200807Chen

 This

has traditionally been carried out using gas chromatography (GC) and highperformance liquid chromatography (HPLC).

200807Chen

 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 separation where the analyte in question behaves poorly under GC analysis.
200807Chen 3

Figure 30.3. A CE system.

200807Chen

 Capillary

zone electrophoresis (CZE), in which ionic species are separated according to their mobility and polarity in aqueous solution, is the most frequently used option in CE.

200807Chen

Figure 30.1. CZE separation of sulfonamides. Separation of twenty compounds, seventeen sulfonamides, 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.

200807Chen

 The

term capillary electrophoresis describes a family of related techniques in which separations are carried out in arrow bore capillaries under the influence of an electric field. The separations obtained by capillary electrophoresis are highly efficient, rapid, and may be applied to both charged and neutral species.
200807Chen 7

 Anon-line

search of the Scifinder Scholar database revealed in excess of 30,000 eferences to capillary electrophoresis to date with applications in a wide range ofareas, including pharmaceuticals, food and beverages, environmental and clinical analysis.
8

200807Chen

 The

potential of this technique for forensic analysis was first demonstrated in 1991 by Weinberger and Lurie, who applied it to the analysis of a wide range of illicit drugs in synthetic mixtures.

200807Chen

 The

exceptional power of separation and resolution, rapid analysis time, low mass detection limits, economy of reagents, and minimum sample requirements make capillary electrophoresis an attractive methodology for forensic laboratories.

200807Chen

10

Capillary electrophoresis
Electrophoresis can been defined as the differential migration of charged species (ions) in an electric field, and was first described as a separation 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 the Nobel Prize for Chemistry in 1948.

200807Chen 11

 Electrophoresis

using such media has become a standard technique for sizedependant separation of biomolecules. Separations carried out in this format are characterised by long analysis times and low efficiencies, when compared to other analytical separation techniques, such as HPLC.
200807Chen 12

 Attempts

to improve analysis time and efficiency in the slab or planar format by increasing the voltage applied are limited due to the effects of Joule heating (Altria,1996). This problem can be addressed by using narrow-bore tubes or capillaries, which allows rapid dispersion of any heat generated.
200807Chen 13

electro-osmotic flow. The term electro-osmotic flow (or electroendoosmotic flow) describes the movement of a conducting liquid against a charged surface when an electric field is applied; this was seen as a problem to be eliminated for free-solution electrophoresis in tubes.
200807Chen 14

 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.

200807Chen

15

Separation modes
Capillary

electrophoresis involves the separation of charged analytes, based on the difference in their electrophoretic mobilities, resulting in different migration velocities. These separations are carried out in fused silica capillaries, typically 2575 m i.d. and 50100 cm in length, filled with a background electrolyte (Heiger,1992).
200807Chen 16

capillary zone electrophoresis (CZE)

Electroosmotic

flow can ensure that both negatively and positively charged species migrate towards the same end of the capillary, where under typical conditions, is towards the cathode end, with neutral species not being separated and migrating with the electro-osmotic flow.
17

200807Chen

 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 weakly acidic analytes.
200807Chen 18

The background electrolyte requires a good buffering capacity at a chosen pH for reproducible 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.

200807Chen

19

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 at concentrations greater than the critical micelle concentration (CMC).
200807Chen 20

 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 using different types of surfactants.
200807Chen 21

 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 of a run.
200807Chen 22

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

200807Chen

23

Forensic analysis of drug seizures and non-biological samples

Capillary

electrophoresis has been successfully applied to the determination of various analytes in drug seizure samples using UV, fluorescence, and laser-induced fluorescence (LIF) methods of detection, which have been summarised in Table 1.

200807Chen

24

Forensic toxicology and biological samples

Capillary

electrophoresis has been used to identify many drugs in a variety of biological samples. Blood and urine serve most frequently as sources of biological specimens for analysis analysis can be extended to other specimens, such as saliva, vitreous humor, hair etc.
200807Chen 25

 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.

200807Chen

26

Analysis of Amphetamines in Urine and Blood by Capillary Electrophoresis with Photodiode Array Detection

200807Chen

27

 In

Taiwan, illegal drugs are not always the same regulation concerning for procession and use. as methamphetamine (MA), it could become similar structure compounds in human metabolism.

Such

200807Chen

28

 Amphetamine-type

stimulants ( ATS ) include amphetamine (A), methamphetamine (MA), methylenedioxy-amphetamine (MDA), methylenedioxy-methamphetamine (MDMA). method is powerful to analyze ATS .

CE

200807Chen

29

Fig.1 Amphetamine-type structure

200807Chen

.
30

CE Analysis

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
31

200807Chen

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

200807Chen

32

Drugs-Urine
200l Sat. K2CO3 2ml Hexane/ CH2Cl2 solution (3:1 v/v)

Drugs-Blood
20l Phosphoric acid (dil. 1/5X)

Drugs-M/H solution

2ml Acetonitrile

1 min on a vortex mixer 3500 rpm, 15min 3000rpm, 10min Analyzed by CE

33

200807Chen

Evaporated to dryness with N2 and add 100l methanol/water (1:1 v/v)

0.225

0.225

0.25

0.25

0.200

0.175

(A)

0.200 0.20 0.175

(MA)

0.20

0.150

0.150 0.15 0.15

0.125

0.125

AU

AU

AU

0.100

0.100

0.10 0.075 0.075

0.10

0.050

0.050

0.05

0.05

0.025

0.025

0.000

0.000

0.00

0.00

-0.025 0 1 2 3 4 5 6 7 8 9 10 11

-0.025 12 0 1 2 3 4 5 6 7 8 9 10 11 12

Minutes

Minutes

0.35

0.35

0.40

0.40

0.30

0.25

0.20

(MDA )

0.30

0.35

0.30 0.25 0.25 0.20

(MDMA)

0.35

0.30

0.25

AU

AU

AU

0.15

0.15 0.15 0.15

0.10

0.10

0.10

0.10

0.05

0.05

0.05

0.05

0.00

0.00

0.00

0.00

10

11

12

10

11

12

Minutes

Minutes

Fig.2 The drug in M/H solution that are the same migration time in different concentration standard compounds.
200807Chen 34

AU

0.20

0.20

AU

0.10

0.08

(A )

0.10 0.35

0.08

0.30

0.25 0.06 0.20

(MA (MA) )

0.35

0.30

0.25

0.06

0.20
AU

AU

AU
0.15

AU

0.04

0.04

0.15

0.10 0.02 0.02 0.05

0.10

0.05

0.00

0.00

0.00

0.00

10

11

12

10

11

12

Minutes
0.08 0.08 0.225

Minutes
0.225

0.200 0.07

0.06

(MDA)

0.07 0.175 0.06 0.150

(MDMA)

0.200

0.175

0.150

0.05

0.05 0.125 0.125

AU

AU

0.04

0.04

AU

0.100

0.100

0.03

0.03 0.075 0.075

0.02

0.02

0.050

0.050

0.01

0.01

0.025

0.025

0.00

0.00

0.000

0.000

-0.025 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11

-0.025 12

Minutes

Minutes

Fig.3 The drug in urine that are the same migration time in
different concentration standard compounds.
200807Chen 35

AU

0.6

0.6

0.8

0.8

0.5

0.4

(A )

0.5

0.7

0.6 0.4 0.5

(MA)

0.7

0.6

0.5

AU

AU

AU

0.3 0.2 0.2 0.2 0.1 0.1 0.1

0.3

0.2

0.1

0.0

0.0

0.0

0.0

10

11

12

10

11

12

Minutes
1.0

Minutes
1.0

1.0

(MDA)

1.0

0.8
0.8

0.8

(MDMA)

0.8

0.6
0.6 0.6

0.6

AU

AU

AU

0.4
0.4 0.4

0.4

0.2

0.2

0.2

0.2

0.0

0.0

0.0

0.0

10

11

12

Minutes

10

11

12

Minutes

Fig.4 The drug in blood that are the same migration time in different concentration standard compounds.
200807Chen 36

AU

AU

0.3

0.3

0.4

0.4

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 Methamphetamin e MDA MDMA MDEA
200807Chen

7.19 7.83 8.32 8.64 9.72

7.29 8.65 8.97 9.19 9.96

7.43 8.72 9.28 9.49 9.96

37

MDA MDA MDMA MDMA MA MDEA MDEA A MA A

(A (A )) (B (B )) (C) (C)

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). 200807Chen

38

Table.3 Correlation coefficiency of standard curve of amphetamine, methamphetamine, MDA, MDMA and MDEA from M/H solution.

R
Amphetamine Methamphetamine MDA MDMA MDEA
200807Chen

CV(%) 4.38 3.83 3.89 4.25 3.13

39

0.9989 0.9992 0.9998 0.9998 0.9963

Table.4 Correlation coefficiency of standard curve of amphetamine, methamphetamine, MDA, MDMA and MDEA from urine.

R
Amphetamine Methamphetamine MDA MDMA MDEA
200807Chen

CV(%) 5.41 6.14 4.71 8.09 4.94

40

0.9995 0.9996 0.9987 0.9987 0.9963

Table.5 Correlation coefficiency of standard curve of amphetamine, methamphetamine, MDA and MDMA from blood.

R
Amphetamine Methamphetamine MDA MDMA MDEA
200807Chen

CV(%) 2.64 4.84 4.82 3.97 6.42

41

0.9960 0.9984 0.9977 0.9993 0.9967

Table.6 Recovery (%) of amphetamine, methamphetamine, MDA and MDMA from M/H solution, urine and blood .

Recovery (%)
Drugs-M/H Amphetamine Methamphetamine MDA MDMA MDEA
200807Chen

Drugs-urine Drugs-blood

97.70 102.50 102.40 98.98 105.07

91.30 106.60 91.30 100.25 101.59

102.50 106.00 106.25 102.50 100.53

42

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

LOD
Drugs-M/H Amphetamine Methamphetamine MDA MDMA MDEA
200807Chen

Drugs-urine Drugs-blood

0.10 0.10 0.10 0.10 0.10

0.10 0.10 0.10 0.10 0.10

0.10 0.20 0.10 0.10 0.50

43

 Clean

electropherograms were obtained showing symmetric electropherographic peaks, and all drugs were resolved in blood and urine. CE method providing reproducible results of Amphetamines in blood and urine sample is developed recently.

The

200807Chen

44

 The

CE method is able to analysis different drugs in one separation step through using uncoated fused-silica capillary and Tris buffer. CE method may be applied for analysis of ATS in clinical and forensic samples.

The

200807Chen

45

Chiral Separation of Amphetamine and Ephendrine in Urine by Capillary Electrophoresis with Photodiode Array Detection

200807Chen

46

The increasing distribution of amphetamine (A), methamphetamine (MA), and their methylenedioxy-derivatives, such as methylenedioxy-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.
47

200807Chen

 All

of these amphetamine-type stimulants (ATS) are chiral drugs and one of the two enantiomers is pharmacologically more active than the other. S-(+) enantiomers of A and MA have approximately five-fold more psychostimulant activity than the R-() enantiomers, and would be useful in forensic analysis to identify the synthetic pathways.

The

200807Chen

48

 1R,2S-()-E

is a popular precursor for the illicit manufacture of S-(+)-MA. most common manufacturing method of illicit S-(+)-MA is the reduction of R-()E, which is extracted from natural resources in the form of the ephedra plant (Ma Huang).

The

200807Chen

49

Several methods are used for separation of amphetamine-like drugs, such as highperformance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and capillary electrophoresis (CE). Capillary electrophoresis (CE) has been reported as a powerful tool for a chiral and chiral separations and offers a number of distinct advantages, such as high resolution, speed of analysis and the feasibility of method development.
50

200807Chen

Tris-phosphate buffer (pH 2.5) was used for the separation of compounds related to amphetamine 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 for determination of the enantiomers of the compounds studied.

200807Chen

51

 All

experiments were performed on a Beckman ProteomeLab PA 800 capillary electrophoresis system (Beckman Coulter, Fullerton, CA, USA) equipped with a photodiode-array detector (DAD) (190600 nm) and detection at 200 nm.

200807Chen

52

 All

electrophoretic runs were carried out using fused-silica capillaries of 50- m internal 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.

200807Chen

53

Each reference solution was prepared from each standard stock solution in methanol/water (1:1 v/v) and then stored at 20 C. Drug-spiked urine was made alkaline by the addition of 20 l of saturated K2CO3 and was extracted with 2 ml of hexane/CH2Cl2 solution (3:1 v/v). For chiral separation of the samples, 20 mM OHP- -CD was dissolved in the same running buffer.

200807Chen

54

0.200

0.200

0.175

0.175

0.150

(1) (2) (3)

0.150

0.125

0.125

0.100

0.100

(4)
0.075

AU

(5) (6) (7) (8)

0.075

0.050

0.050

0.025

0.025

0.000

0.000

6 Minutes

10

11

12

Fig. 1 Electropherograms of eight amphetamines and ephedrines with DAD detection. (1) A, (2) MA, (3) MDA, (4) MDMA, (5) MDEA, (6) E, (7) PE, and (8) cathinone.
200807Chen 55

AU

Table. 1 Repeatability of instrument for the analyzed amphetamines and ephdrines.

Analyte Cathinone Amphetamine MA MDA MDMA Pseudoephedrine Ephedrine MDEA migration time 7.186 7.188 7.533 7.833 8.042 8.059 8.208 8.613 SD (%) 0.232 0.235 0.261 0.253 0.242 0.239 0.233 0.241

Mean of 5 replicate sample injections.

200807Chen 56

0.08

0.08

3
0.07

4 5
0.07

1+8
0.06 0.06

0.05

0.05

0.04 AU

0.04

7
0.03

0.03

6
0.02

0.02

0.01

0.01

0.00

0.00

5.0

5.5

6.0

6.5

7.0

7.5

8.0 Minutes

8.5

9.0

9.5

10.0

10.5

11.0

Fig. 2 Separation of amphetamine and related compounds in a mixture by CEDAD detection. (1) A, (2) MA, (3) MDA, (4) MDMA, (5) MDEA, (6) E, (7) PE, and (8) cathinone.
200807Chen 57

AU

0.16

0.16

0.14

(1) (2) (3) (4)

0.14

0.12

0.12

0.10

0.10

AU

(5)
0.06 0.06

(6)
0.04 0.04

(7)
0.02 0.02

0.00

(8)

0.00

-0.02 0 2 4 6 8 10 Minutes 12 14 16 18 20

-0.02

Fig. 3 Capillary electrophoretic enantioresolution of the amphetamines and ephedrines studied with DAD detection. (1) A, (2) MA, (3) MDA, (4) MDMA, (5) MDEA, (6) E, (7) PE, and (8) cathinone. Running buffer containing 20 mM OHP- -CD.
200807Chen 58

AU

0.08

0.08

0.05

0.05

0.04

0.02

0.01

0.00

10

11

12

Fig. 4 Electropherogram of a mixture of standards of racemic A, MA, MDA, MDMA, MDEA, E, PE and cathinone (approx. 20 g/ml of each enantiomer).
200807Chen 59

S,S-(+)-PE

S-(-)-MA R-(+)-MA R,R-(-)-PE R-(+)-A S-(-)-A

AU

0.02

0.01

0.00

13 Minutes

14

15

16

17

18

19

AU

1R,2S-(-)-E

0.03

S-(-)-MDEA S-(-)-MDMA R-(+)-MDEA R-(+)-MDMA R-(+)-MDA S-(-)-MDA

1S,2R-(+)-E

S-(-)-cathi R-(+)-cathinonenone

0.04

0.03

0. 08

0.08

0. 07

0.07

0. 06

0.06

5
0.05

0. 05

0. 04 AU

0.04 AU

1+8
0. 03 0.03

0. 02

7 6

0.02

0. 01

0.01

0. 00

0.00

5. 0

5.5

6.0

6.5

7. 0

7.5

8.0 Minut es

8. 5

9. 0

9.5

10.0

10.5

11. 0

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.
200807Chen 60

0.05

0.05

0.04

0.04

0.03

AU

0.02

0.02

0.01

0.01

0.00

0.00

10

11

12

13 Minutes

14

15

16

17

18

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 each enantiomer. The CE buffer with 20 mM OHP- -CD.
200807Chen 61

AU

S-(-)-MDMA S-(-)-MDEA R-(+)-MDMA R-(+)-MDEA S-(-)-MDA R-(+)-MDA

0.03

S,S-(+)-PE

R-(+)-MA S-(-)-MA

1R,2S-(-)-E 1S,2R-(+)-E R-(+)-A S-(-)-cathinone S-(-)-A R-(+)-cathinone R,R-(-)-PE

The CE-DAD method, using a buffer of 100 mM Tris base adjusted to pH 2.5 with phosphoric acid, constitutes a reliable and practical approach to the analysis of amphetamines and ephedrines. Application of OHP- -CD was suitable for the enantiomer separation of all eight compounds examined. The method was useful for the analysis of urine samples.

200807Chen

62