Chemical identification and optimization of the 4-aminophenol colorimetric test for the differentiation between hemp-type and marijuana-type cannabis plant samples.

The 4- Aminophenol (4-AP) colorimetric test is a fast, easy-to-use, and cost-effective presumptive assay of cannabis plant material producing different chromophores with THC-rich cannabis (blue color) and with CBD-rich cannabis (pink color). The main drawback of the 4-AP test is a brief observation window where the color rapidly changes to black, limiting the utility of the test. We now report for the first time, the identification of the product chromophores between 4-AP and CBD/THC as well as propose an explanation and a solution for the color degradation of the chromophores. The identification of the chromophores is provided by spectroscopic (UV-Vis), chromatography, and mass spectrometry (TLC and LC-QToF-MS). Oxidation of excess 4-AP (Reagent A) in the presence of NaOH (Reagent B) produces the black color observed for the previously reported 4-AP tests and reported in the literature. The adjustment of reactants concentrations and volumes of 4-AP:THC/CBD to a 1:1 ratio significantly reduces the black oxidation by-product and increases the observation window up to 2 h instead of the previously reported 5-10 min. For the first time, mass spectrometry and chromatography confirmed that the reaction of THC and CBD with 4-AP produced chromophores with m/z (M + H) = 420, consistent with proposed indophenol structures. The TLC method developed confirmed the separation between CBD and THC chromophores. The specificity of the test is also reported, showing false positive results for the presence of THC (blue color) for samples of thyme and oregano. LDA and SIMCA models showed that the optimized 4-AP procedure performs better than the previously reported 4-AP color test.

Healing activity of hydrogel containing nanoemulsified ß-caryophyllene.

ß-caryophyllene is a sesquiterpene present in the oil of many plant species, such as Copaifera sp., which has been shown to possesses potent anti-inflammatory action; however, its healing activity remains under study. The objectives of the present study were to produce a nanoemulsion containing ß-caryophyllene followed by a hydrogel containing nanoemulsified ß-caryophyllene, to evaluate the permeation profile in vitro, and to assess the in vivo healing activity, which is so far unexplored in the literature for pure ß-caryophyllene and in pharmaceutical formulation. The nanoemulsion was obtained through high-pressure homogenization and the hydrogel by direct dispersion with hydroxyethylcellulose. Both formulations were characterized according to droplet size, polydispersity index, volume-weighted mean diameters, particle distribution, droplets diameters tracking, zeta potential, viscosity and bioadhesion behavior. ß-caryophyllene content was determined by gas chromatography coupled with mass spectrometry (GC/MS). Both formulations presented a nanometric droplet size, negative zeta potential, high ß-caryophyllene content, and were stable for 60 days. In agreement with the viscosity results, the hydrogel containing the ß-caryophyllene nanoemulsion showed superior bioadhesiveness than the nanoemulsion. The skin permeation study in Franz cells demonstrated that isolated ß-caryophyllene was unable to cross the stratum corneum and that its nanoemulsification promoted its permeation. On the other hand, in the simulated deeply wounded skin (dermis), no significant differences were observed between the formulations and isolated ß-caryophyllene with respect to the amount of marker retention in the dermis, suggesting saturation of this skin layer. For the study of healing activity, the dorsal wound model was performed with an evaluation of the lesion size, anti-inflammatory markers, and antioxidant activity. The initial closure of the wound was achieved sooner in the group treated with the hydrogel containing the ß-caryophyllene nanoemulsion, indicating its anti-inflammatory effect. The histological analysis indicated that on day 12 day of the lesion, the hydrogel presented similar results to those of the positive control group (Dersani® oil), proving effectiveness in cutaneous tissue repair.

Comparison between counterfeit and authentic medicines: A novel approach using differential scanning calorimetry and hierarchical cluster analysis.

Erectile dysfunction medicines such as Cialis and Viagra are very popular worldwide and are between the most prevalent counterfeit medicines in Brazil. A range of analytical methods has been used to analyze Cialis and Viagra, such as ATR-FTIR, GCMS and UPLC-MS. Until now, there are no data available of DSC methods for analysis of counterfeit medicines of Cialis and Viagra. DSC is a thermal analysis that provides useful information of physico-chemical events, and however is almost not used for forensic purposes. In this study, thermal analysis of 25 counterfeit Viagra and Cialis seized by Brazilian Federal Police were performed by DSC and compared to their authentic medicines and analytical standards, along with chemometric tools. Authentic samples of Viagra displayed a similar thermal profile with the API, while Cialis were different with additional endothermic peaks, that could be related to excipients interference. Thermograms of Viagra counterfeit samples were similar to authentic samples, while Cialis showed an enlargement and displacement of endothermic peaks. Also, some Cialis counterfeit samples showed melting peaks attributed to sildenafil, the API of Viagra, instead tadalafil, confirming previous results obtained by UPLC-MS. Multivariate analysis with application of Hierarchical Cluster Analysis classified different groups of samples, including a cluster with counterfeit Cialis and Viagra, indicating the use of same API for both counterfeit medicines and possibly the same illicit production; and a cluster with authentic Viagra and counterfeit Cialis, confirming the addition of sildenafil instead tadalafil to Cialis counterfeit samples. Here for the first time we described the use of DSC for chemical profiling of Cialis and Viagra and showed that even when applied to a small group of samples, DSC along with chemometric tools can be considered as a good auxiliary method in forensic casework samples. DSC provided useful data to perform the identification of counterfeit and authentic medicines, with low cost and a simple method.

Citrus essential oils inhalation by mice: Behavioral testing, GCMS plasma analysis, corticosterone, and melatonin levels evaluation.

The use of orange essential oils (EOs) as a complementary treatment is very common in Brazilian popular culture. The levels of melatonin (MEL) and corticosterone (CORT) hormones were investigated simultaneously, by the Luminex™ immunoassay system in mice plasma, after Citrus aurantium and Citrus sinensis EOs inhalation for 30 min. The plasma was analyzed by headspace through gas chromatography coupled to mass spectrometry for investigation of the EO components. Mice were submitted to behavioral testing to research anxiolytic-like, sedative, and antidepressant-like effects. The inhalation of atmosphere obtained from vaporization of 10% solution of this Citrus EO separately did not affect MEL or CORT plasma levels; that is, the MEL and CORT levels did not present variation in function of the EO in the schedule used. On the other hand, the imipramine positive control used altered the level of MEL as expected. The EO constituents were detected in plasma at different ratios that is present in inhaled EO. Behavioral tests showed that the inhalation of 10% C. sinensis EO presents an anxiolytic-like and sedative effect. Thus, C. sinensis EO can be a valuable tool for treatment of the anxiety disturbs, apparently without interference with MEL and CORT physiological levels.

Prescription and illicit psychoactive drugs in oral fluid--LC-MS/MS method development and analysis of samples from Brazilian drivers.

This study is part of a larger project designed to investigate the prevalence of psychoactive drug (PAD) use among Brazilian drivers. In this paper we describe the development and validation of an analytical method to analyze 32 prescription and illicit PADs (amphetamines, benzodiazepines, cocaine, cannabis, opioids, ketamine and m-CPP) and metabolites in oral fluid samples collected with a Quantisal™ device. Samples were extracted with ethyl acetate:hexane and analyzed by LC-MS/MS. Instrumental LOD ranged from 0.26 to 0.65 ng/mL. Mean procedural recoveries at 1.3 ng/mL (LLOQ) ranged from 50% to 120% for 24 compounds. Recoveries were concentration independent, with the exception of femproporex, heroin and ecgonine methyl-ester (EME) for which the recovery decreased significantly at higher levels (13 and 52 ng/mL). RSD was <20% for all compounds at all spiking levels. Ion suppression due to the matrix was <20% for most compounds, and higher than 60% for EME and diethylpropion. Analysis was performed against a in-matrix standard curve. About 10% of the 2235 oral fluid samples collected from drivers on Brazilian Federal highways were positive (≥LOD) for at least one analyte investigated. Alone or in combination with other drugs, cocaine/metabolites were the analytes most detected in the samples (129; 5.8%), followed by amphetamines/metabolite (69; 3.1%), benzodiazepines (28; 1.2%), cannabinoids (23; 1.1%) and opioids (8; 0.4%). Detection of at least two PADs from different classes accounted for 9.3% of the 236 positive samples. Cocaine was found at higher levels in the samples (up to 1165 ng/mL). Preventive measures aimed at reducing the use of PADs by drivers in Brazil will certainly contribute to decrease the country's highway death rates.