Analysis of 4-aminobiphenyl in smoker’s and nonsmoker’s urine by tandem mass spectrometry

The aromatic amine 4-aminobiphenyl (4-ABP) is present in tobacco smoke. In humans, it is also a known bladder carcinogen. We describe here a method for the quantification of total 4-ABP in urine using capillary gas chromatography/tandem mass spectrometry, with an effective detection limit in urine samples of approximately 0.87 pg/mL. We also examined the efficiency of chemical or enzymatic hydrolysis of urinary aromatic amine metabolites. Although we found acidic or basic hydrolysis effective, we found enzymatic hydrolysis (β-glucuronidase with either Escherichia coli or Helix pomatia) ineffective. As part of this work, we also confirm the presence of N-acetyl-4-ABP and 4-ABP glucuronide in human urine samples from smokers. These metabolites have been reported in animal studies, but previously they have not been identified in human samples. These metabolites, however, were found to be unstable and thus infeasible for biomonitoring. The final validated urinary total 4-ABP assay was applied to the analysis of samples from smokers and nonsmokers, whose status was confirmed from cotinine EIA measurements. Among 41 confirmed nonsmokers, the geometric mean (95% CI) of 4-ABP concentration was 1.64 pg/mg creatinine (1.30–2.07). Conversely, in 89 smokers, the geometric mean of 4-ABP concentration was significantly greater, at 8.69 pg/mg creatinine (7.43–10.16), p?<?0.001. Our results indicate that following tobacco smoke exposure, total urinary 4-ABP is a reliable biomarker for exposure to this carcinogen.     

Determination of selected monohydroxy metabolites of 2-, 3- and 4-ring polycyclic aromatic hydrocarbons in urine by solid-phase microextraction and isotope dilution gas chromatography-mass spectrometry

Eighteen monohydroxy polycyclic aromatic hydrocarbon metabolites (OH-PAHs) representing polycyclic aromatic hydrocarbons (PAHs) containing up to four rings in human urine have been measured. The method includes the addition of carbon-13 labeled internal standards, enzymatic hydrolysis, and solid-phase microextraction followed by gas chromatography with high-resolution mass spectrometry. By using response factors calculated with the carbon-13 labeled standards, results are presented for calibration, relative standard deviations and analyte levels from an unspiked human urine pool. The method detection limits ranged from 0.78 ng/l for hydroxyphenanthrenes to 15.8 ng/l for 1-hydroxynaphthalene, and the recoveries ranged between 6% for hydroxychrysene and 47% for 1-hydroxypyrene. The relative standard deviation was lowest for 3-hydroxyphenanthrene at 2.4% and went up to 18.7% for 6-hydroxychrysene. The method was calibrated from 10 to 1200 ng/l. Eleven of the 18 metabolites were found in background pooled urine samples. This validated method is a convenient and reliable tool for determining urinary OH-PAHs as biomarkers of exposure to eight PAHs.     

Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human biomonitoring using isotope-dilution gas chromatography high-resolution mass spectrometry

In order to perform comprehensive epidemiological studies where multiple metabolites of several PAHs are measured and compared in low-dose urine samples, fast and robust methods are needed to measure many analytes in the same sample. We have modified a previous method used for measuring polycyclic aromatic hydrocarbon (PAH) metabolites by automating the solid-phase extraction (SPE) and including an additional eight metabolites. We also added seven new carbon-13 labeled standards, which improves the use of isotope-dilution calibration. Our method included enzyme hydrolysis, automated SPE and derivatization with a silylating reagent followed by gas chromatography (GC), coupled with high-resolution mass spectrometry (HRMS). Using this method, we measured 23 metabolites, representing 9 parent PAHs, with detection limits in the low pg/mL range. All steps in the clean-up procedure were optimized individually, resulting in a method that gives good recoveries (69-93%), reproducibility (coefficient of variation for two quality control pools ranged between 4.6 and 17.1%, N>156), and the necessary specificity. We used the method to analyze nearly 3000 urine samples in the fifth National Health and Nutrition Examination Survey (NHANES 2001-2002).     

Gas and liquid chromatography-mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures

Prohibition of some synthetic cannabimimetics (e.g., JWH-018, JWH-073 and CP 47497) in a number of countries has led to a rise in new compounds in herbal mixtures that create marijuana-like psychotropic effects when smoked. The cannabimimetic JWH-250 (1-pentyl-3-(2-methoxyphenylacetyl)indole) was identified in May 2009 by the German Federal Criminal Police as an new ingredient in herbal smoking mixtures. The absence or low presence of the native compound in urine samples collected from persons who had consumed JWH-250 necessitates a detailed identification of their metabolites, which are excreted with urine and present in blood. Using gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS/MS), we identified a series of metabolites in urine samples and serum sample from humans and urine samples from rats that were products of the following reactions: (a) mono- and dihydroxylation of aromatic and aliphatic residues of the parent compound, (b) trihydroxylation and dehydration of the N-alkyl chain, (c) N-dealkylation and (d) N-dealkylation and monohydroxylation. The prevailing urinary metabolites in humans were the monohydroxylated forms, while N-dealkylated and N-dealkyl monohydroxylated forms were found in rats. The detection of the mono- and dihydroxylated metabolites of JWH-250 in urine and serum samples by GC-MS and LC-MS/MS proved to be effective in determining consumption of this drug.     

Elucidation of the structure of talinolol metabolites in man determination of talinolol and hydroxylated talinolol metabolites in urine and analysis of talinolol in serum

The objective of this study was to determine the structure of talinolol metabolites formed and the amounts excreted in urine. Talinolol metabolites in urine were identified by comparing their HPLC retention times and their GC—MS profile with those of previously characterized reference compounds. The metabolites were quantified by HPLC with a normal-phase silica column, a single chloroform extraction and UV detection. Less than 1% of an administered dose was found in urine as hydroxylated talinolol. Other metabolites could be excluded. A sensitive method to determine talinolol in serum and a simple method for analysis of talinolol in urine are described. These methods were found to be precise and accurate for the measurement of talinolol in samples obtained from patients during chronic talinolol treatment as well as from healthy volunteers after a single dose of talinolol.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Chromatography-mass spectrometry studies on the metabolism of synthetic cannabinoids JWH-018 and JWH-073, psychoactive components of smoking mixtures

The Russian Federation prohibited the distribution of herbal mixtures with synthetic aminoalkylindoles JWH-018 and JWH-073, agonist cannabinoid receptors, on January 22, 2010. The lack or low content of their native compounds in urine requires detailed identification of their metabolites, which are excreted with urine and are present in blood. Using gas and liquid chromatography-mass spectrometry, we identified a series of metabolites in urine samples from humans and rats that were products of the following reactions: (a) mono- and dihydroxylation of the parent compounds with hydroxyl groups located at aromatic and aliphatic residues, (b) carboxylation, (c) N-dealkylation and (d) N-dealkylation and hydroxylation. The prevailing urinary metabolites in humans are monohydroxylated forms, while N-dealkylated and N-dealkyl monohydroxylated forms are found in rats. Twenty-six samples of herbal smoking mixtures with JWH-018, purchased in Russia, were analysed.     

Determination of aromatic metabolites in ruminant urine by high-performance liquid chromatography

A method based on reversed-phase HPLC is reported for the separation and quantification of various urinary aromatic metabolites: hippuric, phenylaceturic, salicyluric, benzoic, phenylacetic, salicylic. 3-phenylpropionic and cinnamic acids and several phenols in ruminant urine. In this method, a Nova-Pak C₁₈ (4 μm) 150×3.9 mm I.D. column, two solvents [A: 15°b methanol in 20 mM acetic acid (pH 3.3); B: methanol] in a gradient mode at a flow-rate of 0.8 ml/min, and UV detection at 210 nm were used. Quantification of the total (free and conjugated) benzoic, phenylacetic and salicylic acids present in urine was achieved by hydrolysis of the samples in 3 M HCl at 100°C for 24 h prior to HPLC analysis. The lowest detection concentration was 50 μmol/I. This method is useful for scanning the profile of aromatic metabolites in urine of ruminants, which provides information on the diets the animals receive.     

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.     

Analysis of amphetamine-type stimulants and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry

The aim of this work was to develop and validate a method for analysing amphetamine-type stimulants (ATSs) and their metabolites in plasma, urine and bile by liquid chromatography with a strong cation-exchange column-tandem mass spectrometry, and to apply it to the pharmacokinetic study of ATSs. 3,4-Methylenedioxymethamphetamine, methamphetamine, ketamine and their main metabolites, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, p-hydroxymethamphetamine, amphetamine and norketamine, were simultaneously quantified by the new method (50-5000 ng/ml). The coefficients of variation and the percent deviations for the eight compounds were in the range of 0.2 to 5.3% and -9.4 to +12.8%, respectively. The recoveries were over 90% in all biological samples tested. This method was effective for the separation and the identification of ATSs and their main metabolites having amine moieties in plasma, urine and bile, and was applicable to pharmacokinetic analysis of methamphetamine, ketamine and their main metabolites in biological samples. This analytical method should be useful for the pharmacokinetic analysis of ATSs.     

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.     

Determination of the anti-platelet-activating factor BN-50727 and metabolites in human urine by high-performance liquid chromatography using solid-phase extraction

A sensitive and selective HPLC solid-phase extraction procedure was developed for the determination of platelet-activating factor antagonist BN-50727 and its metabolites in human urine. The procedure consisted in a double solid-phase extraction of the urine samples on cyanopropyl and silica cartridges, followed by an automated solid-phase extraction of the drug and metabolites on CBA cartridges and posterior elution on-line to the chromatographic system for its separation. The method allowed quantitation in the concentration range 10–2400 ng/ml urine for both BN-50727 and the main metabolite, the O-demethylated BN-50727 product. The limit of quantitation for both compounds was 10 ng/ml. The inter-assay precision of the method, expressed as relative standard deviation, ranged from 1.9 to 4.5% for BN-50727 and from 2.5 to 9.0% for the metabolite. The accuracy, expressed as relative error, ranged from −2.4 to 4.2% and from 0.2 to 6.2%, respectively. This paper describes the validation of the analytical methodology for the determination of BN-50727 in human urine and also for its metabolites. The method has been used to follow the time course of BN-50727 and its metabolites in human urine after single-dose administration.     

Sensitive method for the quantification of urinary pyrimidine metabolites in healthy adults by gas chromatography-tandem mass spectrometry

Enzyme deficiencies in pyrimidine metabolism are associated with a risk for severe toxicity against the antineoplastic agent 5-fluorouracil. To assess whether urinary levels of pyrimidines and their metabolites can be used for predicting patients' individual phenotype, a new gas chromatographic-tandem mass spectrometric method was developed which allows the simultaneous determination of uracil and thymine and their metabolites dihydrouracil, dihydrothymine, beta-ureidopropionic acid, beta-ureidoisobutyric acid, and the amino acids beta-alanine and beta-aminoisobutyric acid in human urine. Small aliquots (2-20 microl) of the urine samples were evaporated and derivatized to the tert.-butyldimethylsilyl derivatives before quantification, using the respective stable isotope-labelled analogues as internal standards. Analytical variation was acceptable with an intra-day imprecision (RSD) below 10%, for beta-ureidoisobutyric acid below 15%. The method was used for investigating the stability of urine samples and the influence of urine collection at different times.     

Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose

Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.     

Direct quantification of monohydroxy-polycyclic aromatic hydrocarbons in synthetic urine samples via solid-phase extraction-room-temperature fluorescence excitation-emission matrix spectroscopy

A screening method for six biomarkers from polycyclic aromatic hydrocarbons (PAH) exposure in urine samples is presented. Solid-phase extraction is carried out on commercial C18 cartridges via an optimized procedure that minimizes metabolite loss. PAH metabolites are directly determined in the eluting solvent (3 mL of methanol) without the need of previous solvent evaporation. Spectral overlapping is resolved with the combination of unfolded partial least squares and residual bilinearization. Excellent analytical figures of merit were obtained for all the studied metabolites. Analytical recoveries varied between 87.9% (9-hydroxyphenanthrene) and 99.4% (3-hydroxybenzo[a]pyrene). For 10 mL of urine sample, the limits of detection varied between 0.01 ng.mL⁻¹ (3-hydroxybenzo[a]pyerene and 1-hydroxybenzopyrene) and 0.3 ng.mL⁻¹ (2-hydroxynaphthalene). Because the chemometric algorithm is capable of handling more than six metabolites at once, the application of this approach to a larger number of metabolites is feasible.     

GC/MS analysis of the rat urine for metabonomic research

In this paper, an optimized protocol was established and validated for the metabonomic profiling in rat urine using GC/MS. The urine samples were extracted by methanol after treatment with urease to remove excessive urea, then the resulted supernatant was dried, methoximated, trimethylsilylated, and analyzed by GC/MS. Forty-nine endogenous metabolites were separated and identified in GC/MS chromatogram, of which 26 identified compounds were selected for quantitative analysis to evaluate the linearity, precision, and sensitivity of the method. It showed good linearity between mass spectrometry responses and relative concentrations of the 26 endogenous compounds over the range from 0.063 to 1.000 (v/v, urine/urine+water) and satisfactory reproducibility with intra-day and inter-days precision values all below 15%. The metabonomic profiling method based on GC/MS was successfully applied to urine samples from hyperlipidemia model rats. Obviously, separated clustering of model rats and the control rats were shown by principal components analysis (PCA); time-dependent metabonomic modification was detected as well. It was suggested that metabonomic profiling based on GC/MS be a robust method for urine samples.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone ®) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C₁₈, 250x4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m² of mitoxantrone by intravenous infusion for 10 min.     

Determination of a major metabolite of tipredane in rat urine by high-performance liquid chromatography with column switching

An automated method, based on column-switching reversed-phase high-performance liquid chromatography, has been developed for the determination of a major metabolite of tipredane in rat urine. Samples are injected directly onto a cyanopropyl extraction column. The portion of eluate containing the metabolite is switched, via an injection loop, onto an octadecylsilane analytical column. The limit of quantification of the method was 25 ng/ml for a 20 μl injection volume of urine. The intra-assay precision (0.7–4.8%) and accuracy (94–105%), and the inter-assay precision (2.7–12.6%) and accuracy (94–105%), were acceptable. The analyte was found to be stable in rat urine when stored at room temperature for six days, in a freezer at or below −20°C for twelve weeks, and when the samples were subjected to two freeze–thaw cycles. No significant interference was observed from tipredane and its major human metabolites, or urine constituents in male and female rats. The method was successfully used to analyse samples from a long-term toxicology study.     

Quantitative profiling of bile acids in biofluids and tissues based on accurate mass high resolution LC-FT-MS: compound class targeting in a metabolomics workflow

We report a sensitive, generic method for quantitative profiling of bile acids and other endogenous metabolites in small quantities of various biological fluids and tissues. The method is based on a straightforward sample preparation, separation by reversed-phase high performance liquid-chromatography mass spectrometry (HPLC-MS) and electrospray ionisation in the negative ionisation mode (ESI-). Detection is performed in full scan using the linear ion trap Fourier transform mass spectrometer (LTQ-FTMS) generating data for many (endogenous) metabolites, not only bile acids. A validation of the method in urine, plasma and liver was performed for 17 bile acids including their taurine, sulfate and glycine conjugates. The method is linear in the 0.01-1muM range. The accuracy in human plasma ranges from 74 to 113%, in human urine 77 to 104% and in mouse liver 79 to 140%. The precision ranges from 2 to 20% for pooled samples even in studies with large number of samples (n>250). The method was successfully applied to a multi-compartmental APOE*3-Leiden mouse study, the main goal of which was to analyze the effect of increasing dietary cholesterol concentrations on hepatic cholesterol homeostasis and bile acid synthesis. Serum and liver samples from different treatment groups were profiled with the new method. Statistically significant differences between the diet groups were observed regarding total as well as individual bile acid concentrations.     

Validated method for quantitation of biomarkers for benzene and its alkylated analogues in urine

A validated gas chromatography-mass spectrometric method for the analysis of the metabolites of benzene and its alkylated analogues in urine is reported. A number of metabolites, as required by authorities for biomonitoring of industrial exposure to aromatic vapour, were analysed simultaneously with preservation of quantitative information concerning positional isomers. The use of this method replaces a combination of analytical methods required for the analysis of all these metabolites. Urine samples were subjected to acidic deconjugation followed by a derivatization step. Phenol, ortho-, meta-, para-cresol, mandelic acid, and ortho-, meta-, para-methylhippuric acid were analysed as their corresponding ethoxycarbonyl derivatives, with single ion monitoring. The mass-to-charge ratios (m/z) of the ions used for quantitation by single ion monitoring of the metabolites were: phenol, 94 m/z; cresols, 108 m/z; mandelic acid, 206 m/z; hippuric acid, 105 m/z; methylhippuric acids, 119 m/z. The mass-to-charge ratios for the internal standards were: [(2)H(6)]phenol, 99 m/z; p-chlorophenol, 128 m/z and 3-chloro-4-hydroxyphenyl acetic acid, 214 m/z. The limits of detection for phenol and the cresols were below 0.4 micromol/l and below 0.05 micromol/l for mandelic acid and the hippuric acids. Within-run precision for mandelic acid was 6.2%, for hippuric acid was 7.32% and was below 5% for the rest of the analytes.