HPLC Method Validation and Determination of Synthetic Phenolic Antioxidants in Various Food Items

Hong, Wai Lun (2017) HPLC Method Validation and Determination of Synthetic Phenolic Antioxidants in Various Food Items. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

The purpose of the research is to validate the HPLC method for synthetic phenolic antioxidants (SPAs) analysis, and determine SPAs contents in various food samples. The four widely used SPAs in food industry including butylated hydroxytouluene (BHT), butylated hydroxyanisole (BHA), tertiary butyl hydroquinone (TBHQ) and propyl gallate (PG). SPA is a type of food additives that added into foods containing lipids to prevent fatty acid of foods turns rancid. However, it is crucial to use a validated method to detect SPAs content in foods because they can endanger human health if they exceed the legal limit that had been set by Ministry of Health Malaysia. The research was separated into two parts which are method validation and determination of SPAs content in various food items. In method validation, acetonitrile with 1% acetic acid and water with 1% acetic acid are used as mobile phase while C18 column serves as stationary phase of HPLC. The UV detector was set at 285 nm as it expressed the most intense chromatogram compared with other wavelength. The flow rate used is 2 ml/min while injection volume of sample is 10 ul. Gradient elution that range from 40% to 100% acetonitrile with 1% acetic acid was used instead of isocratic elution as each SPAs possessed different polarity, polarity of mobile phase must vary throughout the analysis in order to elute all SPAs in shorter time. Besides, several method validation tests were performed to determine the linearity, limit of detection (LOD), limit of quantitation (LOQ), precision and accuracy of the HPLC method used. Linearity for four distinct SPAs is from the range 1 ppm-300 ppm. LOD for PG, TBHQ, BHA and BHT is 0.0934 mg/L, 0.0420 mg/L, 0.6694 mg/L and 0.0197 mg/L respectively while LOQ for PG, TBHQ, BHA and BHT is 0.2831 mg/L, 0.1272 mg/L, 2.0287 mg/L and 0.0597 mg/L respectively. Apart from that, Inter-day and Intra-day % relative standard deviation (RSD) are below 5%, and hence it indicated high precision of the method. 10 ppm, 50 ppm and 100 ppm of SPAs standard were also spiked in the food samples to determine the % recovery of SPAs standard. The results showed that it felt in the range of 86.87%-105.25% for PG, 92.12%-106.69% for TBHQ, 97.97%-119.00% for BHA, 94.18%-112.13% for BHT. At the end, twenty five food samples (three noodles, seven potato snacks, three cereals, seven oats, and five biscuits) were analysed using the validated methods to determine SPAs contents. The levels of SPAs in positive samples are all below the legal limit of Malaysia, total SPAs content ≤ 200mg/L (Saad et al., 2007).