Note 35: Volatile Organics Composition of Cranberries

INTRODUCTION

Volatile and semi-volatile organic compounds present both in the sample matrix and in the headspace aroma exhibit a great influence on the flavor/fragrance qualities of cranberries. There is a concern in the cranberry industry as to what constitutes the optimum conditions for harvesting cranberries so as to provide a consistent quality and flavor of cranberry to the consumer. An additional concern also exists as to the level of pesticide residue that may be present in the fruit. Several investigations have reported that the concentrations of volatile constituents increased with the maturation of cranberries. The major volatiles of cranberries 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 cranberry volatiles from promising cultivars under development. However, static headspace techniques are limited in their detection and identification of many organic volatiles and especially the semi-volatiles organics. Other analytical techniques are needed to profile a wider range of volatile and semi-volatile organics in cranberries and to identify the flavors, fragrances, off-flavors, off-odors and potential contaminants that may be present. The purpose of this investigation was to develop an analytical technique that could detect and identify a wide range of volatile and semi-volatile organic compounds in cranberry. For this study, volatile organic compounds were purged from cranberry samples followed by trapping on Tenax® TA adsorbent resin using a dynamic purge and trap technique (P&T). The adsorbent traps were 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

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 a 10 ml test tube. A dry purge gas inlet is located at a right angle to the sparge gas inlet at the top of the apparatus. This can be left in the closed or open position. 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 glass-lined stainless steel (GLT) desorption tube containing the adsorbent resin. The Purge and Trap System also contains two ball rotameters with adjustable needle valves mounted on a stationary base and permits the visual indication and independent adjustment of the carrier gas flow to each of the gas inlets.

All experiments were conducted using a Scientific Instrument Services model TD-3 Short Path Thermal Desorption System accessory connected to the injection port of an HP 5890 Series II GC interfaced to an HP 5971 Mass Selective Detector. The mass spectrometer was operated in the electron impact mode (EI) at 70 eV and scanned from 35 to 550 daltons during the GC run for the total ion chromatogram.

A short 0.5 meter by 0.53 mm diameter fused silica precolumn was attached to the injection port end of a 30 meter x 0.25 mm i.d. DB-5MS capillary column containing a 0.25 um film thickness. The GC injection port was set to 260 degrees C and a 10:1 split was used. The head of the 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, after which the 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.

Experimental

Four varieties of cranberry (Early Black, Howes, Wilcox & Franklin) harvested on August 15, 1995 and September 15, 1995 were analyzed by a dynamic P&T technique to compare and quantitate the volatile organics present during different times of the growing season. For quantification, an internal standard was spiked into the adsorbent traps after the sample had been isolated. 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 cranberry in 100 ml of ultra pure water were homogenized for 3 minutes using a Waring commercial blender. Sample sizes of 10-11 g of cranberry homogenate were then transferred into a 10 ml test tube and heated to 60 degrees C for 45 minutes. Samples were sparged with high purity helium at 20 ml/min with an additional 25 ml/min dry purge using an S.I.S. Purge and Trap System. Volatile analytes were gas extracted and carried to a preconditioned 4.0 mm i.d. glass-lined stainless steel desorption tube packed with 200 mg of Tenax TA. Once the samples were collected, they were purged with helium at 50 ml/min for 5 minutes, spiked with 100 ng of d-14 cymene internal standard by injecting 1 ul of a 100 ng/ml of a d-14 cymene stock solution in methanol by syringe injection into the Tenax matrix, and then purged for an additional 5 minutes 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 at desorption block temperatures of 220 degrees C for 5 minutes.

Results and Discussion

Four varieties of cranberry harvested at different times during the growing season were analyzed to identify, compare and quantify the volatile organics present, and from the data, determine the most sensitive method of analysis. Over 100 volatile organics were identified in the cranberries studied. Most of the cranberries studied produced 50 or more volatile organics which were identified in addition to many more that were either too weak to identify or in which a good NBS library match was not achievable. The cranberries possessed numerous mono- and sesquiterpenoid compounds as well as numerous straight and branched chain hydrocarbons, aldehydes, alcohols, ketones and esters (Figs. 2-9). Although they posess many common compounds, each cranberry variety had its own distinct fingerprint chromatograph.

The cranberries harvested on August 15 were found to contain the flavor compound benzaldehyde which has an almond-like odor and the aliphatic compounds hexanal, heptanal, octanal and nonanal (Figs. 2-5). 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 cranberries contribute to the fruity flavor notes as well as reflect the microbial quality of the cranberry. The predominant terpenes included 1, 8-cineole, 1 terpineol and linalool with trace amounts of cymene, limonene, terpinolene and carene. In addition, the Howes and Wilcox varieties both contained trace amounts of a-pinene (Figs. 3&4). Citral, which has a strong lemon odor, and the sesquiterpene, humulene, were also identified in the Wilcox variety (Fig. 4). Trace amounts of benzeneacetonitrile or benzyl cyanide which exhibits an aromatic odor were detected in each of cranberry varieties early in the growing season.

Increasing concentrations of the flavor compound benzaldehyde were observed in the cranberry varieties harvested later in the growing season on September 15 (Figs. 5-9). It has been previously reported that benzaldehyde, linalool and C-10 lactones increased during fruit maturation, while C-6 aldehydes decreased. The major esters found in the maturing fruit were ethyl acetate, ethyl propionate, ethyl benzoate and ethyl phenylacetate. It is generally considered that esters primarily contribute to the fruity and floral notes. Ethanol and the aliphatic compound 1-octen-3-ol were also identified in the cranberries harvested later in the season. The production of ethanol is characteristic of fruit during maturation whereas the presence of 1-octen-3-ol, which was also detected earlier in the growing season, suggests that the activity of lipoxygenase and hydroperoxide lyase producing C8 compounds from linoleic acid was occurring. Linalool oxide, an isomer of lilac alcohol, and tetrahydro-furfuryl-(2)-alcohol were identified in each of the cranberry varieties while the higher molecular weight compound decadienal was found in the Howes and Franklin varieties (Figs. 7&9). This compound contributes to the flavor and aroma of the cranberries and is the result of fatty acid decomposition. Benzeneacetonitrile was no longer detectable later in the season. In addition to the terpene compounds previously identified, the Early Black and Wilcox varieties were found to contain the flavor -damascenone which has a nutmeg-like odor (Figs. 6&8).

The Wilcox variety possessed numerous furan derivatives which reflect the microbiological purity and storage conditions of the cranberries (Fig. 8). The antioxidants butylated hydroxytoluene and ionol 2 were detected in each of the cranberry varieties during both collection periods and are probably derived from the packaging material during storage. Other middle-chain aliphatic alcohols, aldehydes and ketone derivatives were also found in each of the varieties of cranberry.

Conclusion

The Short Path Thermal Desorption System used in conjunction with a dynamic purge and trap technique permits the identification and quantification of trace levels of volatile organics in cranberries. The method described above permits both the qualitative and quantitative analysis of cranberries at various stages of maturity to identify the volatile organics, flavoring compounds, contaminates 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.