INTRODUCTION:

HILIC or Hydrophilic Interaction Liquid Chromatography is a high-performance liquid chromatographic (HPLC) technique for separation of polar and hydrophilic compounds. Originally the separation technique was called "Hydrophilic-Interaction Chromatography", and occasionally the expression "Aqueous Normal Phase" has also been used. Hydrophilic interaction chromatography (or Hydrophilic interaction liquid chromatography, HILIC) is a version of normal phase liquid chromatography. The name was suggested by Dr. Andrew Alpert in his 1990 paper on the subject.¹He described the chromatographic mechanism for it as liquid-liquid partition chromatography.

Mechanism:

1. Partitioning Into a Hydrophilic Environment:

Present HILIC theory dictates that HILIC retention is caused by a partitioning of the injected analyte solute molecules between the mobile phase eluent and a water-enriched layer in the hydrophilic HILIC stationary phase. The more hydrophilic the analyte is, the more is the partitioning equilibrium shifted towards the immobilized water layer in the stationary phase, and thereby, the more is the analyte retained.

2. Mechanism Still Debated:

Although it is well established that a hydrophilic surface holds water when exposed to mixtures of organic solvent and water, the HILIC partitioning theory is only based on circumstantial evidence. There are studies pointing towards a more multimodal separation mechanism, involving hydrogen bonding as well as dipole-dipole interactions as important contributions.

3. Adjusting the Retention:

The retention and selectivity in HILIC is affected by adjusting the eluent by varying the fraction (and type) of organic solvent, the concentration (and type) of buffer, and the pH. Retention increases with increasing fraction of organic solvent. The pH affects retention since an ionized molecule is more hydrophilic and is stronger retained in HILIC, compared to its neutral state. The effect of column temperature in HILIC separations is often rather small though (typically less than in RPLC), but ultimately this depends on the nature of the retained molecule.

Surface used:

Any polar chromatographic surface can be used for HILIC separations. Even nonpolar bonded silica have been used with extremely high organic solvent composition, when the silica used for the chromatographic media was particularly polar. With that exception, HILIC phases can be grouped into five categories of neutral polar or ionic surfaces:

· Simple unbounded silica silanol or diol bonded phases

· Amino or anionic bonded phases

· Amide bonded phases

· Cationic bonded phases

· Zwitterionic bonded phases.

Mobile phase:

A typical mobile phase for HILIC chromatography includes acetonitrile ("MeCN", also designated as "ACN") with a small amount of water. However, any aprotic solvent miscible with water (e.g. THF or dioxane) can be used. Alcohols can also be used; however, their concentration must be higher to achieve the same degree of retention for an analyte relative to an aprotic solvent - water combination. See also Aqueous Normal Phase Chromatography.

It is commonly believed that in HILIC, the mobile phase forms a water-rich layer on the surface of the polar stationary phase vs. the water-deficient mobile phase, creating a liquid/liquid extraction system. The analyte is distributed between these two layers. However, HILIC is more than just simple partitioning and includes hydrogen donor interactions between neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention. This distinguishes HILIC as a mechanism distinct from ion exchange chromatography. The more polar compounds will have a stronger interaction with the stationary aqueous layer than the less polar compounds. Thus, a separation based on a compound's polarity and degree of salvation takes place.

Stationary Phases:

In principle HILIC stationary phases can be divided into three different groups.

1) Neutral: No electrostatic interactions, Diol phases, amide phases.

2) Charged: strong electrostatic interactions, Plain silica phases, aminopropyl phases.

3) Zwitterionic: weak electrostatic interactions, SeQuant ZIC®-HILIC and ZIC®-pHILIC phases.

Additives:

Ionic additives, such as ammonium acetate and ammonium formate, are usually used to control the mobile phase pH and ion strength. In HILIC they can also contribute to the polarity of the analyte, resulting in differential changes in retention. For extremely polar analytes (e.g. aminoglycoside antibiotics (gentamicin) or Adenosine triphosphate), higher concentrations of buffer (ca. 100mM) are required to assure that the analyte will be in a single ionic form. Otherwise asymmetric peak shape, chromatographic tailing, and/or poor recovery from the stationary phase will be observed. For the separation of neutral polar analytes (e.g. carbohydrates), no buffer is necessary.

Use of other salts such as 100-300 mM sodium perchlorate, which are soluble in high-organic solvent mixtures (ca. 70% acetonitrile), can be used to increase the mobile phase polarity to effect elution. These salts are not volatile, so this technique is less useful with a mass spectrometer as the detector. Usually a gradient (to increasing amounts of water) is enough to promote elution. All ions partition into the stationary phase to some degree, so an occasional washing with water is required to ensure a reproducible stationary phase.

Electrostatic Repulsion Hydrophilic Interaction Chromatography (ERLIC):

In 2008, Alpert coined the term, ERLIC²(Electrostatic Repulsion Hydrophilic Interaction Chromatography), for HILIC separations where an ionic column surface chemistry is used to repel a common ionic polar group on an analyte or within a set of analytes, to facilitate separation by the remaining polar groups. This allows one to minimize the influence of the common, ionic group within the set of molecules; or to reduce the degree of retention from these more polar functional groups, enabling isocratic separations.

Choice of pH:

With surface chemistries that are weakly ionic, the choice of pH can affect the ionic nature of the column chemistry. Properly adjusted, the pH can be set to reduce the selectivity toward functional groups with the same charge as the column, or enhance it for oppositely charged functional groups. Similarly, the choice of pH affects the polarity of the solutes. However, for column surface chemistries that are strongly ionic, and thus resistant to pH values in the mid-range of the pH scale (pH 3.5-8.5), these separations will be reflective of the polarity of the analytes alone, and thus might be easier to understand when doing methods development.

Cation exchange:

Cation exchange surface (negatively charged) chemistry for ERLIC separations to reduce the influence of the anionic (negatively charged) groups (phosphates of nucleotides or of phosphonyl antibiotic mixtures, or sialic acid groups of modified carbohydrates) to allow discrimination based on the basic and/or neutral functional groups of these molecules. Alternatively, one could use a pH 9.2 mobile phase on a polymeric, zwitterionic, betain-sulphonate surface to enhance the influence of its sulphonic acid functional group over the, now diminished, quaternary amine of this surface chemistry to separate the phosphonyl antibiotic mixtures. Commensurate with this, these analytes will show a reduced retention on the column chemistry and will elute earlier and in higher amounts of organic solvent than if a neutral polar HILIC surface were used. This then increases their detection sensitivity by mass spectrometry.

Anion exchange:

By analogy, one could use an anion exchange column surface (positively charged) chemistry to reduce the influence of cationic (positively charged) functional groups on a set of analytes, as when selectively isolating phosphorylated peptide molecules. Use of a pH between 1 and 2 pH units will reduce the polarity of two of the three ionizable oxygens of the phosphate group, and thus will allow easy desorption from the (oppositely charged) surface chemistry. It will also reduce the influence of negatively charged carboxyl in the analytes, since they will be protonated at this low a pH value, and thus contribute less overall polarity to the molecule. The common, positively charged amino group will be repelled from the column surface chemistry and thus these conditions will enhance the role of the phosphate's polarity (as well as other neutral polar groups) in the separation.

Why use HILIC?

1. Rapidly Increasing Interest for HILIC:

The HILIC separation technique is gaining much interest because it solves many of the previously difficult separation problems, including separation of small organic acids, basic drugs, cations, anions and many other neutral and charged substances.

2. Unattractive Alternatives:

Due to the robustness and reproducibility of current bonded RPLC stationary phases, a common strategy has been to "tweak" the RPLC separation to encompass also polar and hydrophilic compounds. The techniques involved to accomplish this have been ion-pairing or micellar chromatography, polar embedded phases, and derivatization. These twists of RPLC have sometimes been able to get the job done, but they are also haunted by a number of limitations such as; low MS compatibility, low stationary phase stability and column bleeding, and time consuming with several interferences.

3. Robust Separation of Hydrophilic Compounds:

HILIC is the most straightforward, versatile and robust separation technique for polar and hydrophilic compounds, compared to the above options. Dedicated, bonded stationary phases with high stability and reproducibility are since a few years available for HILIC separations, overcoming any previous limitations of HILIC relative to other separation techniques. In favour of HILIC is also the good solubility of polar and hydrophilic compounds in the water-containing HILIC eluents.

4. Sensitive Detection by LC-UV and LC/MS:

HILIC separations are very easy to combine with several detection techniques, such as ultraviolet light absorbance (UV), fluorescence (FL), refractive index (RI), evaporative light scattering (ELSD), charged aerosol (CAD), and mass spectrometry (MS).

When HILIC is used with MS detection will the ESI sensitivity be much higher (10-100 times) compared to in RPLC. This is due to the high content of organic solvent in the mobile phase, which lowers surface tension, thereby simplifying the drop formation during the spray process. This significantly improves the formation of ions in the gas phase and thereby the sensitivity. Often, sample preparation can be simplified and made more efficient, which results in a further increase in sensitivity.

Application of HILIC:

1) HILIC stationary phase to determination of dimethindene maleate in topical gel:

Dimethindene maleate, chemically N, N-dimethyl- 2-{3-[(RS)-1-(pyridine-2-yl) ethyl]-1H-inden-2-yl} ethanamin-(Z)- butendioate, is derivate of fenindene reported to be strong antagonist of histamine on H1 receptors. Dimethindene decreases hyper permeability of capillaries during reactions of early sensitivity. It reduces prurience and irritation by different eruptions.^{3, 4}

The application of HILIC stationary phase to the area of pharmaceutical analysis has a raising potential. Recently HILIC approach has been applied for the analysis of polar molecules peptides, nucleotides, nucleosides and amino acids^5–8, cytostatics⁹ and ascorbic acid¹⁰.

HILIC was ﬁrstly introduced for the determination of dimethindene maleate in topical gel. Diltiazem hydrochloride was used as internal standard. The separation was carried out using ZIC®–HILIC analytical column (50 mm × 2.1 mm I.D.; 5 m, SeQuant, Umeå, Sweden) and mobile phase consisting of acetonitrile and aqueous solution of acetic acid (25 mM) and ammonium acetate (2.5 mM) (87.5:12.5, v:v) at a ﬂow rate of 0.3 ml min−1. UV detection was accomplished at 258 nm.

2) Sensitivity loss by triﬂuoroacetic acid (TFA) mobile phases in the hydrophilic interaction chromatography:

Triﬂuoroacetic acid (TFA) has traditionally been used in the HPLC analyses of basic compounds extensively.¹¹ It not only controls the pH of the mobile phases, but also acts as an ion-pair agent to improve peak shapes of basic compounds on silica-based columns. TFA is volatile and therefore can be used in LC–MS, and it has been used in the LC–MS analyses of peptides¹² as well as small molecules.¹³

The major drawback of using TFA in LC–MS, however, is that TFA is known to suppress the ESI signals of analytes and reduce assay sensitivity.^14,15 This is primarily due to the ability of TFA to form gas-phase ion pairs with positively charged analyte ions.¹⁶

Use silica columns operated under HILIC conditions for the analysis of polar, basic compounds. TFA has been used extensively in the HILIC–MS/MS methods at concentrations between 0.01 and 0.05%, mostly due to its ability to obtain excellent peak shapes even from extracted biological samples. However, the reduction in sensitivity caused by TFA oftentimes precluded its use in assays that require high sensitivity. Therefore, we have been trying to investigate methods to minimize the suppression effect of TFA in the HILIC–MS/MS bioanalysis. The so-called “TFA-Fix” approach originally developed by Kuhlmann et al., involved the infusion of propionic acid and isopropanol post-column and before the ion source.¹⁶

3) Determination of zanamivir in rat and monkey plasma by positive ion hydrophilic interaction chromatography (HILIC)/tandem mass spectrometry:

Zanamivir (ZAM) is a first in class neuraminidase inhibitor used to treat all strains of the influenza virus. Zanamivir has been shown to interact with a group of amino acids in the active site of neuraminidase which blocks its action, preventing release and spread of the newly formed virons.¹⁷ Zanamivir has been shown to be effective in preventing, controlling, or rapidly reducing: illness with fever¹⁸, influenza in family contacts ¹⁹, nursing home outbreaks ²⁰, elevated body temperature,²¹ and viral load.²²

These methods employ techniques such as SCX solid phase extraction, pre-column fluorescence derivatization, UV detection, and protein precipitation with 10% trichloroacetic acid (TCA) or acetonitrile. This method incorporates hydrophilic interaction chromatography (HILIC) silica, a stationary phase capable of retaining very polar compounds and improving sensitivity over reverse phase columns.²³

4) For the analysis of tromethamine as the counter ion in an investigational pharmaceutical salt:

2-Amino-2-hydroxymethyl-propane-1, 3-diol (tromethamine) is a weak base with pKa /8 (25 ⁰C) and is readily soluble in water.²⁴ It is commonly used as a buffering or emulsifying agent in pharmaceutical and cosmetic products, or as a counter ion for acidic pharmaceutical compounds suitable to form desired salt forms.

The HILIC approach was employed for tromethamine separation based on the fact that it had been shown to provide sufficient retention for very polar compounds such as sugars, uracil, acetamide, etc.^25-27

In addition to tromethamine, another structurally related compound, AEPD was also selected to aid method development. In this study, only amino stationary phase was used and the mobile phase was a simple mixture of water and organic solvent (i.e. acetonitrile and methanol).

This method was used to analyze seven small research batches of the investigational drug substance in support of process research on salt formation. Table 1 presents the analytical results for the tromethamine content (%w/w) in the investigational API as well as the potency of the drug substance in the form of free acid (%w/w) using a separate reverse-phase HPLC method.

Sample number	Tromethamine (%w/w)	Free acid (% w/w)	Molar ratio^a	Tromethamine added.^b
1	24.9	73.4	1.9	2 Equivalents
2	24.8	76.5	1.9	3 Equivalents
3	27.0	76.2	2.0	4 Equivalents
4	14.8	82.4	1.0	1 Equivalents
5	25.4	77.3	1.8	2 Equivalents
6	25.4	77.2	1.8	2 Equivalents

The ratio of moles of tromethamine to free acid.^a

Amount of tromethamine added in the salt formation step of the synthesis^b

CONCLUSION:

Hydrophilic interaction liquid chromatography (HILIC) is a technique suitable for separation of very polar, basic and hydrophilic compounds. HILIC is the alternative as the elution order is likewise inverted to RPLC (reversed phase liquid chromatography). This means that solutions that have little or no retention on RPLC columns generally show strong retention on HILIC columns. The HILIC technique thus bears similarities with traditional NPLC (normal phase liquid chromatography), but with the important difference that HILIC employs semi-aqueous mobile phases. Hydrophilic interaction liquid chromatography (HILIC) is a technique suitable for separation of very polar, basic and hydrophilic the application of HILIC stationary phase to the area of pharmaceutical analysis has a raising potential. Recently HILIC approach has been applied for the analysis of polar molecules, peptides, nuceleotides, nucleosides and amino acids, cytostatics and ascorbic acid. HILIC approach demonstrated distinct advantages over conventional reverse- phase HPLC for the separation of small polar compounds.

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Received on 21.12.2009 Modified on 23.01.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 640-643

Hydrophilic Interaction Liquid Chromatography (HILIC)