Rapid Quantitative Determination of Squalene in Shark Liver Oils by Raman and IR Spectroscopy

Squalene is sourced predominantly from shark liver oils and to a lesser extent from plants such as olives. It is used for the production of surfactants, dyes, sunscreen, and cosmetics. The economic value of shark liver oil is directly related to the squalene content, which in turn is highly variable and species‐dependent. Presented here is a validated gas chromatography‐mass spectrometry analysis method for the quantitation of squalene in shark liver oils, with an accuracy of 99.0 %, precision of 0.23 % (standard deviation), and linearity of >0.999. The method has been used to measure the squalene concentration of 16 commercial shark liver oils. These reference squalene concentrations were related to infrared (IR) and Raman spectra of the same oils using partial least squares regression. The resultant models were suitable for the rapid quantitation of squalene in shark liver oils, with cross‐validation r² values of >0.98 and root mean square errors of validation of ≤4.3 % w/w. Independent test set validation of these models found mean absolute deviations of the 4.9 and 1.0 % w/w for the IR and Raman models, respectively. Both techniques were more accurate than results obtained by an industrial refractive index analysis method, which is used for rapid, cheap quantitation of squalene in shark liver oils. In particular, the Raman partial least squares regression was suited to quantitative squalene analysis. The intense and highly characteristic Raman bands of squalene made quantitative analysis possible irrespective of the lipid matrix.     

Similarities in the lipid class profiles of oils from Atlantic and Pacific dogfish livers

Liver oils from Atlantic and Pacific dogfish (Squalus acanthias) have been compared for lipid classes, fatty acids of the total oil and of important lipid classes, and details of the alkyl chains in the 1-O-alkyl-2,3-diacylglycerol ethers (DAGE). In general there were few striking differences, confirming the view of biologists that these sharks are one species. The Pacific dogfish liver oil had a higher content (41.2%) of DAGE than the oil from Atlantic dogfish (18.2%). Both oils had all common and expected fatty acids in the proportions usual for marine oils, but they differed in the eicosenoic chains of the glycerol ethers (GE). The Pacific oil was unusual in having low but similar proportions of two alkyl chain isomers, 20:1n-11 and 20:1n-9. The Atlantic oil was very high in the 20:1n-11 isomer, which is usually lower than 20:1n-9 in the fatty acids of most regional marine oils. Unexpectedly, the DAGE of both oils had further unusual 20:1 isomer proportion in the GE chain, with 20:1n-7 >20:1n-9. Minor oddities in the fatty acids may reflect different basic food sources. Presented in part at the 84th Annual Meeting of the American Oil Chemists’ Society, Atlanta, GA, May 8–12, 1994.     

Purification and characterization of deep sea sharkCentrophorus squamosus liver oil 1-O-aklylglycerol ether lipids

The 1-O-alkylglycerol composition of the liver oil of the deep sea sharkCentrophorus squamosus, a species which provides edible flesh, has been determined. After various fractionations of the oil, the unsaponifiable fraction was characterized by means of gas chromatography/mass spectrometry, electron impact, and positive-ion chemical ionization. The oil is composed of 60% unsaponifiable matter, containing 45% squalene, 4.5% cholesterol, and 10% of linear saturated and monounsaturated glycerol ethers with 14–18 carbon atoms. After a first separtion by chromatography on silicic acid, monounsaturated glycerol ethers have been separated from the saturated homologues, in particular from 1-O-octadecylglycerol (batyl alcohol) and 1-O-hexadecylglycerol (chimyl alcohol)via urea complexation. This newer application of the urea method, already used in the past to extract saturated from polyunsaturated fatty acids, allowed the purification of the main components of the complex unsaturated glycerol ether fraction, namely, 1-O-octadecen-9′ylglycerol (selachyl alcohol) and 1-O-hexadecen-9′ylglycerol.     

Supercritical fluid chromatographic analysis of fish oils

Various natural and processed fish oil triglyceride mixtures have been analyzed by capillary supercritical fluid chromatography (SFC). The analyses were performed on nonpolar columns to separate the components by lipid class and by the number of carbon atoms. The compounds separated included free fatty acids, squalene, α-tocopherol, cholesterol, wax esters, cholesteryl esters, di- and triglycerides. This kind of analysis is not possible by gas chromatography or high-performance liquid chromatography methods without prior treatment of the fish oil, making SFC superior for this application. Applications of SFC to fish oils are given, including a control analysis of the various process steps in the refining of a fish oil, analysis of a lipase-catalyzed transesterification of a fish oil and the detection of polymeric artifacts.     

Chemical Compositions of Walnut (Juglans regia L.) Oils from Different Cultivated Regions in China

This study is a comprehensive report on the quality of Chinese walnut oil, which enriches the research of oil resources. A total of 16 walnut samples from China were selected, and walnut oils were obtained using the pressing process. The lipid compositions and micronutrient contents were analyzed. The fatty acids corresponded to palmitic acid (3.05–8.25%), oleic acid (12.56–26.03%), linoleic acid (51.21–68.97%), and linolenic acid (6.83–15.01%), and the main triacylglycerols were trilinolein (27.87–39.47%), followed by oleoyl‐linoleoyl‐linolenoyl‐glycerol (17.07–24.18%), dilinoleoyl‐oleoyl‐glycerol (9.65–15.46%), palmitoyl‐dilinoleoyl‐glycerol (5.96–14.98%), and dilinoleoyl‐linolenoyl‐glycerol (6.42–12.43%). In addition, high amounts of micronutrients, including phytosterol, squalene, tocopherol, and total phenolic content, were found in walnut oils ranging from 540 to 1594, 17 to 131, 345 to 1280, and 1.04 to 20.39 mg kg^?1 among different samples, respectively. The differences in the geographical location and climate caused different regions of cultivation, which resulted in the differences in the chemical composition of walnut oil. Further multiple linear regression analyses between oxidative stability indices, fatty‐acid compositions, and micronutrients revealed that linoleic acid (R = ?0.891; P < 0.05), α‐tocopherol (R = 0.713; P < 0.05), and total phenolic content (R = 0.369; P < 0.05) were the main factors that affect the oxidative stability of the walnut oil.     

Regiospecific Analysis of Shark Liver Triacylglycerols

The liver oils of six shallow-water shark species, silky (Carcharhinus falciformis), thresher (Alopias superciliosus), oceanic whitetip (Carcharhinus longimanus), blue (Prionace glauca), hammerhead (Sphyrna lewini) and salmon (Lamna ditropis) were analyzed with particular attention to the regioisomeric composition of triacylglycerols (TAG). The TAG compositions were analyzed by using an HPLC-evaporative light scattering detector and each molecular species identified by HPLC-atmospheric pressure chemical ionization/mass spectrometry. Major lipid components of all sharks’ oils were TAG (~80 %) made up of omega-3 polyunsaturated fatty acids at 26–40 % and 20–25 % docosahexaenoic acid (DHA). Forty different molecular species were detected in the TAG fractions. TAG consisting of one palmitic acid, one DHA and one oleic acid (12.5–19.9 %) and TAG consisting of two palmitic acids and one DHA (8.4–15.4 %) were the predominant form while 30–50 % TAG molecular species were bound to one or more DHA. Distribution of fatty acids in the primary (sn-1 and sn-3) and secondary (sn-2) position of the glycerol backbones was examined by regiospecific analysis by using pancreatic lipase and it was found that DHA was preferentially positioned at sn-2. These findings greatly extend the utilization of shark liver oils in food productions and may have a significant impact on the future development of the fish oil industry.     

Analysis of seed oil from ricinus communis and dimorphoteca pluvialis by gas and supercritical fluid chromatography

The seed oils from Dimorphoteca pluvialis and Ricinus communis contain hydroxy fatty acids. Dimorphoteca pluvialis contains Δ-9-hydroxy-10t, 12t-octadecadienoic acid (dimorphecolic acid) and R. communis contains Δ-12-hydroxy-9c-octadecenoic acid (ricinoleic acid). The oils were derivatized and analyzed to determine the content of hydroxy fatty acids. The trimethylsilyl fatty acid methyl ester (TMS-FAME) derivatives were analyzed by capillary gas chromatography (GC), and the free fatty acid (FFA) derivatives and the oils were analyzed by capillary supercritical fluid chromatography (SFC). Further, mass spectroscopy of the TMS-FAME derivatives was performed to check the purity of the derivatives. The results from the GC analyses of TMS-FAME corresponded to the results found by SFC analysis of the FFA. The content of ricinoleic acid in the glycerolipids of R. communis was 87.7 wt%, and the content of dimorphecolic acid in D. pluvialis was 54.0 wt%. The methods were evaluated with respect to the cost, ease, and time needed for sample preparation and analysis.     

Anomalous high iodine value of squalene and the impact on iodine values of shark liver oils

Squalene has six ethylenic bonds, but the experimental iodine values in two different solvent systems—chloroform and cyclohexane/acetic acid—were 25% higher than the theoretical values. We propose that this results from an additional halogen adding at each of the two terminal ethylenic bonds carrying two methyl groups. In the solvent system of cyclohexane alone, the excess is only 3–4% greater than the theoretical. Mixtures of squalene in seal oil confirmed the additivity of the experimental squalene high iodine value and the seal oil fatty acid iodine value with reasonable accuracy but depended on the skill of the operator in obtaining the titration end point for cyclohexane/acetic acid. This observation has particular relevance for shark liver oils and olive oils.     

Anomalous high iodine value of squalene and the impact on iodine values of shark liver oils

Squalene has six ethylenic bonds, but the experimental iodine values in two different solvent systems—chloroform and cyclohexane/acetic acid—were 25% higher than the theoretical values. We propose that this results from an additional halogen adding at each of the two terminal ethylenic bonds carrying two methyl groups. In the solvent system of cyclohexane alone, the excess is only 3–4% greater than the theoretical. Mixtures of squalene in seal oil confirmed the additivity of the experimental squalene high iodine value and the seal oil fatty acid iodine value with reasonable accuracy but depended on the skill of the operator in obtaining the titration end point for cyclohexane/acetic acid. This observation has particular relevance for shark liver oils and olive oils.     

Heated fat-based oil substitutes, oleic and linoleic acid-esterified propoxylated glycerol

Four oils [triolein, trilinolein, oleic acid-esterified propoxylated glycerol (EPG-08 oleate), and linoleic acid-esterified propoxylated glycerol (EPG-08 linoleate)], each without added antioxidants, were heated for 12 h/d at approximately 190°C in a small deep-fat fryer until the polymer concentration exceeded 20%, as determined by high-performance size-exclusion chromatography. Increases in the free fatty acid content, total acid value, food oil sensor value, and p-anisidine value during heating indicated that significant thermal oxidation had occurred in each oil. Capillary supercritical fluid chromatography (SFC) was used to determine the substrate concentration of each oil after each heating interval. The average, apparent first-order reaction rate constant (as determined by SFC) for trilinolein was 0.0348±0.0034 h⁻¹, while the rate for EPG-08 linoleate was 0.0253±0.0032 h⁻¹. The average apparent reaction rate constant for triolein was 0.0256±0.0011 h⁻¹, while the rate for EPG-08 oleate was 0.0252±0.0008 h⁻¹. Triolein contained >20% polymer after 60 h of heating, EPG-08 oleate contained >20% polymer after 36 h of heating, and both trilinolein and EPG-08 linoleate contained >20% polymer after 24 h of heating.     

The hydrocarbon fraction of virgin olive oil and changes resulting from refining

In numerous Spanish virgin olive oils, 6,10-dimethyl-1-undecene, various sesquiterpenes, the series ofn-alkanes from C14 to C35, n-8-heptadecene and squalene are the only less volatile components detected by gas chromatography in the hydrocarbon fraction. In oils from olives of the Arbequine variety, a series ofn-9-alkenes has also been found. In refined oils, notable features are the absence of the most volatile compounds and the appearance of other hydrocarbons produced during the refining process. Among these,n-alkanes, alkadienes (mainlyn-hexacosadiene), stigmasta-3,5-diene, isomerization products of squalene, isoprenoidal polyolefins coming from hydroxy derivatives of squalene and steroidal hydrocarbons derived from 24-methylene cycloartanol were identified. Physical refining produces larger amounts of degradation products and greater losses ofn-alkanes than chemical processing. Squalene is the major hydrocarbon component in all oils, both virgin and refined. The ranges of concentration for the different hydrocarbons found in Spanish virgin olive oils are presented.     

Squalene from Fish Livers Extracted by Ultrasound-Assisted Direct In Situ Saponification: Purification and Molecular Characteristics

An ultrasound-assisted direct in situ saponification (U-DS) process was developed to extract unsaponifiable matter (rich in squalene) from the livers of four fish species, sea bass, skipjack tuna, gray bamboo shark, and spot-tail shark. The conventional solvent extraction method was used for comparison. Box–Behnken experimental design (BBD) was adopted to optimize the independent variables including the biomass/methanol ratio (1:3 to 1:9 w/v), 50% KOH volume (1–12 mL), and sonication time (0–30 min) over the unsaponifiable yield. With the conventional process, the yield of squalene was 0.10 ± 0.02 to 5.52 ± 0.06 g (100 g)⁻¹, whereas the U-DS process rendered the yield of 0.13 ± 0.03 to 6.86 ± 0.05 g (100 g)⁻¹, depending on the fish species. After extraction, squalene was further concentrated (purity ≥60%) from all fish species via fractional crystallization (yield of squalene concentrate ranged from 48.35% to 74.49%) and purified using a silica gel column with a maximum recovery up to 98%. For all the fractions, components were examined using thin layer chromatography (TLC). Squalene was qualitatively and quantitatively analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). Entire extraction processes yielded squalene with a purity of ≥94%. Fourier transform infrared (FTIR) analysis confirmed the native structure of squalene with six nonconjugated bonds, suggesting no degradation of squalene taken place during the U-DS process. Thus, the U-DS process along with fractionation and purification could be used for recovering the squalene from fish livers.     

Digestion of the 1-O-alkyl diacylglycerol ethers of atlantic dogfish liver oils by Atlantic salmon Salmo salar

Dogfish (Squalus acanthias) liver poses a waste disposal problem in Canada because it is not utilized for any commercial purpose. The liver of Atlantic dogfish, which is often up to 20% of the weight of the fish, contains 40–70% oil. The oil contains about 30–40% 1-O-alkyl diacylglycerol ethers (DAGE) which render it unacceptable for human use, and it has also not been considered satisfactory for animal feed use. Polyunsaturated fatty acids (20∶5n−3 and 22∶6n−3) are present in dogfish liver oils at levels comparable to those in herring oil. Dogfish liver oil could be a source of essential fatty acids for Atlantic salmon (Salmo salar), but their ability to hydrolyze DAGE from dogfish oil has not been examined. Experiments were designed to measure the digestibility of fatty acids of DAGE in salmon. The fatty acid moieties were liberated by the digestive enzymes of the fish and made readily available as a source of energy. The 1-O-alkylglycerol ether moiety was absorbed to a small extent but should not constitute a health problem in either the fish or the human fish consumer. The long-chain polyunsaturated fatty acids were particulary well absorbed, with an apparent digestibility in salmon of 87–95% when feeding on dogfish liver oil. The total fatty acids and other lipids were in fact both absorbed to the extent of approximately 85%. Presented in part at the Annual Meeting of the American Oil Chemists’ Society, Atlanta, Georgia, May 1994.     

Quantitative determination of diesel oil in contaminated edible oils using high-performance liquid chromatography

A simple and reliable high-performance liquid chromatography method for the analysis of diesel oil in contaminated edible oils is described. Analysis performed using a diol column with a mobile phase of heptane and isopropanol (94∶6, vol/vol). Although baseline separation between diesel and other background fluorescent components was not achieved, quantitation was still possible using baseline integration. The method is linear over the range of 5–1000 μg/g with a correlation coefficient (r ²) of 0.9984. Average recoveries from spiked edible oils were 94.4–101.3%, with a limit of quantitation (LOQ) of 5 μg/g for sunflower oil, palm olein, and groundnut oil. Corn oil has a higher content of ester components, thus, LOQ was slightly worse (40 μg/g). The applicability of the method was confirmed by gas chromatography-mass spectroscopic detection to show the presence of diesel hydrocarbons in the suspected contaminated crude palm oil. This procedure provides a simple and sensitive method for determining diesel oil concentration in contaminated edible oils without prior sample cleanup or extraction.     

Supercritical fluid chromatographic analysis of new crop seed oils and their reactions

Supercritical fluid chromatography (SFC) with an open tubular column of nonpolar stationary phase separated triglycerides from crambe, meadowfoam,Euphorbia lagascae, and vernonia oils based on their molecular weight. The triglyceride compositions were consistent with the literature. SFC proved also to be a valuable tool in analyzing lipase-catalyzed transesterification reactions where lesquerella oil and estolides were among the substrates employed. Analyte molecular weights could be estimated from a retention time- (or elution density-) molecular eeight calibration curve. An increase in isothermal column temperature during SFC pressure or density programming improved the resolution of high-molecular-weight (>600 Da) analytes but yielded poorer resolution for analytes of molecular weight <200. A simultaneous pressure and temperature ramping program proved superior in enhancing resolution in several instances. Presented at the AOCS Annual Meeting & Expo, May 1995, San Antonio, Texas. Retired     

Squalene in oils determined as squalane by gas—liquid chromatography after hydrogenation of methyl esters

Low levels (≤0.1%) of squalene were anticipated in oils from the blubber of the harp seal Phoca groenlandica. The traditional roule of saponification and analytical examination of the total unsaponifiables was unattractive. A method developed for squalene in olive oil, reportedly present in the range of 0.3–0.7%, was based on total conversion of the oil to methyl ester of fatty acids by alkali transesterification, followed by hydrogenation over Adam's catalyst (PtO₂). The analysis of the fully saturated methyl esters and any squalane produced concurrently was by gas—liquid chromatography. This method was satisfactory for the small amounts, 0.03% or less, of squalene in seal oil and is also illustrated for olive oil. A flame-ionization detector excessive response of approximately 25% was observed for all levels of squalene tested. The calculated factor of 1.22 should be applied to the peak area for squalane due to the higher response of hydrocarbons relative to the methyl esters of fatty acids and the system of oil components if reporting as fatty acids. Presented in part at the Annual Meeting of the Canadian Section of the American Oil Chemists' Society, London, Oct. 15–18, 1999.     

Hydrocarbons and alcohols of basking shark and pig liver lipids

Four samples of the unsaponifiables of basking shark liver oil were adsorbed on alumina and eluted to yield Fractions 1–5, inclusive. Analyses by temperature programmed GC and by silica gel chromatography showed hydrocarbons in the first four fractions with squalene increasing to Fraction 3 and the pristane level being highest in Fraction 1. Aside from pristane and squalene, other hydrocarbons occurred at levels of 420–750 mg% in the oils on a weight basis, of which about 60% constituted a series of n-paraffins (relative carbon number range: 15.0–38.0) together with smaller amounts of at least one branched chain saturated group. Unsaturated hydrocarbons eluted mainly after squalene. The oils contained up to 460 mg% sterol and 78–270 mg% alcohols of C₁₀ to C₃₀, the ratio of saturated to unsaturated members being about 1.6. The composition of the unsaponifiable lipids of pig liver was quite different from that of the marine oils. It contained 10.6% sterol in addition to 400 mg% alcohols, the latter consisting of 81.8% saturated components (C₁₂ to C₃₁; ratio of saturated: unsaturated members, 4.4). The hydrocarbons comprised 450–700 mg% of the unsaponifiable mixture and squalene, paraffins and additional unsaturated components occurred at levels of 20.6, 24.4 and 11.9 mg%, respectively. The saturated hydrocarbons were high in normal homologs of relative carbon number range, 15 to 36; pristane could not be detected.     

High-performance liquid chromatography of the triacylglycerols ofVernonia galamensis andCrepis alpina seed oils

The triacylglycerols ofVernonia galamensis andCrepis alpina seed oils were characterized because these oils have high concentrations of vernolic (cis-12,13-epoxy-cis-9-octadecenoic) and crepenynic (cis-9-octadecen-12-ynoic) acids, respectively. The triacylglycerols were separated from other components of crude oils by solid-phase extraction, followed by resolution and quantitation of the individual triacylglycerols by reversed-phase high-performance liquid chromatography with an acetonitrile/methylene chloride gradient and flame-ionization detection. Isolated triacylglycerols were characterized by proton and carbon nuclear magnetic resonance and by capillary gas chromatography of their fatty acid methyl esters. The locations of the fatty acids on the glycerol moieties in the oils were obtained by lipolysis. TheVernonia galamensis oil contained 50% trivernoloyl and 21% divernoloyllinoleoyl glycerols along with 20% triacylglycerols with one vernolic and two other fatty acids. TheCrepis alpina oil contained 36% tricrepenynoyl and 33% dicrepenynoyllinoleoyl glycerols, 17% triacylglycerols with two crepenynic and one other fatty acid and 7% triacylglycerols with one crepenynic acid and two other fatty acids. Vernolic acid was found at both the 1(3)- and 2-glycerol carbons but was more abundant at the 1,(3)-position in theVernonia galamensis oil. Crepenynic acid was found at both glycerol carbon positions but was more abundant at the 2-position in theCrepis alpina oil. Visiting scientist from Technical Research Institute, Snow Brand Milk Company, Ltd., Saitana, Japan.     

Differentiation of Mechanically and Chemically Extracted Hazelnut Oils Based on their Sterol and Wax Profiles

The sterol and wax content of solvent extracted (SEHO) and cold pressed hazelnut oils (CPHO) were compared. A total of 48 samples from 19 hazelnut varieties were collected for two successive crop years from four different geographical districts in Turkey. Hazelnuts were processed to oil with a laboratory scale press, than the remaining oil in cake was extracted with n‐hexane. CPHO and SEHO were evaluated for their wax, sterol and squalene contents. Results showed that sterol, squalene and wax contents of all individual cultivars were higher in SEHO than those of CPHO, indicating the higher solubility of these compounds in solvent. Total sterol contents ranged between 1088.56 (Kargalak)—1609.39 mg/kg (Mincane) for CPHO and 1590.86 (Çak?ldak)—2897.26 mg/kg (Mincane) for SEHO. Hazelnut oils were found to be richer of C36‐38 esters than C40‐46 group. Total wax content was between 24.19 (Kargalak)—94.58 mg/kg (Ku?) for CPHO and 81.46 (Kargalak)—160.92 mg/kg (Akçakoca) for SEHO. The squalene amounts of the samples obtained by hexane extraction were between 499.75 (Allahverdi)—885.36 mg/kg (Cavcava), while it varied between 288.55 (Kargalak)—647.68 mg/kg (Mincane) in cold pressed oils. Significant and obvious variations between SEHO and CPHO were verified by principal component and hierarchical cluster analysis. Geographical discrimination was also achieved by discriminant analysis.     

Properties of Oleogels Formed With High‐Stearic Soybean Oils and Sunflower Wax

In an effort to develop alternatives for harmful trans fats produced by partial hydrogenation of vegetable oils, oleogels of high‐stearic soybean (A6 and MM106) oils were prepared with sunflower wax (SW) as the oleogelator. Oleogels of high‐stearic oils did not have greater firmness when compared to regular soybean oil (SBO) at room temperature. However, the firmness of high‐stearic oil oleogels at 4 °C sharply increased due to the high content of stearic acid. High‐stearic acid SBO had more polar compounds than the regular SBO. Polar compounds in oil inversely affected the firmness of oleogels. Differential scanning calorimetry showed that wax crystals facilitated nucleation of solid fats of high‐stearic oils during cooling. Polar compounds did not affect the melting and crystallization behavior of wax. Solid fat content (SFC) showed that polar compounds in oil and wax interfered with crystallization of solid fats. Linear viscoelastic properties of 7% SW oleogels of three oils reflected well the SFC values while they did not correlate well with the firmness of oleogels. Phase‐contrast microscopy showed that the wax crystal morphology was slightly influenced by solid fats in the high‐steric SBO, A6.