Quantification of main phenolic compounds in sweet and bitter orange peel using CE–MS/MS

The food and agricultural products processing industries generate substantial quantities of phenolics-rich subproducts, which could be valuable natural sources of polyphenols. In oranges, the peel represents roughly 30% of the fruit mass and the highest concentrations of flavonoids in citrus fruit occur in peel. In this work we have carried out the characterisation and quantification of citrus flavonoids in methanolic extracts of bitter and sweet orange peels using CE–ESI–IT–MS. Naringin (m/z 579.2) and neohesperidin (m/z 609.2) are the major polyphenols in bitter orange peels and narirutin (m/z 579.2) and hesperidin (m/z 609.2) in sweet orange peels. The proposed method allowed the unmistakable identification, using MS/MS experiments, and also the quantification of naringin (5.1 ± 0.4 mg/g), neohesperidin (7.9 ± 0.8 mg/g), narirutin (26.9 ± 2.1 mg/g) and hesperidin (35.2 ± 3.6 mg/g) in bitter and sweet orange peels. CE coupled to MS detection can provides structure-selective information about the analytes. In this work we have developed a CE–ESI–IT–MS method for the analysis and quantification of main phenolic compounds in orange peels.     

Rapid quantitation of curcumin in turmeric via NMR and LC–tandem mass spectrometry

A rapid, sensitive and accurate ¹H NMR method has been developed for the quantitation of curcumin isolated from Curcuma longa rhizome (turmeric) extract, the results of which were compared with a validated LC–MS/MS method. The relative standard deviations of the methods were found to be 2.49% and 3.48% for the ¹H NMR and LC–MS/MS methods, respectively. The correlation coefficients were 0.998 for ¹H NMR and 0.995 for LC–MS/MS assay in the calibration range. The recoveries at 5 mg mL⁻¹ and 50 μg mL⁻¹ concentrations averaged to 99–101% for both techniques, respectively. The uncertainty of the measurement of curcumin via ¹H NMR spectroscopy was determined to be 5.80% while in LC–ESI-MS/MS method was 7.38%.     

Screening assay of angiotensin-converting enzyme inhibitory activity from complex natural colourants and foods using high-throughput LC-MS/MS

Inhibition of angiotensin-converting enzyme (ACE) by various foods decreases the blood pressure. ACE inhibitors derived from natural components may be of therapeutic value in preventive medicine. In this study, we report a novel screening assay of ACE inhibitors from complex natural colourants and foods that employ solid phase extraction (SPE), high-throughput liquid chromatography (LC) separation, and stable isotope dilution electrospray tandem mass spectrometry (SID-ESI-MS/MS). When a target sample was subjected to N-Hippuryl-His-Leu (HHL) and ACE in phosphate buffer (pH 7.4), generated hippuric acid (HA) was extracted by SPE. LC/SID-ESI-MS/MS detection of HA allowed us to accurately identify the effects of complex substances such natural colourants and foods that inhibit the ACE of HHL. The major HA and HA-d₅ fragment ions at m/z 180 → 105 and 185 → 110 in the multiple reaction monitoring (MRM) mode can quantify levels that are lower than other methods. The LC/SID-ESI-MS/MS method described here is a rapid, selective, sensitive, and highly reproducible method for the determination of HA in various samples. Based on the assay developed, all samples such as natural colourants, infant formula, soy paste, ketchup, mayonnaise, wheat flour, orange juice, supplement drink, tea, and coffee could be accurately measured for ACE inhibition in various matrices. High-throughput LC/SID-ESI-MS/MS assay has no limitations in the evaluation of inhibition activity in various natural samples such as colour, high-matrix, and processed foods.     

Stereochemical and pharmacological differences between naturally occurring p-synephrine and synthetic p-synephrine

p-Synephrine, the primary protoalkaloid in Citrus aurantium (bitter orange) and some other Citrus species, exists in nature in the l- or [R-(?)]-enantiomeric form, whereas synthetic p-synephrine is a racemic mixture of the l- and d-enantiomeric forms. Based on receptor binding, the synthetic form is believed to exert approximately half the pharmacological activity of the naturally occurring protoalkaloid. This difference occurs because the d- or [S-(+)]-form provides little or no binding to adrenergic receptors in contrast to the l-form. Receptor binding studies also provide an explanation for the differences in pharmacological effects between the isomers p-synephrine and m-synephrine.     

Determination of flavonoids in a Citrus fruit extract by LC–DAD and LC–MS

Flavonoids are a group of polyphenolic compounds with health-related properties. Citrus fruits are rich in flavonoids and their extracts are being used as functional ingredients for several industrial products. A new high performance liquid chromatography technique with an UV photodiode-array detector was used to analyze flavonoids of an extract of Citrus species. To our knowledge this is the first study that reports isoquercitrin presence at a level of 77.3 mg/100 g in a sample made of Citrus fruits; four other flavonoids were quantified as rutin (326.59 mg/100 g), naringin (338.36 mg/100 g), quercetin (96.35 mg/100 g) and naringenin (2.35 mg/100 g). Identification was confirmed by a liquid chromatography mass spectrometer system. Method validation was achieved, providing an analytical technique that can be used to detect trace amounts of these compounds in Citrus extracts with an extremely rapid sample preparation.     

GC–MS quantification and sensory thresholds of guaiacol in orange juice and its correlation with Alicyclobacillus spp.

Guaiacol is a profoundly negative taint/off-flavour produced by the increasingly common microbial contamination of fruit juices with Alicyclobacillus spp. The objectives of this study included: determining sensory thresholds for guaiacol in orange juice (OJ), developing an analytical method whose detection limits were equivalent to sensory thresholds and determining levels of Alicyclobacillus spp. that would produce detectable levels of guaiacol. A 12 member trained panel was used to establish guaiacol detection and recognition thresholds. Guaiacol ortho and retro nasal detection thresholds in OJ were 0.70 and 0.53 μg/l respectively. Odour recognition threshold was 2.0 μg/l. A SPME GC–MS Selected Ion Monitoring (SIM) procedure was optimised to achieve analytical detection limits of 0.5 μg/l. Optimum guaiacol detection limit was achieved using only responses from m/z 109 and 124. Ion ratios (m/z 109/124) and linear retention index value matching were used to confirm the identification of guaiacol. Quantification was achieved using peak areas from standard guaiacol additions in orange juice between 0.5 and 100 μg/l. Alicyclobacillus growth of 2.2 log CFU/ml in OJ produced just detectable levels (0.7 μg/l) of guaiacol.     

Accumulation and antioxidant activity of secondary carotenoids in the aerial microalga Coelastrella striolata var. multistriata

The growth and nitrate uptake of the aerial microalga Coelastrella striolata var. multistriata, which was isolated from the surface of rocks, were characterized under a variety of conditions in this study. The maximum specific growth rate of the alga, having prominent inorganic nitrogen uptake in the fresh medium, was 0.30 d⁻¹, as calculated in the growth logarithmic phase. It was also shown that the alga had abilities to be reddish orange to green colour (depending on the nitrogen source concentration in the medium) and to synthesize very high amounts of a complex mixture of carotenoids, such as canthaxanthin, astaxanthin and β-carotene. The reddish orange cells of the alga could accumulate 56.0 mg of major secondary carotenoids per g biomass. In the content of carotenoids, canthaxanthin, astaxanthin and β-carotene in the cells were 47.5, 1.5, and 7.0 mg/g dwc, respectively. Additionally, it was shown that the algal extract containing those carotenoids had an antioxidant potential in lipid foods. In the near future, the aerial microalga C. striolata var. multistriata will be used as a functional material in a variety of commercial applications, such as feed supplements, natural antioxidants and food dyes.     

Triglycerides constituted of short and medium chain fatty acids and dicarboxylic acids in Momordica charantia, as well as capric acid,inhibit PGE2 production in RAW264.7 macrophages

This study was aimed to identify compounds in bitter gourd (Momordica charantia) ethyl acetate extract (EAE), which inhibit lipopolysaccharide-induced prostaglandin E₂ (PGE₂) production in RAW264.7 cells. Bitter gourd EAE was partitioned between n-hexane and methanol/H₂O (90/10). The hexane fraction was further separated by repeated silica gel chromatographies, and a reverse phase (RP) C18 chromatography. Fraction RP-10 showed the highest inhibition effect on PGE₂ production (Max inhibition = 96%, IC₅₀ = 2.3 μg/ml) and was identified to be triglycerides constituted of short and medium chain fatty acids by ¹H NMR, IR and H–HCOSY, and dicarboxylic acids by GC/MS. Fatty acids with 3–20 carbons were tested for the inhibitory activity, and capric acid exhibited the highest effect (Max inhibition = 99%, IC₅₀ = 6.5 μM). In conclusion, triglycerides composed of short and medium chain fatty acids and dicarboxylic acids in bitter gourd inhibit PGE₂ production, and capric acid is the most potent inhibitor among the fatty acids.     

The use of photochemiluminescence for the measurement of the integral antioxidant capacity of baobab products

The number of methods to measure antioxidants in botanicals, foods, nutraceuticals and other dietary supplements are increased considerably in the last 10 years. However most techniques require long experimental times and high costs to determine antioxidant capacity of hydrophilic or lipophilic compound in a food mixture. By means of a photochemiluminescence method, we assessed the Integral Antioxidant Capacity (IAC) which represents the sum of the antioxidant capacity of hydrophilic and lipophilic antioxidants. In this study the IAC of extracts from Adansonia digitatata (i.e. red fiber, fruit pulp and leaves), was assessed in comparison to those deriving from other natural sources of antioxidants (i.e. orange, kiwi, apple and strawberry). When compared, IAC values for the examined product resulted as follows: Adansonia digitata red fibre ? Adansonia digitata fruit pulp ? Adansonia digitata fresh leaves ? Adansonia digitata seeds ? Adansonia digitata radix cuticle ? orange fresh pulp ? strawberry fresh fruit pulp > Adansonia digitata radix > bilberry fresh pulp ? kiwi fruit pulp. Results clearly indicate the interesting antioxidants properties of Adansonia digitata red fibre, in particular the IAC value of baobab red fibre was 66 time higher than that of orange pulp, with value of 1617.3 μmol/g and 24.3 μmol/g, respectively.     

Production of R-(+)-α-terpineol by the biotransformation of limonene from orange essential oil,using cassava waste water as medium

The use of two agro-residues (liquid cassava waste and orange essential oil) in the biotransformation of R-(+)-limonene was investigated. The main components of orange essential oil were determined by GC–MS and R-(+)-limonene was shown to be the predominant constituent, accounting for more than 94% of the total content. Cassava wastewater proved to be a suitable substrate for mycelial growth, leading to good, rapid growth with all the fungal strains tested, reaching 29.4 g/l (dry weight) after 3 days of growth (Penicillium sp. 2025). The best R-(+)-α-terpineol yields were achieved when the strains were grown in cassava media and the mycelia then transferred to a new flask containing mineral medium and orange essential oil as the sole C- and energy source. One of the strains tested, Fusarium oxysporum 152B, converted R-(+)-limonene to R-(+)-α-terpineol, yielding nearly 450 mg/l after 3 days of transformation. Growth in the presence of a solution of 1% orange essential oil in decane did not increase the transformation yields.     

LC–MS analysis of the cis isomers of chlorogenic acids

The behaviour of cis isomers of selected mono- and di-acyl chlorogenic acids produced by UV-irradiation has been investigated by LC–MSⁿ. cis Isomers fragment identically to the more common trans isomers. cis-5-Acyl chlorogenic acids are more hydrophobic and elute later than their mono- or di-trans counterparts whereas the reverse is true for cis-3-acyl and cis-4-acyl chlorogenic acids. The cis isomers of 1,3-dicaffeoylquinic acid, the only 1-acyl chlorogenic acid investigated, are also more hydrophobic than the di-trans isomer. Coffee leaves had a proportionately greater content of cis isomers relative to trans isomers compared with coffee beans suggesting that UV-irradiation in vivo may also cause geometric isomerisation.     

Further data on the levels of emerging Fusarium mycotoxins enniatins (A,A1, B,B1), beauvericin and fusaproliferin in breakfast and infant cereals from Morocco

Sixty-eight samples of cereals products, including breakfast cereals (n = 48) and infant cereals (n = 20), purchased from supermarkets and pharmacies in Rabat-Salé area from Morocco were analysed for the determination of six emerging mycotoxins: four enniatins ENs (ENA, ENA1, ENB and ENB1), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of acetonitrile:water (85:15, v/v), using an Ultra-Turrax® homogeniser. Mycotoxins were then identified and quantified by liquid chromatography (LC) with diode array detection (DAD). Positive samples were confirmed by LC–MS/MS.     

Removal of Escherichia coli, Enterococcus fecalis, coliphage MS2, poliovirus, and hepatitis A virus from oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) by depuration

Filter-feeding bivalve mollusks (shellfish) can bioaccumulate pathogenic microorganisms in up to 1000-fold higher levels than overlying waters, and therefore disease risks are associated with consuming raw or partially cooked shellfish. Many of these shellfish-borne diseases are due to enteric bacteria and viruses associated with fecal contamination. To control shellfish-borne diseases, guidelines for shellfish harvest waters and shellfish meat have been devised, which include cleansing of contaminated shellfish by depuration in controlled systems, heat pasteurization, or relay to clean waters. This study examines the depuration of oysters (Crassostrea virginica) and hard shell clams (Mercinaria mercinaria) in a flow-through depuration system under variable temperature (12 °C, 18 °C, and 25 °C), salinity (8 ppt, 18 ppt, and 28 ppt), turbidity (< 1 NTU, 10 NTU, and 20 NTU), pH (pH 7 and pH 8), and algae conditions (0 cells/mL and 50,000 cells/mL), with constant dissolved oxygen (5-7 mg/L). Oysters and hard shell clams were artificially contaminated with enteric microorganisms: Escherichia coli, Enterococcus faecalis, coliphage MS2, Poliovirus type-1 and Hepatitis A virus HM-175 (HAV), then depurated in 5-day trials with daily sampling. In oysters, optimizing environmental parameters of water temperature improved E. coli, MS2, poliovirus and HAV depuration, and optimized salinity improved E. coli, E. faecalis, and MS2 depuration rates. In hard shell clams, salinity improved E. coli and E. faecalis depuration rates. Adjusting turbidity, pH or algae did not improve microorganism depuration in either oysters or hard shell clams, with the exception of turbidity on E. faecalis in hard shell clams. Microorganism depuration rates in oysters from greatest to least were: MS2 > E. coli > E. faecalis > poliovirus > HAV, and in clams depuration rates from greatest to least were: E. coli > E. faecalis > HAV > MS2 > poliovirus. Because E. coli and E. faecalis were removed at faster rates than HAV and poliovirus, these fecal bacteria appear to be poor process indicators of the virological quality of depurated oysters and hard shell clams.     

Quantification of (+)-catechin and (−)-epicatechin in coconut water by LC–MS

An analytical technique has been developed to detect trace amounts of both (+)-catechin and (−)-epicatechin in the coconut water extract. Both (+)-catechin and (−)-epicatechin in the coconut water were found for the first time by the solid-phase extraction, and they were further analysed using liquid chromatography (LC)–ion trap mass spectrometry (MS) equipped with positive atmospheric pressure chemical ionisation interface on multiple reaction monitoring mode. The limit of detection and quantification for (+)-catechin were 7.8 and 15.6 μg/ml, respectively, while those for (−)-epicatechin were 3.9 and 7.8 μg/ml, respectively. The average concentration of (+)-catechin and (−)-epicatechin in the coconut water was 0.344 and 0.242 μg/ml, respectively. The LC–MS/MS analysis accelerated the quantitative analysis of (+)-catechin and (−)-epicatechin in the coconut water extract with high accuracy, precision and recovery. Results obtained in this study will serve as quality control and useful reference for drug development.     

Effect of weaning status on lipids of Galician Blond veal: Total fatty acids and 18:1 cis and trans isomers

The effect of weaning at different ages (NW = not weaned, W5 = 5.5 months and W2 = 2 months) on fatty acids (FA) of the Longissimus thoracis (LT) muscle was studied in 36 Galician Blond (GB) calves. Total FA (TFA) were determined by gas chromatography (GC) and 18:1 isomers by a combination of reversed-phase high performance liquid chromatography (HPLC) and GC. NW group showed higher (P < 0.001) values of n-3 polyunsaturated FA (PUFA), conjugated linoleic acid (CLA) and 18:1trans-11 compared to LT from W5 and W2 calves. W2 calves showed the highest levels of n-6 PUFA (P < 0.01), 18:1trans-10 and 18:1trans-6/7/8 (P < 0.001). Generally, W5 calves had intermediate values for TFA and 18:1 isomers. As the suckling period was longer, GB milk and veal FA profiles became more similar, it seems that muscle FA were partially transmitted by the milk FA intake due to the persistence of the reticular grove closing reflex.     

Simultaneous characterisation and quantitation of flavonol glycosides and aglycones in noni leaves using a validated HPLC-UV/MS method

The leaves of Morinda citrifolia L. (noni) have been utilized in a variety of commercial products marketed for their health benefits. This paper reports on a rapid and selective HPLC method for simultaneous characterization and quantitation of four flavonols in an ethanolic extract of noni leaves by using dual detectors of UV (365 nm) and ESI-MS (negative mode). The limits of detection and quantitation were between 0.012 and 0.165 μg/mL. The intra- and inter-assay precisions, in terms of percent relative standard deviation, are less than 4.38% and 3.50%, respectively. The accuracy, in terms of recovery percentage, ranged from 96.66% to 100.03%. Good linearity (correlation coefficient >0.999) for each calibration curve of standards was achieved in the range investigated. The contents of four flavonoids in the noni leaves varied from 1.16 to 371.6 mg/100 g dry weight.     

Determination of PDE-5 inhibitors and appetite suppressants in adulterated dietary supplements using LC/PDA and LC/MS

There have been a number of reports of dietary supplements contaminated with illegal adulterants that threaten consumers’ health because of their adverse pharmacological effects. In the present study, a convenient and economic method was developed to detect illegal pharmaceutics, such as PDE-5 inhibitor and appetite suppressants, using liquid chromatography (LC)/photodiode array (PDA) for screening and LC/mass spectrometry (MS) for successive confirmation. Target peaks were identified by comparison of their chromatographic retention times and PDA spectra with those of synthetic standards and finally confirmed by LC/MS. As a result, tadalafil, a PDE-5 inhibitor, and N-desmethylsibutramine, a derivative of sibutramine, were detected in various dietary supplements at concentrations of 13.5–21.9?mg and 3.0?mg per single dose, respectively. The present study will contribute to the development of an analytical method enabling rapid screening of a variety of health foods, and the result suggests that consumers should be aware of serious health risks related to these illegal compounds.     

LC/ESI-MS/MS characterisation of procyanidins and propelargonidins responsible for the strong antioxidant activity of the edible halophyte Mesembryanthemum edule L.

Mesembryanthemum edule is used as a food ingredient and in traditional medicine. In this study, we investigated antioxidant activities of several extracts (methanol/acidified water, v/v: 20/80; 40/60 and 60/40) obtained from M. edule leaf, stem and root. Then, individual phenolics were characterised by reverse-phase HPLC coupled to electrospray ionisation mass spectrometry and multi-stage MS fragment analysis. Results showed that 40% methanol leaf extract, 40% methanol root extract and 20% methanol stem extract displayed the highest scavenging activity against DPPH and ABTS radicals. Regarding LC/ESI-MS/MS identification of active phenols, there were significant differences among the fractions of interest. In fact, 40% leaf extract mainly contained procyanidins, whereas propelargonidins were the major phenolics in 20% methanol stem extract, while, in 40% root extract, the active compounds remained unidentified. These results indicate that edible M. edule can be used as a nutraceutical in the pharmaceutical industry.     

GC/MS analysis and in vitro antioxidant activity of essential oil and methanol extracts of Thymus caramanicus Jalas and its main constituent carvacrol

Chemical composition of the essential oil, antioxidant activity (DPPH and β-carotene/linoleic acid assays), and total phenolic content (Folin–Ciocalteu assay) of aerial parts of Thymus caramanicus were determined. The highest radical-scavenging activity (DPPH test) was shown by the polar subfraction of the methanol extract (IC₅₀ = 43.0 μg/ml) which was also higher than that of butylated hydroxytoluene (BHT, IC₅₀ = 19.7 μg/ml). However, it was the nonpolar subfraction of the methanol extract that showed the highest inhibition (84.4%), as assessed by the β-carotene/linoleic acid assay, which was only slightly lower than that shown by BHT (93.3%). The antioxidant activities of the essential oil main component (carvacrol) were also evaluated for comparison. Total phenolic content of the polar subfraction, as gallic acid equivalents, was 124.3 μg/mg. Essential oil extracted from the aerial parts by hydrodistillation was analysed by GC and GC/MS. Fifteen constituents, representing 99.3% of the oil, were identified, of which the major ones, carvacrol (85.9%), thymol (3.3%), p-cymene (3.2%), γ-terpinene (1.8%) and borneol (1.3%), accounted for 95.6% of the oil.     

Phenolic acid analysis and antioxidant activity assessment of oil palm (E. guineensis) fruit extracts

Phenolic compounds in oil palm fruit (E. guineensis) were extracted in soluble free (SFP), insoluble-bound (ISBP) and esterified (EFP) forms. The total phenolic content (TPC) of the oil palm fruit extracts was determined using the Folin–Ciocalteu method and found to range from 5.03 to 9.04 g/L per g of dried weight (DW). The antioxidant activities of oil palm phenolic extracts were analysed using free radical scavenging assays and results showed that oil palm phenolic extracts contained antioxidant activities in the order of ISBP > EFP > SFP. Eight different phenolic acids were identified and quantified using a simple reversed-phase high performance liquid chromatography (HPLC) with a diode array detector (DAD) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Ferulic, p-hydroxybenzoic and p-coumaric acid were the dominant phenolic acids found in oil palm fruit extracts and ranged from 55 to 376 μg/g of DW.