Mercè Balcells

Ultra performance liquid chromatography analysis to study the changes in the carotenoid profile of commercial monovarietal fruit juices

We have developed an analytical method that allows the simultaneous determination of epoxycarotenoids, hydroxycarotenoids and carotenes in monovarietal fresh homemade and industrially processed fruit products. Analyses were carried out using ultra performance liquid chromatography (UPLC). The extraction method was optimized using methanol as the first extraction solvent for lyophilized samples followed by a saponification step. Recoveries ranged between 75% and 104% depending on the compound. Repeatability was better than 10% for all compounds (%RSD, n=3). The chromatographic analysis takes less than 17min. In this short period, up to 27 carotenoids were identified in apple, peach and pear products. The developed method allowed us to differentiate juice from six varieties of apple by their carotenoid profile. Moreover, the methodology allows us to differentiate the carotenoid profiles from commercial juices and homemade fresh peach and pear juices, as well as to study the rearrangements of 5,6- to 5,8-epoxycarotenoids.

Synthesis of allyl esters of fatty acids and their ovicidal effect on Cydia pomonella (L.).

Eight allyl esters of fatty acids were synthesized in moderate to high yields with a novel two-step procedure using glycerol as a starting material. The two-step methodology avoids the use of allyl alcohol. The first step consisted of heating at 80 degrees C for 48 h a 2:1:5 mmol mixture of glycerol, a fatty acid, and chlorotrimethylsilane in a solvent-free medium. The crude compound was then dissolved in butanone and heated at 115 degrees C in the presence of NaI. A tandem Finkelstein rearrangement-elimination reaction occurs, producing the corresponding allyl ester. The activity of these esters against Cydia pomonella (L.) (Lepidoptera: Tortricidae) eggs was tested in the laboratory by topical application of one 0.1 microL drop. All of the compounds showed a concentration-mortality response and caused 100% mortality at the highest concentration tested (10 mg/mL). There was an inverse relationship between the alkyl chain length and the ovicidal activity of the allyl ester; the LC(50) and the LC(90) of the two compounds that have the longer alkyl chains were significantly higher than those of the rest of the compounds. The ovicidal and IGR activities of this kind of compound appear to be unprecedented.

Bactericidal and fungicidal activity of Aspergillus ochraceus metabolites and some derivatives

The bactericidal and fungicidal activities of aspyrone and asperlactone, secondary metabolites of Aspergillus ochraceus, and some aspyrone derivatives were studied. In general, aspyrone exhibited better activity than asperlactone or aspyrone derivatives. The inhibition patterns of the assayed compounds were different. Helminthosporium monoceras was the tested mould most inhibited by the studied compounds. The comparative study of the activity of the different compounds showed that the epoxy group seems to be necessary for activity against some micro-organisms.

Characterisation of phenolic compounds in processed fibres from the juice industry.

The content of phenolic compounds was determined in nine industrially processed fibres derived from the juice industry. Apple, peach, and pear as non-citrus fruit fibres were examined, as well as orange peel and flesh, tangerine peel and flesh, and lemon flesh as citrus fruit fibres, and carrot as vegetable fibre. The extractable phenolic profile of all fibres was obtained by UPLC-PDA-FLR-MS/MS. Forty phenolic compounds were identified and their concentrations determined. In addition, bound phenolic acids and proanthocyanidins were measured in solid residues in order to determine the phenolic compounds remaining. Also, to allow the comparison of the profiles and contents in the fresh fruit and fibres, we analysed extractable and bound phenolic compounds in lyophilized peel and pulp from fresh fruit. The profile and phenolic content of the fibres was similar to that of the fresh fruit, except for flavan-3-ols, which registered lower values.

A rapid and reliable direct method for quantifying meat acylglycerides with monomode microwave irradiation

A rapid methodology for direct analysis of meat acylglycerides is proposed. A transesterification is carried out in a microwave reactor consisting of a monomode oven using chlorotrimethylsilane (CTMS) and methanol. High-temperature gas chromatography was used to check the absence of underivatized acylglycerides. Whereas transesterification is complete after 30s at 90 degrees C in the microwave method, the reference method needs 2h to complete this process. Moreover, the CTMS-microwave method shows higher recoveries of individual saturated, monounsaturated and polyunsaturated fatty acids. No influence of microwave irradiation on the composition of the fatty acids was observed.

Dispersive liquid–liquid microextraction and injection-port derivatization for the determination of free lipophilic compounds in fruit juices by gas chromatography-mass spectrometry

A method consisting of dispersive liquid-liquid microextraction (DLLME) followed by injection-port derivatization and gas chromatography-mass spectrometry (GC-MS) for the analysis of free lipophilic compounds in fruit juices is described. The method allows the analysis of several classes of lipophilic compounds, such as fatty acids, fatty alcohols, phytosterols and triterpenes. The chromatographic separation of the compounds was achieved in a chromatographic run of 25.5min. The best conditions for the dispersive liquid-liquid microextraction were 100μL of CHCl3 in 1mL of acetone. For the injection-port derivatization, the best conditions were at 280°C, 1min purge-off, and a 1:1 sample:derivatization reagent ratio (v/v) using N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA):pyridine (1:1) as reagent. Quality parameters were assessed for the target compounds, giving a limits of detection (LODs) ranging from 1.1 to 5.7ng/mL and limits of quantification (LOQs) from 3.4 to 18.7ng/mL for linoleic and stearic acid, respectively. Repeatability (%RSD, n=5) was below 11.51% in all cases. In addition, the method linearity presented an r2 ≥0.990 for all ranges applied. Finally, the method was used to test the lipophilic fraction of various samples of commercial fruit juice.

A rapid gas chromatographic injection-port derivatization method for the tandem mass spectrometric determination of patulin and 5-hydroxymethylfurfural in fruit juices.

A novel method consisting of injection-port derivatization coupled to gas chromatography-tandem mass spectrometry is described. The method allows the rapid assessment of 5-hydroxymethylfurfural (HMF) and patulin content in apple and pear derivatives. The chromatographic separation of the compounds was achieved in a short chromatographic run (12.2min) suitable for routine controls of these compounds in the fruit juice industry. The optimal conditions for the injection-port derivatization were at 270°C, 0.5min purge-off, and a 1:2 sample:derivatization reagent ratio (v/v). These conditions represent an important saving in terms of derivatization reagent consumption and sample preparation time. Quality parameters were assessed for the target compounds, giving LOD of 0.7 and 1.6μg/kg and LOQ of 2 and 5μg/kg for patulin and HMF, respectively. These values are below the maximum patulin concentration in food products intended for infants and young children. Repeatability (%RSD n=5) was below 12% for both compounds. In addition, the method linearity ranged between 25 and 1000μg/kg and between 5 and 192μg/kg for HMF and patulin, respectively. Finally, the method was applied to study HMF and patulin content in various fruit juice samples.

Injection-port derivatization coupled to GC–MS/MS for the analysis of glycosylated and non-glycosylated polyphenols in fruit samples

Polyphenols, including glycosylated polyphenols, were analyzed via a procedure based on injection-port derivatization coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS). The polyphenols in lyophilized fruit samples were extracted with an acidified MeOH mixture assisted by ultrasound. Samples were dried under vacuum, and carbonyl groups were protected with methoxylamine. Free hydroxyl groups were subsequently silylated in-port. Mass fragmentations of 17 polyphenol and glycosylated polyphenol standards were examined using Multiple Reaction Monitoring (MRM) as the acquisition mode. Furthermore, in-port derivatization was optimized in terms of optimal injection port temperature, derivatization time and sample: N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) volume ratio. A C18 solid-phase-extraction clean-up method was used to reduce matrix effects and injection liner degradation. Using this clean-up method, recoveries for samples spiked at 1 and 10μg/g ranged from 52% to 98%, depending on the chemical compound. Finally, the method was applied to real fruit samples containing the target compounds. The complete chromatographic runtime was 15min, which is faster than reported for recent HPLC methods able to analyze similar compounds.

Toxicity of allyl esters in insect cell lines and in Spodoptera littoralis larvae

We investigated the effects of five allyl esters, two aromatic (allyl cinnamate and allyl 2-furoate) and three aliphatic (allyl hexanoate, allyl heptanoate, and allyl octanoate) in established insect cell lines derived from different species and tissues. We studied embryonic cells of the fruit fly Drosophila melanogaster (S2) (Diptera) and the beet armyworm Spodoptera exigua (Se4) (Lepidoptera), fat body cells of the Colorado potato beetle Leptinotarsa decemlineata (CPB) (Coleoptera), ovarian cells of the silkmoth Bombyx mori (Bm5), and midgut cells of the spruce budworm Choristoneura fumiferana (CF203) (Lepidoptera). Cytotoxicity was determined with use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and trypan blue. In addition, we tested the entomotoxic action of allyl cinnamate against the cotton leafworm Spodoptera littoralis .The median (50%) cytotoxic concentrations (EC₅₀s) of the five allyl esters in the MTT bioassays ranged between 0.25 and 27 mM with significant differences among allyl esters (P = 0.0012), cell lines (P < 0.0001), and the allyl ester-cell line interaction (P < 0.0001). Allyl cinnamate was the most active product, and CF203 the most sensitive cell line. In the trypan blue bioassays, cytotoxicity was produced rapidly and followed the same trend observed in the MTT bioassay. In first instars of S. littoralis, allyl cinnamate killed all larvae at 0.25% in the diet after 1 day, while this happened in third instars after 5 days. The LC₅₀ in first instars was 0.08%. In addition, larval weight gain was reduced (P < 0.05) after 1 day of feeding on diet with 0.05%. In conclusion, the data provide evidence of the significant but differential cytotoxicity among allyl esters in insect cells of different species and tissues. Midgut cells show high sensitivity, indicating the insect midgut as a primary target tissue. Allyl cinnamate caused rapid toxic effects in S. littoralis larvae at low concentrations, suggesting further potential for use in pest control.

Influence of Carboxylic Acids on the Synthesis of Chlorohydrin Esters from 1,3‐Butanediol

Abstract The influence of diverse carboxylic acid on the preparation of chlorohydrin esters using a one‐pot esterification–chlorination reaction, in which one of the reagents (chlorotrimethylsilane) acts as solvent, is described. Whereas the acid with low pKa provided higher amounts of the 2‐chloro regioisomer, the ones with higher pKa rendered the 1‐chloro regioisomer in 80% yield. These results are in accordance with the mechanism proposed in a previous article.