KODIAK 1200

17880 points1792698.561*13445239.21*4481746.402*8963492.803*2689047.841*1792698.561*

5.6368 13.976

Am

phet

amin

e

Met

ham

phet

amin

e

MDABDB

MDMA

MDEA

MBDB

135>79

154>110

140>93

135>79

154>110

173>141

172>113

Headspace Solid-Phase Headspace Solid-Phase Dynamic Extraction Dynamic Extraction (SPDE) (SPDE) and GC/MS/MS and GC/MS/MS for the for the determinationdetermination of of AmphetaminesAmphetamines

and and Synthetic Synthetic Designer Designer DrugsDrugs in in Hair Hair SamplesSamplesD.W. Lachenmeier, L. Kröner, F. Musshoff, B. Madea

Institute of Legal Medicine, University of Bonn, Stiftsplatz 12, D-53111 Bonn, Germany

INTRODUCTION

During the past few years solid-phase microextraction (SPME), discoveredand developed by Pawliszyn and co-workers (1), has emerged as a versatilesolvent-free alternative to conventional liquid-liquid extraction and solid-phase extraction procedures. SPME in conjunction with GC-MS analysis hasbeen employed for a variety of organic compounds, especially of volatile andsemi-volatile agents using the headspace technique. The main disadvantagesof SPME are the fragility of the fused silica and the unprotected stationaryphase coating on the outer surface of the fibre when extended through thesyringe needle. The limited flexibility regarding surface area and filmthickness is another problem of SPME. There were several efforts toovercome these disadvantages. All attempts aimed at developing a devicewith the coating on the interior of a needle or capillary instead of a fibre. Theadvantages are greater capacity, higher extraction speed and stability of thedevice. A technique using internally coated hollow needles was described byMurphy (2). In 1997 an inside needle capillary adsorption trap (INCAT)technique was developed (3).

The solid-phase dynamic extraction (SPDE) developed by Chromtech(Idstein, Germany) in 2000 is the first commercially available inside-needledevice. It uses coated stainless steel needles (8 cm) with a 50 µm film ofpolydimethylsiloxane (PDMS) and 10 % activated carbon (BGB Analytik,Anwil, Switzerland). A diagram of a SPDE device in comparison with aSPME fibre is given in Fig. 1. The volume of the stationary phase of theSPDE needle is approximately 5.99 mm3 compared to a 100 µm PDMSSPME fibre with 0.94 mm3. SPDE was successfully applied to the analysis ofpesticides in water by Lipinski (4).

RESULTS AND DISCUSSION

By routine analyses of authentic samples no interferences were observed.Peak purity and selectivity are ensured. Further validation data aredemonstrated in Tab. 1. As shown in Fig. 4 the sensitivity was increased byusing the triple quadrupole MS/MS-mode (B) in comparison to singlequadrupole MS-SIM (A). The limits of quantitation and detection weremarkedly enhanced compared to SPME or SPDE with conventional GC/MS,e.g. the limits of MDA were lowered tenfold (5,6). Regarding the validatingdata, the procedure is sensitive, selective and reproducible.

A large advantage of the SPDE technique in relation to SPME is therobustness of the capillary. It is nearly impossible to damage the SPDEdevice mechanically in contrast to the fragile SPME fibres. The SPDEcapillary lasted more than twice the time of an SPME fibre. The absoluteextraction yield with SPDE was 50% higher compared to a SPME fibre.

Fig. 1: Schematic representation of the SPME device (a) in comparison to the SPDE device (b)

a) SPME b) SPDE

needle

fibreattachment

fused silica fibrewith 100µmPDMS coating

2.5mlHS-syringe

internal coating(50µm PDMS with 10% AC)

1cm

conical tip with side port

EXPERIMENTAL

The hair samples were washed for 5 min in deionised water, petroleumbenzine and dichloromethane, respectively. Ten mg of hair, 1 mL NaOH (10M) and 80 µL internal standard mixture (deuterated analytes, 250 ng/mL)were placed into a 10 mL headspace vial. A second vial was filled with 25 µLMBTFA and sealed in the same way. The fully automated SPDE procedureusing a CTC Combi PAL autosampler is shown in Fig. 2.

The following SPDE specific parameters were successively optimised:temperature of agitator and headspace syringe, number of filling cycles forextraction and derivatisation, speed of aspirating the syringe for extractionand desorption, flush gas volume for desorption, predesorption time in GCinjection port and desorption temperature (Fig. 3).

For analysis an Agilent model 6890 Series Plus gaschromatograph incombination with a Bear Kodiak 1200 GC/MS/MS Triple Quadrupolemasspectrometer and a CTC-Combi-PAL-Autosampler were used(Chromtech, Idstein, Germany).

Fig. 2: Procedure for fully-automated hair-analysis with alkaline hydrolysis, solid-phase dynamic extraction (SPDE), derivatisation and GC-MS analysis.

REFERENCES• Z. Zhang, J. Pawliszyn: Headspace solid-phase microextraction. Anal.

Chem. 65 (1993) 843-852• G.E. Murphy. United States Patent (1996) 5,565,622• M.E. McComb, R.D. Oleschuk, E. Giller, H.D. Gesser: Microextraction

of volatile organic compounds using the inside needle capillaryadsorption trap (INCAT) device. Talanta 44 (1997) 2137-2143

• J. Lipinski: Automated solid phase dynamic extraction - extraction oforganics using a wall coated syringe needle. Fresenius J. Anal. Chem. 369(2001) 57-62

• F. Musshoff, H.P. Junker, D.W. Lachenmeier, L. Kroener, B. Madea:Fully Automated Determination of Amphetamines and SyntheticDesigner Drugs in Hair Samples using Headspace Solid-PhaseMicroextraction and Gas Chromatography – Mass Spectrometry, J.Chromatogr. Sci. (2002) in press.

• F. Musshoff, D.W. Lachenmeier, L. Kroener, B. Madea: AutomatedHeadspace Solid-Phase Dynamic Extraction for the Determination ofAmphetamines and Synthetic Designer Drugs in Hair Samples, J.Chromatogr. A (2002) in press.

Fig. 4: Chromatograms of spiked hair samples (0.5 ng/mg) measured using the Q1-SIM mode (A) and the MS/MS multiple reaction monitoring mode (B)

Tab. 1: Extraction yield, limit of detection and quantitation (LOD/LOQ), precision, linear range and correlation coefficient of the calibration curves

CONCLUSIONS

The application of fully automated headspace solid phase dynamic extraction(HS-SPDE) with following GC/MS/MS for the determination ofamphetamines and synthetic designer drugs in hair was tested. The methodwas successfully applied to the analysis of hair samples from drugconsumers. The SPDE as a further development of SPME turned out to beequally suitable for the requirements of clinical and forensic toxicologyregarding sensitivity and selectivity. The main advantages are the robustnessof the device and the greater capacity.

ACKNOWLEDGMENTS

The authors would like to give special thanks to Chromtech (Idstein,Germany) for the support in establishing the SPDE method.

0 500 1000 1500 2000 25005

10

15

20

25

30

35

40

45

Pea

k ar

ea (

⋅105 )

Desorption volume [µl]

10 µl/sec 50 µl/sec

8.90 9.00 9.10 9.20 9.30 9.40 9.50 9.60 9.70 9.80 9.90 0

1

2

3

4

5

6

7

Time-->

Abundance (⋅105)

MA

MA-D11

100 extraction cycles

50 extraction cycles

10 extraction cycles

50 100 150 200 2500

1

2

3

4

5

6

7

8

9

Pea

k ar

ea (

⋅104 )

Extraction flow speed [µl/sec]

Amphetamine (1:20) Methamphetamine (1:20) MDA MDMA MDEA BDB MBDB

0 1 2 3 4 5 6 7 8 9

1

2

3

4

5

6

7

8

9

Pea

k ar

ea (

⋅104 )

Number of derivatisation cycles

MDA MDMA MDEA MBDB

Fig. 3: Optimisation of the SPDE procedure

30 40 50 60 70 80 900

5

10

15

20

25

Pea

k ar

ea (

⋅105 )

Temperature of agitator and headspace syringe for extraction and derivatisation [°C]

Amphetamine Methamphetamine

Precision Regression line Extraction

yield [%] LOD

[ng/mg] LOQ

[ng/mg] Intraday

[%] Interday

[%] Linear range

[ng/mg] correlation coefficient

Amphetamine 12.9 0.03 0.12 3.5 4.9 0.05-20 0.999 Methamphetamine 10.2 0.03 0.11 4.5 7.6 0.05-20 0.999 MDA 15.0 0.01 0.04 3.0 6.1 0.05-20 0.996 MDMA 16.7 0.03 0.10 1.4 3.7 0.05-20 0.999 MDEA 14.7 0.04 0.33 2.6 6.2 0.1-20 0.999 BDB 12.7 0.02 0.08 5.3 6.5 0.05-20 0.998 MBDB 11.6 0.03 0.11 1.9 3.6 0.05-20 0.998

N2

d) Aspiration of nitrogen ingas station

(1 min)

Headspace syringein heated syringe adapter (50°C)

SPDE-needle

a) Alkaline hydrolysis

(5 min)

10 mghair

+ ISTD+ NaOH

Agitator withheater (50°C)sample: position 1MBTFA: position 2

200 µl/sec 10 µl/sec

e) Desorptionin GC injector(4 min, 250°C)

b) Dynamicextraction

(9 min)

50x

25 µlMBTFA

c) On-coating derivatization

(1 min)

6x

septum

KODIAK 1200

17898 points10000000*9001413.6*

10718026.6*936053.2*730961.6*

1071941.3*

5.6038 13.956

Am

phet

amin

e

Met

ham

phet

amin

e

MDA

BDB

MDMA

MDE

A

MBDB140

154

135

135

154

173

172

A

B