CN115448996A

CN115448996A - Sugammadex sodium related substance and sugammadex sodium or free acid purity detection method based on sugammadex sodium related substance

Info

Publication number: CN115448996A
Application number: CN202210642533.4A
Authority: CN
Inventors: 郭辉; 王刚; 余飞; 杜庆然; 周文莉
Original assignee: Hefei Bosikc Pharmtech Co Ltd
Current assignee: Hefei Bosikc Pharmtech Co Ltd
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2022-12-09

Abstract

The invention discloses sugammadex sodium related substances, which comprise one or more of impurities RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b, wherein the six impurities are sugammadex sodium and byproducts or degradation impurities in the synthesis process of free acid thereof. The invention also discloses a method for preparing the impurities, and the prepared impurities are used as reference standards or reference substances for quality control of sugammadex sodium and free acid thereof.

Description

Sugammadex sodium related substance and sugammadex sodium or free acid purity detection method based on sugammadex sodium related substance

Technical Field

The invention relates to the technical field of impurity analysis of sugammadex sodium, in particular to sugammadex sodium related substances and a method for detecting purity of sugammadex sodium or free acid thereof based on the sugammadex sodium related substances.

Background

Sugammade sodium (Sugamma)x Sodium),

chemical name

6 ^A ,6 ^B ,6 ^C ,6 ^D ,6 ^E ,6 ^F ,6 ^G ,6 ^H -octa-S- (2-carboxyethyl) -6 ^A ,6 ^B ,6 ^C ,6 ^D ,6 ^E ,6 ^F ,6 ^G ,6 ^H Octathio- γ -cyclodextrin octasodium salt (1):

the sugammadex sodium plays a role in the molecular structure of gamma-cyclodextrin, is the first and only selective muscle relaxation antagonist developed successfully for 20 years, can form a compound with rocuronium bromide, and reduces the concentration of free drugs in blood, so that the neuromuscular blockade effect of the rocuronium bromide is reversed, and the side effect is small.

The sugammadex sodium or the free acid related substances thereof refer to starting materials, intermediates, polymers, side reaction products, degradation products in the storage process and the like brought by the sugammadex sodium in the production process. At present, no document reports at home and abroad adopt a high performance liquid chromatography method to effectively separate co-elution components of sugammadex sodium or free acid thereof, and the co-elution components mainly comprise RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b.

The existence of the sugammadex sodium or the free acid related substance thereof is directly related to the quality and safety of the medicine, and the structural confirmation, the source analysis, the impurity definition confirmation, the purity detection and the like are required, so that the sugammadex sodium related substance, and the sugammadex sodium containing the related substance or the free acid thereof are obtained. Therefore, the synthesis of the sugammadex sodium related substance, the sugammadex sodium containing the related substance or the free acid thereof and the purity detection have important significance for the quality control of the sugammadex sodium intermediate, the bulk drug and the preparation thereof.

The sugammadex sodium related substance, and the structural information and the source of the sugammadex sodium or the free acid thereof containing the related substance are shown in the following table 1.

TABLE 1

In the prior art, the impurities (RRT0.35a and RRT0.35b, RRT1.44a and RRT1.44b, and RRT1.59a and RRT1.59b) contained in sugammadex sodium cannot realize baseline separation and accurate quantification. The separation degree between the impurities does not meet the requirement of specificity under the verification item of related substance methodology in ICH Q3A, namely the separation degree between the impurities is less than 1.5.

The prior art does not disclose a quantitative and qualitative detection method capable of separating the impurities RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b. In conclusion, the detection result of sugammadex sodium obtained by the prior art cannot truly reflect the quality of the medicine.

Disclosure of Invention

In order to better perform quality control on the sugammadex sodium, the invention provides a sugammadex sodium related substance and a method for detecting the purity of the sugammadex sodium or free acid thereof based on the sugammadex sodium related substance, which comprises the step of detecting the purity of a solid/liquid composition of a medicament containing the sugammadex sodium or the free acid thereof, wherein the solid/liquid composition refers to a bulk drug containing the sugammadex sodium or the free acid thereof and one or more pharmaceutically acceptable excipients or solvents.

The sugammadex sodium related substance provided by the invention comprises one or more of impurities RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b;

rrt0.35a has the following structure:

rrt0.35b has the following structure:

rrt1.44a has the following structure:

rrt1.44b has the following structure:

rrt1.59a has the following structure:

rrt1.59b has the following structure:

the production pathways of the above six impurities were analyzed below.

1. The impurity RRT0.35a is generated by the following way:

the reaction solvent DMF decomposes under alkaline heating conditions to form free dimethylamine which can undergo a substitution reaction with the iodine group of the mono-iodo impurity (formed by incomplete reaction of the intermediate iodo-gamma cyclodextrin) during the reaction to produce the tertiary amino compound impurity RRT0.35a.

2. The impurity RRT0.35b is generated by the following way:

thioether on a side chain of the sugammadex sodium is easily oxidized and degraded to generate two diastereoisomer impurities with similar sulfoxide structures; the two impurities are continuously oxidized under the condition of illumination to generate photodegradable impurity RRT0.35b.

3. The impurity RRT1.44a is generated by the following way:

cleaving a single side chain of the monohydroxy sugammadex sodium to form the monohydroxy sugammadex sodium; when oxygen participates in the reaction, the mercapto group on the monothiol impurity and the 6-position hydroxyl group of the adjacent sugar ring may undergo dehydration reaction under heating condition to form thioether bond spanning the adjacent two sugar rings, and the impurity RRT1.44a is generated.

4. The impurity RRT1.44b is generated by the following way:

the impurity heptaiodo-gamma cyclodextrin in the intermediate iodo-cyclodextrin is subjected to pinnacle rearrangement on a non-iodo glucose unit under the catalysis of hydroiodic acid generated by the reaction, so that the hexose ring condensation and 6-hydroxymethyl are dehydrated to form a propisocyclic ring at the same time of rearrangement, and the impurity RRT1.44b is generated.

5. The impurity RRT1.59a is generated by the following way:

if the starting material 3-mercaptopropionic acid contains 2-mercaptopropionic acid, the starting material may react with sodium monoiodosulgamate during the synthesis to generate an impurity RRT1.59a.

6. The impurity RRT1.59b is generated by the following way:

under the condition of alkaline heating, the iodine group in the monoiodo sugammadex sodium can generate an E2 elimination reaction, lose one molecule of HI and generate an impurity RRT1.59b with an olefinic bond structure.

Through the generation routes of the six impurities, the sugammadex sodium and the free acid thereof are all byproducts or degradation impurities in the synthesis process, so that the sugammadex sodium and the free acid thereof are suitable for serving as reference standards or reference substances and used for quality control of the sugammadex sodium and the free acid thereof.

That is, the present invention can provide a method for detecting the purity of sugammadex sodium or its free acid sample, which comprises determining the presence of one or more of the impurities rrt0.35a, rrt0.35b, rrt1.44a, rrt1.44b, rrt1.59a and rrt1.59b in the sample by HPLC analysis, comparing with one or more of the known impurities rrt0.35a, rrt0.35b, rrt1.44a, rrt1.44b, rrt1.59a and rrt1.59b in the reference standard solution or control solution, and determining the purity detection of the sugammadex sodium or its free acid sample.

The above are the production routes of six impurities in the synthesis process of sugammadex sodium and free acid thereof. For the convenience of the test, the impurities were extracted from the crude sugammadex sodium, and the specific extraction process is described below.

Preparation of impurity RRT0.35a: taking a sugammadex sodium crude product for column chromatography purification, wherein the stationary phase is ADME, the elution solvent is a sodium dihydrogen phosphate-water-acetonitrile system, and the elution ratio is a sodium dihydrogen phosphate water solution, namely acetonitrile = 83; discarding 10mL of the eluent before, collecting 10mL of the eluent after collection, or desalting, concentrating and drying in vacuum to obtain the impurity RRT0.35a.

Preparation of impurity RRT0.35b: carrying out column chromatography purification on the crude sugammadex sodium product, wherein the stationary phase is ADME, and the elution solvent is a sodium dihydrogen phosphate-water-acetonitrile system; the elution ratio is sodium dihydrogen phosphate water solution and acetonitrile =83, 15mL of eluent is collected or desalted and concentrated and dried in vacuum, and the impurity RRT0.35b is obtained.

Preparation of impurity RRT1.44a: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting ratio is sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =10:90, the sodium dihydrogen phosphate solution: acetonitrile =83:20 in the sodium dihydrogen phosphate-acetonitrile solution, discarding 30mL of the eluent before, collecting 15mL of the eluent after collection, or desalting, vacuum concentrating and drying to obtain the impurity RRT1.44a.

Preparation of impurity RRT1.44b: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting ratio is sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =10 90, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution: acetonitrile = 83.

Preparation of impurity RRT1.59a: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting proportion is that a sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =1 99, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =83, 45mL of the eluent before discarding is carried out, 15mL of the eluent after collecting is carried out, or desalting is carried out, vacuum concentration and drying are carried out, and the impurity RRT1.59a is obtained.

Preparation of impurity RRT1.59b: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting proportion is that sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =1 99, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =83, 30mL of eluent before discarding is performed, 15mL of eluent after collecting is performed, or desalting is performed, vacuum concentration and drying are performed, and the impurity RRT1.59b is obtained.

In order to characterize the six impurities mentioned above, rrt0.35a, rrt0.35b, rrt1.44a, rrt1.59a, rrt1.59b, HPLC analysis was performed on them, including the following steps:

step 1, preparing 1-10 mg/mL sugammadex sodium sample solution;

step 2, preparing a reference standard solution/reference substance solution of 0.5-50 mug/mL by using any one or more of impurities RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b;

step 3, performing HPLC analysis on the sample solution and the reference standard solution/control solution, wherein the chromatography column uses an ADME column bonded with adamantyl by using a silicone polymer coated silicon substrate, the mobile phase a is 0.01 to 0.10mol/L (preferably 0.025 mol/L), pH is 2 to 4 (preferably pH = 3.0), and the mobile phase a is sodium dihydrogen phosphate aqueous solution/acetic acid-ammonium acetate aqueous solution/formic acid-ammonium formate aqueous solution/acetic acid-sodium acetate aqueous solution, acetonitrile =70 to 95 (preferably 83):

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
21	100	0
			35	98	2
50	92	8
			55	75	25
60	50	50
			65	30	70
80	30	70
			85	100	0
105	100	0

The column temperature of the chromatographic column is 35-45 ℃, and preferably 40 ℃; the flow rate of the quaternary pump is 0.15mL/min to 0.35mL/min, preferably 0.27mL/min; the detection wavelength of the ultraviolet detector is 190-210 nm, preferably 200nm;

and 4, comparing typical chromatograms of the sample solution and the reference standard solution/reference substance solution to obtain the purity of the sugammadex sodium or the free acid thereof.

Preferably, the ADME column is connected in series by two chromatographic columns, and a ghost peak trapping small column is connected in series in a flow path between the quaternary pump and the sample injector; the model of the ghost peak trapping column is 2 mm-5 mm multiplied by 100 mm-300 mm, the grain diameter of the filler is 1-5 mu m, wherein the filler material is consistent with the ADME column or has the same polarity.

The invention has the beneficial effects that: 1. providing six sugammadex related substances which can be used for qualitative and quantitative analysis of sugammadex; 2. compared with the traditional reversed-phase retention mechanism chromatographic column mainly based on C18 functional group construction, the ADME column not only maintains hydrophobicity, but also improves surface polarity, can well separate the related substances of sugammadex sodium, has the advantages of remarkable separation effect, strong practicability, low instrument requirement, small instrument loss, low cost, convenient operation, stable separation base line, high sensitivity, reliable analysis result and wide application prospect.

Drawings

FIG. 1 is a diagram of impurity RRT0.35a 1H-NMR;

FIG. 2 is a diagram of impurity RRT0.35a 13C-NMR;

FIG. 3 is a diagram of impurity RRT0.35aMS;

FIG. 4 is a graph of the impurity RRT0.35b 1H-NMR;

FIG. 5 is a graph of the impurity RRT0.35b 13C-NMR;

FIG. 6 shows the diagram (1/2) of impurity RRT0.35b MS;

FIG. 7 shows the impurity RRT0.35b MS pattern (2/2);

FIG. 8 is a graph of the impurity RRT1.44a 1H-NMR;

FIG. 9 is a diagram of the impurity RRT1.44a 13C-NMR;

FIG. 10 shows the impurity RRT1.44a MS pattern (1/2);

FIG. 11 shows the impurity RRT1.44a MS pattern (2/2);

FIG. 12 is a graph of the impurity RRT1.59b 1H-NMR;

FIG. 13 is a graph of the impurity RRT1.59b 13C-NMR;

FIG. 14 shows the impurity RRT1.59b MS;

FIG. 15 is a graph of the impurity RRT1.59a 1H-NMR;

FIG. 16 is a diagram of impurity RRT1.59a 13C-NMR;

FIG. 17 shows a diagram of impurity RRT1.59a MS;

FIG. 18 is a typical chromatogram of a system suitability solution;

FIG. 19 is a typical chromatogram (UV) of a mixed control of RRT0.35b, RRT0.35a, sugammadex sodium, RRT1.44b, RRT1.44a, RRT1.59b, RRT1.59a;

FIG. 20 is a typical Chromatogram (CAD) of a mixed control of RRT0.35b, RRT0.35a, sugammadex, RRT1.44b, RRT1.44a, RRT1.59b, RRT1.59a;

FIG. 21 is a typical chromatogram (ELSD) of a mixed control of RRT0.35b, RRT0.35a, sugammadex, RRT1.44b, RRT1.44a, RRT1.59b, and RRT1.59a.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments. The embodiments of the present invention have been presented for purposes of illustration and description, and are not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to practitioners skilled in this art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

EXAMPLE 1 preparation of sulgammadex sodium related impurities

1. The raw materials are as follows:

the method comprises the following steps of acetonitrile: HPLC grade, supplied by SIMARK corporation, LOT number LOT: s62105AC01H, the content is more than or equal to 99.9%;

two hydrate sodium dihydrogen phosphate: AR grade, provided by the national drug group chemical agents limited, LOT number LOT:20210806;

and c, phosphoric acid: AR grade, supplied by metropolis chemicals ltd, LOT number LOT:2021110801, the content is more than or equal to 85.0 percent;

sodium sumbiglucose: provided by Hefei Bo Si Ke Chuang medicine science and technology Limited company, the batch numbers are respectively: a003-22001, A003-22002 and A003-22003 in the contents of 98.8%, 99.1% and 99.5%, respectively.

2. Preparation of crude sugammadex sodium:

firstly, adding 2.1kg of DMSO (dimethyl sulfoxide) into a 10L reaction kettle, and adding 300g of 3-mercaptopropionic acid while stirring;

dissolving 600g of iodocyclodextrin in 4.8kg of DMSO, and dissolving 235g of sodium hydroxide in 500g of water for later use;

thirdly, slowly dripping a sodium hydroxide aqueous solution into the reaction kettle under the protection of nitrogen, dripping a DMSO solution dissolved with the iodo-cyclodextrin at the temperature of 10-20 ℃ after dripping, and heating for reacting for 14-16h after dripping;

and after the reaction is finished, cooling to 10-30 ℃, slowly adding 3kg of anhydrous methanol into the reaction kettle for quenching reaction, and centrifugally collecting solid materials to obtain the crude sugammadex sodium.

3. Preparation of impurity RRT0.35a reference substance: taking the crude sugammadex sodium product for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, and the eluting proportion is sodium dihydrogen phosphate water solution, namely acetonitrile = 83; discarding 10mL of the eluent before, collecting 10mL of the eluent after collection, or desalting, concentrating and drying in vacuum to obtain the impurity RRT0.35a.

LOT number LOT:0.35A-20201116, the content is 99.82%.

¹ H-NMR (500M Hz, DMSO) nuclear magnetization is shown in FIG. 1, ¹³ the nuclear magnetism of C-NMR (500M Hz, DMSO) is shown in FIG. 2. High resolution mass spectrum shows that molecular ion peak m/z =1940.4552[ M + H ]] ⁺ The results are in accordance with theoretical calculations, as shown in fig. 3.

4. Preparation of impurity RRT0.35b reference: taking the crude sugammadex sodium product for column chromatography purification, wherein the stationary phase is ADME, and the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system; the elution ratio is sodium dihydrogen phosphate water solution and acetonitrile =83, 15mL of eluent is collected or desalted and concentrated and dried in vacuum, and the impurity RRT0.35b is obtained.

LOT number LOT: sug-B-20210617, content 98.9%.

¹ H-NMR (400M Hz, DMSO) nuclear magnetism is shown in FIG. 4, ¹³ C-NMR (400M Hz, DMSO) nuclear magnetic resonance is shown in FIG. 5. The high resolution mass spectrum shows that the molecular ion peak m/z =1015.1910 2[ M + H ]] ⁺ The results are consistent with theoretical calculations, as shown in fig. 6-7.

5. Preparation of impurity RRT1.44a reference substance: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting ratio is sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =10:90, the sodium dihydrogen phosphate solution: acetonitrile =83:20 in the sodium dihydrogen phosphate-acetonitrile solution, discarding 30mL of the eluent before, collecting 15mL of the eluent after collection, or desalting, vacuum concentrating and drying to obtain the impurity RRT1.44a.

LOT number LOT: SG-RRT1.44-20190718, the content is 97.47%.

¹ H-NMR (500M Hz, DMSO) nuclear magnetization is shown in FIG. 8, ¹³ C-NMR (500M Hz, DMSO) nuclear magnetization is shown in FIG. 9. High resolution mass spectrum shows that molecular ion peak m/z =1846.3678 2[ M + Na ]] ⁺ The results are consistent with theoretical calculations, as shown in FIGS. 10-11.

6. Preparation of impurity RRT1.44b reference substance: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting ratio is sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =10 90, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution: acetonitrile = 83.

7. Preparation of impurity RRT1.59b reference substance: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting proportion is that sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =1 99, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =83, 30mL of eluent before discarding is performed, 15mL of eluent after collecting is performed, or desalting is performed, vacuum concentration and drying are performed, and the impurity RRT1.59b is obtained.

LOT number LOT: SG-1.59-20190130 with the content of 99.12 percent.

¹ H-NMR (500M Hz, DMSO) nuclear magnetism is shown in FIG. 12, ¹³ the nuclear magnetism of C-NMR (500M Hz, DMSO) is shown in FIG. 13. The high resolution mass spectrum shows that the molecular ion peak m/z =1894.39018[ M + H ]] ⁺ The results are in accordance with theoretical calculations, as shown in fig. 14.

8. Preparation of impurity RRT1.59a reference substance: taking the crude sugammadex sodium for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting proportion is that a sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =1 99, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =83, 45mL of the eluent before discarding is carried out, 15mL of the eluent after collecting is carried out, or desalting is carried out, vacuum concentration and drying are carried out, and the impurity RRT1.59a is obtained.

LOT number LOT: SG-1.59A-20190325, and the content is 96.95%.

¹ H-NMR (500M Hz, DMSO) nuclear magnetization is shown in FIG. 15, ¹³ the nuclear magnetism of C-NMR (500M Hz, DMSO) is shown in FIG. 16. The high resolution mass spectrum shows that the molecular ion peak m/z =2001.41841[ M ] +H] ⁺ The results were in accordance with the theoretical calculations, as shown in fig. 17.

9. Sugammadex sodium test article: provided by the science and technology ltd of science and technology of the family mixibusco, LOT number LOT: WS20001-2, content 91.8%.

10. System applicability solution preparation: the solution containing about 5 mug of each of the impurities RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b control substances and 5mg of the sugammadex sodium test sample in every 1mL is prepared.

11. Preparation of mixed control solution:

mixed control stock solution (1): precisely weighing 2mg of each of an RRT0.35b reference substance, an RRT0.35a reference substance, a sodium sugammadex test substance, an RRT1.44a reference substance, an RRT1.59b reference substance and an RRT1.59a reference substance, placing the reference substances into a20 mL measuring flask, adding a solvent 1 (0.05 mol/L sodium dihydrogen phosphate solution (pH3.0): acetonitrile = 1) to dissolve and dilute the reference substances to a scale, and shaking up the reference substances to obtain the final product.

Mixing the reference solution: precisely transferring the mixed reference stock solution (1): 1mL into a20 mL measuring flask, diluting with solvent 2 to the scale, and shaking up to obtain the final product.

EXAMPLE 2 sample testing

1. The instrument and HPLC chromatographic conditions were as follows:

the method includes the steps of firstly, performing high performance liquid chromatography (Saimeishi fly U3000);

an ultraviolet detector, a CAD detector or an ELSD detector;

one in ten thousandth electronic balance (shanghai liangping instruments ltd., FA 2004);

a one-hundred-thousandth electronic balance (BT 25S, seudolis scientific instruments (beijing) limited);

fifthly, a millionth electronic balance (MettlerToledo, XP 6);

sixthly, using a pH meter (MettlerToledo, FE 20) in a laboratory;

chromatographic column is either: two CAPCELLPAKAKDME TYPE HR 3 μm,2.1mm × 150mm, two CoI.No. V44H2B 01095 and two CoI.No. V44H2B 01096 are connected in series;

and impurity trapping pillars: welchGhost-Buster Column 4.6 × 50mm 06100-3100061900953 G160027-05;

self-hairy mobile phase A:0.025mol/L sodium dihydrogen phosphate aqueous solution (ph 3.0) acetonitrile =83,

or 0.1% aqueous formic acid (ph 3.0) acetonitrile = 83;

the mobile phase B is the following: and (3) acetonitrile.

The gradient elution time versus flow is shown in table 2 below, for example.

TABLE 2

Test solution of sodium sucrose: precisely weighed and prepared into a solution containing about 5mg of sugammadex sodium test sample in every 1 mL.

2. HPLC analysis of the system suitability solution and the mixed control solution resulted in chromatograms shown in FIGS. 19-21.

3. Detection of sugammadex sodium related substances

The method comprises the steps of injecting 2.5 mu L of sugammadex sodium solution with batch numbers of A003-22001, A003-22002 and A003-22003 into a liquid chromatograph, recording a chromatogram, substituting measured peak areas, and calculating the content by using a correction factor method, wherein the result is shown in Table 3.

TABLE 3

Verification of methodology of Suffraction (analysis and verification of effective detection of impurities)

(1) Linearity

The concentration of the impurity RRT0.35b is in the range of 0.4349-6.5237 mug/ml, y =0.0459x-0.0001, r =0.9997;

the concentration of the impurity RRT0.35a is in the range of 0.5153-7.7298 mug/ml, y =0.0467x-0.0010, r =0.9997;

the concentration of the sugammadex sodium is in the range of 0.4714-7.0709 mug/ml, y =0.0502x +0.0006, r =0.9991;

the concentration of the impurity RRT1.44a is in the range of 0.4925-7.3870 μ g/ml, y =0.0484x +0.0004, r =0.9999;

the concentration of the impurity RRT1.59b is in the range of 0.5826-8.7387 mu g/ml, y =0.0543x-0.0082, r =0.9998;

the concentration of the impurity RRT1.59a is in the range of 0.5027-7.5403 mu g/ml, y =0.0549x-0.0069, r =0.9999.

It can be seen that the correlation coefficient r is not less than 0.990, and the peak area and the concentration thereof have a good linear relationship.

(2) Repeatability of sample introduction

The retention time RSD of the impurity RRT0.35b is 0.23 percent, and the peak area RSD is 4.97 percent;

the retention time RSD of the impurity RRT0.35a is 0.24 percent, and the peak area RSD is 4.50 percent;

the retention time RSD of the sugammadex sodium is 0.07%, and the peak area RSD is 0.37%;

the retention time RSD of the impurity RRT1.44a is 0.04 percent, and the peak area RSD is 1.66 percent;

the retention time RSD of the impurity RRT1.59b is 0.04 percent, and the peak area RSD is 0.53 percent;

the retention time RSD of the impurity RRT1.59a is 0.04 percent, and the peak area RSD is 0.71 percent.

Therefore, the RSD of the retention time of each component is less than 20.0%, the RSD of the peak area is less than 20.0%, and the sample injection repeatability is good.

(3) Stability of

The control solution was mixed within 48 hours of mixing,

the peak area RSD of the impurity RRT0.35b is 3.81 percent;

the peak area RSD of the impurity RRT0.35a is 2.82 percent;

the peak area RSD of the sugammadex sodium is 4.70 percent;

the peak area RSD of the impurity RRT1.44a is 3.70 percent;

the peak area RSD of the impurity RRT1.59b is 4.87%;

the peak area RSD of the impurity RRT1.59a is 2.61%.

Within 48 hours of the test solution of sugammadex sodium,

the peak area RSD of the impurity RRT0.35b is 1.77 percent;

the peak area RSD of the impurity RRT0.35a is 3.00 percent;

the peak area RSD of the sugammadex sodium is 0.16%;

the peak area RSD of the impurity RRT1.44a is 1.70 percent;

the peak area RSD of the impurity RRT1.59b is 1.61 percent;

the peak area RSD of the impurity RRT1.59a is 0.43 percent.

From this, it can be seen that the RSD of the peak area of each component was less than 5.0%, and the solution stability was good.

(4) Recovery rate

Mix control stock solution (2): precisely transferring the mixed reference stock solution (1): 5mL into a 50mL volumetric flask, adding solvent 2 (0.05 mol/L sodium dihydrogen phosphate solution (pH3.0)) to dilute to scale, and shaking up to obtain the final product.

Taking 100mg of the crude sugammadex sodium, precisely weighing, placing in a20 mL measuring flask, precisely transferring a mixed reference stock solution (2): 1mL, adding a solvent 2 to dissolve and dilute to a scale, shaking uniformly to obtain a recovery rate LOQ solution, and preparing 3 parts.

50mg of the crude sugammadex sodium product is precisely weighed and placed in a 10mL measuring flask, and then a mixed reference substance stock solution (2) is precisely transferred and taken, and solvent 2 is added to dissolve and dilute the mixed reference substance stock solution to the scale, so that a solution with the recovery rate of 100% is obtained, and 3 parts of the solution is prepared.

Taking 50mg of the crude sugammadex sodium, precisely weighing, placing in a 10mL measuring flask, precisely transferring 6mL of mixed reference stock solution (2), adding a solvent 2 to dissolve and dilute to a scale, and obtaining a solution with the recovery rate of 120%, and preparing 3 parts.

The recovery test results of the impurity RRT0.35a in the sugammadex sodium related substance are shown in Table 4.

Table 4 results of the recovery test of the impurity rrt0.35b from sugammadex related substances are shown in table 5.

Table 5 results of the recovery test of the impurity rrt1.44a from sugammadex related substances are shown in table 6.

Table 6 results of the recovery test of the impurity rrt1.59a from sugammadex related substances are shown in table 7.

TABLE 7

(5) Limit of quantification

Injected into a liquid chromatograph, the chromatographic peak signal-to-noise ratio is 10, and the quantitative Limit (LOQ) of each impurity is obtained, and the results are shown in the following table 8.

Name (R)	Limit of quantitation ug/ml	The quantitative limit is equal to the percentage of the test solution
			Impurity RRT0.35b	0.5088	0.0102
Impurity RRT0.35a	0.5153	0.0103
			Shugeng glucose sodium salt	0.4714	0.0094
Impurity RRT1.44a	0.4925	0.0098
			Impurity RRT1.59b	0.5060	0.0101
Impurity RRT1.59a	0.5027	0.0101

TABLE 8

(6) Repeatability and intermediate precision

The content of impurities RRT0.35b is 0.0730 percent, and the RSD is 2.37 percent;

the content of the impurity RRT0.35a is 0.1190 percent, and the RSD is 2.31 percent;

the content of impurities RRT1.44a is 0.1125 percent, and the RSD is 2.29 percent;

the content of the impurity RRT1.59b is 0.0288 percent, and the RSD is 6.97 percent;

the content of the impurity RRT1.59a is 0.0057 percent, and the RSD is 4.86 percent.

Compared with the repeatability of the standard solution of the sample with 12 parts of intermediate precision,

the content of the impurity RRT0.35b is 0.0831 percent, and the RSD is 12.93 percent;

the content of the impurity RRT0.35a is 0.1162 percent, and the RSD is 3.64 percent;

the content of the impurity RRT1.44a is 0.1128 percent, and the RSD is 1.75 percent;

the content of the impurity RRT1.59b is 0.0275 percent, and the RSD is 7.30 percent;

0.0055 percent of impurity RRT1.59a, 7.70 percent of RSD,

therefore, the RSD of each impurity content is less than 10.0%, and the repeatability and intermediate precision result are good.

It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by one of ordinary skill in this and related arts based on the embodiments of the present invention without creative efforts, shall fall within the protection scope of the present invention.

Claims

1. The sugammadex sodium related substance is characterized by comprising one or more of impurities RRT0.35a, RRT0.35b, RRT1.44a, RRT1.44b, RRT1.59a and RRT1.59b;

rrt0.35a has the following structure:

rrt0.35b has the following structure:

rrt1.44a has the following structure:

rrt1.44b has the following structure:

rrt1.59a has the following structure:

rrt1.59b has the following structure:

2. the sugammadex-related substance according to claim 1,

preparation of impurity RRT0.35a: taking the crude sugammadex sodium product for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, and the eluting proportion is sodium dihydrogen phosphate water solution, namely acetonitrile = 83; discarding 10mL of the eluent before, collecting 10mL of the eluent after collection, or desalting, concentrating and drying in vacuum to obtain an impurity RRT0.35a;

preparation of impurity RRT0.35b: carrying out column chromatography purification on the crude sugammadex sodium product, wherein the stationary phase is ADME, and the elution solvent is a sodium dihydrogen phosphate-water-acetonitrile system; the elution proportion is sodium dihydrogen phosphate water solution acetonitrile =83, 15mL of eluent is collected, or desalinization, vacuum concentration and drying are carried out, and impurity RRT0.35b is obtained;

preparation of impurity RRT1.44a: taking a sugammadex sodium crude product for column chromatography purification, wherein the stationary phase is ADME, the elution solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the elution ratio is sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =10:90, the sodium dihydrogen phosphate water solution in the sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =83: 30mL of eluent before discarding, and collecting 15mL of eluent after collecting or desalting, and performing vacuum concentration and drying to obtain an impurity RRT1.44a;

preparation of impurity RRT1.44b: taking a sugammadex sodium crude product for column chromatography purification, wherein the stationary phase is ADME, the elution solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the elution ratio is sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =10:90, the sodium dihydrogen phosphate water solution in the sodium dihydrogen phosphate-acetonitrile solution: acetonitrile =83:20, discarding 20mL of the eluent before discarding, and collecting 10mL of the eluent after collecting or desalting, and performing vacuum concentration and drying to obtain an impurity RRT1.44b;

preparation of impurity RRT1.59a: taking the crude sugammadex sodium product for column chromatography purification, wherein the stationary phase is ADME, the eluting solvent is a sodium dihydrogen phosphate-water-acetonitrile system, the eluting proportion is that a sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =1 99, the sodium dihydrogen phosphate aqueous solution in the sodium dihydrogen phosphate-acetonitrile solution is acetonitrile =83, 45mL of the eluent before discarding is carried out, 15mL of the eluent after collecting is carried out, or desalting is carried out, vacuum concentration and drying are carried out, and the impurity RRT1.59a is obtained;

3. The method for detecting the purity of sugammadex sodium or free acid thereof of the sugammadex sodium related substance according to claim 1, comprising the steps of:

step 1, preparing 1-10 mg/mL sugammadex sodium sample solution;

step 3, carrying out HPLC analysis on the sample solution and the reference standard solution/reference solution, wherein an ADME column which is formed by bonding adamantyl with an organic silicon polymer coated silicon substrate is adopted as a chromatographic column, the mobile phase A is 0.01-0.10 mol/L sodium dihydrogen phosphate aqueous solution/acetic acid-ammonium acetate aqueous solution/formic acid-ammonium formate aqueous solution/acetic acid-sodium acetate aqueous solution with the pH of 2-4, acetonitrile = 70-95, the mobile phase B is acetonitrile, and the gradient elution time and the mobile phase ratio are as follows:

The column temperature of the chromatographic column is 35-45 ℃, the flow rate of the quaternary pump is 0.15-0.35 mL/min, and the detection wavelength of the ultraviolet detector is 190-210 nm;

4. The HPLC analysis method for sugammadex sodium-related substances according to claim 3, wherein ADME column is composed of two chromatographic columns connected in series, and ghost peak trapping small columns are connected in series in the flow path between the quaternary pump and the sample injector.

5. An HPLC analysis method of sugammadex sodium related substances according to claim 4, wherein the type of ghost capturing column (2 mm-5 mm) x (100 mm-300 mm) and the particle size of the filler is 1-5 μm, wherein the filler material is identical or similar in polarity to ADME column.