Development of a urinary biomarker for exposure to the organophosphate propetamphos: data from an oral and dermal human volunteer study.

Propetamphos is a member of the vinyl phosphate group of insecticides and is mainly used for sheep dipping. There have been no published metabolic studies on the effect of propetamphos in man to date, although the present authors have published the identification of a metabolite. The present paper presents data from a human volunteer study investigating the toxicokinetics of the organophosphorus pesticide propetamphos following oral and dermal exposure. Five volunteers ingested a propetamphos dose of 10 micrograms kg-1 (35 nmol kg-1) body weight. Following a washout of 4 weeks, a 100 mg (356 mumol) dermal dose of propetamphos was applied, occluded to 80 cm2 of the inner forearm, for 8 h to the same five volunteers. In a pilot study (several weeks before the main study), one volunteer also received an occluded dermal dose of 50 mg (178 mumol) propetamphos. Unabsorbed propetamphos on the skin was washed off after 8 h and collected. Blood and urine samples were collected over 30 and 54 h for the oral and dermal exposures respectively. Blood samples were analysed for plasma and erythrocyte cholinesterase. Urine samples were analysed for a urinary metabolite of propetamphos: methylethylphosphoramidothioate (MEPT). Following oral and dermal exposure, peak urinary MEPT levels occurred at 1 and 10-12 h respectively. The apparent urinary elimination half-lives of MEPT had means of 1.7 h (oral exposure) and 3.8 h (dermal exposure). Approximately 40% of the oral dose and 1% of the dermal dose were recovered as urinary MEPT or metabolites, which could be hydrolysed to MEPT. Approximately 90% of the dermal dose was recovered from the skin washings. Data from a volunteer showed that a doubling of the dermal dose resulted in approximately double the concentration of total MEPT. Alkaline hydrolysis of urine samples increased the level of MEPT detected after both oral and dermal doses. The increase was greater and statistically significant (p < 0.001, paired t-test) for the dermal dose. This increase in MEPT suggests the presence of other MEPT-containing metabolites or conjugates. The difference in the increase between oral and dermal doses raises the question of a difference in metabolism between the two routes. No individual showed a significant depression compared with their pre-exposure levels of erythrocyte acetyl cholinesterase or plasma cholinesterase activity for either dosing route. However, on a group basis, there was a statistically significant mean depression in plasma cholinesterase activity at 8 and 24 h for oral exposure, with a maximum mean depression of 7% from pre-exposure levels at 8 h. Hydrolysis of urine samples had the effect of reducing the interindividual coefficient of variation (CV) for total excretion of MEPT following both oral (CV reduced from 36 to 8%) and dermal (CV reduced from 40 to 17%) exposure. The ability to detect and follow the elimination of low doses of propetamphos by measurement of 'total' (after hydrolysis) urinary MEPT suggests it is a suitable biomarker of propetamphos exposure. The comparatively short elimination half-lives suggest a strategy for biological monitoring of occupational exposure based on samples collected at the end of the shift.     

Development of a urinary biomarker for exposure to the organophosphate propetamphos: data from an oral and dermal human volunteer study

Propetamphos is a member of the vinyl phosphate group of insecticides and is mainly used for sheep dipping. There have been no published metabolic studies on the effect of propetamphos in man to date, although the present authors have published the identification of a metabolite. The present paper presents data from a human volunteer study investigating the toxicokinetics of the organophosphorus pesticide propetamphos following oral and dermal exposure. Five volunteers ingested a propetamphos dose of 10 μg kg^-1 (35nmol kg^-1) body weight. Following a washout of 4 weeks, a 100mg (356 μmol) dermal dose of propetamphos was applied, occluded to 80cm² of the inner forearm, for 8 h to the same five volunteers. In a pilot study (several weeks before the main study), one volunteer also received an occluded dermal dose of 50 mg (178 μmol) propetamphos. Unabsorbed propetamphos on the skin was washed off after 8 h and collected. Blood and urine samples were collected over 30 and 54 h for the oral and dermal exposures respectively. Blood samples were analysed for plasma and erythrocyte cholinesterase. Urine samples were analysed for a urinary metabolite of propetamphos: methylethylphosphoramidothioate (MEPT). Following oral and dermal exposure, peak urinary MEPT levels occurred at 1 and 10-12 h respectively. The apparent urinary elimination half-lives of MEPT had means of 1.7h (oral exposure) and 3.8 h (dermal exposure). Approximately 40% of the oral dose and 1% of the dermal dose were recovered as urinary MEPT or metabolites, which could be hydrolysed to MEPT. Approximately 90% of the dermal dose was recovered from the skin washings. Data from a volunteer showed that a doubling of the dermal dose resulted in approximately double the concentration of total MEPT. Alkaline hydrolysis of urine samples increased the level of MEPT detected after both oral and dermal doses. The increase was greater and statistically significant (p < 0.001, paired t-test) for the dermal dose. This increase in MEPT suggests the presence of other MEPT-containing metabolites or conjugates. The difference in the increase between oral and dermal doses raises the question of a difference in metabolism between the two routes. No individual showed a significant depression compared with their pre-exposure levels of erythrocyte acetyl cholinesterase or plasma cholinesterase activity for either dosing route. However, on a group basis, there was a statistically significant mean depression in plasma cholinesterase activity at 8 and 24 h for oral exposure, with a maximum mean depression of 7% from pre-exposure levels at 8 h. Hydrolysis of urine samples had the effect of reducing the interindividual coefficient of variation (CV) for total excretion of MEPT following both oral (CV reduced from 36 to 8%) and dermal (CV reduced from 40 to 17%) exposure. The ability to detect and follow the elimination of low doses of propetamphos by measurement of 'total' (after hydrolysis) urinary MEPT suggests it is a suitable biomarker of propetamphos exposure. The comparatively short elimination half-lives suggest a strategy for biological monitoring of occupational exposure based on samples collected at the end of the shift.     

Use of a homologous internal standard for the quantification of the major metabolite of prostaglandin F2 alpha in human urine by multiple ion analysis

A gas chromatographic-mass spectrometric method for quantitative determination of 9 alpha, 11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid, the major urinary metabolite of prostaglandin F2 alpha (PGF-M), was developed. The metabolite was analyzed as the dimethyl ester-O-methyloxime-bis-trimethylsilyl ether derivative. The internal standard consisted of a mixture of diethyl ester + monoethyl ester-delta-lactone of PGF-M. Those two species were converted to the 1-methyl-20-ethyl ester derivative during the analytical process. Linear standard curves were developed in the range 0 to 100 ng of injected prostaglandin. The method comprised extraction with Amberlite XAD-2, methylation, chromatography over octadecasilyl-silica, delactonization, remethylation, and chromatography over silicic acid and Lipidex-5000, followed by methoximation, trimethylsilylation, and instrumental analysis. Interassay coefficient of variation, for the analysis of four identical urine specimens, was 7% and intraassay coefficient of variation, when each sample was injected four times, ranged from 3.2 to 6.0%. Specificity, accuracy, and precision of the method were verified by recovery of the metabolite from two different urine pools. The recovery of authentic, underivatized PGF-M added to urine was 99.1 +/- 2.4% (mean +/- SE, N = 6). The plot of recovered versus added metabolite followed the equation y = 0.936 x + 25.8, with r = 0.9918.     

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.     

Enantioselective high-performance liquid chromatographic method for the determination of methadone and its main metabolite in urine using an AGP and a C8 column coupled serially

A simple and sensitive method for the enantioselective high-performance liquid chromatographic determination of methadone and its main metabolite, EDDP, in human urine is described. (−)-(R)-Methadone, (+)-(S)-methadone, (+)-(R)-EDDP, (−)-(S)-EDDP and imipramine as an internal standard are detected by ultraviolet detection at 200 nm. The enantiomers of methadone and EDDP were extracted from human urine by a simple liquid–liquid extraction procedure. The extracted sample was reconstructed in mobile phase and the enantiomers of methadone and EDDP were quantitatively separated by HPLC on a short analytical LiChrospher RP8 column coupled in series with a chiral AGP column. Determination of all four enantiomers was possible in the range of 0.03 to 2.5 μM. The recoveries of methadone enantiomers and EDDP enantiomers added to human urine were about 90% and 80%, respectively. The method was applicable for determination of methadone enantiomers and the enantiomers of its main metabolite in urine samples from methadone maintenance patients and patients suffering from severe chronic pain.     

Quantitative analysis of a model opioid peptide and its cyclic prodrugs in rat plasma using high-performance liquid chromatography with fluorescence and tandem mass spectrometric detection

An isocratic reversed-phase HPLC method for the simultaneous quantitation of alpha-lipoic acid and five of its metabolites in human plasma as well as in human urine employing solid-phase extraction and pulsed amperometric detection was developed and validated. The method was found to be sufficiently precise and accurate for the measurement of alpha-lipoic acid and its metabolites 6,8-bis(methylthio)octanoic acid, 4,6-bis(methylthio)hexanoic acid and 2,4-bis(methylthio)butanoic acid in plasma and urine samples, obtained from patients suffering from diabetic neuropathy as well as from healthy volunteers following daily oral administration of 600 mg alpha-lipoic acid. The quantitation of the metabolite bisnorlipoic acid was often impaired by interferences caused by an unidentified metabolite. However, bisnorlipoic acid was detected in few test samples and the concentrations were consistently low. Despite the poor recovery of the metabolite tetranorlipoic acid from plasma, reproducibility and accuracy were found to be from acceptable magnitude to assess concentration time courses. Thus, the obtained analytical results are considered as reliable and well suited for pharmacokinetic studies of alpha-lipoic acid and its metabolites.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene—divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine in serum and urine using solid-phase extraction and high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatography method for the determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine is described. The samples are purified by reversed-phase solid-phase extraction. The components are separated on a C₁₈ column with a mobile phase containing 18% acetonitrile, 5 mM dodecyl sulphate and 30 mM phosphate buffer, pH 2.1, and measured by fluorescence detection using an excitation wavelength of 285 nm and an emission wavelenght of 380 nm. Detection limits are 0.12 μM (plasma)) and 0.60 μM (urine) for acyclovir, and 0.26 μM (plasma) and 1.3 μM (urine) for metabolite. Correlation coefficients that were better than 0.998 were obtained normally. This analytical method, which enables simultaneous measurement of parent compound and metabolite, has been used in kinetics studies and for therapeutic drug monitoring in different patient groups with variable degrees of renal dysfunction.     

Pharmacokinetics of propetamphos following intravenous administration in the F344 rat

Propetamphos [(E)-l-methylethyl 3[[(ethylamino)methoxyphosphinothioyl]oxy]-2-bu-tenoate], the active ingredient in Safrotin,® is an organophosphate developed by Sandoz, Ltd.® (Switzerland) as an insecticide (1). Although metabolism of propetamphos has been previously investigated (2,3), there is no pharmacokinetic data available in the literature. The current studies were undertaken to investigate the pharmacokinetics of propetamphos following intravenous administration in male and female Fischer 344 (F344) rats. Rats were dosed via an indwelling jugular cannula at a dose of 12 mg/kg (one-tenth the oral LD-50). Blood samples were withdrawn via the cannula at predetermined timepoints to quantitate plasma concentrations of propetamphos over time. Propetamphos is highly bound to plasma proteins (free fraction = 0.06). Free propetamphos concentration in plasma vs. time data were analyzed by noncompartmental methods. The terminal elimination rate constant, λ, was significantly different for males versus females (0.015 min^?1 for males and 0.037 min^?1 for females, p = 0.001). Plasma was cleared of unbound propetamphos at rates of 0.559 ± 0.069 and 0.828 ± 0.181 L/min/kg for males and females (mean ± standard error). Mean residence times (MRTs) for propetamphos in the body for males and females were 28.3 ± 5.7 and 14.4 ± 3.5 min, and the volume of distribution at steady state (Vss) was 14.7 ± 2.6 and 12.3 ± 4.5 L/kg. The differences in these parameters, clearance (CI), MRT, and Vss, were not statistically significant at the p < 0.05 level for males versus females, but MRT was nearly significantly different (p = 0.08). Because of the rapid elimination of propetamphos from plasma following intravenous administration, it is unlikely that propetamphos would bioaccumulate in environmentally exposed animals. Although the pharmacokinetic parameters were not statistically different for males and females in these studies, there was a clear clinical difference in their susceptibility to propetamphos toxicity. Female rats presented with overt signs of organophosphate intoxication, whereas males were only slightly effected. The observed gender-related clinical difference in susceptibility to toxicity suggests that there may be a difference in the extent of elimination due to activation versus detoxication of propetamphos in males and females. Another possible explanation for the clinical difference in propetamphos toxicity is that inhibition of acetyl-cholinesterase by the activated, oxygenated form of propetamphos (propetamphos oxon) may be greater in females than in males.     

Evaluation of urinary dihydrocodeine excretion in human by gas chromatography–mass spectrometry

Urinary metabolic pattern after the therapeutic peroral dose of dihydrocodeine tartrate to six human volunteers has been explored. Using the GC–MS analytical method, we have found that the major part of the dose administered is eliminated via urine within the first 24 h. However, the analytical monitoring of dihydrocodeine and its metabolites in urine was still possible 72 h after the dose was administered. The dihydrocodeine equivalent amounts excreted in urine in 72 h ranged between 32 and 108% of the dose, on average 62% in all individuals. The major metabolite excreted into urine was a 6-conjugate of dihydrocodeine, then in a lesser amount a 6-conjugate of nordihydrocodeine (both conjugated to approximately 65%). The O-demethylated metabolite dihydromorphine was of a minor amount and was 3,6-conjugated in 85%. Traces of nordihydromorphine and hydrocodone were confirmed as other metabolites of dihydrocodeine in our study. This information can be useful in interpretation of toxicological findings in forensic practice.     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.     

Determination of Salvinorin A in body fluids by high performance liquid chromatography-atmospheric pressure chemical ionization

Salvinorin A was quantitated in human and rhesus monkey plasma, rhesus monkey cerebrospinal fluid, and human urine by negative ion LC-MS/APCI. The method for Salvinorin A has been fully validated, the LLOQ using FDA guidelines is 2 ng/mL for 0.5 mL plasma samples. The linear range was from 2 to 1000 ng/mL. Several derivatives in the Salvinorin family can also be analyzed by this method; d(3)-Salvinorin A was prepared and used as internal standard. The metabolite Salvinorin B can be semi quantitatively determined. The method has been used to establish that Salvinorin B is the principal metabolite of Salvinorin A ex vivo and to establish the analytical method to study in vivo samples.     

Capillary Zone Electrophoresis method for determination of (+)-S clopidogrel carboxylic acid metabolite in human plasma and urine designed for biopharmaceutic studies

Fast and reproducible Capillary Zone Electrophoresis (CZE) method for the quantification of (+)-S clopidogrel carboxylic acid metabolite in human fluids was elaborated for the first time. Optimal buffer and CZE conditions were established to obtain the complete separation of clopidogrel, its metabolite and piroxicam (internal standard), during one analytical run. Finally, resolution of the analytes was obtained in an uncoated silica capillary filled with a phosphate buffer of pH 2.5. The analytes were isolated from plasma and urine samples using solid phase extraction (SPE). Validation of the CZE method was carried out. The calibration curve of clopidogrel was linear in the range of 0.5–10.0 mg/L in plasma and urine, whereas for (+)-S carboxylic acid metabolite linearity was confirmed in the range of 0.25–20.0 mg/L in plasma and 0.25–10.0 mg/L in urine. Intra- and inter-day precision and accuracy were repeatable. LOD and LOQ were also estimated. SPE recovery of the analytes from plasma and urine was comparable and greater than 80%. The validated method was successfully applied in pharmacokinetic investigations of (+)-S carboxylic acid metabolite of clopidogrel following the oral administration of clopidogrel to patients prior to percutaneous coronary intervention.     

Novel urinary metabolite of d-delta-tocopherol in rats

A novel metabolite of d-delta-tocopherol was isolated from the urine of rats given d-3,4-[3H2]-delta-tocopherol intravenously. The metabolite was collected from the urine of rats given d-delta-tocopherol in the same manner as that of the labeled compound. It was found that the metabolites consisted of sulfate conjugates. The portion of the major metabolite released with sulfatase was determined to be 2,8-dimethyl-2-(2'-carboxyethyl)-6-chromanol by infrared spectra, nuclear magnetic resonance spectra, and mass spectra. The proposed structure was confirmed by comparing the analytical results with those of a synthetically derived compound. As a result of the structural elucidation of this novel metabolite, a pathway for the biological transformation of delta-tocopherol is proposed which is different from that of alpha-tocopherol. A characteristic feature of the pathway is the absence of any opening of the chroman ring throughout the sequence.     

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV

Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery>89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.     

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.     

Determination of 2-isopropoxyphenol in urine by capillary gas chromatography and mass-selective detection

An analytical method for the assessment of the exposure of workers to the pesticide propoxur through biological monitoring has been developed. This study was part of a survey of occupational exposure to pesticides used in greenhouses for the growing of ornamental plants. In order to assess the actual absorbed amount of propoxur in the body, an analytical method for its metabolite 2-isopropoxyphenol in urine was required. This led to the development of a gas chromatographic—mass spectrometric assay involving hydrolysis and solvent extraction. A mass-selective detector, operated in single-ion mode, provides a selective and sensitive quantification of 2-isopropoxyphenol with a detection limit of 6 μg/l. The method has been validated with respect to the hydrolysis of urine samples, analytical recovery of 2-isopropoxyphenol, stability of its conjugates, limit of detection, background and precision. The analytical recovery from spiked urine was over 95%. 2-Isopropoxyphenol was excreted in urine as a conjugate and was stable for at least seven months when stored at − 20°C. It was not detected in urine from non-exposed persons. Between-day coefficients of variation were 20, 10, 7 and 4% for concentrations of 15, 29, 150 and 213 μg/l, respectively. Measured as 2-isopropoxyphenol, ca. 80% of an orally administered dose of propoxur was excreted in urine within 10 h.     

Simultaneous detection of methylphenidate and its main metabolite, ritalinic acid, in doping control

Two analytical methods for the simultaneous detection in urine of methylphenidate and its main metabolite, ritalinic acid, are described. Both procedures are based on solid-phase extraction of urine samples on Bond Elut Certify columns, and capillary gas chromatographic—mass spectrometric detection of O-trimethylsilyl, N-trifluoroacetyl derivatives. The former method is used as a general screening procedure for the detection of basic polar nitrogen-containing compounds in urine such as stimulants, narcotic and adrenergic drugs. The latter procedure is proposed as a specific method to confirm methylphenidate ingestion. The two methods are sensitive enough to detect methylphenidate and ritalinic acid in urine at least for 24 h after administration of a therapeutic dose (20 mg oral dose) of methylphenidate.     

Analytical method for the determination of urinary 3-phenoxybenzoic acid in subjects occupationally exposed to pyrethroid insecticides

The determination of urinary 3-phenoxybenzoic acid enables exposure to pyrethroid insecticides to be evaluated. A method for the quantitative determination of this metabolite in urine is described. The compound and the internal standard (2-phenoxybenzoic acid) are derivatized with pentafluorobenzylbromide and transformed into pentafluorobenzyl esters, which are determined by gas chromatography with an intermediate polarity capillary column and an electron-capture detector. Before GC analysis, the urinary extracts are purified on LC-Si SPE columns. The proposed method has a detection limit of 0.5 μg/l and a mean recovery of 91.3%. The coefficient of variation of the analytical procedure, evaluated at a concentration of 24.96 μg/l, was 9.58%. Storage of the urine samples for 3 months at −18°C did not lead to significant changes in the concentration of analyte. The method was tested analysing the urine of a farm worker with symptoms of pyrethroid poisoning, occupationally exposed to esfenvalerate.