Mechanistic Studies on Thiazolidine Formation in Aldehyde/ Cysteamine Model Systems

224 J. Agric. Food Chem. 1998, 46, 224−227 Mechanistic Studies on Thiazolidine Formation in Aldehyde/ Cysteamine Model Systems Tzou-Chi Huang,*,† Lee-Zen Huang,† and Chi-Tang Ho‡ Department of Food Science and Technology, National Pingtung University of Science and Technology, 912 Pingtung, Taiwan, and Department of Food Science, Rutgers University, New Brunswick, New Jersey 08903 A mechanism was proposed to elucidate the formation of a thiazolidine in aldehyde/cysteamine model systems. Buffer dramatically promotes thiazolidine formation from formaldehyde and cysteamine. Phosphate tends to stabilize the primary carbocation formed, and this may lead to completion of the cyclization by attack of the amino nitrogen on the activated carbon. Protic solvent, by removing the water molecule, further enhances thiazolidine formation. Redox reaction catalyzed by phosphate ions results in the conversion of thiazolidine to the corresponding thiazoline through hydride transfer. Keywords: Cysteamine; aliphatic aldehyde; formaldehyde; thiazoline; thiazolidine; redox reaction INTRODUCTION dehyde (bp -19 °C) and acetaldehyde (bp 20.8 °C). It is well documented that a Schiff base formed between The volatile carbonyl compounds, especially alde- the amino group of cysteamine and the aldehyde group hydes, are important contributors to rancid and un- of formaldehyde (Hayashi et al., 1986). In our previous pleasant flavors in various lipid-containing foods. They studies, a significant combined effect of buffer and protic are formed from oxidation or decomposition of lipid solvent on Schiff base formation was observed (Huang, during the storage of food. Because of their detrimental 1997). The objective of this experiment is, therefore, effects to the nutritional value of food or their potential to investigate mechanistically the formation of thiazo- carcinogenic property, it is necessary to measure the lidine between cysteamine and aldehydes. levels of volatile aldehyde compounds in food. Quantitative analysis of highly volatile and reactive EXPERIMENTAL PROCEDURES aldehydes such as formaldehyde and acetaldehyde has Materials. Formaldehyde, acetaldehyde, propionaldehyde, been the subject of several studies (Gray, 1978; Frankel, butyraldehyde, and valeraldehyde were purchased from Ald- 1987). Most commonly used analysis methods for these rich Chemical Co. (Milwaukee, WI). Disodium hydrogen aldehydes involved derivatization with 2,4-dinitrophen- phosphate, sodium dihydrogen phosphate dihydrate, sodium ylhydrazine (Stanley et al., 1975; Caporaso and Sink, carbonate, sodium hydrogen carbonate, trisodium citrate di- 1978). However, this derivatization requires a strong hydrate, citric acid monohydrate, sodium acetate, and sodium acidic condition which may cause undesirable reactions hydroxide were of chemical grade and obtained from Nacalai such as decomposition of trimethyl oxide, carbohydrate, Tesque, Inc. (Kyoto, Japan). Ethanol, chloroform, and cysteamine were purchased from Sigma Chemical Co. (St. Louis, lipid, and protein. MO). Recently, a thiazolidine derivative method for the Reaction Procedure. Reaction mixtures were composed determination of trace aldehydes in foods and beverages of 0.6 g of cysteamine and ∼0.02 g of aldehydes (formaldehyde, has been developed (Hayashi et al., 1986; Miyashita et 0.667 mmol; acetaldehyde, 0.455 mmol; propionaldehyde, 0.345 al., 1991). This method is based on the reaction of vola- mmol; butyraldehyde, 0.278 mmol; and valeraldehyde, 0.233 tile carbonyl compounds with cysteamine (2-aminoeth- mmol), dissolved in 50 mL of deionized water or buffer anethiol) to form stable thiazolidine derivatives under solutions. Buffers involving carbonate, phosphate, citrate, and mild conditions (room temperature and neutral pH). The acetate were used, and the final pH value of the reaction thiazolidine derivatives formed were subsequently de- mixture was adjusted to the pH equivalent to the pKa value of each salt with sodium hydroxide; the mixtures were then termined by gas chromatography. However, the forma- made up to a final volume of 100 mL. To study the effect of tion pathways of thiazolidines are not yet well docu- buffer capacity on thiazolidine formation, different proportions mented. of anion phosphate (0.025, 0.05, 0.1, and 0.2 M) and an initial Yasuhara and Shibamoto (1991) reported that it was pH of 7.2 were used to replace the aqueous medium described difficult to obtain consistent results from replicate previously. Potassium chloride was utilized to maintain a experiments on formaldehyde and acetaldehyde analy- constant ion strength for all three model systems following sis. Only limited amounts of formaldehyde and acetal- the method of Reynolds (1959). All of the reactions were run dehyde were able to be trapped by cysteamine. They at 25 °C for 30 min, and then 1 mL of tributylamine (0.2 mg/ attributed this mainly to the high volatility of formal- 10 mL chloroform) was added as an internal standard for GC analysis. The reaction mixtures were extracted two times with 10 mL of chloroform each time, and the extract was made up * Author to whom correspondence should be addressed (fax to 25 mL with chloroform. After drying over Na2SO4,10µL 886-87740213; e-mail [email protected]). of the chloroform extract was subjected to GC analysis. † National Pingtung University of Science and Technology. Effect of Ethanol Content on Thiazolidine Formation. ‡ Rutgers University. Different percentages of ethanol (0, 20, 40, 60, and 80%) in S0021-8561(97)00563-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/19/1998 Thiazolidine Formation J. Agric. Food Chem., Vol. 46, No. 1, 1998 225 Table 1. MS Spectra of Thiazolidine and Thiazoline no. compound RIa MS 1 2-methylthiazoline 804 M+ ) 101 (65), 69 (46), 60 (100), 59 (80), 45 (70) 2 thiazolidine 816 M+ ) 89 (95), 59 (23), 43 (100) 3 2-methylthiazolidine 889 M+ ) 103 (80), 88 (95), 56 (100), 43 (75) 4 2-ethylthiazoline 924 M+ ) 115 (50), 89 (23), 69 (26), 60 (100), 59 (63), 45 (60) 5 2-ethylthiazolidine 983 M+ ) 117 (15), 88 (100), 70 (35), 56 (20), 43 (10) 6 2-propylthiazoline 1041 M+ ) 129 (5), 114 (7), 101 (68), 88 (18), 60 (100), 59 (50), 45 (30) 7 2-propylthiazolidine 1081 M+ ) 131 (25), 88 (100), 70 (40), 61 (55), 56 (70) ,43 (57) 8 2-butylthiazoline 1123 M+ )143 (5), 114 (13), 101 (100), 88 (12), 69 (8), 60 (64), 59 (35), 45 (24) 9 2-butylthiazolidine 1182 M+ ) 157 (5), 142 (2), 128 (8), 114 (22), 101 (100), 88 (4), Figure 1. Gas chromatogram of derivatives from aldehydes: 68 (4), 60 (74), 59 (24), 46 (2) peak 1, 2-methylthiazoline from acetaldehyde; peak 2, thiazolidine from formaldehyde; peak 3, 2-methylthiazolidine from a Kovats index. acetaldehyde; peak 4, 2-ethylthiazoline from propionaldehyde; peak 5, 2-ethylthiazolidine from propionaldehyde; peak 6, 2-propylthiazolidine from butyraldehyde; peak 7, 2-propylthiazolidine from butyraldehyde; peak 8, 2-butylthiazoline from valeraldehyde; and peak 9, 2-butylthiazolidine from valeraldehyde. phosphate buffer, 0.2 M, pH 7.2, were used as reaction medium to investigate the combined effect of buffer and solvent on thiazolidine formation. The reacted mixtures were extracted with chloroform as described previously. Thiazolidines were quantified against an internal standard (tributylamine). Gas Chromatography. A HP 5890 A gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a fused silica column (60 m × 0.32 mm i.d., film thickness, 0.25 µm; SPB-1 Supelco Co.) and a flame ionization detector was used to analyze the chloroform extracts. The operation conditions were as follows: injector and detector temperatures, 250 °C; extrapure helium carrier flow rate, 2.0 mL/min; temperature program, 70-180 °C at 4 °C/min. Gas Chromatography/Mass Spectrometry. GC/MS analysis was accomplished by using a HP 5890 A gas chromatograph coupled to a 5972 mass selective detector. Mass spectra were obtained by electron ionization and chemical ionization at 70 eV and a source temperature of 250 °C. The filament emission current was 1 mA, and the spectra were recorded and analyzed with HP G111034C MS Chemstation software installed in an IBM personal computer. The operation conditions were the same as those used in the GC analysis described above. RESULTS AND DISCUSSIONS GC Separation of Thiazolidines. Under the ex- Figure 2. Mass spectra of thiazolines from aldehydes: (A) perimental conditions described, complete resolution 2-ethylthiazoline from propionaldehyde; (B) 2-butylthiazoline was obtained for a standard mixture composed of from valeraldehyde. formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, and valeraldehyde that was reacted with cys- propionaldehyde (Figure 2). The McLafferty rearrange- teamine. Elution order increased according to chain ment takes place on the longer side chain containing length as shown in Figure 1. The structures of all an n-propyl or longer alkyl group, giving a characteristic thiazolidines were confirmed by both EI and CI GC/MS. peak of m/z 101. Two volatile compounds, 2-acetyl-2- The mass spectra are listed in Table 1. The mass thiazoline and 2-acetylthiazole, were characterized in spectra of the thiazolidines were in good agreement with a heated aqueous L-cysteine solution (Sheldon and the findings of Yasuhara and Shibamoto (1989). The Shibamoto, 1987). These authors postulated that these series of thiazolines were characterized in the same two acetyl derivatives were the dehydrogenation prod- model system. These compounds eluted before the ucts of 2-acetylthiazolidine.

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