Progress in Chiral Stationary Phases Based on Proteins and Glycoproteins.

A lot of chiral stationary phases (CSPs) have been introduced for the purpose of analytical and preparative separations of enantiomers. CSPs based on proteins and glycoproteins have unique properties among those CSPs. This review article deals with the preparation of CSPs based on proteins and glycoproteins, their chiral recognition properties and mechanisms, focusing on the CSPs investigated in our group. The dealt proteins and glycoproteins are including bovine serum albumin, human serum albumin, lysozyme, pepsin, human α1-acid glycoprotein (AGP), chicken ovomucoid and chicken ovoglycoprotein (named chicken AGP).

Preparation of molecularly imprinted polymers for strychnine by precipitation polymerization and multistep swelling and polymerization and their application for the selective extraction of strychnine from nux-vomica extract powder.

Monodisperse molecularly imprinted polymers for strychnine were prepared by precipitation polymerization and multistep swelling and polymerization, respectively. In precipitation polymerization, methacrylic acid and divinylbenzene were used as a functional monomer and crosslinker, respectively, while in multistep swelling and polymerization, methacrylic acid and ethylene glycol dimethacrylate were used as a functional monomer and crosslinker, respectively. The retention and molecular recognition properties of the molecularly imprinted polymers prepared by both methods for strychnine were evaluated using a mixture of sodium phosphate buffer and acetonitrile as a mobile phase by liquid chromatography. In addition to shape recognition, ionic and hydrophobic interactions could affect the retention of strychnine in low acetonitrile content. Furthermore, molecularly imprinted polymers prepared by both methods could selectively recognize strychnine among solutes tested. The retention factors and imprinting factors of strychnine on the molecularly imprinted polymer prepared by precipitation polymerization were 220 and 58, respectively, using 20 mM sodium phosphate buffer (pH 6.0)/acetonitrile (50:50, v/v) as a mobile phase, and those on the molecularly imprinted polymer prepared by multistep swelling and polymerization were 73 and 4.5. These results indicate that precipitation polymerization is suitable for the preparation of a molecularly imprinted polymer for strychnine. Furthermore, the molecularly imprinted polymer could be successfully applied for selective extraction of strychnine in nux-vomica extract powder.

Maintenance of luminal pH and protease activity in lysosomes/late endosomes by vacuolar ATPase in chlorpromazine-treated RAW264 cells accumulating phospholipids.

Cationic amphiphilic drugs (CADs) inhibit phospholipases competitively/uncompetitively. It has also been reported that CADs spontaneously accumulate in acidic organelles and increase their luminal pH, which may lead to deactivation of phospholipid-metabolising enzymes, causing cellular phospholipid accumulation. Recently, however, contradictory results have also been reported in that the luminal pH is not increased by CAD treatment. In this study, we examined whether the lysosomal/late endosomal acidic pH was maintained by vacuolar ATPase (v-ATPase) after treatment with chlorpromazine (CPZ) as a model CAD. The activity of lysosomal protease after CPZ treatment was also measured. Oregon Green-dextran-tetramethylrhodamine conjugate was employed to determine the luminal pH of the lysosomes/late endosomes in RAW264 cells. The luminal pH remained acidic after treatment with CPZ for 23 h, and the lysosomal protease activity was not decreased by 5-min CPZ treatment. Co-treatment with CPZ and bafilomycin A1 (v-ATPase inhibitor) raised the luminal pH. These results suggest that the lysosomal/late endosomal pH is not affected by a 23-h CPZ treatment. In addition, lysosomal enzymes presumably maintain their activity when CPZ accumulates. Our results imply that the pH homeostasis in lysosomes/late endosomes is strictly maintained even after a longer treatment with CADs.

Development and validation of a sensitive GC-MS method for the determination of alkylating agent, 4-chloro-1-butanol, in active pharmaceutical ingredients.

The analysis of genotoxic impurities (GTIs) in active pharmaceutical ingredients (APIs) is a challenging task. The target detection limit (DL) in an API is typically around 1 ppm (1 µg/g API). Therefore, a sensitive and selective analytical method is required for their analysis. 4-Chloro-1-butanol, an alkylating agent, is one of the GTIs. It is generated when tetrahydrofuran and hydrochloric acid are used during the synthesis of the APIs. In this study, a sensitive and robust gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the identification of 4-chloro-1-butanol in APIs. In the GC-MS method, 3-chloro-1-butanol was employed as an internal standard to ensure accuracy and precision. Linearity was observed over the range 0.08 to 40 ppm (µg/g API), with a R(2) value of 0.9999. The DL and quantitation limit (QL) obtained were 0.05 ppm and 0.08 ppm (0.13 ng/mL and 0.20 ng/mL as the 4-chloro-1-butanol concentration), respectively. These DL and QL values are well over the threshold specified in the guidelines. The accuracy (recovery) of detection ranged from 90.5 to 108.7% between 0.4 ppm and 20 ppm of 4-chloro-1-butanol. The relative standard deviation in the repeatability of the spiked recovery test was 6.0%. These results indicate the validity of the GC-MS method developed in this study. The GC-MS method was applied for the determination of 4-chloro-1-butanol in the API (Compound A), which is under clinical trials. No 4-chloro-1-butanol was found in Compound A (below QL, 0.08 ppm).

Identification of novel metabolites of abiraterone in human serum and their metabolic pathways.

Two novel abiraterone (Abi, 3ß-OH-Abi) metabolites in human serum were identified as 3α-OH-Abi and Δ5-Abi (D5A). Both metabolites were confirmed by their retention times on LC/MS and their product-ion mass spectra on LC-MS/MS compared to those of authentic compounds, which were chemically synthesized. The plausible metabolic pathways of these two metabolites are as follows: Abi is first oxidized to D5A by 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and then irreversibly converted to Δ4-Abi (D4A) by ∆5-∆4 isomerase. Presumably, D5A detection is difficult because of its rapid conversion to D4A and its low concentration in serum samples. In contrast, the low concentration 3α-OH-Abi was generated by reducing the remaining D5A using 3α-hydroxysteroid dehydrogenase (3α-HSD).

Interaction of cepharanthine with immobilized heat shock protein 90α (Hsp90α) and screening of Hsp90α inhibitors.

Heat shock protein 90α (Hsp90α) immobilized on aminopropyl silica gels was prepared via the N- or C-terminal, which was termed Hsp90α-NT or Hsp90α-CT, respectively. Binding interactions of biscoclaurine alkaloids (cepharanthine (CEP), berbamine (BBM), isotetrandrine (ITD), and cycleanine (CCN)) with Hsp90α were examined using the Hsp90α-NT or -CT columns by frontal and zonal chromatography studies. The dissociation constants of CEP, BBM, ITD, and CCN to Hsp90α-NT were estimated to be 5.3, 18.6, 46.3, and 159 µM, respectively, by frontal chromatography techniques. Similar results were obtained with the Hsp90α-CT column. These data suggest that these biscoclaurine alkaloids interact with the middle domain of Hsp90α. This was confirmed by demonstrating that CEP competed with endothelial nitric oxide synthase at the middle domain of Hsp90α, where it was shown to have a dissociation constant of 15 nM. Furthermore, the Hsp90α-NT column was applied for preliminary screening of natural Hsp90α inhibitors by zonal chromatography studies.

Preparation of monodisperse curcumin-imprinted polymer by precipitation polymerization and its application for the extraction of curcuminoids from Curcuma longa L.

A monodisperse molecularly imprinted polymer (MIP) for curcumin was first prepared by precipitation polymerization using methacrylamide (MAM) and 4-vinylpyridine as functional co-monomers, divinylbenzene as a crosslinker, and a mixture of acetonitrile and toluene as a porogen. The use of MAM as the co-monomer resulted in the formation of a monodisperse MIP and non-imprinted polymer (NIP). MIP and NIP, respectively, were monodispersed with a narrow particle size distribution (3.3 ± 0.09 and 3.5 ± 0.10 µm). In addition to shape recognition, hydrophobic and hydrogen-bonding interactions affected the retention and molecular-recognition of curcumin on the MIP. The MIP for curcumin could extract curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) in Curcuma longa L.

Comparison of the dissolution rate of ceftriaxone sodium preparations for injection.

This study aimed to compare the dissolution rate of eight different formulations of ceftriaxone sodium preparation for injection, the original product and seven generic versions. The dissolution time was measured precisely as the point at which the freeze-dried ceftriaxone sodium preparation became a transparent solution on the addition of 0.9% sodium chloride solution. To investigate whether differences in the crystalline structure may explain the differences in dissolution rates, the eight products were subjected to X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Powder surface characteristics were examined, including surface area, amount of water adsorbed, water interactions and morphology. The measurement of near-infrared spectroscopy of powder preparations was conducted, and we predicted dissolution time by partial least squares (PLS). The dissolution time of the eight products were different. There were no differences in XRD and DSC findings between the original and generic products, surface characteristics, i.e., surface area, morphology etc., were different between preparations. On near-infrared (NIR) spectroscopy, a good relationship was demonstrated between the actual and predicted dissolution time for each ceftriaxone preparation. The difference in dissolution time between the eight products was due to differences in powder surface characteristics, such as water interaction and crystal shape. Finally, it was shown that the dissolution rates of the products could be predicted by NIR analysis.

A Phase II Trial of Abiraterone With Dutasteride for Second-Generation Antiandrogen- and Chemotherapy-Naïve Patients With Castration-Resistant Prostate Cancer.

The development of a novel therapy to overcome primary and acquired resistance to abiraterone is an unmet need. This study aimed to evaluate the efficacy and safety of adding 5α-reductase inhibitor dutasteride to abiraterone, explore proof of concept, and identify candidates suitable for combination therapy. This phase II, single-arm, and open-label study enrolled second-generation antiandrogen- and chemotherapy-naïve patients with castration-resistant prostate cancer. Patients received abiraterone and prednisolone for 4 weeks, followed by adding dutasteride. The primary end point was a 50% prostate-specific antigen response rate. Serum concentrations of abiraterone and its metabolites as well as HSD3B1 and SRD5A2 genotypes were measured. The association between drug metabolism and genotypes and their impact on the efficacy of combination therapy were assessed. Among 21 patients, 18 (85.7%) achieved ≥50% PSA reduction. Median time to treatment failure was not reached during the median follow-up of 15.4 months. No patients experienced grade ≥3 adverse events. Although dutasteride reduced serum 3-keto-5α-abiraterone concentrations, higher serum 3-keto-5α-abiraterone concentrations on combination therapy were associated with a shorter time to treatment failure. HSD3B1 and SRD5A2 genotypes were associated with serum Δ4-abiraterone and 3-keto-5α-abiraterone concentrations before adding dutasteride, respectively. Time to treatment failure was longer in patients with homozygous wild-type HSD3B1, but comparable between those with the SRD5A2 genotype. The promising outcomes of this study warrant further investigation of combination therapy in a randomized trial. Stratification by HSD3B1 and SRD5A2 genetic profiles might identify patients suitable for combination therapy.

Role of bis(monoacylglycero)phosphate in propranolol binding to phospholipid membranes under acidic conditions as measured by high-performance frontal analysis/capillary electrophoresis.

Bis(monoacylglycero)phosphate (BMP) is localized in acidic organelles such as late endosomes or lysosomes. It has been reported that BMP levels increase under phospholipidosis induced by cationic amphiphilic drugs. In the present study, the effect of BMP on the binding of propranolol (PRO) to phospholipid liposomes under acidic conditions was investigated. Binding experiments were conducted by high-performance frontal analysis/capillary electrophoresis. PRO showed nonspecific binding to BMP-containing liposomes (BMP:phosphatidylcholine = 1:4), when numbers of bound drug molecules per lipid molecule (r) ranged 0.01-0.06. Total binding affinity increased depending on the BMP content. Binding affinity was decreased by low ionic strength, or by substitution of BMP with diacylglycerol, suggesting that electrostatic interactions were involved. The binding-enhancement effect of BMP was almost equivalent to that of phosphatidylglycerol, and slightly larger than that of phosphatidylserine. An acidic environment (pH 5.0) decreased total binding affinity to BMP-containing liposomes. This could be explained by the pH-partition theory (i.e., the loss in affinity was caused by a decrease in the neutral form of the drug accessible to the membrane core). These results suggest that PRO binding is enhanced by BMP in late endosomes or lysosomes, whereas an acidic environment weakens such binding.

Synthesis of Molecularly Imprinted Polymers by Two-Step Swelling and Polymerization.

Synthesis of a molecularly imprinted polymer (MIP) by two-step swelling and polymerization is described. Monodisperse, spherical MIP particles, whose diameters are ca. 5-9µm, are prepared using a polystyrene particle as a shape template and dibutyl phthalate as an activating solvent. The obtained MIPs are suitable for separation media in liquid chromatography or solid-phase extraction media. Procedures for synthesis of MIPs and restricted access media (RAM)-MIP, packing of MIPs and RAM-MIPs, and application of MIPs and RAM-MIPs for selective separation and extraction of a target compound(s) are described.

Retention and molecular-recognition mechanisms of molecularly imprinted polymers for warfarin derivatives and their application for the determination of warfarin in human serum.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF), 4'-chlorowarfarin (CWF), 4'-bromowarfarin (BWF), 4'-nitrowarfarin (NWF) and 4'-methylwarfarin (MWF) (MIPWF, MIPCWF, MIPBWF, MIPNWF and MIPMWF, respectively) were prepared using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate as a functional monomer and crosslinker, respectively, by multi-step swelling and polymerization. The retention and molecular-recognition properties of those MIPs were evaluated in HILIC, and reversed- and normal-phase modes. According to 1H NMR studies, one-to-three complex formation of one WF or CWF molecule with three 4-VPY molecules occurred. Via computational approaches, the intermolecular interaction modes and energies between WF derivatives and 4-VPYs were evaluated by semi-empirical quantum chemistry methods and density functional theory calculations. Three major possible hydrogen bonding interaction modes were identified: the interactions between the 4-hydroxy group, α-proton (methylene C-H) and α-proton (methyl C-H) of the WF derivative and the nitrogen atoms of 4-VPYs. In HILIC and normal-phase modes, the interaction energies showed satisfactory correlations with the retention factors of the WF derivatives. In reversed-phase mode, the retention factors of the WF derivatives were described by the hydrophobicity and the acidity of the 4-hydroxy groups of the WF derivatives. These results demonstrate that three hydrogen bonding interactions in HILIC and normal-phase modes, and hydrogen bonding or ionic interactions and hydrophobic interactions in reversed-phase mode play important roles in the retention and molecular-recognition of the WF derivatives on MIPs. Furthermore, MIPBWF was successfully applied to the determination of WF in human serum by column-switching LC with high accuracy, precision and selectivity and without template-leakage problems.

Molecularly imprinted polymers for arbutin and rutin by modified precipitation polymerization and their application for selective extraction of rutin in nutritional supplements.

Molecularly imprinted polymers (MIPs) for glycosides, arbutin (ARB) and rutin (RUT), were prepared using methacrylamide (MAM) and 4-vinylpyridine (4-VPY) as functional monomers and divinylbenzene as a crosslinker by modified precipitation polymerization. The template molecule, ARB or RUT, was first dissolved in methanol, followed by precipitation polymerization using a mixture of acetonitrile and toluene as a porogenic solvent. The molar ratios of the template molecule, MAM and 4-VPY were optimized to achieve a high molecular recognition ability for ARB and RUT. The retention and molecular recognition properties of these MIPs were evaluated in HILIC or normal-phase mode. With an increase in the acetonitrile content in the mobile phase, the retention factor of ARB or RUT was increased. Furthermore, the MIPs for ARB and RUT showed the highest imprinting factors of 3.65 and 66.5 for the template molecules, respectively. Hydrogen bonding interactions such as N⋯H-O, C=O⋯H-O and NH⋯O-H between 4-VPY or MAM and hydroxy groups of d-glucose or d-rutinose could function in the recognition of a glycone. Furthermore, hydrogen bonding interactions between functional monomers and the hydroxy group(s) of hydroquinone or quercetin could function in the recognition of an aglycone. These results suggest that the MIPs could recognize both a glycone and aglycone via hydrogen bonding interactions. Furthermore, MIPs for RUT were successfully applied to extract RUT in nutritional supplements.

Determination of Abiraterone and Its Metabolites in Human Serum by LC-ESI-TOF/MS Using Solid-phase Extraction.

We developed and validated a liquid chromatography-electrospray ionization-time of flight/mass spectrometry method for the determination of abiraterone (Abi) and its metabolites (Δ4-Abi, 3-keto-5α-Abi, 3α-OH-5α-Abi and 3ß-OH-5α-Abi) in human serum using Abi-d4 as the internal standard. As a pretreatment procedure of serum samples, solid-phase extraction based on a silica-gel cartridge was used. The relative recovery of Abi and its metabolites was over the ranges of 84.5 - 109.2% at a concentration of 6.0 ng mL-1 for Abi and 0.6 ng mL-1 for its metabolites. The method was free from matrix effects. The calibration curve of Abi was linear over the range of 2.0 - 400 ng mL-1 and those of its metabolites over the ranges 0.2 - 40 ng mL-1. The results of the intra- and inter-day accuracy and precision data were within the FDA acceptance criteria. The optimized method was applied for the determination of Abi and its metabolites in human serum after oral administration of Abi acetate.

Enantioseparation of warfarin derivatives on molecularly imprinted polymers for (S)- and (R)-chlorowarfarin.

Monodisperse molecularly imprinted polymers (MIPs) for warfarin (WF), 4'-chlorowarfarin (CWF), (S)-CWF and (R)-CWF (MIPWF, MIPCWF, MIP(S)-CWF and MIP(R)-CWF, respectively) were prepared using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate (EDMA) as a functional monomer and a crosslinker, respectively, by multi-step swelling and polymerization. The molar ratio of a template molecule, 4-VPY to EDMA was 6:18:25 or 4:18:25. The retention and molecular recognition properties of MIPWF and MIPCWF were evaluated using a mixture of sodium phosphate buffer or ammonium formate and acetonitrile in reversed-phase LC. WF and CWF on these MIPs gave the maximal retentions at mobile phase pH 7, and those retentions were decreased with an increase of acetonitrile content. The retention and imprinting factors were in the order of WF < CWF < 4'-bromowarfarin (BWF) on MIPWF and MIPCWF in neutral mobile phases. On the other hands, in acidic mobile phases the retention factors were in the same order with those in neutral mobile phases, while the imprinting factors of WF and CWF were higher on the respective MIPs. These results suggest that ionic or hydrogen bonding interactions, hydrophobic interactions and π-π interactions could work for the retention and molecular recognition of WF, CWF and BWF on these MIPs in a reversed-phase mode. Furthermore, MIP(S)-CWF and MIP(R)-CWF could separate WF, CWF and BWF enantiomers in acidic mobile phases.

Novel bone microenvironment model of castration-resistant prostate cancer with chitosan fiber matrix and osteoblasts.

The interaction between prostate cancer cells and osteoblasts is essential for the development of bone metastasis. Previously, novel androgen receptor axis-targeted agents (ARATs) were approved for metastatic castration-naïve and non-metastatic castration-resistant prostate cancer (CRPC); both of which are pivotal for investigating the association between the bone microenvironment and tumors. The present study established a novel in vitro 3D microenvironment model that simulated the bone microenvironment of CRPC, and evaluated the drug susceptibility of ARATs and the efficacy of the combination of abiraterone and dutasteride. Green fluorescent protein-transferred C4-2 cells (a CRPC cell line) and red fluorescent protein-transferred human osteoblasts differentiated from human mesenchymal stem cells were co-cultured in chitosan nanofiber matrix-coated culture plates to simulate the 3D scaffold of the bone microenvironment. The growth of C4-2 was quantified using live-cell imaging and the Cell3 iMager duos analysis system. The growth of C4-2 colonies were quantified for a maximum of 30 days. The expression of TGF-ß increased and promoted EMT in C4-2 cells co-cultured with osteoblasts, indicating resistance to ARATs. The IC50 of each drug and the combination effect of abiraterone and dutasteride were evaluated using this model. Combination treatment with abiraterone and dutasteride synergistically inhibited the growth of C2-4 colonies compared with individual investigational agents. This could be attributed to the reduction of 3-keto-5α-abiraterone, an androgen receptor agonist. The bone microenvironment model of the present study is unique and useful for evaluating new drug susceptibility testing in prostate cancer cells. This model may help to reveal the unknown mechanisms underlying micro- to clinical bone metastasis in prostate cancer.

Molecularly imprinted polymers as affinity-based separation media for sample preparation.

This review article deals with molecularly imprinted polymers (MIPs) as affinity-based separation media for sample preparation. An over view of two types of MIPs (molecularly imprinted particle and monolith) used for the sample preparation and modes of molecularly imprinted SPE (online mode, offline mode, on-column extraction, SPME, and microextraction in packed syringe) is given, focusing on the advantages and disadvantages of these types and modes. Next, problems (template leakage and incompatibility with aqueous conditions) associated with molecularly imprinted SPE and how to overcome those problems are described. Finally, pharmaceutical, food, bioanalytical, and environmental application of molecularly imprinted SPE will be discussed.

Preparation and Evaluation of Molecularly Imprinted Polymers for Promazine and Chlorpromazine by Multi-step Swelling and Polymerization: the Application for the Determination of Promazine in Rat Serum by Column-switching LC.

Molecularly imprinted polymers (MIPs) for promazine (PZ) and chlorpromazine (CPZ), MIPPZ and MIPCPZ, were prepared by multi-step swelling and polymerization using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker. The retention and molecular-recognition properties of MIPPZ and MIPCPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile, or a mixture of ammonium formate and acetonitrile as the mobile phase in LC. PZ and CPZ gave the maximal retentions on MIPPZ and MIPCPZ at an apparent pH 8.2 using a mixture of potassium phosphate buffer and acetonitrile as the mobile phase. The retentions of PZ and CPZ decreased with an increase of acetonitrile contents from 70 to 90 vol% using a mixture of ammonium formate and acetonitrile as the mobile phase. The template molecules (PZ and CPZ, respectively) were recognized the most on the respective MIPs, and the imprinting factor of PZ was higher on MIPCPZ than on MIPPZ. These results indicate that in addition to shape recognition, ionic and hydrophobic interactions seem to work for the retention and molecular-recognition of PZ and CPZ on the MIPs. MIPCPZ was successfully utilized for the selective extraction of PZ in rat-serum samples in column-switching LC with fluorescence detection.

Evaluation of molecularly imprinted polymers for chlorpromazine and bromopromazine prepared by multi-step swelling and polymerization method-The application for the determination of chlorpromazine and its metabolites in rat plasma by column-switching LC.

Monodisperse molecularly imprinted polymers (MIPs) for chlorpromazine (CPZ) and bromopromazine (BPZ), MIPCPZ and MIPBPZ, were prepared using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of MIPCPZ and MIPBPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile or a mixture of water and acetonitrile including ammonium formate as a mobile phase in reversed-phase LC. On MIPBPZ, CPZ, BPZ and imipramine (IMP) gave the maximal retention factors at a mobile-phase pH 8, while the maximal imprinting factors were obtained at a mobile-phase pH 7. Each MIP recognized a template molecule the most, while CPZ metabolites, desmethyl CPZ (DM-CPZ), CPZ sulfoxide (CPZ-SO) and 7-hydroxy CPZ (7-OH-CPZ), were moderately recognized on MIPCPZ and MIPBPZ. Furthermore, both MIPs gave the similar retention and molecular-recognition for CPZ and its metabolites. For avoiding the template-leakage problems, MIPBPZ was used as the pretreatment column for the determination of CPZ and its metabolites in rat plasma in column-switching LC with UV detection. In addition to DM-CPZ and CPZ-SO, didesmethyl CPZ (DDM-CPZ) and CPZ N-oxide (CPZ-NO) were speculated as the metabolite in rat plasma after administration of CPZ using LC-ESI-TOF-MS, while 7-OH-CPZ was not detected. The column-switching LC method was validated and applied for the determination of CPZ and its metabolites, DM-CPZ, DDM-CPZ, CPZ-SO and CPZ-NO, in rat plasma after intravenous and oral administration of CPZ using IMP as an internal standard.

Retention and molecular-recognition mechanisms of molecularly imprinted polymers for promazine derivatives.

Monodisperse molecularly imprinted polymers (MIPs) for promazine derivatives [promazine (PZ), methylpromazine (MPZ), chlorpromazine (CPZ) and bromopromazine (BPZ)], MIPPZ, MIPMPZ, MIPCPZ and MIPBPZ, were prepared using methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of the obtained MIPs were evaluated using LC in hydrophilic interaction chromatography (HILIC) and reversed-phase modes. In computational approaches, intermolecular interaction modes and energies between PZ derivatives and MAAs were evaluated at the HF/6-311G(d,p) level. The interaction energies of PZ, MPZ, CPZ and BPZ with 4 equivalents of MAAs were calculated. The results indicated that the interaction of the aliphatic amine moiety of a PZ derivative with MAA gave almost similar interaction energies at the HF/6-311G(d,p) level, and that the interaction of the sulfur atom of a phenothiazine scaffold with MAA was also the case. The third interaction of the aromatic amine of a PZ derivative with MAA was in the order of MPZ > PZ > CPZ > BPZ presumably due to the change of basicity by the electron-donating or electron-withdrawing effect of a subsituent. Furthermore, the fourth attractive modes of CPZ and BPZ were suggested to be the interaction of their halogen atoms with MAA through both halogen bonding and hydrogen bonding, while PZ and MPZ were suggested to have the weak C-H ⋅⋅⋅ π interaction with MAA. In HILIC mode, the interaction energies at the HF method had good correlation with the retention factor of a PZ derivative on each MIP, indicating that in addition to the shape recognition, the attractive electrostatic interactions would be more responsible for its retention rather than the dispersion energies. Furthermore, in addition to the shape recognition, ionic and hydrophobic interactions, and halogen bonding and hydrogen bonding (the last interaction seems to be weak) seem to work for the retention and molecular-recognition of PZ derivatives on the MIPs in reversed-phase mode.