Studies on in vitro antipyrine metabolism by 13C,15N double labeled method

The capacities of forms of cytochrome P-450 to oxidize antipyrine were compared. An isotope dilution gas chromatography/mass spectrometry/selected ion monitoring assay was developed to quantify the three main metabolites, norantipyrine, 3-hydroxymethylantipyrine and 4-hydroxyantipyrine. 13C,15N-Double labeled antipyrine was used as a substrate and the metabolites were analyzed as their trimethylsilyl derivatives. Among forms of cytochrome P-450 examined, a male-specific form of P-450, namely P-450-male, showed higher activity to form all the three metabolites. The other forms were responsible only for the formation of norantipyrine and 4-hydroxyantipyrine. The activities of liver microsomes from untreated male and female rats and rats treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyl were expressed dependent on the activities of forms of cytochrome P-450 examined.     

Automated high-performance liquid chromatographic method for the determination of antipyrine and its metabolites in urine : Some preliminary results obtained from smokers and non-smokers

A simple, accurate and fully automated high-performance liquid-chromatographic method was developed for the simultaneous determination of antipyrine (AP), 3-hydroxymethylantipyrine (3HMA), 4-hydroxyantipyrine (4OHA) and norantipyrine (NORA) in urine. This method requires no extraction step and only one chromatographic run with the use of a reversed-phase system. The coefficient of variation (%) (n = 8 each) was: 4.14 for AP, 2.31 for 3HMA, 3.48 for 4OHA, and 2.71 for NORA. The method was applied to studies on AP metabolism in three smokers and three non-smokers who received an oral 10 mg/kg dose of AP. These preliminary results suggest that smokers appear to excrete more 4OHA and NORA in the urine than non-smokers.     

Antipyrine metabolism in man

The metabolism of antipyrine (AP) has been investigated in 15 healthy subjects by relating the saliva AP elimination rates to the urinary excretion rates of 4-hydroxyantipyrine (AP-40H), 3-hydroxymethylantipyrine (AP-CH₂OH) and N-demethylantipyrine (AP-NH₂). Highly significant correlation coefficients have been obtained for AP-40H (r = 0.94). AP-CH₂OH (r = 0.85) and for AP-NH₂ (r = 0.86). While little interindividual variation occured in the urinary concentrations of AP-4OH and AP-CH₂OH, two-fold variation was found for AP-NH₂. The ratios of the three metabolites were highly constant over a period of several months in some subjects but slightly variable in others indicating their control by genetic and environmental factors.     

A radiometric high-pressure liquid chromatography assay for the simultaneous determination of the three main oxidative metabolites of antipyrine in studies in vitro

A quantitative radiometric high-pressure liquid chromatography assay for the estimation of the three main oxidative metabolites of antipyrine in vitro using [3-¹⁴C]antipyrine as substrate is described. Baseline separation of antipyrine, 3-hydroxymethylantipyrine, 4-hydroxyantipyrine, and norphenazone was achieved, after methylation, using a reverse-phase μBondapak C₁₈ column with a mobile phase of 17% acetonitrile in water. The metabolites could be estimated free of interference as confirmed by gas chromatography-mass spectrometry. Unlabeled metabolites were used as recovery standards. Activity could be determined with as little as 100 μg human liver microsomal protein. Maximum velocities for the formation of the three metabolites ranged from 1 to 3.5 nmol product mg^?1 min^?1 with rat liver and from 0.3 to 0.6 nmol product mg^?1 min^?1 with human liver.     

Simultaneous gas chromatographic determination of methamphetamine, amphetamine and their p-hydroxylated metabolites in plasma and urine

We report a method for the simultaneous determination of methamphetamine, amphetamine and their hydroxylated metabolites in plasma and urine samples using a GC-NPD system. The analytical procedures are: (1) adjust the sample to pH 11.5 with bicarbonate buffer, saturate with NaCl and extract with acetate; (2) back-extract the amines in the ethyl acetate fraction with 0.1 M HCl; (3) adjust the pH of the acid fraction to 11.5 and follow by extraction in ethyl acetate; (4) reduce the volume of ethyl acetate under nitrogen and derivatize the concentrate with trifluoroacetic anhydride or heptaflourobutyric anhydride before the GC analysis. The derivatives were separated on a GC-NPD system equipped with a HP-5 column of 25 m×0.32 m I.D. and a 0.52 μm film of 5% phenylmethylsilicone. The detection limit (taking a signal-to-noise ratio of 2) of heptafluorobutyl derivatives of methamphetamine and its metabolites in plasma and the trifluoroacetyl derivatives in urine was 1 ng/ml (22 pg on column). The limit of quantitation of the heptafluorobutyl derivatives in the plasma was 1 ng/ml (22 pg on column), and that of the trifluoroacetyl derivatives in urine was 20 ng/ml (73 pg on column). The between-day variation was from 0.9 to 17.4% and within-day variation from 0.9 to 8.3%. This method was used successfully in the quantitative determination of methamphetamine and its p-hydroxylated metabolites in the plasma and urine of human subjects.     

The effects of malotilate on hepatic drug metabolizing systems in different strains of rats

1. In Sprague-Dawley (SD) rats treated for 7 days with malotilate (MAL:250 mg/kg, p.o.), cytochrome P-450 and b5 contents, aminopyrine N-demethylase and heme oxygenase activities were significantly increased. In Wistar rats, cytochrome b5 content and heme oxygenase and delta-aminolevulinic acid synthetase activities were found to be significantly increased. 2. Among the antipyrine metabolites excreted in urine during the 24 hr after antipyrine (100 mg/kg, i.p.) administration, norantipyrine increased significantly in Sprague-Dawley rats, while a significant increase of 4-hydroxyantipyrine was observed in Wistar rats. 3. The serum dimethadione/trimethadione ratio was only found to be significantly increased in Sprague-Dawley rats. 4. These results indicate that malotilate may have inducible effects on hepatic drug metabolizing enzymes, and that it affects the various cytochrome P-450 isozymes from different strains of rat in different ways.     

Antipyrine metabolism in the rat by three hepatic monooxygenases

The relative contribution of 3 oxidative reactions of antipyrine to its metabolism in vivo were assessed by comparing male and female rats, and by studying the effects of phenobarbital (PB) and 3-methylcholanthrene (MC). After [N¹⁴CH₃] antipyrine, 11–15% of the dose was excreted as ¹⁴CO₂ in both sexes as a consequence of N-demethylation. PB pretreatment had no effect but MC doubled ¹⁴CO₂ excretion. The other metabolites, 4-hydroxyantipyrine (4OHA) and 3-hydroxymethylantipyrine (3HMA), in urine were determined by thin-layer chromatography. Of the total activity in the urine, 4OHA represented 30% in male and 40% in female rats; 3HMA represented 35% in male and 20% in female rats. The ratio of 4OHA to 3HMA in both sexes increased after PB and MC, the effect being more pronounced with the latter. The results show that the 3 major oxidative pathways of antipyrine metabolism are mediated by different enzymes, almost certainly different forms of cytochrome P450.     

Simultaneous determination of lansoprazole enantiomers and their metabolites in plasma by liquid chromatography with solid-phase extraction

A simple and highly sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of lansoprazole enantiomers and their metabolites, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone, in human plasma have been developed. Chromatographic separation was achieved with a Chiral CD-Ph column using a mobile phase of 0.5M NaClO(4)-acetonitrile-methanol (6:3:1 (v/v/v)). The analysis required only 100 microl of plasma and involved a solid-phase extraction with Oasis HLB cartridge, with a high extraction recovery (>94.1%) and good selectivity. The lower limit of quantification (LOQ) of this assay was 10 ng/ml for each enantiomer of both lansoprazole and 5-hydroxylansoprazole, and 5 ng/ml for lansoprazole sulfone. The coefficient of variation of inter- and intra-day assay was <8.0% and accuracy was within 8.4% for all analytes (concentration range 10-1000 ng/ml). The linearity of this assay was set between 10 and 1000 ng/ml (r2>0.999 of the regression line) for each of the five analytes. This method is applicable for accurate and simultaneous monitoring of the plasma levels of lansoprazole enantiomers and their metabolites in the renal transplant recipients.     

Quantification of paclitaxel metabolites in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic (HPLC) method has been validated for the quantitative determination of the three major paclitaxel metabolites (6α-hydroxypaclitaxel, 3′-p-hydroxypaclitaxel, 6α,3′-p-dihydroxypaclitaxel) in human plasma. The HPLC system consists of an APEX-octyl analytical column and acetonitrile-methanol-0.02 M ammonium acetate buffer pH 5 (AMW; 4:1:5, v/v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The sample pretreatment of the plasma samples involves solid-phase extraction (SPE) on Cyano Bond Elut columns.The concentrations of the metabolic products could be determined by using the paclitaxel standard curve with a correction factor of 1.14 for 6α,3′-p-dihydroxypaxlitaxel. The recoveries of paclitaxel and the metabolites 6α,3′-p-dihydroxypaclitaxel, 3′-p-hydroxypaclitaxel and 6α-hydroxypaclitaxel in human plasma were 89, 78, 91 and 89%, respectively. The accuracy of the assay for the determination of paclitaxel and its metabolites varied between 95 and 97%, at a 50 ng/ml analyte concentration. The lower limit of quantitation was 10 ng/ml for both the parent drug and its metabolites.     

Determination of hydroxy metabolites of polychlorinated biphenyls in plasma and tissue by gas chromatography/mass spectrometry

This study examines a novel sample preparation method for the determination of 11 hydroxy metabolites of polychlorinated biphenyls (PCBs) in plasma and organ tissues, followed by gas chromatography with mass spectrometric detection (GC/MS). The clean-up method was optimized to eliminate the interference matter by using a silica column and 10 mL of n-hexane/dichloromethane (4:6, v/v) as an eluent. Solid-phase and solvent extraction procedures were used for the plasma and tissues samples, respectively. Compared to C(18) and C(8) solid-phase, C(2) showed higher extraction efficiency with n-hexane as the eluent for plasma. The hydroxy-PCB extraction recoveries achieved with this combined extraction and clean-up procedure from plasma ranged from 87 to 117%, while those from tissues ranged from 82 to 111%. The linear detector responses for propyl derivatives of hydroxy-PCBs were obtained with the coefficients of determination varying from 0.992 to 0.998 in the concentration range of 0.1-20 ng mL(-1). The method detection limits ranged from 0.1 to 0.5 ng mL(-1) in 1 mL of plasma and from 0.1 to 0.5 ng g(-1) in 1g of tissues. This procedure was successfully applied to the study of 3-OH-2,3',4,4',5-PeCB in rat plasma and liver samples after intraperitoneal injection (20 mg/kg) of 2,3',4,4',5-PeCB.     

Determination of vinorelbine in rabbit plasma by high-performance liquid chromatography with coulometric detection

A high-performance liquid chromatographic method was developed for the determination in plasma (400-μl sample) of a vinca alkaloid, vinorelbine. The analysis was performed by using an octadecylsilane column and heptanesulfonic acid as ion-pairing agent. This method used a new internal standard, teniposide, that permitted a good compromise between sensitivity and retention times (10.6 and 15.5 min for teniposide and vinorelbine, respectively). After a liquid-liquid extraction with diethyl ether, the extracts were injected into a reversed-phase system. The extraction efficiency was approximately 80% for both vinorelbine and the internal standard. The mobile phase was phosphate buffer (pH 3)-acetonitrile-methanol (50:30:20, v/v/v). Using coulometric detection, the limit of detection in plasma (400 μl) was 1 ng.ml. The intra-assay coefficients of variation were 10.95, 3.80 and 5.71% for 5, 500 and 1000 ng/ml, respectively, and the inter-assay coefficients of variation were 20.14, 14.27 and 10.67% for 5, 500 and 1000 ng/ml, respectively. A linear response was observed for the plasma calibration graph in the ranges 2.5–50 and 50–1000 ng/ml. This method was used to follow the time course of the concentration of vinorelbine in rabbit plasma after a single intravenous dose of vinorelbine (30 mg/m²) and seems to be suitable for studying the pharmacokinetics of vinorelbine in rabbit.     

Solid-phase extraction of isosorbide dinitrate and two of its metabolites from plasma for gas chromatographic analysis

A rapid, accurate and selective method for the determination of isosorbide dinitrate and its 2- and 5-isosorbide mononitrate metabolites in 1.0 ml of human plasma has been developed. Before chromatographic quantitation by gas-liquid chromatography with electron-capture detection, the compounds are subjected to solid-phase extraction, using ENVI 18 cartridges (Supelco). The intra-day and inter-day coefficients of variation are less than 10%, except the inter-day coefficient of variation for the assay of 5-isosorbide dinitrate which is less than 15%. Limits of quantitation are 10, 10 and 20 ng/ml for isosorbide dinitrate, 2-isosorbide mononitrate and 5-isosorbide mononitrate, respectively. Recoveries are in excess of 90% for isosorbide dinitrate and 70% for its two metabolites.     

Simultaneous quantification of alprazolam, 4- and alpha-hydroxyalprazolam in plasma samples using liquid chromatography mass spectrometry

A sensitive and specific method was developed for quantification of alprazolam and its two metabolites 4-hydroxyalprazolam and alpha-hydroxyalprazolam in plasma. The work up procedure was solid phase extraction. Liquid chromatography-mass spectrometry (LC-MS) was used for separation, detection and quantification of the analytes. The limit of quantitation (LOQ) was 0.05 ng/mL for alprazolam and the two metabolites. The extraction recovery was more than 82% for alprazolam and its metabolites. The within- and between-assay coefficients of variation were in the range of 1.9-17.9%. The method was used for determination of the pharmacokinetics parameters of alprazolam and its two metabolites in healthy Caucasian subjects who ingested 1mg of alprazolam.     

LC-MS/MS method for simultaneous determination of valproic acid and major metabolites in human plasma

A rapid and sensitive method using liquid chromatography-tandem mass spectroscopy (LC-MS/MS) was developed and validated for simultaneous quantitative determination of valproic acid and three major metabolites (3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid) in human plasma. The analytes and internal standard were isolated from 200 μL samples by solid phase extraction using a ZORBAX SB-C? column (3.5 μm, 2.1×100 mm) with an isocratic mobile phase consisting of methanol-10mM ammonium acetate (80:20, v/v) containing 0.1% formic acid at a flow rate of 0.3 mL/min. The method had a chromatographic total run time of 2.0 min. The lower limit of quantification of valproic acid, 3-OH-valproic acid, 4-ene-valproic acid and 5-OH-valproic acid of the method was 2030, 51.5, 50.15 and 51.25 ng/mL, respectively. The method was linear for valproic acid and the three metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 15.0%. This analytical method was successfully used to assay plasma concentrations of valproic acid and the three metabolites in human plasma from epileptic patients.     

High-performance liquid chromatographic method for the simultaneous determination of the camptothecin derivative irinotecan hydrochloride, CPT-11, and its metabolites SN-38 and SN-38 glucuronide in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT

We established a high-performance liquid chromatography (HPLC) method for the simultaneous determination of the camptothecin (CPT) derivative, irinotecan hydrochloride (CPT-11) and its metabolites, 7-ethyl-10-hydroxycamptothecin (SN-38) and SN-38 glucuronide (SN-38G) in rat plasma with a fully automated on-line solid-phase extraction system, PROSPEKT. Plasma samples were pretreated with 0.146 M H₃PO₄ to inactivate carboxylesterase and β-glucuronidase in rat plasma, and added with the internal standard solution (0.146 M H₃PO₄ containing 1 μg/ml CPT) and then analyzed. The method was validated for CPT-11 (5 to 25 000 ng/ml), SN-38 (5 to 2500 ng/ml) and SN-38G (2.5 to 500 ng/ml). This method enabled the determination of many samples within a relatively short time with easy sample preparation. It also had four advantages compared with conventional determination methods, i.e. automation of a complicated sample preparation, time-saving by the simultaneous determination of three compounds, the direct determination of SN-38G, and the small amount of plasma required for the determination.     

Sensitive determination of omeprazole and its two main metabolites in human plasma by column-switching high-performance liquid chromatography: application to pharmacokinetic study in relation to CYP2C19 genotypes

A simple and sensitive column-switching high-performance liquid chromatographic method was developed for the simultaneous determination of omeprazole and its two main metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in human plasma. Omeprazole, its two metabolites and lansoprazol as an internal standard were extracted from 1 ml of alkalinized plasma sample using diethyl ether-dichloromethane (45:55, v/v). The extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mm x 4.6 mm i.d.) for clean-up and column II (Inertsil ODS-80A column, 5 microm, 150 mm x 4.6mm i.d.) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (92:8 v/v, pH 7.0) for clean-up and phosphate buffer-acetonitrile-methanol (65:30:5 v/v/v, pH 6.5) for separation, respectively. The peak was detected with an ultraviolet detector set at a wavelength of 302 nm, and total time for chromatographic separation was approximately 25 min. The validated concentration ranges of this method were 3-2000 ng/ml for omeprazole, 3-50 ng/ml for 5-hydroxyomeprazole and 3-1000 ng/ml for omeprazole sulfone. Mean recoveries were 84.3% for omeprazole, 64.3% for 5-hydroxyomeprazole and 86.1% for omeprazole sulfone. Intra- and inter-day coefficient variations were less than 5.1 and 6.6% for omeprazole, 4.6 and 5.0% for 5-hydroxyomeprazole and 4.6 and 4.9% for omeprazole sulfone at the different concentrations. The limits of quantification were 3 ng/ml for omeprazole and its metabolites. This method was suitable for use in pharmacokinetic studies in human volunteers, and provides a useful tool for measuring CYP2C19 activity.     

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.     

Development and validation of efficient stable isotope dilution LC-HESI-MS/MS method for the verification of β-lyase metabolites in human urine after sulfur mustard exposure

As a part of the project for screening unequivocal biomarkers after sulfur mustard exposure, a quantitative method for the determination of β-lyase metabolites 1,1'-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) was validated. Full validation was conducted according to the FDA guidelines for method validation using pooled human urine as a sample matrix. The metabolites were extracted from urine with an optimized sample preparation procedure using ENV+ solid phase extraction cartridge with reduced volume of sample and solvents. Metabolites were detected by improved and faster liquid chromatography-heated electrospray ionization-tandem mass spectrometry (LC-HESI-MS/MS) method with two transitions of each chemical using non-buffered eluents, post-column splitter and higher flow-rate. These provided over five times faster analysis than previously published method providing 4.5 min/sample cycle time, to achieve up to 200 samples per day (24 h). Quantification was performed using deuterium labelled internal standard of SBMSE. The method was linear over the concentration range of 5-200 ng/ml (average correlation coefficients were R(2)=0.997 and R(2)=0.989) for both β-lyase metabolites, SBMSE and MSMTESE, respectively. The average retention times for SBMSE and MSMTESE were 1.96±0.01 min and 3.24±0.03 min (n=54). Calculated limits of detection were 4 ng/ml for both SBMSE and MSMTESE, respectively. Lower limits of quantification were 10 ng/ml and 11 ng/ml for SBMSE and MSMTESE, respectively. This validated method was successfully used in the First Confidence Building Exercise on Biomedical Samples Analysis organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Identification criteria for analysing unequivocal biomarkers of sulfur mustard with LC-MS/MS after alleged use is discussed and proposed based on the validation and exercise results.     

Determination of lonazolac and its hydroxy and O-sulfated metabolites by on-line sample preparation liquid chromatography with fluorescence detection

A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20 x 4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C18-HT (100 x 3 mm I.D., 3.5 microm particles) analytical column in the back-flush mode. Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission. Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC-MS-MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode. Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2 +/- 3.5, 96.7 +/- 2.2, and 100.9 +/- 3.5%. The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 microl. Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10-600 ng/ml. The mean values of the coefficients of variation (CV) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46 +/- 1.15, 3.94 +/- 2.13 and 4.79 +/- 2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate. The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.     

Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.