SE452805B - METHOD OF DETERMINING LIGHT CONTENT AND TOTAL PROTEIN CONTENT IN PHOTO SUBSTANCES WITH HPLC TECHNOLOGY - Google Patents

Description

452 805 10 15 2o_ 25 30 35 2 demanding (analysis time: approx. 90 min) and thus costly.

However, this method is well tested and the repeatability is good. Conventional amino acid analysis is performed according to this method with so-called amino acid analyzers, which is a term for devices where the above steps are performed.

In the context of plant breeding, however, Stein'S 0Ch Moore's method is too slow and expensive. One can more indirectly determine the lysine content in a sample by using the so-called The DBC method is based on estimating the amount of a dye that binds to basic amino acids (lysine, histidine, arginine).

The disadvantages of the above method are that sample amounts of about 1 g are required and that the analysis must be supplemented with a protein determination, eg Kjeldahl analysis, where the protein content is measured via the total amount of nitrogen in the sample.

Another technique for amino acid analysis involves the separation of derivatized amino acids by high pressure chromatography (HPLC). The advantage of HPLC is that the analysis time can be made much shorter than when using amino acid analyzers, ie an analysis time of about 20-40 minutes.

For example, in the article by S. Einarsson, B. Josefsson and S. Lagerkvist in Journal of Cromatography, 282 (1983) 609-618, amino acids from seawater are derivatized with a suitable reagent, 9-fluorenylmethyl chloroformate (FMOC), for later detection. by fluorescence spectrophotometry. After extraction, the amino acid derivatives are separated by HPLC. A disadvantage of this method is that the peak representing double-derivatized histidine interferes with the lysine peak in the chromatogram. Sample time for this method is about 30 min. The object of the present invention is to provide a fast, sensitive and precise process for the simultaneous determination of lysine content and total protein content in a proteinaceous sample, which process comprises pretreatment of the product by hydrolysis, addition of a internal standard and derivatization of the amino acids included in the sample, after which the pretreated product is analyzed by separation with phase "- HPLC and integration of the obtained chromatogram.

This object is achieved according to the invention by (a) that the derivatization is carried out with a derivatizing agent in the form of 9-fluorenylmethyl chloroformate and that detection is carried out by fluorescence measurement, (b) that the pretreatment further comprises heating the sample, if it contains histidine, to eliminate double derivatized histidine, (c) using as an internal standard a compound not naturally occurring in the sample with the structural formula HOOC-R-NH 2, wherein R is a straight or branched, aliphatic, aromatic or alicyclic carbon chain having 6 to 12 carbon atoms, ( d) that in the HPLC separation an eluent consisting of acetonitrile is used in a concentration of 40-65 vol% and 35-60 vol% of a buffer with a pH of 5.5-8.0.

The difference between this method and existing HPLC methods for lysine and protein determination is that the amino acids present in the sample, except for lysine, do not need to be separated from each other. Under certain conditions, a protein content can be obtained from such a compressed chromatogram, and in addition, under these conditions, the lysine and total protein content can be determined simultaneously using existing HPLC technology in an analysis time of 2-5 minutes from sample to sample. The advantages of the method according to the invention are that the reproducibility is very good, that the sensitivity is very high and that the instrument cost is relatively low considering that the number of analyzes per unit time is high. Furthermore, the sample preparation is quick and easy, and the derivatives are stable for several days.

The conditions that must be met to carry out the analysis and which are exclusive to this procedure follow below. 10 15 20 25 30 35 452 805 4 (1) To determine the protein content without separating all the individual derivatized amino acids, the reagent used is required to give amino acid derivatives with almost the same response for different amino acids in the detection method used. This does not apply to lysine, as it is separated and analyzed separately. A requirement is also that all the amino acids present in the sample are derivatized. A preferred derivatizing agent is 9-fluorenylmethyl chloroformate (FMOC). Use of FMOC requires the use of a fluorescence spectrometer for detection. (2) The lysine derivative must be easily distinguishable from the other amino acids in the chromatogram. With the reagent used, lysine, histindine and tyrosine become double-derivatized and thus more non-polar than other amino acids. These separate rapidly from the single derivatized amino acids by reverse phase chromatography using an eluent which is preferably 42% by volume of acetonitrile and 58% by volume of a pH 6.8 buffer. The tyrosine derivative separates well from lysine under the given conditions. Double-derivatized histidine interferes with the lysine peak, as it has retention properties similar to those of lysine; Therefore, the sample must preferably be heated at 60 ° C and for 100 minutes during the pre-treatment before the separation, the double derivatized histidine switching to single derivatized histidine, which does not interfere with the chromatogram. (3) An internal standard is used to make sample preparation quick and easy, and to enable high reproducibility to be achieved. This internal standard must be stable, give a single derivative with the reagent, and be separated from the other peaks in the chromatogram.

Such a compound is preferably a primary amino acid of lower polarity than the amino acids present, and has the structural formula HOOC-R-NH 2, wherein R is a straight or branched, aliphatic, aromatic or alicyclic carbon chain having 6 carbon atoms. -12 carbon atoms. In a preferred embodiment, u-aminocaprylic acid is used, since its peak lies between that of the lysine and the other amino acids.

The process according to the invention fully corresponds to the requirements that exist in the context of plant breeding with regard to speed, sensitivity and accuracy in determining lysine content and total protein content.

With the method according to the invention the following results can be achieved: Speed - sample preparation approx. 8 min - analysis time maximum 5 min Sensitivity - a grain kernel (approx. 50 mg) can be analyzed 1000 times Accuracy - lysine content and protein content are determined with a repeatability of approx. 2% RSO (100 x the standard deviation / mean value) The HPLC chromatogram that is sought and allows the analysis to be performed quickly is such that first all amino acids, except the lysine, come close together as a "forest", after which the stable internal standard comes, followed by a reagent residue and finally of lysine.

The total protein content of the sample can be determined by adding the top regions of the respective amino acids in the chromatogram.

Thus, the sum of all constituent amino acid contents becomes a measure of the total protein content of the sample.

In a preferred embodiment of the invention, the pretreatment comprises the steps of first hydrolyzing in a hydrolysis tube containing 6 M HCl and an internal standard in the form of Q-aminocaprylic acid a suitable amount, eg 80-120 mg, of the sample, which may be in fresh or dried, unpainted or ground condition, after cooling then filtering the sample through filter paper, then diluting and mixing the sample, derivatizing the sample by adding a suitable derivatizing agent, eg FMOC, after which the aqueous phase is isolated in sample bottles and heated at 60 ° C in a water bath in in order to eliminate double derivatized histidine which would otherwise interfere with the chromatogram, after which the amino acids included in the sample are separated by means of "reversed-phase" HPLC and analyzed by means of the obtained chromatogram.

In the accompanying drawing, Fig. 1 shows a chromatogram and integrated values for analysis of lysine content and protein content in samples of cereals, and Fig. 2 a chromatogram and integrated values for analysis of lysine content and protein content in samples of oil plant.

The following describes detailed embodiments of the invention, as well as the chemicals and equipment used.

Embodiments Chemicals: 1. Hydrochloric acid 37% (pa Merck) Z. u-aminocaprylic acid (Sigma A-2629) 3. Boric acid (pa Merck) 4. Sodium hydroxide 19 M oil liquor (pa Merck) 5. Fluorenylmethyl chloroformate (97% Sigma) 6. Acetone (by Merck) 7. Sodium acetate (by Janssen Chimica) 8. Acetic acid 100% (by Merck) Petroleum ether 40-600; distilled before use 10. Calibration solution with the following composition in 0,1 M hydrochloric acid L-Alanine L-Methionine Ammonium chloride L-Phenylalanine L-Arginine L-Proline L-Aspartic acid L-Serine L-Cystine L-Threonine L-Glutamic acid L-Tyrosine Glycine L-Valine L-Histidine L-Isoleucine L-Lysine content of L {yStüiär1,25; mm1hM of the other 2.5 pmol / ml 11. Acetonitrile (HPLC grade Lab Scan) 12. Distilled water, further purified with a Gelman Water-I. 452 805 Solutions: I.

II. 6 M hydrochloric acid mixed with equal parts hydrochloric acid 37 2 (1) and water (12) Hydrolysis solution for cereals: 100 mg u-aminocaprylic acid (2) per liter 6 M hydrochloric acid (I) Hydrolysis solution for oil plants: 200 mg u-aminocaprylic acid (2) per liter of 6 M hydrochloric acid (I) 6 H sodium hydroxide solution: Dilute 316 ml of oil liquor (4) to 1 l with water (12) Boric acid buffer stock solution: 61.83 g of boric acid (3) are dissolved in water (12). Adjust to pH 6.2 with 6 H sodium hydroxide solution (IV) and dilute to 1 liter Dilution solution: Mix 200 ml of boric acid buffer (V) with 12 ml of 6 M sodium hydroxide solution (IV) and dilute to 1 liter with water (12). PH of the solution = 8.8-9.0. 15 mM fluorenylmethyl chloroformate solution - stock solution: Dissolve 776.1 mg of fluorenylmethyl chloroformate FHOC (5) in 200 ml of acetone (6).

Reagent solution: Dilute 10 ml of stock solution (V11) with acetane (6) to 100 ml.

Acetate buffer: Dissolve 5 g of sodium acetate (7) in water (12). Adjusted to pH 6.8 with acetic acid (8) and diluted to 1 liter Apparatus and material s lll. lv. v. 10 vl. 15 vll. vlll. lx. 20 25 A_ B. c. N.

E. 30 F. e.

H I. 35 Hydrolysis tubes. Glass tube 14 ml with screw cap and Teflon gasket Hydrolysis cabinet: Heating cabinet with attached device for mixing (cradle) Filter paper 00R 7 cm diam.

Test tube with screw cap Rotary sample mixer water bath with shaker additive (Gallenkamp) Test bottles, 1.5 ml, suitable for automatic injector HPLC equipment: Hewlett-Packard HPLC 1090 Detector: Perkin-Elmer LS-4 Fluorescence Spectrometer Integrator: Shimadzu C-R3A Chromatopac 10 15 20 Example 30 Simultaneous determination of the lysine and protein content of whole grain kernels The kernels are weighed to the nearest 0,1 mg, crushed and placed in hydrolysis tubes (A). 10.0 ml of hydrolysis solution (II) are added, the tubes are closed and placed in the hydrolysis cabinet (B), where the samples are rocked for 15 minutes and left to stand for 45 minutes.

The rocking is repeated throughout the hydrolysis time, ie 24 hours.

The hydrolysis temperature is 10 ° C.

Then measure 10 μl of hydrolyzate and mix with 1.2 ml of dilution solution (VI) in 10 ml test tubes with screw caps (D). The tubes are placed in a rotating sample mixer (E), and the samples are mixed for 2 minutes 20 rpm. 1 ml of reagent solution (VIII) is added and the samples are rotated for 6 minutes. 3 ml of petroleum ether (9) are added and extracted for 2 minutes. The petroleum ether phase is sucked off. The extraction is repeated with another 3 ml of petroleum ether.

The aqueous phase is transferred to test bottles, 1.5 ml, (G).

The bottles are placed in a 60 ° water bath (F) and shaken for 100 minutes. The samples are now ready for HPLC analysis.

If the derivatives are not analyzed immediately, they should be stored in a freezer. 100 μl of standard solution are mixed with 60 ml of dilution solution (VI) and 600 μl of hydrolysis solution (II) are added. 750 μl of this mixture is derivatized in the same way as the samples.

The sample is then analyzed by HPLC under the following conditions: Column: 60 x 4.6 mm, packed with Hypersil ODS (octadecyl silica), and with a particle size of 3 μm (Hewlett-Packard) Temp: 40 ° C Flow: 1 , 5 ml / min Eluent: A. Acetate buffer (IX), B. acetonitrile (II) Gradient: Min% A% B 0 58 42 3 58 42 3.1 S0 50 4 50 50 4.2 58 42 10 15 20 25 452 805 9 A new sample is injected after 5.2 min.

Detector: Fluorescence spectrometer 260 nm column 10 310 nm column 5 The chromatograms obtained are drawn and integrated Exitation Emission using an integrator.

Using the chromatogram shown in Fig. 1, the results could be evaluated. The amount of lysine was 5.6 mg of lysine per g of sample, and the total amount of protein was 136.3 mg of protein per g of sample.

Example 2 Simultaneous determination of lysine and protein content for samples from oil plants 50.0 mg of defatted flour are weighed into hydrolysis tubes (A), 10.0 ml of hydrolysis solution (III) are added, the tubes are sealed and placed in a heating cabinet at 110 ° C 24 tim.

The samples are mixed only once during the hydrolysis, namely 30 minutes after the start of hydrolysis.

After cooling, the samples are filtered through filter paper OOR, 7 cm (C).

Thereafter, the sample is treated and analyzed according to Example 1.

The chromatogram is shown in Figure 2. The amount of lysine obtained was 19.6 mg of lysine per g of sample, and the total amount of protein was 280.7 mg of protein per g of sample.

Claims (4)

452 805 10 PATENT CLAIMS A process for determining lysine content and total protein content of a proteinaceous sample, which process comprises pretreating the sample by hydrolysis, adding an internal standard and derivatizing the 5 amino acids contained in the sample, after which the pretreated product is analyzed by separation with " reversed-phase "HPLC and integration of the obtained chromatogram, characterized by, a) that the derivatization is carried out with a derivatizing agent in the form of 9-fluorenylmethyl chloroformate (FMOC) and that detection is carried out by fluorescence measurement. b) that the pretreatment further comprises heating the sample, if it contains histidine, to eliminate double derivatized histidine; c) using as internal standard a compound not naturally occurring in the sample with the structural formula HOOC-R-NH 2, where R is a straight or branched, aliphatic, aromatic or alicyclic carbon chain having 6-12 carbon atoms, d) using in the HPLC separation an eluent consisting of acetonitrile in a concentration of 40-65 vol% and 35-60 vol% of a buffer with a pH of 5.5-8.0. 2. A process according to claim 1 or 2, characterized in that the heating in step b) is carried out at 60 ° C for 100 minutes. 25 3. A process according to any one of the preceding claims, characterized in that α-aminocaprylic acid is used as the internal standard. 4. A process according to any one of the preceding claims, characterized in that the eluent consists of 42% by volume of acetonitrile and 58% by volume of a buffer of pH 6.8.