Determination of dichloromethane, trichloroethylene and perchloroethylene in urine samples by headspace solid phase microextraction gas chromatography-mass spectrometry

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 75 microm 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 degrees 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 0.005 microg/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, 9.32 microg/l and PCE, n=39; median, 0.58 microg/l) and non-exposed individuals (n=120; median concentrations, 0.64, 0.22 and 0.11 microg/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 8.1 h for one subject and 3.8 and 4.3 h for the other.     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

Plasma and urinary selenium in Saudi Arabian patients with dilated cardiomyopathy

We measured selenium (Se) levels in the urine and blood plasma samples of 72 Saudi Arabian patients with dilated cardiomyopathy (DCM) and 70 control subjects of the same origin. To correct for differences in the hydration state of the subjects, the selenium concentration for each urine sample was normalized by dividing it by the concentration of creatinine (CREAT) in the same sample. The median (and range) of the values found for the concentration of Se in plasma, urine, and normalized concentration in urine for the control subjects was 1.306 (0.66–2.50) μM, 0.478 (0.05–2.00) μM, and 56.7 (10.6–426.5) μM Se/M CREAT, respectively, whereas, for the patients, it was 1.246 (0.53–2.45) μM, 0.39 (0.05–1.90) μM, and 75.1 (4.9–656.2) μM Se/M CREAT, respectively. Additionally, the patients were separated into three subgroups according to the severity of their disease state as judged by NYHA procedure, and were then compared to the control group. Only group 4 (the most severe state of the disease) had a significantly lower concentration of urinary Se than the control group. However, the difference became nonsignificant when normalized for CREAT levels. There was no significant difference in the plasma Se levels between the controls and any of the patient groups. As the plasma Se in the control group and in the DCM patients both fell on the low end of the “normal” range, with the patients being marginally lower than the controls, there is no firm evidence from this study to suggest that Se is related to the high incidence rate of DCM found in Saudi Arabia.     

Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels

A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o-phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C₈ column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.     

Urine levels of aluminum after drinking tea

A microwave-assisted acid digestion procedure coupled with a graphite furnace atomic absorption method has been applied in the determination of aluminum (Al) in urine to verify the correlation of free forms of Al in tea infusions and urinary excretion of Al. Significant urinary Al excretion has been found in 24-h urine of four volunteers after tea drinking. However, the difference in amount of Al excretion in urine between the consumption of Oolong (black tea) and Long-Jin (green tea), each of them with unique Al contents and species, was not significant. These findings indicated that the high levels of free Al species in tea infusions did not result in significant change in urinary excretion of the metal, possibly owing to the transformation by ligands present in food and the gastrointestinal tract (GIT). However, it could not be assumed that there was no big difference in absorption of the metal in the human body if fractions of consumed Al retained in the body or excreted by bile or feces were considered.     

Liquid chromatography-tandem mass spectrometric quantification of the dehydration product of tetranor PGE-M, the major urinary metabolite of prostaglandin E(2) in human urine

A LC-MS/MS method has been developed to analyze tetranor PGE-M, the major urinary metabolite of PGE(2), that involves the acid-catalyzed dehydration of tetranor PGE-M and its deuterated (d(6)) analog followed by LC-MS/MS measurement of the dehydrated tetranor PGE-M product (tetranor PGA-M). We also report a method for quantification of creatinine in urine by LC-MS/MS to normalize tetranor PGE-M concentrations with that of urinary creatinine. These methods were used to study the effect of aspirin on urinary tetranor PGE-M levels in healthy male volunteers. Aspirin did not affect urinary creatinine concentrations but decreased urinary tetranor PGE-M concentrations by approximately 44%.     

LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.     

An improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein. Investigation and resolution of factors affecting its quantification.

A rapid, specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. A dilution of 1 in 100 was chosen for routine assay. Whole urine was centrifuged and the dissolved precipitate and supernatant assayed to quantify the proportion of TH glycoprotein of TH glycoprotein initially present in highly aggregated form. This correlated positively and significantly with increasing osmolality, decreasing pH and increasing TH glycoprotein concentration. When the urine was diluted 1 in 100 in water, no TH glycoprotein was precipitated by centrifugation and the measured concentrations were unaffected by alterations of urine pH or calcium concentration or by addition of sodium dodecyl sulphate. Parallelism was demonstrated between the diluted samples and the disaggregated standard preparation. Recovery of added standard to diluted urine varied between 96 and 114%. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4 degrees C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22--56 mg/24 h, based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance.     

Fast determination of urinary S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acid (S-BMA) by column-switching liquid chromatography-tandem mass spectrometry

Benzene and toluene are important industrial chemicals and ubiquitous environmental pollutants. The urinary mercapturic acids of benzene and toluene, S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acids (S-BMA) are specific biomarkers for the determination of low-level exposures. We have developed and validated a fast, specific and very sensitive method for the simultaneous determination of S-PMA and S-BMA in human urine using an automated multidimensional LC-MS-MS-method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry using isotopically labelled S-PMA as internal standard. The lower limit of quantification (LLOQ) for both analytes was 0.05 microg/L urine and sufficient to quantify the background exposure of the general population. Precision within series and between series for S-PMA and S-BMA ranged from 1.0% to 12.2%, accuracy was 108% and 100%, respectively. We applied the method on spot urine samples of 30 subjects of the general population with no known exposure to benzene or toluene. Median levels (range) for S-PMA and S-BMA in non-smokers (n=15) were 0.14 microg/L (<0.05-0.26 microg/L) and 8.2 (1.6-77.4 microg/L), respectively. In smokers (n=15), median levels for S-PMA and S-BMA were 1.22 microg/L (0.17-5.75 microg/L) and 11.5 microg/L (0.9-51.2 microg/L), respectively. Due to its automation, our method is well suited for application in large environmental studies.     

Monitoring of urine nitric oxide (NO) related substrates and immunological competence in hematological malignancy

It has been reported that concentrations of neopterin in the urine are changed according to the host immunological conditions. In the present study, we measured urinary concentration of neopterin in patients with malignant hematological disorders and investigated the relationship between urinary neopterin levels and laboratory indices for cellular immunity. Urine neopterin levels were correlated with serum sIL-2R levels in the patients with malignant lymphoma, and inversely correlated with lymphocyte reactivity with ConA in the patients with acute myelocytic leukemia. However, no significant correlation was observed between urine neopterin levels and lymphocyte reactivity with phytohemagglutinin and pokeweed mitogen, CD4/8 ratio, CD56+ 16+ subset or serum IFN-gamma levels. In the patients with malignant lymphoma, parallel changes in serum sIL-2R and urine neopterin were observed. The presented results suggest that urine neopterin levels are related to the activation of T cells in malignant lymphoma.     

Mutagens in human urine: effects of cigarette smoking and diet

Human urine from smokers and nonsmokers on strictly controlled diets was assayed for mutagenic activity. Two distinct diets were employed in this study. Diet study A consisted of a high-meat, high-fat diet, observed for 5 days, followed by a vegan diet, adhered to for the next 5 days. The vegan diet contained no meat, fish, eggs, or dairy products. It was comprised of soy products, prepackaged vegan dinners, seeds, nuts, fruits, vegetables, beans and herbal teas. Diet study B consisted of 3 days on a typical western diet followed by a macrobiotic diet of grains and fresh vegetables for 5 days. Portions of 24-h urine samples were assayed in Salmonella typhimurium TA1538. The levels of urinary creatinine and cotinine were measured. Mutagenic activity was observed in the urine of most smokers. However, the levels of mutagens in the urine of light smokers were similar to those of nonsmokers. For both nonsmokers and smokers there was a significant increase in urine mutagenicity when volunteers were on the vegan diet. Several nonsmokers on the vegan diet in diet study A had pronounced mutagenic activity in their urine samples, in some instances at higher levels than that in the urine of smokers on a meat diet. In diet study B no clear differences were observed between the meat diet and the macrobiotic diet. In diet studies A and B the mutagenic potency of smokers' urine could not be correlated with cotinine levels alone or with urinary pH. These data suggest that dietary factors can play a dominant role in the mutagenicity of urine concentrates.     

Determination of N-acetylator phenotype using caffeine as a probe compound: a comparison of high-performance liquid chromatography and capillary electrophoresis methods

A test for determining N-acetylator metabolic phenotype has been developed using caffeine as a probe drug. A spot sample of urine is taken, and the unextracted urine is then analysed by micellar electrokinetic capillary chromatography. Phenotype is determined from the peak-area ratio of urinary 5-acetylamino-6-formylamino-3-methyluracil to 1-methylxanthine. Phenotype assignments using this method were compared with those made using a standard high-performance liquid chromatography assay, with good agreement between the two methods. The advantage of the capillary electrophoresis analysis is that no sample extraction is necessary, resulting in a total analysis time of around 20 min, and removing a potential source of error.     

Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract Background. Measurement of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p 相似文献   

172名上海地区成人饮食及尿雌马酚和代谢产物的调查研究

目的确定上海地区成人雌马酚代谢表型及雌马酚的生理范围;把握由于大豆异黄酮负荷而产生的雌马酚产生者比例;调查雌马酚表型和食物摄取频率及有关激素间的关系。方法应用现状调查方法,筛选出172名居住在上海市区健康成年男女。填写问卷获得研究对象日常饮食频率,检测研究对象血清获得血液激素浓度,采用HPLC法分析负荷大豆异黄酮前后尿中雌马酚等大豆异黄酮24 h排泄量,统计产雌马酚者比例及其与摄食频率和激素的关系。结果负荷前雌马酚生理范围0~33.74μmol/24 h,产雌马酚者比例为30.2%,负荷大豆异黄酮后比例提高至53.5%。产雌马酚者与非产雌马酚者之间日常食品摄取频率的差别无统计学意义(P>0.05)。产Eq者血中游离雌二醇的浓度较非产Eq者低(P<0.05)。结论在通常膳食条件下,约有1/3上海成人尿液中能检测到雌马酚,但负荷大豆异黄酮后,约有1/2能产生雌马酚。     

Two cases of benign methylmalonic aciduria detected during a pilot study of neonatal urine screening

Two cases of benign methylmalonic aciduria (MMAuria) were found among 9780 neonatal screenings using the previously described screening method consisting of urease digestion, ethanol deproteinization and gas chromatography-mass spectrometry. Combining this screening method with the stable isotope dilution technique showed very specific and sensitive measurements of methylmalonic acid in urine. The concentrations of urinary methylmalonic acid were measured at several ages. The levels of urinary methylmalonic acid in two patients varied from 0.27 to 3.04 mol/mol creatinine (control<0.01 mol/mol creatinine). Methylcitrate and homocystine were not increased in the patient's urine or blood. Blood propionylcarnitine was also at normal levels. The urinary methylmalonate excretions were decreased to the levels of about 50% of the start point after vitamin B12 treatment in one patient, but the other patient showed no change. No clinical abnormalities were observed during these periods.     

Amine metabolite profile of normal and uremic urine using gas chromatography—mass spectrometry

A method for the simultaneous analysis of phenolic amines and aliphatic amines in human urine is described. The amine metabolites in urine were extracted using Dowex 50W-X8 cationic resin, derivatized and analyzed by a gas chromatographic—mass spectrometric—computer system. The amine metabolites profile of 5 ml of urine was obtained with good gas chromatographic separation. The gas chromatographic method described here separates urinary phenolic amines, di- and polyamines and methylguanidine in a single chromatographic separation. The urinary levels of methylguanidine, putrescine, cadaverine, spermidine, p-tyramine, dopamine, and 3-methoxytyramine were quantitated by using a mass spectrometric technique. In uremic patients, only the urinary excretion of methylguanidine was increased in comparison with normal subjects, although the urinary excretion of other amines was decreased in uremic patients.     

Simple Disposable Method for Quantitative Cultures of Urine

A disposable kit was tested as a means of detecting significant bacteriuria by quantitative culture of urine. The total error in 3,563 specimens tested by five investigators was less than 1%. The method was very effective in differentiating significant bacteriuria, i.e., more than 100,000 bacteria per ml of urine from uninfected urine. In specimens from patients with urinary tract abnormalities who had mixed bacterial flora, the absolute numbers obtained with the dip-inoculum method had a 10% variation when compared to results obtained by calibrated loop or dilution pour plate methods. Therefore, the main utility of the kit is for screening and following patients after therapy. A significant delay in time between inoculation of the medium in the kit with the freshly voided urine and incubation of the kit to promote growth did not affect the reliability of the kit as a method of doing quantitative urine cultures to detect bacteriuria.     

内毒素和降钙素原在妇科术后尿路感染中的鉴别诊断价值

目的评价中段尿内毒素和血清降钙素原在妇科术后不同种类细菌尿路感染中的鉴别诊断价值。方法收集临床1205例妇科术后患者中段尿进行细菌培养及内毒素检测,同时对患者进行血清降钙素原检测,比较结果对尿路感染的鉴别诊断价值。结果1205份标本中尿培养出阳性350例,感染率为29．04％,其中298例为均存在留置导尿管,而在剩余400例尿培养阴性的患者中仅仅120例留置导尿管。两组之间差异有统计学意义（χ2=26．78,P〈0．05）。其中革兰阴性杆菌189例（54％）,革兰阳性菌112例（32％）,真菌49例（14％）。在三组患者中,中段尿内毒素在革兰阴性菌引起的术后尿路感染较革兰阳性菌和真菌的患者中明显升高,差异均有统计学意义（P〈0．05）。而对于血清降钙素原在革兰阴性菌和革兰阳性菌感染的患者明显高于真菌尿路感染的患者,差异均有统计学意义（P〈0．05）。而在革兰阴性菌和革兰阳性菌感染的患者中差异无统计学意义（P〉0．05）。结论妇科术后尿路感染与留置导尿管密切相关,革兰阴性菌是引起妇科术后尿路感染的主要致病菌,中段尿内毒素有助于鉴别诊断出革兰阴性菌引起尿路感染,而血清PCT升高时则有助于排除真菌尿路感染。     

F2-isoprostane excretion rate and diurnal variation in human urine

8-Iso-PGF2alpha is formed in vivo by non-enzymatic free radical catalysed oxidation of arachidonic acid. Urinary measurement of this compound has previously been shown to reflect the oxidative stress of the body in human and animal studies. To investigate the normal excretion rate and a possible diurnal variation of 8-iso-PGF2alpha excretion in humans urinary samples were collected from ten healthy volunteers of both sexes at different times during a day and as a 24-h urine sample. The samples were analyzed by a newly developed radioimmunoassay with a specific antibody against free 8-iso-PGF2alpha. There was no diurnal variation in the urinary levels of 8-iso-PGF2alpha during the day in this study. Neither was there any statistically significant difference between the 8-iso-PGF2alpha levels at any time of the day or in the morning urine samples compared to the 24-h urine samples. In conclusion, all urine samples collected at any time of the day, preferably a morning urine sample (representative urine from 6-8 hours), can thus be used to obtain a reliable and adequate value of the amount of the 8-iso-PGF2alpha excretion in urine in healthy individuals.