Cinchona-alkaloid-based zwitterionic chiral stationary phases as potential tools for high-performance liquid chromatographic enantioseparation of cationic compounds of pharmaceutical relevance.

Enantiomers of cationic compounds of pharmaceutical relevance, namely tetrahydro-ß-carboline and 1,2,3,4-tetrahydroisoquinoline analogs, were separated by high-performance liquid chromatography. Separations were performed on Cinchona-alkaloid-based zwitterionic ion exchanger type chiral stationary phases applied as cation exchangers using mixtures of methanol and acetonitrile or tetrahydrofuran as bulk solvent components containing triethylammonium acetate or ammonium acetate as organic salt additives. On the zwitterionic ZWIX(+) and ZWIX(-) columns investigated, retention and enantioseparation of the studied basic analytes were influenced by the nature and concentration of the organic components of the mobile phase. The effect of organic salt additives on the retention behavior of the studied analytes can be described by the stoichiometric displacement model related to the counterion concentration. Investigations on the structure-retention relationships were performed applying different mobile phase systems for the two types of cationic analytes. For the thermodynamic characterization, parameters such as changes in standard enthalpy (Δ(ΔH°)), entropy (Δ(ΔS°)), and free energy (Δ(ΔG°)) were calculated on the basis of van't Hoff plots derived from the ln α versus 1/T curves. In most cases, enthalpy-driven enantioseparations were observed, with a consistent dependence of the calculated thermodynamic parameters on the mobile phase composition. Elution sequences of the studied compounds were determined in all cases.

Liquid chromatographic enantiomer separations applying chiral ion-exchangers based on Cinchona alkaloids.

As the understanding of the various biological actions of compounds with different stereochemistry has grown, the necessity to develop methods for the analytical qualification and quantification of chiral products has become particularly important. The last quarter of the century has seen a vast growth of diverse chiral technologies, including stereocontrolled synthesis and enantioselective separation and analysis concepts. By the introduction of covalently bonded silica-based chiral stationary phases (CSPs), the so-called direct liquid chromatographic (LC) methods of enantiomer separation became the state-of-the-art methodology. Although a large number of CSPs is available nowadays, the design and development of new chiral selectors and CSPs are still needed since it is obvious that in practice one needs a good portfolio of different CSPs and focused "chiral columns" to tackle the challenging tasks. This review discusses and summarizes direct enantiomer separations of chiral acids and ampholytes applying anionic and zwitterionic ion-exchangers derived from Cinchona alkaloids with emphasis on literature data published in the last 10 years. Our aim is to provide an overview of practical solutions, while focusing on the integration of molecular recognition and methodological variables.

Dedicated comparisons of diverse polysaccharide- and zwitterionic Cinchona alkaloid-based chiral stationary phases probed with basic and ampholytic indole analogs in liquid and subcritical fluid chromatography mode.

Normal phase (NP) high-performance liquid and sub- and supercritical fluid chromatographic (both acronymed as SFC) methods have been developed for the enantiomer separation of three basic and three ampholytic structurally related C-3-substituted indole analogs on seven non-ionic (neutral) polysaccharide-based and two chemically entirely different zwitterionic Cinchona alkaloid- and sulfonic acid-based chiral stationary phases (CSPs). In a systematic fashion the effect of the composition of the mobile phase, the nature of the alcohol and amine additives on the retention characteristics and enantioselectivity of the ionizable analytes were investigated. On all studied polysaccharide-based CSPs the three ampholytes remained unretained in NP-LC mode, while they were nicely retained and resolved in SFC mode. These unexpected results underline a specific property of liquid CO2 as bulk solvent in combination with alcohols as co-solvents and amine additives thus creating an environment around the chiral selector sites which support the retention of ampholytes. The zwitterionic CSPs worked equally well for the resolution of the basic and ampholytic analytes using a polar ionic mobile phase in both LC and SFC modes. Results acquired by studying the effect of temperature were used to calculate the changes in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°) applying van't Hoff plots. The values of the thermodynamic parameters depended on the nature of selectors, the structure of analytes and the properties of the mobile phases. On polysaccharide-based CSPs and columns operated in NP-LC mode enthalpically-, whereas in SFC mode both enthalpically- and entropically-driven enantiomer separations were observed.

Quantitative determination of major alkaloids in Cinchona bark by Supercritical Fluid Chromatography.

Chinoline alkaloids found in Cinchona bark still play an important role in medicine, for example as antimalarial and antiarrhythmic drugs. For the first time Supercritical Fluid Chromatography has been utilized for their separation. Six respective derivatives (dihydroquinidine, dihydroquinine, quinidine, quinine, cinchonine and cinchonidine) could be resolved in less than 7 min, and three of them quantified in crude plant extracts. The optimum stationary phase showed to be an Acquity UPC2 Torus DEA 1.7 µm column, the mobile phase comprised of CO2, acetonitrile, methanol and diethylamine. Method validation confirmed that the procedure is selective, accurate (recovery rates from 97.2% to 103.7%), precise (intra-day ≤2.2%, inter-day ≤3.0%) and linear (R2 ≥ 0.999); at 275 nm the observed detection limits were always below 2.5 µg/ml. In all of the samples analyzed cinchonine dominated (1.87%-2.30%), followed by quinine and cinchonidine. Their total content ranged from 4.75% to 5.20%. These values are in good agreement with published data, so that due to unmatched speed and environmental friendly character SFC is definitely an excellent alternative for the analysis of these important natural products.

Chiral determination of cinchonine using an electrochemiluminescent sensor with molecularly imprinted membrane on the surfaces of magnetic particles.

A novel molecular imprinting electrochemiluminescence sensor for detecting chiral cinchonine molecules was developed with a molecularly imprinted polymer membrane on the surfaces of magnetic microspheres. Fe3 O4 @Au nanoparticles modified with 6-mercapto-beta-cyclodextrin were used as a carrier, cinchonine as a template molecule, methacrylic acid as a functional monomer and N,N'-methylenebisacrylamide as a cross-linking agent. Cinchonine was specifically recognized by the 6-mercapto-beta-cyclodextrin functional molecularly imprinted polymer and detected based on enhancement of the electrochemiluminescence intensity caused by the reaction of tertiary amino structures of cinchonine molecules with Ru(bpy)32+ . Cinchonine concentrations of 1 × 10-10 to 4 × 10-7  mol/L showed a good linear relationship with changes of the electrochemiluminescence intensity, and the detection limit of the sensor was 3.13 × 10-11  mol/L. The sensor has high sensitivity and selectivity, and is easy to renew. It was designed for detecting serum samples, with recovery rates of 98.2% to 107.6%.

Macromolecular crowding-assisted fabrication of liquid-crystalline imprinted polymers.

A macromolecular crowding-assisted liquid-crystalline molecularly imprinted monolith (LC-MIM) was prepared successfully for the first time. The imprinted stationary phase was synthesized with polymethyl methacrylate (PMMA) or polystyrene (PS) as the crowding agent, 4-cyanophenyl dicyclohexyl propylene (CPCE) as the liquid-crystal monomer, and hydroquinidine as the pseudo-template for the chiral separation of cinchona alkaloids in HPLC. A low level of cross-linker (26%) has been found to be sufficient to achieve molecular recognition on the crowding-assisted LC-MIM due to the physical cross-linking of mesogenic groups in place of chemical cross-linking, and baseline separation of quinidine and quinine could be achieved with good resolution (R(s) = 2.96), selectivity factor (α = 2.16), and column efficiency (N = 2650 plates/m). In contrast, the LC-MIM prepared without crowding agents displayed the smallest diastereoselectivity (α = 1.90), while the crowding-assisted MIM with high level of cross-linker (80%) obtained the greatest selectivity factor (α = 7.65), but the lowest column efficiency (N = 177 plates/m).

A novel hydroquinidine imprinted microsphere using a chirality-matching N-acryloyl-L-phenylalanine monomer for recognition of cinchona alkaloids.

Using a combination of molecular imprinting technology and traditional chiral stationary phases, the synergistic effect between chiral monomer and chiral cavity of molecularly imprinted polymers in stereoselective recognition was investigated. We designed and synthesized an amino acid derivative to be used as a novel chiral functional monomer. Monodisperse molecularly imprinted core-shell microspheres using surface imprinting method on silica gel were prepared with hydroquinidine as the pseudo-template molecule for the resolution of cinchona alkaloids. The results showed a significant synergistic effect in stereoselective recognition, confirming our initial hypothesis. Furthermore, our computational simulation and experiments intensively support the hypothetical chiral recognition mechanism for the imprinted microspheres.

Templating effect in DNA proximity ligation enables use of non-bioorthogonal chemistry in biological fluids.

Here we describe the first example of selective reductive amination in biological fluids using split aptamer proximity ligation (StAPL). Utilizing the cocaine split aptamer, we demonstrate small-molecule-dependent ligation that is dose-dependent over a wide range of target concentrations in buffer, human blood serum and artificial urine medium. We explore the substrate binding preferences of the split aptamer and find that the cinchona alkaloids quinine and quinidine bind to the aptamer with higher affinity than cocaine. This increased affinity leads to improved detection limits for these small-molecule targets. We also demonstrate that linker length and hydrophobicity impact the efficiency of split aptamer ligation. The ability to carry out selective chemical transformations using non-bioorthogonal chemistry in media where competing reactive groups are present highlights the power of the increased effective molarity provided by DNA assembly. Obviating the need for bioorthogonal chemistry would dramatically expand the repertoire of chemical transformations available for use in templated reactions such as proximity ligation assays, in turn enabling the development of novel methods for biomolecule detection.

Rapid and green analytical method for the determination of quinoline alkaloids from Cinchona succirubra based on Microwave-Integrated Extraction and Leaching (MIEL) prior to high performance liquid chromatography.

Quinas contains several compounds, such as quinoline alkaloids, principally quinine, quinidine, cinchonine and cichonidine. Identified from barks of Cinchona, quinine is still commonly used to treat human malaria. Microwave-Integrated Extraction and Leaching (MIEL) is proposed for the extraction of quinoline alkaloids from bark of Cinchona succirubra. The process is performed in four steps, which ensures complete, rapid and accurate extraction of the samples. Optimal conditions for extraction were obtained using a response surface methodology reached from a central composite design. The MIEL extraction has been compared with a conventional technique soxhlet extraction. The extracts of quinoline alkaloids from C. succirubra obtained by these two different methods were compared by HPLC. The extracts obtained by MIEL in 32 min were quantitatively (yield) and qualitatively (quinine, quinidine, cinchonine, cinchonidine) similar to those obtained by conventional Soxhlet extraction in 3 hours. MIEL is a green technology that serves as a good alternative for the extraction of Cinchona alkaloids.

Liquid chromatographic analysis of cinchona alkaloids in beverages.

A method for the determination of Cinchona extract (whose main components are the alkaloids cinchonine, cinchonidine, quinidine, and quinine) in beverages by liquid chromatography was developed. A beverage with an alcohol content of more than 10% was loaded onto an OASIS HLB solid-phase extraction cartridge, after it was adjusted to pH 10 with 28% ammonium hydroxide. Other beverages were centrifuged at 4000 rpm for 5 min, and the supernatant was loaded onto the cartridge. The cartridge was washed with water followed by 15% methanol, and the Cinchona alkaloids were eluted with methanol. The Cinchona alkaloids in the eluate were chromatographed on an L-column ODS (4.6 mm id x 150 mm) with methanol and 20 mmol/L potassium dihydrogen phosphate (3 + 7) as the mobile phase. Cinchona alkaloids were monitored with an ultraviolet (UV) detector at 230 nm, and with a fluorescence detector at 405 nm for cinchonine and cinchonidine and 450 nm for quinidine and quinine (excitation at 235 nm). The calibration curves for Cinchona alkaloids with the UV detector showed good linearity in the range of 2-400 microg/mL. The detection limit of each Cinchona alkaloid, taken to be the concentration at which the absorption spectrum could be identified, was 2 microg/mL. The recovery of Cinchona alkaloids added at a level of 100 microg/g to various kinds of beverages was 87.6-96.5%, and the coefficients of variation were less than 3.3%. A number of beverage samples, some labeled to contain bitter substances, were analyzed by the proposed method. Quinine was detected in 2 samples of carbonated beverage.

Bioactive cinchona alkaloids from Remijia peruviana.

Three known Cinchona alkaloids of the quinine type, quinine (1), cupreine (2), cinchonine (3), and the possible artifact cinchonine-HCl (3-HCl), along with two new ones, acetylcupreine (4) and N-ethylquinine (5), have been isolated from the bark of Remijia peruviana (Rubiaceae). Their stereochemical structures were established by high resolution NMR spectroscopy. Alkaloids 2-4 had antifeedant effects on Leptinotarsa decemlineata with varying potencies. Compound 4 was cytotoxic to both insect Sf9 and mammalian CHO cells after 48 h of incubation, while 3-HCl had stronger and selective cytotoxicity to Sf9. Quinine 1 had a moderate to low effect on Trypanosoma cruzi. Tumoral cells were also affected by these alkaloids, with 4 and 3-HCl being the most cytotoxic to all the cell lines tested. Overall, the 8R, 9S configurations, as in 3 and 3-HCl, as well as the C-6'acetylated alkaloid 4, with an 8S, 9R configuration, showed stronger biological effects.

Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques.

The Cinchona alkaloids, which include the pharmaceuticals quinine and quinidine, continue to have a wide variety of important uses. A number of different chromatographic procedures have been developed for the qualitative and quantitative analysis of these compounds in a variety of sample matrices. Reversed-phase HPLC using ODS columns in combination with acidic mobile phases, and UV detection, is the most widely used method. Nevertheless, precautions need to be taken due to the strong silanophilic interactions which can occur with these analytes and the column surface, which can lead to poor peak shape and resolution. Different selectivity may be achieved in HPLC separations by use of alternative stationary phases, or by varying mobile phase pH. The specificity of detection systems may be improved by use of photodiode array UV detectors, or especially mass spectrometers. Thin-layer chromatography (TLC) provides a cheap alternative analytical method, which is especially useful for qualitative analysis. High-performance TLC, gas chromatography, capillary electrophoresis and capillary electrochromatography are all methods which after some development, could prove useful for Cinchona alkaloid separations.

A selective PVC membrane for di- or trinitrophenol based on N,N-dibenzyl-3,3',5,5'-tetramethylbenzidine.

A new fluorophore, N,N-dibenzyl-3,3',5,5'-tetramethylbenzidine (NBTMB), was prepared and shown to exhibit significant and analytical usefulness for optical sensing toward 2,4-dinitrophenol or 2,4,6-trinitrophenol (picric acid) when it was immobilized in a plasticized poly(vinyl chloride) (PVC) membrane. When the membrane was applied to aqueous nitrophenol solution, NBTMB was able to extract selectively nitrophenol into the membrane phase. Since the extraction equilibrium was accompanied by fluorescence quenching of NBTMB, the chemical recognition process could be directly translated into an optical signal. The sensor showed reversible response in the concentration range from 2.0 x 10(-7) to 6.0 x 10(-5) mol L(-1) for the detection of 2,4-dinitrophenol in NaOAc-HOAc buffer at pH 4.0. It also showed a fast response time (t95% < 1.5 min) when the sensor was applied to 2,4-dinitrophenol solution at concentration levels of 5.26 x 10(-6) and 2.10 x 10(-5) mol L(-1) alternatively. A working principle is proposed and the responses of this sensor to various kinds of nitrophenol were studied. The sensor was applied to the direct determination of 2,4-dinitrophenol in prepared water samples and the indirect assay of the drug cinchonine and the results obtained were satisfactory.

Identification of ethyl pyruvate and dihydrocinchonidine adducts by electrospray ionization mass spectrometry.

Several ethyl pyruvate and dihydrocinchonidine adducts, formed by non-covalent interactions with alkali cations, have been identified for the first time using electrospray ionization mass and tandem mass spectrometry. This type of adduct may have an important role in asymmetric reactions of pyruvates in the presence of cinchonas.

Atomic emission spectrometric determination of ephedrine, cinchonine, chlorpheniramine, atropine and diphenhydramine based on formation of ion associates with ammonium reineckate.

Ion-associate complexes of ephedrine HCl (I), cinchonine HCl (II), chlorpheniramine maleate (III), atropine sulphate (IV) and diphenhydramine HCl (V) with ammonium reineckate were precipitated and their solubilities were studied as a function of pH, ionic strength and temperature. Saturated solutions of each ion-associate under the optimum precipitation conditions were prepared and the Cr ion content in the supernatant was determined. The solubility products were thus elucidated at different temperatures. A new accurate and precise method using direct current plasma-atomic emission spectrometry for the determination of the investigated drugs in pure solutions and in pharmaceutical preparations is described. The drugs can determined by the present method in the ranges 1.6-52,2.64-85.8,3.12-101.4,5.52-180.4 and 2.72-75.85 microg/ml solutions of I, II, III, IV and V, respectively.

Optosensor for cinchona alkaloids with C18 silica gel as a substrate.

A flow-through optosensor for cinchona alkaloids with C18 silica gel as a substrate is proposed. The sensor is developed in conjunction with a flow-injection analysis system and is based on the retention of the cinchona alkaloids on a C18 column and the enhancement of their fluorescence. The analytical performance characteristics of the proposed sensor for the detection and quantification of these alkaloids were as follows: the detection limits of quinine, cinchonine, quinidine and cinchonidine were 2.3, 31.6, 2.3 and 31.6 ng ml-1, respectively, with relative standard deviations of 0.9% for quinine and quinidine (20 ng ml-1, n = 7) and 1.1% for cinchonine and cinchonidine (4.0 micrograms ml-1, n =7), respectively. Most of the common species did not interfere. The recommended method has been successfully tested for determination of quinine in pharmaceutical preparations and soft drinks.

The separation and determination of quinine in bitter drinks by micellar electrokinetic capillary chromatography.

A rapid method for the determination of quinine in bitter drinks by micellar electrokinetic capillary chromatography (MECC) with ultraviolet (UV) detection is described. The beverage is simply diluted with deionized water, filtered, and analyzed using a 75 cm x 75 micrometer uncoated fused-silica capillary column with a buffer consisting of 15% methanol and 85% of a mixture of 0.05 M cetyltrimethylammonium bromide (CTAB), 0.01 M sodium tetraborate, and 0.01 M potassium dihydrogen orthophosphate, pH 8.6, with an operating voltage of -25 kV. The levels of quinine determined by MECC were in good agreement with those determined by HPLC. The CVs for area calculation (2.1%, n = 7) and migration time variation (1.3%, n = 20) for multiple injections of a sample solution by MECC were satisfactory.

Automated multiple development thin layer chromatography of some plant extracts.

The separation of ten plant extracts using automated multiple development thin-layer chromatography (AMD -TLC) is described. Alcoholic extracts were obtained from Cinchona succirubra, Aesculus hippocastanum, Berberis vulgaris. Artemisia abrotanum, Carduus marianus, Thuja occidentalis, Baptisia tinctoria, Paulinia cupana, Lycopus europaeus and Echinacea angustifolia. The separation was performed on silica plates (Sil G-50 UV 254 (Macherey-Nagel), 10 x 20 cm). AMD was achieved in 25 steps using methanol, ethyl acetate, toluene, 1,2-dichloroethane, 25% ammonia solution and anhydrous formic acid as modifiers. The chromatograms were evaluated with a Shimadzu CS-9000 dual-wavelength flying-spot scanner. Better separations were obtained using AMD than isocratic elution.

Spectrophotometric determination of ephedrine HCl, cinchonine HCl, chlorpheniramine maleate, atropine sulphate and diphenhydramine HCl by solvent extraction of reineckate complexes.

A spectrophotometric method for the determination of some alkaloids (namely ephedrine HCl, cinchonine HCl, chlorpheniramine maleate, atropine sulphate and diphenhydramine HCl) as separate compounds as well as in pharmaceutical preparations through the formation of their ion-pair (reineckate complexes) is described. These complexes were prepared and the final products were extracted with nitrobenzene in situ. The optimum conditions for complete extraction were evaluated by investigating the nature of solvent, pH, time of shaking, temperature and reineckate concentrations. The absorption spectra of the extracted organic layer were measured at 520 nm. The extraction constants were calculated and found to have a mean of log KE congruent to 2. The application of the present method was compared with the accepted pharmacopoeia method and the effects of some interfering ions were also studied.

High-performance liquid chromatographic-mass spectrometric assay of high-value compounds for pharmaceutical use from plant cell tissue culture: Cinchona alkaloids.

The use of high-performance liquid chromatography (HPLC) interfaced with thermospray (TSP) mass spectrometry is described for the separation and identification of various alkaloids from Cinchona ledgeriana extracts. The use of water-acetonitrile-acetic acid (71:25:4) with 0.01 M ammonium acetate (pH 3.0) as the mobile phase gave good HPLC separation and good TSP sensitivity. The specificity obtained by single-ion monitoring allowed the analysis of commercially important alkaloids such as quinine and quinidine in plant material, transformed roots and in cells from tissue culture, with relatively simple extraction and work-up procedures. TSP gave protonated species with few fragment ions but collision-induced dissociation offers the promise of increased analytically specificity from the fragment ion data. This work has important implications for the biotechnological production of pharmaceuticals normally obtained from plant sources.