applied

sciences
Article
Commercial Hemp Seed Oils:
A Multimethodological Characterization
Mattia Spano 1,† , Giacomo Di Matteo 1,† , Mattia Rapa 2 , Salvatore Ciano 2 ,
Cinzia Ingallina 1, * , Stefania Cesa 1 , Luigi Menghini 3 , Simone Carradori 3 ,
Anna Maria Giusti 4 , Antonella Di Sotto 5 , Silvia Di Giacomo 5 , Anatoly P. Sobolev 6 ,
Giuliana Vinci 2 and Luisa Mannina 1
1 Department of Chemistry and Technology of Drugs, Sapienza University of Rome, P.le Aldo Moro 5,
00185 Rome, Italy; [email protected] (M.S.); [email protected] (G.D.M.);
[email protected] (S.C.); [email protected] (L.M.)
2 Department of Management, Sapienza University of Rome, via del Castro Laurenziano 9, 00161 Rome, Italy;
[email protected] (M.R.); [email protected] (S.C.); [email protected] (G.V.)
3 Department of Pharmacy, University “G. d’Annunzio” of Chieti-Pescara, Via dei Vestini 31,
66100 Chieti, Italy; [email protected] (L.M.); [email protected] (S.C.)
4 Department of Experimental Medicine, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy;
[email protected]
5 Department of Physiology and Pharmacology “V. Ersparmer”, Sapienza University of Rome, P.le Aldo
Moro 5, 00185 Rome, Italy; [email protected] (A.D.S.); [email protected] (S.D.G.)
6 Institute for Biological Systems, Magnetic Resonance Laboratory “Segre-Capitani”, CNR, Via Salaria
Km 29.300, 00015 Monterotondo, Italy; [email protected]
* Correspondence: [email protected]
† These authors contributed equally to this work.




Received: 10 September 2020; Accepted: 1 October 2020; Published: 3 October 2020


Abstract: Nine commercial hemp seed oils from different countries were studied using a
multimethodological approach to obtain information about their quality and chemical composition.
Due to the lack of a specific regulation for hemp seed oils, quality parameters used in the case of olive oils
(free acidity, peroxides number, spectrophotometer parameters) and anisidine number were measured
and compared with those reported for extra virgin olive oil (EVOO). Free acidity and peroxides number
showed a great variability, ranging from 0.4 to 17.24% and from 4.32 to 22.14 meqO2 /kg, respectively,
whereas the anisidine number ranged from 0.11 to 3.58. K232 value turned out to be generally below
the limit reported for EVOO, whereas K270 and ∆K values were higher, with respect to EVOO limits,
due to the high amount of tri-unsaturated fatty chains. Colorimetric analysis showed a peculiar curve
trend that could represent the fingerprint of this product. Untargeted nuclear magnetic resonance
methodology allowed to measure the amount of fatty chains, ω-6:ω-3 ratio, β-sitosterol, and aldehydes.
The ω-6:ω-3 ratio turned out to be, in some cases, different from that reported on the bottle labels. Finally,
lipoperoxidation assays were also carried out under different storage (light and temperature) and time
exposure conditions, confirming that the exposure to direct light is the condition that interferes more
with the product quality.

Keywords: hemp seed oil; quality parameters; NMR; colorimetric analysis; lipoperoxidation

1. Introduction
Cannabis sativa L. is an annual plant belonging to the family of Cannabinaceae, widely spread in
all five continents. Although its name is frequently associated with drugs of abuse (marijuana), due to
the presence of psychotropic ∆9 -tetrahydrocannabinol (THC) [1], the recent evidence of Cannabis sativa

Appl. Sci. 2020, 10, 6933; doi:10.3390/app10196933 www.mdpi.com/journal/applsci

Appl. Sci. 2020, 10, 6933 2 of 15

L. cultivars characterized by low amounts of ∆9 -THC (known as industrial hemp) raised a growing
interest toward this plant in different fields, including pharmaceutical, food, and cosmetic sectors.
In Europe, hemp cultivars with an amount of ∆9 -THC lower than 0.2% (in dry matter basis) are allowed
for cultivation, and are reported in the EU approved list [2].
Among the different parts of the plant, hemp seeds are the only one to be authorized for food
applications [3], as cannabinoids are present mainly due to cross-contamination during harvesting
and processing [4]. Since ancient times, hemp seeds were consumed in the human diet; however,
they were replaced by other seeds and used as feed materials for all animal species. Only in recent
times the discovery of hemp seeds’ high nutritional value led to a revaluation of this source for
human consumption. Literature regarding the investigation of hemp seeds’ composition has increased,
making them an emerging source for value-added functional food ingredients and nutraceuticals [5,6].
Hemp seeds are consumed shelled in salad dressings, and in the production of snack bars, or they can
be cold pressed to obtain edible hemp seed oil, and the residual cake is grinded to have a fiber-rich
flour used in processed food (such as bread, biscuits, and pasta). The high nutritional value lies in the
presence of secondary metabolites and the characteristic fatty acids profile, which stands out for the
higher levels of polyunsaturated fatty acids (PUFAs) with respect to saturated ones. The two essential
fatty acids, linoleic acid (C18:2 ω-6) and α-linolenic acid (C18:3 ω-3), account for more than 50% and
15–25% respectively, resulting in a perfect balanced 3:1 ratio ω-6:ω-3, optimal for healthy human
nutrition [5]. Moreover, the presence of secondary metabolites such as phenolic compounds and
tocopherols [7–11], with antioxidant properties, improves the health benefits and helps to prevent the
PUFA oxidation process, improving hemp seed oil shelf-life. Some studies recommend a dietary intake
of hemp seed oils [12–14], nowadays largely present in the markets of different countries (Canada,
the European Union, Australia, and China) [15].
It is important to underline that a specific regulation concerning the analytical parameters for the
quality assessment of hemp seed oils is still lacking, although the increasing interest towards hemp
food products for human consumption has prompted few countries to issue guidelines about the
acceptable level of ∆9 -THC in hemp seeds, hempseed oil, or processed food [4,16–18]. Indeed, some
studies discuss the development of a proper analytical procedure for the detection of cannabinoids
levels and ratio in hemp seeds and hempseed oils [4,19–22].
However, a plethora of different factors could influence the oil quality, such as the area of
cultivation [23,24], the type of cultivars [25], the origin of seeds [26], agronomic practices, and extraction
processing [27,28] that usually indicate the cold pressing as the preferred method to preserve the
ω-3:ω-6 ratio [11]. Moreover, the price of commercial hempseed oil ranges from 20 to 100 €/L, making
it an expensive food product sold on the market without any quality assurance. Taking into account
the variability in composition and the price, quality parameters need to be established in order to both
valorize products and preserve consumer safety and awareness.
Hemp seed oils are sold as food, and labeled accordingly [29]. In this context, the final product
has to guarantee the safety standard for consumers, but relevant information, such as plant identity,
variety, chemical characterization in terms of unsaturated fatty acid, secondary metabolites, and similar,
are not mandatory, and therefore not reported in the bottle labels.
In the present study, nine commercial hemp seed oils were analyzed, by a multimethodological
approach, to provide information regarding the quality of the products present on the markets. Due to
the lack of a specific regulation for hemp seed oils, quality criteria reported in the olive oil Regulation
(EU) 2015/1830 (European commission), namely free acidity, peroxides number, and spectrophotometric
parameters such as K270 , ∆K, and K232 , were measured and compared with those reported for extra
virgin olive oil (EVOO). Moreover, anisidine number, colorimetric analysis, and lipoperoxidation
assays were also evaluated. Untargeted nuclear magnetic resonance (NMR) methodology was applied
to identify and quantify simultaneously different classes of compounds [30,31].
Appl. Sci. 2020, 10, 6933 3 of 15

2. Materials and Methods

2.1. Reagents and Materials

All chemical reagents (potassium hydroxide (KOH), phenolphthalein, potassium iodide (KI),
acetic acid (CH3 COOH), sodium thiosulfate (Na2 S2 O3 ), p-anisidine, glacial acetic acid, linoleic acid,
iron (II) chloride (FeCl2 ), potassium thiocyanate (KSCN), and solvents (ethanol (EtOH), methanol
(MeOH), chloroform (CHCl3 ), diethyl ether (Et2 O), iso-octane, and cyclohexane) were purchased from
Sigma-Aldrich Co (St. Louis, MO, USA) (all purity ≥98%). Deionized water, purified with a Milli-Q
system (Millipore, Bedford, USA), was used. Dimethyl sulfoxide D6 99.80% and chloroform D 99.80%
were purchased from Euriso-Top (Saclay, France).

2.2. Sampling
Nine commercial hemp seed oils were purchased in organic foods stores and in the e-marketplace,
chosen as the best representatives of the consumer’s choices (the most common hempseed oils from
greatly organized distribution, and the ones with top customer reviews from the e-marketplace).
Hemp seed oils are usually sold in 250 mL bottles. Provenience and prices (€/L) are reported in Table 1.
The labels of samples 2, 4, 5, 7, 8, and 9 stated that the preparation was performed using cold extraction.
In all labels, the ω-6:ω-3 ratio was indicated. Before carrying out analysis, samples were stored in
a cool and dry place, away from sources of light and heat, in order to prevent oxidation reactions.
All analyses were carried out 6 months before the expiry date. The original bottle was opened just
before analysis and divided in 10 mL tubes, completely fulfilled, hermetically closed, and stored in a
cold place in the dark, in order to prevent oil–air and oil–light interactions.

Table 1. Geographical provenience and price of the commercial hemp seed oils.

Sample 1 2 3 4 5 6 7 8 9
Origin Italy EU EU NON-EU NON-EU Italy NON-EU NON-EU Italy
Price (€/L) 44 20 24 25 26 30 52 64 80

2.3. Quality Parameters

Hemp seed oil quality was evaluated measuring free acidity, peroxides number, anisidine number,
and spectrophotometer parameters (K232 , K270 , and ∆K). Since the methodologies usually applied to
determine all these parameters require great quantities of samples and reagents, specific miniaturized
methods were applied to analyze the hemp seed oil, according to Rapa et al. [32]. All the experiments
were performed three times, and the results were expressed as the mean value ± SD (standard deviation).

2.3.1. Free Acidity

An aliquot of 3.33 g of hemp seed oil was dissolved in 10 mL of diethyl ether/ethanol (60/40 v/v)
mixture. Then, free fatty acids were titrated with KOH 0.01 N until the tuning of the phenolphthalein
indicator. Free acidity (A%) was determined, following Equation (1):

A% = (V × C × M)/(10 × m) (1)

where: V is the titrant volume (expressed in mL); C is the exact concentration of KOH (expressed
in mol/L); M is the molar weight of the oleic acid (282 g/mol) used for the expression of the result;
and m is the weight of the analyzed substance (expressed in g) [33] (Table 2).

2.3.2. Peroxides Number (P.N.)

An aliquot of 0.5 g hemp seed oil was introduced in a flask with 2 mL of chloroform. After shaking
(30 s), 3 mL of glacial acetic acid and 0.1 mL of potassium iodide saturated aqueous solution were
Appl. Sci. 2020, 10, 6933 4 of 15

added. The flask was shaken for 1 min and then left, protected from light, at a temperature between
15 and 25 ◦ C. After 5 min, 75 mL of distilled water was added. The released iodine was titrated with
a sodium thiosulfate solution (0.0005 N) by vigorously shaking, using a starch solution as indicator.
The peroxides number (P.N.), expressed as meqO2 /kg, and was calculated as follows (Equation (2)):

P.N. = (V × T)/m × 1000 (2)

where: V is the volume of titrant (expressed in mL); T is the normality of the sodium thiosulfate
solution (expressed in n.eq./L); and m is the weight in g of the analyzed sample [33].

Table 2. Free acidity (A%), peroxides number (P.N.), anisidine number (A.N.), and spectrophotometric
parameters (K232 , K270 , and ∆K) of the analyzed hemp seed oil samples.

P.N.
Sample A% A.N. K232 K270 ∆K
(meqO2 /kg)
1 17.24 ± 0.63 4.31 ± 0.37 3.58 ± 0.17 2.65 ± 0.09 0.69 ± 0.01 0.03
2 1.86 ± 0.07 16.68 ± 1.28 1.14 ± 0.04 2.04 ± 0.02 0.41 ± 0.01 0.01
3 0.40 ± 0.01 16.42± 1.39 2.84 ± 0.11 2.35 ± 0.04 0.57 ± 0.01 0.02
4 4.15 ± 0.12 8.36 ± 0.75 1.60 ± 0.07 2.12 ±0.03 0.47 ± 0.01 0.01
5 1.47 ± 0.08 10.69 ± 0.96 1.17 ± 0.03 2.01 ± 0.17 0.39 ± 0.01 0.01
6 2.22 ± 0.02 22.14± 1.88 3.30 ± 0.28 2.26 ± 0.05 0.49 ± 0.01 0.01
7 0.76 ± 0.03 10.50 ± 1.26 0.11 ± 0.02 1.85 ± 0.02 0.26 ± 0.01 0.01
8 0.43 ± 0.02 17.13 ± 0.89 0.12 ± 0.01 1.72 ± 0.01 0.19 ± 0.01 0.01
9 1.50 ± 0.06 17.03 ± 1.17 2.80 ± 0.02 2.21 ± 0.02 0.46 ± 0.01 0.02

2.3.3. Anisidine Number (A.N.)

An amount of 0.2 g of hemp seed oil was dissolved in 10 mL of iso-octane (2,2,4-trimethylpentane)
(solution A). Then, the extinction at 350 nm was determined in a 10 mm quartz cuvette. A 2.5% (w/v)
solution of p-anisidine was prepared in glacial acetic acid. An aliquot of 2.5 mL of solution A was
added to 0.5 mL of p-anisidine (2.5% in glacial acetic acid). The mixture (solution B) was shaken and
set to rest for 10 min. Then, the extinction at 350 nm was measured. To obtain the p-anisidine number
(A.N.), the following expression (Equation (3)) was applied:

A.N. = [25 × (1.2 Ea − Eb)]/m (3)

where: Ea is the extinction of solution A; Eb is the extinction of the solution B; and m is the weight of
the sample (expressed in g) [34].

2.3.4. Spectrophotometric Investigation in the Ultraviolet

An amount of 0.25 g of hemp seed oil was dissolved in 25 mL of cyclohexane solvent and
homogenized to obtain a 1% solution (w/v). The extinction coefficients were measured at 232 nm,
266 nm, 270 nm, and 274 nm, with respect to the pure solvent as reference in a 10 mm quartz cuvette.
The extinction absolute value (∆K) was calculated as follows (Equation (4)):

∆K = K270 × (K266 + K274 )/2 (4)

where K270 , K266 , and K274 are the wavelength-specific extinction [33].

2.4. Colorimetry
The hemp seed oils were submitted to colorimetric analyses. CIEL*a*b* parameters (L*, a*, b*,
C*ab , and hab ) were obtained using a colorimeter X-Rite SP-62 (X-Rite Europe GmbH, Regensdorf,
Switzerland), equipped as previously described [35]. The color description is based on three parameters:
L* (lightness), a* (greenness for negative, or redness for positive, values) and b* (blueness for negative,
Appl. Sci. 2020, 10, 6933 5 of 15

or yellowness for positive, values). Cylindrical coordinates C*ab and hab were calculated from the
parameters a* and b*, as described in our previous works [36]. All the experiments were performed
four times, and the results were expressed as the mean value ± SD (Table 3).

Table 3. Colorimetric parameters obtained by CIELAB analyses performed on hemp seed oils 1–9.

Sample L* a* b* C*ab hab

1 29.33 ± 0.15 3.54 ± 0.04 7.30 ± 0.22 8.11 ± 0.21 64.12 ± 0.56
2 25.40 ± 0.02 0.82 ± 0.00 1.19 ± 0.01 1.44 ± 0.01 55.68 ± 0.19
3 33.52 ± 0.04 3.96 ± 0.02 15.11 ± 0.09 15.62 ± 0.08 75.32 ± 0.09
4 46.32 ± 0.05 0.40 ± 0.02 24.60 ± 0.02 24.61 ± 0.02 89.08 ± 0.06
5 42.71 ± 0.88 1.50 ± 0.08 23.66 ± 0.17 23.71 ± 0.16 86.38 ± 0.20
6 39.20 ± 0.53 0.96 ± 0.05 6.28 ± 0.14 6.36 ± 0.13 81.27 ± 0.58
7 44.52 ± 0.09 0.72 ± 0.02 9.89 ± 0.15 9.92 ± 0.15 85.85 ± 0.09
8 52.33 ± 0.08 -0.62 ± 0.02 2.91 ± 0.09 2.98 ± 0.09 102.03 ± 0.47
9 40.13 ± 0.27 5.17 ± 0.10 25.80 ± 0.57 26.32 ± 0.57 78.68 ± 0.15
1 L* = lightness; 2 a* = greenness for negative, or redness for positive, values; 3 b* = blueness for negative,
or yellowness for positive, values; 4 C*ab and 5 hab = cylindrical coordinates.

2.5. NMR Analysis

Each hemp seed oil sample (20 µL) was dissolved in DMSO-d6 (20 µL) and CDCl3 (700 µL)
in a 5 mm NMR tube, which was flame-sealed. The NMR spectra were recorded at 27 ◦ C on a
Bruker AVANCE 600 spectrometer (Milan, Italy) operating at the proton frequency of 600.13 MHz and
equipped with a Bruker multinuclear z-gradient 5 mm probe head. 1 H spectra were acquired using the
following conditions: number of scans 1024, 90◦ pulse 9.5 µs, time domain 64 K data points, relaxation
delay plus acquisition time 3.5 s, and spectral width 10,802 Hz. 1 H NMR spectra were obtained by
the FT (Fourier transform) of the free induction decay, applying an exponential multiplication with
a line-broadening factor of 0.3 Hz and a zero filling (size 64 K) procedure. 1 H NMR spectra were
manually phased. Chemical shifts were reported with respect to the residual CHCl3 signal set at
7.26 ppm. The baseline was corrected using the Cubic Spline Baseline Correction routine in the Bruker
Topspin software. The integrals of seven selected signals were measured. In particular, the selected
proton resonances were due to hexanal (CH) at 9.699 ppm, trans-2-hexenal (CH) at 9.448 ppm, diallylic
protons of tri-unsaturated fatty acids (CH2 ) at 2.778 ppm, diallylic protons of di-unsaturated fatty
acids (CH2 ) at 2.729 ppm, α-methylene protons of all fatty acids (CH2 ) at 2.251 ppm, allylic protons
of all unsaturated fatty acids (CH2 ) at 1.999 ppm, and β-sitosterol (CH3 ) at 0.622 ppm. The integrals
of the selected resonances were normalized with respect to the resonance at 2.251 ppm set to 100.
β-Sitosterol, fatty acids, and aldehydes were expressed as molar %, following the Equations previously
described [37], with some modifications:

%β-SIT = 100 × 0.66Iβ-SIT /Itot (5)

%TRI = 100 × 0.5ITRI /Itot (6)

%DI = 100 × IDI /Itot (7)

%MONO = 100 × 0.5(IUNS − 2IDI − ITRI )/Itot (8)

%SAT = 100 × (IFA − IDI − 0.5ITRI − %MONO )/Itot (9)

%INS = %MONO + %DI + %TRI (10)

%t-2-HEX = 100 × 2It-2-HEX /Itot (11)

%HEX = 100 × 2IHEX /Itot (12)

Appl. Sci. 2020, 10, 6933 6 of 15

where %β-SIT , %TRI , %DI , %MONO , %SAT , %INS , %t-2-HEX, and %HEX are molar % of β-sitosterol,
tri-unsaturated fatty acids, di-unsaturated fatty acids, mono-unsaturated fatty acids, saturated fatty
acids, unsaturated fatty acids, trans-2-hexenal, and hexanal, respectively. Iβ-SIT , ITRI , IDI , IUNS , IFA ,
It-2-HEX , and IHEX are integrals of β-sitosterol, tri-unsaturated fatty acids, di-unsaturated fatty acids,
allylic protons of all unsaturated fatty acids, α-methylene protons of all fatty acids, trans-2-hexenal,
and hexanal, respectively. Itot was calculated according to Equation (13).

Itot = IFA + 0.66Iβ-SIT + 2It-2-HEX + 2IHEX (13)

2.6. Lipoperoxidation
The assay was carried out by the ferric thiocyanate method according to Di Sotto et al. [38],
with minor changes. Preliminarily, both linoleic acid and the oil samples were subjected to storage for
24 h under different temperature conditions, including room temperature (RT), room temperature plus
light exposure (RT plus white light; distance of lamp, 30 cm), refrigerated temperatures of +4 ◦ C and
−20 ◦ C, and warm temperature of +60 ◦ C. After storage sodium phosphate buffer (625 µL; 0.2 M, pH 7.0)
was added to linoleic acid or the hemp seed oil samples (625 µL; 2.5% v/v in pure EtOH) and incubated
at 37 ◦ C for 72 h. Various aliquots (10 µL) were taken at different incubation times (0, 24, 48, 72 h) and
mixed with ethanol (970 µL; 75% v/v), FeCl2 (10 µL; 200 mM in 3.5% w/v HCl) and KSCN (10 µL; 30% w/v
in deionized water). Peroxides, generated during fatty acid peroxidation, oxidize Fe+2 to Fe+3 ; the latter
ion forms a complex with thiocyanate that can be measured spectrophotometrically at 500 nm.
The percentage of the Lipid Peroxidation (LP) was calculated, as follows (Equation (14)):

LP = Asample /Astandard × 100 (14)

where Astandard is the highest absorbance of linoleic acid used as reference, whereas Asample is the
absorbance of the tested sample. The experiments were repeated two times, and, in each experiment,
three technical replicates were assayed for each storage condition.

3. Results and Discussion

3.1. Quality Parameters

Due to the lack of a specific regulation on hemp seed oils, quality parameters established for olive
oils [33], namely free acidity, peroxides number, anisidine number, and spectrophotometric parameters
(K232 , K270 , and ∆K) (ISO 6885:2016), were evaluated.

3.1.1. Free Acidity

Free Acidity (A%), expressed as oleic acid, points out the degree of fat hydrolytic alteration.
Triglycerides hydrolysis occurs in the presence of water and enzymes (lipases), naturally present in
raw vegetable matrix. In the Regulation (EU) 2015/1830 for virgin oils, a maximum limit of 2.0% is
provided [33]. However, the Pharmacopoeia provides a maximum value of 6% in A% for seeds oils [39].
Among the commercial hemp seed oils here investigated (Table 2), samples 3, 7, and 8 showed an
A% less than 1% (0.40%, 0.76% and 0.43%, respectively), whereas samples 1, 4, and 6 showed a value
above the 2.0% threshold (17.24%, 4.15% and 2.22%, respectively). Considering the limit established by
the Pharmacopoeia for seed oils, only sample 1 exceeds the 6% limit. It possible that this commercial
product was not stored using adequate temperature and light conditions.
In literature, few studies have investigated the free acidity of hemp seed oils, and a great variability
of values was found. A% of 0.074–0.890% has been reported for noncommercial hemp seed oils [32,40],
whereas for Italian commercial hemp seed oils, the maximum value of 9.9% has been reported [7].
Appl. Sci. 2020, 10, 6933 7 of 15

3.1.2. Oxidation Parameters: Peroxides Number and Anisidine Number

Fatty acids oxidation can occur in two steps. First, unsaturated fatty acids react with oxygen,
producing odorless peroxides that, in the second step, decompose in aldehydes, responsible for rancid
taste. The degree of the first oxidation step is evaluated by peroxides number (P.N.) (EU Regulation (EU)
2015/1830); a limit of 20 meqO2 /kg P.N. is reported for virgin and extra virgin olive oils. The peroxides
value is useful for assessing the spoilage progression, especially in fresh oils or oil produced with
low temperature technologies [41]. On the other hand, the anisidine number (A.N.) provides the
assessment of the second oxidation step: an acceptable number for fat matrices is 10.
The nine hemp seed oils here investigated showed a relevant variability for P.N. values, ranging
from 4.32 to 22.14 meqO2 /kg. Only sample 6 showed a P.N. value above 20 meqO2 /kg. Again, as in the
case of A%, these values are higher than the values from 1.94 to 7.20 meqO2 /kg reported in the case of
noncommercial samples [40,42,43] and Italian commercial hemp seed oils [7].
All the investigated samples showed A.N. values ranging from 0.11 to 3.58 (below the limit),
in agreement with literature data [44].

3.1.3. K232 , K270 , and ∆K Spectroscopic Values

Regarding the spectroscopic measurements, the K232 value evaluates the presence of conjugated
dienes, whereas K270 and ∆K indicate the presence of conjugated trienes. Almost all the samples
showed a K232 value below the EVOO limit (2.6 L/g·cm), with sample 8 showing the lowest value
(1.72 L/g·cm). The only exception was sample 1, with a value of 2.65 L/g·cm. Generally, the values here
reported were lower than those reported by Anwar et al. for Pakistan samples [23].
The K270 and ∆K values turned out to be higher than the limits reported in the Regulation
(0.25 L/g·cm, 0.01), in agreement with the previously reported data [45]. The higher conjugated trienes
content, with respect to olive oils, is due to the high amount of PUFAs in hemp seed oils.

3.2. Colorimetry
The color of a product plays a preeminent role in the consumer acceptance and, consequently,
in the choice of a food. In the case of oils, color could represent an index of good manufacturing
practice, a warranty of quality and genuineness, and, not least important, of the right storage conditions.
To evaluate the color properties of hemp seed oils, the tristimulus colorimetry was employed. To our
knowledge, only one work is available in literature regarding colorimetric studies on hemp seed oil
samples, reporting an L* value of 18.13, an a* of 2.40 and a b* of 30.87 [46], so that a significant yellow
parameter (+b*) can be shown, compared to a very weak red value (+a*) and a quite dark sample.
In a previous work [46], a comparison among medium reflectance profiles of hemp seed, olive,
and neem oils has been carried out: a similarity among olive oils and hemp oils has been observed,
although hemp seed oils turned out to be darker.
The analyses, performed on the selected nine samples, only in part confirm these data (Table 3).
Two samples (6 and 7) were turbid, and were analyzed before and after filtration. All samples covered a
very wide range between 25 to 46 of lightness, −0.6–5.4 of a* and 1.2–33 of b*, substantially confirming
a yellow color completely dominant on the slight reddish nuance, but also showing significant
differences in the lightness and the tonality, hab = tan−1 (a*/b*), most likely due to the different cultivars,
the work-up, and the purification steps applied by the producers. All the obtained reflectance curves,
shown in Figure 1, account for the relevant differences found among the samples.
The profile of sample 2 corresponded to a very dark oil, whereas the profile of sample 8 corresponded
to a very faded oil. In samples 6 and 7, turbidity and sediments were present. Therefore, when samples
2, 8, 6, and 7 were discarded, a clear reflectance profile was observed (Figure 1), accounting for a
different lightness, corresponding to a higher b*/a* ratio, that indicates a dominant yellow color with
respect to a reddish nuance. Samples 4 and 5, characterized by a quite different profile in the region
between 450 and 500 nm, showed the lowest a* values and, consequently, the highest b*/a* ratios.
 Appl. Sci. 2020, 10, x 8 of 16

different profile in the region between 450 and 500 nm, showed the lowest a* values and,
consequently, the highest b*/a* ratios.
Therefore, with respect to the reflectance profiles of other commercially available oils, such as
Appl. Sci.
corn,2020, 10, 6933
rice, and EVOO, hemp seed oils showed a peculiar curve trend that could represent a8 of 15
Appl. Sci. 2020, 10, x 8 of 16
fingerprint for this product [40].
different profile in the region between 450 and 500 nm, showed the lowest a* values and,
consequently, the highest b*/a* ratios.
Therefore, with respect to the reflectance profiles of other commercially available oils, such as
corn, rice, and EVOO, hemp seed oils showed a peculiar curve trend that could represent a
fingerprint for this product [40].

Figure 1. Spectral reflectance curves obtained by CIEL*a*b* analyses applied to 1–9 hemp seed oils.
Figure 1. Spectral reflectance curves obtained by CIEL*a*b* analyses applied to 1–9 hemp seed oils.
Samples 6 and 7 were measured after filtration.
Samples 6 and 7 were measured after filtration.
3.3. NMR Analysis
Therefore, with respect to the reflectance profiles of other commercially available oils, such as
corn, rice,Inand
Figure
EVOO,2, thehemp
1H spectrum of a hemp seed oil is reported. Hemp seed oils spectra, similarly to
seed oils showed a peculiar curve trend that could represent a fingerprint
other
for this vegetable
Figure
product oils, reflectance
1.[40].
Spectral were characterized by strong
curves obtained signals,analyses
by CIEL*a*b* due to applied
the fatty chains
to 1–9 onseed
hemp the oils.
glycerol
moiety and minor
Samples 6 and 7compounds.
were measuredThe amount
after of β-sitosterol, hexanal, trans-2-hexenal, saturated fatty
filtration.
chains, and
3.3. NMR Analysis unsaturated fatty chains (including mono-unsaturated, di-unsaturated, and
3.3. NMR Analysis
tri-unsaturated fatty chains) are reported as molar percentages in Figure 3.
In Figure 2, the 1 H spectrum of a hemp seed oil is reported. Hemp seed oils spectra, similarly to
In Figure 2, the 1H spectrum of a hemp seed oil is reported. Hemp seed oils spectra, similarly to
other vegetable oils, were characterized by strong signals, due to the fatty chains on the glycerol moiety
other vegetable oils, were characterized by strong signals, due to the fatty chains on the glycerol
and minor compounds. The amount of β-sitosterol, hexanal, trans-2-hexenal, saturated fatty chains,
moiety and minor compounds. The amount of β-sitosterol, hexanal, trans-2-hexenal, saturated fatty
and unsaturated
chains, and fatty chains (including
unsaturated mono-unsaturated,
fatty chains di-unsaturated, and
(including mono-unsaturated, tri-unsaturated
di-unsaturated, and fatty
chains) are reported as molar percentages in Figure 3.
tri-unsaturated fatty chains) are reported as molar percentages in Figure 3.

Figure 2. 600.13 MHz 1H NMR spectrum of a hemp seed oil. Quantified selected NMR signals are
reported in expanded regions. (A) β-sitosterol (0.622 ppm); (B): allylic protons of all fatty chains

FigureFigure 2. 600.13
2. 600.13 MHz MHz
1H H 1
NMRNMRspectrum
spectrum of
of aa hemp
hemp seed
seedoil.
oil.Quantified
Quantified selected NMR
selected NMRsignals are are
signals
reported in expanded regions. (A) β-sitosterol (0.622 ppm); (B): allylic protons of all fatty chains
reported in expanded regions. (A) β-sitosterol (0.622 ppm); (B): allylic protons of all fatty chains
(1.999 ppm); (C): α-methylene protons of all acyl chains (2.251 ppm); (D): diallylic protons of linoleic
fatty chains (2.729 ppm); (E): diallylic protons of linolenic fatty chains (2.778 ppm); (F): trans-2-hexenal
(9.448 ppm); (G): hexanal (9.699 ppm).
 As expected, fatty acid chain profiles of all nine hemp seed oils were characterized by a higher
content of unsaturated fatty acid chains with respect to saturated ones, and, among them,
di-unsaturated fatty chains resulted in more abundance (35–48%), compared to mono- (15–28%) and
tri-unsaturated ones (8–20%). In particular, sample 1 was characterized by the lowest tri-unsaturated
Appl. Sci. 2020, 10, 6933 9 of 15
fatty chain content.

Figure 3. Histograms resulting from the quantitative NMR analysis of (A) β-sitosterol and aldehydes,
Figure 3. Histograms resulting from the quantitative NMR analysis of (A) β-sitosterol and aldehydes,
and (B) fatty acids. The relative molecular abundances (molar percentage) are reported.
and (B) fatty acids. The relative molecular abundances (molar percentage) are reported.
As expected, fatty acid chain profiles of all nine hemp seed oils were characterized by a higher
contentHemp seed oils are
of unsaturated fattyrenowned
acid chainsfor
withtheir optimal
respect 3:1 ratio
to saturated in ω-6:ω-3.
ones, and, among However, on the bottle
them, di-unsaturated
labels of samples 2, 6, and 9 (see Table 4), a ratio lower than 3:1 was reported,
fatty chains resulted in more abundance (35–48%), compared to mono- (15–28%) and tri-unsaturated and, in some cases, the
ω-6:ω-3
ones ratio measured
(8–20%). bysample
In particular, NMR was 1 wasdifferent from the
characterized ratio
by the reported
lowest on the labels.
tri-unsaturated fatty For instance,
chain 2,
content.
3, and 4 hemp seed oil samples showed an ω-6:ω-3 ratio lower than that declared
Hemp seed oils are renowned for their optimal 3:1 ratio in ω-6:ω-3. However, on the bottle labels in the labels. Only
7, 8, 5, and2,1 6,
of samples hemp
and 9seed
(see oil samples,
Table with
4), a ratio values
lower thanof3:1
3.6,was
3.6,reported,
3.3, and and,
4.7, respectively,
in some cases,turned out to
the ω-6:ω-3
have an optimal ω-6:ω-3 ratio. In particular, sample 1 presented a particularly
ratio measured by NMR was different from the ratio reported on the labels. For instance, 2, 3, and high ratio compared
4tohemp
otherseedones.oilAccording to NMRandata,
samples showed samples
ω-6:ω-3 ratio4,lower
2, 9, than
and 6thatdiddeclared
not reach in the 3:1 ω-6:ω-3
the labels. Onlyratio
7, 8,
value, with sample 3 having a particularly low value.
5, and 1 hemp seed oil samples, with values of 3.6, 3.6, 3.3, and 4.7, respectively, turned out to have
an optimal ω-6:ω-3 ratio. In particular, sample 1 presented a particularly high ratio compared to
Table 4. Comparison between ω-6:ω-3 ratios reported on the bottle labels and the ones measured by
other ones. According to NMR data, samples 4, 2, 9, and 6 did not reach the 3:1 ω-6:ω-3 ratio value,
NMR analysis. Integrals of signals D (diallylic protons of linoleic fatty chains at 2.729 ppm) and E
with sample 3 having a particularly low value.
(diallylic protons of linolenic fatty chains at 2.778 ppm) of the nine commercial hemp seed oils
analyzed were measured.
Table 4. Comparison between ω-6:ω-3 ratios reported on the bottle labels and the ones measured by
NMR analysis. Integrals of signals D (diallylic protonsω-6:ω-3ofRatio
linoleic fatty chains at 2.729 ppm) and E
(diallylic protons of linolenic Samples
fatty chains at 2.778 ppm)
Bottle Labelof the
NMRnine Results
commercial hemp seed oils analyzed
were measured. 1 4.20 4.71
2 2.80 ω-6:ω-3 Ratio
2.40
3 3.37 1.99
Samples Bottle Label NMR Results
4 3.17 2.30
1 4.20 4.71
5 3.21 3.36
2 2.80 2.40
36 2.39
3.37 2.27
1.99
47 3.11
3.17 3.64
2.30
58 3.37
3.21 3.61
3.36
69 2.39
2.57 2.27
2.86
7 3.11 3.64
8 3.37 3.61
9 2.57 2.86

It is interesting to note that sample 9, with an ω-6:ω-3 ratio of 2.86, was sold at 80 €/L,
whereas sample 5, with a 3.36 ratio value, was sold at 26 €/L, a much more affordable price.
Regarding the content of minor compounds, the β-sitosterol amount ranged from 0.15% (sample 8)
to 0.33% and 0.45% (samples 6 and 5, respectively).
Appl. Sci. 2020, 10, 6933 10 of 15

The content of trans 2-hexanal and hexanal was generally low in all the samples (between 0 and
0.5%). However, sample 1 showed the highest content of hexanal (0.34%) and trans-2-hexenal (0.17%).

3.4. Evaluation of Oxidation Susceptibility

In order to evaluate the susceptibility of hemp seed oils to lipid peroxidation, the samples were
subjected to different temperatures of storage and to a specific light exposure condition under laboratory
testing [7,47]. The results showed peculiar stability profiles of the oils with respect to linoleic acid,
used as reference. Lipoperoxidation of hemp seed oils was not significantly increased over 24 h of
incubation time, since the susceptibility to lipid peroxidation after 48 and 72 h was the same as of 24 h
(Figure 4). Differences among the percentage of lipoperoxidation were estimated at time zero and after
24 h of exposure of the samples to different storage conditions (Table 5).

Table 5. Lipoperoxidation (%) of linoleic acid, used as standard, and the hemp seed oils 1–9 under
different storage conditions.

Lipoperoxidation (%)
RT * RT/light +4 ◦ C −20 ◦ C +60 ◦ C
Linoleic acid
t0 40.1 ± 0.3 61.3 ± 1.1 *** 41.8 ± 0.8 37.3 ± 0.4 54.2 ± 0.3 **
§§§ 96.6 ± 0.8 §§§
t24 76.6 ± 1.0 76.6 ± 1.0 69.1 ± 1.3 *§§ 77.0 ± 0.3 §§
***§§
1
t0 27.4 ± 0.4 88.0 ± 2.4 *** 25.9 ± 1.2 21.5 ± 0.1 * 41.8 ± 1.9 ***
t24 21.7 ± 0.5 98.2 ± 0.5 ***§ 21.2 ± 1.1 19.5 ± 0.3 * 23.7 ± 1.4
2
t0 16.6 ± 0.3 26.1 ± 1.1 ** 16.5 ± 0.3 17.1 ± 0.3 17.4 ± 0.5
t24 48.9 ± 0.7 §§§ 56.0 ± 3.1 *§§§ 49.3 ± 0.4 §§§ 48.4 ± 2.2 §§§ 50.8 ± 4.6 §§§
3
t0 34.5 ± 1.1 37.8 ± 1.0 34.6 ± 0.7 26.4 ± 1.4 32.4 ± 0.8
t24 25.6 ± 1.3 29.6 ± 1.3 24.5 ± 0.2 24.0 ± 2.3 24.7 ± 0.2
4
t0 37.6 ± 0.9 49.2 ± 1.1 * 31.8 ± 0.5 33.4 ± 2.4 * 28.5 ± 0.2
t24 24.2 ± 0.3 32.8 ± 1.6 * 23.9 ± 0.4 21.9 ± 3.4 24.9 ± 0.3
5
t0 25.6 ± 0.1 40.4 ± 1.0 *** 25.8 ± 0.8 26.4 ± 3.4 26.5 ± 0.3
t24 21.9 ± 1.2 23.2 ± 0.6 21.1 ± 1.1 20.8 ± 2.4 21.0 ± 0.4
6
t0 18.7 ± 0.3 29.6 ± 0.5 ** 21.9 ± 0.3 * 16.5 ± 0.2 * 18.7 ± 0.2
t24 54.2 ± 1.6 §§§ 53.8 ± 0.7 §§§ 55.7 ± 0.1 §§§ 53.8 ± 0.5 §§§ 49.7 ± 4.4 §§§
7
t0 15.9 ± 0.4 22.7 ± 0.2 ** 16.8 ± 0.1 15.6 ± 0.2 16.4 ± 0.3
t24 51.0 ± 0.6 §§§ 48.9 ± 1.4 §§§ 49.3 ± 0.4 §§§ 45.8 ± 0.1 §§§ 47.6 ± 3.1 §§§
8
t0 14.4 ± 0.4 20.4 ± 0.1 ** 13.2 ± 0.5 13.6 ± 0.3 14.5 ± 0.5
t24 48.6 ± 0.8 §§§ 46.4 ± 1.3 §§§ 45.8 ± 1.1 §§§ 47.1 ± 0.6 §§§ 46.7 ± 0.6 §§§
9
t0 28.3 ± 0.5 34.7 ± 0.4 * 26.4 ± 0.4 22.7 ± 0.4 22.5 ± 0.3
t24 21.6 ± 0.7 24.2 ± 1.6 * 21.0 ± 0.3 21.2 ± 0.2 21.5 ± 0.7
* RT: room temperature; RT/light: room temperature with direct exposure to light; +4 ◦ C: cold temperature; −20 ◦ C:
ultra-frost temperature; + 60 ◦ C: warm temperature. * p < 0.05, ** p < 0.01 and *** p < 0.001 denote a statistically
significant difference with respect to RT (ANOVA followed by Dunnet’s Multiple Comparison Post Test). § p < 0.05,
§§ p < 0.01, §§§ p < 0.001 denote a statistically significant increase with respect to time zero under the same storage

conditions (ANOVA followed by Dunnet’s Multiple Comparison Post Test).

Appl. Sci. 2020, 10, 6933 11 of 15
Appl. Sci. 2020, 10, x 11 of 16

Figure 4. Absorbance (λ 500 nm) of lipoperoxides produced in different hemp seed oils, with respect
Figure
to linoleic 4. Absorbance
acid (A) under(λ 500 nm) storage
multiple of lipoperoxides produced
conditions in different hemp
and incubation (t = oils,
timesseed with
0, 24, 48,respect
and 72 h).
to linoleic acid (A) under multiple storage conditions and incubation
Hemp seed oils: (B) 1, (C) 2, (D) 3, (E) 4, (F) 5, (G) 6, (H) 7, (I) 8, (J) 9. times (t = 0, 24, 48, and 72 h).
Hemp seed oils: (B) 1, (C) 2, (D) 3, (E) 4, (F) 5, (G) 6, (H) 7, (I) 8, (J) 9.
Linoleic acid displayed a very low stability, being significantly oxidized under all storage
conditions (Figure 4A): the maximum percentage of peroxidation was achieved after 24 h incubation at
RT/light (about 2.4-fold higher than RT at time zero), followed by storage at 60 ◦ C warm temperature
Appl. Sci. 2020, 10, 6933 12 of 15

(1.5-fold higher than the RT oxidation at time zero). Conversely, an oxidation reduction (almost twofold,
compared to RT oxidation at time zero) occurred under ultra-frost temperature (−20 ◦ C).
Sample 1 exhibited a high stability under almost all the experimental conditions, except for
RT/light, wherein it resulted in being strongly oxidized already at time zero (Figure 4B), with about
a 3.3- and 4.5-fold peroxidation increase, compared to the RT oxidation at time zero and after 24 h,
respectively. Moreover, after warm storage (60 ◦ C), it showed about a twofold increase in oxidation
with respect to RT at time zero, although the basal level was recovered during incubation (Figure 4B).
Conversely, the ultra-frost temperature (−20 ◦ C) weakly (about 1.2-fold) affected the basal oxidation of
sample 1.
The other commercial hemp seed oils displayed a different behavior compared to sample 1.
Samples 3, 4, 5, and 9 were only slightly affected by storage at warm and cold temperatures, or by light,
at all incubation times (Figure 4D–F,L).
Regarding the hemp seed oils 4, 5, and 9, the storage at RT/light induced a weak but significant
lipoperoxidation increase (1.3–1.6-fold), compared RT at time zero, which progressively disappeared
during incubation time. Conversely, sample 3 turned out to be stable, also, to light exposure.
On the basis of this evidence, hemp seed oils appear to be stable under the tested storage
conditions, although the direct exposure to light should be considered with caution, because of the
higher susceptibility of some samples to oxidation, likely due to a high content of polyunsaturated
fatty acids or to the presence of antioxidant constituents. Further studies could clarify this issue.

4. Conclusions
In this paper, nine commercial hemp seed oils were analyzed, by both targeted and untargeted
analyses, to determine their chemical composition and, therefore, to obtain information regarding the
quality of the products present in the markets. The results show a great variability between the samples.
Applying the parameters used for EVOO, the quality was poor in above 50% of the samples, as showed
by A%, P.N., and ω-6:ω-3 ratio, although price was very high. Lipoperoxidation assay confirmed that
storage conditions can affect the shelf life of the product, however, the quality, in terms of healthy
properties, should be assured at least until the expiration date. It is well established that several
parameters (cultivars, pedoclimatic conditions, and agronomical practices) do affect the chemical
composition and sensorial features of foodstuffs, however, the great variability of data obtained on the
commercial hemp seed oil matrix highlights the urgent need to find reference parameters that define
the quality of this product. Moreover, although in some European countries hemp seed oil has been a
product on the market for years, in other countries, such as Italy, hemp seed oil can be considered a
young product, with no history of production.
These results, together with other data recently reported on hemp oils [7], can constitute the
starting point for development, and drawing up, of harmonized guidelines with suitable quality and
safety parameters specific for hemp seed oils. Furthermore, these studies might drive producers to
standardize procedures of hemp seed oil production, guaranteeing the achievement of a good food
objective, consumer safety, and the further expansion of the hemp food industry.

Author Contributions: L.M. (Luisa Mannina) and G.V.; methodology: A.P.S., A.D.S., S.D.G., A.M.G., S.C.
(Salvatore Ciano), S.C. (Simone Carradori), M.R., S.C. (Stefania Cesa), and C.I.; validation: M.S. and G.D.M.; formal
analysis: M.R. and S.C. (Salvatore Ciano); investigation: A.P.S., C.I., A.D.S., S.C. (Salvatore Ciano), L.M. (Luigi
Menghini), and G.V.; data curation: A.P.S., C.I., A.D.S., S.C. (Stefania Cesa), A.M.G., and G.V.; writing—original
draft preparation: G.V.; writing—review and editing: G.V. and L.M. (Luisa Mannina); visualization: M.S., G.D.M.,
A.D.S., S.C. (Salvatore Ciano), S.C. (Simone Carradori), and G.V.; supervision: L.M. (Luisa Mannina) and G.V.,
project administration and funding acquisition: L.M. (Luisa Mannina). All authors have read and agreed to the
published version of the manuscript.
Funding: This work has been realized with funds received from the following agencies: Italian Ministry of
Education, Universities and Research—Dipartimenti di Eccellenza—L. 232/2016; Regione Lazio, “LACanapa”
Project (Progetto di Ricerca, finanziato ai sensi della L.R. 13/08 -Protocol 85-2017-15069 CUP: B86C18000730002).
Acknowledgments: This work is part of a project supported by Lazio Region entitled “La Canapa industriale:
sviluppo e valorizzazione di una nuova filiera agroalimentare ecosostenibile”.
Appl. Sci. 2020, 10, 6933 13 of 15

Conflicts of Interest: The authors declare no conflict of interest.

References
1. TNI. The UN Drug Control Conventions. Available online: https://www.tni.org/en/publication/the-un-drug-
control-conventions#box3 (accessed on 6 September 2020).
2. European Commission. EU Plant Variety Database. Available online: https://ec.europa.eu/food/
plant/plant_propagation_material/plant_variety_catalogues_databases/search/public/index.cfm?event=
SearchVariety&ctl_type=A&species_id=240&variety_name=&listed_in=0&show_current=on&show_
deleted= (accessed on 6 September 2020).
3. Ministero della Salute. Produzione e Commercializzazione di Prodotti a Base di Semi di Canapa Per L’utilizzo nci
Settori Dell’alimentazione Umana; Ministero della Salute: Roma, Italy, 2009; pp. 1–4.
4. Jang, E.; Kim, H.; Jang, S.; Lee, J.; Baeck, S.; In, S.; Kim, E.; Kim, Y.; Han, E. Concentrations of THC, CBD,
and CBN in commercial hemp seeds and hempseed oil sold in Korea. Forensic Sci. Int. 2020, 306, 110064.
[CrossRef] [PubMed]
5. Farinon, B.; Molinari, R.; Costantini, L.; Merendino, N. The seed of industrial hemp (Cannabis sativa L.):
Nutritional Quality and Potential Functionality for Human Health and Nutrition. Nutrients 2020, 12, 1935.
[CrossRef] [PubMed]
6. Xu, Y.; Li, J.; Zhao, J.; Wang, W.; Griffin, J.; Li, Y.; Bean, S.; Tilley, M.; Wang, D. Hempseed as A Nutritious and
Healthy Human Food or Animal Feed Source: A Review. Int. J. Food Sci. Technol. 2020, 14755. [CrossRef]
7. Izzo, L.; Pacifico, S.; Piccolella, S.; Castaldo, L.; Narváez, A.; Grosso, M.; Ritieni, A. Chemical Analysis of
Minor Bioactive Components and Cannabidiolic Acid in Commercial Hemp Seed Oil. Molecules 2020, 25,
3710. [CrossRef] [PubMed]
8. Smeriglio, A.; Galati, E.M.; Monforte, M.T.; Lanuzza, F.; D’Angelo, V.; Circosta, C. Polyphenolic Compounds
and Antioxidant Activity of Cold-Pressed Seed Oil from Finola Cultivar of Cannabis sativa L. Phytother. Res.
2016, 30, 1298–1307. [CrossRef]
9. Crescente, G.; Piccolella, S.; Esposito, A.; Scognamiglio, M.; Fiorentino, A.; Pacifico, S. Chemical composition
and nutraceutical properties of hempseed: An ancient food with actual functional value. Phytochem. Rev.
2018, 17, 733–749. [CrossRef]
10. Pojić, M.; Mišan, A.; Sakač, M.; Hadnad̄ev, T.D.; Šarić, B.; Milovanović, I.; Hadnad̄ev, M. Characterization of
Byproducts Originating from Hemp Oil Processing. J. Agric. Food Chem. 2014, 62, 12436–12442. [CrossRef]
11. Ustun-Argon, Z. Phenolic Compounds, Antioxidant Activity and Fatty Acid Compositions of Commercial
Cold-Pressed Hemp Seed (Cannabis Sativa L.) Oils from Turkey. Int. J. Sci. Eng. Res. 2019, 10, 166–173.
12. Callaway, J.; Schwab, U.; Harvima, I.; Halonen, P.; Mykkänen, O.; Hyvönen, P.; Järvinen, T. Efficacy of dietary
hempseed oil in patients with atopic dermatitis. J. Dermatol. Treat. 2005, 16, 87–94. [CrossRef]
13. Rodriguez-Leyva, D.; Pierce, G.N. The cardiac and haemostatic effects of dietary hempseed. Nutr. Metab.
2010, 7, 32. [CrossRef]
14. Ghirga, F.; Quaglio, D.; Ghirga, P.; Berardozzi, S.; Zappia, G.; Botta, B.; Mori, M.; D’Acquarica, I. Occurrence of
Enantioselectivity in Nature: The Case of (S)-Norcoclaurine. Chirality 2016, 28, 169–180. [CrossRef] [PubMed]
15. Carus, M.; Karst, S.; Kauffmann, A. The European Hemp Industry: Cultivation, Processing and Applications for
Fibres, Shivs and Seeds; European Industrial Hemp Assocaition: Hürth, Germany, 2016.
16. Skoczinski, P.; Carus, M.; Grotenhermen, F.; Beitzke, B.; Kruse, D. Limit and Guideline Values for THC
(Tetrahydrocannabinol) in Hemp Foods; European Industrial Hemp Assocaition: Hürth, Germany, 2019.
17. Ministero della Salute. DECRETO 4 Novembre 2019 Definizione di Livelli Massimi di Tetraidrocannabinolo (THC)
Negli Alimenti; Ministero della Salute: Roma, Italy, 2019; pp. 1–5.
18. Berardozzi, S.; Bernardi, F.; Infante, P.; Ingallina, C.; Toscano, S.; De Paolis, E.; Alfonsi, R.; Caimano, M.;
Botta, B.; Mori, M.; et al. Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli
antagonists bearing the isoflavone scaffold. Eur. J. Med. Chem. 2018, 156, 554–562. [CrossRef] [PubMed]
19. Di Marco Pisciottano, I.; Guadagnuolo, G.; Soprano, V.; De Crescenzo, M.; Gallo, P. A rapid method
to determine nine natural cannabinoids in beverages and food derived from Cannabis sativa by liquid
chromatography coupled to tandem mass spectrometry on a QTRAP 4000. Rapid Commun. Mass Spectrom.
2018, 32, 1728–1736. [CrossRef] [PubMed]
Appl. Sci. 2020, 10, 6933 14 of 15

20. Meng, Q.; Buchanan, B.; Zuccolo, J.; Poulin, M.-M.; Gabriele, J.; Baranowski, D.C. A reliable and validated
LC-MS/MS method for the simultaneous quantification of 4 cannabinoids in 40 consumer products. PLoS ONE
2018, 13, e0196396. [CrossRef]
21. Christinat, N.; Savoy, M.-C.; Mottier, P. Development, validation and application of a LC-MS/MS method for
quantification of 15 cannabinoids in food. Food Chem. 2020, 318, 126469. [CrossRef]
22. Citti, C.; Linciano, P.; Panseri, S.; Vezzalini, F.; Forni, F.; Vandelli, M.A.; Cannazza, G. Cannabinoid Profiling of
Hemp Seed Oil by Liquid Chromatography Coupled to High-Resolution Mass Spectrometry. Front. Plant Sci.
2019, 10, 120. [CrossRef]
23. Anwar, F.; Latif, S.; Ashraf, M. Analytical characterization of hemp (Cannabis sativa) seed oil from different
agro-ecological zones of Pakistan. J. Am. Oil Chem. Soc. 2006, 83, 323–329. [CrossRef]
24. Chen, T.; He, J.; Zhang, J.; Zhang, H.; Qian, P.; Hao, J.; Li, L. Analytical Characterization of Hempseed (Seed
of Cannabis sativa L.) Oil from Eight Regions in China. J. Diet. Suppl. 2010, 7, 117–129. [CrossRef]
25. Dimić, E.; Romanić, R.; Vujasinović, V. Essential fatty acids, nutritive value and oxidative stability of cold
pressed hempseed (Cannabis sativa L.) oil from different varieties. Acta Aliment. 2009, 38, 229–236. [CrossRef]
26. Abdollahi, M.; Sefidkon, F.; Calagari, M.; Mousavi, A.; Mahomoodally, M.F. A comparative study of seed
yield and oil composition of four cultivars of Hemp (Cannabis sativa L.) grown from three regions in northern
Iran. Ind. Crop. Prod. 2020, 152, 112397. [CrossRef]
27. Devi, V.; Khanam, S. Optimization of the Ratio of ω-6 Linoleic and ω-3 α-Linolenic Fatty Acids of Hemp
Seed Oil with Jackknife and Bootstrap Resampling. Chem. Prod. Process. Model. 2019, 15. [CrossRef]
28. Da Porto, C.; Decorti, D.; Tubaro, F. Fatty acid composition and oxidation stability of hemp (Cannabis sativa L.)
seed oil extracted by supercritical carbon dioxide. Ind. Crop. Prod. 2012, 36, 401–404. [CrossRef]
29. European Parliament and Council. Regulation (EC) No 178/2002 Reguarding the General Principles and
Requirements of Food Law, Establishing the European Food Safety Authority and Laying down Procedures
in Matters of Food Safety. Off. J. 2002, 31.
30. Mannina, L.; Sobolev, A.P.; Viel, S. Liquid state 1H high field NMR in food analysis. Prog. Nucl. Magn.
Reson. Spectrosc. 2012, 66, 1–39. [CrossRef]
31. Mannina, L.; D’Imperio, M.; Gobbino, M.; D’Amico, I.; Casini, A.; Emanuele, M.C.; Sobolev, A.P.
Nuclear magnetic resonance study of flavoured olive oils. Flavour Fragr. J. 2012, 27, 250–259. [CrossRef]
32. Rapa, M.; Ciano, S.; Rocchi, A.; D’Ascenzo, F.; Ruggieri, R.; Vinci, G. Hempseed Oil Quality Parameters:
Optimization of Sustainable Methods by Miniaturization. Sustainability 2019, 11, 3104. [CrossRef]
33. European Commission. COMMISSION DELEGATED REGULATION (EU) 2015/1830 of 8 July 2015 amending
Regulation (EEC) No 2568/91 on the characteristics of olive oil and olive-residue oil and on the relevant
methods of analysis. Off. J. 2015, L266, 9–13.
34. British Standards Institution Animal and Vegetable Fats and Oils. Determination of Anisidine Value.
Available online: https://www.iso.org/standard/40052.html (accessed on 6 September 2020).
35. Patsilinakos, A.; Ragno, R.; Carradori, S.; Petralito, S.; Cesa, S. Carotenoid content of Goji berries: CIELAB,
HPLC-DAD analyses and quantitative correlation. Food Chem. 2018, 268, 49–56. [CrossRef]
36. Cairone, F.; Carradori, S.; Locatelli, M.; Casadei, M.A.; Cesa, S. Reflectance colorimetry: A mirror for food
quality—A mini review. Eur. Food Res. Technol. 2020, 246, 259–272. [CrossRef]
37. Ingallina, C.; Sobolev, A.P.; Circi, S.; Spano, M.; Giusti, A.M.; Mannina, L. New Hybrid Tomato Cultivars:
An NMR-Based Chemical Characterization. Appl. Sci. 2020, 10, 1887. [CrossRef]
38. Di Sotto, A.; Di Giacomo, S.; Toniolo, C.; Nicoletti, M.; Mazzanti, G. Sisymbrium Officinale (L.) Scop. and its
Polyphenolic Fractions Inhibit the Mutagenicity of Tert-Butylhydroperoxide in Escherichia Coli WP2 uvr AR
Strain. Phytother. Res. 2016, 30, 829–834. [CrossRef] [PubMed]
39. European Directorate for the Quality of Medicines & HealthCare. Council of Europe European Pharmacopoeia,
9th ed.; European Directorate for the Quality of Medicines & HealthCare: Strasbourg, France, 2016.
40. Teh, S.-S.; Birch, J. Physicochemical and quality characteristics of cold-pressed hemp, flax and canola seed
oils. J. Food Compos. Anal. 2013, 30, 26–31. [CrossRef]
41. Lutterodt, H.; Luther, M.; Slavin, M.; Yin, J.-J.; Parry, J.; Gao, J.-M.; Yu, L. Fatty acid profile, thymoquinone
content, oxidative stability, and antioxidant properties of cold-pressed black cumin seed oils. LWT Food
Sci. Technol. 2010, 43, 1409–1413. [CrossRef]
42. Borhade, S.S. Chemical Composition and Characterization of Hemp (Cannabis sativa) Seed oil and essential
fatty acids by HPLC Method. Arch. Appl. Sci. Res. 2013, 5, 5–8.
Appl. Sci. 2020, 10, 6933 15 of 15

43. Rezvankhah, A.; Emam-Djomeh, Z.; Safari, M.; Askari, G.; Salami, M. Microwave-assisted extraction of
hempseed oil: Studying and comparing of fatty acid composition, antioxidant activity, physiochemical and
thermal properties with Soxhlet extraction. J. Food Sci. Technol. 2019, 56, 4198–4210. [CrossRef]
44. Oomah, B.D.; Busson, M.; Godfrey, D.V.; Drover, J.C. Characteristics of hemp (Cannabis sativa L.) seed oil.
Food Chem. 2002, 76, 33–43. [CrossRef]
45. Da Porto, C.; Decorti, D.; Natolino, A. Potential Oil Yield, Fatty Acid Composition, and Oxidation Stability of
the Hempseed Oil from FourCannabis sativaL. Cultivars. J. Diet. Suppl. 2015, 12, 1–10. [CrossRef]
46. Cesa, S.; Sisto, F.; Zengin, G.; Scaccabarozzi, D.; Kokolakis, A.K.; Scaltrito, M.M.; Grande, R.; Locatelli, M.;
Cacciagrano, F.; Angiolella, L.; et al. Phytochemical analyses and pharmacological screening of Neem oil.
S. Afr. J. Bot. 2019, 120, 331–337. [CrossRef]
47. Singh, A.P.; Fathordoobady, F.; Guo, Y.; Singh, A.; Kitts, D.D. Antioxidants help favorably regulate the kinetics
of lipid peroxidation, polyunsaturated fatty acids degradation and acidic cannabinoids decarboxylation in
hempseed oil. Sci. Rep. 2020, 10, 1–12. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).