Method for the determination of natural ester-type gum bases used as food additives via direct analysis of their constituent wax esters using high-temperature GC/MS

Natural ester-type gum bases, which are used worldwide as food additives, mainly consist of wax esters composed of long-chain fatty acids and long-chain fatty alcohols. There are many varieties of ester-type gum bases, and thus a useful method for their discrimination is needed in order to establish official specifications and manage their quality control. Herein is reported a rapid and simple method for the analysis of different ester-type gum bases used as food additives by high-temperature gas chromatography/mass spectrometry (GC/MS). With this method, the constituent wax esters in ester-type gum bases can be detected without hydrolysis and derivatization. The method was applied to the determination of 10 types of gum bases, including beeswax, carnauba wax, lanolin, and jojoba wax, and it was demonstrated that the gum bases derived from identical origins have specific and characteristic total ion chromatogram (TIC) patterns and ester compositions. Food additive gum bases were thus distinguished from one another based on their TIC patterns and then more clearly discriminated using simultaneous monitoring of the fragment ions corresponding to the fatty acid moieties of the individual molecular species of the wax esters. This direct high-temperature GC/MS method was shown to be very useful for the rapid and simple discrimination of varieties of ester-type gum bases used as food additives.


Introduction
Natural ester-type gum bases are used worldwide as food additives to provide, for example, specific textures to chewing gums and surface coatings for chocolates and fruits. There are various kinds of natural ester-type gum bases, and each is comprised of a characteristic mixture of wax esters composed of a specific combination of longchain fatty acids and long-chain fatty alcohols, and thus they each have different properties with respect to their elasticity and flexibility. To assess the safety of these estertype gum bases and to appropriately manage their quality control, it is necessary to establish their specifications.
Specifications for some ester-type gum bases such as beeswax, candelilla wax, and carnauba wax have been adopted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA; FAO: Food and Agriculture Organization of the United Nations; WHO: World Health Organization), the EU, and the USA. In Japan, the use of many varieties of ester-type gum bases as natural food additives is allowed. However, for the most ester-type gum bases, there is no specification in the Japanese food additive regulation. To establish official specifications for these ester-type gum bases, a simple analytical method for confirmation and discrimination of the different estertype gum bases is required. We previously reported (Tada et al. 2007) an analytical method for 10 types of food additive gum bases such as lanolin, beeswax, and jojoba wax using gas chromatography/mass spectrometry (GC/ MS) following hydrolysis and derivatization of the wax esters. Using the method, major constitutive fatty acids and alcohols of the food additive gum bases were identi-fied, and it was clarified that each ester-type gum base has a characteristic composition of these constituent fatty acids and alcohols. However, this is a time-consuming and complicated procedure. In addition, it does not provide information on the composition of the wax ester species in the gum bases or the combination of fatty acids and fatty alcohols in each wax ester.
Several direct analytical methods for wax esters have been reported (Moldovan et al. 2002a;Regert et al. 2006;Str anskz y et al. 2006;Fitzgerald and Murphy 2007;Vrkoslav et al. 2010Vrkoslav et al. , 2011Zhang et al. 2010). However, GC/ MS methods using conventional temperatures (Str anskz y et al. 2006;Fitzgerald and Murphy 2007;Zhang et al. 2010) can be applied only for wax esters with carbon numbers of~40 and are not suitable for the detection of wax esters with carbon numbers of 50-60. Because the volatilities of long-chain wax esters are extremely low, they are not sufficiently detectable via GC using conventional column temperatures, which are typically less than 330°C. Although liquid chromatography/atmospheric-pressure chemical ionization-mass spectrometry (LC/APCI-MS) and LC/APCI-MS/MS methods (Vrkoslav et al. 2010(Vrkoslav et al. , 2011 have been applied for the detection of wax esters with carbon numbers of 52 and 54 and are more suitable for the detection of unstable highly unsaturated wax esters (Vrkoslav et al. 2010), these methods take longer than 100 min for the detection of wax esters with carbon numbers greater than 52. Furthermore, some direct high-temperature GC/MS for the detection of wax esters have been reported (Moldovan et al. 2002a;Regert et al. 2006;Vrkoslav et al. 2010), but the target materials for these analyses were mainly wax ester standards or sculptures. To the best of our knowledge, development of a direct GC/MS method for the determination of many varieties of gum bases used as food additives has not yet been achieved.

Instrumentation
The GC/MS system (Shimadzu Co. Ltd., Kyoto, Japan) consisted of a GC-17A gas chromatograph equipped with an MS-QP5050 mass spectrometer run in electron ionization (EI) mode using an AOC-20i auto injector.

GC/MS analysis of wax esters in gum bases
GC/MS analysis of the wax esters was performed on a DB-1 HT fused-silica capillary column (15 m 9 0.25 mm, film thickness of 0.10 lm; J&W Scientific, Folsom, CA). The injector and detector temperatures were set at 390°C, and the column temperature was programed from 120°C to 240°C at 15°C/min and then from 240°C to 390°C at 8°C/ min and finally maintained at 390°C for 6 min. Samples (1 lL) were injected through a split-injector (1/5). MS spectra were detected in EI mode by scanning m/z values ranging 50-920. Samples and standards were dissolved in hexane, toluene, or ethanol (0.1-1.0 mg/mL). Each sample solution was injected in duplicate, and reproducibility of the results was confirmed.

Results and Discussion
Establishment of a direct analytical method for wax esters via high-temperature GC/MS on a capillary column The major constituents of gum bases used as food additives are wax esters. They are composed of long-chain fatty acids and long-chain alcohols. Since the volatilities of these wax esters are very low, they are not sufficiently detected via GC using conventional column temperatures (<330°C). To overcome this problem, a GC/MS method was developed using a capillary column that enables hightemperature analysis for the direct detection of wax esters. The appropriate temperatures for the capillary column, injector, and detector were examined using standard wax ester mixtures. It was observed that, considering the sensitivity, the suitable temperature for the injector and detector was 390°C. In addition, an increasing program for the column temperature from 120°C to 390°C provided good separation of the structural isomers of the various triglycerides in the standard mixtures. Figure 1A shows the GC/MS total ion chromatogram (TIC) of a standard mixture of various esters analyzed using the established method. This standard mixture contained straight-chain esters, branched-chain esters, saturated esters, unsaturated esters, and hydroxy esters with carbon numbers ranging from 19 to 44. All of the standard esters were well-separated within 19 min. As shown in Figure 1B, retention times of the standard esters approximately correlate with their carbon numbers, regardless of their structural type (Str anskz y et al. 2006;Zhang et al. 2010). These results suggest that the carbon number of esters can be estimated from the retention times of their peaks. Next, to assess the linearity of the relationship between peak areas in the TIC and the concentrations of the standard esters, calibration curves were constructed. As can be seen in Figure 1C, sufficient linearity for rough determination of the concentrations of wax esters with carbon numbers ranging from 29 to 44 is observed over the concentration range 0.1-0.5 mg/mL (correlation coefficient R 2 = 0.9876). These results indicate that analysis of the TIC peak areas of the wax esters can be used to roughly determine the concentrations of the corresponding esters in the gum bases.

Application of the established GC/MS method to the analysis of wax esters in food additive gum bases and experimental reagents
To investigate the types and quantities of esters contained in various gum base samples, TIC patterns of food additive gum bases and experimental reagents were determined using the established GC/MS method. As shown in Figure 2, similar TIC patterns were observed for gum bases derived from the same waxes. It was also confirmed that MS spectra of the TIC peaks were consistent with those of TIC peaks observed at identical retention times for gum bases derived from the same types of wax (data not shown). These results confirm that the gum bases formulated with the same types of wax have specific and characteristic TIC patterns and ester compositions.
Next, GC/MS TIC patterns of 10 types of food additive gum bases derived from different types of wax were obtained (Fig. 3). Based on the results for the molecular ions and correlation between the retention times and ester carbon numbers (Fig. 1B), it was possible to estimate the carbon numbers of the wax esters represented by the individual TIC peaks. In addition, the other constituents were also identified using previously reported data (Asano 1977;Lawrence et al. 1982;Tonogai et al. 1985;Tachibana et al. 1992;Jover et al. 2002;Moldovan et al. 2002b;Bonaduce and Colombini 2004;Jim enez et al. 2004;Jin et al. 2006) and by comparison to the analyses results described above for the standards and libraries of MS spectra such as NIST 147, NIST 27, and Wiley 7. As shown in Figure 3, each TIC of the 10 types of food additive gum bases has a characteristic pattern. In addition, it was observed that the composition of major esters in the food additive gum bases was nearly the same as those previously reported (Asano 1977;Lawrence et al. 1982;Tonogai et al. 1985;Tachibana et al. 1992;Jover et al. 2002;Moldovan et al. 2002b;Bonaduce and Colombini 2004;Jim enez et al. 2004;Jin et al. 2006  ( Fig. 3A), wax esters with carbon numbers ranging from 48 to 52 were detected along with free cholesterols and sterol esters (Jover et al. 2002;Moldovan et al. 2002b), while in the chromatogram of beeswax (Fig. 3B), wax esters with carbon numbers ranging from 40 to 48 were detected along with hydrocarbons such as C 27 H 56 (Bonaduce and Colombini 2004;Jim enez et al. 2004). For candelilla wax (Fig. 3D), saturated hydrocarbons such as C 31 H 64 were the major constituents in accordance with a previous report (Lawrence et al. 1982;Tonogai et al. 1985). The shellac wax (Fig. 3E) contained wax esters with carbon numbers ranging from 44 to 50 along with free alcohols, free fatty acids, and hydrocarbons (Lawrence et al. 1982). As shown in Figure 3F-H, carnauba wax, rice bran wax, and montan wax contained similar wax esters in terms of their carbon numbers. However, these results suggest that the three gum bases may be distinguished by evaluating the TICs for the peaks of other characteristic constituents such as the alcohol with carbon numbers 32 in carnauba wax (Lawrence et al. 1982;Tonogai et al. 1985) and hydrocarbons in montan wax (Asano 1977;Lawrence et al. 1982). As previously reported (Tachibana et al. 1992;Jin et al. 2006), major compo-nents of urushi and Japan waxes were found to be triglycerides ( Fig. 3I and J). As can be seen in the magnified chromatograms of urushi and Japan waxes ( Fig. 4A and B, respectively). The ratio of glycerol 1,2-dipalmitate 3oleate (PPO) to glycerol tripalmitate (PPP) (table in Fig. 4C for abbreviation definitions) for urushi wax (Fig. 4A) is higher than that for the Japan wax ( Fig. 4B) (Tachibana et al. 1992;Jin et al. 2006). Accordingly, these two gum bases can be distinguished using this ratio. These results thus demonstrate that food additive gum bases can be distinguished from the other based on the TIC patterns obtained using the established direct GC/MS analysis (without hydrolysis and derivatization) of the esters in these food additives.
Comparison of the MS chromatograms of carnauba wax, rice bran wax, and montan wax As shown in Figure 3F-H, it was difficult to differentiate carnauba wax, rice bran wax, and montan wax using the TICs alone. Therefore, the MS chromatograms were analyzed. As shown in Figure 5, the MS spectrum of a stan-  dard of the saturated straight-chain ester, behenyl stearate), obtained using the established GC/MS method contained product ions derived from the fatty acid moiety of the ester ([R 1 COO] + , [R 1 CO] + , and [R 1 ] + ) and product ions derived from the alcohol moiety ([R 2 ] + and [R 2 OCO] + ). It was observed that the product ions corresponding to the fatty acid and alcohol moieties of standard wax esters are generally observed in their MS spectra under these conditions. These results suggest that analysis of the MS spectra obtained using the established method can provide information on the constitutive fatty acids and alcohols of the esters. Therefore, to more clearly distinguish between carnauba wax, rice bran wax, and montan wax, the MS chromatograms of the product ions derived from the constitutive fatty acids of the esters in these three waxes were compared. As can be seen in Figure     C24:0, regardless of the carbon number of the esters (Fig. 6B). In addition, as can be seen in Figure 6C, the esters detected in montan wax consisted of fatty acids with longer chains (C24:0-C32:0) than those of the esters detected in the other two gum bases. On the basis of these results, compositions of the constitutive fatty acids in the C54 and C56 esters in the three gum bases were then compared ( Table 1). As shown in Table 1, among the three gum bases, composition of the constitutive fatty acids in the esters with identical carbon numbers were clearly different. These results indicate that these three gum bases can be distinguished by comparing the mass chromatograms and TICs obtained using the newly developed direct GC/MS method. We previously reported (Tada et al. 2007) an analytical method for the determination of food additive gum bases using GC/MS after hydrolysis and derivatization, and identified the major constitutive fatty acids and alcohols of the gum bases. However, the method does not provide information on the constitutive fatty acid for each respective ester in the gum bases. With the present analytical method, the esters in the gum bases can be directly analyzed with simultaneous prediction of the constitutive fatty acids of the corresponding esters using the MS spectra of the ester peaks. In addition, this direct GC/MS method is simple, clear, and particularly useful for the rapid analysis of various types of food additive gum bases without the need for hydrolysis and derivatization of the esters in the gum bases.