Determination of carbamazepine in fingerprints: a feasibility study to evaluate adherence in epilepsy patients.

Background: Fingerprint drug concentrations can be used as a noninvasive and convenient alternative to evaluate adherence to pharmacotherapy. Methods: Fingerprints were applied over glass slides, extracted and analyzed by ultra-high performance LC-MS/MS. The assay and drug adherence questionnaires were applied to 30 epilepsy patients. Results: The assay had linearity in the range 0.05-10 ng fingerprint-1, with precision of 2.16-7.9% and accuracy of 95.0-102.8%. Carbamazepine (CBZ) levels in fingerprints were stable at 45°C for 15 days. Concentrations in patient samples were 0.06-9.28 ng fingerprint-1. A significant difference (p = 0.003) was found between CBZ concentrations in fingerprints between patient groups divided as low and medium/high adherence. Conclusion: This method can potentially be applied to the identification of epilepsy patients with low adherence to CBZ pharmacotherapy.

Determination of meropenem in capillary plasma microsamples using LC-MS/MS.

Background: The measurement of meropenem plasma concentrations is employed for dosing regimen individualization. The aim of this study was to develop and validate a LC-MS/MS assay for quantification of meropenem in capillary plasma microsamples. Methods: Samples were prepared by protein precipitation with acetonitrile, followed by clean-up with dichloromethane. The method was validated and applied to 12 paired samples of venous and capillary plasma. Results: The method was linear in the range of 0.5-50 µg/ml. Matrix effects were minimal. Inter- and intra-assay were 3.8-7.9% and 2.7-5.5%, respectively, while accuracy was 91.7-100.6%. Concentrations in capillary and venous plasma were highly correlated. Conclusion: An assay for the quantification of meropenem in capillary plasma microsamples was fully validated, showing potential for clinical application.

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.

rAAV-compatible human mini promoters enhance transgene expression in rat retinal ganglion cells.

Recombinant adeno-associated viral vectors (rAAV) are the safest and most effective gene delivery platform to drive the treatment of many inherited eye disorders in well-characterized animal models. The use in rAAV of ubiquitous promoters derived from viral sequences such as CMV/CBA (chicken ß-actin promoter with cytomegalovirus enhancer) can lead to unwanted side effects such as pro-inflammatory immune responses and retinal cytotoxicity, thus reducing therapy efficacy. Thus, an advance in gene therapy is the availability of small promoters, that potentiate and direct gene expression to the cell type of interest, with higher safety and efficacy. In this study, we used six human mini-promoters packaged in rAAV2 quadruple mutant (Y-F) to test for transduction of the rat retina after intravitreal injection. After four weeks, immunohistochemical analysis detected GFP-labeled cells in the ganglion cell layer (GCL) for all constructs tested. Among them, Ple25sh1, Ple25sh2 and Ple53 promoted a widespread reporter-transgene expression in the GCL, with an increased number of GFP-expressing retinal ganglion cells when compared with the CMV/CBA vector. Moreover, Ple53 provided the strongest levels of GFP fluorescence in both cell soma and axons of retinal ganglion cells (RGCs) without any detectable adverse effects in retina function. Remarkably, a nearly 50-fold reduction in the number of intravitreally injected vector particles containing Ple53 promoter, still attained levels of transgene expression similar to CMV/CBA. Thus, the tested MiniPs show great potential for protocols of retinal gene therapy in therapeutic applications for retinal degenerations, especially those involving RGC-related disorders such as glaucoma.

Risk assessment of a Brazilian urban population due to the exposure to pyrethroid insecticides during the COVID-19 pandemic using wastewater-based epidemiology.

Pyrethroids are synthetic insecticides commonly used in agriculture and homes due to their low toxicity to mammals and effectiveness at low doses. However, exposure to pyrethroids can cause various symptoms, depending on the route of exposure. To measure human exposure to pyrethroids, researchers used wastewater-based epidemiology (WBE) with polar organic chemical integrative samplers (POCIS) sampling. This approach is a cost-effective and efficient way to assess exposure to pyrethroids. The study aimed to evaluate the exposure of an urban population in Brazil to pyrethroids during the COVID-19 pandemic using WBE with POCIS sampling. Researchers analyzed 3-phenoxybenzoic acid (3-PBA) in wastewater using passive sampling with POCIS, which was extracted with methanol and analyzed using UPLC-MS/MS. The range of CTWA concentrations of 3-PBA in wastewater was 24.3-298.2 ng L-1, with a mean value of 134 ± 76.5 ng L-1. The values were used to estimate the exposure of the population to pyrethroid insecticides. Three different conversion factors were applied to determine the range of exposure to at least 20 different pyrethroid insecticides. The exposure values ranged from 18.08 to 1441.49 mg day-1 per 1000 inhabitants. The toxicological risk posed to the exposed population was evaluated by calculating the WBE toxicological level (WBE-TL). Lambda-cyhalothrin was used as a reference for risk assessment, and the WBE-TL values for lambda-cyhalothrin ranged from 0.5 to 8.29 (considering the high CF). We compared mobility trends to 3-PBA exposure during the COVID-19 pandemic. The study highlighted the effectiveness of POCIS sampling in WBE and provided useful information for policymakers and regulatory agencies. POCIS sampling has practical advantages, including analyte pre-concentration, low operational cost, and ease of use. Overall, the study shows the importance of monitoring and understanding the exposure of the population to pyrethroid insecticides, especially during the pandemic when people may be spending more time at home.

HTLV-1 p12 modulates the levels of prion protein (PrPC) in CD4+ T cells.

Introduction: Infection with human T cell lymphotropic virus type 1 (HTLV-1) is endemic in Brazil and is linked with pro-inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neuroinflammatory incapacitating disease that culminates in loss of motor functions. The mechanisms underlying the onset and progression of HAM/TSP are incompletely understood. Previous studies have demonstrated that inflammation and infectious agents can affect the expression of cellular prion protein (PrPC) in immune cells. Methods: Here, we investigated whether HTLV-1 infection affected PrPC content in cell lines and primary CD4+cells in vitro using flow cytometry and western blot assays. Results: We found that HTLV-1 infection decreased the expression levels of PrPC and HTLV-1 Orf I encoded p12, an endoplasmic reticulum resident protein also known to affect post-transcriptionally cellular proteins such as MHC-class I and the IL-2 receptor. In addition, we observed a reduced percentage of CD4+ T cells from infected individuals expressing PrPC, which was reflected by IFN type II but not IL-17 expression. Discussion: These results suggested that PrPC downregulation, linked to both HTLV-1 p12 and IFN-γ expression in CD4+ cells, may play a role in the neuropathogenesis of HTLV-1 infection.

Evaluation of dried blood spots as an alternative sampling strategy for 5-fluorouracil monitoring: From method development to clinical application.

Therapeutic drug monitoring (TDM) of 5-Fluorouracil (5-FU) is strongly recommended because of its large inter-individual pharmacokinetic variability, narrow therapeutic window, and incidence of toxicity. However, there are several factors that limit the application of TDM in clinical settings. Considering the intrinsic advantages of dried microsamples, such as minimally invasive sampling, analyte stability, and cost-effective logistics, this study aimed to develop a method for the determination of 5-FU in dried blood spots (DBS) using ultra-high liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and to evaluate its clinical application. Sample preparation was based on an aqueous extraction followed by protein precipitation. Separation was performed in an Acquity UPLC® HSS C18 (150 ×2.1 mm, 1.8 µm), and the mobile phases were water and acetonitrile with 0.5% acetic acid. The total run time was 5.5 min. The method was linear from 100 to 2000 ng/mL, precise (maximum CV% of 7.5%), and accurate (98.3-115.4%). The average recovery was 70%. Blood hematocrit had a minimal impact on the assay. DBS samples were stable for 21 days at 4, 25, and 45 °C. A total of 40 paired samples of plasma, capillary DBS, and venous DBS were analyzed. Median 5-FU concentrations were 444.7, 637.0, and 499.7 ng/mL for plasma, capillary DBS, and venous DBS, respectively. Capillary and plasma concentrations were significantly correlated (r > 0.90), but there was a lack of agreement between the methods, as capillary DBS levels were on average 146% of plasma. Venous DBS corresponded to 110% of the measured plasma concentrations, with a strong correlation (r > 0.97) and agreement between the methods. Our study is the first to report the use of DBS samples to quantify 5-FU. Further studies are needed to establish whether capillary samples can replace plasma.

Evaluation of oxidative stress and its association with drug therapy in inpatients treated for cocaine dependence.

The use of cocaine affects several systems and organs of the human body and the consumption of this substance leads to an increase in the production of reactive oxygen species, and to the reduction of antioxidant defenses. The aim of this study was to evaluate the oxidative stress (OS), biochemical and hematological parameters in patients hospitalized for treatment of cocaine addiction, comparing levels at hospital admission and discharge. Forty patients were included in the study. OS was evaluated using catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GPx), total antioxidant power (FRAP), malondialdehyde (MDA), and sulfhydryl group (GS). The medications used during hospitalization were registered and their influence on the parameters of OS was analyzed. After the hospitalization period, there was an increase in GGT levels, a reduction in SOD activity, and an increase in GPx activity and FRAP levels. Carbamazepine users had higher SOD values and lower FRAP values at hospital discharge. The use of chlorpromazine caused differences in creatinine and gamma-glutamyltransferase (GGT) serum leves, and the levels of glutamic oxalacetic transaminase (TGO), MDA, and FRAP were increased at hospital discharge. Haloperidol and thiamine during hospitalization interfered with alkaline phosphatase levels. The use of risperidone caused an increase in the levels of SOD, and folic acid use was associated with lower levels of GPx and higher levels of glutamic-pyruvic transaminase (TGP) and alkaline phosphatase. Drug rehabilitation treatment was effective in decreasing oxidative damage represented by the reduction of biological markers.

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.

Evaluation of the HealthID PSD microsampling device for the simultaneous determination of cholesterol, high-density lipoprotein, triglycerides, creatinine, and HbA1c in capillary blood.

INTRODUCTION: The use of dried capillary microsamples for clinical chemistry testing is an interesting alternative to conventional phlebotomy. Sampling devices capable to produce plasma from whole blood application are particularly useful. The aim of this study was to validate theHealthID PSDmicrosampling device for the determination of cholesterol (CHOL), high-density lipoprotein (HDL), triglycerides (TRIG), creatinine (CRE), and glycated hemoglobin (HbA1c) after collection of capillary blood. METHODS: Dried blood and plasma extracts were analyzed using modified methods in an open-channel biochemistry analyzer. The plasma volume in the extracts was corrected by the concentration of chloride (CL). Linearity, imprecision, bias, stability, and comparability to conventional samples were evaluated. RESULTS: Dried plasma assays presented total error (TE) within acceptable limits. The analytes were stable for up to 14 days at 40 °C. Predicted serum concentrations of CHO, HDL, TRI, and CRE and predicted whole blood levels of HbA1c, using dried extracts measurements, did not presented systematic or proportional differences to serum and whole blood levels. CONCLUSIONS: Dried sample extracts obtained with capillary blood applied to the HealthID PSD allowed the determination of CHO, HDL, TRI, CRE, and HbA1c, as well as the calculation of LDL level, using only 5 drops of blood. This sampling strategy can be useful for population screening programs, particularly in Developing Countries.

Dried blood spot sampling for therapeutic drug monitoring: challenges and opportunities.

INTRODUCTION: The use of dried blood spots (DBS) has gained interest in the field of therapeutic drug monitoring (TDM) due to its potential advantages, such as minimally invasive capillary blood collection, potential stabilization of drugs and metabolites at room or high temperatures, and lower biohazard, allowing for inexpensive storage and transportation. However, there are several drawbacks to the clinical use of DBS in TDM, mostly related to hematocrit (Hct) effects, differences between venous and capillary blood concentrations, among others, that must be evaluated during analytical and clinical method validation. AREA COVERED: This review focuses on the most recent publications on the applications of DBS sampling for TDM (2016-2022), with a special focus on the challenges presented by this alternative sampling strategy, as well as the opportunities for clinical applications. Real-life studies presenting clinical applications were reviewed. EXPERT OPINION: With the availability of method development and validation guidelines for DBS-based methods in TDM, higher levels of assay validation standardization have been achieved, expanding the clinical applications of DBS sampling in patient care. New sampling devices that overcome the limitations of classical DBS, such as the Hct effects, will further encourage the use of DBS in routine TDM.

Residual contamination in antineoplastic drug packaging.

INTRODUCTION: The handling of antineoplastic drugs should follow strict supervision and safety rules to minimize the occupational exposure risks to professionals involved. The external surface contamination of drug vials is recognized as a health risk. So, our goal was to determine if there is residual contamination on the vials and containers surface of the antineoplastic drugs doxorubicin (DOX) and cyclophosphamide (CP). METHODS: A cross-sectional study was conducted. Samples were collected using a uniform sampling procedure on the inner surfaces of the packages/boxes and the outer surfaces of the vials. The analyzes were executed by high-performance liquid chromatography/mass spectrometry (UHPLC-MS/MS). RESULTS: A total of 209 samples were analyzed, 66 of CP and 143 of DOX. CP levels were detected in nine samples (13.63%), three were below the lower limit of quantification (LLQ) and the other six had contamination levels ranging from 1.24 to 28.04 ng/filter. DOX levels were detected in 36 samples (25.17%), two were below the LLQ and the others had levels between 1.32 and 664.84 ng/filter. The majority of samples with residual contamination were in vials (80.0%), however, boxes also showed contamination. CONCLUSIONS: The results revealed the presence of residual contamination in the vials and packages of CP and DOX drugs. Although the residues found in each sample are small, special care should be taken in the handling and disposal of the antineoplastic drugs. The use of personal protective equipment is fundamental while handling the vials and packaging of cytotoxic drugs.

Determination of Ethyl Glucuronide and Ethyl Sulfate in Dried Blood Spots by UHPLC-MS-MS: Method Validation and Assessment of Ethanol Exposure in Postmortem Samples from Road Traffic Victims.

The determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood has been proposed in clinical and forensic applications to identify recent alcohol consumption. Also, there is a growing interest on the use of dried blood spots (DBS) in toxicological analysis, allowing increased stability of the analytes and simplifying sample transportation and storage. This study presents the development and validation of a method for quantifying EtG and EtS in DBS using ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). The DBS samples were extracted with a mixture of methanol and acetonitrile (80:20 v/v) and analyzed using UHPLC-MS-MS with electrospray source in negative mode, after separation with a fluoro-phenyl stationary phase. Validation was performed according to the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, with calibrations ranging from 0.10 to 18 µg/mL for EtG and 0.02 to 6 µg/mL for EtS. The analytes were stable in DBS stored from -20 to 45°C for 21 days. The method was successfully applied to capillary and venous DBS samples from 20 volunteers after ethanol ingestion and to DBS samples from 99 fatal victims of road traffic injuries. Capillary DBS was comparable to venous DBS and fresh whole blood in Passing-Bablok and Bland-Altman analysis, with correlation coefficients >0.91 (P < 0.001) for all comparisons. In postmortem application, the DBS EtG and EtS analysis indicated positive exposure to ethanol in 72.7% of the cases (EtG: 0.10-24.0 µg/mL and EtS: 0.03-4.11 µg/mL). The identification of ethanol consumption from blood alcohol concentrations (BACs) and EtG/EtS in DBS was in agreement in 98.6% of positive and 96.3% of negative cases (kappa 0.877, P < 0.001), indicating a high level of concordance with BAC in assessing alcohol use in postmortem samples.

Evaluation of UGT1A1 and CYP3A Genotyping and Single-Point Irinotecan and Metabolite Concentrations as Predictors of the Occurrence of Adverse Events in Cancer Treatment.

PURPOSE: The variability on irinotecan (IRI) pharmacokinetics and toxicity has been attributed mostly to genetic variations in the UGT1A1 gene, responsible for conjugation of the active metabolite SN-38. Also, CYP3A mediates the formation of inactive oxidative metabolites of IRI. The association between the occurrence of severe adverse events, pharmacokinetics parameters, and UGT1A1 and CYP3A4 predicted phenotypes was evaluated, as the evaluation of [SN-38]/IRI dose ratio as predictor of severe adverse events. METHODS: Forty-one patients undergoing IRI therapy were enrolled in the study. Blood samples were collected 15 min after the end of drug the infusion, for IRI, SN-38, SN-38G, bilirubin concentrations measurements, and UGT1A1 and CYP3A genotype estimation. Data on adverse event was reported. RESULTS: Fifteen patients (36.5%) developed grade 3/4 adverse events. A total of 9.8% (n = 4) of the patients had UGT1A1 reduced activity phenotype, and 48.7% (n = 20) had UGT1A1 and 63.4% (n = 26) CYP3A intermediary phenotypes. Severe neutropenia and diarrhea were more prevalent in patients with reduced UGT1A1 in comparison with functional metabolism (50% and 75% versus 0% and 13%, respectively). SN-38 levels and its concentrations adjusted by IRI dose were significantly correlated to toxicity (rs = 0.31 (p = 0.05) and rs = 0.425 (p < 0.01)). The [SN-38]/IRI dose ratio had a ROC curve of 0.823 (95% CI 0.69-0.956) to detect any severe adverse event and 0.833 (95% CI 0.694-0.973) to detect severe diarrhea. The cut-off of 0.075 ng mL-1 mg-1 had 100% sensitivity and 65.7% specificity to predict severe diarrhea. CONCLUSION: Our data confirmed the relevance of the pre-emptive genotypic information of UGT1A1. The [SN-38]/IRI ratio, measured 15 min after the end of the IRI infusion, was a strong predictor of severe toxicity and could be applied to minimize the burden of patients after IRI administration.

Volumetric dried blood microsampling for monitoring imatinib mesylate therapy: Method development and clinical application in patients with chronic myeloid leukemia.

Chronic Myeloid Leukemia (CML) is a hematologic neoplasia, characterized as a proliferative disease of the hematopoietic system. Imatinib mesylate (IM), a selective tyrosine kinase inhibitor, is considered a first-line therapy for CML, indicated for both adult and pediatric patients presenting the Philadelphia chromosome (Ph+). However, patients in treatment with IM may show different responses due to interindividual pharmacokinetic variability. Therapeutic drug monitoring should be routinely performed to identify treatment response profile, adherence to treatment, or possible drug interactions, thus supporting better treatment management. Volumetric absorptive microsampling (VAMS) are innovative devices for blood collection whose advantages include the possibility of home collection by the patient or at the physician's office. The assay was fully validated according to bioanalytical validation guidelines. Estimated plasma concentrations of IM were not statistically different between groups according to adherence (p = 0.15), with median of 789 ng ml-1 in the group with some level of non-adherence versus 1141.9 ng ml-1 in the group with adherence, classified with the Morisky-Green questionnaire. This study included 33 patients with CML in treatment with IM. These patients answered socioeconomic, sociodemographic, and adherence profile (Morisky-Green) questionnaires. Patients also received instructions for home blood collection with VAMS devices. Afterwards, the samples were analyzed by LC-MS/MS. The mean age of the patients was 52 years, 84.8% were ingesting doses of 400 mg/day and the majority were male (69.7%). IM and its metabolite NIM were extracted from VAMS with an aqueous solution with 0.1% formic acid, followed by protein precipitation with acetonitrile. The methodology developed in this study was satisfactory for the determination of IM and NIM in VAMS and can be used in hospital and office routines for the therapeutic monitoring of patients with CML.

Evaluation of hemaPEN® sampling device for measurement of cocaine and metabolites in capillary blood by LC-MS/MS.

Background: Dried blood spot sampling has been reported for on-site collection of specimens, but measurements are affected by blood hematocrit, and special handling is required, especially for forensic applications. The hemaPEN® blood collection device was developed to produce spots with constant volume. Results: Linearity between 1 and 500 ng/ml was shown for cocaine and the metabolites benzoylecgonine and cocaethylene. The assay demonstrated acceptable precision and accuracy, and analytes were stable for 7 days when kept inside hemaPEN devices. Accuracy of the assay was affected by hematocrit but was within acceptable limits. Conclusion: Use of the hemaPEN, which retains dried blood within the device, could be advantageous for the quantification of illicit drugs in capillary blood compared with conventional dried blood spot collection.

Evaluation of the Tasso-SST® capillary blood microsampling device for the measurement of endogenous uracil levels.

BACKGROUND: Uracil (U) plasma or serum levels can be used as surrogates of dihydropyrimidine dehydrogenase (DPD) activity, which is strongly related to the occurrence of severe or fatal toxicity after administration of fluoropyrimidines (FP) chemotherapy. Obtaining blood plasma or serum for U measurement usually requires a phlebotomy procedure by a qualified professional. An alternative to conventional blood drawn is the use of the Tasso-SST® device, which allows the collection of a small blood volume from skin capillaries. This study aimed to implement a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the determination of U in small serum samples and to compare U concentrations measured in venous plasma, obtained after phlebotomy, and serum obtained with the Tasso-SST® device. METHODS: Fifty microliter samples were prepared through simple protein precipitation with trichloroacetic acid. Chromatographic separation was performed with a porous graphitic carbon stationary phase and mass spectrometric detection used positive electrospray ionization. The assay was validated according to international guidelines. RESULTS: The linear range of the assay was 5-250 ng/mL. Measurement accuracy was in the range of 98.8-108.2%, inter-assay precision was 4.3-7.3%, and intra-assay precision was 3.4-6.1%. The average matrix effect was -6.42%. The extraction yield was 95-103.3%. U concentrations measured in serum obtained with the Tasso-SST® device and venous blood plasma were highly correlated (rs = 0.910, P < 0.0001), and no systematic or proportional bias between U levels measured in both matrices was found. CONCLUSIONS: The use of blood microsampling with the Tasso-SST® device is a useful alternative for the measurement of U and the identification of patients with DPD deficiency.