Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

气相色谱全分析海水和淡水养殖南美白对虾组织中脂肪酸组成与含量

采用超声提取-毛细管气相色谱法分析测定了海水养殖和淡水养殖的南美白对虾的肌肉和肝胰腺(中肠腺)中脂肪酸的组成与含量,以用于对虾繁殖与营养的研究。采用Folch 液超声提取对虾组织中的脂质,脂质经氢氧化钾-三氟化硼法甲酯化后上机分析。采用BPX70(60 m×0.25 mm i.d.)石英毛细管柱分离,火焰离子化检测器检测。以线性程序升温等当碳长度(ECLT)值结合标准品对照的方法对17种脂肪酸定性,用面积归一化法定量。结果表明,两种对虾的脂肪酸组成均以不饱和脂肪酸为主,而且不饱和多烯酸的含量明显高于不饱和单烯酸。两种对虾的肌肉脂肪酸中高度不饱和脂肪酸,如二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)的总含量均较高,达20%以上;肝胰腺中EPA和DHA的总含量较低,为11%～17%。淡水虾中n-6族不饱和脂肪酸含量比海水虾高,海水虾中n-3族与n-6族脂肪酸含量之比值比淡水虾高。     

Simultaneous quantification of total eicosapentaenoic acid,docosahexaenoic acid and arachidonic acid in plasma by high‐performance liquid chromatography–tandem mass spectrometry

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC–tandem mass spectrometry (HPLC‐MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid–liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r² > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30–90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega‐3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre‐treatment concentrations varied between subjects for EPA (17–68 mg/L), DHA (36–63 mg/L) and AA (121–248 mg/L). During the dosing period EPA increased 460–480% from the baseline concentration, while DHA increased 150–160%. The EPA–AA ratio increased from 0.07–0.56 to 0.3–3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of EPA- and DHA-Enriched Structured Acylglycerols at the sn-2 Position Starting from Commercial Salmon Oil by Enzymatic Lipase Catalysis under Supercritical Conditions

There is consistent evidence that long-chain polyunsaturated fatty acids (LCPUFA) belonging to the n-3 series, i.e., eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids, decrease the risk of heart, circulatory and inflammatory diseases. Furthermore, the bioavailability of such fatty acids has been shown to depend on their location in triacylglycerol (TG) molecules at the sn-2 position. Consequently, great attention has been accorded to the synthesis of structured acylglycerols (sAG), which include EPA or DHA at the sn-2 position. The aim of this work was to synthesize sAG starting from deodorized refined commercial salmon oil. For this, immobilized lipase B from Candida antarctica (nonspecific) was used as a catalyst for the intra–interesterification process under CO₂ supercritical conditions (CO₂SC). According to the CO₂SC reaction time, three different fractions including sAG compounds were obtained. The location of EPA and DHA at the sn-2 position in the resulting glycerol backbone was identified by mass spectrometry (MALDI-TOF) analysis. In all fractions obtained, a marked decrease in the starting TG content was observed, while an increase in the DHA content at the sn-2 position was detected. The fraction obtained after the longest reaction time period (2 h) led to the highest yield of sn-2 position DHA in the resulting sAG molecule.     

Aalysis of the fatty acid composition of the lipid classes in human blood serum under normal diet and when supplemented with fish oil

Total lipids have been extracted from human serum with chloroform–methanol 2:1 (v/v) and separated into individual classes by TLC. After transesterification the fatty acid methyl esters were analyzed by capillary gas chromatography on an FFAP column. The quantitation of ω-3 fatty acids has been performed using internal and external standards. Internal lipid standards for each lipid class were carried throughout the entire analytical procedure. Under normal diet eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are incorporated into the lipid classes to different extents: cholesterol esters; EPA, 6.5 ± 1.9 γ/ml serum; DHA, 4.3 ± 1.9 μg/ml: phospholipids; EPA, 5.9 ± 2.7 μg/ml; DHA, 31.8 ± 8.1 μg/ml. Fish oil supplementation leads to a 4 to 6-fold rise in EPA and to an approximately 2-fold rise in DHA.     

Optimization of Fatty Acid Determination in Selected Fish and Microalgal Oils

The use of ten fatty acid methyl ester reference standards coupled with a detailed quantification method was shown to significantly optimize the fatty acid determination of selected fish and microalgal oils when compared to methods that use only one reference standard (C19:0 or C23:0) as a relative response factor. When using the mixture of ten reference standards after transesterifying oils with NaOH/BF₃, determination of total fatty acids, eicosapentaenoic acid and docosahexaenoic acid improved by an average of 7.3, 11.5 and 8.4%, respectively. Furthermore, improvements of 13.9, 18.9 and 6.8% of total fatty acids, EPA and DHA, respectively, were obtained when using the mixture of reference standards for fatty acid determination after directly extracting and transesterifying oil contained in microalgal cells with a mixture of methanol, HCl and chloroform. Fatty acid methyl ester standards dissolved in isooctane showed <5% variability throughout 130 days of stability testing when stored at ?20 °C. The optimized method can be used for improving the quantification of fatty acids in both oils (fish and microalgal oils) and dry microalgal cells.     

Pythium irregulare Fermentation to Produce Arachidonic Acid (ARA) and Eicosapentaenoic Acid (EPA) Using Soybean Processing Co-products as Substrates

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.     

深海鱼油中脂肪酸的分析

用氢氧化四甲胺／甲醇酯化法使深海鱼油内的三酸甘油酯中的脂肪酸和游离脂肪酸转化成脂肪酸甲酯,用气相色谱和气相色谱／质谱进行分析,鉴定出４１个组分,并对其中的主要组分顺式－５,８,１１,１４,１７－十二碳五稀酸（ＥＰＡ）和顺式－４,７,１０,１３,１６,１９－二十二碳六烯酸（ＤＨＡ）建立了定量分析方法。方法的相对标准偏差分别为４．３％（ＥＰＡ）和４．７％（ＤＨＡ）。ＥＰＡ和ＤＨＡ的回收率分别为１００．     

Control Mechanism for Carbon‐Chain Length in Polyunsaturated Fatty‐Acid Synthases

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KS_A and KS_C) and acyl‐acyl carrier protein (ACP) substrates showed that KS_A accepted short‐ to medium‐chain substrates while KS_C accepted medium‐ to long‐chain substrates. Unexpectedly, condensation from C₁₈ to C₂₀, the last elongation step in EPA biosynthesis, was catalyzed by KS_A domains in both EPA and DHA synthases. Conversely, condensation from C₂₀ to C₂₂, the last elongation step for DHA biosynthesis, was catalyzed by the KS_C domain in DHA synthase. KS_C domains therefore determine the chain lengths.     

Quantification of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization by gas-liquid chromatography

Long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas-liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100 m) operating under optimal conditions was used. Method selectivity was studied by GC-mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220 degrees C for 3 h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220 degrees C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56 g/100 g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.     

Use of Isopropanol as a Modifier in a Hexane-Acetonitrile Based Mobile Phase for the Silver Ion HPLC Separation of Positional Isomers of Triacylglycerols Containing Long Chain Polyunsaturated Fatty Acids

A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs. For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).     

Biosynthetic production of universally <Superscript>13</Superscript>C-labelled polyunsaturated fatty acids as reference materials for natural health product research

Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope ¹³C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, ¹³C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, ¹³C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-¹³C-labelled fatty acids using U-¹³C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of ¹³C-labelled DHA has been achieved with high chemical purity ( >99%) and high ¹³C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-¹³C-DHA was enzymatically re-esterified to glycerol to yield a ¹³C-labelled tridocosahexaenoin.     

Analysis of polyunsaturated fatty acids in blood serum after fish oil administration.

Free and total fatty acids in the blood serum of patients with hyperlipoproteinemia have been analysed as their methyl esters by capillary gas chromatography using an FFAP column. In one-step reactions the free fatty acids in serum react with methanol-acetyl chloride (50:1, v/v) at 25 degrees C, the total fatty acids (free plus esterified) are transesterified with methanol-toluene-acetyl chloride (8:2:1, v/v) at 100 degrees C. The quantification of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is based on an internal standard (13,16,19-docosatrienoic acid) and on calibration standards. Under normal diet the concentrations of EPA and DHA are as follows (mean +/- S.D., n = 27): free EPA, 0.2 +/- 0.1 mg/dl; free DHA, 0.6 +/- 0.2 mg/dl; total EPA, 3.6 +/- 2.1 mg/dl; total DHA 11.4 +/- 3.1 mg/dl. Under a fish oil intake of 9 g per day, free and total EPA concentrations rise by ca. five- to six-fold, and free and total DHA concentrations by ca. two-fold.     

Zinc Ion Doped Solid-Phase Extraction of Eicosapentaenoic Acid and Docosahexaenoic Acid from Antarctic Krill

Antarctic krill crude extracts contain high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Accordingly, the solid phase extraction of EPA and DHA from Antarctic krill crude extracts has attracted significant research interest. This study compared the extraction of EPA and DHA from Antarctic krill crude extracts using an aminopropyl, zinc ion-doped silica, and C₁₈ and zinc ion-doped C₁₈ solid-phase column. The best extraction effect was obtained using the zinc ion-doped C₁₈ SPE with water containing methanol as the eluant. The efficiency increased gradually with increasing methanol concentration from 12.5 to 25% in the washing stage, and when pure methanol (5.0 mL) or acetonitrile (3.0 mL) was used as the eluant. To detect EPA and DHA, the acids were first converted to their methyl esters and detected by gas chromatography with flame ionization detection (GC–FID). In the zinc ion-doped C₁₈ elution fractions, EPA and DHA were isolated from the crude extracts in high yield (85–91% (r² = 4.8–6.3%)).     

Separation of triacylglycerols and free fatty acids in microalgal lipids by solid-phase extraction for separate fatty acid profiling analysis by gas chromatography

Microalgal lipids were separated into two fractions, triacylglycerols (TAGs) and free fatty acids (FFAs), by solid-phase extraction employing sodium carbonate as the sorbent and dichloromethane (20% by volume) in n-hexane as the extracting solvent. The TAG fraction was then saponified, followed by acidification, extraction and tert-butyldimethylsilyl esterification. The FFA fraction was directly acidified, extracted and derivatized. From the lipid extracts of eight microalgal species examined, a total of 13 fatty acids were detected in the TAG fractions and nine were found in the FFA fractions, with at much higher total TAG content in all microalgae. Oleic acid was the most prominent fatty acid in three species, α-linolenic acid was more abundant in two others, and palmitic acid was present in highest concentration in the remaining three species.     

Fatty acid composition and volatile compounds of caviar from farmed white sturgeon (Acipenser transmontanus)

The present study was conducted to characterize caviar obtained from farmed white sturgeons (Acipenser transmontanus) subjected to different dietary treatments. Twenty caviar samples from fish fed two experimental diets containing different dietary lipid sources have been analysed for chemical composition, fatty acids and flavour volatile compounds. Fatty acid make up of caviar was only minimally influenced by dietary fatty acid composition. Irrespective of dietary treatments, palmitic acid (16:0) and oleic acid (OA, 18:1 n-9) were the most abundant fatty acid followed by docosahexaenoic acid (DHA, 22:6 n-3) and eicopentaenoic (EPA, 20:5 n-3). Thirty-three volatile compounds were isolated using simultaneous distillation-extraction (SDE) and identified by GC-MS. The largest group of volatiles were represented by aldehydes with 20 compounds, representing the 60% of the total volatiles. n-Alkanals, 2-alkenals and 2,4-alkadienals are largely the main responsible for a wide range of flavours in caviar from farmed white surgeon.     

Reversed-phase high-performance liquid chromatography purification of methyl esters of C(16)-C(28) polyunsaturated fatty acids in microalgae, including octacosaoctaenoic acid [28:8(n-3)

A preparative reversed-phase (RP; C(18)) high-performance liquid chromatography (HPLC) method with gradient elution using acetonitrile (MeCN)-chloroform (CHCl(3)) (or dichloromethane (DCM)) and evaporative light-scattering detection (ELSD) with automatic multiple injection and fraction collection was used to purify milligram quantities of microalgal polyunsaturated fatty acids (PUFA), separated as methyl esters (ME). PUFA-ME purified included methyl esters of docosahexaenoic acid (DHA; 22:6(n-3)), eicosapentaenoic acid (EPA; 20:5(n-3)) and the unusual very long-chain (C(28)) highly unsaturated fatty acid (VLC-HUFA), octacosaoctaenoic acid [28:8(n-3)(4, 7, 10, 13, 16, 19, 22, 25)] from the marine dinoflagellate Scrippsiella sp. CS-295/c. Other PUFA purified from various microalgae using this RP-HPLC method to greater than 95% purity included 16:3(n-4), 16:4(n-3), 16:4(n-1) and 18:5(n-3). The number of injections required was variable and depended on the abundance of the desired PUFA-ME, and resolution from closely eluting PUFA-ME, which determined the maximum loading. The purity of these fatty acids was determined by electron impact (EI) GC-MS and the chain length and location of double bonds was determined by EI GC-MS of 4,4-dimethyl oxazoline (DMOX) derivatives formed using a low temperature method. Advantages over silver-ion HPLC for purifying PUFA-ME is that separation occurs according to chain length as well as degree of unsaturation enabling separation of PUFA-ME with the same degree of unsaturation but different chain length (i.e. between 18:5(n-3) and 20:5(n-3)). In addition, PUFA-ME are not strongly adsorbed, but elute earlier than their more saturated corresponding FAME of the same chain length. This method is robust, simple, and requires only a short re-equilibration time. It is a useful tool for preparing milligram quantities of pure PUFA-ME for bioactive screening (as free fatty acids), although many multiple injections may be required for minor PUFA-ME. It also enabled dose-response and structure-activity studies to be carried out. It can be used for the enrichment of low levels of VLC-HUFA-ME to facilitate elucidation of their chemical structure and so is a useful adjunct to EI GC-MS of DMOX derivatives and other techniques such as NMR, which requires milligram quantities of purified compounds.     

Production of Human Milk Fat Replacement Rich of 1,3-dioleoyl-2-palmitoilglycerol From Enzymatic Interesterification Tripalmitin,Ethyl Oleate And Mixture of VCO,Soybean Oil And Fish Oil

Human Milk is naturally the only source of food for infant in their early life. It contains 2-6% lipid which provides about 50% of the total energy needed by the infant. Human milk fat (HMF) mainly as TAG with the specific fatty acid composition, palmitic acid (C16:0) (20-25%), which is primary located at sn-2 of glycerol bonds (70%) and oleic acid (C18:1), located at sn-1,3 (35%). HMF also provide fatty acids such as linoleic acid, linolenic acid, EPA, DHA and lauric acid that are very important for infant. The purposes of this research are to synthesize of 1,3-dioleoyl-2-palmitoilglycerol (OPO) and to determine the best composition of OPO, VCO, soybean oil and fish oil for HMFS production for infant formula. Interesterification of tripalmitin and ethyl oleate using immobilized lipase from Rhizomucor miehei (Lipozym RM IM) were used to synthesize of OPO. Interesterification product of mixed VCO, soybean oil and fish oil that are source of lauric acid, linoleic acid, α-linolenic, EPA and DHA, were formulated in mass ratio (58:20:20:2) and (70:18:10:2) for obtaining HMFS which have fatty acids composition similar or close to HMF. Composition of fatty acids from product were analyzed by GCMS. From this research, were obtained HMFS containing palmitic acid as much as 28.89% where 84.49% of that are located at sn-2 while sn-1,3 position are dominated by oleic acid as much as 55.11% from the total 38.7% and 70:18:10:2 w/w is the best composition of interesterification product, VCO, soybean oil and fish oil to obtain HMFS similar to HMF.     

Determination of docosahexaenoic acid and n-3 fatty acids in refined fish oils by 1H-NMR spectroscopy: IUPAC interlaboratory study

A high-resolution proton nuclear magnetic resonance (NMR) method for determining the concentration (mg/g) of docosahexaenoic acid (DHA), the molar proportion (mol%) of DHA, and the molar proportion of total n-3 fatty acids in fish oils was validated by an IUPAC interlaboratory study (the Commission VI-6 on Oils, Fats, and Derivatives WG 3/98). Thirteen laboratories from 5 countries tested 6 pairs of blind duplicate fish oils: a refined tuna oil, 2 extracted tuna oils, an extracted bonito oil, an extracted salmon oil, and an extracted sardine oil ranging from 9 to 30 mol% DHA and from 20 to 35 mol% n-3 fatty acids. Before 1 D-proton NMR measurements with 300-500 MHz instruments, oil samples were weighed and diluted with deuterochloroform solution containing ethylene glycol dimethyl ether as internal standard. To achieve precise performance, a detailed procedure for signal area measurement was described in the protocol, and all participants were instructed about the critical importance of following the protocol. Statistical performances with invalid and outlier data removed were as follows: repeatability relative standard deviations (RSDr) ranged from 0.91 to 2.62% and reproducibility relative standard deviation (RSDR) ranged from 1.73 to 4.27% for DHA concentration (mg/g); RSDr ranged from 0.39 to 2.06%, and RSDR ranged from 0.59 to 3.46% for mol% DHA; RSDr ranged from 0.23 to 0.90% and RSDR ranged from 0.85 to 2.01 % for mol% total n-3 fatty acids. The method is expected to be recommended by IUPAC.