(d)7-10,11l ce for amphetamines 200807

8/6/2019 (D)7-10,11L CE for Amphetamines 200807

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This has traditionally been carried out using

gas chromatography (GC) and high-

performance liquid chromatography (HPLC).


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Capillary electrophoresis has been

investigated as an alternative separation

technique for forensic analysis. It has the potential to provide far more rapid

separations than are generally achievable

with HPLC, and can provide a separationwhere the analyte in question behaves

poorly under GC analysis.


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Figure 30.3. A CE system.


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Capillary zone electrophoresis (CZE), in

which ionic species are separated according

to their mobility and polarity in aqueoussolution, is the most frequently used option

in CE.


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Figure 30.1. CZE separation of sulfonamides. Separation of twenty compounds, seventeensulfonamides, levamisole, trimethoprim, and pyrimethamine (all 50 M); separation with 30 mM sodium

dihydrogen phosphate/10 mM sodium tetraborate pH 6.75, 15 kV, HD 1.8 s at 50 mbar; hydrodynamic

injection, 60(47) 50 m, 200 nm. Key: PST, phthal-sulfathiazole; PY, pyrimethamine; SA, sulfanilic

acid; SAA, sulfanilamide; SAC, sulfacetamide; SDI, sulfadiazine; SDIM, sulfadimethoxine; SG,

sulfaguanidine; SIOX, sulfaisoxazole; SM, sulfameter; SMOP, sulfamethoxypyridine; SMOZ,

sulfamethoxaxole; SMR, sulfamerazine; SMZ, sulfamethazine; SP, sulfapyridine; SQ, sulfaquinoxaline;

SST, succinyl-sulfathiazole; ST, sulfathiozole; and TRI, trimethoprim.


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The term capillary electrophoresisdescribes a family of related techniques in

which separations are carried out in arrowbore capillaries under the influence of anelectric field.

The separations obtained by capillary

electrophoresis are highly efficient, rapid,and may be applied to both charged andneutral species.


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Anon-line search of the Scifinder Scholar

database revealed in excess of 30,000

eferences to capillary electrophoresis todate with applications in a wide range

ofareas, including pharmaceuticals, food

and beverages, environmental and clinicalanalysis.


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The potential of this technique for forensic

analysis was first demonstrated in 1991 by

Weinberger and Lurie, who applied it to theanalysis of a wide range of illicit drugs in

synthetic mixtures.


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The exceptional power ofseparation and

resolution, rapid analysis time, low mass

detection limits, economy of reagents, andminimum sample requirements make

capillary electrophoresis an attractive

methodology for forensic laboratories.


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Capillary electrophoresis

Electrophoresis can been defined as the

differential migration ofcharged species (ions) in

an electric field, and was first described as aseparation technique by Tiselius in 1937.

His work, involving the separation of proteins,

placed between buffer solutions in a tube across

which an electric field was applied, earned him theNobel Prize for Chemistry in 1948.


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Electrophoresis using such media has

become a standard technique for size-

dependant separation of biomolecules.Separations carried out in this format are

characterised by long analysis times and

low efficiencies, when compared to otheranalytical separation techniques, such as

HPLC.


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Attempts to improve analysis time andefficiency in the slab or planar format by

increasing the voltage applied are limiteddue to the effects of Joule heating(Altria,1996).

This problem can be addressed by using

narrow-bore tubes or capillaries, whichallows rapid dispersion of any heatgenerated.


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electro-osmotic flow.

The term electro-osmotic flow (or electro-

endoosmotic flow) describes the movementof a conducting liquid against a charged

surface when an electric field is applied; this

was seen as a problem to be eliminated forfree-solution electrophoresis in tubes.


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Capillary electrophoresis is an instrumental

evolution of traditional slab gel

electrophoretic techniques (Tagliaro1998).Capillary electrophoresis has shown great

potential in a range of applications, such as

the separation of small ions to drug analyis.


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Separation modes

Capillary electrophoresis involves theseparation of charged analytes, based on

the difference in their electrophoreticmobilities, resulting in different migrationvelocities.

These separations are carried out in fused

silica capillaries, typically 2575 m i.d. and50100 cm in length, filled with abackground electrolyte (Heiger,1992).


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capillary

zone electrophoresis (CZE)Electroosmotic flow can ensure that both

negatively and positively charged species

migrate towards the same end of thecapillary, where under typical conditions, is

towards the cathode end, with neutral

species not being separated and migratingwith the electro-osmotic flow.


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The key factor effecting selectivity, when

using CZE for separations, is charge to size

ratio and pH.The later parameter will determine the

degree of ionisation for moderate and

weakly basic, or moderate and weaklyacidic analytes.


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The background electrolyte requires a good buffering

capacity at a chosen pH forreproducible separations,

and low conductivity not to generate a high current,

which leads to excessive Joule heating.

The most commonly employed background

electrolytes have been derived from the large body of

work with gel electrophoresis, and include phosphate,

borate, phosphate/borate, and citrate buffers.


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micellar

electrokinetic chromatography (MEKC)This combination of electrophoresis and

chromatography allows for the separation of

both neutral and charged solutes(Nishi,1996).

It is achieved by the addition of surfactants

to the background electrolyte atconcentrations greater than the critical

micelle concentration (CMC).


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Separation is based upon interaction of the

analytes with the micelles, which can be

considered as a pseudostationary phase(Tagliaro et al.,,2000).

The nature of the interaction between the

solute and micelle can be altered by usingdifferent types of surfactants.


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The most commonly employed surfactants

are sodium dodecyl sulphate (SDS), bile

salts, and quaternary ammonium salts [3].The presence of organic solvents, such

as methanol and acetonitrile may be used

as organic modifiers to alter the selectivity ofa run.


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Applications

The application of capillary electrophoresis

to the forensic analysis of drugs can be

divided into two main areas:

the analysis of drug seizures

toxicology


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Forensic analysis of drug seizures and

non-biological samplesCapillary electrophoresis has been

successfully applied to the determination of

various analytes in drug seizure samplesusing UV, fluorescence, and laser-induced

fluorescence (LIF) methods of detection,

which have been summarised in Table 1.


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Forensic toxicology and biological samples

Capillary electrophoresis has been used toidentify many drugs in a variety of biological

samples.Blood and urine serve most frequently assources of biological specimens for analysis

analysis can be extended to otherspecimens, such as saliva, vitreous humor,hairetc.


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Capillary electrophoresis has been

successfully applied to the determination

of various analytes in biological samples,using UV and fluorescence spectroscopy

methods of detection, which have been

summarised in Table 2.


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Analysis of Amphetamines in Urine

and Blood by Capillary

Electrophoresis with Photodiode

Array Detection


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In Taiwan, illegal drugs are not always the

same regulation concerning forprocession and use.

Such as methamphetamine (MA), it couldbecome similar structure compounds in

human metabolism.


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Amphetamine-type stimulants ( ATS )

include amphetamine (A),methamphetamine (MA),

methylenedioxy-amphetamine (MDA),

methylenedioxy-methamphetamine(MDMA).

CE method is powerful to analyze ATS .


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Fig.1 Amphetamine-type structure .


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CE: Beckman Proteome Lab PA 800

Detector : Photo Diode Array (PDA

190600 nm) and detector at 200 nm.

Capillary Tubing: 50m*50.2cm

Cartridge Temp : 20

Sample storage Temp : 10

Electropherogram scan range:

190~300 nm

CE Analysis


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CE Time Program

1.Rinse-pressure : 15.0 psi 3.0 min. with Tris(hydroxymethyl) aminomethane buffer (pH 2.5)

2.Inject- pressure: 1.0 psi 10.0 sec.

3.Autozero

4.Separate-Voltage: 20.0 KV 12.0 min.5.Stop data

6.Rinse-pressure : 15.0 psi 2.0 min. with 0.1N NaOH

7.Rinse-pressure : 15.0 psi 2.0 min. with D-D water

8.End


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Drugs-Urine Drugs-Blood Drugs-M/H solution

200l

Sat. K2CO3

20l Phosphoric

acid (dil. 1/5X)

2ml Hexane/ CH2Cl2solution (3:1 v/v)

2ml Acetonitrile

3500 rpm, 15min 3000rpm, 10min

Evaporated to dryness with N2 and

add 100l methanol/water (1:1 v/v)Analyzed by CE

1 min on a vortex mixer


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Fig.2 The drug in M/H solution that are the same migration

time in different concentration standard compounds.

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(MDMA)(MDA)

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Fig.3 The drug in urine that are the same migration time indifferent concentration standard compounds.

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)

(MDA)

(MA

)

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Fig.4 The drug in blood that are the same migration

time in different concentration standard compounds.

(A

)

(MDMA)(MDA)

(MA)

( ) f


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Table.1 Migration time (min) of amphetamine,

methamphetamine, MDA, MDMA and MDEA from M/H, urine

and blood.

Migration time (min)Drugs-M/H Drugs-urine Drugs-blood

Amphetamine 7.19 7.29 7.43

Methamphetamine

7.83 8.65 8.72

MDA 8.32 8.97 9.28

MDMA 8.64 9.19 9.49

MDEA 9.72 9.96 9.96


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Fig.5 Mixture of A, MA, MDA, MDMA and MDEA in one

separation step: in M/H (1:1) solution (A), urine (B) and

blood (C).

(A

)

(C)

(B

)

A MA

MDA

MDMAMDEA

(B

)

(C)

(A

)

A

MA

MDAMDMA

MDEA


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Table.3 Correlation coefficiency of standard curve of

amphetamine, methamphetamine, MDA, MDMA and

MDEA from M/H solution.

R CV(%)

Amphetamine 0.9989 4.38

Methamphetamine 0.9992 3.83

MDA 0.9998 3.89

MDMA 0.9998 4.25

MDEA 0.9963 3.13


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amphetamine, methamphetamine, MDA, MDMA and

MDEA from urine.

R CV(%)



MDA 0.9987 4.71

MDMA 0.9987 8.09

MDEA 0.9963 4.94

Table 5 Correlation coefficiency of standard curve of


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amphetamine, methamphetamine, MDA and MDMA

from blood.

R CV(%)Amphetamine 0.9960 2.64


MDA 0.9977 4.82

MDMA 0.9993 3.97

MDEA 0.9967 6.42

T bl 6 R (%) f h t i


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Table.6 Recovery (%) of amphetamine,

methamphetamine, MDA and MDMA from M/H solution,

urine and blood .

Recovery (%)

Drugs-M/H Drugs-urine Drugs-blood

Amphetamine 97.70 91.30 102.50

Methamphetamine 102.50 106.60 106.00

MDA 102.40 91.30 106.25

MDMA 98.98 100.25 102.50

MDEA 105.07 101.59 100.53


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Table.7 Limit of detection of drugs (g/ml).

LODDrugs-M/H Drugs-urine Drugs-blood

Amphetamine 0.10 0.10 0.10

Methamphetamine 0.10 0.10 0.20

MDA 0.10 0.10 0.10

MDMA 0.10 0.10 0.10

MDEA 0.10 0.10 0.50


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Clean electropherograms were obtained

showing symmetric electropherographicpeaks, and all drugs were resolved inblood and urine.

The CE method providing reproducibleresults of Amphetamines in blood andurine sample is developed recently.


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The CE method is able to analysis different drugs inone separation step through using uncoatedfused-silica capillary and Tris buffer.

The CE method may be applied for analysis ofATS in clinical and forensic samples.


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Chiral Separation of Amphetamine

and Ephendrine in Urine byCapillary Electrophoresis with

Photodiode Array Detection


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The increasing distribution of amphetamine (A),

methamphetamine (MA), and their

methylenedioxy-derivatives, such asmethylenedioxy-amphetamine (MDA),

methylenedioxy-methamphetamine (MDMA) and

methylenedioxyethyl-amphetamine (MDEA), has

became a serious social problem in Taiwan.

Ephedrine (E) and pseudoephedrine (PE) are

common over-the-counter (OTC)pharmaceuticals and are also frequently used as

adulterants in packing drugs of abuse.


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All of these amphetamine-type stimulants(ATS) are chiral drugs and one of the twoenantiomers is pharmacologically more activethan the other.

The S-(+) enantiomers of A and MA have

approximately five-fold more psychostimulantactivity than the R-() enantiomers, and wouldbe useful in forensic analysis to identify thesynthetic pathways.


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1R,2S-()-E is a popular precursor for the

illicit manufacture ofS-(+)-MA.

The most common manufacturing method

of illicit S-(+)-MA is the reduction ofR-()-E, which is extracted from natural

resources in the form of the ephedra plant

(Ma Huang).


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Tris-phosphate buffer (pH 2.5) was used for the

separation of compounds related toamphetamine and ephedrine (A, MA, MDA,

MDMA, MDEA, E, PE and cathinone by CE with

photodiode array detection (DAD).

CE method using 2-hydroxylpropyl- -cyclodextrin (OHP- -CD) as a chiral selector fordetermination of the enantiomers of the

compounds studied.


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All experiments were performed on a

Beckman ProteomeLab PA 800capillary electrophoresis system

(Beckman Coulter, Fullerton, CA, USA)

equipped with a photodiode-array

detector (DAD) (190600 nm) anddetection at 200 nm.


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All electrophoretic runs were carried out

using fused-silica capillaries of 50- minternal diameter, with a total length of 50

cm (40 cm to detector).

Samples were injected by positive pressure

of 1 psi for 10 s; a constant voltage of 20

kV was applied (current approx. 4045

A) and the temperature of the cartridge

was maintained at 20 C.

Each reference solution was prepared from each


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Each reference solution was prepared from eachstandard stock solution in methanol/water (1:1v/v) and then stored at 20 C.

Drug-spiked urine was made alkaline by the

addition of 20 l of saturated K2CO

3and was

extracted with 2 ml of hexane/CH2Cl

2solution

(3:1 v/v).

For chiral separation of the samples, 20 mM

OHP- -CD was dissolved in the same runningbuffer.

0.200 0.200


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Fig. 1 Electropherograms of eight amphetamines and ephedrines with DADdetection. (1) A, (2) MA, (3) MDA, (4) MDMA, (5) MDEA, (6) E, (7) PE,and (8) cathinone.

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(8)

(7)

(6)

(5)

(4)

(3)

(2)

(1)


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Table. 1 Repeatability of instrument for the analyzed amphetaminesand ephdrines.

Analyte migration time SD (%)

Cathinone 7.186 0.232


MA 7.533 0.261

MDA 7.833 0.253

MDMA 8.042 0.242

Pseudoephedrine 8.059 0.239

Ephedrine 8.208 0.233

MDEA 8.613 0.241

Mean of 5 replicate sample injections.

0.08 0.08


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Fig. 2 Separation of amphetamine and related compounds in a mixture by CE-

DAD detection. (1) A, (2) MA, (3) MDA, (4) MDMA, (5) MDEA, (6) E,

(7) PE, and (8) cathinone.

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Fig. 3 Capillary electrophoretic enantioresolution of the amphetamines andephedrines studied with DAD detection. (1) A, (2) MA, (3) MDA, (4)MDMA, (5) MDEA, (6) E, (7) PE, and (8) cathinone. Running buffercontaining 20 mM OHP- -CD.

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0.08 0.08


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Fig. 5 Electropherograms of a mixture of (1)A, (2)MA, (3)MDA, (4)MDMA,

(5)MDEA, (6)E, (7)PE and (8)cathinone after liquid/liquid extraction of

blank urine fortified with 10 g/ml of each compounds.

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Fig. 6 Electropherograms of a mixture of A, MA, MDA, MDMA, MDEA, E, PE and

cathinone after liquid/liquid extraction of blank urine fortified with 10 g/ml of eachenantiomer. The CE buffer with 20 mM OHP- -CD.

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S-(-)-cathinoneR-(+)-cathinone

R,R-(-)-PE

R-(+)-A S-(-)-A

1S,2R-(+)-E

1R,2S-(-)-E

R-(+)-MA

S-(-)-MA

S,S-(+)-PE

R-(+)-MDA

S-(-)-MDA

R-(+)-MDMA

S-(-)-MDMA

R-(+)-MDEA

S-(-)-MDEA

The CE DAD method using a buffer of 100 mM


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The CE-DAD method, using a buffer of 100 mMTris base adjusted to pH 2.5 with phosphoricacid, constitutes a reliable and practical

approach to the analysis of amphetamines andephedrines.

Application of OHP- -CD was suitable for theenantiomer separation of all eight compoundsexamined.

The method was useful for the analysis of urinesamples.

(d)7-10,11l ce for amphetamines 200807

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