Article pp.173-184 du Vol.24 n°2 (2004)

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ARTICLE ORIGINAL ORIGINAL PAPER

Characterization and identification of some phenolic compounds in Apricot fruit (Prunus armeniaca L .)

M. Radi^1*, M. Mahrouz¹, A. Jaouad¹, M. J. Amiot²

RÉSUMÉ

Caractérisation et identification des composés phénoliques de l’Abricot (Prunus armeniaca L.)

L’Abricot est un fruit sensible au brunissement enzymatique, dû à l’oxydation des composés phénoliques, produite lors de traumatismes mécaniques et thermiques. L’apparition de couleurs brunes indésirables déprécie les qualités organoleptiques et nutritionnelles des produits transformés. Grâce à différen- tes méthodes analytiques (chromatographie sur couche mince, détection UV- visible, chromatographie liquide haute performance et spectrométrie de masse), nous avons caractérisé et identifié les principaux composés phénoli- ques de l’abricot. Huit composés ont été isolés et identifiés en comparant leurs caractéristiques avec des étalons commerciaux, à savoir, l’acide proto- catéchique, la (+)-catéchine, l’acide chlorogénique, la (-)-épicatéchine, la naringénine-7-glucoside (ou prunine), la quercétine-3-glucoside, la quercé- tine-3-rhamnoglucoside (ou rutine) et le kaempférol-3-rutinoside. Plusieurs autres composés ont été caractérisés dans ce travail. Les résultats obtenus montrent que les acides chlorogénique et néochlorogénique, la (+)-catéchine, la (-)-épicatéchine et la rutine sont les composés majoritaires parmi les poly- phénols de l’Abricot. Par ailleurs, l’acide protocatéchique, la prunine et les procyanidines B₂, B₃ et C₁ sont signalés pour la première fois dans le fruit de l’abricotier.

Mots clés

Abricot, composés phénoliques, caractérisation, identification.

1. Faculté des Sciences Semlalia, Département de Chimie, B.P. 2390, Marrakech, 40000 Maroc.

2. I.N.R.A., UMR A 408, Unité Sécurité et Qualité des Produits d’Origine Végétale, Domaine Saint-Paul, Site Agroparc, 84914 Avignon Cedex 9, France.

*Correspondance : Mohamed RADI, Docteur – Délégation Régionale d’Industrie et du Commerce – Q. Admi- nistratif – B.P. 537 BéniMellal, 23000 Maroc – E-mail : [email protected].

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SUMMARY

Apricot is a climacteric fruit that could develop undesirable color, flavor and a loss of nutrients when submitted to mechanical and technological treat- ments through browning reactions. Enzymatic browning results from the oxidation of polyphenols by polyphenol oxidases in the presence of oxygen.

Identification and characterization of polyphenolic composition of apricot fruits were carried out by chromatography on cellulose plates, high-performance liquid chromatography (HPLC), UV-visible spectral analysis and fast atom bombardment mass spectrometry (FAB-MS). Eight major compounds were isolated and identified as protocatechuic acid, (+)-catechin, 3’-caffeoylquinic (or chlorogenic acid), (-)-epicatechin, naringenin-7-glucoside (or prunin), quercetin-3-glucoside, quercetin-3-rhamnoglucoside (or rutin) and kaempferol-3-rutinoside, by the comparison of characteristics with commercial standards, analyzed with the same conditions. Other compounds were characterized in apricot fruits using several methods. Data indicated that chlorogenic and neochlorogenic acids, (+)-catechin and (-)-epicatechin and rutin were the major phenolic compounds in apricots. Protocatechuic acid, prunin and procyanidins B₂, B₃ and C₁ were characterized for the first time in apricot fruits.

Key words

Apricot, phenolic compounds, characterization, identification.

1 – INTRODUCTION

Phenolic compounds of apricot fruits have been shown to have a major role in tissue browning, and the colour characteristics of fruits and derived products (RADI et al., 1997; AMIOT et al., 1995; AMIOT et al., 1992; CHEYNIER et al., 1991;

OLESZEK et al., 1989).

A better knowledge of the phenolic composition in fresh apricot fruits appeared necessary to determine the suitability of fruits for processing with respect to the color of apricot-derived products.

Apricot phenolics have been characterized by few investigators as summa- rized by MACHEIX et al. (1990). Apricot phenolics can be divided in three main classes: flavan-3-ols, hydroxycinnamic esters and flavonols (RADI et al., 1997).

The proportion of these three classes greatly varied with apricot cultivar (RADI et al., 1997).

Hydroxycinnamic derivatives were identified as esters of caffeic, coumaric and ferulic acids (HERRMANN, 1973). Coumarin and scopoletin have been also identified (RESCHKE and HERRMANN, 1981). Apricot fruit was characterized by the predominance of chlorogenic acid also named 5’-caffeoylquinic acid (GARCIA- VIGUERA et al., 1994; DIJKSTRA and WALKER, 1991; GAJZAGO et al., 1977). Cou- maroyl and feruloyl derivatives were identified as glucose esters and quinic isomers (RISCH and HERRMANN, 1988; RESCHKE and HERRMANN, 1981). The presence of (+)-catechin and (-)-epicatechin has also been reported (RISCH and

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HERRMANN, 1988; MOSEL and HERRMANN, 1974). Flavonols in apricots have been characterized as glucosides and rutinosides of quercetin and of kaempferol (GARCIA-VIGUERA et al., 1994; HERRMANN, 1979).

In regard to the literature data on the phenolic composition of apricot remain limited. Thus, the aim of the present work was to purify, isolate and identify the main phenolic compounds in apricot using modern analytical techniques as HPLC and mass spectrometry.

2 – MATERIALS AND METHODS

2.1 Plant materials and chemicals

Apricot trees were grown at Institut National de la Recherche Agronomique in Manduel (Gard, France). Fruits (cv Canino) were picked at commercial maturity. They were cut into small pieces, immediately frozen in liquid nitrogen, freeze-dried and stored at –20˚C until use.

Phenolic compounds were identified by the comparison of their retention times, UV-visible spectra, their relative mobility on cellulose plates with co- eluted commercial standards (Extrasynthèse, Genay, France) for protocatechuic acid, (+)-catechin, chlorogenic acid, (-)-epicatechin, naringenin-7-glucoside, quecetin-3-glucoside, quercetin-3-rhamnoglucoside and kaempferol-3-rutinoside.

The 4’-caffeoylquinic and 5’-caffeoylquinic standards were obtained by trans-esterification of 3’-caffeoylquinic according to the method described by ADZET and PUIGMACIA (1985). The co-chromatography of each compound with a phenolic extract of apple containing the two isomers (AMIOT et al., 1992) gave the characteristics (R_t, R_f) of each isomer.

Standards of sugars were purchased from Sigma (Saint-Quentin-Fallavier, France).

2.2 Extraction and purification of phenolic compounds

The dried material was finely powdered and a 20 grams portion was blended with 1L of cold ethanol (80%) containing sodium metabisulfite (0.5%). Three successive extractions were carried out at 4˚C for 15 min. After removal of alcohol under vacuum at 35˚C, pigments were eliminated by three successive extractions with petroleum ether (2:1, V/V) (water extract). After addition of amonium sulfate (20%) and metaphosphoric acid (2%) to the aqueous phase, phenolic compounds were three times extracted with ethyl acetate (1:1, V/V).

The three organic phases were collected, combined, dried and evaporated under vacuum. Residue was dissolved in methanol (10ml) (fraction I). Methan- olic extract was filtered through Acrodisc filter (0.45µm) before analyses by high performance liquid chromatography (HPLC).

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2.3 Separation of phenolic acids and neutrals

A water extract obtained as described above, was adjusted at pH 7.0 with NaOH 30% ; then neutral phenolics were extracted three times with ethyl acetate (V/V). The three organic phases collected was purified as described above to obtain neutral fractions (fraction II).The aqueous phase deprived from neutral phenolics, was adjusted to pH 3 with trifluoroacetic acids, and then phenolic acids were extracted three times with ethyl acetate (V/V). The organic phase collected was also purified as previously described to obtain phenolic acid fractions (fraction III).

2.4 Chromatography on cellulose plates

Phenolic compounds (fractions I, II, III) were separated using cellulose plates (Merck, 5577) in two dimensions. Compounds were eluted with acetic acid 15%

(first dimension) and butanol-acetic acid-water (14-2-5; V/V/V) (second dimension). The R_f (frontal ratio) of each compound was measured (Table 1).

Flavan-3-ols were non-fluorescent under UV light and were reveals as red spots by spraying the plates with a solution of vanillin in HCl (RIBEREAU-GAYON, 1968).

Phenolic acids were monitored under UV light (366/254 nm) before and after spraying with Benedikt reagent (REZNIK and EGGER, 1961) or Neu reagent (ANDARY, 1975). Caffeoylquinic and its derivatives (fluorescent under UV) became non-fluorescent with Benedikt reagent, while mono-hydroxylated phenolic acid became blue fluorescent under UV. In contrast, caffeoyl derivatives became green-fluorescent with Neu reagent while feruloyl derivatives were not modified with this spray. Coumaroylquinic acids were fluorescent under UV after exposure to ammonia vapor, and became non-fluorescent with Neu reagent spray. Benzoic acid derivatives were blue fluorescent under UV (254nm) with or without Benedikt or Neu reagents spray. Flavonols and flavanone were monitored under UV at 365 nm after exposure to ammonia vapor or AlCl₃.

2.5 HPLC/UV analyses of phenolics

The purity of the separated compounds, their retention times (R_t), and their general phenolic composition in apricot extracts were determined with HPLC apparatus (Varian 5500 connected to a diode array detector Waters 900) using an chrompack C₁₈ 7µm (200 mm × 3mm i.d., Alltech) column. The UV spectra (200-400 nm) and bathochromic shifts were recorded and assigned according to the method of MABRY et al. (1970). The separation and characterization condi- tions were described in a previous paper (RADI et al., 1997).

2.6 Isolation of phenolics by semi-preparative HPLC

Phenolic compounds (fractions II and III) were isolated by semi-preparative HPLC using the same apparatus described above, but with a Licrosorb RP 18 column (10µm packing, 250mm × 6.2 mm i.d., Altech). The solvent system used was a gradient of A (water, acetic acid; 98:2) and B (MeOH). The following gradient was applied: 0min, 10%B; 30min, 10%B isocratic; 30-42min, 18%B; 42-65min, 18%B isocratic; 80min, 40%B; 80-100min, 40%B isocratic.

The solvent flow rate was 1.3 ml min^-1; and the separation was performed at 35˚C. The injected sample was 100µL.

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Each fraction collected was checked by analytical HPLC, and fractions dis- playing the presence of a single compound with identical spectral characteristics were combined. After removing alcohol under vacuum at 35˚C, phenolic compounds were extracted with ethyl acetate and evaporated to dryness under vacuum at 38˚C. The residue was dissolved in methanol and filtered through an Acrodisc filter (0.45 µm) before analysis and FAB-MS.

2.7 Analysis of hydrolysis products

Compounds (flavonols) isolated by semi-preparative HPLC were hydrolyzed with 4N HCL at 100˚C for 10 min. After cooling, the mixture was treated three times with ethyl acetate, and the organic solutions were dried and evaporated under vacuum at 38˚C. The residue was dissolved in methanol.

Flavonol aglycones were analyzed by chromatography on cellulose plate together with commercial standards and using the same conditions as described above. Sugars were extracted out from aqueous solution with 10% dioctylmethyl- amine reagent in chloroform, and were analyzed by HPLC. Sugars were separated by HPLC along a sugar pak column (Interchim) and detected by refractometry (HPLC VARIAN 9010 with a refractometric differential VARIAN RI-4 connected to a computer PC-CIT). The mobile phase (flow rate 0.5 ml min^-1) consisted of water with 50 mg/l EDTA (calcium di-sodium ethylene diamine tetra-acetate).

2.8 Fast Atom Bombardment Mass Spectrometry (FAB-MS)

Negative-ion ESI-MS spectra were recorded on a Sciex API I Plus (Sciex, Thomhill, Ontario, Canada) simple quadrupole mass spectrometer with a nomi- nal mass range up to m/z = 2400, equipped with an ion spray source. The mass spectrometer was operated with a -400 V voltage applied to the electrospray needle and -60 V to the orifice. The mass spectrometer was scanned to m/z 2400, in step of 0.1 msec unit and with a dwell time of 25 msec at each step.

The instrumentation was calibrated using a solution of polypropylene glycol oli- gomers. The spectrometer was coupled with an HPLC unit ABI 140 B (Applied Biosystems, Weiterstadt, Germany) with a diode array detector (ABI 785 A), using an Superspher 100-RP C₁₈ (3µm packing, 125 × 2mm i.d., Merk, Darms- tadt, Germany) column. Methanol extracts were filtered through an Acrodisc filter (0.45 µm) and 20 µl were injected into HPLC column. The solvent system used was the gradient of A (H₂O / HCOOH; 98/2), and B (CH₃CN / H₂O / HCOOH; 80/18/2). The solvent flow rate was 200 µl/min. The following gradient was applied : 0-3 min, 5%B isocratic; 3-10 min, 20%B linear; 10-30 min, 25%B linear; 30-32 min, 100%B linear; 32-35min, 100%B isocratic.

3 – RESULTS

According to the UV spectroscopy and two-dimensional chromatography on cellulose plates, the phenolics of apricots have been characterized by the predominance of three main classes : flavan-3-ols, hydroxycinnamic esters and flavonols.

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All phenolics of these three families were easily detected on chromatography on cellulose plates. Spots were immediately detectable under UV before and after spraying different reagents.

All the major phenolics were identified by comparing the retention times given by analytical HPLC (figure 1) and the spectral characteristics of standards. Mass spectrometry confirmed the structures by giving the molecular weight and the major associated fragments. The specific characteristics of each compound were described in table 1.

Figure 1

Reversed phase HPLC chromatogram of phenolic compounds of apricot fruit (Detection at 280nm).

Séparation par HPLC (phase inverse) des composés phénoliques de l’Abricot (Détection à 280nm).

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© Lavoisier – La photocopie non autorisée est un délit Table 1 Characterization of apricot phenolics by UV light, RP-HPLC/UV-vis (diode array detector) and FAB-MS Tableau 1 Caractérisation des composés phénoliques de l’Abricot par lumière UV, HPLC phase inverse et Spectrométrie de masse Peak noRf * 100Spot appearance UV light Rt (min) spectra (MeOH) Absorption (nm)m/z compound identifiedclass AA15% BAEUVUV+NH3[M-H]- fragments 16561blue-violetblue-violet07,9252-291n.d.n.d.protocatechic acidbenzoic acid 25338blue-clearyellow-green11,4225-290353191-179neochlorogenic acidHCE 33923darkn.d.14,3280577289procyanidin B3FLA 44717darkn.d.16,6280577289dimeric formsFLA 54348darkn.d.18,3280289(+)-catechinFLA 74118darkn.d.20,7280865577-289trimeric formsFLA 94962blue-clearyellow-green25,9225-290353191-179cryptochlorogenic acidHCE 105249blue-clearyellow-green27,8225-290353191-179chlorogenic acidHCE 114626darkn.d.29,3280577289procyanidin B2FLA 123437darkn.d.33,7280289(-)-epicatechinFLA 135758n.d.blue-bright35,8315337191-1633'-p-coumaroylquinicHCE 145031darkn.d.36,8280865577-289procyanidin C1FLA 154413darkn.d.48,4280577289dimeric formsFLA 165746n.d.yellow50,8283-330433271naringenin-7-glucosideflavanone 176777blueblue-green53,4323-290367feruloylquinic acidHCE 182539darkyellow-brun54,8356-290463301quercetin-3-glucosideFLO 194330darkyellow-brun55,4256-296-358609301quercetin-3- rhamnoglucosideFLO 203652darkyellow-brun59,3350-295593285kaempferol-3-rutinosideFLO 216890blueblue-green60,0323-290367feruloylquinic acidHCE n.d. : not detected. HCE: hydroxycinnamic esters; FLA: flavan-3-ols; FLO: flavonols. n.d. : non détecté HCE: esters hydroxycinnamiques; FLA: flavan-3-ols; FLO: flavonols.

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Flavan-3-ols have been characterized by their spectra with a maximum at 280 nm. The structures of (+)-catechin, (-)-epicatechin, procyanidins B₂, B₃, and C₁ were attributed respectively to the compounds 5, 12, 11, 3 and 14. Com- pounds 4, 7 and 15 were attributed to two dimeric and one trimeric procyanidins according to the molecular weight m/z and fragments. The co- chromatography with commercial standards (isolated from apple and grapes for B₂, B₃, and C₁ ; AMIOT et al., 1992; OSMIANSKI and SAPIS, 1989), confirmed the structures identified by comparing the data obtained by R_f, R_t and UV spectra.

Phenolics acids were present in apricot as hydroxycinnamic esters and one benzoic acid derivative. Among hydroxycinnamic esters, caffeoylquinic acids represented the major class and were identified as 5’-caffeoylquinic (compound 10), 3’-caffeoylquinic (compound 2) and 4’-caffeoylquinic (compound 9). These three isomers have got the main signal with FAB-MS at m/z 353 related respectively to the [M-H]^-. The cleavage of ester bond gave two peaks at m/z 191 and m/z 179 corresponding to quinic acid and caffeic acid respectively. Compound 13 was attributed to p-coumaroylquinic acid with a spectra maximum at 315 nm and a shoulder at 300 nm. The structure of this compound was confirmed by FAB-MS with a signal at m/z 337 related to [M-H]^- and two fragments at m/z 191 and m/z 163 attributed to quinic acid and coumaric acid respectively. The co-chromatography with a phenolic extract of apple fruit containing the two isomers 4’- and 5’-p-coumaroylquinic showed that the compound 13 could be attributed to the 3’-p-coumaroylquinic.

Compounds 17 and 21 displayed similar spectral absorption UV as caffeoylquinic esters, but their fluorescence was modified by Benedikt reagent indicat- ing the presence of a monophenolic function. These two phenolic compounds have got the main signal on FAB-MS at m/z 367 related to the [M-H]^- peak of feruloylquinic acid. In regard to the literature, only two isomers 3’- and 5’-feruloylquinic have been reported (MACHEIX et al., 1990). Consequently, compounds 17 and 21 were supposed to be 3’- and 5’-feruloylquinic acids.

Finally, compound 1 was identified according to its characteristics R_f, UV and R_t by comparing the data obtained with standards. The polarity of this compound was higher than the others compounds. The co-chromatography with standards revealed that compound 1 was protocatechuic acid.

Concerning flavonoids, these compounds displayed the lowest polarity and were eluted by reverse-phase HPLC after hydroxycinnamic esters (figure 1).

Four compounds were identified in apricot fruits as flavonoids and were num- bered 16, 18, 19 and 20. The structures of these compounds were determined using the different analytical methods. Compounds 18, 19 and 20 were not detected under UV light and appeared as yellow spots after exposure to ammonia vapour or spraying AlCl₃. Their spectral absorption maxima were at 350 nm with a shoulder near 295 nm. Acid hydrolysis of compound 18 yielded quercetin and glucose. The FAB-MS of compound 18 showed a prominent peak at m/z 463 related to the [M-H]^- with a fragment at m/z 301 specific for quercetin.

Acid hydrolysis of compound 19 yielded quercetin. FAB-MS gave a molecular ion [M-H]^- at m/z 609 and a fragment at m/z 301 corresponding to quercetin.

Compound 20 was characterized using FAB-MS by a prominent peak at m/z 593 corresponding to [M-H]^- and a main signal at m/z 285 specific to kaempferol. These three compounds (18, 19, 20) were readily identified with commercial standards as quercetin-3-glucoside, quercetin-3-rhamnoglucoside and kaempferol-3-rutinoside respectively.

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The structure of compound 16 was characterized using FAB-MS by a molecular ion [M-H]^- at m/z 433 and a fragment at m/z 271. The spectral absorption maximum was at 283 and shoulder near 330 nm. This compound was revealed with Neu reagent under UV light. The behavior of this compound was similar to those obtained with flavanones. The co-chromatography with standards indicated that compound 16 was attributed to naringenin-7-glucoside (or prunin).

Finally, compounds 6 and 8 displayed the same spectral characteristics as isomers with a maximum at 284 nm and a shoulder at 325 nm, and with the main signal by FAB-MS at m/z 329 related to the [M-H]^-, but their structures remained unknown.

4 – DISCUSSION AND CONCLUSIONS

Twenty-one phenolic compounds were characterized. Some peaks were characterized by diode array detection by comparing their spectral characteristics with commercial standards. The other techniques used in this work were necessary for identification.

Phenolic compounds of apricot fruits were partitioned into three major families according to their fluorescence under UV and their spectral characteristics.

Flavan-3-ols are presented as monomerics and polymerics forms. Since (+)-catechin and (-)-epicatechin were signaled in high concentrations by several authors (RISCH and HERRMANN, 1988; MOSEL and HERRMANN, 1974), procyanidins were not reported in apricot fruit. Procyanidins B₂, B₃, and C₁ were identified comparing their R_f and R_t with known standards. All flavan-3-ols detected in this work were present as free forms. These results are in good agreement with data reported for other fruits as apple (AMIOT et al., 1992) and pear (OLESZEK et al., 1994), but differ from results published in grape where procyanidins were presented as esterified forms (KALLITHRAKA et al., 1994).

Concerning phenolic acids, chlorogenic and neochlorogenic acids were the main compounds. The presence of these two phenolic acids in high amounts in apricot fruits has been previously reported by several authors (GARCIA-VIGUERA et al., 1994; DIJKSTRA and WALKER, 1991; MOLLER and HERRMANN, 1983; GAJZAGO et al., 1977). Besides these major compounds, two isomers of feruloylquinic acids were detected. Previous work reported that apricot leaves contained three isomers of position of feruloylquinic acid (POESSEL, 1983).

Among hydroxycinnamic esters, one p-coumaric ester was identified. This compound was tentatively attributed to 3’-p-coumaroylquinic acid structure. All isomers of this compound were reported in apricot fruits by MOLLER and HERRMANN (1982).

In this work, no glucosides of p-coumaric and ferulic acids were detected as already reported in apricot (RESCHKE and HERRMANN, 1981).

To our knowledge, protocatechuic acid has not been reported in apricot fruits. This compound was detected as free form. Phenolic acids are rarely present as free form, except for few compounds such as p-coumaric acid which was detected in cherries (RUNICKI et al., 1973) and in peaches (RAMINA

and MASIA, 1982).

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Some of flavonoids identified in this work have been previously reported in apricot fruits (GARCIA-VIGUERA et al., 1994; HENNING and HERRMANN, 1980; HERRMANN, 1979). Two glycosides of quercetin were characterized. Quercetin-3-rutinoside (rutin) was particularly found at high concentrations in apricot fruits. One kaempferol glycoside was found as kaempferol-3-rhamnoglucoside. These results were in good agreement with previously reported data for other apricot varieties (HENNING

and HERRMANN, 1980), but differed from results published on American canned apricots where no kaempferol derivatives were detected (EL-SAYED et al., 1965).

No aglycones of flavonoids were detected in this study (quercetin or kaempferol), as previously reported in several Prunus by WOLLENWEBER and DIETZ (1981).

This is the first time that prunin has been reported in apricot fruits. This flavanone was previously found in apricot leaves (POESSEL, 1983).

The better characterization of all the phenolics in apricot fruits is of great inter- est to food technologists because it could contribute to understand the enzymatic browning reaction occurring in apricot during processing. Recently, GOUPY

et al. (1995) have specified the role of different phenolics in browning. It has been shown that the phenolic composition is an important factor determining the browning intensity in different fruits (AMIOT et al, 1997) ; it is the case of apples (AMIOT et al., 1992), pears (AMIOT et al., 1995) and grapes (CHEYNIER et al., 1991).

5 – ACKNOWLEDGEMENTS

We thank l’Ambassade de France au Maroc for financial support for the first author, Mr. Max Tacchini for technical assistance, and Mr. Thierry Doco of INRA Montpellier, for help in Mass Spectrometry.

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