Targeted screening and quantification of synthetic cathinones and metabolites in hair by UHPLC-HRMS.

OBJECTIVE: Synthetic cathinones (SCs) are new psychoactive substances with sympathomimetic effects, which emerged into the illegal drug market to replace controlled stimulants. Since every year more powerful and toxic substances enter the illicit market, there is the need for analytical methodologies able to detect these new compounds in conventional and non-conventional biological matrices. We sought to develop and validate a targeted screening and quantification method for thirty-two parent SCs and two metabolites in hair samples by ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS). MATERIALS AND METHODS: 20 mg hair samples were soaked in 250 µL of 2 mM ammonium formate, methanol and acetonitrile mixture (50/25/25, v/v/v) and incubated overnight at 40°C. After incubation, the samples were evaporated to dryness under nitrogen stream and reconstituted with 100 µL of mobile phase mix (A:B, 80:20) and 10 µL were injected into UHPLC-HRMS. A Q ExactiveTM Focus Orbitrap Mass spectrometer with full scan and targeted data-dependent MS/MS scan acquisition was used for the screening and quantitation analysis. RESULTS: The assay was linear from 5 to 500 pg/mg hair for all the analytes under investigation. Intra-day and inter-day precision were always < 15% and matrix effect and analytical recovery were always within acceptable criteria (±25% and >50%, respectively). The developed method was applied to authentic hair samples from SCs consumers. The most prevalent found SCs were 3,4-Methylenedioxy-α-Pyrrolidinohexanophenone with a concentration range of 6.0-1,000.0 pg/mg along with α-Pyrrolidinohexiophenone (54.0 and 554.0 pg/mg, respectively), 3-Methylmetcathinone (556.0 and 5,000.0 pg/mg) and 4-Methylethcathinone (11.5 and 448.0 pg/mg) CONCLUSIONS: The developed method showed good selectivity, specificity, an easy and low-cost sample preparation and an analysis time compatible with a high throughput laboratory.

New Psychoactive Substances consumption and their monito- ring during Covid-19 pandemic.

ABSTRACT: Currently, the world is facing an unprecedent change of everyday life, due to the Covid-19 pandemic that has been affecting all the nations for more than one year. The public health systems were restructured in all the countries as a response to the constant emergency status, ne-glecting some services like toxicological analyses. In this scenario, the current spread of the New Psychoactive Substances is less controlled than before and the data on its expected mutation come from seizures analyses. Where the global distribution of drugs of abuse was affected by the restriction, fentanyl seizures did not drop during the pandemic. Moreover, new synthesis of fentanyl analogues resulted in new toxic adulterants as by products. Furthermore, diversion of benzodiazepines and new designer benzodiazepines were reported during the pandemic period. In this scenario, the scientific community and the international agencies should tighten their collaboration in order to monitor the emerging of new unknown substances.

Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat.

The induction of heme oxygenase-1 (HO-1) was studied in intact spinal cords and injured spinal cords after a moderate, thoracic contusion injury. HO-1 was immunolocalized in the normal cord and along the axis of the cord at 1, 2, 3 and 4 days after contusion. Induction of this enzyme in astrocytes and microglia/macrophages was evaluated using immunofluorescent double labeling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor. HO-1 was expressed in neurons in the normal spinal cord. After contusion, HO-1 was induced in both gray and white matter at the impact site. In segments of cord that were 1 cm proximal or distal to the injury, HO-1 was primarily induced in the dorsal columns and occasionally in the lateral white matter. This pattern of induction was noted at all time points. The HO-1 was induced primarily in microglia/macrophages. The distribution of the HO-1 positive cells closely correlated with the pattern of intraparenchymal hemorrhage. These findings demonstrate acute induction of HO-1 in non-neuronal cells in the injured spinal cord. Induction of HO-1 in glia may be a consequence of multiple factors including exposure to heme proteins, hypoxia and oxidative stress.

Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier.

The induction of the stress protein heme oxygenase-1 (HO-1) was studied in the rat brain after intracarotid administration of hyperosmolar mannitol. HO-1 was immunolocalized in fixed sections of brain 24 h to 7 days after injection. Immunoglobulin G (IgG) was immunolocalized in adjacent sections to demonstrate areas of breakdown of the blood-brain barrier. Induction of HO-1 was also evaluated by Western immunoblots, performed at 24 h after the insult. Immunofluorescent double labelling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor was used to determine if glia/macrophages expressed HO-1. There was pronounced, widespread induction of HO-1 in the ipsilateral hemisphere and cerebellum by 24 h both by immunocytochemistry and by Western blots. This induction was markedly attenuated at later times. HO-1 was induced in astrocytes and microglia/macrophages in the ipsilateral hemisphere. In addition, the protein was induced in Bergmann glia and scattered microglia/macrophages in the cerebellum. The mechanism of induction of HO-1 in glia after opening of the blood-brain barrier could include exposure to heme proteins, denatured proteins and other plasma constituents known to induce HO-1. This glial induction may reflect a protective response of these cells.

Prevalence of Trichomonas vaginalis in a male sexually transmitted disease clinic population by interview, wet mount microscopy, and the InPouch TV test.

OBJECTIVE: To determine the prevalence of trichomoniasis in male patients from their urine at a Sexually Transmitted Disease (STD) Clinic using the InPouch TV culture system. METHODS: Two hundred and four patients were examined for STD infections. Their ages ranged between 17 and 72 years. Depending on their clinical symptoms tests were ordered for Neisseria gonorrhoeae, chlamydia, and for syphilis. Each patient submitted a clean catch urine specimen for trichomonas testing. A 15 ml aliquot of urine was centrifuged and a drop of the sediment examined microscopically. The remainder was cultured in the InPouch TV test. Each pouch was examined at 24 h, 48 h, and 5 days. RESULTS: Twenty-four of the 204 patients (12%) were culture positive for Trichomonas vaginalis and only three of these were wet mount positive. CONCLUSION: The InPouch TV test demonstrated an epidemiological important infected male population that was not indicated by wet mount microscopy.

A clinical evaluation of trichomoniasis in San Jose, Costa Rica using the InPouch TV test.

OBJECTIVE: to determine the prevalence of trichomoniasis in San Jose, Costa Rica, comparing two methods, the InPouch TV test and the saline wet mount. METHODS: One hundred symptomatic and asymptomatic female patients at two hospitals and at a sexually transmitted disease (STD) clinic were evaluated. Vaginal discharge was the most prevalent genitourinary abnormality among symptomatic patients. The patients were between 18 and 70 years old. Fifty-seven were from the STD clinic, 43 from the two hospitals. A saline wet mount and a culture were taken from each patient. The culture employed a new procedure for diagnosis of trichomonads, the InPouch TV test (BioMed Diagnostics, San Jose, CA). RESULTS: Thirteen of the 100 patients were culture positive, two of whom were wet mount positive. No wet mount positives were culture negative. Eleven of the positive tests were from the STD clinic and two were from the hospitals. CONCLUSIONS: The results of this initial epidemiologic study indicate a prevalence of 19% for trichomoniasis in the STD clinic population and 4.6% in the hospitals group. Trichomonas vaginalis was not diagnosed by laboratory methods prior to this study. The InPouch TV test has a selective fungicidal and bactericidal, enriched proteose-peptone medium which provides a sensitivity of 4 organisms per ml and a 1 year shelf life at room temperature. This in vitro culture test demonstrated unique capabilities as a transport and culture medium. Its procedure offers simplicity in application and an excellent visualisation of trichomonads.