Detection of phenolic oxidation products in cider apple juice by high-performance liquid chromatography electrospray ionisation ion trap mass spectrometry

Juice was prepared from cider apples of the cultivar "Kermerrien" under oxidative conditions. After isolation by solid-phase extraction, the phenolic fraction was subjected to high-performance liquid chromatography/electrospray ionisation mass spectrometry. SIM scans were performed at m/z values obtained in model solutions. The oxidation products, resulting from coupling between a molecule of caffeoylquinic acid and caffeoylquinic acid, catechin or dimeric flavan-3-ol, were detected.     

Characterisation by liquid chromatography coupled to electrospray ionisation ion trap mass spectrometry of phloroglucinol and 4-methylcatechol oxidation products to study the reactivity of epicatechin in an apple juice model system

The reactivity of the (-)-epicatechin structure towards caffeoylquinic acid o-quinones was studied in an apple juice model solution. The approach consisted in considering separately the reactivities of the two phenolic moieties of an (-)-epicatechin molecule: phloroglucinol and 4-methylcatechol were chosen to represent A- and B-rings, respectively. The oxidation products were characterised by RP-HPLC coupled with electrospray ionisation Mass spectrometry (MS). The reactivities of the A- and B-rings were clearly different on the basis of the oxidation products formed. Both A- and B-rings could be involved in covalent bond formation, but electron transfers only occurred with the B-ring. Most of the (-)-epicatechin oxidation products were linked by A/B-ring linkage ("head-to-tail" intermolecular coupling). After this first dimerisation step, intramolecular reactions seemed to be favoured. Therefore, the complexity of oxidation products in apple juice does not only result from an extensive polymerisation of native phenolic compounds, but also from a multiplicity of small molecules in different oxidation states and isomeric forms.     

Enhanced recovery of Salmonella from apple cider and apple juice with universal preenrichment broth

A comparison was made of the relative efficiencies of Universal Preenrichment (UP) broth and lactose broth for the recovery of a variety of Salmonella serovars from pasteurized and unpasteurized apple cider and pasteurized apple juice. Bulk portions of juice were contaminated with single Salmonella serovars at high and low levels of 0.4 and 0.04 CFU/mL, respectively. The juice was aged for a minimum of 5 days at 2-5 degrees C. On the day analysis was initiated, each of 20 test portions (25 mL) of the contaminated juice was preenriched in UP broth and in lactose broth. The Bacteriological Analytical Manual Salmonella culture method was followed thereafter. For pasteurized apple cider, UP broth recovered significantly (p < 0.05) more Salmonella-positive test portions than did lactose broth (112 and 75, respectively). For unpasteurized apple cider, UP broth recovered significantly more Salmonella-positive test portions than did lactose broth (326 and 221, respectively). For pasteurized apple juice, UP broth recovered more Salmonella-positive test portions than did lactose broth (93 and 81, respectively). However, this difference was not statistically significant. These results indicate that UP broth should replace lactose broth for the analysis of pasteurized and unpasteurized apple cider and pasteurized apple juice.     

Characterization of covalent addition products of chlorogenic acid quinone with amino acid derivatives in model systems and apple juice by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

High-performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS(n)) was used to study the covalent interactions between chlorogenic acid (CQA) quinone and two amino acid derivatives, tert-butyloxycarbonyl-L-lysine and N-acetyl-L-cysteine. In a model system at pH 7.0, the formation of covalent addition products was demonstrated for both derivatives. The addition product of CQA dimer and tert-butyloxycarbonyl-L-lysine was characterized by LC/MS(n) as a benzacridine structure. For N-acetyl-L-cysteine, mono- and diaddition products at the thiol group with CQA quinone were found. In apple juice at pH 3.6, covalent interactions of CQA quinone were observed only with N-acetyl-L-cysteine. Taking together these results and those reported by other groups it can be concluded that covalent interactions of amino side chains with phenolic compounds could contribute to the reduction of the allergenic potential of certain food proteins.     

Proteomic fingerprinting of apple fruit,juice, and cider via combinatorial peptide ligand libraries and MS analysis

Combinatorial peptide ligand libraries coupled to MS was applied to extensively map the proteome of apple fruit, and to detect its presence in commercial apple juice and cider to evaluate their authenticity and genuineness. Using the Uniprot_Malus database, 96 proteins were detected in apples, among which 30 proteins were specifically captured via combinatorial peptide ligand libraries. Next, three proteins, previously recognized in fruits, were found in apple juice, which were involved in cellular metabolism of fruit maturation and in allergenic reactions. On the other hand, only one Malus allergen was identified in cider beads eluate, demonstrating that the industrial processes did not prevent any negative effects in sensitive subjects. Thus, the present study not only increases the knowledge of the apple proteome but also offers a reliable analytical method to assess quality and genuineness of commercial products, which could be also used to inform consumers about the presence of allergens.     

Determination of patulin in apple juice by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

An HPLC-MS-MS method with selected reaction monitoring (SRM) for the determination of patulin in apple juice samples is described. Mass spectrometric detection was accomplished following atmospheric pressure chemical ionization (APCI) in both positive and negative ion modes. Collision induced dissociation (CID) of the protonated molecular ion led initially to the loss of H2O (fragment m/z 137). At higher energies CO is lost from both the protonated parent molecule (fragment m/z 127) and the dehydrated molecular ion (fragment m/z 109). In contrast, CID of the deprotonated molecular ion led initially to the fragment at m/z 109 corresponding to the loss of either CO2 or acetaldehyde, followed at higher CID energy by the loss of H2O (fragment m/z 135) and CO (fragment m/z 125) from the deprotonated molecular ion. Detection in the negative ion mode proved superior and a linear response was observed over the injected range from 6 to 200 ng patulin. Apple juice samples spiked with patulin between 10 and 135 microg/l were analyzed following liquid-liquid extraction with ethyl acetate and clean up with sodium carbonate. Utilizing reversed-phase HPLC with acetonitrile-water (10:90) at 0.5 ml/min, levels down to 10 microg/l were readily quantified and a detection limit of 4 microg/l was attainable at a signal-to-noise (SIN) ratio of 4. The MS data for the spiked samples compared well to the UV data and when plotted against each other displayed a correlation coefficient (R) of 0.99.     

Electrospray ionisation mass spectral studies on hydrolysed products of sulfur mustards

Off-site detection of the hydrolysed products of sulfur mustards in aqueous samples is an important task in the verification of Chemical Weapons Convention (CWC)-related chemicals. The hydrolysed products of sulfur mustards are studied under positive and negative electrospray ionisation (ESI) conditions using an additive with a view to detecting them at trace levels. In the presence of cations (Li(+), Na(+), K(+) and NH(4) (+)), the positive ion ESI mass spectra of all the compounds include the corresponding cationised species; however, only the [M+NH(4)](+) ions form [M+H](+) ions upon decomposition. The tandem mass (MS/MS) spectra of [M+H](+) ions from all the hydrolysed products of the sulfur mustard homologues were distinct and allowed these compounds to be characterised unambiguously. Similarly, the negative ion ESI mass spectra of all the compounds show prominent adducts with added anions (F(-), Cl(-), Br(-), and I(-)), but the [M-H](-) ion can only be generated by decomposition of an [M+F](-) ion. The MS/MS spectra of the [M-H](-) ions from all the compounds result in a common product ion at m/z 77. A precursor ion scan of m/z 77 is shown to be useful in the rapid screening of these compounds in aqueous samples at trace levels, even in the presence of complex masking agents, without the use of time-consuming sample preparation and chromatography steps. An MS/MS method developed to measure the detection limits of the hydrolysed products of sulfur mustards found these to be in the range of 10-500 ppb.     

Characterization and quantification of soy isoflavone metabolites in serum of renal transplanted patients by high-performance liquid chromatography/electrospray ionization mass spectrometry

During a dietary intervention study on 16 renal transplanted patients, in which 25 g/day of animal proteins were replaced with 25 g of soy proteins, the metabolic profile of soy isoflavones in serum was characterized. This paper describes a reliable and fast liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method, in negative ion mode, allowing the characterization and simultaneous quantification of several soy isoflavone metabolites. Six metabolites were identified and quantified: daidzein ([M-H](-) at m/z 252.8), dihydrodaidzein (DHD, [M-H](-) at m/z 254.8), equol ([M-H](-) at m/z 240.9), O-desmethylangolensin (O-DMA, [M-H](-) at m/z 256.8), genistein ([M-H](-) at m/z 268.8), and dihydrogenistein (DHG, ([M-H(+)](-) at m/z 270.8). Quantification was assessed using two deuterated internal standards, D(3)-daidzein and D(4)-genistein. This method permitted a limit of quantification (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.05 microM and 0.005 microM for all analytes, except for genistein, where the LOQ and LOD were 0.005 microM and 0.001 microM, respectively. The linearity ranges were from 0.005 to 1.5 microM for genistein, from 0.05 to 1.5 microM for DHG, and from 0.05 to 0.7 microM for the other metabolites. The relative standard deviations (RSDs) were between 0.19% and 13.9% at the LOQ concentration for all metabolites, and between 0.6% and 4.8% at the maximum concentration. On the basis of the results obtained in the dietary intervention study, it was possible to split the patients into five groups characterized by different metabolic pathways.     

超高效液相色谱-串联质谱法测定浓缩苹果汁中的熊果苷

建立浓缩苹果汁样品中熊果苷的固相萃取-超高效液相色谱-串联质谱(SPE-UPLC-MS/MS)检测方法。浓缩苹果汁样品用水溶解、过滤后,用聚苯乙烯-二乙烯基苯共聚物(PS-DVB)固相萃取柱净化,外标法定量。测定时用Eclipse Plus C18色谱柱(100 mm×2.1 mm, 1.8 μm)分离,甲醇-水系统梯度洗脱;MS测定采用多反应监测(MRM)模式。熊果苷的检出限为0.02 mg/L,在0.04～2.0 mg/L的范围内标准溶液的峰面积与质量浓度呈良好的线性关系,回收率为75.2%～102.7%,相对标准偏差(RSD)低于8.9%。该方法简便、快速、灵敏,可用于浓缩苹果汁样品中熊果苷的检测和确证。     

Ultrasound-assisted extraction, HPLC analysis, and antioxidant activity of polyphenols from unripe apple

The polyphenols were extracted from the unripe apple assisted by a highly efficient and simple method of the ultrasound. Response surface methodology was used to investigate the effects of processing parameters, including ultrasound power, extraction time, temperature, and ethanol concentration on total polyphenols yield and polyphenols composition was analyzed by HPLC. Antioxidant activity of the polyphenols was evaluated as 2, 2-diphenyl-1-picrylhydracyl scavenging activity and inhibition activity of lipid peroxidation. The results showed that 10-100 times higher total polyphenols yield was obtained from the unripe apple than those from the reported apple pomace. The optimum extraction conditions were ultrasonic power of 519.39 W, extraction time of 30 min, extraction temperature 50°C, ethanol concentration of 50% gave the total polyphenols yield of 13.26 ± 0.56 mg GAE/g. HPLC analysis indicated that (-)-epicatechin, procyanidin B2, chlorogenic acid, and procyanidin B1 were the predominant polyphenols in unripe apple, which contributed to the higher antioxidant activity to 2, 2-diphenyl-1-picrylhydracyl of unripe apple polyphenols than other apple polyphenols. The extracted polyphenols had higher ability to inhibit lipid peroxidation than butylated hydroxy toluene, which demonstrated that the unripe apple polyphenols have the potential to be used as a substitute of some synthetic antioxidants.     

Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry

Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane–ethyl acetate–1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.     

Solid-phase extraction method for patulin in apple juice and unfiltered apple juice.

Patulin, a mold metabolite, is commonly found in rotting apples. Some countries regulate patulin at levels ranging from 30 to 50 micrograms/L. Most analytical methods for patulin in apple juice include liquid-liquid partitions. A solid-phase extraction method has been developed for apple juice and unfiltered apple juice in the United States. A portion of the test sample (5 mL) was passed through a macroporous copolymer cartridge and was washed with 1 mL 1% sodium bicarbonate and then with 1 mL 1% acetic acid. Patulin was eluted with 3 mL 2% acetonitrile in anhydrous ethyl ether and was determined by reversed-phase liquid chromatography with UV detection at 276 nm. Recoveries ranged from 93 to 104% in test samples spiked at 20-100 micrograms/L.     

Characterization of alpha- and gamma-glutamyl dipeptides by negative ion collision-induced dissociation

The low-energy CID mass spectra of the [M-H](-) ions of a variety of dipeptides containing glutamic acid have been obtained using cone-voltage collisional activation. Dipeptides with the gamma-linkage, H-Glu(Xxx-OH)-OH, are readily distinguished from those with the alpha-linkage, H-Glu-Xxx-OH, by the much more prominent elimination of H-Xxx-OH from the [M-H](-) ions of the former isomers, resulting in formation of m/z 128, presumably deprotonated pyroglutamic acid. Dipeptides with the reverse linkage, H-Xxx-Glu-OH, show distinctive fragmentation reactions of the [M-H](-) ions including enhanced elimination of CO(2) and formation of deprotonated glutamic acid. Exchange of the labile hydrogens for deuterium has shown that there is considerable interchange of C-bonded hydrogens with labile (N- and O-bonded) hydrogens prior to most fragmentation reactions. All dipeptides show loss of H(2)O from [M-H](-). MS(3) studies show that the [M-H-H(2)O](-) ion derived from H-Glu-Gly-OH has the structure of deprotonated pyroglutamylglycine while the [M-H-H(2)O](-) ions derived from H-Glu(Gly-OH)-OH and H-Gly-Glu-OH show a different fragmentation behaviour indicating distinct structures for the fragment ions.     

Investigation of the electrochemical oxidation products of zotepine and their fragmentation using on-line electrochemistry/electrospray ionization mass spectrometry

When zotepine, an antipsychotic drug, was electrochemically oxidized using electrospray ionization mass spectrometry (ESI-MS) coupled with a microflow electrolytic cell, [M + 16 + H]+ (m/z 348), [M-H]+ (m/z 330) and [M-14 + H]+ (m/z 318) were observed as electrochemical oxidation product ions (M represents the zotepine molecule). Although a major fragment ion that was derived from the dimethyl aminoethyl moiety was observed only at m/z 72 in the collision-induced dissociation (CID) spectrum of zotepine, new fragments such as m/z 315 and 286 ions could be generated in the CID spectrum by combining electrochemical oxidation and CID. Since these fragments were relatively specific with high ion strength, it was thought that they would be useful for developing a sensitive LC-MS/MS assay. The S-oxide and N-demethylated products were detected by electrolysis assuring that a portion of P450 metabolites of zotepine could be mimicked by the electrochemistry/electrospray ionization mass spectrometry (EC/ESI-MS) system.     

Electrospray ionization mass spectrometry monitoring of indigo carmine degradation by advanced oxidative processes

The degradation of the dye indigo carmine in aqueous solution induced by two oxidative processes (H(2)O(2)/iodide and O(3)) was investigated. The reactions were monitored by electrospray ionization mass spectrometry in the negative ion mode, ESI(-)-MS, and the intermediates and oxidation products characterized by ESI(-)-MS/MS. Both oxidative systems showed to be highly efficient in removing the color of the dye aqueous solutions. In the ESI(-)-MS of the indigo carmine solution treated with H(2)O(2) and H(2)O(2)/iodide, the presence of the ions of m/z 210 (indigo carmine in its anionic form, 1), 216, 226, 235, and 244 was noticeable. The anion of m/z 235 was proposed to be the unprecedented hydroperoxide intermediate 2 formed in solution via an electrophilic attack by hydroxyl and hydroperoxyl radicals of the exocyclic C=C bond of 1. This intermediate was suggested to be rapidly converted into the anionic forms of 2,3-dioxo-1H-indole-5-sulfonic acid (3, m/z 226), 2-amino-alpha-oxo-5-sulfo-benzeneacetic acid (4, m/z 244), and 2-amino-5-sulfo-benzoic acid (5, m/z 216). In the ESI(-)-MS of the indigo carmine solution treated with O(3), two main anions were detected: m/z 216 (5) and 244 (4). Both products were proposed to be produced via an unstable ozonide intermediate. Other anions in this ESI(-) mass spectrum were attributed to be [4 - H + Na](-) of m/z 266, [4 - H](2-) of m/z 121.5, and [5 - H](2-) of m/z 107.5. ESI-MS/MS data were consistent with the proposed structures for the anionic products 2-5.     

A new solid-phase extraction and HPLC method for determination of patulin in apple products and hawthorn juice in China

A new solid‐phase extraction (SPE) pretreatment method using a home‐made polyvinylpolypyrrolidone‐florisil (PVPP‐F) column was developed for the analysis of patulin in apple and hawthorn products in China. Fifty samples (25 apple juices, 12 apple jams, and 13 hawthorn juices) were prepared using the new method and then analyzed by high performance liquid chromatography with diode array detection (HPLC‐DAD) on an Agela Venusil MP C₁₈ reversed‐phase column (4.6 mm × 250 mm, 5 μm). The cleanup results for all samples using home‐made PVPP‐F column were compared with those obtained using a MycoSep®228 AflaPat column. The correlation coefficient R (0.9998) fulfilled the requirement of linearity for patulin in the concentration range of 2.5–250 μg/kg. The limits of detection (LODs) and quantification (LOQs) of patulin were 3.99 and 9.64 μg/kg for PVPP‐F column, and 3.56 and 8.07 μg/kg for MycoSep®228 AflaPat column, respectively. Samples were spiked with patulin at levels ranging from 25 to 250 μg/kg, and recoveries using PVPP‐F and MycoSep®228 AflaPat columns were in the range of 81.9–100.9% and 86.4–103.9%, respectively. Naturally occurring patulin was found in 2 of 25 apple juice samples (8.0%) and 1 of 13 hawthorn juice samples (7.7%) at concentrations ranging from 12.26 to 36.81 μg/kg. The positive results were further confirmed by liquid chromatography electrospray ionization mass spectrometry (LC‐ESI‐MS).     

Optimization of microwave-assisted extraction of polyphenols from apple pomace using response surface methodology and HPLC analysis

A simple and efficient microwave-assisted extraction of polyphenols from industrial apple pomace was developed and optimized by the maximization of the yield using response surface methodology. A Box-Behnken design was used to monitor the effect of microwave power, extraction time, ethanol concentration and ratio of solvent to raw material (g/mL) on the polyphenols yield. The results showed that the optimal conditions were as follows: microwave power 650.4?W, extraction time 53.7?s, ethanol concentration 62.1% and ratio of solvent to raw material 22.9:1. Validation tests indicated that the actual yield of polyphenols was 62.68±0.35?mg gallic acid equivalents per 100?g dry apple pomace with RSD=0.86% (n=5) under the optimal conditions, which was in good agreement with the predicted yield and higher than those of reflux and ultrasonic-assisted extraction methods. HPLC analysis indicated that the major polyphenols of apple pomace consisted of chlorogenic acid, caffeic acid, syrigin, procyanidin B2, (-)-epicatechin, cinnamic acid, coumaric acid, phlorizin and quercetin, of which procyanidin B2 had the highest content of 219.4?mg/kg.     

Capillary gas chromatography/mass spectrometry with chemical ionization and negative ion detection for confirmation of identity of patulin in apple juice

Gas chromatography/mass spectrometry (GC/MS) with negative ion chemical ionization permits detection of underivatized patulin in apple juice extracts while minimizing co-extractive responses. The technique has been used with a variety of capillary columns in quadrupole, ion trap, and magnetic sector GC/MS instruments to confirm presumptive findings of patulin in apple juice at concentrations ranging from 68 to 3700 micrograms/L. The demonstrated ability to use any of these 3 mass spectrometers and several capillary columns to confirm the identity of patulin are significant strengths of the technique.     

Liquid chromatography/mass spectrometric determination of patulin in apple juice using atmospheric pressure photoionization

This paper describes a comparison between atmospheric pressure chemical ionization (APCI) and the recently introduced atmospheric pressure photoionization (APPI) technique for the liquid chromatography/mass spectrometric (LC/MS) determination of patulin in clear apple juice. A column switching technique for on-line extraction of clear apple juice was developed. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for both APCI and APPI. The results indicated that APPI provides lower chemical noise and signal suppression in comparison with APCI. The linear range for patulin in apple juice (correlation coefficient >0.999) was 0.2-100 ng mL(-1). Mean recoveries of patulin in three apple juices ranged from 94.5 to 103.2%, and the limit of detection (S/N = 3), repeatability and reproducibility were 1.03-1.50 ng mL(-1), 3.9-5.1% and 7.3-8.2%, respectively. The total analysis time was 10.0 min.     

Determination of sugars and alcohols in apple juice and cider by high performance liquid chromatography

Summary The application of a high-performance liquid chromatographic produre to the separation and determination of major sugars, sorbitol, glycerol and ethanol in apples, apple juice and cider is described. The HPLC system consisted of a cation-exchange resin column in calcium form, a solvent system of an aqueous solution with calcium EDTA and a refractive index detector. The analysis was completed after 16 minutes. A recovery greater than 91% was observed for all compounds with most recoveries nearing 100%. The coefficients of variation ranged from 2% to 6%.