TWI479150B - 用於檢測硫酸化寡糖之分析方法 - Google Patents
用於檢測硫酸化寡糖之分析方法 Download PDFInfo
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- 229920001542 oligosaccharide Polymers 0.000 title claims description 37
- 150000002482 oligosaccharides Chemical class 0.000 title claims description 37
- 238000004458 analytical method Methods 0.000 title description 12
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 54
- 238000000034 method Methods 0.000 claims description 36
- 150000003839 salts Chemical class 0.000 claims description 30
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 25
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 17
- 230000005526 G1 to G0 transition Effects 0.000 claims description 17
- 229960001318 fondaparinux Drugs 0.000 claims description 15
- KANJSNBRCNMZMV-ABRZTLGGSA-N fondaparinux Chemical compound O[C@@H]1[C@@H](NS(O)(=O)=O)[C@@H](OC)O[C@H](COS(O)(=O)=O)[C@H]1O[C@H]1[C@H](OS(O)(=O)=O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O4)NS(O)(=O)=O)[C@H](O3)C(O)=O)O)[C@@H](COS(O)(=O)=O)O2)NS(O)(=O)=O)[C@H](C(O)=O)O1 KANJSNBRCNMZMV-ABRZTLGGSA-N 0.000 claims description 15
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- 229940043376 ammonium acetate Drugs 0.000 claims description 6
- 235000019257 ammonium acetate Nutrition 0.000 claims description 6
- FDTUVFSBEYKVAP-UHFFFAOYSA-N formic acid;pyridine Chemical compound OC=O.C1=CC=NC=C1 FDTUVFSBEYKVAP-UHFFFAOYSA-N 0.000 claims description 6
- VZXTWGWHSMCWGA-UHFFFAOYSA-N 1,3,5-triazine-2,4-diamine Chemical group NC1=NC=NC(N)=N1 VZXTWGWHSMCWGA-UHFFFAOYSA-N 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000001195 ultra high performance liquid chromatography Methods 0.000 claims description 3
- XEKSTYNIJLDDAZ-JASSWCPGSA-D decasodium;(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5r,6r)-6-[(2r,3s,4s,5r,6r)-2-carboxylato-4-hydroxy-6-[(2r,3s,4r,5r,6s)-4-hydroxy-6-methoxy-5-(sulfonatoamino)-2-(sulfonatooxymethyl)oxan-3-yl]oxy-5-sulfonatooxyoxan-3-yl]oxy-5-(sulfonatoamino)-4-sulfonatooxy-2-(sul Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].O[C@@H]1[C@@H](NS([O-])(=O)=O)[C@@H](OC)O[C@H](COS([O-])(=O)=O)[C@H]1O[C@H]1[C@H](OS([O-])(=O)=O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](OS([O-])(=O)=O)[C@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](COS([O-])(=O)=O)O4)NS([O-])(=O)=O)[C@H](O3)C([O-])=O)O)[C@@H](COS([O-])(=O)=O)O2)NS([O-])(=O)=O)[C@H](C([O-])=O)O1 XEKSTYNIJLDDAZ-JASSWCPGSA-D 0.000 description 14
- 238000004949 mass spectrometry Methods 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
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- 230000000694 effects Effects 0.000 description 7
- 239000012535 impurity Substances 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 7
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 6
- 229960002897 heparin Drugs 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- -1 BEH Amide Chemical class 0.000 description 5
- 238000012369 In process control Methods 0.000 description 5
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- 229920000669 heparin Polymers 0.000 description 5
- 238000010965 in-process control Methods 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
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- 238000003556 assay Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
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- 238000004366 reverse phase liquid chromatography Methods 0.000 description 3
- 238000002305 strong-anion-exchange chromatography Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- 229920001499 Heparinoid Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000010378 Pulmonary Embolism Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 229940104697 arixtra Drugs 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- XFQYJNINHLZMIU-UHFFFAOYSA-N cataline Natural products CN1CC(O)C2=CC(OC)=C(OC)C3=C2C1CC1=C3C=C(OC)C(OC)=C1 XFQYJNINHLZMIU-UHFFFAOYSA-N 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 229910000420 cerium oxide Inorganic materials 0.000 description 1
- 150000005829 chemical entities Chemical class 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- BMMGVYCKOGBVEV-UHFFFAOYSA-N oxo(oxoceriooxy)cerium Chemical compound [Ce]=O.O=[Ce]=O BMMGVYCKOGBVEV-UHFFFAOYSA-N 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
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- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
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- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Substances C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
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- 230000001105 regulatory effect Effects 0.000 description 1
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- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
-
- 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
- G01N2030/8813—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 biological materials
- G01N2030/8836—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 biological materials involving saccharides
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/18—Sulfur containing
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Saccharide Compounds (AREA)
Description
類肝素(肝素及硫酸乙醯肝素(Heparan sulfate))係多個重要生物過程中已知的調節介質。類肝素及其衍生物低分子量肝素(low-molecular weight heparin,LMWH)已在外科手術及腎透析期間用作臨床抗凝血藥物。舉例而言，磺達肝素鈉(CAS 114870-03-0)係化學名稱為O-[2-脫氧-6-O-磺酸基-2-(磺醯胺基)-α-D-吡喃葡萄糖]-(1--4)-O-(β-D-吡喃葡萄糖醛酸)-(1--4)-O-[2-脫氧-3,6-二-O-磺酸基-2-(磺醯胺基)-α-D-吡喃葡萄糖]-(1--4)-O-(2-O-磺酸基-α-L-吡喃艾杜糖醛酸)-(1--4)-O-[2-脫氧-1-O-甲基-6-O-磺酸基-2-(磺醯胺基)-α-D-葡萄吡喃糖苷]十鈉鹽之寡糖/肝素之一，且由Choay,S.A.研發(參見美國專利第4,818,816號)。該化合物係一合成之五碳糖因子Xa抑制劑，其用作預防經骨外科手術之患者之深層靜脈栓塞以及治療深層靜脈栓塞及肺栓塞之抗凝血藥物。磺達肝素鈉於2001年經美國食品藥物管理局(United States Food and Drug Administration)批准，以商品名ArixtraTM
出售。磺達肝素鈉係皮下投與。
肝素及硫酸乙醯肝素分析方法在傳統上涉及逆相層析及質譜(MS)技術，但因硫酸乙醯肝素之高極性、結構多樣性及硫酸鹽不穩定性而具有限制。舉例而言，使用MS定量合成多硫酸化寡糖受到限制，因為多硫酸化寡糖離子化時易於形成多種類型硫酸根基團損失之
片段及金屬陽離子偶合加成物，此會導致更大之質譜複雜性及訊號分裂
。另外，在分析期間難以顯示硫酸根基團之損失程度，因為其取決於硫酸化寡糖之濃度及電荷態。多硫酸化寡糖分析方法之改良係多組研究團隊之目標。
Catalin等人(Anal.Chem.2009,81,3485)及Tatiana等人(Anal.Chem.2006,78,1774)已各自闡述藉由使用電噴灑游離質譜(ESI-MS)及基質輔助雷射脫附游離質譜(MALDI-MS)來表徵多硫酸化寡糖。然而，目前使用液相層析(LC)與質譜偶聯之方法無法提供線上製程時層析峰之解析/分離且因此在合成多硫酸化寡糖期間無法建立結構相關雜質之鑑定及/或多硫酸化寡糖之定量。
Imanari等人(J.Chromatogr.,A 1996,720,275.)及Rice等人(J.Anal.Biochem.1985,150,325.)發表了藉助強陰離子交換層析(SAX)之多硫酸化寡糖分析方法。此方法經由電荷密度差異來分離高度硫酸化寡糖，但因在流動相組合物中使用非揮發性鹽類，故仍難以直接偶聯SAX與MS。
與多硫酸化寡糖分析方法相關之另一些問題係因多硫酸化寡糖之非發色特性(極低UV吸收)所致，此會限制傳統UV檢測器之使用。其他通用檢測器(例如折射率(RI)檢測器及蒸發光散射檢測器(ELSD))亦對多硫酸化寡糖缺乏足夠檢測靈敏性。
儘管已揭示一些多硫酸化寡糖檢測方法，但仍存在多種限制。因此，業內仍需要改良用於多硫酸化寡糖之分離、定量及質量鑑定之方法。本文所揭示之穩定、靈敏且製程中控制(in-process control,IPC)方法解決了此需要及其他需要。
本文提供使用與電離子檢測器(CAD)或質譜儀(MS)偶聯之親水性相互作用超高效液相層析儀(HILIC-UPLC)檢測多硫酸化寡糖之方
法。本文所提供方法允許改良之峰解析度，藉此允許樣品中多硫酸化寡糖及/或雜質之後續定量。
HILIC之使用克服了滯留及分離極端極性寡糖之挑戰。HILIC之滯留機制極為複雜且係液液分配、吸附、離子相互作用及疏水性相互作用之多模態組合。因此，與逆相液相層析(RPLC)相比，HILIC提供獨特選擇性及滯留特性。
如本文所提供，在一組實施例中，用於HILIC管柱中之固定相係醯胺鍵合固定相。
在另一實施例中，用於HILIC管柱中之流動相包含鹽類。在一組實施例中，該鹽類係甲酸銨。與甲酸吡啶鎓及乙酸銨相比，甲酸銨之使用對滯留、選擇性提供更佳之性能及低雜訊基線。
在一些實施例中，鹽類濃度高於50mM。在一些所選實施例，濃度高於100mM。通常，流動相組合物中鹽添加劑之莫耳強度會對層析滯留、選擇性及靈敏性具有顯著影響。隨著鹽添加劑之莫耳濃度增加，液液分配相互作用壓制了流動相與溶質之離子強度，而非以離子交換作用機制滯留溶質。然而，現已發現在酸性分析物(例如多硫酸化寡糖)之情形下，滯留隨著鹽添加劑之莫耳濃度增加而增強。具體而言，峰解析度因鹽濃度自50mM增加至約200mM而得以進一步改良。
在一組實施例中，用於HILIC管柱中之流動相之溶劑係乙腈。
在一些實施例中，用於定量多硫酸化寡糖之檢測器係電離子檢測器(CAD)。在使用CAD之分析期間，用電離氣體(通常為氮氣)使霧化粒子帶電。在去除高流動性粒子(主要為過量N2
離子)後，對霧化粒子實施電子檢測。最重要的是，已證實該方法對非揮發性分析物提供獨立於其性質之均一偵測強度。因此，(1)使用HILIC或HILIC-UPLC之分離技術與(2)諸如MS或CAD之檢測技術之組合允許多硫酸化之檢
測、鑑定及/或定量，藉此提供用於分析合成多硫酸化寡糖之有效方式。
根據本發明之一所選實施例，藉由本文所述方法檢測及/或定量之多硫酸化寡糖係磺達肝素鈉。
圖1(a)
提供使用Merck,Sequant Zic®-Hilic(3.5μm 2.1×250mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(b)
提供使用Merck,Sequant Zic®-pHilic(5μm 4.6×150mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(c)
提供使用Phenomenex,Synergi Polar-RP(4μm 4.6×250mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(d)
提供使用Phenomenex,Synergi Fusion-RP(4μm 4.6×150mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(e)
提供使用Sepax Polar-Pyridine(1.8μm 2.1×150mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(f)
提供使用ES,Epic Diol(1.7μm 2.1×150mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(g)
提供使用Waters,Acquity BEH HILIC(1.7μm 2.1×150mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖1(h)
提供使用Waters,Acquity BEH Amide(1.7μm 2.1×150mm)之磺達肝素鈉之HILIC-CAD層析圖。
圖2
提供使用Waters,BEH Amide管柱(a)全圖及(b)局部放大圖之磺達肝素鈉之層析圖。
圖3
提供使用不同類型鹽(a)50mM甲酸銨(b)100mM甲酸銨(c)100mM甲酸吡啶鎓鹽及(d)50mM乙酸銨之藥物分析之層析圖(以局部放大圖表示)。
圖4
提供使用不同濃度甲酸銨(以局部放大圖表示)(a)50mM(b)100mM(c)125mM(d)150mM(e)175mM及(f)200mM之藥物分析之層析圖(以局部放大圖表示)。
圖5
提供對流動相使用不同有機溶劑(a)流動相A：200mM甲酸銨；流動相B：丙酮及乙腈，1/1(b)流動相A：200mM甲酸銨；流動相B：乙腈之藥物分析之層析圖(以局部放大圖表示)。
本文提供針對多硫酸化寡糖研發之簡明製程中分析方法，其涉及分別應用HILIC-UPLC、CAD及MS進行分離、定量及質量鑑定。在製造期間之製程中控制(in process control,IPC)對於在高度硫酸化寡糖之全合成中確保品質控制至關重要。本文所述分析方法可用作IPC方法。本文所述分析方法穩定、易於使用、靈敏，且確保有品質的化學實體之製造在期望產率內。
鑒於上文，本文提供用於檢測及定量樣品中一或多種多硫酸化寡糖之方法，該方法包含：(a)在與電離子檢測器(CAD)或質譜儀(MS)偶聯之親水性相互作用超高效液相層析儀(HILIC-UPLC)管柱上對該樣品實施層析，其中用於HILIC管柱中之固定相係醯胺鍵合固定相；及(b)測定該樣品中多硫酸化寡糖之量。
用於本方法中之樣品通常係多硫酸化寡糖之合成製造方法之產物。因此，可根據本方法藉由選擇反應混合物之試樣來分析合成程序中最終步驟之樣品。反應混合物之取樣允許雜質及/或多硫酸化寡糖之檢測及/或鑑定及/或定量。反應混合物之取樣亦允許反應之完成程度之測定。另一選擇為，可對最終產物實施本方法以確定是否需要進
一步純化。
使用親水性相互作用高效液相層析儀之層析條件將通常涉及熟習此項技術者已知之彼等條件，包括(但不限於)管柱選擇(大小、長度及固定相)以及流動相及/或流動相之pH。
管柱之選擇將通常涉及自市售管柱(例如彼等購自Waters、ThermoFisher、Merck、Phenomenex、Shodex、Nucleosil及Sepax者)選擇。在一組實施例中，管柱具有中性電荷之固定相(例如，二醇相或醯胺相)、帶電固定相(例如，二氧化矽相、胺基丙基相)或兩性離子固定相。在一組所選實施例中，固定相係醯胺鍵合固定相。實例1至10說明使用本文所述方法使用不同固定相獲得之結果。
熟習此項技術者應瞭解，流速亦會影響分離及所獲得之解析度。
用於流動相中之溶劑通常係極性非質子有機溶劑或極性非質子有機溶劑之混合物。在一組實施例中，用於HILIC管柱中之流動相之溶劑係乙腈、丙酮或乙腈與丙酮之混合物。實例13a至13d說明在使用本文所述方法之多硫酸化寡糖之檢測及/或定量中不同溶劑及/或溶劑混合物之影響。
在其他實施例中，流動相亦將包含鹽類，通常選自甲酸銨、甲酸吡啶鎓鹽及乙酸銨、及其混合物之鹽類。在某些所選實施例中，流動相將包含甲酸銨。在另一組實施例中，流動相包含選自檸檬酸銨及/或草酸銨之鹽類。實例11a至11d說明在使用本文所述方法之多硫酸化寡糖之檢測及/或定量中流動相中之不同鹽類之影響。
用於流動相中之鹽之濃度將通常係25mM至約400mM，但當鹽係以50mM至約200mM之濃度存於流動相中時發現一些最佳結果。在一些實施例中，鹽係以約50mM至100mM、約100mM至200mM及約75mM至175mM之濃度存於流動相中。實例12a至12d說明當使用
本文所述方法時藉由使用甲酸銨作為鹽在多硫酸化寡糖之檢測及/或定量期間不同鹽濃度對峰解析度及峰寬度之影響。
在特定實施例中，上文所述方法可用於具有以下結構之多硫酸化寡糖磺達肝素之檢測及/或定量：
在另一態樣中，本文提供用於檢測及定量樣品中磺達肝素之方法，該方法包含：(a)在與電離子檢測器(CAD)或質譜儀(MS)偶聯之親水性相互作用超高效液相層析儀(HILIC-UPLC)管柱上對該樣品實施層析，其中用於HILIC管柱中之固定相係醯胺鍵合固定相，用於該層析中之流動相係乙腈，且該流動相以約100mM至約200mM之濃度包含甲酸銨；及(b)測定該樣品中磺達肝素之量。
如上文及此處所用，樣品中磺達肝素之量之「測定」或「磺達肝素之定量」係以一或多種熟習此項技術者容易達成之方式實施。通常，UPLC-MS-及/或CAD之儀器設有之可計算樣品中物質之相對量(例如，藉由計算峰下方面積及/或量測峰之相對強度)之程式及/或演算法。根據儀器之反應及一系列外部標準物之濃度，可藉由回歸分析來獲得外部校準曲線。然後可藉由外部校準曲線來確定樣品濃度。如上文及此處所用，在例示性實施例中，在樣品中之「檢測」(例如，
磺達肝素及/或雜質之檢測)包含記錄使用本文所述方法(例如，藉由使用HILIC-UPLC)之層析圖及使用偶聯技術(例如CAD或MS)之峰鑑定，藉此確認(檢測)樣品中存在或不存在磺達肝素及/或雜質。
給出以下實例以進一步詳細闡述本發明。然而，本發明決不受限於本文所述特定實施例。
參與UPLC-MS研究之儀器係分別由Thermo Fisher Dionex及Bruker Daltonics製造之Ultimate 3000(UPLC及micrOTOF-Q IITM
(MS)。參與UPLC-CAD之儀器係分別由Waters公司及Thermo Fisher Dionex製造之ACQUITY UPLC®系統及Thermo Scientific Dionex Ultra CAD。
下文陳述分析條件。
1.使用醯胺-HILIC型管柱作為用於分析多磺化寡糖之分析管柱。
2.用於流動相中之甲酸銨之濃度係100mM或高於100mM。
3.流動相組合物中甲酸銨：乙腈之體積比例係在95%-5%：5%-95%範圍內。
4.用於下文實例中之流速範圍係0.4mL/min至1mL/min。
5.用於本文所述方法中之管柱溫度範圍係10℃至70℃。
6.用於此方法中之電離子檢測器之霧化溫度範圍係10℃至30℃。
7.測試樣品之濃度係15μg/mL至30mg/mL。
8.測試樣品之注射體積係1μL至5μL。
將樣品溶解於水與乙腈之混合物(1：1；30mg/mL)中。
藉由如表1
所顯示之不同類型HILIC管柱來分析如上文製備之樣品。使用不同管柱記錄之層析圖顯示於圖1(a)至(h)中且藉由LC-MS來證實磺達肝素之峰鑑定。
在實例1至10中之大多數中，發現磺達肝素之峰因與雜質之不良分離而不對稱，BEH AMIDE管柱之情形除外。優化過之Waters BEH AMIDE LC條件之典型層析圖顯示於圖2中。此研究顯示，在不同類型HILIC管柱中，醯胺型HILIC管柱對此合成多硫酸化五碳糖之分析提供相對更佳之選擇。
比較三種不同鹽甲酸銨、甲酸吡啶鎓鹽及乙酸銨。分析條件顯示於表2中。結果顯示於圖3中。
就滯留、選擇性及低雜訊基線而言，最佳層析性能係含有50mM及100mM濃度二者之甲酸銨鹽之流動相(分別為圖3a及3b)。甲酸吡啶鎓鹽(圖3c)及乙酸銨(圖3d)對所關注分析物顯示較高基線雜訊及較少滯留。基於該研究，將流動相組合物中之鹽添加劑選擇為甲酸銨。
對含有不同濃度甲酸銨鹽之流動相組合物進行研究以優化LC條件。實例12a至12f甲酸銨之濃度提供於表3
中。各個層析圖顯示於圖4中。
發現雜質概況極類似於使用50mM及100mM鹽濃度獲得之層析圖(圖4a及4b)。隨著鹽濃度增加，觀察到剛好位於主峰(磺達肝素)前面之小峰(用黑色三角形標記，參見圖4d、4e及4f)。隨著鹽濃度自150mM甲酸銨增加至200mM甲酸銨，此新峰與主峰之解析度得以進一步改良。另外，顯然，當鹽濃度增加至175mM時，主峰之帶寬變窄；觀察到當鹽濃度增加超過175mM時對解析度及峰形狀無進一步改良。
實例13a及13b分別使用乙腈與丙酮之混合物及乙腈作為用於分析本發明磺達肝素鈉之流動相之溶劑。儘管顯示於圖5中之結果指示在乙腈系統與混合物(乙腈及丙酮)系統之間選擇性無明顯差異，但觀察到在包含丙酮/乙腈混合物之系統中背壓高於在乙腈系統中。
儘管出於清晰瞭解之目的已藉助說明及實例相當詳細地闡述了上述發明，但熟習此項技術者應瞭解，可在隨附申請專利範圍之範圍
內實踐某些變化及修改。另外，本文所提供之每一參考文獻之全文皆以引用方式併入，其併入程度如同每一參考文獻個別地以引用方式併入一般。倘若本申請案與本文所提供參考文獻之間存在衝突，則應以本申請案為准。
Claims (10)
- 一種用於檢測及定量樣品中一或多種多硫酸化寡糖之方法，該方法包含：(a)在與電離子檢測器(CAD)偶聯之親水性相互作用超高效液相層析儀(HILIC-UPLC)管柱上對該樣品實施層析，其中用於HILIC管柱中之固定相係醯胺鍵合固定相；及(b)測定該樣品中多硫酸化寡糖之量。
- 如請求項1之方法，其中用於HILIC管柱中之流動相包含鹽。
- 如請求項2之方法，其中該鹽係選自甲酸銨、甲酸吡啶鎓鹽及乙酸銨。
- 如請求項3之方法，其中該鹽係甲酸銨。
- 如請求項3之方法，其中該鹽係甲酸銨且以約50mM至300mM之濃度存於該流動相中。
- 如請求項5之方法，其中該流動相中甲酸銨之濃度係約100mM至200mM。
- 如請求項1之方法，其中用於HILIC管柱中之該流動相之溶劑係乙腈、丙酮或乙腈與丙酮之混合物。
- 如請求項1之方法，其中用於HILIC管柱中之該流動相係乙腈。
- 如請求項1之方法，其中該多硫酸化寡糖係具有以下結構之磺達肝素(fondaparinux)：
- 一種用於檢測及定量樣品中磺達肝素之方法，該方法包含：(a)在與電離子檢測器(CAD)偶聯之親水性相互作用超高效液相層析儀(HILIC-UPLC)管柱上對該樣品實施層析，其中用於HILIC管柱中之固定相係醯胺鍵合固定相，用於該層析中之流動相係乙腈，且該流動相以約100mM至約200mM之濃度包含甲酸銨；及(b)測定該樣品中磺達肝素之量。
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