2009/09/17 by Ken’ichi Ichihara, K. Ichihara, Yumeto Fukubayashi · 26 citations
Chemistry · Nursing · #Analytical Chemistry and Chromatography #Fatty Acid Research and Health #Biochemical Analysis and Sensing Techniques
paper · pdf · doi:10.1194/jlr.d001065
A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC. A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC. Fatty acids are the major component of lipids, and the physical, chemical, and physiological properties of a lipid class depend primarily on its fatty acid composition. The fatty acid composition is determined as the methyl esters of fatty acids by gas-liquid chromatography (GC) (1Sheppard A.J. Iverson J.L. Esterification of fatty acids for gas-liquid chromatographic analysis.J. Chromatogr. Sci. 1975; 13: 448-452Crossref Google Scholar, 2Liu K-S. Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials.J. Am. Oil Chem. Soc. 1994; 71: 1179-1187Crossref Scopus (344) Google Scholar, 3Christie, W. W., 2003. Lipid Analysis. 3rd edition. The Oily Press, Bridgwater, UK.Google Scholar). Saponification followed by methylation is a classical method for preparation of fatty acid methyl esters (FAMEs) from glycerolipids and sterol esters (SEs). Conventionally, FAMEs are prepared by base- or acid-catalyzed esterification. Base-catalyzed methanolysis proceeds much more rapidly under mild temperature conditions than acid-catalyzed reactions (3Christie, W. W., 2003. Lipid Analysis. 3rd edition. The Oily Press, Bridgwater, UK.Google Scholar, 4Carrapiso A.I. Garcia C. Development in lipid analysis: some new extraction techniques and in situ transesterification.Lipids. 2000; 35: 1167-1177Crossref PubMed Scopus (157) Google Scholar), and KOH- or NaOH-catalyzed methanolysis completes within 2 min at room temperature for glycerolipids (5Ichihara K. Shibahara A. Yamamoto K. Nakayama T. An improved method for rapid analysis of the fatty acids of glycerolipids.Lipids. 1996; 31 ([Erratum. 1996. Lipids 31: 889].): 535-539Crossref PubMed Scopus (350) Google Scholar, 6Ichihara K. Waku K. Yamaguchi C. Saito K. Shibahara A. Miyatani S. Yamamoto K. A convenient method for determination of the C20–22 PUFA composition of glycerolipids in blood and breast milk.Lipids. 2002; 37: 523-526Crossref PubMed Scopus (37) Google Scholar) and within 1 h at 37°C for SE (7Ichihara K. Yamaguchi C. Nishijima H. Saito K. Preparation of FAME from sterol esters.J. Am. Oil Chem. Soc. 2003; 80: 833-834Crossref Scopus (7) Google Scholar). However, bases cannot catalyze the esterification of FFAs. BF3 is a commonly used acid catalyst for methylation and methanolysis (8Ackman R.G. Remarks on official methods employing boron trifluoride in the preparation of methyl esters of the fatty acids of fish oils.J. Am. Oil Chem. Soc. 1998; 75: 541-545Crossref Scopus (111) Google Scholar), but it is harmful, and boron and fluorine are also both restricted by local drainage laws. In addition, the methanolic BF3 reagent has a limited shelf life (9Christie W.W. Preparation of fatty acid methyl esters.Inform. 1992; 3: 1031-1034Google Scholar). H2SO4 is also an effective acid catalyst for FAME synthesis, but it is a very corrosive viscous liquid and must be handled with care. When the acid catalysts, BF3 and H2SO4 are used at high concentrations or at high temperatures, artifacts derived from fatty acids can be produced (1Sheppard A.J. Iverson J.L. Esterification of fatty acids for gas-liquid chromatographic analysis.J. Chromatogr. Sci. 1975; 13: 448-452Crossref Google Scholar, 2Liu K-S. Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials.J. Am. Oil Chem. Soc. 1994; 71: 1179-1187Crossref Scopus (344) Google Scholar, 3Christie, W. W., 2003. Lipid Analysis. 3rd edition. The Oily Press, Bridgwater, UK.Google Scholar, 10Hansen R.P. Smith J.F. The occurrence of methyl methoxystearate isomers in the methyl esters prepared from sheep perinephric fat.Lipids. 1966; 1: 316-321Crossref PubMed Scopus (11) Google Scholar, 11Klopfenstein W.E. On methylation of unsaturated acids using boron trihalide-methanol reagents.J. Lipid Res. 1971; 12: 773-776Abstract Full Text PDF PubMed Google Scholar). HCl is most widely used as an acid catalyst because it is a relatively mild reagent and gives almost quantitative yields (1Sheppard A.J. Iverson J.L. Esterification of fatty acids for gas-liquid chromatographic analysis.J. Chromatogr. Sci. 1975; 13: 448-452Crossref Google Scholar, 2Liu K-S. Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials.J. Am. Oil Chem. Soc. 1994; 71: 1179-1187Crossref Scopus (344) Google Scholar, 3Christie, W. W., 2003. Lipid Analysis. 3rd edition. The Oily Press, Bridgwater, UK.Google Scholar). Anhydrous methanolic HCl can be prepared from acetyl chloride and methanol (2Liu K-S. Preparation of fatty acid methyl esters for gas-chromatographic analysis of lipids in biological materials.J. Am. Oil Chem. Soc. 1994; 71: 1179-1187Crossref Scopus (344) Google Scholar, 3Christie, W. W., 2003. Lipid Analysis. 3rd edition. The Oily Press, Bridgwater, UK.Google Scholar, 4Carrapiso A.I. Garcia C. Development in lipid analysis: some new extraction techniques and in situ transesterification.Lipids. 2000; 35: 1167-1177Crossref PubMed Scopus (157) Google Scholar, 9Christie W.W. Preparation of fatty acid methyl esters.Inform. 1992; 3: 1031-1034Google Scholar), but the acid chloride is volatile (bp 52°C) and is an extreme irritant to the eyes. It also reacts violently with methanol. Many researchers purchase commercial expensive anhydrous methanolic HCl reagents. However, anhydrous methanolic HCl is unstable, and HCl reacts nucleophilically with methanol to produce chloromethane and water (12Kishimoto Y. Radin N.S. A reaction tube for methanolysis; instability of hydrogen chloride in methanol.J. Lipid Res. 1965; 6: 435-436Abstract Full Text PDF PubMed Google Scholar); consequently, the concentrations of HCl in commercial anhydrous HCl/methanol reagents decrease during storage. Commercial products of methanolic HCl contain considerable amounts of water that are probably formed during storage or are a contaminant introduced in the process of production. In this study, we have developed a convenient reagent for preparation of FAMEs from acyl lipids, including SEs, triacylglycerols (TGs), phospholipids (PhLs), and FFAs. The reagent is composed of commercial concentrated HCl (conc. HCl), methanol, and toluene and is superior to other reagents in terms of convenience, safety, and cost. Here, we propose two procedures for derivatization of fatty acyl residues with the reagent: one is for mild reaction, and the other is for rapid reaction. All types of fatty acids with O-ester linkages and FFAs were converted almost quantitatively into the corresponding methyl esters in one-step reactions. Fatty acids, TG, and cholesterol esters were purchased from Avanti Polar Lipids (Alabaster, AL), Matreya (Pleasant Gap, PA), Nu-Chek-Prep (Elysian, MN), and Sigma-Aldrich (St. Louis, MO). Phosphatidylcholine (PC; dioleoyl) was synthesized according to Ref. 13Ichihara K. Iwasaki H. Ueda K. Takizawa R. Naito H. Tomosugi M. Synthesis of phosphatidylcholine: an improved method without using the cadmium chloride complex of sn-glycero-3-phosphocholine.Chem. Phys. Lipids. 2005; 137: 94-99Crossref PubMed Scopus (41) Google Scholar. Glass-distilled solvents were purchased from Sigma-Aldrich and Wako Pure Chemical Industries (Osaka, Japan). Anhydrous methanolic HCl reagents, HCl (35%, w/w), acetyl chloride, methyl acetate, and 50% BF3 in methanol were of reagent grade. Reaction products of methanolysis/methylation were analyzed by TLC on silica gel. Lipids separated were visualized by spraying 50% (w/w) sulfuric acid and then heating at 135°C. FAMEs were analyzed with a Shimadzu 2014 gas chromatograph equipped with a column of SUPELCOWAX 10 (0.53 mm × 30 m) at a column temperature of 215°C or 225°C. FAMEs were prepared from 1 mg of cholesteryl oleate, 1 mg of glyceryl trioleate, 1 mg of dioleoyl PC, 1 mg of oleic acid, 0.05 mg of cis-9,10-methyleneocatadecanoic acid (a cyclopropane fatty acid), 0.05 mg of conjugated linoleic acids that were mainly composed of cis-9,trans-11 and trans-10,cis-12 isomers, 1 mg of olive oil, 1 mg of soybean oil, 1 mg of linseed oil, 0.5 mg of fish oil (Pacific saury), 0.3 mg of blood lipids that had been extracted by the method of Bligh and Dyer (14Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar) in the presence of 0.05% (w/v) 2,6-di-tert-butyl-p-cresol as an antioxidant, and 0.025 ml of whole blood. The formation of FAMEs was mainly investigated with cholesteryl oleate because SEs are the most resistant to transesterification of lipid classes having ester linkages (7Ichihara K. Yamaguchi C. Nishijima H. Saito K. Preparation of FAME from sterol esters.J. Am. Oil Chem. Soc. 2003; 80: 833-834Crossref Scopus (7) Google Scholar). The internal standard was methyl heptadecanoate or methyl tricosanoate. FAMEs formed from biological materials were purified on cartridge columns packed with 200 mg of silica gel (No.102021; Merck, Darmstadt, Germany) prior to GC. The silica gel cartridge was conditioned with 3 ml of hexane, charged with FAMEs dissolved in 1 ml of hexane, and washed with 3 ml of hexane. FAMEs were eluted with 3 ml of 1.5% (v/v) methyl acetate in hexane. A lipid sample in a screw-capped glass tube (16.5 × 105 mm) was hydrolyzed with 1 ml of 1 M KOH in 70% ethanol at 90°C for 1 h. The reaction mixture was acidified with 0.2 ml of 6 M HCl, and then 1 ml of water was added. FFAs released were extracted with 1 ml of hexane. After evaporation of the hexane in vacuo, the FFAs were methylated with 1 ml of 10% BF3 in methanol at 37°C for 20 min. Water was added to the solution, and then FAMEs were extracted with 1 ml of hexane. HCl concentrations of commercial anhydrous solutions of HCl/methanol were 5% (w/v) according to the manufacturers’ specifications, but the precise concentrations determined by titration were 2.3–3.2%. Karl-Fischer titration indicated that some of these commercial reagents contained 1–4% water. Anhydrous methanolic 5% (w/v) HCl was also prepared by mixing acetyl chloride with methanol (3Christie, W. W., 2003. Lipid Analysis. 3rd edition. The Oily Press, Bridgwater, UK.Google Scholar, 9Christie W.W. Preparation of fatty acid methyl esters.Inform. 1992; 3: 1031-1034Google Scholar). To a lipid sample in a screw-capped glass test tube was added 2.0 ml of anhydrous methanolic HCl, and the mixture was heated at 100°C for 1 h in a boiling water bath. After cooling, 1 ml of water was added, and then FAMEs were extracted with 1 ml of hexane. Commercial conc. HCl (35%, w/w; 9.7 ml) was diluted with 41.5 ml of methanol to make 50 ml of 8.0% (w/v) HCl. This HCl reagent contained 85% (v/v) methanol and 15% (v/v) water that was derived from conc. HCl and was stored in a refrigerator. A lipid sample was placed in a screw-capped glass test tube (16.5 × 105 mm) and dissolved in 0.20 ml of toluene. To the lipid solution, 1.50 ml of methanol and 0.30 ml of the 8.0% HCl solution were added in this order. The final HCl concentration was 1.2% (w/v) or 0.39 M, which corresponded to 0.06 ml of conc. HCl in a total volume of 2 ml. Addition of 1.2% solution to SE or be because of the of these lipids in methanol. The tube was and then incubated at 45°C overnight h or for mild methanolysis/methylation or heated at 100°C for 1 h for rapid reaction. The reaction at 100°C was to h for After to room 1 ml of hexane and 1 ml of water were added for extraction of The tube was and then the hexane was analyzed by or a silica gel FAMEs were also prepared from one of whole blood on with 1.2% under the mild The was a of a method K. Waku K. Yamaguchi C. Saito K. Shibahara A. Miyatani S. Yamamoto K. A convenient method for determination of the C20–22 PUFA composition of glycerolipids in blood and breast milk.Lipids. 2002; 37: 523-526Crossref PubMed Scopus (37) Google Scholar), which had been developed for fatty acid analysis of blood ml) was a of (1.5 × that had been washed with 0.05% it had was in a screw-capped test to which 0.2 ml of ml of methanol, and 0.3 ml of the 8% HCl reagent were added sequentially and then incubated at 45°C To the reaction mixture were added 1 ml of hexane and 0.2 ml of and the tube was FAMEs in the hexane were purified a silica gel cartridge Reaction conditions were investigated for mild methanolysis and that the methanolysis of cholesteryl oleate at 45°C for h almost quantitatively in the presence of HCl, which corresponded to ml of conc. HCl in a total volume of 2 ml. The HCl concentration of the amount of cholesteryl oleate that by the of the The formation of FFA was as the concentration of HCl This be to an in water In the presence of 1.2% HCl, cholesteryl oleate reaction than h for of methanolysis at 45°C conditions of methanolysis of at the HCl concentration of The concentration of HCl a water of the reaction and in a decrease in FFA formed by and were converted into FAME within h at 45°C in 1.2% HCl and In to methanolysis of esters, methylation of FFA very The formation of FAME from FFA was within 20 min in 1.2% HCl and at much concentrations of HCl and The reaction conditions for methylation of FFA were a reaction temperature of reaction of and HCl concentration of The conditions HCl also of The reactions of glycerolipids and FFA were more rapid than that of and the of of acyl lipids with HCl/methanol was FFA The reaction conditions h or 1.2% h yields of FAME for lipid composed of glycerolipids and the reaction conditions 1.2% h are for lipid fatty acids are and of and derivatization to methyl esters had been by methanolysis or formation in lipids of PubMed Scopus Google Scholar). A cyclopropane fatty acid, acid, was almost quantitatively as the methyl ester with amounts of artifacts under mild temperature conditions of 45°C under which glycerolipids can be converted into FAME linoleic acids are under and of methyl esters in the formation of 5% artifacts under the mild conditions of 1.2% h. However, artifacts were in FAME prepared by the of methanolysis at room temperature or at 37°C (5Ichihara K. Shibahara A. Yamamoto K. Nakayama T. An improved method for rapid analysis of the fatty acids of glycerolipids.Lipids. 1996; 31 ([Erratum. 1996. Lipids 31: 889].): 535-539Crossref PubMed Scopus (350) Google Scholar, K. Yamaguchi C. Nishijima H. Saito K. Preparation of FAME from sterol esters.J. Am. Oil Chem. Soc. 2003; 80: 833-834Crossref Scopus (7) Google Scholar) and acid-catalyzed methylation for 20 min as in a acid and in the methylation of and fatty acids with on conjugated and total fatty PubMed Scopus Google Scholar). oleate was formed from cholesteryl oleate SE was heated at 100°C for min with 1.2% HCl in methanol and but two major artifacts were produced under temperature conditions artifacts were probably to and cholesteryl methyl of cholesterol during transesterification of cholesterol Chromatogr. 1965; PubMed Google Scholar, produced during acid-catalyzed methanolysis of sterol esters.J. Lipid Res. Full Text PDF PubMed Google Scholar), and the could be separated from FAME by with a silica gel column for GC. into the of an overnight reaction at 45°C is for blood lipids that contain cholesterol and its of at 100°C produced artifacts FFA The reaction for methanolysis of was 30 min with 1.2% HCl or min with HCl, in which the amount of FFA released was than that for 1.2% HCl. The reaction for methanolysis of and methylation of FFA were than min in the presence of 1.2% HCl, and methylation of oleic acid with HCl was within min at Reaction conditions of 1.2% min are for lipid 30 min is a reaction to yields of FAME for lipid the reagent water derived from aqueous conc. HCl as a component and the acid-catalyzed methanolysis is a reaction, the formation of FFA is as in To the of of FAME methyl oleate was incubated in methanol HCl and water at 100°C for 1 h. of FAME was a of water and the of methyl oleate with water The 2 ml reaction mixture of 1.2% HCl ml of water derived from conc. HCl, and it was estimated that more than of FAME produced can be hydrolyzed during that the presence of water the of FAME formed but the of and M. method for the fatty acid composition of Am. Oil Chem. Soc. 1992; Scopus Google Scholar) that the formation of FAME was by of to water in a reaction and a that 5% water was for methanolysis was also by and transesterification of classes of lipids in a one-step Lipid Res. Full Text PDF PubMed Google Scholar). of commercial methanolic HCl reagents contained more than and HCl concentrations were at the of It must be in that HCl reacts with methanol to chloromethane and water. the HCl in methanol is within at room and a considerable amount of HCl is during methanolysis at 100°C (12Kishimoto Y. Radin N.S. A reaction tube for methanolysis; instability of hydrogen chloride in methanol.J. Lipid Res. 1965; 6: 435-436Abstract Full Text PDF PubMed Google Scholar). The presence of water also gives to of ester in TG, and and it is that FAMEs are synthesized transesterification methanolysis or in methanolic HCl solutions that are FFA by be as acid-catalyzed methylation of FFA of reaction from to FAME in 1.2% HCl/methanol was by a in the amount of FFA which that the to some amounts of water the formation of FAME and be a with to the of FAME production. is a for SE and TG, and the of these lipids in methanol with in the concentration of water dissolved at a concentration of mg in 2 ml of methanol at but in the presence of the was 2 ml. Addition of 10% toluene to methanol the of in the The of glyceryl in 2 ml of methanol solution ml of water and 0.2 ml of toluene is to that of methanol ml of water. both water and 10% toluene is almost to anhydrous methanol with to the of glyceryl In the of methanolysis of SE and was and considerable amounts of these lipids The of water derived from conc. HCl on the of lipids was by of 10% toluene. The reaction of or FFA the of toluene. Reaction conditions were investigated for methanolysis/methylation of lipid TG, and 1 the most conditions for mild reaction at 45°C and rapid reaction at and yields of FAMEs were synthesized in yields >96% for these lipid classes by the mild and rapid reactions. 1 that lipid as blood lipids, be at 45°C for h or at 100°C for min in 1.2% and that lipid as vegetable oils, can be at 1.2% or concentrations of HCl and with reaction conditions for preparation of FAMEs from lipid classes with conc. of reaction of lipid a hexane solution of methyl heptadecanoate as an internal standard and water were added to the reaction The of FAME in the hexane was determined by GC. of is the of methanolysis/methylation at of with HCl for h at 45°C also a was obtained with 1.2% methanolysis/methylation at were produced from A reaction a of anhydrous methanolic 5% HCl was prepared prior to by mixing 2 ml of acetyl chloride and 20 ml of methanol. SE and were dissolved in toluene and heated at 100°C for and with the anhydrous The HCl concentration of the reaction mixture was The yields of FAME were for SE and for anhydrous methanolic 5% HCl was prepared prior to by mixing 2 ml of acetyl chloride and 20 ml of methanol. SE and were dissolved in toluene and heated at 100°C for and with the anhydrous The HCl concentration of the reaction mixture was The yields of FAME were for SE and for of lipid classes analyzed were cholesteryl oleate as glyceryl as TG, dioleoyl as and oleic acid as After reaction of lipid a hexane solution of methyl heptadecanoate as an internal standard and water were added to the reaction The of FAME in the hexane was determined by GC. of is the of of with HCl for h at 45°C also a A was obtained with 1.2% were produced from A reaction a of anhydrous methanolic 5% HCl was prepared prior to by mixing 2 ml of acetyl chloride and 20 ml of methanol. SE and were dissolved in toluene and heated at 100°C for and with the anhydrous The HCl concentration of the reaction mixture was The yields of FAME were for SE and for in a new of lipid classes analyzed were cholesteryl oleate as glyceryl as TG, dioleoyl as and oleic acid as In to methanolysis (5Ichihara K. Shibahara A. Yamamoto K. Nakayama T. An improved method for rapid analysis of the fatty acids of glycerolipids.Lipids. 1996; 31 ([Erratum. 1996. Lipids 31: 889].): 535-539Crossref PubMed Scopus (350) Google Scholar, 6Ichihara K. Waku K. Yamaguchi C. Saito K. Shibahara A. Miyatani S. Yamamoto K. A convenient method for determination of the C20–22 PUFA composition of glycerolipids in blood and breast milk.Lipids. 2002; 37: 523-526Crossref PubMed Scopus (37) Google Scholar, K. Yamaguchi C. Nishijima H. Saito K. Preparation of FAME from sterol esters.J. Am. Oil Chem. Soc. 2003; 80: 833-834Crossref Scopus (7) Google Scholar), acid-catalyzed methanolysis has two major for amounts of lipids to be and reaction lipid 1 mg of SE or 2 mg of could be in 2 ml of the 1.2% HCl reagent used in the and these 1 and 2 were the for methanolysis of SE and TG, In methylation of at 20 mg could be converted into The and of the methanolysis/methylation method using conc. HCl were with a of lipid The mild h conditions were used for blood lipids that contain considerable amounts of and FAMEs were prepared from one of blood a of TG, PC, and were converted to which were purified to a on TLC a silica gel cartridge column for vegetable in TG, FAMEs were prepared by the rapid methanolysis at have fatty acid in TG, and are to FAMEs were prepared by both the mild and rapid in fatty acid composition were the method using conc. HCl and and also h and min of the method for the lipid 2 fish oil fatty acid obtained by methods blood fatty acid for of FAMEs prepared from fish oil under the mild of fish oil fatty acid determined by a by a anhydrous methanolic HCl and by the conc. acids are by of of is the of Fatty acids are by of of in a new is the of The solution of conc. HCl, methanol, and toluene is a convenient reagent for methanolysis and methylation of acyl The reagent can be prepared in from of FAME prepared and fatty acid obtained using this reagent are almost to those determined with other reagents of acid or Anhydrous HCl/methanol reagents for methanolysis and methylation of acyl lipids can be by conc. which can also reagents as or for lipid of order. The Yamamoto for on with fatty acid methyl ester gas-liquid chromatography followed by methylation sterol ester