A rapid HPLC method for the determination of carboxylic acids in urine samples using a Chromolith Performance RP/18e 100/4.6 with Chromolith Guard Cartridge RP/18e 10/4.6 (Merck KgaA, Darmstadt, Germany) was developed. The method facilitates the simultaneous determination of aromatic hydrocarbon metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene, hippuric acid (HA) from toluene and 2-, 3-, 4-methylhippuric acids (MHA) from xylene. 3-hydroxybenzoic acid (3-HBA) was used as internal standard. A chromatographic run is completed within less than 5 min for styrene, ethylbenzene and toluene metabolites, and within 10 min for xylene metabolites. The detection limits are 9 mg L(-1) urine for MA, 1.25 mg L(-1) urine for PGA, 4.9 mg L(-1) urine for HA, 22 mg L(-1) urine for 2-MHA, and 18.5 mg L(-1) urine for 3-MHA. No significant differences of the MA, PGA and HA concentrations in human urine samples obtained by HPLC chromatography on LiChrosorb RP 18 and on Chromolith RP/18e columns were found. The results were evaluated by using ANOVA.

Styrene 7,8-oxide (SO), a reactive metabolic intermediate of the industrial chemical styrene, binds covalently at nucleophilic amino acid residues of blood proteins in vivo and in vitro. In this study, SO adducts with cysteine, lysine, and histidine were synthesized, characterized, and then used as authentic standards to assign and quantitate the SO adducts in globin incubated with SO. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine were prepared by direct alkylation of cysteine with (R)-SO or (S)-SO. To prepare the SO adducts with lysine and histidine, Nalpha-Boc-protected amino acids were alkylated with (R)-SO or (S)-SO followed by deprotection of the Boc group to obtain Nepsilon-(2-hydroxy-1-phenylethyl)lysine and Nepsilon-(2-hydroxy-2-phenylethyl)lysine as well as Npi-(2-hydroxy-1-phenylethyl)histidine, Npi-(2-hydroxy-2-phenylethyl)histidine, Ntau-(2-hydroxy-1-phenylethyl)histidine, and Ntau-(2-hydroxy-2-phenylethyl)histidine. The individual regioisomers were isolated from their mixtures by semipreparative HPLC, and their structure was assigned using NMR techniques. The SO-modified globin, isolated from human hemoglobin incubated in vitro with racemic SO at a molar ratio SO/globin of 100:1 or 10:1, was digested with pronase and subjected to LC/MS and GC/MS analysis. All known regioisomers of the SO adducts were detected, with S-(2-hydroxy-1-phenylethyl)cysteine, Nepsilon-(2-hydroxy-1-phenylethyl)lysine, and Ntau-(2-hydroxy-2-phenylethyl)histidine being the most abundant in the modified globin. Deuterated analogues of the SO adducts were employed as internal standards. The SO-amino acid adducts described here appear to be suitable biomarkers for long-term exposures to styrene or SO.

A reference material for the biological monitoring of occupational exposure to toluene, benzene and phenol was prepared. O-cresol and hippuric acid (metabolites of toluene) are used for the biological monitoring of occupational exposure to toluene. Phenol, a metabolite of benzene, is used for the biological monitoring of exposure to benzene, but phenol can of course also be used as an indicator of exposure to phenol as well. The reference material (RM) used for the determination of these metabolites was prepared by freeze-drying pooled urine samples obtained from healthy persons occupationally exposed to toluene and those taking part in an inhalation experiment. Tests for homogeneity and stability were performed by determining urine concentrations of o-cresol, hippuric acid, creatinine and phenol. To investigate the stability of the RM, the urinary concentrations of o-cresol and phenol were monitored for eighteen months using GC and HPLC, while those of hippuric acid and creatinine were followed for five and six years, respectively, using HPLC. Analysis of variance showed that the concentrations did not change. The certified concentration values (and their uncertainties) of the substances in this reference material (phenol concentration c=6.46+/-0.58 mg l(-1); o-cresol concentration c=1.17+/-0.15 mg l(-1); hippuric acid concentration c=1328+/-30 mg l(-1); creatinine concentration c=0.82+/-0.10 g l(-1)) were evaluated via the interactive statistical programme IPECA.

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Ethylene glycol monobutyl ether (EGBE), an industrial solvent, is absorbed by the body not only by inhalation but also by dermal absorption (liquid or vapour). EGBE is metabolized to butoxyacetic acid (BAA). Pooled freeze-dried urine candidate reference material (RM) was prepared from urine obtained from persons occupationally exposed to EGBE. This material has the advantage of containing butoxyacetic acid in both the free and conjugated (glutamine and glycine) forms, as found in native urine. In all GC method modifications used, acid hydrolysis was used to release BAA from its conjugated form. The amount of butoxyacetic acid in homogeneity and stability testing was measured by GC after derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Detection was by MS in EI mode, in the authors' laboratory. For interlaboratory comparison of the reference material GC methods with MS, FID, and ECD were used. Different extraction solvents (dichloromethane-isopropanol 2:1, ethyl acetate, or dichloromethane) and derivatisation reagents (trimethylsilyldiazomethane, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide) were used. Using ANOVA (at the statistical level alpha = 0.05) no changes were found in the concentration of butoxyacetic acid during fifteen month isochronous stability testing, or in homogeneity testing. The uncertainty contributions were u (h) = 8.8 mg L(-1) and u (s) = 6.5 mg L(-1). The concentration of butoxyacetic acid in freeze-dried urine RM was evaluated from the results of eight laboratory data sets within an interlaboratory comparison by use of the interactive statistical software IPECA. The contribution to total uncertainty derived from interlaboratory comparison was u (i) = 12.7 mg L(-1). The reference value (c = 273 +/- 33 mg L(-1)) is an unweighted arithmetic average of accepted results. The value is traceable to the pure butoxyacetic acid (98% w/w; Acros Organic #257760010) used as calibrant. The uncertainty given is combined expanded uncertainty derived from the results from interlaboratory comparison, and from homogeneity and stability tests (k = 2). The reference material will be used to verify method performance in the biological monitoring of occupational exposure to EGBE.

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