Analysis of conjugated bile acids by high performance liquid chromatography and mass spectrometry

Because of the known advantages of coupling high performance liquid chromatography with mass spectrometry (HPLC-MS) in biological fluids, studies on the reversed-phase HPLC-MS system for direct analysis of conjugated bile acids in human bile samples are described. Ten samples of gallbladder bile of apparently healthy subjects were examined. The amounts of each tauro- and glycoconjugated bile acid as trifluoracetate were determined by mass fragmentography. Quantitation of at least 1 ng of each bile acid was possible.     

Analysis of conjugated octadecatrienoic acids inmomordica balsamina seed oil by GLC and13C NMR Spectroscopy

Separation of conjugated octadecatrienoic acids by open tubular gas liquid chromatography (GLC) was performed using glass capillary columns coated with Carbowax 20 M and with OV-1. The equivalent chain length of geometrical isomers of the conjugated octadecatrienoic acids belonging to the two series C_18:3Δ^8.10.12 and C_18:3Δ^9.11.13 were determined. The application of these results to the study of theMomordica balsamina seed oil shows that this oil contains two conjugated octadecatrienoic fatty acids in appreciable amounts, punicic acid (50%) and α-eleostearic acid (13%). The isomerization of conjugated acids inM. balsamina seed oil was followed for one year. Quantitation of octadecatrienoic acids using GLC gave results similar to those obtained with¹³C NMR.     

Autoxidation of methyl linolenate: Analysis of methyl hydroxylinolenate isomers by high performance liquid chromatography

The mixture of methyl hydroxylinolenates obtained by reduction of the hydroperoxide isomers formed by autoxidation of methyl linolenate was resolved by high performance liquid chromatography into eight major components. These are positional isomers with the hydroxyl group at positions 9, 12, 13, and 16. Two geometrical isomers of each positional isomer are present; these differ in the configuration of the conjugated double bonds (cis-trans andtrans-trans). Autoxidation of methyl linolenate is regioselective and favors the formation of positional isomers 9 and 16.     

Rapid determination of free fatty acids in vegetable oils by gas liquid chromatography

Determination of the free fatty acids in small quantities of vegetable oil is accomplished by gas liquid chromatography. The free fatty acids are isolated from a hexane solution of the vegetable oil into an aqueous solution of trimethylphenylammonium hydroxide (TMPH). Due to the alkalinity of TMPH, the free fatty acids readily partition into this aqueous phase. Injection of the free fatty acid-TMPH salts into a gas chromatograph results in pyrolytic methylation of the free fatty acid salts—yielding the methyl esters. Excellent results were obtained when this new procedure was used on neutral lipid oils containing known amounts of free fatty acids and compared with the results obtained by a modified BF₃/MeOH esterification procedure. When compared to the AOCS titration procedure, this new procedure gave comparable results. This new procedure has advantages over the AOCS procedure: it is more sensitive and gives quantitative results for individual free fatty acids. This new procedure also has several advantages over the modified BF₃/MeOH esterification procedure: it is easily and more rapidly performed, there is no deposition of glyceride on the column when the sample is injected, and because there is quantitative recovery, the new procedure is more sensitive and can be used on oils with a low weight percentage of free fatty acids.     

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed. Part of this work has been presented at the ISF/AOCS World Congress, New York (1980)JAOCS 58, (4), 1981, abstr. no. 184.     

Separation of surfactant mixtures and their homologs by high performance liquid chromatography

A technique for separating similar and diverse ionogenic mixtures into their individual homologs was studied by high performance liquid chromatography on an octadecyl-silica (TSK Gel LS-410) column. Conditions used for the separation were: column size, 6 mm id×200 mm; mobile phase, water/methanol (15:85, v/v) containing (a) 1.0 M sodium perchlorate and adjsting to pH 2.5 with phosphoric acid, (b) 0.1 M and pH 3.5; column temp., 50 C. By using 2 mobile phase conditions, various surfactant mixtures were separated into their individual homologs and simultaneously distinguished from each other, regardless of their ionogenic properties.     

Identification of conjugated linoleic acid isomers in cheese by gas chromatography, silver ion high performance liquid chromatography and mass spectral reconstructed ion profiles. Comparison of chromatographic elution sequences

Commercial cheese products were analyzed for their composition and content of conjugated linoleic acid (CLA) isomers. The total lipids were extracted from cheese using petroleum ether/diethyl ether and methylated using NaOCH₃. The fatty acid methyl esters (FAME) were separated by gas chromatography (GC), using a 100-m polar capillary column, into nine minor peaks besides that of the major rumenic acid, 9c, 11t-octadecadienoic acid (18∶2), and were attributed to 19 CLA isomers. By using silver ion-high performance liquid chromatography (Ag⁺-HPLC), CLA isomers were resolved into seven trans, trans (5–9%), three cis/trans (10–13%), and five cis, cis (<1%) peaks, totaling 15, in addition to that of the 9c, 11t-18∶2 (78–84%). The FAME of total cheese lipids were fractionated by semipreparative Ag⁺-HPLC and converted to their 4,4-dimethyloxazoline derivatives after hydrolysis to free fatty acids. The geometrical configuration of the CLA isomers was confirmed by GC-direct deposition-Fourier transform infrared, and their double bond positions were established by GC-electron ionization mass spectrometry. Reconstructed mass spectral ion profiles of the m+2 allylic ion and the m+3 ion (where m is the position of the second double bond in the parent conjugated fatty acid) were used to identify the minor CLA isomers in cheese. Cheese contained 7 t,9c-18∶2 and the previously unreported 11t, 13c-18∶2 and 12c, 14t-18∶2, and their trans,trans and cis,cis geometric isomers. Minor amounts of 8,10-, and 10, 12–18∶2 were also found. The predicted elution orders of the different CLA isomers on long polar capillary GC and Ag^*-HPLC columns are also presented.     

高效液相色谱分析乙酰甲胺磷原药

采用反相高效液相色谱法测定乙酰甲胺磷原药中的有效成分,以甲醇∶水=15∶85(体积比)作为流动相,在波长为215 nm条件下检测乙酰甲胺磷原药有效成分含量,本方法的线性相关系数为0.9999,标准偏差为0.21,变异系数为0.22%,平均回收率为99.75%。     

Glyceride studies: Part IX: Intraglyceride distribution of vernolic acid and of five conjugated octadecatrienoic acids in seed glycerides

Vernolic (epoxyoleic) acid, present in six seed oils over the range 19–72%, appears to be preferentially attached to the secondary glycerol hydroxyl group. The distribution of five isomeric conjugated octadecatrienoic acids (8, 10, 12 and 9, 11, 13) in 18 seed oils has been examined by lipase-catalyzed deacylation. The results are not entirely consistent and more species must be examined before a rational distribution pattern becomes apparent.     

Identity and configuration of conjugated fatty acids in certain seed oils

Punicic acid was found in the seed oils ofCayaponia grandifolia, Trichosanthes cordata andT. cucumerina. α-Eleostearic acid was found inMomordica cochinchinensis, M. cochinchinesis varietymixta andM. cymbalaria. The identity of the conjugated triene acid ofAleurites trisperma, Garcia nutans andCyclandrophora laurina was confirmed as α-eleostearic acid (cis, trans, trans configuration). The configuration of kamlolenic acid was proved to becis-9,trans-11,trans-13. The oils ofAleurites remyi andLicania platypus did not contain any conjugated acid. Issued as N.R.C. No. 10589.     

Kamlolenic acid and other conjugated fatty acids in certain seed oils

The major fatty acid of the seed oil ofTrewia nudiflora is shown to be α-kamlolenic acid, not α-eleostearic acid as believed earlier. Other conjugated acids were found and identified in seed oils not previously studied, viz., α-eleostearic acid inParinari insularum andRicinodendron rautanenii; trans,8-trans,10-cis,12-octadecatrienoic acid inCalendula stellata. The identity of the conjugated acids in four other seed oils was established, viz., α-eleostearic acid inPrunus yedoensis andValeriana officinalis; punicic acid inCucurbita digitata andC. palmata. Issued as N.R.C. No. 9063.     

Synthesis and nuclear magnetic resonance properties of all geometrical isomers of conjugated linoleic acids

Pure geometric isomers of conjugated linoleic acid were prepared from castor oil as the primary starting material. Methyl octadeca-9Z, 11E-dienoate (2) and methyl octadeca-9Z, 11Z-dienoate (4) were obtained by zinc reduction of methyl santalbate (1, methyl octadec-11E-en-9-ynoate) and methyl octadec-11 Z-en-9-ynoate (3), respectively, as the key intermediates. Methyl octadeca-9E, 11E-dienoate (8) and methyl octadeca-9E, 11Z-dienoate (9) were prepared by demesylation of the mesyloxy derivative of methyl ricinelaidate (6, methyl 12-hydroxy-octadec-9 E-enoate). A study of the nuclear magnetic resonance spectral properties was carried out and the shifts of the olefinic carbon atoms of 18:2(9Z, 11E) (2) and 18:2(9E, 11Z) (9) were readily identified by a combination of incredible natural abundance double quantum transfer experiment, heteronuclear multiple bond correlation, and ¹H−¹³C correlation spectroscopy correlation techniques. Doubts remain in the absolute identification of the individual olefinic carbon atoms of the 18:2(9Z, 11Z) (4) and 18:2(9E, 11E) (8), except the fact that the shifts of the “inner” (C-10 and C-11) and “outer” (C-9 and C-12) positioned olefinic carbon atoms of the conjugated diene system are distinguishable.     

抗氧化剂168的高效液相色谱法分析

探讨了抗氧化剂168的高效液相色谱分析方法,采用甲醇/乙酸乙酯作流动相。实验表明,在0.2～0.8 mg/mL具有较好的线性,平行测定5组数据,其标准偏差为0.011,质量回收率99.7%～102.7%。     

Detection of adulteration of olive oil with seed oils by a combination of column and gas liquid chromatography

Samples of virgin olive oil and refined seed oils, as well as mixtures of olive oil with 10 and 5% seed oils were fractionated by column chromatography on silicic acid impregnated with ammoniacal silver nitrate. It was possible to isolate a characteristic fraction enriched in polyunsaturated triglycerides. Its linoleic acid content in pure olive oil never exceeds 9.3%, whereas in pure seed oils, it varies between 38.1 and 70.1%; in mixtures of olive oil with 10 and 5% of seed oils, the respective values are 22.3–38.2% and 15.6–32.1%. The oleic-to-linoleic acid ratios of the same fraction are more than 7.6 (olive oil), 0.2–0.8 (seed oils), 1.1–2.0 (olive oil with 10% seed oils) and 1.4–3.6 (olive oil with 5% seed oils). These analytical values may be used as a safe criterion for the eventual adulteration of olive oil with seed oils. This work was taken in part from the doctoral dissertation of S. Passaloglou-Emmanouilidou.     

Fatty acid composition of capsicum oils by gas liquid chromatography

The fatty acid composition of oils extracted from various parts (pericarp, seeds and stem) of the fruit ofCapsicum and from samples of Greek red pepper itself were obtained by gas liquid chromatography. Samples from three different varieties ofCapsicum annuum, cultivated in the region of Almopia, Greece, were taken from two crops (1967, 1968) and examined. The results are discussed. No characteristic differences in fatty acid composition of the corresponding samples of the parts ofCapsicum fruit of the three Greek varieties were noticed and there was no difference between the two crops. However, considerable differences in fatty acid composition between Greek and American varieties were observed, probably due to climatic conditions. Presented at the Association of Greek Chemists Meeting, Athens, 1970.     

Analysis of alpha-linolenic acid geometrical isomers in deodorized oils by capillary gas-liquid chromatography on cyanoalkyl polysiloxane stationary phases: A note of caution

Analysis of alpha-linolenic acid geometrical isomers in deodorized or heated oils by capillary gas-liquid chromatography (GLC) on polar cyanoalkyl polysiloxane stationary phases requires some care to avoid interferences with other fatty acids. Depending on the temperature of the column, thecis-11 20∶1 acid may elute before, with or after thecis-9,cis-12,cis-15 18∶3 acid during GLC. In some instances [temperature higher than 180°C with a CP Sil 88 column (Chrompack, Middelburg, The Netherlands)], the 20∶1 acid coelutes with thetrans-9,cis-12,cis-15 18∶3 acid, leading to abnormally high levels of this last isomer. Consequently, the degree of isomerization of alpha-linolenic acid will be over-estimated under such conditions. It is recommended that the behavior ofcis-11 20∶1 acid relative to temperature be checked carefully prior to the determination of alpha-linolenic acid geometrical isomers by GLC. Temperatures lower than 160°C seem appropriate to separate all of these components from each other and fromcis-11 20∶1 acid in a 50 m×0.25 mm i.d. CP Sil 88 capillary column.     

高效液相色谱法检测氨基酸表面活性剂中脂肪酸的含量

建立了一种快速检测氨基酸表面活性剂中脂肪酸残留的高效液相色谱分析方法。采用Welch Ultimate AQ-C18色谱柱(250 mm×4.6 mm,5μm)分离,以乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱,样品经过酸化、衍生化处理后用紫外可见可变波长检测器(VWD)检测。结果表明,该方法的线性范围为5~500 mg/L,相关系数均大于0.999,平均回收率为93.2%~103.0%,检出限为0.03%,定量限为0.1%。方法耗时短、操作简单、准确度高、通用性强,适用于氨基酸表面活性剂产品的脂肪酸残留检测。     

柱前衍生化-HPLC测定蓝圆鲹蛋白的氨基酸组成

采用柱前衍生化反相高效液相色谱法测定蓝圆鲹蛋白的氨基酸组成,以邻苯二甲醛-9-芴甲基氯甲酸酯为衍生化试剂,醋酸钠缓冲液,甲醇及乙腈为流动相,用Hypersil ODS C18柱和二极管阵列检测器。结果表明,18种氨基酸在38 min内分离效果良好,在一定浓度范围内,氨基酸的峰面积与浓度线性相关系数在0.993 2～0.999 8,相对标准偏差为1.93%～4.89%(n=5)。并测定了蓝圆鲹蛋白的氨基酸组成。该方法简便快速、准确可靠,蓝圆鲹蛋白中含有至少18种氨基酸,其中8种必须氨基酸占总含量的42.68%。     

柱前衍生化-HPLC测定蓝圆鲮蛋白的氨基酸组成

采用柱前衍生化反相高效液相色谱法测定蓝圆骖蛋白的氨基酸组成,以邻苯二甲醛-9-芴甲基氯甲酸酯为衍生化试剂,醋酸钠缓冲液,甲醇及乙腈为流动相,用HypersilODSC18柱和二极管阵列检测器。结果表明,18种氨基酸在38min内分离效果良好,在一定浓度范围内,氨基酸的峰面积与浓度线性相关系数在0．9932-0．9998,相对标准偏差为1．93％。4．89％（n=5）。并测定了蓝圆够蛋白的氨基酸组成。该方法简便快速、准确可靠,蓝圆够蛋白中含有至少18种氨基酸,其中8种必须氨基酸占总含量的42．68％。     

Determination of brominated vegetable oils in soft drinks by gas liquid chromatography

A method is described for the qualitative and quantitative determination of brominated vegetable oils in soft drinks. The procedure involves treatment of the brominated oils with sodium methoxide followed by GLC analysis of the resulting methyl esters using methyl pentadecanoate as internal standard. Recoveries on known amounts of these oils ranged between 93.7% and 102.4%. The technique, applied to several commercial soft drinks, has shown the content of brominated oil to be in the range 10–45 mg per 10 fluid oz of drink.