Simultaneous determination of urinary creatinine and metabolites of toluene,xylene, styrene,ethylbenzene and phenol by automated high performance liquid chromatography

Summary An attempt was made to establish a method for the direct determination of urinary concentrations of creatinine, hippuric acid, methylhippuric acid and mandelic acid by automated high performance liquid chromatography (HPLC). Urine was diluted with distilled water or mobile phase, then the mixture was centrifuged and the supernatant was injected into HPLC. A stainless-steel column packed with octadecyl silanized silicate was used, and the mobile phase was a solution of [20 mM potassium phosphate monobasic containing 3 mM sodium 1-decanesulfonate]/acetonitorile (85/15). Another HPLC method for the determination of urinary concentration of phenol, metabolites of benzene and/or phenol is also described. Phenyl sulfate and phenyl glucuronide in urine were hydrolyzed enzymatically into phenol. The hydrolyzed mixture was injected into HPLC with the ODS column. The mobile phase was a solution of [20 mM potassium phosphate monobasic containing 1 mM sodium 1-decanesulfonate]/acetonitorile (85/15). The ratio of hippuric acid (HA) concentration to creatinine concentration determined by the urine of students after physical exercise was similar to that before exercise. Moreover, the coefficient of correlation found between the toluene concentration in a workshop and the HA concentration in workers' urine, corrected for creatinine, was higher than that obtained between the toluene concentration and the uncorrected HA concentration. For assays on stored urine samples, urine was spotted on filter paper, dried and kept several weeks, and then MA, HA, o-MHA, m-MHA and creatinine in the filter paper were eluted with 50% methanol and their concentrations determined by HPLC.     

Quantitation of urinary metabolites of toluene,xylene, styrene,ethylbenzene, benzene and phenol by automated high performance liquid chromatography

Summary An automated high performance liquid chromatographic method (HPLC) for the direct determination of urinary concentrations of hippuric acid (HA), and o-, m- and p-methyl hippuric acids (MHAs), metabolites of toluene and o-, m-, and p-xylenes, and of urinary phenyl glyoxylic acid (PGA) and mandelic acid (MA), metabolites of styrene or ethylbenzene, is described. Methanol was added to urine, the mixture was centrifuged and the supernatant was injected into HPLC. A stainless-steel column packed with octadecyl silanized silicate was used and the mobile phase was a mixed solution of 5 mM potassium phosphate monobasic/acetonitrile (90/10). The method is simple and specific. Urine can be analyzed without solvent extraction. Analysis can be performed satisfactorily within 45 min for samples containing HA, MHAs, PGA and MA, and within 15 min for those containing HA, PGA and MA. Another automated HPLC method for the determination of urinary concentrations of phenylsulfate (PhS) and phenylglucuronide (PhG), metabolites of benzene and phenol, is also described. Urine was centrifuged and the supernatant was injected into HPLC. A column packed with octadecyl silicate and a mobile phase of 50 mM of potassium phosphate monobasic/acetonitrile (85/15) were used. The whole analyses and quantitative determination can be performed within 15 min for samples containing PhS and PhG in the worker's urine with a simple mobile phase. The accuracy and precision in the present methods by the use of automated HPLC were satisfactory.     

Urinary excretion of phenols as an indicator of occupational exposure in the coke-plant industry

Objective: In the present study the relationship between the level of exposure to o-cresol and of 2,4- +2,5-, 3,4-, and 3,5-xylenols and the urinary excretion of their metabolites was examined. The mixed exposure to phenolic derivatives of exposed workers during their work shift was monitored by personal air sampling of the breathing-zone air and by measurements of phenol, o-cresol, and xylenol isomer concentrations in shift-end urine. Methods: The study subjects were 76 men working at a coke plant who were 22–58 years old and 34 nonexposed subjects. Concentrations of phenolic compounds were determined in the breathing-zone air during the work shift, whereas concentrations of phenol, cresol, and xylenol isomers were measured in urine collected after the work shift. Concentrations of phenols in air and urine were determined by gas chromatography with flame-ionization detection. Urine samples were extracted after acid hydrolysis of glucuronides and sulfates by solid-phase extraction. The gas chromatography-mass spectrometry method was applied to identify metabolites in urine samples. Results: The time-weighted average concentrations of phenol, cresol, and xylenol isomers detected in breathing-zone air showed that the exposure level of the workers was relatively low. The geometric mean values were as follows: 0.26 mg/m³ for phenol, 0.09 mg/m³ for o-cresol, 0.13 mg/m³ for p- and m-cresol, and 0.02–0.04 mg/m³ for xylenols at the tar-distillation process. Corresponding urinary concentrations were 10.39, 0.53, and 0.25–0.88 mg/g creatinine for phenol, o-cresol, and xylenol isomers, respectively. The correlation coefficients between the o-cresol and 2,4-, 2,5-, 3,4-, and 3,5-xylenol concentrations measured in urine and in the breathing-zone air were statistically significant, varying in the range of 0.54–0.74 for xylenol isomers and being 0.69 for o-cresol. Conclusion: We have found that the presence of o-cresol and xylenol isomers in urine can be used as a biomarker for phenol exposure. Analysis performed on workers at the tar-distillation process showed that they were exposed to relatively low concentrations of phenolic compounds. Received: 15 October 1996 / Accepted: 5 May 1997     

A simple determination of phenol in urine by high performance liquid chromatography

A simple analytical method for urinary phenol by high performance liquid chromatography was tested. This procedure consists of the following three steps: hydrolysis of urine with hydrochloric acid, extraction of phenols with isopropylether, separation and determination of phenol by high performance liquid chromatography, under the following operating conditions: column, LiChrosorb RP 18 (250 mm X 4 mm, I.D.); mobile phase, acetonitrile-water (3: 7, v/v) with a flow rate of 0.8 ml/min; column temperature, 40 degrees C; detection, UV monitor at 270 nm. The detection limit (signal-to-noise ratio = 2) was 1 ng. The analytical results by this method were compared with those obtained by another method used for gas chromatography. The analytical results obtained by the two methods were in good agreement (r = 0.998) for 30 normal urine samples tested. The geometrical average value for phenol in normal urine samples (n = 30) was 8.2 micrograms/ml. Therefore this method was proved to be useful for biological monitoring of benzene exposure.     

High performance liquid chromatography for the quantitative determination of urinary phenylsulfate and phenylglucuronide as indices of benzene and phenol exposure in rats

Summary A high performance liquid chromatographic method for the determination of phenylsulfate and phenylglucuronide, metabolites of benzene, or phenol in urine is described. For the determination of urinary phenylsulfate, a stainless-steel column packed with octadecyl-silanized silica gel was used, and the mobile phase was mixed with a solution of methanol/water acetic acid (20/80/0.2) containing 0.05 M tetra-n-butylammonium bromide. For the exact determination of urinary phenylglucuronide, a stainless-steel column packed with the same resin was used and the mobile phase was a solution of methanol/water/acetic acid (50/50/0.2) containing 0.05 M tetra-n-butylammonium bromide. The method is simple and specific. Urine can be analyzed directly, without solvent extraction or pretreatment. The method has a lowest detection limit of 0.2 g on the column. The whole analysis and quantitative determination can be performed within about 15 min for samples containing phenylsulfate and phenylglucuronide in the urine of rats.The phenylsulfate excreted was equivalent to 36.1% and phenylglucuronide was 51.8% of phenol administered to rats intraperitoneally.     

Determination of dimethylhippuric acid isomers in urine by high-performance liquid chromatography

 In order to analyse metabolites in urine after trimethylbenzene (TMB) exposure a method based on high-performance liquid chromatography (HPLC) for determination of the six dimethylhippuric acids (2,3-DMHA, 2,6-DMHA, 2,5-DMHA, 2,4-DMHA, 3,4-DMHA and 3,5-DMHA) in urine has been developed. In contrast to earlier published methods, the present method allows detection of all possible isomers of DMHA in a single analysis. The DMHAs were extracted from urine with dichloromethane. After evaporation, the residue was dissolved in mobile phase and analysed by a stepwise gradient HPLC system with ultraviolet (UV) detection at 225 nm. Mobile phase A (1.25% acetonitrile and 0.3% acetic acid in water) was used up to a retention time of 59.5 min and mobile phase B (5% acetonitrile in water containing 0.3% acetic acid) was used for completion of the analysis at approximately 90 min. The DMHA isomers were chromatographed on a reversed phase Radial-Pak C₁₈ column (4 μm; 100 mm×5 mm inner diameter). The detection limit for the six isomers was 1.5 μg/ml (range 0.5–3.4, 100 μl injection volume). The precision of the method was 4.2% relative standard deviation (range 3.8–4.4; 100 μg/ml). Standard curves of the DMHAs were linear over the interval 10–500 μg/ml in human urine. Individual DMHAs or the sum of DMHA isomers may be used as biological indicators of occupational exposure to TMBs. Received: 18 January 1996/Accepted : 7 June 1996     

高效液相色谱法测定工人尿中三硝基甲苯及五种代谢物(二)分析方法的建立及应用

本文建立了高效液相色谱(HPLC)测定工人尿中TNT及五种代谢物的方法.TNT、4-A、2-A、2,6-DA和2,4-DA的平均回收率66.6～95.0%,变异系数在2.1～10.6%之间,最小检测限为3.0～8.1μg/L尿.本方法已用于35个接触TNT工人尿样的分析.     

高效液相色谱法测定工人尿中三硝基甲苯及五种代谢物(二)——分析方法的建立及应用

本文建立了高效液相色谱(HPLC)测定工人尿中TNT及五种代谢物的方法。TNT、4-A、2-A、2,6-DA和2,4-DA的平均回收率66.6～95.0％,变异系数在2.1～10.6％之间,最小检测限为3.0～8.1μg/L尿。本方法已用于35个接触TNT工人尿样的分析。     

Improved high performance liquid chromatography determination of hippuric acid and methylhippuric acid isomers in urine

A study was made on the effect of detection wavelength and separation mode of HPLC on determination of urinary hippuric acid (HA) and three isomers of methylhippuric acid (MHA). The interference of other constituents of urine in the determination was effectively decreased by detection at a short wavelength of 227.6 nm. Meta and para MHAs were separated by the addition of beta-cyclodextrine to the mobile phase. Four metabolites were successfully separated from other components of urine by the combination of ODS-silica packed-column and mobile phase (method F). The detection limits were found to be 50 and 5 mg/l for HA and MHAs, respectively. MHAs could not be detected in the non-exposed subjects. Average levels (+/- SD) of HA in non-exposed males and females were 272.2 (+/- 210.8) and 393.0 (+/- 269.8) mg/l, respectively. The urinary levels of HA in females were significantly higher than those in males.     

高效液相色谱与液质联用测定尿中苯巯基尿酸的方法比较

[目的]通过高效液相色谱（HPLC）与液质联用（LC/MS）检测尿中苯巯基尿酸（SPMA）的方法比对,评价HPLC方法的可行性。[方法]尿样经50%硫酸酸化、氯仿：异丙醇（体积比为5：1）萃取,挥干残渣用甲醇溶解后经十八烷基硅烷（ODS）柱分离,分别采用紫外检测器和质谱检测器检测;对86名职业苯接触者尿样采用两种方法比对分析,以LC/MS分析数据评定HPLC测定结果的有效性。[结果]HPLC方法和LC/MS方法对SPMA的检出限（LOD）分别为40μg/L和10μg/L,SPMA保留时间分别为31 min和7.8 min左右。86份尿样中有13份为假阳性,两方法检测结果的相关系数为0.915,但HPLC检测数据高估LC/MS数据8%-21%。[结论]HPLC法可作为职业苯接触者生物监测的初步筛检方法,LC/MS可准确定量测定尿中SPMA浓度。     

A method for the collection and determination of phenol and bisphenol A in air

An analytical method has been developed for the determination of phenol and bisphenol A in air. Air samples are concentrated on commercial silica by the well established tube-pump method. The trapped components are desorbed and analyzed by HPLC using UV detection at 275 nm. The method has been validated for a concentration range of 0.5-50 mg m-3 (based on a 10-l air sample) for both compounds. Linearity of response, adsorption/desorption efficiency, influence of humidity on sample collection and influence of temperature and time on storage of collected samples were studied. The reported technique provides a selective and sensitive method for environmental monitoring in industrial atmospheres as well as for personnel exposure measurement.     

高效液相色谱法检测化妆品中氢醌和苯酚

目的:为快速、灵敏、简便的检测祛斑类化妆品中禁用物质氢醌、苯酚的含量,建立二极管阵列检测器高效液相色谱分析法。方法:用甲醇溶解,超声波提取样品后,在适当的色谱条件下,用紫外吸收谱图和保留时间定性测定。结果:该法线性良好,相关系数氢醌为r1=0.9997,苯酚r2=0.9999;氢醌回收率为94.5%-95.7%,苯酚回收率为98.8%-100%。结论:方法分离效果好,简便快捷,灵敏度高,重复性好,适用于各类化妆品中氢醌、苯酚测定,为首选方法。     

尿中双酚S测定的涡旋辅助液液微萃取-高效液相色谱法

目的:建立涡旋辅助液液微萃取-高效液相色谱法测定尿中双酚S的方法。方法:以乙腈为分散剂,正辛醇为萃取剂,萃取尿中双酚S。采用单因素轮换实验确定最佳萃取条件,并评估方法学性能指标。结果:尿中双酚S在0.0~160 μg/L范围线性相关系数大于0.999,检出限为0.76 μg/L,样品加标回收率为88.06%~103.8...     

Determination of Thio Compounds in Urine of Workers Exposed to Carbon Disulfide

Metabolites of carbon disulfide excreted into the urine of viscose workers are detectable by application of a nonselective assay for the determination of thio compounds. Enhanced secretion of thio compounds occurred especially at the end of the workday in exposed workers. A classification of urine samples according to the degree of exposure gave evidence for a relation between exposure to carbon disulfide and excretion of thio compounds in urine. The presence of 2-thiothiazolidine-4-carboxylic acid in urine of viscose workers was indicated by TLC, UV-spectrometry, and HPLC.     

Benzene and leukemia,Pliofilm revisited: II. Take-home leukemia

Abstract

A highly sensitive high-pressure liquid chromatography (HPLC) method was developed for determination of mandelic acid (MA) in urine as a marker of exposure to ethylbenzene at low concentrations. This was achieved by the use of a mobile phase (acetonitrile: 60% perchloric acid: water = 50 ml: 100 μl: 950 ml) that has no absorption at 200 nm, the wavelength at which MA shows intense absorption. The detection limit was 0.07 mg/L urine, and the recovery rate was 101% (CV: 2.7%) when MA was added at 250 mg/L to ten urine samples from non exposed subjects. The method was applied for MA determination, in parallel to a method previously developed, to 641 urine samples (from 360 ethylbenzene-exposed workers and 281 nonexposed subjects), and the results were analyzed by regression analysis to examine the relationship between ethylbenzene exposure concentrations and urinary MA concentrations. A smaller intercept on the axis (essentially zero) and a larger correlaiion coefficient (about 0.7) indicate that the present method is more sensitive and selective to MA than the previous method. Further analysis showed that smoking reduces MA excretion in urine, whereas the effect of drinking was not clear.     

Quantitative determination of conjugated linoleic acid isomers by silver ion HPLC in ewe milk fat

The aim of this research was to validate a method for the quantification of conjugated linoleic acid (CLA) isomers in the fat of ewe's milk. The isomers in the form of methyl ester (FAME) are separated by silver ion high performance liquid chromatography (Ag⁺HPLC) and the quantification was based on the measurement of the integrated area under the 231 nm peaks, using external CLA reference standards. The response linearity is maintained in a wide range of concentrations and the quantification limit estimated was of 0.005 μg/injection for the cis-9,trans-11 and trans-10,cis-12 isomers. Referred to the samples this limit would be 0.008 mg/g of fat in the usual working conditions. In the precision assay an RSDr of 2.14% for the isomer cis-9,trans-11 was calculated and for the rest of the CLA isomers varied between 2.08 and 7.5%. The recovery assays were performed adding CLA cis-9,trans-11 triglyceride and the mean recovery was 96%. The method proposed allows the determination of the individual content of different CLA isomers using one single technique, HPLC and supposes saving time and resources.     

Measurement of urinary delta-aminolevulinic acid (ALA) by fluorometric HPLC and colorimetric methods.

We devised a fluorometric HPLC method for determining delta-aminolevulinic acid (ALA) in the urine of lead-exposed workers. With this fluorometric HPLC method and the conventional colorimetric method, the concentrations of urinary ALA in 84 lead workers were determined and compared. In the measurement of urinary ALA at lower levels (< or = 5 mg/1), the value of urinary ALA obtained by the fluorometric HPLC method was much lower than that obtained by the conventional colorimetric method, indicating that the colorimetric method also measures urinary ALA-like compounds such as aminoacetone. On the other hand, the measurement of urinary ALA at higher levels (> 5 mg/1) demonstrated that the ALA value obtained by the fluorometric HPLC method corresponded well with that of the conventional colorimetric method. A correlation coefficient between the fluorometric HPLC method and the colorimetric method was 0.856 for 60 urine samples with ALA < or = 5 mg/1, and 0.996 for 24 urine samples with ALA > 5 mg/1.     

Simple rapid determination of thiamin by a HPLC method in foods, body fluids, urine and faeces

A direct assay for the determination of thiamin in food, faeces, whole blood, serum and urine samples is described. After enzymatic digestion of the thiamin derivates to free thiamin, the whole thiamin content of the sample is detected by converting the thiamin to fluorescent thiochrome. After the extraction of thiochrome with butanol-2, separation by an automated HPLC technique is performed. Reference values in serum, blood, urine, faeces and for food samples are reported.     

抗坏血酸保存连续流动注射法用于生活饮用水中挥发酚和氰化物的测定

目的 对《生活饮用水标准检验方法水样的采集与保存》(GB/T 5750.2-2006)中挥发酚、氰化物水样的保存方法进行优化.方法 水样使用浓度为2.0 g/L抗坏血酸溶液去除残留余氯后,在连续流动注射系统上测定挥发酚、氰化物.结果 该方法预处理水样后,挥发酚和氰化物高低浓度的加标回收率为93.6％ ～105.0％,相...     

人尿中酚,粘糠酸,苯巯基尿酸的生物监测

为了探索接触低浓度苯的生物监测指标，在建立了灵敏、特异的尿中反，反－粘糠酸（ｔ，ｔ－ＭＡ）的高效液相色谱（ＨＰＬＣ）、苯巯基尿酸（Ｓ－ＰＭＡ）的色谱／质谱／质谱（ＬＣ／ＭＳ／ＭＳ）测定方法的基础上，对４９位接触低浓度苯的工人及２０位非职业接触者进行了生物监测。结果表明：在苯接触浓度低于３．２ｍｇ／ｍ３（１ｐｐｍ）的情况下，作业工人的尿ｔ，ｔ－ＭＡ和Ｓ－ＰＭＡ浓度与空气中苯的ＴＷＡ浓度显著相关；尿酚与空气中苯的ＴＷＡ浓度之间的相关性很差。吸烟能增加尿ｔ，ｔ－ＭＡ和Ｓ－ＰＭＡ的浓度。