Acclaim c18 column

Five-milliliter blood samples were collected by brachial venipuncture into amber tubes with gel, centrifuged, and stored in dry ice at a temperature of −20 °C for transportation to IMIP’s translational research laboratory. Serum retinol measurement (a dependent variable) was analyzed using high-performance liquid chromatography (HPLC), as recommended by the WHO [9 ,19 ], according to the technique established by Furr et al. [20 ]. The analysis was performed using a Dionex UltiMate 3000 HPLC system (Sunnyvale, CA, USA) with an Acclaim C18 column (5 µm × 4.6 mm × 250 mm) coupled with a C18 Nucleosil guard column, and an injection volume of 20 µL. Detection was performed using a UV detector (Dionex UltiMate 3000 Variable Wavelength Detector) at 325 nm. Retinyl acetate obtained from Sigma was used as a standard, peak retention was 3.6 min, and the mobile phase flow was 1.5 mL/min. The cut-off points used for the classification of retinol levels were: deficient: <0.35 μmo/L; low: ≥0.35 and <0.70 μmol/L; acceptable: ≥0.70 and <1.05 μmol/L; and adequate ≥1.05 μmol/L [14 ]. VAD was defined as retinol levels <0.70 μmol/L, as recommended by the WHO [9 ,19 ]; therefore, the cumulative prevalence of vitamin A levels classified as deficient or low (serum retinol levels <0.70 μmol/L) was adopted as the endpoint for the univariate and multivariate analyses.

Erez extract (15% THC) and Avidekel extract (30% CBD) were provided by Tikun Olam Ltd. (Tel-Aviv, Israel). Avidekel and Erez oil preparations used in the study were analyzed for phytocannabinoids (Supplementary Figures S1 and S2, respectively) and terpenes (Supplementary Tables S2 and S3, respectively). CoA Terpene profile was provided by Tikun Olam Ltd. (Tel-Aviv, Israel), and phytocannabinoid analysis was performed by the laboratory for the Mass Spectrometry and Chromatography, the Hebrew University, Rehovot, Israel. Briefly, quantitative analyses of phytocannabinoids in plants and cannabis preparations were carried out using the Dionex Ultimate 3000 RS HPLC, which consisted of a quaternary pump, autosampler, column department, and diode-array detector. Analytes were separated on Acclaim C18 column (250 × 3 mm, 3 µm, Dionex, Sunnyvale, CA, USA) using acetonitrile/water with 0.1% acetic acid gradient. Standards of phytocannabinoids used for identification and quantitative analysis were purchased from Restek (Bellefonte, PA, USA).

One microlitre of sample (1 μl) was analysed on a UHPLC system of the Ultimate 3000 series RSLC (Dionex, Dionex Softron GmbH, Germering, Germany) connected to a Q Exactive Hybrid Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Separation in the UHPLC system was achieved on an Acclaim C18 column (150 × 2.1 mm, 2.2 μm, Dionex) with a flow rate of 300 μl min⁻¹ in a binary solvent system of water and acetonitrile (Merck, hypergrade for LC‐MS), both containing 0.1% (v/v) formic acid (eluent additive for LC‐MS, Sigma‐Aldrich). ESI source parameters were set to 3 kV for spray voltage at a sheath gas flow of 35 and Aux gas flow of 7 l h⁻¹. The voltage in the transfer capillary was set to 35 V at a capillary temperature of 325°C. The samples were measured in positive ion mode in the mass range of m/z 100–1200 using 140 000 m/Δm resolving power in the Orbitrap mass analyser. Data were interpreted using xcalibur software (Thermo Fisher Scientific). For statistical analysis, the raw spectra were converted to mzXML format using the MS Convert feature of proteowizard 3.0.3750. Subsequently, data processing was carried out with r studio 0.96.316 using the Bioconductor XCMS package. The resulting list (mz, mzmin, mzmax, rt, rtmin, rtmax and peak intensities/areas) was saved in csv file format and uploaded to metaboanalyst V3.0 (www.metaboanalyst.ca).