HPLC Determination of Capsaicinoids with Cross-Linked C18 Column and Buffer-Free Eluent

Transcription

1 Journal of Chromatographic Science 2015;53: doi: /chromsci/bmu030 Advance Access publication May 17, 2014 Article HPLC Determination of Capsaicinoids with Cross-Linked C18 Column and Buffer-Free Eluent Hussein G. Daood 1 *, Ga bor Halasz 1,Ga bor Palota s 2, Gabriella Palota s 2, Zsolt Bodai 3 and Lajos Helyes 1 1 Regional Science Centre, Szent Istva n University, Pa ter u.1, Go do lló H-2103, Hungary, 2 Univer Product Psi., Szolnoki u.35, Kecskeme t H-6000, Hungary, and 3 Joint Research and Training Laboratory on Separation Techniques, Eo tvo s Lora nd University, Pa zma ny Pe ter Se ta ny 1/A, Budapest H-1117, Hungary *Author to whom correspondence should be addressed. hussein.daood@fh.szie.hu; hdaood682@gmail.com Received 7 August 2013; revised 10 March 2014 A simple and efficient high-performance liquid chromatographic method was developed and validated for the separation and determination of capsaicin and its major dihydro- and homoderivatives in spice paprika products in 20 min with fluorescent and 35 min with mass-spectrometric detection. The separation was performed on reversed-phase chromatographic adsorbent of cross-linked endcapping with eluent consisting of 1:1 acetonitrile water or acetonitrile 0.1% formic acid under isocratic conditions. Excellent separation of all the major and minor capsaicinoids with resolution index between 1.08 and 7.34 was achieved. The detection and quantification limits of capsaicinoids in standard material solutions were between 2 and 10 ng/ml. The lowest detectable amount of capsaicin, with fluorescent detection, was found to be <1 mg/g non-pungent spice paprika powder. The naturally occurring capsaicinoids could be distinguished from the non-capsaicinoids compounds appeared on liquid chromatography-fluorescence profile of extract from drastically processed paprika by applying mass spectroscopic detection. Hungarian spice paprika were evaluated as mild to very hot (capsaicinoid content: 334 1,660 mg/g) and chili products as very or extremely hot products (1,543 2,818 mg/g). Introduction In all varieties of pungent peppers Capsicum sp., the pods contain capsaicin as the main heat principle, followed by dihydrocapsaicin (DC). These two compounds are also about twice as potent to the taste and nerves as the minor capsaicinoids such as nordihydrocapsaicin (NDC), homodihydrocapsaicin (HDC) and homocapsaicins (HCs). The chemical structure of the capsaicinoid analogs, their pungency power in terms of Scovill heat unit and relevant proportion are shown in Table I. As confirmed by mass-spectrometric imaging (1), capsaicinoids are synthesized in the epidermal cell of the placenta in chili peppers by addition of a branched-chain fatty acid to vanillylamine. Besides the six natural capsaicinoids, nonivamide, as a synthetic member of the capsaicinoid family (vanillylamide of n-nonanoic acid), exist, but used as a reference substance for determining the relative pungency of capsaicinoids (2). In addition to their sensory properties, capsaicinoids have been reported to have important pharmaceutical, biological and ecological roles. Antimicrobial activity of capsaicinoids has been the objective of many researches during the last 10 years (3 6). They have also been found to have protective effect against mutagenesis and carcinogenesis (7, 8) and exhibit against pain effect (9, 10). Capsaicinoids are also used as repellents to deter voles, deer, rabbits, squirrels, insect and attacking dogs (11 13). High-performance liquid chromatography (HPLC) alone (14 16) or hyphenated with selective techniques such as mass spectrometry (MS) (17 20) has been used for the determination of capsaicinoids from pungent pepper products. In general, mixtures of either acetonitrile or methanol and water acidified with formic, acetic or phosphoric acid have been used as the eluents in the HPLC MS determination of capsaicinoids. Recently, simultaneous quantification of the major capsaicinoids and vitamin C has been achieved (21). In the aforementioned methods, separation has been performed on reversed-phase columns having conventional endcapping of C18 phase and with buffer-free elution systems. Despite the long-time taken, in some of those methods, to achieve complete run of capsaicinoids, the different isomers of HC could not be separated from each other. Most recently, in order to perform rapid separation of capsaicinoids, ultraperformance liquid chromatography (UPLC) hyphenated with MS has been used (22). The runtime of 10 min has been found enough to fractionate the pepper extract into NDC, C, DC, HC and HDC, but the satisfactory separation of homoanalogs could not be achieved. Therefore, it has become necessary to work out an efficient protocol for the separation of all analogs and isomers of capsaicin from spice red pepper products with relatively short runtime. The main objectives of the present work were to develop and validate new HPLC method for the separation of capsaicinoids using reversed-phase C18 materials having cross-linked endcapping and buffer-free elution system and to identify, by liquid chromatography mass spectroscopy (LC MS/MS) detection the individual compounds appear in the liquid chromatographyfluorescence (LC-FL) profile of spice red pepper extracts including drastically processed ground paprika. Experimental Chemicals and materials used Standard capsaicinoids were from Sigma-Aldrich (Budapest, Hungary). All analytical and HPLC grade organic solvents and formic acid were purchased from VWR (Debrecen, Hungary). The analytical columns were purchased from Machery-Nagel (Düren, Germany). Different blends of ground spice red pepper (spice paprika) were obtained from Rubin Spice Paprika Processing Ltd (Szeged-Szüreg, Hungary) by mixing the Hungarian SZ-178 cultivar of Capsicum annum L. with different non-pungent cultivars. The samples of pure spice paprika or paprika blends were taken directly after processing and stored in vacuumed nylon sacs at 2208C until analysis. # The Author [2014]. Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oup.com

2 Table I General Information on Capsaicinoids Found in Pungent Pepper Fruits Capsaicinoids Abbreviation Typical relative amount (%) Scoville heat units Chemical structure Capsaicin CAP 69 16,000,000 Dihydrocapsaicin DHC 22 15,000,000 Nordihydrocapsaicin NDHC 7 9,100,000 Homodihydrocapsaicin HDHC 1 8,600,000 Homocapsaicin HC 1 8,600,000 Nonivamide PAVA 9,200,000 Sample preparation Capsaicinoids from spice paprika samples were extracted by methanol according to Hungarian National Standard MSZ ISO 7540 (23) and Chiang (24). From well-homogenized samples, 0.5 g was taken and 25 ml methanol was added in a 100-mL Erlenmeyer flask. The mixture was subjected to ultrasonication in a water bath device for 20 min and then shaken for 15 min. After filtration on a filter paper, the extract was cleaned up through a 0.45-mm Teflon (PTEF) syringe filter. As for stock solution of standard material, 2 mg was dissolved in 10 ml HPLC grade methanol, and working solutions of 0, 2, 4, 6, 8 and 10 mg/ml concentration were prepared by dilution with methanol to conduct calibration of different capsaicinoids. Recovery (%) was performed by spiking known amount (50 mg) of capsaicinoid from stock solutions in 1 g of nonpungent spice paprika samples followed by extraction and HPLC determination. Precision (%RSD) test was carried out by repeating five times the analysis of the same well-homogenized pungent spice paprika sample. Limit of detection (LOD) and limit of quantification (LOQ) were calculated as the concentration of the capsaicinoids at a peak high/noise of 3 and 10, respectively, using spiked non-pungent spice paprika. Before spiking the non-pungent ground paprika was extracted three times by the same method applied for the extraction of pungent samples to remove traces of capsaicinoids. The residues were dried with nitrogen gas, spiked with stock solution of capsaicinoids and dried again to remove the solvent. HPLC and HPLC MS conditions A Merch-Hitachi Chromaster analytical HPLC consisting of a Model 5110 gradient pump, a Model 5210 auto sampler and a Model 5440 Fluorescent Detector was used. The instrument was operated, and the data were processed by EZChrom software. The HPLC-FL separation was performed on three different reversed-phase columns including conventional ODS-3, bifunctional C18-phenyl (Sphinx) and cross-linked C18 endcapping (ISIS) (Figure 1). The different columns had the same dimensions of mm and chromatographic adsorbents with particle size of 3 mm. The isocratic elution of acetonitrile phosphate buffer ( ph 4), acetonitrile water and acetonitrile 0.1% formic acid in water was applied at different ratios and a flow rate of 0.8 ml/min at room temperature. The fluorescent detector was adjusted at extinction and emission wavelength of 280 and 320 nm, respectively, to maximally detect the capsaicinoids. LC MS/MS analysis was performed on an Agilent 1100 HPLC equipped with a degasser (G1322A), a binary pump (G1312A), an auto sampler (G1313A), a column thermostat (G1316A) and a variable wavelength detector (G1314A). Isocratic elution was carried out at a flow rate of 0.5 ml/min using 50% Millipore water with 0.1% formic acid and 50% acetonitrile with 0.1% formic acid. The separation was finished at 35 min. The injection volume was 15 ml and the thermostat was at 308C duringthe analysis. The MS detection was carried out an Applied Biosystems API 2000 triple quadrupole. Data acquisition and evaluation were 136 Daood et al.

3 Figure 2. HPLC-FL profile of standard capsaicinoids separated on Prodigy ODS-3 column with 1:1 CN water isocratic elution. For more details, see text. 1: NDC, 2: CAP, 3: DC, 4: HCAP-1, 5: HCAP-2, 6: HDC-1 and 7:HDC-2. Table II Chromatographic Parameters of HPLC Separation of Capsaicinoids on Different Reversed-Phase Endcapping and Different Elution Systems Figure 1. Chemical structure of endcapping of reversed-phase adsorbents used. performed using Analyst software. Positive electrospray ion source was used with the following parameters: IS 5000, CUR 20, TEM 550, GS1 60, GS2 70, CAD 7. Nitrogen was used as collision gas for collision-induced dissociation (CID), curtain, nebulizing and drying gas. The product ion spectrums were acquired at 25 and 70 ev from 30 to 600 m/z according to Schweiggert and coworkers (15). Results HPLC separation The best isocratic HPLC separation of capsaicinoids could be achieved on RP-ODS-3 column eluted with 45: M KH 2 PO 4 acetonitrile (ph 4) (Figure 2). The chromatographic adsorbent was able to separate, in 20 min, all the compounds of the standard solution injected with resolution factor (R s ).1 accept for isomers of HC, which were separated from each other with R s of 0.45 (Table II). Since our main objective was to achieve efficient separation of all derivatives of capsaicin without use of buffering materials, which do not suit the HPLC MS determination, the separation was performed on different RP adsorbents with different mobile phases consisting of either water acetonitrile or formic acid acidified water acetonitrile. Capsaicinoids Retention time (min) Retention factor K 0 Resolution factor a Conventional ODS-3 NDC CAP DCAP HCAP HCAP HDCAP HDCAP Bi-functional Sphinx NDC CAP DCAP HCAP HCAP HDCAP HDCAP Cross-linked ISIS NDC CAP DCAP HCAP HCAP HDCAP HDCAP Resolution Index R s a a Resolution between each compound and the subsequent one on the HPLC profile (see text). Conventional RP-ODS-3 column was unable to separate the minor capsaicinoids of the standard or sample materials when the HPLC was performed with water acetonitrile eluent. Values obtained for R s and a for HC1 and HC2 and HDC1and HDC2 analogs were close to 0 and 1, respectively. Also, NDC could be separated from CAP with R s value of 0.78 (data not shown in Table II), which is not enough for accurate and reproducible analyses. Parameters of chromatographic separation of the major and minor capsaicinoids were improved when bifunctional Sphinx Capsaicinoids with Cross-Linked C18 Column 137

4 column was used with ACN water in the separation method. However, HCs and HDCs eluted together even when the proportion of water was increased to 55% and the runtime extended to 25 min. This is indicated in the value of 0 for R s and 1 for a determined for homocapsaicinoid isomers. With C18 column having cross-linked endcapping (ISIS) and methanol water was used, as the mobile phase, a rapid elution was achieved, but with unsatisfactory resolution of the minor compounds particularly HDCs. With increased water proportion marked peak broadening happened with high increase in the back pressure of the column. Excellent separation of all of the injected individuals of standard capsaicinoids could be achieved with cross-linked column and 1:1 ACN water as the eluent (Figure 3). As shown in Table II, the R s values for all of the capsaicinoids ranged between 1.08 and The runtime for the complete separation was 20 min. The resolution index of separation of HC1 and 2 and HDC1 and HDC2 were 1.22 and 1.54, respectively. Composition of capsaicinoid extracts from all spice red pepper products analyzed was similar to that of standard materials (originally prepared from pungent peppers) except that from drastically processed spice paprika (dried over 1008C for.6 h),in which an unknown compounds were detected immediately after homocapsaicins on chromatogram (Figure 4). In case of HPLC MS separation the same profile was obtained with extended runtime to min by performing the separation with 1:1 acidified water acetonitrile at a flow rate of 0.5 ml/min that was required for correct ESI and CID in a quadrupole MS/MS detection of capsaicinoids. Such a modification resulted in a slight peak broadening, but with no change in the peaks symmetry. Furthermore, use of acidified water with acetonitrile improved sensitivity of FL and MS detection of capsaicinoids. Method validation Table III shows the results obtained for validation of the developed HPLC-FL method that includes the use of C18 RP column having cross-linked endcapping and isocratic elution with 1:1 water acetonitrile. The linearity range obtained when the concentration of capsaicinoids was plotted versus integrated areas was between 1 and 10 mg/ml with regression coefficient R 2 of 0.994, 0.965, 0.997, 0.998, and for homocapsaicin (y ¼ 2Eþ07x þ 6Eþ06), HDC (y ¼ 2Eþ07x þ 2E206) nordihydrocapsaicin (y ¼ 2Eþ07x þ 3Eþ06), capsaicin (y ¼ 2Eþ07x þ 3Eþ06) and dihydrocapsaicin (y ¼ 2Eþ07x þ 3Eþ06), respectively (see Supplementary data). The LOD and LOQ ranged between and mg/ml and between and 0.01 mg/ml, respectively. The range of precision, in terms of RSD%, was between 1.8 and 2.3 for the major NDC, CAP and DC. Recovery of the capsaicinoids after spiking non-pungent ground spice red pepper samples with known quantities of capsaicinoids was 94 96%. Mass-spectrometric identification LC MS/MS detection was applied for the conformation of structural identification of capsaicinoids in standard solution and pungent spice pepper extracts. Under the conditions described in experimental part, the MS/MS detector provided the spectra, in terms of m/z, for the whole molecules and for the fragments obtained after fragmentation at75 ev as well. Values of m/z of 294, 304, 306, 320 and 322 confirmed the identification of NDC, CAP, DC, HCs and HDCs, respectively, in both standard materials and pungent spice paprika. The two peaks eluted after DC were found to be isomers of HC and the two peaks appeared at the chromatogram with the longest retention time were identified as isomers of HDC. All the capsaicinoids detected gave the characteristic fragment of m/z of 137.2, which represents the principal fragment of capsaicin and its derivatives and isomers (Figure 5). In a drastically processed pungent spice paprika sample having brownish-red color, the HPLC-FL profile contained two extra peaks, one eluted after HCs and the other appeared after HDCs Figure 3. HPLC-FL profile of standard capsaicinoids separated on cross-linked C18 column with 1:1 CN water eluent. For more details, see text. 1: NDC, 2: CAP, 3: DC, 4: HCAP-1, 5: HCAP-2, 6: HDC-1 and 7: HDC-2. Figure 4. HPLC-FL profile of standard capsaicinoids of drastically processed spice paprika separated on cross-linked C18 column with 1:1 CN 0.1% formic acid eluent. For more details, see text. 1: NDC, 2: CAP, 3: DC, 4: HCAP-1, 5: HCAP-2, 6: HDC-1, 7: HDC-2 and X and Y: unidentified degraded products. 138 Daood et al.

5 on the chromatogram. HPLC MS/MS analysis of the main extra peak eluted with 24.6 min (with extended run time) showed that it contains three compounds having molecular weights of 312, 316 and 520. The principal fragments of these compounds had different m/z values from that of CAP (Figures 6 8). All three unknown compounds contain nitrogen in their molecules. Capsaicinoid content in red pepper products To demonstrate the applicability of the developed method, capsaicinoids in different Hungarian spice red pepper products were determined (Table IV). The concentration of the capsaicin, the dominant analog, ranged from 146 to 990 and 1,076 to 1,582 mg/g in ground paprika and chili samples analyzed, respectively. If total capsaicinoid content to be considered as the index of hotness ( pungency) degree, the ground spice paprika blends produced by Rubin company varied from mild with total capsaicinoids level of 334 mg/g to very hot with capsaicinoid level of 1,660 mg/g, whereas ground chili samples examined can be classified as very hot containing a level of capsaicinoids of 1,543 mg/g to extremely hot ones distributing heat principles between 2,162 and 2,818 mg/g. As concern non-pungent (sweet) ground paprika samples, the lowest content capsaicin was found to be as low as 1 mg/g ground paprika. Table III Parameters of Validation of the New Method of Separation of Capsaicinoids on Cross-Linked Column and Fluorescent Detection Parameters Capsaicinoids NDC C DC Linearity range (mg/ml) Precision (%RSD) Recovery (%) LOD (mg/ml) LOQ (mg/ml) Discussion According to the literature data, pungent vegetable or spice peppers contain three major and, at least, nine minor capsaicinoids (19) vegetable or spice red pepper products. The ODS-3 column of the same dimension ( mm, 3 mm particle s size) provided a good separation of capsaicinoids of the standard solution except that HCs that had R s value,0.5, which points to inefficient separation. This was the best capsaicinoid profile obtained with ODS-3 RP adsorbent, which was expected to be applicable to the separation of molecules having low polarity due to the large size of pores (A8) within the silica particles. Whatever good is the separation achieved with ODS-3 column, under the conditions applied, the use of buffering materials shouldbeexcludedfromthemobilephasesincetheydisturb ESI and CID in the MS analysis. Replacement of buffer with acidified water or even with water in combination with acetonitrile was necessary to approach satisfactory separation of capsaicinoid and suits the HPLC MS determinations. The first attempt to use buffer-free elution was conducted on conventional nucleosil or nucleodur C18, 100-3, 150 mm column under isocratic or gradient conditions. Resolution of NDC, HCs and HDCs was unsatisfactory even with gradient elution systems elaborated for this purpose. Separation of capsaicinoids on RP column with buffer-free eluents has been performed on Symmetry C18 column of mm, 5 mm dimensions (19), Metasil Basic 100-3, 3 mm (17) and Zorbax Eclipse C18, , 3.5 mm columns (20). These methods had some limitations concerning either long runtime or low efficiency to separate the minor HC and HDC analogs. The second attempt made to separate capsaicinoids with buffer-free elution system was on bifunctional column having endcapping with C18 and phenylpropyl moieties that ensure partitioning of compounds with aromatic and multiple bond systems on the basis of hydrophobic and p p interactions. From the obtained results, it was clear that the dual functionality of the column improved substantially the resolution and peak symmetry of most of capsaicinoids, but unfortunately the two interaction Figure 5. LC MS ion MS spectrum for capsaicin from standard solution. Capsaicinoids with Cross-Linked C18 Column 139

6 Figure 6. LC MS ion MS spectrum for unknown compound 1 of an extra peak detected in the extract of drastically processed paprika. For more details, see text. Figure 7. LC MS ion MS spectrum for unknown compound 2 of an extra peak detected in the extract of drastically processed paprika. For more details, see text. modes had no impact on the partitioning of the individual HCs and HDCs, which eluted together in on broad peak. Such separation is suitable and may be applicable when the focus is being given to the major capsaicinoids and total HCs and HDCs. The high steric activity, phase stability and relatively low polarity of Nucleosil or Nucleodur adsorbents having cross-linked endcapping encouraged us to use it as the third alternative solution. The first trait was conducted using isocratic elution with 1:1 water methanol as the mobile phase. This resulted in a longtime run exceeded 50 min, broad peaks and low sensitivity of FL detector particularly with the minor constituents. With gradient elution starting with 20% methanol in water and ending with 70% methanol the elution profile showed marked improvement, but with low efficiency to fractionate the minor compounds and a marked trouble in the base line. The best and relatively most rapid separation was achieved with water methanol elution under isocratic conditions using a ratio of 70% for methanol in water, which gave similar profile to that obtained with bifunctional Sphinx column, but with less efficiency to separate individuals of both HCs and HDCs. As shown in Figure 3 cross-linked, endcapped column exhibited its high separation efficiency for both major and minor individuals of both standard materials and pungent red pepper extracts when eluted with 1:1 water acetonitrile. Additionally, with the use of cross-linked C18 column extra peaks appeared on HPLC-FL chromatogram of some capsaicinoid extracts particularly from drastically processed paprika samples (Figure 4). The extra peaks eluted with retention times higher than those of HCs 140 Daood et al.

7 Figure 8. LC MS ion MS spectrum for unknown compound 3 of an extra peak detected in the extract of drastically processed paprika. For more details, see text. Table IV Capsaicinoid Content and Composition in Different Spice Red Pepper Products as Determined by the Newly Elaborated Method Using Cross-Linked Column and Isocratic Elution of 50:50 Water Acetonitrile Samples Capsaicinoids (mg/g) NDC C DC HC þ HDC Total Pungent Hungarian paprika Rubin blend ,660 Rubin blend ,008 Rubin blend Rubin blend Rubin mild blend Organic Hungarian ,293 LSD 5% Chili samples Chili blend , ,162 Chili blend , ,317 Organic chili , ,543 Organic chili ,582 1, ,818 LSD% Non-pungent (sweet) paprika Rubin blend Rubin blend Rubin blend LSD 5% and HDCs revealing their higher retention in C18 phase due to either less polarity or higher molecular weight than that of CAP, DC and HCs as discussed later. In case of HPLC MS/MS analysis, it was necessary to reduce the flow rate to 0.5 ml/min that extended the run time to 30 35min.However,thistimeismuchshorterthan60or 160 min took to fractionate and determine the capsaicinoid extracts by HPLC using conventional analytical C18 or ODS-3 columns ( mm and 5 mm particle size), respectively. Furthermore, with such conventional separations, HCs could not be efficiently separated with satisfactory resolution (11, 17). It is important to mention that acidification of water with formic acid up to 0.1% increased the sensitivity of both fluorescent and MS detector with no change in the retention time and elution order of the major and minor capsaicinoids. The linear concentration responses of peak areas, high recovery and low trouble in base line revealed the validity of the developed method. Furthermore, the high reproducibility, expressed as high precision, and low LOD and LOQ values indicate that the method is enough accurate and sensitive to measure capsaicinoids even at very low levels. As regards the utilization of MS and MS/MS data for structural elucidation of the compounds of the main extra peak detected in the extract of drastically processed paprika sample it can be said that no one of the three compounds gave the m/z [MþH] þ, the characteristic fragment of CAP and dihydro- or homo-type analogs. The MS/MS data on fragments obtained for compound having molecular weight of 316 gave spectra at m/z and 168.3, which are less by 2 H atoms than the and as spectra for fragment of capsaicin after thermal degradation (25). This may points out to a further heat-initiated unsaturation (dehydrogenation) taking place on both aromatic vanillylamide and the hydrocarbon chain, 8-methyle-6-nonenoic acid. The high-molecular-weight compound detected may be formed by possible heat-induced interaction of CAP degraded products with fatty acids as an advanced step in thermal degradation of the main pungency principle (25). It is important to mention that since no exact identification achieved for such extra compounds they should not be included in the calculation of individual heat principles in paprika products. Appearance of such extra peaks in the HPLC-FL profile can be used an index or marker for estimation how drastic was the thermal processing of paprika. As an application of the developed method various spice paprika blends prepared by mixing semi-final products (dried pods) of Hungarian traditional varieties with semi-final products of either non-pungent or mild pungent paprika to produce spice paprika with different pungency levels according to consumer s demand. Different pungent peppers and chilies showed great differences in the level of capsaicinoids. According to level of total capsaicinoids blends from Hungarian spice paprika varied between mild hot and hot except blend 1, which was a ground product of the Capsaicinoids with Cross-Linked C18 Column 141

8 raw material (SZ-178 cultivar) used in Rubin company to produce the different blends. The content of composition of capsaicinoids in SZ-178 variety was very similar to that previously reported (26) despite the fact that in the previous work the variety had been cultivated in different seasonal conditions and different location (Kalocsa, Hungary). It is interesting that the capsaicinoid content of SZ-178 variety decreased significantly (P, 0.05) when cultivated under organic farming conditions when compared with the content measured in the same variety, but cultivated under conventional conditions at the same season. Also of interest is the change in the ratio of CAP/DC, which was found to be 1.96 and in spice red pepper cultivated under conventional and organic farming conditions, respectively. This great change indicates that biochemical hydrogenation was activated, to a high extent, in the fruits of plants grown with organic farming. The ratio of CAP/DC is useful parameter in the authenticity of bioproduct of Hungarian pungent spice paprika products. As concerns the chili products, the level of heat principles determined in the samples possessed was so high that all the products analyzed can be considered as very hot or even extreme hot. Unlike Hungarian spice paprika, products from organic chili peppers had higher capsaicinoid content than conventional ones, andtheratioofcap/dc either significantly increased (P, 0.01) or stayed unchanged depending on the variety of chili peppers. To give an answer to the question whether sweet spice peppers are free of capsaicinoids or not, various non-pungent blends were analyzed also. The level of 4 50 mg/g found in such blends conformed that capsaicinoids occur even in sweet spice pepper, but at undetectable level for the test glands. This also indicates that the genes responsible for pungency in spice red peppers could not be removed entirely by traditional breeding and biotechnological methods applied for.100 years to produce sweet (non-pungent) spice paprika from the pungent origin brought to Europe from Latin America particularly from Mexico (27). Conclusion A new HPLC method that able to separate efficiently all the analogs of capsaicinoids occur in pungent vegetable or spice pepper products with a relatively short runtime. The method suits the LC MS conditions that assist in achieving better and more accurate identification of the usual capsaicinoids and those formed as a result of contamination or processing, thereby, avoiding any mistake in quantitative and qualitative determination of pungent materials of spice pepper product. The method is also applicable to have correct fingerprint of paprika product and is useful for traceability and authenticity purposes. Accurate evaluation of pungency in different products and following the biochemical changes in the major and minor capsaicinoids as a function of agronomical, genetic and technological factors are the other application fields of the developed method. Supplementary data Supplementary data are available at Journal of Chromatographic Science online. Funding This work was financially supported by Hungarian NFU as a part of USOK-2009 and TA MOP B-11/2/KMR Project. This study was funded in part by Research Centre of Excellence /2014/TUDPOL Szent Istva n University and KTIA_AIK_ projects. References 1. 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