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A new method for high-resolution and high-precision analysis of flunitrazepam and 7-aminoflunitrazepam in human body fluids using a Monolithic SPE SpinTip and UPLC–Q-ToF–MS

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Abstract

Purpose

Forensic toxicological analyses of drugs and their metabolites in human specimens usually require extractive pretreatment for successful analysis of substances from the matrix. In the present study, a high-throughput method was developed to analyze flunitrazepam, 7-aminoflunitrazepam, 7-acetamidoflunitrazepam, 7-acetamido-3-hydroxyflunitrazepam, and 3-hydroxyflunitrazepam in human plasma and urine samples using a new Monolithic C18 gel-packed SpinTip and ultra-performance liquid chromatography (UPLC)–quadrupole time-of-flight (Q-ToF) mass spectrometry (MS).

Methods

Plasma (20 µL) or urine (100 µL) samples spiked with each component were extracted using a Monolithic C18 SPE SpinTip and quantified by UPLC–Q-Tof–MS with positive-ion electrospray ionization (ESI).

Results

Good separation, with clear peak shapes of flunitrazepam and its metabolites, was achieved within an analysis time of 6 min, including the extraction time. Recoveries of flunitrazepam and 7-aminoflunitrazepam for plasma and urine samples were 93.5–118% and 97.7–109%, respectively. The regression equations for flunitrazepam and 7-aminoflunitrazepam showed excellent linearity in the range of 0.5–250 ng/mL for plasma and 0.4–500 ng/mL for urine, with detection limits of 0.2–0.5 ng/mL. Intra- and inter-day coefficients of variations for two drugs are smaller than 13.5%. The accuracy of quantitation was 89–110%.

Conclusions

The method was successfully applied to determine the level of flunitrazepam and its metabolites in human plasma and urine, respectively, after oral administration to a volunteer.

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Acknowledgements

This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS), KAKENHI Grant (C) 24590865.

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Authors and Affiliations

  1. Department of Legal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555, Japan

    Masaya Fujishiro, Ai Noguchi, Xiao-Pen Lee, Mari Hashimoto, Keizo Sato & Takaaki Matsuyama

  2. Department of Legal Medicine, Toho University School of Medicine, Tokyo, Japan

    Chika Hasegawa

  3. Seirei Christopher University School of Nursing, Hamamatsu, Japan

    Takeshi Kumazawa

  4. GL Sciences Inc., Tokyo, Japan

    Shota Miyazaki

  5. Department of Neurology, Showa University Kototoyosu Hospital, Tokyo, Japan

    Ayako Kuriki

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  1. Masaya Fujishiro
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Correspondence to Xiao-Pen Lee.

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Ethical approval

This study was approved by the Ethics Committee of Showa University School of Medicine (no. 861). The obtainment of blood and urine samples from healthy volunteers was approved by the Ethics Committees of Showa University School of Medicine (no. 1249).

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Supplementary material 1 (PDF 387 kb)

11419_2019_471_MOESM2_ESM.doc

Fig. S1. Appearance of a Monolithic C18 gel-packed SPE SpinTip and electron micrograph of the monolithic silica gel. The electron micrograph of the monolithic silica gel is used with permission from GL Sciences, Tokyo, Japan (DOC 57 kb)

11419_2019_471_MOESM3_ESM.doc

Fig. S2. Comparison of XICs at different pH from human plasma and urine 1 h after oral administration of flunitrazepam (1 mg). The pH was adjusted to 2.4 with 10 µL of 1M HCl, 6.9 with 10 µL of ultra-pure water, and 10.1 with 10 µL of 10 mM NH4OH (DOC 53 kb)

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Fujishiro, M., Noguchi, A., Lee, XP. et al. A new method for high-resolution and high-precision analysis of flunitrazepam and 7-aminoflunitrazepam in human body fluids using a Monolithic SPE SpinTip and UPLC–Q-ToF–MS. Forensic Toxicol 37, 387–397 (2019). https://doi.org/10.1007/s11419-019-00471-4

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