Determination of 2 , 5-diketopiperazines in Greek Processed Olives by Liquid Chromatography / Mass Spectrometry Analysis

1Laboratory of Chemistry, Analysis & Design of Food Processes, Instrumental Food Analysis, Department of Food Technology, Technological Educational Institution of Athens, Ag. Spyridonos, 12210, Egaleo, Greece. 2Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Vas. Constantinou Ave., 11636, Athens, Greece. 3Laboratory of Food Chemistry and Technology, Department of Chemistry, University of Ioannina, 45110, Ioannina, Greece.

plantarum 6 and fungi, e.g., Aspergillus flavus 7 or Alternaria alternata 8 as well as marine sponges 9 .Recently, the interest in this substantial class has increased due to their immense bioactivities, including antitumor, antiviral, antibacterial, antifungal, anthelmintic and anticancer 10,11,12,13,14 .Diketopiperazines have the ability to bind to a wide range of enzymes and receptors making them attractive scaffolds for drug discovery 15 .
Fermentation process for selected cultivars includes packaging of the fruits into airtight and food-grade containers covered with brine.The lids of the containers were closed loosely and the filled containers were stored at 25 o C.

isolation of 2,5-diketopiperazines from olive pastes
Olive's pastes were prepared by removing the stone and homogenised the rest of the fruit using a blender.Pastes (30 g) were extracted by solid-liquid extraction according to Ryan et al. 16 with the following modifications.For pigments and lipids removal n-hexane extraction was applied four times (4 x 50 mL) at room temperature for 30 min.After centrifugation, the residual material was extracted 4 times with acetone/water (70/30, v/v) for 45 min at room temperature under stirring.The procedure was repeated thrice (3 x 50 mL), the samples were centrifuged and the combined supernatants were dried over anhydrous sodium sulphate, filtered and acetone was removed using a rotary evaporator at 30 o C.. The 2,5-diketopiperazines were extracted from the aqueous solution obtained with dichloromethane (DCM) by liquid/liquid extraction.The procedure was repeated thrice (3 x 30 mL) and the combined extracts dried over anhydrous sodium sulphate, filtered and concentrated in pre-weighed vials in a rotary evaporator to constant weight, to determine the extractable content.Afterwards, the extracted 2,5-diketopiperazines were redissolved in DCM, filtered through a 0.45 ìm filter and stored at -20 ºC until used for LC-MS n analysis.

Liquid Chromatography -Mass Spectrometry (LC-MS) Chromatographic conditions
LC analyses were performed using an a Thermo Accela U-HPLC injecting 1 µL sample from a cooled tray (10°C) directly onto an Kromasil column (2.1 mm × 100 mm, 1.8 µm particles, equilibrated in 90% solvent A (0.1% aqueous solution of acetic acid) and 10% solvent B (acetonitrile containing 0.1% acetic acid).The compounds were eluted using flow rate 250 µL/min by linearly increasing solvent B concentration from 10% to final 60% over 7 min .The column was then washed increasing solvent B up to 100% (3 min) and re-equilibrated in 90% solvent A, 10% solvent B. The total run time, including column wash and equilibration, was 15 min.

Mass spectrometry analysis
For LC/ESI-MS experiments, a Thermo Scientific LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was connected to the UHPLC instrument via an HESI interface.Centroided mass spectra were acquired with AGC target value of 1E6, resolution of 30,000 (FWHM as defined at m/z 400), and maximum ion injection time (IT) of 100 ms, for full scan analysis in the mass range of m/z 80 to 380 Da followed by data dependent MS/MS on the linear ion trap on the most intense ion from parent list.The normalized collision energy for the high efficiency collision-induced dissociation (CID) was adjusted to 35%, and the isolation width of precursor ions was m/z 2.0 The mass spectrometer was operated in the positive ion mode using a source voltage of 3.50 kV.The capillary temperature was 300 o C and the source heater temperature was 200 o C. The auxillary gas and sheath gas flow rates were set at 35 Arb and 15 Arb, respectively.The instrument was calibrated daily using the manufacturer's calibration mixture ProteoMass LTQ/FT-Hybrid ESI Pos.Mode Cal Mix (SUPELCO, Bellefonte, PA, USA).Mass Frontier 6.0 (High Chem Ltd.) was implemented to produce possible fragments and mechanisms using standard databases (HighChem ESI Pos 2008 and HighChem Fragmentation Library).Liquid Chromatography -Mass Spectrometry (LC-MS)  The presence of DKPs in the studied olives is the result of fermentation from microorganisms.Their presence increases the olives' nutritional value and contributes to their organoleptic characteristics, giving bitter and metallic taste.The identification of the 2,5-diketopiperazines(2,5-DKPs) in the 14 different samples was performed by comparison of their retention times and mass spectra with those of reference compounds.The retention times, the molecular masses of the protonated DKP standards as identified by ESI-MS 1 and expressed in mono-isotopic basis as well as their mass spectral characteristics are specified in Table 1.The highresolution mass data of protonated 2,5-DKPs confirmed their elemental composition, given a mass error of below 1.7 ppm with their theoretical molecular mass.The complete chromatogram of the 19 standard DKPs is provided in Figure 1 and the total ion chromatograms of olive samples are presented in Figure 2.
Fragment ions with intensities <5% of the base peak were mentioned only in the case they were needed for identification or comparison.The 2,5-diketopiperazines showed similar fragmentation patterns and revealed the characteristic mass spectrometric behavior in ESI( + ) experiments, producing M + ions in their MS 1 spectra and the product ions [MH + -28], [MH + -17] and [MH + -45] in their MS 2 spectra 17 , with different intensities of the fragments.
Results showed that only 8 out of 19 standard DKPs were identified in the 14 samples (Tables 3  and 4). Figure 3 presents the chromatograms of the 8 standard DKPs found and their corresponding peaks in the olive samples.One common identified DKP in all 14 samples is cyclo(Phe-Phe) followed by cyclo(Leu-Phe) found in 7 samples and cyclo(Pro-Val), cyclo(Phe-Pro) together with cyclo(Leu-Pro) were found in 6 samples (Tables 2 and 3).
Cyclo(Phe-Phe) has also been detected in a wide variety of beverages and foods such as beef, cheddar cheese, cocoa, white wine, yeast extract, etc.It is a naturally occurring 2,5-diketopiperazine and is dual inhibitor of the serotonin transporter (SERT) and acetylcholinesterase (AChE) in vitro 18 .

Fig. 2: total ion chromatograms of olive extracts' samples
The asymmetric and aromatic DKPs cyclo(Leu-Phe) and cyclo(Phe-Pro) have also been detected in cheddar cheese, chicken, coffee, cocoa, roasted pork, red wine, white wine and balsamic vinegars.Cyclo(Leu-Phe) has exhibited an important antiradical activity against the free hydroxyl radicals 10,18 .
The asymmetric and aromatic cyclo(Leu-Phe) was detected in most of the studied varieties (Table 3) and more specifically in samples 2-7 and 14.Cyclo(Leu-Phe) its (MH + ) ion at m/z 261.1598 (Table 1 and 4) released fragments at m/z 233.1786, 244.1782, 216.1437 and 188.1208, resulting from the loss of (C=O), (NH 3 ), (HCONH 2 ) and (C=O + HCONH 2 ) moieties, respectively.The ions at m/z 148.1770 and 120.0578 represented the Phe amino acid after OH and CO 2 H elimination, respectively, whereas the ion at m/z 86.0968 the Leu amino acid after CO 2 H moiety loss.Regarding cyclo(Phe-Pro), the presence of an ion at m/z 172.1002 after the loss of 73 amu only in its MS 3 spectrum, pointed that this compound is a proline-based DKPs analogue 17 .

Fig. 3: Chromatograms of the 8 standard DkPs found and their corresponding peaks in the olive samples
Cyclo(Leu-Tr p) exhibited the parent ion (MH + ) at m/z 300.1707 and cyclo(Trp-Tyr) at m/z 350.1499 (Table 1 and 4).Both compounds produced fragment at m/z 130 in relative abundance 100% which is the typical fragment pointing to the tryptophan side chain (3-methylene-indole positive ion) in the MS 2 spectra 17 .Furthermore, the characteristic fragments for Trp amino acid at m/z 132 and 170 were also observed, corresponding to 3-methyl-indole protonated and 3-(3-indol)-propenal protonated ions, respectively 17 .
Cyclo (Leu-Trp) has also been isolated from marine fungus Acremonium strictum and sponge Callyspongia sp.cyclo (Leu-Trp) displays important antiradical activity against the free hydroxyl radical and higher antioxidant activity than vitamin E 10,18 .

i d e n t i f i c a t i o n o f n o n -a r o m a t i c 2 , 5diketopiperazines
The diketopiperazines, cyclo(Leu-Pro), cyclo(Pro-Val) and cyclo(D-Ala-Pro) presented their parent ions (MH + ) at m/z 211.1441, 197.1285 and 169.0972, respectively (Table 1 and 4).They also exhibited similar fragmentation pattern via the loss of 56 amu, which is resulted by the consequent elimination of C 2 H 2 and C=O moieties from the parent ion 17 , yielding peaks at m/z 155.0913, 141.1104 and 113.1010, respectively.This fragmentation pathway allowed assigning them as proline-based DKPs analogues.Furthermore, the ions at m/z 98 and 70 (Table 1) represented the Pro amino acid after OH and CO 2 H elimination, respectively.These proline containing DKPs are also found in a wide variety of food and beverages as well as in cultures of bacteria and fungi.Cyclo(Pro-Val) was identified as the most important 2,5-diketopiperazine contributing to the bitter taste of several foods 10,16,18,19 .
To a further extent, the symmetric aliphatic DKP cyclo(Val-Val) was detected only in one olive variety (Table 3).Its (MH + ) ion at m/z 199.1441 (Tables 1 and 4) released in the MS 2 spectrum, fragments at m/z 171.1223, 154.1027 and 126.1556, resulting from the loss of (C=O), (HCONH 2 ) and (C=O + HCONH 2 ) moieties, respectively.The ion at m/z 72.0649 represented the Val amino acid after CO 2 H elimination.Cyclo(Val-Val) has also been detected in beef, bread, chicken, cocoa and yeast extract.It has also been isolated from marine microorganism Bacillus subtilis and has been shown to have antimalarial activity 18,20 .

COnCLuSiOnS
LC/ESI-MS analysis identified 8 aromatic and aliphatic 2,5-diketopeperazines firstly reported for Greek processed olives.Two of the richest varieties were 'Kothreiki' medium sized fruit (Sample 2) and 'Hondroelia' large sized fruit (Sample 6) both from Amphissa, with 7 common DKPs.This fact may indicate the importance of the geographical origin and cultivation practices to the metabolic profile of the fruit.Seven DKPs were also found in 'Kalamon' medium sized fruit from Ilia (Sample 4) which is the only sample where cyclo(Val-Val) was identified.Regarding DKPs, the most abundant were those based on phenylalanine-and proline-aminoacids with cyclo(Phe-Phe) being the only diketopiperazine found in all 14 samples.Overall, the different profile of 2,5-diketopiperazines, obtained for the studied varieties is probably related to genetic and geographical factors, cultivation conditions, as well as the handling and storage methods before and during fermentation process.

table 4 : DkPs detected in Greek olive varieties
A: Aromatic DKP; NA: Non aromatic DKP