Application of CE in forensic analysis

Michał Woźniakiewicz

Laboratory for Forensic Chemistry, Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University in Kraków

Jagiellonian University in Kraków

Aim of work

• Present the applicability of the capillary electrophoresis in criminalistics and forensic toxicology

• Demonstration of achievements in CE accomplished in the Laboratory for Forensic Chemistry at Jagiellonian University in Kraków

Electrophoresis

µe

q

Rη

Charge

Stoke’s radius

(~mass)

Viscosity

pH

Ionic

strength

𝜇𝑒 = 6𝜋𝑅𝜂

Surfactants

Organic modifiers

Chiral selectors

Electroosmosis

µEOF

ξ

εη

𝜇𝐸𝑂𝐹 =𝜀𝜉

𝜂

Surfactants

Organic modifiers

Capillary coating

- dynamic

- permanent

CE in forensic analysis

• Low sample consumption

▪ Possible to repeat measurements with other methods, e.g. chromatography

▪ Semi-destructive

• Easy tuning of the method to face a new problem

▪ Low cost of chemicals and consumables

▪ Chemical modification of background electrolyte less expensive than e.g. new columns

• Compatible with chromatography

▪ No special sample pre-treatment needed

CE in forensic investigation

• Questioned documents – inks and dyes

• Forensic toxicology

• Investigation of psychoactive plants and designer drugs

• Simulation of biotransformation

• Drug profiling

• Gun-shot residues

• Explosives and post-blast residues

• Determination of carbohydrate-deficient transferrin

• DNA

CE in forensic investigation

• Questioned documents – inks and dyes

• Forensic toxicology

• Investigation of psychoactive plants and designer drugs

• Simulation of biotransformation

• Drug profiling

• Gun-shot residues

• Explosives and post-blast residues

• Determination of carbohydrate-deficient transferrin

• DNA

Issues of capillary electrophoresis

• Poor repeatability of migration times (identification parameter)

• Poor repeatability of analytical signal (e.g. peak area)

• High detection limits

• No real application in forensic science –it is just a fancy tool for crazy scientists.

Questioned documents

Analysis of questioned documents

Covering materials- Ink jet printing inks and replacements

- Fountain-pen inks

- Ball point pens and gels

- Stamp inks

- toners

PigmentsDyes

1) dye, 2) paper

2

1

1) Pigment particles, 2) paper

CE techniques used in the analysis of questioned documents

• CE-DAD1

• CE-LIF2

• CE-MS3

Application of CE in questioned documents examination

Model samples - printouts

Analysis of black ink jet print-outs by MECC-DAD

• Requires extraction of 25 dots of 0.3 mm

• Repeatability of migration time CV<3% or CV<6% after capillary exchange

(a) Deskjet F4280

(b) Deskjet 3740

(c) Photosmart C4280

M. Król, A. Kula, R. Wietecha-Posłuszny, M. Woźniakiewicz, P. Kościelniak, Examination of black inkjet

printing inks by capillary electrophoresis (2012) Talanta 96, 236-242

Questioned invoice

• BGE: 40 mM borate buffer, 20 mM SDS and 10% ACN

• Capillary I.D.=75 μm, temperature: 25 °C

• Sample garage: 10 °C,

• Extracting solvent :DMSO

• Sample solution: BGE:water (1:1, v/v)

Original document

Questioned

document part

Questioned post stamp

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

mA

U

0

2

4

6

8

10

12

a

b

M

*

M

Y

C

C

1

3

6

24

5

7

Original stamp

Printout

HP Business Inkjet 1200

CE-MS

HV

Det.

-+

Sheath

liquidHV

+

MS

Pump

ESI

microTOF, Bruker

Mass calibration in CE-TOF-MS

• Sample injection

• Electrophoretic separation

• Rinsing – 0.1 M NaOH

• Rinsing – background electrolyte

Sheath

liquidHV

+

MS

Pump

ESI

Isopropanol:

0.1%formic acid

Mass calibration in CE-TOF-MS

• Sample injection

• Electrophoretic separation

• Rinsing – 1 M NaOH

• Rinsing – background electrolyte

Sheath

liquidHV

+

MS

Pump

ESI

Isopropanol:

0.1%formic acid

Mass calibration with

sodium formate

clusters

Mass calibration in CE-TOF-MS

Sheath

liquid

HV

+

TOF-MS

LC Pump

ESI

W

Syringe pump

calibrator

Sodium formate

clusters

Isopropanol:0.1%formic acid

Mass calibration in CE-TOF-MS

301.1424

362.9262

402.3566

430.9141

465.2208

498.9006

566.8892

579.2942

600.8826

634.8756

668.8696

702.8637

770.8508

0

1

2

3

5x10

Intens.

300 350 400 450 500 550 600 650 700 750m/z

36.5 37.0 37.5 38.0 38.5 39.0 Time [min]

0.25

0.50

0.75

1.00

1.25

1.50

6x10

Intens.

1:1 (v/v) isopropyl alcohol/water containing

0.2% (v/v) formic acid (as sheath liquid) and sodium

hydroxide (from CE)

Analysis of black ink jet print-outs by CE-MS

50 mM Ammonium Acetate

0.1 Acetic acid

25% (v/v) ACN, pH*=5.31

5 mM Ammonium Acetate

0.1 Acetic acid

5% (v/v) ACN, pH*=4.23

Kula, A., Król, M., Wietecha-Posłuszny, R., Woźniakiewicz, M., Kościelniak, P., Application of CE-MS to

examination of black inkjet printing inks for forensic purposes, Talanta 128 (2014) 92-101.

5 mM Ammonium Acetate

0.01 Acetic acid

25% (v/v) ACN, pH*=5.41

Differentiation of polymer additives in ink jet printing inks

Hewlett Packard

Brother

Canon

Analysis of black ink jet print-outs by CE-MS

• Increasing sensitivity and reduction of sample size (10 dots of 0.63 mm)

• Increasing selectivity

• Detection of UV-transparent analytes (e.g. polyethylene glycol)

• Repeatability of migration time CV<6%)

Canon I 965

Canon MP 240

Canon IP 1900

Kula, A., Król, M., Wietecha-Posłuszny, R., Woźniakiewicz, M., Kościelniak, P., Application of CE-MS to

examination of black inkjet printing inks for forensic purposes, Talanta 128 (2014) 92-101.

Analysis of dyes extracted from fibers from Krakow Location Act (1257)

S1 S4S3

S2

Photographer: Andrzej Banaś

Source: http://krakow.naszemiasto.pl/galeria/opis/1888330,zobacz-akt-lokacyjny-krakowa-zdjecia,galeria,id,t,tm.html

carminic acid

?

BrnoLarge and small city privileges (1243)

CE-ESI-MS-TOF analysis of dyes extracted from fibres

m/z Molecular formulaChemical

compoundMigration time

[min]:

Sample

1 2 3 4

Ions

241.0483 C14H8O4 alizarine 10 + + + +

317.0552 - m.c.p. 17 + + + +

334.0820 - m.c.p. 17 + + + +

455.1182 - m.c.p. 24 - + - -

477.0994 - m.c.p. 24 - + - -

499.0802 - m.c.p. 24 - + - -

293.0975 C10H16N2O2 m.c.p. 27 + + + +

315.0797 C8H10N8O6 m.c.p. 27 + + + +

331.0463 C16H10O8 kermesic acid 27 + + + +

354.0127 - m.c.p. 28 + + + +

493.0997 C22H20O13 carminic acid 16 - - - -

303.0861 C16H14O6 hematoxylin 10 - - - -

301.0740 C16H12O6 hematein 10 - - - -

m.c.p. – many compounds are possible

Photographer: Andrzej Banaś

Source: http://krakow.naszemiasto.pl/galeria/opis/1888330,zobacz-akt-lokacyjny-krakowa-zdjecia,galeria,5666856,id,t,tm,zid.html

S2

Analysis of fibres extracts using CE-ESI-TOF-MS technique

in the negative ion mode confirmed the results.

Discrimination of inks by CE-LIF

Discrimination of red point ball gel pens by CE-LIF

• BGE: 40 mM borate buffer, 20 mM SDS and 10% ACN

• capillary I.D.=75 μm, temperature: 25 °C, LIF, αex =488 nm and 405 nm

• Extracting solvent :DMSO, Sample solution: BGE:water (1:1, v/v)

Minutes

1 2 3 4 5 6 7 8 9 10 11 12 13 14

RF

U

0

5

10

15

20

25

30

35

40

45

50

55

BL G1 5T R (Pilot)

V5 HI – TECPOINT (Pilot)

GEL SALSA (Handy)

Król, M., Gondko, K., Kula, A. and Kościelniak, P. (2016), Analysis of red inks by micellar electrokinetic

capillary chromatography with laser-induced fluorescence detection. ELECTROPHORESIS, 37: 372–380.

Discrimination of red stamps by CE-LIF

• 87% of comparisons are conclusive (discrimination power)

• Requires 20 dots of 0.5 mm

• Repeatability of migration times CV<0.8%

Król, M., Gondko, K., Kula, A. and Kościelniak, P. (2016), Analysis of red inks by micellar electrokinetic

capillary chromatography with laser-induced fluorescence detection. ELECTROPHORESIS, 37: 372–380.

Investigation of

psychoactive plants

Psychoactive Solanaceae plants

Solanaceae

(Nightshades)

Psychoactive substances:

Atropine

Scopolamine

tomato

pepper

potato

eggplant

Hallucinogenicplants

2,800 species of woody and herbaceous

plants

atropine

scopolamine

Psychoactive Solanaceae plants

Solanaceae

(Nightshades)

Psychoactive substances:

Atropine

Scopolamine

tomato

pepper

potato

eggplant

Hallucinogenic plants

2,800 species of woody and herbaceous

plants

Direct influence on the central nervous system → difficulty in breathing

and even death

Widely used in medicine in a controlled

dose

Deadly dose: approx. 100 mg for

an adult, a few milligrams for a

child

Psychoactive Solanaceae plants

Atropa belladonna L. Datura stramonium L.Brugmansia

(Angel`s Trumpet)

Hyoscyamus niger Scopolia carniolica

Ipomoea

Ipomoea

(Morning glories)

Psychoactive substances:

Ergine

Ergometrine

Lisergol

structural similarity to LSD

500 - 600 species

The largest genus in the clam family

(Convolvulaceae)

Psychoactive plants

Sweet potatoes (Ipomoea batatas)

Ergine (LSA)

Ergometrine

Ipomoea

Ipomoea

(Morning glories)

Psychoactive substances:

Ergine

Ergometrine

Lisergol

structural similarity to LSD

500 - 600 species

The largest genus in the clam family

(Convolvulaceae)

Psychoactive plants

Sweet potatoes (Ipomoea batatas)

Ergometrine:spasm of the blood vessels, uterus (can cause miscarriage)

Ergine:

extremeeuphoria, fatigue,

flashbacks

Lysergol:

Unprovenpsychoactive

activity

5 mM

CH3COONH4

+ CH3COOH

pH 8.0

20% MeOH

10 mM

HCOONH4

+ HCOOH

pH 4.5

20% MeOH

Real plant samples

LD LB NDMAE/QuEChERS/GC–MS

atropina skopolamina atropina skopolamina atropina skopolamina

stężenia wyznaczone 52,69 1350,02 50,38 821,52 3017,15 2431,15

stężenia z literatury 69,87 840,40 64,67 448,20 2788,00 2020,00

0,00

500,00

1000,00

1500,00

2000,00

2500,00

3000,00

Conce

ntr

ation

[µg/g

]

Tropane alkaloids

determined

Reference

Atropine Scopolamine Atropine Scopolamine Atropine Scopolamine

Real plant samples

Ipomoea Tricolor Ipomoea PurpureaUAE/LC-Q-TOF-MS

ergina ergometryna lizergol ergina ergometryna lizergol

stężenia wyznaczone 493,20 131,07 14,42 425,10 142,10 12,42

stężenia z literatury 297,00 40,00 300,00 50,00

0,00

100,00

200,00

300,00

400,00

500,00

stęże

nie

[µg/g

]

Ergot alkaloids

Determined

Reference

Ergine Ergometrine Lysergol Ergine Ergometrine Lysergol

Application of CE in forensic toxicological analysis

Application of CE in forensic toxicological analysis

• Tricyclic antidepressants (TCAs)

• Phenothiazine derivatives

ImipramineDoxepineNortriptyline DesipramineNordoxepine

N

N

CH3

H

N

N

CH3

CH3

O

N

CH3

H

N

CH3

CH3

O

N

CH3

CH3

N

CH3

H

Amitryptyline

Promazine Chlorpromazine Perazine Levomepromazine

S

N Cl

N

N

CH3

S

N

N

CH3

CH3

S

N Cl

N

CH3

CH3

S

N OCH3

N

CH3

CH3

CH3

S

N

N

CH3

SCH3

Thioridazine

Application of nonaqueous capillary electrophoresis (NACE)

5 6 7 8

0,000

0,002

0,004

0,006

0,008

0,010

Tfp

A [a

u]

t [min]

IS

Lev

Pro

ThiChlPrm Per

IS

IS

TfpLev

Pro

ThiChl

Prm Per

Standard drug mixture

Spiked blood (LL extraction)

Blank blood (LL extraction)

BGE: 20 mM CH3COONH4 in MeOH/ACN/CH3COOH (49:50:1)

Madej, K., Kala, M., Woźniakiewicz, M. (2005) LC and non-aqueous CE determination of

phenothiazines in autopsy samples, Chromatographia, 62: 533-538

Analysis of forensic autopsy samples

4 6 8

0,0004

0,0006

0,0008

0,0010

0,0012

0,0014

0,0016

0,0018

0,0020

Pro

A [A

]

t [min]

IS

PerPrzypadek II

Case II – complex

suicidal intoxication with

promazine and perazine

0,86

4,02

17,01

0,80

4,38

16,56

0

2

4

6

8

10

12

14

16

18

Promazine, Case I Promazine, Case II Perazine, Case II

C [u

g/m

l]

NACE HPLC

Madej, K., Kala, M., Woźniakiewicz, M. (2005) LC and non-aqueous CE determination of

phenothiazines in autopsy samples, Chromatographia, 62: 533-538

Blood screening for antidepressants and phenothiazines using NACE

• BGE: 20 mM CH3COONH4 in MeOH/ACN (1:1)

• Repeatability of relative migration time CV<1.4%

• Repeatability of relative peak area (independent extractions) CV<10%

• Limit of detection 0.15 µg/mL

Whole blood sample

extract

Standard mixture

Madej, K., Marczyk, A., Woźniakiewicz, M., Non-aqueous CE screening method for 14 psychotropic

drugs in whole blood samples, (2007) Chromatographia, 65 (5-6), pp. 313-317.

LL extraction

Blood screening for antidepressants and phenothiazines using NACE

• BGE: 20 mM CH3COONH4 in MeOH/ACN (1:1)

• Repeatability of relative migration time CV<1.4%

• Repeatability of relative peak area (independent extractions) CV<10%

• Limit of detection 0.15 µg/mL

Whole blood sample

extract

Standard mixture

Madej, K., Marczyk, A., Woźniakiewicz, M., Non-aqueous CE screening method for 14 psychotropic

drugs in whole blood samples, (2007) Chromatographia, 65 (5-6), pp. 313-317.

LL extraction

HPLC and NACE

• Quantification results were similar for both methods.

• Application of NACE resulted in significant reduction of analysis time (up to 3x, comparing to HPLC).

• Both HPLC and NACE were characterized by similar precision.

Madej, K., Kala, M., Woźniakiewicz, M., LC and non-aqueous CE determination of

phenothiazines in autopsy samples (2005) Chromatographia 62, 533-538

Analysis of hair and serum samples by MAE/CE-TOF-MS

• BGE: 100 mM acetic acid, 20% acetonitrile

• Detection limits: 0.4–1.2 ng/mL - serum, 6.0–23.0 pg/mg - hair

• Repeatability: 3.0–11.3% - serum, 2.4–14.2% - hair

A. Woźniakiewicz, R. Wietecha-Posłuszny, M. Woźniakiewicz, E. Bryczek, P. Kościelniak, A quick method for

determination of psychoactive agents in serum and hair by using capillary electrophoresis and mass

spectrometry (2015) Journal of Pharmaceutical and Biomedical Analysis, 111, 177-185.

Microwave-assisted

extraction

Analysis of hair samples using CE-TOF-MS

Case #1• 48-year-old male with dark hair

2 mg of clonazepam daily

• The found concentration:

• Clonazepam 136.7 2.8 pg/mg

• 7-aminoclonazepam 801.7 72.5pg/mg

Clo

Clo-d4

7-

aClo

7-aClo-d4

10 12 14 16 18Time [min]

1000

3000

5000

Inte

ns.

Clo

Clo-d4

7-aClo

7-aClo-d4

10 12 14 16 18 Time [min]

0.2

0.6

1.0

Inte

ns. x 1

04

Case #2• 53-year-old male with dark hair

4 mg of clonazepam per day

• The found concentration:

• Clonazepam 121.11.4 pg/mg

• 7-aminoclonazepam 1.770.02 ng/mg

Analysis of hair samples using CE-TOF-MS

Case #3

• 34-year-old male with dark hair, treated with 50 mg of tetrazepamdaily for two weeks. The sample was collected three times:

• 28 days (segment #1)

• 51 days (segment #2)

• 77 days (segment #3), after the last intakes.

11 12 130

2

4

6

Inte

ns. x 1

04

Time [min]

Tetr

Dia-

d5

• Segment #1

− 1.180.09 ng/mg

• Segment #2

− 8.530.53 ng/mg

• Segment #3

− 1.630.16 ng/mg

Chiral analysis of warfarin and its metabolites

A - warfarin

B – 7-OH-warfarin

ww

w.c

hro

mato

gra

phy-o

nlin

e.o

rg

Me-β-CD

Nowak, P., Garnysz, M., Woźniakiewicz, M., Koscielniak, P., Fast separation of warfarin and 7-

hydroxywarfarin enantiomers by cyclodextrin-assisted capillary Electrophoresis (2014) Journal of

Separation Science, 37 (18), 2625-2631.

Chiral analysis of warfarin and its metabolites

pH 5.8

pH 9.16

1% (w/v) Me-β-CD

• S and R isomers are differently metabolized – S isomer is 5x more potent

• Warfarin’s main metabolites are (S)-7-OH-warfarin, (R)-10-OH-warfarin

Investigation of psychochemical properties of designer dugs

• The ADME investigation describes the disposition of a chemical entities within the organism.

▪ Physicochemical properties (pKa, log P, log D, solubility, stability)

▪ Metabolism

▪ Protein binding

▪ Membrane permeability (e.g. parallel artificial membrane permeability assay, Caco-2 cells)

▪ Toxicity (genotoxicity, cytotoxicity, pro-apoptotic properties)

Investigation of psychochemical properties of designer dugs

• The ADME investigation describes the disposition of a chemical entities within the organism.

▪ Physicochemical properties (pKa, log P, log D, solubility, stability)

▪ Metabolism

▪ Protein binding

▪ Membrane permeability (e.g. parallel artificial membrane permeability assay, Caco-2 cells)

▪ Toxicity (genotoxicity, cytotoxicity, pro-apoptotic properties)

Designer drugs in Poland and European Union

Number of cathinones spoted for the first time in UE within 2009-2015

Number of drugs registered for the first time in UE in 2015

Number of suspected poisoning related to designer drugs per 100 thousand residents

4

14

74

7

31

26

0

5

10

15

20

25

30

35

2009 2010 2011 2012 2013 2014 2015

CE-based pKainvestigationof designer drugs

NH

O

NH

O

NH

O

O

N

O

N

OO

O

NH Cathinone

2-MMC

3-MMC

4-MMC

Ephedron

α-PVP

MDPV

O

NH2

pKa determination by capillary electrophoresis

pKa determinationby CE

Standard IS-based OVM TVM

pKa determination by CEStandard method

• Based on regression model describing relation between effective

electrophoretic mobility (µeff) and pH.

For monoprotic acids: For monoprotic bases :

pH

µeffpKa

65

𝜇𝑒𝑓𝑓 =𝜇𝐴− ∙ 10

−𝑝𝐾𝑎

10−𝑝𝐾𝑎 − 10−𝑝𝐻𝜇𝑒𝑓𝑓 =

𝜇𝐶+ ∙ 10−𝑝𝐻

10−𝑝𝐾𝑎 + 10−𝑝𝐻

pH

µeffpKa

Two values and One value methods

One-value method (OVM) Two-values method (TVM)

Aaeff pHpK

pH

1010

10

effA

eff

a pHpK

log

BA M

zA

160 170 180 190 200 210 220 230 240 250 260 270 280 290

14

15

16

17

18

19

20

c+ [10

-9m

2/Vs]

M [g/mol]

PVP

sV

m

Mc

29

468,010

125,2

6 7 8 9 10 11 12

0

4

8

12

16

e

ff [

10

-9m

2/ Vs

]

pH

T T T

P

PP

μA+

μeff

pKa determinationStandard method

2-MMC 8,59 ± 0,03

3-MMC 8,74 ± 0,04

4-MMC 8,78 ± 0,03

α-PVP 8,93 ± 0,02

MDPV 9,10 ± 0,02

Ephedron 8,77 ± 0,03

pKa determinationOne-value method

pKa determinationResults

8,5 8,5 8,5

8

7,9

8,5

8,59

8,748,78

9,1

8,93

8,77

8,58

8,71 8,71

9,14

9,01

8,72

8,638,7

8,74

9,15

9

8,748,69

7,2

7,4

7,6

7,8

8

8,2

8,4

8,6

8,8

9

9,2

9,4

2-MMC 3-MMC 4-MMC MDPV α-PVP Ephedron

pK

a

ACD/LABs I-Lab 2.0 Standard Method OVM TVM Reference

Reference: Santali et al., Synthesis, full chemical characterisation and development of validated methods for the quantification of (±)-4’-

methylmethcathinone (mephedrone): A new „legal high”, J. Pharm. Biomed. Sci. 56 (2011) 246-255.

NH

O

NH

O

NH

O

O

N

OO

O

N

O

NH

Acidic-base equilibrium and separation in CE

Ele

ctr

ophore

tic m

obili

ty

pH

Considering three isomeric amines𝑿 −𝑵𝑯𝟐 +𝑯+ ⇄ 𝑿−𝑵𝑯𝟑

+

Separation in optimal conditions

2,2 2,4 2,6 2,8 3,0 3,2 3,4 3,6 3,8 4,0 4,2

0

1

2

3

4

2,5

5

α-PVP

(2,71 min)

MDPV

(2,75 min)

DMSO

(3,71 min)

Absorb

ance [m

AU

]

t [min]

Ephedrone

(2,46 min)

2-MMC

3-MMC

4-MMC

2,6

0

pH=6.0

Ionic strength 50 mM

Determination of logP/logDby capillary electrophoresis

ps

obseof

eofobs

t

tt

ttk

1

bkaP loglog

k – retention factortobs – migration time of an analyteteof – EOF migration timetps –migration time of a marker

logP

T=const.

EOF

pHcs>CMC

Marker of pseudostationary

phase

Organic solvent

𝑙𝑜𝑔𝑃 = 𝑙𝑜𝑔D + log(1 + 10(𝑝𝐾𝑎−𝑝𝐻))

Instrumental setup and chemicals

Capillary electrophoresis system MDQ/PACE (Beckman-Coulter)

Capillary Fused silica,Ltot = 60 cm; Leff = 50 cm; ø = 75 μm

Voltage 30 kV (MEKC) i 12 kV (MEEKC)

Detection DAD, λ = 210 nm

BGE pH = 8.7 (MEKC)

pH = 6.9 (MEEKC)

Samples Investigated cathinones at concetration 50 μg/mL) DMSO – EOF marker (0.2% v/v) and α-

tocopherol (150 μg/mL)

Prepared in BGE

logP values were predicted using ACD/LABs I-Lab 2.0 software

Determination of logP/logD

1.5

3

2.1

9

2.1

8

2.2

2

3.1

5

3.2

4

1.9

1

2.4

7

2.5

5

2.5

5

2.8

8 3.0

2

1.3

9

1.8

6

1.8

6

1.8

6

3.6

5

3.0

6

3.2

7.2

8

7.9

7

8.1

5

20.4

4

30.3

2ephedron 2-MMC 4-MMC 3-MMC a-PVP MDPV

1.0

1.5

2.0

2.5

3.0

3.5

4.0

MEKC MEEKC logP ACD/Labs LC-MS

logD

/ logP

0

5

10

15

20

25

30

LC

-MS

rete

ntio

n tim

e [m

in]

Separation of NPSs by MEKC and MEEKC

MEKC

MEEKC

Ab

so

rba

nce [m

AU

]

Ab

so

rba

nce [m

AU

]

11.5 mM borax,

11 mM phosphoric

acid, 30 mM SDS

20% (v/v) isopropanol

3.88% (w/w) SDS

0.82% (w/w) heptan

6.49% (w/w) 1-butanol

Phosphoric buff. pH=7

Simulation of biotransformation in the CE system

• Electrophoretically mediated microanalysis as a cost and time effective method for investigation of drug metabolic pathways and enzymatic activity.

• Variety of modes:

▪ Pre-capillary (in vial)

▪ In-capillary

▪ Post –capillary

• Widely applied in studies of drugs of forensic interests (e.g. amphetamines) and other compounds

Cost-effective assessment of the activityof plant membrane enzyme chlorophyllase

• The seasonal loss of chlorophylls is the only biological process visible from the Space

Chlorophylls

Enzyme

Chlorophyllase

Chlorophillides

BGE: 12.5 mM borate buffer, pH 9.5, 0.4% LDAO

Capillary temperature: 37°C

Sample garage temperature: 37°C

Simulation of biotransformation in the CE system

• Low-cost simulation of biotransformation – limited use of expensive reagents

• Process automation

• Could be compatible with the mass spectrometry

In-vial In-capillary

Nowak, P., Michalik, M., Fiedor, L., Woźniakiewicz, M., Kościelniak, P., Capillary electrophoresis

as a tool for a cost-effective assessment of the activity of plant membrane enzyme chlorophyllase,

Electrophoresis 34 (2013) 3341-3344

Issues of capillary electrophoresis

Poor repeatability of migration times☺ Modern methods and systems provides CV<3 - 5%

☺ Application of internal standard - CV<1%

Poor repeatability of analytical signal☺ The precision of CE methods fulfils requirement for testing of

biological material – CV <15%

High detection limits☺ Coupling CE with sensitive detectors (MS, LIF)

☺ Tuning of the analytical methods

No real application in forensic science – it is just a fancy tool for crazy scientists.☺ CE is now introduced into the practice of forensic laboratories

☺ There are plenty new, scientific applications of the capillary electrophoresis.

Department of Analytical ChmistryFaculty of Chemistry

Prof. Paweł Kościelniak

Head of Department of Analytical Chemistry

Leader and Staff of Laboratory for Forensic Chemistry

Special thanks to

• Małgorzata Król, PhD

• Katarzyna Madej, PhD, DSc

• Renata Wietecha-Posłuszny, PhD, DSc

• Agnieszka Kula, PhD

• Aneta Woźniakiewicz, PhD

• Paweł Nowak, PhD

• Marta Gładysz, MSc

• Magdalena Snamina, MSc

• Małgorzata Gołąb, MSc

• Maria Dobek, MSc