Method for calculating molar substitution degree of hydroxyethyl starch by applying nuclear magnetic resonance carbon spectrum

Abstract

The invention discloses a method for calculating the molar substitution degree of hydroxyethyl starch by applying a nuclear magnetic resonance carbon spectrum, and belongs to the technical field of analysis and detection. The method comprises the following steps: carrying out nuclear magnetic resonance test on a sample to be detected to obtain a nuclear magnetic resonance 13C spectrum, and determining the molar substitution degree of hydroxyethyl starch according to the peak integral ratio of hydroxyethyl alpha carbon or beta carbon in hydroxyethyl starch glucose to glucose 1-position heterogenous carbon; compared with a traditional Morgan method and a traditional gas chromatography method, the method has the advantages that the operation process is simplified, time and labor are saved, the consumption of experimental consumables and reagents is greatly reduced, the molar substitution degree can be intuitively calculated on the basis of not damaging the structure of a sample, and the method is simpler, more convenient, high in accuracy, simple and convenient to operate and high in detection speed; the method can be used for detecting the molar substitution degree of hydroxyethyl starch.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for calculating the molar substitution degree of hydroxyethyl starch by using carbon nuclear magnetic resonance spectroscopy. Background Art

[0002] Hydroxyethyl starch (HES) is formed by hydrolyzing starch to a certain molecular weight under acidic conditions and then hydroxyethylating it under alkaline conditions. This means that the 2, 3, and 6 hydroxyl groups on the glucose moiety are replaced with hydroxyethyl groups. This substituted starch is less susceptible to hydrolysis by amylases in the body and can maintain osmotic pressure for a longer period of time. HES is a widely used blood volume expander in clinical practice. Used as a plasma substitute, intravenous administration can increase blood volume and temporarily maintain blood pressure. It is used to treat shock or hemorrhagic symptoms caused by massive blood loss, burns, or other trauma, and to prevent microcirculatory impairment caused by hypovolemia, which can lead to reduced organ blood flow and oxygen supply. Studies have found that the degree of hydroxyethylation (molar substitution DS), the site of hydroxyethylation (C2 / C6 ratio), and molecular weight are three key factors influencing the volume expansion effect. The degree of hydroxyethylation (molar substitution DS) determines the residence time of hydroxyethyl starch in the body. Hydroxyethyl starch with low molar substitution has low expansion strength, while hydroxyethyl starch with high molar substitution may cause damage to the coagulation mechanism and accumulation in the body if it stays in the body for too long.

[0003] Currently, the molar substitution degree of hydroxyethyl starch (HES) is mostly determined using the Morgan method and gas chromatography. The Morgan method is cumbersome and has large detection errors. Gas chromatography is more accurate than the Morgan method. Du Zhonghai et al. published a study on the GC method for determining the molar substitution degree of HES (Qilu Pharmaceutical Affairs, 2012, Vol. 31, No. 2). The chromatographic column was a DB-624 quartz capillary column and an FID detector. The inlet temperature was 200°C, and the detector temperature was 280°C. The column temperature was programmed, and the carrier gas was nitrogen at a flow rate of 8 mL / min. -1 , with a split ratio of 1:20. Toluene was used as the internal standard, and iodoethane was used as the reference. The sample to be tested was reacted at 135°C for 15 hours. Results showed that the GC method was more accurate than the Morgan method and could be used as a method for determining the molar substitution of hydroxyethyl starch. Hydroxyethyl starch was hydrolyzed and reacted with hydroiodic acid to produce iodoethane, and the molar substitution of hydroxyethyl starch was indirectly calculated by quantitatively measuring iodoethane. This method required weighing the sample before and after the reaction, with the weight required to be no more than 5 mg. The sample was then reacted at 135°C for 15 hours, making the procedure cumbersome and time-consuming. The use of organic solvents during the test precluded rapid detection. Summary of the Invention

[0004] In view of the problems of high organic solvent consumption and long time consumption in the existing methods for detecting the molar substitution degree of hydroxyethyl starch, the present invention provides a method for calculating the molar substitution degree of hydroxyethyl starch using nuclear magnetic resonance carbon spectroscopy. The method is simple to operate, has a fast detection speed and high accuracy.

[0005] The present invention is achieved through the following technical solutions: The present invention provides a method for calculating the molar substitution degree of hydroxyethyl starch by using nuclear magnetic resonance carbon spectrum. The hydroxyethyl starch sample to be tested is subjected to nuclear magnetic resonance test to obtain the nuclear magnetic resonance 13 C spectrum, the molar substitution degree of hydroxyethyl starch is determined by the peak integral ratio of the hydroxyethyl α carbon or β carbon in hydroxyethyl starch glucose to the anomeric carbon at position 1 of glucose; The method for determining the ratio of the peak integral of the hydroxyethyl α carbon to the anomeric carbon at position 1 of glucose in hydroxyethyl starch glucose is as follows: integrate the carbon peak attributed to the anomeric carbon at position 1 of glucose and set the integral ratio to 1. On this basis, integrate the carbon peaks attributed to the carbon at position 6 of glucose and the hydroxyethyl α carbon. The integral of the carbon peaks attributed to the carbon at position 6 of glucose and the hydroxyethyl α carbon minus 1 is the molar substitution degree of hydroxyethyl starch. The method for determining the peak integral ratio of the hydroxyethyl β carbon to the glucose anomeric carbon at position 1 in hydroxyethyl starch glucose is as follows: integrate the carbon peak attributed to the glucose anomeric carbon at position 1 and set the integral ratio to 1. On this basis, integrate the carbon peak attributed to the hydroxyethyl β carbon. The integral value of the hydroxyethyl β carbon is the molar substitution degree of hydroxyethyl starch. The structural formula of hydroxyethyl starch glucose is: ; R2 is H or -CH2CH2OH, R3 is H or -CH2CH2OH, R6 is H, -CH2CH2OH or the branching point of 1,6 glycosidic bond, 1, 2, 3, 4, 5, 6 represent the position numbers of carbon in hydroxyethyl starch glucose, and n is the repeating structure number of hydroxyethyl glucose unit.

[0006] Furthermore, the nuclear magnetic resonance test 13 The C spectrum detection conditions are as follows: sampling temperature 298 K, spectral width 236 ppm, center frequency 100 ppm, TD 64 K, relaxation time 2 s, pulse program zgig30, scan number 1024, blank scan number 4, line width 1 Hz.

[0007] Furthermore, the positions of the carbon peaks at the 6-carbon of glucose, the α-carbon of hydroxyethyl group, and the β-carbon of hydroxyethyl group were corrected by the chemical shifts of the negative peaks at the same positions in the DEPT135 spectrum; The test conditions of the DEPT135 spectrum are: sampling temperature 298K, spectral width 236ppm, center frequency 100ppm, TD64K, relaxation time 2s, pulse program deptsp135, scan number 256, blank scan number 8, line width 1Hz.

[0008] Furthermore, the carbon peak position of the anomeric carbon at position 1 of glucose is located at 13 The carbon peaks of glucose 6-carbon and hydroxyethyl α-carbon are located at 100.6~96.0ppm in the C spectrum. 13 C spectrum 61.15~60.10ppm; the carbon peak position of hydroxyethyl β carbon is located at NMR 13 C spectrum at 72.40~70.07ppm.

[0009] Furthermore, the sample to be tested is hydroxyethyl starch sodium chloride injection.

[0010] Furthermore, the hydroxyethyl starch sodium chloride injection is pretreated by the following method: the hydroxyethyl starch sodium chloride injection is freeze-dried and then re-dissolved in heavy water, and then freeze-dried after re-dissolution, and the re-dissolution and freeze-drying steps are repeated 2 to 4 times.

[0011] Beneficial effects (1) Compared with the traditional Morgan method and gas chromatography method, the method of calculating the molar substitution degree of hydroxyethyl starch by using nuclear magnetic resonance carbon spectroscopy in the present invention simplifies the operation process, saves time and labor, and greatly reduces the amount of experimental consumables and reagents used; (2) The traditional gas chromatography method indirectly determines the molar substitution by calculating the amount of iodoethane, and the calculation process is cumbersome. The method of the present invention can intuitively calculate the molar substitution without destroying the structure of the sample itself. It is more concise and convenient, and has high accuracy, simple operation and fast detection speed. It can be used as a method for detecting the molar substitution of hydroxyethyl starch. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Hydroxyethyl starch 130 / 0.4 sodium chloride injection 13 C NMR spectrum; Figure 2 Hydroxyethyl starch 130 / 0.4 sodium chloride injection 13 Comparison of C NMR spectrum and DEPT135 spectrum; Figure 3 It is the 6th carbon of glucose and α carbon of hydroxyethyl starch 130 / 0.4 sodium chloride injection. 13 C NMR spectrum and a partial enlarged view of the DEPT135 spectrum; Figure 4 Hydroxyethyl starch 130 / 0.4 sodium chloride injection hydroxyethyl beta carbon 13C NMR spectrum and a partial enlarged view of the DEPT135 spectrum; Figure 5 Hydroxyethyl starch 200 / 0.5 sodium chloride injection 13 C NMR spectrum; Figure 6 Hydroxyethyl starch 200 / 0.5 sodium chloride injection 13 Comparison of C NMR spectrum and DEPT135 spectrum; Figure 7 Hydroxyethyl starch 200 / 0.5 sodium chloride injection glucose 6 carbon and hydroxyethyl α carbon 13 C NMR spectrum and a partial enlarged view of the DEPT135 spectrum; Figure 8 Hydroxyethyl starch 200 / 0.5 sodium chloride injection hydroxyethyl beta carbon 13 C NMR spectrum and a partial enlargement of the DEPT135 spectrum. DETAILED DESCRIPTION

[0013] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0014] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0015] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0016] In the following examples and comparative examples, hydroxyethyl starch 130 / 0.4 sodium chloride injection was produced by Shandong Qidu Pharmaceutical Co., Ltd. with batch number 12C24050201; hydroxyethyl starch 200 / 0.5 sodium chloride injection was produced by Shandong Qidu Pharmaceutical Co., Ltd. with batch number 12C24042601.

[0017] Example 1 (1) Pretreatment of the sample to be tested: Take 0.5 ml of hydroxyethyl starch 130 / 0.4 sodium chloride injection, freeze-dry, add D2O solvent to re-dissolve, and freeze-dry again after re-dissolution. Repeat three times to remove residual solvent interference; (2) Nuclear magnetic resonance detection: The sample after pretreatment in step (1) was dissolved in 0.7 ml of D2O solvent and then subjected to nuclear magnetic resonance testing to obtain nuclear magnetic resonance 13 C( 13 C NMR) spectrum and DEPT135 spectrum; The detection conditions of the NMR test are: Nuclear magnetic resonance imaging 13 C spectrum: sampling temperature 298K, spectral width 236ppm, center frequency 100ppm, TD 64K, relaxation time 2s, pulse program zgig30, scan number 1024, blank scan number 4, line width: 1Hz; DEPT135 spectrum: sampling temperature 298 K, spectral width 236 ppm, center frequency 100 ppm, TD 64 K, relaxation time 2 s, pulse program deptsp135, scan number 256, number of empty scans 8, line width 1 Hz; Hydroxyethyl starch 130 / 0.4 sodium chloride injection 13 C NMR spectrum Figure 1 As shown, hydroxyethyl starch 130 / 0.4 sodium chloride injection 13 The comparison between the C NMR spectrum and the DEPT135 spectrum is shown in the figure below. Figure 2 As shown, the blue is the DEPT135 spectrum; the 6-carbon of glucose and the α-carbon of hydroxyethyl starch 130 / 0.4 sodium chloride injection 13 The C NMR spectrum and the enlarged part of the DEPT135 spectrum are shown in Figure 3 As shown; Hydroxyethyl starch 130 / 0.4 sodium chloride injection hydroxyethyl beta carbon 13 The C NMR spectrum and the enlarged part of the DEPT135 spectrum are shown in Figure 4 As shown: The integral values ​​of the glucose anomeric carbon 1 at 100.6-96.0 ppm were integrated and the integral ratio was set to 1 (corrected to 1.00). Based on this, the integral values ​​of the peaks at the glucose 6 carbon and the hydroxyethyl α carbon at 61.15-60.10 ppm were 1.4022 (corrected to 1.40). The integrals of the glucose 6 carbon and hydroxyethyl α carbon peaks were subtracted by 1 to obtain a molar substitution degree of 0.40 for hydroxyethyl starch 130 / 0.4 sodium chloride injection. The signal-to-noise ratios of all peaks involved in the calculation were greater than 10. The peak integral value of the hydroxyethyl β-carbon at 72.40-70.07 ppm was 0.4029 (corrected to 0.40), indicating that the molar substitution degree of hydroxyethyl starch 130 / 0.4 sodium chloride injection was 0.40; the signal-to-noise ratios of all the peaks involved in the calculation were greater than 10.

[0018] Example 2 (1) Pretreatment of the sample to be tested: Take 0.5 ml of 200 hydroxyethyl starch / 0.5 sodium chloride injection, freeze-dry, add D2O solvent to re-dissolve, and freeze-dry again after re-dissolution. Repeat three times to remove residual solvent interference; (2) Nuclear magnetic resonance testing: The sample pretreated in step (1) was subjected to nuclear magnetic resonance testing, and the testing conditions of the nuclear magnetic resonance testing were the same as those in Example 1; Nuclear Magnetic Resonance Imaging of Hydroxyethyl Starch 200 / 0.5 Sodium Chloride Injection 13 CNMR spectrum Figure 5 As shown, hydroxyethyl starch 200 / 0.5 sodium chloride injection 13 The comparison between the C NMR spectrum and the DEPT135 spectrum is shown in the figure below. Figure 6 As shown, the blue is the DEPT135 spectrum; the 6-carbon of glucose and the α-carbon of hydroxyethyl starch 130 / 0.4 sodium chloride injection 13 The C NMR spectrum and the enlarged part of the DEPT135 spectrum are shown in Figure 7 As shown; Hydroxyethyl starch 200 / 0.5 sodium chloride injection hydroxyethyl beta carbon 13 The C NMR spectrum and the enlarged part of the DEPT135 spectrum are shown in Figure 8 shown.

[0019] The integral values ​​of the anomeric carbon 1 of hydroxyethyl starch at 100.6-96.0 ppm were integrated and the integral ratio was set to 1 (corrected to 1.00). On this basis, the integral values ​​of the peaks at the 6-carbon of glucose and the α-carbon of hydroxyethyl at 61.15-60.10 ppm were 1.4987 (corrected to 1.50). The integral values ​​of the peaks at the 6-carbon of glucose and the α-carbon of hydroxyethyl were subtracted by 1 to obtain the molar substitution degree of hydroxyethyl starch 200 / 0.5 sodium chloride injection as 0.50. The signal-to-noise ratios of all the peaks involved in the above calculations were greater than 10.

[0020] The integral values ​​of the anomeric carbon 1 of hydroxyethyl starch from 100.6 to 96.0 ppm were integrated and the integral ratio was set to 1 (corrected to 1.00). The integral of the values ​​from 72.40 to 70.07 ppm was 0.4990 (corrected to 0.50), indicating that the molar substitution degree of hydroxyethyl starch 200 / 0.5 sodium chloride injection was 0.50. The signal-to-noise ratios of all peaks involved in the calculation were greater than 10.

[0021] Comparative Example 1 (1) Preparation of internal standard solution: Accurately measure 1 ml of toluene, place it in a 200 ml volumetric flask, dilute to the mark with o-xylene, and shake well to obtain the internal standard solution; (2) Preparation of reference solution: Weigh 5 portions of 0.13 g of adipic acid and place them in 5 headspace bottles respectively. Accurately add 1 ml of the internal standard solution prepared in step (1) and 2 ml of hydroiodic acid to each bottle. Seal the bottle immediately. Use a syringe to puncture and add 10 mg, 20 mg, 30 mg, 40 mg, and 50 mg of ethyl iodide, respectively. Shake well, accurately weigh, and record the weight m1. Place the headspace bottle at 150 ° C for 10 h. After cooling to room temperature, accurately weigh and record the weight m2. If the difference between m1 and m2 is greater than 5 mg, re-prepare; accurately measure 100 μl of the supernatant, place it in a sample bottle, accurately add 1 ml of o-xylene, and shake well to obtain the reference solution; (3) Preparation of test solution: accurately measure 1 ml of the sample to be tested (hydroxyethyl starch 130 / 0.4 sodium chloride injection or hydroxyethyl starch 200 / 0.5 sodium chloride injection), place it in an empty bottle, concentrate it in a water bath to dryness, and then dry it at 105°C to constant weight, add 0.13 g of adipic acid, accurately add 1 ml of the internal standard solution prepared in step (1) and 2 ml of hydroiodic acid, immediately seal it, accurately weigh it, and record the weight m1. After reacting at 150°C for 10 hours, cool it to room temperature, accurately weigh it, and record the weight m2. If the difference between m1 and m2 is greater than 5 mg, it needs to be re-prepared; accurately measure 100 μl of the supernatant, place it in a sample bottle, accurately add 1 ml of o-xylene and shake well to obtain the test solution; (4) Experimental methods: 1) Take 1 μl of each reference solution and inject it into the gas chromatograph. Record the chromatogram. Calculate the ratio of the iodine ethane peak area to the internal standard peak area in each chromatogram. Draw a standard curve based on the amount of iodine ethane (mg) and the peak area ratio. The standard curve equation is Y = 0.349X - 0.125, R 2 =0.997, X is the peak area of ​​ethyl iodide / toluene peak area, Y is the weight of ethyl iodide in the solution to be tested, mg; 2) Take 1 μl of the test solution, inject it into the gas chromatograph, record the chromatogram, and calculate the molar substitution; The gas chromatography conditions are as follows: Agilent 8890 gas chromatograph; 30m DB-624 column, 0.53mm inner diameter, 3μm liquid film thickness; FID detector; direct injection mode; programmed temperature: initial column temperature 50°C, maintain for 4 minutes, then increase to 230°C at a rate of 15°C / min, maintain for 4 minutes; gas flow rates: hydrogen 60ml / min, air 400ml / min, carrier gas nitrogen 4ml / min.

[0022] The calculation method of the molar substitution degree is: Molar substitution = (44.05 × T × 100 × 162.14) / 155.97 × m (100 - 44.05 × T × 100 / 155.97 / m) × 44.05; m is the weight of hydroxyethyl starch in the test solution, mg; T is the weight of ethyl iodide in the test solution, T=(AB) / M; Where A is the ratio of the peak area of ​​ethyl iodide in the test solution to the peak area of ​​the internal standard (toluene), B is the intercept of the standard curve with the Y-axis, and M is the slope of the standard curve.

[0023] Hydroxyethyl starch 130 / 0.4 sodium chloride injection was used as the sample to be tested. The weight of hydroxyethyl starch was 60 mg. The iodine ethane peak area (AX) was 238.457; the toluene peak area (AS') was 378.862; T was 21.6161; and the calculated molar substitution degree was 0.41.

[0024] Hydroxyethyl starch 200 / 0.5 sodium chloride injection was used as the sample to be tested. The weight of hydroxyethyl starch was 60 mg. The iodine ethane peak area (AX) was 269.751, the toluene peak area (AS') was 377.435, and T was 24.5525. The calculated molar substitution degree was 0.49.

[0025] When the test solutions were 130% hydroxyethyl starch / 0.4% sodium chloride injection and 200% hydroxyethyl starch / 0.5% sodium chloride injection, the molar substitutions obtained by the gas chromatography analysis method were 0.41 and 0.5, respectively, which were consistent with the results of Examples 1 and 2. However, the method of the present invention significantly shortens the detection time, simplifies the detection steps, and does not affect the accuracy of the test results.

Claims

1. A method for calculating the molar substitution degree of hydroxyethyl starch using carbon nuclear magnetic resonance spectroscopy, characterized in that: The hydroxyethyl starch sample to be tested is subjected to nuclear magnetic resonance testing to obtain nuclear magnetic resonance 13 C spectrum, the molar substitution degree of hydroxyethyl starch is determined by the peak integral ratio of the hydroxyethyl α carbon or β carbon in hydroxyethyl starch glucose to the anomeric carbon at position 1 of glucose; The method for determining the ratio of the peak integral of the hydroxyethyl α carbon to the anomeric carbon at position 1 of glucose in hydroxyethyl starch glucose is as follows: integrate the carbon peak attributed to the anomeric carbon at position 1 of glucose and set the integral ratio to 1. On this basis, integrate the carbon peaks attributed to the carbon at position 6 of glucose and the hydroxyethyl α carbon. The integral of the carbon peaks attributed to the carbon at position 6 of glucose and the hydroxyethyl α carbon minus 1 is the molar substitution degree of hydroxyethyl starch. The method for determining the peak integral ratio of the hydroxyethyl β carbon to the glucose anomeric carbon at position 1 in hydroxyethyl starch glucose is as follows: integrate the carbon peak attributed to the glucose anomeric carbon at position 1 and set the integral ratio to 1. On this basis, integrate the carbon peak attributed to the hydroxyethyl β carbon. The integral value of the hydroxyethyl β carbon is the molar substitution degree of hydroxyethyl starch. The structural formula of hydroxyethyl starch glucose is: ; R2 is H or -CH2CH2OH, R3 is H or -CH2CH2OH, R6 is H, -CH2CH2OH or the branching point of 1,6 glycosidic bond, 1, 2, 3, 4, 5, 6 represent the position numbers of carbon in hydroxyethyl starch glucose, and n is the repeating structure number of hydroxyethyl glucose unit.

2. The method for calculating the molar substitution degree of hydroxyethyl starch using carbon nuclear magnetic resonance spectroscopy according to claim 1, wherein: The nuclear magnetic resonance test 13 The C spectrum detection conditions are as follows: sampling temperature 298 K, spectral width 236 ppm, center frequency 100 ppm, TD 64 K, relaxation time 2 s, pulse program zgig30, scan number 1024, blank scan number 4, line width 1 Hz.

3. The method for calculating the molar substitution degree of hydroxyethyl starch using carbon nuclear magnetic resonance spectroscopy according to claim 1, wherein: The positions of the carbon peaks at the 6-carbon of glucose, the α-carbon of hydroxyethyl group, and the β-carbon of hydroxyethyl group were corrected by the chemical shifts of the negative peaks at the same positions in the DEPT135 spectrum; The test conditions of the DEPT135 spectrum are: sampling temperature 298K, spectral width 236ppm, center frequency 100ppm, TD 64K, relaxation time 2s, pulse program deptsp135, scan number 256, blank scan number 8, line width 1Hz.

4. The method for calculating the molar substitution degree of hydroxyethyl starch using carbon nuclear magnetic resonance spectroscopy according to claim 3, wherein: The carbon peak of glucose anomeric carbon 1 is located at the NMR 13 The carbon peaks of glucose 6-carbon and hydroxyethyl α-carbon are located at 100.6~96.0ppm in the C spectrum. 13 C spectrum 61.15~60.10ppm; the carbon peak position of hydroxyethyl β carbon is located at NMR 13 C spectrum at 72.40~70.07ppm.

5. The method for calculating the molar substitution degree of hydroxyethyl starch using carbon nuclear magnetic resonance spectroscopy according to claim 1, wherein: The sample to be tested is hydroxyethyl starch sodium chloride injection.

6. The method for calculating the molar substitution degree of hydroxyethyl starch using carbon nuclear magnetic resonance spectroscopy according to claim 5, wherein: The hydroxyethyl starch sodium chloride injection is pretreated by the following method: the hydroxyethyl starch sodium chloride injection is freeze-dried and then re-dissolved in heavy water, and then freeze-dried, and the re-dissolution and freeze-drying steps are repeated 2 to 4 times.

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