Automated mass spectrometric analysis of urinary and plasma serotonin

Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography–tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 μL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0–7300 μmol/L calibration range was excellent (R² > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10–78 μmol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders.     

Evaluation of a novel ELISA for serotonin: urinary serotonin as a potential biomarker for depression

Depression is a common disorder with physical and psychological manifestations often associated with low serotonin. Since noninvasive diagnostic tools for depression are sparse, we evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. We developed a competitive ELISA for direct measurement of serotonin in derivatized urine samples. Assay performance was evaluated and applied to clinical samples. The analytical range of the assay was from 6.7 to 425 μg serotonin/g creatinine (Cr). The limit of quantification was 4.7 μg/g Cr. The average recovery for spiked urine samples was 104.4%. Average intra-assay variation was 4.4%, and inter-assay variation was <20%. The serotonin analysis was very specific. No significant interferences were observed for 44 structurally and nonstructurally related urinary substances. Very good correlation was observed between urinary serotonin levels measured by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS; ELISA = 1.16 × LC-MS/MS − 53.8; r = 0.965; mean % bias = 11%; n = 18). Serotonin was stable in acidified urine for 30 days at room temperature and at −20 °C. The established reference range for serotonin was 54–366 μg/g Cr (n = 64). Serotonin levels detected in depressed patients (87.53 ± 4.89 μg/g Cr; n = 60) were significantly lower (p < 0.001) than in nondepressed subjects (153.38 ± 7.99 μg/g Cr). Urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by 5-hydroxy-tryptophane and/or selective serotonin re-uptake inhibitor (p < 0.01). The present ELISA provides a convenient and robust method for monitoring urinary serotonin. It is suitable to monitor serotonin imbalances and may be particularly helpful in evaluating antidepressant therapies.     

Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-d-glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.     

Development and validation of a sensitive,simple, and rapid method for simultaneous quantitation of atorvastatin and its acid and lactone metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The aim of the proposed work was to develop and validate a simple and sensitive assay for the analysis of atorvastatin (ATV) acid, ortho- and para-hydroxy-ATV, ATV lactone, and ortho- and para-hydroxy-ATV lactone in human plasma using liquid chromatography-tandem mass spectrometry. All six analytes and corresponding deuterium (d5)-labeled internal standards were extracted from 50 μL of human plasma by protein precipitation. The chromatographic separation of analytes was achieved using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase consisted of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). All analytes including ortho- and para-hydroxy metabolites were baseline-separated within 7.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode, with multiple-reaction monitoring scan. The calibration curves for all analytes were linear (R ² ≥ 0.9975, n = 3) over the concentration range of 0.05–100 ng/mL and with lower limit of quantitation of 0.05 ng/mL. Mean extraction recoveries ranged between 88.6–111%. Intra- and inter-run mean percent accuracy were between 85–115% and percent imprecision was ≤ 15%. Stability studies revealed that ATV acid and lactone forms were stable in plasma during bench top (6 h on ice-water slurry), at the end of three successive freeze and thaw cycles and at −80 °C for 3 months. The method was successfully applied in a clinical study to determine concentrations of ATV and its metabolites over 12 h post-dose in patients receiving atorvastatin.     

A simple assay for the simultaneous determination of rosuvastatin acid,rosuvastatin-5<Emphasis Type="Italic">S</Emphasis>-lactone,and <Emphasis Type="Italic">N</Emphasis>-desmethyl rosuvastatin in human plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS)

A simple and sensitive assay was developed and validated for the simultaneous quantification of rosuvastatin acid (RST), rosuvastatin-5S-lactone (RST-LAC), and N-desmethyl rosuvastatin (DM-RST), in buffered human plasma using liquid chromatography–tandem mass spectrometry (LC-MS/MS). All the three analytes and the corresponding deuterium-labeled (d6) internal standards were extracted from 50 μL of buffered human plasma by protein precipitation. The analytes were chromatographically separated using a Zorbax-SB Phenyl column (2.1 mm × 100 mm, 3.5 μm). The mobile phase comprised of a gradient mixture of 0.1% v/v glacial acetic acid in 10% v/v methanol in water (solvent A) and 40% v/v methanol in acetonitrile (solvent B). The analytes were separated at baseline within 6.0 min using a flow rate of 0.35 mL/min. Mass spectrometry detection was carried out in positive electrospray ionization mode. The calibration curves for all three analytes were linear (R ≥ 0.9964, n = 3) over the concentration range of 0.1–100 ng/mL for RST and RST-LAC, and 0.5–100 ng/mL for DM-RST. Mean extraction recoveries ranged within 88.0–106%. Intra- and inter-run mean percent accuracy were within 91.8–111% and percent imprecision was ≤15%. Stability studies revealed that all the analytes were stable in matrix during bench-top (6 h on ice–water slurry), at the end of three successive freeze and thaw cycles and at −80°C for 1 month. The method was successfully applied in a clinical study to determine the concentrations of RST and the lactone metabolite over 12-h post-dose in patients who received a single dose of rosuvastatin.     

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R ² > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.     

Validated method for the determination of ethylglucuronide and ethylsulfate in human urine

Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d ₅ was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R ² > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between −5.0% and −11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.     

Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography–tandem mass spectrometry (GC–MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC–MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R ² > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 μmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.     

Simultaneous quantification of loureirin A and loureirin B in rat urine, feces, and bile by HPLC-MS/MS method and its application to excretion study

A simple HPLC-MS/MS method for simultaneous determination of loureirin A and loureirin B in rat urine, feces, and bile after oral administration of 10.6 g/kg of longxuejie (one rare traditional Chinese medicinal herb) was developed for the first time. The analytes and buspirone (internal standard) were separated on a C₅ column with acetonitrile–water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.4 min/mL. The detector was a Q-trap™ mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. Calibration curves of loureirin A in rat urine, feces, and bile were linear over the concentration range of 1.00–5,000 ng/mL. Loureirin B in rat urine, feces, and bile ranged between 0.08 and 20, 0.20 and 20, and 0.10 and 500 ng/mL, respectively. Validation revealed that the method was specific, accurate, and precise. The fully validated method was applied to the excretion study of loureirin A and loureirin B in rats. After oral administration of 10.6 g/kg longxuejie, cumulative excretion amount of loureirin A and loureirin B in rat urine were 2.94 ± 0.81 and 0.36 ± 0.16 μg at 72 h, respectively. Of the total dose, 5.35% of loureirin A and 5.46% of loureirin B were excreted from feces at 60 h. The cumulative amounts of loureirin A and loureirin B in rat bile reached 4.49 ± 0.98 and 5.11 ± 0.83 μg, respectively, at 36 h after dosing, accounting for 0.054% and 0.056% of the total dose.     

Water-compatible molecularly imprinted polymer for the selective recognition of fluoroquinolone antibiotics in biological samples

A novel water-compatible molecularly imprinted polymer (MIP), prepared with enrofloxacin (ENR) as the template, has been optimised for the selective extraction of fluoroquinolone antibiotics in aqueous media. The results of a morphological characterisation and selectivity tests of the polymer material for ENR and related derivatives are reported. High affinity for the piperazine-based fluoroquinolones marbofloxacin, ciprofloxacin, norfloxacin and ofloxacin was observed, whereas no retention was found for nonrelated antibiotics. Various parameters affecting the extraction efficiency of the polymer have been optimised to achieve selective extraction of the antibiotics from real samples and to reduce nonspecific interactions. These findings resulted in a MISPE/HPLC-FLD method allowing direct extraction of the analytes from aqueous samples with a selective wash using just 50% (v/v) organic solvent. The method showed excellent recoveries and precision when buffered urine samples fortified at five concentration levels (25–250 ng mL⁻¹ each) of marbofloxacin, ciprofloxacin, norfloxacin, enrofloxacin and sarafloxacin were tested (53–88%, RSD 1–10%, n = 3). Moreover, the biological matrix of the aqueous samples did not influence the preconcentration efficiency of the fluoroquinolones on the MIP cartridges; no significant differences were observed between the recovery rates of the antibiotics in buffer and urine samples. The detection limits of the whole process range between 1.9 and 34 ng mL^–1 when 5-mL urine samples are processed. The developed method has been successfully applied to preconcentration of norfloxacin in urine samples of a medicated patient, demonstrating the ability of the novel MIP for selective extraction of fluoroquinolones in urine samples.     

Molecularly imprinted monolith in-tube solid-phase microextraction coupled with HPLC/UV detection for determination of 8-hydroxy-2′-deoxyguanosine in urine

Urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) has been widely used as a biomarker of oxidative DNA damage. Measurements of 8-OHdG in urinary samples are challenging owing to the low level of 8-OHdG and the complex matrix. In this study, a novel molecularly imprinted polymer (MIP) monolithic column was synthesized with guanosine as a dummy template which was used as the medium for in-tube solid-phase microextraction (SPME). In-tube SPME coupled with HPLC/UV detection for extraction and determination of urinary 8-OHdG was developed. The synthesized MIP monolithic column exhibited high extraction efficiency owing to its greater phase ratio with convective mass transfer and inherent selectivity. The enrichment factor for 8-OHdG was found to be 76 and the limits of detection and quantification of the method for urinary samples were 3.2 nmol/L (signal-to-noise ratio 3) and 11 nmol/L (signal-to-noise ratio 10), respectively. The MIP^’s selectivity also made the sample preparation procedure and chromatographic separation much easier. The linear range of the proposed method was from 0.010 to 5.30 μmol/L (r = 0.9997), with a relative standard deviation of 1.1–6.8%, and the recovery for spiked urine samples was 84 ± 3%. The newly developed method was successfully applied to determine urinary samples of healthy volunteers, coking plant workers, and cancer patients. The 8-OHdG level in cancer patients was significantly higher than that in healthy people.     

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN^–) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN^– from methemoglobin. CN^– was then reacted with NaAuCl₄ to produce dicyanogold, Au(CN)₂^–, that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN^– was performed by selected reaction monitoring of the product ion CN^– at m/z 26, derived from the precursor ion Au(CN)₂^– at m/z 249. CN^– could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN^– in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN^– in the blood of a victim who ingested NaN₃ was confirmed using MS-MS for the first time, and the concentrations of CN^– in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.     

Determination of pyrethroid metabolites in human urine using liquid phase microextraction coupled in-syringe derivatization followed by gas chromatography/electron capture detection

Metabolites of synthetic pyrethroids such as cis-3-(2,2-dibromovinyl)-2,2-di-methylcyclo-propane-1-carboxylic acid, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid), 3-phenoxybenzoic acid (3-PBA), and 4-fluoro-3-PBA are biomarkers for exposure to phenothrin, tetramethrin, cyfluthrin, cypermethrin, deltamethrin, and permethrin. In this study, the pyrethroid metabolites in workers’ urine samples were monitored for the first time with a novel sample pretreatment process combining hollow fiber liquid phase microextraction (HF-LPME) and in-syringe derivatization (ISD) followed by gas chromatography–electron capture detector (GC-ECD) analysis. A micro-syringe pre-filled with derivatizing agents and syringe needle connected to an extracting solvent impregnated hollow fiber segment was used as the LPME probe. Pyrethroid metabolites were extracted and enriched simultaneously from urine samples by HF-LPME sampling and acid hydrolysis at 70 °C for 10 min. After sampling, the ISD was performed by mixing the extracting solution and derivatizing agents through plunger movements, followed by GC-ECD analysis. Parameters influencing the HF-LPME efficiency and ISD were investigated and optimized. Under optimum conditions, the method provided enrichment factors of 69.8–154.6, repeatability from 5.0 to 12% (n = 5), and good linearity (R ² = 0.9980–0.9998) for interested analytes spiked in urine samples. The method detection limits ranged from 1.6 to 17 ng/mL. A comparison was performed between the proposed method and conventional methods. The proposed method was applied to analyze pyrethroid metabolites in the urine samples collected from workers of pesticide formulation plants. The results suggested that the proposed HF-LPME coupled ISD method was a rapid, simple, efficient, and eco-friendly technique in the biomonitoring of metabolites of pyrethroids in workers’ urine.     

Quantification of luminally released serotonin in rat proximal colon by capillary electrophoresis with laser-induced fluorescence detection

Serotonin (5-hydroxytryptamine, 5-HT) plays vital roles in regulating gastrointestinal functions. Thus, the detection of 5-HT in the gastrointestinal tract is of great importance for biomedical research, medical diagnosis, and pharmaceutical therapy. This paper presents a simple, sensitive, and fast method for the quantification of luminally released serotonin in the feces and tissues of the rat proximal colon by means of capillary electrophoresis with laser-induced fluorescence detection. 5-Carboxyfluorescein N-succinimidyl ester was used for precolumn derivatization of serotonin. The optimal separation and detection conditions were obtained with an electrophoretic buffer containing 60 mM borate (pH 8.90) and an air-cooled argon-ion laser (excitation at 488 nm, emission at 520 nm). The serotonin concentrations in the feces and tissues of proximal colons were analyzed with this method, and the average values of serotonin in the feces samples were 1.951 ± 0.446 ng/mg (male) and 2.095 ± 0.533 ng/mg (female) and 1.397 ± 0.267 ng/mg in rat proximal colon tissues. The results demonstrate that this method can accurately determine luminally released 5-HT in rats.     

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

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

Semi-automated liquid chromatography-mass spectrometry (LC-MS/MS) method for basic pesticides in wastewater effluents

Effluent from wastewater treatment plants have been identified as an important source of micro-organic contaminants in the environment. An online high-performance liquid chromatography–heated electrospray ionization tandem mass spectrometric method was developed and validated for the determination of basic pesticides in effluent wastewaters. Most available methods for pesticide analysis of wastewater samples are time-consuming, require complex clean-up steps and are difficult to automate. The method developed used a simple solid-phase extraction clean-up for salt and lipid reduction. On-line sample pre-concentration was performed using a reversed phase (C₁₈) column, and analytes were separated by back-flushing onto an analytical column (C₈) with detection using QqQ MS. An option to increase MS resolution was exploited to minimize interference from endogenous compounds in the matrix. A better than unit mass resolution was used (Q1 full width half maximum (FWHM) = 0.2 Da and Q3 FWHM = 0.7 Da), which was as rugged as a unit resolution method, and improved signal/noise and better detection limits were achieved for the targeted basic pesticides. This method was applied to the determination of 11 pesticides, including methoxytriazine, chlorotriazines, chloroacetanilides, phenylurea and carbamate pesticides. The percentage recovery values for these pesticides using the online trapping column were within the range, 73–95%, with relative standard deviation (RSD) values <8.9%. The highest concentrations of these pesticides in wastewater effluents in County Cork, Ireland, were simazine (0.51 μg/L), prometon (0.14 μg/L), diuron (0.21 μg/L) and atrazine (0.19 μg/L).     

Optimisation of stir bar sorptive extraction and in-tube derivatisation-thermal desorption-gas chromatography-mass spectrometry for the determination of several endocrine disruptor compounds in environmental water samples

The analysis of organic pollutants in environmental water samples requires a pre-concentration step. Pre-concentration techniques such as stir bar sorptive extraction (SBSE) have gained popularity since they minimise the use of toxic organic solvents and can be considered as green analytical techniques. Similar to other pre-concentration techniques, one of the problems when SBSE is used is the matrix effect, which often occurs during the analysis of environmental water samples such as estuarine or wastewater samples. The present work studied the matrix effect during SBSE coupled to in-tube derivatisation–thermal desorption (TD)–gas chromatography–mass spectrometry for the determination of several endocrine disruptor compounds, such as alkylphenols, bisphenol A, estrogens and sterols, in environmental water samples, after optimisation of the major variables affecting the determination. Variables such as the addition of methanol or an inert salt to the donor phase, the extraction temperature, the volume of the donor phase, the stirring rate and the extraction time were studied during the SBSE optimisation. In the case of the in-tube derivatisation and TD step, the volume of the derivatisation reagent (N,O-bis(trimethylsilyl)triufloroacetamide with 1% of trimethylchlorosilane (BSTFA + 1% TMCS)) and the cryo-focusing temperature were fixed (2 μL and −50 °C, respectively) according to a consensus between maximum signal and optimal operation conditions. Good apparent recovery values (78–124%) were obtained for most of the analytes in Milli-Q water, except for 4-tert-octylphenol (4-tOP), which showed apparent recovery values exceeding 100%. Precision (n = 4) was in the 2–27%, and method detection limits were in the low nanogrammes per litre level for most of the analytes studied. The matrix effect was studied using two different approaches. On the one hand, Milli-Q water samples were spiked with humic acids, and apparent recovery values were studied with and without correction with the corresponding deuterated analogue. On the other hand, estuarine water and wastewater samples were spiked with known concentrations of target analytes, and apparent recoveries were studied as explained above. In general, the matrix effect could be corrected with the use of deuterated analogues, except for 4-tOP and nonylphenols for which [²H₄]-n-nonylphenol did not provide good corrections.     

A sensitive liquid chromatographic-mass spectrometric method for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma

A liquid chromatographic–mass spectrometric (LC–MS) method has been developed and validated for simultaneous determination of dehydroevodiamine and limonin from Evodia rutaecarpa in rat plasma. After addition of the internal standard, domperidone, plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on an Apollo C₁₈ column (250 mm × 4.6 mm, 5 μm), with methanol–0.01% formic acid water (60:40, v/v) as mobile phase, within a runtime of 12.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 1.0–500 ng mL⁻¹ for dehydroevodiamine and 2.0–1,000 ng mL⁻¹ for limonin, with lower limits of quantitation of 1.0 and 2.0 ng mL⁻¹, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, respectively, for both analytes, and the accuracy (relative error, RE, %) was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to a comparative pharmacokinetic study of dehydroevodiamine and limonin in rat plasma after oral administration of dehydroevodiamine, limonin, and an aqueous extract of Evodiae fructus. The results indicated there were obvious differences between the pharmacokinetic behavior after oral administration of an aqueous extract of Evodiae fructus compared with single substances.     

Plasma and urine profiles of Δ9-tetrahydrocannabinol and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol after cannabis smoking by male volunteers to estimate recent consumption by athletes

Since 2004, cannabis has been prohibited by the World Anti-Doping Agency for all sports competitions. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In doping urine analysis, the target analyte is 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), the cutoff being 15 ng/mL. However, the wide urinary detection window of the long-term metabolite of Δ⁹-tetrahydrocannabinol (THC) does not allow a conclusion to be drawn regarding the time of consumption or the impact on the physical performance. The purpose of the present study on light cannabis smokers was to evaluate target analytes with shorter urinary excretion times. Twelve male volunteers smoked a cannabis cigarette standardized to 70 mg THC per cigarette. Plasma and urine were collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-Δ⁹-tetrahydrocannabinol (THC-OH), and THC-COOH were determined after hydrolysis followed by solid-phase extraction and gas chromatography/mass spectrometry. The limits of quantitation were 0.1–1.0 ng/mL. Eight puffs delivered a mean THC dose of 45 mg. Plasma levels of total THC, THC-OH, and THC-COOH were measured in the ranges 0.2–59.1, 0.1–3.9, and 0.4–16.4 ng/mL, respectively. Peak concentrations were observed at 5, 5–20, and 20–180 min. Urine levels were measured in the ranges 0.1–1.3, 0.1–14.4, and 0.5–38.2 ng/mL, peaking at 2, 2, and 6–24 h, respectively. The times of the last detectable levels were 2–8, 6–96, and 48–120 h. Besides high to very high THC-COOH levels (245 ± 1,111 ng/mL), THC (3 ± 8 ng/mL) and THC-OH (51 ± 246 ng/mL) were found in 65 and 98% of cannabis-positive athletes’ urine samples, respectively. In conclusion, in addition to THC-COOH, the pharmacologically active THC and THC-OH should be used as target analytes for doping urine analysis. In the case of light cannabis use, this may allow the estimation of more recent consumption, probably influencing performance during competitions. However, it is not possible to discriminate the intention of cannabis use, i.e., for recreational or doping purposes. Additionally, pharmacokinetic data of female volunteers are needed to interpret cannabis-positive doping cases of female athletes.     

Analysis of the flame retardant metabolites bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl phosphate (DPP) in urine using liquid chromatography–tandem mass spectrometry

Organophosphate triesters tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate are widely used flame retardants (FRs) present in many products common to human environments, yet understanding of human exposure and health effects of these compounds is limited. Monitoring urinary metabolites as biomarkers of exposure can be a valuable aid for improving this understanding; however, no previously published method exists for the analysis of the primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), in human urine. Here, we present a method to extract the metabolites BDCPP and diphenyl phosphate (DPP) in human urine using mixed-mode anion exchange solid phase extraction and mass-labeled internal standards with analysis by atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry. The method detection limit was 8 pg mL⁻¹ urine for BDCPP and 204 pg mL⁻¹ for DPP. Recoveries of analytes spiked into urine ranged from 82 ± 10% to 91 ± 4% for BDCPP and from 72 ± 12% to 76 ± 8% for DPP. Analysis of a small number of urine samples (n = 9) randomly collected from non-occupationally exposed adults revealed the presence of both BDCPP and DPP in all samples. Non-normalized urinary concentrations ranged from 46–1,662 pg BDCPP mL⁻¹ to 287–7,443 pg DPP mL⁻¹, with geometric means of 147 pg BDCPP mL⁻¹ and 1,074 pg DPP mL⁻¹. Levels of DPP were higher than those of BDCPP in 89% of samples. The presented method is simple and sufficiently sensitive to detect these FR metabolites in humans and may be applied to future studies to increase our understanding of exposure to and potential health effects from FRs.