Food analysis derivatization reaction

Detection of amino acids is typically by UV absorption after postcolumn reaction with nin-hydrin. Precolumn derivatization with ninhydrin is not possible, because the amino acids do not actually form an adduct with the ninhydrin. Rather, the reaction of all primary amino acids results in the formation of a chromophoric compound named Ruhemann s purple. This chro-mophore has an absorption maximum at 570 nm. The secondary amino acid, proline, is not able to react in the same fashion and results in an intermediate reaction product with an absorption maximum at 440 nm. See Fig. 5. Detection limits afforded by postcolumn reaction with ninhydrin are typically in the range of over 100 picomoles injected. Lower detection limits can be realized with postcolumn reaction with fluorescamine (115) or o-phthalaldehyde (OPA) (116). Detection limits down to 5 picomoles are possible. However, the detection limits afforded by ninhydrin are sufficient for the overwhelming majority of applications in food analysis. 

Methods for the analysis of aromatic amines in synthetic azo dyes and in food and beverages colored with these dyes have been developed. High-performance LC or GC methods are generally employed, often with the help of derivatization reaction and fluorimetric detection, with HPLC generally regarded as the best technique for the determination of aromatic amines. 

SBSE can be successfully used in the analysis of environmental samples [93-97] and for food analysis [98, 99]. PDMS is the most commonly used polymer, primarily because of its thermal stability and durability. SBSE has been modified by application of derivatization with different reagents (acetic anhydride, BSTFA, etc) [100-104]. This approach is suitable for the extraction of compounds requiring derivatization. The use of multistep derivatization with several extraction elements (each reaction is performed on a different stir bar) allows efficient extraction, desorption, and chromatographic analysis of compounds with different functional groups (e.g., phenols, steroids, amines, thiazoles, ketones). Acetic anhydride (ester formation), ethyl chloroformate (reaction of acids and amines), tetraethyloborane, and sodium bis-trimethylotrifluoroacetamide have been used for extraction and simultaneous derivatization [105]. 

Fluorescence spectroscopy plays an important function in modern food analysis as can be seen from its wide use in the determination of numerous food components, contaminants, additives, and adulterants. This technique has made available very sensitive and selective methods that satisfy the requirements of food analysis, which are usually very complex, taking into account the large number of species to be determined, frequently at very low concentrations, and the wide variety of foodstuffs available. Initially, the use of fluorescence spectroscopy in food analysis was limited to the determination of species with intrinsic fluorescence (e.g., vitamins, aflatoxins, and some polycyclic aromatic hydrocarbons (PAHs)), but now it is widely applied to nonfluorescent species, using several physicochemical means such as chemical or photochemical derivatization reactions. Numerous techniques involve fluorescence detection in liquid chromatography (LC), frequently using pre- or postcolumn derivatization. In addition to conventional fluorime-try, which is commonly chosen for this purpose, other fluorimetric techniques such as laser-induced... 

Ammoniacal nitrogen is usually determined by flow methods involving derivatization reactions for spectrophotometric determination. Berthelot (or indophenol blue) [99,115-119] and Nessler methods [120] are the two most important examples, both adopted for the quantification of ammonium ion in matrices as diverse as natural waters, wastewaters, atmospheric samples, food, soil extracts, plant material, and biological fluids. Measuring the fluorescence intensity produced by the reaction of the ammonium ion with OPA is another widespread method for the analysis of this ion [121-125]. 

The development of flow analysis methods for AA is important because of the presence and significance of this analyte in foodstuffs, pharmaceuticals, and biological fluids, with implications in redox processes, human health, and food quality. However, so far there are few flow analysis methods for simultaneously determining AA and DHAA, or simultaneously determining several vitamins, including vitamin C (the sum of the contents of AA plus DHAA). Furthermore, most flow analysis methods do not include online sample preparation, except for analytical separations and derivatization reactions, and only two online sample dilution methods allow the fully automatic determination of AA. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

UV absorption occurs only below 220nm, thereby it is affected by the interference from mobile phase and from artifacts in complex foods. A multiwavelength UV detection has been experimented successfully for unambiguous evaluation of pantothenic acid [609]. However, UV detection presents a low sensitivity, compared to other techniques, like FLD or MS. FLD is applied by using a postcolumn derivatization. Pantothenic acid is converted to 3-alanine by hot alkaline hydrolysis and a reaction with OPA [610]. Also MS is successfully applied to increase the sensitivity of pantothenic acid analysis. 

Hwang et al. developed a rapid and sensitive HPLC-UV method for the analysis of nine derivatized BAs, with benzoyl chloride as the derivatization agent. The reaction is faster than with tosyl chloride and leads to stable products with shorter elution times than do dansyl derivatives. The amines were previously extracted after acidification with TCA. The method was applied to detect BAs in fried marlin fillet, implicated in a food poisoning incident (in Taipei City in 1996) and indicated that a high level of His (84.1 mg/100 g) was present in the sample (80). 

Methylglyoxal (MG) is a highly reactive a-dicarbonyl formed endogenously in numerous enzymatic and nonenzymatic reactions. MG is very volatile and often coelutes with the solvent peak when the mixture is prepared for GC analysis. Hence, many papers focusing on environmental, clinical, or food studies have been published on its detection and quantification using chemical derivatization. After derivatization, the specificity of analyte, which carries a unique functional group. 

GC is regularly used to identify and quantify the presence of aflatoxins in food samples, and many protocols have been developed for these materials. Normally, the system is linked to MS, flame ionization detector (FID), or Fourier transform infrared spectroscopy (FTIR) detection techniques in order to detect the volatile products [81-83]. Most aflatoxins are not volatile and therefore have to be derivatized for analysis using GC [83]. Several techniques have been developed for the derivatiza-tion of aflatoxins. Chemical reactions such as silylation or polyfluoroacylation are employed in order to obtain a volatile material [32]. 

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