Classic advanced vortex mixer

Several NE formulations loaded with FA were developed according to the method previously reported by Elsewedy et al., using the quantified amounts of ingredients as presented in Table 1. Primarily, 500 mg of FA was dissolved in various concentrations of myrrh essential oil and Transcutol^® P, and subjected to vortexing using a classic advanced vortex mixer (VELP Scientifica, Usmate, Italy) to provide the oily phase. Afterwards, different concentrations of surfactant (Tween 80) were added to distilled water, representing the aqueous phase, and were vortexed well. The aqueous phase was gradually added to the oily phase, homogenized using a high-shear homogenizer (T 25 digital Ultra-Turrax, IKA, Staufen, Germany), and kept for 15 min at 20,000 rpm. Following the homogenization process, the emulsion developed instantly, and was exposed to 30 s of sonication using a probe sonicator (XL-2000, Qsonica, Newtown, CT, USA) until homogeneous NE was obtained [50 (link)].

Prior to analysis, samples were thawed and thoroughly mixed (Classic Advanced Vortex Mixer, Velp Scientifica, Italy). Then, 50 µL of each sample was treated with 10 µL of sodium metabisulfite solution, 950 µL of mobile phase B, 2.5 µL of deuterated histamine d4 (internal standard, IS), at a concentration of 1 µg/mL and 2.5 µL of a 1 µg/mL deuterated serotonin d4 solution, mixed and centrifuged (Centrifuge 5417R, Eppendorf, Germany) at RCF 24,104 × g for 10 min. Next, 500 µL of supernatant was taken for LC–MS/MS analysis. Deuterated histamine and deuterated serotonin solutions were prepared by diluting stock standard solutions in methanol (Serotonin d4 hydrochloride, Toronto Research Chemicals, Canada, and Histamine- α,α,β,β d4 dihydrochloride Sigma Aldrich, US).