The chiral separation of alkaloids in natural product was conducted by direct method which is based on diastereomer formation by using a chiral stationary phase (CSP) (Haginaka 2002). HPLC using CSPs has demonstrated to be extremely useful, accurate, versatile, and it has been a widely used technique in diverse fields and applications, emphasizing (Table 1). The CSP mode is generally the most straightforward and convenient means for chromatographic enantiomer separation; it is the method of choice for both analytical and preparative applications (Lämmerhofer 2010; Kalíková et al. 2011; Scriba 2016; Fernandes et al. 2017). There are more than a hundred commercially available CSPs that have been developed over the past thirty years, (Ali et al. 2017) not to mention the even larger number of “home-made” CSPs. HPLC CSPs are classified by Lammerhofer into nine major types according to the interactions between CSPs and analytes (Lammerhofer et al. 2010).

Polysaccharide selectors have a long tradition in enantioselective liquid chromatography. In 1973, Hesse and Hagel introduced microcrystalline cellulose triacetate (MCTA) as a polymeric selector material (without supporting matrix) for enantioselective liquid chromatography. While MCTA exhibits widely applicable enantio-recognition and favorable loading capacities for preparative separations, it suffers from poor pressure stability, slow separations, and low chromatographic efficiency. A solution to the mechanical stability problem of MCTA was proposed by Okamoto and co-workers in 1984. The cellulose derivatives were coated at about 20 wt% onto the surface of macro-porous silica beads (100 or 400 nm pore size). These materials exhibited considerably improved mechanical stability and much better efficiencies, and permitted HPLC enantiomer separations. Such coated polysaccharide-based CSPs were state-of-the-art for several decades (Lammerhofer et al. 2010).

Until now, they have prepared about 200 kinds of polysaccharide derivatives based on different polysaccharides including cellulose, amylose, chitin, chitosan, galactosamine, curdlan, dextran, xylan, and inulin (Fig. 2) (Aboul-Enein et al. 2003). These derivatives have been coated on a macro-porous silica gel as CSPs, and their chiral recognitions were then evaluated by HPLC.

Figure 2. 

Structures of the various kinds of polysaccharides: (1) Cellulose; (2) Amylose; (3) Chitin; (4) Chitosan; (5) Galatosamine; (6) Curdlan; (7) Dextran; (8) Xylan; (9) Inulin.

The enantioselectivity and the elution order of the various enantiomers differed among these polysaccharides depending on the sugar units, linkage position, and linkage type. Among them, the derivatives of cellulose and amylose usually exhibit higher recognition abilities than the others, although it depends on the structure of a specific racemate. Triesters and tricarbamate are the most useful and successful cellulose and amylose’s derivatives. It has been claimed by Aboul-Enein and Ali that for the resolution of about 500 test racemates, about 80% of them have been successfully resolved on only two kinds of polysaccharide derivative-based CSPs (cellulose and amylose tris (3,5-diphenylcarbamate) CSPs) (Aboul-Enein et al. 2003). The application of coated polysaccharide-derived CSPs has been reviewed in Table 2 including the names of 20 CSPs and their most frequent applications. More specifically, three famous commercially available CSPs, CHIRALCEL OD, OJ, and CHIRALPAK AD, have fully or partially resolved 70% racemates among over 100 racemates tested (Lammerhofer et al. 2010).

Chiral analysis is one of the main fields of the chemical quality control of pharmaceuticals in which CE plays a pivotal role (Pellati et al. 2007). Because of the versatility and the high separation power, capillary electrophoresis (CE) meets the requirements for the herbal drugs’s quality control. The wide opportunity for selectivity tuning allows the analysis of molecules with a wide range of polarity and molecular weight (Gotti 2011). CE is considered a miniaturized and environmentally friendly technique, as it requires very small volumes of sample and solvents (Xie et al. 2010). Moreover, HPLC chromatographic chiral columns are quite expensive, while CE does not require any column. Additionally, CE provides high efficiency, versatility, simplicity, and feasibility enabling fast method development and high success rate, since the type and the concentration of the chiral selector can be easily optimize (Fejos et al. 2020), which is impossible in HPLC where, in general, chiral stationary phases (CSP) are usually used for chiral analysis. Also, different chiral selectors can be combined in CE, whereas this is a difficult approach in chromatographic techniques (Saz et al. 2016).

A particular mode of fast capillary zone electrophoresis (CZE) is non-aqueous capillary electrophoresis (NACE), which employ non aqueous buffer system. Using organic solvents instead of water not only helps increase the hydrophobic analytes solubility but also improves selectivity. Actually, NACE widened the set of physicochemical characteristics of the solvents, which affect the electrophoretic characteristic by their influence on solute–solvent, solute–additive and ion–ion interactions. Most importantly, pKa values of basic analytes in organic solvents are notable different from those in water allowing separations which are difficult to be obtained in water. Moreover, NACE can be ideally suited for online coupling with mass spectrometry thanks to the high volatility and low surface tension of many organic solvents. A chiral microemulsion electrokinetic chromatography method has been developed to split up the enantiomers of the phenethylamines ephedrine, N-methylephedrine, norephedrine, pseudoephedrine, adrenaline (epinephrine), 2-amino-1-phenylethanol, diethylnorephedrine, and 2-(dibutylamino)-1-phenyl-1-propanol, respectively. The separations were achieved using an oil-in-water microemulsion consisting of the oil-component ethyl acetate, the surfactant sodium dodecylsulfate, the cosurfactant 1-butanol, the organic modifier propan-2-ol and 20 mM phosphate buffer pH 2.5 as aqueous phase. For enantio-separation sulphated -cyclodextrin was added. The developed method was successfully applied to impurity analysis (Borst et al. 2010). Electrokinetic chromatography (EKC), and in particular micellar electrokinetic chromatography (MEKC) has been introduced in 1984 by Terabe and proved to be not only the method of choice in analysis of neutral compounds but also one of the most versatile separation approach among the electromigration methods. In MEKC a surfactant (often sodium dodecyl sulfate, SDS) is introduced into the background electrolyte (BGE) at concentration above the critical micelle concentration in order to generate a micellar pseudo-stationary phase. Separation mechanism is a combination of chromatographic partitioning of solutes between pseudo-stationary phase and continuous phase and the electrophoretic mechanism.

In MEKC, the separation selectivity can be modulated not only by variation of BGE type, pH and concentration, but also by benefit of the proper surfactant selected with the optimal concentration. Because phyto-markers to be simultaneously analyzed in plant materials are often acidic, basic and neutral compounds, MEKC is widely applied. The polymeric chiral surfactant as a pseudo-stationary phase or chiral selector in MEKC proved to be a powerful and useful technique for the simultaneous quantitative analysis of chiral alkaloids with high sensitivity.

Microemulsion electrokinetic chromatography (MEEKC), which is similar to MEKC, is used as separation environment with pseudo-stationary phases represented by microemulsions. Typically, the microemulsions are composed of nanometer-sized oil droplets suspended in an aqueous buffer (oil-in-water microemulsions, O/W). These systems are stabilized by using surfactant i.e. SDS and a co-surfactant - a short-chain alcohol such as butanol. The oil droplets are collected by dispersing n-octane or other types of hydrophobic solvents (Hoffmann et al. 2009). Capillary electrochromatography (CEC), likes other electrokinetic techniques, combines the charged solutes electromigration with chromatographic partition; but then, the latter event is different to MEKC and MEEKC, the establishment between a liquid mobile phase and a solid stationary phase is packed in a fused-silica capillary (small particles, ~3 µm) which is fixed by silica sintering frits. CEC can also be used in monolithic stationary phases formed by the in situ (in capillary) polymerization of monomers. The mobile phase in CEC is driven through the stationary phase by electroosmotic flow (EOF), can leads to high separation efficiency by assuming a flat and homogeneous profile.

The analytical separation of chiral analytes is one of the most popular applications in capillary electrophoresis (CE), commonly achieved by adding chiral selectors, most frequently cyclodextrins (CDs) to the background electrolyte (BGE). These popular selectors are composed of (1,4)-linked α-D-glucopyranose units forming a truncated cone shape and contain a hydrophilic outer surface surrounding a rather lipophilic cavity (Saz et al. 2016).

It is worth mentioning due to the different complexity of the matrices in natural pant, different sample preparation procedures have been evaluated including ultrasound assisted extraction (UAE), microwave assisted extraction (MAE), pressurized solvent extraction (PSE), and supercritical fluid extraction (SFE) (see in Table 3).

Due to the complexity of sample matrices (as plant and biological samples) and the low concentration of existed alkaloids, samples should be purified and enriched right after extraction step to facilitate the identification and/or quantification process. Practically, the most popular clean-up methods utilized for sample purification of alkaloids are liquid-liquid extraction (LLE) and solid phase extraction (SPE). Besides, new techniques such as Liquid Membrane Extraction (LME) and Solid-Phase Micro Extraction (SPME) have also been developed based on LLE and SPE, respectively.

Analytical applications, including analyte, separation conditions and CSPs, are summarized in Table 5. A review sheds light on the most novel examples of chiral alkaloids separations on CSPs bring back efficient analyses was prepared.

New column materials also improved the ability of tropane alkaloids enantiomer separation. Separation of (R, S)-hyoscyamine was achieved using chiral stationary phase with immobilizing α-1-acid glycoprotein (Chiral AGP) (Soares et al. 2009), alternatively, enantiomer separation of atropine could be achieved by a chirobiotic V column packed with vancomycin as chiral selector (Aehle et al. 2010). The 6β-hydroxyl derivative of (S)-hyoscyamine called anisodamine was separated from the mixture including synthetic enantiomer and diastereomers by a Chiralpak AD-H column as chiral stationary phase, combined with an amylose derivative as a chiral selector (Yang et al. 2007). Satropane, 3α-paramethylbenzenesulfonyloxy-6β-acetoxy-tropane, could be resolved in lesatropane (3S, 6S-isomer) and desatropane (3R, 6R-isomer). A novel muscarinic agonist called lesatropane is in under preclinical development in China for the treatment of primary glaucoma as a single enantiomer drug. The separation of lesatropane from desatropane was conducted by both Chiralpak AD-H and Chiralpak AS-RH column (Yang et al. 2011).

Chiral separation of isoquinoline alkaloid has also achieved by using chiral stationary phase, for example with tetrahydropalmatine (THP) which was analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel. Quantification of OJ column by UV at 230 nm. The method was used to determine the pharmacokinetics of THP enantiomers in rats and dogs after oral administration of racemic THP or (-)-THP (Hong et al. 2005). Another report discusses sanguinarine derivatives. Chiral determination of benzophenanthridine alkaloids of Hylomecon that are extracted with methanol was proceeded by Chiralcel OD (4.6 × 250 mm) column with mobile phase is isopropanol-hexane-diethylamine (20/80/0.1, v/v/v) (Kang et al. 2003). The stereochemistry of l-isocorypalmine and the d/l ratio of tetrahydropalmatine, stylopine, and corydaline were established unambiguously by using a chiral Chiralcel OD (4.6 × 250 mm) column; 50% ethanol as mobile phase; wavelength 230 nm (Ma et al. 2008). Cularinoids are a group of about 60 compounds of isoquinoline alkaloids. The HPLC enantiomeric separation of the racemic cularinoid alkaloids N-p-methoxy-1,α-dihydroaristoyagonine and 4’,5’demethoxy-1,α-dihydroaristoyagonine was accomplished by five chiral stationary phases (CSPs), in which there is a polysaccharide-derived CSP called Chiralpak AD, leading to the good enantioselectivity and resolution factor (Caccamese et al. 2006).

Indole derivatives are popular in chiral synthesis, chemical asymmetric catalysis, biological and medicinal chemistry. Lately, there were reports about the enantiomeric separation of several chiral plant growth regulators and related compounds, such as 3-(3-indolyl)-butyric acid, abscisic acid and structurally related molecules including a variety of substituted tryptophan compounds. Recently, Zhao et al., reported that a coated and immobilized chiral stationary phases were suitable for the separation of indole derivatives; however, the coated CSP possesses a higher resolving power than the immobilized one (Zhao et al. 2009). A biogenetically interesting indole alkaloid was found in the leaves of Nitraria tangutorum in 1999 named Tangutorine. It was separated from its synthetic enantiomer by chiral stationary phases two polysaccharide-derived CSP (Chiralcel OD and Chiralpak AD) and a network polymer incorporating a bifunctional C2-symmetric chiral selector (Kromasil CHI- (DMB) (Putkonen et al. 2003). Chiralpak AD and Chiralcel OD as chiral stationary phases were used for the HPLC enantiomeric separation of racemic indole alkaloids tacamonine, 17α-hydroxytacamonine, deethyleburnamonine, and vindeburnol (Caccamese et al. 2001).

One of the most popular applications in capillary electrophoresis (CE) is the analytical scale separation of chiral analytes which is commonly succeeded by adding chiral selectors, most frequently cyclodextrins (CDs) to the background electrolyte (BGE). Since decades, cyclodextrins (CDs) are one of the most powerful selectors in chiral capillary electrophoresis for the enantio-separation of diverse organic compounds. A wide range of different CD derivatives (such as methylated, sulfated, carboxymethylated, sulfobutylated ones) were available as randomly substituted derivatives in order to meet the urgent need of market (Zhu et al. 2016). CDs still continue to be the most frequently used chiral selectors in CE as can be seen from the large number of applications even in the relative short period of time covered by this review (Table 6). An increasing number of publications has appeared describing CD-meditated capillary EKC enantioseparations in nonaqueous CE (NACE) with organic solvent-based electrolytes. Organic solvents often offer different selectivities compared to aqueous BGEs.

A CZE method using dimethyl--cyclodextrin (DM--CD) as modifier with tetrabutylammonium chloride (TBAC) as addition has been developed for the chiral separation of ()-ephedrine, ()-pseudoephedrine, ()-N-methylephedrine, and ()-norephedrine in Ephedra sinica and its medicinal preparation (Jiang et al. 2007). Medicinal herbs containing phenylethylamine alkaloids including Citrus species and Ephedra sinica are widely used because of their effects on human metabolism especially thanks to their lipolysis stimulation, therefore promoting the fat mass reduction in obesity treatment. Specifically, ephedra extracts such as Ephedra Herba (Ma Huang) contain alkaloids such as: (1R,2S)-(−)-ephedrine, (1S,2S)-(+)-pseudoephedrine, (1R,2S)-(−)-norephedrine, (1S,2S)-(+)-norpseudoephedrine, (1R,2S)-(−)-N-methylephedrine, and (1S,2S)-(+)-N-methylpseudoephedrine (Gotti 2011). The U.S. Food and Drug Administration (FDA) ban dietary supplements containing ephedra depending on the dosage due to the possible adverse effects while using these products. In addition, the National Institute of Standards and Technology (NIST) issued standard reference materials with certified values for ephedra alkaloids, synephrine and caffeine. Chiral analysis provides a useful tool in order to characterize Ephedra sinica and its medicinal phytopreparations because only (1R,2S)-(−)- ephedrine, (1S,2S)-(+)-pseudoephedrine enantiomers are found in nature. Simple CZE analysis of ephedrine and pseudoephedrine in herbal drugs (tablets) was carried out in a 25 mM triethanolamine phosphate buffer (pH 2.5) in condition of reversed EOF. Highly sulphated -cyclodextrin was added as the chiral additive to obtain the enantioselectivity in need as well as separate ephedrine and pseudoephedrine enantiomers from other herbal drug ingredients (Amini et al. 2006). A chiral system using three neutral cyclodextrins, namely -CD, hydroxypropyl--cyclodextrin (HP--CD) and Heptakis (2,6-di-O-methyl)--cyclodextrin (DM-CD) under acidic conditions was achieved the simultaneous enantio-resolution of octopamine and synephrine enantiomers in Citrus species dietary supplements_ a mixtures with the presence of tyramine and N-methyl tyramine. Particularly, another application of chiral analysis was the thermal racemization evaluation of l-synephrine. Although information on the kinetics of the conversion was not available, the research showed that the real samples extraction process did not have any consequences about chiral artifacts become racemic of synephrine effectively at relatively high temperature (i.e. 1000C) (Avula et al. 2005). Three CE-UV methods developed by Phinney and co-workers that allowed the enantioseparation of ephedrine and pseudoephedrine by using chiral selectors including neutral HP--CD, DM--CD and charged sulfated CD. The combination of negatively charged sulfated -CD (2.8%) and DM--CD (1.2%) under acidic conditions (pH 2.5) provided the best separation of the two couples of enantiomers. NIST is developing the standard reference materials (SRMs) containing ephedra by using each of three CE method to analyze. The SRM samples are suitable in product adulteration detection by its potential in specific stereoisomers identification, although they only found the naturally occurring enantiomers (−)-ephedrine and (+)-pseudoephedrine (Phinney et al. 2005).

Tropane alkaloids are obtained from Solanaceae, e.g., Atropa belladonna, Hyoscyamus niger, and Datura stramonium (Aehle et al. 2010). The first pure compounds were atropine isolated from Atropa belladonna and hyoscyamine from Hyoscyamus niger (Kodama et al. 2009). Today, atropine” is defined as the racemic mixture of (S)- hyoscyamine and (R) hyoscyamine. (S)-hyoscyamine is found in plants while (R)-hyoscyamine can be formed under alkaline conditions. (S)-hyoscyamine is a strong acetylcholine-inhibitor as a muscarine receptor- blocker, while the (R)-hyoscyamine is mostly inactive. Although atropine exhibits approximately half of the (S)-hyoscyamine pharmacological activity, it is still prefered to (S)-hyoscyamine. Baseline separation of atropine in sulfated--cyclodextrin enabled the quantitation of 0.5% (R)-hyoscyamine (45 µg/ml) in (S)-hyoscyamine (Heine et al. 2003). The same sulfated -cyclodextrin proved useful for separation of cocaine enantiomers and diastereomers, i.e. (-)-cocaine (= (2R, 3S)-cocaine, the active enantiomer formed in coca plants), (+)-cocaine, (-)-pseudococaine, and (+)-pseudococaine (Cabovska et al. 2003).

Enantio-separations with high resolution and short migration times of all tropane alkaloids were achieved by using heptakis (2, 3-di-O-methyl-6-O-sulfo)-β-CD and sulfated β-CD in the microemulsion BGE and were superior to corresponding CD modified CE methods (Yaser et al. 2007). The enantiomeric separation of three vinca alkaloid enantiomers (vincamine, vinpocetine and vincadifformine) has achieved in an aqueous capillary electrophoresis (CE) system using Methylated-β-cyclodextrin and (2-hydroxy)propyl- β -CD of CD (Sohajda et al. 2010).

Camptothecins (CPT) are valuable quinoline alkaloids present in Camptotheca acuminata, Nothapodytes foetida, Ophiorrhiza pumila and Tabernaemontana heyneana. It occurs in different plant parts such as the roots, twigs, and leaves. It consists of a pentacyclic ring structure that includes a pyrrole quinoline moiety and one asymmetric center within the -hydroxy lactone. Only the (S)-enantiomers of CPTs exhibit antitumor activity (Caccamese et al. 2006). The interest towards these compounds is grown because of the demonstrated activity in the treatment of colorectal and ovarian cancer. Camptothecin used for the clinical activities as well as for the downstream synthesis of the derivatives is currently obtained from natural sources. The main issue in analytical methods development in order to determine camptothecin in plant materials is the poor water solubility. The enantiomeric resolution of homocamptothecin derivatives was obtained using highly S--CD as chiral selectors at acidic pH (Goossens et al. 2006).