You are currently viewing a new version of our website. To view the old version click .
MDPIMDPI
Search all articles
Molecules
Download PDF
  • Article
  • Open Access

23 September 2022

Authentication of Citrus spp. Cold-Pressed Essential Oils by Their Oxygenated Heterocyclic Components

,
and
1
Aromatic Plant Research Center, Lehi, UT 84043, USA
2
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA
*
Author to whom correspondence should be addressed.
Molecules2022, 27(19), 6277;https://doi.org/10.3390/molecules27196277
This article belongs to the Special Issue Application of Spectroscopy and Chemometrics for Authentication of Foods and Drugs
Version Notes
Order Reprints

Abstract

Citrus essential oils are routinely adulterated because of the lack of regulations or reliable authentication methods. Unfortunately, the relatively simple chemical makeup and the tremendous price variations among Citrus varieties encouraged the interspecies adulteration of citrus oils. In this study, a sensitive UPLC-MS/MS method for the quantitation of 14 coumarins and furanocoumarins is developed and validated. This method was applied to screen the essential oils of 12 different Citrus species. This study, to our knowledge, represents the most comprehensive investigation of coumarin and furanocoumarin profiles across commercial-scale Citrus oils to date. Results show that the lowest amount was detected in calamansi oil. Expressed oil of Italian bergamot showed the highest furanocoumarin content and the highest level of any individual furanocoumarin (bergamottin). Notable differences were observed in the coumarin and furanocoumarin levels among oils of different crop varieties and origins within the same species. Potential correlations were observed between bergapten and xanthotoxin which matches with known biosynthetic pathways. We found patterns in furanocoumarin profiles that line up with known variations among the Citrus ancestral taxa. However, contrary to the literature, we also detected xanthotoxin in sweet orange and members of the mandarin taxon. Using multivariate analysis, we were able to divide the Citrus oils into 5 main groups and correlate them to the coumarin compositions.

1. Introduction

Citrus essential oils (EOs) have several applications in cosmetics, the food industry, and the flavor and fragrance industry. They are also utilized as natural preservatives because of their wide range of biological activities, which include antioxidant and antimicrobial actions [1]. These strong biological activities are attributed to the presence of terpenes, flavonoids, carotenes, and coumarins [2]. Several studies have investigated the volatile makeup of various parts of Citrus species due to their significant economic importance. All cold-pressed Citrus oils contain a portion of non-volatiles fundamentally made of simple coumarins, psoralens, and methoxy-flavones [3]. Coumarins (1,2-benzopyrones) are a huge family of naturally occurring secondary metabolites. Psoralens, also known as furanocoumarins (FCs), are a large family of compounds commonly found in Rutaceae, Apiaceae, and Fabaceae, with Rutaceae containing the highest concentrations [4,5]. FCs contain a furan ring fused to a coumarin core [6]. The fusion helps separate the FCs into linear or angular structural forms. FCs have shown a potential to elicit variable degrees of phototoxic skin reactions. In comparison to angular FCs, linear FCs have often been proven to cause phototoxic responses at lower doses [7].
While studying the volatile composition of Citrus oils, the nonvolatile fractions are hard to detect under standard gas chromatography conditions because of their limited volatilities, relatively polar or heat-liable nature. These nonvolatile ingredients may hold the secret to constructing a perfect analytical strategy for interspecies adulteration detection. This essential fraction of the cold-pressed oil can be used to identify species-specific patterns and establish Citrus species fingerprinting. For instance, creating synthetic bergamot oils or adulterating bergamot oils with similar Citrus oils like bitter orange are simple strategies to boost profits. Both strategies make it essentially impossible for consumers to detect the difference. The non-volatile fraction contributes very little to the Citrus oils’ aroma, but because of its high complexity, commercial unavailability, or extremely high cost in comparison to the Citrus oils themselves, it is more difficult to manipulate. Previous studies report the separation and identification of coumarins and FCs in Citrus peel extracts and oils using gas chromatography–mass spectroscopy (GC-MS) after derivatization [8], high-performance liquid chromatography (HPLC) [8], enzyme-linked immunosorbent assay (ELISA) [9], reversed-phase (RP)-HPLC [10,11], HPLC-diode array detector (DAD) [12], HPLC-nuclear magnetic resonance (NMR) [13], ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) [14,15], LC-MS [16], and HPLC-UV-MS [17].
The objective of the present study was to develop a sensitive UPLC-MS/MS method to quantify 14 selected coumarins and FCs (Figure 1). This validated method was then applied to the cold-pressed essential oils of bergamot (Citrus bergamia Risso & Poit), bitter orange (C. aurantium L.), calamansi (C. × microcarpa (Bunge) Wijnands), clementine (C. clementina Hort. ex Tanaka), grapefruit (C. × paradisi Macfady), kumquat (C. japonica Thunb.), lemon (C. limon Osbeck), lime (C. aurantifolia (Christm.) Swingle), mandarin (C. reticulata Blanco), sweet orange (C. sinensis L.), tangerine (C. tangerina Hort. ex Tanaka), and yuzu (C. junos Sieb. ex Tanaka) as well as petitgrain EO.
Figure 1. Chemical structure of key non-volatile components in expressed Citrus essential oils.

2. Results and Discussions

2.1. Method Validation

The LC-MS/MS chromatogram of 14 coumarins using the MRM acquisition mode is shown in Figure 2. Specificity, precision, accuracy, linearity, intermediate precision, and LOQ results are summarized in Table 1. The method proved specific to the target compounds since no interferences were found in any of the processed blanks. All compounds met the acceptance criterion of RSD% ≤ 10 based on the precision and intermediate precision results. Compound recovery percentages ranged from 94.07 to 114.53% of the expected value. The linearity of the calibration curve of the 14 compounds was well correlated (r ≥ 0.98) within a range of 0.0001–0.1 ppm. The LOQ values ranged from 0.0001 to 0.005 ppm. These findings demonstrate that the developed method is suitable for analyzing the 14 targeted compounds in EOs.
Figure 2. LC-MS/MS chromatogram (MRM acquisition mode) of 14 targeted coumarins using a Shimadzu LCMS8060.
Table 1. Linearity of the UPLC-MS Method (Equation and Coefficient of Determination, r2), Limit of Quantitation (LOQ), and Accuracy of the UPLC-MS Method of 14 Coumarins and Furanocoumarins.

2.2. Comparison of Citrus EO Coumarin and Furanocoumarin Content

Citrus EOs used in this study were produced by expression in industrial settings. A total of 374 Citrus EOs were screened for coumarins using a 20 min UPLC-MS/MS method targeting 14 coumarins, of which 10 are linear furanocoumarins. The compositions of target compounds greatly differed among the tested Citrus EOs (Table 2). The least quantity of coumarins and FCs was detected in calamansi EO (0.15 ± 0.02 ppm). In comparison, the largest presence of coumarins and FCs was found in Italian bergamot EO (171,453.11 ± 9227.11 ppm), followed by the Brazilian bergamot EO (52,473.90 ± 1775.63 ppm). Expressed oil of Italian bergamot showed the highest FC content (167,281.60 ± 1017.74 ppm) and the highest level of any individual FC (109,730.67 ± 3150.55 ppm bergamottin). Notable differences were observed in the coumarin and FC levels among EOs of different crop varieties and origins within the same Citrus species. There have been several previous investigations on the non-volatile components of Citrus essential oils reported in the literature (Table 3). The non-volatile components are far more species-specific than the volatile components, which have comparable patterns in different Citrus oils. We found patterns in FC profiles that correspond with published differences among the Citrus ancestral taxa [15,18]. Our findings are in line with previous reports that found a mixture of FCs from the bergapten, xanthotoxin, and isopimpinellin clusters in EOs derived from the citron (C. medica) and papeda (C. micrantha) ancestral taxa [15]. In this study, EOs derived from fruits of the mandarin taxa (mandarin, clementine, and tangerine) showed low total coumarin (2.44–149.45 ppm) and FC levels (2.44–149.45 ppm), not aligning with a previous report that this taxon is nearly devoid of FCs [14,15]. Interestingly, trioxsalen and toncarine were not detected in any of the Citrus EOs. Epoxybegamottin was absent from calamansi, clementine, mandarin, kaffir lime, and petitgrain oils. The content of psoralen was almost negligible in most of the Citrus EOs but was relatively high in white grapefruit EO (82.65 ± 0.76 ppm). Furthermore, large amounts of xanthotoxin were detected in bergamot and lime EOs. Previous reports indicate that xanthotoxin is absent from sweet orange (C. sinensis, pummelo taxon) and the mandarin taxa [14,15] while we found 4.85 ± 0.32 ppm in sweet orange EO and 0.33–15.24 ppm xanthotoxin in the mandarin taxa EOs. Bergamottin and 5-geranyloxy-7-methoxycoumarin were reported in mandarin, lemon, and lime oils but not in orange oil [17]. The differences between our findings and previous studies could be due to genetic and/or environmental impacts on FC biosynthesis [19]. Our LOQ, however, may be lower than that of other reports because it was based on the weight of EO rather than the weight of fresh fruit peel. Alternative explanations for the differences in our findings include genetic admixture in Citrus varieties or contamination during processing and handling.
Table 2. Total coumarin, total furanocoumarins, and coumarin distribution of the tested Citrus oils.
Table 3. Non-volatile components of cold-pressed Citrus oils that are reported in the literature.

2.3. Multivariate Analysis

In order to examine the similarities and relationships between the coumarin compositions and the Citrus essential oils, AHC and PCA were carried out based on a data matrix comprised of 28 Citrus “types” and 12 coumarin components. Based on > 25% similarity, the AHC shows five groups (Figure 3): Group 1 (bergamot from Italy and bergamot from Brazil), Group 2 (lime and lemon from Germany), Group 3 (yuzu, red and white grapefruit), Group 4 (a large group composed of oranges, tangerines, clementines, mandarins calamansi, and petitgrains), and Group 5 (lemons). The PCA analysis (Figure 4) of the Citrus essential oils indicates that F1 and F2 explain 78.43% of the variation in coumarin compositions among the Citrus types. The bergamot group (Group 1) is positively correlated with bergamottin, bergapten, and xanthotoxin; the lemon group (Group 5) positively correlates with biacangelicol and oxypeucedanin as well as citropten and 5-geranyloxy-7-methoxycoumarin. The grapefruit and yuzu group (Group 3) correlate with 6ʹ,7ʹ-epoxybergamottin and psoralen. Group 4 (oranges, mandarins, clementines, etc.) are characterized as having relatively low levels of coumarins. A positive correlation was found between bergapten and xanthotoxin (2 structures related by a common precursor in biosynthesis [15]).
Figure 3. Dendrogram obtained by cluster analysis of the coumarin composition of Citrus essential oils, based on correlation and using the unweighted pair-group method with arithmetic average (UPGMA).
Figure 4. Principal component biplot of PC1 and PC2 scores and loadings indicating the coumarin chemical relationships of Citrus essential oils.

3. Materials and Methods

3.1. Chemicals

Xanthotoxin, herniarin, toncarine, bergamottin, oxypeucedanin, biacangelicol, psoralen, isopimpinellin, bergapten, and imperatorin (purity ≥ 98%) were purchased from Chengdu Alfa Biotechnology (Chengdu, China). 5-Geranyloxy-7-methoxycoumarin (purity ≥ 99%) was bought from Extrasynthese (Genay, France). Trioxsalen and 6′,7′-epoxybergamottin (purity ≥ 98%) were obtained from Cayman Chemical Company (Michigan, USA). Citropten (purity ≥ 99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). LCMS-grade methanol, LCMS-grade water, and HPLC-formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of each standard at a concentration of 10 ppm were prepared by diluting the powder in methanol.

3.2. Essential Oil Samples

Citrus volatile oils from trusted suppliers were obtained from the collection of the Aromatic Plant Research Center (APRC, Lehi, UT, USA). A total of 374 cold-pressed Citrus oil samples from the APRC collection are listed in Table 4. A simple dilute and shoot technique (1 μL oil in 999 μL of methanol) was used for sample preparation. Further dilution was performed whenever needed.
Table 4. Sample information of citrus essential oil samples from the APRC collection.

3.3. UPLC-MS/MS Analyses

Coumarins were quantified using a NEXERA UPLC system (Shimadzu Corp., Kyoto, Japan) equipped with a mass spectrometer (Triple quadrupole, LCMS8060, Shimadzu, Kyoto, Japan). Target compounds were chromatographed on a Shimadzu Nexcol C18 column (1.8 µm, 50 × 2.1 mm) with a C18 guard column (Restek, Bellefonte, PA, USA) at 40 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B). The compounds were eluted using the following gradient: %10 B at 0 min, %20 B at 0.74 min, %60 B at 5.88 min, %90 B at 10 min, held at %100 B for 4 min, and %10 for 4 min before the next injection. The flow rate was maintained at 0.2 mL/min, and the injection volume was 1 μL. The UPLC system was connected to the MS by electrospray ionization (ESI) operating in positive ion mode. The interface, desolvation line, and heating block temperatures were 350, 250, and 400 °C, respectively. The capillary voltage was 4.5 kV, and CID gas was set at 350 kPa. Nebulizing gas flow was set at 3.0 L/min, and heating and drying gas were set at 10.0 L/min. The detection was completed in multiple reaction monitoring mode (MRM) (Table 5). Samples were run in triplicates with external standards in between. Each run contained a quality control (QC) standard, and at least one QC standard was run at the beginning and the end of the run. The acquired chromatographic results were processed in LabSolutions Insight software version 3.2 (Shimadzu). For each compound, calibration curves (0.005, 0.001, 0.0025, 0.005, 0.01, 0.025, 0.05, and 0.1 ppm) were drawn by linking its peak area and its concentration.
Table 5. Multiple reaction monitoring mode (MRM) parameters.

3.4. Method Validation

Method validation was executed according to the USP<1225> Validation of compendial procedures [31] and ICH harmonized tripartite guideline validation of analytical procedures: text and methodology Q2(R1) [32]. Specificity, precision, accuracy, linearity, intermediate precision, and limit of quantification (LOQ) were determined using standard solutions. Distilled yuzu essential oil was used as a matrix (total coumarins < 0.001 ppm). To prove the specificity of the method, standard solution mixtures and at least three blanks were processed to demonstrate the absence of interferences with the elution of the analytes. Precision and repeatability were determined by injecting six sample preparations spiked to a final concentration of 0.04 ppm and then calculating the RSD% between injections which may reach 10% for each. For the intermediate precision, the repeatability experiment was repeated on a second day and performed by a second analyst with the acceptance criterion of RSD ≤ 10 for each compound and each analyst. To determine the recoveries (accuracy) of the target compounds, three individually prepared samples of yuzu oil were spiked with three concentrations of the standard (LOQ, 0.04, and 0.05 ppm in triplicates). Recoveries were calculated by comparing the absolute peak areas with a reference measurement which must be within 80–120% of the expected value. Five concentrations from 0.001 to 0.1 ppm were used to determine linearity and a coefficient of determination (r) higher than 0.98 was needed. The data obtained during the linearity, precision, and accuracy studies were used to assess the range of the method for the target compounds. The acceptable range was defined as the concentration interval over which linearity, precision, and accuracy are acceptable. To estimate the LOQ, standard mixtures at low concentrations (0.0005 to 0.01 ppm) were analyzed. The calculated LOQ was determined using the signal-to-noise (S/N) ratio (10:1) and then injected 6 times. The acceptance criterion for the LOQ was RSD ≤ 15%. A calibration curve based on the linear range was prepared and injected to estimate the quantity of coumarins in the oil samples. Additionally, QC standards at low (0.05 ppm) and high (0.1 ppm) concentrations were used.

3.5. Multivariate Analysis

The average coumarin concentrations (12 compounds) in the Citrus samples were used as variables in the multivariate analysis. First, the data matrix was standardized by subtracting the mean for each compound concentration and dividing it by the standard deviation. For the agglomerative hierarchical cluster (AHC) analysis, the 24 Citrus samples were treated as operational taxonomic units (OTUs). Pearson correlation was selected as a measure of similarity, and the unweighted pair group method with arithmetic average (UPGMA) was used for cluster definition. Principal component analysis (PCA) was performed for the visual comparison of the coumarin compositions of the different Citrus groups using the 12 coumarin components as variables, with a Pearson correlation matrix. The AHC and PCA analyses were performed using XLSTAT v. 2018.1.1.62926 (Addinsoft, Paris, France).

4. Conclusions

In this study, we developed and validated a simple and sensitive UPLC-MS/MS method for the detection and quantification of 14 selected oxygen heterocyclic compounds (coumarins and furanocoumarins). Targeted screening using this method was successfully completed for the essential oils of 12 different Citrus species. To our knowledge, this is the most comprehensive investigation of coumarin and furanocoumarin profiles across commercial-scale Citrus oils to date. The lowest amount was detected in calamansi oil. Expressed oil of Italian bergamot showed the highest furanocoumarin content and the highest level of any individual furanocoumarin (bergamottin). Remarkable differences were observed in the coumarin and furanocoumarin levels among oils of different crop varieties and origins within the same species. We found potential correlations between bergapten and xanthotoxin which matches with known biosynthetic pathways. Patterns in furanocoumarin profiles lined up with known variations among the Citrus ancestral taxa. Using multivariate analysis, we were able to divide the Citrus oils into 5 main groups (bergamots; lime and German lemon; yuzu and grapefruit; oranges, tangerines, clementines, mandarins, calamansi, and petitgrains; and lemons) and correlate them to the coumarin compositions.

Author Contributions

Conceptualization, N.S.D. and P.S.; methodology, N.S.D.; software, N.S.D. and W.N.S.; validation, N.S.D.; formal analysis, N.S.D. and W.N.S.; investigation, N.S.D. and P.S.; data curation, W.N.S.; writing—original draft preparation, N.S.D.; writing—review and editing, N.S.D., P.S. and W.N.S.; supervision, W.N.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data are available upon reasonable request from the corresponding author (N.S.D.).

Acknowledgments

This work was carried out as part of the activities of the Aromatic Plant Research Center (APRC, https://aromaticplant.org/, accessed on 18 September 2022).

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AHCagglomerative hierarchical cluster analysis
DADdiode array detector
ELISAenzyme-linked immunosorbent assay
EOessential oil
FCfuranocoumarin
GC-MSgas chromatography–mass spectroscopy
HPLChigh-performance liquid chromatography
LC-MSliquid chromatography- mass spectrometry
LOQlimit of quantification
MRMmultiple reaction monitoring mode
NMRnuclear magnetic resonance
OTUsoperational taxonomic units
PCAPrincipal component analysis
ppmparts per million
QCquality control
RP-HPLCreversed-phase-high-performance liquid chromatography
UPGMAunweighted pair group method with arithmetic average
UPLC-MS/MSultra-performance liquid chromatography-tandem mass spectrometry

References

  1. Mitropoulou, G.; Fitsiou, E.; Spyridopoulou, K.; Tiptiri-Kourpeti, A.; Bardouki, H.; Vamvakias, M.; Panas, P.; Chlichlia, K.; Pappa, A.; Kourkoutas, Y. Citrus medica essential oil exhibits significant antimicrobial and antiproliferative activity. LWT-Food Sci. Technol. 2017, 84, 344–352. [Google Scholar] [CrossRef]
  2. Viuda-Martos, M.; Ruiz-Navajas, Y.; Fernández-López, J.; Perez-Álvarez, J. Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food Control 2008, 19, 1130–1138. [Google Scholar] [CrossRef]
  3. Tisserand, R.; Young, R. Essential Oil Safety, 2nd ed.; Elsevier: Amsterdam, The Netherlands, 2014. [Google Scholar]
  4. Pathak, M.A.; Daniels, F.; Fitzpatrick, T.B. The presently known distribution of furocoumarins (psoralens) in plants. J. Invest. Dermatol. 1962, 39, 225–239. [Google Scholar] [CrossRef] [PubMed]
  5. Murray, R.D.H.; Méndez, J.; Brown, S.A. The Natural Coumarins: Occurrence, Chemistry, and Biochemistry; Wiley: Chichester, UK, 1982. [Google Scholar]
  6. Nitao, J.K.; Berhow, M.; Duval, S.M.; Weisleder, D.; Vaughn, S.F.; Zangerl, A.; Berenbaum, M.R. Characterization of furanocoumarin metabolites in parsnip webworm, Depressaria pastinacella. J. Chem. Ecol. 2003, 29, 671–682. [Google Scholar] [CrossRef] [PubMed]
  7. Bourgaud, F.; Hehn, A.; Larbat, R.; Doerper, S.; Gontier, E.; Kellner, S.; Matern, U. Biosynthesis of coumarins in plants: A major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem. Rev. 2006, 5, 293–308. [Google Scholar] [CrossRef]
  8. Ziegler, H.; Spiteller, G. Coumarins and psoralens from Sicilian lemon oil (Citrus limon (L.) Burm. f.). Flavour Fragr. J. 1992, 7, 129–139. [Google Scholar] [CrossRef]
  9. Saita, T.; Fujito, H.; Mori, M. Screening of furanocoumarin derivatives in Citrus fruits by enzyme-linked immunosorbent assay. Biol. Pharm. Bull. 2004, 27, 974–977. [Google Scholar] [CrossRef]
  10. Thompson, H.J.; Brown, S.A. Separations of some coumarins of higher plants by liquid chromatography. J. Chromatogr. A 1984, 314, 323–336. [Google Scholar] [CrossRef]
  11. Kamiński, M.; Kartanowicz, R.; Kamiński, M.M.; Królicka, A.; Sidwa-Gorycka, M.; Łojkowska, E.; Gorzeń, W. HPLC-DAD in identification and quantification of selected coumarins in crude extracts from plant cultures of Ammi majus and Ruta graveolens. J. Sep. Sci. 2003, 26, 1287–1291. [Google Scholar] [CrossRef]
  12. Frérot, E.; Decorzant, E. Quantification of total furocoumarins in Citrus oils by HPLC coupled with UV, fluorescence and mass detection. J. Agric. Food Chem. 2004, 52, 6879–6886. [Google Scholar] [CrossRef]
  13. Sommer, J.; Bertram, H.J.; Krammer, G.; Kindel, G.; Kuhnle, T.; Reinders, G.; Reiss, I.; Schmidt, C.O.; Schreiber, K.; Stumpe, W.; et al. HPLC–NMR—a powerful tool for the identification of non-volatiles in lemon peel oils. Perfum. Flavor 2003, 38, 18–34. [Google Scholar]
  14. Dugrand, A.; Olry, A.; Duval, T.; Hehn, A.; Froelicher, Y.; Bourgaud, F. Coumarin and furanocoumarin quantitation in Citrus peel via ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-MS). J. Agric. Food Chem. 2013, 61, 10677–10684. [Google Scholar] [CrossRef] [PubMed]
  15. Dugrand-Judek, A.; Olry, A.; Hehn, A.; Costantino, G.; Ollitrault, P.; Froelicher, Y.; Bourgaud, F. The distribution of coumarins and furanocoumarins in Citrus species closely matches Citrus phylogeny and reflects the organization of biosynthetic pathways. PLoS ONE 2015, 10, e0142757. [Google Scholar] [CrossRef] [PubMed]
  16. Dugo, P.; Mondello, L.; Dugo, L.; Stancanelli, R.; Dugo, G. LC-MS for the identification of oxygen heterocyclic compounds in Citrus essential oils. J. Pharm. Biomed. Anal. 2000, 24, 147–154. [Google Scholar] [CrossRef]
  17. Fan, H.; Wu, Q.; Simon, J.E.; Lou, S.-N.; Ho, C.-T. Authenticity analysis of Citrus essential oils by HPLC-UV-MS on oxygenated heterocyclic components. J. Food Drug Anal. 2015, 23, 30–39. [Google Scholar] [CrossRef]
  18. Wu, G.A.; Terol, J.; Ibanez, V.; López-García, A.; Pérez-Román, E.; Borredá, C.; Domingo, C.; Tadeo, F.R.; Carbonell-Caballero, J.; Alonso, R.; et al. Genomics of the origin and evolution of Citrus. Nature 2018, 554, 311–316. [Google Scholar] [CrossRef]
  19. Bruni, R.; Barreca, D.; Protti, M.; Brighenti, V.; Righetti, L.; Anceschi, L.; Mercolini, L.; Benvenuti, S.; Gattuso, G.; Pellati, F. Botanical sources, chemistry, analysis, and biological activity of furanocoumarins of pharmaceutical interest. Molecules 2019, 24, 2163. [Google Scholar] [CrossRef]
  20. Lawrence, B.M. Progress in Essential Oils. Perfum. Flavorist 2004, 29, 44–59. [Google Scholar]
  21. Dugo, P.; Mondello, L.; Sebastiani, E.; Ottanà, R.; Errante, G.; Dugo, G. Identification of minor oxygen heterocyclic compounds of Citrus essential oils by liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry. J. Liq. Chromatogr. Relat. Technol. 1999, 22, 2991–3005. [Google Scholar] [CrossRef]
  22. Verzera, A.; la Rosa, G.; Zappala, M.; Cotroneo, A. Essential oil composition of different cultivars of bergamot grown in Sicily. Ital J. Food Sci. 1999, 12, 493–501. [Google Scholar]
  23. Bonaccorsi, I.; Sciarrone, D.; Cotroneo, A.; Mondello, L.; Dugo, P.; Dugo, G. Enantiomeric distribution of key volatile components of Citrus essential oils. Rev. Bras. Farmacogn. (Brasil J. Pharmacogn.) 2011, 21, 841–849. [Google Scholar] [CrossRef]
  24. Russo, M.; Torre, G.; Carnovale, C.; Bonaccorsi, I.; Mondello, L.; Dugo, P. A New HPLC method developed for the analysis of oxygen heterocyclic compounds in Citrus essential oils. J. Essent. Oil Res. 2012, 24, 119–129. [Google Scholar] [CrossRef]
  25. Dugo, P.; Mondello, L.; Proteggente, A.R.; Cavazza, A.; Dugo, G. Oxygen heterocyclic compounds of bergamot essential oils. Rivista Italiana EPPOS 1999, 27, 31–41. [Google Scholar]
  26. Kawaii, S.; Tomono, Y.; Katase, E.; Ogawa, K.; Yano, M. Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds. J. Agric. Food Chem 1999, 47, 4073–4078. [Google Scholar] [CrossRef] [PubMed]
  27. Mangiola, C.; Postorino, E.; Gionfriddo, F.; Catalfamo, M.; Manganaro, R. Evaluation of the genuineness of cold-pressed bergamot oil. Perfum. Flav. 2009, 34, 26–32. [Google Scholar]
  28. Costa, R.; Dugo, P.; Navarra, M.; Raymo, V.; Dugo, G.; Mondello, L. Study on the chemical composition variability of some processed bergamot (Citrus bergamia) essential oils. Flav. Fragr. J. 2010, 25, 4–12. [Google Scholar] [CrossRef]
  29. Menichini, F.; Tundis, R.; Loizzo, M.R.; Bonesi, M.; Provenzano, E.; Cindio, B.D.; Menichini, F. In vitro photo-induced cytotoxic activity of Citrus bergamia and C. medica L. cv. diamante peel essential oils and identified active coumarins. Pharm. Biol. 2010, 48, 1059–1065. [Google Scholar] [CrossRef]
  30. Dugo, G.; Bonaccorsi, I.; Sciarrone, D.; Schipilliti, L.; Russo, M.; Cotroneo, A.; Dugo, P.; Mondello, L.; Raymo, V. Characterization of cold-pressed and processed bergamot oils by using GC-FID, GC-MS, GC-C-IRMS, Enantio-GC, MDGC, HPLC and HPLC-MS-IT-TOF. J. Essent. Oil Res. 2012, 24, 93–117. [Google Scholar] [CrossRef]
  31. USP <1225> Validation of Compendial Procedures. 2021. Available online: https://latam-edu.usp.org/wp-content/uploads/2021/08/1225.pdf (accessed on 18 September 2022).
  32. ICH Expert Working Group, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Ich Harmonised Tripartite Guideline Validation of Analytical Procedures: Text and Methodology Q2(R1). 2005. Available online: https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf (accessed on 18 September 2022).
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Article Metrics

Citations

Article Access Statistics

Journal Statistics
Multiple requests from the same IP address are counted as one view.
Download PDF
Synthesis of Isomeric 3-Benzazecines Decorated with Endocyclic Allene Moiety and Exocyclic Conjugated Double Bond and Evaluation of Their Anticholinesterase Activity
Previous
Renewable Schiff-Base Ionic Liquids for Lignocellulosic Biomass Pretreatment
Next