Influence of Organic Solvents and β-cyclodextrins on Capillary Zone Electrophoresis Separation of Five Biogenic Amines and Two B Vitamins

The CZE experiments were performed with the use of a modular CE analyser EA-102 (Villa Labeco, Spisska Nova Ves, Slovakia) in the CZE mode arrangement operated in a hydrodynamically (membrane) closed system. The sample was injected by a 200 nL internal sample loop that was a part of the injection valve of the analyser. The separation column was provided with a 300 μm I.D. capillary tube made of polytetrafluorethylene (PTFE) of a 160 mm total length. A contactless conductivity detector was an integral part of the CZE separation column. An ECOM photometric detector (ECOM, Prague, Czech Republic) was connected to an on-column photometric detection cell, integrated in the CZE column, via optical fibers. The detector operated under stable wavelength conditions set at 260 nm. This detection wavelength was chosen as a compromise between UV absorption maxima of observed analytes. The ITP Pro 32 software (Villa Labeco, Spisska Nova Ves, Slovakia) was used to control the separation process and to acquire data from the detectors. The experiments were performed in constant current mode at 20 °C. The applied current was 50 μA.

Prior to use, the capillary was not treated by any rinsing procedures to suppress an electroosmotic flow (EOF). Dynamic coating of the capillary wall was performed with the use of methyl-hydroxyethylcellulose (m-HEC, 30000; Serva, Heidelberg, Germany) at a 0.1% (w/v) concentration in separation buffer and serves as an EOF suppression solution. This procedure is very well described in the literature, and it is frequently used in hydrodynamically closed CE systems (Kaniansky et al., 1997).

Analytical-grade 5-hydroxytryptamine hydrochloride (serotonin, 5-HT), dopamine hydrochloride (DOP), tyramine hydrochloride (TYR), adrenaline hydrochloride (ADR), noradrenaline hydrochloride (NOR), thiamine hydrochloride (THI), and pyridoxine hydrochloride (PYR), ɛ-aminocaproic acid (EACA), and γ-aminobutyric acid (GABA) were obtained from Sigma Aldrich (Steinheim, Germany). LC-MS grade acetic acid (HAc), ammonium acetate (NH₄Ac), formic acid (HFo), ammonium formate (NH₄Fo), isopropanol (IP), methanol (MeOH), and acetonitrile (ACN) were purchased from Sigma Aldrich. Tetrahydrofuran (THF) p.a. quality was purchased from Centralchem (Bratislava, Slovakia). Carboxyethyl-β-cyclodextrin (DS 3.5, CE purity), CE-β-CD, was obtained from Cyclolab (Budapest, Hungary) and (2-hydroxypropyl)-β-cyclodextrin (DS 4.5), HP-β-CD, was obtained from Sigma Aldrich. Demineralised water from a Millipore Simplicity 185 (UV) (Millipore, Molsheim, France) water purification system was used for the preparation of electrolyte solutions and samples. All solutions were filtered prior to use with disposable membrane filters (0.45 μm pore size Millipore) and were stored under refrigeration before analysis.

The BGEs were prepared by dissolving of solid substances (NH₄Ac, NH₄Fo, EACA, GABA) or diluting of concentrated liquids (HFo, HAc) in demineralised water. The pH of the prepared electrolyte systems (water solutions) was measured by the SevenExcellence pH meter S400 (Mettler Toledo, Bratislava, Slovakia). The BGE with organic modifier was prepared by appropriate dilution of more concentrated original BGE (twice as concentrated as required) with demineralised water and the demanded amount of organic solvent. The BGE with cyclodextrins was prepared by dissolving the required amount of the selector in original BGE.

The stock solutions of 5-HT, DOP, TYR, ADR, NOR, THI, and PYR reference substances were prepared by dissolving 10 mg of their powder in 10 mL of demineralised water. Working solutions of 5-HT, DOP, TYR, ADR, NOR, THI, and PYR were made by a proper dilution of the stock solutions with demineralised water.

The concentration levels of 5-HT, DOP, TYR, ADR, NOR, THI, and PYR in the injected calibration solutions, prepared in demineralised water were in the range of 0.5–50 μg/mL⁻¹ (0.5, 1, 2.5, 5, 10, 25 and 50 μg/mL⁻¹).

The samples for the stability study were prepared at three concentration levels (2.5, 15, and 30 μg/mL⁻¹) by an appropriate dilution of the standard stock solution with demineralised water. The samples were stored for 24 h under the following temperature conditions: laboratory temperature (i.e., 20 °C), 4 °C, −20 °C, and −80 °C. After the incubation period, the samples were directly (after thawing of the frozen samples) injected into the CE apparatus.

The optimisation of the CZE separation conditions of five biogenic amines and two B vitamins was the crucial step of the method development. The main optimised operating parameters were carrier cation and counter ion, pH of the buffer, type and concentration of the buffer, and type and amount of an additive (i.e., organic solvent, cyclodextrin, or both). These parameters were optimised to obtain (1) the maximal separation efficiency; (2) minimisation of thermal, adsorption, and electromigration dispersion effects; and (3) sufficient resolution of the analytes. The selection of carrier cation and counter ion was made with the goal of simplicity and potential applicability of further connection of the developed CE method with mass spectrometry. Therefore, low molecular weight and volatile buffer constituents such as NH₄Ac, NH₄Fo, Hac, or HFo were tested at first. Hence, the effect of electrolytes composed of EACA and GABA was also investigated, according to the very promising results obtained in analysis of some drugs, metabolites, and endogenous substances (Ginterová et al., 2012; Piešťanský et al., 2015; Piešťanský et al., 2017). The initial experiments have shown that the main problem of the simultaneous separation of the selected analytes was associated with the similar electromigration behaviour of two biogenic amines—dopamine and serotonin—which migrate as one peak. The BGE composed only from NH₄Ac or NH₄Fo was not capable of solving the observed separation problem and similar results were obtained with the use of a combination of NH₄Ac with HAc or NH₄Fo with HFo. The use of BGE composed of two components was more beneficial from the stability of migration time and analytical signal point of view (RSD_tm ≤ 1%; RSD_area ≤ 10%). Moreover, higher stability of the analytical signal was obtained when the combination of NH₄Ac with HAc was used. Higher concentrations of HAc in such a type of BGE led not only to the high stability of analytical signal during the separation, but also to acceptable and very stable levels of separation voltage (≤ 5kV), which are associated with acceptable levels of Joule heating generation. Higher concentration of HAc in BGE was associated with higher buffering capacity and stable pH value of the separation electrolyte. The same effect was also observed when EACA⁺ or GABA⁺ were used as carrier cations. Finally, three BGEs were compared:10 mM NH₄Ac + 30 mM HAc (pH = 5.18) + 0.1% m-HEC (w/v); 25 mM EACA + 50 mM HAc (pH = 4.30) + 0.1% m-HEC (w/v); and 25 mM GABA + 50 mM HAc (pH = 4.10) + 0.1% m-HEC (w/v), where the m-HEC served as an EOF suppressor. Comparison of electropherograms obtained from the CZE-UV analysis of all monitored analytes under the aforementioned BGE composition conditions is illustrated in Figure 1. BGEs composed of 10 mM NH₄Ac + 30 mM HAc and 25 mM EACA + 50 mM HAc showed very similar results. No significant difference in migration time and resolution of the analytes was observed. The use of 25 mM GABA + 50 mM Hac as BGE was associated with elongation of the migration time of the analytes; on the other hand, better parameters of resolution, separation efficiency (N > 13,000), peak shape, and peak intensity were obtained. This BGE composition was therefore used for further experiments. Next optimisation steps were performed to find operational conditions leading to satisfactory separation of all seven analytes from each other. The experimental conditions were systematically changed and optimised by investigating the effects of main CZE parameters of separation.

Figure 1

The effect of addition of organic solvents—acetonitrile (ACN), tetrahydrofuran (THF), and two alcohols (methanol [MeOH] and isopropanol [IP], namely—at 5% to 30% level to the previous selected BGE on separation of five biogenic amines and two B vitamins was investigated. The upper limit—30% of the organic solvent modification—was chosen according to the specification of the electrophoretic analyser. The implementation of methanol into the separation environment brings no effective separation of DOP and 5-HT from each other. With higher amounts of methanol, it shortens of the migration time, but also reduces separation efficiency. The migration order of the analytes was not affected, clearly demonstrated by the electropherograms in Figure 2a. The same behaviour of the monitored analytes was observed in the case of the addition of ACN to the BGE. Hence, a higher amount of ACN led to the creation of a new mixed zone of THI and TYR and probably partial separation of DOP from the mixed zone with 5-HT (Figure 2b). On the contrary, the addition of IP was accompanied by partial separation of DOP and 5-HT. Promising results were obtained when IP at the 20% level was present in the BGE. However, formation of a new mixed zone of THI and TYR was observed. Further increase of IP amount in BGE was superior in the case of enhanced resolution of the main problematic analytes—DOP and 5-HT. On the other hand, it caused formation of a new mixed zone of THI and DOP. These findings are illustrated and summarised in Figure 2c. Very interesting results were obtained with the use of THF. An increased amount of THF in the BGE led to improved separation of the comigrating analytes (i.e., DOP and 5-HT). Addition of 10% of that modifier was able to separate all seven analytes with sufficient resolution. Further increase of THF in the BGE was accompanied with improved resolution between DOP and 5-HT, but also with the creation of a new mixed peak of 5-HT and NOR (Figure 2d).

Figure 2

Further, simultaneous addition of two organic solvents into the BGE was tested. The combination of THF and IP was selected according to the ability to resolve DOP and 5-HT peaks from each other (see Figure 2c and 2d). The best results were obtained when 5% THF was selected as a starting point and the amount of IP was systematically increased from 5% to 15%. Under such conditions, the increase of IP portion in the BGE was accompanied with improved resolution between DOP and 5-HT. The optimum conditions that enabled sufficient resolution of all investigated analytes were obtained when BGE with 5% THF and 10% IP was used (Figure 3). These separation conditions were characterised by high levels of signal stability (constant baseline noise without fluctuations) and reproducibility (RSD_tm ≤ 0.5%, RSD_area ≤ 7%).

Figure 3

Modified β-cyclodextrins were used in the next step to discover their influence on separation of selected biogenic amines and B vitamins. This step was necessary because cyclodextrins are widely used additives in the electromigration environment for selectivity (especially enantioseparation) enhancement. Such investigation was able to provide a comprehensive view of the selectivity enhancement possibilities. Negatively charged CE-β-CD, and uncharged HP-β-CD were tested under the same experimental conditions. Both types of cyclodextrins showed good solubility in the BGE in the range of 1 to 2.5 mg/mL⁻¹ (CE-β-CD) and 5 to 15 mg/mL⁻¹ (HP-β-CD), respectively. Lower concentration range of the CE-β-CD was chosen due to the charged character of the modifier, which significantly affects the separation in terms of elongation of the migration time. It was demonstrated that CE-β-CD at the concentration of 1 mg/mL⁻¹ is responsible for creation of a new mixed peak of ADR and PYR. Further increase of the chiral selector concentration led to peak merging of TYR, DOP, and 5-HT. These findings are illustrated in Figure 4a.

Figure 4

The addition of uncharged HP-β-CD at low concentration (5 mg/mL⁻¹) level was beneficial in terms of partial separation of DOP and 5-HT from each other (Figure 5a). A further increase in resolution between DOP and 5-HT was achieved with the application of a higher concentration of the chiral selector. On the other hand, increased concentration of HP-β-CD formed new mixed peaks of 5-HT and NOR, ADR, and PYR; and TYR and DOP (Figure 5a).

Figure 5

Simultaneous effect of modified β-cyclodextrins and organic solvent on migration behaviour of selected biogenic amines and B vitamins was also investigated. According to favourable parameters obtained during the previous tests, IP as organic solvent was selected. The effect on separation of the selected analytes was discovered by simultaneous addition of chiral selector in the concentration range of 1 to 2.5 mg/mL⁻¹ (CE-β-CD), or 5 to 15 mg/mL⁻¹ (HP-β-CD) and IP in the range of 10% to 30%. According to the obtained results (Figure 4b–d and Figure 5b–d), it can be stated that the addition of IP enhances the resolution of DOP and 5-HT. Unfortunately, there also is a negative effect of the resolution of other biogenic amines and it comes to creation of mixed peaks of THI with TYR, or 5-HT with ADR.

The developed and optimised CZE-UV method using the BGE composed of 25 mM GABA + 50 mM HAc + 0.1% m-HEC + 5% THF + 10% IP was validated according to the International Conference on Harmonisation (ICH) Q2(R1) guideline (ICH 2005) with the use of a model mixture of five biogenic amines and two B vitamins prepared in demineralised water. All resulting statistical data and performance parameters of the method are given in Table 1. Parameters of calibration curves in the water matrix were calculated using Microsoft Excel 2007 (Microsoft Corporation, Redmond, WA, USA).

The hydrodynamically closed CZE separation system was characterised with enhanced sample loadability (200 nL) in comparison with commercially preferred hydrodynamically open apparatus (obviously tens of nL). Therefore, the sensitivity of such an analytical approach was also increased. The predicted limit of detection (LOD) and limit of quantification (LOQ) values calculated from the calibration equations were in the range of 0.15 to 1.25 μg/mL⁻¹ and 0.5 to 2.5 μg/mL⁻¹, respectively. The results clearly show applicability of the developed method in the field of trace quantitative determination of these analytes in, for example, pharmaceutical matrices.

The coefficients of determination (r²) of each analyte were higher than 0.99. It reflects good linearity in a 0.5 to 50 μg/mL⁻¹ (THI, 5-HT, PYR) and 2.5 to 50 μg/mL⁻¹ (TYR, DOP, NOR, ADR) concentration range of selected biogenic amines and B vitamins. The sufficient linearity of the method for investigated analytes in selected concentration ranges was approved by the regression analysis. Moreover, statistical significance of the slope was evaluated. Evaluated repeatability is acceptable as clearly shown by the values of RSDs of migration times (RSD_tm), and peak areas (RSD_area) of selected biogenic amines and B vitamins measured at LOQ concentration level.

Precision of the developed method was determined as intraand interday precision (Table 2). The RSD values were in the range of 0.4% to 15.5% for the intraday assay and in the range of 0.9% to 18.3% for the interday assay. Accuracy of the method was measured as the percent of analyte recovered by the assay (expressed as %Nom). The determined values during the accuracy test were in the range of 82.9% to 117.8% for the intraday measurements and in the range of 87.6% to 119.2% for the interday measurements. All of the observed results were within the acceptance criteria of the ICH guideline.

The small, but deliberate variation of the operational parameters in the robustness test resulted in fluctuations of the migration time less than 1.5% of the value obtained under the standard conditions. Therefore, it can be supposed that small changes in operational parameters should not influence the migration time significantly, the method is robust enough, and indicates its reliability during normal usage.

Finally, the developed CZE-UV approach in a hydrodynamically closed separation system was applied to investigate the stability of the analytes in model water samples. Short-term stability studies under various temperature conditions were investigated. The samples at three concentration levels of the analytes (2.5, 15, and 30 μg/mL⁻¹) were stored for 24 h at laboratory temperature (20 °C), 4 °C, −20 °C, and −80 °C. The results summarised in Table 3 clearly demonstrate appropriate stability of the tested analytes.