WO2022143853A1 - 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用 - Google Patents

一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用 Download PDF

Info

Publication number
WO2022143853A1
WO2022143853A1 PCT/CN2021/142793 CN2021142793W WO2022143853A1 WO 2022143853 A1 WO2022143853 A1 WO 2022143853A1 CN 2021142793 W CN2021142793 W CN 2021142793W WO 2022143853 A1 WO2022143853 A1 WO 2022143853A1
Authority
WO
WIPO (PCT)
Prior art keywords
glycosaminoglycan
compound
carboxylated
solution
mobile phase
Prior art date
Application number
PCT/CN2021/142793
Other languages
English (en)
French (fr)
Inventor
王景文
任丽鸽
林森茂
李锂
Original Assignee
深圳市海普瑞药业集团股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市海普瑞药业集团股份有限公司 filed Critical 深圳市海普瑞药业集团股份有限公司
Priority to EP21914584.4A priority Critical patent/EP4273540A1/en
Priority to CN202180088157.2A priority patent/CN116710772B/zh
Priority to KR1020237026204A priority patent/KR20230128095A/ko
Priority to US18/259,935 priority patent/US20240085383A1/en
Priority to JP2023540536A priority patent/JP2024505364A/ja
Publication of WO2022143853A1 publication Critical patent/WO2022143853A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/44—Sample treatment involving radiation, e.g. heat
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/12—Preparation by evaporation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/12—Preparation by evaporation
    • G01N2030/126—Preparation by evaporation evaporating sample
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient

Definitions

  • the application belongs to the technical field of analytical chemistry, and in particular relates to a method for detecting the content of glycosaminoglycan carboxylated derivatives in a sample and its application, in particular to a method with strong specificity, high accuracy, good precision and quantitative limit A method for detecting the content of glycosaminoglycan carboxylated derivatives in a sample with low detection limit and application thereof.
  • the carboxylated derivative of glycosaminoglycan is a dicarboxylic acid derivative containing uronic acid vicinal diol structure obtained after two-step oxidation reaction of unfractionated heparin (UFH), namely (1) in the glycosaminoglycan
  • UHF unfractionated heparin
  • the adjacent diols on the uronic acid are oxidized to open the ring to form a dialdehyde structure, and (2) the dialdehyde structure is further oxidized to obtain a dicarboxylic acid structure; it is a heparin derivative, and is a chain-like, non-uniform structure. polysaccharides.
  • the purpose of this application is to provide a method for detecting the content of glycosaminoglycan carboxylated derivatives in a sample and its application, especially to provide a kind of strong specificity, high accuracy and good precision , A method for detecting the content of glycosaminoglycan carboxylated derivatives in a sample with a low limit of quantification and a low limit of detection and its application.
  • the present application provides a method for detecting the content of glycosaminoglycan carboxylated derivatives in a sample, the method comprising the following steps:
  • 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
  • n is 0, 1, 2, 3, 4 or 5;
  • step (2) adopt liquid chromatography tandem mass spectrometry to detect the hydrolyzate obtained in step (1);
  • glycosaminoglycan carboxylated derivative is a glycosaminoglycan compound comprising a structural unit represented by formula (II) and an optional structural unit represented by formula (III):
  • each R a is independently -SO 3 H or -H
  • R b is independently H, -SO 3 H or -C(O)CH 3
  • R c is independently -SO 3 H or -H.
  • the glycosaminoglycan is heparin or heparan sulfate; the glycosaminoglycan carboxylated derivative is obtained through a two-step oxidation reaction: (1) the adjacent diol on the uronic acid in the glycosaminoglycan It is oxidized to open the ring to form a dialdehyde structure, and (2) the dialdehyde structure is further oxidized to obtain a dicarboxylic acid structure.
  • the compound represented by the formula (I) has at least one structure in the following structural formula:
  • the mass spectrum signal of compound (a) is MS (ESI, neg.ion) m/z: 432.0 [MH] ⁇ ;
  • the mass spectrum signal of compound (b) is MS (ESI, neg.ion) m/z: 390.0 [MH] ⁇ ;
  • the glycosaminoglycan carboxylated derivative involved in the present application comprises the structural unit represented by the above formula (II) and the optional structural unit represented by the formula (III), that is, the hexaldehyde in the glycosaminoglycan compound Partial or complete ring opening of the acid structure.
  • the glycosaminoglycan carboxylated derivative involved in the present application can be hydrolyzed to obtain the compound represented by formula (I).
  • the reaction mechanism is shown in Schemes 1 and 2, wherein each R a is independently -SO 3 H 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 :
  • the disaccharide structural units are arranged in any order.
  • the weight average molecular weight of the glycosaminoglycan carboxylated derivative is 3000-20000Da, such as 3000Da, 5000Da, 7000Da, 8000Da, 9000Da, 10000Da, 11000Da, 12000Da, 13000Da, 13500Da, 14000Da, 16000Da, 18000Da or 20000Da etc., other specific point values within the numerical range can be selected, which will not be repeated here.
  • 7000-14000Da more preferably 8000-13500Da.
  • the degree of ring opening of the glycosaminoglycan carboxylated derivative is 10-100%, such as 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% Or 100%, etc., other specific point values within the numerical range can be selected, and will not be repeated here. It is preferably 25-80%, more preferably 25-60%.
  • degree of ring opening in this application refers to the ratio of the number of uronic acid residues opened to the total number of uronic acid residues, which was detected and calculated by reference 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.
  • the treatment method for hydrolyzing the sample containing the carboxylated derivative of glycosaminoglycan in step (1) is heating.
  • the heating temperature is 70-100°C, such as 70°C, 75°C, 80°C, 82°C, 85°C, 87°C, 95°C, or 100°C, etc., and other specific point values within this numerical range are all It is optional and will not be repeated here.
  • 85-95°C Preferably 85-95°C.
  • the heating time is 12-168h, such as 12h, 16h, 24h, 36h, 48h, 55h, 60h, 70h, 72h, 75h, 78h, 80h, 90h, 96h, 120h, 144h or 168h, etc. Other specific point values within the numerical range can be selected, which will not be repeated here. Preferably 12-120h.
  • the hydrolysis of the glycosaminoglycan carboxylated derivatives involved in the present application can obtain compound (a), compound (b) or compound (c) with high mass spectrum abundance.
  • the temperature and time of the reaction are selected based on detection efficiency, detection accuracy and precision.
  • the hydrolyzate obtained in step (1) needs to be detected and pretreated, and the pretreatment includes: It was mixed with trifluoroacetic acid solution and acetonitrile-methanol solution, then stood and centrifuged, and the supernatant was dried and reconstituted with water.
  • the added amount of the trifluoroacetic acid solution is 0.5-1.5% by volume of the hydrolyzed solution, such as 0.5%, 0.8%, 1.0%, 1.2% or 1.5%, etc., and other specific values within this numerical range can be selected, and will not be repeated here.
  • the concentration of the trifluoroacetic acid solution is 4-6%, for example, 4%, 5%, or 6%, and other specific values within this range can be selected, which will not be repeated here.
  • the added amount of the acetonitrile-methanol solution is 1-5 times the volume of the hydrolyzate, such as 1 time, 1.5 times, 1.8 times, 2.0 times, 2.2 times, 2.5 times, 3 times, 4 times, 5 times, etc., other specific point values within this numerical range can be selected, and will not be repeated here. It is preferably 1-3 times, more preferably 1.5-2.5 times.
  • the volume ratio of acetonitrile to methanol in the acetonitrile-methanol solution is 1:0.5-1:1.5, such as 1:0.5, 1:0.8, 1:1, 1:1.2 or 1:1.5, etc.
  • Other specific point values in the above can be selected, and will not be repeated here.
  • the standing temperature is -25 to -15°C, such as -25°C, -20°C, -15°C, etc.; the time is 15-25min, such as 15min, 18min, 20min, 22min, 25min, etc., the above Other specific point values within each numerical range can be selected, which will not be repeated here.
  • the liquid chromatography is reverse phase chromatography, size exclusion chromatography or hydrophilic chromatography.
  • 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 amylamine;
  • the mobile phase B is a mixture of hexafluoroisopropanol and amylamine Acetonitrile-aqueous solution;
  • the mobile phase A is an aqueous solution containing 45-55mM (eg 45mM, 48mM, 50mM, 52mM, 55mM, etc.) hexafluoroisopropanol and 13-17mM (eg 13mM, 15mM, 17mM, etc.) amylamine;
  • the mobile phase B is an acetonitrile-water solution containing 45-55 mM (eg 45 mM, 48 mM, 50 mM, 52 mM, 55 mM, etc.) hexafluoroisopropanol and 13-17 mM (eg 13 mM, 15 mM, 17 mM, etc.) amylamine;
  • the volume ratio of water is 70:30-80:20 (eg 70:30, 75:25, 80:20, etc.). Other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.
  • the mobile phases of the liquid chromatography are mobile phase A and mobile phase B, as shown in the following table.
  • the elution process of the liquid chromatography is shown in the following table:
  • mass spectrometry conditions can be exemplarily selected from the following conditions:
  • Conditional content name/indicator mass spectrometer Waters Xevo G2-S QTOF model Negative Resolution Mode set quality 432.0Da Capillary voltage 1.5kV Sampling vertebra 25V Source compensation voltage 80V source temperature 120°C Desolvation temperature 500°C Cone airflow 50L/Hr Desolventizing gas stream 800L/Hr Collect starting molecular weight 200 Collection cut-off molecular weight 2000 Collection start time 1.20mins Collection end time 6.0mins
  • the application also provides a new compound, the specific content is as follows:
  • 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 , where n is 0, 1, 2, 3, 4 or 5.
  • the compound has one of the following structures:
  • the present application provides an application of the detection method for glycosaminoglycan carboxylated derivatives according to the first aspect in the pharmacokinetic study of glycosaminoglycan carboxylated derivatives.
  • the present application provides an application of the method for detecting glycosaminoglycan carboxylated derivatives according to the first aspect in quality detection of a pharmaceutical preparation of glycosaminoglycan carboxylated derivatives.
  • glycosaminoglycan carboxylated derivatives are heterogeneous substances, it is not easy to directly detect the complete structure in biological samples.
  • the inventors of the present application found that the glycosaminoglycan carboxylated derivatives can be stably obtained as described above through hydrolysis.
  • the hydrolyzate of the structure compound (a), compound (b) or compound (c), such compounds can be detected by mass spectrometry.
  • the mass spectrum peak area of compound (a), compound (b) or compound (c) with different concentrations of glycosaminoglycan carboxylated derivatives standard Establish a standard curve; then hydrolyze the glycosaminoglycan carboxylate derivative in the sample and detect the mass spectrum peak area of compound (a), compound (b) or compound (c); the sample containing glycosaminoglycan carboxylate derivative is subjected to After hydrolysis, the mass spectrum peak area of compound (a), compound (b) or compound (c) is determined by liquid mass spectrometry, and the amount of glycosaminoglycan carboxylated derivative in the sample can be indirectly calculated according to the standard curve.
  • the detection method has strong specificity, high accuracy, good precision, low limit of quantification and low limit of detection.
  • Fig. 1 is the mass spectrum of compound (a);
  • Fig. 2 is the secondary mass spectrum of compound (a);
  • Fig. 3 is the hydrogen spectrum of compound (a);
  • Fig. 4 is the carbon spectrum of compound (a);
  • Figure 5 is the 13 C DEPT 135° spectrum of compound (a);
  • Fig. 6 is the 1 H- 1 H COSY spectrum of compound (a);
  • Fig. 7 is the TOCSY spectrum of compound (a);
  • Fig. 8 is the HSQC spectrum of compound (a).
  • Fig. 9 is the HMBC spectrum of compound (a).
  • Figure 10 is a mass spectrum of compound (c).
  • Figure 11 is a secondary mass spectrum of compound (c).
  • Figure 12 is a hydrogen spectrum of compound (c).
  • Figure 13 is the carbon spectrum of compound (c).
  • Figure 14 is the 13 C DEPT 135° spectrum of compound (c);
  • Figure 15 is the 1 H- 1 H COSY spectrum of compound (c);
  • Figure 16 is the TOCSY spectrum of compound (c).
  • Figure 17 is the ROESY spectrum of compound (c).
  • Figure 18 is the HSQC spectrum of compound (c).
  • Figure 19 is the HMBC spectrum of compound (c).
  • the SD rats involved in the following examples were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • glycosaminoglycan carboxylated derivative H1011 involved in the following examples is prepared by the preparation method disclosed in Example 3 of patent CN111670038A, and its weight average molecular weight is 9161 Da and the ring opening degree is 43.1%.
  • the enriched compounds (a), (b) and (c) were obtained by mass spectrometry and nuclear magnetic resonance spectroscopy (one-dimensional 1 H-NMR, one-dimensional 13 CNMR, 13 C DEPT 135°, 1 H- 1 H COSY , two-dimensional TOCSY, HSQC, HMBC, two-dimensional DOSY) to identify the structure.
  • the detection method of the glycosaminoglycan carboxylated derivatives involved in the application is applied to the pharmacokinetic study (with compound (a) as the detection object), and the specific contents are as follows:
  • the hydrolyzed standard solution is pre-treated for detection.
  • To 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), mix well, then place it at -20°C for 20 minutes, centrifuge; take the supernatant and dry it and then reconstitute it with ultrapure water;
  • step (1.5.1) Hydrolyze the plasma sample obtained in step (1.3) at 85°C for 72h, and then add 1% by volume of trifluoroacetic acid (5%, v/v) and twice the volume of acetonitrile / methanol (v/v, 50/50), mix well, then place at -20 °C for 20 min, centrifuge; take the supernatant and dry it and then reconstitute it with ultrapure water;
  • the limit of quantification of this method was 0.8 ⁇ g/mL, and the limit of detection was 0.2 ⁇ g/mL.
  • H1011 plasma solution 50 ⁇ g/mL H1011 plasma solution was selected and tested 6 times by two different inspectors.
  • the RSD of the first inspector's 6 experimental results was 2.0%, and the RSD of the second inspector's 6 experimental results was 1.8%.
  • the RSD of the 12 test results by one inspector was 2.1%, all satisfying the acceptance criterion of ⁇ 10.0%. The method has good precision.
  • H1011 plasma solution 50 ⁇ g/mL H1011 plasma solution was selected, hydrolyzed and pretreated.
  • the detection result of the sample solution on the 5th day was 97.2% of the 0 time, which met the standard.
  • glycosaminoglycan carboxylated derivatives involved in this application is applied to pharmacokinetic research (with compound (b) as the detection object), and the specific contents are as follows:
  • the hydrolyzed standard solution is pre-treated for detection.
  • To 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), mix well, then place at -20°C for 20min, centrifuge; take the supernatant to dry and then reconstitute it with ultrapure water;
  • step (1.5.1) Hydrolyze the plasma sample obtained in step (1.3) at 90°C for 48h, and then add 1% by volume of trifluoroacetic acid (5%, v/v) and twice volume of acetonitrile / methanol (v/v, 50/50), mix well, then place at -20 °C for 20 min, centrifuge; take the supernatant and dry it and then reconstitute it with ultrapure water;
  • the limit of quantification of this method was 1.1 ⁇ g/mL, and the limit of detection was 0.55 ⁇ g/mL.
  • H1011 plasma solution 50 ⁇ g/mL H1011 plasma solution was selected and tested 6 times by two different inspectors.
  • the RSD of the first inspector's 6 experimental results was 1.6%
  • the RSD of the second inspector's 6 experimental results was 2.6%
  • the RSD of the results of 12 experiments by one inspector was 2.2%, which all met the acceptance criterion of ⁇ 10.0%.
  • the method has good precision.
  • H1011 plasma solution 50 ⁇ g/mL H1011 plasma solution was selected, hydrolyzed and pretreated.
  • the detection result of the sample solution on the 5th day was 97.8% of the 0 time, which met the standard.
  • glycosaminoglycan carboxylated derivatives involved in this application is applied to pharmacokinetic research (with compound (c) as the detection object), and the specific contents are as follows:
  • the hydrolyzed standard solution is pre-treated for detection.
  • To 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), mix well, then place it at -20°C for 20 minutes, centrifuge; take the supernatant and dry it and then reconstitute it with ultrapure water;
  • step (1.5.1) The plasma sample obtained in step (1.3) was hydrolyzed at 90°C for 36h, and then 1% by volume of trifluoroacetic acid (5%, v/v) and twice the volume of acetonitrile were added to it / methanol (v/v, 50/50), mix well, then place at -20 °C for 20 min, centrifuge; take the supernatant and dry it and then reconstitute it with ultrapure water;
  • the limit of quantification of this method was 2.0 ⁇ g/mL, and the limit of detection was 1.0 ⁇ g/mL.
  • H1011 plasma solution 50 ⁇ g/mL H1011 plasma solution was selected and tested 6 times by two different inspectors.
  • the RSD of the first inspector's 6 experiments results was 6.5%
  • the RSD of the second inspector's 6 experiments results was 7.2%
  • the RSD of the results of 12 experiments by one inspector was 7.4%, which all met the acceptance criterion of ⁇ 10.0%.
  • the method has good precision.
  • H1011 plasma solution 50 ⁇ g/mL H1011 plasma solution was selected, hydrolyzed and pretreated.
  • the detection result of the sample solution on the 5th day was 90.2% of the 0 time, which met the standard.

Landscapes

  • General Health & Medical Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

本申请涉及一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用，所述方法包括(1)将样品进行水解，得到含有式(I)所示化合物的水解液；(2)采用液相色谱串联质谱检测水解液；(3)以糖胺聚糖羧酸化衍生物作为标准品，按照步骤(1)的方法水解其不同梯度浓度的溶液，按照步骤(2)的方法检测不同浓度标准品溶液的水解液中式(I)所示化合物的质谱信号峰面积，以质谱信号峰面积对糖胺聚糖羧酸化衍生物标准品的量做标准曲线，根据该标准曲线，通过步骤(2)测定的式(I)所示化合物的质谱峰面积计算样品中糖胺聚糖羧酸化衍生物的含量。本申请所涉及的方法通过对糖胺聚糖羧酸化衍生物进行水解，可以稳定得到特定结构的水解产物，该结构可以通过MS检测到并选择其中具有较高质谱丰度的水解产物，因此可以间接计算糖胺聚糖羧酸化衍生物的量。且该检测方法的专属性强、准确度高、精密度好、定量限低、检测限低。

Description

一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用 技术领域
本申请属于分析化学技术领域，具体涉及一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用，尤其涉及一种专属性强、准确度高、精密度好、定量限低、检测限低的用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用。
背景技术
现有技术例如CN105744940A和CN111670038A两篇专利公开报道了糖胺聚糖羧酸化衍生物作为药物具有抗肿瘤及抗肿瘤转移活性，具有广泛的应用前景。糖胺聚糖羧酸化衍生物为未分级肝素(UFH)经过两步氧化反应后得到的含有糖醛酸邻二醇结构氧化开环的双羧酸衍生物，即(1)糖胺聚糖中糖醛酸上相邻的二醇被氧化开环形成二醛结构，(2)二醛结构进一步被氧化得到双羧酸结构；其属于肝素衍生物，是一种链状、结构不均一的粘多糖物质。在药物代谢研究中，需要测定生物样品中药物的含量以评价该药物的药物代谢动力学性质。在生物样品中直接检测完整的结构不容易实现，而且由于生物样品中存在多种内源性物质，例如蛋白、磷脂等，常规的多糖测试方法易受到内源性物质的干扰而无法对糖胺聚糖羧酸化衍生物进行定量测定。因此，如何提供一种准确的糖胺聚糖羧酸化衍生物的检测方法，成为了亟待解决的问题。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
针对现有技术的不足，本申请的目的在于提供一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用，尤其提供一种专属性强、准确度高、精密度好、定量限低、检测限低的用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用。
为达到此目的，本申请采用以下技术方案：
第一方面，本申请提供一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法，所述方法包括如下步骤：
(1)将含糖胺聚糖羧酸化衍生物的样品进行水解，得到含有式(I)所示化合物的水解液：
Figure PCTCN2021142793-appb-000001
其中，各R a独立地为-SO 3H或-H，各R b独立地为H、-SO 3H或-C(O)CH 3，各R c独立地为-SO 3H或-H，n为0、1、2、3、4或5；
(2)采用液相色谱串联质谱检测步骤(1)得到的水解液；
(3)以糖胺聚糖羧酸化衍生物作为标准品，按照步骤(1)的方法水解其不同梯度浓度的溶液，按照步骤(2)的方法检测不同浓度标准品溶液的水解液中式(I)所示化合物的质谱信号峰面积，以质谱信号峰面积对糖胺聚糖羧酸化衍生物标准品的量做标准曲线，根据该标准曲线，通过步骤(2)测定的式(I)所示化合物的质谱峰面积计算样品中糖胺聚糖羧酸化衍生物的含量；
所述糖胺聚糖羧酸化衍生物为包含式(II)所示结构单元和任选的式(III)所示结构单元的糖胺聚糖类化合物：
Figure PCTCN2021142793-appb-000002
其中，各R a独立地为-SO 3H或-H，R b独立地为H、-SO 3H或-C(O)CH 3，R c独立地为-SO 3H或-H。
优选地，所述糖胺聚糖为肝素或硫酸乙酰肝素；所述糖胺聚糖羧酸化衍生物经过两步氧化反应获得：(1)糖胺聚糖中糖醛酸上相邻的二醇被氧化开环形成二醛结构，(2)二醛结构进一步被氧化得到双羧酸结构。
优选地，所述式(I)所示化合物具有以下结构式中至少一种的结构：
Figure PCTCN2021142793-appb-000003
Figure PCTCN2021142793-appb-000004
其中，化合物(a)的质谱信号为MS(ESI,neg.ion)m/z:432.0[M-H] -；
化合物(b)的质谱信号为MS(ESI,neg.ion)m/z:390.0[M-H] -；
化合物(c)的质谱信号为MS(ESI,neg.ion)m/z:522.98[M-2H] 2-。
本申请所涉及的糖胺聚糖羧酸化衍生物包含如上所述式(II)所示的结构单元和任选的式(III)所示的结构单元，即糖胺聚糖化合物中己糖醛酸结构部分或全部开环。
本申请所涉及的糖胺聚糖羧酸化衍生物经过水解后可以得到式(I)所示化合物，反应机理如方案1和2所示，其中，各R a独立地为-SO 3H或-H，各R b独立地为H、-SO 3H或-C(O)CH 3，各R c独立地为-SO 3H或-H，n为0、1、2、3、4或5：
方案1：
Figure PCTCN2021142793-appb-000005
方案2：
Figure PCTCN2021142793-appb-000006
在本申请所涉及的糖胺聚糖羧酸化衍生物的每条多糖链中，各双糖结构单元以任意顺序排列。
优选地，所述糖胺聚糖羧酸化衍生物的重均分子量为3000-20000Da，例如3000Da、5000Da、7000Da、8000Da、9000Da、10000Da、11000Da、12000Da、13000Da、13500Da、14000Da、16000Da、18000Da或20000Da等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。优选7000-14000Da，进一步优选8000-13500Da。
所述糖胺聚糖羧酸化衍生物的开环度为10-100％，例如10％、20％、25％、30％、40％、50％、60％、70％、80％、90％或100％等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。优选25-80％，进一步优选25-60％。
本申请中的术语“开环度”是指开环的糖醛酸残基数量与总的糖醛酸残基数量之比，其检测和计算参考文献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中的核磁方法进行。
优选地，步骤(1)所述将含糖胺聚糖羧酸化衍生物的样品进行水解的处理方式为加热。
优选地，所述加热的温度为70-100℃，例如70℃、75℃、80℃、82℃、85℃、87℃、95℃或100℃等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。优选85-95℃。
优选地，所述加热的时间为12-168h，例如12h、16h、24h、36h、48h、55h、60h、70h、72h、75h、78h、80h、90h、96h、120h、144h或168h等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。优选12-120h。
在上述加热温度和加热时间的条件下，本申请所涉及的糖胺聚糖羧酸化衍生物水解可以得到质谱丰度较高的化合物(a)、化合物(b)或化合物(c)，综合考虑检测效率、检测准确度精密度等选择反应的温度和时间。
当所述含糖胺聚糖羧酸化衍生物的样品为生物样品(如血液、尿液)时，需要对步骤(1)得到的水解液进行检测预处理，所述预处理包括：将水解液与三氟乙酸溶液和乙腈-甲醇溶液混合，之后静置、离心，取上清液干燥后再用水复溶。
优选地，所述三氟乙酸溶液的加入量以体积计为水解液的0.5-1.5％，例如0.5％、0.8％、1.0％、1.2％或1.5％等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述三氟乙酸溶液的浓度为4-6％，例如4％、5％或6％等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述乙腈-甲醇溶液的加入量以体积计为水解液体积的1-5倍，例如1倍、1.5倍、1.8倍、2.0倍、2.2倍、2.5倍、3倍、4倍、5倍等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。优选地为1-3倍，更优选地为1.5-2.5倍。
优选地，所述乙腈-甲醇溶液中乙腈与甲醇的体积比为1:0.5-1:1.5，例如1:0.5、1:0.8、1:1、1:1.2或1:1.5等，该数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述静置的温度为-25～-15℃，例如-25℃、-20℃、-15℃等；时间为15-25min，例如15min、18min、20min、22min、25min等，上述各项数值范围内的其他具体点值均可选择，在此便不再一一赘述。
优选地，所述液相色谱为反相色谱、尺寸排阻色谱或亲水色谱。
优选地，所述液相色谱的流动相为流动相A和流动相B；所述流动相A为六氟异丙醇和戊胺的水溶液；所述流动相B为六氟异丙醇和戊胺的乙腈-水溶液；
所述流动相A为含有45-55mM(例如45mM、48mM、50mM、52mM、55mM等)六氟异丙醇和13-17mM(例如13mM、15mM、17mM等)戊胺的水溶液；所述流动相B为含有45-55mM(例如45mM、48mM、50mM、52mM、55mM等)六氟异丙醇和13-17mM(例如13mM、15mM、17mM等)戊胺的乙腈-水溶液；所述流动相B中乙腈与水的体积比为70:30-80:20(例如70:30、75:25、80:20等)。上述各项数值范围内的其他具体点值均可选择，在此便不再一一赘述。
进一步优选地，所述液相色谱的流动相为流动相A和流动相B，具体如下表所示。
流动相A 50mM六氟异丙醇，15mM戊胺，H 2O
流动相B 50mM六氟异丙醇，15mM戊胺，乙腈/H 2O(75/25,v/v)
优选地，所述液相色谱的洗脱流程如下表所示：
时间(min) 流速(mL/min) ％A ％B 曲线
初始 0.36 98.0 2.0 初始
3.00 0.36 98.0 2.0 6
10.00 0.36 80.0 20.0 6
15.00 0.36 60.0 40.0 6
15.10 0.36 10.0 90.0 6
18.00 0.36 10.0 90.0 6
18.10 0.36 98.0 2.0 6
22.00 0.36 98.0 2.0 6
在本申请中，所述质谱条件示例性地可以选择如下条件内容：
条件内容 名称/指标
质谱仪 Waters Xevo G2-S QTOF
模式 Negative Resolution Mode[负离子分辨率模式]
设定质量 432.0Da
毛细管电压 1.5kV
取样椎 25V
源补偿电压 80V
源温度 120℃
脱溶剂温度 500℃
锥孔气流 50L/Hr
脱溶剂气流 800L/Hr
采集起始分子量 200
采集终止分子量 2000
采集起始时间 1.20mins
采集终止时间 6.0mins
基于第一方面的内容，本申请还提供了一种全新的化合物，具体内容如下：
第二方面，本申请提供一种化合物，所述化合物的结构如式(I)所示：
Figure PCTCN2021142793-appb-000007
其中，各R a独立地为-SO 3H或-H，各R b独立地为H、-SO 3H或-C(O)CH 3，各R c独立地为-SO 3H或-H，n为0、1、2、3、4或5。
优选地，所述化合物具有以下其中之一的结构：
Figure PCTCN2021142793-appb-000008
第三方面，本申请提供一种如第一方面所述的糖胺聚糖羧酸化衍生物的检测方法在糖胺聚糖羧酸化衍生物的药代动力学研究中的应用。
第四方面，本申请提供一种如第一方面所述的糖胺聚糖羧酸化衍生物的检测方法在糖胺聚糖羧酸化衍生物药物制剂质量检测中的应用。
相对于现有技术，本申请具有以下有益效果：
由于糖胺聚糖羧酸化衍生物为非均一物质，在生物样品中直接检测完整的结构不容易实现，本申请发明人发现糖胺聚糖羧酸化衍生物经过水解可以稳定地得到如上所述特定结构的水解产物化合物(a)、化合物(b)或化合物(c)，此类化合物可以通过质谱检测到。通过测定化合物(a)、化合物(b)或化合物(c)，将不同浓度的糖胺聚糖羧酸化衍生物标准品对化合物(a)、化合物(b)或化合物(c)的质谱峰面积建立标准曲线；再通过水解样品中糖胺聚糖羧酸化衍生物并检测化合物(a)、化合物(b)或化合物(c)的质谱峰面积；含糖胺聚糖羧酸化衍生物的样品经过水解后用液相质谱测定化合物(a)、化合物(b)或化合物(c)的质谱峰面积，根据标准曲线可以间接计算出样品中糖胺聚糖羧酸化衍生物的量。该检测方 法的专属性强、准确度高、精密度好、定量限低、检测限低。
在阅读并理解了详细描述后，可以明白其他方面。
附图说明
图1是化合物(a)的质谱图；
图2是化合物(a)的二级质谱图；
图3是化合物(a)的氢谱图；
图4是化合物(a)的碳谱图；
图5是化合物(a)的 13C DEPT 135°谱图；
图6是化合物(a)的 1H- 1H COSY谱图；
图7是化合物(a)的TOCSY谱图；
图8是化合物(a)的HSQC谱图；
图9是化合物(a)的HMBC谱图；
图10是化合物(c)的质谱图；
图11是化合物(c)的二级质谱图；
图12是化合物(c)的氢谱图；
图13是化合物(c)的碳谱图；
图14是化合物(c)的 13C DEPT 135°谱图；
图15是化合物(c)的 1H- 1H COSY谱图；
图16是化合物(c)的TOCSY谱图；
图17是化合物(c)的ROESY谱图；
图18是化合物(c)的HSQC谱图；
图19是化合物(c)的HMBC谱图。
具体实施方式
下面通过具体实施方式来进一步说明本申请的技术方案。本领域技术人员应该明了，所述实施例仅仅是帮助理解本申请，不应视为对本申请的具体限制。
下述实施例所涉及的SD大鼠购自北京维通利华实验动物技术有限公司。
下述实施例所涉及的糖胺聚糖羧酸化衍生物H1011由专利CN111670038A中实施例3公开的制备方法制备得到，其重均分子量为9161Da，开环度为43.1％。
实施例1
化合物(a)、化合物(b)和化合物(c)的制备：
称取H1011(400mg)溶于水(4.0mL)中，将H1011水溶液加热至85℃反应72h，然后冷却至25℃；反应产物通过色谱技术分离纯化(色谱柱为Dionex IonPac AS11-HC，洗脱液M和N(具体组分见表1))，然后脱盐冻干得到化合物(a)、化合物(b)和化合物(c)。
Figure PCTCN2021142793-appb-000009
表1
Figure PCTCN2021142793-appb-000010
富集得到的化合物(a)、化合物(b)和化合物(c)通过质谱和核磁共振波谱法(一维 1H-NMR、一维 13CNMR、 13C DEPT 135°、 1H- 1H COSY、二维TOCSY、HSQC、HMBC、二维DOSY)鉴定结构。
其中化合物(a)的表征数据如下：
MS(ESI,neg.ion)m/z:432.0[M-H] -；
1H NMR(600MHz,D 2O):δ4.87(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).
13C NMR(151MHz,D 2O):δ24.79,56.23,69.39,71.95,73.04,74.14,76.51,83.25,99.56,177.34,178.79,179.99.
化合物(a)的表征图谱如图1-图9所示。
其中化合物(b)的表征数据如下：
MS(ESI,neg.ion)m/z:390.0[M-H] -；
其中化合物(c)的表征数据如下：
MS(ESI,neg.ion)m/z:522.98[M-2H] 2-；
1H NMR(600MHz,D 2O):δ5.37(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).
13C NMR(151MHz,D 2O):δ60.45,60.63,68.92,69.10,70.44,70.60,71.82,72.44,72.80,73.05,73.50,74.54,77.54,78.74,79.51,81.24,100.90,101.38,101.69,175.69,176.18,177.23.
化合物(c)的表征图谱如图10-图19所示。
实施例2
本实施例将本申请所涉及的糖胺聚糖羧酸化衍生物的检测方法应用于药代动力学研究(以化合物(a)作为检测对象)，具体内容如下：
(1)试验方法
(1.1)实验动物：健康成年雄性SD大鼠6只，其中3只用于取空白血浆制作标准曲线，3只用于进行大鼠单次给药后血药浓度检测。
(1.2)药物配制：称取糖胺聚糖羧酸化衍生物H1011用水配置60mg/kg的药物溶液。
(1.3)给药和样品采集：皮下注射给药60mg/kg后，分别在时间点0、0.25、0.5、1、2、4、6、8、24h采血，采集全血后置于K2EDTA抗凝试管中，随后离心15min，分离得到血浆样品。
(1.4)标准曲线的绘制：
(1.4.1)称取糖胺聚糖羧酸化衍生物H1011并将其配成1mg/mL的水溶液，以上述空白血浆作为稀释液逐级稀释H1011水溶液，配置2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、 64μg/mL、128μg/mL、256μg/mL的标准溶液，在85℃条件下水解72h；
(1.4.2)水解后的标准溶液进行检测前处理，向水解后的标准溶液中加入百分之一体积比的三氟乙酸(5％，v/v)和两倍体积的乙腈/甲醇(v/v，50/50)，混匀，然后置于-20℃静置20min，离心；取上清液干燥后再用超纯水复溶；
(1.4.3)进行液相色谱串联质谱检测，测得谱图，色谱条件如表2所示，质谱条件如表3所示：
表2
Figure PCTCN2021142793-appb-000011
表3
条件内容 名称/指标
质谱仪 Waters Xevo G2-S QTOF
条件内容 名称/指标
模式 Negative Resolution Mode[负离子分辨率模式]
设置分子量 432.0Da
毛细管电压 1.5kV
取样椎 25V
源补偿电压 80V
源温度 120℃
脱溶剂温度 500℃
锥孔气流 50L/Hr
脱溶剂气流 800L/Hr
采集起始分子量 200
采集终止分子量 2000
采集起始时间 1.20mins
采集终止时间 7.0mins
(1.4.4)以H1011标准溶液与化合物(a)质谱峰面积线性关系作标准曲线，化合物(a)质谱信号为MS(ESI,neg.ion)m/z:432.0[M-H] -；线性方程为：y＝36.2154x-0.3216；相关系数为：R 2＝0.9987，其中，y为质谱峰面积，x为标准溶液浓度。
(1.5)检测血浆样品中H1011的含量：
(1.5.1)将步骤(1.3)获得的血浆样品在85℃条件下水解72h，然后向其中加入百分之一体积比的三氟乙酸(5％，v/v)和两倍体积的乙腈/甲醇(v/v，50/50)，混匀，然后置于-20℃静置20min，离心；取上清液干燥后再用超纯水复溶；
(1.5.2)进行液相色谱串联质谱检测，测得化合物(a)质谱峰面积，色谱条件如上表2所示，质谱条件如上表3所示；
(1.5.3)根据化合物(a)质谱峰面积结合步骤(1.4)中绘制的标准曲线，计算得到各时间点血浆样品中H1011的浓度，并根据药物浓度-时间曲线计算药代动力学参数。
(2)试验结果如表4所示：
表4
给药途径 剂量 AUC 0-24(h*μg/ml) T 1/2(h) CL(ml/min/kg)
i.h. 60mg/kg 537.47 3.75 0.110
(3)方法学验证
(3.1)专属性
配制超纯水对照溶液和空白血浆对照溶液，经过与上述相同的高温水解、前处理过程，然后经过上述相同条件的液相色谱串联质谱检测，在超纯水对照和血浆对照溶液中未检测到432.0Da的信号峰，即化合物(a)的质谱信号峰，表明超纯水和空白血浆对检测无干扰，本检测方法专属性强。
(3.2)定量限和检测限
经计算，本方法的定量限为0.8μg/mL，检测限为0.2μg/mL。
(3.3)准确度
设置1μg/mL、50μg/mL、120μg/mL三个浓度，测试H1011血浆样品，经计算回收率为82.6％-110.8％，且各个浓度6次实验结果的RSD值依次为4.2％、2.2％和1.3％。
(3.4)精密度
选取50μg/mL H1011血浆溶液，由两名不同检测员各检测6次，第一名检验员6次实验结果的RSD为2.0％，第二名检验员6次实验结果的RSD为1.8％，两名检验员12次实验结果的RSD为2.1％，均满足≤10.0％的可接受标准。方法精密度良好。
(3.5)溶液稳定性
选取50μg/mL H1011血浆溶液，水解并进行前处理，样品溶液第5天的检测结果为0时间的97.2％，符合标准。
实施例3
本实施例将本申请所涉及的糖胺聚糖羧酸化衍生物的检测方法应用于药代动力学研究(以化合物(b)作为检测对象)，具体内容如下：
(1)试验方法
(1.1)实验动物：健康成年雄性SD大鼠6只，其中3只用于取空白血浆制作标准曲线，3只用于进行大鼠单次给药后血药浓度检测。
(1.2)药物配制：称取糖胺聚糖羧酸化衍生物H1011用水配置60mg/kg的药物溶液。
(1.3)给药和样品采集：皮下注射给药60mg/kg后，分别在时间点0、0.25、0.5、1、2、4、6、8、24h采血，采集全血后置于K2EDTA抗凝试管中，随后离心15min，分离得到血浆样品。
(1.4)标准曲线的绘制：
(1.4.1)称取糖胺聚糖羧酸化衍生物H1011并将其配成1mg/mL的水溶液，以上述空白血浆作为稀释液逐级稀释H1011水溶液，配置2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、64μg/mL、128μg/mL、256μg/mL的标准溶液，在90℃条件下水解48h；
(1.4.2)水解后的标准溶液进行检测前处理，向水解后的标准溶液中加入百分之一体积比的三氟乙酸(5％，v/v)和两倍体积的乙腈/甲醇(v/v，50/50)，混匀，然后置于-20℃静置20min，离心；取上清液干燥后再用超纯水复溶；
(1.4.3)进行液相色谱串联质谱检测，测得谱图，色谱条件如上表2所示，质谱条件如上表3所示，设置分子量为390.0Da；
(1.4.4)以H1011标准溶液与化合物(b)质谱峰面积线性关系作标准曲线，化合物(b)质谱信号为MS(ESI,neg.ion)m/z:390.0[M-H] -；做出线性方程为：y＝26.0235x；相关系数为0.9981，其中，y为质谱峰面积，x为标准溶液浓度。
(1.5)检测血浆样品中H1011的含量：
(1.5.1)将步骤(1.3)获得的血浆样品在90℃条件下水解48h，然后向其中加入百分之一体积比的三氟乙酸(5％，v/v)和两倍体积的乙腈/甲醇(v/v，50/50)，混匀，然后置于-20℃静置20min，离心；取上清液干燥后再用超纯水复溶；
(1.5.2)进行液相色谱串联质谱检测，测得化合物(b)质谱峰面积，色谱条件如上表2所示，质谱条件如上表3所示，设置分子量为390.0Da；
(1.5.3)根据化合物(b)质谱峰面积结合步骤(1.4)中绘制的标准曲线，计算得到各时间点血浆样品中H1011的浓度，并根据药物浓度-时间曲线计算药代动力学参数。
(2)试验结果如表5所示：
表5
给药途径 剂量 AUC 0-24(h*μg/ml) T 1/2(h) CL(ml/min/kg)
i.h. 60mg/kg 483.87 3.04 0.089
(3)方法学验证
(3.1)专属性
配制超纯水对照溶液和空白血浆对照溶液，经过与上述相同的高温水解、前处理过程，然后经过上述相同条件的液相色谱串联质谱检测，在超纯水对照和血浆对照溶液中未检测到 390.0Da的信号峰，即化合物(b)的质谱信号峰，表明超纯水和空白血浆对检测无干扰，本检测方法专属性强。
(3.2)定量限和检测限
经计算，本方法的定量限为1.1μg/mL，检测限为0.55μg/mL。
(3.3)准确度
设置1μg/mL、50μg/mL、120μg/mL三个浓度，测试H1011血浆样品，经计算回收率为81.2％-115.8％，且各个浓度6次实验结果的RSD值依次为5.4％、1.8％和2.0％。
(3.4)精密度
选取50μg/mL H1011血浆溶液，由两名不同检测员各检测6次，第一名检验员6次实验结果的RSD为1.6％，第二名检验员6次实验结果的RSD为2.6％，两名检验员12次实验结果的RSD为2.2％，均满足≤10.0％的可接受标准。方法精密度良好。
(3.5)溶液稳定性
选取50μg/mL H1011血浆溶液，水解并进行前处理，样品溶液第5天的检测结果为0时间的97.8％，符合标准。
实施例4
本实施例将本申请所涉及的糖胺聚糖羧酸化衍生物的检测方法应用于药代动力学研究(以化合物(c)作为检测对象)，具体内容如下：
(1)试验方法
(1.1)实验动物：健康成年雄性SD大鼠6只，其中3只用于取空白血浆制作标准曲线，3只用于进行大鼠单次给药后血药浓度检测。
(1.2)药物配制：称取糖胺聚糖羧酸化衍生物H1011用水配置20mg/kg的药物溶液。
(1.3)给药和样品采集：皮下注射给药20mg/kg后，分别在时间点0、0.25、0.5、1、2、4、6、8、24h采血，采集全血后置于K2EDTA抗凝试管中，随后离心15min，分离得到血浆样品。
(1.4)标准曲线的绘制：
(1.4.1)称取糖胺聚糖羧酸化衍生物H1011并将其配成1mg/mL的水溶液，以上述空白血浆作为稀释液逐级稀释H1011水溶液，配置2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL、64μg/mL、128μg/mL、256μg/mL的标准溶液，在90℃条件下水解36h；
(1.4.2)水解后的标准溶液进行检测前处理，向水解后的标准溶液中加入百分之一体积比的三氟乙酸(5％，v/v)和两倍体积的乙腈/甲醇(v/v，50/50)，混匀，然后置于-20℃静置20min，离心；取上清液干燥后再用超纯水复溶；
(1.4.3)进行液相色谱串联质谱检测，测得谱图，色谱条件如上表2所示，质谱条件如上表3所示，设置分子量为522.98Da；
(1.4.4)以H1011标准溶液与化合物(c)质谱峰面积线性关系作标准曲线，化合物(c)质谱信号为MS(ESI,neg.ion)m/z:522.98[M-2H] 2-；做出线性方程为：y＝23.6225x；相关系数为0.9986，其中，y为质谱峰面积，x为标准溶液浓度。。
(1.5)检测血浆样品中H1011的含量：
(1.5.1)将步骤(1.3)获得的血浆样品在90℃条件下水解36h，然后向其中加入百分之一体积比的三氟乙酸(5％，v/v)和两倍体积的乙腈/甲醇(v/v，50/50)，混匀，然后置于-20℃静置20min，离心；取上清液干燥后再用超纯水复溶；
(1.5.2)进行液相色谱串联质谱检测，测得化合物(c)质谱峰面积，色谱条件如上表2所示，质谱条件如上表3所示；
(1.5.3)根据化合物(c)质谱峰面积结合步骤(1.4)中绘制的标准曲线，计算得到各时间点血浆样品中H1011的浓度，并根据药物浓度-时间曲线计算药代动力学参数。
(2)试验结果如表6所示：
表6
给药途径 剂量 AUC 0-24(h*μg/ml) T 1/2(h) CL(ml/min/kg)
i.h. 20mg/kg 237.44 4.04 0.073
(3)方法学验证
(3.1)专属性
配制超纯水对照溶液和空白血浆对照溶液，经过与上述相同的高温水解、前处理过程，然后经过上述相同条件的液相色谱串联质谱检测，在超纯水对照和血浆对照溶液中未检测到522.98Da的信号峰，即化合物(c)的质谱信号峰，表明超纯水和空白血浆对检测无干扰，本检测方法专属性强。
(3.2)定量限和检测限
经计算，本方法的定量限为2.0μg/mL，检测限为1.0μg/mL。
(3.3)准确度
设置2μg/mL、50μg/mL、120μg/mL三个浓度，测试H1011血浆样品，经计算回收率为80.3％-108.8％，且各个浓度6次实验结果的RSD值依次为8.9％、6.5％和5.4％。
(3.4)精密度
选取50μg/mL H1011血浆溶液，由两名不同检测员各检测6次，第一名检验员6次实验结果的RSD为6.5％，第二名检验员6次实验结果的RSD为7.2％，两名检验员12次实验结果的RSD为7.4％，均满足≤10.0％的可接受标准。方法精密度良好。
(3.5)溶液稳定性
选取50μg/mL H1011血浆溶液，水解并进行前处理，样品溶液第5天的检测结果为0时间的90.2％，符合标准。

Claims (11)

  1. 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法，其特征在于，所述方法包括如下步骤：
    (1)将含糖胺聚糖羧酸化衍生物的样品进行水解，得到含有式(I)所示化合物的水解液：
    Figure PCTCN2021142793-appb-100001
    其中，各R a独立地为-SO 3H或-H，各R b独立地为H、-SO 3H或-C(O)CH 3，各R c独立地为-SO 3H或-H，n为0、1、2、3、4或5；
    (2)采用液相色谱串联质谱检测步骤(1)得到的水解液；
    (3)以糖胺聚糖羧酸化衍生物作为标准品，按照步骤(1)的方法水解其不同梯度浓度的溶液，按照步骤(2)的方法检测不同浓度标准品溶液的水解液中式(I)所示化合物的质谱信号峰面积，以质谱信号峰面积对糖胺聚糖羧酸化衍生物标准品的量做标准曲线，根据该标准曲线，通过步骤(2)测定的式(I)所示化合物的质谱峰面积计算样品中糖胺聚糖羧酸化衍生物的含量；
    所述糖胺聚糖羧酸化衍生物为包含式(II)所示结构单元和任选的式(III)所示结构单元的糖胺聚糖类化合物：
    Figure PCTCN2021142793-appb-100002
    其中，各R a独立地为-SO 3H或-H，R b独立地为H、-SO 3H或-C(O)CH 3，R c独立地为-SO 3H或-H。
  2. 如权利要求1所述的方法，其特征在于，所述糖胺聚糖为肝素或硫酸乙酰肝素；
    所述糖胺聚糖羧酸化衍生物经过两步氧化反应获得：(1)糖胺聚糖中糖醛酸上相邻的二醇被氧化开环形成二醛结构，(2)二醛结构进一步被氧化得到双羧酸结构。
  3. 如权利要求1所述的方法，其特征在于，所述式(I)所示化合物具有以下结构式中至少一种的结构：
    Figure PCTCN2021142793-appb-100003
  4. 如权利要求1所述的方法，其特征在于，所述糖胺聚糖羧酸化衍生物的重均分子量为3000-20000Da，优选7000-14000Da，进一步优选8000-13500Da；
    所述糖胺聚糖羧酸化衍生物的开环度为10-100％，优选25-80％，进一步优选25-60％。
  5. 如权利要求1-4中任一项所述的方法，其特征在于，步骤(1)所述将含糖胺聚糖羧酸化衍生物的样品进行水解的处理方式为加热；
    优选地，所述加热的温度为70-100℃，优选85-95℃；
    优选地，所述加热的时间为12-168h，优选12-120h；
  6. 如权利要求1-5中任一项所述的方法，其特征在于，所述液相色谱为反相色谱、尺寸排阻色谱或亲水色谱；
    优选地，所述液相色谱的流动相为流动相A和流动相B；所述流动相A为六氟异丙醇和戊胺的水溶液；所述流动相B为六氟异丙醇和戊胺的乙腈-水溶液；
    优选地，所述流动相A为含有45-55mM六氟异丙醇和13-17mM戊胺的水溶液；所述流动相B为含有45-55mM六氟异丙醇和13-17mM戊胺的乙腈-水溶液；所述流动相B中乙腈与水的体积比为70:30-80:20；
    优选地，所述液相色谱的流动相为流动相A和流动相B，具体如下表所示。
    流动相A 50mM六氟异丙醇，15mM戊胺，H 2O 流动相B 50mM六氟异丙醇，15mM戊胺，乙腈/H 2O(75/25,v/v)
  7. 如权利要求1-6中任一项所述的方法，当所述含糖胺聚糖羧酸化衍生物的样品为生物样品时，需对步骤(1)得到的水解液进行检测预处理；所述预处理包括：将水解液与三氟乙酸溶液和乙腈-甲醇溶液混合，之后静置、离心，取上清液干燥后再用水复溶；
    优选地，所述生物样品包括血液、尿液；
    优选地，所述三氟乙酸溶液的加入量以体积计为水解液体积的0.5-1.5％；
    优选地，所述三氟乙酸溶液的浓度为4-6％；
    优选地，所述乙腈-甲醇溶液的加入量以体积计为水解液体积的1-5倍，优选地为1-3倍，更优选地为1.5-2.5倍；
    优选地，所述乙腈-甲醇溶液中乙腈与甲醇的体积比为1:0.5-1:1.5；
    优选地，所述静置的温度为-25～-15℃，时间为15-25min。
  8. 一种化合物，其特征在于，所述化合物的结构如式(I)所示：
    Figure PCTCN2021142793-appb-100004
    其中，各R a独立地为-SO 3H或-H，各R b独立地为H、-SO 3H或-C(O)CH 3，各R c独立地为-SO 3H或-H，n为0、1、2、3、4或5。
  9. 如权利要求8所述的化合物，其特征在于，所述化合物具有以下其中之一的结构：
    Figure PCTCN2021142793-appb-100005
  10. 如权利要求1-7中任一项所述的糖胺聚糖羧酸化衍生物的检测方法在糖胺聚糖羧酸化衍生物的药代动力学研究中的应用。
  11. 如权利要求1-6中任一项所述的糖胺聚糖羧酸化衍生物的检测方法在糖胺聚糖羧酸化衍生物药物制剂质量检测中的应用。
PCT/CN2021/142793 2020-12-31 2021-12-30 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用 WO2022143853A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP21914584.4A EP4273540A1 (en) 2020-12-31 2021-12-30 Method for detecting content of glycosaminoglycan carboxylated derivative in sample, and application thereof
CN202180088157.2A CN116710772B (zh) 2020-12-31 2021-12-30 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用
KR1020237026204A KR20230128095A (ko) 2020-12-31 2021-12-30 샘플 중의 글리코사미노글리칸 카르복실화 유도체 함량을검출하기 위한 방법 및 그의 응용
US18/259,935 US20240085383A1 (en) 2020-12-31 2021-12-30 Method for detecting content of glycosaminoglycan carboxylated derivative in sample, and application thereof
JP2023540536A JP2024505364A (ja) 2020-12-31 2021-12-30 サンプル中のカルボン酸化したグリコサミノグリカン誘導体の含有量を検出するための方法及びその使用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011624408.8 2020-12-31
CN202011624408.8A CN114689722A (zh) 2020-12-31 2020-12-31 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用

Publications (1)

Publication Number Publication Date
WO2022143853A1 true WO2022143853A1 (zh) 2022-07-07

Family

ID=82133842

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/142793 WO2022143853A1 (zh) 2020-12-31 2021-12-30 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用

Country Status (7)

Country Link
US (1) US20240085383A1 (zh)
EP (1) EP4273540A1 (zh)
JP (1) JP2024505364A (zh)
KR (1) KR20230128095A (zh)
CN (2) CN114689722A (zh)
TW (1) TWI807565B (zh)
WO (1) WO2022143853A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114689722A (zh) * 2020-12-31 2022-07-01 深圳市海普瑞药业集团股份有限公司 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102329397A (zh) * 2011-10-19 2012-01-25 中国科学院昆明植物研究所 一种岩藻糖化糖胺聚糖衍生物及其制备方法
CN105744940A (zh) 2013-11-06 2016-07-06 ＂G.龙佐尼＂ S.R.生化高科技研究中心 葡糖胺聚糖的羧基化衍生物以及作为药物的用途
CN106188171A (zh) * 2016-07-18 2016-12-07 山东师范大学 一种利用生产谷氨酸的菌渣制备胞壁酸的方法
US20170159237A1 (en) * 2015-12-07 2017-06-08 Clean Chemistry Methods of pulp fiber treatment
WO2019149179A1 (zh) * 2018-02-02 2019-08-08 深圳市海普瑞药业集团股份有限公司 糖胺聚糖衍生物及其制备方法和用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1582531A1 (en) * 2004-03-24 2005-10-05 Aventis Pharma S.A. Process for oxidizing unfractionated heparins and detecting presence or absence of glycoserine in heparin and heparin products
WO2021231842A1 (en) * 2020-05-15 2021-11-18 Academia Sinica Flow chemistry system and method for carbohydrate analysis
CN114689722A (zh) * 2020-12-31 2022-07-01 深圳市海普瑞药业集团股份有限公司 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102329397A (zh) * 2011-10-19 2012-01-25 中国科学院昆明植物研究所 一种岩藻糖化糖胺聚糖衍生物及其制备方法
CN105744940A (zh) 2013-11-06 2016-07-06 ＂G.龙佐尼＂ S.R.生化高科技研究中心 葡糖胺聚糖的羧基化衍生物以及作为药物的用途
US20170159237A1 (en) * 2015-12-07 2017-06-08 Clean Chemistry Methods of pulp fiber treatment
CN106188171A (zh) * 2016-07-18 2016-12-07 山东师范大学 一种利用生产谷氨酸的菌渣制备胞壁酸的方法
WO2019149179A1 (zh) * 2018-02-02 2019-08-08 深圳市海普瑞药业集团股份有限公司 糖胺聚糖衍生物及其制备方法和用途
CN111670038A (zh) 2018-02-02 2020-09-15 深圳市海普瑞药业集团股份有限公司 糖胺聚糖衍生物及其制备方法和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GUERRINI, MGUGLIERI, SNAGGI, ASASISEKHARAN, R: "Low molecular weight heparins: Structural differentiation by bidimentional nuclear magnetic resonance spectroscopy", SEMINARS IN THROMBOSIS AND HEMOSTASIS, vol. 33, 2007, pages 478 - 487

Also Published As

Publication number Publication date
TWI807565B (zh) 2023-07-01
CN114689722A (zh) 2022-07-01
JP2024505364A (ja) 2024-02-06
EP4273540A1 (en) 2023-11-08
CN116710772A (zh) 2023-09-05
CN116710772B (zh) 2024-04-02
KR20230128095A (ko) 2023-09-01
US20240085383A1 (en) 2024-03-14
TW202227815A (zh) 2022-07-16

Similar Documents

Publication Publication Date Title
Raetz et al. Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4
Zhao et al. UPLC–MSE application in disease biomarker discovery: the discoveries in proteomics to metabolomics
Lin et al. A sensitive and specific HPGPC‐FD method for the study of pharmacokinetics and tissue distribution of Radix Ophiopogonis polysaccharide in rats
Gao et al. Integrated GC–MS and LC–MS plasma metabonomics analysis of ankylosing spondylitis
CN110542735A (zh) 超高效液相质谱联用高通量测定多种脂溶性维生素的方法
WO2022143853A1 (zh) 一种用于检测样品中糖胺聚糖羧酸化衍生物含量的方法及其应用
Sun et al. Hydrophilic interaction chromatography-multiple reaction monitoring mass spectrometry method for basic building block analysis of low molecular weight heparins prepared through nitrous acid depolymerization
Chi et al. Characterization of a protein-bound polysaccharide from Herba Epimedii and its metabolic mechanism in chronic fatigue syndrome
CN110208393A (zh) 一种检测血清中5种雄激素的方法
Guo et al. Internal exposure of Chinese children from a typical coastal city to bisphenols and possible association with thyroid hormone levels
Jansen et al. A novel, quantitative assay for homocarnosine in cerebrospinal fluid using stable-isotope dilution liquid chromatography–tandem mass spectrometry
CN104897835A (zh) 一种利用uplc-q-tof/ms技术快速测定寡聚古罗糖醛酸的方法
Malchow-Møller et al. Enzymatic determination of total 3α-hydroxy bile acids in faeces: Validation in healthy subjects of a rapid method suitable for clinical routine purpose
WO2020020152A1 (zh) 一种达肝素钠亚硝酸降解产物的分析方法及其应用
Tan et al. Simultaneous determination of neutral and acidic human milk oligosaccharides (HMOs) by liquid chromatography with tandem mass spectrometry (LC-MS/MS)
Zhang Biodistribution evaluation of icaritin in rats by ultra-performance liquid chromatography–tandem mass spectrometry
CN117092228A (zh) 一种基于hplc-ms/ms联用检测生物样品中水黄皮素的方法
Silvestro et al. High-performance liquid chromatographic-mass spectrometric analysis of oligosaccharides from enzymatic digestion of glycosaminoglycans: Application to human samples
Chifang et al. Determination of anabolic steroid residues (medroxyprogesterone acetate) in pork by ELISA and comparison with liquid chromatography tandem mass spectrometry
CN111413439A (zh) 一种快速亲水相互作用色谱-串联质谱法测定血浆中二甲双胍的方法
Chen et al. Inhibition of catechol-O-methyltransferase (COMT) by heparin oligosaccharides with specific structures
Licea-Perez et al. Development of an ultra-sensitive assay for the determination of an aminoalkyl glucosaminide 4-phosphate, GSK1795091, in plasma to support a first time in human study
CN108828096B (zh) 一种同步检测食用菌中麦角固醇与维生素d2的方法与应用
CN107121508A (zh) 一种蛇胆掺伪鉴别的方法
CN109946408B (zh) 测定人血浆中连翘苷及代谢物苷元葡萄糖醛酸结合物、苷元硫酸结合物的检测方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21914584

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202180088157.2

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 18259935

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2023540536

Country of ref document: JP

ENP Entry into the national phase

Ref document number: 20237026204

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021914584

Country of ref document: EP

Effective date: 20230731