CN114113426A

CN114113426A - Method for detecting phospholipid in hemoglobin oxygen carrier

Info

Publication number: CN114113426A
Application number: CN202111616768.8A
Authority: CN
Inventors: 李建军; 严坤平; 谢于斗; 杨鹏飞; 贺迎娣; 柏晓丽; 李鹏云; 卓丹丹; 常娟娟; 陈超
Original assignee: Xi'an Blood Oxygen Biotechnology Co ltd
Current assignee: Xi'an Blood Oxygen Biotechnology Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-03-01
Anticipated expiration: 2041-12-27
Also published as: CN114113426B

Abstract

The invention relates to the technical field of phospholipid detection, and particularly relates to a method for detecting phospholipid in a hemoglobin oxygen carrier. The phospholipid in a sample to be tested is extracted and then is subjected to alkaline saponification, reaction products of ethanolamine and serine and p-toluenesulfonyl chloride (PTSC) are subjected to derivatization reaction, and the derivatized products are separated by reversed-phase high performance liquid chromatography and then are detected at 240 nm.

Description

Method for detecting phospholipid in hemoglobin oxygen carrier

Technical Field

The invention relates to the technical field of phospholipid detection, and particularly relates to a method for detecting phospholipid in a hemoglobin oxygen carrier.

Background

Phospholipids have a major impact on human health and development of disease. Accurate and efficient measurement of in vivo phospholipid is helpful for understanding the metabolism condition of phospholipid in vivo and the role of phospholipid in life activity, thereby effectively diagnosing and preventing and treating diseases.

The hemoglobin oxygen carrier is a blood substitute prepared by using animal or human red blood cells as raw materials and chemically modifying separated and purified hemoglobin by adopting the modes of polymerization, crosslinking, coupling and the like. The erythrocyte membrane phospholipids are mainly Phosphatidylethanolamine (PE) and Phosphatidylserine (PS). During the purification of hemoglobin, the release of hemoglobin causes the hemoglobin purified product to also contain phospholipids, which have obvious toxicity and can cause blood coagulation, and PS is an activator of prothrombin, which can promote the agglutination of erythrocytes and adhere to vascular endothelial cells to form thrombus, so that the content of PE and PS must be controlled.

The phospholipid is detected mainly by normal phase High Performance Liquid Chromatography (HPLC). Normal phase high performance liquid chromatography (NPLC) can achieve better separation between phospholipid classes, with weaker polarity being eluted first, but the mobile phase often used in NPLC methods is of weak polarity, and bubbles are often generated when mixed with solutions of relatively stronger polarity, thus causing problems of drift of retention time and uneven baseline, and its use of organic flow in large quantities is relatively environmentally unfriendly. Due to the lack of chromophore in the molecular structure of phospholipid, detection is usually performed in the range of 200-214nm, the absorbance coefficient of phospholipid in this wavelength range is small, so that the detection sensitivity is low, and some common solvents generally absorb light strongly in this wavelength range, thus interfering with phospholipid detection. Mass Spectrometry (MS) is the most sensitive and specific detection technique, can be used simultaneously for phospholipid separation, characterization and quantification in combination with HPLC, and is commonly used in phospholipid omics analysis with the disadvantage that detection is relatively expensive and is not suitable for routine analysis.

Disclosure of Invention

The present invention is made to solve the above problems and an object of the present invention is to provide a method for detecting phospholipid in hemoglobin-based oxygen carriers.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting phospholipid in hemoglobin oxygen carrier comprises the following steps:

1) extracting phospholipids to be detected from a test sample, said phospholipids comprising phosphatidylethanolamine and phosphatidylserine;

2) using strong base solution to carry out saponification hydrolysis on phospholipid in a test sample to obtain a hydrolysate, wherein the hydrolysate comprises ethanolamine and serine;

3) performing derivatization reaction on a tosyl chloride solution and a hydrolysate, introducing a chromogenic group into a molecular structure of the hydrolysate to obtain a sample derivatization product, passing through a water system filter membrane, and performing sample injection detection by chromatography;

4) and (3) processing the phospholipid standard solution in the steps 2) -3) to obtain a standard derivative product, detecting the standard derivative product and a sample derivative product at 240nm by using reverse phase chromatography, mapping the chromatographic peak area and correlating the amount of phospholipid in the phospholipid standard solution to obtain a working curve equation, and substituting the peak area of the sample derivative product into the working curve equation to obtain the amount of different phospholipids in the sample.

Further, in S1, the extracting the phospholipid to be detected specifically includes:

11) adding chloroform with the same volume to a sample to be tested, shaking and mixing uniformly, adding methanol with the volume 2 times that of the sample, shaking and mixing uniformly, continuing adding chloroform and normal saline with the same volume as the sample, shaking and standing, and extracting a supernatant for later use after layering;

12) repeating the supernatant twice under the same conditions of the step 11), combining the lower clear liquid, concentrating the lower clear liquid to 1/10 of the sample volume, and continuously drying by blowing to obtain the phospholipid to be detected.

Furthermore, in S2, the strong base is NaOH or KOH solution, the concentration is 0.05-0.5 mol/L, and the volume is 0.03-0.06 times of the volume of the sample to be tested.

Further, in S2, the saponification hydrolysis is performed for 80-150 min at 70-90 ℃.

Further, in S2, after completion of saponification hydrolysis, HCl or H is used₂SO₄Neutralizing excess strong base, HCl or H, in solution₂SO₄The concentration of the solution is 0.5-3 mol/L.

Furthermore, in S3, Na with a concentration of 0.05-0.5 mol/L is added in the derivatization reaction₂CO₃-NaHCO₃Buffer solution, the volume of which is 0.06-0.16 times of the volume of the sample to be tested.

Furthermore, in S3, the volume of the solution of tosyl chloride is 0.02-0.1 times of the volume of the sample to be tested, the concentration is 1.0-5.0 mg/ml, and the solution is prepared by acetonitrile solution.

Furthermore, in S3, the reaction conditions are 15-65 ℃ and 3-10 min.

Further, in S3, the pore diameter of the aqueous filter was 0.22 μm or 0.45. mu.m.

The derivatization principle of the invention is as follows: after the phospholipid such as PE and PS is saponified and hydrolyzed, the molecular structures of the ethanolamine and serine products do not have chromogenic groups, but the product belongs to primary amine, and the product is easy to have Hisberg reaction with p-toluenesulfonyl chloride, and the chromogenic groups are introduced into the structures, so that the ultraviolet absorption detection is realized. The specific reaction principle is shown in figure 1. The maximum absorption wavelength of the reaction product of derivatization was 240nm and the maximum absorption wavelength of p-toluenesulfonyl chloride was 230nm, as shown in FIG. 2, and thus the detection wavelength was set to 240 nm.

The invention has the beneficial effects that:

the method of the invention saponifies the extracted phospholipid under the alkaline condition to generate micromolecules (ethanolamine, serine and the like), then the micromolecules are chemically derived from p-toluenesulfonyl chloride, and the derivatives are detected at 240nm after being separated by reversed phase chromatography. A large amount of organic solvents which are not friendly to the environment when the normal phase chromatography is used for measuring the phospholipid are abandoned, the conventional reversed phase liquid chromatography mode with high separation degree is used, and the problems that the normal phase chromatography lacks chromophores and has low sensitivity are solved, so that the method can be used for detecting the conventional items in a laboratory.

Drawings

FIG. 1 shows the reaction principle of ethanolamine, serine and p-toluenesulfonyl chloride.

FIG. 2 shows UV absorption spectra of p-toluenesulfonyl chloride and derivatives thereof.

FIG. 3 is a chromatogram for sample measurement, in which a: a blood substitute; b: whole pig blood; c: pig serum.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Instruments and reagents used in the examples

The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph (Agilent corporation, usa); Milli-Q ultra-pure water devices (Millipore, USA); MTN-2800D nitrogen blower (Beijing Hua Reubo Tech manufacturing Ltd.); MS3 basic model vortex mixer (IKA, germany). BT 125D electronic balance (Sartorius, germany); constant temperature water bath pot and the like

Reagent: acetonitrile, methanol (chromatographically pure, Sigma company, usa), KOH, concentrated sulfuric acid, chloroform (analytically pure, chemical reagents ltd, national drug group); ultrapure water (Milli-Q ultrapure water preparation).

Standard substance: phosphatidylethanolamine, phosphatidylserine standard (sigma usa).

Example 1

1) Sample information: pig whole blood, pig serum and blood substitute (glutaraldehyde polymerization hemoglobin solution, protein concentration: 115 g/L; pH: 7.45).

2) Extracting phospholipid: transferring 5ml of a sample into a 60ml separating funnel (a piston made of polytetrafluoroethylene), slowly adding 5ml of chloroform into the sample while shaking, adding 10ml of methanol, shaking to mix uniformly, continuously adding 5ml of chloroform and 5ml of normal saline into the separating funnel, shaking, standing for layering, and extracting the supernatant into a 50ml centrifuge tube. After extraction is finished, adding 5ml of chloroform into the sample again, repeating the above steps twice, and combining the supernate; and blowing the supernatant to about the residual 0.5ml by using a nitrogen blower, carefully transferring the supernatant into a 2ml centrifuge tube (rinsing a 50ml centrifuge tube by using a small amount of chloroform, transferring the rinsing solution into a 2ml centrifuge tube, repeating the steps twice), and continuously drying the supernatant for later use.

3) Saponification: adding 150 μ L of 1mol/L KOH solution into the centrifuge tube, reacting at 90 ℃ for 100min, and shaking while heating to fully react. After completion of saponification, 1mol/L sulfuric acid was added to adjust the neutrality.

4) Derivatization: to the saponified solution was added 700. mu.l of 0.1mol/L Na₂CO₃-NaHCO₃(pH 9.0) buffer, pH 9.0 was adjusted. Mu.l of 5.0mg/ml p-toluenesulfonyl chloride was added and the derivatization was carried out at 60 ℃ for 5 minutes, with shaking being noted during heating to ensure complete reaction. After the reaction is finished, the mixture is filtered through a 0.22 mu m water system filter membrane to be injected.

5) Liquid chromatography conditions: a chromatographic column: inertsil ODS-3(5 μm, 4.6X 250 mm); mobile phase A: 10mM ammonium acetate (pH 4.00); mobile phase B: chromatographically pure acetonitrile; elution gradient: isocratic elution with 19% solution B; temperature of the chromatographic column: 30 ℃; detection wavelength: 240 nm; flow rate: 1.0 ml/min; operating time: 20 min; sample introduction amount: 20 μ l.

To verify the effectiveness of the method of the invention, the following observations were made:

1. investigation of Linear relationships

Method validation was performed by determining the following parameters: linearity, precision, accuracy, detection limit, and quantitation limit. The method uses an external standard method for quantification, and a working curve equation is obtained by plotting the concentration of a phospholipid standard substance to the chromatographic peak area after saponification and derivatization. The relative standard deviation RSD of the samples is measured in parallel to evaluate the precision of the method; the recovery of the spiked sample was used to assess the accuracy of the method;

the method uses an external standard method for quantification, and a working curve equation is obtained by plotting the concentration of a phospholipid standard substance to the chromatographic peak area after saponification and derivatization. The linearity of the method is shown in Table 1, the linear range is 0.5-50 mug/mL, a good linear relation exists between the phospholipid concentration and the target peak area, and a correlation coefficient R²0.9927(PS) and 0.9995(PE), respectively.

The detection limit and the quantification limit were evaluated at 3-fold and 10-fold signal-to-noise ratio, respectively. As a result, the detection limit of PS was 0.2. mu.g/mL and the quantification limit was 0.5. mu.g/mL, as shown in Table 1; the detection limit of PE was 0.1. mu.g/mL, and the quantification limit was 0.3. mu.g/mL.

TABLE 1 calibration curves, detection limits and quantitation limits

2. Examination of degree of repetition and precision

In order to test the precision of the method, a pig serum sample is taken, the PS content and the PE content are determined under optimized experimental conditions and are repeated for six times, and the determined data and results are shown in table 2. The RSD of 6 times of parallel measurement is 3.7 percent and 4.4 percent respectively, which shows that the precision of the method meets the requirement.

Table 2 repeatability test results (n ═ 6)

3. Recovery and detection limit investigation

The method was used to determine the PS and PE in porcine whole blood, porcine serum and blood substitutes, and the results are shown in FIG. 3 and Table 3. As can be seen from FIG. 3, the method has stable baseline in chromatographic separation, symmetrical target peak pattern, and basically achieves baseline separation, and is suitable for analysis and determination of PS and PE in blood samples. Wherein the PS content and the PE content in the pig whole blood are 258.6 mug/mL and 466.7 mug/mL respectively; the PS content and the PE content in the pig serum are respectively 14.1 mug/mL and 31.2 mug/mL; no PS and PE were detected in the blood substitute. The recovery rate of the sample with different levels of standard addition is 76.0% -89.4%, which shows that the accuracy of the method meets the requirement.

TABLE 3 recovery and sensitivity test results

^aInitial concentration of phospholipids in the raw sample.

In conclusion, in the invention, the phospholipid in the test sample is preliminarily separated from some hydrophilic impurities in the sample through liquid-liquid extraction, and the phospholipid can be separated from some non-saponified hydrophobic components in the saponification process, so that matrix interference in the test sample is greatly reduced after two times of pre-separation, and the sensitivity and the accuracy of the method are improved. The verification result shows that the method can be used for accurately determining the PS and the PE in the blood sample

It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. A method for detecting phospholipids in hemoglobin oxygen carriers is characterized by comprising the following steps:

2. The method according to claim 1, wherein the step of extracting phospholipids to be detected in S1 specifically comprises:

3. The method according to claim 2, wherein the strong base in S2 is NaOH or KOH solution, and the concentration of the strong base is 0.05 to 0.5mol/L, and the volume of the strong base is 0.03 to 0.06 times the volume of the sample.

4. The method according to claim 3, wherein the saponification hydrolysis in S2 is performed at 70-90 ℃ for 80-150 min.

5. The method of claim 4, wherein the step of hydrolyzing S2 with HCl or H₂SO₄Neutralizing excess strong base, HCl or H, in solution₂SO₄The concentration of the solution is 0.5-3 mol/L.

6. The method of claim 5, wherein Na is added to the S3 in an amount of 0.05-0.5 mol/L in the derivatization reaction₂CO₃-NaHCO₃Buffer solution, the volume of which is 0.06-0.16 times of the volume of the sample to be tested.

7. The method according to claim 6, wherein the volume of the p-toluenesulfonyl chloride solution in S3 is 0.02 to 0.1 times the volume of the sample, and the concentration is 1.0 to 5.0mg/ml, and the solution is prepared as an acetonitrile solution.

8. The method of claim 7, wherein the reaction conditions in S3 are 15-65 ℃ for 3-10 min.

9. The method according to claim 8, wherein the pore size of the aqueous filter membrane in S3 is 0.22 μm or 0.45 μm.