Grasas y Aceites Vol. 51. Fase. 6 (2000), 447-456 4a7 e Methods of preparation of fatty acid methyl esters (FAME). Statistical assessment of the precision characteristics from a collaborative trial By A. Cert, W. Moreda and M.C. Pérez-Camino Instituto de la Grasa (C.S.1.C.), Av, Padre Garcia Tejero, 4 41012 - Sevilla, Spain, RESUMEN Métodos de preparacién de ésteres metilicos de doldos ‘grasos (FAME). EvaluaciGn estadistica de la precisién del método mediante un estudio colaborativo. Los métodos ofliales para el contol del aceite de ova y do ‘jo do clva dela Unién Europea (UE) y dl Comité Clelcolain- temacional (COI) ncluyen la determinactén de acidos grasos en la aplcacion de varios criterios de pureza ‘La determinacion de dos grasos requiere la preparacién de {os ésteres metlioos de os acidos grasos (FAME) y su posterior andlisis mediante oromatografa de gases con una buena repel bildad y reproducblidad, Enire los muchos métodos usados por ls laboratorios dela industria y de los organiemos oficiales de contro, so solecciona- ron|oe siguientes: 1) motfacion an fo con potasa metandlicay 2) ‘matilacion en calito con meta sddico seguldo de acidic: €n con desde sullrico en metancly ealentamient Se realiz6 una eveluacion estadistica de a precision de a ‘composiion de écidos grasos obienidos usando ambos metodos {de metiacion, mediante un estudio colaboratvo siguiendo las in ‘leaciones recogidas por la AOAC (AOAC 1988). En aceles con baja acidez, 1s resultados obtenidos usando ambos métodos de motacién fueron equivalentos. Sin embargo, ‘en la muestra de aceite de oryo crudo do olva(acidez 15.5%) 66 aprecaron diferencia signfeatvas en la composicion de actos ‘Fas08 obtenigos usando ambos métodos. Finalmente, o uso del método de metiacién en callonta no do lugar a un aumento de la concentracion de isémeros trans. PALABRAS-CLAVE: Acolte de oliva - Andlsisestadistico - stares Motficos Acidos Grasos = Estualo colaboratvo - Méto- dos de preparacién. SUMMARY [Methods of preparation of fatty acid methyl esters (FAME). Statistical assessment of the precision characteristics from collaborative tral. “Tho official regulations for the control ofthe olive and olive oils of the European Union (EU) and Intemational Ove (O11 Counei (1000) include the determination of fatty acids in ordor to be applied to savoral pury rar “The determination of fatty acids require the preparation of the fatty acid methyl esters (FAME) for the subsequent analysis by {988 chromatography with good precision and reproducty. ‘Among the methods used in the laboratories of both the Industries and the offal institutions looking after the olve ot contol, the ones selected were: 1} cold methylation with methanol Potash and 2) hot mothyaton with sodium methyfats folowed by ‘ackifcaion with suiphurc acd in methanol and heating, ‘Aslatistical assessment ofthe precision charactorstios were performed on the determination of fatty acids using both methods by a collaborative tral following the directions included in te ‘AOAC regulation (AOAC 1985. In ols with low aciditos, the results obtained for both methylation methods were equivalent. However, the olive- Pomace oll sample (acidity 16.5%) showed significative Gifferences between the fatty acid compositions obtained using both methylation methods. Finally, the methylation withthe aciloybasio mothod ald not vield an nerease ofthe transizomars oft fay acts, KEY-WORDS: Collaborative analysis - Fatly Acid Methyl Esters (FAME) - Methods of preparation - Ove oil - Statistical analysis. 1. INTRODUCTION ‘The official regulations for the control of the olive and olive pomace oils of the European Union (EU) (EEC_1991) and International Olive Oil Council (100C) (1OOC 1998) include the tatty acid Composition from C140 to C240 and the transisomers of the fatty acid (FC18:1 and #C18:2+#C18:3) as criteria for the oil genuineness. Furthermore, the fatty acid composition, in particular the C16:0, C16:1, C18:0, C18:1, C18:2 and Ct are used in the determination of a purity criterion, that is the difference between the experimental value of the triaoyiglycerols of equivalent carbon number 42 (ECN42) determined experimentally by HPLC and the theoretical one obtained from the fatty acid composition. The determination of fatty acid requires the preparation of the fatty acid methyl esters (FAME), in ‘order to improve volatility and to reduce peak talling, analysis by gas. chromatography in and reproducibility. Usually, FAME can be conveniently prepared by reaction of lipids with a large excess of methanol with either acid- or base-catalytic reagents. There are several described methods to prepare FAME (EEC. 1991), IUPAC 1987), (ISO 1998) from fats and oils. These methods are based in different chemical principles and therefore, the results are not completely equivalent. We can divide the methods in two main groups: a) methods causing only transesterifcation of glyoeridic compounds and waxes and which do not usually form FAME from free fatty acids (Glass 1971) (basic methylation, (CH:).S0. and cold methanolic potash) and b)methods that methylate glyceridic compounds and free fatty acids (Christie 1992) (F:B, acidic methylation, ‘rimethyisulfonium hydroxide, basic + acidic methylation and 1,1,3,3-tetramethyiguanidine (Schuchardt et al. 1988)). However, in virgin olive oils and specifically in crude pomace oils, there are fatty acid ethyl esters which behaviour in the different methylation methods are not completely studied. With the aim of standardizing the FAME preparation method for olivé’ and olive pomace oils, to obtain repeatable and reproducible measures, the Expert Chemist Group of the Intemational Olive Oil Council ‘chose one method of each group bearing in mind at it easiness and low toxicity of the reactants and vents used. The methods selected for being used in the laboratories of both the industries and the official institutions looking after the olive oil control were: 1) cold methylation with methanolic potash and 2) hot methylation with sodium methylate followed by acidification with sulphuric acid in methanol and heating, To standardize these methods the 100C organized a collaborative trial wth the participation of laboratories of different countries. The results obtained are exposed. 2. EXPERIMENTAL 2.41. Sample descriptions The oil samples are as follows: 4) Extra virgin olive oil (acidity 0.18%) 2) Virgin olive oll (acidity 2.0%) 3) Virgin olive oll acicity 3.9%) 4) Olive oil (acidity 0.88%) 5) Crude olive-pomace oil (acidity 15.8%) 2.2. Instructions. ‘Samples were sent, together with the following instructions. The methyl esters solution should be analysed as soon as possible. If itis necessary, the heptane solution may be stored under an inert gas in a refrigerator to protect the methyl esters from autoxidation, ‘The gas chromatographic analysis of the heptane solutions must be done according to the method for the determination of fatty acid trans-isomers (EEC 1992). Each sample must be analysed using both A and B methylation methods. Each method must be done by duplicate. In total, four analyses by each oil sample, The cisisomers of the FAME ftom C14:0 up to ©24:0 and the trans-isomers of the C18:1, C18:2 and C18:3 must be considered. Likewise, the ethyl esters, of C160, C18:1 and C182 must be considered Grasas y Aceites (Figure 1). The results must be expressed as the percentages on total area of peaks, exciuding the area of squalene, with two decimal figures. 2.3, Methods description A). Methylation with cold methanolic solution of potassium hydroxide (IUPAC 1987) In a 5 -mi screw top test tube, weigh 0.10 g of the oll sample, Add 2 ml of heptane and stir. Add 0.20 ml of 2N methanolic potassium hydroxide solution, put on the cap provided with a PTFE (polytetrafluoroethylone)-joint, tighten the cap, and shake vigorously for 15 seconds. Leave to stratiy until the upper solution becomes clear. Decant the upper layer containing the methyl esters. B) Methylation by heating with sodium ‘methylate in methanol followed by heating in acidic medium (IUPAC 1987) Transfer about 0.25 g of the oll sample into a 50-ml ground-necked volumetric flask. With the ald of a funnel, add 10 mi of 0.2N sodium methylate in metanol and bolling chips. Fit reflux condenser, str, and bring to the Boil. The solution should become clear, which usually occurs in about 10 minutes. The reaction is complete after 15 minutes. Take away the flask from heating, wait until the reflux stops, remove the condenser, and add two drops of 1% of henolftalein solution in methanol. Add a solution of IN sulphuric acid in methanol until the solution becomes colourless and, then, add 1 ml in excess, Fit the condenser and boil again for 20 minutes. Withdraw the source of heat and cool the flask under running water. Remove the condenser, add 20 ml of Figure 1 ‘Gas chromatographic prolie of fatty acid esters obtained by ‘the method A rom a mixture of rude olve-pomace oll and refined ove of, The peaks oorreepond to the methyl esters ‘obiorwise indeated.Vol. 51. Fasc. 6 (2000) saturated sodium chloride aqueous solution, and stir. ‘Add 5 mi of heptane, plug the flask, and shake vigorously for 15 seconds. Leave to settle until the two phases have separated. Add again saturated sodium chloride solution until the aqueous layer reaches the lower end of the flask neck. The upper layer containing the methyl esters fils the flask neck. 2.4, Results organization 24.1. Fatty acid composition For caloulation of the fatty acid composition, the following peaks were considered: C14:0 methyl ester, C16:0 as sum of methyl and ethyl esters, 16:1 as sum of two cis-methy| esters (C16:109 and €16:107), C17:0 methyl ester, C17:1 methyl ester, 18:0 methyl ester, C18:1 as the sum of two cis-methyl esters (C18:109 and C18:107), ethyl ester and trans-methyl ester, C18:2 as sum of cis-methyl, ethyl esters and trans-methyl esters, 18:3 as sum of ols- and trans-methyl esters, 20:0 methyl ester, C20:1 methyl ester, C22:0 methyl ester and C24:0 methyl ester. 243 Fatty acid trans-isomers The trans-isomers of the fatty acids C18:1, C18:2 {as sum of the trans-cis-isomer, cis-trans-Isomer and trans-transisomer) and Ci8:3 (as sum of trans-cis-cisisomer, cis-trans-oisisomer, _cis-cis- trans-isomet and trans-cis-trans-somer) were determined following the method included in the Regulation EEC/1492/92 (EEC 1992). 2.5. Statistical interlaboratory study ‘The collaborative trial was set up following the directions gave by W. Horwitz (Horwitz 1988). The statistical analysis of repeatability and reproducibility was performed following the 1SO5725 (ISO 1986) and AOAC Regulation (AOAC 1995) where the Procedures of identification of outliers and mathematical procedures are described, using a ‘computer program developed by the authors. The criterion used to identify outliers was the Cochran and Grubbs test, determining respectively the laboratories that gave results quite different among replicates and those with outstandingly high and low values. The statistical parameters used were the followings: — S, Standard deviation of the repeatability — Repeatability (2.8 VS) — RSD; Relative standard deviation of the repeatability = Sp: Standard deviation of the reproducibility 449 Ri: Reproducibility (2.8 15%) = RSDp! Relative standard deviation of the reproducibility = Hon: Horwitz ratio RSD 5 FSDaw, ver RSDap = 2"*5"°9, being C the concentration of the analytes expressed in 10 power. ‘Among these parameters is important to point up the statistical meaning of the repeatability, within-laboratory variance, which indicate that the values obtained in two successive determinations of the same sample, using the same analytical method, ‘Wo not differ more than the value of +. Likely, the reproducibility, interlaboratory variance, means that the results obtained by two laboratories using the same sample and analytical method, do not differ in more of the F value. Besides, it must be highlighted the meaning of the Horwitz ratio (Hor) that take in account the analytes concentration figures. The value is obtained by the ratio between the RSDs experimental and theoretical in such way that a value equal or less than 1 means that the analytical method has a good reproducibility (Pocklington 1991). The comparison between both methylation methods was done by an analysis of variance (ANOVA) with repeated measures, being significative differences higher than 95%, The analysis was performed using the statistical package STATISTICA {Statsoft Inc., USA). 2.6. Operating conditions Data shown in Table 1 were reported by collaborators on the operating conditions for the GC analysis. The majority of laboratories used columns of 50 or 60 m length, 0.25 um of intomal diameter, coated with cyanopropyipolysiloxane or ‘eyanopropylphenyisiloxane (0.20-0.25 jum of film thickness). 3, RESULTS AND DISCUSSION The collaborative study was performed with the results of 17 laboratories of different countries for evaluating the reproducibilty and repeatability of the FAME preparation methods. The results sent by the laboratory 17 wore discarded, because the application of response factor for each individual FAME. The determination performed by the laboratory 16 using the method A was also discarded, because the lack of identification of the ethyl esters, neither the FAME C22:0 obtained by the method B. From the results sent by the laboratory 4, the results corresponding to the trans-isomers obtained by the method B were also discarded, because the use of a gas450 Grasas y Aceites Table 1 GC Operating condition of the laboratories Fim inital Fal tin benght Cari alo —_njector Dette (dboratery Col (mxmm) — ‘Gas cininy Temp. °C) Temp. (°c) Temp. (0) Tine(in) Temp.) Tine (nin 1 SP2380 60x05 rT ee er en ee) 70250 2 -BPK70 80x0.30 ca 3 $P-2360 60x0.25 He 101390 ce 4 GPSiL-8 650x025 He 160 80 a a) 4 Gwe0” 35x028 He 170 220 3 250280 5 P2980 600. He e778 5 ot 240 260 3 $2380 60x0. He 1675200 8 2 26260 8 SP240 eox0s 020 He = 15018178 Heed bo 280 8 Sp2sd0 s0x0s2 020 He 180 18 20 1% 8 bs 250 7 Ress s0x0s2 020 He = 170, 7 19 = 2 240 a0 7 Ress Sox0s2 020 He = 170 7 20 Ses) 240 40 8 —CPSIL-8S 100x020 020 He 15 20 12] 8 Gps soxo2 10 Ares 60x025 020 «He | 17D ata 5 250 260 i SP.z300 60x028 020 He «1708210 5 250 260 12 © GPSiLes 60x028 020 «He = 15120 5 260 280 13 SP2590 soxos2 020 He 180 15 210 5 20 0 a0 44 -BPK70 50x09 025 «He «= 180 sO 250 280 15 BPX7O 30x05 0250 He DB 18 © BPX-70 50x02 028 tO a rr] 220 350 17__Spzso0 s0x02s 028 = Me 220250 chromatographic stationary phase quite different (Carbowax 20M) ‘The determination of the ethyl esters of the fatty acids by the method A (Table 2) showed resuits with a great variability of values, in both within-laboratory and inter-laboratory determinations, indicating that transesterification of the ethyl esters of the fatty acids is a slow reaction and hence, the results depend on. the operating mode. Therefore, to calculate the percentages of C16:0, C18:1 and C182 the sum of ateas corresponding to the peaks of both the methyl Table 2 Results of ethyl esters (%) determination by cold methanolic KOH method (A) in crude olive-pomace oll sample (5). Parlgant —C1G:DEthyl © CIBSAEthyt CBZ thy 1 003 003 025 028 003 003 2 062 085 349 381 080 065 3 019 045 088 224 014 035 4 028 027 143 145 022 023 5 050 048 255 247 040 038 8 012 013 076 080 008 0.09 7 010 O11 085 085 009 0.09 B 047 039 246 211 058 048 9 078 O77 343 372 O87 O57 10 043 080 243 322 020 023 M1 © o42 042 208 208 038 038 12 078 127 331 638 083 1.08 13 O67 014 010 O59 4d 44 O18 012 08s Os O14 Out 45000000 085 078 015 O12 and the ethyl esters and trans-isomers were taken in account (On the other hand, methylation by the method B yielded negligible amounts of ethyl esters, indicating that transestertication was completed. ‘The results obtained were analysed statistically as stated in the experimental section and the results showed in Tables 3 to 16 for each fatty acid. From the preliminary examination of the results, it can be pointed out that in general there are good acceptances of the results, except for the fatty acid C1620 (Table 4) and for the trans-C'18:2+trans-C18:3 (Table 16), in which there are more outliers than ‘expected, probably due in the former to the mass discrimination produced during the GC spit injection and in the latter to the different gas chromatographic ‘operating modes result in erroneous assignments. Regarding the repeatability, the results are acceptable, although the C14:0 (Table 3), C24:0, (Table 13), trans-C18:1 (Table 14), trans-C18:2 and trans-C18:2+trans-C'18:3 (Tables 16 and 16) showed high RSDr values, due to that in olive oils these FAME gave very small peaks (less than 0.05%), ‘lose to the detection limit. Similarly, the reproducibility is good for the major ‘components since their Hon are lower than 0.27, meaning that the variance is according to the ‘concentration. For those components with values olose to the detection limit: C14:0 (Table 8), C24:0 (Table 13), trans-C18:1 (Table 14), trans-C18:2 (Table 15) and trans-C18:2+trans-C18:3 (Table 16); the ASDxVol. 51. Fasc. 6 (2000) 451 Table 3 Statistical parameters from C14:0 acid determination in olive and olive-pomace oils face Extavirgin Vegin Lampant ‘te at Crude ove pomace ot Method KOH. Baslovacldle KOHin Baslovaeéle KOHIn Basiesacidle “ KOHIn Basicsacsle KOH In Basicsacc ‘methanol methyation methanol methylation methanol mathylaton methanol methylation methanol methylation Paricipants 15 16 16 18 16 15 16 15 16 Outiors 0 1 9 1 3 1 ° 3 3 Mean(%) 0.0090 0.0087 0.0138 0.0118 0.0098 0.0100 0.0100 ote! 0.0208, Fepeatabiity r 0018 0.0028 0.0043 9.0042 0.0034 0.0088 0.0043 0.0059 r 0.0051 0.0072 Oot 0.9118 0.0092 0.0108 o.0121 ‘0.0165 ASDr(%) 20 30 at 36 37 38 43 20 Reproductity 0.0081 0044 0.0071 0.9062 0.0060 0.0047 0.0057 0.0080 a ood 0012s 0200 00173 ore! o.01s3. 0.0160 0.0168 FsDa(%) 45 st 52 52 ‘et a2 87 23 Hor 05 06 08 08 oredr OS mee 03 Table 4 ‘Statistical parameters from C16:0 acid determination in olive and olive-pomace oils —s tra vigin Vigin ampant ove Crude olve-pomace ot Method —_KOHIn Baslovecdic KOHin Baslevactic KOH In Basicvacidle KOHin Baslvacelc KOHIn Basicracidle rmathancl methylaion methanol methylation methanol methylation methanol methyaion methanet methylation Partkipants 15 18 15 16 16 18 18 18 16 18 Outiors 2 3 3 4 1 3 0 2 0 4 Mean (%) 798 808 ts2 1081108502) 105101871028 Repeatabity or 004 009 015 043-010 os oth r 012 0.28 042 035s oe ase sor) 053 tt 15 re tr io ee Roproducibitty Sr 024 ong oss ot7 Bd i oes = 040 oss 04s 13 12 13 (at RSDa(%) 3.0 18 32 16 4440 47 14 Hr 0090.05 O10 005 ts tS tS 005 Table 5 ‘Statistical parameters from C16:1 acid determination in olive and olive-pomace oils — ara viegin Vega Lampant tv a Cruse ove pomace ot Method KOH Baslosseele _KOHin Baslsacdle KOMI Basictaide KOHin Basicvactle KOHIn Basicvacide ‘methanol methylation methanol methylation methanol methylalon methanol metiyaton methanol methylation Parkipants 18 16 18 16 18 16 15 16 18 16 Outliers 9 1 2 2 ° 4 4 1 1 2 Mean(%) 0.504 «S01 «0.675. 0.882078 O78 «0808-0870 088e 7A ealablty e 001s 0017 Oona ©0268 ong0 mieten ote r Oost = 0048002777074 O08? 0034 0.034 ©0040 0.080 2888 1a 44 38 28 13 1a 23 27 0038 0084 0027-707 © 044 dd 0057 048048 8 0986 00850077 tS1 = 0.1s2 0.122 0.123 0.188 0.128 0.198 RSDa(%) 88 8B 41 70 ea 8A 43 85 72 73 Hoe O14 one 008 StS OTs OB. Rsou(%) ‘oproducitty &452 Grasas y Aceites Table 6 ‘Statistical parameters from C18:0 acid determination in olive and olive-pomace oils ee xa virgin Vigin Lampant otve ot ‘rude ove pomace ol ethod KOMI Baslevacle KOHin Baslvacsle KOMI Basicsscidle KOHIn Bascvacdle _KOHin Basiciacicle methanol methylation methanol methylavon methanol methylation methancl methylation methanol methylation Paricpants 15 16 15 16 18 16 16 16 16 16 ators 2 1 °o 4 0 1 ° ° 1 ° ‘ean (5) 2883 2871 249025082818, «2676 3.402 3.405.118 8.258, Ropesiabity 0032 0018 «= ont2 0017-0030 0.0eF = 0.084035) 0ns8 (a7 r 0089 0.08904 Ry) .0es L075 ned) eT) Oto. Aso) 140810 O8G A 10 © 0980832 15 eprodutoity Sr 0081 0.110 00s2 09 nests ost tar ont? ota a or71 00a = 0259006) 024s «0207 OST) Oat] «O28 (0.388 SOR (%) Ba 38 37 44 34 aoe sa eeas 38 39 Hor 005 00 eof. Table 7 ‘Statistical parameters from C18:1 acid determination in olive and olive-pomace oils a xtra viegin Virgin Lampant ote ait (ruse lve-pomace ot Welnod KOH In Basictacdic KOHin Basiciacdle KOH in Basicacile KOHIn Basloracdle KOHIn Basictacide ‘methanol methyation methanol methyaion methanol methylation methanol methylation methanol methylation Pantcipants 18 18 8 16 18 16 1 16 16 16 Outiors 0 1 0 1 1 1 1 1 0 1 Mean(%) 7942 «7939 «7455 «= 7456 75SB 75.41 «76.14 «76227580 75.02 Ropeatabity sr 015 oto Otek. r 04 022 «030 Os 23 OD Aso) 018 «ONS. OS ot OS OBO Ot ts Reproductoty Se 04g 0420S] MSM OKT BAST. a 17 te 8 1250013413333 tO Rson(%) O81 083 ost ose «= 8DsSszS Sr Ho 003 oz oss sms ss Table 8 Statistical parameters from C18:2 acid determination in olive and olive-pomace oils, = Baravirg Virgin Lampant ove ot Crude ove pomaee ot Matbod —_KOHn Basictacsio KOHin Baslvactle KOMInBaslctacidle KOHin Baslevactlo KOHin Basicracidl rmothanol methylation methanol methylation methanol methylation methanol metylaon methanol methylation Partcipants 15 16 18 16 18 16 15 16 15 16 Outlors 2 2 1 2 ° 1 1 1 0 1 Mean (%) 783 «725 «968 980) Se 8dk 7.18712 BSS Fpeatabilty or 092 004 003 0.08 = 0.08. 008-05 6 O07 on2 = 08st att2 OS Rso(%) 033 ««O8B.S028SCODCOTD 08002 SBOE Cat Roprodveibaty Sa 012 017 O90] tBtSte ote ota B ost 047 oes DCR Rsoa(%) 1738 19 20 24 oat aa 16 24 ta Flor 00s 007 0080808Vol. 51. Fasc. 6 (2000) 453, Table 9 Statistical parameters from C18:3 acid determination In olive and olive-pomace oils angle ata vigin Vigin Lampant veo Crude ove pom ot Wethed ——_KOHIn Basievacie KOHin Baslvacdle KOHIn Baslcrscldle KOHIn Baslovaccle KOHIn Baslevaidle methanol methylation methanol methylafon methanol methylation methanol methylation methanol methylation Partcpants 15 16 15 16 15 16 18 16 16 16 Outors 2 1 9 2 0 0 ° 4 4 1 Mean(%) 0730 ««O719 «0.886.876 (ORG) O44 ©0720 ©7352 Flopeatabilty or 0013 0012 O17 OOS 001000014 0.01020 r 0035 0.025 0049 0.037 O.ug8 0.02) 0.038 0.029.055 O05 ASO) 18 7 19 15 42 18 18 14 26 22 produeibity Sr 0029 003200410089 0.038043 0.028080 oom 05 R 008 ©0089) OID «= OtOT 0.120 0.078 0aS ts (0.087 Asoa(%) «39k 46 45 42 og ee soee ra 4 44 Hor bos 00) oto oto) Table 10 ‘Statistical parameters from €20:0 acid determination in olive and olive-pomace oils a tra virgin Vig ‘Lampant otve ol Cruse olv-pomace ot Wettod _KQHin Basictacdle KOMIn Basictacidle KOH In Basictaidc KOMIn Baslcracdle KOH in Basioracido ‘mathanot melhyaion methanol methylation methanol methylaion methanol methylation mettanol methylation Parcipants 18 5 8 5 15 15 18 18 18 18 ‘Outlrs 1 + ° 7 ° 1 4 1 o + Mean(%) 0.304 0.405 O4s1 04850440) oot 0Az4 «0.423045 0.420 Ropeatabity & 0015 0016001. OIS NIST nists omens r Bost 0044 ©0080 00K) 037 OAT) 037 003700830088 Rsor%) 38 88 40 32 30 378 ‘31 44 3a FReproducibity Se 0029 0037 002 ons100a10n39 onde 00980088 oa ia 01080 0.10 ©0088 O.414 0.086108 O.1I7 0.107 0.102.080 RS0a(%) 738 72 a8 79 a4 ar) 88 66 Hoe oi4 ote odo? ota te teste? ts Table 11 Statistical parameters from €20:1 acid determination in olive and olive-pomace oils — ara virgin Vig Lampant te ot (Cue ove pomace of Method KOH Basiotacidle _KOHin Boslotacdle KOHin Basiosaide KOHIn Basictactle KOHin Basicraidle ‘methanol methylation methanol methylation methanol methylaton methanol methylation methanol methylation Partépants 15 16 15 16 18 16 18 16 15 16 Outiere 1 1 i ° 1 2 ° 1 1 4 Mean(%) 0.372 «S78. «(0888 «= «400 «0370 ©0373-0280 ©0284 «0.208. .208 Repeatability & Cn oY a Tr r ces 0038 .0se OMG 0.036 «nek md7 ose) 0073.07 Rso%) 7888 30 re 35 2360 AO a9 44 epedicby 0.029 00320084 0032 0.028 00270028 002k «omar as a 0082 0.081 0085 0st © 0.088)» 0.077 00780088) 07?) 0.085 FSDa(%) 79 BT 87 at 62 ies oe Tt} 93. 54 7. Hor ois 07d? et Bt? to454 Grasas y Aceites Table 12 ‘Statistical parameters from €22:0 acid determination in olive and olive-pomace oils: so Extra viegin Vig Lampant ove ot Crude otve-pomace ot Method _KOHInBaslovacdle KOHIn Baslossedle KOH In Basiosacidlc KOH in Baslosacidle KOM in Basictacile ‘methanol methyation methanol methylation methanol methylation methanol methyaton methanol methylation Partcipants 15 15 15 15 18 18 15 15 8 5 Outliers 0 1 1 ° 1 1 1 1 a 2 Mean (%), ot ott4 0.135.405 ats OBO. 0.850205 opoatabiny Sr 0.008 0011 0013008014 © 0080s onto 01s. © 0022 ©0002-0038» 022 «Ong Onat OMS 0.028 0.038 (Ona Rp) 7088 86 65 100 BS 14080 68 72 Foproduetoty $e 001s 0.014 = 0018. 0.02 001Bn1B ong om1T = nIsO.0R4 z 0033 0038-0048" 0.088 0.050 «0500880047 ©1043. (O.087 Aspa(%) 120 120 120 140 «©6180 «S120 17015083120 Hor og 02 ois ose oT sh O20 Table 13, ‘Statistical parameters from €24:0 acid determination in olive and olive-pomace oils Extavirgin Vigin Lampant One ot ude olive pomace cil ‘Sample zs igi per en Method KOHIn Baslosscdle KOHin Basioiacidic KOH In Basicsace KOH In Baslcsacdle KOHIn Basioeacic ‘methanol methyation methanol methylation methanol metylalon methanol methyation methanol methylation Partoipants 15 16 8 16 18 16 1 16 18 16 Outiere 1 4 0 2 ° 2 ° 2 3 3 Mean(%) 0.040 «0.047.082 «0.078.008 07S 0049 «00S 07S 0.125 opeatabity x 0008 0014 +0005 0004-12 nIDOMI2 ons mtg (On r 0017 0039 O01s «O12 ©0332) css IT) Ondo (0.036 Rs) 15080089 58 20001402010 198.0S 1000, RReproducbity 5p 0020 0021 002500140026 «Ons ots mtd O.0at 8 0058 0.059 «0.078 «00d ©=—«a72 © 0a5 OA © O.0ds 0.0.06 Ason(x) 490 440 420s i905 5.027.018.0180 Hor os7oez ost 028 oat se SDS Table 14 Statistical parameters from trans-C18:1 acid determination in olive and olive-pomace oils ta virgin Vein Lampant lve ot Cruse olv-pomace ol Sample ethod KOH Baslvacdle KOHIn Baslotacdlec KOH in Basicsace KOHIn Basicractlc KOHIn Basloraic methanol methylation methanol methylation methanol methylation methanol methylalon methanol methylation Panpants 15 18 15 8 15 15 18 18 18 18 ations 1 2 1 1 1 1 4 3 2 4 Mean(%), 0.0100 0.0115. 0.0114 at29§ O0107 até © aoteT§0tIT§o.tt78 0961 opoatabilty Sr 0.0098 0.078.004 0.0027 0.0027 0.0046 0.0045 0.0050 0.0158 0.0108 r O10 040220 00130 0.0078 O.0075 | O.010 0.0125 OOT4 © 0.0443 O00 MO; CRO 41D) BO) 15D: a1) BOY aS 180, 11.0 0.0098 0.0087 0.0098 O.0103 00107 0.018 D013 0088 0.0559 0.0270 a 0.0268 0.0273 0.0276 © 0.0289 © 0.0300 O.ces7 © O.0g1e © 00247 © 0.1888 0.0756 Asoa(%) 960 840 © 880 © 80.0 «10000890780 4808.0 Flr 140095 sr se 110110100 0.85 78S sort) FRoproducibitty SrVol. 51. Fasc. 6 (2000) 455 Table 15 Statistical parameters from trans-C18:2 acid determination in ollve and ollve-pomace olls a tra vgin Vigin Lampant lve ot ‘rude ove-pomace oi Method OH In Basioacsle KOHIn Basietacle KOHn Bascréle KOHIn Basoicile KOHIn Basiacsle ‘moihanol metiyaion methanol mthylaion mehanel_metyiaion methanol metiyiaon methanol matvaion Partopants = 1515 18 15 6 1% 6 15 15 6 utr t 2 ° + 2 2 $ ° t Mean), 00061 0008 ooTt! e088 ote adee acors acbes ootss —_ovtz8 pea ‘sr 0.0042 0.0094 0.0050 0019 9.0020 © a0019 0.0020 .0053 0.0045 0.0038, r SoNs 00098 © .otdo 0.0083 onss © O.poRs | 0.008 OIG LOTS 0.0120 sory) “700 5804502002470“ 40280 Reproducity Sr 2.0064 0.0051 00079 © o.0078 0.0058 .0085 0.0068 0.0008 9.0120 0.0000 cA Dorr oo14s 0.0228 Ooat2 © ote © 0.0257 «O.0180 Oded © 00887 © D.0278 Asoa(%) 1080830720780“ 850 9a” ton0 ©0077 Hor tee 0.00| ee 08 ge 0.88 006g fe ge 09 e108 este 080 Table 16 Statistical parameters from trans-C18:2 +trans-C18:3 acid determination in olive and olive-pomace oils 7 xtaviegn Vein Lampant ‘ove a Crude otve-pomas ot Welnod KOHin Basictactle KOHin Basictecidle KOH in Basiovcidle KOHIn Basiovacdle KOHIn Baslosaidc ‘methanol methylation methanol methylation methanol methylation methanol methylation methanol methylation Panicpants 15 18 18 15 18 18 18 15 18 18 Outiors 3 3 3 3 4 2 2 2 3 4 Mean(%) 0.0054 0.0054 0.0100 0.0088 0.0077 0.0002 0.0088 0.0081, 00254 © antes. Repeatability o 0.0088 "0.0035 0.0050 0.0088 0.0021 0.0028 0.0102 004s © 0.0061 0.088 5 ©0128 0.0099 oT40 © 0.0128 0.0080 0.0078 0.0285 00128 OTT! 0.0108 spr) 840 «65050020 BDsC«DSCi«iISS SKDC. ‘produciity Sr 0.0087 0.0081 0.0079 0.0069 0.0063 0.0095 OMNIS 00088 © ona .n15t 8 0.018 0.0144 ©0221 0.0183 0.0175 0.0287 O.USe1O.N2s1 © 0.0500. 0.0423 Fson(%) 1230 950 70s7BOsBOSCs«ét0BOSst300—s tt.) 0B. Hor 20 088087085080 1401.01.10 088 values range between 43 to 123%, although the Hor was close to 1. In respect to the comparison of the FAME preparation methods, the fatty acid compositions were similar in samples 1 to 4. However, in sample 5 (crude olive-pomace oil) the variance analysis showed that there are significative differences (p-level < 0.005) between both methods in the FAME percentages, probably due that by method A the abundant free fatty acids are not methylated. Finally, in reference to the trans-isomers, there are not ‘significative differences between both ‘methods, indicating that the acidic methylation does not yield transsisomers in this conditions, 4, CONCLUSIONS ‘The results obtained showed a good repeatability and reproducibility in both methods. Coneluding that, in olive olls with low acidity (virgin, olive, refined olive oil, refined pomace oil and olive-pomace oil) is recommended to use the method A, bearing in mind that the ethyl esters have to be added to the corresponding fatty acid methyl esters. On the other hand, in olive oils with high acidity is recommended to use the method B, in order to assume the methylation of free fatty acids. In respect to the trans-isomers determination, the method A is the most appropriate method, although, the basic+acidic ‘method can be also used.456 ACNOWLEDGEMENT The authors wish to thank to the laboratories mentioned as follows: Bundesanstalt fOr Getreide Kartoffel und Fettforschung (B.A.G.K.F) (Germany); Centro de Asistencia Técnica ¢ Inspeccién del Comercio Exterior (SOIVRE) (Spain); Chemical Laboratories of G.S. of Consumer, Ministry of Development (Greece); Chemiservice S.A ({taly); Diipartamento di Scienze degli Aimenti, Univ. di Udine (ttaly); Direceao Geral de Fiscalizagao e Controlo da Qualidade Alimentar (OGFCQA) (Portugal); Direzione Centrale per Lnalisi Merceoiogice il Laboratorio Chimico (DCAMLC) (tly); General Chemical State Laboratory (Greece); Institut des Corps Gras (ITER) (France); Instituto de la Grasa (C.S.L.C.) (Spain); Laboratoires Wolf (France); Laboratoire Otficiel Dianalyses et de Recherches Chimiques (Morocoo); Laboratorio Agroalimentario (Spain); Laboratorio Agroalimentario Industrial S.L. (Spain); Ministere de Agriculture, Office National de LHuile (Tunisia); Ministerio de Agricultura, Pesca y Alimentacién (MAPA), Subdireccién General de Andlisis (Spain) and Stazione Sperimentale per le Industrie degli Oil @ dei Grass (italy). They also wish to thank to Mr. M. Rodriguez Agullar for its technical assistance. REFERENCES AOAC. (1995)—«Collaborative study guidelines» —V. ‘Assoc. Off. Anal. Chem, Int. 78, 143A-160A. Christie, W. W. (1992) —«Preparation of fatty acid methyl esters» —inform 3, 1031-1034. Grasas y Aceites EEC (1991)—«Rogulation EEC/256/1 on the characteristics of olive and olive pomace alls and on theit analytical mothods».—Off J; Eur, Commun, L248, 1-5. [EEC (1982) —

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