Separation and determination of lipids by one-dimensional micro-thin-layer chromatography followed by densitometry

A method is described for the rapid analysis of a mixture of phospholipids and neutral lipids, which was used for the analysis of extracts obtained from a nuclear fraction isolated from rat liver. The lipids are separated by one-dimensional thin-layer chromatography on microchromatoplates (48 X 24 mm), using three solvents for development. After spraying the plates with phosphoric acid and heating, the amount of carbon from the charred compounds is measured densitometrically. Only 2-10 microgram of lipid mixture are needed for the determination of the relative amounts of the separated compounds.     

Faecal lipid chromatography. I. Quantitative determination with chromarods

A new and original method is proposed for the qualitative and quantitative analysis of faecal lipids by thin-layer chromatography and detection through the flame ionization detector of an analyser (the Iatroscan TH 10). This method enables the rapid quantification of the different faecal lipid classes, including cholesterol, with great accuracy and reproducibility. In-series operations are possible with easy manipulation.     

Quantitative high-performance thin-layer chromatography of lipids in plasma and liver homogenates after direct application of 0.5-microliter samples to the silica-gel layer.

Lipid profiles were determined by high-performance thin-layer chromatography (HPTLC) after direct application of 0.5 microliter plasma from capillary blood to the silica-gel layer. Coefficients of variation for the fluorescence measurements were 2.1% for phosphatidylcholine. The recovery of known amounts of lipid was 96--100%. A linear relationship between peak area and amount of lipid was found in the nmole range, corresponding to the amount of lipid in 0.125--0.75 microliter Lipid-Trol, which served as the standard reference sample. The plasma lipids of healthy subjects and of patients suffering from various illnesses were analyzed using reference methods and HPTLC. Identical values were obtained for cholesterol esters, triacylglycerols and phosphatidylcholine. Free cholesterol values determined by HPTLC were slightly lower (7%). The correlation between data obtained by reference methods and HPTLC was as follows: cholesterol, r = 0.938; cholesterol esters, r = 0.964; triacylglycerols, r = 0.985; phosphatidylcholine, r = 0.938. The separaiton and quantitation of liver lipids using HPTLC after direct application of the tissue homogenate to the silical-gel layer was carried out. Comparison with reference methods revealed that HPTLC gave higher cholesterol values (24%). The triacylglycerol concentrations, however, were identical under both methods and correalted satisfactorily (r = 0.959).     

Quantitation of polyamines using thin-layer chromatography and image analysis

Efficient separation of dansylated polyamines can be achieved by thin-layer chromatography (TLC). Quantitation, however, can be laborious because it requires removal of the silica gel and the fluorescing derivative from the glass plates, elution in a suitable solvent, and estimation with a fluorescence spectrophotometer. We report here a relatively simple and rapid method for the quantitation of dansylated polyamines that employs an image analyzer without removal from the glass TLC plates.     

Densitometric quantitation of neutral lipids on ammonium sulfate impregnated thin-layer chromatograms.

A procedure is described which extends the densitometric quantitation of phospholipids on ammonium sulfate impregnated thin-layer chromatograms by Gluck et al. to include total lipid, free and esterified cholesterol, free fatty acid and triglyceride. Lipids separated on thin-layer plates containing silica gel G impregnated with ammonium sulfate were charred upon heating and absorbance was measured densitometrically. Thus, the necessity of spraying or submersing in a charring agent was eliminated, uniform charring became possible, and quantitation over a wider range of sample sizes than most densitometric procedures was obtained. One linear relationship existed for concentrations of standards over the range of 0.0-3.0 mug and another line from 4.0-50.0 mug. Both accuracy and precision of the method were highly reliable.     

Class separation of bile lipids by thin-layer chromatography

A thin-layer chromatography technique is described that permits separation of each class of bile lipid, such as cholesterol, free (unconjugated) bile acids, glycine- and taurine-conjugated bile acids and phospholipids, in a single run. The use of silica gel G-aluminium pre-coated sheets facilitates further processing, such as the extraction in situ of each class of separated bile lipids for determination by conventional methods.     

Rapid analysis of simple carbohydrates by means of vapour-programmed thin-layer chromatography and densitometry and using a new spotting device

A rapid method is described for the separation of common naturally occurring mono- and disaccharides by means of vapour-programmed thin-layer chromatography; the development time is 3 h, and quantitation is obtained by in situ reflectance spectrometry. A new spotting apparatus with syringes in the horizontal position has been developed, which allows perfectly reproducible sample delivery in the microlitre range. The coefficient of variation for the total procedure is about 4–8 %. The total analysis time is 5 hours.     

Separation and quantitation of fatty acids, sterols and bile acids in feces by gas chromatography as the butyl ester-acetate derivatives

A system allowing the separation and quantitation of individual species of fecal fatty acids, sterols and bile acids in a single chromatographic step is described. The system is based on the butylation of carboxyl groups and acetylation of free hydroxyls of the compounds in fecal lipid extracts, followed by their resolution by temperature-programmed gas chromatography. As the butyl ester-acetate derivatives, fatty acids, sterols and bile acids elute separately and with no overlap on a variety of chromatographic columns, obviating the need for prior separation of each class by thin-layer or column chromatography. All common bile acids, a wide variety of sterols and keto-steroids, as well as saturated and unsaturated fatty acids may be routinely resolved. Quantitation is facilitated by the addition of the internal standards, heptadecanoic acid and nor-deoxycholic acid to each sample. With an automatic sample injector, the rapid assessment of a wide range of potential risk factors for colorectal cancer may be carried out in large numbers of samples.     

Assay of lipids in dog myocardium using capillary gas chromatography and derivatization with boron trifluoride and methanol

The fatty acids of three lipid classes (free fatty acids, triglycerides, and cholesteryl esters) from dog heart were analysed by gas chromatography. Samples of the left ventricle were homogenized and total lipids were extracted. After separation by thin-layer chromatography, the bands of the lipid classes studied were scraped off, transmethylated according to the boron trifluoride-methanol procedure, and the fatty acid methyl esters were extracted and analysed. The problems related to the quantitation of fatty acids were investigated, namely transmethylation procedure, thin-layer chromatography, and gas chromatographic conditions. Fatty acid methyl esters were separated on capillary columns coated in the laboratory with SP 2340 stationary phase. The high performance of the separation ensured the reliability and the precision of the analysis.     

Chromatographic analysis of blood lipids. Comparison between gas chromatography and thin-layer chromatography with flame ionization detection

Intact human blood plasma lipids of different composition were analyzed by gas chromatography and thin-layer chromatography with flame ionization detection. The reproducibility of the results obtained by gas and thin-layer chromatography was compared. The main advantages and disadvantages of both methods for lipid analysis are discussed. Generally, the variability of the results measured by thin-layer chromatography in series and from day to day was greater than that obtained by gas chromatography.     

Separation of androsterone from epiandrosterone, and dehydroepiandrosterone from its 3-hydroxy epimer by thin-layer chromatography.

The application of thin-layer chromatography to the separation of 13 steroids, including androstanes, 4-androstenes and 5-androstenes, using silica gel and 1,2-propanediol-impregnated cellulose is described. After group-wise separation of various C19 steroids on silica gel, the 3-hydroxy epimers of 5alpha-androstanes and 5-androstenes can be separated by thin-layer chromatography on impregnated cellulose plates. The chromatographic procedure is rapid and makes the prior formation of steroid derivatives unnecessary.     

Quantitation of hexadecylphosphocholine by high-performance thin-layer chromatography with densitometry

A method for the determination of the antineoplastic ether phospholipid hexadecylphosphocholine (HePC) is presented, based on the separation of the lipids by high-performance thin-layer chromatography charring with a cupric sulphate reagent and quantitation by in situ densitometry. The lower limit of determination is ca. 25 pmol. Concentrated hexane-isopropanol extracts of plasma samples can be applied to the plate without further clean-up, making this method useful for clinical drug monitoring. Additional ion-exchange chromatography and removal of the salt contaminants by gel filtration permits the study of endogenous phospholipids together with HePC from the same sample.     

Determination of methyl nicotinate in pharmaceutical creams by high-performance thin-layer chromatography

Summary A rapid, simple and specific high-performance, thin-layer chromatographic, photodensitometric method is described for the quantitative determination of methyl nicotinate. The HPTLC plates, coated with silica gel, are developed with a mobile phase which allows the separation of several active components in pharmaceutical creams. After quantitation of methyl nicotinate, a second solvent can be used for the identification of the cream base excipients.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Automated quantitative gas-liquid chromatography of intact lipis. II. Accuracy, precision and reproducibility of results

The effect of various factors on the precision and accuracy of the gas chromatographic determination of neutral lipids was studied in the concentration range where the correction factors are dependent on the amount analyzed. The mutual effect of individual components of the neutral lipid spectrum on the recovery was examined. A method is described which provides the stable recovery of the components present at low concentrations, using the addition of high-molecular-weight triglyceride (triarachidin) which does not interfere in the determination of the usual triglycerides. The validity of the correction factors measured with pure compounds was verified by hydrogenation of biological samples of various compositions. Hydrogenation of the sample also solves the problem of the determination of the triglyceride fraction of carbon number 46, which interferes under normal conditions with the determination of the cholesteryl ester fraction of carbon number 47. A method for the standardization of the gas chromatographic determination of neutral lipids is given using pure compounds instead of lyophilized biological samples. Long-term quality control was carried out using synthetic control samples. The results show sufficiently low values of the variation coefficients over the whole period. The values of the variation coefficients measured over an interval of 25 weeks are about 4% for the main components of the neutral lipid spectrum and 6.3% for the components present at concentrations up to 5%. Thw within-day variation for the most neutral lipid fractions and for lipid classes attains a value of 40-75% of the day-to-day variation. The most satisfactory values are obtained for the variation within a single series which amount to less than 2% for all substances except for triglyceride fractions 48 and 54. The correlation of the determination of total cholesterol and triglycerides by gas chromatography and by enzymatic methods shows a very good agreement between the results obtained by the two methods. Using quality control, it is possible to follow the accuracy of the calibration and to demonstrate objectively the necessity for system recalibration.     

Determination of cortisol in human plasma by thin-layer chromatography and fluorescence derivatization with isonicotinic acid hydrazide

The present work describes a specific and rapid determination of cortisol in human plasma. The method includes liquid-liquid extraction of plasma samples, thin-layer chromatography (TLC) of ethanolic extracts on aluminium foil-backed silica gel 60 TLC plates, derivatization of cortisol with isonicotinic acid hydrazide, and densitometric measurement of the fluorescence intensity of cortisol hydrazone. The fluorescence was linearly related to cortisol amounts; the correlation coefficients of standard curve plots were r>0.99. The coefficient of variation ranged between 2.8-7.9% (20 ng, within-assay/between assay variation) and 1.6-6.8% (80 ng, within-assay/between assay variation). The recovery of cortisol from plasma spiked with 21-deoxycortisol was 85%+/-4%. Cortisol concentration in the plasma was 66+/-32 ng/mL (mean+/-standard deviation, n=24). The advantage of this method is its simplicity to separate cortisol from other steroids by TLC, its specificity (formation of cortisol hydrazone), and the rapid quantitation of cortisol by densitometry.     

A quantitative densitometric method for the rapid separation and quantitation of the major lipids of tissues and lipoproteins by high-performance thin-layer chromatography. II. Reduction of the densitometric data

A BASIC program is described for acquisition of data and data reduction for an automated densitometric system for quantitation of lipids separated by high-performance thin-layer chromatography. The program allows calculation of mass of samples from log/log calibration curves computed from standards. The calculated masses are reported as nmol/volume or nmol/mg protein. The program contains a flexible dialog system which permits its use for a variety of applications in addition to the system described for quantitation of lipids.     

Lipid class analysis by normal phase high performance liquid chromatography,development and optimization using multivariate methods

Summary A general method for the analysis of lipid classes by liquid chromatography has been developed using a multivariate optimization strategy to target optimal system conditions. The method was validated using a ruggedness test in the form of a Plackett-Burman design thereby exploring the immediate region around the optimum to ensure stable analytical conditions. Detection and quantitation were optimized by a factorial design in the light scattering detector parameters thus ensuring maximum detector response. This method was found to be suitable for a broad range of lipid sources from vegetable and animal origin, examples of the separation achieved are given for oat kernel, soybean and bovine milk lipids.     

Application of different modes of thin-layer chromatography and mass spectrometry for the separation and detection of large and small biomolecules

Biomolecules are widespread throughout the world. A biomolecule is any organic molecule produced by a living organism, including large polymeric molecules such as proteins, polysaccharides and nucleic acids. Many sample preparation techniques are used in biomolecule analysis; the method selected depends on the complexity of the sample, the nature of the matrix and the analytes, and the analytical technique available. This review covers the current state of knowledge on thin-layer chromatography and mass spectrometry for qualitative analysis of biomolecules. In the first part of the paper the reader will gain useful information to avoid some problems about performing various modes of thin-layer chromatography combined with mass spectrometry experiments and in the second part he will find useful information for application of these techniques for separation, detection, and qualitative investigation of structures and quantitative determination of biomolecules such as proteins, peptides, oligonucleotides, amino acids, DNA, RNA, and lipids.     

Automated separation and quantitation of lipid fractions by high-performance liquid chromatography and mass detection

This report describes an improved separation and quantitation of lipid fractions in a total lipid extract by high-performance liquid chromatography using a modified solvent and gradient system delivered by dual pumps and incorporating a mass detector and autosampler. The detector responses for various lipid fractions (cholesteryl esters, triacylglycerols, free cholesterol, and seven major phospholipid classes) were fitted to a quadratic equation, y = ax2 + bx + c, and quantified after detector calibration by a computer. This new system has the advantage of automation and reproducible separation. The present method was applied to rat liver analysis.