Optimization of the derivatization conditions in samples
The amino groups of MC, EC, and BC undertook the substitution reaction with 9-xanthydrol under acidic conditions in order to produce xanthyl methylcarbamate, xanthyl ethylcarbamate, and xanthyl butylcarbamate as shown in Figure 1, and it was possible to directly analyze the product by the GC-MS.
The optimal reaction conditions for the simultaneous determination of MC and EC in solid fermented foods was also tested. For the first test, the minimum amount of 9-xanthydrol for the derivatization was studied. The derivatization was performed for various 9-xanthydrol concentrations (1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mM of 9-xanthydrol). The yield stayed continuously beyond 4.0 mM of 9-xanthydrol and the optimal 9-xanthydrol amount was 4.0 mM (Figure 2). The effect of the acid concentration on the reaction of MC, EC and BC with 9-xanthydrol was also studied. The derivative was tested at HCl concentrations of 0.01, 0.05, 0.1, 0.2, 0.3, and 0.5 M. The other reaction conditions were set to have a reaction time of 10 min at a temperature of 20°C. The results showed good recovery at the HCl concentration value of 0.2 M (Figure 3). The reaction rate of MC, EC and BC with 9-xanthydrol was also studied. The reaction rate of the derivative was analyzed at reaction temperatures of 20, 30, 40, and 50°C and the reaction time was analyzed in at 5, 10, 20, 30, and 60 min. From the experiment, the optimal reaction temperature and time was 10 min at 20°C (Figures 4 and 5). The recovery was declined slowly beyond the reaction time of 10 min.
As a result, the optimal reaction conditions of MC, EC, and BC with 9-xanthydrol were 4.0 mM 9-xanthydrol, 0.2 M HCl concentration, the reaction time of 10 min at an ambient temperature.
The selection of the extraction solvent was of great importance in order to achieve satisfactory extraction efficiency for the target compounds. Based on the consideration for the solvent strength, methylene chloride, ethyl acetate, ethyl ether and hexane were selected as potential extraction solvents for use in this study. As a result, ethyl acetate gave the highest extraction efficiency, and ethyl acetate was selected as an extraction solvent of the analyte derivatives from samples.
Chromatography and mass spectrometry
The optimum derivatization conditions were applied to the analysis of MC, EC, and BC in fermented food and beverages by GC-MS. Figure 6 shows the GC-MS chromatogram after the derivatization of MC, EC, and BC. For the GC separation of the derivative, the use of a nonpolar stationary phase was found to be efficient. The derivatives of MC, EC, and BC showed a sharp peak, and the compound was quantified as an integration of the peak area. The retention times of xanthyl methylcarbamate, xanthyl ethylcarbamate and xanthyl butylcarbamate are shown in Figure 6. Extraneous peaks were not observed in the chromatograms near the retention times of the analytes.
The mass spectra of xanthyl methylcarbamate, xanthyl ethylcarbamate and xanthyl butylcarbamate by electron ionization at 70 eV have similar fragmentation pattern as shown in Figure 7. The molecular ions at m/z 255, m/z 269 and m/z 297 were appeared in mass spectra of three compounds. The fragment of m/z 240 was accounted for by the loss of [CH3], [C2H5] and [C4H9] from the each molecular ion and that of m/z 196 was accounted for by the loss of [COOCH3], [COOC2H5] and [COOC4H9], and m/z 222 were accounted for by the loss of [H2OCH3], [H2OC2H5] and [H2OC4H9] from the each molecular ion. The fragment of m/z 181 was a result of the xanthyl group.
Validation of the assay
The combination of a high derivatization yield and the high sensitivity of the derivative by EI-MS (SIM) allowed the detection of MC and EC at concentrations well below those reported previously. The limit of detection (LOD) and the limit of quantification (LOQ) were defined as the analyte concentration corresponding to a signal/noise ratio of 3 and 10 in the control food, in which MC and EC were not detected. The LODs in this study were 0.11 μg/kg for MC, and 0.12 μg/kg for EC, and the LOQs were 0.35 μg/kg for MC and 0.38 μg/kg for EC using a 2.0 g sample. Table 1 compares various analytical methods for determining the MC and EC in fermented food and beverages. The method permits the determination of two analytes below that detected previously using the GC-MS method, which was otherwise slightly higher than GC-HRMS or GC-MS/MS methods.
The calibration curves of the MC and EC were constructed by the reaction and extraction of the spiked food samples. Examination of the standard curve by computing a regression line of the peak area ratios for the MC and EC to the internal standard on concentrations using a least-squares fit demonstrated a linear relationship with correlation coefficients of 0.998 and 0.996, respectively. The line of best fit for the MC was y = 4.191 x - 0.0001 over a range of 1.0-100 μg/kg and that for EC was y = 13.46 x + 0.0051 over a range of 1.0-100 μg/kg, where x is the analyte concentration (mg/kg) and y is the peak area ratio of the analyte to the internal standard.
The accuracy can be assessed by determining the recovery in spiked samples: Intra-day accuracy was evaluated using five spiked samples at concentrations of 0.05 and 0.002 μg/kg for MC and EC, respectively. The inter-day accuracy was determined using the sample recovery on three different days. The accuracy was in range of approximately 90- to 109% and the precision of the assay was less than 12%, as shown in Table 2.
This paper was designed to describe a method to detect MC and EC in solid and liquid state matrices using GC-MS. Generally, many traditional Korean foods are made through fermentation of a mixture of various food materials, and therefore these foods have complicated matrix properties. When the proposed method was applied to the food items, interfering peaks were not observed in the chromatograms near the retention times of the analytes.
Using the proposed method, the levels of MC and EC were analyzed in sixteen traditional fermented Korean foods, including soybean paste, red pepper paste, and soy sauce, and eleven beverages and the results were shown in Table 3. MC was detected in a range from 0.4 to 0.8 μg/L in mainly fruit liquors. Most samples had detectable levels of EC in a range from 0.4 to 85.8 μg/L or μg/kg. The concentration range of the EC of each food or beverage type was found for soybean paste (0.9-2.7 μg/kg), red pepper paste (0.7-2.3 μg/kg), soy sauce (0.4-8.9 μg/L), and beverages (not detected-85.8 μg/L). From the results shown in Table 3, the prolonged mean storage time had no relationship with the detected content of EC.
The correlations between the levels of EC and MC in beverages also correlated well with each another (r2=0.69, P=0.001) due to the similar formation mechanisms. It is suggested that MC is also formed by the reaction of urea with methanol.