Use of solid-phase microextraction (SPME) for the determination of methadone and EDDP in human hair by GC–MS

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Abstract

Solid-phase microextraction (SPME) is a new extraction technique with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples. In this paper the use of SPME is proposed for the determination of methadone and its main metabolite EDDP in hair by GC–MS. The hair samples were washed, cut into 1-mm segments, and incubated with Pronase E® for 12 h. A 100-μm polydimethylsiloxane (PDMS) film fibre was submerged for 30 min in a diluted solution of the hydrolysis liquid (1:4 with borax buffer) containing methadone-d3 and EDDP-d3 as internal standards. Once the microextraction was concluded the fibre was directly inserted into the CG injection port. Linearity was found for methadone and EDDP in the range studied, 1.0–50 ng/mg hair, with correlation coefficients higher than 0.99. Interassay relative standard deviation (R.S.D) was determined to be less than 13.30% for methadone and less than 8.94% for EDDP, at 3.0 and 30.0 ng/mg. Analytical recoveries were close to 100% for both compounds on spiked samples. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme. The concentration of methadone in hair ranged from 2.45 to 78.10 ng/mg, and for EDDP from 0.98 to 7.76 ng/mg of hair.

Introduction

The analysis of drugs in hair is of particular interest because it can provide information about long-term use and is useful for many purposes including criminal investigations and monitoring the compliance on drug maintenance programmes.

Immunological methods are normally used for the screening of drugs in hair, and their results must be confirmed by more sensitive and specific methods.

Various methods have been applied to the determination of methadone in hair: radioimmunoassay [1], [2], high-performance liquid chromatography [3], capillary electrophoresis [4], and gas chromatography–mass spectrometry (GC–MS) [5], [6], [7]. Confirmation analysis generally consists of a pretreatment of the sample, and hair hydrolysis followed by liquid–liquid or a solid-phase extraction.

Solid-phase microextraction (SPME) is a new technique of extraction with many advantages: small sample volume, simplicity, quickness and solvent-free. It is mainly applied to environmental analysis, but is also useful for the extraction of drugs from biological samples [8].

Strano-Rossi and Chiarotti [9] demonstrated the suitability of SPME for the determination of methadone in hair samples. Recently, we proposed the use of SPME for the determination of methadone and its main metabolite (EDDP) in plasma, in a comparative study with the classical liquid–liquid extraction [10].

In the present work the use of SPME in the determination of methadone and EDDP in hair by GC–MS is described. The method was applied to the analysis of real hair samples from eight patients of a methadone maintenance programme (MMP).

Section snippets

Hair samples

Drug-free black hair, taken from a laboratory volunteer, was used for preparation of standards and calibrators. Hair samples from eight patients of a MMP from the Autonomic Community of Galicia (Spain) were analyzed.

Chemicals and reagents

Methadone hydrochloride and methadone-d3 hydrochloride were obtained from Sigma (St. Louis, MO); 2-ethylene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) perchlorate and EDDP-d3 perchlorate were obtained from Radian (Austin, TX). Methanol, borax and sodium chloride (analytical grade),

Analytical method

The gas chromatographic method used in this work was efficient to separate methadone and EDDP. The retention times (RT) were 7.21 min for EDDP-d3, 7.24 min for EDDP, 8.69 min for methadone-d3 and 8.72 min for methadone, as can be seen in Fig. 1 that shows the extracted ion chromatogram from patient 6.

No carry-over with the desorption time of 5 min at 250°C and no degradation of the fibre were observed after at least 90 runs.

The standard calibration curves were obtained in a triple run. Simple

Conclusion

This work describes the application of solid-phase microextraction (SPME) for the determination of methadone and EDDP in hair by GC–MS. The method was successfully applied to the analysis of hair samples from patients of a methadone maintenance programme.

The microextraction was performed directly on a small aliquot of the enzymatic hydrolysis liquid, commonly used for the screening techniques for drugs in hair, allowing the use of the remaining sample for confirmation analysis of other drugs,

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