“SAMPLING AND ANALYSIS OF COMMERCIAL FATS AND OLS AOCS Official Method Ce 1b-89 Reapprowed 1957 Fatty Acid Composition by GLC Marine Oils DEFINITION ‘This method determines the fatty acid composition of marine oils and marine oil esters by capillary ‘column gas-liquid chromatography. SCOPE ‘This method is designed to determine the fatty acid composition of marine oils and marine oil esters in relative (area %) values, and eicosapentaenoic acid absolute (ng) values using & bonded poyglycol liquid phase in a flexible fused silica capillary col ‘umn. Czy filly cid is used as the internal standard ‘marine oi or marine ol ethyl or methyl esters, capsules ring polyunsaturated fatty acids (see Notes, 1) (EPA) ond dacoschexaenoic acid (DHA) in ‘The method is applicable to the analysis of of EPA and DHA and minor naturally occur APPARATUS 1 Gas chromatograph—with capillary injection system 1 REAGENTS Sodium hydroxide—teayent grade. Gplit mode prefered, operted at a split ratio of 1:50; 2. Methyl alcohol—reagemt grade (see Notes, Caution See Notes, 2) and flame-ionization detector (FID; see 3. BFy—12% in methanol, 2-mL ampules (Supelco, Inc., Notes, 3. capable of meeting the following minimum eatalog no, 3-3020, or equivalent). requirements (see Notes, 4) 4. Isooctane—reagentarade (see Noles, Caution} Tajection por, 250°C 5. Sodium chloride—reagent grade, Detector, 270°C 6 Cayo metiyl ester—see Notes, 5. 2 (Oven temperate profile 7. Coygethylester—see Noves, § = Initial temperature, 170°C 8. Solations— : Initial hold time, 0 min {a} Alcoholic soxium hydroxide (NaOH), 0.5 N-tise= Program rate, 1.0°C/ini Solve 2.0 g NaOH in methanol and make 1 100 Final temperature, 225°C mL with methanol Final hold time, 0 min (b) Sodium chloride (NaCl)—saturated solution, dis ‘The capillary GLC coluran should be of flexible fused solve 36 g NaC! in 100 mi distilled water, Silica, 25.m or moce in length and 0.20-0.35 mm iu, The liquid phase mus be bonded Carbowax-20M oran PROCEDURE equivalent polyglycol. Examples of the latter are 1 Supeleowax-10, 30 m x 0.25 mm (or 0.32 mm) with 0.25 jim coating (Supelco, Inc. Bellefonte, PA, USA, catalog n0, 2-4079 or no, 2-4080) and Omnegawax 321 30 m x 0.32 mm id. with 0.25 pm coating (Supeleo, Inc, catalog no, 2-4152). No constraints are placed on the column supplier; however, the manufactarer's claim for the upper temperature limit of the column shouldbe noted and verified. Carrer aas—hydrogen or helium, 98.99% pure or bet ter is required. ‘Note—An oxygen scrubber is mandatory with Carbowax-20M and similar columns, Any suitable amplifier, recorder, integrator or data processor may be used Constant-temperature water bath or dry heating block—mainiained at 100°C. 3. Serew-cap tubes—16 x 125 mm, with leak-tight Teflon ined caps. Vial—serew- or erimp-cap, 12 ml Volumetric pipets—I and 2 mL. Pasteur-type pipes ‘Volumetric Masks—25 and 100 mb. Analytical balance—+ 0.0001 g. Source of dry nitrogen. Page tof 5 Oils (a) Accurately weigh (20.1 mg) approximately 25 mg of Cys methylester imteral standard (1S) into a 25.miL volumetric Aask and make to volume with isooctane Pipe | -m. aliquots of 15 (see Notes, 6) into cul ture tubes and evaporate the solvent. Stoce the tubes ine freezer if they will not be used immediatly Accurately weigh («0.1 m) approximately 25 mg of marine oil sample (may he from fish oil cape Sule nto the culture tube containing the IS. Add 1.5 mL of 05 N NaOH. Blanket with ntro- en, cap tly, mix and heat at 100°C for 5 min Cool, add 2 mL BFy/methanol reagent, blanket will nitrogen, cap tighly, mix and heat at 100°C for30 min Coo! to 30-40°C, add 1 ml. of isooctane, blanket with nitrogen, cap and vortex or shake vigorously for 30 sec while stil warm Tinmediately add 5 mL of saturated NaCl solution, blanket with nitrogen, cap and agitate thoroughly Cool to room temperature. When isooctane layer sepacates from the aqueous layer, transfer the ‘sooctane layer to a clean glas tube, blanket with nitrogen and cap. ) © @ © oO @ hy[SQHPENG AND ANALSE OF COMMERCIAL ATS AND OWS Ce 1b-89 + Fatty Acid Composition by GLC (i) Extract the methanolAwater phase again with an ditional 1 mL. of isooctane. Combine isooctane extracts (see Notes, 7) and cncentrate to approximately | mL. with a stream of dry nitrogen, AK) Inject (see Notes. 8) 1-2 pi. under appropriate gas chromatographic conditions (see Apparatus. 1). Methyl or ethyl esters— (a) Accurately weigh (+ 0.1 mg) approximately 25 ig of C,,.) methyl or ethyl ester (appropriate to the substrate to be analyzed) IS into a 25-mL volumetric flask and make to volume with isooctane (b) Pipet a I.O-mL aliquot (see Notes, 6) of the IS into aaculture tube (€)_ Evaporatethe solvent (@) Accurately weigh («0.1 mg) no more than 15 mg of esters into the tube containing the IS, add | mL. ieee $86. ss wean 3 Response of isooctane, blanket with nitrogen, cap, and mix thoroughly () Inject 1-2 pL under appropriate gas chromato- _Braphie conditions (see Apparatus, 1) See Figures 1, 2 and 3 for chromatograms of methyl and ethyl esters of various marine oils; see Tables | and 2 for retention times of marine oi mathyl esters CALCULATIONS, IL Area percentages—Calculate the area percent for fatty acids by the formula Area fatty acids = 10RD aA, Where— ‘area counts for fatty acid x = toll are counts forthe chromatogram ‘Ajg = atea counts ofthe internal standard & 2260-9 Figure 1, Fatty acid methyl esters of sot geatn-encapsulated,steam-deodorized menhaden cil (olaborative study sample 1 analyzed on a lexible fed sca caplary column (30 m 0.25 mm), coated with Supeleowax-10, under the conditions noted in the text. Figure 2. Fatty acid ethyl esters of a soft-gelatin-encapsulated commercial product, SuperEPA 500 (collaborative study sample 2) analyzed on flexible fused silica capillary column (30.m x 0.25 mm), coated with Supelcowax-10, under the conditions noted in the text. The location of added saturated methyl esters is indicated for reference. Page 2 of 5SAMPLNG AND ANALIS OF COMMERGAL FATS INO ONS Ce 1b-89 * Fatty Acid Composition by GLC 140 Response Tine Figure 3. Chromatogram of cod fiver oil (collaborative study sample 3) methyl esters analyzed on a flexible fused si -a capillary column (30 m x 0.25 mv, coated with ‘Supelcowax-10, under the conditions noted inthe text. 2, Calculation of mg of EPA and DHA per g of sample— (a) For marine oils and fish oil capsules (methyl ‘esters}—weight of EPA and DHA (soe Notes, 9). ‘expressed as mg of EPA/DHA fatty acid per gof of: CAO NCE (Aig. XLOH (by For methyl or ethyl esters—weight of EPA and DHA (see Notes, 9), expressed as mg of EPA/DHA fatty acid per g of saraple EPA or DHA. mgig = (AW sKCF) EPA or DHA, mfg =! x 1000 (ApghOW)1108) Where— ‘A, = afea counts for EPA (20:5n-3) or DHA (en) Ag = aed eunts for reral standard CH = theoretical correction factor for EPA or DHA mass of intemal stundard added to the sm: ple We = sample mass, in mg 3. Detector correction factors—Theorelical detector cor- rection factors relative to Cy (the internal standard IS) for Cap.sq-s (0.99) and ay gs (0.97) should be applied to the analytical data far optimum accuracy. PRECISION 1. Collaborative study statistics, based on results from 19 laboratories that participated in the study, appear in Table 3 NoTEs Caution 7 Methanol (methyl algobol) is flammable and toxic. Avoid contact with eyes. Avoid breathing vapors. Use effective fume-removal device. Can react vigorously with sodium hydroxide Isooctane is highly lammable. Aa effective fame hood should be used at all times when working with isooctane NUMBERED NOTES 1, thas been shown thatthe alkali-catalyzed methanoly- sis step for the determination of long-chain marine of fatty aids produces dimethyl and mixed-alcohol esters fcom the plasticizer dioetyl [actually di(2-ethylhexy!)] phihalate An independent boron taifluoride-catalyzed methanolysis step produced a lower level of anfacts, but the official two-step process produced & higher conversion than either catalyst did independently. The retention times for the dimethyl, mixed-alcohol and dioetyl phthalates are discussed (References, |) in rel tion to methyl esters of common fatty acids on poly. _glyeo! wallcoated open-tubular GLC columns. 2. There is usually suficient sample in marine ols analy: sis t permit operation in the spit mode 3. The column end may he inserted directly in the detec- {or jt, oF there: may be a connection at the base ofthe jet, with of without makeup gas, Ifthe FID requires ‘makeup gas, nitrogen is satisfactory. 4, These are suggested operating conditions for 8 30-m column, S. Authentic standards of saturated fatty acid esters, if needed for peak identification, are widely available. Upon request, the Charleston Laboratory, Southeast Fisheries Center, National Marine Fisheries Service, Page 30f5SENPUNG AND ANADSS OF COMBA FATS AND OWS Ce tb-B9 + Fatty Acid Composition by GLC Tablet Table2 Retention times and area percent of fatty acid methyl esters Retention times and area percent of cod liver oil methyl ‘of steam-deodorized menhaden oil (collaborative study sam- esters (collaborative study sample 3). See Figure 3. ple 1, See Figure 1, Tetcnion | — Relate Reenon ] — Retave time | retention | Area time | reteion | Area Fay acid | (nia) tine percent Fayaid | ain) dine percent 40 aid 0.335 & 40 “7 0335 37 160 23 0578 187 160 17 0576 133 16187 186 21 14 i6:Ind 7 08m a8 180 4 100 3 180 ra 100 23 18:In9 1297 105 oe 1nd 108 10 ia int Bz 107 sind BR ior 1820-6 1457 1 2 18.206 6a Lig 20 18303 1708 Ls 08 18a 1720 138 10 in 1838 a0 2 18ed 1850 19 20 200 2032 16s 02 209 2047 02 aint 2091 169) PS 20st 2108 | aie a Bln a un 20:09 2128 oan 237 189 a 20206 as 03 aman 2866 200 02 206 24381 199 ou 20303 2686 26 02 20303 26386 26 on 2oetns 2574 208 ox Being 2591 20% oa a} a0 2 13 20:in 2825 at 06 20503 2029 237 8 205503 29483 2st 1s 20 mm 26 02 20 3050 2s 03 Biwtisis | 3427 23 Th Baintt | _ 3150 353 a Bind 3188 236 Zing 318 256 Bites wT 303 02 aang 3930 308 02 2506 383 3 02 Bsns wu ane ao 20-3 ai) 333 2 Bind a3 32 10 ibn 286 aa? 79 Zion a7 352 70 240 Ras ou 20 2a 33 a 2nd Be 03 2 a7 352 a9 Pagedol 5“SHAPING AND ANALIGE OF COMMING FATS ANDONS Ce th-89 + Fatty Acid Composition by GLC| Toble 3 Statistical analysis of results for 6 fish oils, based on results received from 21 labora met 23 ge i40_| #3] oa] 37] 70] sales 160 | 187] 09] 135 | 130] 28] 90 iso | 31] 2) 23] 29[ oa) a wi _L uy] a7] veo [iar] 02) 119 ie [isae3] 29 19] 207 20] 4a] 29 moi | 18 [42] 190 | 32] 13] 18 ose] a8] 264) 78 ro] 73) 3a mi_[ 14] 106] 90] 24] 10] 13 mse] 20] 43) 10 | 21] 22] 20 nate] 79| a7] 20 | 23] 12s] 7a wo [ua] mi} aif 83] 120] a6 60 | 70] 122] 75 69] 57] 100 iso | 36] 70| 31 | 14a] sa] 57 is1 | 19] 57] 28 [30] 67] 36 lg [isens[ a4] 76 42] 63] ss] 45 @ [aon | so| 32] as] 97] wi 162 mosea| sa) se] ss [gal 75] 98 ma1_[ m2] a7] a9 Page| sa) Tar zsn3| 146] 85] 9.7 Foma] 88] 162 mans] isi [73] 99 | 1g] 7s] ss | 20:sn-3 | 115.3] 223.1 | 683 | 1975] 241.4] 117.7 [220031 cool so] sot [ross] ross] 672 If [2osn3] 0] _92| 77[ 9s] sa] 79 B - beeens_aal- alas [asl] ee Ea B50, | 33] 58] v0 | 1a7] 29 {o> a1} 18 Fos nis] 4 Zhisaal, is Boo [isl aa Blrose3| 8] 19 mi ial 79 meses] 20] 62 a3 [ a9] 37 feo. mug JF [ages [mes] so ("| 22:6n-3| 665] 5.3 “All samples were soi-gelaia encapwulated. |, Steam Jeodorized] ‘mentaden ol; 2, commercial thy ester preparation: 3, cd liv oil; 4, commercial fish ol; 5, commercial fish oil enriched in EPA and DHA:.6, team-deoderized menhaden oi (lind dupl- cat of sample 1. P.O. Bot 12607, Charleston, SC 29412-0607, USA, will provide capsules of collaborative study sample | for use in optimizing GLC equipment. Cy, should be baseline separated from Cy, (1S), and Cy should be baseline separated from Co, (See Fig. 1). 6. Ifthe peak height of the 1S is°= one-half that of the EPA or DHA peaks, cepeat the analysis using 2.0 ml, of IS, 7. Optionsl—Wash isooctane with 2 mL of water and dry ‘over anttydrous sodium sulfate. 8. Manual injection with a microsyringe is preferred; however, an autoinjection may be used, 9. Please note that this calculation gives results for fatty acids and not for esters of fatty acids, The analyst can confirm detector correction factors with pure reference standards on individual instruments and capillary columns. The weigit % calculation should always be based on theoretical FID correction factors. In case of strongly deviating experimental factors (>5% deviation from the theoretical factor), the analyst should locate the cause of the deviation, rather than applying the experimental factor REFERENCES I, J. Chromatogr 587:263 (1991). OTHER REFERENCES INFORM 1:987 (1990), J. Am, Oil Chem. Soc. 64:499 (1987). Ibid. 66:1822 (198), General referee repor: on oils and fas, J. Assoe. Off. Anal, Chem, 73:105 (1990). J. Chromatogr. Biomed. Appl. $33:1 (1990). J.AOAC int 75:488 (1993) AOAC International, 16th ed; Vol. II, Gaithersburg, MD, 1995, Chapt. 41, p. 20, Method 991.39, Page 5 of §