Caenorhabditis elegans as a suitable model to evaluate the toxicity of water from Rolante River, southern Brazil.

Urbanization and agricultural activities increased environmental contaminants. Integrated analysis of water parameters and bioassays represents an essential approach to evaluating aquatic resource quality. This study aimed to assess water quality by microbiological and physicochemical parameters as well as the toxicological effects of water samples on the Ames test and Caenorhabditis elegans model. Samples were collected during (collection 1) and after (collection 2) pesticide application in the upper (S1), middle (S2), and lower (S3) sections of the Rolante River, southern Brazil. Metals were determined by GFAAS and pesticides by UPLC-MS/MS. Bioassays using the Ames test and the nematode C. elegans were performed. Levels of microbiological parameters, as well as Mn and Cu were higher than the maximum allowed limits established by legislation in collection 2 compared to collection 1. The presence of pesticide was observed in both collections; higher levels were found in collection 1. No mutagenic effect was detected. Significant inhibition of body length of C. elegans was found in collection 1 at S2 (P < 0.001) and S3 (P < 0.001) and in collection 2 at S2 (P = 0.004). Comparing the same sampling site between collections, a significant difference was found between the site of collection (F(3,6)=8.75, P = 0.01) and the time of collection (F(1,2)=28.61, P = 0.03), for the S2 and S3 samples. C. elegans model was useful for assessing surface water quality/toxicity. Results suggest that an integrated analysis for the surface water status could be beneficial for future approaches.

Determination of amphetamines, opioids, cocaine metabolites and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in urine using biocompatible solid-phase microextraction and ultra-performance LC-MS/MS.

Background: Workplace drug testing primarily relies on urine analysis, targeting multiple compounds with varying physicochemical characteristics. Biocompatible solid-phase microextraction (BioSPME) is a miniaturized solid-phase extraction technique that enables the simultaneous extraction and preconcentration of analytes directly from the biological matrix. Methods: The BioSPME procedure consisted of the sequential extraction of 50-µl urine samples using LC Tips C18 in basic and acidic pH, followed by desorption with methanol and n-hexane, respectively. The extracts were analyzed by ultra-performance LC-MS/MS. Results: Intra-day precision was 1.2-8.6% and inter-day precision was 1.8-14.2%. Accuracy was 96.8-107.4%. The extraction yields were 62.8-109.4%. The matrix effects were -3.98% to 1%. Conclusion: BioSPME shows promise as an alternative method for preparing urine samples prior to drug measurement by ultra-performance LC-MS/MS.

Long-term monitoring of drug consumption patterns during the COVID-19 pandemic in a small-sized community in Brazil through wastewater-based epidemiology.

The abuse of legal and illegal drugs is a global public health problem, also affecting the social and economic well-being of the population. Thus, there is a significant interest in monitoring drug consumption. Relevant epidemiological information on lifestyle habits can be obtained from the chemical analysis of urban wastewater. In this work, passive sampling using polar organic chemical integrative samplers (POCIS) was used to quantify licit and illicit drugs biomarkers in wastewater for the application of wastewater-based epidemiology (WBE). In this WBE study, a small urban community of approximately 1179 inhabitants was monitored from 18 March 2020 to 3 March 2021, covering the mobility restriction and flexibilization periods of the COVID-19 pandemic in Brazil. Consumption was estimated for amphetamine, caffeine, cocaine, MDMA, methamphetamine, nicotine, and THC. The highest estimated consumption among illicit drugs was for THC (2369 ± 1037 mg day-1 1000 inh-1) followed by cocaine (353 ± 192 mg day-1 1000 inh-1). There was a negative correlation between consumption of caffeine, cocaine, MDMA, nicotine, and THC with human mobility, expressed by cellular phone mobility reports (P-value = 0.0094, 0.0019, 0.0080, 0.0009, and 0.0133, respectively). Our study is the first long-term drug consumption evaluation during the COVID-19 pandemic, with continuous sampling for almost a whole year. The observed reduction in consumption of both licit and illicit drugs is probably associated with stay-at-home orders and reduced access, which can be due to the closure of commercial facilities during some time of the evaluated period, smaller drug supply, and reduced income of the population due to the shutdown of companies and unemployment. The assay described in this study can be used as a complementary and cost-effective tool to the long-term monitoring of drug use biomarkers in wastewater, a relevant epidemiological strategy currently limited to short collection times.

Potential use of distinct biomarkers (trace metals, micronuclei, and nuclear abnormalities) in a heterogeneous sample of birds in southern Brazil.

The analysis of metal concentrations in bird feathers and genotoxicity tests are tools used to evaluate anthropogenic impacts on ecosystems. We investigated the response of birds, used as bioindicators, to disturbances observed in three areas with distinctive environmental characteristics (natural, agricultural, and urban) in southern Brazil. For this purpose, we quantified metals (Mn, Cu, Cr, and Zn) in feathers and determined the number of micronuclei (MN) and other nuclear abnormalities (NA) in 108 birds from 25 species and 17 families captured in the study area. No significant differences was found in the metal concentrations and the number of MN and NA between the sampling areas. Zn and Cu concentrations were significantly higher in insectivorous than those in omnivorous birds. The Zn concentration was significantly different between some species, and the Cu concentration was significantly higher in juveniles than that in adults. The best generalized linear models showed that omnivorous birds had more MN and NA and that juveniles and birds with better body condition index had increased NA numbers. This study demonstrates that the analyzed variables contribute in different ways to the result of each biomarker, mainly due to particular ecological and physiological characteristics of each species. We conclude that wild birds have the potential to be used as environmental bioindicators in the study area, but future studies should focus on one or a few species whose ecological and physiological habits are well known.

Development and validation of an assay for the measurement of gentamicin concentrations in dried blood spots using UHPLC-MS/MS.

Gentamicin sulfate (GEN) is an aminoglycoside antibiotic with a narrow therapeutic range of plasma concentrations. The collection of venous blood represents a significant burden for patients, especially in neonatology. Dried blood spots (DBS) obtained from capillary blood can be an alternative for drug measurements in this particular population. This study aimed to develop and validate an assay for the quantification of GEN in DBS using UHPLC-MS/MS. Total GEN concentrations were obtained by adding the individual concentrations of the GEN forms C1, C1a, and C2. The assay used a DBS disk containing approximately 17 µL of blood for GEN quantitation in the range of 0.1-40 mg L-1. Measurement accuracy for total GEN was in the range of 102.6-108.6%, inter-assay precision was 11.3-13.1% and intra-assay precision was 9.1-12.8.% GEN was stable for 21 days at - 20 and 8 °C, but only for 24 h at room temperature. Blood Hct affected the accuracy within acceptable limits (93.8-95% at Hct% of 30, 104.3-113% at Hct% of 50). Blood spotted volume did not affect GEN measurement accuracy. Concentrations of GEN in DBS obtained after heel pricks were correlated to plasma levels in a small cohort of neonatal patients. However, percentual differences between estimated plasma concentrations and actual plasma levels presented values between - 64-35.3% (average difference of - 1.9%). The use of DBS for the measurement of GEN concentrations can increase access to TDM of this antibiotic due to the ease of sample collection and the facilitated specimen transportation logistics when testing is not available onsite.

Sensitive determination of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and complementary cannabinoids in hair using alkaline digestion and mixed-mode solid phase extraction followed by liquid-chromatography-tandem mass spectrometry.

Hair drug testing can be used for the evaluation of cannabis use with a large detection window, and is required for professional driving license granting in Brazil. A positive hair result for cannabis use requires quantification of the metabolite THC-COOH above the cutoff value of 0.2 ng/g. The achievement of such lower limit of quantification is challenging, particularly with the use of liquid chromatography coupled to triple quadrupole mass spectrometers (LC-MS/MS). In this study, a very sensitive LCMS/ MS assay for the simultaneous quantification of THC-COOH along with THC, CBD, and CBN was developed and validated. Sample preparation was based on hair hydrolysis, followed by selective ion-exchange solid-phase extraction. The extraction yield was 101.5-101.6% for THC-COOH, 92.3-97.4% for THC, 89.7-95.2% for CBN, and 104.9-121.1% for CBD. Internal standard corrected matrix effects were - 2.7 to - 1,1 for THCCOOH and - 11.5 to - 0.1% for the other analytes. The lower limit of quantification was 01 ng/g for THC-COOH and 25 ng/g for THC, CBD, and CBN. The assay fulfilled validation guidelines acceptance criteria. The measurement uncertainties were determined and the assay was ISO17025 accredited, being currently used in routine testing.

Determination of cortisol in hair using UHPLC-MS/MS: application to patients admitted for ethanol dependence treatment.

Aim: Cortisol hair levels can be used to evaluate chronic stress status. In this context, an improved UHPLC-MS/MS assay for the determination of cortisol in hair was developed and validated. Materials & methods: Hair was extracted with methanol for 4 h at 25°C. Chromatographic run time was 5.5 min. The assay was linear in the range of 1-250 pg mg-1. Precision was 3.6-12.2% and accuracy 97.1-103.8%. The method was applied in hair from 19 volunteers admitted at a rehabilitation clinic, with ethanol consumption classified using ethyl glucuronide hair levels. Conclusion: Abstinent/chronic moderate ethanol consumers had significantly lower cortisol hair levels than chronic excessive consumers. This is the first study evaluating cortisol hair levels in ethanol abuse patients using an objective marker for ethanol consumption.

Sensitive determination of gentamicin in plasma using ion-exchange solid-phase extraction followed by UHPLC-MS/MS analysis.

BACKGROUND: Therapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS. OBJECTIVE: This study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma. RESULTS: The use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2-40 mg L-1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8-10.0%, intra-assay CV of 10.2-11.0%, and accuracy of 96.8-104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15-3.57 mg L-1 for GEN C1, 0.12-3.55 mg L-1 for GEN C1a, 0.20-5.77 mg L-1 for GEN C2, and 0.47-12.88 mg L-1 for the GEN complex, all within the linear range of the assay. CONCLUSION: A sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.

Simple determination of valproic acid serum concentrations using BioSPME followed by gas chromatography-mass spectrometric analysis.

Valproic acid (VA) is a drug widely used on the treatment of epilepsy and bipolar affective disorders, with stablished therapeutic concentration ranges in serum. The measurement of VA serum concentrations using chromatographic methods requires a sample preparation step. In this context, this study aims to describe the development and validation of an assay for VA measurement in serum using a new microextraction strategy, known as BioSPME, followed by GC-MS analysis. The extraction procedure was very simple based on direct immersion of the BioSPME tips on acidified serum, followed by agitation and desorption in methanol. The methanolic extracts were directly injected into the chromatograph. Extraction yield was 95.6 to 101.3%. The assay was linear from 10 to 150 mg L-1. Precision, accuracy and stability assays were acceptable according to bioanalytical validation guidelines. The method was applied to 41 clinical serum samples also tested with a previously GC-MS validated assay, which used liquid-liquid extraction as sample preparation. Measurements obtained with both methods were comparable. This study is the first description of the use of BioSPME tips for a therapeutic drug. BioSPME is a promising alternative for the preparation of biological specimens prior to the determination of therapeutic drugs by GC-MS.

Validation of an analytical method for the simultaneous determination of 16 drugs and metabolites in hair in the context of driving license granting.

The use of psychoactive substances has been associated with increased risk for traffic accidents. Hair testing has become a routine practice in clinical and forensic toxicological laboratories, with a unique perspective in the investigation of drug consumption. The study aimed to develop and validate a UHPLC-MS/MS method for the determination of multiple drugs in hair, to be used for toxicological examination in driving license granting. Sample preparation was a one-step liquid extraction of milled hair with methanol, which was incubated for 15h at 50°C. The chromatographic separation was performed in a reversed phase column, with a run time of 2.2min. Measured compounds were cocaine, benzoylecgonine, norcocaine, anhydroecgonine methyl ester, cocaethylene, amphetamine, methamphetamine, methylenedioxyamphetamine, methylenedioxymethamphetamine, fenproporex, amfepramone, mazindol, codeine, morphine, 6-monoacetylmorphine, and tetrahydrocannabinol. The assay was linear for all substances (r>0.99), accurate (86.63-105.87 %), and precise, with a cv ranging from 1.9-13.5 % for intra-assay and 3.3-14.3 % for inter-assay. There was no significant carry over effect and the internal standard corrected matrix effect was minimal. The relative uncertainty percentages were below 9% for all the substances at cut-off values. The method was successfully applied to 50 hair samples from injured drivers, with 12% of positivity, including cocaine, MDMA and THC.

Increase of global DNA methylation patterns in beauty salon workers exposed to low levels of formaldehyde.

Formaldehyde (FA) is a carcinogenic aldehyde illegally added to creams as a hair straightening agent for the Brazilian blowout (BB). This study aimed to investigate the possible effects of occupational exposure to FA on global DNA methylation in salon workers with different exposure levels. FA exposure was monitored using environmental and biological measurements. The study included 49 salon workers divided by FA levels in the workplace into group A (FA < 0.01 ppm; n = 8), group B (0.03 ppm < FA < 0.06 ppm; n = 15), and group C (0.08 ppm < FA < 0.24 ppm; n = 26). The global DNA methylation levels were 3.12%, 4.55%, and 4.29% for groups A, B, and C, respectively, with statistically higher values for groups B and C compared to group A (p = 0.002). A correlation was found between FA in passive samplers and global DNA methylation (rs = 0.307, p = 0.032). Additionally, when only taking into account the hairdressers that performed the BB on clients instead of the whole group, a stronger correlation was observed between FA in personal passive samplers and global DNA methylation (rs = 0.764, p = 0.006). For the first time, an increase in DNA methylation was observed in subjects occupationally exposed to FA. In conclusion, our results indicated that even low levels of FA exposure could cause a disturbance in DNA methylation, leading to epigenetic changes, which is associated with cancer development. These data suggest a possible contribution of FA to cancer development through occupational exposure.

Determination of irinotecan and its metabolite SN-38 in dried blood spots using high-performance liquid-chromatography with fluorescence detection.

Irinotecan (IRI) is an antineoplastic drug widely used for the treatment of colorectal and advanced pancreatic cancer. Despite its clinical utility, the clinical use of IRI is associated with potentially severe hematopoietic and gastrointestinal toxicities. The quantification of IRI and its active metabolite SN-38 in dried blood spots (DBS) may be an alternative to individualize the drug dose through a minimally invasive and easy collection method. The aim of this study was to develop and validate a simple and fast HPLC-FL assay for simultaneous IRI and SN-38 measurement in DBS, with adequate analytical performance for clinical use. The method employs liquid extraction of one 8mm disk of whole blood, followed by separation in a reversed phase Eclipse Plus C8 column (150×4.6mm, 5µm). Detection was performed with a fluorescence detector, with excitation wavelength of 370 and emission of 420 for IRI and 540nm for SN-38 and internal standard (camptothecin). Total analytical run time was 17min. Mobile phase was a mixture of 0.1M phosphate buffer pH 4.0 and acetonitrile (80:20, v/v), at 1mLmin-1. The assay was linear in the range 10-3,000ngmL-1 and from 0.5 to 300ngmL-1 for IRI and SN-38, respectively. Precision assays presented CV% of 2.71-5.65 and 2.15-10.07 for IRI and SN-38, respectively, and accuracy in the range of 94.26-100.93 and 94.24-99.33%. IRI and SN-38 were stable at 25 and 42°C for 14days in DBS samples. The method was applied to DBS samples obtained from fingerpicks from 19 volunteers receiving IRI in single or combined chemotherapy regimens, collected 1 and 24h after beginning of the infusion. The estimated plasma concentration of IRI and SN-38 in sample collected 1h after star of infusion had 16 of 19 values within the ±20% range of the measured plasma concentrations. On the other hand, predictions of IRI and SN-38 plasma concentrations from DBS measurements obtained 24h after the beginning of the infusion were poor. AUC of IRI that was calculated using plasma and DBS-estimated concentrations, with a high correlation (r=0.918). The method presented suitable characteristics for the clinical use. However, translation of IRI and SN-38 DBS to plasma concentrations is challenging due to the compound's variable plasma/blood partition.

Improved determination of uracil and dihydrouracil in plasma after a loading oral dose of uracil using high-performance liquid chromatography with photodiode array detection and porous graphitic carbon stationary phase.

OBJECTIVES: The aim of this study was to develop and validate a high-performance liquid chromatographic method for the measurement of plasma concentrations of uracil and dihydrouracil after administration of an oral loading dose of uracil in the context of evaluation of DPD enzyme activity. DESIGN AND METHODS: Analytes were extracted from 500µL plasma sampler with a mixture of ethyl acetate isopropanol (85:15, v/v) after protein precipitation with solid ammonium sulfate. The extract was inject in the porous graphitic carbon stationary phase, eluted with water and acetonitrile in gradient mode, allowing complete separation of uracil, dihydrouracil and the internal standard (5-fluorouracil). Chromatograms were monitored at 210 and 260nm. RESULTS: Total chromatographic run time, including reequilibration, was 30min. The assay was linear in the concentration range of 0.2 to 20µgmL(-1). Accuracy was 98.4-105.3%, intra-assay precision was 5.1-12.1% and between-assay precision was of 5.3-10.1%. Analytes were stable in plasma at room temperature up to 6h and for three freeze and thaw cycles. Processed samples are stable up to 12h. CONCLUSIONS: The developed method was fully validated and has significantly reduced running time when compared to previous assay using porous graphitic stationary phase, allowing complete resolution of uracil, dihydrouracil and internal standard. This assay might be suitable to investigate the eventual correlation between concentrations of uracil and dihydrouracil in plasma after an oral loading dose and DPD enzyme activity, with potential contribution to therapeutic drug monitoring.