Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

SOP TRANSACTIONS ON ANALYTICAL CHEMISTRY VOLUME 1, NUMBER 1, JUNE 2014 SOP TRANSACTIONS ON ANALYTICAL CHEMISTRY Analysis of Intact Triacylglycerols ...
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SOP TRANSACTIONS ON ANALYTICAL CHEMISTRY VOLUME 1, NUMBER 1, JUNE 2014

SOP TRANSACTIONS ON ANALYTICAL CHEMISTRY

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS Tanyaradzwa E. Mungure, Edward J. Birch* Department of Food Science, University of Otago, PO Box 56, Dunedin, 9054, New Zealand. *Corresponding author: [email protected]

Abstract: Cold-pressed canola, hemp and flax seed oils were separated into their respective intact triglycerides via high pressure liquid chromatography (HPLC) and their fatty acids analysed. Molecular weights of the triglycerides were determined by electrospray ionization-mass spectroscopy (ESI-MS). Seven triglycerides were collected for flax seed, and trilinolenoyl-glycerol was the most abundant at 25.9%. Seven triglycerides were separated and collected for canola seed oil with dioleoyl-linolenoyl-glycerol as the most abundant (32.5%). Eight triglycerides were separated and collected from hemp seed oil with trilinoleoyl-glycerol as the most abundant (21.9%). The fatty acid profiles of the separated triglycerides for the three oils were determined to confirm the assignments. Hempseed was found to have the highest content of palmitic acid in the triglyceride fractions collected (5.6%); flax and canola had 4.1% and 3.1% respectively. The results of fatty acid composition analysis of the cold pressed oils showed oleic acid to be the most abundant fatty acid in canola oil at 57.56 ± 0.13%. Linoleic acid was the most abundant in hemp seed oil at 55.64 ± 1.21%, and linolenic acid in flax seed oil at 58.84 ± 0.91%. Enzymatic hydrolysis using pancreatic lipase treatment was performed to analyse positional distribution of fatty acids in the oils. Thin layer chromatography (TLC) was used for separation of 2-monoglycerides and free fatty acids. For all three oils, unsaturated fatty acids marginally preferred the sn-2 position on the glycerol backbone. Flaxseed had 59.1% of linolenic acid on sn-2 position. At 58.6%, oleic acid was the most abundant fatty acid at the sn-2 position in canola oil, and for linoleic acid in hemp seed oil, 58.2%. Higher ratios of saturated fatty acids to unsaturated fatty acids were located on sn-1, 3 positions. Keywords: Canola; ESI-MS; Flax; Hemp; HPLC; Triglyceride Analysis

1. INTRODUCTION Abundant information has been gathered on the physicochemical and quality characteristics of canola, hemp and flax seed oils, however information on the positional distribution of fatty acids on intact triglycerides has been very limited. Characterization of the triglycerides in plant oils is important for nutritional and industrial use [1]. Determination of the level of unsaturation in these oils has been done 48

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

previously [2] but it is also important to establish the link with positional distribution of fatty acids in the triglycerides. Increased understanding of this will assist in determining other characteristics of the oils; for example purity, stability in deep frying and other food preparation purposes. Knowledge of the fatty acids involved will also help explain variable thermal properties such as melting, boiling points and crystallization of the oils [3]. When the fatty acid profile of intact triglycerides has been established, it can be used as a reference in investigative analysis of oil adulteration. These three cold pressed oils have been used in the food and industrial sectors for many years. Flaxseed oil (Linum usitatissimum) has been used for 8000 years and the people of Neolithic tribes in Europe are credited for its establishment [4]. Flaxseed popularity emanates from its known health benefits and healing abilities as recorded in ancient Greek and Roman literature [5]. Besides its use as a food supplement, flaxseed has been used for various purposes including surface coating, paint production and oleo chemicals [6]. The composition of flaxseed shows that it has 35% lipids, 20-30% proteins, 35-50% fibre and 6% ash by weight [6]. Flaxseed oil is one of the richest sources of a-linolenic acid making over 50% of the total fatty acid composition [6]. Linolenic with linoleic acid are identified as essential fatty acids in the human diet; consumption of these is essential as the body cannot synthesise them [7]. Canola oil is a low erucic acid rapeseed oil (LEAR) product. It has high content of oleic acid and a favourable linoleate / linolenate ratio with low saturated fatty acid content. Canola oil contains approximately 58% oleic acid and 10% linoleic acid [8] which could explain its health benefits [9]. Hemp seed can be tracked to 6500 years ago in central Asia [10]. It has been used extensively in food sources and utilized in other sectors such as clothing and making of fish nets [6]. Hemp seed is an achene containing 25-35% lipid, 20-30% carbohydrates, 20-30% protein and 10-15% insoluble fibre [6]. Hemp seed oil is known to have a 2:1 ratio of linoleic acid (C18:2; n-6) and a-linolenic acid (C18:3; n-3) [10]. The objectives of this research were to separate the oil (canola, flax and hemp seed) into their triglycerides and establish the fatty acid distribution and analyse the positional distribution of fatty acids in the oils.

2. METHODS 2.1 HPLC Separation of Triglycerides in Oils The oil samples were diluted to a ratio of 1:10 with 2-propanol (CH3 CHOHCH3 ). The separation of triglycerides was performed on a C18 Phenosphere-Next column (250⇥ 4.6, 5µm; Phenomenex, Torrance, CA, USA) using an Agilent 1260 infinity Bio-inert Quaternary HPLC with a Diode array detector G4212A with bio-inert flow cells 10mm G4212-60008. Collection of separated triglycerides was done using the Agilent 1260 Infinity Analytical Bio-inert fraction collector (G5664A). Detection of components was achieved at wavelengths of 205nm for hempseed oil, and 215nm for canola and flaxseed oils. An injection of 2µl sample was introduced with gradient system using an acetonitrile/methanol (HPLC-grade) mixture of ratio 7:5 (volume: volume) in solvent line A and 2-propanol as solvent line B. The flow rate was set at 1ml/min. Elution commenced with an initial flow of 100% solvent line A for 10 minutes followed by 20 minutes linear gradient to 100% solvent line B. This gradient was upheld for a further 10 minutes before the run completed when the column was flushed for 10 minutes with mobile phase B. ChemStation (Agilent Technologies, Santa Clara, CA, USA) was used as the analyzing software. The above was adopted with modifications from Choo [11]. The collected fractions were analyzed using the Electrospray ionization-mass spectrometry (ESI-MS) system Bruker microTOF-Q (Bruker Daltronics, Bremen, Germany). Fractions were introduced by direct infusion into the ESI source in a positive mode with 49

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collision energy of 10eV. Dry N2 gas at 99%, 180 C with -4.5kV capillary voltage was used. Sampling was averaged for 3 minutes over an m/z range of 100-1000amu. An external calibrant of formate clusters was used to calibrate the mass. Compass software version 1.3 (Bruker Daltronics, Bremen, Germany) was used to analyze and process spectra.

2.2 Total Fatty Acid Composition of Canola, Hempseed and Flaxseed Oils A modified Van Wijngaarden (1967) method [12] was used for fatty acid methylation. Oil sample (0.1 ml) was thoroughly dissolved in 10ml of hexane, and 2ml transferred to a glass tube. 0.5N methanolic KOH (2ml) (prepared by adding 5.6g of KOH into 200ml methanol with 15minutes stirring) was added into the glass tube and heated for 20 minutes at 80 C. Diethyl ether (2ml) and 5ml of water were added to the glass tube and the two phases separated. The diethyl ether layer (upper phase) was discarded. 5-7 drops of HCI (32%, Fisher Scientific, Leistershire, England) and 2ml of diethyl ether were added into the glass tube. Two layers of separation were formed and the top diethyl ether layer collected. 1ml of boron trifluoride (BF3 14% in methanol; Sigma-Aldrich, Inc., USA) was added into the new glass tube and heated for 20 minutes at 80 C. 5ml of NaCl (BDH Laboratory Supplies, England) were added to the heated solution and vortexed for 1 minute. The two phases were separated and the FAME (Fatty Acid Methylesterification) phase (upper phase) was collected into a vial for GC analysis.

2.3 Pancreatic Lipase Treatment A modified method from Lawson and Hughes [13] was used to analyse the positional distribution of fatty acids. Oil sample (0.1ml) was added into a test tube and mixed with 3ml of 1M of tris (hydroxymethyl)amino methane pH 8.0,0.15M calcium chloride and 0.01% bile salts (DILFCO Laboratories, USA).200mg pancreatic lipase, Type II (Sigma-Aldrich Inc., USA) was mixed with 5ml of tris buffer ,and 0.5ml of this mixture was added into the test tube. The test tube was incubated in shaking incubator (Ratex Orbital Mixer Incubator, Ratek Instruments Private Limited, Australia) at 37 C, 250rpm for 8 minutes. 3ml of diethyl ether (Riedel-de Haen, Seelze, Germany) was added to the test tube and left to settle into two layers. The top layer (diethyl ether layer) was recovered and the extraction process repeated twice with 3ml of diethyl ether. The combined diethyl ether extract was backwashed with 2ml of milli-Q water and transferred into another test tube and concentrated under a stream of nitrogen gas. A glass silica gel plate (silica gel 60 F254 ; Merck KGaA, Germany) was washed with hexane-diethyl ether solution with volume ratios 50:50 before spotting it with samples. Separation of oil components was done in a TLC vessel with hexane-diethyl ether-acetic acid with volume ratios of 50:50:1. A solvent pad was placed on the TLC vessel to bring the system to equilibrium and aid the solvent flow. After running the full TLC plate, it was heated with a blow dryer to visualise the separated components. The TLC plate was placed under UV light to observe the fractions. The Monoglycerides and free fatty acids were encircled with a pencil and scraped off the plate for methyl esterification process and GC analysis.

2.4 Fatty Acid Methyl Ester Preparation for Monoglycerides (MGs) from Thin Layer Chromatography (TLC) 3 ml of a 6% (by volume) sulphuric acid in methanol solution were added to the scraped MG silica band as a methylating reagent. Fatty acids were methylated by acid-catalysed transesterification at 80o C 50

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

Table 1. Fatty Acid Composition of Cold Pressed Flax Seed Oil by FAME Analysis on GC-MS Flaxseed oil fatty acids

Percentage (%)

C16:0

Palmitic

C16:1 n7

Palmitoleic

5.76 ± 0.09

C18:0

Stearic

C18:1n9c

Oleic

C18:2n6t

Linolelaidic

C18:2n6c

Linoleic

C18:3n6

gamma-Linolenic (cis 6, 9, 0.23 ± 0.16 12)

C18:3n3

alpha-Linolenic (cis 9, 12, 58.84 ± 0.91 15)

C20:0

Arachidic

0.11 ± 0.02 2.70 ± 0.18

15.13 ± 0.41 0.67 ± 011

16.20 ± 0.68

0.37 ± 0.02

Unsaturated fatty acids 91.18 ± 0.39 Values are mean ± standard deviations of three measurements

Table 2. Percentage of Fatty Acid and Their Positional Distribution in Flax Seed Oil After Pancreatic Lipase Treatment Fatty acid

Flaxseed oil sn-2 (%) sn-1,3 (%) (%)

C16:0

5.8

3.4

C18:0

2.7

1.6

3.25

C18:1n-9

15.1

19.0

13.15

C18:2n-6

17.3

16.8

18.45

C18:3n-3

58.8

59.1

58.35

7.00

for 12 hours. After cooling to room temperature, 2 ml of hexane followed by 1 ml of water were added to the sample with 2 minutes of vortexing separating each addition. The upper hexane layer containing the fatty acid methyl esters (FAMEs) was removed and stored at -20o C until gas chromatographic analysis. For the oil triglyceride fractions,3.4.3 Fatty acid methyl ester preparation 3 ml of 6% (by volume) sulphuric acid in methanol solution was added to each fraction as a methylating agent. Fatty acids were methylated by acid-catalysed transesterification at 80 C for 4 hours. After cooling to room temperature, 2 ml of hexane followed by 1 ml of water were added to the sample with 10 seconds of vortexing separating each addition. The upper hexane layer containing the fatty acid methyl esters (FAMEs) was removed and stored at -20 C until gas chromatographic analysis.

2.5 Fatty Acid Methyl Ester Preparation for Free Fatty Acids (FFAs) from Thin Layer Chromatography (TLC) Methanol (2ml) and 1 ml of Boron trifluoride (BF3 14% in methanol; Sigma-Aldrich, Inc., USA) were added to scraped FFA silica band in a test tube with screw cap. The mixture was heated for 20 minutes at 80 C using a heating block. Diethyl ether (2ml) and 5ml of NaCl (BDH Laboratory Supplies, England) were added to the heated solution and vortexed for 1 minute before the solution was left to cool and settle into two layers. The top layer (diethyl ether layer) was extracted and transferred into 2ml vials for GC analysis.

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Figure 1.

The Seven Triglycerides Separated and Collected from Flaxseed Oil Via HPLC

2.6 Gas Chromatography Analysis on FAMEs The hexane in which the FAMEs were dissolved was evaporated under a stream of nitrogen and the FAMEs reconstituted in 45 µl of hexane for analysis of fatty acid composition by gas chromatography (GC). FAMEs were separated using a BPX-70 capillary column, 100m⇥0.22 mm i.d, 0.25 µm films (SGE, Melbourne, Australia). The gas chromatographic system consisted of a 6890N GC equipped with an autosampler (HP7673) and ChemStation integration (all Hewlett Packard, Avondale, PA).The column oven was held at an initial temperature of 165 C for 52 minutes, then increased at a rate of 5 C per minute to a final temperature of 210 C for 59 minutes (total run time 120 minutes). Both the injector and flame ionization detector ports were at 250 C. Carrier gas flow (helium) was maintained at 1.0 ml per min (linear gas velocity 20 cm per sec) throughout the temperature program with an inlet split ratio of 30:1. Fatty acid peaks were identified by retention time matching with authentic standards. A composite standard was made from commercially available methyl esters (NuCheck Prep, Elysian, Minnesota and Sigma, St. Louis, Missouri)

3. RESULTS and DISCUSSION 3.1 Flax seed oil triglyceride characterisation (1) Total Fatty Acid Profile for Cold Pressed Flax Seed Oil a-Linolenic acid (C18:3n-3) was the most dominant fatty acid in flaxseed oil with 58.84% abundance. (Table 1). Linoleic acid was found to be the second most abundant fatty acid with 16.20%. The fatty acid profile of flax seed oil has been determined in the past [1] and the following results conform to previous findings. (2) Positional Distribution of Fatty Acids in Cold-pressed Flax Seed Oil On the sn-2 position, a-linolenic acid was the most abundant. The results of the GC analysis of fatty 52

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

Figure 2. Mass Spectrograms for the First Triglyceride Peak Eluting at 28.048min from Flaxseed Oil. The Lower Spectrogram Shows Magnification of the Molecular Ion Region

acids on 2-monoglycerides showed that C18:1, C18:2 and C18:3 (the unsaturated fatty acids) made up over 94 % made of the fatty acid present. The Table 2 below highlights the distribution. This data shows that oleic acid in flax seed oil has a preference for the sn-2 position with the PUFA being similar. Previous studies on other seed oils suggest the same trend for monounsaturated fatty acids [14]. The saturated fatty acids in flaxseed oil preferred position 1, 3. The ratio of saturated fatty acids on sn-2 to sn-1, 3 was almost 1:2. (3) Flax Seed Oil Triglyceride Separation Via HPLC and ESI-MS Seven separated triglyceride peaks were detected at l = 215nm. The chromatogram in Figure 1 shows the distribution of the triglyceride peaks and Table 3 summarizes their identities. Elution order was characterised according to partition number (PN) (PN = CN-2n). CN (carbon number) is the total number of fatty acid carbons and n is the double bond number. The smaller the PN is, the shorter the elution time [15]. ESI-MS results and Choo et al [11] work on unreacted OSELO flaxseed triacylglycerols were used as a reference to identify the triglyceride peaks. An example of the mass spectrogram is illustrated in Figure 2 with a single (M+23) + ion from mass spectral analysis. Secondary peaks with a second sodated molecular ion were observed on mass spectrograms as in Table 3. Figure 3 shows a spectrogram for triglycerides eluting at HPLC retention time 30.278 minutes. The two triglycerides (trilinoleoyl-glycerol and linolenoyl-linoleoyl-palmitoyl-glycerol) have the same PN of 42 explaining the co-elusion [16]. Any stearic acid present would be in a corresponding minor peak of lesser saturation having the same molecular ion and PN number as the major peak component. This could occur from PN 42 onwards.

3.2 Canola Oil Triglyceride Characterisation (1) Total Fatty Acid Profile of Cold Pressed Canola Oil The FAME process showed that oleic acid was the most abundant fatty acid in canola oil constituting 57.56%. This is supports previous work [2] which focused on determining the physicochemical properties 53

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Table 3. Summarises Details for Retention Times of the Flax Seed Oil Triglycerides and Their Respective Molecular Weights from Mass Spectral Analysis HPLC Triglyceride name retention time (min)

% abun- Molecular ESI-MS Partition Double dance weight (M+23)+ Number bonds (PN) (n)

28.048

Trilinolenoyl-glycerol LnLnLn*

25.9

872

895

36

9

28.794

Dilinolenoyl-linoleoyl-glycerol LnLnL*

17.1

874

897

38

8

29.616

Dilinolenoyl-oleoyl-glycerol LnLnO*

21.4

876

899

40

7

30.278

Trilinoleoyl-glycerol LLL* (linolenoyl-linoleoylpalmitoylglycerol LnLP*)

14.0

878

901

42

6

30.977

Dioleoyl-linolenoyl-glycerol LnOO* (linolenoyl-palmitoyl-oleoylglycerol LnPO*)

9.8

880

903

44

5

31.660

Dioleoyl-linoleoyl-glycerol LOO* 2.7 (linoleoyl-palmitoyl-oleoylglycerol LPO*)

882

905

46

4

32.168

Trioleoyl-glycerol OOO* (dioleoyl-palmitoyl-glycerol POO*)

884

907

48

3

2.3

*P is Palmitic acid C16:0, *S is Stearic acid C18:0, * O is Oleic acid C18:1, L is Linoleic acid C18:2 and Ln is Linolenic acid C18:3. Note: (M+23)+ represents protonated molecular ion with a sodium (mass = 23). ( ) = secondary peaks

Figure 3. Mass Spectrogram for the Fourth Triglyceride Peak Eluting at 30.278min from Flaxseed Oil with Multiple Sodated Molecular Ions 54

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

Table 4. Fatty Acid Composition of Cold Pressed Canola Oil by FAME Analysis on GC Canola oil fatty acids

Percentage (%)

C16:0

Palmitic

C16:1n7

Palmitoleic

4.75 ± 0.11

C18:0

Stearic

C18:1n9c

Oleic

C18:2n6c

Linoleic

C18:3n3

a-Linolenic (cis 9, 12, 15) 11.47 ± 0.02

C20:0

Arachidic

C20:1n9

Eicosenoic

0.26 ± 0.06

1.43 ± 0.01

57.56 ± 0.13 22.67 ± 0.09 0.53 ± 0.09

1.12 ± 0.16

Unsaturated fatty acids 93.02 ± 0.24 Values are mean ± standard deviations of three measurements

Table 5. Percentage of Fatty Acid and Their Positional Distribution in Canola Oil after Pancreatic Lipase Treatment Fatty acid

Canola oil (%)

sn-2 (%) sn-1,3 (%)

C16:0

4.75

2.3

5.98

C18:0

1.43

2.3

1.00

C18:1n-9

57.56

58.6

57.04

C18:2n-6

22.67

24.7

21.65

C18:3n-3

11.47

12.1

11.16

of canola oil. Canola oil is therefore predominantly made of monounsaturated fatty acids (MUFA). The distribution of fatty acids on the various triacylglycerols is summarized in Table 4. Canola oil has more unsaturated fatty acids (93.02%) compared to flax seed (91.19%) however flax seed oil is higher in polyunsaturated fatty acids (PUFA) rather than MUFA. (2) Positional Distribution of Fatty Acids in Cold-pressed Canola Oil Canola oil preferred oleic acid on sn-2 position at 58.6% (Table 5). The unsaturated fatty acids all showed slightly higher percentages on sn-2 position compared to the sn-1, 3 positions. It is interesting to note that stearic acid was higher on sn-2 position than on sn-1, 3 positions. This is an unusual phenomenon in seed oils as saturated fatty acids are expected to be higher on the outer positions [14]. (3) Canola Seed Oil Triglyceride Separation Via HPLC and ESI-MS Figure 5 shows the triglycerides separated and collected from canola seed oil, summarized in Table 6. A similar range as was found for flax seed oil was determined. The differences in peak intensities reflect the relative ratios of fatty acids present.

3.3 Hemp Seed Oil Triglyceride Characterisation (1) Total Fatty Acid Profile of Cold Pressed Hemp Seed Oil The fatty acid composition (Table 7) shows that linoleic acid (C18:2n-6c) was the most abundant at 55.64% and a-linolenic (C18:3n-3) the second most abundant at 18.02%. Teh &Birch [2] performed this analysis with similar results. It is interesting to note that gamma-linolenic acid (C18:3n-6) (an isomer) was fairly abundant making up 5.30% of the fatty acid profile. (2) Positional Distribution of Fatty Acids in Cold-pressed Hemp Seed Oil Table 8 shows the positional distribution on hemp seed oil triglycerides. Oleic and linoleic acids were enriched at the sn-2 position as expected [15] however linolenic acid was not. Saturated fatty acids commonly show preference for sn-1 & 3 positions as observed here. 55

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Figure 4. Mass Spectrogram for the Fourth Triglyceride Peak Eluting at 30.278min from Flaxseed Oil with Multiple Sodated Molecular Ions

Figure 5. The Seven Triglycerides Separated and Collected from Canola Seed Oil Via HPLC

(3) Hemp seed oil triglyceride separation via HPLC and ESI-MS The 8 triglyceride fractions collected for mass spectral analysis are depicted in Figure 6 and summarised in Table 9. The identities followed a similar pattern to flax and canola oils although the last two peaks contained different components. The double peaks observed (Figure 6) were probably due to the high presence of linolenic acid in the glycerol with a mix of a- and g-linolenic acid leading to slight differences in retention times due to differences in polarity [9]. Triacylglycerols containing fatty acids with a- and g-linolenic acids cannot be distinguished by ESI-MS detection due to their similar molecular weights but differences in polarity can be separated using silver ion chromatography [17]. 56

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

Table 6. Summary of HPLC and ESI-MS Data for Canola Oil Triglycerides HPLC Triglyceride retention time

% abundance Molecular weight

ESI-MS Partition Double (M+23)+ Number bonds (PN) n

26.162

Trilinolenoyl-glycerol (LnLnLn)* 1.8

872

895

36

9

27.140

Dilinolenoyl-oleoyl-glycerol (LnLnO)*

10.9

876

899

40

8

27.977

Trilinoleoyl-glycerol (LLL)* (linolenoyl-linoleoyl-palmitoylglycerol) LnLP*

19.1

878

901

42

7

28.898

Dioleoyl-linolenoyl-glycerol (LnOO)* (Linolenoyl-oleoyl-glycerol) LnOP*

32.5

880

903

44

6

29.755

Dioleoyl-linoleoyl-glycerol 18.6 (LOO)* (linoleoyl-oleoyl-glycerol) LOP*

882

905

46

5

30.543

Trioleoyl-glycerol (OOO)* (dioleoyl-palmitoyl-glycerol) POO*

10.0

884

907

48

4

31.264

Dioleoyl-stearoyl-glycerol (SOO)*

3.5

886

909

50

3

*P is Palmitic acid, *S is Stearic acid C18:0, O is Oleic acid C18:1, L is Linoleic acid C18:2 and Ln is Linolenic acid C18:3.Note: (M+23) + represents protonated molecular ion with a sodium (mass =23). ( ) = secondary peaks.

Table 7. Fatty Acid Composition of Hemp Seed Oil by FAME Analysis on GC-MS Hempseed oil fatty acids

Percentage (%)

C16:0

Palmitic

C18:0

Stearic

5.98 ± 0.02

C18:1n9c

Oleic

C18:2n6t

Linolelaidic

C18:2n6c

Linoleic

C18:3n6

gamma-Linolenic

C18:3n3

a-Linolenic (cis 9)

C20:0

Arachidic

C20:1n9

Eicosenoic

2.41 ± 0.09 9.15 ± 0.17 0.86 ± 0.15

55.64 ± 1.21 5.30 ± 0.12

18.02 ± 0.19 0.93 ± 0.01

1.71 ± 0.16

unsaturated fatty acids 90.68 ± 0.51 Values are mean ± standard deviations of three measurements

3.4 Confirmation of Triglyceride Composition by FAME Analysis of Triglyceride Fractions (1) Flax Seed Triglyceride Fractions Linolenic acid is present on all three positions of the glycerol for vial one (Table 10) and after mass spectral analysis, flaxseed fraction 1 was determined to be trilinolenoyl-glycerol. The other fatty acids observed in the fraction may be a result of ghost/background peaks from column pre-use when operating at minimal detection limits. The PN number does not support their presence. 57

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Table 8. Percentage of Fatty Acid and Their Positional Distribution in Hemp Seed Oil after Pancreatic Lipase Treatment Fatty acid

Hemp seed oil (%) sn-2 (%) sn-1,3 (%)

C16:0

5.98

4.80

6.57

C18:0

2.41

1.30

2.97

C18:1n-9

9.15

18.01

3.88

C18:2n-6

56.51

58.28

55.67

C18:3n-3

18.02

11.52

21.28

C18:3n-6

5.13

4.31

5.66

C20:0

0.93

0.59

2.00

C20:1

1.71

1.11

1.97

Figure 6. The 8 Hemp seed oil triglyceride peaks separated via HPLC

The second fraction shows a ratio of 2:1 for linolenic acid to linoleic acid in the triglycerol and the mass spectral data confirmed this. The second fraction was found to contain dilinolenoyl-linoleoyl-glycerol. Flax fraction 5 showed a ratio of 2:1 for oleic acid to linolenic acid which was confirmed by mass spectrometry with the triglyceride identified as dioleoyl-linolenoyl-glycerol. The other flax fractions were not conclusive due to the low concentrations present in the methylating process. (2) Hemp Seed Triglyceride Fractions Two hempseed fractions were able to be methylated and taken through GC (Table 11). The other fractions were not conclusive due to their low concentrations. The methylated hemp seed fraction 5 was found to have a 2:1 ratio of linoleic acid to oleic acid. From the mass spectral analysis the major fatty acid in the triacylglycerol fraction was found to be dilinoleoyl-oleoyl-glycerol. Although fraction 6 did not have a clear 2:1 ratio of oleic acid to linoleic acid, the results suggest that the two fatty acids are the more dominant in that particular peak. Mass spectrometry showed the peak to be mainly dioleoyl-linoleoyl-glycerol. (3) Canola Triglyceride Fractions Canola fraction 1 collection (Table 12) showed a high percentage of linolenic acid. The triglyceride 58

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

Table 9. Summary of HPLC and ESI-MS Data for Hemp Seed Oil Triglycerides HPLC Triglyceride retention time

% abun- Molecular ESI-MS Partition Double dance weight (M+23)+ Number bonds (PN) n

25.259

Trilinolenoyl-glycerol (LnLnLn)* 6.0

872

895

36

9

26.188

Dilinolenoyl-linoleoyl-glycerol (LnLnL)*

15.3

874

897

38

8

27.070

Dilinoleoyl-linolenoyl-glycerol (LnLL)*

18.7

876

899

40

7

27.915

Trilinoleoyl-glycerol (LLL)* 21.9 (Linolenoyl-linoleoyl-palmitoylglycerol) LnLP*

878

901

42

6

28.898

Dilinoleoyl-oleoyl-glycerol 13.8 (LLO)* (Dilinoleoyl-palmitoyl-glycerol) LLP*

880

903

44

5

29.755

Dioleoyl-linoleoyl-glycerol (LOO)* (Linoleoyl-oleoyl-palmitoylglycerol) LOP*

9.5

882

905

46

4

30.543

Trioleoyl-glycerol (OOO)*

2.6

884

907

48

3

31.264

Dioleoyl-stearoyl-glycerol (SOO)*

2.6

886

909

50

2

*S is Stearic acid C18:0; O is Oleic acid C18:1; L is Linoleic acid C18:2 Ln is Linolenic acid C18:3 and Eicosenoic acid is C20:1.Note: (M+23) + represents protonated molecular ion with a sodium (mass =23).C20:0 must be in the later smaller peaks on chromatogram. ( ) = secondary peaks.

Table 10. Abundance of Fatty Acids in Flaxseed Triglyceride Fractions after the Methylation Process Fraction/retention Flaxseed Fractions (information C16:0 C18:0 C18:1 C18:2 C18:3 time from HPLC-MS) (%) (%) (%) (%) (%) Vial 1/28.048 min Trilinolenoyl-glycerol LnLnLn*

6.7

4.6

0.4

4.4

84.1

Vial 2/28.794 min Dilinolenoyl-linoleoyl-glycerol LnLnL*

5.5

2.1

0.3

32.3

59.4

Vial 3/29.616 min Dilinolenoyl-oleoyl-glycerol LnLnO*

10.9

0.4

24.4

10.2

49.1

Vial 4/30.278 min Trilinoleoyl-glycerol LLL* (linolenoyl-linoleoyl-palmitoylglycerol LnLP)

12.9

1.9

17.6

47.1

20.4

Vial 5/30.977 min Dioleoyl-linolenoyl-glycerol LnOO* (linolenoyl-palmitoyl-oleoylglycerol LnPO)

10.8

4.2

49.6

11.2

24.1

Values in bold show the most abundant fatty acid in the fraction 59

SOP TRANSACTIONS ON ANALYTICAL CHEMISTRY

Table 11. Abundance of Fatty Acids in Flaxseed Triglyceride Fractions after the Methylation Process Fraction/retention time

Hempseed Fractions (infor- C16:0 C18:0 C18:1 C18:2 C18:3 mation from HPLC-MS) (%) (%) (%) (%) (%)

5 / 28.898 min

Dilinoleoyl-oleoyl-glycerol 14.2 (LLO)* (Dilinoleoyl-palmitoylglycerol) LLP*

2.8

20.7

55.6

5.4

6 / 29.755min

Dioleoyl-linoleoyl-glycerol 10.1 (LOO)* (Linoleoyl-oleoyl-palmitoylglycerol) LOP*

13.9

41.9

28.6

3.1

Table 12. Abundance of Fatty Acids in Canola Seed Triglyceride Fractions after Methylation Process Fraction/retention Canola Fractions (informa- C16:0 C18:0 C18:1 C18:2 C18:3 time tion from HPLC-MS) (%) (%) (%) (%) (%) 1/ 26.162 min

Trilinolenoyl-glycerol (LnLnLn)*

2 / 27.140 min

15.2

8.1

11.4

-

66.6

Dilinolenoyl-oleoyl-glycerol 6.3 (LnLnO)*

9

31.4

-

45.6

4 / 28.898 min

Dioleoyl-linolenoyl-glycerol 6.7 (LnOO)* (Linolenoyl-oleoyl-glycerol) LnOP*

0.7

59.7

18.6

13.9

5 / 29.755 min

Dioleoyl-linoleoyl-glycerol (LOO)* (linoleoyl-oleoyl-glycerol) LOP*

7.8

0.2

61.9

26.8

0.9

6/ 30.543 min

Trioleoyl-glycerol (OOO)* 8.1 (dioleoyl-palmitoyl-glycerol) POO*

1.3

85.9

3.8

-

7 /31.264 min

Dioleoyl-stearoyl-glycerol (SOO)*

35.2

54.1

-

-

10.7

was identified as trilinolenoyl-glycerol through mass spectral analysis. The second fraction had a low yield but supports linolenic as the major fatty acid, consistent with the mass spectral finding. The third fraction was not successful at methylation due to low concentrations. The triglyceride fraction in peak 6 (retention time 30.543min) showed high content of oleic acid (85.9%) and the mass spectral analysis determined it to be trioleoyl-glycerol. The last triglyceride peak, fraction 7, showed a relatively high presence of stearic acid (35.2%). The triglyceride was found to be dioleoyl-stearoyl-glycerol. In conclusion, the FAME analysis has supported the intact triglyceride information for the most abundant fatty acids present, despite the low concentrations recovered due to trapping the peak from the HPLC. More concentrated samples should lead to a more conclusive confirmation.

60

Analysis of Intact Triacylglycerols in Cold Pressed Canola, Flax and Hemp Seed Oils by HPLC and ESI-MS

References [1] C. Wijesundera, “Synthesis of regioisomerically pure triacylglycerols containing n-3 very long-chain polyunsaturated fatty acids,” European Journal of Lipid Science and Technology, vol. 107, no. 11, pp. 824–832, 2005. [2] S.-S. Teh and J. Birch, “Physicochemical and quality characteristics of cold-pressed hemp, flax and canola seed oils,” Journal of Food Composition and Analysis, vol. 30, no. 1, pp. 26–31, 2013. [3] S. Sathivel, “Thermal and flow properties of oils from salmon heads,” Journal of the American Oil Chemists’ Society, vol. 82, no. 2, pp. 147–152, 2005. [4] J. M. Renfrew, Flax. In Palaeoethnobotany. The Prehistoric Food Plants of the Near East and Europe. Columbian University Press, 2002. [5] H. Schiefer, “In pursuit of GRAS status for flaxseed: some practical and hypothetical considerations,” Proceedings of the 54th Rax Institute of the US, Fargo, North Dakota, pp. 67–72, 1992. [6] J. K. Daun, V. J. Barthet, T. Chornick, S. Duguid, L. Thompson, S. Cunnane, et al., “Structure, composition, and variety development of flaxseed,” Flaxseed in human nutrition, no. Ed. 2, pp. 1–40, 2003. [7] de Deckere, E. AM, O. Korver, P. M. Verschuren, and M. B. Katan, “Health aspects of fish and n-3 polyunsaturated fatty acids from plant and marine origin,” in European Journal of Clinical Nutrition, pp. 749–753, Springer, 1998. [8] C. Batista, L. Barros, A. M. Carvalho, and I. C. Ferreira, “Nutritional and nutraceutical potential of rape ( Brassica napus L. var.napus) and tronchuda cabbage (Brassica oleraceae L. var. costata) inflorescences,” Food and Chemical Toxicology, vol. 49, no. 6, pp. 1208–1214, 2011. [9] A. Kuksis and K. Shibamoto, Lipid chromatographic analysis. Marcel Dekker: New York, 1994. [10] A. Simopoulos, “Commentary on the workshop statement,” Prostaglandins, Leukotrienes and Essential Fatty Acids, vol. 63, no. 3, pp. 123–124, 2000. [11] W.-S. Choo, E. J. Birch, and I. Stewart, “Radical scavenging activity of lipophilized products from transesterification of flaxseed oil with cinnamic acid or ferulic acid,” Lipids, vol. 44, no. 9, pp. 3334–3342, 2009. [12] D. Van Wijngaarden, “Modified rapid preparation of fatty acid esters from lipids for gas chromatographic analysis,” Analytical Chemistry, vol. 39, no. 7, pp. 848–849, 1967. [13] L. D. Lawson and B. G. Hughes, “Triacylglycerol structure of plant and fungal oils containing y-linolenic acid,” Lipids, vol. 23, no. 4, pp. 313–317, 1988. [14] W. W. Nawar, 1996. [15] N. Andrikopoulos, H. Brueschweiler, H. Felber, and C. Taeschler, “HPLC analysis of phenolic antioxidants, tocopherols and triglycerides,” Journal of the American Oil Chemists Society, vol. 68, no. 6, pp. 359–364, 1991. [16] P. Laakso, “Mass spectrometry of triacylglycerols,” European journal of lipid science and technology, vol. 104, no. 1, pp. 43–49, 2002. [17] P. Laakso and P. Voutilainen, “Analysis of triacylglycerols by silver-ion high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry,” Lipids, vol. 31, no. 12, pp. 1311–1322, 1996.

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