Elsevier

Journal of Chromatography B

Volume 784, Issue 1, 25 January 2003, Pages 169-182
Journal of Chromatography B

On-line clean-up by multidimensional liquid chromatography–electrospray ionization tandem mass spectrometry for high throughput quantification of primary and secondary phthalate metabolites in human urine

https://doi.org/10.1016/S1570-0232(02)00785-7Get rights and content

Abstract

We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher® ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5–2.0 μg/l urine). First results on a small non-exposed group (n=8) ranged for 5OH-MEHP from 0.59 to 124 μg/l, for 5oxo-MEHP from <LOQ to 73.0 μg/l, and for MEHP from <LOQ to 41.1 μg/l. The other short chain monoester metabolites were detectable in every sample with mean concentrations for MnBuP of 36.5 μg/l, for MBzP of 7.19 μg/l and MEP of 1.0 mg/l. With this rapid and economic method we can determine the internal exposure of the general population to DEHP and other phthalates as well as the body burden of occupationally and medically exposed subjects. The results can help to rank the risks of phthalates in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption. Since secondary, functionalized metabolites of DEHP are included in the method an enduring problem of the past is excluded: sample contamination in the pre-analytical and analytical phase by both di- and monoesters.

Introduction

Plastics have become an almost irreplaceable component of our modern world. And so have the plasticizers giving the synthetic material its desired flexibility. Phthalates, the dialkyl or alkyl aryl esters of phthalic acid have with 93% the lion’s share in the plasticizers’ segment. Each year 900 000 t are produced in Western Europe [1]. In Germany di(2-ethylhexyl)phthalate (DEHP) with ∼250 000 t accounts for 60% of phthalate production. It is estimated that an additional 100 000 t of DEHP alone is emitted into the environment through DEHP containing waste every year in Germany [2], [3], [4]. Other important phthalates application- and production-wise are diethylphthalate (DEP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP), dinonylphpthalate (DNP) and dioctylphthalate (DOP). In addition to being used as plasticizers, phthalates are also used as industrial solvents and lubricants, additives in the textile industry and pesticide formulations, and as components in consumer products such as deodorants and perfumes [5], [6], [7], [8].

Humans are exposed to phthalates in numerous ways, e.g. by migration of phthalates into foodstuffs, by dermal absorption of cosmetics or by inhaling phthalate containing air [9], [10], [11]. In addition, specific groups of the population such as workers in the PVC-industry or medical patients undergoing dialysis, blood transfusions or having implants are potentially more heavily exposed [12], [13], [14], [15], [16], [17].

Several phthalates and some of their monoester metabolites have shown teratogenic, reproductive and developmental effects, toxicity to the testes and liver carcinoma in rodents [18], [19], [20]. Effects on humans in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption are controversial [5], [21], [22], [23], [24], [25], [26], [27], [28].

External exposure scenarios have always been difficult to evaluate. This is partly due to the fact that phthalates have become ubiquitous in the environment and so phthalate diester measurements have been hampered. It is also due to the fact that it has always been difficult to deduce from measurements of environmental contamination to actual individual intake. An unambiguous assessment of the exposure of the population to phthalates can only be achieved by measuring specific metabolites of the phthalates, preferably in urine, in a biological monitoring study [29], [30], [31], [32], [33], [34]. In humans, phthalates are rapidly cleaved to their respective monoesters and a portion is further metabolized to oxidation products. These products are then mainly excreted through the urine [35], [36], [37], [38], [39].

Analytical methods determining diesters in blood or even monoesters in urine have been flawed in the past due to the contamination factor. Sjöberg [38] and Dirven et al. [39] performed biological monitoring studies after DEHP exposure and determined four DEHP metabolites. Detection limits however were not suitable for the determination of baseline excretion in the general population. Neither were the metabolite standards made available nor their synthesis published. Since then no ongoing studies have been performed on phthalate exposure by unequivocal determination of secondary phthalate metabolites. No reference values exist today for the excretion of secondary metabolites of DEHP or other phthalates for the general population. Blount et al. [40] determined only the monoester metabolites of the most applied phthalates. They circumvented a major contamination problem of monoester analysis by introducing a β-glucuronidase enzyme with no non-specific lipase activity. Conjugated phthalate monoesters in urine could be deconjugated even in the presence of phthalate diesters (being ubiquitously present) without generating monoesters out of the diesters. However, to definitely rule out contamination in the pre-analytical phase during sample collection and transportation by phthalate monoesters generated through simple chemical or microbiological ester cleavage of the diesters and not human metabolism, we decided to focus our method on the secondary metabolites of the most applied phthalate diester, di(2-ethylhexyl)phthalate (DEHP). Those secondary metabolites are 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP). They are generated in human metabolism by ω-1 oxidation of the monoester alkyl chain. Only by implementing those secondary metabolites of DEHP in the analytical method is the risk of external contamination ruled out. Synthesis of those secondary metabolites was published by Gilsing et al. [41] and the standards are also available D4-isotopically labelled for internal standard use. Fig. 1 illustrates the general human metabolism of phthalates and the resulting metabolites analysed in our method.

With our method and subsequent results we hope to provide insights into the actual exposure of the general population and certain risk groups to phthalates.

Section snippets

Chemicals

5-Hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP), 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) and their D4-ringlabelled analogues were synthesized in cooperation with the “Institut für Dünnschichttechnologie und Mikrosensorik e.V. (IDM)” according to Gilsing et al. [41]. All four compounds had chemical purity >95%. The isotopic purity of each labelled internal standard was tested by LC–MS–MS and contained no measurable unlabelled or partially labelled (D3 or D2) compound.

General considerations

Method development was led by three requirements: minimization of contamination factors, coverage of the most important phthalates and effectiveness.

In order to build a fast and easy method we applied novel HPLC on-line enrichment and column-switching techniques and combined them with state-of-the-art MS–MS detection. The applied RAM-phase enabled us to extract the analytes online out of the matrix and transfer them with a switching valve directly onto the analytical column. This effective

Conclusions

We have developed a reliable, easy and fast analytical procedure for the determination of various phthalates in human urine samples. To our knowledge this is the first time that secondary phthalate metabolites have been used to unequivocally determine the exposure of the general population. Within-day and between-day repeatability is very good, even at low concentrations but also over the whole concentration range. The on-line clean-up procedure is very efficient, so that no interfering effect

Acknowledgements

We would like to thank the DFG for their financial support (AN 107/16-1). We would also like to acknowledge the U.S. Centers for Disease Control and Prevention’s National Center for Environmental Health for sharing phthalate metabolites used in preliminary method development.

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