28 February / March 2020
Elimination of the Sample Solvent Effect when Analysing Water Solutions of Basic Peptides by HILIC
by Mattias Malm*, Johan Kjellström, Global Pharmaceutical R&D, Ferring Pharmaceuticals A/S, Copenhagen, Denmark
The Hydrophilic Interaction Chromatography (HILIC) separation mode offers many advantages for the analysis of basic peptides, such as good peak shape and separation efficiency, compared to, e.g., reversed-phase chromatography. However, the need to match the sample solvent with the highly organic mobile phase is limiting the use of HILIC. This article explores the suitability of a column-switch approach to enable the use of HILIC for large injection volumes of aqueous samples.
Introduction
The determination of potency and purity of the active pharmaceutical components is important in many stages of drug development, such as formulation development and stability studies for determination of shelf-life. Reversed-phase liquid chromatography (RPLC) is commonly used for this type of determinations. RPLC is a rugged and well-established technique for both small molecules and biomolecules. However, basic molecules, such as peptides containing the amino acids lysine or arginine, are difficult to analyse by RPLC, due to unwanted silanol interactions with the analyte, which can cause peak tailing and peak broadening [1].
An ion-pair reagent (such as TFA), or high amounts of salt in the mobile phase, can reduce such unwanted interactions, but can, on the other hand, cause problems if MS- detection is used. TFA causes ion suppression [2] and thus a decreased MS response, and
non-volatile buffer salts are directly unsuitable to use in combination with MS.
Based on our experience, HILIC is an attractive technique for basic peptides since it provides good peak shape with MS- compatible mobile phases.
It is generally assumed that the retention in HILIC is mainly caused by partitioning of the analytes between the mobile phase and a water-enriched solvent layer close to the hydrophilic column surface [3,4]. This mechanism could explain the reduced secondary interactions in HILIC since the analytes are not interacting with the column material itself, or at least to a lesser extent, compared to RPLC.
What is limiting the use of HILIC is the need to match the sample solvent to the highly organic mobile phase. Since water is the strong solvent in HILIC, injection of aqueous solutions will lead to partial elution of the analytes at time of injection. This is called
the sample solvent effect, which can lead to peak distortion and loss of retention and efficiency [5]. Thus, peptides formulated as aqueous solutions are not suitable for direct analysis by HILIC.
This article presents a fully automated way to eliminate the sample solvent effect also for large injection volumes of aqueous peptide samples in HILIC, by using a column-switch approach.
Experimental (HILIC method)
Acetonitrile (JT Baker, Ultra Gradient Grade) was used as HILIC mobile phase A. HILIC mobile phase B was prepared by adding 7.7 g ammonium acetate (Merck, p.a.) and 2000 µl glacial acetic acid (Merck, p.a.) to 1000 ml of water (measured pH 5.1). The trapping mobile phase was 5% acetonitrile in water.
[Lys8 ]vasopressin and [Arg8 ]vasopressin
Figure 1. Configuration of the switching valve.
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