CN114062564A - Detection method of taffeta and variants thereof - Google Patents

Abstract

The invention provides a detection method of taffeta and variants thereof, which is a high performance liquid chromatography and adopts a C18 chromatographic column; mobile phase a is 0.03% heptafluorobutyric acid aqueous solution, mobile phase B is acetonitrile, mobile phase a: the volume ratio of B is 80-90: 20-10; the flow rate of the mobile phase is 0.8-1.2 ml/min, the detection wavelength is 214nm, the temperature of the chromatographic column is 25-35 ℃, and the sample injection amount is 5-20 mu l. The high performance liquid chromatography detection method adopted by the invention is simple and rapid, can accurately detect the existence of the taffeta and the variant thereof in the sample, and has important significance for quality control of products containing the taffeta and the variant thereof.

Description

Detection method of taffeta and variants thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a detection method of taffeta and a mutant thereof.

Background

Taffsin (TKPR) is a bioactive polypeptide secreted from spleen and having the effects of promoting phagocytosis of leukocyte and regulating immune response of organism. The tafferin can improve phagocytic and bactericidal activity of macrophages, has chemotactic effect on polymorphonuclear leukocytes, and can improve cytotoxicity of natural killer cells; it also has anticancer effect, and can inhibit tumor cell proliferation in vitro and in vivo.

Although the existence of natural variants of taffysin (TKP and TKPPR) has long been known, for example, the document "structure and function of Tuftisn" [ national medical biochemistry and laboratory Manual ] Vol.11, No. 6, 13-16, 1990 has been reported; the detection of teffsin was also performed by "reverse phase-high performance liquid chromatography (RP-HPLC) for serum tuftsin and its clinical application" proceedings of third military medical science 1995, volume 171, phase 5, 445-447, but we verified experimentally that the method could not distinguish teffsin and its natural variant TKPPR.

As reported in THE literature (THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL 281, number 9, pp. 5702-.

The prior art does not relate to the detection of the content of the natural mutant of the taffycin in vivo, and does not relate to the detection of the differentiation and identification of the taffycin and the mutant thereof under the same condition.

The taffrin and the natural variant TKPPR are very similar in structure, only have the difference of one nonpolar amino acid proline, so that the taffrin and the natural variant TKPPR are basic amino acids and have the same charge and similar polarity. Therefore, the simultaneous detection of the three components of the taffeta and the natural variants TKP and TKPPR thereof has great difficulty. At present, no literature reports a detection method for distinguishing the three components.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a method for rapidly detecting taffeta and a mutant thereof.

The technical scheme adopted by the invention is as follows:

a detection method of taffeta essence and its variant is high performance liquid chromatography, which adopts C18 chromatographic column; mobile phase a is 0.03% heptafluorobutyric acid aqueous solution, mobile phase B is acetonitrile, mobile phase a: the proportion of B is (80-90): (20-10); the flow rate of the mobile phase is 0.8-1.2 ml/min, the detection wavelength is 214nm, the temperature of the chromatographic column is 25-35 ℃, and the sample injection amount is 5-20 mu l.

Further, the mobile phase a: the proportion of B is 90: 10.

further, the flow rate of the mobile phase is 1.0ml/min, the detection wavelength is 214nm, and the temperature of the chromatographic column is 30 ℃.

The detection method comprises the following steps:

s1, sample solution preparation: dissolving a sample to be tested in a methanol solution, wherein the mass volume ratio of the sample to the methanol is 1mg:1 ml;

s2, preparing a reference substance solution: respectively dissolving a reference substance of the taffysin TKPR, the variant TKP and the variant TKPPR in a methanol solution, wherein the mass volume ratio of the reference substance to the methanol is 1mg:1 ml;

s3, detection: measuring 5 mul of reference solution, injecting into a chromatograph, and recording chromatogram; a sample of 20. mu.l was measured, injected into a chromatograph, and the chromatogram was recorded.

The beneficial effects obtained by the invention are as follows: the high performance liquid chromatography detection method adopted by the invention is simple and rapid, can accurately detect the existence of the taffeta and the variant thereof in the sample, and has important significance for quality control of products containing the taffeta and the variant thereof.

Drawings

FIG. 1 is a literature chromatogram of a reference substance of Tafuxin (TKPR);

FIG. 2 is a chromatogram obtained by detecting TKP reference substance by literature method;

FIG. 3 is a detection chromatogram obtained by the TKPPR reference substance literature method;

FIG. 4 is a chromatogram obtained by assaying TKP + TKPR + TKPPR mixed control by literature method;

FIG. 5 shows the new detection chromatogram and comparison results of TKP, TKPR, and TKPPR reference substances;

FIG. 6 is a new method detection chromatogram of TKP control;

FIG. 7 is a new method detection chromatogram of TKPR control;

FIG. 8 is a new method detection chromatogram of TKPPR control;

FIG. 9 is a chromatogram obtained by detecting with a new methanol solvent control method;

FIG. 10 shows the comparison results of the new method for detecting the reference substance and the sample.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

In the research process, firstly, according to a high performance liquid chromatography method reported in a reference literature of 'reverse phase-high performance liquid chromatography (RP-HPLC) for measuring serum tuftsin and clinical application thereof', the Tafferin (TKPR) and the natural variants TKP and TKPPR thereof are respectively detected, and the experimental results are shown in figures 1-3. Through comparison of experimental results, it was found that the retention time of the Taff base (TKPR) and the variant TKPPR is the same after detection by the method reported in the literature. Therefore, the three components (the taffeta, the TKP and the TKPPR) are mixed and detected, and the result shows that the detection method cannot distinguish the taffeta and the TKPPR, and the experimental result is shown in FIG. 4.

The application identifies a new detection condition, and can successfully separate and locate the taffeta element (TKPR), the variant TKP and the variant TKPPR. Meanwhile, the spleen ammonia peptide spray-dried powder which is the low molecular weight extract of the animal spleen is detected, and the TKPPR component is confirmed to be contained in the spray-dried powder, so that the TKPR and the TKPPR in the biological product from the animal spleen are detected under the same condition for the first time, and finally the following detection method is formed:

a detection method of taffeta essence and variants thereof is high performance liquid chromatography, and the detection conditions are as follows:

a chromatographic column: agilent ZORBAX 4.6 x 250mm 5 μm 300 SB-C18;

mobile phase A: 0.03% aqueous solution of heptafluorobutyric acid;

mobile phase B: acetonitrile;

mobile phase A: the volume ratio of B is 90: 10;

flow rate of mobile phase: 1.0ml/min, detection wavelength 214nm

Chromatographic column temperature 30 ℃, sample injection amount: 5 mul of standard substance; 20. mu.l of the sample to be tested.

Acetonitrile: batch number: 203022 Saimer Feishell science & technology (China) Co., Ltd 99.95%;

purifying water: self-made in a laboratory;

heptafluorobutyric acid: maxin 98%

Tafferin (TKPR): purity: 98% Nanjing Laong Kai Biotech Co., Ltd

Variant (TKP): purity: 98% Nanjing Laong Kai Biotech Co., Ltd

Variant (TKPPR): purity: 98% Nanjing Laong Kai Biotech Co., Ltd

A sample to be tested: spleen aminopeptide spray dry powder laboratory self-made

In the specific implementation:

the detection method comprises the following steps:

s1, sample solution preparation: dissolving 10mg of spleen aminopeptide spray-dried powder of a sample to be tested in 10ml of methanol solution;

s2, preparing a reference substance solution: dissolving 10mg of a taffysin TKPR + isomer TKP + isomer TKPPR taffysin TKPR mixed reference substance, 10mg of the taffysin TKPR, 10mg of the isomer TKP and 10mg of the isomer TKPPR reference substance in 10ml of methanol solution respectively;

s3, detection: measuring 5 μ l of the reference solution, injecting into chromatograph, and recording chromatogram according to the above detection conditions; a sample (20. mu.l) was measured and injected into a chromatograph, and a chromatogram was recorded according to the above detection conditions.

As shown in FIG. 5, the sample mixture A, TKP, TKPR and TKPPR are shown from top to bottom; b: a TKP sample; c: TKPR samples; TKPPR sample; e: methanol solvent control. Experimental results show that the detection method can well distinguish the taffeta pigment (TKPR) and the variant TKP and TKPPR thereof.

As shown in fig. 6, 7, 8 and 9, the detection chromatograms of the isomer TKP, the taffoltkpr, the isomer TKPPR and the methanol solvent control are respectively shown. Under the detection condition, the three components peak at different retention times, can be well separated and positioned, and have no mutual interference with the contrast of the methanol solvent.

As shown in FIG. 10, the A is TKP + TKPR + TKPPR mixed control in the order from top to bottom; b: spraying dry powder of spleen aminopeptide; c: a + B (mixed control + splenopeptide spray-dried powder).

And (4) conclusion: experiments show that the high performance liquid chromatography detection method adopted by the invention can well distinguish the Taffysin (TKPR) and the variant TKP and TKPPR thereof, and fills the blank of the technology in the field.

Meanwhile, the spleen ammonia peptide spray-dried powder, which is a low molecular weight extract of the spleen of the animal, is found to have a peak at the same retention time as that of the TKPPR control product, and is presumed to contain the TKPPR component. To more accurately confirm the presence of TKPPR component in the sample, we added a control solution to the sample. According to the chromatogram, when the reference substance is added, a chromatographic peak is formed at the peak position of TKPPR, and the peak area is increased. The chromatographic condition is proved to be capable of identifying the existence of the TKPPR component in the spleen ammonia peptide spray-dried powder.

In addition, the invention has more important application in detecting the content and proportion of the taffeta and biological variants thereof in organisms, thereby being capable of confirming the source of the biological activity exerted by the taffeta and the biological variants thereof, being helpful for clarifying the material basis of the biological activity source for improving the immunity through phagocytosis in the organisms and providing technical support for further research and development of TKPPR.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (4)

1. A detection method of taffeta and variants thereof is characterized in that: the method is high performance liquid chromatography, and adopts a C18 chromatographic column; mobile phase a is 0.03% heptafluorobutyric acid aqueous solution, mobile phase B is acetonitrile, mobile phase a: the proportion of B is (80-90): (20-10); the flow rate of the mobile phase is 0.8-1.2 ml/min, the detection wavelength is 214nm, the temperature of the chromatographic column is 25-35 ℃, and the sample injection amount is 5-20 mu l.

2. The method for detecting taffysin and variants thereof according to claim 1, wherein: the mobile phase A: the proportion of B is 90: 10.

3. the method for detecting taffysin and variants thereof according to claim 1, wherein: the flow rate of the mobile phase is 1.0ml/min, the detection wavelength is 214nm, and the temperature of a chromatographic column is 30 ℃.

4. The detection method according to any one of claims 1 to 3, characterized in that: the method comprises the following steps:

s1, sample solution preparation: dissolving a sample to be tested in a methanol solution, wherein the mass volume ratio of the sample to the methanol is 1mg:1 ml;

s2, preparing a reference substance solution: respectively dissolving a reference substance of the taffysin TKPR, the variant TKP and the variant TKPPR in a methanol solution, wherein the mass volume ratio of the reference substance to the methanol is 1mg:1 ml;

s3, detection: measuring 5 mul of reference solution, injecting into a chromatograph, and recording chromatogram; a sample of 20. mu.l was measured, injected into a chromatograph, and the chromatogram was recorded.

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