Chiral Resolution Techniques by High Performance Liquid Chromatography (HPLC)

With the maturity of asymmetric synthesis technology and chiral separation technology, chiral drug research has become a hot spot in new drug development. Due to the large differences in pharmacodynamics and pharmacokinetics among the enantiomers of chiral drugs, it is very important to establish a quality control method for chiral separation. Chiral enantiomers usually have the same physical, thermodynamic and chemical properties, such as melting point, boiling point, solubility, refractive index, acidity, density, free energy, enthalpy, entropy, etc. Enantiomers show differences in physical and chemical properties only in chiral environments such as chiral reagents and chiral solvents. Enantiomers act differently on polarized light, with the same specific rotation values but in opposite directions.

A mixture of equal amounts of levorotatory and dextrorotatory forms the racemate. The method of separating a single optical isomer from the enantiomer is called chiral resolution. The most common chiral resolution method is that the racemate reacts with ions with opposite optical activity (called resolution agent) to form diastereomers. The methods for enantiomeric separation of chiral drugs mainly include non-chromatography and chromatography. Non-chromatography (mainly including crystallization method, microbial digestion method, etc.) is time-consuming and cannot prepare high-purity enantiomers due to the cumbersome process. Chromatography is based on converting the mixture of enantiomers into diastereomers, and then using their differences in chemical or physical properties for separation. Chromatography mainly includes gas chromatography (GC), supercritical fluid chromatography (SFC), capillary electrophoresis (CE) and capillary electrochromatography (CEC). Table 1 lists the development history of chromatographic chiral resolution. Among them, high performance liquid chromatography (HPLC) has become the most commonly used technique in the field of chiral analysis due to its unique advantages.

YearsMilestone
1939Chromatographic separation of racemic camphor derivatives on lactose by Henderson and Rule
1952Dalliesh proposed a three-point role hypothesis for the optical separation of amino acids on paper chromatography
1966Direct separation of enantiomers by gas chromatography by Gil-Au et al
1971Davankov and Rogozhin introduced chiral ligand exchange chromatography
1972Enzyme-mimicking polymers of chiral liquid chromatography prepared by Wulff and Sarhan
1973Chiral resolution of cellulose triacetate prepared by Hesse and Hagel
1973Stewart and Dherty used agarose-bonded bovine serum albumin (BSA) for chiral resolution
1974Blaschke synthesized chiral polymers of chiral liquid chromatography from optically active monomers
1975Gram et al developed host guest chromatography with chiral crown ethers
1979Dirkle and House synthesized the first silica-bonded chiral stationary phase and apply it to chiral liquid chromatography separations
1979Okamoto et al synthesized helical polymers of chiral liquid chromatography
1982Allenmark et al used agar-bonded BSA for chiral liquid chromatography
1983Hermansson used silica-bonded a1-acid glycoproteins for chiral resolution
1984Silica-bonded cyclodextrin stationary phase prepared by Armstrong and DeMond

Table 1. Development history of chromatographic chiral resolution.

HPLC Chiral Resolution Methods

The HPLC resolution of chiral drugs is usually divided into direct method and indirect methods. The common feature of the two methods is that both are based on modern technology and introduce asymmetric centers or photoactive molecules. The difference is that the indirect method is to introduce the optical center into the molecular solute, while the direct method is to introduce it between molecules. The difference of physical properties between enantiomers caused by the introduction of chiral environment is the basis for the separation of chiral optical isomers.

  • Indirect Methods

Chiral derivatization reagent method (CDR) refers to the method of reacting enantiomers with chiral derivatization reagents to form diastereomeric complexes. The difference from chemical methods is that it does not use physical and chemical methods such as crystallization, distillation, extraction, etc. to achieve separation, but uses ordinary chromatographic columns to separate diastereomers. The method has low cost and wide application range, but is complicated in operation and prone to racemization, so the derivatization reagents are required to have high purity. In the separation study of (±)-gossypol in cottonseed kernel, R-(-)-2-amino-1-propanol with a purity of 98 % was used as a derivatization reagent to react with the aldehyde group in (±)-gossypol to convert it into an enantiomer, so that it can be better separated on a common C18 column. In addition, the derivatization reagents with chromophores or fluorescence structures commonly used in chiral derivatization methods can also improve the detection ability. Table 2 shows commonly used chiral derivatizing agents.

Chiral Derivatization ReagentsIntroduction
Isothiocyanate (ITC), Isocyanate (IC)Reacts with most alcohols and amines to form the corresponding diastereomers of carbamates or ureas, such as ephedrines, epinephrines, epinephrine anticaking agents, catechins
Naphthalene DerivativesIts structural characteristics are conducive to improving stereoselectivity. Meanwhile, such compounds have strong UV absorption, and UV detector can greatly improve the detection sensitivity, which is widely used in chiral derivatization reagent method
Carboxylic Acid DerivativesMainly include acid chlorides, sulfonyl chlorides, and acid anhydrides. Their chiral carbons are located at the alpha position of the carboxyl group, and they can react with amines, amino acids and alcohols to form diastereomers
AminesMainly used for derivatization of carboxylic acids, N-protected amino acids, alcohol drugs

Table 2. Commonly used chiral derivatives.

  • Direct Methods

The direct method is to introduce a "chiral recognizer" or chiral environment in the HPLC system to form a temporary diastereoisomer complex, which are separated according to the stability coefficient. Direct methods are divided into chiral mobile phase method (CMP) and chiral stationary phase method (CSP).

(1) Chiral mobile phase method refers to the addition of chiral additives that can form diastereomers with chiral drugs in the mobile phase, and the difference in the solubility of the substance in the mobile phase and the combination with the stationary phase is used to achieve enantiomers separation. The chiral mobile phase method is suitable for achiral stationary phases and does not require pre-column derivatization, so the operation is simpler. The key to its separation is mainly chiral additives. Commonly used chiral additives include cyclodextrin compounds, chiral metal complexes, chiral ion pair reagents, chiral hydrogen bonding reagents, and the like (Table 3).

Chiral AdditivesIntroduction
Ligand Exchange Chiral AdditivesAmong the many chiral additives, the basic theoretical research of ligand-exchanged chiral additives is relatively mature and widely used. Chiral ligands are mostly photoactive amino acids or their derivatives. They chelate with divalent metal ions and are distributed in the mobile phase at appropriate concentrations. When encountering drug racemates, it coordinates to form complexes, which are resolved on reversed-phase or normal-phase columns.
Cyclodextrin AdditivesCyclodextrins are cyclic oligosaccharides composed of glucopyranose linked by α-(1,4). Its molecule is frustoconical, with many hydroxyl groups lining the edges and a relatively hydrophobic cavity inside. When the molecular size of the compound to be analyzed is consistent with the cavity, a cyclodextrin inclusion complex can be formed, thereby achieving chiral separation.
Chiral Ion Pair AdditivesChiral ion-pairing reagents refer to chiral additives with opposite charges to enantiomeric ions, which are added to the mobile phase to combine with enantiomers to form diastereomeric ions to achieve chiral separation.

Table 3. Commonly used chiral additives.

(2) Chiral stationary phase method refers to the separation of enantiomers by using the difference of chiral interaction between the chiral stationary phase and the enantiomers. The commonly used chiral stationary phases include polysaccharide chiral stationary phases, cyclodextrin chiral stationary phases, protein chiral stationary phases, and molecularly imprinted chiral stationary phases, among which polysaccharide chiral stationary phases are the most common (Table 4).

TypesIntroduction
Polysaccharide Chiral Stationary PhaseThe polysaccharide chiral stationary phase is based on amylose or cellulose as the chiral source, and can be divided into coated chiral stationary phase and bonded chiral stationary phase. Hydrogen bonds or dipole-dipole interactions are formed between the carbamates of the polysaccharide chiral stationary phase and the molecules to be resolved, resulting in chiral separation. It is mainly used to separate barbiturates, anesthetics and antidepressants.
Cyclodextrin Chiral Stationary PhaseDue to the structural characteristics, cyclodextrins can form inclusion complexes with many drug molecules through van der Waals forces. The separation of enantiomers can thus be achieved by adding cyclodextrins to the mobile phase or incorporating cyclodextrins on the stationary phase. β-cyclodextrin is suitable for most drugs, γ-cyclodextrin is suitable for drugs with larger molecular weight, and α-cyclodextrin is suitable for drugs with relative molecular weight less than 200.
Protein Chiral Stationary PhaseProtein is a high molecular polymer composed of amino acids, mainly L-type amino acids, which can specifically recognize some chiral molecules, and α1-acid glycoprotein and bovine serum albumin are the most commonly used.
Pirkle TypesChiral precursors containing terminal carboxyl or isocyanate groups were condensed with amino bonded silica to form amide or urea-type structures, respectively. It is applied to the enantiomers of amino acids, hydantoin, lactam, amines, alcohols and thiols.

Table 4. Types of chiral stationary phases.

HPLC has the characteristics of rapidity, good separation effect, high detection sensitivity, and can be equipped with different detectors with strong selectivity and high sensitivity, and detection automation. The future development of HPLC in the field of chiral analysis is expected to focus on the in-depth study of chiral recognition mechanism and provide a clear theoretical basis for rapid screening of suitable analysis or preparation methods. In chiral chromatography, both the indirect method and the direct method have their own advantages and limitations (Table 5), and their applications in pharmaceutical science are complementary to each other and have their own characteristics. Researchers should select appropriate methods to effectively separate the tested drugs according to their structure and properties, so as to provide reliable guarantee for the development and quality control of chiral drugs and continuously promote the development of chiral drugs.

MethodsAdvantagesLimitation
CDRThe existing achiral stationary phases can be used with low cost. The detection sensitivity can be improved by selecting chiral reagents with strong ultraviolet absorption or fluorescence absorption. Most of the derivatization reagents have good stability to heat and water.The chiral reagent needs to have high optical purity, and the derivatization rate and equilibrium constant of each enantiomer should be consistent. The derivatization reaction should be rapid and thorough. The resolution steps are cumbersome and the derivatization reagents are toxic, making it difficult to automate the analytical method.
CMPNo need to do pre-column derivatization, no special requirements for stationary phase, the diastereoisomerization complexation of the sample is reversible and easy to prepare.Limited application range, some additives are not stable enough and can interfere with detection.
CSPCan be widely applied to all kinds of compounds, suitable for conventional and biological sample analysis and determination, preparation and separation is convenient, high reliability of quantitative analysis.Some samples require pre-column derivatization, which has certain restrictions on the sample structure. Its applicability is not as broad as that of HPLC stationary phases.

Table 5. Comparison of three chiral separation methods.

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