PubMed · 14082761
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S P BYSTROV. [INDICATORS FOR IODOMETRY].. https://pubmed.ncbi.nlm.nih.gov/14082761/
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A two-phase system is developed here for converting: (1) benzene to phenol and (2) naphthalene to 2-naphthol, using whole cells expressing wild-type toluene 4-monooxygenase (T4MO) and the alpha subunit variant TmoA I100A from Pseudomonas mendocina KR1. Using the T4MO TmoA I100A variant, the solubility of naphthalene was enhanced and the toxicity of the naphthols was prevented by the use of a water/dioctyl phthalate (80:20, vol%) system which yielded 21-fold more 2-naphthol. More than 99% 2-naphthol was extracted to the dioctyl phthalate phase, dihydroxynaphthalene formation was prevented, 92% 2-naphthol was formed, and 12% naphthalene was converted. Similarly, using 50 vol% dioctyl phthalate, an initial concentration of 3.0 g l(-1) (39 mM), and wild-type T4MO, a 51+/-9% conversion of benzene was obtained and phenol was produced at a purity of 97%. Relative to the one-phase system, there was a 12-fold reduction in the formation of the byproduct catechol.
Benzene is a human carcinogen and its metabolite, urinary trans,trans-muconic acid (ttMA), is a biomarker for risk assessment. However, most of the existing methods were not sensitive enough for monitoring of low level exposure. This paper describes a HPLC-UV method for ttMA determination with enhanced selectivity and sensitivity. A 30 mg OasisMAX cartridge was used to clean-up 50 microl of urine sample and gradient elution was performed on a Zorbax SB-C(18) column (30 degrees C). ttMA was detected at wavelength 263 nm using a UV diode array detector (DAD). The two mobile phases used were (A) 150 mM ortho-phosphoric acid containing of 9% (v/v) methanol; and (B) 125 mM ortho-phosphoric acid containing 30% (v/v) acetonitrile. The method was validated with 61 urine samples collected from non-occupationally benzene exposed individuals and 14 quality control specimens from an international quality assessment scheme. The urinary ttMA concentrations (mean+/-S.D.microg/g creatinine) were 90+/-34 for smokers (n=26), 49+/-39 for non-smokers (n=21) and 23+/-18 for non-smoking hospital staff (n=14). A correlation coefficient, r=0.99 was found with 14 external quality specimens for ttMA ranged from 0.4 to 6.8 mg/l. The recovery and reproducibility were generally over 90% and the detection limit was 5 microg/l.
Urinary benzene is used as biomarker of exposure to evaluate the uptake of this solvent both in non-occupationally exposed population and in benzene-exposed workers. The quantitative determination of benzene in urine is carried out in a three steps procedure: urine collection, sample analysis by head space/solid phase microextraction/gas chromatography/mass spectrometry and analyte quantification. The adopted quantification method influences the initial step, hence the whole procedure. Two quantification approaches were compared as regards precision and accuracy: the calibration curves and the standard addition method. Even if calibration curves obtained by using urine samples from different subjects were always linear, their slopes and intercepts showed noteworthy variations, attributable to the influence of the biological matrix on benzene recovery. The standard addition method showed to be more suitable for compensating matrix effects, and a three-point standard addition protocol was used to quantify benzene in urine samples of 11 benzene-exposed workers (smokers and non-smokers). Urine from occupationally exposed workers was collected before and after work-shift. Besides urinary benzene, the applicability of the method was verified by measuring the urinary concentration of the S-phenylmercapturic acid, a specific benzene metabolite, generally adopted as biomarker in biological monitoring procedures. A similar trend of concentration levels of both analytes measured in urine samples collected before work-shift with respect to the after work-shift ones was found, showing the actual applicability of the standard addition method for biological monitoring purposes.