Dual 2-Hydroxypropyl-β-Cyclodextrin and 5,10,15,20-Tetrakis (4-Hydroxyphenyl) Porphyrin System as a Novel Chiral-Achiral Selector Complex for Enantioseparation of Aminoalkanol Derivatives with Anticancer Activity in Capillary Electrophoresis

In this study, a complex consisting of 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin, (named dual chiral-achiral selector complex) was used for the determination of two novel potential anticancer agents of (I) and (II) aminoalkanol derivatives. This work aimed at developing an effective method that can be utilized for the determination of I (S), I (R), and II (S) and II (R) enantiomers of (I) and (II) compounds through the use of a dual chiral-achiral selector complex consisting of hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin system by applying capillary electrophoresis. This combination proved to be beneficial in achieving high separation selectivity due to the combined effects of different modes of chiral discrimination. The enantiomers of (I) and (II) compounds were separated within a very short time of 3.6–7.2 min, in pH 2.5 phosphate buffer containing 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin system at a concentration of 5 and 10 mM, respectively, at 25 °C and +10 kV. The detection wavelength of the detector was set at 200 nm. The LOD for I (S), I (R), II (S), and II (R) was 65.2, 65.6, 65.1, and 65.7 ng/mL, respectively. LOQ for I (S), I (R), II (S), and II (R) was 216.5, 217.8, 217.1, and 218.1 ng/mL, respectively. Recovery was 94.9–99.9%. The repeatability and reproducibility of the method based on the values of the migration time, and the area under the peak was 0.3–2.9% RSD. The stability of the method was determined at 0.1–4.9% RSD. The developed method was used in the pilot studies for determining the enantiomers I (S), I (R), II (S), and II (R) in the blood serum.


Introduction
Separation of chiral compounds is very important in medical and pharmaceutical research as it allows studying the content of individual enantiomers in drugs and their concentrations in the human body and thus to determine the pharmacokinetic and pharmacodynamic parameters. Chiral compounds play a major role in drug-receptor interactions, in which one enantiomer of a racemic pair confers the desired pharmacological activity, while the other may exhibit negative side effects in the worst case [1][2][3]. In chiral compounds, numerous drugs, which have one or multiple asymmetric centers, occur as an enantiomer pair [4][5][6][7][8][9][10]. Therefore, it is necessary for the researchers to have knowledge of the composition of enantiomeric medicinal substances [11]. The most popular technique used for the determination of biological substances is the high-performance liquid chromatography. However, capillary electrophoresis (CE) is becoming an appealing alternative chromatography. However, capillary electrophoresis (CE) is becoming an appealing alternative as it possesses a number of benefits such as high-speed analysis, low sample consumption, and high separation efficiency. CE has already been demonstrated to have high potential in chiral separations [12], yielding very good results in the analyses of various pharmaceutically active substances. As we know, interactions occurring between chiral selectors and an enantiomer are the basis for separation in CE. For achieving effective chiral separation in CE, the chiral selector should be added to the run buffer. Among the great variety of compounds that can be used as a chiral selector in CE are cyclodextrins (CDs), antibiotics, crown ethers, cyclofructans, and polysaccharides. CDs are one of the most commonly used chiral selectors [13][14][15]. Antibiotics [16][17][18][19][20][21], CD derivatives [22][23][24][25][26][27], chiral ligand exchangers [28][29][30][31][32][33][34], crown ethers [35][36][37][38], polysaccharides [39][40][41][42], and proteins [43][44][45] have also been employed depending on the properties of the enantiomers to be separated.
This study aimed to develop a new CE method characterized by high sensitivity and having the potential for rapid separation of the enantiomers I(R), I(S), and II(R), II(S) of the patented 4-[2-hydroxy-3-(propan-2-ylamino)propyl]-1,7-diethyl-8,9-diphenyl-4azatricyclo-[5.2.1.02,6]dec-8-ene-3,5,10-trione hydrochloride (I) and 4-[3-(dimethylamino)-2-hydroxypropyl]-1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5,10-trione hydrochloride (II) (Figure 1) exhibiting anticancer activity [46]. These compounds came from research into the cytotoxicity and anticancer properties notificated in a patent in 2012 [46]. Determination of compounds (I) and (II) using a CD derivative as a chiral selector has already been described in the previous work [47]. In the present study, we used the dual 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin ( Figure 2) chiral-achiral selector complex to investigate the combined effect of these two types of selectors on the enantioseparation of the model compounds. The developed method was found to be very effective and was used in the pilot studies for the determination of enantiomers in the blood serum. These compounds came from research into the cytotoxicity and anticancer properties notificated in a patent in 2012 [46]. Determination of compounds (I) and (II) using a CD derivative as a chiral selector has already been described in the previous work [47]. In the present study, we used the dual 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin ( Figure 2) chiral-achiral selector complex to investigate the combined effect of these two types of selectors on the enantioseparation of the model compounds. The developed method was found to be very effective and was used in the pilot studies for the determination of enantiomers in the blood serum.

Chemicals and Reagents
The synthesis of the compounds as well as their spectroscopic analysis (nuclear magnetic resonance and mass spectrometry) has been explained in patent application [46]. The purity of reference compounds was greater than 98%, while other analytes used in separations had 99% purity [47]. Phosphate buffer used in the study was obtained from Beckman-Coulter (Brea, USA), while the other chemicals were purchased from Sigma-Aldrich: γ-, α-, β-, and 2-hydroxypropyl-β-cyclodextrin (St. Louis, USA); deionized water and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin (Darmstadt, Germany); and ethyl acetate and hexane (Steinheim, Germany). The serum samples were obtained from 10 healthy drug-free volunteers.

Instrumentation
A Beckman Coulter P/ACE MDQ CE system, which contained an autosampler as well as a UV/Visible detector, was used for the separation. Karat software version 32 was used for controlling the CE parameters [47]. An eCAP fused-silica capillary (total length: 30 cm, effective length: 20 cm, ID: 50 μm, OD: 375 μm) was employed.

Sample Preparation
Stock solutions for the I (S), I (R), and II (S) and II (R) enantiomers, at the concentration of 10,000 ng/mL were prepared by dissolving of these substances in methanol. Then, from these solutions, calibration solutions with specific concentrations were prepared. These solutions were stored at −15°C. The analytes were found to be stable in the solution for around 30 days in the previous studies. Samples for the preparation of the working standards and the quality controls were prepared from the serum obtained from 10 healthy drug-free volunteers. The calibration solutions of 50, 100, 500, 1000, 5000, and 10,000 ng/mL were prepared in water and in serum, by adding appropriate amounts of the analyte stock solutions to the blank serum samples. Serum from healthy volunteers was a medium for dilution of the compounds and did not contain the tested compounds. As described previously [47], extraction was carried out with 500 μL of serum, 100 μL of I (S) and I (R), 100 μL of II (S) and II (R), and 5 mL of the n-hexane-ethyl acetate mixture (90:10, % v/v). These solutions were mixed and shaken well for around 4 min. After centrifugation (at 5 min, 1000× g), the organic layer was removed and subjected to evaporation under a nitrogen stream at 37 °C. Then, the obtained sample was mixed with deionized water (100 μL) and introduced on the capillary [47].

Chemicals and Reagents
The synthesis of the compounds as well as their spectroscopic analysis (nuclear magnetic resonance and mass spectrometry) has been explained in patent application [46]. The purity of reference compounds was greater than 98%, while other analytes used in separations had 99% purity [47]. Phosphate buffer used in the study was obtained from Beckman-Coulter (Brea, USA), while the other chemicals were purchased from Sigma-Aldrich: γ-, α-, β-, and 2-hydroxypropyl-β-cyclodextrin (St. Louis, USA); deionized water and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin (Darmstadt, Germany); and ethyl acetate and hexane (Steinheim, Germany). The serum samples were obtained from 10 healthy drug-free volunteers.

Instrumentation
A Beckman Coulter P/ACE MDQ CE system, which contained an autosampler as well as a UV/Visible detector, was used for the separation. Karat software version 32 was used for controlling the CE parameters [47]. An eCAP fused-silica capillary (total length: 30 cm, effective length: 20 cm, ID: 50 µm, OD: 375 µm) was employed.

Sample Preparation
Stock solutions for the I (S), I (R), and II (S) and II (R) enantiomers, at the concentration of 10,000 ng/mL were prepared by dissolving of these substances in methanol. Then, from these solutions, calibration solutions with specific concentrations were prepared. These solutions were stored at −15 • C. The analytes were found to be stable in the solution for around 30 days in the previous studies. Samples for the preparation of the working standards and the quality controls were prepared from the serum obtained from 10 healthy drug-free volunteers. The calibration solutions of 50, 100, 500, 1000, 5000, and 10,000 ng/mL were prepared in water and in serum, by adding appropriate amounts of the analyte stock solutions to the blank serum samples. Serum from healthy volunteers was a medium for dilution of the compounds and did not contain the tested compounds. As described previously [47], extraction was carried out with 500 µL of serum, 100 µL of I (S) and I (R), 100 µL of II (S) and II (R), and 5 mL of the n-hexane-ethyl acetate mixture (90:10, % v/v). These solutions were mixed and shaken well for around 4 min. After centrifugation (at 5 min, 1000× g), the organic layer was removed and subjected to evaporation under a nitrogen stream at 37 • C. Then, the obtained sample was mixed with deionized water (100 µL) and introduced on the capillary [47].
New capillaries were flushed with 1 M NaOH (15 min, 10 psi), water (15 min, 10 psi), and BGE (20 min, 10 psi). Finally, the system was electroconditioned with the running buffer by applying the separation voltage (+10 kV) for 20 min. Between workdays, the capillary was conditioned by rinsing successively with 0.1 M NaOH (5 min, 10 psi), water (5 min, 10 psi), and BGE (10 min, 10 psi). Samples were hydrodynamically injected at a pressure of 2 psi for 6 s. A separation voltage of 10 kV (normal polarity anode at the injection capillary end) was applied for performing the electrophoretic separations at 25 • C. Between runs, the capillary was rinsed for 2 min with water and for 3 min with BGE. At the end of every analysis, the working BGE was refreshed to ensure optimum separation repeatability. After extraction with n-hexane-ethyl acetate mixture (90:10, % v/v) and dissolving in 0.1 mL of aqueous solution, the serum samples were injected automatically using pressure (2 psi on sample solution for 5 s). The vials having both cathode and anode buffers were emptied late in the day, and the running buffer was refilled at the start of the following working day, before analysis, as indicated by the earlier described procedure [47].

Optimization
In this study, three kinds of CDs, namely 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropylβ-cyclodextrin, and 2-hydroxypropyl-γ-cyclodextrin, and one porphyrin derivative (5,10,15,20tetrakis (4-hydroxyphenyl) porphyrin) were screened at different pH values as potential chiral selector candidates, either alone or in combinations. Among the tested CDs, only 2-hydroxypropylβ-cyclodextrin allowed a baseline separation of the enantiomers of compounds (I) and (II). The best results were achieved when 2-hydroxypropyl-β-cyclodextrin was used at a concentration of 5 mM ( Figure 3A). Other phosphate buffers, containing 2-hydroxypropyl-αcyclodextrin and 2-hydroxypropyl-γ-cyclodextrin at a concentration of 5 mM, did not allow satisfactory separation of compounds (I) and (II) into individual enantiomers. Therefore, for the initial separation of the enantiomers I (S), I (R), II (S), and II (R), 2-hydroxypropylβ-cyclodextrin was chosen as the first chiral selector.
The resolution was tested by introducing the second selector-(5,10,15,20-tetrakis (4hydroxyphenyl) porphyrin) into the system. Maintaining the concentration of 2-hydroxypropyl-βcyclodextrin in the tested system at 5 mM, the concentration of 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin was gradually increased while examining the resolution of the enantiomers I (S), I (R), II (S), and II (R). The best resolution and absorbance were achieved for the system containing 2-hydroxypropyl-β-cyclodextrin at a concentration of 5 mM and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin at a concentration of 10 mM (Figure 4). The resolution was tested by introducing the second selector-(5,10,15,20-tetrakis (4hydroxyphenyl) porphyrin) into the system. Maintaining the concentration of 2hydroxypropyl-β-cyclodextrin in the tested system at 5 mM, the concentration of 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin was gradually increased while examining the resolution of the enantiomers I (S), I (R), II (S), and II (R). The best resolution and absorbance were achieved for the system containing 2-hydroxypropyl-βcyclodextrin at a concentration of 5 mM and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin at a concentration of 10 mM (Figure 4). The influence of BGE concentration on the resolution and migration time was investigated at 10, 15, 20, 25, and 30 mM. As the concentration of phosphate buffer increased, the resolution increased, the peak shapes sharpened, and the migration time of the determined compounds I (S), I (R), II (S), and II (R) decreased. The best results were observed at the concentration of 25 mM ( Figure 5).  Considering the resolution, peak shape, and migration times of (I) and (II) compounds, the final BGE conditions were determined as follows: concentration: 25 mM and pH: 2.5.
The effect of pH on the separation process was analyzed at pH values from 7.0 to 2.5. A decrease in pH led to an increase in the resolution between (I) and (II) enantiomers. At pH 7.0, no resolution was found between the tested compounds. At pH 3.5, the resolution was insufficient between derivatives I (S), I (R), and II (S), II (R). The best resolution and absorbance of these compounds were observed with phosphate buffer at a concentration of 25 mM and pH 2.5 ( Figure 6). The effect of pH on the separation process was analyzed at pH values from 7.0 to 2.5. A decrease in pH led to an increase in the resolution between (I) and (II) enantiomers. At pH 7.0, no resolution was found between the tested compounds. At pH 3.5, the resolution was insufficient between derivatives I (S), I (R), and II (S), II (R). The best resolution and absorbance of these compounds were observed with phosphate buffer at a concentration of 25 mM and pH 2.5 ( Figure 6).  The influence of voltage on the separation and migration time was assessed at 5, 7, 10, 15, 20, and 25 kV. An increase in voltage to 10 kV increased the resolution. After 10 kV was exceeded, the resolution decreased, while the migration time decreased with the increasing voltage (Figure 7). The influence of the temperature of the separation process was investigated at 18, 20, 22, 25, and 30°C. An increase in the temperature caused an increase in absorbance and resolution. The best results were achieved at 25°C (Figure 8).  The influence of the temperature of the separation process was investigated at 18, 20, 22, 25, and 30°C. An increase in the temperature caused an increase in absorbance and resolution. The best results were achieved at 25°C (Figure 8). The influence of BGE concentration on the resolution and migration time was investigated at 10, 15, 20, 25, and 30 mM. As the concentration of phosphate buffer increased, the resolution increased, the peak shapes sharpened, and the migration time of the determined compounds I (S), I (R), II (S), and II (R) decreased. The best results were observed at the concentration of 25 mM ( Figure 5).
Considering the resolution, peak shape, and migration times of (I) and (II) compounds, the final BGE conditions were determined as follows: concentration: 25 mM and pH: 2.5.
The effect of pH on the separation process was analyzed at pH values from 7.0 to 2.5. A decrease in pH led to an increase in the resolution between (I) and (II) enantiomers. At pH 7.0, no resolution was found between the tested compounds. At pH 3.5, the resolution was insufficient between derivatives I (S), I (R), and II (S), II (R). The best resolution and absorbance of these compounds were observed with phosphate buffer at a concentration of 25 mM and pH 2.5 ( Figure 6).
The influence of voltage on the separation and migration time was assessed at 5, 7, 10, 15, 20, and 25 kV. An increase in voltage to 10 kV increased the resolution. After 10 kV was exceeded, the resolution decreased, while the migration time decreased with the increasing voltage ( Figure 7).
The influence of the temperature of the separation process was investigated at 18, 20, 22, 25, and 30 • C. An increase in the temperature caused an increase in absorbance and resolution. The best results were achieved at 25 • C (Figure 8).
Because the best separation of I (S), I (R), II (S), and II (R) was achieved using 2hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin system at concentrations of 5 and 10 mM, respectively, in 25 mM phosphate buffer at a pH of 2.5, separation voltage of 10 kV, and temperature of 25 • C, this method was used for the preliminary studies of the enantiomers I (S), I (R), and II (S) and II (R) of the newly patented aminoalkanol derivatives in the blood serum ( Figure 3D).

Method Development
CE enables us to selectively monitor I (S), I (R), and II (S) and II (R) and to eliminate interference with the endogenous components that can be co-eluted from serum samples. The enantiomers I (S), I (R), and II (S), II (R) were separated using 2-hydroxypropyl-βcyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin system at the concentration of 5 and 10 mM, respectively. For developing this method as well as determining the validation parameters, the buffer pH, separating BGE concentration, chiral selector concentration, concentration and composition of extraction phase, wavelength, voltage, and temperature were optimized. During extraction, 500 µL of the serum solution was added in a glass tube, followed by which 100 µL of I (S) and I (R) and 100 µL of II (S) and II (R) were added, and the solutions were mixed together. The mixture was set aside for 5 min, and then, 5 mL of extraction solvent (n-hexane-ethyl acetate, 90:10, % v/v) was added. The mixture was shaken vigorously (vortexed) for 4 min. Subsequently, the mixture was centrifuged for 4 min at 1000× g. After centrifugation, the separated organic layer was removed and added to another glass tube, and was placed in a heating mantle, at a temperature of 37 • C. Then, it was subjected to evaporation at 37 • C under a nitrogen stream. The obtained residue was mixed with deionized water at 37 • C. Finally, the dissolved sample was moved to a capillary vial and then injected on the capillary. In this study, phosphate buffer containing 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin system at a concentration of 5 and 10 mM, respectively, was chosen as a BGE because of its good separation performance. Considering the analysis time and resolution comprehensively, +10 kV was chosen as the separation voltage. Based on the above study, CE was applied for separation, with the separation conditions set as follows: BGE: 25 mM phosphate buffer at pH 2.5 containing 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin system at 5 and 10 mM, respectively; voltage: +10 kV; detection temperature: 25 • C; and detection wavelength: 200 nm. The measurements were performed with a fused-silica capillary (effective length: 20 cm; diameter: 50 µm). The samples to be tested were hydrodynamically injected on the capillary [47]. Under these separation conditions, compounds (I) and (II) were eluted and separated in less than 7 min. The peaks were identified by injecting individual analytes based on migration time matching.
The preliminary analysis of the studied drugs exhibiting biological activity is described elsewhere [46,47]. The extraction procedure developed in this study required using an extraction solvent made of n-hexane and ethyl acetate, in 90:10 (% v/v) proportions. The optimum time of sample shaking was determined as 4 min. Appropriate organic layer separation was performed by centrifuging at 1000× g for 5 min. The separated organic layer was subjected to evaporation under a nitrogen stream at 37 • C. Deionized water was added to dissolve the samples, after which the samples were injected on the capillary. The developed extraction procedure is a simple, time-saving, and single-step process.

Method Validation
The linearity, precision, specificity, accuracy, carry-over extraction recoveries, and limit of detection (LOD) as well as limit of quantification (LOQ) of the developed method were validated by following the guidelines of the International Council on Harmonization (ICH) [48] and the previously presented procedure [47].
The first step before the validation process involved the screening of selectors, as described in Section 3.1. Optimization.
The specificity of the method was defined as its ability to differentiate and quantify an analyte of interest and an internal standard from the endogenous components in the matrix, or other components in the sample. The presence of potential endogenous interference was assessed by analyzing 10 serum samples obtained from the free-drug volunteers, which were fortified with the I (S), I (R), and II (S), II (R) compounds. The measurement results ( Figure 3C) showed the absence of endogenous interferences for every analyte of interest. The LODs and LOQs were determined by measuring the I (S), I (R), and II (S), II (R) compounds in a series of decreasing concentrations in the serum samples. In line with the ICH guidelines, LODs were determined at the lowest concentrations of I (S), I (R), and II (S), II (R), with a signal-to-noise ratio of at least 3 for all the compounds. The LOQ values were determined by quantitative measurement of the lowest compound concentration with a stated and acceptable accuracy and precision (CV% <20). The LODs and LOQs were estimated by analyzing the fortified serum samples of 500 µL volume. All the calibration curves indicated an excellent linear regression (with an R 2 of 0.9969-0.9998). The LOD values for I (S), I (R), II (S), and II (R) were estimated at 65.2, 65.6, 65.4, and 65.7 ng/mL, respectively, while the LOQ values for these compounds were 216.5, 217.8, 217.1, and 218.1 ng/mL, respectively (Table 1). Recovery of the compounds I (S), I (R), II (S), and II (R) was assessed by comparing the mean relative peak area for the samples containing these compounds at three different levels of concentration (high, medium, and low) in relation to the true concentrations of the pure standards, with the mean relative peak area for those compounds after extraction from the matrix. The recoveries determined for the analytes from serum solution and water were satisfactory in a range of 94.6-99.9% ( Table 2). The results presented in Table 2 show that the extraction method is suitable for measuring the drug levels in patients' serum.
The precision of the proposed method in terms of repeatability was determined by conducting a replicate analysis (n = 6) of serum extracts that were spiked with 100 and 1000 ng/mL of each compound. The relative standard deviation for migration time and relative peak area was less than 3.0% (Table 3).
Stability tests were performed under high-quality control. The analytes were stable in a serum blank at room temperature for 6 h and under repeated freeze-thaw conditions (totally three cycles). The samples to be tested were kept at −25 • C to evaluate their stability in a short-term experiment. The findings presented in Table 4 confirm the stability of the analytes for around 30 days.  Table 3. Intra-day and inter-day precision of compounds I (S), I (R) and II (S), II (R) analytes.

Conclusions
This paper described the procedure that uses the dual chiral-achiral selector complex consisting of 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin in CE for the concomitant detection of the four enantiomers of anticancer drugs, the newly patented I and II derivatives of 1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.0 2,6 ]dec-8ene-3,5,10-trione. The present study showed that the developed method allows separating a mixture of molecules I (S), I (R), and II (S), II (R), and is stereospecific, accurate and precise, and suitable for studying the pharmacokinetics, bioavailability, and optical purity of these enantiomers. In addition, the method enables a good separation of the enantiomers I (S), I (R), and II (S), II (R), in a very short time, with a lower amount of the injected sample. The single-step method of extraction offers an excellent recovery at a range of 94.6-99.9%. Furthermore, the extraction procedure is easy to perform and time saving, and hence can be used in clinical trial studies. The LOQs were 216.5 ng/mL for compound I (S), 217.8 ng/mL for compound I (R), 217.1 ng/mL for compound II (S), and 218.1 ng/mL for compound II (R). The run time was found to be only 8 min for each sample. Among the three CDs tested as chiral selectors, only 2-hydroxypropyl-β-cyclodextrin allowed for the separation of I (S), I (R), and II (S), II (R) enantiomers. The introduction of 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin as the second selector and creation of the dual 2-hydroxypropyl-β-cyclodextrin and 5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin chiral-achiral selector system led to a significant improvement in the separation process and sensitivity of the method, and thus allowed achieving very good LODs and determining the enantiomers of compounds (I) and (II).
The porphyrin derivative used in this study most likely creates additional connections with the previously formed complexes of individual enantiomers of compounds I and II with 2-hydroxypropyl-β-cyclodextrin, creating a kind of additional molecular sieve increasing the molecular weight of the complexes, their absorbance, and resolution.
In addition, the developed CE method was applied in the pilot studies of I (S), I (R), II (S), and II (R) enantiomers in the blood serum and yielded great results.