Simultaneous derivatization and extraction of amphetamine-like drugs in urine with headspace solid-phase microextraction followed by gas chromatography–mass spectrometry

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Abstract

A sensitive and solvent-free procedure for the determination of amphetamine-like drugs, amphetamine (AM), methamphetamine (MeAM), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA) and methylen-dioxyethylamphetamine (MDEA) in urine samples was developed using solid-phase microextraction (SPME) with an on-fiber derivatization device. In this study, the amphetamine-like drugs were derivatized with heptafluorobutyric anhydride (HFBA) and heptafluorobutyric chloride (HFBCl) and determined by means of a gas chromatography–mass spectrometer with selected ion storage (SIS) detection. An improved design was used for sample preparation. The test specimen was placed in an 8 ml vial along with additives (KOH and NaCl), a glass insert with twelve holes containing the derivatizing reagents was inserted into the vial, and the vial was then sealed tightly. A SPME device with a 100 μm polydimethylsiloxane fiber was inserted into the vial and the fiber was exposed to the headspace in the insert, then the vial was heated and stirred at 95 °C and 600 rpm for 30 min for evaporation/adsorption/derivatization. The SPME needle was finally removed and inserted into the GC injection port to desorb the analytes.

The influence of several parameters on the efficiency of microextraction (type of fiber coating, extraction time, extraction temperature, etc.) was investigated. The SPME method developed provides good sensitivity with a calibration range from 0.5 to 1000 ng ml−1 for MDA, and 0.1–1000 ng ml−1 for AM, MA, MDMA and MDEA. The method detection limits (MDLs) are in the range 0.016–0.193 ng ml−1 with the studied drugs. This method was also successfully tested on real samples of urine from people who were suspected of using amphetamines.

Introduction

During the past few years, solid-phase microextraction (SPME), discovered and developed by Pawliszyn and co-workers [1], [2], has emerged as a versatile solvent-free alternative to conventional liquid–liquid extraction and solid-phase extraction procedures. It is a simple approach, involving the partitioning of the analytes between the fiber coating and the sample matrix, and was widely applied to analysis of biological samples [3], [4], [5], [6]. The conjunction of SPME with gas chromatography–mass spectrometry (GC–MS) analysis has been employed for a variety of organic compounds, especially for volatile and semi-volatile agents using the headspace technique [7], [8], [9], [10].

Amphetamine (AM), methamphetamine (MeAM) and their methylenedioxy derivatives are a major class of central nervous system stimulants and are often abused by drug addicts and recreational users. A number of methods for the determination of stimulants in biological samples have been reported [11], [12], [13], [14]. Among these methods, GC–MS has been the most widely used analytical method because of its sensitivity and selectivity. For the analysis of these polar compounds by gas chromatography, it is necessary to derivatize the amphetamines to increase chromatographic efficiency. Several derivatization techniques in SPME have been developed, and reviews on the development and application of derivatization with SPME have been published [15], [16]. An on-column derivatization procedure [17], [18] was reported in which the derivatizing reagent was injected into the GC injection port immediately prior to insertion of the analyte-carrying fiber. This method, however, may cause unnecessary contamination to the GC–MS system and result in shortened column life and additional maintenance requirements. An on-fiber derivatizating approach [19] has also been developed, in which the adsorption and the derivatization processes were conducted in two separate vials.

In previous work in our laboratory, a SPME method with one-step derivatization and extraction of amphetamines was developed and yielded good results [20]. It explored an approach in which the derivatization and extraction steps are allowed to occur simultaneously in the headspace with a derivatizing reagent-containing insert placed in the sample vial; thus being isolated from the sample matrix.

In the current study, a modified device was built that gave better sensitivity compared to the previous report. In the present work, the number of holes (2-mm diameter) in the glass insert containing derizatizing reagent was increased from 2 to 12. This approach enhances the contact between the exposed fiber coating and the sample in headspace area, thus the extraction efficiencies were enriched. These improvements were also discussed in this study. The detection limits obtained in this improved method, compared with the previous report from our laboratory [20], were decreased from 0.3 to 0.053 ng ml−1 and 0.3–0.016 ng ml−1 with amphetamine and methamphetamine, respectively. The new approach proved to be a fast and an economic method, with good sensitivity and reproducibility. In this work the five common stimulants are analyzed, and this developed method applicable to examine real urine samples from drug abuser practically.

Section snippets

Reagents and materials

All chemicals were of research grade and used without further purification. Amphetamine, methamphetamine, methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA) and methylen-dioxyethylamphetamine (MDEA) were bought from Cerilliant (Austin, TX, USA). The isotopically substituted compounds used as internal standards, AM-d8, MA-d11, MDA-d5, MDMA-d5 and MDEA-d5, were also purchased from Cerilliant. Stock standard solutions of the studied drugs were prepared in methanol and maintained

Fiber evaluation and the effect of added derivatizing reagent

Fiber coatings are dominant factors in the recoveries of analytes. According to the rule of “like dissolves like”, three types of commercial fibers were selected for investigation: PDMS, CAR/PDMS and PDMS/DVB fibers. Fig. 2 shows the extraction efficiencies of the five drugs. It is clear from the graph that CAR/PDMS fiber exhibited the lowest extraction. With the three studied drugs, MDA, MDMA and MDEA, PDMS fiber demonstrated better sensitivity than PDMS/DVB fiber. Although the extraction

Conclusion

The proposed sample preparation procedure is much simpler than the conventional LLE, and SPE approaches and does not require the use of organic solvent. By combining HFBCl and HFBA as derivatizing reagents and placing them in an insert (instead of mixing them in the sample matrix), the HS-SPME method achieves excellent sensitivity for the analysis of these five stimulants in urine specimens. Because the water hydrolysis of the derivatizing reagent is much faster than the acylation reaction of

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