In liquid chromatography (LC), separation modes like reversed phase chromatography (RP), hydrophilic interaction liquid chromatography (HILIC), and ion chromatography (IC) are well-established. However, it is still challenging to analyze very polar, i. e., highly charged, low molecular weight organic molecules with these separation methods, as either retention, elution, peak shapes, separation, or sensitivity are in most cases not satisfying [14,17]. Nonetheless, approaches such as metabolomics and untargeted environmental screening are becoming increasingly relevant and therewith also the need for analyzing the whole subset of polar compounds, including highly charged, low molecular weight organic compounds [19].

One example for the analysis of highly charged, low molecular weight organic compounds relevant for several biological topics, but also for food analytical questions is the determination of polyamines such as cadaverine, putrescine, spermidine, and spermine. These small aliphatic amines possess between two and four amine groups (Fig. 1) and are completely protonated at physiological pH values. Thus, they can carry a broad variety of charges, for example, from +2 for cadaverine and putrescine to +4 for spermine [15]. These polyamines are essential constituents of eukaryotic and prokaryotic cells and are found in all living organisms such as bacteria, fungi, animals, as well as higher plants [1]. They are involved in various biological processes such as cell proliferation and differentiation, stabilization of negative charges of the DNA, RNA transcription, protein synthesis, and many more [15]. As a byproduct of the ornithine cycle, putrescine, spermidine, and spermine are synthesized endogenously in human cells, while all four polyamines are also ingested and absorbed from food sources in the human gut [15]. Due to their ubiquity in biological matrices, analysis of these compounds in various matrices is of importance to scientists in various fields. In food analysis, cadaverine, putrescine, and spermidine are regarded as potential spoilage markers in fish [11]. Additionally, putrescine, spermidine, and spermine affect taste and flavor in the fruit development process of plants [13].

Multiple LC methods have been reported for the separation and elution of cadaverine, putrescine, spermidine, and spermine. However, all of them suffer from imperfections in separation. RP shows little to no retention for such analytes, and thus, it is needed to resort to other means like derivatization or ion-pairing, which are either laborious or costly or both [12]. Also, ion-pairing agents are costly and may lead to memory effects, ion-suppression, ion source contamination, and hence, reduction of instrumental sensitivity in MS detection [1,22]. IC on the other hand suffers from long run-times, lower sensitivity, and low separation efficiency for organic ions [14]. While retention and sensitivity are good in HILIC, peaks of highly charged analytes often show tailing, especially for organic bases [5,10,18,22]. Additionally, HILIC of highly charged compounds often leads to carry-over of analytes into the next run due to their strong adsorption onto the hydrophilic stationary phase [7,20]. This is especially the case with basic compounds, which are normally the best retained compounds in HILIC [2,3].

Electrostatic repulsion hydrophilic interaction liquid chromatography (ERLIC) is a mixed mode liquid chromatographic separation method that may be able to improve on the shortcomings of the existing methods laid out in the previous paragraph. ERLIC uses an ion exchange column with the same charge as the analytes, along with a HILIC mobile phase (i.e., highly organic with low water content) [3]. Similarly to HILIC, the aqueous part of the mobile phase firstly hydrates the hydrophilic stationary phase, and thus, forms a stagnant aqueous layer on the stationary phase surface [3]. Excess water accumulates towards the stagnant aqueous layer, forming a diffuse aqueous-organic layer. The residual organic solvent forms an organic layer in the middle of the column [4]. In ERLIC, retention and elution are affected by only two effects working antagonistically: electrostatic repulsion and hydrophilic interaction [3]. That is in contrast to HILIC, where partitioning, adsorption, hydrogen bonding, dipole-dipole interactions, and weak hydrophobic interactions influence retention and elution and thus, peak shape [3,19]. As a result, retention and elution are more straightforwardly manipulated and the effects of these manipulations are more predictable as well as controllable. At low water shares, all available water hydrates the stationary phase. In this case, the hydrophilic interaction between the charged molecules and the stagnant water layer is stronger than the electrostatic repulsion between the charged molecules and the stationary phase [3]. Hence, the analytes are retained. By increasing the water content of the mobile phase, a diffuse mobile water layer forms and hydrophilic interactions between the analytes and the stagnant water layer are less prevalent. With decreasing hydrophilic interaction, the effect of electrostatic repulsion becomes stronger. Hence, analytes of the same charge are repelled from the stationary phase and the stagnant water layer towards the mobile diffuse aqueous layer. This leads to the elution of the analytes [3].

Although the retention and elution mechanisms are simple and well suited for charged organic molecules, ERLIC has been until now almost exclusively applied qualitatively to the separation of biomolecules such as peptides, with few applications for nucleotides and oligosaccharides [8,16,21,23]. Nonetheless, there is no apparent reason for ERLIC not to be applied for quantitative analysis of highly charged low molecular weight compounds. As a proof of concept, the polyamines cadaverine, putrescine, spermidine, and spermine are a good benchmark due to their low molecular weight, high polarity, and broad variety of possible charges. It was hypothesized that by applying ERLIC, an improved separation between analytes of different charges with proper retention and symmetrical peak shapes and without carry-over can be reached. Furthermore, hyphenation with tandem mass spectrometry (MS/MS), will additionally enable achieving good selectivity and sensitivity. Consequently, the aim of the present study was to develop an ERLIC-MS/MS method for the analysis of highly charged low molecular weight molecules by the example of polyamines.