WO2016051170A1

WO2016051170A1 - Hydrophilic interaction liquid chromatography

Info

Publication number: WO2016051170A1
Application number: PCT/GB2015/052854
Authority: WO
Inventors: Lingzhi GONG
Original assignee: Isis Innovation Limited
Priority date: 2014-10-01
Filing date: 2015-09-30
Publication date: 2016-04-07
Also published as: GB201417341D0

Abstract

The present invention relates to a chromatographic process, which process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises: a)one or more analytes; b)water; c)an organic solvent; and d)an ion-pairing reagent; wherein the concentration of the ion-pairing reagent in the composition is less than or equal to 50 mM. Also described is a hydrophilic interaction liquid chromatographic process, which process comprises contacting a hydrophilic stationary phase with a composition comprising one or more analytes and an ion-pairing reagent, wherein the concentration of the ion-pairing reagent is less than or equal to 50 mM. The invention also relates to a chromatographic process, which process comprises (i) contacting a composition with a hydrophilic stationary phase, which composition comprises: a) one or more analytes; b) water; c) an organic solvent; and d) an ion-pairing reagent; and (ii) a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection(ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection. Uses and an apparatus are also described.

Description

HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY

FIELD OF THE INVENTION

The present invention relates to liquid chromatography and a chromatographic process. In particular, the invention relates to hydrophilic interaction liquid chromatography. An apparatus for performing a chromatographic process and the use of an ion-pairing reagent are also described.

BACKGROUND OF THE INVENTION

Liquid chromatography is an important process for the separation of a variety of analytes. A number of different forms of liquid chromatography are available. These include normal- phase high performance liquid chromatography (NP-HPLC), reversed-phase high performance liquid chromatography (RP-HPLC), size-exclusion chromatography and ion- exchange chromatography. NP-HPLC makes use of a polar stationary phase (for instance silica) and a non-polar, or moderately polar mobile phase (for instance chloroform). RP- HPLC involves a non-polar stationary phase (for instance silica modified with alkyl groups) and an aqueous, moderately polar mobile phase (for instance water and acetonitrile). Liquid chromatography is often coupled with mass-spectrometry as this allows mass-analysis of the analytes separated by the chromatography.

One important example of an analyte is that of oligonucleotides. Sensitive and selective methods have always been demanded for the characterization of oligonucleotides when the oligonucleotides are applied as therapeutics. Chromatographic techniques such as high- performance liquid chromatography (HPLC) and electrophoresis have been frequently used in the purification and analysis of oligonucleotides.

Electrospray ionization mass spectrometry (ESI-MS) has shown its important applications in the sequencing, identification, and characterization of oligonucleotides and other analytes. However, mass measurement of some analytes, and particularly oligonucleotides, faces a serious problem of adduct formation, as moieties in the analyte, for instance the deprotonated phosphate backbone of oligonucleotides, may have high affinities for various cations, in particular sodium and potassium ions which are often present in analyte solutions (Oberacher H (2008), On the use of different mass spectrometric techniques for characterization of sequence variability in genomic DNA, Anal. Bioanal. Chem. 391 : 135-149). These cations lower the MS signal sensitivity of oligonucleotide by dispersing the ion abundance among multiple adducted ions. Hence, sample pre-treatment by removing adducts off-line or on-line is essential in order to obtain high-quality mass spectra.

HPLC has been widely used as an on-line sample preparation technique, as it has the advantage of easily coupling to mass spectrometry (LC-MS) combining desalting, separation, and identification. In the early 1990s, a few research groups attempted using reversed-phase (RP) LC to separate analytes such as single-stranded oligonucleotides (Huang G, Krugh TR (1990), Anal. Biochem. 190:21-25). In most of the applications, advantage was taken of the attachment of the hydrophobic protecting group (trityl-, dimethoxytrityl groups) to the investigated oligonucleotides, which increased their retention in reversed-phase HPLC. In the late 1990s, the separation of analytes was largely improved by adding ion-pairing reagent in the reversed-phase mobile phase, i.e. ion-pair reversed-phase HPLC (A. Apffel, J.A.

Chakel, S. Fischer, K. Lichtenwalter, W.S. Hancock, Anal. Chem. 69 (1997) 1320). Ion-pair reversed-phase liquid chromatography has been widely used for the analysis of

oligonucleotides since then (Lei B, Li S, Xi L, Li J, Liu H, Yao X (2009), J. Chromatogr. A 1216:4434-4439).

An alternative chromatographic approach, hydrophilic interaction liquid chromatography (HILIC), has also been employed to the separation and analysis of oligonucleotides (L. Gong, J.S.O. McCullagh, J. Chroatogr. A 1218 (2011) 5480). Hydrophilic interaction liquid chromatography is a version of normal phase liquid chromatography. The name of HILIC was suggested by Alpert (A.J. Alpert, J. Chromatogr. 499 (1990) 177) who proposed that the HILIC mechanism involves the mobile phase forming a water-rich layer on the surface of the polar stationary phase which forms an interface with the water-deficient mobile phase, creating a liquid/liquid extraction system. Polar analytes are retained and separated primarily by partitioning between these two layers. The high organic content of the mobile phase makes HILIC particularly compatible with electrospray (ESI) ionization mass spectrometry. HILIC HPLC has been increasingly used for separating small and large polar compounds.

Largely improved chromatographic performance for the ion-pair reversed-phase (IP-RP) separation of oligonucleotides has been observed with the addition of ion-pairing reagents in the mobile phase of reversed-phase chromatography. The addition of an ion-pairing agent to a HILIC process at high concentrations (100 mM) has also been performed (P.

Holdsvendova, J. Suchankova, M. Buncek, V. Backovska, P. Coufal, J. Biochem. Biophys. Methods 70 (2007) 23). However, improvements in the separation of analytes by a HILIC process are still needed. Furthermore, there has been no discussion of how ion-pairing reagents may be used in a HILIC process coupled with mass spectrometry.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that the use of HILIC to separate analytes can be greatly improved by using a low concentration of an ion-pairing reagent. The inventors have improved resolution for chromatography whilst maintaining or improving the sensitivity that can be achieved for mass spectrometry by including an ion-paring reagent in the process. The ion-pair (IP) HILIC approach produces lower retention capacity and has the added benefit of allowing for the provision of simpler MS spectra with fewer charge states when compared with traditional HILIC.

The development of this ion-pair hydrophilic interaction liquid chromatography (IP-HILIC) process allows for effective separation of analytes when ion-pairing reagent concentrations of less than or equal to 50 mM are used. In particular, this new IP-HILIC method is suitable for coupling with mass spectrometry, for instance electrospray mass spectrometry. Thus, the inventors have also developed the ion-pair hydrophilic interaction liquid chromatography electrospray mass spectrometry (IP-HILIC LC-ESI-MS) method which uses an ion-pairing reagent at a concentration that has been found to be MS compatible, for instance for the analysis of unmodified and chemically modified oligonucleotides. The described process has been shown to be capable of separating unmodified and chemically modified

oligonucleotides. The concept of ion-pair hydrophilic interaction liquid chromatography (IP- HILIC) is suggested for the first time.

The invention therefore provides a chromatographic process, which process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises: a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

wherein the concentration of the ion-pairing reagent in the composition is less than or equal to 50 mM. The invention also provides a hydrophilic interaction liquid chromatographic process, which process comprises contacting a hydrophilic stationary phase with a composition comprising one or more analytes and an ion-pairing reagent, wherein the concentration of the ion-pairing agent in the composition is less than or equal to 50 mM.

The effects of the concentration of ion-pairing reagent on the separation of analytes has also been studied. The inventors have surprisingly found that the use of unprecedentedly low concentrations of the ion-pairing reagent (for instance less than 10 mM) allow for an increase in resolution and strength of mass spectrometry (MS) signals from the IP-HILIC LC-ESI-MS process compared to HILIC LC-ESILMS process without ion-pairing reagent (L. Gong, J. McCullagh, J. Chromatogr. A 1218 (2011), 5480).

The invention also therefore provides a chromatographic process, which process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises: a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

wherein the concentration of the ion-pairing reagent in the composition is less than or equal to 10 mM, for instance from 0.01 mM to 9 mM.

The invention also provides an apparatus for performing a chromatographic process, which apparatus comprises a hydrophilic stationary phase and a composition, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

wherein the concentration of the ion-pairing reagent in the composition is less than or equal to 50 mM.

The invention also provides use of an ion-pairing reagent for increasing resolution in a chromatographic process, which chromatographic process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises:

a) one or more analytes; b) water;

c) an organic solvent; and

d) said ion-pairing reagent;

wherein the concentration of said ion-pairing reagent in the composition is less than or equal to 50 mM.

The invention also provides use of a composition comprising less than or equal to 50 mM of an ion-pairing reagent for increasing resolution in a hydrophilic interaction liquid

chromatographic process.

The invention also provides a chromatographic process, which process comprises

(i) contacting a composition with a hydrophilic stationary phase, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent; and

(ii) a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection (ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection. Often, step (ii) comprises performing online mass spectrometry.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows evaluation of six HILIC columns for IP-HILIC. a). PolyHYDROXYETHYL^® A 90-10% ACN, lmL/min; b). YMC^® pack silica, 75-30% ACN, 0.2mL/min; c). Kinetex^® HILIC, 90-50% ACN, 0.2mL/min; d). ZIC^®-HILIC, 75-50% ACN 0.2mL/min; e). Luna^® HILIC, 80-65% ACN, 0.2mL/min; f). Zorbax^® RRHD HILIC, 75-60% ACN, 0.2mL/min. Mobile phase A: H₂0, mobile phase B: ACN, mobile phase C: lOOmM TEAA pH7 (kept at 10%) through whole gradient).

Figure 2 shows comparison of retention of oligonucleotides between IP-HILIC and HILIC chromatography, a). Luna HILIC, lOmM TEAA (pH7) 80-50% ACN in 7.5min, 0.5mL/min; b). Luna HILIC, lOmM ammonium acetate (pH7) 80-50% ACN in 7.5min, 0.5mL/min; c). ZIC-HILIC, lOmM TEAA (pH7) 70-50% ACN in 7.5min, 0.2mL/min; d). ZIC-HILIC, lOmM ammonium acetate (pH7) 70-50% ACN in 7.5min, 0.2mL/min; e). YMC pack silica, lOmM TEAA (pH7) 75-30% ACN in 7.5min, lmL/min; f). YMC pack silica, lOmM ammonium acetate (pH7) 75-30% ACN in 7.5min, lmL/min.

Figure 3 shows the effect of the concentration of ion-pairing reagent on the separation of oligonucleotides (extracted ion chromatogram). Zorbax^® RRHD HILIC, 100 mm><2.1 mm, 1.8 μιη column. Mobile phase A: Milli-Q H₂0; B: acetonitrile; C: 100 mM TEAA, pH 7. Gradient from 75% to 60% B in 7.5 min, with constant 5, 10, 15% C, flow rate, 0.2 mL/min, temperature, 40°C.

Figure 4 shows the effect of organic modifier on the separation of oligonucleotides. Zorbax^® RRHD HILIC column, 100 mm X 2.1 mm, 1.8 μιη. Mobile phase A: Milli-Q H₂0; B:

acetonitrile/methanol; C: 100 mM TEAA, pH 7. Gradient from 75% to 60% B in 7.5 min for ACN and 80% to 65% B in 7.5 min for methanol, with constant 10% C, flow rate, 0.2 mL/min, temperature, 40°C.

Figure 5 shows: - (A) Separation of unmodified oligonucleotide (MIXl). Zorbax^® RRHD HILIC column, 100 mmx2.1 mm, 1.8 μιη. Mobile phase A: Milli-Q H₂0; B: acetonitrile;

C: 100 mM TEAA, pH 7. Gradient from 75% to 60% B in 7.5 min, with constant 10% C, flow rate, 0.2 mL/min, temperature, 40 °C. (a) MIXl & MIXIT; (b) MIXl & MIXIG; (c) MIXl & MIX1C; (d) MIXl & MIX1A; (e) MIXl & MIX2; - (B) Separation of unmodified oligonucleotide (MIX2). Zorbax^® RRHD HILIC column, 100 mmx2.1 mm, 1.8 μιη. Mobile phase A: Milli-Q H₂0; B: acetonitrile; C: 100 mM TEAA, pH 7. Gradient from 75% to 60% B in 7.5 min, with constant 10% C, flow rate, 0.2 mL/min, temperature, 40 °C. (a) MIX2 & MIX2T; (b) MIX2 & MIX2G; (c) MIX2 & MIX2C; (d) MIX2 & MIX2A.

Figure 6 shows separation of chemically modified oligonucleotides, (a). Phosphorylated oligonucleotide; (b). Phosphorothioated oligonucleotide; (c). Fluorescent labeled

oligonucleotide; (d). Locked Nucleic Acid. Zorbax^® RRHD HILIC column, 100 mmx2.1 mm, 1.8 μιη. Mobile phase A: Milli-Q H₂0; B: acetonitrile; C: 100 mM TEAA, pH 7.

Gradient from 75% to 60% B in 7.5 min, with constant 10% C, flow rate, 0.2 mL/min, temperature, 40 °C.

Figure 7 shows the mass spectrum of oligonucleotide EVEN and inset to Figure is its deconvoluted mass spectrum. DETAILED DESCRIPTION OF THE INVENTION

The invention provides a chromatographic process, which process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

wherein the concentration of the ion-pairing reagent in the composition is less than or equal to 50 mM. The composition typically comprises greater than or equal to 80 wt% of components (a) to (d). For instance, the composition may comprise greater than or equal to 90 wt% of components (a) to (d), or greater than or equal to 95 wt% of components (a) to (d). The composition may in some cases consist essentially of components (a) to (d). "Consisting essentially of, as used herein, refers to a composition which comprises the listed components and does not comprise any further components which materially effect the properties of the composition.

The general features of chromatographic processes are well known to the skilled person. Chromatographic processes are processes which separate components in a mixture. In general, a composition comprising several analytes in a mobile phase will flow, or be pumped, through a stationary phase. Due to variable interactions between different analytes with the stationary phase, different analytes will flow at different rates though the stationary phase. This allows analytes to be separated, for instance based on the time taken for the analyte to pass through the stationary phase.

Typically, a chromatographic process comprises pumping a mobile phase (for instance the composition described herein) through a column containing a stationary phase. The mobile phase is often pumped through at pressure, for instance a pressure of from 20 bar to 1000 bar. The composition leaving the chromatographic column may be analysed, for instance by passing through a detector. Signals can be detected as analytes with different retention times exit the column. The chromatographic process of the invention is particularly suited for coupling with mass spectrometry. Thus, the process may comprise contacting a composition with a hydrophilic stationary phase, which a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent; and

(ii) performing mass spectrometry on the composition.

The concentration of the ion-pairing reagent in the composition is typically less than or equal to 50 mM. The volume of the organic solvent in the composition is typically greater than the volume of water in the composition. The volumes of organic solvent and water may vary with position within the chromatographic column, but typically in a volume of the chromatographic column (for instance the volume in 2 cm length of the column), the volume of the organic solvent in the composition is typically greater than the volume of water in the composition at a time during the chromatographic process. As discussed below, the ratio of the volume of the water and the organic solvent is typically varied during the chromatographic process. In some cases, the volume of the organic solvent in the composition is greater than the volume of water for the entirety of the chromatographic process (i.e. the ratio of (volume organic solvent): (volume water) is always from 51 :49 to 100:00). Typically, however, the volume of the organic solvent in the composition is greater than the volume of water in the composition for greater than or equal to 1 minute during the chromatographic process (e.g. the ratio of (volume organic solvent): (volume water) is from 51 :49 to 100:00 for greater than or equal to 1 minute). More typically, the volume of the organic solvent in the composition is greater than the volume of water in the composition for greater than or equal to 3 minutes, or greater than or equal to 5 minutes, during the chromatographic process. The concentration of the ion-pairing agent is often fixed during the process, however. For instance, the concentration of the ion-pairing agent may be from 1 to 20 mM for the majority of the chromatographic process (i.e. for more than 80% of the duration of the process).

Typically, the amount of water in the composition is greater than or equal to 5% by volume and the volume of the organic solvent in the composition is greater than the volume of water in the composition. For instance, the amount of water in the composition may be greater than or equal to 20% by volume and the volume of the organic solvent in the composition is then greater than the volume of water in the composition.

Typically, the chromatographic process comprises allowing the composition to elute through the hydrophilic stationary phase. Elution may be achieved by allowing the composition to flow through the stationary phase under gravity, or, more typically, may be achieved by pumping the composition through the stationary phase. The term "elute" is well understood by the skilled person and describes a process whereby the composition or solvents within the composition pass through the chromatographic column.

The water is typically more strongly eluting that the organic solvent. "Eluting strength" is a well known property. An example of a list of solvents by increasing eluting power could be hexane < cyclohexane < benzene < dichloromethane <chloroform < ether (anhydrous) < ethyl acetate (anhydrous) < acetone (anhydrous) < ethanol < methanol < water < pyridine < acetic acid.

Chromatography can separate analytes by a number of mechanisms, for instance by adsorption of the analyte to the stationary phase. Typically, the chromatographic process of the invention comprises retention of analytes by liquid-liquid partitioning. With liquid-liquid partitioning, the analytes partition between two liquids, in this case typically water and said organic solvent. One of the liquids adheres more strongly to the stationary phase, typically water, and this effectively creates a liquid stationary phase. As discussed above, low concentrations of ion-pairing reagent (IPR) mean that the process of the invention provides good resolution and is well suited for coupling with mass

spectrometry. Typically, the concentration of the ion-pairing reagent in the composition is less than 40 mM, for instance from 0.01 mM to 40 mM. For instance, the concentration of the IPR may be from 0.01 mM to 30 mM. More typically, the concentration of the of the IPR may be from 0.1 mM to 25 mM. For instance, the concentration of the IPR may be from 1 mM to 20 mM, or from 3 mM to 15 mM.

When the concentration of the IPR is very low, the process of the invention may surprisingly be coupled with mass spectrometry and produce well resolved MS peaks with strong signals. Often, therefore, the concentration of the ion-pairing reagent in the composition is less than 10 mM. For instance, the concentration of the ion-pairing reagent in the composition may be from 0.1 mM to 9 mM. When using low IPR concentrations, the concentration of the IPR in the composition may be from 1 mM to 8 mM, for instance from 1 mM to 7 mM. In some cases, the concentration of the IPR may be about 5 mM, for instance from 4 mM to 6 mM.

A number of different ion-pairing agents may be used in the process of the invention.

Typically, the ion-pairing reagent is a primary alkyl amine, a secondary alkyl amine, a tertiary alkyl amine, an ammonium salt, a primary ammonium salt, a secondary ammonium salt, or a tertiary ammonium salt. For instance, the primary alkyl amine, the secondary alkyl amine or the tertiary alkyl amine may be selected from compounds of formula R₃ wherein each R is independently selected from H and unsubstituted and substituted alkyl. The term "alkyl", as used herein, refers to a linear or branched chain saturated hydrocarbon radical. An alkyl group may be a C_1-20 alkyl group, a C_1-14 alkyl group, a Ci-io alkyl group, a Ci-6 alkyl group or a Ci-4 alkyl group. Examples of a Ci-io alkyl group are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl. Examples of Ci-6 alkyl groups are methyl, ethyl, propyl, butyl, pentyl or hexyl. Examples of Ci-4 alkyl groups are methyl, ethyl, i-propyl, n- propyl, t-butyl, s-butyl or n-butyl. The term "substituted", as used herein in the context of substituted organic groups, refers to an organic group which bears one or more substituents selected from aryl (for instance phenyl or pyridinyl), cyano, amino, nitro, hydroxy, oxo (=0), halo, -COOH, ester (e.g. -COORa, where Ra is C1-5 alkyl), acyl (e.g. -C(=0)Rb, where Rb is C1-5 alkyl), Ci-10 alkoxy, sulfonic acid, thiol, sulfonyl, phosphoric acid ester. Typically, a substituent is selected from hydroxyl, amino, oxo and halo. Examples of substituted alkyl groups include haloalkyl, perhaloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups. When a group is substituted, it may bear 1, 2 or 3 substituents. For instance, a substituted group may have 1 or 2

substitutents.

Typically, the primary alkyl amine, the secondary alkyl amine or the tertiary alkyl amine is selected from compounds of formula NR₃ wherein each R is independently selected from H and unsubstituted and substituted Ci-10 alkyl. More typically, the primary alkyl amine, the secondary alkyl amine or the tertiary alkyl amine may be selected from compounds of formula R₃ wherein each R is independently selected from H and unsubstituted Ci-10 alkyl.

Examples of a primary alkyl amine include butylamine, pentylamine, hexylamine and heptylamine. Examples of a secondary alkyl amine include dipropylamine, dibutylamine, dipentylamine and dihexylamine. Examples of a tertiary alkyl amine include triethylamine, tripropylamine, triethanolamine, tripropanolamine, tributylamine, dimethylbutylamine, and diisopropylethylamine.

More typically, the ion-pairing reagent is an ammonium salt, a primary ammonium salt, a secondary ammonium salt, or a tertiary ammonium salt. Thus, the IPR may be a compound of formula (NR'₄)X, wherein each R' is independently selected from H and unsubstituted and substituted alkyl, and X is a suitable anion. Typically, at least one R' is H. Each R' is typically selected from H and unsubstituted and substituted C_1-10 alkyl, for instance from H and unsubstituted and substituted Ci-6 alkyl. More typically, the IPR may be a compound of formula (NR'₄)X, wherein each R' is independently selected from H and unsubstituted Ci-io alkyl, and X is a suitable anion.

X may be any anion suitable for use in an ion pairing agent. For instance, X may be a halide anion (e.g. Γ, Br^", CI^" and F^"), a carbonate anion, a bicarbonate anion, a nitrate anion, a carboxylate anion, or a sulfate anion. Examples of carboxylate anions include anions of formula R"COO^" wherein R" is selected from H and unsubstituted and substituted C_1-10 alkyl. Typically, R" is selected from H and unsubstituted Ci-io alkyl. Examples of carboxylate anions include formate, acetate, propanoate, butanoate, pentanoate and hexanoate. Examples of sulfate anions include anions of formula SO4^2" and R'"OS03^" wherein R'" is selected from H and unsubstituted and substituted Ci-20 alkyl. Typically, R'" is selected from H and unsubstituted Ci-20 alkyl. Examples of sulfate anions include sulfate, methyl sulfate, ethyl sulfate, propyl sulfate, butylsulfate, pentyl sulfate, hexylsulfate, decylsulfate and dodecylsulfate. Preferably, the anion is a carboxylate anion, for instance acetate.

Examples of ammonium salts include H4X where X is selected from formate, acetate, propanoate, butanoate, pentanoate, hexanoate, methyl sulfate, ethylsulfate, propyl sulfate, butylsulfate, pentyl sulfate, hexylsulfate, decylsulfate and dodecylsulfate, for instance acetate. Examples of primary ammonium salts include (NH₃(CH₃))X, ( H₃(C₂H₅))X, ( H₃(C₃H₇))X, ( H₃(C₄H₉))X, ( H₃(C₅Hii))X, ( H₃(C₆Hi₃))X and ( H₃(C₇Hi₅))X where X is selected from formate, acetate, propanoate, butanoate, pentanoate, hexanoate, methyl sulfate, ethylsulfate, propyl sulfate, butylsulfate, pentyl sulfate, hexylsulfate, decylsulfate and dodecylsulfate, for instance acetate. Examples of secondary ammonium salts include ( H2(CH₃)2)X, ( H2(C2H5)2)X,

( H₂(C₃H₇)₂)X, ( H2(C₄H₉)2)X, ( H₂(C₅Hn)2)X, ( H₂(C₆Hi₃)2)X and ( H₂(C₇Hi₅)₂)X where X is selected from formate, acetate, propanoate, butanoate, pentanoate, hexanoate, methyl sulfate, ethyl sulfate, propyl sulfate, butyl sulfate, pentyl sulfate, hexyl sulfate, decylsulfate and dodecyl sulfate, for instance acetate.

Preferably, the ion-pairing agent is a tertiary ammonium salt. For instance the IPR may be selected from: trimethylammonium X, i.e. (FIN(CiI₃)₃)X; triethylammonium X, i.e.

(HN(C₂H₅)₃)X; tripropylammonium X, (HN(C₃H₇)₃)X; tributylammonium X, (HN(C₄H₉)₃)X; dimethylbutylammonium X, and diisopropylethylammonium X, where X is a carboxylate anion of a sulfate anion as described herein. X is typically formate, acetate, propanoate, butanoate, pentanoate, or hexanoate. For instance, the IPR may be selected from trimethylammonium acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium acetate, dimethylbutylammonium acetate and diisopropylethylammonium acetate. Preferably, the ion-pairing reagent is triethylammonium acetate.

As mentioned above, in the process of the invention, elution is normally a gradient elution, i.e. the volume of the eluents (water and the organic solvent) vary during the

chromatography. Typically, the ratio by volume (organic solvent): (water) is varied from a first ratio to a second ratio during the chromatographic process, wherein the first ratio is from 95:5 to 60:40 and the second ratio is from 70:30 to 20:80. Thus, at the beginning of the process, for instance at the time when the composition comprising the one or more analytes first enters the chromatography column, the (first) ratio by volume (organic solvent): (water) may be from 95:5 to 60:40. Towards the end of the chromatographic process, for instance when greater than 80 wt% of the one or more analytes have left the chromatographic column (e.g. are no longer in contact with the stationary phase) the (second) ratio by volume (organic solvent): (water) may be from 70:30 to 20:80, for instance from 65:35 to 20:80. Often, the first ratio by volume (organic solvent): (water) is from 95:5 to 70:30. For instance, the first ratio by volume (organic solvent): (water) is from 80:20 to 70:30. Often, the second ratio by volume (organic solvent): (water) is from 65:45 to 40:60. For instance, the second ratio by volume (organic solvent): (water) is from 60:40 to 50:50.

If the first ratio is 80:20 and the second ratio is 50:50, this may be described as a 80-50% organic solvent gradient. The organic solvent gradient may for instance be (from 90 to 65)- (from 60 to 40)%, i.e. the from a broad gradient of 90-40%) to a narrow gradient of 65-60%). With an elution gradient, the ratio of eluents will vary over a certain time from a first ratio to a second ratio. Typically, the ratio by volume (organic solvent): (water) is varied from the first ratio to the second ratio over a time of from 5 to 120 minutes. More typically, the ratio by volume (organic solvent): (water) is varied from the first ratio to the second ratio over a time of from 5 to 60 minutes, for instance from 5 to 20 minutes. Often, the ratio by volume (organic solvent): (water) in the composition is varied from the first ratio to the second ratio over a time of from 5 to 10 minutes. For instance, if the composition contacting part of the stationary phase at time 0 has a first volume ratio as described above (e.g. 80:20) and the gradient time is 7.5 minutes, at time 7.5 minutes the composition contacting the same part of the stationary phase has a second volume ratio as described above (e.g. 60:40).

The organic solvent may be any suitable organic solvent. Typically, the organic solvent is a polar organic solvent. For instance, the organic solvent may be less hydrophobic than hexane. The solvent may be a protic polar solvent or an aprotic polar solvent. The organic solvent may be an alcohol (for instance methanol, ethanol, propanol or butanol), a ketone (for instance acetone or methylethylketone), an aldehyde (for instance ethanal), a halogenated solvent (for instance chloroform, dichloromethane, chlorobenzene), an ether (for instance diethyl ether or tetrahydrofuran (TFIF)), an amide (for instance dimethyl formamide (DMF)), and ester (for instance ethylacetate), a nitrile compound (for instance acetonitrile (ACN)) or a sulfoxide (for instance dimethyl sulfoxide). Often, the organic solvent is acetonitrile, methanol, ethanol, dichloromethane, chlorobenzene, tetrahydrofuran, ethyl acetate, acetone, dimethyl formamide or dimethyl sulfoxide. For instance, the organic solvent may be selected from acetonitrile, methanol, ethanol, acetone, dimethyl formamide or dimethyl sulfoxide. Typically, the organic solvent is methanol or acetonitrile. More typically, the organic solvent is acetonitrile. The process conditions for the process according to the invention may be varied as appropriate for a chromatographic process, as the skilled person would understand. The composition is typically held at a temperature of from 10°C to 80°C. For instance, the temperature may be from 30°C to 45°C. The flow rate of the composition may be from 0.01

The process of the invention may be used to separate any suitable analyte. These include small organic molecules (for instance drugs and amino acids) and biomolecules such as peptides, saccharides and nucleotides. The process of the invention is well suited to separating charged molecules. For instance, the one or more analytes may comprise one or more charged molecules. The process of the invention has been shown to work well with oligonucleotides. Thus, in one embodiment, the one or more analytes comprise one or more oligonucleotides. Oligonucleotides are short single stranded molecules of DNA or RNA. Oligonucleotides typically comprise from 5 to 50 nucleotides (i.e. A, G, C, T and U). An oligonucleotide may be unmodified or chemically modified.

The total concentration of the one or more analytes may be small, for instance less than or equal to 0.1 mM or less than or equal to 0.1 μΜ. The chromatographic process of the invention makes use of a hydrophilic stationary phase. Any hydrophilic stationary phase suitable for chromatography may be used. These are well known to the skilled person. Examples of hydrophilic stationary phases include simple unbonded silica, silanol or diol bonded phases, amino or anionic bonded phases, amide bonded phases, cationic bonded phases and zwitterionic bonded phases. Typically, the hydrophilic stationary phase comprises silica, zirconia, porous graphitic carbon or a polymer. Examples of polymers include methyl methacrylate, polyvinyl alcohol gel, PVA copolymer, styrene/divinylbenzene, or crosslinked polyacrylamide. More typically, the hydrophilic stationary phase comprises silica. The hydrophilic stationary phase typically comprises underivatised silica or silica functionalised with one or more of a diol, an amine, an amide, a poly(succinimide), a cyclodextrin, a cyanopropyl, and a sulfoalkylbetaine. Often, the stationary phase is underivatized silica, or silica modified with a diol or a polymeric sulfoalkylbetaine. For instance, a column comprising a suitable stationary phase is the Zorbax^® RRHD HILIC column. The stationary phase is typically in the form of particles. The particles often have an average particle size (for instance Dv50) of from 1 to 4 μπι. For instance, the stationary phase may be a stationary phase selected from those in Table A below. - hydrophilic stationary phase

Other examples of suitable stationary phases may be found in Hemstrom and Irgum, Hydrophilic interaction chromatography, J. Sep. Sc. 2006, 29, 1784-1821.

The process of the invention is well suited for coupling to mass spectrometry. This leads to ion-pair hydrophilic interaction liquid chromatography electrospray mass spectrometry (IP- HILIC LC-ESI-MS).

Often, the process further comprises a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection (ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection.

Thus, the process typically further comprises a step of performing mass-spectrometry on the composition which has been contacted with the hydrophilic stationary phase. The mass spectrometry step is typically performed directly on the eluted composition. The chromatographic process is thus typically coupled directly with mass spectrometry. The process often therefore further comprises a step of performing mass spectrometry. The mass spectrometry is typically coupled with the chromatographic process. Thus, mass spectrometry may be performed on a sample of the eluted composition directly after elution or mixed with other added reagents (for instance acetonitrile or imidazole) after elution. MS analysis following liquid chromatography can be performed online, by feeding the composition eluting from the stationary phase directly to a mass spectrometer (for instance an electrospray mass spectrometer), or offline, by collecting fractions of the eluted composition to be later analyzed in a classical nanoelectrospray-mass spectrometry setup. Preferably the mass spectrometry process is performed online following the chromatographic process.

Any suitable mass spectrometry technique may be used. These include matrix-assisted laser desorption/ionization (MALDI) and electrospray mass spectrometry (EMS). Preferably, the mass spectrometry is electrospray mass spectrometry. These terms are well known in the art. Electrospray mass spectrometry uses ions produced using an electrospray in which a high voltage is applied to a liquid to create an aerosol. The electrospray mass spectrometer may be a time-of-flight electrospray mass spectrometer. For instance, an ESI-TOF mass spectrometer (Micromass® LCT Premier XE, Waters Corporation, Manchester, UK) may be coupled with a HP1050 HPLC system (Agilent Technologies UK Ltd., Berkshire, UK) equipped with a degasser, quaternary pump, autosampler and temperature controlled column compartment.

The invention also provides a hydrophilic interaction liquid chromatographic process, which process comprises contacting a hydrophilic stationary phase with a composition comprising one or more analytes and an ion-pairing reagent. The hydrophilic interaction liquid chromatographic process may be as described above for the chromatographic process according to the invention. For instance, the concentration of the ion-pairing agent is typically less than or equal to 50 mM.

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

wherein the concentration of the ion-pairing reagent in the composition is less than or equal to 50 mM. The components of the apparatus may be as defined anywhere above for processes according to the invention. The hydrophilic stationary phase may be as further defined above. The composition may be as further defined above. Often, the apparatus further comprises an apparatus for performing mass spectrometry, which mass spectrometry is as defined above (for instance electrospray mass spectrometry).

The invention also provides use of an ion-pairing reagent for increasing resolution in a chromatographic process, which chromatographic process comprises contacting a

composition with a hydrophilic stationary phase, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) said ion-pairing reagent;

chromatographic process.

In the uses according to the invention, the chromatographic process may be as further defined herein for a process according to the invention. The ion-pairing reagent is typically a primary alkyl amine, a secondary alkyl amine, a tertiary alkyl amine, an ammonium salt, a primary ammonium salt, a secondary ammonium salt, or a tertiary ammonium salt, preferably wherein the ion-pairing reagent is triethylammonium acetate.

The invention also provides a chromatographic process, which process comprises

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent; and

(ii) a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection (ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection.

The invention also provides a hydrophilic interaction liquid chromatographic process, which process comprises

(i) contacting a hydrophilic stationary phase with a composition comprising one or more analytes and an ion-pairing reagent; and

The processes which further comprise an analysis step may be as further defined herein for a process according to the invention. For instance, the concentration of the ion-pairing agent in the composition may be less than or equal to 50 mM. The analysis method is typically mass spectrometry, for instance electrospray mass spectrometry.

The invention also provides use of an ion-pairing reagent for increasing resolution in a chromatographic process, which chromatographic process comprises

a) one or more analytes;

b) water;

c) an organic solvent; and

d) said ion-pairing reagent; and

The invention also provides use of a an ion-pairing reagent for increasing resolution in a hydrophilic interaction liquid chromatographic process, which hydrophilic interaction liquid chromatographic process further comprises a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection (ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection.

The uses of the ion-pairing agent in a process which further comprise an analysis step may be as further defined herein for a process or use according to the invention. For instance, the concentration of the ion-pairing agent in the composition may be less than or equal to 50 mM. The analysis method is typically mass spectrometry, for instance electrospray mass spectrometry.

The invention will be described in further detail with reference to the following Examples.

EXAMPLES Summary

Ion-pair hydrophilic interaction liquid chromatography (IP-HILIC) was successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of unmodified and modified oligonucleotides. Separation was performed on a 2.1 x 100 mm Zorbax^® RRHD HILIC column and a particle size of 1.8 μπι with an average pore diameter of 300 A. A method has been developed which allows separation of unmodified and chemically modified oligonucleotides using a quaternary pumping system containing triethylammonium acetate (TEAA) and water with an acetonitrile gradient. Analyses of oligonucleotides were performed by LC-MS with a detection limit of 2.5 picomole (20-mer). The influence of the concentration of ion-pairing reagent and organic modifier was also evaluated.

1. Introduction

Synthetic oligonucleotides are widely used in the polymerase chain reaction (PCR) as DNA primers or in molecular biology as probes to screen for diseases, viral infections, and to identify genes. Sensitive and selective methods have always been demanded for the characterization of oligonucleotides, especially, when the oligonucleotides are applied as therapeutics. Chromatographic techniques such as high-performance liquid chromatography (HPLC) and electrophoresis have been frequently used in the purification and analysis of oligonucleotides. Electrospray ionization mass spectrometry (ESI-MS) has shown its important applications in the sequencing, identification, and characterization of oligonucleotides, as well as for genotyping applications. However, mass measurement of oligonucleotides faces a serious problem of adduct formation, as the deprotonated phosphate backbone of oligonucleotide has high affinity to various cations, in particular sodium and potassium ions which are often present in analyte solutions. These cations lower the MS signal sensitivity of oligonucleotide by dispersing the ion abundance among multiple adducted ions. Hence, sample pre-treatment by removing adducts off-line or on-line has so far been essential in order to obtain high- quality mass spectra. HPLC has been widely used as an on-line sample preparation technique, as it has the advantage of easily coupling to mass spectrometry (LC-MS) combining desalting, separation, and identification. In the early 1990s, a few research groups attempted using reversed-phase (RP) LC to separate single-stranded oligonucleotides. In most of the applications, advantage was taken of the attachment of the hydrophobic protecting group (trityl-, dimethoxytrityl groups) to the investigated oligonucleotides, which increased their retention in reversed- phase HPLC. In the late 1990s, the separation of oligonucleotides was largely improved by adding ion-pairing reagent in the reversed-phase mobile phase, i.e. ion-pair reversed-phase HPLC. Ion-pair reversed-phase liquid chromatography has been widely used for the analysis of oligonucleotides since then. An alternative chromatographic approach, hydrophilic interaction liquid chromatography (HILIC), has also been employed to the separation and analysis of oligonucleotides.

Hydrophilic interaction liquid chromatography is a version of normal phase liquid

chromatography. The high organic content of the mobile phase makes HILIC particularly compatible with electrospray (ESI) ionization mass spectrometry. HILIC HPLC has been increasingly used for separating small and large polar compounds, however, only a very small number of publications describing HILIC LC-MS analysis of oligonucleotides have been published to date.

As the largely improved chromatographic performance for the IP-RP separation of oligonucleotides mainly results from the addition of ion-pairing reagents in the mobile phase of reversed-phase chromatography, the developed HILIC LC-MS method could also benefit from the addition of ion-pairing reagent in the mobile phase and the resulted HILIC chromatography is termed as ion-pair HILIC (IP-HILIC), a different type of chromatography from HILIC.

Below, an ion-pair hydrophilic interaction liquid chromatography electrospray mass spectrometry (IP-HILIC LC-ESI-MS) method is described using TEAA at an MS compatible concentration of lOmM for the analysis of unmodified and chemically modified

oligonucleotides. The effects of the concentration of ion-pairing reagent and organic modifier on the separation of oligonucletoides were also studied. The described method is shown to be capable of separating unmodified and chemically modified oligonucleotides. Meanwhile, the concept of ion-pair hydrophilic interaction liquid chromatography (IP- HILIC) is suggested for the first time, and the possible mechanism of IP-HILIC

chromatography is also briefly discussed.

2. Experimental

2.1 Chemicals and oligonucleotide samples

Analytical reagent grade glacial acetic acid (VWR international, West Sussex, UK), and HPLC - grade acetonitrile (ACN) and methanol (MeOH) (Fisher Scientific, Leicestershire, UK) were used in all experiments. Water was purified in-house using a Milli-Q system (Millipore, Bedford, MA, USA). Triethylamine (TEA, >99%) was purchased from Sigma- Aldrich (Dorset, UK). The standards of oligonucleotides were purchased as 'standard desalting' from Integrated DNA Technologies (Coralville, IA, USA) without further purification (Table 1).

Table 1 Sequences and molecular properties of oligonucleotides used in the study.

Name Sequence Length Mr (Da)

(mer)

EVEN GGG GGC CCC CAA AAA TTT TT 20 6117.0

EVEN C GGG GGC CCC CAA AAA TTT TTC 21 6406.2

MIX1 GGG GCC CCA AAA TTT T 16 4881.2

MIX1T GGG GCC CCA AAA TTT TT 17 5185.4

MIX1A GGG GCC CCA AAA TTT TA 17 5194.4 MIX1C GGG GCC CCA AAA TTT TC 17 5170.4

MIX1G GGG GCC CCA AAA TTT TG 17 5210.4

MIX2 GTC AGT CAG TCA GTC A 16 4881.2

MIX2T GTC AGT CAG TCA GTC AT 17 5185.4

MIX2A GTC AGT CAG TCA GTC AA 17 5194.4

MIX2C GTC AGT CAG TCA GTC AC 17 5170.4

MIX2G GTC AGT CAG TCA GTC AG 17 5210.4

MIX25 ATT GCT AGT GAA TCT GCT ACT AGT G 25 7686.8

LNA16 GTC AGT CAG XCA GTC A (X=Locked 16 4909.2 thymine)

MIX2PS GYC AGT CAG TCA GTC A 16 4897.3

(Y=phosphorothioated thymine)

MIX2F GTC AGT CAG TCA GTC A* (A* - labeled 16 5450.7 with 6-FAM™ fluorescein)

MIX2P ZTC AGT CAG TCA GTC A 16 4961.2

(Z=phosphorylated guanine)

2.2 Instrumentation

An ESI-TOF mass spectrometer (Micromass LCT Premier XE, Waters Corporation, Manchester, UK) was coupled with a HP1050 HPLC system (Agilent Technologies UK Ltd., Berkshire, UK) equipped with a degasser, quaternary pump, autosampler and temperature controlled column compartment. The LC-MS system was operated by MassLynx™ software, version 4.1 (Waters Corporation, Milford, MA, USA). LC-MS chromatograms were acquired in negative ion mode using an ESI-MS capillary voltage of 2.5-3.0 kV, a sample cone voltage of 40 V, and an MCP detector voltage of 1900 V. Desolvation gas flow rate was maintained at 800 L/h. Cone gas flow rate was set to 30 L/h. Desolvation temperature and source temperature were set to 350 and 150 °C, respectively. The acquisition range was m/z 700-3500. Instrument calibration was performed routinely in negative ion mode prior to LC-MS experiment by direct infusion of Nal/Rbl (2.0/0.05μg/μL) in 50/50 2-propanol/water. The mass range for calibration was 200 - 3500 Da. 2.3 Chromatography

A Zorbax^® RRHD HILIC column [100mm 2.1 mm (i.d.)] with 1.8 μιη particles (average pore diameter 300 A) (Agilent Technologies Inc, Santa Clara, California, United States) was used for all LC-MS experiments. All experiments used a 7.5 minute gradient of either acetonitrile or methanol, and water. For experimental conditions of gradients, mobile phases, and other conditions, see figure captions. All chromatographic resolutions in this paper were calculated according to the following equation: Rs = 1.18 x (tm - tm) W i + Wh2. tm and tm = retention times or baseline distances between the point of injection and the perpendicular dropped from the maximum of each of the two peaks.

and Wm = the respective peak widths determined at half peak height, measured in the same units as t and tm.

2.4 Preparation of buffer and oligonucleotide samples

Ion-pairing reagent, 100 mM TEAA, was prepared by dissolving 1.39 mL of triethylamine in 100 mL of Milli-Q water, and by using glacial acetic acid to bring the pH down to ca. 7. All oligonucleotides were used as purchased. Milli-Q water was used to prepare all stock solutions and then diluted by acetonitrile to make work solutions before injected onto column.

3. Results and discussion

3.1 Method Development

Six HILIC columns, each with subtly different stationary phase design (Table 2), were evaluated with the mobile phases containing constant 10% (by volume) 100 mM

triethylammonium acetate pH 7 with ACN gradient. The retainability of oligonucleotide on each column was tested by injecting oligonucleotide EVEN, and oligonucleotides EVEN and EVEN C were used to check the resolving ability of oligonucleotides for HILIC columns. All columns tested in this study are able to show the peak of oligonucleotide EVEN except a polymer based PolyHYDROXYETHYL^® A HILIC column, which failed to show the peak of oligonucleotide even with a gradient of 90-10% ACN [Figure la)]. Figure If also shows that the Zorbax^® RRHD HILIC column possesses the strongest resolving ability of

oligonucleotides, although the Luna HILIC column has the best peak shape of

oligonucleotide out of the six HILIC columns. As a result of this initial evaluation experiments, the Zorbax^® RRHD HILIC column was chosen for further investigation and the column temperature was fixed at 40°C for all experiments performed on this column due to the manufacturer's operational guidelines. The other remaining columns were not evaluated further in this study but they might be useful for the separation of oligonucleotides under different elution conditions, especially the Luna^® HILIC column.

Table 2. Evaluation of six HILIC columns for IP -HILIC chromatography

Brand name Manufacturer Support Dimension Functionality

ZIC^®-HILIC SeQuant Silica 2.1 Polymeric

100mm, sulfoalkylbetaine 3.5 μιη,

200A

YMC^®-pack silica YMC Silica 4.0 x 50mm, Underivatized

3.0 μιη,

200A

Luna^® HILIC Phenomenex Silica 3.0 x Diol

150mm,

3.0μιη, 20θΑ

Kinetex^® HILIC Phenomenex Silica 2.1 x 50mm, Underivatized

(core 1.7μιη, ΙΟθΑ

shell)

PolyHYDROXYETHYL^® PolyLC Silica 4.6 x Poly(2- A 100mm, hydroxy ethyl

3um, 300 A aspartamide)

Zorbax^® RRHD HILIC Agilent Silica 2.1 x Underivatized

100mm,

1.8μιη, 30θΑ

It is believed that there is a predominant partitioning mechanism for IP -HILIC

chromatography. Like the proposed ion pair model for IP-RP chromatography, TEA⁺ (protonated triethylamine) ions in the mobile phase ion pair with negatively charged phosphate groups of oligonucleotides (Oligo^11"), and then the pair of [TEA⁺-01igo^n"] undergoes hydrophilic interaction between the water-rich layer on the column surface and the mobile phase. Oligonucleotides become more hydrophobic after being ion paired with positively charged triethylamine, and hence they are more soluble in the hydrophobic mobile phase, which results a reduced partition coefficient of oligonucleotides between mobile phase and stationary phase. Moreover, the physical size of TEA⁺ ions is much bigger than that of ammonium ions, therefore, TEA⁺ ions could be more likely to block oligonucleotides interacting with the water-rich layer to some extent compared to the ZIC^®-HILIC LC-MS method (L. Gong, J. McCullagh, J. Chromatogr. A 1218 (2011), 5480).. On the other hand, it is speculated that the pair of [TEA⁺-01igo^n"] also has hydrophobic interaction with the hydrophobic mobile phase and tends to stay in the mobile phase longer, which also results in a shorter retention of oligonucleotide on the HILIC column. As seen from Figure 2, all the three HILIC columns with different stationary phase show a reduced retention of

oligonucleotide with IP -HILIC chromatography than HILIC chromatography without ion- pairing reagents.

Next, the effect of the concentration of TEAA in the mobile phase on the separation of oligonucleotides was examined using the Zorbax^® RRHD HILIC column by injecting a mixture of oligonucleotides MIXl and MIX2. As seen from Figure 3, there are no significant difference of the separation of the two oligonucleotides between 10 mM and 15 mM TEAA. However, higher concentration of TEAA in the mobile phase can cause bigger ion

suppression under MS detection, therefore, 10 mM TEAA was used in the following experiments in this study. Interestingly, higher concentration of TEAA in the mobile phase also leads to stronger retention of oligonucleotides under IP -HILIC condition, which is the same as the effect of salt's concentration on the retention of polar analytes in HILIC chromatography without ion-pairing reagent. Higher concentration of TEAA increases the ionic strength of the mobile phase, which would drive more solvated ions into the water-rich layer on the column surface due to high level of organic content in the mobile phase leading to longer retention of oligonucleotide.

The type of organic modifier was also studied. The solvent strength in HILIC is roughly inverted from what is observed for reversed-phase chromatography. Acetonitrile, one of the weaker solvents in HILIC, provides stronger retention compared to methanol. The effect of organic modifiers (methanol and acetonitrile) as weak eluents, on the retention and separation of oligonucleotides, was examined by injecting the mixture of oligonucleotides MIXl and MIX2. As seen from Figure 4, methanol gave less retention to oligonucleotides with a slightly lower separation and poorer peak shape of the two oligonucleotides than that of acetonitrile, and the MS signal of oligonucleotides is much stronger with acetonitrile (signal intensity of MIX2 after deconvolution : ACN - 129801 accounts; MeOH - 47378 accounts). Acetonitrile, therefore, was chosen as the organic modifier in the following experiments.

3.2 Separation of unmodified oligonucleotides The optimized IP-HILIC LC-MS method was applied to separate unmodified

oligonucleotides (N mer) from sequences with an extra nucleotide (N+l mer), as synthesis of oligonucleotides always produces some impurities such as N-l and N-2, etc., and N + l and N + 2, etc.. The LC conditions were the same as Figure 4a. As seen from Figure 5, the method is able to produce some extent of separation of N-mer from N+A/C/G but not N+T for both sequences of MIX1 and MIX2. Interestingly, the separation of N-mer from

N+A/C/G, respectively, has been improved to a large extent when changed the sequence from MIX1 to MIX2 under the same LC conditions, indicating that the separation of

oligonucleotides for the developed IP-HILIC method is sequence dependent. This separation behavior is the same as that of IP-RP chromatography. It is worth mentioning that the developed IP-HILIC method improved the separation of N from N+A significantly compared to the previous HILIC method without ion-pairing reagent involved (L. Gong, J. McCullagh, J. Chromatogr. A 1218 (2011), 5480). On the other hand, the sequence isomers, MIX1 and MIX2, are nearly separated at baseline, probably because the re-arrangement of the 17 bases changed the hydrophilicity of the sequence significantly, as both oligodeoxyguanylic acid and oligodeoxyadenylic acid were reported to form strong intra- and inter-molecular interactions. However, one may notice that MIX2 is less retained than MIX1 under the IP-HILIC conditions [Figure 5(A)], which is the same retention behavior for the sequence isomers under HILIC conditions without using ion-pairing reagent. The sequences of MIX1 and MIX2 contain exactly the same amount of mononucleotides A, T, C & G but differ in their arrangement throughout the molecule, which further approves that the IP-HILIC method is also sequence dependent for the separation of oligonucleotides. It is also worth pointing out that all separations, probably except N+T, can be optimized further to achieve a baseline separation with the expense of largely extended run time.

3.3 Separation of chemically modified oligonucleotides The optimized IP-HILIC LC-MS method was also used to separate chemically modified oligonucleotides. As seen from Figure 6, the IP-HILIC method separated both phosphorylated and fluorescent labeled oligonucleotides from the unmodified oliognucleotide (MIX2) at baseline as expected, because the addition of a phosphate group and fluorescein to the sequence changed the hydrophilicity of MIX2 significantly. However, one may not expect the separation of phosphorothioated oligonucleotide from MIX2 due to a simple replacement of oxygen with sulfur. Surprisingly, the IP-HILIC method achieved some separation of MIX2 from MIX2PS but with a less retained phosphorothioated

oligonucleotide, which suggests that the exchange of oxygen with less electronegative sulfur has reduced the hydrophilicity of the sequence to the required level of separation. On the other hand, there is no separation between MIX2 and LNA16 by the IP-HILIC method, which might be due to not enough change of hydrophilicity of the sequence after the addition of a 2'-0-4'-C methylene bridge on the sugar of thymidine in MIX2.

3.4 Mass spectrum of oligonucleotide

Figure 7 shows the mass spectrum of oligonucleotide EVEN and its deconvoluted spectrum. As seen from Figure 7, only three charge states (3^", 4^", 5^") are visible, which is far less complex mass spectrum compared to the HILIC method without ion-pairing reagent (L.

Gong, J. McCullagh, J. Chromatogr. A 1218 (2011), 5480) and the ion-pair reversed-phase (IP-RP) method with TEA and hexafluoroisopropanol (HFIP) (M. Gilar et al.,

Oligonucleotides 13 (2003) 229). The less complex mass spectrum of oligonucleotide generated by the IP-HILIC method may result of the acidified TEA buffer, as the more acidic the solution, the more likely acids will donate protons to oligonucleotide anions and reduce the charge states of oligonucleotides. The inset to Figure 6 is the deconvoluted mass spectrum of oligonucleotide EVEN, and it shows a few metal ion adducts (K⁺, Na⁺). The simple MS spectrum and HILIC eluting conditions (high level of organic solvent) make the MS detection of oligonucleotide as low as 2.5 picomole, although there is still some ion suppression due to ion-pairing reagent used in the mobile phase.

4. MS signal intensity

The MS signal intensity of the oligonucleotides at different concentrations of ion-pairing reagent (TEA) was measured. The results are shown in Table 3. This demonstrates that an increase in signal intensity is observed for low concentrations of the IPR, particularly at less than 10 mM. Table 3

5. Conclusions

An IP -HILIC ESI-TOFMS method has been developed for the characterization and identification of unmodified and chemically modified oligonucleotides. The effects of ion- pairing reagent's concentration and organic modifier on the separation of oligonucleotides were studied. The possible mechanism of IP -HILIC chromatography was briefly discussed first time. Using a Zorbax^® RRHD HILIC column with an addition of 10 mM TEAA (pH 7) under gradient of acetonitrile, unmodified oligonucleotides can be separated within 7.5 min with single nucleotide resolution. The same method has also been approved to enable the separation of sequence isomers and chemically modified oligonucleotides such as phosphorylation, phosphorothioation, and fluorescent labeling. The IP-HILIC

chromatography demonstrated that the separation of oligonucleotides is sequence dependent. In addition, the IP-HILIC HPLC enables much simpler mass spectrum of oligonucleotide with very efficient desalting, only a few alkali cation adduction was visible, allowing for accurate mass determination of oligonucleotides. MS detection limits were in the lower picomole range with full-scan mode making the method highly capable of quickly and sensitively identifying and resolving unmodified and chemically modified oligonucleotides.

Claims

1. A chromatographic process, which process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

2. A process according to claim 1, wherein the volume of the organic solvent in the composition is greater than the volume of water in the composition.

3. A process according to claim 1 or claim 2, wherein the amount of water in the composition is greater than or equal to 5% by volume and the volume of the organic solvent in the composition is greater than the volume of water in the composition.

4. A process according to any one of the preceding claims, wherein the chromatographic process comprises allowing the composition to elute through the hydrophilic stationary phase.

5. A process according to any one of the preceding claims, wherein water is more strongly eluting that the organic solvent.

6. A process according to any one of the preceding claims, wherein the chromatographic process comprises retention of analytes by liquid-liquid partitioning.

7. A process according to any one of the preceding claims, wherein the concentration of the ion-pairing reagent in the composition is from 0.01 mM to 40 mM.

8. A process according to any one of the preceding claims, wherein the concentration of the ion-pairing reagent in the composition is less than 10 mM.

9. A process according to any one of the preceding claims, wherein the concentration of the ion-pairing reagent in the composition is from 0.1 mM to 9 mM.

10. A process according to any one of the preceding claims, wherein the ion-pairing reagent is a primary alkyl amine, a secondary alkyl amine, a tertiary alkyl amine, an ammonium salt, a primary ammonium salt, a secondary ammonium salt, or a tertiary ammonium salt.

11. A process according to any one of the preceding claims, wherein the ion-pairing reagent is triethylammonium acetate.

12. A process according to any one of the preceding claims, wherein the ratio by volume (organic solvent): (water) is varied from a first ratio to a second ratio during the

chromatographic process, wherein the first ratio is from 95:5 to 60:40 and the second ratio is from 70:30 to 20:80.

13. A process according to claim 12, wherein the ratio by volume (organic

solvent): (water) is varied from the first ratio to the second ratio over a time of from 5 to 120 minutes.

14. A process according to any one of the preceding claims, wherein the organic solvent is a polar organic solvent.

15. A process according to any one of the preceding claims, wherein the organic solvent is less hydrophobic than hexane.

16. A process according to any one of the preceding claims, wherein the organic solvent is acetonitrile, methanol, ethanol, dichloromethane, chlorobenzene, tetrahydrofuran, ethyl acetate, acetone, dimethyl formamide or dimethyl sulfoxide.

17. A process according to any one of the preceding claims, wherein the organic solvent is acetonitrile.

18. A process according to any one of the preceding claims, wherein the one or more analytes comprise one or more oligonucleotides.

19. A process according to any one of the preceding claims, wherein the hydrophilic stationary phase comprises silica, zirconia, porous graphitic carbon or a polymer.

20. A process according to any one of the preceding claims, wherein the hydrophilic stationary phase comprises silica.

21. A process according to claim 19 or claim 20, wherein the silica is underivatised silica or silica functionalised with one or more of a diol, an amine, an amide, a poly(succinimide), a cyclodextrin, a cyanopropyl, and a sulfoalkylbetaine.

22. A process according to any one of the preceding claims, which process further comprises a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection (ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection.

23. A process according to any one of the preceding claims, which process further comprises a step of performing mass spectrometry.

24. A process according to claim 23, wherein the mass spectrometry is coupled with the chromatographic process.

25. A process according to claim 23 or claim 24, wherein the mass spectrometry is electrospray mass spectrometry.

26. A hydrophilic interaction liquid chromatographic process, which process comprises contacting a hydrophilic stationary phase with a composition comprising one or more analytes and an ion-pairing reagent, wherein the concentration of the ion-pairing reagent is less than or equal to 50 mM.

27. A process according to claim 26, which process is as further defined in any one of claims 1 to 25.

28. An apparatus for performing a chromatographic process, which apparatus comprises a hydrophilic stationary phase and a composition, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent;

29. An apparatus according to claim 28, wherein the hydrophilic stationary phase is as further defined in any one of claims 19 to 21; or wherein the composition is as further defined in any one of claims 2, 3, 7 to 11 and 14 to 18.

30. An apparatus according to claim 28 or claim 29, which apparatus further comprises apparatus for performing mass spectrometry as defined in any one of claims 22 to 25.

31. Use of an ion-pairing reagent for increasing resolution in a chromatographic process, which chromatographic process comprises contacting a composition with a hydrophilic stationary phase, which composition comprises:

a) one or more analytes;

b) water;

c) an organic solvent; and

d) said ion-pairing reagent;

32. Use of a composition comprising less than or equal to 50 mM of an ion-pairing reagent for increasing resolution in a hydrophilic interaction liquid chromatographic process.

33. Use according to claim 31 or claim 32, wherein the chromatographic process is as further defined in any one of claims 2 to 25.

34. Use according to any one of claims 31 to 33, wherein the ion-pairing reagent is a primary alkyl amine, a secondary alkyl amine, a tertiary alkyl amine, an ammonium salt, a primary ammonium salt, a secondary ammonium salt, or a tertiary ammonium salt, preferably wherein the ion-pairing reagent is triethylammonium acetate.

35. A chromatographic process, which process comprises

a) one or more analytes;

b) water;

c) an organic solvent; and

d) an ion-pairing reagent; and

36. A hydrophilic interaction liquid chromatographic process, which process comprises

37. A process according to claim 35 or claim 36, which process is as further defined in any one of claims 1 to 25.

38. Use of an ion-pairing reagent for increasing resolution in a chromatographic process, which chromatographic process comprises

a) one or more analytes; b) water;

c) an organic solvent; and

d) said ion-pairing reagent; and

39. Use of a an ion-pairing reagent for increasing resolution in a hydrophilic interaction liquid chromatographic process, which hydrophilic interaction liquid chromatographic process further comprises a step of analysing the composition which has been contacted with the hydrophilic stationary phase by mass spectrometry, evaporative light scattering detection (ELSD), charged aerosol detection (CAD), fluorescence detection, refractive index detection, chemiluminescence detection, optical rotation detection, or electrochemical detection.

40. Use according to claim 38 or claim 39, wherein the chromatographic process is as further defined in any one of claims 1 to 25.