Methods for the analysis of enantiomers of racemic drugs application to pharmacological and pharmacokinetic studies

JPM Vol. 29, No. 1

February 1993:1-9

REVIEW ARTICLES

Methods for the Analysis of Enantiomers of Racemic Drugs Application to Pharmacological and Pharmacokinetic Studies M a t t h e w R. Wright and F a k h r e d d i n Jamali Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

Although the existence and differences in biological behavior of optical isomers have long been appreciated, there has been an apparent reluctance to address these differences in pharmacology and the pharmaceutical sciences. At least part of this reluctance arises from the belief that the separation of enantiomers requires highly specialized analytical equipment and expertise. The purpose of this review is to present general principles that allow the separation of stereoisomers and demonstrate that these procedures can be accomplished using available and convenient chromatography techniques.

Keywords: Racemic drugs; Enantiomers; Optical isomers; Stereoisomerism

Introduction Although the asymmetry of organic molecules has been known since the time of Pasteur, the implications of optical isomerism, in terms of pharmacological activity and/or pharmacokinetic behavior, have only been seriously addressed in the pharmaceutical sciences during the last 20 years. The differing pharmacological and pharmacokinetic behavior of enantiomers has been relatively well reviewed (Simonyi, 1983; Ariens, 1984; Williams and Lee, 1985; Jamali et al., 1989) and it would be beyond the scope of this manuscript to describe these differences fully. Despite the widespread appreciation of the potential pharmacological and pharmacokinetic differences of enantiomers and the large body of current literature addressing these issues, there is still some apparent reluctance among investigators to examine stereoselective phenomena. Much of this reluctance apparently stems from a gen-

Address reprint requests to Dr. F. Jamali, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2N8. Received October 2, 1992; revised and accepted November 18, 1992. Journal of Pharmacologicaland Toxicological Methods 29, 1-9 (1993) © 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

eral feeling that to separately quantitate the enantiomers of a given drug from a fluid or biological fluid matrix is an analytically difficult process that requires special expertise. This, however, for many drugs is a misconception, and stereospecific analytical methods may be developed that require no more specialized knowledge than does general chromatography and drug extraction. The purpose of this review, therefore, is to present the basic principles that allow the separation of the enantiomers of drugs by chromatographic procedures.

Terminology An object that is not superimposable on its mirror image is said to be chiral. In chemical terms the most common situation giving rise to chirality is an sp 3 hybridized carbon atom to which four different groups are attached. The molecule to which such a carbon atom belongs has no internal plane of symmetry and is called a chiral molecule. A chiral molecule with one chiral carbon atom exists as a pair of enantiomers (i.e., two molecules that are nonsuperimposable mirror images of one another). Enantiomers have similar physicochemical properties to one another (e.g., melting point,

1056-8719/93/$6.00

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JPM Vol. 29, No. 1 February 1993:1-9

partition coefficient) with the exception of the direction in which each will rotate a plane of polarized light. This property gives rise to the term optical activity and, in addition, often leads to enantiomers being called optical isomers. Despite the similarity of the physical properties of enantiomers, it is abundantly clear that their biological properties (e.g., pharmacological activity, rate of metabolism, rate of excretion) may be very different from one another (Hurt and Caldwell, 1984; Jamali et al., 1989). The ability of enantiomers to rotate polarized light gives rise to one of the earliest classification systems. An enantiomer that rotates light in the clockwise direction is said to be dextrorotatory [denoted by d or ( + )], whereas the other enantiomer is said to be levorotatory [denoted by I or ( - ) ] . A mixture or a compound of equal parts of these enantiomers will not rotate polarized light and is termed a racemate [denoted by (___)]. Prior to 1951, optically active compounds were also described by their relative configuration as compared to (+)-glyceraldehyde, the absolute configuration of which had been assumed. As originally developed, this system was used primarily with carbohydrate molecules. The molecule was drawn in Fischer projection and, if the hydroxyl group of the chiral carbon atom farthest away from carbon 1 was to the right, then the molecule was denoted as o. If the hydroxyl of the last chiral carbon was to the left, then the molecule was denoted as L. However, because the direction of rotation of polarized light is a physical property, whereas the absolute configuration is characteristic of the molecular structure, there is no simple relationship between the two. Thus an enantiomer of glyceric acid with the same absolute configuration as (+)-glyceraldehyde is actually levorotatory (Fessenden and Fessenden, 1980). A further limitation of this system is that it does not specify the configuration of any other chiral carbon in the molecule except the one farthest from carbon 1. This system does, however, allow carbohy-

Enantiomers

drate structures to be quickly related to one another. The o and L nomenclature has fallen into much less common use in recent years but still is present in some literature. More recently, the Cahn-Ingold-Prelog system has been used to assign the absolute configuration of a molecule (Cahn et al., 1956). This leads to the designation of molecules as (R) or (S) in addition to allowing the direction of rotation of polarized light to be denoted. Fortunately for the pre- 1951 stereochemical literature, D-glyceraldehyde has an R absolute configuration and, thus o and L compounds have R and S absolute configurations, respectively. It is also possible for molecules to have more than one chiral carbon atom. For such a molecule, the maximum number of optical isomers is 2 n, where n is the number of chiral carbon atoms in the molecule. For simplicity, consider the case of a molecule with two chiral carbon atoms (Figure 1). For such a compound there are four optical isomers, two pairs of enantiomers (R,R & S,S and R,S & S,R). However, within this group of four optical isomers, there are also compounds that are not enantiomers. Stereoisomers that are not mirror images of each other (i.e., that are not enantiomers) are termed diastereomers. The term diastereomer, however, is not restricted to optical isomers but also includes geometric isomers (e.g., cis and trans or E and Z isomerism). In contrast to enantiomers, diastereomers may differ in their physical properties to one another (e.g., melting point, solubility) as well as in their biological properties. The general approach to the chromatographic separation of enantiomers has been to form diastereomers either irreversibly through the covalent reaction with another optically pure compound, or reversibly through interaction with an optically pure component within the mobile phase or through the use of an optically pure chiral stationary phase. Because diastereomers, either transient or permanent, have different

Enantiomers

I CHO

CHO

CHO

I

i

I

H--C--OH

I H--C--OH

HO--C--H

H--C--OH

I

I

HO--C--H

HO--C--H

CHO

I HO--C--H

I H--C--OH

[

I

I

I

CH20H

CH2OH

CH2OH

CH2OH

Diastereomers

I (2R, 3R)

II (2S, 3S)

III (2R, 3S)

IV (2S, 3R)

Figure

1. Fisher droxybutanal.

projection

of

2,3,4-tri-hy-

M. R. WRIGHT AND F. JAMALI KINETICS-DYNAMICS OF ENANTIOMERS

3

physico-chemical properties, their retention on a chromatographic column will be different. The aims of this review are to describe the general methods available for the HPLC separation of enantiomers on an analytical scale and their inherent strengths and weaknesses.

Pharmacological Significance of Stereoisomerism Although most drugs of natural origin are optically active (i.e., they are not created in nature as a racemate), the majority of those synthesized chemically in a laboratory are produced as an optically inactive racemate (i.e., equal proportions of enantiomers) (Drayer, 1986). Certainly the trend in the recent past has been that more chemically synthesized drugs have come to market than have those of natural origin and, as a result, a large number of currently available pharmaceuticals are administered as racemates. Table 1 presents a list of examples of chiral drugs divided by therapeutic category and demonstrates that the influence of stereochemistry may be seen across diverse pharmacological and chemical groups. Perhaps the most readily appreciated influences of stereochemistry are those that affect either the nature or the magnitude of the pharmacological effect(s) measured. The naturally occurring alkaloids quinine and quinidine are optical isomers but have significantly different pharmacological activities (White et al., 1981, 1983). Similarly, the differing affinities for the S-(+) and R-( - ) enantiomers of [3-blocking drugs for binding at the 13-adrenoreceptor have also been widely recognized (Walle et al., 1988). Although these stereochemi-

cal differences may be most readily appreciated by pharmacologists, there are a host of other possible effects of stereochemistry (partially represented in Table 1) that can profoundly affect the interpretation of in vivo studies in particular. Although it would be impossible to exhaustively list all of the possible effects of stereochemistry, some representative examples will illustrate this last point and demonstrate the need for the quantitation of drug by stereospecific methods. The 13-blocker propranolol has been shown to undergo extensive and stereoselective first-pass metabolism in humans (Von Bahr et al., 1982). Furthermore, its pharmacological activity has been largely attributed to the S - ( - ) enantiomer (Barrett and Cullum, 1968). The observation, therefore, of the apparent right shift, following intravenous drug administration, in the dose-response curve as compared to an oral dose is quite difficult to interpret if the plasma concentrations determined are for total (R + S) drug (Figure 2). Using stereospecific assay methods, however, would demonstrate that this apparent incongruity is the result of the S-( - ) enantiomer being more bioavailable following an oral dose than is the R-(+) enantiomer (Von Bahr et al., 1982). Thus, in Figure 2, the total plasma concentrations depicted after the i.v. and oral dose of racemic propranolol, represent different ratios of the pharmacological active [S-(-)] and much less pharmacologically active JR-( + )] enantiomers. The calcium channel blocker, verapamil, is marketed as a racemate and has also received significant attention as a classical example of stereoselective firstpass metabolism (reviewed by Jamali, 1989). The S-(-)-enantiomer which has the greater negative

Table 1. Examples of Chiral Drugs Therapeutic Class

Example

Nonsteroidal antiinflammatories

Ibuprofen Etodolac

13-Blockers

Propranolol Metoprolol

Ca 2÷ channel blockers 13-Adrenergic agonists Antiarrhythmics Analgesics

Verapamil Salbutamol Disopyramide Methadone Propoxyphene

Antibiotics Anticoagulants Barbiturates Diuretics Antimalarials Antineoplastics

Moxalactam Warfarin Hexobarbital Indacrinone Primaquine Cyclophosphamide

Source: Jamali et al., 1989.

Comments Significant unidirectional inversion of enantiometers (R --9 S) noted Plasma concentration of the pharmacologically active enantiomer IS-( + I] "~ than the concentration of the inactive enantiomer [R-(-)] 13-blocking activity of S > R; Spermicidal activity of S = R; stereoselective first-pass metabolism Pharmacological activity of S > R; Stereoselective genetically controlled metabolism Enantiomers of differing pharmacological activities and kinetics Pharmacological activity of R > S Pharmacological activity of S(+) > R ( - ); protein binding of S > R in humans Pharmacological activity of R( - ) > S( + ) S enantiomer has analgesic potency, R enantiomer has antitussive potency; significant pharmacokinetic interaction between enantiomers Pharmacological activity of R > S; Undergoes reversible epimerization Pharmacological activity of S-( - ) > R-( + ) Pharmacological activity of S > R; clearance of S < R Naturetic effects of R > S; Uricosuric effects of S > R; ( + ) and ( - ) equipotent Equipotent enantiomers

4

JPM Vol. 29, No. I February 1993:1-9

100 8O 60

y'/

4o 2o ~, 0

l'o 2;

i;o 2o'o

Plasma propranolol concentration, ng/mL

Figure 2. Apparent rightward shifting of the dose-response

curve for racemic propranolol following intravenous (open circles) and oral (solid circles) administration (from Jamali et al., 1989, reproduced with permission).

dromotropic activity (Echizen et al., 1985) has twoto three-fold lower oral bioavailability than does the R-(+)-enantiomer. However, both enantiomers are equally effective in producing coronary vasodilatation in dogs (Drayer, 1986). Significant stereoselectivity has been also noted in the protein binding, clearance, and volume of distribution of verapamil enantiomers in humans. Clearly, in vivo studies of the pharmacological activity of verapamil must take into account the stereochemical composition of the drug concentrations measured. The metabolism of many drugs, including the [3blocker metoprolol, has been shown to be controlled by debrisoquine:sparteine genetic polymorphism (Francis et al., 1982). If dose-response curves are constructed for poor and extensive metabolizers, respectively, based on total drug (Figure 3), then there is an apparent right shift of the concentration-effect curve

Figure 3. Apparent rightward shift of the dose-response

curve for racemic metoprolol for poor metabolizers (debrisoquine/sparteine) (PM) as compared to extensive metabolizers (EM) (from Jamali et al., 1989, reproduced with permission). 40

for poor metabolizers. This phenomenon occurs due to lack of stereoselectivity in first-pass metabolism of the relatively inactive enantiomer [R-(+)-metoprolol] (Lennard et al., 1983). Again, this example highlights the importance of quantifying individual enantiomers when forming dose-response relationships for racemic drugs with enantiomers of differing potencies and/or activities. Some nonsteroidal 2-arylpropionic acid antiinflammatory drugs (e.g., ibuprofen, fenoprofen) undergo unidirectional inversion from the inactive R-enantiomer to the active S-enantiomer (Jamali, 1988). Thus, not only are nonstereospecific assay methods not useful in interpreting the time course of the individual enantiomers but would be also extremely misleading in relating either therapeutic or toxic effects to the total (racemic) drug concentration. Another nonsteroidal antiinflammatory drug of interest, is etodolac, a pyranocarboxyoxylic acid derivative. Although the pharmacological activity resides predominantly with the S-( + )-enantiomer, the plasma concentrations of S-( + )-etodolac are much lower than those of the R-(-)-enantiomer (Jamali et al., 1988). This difference is attributable to an approximate tenfold larger volume of distribution for the S-( + )-etodolac as compared to the R-( - )-enantiomer (Jamali et al., 1988). For etodolac, therefore, any interpretation of effects based on nonstereospecific measurements can be misleading. A large body of literature has begun to examine the pharmacokinetic and pharmacodynamic influences of stereochemistry (Evans et al., 1988; Ariens et ai., 1988; Eichelbaum, 1988; Drayer, 1986; Walle and Walle, 1986; Mehvar, 1992). It should be noted that, in the absence of stereoselective drug measurement, the observations made previously in this manuscript and those present in the previously cited literature could not be interpreted in a scientifically reasonable manner. Thus, it is of significant importance that the individual concentrations of enantiomers be determined for drugs administered as racemates, particularly when the enantiomers have differing pharmacological activities or potencies.

Chromatographic Methods of Separation of Enantiomers

3O

Chiral Stationary Phases

o~

2O

Y

I0

I

100

I

]OOO

Concentration, ng/mL

The principle of direct resolution of enantiomers using a chirai stationary phase is based on a three-point interaction between solute and stationary phase (Dalgleish, 1952; Pirkle and Finn, 1983; Davankov and Kurganov, 1983). Under this model there are at least three possible interactions between the solute and stationary phase which may include hydrogen bonding, di-

M. R. WRIGHT AND F. JAMALI KINETICS-DYNAMICS OF ENANTIOMERS

D

5

n

I i !,1 Figure 4. Illustration of the "three-point interaction" between enantiomers of drug with functional groups A, B, C, and D and a chiral stationary phase with complementary binding sites A', B', and C'.

pole-dipole, rr-~r, electrostatic, hydrophobic or steric interaction. Because the enantiomers differ in the arrangement of their atoms in space, one enantiomer will have greater interaction with the stationary phase (i.e., will be retained to a greater extent) than the other enantiomer (Figure 4). Using these methods, we note a great many separations have been achieved (Allenmark, 1984). There are four general types of chiral stationary phase available for use in HPLC, roughly based on the types of interactions possible between solute and stationary phase (Krstulovic, 1989). The first commercially successful phases were the (R)-N-(3,5-dinitrobenzoyl) phenylglycine type developed by Pirkle and Finn (1983). The interactive forces are hydrogen bonds, ~r--rr, and dipole-dipole in nature, and these phases often require precolumn derivatization of the solute (Wainer, 1986; Doyle et al., 1986; Wainer et al., 1983a and b; Feibush et al., 1986). These columns are most commonly used under normal-phase conditions (e.g., hexane/isopropranol mobile phases. The second group of phases are those based on either cellulose esters to which various terminal groups have been attached (Chiracel), et- or [3-cyclodextrin (Cyclobond) or polyacrylamide (Chiralpak). There are several different Chiracel phases available with the Chiracel OD (tris (3,5-dimethylcarbamate)-cellulose) phase being extremely popular for the separation of drugs, especially [3-blockers (Krstulovic, 1989). The mechanisms of interaction are not well understood but include hydrogen bonding, "rr-'rr, and dipole-dipole interaction, and occur within the cavities formed by these molecules, in contrast to the Pirkle-type phases where interaction is thought to occur on the surface of the stationary phase (Blaschke, 1986). Similar to the Pirkle-type phase, the polysaccharide-based phases are used under normal-phase conditions. The observation of stereochemical differences in the extent of protein binding of drugs has led to the development of chiral stationary phases based on alphar acid-glycoprotein (AAG) (EnantioPac, Hermansson, 1983) and bovine serum albumin (BSA) (Resolvosil, Allenmark et al., 1982). Numerous effects contributing

to the separation of enantiomers on these phases include hydrogen bonding, electrostatic interaction, hydrophobic interactions, charge-transfer reactions (on BSA) and ion-pairing (on AAG) (Krstulovic, 1989). Generally, polar mobile phases consisting of a buffer with organic modifiers are used for separation with these phases. The final group of HPLC stationary phase, much less commonly used than the three previously mentioned groups, is the ligand exchange phase. In this group an amino acid, such as L-proline, is bonded to silica gel and treated with a copper sulphate solution. During chromatography a ternary complex is formed between the L-proline, copper ion, and the analyte. Separation is based on the differences in stability of the complexes of the various analytes. The usual mobile phase is a buffered 0.25 mM solution of copper sulphate (Mehta, 1988). In general, the separation of enantiomers using a chiral column is very sensitive to changes in temperature and mobile phase composition (i.e, pH, polarity, ionic strength and composition) (Mehta, 1988). This sensitivity is thought to result because chiral recognition requires multiple selective interactions between the analyte and stationary phase (Armstrong, 1987). Without precolumn derivatization to improve resolution and/or improve detection, chiral HPLC columns generally have lower efficiencies than do conventional columns that often limit the resolution of enantiomers (Krstulovic, 1989). The low flow rates employed in analytical methods using chiral columns (<0.8 mL/min) often result in prolonged analysis times and reflect the apparently slow mass transfer between mobile and stationary phases (Mehta, 1988). The chromatography, including enantiomeric separation, of the [3-blockers has been recently well reviewed (Davies, 1990). A number of methods that use chiral stationary phases, both with and without precolumn derivatization, for a wide variety of [3-blockers is presented (Davies, 1990).

Chiral Mobile Phases The addition of an optically active molecule to the mobile phase may allow the separation on conventional (nonchiral), HPLC stationary phases of optically active solutes from a biological sample through the formation of covalent or noncovalent diastereomeric complexes (Schill, 1989). Although it is possible to use covalently bonded complexes, more commonly coordinatively coupled complexes are preferred (Schill, 1989). Similar to the requirements for separation of enantiomers on chiral columns, at least three points of interaction are required between the chiral solute and the chiral mobile-phase additive which will produce diastereomers with different stabilities or

6

strengths of interaction with the column (Pettersson and Schill, 1981). Enantiomers with an acidic functional group may be separated by the addition ofa chiral aminoalcohol to the mobile phase. Schill (1989) demonstrated the use of quinidine (3 x 10 - 4 M) and quinine (3.5 x 10 -4 M) to separate the enantiomers of a number of carboxylicacid-containing compounds, including naproxen and 2phenylpropionic acid, under normal-phase conditions. In general, the mobile phase must be of low polarity so as to allow the diastereomeric ion pairs sufficient stability for separation to occur (Schill, 1989). Further, if the mobile-phase additive has an ultraviolet (UV) or fluorescent chromophore, then it can be used to detect a chiral solute that does not possess such a chromophore. Conversely, amine-containing enantiomers may be separated by the addition of an optically pure acid to the mobile phase. Derivatives of camphorsulphonic acid have been widely used for this purpose (Dennis, 1986; Pettersson and Schill, 1981). Again, the polarity of the mobile phase is usually low such that the diastereomeric ion pairs are stable (Pettersson and Schill, 1981). Other ion-pairing reagents that have been used for the separation of enantiomers include an optically active Cu(II)-proline, largely to separate derivatized or underivatized amino acids (Dennis, 1986). Higher selectivities in the separation of aminoalcohol enantiomers (e.g., propranolol) have also been achieved through the use of Z-glycyl-L-proline (2.5 x 10 -3 M) (Schill, 1989). Using this reagent, we note that a wider choice of mobile phases is possible and the extent of retention can be controlled through the use of a competing amine (e.g., triethylamine) (Schill, 1989).

Chiral Derivatization Techniques Chiral derivatization techniques are based on the formation of a covalent bond between the enantiomers of a chiral molecule and an optically pure reagent such that stable diastereomeric complexes are formed. The diastereomeric complexes may then be separated using conventional normal- or reverse-phase liquid chromatography. The covalent bond is generally formed between a functional group of the molecule of interest and a reactive derivatizing reagent. The nature of the chemical reaction and the covalent bond formed depends upon the molecule of interest and the derivatizing reagent used. The functional groups amenable to derivatization, suitable derivatizing reagents, the chemical reaction and end product of commonly performed reactions for the separation of enantiomers are listed in Table 2. Although in most cases the reactions with chiralderivatizating reagents (CDR) will produce an expected end product, the possibility of more complex

JPM Vol. 29, No. 1 February 1993:1-9

reactions (e.g., where more than one functional group can react) must not be ignored. In these instances it is desirable to confirm the structure of the derivatives by suitable analytical means (e.g., mass spectrometry, NMR, elemental analysis). Further, the derivatization reaction should occur under relatively mild chemical conditions such that degradation of the reactants and epimerization of the chiral components are avoided The derivatization reaction should also proceed until as near completion as possible.

Advantages and Disadvantages of the Methods of Separation of Enantiomers Although there is a relatively large body of literature examining each of the previously mentioned techniques in isolation, very little attention has been given to direct comparison of the methods with one another. The use of chiral stationary phases has been referred to as direct resolution (Allenmark, 1984), whereas the use of either chiral mobile-phase additives or chiralderivatizing reagents has been termed indirect resolution (Gal, 1988). One of the most frequently cited potential problems with the indirect resolution has been the possibility of enantiomeric impurity and/or racemization of the chiral mobile-phase additive or chiral-derivatizing reagent (Gal, 1988). Enantiomeric impurity or racemization of the chiral-derivatizing reagent would be a problem as it would lead to the formation of enantiomeric pairs of molecules which would be resolvable only into two diastereomeric peaks (e.g., R/ S-drug + R/S-CDR ~ R-drug-R-CDR + R-drug-SCDR + S-drug-R-CDR + S-drug-S-CDR; note that two pairs of inseparable isomers would be formed (R,R), (S,S) and (R,S), (S,R). Such problems have been encountered with the use of (S)-N-trifluoroacetylpropyl chloride, which has been used to acetylate amino functional groups (Adams et al., 1982). It should be noted, however, that the majority of cases where racemization has been cited as a problem have occurred when the investigators have synthesized compounds that were not available commercially. It is prudent then to recognize that some chiral reagents do suffer from racemization problems, and these should probably not be used, but most have excellent stereochemical stability (Gal, 1988). Further, although the enantiomeric purity of the chiral reagents may be less than 100%, the stereochemical purity of most is greater than 99% and does not present a serious drawback for most investigations (Gal, 1988). Another frequently suggested problem with chiral derivatization is the possibility of unequal rates of reaction between the chiral-derivatizing reagent and the enantiomers (e.g., Krstulovic, 1989). This potential problem may be overcome by using an excess of the chiral-derivatizing reagent and through characterization of the time course of the derivatiza-

M. R. WRIGHTAND F. JAMALI KINETICS-DYNAMICSOF ENANTIOMERS

7

Table 2. Typical Derivatization Reactions for Enantiomeric Separation using HPLC Functional Group Amine

CDA (1°, 2°) Acid halide Acid anhydride Chloroformate Isocyanate Isothiocyanate 2-oxazolidones

Product

General Reaction R-NH2 + XCOR--* R-NCOR X = CI, Br, I, OOCR R-NH2 + CICOOR' ~ R-NCOOR' R-NH2 + OCNR' ---, R-NCONHR' R-NH2 + SCNR' ~ R-NSCNHR' R-NH2 O ~ O O II O II A II o

)

O

U--C--CI

t

Amide Carbamate Urea Thiourea Allophane

Example Adams et al., 1982 Coleman, 1983 Seeman et al., 1985 Thompson et al., 1982 Gal and Brown, 1986 Pirkle and Simmons, 1983

N--C--NH--R

R Hydroxyl

(1°, 2°, phenol) Acid halide Acid anhydride Acid

R-OH + XCOR' ~ R-OCOR' X = CI, Br, I, or = OOCR', or = HO R-OH + NCCOR' ~ ROCOR' R-OH + C1COOR' ~ ROCOOR' R-OH + R'NCO ~ ROCONHR'

Ester

Banfield and Rowland, 1984 Shimizu et al., 1982

Ester Carbonate Carbamate

Linder et al., 1984 Brash et al., 1985 Williams, 1984

Alcohol Amine

RCOOH + R'OH -* RCOOR' RCOOH + R'NH2--* RCONR' OH

Ester Amide

Lee et al., 1984 Bjorkman, 1985

Thiol

R/L'R

Thioalcohol

Armstrong et al., 1981

Aminoalcohol

Panthananickal et al., 1983

Thiourea

Gal, 1985

Ureide

Pirkle et al., 1984

Nitrile Chloroformate Isocyante" Carboxyl

Epoxide

'

+ HS-R"---~R-RI'-S-R OH

Amine

i

R

R° + R"-NH2 ~ R-R'-NH-R" OH

Alkylamine/ isothiocyante

i

R

R' + R"-NH2 ~ R-R'-NH-R"

R-R'-NH-R"+ SCNR" ~ R-R'-NR"-C(S)-NH-R" ~)H

Lactam

0

II

Isocyanate

R ~ H

+ OCNR' ---~

O

II

O

Abbreviations." CDA, Chiral derivatizing agent. a

Isothiocyanates are less reactive than the corresponding isocyanate with alcohols (March, 1968).

tion r e a c t i o n (Gal, 1988). S i m i l a r c o m m e n t s c a n b e m a d e for the chiral r e a g e n t s a d d e d to the m o b i l e p h a s e to effect e n a n t i o m e r s e p a r a t i o n . I n g e n e r a l , the a s s a y p r o c e d u r e s e m p l o y i n g derivatiz a t i o n with a chiral r e a g e n t are v e r y facile, cost a n d time effective. A t y p i c a l a s s a y u s i n g chiral d e r i v a t i z a tion w o u l d p r o c e e d s i m i l a r to that o f W r i g h t et al. (1992) for the n o n s t e r o i d a l a n t i i n f l a m m a t o r y drug, ibup r o f e n . T h e acidified p l a s m a is e x t r a c t e d into isooct a n e / i s o p r o p a n o l , a n d the o r g a n i c l a y e r is s e p a r a t e d a n d e v a p o r a t e d to d r y n e s s . T h e r e s i d u e is r e c o n s t i t u -

ted in t r i e t h y l a m i n e in a c e t o n i t r i l e a n d d e r i v a t i z e d with e t h y l c h l o r o f o r m a t e for 1 rain f o l l o w e d b y R-(+)-otp h e n y l e t h y l a m i n e for 2 m i n . T h e d e r i v a t i z e d s a m p l e is t h e n acidified a n d e x t r a c t e d i n t o c h l o r o f o r m . A l t h o u g h this p a r t i c u l a r d e r i v a t i z a t i o n r e a c t i o n t a k e s place at r o o m t e m p e r a t u r e , in s o m e c a s e s e i t h e r h e a t i n g or mic r o w a v e t r e a t m e n t o f the r e a c t i o n m i x t u r e m a y b e req u i r e d to c o m p l e t e the d e r i v a t i z a t i o n r e a c t i o n . T h e org a n i c l a y e r is s e p a r a t e d , e v a p o r a t e d to d r y n e s s , a n d t h e n r e c o n s t i t u t e d in m o b i l e p h a s e . A l i q u o t s o f 10-150 txL c a n t h e n b e i n j e c t e d o n t o a C~8 c o l u m n , a n d the

8

ibuprofen enantiomers can be separated using a mobile phase of acetonitrile/water/acetic acid/triethylamine. Using such a procedure 50 biological fluid samples may be prepared for analysis in approximately 2.5 hr. Much attention has been given to the' 'direct" resolution of enantiomers on optically active stationary phases (e.g., Wainer, 1988). The major potential advantage of separation by this method is that, generally, no derivatization is needed and, therefore, sample preparation is simpler than when derivatization is involved. Further, the previously mentioned problems ofenantiomeric purity of derivatizing reagents and stereoselective rates for derivatization are obviously avoided. However, several "direct" assay procedures require preseparation of the racemic drug on a conventional column prior to enantiomeric separation on a chiral phase (e.g., McClachlan et al., 1991; Iredale and Wainer, 1992). This separation prior to the chiral column was required to remove interference from endogenous substances and from drug metabolites formed in vivo. Chiral stationary phases are not without further disadvantages. As mentioned previously, the relatively low efficiency (Krstulovic, 1989) of these columns and the low flow rates used (Mehta, 1988) lead to generally long sample-run times, diminished resolution between the enantiomers, and possibly decreased sensitivity as compared to resolution on conventional columns. In addition, not all mobile phases can be used with chiral columns and these columns do not, in general, last as long as do conventional columns. Further, the preparation of these columns requires the use of optically active chemicals and, therefore, the problems of the optical purity of these substances and the potential for racemization during use should be also addressed. Also, the cost of chiral columns is on the order of up to four times greater than a similar-length conventional column. In theory it should be possible to separate enantiomers of drugs with no functional groups amenable to derivatization on a chiral stationary phase. This, however, would be extremely molecule dependent requiring the presence of relatively large chemical groups (e.g., phenyl ring) close to the site of chirality for separation to occur. An alternative for structures with rings containing heteroatoms would be to cleave the ring chemically in order to create a structure amenable to derivatization. In summary, although all of the commonly used methods for enantiomeric separation have their own set of advantages and disadvantages, all have been used effectively in both stereoselective pharmacokinetic and pharmacological studies involving a wide variety of substances. All the methods presented here can be used without any more extensive chromatographic equipment or expertise than the conventional HPLC techniques. Given the well-documented differences in both pharmacokinetic and pharmacological behavior

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of many stereoisomers and the relative ease of their separation, there is little rationale for not generating stereoselective data. References Adams JD, Jr, Woolf TF, Trevor AJ, Williams LR, Castagnoli N, Jr (1982) Derivatization of chiral amines with (S,S)-N-trifluoroacetyl proline anhydride for estimation of enantiomeric composition. J Pharm Sci 71:658-661.

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