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Article

Quantification of Squalene in Olive Oil Using 13C Nuclear Magnetic Resonance Spectroscopy

Équipe Chimie et Biomasse, UMR 6134 SPE, Université de Corse-CNRS, Route des Sanguinaires, 20000 Ajaccio, France
*
Author to whom correspondence should be addressed.
Magnetochemistry 2017, 3(4), 34; https://doi.org/10.3390/magnetochemistry3040034
Submission received: 11 October 2017 / Revised: 26 October 2017 / Accepted: 31 October 2017 / Published: 6 November 2017
(This article belongs to the Special Issue Nuclear Magnetic Resonance Spectroscopy)

Abstract

:
In the course of our ongoing work on the chemical characterization of Corsican olive oil, we have developed and validated a method for direct quantification of squalene using 13C Nuclear Magnetic Resonance (NMR) spectroscopy without saponification, extraction, or fractionation of the investigated samples. Good accuracy, linearity, and precision of the measurements have been observed. The experimental procedure was applied to the quantification of squalene in 24 olive oil samples from Corsica. Squalene accounted for 0.35–0.83% of the whole composition.

Graphical Abstract

1. Introduction

Squalene—(E)-2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene—is a natural acyclic symmetrical triterpene. It is a key intermediate in the biosynthesis of sterols [1]. In the human body, squalene is synthesized and then converted into cholesterol. In medicine, squalene plays a major role in the reduction of cancer risks, particularly with regard to cancer of the pancreas and colon in rodents [2,3,4]. Squalene increases the stability of various emulsions (vaccines, pharmaceutical formulations) [5,6]. It is also useful at the surface of the skin, playing the role of protective barrier against Ultra-Violet (UV) radiations [7]. Hydrogenated squalene (i.e., squalane) is appreciated in cosmetics as emollient agent in creams and capillary serums [8].
The largest source of squalene for industrial purposes is from animal origin, provided by various species of shark [9]. According to the species, squalene represents up to 80% of the shark liver oil [10]. Various species of shark are now endangered as a result of their overexploitation.
Squalene is also widespread in the vegetable kingdom. Indeed, it is present in oil seeds and in green vegetables [11]. In olive oil, squalene represents 0.3% to 0.7% of the whole mass, accounting for 60–75% of the unsaponifiable fraction [12]. The presence of squalene confers to olive oil a great stability against auto-oxidation and photo-oxidation [13].
The Association of Official Analytical Chemists [14] recommended a method for extraction of squalene from natural matrices. Analytical techniques used in quantification of squalene in edible oils, in the presence of acylglycerols, fatty acids, phytosterols, and tocopherols have been recently reviewed [15]. Methods using a preliminary fractionation of samples, procedure that simplifies the analysis have been developed. Analysis of squalene in edible oils is predominantly achieved by chromatographic techniques (Gas Chromatography (GC) or Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)), after saponification of triglycerides, solvent extraction of the unsaponifiable fraction, and eventually isolation of the hydrocarbon fraction by Column Chromatography (CC) or Thin Layer Chromatography (TLC) [16,17,18,19,20,21]. The direct injection of olive oil in the injector port has been applied [22], as well as HPLC coupled with GC [23] or HPLC coupled to electrospray tandem mass spectrometry [24].
In parallel, 1H and 13C NMR have been widely used for identification and quantitative evaluation of triglycerides in olive oil (saturated fatty acid chains, mono-unsaturated, poly-unsaturated, stereochemistry of the double bonds, etc.) and for quality assessment and authentication [25,26,27]. Using the fingerprint technique, characteristic resonances of individual components of the unsaponifiable fraction (sterols, alcohols, tocopherol) have been identified using 1H NMR, and the results allowed the determination of geographical origin of olive oil [28]. Similarly, the ratio of squalene vs. the other minor components of olive oil has been evaluated, and statistical analysis of the results gave useful information on the quality, authenticity, and origin of the investigated olive oil samples [25,29]. The content of squalene in human sebum (containing low proportion of triglycerides) has been measured using a 600 MHz spectrometer equipped with a cryoprobe [30]. Otherwise, quantitative analyses of two structurally close triterpenoid acids, as well as that of positional and geometric isomers of octadecadienoic acid with conjugated double bonds, have been performed using 2D NMR [31,32].
In previous works carried out in our laboratory, we demonstrated 13C NMR spectroscopy was a powerful tool for the identification and quantitative determination of terpenes in natural matrices, mono and sesquiterpenes in essential oils [33] and fixed oil [34], diterpenes in cedar resins [35], triterpenes in solvent extracts from cork [36], or leaves from olive tree [37]. Taking into account that chromatographic techniques used to quantify squalene in olive oil needed laborious and time-consuming fractionation steps, the aim of the present study was to develop a method, based on 13C NMR, and using a routine spectrometer (9.4 Tesla), that allowed the quantitative determination of squalene in olive oil, avoiding the fractionation steps.

2. Results and Discussion

2.1. 13C NMR Data of Squalene and Olive Oil

The 13C NMR spectrum of squalene displayed 15 resonances belonging to quaternary carbons (135.11; 134.90 and 131.26 ppm), ethylenic methines (124.42; 124.32 and 124.28 ppm), allylic methylenes (39.77; 39.74, 28.29; 26.78; 26.67 ppm) and methyl groups (25.71; 17.69; 16.05 and 16.01 ppm). The chemical shift values of our recorded spectrum (Table 1) fitted perfectly with previous data reported by Pogliani et al. [38]. However, it could be noted a difference of 1 ppm for carbon C3, probably due to a misprint in the paper [38].
The 13C NMR spectrum of a commercially available olive oil is more complex (Figure 1). Four parts may be distinguished: 172–174 ppm, esters; 124–134 ppm, ethylenic carbons; 60–72 ppm, carbons of glycerol; and 13–35 ppm, aliphatic carbons. In that spectrum, all the resonances with high intensity belong to the triglycerides. Twelve out of 15 resonances of squalene were observed. They were perfectly resolved, and therefore they could be used for quantitative determination of squalene in olive oil.

2.2. Validation of the Experimental Procedure for Quantitative Determination of Squalene Using 13C NMR

In order to approach the physico-chemical properties of olive oil (viscosity for instance) the experiments for validation of the experimental procedure have been carried out using know quantities of squalene in trioleine (glyceryl tris octadec-9-enoate) that is the major triglyceride of olive oil accounting for 48–62% of the whole composition [39].
Several techniques have been developed for quantification of individual components of a natural mixture based on 13C NMR spectroscopy. The standard sequence combines a 90° pulse angle, gated decoupling technique and requires waiting a period of 5T1 of the longest T1 value, before applying another pulse. This sequence provides accurate result but is really time consuming. Otherwise, use of a paramagnetic relaxation reagent allows decrease of experimental time but induces a line width broadening. Quantitative determination can be led using a rapid train of short pulses because a small flip angle provides less difference in the steady-state magnetization than a larger one in the presence of carbons having different T1 values.
Owing to our experience in the analysis of complex natural mixtures containing nuclei with different T1 values, a good approach is a compromise between the aforementioned procedures. For instance, quantification of various compounds has been performed in our laboratories, using this approach: carbohydrates in ethanol extract of Pinus species [40], triterpenes in cork extract [36] and olive leaf extract [37], and taxanes in leaf extract of Taxus baccata [41]. Quantitative determination of a component in a natural mixture is achieved by internal standardization by comparison of the areas of the resonances of that compound with those of an internal standard. In these conditions, it is obvious that quantitative estimation will be led from not fully relaxed spectra and that validation of the method should be performed before applying it to the analysis of mixtures [42]. The best conditions for the pulse sequence are those that reduce as far as possible the difference in the steady-state magnetization of nuclei with different T1 values and that simultaneously allow a good S/N ratio in a short period of time. They could be selected using Becker’s equation that allows the calculation of the S/N ratio as a function of the pulse angle and the ratio of longitudinal relaxation time to total recycling time [43].
Then, the theoretical parameters (precision, accuracy, linearity of measures) should be validated using pure squalene in trioleine before application of the method to the quantification of squalene in genuine olive oils. To carry out the validation of the method:
  • CDCl3 has been conserved as solvent and trioleine has been used as a model for olive oil;
  • Longitudinal relaxation times have been measured for carbons of squalene by the inversion-recovery method. They ranged from 0.4 to 10.0 s, the highest values (4.1–10.0 s) being measured, as expected, for quaternary carbons (Table 1). T1 values of vinylic methines and allylic methylenes ranged from 1.2 s to 2.5 s and from 0.4 s to 0.9 s, respectively. Finally, T1s of the four methyl groups ranged from 1.9 s to 4.5 s. Quantitative analysis has been conducted with resonances of carbons not overlapped, perfectly resolved and with T1 values comprised from 0.7 s to 4.5 s;
  • Di-2-methoxyethyl oxide (diglyme) has been chosen as internal standard (T1 value of its methylenes = 3.8 s) since its resonances do not overlap with those of triglycerides contained in olive oil.
The parameters of the pulse sequence have been determined using formula (1) for various T1 values (0.7–4.5 s), and for a repetition delay of 3.7 s (acquisition time = 2.7 s; relaxation delay = 1.0 s) required for a 128 K data table. According to Becker et al. [43], we determined and plotted the percentage of recovered signal, expressed as S/N (%), as a function of the pulse angle α, using formula (1). Using a pulse angle of 30°, this procedure provided a small difference (3.6%) in the steady-state magnetization between carbons exhibiting different T1 values and a reasonable time of analysis in spite of the utilization of a medium field spectrometer (3000 scans in less than 3 h) (Figure 2).
S N = M 0 × [ 1 e ( D / T 1 ) ] × sin α D × [ 1 e ( D / T 1 ) cos α ]
S/N: signal-to-noise ratio, M0: initial magnetization, D: time between two pulses (in seconds), T1: longitudinal relaxation time (in seconds), and α: pulse angle (in degrees).
Accuracy, precision and response linearity of this method have been validated by various experiments carried out on pure squalene by comparing the weighted quantities (0.37–1.66 mg) with those measured by NMR. From the 13C NMR spectrum, the mass of squalene mSQ (mg) was calculated using Formula (2). Relative errors between weighted and calculated masses are comprised between 0.0% and 10.3%, and therefore they demonstrated good accuracy of measurements (Table 2).
m S Q = 2 × A S Q × M S Q × m D A D × M D × p S Q × p D
The area ASQ taken into account was the mean value of the areas of selected protonated carbons. AD is the mean value of the areas of the two methylenes of diglyme. MSQ is the molecular weight of squalene. MD is the molecular weight of diglyme and mD is the amount of diglyme. pSQ and pD: purity of squalene and of diglyme, respectively.
Then, we drew the calibration line for the quantification of squalene. The straight line was plotted by expressing the ratio of the mean value of areas of the resonances of squalene selected carbons (ASQ) with those of diglyme (AD) as a function of the weighed mass of squalene (mw). We observed a good linearity of the measurements because the linear determination factor (R2) is 0.996 (Figure 3).
Finally, the spectrum of the sample containing 0.55 mg of squalene has been recorded five times. The repeatability, calculated with a confidence interval of 99% (Student’s t-test) was equal to 0.56 mg ± 0.04 mg, i.e., 0.56 mg ± 6.8% which indicates a good precision of measurements.
The experimental procedure developed to quantify squalene in triolein exhibited good accuracy, precision and linearity of measurements. Analysis time with a routine spectrometer (9.4 Tesla) is not prohibitive since a single analysis requires three hours. Therefore, this procedure could be applied for quantification of squalene in olive oils of Corsican origin.

2.3. Quantification of Squalene in Various Olive Oil Samples of Corsican Origin

Twenty-four olive oil samples from various localities in Corsica and from various olive varieties have been analyzed using 13C NMR, according to the experimental procedure previously described. In the 13C NMR spectrum of olive oil (Figure 2), eight out of 12 of the protonated carbons of squalene were observed. All of these resonances were perfectly resolved and did not overlap with resonances of other components of olive oil, and their relaxation times were between 0.7 s and 4.5 s. The mass of squalene in every olive oil sample has been calculated using Formula (2), taking into account the mean areas of these resonances. Then, the mass percentages of squalene have been calculated using Formula (3), which are reported in the Table 3.
% C = m S Q m × 100
%C: percentage of squalene; mSQ: calculated mass (mg) of squalene; m: mass of the olive oil sample.
Among the 24 olive oil samples, 18 samples were obtained from olive of a single variety, the last six samples coming from olives of two varieties. From Table 3, it is observed that Corsican olive oils contained appreciable amount of squalene comprised between 0.35% and 0.52% for 22 samples out of 24. The two last samples exhibited higher contents (0.67% and 0.83%). These results are in agreement with those reported in the literature (0.3–0.7%) [12].
Although the number of samples from every locality and from every olive variety is limited, it seems that there is no direct relation between the content of squalene in a given olive oil sample and the variety of the olive. However, it could be observed that zinzala, sabine, and picholine olives produced an oil containing 0.35–0.42% of squalene. The olive oil from Germaine, Cortenaise and Capanacce varieties exhibited a slightly higher content of squalene (0.40–0.83%). Finally, olive oil coming from two varieties of fruits contained 0.37–0.67% of squalene.

3. Materials and Methods

3.1. Chemicals

Squalene, triolein and di-2-methoxyethyloxide (diglyme) were obtained from Sigma-Aldrich (St-Louis, MO, USA), Acros Organics (Geel, Belgium), and Jansen Chimica (Geel, Belgium), respectively. Olive oil samples were supplied by Mrs. Henneman (Chambre d’Agriculture de la Haute Corse, Bastia, Corsica, France).

3.2. NMR Experiments

3.2.1. Quantitative 13C NMR Spectra

Quantitative 13C NMR spectra were recorded on a Bruker (Wissembourg, France) AVANCE 400 Fourier Transform spectrometer operating at 100.13 MHz for 13C, equipped with a 5 mm probe, in CDCl3 with all shifts referred to internal TMS. 13C NMR spectra were recorded with the following parameters: inverse gated decoupling, flip angle 30°, acquisition time = 2.7 s for 128 K data table with a spectral width of 24,000 Hz (240 ppm), a relaxation delay D1 = 1.0 s, composite pulse decoupling of the proton band, and a digital resolution of 0.366 Hz/pt. The internal reference used was diglyme. The number of accumulated scans was 3000 for each sample. Exponential line broadening multiplication (1 Hz) of the free induction decay was applied before Fourier transformation.

3.2.2. T1 Measurements

The longitudinal relaxation times of the 13C nuclei (T1 values) were determined by the inversion-recovery method, using the standard sequence: 180°–τ–90°–D1, with an acquisition time of 0.68 s (for 32 K data table with a spectral width of 25,000 Hz) and a relaxation delay D1 of 20 s. Each delay of inversion (τ) was thus taken into account for the computation of the corresponding T1 using the function Ip = I0 + pe−τ/T1 (Bruker microprogram; Ip and I0 are populations of nuclear spins; p is a constant of integration).

3.2.3. Calibration Line

A weighted amount of 0.37–1.66 mg of squalene was diluted in 0.5 mL of CDCl3 containing 1.49 mg of diglyme.

3.2.4. Quantification of Squalene in Olive Oils

A weighted amount of 140–150 mg of olive oil was diluted in 0.5 ml of CDCl3 containing 1.53 mg of diglyme.

4. Conclusions

An experimental procedure, based on 13C NMR spectroscopic analysis, was developed and allowed for the quantification of squalene in olive oil samples. An optimized pulse sequence (flip angle α = 30°, inverse gated decoupling, total recycling time 3.7 s) was checked and led to reliable quantitative determination of squalene in olive oil samples from Corsica with an analysis time of less than three hours using a medium field NMR spectrometer (9.4 T). In the 24 olive oil samples investigated, squalene accounted for 0.35–0.83% of the whole composition.

Acknowledgments

The authors are indebted to the Collectivité Territoriale de Corse for a research grant (A.-M.N.) and for financial support (FEDER AGRIEX). Thanks to Mrs. Henneman (Chambre d’Agriculture de la Haute Corse) for olive oil samples.

Author Contributions

A.B., F.T. and J.C. conceived and designed the experiments; A.-M.N. and M.P. performed the experiments; A.B. and F.T. analyzed the data; J.C. and M.P. wrote the paper.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Psomiadou, E.; Tsimidou, M. On the role of squalene in olive oil stability. J. Agric. Food Chem. 1999, 47, 4025–4032. [Google Scholar] [CrossRef] [PubMed]
  2. Smith, T.J.; Yang, G.Y.; Seril, D.N.; Liao, J.; Kim, S. Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis by dietary olive oil and squalene. Carcinogenesis 1998, 19, 703–706. [Google Scholar] [CrossRef] [PubMed]
  3. Newmark, H.L. Squalene, olive oil, and cancer risk: A review and hypothesis. Cancer Epidemiol. Biomark. Prev. 1997, 6, 1101–1103. [Google Scholar] [CrossRef]
  4. Rao, C.V.; Newmark, H.L.; Reddy, B.S. Chemopreventive effect of squalene on colon cancer. Carcinogenesis 1998, 19, 287–290. [Google Scholar] [CrossRef] [PubMed]
  5. Reddy, L.H.; Couvreur, P. Squalene: A natural triterpene for use in disease management and therapy. Adv. Drug Deliv. Rev. 2009, 61, 1412–1426. [Google Scholar] [CrossRef] [PubMed]
  6. Fox, C.B. Squalene emulsions for parenteral vaccine and drug delivery. Molecules 2009, 14, 3286–3312. [Google Scholar] [CrossRef] [PubMed]
  7. Auffray, B. Protection against singlet oxygen, the main actor of sebum squalene peroxidation during sun exposure, using Commiphora myrrha essential oil. Int. J. Cosmet. Sci. 2007, 29, 23–29. [Google Scholar] [CrossRef] [PubMed]
  8. Jame, P.; Casabianca, H.; Batteau, M.; Goetinck, P.; Salomon, V. Differentiation of the origin of squalene and squalane using stable isotopes ratio analysis. SOFW J. 2010, 136, 2–7. [Google Scholar]
  9. Deprez, P.; Volkman, J.; Davenport, S. Squalene content and neutral lipids composition of livers from deep-sea sharks caught in Tasmanian waters. Mar. Freshw. Res. 1990, 41, 375–387. [Google Scholar] [CrossRef]
  10. Wetherbee, B.M.; Nichols, P.D. Lipid composition of the liver oil of deep-sea sharks from the chatham rise, New Zealand. Comp. Biochem. Physiol. B Biochem. Mol. Biol. B 2000, 125, 511–521. [Google Scholar] [CrossRef]
  11. Ryan, E.; Galvin, K.; O’Connor, T.P.; Maguire, A.R.; O’Brien, N.M. Phytosterol, squalene, tocopherol content and fatty acid profile of selected seeds, grains, and legumes. Plant Foods Hum. Nutr. 2007, 62, 85–91. [Google Scholar] [CrossRef] [PubMed]
  12. Liu, G.C.; Ahrens, E.H.; Schreibman, P.H.; Crouse, J.R. Measurement of squalene in human tissues and plasma: Validation and application. J. Lipid Res. 1976, 17, 38–45. [Google Scholar] [PubMed]
  13. Grigoriadou, D.; Androulaki, A.; Psomiadou, E.; Tsimidou, M.Z. Solid phase extraction in the analysis of squalene and tocopherols in olive oil. Food Chem. 2007, 105, 675–680. [Google Scholar] [CrossRef]
  14. AOAC (Association of Official Analytical Chemists). Squalene in Oils and Fats. Titrimetric Method, Official Method AOAC; AOAC: Rockville, MD, USA, 1999. [Google Scholar]
  15. Popa, O.; Băbeanu, N.E.; Popa, I.; Niță, S.; Dinu-Pârvu, C.E. Methods for Obtaining and Determination of Squalene from Natural Sources. BioMed Res. Int. 2015. [Google Scholar] [CrossRef] [PubMed]
  16. Bondioli, P.; Mariani, C.; Lanzani, A.; Fedeli, E.; Muller, A. Squalene recovery from olive oil deodorizer distillates. J. Am. Oil Chem. Soc. 1993, 70, 763–766. [Google Scholar] [CrossRef]
  17. De Leonardis, A.; Macciola, V.; De Felice, M. Rapid determination of squalene in virgin olive oils using gas-liquid chromatography. Ital. J. Food Sci. 1998, 10, 75–80. [Google Scholar]
  18. Giacometti, J. Determination of aliphatic alcohols, squalene, α-tocopherol and sterols in olive oils: Direct method involving gas chromatography of the unsaponifiable fraction following silylation. Analyst 2001, 126, 472–475. [Google Scholar] [CrossRef] [PubMed]
  19. He, H.P.; Cai, Y.; Sun, M.; Corke, H. Extraction and purification of squalene from Amaranthus grain. J. Agric. Food Chem. 2002, 50, 368–372. [Google Scholar] [CrossRef] [PubMed]
  20. Nenadis, N.; Tsimidou, M. Determination of squalene in olive oil using fractional crystallization for sample preparation. J. Am. Oil Chem. Soc. 2002, 79, 257–259. [Google Scholar] [CrossRef]
  21. Oueslati, I.; Anniva, C.; Daoud, D.; Tsimidou, M.Z.; Zarrouk, M. Virgin olive oil (VOO) production in Tunisia: The commercial potential of the major olive varieties from the arid Tataouine zone. Food Chem. 2009, 112, 733–741. [Google Scholar] [CrossRef]
  22. Owen, R.W.; Giacosa, A.; Hull, W.E.; Haubner, R.; Wurtele, G.; Spiegelhalder, B.; Bartsch, H. Olive-oil consumption and health: The possible role of antioxidants. Lancet Oncol. 2000, 1, 107–112. [Google Scholar] [CrossRef]
  23. Villén, J.; Blanch, G.P.; Ruiz del Castillo, M.L.; Herraiz, M. Rapid and simultaneous analysis of free sterols, tocopherols, and squalene in edible oils by coupled Reversed-Phase Liquid Chromatography-gas chromatography. J. Agric. Food Chem. 1998, 46, 1419–1422. [Google Scholar] [CrossRef]
  24. Russo, A.; Muzzalupo, I.; Perri, E.; Sindona, G. A new method for detection of squalene in olive oils by mass spectrometry. J. Biotechnol. 2010, 150, 296–297. [Google Scholar] [CrossRef]
  25. Mannina, L.; Sobolev, A.P. High resolution NMR characterization of olive oils in terms of quality, authenticity and geographical origin. Magn. Reson. Chem. 2011, 49, 3–11. [Google Scholar] [CrossRef] [PubMed]
  26. Dais, P.; Hatzakis, E. Quality assessment and authentication of virgin olive oil by NMR spectroscopy: A critical review. Anal. Chim. Acta 2013, 765, 1–27. [Google Scholar] [CrossRef] [PubMed]
  27. Alexandri, E.; Ahmed, R.; Siddiqui, H.; Choudhary, M.I.; Tsiafoulis, C.G.; Gerothanassis, I.P. High resolution NMR spectroscopy as structural and analytical tool for unsaturated lipids in solution. Molecules 2017, 22, 1663. [Google Scholar] [CrossRef] [PubMed]
  28. Alonso-Salces, R.M.; Héberger, K.; Holland, M.V.; Moreno-Rojas, J.M.; Mariani, C.; Bellan, G.; Reniero, F.; Guillou, C. Multivariate analysis of NMR fingerprint of the unsaponifiable fraction of virgin olive oils for authentication purposes. Food Chem. 2010, 118, 956–965. [Google Scholar] [CrossRef]
  29. Mannina, L.; D’Imperio, M.; Capitani, D.; Rezzi, S.; Guillou, C.; Mavromoustakos, T.; Vilchez, M.D.; Fernández, A.; Thomas, F.; Aparicio, R. 1H NMR-based protocol for the detection of adulterations of refined olive oil with refined Hazelnut oil. J. Agric. Food Chem. 2009, 57, 11550–11556. [Google Scholar] [CrossRef] [PubMed]
  30. Robosky, L.C.; Wade, K.; Woolson, D.; Baker, J.D.; Manning, M.L.; Gage, D.A.; Reily, M.D. Quantitative evaluation of sebum lipid components with nuclear magnetic resonance. J. Lipid Res. 2008, 49, 686–692. [Google Scholar] [CrossRef] [PubMed]
  31. Kontogianni, V.G.; Exarchou, V.; Troganis, A.; Gerothanassis, I.P. Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy—Comparison with HPLC methods. Anal. Chim. Acta 2009, 635, 188–195. [Google Scholar] [CrossRef] [PubMed]
  32. Tsiafoulis, C.G.; Skarlas, T.; Tzamaloukas, O.; Miltiadou, D.; Gerothanassis, I.P. Direct nuclear magnetic resonance identification and quantification of geometric isomers of conjugated linoleic acid in milk fraction without derivatization steps: Overcoming sensitivity and resolution barriers. Anal. Chim. Acta 2014, 821, 62–71. [Google Scholar] [CrossRef] [PubMed]
  33. Blanc, M.C.; Bradesi, P.; Casanova, J. Identification and Quantitative Determination of Eudesman-Type Acids from Dittrichia viscosa sp. viscosa Essential Oil using 13C-NMR Spectroscopy. Phytochem. Anal. 2005, 16, 150–154. [Google Scholar] [CrossRef] [PubMed]
  34. Ferrari, B.; Castilho, P.; Tomi, F.; Rodrigues, A.I.; Costa, M.C.; Casanova, J. Direct Identification and Quantitative Determination of Costunolide and Dehydrocostuslactone in Laurus novocanariensis Fixed Oil using 13C-NMR Spectroscopy. Phytochem. Anal. 2005, 16, 104–107. [Google Scholar] [CrossRef] [PubMed]
  35. Nam, A.M.; Paoli, M.; Castola, V.; Casanova, J.; Bighelli, A. Identification and Quantitative Determination of Lignans in Cedrus atlantica Resins using 13C NMR Spectroscopy. Nat. Prod. Commun. 2011, 6, 379–385. [Google Scholar] [PubMed]
  36. Castola, V.; Bighelli, A.; Casanova, J. Direct Qualitative and Quantitative Analysis of Triterpenes Using 13C NMR Spectroscopy Exemplified by Dichloromethanic Extracts of Cork. Appl. Spectrosc. 1999, 53, 344–350. [Google Scholar] [CrossRef]
  37. Duquesnoy, E.; Castola, V.; Casanova, J. Identification and quantitative determination of triterpenes in the hexane extract of Olea europaea L. leaves using 13C NMR spectroscopy. Phytochem. Anal. 2007, 18, 347–353. [Google Scholar] [CrossRef] [PubMed]
  38. Pogliani, L.; Ceruti, M.; Ricchiardi, G.; Viterbo, D. An NMR and molecular mechanics study of squalene and squalene derivatives. Chem. Phys. Lipids 1994, 70, 21–34. [Google Scholar] [CrossRef]
  39. Bronzini de Caraffa, V.; Gambotti, C.; Giannettini, J.; Maury, J.; Berti, L.; Gandemer, G. Using lipid profiles and genotypes for the characterization of Corsican olive oils. Eur. J. Lipid Sci. Technol. 2008, 110, 40–47. [Google Scholar] [CrossRef]
  40. Duquesnoy, E.; Castola, V.; Casanova, J. Identification and quantitative determination of carbohydrates in ethanol extracts of two conifers using 13C NMR spectroscopy. Carbohydr. Res. 2008, 343, 893–902. [Google Scholar] [CrossRef] [PubMed]
  41. Paoli, M.; Bighelli, A.; Castola, V.; Tomi, F.; Casanova, J. Quantification of taxanes in a leaf and twig extract from Taxus baccata L. using 13C NMR spectroscopy. Magn. Reson. Chem. 2013, 51, 756–761. [Google Scholar] [CrossRef] [PubMed]
  42. Günther, H. La Spectroscopie de RMN. Principes de Base, Concepts et Applications de la Spectroscopie de Résonance Magnétique Nucléaire du Proton et du Carbone-13 en Chimie; Masson: Paris, France, 1994. [Google Scholar]
  43. Becker, E.D.; Ferretti, J.A.; Gambhir, P.N. Selection of optimum parameters for pulse Fourier transform nuclear magnetic resonance. Anal. Chem. 1979, 51, 1413–1420. [Google Scholar] [CrossRef]
Figure 1. 13C NMR spectrum of a commercially available virgin olive oil. SQ: squalene.
Figure 1. 13C NMR spectrum of a commercially available virgin olive oil. SQ: squalene.
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Figure 2. S/N (%) vs. flip angle α, plotted from formula (1) according to Becker et al. [43], for selected values of T1 (0.7 s< T1 < 4.5 s) and a minimum total recycling time τ of 3.7 s using a 128 K data table (acquisition time = 2.7 s and relaxation delay = 1.0 s).
Figure 2. S/N (%) vs. flip angle α, plotted from formula (1) according to Becker et al. [43], for selected values of T1 (0.7 s< T1 < 4.5 s) and a minimum total recycling time τ of 3.7 s using a 128 K data table (acquisition time = 2.7 s and relaxation delay = 1.0 s).
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Figure 3. Calibration line of squalene. mw = weighted mass of squalene.
Figure 3. Calibration line of squalene. mw = weighted mass of squalene.
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Table 1. Structure, 13C NMR chemical shifts, and longitudinal relaxation times (T1) of carbons of squalene.
Table 1. Structure, 13C NMR chemical shifts, and longitudinal relaxation times (T1) of carbons of squalene.
Magnetochemistry 03 00034 i001
Cδ (SQ)T1Cδ (SQ)T1
125.711.9939.77 *0.7
2131.2610.010135.115.2
3124.422.511124.32 #1.7
426.780.91228.290.4
539.74 *0.71317.694.5
6134.904.11416.05 2.8
7124.28 #1.21516.01 3.3
826.670.6
δ (SQ): Chemical shift of carbons of squalene (ppm vs. tetramethylsilane (TMS)). Assignment has been done according to Pogliani et al. [38]. *, # and : chemical shifts may be inversed. T1: longitudinal relaxation times in s.
Table 2. Quantitative determination of squalene by 13C NMR spectroscopy using diglyme as internal reference.
Table 2. Quantitative determination of squalene by 13C NMR spectroscopy using diglyme as internal reference.
AD0.99010.99550.99591.00741.00110.98451.01681.0139
ASQ0.07340.12200.16680.19700.22530.26890.31290.3431
mw (mg)0.370.550.740.921.101.291.471.66
mc (mg)0.330.560.741.001.031.221.441.58
ER (%)10.3−0.70.0−9.16.85.52.24.8
AD and ASQ: Mean areas of selected carbons of diglyme and squalene, respectively; Mass of diglyme (mD): 1.49 mg; mw: weighted mass of squalene (mg); mc: calculated mass of squalene (mg) using formula (2); ER: relative error (%) between mc and mw; Molecular weight of squalene: 410.7 g·mol−1.
Table 3. Quantification of squalene in olive oils from Corsica using 13C NMR.
Table 3. Quantification of squalene in olive oils from Corsica using 13C NMR.
SampleOlive VarietySqualene (%)*
1Zinzala0.35
20.37
30.41
4Sabine0.35
50.35
60.40
70.42
8Picholine0.38
9Germaine0.40
100.43
110.44
120.49
130.51
140.51
150.83
16Cortenaise0.42
170.47
18Capannacce0.52
19Germaine/Picholine0.37
20Germaine/Capanacce0.44
210.67
22Germaine/Sabine0.47
230.49
24Sabine/Picholine0.46
*: percentages calculated using formula (3).

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Nam, A.-M.; Bighelli, A.; Tomi, F.; Casanova, J.; Paoli, M. Quantification of Squalene in Olive Oil Using 13C Nuclear Magnetic Resonance Spectroscopy. Magnetochemistry 2017, 3, 34. https://doi.org/10.3390/magnetochemistry3040034

AMA Style

Nam A-M, Bighelli A, Tomi F, Casanova J, Paoli M. Quantification of Squalene in Olive Oil Using 13C Nuclear Magnetic Resonance Spectroscopy. Magnetochemistry. 2017; 3(4):34. https://doi.org/10.3390/magnetochemistry3040034

Chicago/Turabian Style

Nam, Anne-Marie, Ange Bighelli, Félix Tomi, Joseph Casanova, and Mathieu Paoli. 2017. "Quantification of Squalene in Olive Oil Using 13C Nuclear Magnetic Resonance Spectroscopy" Magnetochemistry 3, no. 4: 34. https://doi.org/10.3390/magnetochemistry3040034

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