Elsevier

Journal of Chromatography B

Volumes 1033–1034, 15 October 2016, Pages 321-327
Journal of Chromatography B

Isotope-dilution method for the determination of 1-vinyl-2-pyrrolidone-mercapturic acid as a potential human biomarker for 1-vinyl-2-pyrrolidone via online SPE ESI-LC/MS/MS in negative ionization mode

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Highlights

  • The manuscript provided deals with a new online SPE ESI-LC/MS/MS method for a yet unknown 1-vinyl-2-pyrrolidone (VP) metabolite, that is 1-vinyl-2-pyrrolidone mercapturic acid (VPMA).

  • Comparisons to structural similar substances and their metabolites and analytical methods were made.

  • A fast and simple but specific and sensitive automated online SPE program using restricted access material (RAM) is presented. Mass spectrometric tuning data with according mass transitions of standard and isotope labeled internal standards are presented.

  • Thorough method validation data is presented.

  • Chromatograms of samples from exposure experiments with Sprague-Dawley rats are given for the proof of principle.

Abstract

We established and validated a specific and sensitive analytical method for the determination of 1-vinyl-2-pyrrolidone (VP) as 1-vinyl-2-pyrrolidone-mercapturic acid (VPMA) in urine using an electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) column switching method. An online solid phase extraction (SPE) for sample cleanup was performed by column switching to a restricted access material and back-flushing to the analytical column. A Phenomenex Luna C8 column was used for sample separation (150 mm; ID 4,6 mm; 3 μm). D4-VPMA served as an isotope labeled internal standard and was detected in negative multiple-reaction monitoring (MRM) mode.

The Limit of quantification (LOQ) for VPMA was 1.5 μg/L, the intra-day precision of three concentrations (2 μg/L, 75 μg/L and 400 μg/L) of spiked urine samples ranged from 2.7 to 7.3%, the inter-day precision from 3.4 to 14.4%. The accuracy ranged from 6.2 to 9.0%, for the intra-day experiments and from 0.3 to 6.9% for the inter-day experiments. The method was applied to urines of Sprague-Dawley rats exposed to VP as a proof of principle of VPMA as a potential biomarker.

Introduction

1-Vinyl-2-pyrrolidones main characteristics are its polar and non-polar features as well as its polymerizability to soluble polyvinylpyrrolidone (PVP) or insoluble polyvinylpolypyrrolidone (PVPP). VP based products are used in a wide variety of products and are produced in industrial scale, thus penetrating the society [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. For example polymerized VP is used as a solvent in pharmaceutical products like tablets, as a food additive or in UV-durable inks.

Since 2013 VP is classified as a carcinogenic type 4 by the German Research Foundation, that is as a substance with a non-genotoxic mechanism. The International Agency for Research on Cancer (IARC) has classified VP as not classifiable as to its carcinogenicity to humans (group 3) in 1979 [11]. VP is classified as skin-permeable [12]. A specific biomarker capable of determing a potential background burden of the general population or helping to identify potential health threats of workers exposed to VP, would be desirable.

There is an oxidative metabolic pathway postulated which can be found in [13] by the German Research Foundation. While in acidic environment, which can be found in the stomach in the human body, a cleavage of the vinyl group is postulated due to the molecule structure. In neutral environment occurring in the blood pyrrolidone ring oxidation analogue to that observed in N-methyl-2-pyrrolidone (NMP) or pyrrolidone is assumed. NMP was found to be excreted to 44% as 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP) and to 20% as 2-hydroxy-N-methylsuccinimde in experiments of orally dosed NMP to human volunteers. Similar results were obtained after NMP inhalation for 8 h [14], [15], [16]. However, given the reactivity of the vinyl group it remains questionable if these can be detected as ring oxidized vinyl-pyrrolidone metabolites. Oxidation of the vinyl group seems likely, so various other metabolites could occur which on top do not have to be necessarily specific for VP, and might also be metabolites of N-ethyl-2-pyrrolidone.

It is known that the elimination of exogenous compounds in the human body is supported by functionalization and conjugation reactions. For electrophilic substances, like acrylnitrile, acrylamide or benzene, the conjugation with glutathione is a common reaction. The formed glutathione derivate is metabolized further to an acetylcysteine-derivate or a mercapturic acid respectively. These formations of mercapturic acids and according analytical methods have been described for many solvents within the last decades [17], [18], [19]. Substantial amounts of up to 70% of the exogenous substance have been found to be eliminated from the organism via the conjugation pathway with glutathione [20], [21]. On the basis of this information it is very likely that for VP a 1-vinyl-2-pyrrolidone-mercapturic acid (VPMA) is formed (s. Fig. 1).

The paper provided, describes the established and validated analytical method for the determination of VPMA as a potential biomarker for VP via ESI-LC/MS/MS which has not been described yet elsewhere. The advantage of this approach in contrast to analogue metabolites of the methods described for NMP is alongside its specifity for VP the use of an online SPE method instead of a time consuming offline SPE method which is used for NMP. A brief example of the analysis of urine samples of Sprague-Dawley rats, dermally exposed to VP is given as a proof of principle.

Section snippets

Chemicals

LC-Water and formic acid 100% suprapur were purchased from Merck (Darmstadt, Germany), acetonitrile HPLC-Grade was supplied by J.T. Baker (Germany), ammonium formate p.A. from Fluka (Buchs, Suisse). The standard of VPMA ((S)-2-Acetylamino-3-[2-(2-oxo-pyrrolidin-1-yl)-ethylsulfanyl]-propionic acid) as well as the d4-VPMA internal standard((S)-2-Acetylamino-3-[2-(2-oxopyrrolidin-1-yl)(d4)ethylsulfanyl]-propionic acid) both of a purity of 95% were synthesized by an external laboratory (Chiroblock,

Sample treatment

Under neutral conditions, that is in pure LC-water and acetonitrile, chromatographic results were not satisfying. Thus, urine samples were acidified thoroughly by adding 50 mM ammonium formate buffer pH = 2.5 and 10 μL 100% formic acid in order to protonize the VPMA to gain sharp peaks. Sample cleanup using a RP-8 ADS-RAM-phase was applied. The duration of the cleanup step was rather short with 0.6 min, maybe thus not removing as much matrix as an offline SPE method would. However compared to an

Conclusion

In the present paper a simple, sensitive and specific online SPE ESI-LC/MS/MS method for the determination of VPMA as a metabolite of VP is described, which is to our knowledge unique at this time. A proof of principle for VPMA being a metabolite of VP was presented within the urines of Sprague-Dawley rats exposed to VP dermally.

Ethics

An animal experiment proposal was granted by the State Agency for Nature, Environment and Consumer Protection (LANUV) North Rhine-Westphalia, Germany (File reference: 84-02.04.2014.A421)

Acknowledgment

This research project is supported by the START-Programm of the Faculty of Medicine, RWTH Aachen.

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