The simultaneous determination of hippuric acid, o-, m-, p-methylhippuric acids, mandelic acid and phenylglyoxylic acid in urine by HPLC

A high-performance liquid chromatographic method is described for the simultaneous determination of six metabolites of aromatic hydrocarbons: hippuric acid (HA) from toluene; o-, m-, p-methylhippuric acids (o-, m-, p-MHA) from xylene; mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene. Metabolites were first extracted from urine by solid phase extraction with anion exchange resin, then isocratically separated on a C8 column with 3 microns particle size, 10 cm length and 3 mm internal diameter. Mobile phase was prepared diluting 16 mL of tetrahydrofuran, 14 mL of acetronitrile and 5 mL of methanol to 500 mL with phosphoric acid/potassium dihydrogen phosphate buffer 0.01 M (pH 2.7). The internal standard was 3-hydroxybenzoic acid. Chromatographic runs were completed in about 21 min. The accuracy and reproducibility obtained make this method useful for the biological monitoring of occupational exposure to toluene, xylene, styrene and ethylbenzene.     

Evaluation of hippuric,phenylglyoxylic and mandelic acids in urine as indices of styrene exposure

Summary In addition to those of phenylglyoxylic and mandelic acids, a distinct increase in hippuric acid level was observed in the urine of factory workers who were exposed to styrene at 50 to 200 ppm for 160 min. Comparison of the postexposure levels with respective non-exposure levels and supplements with rat exposure experiments revealed hippuric acid to be a poor indicator of styrene exposure at a moderate degree, while the other two acids are much more adequate indices. At a higher dose of styrene (e.g., 500 mg/kg i.p. or 100 ppm for 8 hrs for rats), however, the urinary levels of phenylglyoxylic and mandelic acids reach plateaus, while hippuric acid level remained proportional to the amount of styrene given.The biological half-life of styrene in human subjects is approximately 8 hrs as measured by the disappearance of phenylglyoxylic and mandelic acids from urine.This work was supported in part by a grant from the Fujiwara Memorial Foundation     

Simultaneous gas chromatographic determination of urinary mandelic and phenylglyoxylic acids using diazomethane derivatization

Summary A new gas chromatographic method is proposed for the simultaneous determination of mandelic and phenylglyoxylic acids in human urine. The two acids are extracted into ethyl acetate from acidified and ammonium sulphate-saturated urine, converted into methyl esters with diazomethane and chromatographed in this form.Gaseous diazomethane is generated in a simple apparatus and introduced by a mild nitrogen flow into the extract of the investigated urine. Completion of esterification is indicated by an external indicator (chloroform solution of iodine). When the pink colouring of the indicator disappears, further supply of diazomethane is stopped. Since only the exact amount of diazomethane needed enters the reaction, any adverse effects of its excess cannot assert themselves. Phenylglyoxylic acid is transformed quantitatively into the corresponding methyl ester without formation of any secondary products. The method permits simple, rapid and reliable determination of the two above named acids in human urine. It is used with success in biological monitoring of industrial styrene exposure.     

Quantitative determination in urine of hippuric acid and m- or p-methylhippuric acid, metabolites of toluene and m- or p-xylene

Ogata, M., Tomokuni, K., and Takatsuka, Y. (1969).Brit. J. industr. Med.,26, 330-334. Quantitative determination in urine of hippuric acid and m- or p-methylhippuric acid, metabolites of toluene and m- or p-xylene. Improved and more specific methods for the quantitative determination of hippuric and m- and p-methylhippuric acids in urine are described. The acids were extracted from urine with ether/ethanol, which was dried with silica gel, or with ethyl acetate. After removing the solvent by evaporation coloured azlactones were formed by reaction with p-dimethylaminobenzaldehyde in acetic anhydride (DAB, only usable after ether/ethanol extraction) or benzenesulphonyl chloride in pyridine, and the absorbances were measured. The sensitivities were about 4 μg./ml. of urine using DAB, and 20 μg./ml. of urine using benzenesulphonyl chloride reagent. Separation of hippuric and methylhippuric acids was achieved by paper and thin-layer chromatography before estimation. A spot test is also described.     

TLC separation of hippuric, mandelic, and phenylglyoxylic acids from urine after mixed exposure to toluene and styrene.

A method using thin-layer chromatography is described to determine the concentration of hippuric acid, mandelic acid, and phenylglyoxylic acid present in the urine after occupational mixed exposure to toluene and styrene. These substances are known metabolites of toluene and styrene, and therefore the evaluation to mixed exposure to toluene and styrene may be carried out separating these metabolites beforehand. Procedures are proposed to separate the metabolites as follows: (1) separation of hippuric acid from mandelic acid, (2) separation of mandelic acid from phenylglyoxylic acid, and (3) separation of hippuric acid and mandelic acid from phenylglyoxylic acid. The developing reagent p-dimethylaminobenzaldehyde in acetic acid anhydride was used after separation on Kieselgel and Silicagel. The sensitivity of the method was 6 microgram of hippuric acid, 10 microgram of mandelic acid, and 7 microgram of phenylglyoxylic acid with an average recovery of 94%.     

高效液相色谱法测定尿中苯乙醛酸和苯乙醇酸

尿样经溶剂提取、C18反相柱分离后,在225nm波长对苯乙醛酸(PGA)和苯乙醇酸(MA)进行检测。PGA的标准曲线线性范围0.04-0.4mg／ml,检出限(3倍噪声)5．7ng,回收率为95．9％-102.0％,批内精密度(RSD)为4．5％-7．1％,批间精密度为4．6％-8．9％。MA的标准曲线线性范围0．1-1．0mg／ml,检出限(3倍噪声)8．6ng,回收率为86.8％-95．5％,批内精密度为3．1％-5．3％。批间精密度为5．5％-9．5％。该方法被应用于测定苯乙烯接触者尿中PGA和MA,具有快速、准确、简便、灵敏度高的特点。     

Quantitative determination of urinary hippuric acid and m- or p-Methylhippuric Acid as Indices of toluene and m- or p-Xylene exposure by high performance liquid chromatography

Summary A high performance liquid chromatographic method for the determination of hippuric and m-(p-)methylhippuric acids in urine, metabolites of toluene and m-(p-)xylene, is described. A stainless-steel column packed with octadecyl silanized silica gel was used and the mobile phase was a mixed solution of methanol/water/acetic acid (20/80/0.2). The method is simple and specific. Urine can be analyzed directly, without solvent extraction or pretreatment. The method has a lower detection limit of 0.2 g on column. All the analysis and quantitative determination can be performed within about 30 min. for samples containing m-(p-)methylhippuric acid.     

高效液相色谱法测定尿中马尿酸的规范化研究

目的:进一步完善和规范化甲苯接触者尿中马尿酸的高效液相色谱测定法。方法:采用ODS C18柱,流动相为甲醇-0.01 mol/L磷酸(40+60),流量为1.0 ml/min,检测波长228 nm。结果:马尿酸在10μg/ml～500μg/ml范围内线性良好,相关系数r为0.9999,平均加标回收率为99%,平均相对标准偏差RSD为1.1%,最低检测浓度0.41 mg/L。结论:该方法快速、准确、灵敏度高,有良好的重现性和稳定性,可用于职业接触甲苯人群尿中马尿酸的测定。     

高效液相色谱法同时测定食品中的苯甲酸、山梨酸、脱氢乙酸及糖精钠

目的:采用0.3%盐酸乙醇提取,低温下沉淀蛋白质,水定容离心过滤的前处理方法,高效液相色谱法同时测定苯甲酸、山梨酸、脱氢乙酸及糖精钠。方法:色谱柱DIONEX Acclaim120 C18(4.6×250 mm,5μm),甲醇-0.02 mol/L磷酸氢二钾溶液pH 7.0(10∶90)为流动相,流速:1.0 ml/min,检测波长:230 nm(苯甲酸、山梨酸、糖精钠)和293 nm(脱氢乙酸)。结果:苯甲酸、山梨酸、脱氢乙酸及糖精钠在0.1μg/ml～20μg/ml浓度范围内线性关系良好(r>0.9999),检出限小于0.4 mg/kg,其加标回收率在92.6%～105.4%之间,重复测定的RSD≤2.0%。结论:该方法操作简单,灵敏度高,准确性好,可应用于大批量样品的快速检测。     

A thin-layer chromatography method for determining the styrene metabolites mandelic and phenylglyoxylic acid in urine

A thin-layer chromatographic method is described for the determination of mandelic and phenyglyoxillic acid on silicagel (Silufol UV 254) after extraction from urine of styrene exposed workers. The quantitative determination was performed after eluting the spots. Phenylglyoxilic acid was measured at 255 nm and mandelic acid by derivative spectroscopically estimation of the .CH(OH).COOH -chromophore at 217 nm or by a three-wavelength mode, respectively. The recovery in urine was 80-104% for phenylglyoxilic acid and 99-105% for mandelic acid.     

Stereometabolism of ethylbenzene in man: gas chromatographic determination of urinary excreted mandelic acid enantiomers and phenylglyoxylic acid and their relation to the height of occupational exposure

Summary Ethylbenzene is an important industrial solvent and a key substance in styrene production. Ethylbenzene metabolism leads to the formation of mandelic acid, which occurs in two enantiomeric forms, and phenyl-glyoxylic acid. To decide which enantiomer is preferably formed, 70 urine samples of exposed workers were taken at the end of shifts and — after 3-pentyl ester derivatisation — gas chromatographically analysed. The R/S ratio of mandelic acid enantiomers in urine amounts to 19:1, which means that R-mandelic acid is a major metabolite and S-mandelic acid is one of the minor urinary metabolites of ethylbenzene in man. The R/S ratio is independent of ambient air concentration of ethylbenzene within the investigated range. Compared to an ethylbenzene monoexposure the height of total mandelic acid excretion is decreased in the case of coexposure to other aromatic solvents.Dedicated to Professor Dr. med. Heinz Weichardt on the occasion of his 75th birthday     

Capillary gas chromatographic method for mandelic and phenylglyoxylic acids in urine

Summary In support of an occupational investigation of styrene exposure, a capillary gas chromatographic method was developed for the quantitation of the styrene metabolites mandelic and phenylglyoxylic acids. The method was based on that of Guillemin and Bauer [7], in which phenylglyoxylic acid was converted to mandelic acid by reduction before instrumental analysis. The earlier method had to be modified for use with capillary columns; the resulting method was sensitive, selective and reproducible. The detection limit was approximately 0.001 mg/ml urine. Approximately <5% relative precision was achieved in the range of 0.05–2 mg/ml urine. Mandelic acid was resolved from other components of urine and from by-products of derivatization.     

高效液相色谱法同时测定赤砂糖中的过氧化苯甲酰和苯甲酸

目的:建立用高效液相色谱同时测定赤砂糖中的过氧化苯甲酰和苯甲酸的方法。方法:采用Shim-pack C18色谱柱(5μm,4.6 mm×250 mm);以0.1%磷酸-甲醇(25∶75)为流动相;流速1.0 ml/min;检测波长为235 nm;柱温:室温。结果:过氧化苯甲酰和苯甲酸的最低检出限分别为0.11 mg/kg和0.15 mg/kg,平均回收率分别为100.71%和98.55%,RSD分别为0.78%和1.22%。结论:该方法具有准确可靠、操作简便的特点,可有效用于赤砂糖中的过氧化苯甲酰和苯甲酸的检测。     

Simultaneous determination of B-vitamins and ascorbic acid in multi-vitamin preparations by reversed-phase HPLC

The tedious and time consuming methods employed for the analysis of individual B-vitamins can now be replaced by ion-pair reversed-phase high-performance liquid chromatographic (HPLC) methods. This laboratory has previously reported the simultaneous determination of eight water-soluble vitamin standards that is, B1, B2, B6, B12, C, niacin, niacinamide and folic acid. The proposed isocratic HPLC method, employing 3 channels of detection, adequately separated all eight vitamins in less than 20 minutes. This study reports another phase of the project whereby the method was employed for the analysis of pharmaceutical preparations. Different extraction procedures were first evaluated, namely acid, acid plus enzyme and alkaline hydrolysis methods, using vitamin standards, individual vitamin tablets and multivitamin preparations. The amounts obtained from the analysis were compared with the declared values. Recovery studies were also carried out. The method of acid hydrolysis with 0.1N sulphuric acid was found suitable for use and was thus adopted as the extraction procedure for the analysis of 10 multivitamin preparations obtained from various pharmaceutical outlets. For most of these preparations, the amount obtained were close to the declared values, except for folic acid and cyanocobalamin. Further trials on folic acid showed that the problem could be resolved by omitting the filtration step in the final extract after acid hydrolysis and diluting with 0.01N sodium hydroxide before processing for chromatography. Vitamin B12 was not detectable using the present chromatography system probably because of its low concentration in the samples studied.     

The time course of mandelic and phenylglyoxylic acid excretion in workers exposed to styrene under model conditions.

Urinary excretion of mandelic and phenylglyoxylic acids by two technicians building glass-reinforced plastic boats has been measured over a 7-day period. Peak excretion of both metabolites occurred several hours after the end of exposure. There was little relationship between urinary mandelic acid concentrations measured at the end of shift and the maximum excretion observed in samples collected after this time. It is suggested that sampling strategies devised to monitor workers exposed to styrene should reflect maximum excretion rates of urinary mandelic acid.     

The time course of mandelic and phenylglyoxylic acid excretion in workers exposed to styrene under model conditions.

Urinary excretion of mandelic and phenylglyoxylic acids by two technicians building glass-reinforced plastic boats has been measured over a 7-day period. Peak excretion of both metabolites occurred several hours after the end of exposure. There was little relationship between urinary mandelic acid concentrations measured at the end of shift and the maximum excretion observed in samples collected after this time. It is suggested that sampling strategies devised to monitor workers exposed to styrene should reflect maximum excretion rates of urinary mandelic acid.     

High performance liquid chromatographic procedure for quantitative determination of urinary phenyl-glyoxylic,mandelic, and hippuric acids as indices of styrene exposure

Summary A method is described for the quantitative determination of phenyl-glyoxylic, mandelic, and hippuric acids in urine are all metabolites of styrene monomer. After o-methylhippuric acid was added to urine as an internal standard, these three acids and o-methylhippuric acid were extracted with a mixture of ethyl ether and methanol (9/1, by vol.).After 1.0 ml of extract was transferred to a test tube and dried, 0.25 ml of methanol was added. An aliquot of this solution was injected into a high performance liquid chromatography equipped with column packed with octadecylsilicate. Phenylglyoxylic acid was estimated from the peak height at wave length 254 nm. Mandelic, hippuric and o-methylhippuric acids were estimated from their peak height at 225 nm.For a micro-determination of urinary mandelic acid, phenacyl esterification of mandelic acid could be performed by combination with phenacyl bromide and thriethylamine. This was then analyzed at 254 nm using the column packed with silica gel.     

Analysis and stability of phenylglyoxylic and mandelic acids in the urine of styrene-exposed people

Summary In this work a high-performance liquid chromatographic method is described that is reliable and practical for use in routine biological monitoring of exposure to styrene. The method uses a modern diode array detection technique by which mandelic and phenylglyoxylic acids can be measured simultaneously using different wavelengths. The liquid chromatographic method was compared to a gas chromatographic method developed for the analysis of mandelic, phenylglyoxylic and para-hydroxymandelic acids. The methods gave results consistent with each other. These two methods were then used to check the stability of the main metabolites of styrene, especially of phenylglyoxylic acid, in urine samples stored at +6°C or at –18°C for periods up to 70 days. None of the frozen samples showed any significant decrease in the phenylglyoxylic acid concentration, whereas at 6°C one of the samples showed a reduction of 46% after 1 month.