IJRPC 2016, 6(3), 476-484 Purushoth Prabhu et al. ISSN: 22312781

INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACY AND

Available online at www.ijrpc.com Review Article

PHARMACEUTICAL REVIEW AND ITS IMPORTANCE OF CHIRAL CHROMATOGRAPHY Purushoth Prabhu T1*, Shinesudev2, Manju kurian2, Manjushsa P2, Minnu PS2, Mohamed Hisham C2 and Sapna Shrikumar2 1C.L.Baid Metha College of Pharmacy, Chennai, Tamil Nadu, India. 2Moulana College of Pharmacy, Angadippuram P.O., Perinthalmanna, Malappuram (DT) – 679321, Kerala, India.

ABSTRACT The separation of chiral compounds has been of. great interest because the majority of bioorganic are chiral. Living organisms, for example, are composed of chiral biomolecules such as amino acids, sugars, proteins and nucleic acids. In nature these biomolecules exist in only one of the two possible enantiomeric forms, e.g., amino acids in the L-form and sugars in the D-form in our body. In this article we had an attempt to review about chiral chromatography and its pharmaceutical importance.

INTRODUCTION materials in plant life, a Russian botanist

The separation of chiral compounds has been invented chromatography in 1903. His name of great interest because the majority of was M.S. T.S.wett. Chromatography is such bioorganic molecules are chiral. Living an important technique that two nobel prizes organisms, for example, are composed of have been awarded to chromatographers. chiral biomolecules such as amino acids, Over 60% of chemical analysis worldwide is sugars, proteins and nucleic acids. In nature currently done with chromatography or a these biomolecules exist in only one of the two variation there on. Chromatography is used in possible enantiomeric forms, e.g., amino acids many different ways. Some people use in the L-form and sugars in the D-form in our chromatography to find out what is in a solid or body. a liquid. It is also used to determine what unknown substances are. The Police, F.B.I., Chromatography and other detectives use chromatography Some materials appear homogenous, but are when trying to solve a crime. It is also used to actually a combination of substances. For determine the presence of cocaine in urine, example, green plants contain a mixture of alcohol in blood, and lead in water. different pigments. In addition, the black ink in Chromatography is used by many different the pens that are used in this experiment is a people in many different ways. mixture of different coloured materials. In Chromatography is based on differential many instances, we can separate these migration. The solutes in a mobile phase go materials by dissolving them in an appropriate through a stationary phase. Solutes with a liquid and allowing them to move through an greater affinity for the mobile phase will spend absorbent matrix, like paper. Chromatography more time in this phase than the solutes that is a method used by scientists for separating prefer the stationary phase. As the solutes organic and inorganic compounds so that they move through the stationary phase they can be analyzed and studied. By analyzing a separate. This is called chromatographic compound, a scientist can figure out what development. makes up that compound. Chromatography is Pharmacologically active compounds (PACs) a great physical method for observing mixtures are widely regarded as emerging and solvents. contaminants and many of them possess at The word chromatography means "colour least one stereogenic centre. The aim of this writing" which is a way that a chemist can test paper is to introduce the subject of liquid mixtures. While studying the colouring within PACs and its implications in

476 IJRPC 2016, 6(3), 476-484 Purushoth Prabhu et al. ISSN: 22312781 environmental contamination. The paper pair of in drugs, pesticides, or describes contemporary techniques utilized in waste compounds, etc. the analysis of chiral contaminants and Chirality is a major concern in the modern provides a critical review of the methods pharmaceutical industry. This interestcan be employed and results gained in the field. attributed largely to a heightened awareness that enantiomers of a racemic drug may have Phenomenon of chirality different pharmacological activities, as well as Enantiomers are molecular entities which are different pharmacokinetic and non-super imposable mirror images. The pharmacodynamic effects. The body being chirality (handedness) of enantiomeric amazingly chiral selective, will interact with molecules is caused by the presence of one or each racemic drug differently and metabolize more chiral elements (chirality axis, chirality each by a separate pathway to plane, or chirality centre, e.g., asymmetric produce different pharmacological activity. carbon atom) in the structure. The chirality and Thus, one isomer may produce the desired optical activity of the enantiomers is therapeutic activities, while the other may be determined by their absolute configuration, inactive or, in worst cases, produce unwanted i.e., the spacial arrangement of the atoms in effects. Consider the tragic case of the the . racemic drug of n-phthalyl-glutamic acid imide that was marketed in the 1960‟s as the Nomenclature sedative Thalidomide. Its therapeutic acitivity IUPAC approved naming system is the Cahn- resided exclusively in the R-(+)-enantiomer. It Ingold and Prelog designation for four- and was discovered only after several hundred six-coordinate stereogenic centres, pre-fixed births of malformed infants that the S-(+)- with R or S, when discussing molecules with enantiomer was teratogenic. planar chirality . Dextro and Levo (+/_) may The U.S. Food and Drug Administration(U.S. also be used to describe enantiomers where F.D.A), in 1992, issued a guideline that for the absolute configuration may not be known chiral drugs only its therapeutically active or to describe the rotation of under isomer be brought to market, and that each prescribed conditions, although the prefixes „d‟ enantiomer of the drug should be studied and „l‟ are discouraged. separately for its pharmacological and metabolic pathways. In addition, a rigorous Chirality in pharmaceuticals justification is required for market approval of a Two enantiomers of the same compound, racemate of chiral drugs. Presently, a majority despite having the same physical and of commercially available drugs are both chemical properties, show different synthetic and chiral. However, a large number interactions with other chiral molecules due to of chiral drugs are still marketed as racemic differences in spacial arrangement of the mixtures. Nevertheless, to avoid the possible atoms and therefore binding affinity. This undesirable effects of a chiral drug, it is phenomenon is particularly significant in imperative that only the pure, therapeutically biological interactions as all proteins, enzymes active form be prepared and marketed. Hence and carbohydrates are chiral . Thus organisms there is a great need to develop the might respond uniquely to each enantiomer, a technology for analysis and separation of phenomenon discovered by Pasteur in 1857 . racemic drugs. This is of particular importance in the case of Chiral compounds are also utilized for chemicals such as PACs and pesticides, asymmetric synthesis, i.e., for the preparation which are designed to illicit biological action of pure optically active compounds. They are also used in studies for determining reaction Chiral Separation mechanisms, as well as reaction pathways. The separation of chiral compounds has been Chiral compounds are also important in the of great interest because the majority of agrochemical industries. Current methods of bioorganic molecules are chiral. Living enantiomeric analysis include such non- organisms, for example, are composed of chromatographic techniques as polarimetry, chiral biomolecules such as amino acids, nuclear magnetic resonance, isotopic dilution, sugars, proteins and nucleic acids. In nature calorimetry, and enzyme techniques. The these biomolecules exist in only one of the two disadvantages of these techniques are the possible enantiomeric forms, e.g., amino acids need for pure samples, and no separation of in the L-form and sugars in the D-form in our enantiomers are involved. Quantitation, which body. Because of chirality, living organisms does not require pure samples, and separation show different biological responses to one of a of enantiomers can be done simultaneously by either gas chromatography (GC) or high

477 IJRPC 2016, 6(3), 476-484 Purushoth Prabhu et al. ISSN: 22312781 performance liquid chromatography (HPLC). difficult. The decision relies mostly on Chiral HPLC has proven to be one of the best empirical data. Most chiral separations methods for the direct separation and analysis achieved on CSPs, however, were obtained of enatiomers. It is more versatile than chiral based upon the accumulated trial-and-error GC, because it can separate a wide variety of knowledge of the analyst, intuition, and often nonvolatile compounds. It provides fast and simply by chance. An alternative way of accurate methods for chiral separation, and choosing a CSP is by using predictive allows on-line detection and quantitation of empirical rules that have been developed both mass and of enantiomers based on empirical structures. Neither scheme if appropriate detection devices are used. of choosing a right CSP offers a guarantee for Current chiral HPLC methods are either direct, a successful enantiomeric separation. which utilizes chiral stationary phases (CSPs) Although enantioseparation is hoped to be and chiral additives in the mobile phase, or achieved by knowing the chemistry of the indirect, which involves derivatization of racemic analytes and the CSP sometimes, samples. Direct chiral separations using CSPs however, it does not work because the are more widely used and are more interactions of the mobile phase with both the predictable, in mechanistic terms, than those racemic analyte and CSP have to be using chiral additives in the mobile phase. considered. All three components, analyte, To date nearly a hundred HPLC CSPs have CSP, and mobile phase, must be taken into been developed and are commercially consideration when developing a chiral available. However, there is no single CSP separation method. The key, therefore, to a that can be considered universal, i.e., has the successfull enantioseparation of a particular ability to separate all classes of racemic class of racemates on a given CSP is the compounds. Choosing the right CSP for the understanding of the possible chiral enantioseparation of a chiral compound is recognition mechanisms.

PRINCPLES OF CHIRAL CHROMATOGRAPHY

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Fig. 1

Separation of optical isomers can be done by and therefore pass through the column more using chiral stationary phases. Different rapidly, under the force of the mobile phase. principle operates for different type of This separates the enantiomers in time and stationary phases and different samples. allows for stereoselective analysis. This The two enantiomers of the same analyte transient diastereomeric relationship can be compound differ in affinity of the single described by the equilibrium enantiomer to stationary phase and they exit the column (CHIRALCEL*, CHIRACEL* OA, Importance of enantiomeric separation CHIRALPAK*IA, CHIRALPAK*AD ) at different This technique is used for the separation of times. The chiral stationary phase can be many enantiomers of drugs like:- prepared by attaching a suitable chiral • Albuterol (anti-asthmatic inhalant) compound to the surface of an achiral support D-albuterol may actually cause airway such as silicagel. constriction Levalbuterol (L-albuterol) avoids side Chiral stationary phases effects Several chiral stationary phases (CSPs) have • Allegra (allergy medication) been developed and successfully used in drug Single enantiomer of Seldane that discovery, a major driving force behind chiral avoids life-threatening heart disorders chromatography. A brief introduction to of Seldane. generic chiral mechanisms and detailed • Fluoxetine ( depression medication) discussion of chiral stationary phases reported R-Fluoxetine – improved efficacy; to be used in environmental analysis of chiral minimizes side effects, i.e. anxiety and pharmaceuticals is given below. There are sexual dysfunction. Other indications many comprehensive reviews, the most up to (eating disorders) date include. Chiral stationary phases S-Fluoxetine – use for treatment of comprise a chiral molecule bonded to a migraines support, usually silica. Chromatography It also usefull in case of thalidomide because, columns rely on a substrate to approach the thalidomide as a chiral centere which allows stationary phase either under a force for two slightly different isomers (differing only propelling, or pulling, it to the stationary phase by the arrangements of their functional surface or through arbitrary collision with it. In groups).The two enantiomers are classified as chiral chromatography one of the analyte‟s either “S” / “R”. The R enantiomer is a enantiomers is then preferentially bound to the relatively safe drugs with sedative attributes, station-ary phase due to the spatial while the S enantiomer can devastating effect. arrangement of the atoms; the other The dangerous out come of thaliodomide in enantiomer may not bind at all or, more likely, the blood is that it recimises creating equal bind more weakly than its counterpart. This amount of both enantiomers, even ois only weaker bound enantiomer will have a shorter one kind was initialy present. transient relationship with the stationary phase

INSTRUMENTATION

Fig. 2: Chiral coloumns

479 IJRPC 2016, 6(3), 476-484 Purushoth Prabhu et al. ISSN: 22312781

Fig.3: Instrumentation of HPLC

Chiral liquid chromatography coupled with OJ-RH, etc. from Chiral Technologies, Inc. mass spectrometry is the method of choice in Chiralpak IA and IB…same chiral selectors as the analysis of chiral drugs. Examples of other AD and OD, respectively, but these are detectors such as Electron Capture Detectors, immobilized on the silica; more robust and has UV detectors or circular dichroism (CD) much greater solvent compatibilities detectors are in the minority even when expanding the search to related fields. Pirkle or Brush-type Phases: (Donor- Detection should be nonstereoselective. Acceptor) However, enantioselective ion suppression or Small chiral molecules bonded to silica enhancement caused by the matrix and their More specific applications; strong 3-point physical separation in time. interactions through 3 classes: • π-donor phases CLASSIFICATION OF CHIRAL • π-acceptor phases STATIONARY PHASES (CSP) • Mixed donor-acceptor phases POLYMER-BASED CARBOHYDRATES Binding sites are π-basic or π-acidic aromatic Chiral polysaccharide derivatives, i.e. amylose rings (π-π interactions), acidic and basic sites and cellulose, coated on a silica support (H-bonding), and steric interaction Enantiomers form H-bonds with carbamate Separation occurs through preferential binding links between side chains and polysaccharide of one enantiomer to CSP backbone steric restrictions at polysaccharide Mostly used with normal phase HPLC, SFC. backbone may prevent access of one of May get less resolution with RP-HPLC; enantiomers to H-bonding site can be used compatible with a broad range of solvents with normal phase HPLC, SFC, RP-HPLC Limitations: only works with aromatic Limitations: Not compatible with a wide range compounds of solvents other than alcohols Available columns: Whelk-O 1, Whelk-O 2, Available columns: i.e. Chiralpak AD, AD-RH, ULMO, DACH-DNB (mixed phases), -Burke AS, AS-RH, and Chiralcel OD, OD-RH, OJ, 2, β-Gem 1 (π-acceptor phases),

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Naphthylleucine (π-donor phases), from Regis Protein-based CSPs Technologies, Inc. Natural proteins bonded to a silica matrix Proteins contain large numbers of chiral Cyclodextrin CSPs centers and interact strongly with small chiral Alpha, beta and gamma-cyclodextrins bond to analytes through: Hydrophobic and silica and form chiral cavities 3-point electrostatic interactions, H-bonding interactions by: Limitations: Requires aqueous based • Opening of cyclodextrin cavity conditions in RP-HPLC Analyte must have contains hydroxyls for H-bonding with ionizable groups such as amine or acid. polar groups of analyte Not suited for preparative applications due to • Hydrophobic portion of analyte fits into low sample capacity non-polar cavity (inclusion complexes) Available columns: One enantiomer will be able to better fit in the Chiral AGP (-glycoprotein) from ChromTech cavity than the other used in RP-HPLC and & HAS (human serum albumin) from polar organic mode ChromTec Limitations: analyte must have hydrophobic or aromatic group to “fit” into cavity APPLICATIONS OF CHIRAL Available columns: Cyclobond (-, -, and - CHROMATOGRAPHY cyclodextrins) from Astec, Inc. ORpak CDA The process have been applied to a wide (), ORpak CDB (), ORpak CDC () from JM variety of natural products such as nucleic Sciences acid, urine, serum, carbohydrates, lipids, amino acids, bile acids, and manufactured Chirobiotic Phases products such as pharmaceuticals, pesticides, Macrocyclicglycopeptides linked to silica herbicides, surfactants and antioxidants.Mainly Contain a large number of chiral centers used for the separation of barbiturates. together with cavities for analytes to enter and Resolution of Single enantiomers simplify the interact human toxicology profile and can have Potential interactions: numerous benefits such as reduced side • π-π complexes, H-bonding, ionic effects and/or reduced dosage requirements. It interactions is used in industry primarily for separation of • Inclusion complexation, steric chiral molecules like Serine, Soman, interactions Glyceraldehydes, Phosphours, Sulfarmetal , Capable of running in RP-HPLC, normal Cobalt , Enkephalins. This technique is more phase, polar organic, and polar ionic modes important in case of drugs, because many Available columns: Chirobiotic V and V2 drugs shows different activities with different (Vancomycin), Chirobiotic T and T2 enantiomers (shown in table 1). (Teicoplanin), Chirobiotic R (Ristocetin A) from Astec

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Thalidomide thalidomide are unstable and spontaneously The use of thalidomide led to a tragedy in the epimerize to form the racemate in in-vivo in 1960sin Europe. The drug was prescribed to humans. The in- studies demonstrated the pregnants womens to counter morning hydrolysis products 5-hydroxy-thalidomide and sickness. Studies later suggested that this 5‟-hydroxy-thalidomide while in-vivo only the effects were caused by the S-enantiomer and 5‟-hydroxy metabolite was found, in low that the R-enantiomer contained the desired concentrations, in plasma samples from eight therapeutic activity. More recently, studies healthy male volunteers who had received have concluded that both enantiomers of thalidomide orally(Meyring et-al. , 2002).

Fig. 4

Amlodipin heart rate. The bronchodialator activity resides Amlodipine exhibit chirality, i.e, it exist as two in (R)-albuterol. (S)-albuterol, however, is not isomers. Moreover, the receptor binding inert, as it indirectly have proinflammatory studies have shown that it the S(-)isomer of effects.There are pharmacokinetic differences amlodipine that has L-type calcium channel between the enantiomer with (S)-albuterol blocking activity. The R(-)isomer exhibits a being cleared more slowly. The (S)- 1000-fold weaker calcium channel blocking enantiomer tends to accumulate in preference activity. Thus, the antihypertensive and to the therapeutically effective (R)-enantiomer. antianginal activity of amlodipine can be These pharmacokinetic and attributed only to S (-) amlodipine, racemic pharmacodynamics differences provided the amlodipine contains R(+) and S(+)isomer in basis for the chiral switch patent of albuterol to 1:1 ratio, purifying the pharmacologically levoalbuterol, (R)-albuterol, which has the active S(-)isomer can reduce the doseof same bronchodilator activity as racemic racemic amlodipine. albuterol, but has a superior side effect profile(Nowak, 2003).

Fig. 5

Fig. 6 Albuterol Albuterol is leading bronchodialator, an adrenoreceptor agonist that can increase bronchial airway diameter without increasing

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COMPARISON OF CHIRAL HPLC & CLASSICAL COLUMN CHROMATOGRAPHY Parameter Classical column chromatography Chiral HPLC Stationary phase particle size Large small Column size Large small Particle operating pressure Low high principle adsorption Vary with stationary phase & sample used Scale of operation Preparative scale Analytical & preparative scale cost Low- few hundreds High- few lakhs Sample load Low- medium Low – very low(µg) Type of column used Ordinary columns Chiral columns Separation of amino acids, dyes, alkaloids Separation of natural products(nucleic acid, Applications etc proteins etc), barbiturates etc.

o Maintenance of equipment is INSTITUTIONS PERFORMING CHIRAL difficult CHROMATOGRAPHY .  Analytical development laboratory CONCLUSION Hyderabad Chiral chromatography gives a new hope for  Institute of science and various extraction processes in technology Jawaharlal Nehru pharmaceutical industry. From this review, we technology university, Hyderabad concluded that chiral chromatography is a  Research and widely used extraction technique of 21st development,magafenepharma(p century. From this study we came to know ), Nasik about several applications of chiral  Daicel chiral technologies (india), chromatography in pharmaceutical industry Hyderabad. such as separation of chiral molecules like  Laboratory of govt. chemist Serine, Soman, Glyceraldehydes etc. Also Queens Road ,Teddington,Middle used for the separation of barbiturates sex TW11 OLY U.K .Resolution of Single enantiomers simplify the  Laboratory of element-organic human toxicology profile and can have chemistry Nankai university numerous benefits such as reduced side 300071,Tianjin P.R china. effects and/or reduced dosage requirements.  LPD lab services company. REFERENCES ADVANTAGES OF CHIRAL 1. File:/// G; /hmmm/chiral/ 20 column/ 20 CHROMATOGRAPHY chromatography/ 20, 20/1/2016.,  Toxicity of various drugs can be 5.16pm reduced 2. https://www.engg.ksu.edu/.../salvador.p  Less amount of solvents is df 3/2/2016., 8.30pm required 3. www.sigmaaldrich.com/.../supelco-4-  Direct sepration of enantiomers chiral... 3/2/2016 9.12pm. achived by changing CPS”s 4. Chromsci.oxfordjournals.org/…/753.full  This technique is used to …. 28/1/2016 8.45pm. eliminate teratogenic effect of S (- 5. www.ncbi.nlm.gov/pubmed/22959844 ) Thalidomide 28/1/2016 6.25pm  Used for the sepration of S(+) 6. Scholar,lib.vt.edu/theses/…/ch_01.pdf barbiturates because they does 1/2/2016 7.30pm. not produce any pharmacological 7. Wnendt S, Finkam M, Winter W, Ossig effect J, Raabe G and Zwingen-berger K.  This usefull for the sepration of Chirality. 1996;8:390–396. pharmacologicaly inactive 8. Hoglund P, Eriksson T and Bjorkman S.  R(-) Citalopram Journal of Pharmacokinetics and Biophar-maceuticals. 1998;26:363–383. LIMITATIONS OF CHIRAL CHROMATOGRPAHY 9. Ramsay CM, Cowan J, Flannery E, o Chiral silica gel is much more McLachlan C and Taylor TR. European expensive than standard Journal of Clinical Pharmacology. silicagel 1999;55(5):353–359. o Need for expensive column 10. Sanchez C, Bogeso KP, Ebert B, with chiral stationary phase Reines EH and Braestrup C. o It requires expensive Psychopharmacology. equipments 2004;174(2):163–176.

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11. Ruffolo RR, Spradlin TA, Pollock GD, 19. European Medicines Agency, Guideline Waddel JE and Murphy PJ. Journal of on the Environmental Risk Assessment Pharmacology and Experimental of Medicinal Products for Human Use, Therapeutics. 1981;219(2):447–452. C.f.M.P.f.H. Use, Editor, London, 2006. 12. https://books.google.co.in/books?isbn=9 20. iupac.org/pubblications/pac/69/7/…/pdf/ 40090861X 3/2/2026 8.12pm. 5/2/2016 5.44pm 13. www.us.edu.pl/.../acta/.../12_AC17.pdf 21. Davies NM and Teng XW. Importance 3/2/2016 7.55pm. of chirality in drug therapy and 14. Kasprzyk-Hordern B and Baker DR. pharmacy practice: Implications for Science of the Total Environment. psychiatry. Advances in Pharmacy. 2012;423:142–150. 2003;1:242–252. 15. https://pooya.um.ac.ir/.../1012689.pdf 22. Wolf C. Dynamic of 4/2/2012 6.50pm chiral compounds: principles and 16. Yacobi A and Levy G. Journal of applications. Chapter 2, Published by Pharmacokinetics and Royal Society of Chemistry. 2008:6. Biopharmaceutics. 1977;5(2):123–131. 23. Stinson SC. Chiral pharmaceuticals. C& 17. Ja¨ger W, Mayer M, Reznicek G and EN 2001; 79: 79-97. Buchbauer G. Journal of Pharmacy and 24. Lib.dr.iastste.edu/cgi/viewcontent.cgi.. Pharmacology. 2001;53(5):637–642. 6/2/2012 7.36pm. 18. Walle T, Webb JG, Bagwell EE, Walle 25. www.clinchem.org/content/45/5/593.full UK, Daniell HB and Gaffney TE. 6/2/2026 7.50pm. Biochemical Pharmacology. 1988;37(1):115–124.

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