Method for identifying whether porcine heparin is adulterated with heparin from ruminants

Abstract

The present application is related to a method for identifying whether porcine heparin is adulterated with heparin from ruminants, comprising: (1) respectively detecting the contents of trisaccharide(4S) and ΔUA2S-GlcNAc6S (ΔIA) in a sample and at least three batches of porcine heparin standards; (2) calculating a ratio of the trisaccharide(4S) to the ΔIA as well as a standard deviation (SD) of the ratio in the porcine heparin standards; when the ratio of the trisaccharide(4S) to the ΔIA in the sample exceeds a maximum value of the ratio in the porcine heparin standards+3SD, where the sample is considered to be mixed or adulterated with heparin from ruminants; wherein the detection method used is hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) or multiple reaction monitoring (MRM). The method can distinguish porcine heparin from ovine and bovine heparin based on the structural differences, regardless of the production process the heparin has undergone.

Claims

1. A method for identifying whether porcine heparin is adulterated with heparin from ruminants, comprising the following steps: (1) respectively detecting contents of trisaccharide(4S) and ΔUA2S-GlcNAc6S (ΔIA) in a sample and at least three batches of porcine heparin standards; (2) calculating a ratio content of the trisaccharide(4S) to the ΔIA as well as a standard deviation (SD) of the ratio in the porcine heparin standards; when the ratio content of the trisaccharide(4S) to the ΔIA in the sample exceeds a maximum value of the ratio in the porcine heparin standards+3SD, the sample is considered to be mixed with heparin from ruminants; wherein, the contents of the trisaccharide(4S) and ΔIA in the sample in the step (1) are detected by hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, a multiple reaction monitoring (HILIC-MRM) method, or a C18-MRM method; wherein: operating conditions of the hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method are as follows: (1) performing exhaustive enzymatic digestion on the sample with a mixed enzyme of heparinase I, heparinase II and heparinase III; (2) performing quantitative analysis on the trisaccharide(4S) and ΔIA in the step (1) by the hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method, wherein an ion detection form of the trisaccharide(4S) is [M−2H].sup.2-, and/or [M−3H+Na].sup.2-; and an ion detection form of ΔIA is [M−H].sup.−, and/or [M−3H+Na].sup.2-; chromatographic conditions of the hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) method are as follows: analytical column: Phenomenex Luna 3 μm HILIC 200 Å (150×2.0 mm); mobile phase: phase A: 5 mmol/L ammonium acetate aqueous solution; phase B: 5 mmol/L ammonium acetate, and 98% acetonitrile solution; flow rate: 0.15 mL/min; injection volume: 20 pL; step gradient: 0-5 min, 95% B; 5-6 min, 90% B; 6-25 min, 90-84% B; 25-27 min, 84-50% B; 27-31 min, 50-50% B; 31-32 min, 50-95% B; 32-40 min, 95-95% B; parameters of mass spectrometry: instrument: Q Exactive plus; sheath gas: 40 psi; Aux gas: 10 psi; spray voltage: 3.8 kV; capillary temperature: 275° C.; S-lens: 50; m/z: 150-800; and acquisition time: 40 min; the HILIC-MRM method comprises the following operating steps: 1 performing exhaustive enzymatic digestion on the sample with a mixed enzyme of heparinase I, heparinase II and heparinase III; 2 performing a quantitative analysis on the trisaccharide(4S) and the ΔIA in a product of the step 1, wherein in the quantitative analysis of the HILIC-MRM method for the trisaccharide(4S), a parent ion mass-to-charge ratio is 415.5, z=−2, and a daughter ion mass-to-charge ratio is 157.0; and in the quantitative analysis of the HILIC-MRM method for the ΔIA, a parent ion mass-to-charge ratio is 268.5, z=−2, and a daughter ion mass-to-charge ratio is 300.0; chromatographic conditions of the HILIC-MRM method are as follows: analytical column: Phenomenex Luna 3 pm HILIC 200 Å (150×2.0 mm); mobile phase: phase A: 5 mmol/L ammonium acetate aqueous solution; phase B: 5 mmol/L ammonium acetate, and 98% acetonitrile solution; flow rate: 0.15 mL/min; injection volume: 10 μL; the step gradient: 0-5 min, 95% B; 5-12 min, 95-50% B; 12-15 min, 50% B; 15-20 min, 95% B; mass spectrometry conditions of the HILIC-MRM method are as follows: spray voltage: −3.7 kV; spray gas flow rate: 30 arb; acquisition time: 15 min; HILIC-MRM channel parameters are as follows: for the ΔIA, a structure is ΔUA2S-GlcNAc6S, a theoretical molecular weight is 539.0251, a parent ion is m/z=268.5, z=−2, and a daughter ion is m/z=300; for the trisaccharide(4S), a structure is ΔUA2S-GlcNS6S-HexA2S, a theoretical molecular weight is 832.9602, a parent ion is m/z=415.5, z=−2, and a daughter ion is m/z=157.0; the C18-MRM method comprises the following operating steps: 1 performing exhaustive enzymatic digestion on the sample with a mixed enzyme of heparinase I, heparinase II and heparinase III; 2 labelling the sample obtained after the step 1 with a 2-aminoacridone solution (AMAC), reducing the labelled sample with sodium cyanoborohydride, centrifuging to take supernatant and preserving the labelled sample at −20° C. for further use; 3 performing a quantitative analysis on the trisaccharide(4S) and the ΔIA in the step 2 with multi-C18-MRM method, wherein in the quantitative analysis derived from the trisaccharide(4S) labelled with the AMAC, a parent ion mass-to-charge ratio is 512.5, z=−2; and a daughter ion mass-to-charge ratio is 432.6; and in the quantitative analysis derived from the ΔIA labelled with the AMAC, a mass-to-charge ratio is chosen to 732.1, z=−2, and a daughter ion mass-to-charge ratio is 652.1; chromatographic conditions of the C18-MRM method are as follows: analytical column: Kinetex 2.6 pm EVO C18 100A (150×2.1 mm); mobile phase: phase A:50 mmol/L ammonium acetate aqueous solution; mobile phase B: methanol solution; flow rate: 0.3 mL/min; injection volume: 1 μL; column temperature: 45° C.; the step gradient: 0-2 min, 5% B; 2-4 min, 26% B; 4-8 min, 40% B; 8-10 min, 100% B; 10-15 min, 5% B; mass spectrometry conditions of the C18-MRM method are as follows: spray voltage: −3.7 kV; spray gas flow rate: 30 arb; acquisition time: 15 min; mass spectrometry conditions: spray voltage: −3.7 kV; spray gas flow rate: 30 arb; acquisition time: 15 min; C18-MRM channel parameters are as follows: for the ΔIA labelled with the AMAC, a structure is ΔUA2S-GlcNAc6S-AMAC, a theoretical molecular weight is 733.1095, a parent ion is m/z=732.1022, z=−1, and a daughter ion is m/z=652.1454; for the trisaccharide(4S) labelled with the AMAC, a structure is ΔUA2S-GlcNS6S-HexA2S-AMAC, a theoretical molecular weight is 1027.0446, a parent ion is m/z=512.5150, z=−2, and a daughter ion is m/z=432.5366.

2. The identification method according to claim 1, wherein for the trisaccharide(4S), the ion [M−2H].sup.2- has a mass-to-charge ratio of 415.4729, and the ion [M−3H+Na].sup.2- has a mass-to-charge ratio of 426.4639; for the ΔIA, the ion [M−H].sup.− has a mass-to-charge ratio of 268.5, and the ion [M−3H+Na].sup.2- has a mass-to-charge ratio of 279.5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is high-resolution mass spectra of (A) ΔIA ([M−2H].sup.2-); (B) trisaccharide(4S) ([M−2H].sup.2-); (C) ΔIA ([M−3H+Na].sup.2-) and (D) trisaccharide(4S) ([M−3H+Na].sup.2-) by HILIC-MS analysis.

(2) FIG. 2 is extracted ion chromatogram (EIC) of trisaccharide(4S) and ΔIA in ovine, bovine and porcine heparin by HILIC-MS analysis.

(3) FIG. 3 shows HILIC-MS results, ratios of trisaccharide(4S) contents to ΔIA contents of ovine, porcine and bovine heparin, where the black horizontal line max_Porcine+3 SD represents the maximum value of trisaccharide(4S)/ΔIA from the six batches of porcine heparin standards plus 3SD.

(4) FIG. 4 shows the ratios (analysed by HILIC-MS) of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of ovine heparin after the full enzymolysis, where Ovine_mix indicates that six batches of ovine heparin were mixed in equal proportions, and Porcine_mix indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% represents that 1% ovine heparin is mixed in the porcine heparin standards, and so on.

(5) FIG. 5 is a tandem mass spectra of ΔIA and trisaccharide(4S), where, fragment ions (m/z=300.0, z=−1) of ΔIA and fragment ions (m/z=156.7, z=−1) of trisaccharide(4S) are used for the establishment of the HILIC-MRM method.

(6) FIG. 6 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of ovine heparin analysed by HILIC-MRM, where Ovine indicates that six batches of ovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% Ovine represents that 1% ovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation.

(7) FIG. 7 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of bovine heparin analysed by HILIC-MRM, where Bovine indicates that six batches of bovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% bovine represents that 1% bovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation.

(8) FIG. 8 is a tandem mass spectra of ΔIA and trisaccharide(4S) after being labelled by AMAC, where, parent ions (m/z=652.3, z=−1) of ΔIA-AMAC and parent ions (m/z=432.5, z=−1) of trisaccharide(4S)-AMAC are used for the establishment of the C18-MRM method.

(9) FIG. 9 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of ovine heparin analysed by C18-MRM, where Ovine indicates that six batches of ovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% Ovinere presents that 1% ovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation.

(10) FIG. 10 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of bovine heparin analysed by C18-MRM, where Bovine indicates that six batches of bovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% bovine represents that 1% bovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(11) In the following description, specific details of the present disclosure are further set forth to provide a thorough understanding of the present disclosure. Terms used in the description of the present disclosure herein are for the purpose of describing advantages and features of the present disclosure only but not construed as limiting the present disclosure.

(12) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this present disclosure belongs. Unless otherwise specified, all the drugs or reagents used herein are used according to the product manual or conventional methods in the art. The technical solutions of the present disclosure will now be further described with reference to the accompanying drawings and detailed embodiments.

Example 1

(13) Instruments and equipment, chemical reagents and experimental procedures used in the present disclosure are specifically as follows:

(14) 1. Instruments

(15) (1) High performance liquid chromatography-mass spectrometer

(16) (2) High resolution mass spectrometer

(17) (3) Triple quadrupole mass spectrometer

(18) 2. Reagents

(19) TABLE-US-00003 Reagents Specification Heparinases I, II, Commercially available heparinases, and III such as Asnail and BIOKANGTAI Ammonium acetate Guaranteed Reagent (GR)/Ultra Pure Acetonitrile, H.sub.2O Analytical Reagent (AR)/chromatographically pure (LC) AMAC Analytical Reagent (AR)/chromatographically pure (LC) DMSO Analytical Reagent (AR)/chromatographically pure (LC) Acetic acid Analytical Reagent (AR)/chromatographically pure (LC) NaBH.sub.3CN Analytical Reagent (AR)/chromatographically pure (LC)

(20) 3. Experimental Operation

(21) 3.1 Detection Method of Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry (HILIC-MS)

(22) 3.1.1 Processing Steps for Heparin Samples

(23) Exhaustive enzymatic digestion: porcine heparin standards and samples were dissolved with water to 20 μg/μL, respectively, each 2.5 μL was taken and added with 8.75 μL sodium acetate/calcium acetate buffer solution (10 mg bovine serum albumin and 32 mg calcium acetate were dissolved into 60 mL water, 580 μL glacial acetic acid was added and mixed well, then pH was regulated to 7.0 with 2 mol/L sodium hydroxide solution, metered to a volume of 100 mL with water) and 12.5 μL mixed solution of heparinases I, II and III (heparinase I, heparinase II and heparinase III were all 0.4 mIU/μL, dissolved by the sodium acetate/calcium acetate buffer solution). The above mixture was incubated for 36 h at 25° C., then 12.5 μL mixed solution of heparinases I, II and III was added to continue the incubation. After the total incubation time reached 72 h, the above product was heated in a water bath at 100° C. for 10 min to inactivate the heparinases, then centrifuged at 12000 r/min for 10 min, and the supernatant was taken and lyophilized.

(24) 3.1.2 The Detection Conditions of HILIC-MS are as Follows:

(25) Prior to the analysis by HILIC-MS, 20 μg sample was dissolved in 80% mobile phase B.

(26) Analytical column: Phenomenex Luna 3 μm HILIC 200 Å (150×2.0 mm); mobile phase A: 5 mmol/L ammonium acetate aqueous solution; mobile phase B: 5 mmol/L ammonium acetate, and 98% acetonitrile solution; flow rate: 0.15 mL/min; injection volume: 20 μL; the step gradient: 0-5 min, 95% B; 5-6 min, 90% B; 6-25 min, 90-84% B; 25-27 min, 84-50% B; 27-31 min, 50-50% B; 31-32 min, 50-95% B; 32-40 min, 95-95% B.

(27) Parameters of mass spectrometry: instrument: Q Exactive plus; sheath gas: 40; Aux gas: 10; spray voltage: 3.8 kV; capillary tmp: 275° C.; S-lens: 50; m/z: 150-800; and acquisition time: 40 min.

(28) 3.2 Multiple Reaction Monitoring (MRM) Detection Method

(29) 3.2.1 Processing Steps for Heparin Samples

(30) The MRM technical operation may be classified into a HILIC-MRM method or a C18-MRM method.

(31) HILIC-MRM sample processing: exhaustive enzymatic digestion: samples and porcine heparin standards were dissolved with water to 20 μg/μL, respectively, each 2.5 μL was taken and added with 8.75 μL sodium acetate/calcium acetate buffer solution (10 mg bovine serum albumin and 32 mg calcium acetate were dissolved into 60 mL water, 580 μL glacial acetic acid was added and mixed well, then pH was regulated to 7.0 with 2 mol/L sodium hydroxide solution, metered to a volume of 100 mL with water) and 12.5 μL mixed solution of heparinases I, II and III (heparinase I, heparinase II and heparinase III were all 0.4 mIU/μL, dissolved by the sodium acetate/calcium acetate buffer solution). The above mixture was incubated for 36 h at 25° C., then 12.5 μL mixed solution of heparinases I, II and III was added to continue the incubation. After the total incubation time reached 72 h, the above product was heated in a water bath at 100° C. for 10 min to inactivate the heparinases, then centrifuged at 12000 r/min for 10 min, and the supernatant was taken and lyophilized.

(32) C18-MRM sample processing: the sample was labelled by AMAC after being subjected to enzymolysis and lyophilization. A certain amount of 2-aminoacridone (AMAC) was weighed and dissolved into the mixed solution of dimethyl sulfoxide and glacial acetic acid (a volume ratio of dimethyl sulfoxide to glacial acetic acid was 17:3) to a final concentration of 0.1 moL/L; 5 μL 0.1 moL/L AMAC was added to the full-enzymolysis sample which had been completely dried for reaction for 15 min at room temperature, and 5 μL 1 moL/L NaBH.sub.3CN aqueous solution was added for 1 h at 45° C. After the reaction, 9 μL supernatant was taken after high-speed centrifugation for 3 min, and placed to a sample bottle.

(33) 3.2.2 The Detection Conditions of the MRM:

(34) (1) HILIC-MRM Detection Conditions:

(35) Prior to the analysis by HILIC-MRM, 5 μg sample was dissolved in 80% mobile phase B.

(36) Analytical column: Phenomenex Luna 3 μm HILIC 200 Å (150×2.0 mm); mobile phase A: 5 mmol/L ammonium acetate aqueous solution; mobile phase B: 5 mmol/L ammonium acetate, and 98% acetonitrile solution; flow rate: 0.15 mL/min; injection volume: 10 μL; 0-5 min, 95% B; 5-12 min, 95-50% B; 12-15 min, 50% B; 15-20 min, 95% B.

(37) Mass spectrometry conditions: spray voltage: −3.7 kV; spray gas flow rate: 30 arb; acquisition time: 15 min.

(38) TABLE-US-00004 TABLE 1 shows the HILIC-MRM channel parameters: Theoretical molecular Parent Daughter # Name Structure weight ion ion 1 ΔIA ΔUA2S- 539.0251  m/z = 268.5, m/z = 300,  G1cNAc6S z = −2 z = −1 2 Trisaccharide ΔUA2S- 832.9602  m/z = 415.5,  m/z = 157.0, (4S) GlcNS6S- z = −2 z = −1 HexA2S

(39) (2) C18-MRM

(40) The amount of sample to be analyzed is 5 μg.

(41) Analytical column: Kinetex 2.6 μm EVO C18 100 Å (150×2.1 mm); mobile phase A: 50 mmol/L ammonium acetate aqueous solution; mobile phase B: methanol solution; flow rate: 0.3 mL/min; injection volume: 1 μL; column temperature: 45° C.; the step gradient: 0-2 min, 5% B; 2-4 min, 26% B; 4-8 min, 40% B; 8-10 min, 100% B; 10-15 min, 5% B;

(42) mass spectrometry conditions: spray voltage: −3.7 kV; spray gas flow rate: 30 arb; acquisition time: 15 min.

(43) TABLE-US-00005 TABLE 2 shows the C18-MRM channel parameters: Theoretical molecular Parent Daughter # Name Structure weight ion ion 1 ΔIA-AMAC ΔUA2S-  733.1095 m/z = m/z = G1cNAc6S- 732.1022, 652.1454, AMAC z = −1 z = −1 2 Trisaccharide ΔUA2S- 1027.0446 m/z = m/z = (4S) GlcNS6S- 512.5150, 432.5366, HexA2S- z = −2 z = −1 AMAC

(44) 4. Verification Test

(45) The six batches of porcine heparin standards, six batches of ovine heparin and six batches of bovine heparin were subjected to relative quantification and ratio analysis of trisaccharide(4S) and ΔIA by the method of the present disclosure, respectively.

(46) (I) Detection Results and Analysis of HILIC-MS

(47) FIG. 1 is high resolution mass spectrums and structure diagrams of trisaccharide(4S) ([M−2H].sup.2) and [M−3H+Na].sup.2-) and ΔIA ([M−2H].sup.2- and [M−3H+Na].sup.2-) analysed by HILIC-MS described in the present disclosure. The extracted ion chromatograms (EIC) are shown in FIG. 2. The content of trisaccharide(4S) in porcine heparin standards is significantly lower than that in bovine and ovine heparin.

(48) The ratios of trisaccharide(4S) to ΔIA in six batches of porcine heparin standards, six batches of ovine heparin and 3 batches of bovine heparin obtained by the HILIC-MS are shown in FIG. 3. The ratio of trisaccharide(4S) to ΔIA is much lower in porcine heparin than in ovine and bovine heparin. Therefore, the ratio is used to distinguish porcine heparin from bovine and ovine heparin. When the maximum ratio of trisaccharide(4S) to ΔIA in porcine heparin standards+3SD serves as the determination condition (the black horizontal line max_Porcine+3SD in FIG. 3), the ratios of bovine and ovine heparin obviously exceed the standard, such that the sample is determined to be ruminant-derived heparin or to contain ruminant-derived heparin.

(49) Furthermore, porcine heparin standards were mixed with different proportions of ovine heparin; and the ratios of trisaccharide(4S) to ΔIA were analysed by HILIC-MS (see FIG. 4). In FIG. 4, Ovine_mix represents six batches of ovine heparin that are mixed in equal proportion, and Porcine_mix represents six batches of porcine heparin standards that are mixed in equal proportion; 50% represents that 50% mixed ovine heparin is mixed in the mixed porcine heparin standards, and so on; the maximum ratio of trisaccharide(4S) to ΔIA in the six batches of porcine heparin standards+3SD serves as the determination condition (the black horizontal line max_Porcine+3SD in FIG. 4); when more than 15% ovine heparin is mixed in porcine heparin, porcine heparin may be judged to be mixed with ovine heparin.

(50) (II) Detection Results of the MRM Method

(51) (1) HILIC-MRM Detection Results

(52) HILIC-MRM method was used to quantitatively analyse the contents of trisaccharide(4S) and ΔIA. FIG. 5 is a tandem mass spectra of ΔIA and trisaccharide(4S), where, fragment ions (m/z=300.0, z=−1) of ΔIA and fragment ions (m/z=156.7, z=−1) of trisaccharide(4S) are used for the establishment of the HILIC-MRM method;

(53) FIG. 6 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of ovine heparin analysed by HILIC-MRM, where Ovine indicates that six batches of ovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% Ovine represents that 1% ovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation. The maximum ratio of trisaccharide(4S) to ΔIA in the six batches of porcine heparin standards+3SD serves as the determination condition (black horizontal line Porcine.sub.max+3SD in FIG. 6), when more than 15% ovine heparin is mixed in porcine heparin, the ratios of trisaccharide(4S) to ΔIA obviously exceed the standard, such that the sample may be determined as heparin from ruminants or to contain heparin from ruminants.

(54) FIG. 7 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of bovine heparin analysed by HILIC-MRM, where Bovine indicates that six batches of bovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% Bovine represents that 1% bovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation. The maximum ratio of trisaccharide(4S) to ΔIA in the six batches of porcine heparin standards+3SD serves as the determination condition (black horizontal line Porcine.sub.max+3SD in FIG. 7), when more than 10% bovine heparin is mixed in porcine heparin, the ratios of trisaccharide(4S) to ΔIA obviously exceed the standard, such that the sample may be determined as heparin from ruminants or to contain heparin from ruminants.

(55) (2) C18-MRM Detection Results

(56) C18-MRM method was used to quantitatively analyse the contents of trisaccharide(4S) and ΔIA as well as their ratios. FIG. 8 shows is a tandem mass spectra of ΔIA and trisaccharide(4S) after being labelled by AMAC, where, fragment ions (m/z=652.3, z=−1) of ΔIA-AMAC and fragment ions (m/z=432.5, z=−1) of trisaccharide(4S)-AMAC are used for the establishment of the AMAC method; and the contents of trisaccharide(4S) and ΔIA and their ratio may be obtained.

(57) FIG. 9 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of ovine heparin analysed by C18-MRM, where Ovine indicates that six batches of ovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% Ovine represents that 1% ovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation. The maximum ratio of trisaccharide(4S) to ΔIA in the six batches of porcine heparin standards+3SD serves as the determination condition (black horizontal line Porcine.sub.max+3SD in FIG. 9), when more than 15% ovine heparin is mixed in porcine heparin, the ratios of trisaccharide(4S) to ΔIA obviously exceed the standard, such that the sample may be determined as heparin from ruminants or to contain heparin from ruminants.

(58) FIG. 10 shows the ratios of trisaccharide(4S) to ΔIA of the porcine heparin standards mixed with different proportions of bovine heparin analysed by C18-MRM, where Bovine indicates that six batches of bovine heparin were mixed in equal proportions, and Porcine indicates that six batches of porcine heparin standards were mixed in equal proportions; 1% bovine represents that 1% bovine heparin is mixed in the porcine heparin standards, and so on; Porcine.sub.max+3SD represents that the maximum ratio of trisaccharide(4S) to ΔIA in the six bathes of porcine plus 3 times the standard deviation. The maximum ratio of trisaccharide(4S) to ΔIA in the six batches of porcine heparin standards+3 SD serves as the determination condition (black horizontal line Porcine.sub.max+3SD in FIG. 10), when more than 15% ovine heparin is mixed in porcine heparin, the ratios of trisaccharide(4S) to ΔIA obviously exceed the standard, such that the sample may be determined as heparin from ruminants or to contain heparin from ruminants.