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Detection of Benzoyl Peroxide in Flour Using Raman Spectroscopy

Benzoyl peroxide (BPO) is a common bleaching agent used in wheat flour. Due to its ability to damage existing nutrients in food and potential adverse effect to health, BPO have been strictly banned as a food additive in several countries and regions, such as China and Europe. However, the United States specifies that BPO is generally recognized as safe (GRAS). So, the WHO/FAO created a Codex Alimentarius Commission (CAC) to regulate the international BPO usage standard. According to the CAC, it is restricted at 75 mg/kg or parts per million (ppm). BPO is very unstable and easily converts to benzoic acid (BA), which places the analytical challenge for accurate BPO quantification. The objective of this study is to develop a reliable method for BPO quantification in flour. Raman spectroscopy was first explored to detect BPO and BA on an aluminum foil slide. The result showed BPO and BA produced distinct Raman peaks that can be discriminated against. However, the sensitivity was not satisfactory to reach the regulation limit. To improve sensitivity, surface-enhanced Raman spectroscopy (SERS) was applied using silver nanoparticles as the substrate. Although the signals did enhance significantly using SERS, the characteristic peaks of BPO disappeared as BPO converted to BA during the sample preparation. We then went back to Raman spectroscopy but focused on optimizing the sample preparation to enhance the signal intensity. Using a hydrophobic surface (i.e., parafilm) which can hold the droplet and minimize the spread, the Raman signal was enhanced significantly after repeating multiple droplets on the same surface. A standard curve was created for BPO from 25 ppm to 250 ppm and for BA from 250 ppm to 1000 ppm, respectively. To detect BPO in wheat flour, we applied a more advanced Raman imaging instrument and focused on the analysis of Raman maps instead of spectra for the analysis of effect flour matrix to BPO extraction and detection. We firstly tried an in situ method, which scanned the pellet of flour spiked with different amounts of BPO without extraction. However, we could not detect BPO at 0.1% or lower in flour samples. We then tried an extraction method using acetonitrile as the solvent, which showed a lower detection limit compared to the in situ method. However, this extraction method yielded inconsistent results for BPO that is under 0.05% in flour. The extraction method developed was further improved with an evaporating step and a C18 solid phase extraction (SPE) spin column. This improved the extraction efficacy and provided a roughly 60% recovery percentage for detecting BPO in wheat flour without decomposing into BA. In conclusion, we developed a simple sample preparation protocol coupled with Raman spectroscopy to quantify BPO in flour without converting to BA, which would meet the regulation requirement. This method also shortened the experiment time including both sample preparation and detection time compared to current methods.