Note 12: Identification of the Volatile and Semi-Volatile Organics In Chewing Gums By Direct Thermal Desorption

INTRODUCTION

A wide variety of volatile and semi-volatile organic flavoring chemicals are formulated into consumer brands of chewing gums. These flavoring components consist of many essential oil plant extracts such as peppermint, spearmint and cinnamon, as well as artificial flavoring compounds. In addition, artificial preservatives, as Butylated Hydroxytoluene (BHT) and Butylated Anisole (BHA), are present in the chewing gums. Previous methods for the extraction and analysis of these compounds in chewing gums, plant materials and essential oils used techniques such as solvent extraction (1, 2, 3 & 4), headspace analysis (5 & ,6), and microdistillation (6, 7, 8) followed by capillary gas chromatography. These methods either require large sample sizes, the use of solvents or considerable time and effort to achieve the analysis.

A new technique entitled Direct Thermal Desorption (DTD) using a Thermal Desorption apparatus attached to the injection port of the GC/MS system permits the direct thermal extraction of the volatile and semi-volatile organics directly from small samples of chewing gums without the need for solvent extraction or other sample preparation. The technique uses a combination of ballistic heating of the sample and GC carrier gas through and over the sample to thermally extract the organics directly from the small solid gum samples (1 to 10 milligram) and onto the front of the GC column for subsequent analysis via GC and GC/MS instrumentation. The method requires no solvent extraction. The thermal extraction is complete in only 10 minutes. Accurate quantification of BHT and other volatiles in various food products (10) and pharmaceutical products (11) using this technique provide for the detection of organics at levels from 20 ppb to 400 ppm with a relative standard deviation of 5%. This method can be used to identify the active components and flavoring compounds in chewing gum and other flavored food products and herbs. It can also be used to evaluate and select flavoring compounds and essential oils to be used for the production of these products. The GC chromatogram profile can be used for both the qualitative fingerprinting of the flavoring compounds, as well as for the quantitative identification of these compounds.

INSTRUMENTATION

A Short Path Thermal Desorption System Model TD-2 (Scientific Instrument Services, Ringoes, NJ) was attached to the injection port of a Hewlett Packard 5890 Series II GC and 5971 Mass Selective Detector. The mass spectrometer was operated in the electron impact mode (EI) at 70 eV and scanned from 35 to 350 daltons during the GC run. The data was interpreted on the H.P. ChemStation software and library searched using the Wiley NBS Library.

A short 0.5 meter by 0.53 mm diameter fused silica guard column was attached to the injection port end of a DB5-MS fused silica capillary column, 30 meter x 0.25 mm I.D., 0.5 micron film thickness (J&W). The GC injection port was set to 250 degrees C and a 4:1 split was used during the injection step. The GC oven was maintained at 0 degrees C during the desorption and extraction process, after which the column oven was temperature programmed from 0 degrees C to 300 degrees C at a rate of 10 degrees per minute.

Figure 1 - Direct Thermal Desorption of Chewing Gum by Thermal Desorption Quantification of BHT

EXPERIMENTAL

The sampling tubes consist of a 1/4 in. O.D. by 4.0 in. long glass lined stainless steel tubes packed with a 3 mm by 10 mm silanized glass wool plug (Figure 1). Before use, each of the sample tubes with glass wool plug inserted was conditioned in the S.I.S. Tube Conditioning System at 350 degrees C for 60 minutes with a 10 ml/min flow of high purity nitrogen through each tube. This high temperature flow conditioning assured that no foreign contamination or cross contamination of samples occurred. In a likewise manner, all needle transfer lines are flow conditioned in the same conditioning oven. After conditioning, the samples and transfer line needles are allowed to cool for approximately 10 minutes before samples are loaded.

The S.I.S. Short Path Thermal Desorption System was used with the Direct Thermal Desorption Technique for the analysis of all samples (Figure 1). The sample tubes are placed on an analytical balance; the balance is tarred. One to 10.0 milligrams of the thinly sliced chewing gum samples are inserted into the sample tubes on top of the glass wool plug, and the weight of the sample was accurately determined on the analytical balance. The sample tube with sample was attached to the desorption system; a transfer line needle was attached to the other end of the sample tube. Helium carrier gas was diverted through the sample tube at a flow rate of 2 ml/min for 30 seconds in order to purge residual air from the inside of the sample tube. Thereafter, the sample was injected into the GC injection port. The two temperature regulated heating blocks were closed around the sample tube to ballistically heat the sample tube and samples within. The sample was thermally extracted for 10 minutes at a heater block temperature of 150 degrees C to extract the volatile components into the GC injection port. This combination of ballistic heating and GC carrier gas flowed through the sample to drive any volatile or semi-volatiles into the hot GC injection port where they were subsequently cryofocused at the front of the GC column in a narrow band. The molecular weight or boiling point range of the compounds extracted was determined by the temperature of the heater blocks (desorption temperature). For the chewing gums analyzed, this temperature was kept at 150 degrees C in order not to decompose the sugar within the gum samples. After the thermal extraction process was complete, the heater blocks were opened, the sample and transfer line needle were withdrawn from the GC injection port and the temperature programming of the GC column was commenced to elute the organic compounds from the GC capillary column. A more detailed description of the theory of operation of the Short Path Thermal Desorption System and the technique of Direct Thermal Desorption has been described elsewhere (12, 13, and 14).

The total ion chromatogram for each of the chewing gums analyzed was plotted and the mass spectrometer was scanned over the mass range 35 to 350 daltons and the Wiley NBS library was searched for each compound to identify each of the components eluted from the column. Blanks were run between samples to assure that no Memory effects occurred.

Results and Discussion

Figure 2 - Peppermint Chewing Gum, 1.8 mg gum int, std., Desorbed for 10 min at 150 Degrees C To Identify Flavoring Compounds

Figure # 2 is the total ion chromatogram of a 1.8 milligram sample of a peppermint chewing gum. The major peaks in the chromatogram were identified as Menthone and Menthol and have been previously reported from essential oil extracts from peppermint oils by other authors (3, 5 and 6). As has been reported in these same publications, a large number of other terpenes, hydroxy terpenes and sesquiterpenes have also been separated and identified with this technique. The importance of these minor flavoring components cannot be over looked. Although minor in relative concentration to the major flavoring compounds, these minor ingredients can add distinctive flavors or off-flavors to the finished product. By making subtle changes in the relative amounts of these minor terpenes such as limonene, pinene and carophyllene, unique flavors can be achieved. Also identified in this chewing gum sample are many hydrocarbon peaks from the gum base. In addition, BHT was identified and quantified (10) as previously reported. Several different manufactures of peppermint gum were analyzed and compared. All the gums analyzed exhibited the same major components identified above with only minor differences in the relative concentration of the various terpenoids. The technique could readily be applied to develop a rapid and accurate method for the qualitative and quantitative comparison of these gum samples as well as a routine quality control procedure to monitor the day-to-day production of these gums in a manufacturing plant.

Figure 3 - Spearmint Chewing Gum, B: 1.9 mg gum + int. std., Desorbed for 10 min At 150 degrees C To Identify Flavoring Compounds

Figure # 3 is the total ion chromatogram of a spearmint flavored chewing gum. The major compound identified in this gum was carvone and a secondary peak for menthyl acetate. Among the other peaks prevalent were some of the same minor peaks present in the peppermint gum samples, mainly menthone and menthol. The presence of small amounts of peppermint in the spearmint gums was identified in many of the spearmint gum samples analyzed. It is used to enhance the flavor of the spearmint. This again points out the importance of the minor components and their effect on the subtle differences in flavor and how they can contribute to the finished product. In addition, several peaks from the hydrocarbon gum base, as well as BHT, were also identified in the spearmint gum samples.

Figure 4 - Cinnamon Chewing Gum, 2.3 mg gum + int. std., Desorbed For 10 min at 150 Degrees C To Identify Flavoring Compounds

Figure # 4 is the total ion chromatogram of a cinnamon flavored chewing gum. The major compounds identified for this gum include cinnamaldehyde, cinnamyl alcohol and eugenol. In addition, smaller amounts of menthone and menthol were identified, which indicate that the gum contained small amounts of peppermint in addition to the cinnamon flavoring compounds. It has become quite evident in the analysis of many gum samples that, even though the gum is labeled as a particular flavor, the manufacturers alter the pure flavoring compounds with other ingredients, in particular peppermint, to enhance or improve the flavor. As in the samples above, this gum sample also contained several hydrocarbons from the gum base, as well as BHT preservative.

Figure 5 - Fruit Flavored Chewing Gum, 1.9 mg gum + int. std., Desorbed For 10 min At 150 Degrees C To Identify Flavoring Compounds

Figure # 5 is the total ion chromatogram of a fruit flavored chewing gum. Again, this gum indicates that a mixture of flavoring compounds were used in its manufacture. Methyl salicylate (wintergreen oil), cinnamaldehyde (cinnamon), limonene (lemon) and citral (lemon flavoring) were identified among the many compounds isolated from this gum. This mixture of various fruit flavors can impart a unique flavor to the gum depending on the relative concentration of the various flavors. In addition, a large number of hydrocarbons and BHT were also identified in this fruit flavored gum.

Most of the gums analyzed contained BHT as a gum preservative. The BHT was quantified using deuterated BHT (d20 -BHT) as an internal standard as previously reported (10). The BHT quantification produced a linear regression calibration curve for levels of BHT from 400 ng down to 0.1 ng, and was thus able to quantify BHT at levels from greater than 400 ppm down to 20 ppb with high accuracy and a relative standard deviation of 5%. In most of the gum samples analyzed, the BHT was present in levels from 50 ppm up to 200 ppm, the legal limit in the United States.

CONCLUSION

A wide number of terpenes, hydroxy terpenes and sesquiterpenes were detected and identified in solid gum samples by thermally extracting these volatile and semi-volatile compounds using the Direct Thermal Desorption technique in conjunction with the Short Path Thermal Desorption System. The method has proven to be a versatile and time-saving technique not only for the qualitative analysis of the various flavoring terpenoids in chewing gums but also for the quantification of BHT preservatives. This quantification could easily be extended to all of the other compounds extracted and isolated from the chewing gums.

This technique offerS several unique advantages over other techniques such as solvent extraction, headspace sampling and microdistillation. First, it is quick and requires no sample preparation. Second, no solvent extraction is required; this eliminates the exposure of laboratory staff to these solvents and the disposal of these solvents. In addition, the thermal extraction of the volatiles and semi-volatiles from the solid sample is thorough and complete, and no trace of foreign contaminates from the extraction technique, solvents or other Memory Effects contribute to the chromatograms. The Direct Thermal Desorption technique makes it possible to identify all the volatiles and semi-volatiles accurately and quantitatively. By selecting the proper desorption temperature, the user can control the complexity of the chromatogram. This same technique has been used to detect and quantify BHT in various food products (10), polynuclear aromatics (PNA) and polycyclic biphenyls (PCB) in soils (15), and naphthalene in pharmaceutical products (11).

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