New Techniques For Sample Preparation

Division for Chemistry
Department of Analytical Chemistry
Zeki Altun
New Techniques for

Sample Preparation in
Analytical Chemistry
Karlstad University Studies

2005:24
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CH3 O
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PPX, Mw 232 g/mol
CH3 O
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N
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Lidocaine, Mw 234 g/mol
2H -ropivacaine,
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Ropivacaine, Mw 274 g/mol
CH3
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Mw 281 g/mol
CH3 O
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Bupivacaine, Mw 288 g/mol
Figure 6. Structures and molecular weights of local anaesthetics utilized in this work.

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Olomoucine (I.S), Mw 298 g/mol
Figure 7. The structures and molecular weights of roscovitine and olomoucine (internal
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Figure 8. Effect of type of sorbents on the recovery of ropivacaine.

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solutions.
Ropivacaine
PPX
3-OH-ropivacaine
Internal Standard
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40%
20%
0%
0.5 mg
1 mg
2 mg
Figure 10. Effect of amount sorbent, benzenesulphonic acid, on the recovery of

ropivacaine and its metabolites PPX and 3-OH-ropivacaine compared to direct injection of
pure standard solutions.

21

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Relative response
0,9
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ate
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Figure 11. Effect of the washing solution on the response of ropivacaine.

66 ++
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+

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23
80%
C8
C2
Recovery
60%
40%
20%
0%
M eOH/ Wat er 8:2
M eOH/ Wat er 9:1(v/ v)
(v/ v) (0.25%NH4OH)
(0.25%NH4OH)
M eOH/ Wat er/ ACN

8:1:1(v/v) (0.25%
NH4OH)
M eOH/Water 95:5
(v/v) (0.25%NH4OH)
Figure 12. Effect of different elution solvents on the recovery of ropivacaine.

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Figure 13. A photographic image of UV-polymerized monolith inside a disposable micro
pipette tip. The polymerization mixture contained GMA 20%, EGDMA 15.5%, BMA 3.5%,
AIBN 1% (with respect to monomers), cyclohexanol 30% and 1-dodecanol 30%.
Polymerization was allowed to proceed for 85 minutes using 254 nm light.

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Figure 14. Packed 96-tips with monolithic methacrylate polymer.

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041005024
MRM of 2 Channels ES+

355.3 > 233
8.58e4
100
Roscovitine 125 nM in plasma sample
Blank plasma
0
1.00
2.00
3.00
4.00
5.00
Time
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8.00
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299 > 91
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6.00
041005086
4.87
100
IS
Blank plasma
0
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2.00
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4.00
5.00
6.00
7.00
8.00
Time
Figure 15. Chromatograms of (A) blank plasma and plasma spiked with 125 nM
roscovitine and (B) blank plasma and plasma spiked with olomoucine (I.S.).
Chromatographic conditions: column Zorbax 50 x 2.1 mm I.D., SB-C8, 3,5 m connected
to a guard column, an Optiguard 10 x 1 mm I.D., C8; mobile phase (A) 0.1 % formic acid in
ACN-Water (10:90, v/v), (B) 0.1% formic acid in ACN-Water (80-20, v/v); linear gradient
0% B for 1 min, 0-80% B in 4 min, 80% B in 1 min; flow rate 150 L/min.
28
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New Techniques for

Sample Preparation in
Analytical Chemistry
Sample preparation is often a bottleneck in systems for chemical analysis. The aim
of this work was to investigate and develop new techniques to address some of the
shortcomings of current sample preparation methods. The goal has been to provide
full automation, on-line coupling to detection systems, short sample preparation
times and high-throughput.
A new technique for sample preparation that can be connected on-line to liquid
chromatography (LC) and gas chromatography (GC) has been developed. Microextraction in packed syringe (MEPS) is a new solid-phase extraction (SPE) technique
that is miniaturized and can be fully automated. In MEPS approximately 1 mg of
sorbent material is inserted into a gas tight syringe (100-250 L) as a plug. Sample
preparation takes place on the packed bed. Evaluation of the technique was done
by the determination of local anaesthetics in human plasma samples using MEPS
on-line with LC and tandem mass spectrometry (MS-MS). MEPS connected to an
autosampler was fully automated and clean-up of the samples took one minute. In
addition, in the case of plasma samples the same plug of sorbent could be used for
about 100 extractions before it was discarded.
A further aim of this work was to increase sample preparation throughput. To do
that disposable pipette tips were packed with a plug of porous polymer monoliths
as sample adsorbent and were then used in connection with 96-well plates and LCMS-MS. When roscovitine in human plasma and water samples was used as model
substance, a 96-plate was handled in two minutes.
Karlstad University Studies

ISSN 1403-8099
ISBN 91-85335-65-7

New Techniques For Sample Preparation

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New Techniques For Sample Preparation

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Division for Chemistry

Department of Analytical Chemistry

New Techniques for

Karlstad University Studies

Figure 2. The four processing steps in the operation of SPE experiment.

 + 4 6+ 0  +<

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Figure 4. Schematic setup of an LC system. 1. Pump, 2. Mobile Phase Mixer, 3. Injector

+6 6 +7 7   0- 4  +

PPX, Mw 232 g/mol

Ropivacaine, Mw 274 g/mol

Roscovitine, Mw 354 g/mol

Olomoucine (I.S), Mw 298 g/mol

Ethyl silica (C2) Octyl silica (C8) Octadecyl silica

Figure 8. Effect of type of sorbents on the recovery of ropivacaine.

Figure 10. Effect of amount sorbent, benzenesulphonic acid, on the recovery of

Figure 11. Effect of the washing solution on the response of ropivacaine.

M eOH/ Wat er/ ACN

Figure 12. Effect of different elution solvents on the recovery of ropivacaine.

 * .+ 

MRM of 2 Channels ES+

Roscovitine 125 nM in plasma sample

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New Techniques for

Karlstad University Studies

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