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Abstract: We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms, respectively “light” and “heavy”, by using light-derivatized standards for the quantification of the heavy-derivatized analytes, and vice versa. To this purpose, 2,4-dinitro-3,5,6-trideuterophenylhydrazine (d 3 -DNPH) has been synthesized and used as “heavy” reagent in combination with commercial “light” DNPH. Using the AIDA method, any unknown concentration of the analyte in the matrix can be calculated without the need of a calibration curve. An external calibration method has been also investigated for comparative purpose. The stability of DNPH and d 3 -DNPH derivatives was verified by excluding any exchange of hydrazones with each other. In the range of concentrations of biological interest, e.g., 2–40nM for MDA and 0.5–10nM for 4-HNE, the derivatization reactions of MDA and 4-HNE with DNPH and d 3 -DNPH showed overlapping kinetics and comparable yields. The MS response of both DNPH and d 3 -DNPH hydrazones was similar. The precision of AIDA, calculated as %RSD, was within 3.2–8% for MDA and 4.5–11% for 4-HNE. Accuracy was tested by analyzing a spiked EBC pool sample and acceptable results (accuracy within 98–108% for MDA and 93–114% for 4-HNE) were obtained by AIDA after subtraction of the blank, which was not negligible. The results of quantitative analysis of MDA and 4-HNE in EBC samples obtained by AIDA assay with four analyses per sample were in good agreement with those obtained by external calibration method on the same samples. [Copyright &y& Elsevier]

Abstract: A method for the determination of volatile chlorinated hydrocarbons, namely dichloromethane (DCM), trichloroethylene (TCE), and perchloroethylene (PCE), in urine samples was developed using headspace solid phase microextraction (HS-SPME) gas chromatography–mass spectrometry (GC–MS). HS-SPME was performed using a 75μm Carboxen-polydimethylsiloxane fiber. Factors, which affect the HS-SPME process, such as adsorption and desorption times, stirring, salting-out effect, and temperature of sampling have been evaluated and optimized. The highest extraction efficiency was obtained when sampling was performed at room temperature (22°C), from samples saturated with salt and under agitation. Linearity of the HS-SPME-GC–MS method was established over four orders of magnitude and the limit of detection was 0.005μg/l for all the compounds. Precision, calculated as %R.S.D. at three different concentration levels, was within 1–8% for all intra- and inter-day determinations. The method was applied to the quantitative determination of TCE and PCE in human urine samples from exposed (TCE, n =5; median, 9.32μg/l and PCE, n =39; median, 0.58μg/l) and non-exposed individuals (n =120; median concentrations, 0.64, 0.22 and 0.11μg/l for DCM, TCE and PCE, respectively. In addition, two cases of acute accidental exposure to DCM are reported, and the elimination kinetics in blood and urine was followed up. The calculated half-lives of urinary and blood DCM were, respectively, 7.5 and 8.1h for one subject and 3.8 and 4.3h for the other. [Copyright &y& Elsevier]

Abstract: Biological monitoring of low level exposure to pollutants is a very challenging analytical activity, and the quality of results is difficult to assess, especially when a certified reference material is unavailable. The aim of this work was to evaluate the reliability of the assays used to measure urinary benzene (Benz-U) and S-phenylmercapturic acid (SPMA), by applying an internal quality control protocol. Urine spot samples from 705 subjects who were either members of the general urban population, gasoline station attendants, or refinery plant workers were assayed for Benz-U and SPMA, using GC/MS and LC/MS/MS, with quantification limits of 15ng/L and 0.10μg/L. The median Benz-U concentration was 263ng/L (60-2789ng/L, 5th-95th percentile), and the median SPMA concentration was 0.19μg/L (<0.1-2.5μg/L, 5th-95th percentile). Linearity of both assays was good, but a less-than-proportional response was found for SPMA concentrations below 1μg/L. Between-run precision and accuracy for Benz-U concentration determination were assessed using quality controls at 120ng/L and 1000ng/L and were 10.3% and 4.8%, and 104.8% and 98.9%, respectively; while the precision and accuracy for SPMA concentration determination at 0.3μg/L, 2.5μg/L, and 20μg/L were 40.3%, 6.2%, and 6.2%, and 48.3%, 96.3%, and 98.8%, respectively. Precision, estimated using duplicates of unknown samples, was 13.4% for Benz-U and 26.5% for SPMA analyses. Control charts for the means of the slope of the linear calibration curve of Benz-U showed good stability of the means over a five-year period. For SPMA, a two-laboratory comparison revealed acceptable agreement between ln-transformed data pairs, with a slope of the linear regression of 0.863 (confidence interval 0.774-0.952), null intercept, and a Pearson''s r value of 0.844. Reliable results were obtained for Benz-U analyses over the entire concentration range, and for high and medium SPMA levels. However, the determination of SPMA concentrations at levels close to the limit of quantification was less reliable. [Copyright &y& Elsevier]