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A selective, highly sensitive, and robust method for determination of a large panel of fentanyls and naloxone in blood was developed and validated. Naloxone was included to monitor use and efficacy of the opioid antidote in cases of fentanyl overdoses. The method demonstrated good ability to separate structural isomers, which is important to differentiate between the numerous available fentanyls with variable potency, toxicity, and legal status. The developed method can be used to identify fentanyls on the drug market to help combat the fentanyl crisis.

The current opioid overdose crisis is being exacerbated by illicitly manufactured fentanyl and its analogs. Carfentanil is a fentanyl analog that is 10,000-times more potent than morphine, but limited information is available about its pharmacology. The present study had two aims: 1) to validate a method for quantifying carfentanil and its metabolite norcarfentanil in small-volume samples, and 2) to use the method for examining pharmacodynamic-pharmacokinetic relationships in rats. The analytical method involved liquid-liquid extraction of plasma samples followed by quantitation of carfentanil and norcarfentanil using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was validated following SWGTOX guidelines, and both analytes displayed limits of detection and quantification at 7.5 and 15 pg/mL, respectively. Male Sprague-Dawley rats fitted with jugular catheters and temperature transponders received subcutaneous carfentanil (1, 3 and 10 μg/kg) or saline. Repeated blood specimens were obtained over 8 h, along with pharmacodynamic measures including core temperature and catalepsy scores. Carfentanil produced dose-related hypothermia and catalepsy that lasted up to 8 h. Carfentanil C max occurred at 15 min whereas metabolite C max was at 1-2 h. Concentrations of both analytes increased in a dose-related fashion, but area-under-the-curve values were much greater than predicted after 10 μg/kg. Plasma half-life for carfentanil increased at higher doses. Our findings reveal that carfentanil produces marked hypothermia and catalepsy, which is accompanied by nonlinear accumulation of the drug at high doses. We hypothesize that impaired clearance of carfentanil in humans could contribute to life-threatening effects of this ultrapotent opioid agonist.

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Our study shows that MPA is found in DUID cases and reveals that NPS are used among drivers and also proven in blood from drivers involved in traffic accidents. More studies are requested regarding the pharmacological and toxicological effects of MPA and other NPS. This is the first article that describes a method for analyzing and quantifying MPA in whole blood samples.

Background: The opioid analgesic fentanyl and its analogues pose a major health concern due to its high potency and increasing number of overdose deaths worldwide. The analogues of fentanyl may differ in potency, toxicity and legal status and it is therefore important to develop analytical methods for correct identification. This can be challenging since many fentanyl analogues are structural isomers. Two fentanyl isomers that have been in the spotlight lately due to difficulties regarding separation and identification are cyclopropylfentanyl and crotonylfentanyl, which have been reported to display nearly identical fragmentation patterns and chromatographic behavior. Methods: Chromatographic separation of cyclopropylfentanyl and crotonylfentanyl by UHPLC was investigated using three different stationary phases (HSS T3, BEH C18 and Kinetex biphenyl) employing gradient elution with a mobile phase consisting of 10 mM ammonium formate pH 3.1 and MeOH. Detection was performed by MS/MS. In addition, the major metabolites of the two compounds formed upon incubation with human liver microsomes (HLM) were identified by UHPLC-QTOF-MS analysis. Results: Baseline separation of cyclopropylfentanyl and crotonylfentanyl was achieved on the BEH C 18 column with retention times of 6.79 and 7.35 min, respectively. The major metabolites of the two analogues formed by HLM differed, with the main biotransformation being N-dealkylation and carboxylation for cyclopropylfentanyl and crotonylfentanyl, respectively. We demonstrated the usefulness of the two approaches by distinguishing between cyclopropylfentanyl and crotonylfentanyl in two authentic post mortem blood samples. Conclusions: In the present study we successfully demonstrated that cyclopropylfentanyl and crotonylfentanyl can be distinguished by methods commonly available in forensic laboratories.

Background: A decline of brain serotonin (5-HT) is held responsible for the changes in mood that can be observed in Alzheimer’s disease (AD). However, 5-HT’ergic signaling is also suggested to reduce the production of pathogenic amyloid-4β (Aβ). Objective: To investigate the effect of targeted inactivation of tryptophan hydroxylase-2 (Tph2), which is essential for neuronal 5-HT synthesis, on amyloidosis in amyloid precursor protein (APP)swe/presenilin 1 (PS1) ΔE9 transgenic mice. Methods: Triple-transgenic (3xTg) APP/PS1 mice with partial (+/-) or complete Tph2 knockout (–/–) were allowed to survive until 6 months old with APP/PS1, Tph2–/–, and wildtype mice. Survival and weight were recorded. Levels of Aβ 42/40/38, soluble APPα (sAβPPα) and sAβPPβ, and cytokines were analyzed by mesoscale, neurotransmitters by mass spectrometry, and gene expression by quantitative PCR. Tph2, microglia, and Aβ were visualized histologically. Results: Tph2 inactivation in APP/PS1 mice significantly reduced viability, without impacting soluble and insoluble Aβ 42 and Aβ 40 in neocortex and hippocampus, and with only mild changes of soluble Aβ 42/Aβ 40. However, sAβPPα and sAβPPβ in hippocampus and Aβ 38 and Aβ 40 in cerebrospinal fluid were reduced. 3xTg–/–mice were devoid of Tph2 immunopositive fibers and 5-HT. Cytokines were unaffected by genotype, as were neocortical TNF, HTR2a and HTR2b mRNA levels in Tph2–/– mice. Microglia clustered around Aβ plaques regardless of genotype. Conclusion: The results suggest that Tph2 inactivation influences AβPP processing, at least in the hippocampus, although levels of Aβ are unchanged. The reduced viability of 3xTg–/–mice could indicate that 5-HT protects against the seizures that can impact the viability of APP/PS1 mice.