Identification and quantitative determination of benproperine metabolites in human plasma and urine by liquid chromatography-tandem mass spectrometry

Two novel metabolites of benproperine (BPP), 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-3-piperidinol (3-OH-BPP) and 1-[1-methyl-2-[2-(phenylmethyl)phenoxy]ethyl]-4-piperidinol (4-OH-BPP), were confirmed by comparison of retention times and mass spectra with those of synthetic standards using liquid chromatography-tandem mass spectrometry. Selective and sensitive procedures were developed for the simultaneous determination of BPP, 3-OH-BPP and 4-OH-BPP in human plasma and urine. The analytes were extracted from plasma sample and enzymatically hydrolyzed urine samples by liquid-liquid extraction, separated through a Diamonsil C(18) column (150 mm x 4.6 mm i.d.) and determined by tandem mass spectrometry with an electrospray ionization interface in selected reaction monitoring mode. Dextromethorphan was used as internal standard. The mobile phase consisted of acetonitrile-water-formic acid (34:66:1, v/v/v), and flow-rate was 0.5 ml min(-1). This method has a lower limit of quantification (LLOQ) of 60, 4.0 and 4.0 nmol l(-1)for BPP, 3-OH-BPP and 4-OH-BPP in plasma, 4.9, 4.7 and 2.4 nmol l(-1) in urine, respectively. The intra- and inter-run precision were measured to be below 9.2%, and the accuracy was within +/-4.3% for the analytes. The method was successfully used to determine BPP, 3-OH-BPP and 4-OH-BPP in plasma and urine for pharmacokinetic investigation. The results indicated residue of 3-OH-BPP in the body at least 192 h after an oral dose of BPP.     

Quantitation of soy-derived phytoestrogens in human breast tissue and biological fluids by high-performance liquid chromatography

A new and reliable HPLC method for the quantitation of daidzein, equol, and genistein in human breast tissue has been developed. The method was applied to biopsies from women undergoing breast reductions, who, prior to surgery, had ingested either a soy isoflavone preparation or a placebo tablet. The results were compared with data collected for urine and serum of the same subjects using standard methods. The limits of detection in the breast tissue homogenate were 24.7 nmol/l for daidzein, 148.0 nmol/l for equol, and 28.4 nmol/l for genistein (S/N of 3). The chromatographic limits of quantitation were 62.5 nmol/l for daidzein and genistein, and 125.0 nmol/l for equol, for which the accuracies were 86.0%, 83.6%, and 81.8%, respectively. The coefficients of variation of these measurements were all below 20% (11.1% for daidzein, 16.4% for genistein, and 13.2% for equol). The sample preparation comprised a concentration step and the absolute limits of quantitation were, therefore, 4.7 nmol/l, 18.8 nmol/l, and 0.94 nmol/l for daidzein and genistein, and 9.4 nmol/l, 37.5 nmol/l, and 1.9 nmol/l for equol in urine, serum, and breast tissue homogenate, respectively. Recoveries were between 70% (+/-5.6%) in breast tissue homogenate and 100% (+/-14.1%) in urine and serum for all three compounds. Equol (less than 1 micromol/l homogenate) was found to be the predominant phytoestrogen in breast tissue and its concentrations exceeded those in serum. The concentrations of phytoestrogens were at least 100-fold higher in urine than in serum and breast tissue.     

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).     

Quantification of apolipoprotein D in human urine by zone immunoelectrophoresis assay: a methodological and clinical study.

A zone immunoelectrophoresis assay (ZIA) has been developed for the quantification of apolipoprotein D (apo D) in unconcentrated native human urine. A standard curve, linear between 1 and 8 mg apo D/l was obtained with ZIA. The relative coefficients of variation for this method were 5-9% (n = 15 x 6) with a mean +/- SD of 7 +/- 1.4% and below 11% (n = 6 x 15) for within-run and between-run reproducibility, respectively. Equal amounts of apo D in unconcentrated and diluted urines, in serum and of the purified protein produced the same zone migration distances indicating parallelism between the immunologic reactions of apo D in different sample matrixes. Storage experiments with normal urines demonstrated good stability of apo D in both acidic and alkalinized urine over at least 2 days at +5 degrees C and during several days at -20 degrees C to -40 degrees C. Using ZIA, urine samples from 50 normal healthy men aged 23-65 years were analyzed for apo D. Mean and SD were: 2.8 +/- 2.1 mg/l, 2.6 +/- 1.8 micrograms/min and 0.24 +/- 0.13 mg/mmol for concentration, rate of excretion and mass/creatinine concentration, respectively.     

Development and validation of a liquid chromatographic method for Casiopeina IIIi in rat plasma

In order to measure changes in physiological CO concentrations in blood with good accuracy, a method was developed using gas chromatography with flame ionisation detection (250 degrees C). A nickel catalyst system was fitted to convert CO to methane at 375 degrees C after separation with a molecular sieve column at 35 degrees C. Helium was used as carrier at 30 ml/min. Porcine or human blood (400 microl) was sampled in gastight tubes and treated with sulfuric acid and saponin (800 microl). Accuracy was 1.4% and 1.5% (RSD), respectively. Precision was 2.8% (porcine blood). Limit of detection was 0.01 nmol/ml gas and limit of quantification 12 nmol/ml blood. Calibration was made in the interval 12-514 nmol/ml blood (corresponding to 0.1-6% COHb). Samples were stable for at least a month at +4 degrees C. This paper describes a method with high sensitivity and good accuracy, suitable for analysis of low CO concentrations.     

Gas chromatographic-mass spectrometric method for the determination of the herbicides paraquat and diquat in plasma and urine samples

In the present work, a method was developed and optimized aiming to determinate the herbicides paraquat (PQ) and diquat (DQ) in human plasma and urine samples. An initial procedure of chemical reduction of the analytes by adding NaBH4 directly in the buffered samples (pH 8.0) was performed. This procedure was necessary to convert the quaternary ammonium substances into more volatile compounds for gas chromatographic analysis. The reduction compounds were extracted with C18 cartridges (solid-phase extraction). Ethyl paraquat (EPQ) was used as internal standard (IS). Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify the analytes in selected ion monitoring (SIM) mode. The limits of detection were 0.05 mg/l for both PQ and DQ. By using the weighted least squares linear regression (1/x1/2 for plasma and 1/y for urine), the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.1 to 50 mg/l; r>0.98). This method can be readily utilized as an important tool to confirm the suspicion of PQ and/or DQ poisoning and evaluate the extent of the intoxication.     

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20 267) in plasma and of letrozole and its metabolite, CGP 44 645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C₈ columns using the ASPEC system. The separation was achieved on an ODS Hypersil C₁₈ column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44 645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.     

Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry

The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 microl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 microm I.D.x 30 mm 10 microm Kromasil C(18) pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 microm I.D.x 100 mm 3.5 microm Kromasil C(18) analytical column. Loading flow rates up to 100 microl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M-H](-) at m/z 217.08. The method was validated over the concentration range 0.2-40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4-7.3 and 7.0-8.1%, respectively, and the recoveries were in the range 96.2-97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.     

Stability studies of amphetamine and ephedrine derivatives in urine

Knowledge of the stability of drugs in biological specimens is a critical consideration for the interpretation of analytical results. Identification of proper storage conditions has been a matter of concern for most toxicology laboratories (both clinical and forensic), and the stability of drugs of abuse has been extensively studied. This concern should be extended to other areas of analytical chemistry like antidoping control. In this work, the stability of ephedrine derivatives (ephedrine, norephedrine, methylephedrine, pseudoephedrine, and norpseudoephedrine), and amphetamine derivatives (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA)) in urine has been studied. Spiked urine samples were prepared for stability testing. Urine samples were quantified by GC/NPD or GC/MS. The homogeneity of each batch of sample was verified before starting the stability study. The stability of analytes was evaluated in sterilized and non-sterilized urine samples at different storage conditions. For long-term stability testing, analyte concentration in urine stored at 4 degrees C and -20 degrees C was determined at different time intervals for 24 months for sterile urine samples, and for 6 months for non-sterile samples. For short-term stability testing, analyte concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing (at room temperature) was also studied in sterile urine for up to three cycles. No significant loss of the analytes under study was observed at any of the investigated conditions. These results show the feasibility of preparing reference materials containing ephedrine and amphetamine derivatives to be used for quality control purposes.     

Highly sensitive gas chromatographic-mass spectrometric screening method for the determination of picogram levels of fentanyl, sufentanil and alfentanil and their major metabolites in urine of opioid exposed workers

Highly sensitive and specific analytical GC-MS procedures were developed and comprehensively validated for the determination of the opioid narcotics fentanyl, sufentanil and alfentanil and their major nor-metabolites in urine of potentially exposed opioid production workers. A simple, one step extraction protocol was developed using commercially available solid phase extraction (SPE) columns to recover all analytes from urine. The secondary amine functionalities of the nor-metabolites were derivatized to form stable, pentafluorobenzamide (PFBA)-derivatives with good chromatographic properties. Using the penta-deuterated analogues as internal standards, a limit-of-detection (LOD) of 2.5 pg fentanyl/ml, 2.5 pg sufentanil/ml and 7.5 pg alfentanil/ml urine was achieved. For the opioid metabolites the LODs were found to be <50 pg/ml urine. The developed analytical procedures show excellent intra-assay accuracy, particularly considering the ultra low levels of the analytes, with relative errors generally below 10%. Overall, an excellent reproducibility was observed with coefficients of variation below 10% at all spike levels for all opioid parent compounds and their metabolites, except for low norfentanyl concentrations. Upon storage at -30 degrees C urine samples were found to be stable for at least 2 months as no significant losses of either compound were observed. The developed analytical procedures have been successfully applied in a biological monitoring survey of fentanyl exposed production workers.     

Sensitive headspace gas chromatography-mass spectrometry determination of trihalomethanes in urine

A sensitive and straightforward method for the determination of trihalomethanes (THMs) in urine by using headspace extraction technique has been developed. Chemical and instrumental variables were studied in order to optimize the method for sensitivity: an excess of KCl (4 g per 12 ml of urine), an oven temperature of 85 degrees C and an equilibration time of 30 min were selected. The use of the mass spectrometer in selected ion monitoring mode allows achieving linear ranges between 10 and 5000 ng/l and detection limits from 3 to 10 ng/l, for 12 ml of urine. The stability of the urine sample during storage at 4 and -20 degrees C was also evaluated: THMs remained stable for up to 2 days and 2 months, respectively. Finally, the method was successfully applied to study the THM uptake from swimmers of an indoor swimming pool, as well as non-swimmers. This study revealed that the concentrations of THMs in urine increased approximately three times for chloroform and bromodichloromethane after swimming activity. In addition, THMs in unchanged form were mainly excreted within 2-3h after the end of exposure.     

Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats

A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of the above analytes. The lower limits of quantification were 2.5 ng/mL for phenacetin and paracetamol, 5 ng/mL for midazolam, 0.5 ng/mL for 1'-hydroxymidazolam, 5 ng/mL for dextromethorphan, 50 ng/mL for dextrorphan and 4'-hydroxymephenytoin, 5 ng/mL for tolbutamide, 50 ng/mL for 4-hydroxytolbutamide and 25 ng/mL for carboxytolbutamide. All the analytes were evaluated for short-term (24 h, room temperature), long-term (3 months, -20 degrees C), three freeze-thaw cycles and autosampler (24 h, 4 degrees C) stability. The stability of urine samples was also prepared with and without beta-glucuronidase incubation (37 degrees C) and measured comparatively. No significant loss of the analytes was observed at any of the investigated conditions. The current method provides a robust and reliable analytical tool for the above five-probe drug cocktail, and has been successfully verified with known CYP inducers.     

Determination of ximelagatran,an oral direct thrombin inhibitor,its active metabolite melagatran,and the intermediate metabolites,in biological samples by liquid chromatography-mass spectrometry

Analytical methods for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and intermediate metabolites, melagatran hydroxyamidine and melagatran ethyl ester, in biological samples by liquid chromatography (LC) positive electrospray ionization mass spectrometry (MS) using selected reaction monitoring are described. Isolation from human plasma was achieved by solid-phase extraction on octylsilica. Analytes and isotope-labelled internal standards were separated by LC utilising a C(18) analytical column and a mobile phase comprising acetonitrile-4 mmol/l ammonium acetate (35:65, v/v) containing 0.1% formic acid, at a flow-rate of 0.75 ml/min. Absolute recovery was approximately 80% for ximelagatran, approximately 60% for melagatran ethyl ester and >90% for melagatran and melagatran hydroxyamidine. Limit of quantification was 10 nmol/l, with a relative standard deviation <20% for each analyte and <5% above 100 nmol/l. Procedures for determination of these analytes in human urine and breast milk, plus whole blood from rat and mouse are also described.     

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.     

Measurement of total mirtazapine and normirtazapine in plasma/serum by liquid chromatography with fluorescence detection

A simple high performance liquid chromatography (HPLC) method for the measurement of the new antidepressant mirtazapine and its N-demethyl metabolite, normirtazapine, in human plasma or serum during low dose mirtazapine therapy has been developed. A Waters Spherisorb S5 SCX column was used with ammonium perchlorate (50 mmol/l) in methanol/water (95 + 5 (v/v)), apparent pH 6.7, as eluent, and fluorescence detection. Only small volumes of sample (0.2 ml) and extraction solvent are used. An interference study found no significant co-elution with drug or metabolite, although paroxetine co-elutes with the internal standard. The recovery of mirtazapine and normirtazapine (mean +/- S.D.) was 79 +/- 2, and 64 +/- 3%, respectively. The LOD was estimated as 0.5 microg/l, LLOQ was 1 microg/l, with a linear response over the concentration range 4-1000 microg/l (both analytes). The analytes were stable in serum for at least 10 months when stored at -20 degrees C. Intra- and inter-day accuracy were in the range 91-107 and 93-103%, respectively. In clinical samples (n = 14, median mirtazapine dose 45 mg per day, range 15-45 mg per day) the median (range) mirtazapine and normirtazapine concentrations were 26 (8-40) and 21 (8-32) microg/l, respectively.     

Determination of melagatran, a novel, direct thrombin inhibitor, in human plasma and urine by liquid chromatography-mass spectrometry

Analytical methods for the determination of melagatran (H 319/68) in biological samples by liquid chromatography (LC)-positive electrospray ionization mass spectrometry using multiple reaction monitoring are described. Melagatran in plasma was isolated by solid-phase extraction on octylsilica, either in separate extraction tubes or in 96-well plates. Absolute recovery of melagatran from plasma was >92%. Melagatran and the internal standard, H 319/68 D2 13C2, were separated from other sample components by LC utilizing a C18 stationary phase and a mobile phase comprising 35% acetonitrile and 0.08% formic acid in 0.0013 mol/l ammonium acetate solution. After dilution, urine was injected directly onto the LC column and subjected to gradient LC. The relative standard deviation was 1-5% for concentrations above the limit of quantification, which was estimated for plasma at 10 or 25 nmol/l for sample volumes of 500 or 200 microl, respectively, and 100 nmol/l for urine.     

Three phase liquid phase microextraction of phenylacetic acid and phenylpropionic acid from biological fluids

Three phase liquid phase microextraction (three phase LPME) technique coupled with HPLC-UV has been applied as a sensitive and efficient sample preparation method to determine phenylacetic acid (PAA) as a biomarker of depressive disorders and phenylpropionic acid (PPA) in biological fluids. The compounds were extracted from 3.0 ml aqueous solution with the adjustment of pH at a fixed value in the range of 2.0-3.5 (donor solution) into an organic phase (1-hexanol) layered on the surface of the donor solution and finally back-extracted into 4.0 microl of the acceptor microdrop (pH 11.1) located at the end of the microsyringe needle. After a prescribed back-extraction time, the acceptor microdrop was withdrawn into the microsyringe and then directly injected into the HPLC system. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. At the optimum conditions (donor solution: 2.3M Na(2)SO(4), pH 2.0-3.5; organic membrane: 95 microl of 1-hexanol; acceptor solution: 4.0 microl of 0.1M NH(3)/NH(4)(+) with pH 11.1; donor solution temperature: 45-50 degrees C; extraction time: 20 min and back-extraction time: 12 min), up to 110-fold enrichment factor was obtained. The calibration curve for these analytes was linear in the range of 1-5000 microg/l with r(2)>0.998. The intraday and interday RSD% were below 6.5% and the limits of detection (LODs) for both analytes were 0.2 microg/l (based on S/N=3). The proposed technique is a low cost, simple and sensitive method with highly clean-up effect. Finally, this technique was successfully utilized for the detection of target analytes in human urine, serum and plasma.     

Determination of melarsoprol in biological fluids by high-performance liquid chromatography and characterisation of two stereoisomers by nuclear magnetic resonance spectroscopy

The analysis of melarsoprol in whole blood, plasma, urine and cerebrospinal fluid is described. Extraction was made with a mixture of chloroform and acetonitrile followed by back-extraction into phosphoric acid. A reversed-phase liquid chromatography system with ultraviolet detection was used. The relative standard deviation was 1% at concentrations around 10 μmol/l and 3–6% at the lower limit of determination (9 nmol/l in plasma, 93 nmol/l in whole blood, 45 nmol/l in urine and 10 nmol/l in cerebrospinal fluid). Melarsoprol is not a stable compound and samples to be stored for longer periods of time should be kept at −70°C. Plasma samples can be stored at −20°C for upt to 2 months. Chromatography showed that melarsoprol contains two components. Using nuclear magnetic resonance spectroscopy the two components were shown to be diastereomers which slowly equilibrate by inversion of the configuration at the As atom.     

Determination of urinary ortho- and meta-cresol in humans by headspace SPME gas chromatography/mass spectrometry

ortho-Cresol (o-C) and meta-cresol (m-C) are minor urinary metabolites of toluene, a widely used chemical with neurotoxicological properties. A new assay for their determination in human urine is here proposed. Urinary cresol sulphates and glucuronates are submitted to acid hydrolysis, urine is neutralized, added with o-cresols-d8, and analytes are sampled in the headspace of urine by SPME using a polydimethylsiloxane fiber. Analysis is performed by GC/MS using, for separation, either a SupelcoWax10 (for o-C) or a chiral CP Cresol (for o-C and m-C) column. The method is very specific, with a range of linearity 0-5.0 mg/l, within- and between-run precision, as coefficient of variation, <15% and <19%, limit of detection of 0.006 mg/l for o-C and 0.007 mg/l for m-C. The procedure is applied to the quantification of cresols in urine from workers exposed to toluene and from subjects belonging to the general population.     

High-performance liquid chromatographic determination of the magnetic resonance imaging contrast agent Gadocoletate ion in human plasma, urine and faeces

Gadocoletate ion is a new paramagnetic intravascular contrast agent for magnetic resonance imaging (MRI). An high-performance liquid chromatographic method for assaying Gadocoletate ion in human plasma, urine and faecal samples is described. The analysis is based on the reversed-phase chromatographic separation of Gadocoletate ion from the endogenous components of the biological matrices and its detection during elution by ultraviolet light absorption at 200 nm. The selectivity of the method was satisfactory. The mean absolute recovery during the analytical sample preparation was greater than 87%. The precision, expressed as coefficient of variation (CV%) ranged from 0.29 to 5.90% and the accuracy, expressed as mean relative error (R.E.%) of the analytical method ranged from -3.7 to +7.1%. The detection limit in plasma and urine was 2.01 and 10.0 microg/mL (0.00203 and 0.0101 micromol/mL), respectively. The detection limit in homogenized faecal samples was 17.7 microg/g (0.0179 micromol/g). Stability studies were performed in human plasma and urine samples during the analytical cycle. Gadocoletate ion was shown to be stable in human plasma and in human urine when stored at about +4 degrees C for up 24 h, and after three freeze-thaw cycles. In addition, it was shown to be stable in samples of processed plasma and in diluted urine at about +4 degrees C for 48 h, and at room temperature for at least 24 h. As regards the long-term stability of Gadocoletate ion, the results of dedicated studies showed that Gadocoletate ion is stable in human plasma samples when stored at +4 degrees C for up to 30 days and at -80 degrees C for up to 90 days. Gadocoletate ion is stable in samples of human urine when stored at +4 degrees C for up to 30 days, and when stored at -20 degrees C and at -80 degrees C for up to 90 days. The method has been successfully validated in human plasma, urine and faeces and it has been shown to be precise, accurate and reliable.