Flavor Analysis - American Chemical Society

Chapter 18

Comparison of HPLC and GC-MS Analysis of Furans and Furanones in Citrus Juices Downloaded by STANFORD UNIV GREEN LIBR on September 29, 2012 | http://pubs.acs.org Publication Date: September 10, 1998 | doi: 10.1021/bk-1998-0705.ch018

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1

R. Rouseff , K. Goodner , H. Nordby , and M . Naim

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1Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850 2Institute of Biochemistry, Food Science and Human Nutrition, Hebrew University of Jerusalem, Rehovot, Israel The lack of quantitative agreement between HPLC and GC-MS values for 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol), furfural and 5hydroxymethylfurfural reported in the literature is due to incomplete HPLC resolution. Furanoids of interest were not resolved from other citrus juice components using reversed phase HPLC with detection at 290 nm. Attempts to develop a rugged chromatographic separation to resolve all components were unsuccessful. A more accurate procedure was developed which employed a second detecting wavelength (335 nm) to provide maximum response from the interfering compounds and minimum response from the furanoids of interest. Reconstructed difference chromatograms (290-335 nm) negated interfering compounds thus providing the spectral selectivity to accurately quantify the furanoids of interest. GC-MS studies demonstrated that contrary to literature reports, Furaneol is thermally stable under GC conditions and can be chromatographically or spectrally resolved from other juice components. Furans can have considerable impact on the aroma and taste of citrus juices that have been stored at elevated temperatures. They are formed to a minor extent as the result of natural biosynthetic pathways during fruit ripening but most are formed as a result of thermal processing and/or storage at elevated temperatures. Furfural and 5-hydroxymethylfurfural are produced during the Maillard reaction in acid environment from the 1,2 enolization of Amadori products formed from pentoses and hexoses respectively. Furaneol is formed in the same reaction from the 2,3 enolization of Amadori products from hexoses under less acid conditions(l). Since citrus juices are highly acidic, furfural and 5-hydroxymethyl furfural will be the favored Maillard reaction products. Even though produced at much lower levels than furfural or 5-HMF, Furaneol (2,5-dimethyl-4-hydroxy-3 (2H)-furanone) is the most important because of its much lower aroma threshold and striking sensory properties. FuraneoFs taste threshold is 0.05 ppm in orange juice (2) whereas the corresponding values for furfural and 5-HMF are 80 and

©1998 American Chemical Society In Flavor Analysis; Mussinan, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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Downloaded by STANFORD UNIV GREEN LIBR on September 29, 2012 | http://pubs.acs.org Publication Date: September 10, 1998 | doi: 10.1021/bk-1998-0705.ch018

212 200 ppm respectively(3). Furaneol or DMHF, has been found in arctic bramble (4-6), raspberry (Rubus idaeus, L.)(5), strawberries, pineapples, mangoes (7), grapefruit (8) coffee (9), grapes and wine (10). Furaneol has also been found as a nonvolatile glycoside in: tomatoes (11), pineapple (Ananas comosusL. Merr.) (12) and strawberries(13). Furaneol, furfural and 5-HMF are all products of the Maillard reaction and as such have been proposed as markers of thermal abuse or excessive elevated temperature storage. Even though the furfural content of orange juice was observed to increase in proportion to increased storage temperature and time (14), it was not until several years later that it was proposed as an index of thermal abuse (15). Mori and coworkers (16) suggested that 5-HMF could also be used as a quality index for citrus juices. They reported that the 5-HMF content of mandarin juices increased during storage, especially at higher temperatures. Furthermore, its content correlated well with sensory acceptance. Tatum and coworkers (2) reported that Furaneol was one of the three major off-flavors produced in temperature abused canned orange juices. As a result of their studies with grapefruit juice, Lee and Nagy (8) proposed that Furaneol could also be used as a quality deterioration index. Furaneol, furfural and 5-MHF have been analyzed using a variety of analytical techniques beginning with colorimetric procedures and currently are usually determined using chromatographic procedures such as GC or HPLC. Colorimetric tests for furfural were based on relatively non specific reactions such as the reaction of aldehydes with aniline (15) or benzidine (17). Since the color forming reactions were non specific, relatively selective sample preparation techniques were required. 5-hydroxymethylfurfural (5-HMF) was usually determined using the Winkler method (18). This procedure was based on reaction of 5-HMF with p-toluidine and barbituric acid reagent to produce a red colored product which was measured spectrophotometrically at 550 nm. However, both furfural and 5-HMF react. Values obtained thus represent the sum of both compounds in citrus juices (19). No colorimetric procedures for Furaneol have been found. Concentrations of these compounds reported in the literature appear to be method dependent. In the case of Furaneol, HPLC values are considerably higher than those obtained using GC or GC-MS. For example, Wu and coworkers reported finding 1.6 ppm free DMHF in Costa Rican pineapple and only 0.7 ppm for the same product using GC MS. In a similar manner, Sanz and coworkers (20) employed H P L C to determine mesifuran and DMHF concentrations in strawberries. They reported concentration values considerably higher than any previously reported for the identical cultivars analyzed using GC or GC-MS. The discrepancy between these results was attributed to the use of a non thermal method of analysis (HPLC). It was implied that the GC results were low because of decomposition of the thermally labile Furaneol in the elevated temperature environment of the gas chromatograph. The purpose of this study was to investigate sources for the apparent lack of quantitative agreement between HPLC and GC methods for furans in food products using orange and grapefruit juices as examples. Our goal was to determine ifHPLC results were high because of inadequate resolution or selectivity which might have quantified co-eluted matrix compounds or if GC results were low due to thermal decomposition of Furaneol in the elevated temperatures of typical gas chromatographs. This study will be limited to the analysis of furfural, 5-hydroxymethyl furfural and Furaneol in orange and grapefruit juices.

In Flavor Analysis; Mussinan, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

213

Materials and Methods


Solvents and Standards. Solvents were of HPLC grade from Fisher Scientific (Pittsburgh, PA). Standard furfural, Furaneol, 5-HMF, and mesifurane were obtained from Aldrich Chemical (Milwaukee, WI). Stock solutions of standards at approx. 3000 ppm were prepared in methanol. Working solutions of standards were prepared as methanolic dilutions. Sample Preparation. Ten mL of orange or grapefruit juice were centrifuged for 5 min at 3000 rpm. Three mL of supernatant juice were passed through a C-18 cartridge (Whatman, SPE ODS 500 mg, Fairfield, NJ) which had been conditioned with methanol and washed with water. The cartridge and juice were washed with 2 mL water to remove sugars and eluted with 2 mL methanol. Samples were filtered through a .45 u filter, and stored in an amber vial until injected into the HPLC. H P L C Instrumentation. A Perkin Elmer (Norwalk, CN) model 410 four solvent low pressure gradient pump, with a Hewlett Packard (Palo Alto, C A) model 1050 autosampler, and either a Waters (Milford, MA) model 490E multiwavelength or 990 photodiode array detector was used to analyze the juice extracts. H P L C Chromatographic Conditions. OJ and GFJ extracts as well as standards (25 nL) were separated on a 5\i Bondesil (Varian, Sugarland, TX) C-18,4.0 mm i.d.x 25 cm column. The solvent gradient is shown below. All concentration changes were linear. The column was equilibrated for at least 15 minutes at initial conditions before each injection. Time % pH 4.0 Buffer Flow % acetonitrile % methanol (min) (mL/min) 0 16 20 40 50 60 62

1.00 1.00 1.00 1.00 1.00 1.00 1.00

7 0 5 5 5 5 7

0 20 20 35 85 85 0

93 80 75 60 10 10 93

G C - M S Instrumental and Chromatographic conditions. All GC-MS data were collected using a Finnigan GCQ Plus system (Finnigan Corp, San Jose, CA). A 30 m x 0.25 mm i.d. DB-5 column was used with helium (99.999%) as the column carrier gas (flow rate 31.9 cm/sec) as well as the collisior^bath gas in the ion trap. All chemical ionization experiments were conducted using 99.99% pure methane at approximately 60 torr. Injector temperature was 250°C. The thermal gradient was as follows: initial temp. 35°C, hold for 3 minutes followed by an 8°C/min temperature ramp to 127°C which was in turn followed by a 40°C/min ramp to 275°C. which was held for at constant temperature 1.8 minutes. The MS transfer line was held at 275°C and the ion source was held at 170°C. The mass spectrometer had a delay of 4 minutes to avoid the solvent peak,


214 and then scanned from m/z 40 to m/z 300 in order to achieve 2 spectra per second with each spectra being the sum of 5 microscans. Injections were 0.2 uL in the splitless mode unless otherwise stated.


Results and Discussion Sample Preparation Techniques. Furan derivatives such as furfural, 5-HMF and Furaneol are relatively polar compounds which are difficult to extract from food matrices with organic solvents. One of the earliest separation techniques was simple distillation of liquid products. Furfural was distilled from orange juice before it was reacted with 10% aniline in acetic acid-ethanol (15). A later HPLC technique (21) which employed this sample preparation procedure found just a single peak. Although the distillation procedure was highly selective in separating furfural from the myriad other components in orange juice, it only recovered 38% of the total furfural. Distillation has also been employed to separate and analyze furfural and 5-HMF in wines using U V absorption spectrophotometry (22). The 5-HMF is determined after the furfural has been distilled off. The unfavorable partition coefficients for these furans can be overcome using continuous liquid-liquid extraction with a slightly polar solvent such as diethyl ether. Buttery and coworkers (23) were able to isolate water-soluble volatile compounds such as Furaneol in tomato juice using this technique. Alternatively, dynamic headspace purge and trap using a solid adsorbent and either solvent elution or thermal desorption (24-26) can be used to separate these compounds from food matrices. Final separation and analysis is usually carried out using GC, often in combination with MS. Another approach has been to use C - l 8 solid phase adsorbent to separate these compounds from citrus juices (8, 27). Sample preparation involved three steps. The first step is to add Carriz reagent to juice and centrifuge. The supernatant is passed through a C-l8 cartridge and eluted with successive washes of ethyl acetate. Finally the ethyl acetate is dried with sodium sulfate and reduced in volume under a stream of nitrogen before injection into the HPLC. In this study we have employed a simplified technique developed earlier for the determination of 4-vinyl guaiacol (28) and later modified for Furaneol (29). In this procedure the centrifuged juice is passed through a C-18 cartridge, washed with water and eluted with a small amount of methanol. It can be injected directly into the HPLC without further concentration. Furfural recoveries using this procedure averaged 97%. Methods of Analysis. Chromatography appears to be the method of choice in determining these compounds in foods. The earlier colorimetric methods have largely been abandoned because of their lack of specificity and the time consuming sample preparation. In terms of chromatography, these compounds can be determined using either HPLC or GC. The choice is often made on the basis of what equipment is available and what other compounds might be of interest. Gas chromatography offers the advantage of being less expensive to purchase and allows for a greater number of other volatile components to be determined. HPLC is more expensive to purchase and maintain but also allows for non volatile components such as flavanone glycosides and hydroxycinnamic acids to be determined. HPLC does not have the resolving power of GC but it is a non thermal separation process which is less subject to artifact formation. In addition, the


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classical concerns for selectivity, resolution, speed, and sensitivity (minimum detectable amounts) for a particular food will influence the final chromatographic choice. H P L C (Non Thermal Analysis). Reverse phase chromatography has been the mode of choice to separate furan derivatives in citrus juices (21, 30, 31). Under reverse phase conditions the elution order is typically, 5-HMF, Furaneol, furfural and finally, mesifurane (the 4-methoxy analog of Furaneol). Shown in Figure 1 is the plot of Log k' vs solvent composition for methanol-water (buffered to pH 4). (The plot is essentially identical for acetonitrile - pH 4.0 acetate buffer combinations). It should be noted that furfural and Furaneol elute at very similar retention times and are not resolved at concentrations greater than 25% methanol. Optimum separation between these two compounds is somewhere between 5-10% methanol. One procedure (8) reported the separation of furfural and Furaneol using a Zorbax ODS column with a mobile phase of 0.05M sodium acetate (pH 4.0) and 30% methanol. This separation was achieved due to the unique composition of the stationary phase. At that time Zorbax C-18 columns were not endcapped. This left polar silanol sites exposed to the mobile phase. The net result was a "mixed mode" separation that allowed small, relatively polar molecules such as furfural to be retained much more than they would under purely reversed phase C-18 chromatography. Whereas the lack of endcapping allowed for some interesting applications, it also produced columns with shorter lifetimes. Currently all reverse phase columns are endcapped and this approach is no longer available. However the same authors have employed a new column which is more polar than normal C - l 8 to separate Furaneol from the other components in orange juice (32). Since only Furaneol was identified in the chromatogram provided, it is not known if Furaneol and furfural can be resolved with their new column. Separation vs Solvent Strength. The separation obtained using a typical C-18 column is shown in Figure 2 with four isocratic solvent conditions (the aqueous portion of each mobile phase was buffered to pH 4.0). It can be seen that at 30% acetonitrile, Furaneol and furfural peaks are merged into a single symmetric peak. This combination peak becomes somewhat asymmetric at 20%, is somewhat resolved at 15% and completely resolved at 10% acetonitrile. Although resolution is improved as solvent strength decreases, analysis time increases. We have chosen to employ a solvent gradient to reduce analysis time and also increase the range of compounds which can be analyzed. The gradient and resulting chromatogram for an orange juice sample stored for 15 weeks at 40 °C are shown in Figure 3. The amount of methanol in the mobile phase remains low throughout the chromatogram and is present to provide additional selectivity to help separate the many components found in orange juice. The resolution shown in Figure 2 demonstrates that a simple 10% acetonitrile solvent composition can resolve Furaneol and furfural. However it is also necessary to resolve these compounds from the many other components in citrus juices which elute in this region. It can be seen that the interfering compounds are numerous and as large or larger than the peaks of interest. Single vs Dual Wavelength Detection. Furaneol and furfural are small peaks that are not resolved at 290 nm from the other components in OJ. Furaneol and furfural have been detected at wavelengths which range from 280 nm (21) to 292 nm (29). Because absorbance maxima can be solvent dependent, we determined the absorbance


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1.5

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mesifurane


^furfural 0.5

O

-0.5

Figure 1. Log of capacity factor (k') versus methanol pH 4 acetate buffer solutions.

10

15

20

Time (min.)

Figure 2. Four chromatograms from various combinations of acetonitrile: pH 4 aqueous buffer showing the affect of changing the solvent strength with (A) 30% C H C N , (B) 20% C H C N , (C) 15% C H C N , and (D) 10% C H C N . Flow rate in all cases was 1 mL/min where (l)-5-HMF, (2)-Furaneol, (3)-furfural, and (4)-mesi-furane. 3

3

3

3



217 spectra for each compound using a photodiode array detector. The advantage of this technique is that it determines the spectra for each compound of interest in the exact solvent composition it elutes in. In the above gradient we found the absorbance maxima to be 278, 287 and 285 nm for 5-HMF, Furaneol and furfural respectively. As shown in Figure 3, 290 nm wavelength is not selective for furanoids because it detects both furanoids and numerous extraneous substances. In an earlier study (29) we found that by taking the difference between two wavelengths, greatly enhanced selectivity could be obtained with little loss in sensitivity. The second wavelength (335 nm) was chosen to provide maximum response from the interfering compounds and minimum response from the furanoids of interest. The region of interest in the previous chromatogram is shown in Figure 4 showing the responses at 290 and 335 nm and the trace due to the difference between the two signals plotted as a third chromatogram. In our earlier studies we had determined the absorbance maxima for some of the surrounding non furanoid peaks and found their average maximum in the region of 335 nm. We also knew from our photodiode array spectra of standards, that the furanoids of interest had essentially no absorbance at 335 nm. It can be seen in Figure 4 that the furfural peak appears to be a well resolved peak but the 335 chromatogram indicates that there is a serious impurity in this peak and that the true peak area is considerably smaller. Furaneol could not be accurately quantified at 290 nm because it appears on the shoulder of a larger impurity and has a second impurity which elutes shortly behind it. However, it can be seen that Furaneol is completely resolved from the impurities in the "difference" chromatogram. Likewise the 5-HMF peak had an impurity that gave the appearance of a tailing peak at 290 nm. The interfering peak can be clearly distinguished at 335 nm. Thus, the "difference" peak produces a single, symmetric peak for 5-HMF which can be accurately quantified. Therefore, it appears that analytical values obtained from single wavelength studies would produce erroneously high values because integration would likely have included coeluting peaks. Whereas it was not possible to chromatographically resolve all the peaks of interest from interfering peaks from stored orange juice, the furanoids of interest could be spectrally resolved using the "difference" chromatogram. G C - M S (Thermal Analysis). Because reports (33) identifying 4-methoxy-2,5-dimethyl3(2H)-furanone (mesifurane) as an aroma constituent of strawberries made special note of the absence of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol), Pickenhagen and coworkers (7) employed high resolution capillary GC to determine if Furaneol was present. They found significant amounts of both compounds in strawberries, pineapple and mango. It was speculated that a possible reason that Furaneol had not been detected in earlier studies was that steam distillation was used as the extraction method, or that capillary columns other than fused silica or acid leached soft glass had been employed. Thermal Stability. Furaneol had been reported to be a thermally unstable compound (34, 35). Thus, we were concerned that it might decompose at the elevated temperatures of most GC injector ports. To determine if decomposition might be a problem, we injected standard Furaneol solutions with injector temperatures of 150, 175, 200, 225,250,275 and 300°C. We found only a single peak in all cases whose height did not diminish with increasing temperature as one might expect from thermal decomposition. The single peak appeared at the proper Kovat's retention index and its resulting mass


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It

J3H4 Buffer acetorjitrjli


o o CO o E c o o> CM CO JQ

Flavor Analysis - American Chemical Society

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