The development and validation of a method to quantify brorphine in saliva by using FPSE and LC-MS/MS.

Brorphine (3-[1-[1-(4-bromophenyl)ethyl]-4-piperidyl]-1H-benzimidazol-2-one) is a substituted piperidine benzimidazolone analogue that retains some structural similarities to fentanyl. It was first identified on the NPS market in the United States in August 2019 and reported from Sweden to the EMCDDA in June 2020. Occasionally it was sold under the street name “purple heroine” and had contributed to hundreds of deaths and adverse events.

FPSE was used for brorphine extraction from saliva. One mL oral fluid after centrifugation and protein precipitation with acetonitrile, was sorptived onto the PEG 300 fabric for 30min under gentle stirring. Back extraction from the FPSE matrix was performed with 150μL methanol for 10min under vortex. Targeted analysis for brorphine was performed on a Dionex UHPLC system (Thermo Scientific) coupled to a QTrap 5500+ mass spectrometer (Sciex, Darmstadt, Germany) in positive scheduled multiple reaction monitoring (sMRM) mode using a Accupore C18, 2.6μm, 50×3mm, (Thermo Scientific) equipped with the respective guard column. The mobile phase consisted of a 10mM NH4Ac, 0.1% formic acid in water and acetonitrile, 0.1% formic acid as organic eluent. The autosampler was operated at 4°C, the injection volume was 5μL and a gradient elution of three steps, starting from 12%B to 100% and return to initial conditions resulting in a total run time of 6.5min was applied. The flow rate was 0.5mL/min, and the column oven was maintained at 30°C. The following ESI inlet conditions were applied: gas 1/2, nitrogen (90 psi; 620.5kPa); ion spray voltage, 5500V in positive mode; ion-source temperature, 550°C; curtain gas, nitrogen (55 psi; 379.2kPa). The MRM detection window was set at 8s, and the target scan time was 0.2s, resulting in at least 8–12 points across the peak above half height. Three MRMs per analyte were monitored as per internationally accepted guidelines. The method, validated through the calculation of all analytical parameters in accordance to International Guidelines, (SWGTOX) in terms of accuracy, sensitivity, selectivity, intra- and interday precision and stability. Quantification of brorphine was performed using spiked saliva donated from volunteers. Seven calibrators were prepared at concentrations of 0.05, 0.25, 1, 5, 10, 25, 50ng/mL (R²=0.9993). Each validation parameter was replicated for three concentration levels, at 0.25, 0.5, 50ng/mL (low, medium and high level).

In a series of preliminary experiments the recovery of brorphine from saliva with different extraction mixtures was tested. Limit of detection (LOD) and limit of quantification (LOQ) were 0.01ng/mL and 0.05ng/mL, respectively. Extraction recoveries ranged from 65 to 75%; intra-/inter-day precision (% coefficient of variation) was in the range 6.4–9.9% and accuracy was less than 12%, for the medium and high concentration levels. Post-preparative stability of analyte was higher than 95%. No interferences were observed in the selectivity experiments and no carry over was measured between analyses.

This relatively simple methodology is effective and reliable for routine identification and quantitation of brorphine in saliva in clinical casework.

Brorphine acts as a strong μ-opioid receptor agonist and has been involved in many serious intoxications. The identification of brorphine has not been previously reported in saliva. Although, we are not aware of any brorphine case in Greece yet, the reported implication in emergent intoxication cases and the possibility of widespread use, stress the importance of being able to detect this potent NPS in clinical cases.