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During this study, a simple and easy-to-prepare electrophoretic method was developed for the enantioseparation of amphetamine and cathinone derivatives. Different types of β-cyclodextrin and cyclofructan-based chiral selectors (CSs), both native and derivatized, were utilized, and the most effective ones, in terms of resolution and analysis time, were identified. In addition, several electrophoretic parameters, such as background electrolyte concentration and pH, and CS concentration, were examined to optimize the separation conditions. Under the optimal electrophoretic conditions, 10 psychoactive substances were enantiomerically separated using 1 mM sulfated cyclofructan-6 (SCF-6) for the amphetamine derivatives and 1 mM sulfated cyclofructan-7 (SCF-7) for the cathinone derivatives dissolved in an aqueous solution of 20-mM monobasic sodium phosphate at pH 2.5, a temperature of 25°C, and an applied voltage of 25 kV. In addition, the method was validated by estimating the intra- and interday precision.

The enantioselective potential of two macrocyclic glycopeptide-based chiral stationary phases for analysis of 28 structurally diverse biologically active compounds such as derivatives of pyrovalerone, ketamine, cathinone, and other representatives of psychostimulants and antidepressants was evaluated in sub/supercritical fluid chromatography. The chiral selectors immobilized on 2.7 μm superficially porous particles were teicoplanin (TeicoShell column) and modified macrocyclic glycopeptide (NicoShell column). The influence of the organic modifier and different mobile phase additives on the retention and enantioresolution were investigated. The obtained results confirmed that the mobile phase additives, especially water as a single additive or in combination with basic and acidic additives, improve peak shape and enhance enantioresolution. In addition, the effect of temperature was evaluated to optimize the enantioseparation process. Both columns exhibited comparable enantioselectivity, approximately 90% of the compounds tested were enantioseparated, and 30% out of them were baseline enantioresolved under the tested conditions. The complementary enantioselectivity of the macrocyclic glycopeptide-based chiral stationary phases was emphasized. This work can be useful for the method development for the enantioseparation of basic biologically active compounds of interest.

The misuse of so called novel psychoactive substances is still a challenging problem worldwide. A special attribute of a lot of these compounds is a chiral centre enabling two possible enantiomers probably related to different pharmacological and toxicological properties. The goal of the present study was to present a simple and isocratic HPLC–UV method for enantioseparation of mainly cathinone and pyrovalerone derivatives as well as selected representatives of amphetamines, ketamines, benzofuries, phenidines, phenidates, morpholines and thiophenes. A Waters Acquity UPC2® Trefoil™ CEL1 2.5 µm, 3.0 × 150 mm column served as chiral stationary phase by means of cellulose tris-(3,5-dimethylphenylcarbamate) as chiral selector. Mobile phases consisted either of n-hexane/n-butanol/diethylamine (100:0.3:0.2) or n-hexane/diethylamine (100:0.2). The method was found to be applicable for rapid simultaneous chiral separations of cathinone derivatives, to determine enantiomeric elution orders, to detect positional isomers and to identify real-life samples. Also, a repeatability study was performed successfully. 78 out of 95 compounds were separated in their enantiomers successfully, 51 of them within 6 min. It was shown that all NPS bought from online vendors or seized by police were traded as racemic mixtures.

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as "Legal Highs". These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010-2018. The CSP, namely Lux® 5 μm i-Amylose-1, LC Column 250 x 4.6 mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245 nm and 230 nm, injection volume was 10 μl and flow rate was 1 ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.

New psychoactive substances are defined as compounds with consciousness-changing effects and have been developed simultaneously with classical drugs. They arise through structural modifications of illegal substances and are mainly produced to circumvent laws. Availability is simple, since new psychoactive substances can be purchased from the Internet. Among them many chemical drug compound classes are chiral and thus the two resulting enantiomers can differ in their effects. The aim of this study is to develop a suitable chiral high-performance liquid chromatography separation method for a broad spectrum of new psychoactive substances using cellulose tris(3,5-dichlorophenylcarbamate) as a chiral selector. Experiments were performed by high-performance liquid chromatography in normal-phase mode under isocratic conditions using ultraviolet detection. Direct separation was carried out on a high-performance liquid chromatography column (Lux® i-Cellulose-5, 3.5 μm, Phenomenex®), available since 2016. Excellent separation results were obtained for cathinones. After further optimization, even 47 instead of 39 out of 52 cathinones showed baseline separation. For amphetamine derivatives, satisfactory results were not achieved. Further, new psychoactive substances from other compound classes such as benzofuranes, thiophenes, phenidines, phenidates, morpholines, and ketamines were partially resolved, depending on the polarity and degree of substitution. All analytes, which were mainly purchased from the Internet, were proven to be traded as racemates.

A cellulose tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase was studied as a tool for the enantioselective separation of 21 selected analytes with different pharmaceutical and physicochemical properties. The enantioseparations were performed using supercritical fluid chromatography. The effect of the mobile phase composition was studied. Four different additives (diethylamine, triethylamine, isopropylamine, and trifluoroacetic acid) and isopropylamine combined with trifluoroacetic acid were tested and their influence on enantioseparation was compared. The influence of two different mobile phase co-solvents (methanol and propan-2-ol) combined with all the additives was also evaluated. The best mobile phase compositions for the separation of the majority of enantiomers were CO2 /methanol/isopropylamine 80:20:0.1 v/v/v or CO2 /propan-2-ol/isopropylamine/trifluoroacetic acid 80:20:0.05:0.05 v/v/v/v. The best results were obtained from the group of basic β-blockers. A high-performance liquid chromatography separation system composed of the same stationary phase and mobile phase of similar properties prepared as a mixture of hexane/propan-2-ol/additive 80:20:0.1 v/v/v was considered for comparison. Supercritical fluid chromatography was found to yield better results, i.e. better enantioresolution for shorter analysis times than high-performance liquid chromatography. However, examples of enantiomers better resolved under the optimized conditions in high-performance liquid chromatography were also found.

The worldwide consumption of illicit drugs presents a big problem in terms of health care and prosecution. In the recent years, hundreds of novel psychoactive substances came up and were traded via the Internet, but there is still a big demand for classic illicit drugs such as cocaine, heroin, cannabis and ecstasy. Among these, cocaine particularly is frequently altered not only with excipients, but also with other physiologically active substances. The purpose of this work was to estimate the trend of cocaine purity and abundance of its adulterants in samples seized by Austrian police from 2012 to 2017. A micro-HPLC method for quantification of cocaine and its most common adulterants was developed and validated using gradient elution and UV detection at four wavelengths. 110 cocaine samples were analysed. In all the samples cocaine was present as hydrochloric salt. Caffeine, procaine, levamisole, phenacetin, lidocaine and benzocaine were the most abundant adulterants.

Besides the abuse of well‐known illicit drugs, consumers discovered new synthetic compounds with similar effects but minor alterations in their chemical structure. Originally, these so‐called novel psychoactive substances (NPS) have been created to circumvent law of prosecution because of illicit drug abuse. During the past decade, such compounds came up in generations, the most popular compound was a synthetic cathinone derivative named mephedrone. Cathinones are structurally related to amphetamines; to date, more than 120 completely new derivatives have been synthesized and are traded via the Internet. Cathinones possess a chiral center; however, only little is known about the pharmacology of their enantiomers. However, NPS comprise further chiral compound classes such as amphetamine derivatives, ketamines, 2‐(aminopropyl)benzofurans, and phenidines. In continuation of our project, a cheap and easy‐to‐perform chiral capillary zone electrophoresis method for enantioseparation of cathinones presented previously was extended to the aforementioned compound classes. Enantioresolution was achieved by simply adding native β‐cyclodextrin, acetyl‐β‐cyclodextrin, 2‐hydroxypropyl‐β‐cyclodextrin, or carboxymethyl‐β‐cyclodextrin as chiral selector additives to the background electrolyte. Fifty‐one chiral NPS served as analytes mainly purchased from online vendors via the Internet. Using 10 mM of the aforementioned β‐cyclodextrins in a 10 mM sodium phosphate buffer (pH 2.5), overall, 50 of 51 NPS were resolved. However, chiral separation ability of the selectors differed depending on the analyte. Additionally, simultaneous enantioseparations, the determination of enantiomeric migration orders of selected analytes, and a repeatability study were performed successfully. It was proven that all separated NPS were traded as racemic mixtures.

The enantioseparation of basic compounds represent a challenging task in modern SFC. Therefore this work is focused on development and optimization of fast SFC methods suitable for enantioseparation of 27 biologically active basic compounds of various structures. The influences of the co-solvent type as well as different mobile phase additives on retention, enantioselectivity and enantioresolution were investigated. Obtained results confirmed that the mobile phase additives, especially bases (or the mixture of base and acid), improve peak shape and enhance enantioresolution. The best results were achieved with isopropylamine or the mixture of isopropylamine and trifluoroacetic acid as additives. In addition, the effect of temperature and back pressure were evaluated to optimize the enantioseparation process. The immobilized amylose-based chiral stationary phase, i.e. tris(3,5-dimethylphenylcarbamate) derivative of amylose proved to be useful tool for the enantioseparation of a broad spectrum of chiral bases. The chromatographic conditions that yielded baseline enantioseparations of all tested compounds were discovered. The presented work can serve as a guide for simplifying the method development for enantioseparation of basic racemates in SFC.

The screening part of an earlier defined chiral separation strategy in capillary electrochromatography (CEC) was used for the separation of ten cathinone- and amphetamine derivatives. They were analyzed using 4 polysaccharide-based chiral stationary phases (CSPs), containing cellulose tris(3,5-dimethylphenylcarbamate) (ODRH), amylose tris(3,5-dimethylphenylcarbamate) (ADH), amylose tris(5-chloro-2-methylphenylcarbamate) (LA2), and cellulose tris(4-chloro-3-methylphenylcarbamate) (LC4) as chiral selectors. After applying the screening to each compound, ADH and LC4 showed the highest success rate. In a second part of the study, a comparison between CEC and other analytical techniques used for chiral separations i.e., supercritical fluid chromatography (SFC), polar organic solvent chromatography (POSC), reversed-phase (RPLC) and normal-phase liquid chromatography (NPLC), was made. For this purpose, earlier defined screening approaches for each technique were applied to separate the 10 test substances. This allowed an overall comparison of the success rates of the screening steps of the 5 techniques for these compounds. The results showed that CEC had a similar enantioselectivity rate as NPLC and RPLC, producing the highest number of separations (9 out of 10 racemates). SFC resolved 7 compounds, while POSC gave only 2 separations. On the other hand, the baseline separation success rates for NPLC and RPLC was better than for CEC. For a second comparison, the same chiral stationary phases as in the CEC screening were also tested with all techniques at their specific screening conditions, which allowed a direct comparison of the performance of CEC versus the same CSPs in the other techniques. This comparison revealed that RPLC was able to separate all tested compounds, and also produced the highest number of baseline separations on the CSP that were used in the CEC screening step. CEC and NPLC showed the same success rate: nine out of ten substances were separated. When CEC and NPLC are combined, separation of the ten compounds can be achieved. SFC and POSC resolved eight and three compounds, respectively. POSC was the least attractive option as it expressed only limited enantioselectivity toward these compounds.

Novel Psychoactive Substances (NPS) represent an alternative to established illicit drugs. They are traded via the internet and exhibit small alterations in their chemical structure to circumvent law, however, their psychotropic effects are comparable. There is still poor knowledge about side effects and health risks. By the end of 2018, 730 NPS were reported to EMCDDA (European Monitoring Centre for Drugs and Drug Addiction). Among different compound classes, many NPS are chiral and few publications deal with the different pharmacological and toxicological properties of their pure enantiomers. Therefore, analytical method development concerning enantioseparation of NPS is of great interest. Chiral separation protocols of established illicit drugs have been transferred for NPS, selected examples are given as well. Different methods for enantioseparation of NPS comprising mainly stimulating drugs such as cathinones, pyrovalerones, amphetamines, ketamines, (2-aminopropyl) benzofuranes, phenidines, phenidates, morpholines and thiophenes are reviewed. Moreover, chiral resolution of some cannabinomimetics by HPLC is presented. Chromatographic and electrophoretic techniques such as GC, HPLC, SFC, CE and CEC are discussed and in some cases compared. Mainly, solid samples either purchased from internet vendors, seized by police or collected from patients in hospitals are subject to analysis. Chiral selectors used for HPLC are listed in a Table. It was shown that particularly stimulating drugs are traded as racemic mixtures, which is not the case with cannabinomimetics. Mainly, HPLC and CE were used for enantioseparation of NPS.

Recently, novel psychoactive drugs for human abuse such as amphetamines, phenethylamines, benzofuries, and tryptamines, cathinones have gained high popularity. These designer drugs are mainly sold via online stores as “bath salts” and are labeled “not for human consumption.” Due to the novelty of the compounds, only a little information about pharmacology, toxicology, and the long-term damage they may cause is available. Moreover, there are only few analytical methods for their identification and analysis. Among new cathinone derivatives, 1-(3,4-dimethoxyphenyl)-2-(ethylamino)pentan-1-one (DL-4662), became available via an internet shop. A sample of this compound was purchased and investigated. The first aim of our study was an identity check by NMR spectroscopy and gas chromatography with mass spectrometry. As many of the recreational drugs are chiral and are mainly sold as racemates, a further goal of our research was enantioseparation by gas chromatography with mass spectrometry and high-performance liquid chromatography with UV detection, to prove whether DL-4662 was traded enantiomerically pure or as racemic mixture. Both chiral separation methods showed the presence of a racemate.

Introduction Cannabis sativa L. is a powerful medicinal plant and its use has recently increased for the treatment of several pathologies. Nonetheless, side effects, like dizziness and hallucinations, and long-term effects concerning memory and cognition, can occur. Most alarming is the lack of a standardised procedure to extract medicinal cannabis. Indeed, each galenical preparation has an unknown chemical composition in terms of cannabinoids and other active principles that depends on the extraction procedure. Objective This study aims to highlight the main differences in the chemical composition of Bediol® extracts when the extraction is carried out with either ethyl alcohol or olive oil for various times (0, 60, 120 and 180 min for ethyl alcohol, and 0, 60, 90 and 120 min for olive oil). Methodology. Cannabis medicinal extracts (CMEs) were analysed by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC–MS/MS) using an untargeted metabolomics approach. The data sets were processed by unsupervised multivariate analysis. Results Our results suggested that the main difference lies in the ratio of acid to decarboxylated cannabinoids, which dramatically influences the pharmacological activity of CMEs. Minor cannabinoids, alkaloids, and amino acids contributing to this difference are also discussed. The main cannabinoids were quantified in each extract applying a recently validated LC–MS and LC-UV method. Conclusions Notwithstanding the use of a standardised starting plant material, great changes are caused by different extraction procedures. The metabolomics approach is a useful tool for the evaluation of the chemical composition of cannabis extracts. Copyright © 2017 John Wiley & Sons, Ltd.

Application of the superficially porous particles (SPPs) grafted with chiral selectors can substantially improve resolution in chromatographic techniques. In this work, we carried out a deeper study on supercritical fluid chromatography systems with 2.7 µm SPPs bonded with teicoplanin and vancomycin. Fast separations of the majority of enantiomers of phytoalexins, substituted tryptophans, and ketamine derivatives, as representatives of important biologically active and structurally diverse chiral compounds have been achieved. The chromatographic behavior of the structurally different analytes served to characterize these separation systems. The influence of separation conditions, namely mobile phase composition, i.e. type of co-solvent and additive on retention, enantioselective resolution and enantioselectivity was examined. The success rate of baseline and partial separations in individual groups of compounds differed with the chiral stationary phase and also with mobile phase composition. The best, baseline separations for the phytoalexins were achieved on the TeicoShell column using methanol as a co-solvent and trifluoroacetic acid as an additive if used. Mostly partial separations were achieved on the vancomycin-based column for all groups of analytes. Complementary separation behavior of these CSPs was confirmed for the majority of the chiral compounds examined in this work.

The abuse of Novel Psychoactive Substances (NPS) still cause severe problems. A lot of them contain a stereogenic centre which leads to possible different pharmacological effects of the enantiomers. The aim of this study was to establish an enantioselective HPLC-UV method with applicability to a broad spectrum of NPS. Different compound classes including mainly amphetamines, cathinones, ketamines, pyrovalerones and benzofuries were tested for successful enantioseparation by one common chromatographic condition. All NPS samples were bought in various internet stores, were sythesized inhouse or were seized by the Austrian police. A commercially available Phenomenex LUX® AMP 3 μm, 150 × 4,6 mm column served as chiral stationary phase (CSP) for the enantiomeric separation experiments. This CSP has been developed particularly for enantioresolution of high abundant synthetic chiral drugs such as MDMA and amphetamine. All measurements were performed under isocratic conditions. For method optimisation, the effects of different mobile phase compositions, different pH values and temperatures on enantioseparation were investigated. Final mobile phase consisted of ammonium bicarbonate solution (5 mM) adjusted with concentrated ammonia to a pH of 11.3 mixed with acetonitrile in a ratio of 70:30. All in all, 83 of 95 analysed NPS were separated in their enantiomers successfully within 40 min. Furthermore, the method was found to be suitable for simultaneous enantioseparations, for enantiomeric elution order determinations, enantiomeric purity checks and for positional isomer separations. To prove the repeatability of the method, an intra- and an interday validation was performed successfully. By means of the wide applicability of the chosen column all separated NPS were shown to be traded as racemic mixtures.

New recreational drugs such as amphetamine-, cathinone, and benzofury derivatives gained high popularity on the drug market in recent years. They can be purchased via the Internet from different providers and online portals. Most of these compounds are chiral, which makes the development of chiral separation methods necessary. Besides this, it is useful to find out if the compounds were sold as racemic mixtures. Also, it is important to check whether the new psychoactive compounds contain further ingredients or impurities. The aim of this research was the continuation of the application of a method for indirect chiral separation of 24 new psychoactive compounds recently purchased via the Internet. After derivatization with the chiral derivatization reagent trifluoroacetyl-L-prolyl chloride, chromatographic separation of diastereomers was achieved using a 30 m HP5-MS capillary column. As carrier gas, helium was used with a constant flow of 1.0 ml/min. Three different column temperature programs were tested. Under optimum conditions 13 out of 24 compounds were successfully resolved into their enantiomers obtaining Rs values up to 7.0. The use of a single quadrupole mass spectrometer as the detector allowed the identification of the compounds in multicomponent samples. Chirality 27:211–215, 2015. © 2014 Wiley Periodicals, Inc.

Derivatized polysaccharide-based columns have high enantiodiscrimination potential mainly in normal phase separation mode. In this work chiral recognition ability of four immobilized polysaccharide-derived chiral stationary phases was evaluated under reversed phase conditions. A set of 30 chiral compounds, particularly drugs possessing various functional groups was used for testing. Baseline enantioseparation was achieved for 17 of them. In general, amylose-based chiral stationary phases showed higher enantioselectivity than the cellulose-based ones, mainly for acidic and bifunctional compounds. The influence of the type and pH of the aqueous mobile phase constituents as well as the role of the organic modifier on the enantioselective separation ability of the stationary phases were also investigated and compared. Complementary separations were obtained on the amylose- and cellulose-based columns. The immobilized polysaccharide-based chiral stationary phases were shown to be useful tool for the enantioseparation of a broad spectrum of chiral analytes in reversed phase separation mode.

In this study, a chiral CEC method for the enantiomeric separation of ten cathinone derivatives, by means of a polysaccharide-based chiral stationary phase, has been developed. Capillary columns of 100 μm id packed with amylose tris(5-chloro-2-methylphenylcarbamate) coated on silica, also called Sepapak 3 or Lux Amylose-2, were used to achieve the enantioseparation of the studied designer drugs. Enantioresolution, chromatographic retention, and separation efficiency were evaluated in dependence of mobile-phase composition in terms of the content of the organic modifier, nature, and pH buffer. To obtain a sensitivity improvement, a field-amplified sample injection was evaluated optimizing the sample solvent composition and injection time. The LODs and LOQs values were in the range 25-100 and 50-150 ng/mL, respectively, for all the racemic compounds. Good results in terms of resolution (Rs ), separation efficiency (N/m), and short analysis times were obtained using a mixture of ACN/methanol/sodium acetate pH 9 (89/10/1, v/v/v). Applying a voltage of 10 kV and a temperature of 20°C, the analyzed cathinone derivatives were separated in their enantiomers in less than 10 min. A study, concerning the method precision, in terms of intra- and interday repeatability and column-to-column reproducibility was carried out in accordance with the analytical procedures for method validation. Intra- and interday repeatability provided RSD values in the ranges 1.1-1.7, 1.3-2.3% for retention time and 1.3-2.6, 2.1-3.4% for peak area, respectively.

In the last years the identification of new legal and illegal highs has become a huge challenge for the police and prosecution authorities. In an analytical context, only a few analytical methods are available to identify these new substances. Moreover, many of these recreational drugs are chiral and it is supposed that the enantiomers differ in their pharmacological potency. Since nonenantioselective synthesis is easier and cheaper, they are mainly sold as racemic mixtures. The goal of this research work was to develop an inexpensive method for the chiral separation of cathinones and amphetamines. This should help to discover if the substances are sold as racemic mixtures and give further information about their quality as well as their origin. Chiral separation of a set of 6 amphetamine and 25 cathinone derivatives, mainly purchased from various Internet shops, is presented. A LiChrospher 100 RP-18e, 250 x 4 mm, 5 µm served as the stationary phase. The chiral mobile phase consisted of methanol, water, and sulfated s-cyclodextrin. Measurements were performed under isocratic conditions in reversed phase mode using UV detection. Four model compounds of the two substance classes were used to optimize the mobile phase. Under final conditions (methanol:water 2.5:97.5 + 2% sulfated s-cyclodextrin) enantiomers of amphetamine and five derivatives were baseline separated within 23 min. In all, 17 cathinones were completely or partially chirally separated. However, as only 3 of 25 cathinones were baseline resolved, the application of this method is limited for cathinone analogs. Additionally, the results were compared with an RP-8e column. Chirality 26:411–418, 2014. © 2014 Wiley Periodicals, Inc.

5-Hydroxymethylfurfural (5-HMF) is a natural occurring substance taken up by everyday food. In former studies it was shown that 5-HMF is completely decomposed in the body after oral or intravenous application resulting in three main metabolites named 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine, and possibly a forth metabolic substance, termed 5-sulphoxymethylfurfural, is formed. Determination is possible via HPLC using a hydrophilic interaction chromatography (HILIC) column with an appropriate gradient system (ACN/ammonium formate 100 mM, pH 2.35). Urine samples were purified by use of an SPE method beforehand working with ScreenA cartridges. This cleaning procedure was validated based on ICH guidelines in terms of linearity, quantification, and detection limit, as well as precision, repeatability, and accuracy. Analysis of real-life samples coming from two healthy probands and one cancer patient, who all received 240 mg 5-hydroxymethylfurfural orally once a day, showed dicarboxylic acid and the glycine conjugate in their urine samples. Recovery of the initial compound in form of transformed metabolites was up to 90% within 48 h. Potentially toxic 5-sulphoxymethylfurfural could not be found.

This review gives an overview about chiral separation by capillary electrophoresis and capillary electrochromatography using different chiral metal-ion complexes. The topic enantioseparation is still of big interest for chiral drugs and natural compounds. Regarding chiral drugs it is often the case that the enantiomers differ in activity. The chiral separation principle of ligand-exchange (LE) can be enabled for liquid chromatography, capillary electrophoresis as well as for capillary electrochromatography. Ligand-exchange can be applied particularly for chiral amino acids, amino alcohols or α-hydroxy acids. Examples and applications are given along with the latest developments.

The food component 5-hydroxymethylfurfural is supposed to have antioxidative properties and is therefore used as an acting agent in a novel anticancer infusion solution, named Karal®, and an oral supplementation. Previous studies showed that after oral and intravenous application, the substance is completely decomposed to its metabolites: 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine. The formation of a fourth metabolite, namely 5-sulphoxymethylfurfural, is still not clarified according to literature. Due to commercial unavailability, synthesis of 5-sulphoxymethylfurfural was conducted and a synthesis procedure for N-(hydroxymethyl)furoyl glycine had to be developed. Identification of the synthesised compounds was proven by LC-MS and NMR. An appropriate HPLC method was established to obtain good separation of the four possible metabolic substances and 5-hydroxymethylfurfural within 12 min via a HILIC column (150 × 4.6 mm, 5 μm) using a gradient grade system switching from mobile phase A (ACN/ammonium formate 100 mM, pH 2.35, 95:5, v/v) to mobile phase B (ACN/ammonium formate 100 mM, pH 2.35, 85:15, v/v). The procedure was afterward validated following ICH guidelines in terms of selectivity, linearity, precision, LOD, and LOQ.

Cathinone derivatives gained high popularity on the recreational drugs market during the past 10 years. All these compounds are chiral, and the pharmacological potency of the enantiomers of these stimulants is supposed to differ. The goal of this research was to develop a reliable and easy-to-perform high-performance liquid chromatography ultraviolet method for the chiral separation of a set of 24 cathinone derivatives. A commercially available CHIRALPAK® AS-H column consisting of amylose tris [(S)-α-methylbenzylcarbamate] coated on 5-µm silica gel was found to be suitable to resolve a majority of the tested compounds. High-performance liquid chromatography measurements were performed in normal phase mode under isocratic conditions with a mobile phase consisting of hexane, isopropanol, and triethylamine at a flowrate of 1 ml/min. The ratio between hexane and isopropanol was optimized by means of three model substances. Under final conditions with a mobile phase of hexane, isopropanol, and triethylamine (97:3:0.1), 19 out of 24 compounds were successfully resolved into their enantiomers and detected at a wavelength of 254 nm. A correlation between the substituents of the nitrogen atom and the separation results are shown. Furthermore, enantiomer separation results of four cathinone derivatives were compared with the results of their amphetamine analogs.

The principle of chiral ligand-exchange, introduced by Davankov, has become a widely used technique for chiral separation in both chromatography and in electromigration techniques. This simple technique makes use of the formation of mixed metal chelate complexes between a chiral selector and both enantiomers of an analyte. In HPLC, the chiral selector can be either bonded to the stationary phase or added to the mobile phase. In CE the chiral selector is simply added to the electrolyte. A relatively new approach represents CEC, where capillaries contain a chiral stationary phase. More than thousand papers appeared in the field of chiral ligand-exchange. To cite all papers would require several books.The present article gives an overview of both milestones and our activities on chiral separation using the principle of ligand-exchange. Recent advances in chip technology for chiral separations and new approaches regarding improvement of detection sensitivity are mentioned.

This article gives a short overview of the application of the principle of chiral ligand-exchange in HPLC, CE, and CEC. Since its introduction by Davankov, more than thousand articles have appeared in this field. Citing all these papers would extend the scope of this review-it would fill several books. Therefore only some milestones are mentioned in this article and it will focus on our own activities in this field. Some new developments are mentioned, and selected biochemical and biomedical application are presented.

A new chiral stationary phase based on continuous bed (CB) technology using L-prolinamide as a chiral selector was prepared. Its ability for enantioseparation of amino acids and alpha-hydroxy acids by ligand-exchange CEC was compared with that of a CB containing L-4-hydroxyproline as chiral selector. Preparation was done by a one-step in situ copolyermerization procedure using methacrylamide as monomer, vinylsulfonic acid as charge providing agent and piperazine diacrylamide as crosslinker. L-Prolinamide showed marked enantioselective properties for the separation of amino acids and alpha-hydroxy acids. Furthermore, we show the possibility of simultaneous enantioseparation of amino acids and alpha-hydroxy acids using the CB with L-4-hydroxyproline as chiral selector.

This work deals with the application of silica-based ligand-exchange chiral stationary phases (CSPs) for the enantioseparation of underivatized amino acids, alpha-hydroxy acids, and dipeptides with packed CEC. Two different possibilities of preparing silica-based CSPs are presented. One phase contains L-4-hydroxyproline chemically bonded via a spacer to 3 mum silica material. The other approach makes use of N-decyl-L-4-hydroxyproline dynamically coated on a reversed-phase packed capillary. Dynamical coating of reversed-phase material represents a simple alternative to prepare CSP. A comparison of the chemically bonded phase with the dynamically coated CSP by means of resolution of complex-forming analytes is presented. The chemically bonded phase was found to be superior to the dynamically coated phase in terms of resolution of amino acids and dipeptides. However, the dynamically coated CSP was found to be especially suitable for the separation of alpha-hydroxy acids. Both techniques are applicable for enantiomer purity tests.

This review gives an overview of chiral separation principles and their applications in capillary electromigration techniques. The basic chiral separation principles are explained and the mechanisms discussed. Recent developments and new techniques in CZE and capillary electrochromatography (CEC) are highlighted. New chiral selectors among cyclodextrins, crown ethers, carbohydrates, macrocyclic antibiotics, proteins, chiral ion-pairing reagents, chiral surfactants and chiral metal ion complexes and their chiral recognition ability are discussed. Recent advances in chip technology for chiral separation and new approaches regarding improvement of detection sensitivity are presented. Due to the tremendous number of publications dealing with applications, in this review only recent applications are summarized.

This paper deals with comparative studies on the use of copper(II), cobalt(II), nickel(II) and zinc(II) complexes of d-gluconic acid, d-saccharic acid and l-threonic acid as chiral selectors for the enantioseparation of aromatic amino acids and glycyl dipeptides using the principle of ligand-exchange capillary electrophoresis. Although copper(II) is the most frequently used central ion in ligand-exchange capillary electrophoresis, in the case of d-gluconic acid cobalt(II) was shown to be an alternative for the enantioseparation of amino acids. Glycyl dipeptides, however, were resolved only with copper(II) complexes. Zn(II) as a central ion was not effective in all cases and with Ni(II) only some partial separations were achieved.

This paper deals with the use of Cu(II) complexes of L-tartaric acid or L-threonine as selectors for the chiral separation of drugs containing amino alcohol structure by ligand-exchange CE. Using Cu(II) ions as a complexing agent, a series of sympathomimetics and beta-blockers were resolved. It was found that the resolution strongly depends on selector concentration and pH. The optimum pH for complexation was 12.

This review gives an overview of recent developments in CZE, EKC, and CEC covering the literature since the year 2004. Since there appeared a special issue on applications, this review focuses on the progress in electromigration techniques and new methodological developments. New techniques, new chiral selectors as well as new chiral stationary phases for CEC are discussed.

This work deals with the application of BSA and canine serum albumin (CSA) for enantioseparation of tryptophan derivatives with CE. The aim of this work was the investigation of the influence of different functional groups of tryptophan derivatives on enantioseparation. CSA as a chiral selector was tested to compare its selector properties with those of BSA. The enantiomers of the tryptophan derivatives were separated by adding BSA or CSA to the BGE. The influence of pH, temperature, BSA and CSA concentration and organic modifiers was investigated. It was found that the stereoselectivity for the different tryptophan derivatives is dependent on the albumin species. It turned out that the different functional groups of the derivatives showed a significant influence on stereoselectivity.

Three flavonolignans derived from the flavone tricin were isolated from Avena sativa herb. This is the first report of the presence of flavonolignans in A. sativa. In the known compounds 1a and 1b, a coniferyl alcohol moiety is linked to the flavone by an ether bond; in the new natural product 2, it is linked by C-C bonds. Structure elucidation of compound 2 was performed by 1D and 2D NMR experiments, and the absolute configuration was determined from circular dichroic data.

Fritless particle-loaded monoliths for chiral capillary electrochromatographic (CEC) separation were prepared. Silica particles containing a chiral selector are suspended in a monomer solution, which is drawn into the capillary followed by in situ polymerization. Thereby the silica-based particles containing the chiral selector are embedded in a nonchiral continuous bed. This kind of chiral stationary phase is inexpensive, easy, and reproducible to prepare and circumvents the preparation of frits. As a model, teicoplanin aglycone as chiral selector bonded to 3 microm silica particles was used. The applicability of this approach is demonstrated by means of the chiral separation of aliphatic and aromatic amino acids and dipeptides. As a further application, the chiral selector ristocetin A bonded to 3 microm silica particles was used for the enantiomeric separation of chiral alpha-hydroxy acids. Since alpha-hydroxy acids migrate toward the anode, a cationic charge-providing agent was copolymerized with the matrix. This served to reverse the direction of the electroosmatic flow (EOF).

A method for the quantitative determination of S‐adenosyl‐L‐methionine (SAM) in dietary supplement preparations using capillary zone electrophoresis (CZE) is described. Linearity was observed in a concentration range of 10–200 µg mL−1 with r = 0.9997. The limit of detection was 0.5 µg and the limit of quantification 2 µg. The intra‐day and the inter‐day precision was in the range of 0.52–0.89 and 1.15–1.60% RSD, respectively. The mean recovery determined by spiking with known amounts of SAM was 97.9%. Furthermore, stability investigations of SAM were carried out using this method.

Chiral separation of amino acids, drugs with amino alcohol structure, and other drugs has been investigated by capillary electrochromatography (CEC), pressure supported CEC, and micro-HPLC on a teicoplanin aglycone stationary phase. The teicoplanin aglycone lacks the D-glucosamine and the D-mannose moieties and shows different separation behavior compared to a teicoplanin stationary phase. The selector is bonded to 3.5-μm silica gel and separations are carried out either in the reversed phase or the polar organic mode. This chiral stationary phase (CSP) shows high enantioselectivity especially for amino acids, e. g. for DOPA a Rs value of 10.5 was achieved. Although the chiral recognition ability for drugs is lower than that of intact teicoplanin, out of 22 β-blockers investigated, 16 are baseline separated by CEC.

The dependence of the chiral recognition ability and enantiomer migration order on the structure, substitution pattern and chirality of chiral selectors used in ligand-exchange capillary electrophoresis is investigated. As chiral selectors different N-alkyl derivatives of proline and hydroxyproline as their copper(II) complexes are used. The influence of the position and conformation of the hydroxy group in the hydroxyproline derivatives and of the structure and chirality of the side chain on enantioselectivity is investigated. Furthermore, the effect of surfactants such as sodium dodecyl sulfate and cetyl trimethyl ammonium bromide on resolution and enantiomer migration order is studied. The investigations were carried out with three aromatic amino acids as model compounds.

This study deals with the development of enantioselective flow-through immunosensors for triiodothyronine (T 3) and tetraiodothyronine (thyroxine, T4) on the basis of a competitive assay using enantioselective antibodies. The instrumental set-up is based on a simple sequential-injection system equipped with a chemiluminescence detector and an immunoreactor, which consists of a flow-cell packed with immobilized haptens. As haptens, 4-amino- l-phenylalanine (4-amino-l-Phe), 4-amino-d-Phe or l-T3 were used. Antibodies directed against 4-amino-l -o rd-Phe or l-T3 were labeled with an acridinium ester. Three different approaches for immobilizing the haptens were investigated including simple adsorption on polystyrene, chemical binding to an activated methacrylate polymer and binding via the biotin–streptavidin binding (BSB) system. The latter approach showed the best results regarding repeatability and sensivity. Using biotinylated l-T3 immobilized onto a streptavidin-derivatized trisacryl support and labeled anti- l-T3 antibodies, a detection limit of 15.5 fmol/ml for l-T3 was obtained. One assay cycle including regeneration takes only about 5 min. This approach was applied to detect l-T3 in plasma samples without any sample pre-treatment. The average recovery from spiked plasma sample was about 93% with a R.S.D. below 5%. © 2002 Elsevier Science B.V. All rights reserved.

The illicit drug market of psychoactive substances for human abuse is continuously expanding and developing. Besides already known substance classes like cathinones, amphetamines or synthetic cannabinoids, further derivatives such as benzofurys, thiophenes, and structural analogues of methylphenidate entered the global market recently. As many of these new compounds contain a stereogenic centre it is supposed that their isomers may differ in their pharmacological effects as it is the case with amphetamines or several chiral active pharmaceutical ingredients, for instance. In the course of this study, a method for the chiral separation of a set of 16 recreational drugs by CE was developed. The aim was to separate the analytes into their enantiomers at equal conditions within short time. Sulfobutylether β-cyclodextrin served as chiral selector in an aqueous ammonium acetate solution containing ACN. For method optimization, methedrone and ethylphenidate were used as model compounds to find the appropriate concentration of chiral selector. Moreover, the influence of the pH value on enantioseparation was tested. Fourteen or 16 mM sulfobutylether β-cyclodextrin, 50 mM ammonium acetate solution (pH 4.5) with 10% ACN were found to be optimal for enantioseparation of seven benzofurys, four cathinones, two diphenidines, ethylphenidate, methiopropamine, and thiothinone. Most of them were baseline resolved at migration times below 25 min.

This review gives a survey of different chiral separation principles and their use in high-performance liquid chromatography (HPLC), gas chromatography (GC), supercritical fluid chromatography (SFC), thin-layer chromatography (TLC), capillary electrophoresis (CE) and capillary electrochromatography (CEC) highlighting new developments and innovative techniques. The mechanisms of the different separation principles are briefly discussed and some selected applications are shown.

The chiral separation of β-blockers on cellulose tris (3,5-dimethylphenylcarbamate) columns is described. The separation power of a Chiracel OD normal phase column, a Chiracel OD-RH reversed phase column, and a Chiracel ODRH reversed phase narrow-bore column were compared. Hexane–2-propanol–diethylamine mixtures were used as mobile phase in the normal phase mode and sodium perchlorate buffer containing different amounts of acetonitrile in the reversed phase mode. Sixteen μ-blockers were resolved under normal phase conditions and eleven under reversed phase conditions. The values of α and Rs on normal phase column ranged from 4.46 to 1.10 and 10.04 to 0.97, respectively. The values of α and Rs on reversed phase columns varied from 4.04 to 1.04 and 3.98 to 0.40(on normal bore column) and 4.36 to 1.06 and 2.26 to 0.40(on narrow bore column).

We report on the chiral separation of pharmacologically active dihydropyrimidinones by capillary electrophoresis (CE) using carboxymethyl-beta-cyclodextrin as chiral selector. The influence of selector concentration, pH, and the addition of varying amounts of methanol is investigated. Out of 21 compounds investigated, 19 were resolved, 13 with baseline separation.

The state of art in chemiluminescence flow-injection immunoassays is reviewed. Different approaches and manifolds are described. Advantages of chemiluminescence flow-injection immunoassays, which are discussed in detail, are speed, selectivity, and sensitivity. The major benefit of such systems is the simple and economical set up, which is easily adapted to automation.

This paper deals with the enantioseparation of hydroxy acids by ligand-exchange capillary electrochromatography. A chiral continuous bed was easily prepared by in situ polymerization of monomers, including an L-4-hydroxyproline derivative. This phase showed chiral recognition for several hydroxy acids, in addition to amino acids.

A method for determining the enantiomers of 10 therapeutically relevant aminoalcohols using HPLC and precolumn derivatization was developed. Naphthyl isocyanate reacted with racemic aminoalcohols to form urea derivatives which were separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column, and detected fluorometrically in the lower ng mL(-1) range. The effluents can also be monitored at lower sensitivity, using an ultraviolet detector operated at 220 nm.

The chiral resolution of 1-[(benzofuran-2-yl)-5-nitrophenylmethyl] tetrazole (I) and 1-[(benzofuran-2-yl)-5-bromophenylmethyl] tetrazole (II), the novel aromatase inhibitors, has been achieved on Chiracel OD, Chiracel OD-RH columns which are coated with cellulose [tris (3,5-dimethylphenyl carbamate)]and Chiracel OJ-R column coated with cellulose [tris(4-methyl benzoate)]. The mobile phases used were hexane-2-propanol (95:5) on Chiracel OD and water-acetonitrile(45:55) on Chiracel OD-RH and Chiracel OJ-R columns respectively. Compound I has been resolved on Chiracel OD(α = 1.30 and Rs = 3.13) and OD-RH (α = 1.15 and Rs = 1.20)columns while compound II has been resolved on OJ-R(α = 1.31 and Rs = 1.80) column only. The effect of acetonitrile concentration in water-acetonitrile mobile phase was studied on the retention and resolution of both compounds.

The chiral separation of several thiobarbiturates on a tris(4-methylbenzoate) phase (Chiralcel OJ-R, 15 × 0.46 cm and Chiralcel OJ-R 15 × 0.20 cm) under reversed-phase conditions is described. The separation properties of a normal sized and a newly developed narrow-bore column were compared. The influence of buffer and organic modifiers on enantioselectvity was investigated. The three mobile phases used were water/acetonitrile (70:30,v/v), water/acetonitrile/methanol (50:25:25, v/v), and water/acetonitrile/methanol (55:15:30, v/v). Of seven thiobarbiturates investigated, five have been resolved. The values of α (1.11–1.85) and R s (0.71–5.75) have been reported.

This review summarizes recent developments in the field of chiral separations by electromigration techniques including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), isotachophoresis (ITP), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC). This overview focuses on the development of new chiral selectors and the introduction of new techniques rather than applications of already established selectors and methods. The mechanisms of the different chiral separation principles are discussed.