MATERIALS AND METHODS FOR MIXED MODE, ANION EXCHANGE REVERSED PHASE LIQUID CHROMATOGRAPHY

Abstract

In various aspects, the present disclosure pertains to high purity chromatographic materials that comprise a chromatographic surface wherein the chromatographic surface comprises a hydrophobic modifier and an ionizable modifier comprising one or more anion exchange moieties that are positively charged when ionized, as well as devices containing such materials. In other aspects, the present disclosure provides methods for mixed mode, anion exchange reversed phase liquid chromatography comprising: (a) loading a sample comprising a plurality of acidic analytes (e.g., acidic glycans) onto a chromatographic separation device comprising such a high purity chromatographic material and (b) eluting adsorbed acidic analytes from the high purity chromatographic material with a mobile phase comprising water, organic solvent, and an organic acid salt, wherein during the course of elution a pH of the mobile phase, an ionic strength of the mobile phase, and a concentration of the organic solvent are altered over time.

Claims

1. A chromatographic material that comprises a chromatographic surface wherein the chromatographic surface comprises a hydrophobic modifier and an ionizable modifier comprising one or more anion exchange moieties, which are positively charged when ionized.

2. The chromatographic material of claim 1, wherein the high purity chromatographic material is hydrolytically stable over a pH range of about 3 to about 10.

3. The chromatographic material of claim 1, wherein the chromatographic material is an inorganic material, a hybrid organic/inorganic material, an inorganic material with a hybrid surface layer, a hybrid material with an inorganic surface layer, or a hybrid material with a different hybrid surface layer.

4. (canceled)

5. The chromatographic material of claim 1, wherein the high purity chromatographic material comprises a hybrid organic/inorganic material that comprise ≡Si—(CH.sub.2).sub.n—Si≡ moieties and/or (CH.sub.2).sub.mCH.sub.3 moieties, where n is an integer equal to 1, 2, 3, or 4 and m is an integer equal to 0, 1, 2 or 3.

6. The chromatographic material of claim 1, wherein the chromatographic material may be formed by hydrolytically condensing (a) one or more silane compounds of the formula SiZ.sub.1Z.sub.2Z.sub.3Z.sub.4, where Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 are independently selected from Cl, Br, I, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, and C.sub.1-C.sub.4 alkyl, although at most three of Z.sub.1, Z.sub.2, Z.sub.3 and Z.sub.4 can be C.sub.1-C.sub.4 alkyl, and/or (b) one or more compounds of the formula Si—Z.sub.1Z.sub.2Z.sub.3—R—SiZ.sub.4Z.sub.5Z.sub.6, where Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from Cl, Br, I, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, and C.sub.1-C.sub.4 alkyl, although at most two of Z.sub.1, Z.sub.2 and Z.sub.3 can be C.sub.1-C.sub.4 alkyl, and where Z.sub.4, Z.sub.5 and Z.sub.6 are independently selected from Cl, Br, I, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, and C.sub.1-C.sub.4 alkyl, although at most two of Z.sub.4, Z.sub.5 and Z.sub.6 can be C.sub.1-C.sub.4 alkyl, and where R is an organic radical.

7. (canceled)

8. The chromatographic material of claim 1, wherein the hydrophobic modifier comprises a C.sub.4-C.sub.30 aliphatic moiety, C.sub.4-C.sub.30 aromatic moiety, a phenylalkyl moiety, or a fluoro-aromatic moiety.

9. (canceled)

10. The chromatographic material of claim 1, wherein the hydrophobic modifier is present in a surface concentration ranging from 0.1 to 5 micromoles per square meter.

11. The chromatographic material of claim 1, wherein the ionizable modifier comprises an anion exchange moiety having a pKa ranging from 4 to 13.

12. The chromatographic material of claim 1, wherein the ionizable modifier comprises an anion exchange moiety selected from an amine-containing moiety, a guanidine-containing moiety, an amidine-containing moiety, a pyridyl-containing moiety, an imidazolyl-containing moiety, a carbazolyl-containing moiety, an isocyanurate-containing moiety and/or a semicarbazidyl-containing moiety.

13. The chromatographic material of claim 11, wherein the anion exchange moiety is attached to the chromatographic surface by six or more siloxy bonds.

14. The chromatographic material of claim 1, wherein the ionizable modifier comprises an anion exchange moiety selected from an amine-containing moiety, a guanidine-containing moiety, an amidine-containing moiety, a pyridyl-containing moiety, an imidazolyl-containing moiety, a carbazolyl-containing moiety, an isocyanurate-containing moiety and/or a semicarbazidyl-containing moiety that bridges two or more siloxy groups, wherein each siloxy group is attached to the chromatographic surface by three siloxy bonds.

15. The chromatographic material of claim 1, wherein a molar ratio of hydrophobic modifier:ionizable modifier is from about 2:1 to about 100:1.

16. The chromatographic material of claim 1, wherein a surface concentration of the ionizable modifier is from about 0.01 μmol/m.sup.2 to about 1.0 μmol/m.sup.2.

17. The chromatographic material of claim 1, wherein the chromatographic surface is derivatized by reacting the chromatographic material with reactive hydrophobic modifying reagent that comprises (a) a hydrophobic moiety and (b) one or more reactive silane groups.

18. The chromatographic material of claim 17, wherein the ionizable modifying reagent is of the formula M(SiZ.sub.1Z.sub.2Z.sub.3).sub.n where n=1, 2, 3, or more, M designates a hydrophobic moiety, and Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from Cl, Br, I, C.sub.1-C.sub.4 alkoxy and C.sub.1-C.sub.4 alkylamino.

19. (canceled)

20. (canceled)

21. The chromatographic material of claim 1, wherein the chromatographic surface is derivatized by reacting the chromatographic material with an ionizable modifying reagent that comprises (a) one or more moieties selected from an amine-containing moiety, a guanidine-containing moiety, an amidine-containing moiety, a pyridyl-containing moiety, an imidazolyl-containing moiety, a carbazolyl-containing moiety, an isocyanurate-containing moiety and/or a semicarbazidyl-containing moiety and (b) two or more reactive silane groups.

22. (canceled)

23. The chromatographic material of claim 1, wherein the chromatographic surface is derivatized by reacting the chromatographic material with an ionizable modifying reagent of the formula A(SiZ.sub.1Z.sub.2Z.sub.3).sub.n where n=1, 2, 3, 4 or more, where A designates an amine-containing moiety, a guanidine-containing moiety, an amidine-containing moiety, a pyridyl-containing moiety, an imidazolyl-containing moiety, a carbazolyl-containing moiety, an isocyanurate-containing moiety and/or a semicarbazidyl-containing moiety, where Z.sub.1, Z.sub.2 and Z.sub.3 are independently selected from Cl, Br, I, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, and C.sub.1-C.sub.4 alkyl, although at most two of Z.sub.1, Z.sub.2 and Z.sub.3 can be C.sub.1-C.sub.4 alkyl, and where when n=2 or more, each of the —SiZ.sub.1Z.sub.2Z.sub.3 groups may be the same as each other, or each of the SiZ.sub.1Z.sub.2Z.sub.3 groups may be different from one another.

24. (canceled)

25. (canceled)

26. (canceled)

27. The chromatographic material of claim 17, wherein the chromatographic surface is further derivatized by reacting the chromatographic material with a silane capping reagent.

28. The chromatographic material of claim 27, wherein the silane capping reagent is a silane compounds of the formula SiZ.sub.1Z.sub.2Z.sub.3Z.sub.4, where Z.sub.1 is selected from Cl, Br, I, C.sub.1-C.sub.4 alkoxy and C.sub.1-C.sub.4 alkylamino and wherein Z.sub.2, Z.sub.3 Z.sub.4 are independently selected from C.sub.1-C.sub.2 alkyl.

29. The chromatographic material of claim 1, wherein the chromatographic material is in the form of a particle, a monolith, a superficially porous material, a superficially porous particle, a superficially porous monolith, or a superficially porous layer for open tubular chromatography.

30. The chromatographic material of claim 1, wherein the high purity chromatographic material has a pore diameter of about 20 to 1500 Å.

31. (canceled)

32. (canceled)

33. A chromatographic separation device that contains chromatographic material of claim 1.

34. (canceled)

35. (canceled)

36. (canceled)

37. (canceled)

38. A method for mixed mode, anion exchange reversed phase liquid chromatography comprising: (a) loading a sample comprising a plurality of acidic analytes onto a chromatographic separation device comprising a chromatographic material in accordance with claim 1 such that the acidic analytes are adsorbed onto the high purity chromatographic material and (b) eluting the adsorbed acidic analytes from the high purity chromatographic material with a mobile phase comprising water, organic solvent, and an organic acid salt thereby separating the acidic analytes, wherein eluting the acidic analytes from the chromatography material with the mobile phase comprises a course of elution in which a pH of the mobile phase is altered over time, an ionic strength of the mobile phase is altered over time, and a concentration of the organic solvent is altered over time.

39-59. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0062] FIG. 1 is a chromatogram showing the results of anion exchange reversed phase liquid chromatography of fetuin N-glycans. The chromatogram is the result of 2.1×150 mm column packed with a 1.7 μm 100 Å high purity chromatographic material using the methods described in Example 9.

[0063] FIG. 2 is a chromatogram showing the results of anion exchange reversed phase liquid chromatography of RapiFluor-MS® labeled N-glycans from glucuronidase, illustrating separation of glucuronidase N-glycans The chromatogram is a result of a 2.1×150 mm column packed with a 1.7 μm 100 Å high purity chromatographic material using the methods described in Example 10.

DETAILED DESCRIPTION

[0064] In specific embodiments, the present disclosure provides a method for mixed mode, anion exchange reversed phase liquid chromatography and the selective retention of acidic glycan species, including but not limited to sialylated, phosphorylated and sulfated glycans. These glycan species can be analyzed in the form of released and labeled derivatives, unlabeled native glycans, unlabeled, reduced glycans, or glycopeptides. The method advantageously combines volatile mobile phases with gradients of increasing concentrations of ammonium salt and organic solvent and columns packed with high purity chromatographic materials that are designed to minimize interference during electrospray ionization.

[0065] The present disclosure highlights the utility of materials prepared with ionizable modifying reagents having two, three or more silyl groups, including bis and tris silyl ionizable modifying reagents.

[0066] By selecting suitable ionizable modifiers, a surface chemistry can be created that is ideally suited to separating acidic glycans derivatized with MS-enhancing labels, including but not limited to amphiphilic, strongly basic moieties such as Waters RapiFluor-MS®, Prozyme Instant Procaine (InstantPC™), procaine, and procainamide. Ionizable modifiers described herein uniquely provide deeply embedded ionizable surface residues and thus show enhanced chromatographic selectivity. Because these ionizable modifiers have multiple points of attachment to their substrate, their corresponding high purity chromatographic materials exhibit comparatively low levels of so-called column bleed, which is of benefit to online mass spectrometric detection.

[0067] In various preferred embodiments, the present disclosure provides an LC-MS method for glycan profiling that achieves enhanced chromatographic resolving power with high sensitivity detection, as enabled by improved chromatographic materials that provide low levels of interference when used with mass spectrometric detection.

Example 1

[0068] BEH porous hybrid particles (prepared following the method as described in U.S. Pat. No. 6,686,035) were fully dispersed in toluene (5 mL/g), then azeotropically stripped (reflux, 1 h) to ensure anhydrous conditions. An ionizable modifying reagent as shown in Table 1, S1-S10 (0.1-0.3 mol/m.sup.2) was added to the BEH/toluene slurry, then stirred at room temperature for 1 h.

TABLE-US-00001 TABLE 1 Designation Silane Name S1 3-Aminopropyldiisopropylethoxysilane S2 N-(2-aminoethyl)-11-aminoundecyltrimethoxysilane S3 Methanamine, 1-(triethoxysilyl)-N,N-bis[(triethoxysilyl)methyl]- S4 N-Cyclohexylaminomethyltriethoxysilane S5 2-(4-Pyridylethyl)triethoxysilane S6 1-Butanamine, 2,2-dimethyl-4-(trimethoxysilyl) S7 1-Propanamine, N-methyl-3-(trimethoxysilyl)-N-[3-(trimethoxysilyl)propyl]- S8 1-Hexanamine, N,N-dimethyl-6-(trimethoxysilyl)- S9 Ethanol, 2-[bis[3-(trimethoxysilyl)propyl]amino]- S10 2-Oxa-7,10-diaza-3-siladodecan-12-ol, 7-(2-hydroxyethyl)-3,3-dimethoxy-10- [3-(trimethoxysilyl)propyl]

[0069] The slurry was then allowed to stir at reflux for 2 h under an inert atmosphere. The reaction was then cooled to room temperature and the particles were isolated via filtration. The particles were washed successively with toluene, acetone/water (1:1 v/v), and acetone. The isolated particles were then dried for 16 h at 80° C. under 25 mm vacuum. Once dry, the particles were fully dispersed in toluene (10 mL/g), then azeotropically stripped (reflux, 3 h) to ensure anhydrous conditions. A base catalyst (pyridine or imidazole: 3.2 mol/m.sup.2) was added to the particles/toluene slurry, then a hydrophobic modifying reagent (octadecyltrichlorosilane (tC.sub.18): 1.6 mol/m.sup.2) was added to the particles/toluene slurry. The slurry was stirred at reflux for 20 h under an inert atmosphere. The reaction was cooled to room temperature and the particles were isolated via filtration. The particles were washed successively with toluene, acetone acetone/water (1:1 v/v), and acetone. The acetone-wet particles were transferred into a clean reactor, dispersed in acetone/0.12 M ammonium acetate (8.2:1.8 v/v) and hydrolyzed (59° C., 2 h). The particles were then isolated via filtration and washed successively with toluene, acetone, acetone/water (1:1 v/v), and acetone. Finally, the isolated surface modified particles were dried for 16 h at 70° C. under 25 mm vacuum and characterized. Results are shown in Table 2.

TABLE-US-00002 TABLE 2 Ionizable Ionizable Modifier (I) Hydrophobic Group (H) Charge Modifier Final Total Particles Designated Charge Charge Molar Coverage Coverage Product (g) Name (μmol/m.sup.2) Type (μmol/m.sup.2) Ratio H/I (μmol/m.sup.2) (μmol/m.sup.2) 1a 10 S1 0.3 tC18 1.61 5.4 0.24 2.00 1b 10 S2 0.3 tC18 1.60 5.3 0.24 1.96 1c 10 S3 0.3 tC18 1.60 5.3 0.24 1.65 1d 10 S4 0.3 tC18 1.59 5.3 0.16 1.62 1e 10 S5 0.3 tC18 1.60 5.3 0.04 1.69 1f 10 S6 0.3 tC18 1.61 5.4 0.20 1.73 1g 20 S7 0.3 tC18 1.60 5.3 0.28 1.71 1h 10 S7 0.2 tC18 1.60 8.0 0.20 1.71 1i 10 S7 0.1 tC18 1.60 16.0  0.08 1.66 1j 10 S8 0.3 tC18 1.60 5.3 0.24 1.88 1k 10 S9 0.3 tC18 1.60 5.3 0.28 1.82 1l 10  S10 0.3 tC18 1.60 5.3 0.26 1.85

[0070] The surface coverage of ionizable modifier was determined by the difference in particle % N after surface modification as measured by elemental analysis. The surface coverage of the C.sub.18 hydrophobic modifier was determined by the difference in particle % C before and after the surface modification as measured by elemental analysis. % N values were measured by combustion analysis (CE-440 Elemental Analyzer; Exeter Analytical Inc., North Chelmsford, Mass.) or % C by Coulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet, Ill.).

Example 2

[0071] Surface modified particles from Example 1 were fully dispersed in toluene (10 mL/g) and then azeotropically stripped (reflux, 1 h) to ensure anhydrous conditions. Triethylchlorosilane (TES) or (N,N-dimethylamino)triethylsilane was charged (TES: 5 mol/m.sup.2) to the surface modified particles/toluene slurry, then stirred at reflux for 4 h under an inert atmosphere. The reaction was then cooled to room temperature and trimethylchlorosilane or (N,N-dimethylamino)trimethylsilane was charged (TMS: 8 mol/m.sup.2) to the surface modified particles/toluene slurry, then stirred at reflux for 16 h under an inert atmosphere. A base catalyst (pyridine or imidazole: 3.2 mol/m.sup.2) was added to the reaction slurry if chlorosilanes were used. The reaction was cooled to room temperature and the particles were subsequently isolated via filtration and washed successively with toluene, acetone, acetone/water (1:1 v/v), and acetone. Finally, the isolated surface modified particles were dried for 16 h at 70° C. under 25 mm vacuum. Particles were characterized and the results presented in Table 3.

TABLE-US-00003 TABLE 3 Particles TES TMS Final Carbon Product (g) Precursor Mass (g) Mass (g) (% C) 2a 8 1a 1.2 1.3 14.40 2b 8 1b 1.1 1.3 14.47 2c 8 1c 1.1 1.3 13.63 2d 8 1d 1.1 1.3 13.65 2e 8 1e 1.1 1.3 13.79 2f 8 1f 1.1 1.3 13.85 2g 19 1g 2.6 2.9 13.84 6h 8 1h 1.1 1.3 13.77 6i 8 1i 1.1 1.2 13.77 2j 8 1j 1.1 1.3 14.19 2k 8 1k 1.1 1.3 14.08 2l 8 1l 1.1 1.3 14.13

Example 3

[0072] The method as described in Example 1 is expanded to include an ionizable modifier coverage of 0.03-1.0 μmol/m.sup.2 and a hydrophobic modifier to ionizable modifier ratio of 2.5:1 to 67:1.

Example 4

[0073] The surface modified particles from Example 3 are further modified using the method as described in Example 2.

Example 5

[0074] The methods as described in Examples 1 and 3 are expanded to include other ionizable modifiers of interest, such as, but not limited to S11-S42 in Table 4 in combination with a hydrophobic group to yield a hydrophobic phase to ionizable modifier ratio of 2.5:1 to 67:1.

TABLE-US-00004 TABLE 4 Designation Silane Name S11 11-Aminoundecyltriethoxysilane S12 4-Aminobutyltriethoxysilane S13 1,2-Ethanediamine, N.sup.1,N.sup.2-bis[(diethoxymethylsilyl)methyl]-N.sup.1,N.sup.2-dimethyl- S14 (N,N-Diethylaminopropyl)trimethoxysilane S15 (3-Aminopropyl)triethoxysilane S16 1,2-Ethanediamine, N.sup.1-(3-phenyl-2-propen-1-yl)-N.sup.1-[3-(trimethoxysilyl)propyl]- S17 1-Propanamine, 3-(dimethoxyphenylsilyl)- S18 1-Propanamine, N-ethyl-2-methyl-1-(trimethoxysilyl)- S19 1-Naphthalenamine, N,N-dimethyl-4-(triethoxysilyl)- S20 9H-Carbazole, 9-[2-(triethoxysilyl)ethyl]- S21 1,2-Ethanediamine, N.sup.1,N.sup.1,N.sup.2-trimethyl-N.sup.2-[3-(trimethoxysilyl)propyl]- S22 (Aminoethylaminomethyl)phenethyltrimethoxysilane S23 N-3-[(Amino(polypropylenoxy)]aminopropyltrimethoxysilane S24 1,3,5-Triazine-2,4,6(1H,3H,5H)-trione, 1,3,5-tris[3-(trimethoxysilyl)propyl]- S25 1-Propanamine, 3-(triethoxysilyl)-N-[3-(triethoxysilyl)propyl]- S26 1,2-Ethanediamine, N.sup.1,N.sup.1-bis[3-(dimethoxymethylsilyl)propyl]- S27 1,2-Ethanediamine, N.sup.1,N.sup.2-bis[3-(trimethoxysilyl)propyl]- S28 N,N'-bis(2-hydroxyethyl)-N,N′-bis(trimethoxysilylpropyl)ethylenediamine S29 N-(2-Aminoethyl)-3-aminopropyltrimethoxysilane S30 N-Cyclohexyl-3-aminopropyltrimethoxysilane S31 N-(2-Aminoethyl)-3-Aminopropylmethyldimethoxysilane S32 3,3,15,15-Tetramethoxy-2,7,16-trioxa-11-aza-3,15-disilaheptadecan-9-ol S33 3-(m-Aminophenoxy)propyltrimethoxysilane S34 3-(4-Semicarbazidyl)propyltriethoxysilane S35 (17-Aminoheptadecyl)trimethoxysilane S36 16-Aminohexadecyltrimethoxysilane S37 N.sup.1-[12-(Triethoxysilyl)dodecyl]-1,2-ethanediamine S38 1,7-Heptanediamine, N1,N7-bis[9-(triethoxysilyl)nonyl]- S39 1,7-Heptanediamine, N1,N1-bis[7-(trimethoxysilyl)heptyl]- S40 1,10-Decanediamine, N1,N10-bis[6-(dimethoxymethylsilyl)hexyl]-N1,N10-dimethyl- S41 1,2-Ethanediamine, N2-[3-(trimethoxysilyl)propyl]-N1,N1-bis[2-[[3-(trimethoxysilyl) propyl]amino]ethyl]- S42 1,2-Ethanediamine, N,N,N'-tris[3-(trimethoxysilyl)propyl]-(9Cl)

Example 6

[0075] The surface modified particles from Example 5 are further modified using the method as described in Example 2.

Example 7

[0076] The method along the lines of Example 1 is expanded to include other modifying reagents of interest, which provide the resulting product with both an ionizable modifier and a hydrophobic modifier. Such modifying reagents include but are not limited to not limited to S43-S48 in Table 5. Because the modifying reagent includes a hydrophobic modifier, the steps in Example 1, wherein a hydrophobic modifying reagent (Octadecyltrichlorosilane (tC.sub.18) is added to the particles/toluene slurry, refluxed and cooled can be dispensed with. In other embodiments, the steps in Example 1, wherein a hydrophobic modifying reagent (Octadecyltrichlorosilane (tC.sub.18) is added to the particles/toluene slurry, refluxed and cooled are conducted.

TABLE-US-00005 TABLE 5 Designation Silane Name S43 1-Octadecanamine, N-[3-(trimethoxysilyl)propyl]- S44 1-Docosanamine, N-[3-(trimethoxysilyl)propyl]- S45 1,3-Propanediamine, N-octadecyl-N'-[3-(trimethoxysilyl)propyl]- S46 3-Octadecanamine, 1-(trimethoxysilyl)- S47 1-Hexadecanamine, N,N-bis[3-(trimethoxysilyl)propyl]- S48 3,8-Dioxa-4,7-disiladecan-5-amine, 4,4,7,7-tetraethoxy-N-hexadecyl-N-propyl

Example 8

[0077] The surface modified particles from Example 8 are further modified using the method as described in Example 2.

Example 9

[0078] Anion Exchange/Reversed Phase Liquid Chromatography (RPLC) of Sialylated Glycans. Separations of sialylated glycans have been achieved with a material in accordance with the present disclosure, and they have been found to exhibit remarkably high resolution. An exemplary embodiment is provided in which RapiFluor-MS labeled glycans from bovine fetuin (Sigma) have been separated with a dual ammonium formate/acetonitrile gradient using a column packed with 1.7 μm bridged ethylene organosilica 100 Å fully porous particles modified with a bis-silyl tertiary amine containing ionizable modifier along with a trifunctionally bonded C.sub.18 as described above.

[0079] A chromatogram from this separation is displayed in FIG. 1. Glycan assignments have been made using Oxford notation. For example, A3G3S3 denotes a glycan comprised of a triantennary core (A3) that is modified with three galactose residues (G3) and three sialic acid residues (S3). Some annotations include a parenthesis marking with NGNA to denote where sialic acid residues are present in the form of N-glycol versus N-acetyl moieties. In this, it can be seen that it was possible to resolve isomer and glycan charge heterogeneity from bovine fetuin, as is exemplified by the numerous, baseline resolved peaks and their annotations. In turn, these results suggest there to be great promise in this disclosure and its application to the structural characterization of sialylated glycoproteins, including recombinant protein therapeutics ranging from blood factors to enzyme replacement therapies, which are typically designed to exhibit high levels of sialylation in order to extend circulation half-life or affect inflammatory responses.

TABLE-US-00006 TABLE 6 Conditions for Anion Exchange RPLC with Fluorescence and MS Detection ACQUITY UPLC I-Class System: and Xevo G2-S QTof Data Acquisition UNIFI and Analysis: Temperature:  60° C. Seal Wash: 10% HPLC grade Methanol/90% HPLC grade water v/v (Seal Wash interval set to 5 min) Sample Manager HPLC grade water Wash: Mobile Phase A1 Water and A2: Mobile Phase B1 100 mM formic acid/100 mM ammonium and B2: formate in 40:60 water/acetonitrile Flow Rate: 0.40 mL/min Sample Temp.  10° C. Sample: RapiFluor-MS labeled N-glycans from bovine fetuin (glycans from 0.17 μg of fetuin/μL) Blank: Milli-Q water Injection Volume:   3 μL Fluorescence Ex 265 nm/Em 425 nm Detection: Mode: ESI+ sensitivity Capillary voltage:  3.0 kV Sampling Cone   30 V voltage: Source temp.: 100° C. Desolvation temp.: 300° C. Cone gas flow:   50 L/h Desolvation gas  800 L/h flow:

Example 10

[0080] Anion Exchange RPLC of Phosphorylated Glycans. Glycans released from recombinant human β-glucuronidase (Novus Biologics, 6144-GH) have also been prepared, labeled with RapiFluor-MS® and subjected to chromatographic analysis using a column packed with 1.7 μm bridged ethylene organosilica 100 Å fully porous particles modified with a bis-silyl tertiary amine containing ionizable modifier along with a trifunctionally bonded C.sub.18 as described above. Glycans can be found in this type of sample that contain mannose-6-phosphate (M6P) residues. In the case of enzyme replacement therapies that address lysosomal storage disorders, the amount of this residue and the relative amounts of the corresponding glycan structures are critically important. A multitude of enzymes have been brought to market for patient treatments, including recombinant glucuronidase, galactosidase, glucosidase, heparin sulfatase, and each of these must be post-translationally modified with M6P in order for cellular uptake to be properly facilitated. Accordingly, it is important for there to be robust assays for the detection and quantitation of M6P containing glycans.

[0081] A chromatogram from this separation is displayed in FIG. 2. Glycan assignments have been made according to their compositional information (Hex=hexose, HexNAc=N-acetyl hexosamine). M5, M6, and M9 correspond to high mannose glycans. FA1G1 is assigned based on Oxford notation. Importantly, glycans containing a phospho group are denoted with a “P”; singly phosphorylated glycans are assigned “-P” and doubly phosphorylated are assigned “-2P”.

[0082] With these analytical results, enhanced resolution is achieved on top of charge-based speciation of the glycan species. That is, the N-glycans are seen to elute into distinct pools of uncharged, singly charged, doubly charged, and triply charged species. Within each of the chromatographic regions, glycan heterogeneity is further elucidated by their isomerization and individual composition of charge bearing residues, e.g., one phosphorylated residue and two sialylated residues. There is consequently an abundance of information produced in a short amount of time and each species can be detected via an optical detector or with online mass spectrometric detection.