**4. High performance liquid chromatography in chiral separation**

High performance liquid chromatography (HPLC) is currently the most widely used chromatographic enantio-separation technique [65]. HPLC has become one of the most common modern chemical analysis techniques because of its versatility, efficiency, stability, reproducibility and sensitivity. With these advantages, HPLC continues to be one of the best choices for chiral analysis and separation. Basically, chiral separation by HPLC techniques included direct and indirect methods. The indirect method is based on diastereomer formation by the derivatization reaction of analytes and a chiral reagent, then the separation of diastereomeric derivatives is performed by using a column having an achiral stationary phase. In addition, the direct method uses a chiral stationary phase for chiral separation or forms diastereomer by using a chiral mobile phase additive (CMPA).

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 3**). 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 [66–69]. A hundred CSPs have been developed and commercialized thirty years ago [70]. Besides, the larger number of CSPs are made in laboratory for specialized separation. CSPs are divided into nine major types by Snyder basing on the interaction mechanism between stationary phase and analytes [71].


**51**

*Chiral Alkaloid Analysis*

II Synthetic-Polymer CSPs

V Macrocyclic Antibiotic

VII Donor-Acceptor Phases

Ion-Exchangers

Ligand-Exchange

VIII Chiral

IX Chiral

**Table 3.**

*DOI: http://dx.doi.org/10.5772/intechopen.96009*

**Type CSP Typical column trade** 

I Polysaccharide AD, OD, OJ, AS, IA,

III Protein Phases Chiral HSA, Chiral

VI Chiral Crown-Ether ChiroSil RCA(+);

**name**

IB, IC

Kromasil CHI-DMB and CHI-TBB

AGP, Ultron ES-OVM,

Chirobiotic V, T, R, TAG; vancomycin

Whelk-O 1, ULMO, Sumichiral 2500, Sumichiral OA 4900

Chiralpak QN-AX; Chiralpak QD-AX

Chiralpak MA+, Nucleosil Chiral-1

Chiral CBH

IV Cyclodextrin Cyclobond I, II, III Beta blockers

SCA(−); ChiralHyun-CR-1 **Application**

beta blockers

amino acids

alcohols

Alkaloids, tropines, amines, beta blockers, aryl methyl esters, aryl methoxy esters

Benzodiazepine, Warfarin and oxazepam,

Polar compounds such as underivatized

Amino acids, amino acid esters, amino

Chiral carboxylic, sulfonic, phosphonic,

Amides, epoxides, esters, ureas, carbamates, ethers, aziridines, phosphonates, aldehydes,ketones, carboxylic acids, alcohols

and phosphoric acids

Amino acids

Acidic, neutral, and basic compounds

**4.1 Type I polysaccharide-derived CSPs in HPLC**

*Application of nine major types of CSP and their commercial CSP [72].*

*4.1.1 Coated polysaccharide-derived CSPs*

Polysaccharide-derived CSPs are widely used in enantio-separation of a large number of chiral compounds [71]. The development of polysaccharide-derived CSPs has continued for about three decades. It can be roughly divided into two

Polysaccharide selectors have been used for enantioselective liquid chromatography technique for a long time. In 1973, a polymeric selector (without supporting matrix) was introduced by Hesse and Hagel named as microcrystalline cellulose triacetate (MCTA) used for enantioselective liquid chromatography. MCTA are widely applied in enantio-recognition and preparative separations due to its ideal loading capacities. However, this material has major disadvantages including poor pressure stability, slow separations, and low chromatographic efficiency. To overcome the mechanical stability problem of MCTA, a solution was found by Okamoto and co-workers in 1984, in which the surface of macro-porous silica beads (100 or 400 nm pore size) was coated by the cellulose derivatives at about 20 wt%. Thanks to this coating, the mechanical stability of this material was remarkably improved resulting in better efficiencies qualified for HPLC enantiomer separations. Such coated polysaccharide-based CSPs were the high-

stages (i) the coated CSPs stage, and (ii) the immobilized CSPs stage.

est level of polymeric selector developments for several decades [71].

Nowadays, about 200 kinds of polysaccharide derivatives were introduced by using different polysaccharides which includes cellulose, amylose, chitin, chitosan, galactosamine, curdlan, dextran, xylan, and inulin [72] (**Figure 13**).



**Table 3.**

*Current Topics in Chirality - From Chemistry to Biology*

cartridges [64].

additive (CMPA).

phase and analytes [71].

(poly-tartramides)

type CSPs)

exchange

functional group which has pKa value about 4.8, so these sorbents should be conditioned by solutions having pH above 6.8 for sorbent ionization. In addition, the loading and washing solvent pH should be adjusted at the value above 6.8 and below 2 value of analytes's pKa to maintain the ionized state of both sorbent and analytes. Finally, the alkaloids will be eluted by the acidic solutions [63]. Besides, the C18 and C8 sorbents are also applied to extract aromatic alkaloids and eliminate hydrophilic interferences from matrices. In some case, those sorbents could be used for eliminating hydrophobic interferences by loading unretained alkaloids through

**4. High performance liquid chromatography in chiral separation**

High performance liquid chromatography (HPLC) is currently the most widely used chromatographic enantio-separation technique [65]. HPLC has become one of the most common modern chemical analysis techniques because of its versatility, efficiency, stability, reproducibility and sensitivity. With these advantages, HPLC continues to be one of the best choices for chiral analysis and separation. Basically, chiral separation by HPLC techniques included direct and indirect methods. The indirect method is based on diastereomer formation by the derivatization reaction of analytes and a chiral reagent, then the separation of diastereomeric derivatives is performed by using a column having an achiral stationary phase. In addition, the direct method uses a chiral stationary phase for chiral separation or forms diastereomer by using a chiral mobile phase

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 3**). 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 [66–69]. A hundred CSPs have been developed and commercialized thirty years ago [70]. Besides, the larger number of CSPs are made in laboratory for specialized separation. CSPs are divided into nine major types by Snyder basing on the interaction mechanism between stationary

• Macromolecular selectors of semisynthetic origin (polysaccharides)

• Macromolecular selectors of natural origin (proteins)

cyclic antibiotics, chiral crown ethers)

• Macromolecular selectors of synthetic origin (poly(meth)acrylamides),

• Macrocyclic oligomeric or intermediate-sized selectors (cyclodextrins, macro-

• Synthetic, neutral entities of low molecular weight (Pirkle-type phases, brush-

• Synthetic, ionic entities of low molecular weight that provide for ion

• Chelating selectors for chiral ligand-exchange chromatography.

**50**

*Application of nine major types of CSP and their commercial CSP [72].*

## **4.1 Type I polysaccharide-derived CSPs in HPLC**

Polysaccharide-derived CSPs are widely used in enantio-separation of a large number of chiral compounds [71]. The development of polysaccharide-derived CSPs has continued for about three decades. It can be roughly divided into two stages (i) the coated CSPs stage, and (ii) the immobilized CSPs stage.

## *4.1.1 Coated polysaccharide-derived CSPs*

Polysaccharide selectors have been used for enantioselective liquid chromatography technique for a long time. In 1973, a polymeric selector (without supporting matrix) was introduced by Hesse and Hagel named as microcrystalline cellulose triacetate (MCTA) used for enantioselective liquid chromatography. MCTA are widely applied in enantio-recognition and preparative separations due to its ideal loading capacities. However, this material has major disadvantages including poor pressure stability, slow separations, and low chromatographic efficiency. To overcome the mechanical stability problem of MCTA, a solution was found by Okamoto and co-workers in 1984, in which the surface of macro-porous silica beads (100 or 400 nm pore size) was coated by the cellulose derivatives at about 20 wt%. Thanks to this coating, the mechanical stability of this material was remarkably improved resulting in better efficiencies qualified for HPLC enantiomer separations. Such coated polysaccharide-based CSPs were the highest level of polymeric selector developments for several decades [71].

Nowadays, about 200 kinds of polysaccharide derivatives were introduced by using different polysaccharides which includes cellulose, amylose, chitin, chitosan, galactosamine, curdlan, dextran, xylan, and inulin [72] (**Figure 13**).

**Figure 13.**

*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.*

These materials have been coated on a surface of macro-porous silica gel to create CSPs and followed by the evaluation of chiral recognitions on HPLC.

Each of coated polysaccharide-based CSPs exhibit the different enantioselectivity and elution order of the various enantiomers due to the structural differences of CSPs including sugar units, linkage position, and linkage type. In particular, the derivatives of cellulose and amylose usually perform higher recognition abilities than the others, though this property also depends on the structure of a specific racemate. The most useful and successful derivatives of cellulose and amylose are triesters and tricarbamate. 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) [72]. 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 [71].

A current strategy introduced by Snyder for chiral separation method development includes trial-and-error experiments of various polysaccharidetype CSPs under multiple respective mobile-phase conditions using fully automated column- and solvent-switching. Nowadays, more and more studies have focused on developing a more efficient screening procedure to enhance the chance for success and shorten the experiment time: the most favorable CSP in the normal phase mode is

$$\text{Chiralpak AD} -- > \text{Chiralcel OD} -- - > \text{Chiralcel O}\_2$$

The separation efficiency of column should be tested before conducting screening experiment if serial instead of parallel screening is utilized. The application of coated polysaccharide-derived CSPs has been reviewed in **Table 4** including the names of CSPs and their most frequent applications.

#### *4.1.2 Immobilized polysaccharide-derived CSPs*

The coated CSPs are formed by coating the polysaccharide derivatives onto surface of silica gel. Due to the weak linkages and interactions between the polysaccharide derivatives (chiral selector) and silicagel (substrate), a number of organic solvents including chloroform, dichloromethane, tetrahydrofuran and ethyl acetate which can dissolve or swell the chiral selector are not allowed

**53**

*a*

*b*

*c*

*d*

**Table 4.**

*particle size 10*

*Chiral Alkaloid Analysis*

Cellulose CSPs

*DOI: http://dx.doi.org/10.5772/intechopen.96009*

Chiralcel OD Cellulose

Chiralcel OD-Hb Cellulose

Chiralcel OD-Rc Cellulose

Chiralcel OD-RHd Cellulose

Chiralcel OF Cellulose

Chiralcel OG Cellulose

Chiralpak AD Amylose

Chiralpak AD-R<sup>a</sup> Amylose

Chiralpak AD-RHb Amylose

Chiralpak AD-H Amylose

Chiralpak AR Amylose

Chiralpak AS Amylose

*m, except as noted.*

*Various polysaccharide-based commercial CSP [72, 90, 91, 93].*

*Column size 25 cm X 0,46 cm, particle size 5*

*Column size 15 cm X 0,46 cm, particle size 10*

*Column size 15 cm X 46 cm, particle size 5*

µ

Amylose CSPs

**Trade name Chemical name Applications**

Chiralcel OC Cellulose trisphenylcarbamate Cyclopentanones

tris(3,5-dimethylphenylcarbamate)

tris(3,5-dimethylphenylcarbamate)

tris(3,5-dimethylphenylcarbamate)

tris(3,5-dimethylphenylcarbamate)

tris(4-chlorophenylcarbamate)

tris(4-methylphenylcarbamate)

tris(3,5-dimethylphenylcarbamate)

tris(3,5-dimethylphenylcarbamate)

tris(3,5-dimethylphenylcarbamate)

tris-(3,5-dimethylphenylcarbamate)

tris(R)-1-phenylethylcarbamate

tris(S)-1-methylphenylcarbamate

µ*m.*

µ*m.*

µ*m.*

Chiralcel OA Cellulose triacetate on silica gel Small aliphatie compounds

Chiralcel OB Cellulose trisbenzoate Small aliphatic and aromatic

Chiralcel OJ Cellulose tris(4-methyl benzoate) Aryl methyl esters, aryl methoxy esters

compounds

blockers

blockers

blockers

blockers

alkaloids

β

β

β

β

β

Alkaloids

Alkaloids, amines,

Alkaloids, amines,

Alkaloids, amines,

Alkaloids, amines,


Alkaloids, tropines, amines,

Alkaloids, tropines, amines,

Alkaloids, tropines, amines,

Alkaloids, tropines, amines

Alkaloids, tropines, amines




β-adrenergic

β-adrenergic

β-adrenergic

β-adrenergic


using as mobile phase components. Besides, the mixtures of alkanes (n-pentane, n-hexane or n-heptane) and alcohols (2-propanol (IPA), ethanol or methanol (n-pentane, n-hexane or n-heptane)) and alcohols (2-propanol (IPA), ethanol or methanol) are favorable mobile phase solvents used in normal phase mode. The addition of "prohibited solvents" may lead to better separation and greater solubility of racemic analytes than the standard solvent combinations so the performance of separation method is improved. Besides, the better solubility of racemic analytes also facilitates preparative separations. In addition, the spectroscopic techniques used for chiral recognition should ideally be in the "prohibited solvents". As a result, chemical immobilization of polysaccharide derivatives becomes an interesting research topic to overcome the drawbacks of the coated

*Columns supplied by Daicel Chemical Industries, Tokyo, Japan, Dimension are column size 25 cm X 0,46 cm,* 

*Current Topics in Chirality - From Chemistry to Biology*

These materials have been coated on a surface of macro-porous silica gel to create

*Structures of the various kinds of polysaccharides: (1) cellulose; (2) amylose; (3) chitin; (4) chitosan;* 

Each of coated polysaccharide-based CSPs exhibit the different enantioselectivity and elution order of the various enantiomers due to the structural differences of CSPs including sugar units, linkage position, and linkage type. In particular, the derivatives of cellulose and amylose usually perform higher recognition abilities than the others, though this property also depends on the structure of a specific racemate. The most useful and successful derivatives of cellulose and amylose are triesters and tricarbamate. 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) [72]. More specifically, three famous commercially available CSPs, CHIRALCEL OD, OJ, and CHIRALPAK AD, have fully or partially resolved 70% racemates among over 100

A current strategy introduced by Snyder for chiral separation method development includes trial-and-error experiments of various polysaccharidetype CSPs under multiple respective mobile-phase conditions using fully automated column- and solvent-switching. Nowadays, more and more studies have focused on developing a more efficient screening procedure to enhance the chance for success and shorten the experiment time: the most favorable CSP in

Chiralpak AD Chiralcel OD Chiralcel OJ − −− > −−−−>

The separation efficiency of column should be tested before conducting screening experiment if serial instead of parallel screening is utilized. The application of coated polysaccharide-derived CSPs has been reviewed in **Table 4** including the

The coated CSPs are formed by coating the polysaccharide derivatives onto surface of silica gel. Due to the weak linkages and interactions between the polysaccharide derivatives (chiral selector) and silicagel (substrate), a number of organic solvents including chloroform, dichloromethane, tetrahydrofuran and ethyl acetate which can dissolve or swell the chiral selector are not allowed

CSPs and followed by the evaluation of chiral recognitions on HPLC.

*(5) Galatosamine; (6) Curdlan; (7) dextran; (8) Xylan; (9) inulin.*

**52**

racemates tested [71].

**Figure 13.**

the normal phase mode is

names of CSPs and their most frequent applications.

*4.1.2 Immobilized polysaccharide-derived CSPs*


*a Columns supplied by Daicel Chemical Industries, Tokyo, Japan, Dimension are column size 25 cm X 0,46 cm, particle size 10* µ*m, except as noted.*

*b Column size 25 cm X 0,46 cm, particle size 5* µ*m.*

*c Column size 15 cm X 0,46 cm, particle size 10* µ*m.*

*d Column size 15 cm X 46 cm, particle size 5* µ*m.*

#### **Table 4.**

*Various polysaccharide-based commercial CSP [72, 90, 91, 93].*

using as mobile phase components. Besides, the mixtures of alkanes (n-pentane, n-hexane or n-heptane) and alcohols (2-propanol (IPA), ethanol or methanol (n-pentane, n-hexane or n-heptane)) and alcohols (2-propanol (IPA), ethanol or methanol) are favorable mobile phase solvents used in normal phase mode. The addition of "prohibited solvents" may lead to better separation and greater solubility of racemic analytes than the standard solvent combinations so the performance of separation method is improved. Besides, the better solubility of racemic analytes also facilitates preparative separations. In addition, the spectroscopic techniques used for chiral recognition should ideally be in the "prohibited solvents". As a result, chemical immobilization of polysaccharide derivatives becomes an interesting research topic to overcome the drawbacks of the coated

#### **Figure 14.**

*Effect of column type (immobilized vs. coated polysaccharide-based CSP) and mobile phase on enantioselectivity. Enantiomer separations of N-benzyloxycarbonyl-phenylalanine (a–c) with Chiralpak IB (immobilized) and Chiralcel OD (coated). Flowrate: 1 mL/min; temperature: 25°C; UV detection at 230 nm. Note that the mobile phases used in (a) is forbidden mobile phases for the coated version Chiralcel OD [71].*

CSPs (see in **Figure 14**). In 2005, Daicel and Chiral Technologies introduced a set of three immobilized polysaccharide CSPs as [71]:


Finally, it should be noted that polysaccharide CSPs have now also been established as the first-choice of chiral phases for enantiomer separation.

#### **5. Analytical methods of chiral alkaloids in medicinal plants**

Analytical applications, including CSPs, separation conditions and analyte, are summarized in **Table 5**. A review focused mainly on the latest examples of chiral alkaloids separations on CSPs for efficient analyses was prepared.

Novel column materials also improved enantiomer separation of tropane alkaloids. Separation of (R, S)-hyoscyamine was achieved using chiral stationary phase with immobilizing α-1-acid glycoprotein (Chiral AGP®) [83], alternatively, enantiomer separation of atropine could be achieved by a chirobiotic V column packed with vancomycin as chiral selector [84]. Anisodamine, the 6β-hydroxyl derivative of (S)-hyoscyamine was separated from its synthetic enantiomer and diastereomers by a Chiralpak AD-H column as chiral stationary phase, which uses an amylose derivative as chiral selector [85]. Satropane, 3α-paramethylbenzenesulfonyloxy-6β-acetoxy-tropane, could be resolved in 3S, 6S-isomer named lesatropane and 3R, 6R-isomer named desatropane. Lesatropane as a novel muscarinic agonist is being under preclinical development in China as a single enantiomer drug for the treatment of primary glaucoma. The separation of lesatropane from desatropane was conducted by both Chiralpak AD-H and Chiralpak AS-RH column [86].

Chiral separation of isoquinoline alkaloid has also achieved by using chiral stationary phase. For example, the determination of tetrahydropalmatine (THP) was performed by using a Chiralcel OJ column with quantification by UV at

**55**

**Columns** CHIRALPAK AS-H column

CHIRALPAK AD-H column

(12S,22S)-Dihydroxyisoechinulin A (2) and (12R/S)- Neoechinulin A

Mucroniferanine A

(±)-homocrepidine A

(+)-(3R,6R)- and (−)-(3S,6S)-3α,6β-tropanediol

Chirobiotic V, Chiralpak-AY3 column

Chirex 3019 chiral column

CHIRALPAK AGP column

A Phenomenex Lux Cellulose-2

chiral column

Chiralpak IA column

Phenomenex-Chirex-3126 column

**Table 5.**

*Summary of CSPs, mobile phase compositions, and applications.*

dihydrocarneamide A and iso-notoamide B

intermedine and lycopsamine

S-(−)-canadine and R-(+)-canadine

(R)-nicotine; (S)-nicotine; anabasine, and anatabine

(+)- and (−)-5-hydroxyl-8-oxyberberine

Tobacco Coptis chinensis

Symphytum

uplandicum

Paecilomyces variotii

MeCN–H

O (5:95).

> 2

CH

3

CN:H2O (40:60)

ACN/methanol (80:20) and methanol/methyl-t-

[81]

[82]

butyl ether (90:10)

NH

4

OH- methanol (90:10)

[79]

[80]

*Hydrastis Canadensis*

L.

(−) and (+) hyoscyamine

Erythroxylaceae species Solanaceaes seeds

n-hexane and 2-propanol (9:1) with 0.1% of diethylamine

Ethanol, 0.1% DEA

Hexan:DCE:EtOH:TFA (75:40:7:0.1)

[78]

**Analyte**

**Plant** Cannabis sativ L

Corydalis mucronifera Dendrobium crepidatum

n-hexane−2-propanol (70:30)

n-hexane/2-propanol (95:5)

[75] [76] [77]

**Separation conditions**

Hexane/isopropanol/diethylamine (4:1:0.05)

[73]

*Chiral Alkaloid Analysis*

**Ref.**

*DOI: http://dx.doi.org/10.5772/intechopen.96009*

[74]


#### *Chiral Alkaloid Analysis DOI: http://dx.doi.org/10.5772/intechopen.96009*

*Current Topics in Chirality - From Chemistry to Biology*

of three immobilized polysaccharide CSPs as [71]:

**Figure 14.**

• Chiralpak®IB (immobilized Chiralcel OD) [61]

selector that is not available in coated form.

ary phase with immobilizing

Chiralpak AS-RH column [86].

• Chiralpak®IA (immobilized version of Chiralpak AD)

lished as the first-choice of chiral phases for enantiomer separation.

**5. Analytical methods of chiral alkaloids in medicinal plants**

alkaloids separations on CSPs for efficient analyses was prepared.

CSPs (see in **Figure 14**). In 2005, Daicel and Chiral Technologies introduced a set

*enantioselectivity. Enantiomer separations of N-benzyloxycarbonyl-phenylalanine (a–c) with Chiralpak IB (immobilized) and Chiralcel OD (coated). Flowrate: 1 mL/min; temperature: 25°C; UV detection at 230 nm. Note that the mobile phases used in (a) is forbidden mobile phases for the coated version Chiralcel OD [71].*

*Effect of column type (immobilized vs. coated polysaccharide-based CSP) and mobile phase on* 

• Chiralpak®IC based on the cellulose tris (3,5-dichlorophenylcarbamate)

Finally, it should be noted that polysaccharide CSPs have now also been estab

Analytical applications, including CSPs, separation conditions and analyte, are summarized in **Table 5**. A review focused mainly on the latest examples of chiral

α-1-acid glycoprotein (Chiral AGP®) [83], alter

Novel column materials also improved enantiomer separation of tropane alkaloids. Separation of (R, S)-hyoscyamine was achieved using chiral station

natively, enantiomer separation of atropine could be achieved by a chirobiotic V column packed with vancomycin as chiral selector [84]. Anisodamine, the 6β-hydroxyl derivative of (S)-hyoscyamine was separated from its synthetic enantiomer and diastereomers by a Chiralpak AD-H column as chiral station

ary phase, which uses an amylose derivative as chiral selector [85]. Satropane, 3α-paramethylbenzenesulfonyloxy-6β-acetoxy-tropane, could be resolved in 3S, 6S-isomer named lesatropane and 3R, 6R-isomer named desatropane. Lesatropane as a novel muscarinic agonist is being under preclinical development in China as a single enantiomer drug for the treatment of primary glaucoma. The separation of lesatropane from desatropane was conducted by both Chiralpak AD-H and

Chiral separation of isoquinoline alkaloid has also achieved by using chiral stationary phase. For example, the determination of tetrahydropalmatine (THP) was performed by using a Chiralcel OJ column with quantification by UV at





**54**

**Table 5.**

*Summary of CSPs, mobile phase compositions, and applications.*

230 nm. This developed method was used for determining the pharmacokinetics of THP enantiomers in rats and dogs after oral administration [87]. Another report of isoquinoline alkaloid group is sanguinarine derivatives. Chiral determination of Benzophenanthridine alkaloids from methanol extracts of Hylomecon species was conducted by Chiralcel OD (4.6 x 250 mm) column with mixture of isopropanolhexane-diethylamine (20/80/0.1, v/v/v) as a mobile phase [88]. 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 x 250 mm) column; 50% ethanol as mobile phase; wavelength 230 nm [89]. Cularinoids are a group of isoquinoline alkaloids consisting of about 60 members. The HPLC enantiomeric separation of the racemic cularinoid alkaloids N-pmethoxy-1,α-dihydroaristoyagonine and 4′,5'demethoxy-1,α-dihydroaristoyagonine was accomplished using five chiral stationary phases (CSPs), the good enantioselectivity and resolution factor obtained with a polysaccharide-derived CSP (Chiralpak AD) [90].

Indole derivatives are widely used in chiral synthesis, chemical asymmetric catalysis, biological and medicinal chemistry. Recently, 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 was reported. Chiral stationary phases such as coated and immobilized were suitable for the separation of indole derivatives; however, the coated CSP possesses a higher resolving power than the immobilized one [91]. Tangutorine, a biogenetically interesting indole alkaloid, was found in the leaves of Nitraria tangutorum in 1999. 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) [92]. The HPLC enantiomeric separation of racemic indole alkaloids tacamonine, 17α-hydroxytacamonine, deethyleburnamonine, and vindeburnol was accomplished using Chiralpak AD and Chiralcel OD as chiral stationary phases [93].

The enantiomers of homocamptothecin (hCPT) derivatives which constitute a promising series of potent anticancer agents targeting DNA topoisomerase I were separated by using the combination of two silica-based normal phase column including Chiralcel OD-H (celluloses tris-3,5-dimethylphenylcarbamateand) Chiralcel OJ (celluloses tris-methylbenzoate) or Chiralpak AD (amyloses tris-3,5-dimethylphenylcarbamate) and Chiralpak AS (amyloses tris-(S)-1-phenylethylcar-bamate) [94]. A method for the simultaneous determination of eight Cinchona alkaloids (quinine, quinidine, cinchonine, cinchonidine, and their corresponding dihydro analogs) using a novel strong cation-exchange-type chiral stationary phase (cSCX) column in HPLC has been developed and exemplarily applied to impurity profiling of a commercial alkaloid sample [95].

### **6. Conclusion**

Most of alkaloids are chiral compounds and are clinically administered as the racemic mixture, although its enantiomers have been shown to exert different pharmacological activity. The complication of sample matrices and low concentration of chiral alkaloids are major challenges for analytical processes. To improve the performance of analytical procedure, we provide the current state of the art in sample preparation focusing on extraction and purification to remove interferences and enrich analyte concentrations. HPLC using CSPs has demonstrated to be extremely useful, accurate, versatile, its mode is generally the most straightforward

**57**

*Chiral Alkaloid Analysis*

**Acknowledgements**

**Conflict of interest**

**Author details**

Kyeong Ho Kim2

South Korea

Center, Egypt

ntnvan@ctump.edu.vn

provided the original work is properly cited.

Ngoc Van Thi Nguyen1

of CSPs.

assistance.

*DOI: http://dx.doi.org/10.5772/intechopen.96009*

The authors declare no conflict of interest.

and convenient means for chromatographic enantiomer separation. The development of CSPs for HPLC is a continuous and challenger issue covering various types

The authors would like to express their hearty gratitude to Can Tho University of Medicine and Pharmacy. We also thank all of our colleagues for their excellent

\*, Kim Ngan Huynh Nguyen1

1 Can Tho University of Medicine and Pharmacy, Can Tho City, Vietnam

\*Address all correspondence to: nguyenthingocvanct@gmail.com;

2 College of Pharmacy, Kangwon National University, Chuncheon-si, Gangwon-do,

3 Department of Pharmaceutical and Medicinal Chemistry, National Research

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Hassan Y. Aboul-Enein3

, Kien Trung Nguyen1

,

*Chiral Alkaloid Analysis DOI: http://dx.doi.org/10.5772/intechopen.96009*

and convenient means for chromatographic enantiomer separation. The development of CSPs for HPLC is a continuous and challenger issue covering various types of CSPs.
