Method development for simultaneous detection of ferulic acid and vanillin using high-performance thin layer chromatography

Swarali S Hingse 0 Shraddha B Digole 0 Uday S Annapure 0 0 Food Engineering and Technology Department, Institute of Chemical Technology , Nathalal Parekh Marg, Matunga, Mumbai - 400019, India Background: A simple, accurate, and reliable high-performance thin-layer chromatography (HPTLC) method was developed for separation and detection of ferulic acid and vanillin. Methods: Separation of ferulic acid and vanillin was carried out on 20 10 cm thin layer chromatography (TLC) plates using mobile phase containing toluene/1, 4-dioxan/acetic acid in the ratio 9:2.5:0.4 (v/v). The FA and vanillin were scanned at 320 and 312 nm, respectively. Method was validated for linearity, accuracy, precision, robustness, limit of detection, limit of quantification, and specificity. Results: Retention factor (Rf) obtained for ferulic acid and vanillin was 0.48 and 0.56, respectively. The correlation coefficients, 0.9975 and 0.9991 with an average recovery of 98.77% and 98.45% obtained for ferulic acid and vanillin respectively by this method were satisfactory. Conclusion: The optimized method was found to be efficient, precise, accurate, specific, and economic. Therefore, the method would be useful for both qualitative and quantitative routine analysis in pharmaceutical, food industry, and research laboratories. - Background Aromatic compounds are present in natural sources with substantial combinations which are directly responsible for its odor and sensitivity. They can be categorized as volatile organic compounds like aldehydes, alcohols, ketones, esters, lactones, and terpenes (Raisi et al. 2008). They are known to be precursors for the production of numerous products employed in the food, pharmaceutical, and chemical industries and are present at very low concentrations in natural sources. According to US and European legislations, synthetic flavor production is not considered as natural. Alternatively, biotechnology offers microorganisms as production hosts for different types of aromatic compounds in industrial fermentative processes (Lomascolo et al. 1999). The most intensively studied biotransformation using microorganisms is the bio conversion of ferulic acid (FA) to produce natural vanillin (Priefert et al. 2001). FA is an important precursor of vanillin that is available in abundance in plant cell walls linked to polysaccharide by an ester or ether bonds (Xu et al. 2005). FA is a potent antioxidant because it effectively scavenges free radicals and even possesses antimicrobial properties by preventing the lipid peroxidation caused by microbes (Graf 1992). Moreover, it is used in cosmetics for the photo protection of skin and in protection against various inflammatory diseases. Vanillin is widely used in food industry as a flavoring agent but also has applications in some fragrances and pharmaceuticals (Priefert et al. 2001). It is also known to possess anti-metastatic, anticancer (Ho et al. 2009) and anti-inflammatory (Wu et al. 2009) activities. It exhibits antimicrobial properties due to its phenolic nature and hence used to develop antimicrobial films used in packaging of bakery products (Rakchoy et al. 2009). FA and vanillin are generally determined by various chromatographic methods. Different approaches such as UV spectrophotometry (Mabry et al. 1970; Macheix et al. 1990) gas chromatography (GC), capillary electrophoresis (CE), high-pressure liquid chromatography (HPLC), thin layer chromatography (TLC), and high-performance thin layer chromatography (HPTLC) are some of the frequently used methods for the detection, qualitative analysis, and quantification (Sharma et al. 2007). Spectrophotometric methods are used for identification of phenolic acids and are generally carried between a range of 220 to 320 nm (Mabry et al. 1970; Macheix et al. 1990); however, methods such as the Folin Ciocalteu spectrophotometric method results in nonspecific detection of the phenolic compounds and the interference of components such as ascorbic acid in food samples, that behave as reducing agents. Absorption of phenolic compounds is affected by pH, solvents used in the method and the interference of proteins and amino acids (Constantine et al. 2007). Volatile compounds are directly analyzed by gas chromatography, a technique of unsurpassed separation capacity (Sostaric et al. 2000). GC is a major chromatographic technique employed for the analysis of essential phenolic acids in plants. It deals with high sensitivity and selectivity (Chiou et al. 2007) but requires derivatization step of hydroxyl groups in phenolic compounds. They are modified by various reagents to make more volatile compounds by a process such as methylation, conversion into trimethylsilyl (TMS) derivatives. However, problems such as poor separation and low stability after derivatization state are some of the shortcomings of this method. CE is too employed for analysis of phenolic compounds (Huck et al. 2005; Butehorn et al. 1996). Mostly, the method falls in the field of natural product research, including the analysis of plants, vegetables, herbs, and other plant- or fruit-derived products. It results in oxidation of phenolic compounds by dissolved oxygen and increase in migration time of flavonoids due to the increase in buffer concentrations (Constantine et al. 2007). TLC methods have the ability to screen phenolic compounds easily (Tilay et al. 2008). The results obtained by TLC method are generally quantified using more multifaceted techniques like HPTLC (Mabinya et al. 2006). However detection of vanillin by spraying with 2, 4dinitrophenylhydrazine (2, 4-DNPH) is not significant as the peaks are not detected properly. Currently, the main qualitative and quantitative techniques for phenolic compound detection are HPLC (Rao et al. 1999; Zheng et al. 2007). The European pharmacopoeia suggests the development of such analytical method which demands the adequate amount of reagents, solvents, and material (European Pharmacopoeia 2008). HPTLC allows for the simultaneous analysis of large sample size using small quantities of solvents, thus reducing time and cost of the analysis. The sensitivity for phenolic compounds performed by HPTLC is more as compared to HPLC (Prinjaporn et al. 2013). Mobile phase having pH 8 and above can be employed. Sample with turbidity and different combinations of solvent can be directly applied. It facilitates automated application and repeated scanning of the chromatogram with the same or different parameters (Bakshi et al. 2002). Therefore, this technique should be taken into consideration as an alternative to HPLC. HPTLC is a sophisticated instrumental technique which allows a fast and inexpensive method for analysis. Special advantage of HPTLC includes high sample throughput and low cost per analysis. HPTLC offers a great variety of stationary phases with unique selectivity for mixture components and their separation simultaneously. Processing of standards and samples identically (...truncated)