Sensitive monitoring of monoterpene metabolites in human urine using two-step derivatisation and positive chemical ionisation-tandem mass spectrometry

• Sensitive monitoring of 10 metabolites of (R)-limonene, α-pinene, and Δ3-carene in human urine samples.
• Fast and simple sample preparation and derivatisation procedure using two-step silylation for unreactive tertiary hydroxyl groups.
• Synthesis of reference substances and isotopically labelled internal standards of (R)-limonene, α-pinene, and Δ3-carene metabolites.
• Study on (R)-limonene, α-pinene, and Δ3-carene metabolite background exposure of 36 occupationally unexposed volunteers.

A gas chromatographic–positive chemical ionisation-tandem mass spectrometric (GC–PCI-MS/MS) method for the simultaneous determination of 10 oxidative metabolites of the monoterpenoid hydrocarbons α-pinene, (R)-limonene, and Δ3-carene ((+)-3-carene) in human urine was developed and tested for the monoterpene biomonitoring of the general population (n = 36). The method involves enzymatic cleavage of the glucuronides followed by solid-supported liquid–liquid extraction and derivatisation using a two-step reaction with N,O-bis(trimethylsilyl)-trifluoroacetamide and N-(trimethylsilyl)imidazole. The method proved to be both sensitive and reliable with detection limits ranging from 0.1 to 0.3 μg L−1. In contrast to the frequent and distinct quantities of (1S,2S,4R)-limonene-1,2-diol, the (1R,2R,4R)-stereoisomer could not be detected. The expected metabolite of (+)-3-carene, 3-caren-10-ol was not detected in any of the samples. All other metabolites were detected in almost all urine samples.The procedure enables for the first time the analysis of trace levels of a broad spectrum of mono- and bicyclic monoterpenoid metabolites (alcohols, diols, and carboxylic acids) in human urine. This analytical procedure is a powerful tool for population studies as well as for the discovery of human metabolism and toxicokinetics of monoterpenes.

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