.25 mm ID,
0.25 µm film thickness
Helium @ 30 cm/sec
40° C initial, 8° C/ min to 300 °C
HP 5972 MSD
(EI, SIM mode, m/z = 123)
1 µL
For GC
Elute:
8 mL methanol (10% H2SO4)
COOH
O
endothall
25 µg/kg in soil
Endothall
endothall
2.5 µg/L in water
B)
blank soil
4
minutes
8
5
16
15
% Recovery (% RSD) - LC/MS
Tap Water
Spike Level
2.5 µg/L
4 replicates
Tap Water
Spike Level
10 µg/L
4 replicates
81.1% (18%)
99.6% (3.1%)
Alliance, Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
Platform LC is a trademark of Micromass U.K. Ltd. RTX 5 is a trademark of Restek Corporation.
© 2002 Waters Corporation
A) For LC/MS: No derivatization
required. The MTBE* in the eluent is
removed by evaporation and the extract is
adjusted to a final volume of 1.0 mL with
10% methanol in water.
B) For GC: The eluent is heated for 40 min
@ 60° C to convert endothall to the dimethyl
ester. The ester is then extracted with
DCM**. After removal of water by treatment with Na2SO4, the DCM** extract is
evaporated to a final volume of 0.5 mL.
spiked soil
0
Elute:
8 mL 10:90 methanol/MTBE*
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
COOH
A)
For LC/MS
minutes
25
% Recovery (% RSD) - GC
Soil (GC/FID)
Soil (GC/MS)
Spike Level
Spike Level
100 µg/L
25 µg/L
4 replicates
4 replicates
81.8% (20%)
76.2% (9.5%)
Soil Samples: The sample (10 g) is
extracted with 35 mL pH 10 carbonate
buffer (0.1M) followed by 20 mL of water.
The combined extracts are adjusted to pH 2
with phosphoric acid and centrifuged. SPE is
then performed using the same protocol as
water samples.
* methyl butyl ether
** methylene chloride
Fungicides in Apple Juice
Compounds
1. Carbendazim
2. Thiabendazole
Oasis® MCX SPE Method for Fungicides
% Recovery, n=4
82% (2%RSD)
96% (2% RSD)
Conditions for Oasis® MCX Cartridge, 6 cc, 150 mg
Part Number 186000256
(20 ppb spike level)
Prepare Sample:
adjust to pH 10 with NaOH
Condition: 2 mL methanol
Rinse with 3 mL 2% NH4OH
Load Sample
Wash #1:
2 mL 2% NH4OH
Wash #2:
2 mL 30% methanol/4% NH4OH
N
NHCOOCH3
Wash #3:
2 mL 0.1 N HCI
N
H
Carbendazim
N
Wash #4:
2 mL methanol
Elute:
3 mL methanol (4% NH4OH)
N
N
H
S
Evaporate and Reconstitute
Thiabendazole
HPLC Method
Column:
1
2
Mobile Phase:
Flow Rate:
Detection:
Injection:
0
5
Minutes
XTerra® RP18, 4.6 x 100 mm
(3.5 µm DP)
72.5% phosphate buffer, (20 mM pH 6.8)
27.5% acetonitrile
1.0 mL/min
PDA (288nm)
20 µL
10
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
17
Fungicides in Grape Juice
Compounds
1. Carbendazim
2. Thiabendazole
Oasis® MCX SPE Method
% Recovery, n=4
81% (8%RSD)
94% (10% RSD)
Conditions for Oasis® MCX Cartridge, 6 cc, 150 mg
Part Number 186000256
(20 ppb spike level)
Prepare Sample:
adjust to pH 10 with NaOH
Condition: 2 mL methanol
Rinse with 3 mL 2% NH4OH
N
Load Sample
NHCOOCH3
N
H
Wash #1:
2 mL 2% NH4OH
Carbendazim
Wash #2:
2 mL 30% methanol/4% NH4OH
N
N
Wash #3:
2 mL 0.1 N HCI
S
N
H
Wash #4:
2 mL methanol
Thiabendazole
Elute:
3 mL methanol (4% NH4OH)
Evaporate and Reconstitute
HPLC Method
Column:
1
Mobile Phase:
2
Flow Rate:
Detection:
Injection:
0
5
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
18
Minutes
10
XTerra® RP18, 4.6 x 100 mm
(3.5 µm DP)
72.5% phosphate buffer, (20 mM pH 6.8)
27.5% acetonitrile
1.0 mL/min
PDA (288nm)
20 µL
Fungicides in Orange Juice
Compounds
1. Carbendazim
2. Thiabendazole
Oasis® MCX SPE Method
% Recovery, n=4
91% (2%RSD)
94% (3% RSD)
Conditions for Oasis® MCX Cartridge, 6 cc, 150 mg
Part Number 186000256
(20 ppb spike level)
Prepare Sample:
adjust to pH 2 with HCI
Condition: 2 mL methanol
Rinse with 2 mL H2O
N
Load Sample
NHCOOCH3
N
H
Wash #1:
2 mL 0.1 N HCI
Carbendazim
Wash #2:
2 mL methanol
N
N
Wash #3:
2 mL 2% NH4OH
S
N
H
Wash #4:
2 mL 30% methanol/4% NH4OH
Thiabendazole
Elute:
3 mL methanol (4% NH4OH)
Evaporate and Reconstitute
HPLC Method
Column:
1
Mobile Phase:
2
Flow Rate:
Detection:
Injection:
0
5
Minutes
XTerra® RP18, 4.6 x 100 mm
(3.5 µm DP)
72.5% phosphate buffer, (20 mM pH 6.8)
27.5% acetonitrile
1.0 mL/min
PDA (288nm)
20 µL
10
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
19
LC/MS Determination of Fungicides
in Commercial Apple Cider
Oasis® MCX SPE Method
Compounds
1. Carbendazim
2. Thiabendazole
70 µg/L
170 µg/L
Conditions for Oasis® MCX Cartridges, 6 cc 150 mg
Part Number 186000256
Prepare Sample:
adjust to pH 10 with NaOH
NHCOOCH3
Condition: 2 mL methanol
Rinse with 3 mL 2% NH4OH
S
N
H
N
H
Load Sample
Thiabendazole
Carbendazim
UV
N
N
N
0.01
absorbance
2
Wash #1:
2 mL 2% NH4OH
Wash #2:
2 mL 30% methanol/4% NH4OH
PDA
288 nm
Wash #3:
2 mL 0.1 N HCI
1
Wash #4:
2 mL methanol
0
MS
100
Elute:
3 mL methanol (4% NH4OH)
7.31
2
m/z = 202.0
% abundance
Evaporate and Reconstitute
0
5.29
100
1
LC/MS Method
m/z = 192.1
LC Conditions:
Instrument:
0
2
4
6
8
10
12
14
Minutes
Column:
Mobile Phase:
SIR
Group
Time
mins.
Compound
Mass
Cone
Voltage
Dwell
Time
1
0-6.5
Carbendazim
192.1
25V
1.0 secs
2
6-15
Thiabendazole
202.0
35V
1.0 secs
Oasis, XTerra, Alliance and Waters are trademarks of Waters Corporation.
Platform LC is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
20
Flow Rate:
Waters Alliance® Separations Module
with 996 PDA
XTerra® MS18, 2.1 x 100 mm, 5 µL
20% acetonitrile/80% 10 mM NH4HCO3
(pH 8.3)
200 µL/min, split 1:1 through each detector
MS Conditions:
Instrument:
Interface:
Waters/Micromass Platform LC™
Positive Electrospray (ESI+)
Multiple Selected-Ion Recording (SIR)
Determination of Naptalam and
Metabolite in Drinking Water
Generic Oasis® MAX method
Results* (% recovery ± % RSD, n=4)
10 ppb
73 (6)
Conditions for Oasis® MAX Cartridge, 6 cc, 500 mg
Part Number 186000865
50 ppb
76 (8)
SIR
Time
Group Mins. Compound
1
5 - 8 Naptalam
Prepare Sample:
pH 5.5 to 7.5
Cone
Dwell
Voltage Time
17V
0.8 secs.
Mass
144,292,293
Condition:
3 mL methanol/3 mL water
* recovery measured against standards prepared in cucumber matrix
Load:
up to 300 mL sample
Wash 1:
3 mL 50 mM NaOAc (pH 7)
Elute 1 contains the
1-naphthylamine
metabolite residue
O
NH2
HOOC
Elute 2 contains the naptalam residue
COOH
+
N
H
Phthalic Acid
1-Naphthylamine
Phthalic Acid
50 µg/g spike level
(same extract as
LC/PDA example)
naptalam
m/z = 292
% abundance
100
Elute 2:
4 mL methanol (2% formic acid)
Evaporate and Reconstitute
COOH
Naptalam
Elute 1 (Wash 2):
4 mL methanol
HPLC Method
spiked sample
Instrument:
Column:
Injection Volume:
Mobile Phase:
blank sample
0
1
2
3
4
5
6
7
8
9
10
Flow Rate:
MS Conditions:
Instrument:
Interface:
Oasis, Alliance, XTerra and Waters are trademarks of Waters Corporation.
ZMD is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
Waters Alliance® Separations Module
Waters XTerra® MS C18, 2.1 x 100 mm,
20 µL
25% acetonitrile/75% 10mM ammonium
acetate (pH 5.5) to 90% acetonitrile in
6 minutes
200 µL/min
Waters/Micromass ZMD™
Positive Electrospray (ESI+)
Multiple Selected-Ion Recording (SIR)
21
Naptalam in Cucumber (LC/PDA)
Recovery: 86% (± 9% RSD, n=4)
However, there is an interference equivalent to 15 % of the spike level.
(UV analysis indicates this interference is not naptalam). This interference
can be overcome by modification of the LC separation, or by application
of LC/MS (see next application).
Oasis® MAX Extraction Method
Conditions for Oasis® MAX Cartridge, 6 cc, 500 mg
Part Number 186000865
Prepare Sample:
pH 5.5 to 7.5
Condition:
3 mL methanol/3mL water
Load:
up to 300 mL sample
Wash #1:
3 mL 50 mM NaOAc (pH 7)
Wash #2:
4 mL methanol
Elute:
4 mL methanol (2% formic acid)
Evaporate and Reconstitute
50 µg/g spike level
O
N
H
COOH
PDA (290 nm, 0.05 AUFS)
Naptalam
Naptalam
HPLC Method
Instrument:
spiked sample
blank sample
0
10
Minutes
Oasis, Alliance, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
22
20
Waters Alliance® Separations Module
with 996 PDA
Column:
Waters XTerra® RP18, 4.6 x 100 mm,
(3.5 µm dP)
Injection Volume: 50 µL
Mobile Phase:
20% acetonitrile/80% 10mM ammonium
acetate (pH 5.5) to 80% acetonitrile in
20 minutes
Flow Rate:
1.0 mL/min
Naptalam in Cucumber (LC/MS)
Oasis® MAX Extraction Method
Results* (% recovery ± % RSD, n=4)
10 ppb
73 (6)
Conditions for Oasis® MAX Cartridge, 6 cc, 500 mg
Part Number 186000865
50 ppb
76 (8)
SIR
Time
Group Mins. Compound
1
5 - 8 Naptalam
Prepare Sample:
pH 5.5 to 7.5
Cone
Dwell
Voltage Time
17V
0.8 secs.
Mass
144,292,293
Condition:
3 mL methanol/3 mL water
* recovery measured against standards prepared in cucumber matrix
Load:
up to 300 mL sample
Wash #1:
3 mL 50 mM NaOAc (pH 7)
Wash #2:
4 mL methanol
O
Elute:
4 mL methanol (2% formic acid)
N
H
COOH
Evaporate and Reconstitute
Naptalam
Naptalam
HPLC Method
50 µg/g spike level
(same extract as
LC/PDA example)
LC Conditions:
Instrument:
Column:
Injection Volume:
Mobile Phase:
naptalam
m/z = 292
% abundance
100
Flow Rate:
Waters Alliance® Separations Module
Waters XTerra® MS C18, 2.1 x 100 mm,
20 µL
25% acetonitrile/75% 10mM ammonium
acetate (pH 5.5) to 90% acetonitrile in
6 minutes
200 µL/min
spiked sample
blank sample
0
1
2
3
4
5
6
7
8
9
10
MS Conditions:
Instrument:
Interface:
Waters/Micromass ZMD™
Positive Electrospray (ESI+)
Multiple Selected-Ion Recording (SIR)
Oasis, Alliance, XTerra and Waters are trademarks of Waters Corporation.
ZMD is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
23
Paraquat and Diquat in Tap Water
Compounds
% Recovery,
0.5 µg/L
111 + 7
91 + 4
1. Paraquat
2. Diquat
n=5
2.0 µg/L
Oasis® MCX Method for Quaternary
Amines in Drinking Water
110 + 5
96 + 4
Conditions for Oasis® MCX Cartridge, 3 cc, 60 mg
Part Number 186000254
2 mg/L Spike Level
Condition: 3 mL methanol
Rinse with 3 mL H2O
Load:
250 mL sample
CH3
+
+
N
N
Wash:
3 mL 50% methanol/ H2O
CH3
Elute: 2 mL of 1 M NH4CI
in 50% methanol/ H2O
Paraquat
For LC analysis dilute the eluent to 5 mL
with 50 mM sodium octanesulfonate
N+
N
+
Diquat
PDA (0.08 AUFS)
1
HPLC Method
2
Column:
Mobile Phase:
257 nm
Flow Rate:
Detection:
Injection:
Sample:
308 nm
2
4
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
24
Minutes
6
Symmetry® C18, 3.9 x 150 mm
45% 25 mM octanesulfonate,
55% methanol, pH 3.0
0.75 mL/min
PDA
80 µL
200 mL spiked tap water processed
on Oasis® MCX cartridge
Glyphosate and AMPA in Drinking Water
Compounds
Results (5 replicates)
0.2 µg/L
10 µg/L
79% RSD 15% 81% RSD 1%
b.d.l.*
55% RSD 5%
1. Glyphosate
2. AMPA
Oasis® MAX SPE Method for
Glyphosate and Metabolite
Conditions for Oasis® MAX Cartridge, 6 cc, 200 mg
Part Number 186000865
* below detection limit
Prepare Sample:
pH 6-8
Condition: 2 mL methanol,
4 mL 0.5 M NaOH, 2 mL H2O
1
Rinse: 4 mL 0.5 M NaOH
Rinse: 2 mL H2O
1. Glyphosate
2. AMPA
Load:
25 mL sample
3 mV FS
0.2 µg/L spike
100 µL injection
Wash:
2 mL H2O
2
Elute:
4 mL 0.5 M HCI in acetonitrile*
15
5
Minutes
Evaporate and Reconstitute:
adjust to pH 3 for LC analysis
30
* Alternate eluent is 4 mL 0.6 M sodium citrate
Use 6 cc, 500 mg Oasis® MAX
for samples > 50 mL
Part Number 186000865
1
1. Glyphosate
2. AMPA
HPLC Method
20 mV FS
10 µg/L spike
50 µL injection
Column:
Mobile Phase:
Flow Rate:
Detection:
2
5
15
Minutes
30
Waters Ion Exclusion, 7.8 x 150 mm
0.05% phosphoric acid
1.5 mL/min
o-phthalaldehyde post column
derivatization/fluorescence
Waters M474 Fluorescence Detector
excitation - 339 nm
emission - 445 nm
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
25
SPE Methods for Endocrine Disruptors
Aqueous Samples
• Use 250 - 1000 mL of sample for
water analysis
• Use 40 - 200 mL of sample for beverage analysis
• Centrifuge prior to analysis if necessary
Soil and Food Samples
• Extract the homogenized sample
(5-10 gm) with 20-40 mL acetonitrile
or other water miscible solvent
• If desired, concentrate the organic
extract by evaporation
• Dilute the organic extract with water
(85-95% water)
• Perform SPE using the EED procedure
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
26
Generic Oasis® HLB
method for LC/GC
Conditions for Oasis® HLB Glass Cartridge, 5 cc, 200 mg
Part Number 186000683
Prepare Sample:
adjust to pH3
Condition/Equilibrate: 3 mL solvent*/
3 mL methanol/ 3 mL water
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in water
Elute
6 mL 10% methanol/90% MTBE*
For GC analysis,
dry extract
over Na2SO4,
then adjust
to 1 mL
For LC analysis,
exchange to
acetonitrile,
then adjust
to 1 mL
*methyl t-butyl ether
Phenols in Tap Water (Endocrine Disruptors)
Oasis® SPE Method for Endocrine Disruptors
Results
Results are given as % recovery with % RSD in parenthesis
Level 1
Level 2
Level 3
Level 4
Compounds
5 replicates
5 replicates
5 replicates
5 replicates
1. Bisphenol A
101 (17)
97.1 (2.9)
95.4 (1.0)
97.4 (1.2)
2. Nonylphenol
n.a.*
82.9 (6.0)
80.0 (3.1)
78.0 (4.2)
Spike levels:
bisphenol A - 0.24, 0.80, 3.2, 20 µg/L
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Prepare Sample:
adjust to pH 3
Condition:
3 mL MTBE*
nonylphenol - 1.2*, 4.0, 16, 100 µg/L
Estimated LOQs: bisphenol A - 0.1 µg/L
Rinse: 3 mL methanol
Rinse: 3 mL H2O
nonylphenol (mixed isomers) - 2.5 µg/L
* level 1 for nonylphenol, blank value greater than 50 % of spike level
Load:
up to 500 mL sample
Compounds
1. Bisphenol A, 240 ng/L
2. Nonylphenol (isomer mix), 1.2 µg/mL
(estimated concentration of para n-nonyl isomer,100 ng/L)
Wash:
3 mL 5% methanol in H2O
Elute:
6 mL 10% methanol/MTBEt*
CH3
OH
C
CH3
OH
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
Bisphenol A
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
HPLC Method
1
Column:
Mobile Phase:
2
Gradient:
spiked sample
nonspiked sample
0
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
10
Minutes
20
Flow Rate:
Detection:
Injection:
SymmetryShield™ RP18
A: pH 3.0 phosphate buffer (15 mM)
B: acetonitrile
60% A initial, then linear
gradient to 100 % B in 20 min
1.0 mL/min
UV @ 225 nm (0.02 AUFS)
75 µL
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
27
Nonylphenol in Tap Water (Endocrine Disruptors)
Oasis® SPE Method for Endocrine Disruptors
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
CH3
C6H13
C
CH3
OH
Prepare Sample:
adjust to pH 3
Nonylphenol
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in H2O
GC Results
Spike level:
n=5
Recovery = 84.3%
RSD = 9.0%
Elute:
6 mL 10% methanol/MTBEt*
100 µg/L
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
1: 4,4-Difluorobiphenyl
2: Nonylphenol (isomer mix), 100 µg/mL
GC Method
ISTD
Column:
2
spiked sample
nonspiked sample
0
10
Oasis and Waters are trademarks of Waters Corporation.
DB-5 is a trademark of J&W Scientific Inc.
© 2002 Waters Corporation
28
Minutes
20
J&W DB-5 capillary,
30 meters, 0.25 mm ID,
0.25 µm film thickness
Carrier Gas:
Helium @ 30 cm/sec
Temp. Program: 50 °C initial, 30 °C/min to 120 °C
then 8 °/min to 280 °
Detection:
NPD
Injection:
2 µL
Phthalates in Tap Water (Endocrine Disruptors)
Oasis® SPE Method for Endocrine Disruptors
Results
Results are given as % recovery with % RSD in parenthesis
4 µg/L
5 replicates
n.d.
97.4 (3.8)
88.7 (14)
88.0 (17)
66.9 (22)**
64.9 (22)
Compounds
1. Dimethyl phthalate
2. Diethyl phthalate
3. Benzylbutyl phthalate
4. Dibutyl phthalate
5. Bis(ethylhexyl)phthalate
6. Dioctyl phthalate
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
16 µg/L
5 replicates
87.2 (3.2)
92.3 (2.1)
82.2 (1.6)
87.7 (2.2)
72.1 (5.5)
70.6 (5.6)
Prepare Sample:
adjust to pH 3
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
n.d blank value greater than 50 % of spike level
** blank value greater than 20 % of spike level
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in H2O
Elute:
6 mL 10% methanol/MTBEt*
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
O
C
OCH3
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
COOCH3
1
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
Dimethyl Phthalate
HPLC Method
3
4 5
6
Column:
Mobile Phase:
7
2
blank
0
10
Minutes
20
Gradient:
Flow Rate:
Detection:
Injection:
Sample:
SymmetryShield™ RP8
A: water
B: Acetonitrile
50% B linear to 100% B in 10 min
0.8 mL/min
UV @ 196nm (0.03 AUFS)
20 µL
150 mL of surface water
spiked @ 4 ng/L
Oasis, SymmetryShield and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
29
Phthalates From Tap Water
Generic Oasis® HLB
method for LC/GC
GC/FID, 15 µg/L, n = 5
Results
1. Dimethyl phthalate
2. Diethyl phthalate
3. Dibutyl phthalate
4. Benzylbutyl phthalate
5. Bis(ethylhexyl)phthalate
6. Bis(ethylhexyl)phthalate
7. Dioctyl phthalate
% recovery (% RSD)
115 (6)
114 (11)
82 (3)
83 (3)
70 (8)
66 (8)
73 (7)
Conditions for Oasis® HLB Glass Cartridge, 5 cc, 200 mg
Part Number 186000683
Prepare Sample:
adjust to pH3
Condition/Equilibrate: 3 mL solvent*/
3 mL methanol/ 3 mL water
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in water
Elute
6 mL 10% methanol/90% MTBE*
For GC analysis,
dry extract
over Na2SO4,
then adjust
to 1 mL
3
2
5 6
4
1
For LC analysis,
exchange to
acetonitrile,
then adjust
to 1 mL
*methyl t-butyl ether
7
GC/FID Method
Column:
Carrier Gas:
Temp Program:
Detection:
Injection:
0
15
Minutes
Oasis and Waters are trademarks of Waters Corporation.
DB-5 is a trademark of J&W Scientific Inc.
© 2002 Waters Corporation
30
12
30
J&W DB-5 capillary, 30 meters, 0.25 mm ID,
0.25 µm film thickness
Helium @ 30 cm/sec
50 °C initial, 30 °C/min to 120 °C,
then 8 °C/ min to 290 °C, hold 10 min
FID
1 µL
Carbamates in Drinking Water
(Endocrine Disruptors), LC/PCFD
Compounds
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Aldicarb Sulfoxide
Aldicarb Sulfone
Oxamyl
Methomyl
3-Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
Methiocarb
Oasis® SPE Method for Endocrine Disruptors
% Recovery
LC/PCFD
50 ng/L
45.7 (5.1)
101 (3.6)
122 (18)
100 (3.2)
111 (6.5)
104 (5.8)
99.9 (5.8)
104 (7.9)
122 (11)
120 (14)
LC/PCFD*
500 ng/L
54.7 (0.5)
98.7 (4.0)
90.8 (7.0)
99.9 (6.4)
98.7 (2.3)
90.7 (9.3)
97.5 (5.6)
97.2 (4.7)
89.6 (2.2)
91.6 (2.2)
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Prepare Sample:
adjust to pH 3
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Load:
up to 500 mL sample
* The 500 ng/L sample SPE extracts were split and analyzed by each method
LC/PCDF - LC with post column derivitization and fluorescence detection
Wash:
3 mL 5% methanol in H2O
50 ng/L spike level
Elute:
6 mL 10% methanol/MTBE*
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
SCH3
C
H3C
N
O
C
N
CH3
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
O
Methomyl
HPLC Method
11
Column:
Mobile Phase:
5
6
7
8
10
9
4
2
1
3
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Flow Rate:
Detection:
Injection:
Sample:
Waters Carbamate Analysis 3.9 x 150 mm
A: Water
B: Methanol
C: Acetonitrile
1.5 mL/min
Post Column Derivatization, fluorescence
75 µL
200 mL of drinking water spiked @ 50 ng/L
Minutes
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
31
Carbamates in Drinking Water
(Endocrine Disruptors), LC/MS
Compounds
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Oasis® SPE Method for Endocrine Disruptors
% Recovery
LC/MS*
500 ng/L
74.8 (19)
88.7 (16)
83.2 (18)
92.3 (8.0)
101 (8.6)
79.4 (9.3)
103 (13)
95.6 (7.5)
97.7 (14)
81.2 (14)
Aldicarb Sulfoxide
Aldicarb Sulfone
Oxamyl
Methomyl
3-Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
Methiocarb
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Prepare Sample:
adjust to pH 3
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in H2O
50 ng/L spike level
Elute:
6 mL 10% methanol/MTBEt*
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
SCH3
H3C
C
N
O
C
N
CH3
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
O
Methomyl
SIR
Group
Time
(mins)
1
0-9
2
3
4
9-11
0.5-12.5
11.5-14
5
14-20
HPLC Method
Compound
Mass
Cone
Voltage
Dwell
Time
Aldicarb Sulfoxide
Aldicarb Sulfone
Oxamyl
Methomyl
3-OH Carbofuran
Alicarb
Propoxur
Carbofuran
Carbaryl
Methiocarb
207.1
223.2
237.2
163.2
238.2
208.2
210.2
222.2
202.2
226.2
18V
25V
10V
15V
15V
8V
18V
22V
18V
19V
0.5 secs
0.5 secs
0.5 secs
0.5 secs
1.5 secs
1.5 secs
0.4 secs
0.4 secs
0.4 secs
0.6 sec
Instrument:
Column:
Temperature:
Injection Volume:
Mobile Phases:
Gradient:
Flow Rate:
32
Waters Alliance® Separations Module
Waters Symmetry® C18, 1.0 x 150 mm
35 °C
10 µL
A: 10% methanol/10 mM ammonium acetate
B: 90% methanol/10 mM ammonium acetate
90% A initial, linear gradient to
90% B in 10 minutes
75 µL/min
MS Conditions
Instrument:
Interface:
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
Waters/Micromass ZMD4000™
Positive Electrospray (ESI+), Multiple Selected-Ion
Recording (SIR)
Determination of Polar Compounds
(Endocrine Disruptors) in Soil
Compounds
1. benomyl
2. carbaryl
3. atrazine
4. bisphenol A
% RECOVERY ± % RSD
62 ± 6
91 ± 4
84 ± 5
78 ± 6
Oasis® SPE Method for Endocrine Disruptors
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Prepare Sample:
adjust to pH 3
Soil samples (5 g) were spiked with the appropriate compounds and
extracted with 25 mL of acetonitrile (30 minutes on shaker). A 5 mL aliquot
of the acetonitrile extract was diluted to 100 mL with reagent water (MilliQ)
and then processed by SPE.
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
50 ppb spike level
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in H2O
Elute:
6 mL 10% methanol/MTBEt*
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
3
2
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
4
HPLC Method
sample
Column:
Mobile Phase:
1
blank
0
5
Minutes
10
Gradient:
Flow Rate:
Detection:
Injection:
Sample:
Symmetry® C18, 3.9 x 150 mm
A: 10 mM phosphate pH 6.8
B: methanol
40% B linear to 100% B in 20 min
1.0 mL/min
PDA (225 nm extracted, 0.04 AUFS)
100 µL
10 g potting soil extracted with 25 mL
acetonitrile; then SPE on Oasis® HLB
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
33
Determination of Polar Compounds in Pear
Compounds
1. benomyl (interference)
2. carbaryl
3. atrazine
4. bisphenol A
% RECOVERY ± % RSD
Oasis® SPE Method for Endocrine Disruptors
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
99 ± 4
95 ± 1
86 ± 2
Prepare Sample:
adjust to pH 3
100 ppb spike level
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in H2O
Elute:
6 mL 10% methanol/MTBEt*
2
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
3
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
1
4
HPLC Method
Column:
Mobile Phase:
sample
Gradient:
Flow Rate:
Detection:
Injection:
blank
0
5
Minutes
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
34
10
Symmetry® C18, 3.9 x 150 mm
A: 10 mM phosphate pH 6.8
B: methanol
40% B linear to 100% B in 20 min
1.0 mL/min
PDA (225 nm extracted, 0.04 AUFS)
100 µL
Benomyl and Bisphenol A at 283 nm
Compounds
1. benomyl
2. carbaryl
3. atrazine
4. bisphenol A
% RECOVERY ± % RSD
65 ± 10
N/A
N/A
83 ± 2
Oasis® SPE Method for Endocrine Disruptors
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Prepare Sample:
adjust to pH 3
100 ppb spike level in pear
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in H2O
Elute:
6 mL 10% methanol/MTBEt*
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
1. benomyl
2. carbaryl
3. atrazine
4. bisphenol A
HPLC Method
1
2
sample
3
4
Gradient:
Flow Rate:
Detection:
Injection:
blank
0
Column:
Mobile Phase:
5
Minutes
Symmetry® C18, 3.9 x 150 mm
A: 10 mM phosphate pH 6.8
B: methanol
40% B linear to 100% B in 20 min
1.0 mL/min
PDA (283 nm extracted)
100 µL
10
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
35
Estrogens in River Water (Endocrine Disruptors)
Compounds
1. 17β-estradiol
2. 17α-ethynylestradiol
3. Diethylstilbestrol
Oasis® SPE Method for Endocrine Disruptors
% Recovery
95%
93%
85%
Conditions for Oasis® HLB Cartridge, 6 cc, 200 mg
Part Number WAT106202
Prepare Sample:
adjust to pH 3
200 ng/L Spike Level
Condition:
3 mL MTBE*
Rinse: 3 mL methanol
Rinse: 3 mL H2O
Load:
up to 500 mL sample
CH3
OH
Wash:
3 mL 5% methanol in H2O
Elute:
6 mL 10% methanol/MTBEt*
HO
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
17β-estradiol
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
HPLC Method
1
UV @ 283nm
(0.003 AUFS)
2
Column:
Mobile Phase:
Gradient:
Flow Rate:
Detection:
Injection:
3
blank
7
Minutes
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
36
10
XTerra® RP18, 4.6 x 100 mm (3.5 µm DP)
A = water B = methanol
50 % B to 90 % B in 20 min
1.0 mL/min
PDA (283nm)
75 µL
SPE Methods For Endocrine
Disruptors at Low Levels
1-D (LOQ = 50 ppt)
2-D (LOQ < 5 ppt)
Oasis® HLB 200 mg Cartridge
(Glass recommended, Part Number 186000683)
Oasis® HLB 200 mg Cartridge
(Glass recommended, Part Number 186000683)
Prepare Sample
Prepare Sample
Condition/Equilibrate:
3 mL MTBE/3 mL methanol/3 mL water
Condition/Equilibrate:
3 mL MTBE/3 mL methanol/3 mL water
Load:
up to 500 mL sample
Load:
up to 500 mL sample
Wash:
3 mL 5% methanol in water
Wash 1:
3 mL 40% methanol in water
Elute:
6 mL 10%methanol/MTBE
Re-Equilibrate:
3 mL water
Wash 2: 3 mL 10% methanol/
2% NH4OH in water
Elute:
6 mL 10%methanol/MTBE
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
37
Phthalates and Nonylphenol from
River Water, GC/MS, 2-D Method
Compounds
1. Dimethyl phthalate
2. Diethyl phthalate
3. n-nonylphenol
4. Dibutyl phthalate
5. Benzylbutyl phthalate
6. Bis(ethylhexyl)phthalate
7. Dioctyl phthalate
% Recovery
130 (15)
86 (12)
90 (11)
110 (11)
110 (8)
60 (8)
<50
200 ng/L Spike Level, n = 4
2-D (LOQ < 5 ppt)
Oasis® HLB Glass Cartridge 5 cc, 200 mg
Part Number 186000683
Prepare Sample
Condition/Equilibrate:
3 mL MTBE*/3 mL methanol/3 mL water
Load:
up to 500 mL sample
Wash 1:
3 mL 40% methanol in water
Re-Equilibrate:
3 mL water
Wash 2: 3 mL 10% methanol/
2% NH4OH in water
Elute:
6 mL 10% methanol/MTBE*
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
4
5
2
1
3
Oasis and Waters are trademarks of Waters Corporation.
RTX 5 is a trademark of Restek Corporation.
© 2002 Waters Corporation
38
GC/MS:
HP5890 II with 5971 MSD
6
7
Column:
30m x 0.25 mm RTX5ms
Estrogens in River Water at 5 ng/L
(Endocrine Disruptors)
Compounds
Results, n=4
1. Diethylstilbestrol
2. Estrone
3. Ethynylestradiol
4. Estradiol
5. Bisphenol A
75% ± 5%
87% ± 5%
94% ± 12%
93% ± 15%
113% ± 11%
Oasis® SPE Method for Endocrine Disruptors
CH3
Modifications for low ppt analysis by LC/MS and GC/MS
Conditions for Oasis® HLB Glass Cartridge, 5 cc, 200 mg (Appendix A)
Part Number 186000683
OH
Condition:
3 mL MBTE*
5 ng/L Spike level
Rinse: 3 mL methanol
Rinse: 3 mL H2O
HO
17β-estradiol
100
electrospray -, 27 V
m/z = 267
diethylstilbestrol
75 % ± 5 %
%
Load:
up to 800 mL sample
10.70
36
40% methanol
wash removes
organic interferences
Wash:
3 mL 40% methanol in H2O
9.85
100
Re-Equilibrate:
3 mL H2O
m/z = 269
estrone
87 % ± 5 %
%
44
9.63
100
m/z = 295
ethynylestradiol
96 % ± 12%
%
38
pH 11 wash
removes non
organic material
interference
Elute:
6 mL 10% methanol/MBTE*
8.53
100
93 % ± 15 %
%
Wash #2: 3 mL 10% methanol/2%
NH4OH in H2O
m/z = 271
estradiol
39
7.80
100
113 % ± 11%
%
m/z = 227
bisphenolA
10
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
13.00
minutes
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
LC/MS Method
MS Conditions
LC Conditions
Instrument:
Column:
Injection:
Mobile Phase:
Gradient:
Flow Rate:
Waters Alliance® Separations Module
Waters XTerra® MS C18, 2.1 x 100 mm
20 µL
A: pH 10.5 NH4OH in water
B: acetonitrile
30 % B initial, linear to 65 % B in 8 min,
then to 90 % B in 9 min
200 µL/min, plumbed directily to detector
Instrument:
Interface:
Waters/Micromass Platform LC™
Negative Electrospray (ESI-)
Multiple Selected-Ion Recording (SIR)
Core voltage 27 V
Oasis, XTerra, Alliance and Waters are trademarks of Waters Corporation.
Platform LC is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
39
Estrogen in River Water,
Unspiked (Native) Sample
CH3
Oasis® SPE Method for Endocrine Disruptors
OH
Modifications for low ppt analysis by LC/MS and GC/MS
Conditions for Oasis® HLB glass cartridge, 5 cc, 200 mg (Appendix A)
Part Number 186000683
Condition:
3 mL MBTE*
HO
Rinse: 3 mL methanol
Rinse: 3 mL H2O
17β-estradiol
Load:
up to 800 mL sample
17β-estradiol. 15 ng/L
40% methanol
wash removes
organic interferences
271.0
Wash #1:
3 mL 40% methanol in H2O
river sample
Re-Equilibrate:
3 mL H2O
8.66
145.0
pH 11 wash
removes non
organic material
interference
calibration standard
Wash #2: 3 mL 10% methanol/2%
NH4OH in H2O
Elute:
6 mL 10% methanol/MBTE*
271.0
8.67
145.0
m/z
5.00
For GC analysis, dry
extract over Na2SO4,
then adjust to 1 mL
15.00
Time
For LC analysis, exchange
to acetonitrile, then
adjust to 1 mL
* methyl t-butyl ether
diethyl ether can be used as an alternative to MTBE
LC/MS Method
MS Conditions
LC Conditions
Instrument:
Column:
Injection:
Mobile Phase:
Gradient:
Flow Rate:
Waters Alliance Separations Module
Waters XTerra® MS C18, 2.1 x 100 mm
20 µL
A: pH 10.5 NH4OH in water
B: acetonitrile
30 % B initial, linear to 65 % B in 8 min,
then to 90 % B in 9 min
200 µL/min, plumbed directily to detector
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
40
®
Instrument:
Interface:
Waters/Micromass Platform LC™
Negative Electrospray (ESI-)
Multiple Selected-Ion Recording (SIR)
Core voltage 45 V for ion 211
Core voltage 90 V for ion 145
LC Determination of Acidic
Drugs in Horse Urine
Compounds
1. ketoprofen
2. naproxen
3. phenylbutazone
4. ibuprofen
5. meclofamic acid
% RECOVERY ± % RSD
90 (5.0)
90 (6.1)
73 (6.9)
83 (9.5)
71 (6.6)
Oasis® MAX Generic SPE Method
Typical Analysis: Acidic Analytes
Conditions for Oasis® MAX Cartridge, 6 cc, 150 mg
Part Number 186000370
Prepare Sample:
(neutral pH for most samples)
ISTD: Dichlorobenzoic acid
Condition:
3 mL methanol/ 3 mL water
(2 ppb, 10 mL sample)
Load:
up to 300 mL sample
Wash #1:
3 mL 50mM NaOAc (pH 7)
Elute 1 (Wash #2):
4 mL methanol
Elute 2*:
4 mL methanol (2 % formic acid)
2
Evaporate and Reconstitute
*Analytes with pKa < 3 require stronger acid
(i.e trifluoroacetic) at this step
5
ISTD
HPLC Method
1
4
3
Column:
Mobile Phase:
Gradient:
Flow Rate:
Detection:
Injection:
XTerra® MS C18
A: 0.1% Acetic acid in water
B: Methanol
50% B for 2 mIn, then linear
to 60% B in 20 min, then to
90% B in 30 min
0.9 mL/min
UV @ 221 nm
20 µL
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
41
Acidic Analytes in Acidic Matrix
Acidic Herbicides in Red Wine (Page 1 of 5)
OCH3
OH
+
HO
O
OCH3
O-glu
OH
typical grape
grape pigment
typical
pigment
(malvidin-3-glucoside)
(malvidin-3-glucoside)
Chromatogram for Non Extracted Standard
3
4
1
1. picloram
2. chloramben
3. dicamba
4. bentazon
5. 2,4-D
6. MCPA
2
HPLC Method
6
5
Column:
Mobile Phase:
Gradient:
Flow Rate:
Detection:
Injection:
0
5
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
42
Minutes
10
XTerra® MS C18, 100 x 2.1 mm, 3.5 µm
A: 20 mM phosphate pH 3
B: methanol
25% B to 60% B in 6 minutes
0.2 mL/min
UV @ 221 nm
10 µL
Acidic Analytes in Acidic Matrix
Acidic Herbicides in Red Wine (Page 2 of 5)
Compounds
1. picloram
2. chloramben
3. dicamba
4. bentazon
5. 2,4-D
6. MCPA
Oasis® HLB Generic Method
% RECOVERY
?? ( I )
180 % ( I )
130 % ( I )
100 %
95 %
98 %
Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Prepare Sample:
adjust to pH 2
Condition/Equilibrate:
1 mL methanol/1 mL water
I = pigment interference
Load:
up to 20 mL sample
2 ppm
Wash:
1 mL 5% methanol in water
2
1
3
Elute:
2 mL methanol
Evaporate and Reconstitute
4
5
HPLC Method
6
Column:
Mobile Phase:
note:
pigment interference
0
5
Gradient:
Flow Rate:
Detection:
Injection:
Minutes
XTerra® MS C18, 100 x 2.1 mm, 3.5 µm
A: 20 mM phosphate pH 3
B: methanol
25% B to 60% B in 6 minutes
0.2 mL/min
UV @ 221 nm
10 µL
10
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
43
Acidic Analytes in Acidic Matrix
Acidic Herbicides in Red Wine (Page 3 of 5)
Compounds
1. picloram
2. chloramben
3. dicamba
4. bentazon
5. 2,4-D
6. MCPA
Oasis® HLB Optimized Method
% RECOVERY
70 %
120 %
110 %
104 %
90 %
95 %
Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Prepare Sample:
adjust to pH 2
Condition/Equilibrate:
1 mL methanol/1 mL water
Load:
up to 20 mL sample
Wash 1:
1 mL 5% methanol in water
Wash 2:
1 mL 30% methanol, 2% TFA
1
Wash 3:
1 mL 2% NH4OH
Optimization
Steps
3
Elute:
1 mL 10:90 methanol/MTBE
2
Evaporate and Reconstitute
4
6
HPLC Method
5
Column:
Mobile Phase:
Gradient:
Flow Rate:
Detection:
Injection:
0
5
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
44
Minutes
10
XTerra® MS C18, 100 x 2.1 mm, 3.5 µm
A: 20 mM phosphate pH 3
B: methanol
25% B to 60% B in 6 minutes
0.2 mL/min
UV @ 221 nm
10 µL
Acidic Analytes in Acidic Matrix
Acidic Herbicides in Red Wine (Page 4 of 5)
Compounds
1. picloram
2. chloramben
3. dicamba
4. bentazon
5. 2,4-D
6. MCPA
Optimized Oasis® MAX method
% RECOVERY
95 % ( I )
98 %
100 %
102 %
85 %
88 %
Oasis® HLB Cartridge, 3 cc, 60 mg
Part Number WAT094226
Prepare Sample:
adjust to pH 10
Condition/Equilibrate:
2 mL methanol/2 mL water
I = pigment interference
Load:
10 mL sample diluted 1:1 water
Note: Load at pH 10 pigments are not retained at pH 10
Wash 1:
1 mL 50 mM NaOAc
Wash 2:
2 mL methanol
Elute:
3 mL methanol (2% formic acid)
3
Evaporate and Reconstitute
1
4
2
HPLC Method
6
5
Column:
Mobile Phase:
Gradient:
Flow Rate:
Detection:
Injection:
0
5
Minutes
XTerra® MS C18, 100 x 2.1 mm, 3.5 µm
A: 20 mM phosphate pH 3
B: methanol
25% B to 60% B in 6 minutes
0.2 mL/min
UV @ 221 nm
10 µL
10
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
45
Acidic Analytes in Acidic Matrix
Acidic Herbicides in Red Wine (Page 5 of 5)
Comparison of Oasis® SPE Methods
Calibration Standard
Oasis® MAX
Oasis® HLB 2-D
Oasis® HLB Generic
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
46
Determination of flavonoids in Ginkgo,
commercial products and whole leaf
Compounds
1. quercetin
2. kaempferol
3. isorhamnetin
total: 11 mg / tablet
Oasis® HLB method for flavonoids in ginkgo
% RECOVERY
82%
> 90%
> 90%
Conditions for Oasis® HLB cartridge, 3 cc, 60 mg
Part Number WAT094226
Condition/Equilibrate:
1 mL methanol/1 mL water
Sample pre-preparation
1 g sample is refluxed in 50 mL of ethanol:
3M HCl (70:30) for 2.5 hr
The cooled sample is adjusted to exactly 100 mL
0.3 mL of the ethanolic extract is diluted 1:10 with water
Load:
3 mL of diluted sample
• Recovery, measured with certified standards at 100 ppm in reagent water, was 82 % for quercitin
and > 90 % for the other compounds
• All analyses gave results within ± 40 % of the expected values with the exception of the
capsule (+ 60%)
• The selective SPE extraction and cleanup procedure provided a convenient analysis of ginkgo
flavonoids in a complex matrix (Herbal One with 16 herbal ingredients)
Wash 2: 2 ml methanol/
50mM NH4OAc (30:70) pH 5
Dry Cartridge:
air dry for 15 min with vacuum
OH
HO
Wash 1:
2 mL 5% methanol/water
Elute:
3 ml MTBE/methanol (80:20)
O
OH
Kaempferol
OH
Evaporate and Reconstitute:
1 mL mobile phase
O
1
Ginkgo extract
before SPE cleanup
1: quercetin
2: kaempferol
3: isorhamnetin
total: 11 mg / tablet
HPLC Method
2
Column:
Mobile Phase:
Flow Rate:
Detection:
Injection:
Temperature:
3
10
10
Minutes
Ginkgo extract after SPE
cleanup using Oasis® HLB
Symmetry® C18, 4.6 x 250 mm, 5 µm
A: 0.5% Phosphoric acid, 50%
B: Methanol, 50%
1.5 mL/min
UV @ 270nm 0.02 AUFS
10 µL
25° C
20
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
47
Determination of phenolics in Echinacea,
commercial products or whole leaf
Sample pre-preparation
100 mg sample is extracted with 25 mL of ethanol:water (70:30)
1 mL of the ethanolic extract is diluted 1:3 with water (~ pH 7)
Oasis® MAX method for phenolics in echinacea
Conditions for Oasis® MAX Cartridge, 3 cc, 60 mg
Part Number 186000367
• Recovery, measured with certified standards at 100 ppm in reagent water,
was greater than 85 % for all compounds
• All analyses gave results within ± 35 % of the expected values with the
exception of the liquid supplement (see goldenseal analysis)
• The selective SPE extraction and cleanup procedure provided a convenient
analysis of echinacea phenolics in complex matrix such as in herbal tea
Condition/Equilibrate:
1 mL methanol/1 mL water
Load:
3 mL of diluted sample
Wash 1:
2 mL of 50 mM sodium acetate
Wash 2:
2 mL of methanol
Dry Cartridge:
air dry for 15 min with vacuum
HO
Elute:
3 mL MTBE/methanol/TFA (49:49:2)
COOH
O
HO
O
OH
OH
Chlorogenic Acid
OH
Evaporate and Reconstitute:
1 mL mobile phase
3
0
2
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
48
Column:
Mobile Phase:
Gradient:
10
Minutes
Herbal tea extract after SPE
cleanup using Oasis® MAX
HPLC Method
1. cafteric acid
2. chlorogenic acid
3. cichoric acid
total: 5 mg / teabag
1
Tea bag
extract
Herbal tea extract
before SPE cleanup
20
Flow Rate:
Detection:
Injection:
Temperature:
Symmetry® C18, 4.6 x 250 mm, 5 µm
A: 0.1% phosphoric acid
B: Acetonitrile
10% B initial, then linear
gradient to 22% B in 13 min,
to 40 % B in 40 min
1.5 mL/min
UV @ 330 nm
10 µL
35° C
Determination of Alkaloids in Goldenseal,
commercial products or whole leaf
Oasis® HLB method for alkaloids in Goldenseal
Sample pre-preparation
100 mg sample is extracted with 25 mL of ethanol:water (70:30)
1 mL of the ethanolic extract is diluted 1:3 with water (~ pH 7)
Conditions for Oasis® HLB cartridge, 3 cc, 60 mg
Part Number WAT094226
• Recovery, measured with certified standards at 100 ppm in reagent water,
was greater than 85 % for all compounds
• All analyses gave results within ± 35 % of the expected values with the
exception of the liquid supplement (see goldenseal analysis)
• The selective SPE extraction and cleanup procedure provided a convenient
analysis of echinacea phenolics in complex matrix such as in herbal tea
Condition/Equilibrate:
1 mL methanol/1 mL 150 mM K2HPO4
Load:
3 mL of diluted sample
Wash:
1 mL 30% methanol/ 150 mM K2HPO4
Elute:
3 mL methanol
O
O
+
N
H3 CO
H3 CO
Herbal tea extract
before SPE cleanup
1. cafteric acid
2. chlorogenic acid
3. cichoric acid
total: 5 mg / teabag
HPLC Method
Column:
Mobile Phase:
hydrastine
0
Herbal tea extract after SPE
cleanup using Oasis® MAX
5
Minutes
Gradient:
berberine
10
Flow Rate:
Detection:
Injection:
Temperature:
Symmetry® C18, 4.6 x 250 mm, 5 µm
A: 0.1% phosphoric acid
B: Acetonitrile
10% B initial, then linear
gradient to 22% B in 13 min,
to 40 % B in 40 min
1.5 mL/min
UV @ 330 nm
10 µL
35° C
Oasis, Symmetry and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
49
Oasis® HLB Method for Solid Phase Synthesis
Conditions for Oasis® HLB Cartridge,35 cc, 6 g, Part Number 186000118
Goals : Remove DMSO, Cleave Amine and Impurities peaks
After the synthesis 3 ml of amine is added to cleave products (total of 300 mg)
from the resin and all is diluted in DMSO (total sample volume 10 mL)
Condition 18 mL ACN and Equilibrate
50 mL 0.1 % TFA in water
Load:
10 ml sample
Wash 1: 50 mL 0.1 %TFA in water
(to remove DMSO)
Wash 2: 50 mL 15% of ACN in 0.1%
TFA in water (to remove cleave amine)
Elute:
50 ml 90 % ACN in water
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
50
LC/MS and LC/PDA Determination of Pharmaceutical
Residues in Environmental Samples (Page 1 of 3)
Compounds
HO
NH
COOH
O
O
CH 3
Cl
acetaminophen
CH 3
H 3C
clofibric acid
CH 3
H 3C
N
O
CH 3
CH
OH
H
H
H
HO
diphenhydramine
ethynylestradiol
CH 3
CH 3
OH
CH 3
COOH
NH 2
H 3C
ibuprofen
phenylpropanolamine
H 3C
COOH
N CH 3
H 3C
O
OH
salicylic acid
tamoxifen
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
51
LC/PDA Determination of Pharmaceutical Residues in
Environmental Samples (Page 2 of 3)
Results of LC/PDA Analysis, 2.5 ppb
Compound
1. acetaminophen
2. phenylpropanolamine
3. salicylic acid
4. diphenhydramine
5. clofibric acid
6. ethynylestradiol
7. tamoxifen
8. ibuprofen
Spike levels
500, 2500
500, 2500
500, 2500
500, 2500
500, 2500
500, 2500
500, 2500
500, 2500
% Recovery
int, 78
nd, 61
60, 61
89, 86
101, 89
93, 86
76, 76
82, 77
%RSD
7.7, 10
nd, 5.7
20, 10
3.7, 4.6
4.0, 4.2
7.3, 4.1
6.0, 2.1
7.7, 4.7
Optimized SPE Method for LC/MS Determination of
Pharmaceutical Residues in Environmental Samples
Conditions for
Oasis® MCX 6 cc, 150 mg (60 µm) Part Number 186000255
Oasis® HLB Plus Part Number 186000132
Prepare Sample:
acidify to pH 2
Condition:
5 mL methanol/ 1 mL water
Load:
250 mL sample (5 mL/min)
1
1
Wash #1:
2 mL 0.1 N HCl
2
2
Oasis® MCX 6 cc,
150 mg (60 mm)
Part Number 186000255
PDA @ 230 nm (0.05 AUFS)
Wash #2:
2 mL 5 % MeOH/water
Elute: 10 mL MeOH/MTBE/
NH4OH (20:75:5)
205 nm
3
Oasis® HLB Plus
Part Number 186000132
Evaporate and Reconstitute:
250 mL mobile phase
MeOH - methanol MTBE - methyl t-butyl ether
NH4OH - 30% ammonium hydroxide
4
5
HPLC Method
6
Column:
7
Mobile Phase:
8
Gradient:
Flow Rate:
Injection:
0
2
4
6
Minutes
Oasis, XTerra and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
52
8
10
12
XTerra® MS C18 4.6 mm x 100 mm
(3.5 µm dp)
A: 15 mM ammonium formate (pH 4.0)
B: methanol
25 % linear to 90% B in 10 min
1.0 mL/min
40 µL
LC/MS Determination of Pharmaceutical Residues in
Environmental Samples (Page 3 of 3)
Results of LC/MS Analysis, 2.5 ppb
Compound
Response
(area/ng/L)
1. acetaminophen
2. phenylpropanolamine
3. salicylic acid(3 levels)
4. diphenhydramine
5. clofibric acid
6. ethynylestradiol (pH4)
6. ethynylestradiol (pH11)
7. tamoxifen
8. ibuprofen
%RSD/
R2
248.6
635
27.1
3793
62.35
no response
104
1092
19.6
0.9999
0.9996
0.9999
0.9985
0.9998
level*
11, 4.3, 5.1, 3.6
21, 22, 7.0, 3.6
nd, 15, 19, 9.0
35, 4.6, 19, 6.8
2.6, 2.3, 3.2, 3.9
0.9997
0.9990
0.9999
6.2, 5.1, 8.1, 11
15, 11, 13, 7.9
20, 5.2, 6.4, 5.8
* %RSD observed for 5 replicates at 25, 100, 500, 2500 ng/L spike levels
Optimized SPE Method for LC/MS Determination of
Pharmaceutical Residues in Environmental Samples
Conditions for
Oasis® MCX 6 cc, 150 mg (60 µm) Part Number 186000255
Oasis® HLB Plus Part Number 186000132
Prepare Sample:
acidify to pH 2
Condition:
5 mL methanol/ 1 mL water
Load:
250 mL sample (5 mL/min)
Oasis® MCX 6 cc,
150 mg (60 mm)
Part Number 186000255
4
2
ESI+
Wash #1:
2 mL 0.1 N HCl
7
Wash #2:
2 mL 5 % MeOH/water
5
8
pH 10.5
Elute: 10 mL MeOH/MTBE/
NH4OH (20:75:5)
6
ESI-
4
* note: ethynylestradiol
response only @ pH 10.5
7
ESI+ 1 2
Oasis® HLB Plus
Part Number 186000132
Evaporate and Reconstitute:
250 mL mobile phase
MeOH - methanol MTBE - methyl t-butyl ether
NH4OH - 30% ammonium hydroxide
5
pH 4
ESI-
3
HPLC Method
LC/MS Conditions Waters/Micromass ZMD™
Compound
acetaminophen
phenylpropanolamine
salicylic acid
diphenhydramine
clofibric acid
ethynylestradiol
tamoxifen
ibuprofen
MW
151.2
151.2
138.1
255.3
214.7
296.4
371.5
206.3
ESI+
152(40) 110(40)
152(40) 134(40)
ESI150(20)
137(17)
256(25) 167(40)
213(17) 127(17)
295(40) 145(80)
Column:
Mobile Phase:
Gradient:
Flow Rate:
Injection:
XTerra® MS C18 4.6 mm x 100 mm
(3.5 µm dp)
A: 15 mM ammonium formate (pH 4.0)
B: methanol
25 % linear to 90% B in 10 min
1.0 mL/min
40 µL
372(40)
205(17) 159(30)
Oasis, XTerra and Waters are trademarks of Waters Corporation.
ZMD is a trademark of Micromass U.K. Ltd.
© 2002 Waters Corporation
53
LC/MS/MS Analysis of Fusidic Acid in Plasma
Oasis® MAX Method
Results
95% for fusidic acid and ketoprofen
Conditions for Oasis® MAX Cartridge, 1 cc, 30 mg
Part Number 186000366
Prepare sample:
500 µl of plasma + 500 µl
of ammonium acetate buffer
+ 50 µl of fusidic acid solution
+ 50 µl of Ketoprofen (IS).
O
OH
H
OH
Condition: 1ml methanol,
Equilibrate: 1 ml water
O
Load:
1, 1 ml of sample
O
H
Wash 1:
1 ml ammonium acetate buffer
OH
H
Fusidic acid
Wash 2:
1ml methanol
Elute: 1ml methanol:formic acid
(99/1; v/v)R
Evaporate and reconstitute:
with mobile phase
HPLC Method
Column:
Column Temp:
Mobile phase:
C18-CN , 3.5 µm 75 x 4.6 mm
40 °C
Ammonium Acetate buffer
with 0.1% formic acid/ACN
(40/60; v/v)
Flow rate:
0.5 ml/min
Injection volume: 10µl
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
54
Oasis® Sample Extraction Products
Oasis® HLB Sample Extraction Products
Oasis® sample extraction products contain a polymeric, water-wettable sorbent that will allow you to process biologic samples
faster and develop rugged methods to assay acidic, basic and neutral drugs and their metabolites.
Description
Oasis® HLB cartridge
Oasis® HLB cartridge
Oasis® HLB cartridge with Gilson ASPEC™ Adapter
Oasis® HLB cartridge with Gilson ASPC™ Adapter
Oasis® HLB cartridge
Oasis® HLB cartridge with Gilson ASPC™ Adapter
Oasis® HLB cartridge
Oasis® HLB cartridge
Oasis® HLB cartridge
Oasis® HLB cartridge
Oasis® HLB cartridge
Oasis® HLB Plus cartridge
Oasis® HLB Vac RC cartridge
Oasis® HLB Vac RC cartridge
Oasis® HLB Glass cartridge
Oasis® HLB Prospekt™ cartridge*
Oasis® HLB Prospekt™ 2 cartridge
Resevoir 30 cc for Oasis® cartridges
Resevoir 60 cc for Oasis® cartridges
Resevoir Adapter for 1 cc, 3 cc, 6 cc cartridges
Resevoir Adapter for 12 cc, 20 cc, 35 cc cartridges
Resevoir Adapter for 5 cc cartridges, Teflon
Particle Size
Quantity
Part Number
30 µm
30 µm
100/box
100/box
500/box
500/box
100/box
500/box
30/box
30/box
20/box
20/box
10/box
50/box
50/box
50/box
30/box
100/box
96/box
48/box
12/box
10/box
10/box
10/pkg
186000383
WAT094225
186000988
WAT058882
WAT094226
WAT058883
WAT106202
186000115
186000116
186000117
186000118
186000132
186000382
186000381
186000683
186000258
186001196
WAT011390
WAT024659
WAT054260
WAT048160
405000934
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
1/pkg
5/pkg
1/pkg
186001413
186001414
186001792
186000706
186000262
WAT082745
WAT084560
WAT005139
WAT084567
1 cc/10 mg
1 cc/30 mg
1 cc 10 mg
1 cc/30 mg
3 cc/60 mg
3 cc/60 mg
6 cc/200 mg
6 cc/500 mg
12 cc/500 mg
20 cc/1 g
35 cc/6 g
225 mg
20 cc/30 mg
20 cc/60 mg
5 cc/200 mg
2 mm x 10 mm/15 mg
30 µm
30 µm
30 µm
30 µm
60 µm
60 µm
60 µm
60 µm
60 µm
30 µm
30 µm
60 µm
30 µm
3.9 mm x 20 mm
3.9 mm x 20 mm
2.0 mm x 15 mm
2.1 mm x 20 mm
5 µm
15 µm
25 µm
25 µm
96-well
5 mg/96-well
10 mg/96-well
30 mg/96-well
60 mg/96-well
30 µm
30 µm
30 µm
60 µm
24/pkg
1/pkg
1/pkg
1/pkg
1/pkg
186001136
186000309
186000128
WAT058951
186000679
3 cc/60 mg
30 µm
10 each MCX, HLB, MAX
186000867
* For use with Spark Holland Prospekt system
Oasis® HLB cartridge column
Oasis® HLB cartridge column
Oasis® HLB column
Oasis® HLB cartridge column
Holder Kit for 2.1 mm x 20 mm cartridge column
Extraction Column Connector
Inline precolumn filter kit
Replacement filters
Replacement steel gaskets
Oasis® filter plate
Oasis® HLB plate
Oasis® HLB plate
Oasis® HLB plate
Oasis® HLB plate
Oasis® Method Development Kit
Oasis and Waters are trademarks of Waters Corporation.
Aspec is a trademark of Gilson, Inc. Prospekt is a trademark of Spark, Holland.
© 2002 Waters Corporation
55
Oasis® Sample Extraction Products
Oasis® MCX Sample Extraction Products
The Oasis® MCX (mixed-mode: cation-exchange, reversed-phase) sorbent enables the selective retention of basic drugs. It is designed
to extract basic drugs from complex matrices such as whole blood, urine, serum, or plasma.
Description
Particle Size
Quantity
Part Number
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX cartridge
Oasis® MCX Vac RC cartridge
Oasis® MCX Vac RC cartridge
1 cc/30 mg
1 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
20 cc/60 mg
20 cc/60 mg
30 µm
60 µm
30 µm
60 µm
30 µm
60 µm
60 µm
60 µm
60 µm
30 µm
60 µm
100/box
100/box
100/box
100/box
30/box
30/box
30/box
20/box
10/box
50/box
50/box
186000252
186000782
186000254
186000253
186000256
186000255
186000776
186000777
186000778
186000261
186000380
Oasis® MCX plate
Oasis® MCX plate
Oasis® MCX plate
Oasis® MCX Plate
10 mg/96-well
30 mg/96-well
30 mg/96-well
60 mg/96-well
30 µm
30 µm
60 µm
60 µm
1/pkg
1/pkg
1/pkg
1/pkg
186000259
186000248
186000250
186000678
3 cc/60 mg
30 µm
10 each MCX, HLB, MAX
186000867
Oasis® Method Development Kit
Oasis® MAX Sample Extraction Products
Oasis® MAX (mixed-mode: anion-exchange, reversed-phase) sorbent gives you greater selectivity for your acidic drugs. It is designed to
extract acidic drugs from complex matrices such as whole blood, urine, serum or plasma.
Description
Oasis MAX cartridge
Oasis® MAX cartridge
Oasis® MAX cartridge
Oasis® MAX cartridge
Oasis® MAX cartridge
Oasis® MAX cartridge
Oasis® MAX Vac RC cartridge
Oasis® MAX Vac RC cartridge
Oasis® MAX Vac RC cartridge
®
Oasis® MAX plate
Oasis® MAX plate
Oasis MAX plate
Oasis® Method Development Kit
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
56
Particle Size
Quantity
Part Number
1 cc/30 mg
3 cc/60 mg
3 cc/60 mg
6 cc/150 mg
6 cc/150 mg
6 cc/500 mg
20 cc/30 mg
20 cc/60 mg
20 cc/60 mg
30 µm
30 µm
60 µm
30 µm
60 µm
60 µm
30 µm
30 µm
60 µm
100/box
100/box
100/box
30/box
30/box
30/box
50/box
50/box
50/box
186000366
186000367
186000368
186000369
186000370
186000865
186000372
186000371
186000378
10 mg/96-well
30 mg/96-well
60 mg LP/96-well
30 µm
30 µm
60 µm
1/pkg
1/pkg
1/pkg
186000375
186000373
186001205
3 cc/60 mg
30 µm
10 each MCX, HLB, MAX
186000867
Oasis® Sample Extraction Products
Manifold for Extraction Plate
Description
Extraction plate manifold
Extraction Plate Manifold Kit A
(includes extraction plate manifold, reservoir tray, manifold top gasket,
sealing cap and 350 µL sample collection plate)
Extraction Plate Manifold Kit B
(as kit A, with 1 mL sample collection plate)
Extraction Plate Manifold Kit C
(as kit A, with 2 mL sample collection plate)
Quantity
1/box
Part Number
WAT058941
WAT097944
WAT097945
WAT097946
Accessories for Extraction Plate Manifold
Reservoir tray
Sample collection plate, 350 µL
Sample collection plate, 1 mL
Sample collection plate, 2 mL
Sealing cap for 96-well collection plate
Manifold gasket, top
Manifold gasket, white
25/box
50/box
50/box
50/box
50 sheets/pkg
1/pkg
1/pkg
WAT058942
WAT058943
WAT058957
WAT058958
WAT058959
WAT058955
WAT058956
Manifold for Extraction Cartridges
Waters extraction manifold, 20-position without rack (includes 20 needle tips, 25 plugs, and ejector tool)
Waters extraction manifold, 20-position (complete with rack for 13 mm x 75 mm tubes)
Waters extraction manifold, 20-position (complete with rack for 13 mm x 100 mm tubes)
Waters extraction manifold, 20-position (complete with rack for 16 mm x 75 mm tubes)
Waters extraction manifold, 20-position (complete with rack for 16 mm x 100 mm tubes)
Vacuum pump (110V, 60 Hz)
Vacuum pump (220V, 50 Hz)
Vacuum pump (110V, 50 Hz)
WAT200677
WAT200606
WAT200607
WAT200608
WAT200609
WAT085114
WAT085115
WAT085123
Oasis and Waters are trademarks of Waters Corporation.
© 2002 Waters Corporation
57
Appendix A:
Oasis® HLB Glass Cartridge Instruction Sheet
Section 1: Introduction
Note: Once the HLB sorbent has been conditioned and
equilibrated, there is no need to keep the cartridges wet prior
to sample loading. Maintain a continuous vacuum on all
cartridges throughout steps 4a-4d. This convenience will save
you time.
Waters Oasis® HLB glass cartridges are available in 5 cc (200 mg)
configuration with Teflon® frits. The clean glass cartridge is designed
for trace analysis at parts per trillion level including monitoring endocrine
disruptors, such as phenols and phthalates.
Note: For the load and elute steps, the recommended flow
rate is 10 mL/min for 5 cc cartridges. You may need to
momentarily increase the vacuum to start the flow of
aqueous solutions.
Each lot of glass cartridges and Teflon® frits are tested for the presence of
bisphenol A and other phenols and phthalates before packing. These
tests assure that endocrine disruptors, in water samples, can be analyzed
to part per trillion levels.
The Certificate of Analysis [COA] reports recoveries, with RSDs, for
three polar pharmaceutical compounds. The COA displays results from
stringent quality control tests on the batch of polymer sorbent and the lot
of packed cartridges.
Section 2: Quick Start SPE Procedure
for Endocrine Disruptors
1.
If desired, add / mix 10 to 50 µL of internal standard to the
sample (soil, food and other solid samples require pretreatment
before SPE)
2.
Adjust the sample to pH 3.
3.
Place Oasis® HLB extraction cartridges on vacuum manifold
and set vacuum to approximately 5” Hg. The extraction
procedure can also be done by positive pressure
using the 5 cc Teflon® adaptors
(part number 405000934).
4a. Condition: Add to and draw through each cartridge
5-10 mL 10% methanol in methyl tertbutyl ether
(MtBE*) and then 3 mL methanol.
4b. Equilibrate: 3 mL water
4c. Load: Draw sample through the cartridge. The maximum
recommended sample volume is 1 L for 5 cc cartridges.
4d. Wash: Add to and draw through each cartridge 3 mL of
5% methanol in water (v/v).
Release vacuum, remove manifold cover, and discard waste
fluids. Insert rack containing collection vessels, replace cover,
and turn on vacuum.
4e. Elute: Add to and draw through each cartridge
6 mL 10% methanol in MtBE*.
If desired, evaporate eluates to dryness.
5.
Reconstitute in acetonitrile and adjust to the mobile phase
concentration for LC analysis.
6.
For GC analysis dry extract over sodium sulfate and
reconstitute to 1 mL.
No individual stopcocks are necessary.
4.
Solid-Phase Extraction Procedure: The following simple protocol
should be used in preparing and using the cartridges for the
isolation of a wide spectrum of acidic, basic, and neutral
analytes especially classes of endocrine disruptors.
No step should be omitted.
Procedure optimization is discussed in Section 3.
* Dethyl ether can be used as an alternative to MBTE
1
Oasis, Sep-Pak and Waters are trademarks of Waters Corporation.
Teflon is a trademark of E.I. Dupont de Nemours & Co.
© 2002 Waters Corporation
58
U.S. Patent No. 5,882,521
Section 3: Adjustments to Optimize Recoveries (Table 6)
Spike an appropriate volume of reagent water (for general analysis) or
PBS (for biological fluids analysis) with all analytes and internal/surrogate
standards. For preparation of PBS solution see Section 4. Follow steps
4a-4e in Section 2, 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 elution solvent and
collect the eluate. Analyze all four collected fractions. Use the table to
determine adjustments, if necessary, to optimize sample recovery.
Table 6
If the fraction from this
step contains the analyte
Load (4c)
Wash (4d)
First Elution (4e)
Second Elution
(4e repeated)
Make this adjustment for
optimum sample recovery
The Oasis® HLB sorbent has been
found to retain ionized analytes more
strongly than silica-based reversedphased 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.
Recoveries of very polar analytes can
be increased by using only 1 mL of
water (not 5% methanol in water) as
the wash solution.
Section 4: Preparation of PhosphateBuffered Saline (PBS)
(PBS required only when analyzing analytes in serum, plasma, or urine,
not required for water, soil, or food samples)
To make phosphate-buffered saline solution:
1. To a 1-liter flask, add the following anhydrous salts:
a. 200 mg KCl
b. 8000 mg NaCl
c. 200 mg KH2PO4
d. 1150 mg Na2HPO4
2. Add 1 liter of deionized water. Stir to dissolve.
3. Adjust pH to 7.0 with 10% phosphoric acid.
Ordering Information:
Description
Part Number
Oasis® HLB Glass Cartridges
5 cc /200 mg 30/box
186000683
Adaptor, 5cc, Teflon® 10/pkg
405000934
Sep-Pak® Connector Kit
WAT011400
If an acceptable recovery of analyte(s)
is obtained in this fraction (usually
> 90%), no adjustements are
necessary
For very nonpolar analytes, stronger
solvents such as acetonitrile, methylene
chloride or ethyl acetatemay be
substituted, or used in sequence. In
addition, for ionizable analytes,
methanol may needed to be 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, Section 2) 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 1 mL of
ethyl acetate, followed by 1 mL of
methanol and 1 mL of water.
Oasis, Sep-Pak and Waters are trademarks of Waters Corporation.
Teflon is a trademark of E.I. Dupont de Nemours & Co.
© 2002 Waters Corporation
59
Appendix B:
Choosing the Correct Procedure for EPA (SW-846)
As evident by the number of applications included in this notebook many
people have been successful in developing and employing Oasis® solid
phase extraction methods for many compounds covered by SW-846
analytical methods. We at Waters invite you take full advantage of the
enhanced capacity, pH resistance and water wettable of Oasis® SPE
products. For guidance when using Oasis® products to perform SW-846
Methods, we have provided the following excerpts from section 2.1
Guidance regarding Flexibility Inherent to SW-846 Methods and the
Precedence of SW-846 Quality Control Criteria.
This information can be downloaded from:
www.epa.gov/epaoswer/hazwaste/test/main.htm
and or CD-ROM, hard copies from:
U.S. Government Printing Office (GPO)
Superintendent of Documents
Washington, DC 20402
Phone: (202) 512-1800
Publication Number: 955-001-00000-1
CD-ROM TWO - 1 Revision 3
December 1996
CHAPTER TWO
CHOOSING THE CORRECT PROCEDURE
SW-846 analytical methods are written as quantitative trace analytical
methods to demonstrate that a waste does not contain analytes of concern that cause it to be managed as a hazardous waste. As such, these
methods typically contain relatively stringent quality control (QC)
criteria appropriate to trace analyses. However, if a particular application
does not require data of this quality, less stringent QC criteria may be
used. The purpose of this chapter is to aid the analyst in choosing the
appropriate methods for sample analyses, based upon the sample matrix
and the analytes to be determined. The ultimate responsibility for producing reliable analytical results lies with the entity subject to the regulation.
Therefore, members of the regulated community are advised to refer to
this chapter and to consult with knowledgeable laboratory personnel
when choosing the most appropriate suite of analytical methods. In addition, analysts and data users are advised that, except where explicitly
specified in a regulation, the use of SW-846 methods is not mandatory
in response to Federal testing requirements.
Section 2.1 provides guidance regarding the analytical flexibility inherent
to SW-846 methods and the precedence of various QC criteria. Section
2.2 reviews the information required to choose the correct combination
of methods for an analytical procedure. Section 2.3 provides useful information on implementing the method selection guidance for organic analyses. Section 2.4 provides guidance on characteristic analyses and
Section 2.5 provides guidance on the determination of analytes in
ground water.
60
2.1 GUIDANCE REGARDING FLEXIBILITY INHERENT TO
SW-846 METHODS AND THE PRECEDENCE OF SW-846
QUALITY CONTROL CRITERIA
The specific products and instrument settings cited in SW-846 methods
represent those products and settings used during method development or
subsequently evaluated by the Agency for use in the method.
Glassware, reagents, supplies, equipment and settings other than
those listed in this manual may be employed, provided that
method performance appropriate for the intended RCRA application has been documented. Such performance includes consideration
of precision, accuracy (or bias), recovery, representativeness, comparability, and sensitivity (detection, quantitation, or reporting limits) relative to the
data quality objectives for the intended use of the analytical results.
In response to this inherent flexibility, if an alternative analytical
procedure is employed, then EPA expects the labor-atory to
demonstrate and document that the procedure is capable of providing appropriate performance for its intended application. This
demonstration must not be performed after the fact, but as part
of the laboratory’s initial demonstration of proficiency with the
method. The documentation should be in writing, maintained in the laboratory, and available for inspection upon request by authorized representatives of the appropriate regulatory authorities. The documentation should
include the performance data as well as a detailed description of the procedural steps as performed (i.e., a written standard operating procedure).
Given this allowance for flexibility, EPA wishes to emphasize that this
manual also contains procedures for “method-defined parameters,” where
the analytical result is wholly dependant on the process used to make the
measurement. Examples include the use of the toxicity characteristic
leaching procedure (TCLP) to prepare a leachate, and the flash point,
pH, paint filter liquids, and corrosivity tests. In these instances, changes to
the specific methods may change the end result and incorrectly identify a
waste as nonhazardous. Therefore, when the measurement of such
method-defined parameters is required by regulation, those methods are
not subject to the flexibility afforded in other methods.
Analysts and data users are advised that even for those analytes that are
not method-defined, different procedures may produce some difference in
results. Common examples include the differences in recoveries of phenolic compounds extracted from water by separatory funnel (Method 3510)
and continuous liquid-liquid (Method 3520) extraction techniques,
differences in recoveries of many compounds between Soxhlet (Method
3540) and ultrasonic (Method 3550) extraction techniques, and differences resulting from the choice of acid digestion of metals (Method
3050) or microwave digestion (Method 3051). Where practical, the
Agency has included guidance in the individual methods regarding
known potential problems, and analysts are advised to review this
information carefully in choosing or modifying analytical procedures.
Chapter One describes a variety of QC procedures that may be used
to evaluate the quality of the analytical results. Additional QC procedures
may be described in the individual methods. The results of these QC
procedures should be used by the analyst to evaluate if the choice of the
analytical procedures and/or any modifications are appropriate to
generate data of the quality necessary to satisfy the data quality needs of
the intended application.
The performance data included in the SW-846 methods are not intended to be used as absolute QC acceptance criteria for method performance. The data are intended to be guidance, by providing typical
method performance in typical matrices, to assist the analyst in selection
of the appropriate method for the intended application. In addition, it is
the responsibility of the laboratory to establish actual operating parameters and in-house QC acceptance criteria, based on its own laboratory
SOPs and in-house QC program, to demonstrate appropriate performance of the methods used in that laboratory for the RCRA analytical
applications for which they are intended.
The regulated community is further advised that the methods here or from
other sources need only be used for those specific analytes of concern
that are subject to regulation or other monitoring requirements. The fact
that a method provides a long list of analytes does not mean that each of
those analytes is subject to any or all regulations, or that all of those analytes must be analyzed each time the method is employed, or that all of
the analytes can be analyzed using a single sample preparation procedure. It is EPA’s intention that the target analyte list for any procedure
includes those analytes necessary to meet the data quality objectives of
the project, i.e., those analytes subject to monitoring requirements and set
out in a RCRA permit (or other applicable regulation), plus those analytes
used in the methods for QC purposes, such as surrogates, internal standards, system performance check compounds, etc. Additional analytes,
not included on the analyte list of a particular method(s) but needed for a
specific project, may be analyzed by that particular method(s), if appropriate performance can be demonstrated for the analytes of concern in
the matrices of concern at the levels of concern.
Revision 3
December 1996
61
®
Search Waters Applications Library for the
latest references on the use of Oasis® SPE Products.
SAMPLE EXTRACTION PRODUCTS
1. Straightforward solid-phase extraction method for the determination of verapamil and its
metabolite in plasma in a 96-well extraction plate
Apps. Code ung-Fong Cheng, Uwe D. Neue, Laura Bean
Waters Corporation, 34 Maple Street, Milford, MA, USA
J Chromatogr A 1998 828 273-281
Lit. Code: WT078
2. Optimisation and routine use of generic ultra-high flow-rate LC with MS detection for the direct
on-line analysis of pharmaceuticals in plasma
J Ayrton; GJ Dear; WJ Leavens; DN Mallett; RS Plumb GlaxoWellcome R&D, Ware, UK
J Chromatogr A 1998 828 199-207
Apps. Code 990048
3. The Use of High-flow HPLC coupled with Positive and Negative Ion Electrospray Tandem MS for
Quantitative Bioanalysis via Direct Injection of the Plasma/Serum Samples
M Jemal; Yuan-Qing; DB Whigan Bristol-Myers Squibb Pharm Res Inst, New Brunswick, NJ
Rapid Commun Mass Spectrom 1998 12 1389-1399
Apps. Code 981073
4. Use of generic fast gradient LC-tandem MS in quantitative bioanalysis
J Ayrton; GJ Dear; WJ Leavens; DN Mallett; RS Plumb GlaxoWellcome R&D, Ware, UK
J Chromatogr B 1998 709 243-254
Apps. Code 980668
5. Measurement of Plasma S-Adenosylmethionine and S-Adenosylhomocysteine as their Fluorescent
Isoindoles
Antonieta Capdevila and Conrad Wagner Department of Biochemistry, Vanderbilt University, USA
Anal Biochem 1998 264 180-184
6. Simplified procedure for measurement of serum dehydroepiandrosterone and its sulfate with
GC-ion trap MS and selected reaction monitoring
MA Zemaitis; PD Kroboth Dept Pharm Sci, Univ Pittsburgh, PA
J Chromatogr B 1998 716 19-26
Apps. Code 981121
7. Development of a sensitive and quantitative analytical method for 1H-4-substituted imidazole
histamine H3-receptor antagonists utilizing high-performance liquid chromatography and dabsyl
derivatisation
Michael K. Handley, Walter W. Hirth, James G. Phillips, Syed M. Ali, Amin Khan, Leena Fadnis,
Clark E. Tedford Gliatech Inc, Cleveland, USA
J Chromatogr B 1998 716 239-249
8. Simultaneous determination of omeprazole and 5-hydroxyomeprazole in human plasma by
LC-tandem MS
EJ Woolf; BK Matuszewski, Merck Research Labs, West Point, PA
J Chromatogr A 1998 828 229-238
Apps. Code 990047
9. Assay of acetylsalicyclic acid and three of its metabolites in human plasma and urine using
non-aqueous capillary electrophoresis with reversed electroosmotic flow
Steen Honore Hansen, Maj Elgin Jensen, Inga Bjornsdottir; Department of Analytical and
Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Copenhagen, Denmark
J Pharm Biomed Anal 1998 17 1155-1160
10. Comparison of the properties of polymeric and C8 based materials for SPE
P Martin; ID Wilson, Zeneca Pharmaceuticals, Macclesfield, UK
J Pharm Biomed Anal 1998 17 1093-1100
Apps. Code 990035
11. Determination of the Enantiomers of Salbutamol and its 4-O-Sulphate Metabolites in Biological
Matrices by Chiral LC Tandem MS
KB Joyce; AE Jones; RJ Scott; RA Biddlecombe; S Pleasance Dept of International Bioanalysis,
GlaxoWellcome R&D, Ware, UK
Rapid Comm Mass Spectrom 1998 12 1899-1910
Apps. Code 990019
12. Electrospray Ionization and Tandem Ion Trap Mass Spectrometry for the Confirmation of Seven
ß-Lactam Antibiotics in Bovine Milk
David N. Heller and Maureen A. Ngoh, FDA Center for Veterinary Medicine, Laurel, MD, USA
Rapid Commun Mass Spectrom 1998 12 2031-2040
13. Metabolism of 2,4,6-Trinitrotoluene by Pseudomonas sp. JLR11
A Esteve-Nunez; JL Ramos, Estacion Experimental del Zaidin, Granada, Spain
Environ Sci Technol 1998 32 3802-3808
Apps. Code 981392
14. Alkylation of 2-Deoxynucleosides and DNA by the Premarin Metabolite 4-Hydroxyequilenin
Semiquinone Radical
L Shen; S Qiu; Y Chen; F Zhang; RB van Breemen; D Nikolic; JL Bolton Dept Med Chem &
Pharmacognosy, U Illinois, Chicago
Chem Res Toxicol 1998 11 94-101
Apps. Code 980271
62
15. Inhibition of Glutathione S-Transferase Activity by the Quinoid Metabolites of Equine Estrogens
M Chang; F Zhang; L Shen; N Pauss; I Alam; RB van Breemen; SY Blond; JL Bolton; Dept Med
Chem & Pharmacognosy, U Illinois, Chicago
Chem Res Toxicol 1998 11 758-765
Apps. Code 980815
16. Formation of N-(Carboxymethyl)fumonisin B1, Following the Reaction of Fumonisin B1 with
Reducing Sugars
PC Howard; MI Churchwell; LH Couch; MM Marques; DR Doerge; Natl Ctr for Toxicol Res, US FDA,
Jefferson, AK
J Agric Food Chem 1998 46(9) 3546-3557
Apps. Code 981045
17. A Chromatographic and Mass Spectrometric Strategy for the analysis of oligosaccharides:
Determination of the Glycan Structures in Porcine Thyroglobulin
Joanne Charlwood; Helen Birrell; Andrew Organ; Patrick Camilleri, SmithKline Beecham
Pharmaceuticals, Essex, UK
Rapid Comm Mass Spectrom 1999 13 716-723
Apps. code 990482
18. Determination of chlorobenzidines in industrial effluent by solid-phase extraction and
liquid chromatography with electrochemical and mass spectrometric detection
Silvia Lacorte; Marie-Claude Perrot; Daqniel Fraisse; Damia Barcelo CARSO, Centre d’ Analyss de
Traces, Lyon France and Department of Environmental Chemistry, CID-CSIC, Barcelona Spain
J Chromatogr A 1999 833 181-194
Apps. Code 990361
19. Semi-Automated, Solid-Phase Extraction Procedure for Liquid Chromatographic
Determination of Papaverine, Diltiazem, Desipramine and Nicardipine in Urine
A. Bakkali; A. Barranco; R.M. Alonso-Salces; E. Corta; L.A. Berrueta; B. Gallo; F. Vicente; J.I.
Marquez
Department of Analytical Chemistry, University of the Basque Country, Bilbao, Spain
Apps. Code 990390
20. Analysis of Tetrahydrocannabinol and its two major metabolites by APCI-LC/MS.
Pascal Mireault
Laboratoire de Sciences Judiciaries et de Medecine Legale, Montreal, Quebec
Presentation presented at the 1998 ASMS conference
21 Development and validation of an HPLC method for Determination of 2-Hydroxy4 Trifluoromethylbenzoic acid (HTB) and Salicyclic acid in Human Plasma.
C. Nieto; J. Ramis; L. Conte; I. Escamilla; E. Turmo; J. Fron, J. Uriach & Cia Research Centre,
Barcelona, Spain
22. Determination of catecholamines in human plasma by high-performance liquid
chromatography with electrochemical detection.
M.A. Raggi; C. Sabbioni; G. Casamenti; G. Gerra; N. Calonghi; L. Masotti
Dept. of Pharmaceutical Sciences, University of Bologna, Bologna Italy; Ser.T., AUSL of Parma,
Parma Italy; Department of Biochemistry, University of Bologna, Bologna, Italy
J Chromatogr B 1999 730 201-211
Apps. Code 990942
23. The Major Metabolite of Equilin, 4-Hydroxyequilin, Autoxidizes to an o-Quinon Which
Isomerizes to the Potent Cytotoxin 4-Hydroxyequilenin-o-quinone
Fagen Zhang; Yumei Chen; Emily Pisha; Li Shen; Yansan Xiong; Richard B. van Breemen;
Judy Bolton
Department of Medical Chemistry and Pharmacognosy, University of Illinois, Chicago
Chem Res Toxicol 1999 12 204-213
Apps. Code 990303
24. Determination of Indinavir in Plasma by Solid-phase Extraction and Column Liquid
Chromatography
Jean-Marie Poirier; Pascal Robidou; Patrice Jaillon
Department of Pharmacology, Saint-Antoine University Hospital, Paris France
Ther Drug Monit 1999 21 404-410
25. Determination of Acyclovir in Plasma by Solid-phase Extraction and Column Liquid
Chromatography
Jean-Marie Poirier; Pascal Robidou; Patrice Jaillon
Department of Pharmacology, Saint-Antoine University Hospital, Paris France
Ther Drug Monit 1999 21 129-133
26. Induction of UDP-Glucuronosyl-Transferase by the Flavonoids Chrysin and Quercetin
in Caco-2 Cells
Alema Galijatovic; U. Kristina Walle; Thomas Walle
Pharm Res 2000 17
Apps. code WA00245
27. High Performance Liquid Chromatography/Electrospray Tandem Mass Spectrometry for
Phenothiazines with Heavy Side Chaines in Whole Blood
Hiroshi Seno; Hideki Hattori; Akira Ishii; Takeshi Kumazawa; Kanako Watanbe-Suzuki;
Osamu Suzuki
Rapid Commun Mass Spectrom 1999 13 2394-2398
Apps. Code- 991149
®
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latest references on the use of Oasis® SPE Products.
SAMPLE EXTRACTION PRODUCTS
28. Determination of alternariol in tomato paste using solid phase extraction and
high-performance liquid chromatography with fluorescence detection
C.A. Fente; J. Jaimez; B.I. Vazquez; C.M. Franco
Laboratorio de Higiene e Inspeccion de Alimentos, Universidad de Santiago de Compostela,
Lugo Spain
Analyst
Apps. Code 981253
29. Matrix-assisted Laser Desorption/Ionization Mass Spectrometry of Deoxynucleotides
Labeled with IMI Dye
Zhang-Hua Lan; Poguang Wang; Roger Giese
Department of Pharmaceutical Sciences in the Bouve College of Pharmacy, Northeastern University,
Boston, MA
Rapid Comm Mass Spectrom 1999 13 1454-1457
Apps. Code 991134
30. Analysis of Oligosaccharides by Microbore High-Performance Liquid Chromatography
Joanne Charlwood; Helen Birell; Edouard S.P. Bouvier; Jim Langridge; Patrick Camilleri
SmithKline Beecham Pharmaceuticals, Waters Corporation, Micromass UK Ltd.
Anal Chem 2000 72 1469-1474
Apps. code WA00413
31. Broad Spectrum Analysis of 109 Priority Compounds Listed in 76/464/CEE Council Directive
Using Solid-Phase Extraction and GC/EI/MS
Silvia Lacorte; Ingrid Guiffard; Daniel Fraisse; Damia Barcelo
Department of Environmental Chemistry Barcelona Spain; Centre d’Analyse de Traces,
Lyon, France
Anal Chem 2000 72 1430-1440
Apps. code WA00412
32. Identification and Quantification of Cardiac Glycosides in Blood and Urine Samples by
HPLC MS/MS
Fuyu Guan; Akira Ishii; Hiroshi Seno; Kanako Watanabe-Suzuki; Takeshi Kumazawa;
Osamu Suzuki
Department of Legal Medicine, Universities of Hamamatsu and Showa, Japan
Anal Chem 1999 71 4034-4043
Apps. Code 991139
33. Structural Characterization of N-Linked Glycan Mixtures by Precursor Ion Scanning and Tandem
Mass Spectrometric Analysis
Joanne Charlwood; Jim Langridge; Patrick Camilleri
SmithKline Beecham Pharmaceuticals, Waters Corporation, Micromass UK Ltd.
Rapid Comm Mass Spectrom 1999 13 1522-1530
Apps. Code 991131
34. Solid-Phase Extraction Method for Patulin in Apple Juice and Unfiltered Apple Juice
Mary Trucksees; Yifeng Tang
U.S. Food and Drug Administration, World Health Organization
J AOAC Intl 1999 82 1109-1113
Apps. Code WA00064
35. Efficient solid-phase extraction procedures from trace enrichment of priority phenols from industrial effluents with high total organic carbon content
Silvia Lacorte; Daniel Fraisse; Damia Barcelo
Department of Environmental Chemistry, Barcelona Spain, and CARSO, Centre d’Analyse de Traces,
Lyon, France
J Chromatogr A 1999 857 97-106
Apps. Code 991133
39. Sample preparation of biological materials, tricyclic antidepressants.
Neue, U.D.; Altepeter, B.J.
Waters GmbH, 65760 Eschborn, Germany
Labor Praxis 1997 21( 7) 54-56, 61
40. High performance liquid chromatography analysis of chlorocresol using Oasis® HLB extraction
cartridges for extraction from corticosteroid cream
Weiss, Ami and Tapiro, Rachel
Analytical Research and Development Department,
Trima, Israel Pharmaceutical Products, Kibbutz Maabarot, Israel.
41. Novel HPLC and Solid-Phase Extraction Methods for Quantitating Methadone and its
Metabolite in Spiked Human Urine
Yung-Fong Cheng, Uwe D. Neue, and Laura L. Woods, Waters Corporation
J Chromatogr B 1999 729 19-31
42. Straightforward Solid-Phase Extraction Method Development Strategy
Yung-Fong Cheng, Ziling Lu, Uwe Neue, Dorothy Phillips, Laura L. Woods, and Robert Bonin,
Waters Corporation, Proceedings 47th ASMS on Mass Spectrometry and Allied Topics 1999 970
43. Polymeric Reversed-Phase SPE Sorbents. Characterization of a Hydrophilic-Lipophilic
Balanced SPE Sorbent
Edouard S.P. Bouvier, Pamela C. Iraneta, Uwe D. Neue, Patrick D. McDonald, Dorothy J. Phillips,
Mark Capparella and Yung-Fong Cheng, Waters Corporation, LC-GC 1998 S53
44. Simple Extraction Methods for the Determination of Drugs in Serum
Yung-Fong Cheng, Dorothy J. Phillips, Uwe D. Neue, Mark Capparella and Laura L. Bean,
Waters Corporation, Am Biotechnol Lab 14 December1997.
45. A Novel Polymeric Reversed-Phase Sorbent for Solid-Phase Extraction
Edouard S.P. Bouvier, Donna M. Martin, Pamela C. Iraneta, Mark Capparella,
Yung-Fong Cheng, and, Dorothy J. Phillips, Waters Corporation, LC-GC 1997 15(2)
46. Solid-Phase Extraction for the Determination of Tricyclic Antidepressants in Serum
Using a Novel Polymeric Extraction Sorbent
Y.F. Cheng, D.J. Phillips, U. Neue and L. Bean, Waters Corporation
J Liq Chromatogr 1997 20(15) 2461
47. Simple and Rugged SPE Method for the Determination of Tetracycline Antibiotics in Serum by
HPLC Using a Volatile Mobile Phase
Y.F. Cheng, D.J. Phillips and U. Neue, Waters Corporation
Chromatographia 1997 44(3/4) 187
48. A Novel Polymeric Reversed-Phase Sorbent for Solid-Phase Extraction
E.S.P. Bouvier, D.M. Martin, P.C. Iraneta, M. Capparella, Y.F. Cheng and D.J. Phillips, L. Bean,
Waters Corporation, LC-GC 1997 15(2) 152
49. Mixed Mode Solid Phase Extraction and Cleanup Procedures for the LC Determination of
Thiabendazole and Carbendazim in Fruit Juices.
Michael S. Young, P.C. Iraneta, J. Krol, D.J. Phillips
Waters Corporation
Submmited for publication
50. Purification of crude DNA Oligonucleotides by solid-phase extracion and reversed-phase
high-preformance liquid chromatography
Martin Gilar, E.S.P. Bouvier, Waters Corporation
J Chromatogr A 2000 890 167-177
36. The Use of High-Flow High Performance Liquid Chromatography Coupled with Positive and
Negative Ion Electrospray Tandem Mass Spectrometry for Quantitative Bioanalysis via Direct Injection
of the Plasma/Serum Samples
M. Jemal; Yuan-Qing; Daisy Whigan
Bristol-Myers Squibb Pharmaceutical Research Institute
Rapid Comm Mass Spectrom 1998 12 1389-1399
Apps. Code 981073
51. Determination of a "GW Cocktail" of Cytochrome P450 probe Substrates and their metabolites
in plasma and Urine Using Automated Solid Phase Extraction and Fast Gradient Liquid
Chroamtogrphy Tandem Mass Spectrometry
RJ Scott, J. Palmer, I.A.S. lewis; S Pleasance
Dept of International Bioanalysis, GlaxoWellcome R&D, Ware, UK
Rapid Comm Mass Spectrom 1999 13 2305-2319
Apps. Code 991150
37. Ultra-high Flow Rate Capillary Liquid Chromatography With Mass Spectrometric Detection for the
Direct analysis of Pharmaceuticals in Plasma at Sub-nanogram Per Milliliter Concentrations.
J Ayrton; R.A. Clare, GJ Dear; DN Mallett*; RS Plumb International Bioanalysis and Drug metabolism Division, GlaxoWellcome R&D, Park Road, Ware, Herts Sg120DP, UK
Rapid Comm Mass Spectrom 1999 13 1657-1662
52. A Versatile System of High-flow High Performance Liquid Chromatography with Tandem Mass
Spectrometry for Rapid Direct-injection Analysis of Plasma Samples for Quantitation of a ß-lactam
Drug Canadidate and its Open-ring Biotransformation Product.
M Jemal; Yuan-Qing Xia;Z. Ouyang; M. L. Powell
Bristol-Myers Squibb Pharm Res Inst, New Brunswick, NJ
Rapid Comm Mass Spectrom 1999 13 1462-1471
Apps. Code 990992
38. Simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in meat
and fish by HPLC.
Horie, M.; Saito, K.; Hoshino, Y.; Terada, H.; Nakazawa, H.
Saitama Prefectural Inst. Public Health, Saitama 338, Japan
Shokuhin Eiseigaku Zasshi 1997 38(5) 329-334
63
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