Towards a microfluidic H295R steroidogenesis assay-biocompatibility study and steroid detection on a thiol-ene-based chip.

The development of cell-based microfluidic assays offers exciting new opportunities in toxicity testing, allowing for integration of new functionalities, automation, and high throughput in comparison to traditional well-plate assays. As endocrine disruption caused by environmental chemicals and pharmaceuticals represents a growing global health burden, the purpose of the current study was to contribute towards the miniaturization of the H295R steroidogenesis assay, from the well-plate to the microfluidic format. Microfluidic chip fabrication with the established well-plate material polystyrene (PS) is expensive and complicated; PDMS and thiol-ene were therefore tested as potential chip materials for microfluidic H295R cell culture, and evaluated in terms of cell attachment, cell viability, and steroid synthesis in the absence and presence of collagen surface modification. Additionally, spike-recovery experiments were performed, to investigate potential steroid adsorption to chip materials. Cell aggregation with poor steroid recoveries was observed for PDMS, while cells formed monolayer cultures on the thiol-ene chip material, with cell viability and steroid synthesis comparable to cells grown on a PS surface. As thiol-ene overall displayed more favorable properties for H295R cell culture, a microfluidic chip design and corresponding cell seeding procedure were successfully developed, achieving repeatable and uniform cell distribution in microfluidic channels. Finally, H295R perfusion culture on thiol-ene chips was investigated at different flow rates (20, 10, and 2.5 µL/min), and 13 steroids were detected in eluting cell medium over 48 h at the lowest flow rate. The presented work and results pave the way for a time-resolved microfluidic H295R steroidogenesis assay.

New synthetic opioid cyclopropylfentanyl together with other novel synthetic opioids in respiratory insufficient comatose patients detected by toxicological analysis.

Introduction: Fentanyl derivatives like cyclopropylfentanyl have recently appeared on the recreational drug market. Cyclopropylfentanyl is probably a highly potent opioid, but human toxicological data are not available so far. Similar to other fentanyl derivatives the most serious acute health risk due to the use of cyclopropylfentanyl is likely to be respiratory depression. In case of overdose, this may lead to apnoea, respiratory arrest and death. In this paper, we present three cases of severe intoxication with cyclopropylfentanyl. Methods: Observational case series including three intoxications treated in the Emergency Department at the University Medical Centre in 2017. In all cases, the consumption of any drugs was denied by the patients and relatives. Toxicological analyses using GC-MS, LC-QTOF-MS and LC-MS-MS of serum, urine samples and in one case of a powder sample, found in the hospital room, were performed. Medical records were reviewed to obtain clinical data. Results: Clinical effects of severe opioid intoxications comprising loss of consciousness, bradypnea, hypercapnia, arterial hypotension and miosis were recorded. In all cases, the novel fentanyl analogue cyclopropylfentanyl was detected in body fluids. In two cases further synthetic opioids (U-47700, methoxyacetylfentanyl, butyrfentanyl, 2-fluoroiso- or 4-fluoroisobutyrfentanyl) and mitragynine or desoxypipradrol were found. A discovered powder sample contained cyclopropylfentanyl, cyclopropylnorfentanyl, acetylfentanyl, 4-ANPP, U-47700 and caffeine. Except for acetylfentanyl all ingredients could be detected in the respective blood and urine sample. In two cases a cyclopropylfentanyl serum concentration of 51 and 76 ng/ml was determined. Discussion: In three cases of severe potentially life-threatening intoxication, cyclopropylfentanyl was verified using different analytical procedures. The ingested substance, as well as the excreted metabolites, were detected by application of various mass spectrometric techniques. Conclusions: In cases of intoxication without a medical history, the detailed toxicological analysis may reveal new psychoactive substances which are not detected by standard toxicological screening approaches. The high pharmacological potency of new products with unknown toxicological data and unknown synergistic effects may easily lead to a life-threatening overdose.