Optimization and validation of organochlorine compounds in adipose tissue by SPE-gas chromatography

Scientific evidence has shown an association between organochlorine compounds (OCC) exposure and human health hazards. Concerning this, OCC detection in human adipose samples has to be considered a public health priority. This study evaluated the efficacy of various solid-phase extraction (SPE) and cleanup methods for OCC determination in human adipose tissue. Octadecylsilyl endcapped (C18-E), benzenesulfonic acid modified silica cation exchanger (SA), poly (styrene-divinylbenzene (EN) and EN/RP18 SPE sorbents were evaluated. The relative sample cleanup provided by these SPE columns was evaluated using gas chromatography with electron capture detection (GC–ECD). The C18-E columns with strong homogenization were found to provide the most effective cleanup, removing the greatest amount of interfering substance, and simultaneously ensuring good analyte recoveries higher than 70%. Recoveries>70% with standard deviations (SD)<15% were obtained for all compounds under the selected conditions. Method detection limits were in the 0.003–0.009 mg/kg range. The positive samples were confirmed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The highest percentage found of the OCC in real samples corresponded to HCB, o,p′-DDT and methoxychlor, which were detected in 80 and 95% of samples analyzed respectively. Copyright © 2012 John Wiley & Sons, Ltd.

Organochlorine pesticide residues in strawberries from integrated pest management and organic farming

A rapid, specific, and sensitive method based on theQuick Easy Cheap Effective Rugged and Safe (QuEChERS) method and a cleanup using dispersive solid-phase extraction with MgSO4, PSA, and C18 sorbents has been developed for the routine analysis of 14 pesticides in strawberries. The analyses were performed by three different analytical methodologies: gas chromatography (GC) with electron capture detection (ECD), mass spectrometry (MS), and tandem mass spectrometry (MS/MS). The recoveries for all the pesticides studied were from 46 to 128%, with relative standard deviation of <15% in the concentration range of 0.005-0.250 mg/kg. The limit of detection (LOD) for all compoundsmetmaximumresidue limits (MRL) accepted in Portugal for organochlorine pesticides (OCP). A survey study of strawberries produced in Portugal in the years 2009-2010 obtained from organic farming (OF) and integrated pest management (IPM) was developed. Lindane and β-endosulfan were detected above the MRL in OF and IPM. Other OCP (aldrin, o,p0-DDT and their metabolites, and methoxychlor) were found below the MRL. The OCP residues detected decreased from 2009 to 2010. The QuEChERS method was successfully applied to the analysis of strawberry samples.

Multiresidue pesticides analysis in soils using modified QuEChERS with disposable pipette extraction and dispersive solid-phase extraction

QuEChERS original method was modified into a new version for pesticides determination in soils. The QuEChERS method is based on liquid–liquid portioning with ACN and was followed by cleanup step using dispersive SPE and disposable pipette tips. Gas chromatographic separation with MS detection was carried out for pesticides quantification. The method was validated using recovery experiments for 36 multiclass pesticides. Mean recoveries of pesticides at each of the four spiking levels between 10–300 µg/kg of soil ranged from 70–120% for 26 pesticides with RSD values less than 15%. The method achieved low limit of detection less than 7.6 µ g/kg. Matrix effects were observed for 13 pesticides. Matrix effects were compensated by using matrix-matched calibration. The method was applied successfully using d-SPE or DPX in the analysis of the pesticides in soils from organic farming and integrated pest management.

Optimization of QuEChERS procedure coupled to GC-ECD for organochlorine pesticide determination in carrot samples

An optimised version of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for simultaneous determination of 14 organochlorine pesticides in carrots was developed using gas chromatography coupled with electron-capture detector (GC-ECD) and confirmation by gas chromatography tandem mass spectrometry (GC-MS/MS). A citrate-buffered version of QuEChERS was applied for the extraction of the organochlorine pesticides, and for the extract clean-up, primary secondary amine, octadecyl-bonded silica (C18), magnesium sulphate (MgSO4) and graphitized carbon black were used as sorbents. The GC-ECD determination of the target compounds was achieved in less than 20 min. The limits of detection were below the EUmaximum residue limits (MRLs) for carrots, 10–50 μg kg−1, while the limit of quantification did exceed 10 μg kg−1 for hexachlorobenzene (HCB). The introduction of a sonication step was shown to improve the recoveries. The overall average recoveries in carrots, at the four tested levels (60, 80, 100 and 140 μg kg−1), ranged from 66 to 111% with relative standard deviations in the range of 2– 15 % (n03) for all analytes, with the exception of HCB. The method has been applied to the analysis of 21 carrot samples from different Portuguese regions, and β-HCH was the pesticide most frequently found, with concentrations oscillating between less than the limit of quantification to 14.6 μg kg−1. Only one sample had a pesticide residue (β-HCH) above the MRL, 14.6 μg kg−1. This methodology combines the advantages of both QuEChERS and GC-ECD, producing a very rapid, sensitive and reliable procedure which can be applied in routine analytical laboratories.

Analysis of pesticide residues in strawberries and soils by GC-MS/MS, LC-MS/MS and two-dimensional GC-timeof- flight MS comparing organic and integrated pest management farming

This study analysed 22 strawberry and soil samples after their collection over the course of 2 years to compare the residue profiles from organic farming with integrated pest management practices in Portugal. For sample preparation, we used the citrate-buffered version of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. We applied three different methods for analysis: (1) 27 pesticides were targeted using LC-MS/MS; (2) 143 were targeted using low pressure GC-tandem mass spectrometry (LP-GC-MS/MS); and (3) more than 600 pesticides were screened in a targeted and untargeted approach using comprehensive, two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF-MS). Comparison was made of the analyses using the different methods for the shared samples. The results were similar, thereby providing satisfactory confirmation of both similarly positive and negative findings. No pesticides were found in the organic-farmed samples. In samples from integrated pest management practices, nine pesticides were determined and confirmed to be present, ranging from 2 μg kg−1 for fluazifop-pbutyl to 50 μg kg−1 for fenpropathrin. Concentrations of residues in strawberries were less than European maximum residue limits.

Effect of active components from Garlic on AGEs formation

Advanced glycation end-products are Maillard reaction products that are found in thermal processed food. This compounds are often referred as unhealthy for human diet, namely because of their capacity to form amino-acid dimers. There is a broad range of answers to get about how these products are formed, how they interact with the organism and how these reactions can be inhibited to prevent the referred effects. Some compounds from garlic are thought to be able to inhibit these reactions. This study using spectrophotometric, High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) and Fourier transformed infrared spectroscopy (FTIR) analysis, helps to understand better not only not only the effect of some compounds obtained from garlic, diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS), on these AGEs production reaction, but also helped to understand better the reaction itself.

Development of a SPE–UHPLC–MS/MS methodology for thedetermination of non-steroidal anti-inflammatory and analgesicpharmaceuticals in seawaterPaula

An analytical methodology for the simultaneous determination of seven pharmaceuticals and two metabolites belonging to the non-steroidal anti-inflammatory drugs (NSAIDs) and analgesics therapeutic groups was developed based on off-line solid-phase extraction and ultra-high performance liquid chromatography coupled to tandem mass spectrometry (SPE–UHPLC–MS/MS). Extraction conditions were optimized taking into account parameters like sorbent material, sample volume and sample pH. Method detection limits (MDLs) ranging from 0.02 to 8.18 ng/L were obtained. This methodology was successfully applied to the determination of the selected pharmaceuticals in seawater samples of Atlantic Ocean in the Northern Portuguese coast. All the pharmaceuticals have been detected in the seawater samples, with pharmaceuticals like ibuprofen, acetaminophen, ketoprofen and the metabolite hydroxyibuprofen being the most frequently detected at concentrations that can reach some hundreds of ng/L.

Validation of QuEChERS method for organochlorine pesticides analysis in tamarind (Tamarindus indica) products: Peel, fruit and commercial pulp

In this study a citrate-buffered version of QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for determination of 14 organochlorine pesticides (OCPs) residues in tamarind peel, fruit and commercial pulp was optimized using gas chromatography (GC) coupled with electron-capture detector (ECD) and confirmation by GC tandem mass spectrometry (GC–MS/MS). Five procedures were tested based on the original QuEChERS method. The best one was achieved with increased time in ultrasonic bath. For the extract clean-up, primary secondary amine (PSA), octadecyl-bonded silica (C18) and magnesium sulphate (MgSO4) were used as sorbents for tamarind fruit and commercial pulp and for peel was also added graphitized carbon black (GCB). The samples mass was optimized according to the best recoveries (1.0 g for peel and fruit; 0.5 g for pulp). The method results showed the matrix-matched calibration curve linearity was r2 > 0.99 for all target analytes in all samples. The overall average recoveries (spiked at 20, 40 and 60 μg kg−1) have been considered satisfactory presenting values between 70 and 115% with RSD of 2–15 % (n = 3) for all analytes, with the exception of HCB (in peel sample). The ranges of limits of detection (LOD) and quantification (LOQ) for OCPs were for peel (LOD: 8.0–21 μg kg−1; LOQ: 27–98 μg kg−1); for fruit (LOD: 4–10 μg kg−1; LOQ: 15–49 μg kg−1) and for commercial pulp (LOD: 2–5 μg kg−1; LOQ: 7–27 μg kg−1). The method was successfully applied in tamarind samples being considered a rapid, sensitive and reliable procedure.

Eruca sativa: Benefits as antioxidants source versus risks of already banned pesticides

Eruca sativa (rocket salad) has been intensely consumed all over the world, insomuch as, this work was undertaken to evaluate the antioxidant status and the environmental contamination (positive and negative nutritional contribution) of leaves and stems from this vegetable. Antioxidant capacity of rocket salad was assessed by mean of optical methods, such as the total phenolic content (TPC), reducing power assay and DPPH radical scavenging activity. The extent of the environmental contamination was reached through the quantification of thirteen organochlorine pesticides (OCP) by using gas chromatography coupled with electron-capture detector (GC-ECD) and compound confirmations employing gas chromatography tandem mass-spectrometry (GC-MS/MS). The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology.The extent of the environmental contamination was reached through the quantification of thirteen OCP by using gas chromatography coupled with electron-capture detector (GC-ECD) and compound confirmations employing GC-MS/MS. The OCP residues were extracted by using Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) methodology. This demonstrated that leaves presented more antioxidant activity than stems, emphasizing that leaves contained six times more polyphenolic compounds than stems. In what concerns the OCP occurrence, the average recoveries obtained at the three levels tested (40, 60 and 80 µg kg−1) ranged from 55% to 149% with a relative standard deviation of 11%, (except hexachrorobenzene). Three vegetables samples were collected from supermarkets and analysed following this study. According to data, only one sample achieved 16.21 of β-hexachlorocyclohexane, confirmed by GC-MS/MS. About OCP quantification, the data indicated that only one sample achieved 16.21 µg kg−1 of β-hexachlorocyclohexane, confirmed by GC-MS/MS, being the QuEChERS a good choice for the of OCPs extraction. Furthermore, the leaves consumption guaranty higher levels of antioxidants than stems.

Protein adducts from the anti-HIV drug abacavir – possible biomarkers of drug toxicity

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Evidence for Nevirapine Bioactivation in Man: Searching for the First Step in the Mechanism of Nevirapine Toxicity

Despite its efficacy, including in the prevention of vertical transmission, the antiretroviral nevirapine is associated with severe idiosyncratic hepatotoxicity and skin rash. The mechanisms underlying nevirapine toxicity are not fully understood, but drug bioactivation to reactive metabolites capable of forming stable protein adducts is thought to be involved. This hypothesis is based on the paradigm that drug reactive metabolites have the potential to bind to self-proteins, which results in drug-modified proteins being perceived as foreign by the immune system. The aim of the present work was to identify hemoglobin adducts in HIV patients as biomarkers of nevirapine haptenation upon bioactivation. The ultimate goal is to develop diagnostic methods for predicting the onset of nevirapine-induced toxic reactions. All included subjects were adults on nevirapine-containing antiretroviral therapy for at least 1month. The protocol received prior approval from the Hospital Ethics Committees and patients gave their written informed consent. Nevirapine-derived adducts with the N-terminal valine of hemoglobin were analyzed by an established liquid chromatography-electrospray ionization-tandem mass spectrometry method and characterized on the basis of retention time and mass spectrometric fragmentation pattern by comparison with adduct standards prepared synthetically. The nevirapine adducts were detected in 12/13 patient samples, and quantified in 11/12 samples (2.58±0.8 fmol/g of hemoglobin). This work represents the first evidence of nevirapine-protein adduct formation in man and confirms the ability of nevirapine to modify self-proteins, thus providing clues to the molecular mechanisms underlying nevirapine toxicity. Moreover, the possibility of assessing nevirapine-protein adduct levels has the potential to become useful for predicting the onset of nevirapine-induced adverse reactions.

Monitoring Abacavir Bioactivation in Humans: Screening for an Aldehyde Metabolite

The anti-HIV drug abacavir is associated with idiosyncratic hypersensitivity reactions and cardiotoxicity. Although the mechanism underlying abacavir-toxicity is not fully understood, drug bioactivation to reactive metabolites may be involved. This work was aimed at identifying abacavir-protein adducts in the hemoglobin of HIV patients as biomarkers of abacavir bioactivation and protein modification. The protocol received prior approval from the Hospital Ethics Committee, patients gave their written informed consent and adherence was controlled through a questionnaire. Abacavir-derived Edman adducts with the N-terminal valine of hemoglobin were analyzed by an established liquid chromatography-electrospray ionization-tandem mass spectrometry method. Abacavir-valine adducts were detected in three out of ten patients. This work represents the first evidence of abacavir-protein adduct formation in humans. The data confirm the ability of abacavir to modify self-proteins and suggest that the molecular mechanism(s) of some abacavir-induced adverse reactions may require bioactivation.

Determinação de psicofármacos em cabelo humano por técnicas cromatográficas hifenadas: um método alternativo não-invasivo

Dissertação de mestrado em Técnicas de Caracterização e Análise Química

Pharmacokinetics and pharmacogenomics of once-daily raltegravir and atazanavir in healthy volunteers.

Atazanavir inhibits UDP-glucuronyl-transferase-1A1 (UGT1A1), which metabolizes raltegravir, but the magnitude of steady-state inhibition and role of the UGT1A1 genotype are unknown. Sufficient inhibition could lead to reduced-dose and -cost raltegravir regimens. Nineteen healthy volunteers, age 24 to 51 years, took raltegravir 400 mg twice daily (arm A) and 400 mg plus atazanavir 400 mg once daily (arm B), separated by ?3 days, in a crossover design. After 1 week on each regimen, raltegravir and raltegravir-glucuronide plasma and urine concentrations were measured by liquid chromatography-tandem mass spectrometry in multiple samples obtained over 12 h (arm A) or 24 h (arm B) and analyzed by noncompartmental methods. UGT1A1 promoter variants were detected with a commercially available kit and published primers. The primary outcome was the ratio of plasma raltegravir C(tau), or concentration at the end of the dosing interval, for arm B (24 h) versus arm A (12 h). The arm B-to-arm A geometric mean ratios (95% confidence interval, P value) for plasma raltegravir C(tau), area under the concentration-time curve from 0 to 12 h (AUC(0-12)), and raltegravir-glucuronide/raltegravir AUC(0-12) were 0.38 (0.22 to 0.65, 0.001), 1.32 (0.62 to 2.81, 0.45), and 0.47 (0.38 to 0.59, <0.001), respectively. Nine volunteers were heterozygous and one was homozygous for a UGT1A1 reduction-of-function allele, but these were not associated with metabolite formation. Although atazanavir significantly reduced the formation of the glucuronide metabolite, its steady-state boosting of plasma raltegravir did not render the C(tau) with a once-daily raltegravir dose of 400 mg similar to the C(tau) with the standard twice-daily dose. UGT1A1 promoter variants did not significantly influence this interaction.

Proteomic and Metabolomic Analyses of Mitochondrial Complex I-deficient Mouse Model Generated by Spontaneous B2 Short Interspersed Nuclear Element (SINE) Insertion into NADH Dehydrogenase (Ubiquinone) Fe-S Protein 4 (Ndufs4) Gene.

Eukaryotic cells generate energy in the form of ATP, through a network of mitochondrial complexes and electron carriers known as the oxidative phosphorylation system. In mammals, mitochondrial complex I (CI) is the largest component of this system, comprising 45 different subunits encoded by mitochondrial and nuclear DNA. Humans diagnosed with mutations in the gene NDUFS4, encoding a nuclear DNA-encoded subunit of CI (NADH dehydrogenase ubiquinone Fe-S protein 4), typically suffer from Leigh syndrome, a neurodegenerative disease with onset in infancy or early childhood. Mitochondria from NDUFS4 patients usually lack detectable NDUFS4 protein and show a CI stability/assembly defect. Here, we describe a recessive mouse phenotype caused by the insertion of a transposable element into Ndufs4, identified by a novel combined linkage and expression analysis. Designated Ndufs4(fky), the mutation leads to aberrant transcript splicing and absence of NDUFS4 protein in all tissues tested of homozygous mice. Physical and behavioral symptoms displayed by Ndufs4(fky/fky) mice include temporary fur loss, growth retardation, unsteady gait, and abnormal body posture when suspended by the tail. Analysis of CI in Ndufs4(fky/fky) mice using blue native PAGE revealed the presence of a faster migrating crippled complex. This crippled CI was shown to lack subunits of the "N assembly module", which contains the NADH binding site, but contained two assembly factors not present in intact CI. Metabolomic analysis of the blood by tandem mass spectrometry showed increased hydroxyacylcarnitine species, implying that the CI defect leads to an imbalanced NADH/NAD(+) ratio that inhibits mitochondrial fatty acid β-oxidation.