Cannabinoids biosynthesis in phytoplankton has attracted much attention due to the rapid development of genetic tools and the optimization of genetic transformation methods in microalgae. To monitor the biosynthesis process, proper sample preparation and practical instrumental methods are needed to measure the various precursors, intermediates, cannabinoids, and their degradation products. The objective of this study was to develop a sample preparation procedure for the quantification of olivetolic acid (OA), cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), tetrahydrocannabinolic acid (THCA), olivetol (OL), cannabidiol (CBD), and tetrahydrocannabinol (THC) using single-quadrupole gas chromatography-mass spectrometry (GC-MS). Isochrysis galbana was used as the model matrix. After methanol extraction, samples were purified using solid phase extraction (SPE), silylated with N-methyl-N-(trimethylsilyl)trifluoroacetamide, and analyzed using GC-MS in electron ionization mode. A strong anion-exchange SPE efficiently recovered OA, CBGA, CBDA, and THCA. A graphitized carbon black SPE was necessary to purify OL, CBD, and THC. Both columns removed amino acids, sugars, polyols, and pigments from the algae extract and prepared samples that are suitable for silylation and GC-MS analysis. The total protocol, including solvent extraction, SPE, silylation, and GC-MS analysis, was validated in accordance with the ICH guidelines. Performance characteristics of our method are superior to existing protocols with similar complexity in the literature.

American Oil Chemists’ Society (AOCS)’s Official Method Cd 18-90 for p-Anisidine Value (pAV) is commonly used to evaluate secondary oxidation in fish oils. Flavoring agents in fish oil products may interfere with pAV and lead to inaccurate results. The Global Organization for EPA and DHA (GOED) recommends a protocol for calculating pAV of flavored fish oils, based on the assumption that flavors’ contribution to the pAV does not change over the course of oxidation. The objective of this study was to test this assumption. All fourteen flavors evaluated increased the pAV when added to fresh fish oil; chocolate-vanilla and lemon flavors generated the largest increase. Under accelerated oxidation conditions, both chocolate-vanilla and lemon flavors had a similar effect; oxidized flavored fish oils had lower pAV than oxidized fish oils with newly added flavors. This was due to either an antioxidant effect of the flavor or degradation of the flavor during oxidation. Following the GOED recommendation, we would have underestimated the oxidation in the flavored oils. For this reason, pAV of flavored fish oils should be considered with caution and used in combination with other secondary oxidation markers when possible.