A method for measuring hemoglobin content in hemoglobin oxygen carrier

Abstract

The present invention provides a method for measuring the hemoglobin content in a hemoglobin oxygen carrier, comprising the following steps: (1) preparing a plurality of hemoglobin standard working solutions of known concentrations and measuring their absorbances, establishing a standard working curve or a standard working curve equation of absorbance and concentration, wherein the hemoglobin standard working solution contains an acidic substance; (2) preparing a sample solution to be tested and measuring its absorbance, wherein the sample solution to be tested contains an acidic substance; (3) obtaining the hemoglobin content in the sample to be tested according to the absorbance of the sample solution to be tested obtained in step (2) and the standard working curve or the standard working curve equation obtained in step (1). The method of the present invention has accurate measurement results, simple operation, environmental friendliness, and low equipment requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of hemoglobin oxygen carriers, and in particular to a method for measuring the hemoglobin content in a hemoglobin oxygen carrier. Background Art

[0002] Hemoglobin (Hb) in the blood plays an important role in transporting oxygen, carbon dioxide and regulating the acid-base balance of the blood. The determination of hemoglobin is an important indicator for clinical tests such as blood and urine, and has been paid attention to in the field of clinical analysis. Hemoglobin-Based Oxygen Carriers (HBOCs) are an emerging blood substitute with oxygen-carrying function. They have the advantages of long-term storage, no need for matching, easy transportation, and low risk of infection. However, while they show good therapeutic effects, they also have toxic side effects such as oxidative stress, vasoconstriction, and nephrotoxicity. Some studies have prepared PolyHb-SOD-CAT-CA by compounding hemoglobin with superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) to solve the above problems. In order to further optimize the performance of such HBOCs products, it is necessary to measure the hemoglobin concentration in them.

[0003] However, it is still difficult to measure the content of Hb using the absorbance method in the prior art, because the wavelength showing the maximum absorption value is different depending on the state of being bound to oxygen and the state of being unbound. Therefore, it is difficult to accurately determine the amount of Hb even if the absorbance is simply measured. The prior art often stabilizes Hb by denaturation and then measures its absorbance. For example, the international standard HiCN method has been widely adopted. However, this method will produce cyanide-containing waste liquid that pollutes the environment.

[0004] Therefore, there is an urgent need for a method for measuring hemoglobin content that is simple to operate, environmentally friendly, and has accurate results. Summary of the invention

[0005] The purpose of the present invention is to provide a method for measuring hemoglobin content which is simple to operate, environmentally friendly and has accurate results.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] A method for measuring the hemoglobin content in a hemoglobin-based oxygen carrier comprises the following steps:

[0008] (1) preparing a plurality of hemoglobin standard working solutions of known concentrations and measuring their absorbances, and establishing a standard working curve or a standard working curve equation of absorbance versus concentration, wherein the hemoglobin standard working solutions contain an acidic substance;

[0009] (2) preparing a sample solution to be tested and measuring its absorbance, wherein the sample solution to be tested contains an acidic substance;

[0010] (3) Obtaining the hemoglobin content in the sample to be tested based on the absorbance of the sample solution to be tested obtained in step (2) and the standard working curve or the standard working curve equation obtained in step (1).

[0011] According to some embodiments of the present invention, the number of the hemoglobin standard working solutions is 3 or more, preferably 5-10, such as 5, 6, 7, 8, 9 or 10.

[0012] According to some embodiments of the present invention, the hemoglobin standard working solution is a bovine hemoglobin standard working solution.

[0013] According to some embodiments of the present invention, the concentration of the hemoglobin standard working solution is 0.04 mg / mL-1.0 mg / mL, preferably 0.08 mg / mL-0.80 mg / mL, and more preferably 0.08-0.48 mg / ml.

[0014] According to a preferred embodiment of the present invention, the concentration of the hemoglobin standard working solution is 0.08 mg / mL, 0.16 mg / mL, 0.24 mg / mL, 0.32 mg / mL, 0.40 mg / mL, and 0.48 mg / mL.

[0015] According to some embodiments of the present invention, a standard working curve is prepared with the hemoglobin concentration of the standard working solution as the abscissa and the absorbance value as the ordinate.

[0016] According to a preferred embodiment of the present invention, the standard working curve equation is y=1.9859x+0.1038, wherein y represents absorbance, x represents hemoglobin concentration, and the unit of x is mg / mL.

[0017] According to some embodiments of the present invention, in the hemoglobin standard working solution, the mass concentration of the acidic substance is 0.05%-0.20%, preferably 0.08%-0.12%.

[0018] According to a preferred embodiment of the present invention, in the hemoglobin standard working solution, the mass concentration of the acidic substance is 0.1%.

[0019] According to some embodiments of the present invention, in the sample solution to be tested, the mass concentration of the acidic substance is 0.05%-0.20%, preferably 0.08%-0.12%.

[0020] According to a preferred embodiment of the present invention, in the sample solution to be tested, the mass concentration of the acidic substance is 0.1%.

[0021] According to some embodiments of the present invention, the acidic substance is trifluoroacetic acid.

[0022] According to some embodiments of the present invention, the acidic substance is trifluoroacetic acid with a mass concentration of 0.05%-0.20%.

[0023] According to a preferred embodiment of the present invention, the acidic substance is trifluoroacetic acid with a mass concentration of 0.08%-0.12%.

[0024] According to some embodiments of the present invention, the absorbance is measured at a wavelength of 380 nm-410 nm, preferably 390 nm-400 nm.

[0025] According to a preferred embodiment of the present invention, the absorbance is measured at a wavelength of 393 nm-397 nm.

[0026] According to some embodiments of the present invention, the sample solution to be tested is a dilution of bovine blood, and the preferred dilution factor is 250-500 times.

[0027] According to some embodiments of the present invention, the sample solution to be tested is a dilution of a hemoglobin-based oxygen carrier stock solution, and the preferred dilution factor is 250-500 times.

[0028] According to some embodiments of the present invention, the volume ratio of the hemoglobin standard working solution to the acidic substance is 1:(3-25).

[0029] According to some embodiments of the present invention, the volume ratio of the sample solution to be tested to the acidic substance is 1:(490-510).

[0030] According to some embodiments of the present invention, the specific operations of step (1) include:

[0031] a. Prepare six hemoglobin standard working solutions with trifluoroacetic acid at concentrations of 0.06 mg / mL-0.10 mg / mL, 0.14 mg / mL-0.18 mg / mL, 0.22 mg / mL-0.26 mg / mL, 0.30 mg / mL-0.34 mg / mL, 0.38 mg / mL-0.42 mg / mL, and 0.46 mg / mL-0.5 mg / mL, respectively. The mass concentration of trifluoroacetic acid is preferably 0.08% -0.12%;

[0032] b. Using the trifluoroacetic acid described in step a as blank zero adjustment, measure the absorbance of the standard working solution, and establish a standard working curve or standard working curve equation of absorbance and concentration; the measurement wavelength of the absorbance is preferably 390nm-400nm.

[0033] Compared with the prior art, the present invention has the following advantages: the present invention selects a specific acidic substance so that hemoglobin has a maximum absorption peak at a certain wavelength, and by establishing a standard working curve of hemoglobin, the accurate determination of the hemoglobin concentration in the hemoglobin oxygen carrier is achieved, and the operation is simple, environmentally friendly, and has low requirements on equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the standard working curve established in Example 1. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, it should be understood that these embodiments and drawings are only used to illustrate the present invention, but do not constitute any limitation.

[0036] Example 1: Determination of hemoglobin content in HBOCs

[0037] (1) Preparation of standard solution: Accurately weigh 100.0 mg of bovine hemoglobin standard, place in a 50 mL volumetric flask, dissolve in 0.1% trifluoroacetic acid solution and dilute to 50 mL to prepare a standard stock solution with a concentration of 2 mg / mL.

[0038] (2) Establish a standard working curve: Accurately pipette 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 6 mL of the standard solution into a 25 mL volumetric flask, dilute to volume with 0.1% trifluoroacetic acid solution to prepare a series of standard working solutions (STD1, STD2, STD3, STD4, STD5, STD6), pour into a cuvette, and measure the absorbance at a wavelength of 395 nm using a UV spectrophotometer (use 0.1% trifluoroacetic acid solution as the blank to adjust to zero).

[0039] The hemoglobin concentration and absorbance of the standard working solution are shown in Table 1.

[0040] Table 1

[0041]

[0042]

[0043] With the hemoglobin concentration of the standard working solution as the horizontal axis and the absorbance value as the vertical axis, a standard working curve is prepared, such as Figure 1 shown.

[0044] Test conclusion: When the concentration of the standard solution is in the range of 0.08mg / ml-0.48mg / ml, the linear equation is y=1.9859x+0.1038, and the correlation coefficient (R 2)=0.9954, which has a good linear relationship and meets the requirements for confirmation of method linearity and range: correlation coefficient R 2 ≥0.99.

[0045] (3) Sample measurement: Accurately pipette 0.1 mL of sample into a 50 mL volumetric flask, dissolve it with 0.1% trifluoroacetic acid solution and make up to the mark. Pour the sample dilution into a cuvette and measure the absorbance at a wavelength of 395 nm using a UV spectrophotometer (use 0.1% trifluoroacetic acid solution as blank to adjust to zero).

[0046] (4) Calculation of results:

[0047] Substitute the absorbance value of the sample into the standard working curve equation to obtain the hemoglobin concentration in the sample diluent. Calculate the hemoglobin content in the sample according to the following formula:

[0048] X=C×N×0.1

[0049] Where: X—the content of hemoglobin in the sample, g / 100mL;

[0050] C—Hemoglobin concentration of the sample, mg / mL;

[0051] N—sample dilution multiple.

[0052] Example 2: Limit of Detection and Limit of Quantitation

[0053] When the hemoglobin concentration of the present invention is detected under ultraviolet detection conditions, the theoretical detection limit absorbance is 0.005, and the theoretical quantitative limit absorbance is 0.015.

[0054] (1) Concentration of hemoglobin detection limit

[0055] Accurately pipette 10 μL of the standard stock solution in Example 1, and adopt the same preparation method as the standard working solution in Example 1 to prepare a standard solution with a hemoglobin concentration of 1.66 μg / mL, and detect it according to the ultraviolet detection conditions of Example 1, and obtain the absorbance of hemoglobin in the standard solution. It is 0.0053, and its ratio to the absorbance of the theoretical detection limit is between 80-120%, and the concentration of the detection limit is determined to be 1.66 μg / mL.

[0056] (2) Concentration of hemoglobin quantification limit

[0057] Accurately pipette 30 μL of the standard stock solution in Example 1, and adopt the same preparation method as the standard working solution in Example 1 to prepare a standard solution with a hemoglobin concentration of 4.97 μg / mL, and detect it under the ultraviolet detection conditions of Example 1, and obtain the absorbance of hemoglobin in the standard solution is 0.0151, and its ratio to the absorbance of the theoretical quantitative limit is between 80-120%, and the concentration of the quantitative limit is determined to be 4.97 μg / mL.

[0058] Example 3: Precision Test

[0059] (1) Test method

[0060] Six samples were prepared respectively, and the hemoglobin content in the samples was determined according to the method of Example 1, and the RSD (%) thereof was calculated.

[0061] (2) The experimental data are shown in Table 2.

[0062] Table 2

[0063]

[0064] (3) Experimental conclusion

[0065] The repeatability RSD of the 6 samples was 2.1%, which met the confirmation requirements of the methodological precision: Repeatability: RSD ≤ 8%.

[0066] Example 4: Recovery test

[0067] (1) Test method

[0068] Treatment of spiked samples: Accurately pipette 0.1 mL of each of 9 samples, place in a 50 mL volumetric flask, divide into 3 gradients, 3 portions for each gradient, add 3, 4 and 5 mL (standard stock solution 2 mg / mL) to the corresponding samples respectively, dissolve and dilute to the scale with 0.1% trifluoroacetic acid solution, pour into a cuvette and measure at 395 nm (use 0.1% trifluoroacetic acid solution as blank to zero), and calculate the recoveries of the 3 different gradients.

[0069] (2) Test data

[0070] The experimental data obtained are shown in Table 3.

[0071] Table 3

[0072]

[0073]

[0074] Measured added concentration = standard sample concentration - sample concentration

[0075] Recovery rate (%) = measured concentration / actual spiked concentration × 100%

[0076] (3) Experimental conclusion

[0077] The recovery rates of the three different gradients were 92.67%, 93.37% and 95.23%, respectively, which met the confirmation requirements of the methodological recovery rate: 92%-105%. The method was used to detect the hemoglobin content with a certain degree of accuracy.

[0078] In summary, by confirming the linearity and range, detection concentration, quantitative concentration, repeatability and recovery rate of the hemoglobin detection method, the confirmation results can meet the standard requirements of methodological confirmation. The method of the present invention can be used to detect the hemoglobin content in HBOCs.

[0079] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for measuring the hemoglobin content in a hemoglobin-based oxygen carrier, comprising the following steps: (1) preparing a plurality of hemoglobin standard working solutions of known concentrations and measuring their absorbances, and establishing a standard working curve or a standard working curve equation of absorbance versus concentration, wherein the hemoglobin standard working solutions contain an acidic substance; (2) preparing a sample solution to be tested and measuring its absorbance, wherein the sample solution to be tested contains an acidic substance; (3) Obtaining the hemoglobin content in the sample to be tested based on the absorbance of the sample solution to be tested obtained in step (2) and the standard working curve or the standard working curve equation obtained in step (1); In the hemoglobin standard working solution, the mass concentration of the acidic substance is 0.05%-0.20%; in the sample solution to be tested, the mass concentration of the acidic substance is 0.05%-0.20%; The acidic substance is trifluoroacetic acid; The absorbance measurement wavelength is 380nm-410nm; When measuring absorbance in step (1) and step (2), trifluoroacetic acid is used as blank to adjust the zero.

2. The method according to claim 1, characterized in that: In step (1), the number of the hemoglobin standard working solutions is 3 or more.

3. The method according to claim 1, characterized in that: In step (1), the number of the hemoglobin standard working solutions is 5-10.

4. The method according to claim 1, characterized in that: The hemoglobin standard working solution is a bovine hemoglobin standard working solution; and / or the concentration of the hemoglobin standard working solution is 0.04 mg / mL-1.0 mg / mL.

5. The method according to claim 4, characterized in that The concentration of the hemoglobin standard working solution is 0.08 mg / mL-0.80 mg / mL.

6. The method according to claim 4, characterized in that The concentration of the hemoglobin standard working solution is 0.08-0.48 mg / ml.

7. The method according to any one of claims 1 to 6, characterized in that: The standard working curve equation is y=1.9859x+0.1038, wherein y represents absorbance, x represents hemoglobin concentration, and the unit of x is mg / mL.

8. The method according to any one of claims 1 to 6, characterized in that: In the hemoglobin standard working solution, the mass concentration of the acidic substance is 0.08%-0.12%; and / or in the sample solution to be tested, the mass concentration of the acidic substance is 0.08%-0.12%.

9. The method according to any one of claims 1 to 6, characterized in that: The absorbance is measured at a wavelength of 390 nm to 400 nm.

10. The method according to any one of claims 1 to 6, characterized in that: The sample solution to be tested is a dilution of bovine blood or a dilution of a hemoglobin-type oxygen carrier stock solution.

11. The method according to claim 10, characterized in that: The dilution multiple of the bovine blood dilution solution or the hemoglobin oxygen carrier stock solution dilution solution is 250-500 times.

12. The method according to any one of claims 1 to 6, characterized in that: The volume ratio of the hemoglobin standard working solution to the acidic substance is 1:(3-25); and / or the volume ratio of the sample solution to be tested to the acidic substance is 1:(490-510).

13. The method according to any one of claims 1 to 6, characterized in that: The specific operations of step (1) include: a. Use trifluoroacetic acid to prepare six hemoglobin standard working solutions with concentrations of 0.06mg / mL-0.10mg / mL, 0.14mg / mL-0.18mg / mL, 0.22mg / mL-0.26mg / mL, 0.30mg / mL-0.34mg / mL, 0.38mg / mL-0.42mg / mL, and 0.46mg / mL-0.5mg / mL; b. Use the trifluoroacetic acid described in step a as blank zero adjustment, measure the absorbance of the standard working solution, and establish a standard working curve or standard working curve equation of absorbance and concentration.

14. The method according to claim 13, characterized in that: The mass concentration of the trifluoroacetic acid is 0.08%-0.12%.

15. The method according to claim 13, characterized in that: The absorbance is measured at a wavelength of 390 nm to 400 nm.

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