Method for detecting the content of carboxylated glycosaminoglycan derivatives in a sample and use thereof - Patents.com

Abstract

The present application relates to a method for detecting the content of carboxylated glycosaminoglycan derivatives in a sample and its use. The method of the present application can stably obtain hydrolysis products of specific structures by hydrolyzing carboxylated glycosaminoglycan derivatives, and the structures can be detected by MS, and hydrolysis products having high mass spectrometry abundance can be selected from the hydrolysis products, so that the amount of carboxylated glycosaminoglycan derivatives can be indirectly calculated. The detection method is highly specialized, has high accuracy and precision, has a low quantification limit, and a low detection limit. The detection method of the present application includes the steps of: (1) hydrolyzing a sample to obtain a hydrolyzate containing the compound represented by formula (I); (2) detecting the hydrolyzate by liquid chromatography tandem mass spectrometry; and (3) using a carboxylated glycosaminoglycan derivative as a standard, hydrolyzing solutions of different concentration gradients thereof according to the method of step (1), detecting the mass spectrum signal peak area of ​​the compound represented by formula (I) in the hydrolyzate of the standard solution of different concentrations according to the method of step (2), creating a calibration curve for the amount of the carboxylated glycosaminoglycan derivative standard by the mass spectrum signal peak area, and calculating the content of the carboxylated glycosaminoglycan derivative in the sample based on the mass spectrum peak area of ​​the compound represented by formula (I) measured in step (2) based on this calibration curve.

Description

[Technical field]

[0001] The present application belongs to the technical field of analytical chemistry, specifically relates to a method for detecting the content of carboxylated glycosaminoglycan derivatives in a sample and its use, and in particular relates to a method for detecting the content of carboxylated glycosaminoglycan derivatives in a sample, which is highly specialized, has high accuracy, good precision, low quantification limit, and low detection limit, and its use. [Background technology]

[0002] The prior art, for example, the two patents of Patent Document 1 and Patent Document 2, publicly report that carboxylated glycosaminoglycan derivatives have antitumor and antitumor metastasis activities as drugs and have a wide future use. The carboxylated glycosaminoglycan derivatives are dicarboxylic acid derivatives containing oxidative ring-opening of uronic acid adjacent diol structure, which are obtained after unfractionated heparin (UFH) undergoes a two-step oxidation reaction, that is, (1) in glycosaminoglycan, the adjacent diol in uronic acid is oxidatively ring-opened to form a dialdehyde structure, and (2) the dialdehyde structure is further oxidized to obtain a dicarboxylic acid structure. The carboxylated glycosaminoglycan derivatives belong to heparin derivatives and are linear and structurally heterogeneous mucopolysaccharide substances. In the study of drug metabolism, it is necessary to measure the content of the drug in a biological sample in order to evaluate the drug metabolism kinetics of the drug. It is difficult to directly detect the complete structure from biological samples, and biological samples contain multiple endogenous substances, such as proteins, phospholipids, etc., so the conventional polysaccharide testing method is easily affected by endogenous substances and cannot quantitatively measure carboxylated glycosaminoglycan derivatives. Therefore, how to provide an accurate detection method for carboxylated glycosaminoglycan derivatives is an urgent problem to be solved. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] China Patent Publication No. 105744940 [Patent Document 2] China Patent Application Publication No. 111670038 [Non-patent literature]

[0004] [Non-Patent Document 1] Guerrini, M., Guglieri, S, Naggi, A, Sasisekharan, R, (2007). Low molecular weight heparins: Structural differentiation by bidimentional nuclear magnetic resonance spectroscopy. Seminars in Thrombosis and hemostasis, 33, 478-487 Summary of the Invention

[0005] The following is a brief summary of the subject matter described in more detail herein, which is not intended to limit the scope of protection of the claims.

[0006] In response to the shortcomings of the prior art, the objective of the present application is to provide a method for detecting the content of carboxylated glycosaminoglycan derivatives in a sample and its use, in particular to provide a method for detecting the content of carboxylated glycosaminoglycan derivatives in a sample and its use, which is highly specialized, has high accuracy, good precision, low quantification limit and low detection limit.

[0007] To achieve this objective, the present application employs the following technical aspects.

[0008] In a first aspect, the present application provides a method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample, the method comprising: (1) hydrolyzing a sample containing a carboxylated glycosaminoglycan derivative to obtain a hydrolyzate containing a compound represented by formula (I);

[0009] [ka]

[0010] (However, each R a is independently -SO3H or -H, and each R b are independently H, -SO3H, or -C(O)CH3, and each R c are independently -SO3H or -H, and n is 0, 1, 2, 3, 4, or 5. (2) detecting the hydrolyzate obtained in step (1) by liquid chromatography tandem mass spectrometry; (3) Using a carboxylated glycosaminoglycan derivative as a standard, solutions of the standard at different concentrations are hydrolyzed according to the method of step (1), and the mass spectrum signal peak area of ​​the compound represented by formula (I) in the hydrolyzed solutions of the standard at different concentrations is detected according to the method of step (2). ,mosquito Amount of carboxylated glycosaminoglycan derivative standard and mass spectrum signal peak area preparing a calibration curve, and calculating the content of the carboxylated glycosaminoglycan derivative in the sample based on the calibration curve from the mass spectrum peak area of ​​the compound represented by formula (I) measured in step (2); The carboxylated glycosaminoglycan derivative is a glycosaminoglycan compound containing a structural unit represented by formula (II) and an optional structural unit represented by formula (III).

[0011] [ka]

[0012] [ka]

[0013] (However, each R a are independently -SO3H or -H, R b are independently H, -SO3H, or -C(O)CH3, R c are independently -SO3H or -H.

[0014] Preferably ,before The carboxylated glycosaminoglycan derivative is obtained through a two-step oxidation reaction: (1) the adjacent diol in the uronic acid in the glycosaminoglycan is oxidized and ring-opened to form a dialdehyde structure, and (2) the dialdehyde structure is further oxidized to obtain a dicarboxylic acid structure. wherein the glycosaminoglycan is heparin or heparan sulfate. do.

[0015] Preferably, the compound represented by formula (I) has the following structural formula: Selected from the group consisting of do.

[0016] [ka]

[0017] [ka]

[0018] [ka]

[0019] However, the mass spectrum signal of compound (a) is MS (ESI, neg. ion) m / z: 432.0 [MH] - and The mass spectrum signal of compound (b) is MS (ESI, neg.ion) m / z: 390.0 [MH] - and The mass spectrum signal of compound (c) is MS (ESI, neg.ion) m / z: 522.98 [M-2H]2- It is.

[0020] The carboxylated glycosaminoglycan derivative of the present application contains a structural unit represented by the above formula (II) and an optional structural unit represented by the formula (III), i.e., the hexuronic acid structure in the glycosaminoglycan compound is partially or completely ring-opened.

[0021] The carboxylated glycosaminoglycan derivatives according to the present application can be hydrolyzed to obtain the compound represented by formula (I), and the reaction mechanism is as in the embodiments 1 and 2, where each R a is independently -SO3H or -H, and each R b are independently H, -SO3H, or -C(O)CH3, and each R c is independently -SOH or -H, and n is 0, 1, 2, 3, 4, or 5.

[0022] Aspect 1:

[0023] [ka]

[0024] Aspect 2:

[0025] [ka]

[0026] In each polysaccharide chain of the carboxylated glycosaminoglycan derivative according to the present application, the disaccharide structural units may be arranged in any order.

[0027] Preferably, the weight-average molecular weight of the carboxylated glycosaminoglycan derivative is 3000-20000Da, for example, 3000Da, 5000Da, 7000Da, 8000Da, 9000Da, 10000Da, 11000Da, 12000Da, 13000Da, 13500Da, 14000Da, 16000Da, 18000Da or 20000Da, and other specific point values ​​within this numerical range can be selected, and the description is omitted here.Preferably, it is 7000-14000Da, and more preferably, it is 8000-13500Da.

[0028] The degree of ring opening of the carboxylated glycosaminoglycan derivative is 10% to 100%, for example, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, and other specific point values ​​within this numerical range can be selected, and the explanation is omitted here. It is preferably 25% to 80%, and more preferably 25% to 60%.

[0029] The term "degree of ring opening" in the present application refers to the ratio of the number of ring-opened uronic acid residues to the total number of uronic acid residues, and its detection and calculation are carried out with reference to the nuclear magnetic method in the literature, Non-Patent Document 1.

[0030] Preferably, the treatment method for hydrolyzing the sample containing the carboxylated glycosaminoglycan derivatives described in step (1) is heating.

[0031] Preferably, the heating temperature is 70 to 100°C, for example, 70°C, 75°C, 80°C, 82°C, 85°C, 87°C, 95°C, or 100°C, and other specific point values ​​within this range can be selected, and the explanation is omitted here. The temperature is preferably 85 to 95°C.

[0032] Preferably, the heating time is 12 to 168 hours, for example, 12h, 16h, 24h, 36h, 48h, 55h, 60h, 70h, 72h, 75h, 78h, 80h, 90h, 96h, 120h, 144h or 168h, and other specific point values ​​within this numerical range can be selected, and the description here is omitted. Preferably, it is 12 to 120 hours.

[0033] Under the above heating temperature and heating time conditions, the carboxylated glycosaminoglycan derivative of the present application is hydrolyzed to obtain compound (a), compound (b) or compound (c) which is abundant in mass spectrometry. The reaction temperature and time are selected taking into consideration the detection efficiency, detection accuracy, etc.

[0034] When the sample containing the carboxylated glycosaminoglycan derivative is a biological sample (e.g., blood, urine), the hydrolyzed solution obtained in step (1) needs to be subjected to a detection pretreatment, which includes mixing the hydrolyzed solution with a trifluoroacetic acid solution and an acetonitrile-methanol solution, allowing it to stand, centrifuging it, removing the supernatant, drying it, and then redissolving it in water.

[0035] Preferably, the amount of the trifluoroacetic acid solution added is 0.5% to 1.5% by volume of the hydrolysis solution, for example, 0.5%, 0.8%, 1.0%, 1.2% or 1.5%, and other specific values ​​within this range can be selected, and the description thereof will be omitted here.

[0036] Preferably, the concentration of the trifluoroacetic acid solution is 4% to 6%, for example, 4%, 5% or 6%, and other specific point values ​​within this numerical range can be selected, and the description is omitted here.

[0037] Preferably, the amount of the acetonitrile-methanol solution added is 1 to 5 times the volume of the hydrolysis solution, for example, 1, 1.5, 1.8, 2.0, 2.2, 2.5, 3, 4, 5 times, etc., and any other specific point value within this numerical range can be selected, and the description here is omitted. It is preferably 1 to 3 times, and more preferably 1.5 to 2.5 times.

[0038] Preferably, in the acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 1:0.5 to 1:1.5, for example, 1:0.5, 1:0.8, 1:1, 1:1.2 or 1:1.5, and other specific point values ​​within this numerical range can be selected, and the description here is omitted.

[0039] Preferably, the temperature for the standing is −25 to −15° C., for example, −25° C., −20° C., −15° C., etc., and the time is 15 to 25 minutes, for example, 15 minutes, 18 minutes, 20 minutes, 22 minutes, 25 minutes, etc., and any other specific point value within the numerical range of each item above can be selected, and the description thereof will be omitted here.

[0040] Preferably, the liquid chromatography is reverse phase chromatography, size exclusion chromatography or hydrophilic chromatography.

[0041] Preferably, the mobile phase of the liquid chromatography is mobile phase A and mobile phase B, the mobile phase A is an aqueous solution of hexafluoroisopropanol and pentylamine, and the mobile phase B is an acetonitrile-aqueous solution of hexafluoroisopropanol and pentylamine; The mobile phase A is an aqueous solution containing 45 to 55 mM (e.g., 45 mM, 48 mM, 50 mM, 52 mM, 55 mM, etc.) of hexafluoroisopropanol and 13 to 17 mM (e.g., 13 mM, 15 mM, 17 mM, etc.) of pentylamine, the mobile phase B is an acetonitrile-aqueous solution containing 45 to 55 mM (e.g., 45 mM, 48 mM, 50 mM, 52 mM, 55 mM, etc.) of hexafluoroisopropanol and 13 to 17 mM (e.g., 13 mM, 15 mM, 17 mM, etc.) of pentylamine, and the mobile phase In B, the volume ratio of acetonitrile to water is 70:30 to 80:20 (for example, 70:30, 75:25, 80:20, etc.). Any other specific point value within the numerical range of each item above can be selected, and the description here is omitted.

[0042] More preferably, the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, specifically as shown in the table below.

[0043] [Table 1]

[0044] Preferably, the elution procedure for said liquid chromatography is as shown in the table below.

[0045] [Table 2]

[0046] In the present application, the mass spectrum conditions can be selected, for example, from the following condition contents.

[0047] [Table 3]

[0048] Based on the content of the first aspect, the present application further provides a new compound, the specific content of which is as follows:

[0049] In a second aspect, the present application provides a compound having the structure shown in formula (I):

[0050] [ka]

[0051] (However, each R a is independently -SO3H or -H, and each R b are independently H, -SO3H, or -C(O)CH3, and each R c are independently -SO3H or -H, and n is 0, 1, 2, 3, 4, or 5.

[0052] Preferably, the compound has one of the following structures:

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] In a third aspect, the present application provides the use of the method for detecting a carboxylated glycosaminoglycan derivative according to the first aspect in a pharmacokinetic study of a carboxylated glycosaminoglycan derivative.

[0057] In a fourth aspect, the present application provides the use of the method for detecting a carboxylated glycosaminoglycan derivative according to the first aspect in detecting the quality of a pharmaceutical preparation of a carboxylated glycosaminoglycan derivative.

[0058] Compared with the prior art, the present application has the following beneficial effects: Since carboxylated glycosaminoglycan derivatives are non-homogeneous substances, it is difficult to directly detect the complete structure from biological samples. The present inventors have discovered that the carboxylated glycosaminoglycan derivatives can be hydrolyzed to stably obtain the hydrolysis products of the specific structures described above, compound (a), compound (b) or compound (c), and that such compounds can be detected by mass spectrometry. Compound (a), compound (b) or compound (c) is measured, and a calibration curve is prepared for the mass spectrum peak area of ​​compound (a), compound (b) or compound (c) using different concentrations of carboxylated glycosaminoglycan derivative standard samples, and the mass spectrum peak area of ​​compound (a), compound (b) or compound (c) is detected by hydrolyzing the carboxylated glycosaminoglycan derivative in the sample, so that the mass spectrum peak area of ​​compound (a), compound (b) or compound (c) is measured by liquid chromatography mass spectrometry after the sample containing the carboxylated glycosaminoglycan derivative is hydrolyzed, and the amount of the carboxylated glycosaminoglycan derivative in the sample can be indirectly calculated based on the calibration curve. This detection method is highly specialized, has high accuracy, good precision, low quantification limit and low detection limit.

[0059] Other aspects may become apparent upon reading and understanding the detailed description. [Brief description of the drawings]

[0060] [Figure 1] 1 is a mass chromatogram of compound (a). [Diagram 2] This is a secondary mass chromatogram of compound (a). [Diagram 3] This is the hydrogen spectrum of compound (a). [Figure 4] This is the carbon spectrum of compound (a). [Diagram 5] This is the 13C DEPT 135° spectrum of compound (a). [Figure 6] This is the 1H-1H COSY spectrum of compound (a). [Figure 7] 1 is a TOCSY spectrum of compound (a). [Figure 8] HSQC spectrum of compound (a). [Figure 9] 1 is an HMBC spectrum of compound (a). [Figure 10] 1 is a mass chromatogram of compound (c). [Figure 11] This is a secondary mass chromatogram of compound (c). [Figure 12] This is the hydrogen spectrum of compound (c). [Figure 13] This is the carbon spectrum of compound (c). [Figure 14] This is the 13C DEPT 135° spectrum of compound (c). [Figure 15] This is the 1H-1H COSY spectrum of compound (c). [Figure 16] 1 is a TOCSY spectrum of compound (c). [Figure 17] 1 is a ROESY spectrum of compound (c). [Figure 18] HSQC spectrum of compound (c). [Figure 19] 1 is an HMBC spectrum of compound (c). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The technical aspects of the present application are further described below through specific embodiments. It should be clear to those skilled in the art that the above examples are only for assisting in understanding the present application, and should not be regarded as specific limitations on the present application.

[0062] The SD rats in the following examples were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0063] The carboxylated glycosaminoglycan derivative H1011 according to the following Example was prepared by the preparation method disclosed in Example 3 of Patent Document 2, and has a weight-average molecular weight of 9161 Da and a degree of ring opening of 43.1%.

[0064] Example 1 Preparation of Compound (a), Compound (b) and Compound (c): H1011 (400 mg) was weighed and dissolved in water (4.0 mL). The H1011 aqueous solution was heated to 85°C and reacted for 72 h, then cooled to 25°C. The reaction product was separated and purified by chromatography (the chromatography column was Dionex IonPac AS11-HC, and the eluent was M and N (see Table 1 for specific components)), followed by desalting and lyophilization to obtain compound (a), compound (b) and compound (c).

[0065] [ka]

[0066] [ka]

[0067] [ka]

[0068] [Table 4]

[0069] The concentrated compounds (a), (b) and (c) were analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy (one-dimensional 1 H-NMR, one-dimensional 13 CNMR, 13 C DEPT 135°, 1 H- 1The structure is identified by H COSY, 2D TOCSY, HSQC, HMBC, and 2D DOSY).

[0070] The characteristic data of compound (a) are as follows: MS(ESI,neg.ion)m / z:432.0[MH] - ; 1 H NMR(600 MHz,D2O):δ 4.87(d,J=3.5Hz,1H),4.34(dd,J=11.2,3.7Hz,1H),4.29(d,J=4.6Hz,1H),4.27(dd,J=11.0,2.1Hz,1H),4.20(d,J=4.6Hz,1H),4.05 (ddd,J=10.1,3.7,2.2Hz,1H),3.96(dd,J=10.6,3.5Hz,1H),3.81(dd,J=10.5,9.2Hz,1H),3.59(dd,J=10.1,9.2Hz,1H),2.07(s,3H). 13 C NMR(151MHz,D2O):δ 24.79,56.23,69.39,71.95,73.04,74.14,76.51,83.25,99.56,177.34,178.79,179.99.

[0071] The characteristic charts of compound (a) are shown in FIGS.

[0072] The characteristic data of compound (b) are as follows: MS(ESI,neg.ion)m / z:390.0[MH] - ;

[0073] The characteristic data of compound (c) are as follows: MS(ESI,neg.ion)m / z:522.98[M-2H] 2- ; 1H NMR(600MHz,D2O):δ 5.37(d,J=3.5Hz,1H),5.25-5.23(m,1H),5.16(d,J=3.6Hz,1H),5.15-5.12(m,1H),4.38-4 .28(m,5H),4.23(dd,J=11.4,2.1Hz,1H),4.19(dd,J=11.1,2.1Hz,1H),4.13(dd,J=2.9Hz, 1H),3.87(dt,J=9.7,2.7Hz,1H),3.84(dt,J=9.9,2.6Hz,1H),3.78-3.70(m,2H),3.64(q,J =7.1Hz,1H),3.60(dd,J=9.9Hz,1H),3.56(dd,J=9.5Hz,1H),3.25(dd,J=10.1,3.5Hz,3H). 13 C NMR(151MHz,D2O):δ 60.45,60.63,68.92,69.10,70.44,70.60,71.82,72.44,72.80,73.05,73.50,74. 54,77.54,78.74,79.51,81.24,100.90,101.38,101.69,175.69,176.18,177.23.

[0074] The characteristic charts of compound (c) are shown in FIGS.

[0075] Example 2 In this example, the detection method for a carboxylated glycosaminoglycan derivative according to the present application was used in a pharmacokinetic study (with compound (a) as the detection target), and the specific details are as follows.

[0076] (1) Test method (1.1) Experimental animals: Six healthy male adult SD rats, three of which will be used to collect blank plasma and prepare a calibration curve, and three of which will be used to detect blood concentrations after a single dose to the rats.

[0077] (1.2) Drug preparation: Carboxylated glycosaminoglycan derivative H1011 is weighed out and a 60 mg / kg drug solution is prepared in water.

[0078] (1.3) Medication and sample collection: After administration of 60 mg / kg by subcutaneous injection, Blood samples were taken at 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. Whole blood was collected and placed in K2EDTA anticoagulant tubes, then centrifuged for 15 min and separated to obtain plasma samples.

[0079] (1.4) Preparation of calibration curve: (1.4.1) Weigh out the carboxylated glycosaminoglycan derivative H1011 and prepare a 1 mg / mL aqueous solution. Dilute the H1011 aqueous solution stepwise using the blank plasma to prepare standard solutions of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL. Hydrolyze the solution at 85°C for 72 hours. (1.4.2) Perform pre-detection treatment on the hydrolyzed standard solution. Add 1% by volume of trifluoroacetic acid (5%, v / v) and twice the volume of acetonitrile / methanol (v / v, 50 / 50) to the hydrolyzed standard solution, mix uniformly, leave at -20°C for 20 minutes, centrifuge, collect the supernatant, dry it, and then redissolve it in ultrapure water. (1.4.3) Liquid chromatography tandem mass spectrometry detection was performed to obtain spectra. The chromatography conditions are as shown in Table 2, and the mass spectrometry conditions are as shown in Table 3.

[0080] [Table 5]

[0081] [Table 6]

[0082] (1.4.4) Relationship between the mass spectrum peak area of ​​H1011 standard solution and compound (a) Between A calibration curve was created, and the mass spectrum signal of compound (a) was MS (ESI, neg. ion) m / z: 432.0 [MH] -The linear equation is y=36.2154x-0.3216, and the correlation coefficient is R 2 =0.9987, where y is the mass spectrum peak area and x is the standard solution concentration.

[0083] (1.5) Detection of the content of H1011 in plasma samples: (1.5.1) The plasma sample obtained in step (1.3) is hydrolyzed at 85°C for 72 hours, and then 1% by volume of trifluoroacetic acid (5%, v / v) and twice the volume of acetonitrile / methanol (v / v, 50 / 50) are added thereto, mixed uniformly, left to stand at -20°C for 20 minutes, centrifuged, and the supernatant is collected and dried, and then redissolved in ultrapure water. (1.5.2) Liquid chromatography tandem mass spectrometry detection was performed to measure the mass spectrum peak area of ​​compound (a). The chromatography conditions were as shown in Table 2 above, and the mass spectrometry conditions were as shown in Table 3 above. (1.5.3) The concentration of H1011 in the plasma sample at each time point can be calculated from the mass spectrum peak area of ​​compound (a) in conjunction with the calibration curve prepared in step (1.4), and pharmacokinetic parameters are calculated based on the drug concentration-time curve.

[0084] (2) The test results are as shown in Table 4.

[0085] [Table 7]

[0086] (3) Methodological verification (3.1) Expertise An ultrapure water control solution and a blank plasma control solution were prepared and subjected to the same high-temperature hydrolysis and pretreatment processes as described above. After undergoing liquid chromatography tandem mass spectrometry detection under the same conditions as described above, no signal peak at 432.0 Da, i.e., the mass spectrum signal peak of compound (a), was detected in the ultrapure water control and plasma control solutions. This demonstrates that the ultrapure water and blank plasma do not affect the detection, and that the detection method is highly professional.

[0087] (3.2) Quantitation limits and detection limits After calculation, the quantification limit of this method is 0.8 μg / mL and the detection limit is 0.2 μg / mL.

[0088] (3.3) Accuracy Three concentrations of 1 μg / mL, 50 μg / mL, and 120 μg / mL were set and the H1011 plasma samples were tested. After calculation, the recovery rate was 82.6%-110.8%, and the RSD (relative standard deviation) values ​​of the six experimental results at each concentration were 4.2%, 2.2%, and 1.3%, respectively.

[0089] (3.4) Accuracy A 50μg / mL H1011 plasma solution was selected and tested six times by two different testers, and the RSD of the six test results by the first tester was 2.0%, the RSD of the six test results by the second tester was 1.8%, and the RSD of the 12 test results by the two testers was 2.1%, all of which met the acceptable standard of ≦10.0%. The method precision is good.

[0090] (3.5) Solution stability A 50 μg / mL H1011 plasma solution was selected and hydrolyzed for pretreatment, and the detection result of the sample solution on the 5th day was 97.2% of the 0th time, meeting the standard.

[0091] Example 3 In this example, the detection method for a carboxylated glycosaminoglycan derivative according to the present application was used in a pharmacokinetic study (with compound (b) as the detection target), and the specific details are as follows.

[0092] (1) Test method (1.1) Experimental animals: Six healthy male adult SD rats, three of which will be used to collect blank plasma and prepare a calibration curve, and three of which will be used to detect blood concentrations after a single dose to the rats.

[0093] (1.2) Drug preparation: Carboxylated glycosaminoglycan derivative H1011 is weighed out and a 60 mg / kg drug solution is prepared in water.

[0094] (1.3) Medication and sample collection: After subcutaneous administration of 60 mg / kg, blood was collected at 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. Whole blood was collected and placed in a K2EDTA anticoagulant tube, then centrifuged for 15 min to separate and obtain plasma samples.

[0095] (1.4) Preparation of calibration curve: (1.4.1) Weigh out the carboxylated glycosaminoglycan derivative H1011 and prepare a 1 mg / mL aqueous solution. Dilute the H1011 aqueous solution stepwise using the blank plasma to prepare standard solutions of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL. Hydrolyze the solution at 90°C for 48 hours. (1.4.2) Perform pre-detection treatment on the hydrolyzed standard solution. Add 1% by volume of trifluoroacetic acid (5%, v / v) and twice the volume of acetonitrile / methanol (v / v, 50 / 50) to the hydrolyzed standard solution, mix uniformly, leave at -20°C for 20 minutes, centrifuge, collect the supernatant, dry it, and then redissolve it in ultrapure water. (1.4.3) Liquid chromatography tandem mass spectrometry detection was performed to obtain a spectrum. The chromatography conditions were as shown in Table 2 above, the mass spectrometry conditions were as shown in Table 3 above, and the molecular weight was 390.0 Da. (1.4.4) Relationship between mass spectrum peak area of ​​H1011 standard solution and compound (b) Between A calibration curve was created, and the mass spectrum signal of compound (b) was MS (ESI, neg. ion) m / z: 390.0 [MH] - The linear equation generated is y=26.0235x with a correlation coefficient of 0.9981, where y is the mass spectrum peak area and x is the standard solution concentration.

[0096] (1.5) Detection of the content of H1011 in plasma samples: (1.5.1) The plasma sample obtained in step (1.3) is hydrolyzed at 90°C for 48 hours, and then 1% by volume of trifluoroacetic acid (5%, v / v) and twice the volume of acetonitrile / methanol (v / v, 50 / 50) are added thereto, mixed uniformly, left to stand at -20°C for 20 minutes, centrifuged, and the supernatant is collected and dried, and then redissolved in ultrapure water. (1.5.2) Liquid chromatography tandem mass spectrometry detection is performed to measure the mass spectrum peak area of ​​compound (b), the chromatography conditions are as shown in Table 2 above, the mass spectrometry conditions are as shown in Table 3 above, and the molecular weight is 390.0 Da. (1.5.3) From the mass spectrum peak area of ​​compound (b), the concentration of H1011 in the plasma sample at each time point can be calculated by combining it with the calibration curve prepared in step (1.4), and the pharmacokinetic parameters are calculated based on the drug concentration-time curve.

[0097] (2) The test results are as shown in Table 5.

[0098] [Table 8]

[0099] (3) Methodological verification (3.1) Expertise An ultrapure water control solution and a blank plasma control solution were prepared and subjected to the same high-temperature hydrolysis and pretreatment processes as described above. After undergoing liquid chromatography tandem mass spectrometry detection under the same conditions as described above, no signal peak at 390.0 Da, i.e., the mass spectrum signal peak of compound (b), was detected in the ultrapure water control and plasma control solutions. This demonstrates that the ultrapure water and blank plasma do not affect the detection, and that the detection method is highly professional.

[0100] (3.2) Quantitation limits and detection limits After calculation, the quantification limit of this method is 1.1 μg / mL, and the detection limit is 0.55 μg / mL.

[0101] (3.3) Accuracy Three concentrations of 1 μg / mL, 50 μg / mL, and 120 μg / mL were set and H1011 plasma samples were tested. After calculation, the recovery rate was 81.2%-115.8%, and the RSD values ​​of the six experimental results at each concentration were 5.4%, 1.8%, and 2.0%, respectively.

[0102] (3.4) Accuracy A 50μg / mL H1011 plasma solution was selected and tested six times by two different testers. The RSD of the six test results by the first tester was 1.6%, the RSD of the six test results by the second tester was 2.6%, and the RSD of the 12 test results by the two testers was 2.2%, all of which met the acceptable standard of ≦10.0%. The method precision is good.

[0103] (3.5) Solution stability A 50 μg / mL H1011 plasma solution was selected and hydrolyzed for pretreatment, and the detection result of the sample solution on the 5th day was 97.8% of the 0th time, meeting the standard.

[0104] Example 4 In this example, the detection method for a carboxylated glycosaminoglycan derivative according to the present application was used in a pharmacokinetic study (with compound (c) as the detection target), and the specific details are as follows.

[0105] (1) Test method (1.1) Experimental animals: Six healthy male adult SD rats, three of which will be used to collect blank plasma and prepare a calibration curve, and three of which will be used to detect blood concentrations after a single dose to the rats.

[0106] (1.2) Drug preparation: Carboxylated glycosaminoglycan derivative H1011 is weighed out and a 20 mg / kg drug solution is prepared in water.

[0107] (1.3) Medication and sample collection: After subcutaneous administration of 20 mg / kg, blood was collected at 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. Whole blood was collected and placed in a K2EDTA anticoagulant tube, then centrifuged for 15 minutes to separate and obtain plasma samples.

[0108] (1.4) Preparation of calibration curve: (1.4.1) Weigh out the carboxylated glycosaminoglycan derivative H1011 and prepare a 1 mg / mL aqueous solution. Dilute the H1011 aqueous solution stepwise using the blank plasma to prepare standard solutions of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 64 μg / mL, 128 μg / mL, and 256 μg / mL. Hydrolyze the solution at 90°C for 36 hours. (1.4.2) Perform pre-detection treatment on the hydrolyzed standard solution. Add 1% by volume of trifluoroacetic acid (5%, v / v) and twice the volume of acetonitrile / methanol (v / v, 50 / 50) to the hydrolyzed standard solution, mix uniformly, leave at -20°C for 20 minutes, centrifuge, collect the supernatant, dry it, and then redissolve it in ultrapure water. (1.4.3) Liquid chromatography tandem mass spectrometry detection was performed to measure the spectrum, the chromatography conditions were as shown in Table 2 above, the mass spectrometry conditions were as shown in Table 3 above, and the molecular weight was 522.98 Da. (1.4.4) Relationship between the mass spectrum peak area of ​​H1011 standard solution and compound (c) Between A calibration curve was created, and the mass spectrum signal of compound (c) was MS (ESI, neg. ion) m / z: 522.98 [M-2H] 2- The linear equation generated is y=23.6225x with a correlation coefficient of 0.9986, where y is the mass spectrum peak area and x is the standard solution concentration.

[0109] (1.5) Detection of the content of H1011 in plasma samples: (1.5.1) The plasma sample obtained in step (1.3) is hydrolyzed at 90°C for 36 hours, and then 1% by volume of trifluoroacetic acid (5%, v / v) and twice the volume of acetonitrile / methanol (v / v, 50 / 50) are added thereto, mixed uniformly, left to stand at -20°C for 20 minutes, centrifuged, and the supernatant is collected and dried, and then redissolved in ultrapure water. (1.5.2) Liquid chromatography tandem mass spectrometry detection was performed to measure the mass spectrum peak area of ​​compound (c). The chromatography conditions were as shown in Table 2 above, and the mass spectrometry conditions were as shown in Table 3 above. (1.5.3) From the mass spectrum peak area of ​​compound (c), the concentration of H1011 in the plasma sample at each time point can be calculated by combining it with the calibration curve prepared in step (1.4), and the pharmacokinetic parameters are calculated based on the drug concentration-time curve.

[0110] (2) The test results are as shown in Table 6.

[0111] [Table 9]

[0112] (3) Methodological verification (3.1) Expertise An ultrapure water control solution and a blank plasma control solution were prepared and subjected to the same high-temperature hydrolysis and pretreatment processes as described above. After undergoing liquid chromatography tandem mass spectrometry detection under the same conditions as described above, no signal peak at 522.98 Da, i.e., the mass spectrum signal peak of compound (c), was detected in the ultrapure water control and plasma control solutions. This demonstrates that the ultrapure water and blank plasma do not affect the detection, and that the detection method is highly professional.

[0113] (3.2) Quantitation limits and detection limits After calculation, the quantification limit of this method is 2.0 μg / mL and the detection limit is 1.0 μg / mL.

[0114] (3.3) Accuracy Three concentrations of 2 μg / mL, 50 μg / mL, and 120 μg / mL were set. The plasma samples were tested, and the calculated recoveries were 80.3%-108.8%, and the RSD values ​​of the six experimental results at each concentration were 8.9%, 6.5% and 5.4%, respectively.

[0115] (3.4) Accuracy A plasma solution of 50μg / mL H1011 was selected and tested six times by two different testers. The RSD of the six test results by the first tester was 6.5%, the RSD of the six test results by the second tester was 7.2%, and the RSD of the 12 test results by the two testers was 7.4%, all of which met the acceptable standard of ≦10.0%. The method precision is good.

[0116] (3.5) Solution stability A 50 μg / mL H1011 plasma solution was selected and hydrolyzed for pretreatment, and the detection result of the sample solution on the 5th day was 90.2% of the 0th time, meeting the standard.

Claims

1. 1. A method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample, the method comprising: (1) hydrolyzing a sample containing a carboxylated glycosaminoglycan derivative to obtain a hydrolyzate containing a compound represented by formula (I); 【Chemistry 1】 (However, each R ａ is independently -SO ３ H or -H, and each R ｂ are independently H, -SO ３ H or -C(O)CH ３ and each R ｃ is independently -SO ３ H or -H, and n is 0, 1, 2, 3, 4, or 5. (2) detecting the hydrolyzate obtained in step (1) by liquid chromatography tandem mass spectrometry; (3) using a carboxylated glycosaminoglycan derivative as a standard, solutions of the standard with different concentration gradients are hydrolyzed according to the method of step (1), mass spectrum signal peak areas of the compound represented by formula (I) in the hydrolyzed solutions of the standard with different concentrations are detected according to the method of step (2), a calibration curve between the amount of the carboxylated glycosaminoglycan derivative standard and the mass spectrum signal peak area is prepared, and the content of the carboxylated glycosaminoglycan derivative in the sample is calculated based on the mass spectrum peak area of ​​the compound represented by formula (I) measured in step (2) based on the calibration curve. and A method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample, characterized in that the carboxylated glycosaminoglycan derivative is a glycosaminoglycan-based compound containing a structural unit represented by formula (II) and a selective structural unit represented by formula (III). 【Chemistry 2】 【Chemistry 3】 (However, each R ａ is independently -SO ３ H or -H, R ｂ are independently H, -SO ３ H or -C(O)CH ３ and R ｃ is independently -SO ３ H or -H.)

2. The carboxylated glycosaminoglycan derivative is obtained through a two-step oxidation reaction of (1) forming a dialdehyde structure by oxidative ring-opening of adjacent diol in uronic acid in glycosaminoglycan, and (2) further oxidizing the dialdehyde structure to obtain a dicarboxylic acid structure. The method according to claim 1, wherein the glycosaminoglycan is heparin or heparan sulfate.

3. 2. The method of claim 1, wherein the compound represented by formula (I) is selected from the group consisting of the following structural formulas: 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】

4. The weight-average molecular weight of the carboxylated glycosaminoglycan derivative is 3,000 to 20,000 Da; 2. The method according to claim 1, wherein the degree of ring opening of the carboxylated glycosaminoglycan derivative is 10% to 100%.

5. The weight-average molecular weight of the carboxylated glycosaminoglycan derivative is 7,000 to 14,000 Da, 2. The method according to claim 1, wherein the degree of ring opening of the carboxylated glycosaminoglycan derivative is 25% to 80%.

6. The weight average molecular weight of the carboxylated glycosaminoglycan derivative is 8000 to 13 500 Da, 2. The method according to claim 1, wherein the degree of ring opening of the carboxylated glycosaminoglycan derivative is 25% to 60%.

7. 2. The method according to claim 1, wherein the treatment method for hydrolyzing the sample containing the carboxylated glycosaminoglycan derivative described in step (1) is heating.

8. The method according to claim 7, wherein the heating temperature is 70 to 100° C. and the heating time is 12 to 168 hours.

9. The method according to claim 7, wherein the heating temperature is 85 to 95° C. and the heating time is 12 to 120 hours.

10. The liquid chromatography is reverse phase chromatography, size exclusion chromatography or hydrophilic chromatography; 2. The method according to claim 1, wherein the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, the mobile phase A being an aqueous solution of hexafluoroisopropanol and pentylamine, and the mobile phase B being an acetonitrile-water solution of hexafluoroisopropanol and pentylamine.

11. The method according to claim 10, characterized in that the mobile phase A is an aqueous solution containing 45 to 55 mM hexafluoroisopropanol and 13 to 17 mM pentylamine, the mobile phase B is an acetonitrile-aqueous solution containing 45 to 55 mM hexafluoroisopropanol and 13 to 17 mM pentylamine, and in the mobile phase B, the volume ratio of acetonitrile to water is 70:30 to 80:

20.

12. The method according to claim 10, characterized in that the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, specifically as shown in the following table. 【Table 1】

13. The method according to claim 1, characterized in that, when the sample containing the carboxylated glycosaminoglycan derivative is a biological sample, the hydrolyzed solution obtained in step (1) needs to be subjected to a detection pretreatment, the pretreatment including mixing the hydrolyzed solution with a trifluoroacetic acid solution and an acetonitrile-methanol solution, allowing it to stand, centrifuging it, recovering the supernatant, drying it, and then redissolving it in water.

14. The biological sample includes blood and urine. The amount of the trifluoroacetic acid solution added is 0.5% to 1.5% by volume of the hydrolysis solution; The method according to claim 13, wherein the amount of the acetonitrile-methanol solution added is 1 to 5 times the volume of the hydrolysis solution.

15. The concentration of the trifluoroacetic acid solution is 4% to 6%, The amount of the acetonitrile-methanol solution added is 1 to 3 times the volume of the hydrolysis solution, In the acetonitrile-methanol solution, the volume ratio of acetonitrile to methanol is 1:0.5 to 1:1.5; The method according to claim 13, wherein the temperature of the standing is −25 to −15° C. and the time is 15 to 25 min.

16. A compound having the structure of formula (I): 【Chemistry 7】 (However, each R ａ is independently -SO ３ H or -H, and each R ｂ are independently H, -SO ３ H or -C(O)CH ３ and each R ｃ is independently -SO ３ H or -H, and n is 0, 1, 2, 3, 4, or 5.

17. 17. The compound of claim 16, having one of the following structures: 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】

18. A method for studying the pharmacokinetics of a carboxylated glycosaminoglycan derivative, comprising the method for detecting a carboxylated glycosaminoglycan derivative according to claim 1.

19. A method for detecting the quality of a pharmaceutical preparation of a carboxylated glycosaminoglycan derivative, comprising the method for detecting a carboxylated glycosaminoglycan derivative according to claim 1.

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