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Development, validation and application of an analytical method for the determination of fucose in human plasma after oral administration of fucoidan

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Cahyana, A (2016) Development, validation and application of an analytical method for the determination of fucose in human plasma after oral administration of fucoidan. Research Master thesis, University of Tasmania.

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Abstract

Background:
It has been reported that fucoidan, a polysaccharide commonly extracted from brown seaweeds, possesses a wide range of biological activities. Fucoidan is composed of a highly negatively charged and polydispersed oligosaccharides having fucose as the main component. It is not possible to separate various oligosaccharides of intact fucoidan using only one method because of their high negative charge, high molecular weight and polydispersity. Fucoidan is commercially available in the oral dosage form (only available as capsules). However, its bioavailability after oral administration is currently unknown. Therefore, the objective of this study was to develop an analytical technique that can measure the concentration of fucose (the main component of intact fucoidan) after oral administration of fucoidan. The application of newly developed method was demonstrated by analysing the fucose levels in patients’ plasma before and after oral administration of fucoidan.
Method:
Different types of chromatographic methods (reversed-phase chromatography, reversed-phase ion-pairing chromatography and normal phase chromatography) were tried to obtain a method that can separate fucose from other compounds present in plasma. Once this was achieved, several analytical parameters such as mobile phase composition, buffer strength, pH, and other parameters within the detector were also tested to increase the sensitivity of the method. Digestion and precipitation methods were used to breakdown fucoidan into fucose and to precipitate fucose respectively. The developed method was then validated using inter- and intra-day precision, accuracy and reproducibility. The limit of detection (LOD) and limit of quantitation (LOQ) was also calculated using the mean standard deviation obtained from three calibration curves. The developed and validated method was then used to determine the presence of plasma fucose levels in cancer patients before and after oral administration of fucoidan. The patients were taking either Docexatel (maximum dose between 75 and 100 mg every 3 weeks) or Anastrozole (1 mg daily). A 250 mg hard-gelatine capsule containing 187.5 mg of fucoidan was orally administered four times a day for seven days. The patients’ plasma samples were precipitated with acetonitrile and digested using 20% trifluoroacetic acid to depolymerise fucoidan into fucose. The amount of fucose in the plasma samples was then determined using the developed method.
Results:
It was concluded that, although this might be limited to the particularly selected chromatographic parameters (types of column, mobile phase and ion-pairing reagents), normal phase chromatography was more suitable in the analysis of fucose compared to the reversedphase and reversed-phase ion-pairing chromatography tested in this study. The tested normal phase chromatography method performed well in retaining and resolving fucose both in standard solution and plasma samples. Fucose-containing plasma sample was resolved using Shodex Asahipak NH2P-50 4E column employed with a series of Dionex Ultimate Pump, autosampler, column compartment and a Charged Aerosol Detector (CAD). Mobile phase consisted of 5 mM ammonium acetate pH 4.75 and acetonitrile (20:80) with the flow rate of 1.0 mL/minute. Peak to concentration correlation test using 5 different concentrations of fucose solutions (5 μg/mL to 100 μg/mL) gave exponential correlation coefficient (`R^2`) of 0.9999. Fucoidan was digested using 20% trifluoroacetic acid and was found to contain 20.35%±0.87% (n=3) of fucose (w/w). Plasma protein and spiked fucoidan from 1 mL of plasma was precipitated using 4 mL of acetonitrile. Recovery of fucose in fucoidan-spiked blank plasma after precipitation was found to be more than 92% (n=2) based on the calculated fucose concentration. Intra- and Inter-day precision, accuracy and reproducibility values were found to be less than 5% RSD. Limit of detection (LOD) and limit of quantitation (LOQ) of the developed method was calculated to be 2.28 ng and 6.92 ng, respectively. Fucose concentration in patients’ plasma after the administration of fucoidan was elevated by (1.14±0.61) mg/100 mL (n=19). This suggested that the oral absorption of fucoidan was around 14.28%±7.59% (n=19).
Conclusion:
The developed method was suitable for the detection of fucose in patients treated with oral fucoidan. The result obtained in this study showed that less than 15% of orally administered fucoidan was absorbed. Nonetheless, this shows that, to some extent, fucoidan is getting absorbed following oral administration. Moreover, the proposed method can be used in the analysis of fucoidan in human plasma as an alternative option to the currently available ELISA method or those using derivatisation. It offers simplicity in the procedure and specificity in the detection of fucose, the main component of fucoidan.

Item Type: Thesis (Research Master)
Keywords: Fucoidan, Plasma, HPLC, Charged aerosol detector, Fucose, Absorption, Digestion
Copyright Information:

Copyright 2015 the author

Date Deposited: 10 Apr 2017 02:38
Last Modified: 14 May 2017 23:47
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