DE4409987C1 - High pressure liquid chromatography (HPLC) method for phospholipid - Google Patents

Abstract

Method for sepg. and quantifying the main phospholipid classes using high pressure liquid chromatography (HPLC) comprises (i) applying the sample mixt. contg. an internal reference to a first short column connected to a second, taller column and eluting isocratically (ii) separating the phospholipids having a retention time of up to 40 mins. from the second column, and separating the phospholipids with a retention of time of 40-60 mins. from the first column; and (iii) analysing the eluates using UV detection and then fluorescence detection techniques.

Description

So far, column chromatography has been used to analyze phospholipids graphics as well as the one-dimensional and two-dimensional Thin layer chromatography used. These techniques are quite labor intensive and take a lot of time, which is why the High pressure liquid chromatography to separate Phospholipids proposed and the about two decades ago Methodology was continuously improved.

DE 27 37 513 B2 describes a method for determining Phospholipids known in which the sample to be examined with a choline-forming system that is at least phospholipase D, phospholipase C, phosphotase, phospholipase B, glycero- Phosphorylcholine diesterase and a choline detection system brings to implementation, the latter a choline dehydrogenase and contains a hydrogen acceptor. With this procedure NAD consumed proportional to the enzyme reaction and NADH ent educated speaking. Both can be used photometrically for determination the amount of phospholipids can be measured. But it takes place no separate determination of individual phospholipid classes takes place.

Known methods of high pressure liquid chromatography do not work with isocratic elution in combination with one Column switching, but using gradient elution processes and do not separate all major classes of phospholipids. They also work with ultraviolet absorption alone, which with a changing fatty acid pattern of the phospholipids leads to incorrect measured values, and the labor-intensive classic Phosphate determination of the eluted phospholipids, making it impossible makes, phosphorus-containing buffer systems in the eluent to use.  

UV detection is sensitive to fatty acid residues the phospholipids and easy to use. For the analysis of However, it is phospholipid mixtures of biological material difficult to set up suitable calibration curves because the Fatty acid composition of the phospholipids depending on Nutritional parameters, experimental parameters in the laboratory as well the state of health of the tissue or whole organism from which the phospholipids obtained can change drastically. In addition, the fatty acid pattern and thus the content of unsaturated fatty acids of a given tissue phospholipid very different to tissue, so that common calibration curves for UV absorption is generally unusable. This detection The process is independent of the fatty acid composition of the Rehearse.

It is an object of the invention to use a high pressure liquid Chromatography process for simple and quick separation of the To provide major phospholipid classes, it is also an object the invention, information regarding the number of double bonds to get in the fatty acid portion of the phospholipid.

These tasks are solved by a method according to claim 1.

The method according to the invention allows for the first time complete separation of the main classes of phospholipids by High pressure liquid chromatography (HPLC) with comparative precise and quantitative information within a fairly short period Time using an isocratic mixture of eluents in Combination with one arranged between the separation columns Switching valve. Compared to the previously usual in the clinical area separation by thin layer chromatography brings this The method according to the invention saves a considerable amount of time and requires an amount reduced by at least a factor of 5 Sample substance. The ratio of those determined by UV detection Values to those of the fluorescence detection leaves one Conclusion on the number of double bonds in the fatty acid portion of the individual phospholipids. This information can be obtained from the Diagnostics, scientific analysis and the Quality control of commercial phospholipids of considerable Be meaning and is with no other previous HPLC or Thin-film technology possible using the "on-line" process.

Fig. 1 shows the structure of a high-pressure liquid chromatography system according to the invention for the analysis of the phospholipid classes.

Fig. 2 shows the separation of standard and Leberphospholipide by HPLC followed by UV and fluorescence detection.

Fig. 3 shows the fluorescence detection and UV absorption of various phosphatidylcholines and the internal standard.

Fig. 4 shows the fluorescence detection and UV absorption indicates various phospholipids.

Fig. 5 UV / fluorescence ratios displays different Phospholipidmengen.

Fig. 6 shows UV / fluorescence ratios of various phosphatidylcholine derivatives in various combinations.

Fig. 7 shows the correlation between the UV / fluorescence ratios of phosphatidylcholine and phosphatidylethanolamine from different tissues and their content of double bonds.

The method according to the invention is explained in more detail below.

Suitable column materials for the process according to the invention are aminopropyl-derivatized silica columns, which are among the Nucleosil® or Hypersil® are commercially available.

The HPLC system consisted of an eluent pump system from Sykam, Model S 1110, a Shimadzu SIL-9A auto injector, one analytical column made of stainless steel, Schambeck-nucleosil 5 NH₂ 50 mm × 4.6 mm, in front of a protective column of the same material was switched, a switching valve and a second analytical Column (Schambeck-Nucleosil 5NH₂ 175 mm × 4.6 mm). The pillars were operated at a flow of 1 ml / minute eluent at 50 ° C.

The UV detection was carried out at 205 nm, the fluorescence detector was set to 340 nm excitation and 460 nm emission wavelength. For the quantification of sample phospholipids and for standardization of the UV / fluorescence ratio was used N-monomethyl-1,2-dio leoylphosphatidylethanolamine, which is used for this process according to a modification of the Biochimica et  Biophysica Acta, Vol. 960 (1988) 334-341, since this phospholipid is not commercially available. N-Monomethylphosphatidylethanolamine is suitable as an internal Standard because it is only in traces below the detection limit in biological samples examined there occur and because with the UV / fluorescence ratio can be standardized. These Standardization may be necessary for several series of tests compare with each other when fluorescence detection between different approaches of 1,6-diphenyl-1,3,5-hexatriene solution shows fluctuations. The UV / fluorescence ratio of N-Monomethyl-1,2-dioleoylphosphatidylethanolamine is usually 0.11 and can vary between 0.08 and 0.15. Of the other sense of the internal standard is from the defined Amount of N-monomethyl-1,2-dioleoylphosphatidylethanolamine added calculate the amount of other phospholipids in the sample.

A mixture of 1460 ml was preferably used as the eluent Acetonitrile, 500 ml methanol, 30 ml water and 600 µl Methylphosphonic acid (50% in water) used. The The eluent was degassed and the pH value with ammonium hydroxide set to 6.3.

The derivatizing agent was prepared by adding 1 liter double distilled water 250 µl Brÿ 35, 100 mg sodium azide and 150 µl 1,6-diphenyl-1,3,5-hexatriene solution (3 mmol / l Tetrahydroforane) were added. This solution was created using Treated with ultrasound and with the eluent at a river of 4.5 mm / minute thanks to a stainless steel T-piece added after the eluate with the phospholipids Had passed through the detector. The formation of the fluorescent Micelles were reached in a Teflon® tube at 50 ° C.

Phospholipids became from the samples of organic materials extracted quantitatively by a known method and the Samples in the dark in a mixture of ten parts by volume Chloroform and a volume of methanol under nitrogen  stored at minus 20 ° C. For HPLC analysis, the samples were taken under Dried nitrogen and in a mixture of one volume Chloroform and four volumes of methanol dissolved.

To compare the method according to the invention with that in the Clinic used thin layer chromatography procedures the latter was carried out as follows. Two-dimensional Thin layer chromatographies were performed on 20 x 20 cm silica gel 60 Plates made without a fluorescent indicator. The plates were for an hour before applying samples and standards 100 ° C dried in an oven. The solvent for the first Dimension consisted of chloroform, methanol and water in the Ratio 62.5: 25: 2.5 (v / v). After drying for five minutes The separation in the second dimension took place at 90 ° C in one Mixture of chloroform, methanol, glacial acetic acid and water in Ratio 56: 21: 7.5: 1.25 (v / v). Thin layer chromatography was terminated when the solvent front was about 1 cm below the Plate edge was. The phospholipids became visible with iodine staining made, scraped out, transferred into phosphate-free tubes and determined after ashing by a known method phosphate.

For fatty acid analysis, the phospholipid fractions obtained by HPLC were separated from the solvent under nitrogen vaporization and transmethylated with 5% methanolic anhydrous hydrochloric acid. The fatty acid methyl esters were separated by gas chromatography and quantified using a flame ionization detector. Hydrogen was used as carrier gas at a flow of 3 ml / minute at a temperature gradient of 80 to 200 ° C. The peaks were identified according to the retention times of the standard fatty acid methyl esters. The average content of the double bonds of a phospholipid was calculated from the percentage of unsaturated fatty acids and the number of double bonds in the respective fatty acids. This value was plotted against the UV / fluorescence ratio of the phospholipid compounds in order to be able to estimate the content of unsaturated fatty acids in phospholipids from their UV / fluorescence ratio ( FIG. 7).

To check the validity of the HPLC system according to the invention Analysis of biological phospholipids were lipid extracts from Pig, liver, stomach and lung tissues analyzed. The crude phospholipid extracts were both two-dimensional Thin layer chromatography separated from the plates scraped off and the phosphate content of the phospholipids determined as also separated by HPLC and by UV and fluorescence detection quantified. The results are in the table below the two-dimensional thin-layer chromatographic examination and those of HPLC separation listed. The fluorescence Detection of phospholipids, phosphatidylcholine, phosphatidyl glycerol and phosphatidylethanolamine gave similar results as in thin layer chromatography, but this is not the case for UV absorption too. Phosphatidylinositol and However, phosphatidylserine were not separated from each other by HPLC separated by thin layer chromatography. In the table mean PC phosphatidylcholine, LPC lysophosphatidylcholine, SPH Sphyngomyelin, PG Phosphatidylglycerin, PE Phosphatidylethanolamine, PI Phosphatidylinositol and PS Phosphatidylserine. Mean values are given ± Standard error. 1: Phosphatidylserine is in the Thin layer chromatography contained in phosphatidylinositol.

TABLE

According to a preferred embodiment, the HPLC apparatus is described in more detail in FIG. 1: the eluent mixture is made from a reservoir ( 1 ) by means of an HPLC pump ( 2 ) by means of a protective (precolumn) ( 4 ), first separation column ( 5 ) and second Separation column ( 6 ) pumped with a constant flow of approx. 1 ml / min. The sample is fed into the eluent mixture in a small volume (2-100 µl) by the automatic sample dispenser ( 3 ). During the first 30 to 32 minutes, all phospholipids except phosphatidylinositol and phosphatidylserine are separated and eluted over the entire length of the column (23.5 cm in this arrangement). At this point in time, phosphatidylinositol and phosphatidylserine are still on the separation column labeled ( 5 ). The column circuit ( 7 ) now removes the column labeled ( 6 ) from the eluent flow, so that phosphatidylinositol and phosphatidylserine no longer have to go through the entire column length, which causes a strong "peak" broadening and thus a deterioration in the detection limit and an extension of the total elution time (phosphatidylinositol: approx. 140 instead of 35 minutes; phosphatidylserine 215 instead of 55 minutes). After elution of the phospholipids from the separation columns, the phospholipid-containing eluate passes through the flow cell of an ultraviolet absorption detector (wavelength 200-210 nm). Behind the UV detector ( 8 ) from the reservoir ( 9 ) by means of the pump ( 10 ) with a constant flow of 3 to 5 ml / minute a solution of 1,6-diphenyl-1,3,5-hexatriene containing phospholipid The eluate is mixed in and the formation of the diphenylhexatriene-containing phospholipid micelles is achieved in a 2 to 3 m long, thin-lumen Teflon® tube ( 11 ) at 50 ° C. The fluorescence of the micelles is then determined in the fluorescence detector ( 12 ) (excitation wavelength approx. 340 nm; detection wavelength: approx. 460 nm), whereupon the samples are collected for further analysis or flow into the collecting vessel ( 13 ).

In the separation of standard and liver phospholipids in FIG. 2 with the HPLC method and subsequent UV / fluorescence detection, the second column was switched off after 30 (standard phospholipids) or 31 minutes (liver phospholipids). This figure shows not only a good separation of the main phospholipids, phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, but also the other phospholipids. From the total elution times for phosphatidylinositol and phosphatidylserine, it could be calculated that after 30 to 32 minutes, when phosphatidylethanolamine had passed a total column length of 23.5 cm, phosphatidylinositol was 5 cm and phosphatidylserine was 3.5 cm. For this reason, the separation system was divided into two columns and a switching valve interposed. After elution of phosphatidylethanolamine, the two further phospholipids are thus separated according to the invention via the first, shorter column, the retention times being 33 to 35 minutes in the case of phosphatidylinositol and 55 to 60 minutes in the case of phosphatidylserine.

The phospholipids separated in FIG. 3 were applied in amounts of 0.5 to 80 nmol. The mean values ± standard errors (= standard deviation / root of n-1) of 3 to 4 determinations are given. If no bars are shown in the diagram, the standard error is less than the symbol. The samples in FIG. 4 were applied in amounts of 0.5 to 100 nmol, otherwise the statements made in FIG. 3 apply. Figures 3 and 4 show that the fluorescence and UV response correlate well with the amount of phospholipids, but UV absorption varied considerably with the different classes of phospholipids. This was due to the different fatty acid composition of the phospholipids as can be seen from FIG. 3. The different phosphatidylcholines and the internal standard show the same fluorescence. The UV absorption of the phosphatidylcholines is very different depending on the fatty acid composition and shows almost no UV sensitivity in the case of the saturated 1,2-dipalmitoylphosphatidylcholine. Only Dioleoyl phosphatidylcholine and the internal standard show identical UV sensitivity, both of which have two oleic acid residues (= 2 double bonds). With the highest sensitivity of the fluorescence detector, the detection limit was 0.5 nmol for all phospholipids with the exception of lysophosphatidylcholine (2 nmol). With this setting, the response of the fluorescence detector was linear to 60 nmol for all phosphatidylcholine derivatives and 100 nmol for the other phospholipids. Within a phospholipid class, UV absorption depended on the fatty acid composition, but the fluorescence was independent of it.

In Fig. 5, DOPC means dioleoylphosphatidylcholine and DPPC dipalmitoylphosphatidylcholine. The UV / fluorescence ratio of a phospholipid is constant over a wide concentration range and this ratio correlates linearly with its content of unsaturated fatty acids, as can be seen from FIG. 6. Therein P0PC means 1-palmitoyl-2-oleoylphosphatidylcholine, SAPC 1-stearoyl-2-arachidonoylphosphatidylcholine. The values given here are the mean values of 4 to 6 determinations and their standard errors. Figure 6 shows that the linearity of the UV / fluorescence ratio correlated with the unsaturated fatty acid content.

In Fig. 7, the UV / fluorescence ratios of phosphatidylcholine (filled symbols) symbols were and phosphatidylethanolamine (open symbols from liver (squares), stomach (standing on corner squares) and lung (triangles) correlated with their content of double bonds. and bars are mean values from 10 to 11 measurements ± standard errors. "r" is the correlation coefficient. Fig. 7 shows that the method according to the invention allows conclusions to be drawn about the content of double bonds in the fatty acid portion of the phospholipid.

Claims (3)

1. A method for the separation and quantification of the main phospholipid classes by means of high pressure liquid chromatography, characterized in that the sample mixture is applied with a phospholipid which does not influence the measurement as an internal standard to a first shorter column which is connected to a second longer column and isocratically elutes , After elution of the phospholipids having a retention time of up to 40 minutes, the columns are separated from the second longer column and the phospholipids still on the first separation column are eluted from the first column with a retention time of 40 to 60 minutes, and then the eluates are first of all by means of UV detection and then detected after derivatization using fluorescence detection. 2. The method according to claim 1, characterized in that the Derivatization of the phospholipids by reaction with 1,6-diphenyl-1,3,5-hexatriene takes place. 3. The method according to claim 1 or 2, characterized in that as an internal standard N-monomethyl-1,2-di oleoylphosphatidylethanolamine, N-Monomethyl-1-palmitoyl-2- rinoloylphosphatidylethanolamine and N-monomethyl-1,2-dili noleoylphosphatidylethanolamine is used.