Api 3000

To validate that there was a dose-dependent change in CBD levels, serum levels of cannabinoids were analyzed [22 (link)]. In brief, methanolic extracts of 90 µl of serum were partially purified on C18 solid phase extraction columns (Zorbax) and eluants were analyzed using HPLC/MS/MS (API 3000, Applied Biosystems). Deuterium-labeled anandamide elutes in the same fraction as CBD and was used as an internal standard to monitor recovery. Levels of CBD and THC were analyzed using standard curves with Analyst Software as previously described [22 (link)]. During analysis it was discovered that each of the samples contained a small fraction THC in addition to CBD. This can occur during synthesis and is often unknown if the levels are not analyzed. The plasma ratio in each dose was ca 25:1 CBD:THC (Additional file 1: Figure S3B).

After filtration, tartaric acid was analyzed using an Atlantis TE C18, 100 mm × 2.1 mm, 3 µm (Waters, Milford, MA, USA) reversed-phase column coupled for detection to the triple quadrupole mass spectrometer API 3000 (Applied Biosystems, Foster City, CA, USA). The mass spectrometer was operated in negative electrospray ionization mode. The column was maintained at 25 °C throughout the analysis. Mobile phases A and B were 0.5% formic acid in water and 0.5% formic acid in acetonitrile, respectively. The following linear gradient was used: holding at 100%A for 3.5 min, decrease to 10%A over 2 min and holding for 2 min, return to initial conditions for 1.5 min, and re-equilibration for 6 min. The flow rate was set at 350 µL/min and the injection volume was 10 µL. Post-column addition of acetonitrile (250 µL/min) was carried out to improve analyte ionization efficiency. The detection was accomplished in multiple reaction monitoring (MRM) mode, and the following MS/MS transitions were used for quantification and confirmation, respectively: m/z 149/87 and m/z 149/73 for tartaric acid, and m/z 151/88 and m/z 151/74 for the deuterated isotope.

Erinacine S concentration in the samples were analyzed using Agilent 1100 series LC system equipped with a G1376A capillary pump and a G1313A autosampler, according to a previous published method [16 (link)]. Chromatographic separation was performed on an Agilent Eclipse XDB-C18 column (3.5 µm, 4.6 × 100 mm), using a mobile phase consisting of water (A) and acetonitrile (B), at a flow rate of 0.35 mL/min. The injection volume was 10 μL and the gradient elution program was as follows—0 min, 70% B; 0–5 min, 70–100% B; 5–8 min, 100% B; 8–8.1 min, 100–70% B; and 8.1–11 min 70% B. Mass spectrometric detection was carried out on a triple quadrupole mass spectrometer (API 3000; Applied Biosystems, Vaughan, Ontario, Canada), using Turbo Ion spray as source, coupled with electro spray ionization interfaces, operating in a negative ion mode and ion spray voltage at −4500 V. The source turbo spray consisted of a nebulizer gas at 10 psi, collision gas at 2 psi, curtain gas at 7 psi, with a source temperature at 275 °C. Multiple reaction monitoring (MRM) mode was employed for the quantification—m/z 429.3 → 411.3 for Erinacine S (rt = 8.7 min), and m/z 121 → 91.9 for 4-hydroxybenzaldehyde (internal standard). Data processing was performed with the Analyst 1.4.2 software (Applied Biosystems, Concord, ON, Canada).