Chapter 9 Pungent Flavor Profiles and Components of Spices by

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Chapter 9

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Pungent Flavor Profiles and Components of Spices by Chromatography and Chemiluminescent Nitrogen Detection E. M. Fujinari Antek Instruments, Inc., 300 Bammel Westfield Road, Houston, TX 77090-3508

The pungent characteristic of hot flavors is often due to the presence of a class of nitrogen containing compounds such as capsaicinoids. These components can be analyzed by chromatography with chemiluminescent nitrogen detection ( C L N D ) . This sensitive nitrogen-specific detector can simplify complex analyses by eliminating non-nitrogenous components in the sample. This allows the chromatographer to easily focus on the separation of the nitrogen containing components responsible for the "hotness" of spices.

Many kinds of nitrogen containing compounds responsible for pungent or "hot" flavors in spices have been reported. Structures of hot components (I-V) in horseradish oil are shown in Figure 1. The red hot chili peppers contain nitrogenous compounds known as capsaicinoids which are quite similar in structure (Figure 2). Hybrid peppers possess different degrees of hotness, e.g. jalapeno> Indian birds-eye s> Mexican habanero varieties. Piperine (IX) is the hot component in black pepper. Since these analytes contain nitrogen, chromatographic detection using the chemiluminescent nitrogen detector is inherently suitable. Simplified chromatograms are obtained since non-nitrogenous compounds in the samples are transparent to the detector. Historically, capsaicinoids in foods have been analyzed by organoleptic evaluation Q J , colorimetry (2), and U V spectrophotometric methods (3,4). Chromatographic methods have also been used, including thin layer chromatography (TLC) (5,6) and gas chromatography ( G Q (7-9). Typically, G C methods require a T

© 1997 American Chemical Society In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

9. F U J I N A R I

Pungent Flavor Profiles & Components of Spices

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Figure 1. Structure of hot compounds in horseradish oil.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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100

Figure 2. Structure of capsaicinoids and piperine.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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derivatization step for these compounds prior to analysis in order to make them more volatile. However, capsaicins have been analyzed without derivatization by high performance liquid chromatography (HPLC) with UV detection using normal- and reversed-phase techniques (10-17). Better resolution of capsaicins (17) has been reported using reversed-phase (RP) rather than normal-phase (NP) chromatography. Reversed-phase HPLC separation of piperine followed by UV detection has been reported earlier (14,18). Using mass spectrometry (MS) as a means for GC detection provides a powerful tool for structural characterization of flavors, e.g. ginger oil (19). However, quantitation of analytes by MS detection in chromatography may be difficult because of the response variation of the detector due to sample and/or solvent induced matrix effects. On the other hand, the CLND response is stable and not affected by complex sample matrices. Quantitation of capsaicin and dihydrocapsaicin in red pepper by HPLC-CLND was previously reported (20). The linear response of the CLND is shown in Figure 3.

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capsaicin ^g) r=0.99952; m=0.00189; b=-0.00202 Figure 3. HPLC-CLND calibration curve of capsaicin. Reprinted with permission from Ε. M. Fujinari, in "Spices, Herbs and Edible Fungi", G. Charalambous (Ed.), 1994, pp 367-379. with kind permissionfromElsevier Science - NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.

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Benn et. al. (21) demonstrated the use of GC-CLND for the detection of nitrogen containing compounds in flavors and essential oils. The advantage of this technique is illustrated in Figures 4a-b using a CP-Sil 5 CB (Chrompack, 25m χ 0.53mm ID, 1.0 μηι film thickness) column. Figure 4a is the GC-FID profile of a green pepper flavor containing nitrogenous components from the horseradish oil and the three pyrazine compounds. As Figure 4b shows, using GCCLND, the nitrogen-containing compounds are easily detected without interference from the sample matrix. Peak identification was achieved using the horseradish oil and the pyrazine standards. Supercritical fluids (such as supercritical CO2) possess similar viscosities to those of gases, yet their diffusivities are much greater than liquids. These physical properties together provide higher separation efficiencies for SFC, with sharper peaks than for HPLC. Since high molecular weight and thermally labile compounds can be analyzed by this technique, SFC also provides an added advantage over GC. Taylor et. al. (22) reported a feasibility study for supercritical fluid chromatography - chemiluminescent nitrogen detection (SFC-CLND) with open tubular columns. SFC-CLND of hot mustard extract is shown in Figure 5. Hot components were identified as allyl isothiocyanate (peak A) and butyl isothiocyanate (peak B) using corresponding analytical standards. This paper will focus on nitrogen-specific detection for liquid chromatography including HPLC-CLND profiles of chili powder, paprika oleoresin, black pepper, and capsaicins in onion and garlic flavors. Experimental Apparatus. High performance liquid chromatographic separations were achieved on a binary gradient microbore HPLC system: primary pump (A) Model 305, secondary pump (B) Model 306, monometric module Model 805, and a dynamic mixer Model 811C from Gilson Inc. (Middleton, WI). Sample injections were achieved with a 20 \iL loop on a Model EQ-36 injection valve from Valco Instruments Co. Inc. (Houston, TX). A stainless steel Y-splitter also from Valco was used in order to achieve a post-column split of the mobile phase flow to the CLND. A Supelcosil LC-18S analytical HPLC column was purchased from SUPELCO Inc. (Bellefonte, PA). The Y-splitter was attached to the analytical column by a SLIPFREE connector, available from Keystone Scientific Inc. (Bellefonte, PA). Analyses of nor-dihydrocapsaicin, capsaicin and dihydrocapsaicin in spices as well

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Figure 4. GC profile of green pepper flavor with horseradish oil and pyrazine mixtures. a) FID: peaks Ε = 2-methyl-3-methoxypyrazine and G = 2-methoxy-3-ethylpyrazine. b) CLND: peaks A = but-3-enonitrile, Β = allyl thiocyanate, C = allyl isothiocyanate, D = 2-butyl isothiocyanate, Ε = 2-methyl-3-methoxypyrazine, F = 2-methyl-5methoxypyrazine, G = 2-methoxy-3-ethyl pyrazine, and H = phenyl ethyl isothiocyanate. Reprinted with permission from S. M. Benn, K. Myung, and Ε. M. Fujinari, in "Food Flavors, Ingredients and Composition", G. Charalambous (Ed.), 1993, pp 65-73. with kind permissionfromElsevier Science - NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Figure 5. Capillary SFC-CLND profile of hot mustard extract [0.1 gram of mustard powder extracted in 1 mL water (30%) and methanol (70%) solution]. Peaks A = allyl isothiocyanate, Β = 2-butyl isothiocyanate, and C = unknown nitrogen containing compound. Chromatographic conditions: pressure program from 80 atm (hold 5 min), ramp to 150 atm at 10 atm/min, then to 200 atm at 15 atm/min; Cyano (20 m χ 100 mm ID, 0.25 mmfilmthickness) column; time split injection 0.2 sec. Reprinted with permission from H. Shi, J. T. B. Strode III, Ε. M. Fujinari and L. T. Taylor, J Chromatogr. A, 734 (1996) 303. with kind permissionfromElsevier Science - NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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as pipeline in black pepper were accomplished with the nitrogen specific detector, model 7000 HPLC-CLND, from Antek Instruments Inc. (Houston, TX) and Delta chromatography software from Digital Solutions (Margate, Australia) run on an IBM 486 compatible computer. Reagents and Standards. The natural capsaicin standard mixture was composed of 65% capsaicin and 35% dihydrocapsaicin. Piperine (97%) and citric acid (99+%) were also purchased from Aldrich Chemical Co. (Milwaukee, WI). HPLC grade methanol (99+%) was obtained from Fisher Scientific Co. (Fair Lawn, NJ). Sodium free distilled water was obtained from Ozarka Drinking Water Co. (Houston, TX). All standards and reagents were used without further purification. The HPLC mobile phase was filtered through a Millipore Corp. (Bedford, MA) HV filter with a 0.45 μπι pore size. Paprika oleoresin, onion, garlic, black pepper, and chili powders were obtained from commercial sources. Analytical method. The capsaicin/dihydrocapsaicin and piperine reference standards were prepared as 13.19 mg/mL and 4.16 mg/mL solutions in methanol, respectively. Samples were prepared by separately weighing the following and bringing the volume to 25 mL with methanol: red chili powder (2.5136 g), black pepper powder (2.5154 g), onion powder (1.5126 g), and garlic powder (1.5297 g). Each sample (5 mL) was concentrated to 1 mLfinalvolume with a gentle stream of helium. Samples for the hot garlic and onion flavor profiles were prepared using a (1 + 1 v/v) mixture of the capsaicin reference standard and 100 of the concentrated flavors. HPLCCLND analyses were accomplished using a 15 \iL partial filled injection to a 20 μΐ, sample loop into a Supelcosil LC-18S analytical column: 250mm χ 4.6mm ID, 5 μηι particle size, 100 A pore size. An isocratic mobile phase, methanol/water with 0.1% citric acid at pH 3.0 (65:35 v/v), was utilized with a flow rate of 0.650 mL/min. A Ysplittèr was configured post-column and used to deliver a flow of 100 μί/ιηίη to the CLND. The CLND conditions were: 1100° C pyrolysis temperature, PMT voltage 780, range xlO, and 1 volt detector output. Results and Discussion This chemiluminescent nitrogen detector for HPLC was first described in (23). The detection mechanism for nitrogen determination is shown below:

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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Nitrogen containing analytes (R-N) are oxidized in a furnace at high temperatures to nitric oxide (·ΝΟ). Chemiluminescence as shown in the second equation is detected by a photomultiplier tube (PMT). The photons detected are proportional to the amount of nitrogen in the analyte(s). The HPLC-CLND profile of red chili pepper is shown in Figure 6. The capsaicinoids nordihydrocapsaicin (A), capsaicin (B), and dihydrocapsaicin (C) are easily observed without interference from the red chili powder matrix in this chromatogram. This separation was achieved via isocratic reversed-phase chromatography using a mobile phase consisting of MeOH/water with 0.1% citric acid at pH 3.0 (65:35 v/v). A Supelcosil LC-18S (250mm χ 4.6mm ID) column with a mobile phase flow rate of 0.65 mL/min was used. A post-column split was used to direct 100 μί/ππη to the CLND. The three capsaicinoid compounds (A, B, and C) are also very easily detected in onion (Figure 7) and garlic (Figure 8). Several polar nitrogen containing components eluted at the solvent front for the onion, garlic and red chili powder samples. Figure 9 shows the HPLC-CLND profile of black pepper. Paprika oleoresin was extracted by the method reported by Cooper (17) and analyzed by HPLC-CLND (Figure 10) showing the presence of capsaicin (A) and dihydrocapsaicin (B). Conclusion Pungent (hot) components can be separated using gas chromato­ graphy, supercritical fluid chromatography, and high performance liquid chromatography and detected with the chemiluminescent nitrogen detectors (CLND). These nitrogen-specific detectors provide a means of analyzing nitrogen-containing compounds free of matrix interference.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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9. FUJINARI

Pungent Flavor Profiles & Components of Spices

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Figure 6. HPLC-CLND profile of red chili powder. Peaks: A = nordihydrocapsaicin, Β = capsaicin, and C= dihydrocapsaicin.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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SPICES: FLAVOR CHEMISTRY AND ANTIOXIDANT PROPERTIES

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Figure 7. HPLC-CLND profile of capsaicins with onion flavor. Peaks: A = nordihydrocapsaicin, Β = capsaicin, and C= dihydrocapsaicin.

In Spices; Risch, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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9. FUJINARI

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Figure 8. HPLC-CLND profile of capsaicins with garlic flavor. Peaks: A = nordihydrocapsaicin, Β = capsaicin, and C= dihydrocapsaicin.

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SPICES: FLAVOR CHEMISTRY AND ANTIOXIDANT PROPERTIES

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