Art05.pmd Journal of Applied Botany and Food Quality 80, 31 - 35 (2006) 1Federal Centre for Breeding Research on Cultivated Plants, Institute of Plant Analysis, Quedlinburg, Germany 2Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marburg, Germany 3Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany Efficient determination of cysteine sulphoxides in Allium plants applying new biosensor and HPLC-MS² methods K. Ziegert1, W. Schütze1, H. Schulz1, M. Keusgen2, F. Gun2, E.R.J. Keller3 (Received December 14, 2005) Summary Cysteine sulphoxide (CSO) contents of 16 different accessions of garlic (Allium sativum L.) and 15 varieties of onion (Allium cepa L.) were measured using two different rapid analytical methods: a biosensoric approach and a newly developed HPLC-MS2 technique. Both methods allow quantification of naturally occurring cysteine sulphoxides present in Allium plants without time-consuming sample pretreatment such as derivatisation of amino acid derivatives prior to HPLC-separation. It has been found that the amount of alliin, which is the predominant CSO occurring in garlic, varies between 0.2 and 2.2 g/100 g dry matter. Contrary to that, isoalliin representing the main CSO in onion has been detected in significantly lower amounts (0.3 to 1.25 g/100 g dry matter). Introduction Onion (Allium cepa L.) and garlic (Allium sativum L.) are eco- nomically the most important species of the genus Allium. Onion varieties (Allium cepa) can be found in many regions of Europe, Asia, North America and Africa. This species is one of the most important horticultural crops with an annual world-wide production of around 44 million tons (GRIFFITHS et al., 2002). It is well-known that S-alk(en)yl-L-cysteine sulphoxides (CSO), which are mainly present in the bulb, act as flavour precursors generating the typical taste and odour in presence of the enzyme alliinase (e.g. dipropyl disulfide, diallyl disulfide, thiophene). Fig. 1 shows the biotransformation of sulphur compounds in Allium during processing of the plant raw material. The alliinase reaction initiates the production of highly reactive sulphenic acids, which condense with each other to form thio- sulphinates. These compounds are mainly responsible for the flavour of fresh onion or garlic. The thiosulphinates can disproportionate into thiosulphinoates and sulphides presenting the characteristic flavour of cooked onions. Steam-distilled oils obtained from various Allium species such as onion, garlic or leek are widely used for flavouring food and for production of food supplements. Several studies have been performed during recent years in order to evaluate potential Allium wild plants and hybrids with special regard to their aroma properties as well as pharmacological value (SCHULZ et al., 1998; SCHULZ et al., 2000; KEUSGEN et al., 2002; STORSBERG et al., 2004; SCHMITT et al., 2005). Besides sulphur compounds, also pyruvic acid and ammonia were formed in equimolar amounts, which can be used for a rapid biosensoric determination of cysteine sulphoxides (KREST and KEUSGEN, 2002; KREST and KEUSGEN, 1999). In the latter case, alliinase has to be immobilized with an ammonia- detecting device. Further on, cepaenes (α-sulphinyl disulphides) were found in aged extracts of Allium species (RABINOWITCH and CURRAH, 2002) acting as potent inhibitors of platelet aggregation and different enzymes (e.g. cyclooxygenase, lipoxygenase) (BLOCK et al., 1997). The for- mation of particular compounds depends on different reaction conditions (e.g. temperature, solvent). In an ethanolic extract, the thiosulphinate allicin can react with itself (disproportionation) forming compounds with three sulphur atoms in the molecule (ajoens), which are especially known for their antithrombotic properties. The lachrymatory factor, built from 1-propenyl-sulphenic acid, can dimerize to bisulphines and cyclic zwiebelans. As shown above, CSO play an important role in the biochemistry of Allium plants and significantly contribute to the health of men. Therefore, efficient analytical methods are required to determine the individual content of these compounds in plant material as well as to p y r u v a te C H -C O - C O O H3 s ulp h e nic a cid R-S - O HN H 3 s ulp h in e (L F) R = S O+ - p r op a n a l C H - C H -C H O3 2 th ios u lp h ina te R-S -S O - R ’ s u lp h id es R-S -S -R ’ t hios u lp hin o a tes R-S -SO R ’2 ‘a j oe ne s ’ H C S O - C H -S -S -C H5 3 - 3 5 3 5 + + + b i s-p ro p e n yl d is u lp h id e ‘c y c lic zw ie b e la n e s ’ + H O2 d im e r iza tio n c o n d e n s a tio n fo r a llic in d is p ro p o r tio n a tio n + s u lp h e n ic a c id d is p ro p o rtio n a tio n C S O R-SO -C H (N H )- C O O H2 2 a liin a s e ‘cep a en es ’ H C -SO -C H -S -S - C H5 3 3 6 3 5 Fig. 1: Scheme presenting the formation of sulphur compounds starting from cysteine sulphoxides coming in contact with alliinase during processing of plant material. R = methyl, propyl, 2-propenyl and 1-propenyl; LF = lachrymatory factor (RABINOWITCH and CURRAH, 2002). characterise herbal products obtained from Allium plant material. Several HPLC techniques for the determination of CSO, especially of alliin, have been described in the past (ZIEGLER and STICHER, 1989; IBERL et al., 1990; KEUSGEN, 1997) but all these analysis methods generally are time consuming because of extensive sample clean-up steps necessary to separate CSO from interfering matrix components. Therefore, two different sophisticated methods were applied to study the CSO content and distribution in various cultivars of Allium cepa as well as gene bank accessions of Allium sativum: a new biosensoric method based on immobilized alliinase (KREST and KEUSGEN, 2002) and a newly developed HPLC-MS2 method. For the biosensoric analysis immobilized alliinase was used to initiate the degradation of CSO. Pyruvate and ammonia are by-products formed during this process. The amount of enzymatically formed ammonia is proportional to the level of cysteine sulphoxides and can be, therefore, used for an indirect quantification of these sulphur constituents in Allium plant material, if native alliinase was inactivated during extraction (methanol, heat). The new HPLC-MS2 method with selective ion monitoring (SIM) was developed to more rapidly screen extracts of various Allium species. The method was used for the analysis of the four main im- portant CSO in Allium species: methiin, alliin, isoalliin and propiin. The special advantage of MS2-detection should be used to detect the individual analyte molecule ions and their related fragments with high selectivity also in presence of other co-eluting non-sulphur compounds. Finally, we also will discuss the variation of CSO found in different cultivars/accessions of A. cepa and A. sativum. Materials and methods Reagents Alliin was obtained from Carl Roth GmbH & Co KG (Karlsruhe, Germany). Methiin and propiin were prepared following the pro- cedures described earlier (KEUSGEN, 1997; THEODOROPOULOS, 1959; STOLL and SEEBECK, 1951; FREEMAN et al., 1994; KOCH and KEUSGEN, 1998; KREST et al., 2000). Isoalliin, synthesized according to a new efficient procedure (NAMYSLO), was generously provided by the Institute of Organic Chemistry, Clausthal University of Tech- nology, Germany. Alliinase was isolated, stabilized and purified according to methods developed by Krest and Keusgen (KREST and KEUSGEN, 2002; KREST and KEUSGEN, 1999). Plant material The investigated garlic plant material was obtained from the Allium collection of the Institute of Plant Genetics and Crop Plant Research in Gatersleben, Germany. The different onion varieties were cultivated by the „Agrargenossenschaft Calbe“, Calbe, Germany. The plants were grown under comparable conditions to reduce influences caused by environmental factors. The freshly harvested bulbs were directly cooled down (-80 °C) and subsequently freeze-dried in order to pre- vent losses of CSO caused by enzymatic reactions. Sample preparation HPLC-MS2 About 200 mg of an exactly weighed freeze-dried sample were heated under reflux for 10 minutes in 20 mL of methanol. After that, the sample was crushed in a mortar and returned to the methanolic solution with the addition of 20 mL deionised water and heated again for 10 minutes. Then the extract was filtered and the residue was washed with 3x3 mL of methanol/water (50:50). The filtrates were evaporated to dryness under reduced pressure. For HPLC analysis the residue was redissolved in 2.5 mL of deionised water (THEO- DOROPOULOS, 1959). From each variety two samples were taken in order to get an overview about the variability of the CSO content within the analysed plant material. Biosensor For biosensor analyses the freeze-dried plant material (without green parts) was pulverised in a mortar. 200 mg of this material was trans- ferred to a test tube containing 3 mL of methanol. This enzyme- inactivated solution was gently heated for five minutes in a heating block at 100 °C block temperature. Then the test tube was cooled down for three minutes and the sample was heated again for another 15 minutes after addition of 3 mL of deionised water. Finally, the mixture was transferred into a graduated flask (25.0 mL) and filled up with biosensor phosphate buffer (pH = 7). Prior to biosensor analysis, the sample was filtered and diluted with the buffer solution (1:20, v/v). HPLC-MS2 Analysis The analyses were performed on an HP 1100 series HPLC system (Agilent) hyphenated with a Bruker Esquire 3000 mass spectro- meter with electrospray ion source. A ZORBAX SB-C18 column (3 x 150 mm), particle size 3.5 µm, was used for the CSO separation. Sample aliquots of 1-5 µL were injected into the analytical column and the CSO separation was performed using two solvents (A: deionised water, B: methanol). The following elution gradient was applied (flow rate = 0.5 mL/min). Tab. 1: Gradient elution profile for HPLC separation of cysteine sulphoxides. Step Time (Min.) % Eluent B (methanol) 1 0.00 1.0 2 2.00 1.0 3 7.00 6.0 4 7.10 1.0 5 9.00 1.0 The optimisation steps for detection of mass fragments were carried out in the flow injection mode using a scan range of 50-400 m/z for all analysed CSO. The quantification of CSO was performed on the basis of the extracted ion [M+H]+ without further fragmentation within a given time window (Fig. 2). The individual MS conditions were specially adapted to methiin, alliin, isoalliin and propiin. Accordingly the calibration equations were established for these CSO. Flow-through analysis The biosensoric method is based on the alliinase reaction, the enzyme was immobilized and placed inside a flow-through cartridge. De- termination of free ammonia and enzymatically formed ammonia was determined by a flow-through-apparatus (MANA and SPOHN, 2000). Ammonia in equimolar amounts was obtained from cysteine sulphoxides by immobilized alliinase. The enzyme was isolated, stabilized and purified according to the methods already described earlier (KREST and KEUSGEN, 2002). The powdered garlic or onion sample was homogenized and a crude alliinase preparation was obtained by precipitation with ammonium sulphate or by ultra- 32 K. Ziegert, W. Schütze, H. Schulz, M. Keusgen, F. Gun, E.R.J. Keller filtration. Ultra-filtrated alliinase was used for standard experiments. Alliinase was stabilized with 10 % sucrose, 0.17 M NaCl and 25 mM pyridoxal-5’-phosphate (P-5’-P) dissolved in phosphate buffer (pH 7.0, 60 mM). Further purification was done by gel filtration followed by affinity chromatography. Immobilization of alliinase was performed on a ConA/agarose-carrier (Sigma, Ord.-No. C 7555). Briefly, the carrier was filled into a cartridge (60 µL) and rinsed with buffer. A solution containing alliinase was then pumped through this cartridge. Immobilized alliinase may be also renewed. Elution of the enzyme was performed with buffer containing mannose followed by rinsing with a glucose solution and buffer. The carrier may be newly loaded with alliinase as described above. For analysis of cysteine sulphoxides, the cartridge was operated with phosphate buffer (pH 7.0, 0.02 M containing 0.17 M NaCl). Analysis of cysteine sul- phoxides by HPLC and the biosensor was repeated at least three times. Given values for cysteine sulphoxides were corrected by the amount of native ammonia before alliinase reaction. Results and discussion HPLC-MS2 with SIM has been shown to be a suitable method to detect and to quantify naturally occurring CSO (methiin, alliin, isoalliin, propiin) in A. cepa and A. sativum. All four compounds could be individually detected. The newly developed method has been found to be very sensitive, robust, and reliable over a wide concentration range of the targeted compounds. The low detection limit allows to analyse CSO even in concentrations below 1 µg/mL sample. The linear range could be confirmed over three concentration decades. Sensitivity is sufficient even for the analysis of small Allium samples even in the order of a few mg. Fig. 2: The extracted ion chromatograms of CSO occurring in Allium cepa (black line) and Allium sativum (dotted line). Isolation of cysteine sulphoxide ions was performed using different time windows in- dividually adapted to the masses of individual CSO The use of MS2 detection has been proven to be particularly effective in analysing the non-volatile sulphur substances from Allium extracts, because other co-eluting Allium substances do not interfere with the significant key signals of the molecule ion [M+H]+. The ESI inter- face and mass spectrometer parameter were optimised to obtain maximum sensitivity as displayed in Tab. 2 and Fig. 3. For calibration, pure CSO standards were used in different concentration ranges related to the authentic content which is usually measured in both Allium species. Within these considered ranges the individual calibration curves (Fig. 3) were found to be linear (Tab. 2). The biosensoric method is also suitable for a simple and rapid determination of the total amount of CSO. Each analysis needs only less than five minutes. As shown in previous investigations with immobilized alliinase, results obtained by ammonium determination after alliinase digestion of CSO are comparable with those obtained by HPLC (KREST and KEUSGEN, 2002). The method is less sensitive than the HPLC-MS method but concentrations below 5 µg alliin/mL can be accurately determined. Samples described here in this work were diluted as least 20 times to reach the linear calibration range (about 2 µg to 100 ng/µL). So, at any rate the sensivity of the presented HPLC-MS method is high enough for quality control purposes. Analysis of Allium sativum The CSO concentration obtained for Allium sativum gene bank accessions are presented in Fig. 4. These data show the results of the biosensor and of the HPLC method as well. Tab. 2: Characteristics of naturally occurring cysteine sulphoxides based on the individual molecule ions [M+H]+ CSO [M+H]+ Fragment ion Retention time Precision R.S.D. Calibration range Calibration linearity [m/z] [m/z] [min] (n=3) [ng/µL] (R2) methiin 151.8 88.2 1.5 0,17 0.1-20 0.9996 alliin 177.9 88.2 1.8 7.89 0.4-400 0.9994 isoalliin 177.9 88.2 2.0 6.83 0.4-400 0.9998 propiin 179.9 88.2 2.4 0.13 0.2-20 0.9996 S O O NH2 CH2 OH 177.9[M+H] + m/z R el at iv e A b un d an ce R =0.9994 2 Fig. 3: HPLC-MS calibration curve obtained for alliin (based on the in- dividual signal intensity of the protonated molecule ion at m/z 177.9) and MS spectrum of a pure alliin standard Determination of cysteine sulphoxides in Allium plants 33 We found a high variability regarding the CSO content within the measured Allium sativum accessions. Samples with the numbers TAX 1125 and ALL 1288 present highest content of total CSO (approx. 2.2 g/100 g dry matter) contrary to those plants with the numbers ALL 1549 or ALL 781 containing only comparatively low amounts of CSO of approx. 0.23 g/100 g dry matter. The mean difference between the two analysed samples A and B (for HPLC-MS²) was 0.23 g/100 g dry matter. The main CSO present in A. sativum is alliin, whereas methiin was only detected in traces (< 0.1 %). Mean standard deviation for the biosensor method was found to be 0.08 g/100 g dry matter. The biosensor method reflected the same trend of CSO content of samples as described above for the HPLC-MS² method. However, there are some significant differences in detail. We assume that inhomogeneous sample material is the reason for this behaviour. To avoid losses of CSO before sample lyophilisation, cloves were only roughly cut. This causes some problems during lyophilisation. Some parts of the material could not be properly dried because the sample material got amber-like at its surface preventing water evaporation in the tissue under these zones. It can be assumed that this garlic material showed a dramatically reduced alliin content. Because of the limited amount of sample material, only small amounts of each accession could be analysed leading to a big influence of in- homogeneous sample material. In order to truly compare both methods with each other, the individual ratio between the results obtained by the biosensor and those obtained by HPLC-MS was calculated. Because only very low amounts of CSO were detected in ALL 781, this sample was not included in the calculation. The average ratio was determined to be 0.97, de- monstrating that both methods lead to nearly identical results and variations between the two series are highly randomized (as an effect of insufficient homogeneity of sample material). Analysis of Allium cepa The CSO contents determined by both techniques in 15 varieties of A. cepa are shown in Fig. 5. In agreement with the results reported in former studies (RABINOWITCH and CURRAH, 2002) it has been found that the content of total CSO is much lower than in the analysed A. sativum accessions (0.2 – 0.8 g/100 g dry matter). Both series (Biosensor and HPLC) showed a rather good correlation. Especially those samples, which displayed a relatively low CSO concentration obtained by the biosensor (cultivars 'Dacapo', 'Collito', and 'Aranca') were also low by HPLC analysis. Interestingly, values obtained by the biosensor are lower than those obtained by the HPLC- MS² method. These differences may be explained by the fact that CSO are not completely converted to ammonia by the alliinase immobilized in the biosensor. Currently, further studies are under investigation aiming to get more detailed information regarding the alliinase selectivity towards individual CSO in the near future. In the case of A. cepa, sample preparation in terms of drying was much easier. Coarse cut pieces are rather thin and can be sufficient- ly freeze-dried. Therefore, onion sample material is much more homogenous than that obtained from garlic bulbs applying the same method. But nevertheless, because of the good correlation of both methods, the biosensor approach as well as HPLC-MS² seem to be suitable for the quality control of A. cepa material. It could be demonstrated that methods described above are rather helpful for a time-saving and effective quality control of garlic and common onion. In case of common onion, freeze drying seems to be an appropriate method for sample preparation. Contrary to that, drying of garlic samples causes serious problems regarding sample homogeneity. In contrast to onion, garlic cloves consist of only one thick storage leaf covered with a rather thick skin, which makes freeze-drying more difficult. Cutting in smaller pieces probably leads to more homogenous sample material, but will definitely result in lower alliin amounts because of degradation of CSO at the cut surface. Further research studies are necessary to overcome these problems specially related to insufficient sample homogeneity. Both devices used – biosensor and HPLC-MS² – are rather complex tools. However, the biosensor approach is much more inexpensive (a tenth of the price of an HPLC-MS²-equipment). Moreover, only the total amount of CSO can be gained by the biosensor approach. As favourable point of this device, it can be fully operated by aqueous solutions. Operating costs are, therefore, extremely low. On the other hand, the HPLC-MS² device is a rather complex tool, which provides full information about different CSO and can be also used for a broad range of further analytical tasks. Therefore, this technique provides a very useful support to breeding projects as well as fast evaluation of Allium genebank accessions aiming at controlling the content of individual CSO. The HPLC-MS method presented in this study allows to perform an effective selection of single plants and to measure simultaneously the optimal dis- tribution and amount of all naturally occurring CSO in one course of analysis. Furthermore, this method can be applied also for quality control purposes in order to check the level of valuable CSO in certain All 1288 TAX 1125 All 1453 All 937 All 1279 TAX 1337 All 1292 All 1284 All 1550 All 1540 All 290 All 1250 All 781 All 818 All 1536 All 1549 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 2,2 2,4 C o n te n t of C S O ( g / 10 0 g) Biosensor HPLC-MS² Fig. 4: Total CSO content in various garlic accessions analysed by biosensor and HPLC-MS² methods. Arenal Sunskin H ybelle Bonus A vanti D rago Collito Starito Canto D acapo V aleria RX 77796 Aranca Barito 0 0,2 0,4 0,6 0,8 1 1,2 1,4 C on te n t o f C S O ( g /1 00 g ) Biose nsor HPLC-MS² Fig. 5: Total CSO content in different Allium cepa varieties analysed by biosensor and HPLC-MS2 methods. 34 K. Ziegert, W. Schütze, H. Schulz, M. Keusgen, F. Gun, E.R.J. Keller Allium species (e.g. garlic, onion, ramson) used as raw materials for the production of various phytopharmaceutical preparations. References BLOCK, E., GULATI, H., PUTMAN, D., 1997: Allium chemistry: Synthesis of 1-[alk(en)ylsulfinyl]propyl alk(en)yl disulfides (cepaenes), antithrom- botic flavorants from homogenates of onion (Allium cepa). J. Agric. Food Chem. 45, 4414-4422. FREEMAN, F., HUANG, B.G., LIN, R.I.S., 1994: Garlic Chemistry – nitric-oxide oxidation of S-(2-propenyl)cysteine and (+)-S-(2-propenyl)-L-cysteine sulfoxide. J. Org. Chem. 59, 3227-3229. GRIFFITHS, G., TRUEMAN, L., CROWTHER, T., 2002: Onions – A global benefit to health. Phytother. Res. 16, 603-615. IBERL, B., WINKLER, G., MÜLLER, B., KNOBLOCH, K., 1990: Quantitative determination of allicin and alliin from garlic by HPLC. Planta Med. 56, 320-326. KEUSGEN, M., 1997: TLC analysis of Allium sativum constituents. Planta Med. 63, 93-94. KEUSGEN, M., SCHULZ, H., GLODEK, J., KREST, I., KRÜGER, H., HERCHERT, N., KELLER, E.R.J., 2002: Characterization of some Allium hybrids by aroma precursors, aroma profiles, and alliinase activity. J. Agric. Food Chem. 50, 2884-2890. KOCH, I., KEUSGEN, M., 1998: Diastereoselective synthesis of alliin by an asymmetric sulfur oxidation. Pharmazie 53, 668-671. KREST, I., GLODEK, J., KEUSGEN, M., 2000: Cysteine sulfoxides and alliinase activity of some Allium species. J. Agric. Food Chem. 48, 3753-3760. KREST, I., KEUSGEN, M., 1999: Stabilization and pharmaceutical use of alliinase. Pharmazie 54, 289-293. KREST, I., KEUSGEN, M., 2002: Biosensoric flow-through method for the determination of cysteine sulfoxides. Anal. Chim. Acta 469, 155-164. MANA, H., SPOHN, U., 2000: Rapid and selective determination of ammonium by fluorimetric flow injection analysis. Fres. J. Anal. Chem. 366, 825- 829. NAMYSLO, J.C. (private communication). RABINOWITCH, R.H., CURRAH, L., 2002: Allium Crop Science: Recent ad- vances. CABI Publishing, New York. SCHMITT, B., SCHULZ, H., STORSBERG, J., KEUSGEN, M.: Chemical charac- terization of Allium ursinum L. depending on harvesting time, J. Agric. Food Chem. 53, 7288-7294. SCHULZ, H., KRÜGER, H., LIEBMANN, J., PETERKA, H., 1998: Distribution of volatile sulfur compounds in an interspecific hybrid between onion (Allium cepa L.) and leek (Allium porrum L.). J. Agric. Food Chem. 46, 5220-5224. SCHULZ, H., KRÜGER, H., HERCHERT, N., KELLER, E.R.J., 2000: Occurrence of volatile secondary metabolites in selected Allium wild types. J. Appl. Bot. 74, 119-121. STOLL, A., SEEBECK, E., 1951: Über Allium Substanzen. 5. Die Synthese des natürlichen Alliins und seiner drei optisch aktiven Isomeren. Helv. Chim. Acta 34, 481-487. STORSBERG, J., SCHULZ, H., KEUSGEN, M., TANNOUS, F., DEHMER, K.J., KELLER, E.R.J., 2004: Chemical characterization of interspecific hybrids between Allium cepa L. and Allium kermesinum Rchb. J. Agric. Food Chem. 52, 5499-5505. THEODOROPOULOS, D., 1959: Synthesis of certain S-substituted L-cysteines. Acta. Chem. Scand. 13, 383-384. ZIEGLER, S. J., STICHER, O., 1989: HPLC of S-alk(en)yl-L-cysteine derivatives in garlic including quantitative determination of (+)-S-allyl-L-cysteine sulfoxide (Alliin). Planta Med. 372-378. Addresses of the authors: 1 Bundesanstalt für Züchtungsforschung an Kulturpflanzen, Institut für Pflanzenanalytik, Neuer Weg 22-23, D-06484 Quedlinburg, Germany 2 Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, D 35032 Marburg, Germany 3 Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK), Correns- straße 3, D-06466 Gatersleben, Germany Determination of cysteine sulphoxides in Allium plants 35 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /Description << /FRA /ENU (Use these settings to create PDF documents with higher image resolution for improved printing quality. 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