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Rapid evaluation of edible hemp oil quality using NMR and FT-IR spectroscopy
Accepted Manuscript Rapid evaluation of edible hemp oil quality using NMR and FT-IR spectroscopy Paweł Siudem, Iwona Wawer, Katarzyna Paradowska PII:
S0022-2860(18)31141-4
DOI:
10.1016/j.molstruc.2018.09.057
Reference:
MOLSTR 25691
To appear in:
Journal of Molecular Structure
Received Date: 19 July 2018 Revised Date:
18 September 2018
Accepted Date: 21 September 2018
Please cite this article as: Paweł. Siudem, I. Wawer, K. Paradowska, Rapid evaluation of edible hemp oil quality using NMR and FT-IR spectroscopy, Journal of Molecular Structure (2018), doi: https:// doi.org/10.1016/j.molstruc.2018.09.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Rapid evaluation of edible hemp oil quality using NMR and FT-IR spectroscopy
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Paweł Siudem, Iwona Wawer, Katarzyna Paradowska*
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Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, PL-02097
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Warsaw, Poland
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Corresponding Author: Paradowska Katarzyna
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*Corresponding Author's Institution: Medical University of Warsaw, Faculty of Pharmacy, Phone/Fax.: +48 22
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5720 951,
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Corresponding Author’s email: [email protected]
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ACCEPTED MANUSCRIPT Abstract:
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Since Cannabis species include psychoactive varieties containing high levels of ∆9-tetrahydrocannabinol (THC),
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psychotropic properties could be wrongly attributed to hemp-seed oil obtained from Cannabis sativa. Hemp oil
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does not exert any psychotic effect; on the contrary, it may provide significant health benefits, because it has an
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optimum proportion of ω-6 to ω-3 fatty acids (3:1). Six samples of cold pressed hemp-seed oils were studied by
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nuclear magnetic resonance (NMR) and Fourier-transformed infrared spectroscopy (FT-IR). These methods
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were applied to evaluate the ratio of ω-6 to ω-3 fatty acids and to control the quality of hemp oils. FT-IR method
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allowed us to distinguish three oils with the highest level of γ-linolenic acid. Additionally, NMR spectroscopy
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was applied to determine the ω-6 to ω-3 ratio. The outcomes of the NMR experiment are in agreement with GC
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outcomes measures. The results indicate that NMR and FT-IR may be used in routine evaluation of hemp-seed
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oil quality as a fast and reliable method of verification of ω-6 to ω-3 ratio and origin of the oil.
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Key words: hemp-seed oil, fatty acids, NMR, GC, FT-IR
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1.
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Cannabis sativa is an annual plant belonging to the family of Cannabaceae. It has been used as a source of food,
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medicines, oil and fiber since ancient times . Today Cannabis species are known primarily because they contain
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a psychoactive compound THC. Two main varieties should be distinguished: the marijuana and industrial hemp.
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The concentration of THC in the former type is between 1-20 %, whereas in the latter the concentration does not
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exceed 0.2 % [1]. In many Western countries the cultivation of Cannabis indica became illegal in the 20th
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century. Industrial hemp with a low THC content has no psychoactive effects. Nowadays both types of Cannabis
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are topics of social discussion and the researchers’ attention: the marijuana-type because of its possible medical
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use and industrial type as a source of oil and fiber.
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Hemp-seed oil, the main food product obtained from hemp, is most frequently produced by cold pressing of
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seeds. Hemp-seed oil exhibits health benefits due to its high content of essential fatty acids. It is composed in
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approximately 80 % of polyunsaturated fatty acids (PUFA) [2], mainly of ω-6 linoleic acid (LA) and ω-3 alpha-
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linolenic acid (ALA). The LA:ALA ratio is ca. 3:1, as it is recommended in healthy diet [2]. Hemp oil also
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contains γ-linolenic acid (GLA) (which is found only in certain plant seed oils) at the levels of up to 5 % [3]. The
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fat-soluble vitamins D and E are other important constituents of hemp-seed oil. Hence, hemp oil has an excellent
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nutritional value and it seems that the high PUFA content and LA:ALA ratio are the main reasons for its health
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benefits.
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Gas chromatography (GC) is a routine method in fatty acids profiling [3]. Nevertheless, spectroscopic methods
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may also be used in oil examinations. In the last years the role of NMR in the analysis of oils has increased. The
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advantages of NMR over chromatographic methods are: short analysis time and small solvent volumes. Also, in
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NMR no special sample preparation is required, the method is not destructive and permits fast quantitative
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analysis of oils. The 1H NMR method is most commonly used in oil studies [4]. The method allows one to
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determine the LA:ALA ratio which is crucial for hemp-seed oil health benefits. Popescu et al. described the
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possibility of discrimination of vegetable oils using NMR [5].
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Different fatty oils (305 samples) including pharmaceutical lipids were examined by both the classical and the
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1
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and oil matrix on line broadening and the chemical shift of the carboxyl group signal were discussed [6].
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The automated analysis of the fatty acid composition was done by low-field ¹H NMR spectroscopy; this
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technique is promising for high-throughput screening. To demonstrate the applicability of this method, the fatty
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acid composition of extra virgin olive oils from various Spanish olive varieties was determined [7].
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Variations in the LA:ALA ratio may be caused by differences in origin and composition of seeds or the presence
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of adulteration like the addition of cheaper oils [8]. Standard, time consuming methods, like GC or high-
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performance liquid chromatography (HPLC) may be replaced by the fast Fourier-transformed infrared
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spectroscopy technique. The analysis of near infrared (NIR) spectrum (from about 700 nm to 2500 nm), which is
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a “fingerprint” of the examined sample, permits good discrimination between oils. Spectroscopic methods NMR
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and FT-IR have a potential in fast screening of hemp oil quality.
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Quality control becomes especially important at the present time when hemp-seed oil is rediscovered and widely
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used as part of the diet or a dietary supplement.
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2.
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Introduction
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H NMR method. Data obtained by the two methods were in good agreement. Furthermore, the effects of solvent
Material and methods:
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2.1. Materials Six samples of hemp-seed oil (HO1, HO2, HO3, HO4, HO5 and HO6) were purchased on the Polish market
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(two samples at the pharmacy, one sample at a health food store and three at a grocery store). The oils were
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produced by cold pressing of hemp seeds, during the period of validity the oils were stored at a temperature of 2-
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8 °C, according to manufacturer’s recommendations. Two samples of oil were manufactured in Poland, one
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sample in the Czech Republic, one sample in Canada and one sample in New Zealand (Table 1).
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Table 1. Description of tested hemp-seed oils (country of origin and plant species).
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2.2. Spectroscopic analysis
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2.2.1.
NMR studies
For NMR studies, each sample was prepared by dissolving 200 µL of oil in 800 µL of CDCl3. The solvent was
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purchased from ARMAR Chemicals. 1H spectra for CDCl3 solutions were recorded at 300 MHz, , on a Varian
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VNMRS-300 spectrometer using standard Varian pulse program. 2.2.2.
FT-IR analysis
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The FT-IR spectra were recorded from KBr pellets in the 400–4000 cm−1 range using a Spectrum 1000
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spectrometer (Perkin Elmer, Cambridge, UK) with spectral resolution of 4 cm−1 and 32 scans. They were
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processed using GRAMS/AI 8.0 software (Thermo Scientific 2008).
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2.2.3.
GC protocol
Fatty acids profile was analyzed by GC with a capillary column and flame-ionization detection. Three parallel
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samples of 100 µL were trans-esterified. They were hydrolyzed without prior lipid extraction by heating with 2.5
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mL sodium methoxide in methanol (0.5 mol/l) at 80 °C. Fatty acids were converted to methyl esters by heating
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with 2.5 mL of 14 % boron trifluoride-methanol reagent at 80 °C for 3 min. Fatty acids methyl esters (FAME)
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were isolated with hexane (2×0.5 mL) after adding 1.0 mL of the saturated sodium chloride solution. Organic
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extracts were dried with anhydrous sodium sulphate and evaporated to dryness under a stream of nitrogen.
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FAME were diluted in 20 µL of hexane and stored at −20 °C. Separations of FAME were performed on a BPX
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70 capillary column (60 m×0.25 mm i.d., film thickness: 0.2 µm, SGE) with helium as carrier gas. The injector
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was heated to 250 °C and the detector to 270 °C. The initial oven temperature was 140 °C/min, thereafter
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increased by 20 °C/min to 200 °C and held for 20 min and increased by 5 °C/min to 220 °C held for 25 min.
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FAME standards (Supelco 37 Component FAME Mix) and CLA FAME reference standard (Nu-Chek-Prep,
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INC., USA) were used to identify the fatty acids present in the samples.
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3.
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3.1.
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Techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) have been
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generally used in the studies of edible oils. However, they are expensive and time consuming. Infrared
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spectroscopy (IR) and NMR are fast and effective methods. FT-IR spectroscopy is a fast, non-destructive
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technique that requires minimum sample preparation and has become a powerful analytical tool in food research.
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Results
FT-IR experiment
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It is particularly important in the study of edible oils and fats and can be considered as a “green analytical method” [9].
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The FT-IR spectra in the mid-infrared region of the six oil samples were used to identify functional groups and
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the bands corresponding to various stretching and bending vibrations. Fig. 1 shows the IR spectrum of the hemp-
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seed oil sample 1 as an example. The spectrum has a weak band associated with the overtone of the glyceride
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ester carbonyl absorption at 3474 cm-1. The band at 3010 cm-1 is a result of stretching vibrations of the olefinic
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CH double bond. This suggests the presence of a high amount of polyunsaturated acyl groups in the oil. The two
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bands at 2925 and 2854 cm-1 represent stretching vibrations of the methylene group. The esters have two
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characteristic strong absorption bands around 1750–1730 cm-1 and at 1300–1000 cm-1 [10]. The band at 1746
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cm-1 (e) comes from the ester carbonyl (C=O) functional group of triglycerides, and the weak band at 1653 cm-1
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(f) from C=C unsaturated acyl groups. The bands at 1400-1200 cm-1 correspond to bending vibrations of the CH2
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and CH3 aliphatic groups (g). The bands in the region 1120-1093 cm-1 (h) are due to stretching vibrations of C–O
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ester groups (antisymmetric axial stretching and asymmetric axial stretching). The bands at 722 cm-1 (i) arise
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from overlapping of the CH2 rocking vibrations and out-of-plane CH vibrations.
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Fig. 1. FT-IR spectrum of HO1; the region at 1500-500 cm-1 is considered as a “fingerprint”.
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FT-IR spectroscopy can be successfully used to monitor the adulteration of hemp-seed oil with much cheaper
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oils. At first sight, the FT-IR spectra of various vegetable oils are similar. However, they can be distinguished by
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the analysis of frequencies especially in the region at 1500-500 cm-1, considered as a “fingerprint region”. The
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differences between the HO1-HO6 were small but visible, mainly in peak intensities and occurred only in limited
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regions of the spectra. There is a difference, best seen at 1440-1460 cm-1, that allows distinguishing HO3 and
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HO4 from other samples (HO1, HO2, HO5 and HO6) (Fig. 2). The band at 1465 cm-1 for HO1, 2, 5 and 6 is split
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into two, while for HO4 and HO3 it is not. The band at 1465 cm-1 represents bending vibrations of the CH2 and
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CH3 aliphatic groups.
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Rohman et al. described the analysis of virgin coconut oil mixtures with olive oil and palm oil [11]. The
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adulteration of coconut oil with these two oils caused changes in the shape of the bands at the fingerprint region.
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Thus we can suspect that the differences of the band’s shape at 1465 cm-1 for hemp-seed oils may be induced by
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another composition of the hemp-seed oil sample.
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According to manufacturer’s information HO6 is obtained from Cannabis indica. The analysis of the fingerprint
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region for this sample of oil shows another composition of hemp oil. However, the band at 1456 cm-1 is split into
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two, like in HO1, HO2 and HO5, the general shape of the bands in the fingerprint region is different. Fingerprint
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analysis allowed us to extract 3 samples of oil with similar composition (HO1, HO2, HO5) and 3 samples with
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distinct composition (HO3, HO4, HO6).
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Fig. 2. Fingerprint region at FT-IR spectra of HO1-HO6.
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3.2.
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The most popular method of hemp oil receiving is cold-pressing of the seeds. By this method, 60–80 % of oil
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can be extracted from the seed material, depending on the settings of the screw press. To define the fatty acid
GC analysis
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for six hemp-seed oil samples are given in Table 2 as means ±SD, analyzed individually in triplicate.
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The analysis of fatty acid composition by GC shows that HO4 and HO3 differ from the rest of the examined
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hemp oils. HO3, HO4 and HO6 have the highest level of GLA (3.57 – 4.51 %) as compared with HO1, HO2 and
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HO5 (GLA: 0.46 – 0.63 %). However, HO3 and HO4 may be easily distinguished from HO6 by calculations of
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LA:ALA ratio. HO3 and HO4 are two hemp-seed oil samples that have the lowest and the highest LA:ALA
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ratio. Thus, the differences in the fatty acid composition may be proved by GC analysis.
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Table 2. Mean values and standard deviations of the fatty acid compositions (%) of the hemp-seed oil.
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The contents of palmitic and stearic acids in hemp-seed oils ranged from 6.22 to 7.24 and 2.75 to 3.42 %,
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respectively. The oils contain a high degree of unsaturation 88.90 – 89.42 %. The predominant fatty acids are
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linoleic acid (52.13 – 56.80 %) and α-linoleic acid (14.80-19.10 %). The ratio between ω-6 and ω-3 fatty acids is
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between 2.73 and 3.38.
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The contents of palmitic and stearic fatty acids in the examined hemp-seed oils were comparable with those
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described earlier (6.23 – 8.27 and 2.19 – 3.22 %, respectively) [12]. The level of the main fatty acid LA in HO1,
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HO2, HO4, HO5 and HO6 was similar to that reported earlier (56.10 % on average) [13]. However, the content
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of LA in HO3 is significantly lower. Additionally, the ALA value in HO3 is the highest, which affects the
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LA:ALA ratio, reducing it to 2.73.The content of ALA in HO1–HO6 remains in agreement with the values given
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by Anwar et al. (16.85 – 20.00 %) [12].
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In 2009, The European Food Safety Authority (EFSA) published its recommendations for PUFA:
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• an omega-3 fatty acid intake of 2 g/day alpha-linolenic acid (ALA) and 250 mg/day long-chain omega-3 fatty
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acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) • an omega-6 fatty acid intake of 10 g/day linoleic acid (LA).
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The World Health Organization recommends an omega-6 fatty acid intake of 5–8 % of energy and an omega-3
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fatty acid intake of 1–2 % of energy [14].
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In addition to the fatty acids found in most of the commonly used edible oils, hemp seed oil contains remarkable
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amounts of GLA which appears only in certain plant seeds. GC analysis shows that hemp-seed oil contains 0.46-
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4.51 % of γ-linolenic acid. According to Matthäus and Brühl , hemp cultivars contain varying quantities of GLA,
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ranging from 0.34 to 6.80 % [15]. Surprisingly, the content of GLA in HO3, HO4 and HO6 was significantly
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higher (up to 4.51 %). These three oils are a rich source of GLA. Others have significantly smaller amounts of
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GLA (up to 0.63 %). Higher amounts of this fatty acid are present in evening primrose (Oenotheramacrocarpa)
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and borage (Borago officinalis) seeds. Both oils are used in dietary supplements and cosmetics. Certain studies
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suggest that supplementation with GLA, and particularly combinations of GLA with (ω-3) long chain-PUFAs
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have great potential to exert anti-inflammatory effects and may improve symptoms of several inflammatory
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diseases [16]. Instead of borage oil which is rich in GLA but totally lacking ω-3 PUFAs (polyunsaturated fatty
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acids), hemp oil may be considered as a better dietary source of GLA. The samples HO1-HO6 were produced by
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cold pressing of hemp-seed. The profile of fatty acids is related to the method of oil receiving. In 2012 Porto et
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al. described fatty acid composition of hemp-seed oil extracted by supercritical carbon dioxide [17]. It is
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characterized by a higher amount of PUFA: LA (59.23-59.77 %), ALA (17.95-18.20 %) and GLA (3.42-3.58 %)
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and the degree of unsaturation up to 92 %. The edible hemp-seed oil manufactured by extraction using
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supercritical carbon dioxide is so far unavailable on the Polish market.
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3.3.
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The main information given by NMR spectroscopy is chemical shift and intensity of the signals. NMR is
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commonly used in qualitative and quantitative analysis of natural compounds and plant extracts as well as of
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edible oils [6].
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classical AV method, based on the analysis of 420 edible oil samples. Good agreement between both methods
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was obtained. Numerous studies showed the advantages of NMR for the edible oil analysis: NMR spectroscopy
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is reliable, fast and can analyze multiple components with only one measurement. It was shown that NMR is a
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powerful tool to characterize edible oils in many different ways: 1H NMR spectra allow the reliable quantitation
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of the fatty acid content with respect to monounsaturated fatty acids (MUFA), polyunsaturated fatty acids
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(PUFA), and saturated fatty acids (SFA). This process is so straightforward that even a completely automatic
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processing and analysis of the spectra is possible.
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NMR analysis
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H NMR was applied for determination of free fatty acids [18]. The NMR method was compared with the
H spectra were recorded for all hemp-seed oil samples in CDCl3, the spectrum of HO1 is illustrated in Fig. 3a.
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Fig. 3. (a) 1H NMR spectrum of hemp-seed oil (HO1) in CDCl3 and (b) superimposed signals in 1H NMR spectra
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of HO3 and HO4 in CDCl3.
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Ten multiplet resonances of the vegetable oil are visible in the 1H NMR spectrum. The area of these signals is
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proportional to the number of each kind of hydrogen atoms present in the sample. The signals in the NMR
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spectra of vegetable oil were assigned previously [19]. Signal 1 results from the overlapping of the triplets of
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methyl proton signals of all acyl groups, except those of linolenic acid. The resonance 2 is a triplet assigned to
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the methyl protons of linolenic acid. In the spectra of HO1-HO6 the area of resonance 2 varies. The most
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significant difference is observed for HO3 and HO4 (Fig. 3b).
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Multiplet number 7 is formed by the overlapping of signals of methylene protons of linoleic and linolenic acyl
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groups. 1H spectra of six hemp-seed oils were compared and significant differences in shape of the signals are
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visible (Fig. 3b).
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Popescu et al. gave the formula that allows one to determine the composition of ALA and LA by analyzing the
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area of these signals [5].
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(ଶ)
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( = ܣܮܣଶ)ା(ଵ)
= ܣܮ
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ALA and LA compositions obtained from NMR and GC experiments are shown in Fig. 4.
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4.
Disscusion
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ACCEPTED MANUSCRIPT It is known that a balance between omega-6 and omega-3 acids is important for health [20]. Therefore, it is
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relevant to evaluate the ratio of ω-6 to ω-3 fatty acids. GC analysis allows one to establish this ratio, however,
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the NMR technique can also be recommended. The LA:ALA ratio for each hemp-oil is similar when obtained
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either from GC or NMR experiments (Fig 4).
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Fig. 4. Correlation of LA:ALA ratio calculated from GC and NMR experiment (the coefficient of determination
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is 0.98).
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In the past three decades the intake of omega-6 fatty acid increased and the omega-3 fatty acid decreased,
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resulting in a large increase in the ω -6: ω -3 ratio from 1:1 in the past to 20:1 today. It is therefore essential to
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return to a balanced dietary ω-6:ω-3 ratio. Analytical methods are needed to determine the LA:ALA ratio.
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Besides GC experiment, 1H NMR in solution may be considered as a fast and effective method. The technique
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has the potential to substitute a wide range of classical analysis methods in the edible oil industry. The NMR
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method might be useful for pharmaceutical industry and even introduced in Pharmacopeia.
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Conclusions
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Gas chromatography is a routine method in fatty acids profiling. However, two spectral methods: NMR and FT-
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IR are also useful in routine examination of edible oils. NMR spectroscopy surpasses the chromatographic
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methods (GC) for vegetable oils as concerns analysis time and sample preparation.
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The differences of fatty acid composition caused by variability of Cannabis sativa plants or by adulteration of
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hemp-seed oil with other oils should be supervised. Spectral methods described above have a potential in fast
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screening of hemp oil quality.
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Acknowledgements: This work was supported by the Medical University of Warsaw (FW28/PM1/16). The
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authors would like to thank Ms. Agnieszka Białek (Ph.D.) for performing the GC measurements and Ms. Joanna
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Kolmas (Ph.D.) for her assistance in FT-IR experiments.
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Conflicts of Interest: The authors declare no conflict of interest.
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References:
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[1] EFSA, Scientific Opinion on the safety of hemp (Cannabis genus) for use as animal feed, EFSA J. 9 (2011)
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the Hempseed Oil from Four Cannabis sativa L. Cultivars, J. Diet. Suppl. 12 (2015) 1-10.
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P and 1H NMR
ACCEPTED MANUSCRIPT Table 1. Description of tested hemp-seed oils (country of origin and plant species) Sample Country of origin Source HO1 Czech Republic Cannabis sativa HO2 Poland Cannabis sativa HO3 Canada Cannabis sativa HO4 France Cannabis sativa HO5 Poland Cannabis sativa HO6 New Zealand Cannabis indica
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Table 2. Mean values and standard deviations of the fatty acid compositions (%) of the hempseed oil
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Fatty acid composition (g/100g fatty acids) by GC, * LA, ** GLA, *** ALA Fatty acid HO1 HO2 HO3 HO4 HO5 C16:0 7.24±0.60 7.07±0.15 6.22±0.19 6.53±0.12 7.16±0.04 C16:1 0.11±0.01 0.09±0.01 0.10±0.01 0.10±0.02 0.08±0.01 C17:0 0.07±0.01 0.06±0.03 0.06±0.01 0.05±0.01 0.04±0.01 C18:0 3.11±0.09 3.29±0.08 3.05±0.05 3.11±0.21 3.42±0.07 C18:1 12.60±0.06 11.44±0.14 10.20±0.37 13.05±0.18 12.31±0.02 C18:2* 56.80±0.29 56.63±0.21 52.13±0.01 55.17±0.33 56.58±0.14 C18:3** 0.49±0.04 0.46±0.01 0.63±0.09 4.51±0.16 3.57±0.03 C18:3*** 16.80±0.30 18.76±0.05 19.10±0.08 14.80±0.03 17.47±0.04 C20:0 0.23±0.03 0.16±0.05 0.20±0.06 1.77±0.11 0.97±0.02 LA:ALA 3.38±0.05 3.02±0.02 2.73±0.04 3.73±0.03 3.24±0.02 ratio
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HO6 6.41±0.11 0.10±0.02 0.04±0.01 2.75±0.14 8.42±0.04 55.22±0.16 4.42±0.18 18.94±0.30 1.49±0.08 2.92±0.05
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Fig 1. FT-IR spectrum of HO1; the region at 1500-500 cm-1 is considered as a “fingerprint”.
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Fig. 2. Fingerprint region at FT-IR spectra of HO1-HO6.
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Fig. 3. (a) 1H NMR spectrum of hemp-seed oil (HO1) in CDCl3 and (b) superimposed signals in 1H NMR spectra of HO3 and HO4 in CDCl3.
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3.5 3.3 3.1 2.9 2.7 2.5 2.5
3 3.5 LA:ALA ratio GC
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Fig. 4. Correlation of LA:ALA ratio calculated from GC and NMR experiment (the coefficient of determination is 0.98).
ACCEPTED MANUSCRIPT Highlights •
FT-IR spectroscopy is a fast technique to monitor variability of hemp oil composition.
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Spectroscopic methods may be used in a routine control of oil quality.
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H qNMR is proposed as a new and fast method to determine ω-6 to ω-3 ratio.
S0022-2860(18)31141-4
DOI:
10.1016/j.molstruc.2018.09.057
Reference:
MOLSTR 25691
To appear in:
Journal of Molecular Structure
Received Date: 19 July 2018 Revised Date:
18 September 2018
Accepted Date: 21 September 2018
Please cite this article as: Paweł. Siudem, I. Wawer, K. Paradowska, Rapid evaluation of edible hemp oil quality using NMR and FT-IR spectroscopy, Journal of Molecular Structure (2018), doi: https:// doi.org/10.1016/j.molstruc.2018.09.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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𝝎−𝟔 ≈𝟑 𝝎−𝟑
ACCEPTED MANUSCRIPT Rapid evaluation of edible hemp oil quality using NMR and FT-IR spectroscopy
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Paweł Siudem, Iwona Wawer, Katarzyna Paradowska*
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Department of Physical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, Banacha 1, PL-02097
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Warsaw, Poland
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Corresponding Author: Paradowska Katarzyna
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*Corresponding Author's Institution: Medical University of Warsaw, Faculty of Pharmacy, Phone/Fax.: +48 22
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5720 951,
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Corresponding Author’s email: [email protected]
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ACCEPTED MANUSCRIPT Abstract:
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Since Cannabis species include psychoactive varieties containing high levels of ∆9-tetrahydrocannabinol (THC),
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psychotropic properties could be wrongly attributed to hemp-seed oil obtained from Cannabis sativa. Hemp oil
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does not exert any psychotic effect; on the contrary, it may provide significant health benefits, because it has an
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optimum proportion of ω-6 to ω-3 fatty acids (3:1). Six samples of cold pressed hemp-seed oils were studied by
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nuclear magnetic resonance (NMR) and Fourier-transformed infrared spectroscopy (FT-IR). These methods
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were applied to evaluate the ratio of ω-6 to ω-3 fatty acids and to control the quality of hemp oils. FT-IR method
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allowed us to distinguish three oils with the highest level of γ-linolenic acid. Additionally, NMR spectroscopy
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was applied to determine the ω-6 to ω-3 ratio. The outcomes of the NMR experiment are in agreement with GC
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outcomes measures. The results indicate that NMR and FT-IR may be used in routine evaluation of hemp-seed
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oil quality as a fast and reliable method of verification of ω-6 to ω-3 ratio and origin of the oil.
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Key words: hemp-seed oil, fatty acids, NMR, GC, FT-IR
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1.
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Cannabis sativa is an annual plant belonging to the family of Cannabaceae. It has been used as a source of food,
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medicines, oil and fiber since ancient times . Today Cannabis species are known primarily because they contain
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a psychoactive compound THC. Two main varieties should be distinguished: the marijuana and industrial hemp.
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The concentration of THC in the former type is between 1-20 %, whereas in the latter the concentration does not
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exceed 0.2 % [1]. In many Western countries the cultivation of Cannabis indica became illegal in the 20th
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century. Industrial hemp with a low THC content has no psychoactive effects. Nowadays both types of Cannabis
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are topics of social discussion and the researchers’ attention: the marijuana-type because of its possible medical
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use and industrial type as a source of oil and fiber.
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Hemp-seed oil, the main food product obtained from hemp, is most frequently produced by cold pressing of
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seeds. Hemp-seed oil exhibits health benefits due to its high content of essential fatty acids. It is composed in
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approximately 80 % of polyunsaturated fatty acids (PUFA) [2], mainly of ω-6 linoleic acid (LA) and ω-3 alpha-
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linolenic acid (ALA). The LA:ALA ratio is ca. 3:1, as it is recommended in healthy diet [2]. Hemp oil also
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contains γ-linolenic acid (GLA) (which is found only in certain plant seed oils) at the levels of up to 5 % [3]. The
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fat-soluble vitamins D and E are other important constituents of hemp-seed oil. Hence, hemp oil has an excellent
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nutritional value and it seems that the high PUFA content and LA:ALA ratio are the main reasons for its health
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benefits.
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Gas chromatography (GC) is a routine method in fatty acids profiling [3]. Nevertheless, spectroscopic methods
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may also be used in oil examinations. In the last years the role of NMR in the analysis of oils has increased. The
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advantages of NMR over chromatographic methods are: short analysis time and small solvent volumes. Also, in
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NMR no special sample preparation is required, the method is not destructive and permits fast quantitative
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analysis of oils. The 1H NMR method is most commonly used in oil studies [4]. The method allows one to
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determine the LA:ALA ratio which is crucial for hemp-seed oil health benefits. Popescu et al. described the
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possibility of discrimination of vegetable oils using NMR [5].
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Different fatty oils (305 samples) including pharmaceutical lipids were examined by both the classical and the
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1
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and oil matrix on line broadening and the chemical shift of the carboxyl group signal were discussed [6].
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The automated analysis of the fatty acid composition was done by low-field ¹H NMR spectroscopy; this
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technique is promising for high-throughput screening. To demonstrate the applicability of this method, the fatty
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acid composition of extra virgin olive oils from various Spanish olive varieties was determined [7].
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Variations in the LA:ALA ratio may be caused by differences in origin and composition of seeds or the presence
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of adulteration like the addition of cheaper oils [8]. Standard, time consuming methods, like GC or high-
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performance liquid chromatography (HPLC) may be replaced by the fast Fourier-transformed infrared
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spectroscopy technique. The analysis of near infrared (NIR) spectrum (from about 700 nm to 2500 nm), which is
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a “fingerprint” of the examined sample, permits good discrimination between oils. Spectroscopic methods NMR
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and FT-IR have a potential in fast screening of hemp oil quality.
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Quality control becomes especially important at the present time when hemp-seed oil is rediscovered and widely
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used as part of the diet or a dietary supplement.
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2.
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Introduction
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H NMR method. Data obtained by the two methods were in good agreement. Furthermore, the effects of solvent
Material and methods:
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2.1. Materials Six samples of hemp-seed oil (HO1, HO2, HO3, HO4, HO5 and HO6) were purchased on the Polish market
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(two samples at the pharmacy, one sample at a health food store and three at a grocery store). The oils were
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produced by cold pressing of hemp seeds, during the period of validity the oils were stored at a temperature of 2-
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8 °C, according to manufacturer’s recommendations. Two samples of oil were manufactured in Poland, one
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sample in the Czech Republic, one sample in Canada and one sample in New Zealand (Table 1).
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Table 1. Description of tested hemp-seed oils (country of origin and plant species).
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2.2. Spectroscopic analysis
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2.2.1.
NMR studies
For NMR studies, each sample was prepared by dissolving 200 µL of oil in 800 µL of CDCl3. The solvent was
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purchased from ARMAR Chemicals. 1H spectra for CDCl3 solutions were recorded at 300 MHz, , on a Varian
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VNMRS-300 spectrometer using standard Varian pulse program. 2.2.2.
FT-IR analysis
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The FT-IR spectra were recorded from KBr pellets in the 400–4000 cm−1 range using a Spectrum 1000
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spectrometer (Perkin Elmer, Cambridge, UK) with spectral resolution of 4 cm−1 and 32 scans. They were
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processed using GRAMS/AI 8.0 software (Thermo Scientific 2008).
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2.2.3.
GC protocol
Fatty acids profile was analyzed by GC with a capillary column and flame-ionization detection. Three parallel
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samples of 100 µL were trans-esterified. They were hydrolyzed without prior lipid extraction by heating with 2.5
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mL sodium methoxide in methanol (0.5 mol/l) at 80 °C. Fatty acids were converted to methyl esters by heating
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with 2.5 mL of 14 % boron trifluoride-methanol reagent at 80 °C for 3 min. Fatty acids methyl esters (FAME)
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were isolated with hexane (2×0.5 mL) after adding 1.0 mL of the saturated sodium chloride solution. Organic
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extracts were dried with anhydrous sodium sulphate and evaporated to dryness under a stream of nitrogen.
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FAME were diluted in 20 µL of hexane and stored at −20 °C. Separations of FAME were performed on a BPX
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70 capillary column (60 m×0.25 mm i.d., film thickness: 0.2 µm, SGE) with helium as carrier gas. The injector
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was heated to 250 °C and the detector to 270 °C. The initial oven temperature was 140 °C/min, thereafter
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increased by 20 °C/min to 200 °C and held for 20 min and increased by 5 °C/min to 220 °C held for 25 min.
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FAME standards (Supelco 37 Component FAME Mix) and CLA FAME reference standard (Nu-Chek-Prep,
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INC., USA) were used to identify the fatty acids present in the samples.
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3.
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3.1.
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Techniques such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) have been
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generally used in the studies of edible oils. However, they are expensive and time consuming. Infrared
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spectroscopy (IR) and NMR are fast and effective methods. FT-IR spectroscopy is a fast, non-destructive
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technique that requires minimum sample preparation and has become a powerful analytical tool in food research.
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Results
FT-IR experiment
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It is particularly important in the study of edible oils and fats and can be considered as a “green analytical method” [9].
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The FT-IR spectra in the mid-infrared region of the six oil samples were used to identify functional groups and
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the bands corresponding to various stretching and bending vibrations. Fig. 1 shows the IR spectrum of the hemp-
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seed oil sample 1 as an example. The spectrum has a weak band associated with the overtone of the glyceride
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ester carbonyl absorption at 3474 cm-1. The band at 3010 cm-1 is a result of stretching vibrations of the olefinic
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CH double bond. This suggests the presence of a high amount of polyunsaturated acyl groups in the oil. The two
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bands at 2925 and 2854 cm-1 represent stretching vibrations of the methylene group. The esters have two
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characteristic strong absorption bands around 1750–1730 cm-1 and at 1300–1000 cm-1 [10]. The band at 1746
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cm-1 (e) comes from the ester carbonyl (C=O) functional group of triglycerides, and the weak band at 1653 cm-1
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(f) from C=C unsaturated acyl groups. The bands at 1400-1200 cm-1 correspond to bending vibrations of the CH2
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and CH3 aliphatic groups (g). The bands in the region 1120-1093 cm-1 (h) are due to stretching vibrations of C–O
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ester groups (antisymmetric axial stretching and asymmetric axial stretching). The bands at 722 cm-1 (i) arise
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from overlapping of the CH2 rocking vibrations and out-of-plane CH vibrations.
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Fig. 1. FT-IR spectrum of HO1; the region at 1500-500 cm-1 is considered as a “fingerprint”.
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FT-IR spectroscopy can be successfully used to monitor the adulteration of hemp-seed oil with much cheaper
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oils. At first sight, the FT-IR spectra of various vegetable oils are similar. However, they can be distinguished by
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the analysis of frequencies especially in the region at 1500-500 cm-1, considered as a “fingerprint region”. The
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differences between the HO1-HO6 were small but visible, mainly in peak intensities and occurred only in limited
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regions of the spectra. There is a difference, best seen at 1440-1460 cm-1, that allows distinguishing HO3 and
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HO4 from other samples (HO1, HO2, HO5 and HO6) (Fig. 2). The band at 1465 cm-1 for HO1, 2, 5 and 6 is split
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into two, while for HO4 and HO3 it is not. The band at 1465 cm-1 represents bending vibrations of the CH2 and
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CH3 aliphatic groups.
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Rohman et al. described the analysis of virgin coconut oil mixtures with olive oil and palm oil [11]. The
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adulteration of coconut oil with these two oils caused changes in the shape of the bands at the fingerprint region.
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Thus we can suspect that the differences of the band’s shape at 1465 cm-1 for hemp-seed oils may be induced by
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another composition of the hemp-seed oil sample.
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According to manufacturer’s information HO6 is obtained from Cannabis indica. The analysis of the fingerprint
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region for this sample of oil shows another composition of hemp oil. However, the band at 1456 cm-1 is split into
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two, like in HO1, HO2 and HO5, the general shape of the bands in the fingerprint region is different. Fingerprint
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analysis allowed us to extract 3 samples of oil with similar composition (HO1, HO2, HO5) and 3 samples with
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distinct composition (HO3, HO4, HO6).
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Fig. 2. Fingerprint region at FT-IR spectra of HO1-HO6.
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3.2.
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The most popular method of hemp oil receiving is cold-pressing of the seeds. By this method, 60–80 % of oil
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can be extracted from the seed material, depending on the settings of the screw press. To define the fatty acid
GC analysis
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for six hemp-seed oil samples are given in Table 2 as means ±SD, analyzed individually in triplicate.
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The analysis of fatty acid composition by GC shows that HO4 and HO3 differ from the rest of the examined
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hemp oils. HO3, HO4 and HO6 have the highest level of GLA (3.57 – 4.51 %) as compared with HO1, HO2 and
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HO5 (GLA: 0.46 – 0.63 %). However, HO3 and HO4 may be easily distinguished from HO6 by calculations of
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LA:ALA ratio. HO3 and HO4 are two hemp-seed oil samples that have the lowest and the highest LA:ALA
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ratio. Thus, the differences in the fatty acid composition may be proved by GC analysis.
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Table 2. Mean values and standard deviations of the fatty acid compositions (%) of the hemp-seed oil.
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The contents of palmitic and stearic acids in hemp-seed oils ranged from 6.22 to 7.24 and 2.75 to 3.42 %,
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respectively. The oils contain a high degree of unsaturation 88.90 – 89.42 %. The predominant fatty acids are
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linoleic acid (52.13 – 56.80 %) and α-linoleic acid (14.80-19.10 %). The ratio between ω-6 and ω-3 fatty acids is
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between 2.73 and 3.38.
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The contents of palmitic and stearic fatty acids in the examined hemp-seed oils were comparable with those
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described earlier (6.23 – 8.27 and 2.19 – 3.22 %, respectively) [12]. The level of the main fatty acid LA in HO1,
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HO2, HO4, HO5 and HO6 was similar to that reported earlier (56.10 % on average) [13]. However, the content
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of LA in HO3 is significantly lower. Additionally, the ALA value in HO3 is the highest, which affects the
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LA:ALA ratio, reducing it to 2.73.The content of ALA in HO1–HO6 remains in agreement with the values given
156
by Anwar et al. (16.85 – 20.00 %) [12].
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In 2009, The European Food Safety Authority (EFSA) published its recommendations for PUFA:
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• an omega-3 fatty acid intake of 2 g/day alpha-linolenic acid (ALA) and 250 mg/day long-chain omega-3 fatty
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acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) • an omega-6 fatty acid intake of 10 g/day linoleic acid (LA).
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The World Health Organization recommends an omega-6 fatty acid intake of 5–8 % of energy and an omega-3
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fatty acid intake of 1–2 % of energy [14].
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In addition to the fatty acids found in most of the commonly used edible oils, hemp seed oil contains remarkable
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amounts of GLA which appears only in certain plant seeds. GC analysis shows that hemp-seed oil contains 0.46-
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4.51 % of γ-linolenic acid. According to Matthäus and Brühl , hemp cultivars contain varying quantities of GLA,
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ranging from 0.34 to 6.80 % [15]. Surprisingly, the content of GLA in HO3, HO4 and HO6 was significantly
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higher (up to 4.51 %). These three oils are a rich source of GLA. Others have significantly smaller amounts of
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GLA (up to 0.63 %). Higher amounts of this fatty acid are present in evening primrose (Oenotheramacrocarpa)
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and borage (Borago officinalis) seeds. Both oils are used in dietary supplements and cosmetics. Certain studies
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suggest that supplementation with GLA, and particularly combinations of GLA with (ω-3) long chain-PUFAs
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have great potential to exert anti-inflammatory effects and may improve symptoms of several inflammatory
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diseases [16]. Instead of borage oil which is rich in GLA but totally lacking ω-3 PUFAs (polyunsaturated fatty
173
acids), hemp oil may be considered as a better dietary source of GLA. The samples HO1-HO6 were produced by
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cold pressing of hemp-seed. The profile of fatty acids is related to the method of oil receiving. In 2012 Porto et
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al. described fatty acid composition of hemp-seed oil extracted by supercritical carbon dioxide [17]. It is
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characterized by a higher amount of PUFA: LA (59.23-59.77 %), ALA (17.95-18.20 %) and GLA (3.42-3.58 %)
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and the degree of unsaturation up to 92 %. The edible hemp-seed oil manufactured by extraction using
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supercritical carbon dioxide is so far unavailable on the Polish market.
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3.3.
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The main information given by NMR spectroscopy is chemical shift and intensity of the signals. NMR is
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commonly used in qualitative and quantitative analysis of natural compounds and plant extracts as well as of
182
edible oils [6].
183
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classical AV method, based on the analysis of 420 edible oil samples. Good agreement between both methods
185
was obtained. Numerous studies showed the advantages of NMR for the edible oil analysis: NMR spectroscopy
186
is reliable, fast and can analyze multiple components with only one measurement. It was shown that NMR is a
187
powerful tool to characterize edible oils in many different ways: 1H NMR spectra allow the reliable quantitation
188
of the fatty acid content with respect to monounsaturated fatty acids (MUFA), polyunsaturated fatty acids
189
(PUFA), and saturated fatty acids (SFA). This process is so straightforward that even a completely automatic
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processing and analysis of the spectra is possible.
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H NMR was applied for determination of free fatty acids [18]. The NMR method was compared with the
H spectra were recorded for all hemp-seed oil samples in CDCl3, the spectrum of HO1 is illustrated in Fig. 3a.
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Fig. 3. (a) 1H NMR spectrum of hemp-seed oil (HO1) in CDCl3 and (b) superimposed signals in 1H NMR spectra
194
of HO3 and HO4 in CDCl3.
195
Ten multiplet resonances of the vegetable oil are visible in the 1H NMR spectrum. The area of these signals is
196
proportional to the number of each kind of hydrogen atoms present in the sample. The signals in the NMR
197
spectra of vegetable oil were assigned previously [19]. Signal 1 results from the overlapping of the triplets of
198
methyl proton signals of all acyl groups, except those of linolenic acid. The resonance 2 is a triplet assigned to
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the methyl protons of linolenic acid. In the spectra of HO1-HO6 the area of resonance 2 varies. The most
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significant difference is observed for HO3 and HO4 (Fig. 3b).
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Multiplet number 7 is formed by the overlapping of signals of methylene protons of linoleic and linolenic acyl
202
groups. 1H spectra of six hemp-seed oils were compared and significant differences in shape of the signals are
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visible (Fig. 3b).
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Popescu et al. gave the formula that allows one to determine the composition of ALA and LA by analyzing the
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area of these signals [5].
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(ଶ)
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( = ܣܮܣଶ)ା(ଵ)
= ܣܮ
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ALA and LA compositions obtained from NMR and GC experiments are shown in Fig. 4.
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4.
Disscusion
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ACCEPTED MANUSCRIPT It is known that a balance between omega-6 and omega-3 acids is important for health [20]. Therefore, it is
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relevant to evaluate the ratio of ω-6 to ω-3 fatty acids. GC analysis allows one to establish this ratio, however,
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the NMR technique can also be recommended. The LA:ALA ratio for each hemp-oil is similar when obtained
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either from GC or NMR experiments (Fig 4).
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Fig. 4. Correlation of LA:ALA ratio calculated from GC and NMR experiment (the coefficient of determination
214
is 0.98).
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In the past three decades the intake of omega-6 fatty acid increased and the omega-3 fatty acid decreased,
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resulting in a large increase in the ω -6: ω -3 ratio from 1:1 in the past to 20:1 today. It is therefore essential to
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return to a balanced dietary ω-6:ω-3 ratio. Analytical methods are needed to determine the LA:ALA ratio.
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Besides GC experiment, 1H NMR in solution may be considered as a fast and effective method. The technique
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has the potential to substitute a wide range of classical analysis methods in the edible oil industry. The NMR
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method might be useful for pharmaceutical industry and even introduced in Pharmacopeia.
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Conclusions
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Gas chromatography is a routine method in fatty acids profiling. However, two spectral methods: NMR and FT-
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IR are also useful in routine examination of edible oils. NMR spectroscopy surpasses the chromatographic
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methods (GC) for vegetable oils as concerns analysis time and sample preparation.
227
The differences of fatty acid composition caused by variability of Cannabis sativa plants or by adulteration of
228
hemp-seed oil with other oils should be supervised. Spectral methods described above have a potential in fast
229
screening of hemp oil quality.
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Acknowledgements: This work was supported by the Medical University of Warsaw (FW28/PM1/16). The
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authors would like to thank Ms. Agnieszka Białek (Ph.D.) for performing the GC measurements and Ms. Joanna
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Kolmas (Ph.D.) for her assistance in FT-IR experiments.
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Conflicts of Interest: The authors declare no conflict of interest.
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References:
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[1] EFSA, Scientific Opinion on the safety of hemp (Cannabis genus) for use as animal feed, EFSA J. 9 (2011)
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1-41.
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[2] C.D. Porto, D. Decorti, A. Natolino, Potential Oil Yield, Fatty Acid Composition, and Oxidation Stability of
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the Hempseed Oil from Four Cannabis sativa L. Cultivars, J. Diet. Suppl. 12 (2015) 1-10.
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lipids by ¹H NMR and comparison with the classical acid value, J. Pharm. Biomed. Anal. 93 (2014) 43-50.
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[10] M. Ahmad., K. Ullah, M.A. Khan, M. Zafar, M. Tariq, S. Ali, S. Sultana, Physicochemical Analysis of
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ACCEPTED MANUSCRIPT Table 1. Description of tested hemp-seed oils (country of origin and plant species) Sample Country of origin Source HO1 Czech Republic Cannabis sativa HO2 Poland Cannabis sativa HO3 Canada Cannabis sativa HO4 France Cannabis sativa HO5 Poland Cannabis sativa HO6 New Zealand Cannabis indica
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Table 2. Mean values and standard deviations of the fatty acid compositions (%) of the hempseed oil
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Fatty acid composition (g/100g fatty acids) by GC, * LA, ** GLA, *** ALA Fatty acid HO1 HO2 HO3 HO4 HO5 C16:0 7.24±0.60 7.07±0.15 6.22±0.19 6.53±0.12 7.16±0.04 C16:1 0.11±0.01 0.09±0.01 0.10±0.01 0.10±0.02 0.08±0.01 C17:0 0.07±0.01 0.06±0.03 0.06±0.01 0.05±0.01 0.04±0.01 C18:0 3.11±0.09 3.29±0.08 3.05±0.05 3.11±0.21 3.42±0.07 C18:1 12.60±0.06 11.44±0.14 10.20±0.37 13.05±0.18 12.31±0.02 C18:2* 56.80±0.29 56.63±0.21 52.13±0.01 55.17±0.33 56.58±0.14 C18:3** 0.49±0.04 0.46±0.01 0.63±0.09 4.51±0.16 3.57±0.03 C18:3*** 16.80±0.30 18.76±0.05 19.10±0.08 14.80±0.03 17.47±0.04 C20:0 0.23±0.03 0.16±0.05 0.20±0.06 1.77±0.11 0.97±0.02 LA:ALA 3.38±0.05 3.02±0.02 2.73±0.04 3.73±0.03 3.24±0.02 ratio
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HO6 6.41±0.11 0.10±0.02 0.04±0.01 2.75±0.14 8.42±0.04 55.22±0.16 4.42±0.18 18.94±0.30 1.49±0.08 2.92±0.05
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Fig 1. FT-IR spectrum of HO1; the region at 1500-500 cm-1 is considered as a “fingerprint”.
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Fig. 2. Fingerprint region at FT-IR spectra of HO1-HO6.
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Fig. 3. (a) 1H NMR spectrum of hemp-seed oil (HO1) in CDCl3 and (b) superimposed signals in 1H NMR spectra of HO3 and HO4 in CDCl3.
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3.5 3.3 3.1 2.9 2.7 2.5 2.5
3 3.5 LA:ALA ratio GC
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LA:ALA ratio NMR
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Fig. 4. Correlation of LA:ALA ratio calculated from GC and NMR experiment (the coefficient of determination is 0.98).
ACCEPTED MANUSCRIPT Highlights •
FT-IR spectroscopy is a fast technique to monitor variability of hemp oil composition.
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1
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Spectroscopic methods may be used in a routine control of oil quality.
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H qNMR is proposed as a new and fast method to determine ω-6 to ω-3 ratio.