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Note 43: Volatile Organic Composition In Blueberries

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By Santford Overton and John J. Manura
1999

INTRODUCTION

Volatile and semi-volatile organic compounds present both in the sample matrix and in the headspace aroma exhibit a tremendous influence on the flavor/fragrance qualities of blueberries. There is a concern in the blueberry industry as to what constitutes the optimum conditions for harvesting blueberries so as to provide a consistent quality and flavor of blueberry to the consumer. Several investigations have reported that the concentrations of volatile constituents increased with the maturation of blueberries. The major volatiles of blueberries appear to be useful indices for determining maturity, and recently, the determination of flavor precursors and intermediates have become the target of flavor studies. To date, headspace GC analysis, cryofocusing techniques, and high resolution GC have been used for the analysis of blueberry volatiles from promising cultivars under development. However, static headspace techniques are limited in their detection and identification of many organic volatiles and especially semi-volatile organics. Other techniques are needed to profile a wider range of volatile and semi-volatile organics in blueberries and to identify the flavors, fragrances, off-flavors, off-odors and potential contaminants that may be present. The purpose of this investigation is to develop an analytical technique that could detect and identify a wide range of volatile and semi-volatile organic compounds in blueberries. For this study, volatile organic compounds are purged from blueberry samples followed by trapping on Tenax® TA adsorbent resin using a dynamic purge and trap technique (P&T), and then chromatographed on two different columns to compare and quantitate the volatile organics present. The adsorbent traps are subsequently analyzed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). The P&T technique permits the analysis of a wider range of both volatile and semi-volatile organic compounds and is more sensitive as compared to the static headspace technique.

Instrumentation

Purge & Trap of Blueberries

Figure 1 - Purge and Trap Of Blueberries

Samples were collected using a Scientific Instrument Services Purge and Trap System. This apparatus (Fig. 1) consists of a sparge gas inlet connected to a stainless steel purging needle that is inserted through an adaptor fitting into the blueberry liquid at the bottom of the 10 ml purge and trap test tube. A dry purge gas inlet is located at a right angle to the sparge gas inlet at the top of the apparatus. The purpose of the dry purge is to reduce the water vapor condensation on the adsorbent trap. Opposite the dry purge inlet is the connector for the desorption tube. To this fitting a glass-lined stainless steel (GLT) desorption tube containing the adsorbent resin is attached for the trapping of the purged volatiles.

Samples were desorbed into the GC injection port using the SIS Model TD-3 Short Path Thermal Desorption System. The desorption tube blocks were set to 220 degrees C and the desorption system flow rate was set to 12 ml/min. The desorption system timer was set to 5.0 minutes for the desorption system cycle.

For both systems described below, the head of each capillary column was maintained at -70 degrees C using an S.I.S. Cryotrap model 951 during the desorption and extraction process and then ballistically heated to 200 degrees C to release the volatiles after which the GC oven was temperature programmed from 35 degrees C (hold for 5 minutes) to 80 degrees C at 10 degrees C/min, then to 200 degrees C at 4 Degrees C/min and finally to 260 degrees C at a rate of 10 degrees C/min.

System A - The experiments were conducted using a Scientific Instrument Services model TD-3 Short Path Thermal Desorption System accessory connected to the injection port of a HP 5890 Series II GC interfaced to an HP 5971 Mass Selective Detector (MSD). The HP 5890 Series II GC contained a short 0.5 meter by 0.53 mm diameter fused silica precolumn attached to the injection port end of a 30 meter x 0.25 mm i.d. DB-5MS J&W capillary column containing a 0.25 micron film thickness. The GC injection port was set to 260 degrees C and a 10:1 split was used. The mass spectrometers were operated in the electron impact mode (EI) and scanned from 35 to 550 daltons during the GC run for the total ion chromatogram. The HP Chem Station software with the Wiley NBS library was used to analyze the eluted volatiles and identify each compound.

System B. The experiments were conducted using a Scientific Instrument Services model TD-3 Short Path Thermal Desorption System accessory connected to the injection port of a HP 5890 Series II GC with electronic pressure control interfaced to an HP Engine Mass Spectrometer. A SGE (Scientific Glass Engineering, Austin Texas) BPX35 capillary column, 60 meters long by 0.22 mm i.d. x 0.25 micron film thickness was used in the GC oven. The GC injection port was set to 220 degrees C. The mass spectrometers were operated in the electron impact mode (EI) and scanned from 35 to 550 daltons during the GC run for the total ion chromatogram. The HP Chem Station software with the Wiley NBS library was used to analyze the eluted volatiles and identify each compound.

Experimental

Three varieties of blueberry (Jersey lowbush, Bluecrop lowbush & Rancocas lowbush) were analyzed by a dynamic P&T technique and chromatographed on both a 30 meter DB-5MS capillary column and a 60 meter BPX35 capillary column to compare and quantitate the volatile organics present. For quantification, a deuterated cymene internal standard was spiked into the adsorbent traps after the volatiles from the blueberries had been adsorbed onto the adsorbent resin traps. No correction for extraction efficiency of recovery is achieved using this technique; however, it functions as a useful means of quantifying the levels of components present on the adsorbent traps.

Approximately 100 grams of blueberry were homogenized for 3 minutes using a Waring® commercial blender. Sample sizes of 10-11 grams of this blueberry homogenate were then transferred into a 10 ml purge and trap test tube and heated to 60 degrees C in a water bath. Samples were then purged for 45 minutes with high purity helium at 20 ml/min (900 ml total) with an additional 25 ml/min dry purge using the S.I.S. Purge and Trap system. The volatile analytes were purged onto a preconditioned 4.0 mm i.d. glass-lined stainless steel desorption tube packed with 200 mg of Tenax TA. The samples were subsequently spiked with 100 ng of d-cymene internal standard by injecting 1 ul of a 100 ng/ul stock solution of a d-14 cymene in methanol by syringe injection into the Tenax matrix, and then purged for an additional 5 minutes with helium at 50 ml/min to remove the methanol. The desorption tube with sample and internal standard was then attached to the Short Path Thermal Desorption System and a syringe needle attached. The desorption tube was injected into the GC injection port and the volatiles desorbed into the GC at desorption block temperatures of 220 degrees C for 5 minutes and a flow rate of 12 ml/min. The volatiles were cryo-focused at -70 degrees C at the front of the GC column during the desorption process and then released onto the GC column by rapidly heating the cryo-trap to 220 degrees C. The GC column temperature program was initiated and the eluted analytes detected and identified via the mass spectrometer.

Results and Discussion

Blueberry 2

Fig. 2 - Jersey Lowbush analyzed On J&W DB-5MS Column

Blueberry 3

Fig. 3 - Rancocas Lowbush Analyzed On J&W DB-5MS Column

Blueberry 4

Fig. 4 - Bluecrop Lowbush Analyzed On J&W DB-5MS Column

Three varieties of blueberry were analyzed to identify, compare and quantify the volatile organics present using both the SGE BPX35 and the J&W DB-5MS capillary columns, and from the data, determine the most sensitive method of analysis. Over 100 volatile organics were identified in the blueberries studied. The blueberries studied produced 50 or more volatile organics which were identified in addition to many more that were either too weak or in which a good NBS library match was not achievable. The blueberries possessed numerous mono- and sesquiterpenoid compounds as well as numerous straight and branched chain hydrocarbons, aldehydes, alcohols, ketones and esters (Figs. 2-7). Although they possess many common compounds, each blueberry variety had its own distinct fingerprint chromatograph.

The SGE BPX35 capillary column provided better separation and resolution for the three blueberry samples analyzed (Figs. 5-7) than the J&W DB-5MS (Figs. 2-4) capillary column for this application . Due to the slightly higher polarity of the BPX35 column the more polar alcohols, aldehydes and acids produced highly resolved GC peaks which were easily detected and identified. Many of these polar compounds were not resolved and identified with the non polar DB5-MS column.

The blueberries were found to contain the flavor compound benzaldehyde which has an almond-like odor and the aliphatic compounds hexanal, heptanal and nonanal (Figs. 2-7). It has been assumed that unsaturated fatty acids, primarily linoleic and linolenic acids, are the precursors of these aliphatic compounds and may contribute to the development of rancid flavor. The presence of the branched aldehydes 3-methyl-butyraldehyde and 2-methyl-butyraldehyde in each of the blueberries (Figs. 2-4) contribute to the fruity flavor notes as well as reflect the microbial quality of the blueberry. The predominant terpenes included 1,8-cineole and linalool with trace amounts of cymene, limonene, 1-terpineol, trans-caryophyllene (Figs. 2-4) and 3-carene (Figs. 5-7). Citral which has a strong lemon odor was also identified in the Rancocas and Bluecrop varieties (Figs. 3&4).

Blueberry 5

Fig. 5 - Jersey Lowbush Analyzed On SGE BPX-35 Column

Blueberry 6

Fig. 6 - Rancocas Lowbush Analyzed On SGE BPX-35 Column

Fig. 7 - Bluecrop Lowbush Analyzed On SGE BPX-35 Column

The major esters found in the blueberry fruit included ethyl acetate and 3-isopropyl-buyrate. It is generally considered that esters primarily contribute to the fruity and floral notes. SGE's BPX35 capillary column (Figs. 5-7) provided additional information to the volatile organic composition of blueberries with the identification of the alcohols 1-butanol, 1-pentanol, 1-hexanol, 2-ethyl-1-hexanol, 1-heptanol, 1-octanol and 1-nonanol which were not detected with J&W's DB-5MS column. The production of these alcohols may be characteristic of the fruit during maturation. Linalool oxide, an isomer of lilac alcohol, and tetrahydro-furfuryl-(2)-alcohol were identified in the Jersey and Rancocas varieties (Figs. 2&3) while the higher molecular weight compound decadienal was found in each of the varieties (Figs. 2-4). This compound contributes to the flavor and aroma of the blueberries and is the result of fatty acid decomposition.

The Jersey and Bluecrop varieties exhibited several furan derivatives (Figs. 5&7) which reflect the microbiological purity and storage conditions of the blueberries. The antioxidant butylated hydroxytoluene was detected in each of the blueberries and is probably derived from the packaging material during storage. Other middle-chain aliphatic alcohols, aldehyde and ketone derivatives were also found in each of the varieties of blueberry.

Conclusion

The Short Path Thermal Desorption System used in conjunction with a dynamic purge and trap technique along with the appropriate capillary column permits the identification and quantification of trace levels of volatile organics in blueberries. The method described above permits both the qualitative and quantitative analysis of blueberries to identify the volatile organics, flavoring compounds, contaminants and off-odor compounds present. This technique has proven effective in detecting and identifying a larger number of organic compounds at concentrations lower than was previously obtainable via other analysis techniques, such as static headspace GC analysis. It also represents a tremendous improvement over the time-consuming solvent extraction techniques normally used in the laboratory. This technique can be easily incorporated into a troubleshooting technique to detect problems in a wide variety of commercial food products, to compare various competing manufacturers products, as well as a quality control program.

In addition the SGE BPX-35 mid polarity capillary column was found to be superior for the analysis of blueberries and other food products which contain numerous aldehydes and alcohols in the volatile flavor profile.

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