Application of experimental design in HPLC method optimisation for the simultaneous determination of multiple bioactive cannabinoids.

The scientific interest in Cannabis sativa L. analysis has been rapidly increasing in recent years, especially for what concerns cannabinoids, plant secondary metabolites which are well known for having many biological properties. High-performance liquid chromatography (HPLC) is frequently used for both the qualitative and quantitative analysis of cannabinoids in plant extracts from C. sativa and its derived products. Many studies have been focused on the main cannabinoids, such as ∆9-tetrahydrocannabinolic acid (∆9-THCA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA) and their decarboxylated derivatives, such as ∆9-tetrahydrocannabinol (∆9-THC), cannabidiol (CBD) and cannabigerol (CBG). In addition to the abovementioned compounds, the plant produces other metabolites of the same chemical class, and some of them have shown interesting biological activities. In the light of this, it is important to have efficient analytical methods for the simultaneous separation of cannabinoids, which is quite complex since they present similar chemical-physical characteristics. The present work is focused on the use of the Design of Experiments technique (DoE) to develop and optimise an HPLC method for the simultaneous separation of 14 cannabinoids. Experimental design optimisation was applied by using a Central Composite Face-Centered design to achieve the best resolution with minimum experimental trials. Five significant variables affecting the chromatographic separation, including ammonium formate concentration, gradient elution, run time and flow rate, were studied. A multivariate strategy, based on Principal Component Analysis (PCA) and Partial Least Squared (PLS) regression, was used to define the best operative conditions. The developed method allowed for the separation of 12 out of 14 cannabinoids. Due to co-elution phenomena, HPLC coupled with a triple quadrupole mass analyser (HPLC-ESI-MS/MS) was applied, monitoring the specific transitions of each compound in the multiple reaction monitoring (MRM) mode. Finally, the optimised method was applied to C. sativa extracts having a different cannabinoid profile to demonstrate its efficiency to real samples. The methodology applied in this study can be useful for the separation of other cannabinoid mixtures, by means of appropriate optimisation of the experimental conditions.

Comparative Analysis of VOCs from Winter Melon Pomace Fibers before and after Bleaching Treatment with H2O2.

In this study, the trend of Volatile Organic Compounds (VOCs) in dietary fiber samples from the winter melon (Cucumis Melo var. Inodorus, Yellow Canary type) were investigated. This foodstuff, obtained as a by-product of agri-food production, has gained increasing attention and is characterized by many bioactive components and a high dietary-fiber content. As regards fiber, it is poorly colored, but it may be whitened by applying a bleaching treatment with H2O2. The result is a fibrous material for specific applications in food manufacturing, for example, as a corrector for some functional and technological properties. This treatment is healthy and safe for consumers and widely applied in industrial food processes. In this study, a method based on headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was applied for the characterization of the aromatic profile of the dried raw materials. Furthermore, VOC variation was investigated as function of the bleaching treatment with H2O2. The bleached samples were also analyzed after a long storage period (24 months), to assess their stability over time. As a result, the VOC fraction of the fresh raw fiber showed nine classes of analytes; these were restricted to seven for the bleached fiber at t0 time, and further reduced to four classes at the age of 24 months. Alcohols were the main group detected in the fresh raw sample (33.8 % of the total chromatogram area), with 2,3-butanediol isomers as the main compounds. These analytes decreased with time. An opposite trend was observed for the acids (9.7% at t0), which increased with time and became the most important class in the 24-month aged and bleached sample (57.3%).