Michael Bader

GC-MS determination of creatinine in human biological fluids as pentafluorobenzyl derivative in clinical studies and biomonitoring: Inter-laboratory comparison in urine with Jaffé, HPLC and enzymatic assays.

In consideration of its relatively constant urinary excretion rate, creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous biomolecules. Assays based on the reaction of creatinine and picric acid first reported by Jaffé in 1886 still belong to the most frequently used laboratory approaches for creatinine measurement in urine. Further analytical methods for creatinine include HPLC-UV, GC-MS, and LC-MS and LC-MS/MS approaches. In the present article we report on the development, validation and biomedical application of a new GC-MS method for the reliable quantitative determination of creatinine in human urine, plasma and serum. This method is based on the derivatization of creatinine (d(0)-Crea) and the internal standard [methyl-trideutero]creatinine (d(3)-Crea) with pentafluorobenzyl (PFB) bromide in the biological sample directly or after dilution with phosphate buffered saline, extraction of the reaction products with toluene and quantification in 1-μl aliquots of the toluene extract by selected-ion monitoring of m/z 112 for d(0)-Crea-PFB and m/z 115 for d(3)-Crea-PFB in the electron-capture negative-ion chemical ionization mode. The limit of detection of the method is 100 amol of creatinine. In an inter-laboratory study on urine samples from 100 healthy subjects, the GC-MS method was used to test the reliability of currently used Jaffé, enzymatic and HPLC assays in clinical and occupational studies. The results of the inter-laboratory study indicate that all three tested methods allow for satisfactory quantification of creatinine in human urine. The GC-MS method is suitable for use as a reference method for urinary creatinine in humans. In serum, creatine was found to contribute to creatinine up to 20% when measured by the present GC-MS method. The application of the GC-MS method can be extended to other biological samples such as saliva.

Quantitative Risk Analysis for N-Methyl Pyrrolidone Using Physiologically Based Pharmacokinetic and Benchmark Dose Modeling

Establishing an occupational exposure limit (OEL) for N-methyl pyrrolidone (NMP) is important due to its widespread use as a solvent. Based on studies in rodents, the most sensitive toxic end point is a decrease in fetal/pup body weights observed after oral, dermal, and inhalation exposures of dams to NMP. Evidence indicates that the parent compound is the causative agent. To reduce the uncertainty in rat to human extrapolations, physiologically based pharmacokinetic (PBPK) models were developed to describe the pharmacokinetics of NMP in both species. Since in utero exposures are of concern, the models considered major physiological changes occurring in the dam or mother over the course of gestation. The rat PBPK model was used to determine the relationship between NMP concentrations in maternal blood and decrements in fetal/pup body weights following exposures to NMP vapor. Body weight decrements seen after vapor exposures occurred at lower NMP blood levels than those observed after oral and dermal exposures. Benchmark dose modeling was used to better define a point of departure (POD) for fetal/pup body weight changes based on dose-response information from two inhalation studies in rats. The POD and human PBPK model were then used to estimate the human equivalent concentrations (HECs) that could be used to derive an OEL value for NMP. The geometric mean of the PODs derived from the rat studies was estimated to be 350 mg h/l (expressed in terms of internal dose), a value which corresponds to an HEC of 480 ppm (occupational exposure of 8 h/day, 5 days/week). The HEC is much higher than recently developed internationally recognized OELs for NMP of 10-20 ppm, suggesting that these OELs adequately protect workers exposed to NMP vapor.

Quantification of N-(3-chloro-2-hydroxypropyl)valine in human haemoglobin as a biomarker of epichlorohydrin exposure by gas chromatography-tandem mass spectrometry with stable-isotope dilution.

Epichlorohydrin (ECH) is an important industrial intermediate for the production of polymers and surface coatings. Animal experiments support the classification of ECH as a carcinogen, and a significant contribution to the cancer risk of ECH exposed humans has to be considered. Upon uptake, epichlorohydrin reacts with nucleophilic moieties of N- and S-containing macromolecules to form stable adducts, e.g. with haemoglobin. In this article, we describe a GC-tandem MS method for the quantitative analysis of the primary ECH adduct to the N-terminal amino acid of human haemoglobin, i.e. of N-(3-chloro-2-hydroxypropyl)valine (CHPV), using a globin labelled with d(5)-ECH as the internal standard. Incubation of erythrocyte lysate from human blood with ECH or d(5)-ECH yielded two reaction products, with CHPV being the major component. The GC-tandem MS method is based on a modified Edman degradation procedure with subsequent O-acetylation. The limits of detection and quantification of this method are 10 and 25 pmol/g globin, respectively. Intra- and inter-assay imprecision of the method was about 12 and 15%, respectively, and the mean recovery was 105 and 96% at the levels of 25 and 100 pmol of CHPV per g globin, respectively. The present study reports for the first time on the analysis of CHPV as a haemoglobin adduct of ECH using GC-tandem MS and a stable-isotope labelled internal standard. By this method we quantified haemoglobin adducts of ECH in the blood of subjects potentially exposed to ECH after a freight train accident. Our study points to CHPV in human haemoglobin as a possible biomarker for epichlorohydrin exposure.

Assessment of long-term health risks after accidental exposure using haemoglobin adducts of epichlorohydrin.

On September 9th, 2002, two goods trains collided in Bad Münder, Lower Saxony, causing the release of more than 40 metric tonnes of epichlorohydrin (1-chloro-2,3-epoxypropane) into the environment. A human biomonitoring study was performed to evaluate the accidental exposure to epichlorohydrin and to assess the possible long-term, i.e. carcinogenic health effects. This was done on the basis of a biochemical effect monitoring using the N-(3-chloro-2-hydroxypropyl)valine and the N-(2,3-dihydroxypropyl)valine haemoglobin adducts of epichlorohydrin in blood to respond to missing ambient monitoring immediately after the crash. N-(3-chloro-2-hydroxypropyl)valine adduct levels above the LOQ (25 pmol/g globin) ranged from 32.0 to 116.4 pmol/g globin in 6 out of 628 samples. The N-(2,3-dihydroxypropyl)valine adduct was not detected above the LOD (10 pmol/g globin) in any of the blood samples. Based on the quantified N-(3-chloro-2-hydroxypropyl)valine adduct values, the body doses after two days of exposure were estimated to be in the range of 1.7-6.2 nmol/kg body weight. The reverse estimation of the external exposure leads to cumulative additional lifetime cancer risks ranging from 2.61×10(-8) to 9.48×10(-8). The estimated excess lifetime cancer risks have to be assessed as extremely low. Our biomonitoring study facilitated the dialogue between individuals and groups concerned and authorities, because suspected or occurred exposures and risks to human health could be quantified and interpreted in a sound manner.