Method for calculating the degree of branching of icodextrin by nuclear magnetic resonance carbon spectrum

Abstract

This invention discloses a method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy, belonging to the field of pharmaceutical chemical detection technology. The icodextrin sample to be tested is subjected to nuclear magnetic resonance testing to obtain... 13 The C-NMR spectrum is analyzed by integrating the carbon peak at position 1 of the α-1,6-glycosidic bond in the icolodextrin branch and assigning the integral to 1. Then, the carbon peak at position 1 of the α-1,4-glycosidic bond in the icolodextrin main chain is integrated. The ratio of the integral value of the carbon peak at position 1 of the α-1,6-glycosidic bond in the icolodextrin branch to the total integral of the carbon peak at position 1 in the icolodextrin molecule is the branching degree of icolodextrin. This invention provides a direct calculation of the branching degree of icolodextrin without damaging the sample structure. It is simpler, more convenient, more accurate, easier to operate, and faster, making it a suitable method for detecting the branching degree of icolodextrin.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemical detection technology, specifically relating to a method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy. Background Technology

[0002] Icodextrin is a colloidal penetrant derived from maltodextrin, used in aqueous solution for peritoneal dialysis or as an adhesion-reducing agent (a fibrous band formed between tissues and organs) after gynecological laparoscopic surgery. When used in peritoneal dialysis, the icodextrin solution absorbs waste from the blood and is removed from the peritoneum along with the waste after 8–16 hours. When used postoperatively to prevent adhesions, its penetrating activity allows the solution to remain in the peritoneum for three to four days, separating tissues and thus reducing adhesions between tissues when fibrin forms after surgery.

[0003] Icodextrin is metabolized by α-amylase into oligosaccharide polymers (DPs) with a low degree of branching, including maltose (DP2), maltotriose (DP3), maltotetraose (DP4), and higher molecular weight polysaccharides. As the α-1,6-linked products in the glucose polymer increase, they tend to accumulate in vivo and cause harm. Therefore, in the preparation of icodextrin, the icodextrin raw material is a water-soluble glucose polymer of starch derivatives linked by α-1,4- and less than 10% α-1,6-glycosidic bonds. The main chain of icodextrin is an α-1,4-linked glucose polymer, and the branches are α-1,6-linked, with the α-1,6-linked products not exceeding 10%. Therefore, the determination of the α-1,6-linking (branching degree) of icodextrin is extremely important for product safety. However, currently, there is a significant lack of methods for directly testing the branching degree of icodextrin. Patent CN107831227A discloses a method for determining the branching degree of dextran. The method calculates the branching degree of dextran by measuring the amount of formic acid (a) generated from the oxidation of a unit weight of dextran with periodic acid and the amount of PMP consumed (b) in the reaction of a unit weight of dextran with PMP solution. However, obtaining a and b in this method is complex, requiring chromatography and related chemical reactions, is time-consuming, and prone to errors. Furthermore, the background of this patent also mentions that the accuracy of carbon NMR spectroscopy is affected by low carbon atom response values ​​and peak overlap, while hydrogen NMR spectroscopy suffers from unavoidable measurement errors due to differences in the response values ​​of hydrogen atoms at different positions. Patent CN108020576 A discloses a method for determining the branching degree of glucose polymers using nuclear magnetic resonance (NMR). The branching degree is calculated by integrating the signal of glucose residue C6 in the α-1,6 linkage, which effectively improves the accuracy and simplifies the measurement process. However, verification revealed errors in the assignment of the 1,6 glycosidic bonds (including C6), and the low response value of C6, along with large integration errors, necessitates more stringent sampling conditions. These factors contribute to significant errors in the patent's detection. Therefore, a simple and efficient method for testing the branching degree of icodextrin has not yet been found. Summary of the Invention

[0004] To address the problems of complexity and low accuracy in existing methods for testing the branching degree of icodextrin, this invention provides a method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy. This method is simple to operate, fast in detection, and highly accurate.

[0005] This invention is achieved through the following technical solution: This invention discloses a method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy, characterized in that the icodextrin sample to be tested is subjected to nuclear magnetic resonance testing to obtain... 13The C-NMR spectrum is integrated to the carbon peak at position 1 of the α-1,6-glycosidic bond in the icolodextrin branch and the integral is set to 1. Based on this, the carbon peak at position 1 of the α-1,4-glycosidic bond in the icolodextrin main chain is integrated. The ratio of the integral value of the carbon at position 1 of the α-1,6-glycosidic bond in the icolodextrin branch to the total integral of the carbon at position 1 in the icolodextrin molecule is the branching degree of icolodextrin. The total integral of the carbon at position 1 in the icodextrin molecule is the sum of the integral values ​​of the carbon peak at position 1 of the α-1,4-glycosidic bond in the icodextrin main chain and the carbon peak at position 1 of the α-1,6-glycosidic bond in the icodextrin side chain. The structural diagram of the described icodextrin is shown below. Figure 1 As shown.

[0006] Furthermore, the nuclear magnetic resonance test is performed using a nuclear magnetic resonance spectrometer with an ultra-low temperature probe.

[0007] Furthermore, in the nuclear magnetic resonance test 13 The C-NMR spectroscopy detection conditions were as follows: sampling temperature 303K, spectral width 138ppm, center frequency 60ppm, TD 64K, relaxation time 10s, pulse program zgig30, number of scans 4096, number of blank scans 4, and linewidth 1Hz. After performing Fourier transform on the obtained FID signal, the peak position of TSP was chemically shifted to 0ppm.

[0008] Furthermore, the carbon peak position of the α-1,4-glycosidic bond 1-carbon in the icodextrin backbone is located at... 13 In the C-NMR spectrum, at 103.4–101.6 ppm, the carbon peak at the 1-position of the α-1,6-glycosidic bond in the icodextrin branch chain is located at... 13 The C-NMR spectrum integral is approximately 101.4–100.6 ppm.

[0009] Furthermore, the icodextrin is pretreated by the following method: the icodextrin is dissolved in a heavy aqueous solution containing sodium 3-(trimethylsilyl)deuterated propionate to obtain the icodextrin solution to be tested.

[0010] Furthermore, the concentration of the sodium 3-(trimethylsilyl)deuterated propionate is 0.01 wt%.

[0011] Furthermore, the concentration of the icodextrin solution to be tested is 0.2~0.4 g / mL.

[0012] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) The present invention uses nuclear magnetic resonance carbon spectroscopy to calculate the branching degree of icodextrin. Compared with the methods disclosed in the prior art, it simplifies the operation process, saves time and effort, and greatly reduces the amount of experimental consumables and reagents used. It also optimizes the operation steps and reduces operation errors. (2) This invention uses carbon nuclear magnetic resonance spectroscopy to calculate the branching degree of icodextrin. By selecting appropriate carbons and accurately determining the integration position, an ultra-low temperature inverted probe nuclear magnetic resonance spectrometer is used. This can effectively suppress noise while increasing the number of accumulations, effectively improving the response value of carbon atoms and the accuracy of integration. The branching degree of icodextrin can be calculated intuitively without damaging the structure of the sample itself. It is simpler and more convenient, with high accuracy, easy operation and fast detection speed. It can be used as a method for detecting the branching degree of icodextrin. Attached Figure Description

[0013] Figure 1 Here is the structural diagram of icodextrin; Figure 2 For example, icodextrin in Example 1 13 C NMR spectrum; Figure 3 Example 2: Icodextrin 13 C10 NMR spectrum. Detailed Implementation

[0014] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, all materials used in this invention are used in accordance with conventional practices or product instructions.

[0016] Example 1 Weigh 100.21 mg of icodextrin sample (Qingdao Liteng Pharmaceutical Co., Ltd., 20220103). (The structural formula of icodextrin is shown in the figure below.) Figure 1As shown), 0.5 mL of a heavy aqueous solution containing 0.01 wt% sodium 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) was added to dissolve the icolodextrin. After complete dissolution, the solution was transferred to a 5 mm NMR tube, and carbon NMR spectroscopy was performed using a cryogenic probe NMR spectrometer (instrument model: Bruker 600MHz, probe: TCI 600 S3) to obtain the carbon NMR spectrum of icolodextrin. Figure 2 As shown; In the nuclear magnetic resonance test 13 The C-NMR spectroscopy detection conditions were as follows: sampling temperature 303K, spectral width 138ppm, center frequency 60ppm, TD 64K, relaxation time 10s, pulse program zgig30, number of scans 4096, number of blank scans 4, and linewidth 1Hz. After performing Fourier transform on the obtained FID signal, the peak position of TSP was chemically shifted to 0 ppm.

[0017] Based on the structure of icodextrin, it is known that icodextrin is a polysaccharide formed by glucose units linked by glycosidic bonds. The C1 of one glucose unit is connected to the hydroxyl group of another glucose unit via a glycosidic bond, placing C1 in an adjacent position to an oxygen atom. The high electronegativity of the oxygen atom reduces the electron cloud density around C1 through an inductive effect, weakening its electron shielding and causing the chemical shift to move towards a lower field (higher ppm value). Generally, C1 is the lowest field carbon in this structure. Since glucose has a six-membered pyran ring structure, the configuration (α or β) of C1, as the terminal carbon, affects the chemical shift. However, icodextrin primarily uses the α configuration. Combined with the ring strain of the pyran ring and the effect of the oxygen atom, the typical chemical shift of C1 remains stable in the 104–100 ppm range.

[0018] Based on the carbon NMR spectrum of icodextrin, C1 can be identified as the peak at 100.6–103.4 ppm in the figure (under specific parameters and sampling conditions).

[0019] Icodextrin is a starch derivative composed of glucose molecules linked by α-1,4-glycosidic bonds and a small number of α-1,6-glycosidic bonds. Its main chain is primarily linked by α-1,4-glycosidic bonds, supplemented by a small number of α-1,6-glycosidic bonds (less than 10%), forming a branched structure. Because icodextrin contains branched α-1,6-glycosidic bonds, and in these α-1,6-glycosidic bonds, the oxygen atom connected at C1 is at the 6th carbon (methylene carbon, -CH2-), while in the main chain's α-1,4-glycosidic bonds, the oxygen atom connected at C1 is at the 4th carbon (intercyclic methylene carbon). Carbon 6 has a relatively stronger electron-donating ability, and the electron feedback effect of the oxygen atom on C1 is more pronounced, increasing the electron cloud density around C1 and enhancing the shielding effect. Simultaneously, the branched structure formed by the α-1,6-glycosidic bond has less steric hindrance, reducing the deshielding effect of adjacent groups on C1 and causing its chemical shift to move to a higher field (typically 1-3 ppm lower than C1 of the main chain α-1,4-glycosidic bond). Therefore, it can be inferred that C1 of the α-1,6-glycosidic bond carbon spectrum can be determined as... Figure 2 The peak at 101.4–100.6 ppm (under specific parameters and sampling conditions) indicates that the C1 carbon spectrum of the α-1,4-glycosidic bond can be determined as... Figure 2 The peak is located at 103.4~101.6 ppm (under specific parameters and sampling conditions).

[0020] Analyzing the structure of icodextrin, it is composed of n m parts. In each m part, the x and y parts form the main chain of icodextrin, and z is the branched part. By integrating the carbon spectrum of icodextrin, the relative proportions of the C1 integrals of α-1,6-glycosidic bonds and α-1,4-glycosidic bonds are known. Dividing the former by the sum of the two, z / (x+y+z), gives the proportion of the branched z in the m part. Finally, scaling m up to the entire icodextrin molecule, the branching degree is z / (x+y+z)×100%.

[0021] In this embodiment, the integral value of the carbon peak of the α-1,6-glycosidic bond at position 1 of the icodextrin branch chain at 101.4~100.6 ppm was calculated to be 1. Based on this, the integral value of the carbon peak of the α-1,4-glycosidic bond at position 1 of the icodextrin main chain at 103.4~101.6 ppm was 11.88. Therefore, the branching degree of icodextrin in Example 1 was 1 / (1+11.88)×100%=7.76%.

[0022] Example 2 Weigh 100.06 mg of icodextrin sample (Qingdao Liteng Pharmaceutical Co., Ltd., 20250701), add 0.5 mL of a heavy aqueous solution containing 0.01 wt% sodium 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP) to dissolve it. After complete dissolution, transfer the solution to a 5 mm NMR tube and perform carbon NMR spectroscopy using a cryogenic probe to obtain the carbon NMR spectrum of icodextrin. Figure 3 As shown; In this embodiment, the integral value of the carbon peak of the α-1,6-glycosidic bond at position 1 of the icodextrin branch chain at 101.4~100.6 ppm was calculated to be 1. Based on this, the integral value of the carbon peak of the α-1,4-glycosidic bond at position 1 of the icodextrin main chain at 103.4~101.6 ppm was 11.19. Therefore, the branching degree of icodextrin in Example 2 was 1 / (1+11.19)×100%=8.20%.

[0023] Comparative Example 1 The branching degree of the icodextrin sample (Qingdao Liteng Pharmaceutical Co., Ltd., 20220103) in Example 1 of this application was calculated using the method described in CN107831227B (specific implementation method). The amount of formic acid generated per unit weight of dextran was measured to be 6.1350 mmol / g (a), and the amount of PMP consumed by the PMP solution in the neutralization reaction with per unit weight of icodextrin was 5.6473 mmol / g (b). The molar substitution degree ((ab)×0.001×163×100%) of the icodextrin sample (Qingdao Liteng Pharmaceutical Co., Ltd., 20220103) was calculated to be 7.95%, which is close to the branching degree of icodextrin of 7.76% measured by NMR in Example 1, proving the accuracy of the NMR method.

[0024] Comparative Example 2 The branching degree of the icodextrin sample (Qingdao Liteng Pharmaceutical Co., Ltd., 20250701) in Example 2 of this application was calculated using the method described in CN107831227B (specific implementation method). The amount of formic acid generated per unit weight of dextran was measured to be 6.1348 mmol / g (a), and the amount of PMP consumed by the PMP solution in the neutralization reaction with per unit weight of icodextrin was 5.6250 mmol / g (b). The molar substitution degree ((ab)×0.001×163×100%) of the icodextrin sample (Qingdao Liteng Pharmaceutical Co., Ltd., 20220103) was calculated to be 8.31%, which is close to the branching degree of icodextrin of 8.20% measured by NMR in Example 1, proving the accuracy of the NMR method.

Claims

1. A method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy, characterized in that, The sample to be detected is subjected to nuclear magnetic resonance test, and the following results are obtained 13 C-NMR spectrum, the carbon peak of the carbon of the α-1,6-glycosidic bond 1 of the excipient branched chain is integrated and is 1, and on this basis, the carbon peak of the carbon of the α-1,4-glycosidic bond 1 of the excipient main chain is integrated, and the ratio of the integral value of the carbon of the α-1,6-glycosidic bond 1 of the excipient branched chain to the total integral of the carbon of 1 in the excipient molecule is the branching degree of the excipient. The total integral of the carbon at position 1 in the icodextrin molecule is the sum of the integral values ​​of the carbon peak at position 1 of the α-1,4-glycosidic bond in the icodextrin main chain and the carbon peak at position 1 of the α-1,6-glycosidic bond in the icodextrin side chain. In the nuclear magnetic resonance test 13 The C-NMR spectroscopy detection conditions were as follows: sampling temperature 303K, spectral width 138ppm, center frequency 60ppm, TD 64K, relaxation time 10s, pulse program zgig30, number of scans 4096, number of blank scans 4, and linewidth 1Hz. After performing Fourier transform on the obtained FID signal, the peak position of TSP was chemically shifted to 0ppm. The carbon peak position of the α-1,4-glycosidic bond 1 carbon in the icodextrin backbone is located at... 13 In the C-NMR spectrum, at 103.4–101.6 ppm, the carbon peak at the 1-position of the α-1,6-glycosidic bond in the icodextrin branch chain is located at... 13 C-NMR spectra at 101.4~100.6 ppm.

2. The method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, The nuclear magnetic resonance test was performed using a nuclear magnetic resonance spectrometer with an ultra-low temperature probe.

3. The method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy according to claim 1, characterized in that, The icodextrin is pretreated by the following method: the icodextrin is dissolved in a heavy aqueous solution containing sodium 3-(trimethylsilyl)deuterated propionate to obtain the icodextrin solution to be tested.

4. The method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy according to claim 3, characterized in that, The concentration of the sodium 3-(trimethylsilyl)deuterated propionate is 0.01 wt%.

5. The method for calculating the branching degree of icodextrin using carbon nuclear magnetic resonance spectroscopy according to claim 3, characterized in that, The concentration of the icodextrin solution to be tested is 0.2~0.4 g / mL.

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