Agilent 5975 ms

An Agilent 7890 GC equipped with an Agilent 5975 MS (Agilent Technologies, Inc. CA, USA) was used for the detection of MPs. After extraction, injection was conducted in splitless mode with an 8-min thermal desorption at 250 °C. The separation was carried out on a HP-INNOWAX capillary column (60 m × 0.25 mm id, 0.25 μm film thickness, J&W Scientific, Folsom, CA, USA). The flow rate of carrier gas, helium, was 1 mL/min. The GC temperature program was as follows: holding at 50 °C for 1 min, increasing at a rate of 3.0 °C/min to 110 °C, and increasing at a rate of 1.5 °C/min to 131 °C. The after run temperature was set as 220 °C for 10 min. The MSD transfer line heater was set at 250 °C, and the temperature of ion source and quadrupole were 250 °C and 150 °C, respectively. The MSD was operated in electron ionization (EI) mode at 70 eV and selected ion monitoring (SIM) with the selected mass channels for IPMP (m/z 137, 152), SBMP (m/z 124, 138, 151), and IBMP (m/z 124, 151, 166), with a dwell time of 100 ms. The quantification ions for IPMP, SBMP, and IBMP were m/z 137, 124, and 124, respectively.

Malondialdehyde (MDA) was first extracted from 10 to 15 µL of plasma following the protocol described in Eikenaar et al. (2016) (link). Briefly, samples were vortexed with 50 µL buffer (1 mM O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine hydrochloride in 1.5 M sodium acetate buffer pH 5.0) and incubated at room temperature for 1 h (with vortexing at 30 min). To this, 300 µL of heptane with internal standard (1.57 fg µL⁻¹ 1-bromo-3-fluorobenzene) was added. Following vortexing, the lower phase was carefully removed by pipette, leaving the upper phase containing MDA. Extracts subsequently went through two to three washing steps: 200 µL distilled water was added, followed by vortexing and removal of the lower phase. Residual water was removed by the addition of anhydrous sodium sulfate. Extracts were finally dried under nitrogen gas, leaving a final volume of 40–50 µL. MDA was then quantified by gas chromatography–mass spectrometry (GC/MS) using an Agilent 5975 MS coupled to an Agilent 6890 GC with a non-polar capillary column, HP-5MS (30 m, 0.25 mm id, df 0.25 µm; J&W Scientific, USA). The GC oven was programmed to 60°C for 1 min, followed by 15°C/min to 150°C, and then 10°C/min to 270°C, which was held for 5 min.

CBD extracts were analyzed by gas chromatograph (GC)/mass spectrometry (MS) using Agilent 6890N GC and Agilent 5975 MS (Agilent Technologies Inc., Santa Clara, CA, USA) with a Restek Rxi-5Sil MS with integra guard column (15 m, 0.250 mmID, 0.25 µm df) (Restek Corporation, Bellefonte, PA, USA). A solution of Restek Qualitative Retention Time Index Standard (Restek Corporation, Bellefonte, PA, USA) was used to create the retention time index. The temperature of the injection port was set at 280 °C and the helium gas flow was constant at 1.1 mL/min. The samples were injected in split mode (2:1) with a volume of 1 µL of sample. The GC oven temperature was programmed as follows: initial temperature of 70 °C for 4 min, ramp to 200 °C at 20 °C/min, ramp to 300 °C at 8 °C/min, ramp to 325 °C at 50 °C/min with a 5 min hold, thus requiring a total run time of 30.5 min. The MS transfer line was set to 250 °C, source to 230 °C, and quads to 150 °C. The raw data were processed and analyzed using Agilent Enhanced ChemStation software (Agilent Technologies Inc., Santa Clara, CA, USA). The NIST 2.0 library was used with AMDIS for compound identification.