CN113567573B

CN113567573B - Method for separating and analyzing osteopontin

Info

Publication number: CN113567573B
Application number: CN202110780296.3A
Authority: CN
Inventors: 蒋士龙; 王象欣; 解庆刚; 刘莹; 崔东影; 梁爱梅; 冷友斌
Original assignee: Heilongjiang Feihe Dairy Co Ltd
Current assignee: Heilongjiang Feihe Dairy Co Ltd
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2023-06-02
Anticipated expiration: 2041-07-09
Also published as: CN113567573A

Abstract

The invention provides a separation method and an analysis method of osteopontin. The separation method comprises the following steps: and carrying out HPLC (high performance liquid chromatography) separation on an object to be tested containing osteopontin by adopting a strong anion exchange chromatographic column to obtain an osteopontin sample, wherein a filling material in the strong anion exchange chromatographic column comprises surface porous silica gel bonded with trimethyl aminopropyl or microporous ethyl vinyl benzene crosslinked 55% divinylbenzene polymer bonded with trimethyl aminopropyl. The strong anion exchange chromatographic column used in the invention achieves the purpose of effectively separating OPN from other impurities. Compared with the analysis method for analyzing the osteopontin peptide fragment by adopting mass spectrometry in the prior art, the separation method of the OPN in the application ensures that the retention difference of each component in the object to be tested containing the OPN on a chromatographic column is obvious, and the OPN has excellent selectivity and high sensitivity and better separation capacity on the whole, so that the OPN in the object to be tested can be analyzed more accurately.

Description

Method for separating and analyzing osteopontin

Technical Field

The invention relates to the technical field of analysis of osteopontin, in particular to a separation method and an analysis method of osteopontin.

Background

Osteopontin (OPN) is an acidic phosphorylated glycoprotein that serves a variety of functions including maintaining intestinal integrity by affecting tight junctions, which helps maintain intestinal mucosal barrier, maintain intestinal architecture, regulate intestinal inflammation, cell adhesion, bone remodeling, vascular growth, regulation of calcium oxalate formation, and cell signaling, among others. OPN is involved in human immune responses, including opsonin activity in mouse models and aids in inflammatory cell migration to wound sites and cytokine expression. Senger et al identified OPN in breast milk, which in turn identified OPN in milk, although at a lower level than breast milk. In breast milk, OPN accounts for about 100-300 mg/L of total protein, and average OPN is 138mg/L. Cow's milk contains about 15-20 mg/L of OPN (Schack et al, 2009; lonnerdal, 2011) about 5 to 10 times lower than the OPN content in human milk (Lonnerdal, 2011). In addition, some commercial infant formulas (obtained from korea and denmark) also contain 5-13 mg/L of natural OPN, which is much lower than breast milk.

Breast milk contains low levels of a number of different proteins, and these proteins contribute to the overall benefit of breast feeding. The probability of infection (e.g., otitis media, diarrhea) is reduced and gastrointestinal comfort is increased in breast-fed infants compared to formula-fed infants. Breast feeding was preferred over formula feeding during the first six months of infancy, followed by continued breast feeding with the addition of a complementary food, and continued breast feeding for one year or more depending on the combined intent of the mother and infant. However, infants and subsequent formulas that are closest to the composition and function of breast milk in terms of nutritional value are advantageous when breast feeding is inadequate or not as a viable option.

The european commission (EC, 2013) defines infant formulas as: "food for infants during the first months after birth and meeting the nutritional needs of the infant before the introduction of appropriate complementary food". The European Union committee also defines infant formulas as "foods for infants after the introduction of appropriate complementary foods, which constitute the major liquid component in such infant gradually diversified diets". At present, the goal of any infant formula manufacturer is to try to mimic the composition and function of breast milk as much as possible, providing a nutritionally rich, growth-promoting formula for infants from 0 to 12 months old. Over the past few decades, basic research into infant nutrition needs has increased significantly, which helps infant formulas provide nutritional value and biological components that are more consistent with breast milk. In particular, over the last 20 years, there has been a great increase in the assessment of breast milk components, and new components that may be important for infant growth have been characterized, some of which include docosahexaenoic acid (DHA), arachidonic acid and lactoferrin (Lonnerdal, 2014; richard et al 2016). However, the characterization of osteopontin is far from the above-described components.

There are two forms of osteopontin in cow's milk; (1) full length OPN (262 amino acid residues) with a molecular weight of 33.9kDa including modifications (migration of about 60kDa in SDS-PAGE) and (2)N terminal fragment (amino acid residues 1-150) with a molecular weight of 19.8kDa including modifications (migration of 35-40 kDa in SDS-PAGE). The N-terminal fragment results from the inherent proteolytic activity in mammary and milk (Bissonnette et al 2012; christensen et al 2014), and Bissonnette, christensen et al have described similar cleavage of OPN in breast milk, found similar cleavage sites between bovine and human OPN, and in addition, the full length OPN and N-terminal fragment function the same for both OPNs, fragments and integrins can be detected simultaneously.

Currently, three commercial enzyme-linked immunosorbent assay (ELISA) kits for measuring human osteopontin dominate the commercial market and scientific literature. In addition, some laboratories have developed and validated internal ELISA methods for measuring osteopontin, but currently no commercially available ELISA for measuring bovine osteopontin is available. In order to market osteopontin assays, a number of monoclonal antibodies directed against human, mouse and rat OPN have been developed, but only 1-2 of them cross-react with bovine milk OPN, probably due to the heterogeneity of 27 bovine milk osteopontin in terms of modification and proteolytic processing, making it difficult to generate antibodies that capture all forms of OPN, and thus antibody-based methods are not used for quality control or regulation. Furthermore, mass spectrometry detection is currently adopted and is considered to be the best method for OPN-10 quality control for quantitative purposes.

It must be noted that the reliability of IBL-OPN-ELISA was questioned (Plumer et al, 2008; schack et al, 2009). Nagatomo et al analyzed the amount of osteopontin in breast milk using IBL ELISA, by which they found a surprisingly high concentration of OPN in human breast milk, with osteopontin concentrations in early milk (3-7 days) and mature milk (one month after delivery) of 1493.4 mg/l and 896.3 mg/l, respectively. Although the OPN concentration in milk has been reported to be quite high, these values indicate that OPN should account for more than 10% of the total protein in breast milk, and the detection results of the visible side method are relatively high compared to actual results.

The protein of the dairy product is detected by a mass spectrum method, and the first problem is that the Maillard reaction in the production process leads to lactose modification of lysine, so that the pancreatic enzyme action site loses specificity, the obtained specific peptide is reduced, and finally the test content of OPN is lower. Secondly, mass spectrometry from reagent consumable to instrument price to operation method is very tested on the whole capability of the laboratory.

In addition, for the peptide fragment detection method of the osteopontin, the enzyme digestion is more difficult in space due to the specificity of the OPN structure (28 phosphorylation sites and 2O sugar sites), and the two are overlapped, so that the test content of the OPN is lower, and the result is inaccurate.

Disclosure of Invention

The invention mainly aims to provide a separation method and an analysis method of osteopontin, which are used for solving the problems of complex operation, high cost, inaccurate analysis result and the like in the analysis method of the osteopontin in the prior art.

In order to achieve one aspect of the present invention, there is provided a method for separating osteopontin, the method comprising: and carrying out HPLC (high performance liquid chromatography) separation on an object to be tested containing osteopontin by adopting a strong anion exchange chromatographic column to obtain an osteopontin sample, wherein a filling material in the strong anion exchange chromatographic column comprises surface porous silica gel bonded with trimethyl aminopropyl or microporous ethyl vinyl benzene crosslinked 55% divinylbenzene polymer bonded with trimethyl aminopropyl.

Further, the strong anion exchange chromatographic column has the following specification: the length is 50-250 mm, the inner diameter is 2-30 mm, the particle size of the filling material is 2-20 mu m, and the specification of the preferable strong anion exchange chromatographic column is as follows: the length is 50-150 mm, the inner diameter is 2-10 mm, the particle size of the filling material is 2-10 mu m, and more preferably, the specification of the strong anion exchange chromatographic column is as follows: the length is 100mm, the inner diameter is 4.6mm, and the particle size of the filling material is 5 mu m.

Further, the strong anion exchange chromatography column is Waters Protein-Pak Hi Res Q or Thermo Fisher Scientific ProPac WAX-10.

Further, the HPLC separation is carried out by adopting a gradient elution method, and the mobile phase of the gradient elution comprises a mobile phase A and a mobile phase B; preferably, the mobile phase a and the mobile phase B are each independently a buffer solution selected from the group consisting of a tris (hydroxymethyl) aminomethane solution, a tris (hydroxymethyl) aminomethane hydrochloride solution, a tromethamine solution, a dibasic phosphate solution, a monobasic phosphate solution, or a combination of any one or more of them with a sodium chloride solution, a potassium chloride solution, a calcium chloride solution, a magnesium chloride solution, or a lithium chloride solution, more preferably the buffer solution is a mixture of a tris (hydroxymethyl) aminomethane solution and a lithium chloride solution, a sodium chloride solution, or a potassium chloride solution, further preferably the pH of the buffer solution is 7 to 9, preferably 7.05 to 8.5, and more preferably 8.

Further, the mobile phase A comprises 10-50 mmol/L of a tris (hydroxymethyl) aminomethane solution and 5-20 mmol/L of a sodium chloride solution, and preferably the mobile phase A comprises 20mmol/L of a tris (hydroxymethyl) aminomethane solution and 10mmol/L of a sodium chloride solution; the mobile phase B comprises 10-50 mmol/L of tris (hydroxymethyl) aminomethane solution and 100-800 mmol/L of sodium chloride solution, and preferably the mobile phase B comprises 20mmol/L of tris (hydroxymethyl) aminomethane solution and 800mmol/L of sodium chloride solution.

Further, the gradient elution process includes: the volume ratio of the mobile phase A in the initial mobile phase is 20-100%, the volume ratio of the mobile phase A is reduced within 10-50% in 2-7 min, and the volume ratio of the mobile phase A is increased within 10-50% in 7-30 min; the preferred gradient elution procedure includes: the volume ratio of the mobile phase A is reduced from 100% to 15-25% in 0-3 min, from 15-25% to 5-15% in 3-5 min, the volume ratio of the mobile phase A is kept unchanged in 5-15% in 5-7 min, from 5-15% to 45-55% in 7-7.5 min, and the volume ratio of the mobile phase A is kept unchanged in 45-55% in 7.5-15 min.

Further, the flow rate of the mobile phase is 0.1-1 mL/min, preferably 0.4mL/min; the sample injection amount is preferably 1 to 50. Mu.L, more preferably 5. Mu.L; the column temperature is preferably 20 to 40 ℃, more preferably 35 ℃.

Further, the pretreatment of the osteopontin-containing object to be tested is performed by using trichloroacetic acid solution before HPLC separation of the osteopontin-containing object to be tested, so as to obtain a pretreated liquid, wherein the pH value of the pretreated liquid is preferably the same as that of the buffer solution, and the mass concentration of the trichloroacetic acid solution is preferably 40-70 wt%, preferably 55-65 wt%, more preferably 60wt%.

According to another aspect of the present invention, there is provided an analysis method of osteopontin, the analysis method including: and detecting the content of the osteopontin in the osteopontin sample, wherein the osteopontin sample is obtained by the separation method.

Further, the detection is ultraviolet detection, and the wavelength of the ultraviolet detection is 200 to 300nm, preferably 214nm.

By applying the technical scheme, the packing material of the strong anion exchange chromatographic column has strong cations with opposite charges to OPN, so that the packing material can be combined with OPN (with 28 phosphoryl groups) with negative charges in an object to be tested containing osteopontin, so that the OPN is reserved on the strong anion exchange chromatographic column, and the other proteins have weaker binding force with the strong anion exchange chromatographic column and are easy to be eluted. The protein with weak binding force with the strong anion exchange chromatographic column is eluted firstly by the elution of the mobile phase in HPLC separation, so that the OPN is eluted finally, thereby not only effectively retaining two forms of OPN (full-length OPN containing 262 amino acid residues and having the molecular weight of 33.9kDa and N-terminal fragment containing 1-150 amino acid residues and having the molecular weight of 19.8 kDa) at the same peak-out time, but also achieving the purpose of effectively separating other impurities. On the basis, the content of the osteopontin in the separated osteopontin sample is detected and analyzed. Compared with the analysis method for analyzing the osteopontin peptide fragments by adopting mass spectrometry in the prior art, the separation method of the osteopontin in the application ensures that the principle of retention of each component in the to-be-tested object containing the osteopontin on a chromatographic column is obvious in difference, and the method has the advantages of excellent selectivity to OPN, high sensitivity, better separation capability, simplicity and rapidness in operation, lower cost and more accurate analysis of the osteopontin.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 shows a schematic diagram of the chromatographic peak positions of OPN under the gradient elution conditions of example 1 after OPN addition to a base milk powder;

fig. 2 shows a high performance liquid chromatogram of the OPN test solution of example 1;

FIG. 3 shows a standard graph of an OPN standard sample at 214nm;

FIG. 4 shows a standard graph of OPN standard samples at 280nm;

fig. 5 shows a high performance liquid chromatogram of the OPN test solution of example 4; and

fig. 6 shows a high performance liquid chromatogram of the OPN test solution in comparative example 1 and a partial enlarged view thereof.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

As analyzed by the background technology, the method for analyzing the osteopontin in the prior art has the problems of complex operation, high cost and inaccurate analysis result, and the invention provides a method for separating the osteopontin and an analysis method for separating the osteopontin.

In an exemplary embodiment of the present application, there is provided a method for isolating osteopontin, the method comprising: and carrying out HPLC (high performance liquid chromatography) separation on an object to be tested containing osteopontin by adopting a strong anion exchange chromatographic column to obtain an osteopontin sample, wherein a filling material in the strong anion exchange chromatographic column comprises surface porous silica gel bonded with trimethyl aminopropyl or microporous ethyl vinyl benzene crosslinked 55% divinylbenzene polymer bonded with trimethyl aminopropyl.

The packing material of the strong anion exchange chromatographic column used in the invention has strong cations with opposite charges to OPN, so that the packing material can be combined with OPN (with 28 phosphoryl groups) with negative charges in an object to be tested containing osteopontin, so that the OPN is reserved on the strong anion exchange chromatographic column, and the other proteins have weak binding force with the strong anion exchange chromatographic column and are easy to be eluted. The protein with weak binding force with the strong anion exchange chromatographic column is eluted firstly by the elution of the mobile phase in HPLC separation, so that the OPN is eluted finally, thereby not only effectively retaining two forms of OPN (full-length OPN containing 262 amino acid residues and having the molecular weight of 33.9kDa and N-terminal fragment containing 1-150 amino acid residues and having the molecular weight of 19.8 kDa) at the same peak-out time, but also achieving the purpose of effectively separating other impurities. On the basis, the content of the osteopontin in the separated osteopontin sample is detected and analyzed. Compared with the analysis method for analyzing the osteopontin peptide fragments by adopting mass spectrometry in the prior art, the separation method of the osteopontin in the application ensures that the principle of retention of each component in the to-be-tested object containing the osteopontin on a chromatographic column is obvious in difference, and the method has the advantages of excellent selectivity to OPN, high sensitivity, better separation capability, simplicity and rapidness in operation, lower cost and more accurate analysis of the osteopontin.

The separation method of the osteopontin can be used as an important auxiliary means for quality control of products such as infant formula milk powder, prepared milk powder, whey protein raw materials and the like.

The specification parameters of the strong anion exchange chromatographic column, such as length, inner diameter, and particle size of the packing material, affect the HPLC separation of the object to be tested containing osteopontin, and in order to further improve the efficiency of the strong anion exchange chromatographic column in the HPLC separation, the specification of the strong anion exchange chromatographic column is preferably as follows: the length is 50-250 mm, the inner diameter is 2-30 mm, the particle size of the filling material is 2-20 mu m, and the specification of the preferable strong anion exchange chromatographic column is as follows: the length is 50-150 mm, the inner diameter is 2-10 mm, the particle size of the filling material is 2-10 mu m, and the specification of the strong anion exchange chromatographic column is more preferable: the length is 100mm, the inner diameter is 4.6mm, and the particle size of the filling material is 5 mu m.

In order to further enhance the separation effect of the HPLC separation method on osteopontin, the above strong anion exchange column is preferably Waters Protein-Pak Hi Res Q or Thermo Fisher Scientific ProPac WAX-10. And in actual use, waters Protein-Pak Hi Res Q is more preferably used.

In one embodiment of the present application, HPLC separation is performed using a gradient elution method, wherein the gradient eluted mobile phase comprises mobile phase a and mobile phase B; preferably, the mobile phase a and the mobile phase B are each independently a buffer solution selected from the group consisting of a tris (hydroxymethyl) aminomethane solution, a tris (hydroxymethyl) aminomethane hydrochloride solution, a tromethamine solution, a dibasic phosphate solution, a monobasic phosphate solution, or a combination of any one or more of them with a sodium chloride solution, a potassium chloride solution, a calcium chloride solution, a magnesium chloride solution, or a lithium chloride solution, more preferably the buffer solution is a mixture of a tris (hydroxymethyl) aminomethane solution and a lithium chloride solution, a sodium chloride solution, or a potassium chloride solution, further preferably the pH of the buffer solution is 7 to 9, preferably 7.05 to 8.5, and more preferably 8. Wherein the chloride ion is used as strong eluting ion, and the elution time and degree of the osteopontin and other impurity proteins are controlled by adjusting the content of the chloride ion in the mobile phase.

Through a large number of experiments, the application optimizes some chromatographic conditions including the specific mobile phase, gradient conditions and pH value range of the buffer solution, so that the interference impurities can be more effectively removed from the to-be-tested object containing the osteopontin, and the test sensitivity of the osteopontin sample is further improved. Preferably, the pH of the above buffer solution may be adjusted with a sodium hydroxide solution or a hydrochloric acid solution, and the concentration of the solution for adjusting the pH is a concentration conventional in the art, preferably the concentration of the sodium hydroxide solution is 1 to 3mol/L, and preferably the concentration of the hydrochloric acid solution is 1 to 10wt%.

In one embodiment of the present application, the mobile phase A comprises 10-50 mmol/L of a tris (hydroxymethyl) aminomethane solution and 5-20 mmol/L of a sodium chloride solution, preferably the mobile phase A comprises 20mmol/L of a tris (hydroxymethyl) aminomethane solution and 10mmol/L of a sodium chloride solution; the mobile phase B comprises 10-50 mmol/L of tris (hydroxymethyl) aminomethane solution and 100-800 mmol/L of sodium chloride solution, and preferably the mobile phase B comprises 20mmol/L of tris (hydroxymethyl) aminomethane solution and 800mmol/L of sodium chloride solution.

The chloride ions in the sodium chloride solution are used as strong eluting ions, and the concentration of the chloride ions in the mobile phase A and the mobile phase B is regulated to control the coordination relation between the mobile phase A and the mobile phase B in the eluting process, so that the osteopontin is effectively separated from other proteins in the milk powder as much as possible, the separation time is shortened, and the purified osteopontin sample meets the sensitivity requirement of ultraviolet detection.

In one embodiment of the present application, the gradient elution process includes: the volume ratio of the mobile phase A in the initial mobile phase is 20-100%, the volume ratio of the mobile phase A is reduced within 10-50% in 2-7 min, and the volume ratio of the mobile phase A is increased within 10-50% in 7-30 min; the preferred gradient elution procedure includes: the volume ratio of the mobile phase A is reduced from 100% to 15-25% in 0-3 min, from 15-25% to 5-15% in 3-5 min, the volume ratio of the mobile phase A is kept unchanged in 5-15% in 5-7 min, from 5-15% to 45-55% in 7-7.5 min, and the volume ratio of the mobile phase A is kept unchanged in 45-55% in 7.5-15 min.

The gradient elution process is more favorable for achieving good separation of all components in the to-be-tested object containing osteopontin according to respective proper capacity factors. Specifically, in the gradient elution process, firstly the mobile phase A occupies a main proportion, the impurity protein adsorbed on the chromatographic column is eluted by utilizing the characteristic of relatively low anion concentration, and then the osteopontin is eluted along with the increase of the mobile phase B, so that the good separation of the osteopontin is realized.

In order to further improve the efficiency and separation effect of the HPLC separation, the flow rate of the mobile phase is preferably 0.1 to 1mL/min, preferably 0.4mL/min; the sample injection amount is preferably 1 to 50. Mu.L, more preferably 5. Mu.L; the column temperature is preferably 20 to 40 ℃, more preferably 35 ℃.

In one embodiment of the present application, the pretreatment of the test substance containing osteopontin with trichloroacetic acid solution is performed before the HPLC separation of the test substance containing osteopontin, so as to obtain a pretreated liquid, preferably the pH value of the pretreated liquid is the same as the pH value of the buffer solution, preferably the mass concentration of the trichloroacetic acid solution is 40-70 wt%, preferably 55-65 wt%, more preferably 60wt%.

The trichloroacetic acid solution can precipitate casein in the to-be-tested object containing osteopontin, and can reduce the pH value of the to-be-tested object containing osteopontin to about 1, under the condition that the casein is precipitated due to destabilization, and the phosphate groups of OPN are blocked and remain in the solution under the pH condition, the precipitated casein is filtered and removed to obtain pretreated liquid containing OPN, the pH value of the pretreated liquid is regulated to be the same as fluidity A (the solution for regulating the pH value of the pretreated liquid is sodium hydroxide solution with the concentration of 1-3 mol/L in the prior art), and the pretreated liquid is subjected to HPLC separation, so that the damage of the too low pH value to a strong anion exchange chromatographic column is avoided, the ionic strength of the pretreated liquid and the ionic strength of a mobile phase are consistent, and the solvent effect is avoided. In addition, if the concentration of the trichloroacetic acid solution is too high, the pH value of the pretreated liquid is too low, precipitated casein is partially redissolved to cause turbidity of the pretreated liquid, and if the concentration of the trichloroacetic acid solution is too low, casein precipitation is incomplete, OPN cannot be completely neutral, so that casein is combined with OPN, and the measurement result is low, therefore, the treatment of the to-be-tested object containing osteopontin by the trichloroacetic acid solution with the mass concentration is more beneficial to obtaining an osteopontin sample with higher purity in subsequent HPLC separation.

In another exemplary embodiment of the present application, there is provided an assay for osteopontin comprising: and detecting the content of the osteopontin in the osteopontin sample, wherein the osteopontin sample is obtained by the separation method.

The invention utilizes the special filling material of the strong anion exchange chromatographic column to achieve the purpose of effectively separating OPN from other impurities. Compared with OPN obtained by HPLC separation in the prior art, the OPN in the osteopontin sample obtained by the method has higher purity, and the interference effect of impurities on the OPN is weaker, so that the method can directly and quantitatively test the OPN in the osteopontin sample by a conventional detection method and even an ultraviolet detection method of the OPN in the prior art, and has higher accuracy and sensitivity, simple test operation and lower cost.

In some embodiments of the present application, the above-described detection is an ultraviolet detection that is simpler, faster, and less costly than conventional methods of testing OPN in the prior art, such as mass spectrometry. And the wavelength of ultraviolet detection is preferably 200 to 300nm, preferably 214nm, in order to further contribute to the improvement of sensitivity of the detection of osteopontin by ultraviolet.

In order to minimize the influence of the solvent required for dissolving the OPN standard on the standard curve, it is preferable to dissolve the OPN standard using ultrapure water or mobile phase a, and more preferable to dissolve the OPN standard using mobile phase a.

The advantageous effects of the present application will be described below with reference to specific examples and comparative examples.

Example 1

1) Instrument and conditions:

high performance liquid chromatograph: ACQUITY Arc Bio UHPLC system, uv detector;

chromatographic column: waters Protein-Pak Hi Res Q, filler particle size 5 μm, 4.6X100 mm, P/N186004931;

column temperature: 35 ℃;

wavelength: 214nm;

needle washing liquid: 0.1% tfa in water;

sample injection amount: 5. Mu.L;

mobile phase: a: an aqueous solution containing 20mmol/L Tris (Tris solution) and 10mmol/L NaCl at pH=8.0; b: pH=8.0 aqueous solution containing 20mmol/L Tris and 800mmol/L NaCl.

Gradient elution procedure:

TABLE 1

	Time(min)	Flow rate mL/min	％A	％B
					1	0	0.4	50	50
2	3	0.4	20	80
					3	5	0.4	10	90
4	7	0.4	10	90
					5	7.5	0.4	50	50
6	15	0.4	50	50

OPN is added into basic milk powder, the chromatographic peak position of OPN under the gradient elution condition is detected, the detection result is shown in figure 1, the lower curve in figure 1 is a chromatogram of a 5g milk powder sample without OPN, the upper curve is a chromatogram of a 5g milk powder sample with OPN standard added, and a new peak appears in about 4-6 min, so that the peak emergence time of OPN is proved to be about 4-6 min.

2) Preparing an OPN standard sample:

stock solution: 0.1052g (accurate to 0.01 mg) of OPN standard (Sigma-Aldrich O3514, purity 95%) was accurately weighed, dissolved in water, and the volume was set to 10mL to prepare a stock solution with a concentration of 10mg/mL, and the stock solution was kept in a refrigerator at-20 ℃.

Intermediate liquid: accurately transferring 1mL of OPN standard stock solution, fixing the volume to 10mL by using a mobile phase A, and uniformly mixing to prepare OPN standard intermediate solution (prepared in situ) with the concentration of 1 mg/mL.

Standard working solution: accurately sucking 5 mu L,20 mu L, 100 mu L and 200 mu L to 100mL volumetric flasks from OPN standard intermediate liquid respectively, and adding mobile phase A to fix volume to scale respectively to obtain standard samples, wherein the concentrations of the standard samples are as follows: 0.05mg/L, 0.2mg/L, 1.0mg/L, 2.0mg/L (prepared just before use).

3) Preparation of osteopontin samples:

5g of milk powder was taken, 30mL of warm water at 40℃was added, and 5 minutes of 10mL of 60wt% trichloroacetic acid solution was vortexed, shaken well, and filtered through filter paper. Taking 10mL of filtrate, accurately adjusting the pH to 8.0 by using a sodium hydroxide solution with the concentration of 3mol/L, fixing the volume to 25mL, shaking uniformly, filtering to obtain a pretreated liquid, injecting the pretreated liquid into the high performance liquid chromatograph in the step 1), performing gradient elution and ultraviolet detection according to a gradient elution program shown in the table 1, and obtaining a high performance liquid chromatogram of the OPN to-be-detected liquid shown in figure 2.

4) And (3) manufacturing a standard curve:

and respectively injecting the OPN standard samples into a high performance liquid chromatograph to obtain corresponding peak areas, and preparing a standard curve by taking the concentration of OPN in the OPN standard samples as an abscissa and the corresponding peak areas (or peak heights) as an ordinate, wherein as shown in fig. 3, the corresponding standard curve equation is Y= 1150000X-3700, and the fitting degree is 0.999836.

5) And (3) data processing:

the content of OPN in the sample to be measured is calculated according to the concentration X, the unit is mg/100g, and the content is calculated according to the following formula:

wherein: c-OPN concentration of the solution to be measured in milligrams per liter (mg/L);

v-the constant volume, mL, of the sample to be tested;

m-mass of the sample to be measured g;

n-dilution of the sample to be measured;

the result of the calculation retains two significant digits.

6) The method is confirmed by referring to GB/T27417-2017 'inspection and verification guidelines for qualification analysis methods', and all standard requirements are met.

The OPN content in the milk powder is 98.2mg/100g according to the peak area and standard curve calculation obtained by the detection, which is basically the same as the theoretical addition content of OPN in the milk powder composition specification of 100mg/100 g.

Example 2

The test subjects were supplemented with OPN in the base formula without OPN and the OPN content was 0.1mg/L, example 2 was the same test procedure as example 1.

Example 3

The test subjects were the same as in example 2.

Example 3 differs from the test procedure of example 1 in that the chromatographic column was chosen as Thermo Fisher Scientific ProPac WAX-10, P/N:054999; the sample injection amount is 10 mu L; the gradient procedure is:

TABLE 2

	Time(min)	Flow rate mL/min	％A	％B
					1	0	1.0	50	50
2	7	1.0	10	90
					3	10	1.0	10	90
4	10.5	1.0	50	50
					5	20	1.0	50	50

Example 4

The test subjects were the same as in example 2.

The test procedure of example 4 is different from that of example 1 in that the detection wavelength is 280nm, wherein, the concentration of OPN in the OPN standard sample is taken as the abscissa, the corresponding peak area (or peak height) is taken as the ordinate, a standard curve is prepared, the corresponding standard curve equation is y=23800X-217, the fitting degree is 0.999439, as shown in fig. 4, and the obtained high performance liquid chromatogram of the OPN test solution is shown in fig. 5.

It should be noted that, the standard curve of this embodiment is also established based on the detection wavelength, so as to obtain a corresponding standard curve, and then the data processing is performed according to the above data processing manner to convert the content of osteopontin in the sample to be detected.

Example 5

The test subjects were the same as in example 2.

Example 5 differs from the test procedure of example 1 in that,

mobile phase a was an aqueous solution containing 10mmol/L Tris (Tris solution) and 5mmol/L NaCl at ph=7.05; b: pH=7.05 aqueous solution containing 10mmol/L Tris and 100mmol/L NaCl.

It should be noted that, the standard curve of this embodiment is also established based on the mobile phase, so as to obtain a corresponding standard curve, and then the data processing is performed according to the above data processing manner to convert the content of osteopontin in the sample to be measured.

Example 6

The test subjects were the same as in example 2.

Example 6 differs from the test procedure of example 1 in that,

mobile phase a was an aqueous solution containing 50mmol/L Tris (Tris solution) and 20mmol/L NaCl at ph=8.5; b: pH=8.5 aqueous solution containing 50mmol/L Tris and 600mmol/L NaCl.

Example 7

The test subjects were the same as in example 2.

Example 7 differs from the test procedure of example 1 in that,

mobile phase a was an aqueous solution containing 30mmol/L Tris (Tris solution) and 15mmol/L NaCl at ph=7.0; b: pH=7.0 aqueous solution containing 30mmol/L Tris and 400mmol/L NaCl.

Example 8

The test subjects were the same as in example 2.

Example 8 differs from the test procedure of example 1 in that,

mobile phase a was an aqueous solution containing 40mmol/L Tris (Tris solution) and 10mmol/L NaCl at ph=9.0; b: pH=9.0 aqueous solution containing 50mmol/L Tris and 500mmol/L NaCl.

Example 9

The test subjects were the same as in example 2.

Example 9 differs from the test procedure of example 1 in that the mass concentration of the trichloroacetic acid solution is 55% by weight.

Example 10

The test subjects were the same as in example 2.

Example 10 differs from the test procedure of example 1 in that the mass concentration of the trichloroacetic acid solution is 65% by weight.

Example 11

The test subjects were the same as in example 2.

Example 11 differs from the test procedure of example 1 in that the mass concentration of the trichloroacetic acid solution is 70% by weight.

Example 12

The test subjects were the same as in example 2.

Example 12 differs from the test procedure of example 1 in that the mass concentration of the trichloroacetic acid solution is 40% by weight.

Comparative example 1

The test subjects were the same as in example 2.

Comparative example 1 differs from the test procedure of example 1 in that,

1) Instrument and conditions:

high performance liquid chromatograph: ACQUITY UPLC H-Class Bio ultra-high performance liquid chromatography with PDA diode array detector;

chromatographic column: protein BEH 300C 4 Column,1.7 μm, 2.1X100 mm, P/N:186004496;

column temperature: 40 ℃;

wavelength: 214. 280nm;

needle washing liquid: 0.1% tfa in water;

sample injection amount: 5. Mu.L;

mobile phase: c:0.1% (V/V) TFA in ACN; d:0.1% (V/V) TFA in H ₂ O。

Gradient elution procedure:

TABLE 3 Table 3

	Time(min)	Flow rate mL/min	％C	％D
					1	0	0.4	10	90
2	0.5	0.4	10	90
					3	4.5	0.4	35	65
4	5.0	0.4	40	60
					5	5.5	0.4	90	10
6	6.5	0.4	10	90
					7	10.0	0.4	10	90

The high performance liquid chromatogram and the partial enlarged view of the OPN test solution in comparative example 1 are shown in fig. 6.

The peak areas of OPN under the same sample volume conditions of the ultraviolet detection wavelengths and concentrations of 0.1mg/mL in examples 2 to 12 and comparative example 1 are shown in Table 4.

TABLE 4 Table 4

As can be seen from the control of example 2 and example 3, the Thermo Fisher Scientific ProPac WAX-10 column of example 3 was less effective in separating OPN from the solution to be tested than the Waters Protein-Pak Hi Res Q of example 2 under the same gradient elution conditions. From the comparison of example 2 with example 4, it can be seen that the responsivity of the OPN itself is low at 280nm, but the standard deviation of the established standard curve is still within a reliable range, probably less sensitive than at 214nm. As can be seen from the controls of examples 5 to 8, the separation degree of osteopontin and other impurity proteins can be controlled by controlling the concentration of chloride ions and pH value in the eluent, so that the eluent of example 6 and example 8 has better separation effect on OPN than the eluent of examples 5 and 7. As can be seen from the comparison of examples 12 and 2 and examples 9 to 11, in examples 2 and examples 9 to 11, the effect of removing casein and the like in the solution to be tested is relatively good due to the high mass concentration of the trichloroacetic acid solution, and turbidity of the pretreated solution caused by partial re-dissolution of the precipitated casein due to too low pH value of the pretreated solution is avoided, so that the separation effect of OPN in the solution to be tested is better, but the concentration of the trichloroacetic acid solution in example 12 is too low to cause incomplete casein precipitation and the OPN is not completely neutral, so that casein is combined with the OPN, the separation effect of OPN in the solution to be tested is poor, and the measurement result is lower.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of isolating osteopontin, the method comprising: performing HPLC separation on the to-be-tested object containing osteopontin by adopting a chromatographic column to obtain an osteopontin sample,

wherein the chromatographic column is Waters Protein-Pak Hi Res Q or Thermo Fisher Scientific ProPacWAX-10;

performing HPLC separation by adopting a gradient elution method, wherein the mobile phase of the gradient elution comprises a mobile phase A and a mobile phase B; the mobile phase A and the mobile phase B are respectively and independently buffer solutions, wherein the buffer solutions are selected from any one or more of a tris (hydroxymethyl) aminomethane solution, a tris (hydroxymethyl) aminomethane hydrochloride solution, a tromethamine solution, a dibasic phosphate solution and a monobasic phosphate solution, or a combination of any one or more of the buffer solutions and a sodium chloride solution, a potassium chloride solution, a calcium chloride solution, a magnesium chloride solution or a lithium chloride solution;

preprocessing the osteopontin-containing to-be-tested object by adopting trichloroacetic acid solution before carrying out HPLC separation on the osteopontin-containing to-be-tested object to obtain preprocessed solution;

the gradient elution process comprises the following steps: the volume ratio of the mobile phase A is reduced from 100% to 15-25% in 0-3 min, from 15-25% to 5-15% in 3-5 min, the volume ratio of the mobile phase A is kept unchanged in 5-15% in 5-7 min, the volume ratio of the mobile phase A is increased from 5-15% to 45-55% in 7-7.5 min, and the volume ratio of the mobile phase A is kept unchanged in 45-55% in 7.5-15 min.

2. The separation method according to claim 1, wherein the specification of the chromatographic column is: the length is 50-250 mm, the inner diameter is 2-30 mm, and the particle size of the filling material is 2-20 mu m.

3. The separation method according to claim 2, wherein the specification of the chromatographic column is: the length is 50-150 mm, the inner diameter is 2-10 mm, and the particle size of the filling material is 2-10 mu m.

4. A separation method according to claim 3, wherein the chromatographic column has a specification of: the length is 100mm, the inner diameter is 4.6mm, and the particle size of the filling material is 5 mu m.

5. The separation method according to any one of claims 1 to 4, wherein the buffer solution is a mixture of a tris (hydroxymethyl) aminomethane solution and a lithium chloride solution, a sodium chloride solution or a potassium chloride solution.

6. The method according to claim 5, wherein the buffer solution has a pH of 7 to 9.

7. The method according to claim 6, wherein the buffer solution has a pH of 7.05 to 8.5.

8. The method according to claim 7, wherein the buffer solution has a pH of 8.

9. The separation method according to claim 1, wherein the mobile phase a comprises 10 to 50mmol/L of a tris solution and 5 to 20mmol/L of a sodium chloride solution.

10. The separation method according to claim 9, wherein the mobile phase a comprises a solution of tris and 10mmol/L sodium chloride; the mobile phase B comprises 10-50 mmol/L of tris (hydroxymethyl) aminomethane solution and 100-800 mmol/L of sodium chloride solution.

11. The separation method according to claim 10, wherein the mobile phase B comprises a solution of tris and 800mmol/L sodium chloride.

12. The separation method according to claim 1, wherein the flow rate of the mobile phase is 0.1 to 1mL/min.

13. The separation method according to claim 12, wherein the flow rate of the mobile phase is 0.4mL/min.

14. The separation method according to claim 1, wherein the amount of the sample introduced is 1 to 50. Mu.L.

15. The method of claim 14, wherein the sample is introduced at a volume of 5 μl.

16. The separation method according to claim 1, wherein the column temperature is 20 to 40 ℃.

17. The separation process of claim 16, wherein the column temperature is 35 ℃.

18. The separation method according to claim 1, wherein the pH of the pretreated liquid is the same as the pH of the buffer solution.

19. The separation method according to claim 1, wherein the trichloroacetic acid solution has a mass concentration of 40 to 70wt%.

20. The separation method according to claim 19, wherein the trichloroacetic acid solution has a mass concentration of 55 to 65wt%.

21. The separation method according to claim 20, wherein the trichloroacetic acid solution has a mass concentration of 60wt%.

22. A method of analyzing osteopontin, the method comprising: detecting the content of osteopontin in a osteopontin sample, which is obtained by the separation method according to any one of claims 1 to 21.

23. The method according to claim 22, wherein the detection is ultraviolet detection having a wavelength of 200 to 300nm.

24. The method of claim 23, wherein the ultraviolet detection has a wavelength of 214nm.