Theses (MSc. Analytical & Pharmaceutical Chemistry)

A simple, rapid and accurate reversed-phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of sildenafil and tadalafil in twenty herbal dietary supplements sold for enhancing sexual performance in Malaysia. Chromatographic separation was achieved with a Hypersil Gold C18 column (150 mm × 4.6 mm i.d., 5 µm particle size) at 30 °C, using a mobile phase consisting of 20 mM of ammonium acetate (adjusted to pH 5.5 with orthophosphoric acid) and acetonitrile at a flow rate of 1.0 mL/min. The UV detection wavelength was set at 245 nm. Tadalafil and sildenafil eluted out from the column at retention times 5.8 min and 6.3 min, respectively. The method was validated in accordance to specificity, accuracy, linearity and range, precision, limit of detection (LOD) and limit of quantification (LOQ). Calibration curves were linear over the concentration range of 10−30 µg/mL and 1−3 µg/mL for sildenafil and tadalafil, respectively. The LOD values of sildenafil and tadalafil were 1.00 µg/mL and 0.07 µg/mL, respectively while LOQ values were 3.02 µg/mL and 0.21 µg/mL, respectively. The recoveries of both compounds were in the range of 99.11−100.87%. The RSD of repeatability and intermediate precision were less than 2%. The proposed method was successfully applied in the screening and quantification of tadalafil and sildenafil in adulterated herbal dietary samples. Keywords: Herbal dietary supplements; Sildenafil; Tadalafil; HPLC; Adulteration.

Slimming products based on phytotherapeutic formulations are popular due to their affordability and the misconception that they are safe because they are „natural products‟. However, adulterants have been found to be added into these herbal formulations to improve the weight lost effect of the product. Consumption of these adulterated products may lead to health problems, and even death. We hypothesise that over-the-counter (OTC) slimming phythotherapeutic formulations sold in Malaysia are adulterated with synthetic and natural drugs. Caffeine and phenolphthalein are popular adulterants due to their respective properties as an anorexic (appetite suppressor) and laxative. The objective of this study is to develop and validate a simple, precise and specific high-performance liquid chromatography coupled with ultraviolet detector (HPLC-UV) analytical method for the simultaneous detection and measurement of caffeine and phenolphthalein in slimming phytotherapeutic formulations. The method was then applied to analyse eight OTC slimming phythotherapeutic formulations purchased from the Malaysian market. The method was developed using a HPLC connected to a variable-wavelength detector (VWD). Separation was achieved with a C18 reversed-phase column using gradient elution. Mobile phase used were solvent A (aqueous): 25 mM ammonium acetate buffer (pH 5), and solvent B (organic): methanol. Gradient elution was achieved by a ratio of time (minutes) over methanol (%): 0/10, 1/10, 3/40, 5/55, with a run time of 10 minutes. Flow rate was maintained at 1.1 mL/min and the effluents monitored at 254 nm wavelength. The retention time of caffeine and phenolphthalein were approximately 5.5 minutes and 8.8 minutes, respectively. The developed method was validated for linearity, precision, accuracy and selectivity according to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines (Validation of Analytical Procedures: Text and Methodology, Q2(R1)). The calibration curve area versus concentration was found to be linear (coefficient of determination, R2 ≥ 0.999) in the range of 10–100 μg/mL for both caffeine and phenolphthalein. The limit of detection (LOD) which indicates the lowest concentration of caffeine and phenolphthalein that can be detected were 0.77 and 0.47 μg/mL, respectively. The limit of quantification (LOQ) that express the lowest concentration of caffeine and phenolphthalein which can be confidently quantified were 2.35 and 1.44 μg/mL, respectively. The method was found to be accurate for both compounds with percent recovery values of 98–102% for both caffeine and phenolphthalein. The method had good intra-day and inter-day precision with percent relative standard deviation (%RSD) values of less than 2%. The validated method was applied to analysis of eight OTC slimming phytotherapeutic formulations purchased from the Malaysian market. Phenolphthalein was detected and quantified in five of these slimming products, at levels ranging from 0.68–126.83 mg/g. Caffeine was detected in six of these slimming products but only quantified in two products (sample A, 3.39 ± 0.21 mg/g and sample B, 0.99 ± 0.01 mg/g) as most of the products claimed to be of plant origin and hence caffeine detected were likely from natural sources. In conclusion, a simple and fast HPLC-UV method to detect for caffeine and phenolphthalein as adulterants was successfully developed and validated. The method was applied to actual OTC slimming products, and proved the hypothesis that OTC slimming phythotherapeutic formulations sold in Malaysia are adulterated. As future work, the developed method can be used to analyse more variety of slimming products. The analytical technique is also suitable to be used in quality control laboratories for routine detection of phenolphthalein and caffeine in slimming products.

A simple and rapid isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated as per International Conference on Harmonisation (ICH) and United States Pharmacopeia (USP) guidelines for analysis of tiagabine in the presence of its degradation products. The chromatographic separation was achieved on a Vision HT C18 column (150 mm × 4.6 mm, 5 μm) with mobile phase comprising of 11.5 mM sodium dihydrogen phosphate buffer (adjusted to pH 2.0 with orthophosphoric acid)/acetonitrile (50:50, v/v) at a flow rate of 1 mL/min and the UV detection wavelength was set at 254 nm. Stress degradation studies were performed as stated in ICH guidelines on tiagabine bulk drug using acid, base, oxidation, heat and light. The method was validated in accordance to system suitability, linearity and range, specificity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), and robustness. Tiagabine was found to degrade under acidic, photolytic, oxidative and thermal condition, but stable under basic hydrolysis condition. The developed method was found to be linear in the concentration range of 50-150 μg/mL (r2 = 0.9983) and the percentage recovery for the accuracy was 98.86-99.35%. The LOD and LOQ obtained were 31.93 μg/mL and 96.76 μg/mL, respectively while the %RSD for precision, robustness and stability studies were less than 2%. The degradation products formed from the stress degradation studies were well separated from tiagabine and hence the method could be regarded as stability indicating. The proposed method can be used in the analysis of tiagabine bulk drug and pharmaceutical formulations in quality control laboratories. Keywords: Tiagabine, RP-HPLC, Stress degradation studies, Validation.

Everolimus is a mammalian target of a rapamycin inhibitor drug approved for treating advanced-stage, metastatic cancer that is non-responsive to endocrine therapy. Like most chemotherapeutic drugs, everolimus comes with numerous side effects, and the severity of these side effects often varies from patient to patient. In hopes of curbing the side effects during the treatment process, it was reported that some patients default to complementary and alternative medicine (CAM) in the form of natural product-based dietary supplements. One of the natural product supplements that was reported to have been consumed by patients undergoing everolimus treatment is curcumin. Following the consumption of curcumin during everolimus treatment is a significant fall in everolimus blood concentration levels (Cmin.). It was speculated that curcumin induces CYP3A4 activity, thus increasing everolimus metabolism. However, whether the effects of curcumin on everolimus metabolism are CYP3A4-related is yet to be proven, as there are other possible ways for curcumin to be involved in decreasing everolimus Cmin in patients. One of them includes the possibility of physicochemical interaction. The primary objective of this project was to investigate the potential chemical interaction(s) that could occur between everolimus and certain selected natural compounds (rutin, quercetin and curcumin) when present in an aqueous matrix under different physiological pH environments via HPLC. The occurrences of said interactions are depicted by the appearance of unknown peaks in sample chromatograms whereby everolimus was paired with other foreign compounds. These unknown peaks were not detected in the control samples, whereby everolimus stands as the lone compound. Not all peaks present in the sample chromatograms are integrated and quantified. As a pre-requisite, the unknown peaks that are isolated in sample chromatograms mustn’t be present in blanks or controls. Only the unknown peaks that share the same spectral profile as the primary molecule, everolimus are captured and quantified. Based on the findings of this investigation, everolimus is most unstable in the presence of the natural compounds when incubated in pH 6.8 medium. The degree of instability of everolimus was depicted by the number of unknown peaks present. This indicates that more chemical interactions take place between everolimus and the natural compounds at a pH 6.8 environment compared to other pHs. Also, in pH 6.8, everolimus is most unstable and degrades rapidly when paired individually with the natural compound quercetin. The extent of interaction that takes place between everolimus and each natural compound differs. When in the presence of all three natural compounds at once, the rate of degradation of everolimus is highest at pH 6.8. The rate of degradation is interpreted as the formation of unknown peaks per unit time. However, in a pH 4.5 environment, everolimus has the most interaction when individually paired with curcumin. Most of the unknown peaks observed in single-paired samples are not found in the control sample. Based on the evidence gathered, the stability of everolimus in different pH environments is highly affected by the presence of natural compounds.