Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

Molecular analysis of the RHD pseudogene by duplex real-time polymerase chain reaction.

OBJECTIVES: In this study, we aimed to present a strategy for the detection of the RHD pseudogene (RHDψ) based on a real-time polymerase chain reaction (PCR) assay. BACKGROUND: The D-negative phenotype is associated with many genetic alterations. In populations with African ancestry, this phenotype commonly results from the silent variant RHDψ. The evaluation of RHDψ is essential for correct inference of the RhD phenotype in order to avoid false-positive results. METHODS: We utilised a new method for the simultaneous detection of RHDψ and a fragment from exon 5 of the wild-type RHD gene based on duplex real-time PCR assay. RESULTS: The PCR assay allowed specific detection of RHDψ. There was complete agreement between the results generated by the new test and the results generated by molecular analysis performed using end-point PCR methods previously described. CONCLUSIONS: The assay developed is easy to execute and presents the potential for routine use at blood banks and other associated facilities where it is desired to determine the presence of RHDψ.

Tadpoles fed supplemented diet with probiotic bacterium isolated from the intestinal tract of bullfrog Lithobates catesbeianus: Haematology, cell activity and electron microscopy.

The aim of this study is to select and isolate autochthonous bacteria with probiotic potential for use in a supplemented diet for bullfrog tadpoles, Lithobates catesbeianus. A total of 20 strains of lactic acid bacteria were isolated. Nine out of these were used in the following in vitro assays: antagonism against pathogenic bacteria (ANT), antimicrobial activity from extracellular compounds (MIC), tolerance to bile salts (TBS), pH reduction, protease production, sensitivity to antimicrobial tetracycline, cell viability, growth rate and doubling time. Using these data was defined an ideotype (ideal strain) based on the best results. Distances were estimated with the Mahalanobis (D2) test, and the best candidates, presenting the shortest ideotype distances, were considered to be used. The best strain was found to be Lactobacillus plantarum because it presented 10.00 ± 0.50 mm of ANT against Aeromonas hydrophila, 3.99 ± 0.01 of MIC independent of pathogenic bacteria, 85.07 ± 0.01 of TBS, 4.20 ± 0.02 of final pH, 17.67 ± 1.15 of protease production, 13.50 ± 2.00 sensitivity to antimicrobial tetracycline, 9.36 ± 0.04 of cell viability, 0.20 ± 0.00 of growth rate and 3.46 ± 0.00 doubling time. Therefore this probiotic candidate was then supplemented (2.045 ± 1.07 × 107 colony forming unities. g-1) into the diets of bullfrog tadpoles for a period of 42 days. At the end of the trial, samples of blood and intestines were collected to verify the haematological alterations and the intestinal morphology using transmission and scanning electron microscopy. Tadpoles fed the supplemented diet showed successful lactic acid bacterium colonisation, an increased number of circulating thrombocytes, monocytes, eosinophil and LG-PAS+ and also an increase in the length and density of intestinal microvilli. This study shows the feasibility of using probiotics isolated from farmed bullfrogs as a supplement in the diets of tadpoles, providing a promising alternative for modulating the health of these animals.

Fish parasites as indicators of organic pollution in southern Brazil.

Increasing urbanization along riverbanks is a constant source of stressors to the aquatic community, and the use of bioindicators is suitable to detect and monitor the effect of each stressor. We investigated the parasites of the 'cará' fish (Geophagus brasiliensis) as potential bioindicators in a river whose banks are subject to increasing anthropogenic pressure. Samples were taken at four points of the Sangradouro River, in Florianópolis, southern Brazil, bimonthly for 12 months. Water temperature, pH, dissolved oxygen, conductivity and salinity were measured at each point and water samples were taken for nutrient analysis (total nitrogen, nitrite, total ammonia nitrogen, total phosphorus, phosphate), and total and faecal coliforms. A generalized linear model (GLM) was constructed using the abundance of each parasite species as the response variable and biometric characteristic of the fish and water variables as possible predictors. Among the 137 fish examined, 114 (83.2%) were parasitized by at least one parasite species. Two species of ectoparasites (Sciadicleithrum guanduensis and Neascus-type metacercariae) and two species of endoparasites (Pandosentis aff. iracundus and Homalometron pseudopallidum) were observed. This is the first record of the genus Pandosentis in Brazilian waters and the first record of the species G. brasiliensis as a host for this parasite. Among the analysed groups of parasites, monogeneans and acanthocephalans proved to be the most sensitive to the concentrations of dissolved oxygen, faecal coliforms and total ammonia nitrogen in the water. Our study suggests that the abundance of both monoxenic and heteroxenous parasites can be negatively affected by organic pollution in the aquatic environment, reinforcing the potential of fish parasites as indicators of water quality.

Impact of the anatomical location, alcoholism and smoking on the prevalence of advanced oral cancer in Brazil.

BACKGROUND: To evaluate the prevalence of oral cancer in Brazil according to the clinical stage, anatomical location, alcoholism and smoking. MATERIAL AND METHODS: Data referring to 31,217 cases of oral cancer, from 2000 to 2010, were obtained from the Integrator Module of the Hospital Registry of Cancer. Inconsistent data ("non-classified" cases) was eliminated and 21,160 cases were analyzed. The frequency distribution according to clinical stage, anatomical location, alcoholism and smoking was analyzed descriptively and through a binary logistic regression model (α<0.05). The clinical stage (dependent variable) was dichotomized in early stage (I and II) or advanced stage (III and IV). The year of diagnosis, anatomical location and deleterious habits (alcoholism and smoking) were considered independent variables. RESULTS: The most frequent characteristics were: oropharynx location (n=3856, 18.41%), clinical stage IV (n=11924, 56.09%) and combined use of alcohol and tobacco (n=19226; 61.59%). The year 2009 (p<0.01, PR = 1.162, CI-95%=1.053-1.283) and location at the base of tongue (p<0.01, PR = 2.485, CI-95% = 2.182-2.807) presented a higher prevalence ratio for advanced stage oral cancer. The combined use of alcohol and tobacco showed a higher prevalence rate for the advanced clinical stage of cancer (p<0.01, PR =1.449, CI-95%=1.382-1.520) if compared to individuals without habits, or just alcoholics. CONCLUSIONS: Higher prevalence of advanced stage of oral cancer is related to the localization at the base of the tongue and to the concomitant use of alcohol and tobacco. Therefore, it can be suggested that all these characteristics lead to a worse prognosis of oral cancer.

A method for collecting eggs of Pseudocapillaria tomentosa (Nematoda: Capillariidae) from zebrafish Danio rerio and efficacy of heat and chlorine for killing the nematode's eggs.

Pseudocapillaria tomentosa is a common pathogen of zebrafish (Danio rerio) in research facilities. We developed a method to collect and concentrate the nematode eggs using a modified sugar centrifugation method and documented their normal development. Embryonating stages with blastomere formation followed by elongation of the embryo prior to larva formation cumulated in developed larvae inside the eggs and hatching after 5-10 day. We then evaluated the efficacy of heat and chlorine to kill them based on a larva development assay. Eggs were exposed to 40, 50, 60 °C for 30 min and 1 h. Chlorine treatment was performed at 100, 250, 500, 1000, 3000 and 6000 ppm for 10 min. Samples exposed to 40 °C for 30 min or 1 h showed incidences of larvated eggs similar to controls. In contrast, no larvation occurred with eggs exposed to either 50 or 60 °C for 30 min or 1 h. Remarkably, in repeated assays, samples exposed to low doses of chlorine (100, 250, 500 and 1000 ppm for 10 min) showed significantly higher incidence of larvation than controls. Eggs treated with 3000 ppm for 10 min did not develop larvae, and no eggs were found after 6000 ppm treatment.

HIV/AIDS-Associated Cryptococcosis in Portugal Spanning the Pre- to Post-HAART Era: A Retrospective Assessment at the Genotypic Level Based on URA5-RFLP.

Cryptococcosis caused by the fungus Cryptococcus neoformans is an opportunistic mycosis, infecting mainly immunodepressed individuals. Molecular epidemiology studies of cryptococcosis in Europe are limited. This paper presents a retrospective study of cryptococcosis in 105 cryptococcal isolates from two hospitals in Lisbon, Portugal, among HIV/AIDS patients, from 1991 to 2007. Among these patients, the number of cases of cryptococcosis increased from 5.1 to 6.9 cases per year from the pre- to post-highly active antiretroviral therapy (HAART) era. As expected, the median age of the patients increased, from 32 (mean: 33 ± 8) to 39 (mean: 41 ± 10) years, and the ratio of male to female patients remained high (7.7 and 7.6, respectively). Strain genotyping based on restriction fragment length polymorphism of the orotidine monophosphate pyrophosphorylase (URA5-RFLP) gene showed that, in general, the relative frequencies of the genotypes VNI-IV are similar to those from other European countries. These frequencies were, respectively, for the pre- and post-HAART periods: 41.7 and 43.5 % for VNI; 2.8 and 17.4 % for VNII; 38.9 and 30.4 % for VNIII; 16.7 and 7.2 % for VNIV and 0 and 1.4 % for VGII. Some apparent although statistically insignificant differences among these values were observed between both periods. The genotypic frequencies were not also statistically different according to the patients' gender or age range. Of note are the high proportion of VNIII isolates (common in Europe) and the high increase in the frequency of the VNII genotype in the post-HAART. Ultimately, these results may have implications in disease therapy, management and control.

New species of Cucullanus (Nematoda: Cucullanidae), an intestinal parasite of the peacock bass Cichla piquiti (Perciformes: Cichlidae) from the Tocantins River, Brazil.

Cucullanus tucunarensis n. sp. (Cucullanidae) is described parasitizing the peacock bass Cichla piquiti (Cichlidae) from the Tocantins River, Tocantins State, Brazil. The new species is unique and differs from all its congeners by having a tail tip provided with several sclerotized spine-like processes on its ventral side, present in both the male and female. Furthermore, C. tucunarensis n. sp. is compared with other species of the genus recorded in the same zoogeographical region and in the same group of hosts (Perciformes).

Insights on the genotype distribution among Cryptococcus neoformans and C. gattii Portuguese clinical isolates.

This study provides a comprehensive picture of the C. neoformans/C. gattii molecular types most often associated with human cryptococcosis in Portugal and assesses the impact of C. gattii in these infections. One hundred and twenty-two clinical isolates, from distinct patients, were identified as C. neoformans and genotyped by URA5-RFLP, with the molecular types VNI (45.5 %) and VNIII (30.9 %) being the most commonly found ones. The molecular types VNII (11.4 %) and VNIV (11.4 %) were less abundant. One patient was found to be infected with a VGII isolate. This patient exhibited unusual clinical symptoms of cryptococcosis, reinforcing the suspicion for the presence of a different genotypic pattern, as determined afterwards. This case was detected in 2007 and is the first report of a potential autochthonous C. gattii infection case in Portugal, as the patient revealed no historical record of travelling outside the country.

Noninvasive fetal RHD genotyping from maternal plasma in an admixed Brazilian population.

We evaluated the efficacy of noninvasive fetal Rhesus D (RHD) genotyping from maternal plasma in a highly admixed population. Fifty-five blood samples from RhD-negative pregnant women from Brazil were processed for extraction of cell-free plasma DNA. Real-time PCR was performed to amplify segments of exons 5 and 7 from the RHD gene, as well as for detection of the SRY gene to confirm the presence of fetal DNA. Fetal genotyping results were compared with the RhD phenotype determined from newborn cord blood samples obtained at birth. Thirty-two samples were RHD-positive, 18 were RHD-negative and 5 were inconclusive due to amplification of only one RHD exon. In 43 samples, the fetal RHD genotype was compared to the neonatal RhD phenotype, and only one result was discordant, due to false-negative serology. There was one false SRY genotyping negative result. We conclude that noninvasive fetal RHD genotyping from maternal blood provides accurate results and suggests its viability as a clinical tool for the management of RhD-negative pregnant women in an admixed population.

The growth performance and parasite load of angelfish juveniles Pterophyllum scalare kept at different stocking densities in two rearing systems.

This study evaluated the growth performance and parasite load of angelfish juveniles Pterophyllum scalare kept at different stocking densities using two rearing systems. The experiment was conducted in a factorial design (4x2) with four stocking densities (0.1, 0.4, 0.7, and 1.0 g/L), two type of aquarium tanks (glass and ceramic aquariums), and four replicates. The experiment lasted 60 days using 148 juvenile fish (3.05 ± 0.09 g) randomly placed in 32 aquariums (50 L) equipped with filters and aeration. All fish received two meals a day ad libitum (8:00 and 16:00). Water quality parameters such as temperature, dissolved oxygen, pH, and total ammonia were measured. At the end of the experiment, all fish were measured and weighed to determine growth performance and then subjected to parasitological analysis. The data were analyzed with a two-way ANOVA with post-hoc Tukey test (p<0.05). No effects on growth performance at different stocking densities were observed. However, there was an increase in Capillaria pterophylli infestation in the high stocking density within ceramic aquariums. Thus, this study recommends the use of 1.0 g/L for the intensive aquaculture system of freshwater angelfish, and applying cleaning management to avoid parasite infestation, particularly in ceramic aquariums.

Parasitological diagnosis in ornamental freshwater fish from different fish farmers of five Brazilian states.

This study aimed to search parasites in 333 ornamental fish from five Brazilian states (Ceará, Minas Gerais, São Paulo, Paraná and Santa Catarina). Fish were sent from eight farms located in the municipalities of Fortaleza, Patrocínio do Muriaé, São Francisco do Glória, Cascavel, Timbó, Iguape, Jacareí and Mairinque. All fish received anesthesia earlier to euthanasia procedures. After the search for parasites, it was verified that 70.6% (235/333) of fishes were infected by at least one type of parasite, being 12 types of parasites identified: monogeneans, digenean metacercariae, cestodes, nematodes, Lernaea cyprinacea, trichodinids, Piscinoodinium pillulare, Ichthyophthirius multifiliis, diplomonad flagellates, Ichthyobodo sp., Chilodonella sp., and Tetrahymena sp. The proportion of infected fishes among the farms is compared through statistical tests, besides, animal handling adopted in each farm is also discussed. The importance of ensuring fish health in order to make the ornamental freshwater fish industry economically viable and reduce losses in production is highlighted.

Treatment time, pain experience and acceptability of the technique for caries removal in primary teeth using the ART approach with or without Brix3000™ papain gel: a preliminary randomised controlled clinical trial.

PURPOSE: To compare the Atraumatic Restorative Treatment (ART) associated with Brix3000™ to ART considering treatment time, pain experienced, and acceptability to children. METHODS: This study was accepted in Research Ethics Committee in July 2019 (number 3469402). Healthy patients (n = 20) aged 3-9 years, with at least one primary molar with occlusal dentine caries without cusp involvement were randomly allocated to either the ART + Brix3000™ group or the ART-only group. The sample was characterised by sex, age, tooth location and caries experience. Time spent and pain experience scores were recorded at prophylaxis, caries removal and restoration. The pain experience (intense, moderate, or mild) was evaluated by the Face, Legs, Activity, Cry, Consolability-revised scale (FLACC-r). Acceptability was assessed by a five-point hedonic facial scale (dichotomised into 'like' and 'indifferent/dislike' bins) and by an open-question interview. Mann-Whitney, Chi-square, and Fisher's exact tests were applied to discern differences in time, pain/sample characterisation and acceptability, respectively. RESULTS: The ART + Brix3000™ group required 8.6 ± 3.1 min to remove caries tissue, whereas the ART group required only 4.8 ± 2.0 min (p = 0.03). The total time spent with treatments was 13.1 ± 4.0 min for ART + Brix3000™, and 9.8 ± 2.7 min for ART (p = 0.03). There was no difference in pain experience and acceptability found among the groups (p > 0.05). CONCLUSION: Although the ART + Brix3000™ technique demanded more treatment time than the ART alone, there were no differences in either pain experience or acceptability.

Worldwide research trends on the use of chemical-mechanical caries removal products over the years: a critical review.

BACKGROUND: Chemical-mechanical caries removal (CMCR) products are in constant evolution and were recommended during the COVID-19 pandemic as substitutes for conventional caries removal. AIM: Characterize the worldwide scientific literature about CMCR products, over the years, by means of a critical review. DESIGN: Electronic search was performed on Medline/PubMed, Scopus, Web of Science, Cochrane Library, Lilacs, and Embase up to November 2020. Year, journal, country of authors, and type of study were the data extracted from the retrieved studies. Additional data of the clinical studies and systematic reviews were investigated. RESULTS: 2221 records were identified, 397 selected. 2011-2020 period concentrates higher number of publications (n = 169), in the Journal of Dental Research (n = 51), developed in Brazil (n = 45) and India (n = 44). Most studies were in vitro (n = 211) and clinical trials (n = 101). Carisolv™ (n = 48) and Papacarie Duo Gel™ (n = 33) were the most used products, prescript in isolated usage (n = 101), and compared with drills (n = 77). CMCR were more studied in primary teeth (n = 78), receiving glass ionomer cement (GIC) (n = 51) as restorative material. The most evaluated outcomes were time spent (n = 48) and pain (n = 41). Clinical application of CMCR takes more time than other techniques, but can also reduce patient anxiety, pain, and need for anesthesia. CONCLUSION: In vitro and clinical studies with CMCR products have been increasing, mostly carried out in developing countries, evaluating Carisolv™ and Papacarie Duo Gel™. Clinical studies tend to evaluate the time spent and pain compared to drills for removing caries in primary teeth, posteriorly restored with GIC. CMCR clinical application reduces anxiety, pain, and need for anesthesia, despite increase treatments' time.

Temperature effects on immune response and hematological parameters of channel catfish Ictalurus punctatus vaccinated with live theronts of Ichthyophthirius multifiliis.

This study evaluated the influence of temperature on the immune responses and hematological parameters in channel catfish Ictalurus punctatus immunized via intraperitoneal injection with live theronts of Ichthyophthirius multifiliis. Fish were distributed in 18 aquaria and received 9 treatments: 4 groups of fish were vaccinated with live theronts and maintained at constant temperature 15 °C, 20 °C, 25 °C and 30 °C; 3 groups of fish vaccinated and subjected to cycling temperature regime from 15-25 °C, 20-25 °C and 20-30 °C, changed 5 °C each day; 2 groups of fish were not vaccinated and served as controls at 25 °C, one with Ich challenge and the other without challenge. Non vaccinated fish and those vaccinated at 15 °C or 15-25 °C did not show anti-Ich antibodies in the serum 14 and 21 days post-immunization. The antibody levels were significantly higher from fish vaccinated at 25 °C, 30 °C, 20-25 °C and 20-30 °C compared to fish at 15 °C, 20 °C and 15-25 °C both 14 and 21 days post-immunization. At constant water temperature, fish vaccinated at 15 °C showed significantly higher mortality rate (67.8%, P < 0.05) than those vaccinated at 20 °C, 25 °C, and 30 °C (0-10.7% mortalities). At cycling water temperature, fish vaccinated at 15-25 °C showed significantly higher mortality rate (67.8%) than those vaccinated at 20-25 °C and 20-30 °C (P < 0.05). Twenty days after immunization fish vaccinated at 30 °C and 20-30 °C showed significant increase in the red blood cells, white blood cells, thrombocytes and monocytes. Six days after challenge with I. multifiliis theronts the fish showed decreased white blood cells, thrombocytes and monocytes. This study suggests that vaccinated catfish were severely impacted by low temperature, either at 15 °C constant temperature or at 15-25 °C cycling temperature. The fish showed no anti-Ich antibodies and suffered high mortality similar to non vaccinated control fish.

Infection levels of proteocephalidean cestodes in Cichla piquiti (Osteichthyes: Cichlidae) of the Volta Grande Reservoir, Minas Gerais, Brazil, relative to host body weight and gender.

We evaluated the relationship between infection by proteocephalid cestodes and the sex and weight classes of tucunaré (Cichla piquiti) captured between August 1999 and June 2001 in the Volta Grande Reservoir, Minas Gerais, Brazil. A total of 96 fish, 75.9 ± 9.3% males and 88.9 ± 6.4% females, were parasitized by Proteocephalus macrophallus and P. microscopicus, with total mean intensities of 76.6 ± 23.9 and 145.2 ± 36.7, respectively, during this period. In the majority of the months analysed, males showed 71.4-100% prevalence of parasitism and females 80-100%. Although there was no significant difference, females showed a higher mean intensity of infection (145.2 ± 36.7) than males (76.6 ± 23.9). Fish weighing 300-800 g showed a higher mean abundance of parasites (P < 0.05) compared with the biggest specimens weighing 801-2750 g. Analysing both males and females together, the greatest mean intensities of infection were found in October and December (P < 0.05) independent of the year, which coincides with the months of highest rainfall. These results show that fish living in reservoirs may be more susceptible to intermediate hosts than those that live in rivers.

Dynamics of Emim+ in [Emim][TFSI]/LiTFSI Solutions as Bulk and under Confinement in a Quasi-liquid Solid Electrolyte.

Quasi-liquid solid electrolytes are a promising alternative for next-generation Li batteries. These systems combine the safety of solid electrolytes with the desired properties of liquids and are typically formed by solutions of Li salts in ionic liquids incorporated into solid matrices. Here, we present a fundamental understanding of the transport properties in solutions of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) in 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([Emim][TFSI]), either in bulk form or incorporated in a boron nitride (BN) matrix. We performed a series of quasi-elastic neutron scattering experiments that, given the high incoherent neutron scattering cross section of hydrogen, allowed us to focus on the Emim+ dynamics. First, [Emim][TFSI]/LiTFSI solutions (0.5 and 2.5 mol·kg-1) were investigated and we show how the increase in the concentration reduces the Emim+ mobility and increases the activation energy of their long-range motions. Then, the 0.5 mol·kg-1 solution was incorporated into the BN matrix and we report that the diffusivities of the Emim+ cations that remain mobile under confinement are highly accelerated in comparison with the bulk sample and the activation energy of these motions is drastically reduced. We present the experimental evidence that this effect is related to the content of the Emim+ cations immobilized near the surfaces of the BN pores.

Development and validation of a UPLC-MS/MS method with a broad linear dynamic range for the quantification of morphine, morphine-3-glucuronide and morphine-6-glucuronide in mouse plasma and tissue homogenates.

The aim of this study was to develop and to validate a UPLC-MS/MS method for the quantification of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in mouse plasma and tissue homogenates to support preclinical pharmacokinetic studies. The sample preparation consisted of protein precipitation with cold (2-8 °C) methanol:acetonitrile (1:1, v/v), evaporation of the supernatant to dryness, and reconstitution of the dry-extracts in 4 mM ammonium formate pH 3.5. Separation was achieved on a Waters UPLC HSS T3 column (150 × 2.1 mm, 1.8 µm) maintained at 50 °C and using gradient elution with a total runtime of 6.7 min. Mobile phase A consisted of 4 mM ammonium formate pH 3.5 and mobile phase B of 0.1% formic acid in methanol:acetonitrile (1:1, v/v). Detection was carried out by tandem mass spectrometry with electrospray ionization in the positive ion mode. The method was validated within a linear range of 1-2,000 ng/mL, 10-20,000 ng/mL, and 0.5-200 ng/mL for morphine, morphine-3-glucuronide, and morphine-6-glucuronide, respectively. In human plasma, the intra- and inter-run precision of all analytes, including the lower limit of quantification levels, were ≤ 15.8%, and the accuracies were between 88.1 and 111.9%. It has been shown that calibration standards prepared in control human plasma can be used for the quantification of the analytes in mouse plasma and tissue homogenates. The applicability of the method was successfully demonstrated in a preclinical pharmacokinetic study in mice.