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Reverse phase high-pressure liquid chromatography (RP-HPLC) chromatograms of a separation on a C18 Ascentis Express coloumn of a standard mix of coumarins, psoralens and polymethoxyflavones obtained with two different methods: with acetonitrile (A) and with methanol (B). For peak identification, see Table 1.
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In 2009, a temporary crisis in the world production of acetonitrile caused the abnormal increase of the price of this substance, commonly used as a solvent and in high-pressure liquid chromatography (HPLC) separation as a mobile phase. For this reason, a new HPLC method was developed to analyze the oxygen heterocyclic components of Citrus oils, sub...
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53: 4-8.2001 4 Commercial citrus production in Texas is confined mainly to the Lower Rio Grande Valley (LRGV) where mostly grapefruit (C. paradisi Macf.) and sweet orange [C. sinensis (L) Osbeck] on sour orange (C. aurantium L.) rootstock are grown. Cold tolerant varieties on trifoliate rootstocks are grown in small orchards and backyards in other...
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... [1] The minor fraction of 1 to 10 % EO is non-volatile compounds of fatty acids, waxes and hydrocarbons, and Oxygenated Heterocyclic Compounds (HC) (Figure 1). [2,3] It contains Coumarins (Cs), Furocoumarins (Fcs), and Polymethoxyflavones (PMF), where the biological, organoleptic properties and quantitative and qualitative composition of HCOs determine by citrus species. At the same time, HCOs play a vital role characterizing and authenticating perfumery and aromas. ...
Citrus essential oils (EOs) contain a variety of secondary metabolites, including coumarins (Cs), furocoumarins (FCs) and polymethoxyflavones (PMFs), all with different biological activities. While Regulation (EC) No 1334/2008 strictly regulates the use of coumarins, undesirable side effects of consuming furocoumarins remain without established maximum toxicity limits for human dietary intake. This study is based on an in‐silico approach aimed at determining these toxicity limits for the oxygenated heterocyclic compounds of citrus EOs approved for use in food. In addition, an innovative quantitative structure‐property relationship (QSPR) using the linear retention index system was explored to characterize the oxygenated heterocyclic compounds (COHs) present in these essential oils. Molecular docking of the studied compounds provided crucial insights into the structure‐odor relationships, offering insights into their mechanisms of action. In addition, an analysis of the pharmacokinetic and pharmacodynamic properties through ADME‐Tox deconstruction provides insight into the potential side effects of their consumption. The results are consolidated by dynamic simulations, providing crucial data for the development of regulations on the maximum toxicity limits of citrus EOs, given the health implications of the various compounds such as coumarins, furocoumarins and polymethoxyflavones present in these essential oils.
... paradisi) and bitter oranges (C. aurantium) also present other coumarins (e.g., auraptene, osthol) [17], and all the CPW are source of many glycosylated phenolic flavonoids (e.g., hesperidin, narirutin, naringin) [1]. ...
... kg −1 [19]. Many methods have been proposed to evaluate these bioactive compounds extracted from CPW [20,21] or directly from essential oils [17,22]. The former ones are mainly focused on the chemical profile covering the phenolic flavonoids (usually employing LC-MS), while the latter ones are focused on coumarins, furocoumarins, and PMFs (LC-DAD). ...
... Unfortunately, furocoumarins 6 and 7 could not be solved so far, and no condition seemed to indicate any resolution. This situation has been reported in literature in such a way the quantification considered both peaks [26], but also this critical pair was separated at baseline when 5% THF was added to the mobile phase (17). Nonetheless, the chromatographic system used in this work was incompatible with this solvent even in this concentration according to the manufacturer. ...
The growing interest in ingredients from natural sources has expanded the need for quality assessments of plant extracts. Analytical quality-by-design (AQbD) has been increasingly applied in regulated environments such as pharmaceutical industries and, more recently, for the bioactive compounds found in botanical materials. This work aimed to obtain qualitative (overall resolution and maximum peak capacity) and quantitative performances for target analytes using AQbD principles. The analytical target profile was elaborated; critical method parameters (independent variables) that affect the critical method attributes (dependent variables) were selected from a risk assessment for a reversed-phase liquid chromatography with diode array detection (RPLC-DAD) method. YMC-Triart C18 (3.0 × 100 mm, 1.9 μm) and a gradient elution using 0.2% acetic acid and methanol:acetonitrile 1:3 (v/v) were chosen as the stationary and mobile phases, respectively. The optimal and robust conditions (temperature at 33.3 °C, flow rate of 0.68 mL.min-1, and a gradient slope of 4.18%.min-1) were established by the method operable design region (MODR). The validation was performed by accuracy profiles using 90% expectation tolerance intervals for the selected compounds found in Citrus spp. using C. japonica as blank matrix. The lower limits of quantification for hesperidin, bergapten, herniarin, and citropten were 5.32, 0.40, 0.49, and 0.52 mg.L-1, respectively (acceptance limit was set at ± 20%). Nobiletin did not show an adequate quantitative performance.
... High concentrations of coumarins are usually found in the peels of Citrus species [44]. Coumarin compounds such as transferrin, limettin, auraptene, isomeranzin, umbelliferone, herniarin, psoralen, bergamottin, ecxybergamottin, 5hydroxyfurocoumarin, bergapten, osthol, and 8-geranyloxypsoralen have been reported in different Citrus species [45]. Among various groups of coumarins, auraptene (7geranyloxycoumarin) is the key coumarin that is found plentifully in Citrus species. ...
The genus Citrus contains a vast range of antioxidant metabolites, dietary metabolites, and antioxidant polyphenols that protect plants from unfavorable environmental conditions, enhance their tolerance to abiotic and biotic stresses, and possess multiple health-promoting effects in humans. This review summarizes various antioxidant metabolites such as organic acids, amino acids, alkaloids, fatty acids, carotenoids, ascorbic acid, tocopherols, terpenoids, hydroxycinnamic acids, flavonoids, and anthocyanins that are distributed in different citrus species. Among these antioxidant metabolites, flavonoids are abundantly present in primitive, wild, and cultivated citrus species and possess the highest antioxidant activity. We demonstrate that the primitive and wild citrus species (e.g., Atalantia buxifolia and C. latipes) have a high level of antioxidant metabolites and are tolerant to various abiotic and biotic stresses compared with cultivated citrus species (e.g., C. sinensis and C. reticulata). Additionally, we highlight the potential usage of citrus wastes (rag, seeds, fruit peels, etc.) and the health-promoting properties of citrus metabolites. Furthermore, we summarize the genes that are involved in the biosynthesis of antioxidant metabolites in different citrus species. We speculate that the genome-engineering technologies should be used to confirm the functions of candidate genes that are responsible for the accumulation of antioxidant metabolites, which will serve as an alternative tool to breed citrus cultivars with increased antioxidant metabolites.
... Based on this fragmentation pattern, compound 9 that displayed protonated molecules ([M + H] + ) at m/z 369 and product ions at m/z 233 ([M + H -136] + ]) and 218 ([M + H -136 -15] + ) was assigned to a psoralen carrying a geranyloxy and a methoxy group. Compound 9 may correspond to 5-geranyloxy-8methoxypsoralen, as has been tentatively identified earlier in lime juice and lime essential oil (Dugo et al., 2009;Costa et al., 2014;Russo et al.,2012). ...
Mutual adulterations of lemon and lime juices may be detected using coumarins and psoralens as markers. Poor manufacturing practices or legal but mechanically intense processing of lemons were recently suspected to lead to false accusations of deliberate adulterations with lime juices due to potentially unspecific markers. Therefore, we studied coumarin and psoralen profiles in carefully dissected flavedo, albedo, and endocarp of lime and lemon as well as in juices produced under variable mechanical stresses at laboratory and pilot plant scale. Although the marker herniarin was detectable in juices from lime and harshly extracted lemons at low levels, isopimpinellin, bergapten and the herein proposed, tentatively assigned 5–geranyloxy-8-methoxypsoralen represented unambiguously lime-specific markers. Coumarin and psoralen data also allowed differentiating juices produced at differing degrees of mechanical stress. The latter was also possible using quantitative ¹H-NMR spectroscopy, which yielded best results when combined with HPLC data on coumarins and psoralens. In the future, the reported approach may be used for establishing a robust database prior to being used in industrial practice.
... Analytical method kept unchanged both column and the gradient program. Thanks to the method validated by Russo et al. (33) was easy to identify and quantify 35 oxygen heterocyclic compounds (8 polypmethoxyflavones, 10 coumarins, and 17 furocoumarins) in seven cold-pressed Citrus essential oils (mandarin, lemon, lime, bergamot, grapefruit, sweet and bitter orange) under the same experimental conditions. Figure 2(a) shows a RP-HPLC chromatogram of oxygen heterocyclic compounds standard mixture obtained by Russo et al. (33). ...
... Thanks to the method validated by Russo et al. (33) was easy to identify and quantify 35 oxygen heterocyclic compounds (8 polypmethoxyflavones, 10 coumarins, and 17 furocoumarins) in seven cold-pressed Citrus essential oils (mandarin, lemon, lime, bergamot, grapefruit, sweet and bitter orange) under the same experimental conditions. Figure 2(a) shows a RP-HPLC chromatogram of oxygen heterocyclic compounds standard mixture obtained by Russo et al. (33). From an analytical point of view, the new HPLC/PDA method increased the baseline separation of oxygen heterocyclic compounds because peak resolution of bergaptenisopimpinellin and byakangelicol-oxypeucedanin critical pairs was improved (3.08-4.84 and 1.99-5.39, ...
... This type of hybrid instrumentation, combining the high mass accuracy and the possibility to perform tandem MS experiments, greatly enhances the confidence in elucidation of unknown structures. 127 cold-pressed mandarin essential oils and fruits of three different bergamot cultivars (Femminello, Castagnaro and Fantastico) were used to manually recover cold-pressed essential oil that was analyzed at the same experimental condition validated by Russo et al. (33). Authors reported for the first time the presence of four polymethoxyflavones (sinensetin, nobiletin, tetra-O-methyl-scutellarein, and tangeretin) in addition to the well-known coumarins and psoralens in bergamot essential oils and of three additional polymethoxyflavones (isosinensetin, 5-demethylnobiletin, and 5-demethlytangeretin) in mandarin essential oils. ...
Cold-pressed Citrus essential oils namely lemon, bergamot, lime, grapefruit, mandarin, sweet and bitter orange are obtained from the peel of Citrus fruits. They were normally used in food, cosmetic, and pharmaceutical industries as ingredients. Essential oils extraction processes produce oils with different sensory qualities, and often these processes are expensive and have a low yield. For such a reason adulteration process are frequent, so careful attention must be paid to ensure the oils’ genuineness and authenticity. Moreover, psoralen levels are continuously monitored by opinions and regulations released from International Fragrance Association and the European Parliament due to their photoactivity.
This review aims to browse the scientific literature of the past 30 years on the characterization of the non-volatile fraction of cold-pressed Citrus essential oils to discuss results achieved so far and highlight future prospect.
... Citrus fruits are rich sources of bioactive compounds and dietary fibre (Dugo et al. 2009;Russo et al. 2012;Cicero et al. 2015;Salvo et al. 2016); however, information on their distribution in the different by-products is still incomplete, limiting the development of their applications for further uses. The re-use of the food industry wastes has been widely demonstrated that it can lead to reducing the costs and the waste amounts (Ravindran and Jaiswal 2016). ...
This study is part of a wider investigation aimed to sustain the economical value of the by-products generated by the Citrus industry. In particular, the protected geographic indication (PGI) red orange of sicily (known as blood orange) has been analysed by HPLC and by the enzymatic AOAC method. All the by-products contain significant amounts of biologically active compounds (limonoids and flavonoids). The decanted pulps were the most abundant, with the highest amount of flavonoids (130 g/kg) and high amount of limonoids (5.5 g/kg). Seeds were the best source of limonoids with about 10 g/kg. Low amount of anthocyanins were found only in coarse pulps and waste water. The total, the insoluble and the soluble dietary fibre (TDF, IDF and SDF respectively) were also determined. The pulps resulted to be the best source of dietary fibre, based on the amount and on the best insoluble/soluble ratio.
... The first step was the development of a high performance nanoLC method for a satisfactory separation of all the analytes, by using the UV detection system. In particular, differently from a previous work dealing only with polymethoxyflavones in which a common gradient of acetonitrile in water and a sub-optimal flow rate were applied [19] , the trapping configuration and the features of the ultra-high performance LC setup allowed to set the optimal flow rate (300 nL min −1 ) of the employed column (75 μm ID) and a solvent gradient quite similar to a previously developed conventional HPLC method [47] . A ternary solvent gradient was used, consisting of a mixture of water, methanol and tetrahydrofuran without any backpressure or pump instability issues. ...
... Chromatographic peaks were preliminarly identified according to literature data [47] and then confirmed by standard injection, depending on the availability of the standard material in the analytical laboratory (see Section 2.1 ). ...
The present research is focused on the object to improve identification capability in liquid chromatography (LC), by creating a system as similar as possible to gas chromatography (GC), where the combination/complementarity of Linear Retention Index (LRI) and Electron Ionization Mass Spectrometry (EI-MS) data makes the identification process easy, automatic and reliable. Conversely, in LC the untargeted characterization of real-world samples is still a challenge, due to the not repeatable and poorly informative nature of typical atmospheric pressure ionization mass spectrometry, normally hyphenated to LC. In the last decades the miniaturization of LC instrumentation together with the considerable progresses in MS vacuum pump capability has made the LC-EI-MS hyphenation more feasible. In the present work, a prototypal nanoLC-EI-MS system was used for the determination of typically LC-amenable compunds, such as coumarins, furocoumarins and polymethoxyflavones in citrus essential oils. All the compounds provided high quality EI-MS spectra, evaluated by the comparison with thousands of spectra present in commercial EI-MS libraries. Spectral similarities major than 80% were achieved. Furthermore, an LRI system, based on the use of an alkyl aryl ketone homologue reference series, was proposed as additional filter to achieve a univocal identification. Then, a novel dual-filter LRI/EI-MS library was built and resulted very helpful in the case of isomeric compounds characterized by identical EI-MS spectrum, but different retention behaviour. The very low inter-day variability attained for each LRI value, together with the satisfactory chromatographic resolution of the developed method, led to a 100% reliability of the identification process based on LRI.
... There are many research articles regarding the composition of the non-volatile fraction in different cold-pressed Citrus essential oils (Frérot and Decorzant 2004;Russo et al. 2012Russo et al. , 2015aDugrand et al. 2013), but there is a lack of investigation on the content of limonoids. These molecules are present in the peel of Citrus fruits both in glucosidic and aglyconic form (Manners and Breska III 2004). ...
The dual purpose of this research was to investigate the presence of limonoid aglycones in Citrus essential oils and to develop a rapid and reliable SFC-APCI-QqQ MS method for their characterization. In this study, 12 limonoid aglycones were tentatively identified in 11 different citrus essential oils. The developed method allowed a very fast separation in less then 7 min with a very low amount of organic solvent. Calibration curve of limonin was constructed by using a triple quad MS detector in order to quantify the limonin content in the analyzed samples. The content of limonin ranged from 0.5 (clementine essential oil) to 21.2 mg L⁻¹ (bergamot essential oil) in the cold-pressed Citrus essential oils analyzed. Moreover, product ion scan (PIS) acquisition mode was also used for the structure elucidation of isobaric compounds. To the best of our knowledge, this is the first investigation on limonoid aglycones in Citrus essential oils.
... Other tentative phytochemicals were identified by reference to former studies of the chemical constituents isolated from HECA. The precursor ions and fragment patterns of identified and tentative phytochemicals are consistent with previous reports [9,[18][19][20][21][22][23]. In both MS and MS/MS experiments, high resolution and accurate mass measurements using a quadrupole-Orbitrap mass spectrometer offer significant advantages for rapid screening of target analytes in a complex unknown matrix. ...
Although traditionally used to improve indigestion, diarrhea, dysentery, and constipation, the therapeutic effects of Citrus aurantium on intestinal inflammation remain unclear. The aim of this study was to evaluate the beneficial effects and to identify the active components of a hydroalcoholic extract of C. aurantium (HECA) on ulcerative colitis. HECA was prepared with 70% ethanol solution in water and extracted at 37 °C for 12 h in triplicate, filtered through a sieve, and lyophilized. Phytochemical identification of HECA was performed by ultra-performance liquid chromatography-diode array detector-tandem mass spectrometry (UPLC-DAD-MS/MS). Animals were randomly assigned to one of four groups based on the treatment conditions. Ulcerative colitis was induced by administration of 2.5% dextran sodium sulfate (DSS) in drinking water for 5 d. Body weight, clinical signs, colon length, pro-inflammatory cytokine expression levels, and histopathological findings were evaluated. In UPLC-DAD-MS/MS analysis, the identified phytochemical components of HECA included four alkaloids, seven coumarins, 18 flavonoids, two lignans, two phenolics, and 10 terpenoids. HECA markedly protected against body weight loss and colon shortening. In pathological examination, HECA alleviated DSS-related mucosal inflammatory lesions in the colon. Moreover, HECA markedly reduced the expression levels of interleukin-6, interferon-γ, tumor necrosis factor-α, and monocyte chemotactic protein-1 in colonic inflammation. Taken together, HECA has potential to relieve mucosal inflammation in the colon, suggesting that the putative active ingredients are responsible for the anti-ulcerative effects.
... Highperformance liquid chromatography coupled with a diode array detector (HPLC-DAD) is the method of choice generally reported for their analysis. [15][16][17][18][19][20] In 2004, Frérot et al. proposed a reversed phase HPLC method with UV detection at 310 nm to separate and quantify the 15 most common furocoumarins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) in essential oils. 18 This method was validated in a collaborative ring test by the IFRA Analytical Working Group 8 years later. ...
... 27 Kushboo et al. were able to quantify psoralen, in the seeds of Psoralea corylifolia, with a linearity range of 10 to 100 ng spot −1 with a LOD of 8.65 ng and an LOQ 26.2 ng. 38Thus, the aim of this study was (i) to propose a new HPTLC method to analyse the 20 most common furocoumarins(1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) found in perfumery raw materials for screening purposes (Figure 1). and (ii) to propose a validated HPTLC method allowing the quantification of bergapten in essential oils in accordance with regulated limits. ...
... Occurrence of coumarins and furocoumarins in some furocoumarin-containing plants[5][6][7][8][9] ...
The concentration of furocoumarins, known to induce mutagenesis, carcinogenesis and photodermatitis under UV radiation, is limited by the EU cosmetic regulation in consumer products such as cosmetics, sun protection and bronzing formulations. The bergapten concentration is also limited by the International Fragrance Association. Analytical methods have been proposed for their quantification using high-performance liquid chromatography (HPLC) coupled with UV detection or mass spectrometry with limits of detection (LOD) and quantification (LOQ) in accordance with the regulation. These methods are time and solvent consuming for routine quality control analyses. In this context, simple and fast HPLC methods in one- (1-D) and two- (2-D) dimensions were developed for the simultaneous analysis of the 15 most common furocoumarins reported in Citrus essential oils: psoralen, bergapten, xanthotoxin, isopimpinellin, oxypeucedanin, oxypeucedanin hydrate, byakangelicol, byakangelicin, heraclenin, 8-geranyloxypsoralen, bergamottin, imperatorin, isoimperatorin, phellopterin, epoxybergamottin along with five other furocoumarins known to be present in Angelica, bergamot or Heracleum species: angelicin, bergaptol, isobergapten, psora-4 and 5-(4-phenoxybutoxy)psoralen. The 1-D method was validated for the quantification of bergapten using a Camag TLC Scanner as a detector. The LOD was determined at 2 mg/kg. The method was linear from 2 to 100 mg/kg and the method range was defined using accuracy profiles at with an 80% tolerance interval, with a 30% error, to be 5–100 mg/kg in acetonitrile and 13–100 mg/kg in matrix.
