Simultaneous determination of barbiturates in human biological fluids by direct immersion solid-phase microextraction and gas chromatography–mass spectrometry

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Abstract

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI–SPME) with gas chromatography-mass spectrometry (GC–MS) is presented. The main parameters affecting the DI–SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180–0.988 and 0.0156–2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r2) were 0.994–0.999. The detection limits for whole blood were 0.05–1 μg ml−1, and those for urine 0.01–0.6 μg ml−1. Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI–SPME/GC–MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.

Introduction

Solid-phase microextraction (SPME) was first reported by Arthur and Pawliszyn in 1990 [1], and is an organic-solvent-free extraction technique that incorporates sample extraction, concentration and introduction into a single step. Direct immersion (DI) is one of the techniques for SPME, and has been applied to the detection of various drugs and poisons in biological fluids [2], [3], [4], [5], [6].

Barbiturates are one of the most popular sedative hypnotic group. They sometimes cause death in suicidal and accidental cases [7], [8], [9]. However, the reports for analysis of the drugs by SPME are sporadic; Queiroz et al. [10] have reported DI–SPME for phenobarbital and primidone in plasma, and Staerk et al. [11] reported headspace SPME for phenobarbital and secobarbital in urine. Hall et al. [12] reported DI–SPME for eight barbiturates in water solution, but not in biological samples.

In this study, we have optimized various conditions of DI–SPME for simultaneous determination of seven barbiturates in human biological fluids by gas chromatography (GC)–mass spectrometry (MS); and the usefulness of the established method has been evaluated.

Section snippets

Materials

The structures of the seven barbiturates examined are given in Table 1. Primidone was obtained from Dainippon Pharmaceutical Co., Ltd., Osaka, Japan; amobarbital from Nippon Shinyaku Co., Ltd., Kyoto, Japan; pentobarbital calcium from Tanabe Seiyaku Co., Ltd., Osaka, Japan; secobarbital sodium from Yoshitomi Pharmaceutical Ind. Ltd., Osaka, Japan; hexobarbital from Teikoku Chemical Ind. Co., Ltd., Osaka, Japan; mephobarbital from Bayer AG, Leverkusen–Bayerwerk, Germany; and phenobarbital from

Optimization of conditions for DI–SPME

To select the optimal conditions for simultaneous analysis of barbiturates, SPME fibers, salt additives, pHs of the sample solution, extraction temperatures and immersion times were examined for 0.5 ml of whole blood, to which 10 μg each of phenobarbital and primidone and 0.5 μg of other drugs had been added.

At the first step, three types of fibers, such as PDMS, PDMS/DVB and polyacrylate, were tested under the conditions of pH at 6.0–7.0 in the presence of sodium sulfate. Polyacrylate fiber gave

Conclusion

To our knowledge, this is the first report dealing with DI–SPME/GC–MS for simultaneous determination of seven barbiturates from human whole blood and urine. They could be rapidly and simultaneously determined even at the therapeutic levels. The DI–SPME/GC–MS established in this study is recommendable in the fields of the therapeutic drug monitoring, clinical toxicology and forensic toxicology.

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