A 1D High Performance Thin Layer Chromatography Method Validated to Quantify Phospholipids Including Cardiolipin and Monolysocardiolipin from Biological Samples

The aim of this study is to identify high performance thin layer chromatography (HPTLC) conditions allowing the separation and quantification of mammalian cellular phospholipids (PLs) (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and especially phosphatidic acid, cardiolipin, and monolysocardiolipin, these latter two being specifically located in mitochondria membranes). In order to make this method faster and easier, a 1D HPTLC method is chosen, testing several eluents as well as several staining methods. A pre‐conditioning of HPTLC plates with boric acid and a copper staining reagent followed by carbonization are selected for the quality of PL separation and homogeneity of staining. The selected conditions are discussed and the method validation is performed according to the International Conference on Harmonization guidelines. Linearity is effective between 1 and 8 µg and limit of quantification is between 0.5 and 2.3 µg depending on PL classes. Precision measurements show coefficients of variation <6%, and when amounts are close to the detection limit, <12%. Lipid extracts of tumor cell lines or isolated mitochondria are used to assess PL profiles. This shows that the HPTLC method can be used routinely to follow level variations of PLs. Practical Applications: The changes in PL composition play a crucial role in tumor processes and regulate cellular functions modulating cellular signaling or mitochondrial metabolism. The simple and cost‐effective 1D HPTLC method that is developed is applied to lipid extracts of whole tumor cells or hepatocyte‐isolated mitochondria. It is sensitive as well as precise to detect variations of phosphatidic acid or cardiolipin levels linked to physio‐pathological conditions. It can also be used to investigate the composition changes of other membrane PLs. Moreover, with a simultaneous analysis of 14 samples/standards on the same plate (six plates per day), this method is adapted for large series of samples.     

Nanogram quantification of nonpolar lipid classes in environmental samples by high performance thin layer chromatography

A sensitive, simple, and rapid high performance thin layer chromatography (HPTLC) method that quantifies nanogram amounts of complex nonpolar lipid classes in environmental samples with a minimum of sample preparation is presented. The derivatization method is lipid-specific and insensitive to carbon number or the degree of unsaturation of the fatty acids composing the lipid class compounds. Nonpolar lipid classes ranging from 10 to over 500 ng easily may be quantified in the same sample run. The coefficient of variation for sample replicates on different plates ranged from 2.3% to 5.9%. Accuracy of the method is better than 15%. The nonpolar lipid class composition of <53 μm oceanic particles in a vertical depth profile of the upper 600 meters of the Slope Water was determined to illustrate the HPTLC method. The observed changes in particulate lipid class composition indicate that secondary production by deep-living organisms significantly alters suspended particle composition with depth in the water column.     

Suitability of Soxhlet Extraction to Quantify Microalgal Fatty Acids as Determined by Comparison with In Situ Transesterification

To assess Soxhlet extraction as a method for quantifying fatty acids (FA) of microalgae, crude lipid, FA content from Soxhlet extracts and FA content from in situ transesterification (ISTE) were compared. In most cases, gravimetric lipid content was considerably greater (up to sevenfold) than the FA content of the crude lipid extract. FA content from Soxhlet lipid extraction and ISTE were similar in 12/18 samples, whereas in 6/18 samples, total FA content from Soxhlet extraction was less than the ISTE procedure. Re-extraction of residual biomass from Soxhlet extraction with ISTE liberated a quantity of FA equivalent to this discrepancy. Employing acid hydrolysis before Soxhlet extraction yielded FA content roughly equivalent to ISTE, indicating that acidic conditions of ISTE are responsible for this observed greater recovery of FA. While crude lipid derived from Soxhlet extraction was not a useful proxy for FA content for the species tested, it is effective in most strains at extracting total saponifiable lipid. Lipid class analysis showed the source of FA was primarily polar lipids in most samples (12/18 lipid extracts contained <5% TAG), even in cases where total FA content was high (>15%). This investigation confirms the usefulness of ISTE, reveals limitations of gravimetric methods for projecting biodiesel potential of microalgae, and reinforces the need for intelligent screening using both FA and lipid class analysis.     

Improved Butanol–Methanol (BUME) Method by Replacing Acetic Acid for Lipid Extraction of Biological Samples

Extraction of lipids from biological samples is a critical step in lipidomics, especially for shotgun lipidomics where lipid extracts are directly infused into a mass spectrometer. The butanol–methanol (BUME) extraction method was originally developed to extract lipids from plasma samples with 1 % acetic acid. Considering some lipids are sensitive to acidic environments, we modified this protocol by replacing acetic acid with lithium chloride solution and extended the modified extraction to tissue samples. Although no significant reduction of plasmalogen levels in the acidic BUME extracts of rat heart samples was found, the modified method was established to extract various tissue samples, including rat liver, heart, and plasma. Essentially identical profiles of the majority of lipid classes were obtained from the extracts of the modified BUME and traditional Bligh–Dyer methods. However, it was found that neither the original, nor the modified BUME method was suitable for 4‐hydroxyalkenal species measurement in biological samples.     

Egg-yolk lipid fractionation and lecithin characterization

Egg-yolk lecithin has phospholipid (PL) classes and a FA composition that differ from soybean lecithin and may have unique functional properties. The purposes of this research were to develop an effective method for extracting a sufficient amount to lecithin from fresh egg yolks and to evaluate its functional properties. Ethanol was used to dehydrate and partially extract the PL, after which hexane was used to extract the total lipids. A phase separation of the combined extracts resulted in neutral and polar lipid fractions. An acetone precipitation of PL from the final polar lipid fraction was necessary to remove the residual neutral lipids, especially cholesterol. The purity of PL in the lecithin product was 95%. Surface tension reduction, emulsion stability, and oxidative stability studies were conducted to characterize the functional properties of egg-yolk lecithin. Egg-yolk lecithin and soy lecithin had similar surface activities, as evaluated by the surface tension reduction in an aqueous system and the critical micelle concentration. Soybean lecithin created a more stable emulsion than egg-yolk lecithin. However, egg-yolk lecithin was more oxidatively stable than soybean lecithin.     

Development of a Novel High-Performance Thin Layer Chromatography–Based Method for the Simultaneous Quantification of Clinically Relevant Lipids from Cells and Tissue Extracts

Lipids such as cholesterol, triacylglycerols, and fatty acids play important roles in the regulation of cellular metabolism and cellular signaling pathways and, as a consequence, in the development of various diseases. It is therefore important to understand how their metabolism is regulated to better define the components involved in the development of various human diseases. In the present work, we describe the development and validation of a high-performance thin layer chromatography (HPTLC) method allowing the separation and quantification of free cholesterol, cholesteryl esters, nonesterified fatty acids, and triacylglycerols. This method will be of interest as the quantification of these lipids in one single assay is difficult to perform.     

High docosahexaenoic acid levels in both neutral and polar lipids of a highly migratory fish: <Emphasis Type="Italic">Thunnus tonggol</Emphasis> (Bleeker)

The lipid and FA compositions of various organs (light muscle, dark muscle, liver, pyloric cecum, and the orbital region) and of the stomach contents of a highly migratory fish species Thunnus tonggol (Bleeker) were analyzed. TAG and phospholipids (PE and PC) were the major lipid classes in the total lipids of T. tonggol. DHA was characteristically the major FA of all the major classes of all its organs except for only one case of liver TAG. The mean DHA contents of the various organs accounted for more than 20% of the total FA (TFA), even though it is a neutral depot lipid. However, DHA in the stomach contents, originating from their prey, fluctuated and was generally low. DHA levels were generally higher in a year (2000) when water temperatures were colder than in one when it was warmer (1998). Furthermore, DHA levels in muscle TAG were consistently high in spite of the fluctuation of those in the visceral TAG, which might be directly influenced by the prey lipids. This phenomenon suggests the physiological selective accumulation of DHA in the muscle, after the migration of the digested FA in the vascular system and absorption of the prey lipids in the intestine. In contrast, the FA composition of other species is generally variable and their DHA contents of TAG are usually less than 20% of TFA.     

FA determination in cold water marine samples

The determination of FA in cold water marine samples is challenging because of the presence of large proportions of a variety of labile PUFA. This study was undertaken to establish optimal methods for FA analysis in various sample types present in the marine environment. Several techniques used in FA analysis, including lipid fractionation, FAME formation, and picolinyl ester synthesis, were examined. Neutral lipids, acetone-mobile polar lipids, and phospholipids (PL) were readily separated from each other on columns of activated silica gel, but recoveries of PL were reduced. Deactivation of the silica gel with 20% w/w water produced variable recoveries of PL (66±22%). FAME formation with BF₃ gave optimal recoveries, and a method to remove hydrocarbon contamination from these samples before GC analysis using column chromatography was optimized. Picolinyl derivatives of FA are useful in structural determinations with MS, and a new base-catalyzed transesterification method of their synthesis from FAME was developed. Finally, a series of calculations, combining FA proportions with acyl lipid class concentrations, was designed to estimate FA concentrations. In algae and animal samples, these estimates were in good agreement with actual FA concentrations determined by internal standards.     

The simultaneous separation and quantitation of human milk lipids

A protocol using a dry column method was modified for the extraction of total lipids and the simultaneous separation and quantitation of neutral and polar lipids in human milk. The triacylglycerol, cholesterol, phospholipid and vitamin E contents of the lipid extracts were determined and compared with lipids extracted using a modified Folch procedure. Good precision for the extraction of neutral, polar and total lipids, as well as the different lipid classes, was demonstrated. No significant differences were found between the two methods with respect to the amount of cholesterol, phospholipid, total lipid or vitamin E extracted, thus validating the method as an extraction technique. We discuss the relationship between vitamin E and the three major milk lipids as an indicator of the vitamin's place of origin in the mammary gland. Our findings do not support the idea that vitamin E in mature milk has its original location in the apical membrane. Scientific Contribution No. 1254, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06269.     

Enantioselective Analysis of Chiral Anteiso Fatty Acids in the Polar and Neutral Lipids of Food

Anteiso fatty acids (aFA) are substituted with a methyl group on the antepenultimate carbon of the straight acyl chain. This feature leads to a stereogenic center. The 12-methyltetradecanoic acid (a15:0) and the 14-methylhexadecanoic acid (a17:0) are the most common aFA found in food, although they occur only in very small quantities. In this study we used gas chromatography in combination with a chiral stationary phase to determine the enantiomeric distribution of both a15:0 and a17:0 in the neutral and polar lipids of aquatic food samples and cheese. The best suited column was selected out of four custom-made combinations of heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)-β-cyclodextrin (β-TBDM) with different amount and polarity of an achiral polysiloxane. After separation of polar and neutral lipids of the food samples by solid phase extraction, fatty acid methyl esters were prepared and the fatty acid methyl esters were fractionated by reversed phase high performance liquid chromatography. Measurements of fractions high in aFA by enantioselective GC/MS in the selected ion monitoring mode verified the dominance of the (S)-enantiomers of a15:0 and a17:0 in both lipid fractions. However (R)-enantiomers were detectable in all samples. The relative proportion of the (R)-enantiomers was up to fivefold higher in the polar lipids than in the neutral lipids. The higher proportions in the polar lipids indicate that microorganisms might be involved in the formation of (R)-aFA.     

Lipid class separation by HPLC combined with GC FA analysis: Comparison of seed lipid compositions from different <Emphasis Type="Italic">Brassica napus</Emphasis> L. varieties

A reliable HPLC method was established to evaluate the lipid composition of useful plants modified by breeding techniques. This study focused on the polar lipid distribution and polar lipid FA compositions of four rapeseed varieties. Structure and quantity of the distinct lipid classes were compared by HPLC using ELSD followed by a GC FA analysis. A baseline separation of 14 lipid classes could be achieved within one step by using an eluent gradient of hexane, tert-methylbutyl ether, isopropanol, acetonitrile, chloroform, triethylamine, acetic acid, and water supplemented with ammonium sulfate with a polyvinyl alcohol column. After automatic fractionation, the FA compositions of the distinct lipid classes were characterized by a subsequent complementary GC FA analysis through direct acetylchloride methylation. The rape varieties analyzed showed diversity in polar lipid content and distribution, dominated by PC, PE, PI, monoglycosyldiacylglycerols, and phytosterols. Extensive variations were detected in FA within the lipid classes of rape varieties with predominantly oleic acid, linoleic acid, and α-linolenic acid observed followed by palmitic acid and gondoic acid. Oleic acid was mainly connected to PC and linoleic acid to PE, whereas α-linolenic acid and γ-linolenic acid were predominantly linked to PI in all varieties.     

Fatty Acid Profile of Sunshine Bass: II. Profile Change Differs Among Fillet Lipid Classes

Fatty acid (FA) profile of fish tissue mirrors dietary FA profile and changes in a time-dependent manner following a change in dietary FA composition. To determine whether FA profile change varies among lipid classes, we evaluated the FA composition of fillet cholesteryl esters (CE), phospholipids (PL), and triacylglycerols (TAG) of sunshine bass (SB, Morone chrysops x M. saxatilis) raised on feeds containing fish oil or 50:50 blend of fish oil and coconut, grapeseed, linseed, or poultry oil, with or without implementation of a finishing period (100% FO feed) prior to harvest. Each lipid class was associated with a generalized FA signature, irrespective of nutritional history: fillet PL was comprised largely of saturated FA (SFA), long-chain polyunsaturated FA (LC-PUFA), and total n-3 FA; fillet TAG was higher in MC-PUFA and total n-6 FA; and fillet CE was highest in monounsaturated FA (MUFA). Neutral lipids reflected dietary composition in a near-direct fashion; conversely, PL showed evidence of selectivity for MC- and LC-PUFA. Shorter-chain SFA were not strongly reflected within any lipid fraction, even when dietary availability was high, suggesting catabolism of these FA. FA metabolism in SB is apparently characterized by a division between saturated and unsaturated FA, whereby LC-PUFA are preferentially incorporated into tissues and SFA are preferentially oxidized for energy production. We demonstrated provision of SFA in grow-out feeds for SB, instead MC-PUFA which compete for tissue deposition, meets energy demands and allows for maximum inclusion of LC-PUFA within fillet lipids.     

Characterization of Free and Bound Lipids among Four Corn Genotypes as Affected by Drying and Storage Temperatures

For whole grains, the most sensitive components to the environmental changes are the lipids. In the current study, the effects of drying temperature (27 and 93 °C) and storage temperature (4 and 27 °C) on the fatty acid (FA) levels and lipid classes of endosperm lipids on four selected corn genotypes were investigated during a 12-month storage period. Storage temperature indicated greater impact on the FA composition than did the drying temperature. The ratio of saturated to unsaturated fatty acids, which was around 0.20–0.22 levels in the free lipid (FL) fractions of all corn types studied here, did not change significantly due to the drying and storage temperatures. However, in the bound lipid (BL) fractions, it was changed by a change in the drying and storage temperature in some of the corn types. Some changes were also found in the lipid classes within the FL and BL fractions of the studied corn samples. No lysophosphatidylcholine (LPC) was found in the FL fractions. In the BL fractions of two of the corn samples, the level of free fatty acids (FFA) increased more likely due to the deterioration of LPC. The results of the current study indicated a possible migration of triglycerides and FFA between the FL and BL fractions due to drying and storage at higher temperatures.     

Preparation of fatty acid methyl esters from lipoprotein and macrophage lipid subclasses on thin-layer plates

A simple, accurate, and fast procedure for quantitative analysis of fatty acids (FA) in simple lipid subclasses from different biological specimens is presented. Lipid extracts of isolated plasma lipoproteins (very low, low, and high density lipoproteins; VLDL, LDL, and HDL, respectively) and permanent J774 mouse macrophages were fractionated into lipid subclasses by thin-layer chromatography (TLC) on silica gel 60 plates. Bands comigrating with authentic lipid standards were scraped off under argon and subjected to direct,in situ transesterification with BF₃/MeOH in the presence of the TLC adsorbent. Fatty acid methyl esters were subsequently quantitated by capillary gas chromatography. A comparison of the FA content present in total lipid extracts and in lipid subclasses separated by TLC revealed recoveries ranging from 93 (J774 cell extracts) to 99.7% (LDL). The method described is applicable for the measurement of FA in individual lipid subclasses and was successfully applied to quantitatively analyze the FA composition of the phospholipid, triacylglycerol, and cholesteryl ester fraction derived from VLDL, LDL, and HDL. In J774 lipid extracts, the FA composition of the phospholipid-, monoacylglycerol-, diacylglycerol-, free fatty acid-, triacylglycerol-, and cholesteryl ester fraction was quantitated. In addition we have analyzed the time-dependent loss of the major HDL polyunsaturated fatty acids (18:2, 20:4) in the phospholipid and cholesteryl ester fraction during copper-dependent peroxidation of HDL. We have not encountered analytical problems concerning low FA recoveries from CE-rich lipid extracts as indicated by almost quantitative recoveries of FA in LDL, HDL, and J774 extracts.     

Lipid analysis of Greek broad bean oil: Preparation of liposomes and physicochemical characterization

Liposomes were prepared from the isolated phospholipids of mature broad bean [Vicia faba L. (syn. Fabae calabaricae)] oil and their physical properties were studied. The method of preparation was the hydration of the thin lipid film, while the probe sonication methodology was used for reducing the size of the vesicles. The seeds of the broad bean were collected in two different periods of maturity and extracted by the Bligh‐Dyer method, and the lipid classes were studied by HPTLC/FID. The oils were found to be rich in polar lipids (63.1% and 60.2% of total lipids) and low in neutral lipids (36.9% and 39.8% of total lipids) for the immature and mature seed oils, respectively. The neutral lipid fraction consisted mainly of triacylglycerides (34.2% and 32.3%) whereas the polar lipids mainly consisted of phospholipids (60.2% and 54.2%) for the mature and immature seed oils, respectively. Sphingolipids (8.9%) were identified only in the immature seed oil. The overall goal of this study was the preparation of a new liposomal formulation with physicochemical properties such as unique lipid composition, size and ζ‐potential, which are important factors influencing drug delivery to the target tissues.     

An evaluation of supercritical fluid extraction as an analytical tool to determine fat in canola,flax, solin,and mustard

Supercritical fluid extraction (SFE) with carbon dioxide was used to extract oil from soft oilseeds (flax, solin, canola, and mustard). Oil content determinations from the SFE method AOCS Am 3–96, with and without ethanol as a modifier, were compared to results obtained with an exhaustive extraction using petroleum ether (FOSFA as in AOCS Am 2–93). Without the modifier, oil recoveries using SFE were 10 to 15% lower than oil contents by the FOSFA method for the flax and canola samples. For mustard, the oil recoveries by SFE were about 20 to 30% lower than oil contents by the FOSFA method. In the presence of the modifier, oil recoveries for flax and canola were about 3% lower than the FOSFA recoveries. Varying the time, temperature, and amount of modifier (ethanol) showed that recoveries increased with time, pressure, temperature, and amount of modifier independently of the oilseeds tested. Kinetics of the SFE extraction showed that the oil recoveries increased with the extraction time and reached a plateau after 60 min. Multiple extractions (2×30 min), however, gave better recoveries than a single extraction for the same amount of time (60 min). The best results were obtained using multiple extractions without modifier or a combination of multiple extractions first without and then with 15% modifier. Under these last two conditions, oil recoveries were close to 100% for flax, solin, and canola, but mustard oil recoveries were still 10% lower than recoveries using the FOSFA method. Mustard samples gave the lowest oil recovery from SFE when compared to FOSFA method recoveries whatever conditions were tested, suggesting a matrix effect on the oil recovery. The acyl lipid content of the various extracts was studied using the sum of all FA expressed as TAG as a measure of acyl lipid extraction. The acyl lipid contents of the extracts were close to 100% when no modifier was used during the SFE. In the presence of modifier, the acyl lipid contents of the extracts were 10 to 15% lower than the results obtained without modifier. The amount of acyl lipid in the extract decreased as the quantity of modifier increased. This suggests that increasing the ethanol modifier increased the amount of polar compounds extracted without significantly increasing the total amount of lipids. The FA profiles were constant throughout the various extraction procedures.     

Lipid Fractions,Fatty Acid Profiles,and Bioactive Compounds of Lithospermum officinale L. Seeds

Seeds of Lithospermum officinale L. from different climatic zones were analyzed for new sources of γ-linolenic acid (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3). Cultured Borago officinalis was also analyzed for comparative purposes. Analyses were conducted for fatty acid (FA) profiles of the glyceride oils from the seeds and in the neutral and polar lipids by gas chromatography (GC); lipid classes by open column chromatography and preparative thin layer chromatography (TLC); and tocopherols, sterols, and phenolic compounds by high-performance liquid chromatography with diode array detection (HPLC-DAD), and the later compounds were confirmed by liquid-chromatography-mass spectrometry (LC–MS). L. officinale from St. Petersburg Botanical Garden showed the highest percentage of GLA (17.9% of total FA), while wild-growing L. officinale from the Rostov region contained the highest percentage of SDA (17.2% of total FA). Total FA content ranged from 11.3 to 20.8% of seed weight. Neutral and polar lipids accounted for ~98 and 2.27%, respectively, of total lipids. Five neutral lipid classes were identified (% of NL): triterpene esters (1.3), triacylglycerols (93.1), free FA (1.8), diacylglycerols (1.4), and monoacylglycerols (2.4). The highest tocopherol content was found in samples from Chechen Republic (35.7 mg/100 g), in which the δ isomer was the main component. Samples from the Rostov region had the highest amounts of sterols (83.8 mg/100 g), and Δ⁵-avenasterol was the predominant compound in all samples. L. officinale seeds contain high amounts of phenolic compounds (389.9 mg/100 g as upper limit), in which rosmarinic acid is highlighted. Overall, all data suggest the possibility of using L. officinale seed oil in pharmaceutical and cosmetic formulae and as functional food.     

Lipid Estimation of Surfactant-Extracted Microalgae Oil Using Nile Red

Nile red staining has been used as a lipid quantification technique in many microalgae growth and oil accumulation studies. However, its application in lysed microalgae cells is limited. Therefore, this study focused on lysed microalgae cells and utilized the Nile red staining technique to evaluate oil content and extraction. This study aims to provide a rapid and high-throughput alternative method particularly in the microalgae extraction screening process. Potential interferences such as chlorophyll, β-carotene, soluble protein, and phospholipids were evaluated. The hydrophobic Nile red dye was found to quench in water, therefore the fluorometric measurement has to be completed immediately or within 5 min of dye addition. The fluorescence intensity was also found to be Nile red concentration dependent. The optimum Nile red concentration of 656 ppb was used throughout the study. In microalgae samples containing chlorophyll and carotenoids (such as Nannochloropsis sp.), Nile red fluorescence intensity was significantly reduced in comparison to non-chlorophyll microalgae (Schizochytrium limacinum). Soluble proteins from defatted microalgae did not fluoresce significantly relative to lipids, therefore did not interfere with the method to a high degree. Comparing the optimized Nile red staining method with the gravimetric lipid quantification method, a good linear correlation was found in all three materials tested (soybean oil, Nannochloropsis sp., and Schizochytrium limacinum).     

A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction

A modified procedure for extraction of total lipids from whey protein concentrates was developed such that stable emulsion with extracting solvents was avoided and the solvent system remained monophasic. Nonlipid contaminants from the extract were removed using gel filtration instead of traditional aqueous washing to prevent any loss of polar lipids. The extraction of total lipids by the modified procedure was complete and comparable with a reference procedure. Traditional thin-layer chromatography is tedious and more qualitative than quantitative for lipid class separation. Total lipids were further separated into free fatty acids, phospholipids, cholesterol ester, triacylglycerol, cholesterol, diacylglycerol, and monoacylglycerol, using modified solid phase extraction procedure. Columns with 2 g amino propyl packing allowed separation of up to 80 mg of total lipids into lipid classes gravimetrically. The values for anhydrous milk fat for all lipid classes agreed with those in the literature. Separation of total lipids into lipid classes with solid phase extraction is easy, quantitative, and can also be performed on a preparative scale.     

The Spectrophotometric Sulfo-Phospho-Vanillin Assessment of Total Lipids in Human Meibomian Gland Secretions

Human meibomian gland secretions (meibum) are the major lipid component of the human preocular tear film. The predominant lipid classes found in meibum include waxes (WE), cholesteryl esters (CE), and varying amounts of cholesterol (Chl). The classical sulfo-phospho-vanillin assay (SPVA), adapted for a microplate reader, was used to quantitate lipids in meibum. To account for varying reactivities of different lipids in SPVA, a model meibomian lipid mixture (MMx) that approximated the WE/CE/Chl composition of meibum was developed and used to quantitate meibomian lipids. The overall SPV responses of MMx and meibum were found to be close, with similar intermediate and final reaction products for both. Saturated WE that had not been expected to be reactive were found to be SPV-positive. A reaction mechanism for these compounds in SPVA which involves the formation of alkenyl ethers is proposed and discussed. Tested proteins were non-reactive in SPVA. Thus, by comparing the results of gravimetric analyses of meibum samples with the results of a properly calibrated SPVA, it was estimated that the SPV-reactive lipid content of dry meibum in tested samples was about 78 % (w/w). The SPV method can also be adopted for analyzing other types of complex lipids secretions, such as sebum, as well as whole lipid extracts from other lipid-enriched organs and tissues, if proper standards are chosen.