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Application of capillary electrophoresis to the drug analysis of forensic interest

Katarzyna Madej, Micha Woniakiewicz, Pawe Kocielniak


Jagiellonian University, Faculty of Chemistry, Laboratory for Forensic Chemistry, Ingardena St. 3, Krakow, Poland
INTRODUCTION One of the main domain of forensic examinations is the analysis of drugs in biological samples - serum, plasma, whole blood, urine and in nonbiological materials - tablets, powders, liquids, food. Narcotic drugs such as amphetamines, heroine, cocaine, cannabinoids and some medicines which are often used and overdosed involving barbiturates, benzodiazepines, tricyclic antidepressants and phenothiazines are of special forensic interest. Up to now the most frequently utilized analytical technique for the determination of the mentioned above substances has been chromatography with its few variants (TLC, HPLC, GC). The alternative technique to the chromatography seems to be capillary electrophoresis (CE) which has a similar analytical potential. This technique is characterized by high resolution, fast speed and small samples and reagents consumption. Generally, the CE analysis consists in the separation of examined compounds in a capillary (20-100 m ID) under high electric field (up to 30 kV). CE can work in a few separation modes which are modification of the basic one - capillary zone electrophoresis (CZE). The aim of the work: investigations of the possibility of application of two CE modes: CZE and MECC (micellar electrokinetic capillary chromatography) to the analysis of phenothiazines (PHE) and tricyclic antidepressants (TCA) in the whole blood and pharmaceuticals. 11 commonly used psychotropic drugs were included in the examinations. Reagents CAPSO (3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid - Sigma, Germany) STDC (sodium salt of taurodeoxycholic acid - Sigma, Germany) methanol (HPLC gradient grade - Merck, Germany) 85 % ortophosphoric acid (analytical grade POCH, Gliwice) sodium hydroxide (analytical grade POCH, Gliwice) sodium tetraborate (analytical grade POCH, Gliwice) the deionized water Examined drugs and materials PHE: chlorpromazine, levomepromazine, perazine, promazine, thioridazine TCA: amitriptyline, imipramine, clomipramine, desipramine, doxepin, noxiptyline Blood was taken from the local blood bank (Krakw, Poland). The pharmaceutical preparations: Petylyl (Arzneimittelwerk, Dresden, Germany) and Anafranil SR (Novartis Pharma AG Bazylea, Switzerland) were bought in the Polish pharmacy. Preparation of samples for analysis E x p e r i m e n t a l Apparatus A Prince 550 air thermostated capillary electrophoresis system (Prince Technologies, Emmen, Holland) with Lambda 1010 spectrophotometer (Bishop, Leonberg, Germany) as UV-VIS detector was used. The separation of analytes was conducted at 30 kV in a bare fused silica capillary of 50 m ID and 100 cm/66cm length. Samples were injected into the capillary by hydrodynamic mode using the pressure of 100 mbar for 6 seconds. The detection of analytes was performed at 210 and 254 nm. One tablet of Petylyl and one tablet of Anafranil were pulverised (after removing their external layers) in agat mortar, dissolved in appropriate amount of methanol, ultrasonificated and filtered twice. Blood samples were spiked with methanolic drug standards at concentrations corresponding to the toxic and death levels of these drugs in blood, then they were extracted with hexane-isoamyl alcohol (99:1, v/v) from pH 12 solution and reextracted into 0.01% ortophosphoric acid.

RESULTS
CZE
S eparatio n o f 4 TCA and pheno tia zine s tandards : dez ypramine (DEZ), no rtrytpyline (NOR), c hlo rproma zine (CHPRO) and pro ma zine (PRO)
0,007 0,006

MECC
Tablet e xtrac t of anafranil (c lo mipramine ) with inte rnal s ta ndard dox e pine (DOX)
0,0 03

NOR

DOX (IS )
0,005

Determination of Relative Migration Time of analysed drugs in CZE mode

0,004

0,00 25

CLO EOF

A [AU]

0,003

A [AU]

0,002

DEZ

PRO M

CHPRO E OF

0,0 02

0,001

0,00 15
0 0 -0,001 2 4 6 8 10 12 14

S ample
Se paratio n buffe r: 40 mM s odium te tra borate , pH 9,5 with a ddition 10 mM STDC.
0 5 10 15 20 25 30

Analyte

Inte rna l S ta ndard Nortryptyline Imipra mine

Re lative Migration Time 1,16 0,86

Sta nda rd De via tion 0,01 0,01

LOD [mg/l] 0,50 *******

-0,002

Se paratio n Buffe r: 50mM CAP SO in 30% v/v me thanol; pH 9,5

0,0 01

t [min]

Blood Ta blet

P romazine Dezypra mine

Separatio n o f 5 TCA s tandards : no rtry ptyline (NOR), amitryptyline , (AMI), do xe pine (DOX), dezypramine (DEZ) and imipramine (IMI)
0,006

0,00 05

t [min]

AMI
0,005

0,004

NOR DOX EO F

0,003

A [AU]

0,002

DEZ

IMI

S epa ratio n o f drug mixture : amitry ptyline (AMI), c lomipramine (CLO), do xe pine (DOX) and imipra mine (IMI)
0, 006

0,001

0
-0,001

10

12

14

Se paratio n Buffer: 50mM CAP SO in 30% v/v me tha nol; pH 9,5

EOF
0, 005

AMI
0, 004

CONCLUSIONS

CLO S e paratio n buffe r: 40 mM s odium te tra bora te , pH 9,5 with a ddition 10 mM S TDC.

-0,002

t [min]

S e paration o f de zipram ine (DEZ), no rtryptyline (NOR), c hlo rpro m azine (CHPROM) and pro maznine (PROM) in the e xtrac t of blo o d
0, 012

A [AU]

0, 003

DOX

Both CZE and MECC may be used for identification of pharmaceutical preparations.

0, 002

IMI

0 ,01

NOR

0, 008 E OF

Extra ct of blood with drug mixture and IS (NOR) Extra ct of blood with IS (NOR)

The results of CZE blood analysis (spiked with drug standards) show that some drugs may be well separated (e.g.. desipramine, nortriptyline) while the others (e.g.. promazine, chlorpromazine) are not completely resolved from endogenous compounds of blood matrix. However, promazine is resolved enough to be determined in whole blood samples using standard addition method.

0, 001

0 0 5 10 15 20 25 30 35 40

A [AU]

0, 006 NOR 0, 004 CHP ROM P ROM 0, 002 DEZ 0 0 -0, 002 2 4 6 8 10 12 14 EOF

t [min]
S e paratio n Buffer: 50mM CAPS O in 30% v/v me tha nol; pH 9,5

Extract o f blo o d with additio n o f drug s tanda rds


0, 01

The initial examinations on application of MECC to the separation of four TCA showed that using this mode, the protein fraction of blood matrix does not interfere with the examined drugs, however the peaks on electropherograms were broader and their relative times were less repeatable compared to those in the CZE

t [min]

De z ypramine (DEZ) - table t (Pe tylyl) e xtract with interna l s tandrad additio n o f imipramine (IMI)
0,01

CLO
0,0 08

DOX IMI EOF

AMI Blind S ample Blood e xtract

0,0 06

mode. The further examinations in this matter should be carried out. Generally, it may be concluded that for the air thermostated capillary electrophoresis system, CZE mode seems to be preferable rather than MECC, especially in the cases of multi-drug analyses (e.g. screening analyses). However cleaner blood extracts (e.g. using SPE technique) are required. The alternative way for the improvement of the extraction process might be the application of the combined CE modes, for example CZE with addition of appropriate amount of surfactant.

DE Z

0,006

A [AU]

0,008

0,0 04

A [AU]

IMI (IS)
0,004

0,0 02

EOF

0
0,002

S eparation buffe r: 40 mM s odium te tra bora te , pH 9,5 with addition 10 mM S TDC.


20 30 40 50 60

EOF

10

0 0 2 4 6 8 10 12 14

S e paration Buffe r: 50mM CAPS O in 30% v/v me tha nol; pH 9,5

-0,0 02

t [min]

-0,002

t [min]