Development of silica materials of different particle and pore morphology for the application in cannabinoid research and in separation of biomolecules by HPLC

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Analakkattillam, Sandhyarani
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University College Cork
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Currently, pharmaceutical companies have focused their development to large molecule biotherapeutics for the treatment of diseases such as cancer and neurodegenerative diseases. These new protein biopharmaceuticals have large molecular weights and have intricate structures. The complexity of these biopharmaceuticals requires new analysis methodology as compared to those of small molecule drugs. To enable this characterization, new liquid chromatographic stationary phase particle technologies are required. In this project, we developed novel large pore (≥ 1000 Å) and non-porous silica based stationary phases for the separation of biomolecules such as proteins. Both fully porous and superficially porous silica materials were utilized in an alkali hydrothermal pore expansion process. The experimental conditions were optimised for type and concentration of mineralising agent and the treatment temperature. The selection of mineralising agent and the treatment temperature depends on the particle and pore size of silica precursor molecule and the optimum concentration was found to be 0.5% w/v. These silica materials were subsequently chemically bonded with monofunctional alkyl ligands of different chain lengths under optimised reflux conditions. These materials were then packed into stainless steel columns of varying column dimensions and subjected to performance evaluation studies using in-house as well as Tanaka and Walters test methods. The suitability of these silica materials in biomolecular separation was tested using a test mixture containing six proteins of molecular weight ranging from 12 to 97 kDa. The non-porous stationary phases 2 µm C18 columns shows the ability to separate all the protein molecules with lower recovery for larger proteins. Both fully porous and superficially porous ultra-wide pore stationary phases of short and longer alkyl groups (C4, C8 and C18) displayed the ability to separate proteins where the superficially porous materials leads faster separation with low column backpressure. Moreover, the C8 stationary phases achieved to recover the larger proteins as compared to C18 and C4 stationary phases. The use of amorphous silica as drug carrier system in cannabinoid research was also studied. The main focus was made on two cannabinoids namely cannabidiol (CBD) and tetrahydrocannabinol (THC). Hemp Oil Supplements-mainly CBD rich products-have grown in popularity worldwide and have received much positive (due to non-psychoactive CBD) and negative press (because of psychoactive THC), however little is known about the actual strength of CBD and THC in these products. This demands a uniform method to quantify CBD and THC in these products. Hence, a simple and reliable assay test method was developed and validated simultaneous determination of CBD and THC in hemp oil products by high performance liquid chromatography. The assay test method was validated as per International council for harmonisation quality guidelines allowing hemp oil/CBD manufactures to utilize this method for quality control checks for their products. Moreover, for the first time in-vitro dissolution studies were conducted to quantify the CBD release in hemp oil products. Additionally, for the first time stability studies were conducted for quantifying CBD in hemp oil and hemp oil infused silica materials to identify the correct storage and labelling conditions to be followed while handling hemp oil products in market.
High performance liquid chromatography , Cannabinoids , Cannabidiol , Silica materials , Chromatographic column packing , Protein separation , Analytical method validation , Dissolution testing , Stability studies
Analakkattillam, S. 2023. Development of silica materials of different particle and pore morphology for the application in cannabinoid research and in separation of biomolecules by HPLC. PhD Thesis, University College Cork.