Detection method for low molecular weight heparin complete degradation products using hydrophilic interaction chromatography and multiple reaction monitoring tandem mass spectrometry

Abstract

A detection method for low molecular weight heparin complete degradation products using hydrophilic interaction chromatography and multiple reaction monitoring tandem mass spectrometry. Identifying the original reducing end and non-reducing end of enoxaparin sodium by means of reducing the reducing end of enoxaparin sodium, and performing hydrolysis using hydrogen peroxide. Performing quantitative analysis on all component units utilizing hydrophilic interaction chromatography and multiple reaction monitoring tandem mass spectrometry, in particular quantifying low-content special structures and characterizing low molecular weight heparin.

Claims

1. A method for testing completely degraded products from low-molecular-weight heparins using a hydrophilic interaction chromatography combined a multiple reaction monitoring mass spectrometry, comprising: (1) dissolving ammonium acetate in deionized water up to 3 to 10 mM as mobile phase A (MPA); (2) dissolving ammonium acetate in deionized water and adding acetonitrile resulted in mobile phase B (MPB) wherein concentration of ammonium acetate is between 3 and 10 mM and acetonitrile is 90 to 98% by volume; (3) reducing 10 to 50 g of hydrolyzate products, which contains internal standard, of the low-molecular-weight heparins by sodium borohydride for 10 to 12 hours, and hydrolyzing by hydrogen peroxide, then preparing the degraded products from the low-molecular-weight heparins at the concentration between 1 and 10 g/L as a test solution for step (4), if end structures of the low-molecular-weight heparins are desired for further identification; or preparing the hydrolyzate products, which contains the internal standard, at the concentration between 1 and 10 g/L as the test solution for step (4) if the end structures of the low-molecular-weight heparins are not desired for further identification; (4) centrifuging the test solution obtained from step (3), loading the supernatant onto a hydrophilic interaction chromatography column for separation: wherein the flow rate is 0.1 to 0.5 mL/min, and elution gradient are as follows: 0-5 min, 5% MPA, 95% MPB; 5-107 min, 5-23% MPA, 95-77% MPB; 107-112 min, 23-50% MPA, 77-50% MPB; 112-125 min, 50% MPA, 50% MPB; (5) the multiple reaction monitoring mass spectrometry is performed under positive or negative ionization mode on a triple quadrupole mass spectrometry: wherein the parameters are set as follows, spry voltage under positive ionization mode is +4.0 kV, spry voltage under negative ionization mode is 3.2 kV, the sheath gas flow is 20-30 arb, tube lens voltage is 50-150 V, collision energy is 20-50.

2. The method according to claim 1, wherein the hydrolyzate products with the internal standard of the low-molecular-weight heparins in step (3) are dried under vacuum decompression dry for 1 to 3 hours at 30 to 60 C.

3. The method according to claim 1, wherein the centrifugation condition in step (4) is 10,000 to 15,000 rpm for 5 to 15 min at room temperature.

4. The method according to claim 3, wherein the centrifugation condition in step (4) is 12,000 rpm for 15 min at room temperature.

Description

FIGURE LEGENDS

(1) FIG. 1. Extracted ion chromatography of enzymatical digested and chemical degraded enoxaparin reference standard in example 1.

(2) FIG. 2. MS/MS spectra of component 30, 31 of enzymatical digested and RE component 5 of chemical degraded enoxaparin reference standard in example 1. a: MS/MS spectra of component 30 of enzymatical digested enoxaparin reference standard in example 1; b: MS/MS spectra of component 31 of enzymatical digested enoxaparin reference standard in example 1; c: MS/MS spectra of RE component 5 of chemical degraded enoxaparin reference standard in example 1

(3) FIG. 3. Extracted ion chromatography of enzymatical digested dalteparin reference standard in example 2

SPECIFIC IMPLEMENTATION METHOD

(4) Further restrictions will be defined by the figures attached combining with the examples below, but not limited as these.

(5) The examples were performed on an Agilent 1100 series HPLC with a ChemStation workstation online coupling to a Thermo TSQ Quantum Ultra triple quadrupole MS with a Xcalibur workstation.

EXAMPLE 1

(6) The procedure of this novel analytical method for complete degradation products of low molecular weight heparin using hydrophilic interaction chromatography tandem multiple reaction monitoring mass spectrometry is as follows:

(7) 1. Mobile phase A (MPA) is 5 mM of ammonium acetate in DI water.

(8) 2. Mobile phase B (MPB) is 5 mM of ammonium acetate in 95% acetonitrile.

(9) 3. Enoxaparin reference standard was enzymatically digested by heparinase I, II and III at 25 C. for 48 h, and internal standard was added before ultrafiltration using a 30 KDa molecular weight cut off membrane. The digests were vacuum decompression dried. The dried digests is prepared into solutions with the concentration of 10 g/L and go to step 5.

(10) 4. 50 g enoxaparin sample with internal standard was reducted with sodium borohydride for 12 h first and hydrolysis using hydrogen peroxide. The dried hydrolysis product is prepared into solutions with the concentration of 10 g/L and go to step 5.

(11) 5. Solutions prepared in step 3 are centrifuged prior to separation on HILIC column with a particle size of 200 (2.0 mm150 mm) and detection on MRM tandem MS. The flow rate is 0.1 to 0.5 mL/min, and elution gradient are as follows, 0-5 min, 5% MPA, 95% MPB; 5-107 min, 5-23% MPA, 95-77% MPB; 107-112 min, 23-50% MPA, 77-50% MPB; 112-125 min, 50% MPA, 50% MPB;

(12) 6. The MRM is performed on a Thermo TSQ Quantum Ultra triple quadrupole MS under negative ionization mode on a triple quadrupole MS. The parameters are set as follows, spry voltage under negative ionization mode is 3.2 kV, the sheath gas flow is 20-30 arb, tube lens voltage is 75 V, collision energy is 35.

(13) 7. The concentration (c) of each component can be calculated according to the formula below, c=c.sub.IP(A/A.sub.IP), c.sub.IP is the concentration of internal standard, A is the area of internal standard and the A.sub.IP is the area of this component.

(14) 8. The composition analysis of all identified building blocks were performed, the results for building blocks derived via enzymatic digestion are listed in table 1, the results for building blocks derived via chemical degradation are listed in table 2.

(15) TABLE-US-00001 TABLE 1 Theoretical Precursor Daughter Identity Structure WM ions Charge ions 1 IS UA2S-GlcNS6S 576.9713 287.5 2 138 2 IIS UA-GlcNS6S 497.0145 247.5 2 138 3 IIIS UA2S-GlcNS 497.0145 247.5 2 138 4 IVS UA-GlcNS 417.0577 416.0 1 138, 175 5 IA UA2S-GlcNAc6S 539.0251 268.5 2 300 6 IIA UA-GlcNAc6S 459.0683 458.0 1 157, 175 7 IIIA UA2S-GlcNAc 459.0683 458.0 1 157, 175 8 IVA UA-GlcNAc 379.1115 378.0 1 115, 175 9 1,6-anhydro UA-GlcNS-1,6- 399.0471 398.0 1 175 IIS anhydro 1,6-anhydro UA-ManNS-1,6- IIS.sub.epi anhydro 10 1,6-anhydro UA2-GlcNS-1,6- 479.0040 478.0 1 398 IS anhydro 11 1,6-anhydro UA2S-GlcNS6S- 1055.9753 527.0 2 406.5 IS-IS.sub.epi IdoA2S-ManNS-1,6- anhydro 12 I-H UA2S-GlcN6S 497.0145 496.0 1 258, 416 13 II-H UA2S-GlcN 417.0577 416.0 1 157, 175 14 III-H UA-GlcN6S 15 IV-H UA-GlcN 337.1009 336.0 1 115 16 IIA-IIS.sub.glu UA-GlcNAc6S-GlcA- 1036.0396 517.0 2 175, 458, 616 GlcNS3S6S 17 IIS-IIS.sub.glu UA-GlcNS6S-GlcA- 1073.9859 536.0 2 416, 458 GlcNS3S6S 18 NRE dp2 (2S) IdoA2S-GlcNS 515.0251 256.5 2 138, 258 19 NRE dp2 (3S) IdoA2S-GlcNS6S 594.9819 296.5 2 138 20 Linkage UA-Gal-Gal-Xyl-O- 719.2120 358.6 2 218, 337 Ser 21 Linkage.sub.ox UA-Gal-Gal-Xyl-O- 690.1855 344.0 2 189 Ser.sub.ox 22 dp3 (2S) UA-GlcNS6S-HexA 673.0466 335.5 2 157, 339 23 dp3 (2S, 1Ac) UA-GlcNAc6S- 715.0572 356.0 2 97, 157 HexA2S 24 dp3 (3S) UA2S-GlcNS6S- 753.0034 375.5 2 193, 314 HexA 25 dp3 (4S) UA2S-GlcNS6S- 832.9602 415.5 2 97, 157 HexA2S 26 IVS.sub.gal GalA-GlcNS 417.0577 416.0 1 138, 175 27 IIS.sub.gal GalA-GlcNS6S 497.0145 247.5 2 138 28 dp2 (CS) UA2CS-GlcNS6S 560.9764 279.5 2 138 29 Epoxide UA2S-GlcNS6S- 976.0185 478.0 2 258 GlcA-2,3-anhydro-GlcNS 30 NRE dp3 GlcNS6S-HexA2S- 915.9643 457.0 2 240, 258 GlcNS6S 31 dp4 UA-GlcNS- 994.0290 496.0 2 258, 416 (HexA2S3S) HexA2S3S-GlcNS

(16) TABLE-US-00002 TABLE 2 Theoretical Precursor Identity Structure WM ions Charge Daughter ions NREs 1 NRE IS UA2S-GlcNS6S 576.9713 287.5 2 138 2 NRE IIS UA-GlcNS6S 497.0145 247.5 2 138 3 NRE IIIS UA2S-GlcNS 2 4 NRE dp3 (4S) UA2S-GlcNS6S- 832.9602 415.5 2 157, 193, 273 HexA2S 5 NRE dp4 (6S) UA2S-GlcNS6S- 1153.9427 576.0 2 415.5 HexA2S-GlcNS6S 6 NRE dp4 (5S) UA2S-GlcNS6S- 1073.9859 536.0 2 415.5, 375.5 HexA2S-GlcNS REs 1 RE dp2 (3S) HexA2S-GlcNS6S- 596.9976 297.5 2 242, 260 ol 2 RE dp2 (2S) HexA-GlcNS6S-ol 517.0407 257.5 2 242 HexA2S-GlcNS-ol 3 RE dp3 (5S) GlcNS6S-HexA2S- 917.9800 458.0 2 242, 260 GlcNS6S-ol 4 RE dp3 (4S) GlcNS-HexA2S- 838.0232 418.0 2 260 GlcNS6S-ol 5 RE dp3 (4S, HexA2S-GlcNS6S- 852.9865 425.5 2 257 HexA-ol) HexA2S-ol 6 RE dp3 (3S, GlcNAc6S- 800.0769 399.0 2 260 1Ac) HexA2S-GlclNS-ol Note: UA represents unsaturated uronic acid, Hex represents uronic acid, GlcA represents glucuronic acid, IdoA represents iduronic acid, GlcN represents glucosamine, Ac represents acetyl group, S represents sulfo group, -ol represents alditol.

(17) The extracted ion chromatography of building blocks derived via enzymatic digestion are shown in FIG. 1, The extracted ion chromatography of building blocks derived via chemical degradation are shown in FIG. 2. This method successfully identified and quantified all possible complete degradation products in enoxaparin.

EXAMPLE 2

(18) The procedure of this novel analytical method for complete degradation products of low molecular weight heparin using hydrophilic interaction chromatography tandem multiple reaction monitoring mass spectrometry is as follows:

(19) 1. Mobile phase A (MPA) is 5 mM of ammonium acetate in DI water.

(20) 2. Mobile phase B (MPB) is 5 mM of ammonium acetate in 95% acetonitrile.

(21) 3. Dalteparin reference standard was enzymatically digested by heparinase I, II and III at 25 C. for 48 h, and internal standard was added before ultrafiltration using a 30 KDa molecular weight cut off membrane. The digests were vacuum decompression dried.

(22) 4. The dried digests is prepared into solutions with the concentration of 10 g/L and go to step 5.

(23) 5. Solutions prepared in step 3 are centrifuged prior to separation on HILIC column with a particle size of 200 (2.0 mm150 mm) and detection on MRM tandem MS. The flow rate is 0.1 to 0.5 mL/min, and elution gradient are as follows, 0-5 min, 5% MPA, 95% MPB; 5-107 min, 5-23% MPA, 95-77% MPB; 107-112 min, 23-50% MPA, 77-50% MPB; 112-125 min, 50% MPA, 50% MPB;

(24) 6. The MRM is performed on a Thermo TSQ Quantum Ultra triple quadrupole MS under negative ionization mode on a triple quadrupole MS. The parameters are set as follows, spry voltage under negative ionization mode is 3.2 kV, the sheath gas flow is 20-30 arb, tube lens voltage is 75 V, collision energy is 35.

(25) 7. The concentration (c) of each component can be calculated according to the formula below, c=c.sub.IP(A/A.sub.IP), c.sub.IP is the concentration of internal standard, A is the area of internal standard and the A.sub.IP is the area of this component.

(26) 8. The composition analysis of all identified building blocks were performed, the results for building blocks derived via enzymatic digestion are listed in table 3.

(27) TABLE-US-00003 TABLE 3 Theoretical Precursor Daughter Identity Structure WM ions Charge ions 1 IS UA2S-GlcNS6S 576.9713 287.5 2 138 2 IIS UA-GlcNS6S 497.0145 247.5 2 138 3 IIIS UA2S-GlcNS 497.0145 247.5 2 138 4 IVS UA-GlcNS 417.0577 416.0 1 138, 175 5 IA UA2S-GlcNAc6S 539.0251 268.5 2 300 6 IIA UA-GlcNAc6S 459.0683 458.0 1 157, 175 7 IIIA UA2S-GlcNAc 459.0683 458.0 1 157, 175 8 IVA UA-GlcNAc 379.1115 378.0 1 115, 175 9 dp2 (1S) RE UA-Mnt6S 402.0468 401 1 243, 285 10 dp2 (2S) RE UA2S-Mnt6S 482.0036 481 1 243 11 dp4 (3S) RE UA2S-GlcNS-UA- 899.0613 448.5 2 243, 255, 339 Mnt6S 12 dp4 (4S) RE UA2S-GlcNS-UA2S- 979.0182 488.5 2 243, 339 Mnt6S 13 dp4 (5S) RE UA2S-GlcNS6S- 1058.9750 528.5 2 157, 409 UA2S-Mnt6S 14 I-H UA2S-GlcN6S 497.0145 496.0 1 258, 416 15 II-H UA2S-GlcN 417.0577 416.0 1 157, 175 16 III-H UA-GlcN6S 417.0577 416.0 1 157, 175 17 IIA-IIS.sub.glu UA-GlcNAc6S-GlcA- 1036.0396 517.0 2 175, 458, 616 GlcNS3S6S 18 IIS-IIS.sub.glu UA-GlcNS6S-GlcA- 1073.9859 536.0 2 416, 458 GlcNS3S6S 19 NRE dp2 (2S) IdoA2S-GlcNS 515.0251 256.5 2 138, 258 20 NRE dp2 (3S) IdoA2S-GlcNS6S 594.9819 296.5 2 138 21 Linkage UA-Gal-Gal-Xyl-O- 719.2120 358.6 2 218, 337 Ser 22 Linkage.sub.ox UA-Gal-Gal-Xyl-O- 690.1855 344.0 2 189 Ser.sub.ox 23 dp3 (2S) UA-GlcNS6S-HexA 673.0466 335.5 2 157, 339 24 dp3 (3S) UA2S-GlcNS6S-HexA 753.0034 375.5 2 193, 314 25 dp3 (4S) UA2S-GlcNS6S- 832.9602 415.5 2 97, 157 HexA2S 26 IVS.sub.gal GalA-GlcNS 417.0577 416.0 1 138, 175 27 IIS.sub.gal GalA-GlcNS6S 497.0145 247.5 2 138 28 dp2 (CS) UA2CS-GlcNS6S 560.9764 279.5 2 138 29 Epoxide UA2S-GlcNS6S- 976.0185 478.0 2 258 GlcA-2,3-anhydro-GlcNS 30 NRE dp3 GlcNS6S-HexA2S- 915.9643 457.0 2 240, 258 GlcNS6S 31 dp4 UA-GlcNS- 994.0290 496.0 2 258, 416 (HexA2S3S) HexA2S3S-GlcNS Note: UA represents unsaturated uronic acid, Hex represents uronic acid, GlcA represents glucuronic acid, IdoA represents iduronic acid, GlcN represents glucosamine, Ac represents acetyl group, S represents sulfo group, -ol represents alditol.

(28) The extracted ion chromatography of building blocks derived via enzymatic digestion are shown in FIG. 3. This method successfully identified and quantified all possible complete degradation products in dalteparin.