Development of stationary phase materials and methods for analysis of pharmaceuticals via high-performance liquid chromatography and electrochemical detection

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Alghamdi, Huda
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University College Cork
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In recent years there has been a significant research focus on developing more efficient chromatographic materials and methods for pharmaceutical analysis. Determination of pharmaceutically active compounds in pharmaceutical formulations requires the development of efficient, rapid and sensitive analytical methods. In addition to providing an overview of different analytical methods used for pharmaceutical analysis via liquid chromatography (LC) and electrochemical analysis at a boron-doped diamond (BDD) electrode this research explores and characterises a novel polymer immobilised porous silica particle phase for fast LC analysis of selected pharmaceutical actives. In this thesis, a Nafion polymer-coated silica stationary phase material for the fast and efficient separation of pharmaceutical solutes is reported. Initially, quaternary amine functionalised fully porous and non-porous silica particles (3 µm) were prepared for subsequent coating with Nafion perfluorinated resin (2 % w/v in ethanol). Elemental analysis (CHN), thermogravimetric analysis (TGA), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), zeta potential measurement and scanning electron microscopy (SEM) with energy dispersive X-ray detector (EDX) confirmed the stepwise silica surface functionalisation. The Nafion-coated phases were evaluated for ion-exchange retention behaviour after they were slurry-packed into stainless steel columns (2.1 x 30 mm). These columns were applied to the separation of pharmaceutically-active tetracaine hydrochloride (TCH), oxymetazoline hydrochloride (OZH) and benzalkonium chloride (BAK) through the use of high-performance liquid chromatography (HPLC). Attention was then placed on the rapid separation and sensitive detection of the local anaesthetic TCH and the nasal decongestant OZH combining the high separation efficiencies and the short analysis time of core-shell silica particles with the sensitivity of a BDD electrode. The chromatographic separation was carried out using a poroshell 120 EC-C18 (2.1 × 50 mm, 2.7 μm) column, and isocratic elution is followed by ultraviolet (UV) and amperometric detection at the boron-doped diamond electrode. Rapid reversed-phase (RP) separation of TCH and OZH in nasal spray and ophthalmic formulations was achieved within 45 sec, by adjusting the ratio of organic solvent, mobile phase pH and detection potential. Limits of detections (LODs) of TCH and OZH with BDD electrode are 12 ng/mL and 20 ng/mL respectively, lower than that obtained with UV detection (60 and 89 ng/mL respectively). In addition, a sensitive detection method was developed for the detection of BAK (which is of importance as a preservative and antimicrobial agent in the pharmaceutical industry), using direct electroanalysis at a pristine BDD electrode. The detection limit of BAK homologs with the BDD electrode was 0.4 µg/mL lower than that obtained with a glassy carbon (GC) electrode (0.68 µg/mL) in a non-aqueous medium using square wave voltammetry (SWV). The method was extended to the detection of the C12 homolog in three ophthalmic formulations, and the results were validated by HPLC. The chromatographic separation of BAK homologs (C12, C14, C16, and C18) was carried out using reversed-phase HPLC with a poroshell 120 EC-C8 (2.1 × 50 mm, 1.9 μm) column. The HPLC results confirmed the presence of one single homolog (C12) in the three ophthalmic formulations. The research outcomes represent an advancement in the separation and detection of pharmaceuticals of importance in the pharmaceutical industry at a time when the number of samples and sample matrices in the pharmaceutical industry is on the rise.
Stationary phase , High-performance liquid chromatography , Electrochemical detection
Alghamdi, H. 2022. Development of stationary phase materials and methods for analysis of pharmaceuticals via high-performance liquid chromatography and electrochemical detection. PhD Thesis, University College Cork.