Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

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Abstract

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C8 solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.

Introduction

N-Methyl-2-pyrrolidone (NMP; structural formula C5H9NO; CAS number 872-50-4; boiling point 202°C at 101.3 kPa), due to its strong and selective solvent power, is a widely used compound [1]. NMP dissolves most monomers and polymers and catalyses many polymerisation reactions. NMP is used increasingly as a substitute for other solvents of higher inherent toxicity in the occupational and environmental settings, e.g., dichloromethane in paint strippers. The use of NMP as a remover of graffiti has extremely increased [2]. Moreover, NMP is suggested as a skin penetration enhancer in transdermal therapy 1, 3.

Experimental exposure in human volunteers shows an efficient absorption of NMP through the respiratory tract [4]. NMP is readily eliminated from the body, mainly by biotransformation to other compounds: only a minimal fraction is eliminated in urine as NMP. A metabolic pathway, where NMP is first hydroxylised to 5-hydroxy-N-methylpyrrolidone (5-HNMP) and then further oxidised to N-methylsuccinimde (MSI), which in turn is hydroxylised to 2-hydroxy-N-methylsuccinimide (2-HMSI), has been suggested [5].

Studies on reproductive toxicity show that NMP may cause developmental toxicity at doses causing no or mild maternal toxicity 6, 7. The large number of women exposed to NMP in the microelectronics fabrication industry makes it urgent to assess the exposure to NMP. Air monitoring methods have been described 8, 9. However, the extensive percutaneous absorption of NMP makes it necessary to develop methods for biological monitoring. A method aimed at the analysis of NMP itself in urine and plasma has recently been reported by our group [4]. However, the ready biotransformation of NMP to other compounds and the minor excretion of non-metabolized NMP in urine suggest that the metabolites of NMP, rather than NMP itself, can be the most suitable compounds for biological monitoring. No methods for the quantitative analysis of these metabolites have been described so far.

In the present study, we report a method for the determination of 5-HNMP and 2-HMSI in human urine.

Section snippets

Apparatus

A Model 8065 gas chromatograph (Carlo-Erba, Milan, Italy) connected with a VG Trio 1000 quadrupole mass-spectrometer (Fisons, Manchester, UK) and fitted with an A200S auto-sampler (Carlo Erba) was used. The column (30 m×0.25 mm) with a DB-5 MS stationary phase and a film thickness of 0.25 μm was from J&W Scientific (Folsom, CA, USA). For the clean-up procedure, C8 Isolute solid-phase extraction (SPE) columns (500 mg; IST, Hengoed, UK) were used. These disposable micro-columns were used with a

Standards

The identity of the synthesised 5-HNMP was confirmed by proton NMR. In this a multiplet was found at 5.1 ppm (one ring proton next to a hydroxyl group), a doublet at 4.6 ppm (one hydroxyl proton), a singlet at 2.8 ppm (three methyl protons) and three multiplets at 1.9, 2.3 and 2.5 ppm, respectively (four ring protons). The proton purity was found to be better than 90%. No attempts were performed to check the purity by GC since 5-HNMP is a thermo-labile compound which can not even be analysed

Conclusions

The proposed method is simple and rapid and shows high precision for both 5-HNMP and 2-HMSI. The detection limits are sufficiently low for determinations of 5-HNMP and 2-HMSI in urine from workers exposed to NMP within the Swedish occupational exposure limit. Thus, the method is proposed for the biological monitoring of the exposure to NMP.

Acknowledgements

We thank Ms. Karin Paulsson and Ms. Kerstin Olsson for skilful technical assistance. This work was supported by the Swedish Council for Work Life Research and the Medical Faculty at Lund University.

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