Argentation supercritical fluid chromatography for quantitative analysis of triacylglycerols

A method for quantitative analysis of neutral lipids has been developed. Four different techniques have been combined for this purpose—supercritical fluid chromatography (SFC), silver ion chromatography, packed microcolumns and miniaturized evaporative light-scattering detection (ELSD). The development and optimization of the method are discussed. The separation of a series of vegetable, fish and hydrogenated oils was demonstrated. Application of eluent composition programming resulted in excellent separation of complex samples. Packed microcolumn argentation SFC provides at least as high a separation power as corresponding high-performance liquid chromatography methods. The combination of packed microcolumn SFC and miniaturized ELSD constitutes a powerful analytical system for the quantitative analysis of triacylglycerols.     

cis-Vaccenic acid in mango pulp lipids

A peak corresponding to a methyl octadecenoate other than oleate has been detected on the capillary gas chromatogram of the methyl esters of mango pulp fatty acids. This octadecenoate was isolated by silica gel and argentation column chromatography, high performance liquid chromatography and argentation thin layer chromatography, and then analyzed by infrared, nuclear magnetic resonance and gas chromatography-mass spectrometry, chromatographic separations and oxidative degradation. These analytical data proved that the octadecenoic acid wascis-vaccenic acid (cis-11-octadecenoic acid). The concentration of this acid in total octadecenoic acids ranged from 35% to 50% in the pulp of mangoes from Fiji, Mexico, the Philippines and Taiwan.cis-Vaccenic acid was revealed to be one of the major component fatty acids of non-polar lipids (mainly triacylglycerols), glycolipids and phospholipids in mango pulp. The glycolipids containedcis-vaccenic acid (ca. 20%) in higher concentration than oleic acid (ca. 15%). A trace amount ofcis-vaccenic acid (0.5%) was detected in the total lipids of mango seeds. Profile of fatty acid composition of mango pulp lipids (0.2–0.3 wt% of wet pulp) was characterized by the presence of n−7 acid isomers,cis-vaccenic acid and palmitoleic acid, and unusual mono- and dienoic positional isomers.     

Esterified lipids of the freshwater dinoflagellatePeridinium lomnickii

Steryl esters, phytyl esters and triacylglycerols of a naturally occurring freshwater dinoflagellate,Peridinium lomnickii, were identified using capillary gas chromatography-mass spectrometry (GC-MS). Steryl esters differing in degree of unsaturation were separated, prior to analysis, by argentation thin layer chromatography. 5α(H)-Cholestanol was more dominant, relative to 4α-methylstanols, in steryl esters than in the free sterols, but the same sterol moieties occurred in both fractions. Monoenoic fatty acids were enriched in the steryl esters relative to the free fatty acids. Major acyl groups in steryl esters were 16∶0 or 20∶1, with smaller amounts of 14∶0 and 18∶1. In triacylglycerols the acyl moieties were 14∶0, 16∶0, 18∶1, 16∶1 and 12∶0, in order of decreasing abundance. Phytyl esters, previously inferred to occur in a marine dinoflagellate only by analysis of transesterified products, were identified by GC-MS comparison with authentic compounds. Direct analysis of these esterified lipids has not been reported for freshwater phytoplankton. The 4α-methylstanyl esters, 5α(H)-cholestan-3β-yl esters and phytyl esters occurring inP. lomnickii are further features in common with marine dinoflagellates, additional to the 4α-methylsterols reported previously.     

Identification of a novel all-cis-5,9,12-Heptadecatrienoic acid in the cellular slime moldPolysphondylium pallidum

The all-cis-5,9,12-heptadecatrienoic acid was identified in the cellular slime moldPolysphondylium pallidum. The structural elucidation was accomplished by capillary gas chromatography, argentation thin-layer chromatography, and gas chromatography/mass spectrometry. This fatty acid has not been reported previously. Dedicated to Professors Tadao Yoshida and Masakazu Tatewaki on the occasion of their academic retirement.     

An Unprecedented Occurrence of Δ5,9- and Δ9,15-Dienoic Fatty Acids in Ovaries of the Archaeogastropod Limpet Cellana toreuma

A detailed structural diversity of dienoic fatty acids (FA), including non‐methylene‐interrupted dienoic FA, of triacylglycerols and polar lipids in ovaries of Cellana toreuma was clarified for the first time by using capillary gas chromatography–mass spectrometry of their 3‐pyridylcarbinol esters and argentation thin‐layer chromatography. Interestingly, in addition to 5,9‐octadecadienoic (18:2Δ5,9), 5,9‐eicosadienoic (20:2Δ5,9), 5,9‐heneicosadienoic (21:2Δ5,9), 5,9‐docosadienoic (22:2Δ5,9), 5,9‐tricosadienoic (23:2Δ5,9), and 5,9‐tetracosadienoic (24:2Δ5,9) acids, previously identified in ovaries of C. grata, rare FA 5,9‐hexadecadienoic (16:2Δ5,9), 5,9‐nonadecadienoic (19:2Δ5,9), and 21‐methyl‐5,9‐docosadienoic (iso 23:2Δ5,9) were newly recognized in ovaries of C. toreuma. Detectable amounts of four Δ9,15‐dienoic FA were present in the ovary lipids. The FA identified were one novel 9,15‐heneicosadienoic (21:2Δ9,15) acid and known 9,15‐docosadienoic (22:2Δ9,15), 9,15‐tricosadienoic (23:2Δ9,15), and 9,15‐tetracosadienoic (24:2Δ9,15) acids. The findings help to explain the broad evidence of the structural diversity in marine gastropods and suggest biomarkers to evaluate marine food web relations.     

Non-Polar Lipid Components of Human Cerumen

Human cerumen was separated by column chromatography into the following groups of compounds: hydrocarbons, squalene, wax esters and cholesterol esters, triacylglycerols, free fatty acids, free fatty alcohols, monoacylglycerols, free cholesterol, free sterols, and free hydroxy acids. The groups of compounds obtained were examined in detail by gas chromatography and gas chromatography–mass spectrometry. In total, about one thousand compounds have been identified.     

Analysis of tocopherols by capillary supercritical fluid chromatography and mass spectrometry

Tocopherol-containing mixtures were analyzed by gas chromatography (GC) and capillary supercritical fluid chromatography (SFC). GC analysis of tocopherols required the formation of the silyl derivatives, while SFC analysis of the tocopherol-containing mixtures was accomplished on neat samples. SFC analysis conditions were optimized with respect to column type and density/pressure programming. Enhanced resolution of many components was achieved by using inverse temperature programming during the SFC analyses. Both SFC and GC analyses permitted the separation and quantitation of alpha-, beta-, gamma- and delta-tocopherols. In addition, SFC proved particularly applicable for characterizing the composition of a deodorizer distillate and commercial antioxidant formulation. Coupling of a quadrapole mass spectrometer with a supercritical fluid chromatograph was also achieved; the mass spectrometer provided electron impact mass spectra on the underivatized tocopherol and sterol moieties. Both SFC and SFC/mass spectrometry proved effective for the analysis of complex lipid-containing mixtures, requiring minimal sample preparation prior to analysis. Presented at the 82nd Annual Meeting of the American Oil Chemists’ Society, Chicago, IL, May 12–15, 1991.     

Capillary supercritical fluid chromatographic analysis of shark liver oils

The liver oils of six different shark species have been analyzed by supercritical fluid chromatography (SFC). The liver oils were from the species Pseudotriakis microdon (False catshark), Centroscymnus coelepsis (Portuguese dogfish), Centrophorus squanosus (Leafscalp gulper shark), Deanea calceus (Birdbeak dogfish), Etmopterus princips (Greater lantern shark), and Centroscymnus crepidater (Longnose velvet dogfish). The method was capable of direct quantitation of squalene and cholesterol, while quantitation of triacylglycerols, cholesterol esters, and diacylglycerol ethers required thin-layer chromatographic fractionation prior to SFC analysis. The iodine values of the liver oil samples gave a linear correlation when plotted against the squalene content found by SFC. The variation of squalene content within one shark species is large, and there are large differences in squalene content from species to species. The squalene contents varied between 0.22 and 82.54 wt%. The identity of the glycerol ethers was investigated by SFC of the unsaponifiable matter. The major glycerol ethers contained chimyl, batyl, and selachyl alcohol.     

Identification of nine acetylenic fatty acids, 9-hydroxystearic acid and 9,10-epoxystearic acid in the seed oil ofJodina rhombifolia hook et arn. (Santalaceae)

In addition to some usual fatty acids, the seed oil ofJodina rhombifolia (Santalaceae) contains nine acetylenic fatty acids [9-octadecynoic acid (stearolic acid) (1.1%),trans-10-heptadecen-8-ynoic acid (pyrulic acid) (20.1%), 7-hydroxy-trans-10-heptadecen-8-ynoic acid (2.3%),trans-10,16-heptadecadien-8-ynoic acid (0.7%), 7-hydroxy-trans-10,16-heptadecadien-8-ynoic acid (0.1%),trans-11-octadecen-9-ynoic acid (ximenynic acid) (20.3%), 8-hydroxy-trans-11-octadecen-9-ynoic acid (12.2%),trans-11,17-octadecadien-9-ynoic acid (1.5%), 8-hydroxy-trans-11,17-octadecadien-9-ynoic acid (1.3%), 9-hydroxystearic acid (<0.1%) and 9,10-epoxystearic acid (0.7%)]. The fatty acids have been analyzed by gas chromatography/mass spectrometry of their methyl ester and 4,4-dimethyloxazoline derivatives. The hydroxy fatty acid methyl esters have been examined also as trimethyl-silyl ethers. Furthermore, the fatty acid methyl esters (FAME) have been fractionated according to their polarity (FAME-A: nonhydroxy; FAME-B: hydroxy fatty acids) and to their degree of unsaturation (FAME-A1/A2; FAME-B1/B2) by preparative thin-layer chromatography and argentation chromatography, respectively. All of these fractions have been analyzed by ultraviolet and infrared spectroscopy, and the fractions FAME-A and FAME-B have been analyzed further by nuclear magnetic resonance (¹H,¹³C, 2D H/C, attached proton test) spectroscopy and gas chromatography/mass spectrometry. This work is dedicated to the 65th birthday of Prof. Dr. K. Pfeilsticker, Institut of Food Science, University Bonn (Germany).     

Identification of milk fat triacylglycerols by capillary supercritical fluid chromatography-atmospheric pressure chemical lonization mass spectrometry

Identification of milk fat triacylglycerols was accomplished by capillary supercritical fluid chromatography (SFC) combined with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. Supercritical carbon dioxide was the carrier fluid in SFC. Ionization was achieved by introducing vapor of ammonia in methanol into the ionization chamber, which resulted in the formation of abundant [M+18]⁺ and [M-RCOO]⁺ ions of triacylglycerols. These ions defined both the molecular weight and the fatty acid constituents of a triacylglycerol, respectively. SFC on a nonpolar stationary phase provided an efficient separation of triacylglycerols according to the combined number of carbon atoms in the acyl chains of a molecule. In addition to the identification of the major chromatographic peaks representing molecules with 26–54 acyl carbons, minor peaks representing triacylglycerols with an odd number of acyl carbons were separated and identified. Furthermore, compositional information on partially separated isobaric triacylglycerols, which differed substantially in the chain length of the fatty acyl residues, was achieved within some of the peaks. A new finding of the present study was the formation of abundant [M+18]⁺ ions of saturated triacylglycerols in addition to diagnostic fragment ions, being of primary importance in structure elucidation. This extends the applicability of capillary SFC-(APCI)MS in the analysis of both saturated and unsaturated triacylglycerols.     

4-Desmethylsterols from the marine bivalveMacoma balthica

4-Desmethylsterols fromMacoma balthica L. were investigated by gas chromatography and gas chromatography/mass spectrometry after isolation by argentation and column chromatography. Twenty-three Δ⁰- and Δ⁵-sterols with saturated and unsaturated side chains were identified. It is suggested that sterols inM. balthica are derived from dietary sources. The great diversity of minor 4-desmethylsterols is likely to be due to their role as intermediates in the metabolism of dietary sterols.     

Molecular species of triacylglycerols in the hulls of sunflower seeds (Helianthus annuus L.) following microwave treatment

Whole sunflower seeds (Helianthus annuus L.) were exposed to microwaves for 6, 12, 20 or 30 min at a frequency of 2450 MHz. The hulls were then stripped from the seeds. Molecular species and fatty acid distributions of triacylglycerols (TAGs), isolated from total lipids in the hulls, were analyzed by a combination of argentation thin‐layer chromatography (TLC) and gas chromatography. A modified argentation TLC procedure, developed to optimize the separation of the TAGs, provided 10 different groups of TAGs, based on both the degree of unsaturation and the total fatty acid chain‐length. Dilinoleolein (29.5—30.2 wt‐%), trilinolein (18.2—24.2 wt‐%), dilinoleopalmitin and dilinoleostearin (17.0—18.1 wt‐%), palmitoleolinolein and stearoleolinolein (11.4—14.0 wt‐%) and dioleolinolein (7.5—8.6 wt‐%) were the main TAGs detected after microwave roasting. However, roasting caused a significant decrease (p < 0.05), not only in TAG molecular species containing more than four double bonds, but also in the amounts of diene species present in TAGs. These results suggest that microwaves should affect TAGs in the hulls more significantly (p < 0.05) than those in the sunflower kernels.     

Determination of the conjugated linoleic acid-containing triacylglycerols in New Zealand bovine milk fat

Reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection at 233 nm was used to separate, quantify, and identify the triacylglycerols (TAG) of milk fat that contain conjugated linoleic acid (CLA). The absorbance at 233 nm was substantially due to CLA-TAG (chromatography of some representative TAG devoid of CLA, such as tripalmitin and triolein, showed poor responses at 233 nm, 1/800th that of CLA-TAG). A CLA molar extinction coefficient at 233 nm of 23 360 L mol⁻¹ cm⁻¹ and an HPLC UV response factor were obtained from a commercially available cis-9, trans-11-CLA standard. This molar extinction coefficient was only 86% reported literature values. Summation of all chromatographic peaks absorbing at 233 nm using the corrected response factor gave good agreement with independent determinations of total CLA by gas chromatography and UV spectrophotometry. This agreement allowed quantification of individual CLA-TAG peaks in the HPLC separation of a typical New Zealand bovine milk fat. Three CLA-containing TAG, CLA-dipalmitin, CLA-oleoyl-palmitin and CLA-diolein, were prepared by interesterification of tripalmitin with the respective fatty acid methyl esters and used to assign individual peaks in the reversed-phase chromatography of total milk fat, of which CLA-oleoyl-palmitin was coincident with the largest UV peak. Band fractions from argentation thin-layer chromatography of total milk fat were similarly employed to identify five predominant CLA-TAG groups in total milk fat: CLA-disaturates, CLA-oleoyl-saturates, CLA-vaccenyl-saturates, CLA-vaccenyl-olein, and CLA-diolein.     

Determination of vernolic acid content in the oil ofEuphorbia lagascae by gas and supercritical fluid chromatography

Determination of the content of vernolic acid (12,13-epoxy-9c-octadecenoic) in the oil ofEuphorbia lagascae has been performed by gas chromatography of the fatty acid methyl ester derivatives of the triacylglycerols in the oil and by supercritical fluid chromatography (SFC) of the raw oil and the fatty acid derivatives of the oil. The content of vernolic acid was found to be 55 wt%. The three methods were compared, and SFC analysis of the fatty acid derivatives was found to be the most accurate method.     

The effect of lyophilization on the solvent extraction of lipid classes,fatty acids and sterols from the oysterCrassostrea gigas

The lipid class compositions of adult Pacific oysters [Crassostrea gigas (Thunberg)] were examined using latroscan thin-layer chromatography/flame-ionization detection (TLC/FID), and fatty acid compositions determined by capillary gas chromatography and gas chromatography/mass spectrometry (GC/MS). The fatty acid methyl esters were separated using argentation TLC and also analyzed as their 4,4-dimethyloxazoline derivatives using GC/MS. Major esterified fatty acids inC. gigas were 16∶0, 20∶5n−3, and 22∶6n−3. C₂₀ and C₂₂ nonmethylene interrupted (NMI) fatty acids comprised 4.5 to 5.9% of the total fatty acids. The NMI trienoic fatty acid 22∶3(7,13,16) was also identified. Very little difference was found in the proportions of the various lipid classes, fatty acids or sterols between samples of adult oysters of two different sizes. However, significant differences in some of the lipid components were evident according to the method of sample preparation used prior to lipid extraction with solvents. Lyophilization (freeze drying) of samples led to a significant reduction in the amounts of triacylglycerols (TG) extracted by solvents in two separate experiments (7.0 and 52.5% extracted). Extracts from lyophilized samples had less 16∶0, C₁₈ unsaturated fatty acids, and 24-ethylcholest-5-en-3β-ol, while C₂₀ and C₂₂ unsaturated fatty acids comprised a higher proportion of the total fatty acids. There was no significant change in the amounts of polar lipids, total sterols, free fatty acids or hydrocarbons observed in extracts from lyophilized samples relative to extracts from nonlyophilized samples. Addition of water to the freezedried samples prior to lipid extraction greatly improved lipid yields and resulted in most of the TG being extracted.     

Plasma lipid profiling by liquid chromatography with chloride-attachment mass spectrometry

A sensitive high-performance liquid chromatographic assay was developed using chloride attachment negative chemical ionization mass spectrometry for detection of glyceryl esters and ceramides, and positive chemical ionization mass spectrometry for detection of free cholesterol and cholesteryl esters in minimal quantities of plasma. The novel technique was validated by high temperature gas-liquid chromatography with flame ionization detection. Sample preparation was achieved by phospholipase C digestion of whole plasma, total lipid extraction and derivatization of any free carboxyl and hydroxyl groups by trimethyl- ortert-butyldimethyl-chlorosilane. The lipids were separated by reverse phase HPLC with 20–90% propionitrile in acetonitrile containing 1% dichloromethane, which served as the reagent and the source of chloride. Negative chemical ionization with chloride attachment is estimated to provide about 100 times higher response for the triacylglycerols and the trimethylsilyl ortert-butyldimethylsilyl ethers of diacylglycerols, and about 500 times higher response for the trimethylsilyl ortert-butyldimethylsilyl ethers of ceramides than positive chemical ionization mass spectrometry. Determination of the full negative chemical ionization mass spectra showed that each glycerolipid and ceramide species yielded a single ionic species corresponding to the chloride-attachment product of the parent ion. The cholesteryl esters and ethers failed to attach chloride and remained undetected by negative chemical ionization. However, the cholesteryl esters and ethers gave a high response for the steroid nucleus in positive chemical ionization mass spectrometry. Chloride attachment negative chemical ionization mass spectrometry is suitable for the unequivocal identification of plasma glycerolipids and ceramides in high-performance liquid chromatography and for the quantitation of molecular species in any unresolved peaks following appropriate calibration of the instrument response.     

Heart-Cut Two-Dimensional Countercurrent Chromatography for the Isolation of a Furan Fatty Acid Triacylglycerol from Latex Gloves and Identification of Further Lipid Compounds

Hevea brasiliensis latex from specific genotypes such as RRIM501 or PB235 is one of the richest natural sources of furan fatty acid 9-(3-methyl-5-pentylfuran-2-yl)-nonanoic acid (9M5), which is mainly present in the form of triacylglycerols. In this study, we successfully isolated a triacylglycerol esterified with three 9M5 molecules (tri-9M5) with a purity of 97% from the lipid extract of latex gloves by means of countercurrent chromatography (CCC). The gas chromatography with a mass spectrometry (GC/MS) spectrum of tri-9M5 not only featured the diagnostic fragment ion [M-RCOO]⁺ of triacylglycerols but also a fragment ion shifted by 16 Da to higher mass which corresponded with [M-RCO]⁺. [M-RCO]⁺ was only detected in triacylglycerols substituted with at least one furan fatty acid. Additionally, five γ-tocotrienyl fatty acid esters (γ-T₃-FA esters), namely, γ-T₃-16:0, γ-T₃-18:0, γ-T₃-18:1n-9, γ-T₃-18:2n-6 and γ-T₃-20:0, were detected in the sample. Contributions of γ-T₃-FA esters with 18:1n-9 and 18:2n-6 which co-eluted in GC/MS could be calculated after mathematical correction for contributions of the ¹³C₂ isotope peak of γ-T₃-18:2n-6 to γ-T₃-18:1n-9. This was necessary for quantitation of these two γ-T₃-FA esters. Improved separation of the γ-T₃-FA esters could be achieved by the novel heart-cut recycling CCC mode (four cycles). Sterols detected in disposable latex gloves were β-sitosterol, Δ⁵-avenasterol, and stigmasterol along with small quantities of 24-methylene-cycloartenol and Δ⁵-citrostadienol.     

Lipid classes,fatty acid composition and triacylglycerol molecular species of kidney beans (Phaseolus vulgaris L.)

Seed oils from five legume cultivars of Phaseolus vulgaris, grown in Japan, were extracted and classified by thin‐layer chromatography (TLC) into seven fractions: hydrocarbons (HC; 0.7–1.4 wt‐%), steryl esters (SE; 1.7–3.3 wt‐%), triacylglycerols (TAG; 33.8–45.9 wt‐%), free fatty acids (FFA; 0.6–1.5 wt‐%), sn‐1,3‐diacylglycerols (1,3‐DAG; 0.3–1.0 wt‐%), sn‐1,2‐diacylglycerols (1,2‐DAG; 0.4–1.2 wt‐%) and phospholipids (PL; 49.4–58.8 wt‐%). Fatty acids derivatized as methyl esters were analyzed by gas chromatography (GC) and a flame ionization detector. Molecular species and the fatty acid distribution of TAG isolated from the total lipids in the beans were analyzed by a combination of argentation‐TLC and GC. A modified argentation‐TLC procedure, developed to optimize the separation of the complex mixture of total TAG, provided 18 different groups of TAG, based on both the degree of unsaturation and the total length of the three acyl chains of fatty acid groups. SDT (3.2–4.2 wt‐%), M₂T (3.8–5.0 wt‐%), D₃ (4.8–5.9 wt‐%), MDT (8.0–13.9 wt‐%), D₂T (12.5–15.8 wt‐%), MT₂ (19.4–22.7 wt‐%), DT₂ (17.8–23.5 wt‐%) and T₃ (9.2–13.0 wt‐%) were the main TAG components. The dominant fatty acids of TAG were α‐linolenic (48.5–57.8 wt‐%) and linoleic (16.7–25.8 wt‐%) acids, with appreciable amounts of palmitic (8.3–13.2 wt‐%) and oleic (7.8–13.8 wt‐%) acids. The high content of α‐linolenic acid in the cultivars of P. vulgaris could very likely play a beneficial role in reducing the risk of coronary heart disease among the large populations consuming them in Japan.     

Separation of milk fat triacylglycerols by argentation thin-layer chromatography

Argentation thin-layer chromatography was investigated as a method of obtaining detailed compositional information about milk fat. A modified argentation thin-layer chromatography procedure, developed to optimize the separation of the complex mixture of total milk fat triacylglycerols, provided nine different groups of triacylglycerols, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester (FAME) analysis was performed to determine the composition of each band. Separation on the basis of chainlength was most pronounced among the fully saturated triacylglycerol groups, as evidenced by the high level of C_4:0 and C_6:0 in bands 7 and 8, respectively. For the cis-monoenoic triacylglycerols, the separation of C_4:0 and C_6:0 was less distinct. The cis,cis dienes and other dienoic, trienoic, or tetraenoic species were principally observed in two bands of retention factor <0.08 on the chromatography plate. Minimal cross-contamination of bands was observed, with the exception of the lowest of the trisaturate bands, band 7, in which trans-monoenes were found to be present. Three samples from different points of the New Zealand dairy season were separated by argentation thin-layer chromatography, and their FAME distributions were measured. In addition to differences in the masses of band extracts from these samples, levels of C_10:0 and C_12:0 in all bands, and levels of monounsaturates in the dienoic and trienoic bands, were found to differ. These changes were generally consistent with a pattern of decreasing fat hardness over the November to March period of a typical dairy season.     

Techniques for the isolation and identification of steroids in insects and algae

Analytical techniques, methods and instrumentation employed for the extraction, isolation separation, purification and identification of steroids from algae and insects are presented. The techniques include adsorption and argentation column chromatography and counter-current distribution for separating the individual steroids, and thin layer chromatography and gas liquid chromatography for monitoring the purification process. Double bond, steric and alkyl substituent separation factors and relative retention times are reported for a large number of sterols on 4 different column systems. Their use permits the tentative structural assignment of sterols. Ultraviolet, infrared, nuclear magnetic resonance (NMR) and mass spectral analyses are discussed in light of their significance in the isolation and identification of steroids from insects and algae. Numerous examples are presented, including the use of 220 MHz NMR spectrometry, which permits the differentiation and characterization of C-24 epimetric sterols and allows for a semiquantitative estimate of the 24α- and 24β-epimers present in a mixture. Presented at Symposium on “Steroid Analysis” ISF/AOCS World Congress, New York, NY, April 1980.