固相萃取-高效液相色谱-串联质谱法同时测定尿液中的4种巯基尿酸

建立了固相萃取-高效液相色谱-串联质谱(HPLC-MS/MS)同时检测人体尿液中N-乙酰基-S-(3,4-二羟基丁基)-L-半胱氨酸(DHBMA)、N-乙酰基-S-(3-羟基丙基)半胱氨酸(3-HPMA)、N-乙酰基-S-(2-氰乙基)-L-2-氨基-3-巯基羧酸(CEMA)和苯巯基尿酸(SPMA)的检测方法。冰冻的人体24 h尿液在室温下解冻,混合均匀后离心过滤,经C18固相萃取小柱净化富集后在多反应监测模式下采用HPLC-MS/MS进行定量分析。在3个添加水平下,尿液中DHBMA、3-HPMA、CEMA和SPMA的加标回收率分别为105.6%～124.4%、102.7%～106.5%、103.2%～103.9%和101.7%～104.3%,相对标准偏差为2.6%～7.7%。以不低于3倍的信噪比估算DHBMA、3-HPMA、CEMA和SPMA的检出限分别为0.062、0.031、0.020和0.003 μg/L。应用该方法检测了37例吸烟和非吸烟者的24 h尿液样本,结果发现吸烟者尿液中3-HPMA、SPMA和CEMA的平均含量比非吸烟者高3到6倍。     

Determination of N-acetyl-S-(N-alkylthiocarbamoyl)-L-cysteine, a principal metabolite of alkyl isothiocyanates, in rat urine

A simple and rapid analytical procedure is described for N-acetyl-S-(N-alkylthiocarbamoyl)-L-cysteine (alkyl = benzyl, allyl, methyl, ethyl or n-butyl), a mercapturic acid with an unstable dithiocarbamic acid ester structure, which is found in rat urine as the principal metabolite of the corresponding alkyl isothiocyanate. Because such mercapturic acids decompose at pH values greater than 5 to N-acetylcysteine and alkyl isothiocyanate, the free isothiocyanate is converted with n-butylamine into the corresponding disubstituted thiourea, and, after extraction, measured by high-performance liquid chromatography using an ultraviolet detector. The recovery is ca. 100% and the precision is very good. The lower limit of detection is ca. 0.5 microgram of thiourea. The 24-h renal excretion of these mercapturic acids was determined in rats after administration of benzyl, allyl, methyl, ethyl or n-butyl isothiocyanate.     

Simultaneous determination of mercapturic acids derived from ethylene oxide (HEMA), propylene oxide (2-HPMA), acrolein (3-HPMA), acrylamide (AAMA) and N,N-dimethylformamide (AMCC) in human urine using liquid chromatography/tandem mass spectrometry

Mercapturic acids are highly important and specific biomarkers of exposure to carcinogenic substances in occupational and environmental medicine. We have developed and validated a reliable, specific and very sensitive method for the simultaneous determination of five mercapturic acids derived from several high-production chemicals used in industry, namely ethylene oxide, propylene oxide, acrylamide, acrolein and N,N-dimethylformamide. Analytes are enriched and cleaned up from urinary matrix by offline solid-phase extraction. The mercapturic acids are subsequently separated by means of high-performance liquid chromatography on a Luna C8 (2) column and specifically quantified by tandem mass spectrometric detection using isotopically labelled analytes as internal standards. The limits of detection (LODs) for N-acetyl-S-2-carbamoylethylcysteine (AAMA) and N-acetyl-S-2-hydroxyethylcysteine (HEMA) were 2.5 microg/L and 0.5 microg/L urine, while for N-acetyl-S-3-hydroxypropylcysteine (3-HPMA), N-acetyl-S-2-hydroxypropylcysteine (2-HPMA) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC) it was 5 microg/L. These LODs were sufficient to detect the background exposure of the general population. We applied the method on spot urine samples of 28 subjects of the general population with no known occupational exposure to these substances. Median levels for AAMA, HEMA, 3-HPMA, 2-HPMA and AMCC in non-smokers (n = 14) were 52.6, 2.0, 155, 7.1 and 113.6 microg/L, respectively. In smokers (n = 14), median levels for AAMA, HEMA, 3-HPMA, 2-HPMA and AMCC were 243, 5.3, 1681, 41.7 and 822 microg/L, respectively. Due to the simultaneous quantification of these mercapturic acids, our method is well suited for the screening of workers with multiple chemical exposures as well as the determination of the background excretion of the general population.     

Mercapturic acids derived from toluene in rat urine samples: identification and measurement by gas chromatography–tandem mass spectrometry

Toluene is one of the most widely used CMR chemicals in industry. Worker exposure to this compound is regulated in France, but new, more sensitive methods are required to effectively monitor this exposure. A gas chromatography-tandem mass spectrometry (GC-MS/MS) method was developed and fully validated for the simultaneous determination of urinary toluene mercapturic acids derived from side chain and ring oxidation, i.e., benzylmercapturic acid and the three isomers o-, m- and p-toluylmercapturic acids, respectively. The method involves a simple and efficient two-step preparation procedure consisting of liquid-liquid extraction of the urinary acids followed by a microwave-assisted esterification of the isolated compounds using 2-propanol. The method meets all the required validation criteria: high selectivity, intra-day and inter-day precision ranges between 1.0?% and 12.4?%, with close to 100?% recovery. Linearity has been shown over the reduced concentration range 0.03-0.5?mg/L whereas a multiplicative model (ln-ln transformation) had to be used to describe the full range of concentrations 0.03-20?mg/L. The limits of detection for the four analytes, ranging from 2.8 to 5.5?μg/L, made the method suitable for their identification and quantification in urine from rats inhaling toluene in the 2 to 200?ppm concentration range. All urine samples from exposed rats contained measurable amounts of all metabolites. This is the first time that o- and m-toluylmercapturic acids have been shown to occur. Our results confirm the hypothesis that toluene mercapturic acids derived from ring oxidation exist in three forms.     

Simultaneous determination of metabolites of trimethylbenzenes,dimethylbenzylmercapturicacid and dimethylhippuric acid,in human urine by solid-phase extraction followed by liquid chromatography tandem mass spectrometry

We describe a novel method for the determination of three kinds of dimethylbenzylmercapturic acids (DMM) and six kinds of dimethylhippuric acids (DMH), found in urine as metabolites of trimethylbenzenes, based on liquid chromatography/electrospray ionization tandem mass spectrometry. A solid-phase extraction procedure was used for the extractions of DMM and DMH from a urine sample, and the separation was performed on a reversed-phase C(30) column. The analytes were ionized by electrospray in the positive-ion mode. Operating in the multiple reaction monitoring mode, the linearity of the relative mass spectrometric responses to the internal standard versus analyte concentrations were established in the range 0.1-100 ng ml(-1). The extraction procedure was rapid and the relative standard deviations were below 5%. The detection limits of DMM and DMH in the urine by the proposed method were in the ranges 0.26-0.41 and 0.42-2.0 ng l(-1), respectively. Furthermore, DMM and DMH were detected in a urine sample from an individual who did not suffer from occupational exposure to trimethylbenzenes, by using this method.     

Determination of Acrolein‐Derived 3‐Hydroxypropylmercapturic Acid in Human Urine Using Solid‐phase Extraction Combined with Molecularly Imprinted Mesoporous Silica and LC‐MS/MS Detection

A novel method for the analysis of (3‐hydroxypropyl)mercapturic acid (HPMA), a major acrolein metabolite in human urine incorporating a molecularly imprinted solid‐phase extraction (MISPE) process using N‐acetylcysteine ‐imprinted mesoporous silica particles coupled with LC‐MS/MS detection was developed. The molecularly imprinted mesoporous silica particles were synthesized based on the supported material of ordered mesoporous silica SBA‐15 with N‐acetylcysteine (NAC) as template using surface molecular imprinting technology. The condition of MISPE procedures was optimized. The use of MISPE improved the accuracy and precision of the LC‐MS method and lowered the limit of detection (0.23 ng/mL). The recoveries at three spiked levels ranged between 88.5% to 108.6%. The developed MISPE method enabled the selective extraction of HPMA successfully in human urine and could be used as an effective approach for the determination of ultra‐trace HPMA in complex biological matrices. The results in real samples showed that median levels of HPMA were significantly higher (1922.0 ng/mg of creatinine, N = 75) in smokers than in nonsmokers (759.1 ng/mg of creatinine, N = 5), demonstrating the higher acrolein uptake in smokers than in nonsmokers.     

Gas chromatographic analysis of chlorophenylmercapturic acid lindane metabolites

An analytical method for phenols has been adapted for the analysis of chlorophenylmercapturic acids in rat urine. Chlorothiophenols were produced from the mercapturic acids by hydrolytic cleavage with sodium hydroxide. Acetate esters of the chlorothiophenols were formed by addition of acetic anhydride to the aqueous alkaline solution. After acylation, the acetate derivatives were extracted into hexane. Forming the acetate esters of the chlorothiophenols prevented their oxidation to disulfides and significantly improved their chromatographic properties. Electron-capture gas chromatographic analysis of the stable acetate esters was performed on a mixed phase column, 4% SE-30 + 6% OV-210. Recoveries of four chlorothiophenols ranged from 82 to 93%. This method required no sample transfer steps; therefore, sample loss and analysis time were minimized.     

Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry

Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-l-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-l-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography–tandem mass spectrometry method using ¹³C₆-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 μg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 μg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 μg/L (less than 0.3 to 1.1 μg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 μg/L (less than 0.3 to 2.8 μg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.     

Determination of conjugated bile acids in human urine by high-performance liquid chromatography with chemiluminescence detection.

A qualitative and quantitative analysis of the conjugated 1 beta- and 6 alpha-hydroxy bile acids, including common bile acids, in human urine using high-performance liquid chromatography with chemiluminescence detection is described. After extraction of urine with C18 silica cartridges, the bile acids were separated into non-conjugated, glycine, taurine and sulphate fractions by ion-exchange chromatography on a lipophilic gel. Solvolysis of the sulphate was carried out by treatment with trifluoroethanol in acetone containing hydrochloric acid, and the liberated amino acid conjugates were fractionated again. The individual bile acids were separated on a reversed-phase C18 column (Bile Pak II), with detection by an immobilized 3 alpha-hydroxysteroid dehydrogenase enzyme reactor and chemiluminescence reaction of the generated NADH using 1-methoxy-5-methylphenazinium methylsulphate-isoluminol-microperoxidase system. The assay method showed the detection limits ranging from 8 to 250 pmol for the bile acids tested. Analysis of urine samples obtained from newborns, non-pregnant women and women in late pregnancy showed a large difference in bile acid composition and conjugation mode, suggesting that bile acid metabolism is different during fetal and neonatal periods.     

Determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine by liquid chromatography with fluorimetric detection

A highly sensitive and rapid liquid chromatographic method for the determination of free and total phenylacetic and p- and m-hydroxyphenylacetic acids in human urine is described. After extraction of urine with diethyl ether, these acids and phenylpropionic acid (internal standard) are converted into the corresponding fluorescent derivatives by treatment with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated on a reversed-phase column (Radial-Pak cartridge C18) with aqueous 65% (v/v) methanol and detected fluorimetrically. The detection limits for phenylacetic and p- and m-hydroxyphenylacetic acids are 5, 30 and 100 fmol, respectively, at a signal-to-noise ratio of 5 in a 20-microliter injection volume. This sensitivity permits precise determination of the free and total acids in 20 microliter of normal human urine.     

临床化学上氨基酸、有机酸及核苷类的二维薄层色谱多相分离

在同一块薄层板上多相二维分离３０余种氨基酸、４０余种非挥发性有机酸和２０余种碱基。核苷类化合物。应用一对新的展开剂,用紫外灯－吖啶试剂－镉茚三酮试剂组合显色定位分析,改良了尿液非挥发性有机酸的溶剂萃取。尿液中此三类化合物提取物的多相二维色谱分离效果良好。     

Determination of methylmalonic acid and glutaric acid in urine by aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry

In this work, a novel technique of aqueous-phase derivatization followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry was developed for the determination of organic acids in urine. The analytical procedure involves derivatization of organic acids to their ethyl esters with diethyl sulfate, headspace sampling, and GC/MS analysis. The proposed method was applied to the determination of methylmalonic acid and glutaric acid in urine. The experimental parameters and method validation were studied. Optimal conditions were obtained: PDMS fiber, extraction temperature 55 degrees C, extraction time 30 min, and 60 microL of diethyl sulfate as derivatization reagent with 2 mg of the ion pairing agent tetrabutylammonium hydrogensulfate. The method was linear over three orders of magnitude, and detection limits were 21 nM for methylmalonic acid and 34 nM for glutaric acid, respectively. Consequently, in-situ derivatization/HS-SPME/GC/MS is an alternative and powerful method for determination of organic acids as biomarkers in biological fluids.     

Quantitative analysis of amino acids by HPLC in dried blood and urine in the neonatal period: Establishment of reference values

Quantitative analysis of amino acids in blood and urine is primarily indicated for the diagnosis of amino acid disorders. The high-performance liquid chromatography (HPLC) technique is frequently used for this detection. The frequency of sample collection on filter paper has been increasing exponentially, and there are many advantages attributed to processing biological samples in this way. The aim of this study was to validate a quantitative analysis of amino acids by HPLC in blood and urine collected on filter paper and to establish reference values in the neonatal period. Dried blood and dried urine samples of respectively 58 and 45 healthy newborns (2–9 days) were collected. Pre-treatment and extraction of samples were done according to the literature. Separation and analysis of amino acids were carried out by HPLC with fluorescence detection. The developed method demonstrated excellent separation, linearity, limits of detection and quantification, repeatability and recovery. The reference values for 17 amino acids were defined in dried blood and urine samples of newborns. This work presents a simple, fast and effective method for the simultaneous analysis of 17 amino acids in blood and urine collected on filter paper in a single run. The reference values were established and validated.     

Group separation of non-sulphated and 3-sulphated bile acids by disposable anion-exchange cartridges

Summary A rapid and simple method has been developed for the group fractionation of the major unsulphated and mono-sulphated bile acids in human body fluids. After extraction with Bond Elut C₁₈ cartridges, the bile acids are separated into the unconjugated, glycine-, taurine- and sulphate-conjugated forms on pre-packed Bond Elut SAX columns by increasing the ionic strength of the methanol-acetate buffer eluent. The procedure was found to be accurate and reproducible and to give complete resolution between the different groups. The levels of 3-sulphate bile acids in human serum and urine from patients with liver disease were determined by high-performance liquid chromatography, after group separation and solvolysis of the sulphate fraction.     

Rapid detection and identification of <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="Italic">L</Emphasis>-cysteine thioethers using constant neutral loss and theoretical multiple reaction monitoring combined with enhanced product-ion scans on a linear ion trap mass spectrometer

A sensitive and specific liquid chromatography-mass spectrometry (LC-MS) method based on the combination of constant neutral loss scans (CNL) with product ion scans was developed on a linear ion trap. The method is applicable for the detection and identification of analytes with identical chemical substructures (such as conjugates of xenobiotics formed in biological systems) which give common CNLs. A specific CNL was observed for thioethers of N-acetyl-L-cysteine (mercapturic acids, MA) by LC-MS/MS. MS and HPLC parameters were optimized with 16 MAs available as reference compounds. All of these provided a CNL of 129 Da in the negative-ion mode. To assess sensitivity, a multiple reaction monitoring (MRM) mode with 251 theoretical transitions using the CNL of 129 Da combined with a product ion scan (IDA thMRM) was compared with CNL combined with a product ion scan (IDA CNL). An information-dependent acquisition (IDA) uses a survey scan such as MRM (multiple reaction monitoring) to generate "informations" and starting a second acquisition experiment such as a product ion scan using these "informations." Th-MRM means calculated transitions and not transitions generated from an available standard in the tuning mode. The product ion spectra provide additional information on the chemical structure of the unknown analytes. All MA standards were spiked in low concentrations to rat urines and were detected with both methods with LODs ranging from 60 pmol/mL to 1.63 nmol/mL with IDA thMRM. The expected product ion spectra were observed in urine. Application of this screening method to biological samples indicated the presence of a number of MAs in urine of unexposed rats, and resulted in the identification of 1,4-dihydroxynonene mercapturic acid as one of these MAs by negative and positive product ion spectra. These results show that the developed methods have a high potential to serve as both a prescreen to detect unknown MAs and to identify these analytes in complex matrix.     

Liquid chromatography/electrospray tandem mass spectrometry characterization of styrene metabolism in man and in rat

Liquid chromatography with electrospray tandem mass spectrometry was used to characterize the metabolism of styrene in man and in rat. To improve identification and characterization of minor styrene metabolites, rats were co-exposed to styrene and styrene-d(8). In addition to the main styrene metabolites, mandelic acid and phenylglyoxylic acid, and specific mercapturic acids, phenylhydroxyethylmercapturic acids (PHEMAs), other minor metabolites, including phenylglycine, N-acetyl-S-(phenacyl)cysteine, 4-vinylphenol and styreneglycol conjugates (glucuronides and sulfates) were identified and determined both in human and rat urine. Phenylglycine and N-acetyl-S-(phenacyl)cysteine have been hypothesized to occur, but never detected in human or rat urine after styrene exposure. 4-Vinylphenol and styrene glycol had already been recognized as styrene metabolites, but never determined as intact glucuronide and sulfate conjugates. Failure to identify 1- and 2-phenylethanol conjugates suggests that phenylethanol might be an intermediate metabolite, but it is not a conjugated catabolite. A method for the simultaneous determination of mandelic acid, phenylglyoxylic acid, phenyglycine and the four PHEMA diastereoisomers has been developed and validated. For those glucuronide and sulfate conjugates whose standards are not commercially available, a method for semiquantitative analysis, based on the use of structurally similar compounds as standards, has been developed. This approach was found to be valid for the determination of 4-vinylphenol glucuronide and 4-vinylphenol sulfate.     

Studies on the chromatographic fractionation of metabolites of benzo[a]pyrene in faeces and urine from germfree and conventional rats

Rats, germfree and conventional, were dosed with 14C-labelled benzo[a]pyrene. Faeces and urine were collected. Metabolites in faeces were effectively extracted with a new method using a combination of solvents and solid sorbents. Metabolites in urine were extracted with octadecylsilane-bonded silica. The metabolites were fractionated into groups by chromatography on a cation exchanger (SP-LH-20 or SP-Sephadex C-25) and an anion exchanger (TEAP-LH-20). Some of the groups were further purified by column chromatography and analysed by HPLC and TLC. The analyses show a complex pattern of metabolism. A large part of the metabolites (9-24% depending on animal type and route of excretion) had amphoteric properties, e.g. like glutathione and cysteine conjugates. The abundance of conjugates sensitive to beta-glucuronidase and sulphatase was low. The relative amount of acidic conjugates in faeces was much higher in the germfree than in the conventional rats indicating the influence of the intestinal flora on the metabolism. The results support the view that the mercapturic acid pathway is a quantitatively important metabolic route for benzo[a]pyrene in rats. The methods of extraction and group fractionation were designed to be generally applicable to the analysis of lipophilic xenobiotics and their metabolites.     

Simultaneous determination of C2-C22 non-esterified fatty acids and other metabolically relevant carboxylic acids in biological material by gas chromatography of their benzyl esters

A method for the simultaneous determination of non-esterified short-, medium- and long-chain fatty acids and other types of metabolically relevant carboxylic acids such as hydroxy, keto, aromatic and dicarboxylic acids in biological material by capillary gas chromatography of benzyl ester derivatives is described. Sample preparation avoiding incomplete isolation of carboxylic acids consisted of deproteinization and extraction with ethanol, fixation of carboxylic acids as carboxylates, removal of interfering compounds such as neutral lipids by hexane extraction and amino acids, acyl carnitines and other cations by cation-exchange chromatography, derivatization of keto groups of ketocarboxylic acids into O-methyl oximes and benzyl ester formation by reaction of the potassium carboxylates with benzyl bromide via crown ether catalysis. The sample preparation conditions were investigated, showing the usefulness of this method for quantitative determinations. Chromatograms obtained from human serum, human urine and rat heart ventricle and concentrations of carboxylic acids in these specimens are presented.     

Sequential ethoxycarbonylation, methoximation and tert-butyldimethylsilylation for simultaneous determination of amino acids and carboxylic acids by dual-column gas chromatography

Amino acids (AAs) in alkaline solution were first ethoxycarbonylated with subsequent methoximation of keto acids (KAs). After acidification and solid-phase extraction, tert-butyldimethylsilylation was performed for direct analysis by gas chromatography (GC) on dual-columns with different polarities, which provided simultaneous separation of multiple amino acids, carboxylic acids (CAs) and keto acids, facilitating accurate peak confirmation based on matching with retention index sets characteristic of each analyte. The present method was linear (r2 > or = 0.9955) with good precision (0.1-9.4%) and accuracy (-8.6 to 9.9%), allowing simultaneous screening for diagnostic amino acids along with carboxylic acids and keto acids in urine from a phenylketonuria patient.     

尿中有机酸的毛细管气相色谱—质谱轮廓分析—用于苯丙酮尿症的诊断

应用毛细管气相色谱-质谱轮廓分析方法,测定了33例2.5～4.5岁健康儿童尿中有机酸种类及含量和8例拟诊为苯丙酮尿症儿童尿中的有机酸,结果表明患儿尿样中苯丙酮酸、苯乙酸、邻羟基苯乙酸、对羟基苯乙酸高于正常值10～470倍。为苯丙酮尿症的确诊提供了可靠方法。