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EP-4273540-B1 - METHOD FOR DETECTING CONTENT OF GLYCOSAMINOGLYCAN CARBOXYLATED DERIVATIVE IN SAMPLE, AND APPLICATION THEREOF

EP4273540B1EP 4273540 B1EP4273540 B1EP 4273540B1EP-4273540-B1

Inventors

  • WANG, JINGWEN
  • REN, Lige
  • LIN, SENMAO
  • LI, LI

Dates

Publication Date
20260506
Application Date
20211230

Claims (11)

  1. A method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample, comprising the following steps: (1) hydrolyzing a sample containing a carboxylated glycosaminoglycan derivative to obtain a hydrolysate containing a compound of formula (I): wherein, each R a is independently -SO 3 H or -H, each R b is independently H, -SO 3 H or -C(O)CH 3 , each R c is independently -SO 3 H or -H, and n is 0, 1, 2, 3, 4 or 5; (2) detecting the hydrolysate obtained in step (1) by liquid chromatography tandem mass spectrometry; and (3) hydrolyzing solutions containing different gradient concentrations of the carboxylated glycosaminoglycan derivative as a standard according to the method of step (1); detecting mass spectral signal peak areas of the compound of formula (I) in the hydrolysates of the solutions containing different concentrations of the standard according to the method of step (2); establishing a standard curve between the contents of the carboxylated glycosaminoglycan derivative standard and the mass spectral signal peak areas; and calculating the content of the carboxylated glycosaminoglycan derivative in the sample based on the standard curve and the mass spectral signal peak area of the compound of formula (I) determined according to the method of step (2); wherein, the carboxylated glycosaminoglycan derivative is a glycosaminoglycan compound comprising a structural unit of formula (II) and optionally a structural unit of formula (III): wherein, each R a is independently -SO 3 H or -H, R b is independently H, -SO 3 H or -C(O)CH 3 , and R c is independently -SO 3 H or -H.
  2. The method of claim 1, wherein the carboxylated glycosaminoglycan derivative is obtained by a two-step oxidation reaction, comprising: (1) oxidizing two adjacent alcohol groups on the uronic acid of the glycosaminoglycan and ring-opening to form a dialdehyde structure, and (2) further oxidizing the dialdehyde structure to obtain a dicarboxylic acid structure; wherein the glycosaminoglycan is heparin or heparan sulfate.
  3. The method of claim 1, wherein the compound of formula (I) is selected from the group consisting of the following structural formulas: or
  4. The method of claim 1, wherein the carboxylated glycosaminoglycan derivative has a weight average molecular weight of 3000-20000 Da, preferably 7000-14000 Da, and further preferably 8000-13500 Da; the carboxylated glycosaminoglycan derivative has a ring-opening degree of 10-100%, preferably 25-80%, and further preferably 25-60%.
  5. The method of any one of claims 1 to 4, wherein in step (1), a method of hydrolyzing the sample containing a carboxylated glycosaminoglycan derivative is heating; preferably, the heating is performed at 70-100°C, preferably 85-95°C; preferably, the heating is performed for 12-168 h, preferably 12-120 h.
  6. The method of any one of claims 1 to 5, wherein the liquid chromatography is reversed-phase chromatography, size-exclusion chromatography or hydrophilic chromatography; preferably, mobile phases of the liquid chromatography are mobile phase A and mobile phase B; the mobile phase A is an aqueous solution of hexafluoroisopropanol and pentylamine; the mobile phase B is an acetonitrile-water solution of hexafluoroisopropanol and pentylamine; preferably, the mobile phase A is an aqueous solution containing 45-55 mM hexafluoroisopropanol and 13-17 mM pentylamine; the mobile phase B is an acetonitrile-water solution containing 45-55 mM hexafluoroisopropanol and 13-17 mM pentylamine; the mobile phase B has a volume ratio of acetonitrile to water of 70:30-80:20; preferably, the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, which as below in the table. Mobile phase A 50 mM hexafluoroisopropanol, 15 mM pentylamine, H 2 O Mobile phase B 50 mM hexafluoroisopropanol, 15 mM pentylamine, acetonitrile/H 2 O (75/25, v/v)
  7. The method of any one of claims 1 to 6, wherein, when the sample containing a carboxylated glycosaminoglycan derivative is a biological sample, the hydrolysate obtained in step (1) requires pretreatment before detection; the pretreatment comprises: mixing the hydrolysate with a trifluoroacetic acid solution and an acetonitrile-methanol solution, then performing standing and centrifugation, collecting a supernatant and drying, and then re-dissolving with water; preferably, the biological sample comprises blood and urine; preferably, the trifluoroacetic acid solution is added in an amount of 0.5-1.5% of a volume of the hydrolysate; preferably, the trifluoroacetic acid solution has a concentration of 4-6%; preferably, the acetonitrile-methanol solution is added in an amount of 1-5 times of a volume of the hydrolysate, preferably 1-3 times, and more preferably 1.5-2.5 times; preferably, a volume ratio of acetonitrile to methanol in the acetonitrile-methanol solution is 1:0.5-1:1.5; preferably, the standing is performed at -25 to -15°C for 15-25 min.
  8. A compound, which has a structure of formula (I): wherein, each R a is independently -SO 3 H or -H, each R b is independently H, -SO 3 H or -C(O)CH 3 , each R c is independently -SO 3 H or -H, and n is 0, 1, 2, 3, 4 or 5.
  9. The compound of claim 8, wherein the compound has one of the following structures: and
  10. Use of the method for detecting a carboxylated glycosaminoglycan derivative of any one of claims 1 to 7 in a pharmacokinetic study of a carboxylated glycosaminoglycan derivative.
  11. Use of the method for detecting a carboxylated glycosaminoglycan derivative of any one of claims 1 to 6 in a quality test of a carboxylated glycosaminoglycan derivative pharmaceutical preparation.

Description

FIELD OF THE INVENTION The present application belongs to the technical field of analytical chemistry, and specifically relates to a method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample and use thereof, and especially to a method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample and use thereof with high specificity, high accuracy, good precision, low limit of quantitation and low limit of detection. BACKGROUND OF THE INVENTION The prior art, such as patents CN105744940A and CN111670038A, reported that carboxylated glycosaminoglycan derivatives have anti-tumor and anti-metastasis activity as drugs, which have wide application prospects. The carboxylated glycosaminoglycan derivative is a bicarboxylic acid derivative obtained from unfractionated heparin (UFH) by a two-step oxidation reaction, where the uronic acid vicinal diol structure is oxidized and ring-opened, and the two-step oxidation reaction includes (1) two adjacent alcohol groups on the uronic acid of the glycosaminoglycan are oxidized and the ring is opened to form a dialdehyde structure, and (2) the dialdehyde structure is further oxidized to obtain the dicarboxylic acid structure; the carboxylated glycosaminoglycan derivative belongs to heparin derivatives, which is a linear and structurally inhomogeneous mucopolysaccharide substance. In drug metabolism studies, the drug content of biological samples is required to evaluate the pharmacokinetic properties of the drug. It is difficult to directly detect the intact structure in biological samples; additionally, due to the presence of endogenous substances in biological samples, such as proteins and phospholipids, conventional polysaccharide detection methods are susceptible to the interference of endogenous substances, and thus cannot quantitatively determine the carboxylated glycosaminoglycan derivative. Therefore, it has been an urgent problem to be solved about how to provide an accurate method for detecting a carboxylated glycosaminoglycan derivative. SUMMARY OF THE INVENTION The following is a summary of the subject matter described in detail herein. The summary is not intended to limit the protection scope of the claims. In view of the deficiencies of the prior art, an object of the present application is to provide a method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample and use thereof, in particular to a method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample and use thereof with high specificity, high accuracy, good precision, low limit of quantitation and low limit of detection. To achieve the object, the present application adopts the technical solutions described below. In a first aspect, the present application provides a method for detecting the content of a carboxylated glycosaminoglycan derivative in a sample. The method includes the following steps: (1) hydrolyzing a sample containing a carboxylated glycosaminoglycan derivative to obtain a hydrolysate containing a compound of formula (I): wherein, each Ra is independently -SO3H or -H, each Rb is independently H, -SO3H or -C(O)CH3, each Rc is independently -SO3H or -H, and n is 0, 1, 2, 3, 4 or 5;(2) detecting the hydrolysate obtained in step (1) by liquid chromatography tandem mass spectrometry; and(3) hydrolyzing solutions containing different gradient concentrations of the carboxylated glycosaminoglycan derivative as a standard according to the method of step (1); detecting mass spectral signal peak areas of the compound of formula (I) in the hydrolysates of the solutions containing different concentrations of the standard according to the method of step (2); establishing a standard curve between the contents of the carboxylated glycosaminoglycan derivative standard and the mass spectral signal peak areas, and calculating the content of the carboxylated glycosaminoglycan derivative in the sample based on the standard curve and the mass spectral signal peak area of the compound of formula (I) determined according to the method of step (2); the carboxylated glycosaminoglycan derivative is a glycosaminoglycan compound including a structural unit of formula (II) and optionally a structural unit of formula (III): wherein, each Ra is independently -SO3H or -H, Rb is independently H, -SO3H or -C(O)CH3, and Rc is independently -SO3H or -H. Preferably, the carboxylated glycosaminoglycan derivative is obtained by a two-step oxidation reaction, including: (1) oxidizing two adjacent alcohol groups on the uronic acid of the glycosaminoglycan and ring-opening to form a dialdehyde structure, and (2) further oxidizing the dialdehyde structure to obtain a dicarboxylic acid structure; and wherein the glycosaminoglycan is heparin or heparan sulfate. Preferably, the compound of formula (I) is selected from the group consisting of the following structural formulas: or wherein, a mass spectral signal o