Rapid determination of four short-chain alkyl mercapturic acids in human urine by column-switching liquid chromatography-tandem mass spectrometry.

We developed and validated an analytical method for the simultaneous determination of methyl mercapturic acid (MeMA), ethyl mercapturic acid (EtMA), n-propyl mercapturic acid (PrMA) and iso-propyl mercapturic acid (iPrMA) in human urine. These alkyl mercapturic acids are known or presumed biomarkers of exposure to several alkylating agents including methyl bromide, dimethyl sulfate, ethyl bromide, 1-bromopropane and 2-bromopropane. The method involves a column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using liquid chromatography and tandem mass spectrometry. Within day and day-to-day imprecision was determined to range from 4.5 to 12.2%. The analytical method is distinguished by its wide linear working range of up to 2,500 µg/L with detection limits ranging from 2.0 µg/L (for PrMA) to 5.1 µg/L (for MeMA) that render possible the application in various biomonitoring studies regarding exposure to alkylating agents. The results of a pilot study on urine samples of 30 individuals occupationally non-exposed to alkylating agents using the new procedure confirmed the background excretion of MeMA (<5.1-35.6 µg/L) and PrMA (<2.0-95.7 µg/L).

Mercapturic acids as metabolites of alkylating substances in urine samples of German inhabitants.

Hydroxyalkyl mercapturic acids (HAMA) are the main urinary metabolites of several alkylating substances that possess a carcinogenic potential, like acrolein, 1,3-butadiene, ethylene oxide, propylene oxide and glycidol. These alkylating substances are used extensively in industrial processes, but they do also occur environmentally, e.g. in tobacco smoke. The aim of this study was the determination of six HAMA, as biomarkers of exposure, in human urine of smokers and non-smokers. We applied a sensitive analytical method, using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) for the determination of 2-hydroxyethyl mercapturic acid (HEMA, biomarker for ethylene oxide), 2-hydroxypropyl mercapturic acid (2-HPMA, biomarker for propylene oxide), 3-hydroxypropyl mercapturic acid (3-HPMA, biomarker for acrolein), 2,3-dihydroxypropyl mercapturic acid (DHPMA, biomarker for glycidol) as well as 3,4-dihydroxybutyl mercapturic acid and 3-monohydroxybutenyl mercapturic acids (DHBMA and MHBMA, biomarkers for 1,3-butadiene). Background concentrations of four HAMA were detected in each urine sample we analyzed. The mercapturic acids HEMA and MHBMA were detected in 55% and 10% of the samples, respectively. In the urine of non-smokers (n = 54) we observed median levels of 206, 1.6, 12.1, 146, 159, and <5.0 µg/g creatinine for DHPMA, HEMA, 2-HPMA, 3-HPMA, DHBMA and MHBMA, respectively. Among smokers (n = 40) median levels of DHPMA, HEMA, 2-HPMA, 3-HPMA, DHBMA and MHBMA were determined to be 217, 4.9, 46.2, 884, 211 and <5.0 µg/g creatinine, respectively. The excretion rate of the biomarkers HEMA, 2-HPMA and 3-HPMA was distinctly higher in smokers than in non-smokers. Furthermore, our study revealed a comparatively high background level of DHPMA in urine of smokers and non-smokers whose origin is still unknown. The presented data may contribute to the evaluation of reference values for urinary HAMA levels in the general population.

Influence of preservatives on the stability of ethyl glucuronide and ethyl sulphate in urine.

BACKGROUND: Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are specific and sensitive markers of ethanol consumption well established in monitoring withdrawal treatment in patients with chronic alcoholism. Recently, bacterial decomposition as well as in vitro and post-mortem formation of EtG was reported. The aim of this study was to investigate the influence of different preservatives on the stability of EtG and EtS concentrations in urine samples. METHODS: Urine samples were doped with glucuronidase-positive Escherichia coli after sterile filtration. The preservatives used were thymol, chlorhexidine, boric acid and the combination of chlorhexidine, ethylparabene and sodium propionate. Different aliquots of urine samples were stored refrigerated (4-8 degrees C), at room temperature (18+/-1 degrees C) and in an incubator (36+/-1 degrees C) for a period of 9 days with daily sampling. EtG and EtS analyses were performed by LC-ESI-MS/MS. The number of bacteria was detected by counting the colony forming units on Columbia blood agar plates. RESULTS AND CONCLUSIONS: Chlorhexidine on its own as well as in the aforementioned combination, and boric acid proved useful preservatives, while EtG degraded in samples doped with thymol. Addition of these preservatives did not interfere with the LC-MS/MS analysis.

Quantitative determination of urinary N7-ethylguanine in smokers and non-smokers using an isotope dilution liquid chromatography/tandem mass spectrometry with on-line analyte enrichment.

Previous studies demonstrated the presence of unknown direct-acting ethylating agents arising from cigarette smoke. We hypothesized that such agents would also lead to ethylation of guanine in DNA followed by depurination/repair and excretion of N7-ethylguanine (N7-EtG) in urine. In this study, a highly specific and sensitive liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was firstly developed for measuring urinary N7-EtG. With the use of an isotope internal standard (15N5-N7-EtG) and on-line enrichment techniques, the detection limit of this method was estimated as 0.59 pg/ml (0.33 pmol) on-column. This method was then applied to measure urinary samples obtained from 35 non-smokers and 32 smokers with dietary control. The results showed that the mean urinary levels of N7-EtG were 85.5+/-105 and 28.1+/-19.4 pg/mg creatinine for smokers and non-smokers, respectively. Smokers had about three times higher level of N7-EtG than non-smokers (P<0.005). It was further noted that the urinary level of N7-EtG was significantly associated with cotinine for smokers (r=0.49, P<0.005). Taken together, this is the first study that demonstrated the presence of N7-EtG in urine, and that cigarette smoke was highly responsible for the increased urinary excretion of N7-EtG. This non-invasive measurement of urinary N7-EtG would be useful for the surveillance of ethylating agent exposure and its associated cancer risk in the future.

[Clinical aspects of the excretion of alkylating cyclophosphamide metabolites in the urine of tumor bearing patients]. / Untersuchungen zur Ausscheidung alkylierender Cyclophosphamid-Metabolite im Urin von Tumorpatienten unter klinischen Aspekten.

The urinary excretion of alkylating CP metabolites was measured in 33 tumor patients by using NPB-test under consideration of different doses, routes of administration (i.v. and i.m.) and function of the liver and the kidney. There was no limit in activation capacity with increased doses of CP. The estimation of the upper activation limit was hindered by the host's subjective toxicity. CP was activated in a comparable manner after i.v. or i.m. administration. It is of interest that liver involvement by the disease or functionally compensated post-nephrectomy state of patients investigated so far did not diminish the excretion of alkylating CP metabolites.

Pharmacokinetics of divided-dose ifosfamide.

The pharmacokinetics of a divided-dose schedule of ifosfamide was investigated in 3 patients given 1, 600 to 2,400 mg/sq m/day for 3 days, with a second course of treatment 21 days later. In contrast to the biexponential decay seen with single-dose ifosfamide (5,000 mg/sq m), data for divided low-dose plasma ifosfamide are best fitted by a monoexponential decay function compatible with a one-compartment open model. Plasma half-life for ifosfamide given in the divided-dose schedule was 6.9 hr, less than half that previously reported for high single-dose ifosfamide. Renal excretion rates and clearances for unchanged drug were similar in both schedules. The proportion of drug metabolized was larger and amount of unchanged drug excreted in the urine was smaller than after single large doses. The elimination constant for unchanged ifosfamide increased from day 1 to day 2 of treatment and remained relatively stable from day 2 to day 3 in the multidose regimen, with all parameters reverting to the pretreatment values 21days later.