A high-throughput method for determination of metabolites of organophosphate flame retardants in urine by ultra performance liquid chromatography–high resolution mass spectrometry

Organophosphate triesters are common flame retardants used in a wide variety of consumer products from which they can migrate and pollute the indoor environment. Humans may thus be continuously exposed to several organophosphate triesters which might be a risk for human health. An analytical method based on direct injection of 5 μL urine into an ultra performance liquid chromatography system coupled to a time-of-flight mass spectrometry has been developed and validated to monitor exposure to organophosphate triesters through their respective dialkyl and diaryl phosphate metabolites (DAPs). The targeted analytes were: di-n-butyl phosphate (DNBP), diphenyl phosphate (DPHP), bis(2-butoxyethyl) phosphate (BBOEP), bis(2-chloroethyl) phosphate (BCEP), bis(1-chloro-2-propyl) phosphate (BCPP) and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP). Separation was achieved in less than 3 min on a short column with narrow diameter and small particle size (50 mm × 2.1 mm × 1.7 μm). Different mobile phases were explored to obtain optimal sensitivity. Acetonitrile/water buffered with 5 mM of ammonium hydroxide/ammonium formate (pH 9.2) was the preferred mobile phase. Quantification of DAPs was performed using deuterated analogues as internal standards in synthetic urine (averaged DAP accuracy was 101%; RSD 3%). Low method limits of quantification (MLQ) were obtained for DNBP (0.40 ng mL⁻¹), DPHP (0.10 ng mL⁻¹), BDCIPP (0.40 ng mL⁻¹) and BBOEP (0.60 ng mL⁻¹), but not for the most polar DAPs, BCEP (∼12 ng mL⁻¹) and BCPP (∼25 ng mL⁻¹). The feasibility of the method was tested on 84 morning urine samples from 42 mother and child pairs. Only DPHP was found above the MLQ in the urine samples with geometric mean (GM) concentrations of 1.1 ng mL⁻¹ and 0.57 ng mL⁻¹ for mothers and children respectively. BDCIPP was however, detected above the method limit of detection (MLD) with GM of 0.13 ng mL⁻¹ and 0.20 ng mL⁻¹. While occasionally detected, the GM of DNBP and BBOEP were below MLD in both groups.     

Chromatographic analysis of serotonin, 5-hydroxyindolacetic acid and homovanillic acid in dried blood spots and platelet poor and rich plasma samples

An isocratic high-performance liquid chromatographic method has been developed for the measurement of serotonin, 5-hydroxyindolacetic and homovanillic acids in dried blood spots and in platelet poor and rich plasma samples. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 13% methanol and 87% aqueous citrate buffer, containing octanesulfonic and ethylendiaminotetracetic acids. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at −0.200 V and the second analytical cell at +0.400 V. For the pre-treatment of biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange Oasis cartridges, was implemented. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0%. The calibration curve was linear over the on-column concentration range of 0.1–22.5 ng mL⁻¹ for serotonin and 5-hydroxyindolacetic acid and of 0.25–22.5 ng mL⁻¹ for homovanillic acid. The sensitivity was good with a limit of detection of 0.05 ng mL⁻¹ for serotonin and 5-hydroxyindolacetic acid and 0.12 ng mL⁻¹ for homovanillic acid. Results were also satisfactory in terms of precision, selectivity and accuracy. The analytical method was successfully applied to human platelet poor and rich plasma samples and to dried blood spots from volunteers and psychiatric patients.     

Determination of trace element in Italian virgin olive oils and their characterization according to geographical origin by statistical analysis

Multi-element analysis of organic virgin olive oils from different Italian regions was carried out by inductively coupled plasma mass spectrometry (ICP-MS) aiming at developing a reliable method in the traceability of the origin of oils. The data were processed by means of the chemiometric approach of linear discriminant analysis (LDA) that allows classifying unknown samples after checking possible differentiation of samples of known origin.An external calibration curve was build for the quantitative analysis. The calibration curves for each element were linear in the range between 0.01 and 100 ng mL⁻¹ and 0.2 to 2000 ng mL⁻¹, the correlation coefficients were ranging between 0.996 and 0.999. Results from spike and recovery experiments at levels of 30 and 65 ng mL⁻¹ were in the range of 91-119%, whereas the quantitation limits, based on 10 times standard deviation of the blank, were also in the range of 0.009-10.2 ng g⁻¹, for almost all the elements.     

Immunochemical method for sulfasalazine determination in human plasma

Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized.The immunoassay showed a high sensitivity (IC₅₀ = 0.51 ng mL⁻¹) and specificity, a detection limit of 0.02 ng mL⁻¹ and a dynamic range of 0.06-3.75 ng mL⁻¹ (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL⁻¹) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV = 13%).Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses.     

Electro membrane extraction of sodium diclofenac as an acidic compound from wastewater,urine, bovine milk,and plasma samples and quantification by high-performance liquid chromatography

Electro membrane extraction (EME) as a new microextraction method was applied for extraction of sodium diclofenac (SDF) as an acidic compound from wastewater, urine, bovine milk and plasma samples. Under applied potential of 20 V during the extraction, SDF migrated from a 2.1 mL of sample solution (1 mM NaOH), through a supported liquid membrane (SLM), into a 30 μL acceptor solution (10 mM NaOH), exist inside the lumen of the hollow fiber. The negative electrode was placed in the donor solution, and the positive electrode was placed in the acceptor solution. 1-octanol was immobilized in the pores of a porous hollow fiber of polypropylene as SLM. Then the extract was analyzed by means of high-performance liquid chromatography (HPLC) with UV-detection for quantification of SDF. Best results were obtained using a phosphate running electrolyte (10 mM, pH 2.5). The ranges of quantitation for different samples were 8–500 ng mL⁻¹. Intra- and inter-day RSDs were less than 14.5%. Under the optimized conditions, the preconcentration factors were between 31 and 66 and also the limit of detections (LODs) ranged from 2.7 ng mL⁻¹ to 5 ng mL⁻¹ in different samples. This procedure was applied to determine SDF in wastewater, bovine milk, urine and plasma samples (spiked and real samples). Extraction recoveries for different samples were between 44–95% after 5 min of extraction.     

A solid-phase extraction and size-exclusion liquid chromatographic method for polyethylene glycol 25 p-aminobenzoic acid determination in urine: validation for urinary excretion studies of users of sunscreens

No previous publications about percutaneous absorption of polyethylene glycol 25 p-aminobenzoic acid (PEG-25 PABA) have been found in the literature and the expected levels to be found in human urine after sunscreens use are unknown. The method proposed here is suitable to determine PEG-25 PABA in the urine of sunscreens users in order to carry out studies on body accumulation/excretion. It is based on solid-phase extraction (SPE) with size-exclusion liquid chromatography determination. Solid-phase extraction allows the analyte to be retained and subsequently eluted for a clean-up, using a silica-based cartridge. The size-exclusion liquid chromatography of the eluted allows the rest of matrix interferences to be avoided. Fluorescence intensity was measured at λ_em = 350 nm (λ_exc = 300 nm). The sensitivity of the proposed method is in the order of 450 ± 5 mL ng⁻¹ and the detection limit (3 S_y/x/b) in the measured solutions is in the order of 13 ng mL⁻¹, that is 2.6 ng mL⁻¹ in urine samples. The method enables PEG-25 PABA to be determined in both, spiked and unspiked human urine samples. Results obtained for spiked human urine samples (11-100 ng mL⁻¹) demonstrated the accuracy of the method. The mean relative standard deviation of the results was in the order of 3-10%. Three volunteers applied a sunscreen lotion containing a 8% PEG-25 PABA sunscreen cream and their urinary excretion was controlled from the moment of application until the excreted amounts were no longer detectable.     

High throughput screening various abused drugs and metabolites in urine by liquid chromatography-heated electrospray ionization/tandem mass spectrometry

An integrated method of liquid chromatography-heated electrospray ionization/tandem mass spectrometry was evaluated for high throughput screening of various abused drugs in urine. Chromatographic analysis was performed on a C18 reverse phase column using a linear gradient of 10 mM ammonium acetate containing 0.1% formic acid-methanol as mobile phase and the total separation time was 7 min. A simple and rapid sample preparation method used was by passing urine samples through a 0.22 μm PVDF syringe filter. The detection limits of the studied abused drugs in urine were from 0.6 ng mL⁻¹ (ketamine) to 9.0 ng mL⁻¹ (norcodeine). According to the results, the linear range was from 1 to 1200 ng mL⁻¹ with relative standard deviation (R.S.D.s) value below 14.8% (intra-day) and 24.6% (inter-day). The feasibility of applying the proposed method to determine various abused drugs in real samples was examined by analyzing urine samples from drug-abused suspects. The abused drugs including ketamines and amphetamines were detected in suspected urine samples. The results demonstrate the suitability of LC-HESI-MS/MS for high throughput screening of the various abused drugs in urine.     

Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones

Different second-order multivariate calibration algorithms, namely parallel factor analysis (PARAFAC), N-dimensional partial least-squares (N-PLS) and multivariate curve resolution-alternating least-squares (MCR-ALS) have been compared for the analysis of four fluoroquinolones in aqueous solutions, including some human urine samples (additional four fluoroquinolones were simultaneously determined by univariate calibration). Data were measured in a short time with a chromatographic system operating in the isocratic mode. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. The developed approach enabled us to determine eight analytes, some of them with overlapped profiles, without the necessity of applying an elution gradient, and thus significantly reducing both the experimental time and complexity. The study was employed for the discussion of the scopes of the applied second-order chemometric tools. The quality of the proposed technique coupled to each of the evaluated algorithms was assessed on the basis of the figures of merit for the determination of fluoroquinolones in the analyzed water and urine samples. Univariate calibration of four analytes led to limits of detection in the range 20–40 ng mL⁻¹ and root mean square errors for the validation samples in the range 30–60 ng mL⁻¹ (corresponding to relative prediction errors of 3–8%). The ranges for second-order multivariate calibration (using PARAFAC and N-PLS) of the remaining four analytes were: limit of detection, 2–8 ng mL⁻¹, root mean square errors, 3–50 ng mL⁻¹ and relative prediction errors, 1–5%.     

Determination of ketamine and metabolites in urine by liquid chromatography-mass spectrometry

We have developed an analytical method by using liquid chromatography-mass spectrometry (LC-MS) to determine ketamine and its metabolites in urine. The ionization efficiency between two ionization modes (ESI and APCI) of LC-MS was compared to each other. An easy and simple sample preparation of urine samples was made by passing samples through a 0.22 μm PVDF syringe filter. The results indicated that the ionization efficiency of positive APCI mode is better than positive ESI mode for determination of trace ketamines. A wide linearity range of the research is from 5 to 250 ng mL⁻¹ and the detection limits for ketamine, norketamine and dehydronorketamine were 0.95, 0.48 and 0.33 ng mL⁻¹, respectively. The proposed method was tested by analyzing ketamine and metabolites in the urines of volunteers. The concentrations of ketamine, norketamine and dehydronorketamine are ranged of 5.4-131.0, 12.5-74.1 and 22.8-278.9 ng mL⁻¹, respectively and the ketamines concentration profiles in human urine were also determined. The results demonstrate the suitability of liquid chromatography-mass spectrometry approach to analyze trace amount of ketamine and its metabolites in urine.     

A novel stacking method of repetitive large volume sample injection and sweeping MEKC for determination of androgenic steroids in urine

In this research, a novel stacking capillary electrophoresis method, repetitive large volume sample injection and sweeping MEKC (rLVSI-sweeping MEKC) were developed to analyze the presence of three androgenic steroids considered as sport doping drugs, testosterone (T), epitestosterone (E) and epitestosterone glucuronide (EG) in urine. This method provides better sensitivity enhancement than the traditional large volume sample stacking-sweeping strategies due to sensitivity enhancement by repetitive injections. This multiple sampling method enhances sensitivity of monitoring of urine samples by UV detection (254 nm). Firstly, the phosphate buffer was filled into an uncoated fused silica capillary and the samples were injected into the capillary at 10 psi for 20 s, and then stacked at −10 kV for 1 min using phosphate buffer containing SDS. The above injecting and stacking steps were repeated five times. Finally, separation was performed at −20 kV, using phosphate buffer containing methanol, SDS and (2-hydroxypropyl)-β-cyclodextrin. Method validation showed that calibration plots were linear (r ≧ 0.997) over a range of 5-200 ng mL⁻¹ for T, 20-200 ng mL⁻¹ for E and 0.5-500 ng mL⁻¹ for EG. The limits of detection were 1.0 ng mL⁻¹ for T, 5.0 ng mL⁻¹ for E and 200.0 pg mL⁻¹ for EG. When evaluating precision and accuracy, values of RSD and RE in intra-day (n = 3) and inter-day (n = 5) analysis were found to be less than 10.0%. Compared with the simple LVSS-sweeping, which is also a stacking strategy, this method further improves sensitivity up to 25 folds (∼2500 folds with MEKC without preconcentration). This method was applied to monitor 10 athletes’ urine, and did not detect any analyte. The novel stacking method was feasible for monitoring of doping by sportsmen.     

Monolithic silica spin column extraction and simultaneous derivatization of amphetamines and 3,4-methylenedioxyamphetamines in human urine for gas chromatographic-mass spectrometric detection

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d₅ was used as an internal standard. The linear ranges were 0.01-5.0 μg mL⁻¹ for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 μg mL⁻¹ for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation ≧0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 μg mL⁻¹ of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio ≧ 3) in urine was 5 ng mL⁻¹ for MA and MDMA and 10 ng mL⁻¹ for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.     

Determination of cadmium, lead and thallium in highly saline hemodialysis solutions by potentiometric stripping analysis (PSA)

Potentiometric stripping analysis (PSA) was investigated to assay simultaneously cadmium, lead and thallium present as contaminants in highly saline solutions used in hemodialysis. The saline matrices were sodium, potassium, magnesium and calcium chlorides, sodium acetate, sodium bicarbonate and glucose, which constitute concentrates for hemodialysis. A 1000 μg mL⁻¹ Hg(II) solution was used to prepare the mercury film electrode (MFE) and to carry out the stripping step. After a 30 s accumulation interval the analytes were simultaneously detected in the saline matrices without using masking agents. Determination limits of 80 ng L⁻¹ for cadmium and thallium, and 50 ng L⁻¹ for lead were calculated and a R.S.D. ranging from 0.5 to 2.2% (n = 3) was obtained measuring the analytes directly in commercial hemodialysis saline solutions. Recoveries from spiked samples ranging from 94.6 to 102.0% were obtained. The investigated metals were found in concentrations ranging from 2.7 to 5.7 μg L⁻¹ for cadmium, 27.7 to 75.8 μ L⁻¹ for lead and 9.6 to 18.7 μg L⁻¹ for thallium in commercial hemodialysis solutions. The PSA method showed to be adequate to the quality control of saline concentrates for hemodialysis.     

Rapid analysis of captopril in human plasma and pharmaceutical preparations by headspace solid phase microextraction based on polypyrrole film coupled to ion mobility spectrometry

A rapid, simple, and sensitive headspace solid phase microextraction coupled to ion mobility spectrometry (HS-SPME-IMS) method is presented for analysis of the highly specific angiotensin-converting enzyme (ACE) inhibitor, captopril (CAP). Positive ion mobility spectra of CAP were acquired with an ion mobility spectrometer equipped with a corona discharge ionization source. Mass-to-mobility correlation equation was used to identify product ions. A dodecylsulfate-doped polypyrrole (PPy-DS) coating was used as a fiber for SPME. The results showed that PPy-DS based SPME fiber was suitable for successfully extracting CAP from human blood plasma and pharmaceutical samples. The HS-SPME-IMS method provided good repeatability (R.S.D.s < 4%) for aqueous and spiked plasma samples. The calibration graphs were linear in the range of 10-300 ng mL⁻¹ (R² > 0.99) and detection limits were 7.5 ng mL⁻¹ for aqueous and 6.3 ng mL⁻¹ for plasma blank samples. Finally, a standard addition calibration method was applied to HS-SPME-IMS technique for the analysis of blood plasma samples and tablets. Purpose method seemed to be suitable for the analysis of CAP in plasma samples as it is not time consuming (state total time from sample preparation to analysis), it required only small quantities of the sample, and no derivatization was required.     

Determination of the antidepressant mirtazapine and its two main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method for the determination in human plasma of the recent noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine and its two main metabolites, N-desmethylmirtazapine and 8-hydroxymirtazapine, has been developed. Fluorescence detection was used, exciting at λ = 290 nm and monitoring emission at λ = 370 nm. Separation was obtained by using a reversed-phase column (C8, 250 mm × 4.6 mm I.D., 5 μm) and a mobile phase composed of 75% aqueous phosphate buffer containing triethylamine at pH 3.0 and 25% acetonitrile. Melatonin was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with phenyl cartridges (100 mg, 1 mL). The calibration curves were linear over a working range of 5-150 ng mL⁻¹ for mirtazapine and of 2.5-75.0 ng mL⁻¹ for N-desmethylmirtazapine and 8-hydroxymirtazapine. The limit of quantitation (LOQ) was 2.5 ng mL⁻¹ and the limit of detection (LOD) was 1.25 ng mL⁻¹ for all analytes. The method was applied with success to plasma samples from depressed patients undergoing treatment with mirtazapine. Precision data, as well as accuracy results, were satisfactory and no interference from other drugs was found. Hence the method is suitable for therapeutic drug monitoring of mirtazapine and its metabolites in depressed patients’ plasma.     

Development of an immunosensor for the detection of testosterone in bovine urine

In the presented work, a disposable immunosensor for the detection of testosterone, an endogenous steroid hormone, in bovine urine has been developed using screen-printed electrodes (SPEs). Due to concerns over the use of steroid hormones as growth promoters, the EU prohibits their use in food producing animals. Consequently, rigorous screening procedures have been implemented in all member states to detect the illegal administration of such compounds. Competitive immunoassays were developed, initially by enzyme linked immunosorbent assay (ELISA), and subsequently transferred to an electrochemical immunosensor format using disposable screen-printed carbon electrodes. Horseradish peroxidase (HRP) was the enzyme label of choice and chronoamperometric detection was carried out using a tetramethylbenzidine/hydrogen peroxide (TMB/H₂O₂) substrate system, at +100 mV. The EC₅₀ values obtained for the assay in buffer and urine gave relatively comparable results, 710 pg mL⁻¹ and 960 pg mL⁻¹, respectively. The linear range obtained for the assay in buffer extended from 0.03 ng mL⁻¹ to 40 ng mL⁻¹; while that in urine ranged from 0.03 ng mL⁻¹ to 1.6 ng mL⁻¹. The corresponding limits of detection (LOD) in buffer and urine were 26 pg mL⁻¹ and 1.8 pg mL⁻¹. Cross reactivity profiles of the antibody have been examined, with notable cross reactivities with 19-nortestosterone (11.6%) and boldenone (9.86%). Precision studies for the sensor demonstrated adequate reproducibility (CV < 13%, n = 3) and repeatability (CV < 9%, n = 3). Recovery data obtained showed good agreement between spiking studies and known concentrations of analyte. Sensors showed stability for 4 days at +4 °C. A sensitive, highly specific, inexpensive, disposable immunosensor, showing excellent overall performance for the detection of testosterone in bovine urine, has been developed.     

Development of an ICP-MS immunoassay for the detection of anti-erythropoietin antibodies

A sandwich-type immunoassay linked with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the detection of anti-erythropoietin antibodies (anti-EPO Abs). Recombinant human erythropoietin (rhEPO) was immobilized on the solid phase to capture anti-rhEPO Abs specifically. After the immunoreactions with Au-labeled goat-anti-rabbit IgG, a diluted HNO₃ (2%) was used to dissociate Au nanoparticles which was then introduced to the ICP-MS for measurements. Under the optimized conditions, the calibration graph for anti-EPO Abs was linear in the range of 35.6-500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹ (3σ, n = 9). The relative standard deviation (R.S.D.) for three replicate measurements of 30.9 ng mL⁻¹ of anti-EPO Abs was 8.43%. The recoveries of anti-EPO Abs in sera at the spiking level of 50, 100, 150, 200 and 400 ng mL⁻¹ were 99.2%, 101.5%, 95.0%, 94.0% and 102.9%, respectively. For the real sample analysis, 26 samples from healthy people and 53 samples from patients with rhEPO treatments were studied. One sample from patients showed significantly higher anti-EPO Abs from other samples, indicating a possibility of immune response of this patient.     

Development, validation and application of a method based on DI-SPME and GC-MS for determination of pesticides of different chemical groups in surface and groundwater samples

A simple and rapid method based on solid-phase micro extraction (SPME) technique followed by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS, SIM) was developed by the simultaneous determination of 16 pesticides of seven different chemical groups [Six organophosphorus (trichlorfon, diazinon, methyl parathion, malathion, fenthion and ethyon), three pyrethroids (bifenhin, permethrin, cypermethrin), two imidazoles (imazalil and prochloraz), two strobilurins (azoxystrobin and pyraclostrobin), one carbamate (carbofuran), one tetrazine (clofentezine), and one triazole (difenoconazole)] in water. The pesticides extraction was done with direct immersion mode (DI-SPME) of the polyacrilate fiber (PA 85 µm). The extraction temperature was adjusted to 50 °C during 30 min, while stirring at 250 rpm was applied. After extraction, the fiber was introduced in the GC injector for thermal desorption for 5 min. at 280 °C. The method was validated using ultra pure water samples fortified with pesticides at different concentration levels and shows good linearity in the concentrations between 0.05 and 250.00 ng mL^− 1. The LOD and LOQ ranged, from 0.02 to 0.30 ng mL^− 1 and 0.05 to 1.00 ng mL^− 1, respectively. Intra-day and inter-day precisions were determined in two concentration levels (5.00 and 50.00 ng mL^− 1). Intra-day relative standard deviation (%R.S.D.) ranged between 3.6 and 13.6%, and inter-day (%R.S.D.) ranged between 6.3 and 18.5%. Relative recovery tests were carried out spiking the ultra pure sample with standards in three different concentration levels 0.20, 5.00 and 50.00 ng mL^− 1. The recovery at 0.20 ng mL^− 1 level varied from 86.4 ± 9.4% to 108.5 ± 10.5%, at 5.00 ng mL^− 1 level varied from 77.5 ± 10.8% to 104.6 ± 9.6% and at 50.00 ng mL^− 1 level varied from 70.2 ± 4.6% to 98.4 ± 8.5%. The proposed SPME method was applied in twenty-six water samples collected in the “Platô de Neópolis”, State of Sergipe, Brazil. Methyl parathion was detected in five samples with an average concentration of 0.17 ng mL^− 1 and bifenthrin, pyraclostrobin and azoxystrobin residues were found in three samples with average concentrations of 2.28, 3.12 and 0.15 ng mL^− 1, respectively.     

A new high-performance liquid chromatography assay for the determination of sesquiterpene lactone 15-deoxygoyazensolide in rat plasma

A simple, rapid and sensitive high-performance liquid chromatography method was developed for the analysis of the sesquiterpene lactone 15-deoxygoyazensolide (LAC15-D) in rat plasma samples. The chromatographic separation was achieved on a LiChrospher^® RP18 column using methanol:water (50:50, v/v) containing 0.6% acetic acid as mobile phase, at a flow rate of 0.7 mL min⁻¹. UV detection was carried out at 270 nm. Phenytoin was used as internal standard. Prior to the analysis, the rat plasma samples were submitted to liquid-liquid extraction with dichloromethane. The mean absolute recoveries were 73% with R.S.D. values lower than 3.5. The method was linear over the 6.0-2000 ng mL⁻¹ concentration range and the quantification limit was 6.0 ng mL⁻¹. Within-day and between-day assay precision and accuracy were studied at three concentration levels (15, 300 and 480 ng mL⁻¹) and were lower than 15%. The validated method was used to measure the plasmatic concentration of LAC15-D in rats that received a single intraperitoneal dose of 30 mg kg⁻¹.     

Detection of boldenone,its conjugates and androstadienedione,as well as five corticosteroids in bovine bile through a unique immunoaffinity column clean-up and two validated liquid chromatography–tandem mass spectrometry analyses

The presence of β-boldenone II phase metabolites and prednisolone in urine samples, owing to endogenous or natural origin or illicit treatment, is under debate within the European Union. The detection of β-boldenone conjugates, α-boldenone conjugates at concentrations higher than 2 ng mL⁻¹ and prednisolone above the cut-off level of 5 ng mL⁻¹ in urine have been, until now, critical in deciding if illegal drug use has occurred. The use of urine sometimes is not entirely satisfactory, especially when the drug is administrated at low doses or when its metabolic conversion is very fast. This subsequently would hamper its detection in urine. The introduction of a new, advantageous matrix where the illicit treatment can be investigated would be highly appreciated. In this study, we have developed and validated a simple and unique immunoaffinity clean-up procedure, which was applied to bovine bile samples, followed by two different analytical liquid chromatography-electrospray-tandem mass spectrometry methods. The first method tests androstadienedione, α- and β-boldenone sulphate, glucuronate and related free forms, while the other method assays prednisolone, prednisone, dexamethasone, cortisone, and cortisol. The methods were validated according to European Commission Decision 2002/657/EC. The evaluated parameters were linearity, specificity, precision (repeatability and intra-laboratory reproducibility), recovery, decision limit and detection capability. The decision limits (CCα) were between 0.38 and 0.45 ng mL⁻¹ for anabolic steroids, and 0.13 and 0.15 ng mL⁻¹ as far as corticosteroids were concerned. Intra- and inter-day repeatability was below 15.8 and 19.9% for all analytes, respectively. The methods were applied to the analysis of some bile samples collected from untreated young bulls in order to investigate the presence of the studied steroids in this matrix.     

Development and validation of a sensitive enzyme immunoassay for surveillance of Cry1Ab toxin in bovine blood plasma of cows fed Bt-maize (MON810)

The increasing global adoption of genetically modified (GM) plant derivatives in animal feed has provoked a strong demand for an appropriate detection method to evaluate the existence of transgenic protein in animal tissues and animal by-products derived from GM plant fed animals. A highly specific and sensitive sandwich enzyme immunoassay for the surveillance of transgenic Cry1Ab protein from Bt-maize in the blood plasma of cows fed on Bt-maize was developed and validated according to the criteria of EU-Decision 2002/657/EC. The sandwich assay is based on immuno-affinity purified polyclonal antibody raised against Cry1Ab protein in rabbits. Native and biotinylated forms of this antibody served as capture antibody and detection antibody for the ELISA, respectively. Streptavidin-horseradish peroxidase conjugate and TMB substrate provided the means for enzymatic colour development.The immunoassay allowed Cry1Ab protein determination in bovine blood plasma in an analytical range of 0.4-100 ng mL⁻¹ with a decision limit (CCα) of 1.5 ng mL⁻¹ and detection capability (CCβ) of 2.3 ng mL⁻¹. Recoveries ranged from 89 to 106% (mean value of 98%) in spiked plasma.In total, 20 plasma samples from cows (n = 7) fed non-transgenic maize and 24 samples from cows (n = 8) fed transgenic maize (collected before and, after 1 and 2 months of feeding) were investigated for the presence of the Cry1Ab protein. There was no difference amongst both groups (all the samples were below 1.5 ng mL⁻¹; CCα). No plasma sample was positive for the presence of the Cry1Ab protein at CCα and CCβ of the assay.