THE DETELOPMENT OF A STEREOSPECIFIC ASSAY FOR SELEGILINE HM>ROCHLORIDE

Maureen A. McLaughlin

A thesis submitted in conformity with the requirements for the degree of Master of Science in Pharmacy Graduate Department of the Faculty of Pharmacy University of Toronto

O Copyright by Maureen A. McLaughiin (1997) National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et 8ibliographic Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K1A ON4 ûttawaON KIAOiU4 Canada -da Your itk votre reterenm

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or othemise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The Development of a Stereospecific Assay for Selegiline hydrochloride

Maureen A. McLaughlin Master of Science 1997 University of Toronto, Faculty of Pharmacy

Seiegihe ([RI [LI - (-) - N-, a -dirnethyl - N-(prop-2-ynyl) phenylethylarnine hydrochloride) is a potent type B monoamine oxidase inhibitor used in the heatment of

Parkinson's disease. The major metabolites ((-) amphetamine and (-) methamphetamine]

of Çeleghe are devoid of cenbal nervous system stimulation, however a small amount

of (+) Seleghe wodd produce the cenbal nervous system stimulants (+) amphetamine

and (+) methamphetamine. Therefore, a chiral assay for Selegiline drug substance and

tablets has been developed by derivatization with (-) menthyl diloroformate followed by separa tion of the dealkylated carbamates on a silica column.

The reaction of Selegiline with (-) menthyl diloroformate consistently resulted in

the formation of one product Kising from the deavage of the propargyl group. The developed assay procedure has been applied to the determination of (-) SelegiLine in commercial tablet preparations. Analysis of ten tablets showed a recovery of 99.70h

(+ 1.4) of the label daim. The assay is not suffiuently sensitive to alIow determination of

Selegiline in biological samples. In atternpts to potentiaiiy increase sensitivity additional experiments with two achiral chloroformates, ethyl chloroformate and 9-fluorenyl methyl chloroformate were performed. Experiments with these two adiiral reagents resulted in the formation of a mixture of produa carbamates. Acknowledgments

1 would like to express my sincere gratitude to my advisor Dr. J. Barry Robinson for bis patience and advice throughout my research endeavors. In addition 1 wodd like to thank the other members of my thesis cornmittee; Dr. J. 1. Thiessen, Dr. M. Spino and Dr. J. R. Ballinger for their time and interest in my work. The financial support (Grant in Aid) provided by Apotex Inc. was greatly appreciated. The support and friendship of my fellow graduate students: Susan, Gay, Mike and Rosa will always be remembered. Lastly I would like to thank the anonymous Parkinson patient who generously provided me with the Selegiline tablets which were used in this research. Table of Contents Introduction...... ,,...... 1 Stereochemical Concepts...... 6 Stereochemical Techniques ...... 10 Chromatography ...... 14 Ma thematical Principles of Chromatography ...... 21 Chiral Separations-A Direct Approach using Chird Stationary Phases ...... 26 Chiral Separation-Direct Separations using Chiral Mobile Phase Additives ... 35 Chiral Separation via the Indirect Method Using Chiral Derivatizing Agents ...... 37 Chiral Separations of Amines with Particular Reference to Amphetamine and its Analogues ...... 45 Discussion of Experimental ...... 56 Physical. Chemical Characteristics of Selegiline hydrochloride ...... 56 Preliminary Studies of Po tential Assay Procedures ...... 56 Chiral Columns and Chiral Additives ...... 57 Reverse Phase Separa tions ...... 59 5 mg Preparation .Methamphetamine and Selegiline carbama te ...... 64 Reverse Phase Chromatography .Lntemal Standard Selection ...... 65 Normal Phase Separations ...... 3 Normal Phase Chromatography- Interna1 Standard Selection ...... 77 Preparation of Calibration Cumes .Methamphetamine ...... 81 . . Preparation of Calibration Cumes .Seleplme ...... 82 Application of the Assay to Seelegiline hydrochloride Tablets ...... 86 Studies Employing other Chloroformate Reagents ...... eJ37 Studies wi th 9-Fluorenylmethyl Chloroformate (FMOC) ...... 89 Experimental ...... 97 Achiral Separation .Reverse Phase Chromatographie Conditions ...... 98 Chiral Separation .Normal Phase Chromatographie Conditions ...... 98 Preparation of [RI and [SI Methamphetamine Calibration Curves/ Separa tion by Normal Phase Chroma tography ...... 99 Preparation of Initial [RI and [SI Selegiline Calibration Cumes ...... Normal Phase ...... 100 Preparation of [RI Selegiline Standard Calibration Cu~e#3 ...... 101 Extraction Procedure of Selegdine Table ts ...... 101 E thyl Chloroformate Deriva tiza tion Reactions ...... 102 . . FMOC Derivatrzation Reactions ...... -102 Resul ts of Tablet Assays ...... 114 Discussion ...... 118 References...... 123 List of Tables. Charts and Gra~hs Table I- Types of Chiral Stationary Phases...... 28

Table II a Method Development using various solvent systems ...... 61

Table III O Reverse Phase = Interna1 Standards ...... 70

Table IV-Normal Phase = Internal Standards ...... 79 [RI Methamp he tamine Calibration Cume Data ...... 104 [RI Methamphe tamine Calibraiion Graph ...... 105 [SI Methamp he tamine Calibration Curve Da ta ...... 106 [S1 Methamp hetamine Calibration Graph...... =...... 107 [SI Selegiline Calibration Curve Data ...... 108 [SI Selegiline Calibration Curve Graph ...... 109 [RI Selegiline Calibration Cu~e(#1) Data ...... 110 [RI Selegiline Calibration Curve (#1) Grap h ...... 111 [RI Selegiline Calibration Curve (#3) Data ...... 112 [RI Selegiline Calibration Cunre (#3) Graph ...... 113 Chinoin Tablet Data- Lot# 0600289 ...... -115

Chiesi Jurnex Tablets O First Batch .Lot# 118 ...... 116

Chiesi Jumex Tablets O Second Batch O Lot# 118 ...... 117 List of Figures

Figure 1O Selegiline and Metabolites ...... 2

Figure 2 O 2. 3- Dihydroxybutanoic acid ...... 8 Figure 3 .Types of Chromatography ...... 15

Figure 4 = Chernical Structure of Silica...... 18

Figure 5 O Silica "'T'ails" ...... 18

Figure 6 O Chromatogram Depicting the Separation of two Compounds ...... 22

Figure 7 = Resolution of Neighbonng Peaks ...... -22 Figure 8 .Generalized Chiral Recognition Mode1 Between a Chiral Stationary Phase and a chiral Dinitrobenzamide Enantiorner ...... 30 Figure 9 .Structure of B- Cyclodextrin Units ...... 32 Figure 10 .Some Chiral Denvatizing Reagents ...... 39 Figure 11 .2-Aryl Propionic Acid Derivatives ...... 40 Figure 12 .Potential Chiral Derivatizing Reactions for Amines ...... 42 Figure 13 .Stereochemical Precursors of S (4 Amphetamine ...... 48 Figure 14 .Derivatization Reaction of an enantiomer of Methamphetamine with 2r 3,4,6.tetra.O.acetyl.B. D.glucopyranosy1 isothiovanate (GITC)...... 48 Figure 15 .Reaction of 2 -Naphthyl Chloroformate with a Tertiary amine ..... 50 Figure 16 .Tertiary amines Derivatized with 2 .Naphthyl Chloroformate..... 51 Figure 17 .Promethazine derivatized with (-1 Menthyl chloroformate ...... 53 Figure 18 .Possible Products of the denvatization of Selegiline with (-1 Menthyl Chlorof ormate ...... -54 Figure 19 - Chromatogram - (k) Methamphetamine derivatized with (-) Menthyl Chloroformate ...... 62 Figure 20 - Relationship Between Peak Separation and Resolution ...... 63

Figure 21 - Chromatogram - (0) Menthyl carbarnate derivatives of (k) Methamp hetamine, 70130 ,methanollwater ...... -66 Figure 22 - Chromatogram - (0) Menthyl carbamate derivatives of (k) Methamphetamine - Effect of 2% isopropanol ...... -...... 67 Figure 23 - Chromatograrn - (-1 Menthyl carbamate derivatives of (f) Methamp hetamine - Eff ect of 5% isopropanol ...... 68 Figure 24 - Chromatogram - (-) Menthyl carbamate denvatives of (k) Methamphetamine .Peaks starting to tail ...... 71 Figure 25 - (-1 Menthyl carbamate derivatives of [RI Amphetamine Intemal Standard, Peaks are tailing ...... 72 Figure 26 - Chromatograrn - (-) Menthyl carbarnate derivatives of (_+) Selegiline, Nonnal Phase Cluomatography ...... 74 Figure 27 - Chromatogram - (-1 Menthyl carbamate derivatives of (k) Methamp hetamine, Normal Phase Cluomatograp hy, 96/4 ...... *75 Figure 28 - Chromatogram - (-1 Menthyl carbamate derivatives of (&) Methamphetamine, Normal Phase Chrornatograp hy, 95/5 ...... 76 Figure 29 - Chromatogram - (k) Selegiline carbamates with a, a- Dimethylbenzylamine ...... 80 Figure 30 - Chrornatogram - [RI Selegiline and a, a- Dimethylbenzylamine

derivatized with (0) menthyl chloroformate ...... 85 Figure 31 - Chromatogram - (f)Methamphetamine derivatized with Ethyl chloroformate ...... -91 Figure 32 - Chromatogram - (k) Desmethyl selegiline derivatized with Ethyl chloroformate ...... ,, ...... 92 Figure 33 - Chromatogram - (c) Selegiline derivatized with Ethyl chloroformate ...... 93 Figure 34 - Chromatogram - (2) Methamphetamine derivatized with FMOC...... *.....*...... 94 A~pendix Preparation of a - Phenylacetoacetonitrile ...... O...... A Preparation of Benzy 1 Methyl Ketone ...... B Preparation of (f)-N, a -Dimethy l-13-pheny le thylamine ...... C Preparation of (t)- N-propargyl-a-methy 1-B-phenylethylamine...... D Prepara tion of (I)-N-a -Dimethyl-N-propargyl-B-p henylethylamine ...... E Preparation of the Menthyl carbamate of (+) Methamphetamine...... F Preparation of the Menthyl carbarnate of (t)Desmethyl Selegiline...... G Preparation of N-Acetyl-2-Phenylisopropylamine ...... H Preparation of 2-Amino-3 phenylpropane...... I Preparation of N-Formyl-2-phenylisopropylamine ...... J Preparation of a-a-Dimethylbenzylamine ...... K Introduction The work described in this thesis concerns studies aimed at the development of a stereospecific assay for the drug Selegiline ([LI [RI- (-)-N, a- dimethyl-~-(prop-2-~~yl)phenylethylamine hydrochloride) (figure l) which is one of the agents currently employed in the treatment of Parkinson's disease. Parkinson's syndrome or Parkinson's disease, first described by James Parkinson in 1817, is an age related progressive disorder involving the loss of dopaminergic nigrostriatal neurons and is clinically characterized by akinesia and tremor. Biochemically patients with Parkinson's disease show a pronounced deficiency of dopamine in the striatum (caudate nucleus and putamen) (Cobias, G. C., 1971). Selegiline is a potent monoamine oxidase type B inhibitor which has been shown to delay the progression of the disease when taken in its early stages, Le., shortly after diagnosis. Additionally, when used in combination with L-Dopa, [LI-(-)-3-(3,1-dihydroxyphenyl) alanine, it allows for a reduction in the levels of L-Dopa, and therefore a reduction in toxic side effects normally associated with L-Dopa therapy (Birkmayer, W., Riederer, P., Ambrozi, L. and Youdim, M. B. H., 1977) ïhe discovery of Selegiline was prompted by a series of pharmacological studies with racemic methamphetamine, racemic amphetamine and dextroamphetamine which indicated that, depending on the dose, one could achieve an increase in catecholamine or serotonin release. In 1963, the N- propargyl derivative of N-methyl amphetarnine1 was synthesized and found to

l Although the compound employed was the racemic form of Methamphetamine it should be noted that the name Methamphetamine as employed in the USP %XII, designates the (+) enantiomer. Selegiline and Metabolites

Amp he tamine

N-Methyl amphetamine be a potent irreversible inhibitor of monoamine oxidase. in order to possibly diminish the amphetamine like stimulus, the individual enantiomers were prepared and it was found that the (-) enantiomer was approximately 500 times more potent than the (+) enantiomer as a MAO inhibitor in vitro (Knoll, J., 1983) Monoamine oxidase (EC 1.4.3.4.;MAO) is a flavin-adenosine-dinucleotide (FAD) containing enzyme located on the outer mitochondria membrane. The enzyme is thought to be responsible for the regulation of the levels of certain biogenic amines within the brain and peripheral tissues. Specifically the enzyme catalyzes the oxidative deamination of biogenic amines such as dopamine (DA), noradrenaline (NA) and serotonin (5-HT) to their corresponding aldehydes (equation l), and is therefore involved in their biological inactivation in vivo (Kinemuchi, H., Fowler, C.J. and Tipton, K.F., 19û4)

Prior to the advent of Selegiline, certain monoamine oxidase inhibitors had been employed briefly in the treatment of hypertension and depression. However, due to the inhibition of monamine oxidase, patients eating foods rich in certain amines e.g. tyramine found in cheese, red wine, pickled herring etc., were unable to metabolize the amines which resulted in increased adrenergic activity. Studies showed that, although Selegiline inhibited monoamine oxidase, it also inhibited the release of biogenic amines from the nerve terminais and was free of the "cheese effect" in vitro and in vivo (Knoll, J., Vizi, E.S. and Somogyi, G., 1968 and Knoli, J., 1978) In 1968 Johnston differentiated monoamine oxidase into two forms, designated A and B. The differences in the two forms are based on their substrate and inhibitor selectivity. MAO-A preferentially deaminates serotonin and noradrenaline and iç selectively inhibited by clorgyline (approximately 10-8 M) (Johnson, J. P., 1968), harmaline, and LY51641 (Fuller, R. W., 1968), whereas MAO-B deaminates B-phenylethylamine (and the synthetic substrate benzyiamine) and is selectively inhibited by L-Selegiline (approximately 10-8 M) (Knoll, J., and Magyar, K., 1972) Selegiline belongs to a class of inhibi tors known as "suicide" or irreversib le inhibitors. Such inhibitors forrn a covalent bond with the enzyme, resulting in prolonged or irreversible inhibition. Selegiline reacts with MAO-B initiallv in a reversible reaction forming a non covalent complex, followed by a tirne dependent irreversible reaction leading to the reduction of the enzyme bound FAD and simultaneous oxidation of the Selegiline. Oxidized Selegiline reacts covalently with FAD at the N-5 position of the isoalloxazine ring. The following

equation describes the Lnhibition mechanism ;

where [EI] represents the initial enzyme inhibitor complex, [E14] the enzyme activated inhibitor complex and [EI"] the irreversible inhibitor complex (Geriach, M., Riederer, P. and Youdim, M. B. H., 1992)

Selegiline's major metabolites are (-) amphetamine, (-) methamphetamine

and (-) desmethyl selegiline. Presently there are both chiral and achiral methods available to assay methamphetamine and amp hetamine, either in combination or separatelv. In addition there is also an HPLC method that separates racemic amphetamine, methamphetamine, desmethyl selegiline and Selegiline (Beaulieu, N., Cyr, T.D., Graham, S. J. and Lovering, E. G., 1991). Currently, the proposed USP method for Selegiline (in-process revision) is an achiral separation (Convention, U. S. P., 1991) . However, Pharmeuropa has suggested an alternative chiral method to detect (S)-(+)-Selegihe,suggesting the possibility of

(+) Selegiline in (-) Selegiline raw material and tablets (Pharmeuropa, 1996). The

presence of (+) Selegiline in tablets would result in the formation of (+)

amphetamine and (+) methamphetamine as metabolites. Clearly, additional central nervous system stimulation would not be beneficial for a Parkinson's patient. Taking into account the above, together with the fact that Selegiline is

marketed solely as the (-) enantiomer, it seems prudent that a stereospecific assay for Selegiline be developed. Therefore, the main objective of this research has been to develop a chiral assay for Selegiline using high pressure liquid chromatography. The developed assay is based upon the reaction of a tertiary amine (Selegiline)

with a chloroformate derivative [(-) menthyl chloroformate] followed by dealkylation to yield the carbarnate ester of a secondary amine. While the assay is shown to be capable of application to both Selegiline drug substance and Selegiline in its dosage form, it is not of sufficient sensitivity to allow detection of Selegiline in biological fluids. Accordingly, additional studies have been initiated involving the reaction of Selegiline, desmethyl selegiline and methamphetamine with other chloroformate derivatives in an attempt to identify other potential derivatizing reagents which could provide the basis of a more sensitive assay. Stereochemical Concepts There has been an increased interest in the area of stereochemistry in recent years due to recognition of the importance of chiral molecules in both biological and chernical systems. Often biologically active compounds show a high degree of stereospecificity with regard to their affinity toward receptor proteins and their resultant pharmacological activity. Many dmgs in the past were marketed as racemates, with the assumption that there was no difference between enantiomers. Further investigation revealed that there are frequently significant differences between enantiomers, not only in the magnitude of the pharmacological response but also in their distribution, metabolism, elimination and side effects. One example is isoprenaline, (R)-(-)-isopropyl norepinephrine; the (-) isomer is about 800 times more effective as a bronchodilator than the (+) içorner (Allenmark, S. G., 1988). Such differences have required the pharmaceutical indus t~ to provide the regula tory agencies with detailed studies demonstrating either that both enantiomers are equally efficacious and safe, or that one enantiomer is lacking in the required pharmacological activity and devoid of any other activity or toxicity. Stereochemistry is an area of chemistry that looks at molecules in three dimensions. When Louis Pasteur examined the Faces of tartaric acid he noticed some of the crystals had "hemihedral" faces. These hemihedral or asymmetric crystals were different from his racemic acid crystals. The asymmetric crystals were mirror images of each other, some were right handed and others were left handed. These right and left handed crystals were called "chiralM(derivedfrom the Greek word "cheiros" meaning hand). A substance is chiral if its mirror images are not supenmposable (Ramsey, O. B., 1981). Stereoisomers are compounds that contain the same structural formula but differ in the spatial arrangement of the atoms. Classical stereochemistry, as defined by Eliel, divides stereoisomers into optical isomers, diastereomers and geometric (cis -trans) isomers. Mislow has suggested a new system of classification of stereoisomers which is based on symmetry and energv criteria and therefore includes only enantiomers and diastereorners. (Eliel, E. L., 1962) Two stereoisomers that are mirror images of each other and are not superimposable are called enantiomers. Enantiomers have identical physical properties such as melting points, boiling points, molecular weight, solubility and density. The only difference between a pair of enantiomers is the direction in which they rotate plane polarized light and in the rate of reaction with other enantiomenc cornpounds. The magnitude of the rotation is the same for a pair of enantiomers, but opposite in direction. Stereoisomers that are not mirror images of each other, Le., are not enantiomers are called diastereorners. Unlike enantiomers, diastereomers do not have the same physical properties. They have different solubilities, melting points, boiling points and can have both different magnitudes and directions with respect to optical rotation. Diastereomeric molecules contain minimally two chiral centers. Figure (2) shows the various stereoisorners of 2, 3 - dihydroxybutanoic acid. Compounds (1)and (2) and compounds (3) and (4) are enantiomers of each other. Stereoisomer (1) is not a mirror image of (3) or (4) so it is a diastereomer of both (3) and (4). Similarly, stereoisomer (2) is not a mirror image of either (3) or (4) and is also a diastereomer of both. To convert a molecule with two chiral centers to its enantiomer, both chiral centers must be changed. Reversing the configuration at a single chiral carbon results in a diastereomer (Carey, F. A., 1987). 2,3, - Dihydroxybutanoic acid

- enantiomers

dias tereomers dias tereomers I diastereomers I

< enan tio mers

Figure 2 Aside from the classification of stereoisomers there are also a few tenns used to describe the various ratios of enantiomers such as racemic mixtures, enantiomeric excess and enantiomeric composition. Pasteur discovered that paratartaric acid or racemic acid was optically inactive because 5096 of the crystals were of the (+) configuration and 50% were of the (-) configuration, in other words a 1:I enantiomeric mixture. This definition has withstood the test of time and a racemic mixture is still defined as a 50:50 mixture of the (+) and (-) enantiorners. The enantiomeric composition of a sample may be described by a simple dimensionless mole ratio or mole percent of the major enantiomer. Enantiomeric excess is another term often used to describe the excess of one enantiomer over another. The enantiomeric excess (or ee) is described by equation (3) , where [RI and [SI represent the mole fractions of the R and S enantiomers. S tereochemical Techniques There are many techniques available to determine the enantiomeric composition or purity of a compound such as polarimetry, various enzyme based techniques, nuclear magnetic resonance (NMR), and chrornatographic methods. Polarimetry, NMR, calorimetry and enzyme techniques do not require the separation of the enantiomers, whereas chromatography does. Other techniques such as x-ray crystallography, circular dichroism and optical rotary dispersion may be used also to determine the absolute configuration (Allemark, S. G., 1988). One of the oldest techniques used to study optically active compounds is polarimetry. Historically polarirnetry owes its beginnings to the investigations done by physicists on the behavior of light through various crystals. As early as 1669 Erasmus Bartholinus noted the double refraction of light by calcite. Later, in 1809, Malus noted that light which was reflected from a transparent surface sudi as water or glass has the same characteristics as one of the beams produced by the double refraction of calcite. He called this characteristic "polarization". Subsequent to this, the reflection plane itself, Le., plane passing through the incident ray which was normal to the reflecting surface was called the "plane of polarization" (Pasteur, L., 1901). The magnitude of the rotation is dependent on solute concentration, optical path length, solvent, temperature, and wavelength used. In order to compare individual measurements to each other? specific conditions mut be specihed. Thus, the specific rotation [a]is defined by equation (4),

100a [a]:= - Ic where a= measured optical rotation, T= temperature (C), A, the wavelength , [usually the D 1ine of sodium, A = 529 nm], l= path length of the cell (dm), c = concentration of the compound (g/lOOml). If the specific rotation of an optically pure compound is denoted by [a],, then the optical purity, P (%) of a given

sample of a specific optical rotation [a]can be calculated from equation (5).

This equation defines the optical purity which is based on experimentally derived values which in themselves may have errors associated with them . Optical purity is linearly related to enantiomeric purity only when there is no rnolecular association between the enantiomers in solution. Therefore methods that allow for a separation of enantiomers are generally more reliable. Since the precision associated with polarimeter measurements is about 1-2% the optical purity will give only a rough estimate of enantiomeric composition. Additionally, the use of polarimetry for purity determinations requires that the specific rotation of the optically pure compound is known. The optical purity of amino acids can be determined using enzyme techniques that exploit the fact that enzymes are stereoselective (Greenstein, J. P. and Winitz, M., 1961). Using specific enzyme catalyzed reactions high enantiomeric purity can be determined in the presence of a small amount of its antipode. For example it is possible to detect as little as 0.1% of one enantiomer in the presence of 99.9% of its antipode. There are two types of reactions that have been used extensively in this technique, one is an oxidation reaction for which both L- and D- amino acid oxidases are available. The other reaction, a decarboxylation reaction can only be catalyzed by L-amino acid decarboxylase and therefore only the optical purity of D-arnino acids cm be determined.

1 / 2 O2 A4 - oxidase oxidation : H,N-CHR-CO,H -R-CO-C02H +Mi3

AA - decarboxylase decarboxylation : H2N-CHR-C02H 2R-CH2-NH2 + CO,

An NMR spectrum will no' differentiate between enantiomers and racemates but is capable of distinguishing between the resonance of specific atorns within diastereomers or diastereomeric complexes. Enantiomers are potentially differentiated by reaction with a chiral derivatizing agent or complexation with a chiral solvent. Mislow and Mosher developed one of the first chiral derivatizing reagents used for this purpose known as Mosher's acid ;

MITA ((R)- (+)- a-methoxy- cc-trifluromethyl-phenylacetic acid), used to convert chiral alcohols and amines to diastereomeric esters and amides. In addition to converting enantiomers into diastereomers using a chiral derivatizing agent, two direct methods have been also been developed. Pirkle used an optically active solvent, (R)- (-)-2,2,2-trifluro-1-phenylethanol,to induce a chemical shift difference between the enantiomers. The optical purity of the solvent does not influence the integration results, but only the peak separation. The peak splitting resulting kom the solvent induced chemical shift difference is a consequence of the differential interaction of one of the enantiomers with the chiral solvent. This is the same type of recognition that occurs within enantiorneric compounds and chiral stationary phases within chrornatographic processes. These NMR studies actually assisted Pirkle and CO-workersin the design of chiral stationary phases. A more powerful NMR method used to differentiate enantiomers uses the optically active lanthdde shift reagents. This technique combines the high resolution obtained with pseudo contact downfield shifts with splitting of resonance Iines by enantioselective interaction with the chiral lanthanide reagent. Results obtained from NMR using peak integration methods give the concentration ratio of the enantiomers and then enantiomenc excess can be calculated. Although the above rnethods are often used to determine the enantiomeric purity of a compound they all (except for NMR) rely on a measurement from an optically pure compound. Chrornatographic methods which result in the complete separation of enantiomers represent one of the most powerhl techniques available to determine enantiomeric composition and do not necessarily require an optically pure standard, a partiaily purified standard will suffice to allow identification of the individual chromatographic peaks (Allenmark, S. G., 1988). Chrornatoma~hy A Russian botanist named Mikhail Tswett performed the first separation by column chromatography. In 1900 Tswett, working with plant pigments, separated chlorophylls and xanthophylls by passing solutions of the compounds in organic solvents through a column packed with calcium carbonate. As the separation took place, colored bands appeared on the column. The appearance of these colored bands aided Tswett in naming the separation technique "chromatography" from the Greek chroma meaning "color" and graphein meaning "to write" (Skoog, D. A. and Leary, J. J., 1992). Although Tswett's work was dune in the early 1900's, further development in the area of chromatography was not pursued until 1941 when Martin and Synge produced the first mathematical treatment of chrornatographic theory (Martin, A. J. P. and Synge, R.L.M., 1941). Chromatography employs two major components, a stationary and a mobile phase. The classification of the vanous types of chromatography depends on the nature of these two phases. The mobile phase can be either a gas, liquid or supercritical fluid. The stationary phase may be either a column or a plate that has a chemical phase either bonded or adsorbed to it. In al1 chrornatographic separations the sample is dissolved in the mobile phase which is forced through an immiscible stationary phase. The two phases are chosen so that the sample can distribute itself between the mobile and stationary phases. Samples that are strongly retained by the column move slowly with the flow of the mobile phase. Conversely, samples that are weakly retained by the stationary phase, have1 much quicker. ïhese differences in retention are responsïble for the separation of the various sample components and allow for both qualitative and/or quantitative analysis. Figure (3) is a chart of the various types of chroma tography. Liquid chromatography (LC) Liquid-Iiquid, or partition Liquid adsorbcd on a Partition bctwecn immis- (mobiIe phase: liquid) solid cible liquids Liquid-bondcd phase Organic spccics bonded Partition bctween liquid to a solid surfacc and bunded surface Liquid-solid, or adsorp- Adsorption tion Ion exchange Ioncxchange rcsin Ion exchange Sizc exclusion Liquid in interstices of a Partitionlsicving polymeric solid Gas chromatography (GC) Gas-liquid Liquid adsorbed on a Partition betwcen gas and (mobile phase: gas) solid liquid Gas-bonded phase Organic specics bondcd Partition ktwecn liquid to a solid surface and bonded surface Adsorption Supercritical-fluid chroma- Organic species bondtd Partition betwttn super- tography (SFC)(mobile to a solid surface critical fluid and bondcd phase: supercritical fluid) surface

Figure 3 (Skoog, D. A. and Leary, J. J., 1992) In addition to the column and mobile phase, a typical high pressure liquid chromatography (HPLC) system also consists of a pump to deliver the mobile phase through the system , a detector, an injection system (either a manual or automated injection system) and a data collection system. Today most Laboratories use integrators that are run by computer software, but a simple strip chart recorder will also suffice to record the da ta. Chromatography has corne a long way since the days of Tswett, and today perhaps the most common type of chromatography used is high pressure liquid chromatography- High pressure liquid chromatography (HPLC) offers two main modes of separation, termed normal phase or reverse phase chromatography. There are many other types of chromatography such as ion-exchange, size exclusion and affinity chromatography; however for the purpose of considering chiral separations, the discussion will be limited to reverse phase and normal phase systems and how they apply to chiral separatioffi. Since its inception chromatography has gone through tremendous development and irnprovements. Improvements often bring new terminology, leaving the scientist confused over the meaning of the original and/or revised terminology. Thus several different names may be used to describe the same type of chromatography. Normal phase chromatography is also called adsorption and straight phase chromatography, while reverse phase has also been referred to as liquid/liquid or partition chromatography. The term "liquid/liquidWrefers to the type of column that has a liquid film of stationary phase covering the column packing. Due to the inconsistencies associated with liquid/liquid columns, they are rarely used today and have been replaced with bonded phase columns (Wainer, 1. W., 1994). In normal phase chromatography the column is composed of a polar stationary phase such as silica ,and a mobile phase containing non polar solvents such as hexane and ciidilorornethane. It will be noted that it was a normal phase separation which was originally performed by Tswett on the plant pigments. Silica is an absorbent with many uses; it can be used alone as a stationary phase or can be chemically modified to prepare many other types of chrornatographic phases. Silica contains silicon atoms bridged three dimensionaily by oxygen atoms. The silicon-oxygen Iattice is then saturated by OH groups (silanol groups, figure 4). The silano1 groups are the active sites in the stationary phase and can form weak bonds with nearby molecules that are capable of hvdrogen bonding, dipole-induced dipole interactions, dipole-dipole interactions or rc-cornplex bonding. The silica gel in the chrornatographic bed is surrounded on al1 sides by mobile phase molecules. A sample molecule is adsorbed only if it interacts more strongly than the solvent with the adsorbent. Separation is achieved by the differences in adsorption between various componens within a sample. In normal phase separations, both sample and solvent molecules are arranged on the silica surface so that their polar functional groups or double bonds are closer to the silanol groups. Therefore, any hydrocarbon "tails" are diverted away from the silica (figure 5). A silica column cannot distinguish between compounds that are identical apart from their aliphatic moiety. The strength of the interaction, and therefore the separation, depends not only on the functional groups contained on the sample molecule, but also on steric factors. Therefore, molecules with different stenc structures, such as geometncal isomers are suitable for separation by normal phase chromatography. Typically polar compounds are generally best separated by normal phase chromatography (Meyer, V. R., 1994). Chernical Structure of S ilica

Figure 4

Silica "Tails"

Figure 5 In reverse phase chromatography, the stationary phase is less polar than the mobile phase. The most frequently used reverse phase packing is composed of a silica backbone to which n-octadecyl chains (ODS, C-18) have been chemically bonded. The long chain hydrocarbon groups are aligned parallel to one another and perpendicular to the particle surface, resulting in a brush or bristle Like structure. The exact mechanisrn by which these surfaces retaui solute molecules is at present not entirely clear. Some scientists feel that the bmsh like surface acts as a liquid hydrocarbon layer and separation occurs due to the solubility differences of the various solutes for either the mobile phase or the so called "hydrocarbon liquid layer". This theory follows the separation mechanism of a typical liquid/liquid stationa. phase. Other scientists feel that the brush coating acts as a modified surface with which the solute molecules compete along with the mobile phase for adsorption. The mobile phase is usually a combination of polar solvents, such as methanol, water or buffer solutions. In normal phase chromatography, the most non-polar compounds are eluted first, whereas in reverse phase diromatography, a more non-polar compound would interact strongly with the stationary phase and would be eluted last. In addition to the much used C-18 column, several other types of reverse phase packing are available e.g., C-8, phenyl and cyano columns. The decision as to which type of reverse phase column to use is generally dependent on the samples you wish to separate. A more non-polar sample would be best separated by a C-18 or a C-8 column, whereas more polar samples may be more suitable for a phenyl or cyano column. The longer the alkyl group (C-18 vs. C-8) the more non-polar the column, and therefore longer retention times would be expected.

Eluted compounds* are trans~ortedL bv J the mobile L hase to the detector and the response is recorded using either an integrator or strip recorder. The signals that are recorded are Gaussian shaped peaks. Each peak can give both quantitative and qualitative information about the solute. The time required for a substance to elute from a column is known as its retention tirne. ïhis retention time will remain constant under identical chrornatographic conditions. chromatographic conditions are specified by the type and dimensions of a column , mobile phase composition, detection wavelength (for UV or fluorescence detection) and temperature. Therefore, if the retention time of a known substance is determined under certain chromatographic conditions it cm be used as a comparison to aid in the identification of an unknown analyte. Quantitative measurements are possible by comparing the area under the curves. The area of a particular peak is proportional to its concentration. Therefore, by consmicting a calibration curve, the concentration of an unknown sample may be detennined. Ln addition to quantitative and qualitative information about an unknown solute, chromatograms can also give information regarding the efficiency of a separation. In the following paragraphs chromatographic terms such as the capacity factor, resolution and the separation factor will be presented. During method development these expressions allow for a quantitative comparison among different chromatographic separations. Mathematical Principles of Chromatoma~hv The efficiency of a particular separation can be described mathematically by two separate terms, the capacity factor and the separation factor. Different components of a sarnple are identified on a chromatogram by their retention times, tR in minutes. A compound that is not retained by the column has a retention time of t, and is the mean time for the solvent to flow through the column and is the same for al1 non-retained compounds within a particular chromatogram (figure 6). Retention time is a function of mobile phase flow velocity and column length. If the mobile phase flow rate is low or the column is long, both the t and tR would be large and by themselves not suitable for characterizing a separation. Therefore, the capacity factor or k' is used to describe the migration rates of retained analytes on columns and is described by the following equa tion;

The capacity factor, k' is independent of both column length and mobile phase flow rate and represents the molar ratio of the compound in the stationarv and mobile phase. Capacity factors between 1 and 5 are generally preferred. As can be seen from the equation, if the k' values are very low then the separation may be inadequate ( compounds pass too quickly through the column, little column interaction). However, high k' values are accompanied by long analysis times. The capacity factor, k', can also be described by the following equation: Signai

Somple injection

Chromatogram Depicting the Separation of two Compounds (Meyer, V. R., 1994) Figure 6

Pcak- height ratio 1:l

Resolutions of two Neighboring Peaks (Meyer, V. R., 1994) Figure 7 where K is the distribution coefficient of the solute between the stationary and mobile phase, Vs is the volume of stationary phase and Vhf the volume of mobile phase in the column. In other words the capacity factor is directly proportional to the volume occupied by the stationary phase and particularly to the specific surface area of the adsorbents. A column packed with porous layer beads produces lower k' values and therefore shorter retention times. A silica column packed with narrow porous material produces larger k' values than a wide porous material. In order for two components to be separated they must have different k' values (Meyer, V. R., 1994) The extent by which two species, 1 and 2 are separated from one another on a particular column is described by a parameter known as a or the separation factor, a quantity which compares the relative retention times of the hvo peaks and is described by;

In this equation kt2> kVl, therefore tR2 > tR, and a should be greater than 1. If a

=1, then no separation occurs. hother important quantity to be determined, especially with respect to chiral separations is the resolution. The resolution or R5 of a column is a quantitative measure of its ability to separate two analytes. Figure (7) correlates the various R, values with individual separations. The Resolution or R, of two analytes is defined by the following equations; In equation (9),W is the peak width, which can be measured either by the use of an integrator or manually with a mler and A2 is the difference between the two retention times. A cornplete separation between two peaks is obtained when R, =

1.5, however an R, = 1.25 would be sufficient for quantitative analysis with an integrator. Chromatograms can also be used to determine column efficiency. Column efficiency is usually described by two related terms, the plate height, H and the number of theoretical plates, N. The foollowing expression relates the two quantities;

where L is the length in centimeters of the column packmg. The efficiency of a column increases as the number of theoretical plates increases and the plate height decreases. An increase in the number of theoretical plates is a function of a better packing, longer column length and an optimum mobile phase flow rate. A column with a high number of theoretical plates cmseparate a mixture of closely related compounds, Le. similar a values. The term "plate height" and "number of theoretical plates" cornes from the mathematical treamient of Martin and Synge (Martin, A. J. P. and Synge, R. L. hd. 1941). They viewed a chrornatographic column as though it were made up of nurnerous nanow layers called theoretical plates. They proposed that at each plate, equilibration of the analyte between the mobile and stationary phase was established. Movement of a solute through the column was then treated as a series of stepwise transfers of equilibrated mobile phase from one plate to the next. Although this theory accounts for the Gaussian shape of chrornatographic peaks and the rate of movement through a column, it does not account for band broadening. ïhis plate theory was later dismissed and replaced with the rate theory. Although the terms theoretical plates and plate height are still being used as applied to the rate ~eory,it should be noted that the concept that a column contains plates where an equilibrium exists is not accepted. Ln fact, an equilibrium can never be realized with a mobile phase in constant motion2(Skoog, D. A. and Leary, J. J., 1992). Both the plate height H, and the number of theoretical plates N, also can be calculated using the following equa tions;

Another method for calculating N, which some scientists feel is more reliable is to determine the WlI2,the width of the peak at half its maximum height. Using W,, ,equation (12) becomes;

The above equations represent a series of mathematical expressions that are often used to either describe a particular separation, the efficiency of a column or used in method development to improve a separation. With respect to the separation of enantiomers or diastereomers the most important parameters are the separation factor, a and the resolution, R, .

However, plate height or height equivalent to a theoretical plate can be considered as that length of column over which the equivalent of an equilibrium is established. Chiral Seuarations - A Direct Approach usine Chiral Stationam Phases In the 1960's Gil-Av used chiral stationary phases to separate enantiomers by gas liquid chromatogaphy (GLC) (Gil-Av, E., Feibush, B. and Charles-Sigler, R., 1966). Also at this time, enantiomers were converted to diastereomers using chiral derivatizing agents followed by separation by achiral GLC or thui laver chromatography. As the knowledge of chiral recognition interactions increased, chiral derivatizing agents for liquid chromatography (LC) were developed. Eventually by the 1980's chiral stationary phases for liquid chromatography were developed and direct separation of enantiomers was possible. Today, many more chiral stationary phases and chiral derivatizing agents have been developed and studied. Although analysis by gas chromatography is still a useful method, the requirement that the analyte is both volatile and thermally stable often necessitates prior derivatization. Presently the most popular method for analyzing a chiral compound is by high pressure liquid chromatography. High pressure liquid chromatography affords two basic methods to separate chiral compounds, a direct and an indirect method. A direct separation includes the use of a chiral column or of a chiral mobile phase. An indirect separation involves derivatization of the compound using a chiral reagent resulting in the formation of diastereomers followed by separation on an achiral column. In sorne cases a combination of the two methods is used e.g., a solute may be derivatized with a chiral or adiiral reagent prior to separation on a chiral column. Direct separations using chiral stationary phases (CSPs) are the result of the formation of a weak diastereomeric complex between the enantiomeric solute and the chiral selector of the column. The ability to separate a solute on a particular chiral column is dependent on the correct "interaction sites" on the solute. Therefore, the presence of specific functional groups in the correct orientation is required for a solute to be separated on a chiral stationas. phase. In some cases the correct "interaction site" is not part of the solute and needs to be added, by prior derivatization. Dalgliesh has proposed a "three point interaction" model to explain the chiral recognition process for the solute-CSP cornplex. According to this model, there are required minimally three interaction sites between the solute and the chiral selector, with one of these interactions being dependent on the stereochemical structure of the solute (Dalgliesh, C. E., 1952). ïhe differences in free energy between the individual enantiomers/chiral selector will determine the magnitude of the separation. For example, if the (+) isomer forms a more stable (lower energy) bond with the chiral column, this isomer will be retained longer. The actual efficiency of the separation is dependent on the specific interactions between the chiral column and the enantiomeric solutes. The best way to appreciate the different types of chiral stationary phases is to group them according to the type of solute/CSP interaction that takes place. Table 1 (Wainer, 1. W., 1993) lists some of the more popular columns along with their ~referredsubstrate interactions. Interaction sites can be classified as x -basic, rr -acidic, aromatic rings, acidic sites, basic sites , steric sites or sites for electrostatic interactions. Aromatic rings represent a source of rc -n. interactions, whereas acidic sites rnay allow electrostatic interactions or hydrogen bonding to take place. Typically the hydrogen bonding involves such functional groups as amide, carbarnate, urea, amine or alcohols, although ether, sulfinyl or phosphinyl oxygens may also be involved in hydrogen bond formation. Electrostatic interactions may occur at charged groups or with permanent or induced dipoles. Steric interactions occur between large groups. - TVpe Interaction salute chiral selector Derivatizing I hydrogen a lkyl carbinols, (R)-N-(3,5 Dinitro benzoyl) :onvert alcohols, amines 07 bonding, dipnle aryl substitutcd phenyl glycine :arboxylic acids to arornatic slacking, iZ - K liydan toins (S)-N-(3,5 DNBPG) amide, urea or carliamate lactanis, sulfoxides II Combina tion of Contain phenyl Microcrystalline cëllulose hhols to csters, espccially h ydrogen t;roups, amides, triacetate para-nitrobcnzoic esters if Ot- hmding, dipolc iniides, keloncwj, near chiral ccnter, benzyl Type II -cellulose 1 I stacking, A - n esters Cellulose triaceta te amides (or benzyl esters fitting in cavity Cellulose Mbenzoatc 11 1 part of all the phcnytoin and B-cyclodex trin lntroduce phcnyl groups. Cyclobond 1, 11 ,111 solutc rnust fit ir mctab»lites, y- cyclodextrin Chiral aliphatic amines hav the cavlty, mpod a - cyclodex trin wnconverted to benazmide ahhydrogen crmtaining rnultiplc Poly (triphenylmet hyl derivalives bonding and pheny 1 groups such methacrylate) clicarl>«xylicacids have lieen Ir-lt as alcohols, amide, Chiral 18-crown-6-ethcr converted to benzyl esters or ester, ketone amides I + III hydrogen aromatic amines, Type 1- naphthylethyl Type 1 -Non aromalic amines Mixed Mode CSP bond ing alcohols, multiple carbamate and alcohols must be convertec dipole stacking phcnyl group Type 111- cyclodextrin Io dinitrophenyl derivative Ir- lt and aimpounds cavi ty inclusion IV complex with a-amino acids (WE) N-carboxymethyl(lR, derivatizd amino acids transition metnl mino and 2s)-diphenylamino ethanol including N-acetyl, amide, N WE, Wl1, WM (M), chiral dicarboxylic acids (W 11)- L-Praline carbamoy 1, N-carbobenzoxy moleculc (A A) containing a-OH (WM) - L-tert-Leucinc (L) and enantiomer of racemic solute AGP v hydrogen cationic and anioni AGP-ai-Acid protein Por HSA/DSA -most amino OVM Proteins bonding and mmpaunh, OVM - Ovomucoid acids need ptecalurnn HSA hydrophobie ephedrine, a, B- HSA-tiuman serurn albumin derivalizalion wilh N-acety BSA interactions amino acids BSA- Bovine serum albumin benzenesulphonyl, DANSY L bctwcen protein 1-1SA/BSA-anionic Often used in achiral/chiral and çolute. Alsri and neutral systerns electrostatic mmpounds. Necd aromatic and polai moicty One of the most popular CSP's used today is the type 1 or " bmçh type" phase (see Table 1) . Bmsh type phases are based on amino acid derivatives, which contain additional polar groups which can facilitate hydrogen bonding. The most widely used r-electron accep tor columns have a 3,5-dinitrobenzoy l derivatized phenylglycine or leucine group linked to a silica backbone. Type 1 CSP's rely on rc -r interactions, hydrogen bonding and dipole stacking for the recognition process to take place. Figure (8) depicts the chiral recognition mode1 as suggested by Pirkle for the longer retained enantiomer of the N-(3,5- dinitrobenzoyl) derivative of an amine separated on a N-(2-naphthy1)alanine

CSP. The three interactions are the R. -n interactions between the arornatic groups, hydrogen bonding between the acid N-H proton of the amine and the carbonyl group of the CSP and finally hydrogen bonding between the basic site (8)of the amine to the N-H group on the CSP (Pirkle, W. H., 1986). Chiral recognition is usuallv successful when the three chiral recognition sites are adjacent to the stereogenic center. The presence of an aromatic group is required for chiral separation on this type of CSP's ,and on most other CSP's as well. Typical solutes suitable for this type of CSP include aryl-substituted hydantoins, lactams, sulfoxides and amino alcohols. While most solutes contain an aromatic n -basic group not al1 solutes contain a x -acidic group, and must be derivatized. In many cases derivatization with 3, 5 dinitrobenzoyl is necessary. This type of CSP would use a mobile phase similar to that used in normai phase diromatography, e.g. hexane, heptane and a more polar modifier such as tetrahydrafuran or methanol (Pasutto, F. M., 1992). Several types of CSP's rely on the formation of an inclusion complex. In such instances, the solute must be able to fit inside the cavity created by the CSP's structure. Both Cellulose type 1 and II and cyclodextrin CSP's depend on the formation of an inclusion complex. Generalized Chiral Recognition Model Between a Chiral Stationary Phase and a chiral Dinitobenzamide Enantiomer (Pirkk, W. H.,1986)

Figure 8

30 Cellulose 1 CSPs are composed of D-O-glucose units arranged in chahs in either parallel (CTA type 1) or anti parallel directions (CTA type II) . The arrangement of the D-B-glucose rnoieties allow for the formation of cavities between the units and for spaces between the polysaccharide chahs. One of the requirements for this CSP is a phenyl group which must enter the cavity of the CSP to form the solute/CSP complex. Additional interactions include hydrogen bonding and dipole stacking. Typically enantiomeric cyclic amides, imides, esters, ketones, and alkyl substituted indenes are separated on such CSP's. Chiral alcohols are best resolved when they are converted to esters. Cellulose II type CSP's also contain a cavity which appears to be important in the recognition process. Pharmaceutical compounds such as warfarin, verapamil and propranolol have been separated on this type of CSP. Typical solutes contain one or more aromatic ring and a carbonyl, sulfinyl or nitro group. Amines and carboxylic acids are often derivatized to improve the chrornatographic efficiency of the column. Mobile phases for these type of CSP's are usually composed of hexane and isopropanol or a similar modifier. The cyclodextrin type CSP's are cyclic oligosaccharides composed of a -D-(+) glucose units linked at the 1,4 position. The most cornmon forms of this molecule are the a -, B- and y -cyclodextrins, which contain six, seven and eight glucose uni& respectively. The mouth of the cyclodextrin molecule has a larger circumference than its base, giving it the overall shape of a cup. The mouth of the cup is lined with the secondary hydroxyl groups of the C-2 and C-3 atoms of each glucose unit, while the C-6 primary hydroxyl groups line the base of the cup (Figure 9) (Ahuja, S., 1991).The separation mechanism is based on the formation of an inclusion complex within the hydrophobic cavity. The size, shape and polarity of the solute are the most critical factors affecting the stability of the inclusion complex. Similar to the cellulose CSP's, solutes for cyclodextrin Smicture of B- Cyclodextrin Units

Figure 9 CSP's must also contain an aromatic moiety at or adjacent to the stereogenic center. The aromatic portion of the compound must be inserted into the cyclodextrin cavity. The size of the aromatic portion of the molecule, thus determines which cyclodextrin CSP is appropriate. Cyclodextrin CSP's have been used in both reversed and normal phase modes, although the most common mobile phases are composed of water, or buffers modified with methanol. Cyclodextrin CSP's have been used to separate enantiomers of phenytoin along with several aromatic amino acids such as D,L-pheny lalanine, D,L-~N~tophan and D,L-tvrosine (Wainer, 1. W., 1993). The last CSP to be discussed is the type V or protein based chiral stationary phase. Proteins are polymers of hgh molecular weight that are composed of L- amino acids. Since proteins are hvolved in the uptake and transport of dmg substances, it seems logical that such proteins would display stereospecific binding. Experiments using human al- acid glycoproteui (AGP) indicated that Spropranolol is bound io a greater extent than the R-enantiomer. The ability of proteins to stereoselectively bind small molecules fostered the development of a series of protein based CSPs made by immobilizing the particular protein ont0 a sika backbone. In addition to AGP, ovomucoid (OVM), human serum albumin (HSA) and bovine serum albumin (BSA) have al1 been immobilized ont0 silica. Several pharmaceutical compounds have been separated on AGP columns ,e.g., atropine, ephedrine, and verapamil. BSA type phases have separated amino acids, warfarin and sulfur containing cornpounds. Separations with protein phases are normally used with an aqueous mobile phases containing a phosphate buffer at neutral pH. Under these conditions, hydrogen bonding, electrostatic and hydrophobic interactions cmtake place. Although the exact cnteria for separation on a protein type CSP are not fully understood, it has been shown that adjustments in the mobile phase, flow rate or pH tend to affect the s tereoselectivity sugges ting multiple types of interactions may be necessary for a successhl separation (Ahuja, S., 1991). Chiral Seuaration - Direct Se~arationsus in^ Chiral Mobile Phase Additives Aside from the use of a chiral column, a direct separation may be obtained by the addition of a chiral additive to the mobile phase. When a chiral selector is added to the mobile phase conventional non-chiral columns cm be used. The stereoselective separation can be due to the formation of a stereoselective cornplex in the mobile phase, the adsorption of the chiral selector to the stationarv phase creating an in situ chiral column or the formation of diastereomeric complexes with different distribution properties between the mobile and stationary phase. In ail cases, except the formation of diastereomeric complexes (ion pairs), it is necessary to have the typical "three point interaction" for separation to occur. A two point interaction consisting of an electrostatic attraction and hydrogen bonding may be sufficient for diastereomeric complexes. (Pettersson, C., 1988). There are several different types of chiral mobile phase additives (CMPAs) such as L-proline, cyclodextrins, albumin, (R, R)-tartaric acid mono-n- octylamide, quinine and uIacid glycoprotein. Many of these CMPAs have borrowed principles that are used in other types of chromatographv. For example ligand exchange chromatography was originally developed for the separation of enantiomenc amino acids. It involved the use of a stationary phase containing L-proline (ligand) bonded to a resin on which copper was loaded. Subsequently, ligand exchange with the chiral metal complexing agent in the eluent was developed. An injection of an racemic amino acid into a chromatographie system with an optically active ligand (L-Lig) and copper ions u.i the mobile phase results in the formation of two ternary diastereomeric metal complexes (LePage, L., Linder, W., Davies, G., Seitz, D. and Karger, B., 1979). The separation is due to the differences in the structure of the two diastereomeric complexes. Ligand exchange chromatography with a chiral mobile phase additive is used with conventional reverse phase columns and is used to separate racemic amino acids (Pettersson, C., 1988). The use of crown ethers and cyclodextrins in the mobile phase has been used successfully for chiral separations. Separations with mandelic acid and derivatives indicated that the asymmetric carbon atom rnust have functional groups capable of bonding to the hydroxyl groups of the B-cyclodextrin (Debowski, J., Sybilska, D. and Jurczak, J., 1982). Unfortuna tely, relatively low steroselectivity is found when cyclodextrins are used alone as the mobile phase additive. improved selectivity has been shown with the addition of a counter ion such as d-camp hor-10-sulfonic acid. The addition of an ion pairing reagent has often been used in reverse phase chromatography. Similarly, the use of chiral counter ions has been shown to be able to separate chiral acids, amines and alcohols. The separation is based on the formation of diastereomeric ion pairs in the mobile phase. It appears that a two point interaction between the ions is adequate for separation. For a reasonable separation, the counter ion should contain the ionized hction and have a hydrogen bonding group near the chiral center. Quinine and the related cinchona alkaloids have been used for the separation of chiral carboxylic acids (Pettersson, C. and No, K., 1983). Enantiomers of propranolol and similar amino alcohols have been separated using d-camphor-10-sulfonic acid (Pettersson, C. and Schill, G., 1981) or N-benzoxycarbonyl-glycyi-L-proline(ZGP) (Pettersson, C. and Josefesson, M., 1986). The chiral separation is optimized by varying the concentrations of the counter ion in the mobile phase and a competing compound with the same ionized hction as the enantiomers. In many cases low polarity solvents are used to obtain a high degree of ion pair formation. This method is usually used for separation of enantiomeric amines, acids and alcohols. Chiral Separation via the Indirect Method: Usine Chiral Derivatizine Agents Chiral separation by the indirect method involves the use of a chiral derivatizing agent (CDA) to convert enantiomers into diastereomers. In some cases derivatization is done to provide greater sensitivity by the detector, but may also be used to introduce needed interaction sites for subsequent separation on a dural column. CDA's have also found use in asymmetric synthesis (Olofson, R. A., Schnur, R.C., Bunes, L. and Pepe, J.P., 1977). Since diastereomers have different physical chernical characteristics, separation on conventional achiral columns is possible. Despite the expanding availability of chiral stationary phases a wide variety of chiral separations are still performed using CDA's since chiral columns generally are not as rugged and reproducible as haditionai normal and reverse phase columns, and they are also very mu& more expensive. The main consideration in the use of a CDA is its purity and stability under derivatization conditions. If a racemic compound is derivatized with a pure R-reagent the following diastereomers would be formed; S-dmg-R-reag (A) + R-drug-R-reag (B). However if the CDA was contaminated with a small arnount of Sreagent then there would also be formed S-drug-S-reag (C) + R- dmg-S-reag (D) in addition to compounds (A) and (B). Compounds (A) and (B) are diastereomers as are compounds (C) and (D) which can be separated on an achiral column. However, compounds (A) and (D) are enantiomers as are (B) and (C). The use of an achiral column would not allow for the separation of (A) from (D) or (B) from (C) and therefore would result in art inaccurate enantiomeric determination. The precision of the analytical results as well as the optical purity of the collected peaks is thus limited by the purity of the CDA. Further the reaction conditions must not be so harsh as to result in racemization at the chiral centers of either the solute or the CDA. The chiral substances rnost often resolved as diastereomers are amines, alcohols and carboxylic acids. Chiral alcohols are usually derivatized with chiral acids, chloroformates or isocyanates, giving diastereomeric esters, carbonates and carbamates respectively. Esters are usually prepared from an alcohol and an "activated" chiral carboxylic acid, usuaily in the form of a diloride, anhydride or imidazole derivative. Acyl chlorides are highly reactive and are used to derivatize hindered secondary alcohols and amines. Unfortunately acyl chlorides, particularly those used containing a proton attached to the chiral carbon are susceptible to racemization under extreme acidic or basic conditions or at elevated temperatures. The most commonly used reagents are the dilorides of 313-acetoxy-A3 - etienic acid, (-)menthoxyacetic acid and S(-)-N- (triAuoroacetyl)proline. Figure (10) shows the chernical structures of some of the more common CDA's. The formation of carbamates by the reaction of alcohols with chiral isocyanates has become quite popular for the resolution of chiral alcohols and prirnary and secondary amines. Typical reagents include R(+)/S(-) phenylethyl isocyana te and R(+)/S(-)-b(1- naphthy1)ethyl isocyana te. The diastereorneric carbamates are generally stable without racemization. Alcohols react slowly and may require heating for several hours, whereas phenols react rapidly, even at room temperature (Wainer, 1. W., 1993). Many non steroidal anti inflammatory drugs (NSAIDs) such as ibuprofen, fenoprofen, and flurbiprofen contain a 2-arylpropionic acid group in their structure and are chiral due to the asymmetric center a to the carboxyl group (Figure 11). These drugs are marketed in their racemic form , with the full knowledge that most of the desired activity resides in the S - isomer. The R - isomer has little or no activity. In addition, it has been shorvn that many of these NSAIDs undergo in vivo metabolic biotransformation of the inactive R R (+)/S a(-)-a-Methoxy-a- (tnfluromethy1)pheny lacetic acid giucopyranosy l iso thiocyana te (Mosher's acid)

i C-C-OH

3f3- AC^toxy-~--e tienic acid

2,3,4-tri-O-acetyl-a-D- arbinopyranosyl isothiocyanate (AITC)

Some Chiral Derivatiting Reagents

Figure 10 2-Aryi Propionic Acid Derivatives

CHCOOH

Fenoprofen

-CHCOOH

Flurb ip rofen

Figure 11 enantiomer to the active S enantiomer. This interesting finding (ody possible from use of chiral chrornatographic separations) has prompted most of the publications dealing with derivatizing carboxylic acids to be focused on arylpropionic acid derivatives (Hutt, A. J. and Caldwell, J., 19%). Chiral alcohols typically used in such derivatizations are 2-octanol (Johnson, D. M., Rerter, A., Collins, M. and Thompson, G.E., 1979), (-)-2-butanol (Kamerling, J. P., Duran, M., Gerwig, G.I., Ketting, D., Bruinvis, L., Vliegenthart, J.F.G. and Wadman, S.K., 1981) and (-)-menthol (Hasegawa, M. and Matsubara, I., 1975). In addition to carboxylic acids the use of chiral aIcohoIs to Çom diastereomeric esters has also been used for the resolution of amino acids (Hasegawa, M. and Matsubara, I., 1975) hydroxy acids and 2-alkyl-substituted carboxylic acids (Konig, W. A. and Benecke, 1-, 1980). Amines represent one of the easiest functional groups to derivatize, a Çact which is reflected in the number of different reagents available. Figure (12) depicts the various derivatization routes possible for amines. The formation of amides from chiral acylating reagents is one of the most popular derivatizing techniques for prirnary and secondary arnino groups. N-heptafluorobutyryl -L- prolyl chloride and CNitrophenylsulphonyl-L-prolyl chloride have both been used to derivatize amphetamine. Primary and secondary amino groups react with isocyanates and isothiocyanates to give urea and thiourea derivatives respectively. Miller et al compared the use of four chiral reagents; (R)-(+)-1- phenylethyl isocyanate (PEIC), (-) - a -methoxy-a (trifluoromethy1)phenylacetyl diloride (MPTA Cl) ,2,3,4,6-tetra-O-acetyl-E-D glucopyranosyl isothiocyanate (GITC), and 2,3 ,4-tri-o-acetyl - a -D-arabinopyranosyl isothocyanate (AITC)for the derivatization of a series of chiral ring substituted 1-phenyl-2-aminopropanes(amphetamines) and 1-phenylethylamine. Al1 four CDA's formed diastereomeric products with each solute using mild reaction Alkylation Amine

Chloroformate Carbornate

Isothiocyonate Thiourea

Isocyonate Ureo

RlNH2 + R2SH + acHo\ - RJ" CHO ~

OPT/ chiral th101 Isoindole

Acyla tion Amide

Potential Chiral Derivatizing Reactions for Amines

Figure 12 conditions. However, better resolutions (R-values) were seen wi th AITC, GITC and MPTA 4 Cl than with PEIC. Separations were performed using a reversed phase C-18 column with methanol - water as the mobile phase (Miller, K. R., Gal, J. and Ames, M. M., 1984). Primary amines when reacted with O-phthalaldehyde (OPA) in the presence of a thiol form isoindoles (Simons, S. S. and Johnson, D.F., 1976). Ln addition thiols may be assayed by derivatization with OPA in the presence of an excess of a suitable primary amine. This procedure is often used for the determination of amino acids (Deyl, Z., Hyanek, J-, and Horakova, M., 1986). The use of a chiral thiol for the derivatization with OPA results in the formation of diastereomers in the presence of primary amines- Amino alcohols, amino acids and primary amines have been resolved using OPA and N-acetyl-L-cysteine, Boc-L-cysteine and N-acetyl-D-penicillamine as the chiral thiol (Buck,R. H. and Knunmen, K., 1984, Buck, R. H. and Krurnmen, K., 1987). Thus far the reactions discussed for amines are suitable for either prirnary or secondary amines. However, chloroformates are suitable reagents for primary, secondary and tertiary amines. The reaction of a chloroformate with a prirnary or secondary amine results in the formation of the corresponding carbamate and chiral chloroformates are readily synthesized from chiral alcohols and phosgene. One of the unique properties of chloroformates however is their ability to N- dealkylate tertiary amines to produce the carbamate of the correspondine secondary amine ( equation 14).

Witte et al derivatized promethazine with (-)menthyl chloroformate and separated the diastereomers by reverse phase HPLC (Witte, D. K., de Zeeuw, R.A. and Drenth, B. F. H., 1990) - A series of antihistamine derivatives were reacted with 2-Naphthyi chloroformate forming fluorescence carbarnates that could be separated using RP-HPLC (Gubitz, G-, Wintersteiger, R. and Hartinger, A., 1981). The potential use of this reaction as the basis for an assay process for Selegiline will be discussed following consideration of chiral assay procedures for amphetamine and its analogues. Chiral Separations of Amines with Particular Reference to

Amphetamine and its Analogues Many chiral pharmaceutical compounds contain pnmary or secondary amine functions and have provided much of the impetus for the development of enantiomer separations of basic substances using chromatographie methods. The foollowing discussion will center predominantly upon the separation of enantiomers of amphetamine (LW),methamphetamine (MAI') and Selegiline as examples of primary, secondary and tertiary amines respectively and will consider separations utilizing high pressure liquid diromatography specifically. It should be noted however, that (although not discussed here) enantiomer separations of many of these compounds have also been reported using gas/liquid chromatography and capillary electrophoresis.

Amphe tamine Methamp he tamine

CH-

While derivatization prior to enantiomer or diastereomer separation on a chiral or achiral column is undoubtedly the more reliable method for development of a chiral assay for these compounds, some direct enantiomeric separations have been reported. Thus Makino (Makino, Y., Ohta, S. and Hirobe, M., 1996), using a chiral crown ether column ( 15 x 0.4 an Daicel Crovmpack (+), Daicel Chemicals, Japan) reported separation of racemic amphetamine but not racemic methamphetamine using aqueous perchloric acid (pH 1.8) mobile phase. Similarly, using what is a poorly defined B-cyclodextrin coated support, (the colurnn is reported to be awaiting patent approval) the enantiomers of Selegiline were separated (a = 1.095 from reported k' (+)-Selegiline = 1.47 and k' (-)- Seledine = 1.61) using a methanol/50 mM potassium phosphate buffer (60/40) mobile phase (Cserhati, T. and Forgacs, E., 1994). This separation however appears to be very solvent sensitive since replacement of the methanol in the mobile phase with ethanol yields identical values for the capacity factor (kt)of the enantiorners (Forgacs, E., 1995). While the above are examples of direct enantiomer separations using chiral supports, prior derivatization of amphetamine and its analogues would appear to greatly increase the potential for a successful separation of the enantiomers. Thus derivatization of a series of amines (including methamphetamine) by 2-naphthylchloroformate (NCF) has been followed by separation of the product carbarnates on a Pirkle type covalent chiral stationary phase ((Il)-N-(3, 5-dinitrobenzoy1)phenyl glycine bonded to silica). The derivatization introduces the R-basicnaphthyl function whch can interact with the IF acidic 3, 5 -dinitrobenzoyl group of the column leading to a successful "th.ree point attadiment" to the stationary phase (see previous discussion) (Doyle, T. D., Adams, W.A., Fry, F.S. Jr. and Wainer, LW., 1986).Similarly, the N-benzoyl amide derivatives of (2) AMP and (k) MAP have been separated on chiral cellulose based columns (Chiralcel OB (tri-O-benzoylcellulose)and Chiralcel OJ (tri-O-tolylcellulose)). Resolutions for both MAI? and AMF was superior on the Chiralcel OB column when using a mobile phase of hexane/2-propanol (90/10) with a flow rate of 1.0 ml/min (Nagai, T. and Kamiyama, S., 1990). More frequently however, derivatization is accomplished using chiral reagents followed by separation of the product diastereomers upon an achiral column. Several examples exist of this procedure in which chiral acvlating agents, isothiocyanates or chloroformates have been employed. N-acyl-L-prolyl chlorides have been shown to be useful chiral reagents when used prior to separation by gas chromatography (Liu, J. H. and Ku, W. W., 1981). The similar use of such reagents in high pressure liquid chromatography is typified by the reaction of 4-nitrophenylsulfonyl-(S)-prolyl chloride with AMP and MAP. Separations were carried out on a silica column in the case of AMP derivatives using a mobile phase of chloroform/heptane (80/20) whereas the diastereomers of MAP were separated on a Zorbax C-18 column with a mobile phase of methanol/ water (60/40)(Barksdale, J. M. and Clark, CR., 1985). Among the chiral isothiocyanates employed as derivatizing agents, 2,3,4,6-tetra-0-acetyi-E-D-glucopyranoslisothiocyanate (GITC) is frequently used. Noggle et al (Noggle, F. T. J., DeRuiter, J. and Clark, CR., 1986) describe the separation of the diastereomers of (+)-methamphetamine and the use of such a separation in studies of forensic samples containhg (S)-methamphetamine and significant quantities of (IR, 2s)-ephedrine (figure 13). The amines are derivatized in chloroform solution in the presence of a 10 molar excess of reagent followed by separation on a C-18 column using a mobile phase of water/THF/acetic acid (70/35/1). The isothiocyanate group in GITC is very reactive and undergoes nucleophilic attack by the amino group of primary and secondary amines to form thiourea products (figure 14 ). (N. B. The presence of small quantities of (IR, 2s)-ephedrine or (lS, 2s)-pseudoephedrine in samples of (5)-methamphetamine would probably indicate a poorly purified sample derived (IR, 2S) Ephednne S (+) Methamphetamine

Stereochemical precursors of S (+) Amphetamine

Figure 13

CH; I

X = H for Methamphetamine/ X = OH for Ephedrine

Derivatization Reaction of an enantiomer of Methamphetamine with 2,3,4,6-tetra-O-acetyI-i3-D-glucopyranosylisothiocyanate (GITC)

Figure 14

48 from illicit synthesis; s imilarly , the presence of solely (R)-(-)-methamphe tamine in urine samples would be indicative of therapeutic use of Selegiline rather than use of illicit methamphetamine. Such distinctions require the use of chiral assav procedures). The chiral derivatizing agents 2,3,1,-tri-O-acetyl-a-D- arabinopyranosyl isothiocyanate (AITC) and (R)-(+)-1-phenylethyl isocvanate (PEIC) have also been used for the separation of amphetamines (Miller, K. R., Gal, J. and Arnes, M.M., 1984). Mention has already been made of the use of 2-naphthyl chloroformate (NCF) as an acylating agent for primary and secondary amines prior to separation of the enantiomeric carbamates on a chiral colurnn (see separation of amphetamine enantiomers above). Many of the carbamates derived from 2-NCF are fluorescent making detection of the products extremelv sensitive. In addition however, chloroformates react with tertiary amines but the product quatemary carbamate is unstable, dealkylating at slightly elevated temperatures to the carbamate of the secondary amines. Thus, a number of tertiary amines of therapeutic importance have been analyzed as the carbamate of the demethylated secondary amine by this method e-g. diphenhydramine, tenalidine, diphenylpyraline, clemastine (see figures 15 and 16) (Gubitz, G., Wintersteiger, R. and Hartinger, A., 1981). Reaction of 2-Naphthyl Chloroformate with a tertiary amine Figure 15 Diphenhydramine

Diphenyipyraline

Tertiq Amines derivatized with 2-Naphthyl chloroformate Figure 16 Other chloroformates commercially available are (- menthyl chloroformate and the strongly fluorescent compounds 9-fluorenylmethyl chloroformate (FMOC) and (+)-1-(9-F1uorenyl)ethylChloroformate (FLEC). This latter reagent has been used to separate amino acid enantiomers and various chiral amines. The derivatization of amino acids with FLEC occurs at room temperature under basic conditions in an aqueous environment. At the completion of the reaction, excess reagent and its hydrolysis product is removed by pentane extraction and the required diastereomeric products separated on a C-8 column. Enantiomers of Metoprolol, a secondary amine, have been separated using the FLEC reagent (Einarsson, S., Josefsson, B., Moller, P. and Sanchez, D., 1987). The secondary amine was mixed with borate buffer (lM, pH 7.85) and reacted with FLEC (1 mM in acetone) at room temperature for 30 minutes. Excess reagent was removed by reaction with an excess of hydroxyproline and the product diastereoisomeric carbarnate derivatives separated on a C-8 column using a mobile phase of acetonitrile/ water (60 /XI). Recently, a sensitive HPLC rnethod for the determination of the three main metabolites of Selegiline in human plasma has been developed using FMOC as a fluorescent derivatking agent (La Croix, R., Pianezzola, E., Benedetti and Strolin Benedetti, M., 1994). Using FMOC with fluorescence detection considerably enhances the detection of desmethyl selegiline, N-methyl amphetamine and amphetamine. Samples (in 0.1 M HC1) are mixed in borate buffer (pH Il), FMOC (4 mM in acetonihile) is added, the samples allowed to react at 500 C for five minutes. Excess reagent was reacted with proline and the mixture injected directly ont0 a Nova-Pak Phenyl colurnn using a mobile phase of acetonitrile/50 mM phosphate buffer (50/50) (pH 6.0). Detection was by fluorescence using an excitation wavelength of 260nm and an emission wavelength of 315 nm. Linearity of response was obtained over the concentration range of 0.5 - 80.0 ng/ml plasma for both amphetamine and desmethyl- selegiline. While this study was aimed at the detection of the metabolites of Selegiline i-e. primary and secondam amines, no mention is made of studies of the reaction with the tertiary amine Selegiline- Thus, while the reaction conditions are mild, it is uncertain whether all the metabolites detected have corne from the metabolic processes or whether some may have arisen from the reaction of the small amount of unchanged Selegiline in the plasma with FMOC and subsequent chernical dealkylation.

(-) - Menthyl chloroformate is a further chemicallv stable chiral chloroformate which has been employed in chiral assays involving prior derivatization to form diastereorners. The matenal is capable of reacting with primary, secondary or tertiary amines. As previously mentioned, the product from reaction with tertiary amines dealkylates on heating to yield the carbarnate of the secondary amine. An example of the use of this reagent for the determination of tertiary amines is provided by studies on the chiral amine

Promethazine (figure 17 ) (Witte, D. K., de Zeeuw, R. A. and Drenth, B. F. H., 1990). The product formed from dealkylation of Promethazine is unambiguous in structure. However, the application of this dealkylation reaction to the development of a chiral assay for Selegiline could result in either or both of two possible products i.e. that resultuig from dealkylation at the N-methvl function and that resulting fxom dealkylation at the N-propargyl function (figure 18 ).

Figure 18 Thus, if the propargyl group is cleaved from Selegiline, the product formed will be the menthyl carbamate of methamphetamine whereas, if the methyl group is cleaved preferentially, the product will be menthyl carbamate of desmethyl selegiline. Kapnang and Charles (Kapnang, H. and Charles, G., 1983) studied the reactions between various amines and chloroformates in order to examine the ease of cleavage of different substituents on nitrogen. When one of the substituents was a benzyl group, its cleavage was favored over the loss of methyl, cyclohexyl or n-pentyl. Through a series of related experiments they reported the following preference with respect to cleavage at the nitrogen; N- debenzylation > N-deallylation > deamination of cyclohexylamines >N- demethylation. This information would seem to suggest with respect to Selegiline that the N-propargyl group wouid be cleaved more readily than the N- methyl function. However, the nature of the products formed following reaction with menthyl chloroformate with Selegiline and subsequent dealkylation can readily be determined by cornparison with the products of reaction with methamphetamine and with desmethyl-Selegiline. Provided the dealkylation reaction results in only one carbarnate product, the use of (-)-menthyl diloroformate as the derivatking agent should provide a useful starting point to the developrnent of a chiral assay for Selegiline. The work presented in this thesis will report upon this reaction and the studies to develop a chiral assay for Selegiline alone and within its dosage form (tablets), along with

quantitative data of Selegiline tablets from two different lots. In addition, some qualitative results will be reported on studies of the reaction of Selegiline, desmethyl selegiline and methamphetamine with other chloroformates (ethyl chloroformates and 9-fluorenyhethyl chloroformate) since there is only limited information in the literature pertaining to the reaction of tertiary amines with chloroformates. Any additional information could be valuable to scientists interested in chiral separations of iertiary amines or dealkylation reactions. Discussion of Expenmental Phvsical. Chemical Characteristics of Seleeiline hvdrochloride Selegiline hydrochloride is a near white powder, with a calculated molecular weight (Cifii7N HCL) of 223.75 and a reported melting point

between 141 to 144 O C (Ecsery, Z., Kosa, L, Knoll, J., Somfai, E., 1967, Fowler, J.

S., 1977, and Robinson, J. B., 1985). X- ray diffraction studies on (-) Selegiline hydrochloride have determined that the crystals are orthorombic and belong to the P212121 space group (Simon, K., Podanyi, B., Ecsery, 2. and Torok, Z., 1986, Simon, K., Bocskei, Z. and Torok, Z., 1992) . Chafetz et al (Chafetz, D., 1980, L., Desai, M.P. and Sukonik, L., 1994) determined by titration a pKa value of 7.4 at 250 C for Selegiline hydrochloride, a value which agrees with predictions (Robinson, J. B., 1985,, Ullrich, K. J., Rumrich, G., Neiteler, K., and Fritzsch, G., 1992) derived from the known pKa of amphetamine (pKa = 9.92 at 250 C) (Girault-Vexlearschi, G., 1956) and the known base-strengthening and base -weakening effects following successive alkylation of the amine function with methyl and propargyl functions (Perrin, D. D., 1980) Selegiline hydrochloride, as expected is freely soluble in water and methanol but does display an unusually high solubility in chloroform [Chafetz et al report a water/chlorofonn partition coefficient of close to 1 without however reporting the pH of the aqueous phase

(Chafetz, L., Desai, M. P. and Sukonik, L., 1994). As will be shown in a subsequent section, this unusual solubility of Selegiline hydrochloride in chloroform proved advantageous in the extraction of the drug frorn its dosage form. Preliminaw Studies of Potential Assav Procedures While the main objective of ths study was to develop a stereospecific assay procedure for Selegiline Hydrochloride, the availability of achiral assay procedures were employed initially, particularly for confirmatory purposes in the syntheses of (5)-Selegiline, (+)-desmethyl-selegiline and (f) - methamphetamine. The method reported by Beaulieu et al used employing a cyan0 column (Waters pBondapak CN; 150 x 3.9 mm) with a mobile phase of 0.1 M ammonium phosphate dibasic (adjusted to pH = 3. 1 with 85% phosphoric acid) and acetonitrile at a ratio of 8515 with a flow rate of 1.0 ml/min (Beaulieu, N., Cyr, T.D., Graham, S. J. and Lovering, E. G., 1991). Detection was by means of a variable UV detector set at 254 nm and the diluent used for bolfi the samples and standards was 15% acetonitrile in distilled water. For the purposes of confirming the identity of newly synthesized material, both samples and standards were prepared at a concentration of 0.1 pg/ml. The method is capable of separating amphetamine, methamphetamine, desmethyl selegiline and Selegiline. Chiral columns and Chiral Additives As part of the initial investigation of potential chiral separatory methods two chiral columns were also studied, namely a Bakerbond Chiral DNBPG column [chiral discriminator of (R) -N-(3, 5,-dinitrobenzoyl) phenyl glycine covalently bonded to silica; 250 x 4.6 mm; 5 pm ] and a Chiralcel OC column N- phenylcarbamate ester of cellulose which is covalently bonded to silica; 230 x 4.6 mm; 10 pm]. Solutions of (f) Selegiline, (f) desmethyl Selegiline and (2) methamphetamine failed to show enantiomer separation when employing a mobile phase of hexane/isopropyl alcohol (97:3) at a flow rate of 1.0 ml/min. Further, brief studies employing the diastereoisomeric mixtures derived frorn the reaction of (-) menthyl chloroformate with each of the above racemates similarly failed to show separa tion of the dias tereoisomeric compounds. An alternative means of potentially separating racemates is to ernploy a chiral mobile phase with the hope that the product diastereoisomeric complexes (ion pairs) will show separation. Accordingly, using the cyano column previously employed for identification of the various synthetic racemates (see above), the original mobile phase was modified by replacing the ammonium phosphate with either (+) tartaric acid (concentration 0.05 or 0.025 M) or with d- carnphor-10-sulfonic acid. Not unexpectedly, these systems failed to yield a çepara tion of the enan tiomeric forms of Selegiline, desmethyl-çelegiline or methamphetamine- Although, several authors have demonstrated the use of a low polarity solvent such as methylene chloride (separation on a normal phase column) favors the formation of "ion pairs" it should be noted that amphetamine was separated (although not baseline separation) using the above mentioned aqueous mobile phase (Pettersson, C., 1988, Pettersson, C. and Sdiill, G., 1981, Pettersson, C. and Josefçson, M., 1986). As previously rnentioned rnost chiral recognition processes require the usual "three point" interaction , whereas the separation of diastereomeric complexes (ion pairs) can sometimes be achieved by a two point interaction. It appears that the underlying mechanisms responsible for a chiral separation based on selectors in the mobile phase are mudi more cornplex. In addition to the interaction behveen the chiral selector and solute, the entire equlibriurn of the diromatographic system must be taken into account (Pettersson, C. and Schill, G., 1981). Chiral mobile phases were also produced using solutions of chemically modified B-cyclodextrin. Specifically, three forms of hydroxy1propyl-B- cyclodextrin (containing varying degrees of hydroxypropyl substitution) were added at either 1 or 5 mM concentration to a mobile phase of phosphate buffer (M/15; pH 8.2) containing 30% acetonitrile. Chromatography was studied using a silica column ( Brownlee; Spheri-5; 100 x 4.6 mm) at a flow rate of 0.5ml/min with detection at 254 nm. While the retention time of Selegiline hydrochloride was modified by the presence of the hydroxypropyl-D-cyclodextrin,the system failed to show separation of the enantiomers of Selegiline hydrodiloride. Reverse Phase Separations Having established that it was not possible to separate Selegiline without prior derivatization using either a chiral column or a chiral mobile phase additive, separation of the diastereomers formed from reaction with (-) menthyl chioroforma te using a reverse phase system was inves tigated. Separations were started using various C-8 columns with mobile phases containing rnethanol, water and modifiers such as tetrahydrofuran, diethyl ether, or isopropanol.

Experiments with a 150 x 4.6 mm Zorbax C-8 column (5 pM) showed inferior separations as compared to other C-8 columns. Such Zorbax columns are end capped and often used to separate cornpounds that exhibit tailing. The inferior separation seems to indicate that the presence of some untreated silanol groups are needed for the separation. It was also noticed that the addition of either tetrahydrofuran or diethyl ether improved the peak shape. Although, the peaks were not adequately resolved with these mobile phases, thinner peaks were obtained. Table II hts some of the different coiumns and mobile phases studied. Eventually, an Altex C-8 (130 x 4.6 mm) was employed with a mobile phase consisting of 70/30, methanol/water at a 80w rate of 1.3 rnl/rnin. The separation had a resolution l& of 1.12, and a separation factor, a of 1.07. The retention times of the two diastereomers were 27.6 and 29.4 minutes. As can be seen from the chromatogram (figure 19) there are clearly two peaks; however baseline resolution is not achieved (Rs= 1.5). An S value of 1.12 for two peaks with a Peak height ratio of 111represents between a 98.0 O/O to 99.4 Oh resolution (see figure 20). Since it was assumed that adjustments to the chromatography might have to be made for the tablet assay, this separation was considered adequate. Separate injections of individual enantiomers showed that the [SI carbarnate of methamphetamine eluted fïrst followed by the [RI denvative. Since one of the objectives of this work will be to apply the chiral separation to assay Selegiline tablets (expecting only to see the [RI enantiomer) this is the preferred order, as it is always desirable to'have the minor antipode eluted before the major due to the possibility of tailing (Lough, W. J., 1989). Table II Method Development us in^ MehoIWater

Method Develo~rnentUsine MethanoWater / Diethvl ether

MethanoüH,-O/Diethyl ether

Method DeveIo~mentUsing MethanoVWater/TKF

* *Zohau CoIumn ***Flowrate in dmin (-) Menthyl carbamate derivative of (f)Methamphetamine RG = 1.12 COI-: ~ltexC-8 (150 x 4.6-1, Mobile 70/30 methLi/~~o Flow rate: 1.3 ml/min, Detector set at 254 nm, 20 pl manual injection

Figure 19 Fig. 20.4 Resolution of neighbouring peaks, peak-height ratio 1 :1

Resolution

Relationship Between Peak Separation and Resolution (Meyer, V. R., 1994)

Figure 20 5 me Prevarations - Metharn~hetamineand SeIedine carbarnate Having conducted al1 of the preliminary separations on the bulk derivatives of either methamphetamine or desmethyl-selegiline, conditions for the derivatization reaction at a concentration sirnilar to that of Selegiline tablets (5 mg) needed to be determined. In addition, the reaction of (-1 menthyl chioroformate and Selegiline had not been performed on a microscale and it was suspected that harsher reaction conditions may be necessary for the tertiary amine. The derivatization conditions used were a modification of those used by Gubitz et al (Gubitz, G-, Wintersteiger, R. and Hartinger, A., 1981). The reaction was performed in benzene with a 30 fold molar excess of potassium carbonate and a 10 fold molar excess of (-) menthyl chloroformate. Specifically, 5mg of the amine hydrochloride, lOOmg of potassium carbonate, 500 pl of (-) menthyl diloroformate (10% v/v solution in benzene) and 500 pl of benzene were added to a Silli-vap vial. The vials were sealed with appropriate septa and heated. Studies showed that the derivatization reaction with Selegiline had to be conducted at 1000 C, whereas the carbamates of methamphetamine and amphetamine couid be formed in higher vields at 600 C. Therefore, samples were heated for one hour at 1000 C and allowed to cool to room temperature. The liquid layer was poured into a centrifuge tube, allowing the solids to remain in the vial. In order to remove any excess menthyl chloroformate 3 mls of methanolic potassium hydroxide was added and the mixture vortexed. To aid in phase separation 5 mls of distilled water was added and the tube was then centrihged. The benzene layer was removed, dried (molecular sieve), dilrited as necessary and injected onto the HPLC. As expected the yields of the Selegiline carbamates were much lower than those formed from either methamphetamine or desmethyl selegiline. Calculating peak areas of Selegiline against those of methamphetamine, it was estimated that there was only an 8% yield from the Selegiline hydrochloride derivatization reaction. Extending the time for the Selegiline hydrochloride reaction did not seem to improve the yields significantly [It was difficult to tell at this stage in the absence of an interna1 standard if an increase in yield was due to evaporation or the additional reaction tirne]. Although the yields were low it was determined that the N-propargyl group was cleaved from the Selegiline molecule, since the carbarnate retention times of both methamphetamine and Selegiline were identical and different from those of desmethyl Selegiline. Although the reaction worked, with continued column use the separation deteriorated, the peaks widened resulting in a decreased separation, R, = 0.91 (figure 21). To improve the chromatography the mobile phase was adjusted to include 5% isopropanol. Figures 22 and 23 demonstrate the effect of isopropanol on the chromatography. Reverse Phase Chromatoera~hy- Interna1 Standard Selection For the present tirne the separation was adequate and an intemal standard had to be chosen. Generally the use of an intemal standard ensures a high degree of analytical precision, specifically the uncertainties introduced by manual injection can be avoided. Since this separation requires a derivatization reaction, the use of an intemal standard overcomes any slight incowistency in the reaction conditions and extraction process. An ideal internal standard would be either an achiral or optically pure primary or secondary amine. Table III lists the internal standards evaluated along with their retention times. Based on a reasonable retention tirne (15 minutes) and ease of derivatization, R-amphetamine used in the free base forrn was chosen. The extremely low yields of the Selegiline reaction were a concem and it was considered that changing the solvent from benzene to dichloroethane might increase the yield. Dichloroethane was chosen because, it had been used by other (-) Menthyl carbarnate derivatives of (k) Methamphetamine Chromatography deteriorating, Rs = 0.91 Column: Altex C-8 (150 x 4.6mm), Mobile phase: 70/30 methanol/HzO Flow rate: 1.3 mlhin, Detector set at 254 nm, 20 pl rnanual injection

Figure 21 (-) Menthyl carbarnate derivatives of (k) Methamphetamine Effect of 2% isopropanol (PA)on the separation Column: Altex C-8 (150 x 4.6mm), Det'ector set at 254 nm, 20 pl injection Mobile phase: 68/30/2 methanol/H20/IPA, Flow rate: 1.3 ml/min

I Figure 22 (-) Menthyl carbamate derivatives of (k) Methamphetamine Effect of 5% isopropanol (PA) on the separation Column: Altex C-8 (150 x 4.6mm), Detector set at 254 nm, 20 pl injection Mobile phase: 65/30/5 methanol/H20/IPA, Flow rate: 1.3 ml/min Figure 23 investigators in similar reactions and has a high boiling point (830 C) with a W cut off of 230 m. Chloroform was considered but it has boiling point of 610 C and a UV cut off of 245 nrn. Experiments indicated that replacing benzene with dichloroethane increased the yields of the Selegiline carbamates as well as those of methamphetamine and desmethyl selegiline. Although there was a substantial improvement (-35% for Selegiline) using dichloroethane, yields from both methamphetamine and desmethyl selegiline were still greater than those of Selegiline. At this point the amount of isopropanol in the mobile phase was increased to IO%, for a final mobile phase composition of 60:30:10, methanol: water: isopropanol with a flow rate of 1.3 ml/min. Having corne to the point where the conditions of the reaction and chromatography were set, two calibration curves of R-methamphetamine and R- amphetamine were prepared. In one curve the interna1 standard, R- amphetamine was derivatized separately from methamphetamine and then mixed together prior to injection. Ln the second calibration curve, both R- amphetamine and R-methamphetamine were derivatized in the same vial. The slopes and intercepts of the two curves were very similar, indicating that there was enough (-) menthyl chloroformate in the reaction vial to derivatize both compounds. Although the results from the calibration curves were encouraging, by the end of the second calibration curve the chromatograms showed tailing and poor peak shapes (figures 24 and 25). Table LU Reverse Phase - Interna1 Standards

Compound Retention time R- Amphetamine 14.24 minutes

Pargyline (two peaks) 20.61/22 minutes a ,a - Dimethy 1 phenylethylamine 24.20 minutes R-a-Methyl pargyline (peak tails badly) 25.80 minutes S-N-GDimethy 1 N-propargy 1 l3-phenyle thy lamine 36.61 minutes N- a ,a Trimethylpropargyl B-phenylethylamine 50.50 minutes

Deriva tized Metham~hetamine 25.80 minutes (-) Menthyl carbarnate derivatives of [RI Methamphetamine and [RI Amphetamine / Peaks starting to tail Column: Altex C-8 (150 x 4.6mm), Detector set at 254 MI, 20 pl injection Mobile phase: 60/30/10 rnethanol/H20/IFA, Flow rate: 1.3 ml/min

Figure 24 (-) Menthyl carbamate derivative of [RI Amphetamine Interna1 Standard Peaks are Taihg Column: Altex C-8 (150 x 4.6mm), Detector set at 254 nm,20 pl injection Mobile phase: 60 /3O/lO methanol/H20/IFA, Flow rate: 1.3 ml/min

Figure 25 Ofien when peaks are not gaussian in shape it can be due to either the column deteriorating, too much sarnple being injected (sample loading) , solvent incompatibility or an inappropriate pH (Meyer, V. R., 1994). Each standard via1 contained 100 pl of dichloroethane and 200 pl of methanol. Perhaps the 2:l ratio

of methanol : dichioroethane was a problem. Not really sure if it was a solvent incompatibility problem or just column deterioration, normal phase separations were studied. Dichloroethane would be more compatible with a normal phase separation. Normal Phase Separation The C-8 column was replaced with a silica column [Brownlee Spheri- 5,

220 x 4.6 mm 1 and various concentrations of hexane and diethyl ether or isopropanol were evaluated for the mobile phase. Using isopropanol in the mobile phase did not result in any separation. Isopropanol is probably too polar and therefore prevents the derivatized products from interacting with the stationary phase to allow for a separation. Experiments with diethyl ether proved successful and showed a superior separation as compared with that of the reverse phase system (figure 26). While optimizing the amount of diethyl ethyl ether to be used in the mobile phase it was noticed that a slight modification in the amount of diethyl ether (1%) resulted in a significant difference in the retention times of the resulting carbamate derivatives. An example of th& is seen in figures 27 and 28. With a flow rate of 1.5 ml/min and a mobile phase of 96:4 (hexane: diethyl ether) the retention times of the methamphetamine carbamate diastereomers are 25.42 and 26.45 respectively, whereas using a mobile phase of 95:5 (hexane: diethyl ether, 1.5 ml/min) resulted in retention times of 21.03 and 21.84 for the same derivatives. (-) Menthyl carbamate derivatives of (k) Seleghe (-) Selegiline - 18.93 min / (+) Selegdine - 19.71 min Normal Phase Cluomatography Column: Silica Brownlee Spherî 5 (220 x 4.6mm) Mobile phase: 95 /5, hexane/diethyl ether, Flow rate 1Jml/ min Detector set at 254 nm,20 pl manual injection

Figure 26 (-) Menthyl carbarnate derivatives of (k) Methamphetamine (-) Methamphetamine - 21.03 min / (+) Methamphetamine 21.84 min Normal Phase Chromatography 96/4 Hexane/diethyl ether Column: Silica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 96 /4, hexane/diethyl ether, Flow rate 1Sml/ rnin Detector set at 254 nm, 20 yl manual injection

Figure 27 (-) Menthyl carbarnate derivatives of (k) Methamphetamine (-) Methamphetamine - 25.42 min /(+) Methamphetamine - 26.46 min Normal Phase Chromatography, 95/5 Hexane/diethyl ether Column: Silica Brownlee Sphen 5 (220 x 4.6mm) Mobile phase: 95 /5, hexane/diethyl ether, Flow rate 1Sml/ min Detector set at 254 nm, 20 pl manual injection

Figure 28 Typically when working in reverse phase duomatography a larger percentage of

the more non-polar component in the mobile phase is needed for such a change in retention time. Having noticed the pronounced effect that the concentration of diethyl ether had on the retention tirnes, the mobile phase container was tightly sealed preventing evaporation of the ether. However it should be noted that changes in retention times were not totally preventable, factors such as environmental conditions (room temperature) and slight differences in the

hexane ( due to different manufacturers) resulted in minor variations of retention times. At this time a mobile phase of 964 (hexane: diethylether) at a fiow rate of 1.5 ml/min was chosen and such conditions were employed to select an appropriate intemal standard. In addition to improving the chromatography, changing from a reverse phase system to a normal phase system also reversed

the elution order the diastereomers. In the normal phase systern the order is R (-)

followed by S (+). Normal Phase Chrornatoera~hv- Intemal Standard Selection Using the same criteria as previously mentioned in the reverse phase separation various interna1 standards were evaluated. Although [RI amphetamine was used in the reverse phase separation, its retention time of 24 minutes was too close to that of [RI methamphetamine. Table IV lists the compounds that were evaluated using normal phase chromatographic conditions, along with their retention times. Since a ,a -Dirnethylbenzylamine did not interfere with the quantitation of the Selegiline carbarnates it was chosen as the intemal standard. Since the separation of the derivatives and the interna1 standard was suffïcient it was decided to adjust the mobile phase to 95:5 (hexane: diethylether) in order to shorten the total run time of the separation. The flow rate remained the same at 1.5 ml/min. Figure (29) shows a chromatogram of the separation of derivatized Selegiline with a , a -Dimethylbenzylamine as the interna1 standard. Table TV Normal Phase Intemal Standards

Com~ound Retention The' a, a- Dimethylp henylethy lamine 6.18 minutes Aniline 6.22 minutes [RI Arnp hetamine 24.00 minutes [SI - a-Methylbenzylamine 26.92 minutes a,a- Dimethylbenzylamine 34.78 minutes [RI - a-Methyl benzylamine 39.43 minutes

Derivatized Methamrhetamine [RI Methamphetamine 25.42 rninu tes

[SI Me thamp he taamine 26.46 minutes

--

The retention tirnes are based on a mobile phase composition of96:4 (hexane: diethyl ether) with a flow rate of 1.5 ml/rnin. (+) Selegiline Carbarnates with a ,a -Dimethylbenzylamine Retention tirne of Interna1 Standard 24.63 minutes Normal Phase Chromatography, 95/5 Hexane/diethyl ether Column: Silica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 95 /5, hexane/diethyl eh,Flow rate 1.5ml/min Detector set at 254 nm, 20 p1 manual injection

Figure 29 Preparation of Calibration Curves - Metham~hetamine Using normal phase chromatography individual calibration curves for the

(-) menthyl ~Moroformatederiva tives of [SI and [RI methamphetamine and [SI and [RI Selegiline using u , a -Dimethylbenzylamine as an intemal standard were prepared. Having realized the benefit of dichloroethane, both the intemal

standard and the (-) menthy 1 chloroformate were dissolved in dichloroethane. Calibration curves for methamphetamine were prepared by weighing approximately 2.5 to 5.5 mg of an individual enantiomer into a silli-vap vial. To each vial 500~1of (-) menthyl chloroformate (10% v/v in dichloroethane), 500pl

a , a -dimethylbenzylamine (0.5% v/v in dichloroethane) and 100 mg of potassium carbonate was added. The vials were sealed with the appropriate septa and heated for one hour at 1000 C. After allowing the vials to cool, the liquid layer was poured into a l5ml centrifuge tube, leaving the solid behind and 3 ml of methanolic potassium hydroxide was added. The mixture was vortexed for two minutes and 5 ml of distilled water added. The tubes were then centrifuged for ten minutes. The top aqueous layer was removed by a pasteur pipet and discarded. The dichloroethane layer was poured into a clean dry silli- vap vial containing molecular sieves ( type 4A to remove any residual water). The standard solution (100 pl) was diluted to 1.0 ml with hexane. Solutions were then injected until three injections yielded area ratios within 2% of each other. The range of 2.5 to 5.5 mg was diosen because it brackets the expected 5mg dose contained in a tablet. Results of the first calibration curve of [RI methamphetamine showed inconsistent internal standard peak areas. Specifically, as the concentration of methamphetamine was increased the peak areas of a , a -dimethylbenzylamine decreased. This suggested that there was not enough (-) menthyl chloroformate to derivatize both methamphetamine and the internal standard. To alleviate this problem the stock solution of (-) menthyl chloroformate was increased from 10% v/v to 20% v/v. This solved the problem, the intemal standard peak areas were consistent again, and in al1 subsequent reactions 500pl of (-) menthyl chloroformate (20.0 v/v in dichloroethane) was added to the derivatization reactions. The above procedure was conducted for both enantiomers of methamphetamine. Linear regression analysis using the least squares method showed straight lines for both curves with R2 values of 0.99490 and 0.98924 for the [RI and [SI calibration curves respectively. Although the y-intercept (0.15413) of [SI methamphetamine was not optimal the data does indicate that the derivatiza tion process is quantitative. Statistical data and corresponding graphs are presented in the Experimental section. Preparation of Calibration Curves - Seledine Realizing that Selegiline hydrodiloride would have to be extracted from the tablets it was decided that a procedure be developed and used to extract Selegiline hydrodiloride from the tablets as weil as to prepare calibra tion curves. Since Selegiline hydrochloride is freely soluble in chloroform and not as soluble in dichloroethane it was decided to extract the hydrochloride salt into chloroform and then derivatize as usual with bot-the interna1 standard and (-) menthyl chloroformate in dichloroethane. Chloroform with a boiling point of 61.20 C and a UV cut off of 245nm would not be a suitable derivatization solvent, and therefore would only be used as the extraction solvent. Experiments showed that extracting in situ (in the silli-vap vial) resulted in the greatest yields. Attempts at dissolving 5mg of Selegiline hydrochloride in water, making the solution basic and then extracting into chloroform resulted in low yields and large variations possibly due to the known volatility of Selegiline base. The first calibration curves of [RI and [SI Selegiline hydrochloride were prepared by weighing a specific amount of Selegiline hydrodiloride (2.5 to 5.5 mg) into a silli- vap vial, recording the weight and adding Iml of chloroform to each vial. The chloroform layer was evaporated to dryness under a gentle stream of air. To eadi

vial 100 mg of potassium carbonate, 500pl(-) menthyl chloroformate (20% V/V in dichloroethane) and 500pI a , a -dimethylbenzylamine (0.25% v/ v in didiloroethane) was added. The silli-vap vial was heated for one hour at 1000 C , then allowed to cool. As with the methamphe tamine standard preparation the liquid layer was poured into a centrifuge tube, 3ml of methanolic potassium hydroxide solution added and the contents vortexed. Distilled water (5ml) was added and the mixture centnfuged for ten minutes. Using a disposable pasteur pipet the top aqueous layer was removed and discarded. The remaining dichloroethane Iayer was placed into a clean silli-vap vial containing molecular sieve (type 4A) for the removal of residual water. Due to the differences in yields relative to pure methamphetamine, the final standard solution was prepared by adding 100~1of standard solution to 200~1of hexane. Each standard solution was injected until three injections pelded area ratios within 2% of each other. Cornparisons of the two curves showed similar dopes of 0.02329 and 0.02070 for the [RI and [SI enantiomers indicatuig consistent extraction and derivatization of the two enantiomers. Regression analysis showed correlation coefficients of 0.993 and 0.994 for the [RI and [SI calibration curves respectively. Although these results showed reasonable correlation coefficients, one final modification was made. To eliminate errors associated with weighing srnall amounts (2.5 - 5.5 mg) of Selegiline hydrochloride into individual vials, a stock solution of Selegiline hydrochoride in chloroform was prepared. Using an Eppendorf pipet specific amounts ranging from 100 ~1 to 500 pl of Selegiline hydrochloride stock solution were added to individual silli-vap vials, which were then evaporated to dryness under a stream of air. The remainder of the derivatization procedure was carried out exactly as had been done previously. Regression analysis of the [RI curve showed improvernent with a correlation coefficient of 0.999. Figure 30 shows a chromatogram of [RI Selegiline and a ,a -dirnethylbenzylamine standard solution derivatized with (-) menthyl chloroformate. Statistical information and the calibration curves are presented in the Experimental section. Aboorbancm -0.0050 O.OQOQ O-OQSO 0.OIOQ 1 1 I 1t1 Il, 1 1 I I

[RI Selegiline and a ,a -dimethylbenzylamine standard solution derivatized with (-) menthyl chloroformate, Colurnn: Silica Brownlee Spheri 5 (220 x 4.6mrn) Mobile phase: 95 /5, hexane/diethyl ether, Flow rate 1 Sml/min Detector set at 254 m, 20 pl manual injection

Figure 30 Avvlication of the Assav to Seleeiline hvdrochloride Tablets The final portion of this work was to apply the assay to analyze Selegiline hydrochloride tablets. Once again using the favorable solubility of Selegiline hydrochloride in chioroform, experiments were conducted to develop a suitable extraction technique. The major difference between assaying the tablets and the raw material is the presence of the excipients. Each Selegiline hydrochloride tablet contains 5mg of Selegiline hydrochloride, 84 mg of Lactose, 9 mg of Polyvinyl povidone (PVP), 3 mg of talc, 3 mg of magnesium stearate and 16 mg of starch for a total tablet weight of 150 mg'. Experiments showed that a tablet would not dissolve by simply placing a tablet in a solution of chloroform and shaking by means of a mechanical shaker. Therefore it was determined that a tablet (weight recorded) would have to be cmshed uçing a mortar and pestle and then extracted. Once the Selegiline was extracted the derivatization could be carried out as had been done previously. Borrowhg from the technique used to prepare the calibration curves, an individual tablet was cmshed and a known amount (approximately 100 mg) of the powder was placed into a test tube to which 1.0 ml of chiorofom was added. The tube was allowed to shake for 15 minutes on a mechanical shaker. At this time experiments were conducted to separate the excipients from the Selegiline by means of filtering. Filter paper, scintered glass filters and a nail filter were employed for this purpose. Results from these experiments showed very low yields. Realizing that the Selegiline was being absorbed ont0 the various filters it was decided to centrifuge the crushed tablet and pour off the remaining liquid. A single tablet was therefore weighed, crushed using a mortar and pestle and an aliquot added (100 mg) to a centrifuge tube. Chloroform was added (1 ml), the

** Thinformation was listed on package of Selegiline tablets

86 tube was centrihiged for 5 minutes and the liquid layer poured into a silli-vap vial. The chloroform was evaporated to ddryness under a gentle stream of air and

100 mg of potassium carbonate, 500 pl of (-) menthyl chloroformate, and 500 pl of internal standard solution was added. The derivatization procedure was continued as previous. Results showed a mean recovery of 96.5O/0 with a standard deviation of 2.01%- in order to increase the recovery the crushed tablet was extracted twice with two 1 ml fractions of chloroform. Results indicated 102.0Y0 recovery, this procedure was then used to analyze the two different lots of Selegiline hydrochloride tablets. To demonstrate the lack of interference from the excipients, an excipient mix was prepared using the above quantities and assayed using the same procedure (including the internal standard). Results showed no interference. Studies employin~other chloroformate reagents As previously mentioned, a further objective of this study was to investigate the reactions of Selegiline, methamphetamine and desmethyl selegiline with other chloroformate reagents. While the reaction of menthyl chloroformate with the tertiary amine Selegiline yielded the carbarnate derivative of methamphetamine as the sole identified product i.e. was the result of loss of the propargyl function from the quatemary intermediate, it was unknown whether reaction of Selegiline with other chloroformates would similarly yield a single product from the dealkylation reaction. The chloroformate reagents employed in this study were ethyl chloroformate (EC) and 9-fiuorenylmethyl chloroformate (FMOC). Although neither of these reagents are chiral, they are both structurally different from (-) menthyl chloroformate, the one (ethyl chloroformate) being a simple aliphatic reagent while FMOC is the derivative of a highly bulky (planar) and lipophilic aromatic alcohol. The possibility exists that these reagents may yield products which: a) dealkylate more readily than the corresponding menthyl chloroformate derivatives giving rise to the carbamate derivative of a secondas. amine in higher yield b) deaikylate by competing routes giving Ne to a mixture of products

C) dealkylate by an unknown mechanism yielding the carbamate derivative of desrnethyl selegiline as the major or only product. In addition , the results obtained from investigating FMOC as a derivatizing reagent would possibly indicate the potential for employing the

commercially available (but extremely expensive) (+) -1-(9-fluorenyl) ethyl chloroformate (FLEC) as a derivatizing agent for Selegiline. The latter derivatizing agent (and other fiuorenyl derivatives) is highly chromophoric and also strongly fluorescent and would thus provide a far more sensitive assay procedure than when using enantiomers of menthyl chloroformate. The studies with ethyl chloroformate were carried out using the conditions. previously established for reaction of Selegiline with menthyl chloroformate with dichloroethane as the derivatizing solvent and chromatographie separations performed using the normal phase system. Figures 31, 32 and 33 show chromatograms of racemic methamphetamine, racemic desmethyl selegiline and racernic Selegiline. Looking at the chromatogram of racemic Selegiline derivatized with ethyl chloroformate (figure 33) there are three peaks present (13.76, 16.24 and 31.98 minutes). The peak at 31.98 minutes corresponds to the peak obtained from the derivatization of racemic methamphetamine with ethyl chloroformate (figure 32). The peak at 16.24 minutes appears to be the same peak obtained from the derivatization of ethyl chloroformate with desmethyl selegiline which has a retention time of 16.35 minutes (figure 32), while the peak at 13.76 minutes is only seen on this chroma togram and does no t correspond to any ethyl carbama te p roduct. Although further experiments would be needed it does appear that the derivatization of Selegiline with ethyl chloroformate does result in a mixture of products. However the predominant carbamate produced is the methamphetamine carbamate. Interestingly the peak area corresponding to the desmethyl carbamate is slightly higher than that of the methamphetamine carbamate (5mg of both methamphetamine and desmethyl selegiline were derivatized with ethyl chloroformate). As with (-1 menthyl chloroformate the yields are higher (approximately 32.0%) for the methamphetamine ethyl carbamate than the Selegiline ethyl carbamate. This is not surprising since the derivatization reactior.

of methamphetamine with (-) menthyl chloroformate (in dichloroethane, normal phase separation) has a 65% greater yield than that of Selegiline derivatized with

(-) menthyl chloroformate. Studies with 9-fluorenvlmethvl chloroformate (FMOC) Experiments were performed employing both benzene and dichloroethane as the derivatizing solvent. Although the experiments performed in benzene gave confusing results, reactions performed in dichloroethane gave results similar to those of ethyl chloroformate. Derivatization reactions of Selegiline, desmethyl selegiline and methamphetamine with FMOC in dichloroethane showed that the derivatization of Selegiline resulted in the formation of carbamates corresponding to both desrnethyl selegiline and methamphetamine 9-fluorenylmethyl carbama tes. (Figures 34,35 and 36).Once again the major product of the reaction of Selegiline with FMOC was the formation of the methamphetamine ethyl carbamate. In addition to the peaks corresponding to amine carbamates was an additional peak which elutes immediately after the methamphetamine carbamate peak. Derivatization of a blank solution indicate that this additional peak cornes from unreacted 9- fluorenylmethyl chloroformate and is most likely the fluorenyl alcohol. In a similar readion reported by La Croix et al(La Croix, R., Pianezzola, E., Benedetti and Strolin Benedetti, M., 1994) FMOC was used to derivatize and amphetamine, methamphetamine and desmethyl selegiline from plasma. Ln this reaction proline was used to remove any unreacted 9-flurorenyl methyl chioroformate. The use of either a suitable amine as done by La Croix or an extraction could be used to remove any unreacted 9-fluorenyl chloroformate. As seen in ail other derivatization reactions, the carbamate of methamphetamine is produced in much higher quantities than the Selegiline carbamate. As expected due to the highly chromophoric nature of the FMOC reagent, the reactions of FMOC with al1 three amines is a much more sensitive assay than reactions with either (-1 menthyl chloroformate or ethyl chloroformate. (f)Methamphetamine Derivatized with Ethyl Chloroformate Deriva tiza tion in Dichloroethane, Column: Silica Brownlee Spheri 5 (220 x 4.6m) Mobile phase: 95 /5, hexane/diethyl ether, Flow rate 1.5ml/ min Detector set at 254 nm,20 pl manual injection Peak at 30.39 - Methamphetamine ethyl carbarnate

Figure 31 (+_)Desmethylselegiline Deriva tized with Ethyl Chloroforma te Derivatization in Dichloroethane, Separation on Silica Column Column: Silica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 95/5, hexane/diethyl ether, Flow rate 15ml/min Detector set at 254 run, 20 pl manual injection Peak at 16.35 - Desmethyl seleghe ethyl carbarnate

Figure 32 Abeorbanca -amsa a. 300~ a. 0050 o.0100 0.00 l 1 1 I i 11 111 Ill III 1 J 4 --5 -. !:Il 1.5'1 -

10.00 -

*

1o.00 - ,

10.00 - i t - 31.96

* f'

39.99 1 I Iiil K81 1" 1 i 1 1 -0.0050 0.0000 0.0050 0.0100

(f)Selegiline Derivatized with Ethyl Chloroformate Derivatization in Dichioroethane, Separation on Silica Column Colum: SiLica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 95/5, hexane/diethyl ether, Flow rate l.Sml/rnin Detector set at 254 nm, 20 pl manual injection Peak at 16.24 - Desmethyl selegiline ethyl carbamate Peak at 31.98 - Methamphetamine ethyl carbarnate

Figure 33 (+) Methamp hetamine derivatized with FMOC Derivatization in Dichloroethane, Separation on Silica Column Column: Silica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 95 /5, hexane/diethyl ether, Flow rate lSml/ min Detector set at 254 nm, 20 pl manual injection Peak at 48.66 - Methamphetamine FMOC derivative Peak at 52.15 - Excess FMOC reagent

Figure 34 (i)Desmethyl selegiline derivatized with FMOC Derivatization in Didiloroethane, Separation on Silica Column Column: Silica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 95/5, hexane/diethyl ether, Flow rate 1Sml/ min Detector set at 254 nm, 20 pl manual injection Peak at 25.66 - Desmethyl selegiline FMOC derivative Peak at 51.55 - Excess FMOC reagent

Figure 35 Msorbauca O.QldO -o.itaso 0.0000 o.oa!o I fi t III 1 L 1 1 III 1 111 1

1 1 . 1111 I III 1' I III -Q .O050 O.OOQ0 O.0OSO Q.0100

(f)Selegiline Derivatized with FMOC Derivatization in Didiloroethane, Separation on Silica Colurnn Column: Silica Brownlee Spheri 5 (220 x 4.6mm) Mobile phase: 95/5, hexane/diethyl ether, Flow rate 1.hl/ min Detector set at 254 nm, 20 pl manual injection Peak at 25.89 - Desmethyl selegiline FMOC derivative Peak at 48.46 - Methamphetamine FMOC derivative Peak at 52.02 - Excess FMOC reagent

Figure 36 Ex~erhenta1 Al1 weighing were done on a five place Mettler balance, model number

86. (-) Menthyl chloroformate ( 99% ee by GLC), ethyl chloroformate (97% purity) and 9-fluorenyl methyl chloroformate were al1 purchased frorn Aldrich.

(-) Selegdine hydrochloride used to prepare al1 (-) Selegiline standard curves was generously donated by Apotex Inc. Individual enantiomers of amphetamine hydrochloride, methamphetamine hydrochloride and the (+) enantiomer of Selegiline hydrochloride were previously synthesized by J. Barry Robinson. Potassium carbonate (anhydrous), ammonium phosphate dibasic and potassium hydroxide were al1 reagent grade. Synthesis of racemic methamphetarnine, desmethyl selegiline, Selegiline, a, a-dimethylbenzylamine and al1 precursors are listed in the appendix. Al1 solvents used were either reagent or HPLC grade. Samples were derivatized in Silli-vapB vials fi tted with appropriate septa and heated using a heating block equipped with sample wells designed to hold the vials. The heating unit contains a thermometer to monitor the temperature. Infrared Spectra of synthetic materials was performed using a Perkh Elmer Infrared Spectrophotometer model 1330 and were run as liquid films or Nujol mulls between NaCl plates. Melting points determinations were done using a Thomas Hoover Capillary melting point apparatus and are uncorrected. Initial reverse phase chiral and achiral separations were performed using a Waters M-45 solvent delivery system , Waters 490E programmable multiwavelength detector with either a Honeytvell chart recorder or Shimadzu C-RIA Chromatopac integrator. In both cases manual injection was by a Rheodyne valve rnodel7725i fitted with a 20 pl loop . The HPLC used for the later chiral reverse phase separations and al1 chiral normal phase separations was a Beckman system consisting of a programmable pump model 116 and a variable wavelength detector model 166. The HPLC system is controlled via the System Gold software@ package. Injections were made manually using a Rheodyne manual injector mode1 7725i fitted with a 20p1 loop. Chromatograms were printed on a Canon inkjet printer . Chiral reverse phase separations used a variety of columns and mobile

phases. The C-8 columns used include an Altex Ultrasphere 5 ym [150 x 4.6

mm], Brownfee 5 ~IIIcartridge [IO0 x 4.6 mm ] and a Zorbax 5 pm [IO0 x 4.6 mm]. A variety of mobile phases and flow rates were employed. However, in al1 cases the mobile phase was filtered using a 45 pm nyIon filter and degassed by sonicacing for 20 minutes. Distilled water and KPLC grade solvents were used to prepare al1 mobile phases. Achiral Se~aration- Reverse Phase Chromatoaravhic Conditions The achiral separation was done using a Waters pBondapak CN column [150 x 3.9 mm ] with a mobile phase of 82/15, 0.1M ammonium phosphate dibasic (pH = 3.1, adjusted with phosphoric acid, 85%)/acetonitrile at a flow rate of 1.0 ml/min. The detector, a Waters 490E was set at a wavelength of 254 mn and a range of 0.1 AUFS. The Shimadzu C-RIA Chromatopac ùitegrator was nin at a chart speed of 10 mrn/min. Racemic amphetamine hydrochloride, me thamphetamine hydrochoride, desmethyl seiegiline and Selegiline hydrochloride were prepared at a concentration of 0.1 &ml in 15% acetonitrile in \va ter. Chiral Separation -Normal Phase Chrornatoaraphic Conditions Normal phase separations were achieved using a Brownlee Spheri-5 Silica cartridge [220 x 4.6 mm ] with a mobile phase of 95:5, hexane : diethylether at a flow rate of 1.5 ml/min. The mobile phase was filtered through a 45 pm nylon filter and degassed by sonicating for 20 minutes. The detector was set to 254 nm at a range of 0.01 AUFS. Preparation of IR1 and rS1 Methamuhetamine Calibration Curves/ Separation bv Normal Phase Chromatoera~hv Specific amounts ranging from 2.5 to 5.5 mg of an individual enantiomer was weighed into a Silli-vap vial. To each via1 500 pl of (-) menthyl chlorofomate solution (20% v/ v in dichloroethane), 500~1of u , a -dimethylbenzylamine solution (0.5% v/v in dichloroethane) and 100 mg of potassium carbonate was added. The vials were sealed with the appropriate septa and heated for one hour at 100 0 C. After coolinq the liquid contents of the via1 was poured into a 13 ml centrifuge tube and 3 ml of a saturated solution of methanolic potassium hydroxide was added. The mixture was vortexed for two minutes and 5 ml of distilled water added. The tubes were centrifuged for ten minutes and the top aqueous layer removed by pasteur pipet. The remaining dichloroethane layer was poured into a clean dry Silli-vap vial containing molecular sieves (type 4A) to remove any residual water. Each derivatized standard (100 pl) was diluted to 1.0 ml with hexane. Solutions were injected until three injections (20 pl) yielded area ratios within 2% of each other. Chromatographie conditions as listed above for chiral normal phase separations were employed. Graphs and statistical data are presented at the end of this section. Pre~arationof Initial IR1 and [SI Seledine Calibration Curves - Normal Phase Çpecific amounts ranging from 2.5 to 5.5 mg of an individual enantiomer was weighed into a Silli-vap vial. To each vial 1 ml of chloroform was added, the mixture vortexed and the chloroform layer was then evaporated to drvness under a stream of air. Potassium carbonate (100 mg), 500 pl (-) menthyl chloroformate solution (20% v/v in dichloroethane) and 500 pl a,a- dimethylbenzylamine solution (0.250/0 v/v in dichloroethane) was added to each vial. The vials were seaied and heated for one hour at 1000 C. After cooling, 3 ml of methanolic potassium hydroxide solution was added and the contents vortexed. Distilled water (5 ml) was added and the mixture centrifuged for ten minutes. The top aqueous layer was removed by pasteur pipet and the remaining dichloroethane layer was placed into a clean Silli-vap vial containing molecular sieves. Solutions for injection were prepared by adding 100 pl of derivatized standard to 200 pl of hexane. Each standard was injected (20 pl) until three injections yielded area ratios within 2 % of each other. Chroriiatographic conditions as listed above for chiral normal phase separations were employed. Graphs and statistical data are presented at the end of this section. Prevaration of TRI Seledine Calibration Cunre #3 %s calibration curve was prepared exactly as the above curves except instead of weighing specific amounts of [RI Selegiline into each vial an aliquot of stock solution of [RI Selegiline is added into each vial. A stock solution of [RI Selegiline at a concentration of 9.92 mg/ml in chloroform was prepared. Using an Eppendorf pipet 230 pl to 500 pl of stock solution was added to separate Silli- vap vials and the chloroform layer evaporated to dryness under a current of air. The remainder of the derivatization reaction was exactly as described for the initial [RI and [SI calibration curves. The graph and statistical data are presented at the end of this section.

Extraction Procedure of Seleeiline Tablets An individual tablet was weighed, crushed using a mortar and pestle and an aliquot ( approximately 100 mg) added to a centrifuge tube. One ml of diloroform was added to the tube and the tube shaken and then centrifuged for five minutes. The chloroform layer was poured into a silli-vap vial and evaporated to dryness under a Stream of air. To the original centrifuge tube a further 1 ml of chloroform was added and the tube was centrifuged for five minutes. The chloroform layer was added to the original silli-vap via1 and evaporated to dryness. To each vial, 500 p1 of (-) menthyl chloroformate solution

(20 % v/v in dichloroethane), 500 pl of a, a-dimethylbenzylamine solution (0.25% v/v in didiloroethane) and 100 mg of potassium carbonate was added. The vials were sealed and heated for one hour at 1000 C. After cooling, 3 ml of methanolic potassium hydroxide solution was added and the contents vortexed. Distilled water (5 ml) was added and the mixture centrifuged for ten minutes. The top aqueous layer was removed by pasteur pipet and the remaining dichloroethane layer was placed into a clean Silli-vap vial containing molecular sieves. Solutions for injection were prepared by adding 100 p1 of derivatized standard to 200 pl of hexane. Each standard was injected (20 pl) until three injections yielded area ratios within 2 '10 of each other. Chrornatographic conditions as listed above for chiral normal phase separations were employed.

Ethvi Chloroformate Derivatization Reactions To individual Silli-vap vials 5.0 mg of racemic methamphetamine (free base), 5.0 mg desmethyl selegiline (free base) and 5.0 mg of racemic selegiline (free base) was added. To each vial 100 mg potassium carbonate and 500 pl ethyl chloroformate solution (10% v/v in dichloroethane) was added. The vials were sealed and heated for one hour at 1000 C. After cooling, 3 ml of methanolic potassium hydroxide solution was added and the contents vortexed. Distilled water (5 ml) was added and the mixture centrifuged for ten minutes. The top aqueous layer was removed by pasteur pipet and the remaining dichloroethane layer was placed into a clean silli-vap vial containhg molecular sieve. The derivatized methamphetamine and desmethyl selegiline derivatives were diluted (100 pl) to 1.0 ml with hexane, whereas the derivatized Selegiline was diluted (100 pl) to 0.5 ml with hexane. In al1 cases 20 pl was injected and diromatographic conditions as listed above for chiral normal phase separations were employed. FMOC Derivatization Reactions Derivatization reactions in dichloroethane were perfonned by adding 5.0 mg of racemic methamphetamine (Free base), 5.0 mg desmethyl selegiline (free base) and 5.0 mg racemic Selegiline (free base) into individual silli-vap vials. To each vial 100 mg of potassium carbonate and 500 p1 of FMOC solution (4 mM in dichloroethane) was added. The vials were heated for one hour at 1000 C and allowed to cool. The liquid contents of each vial was decanted into individual centrifuge tubes to which 3 ml methanolic potassium hydroxide was added. The tubes were vortexed for two minutes and 5 ml of distilled water was added. The tubes were centrifuged for ten minutes and the top aqueous layer removed by pasteur pipet. The dichloroethane layer was poured into a clean dry silli-vap via1 containing molecular sieves (type 4A). Samples (100 pl) were diluted to 1.0 ml with hexane and 20 pl manual injections were made. Chrornatographic conditions as iisted above for chiral normal phase separations were employed. IR1 Metham~hetamineCalibration Curve

Standard # Concentration R-bWMC* -DMBA** Ratio Mean & pgs înjected Peak Area Peak Area Standard Dev 1

*R-ICILAMC - R-Methamphetamine denvatized with (-) rnenthyl chloroformate "DMBA- Dimethylbenzylamine internai standard derivatized with (-) rnenthyl chloroforma te R - Methamphetamine Calibration Curve

6 9 ugs in 20ul injection [SI- Metham~hetamine- Calibration Curve Data

1 Standard # Concentration S-WIC* DMBA" Ratio Mean & ugs injecteci Peak Area Peak Area Standard Dev

ta tistical Da ta r2 = 0.98924 m = 0.05925 b = 0.15413

* S-MAhfC-SMethamphetamine derivatized with (-) menthyl chioroformate

** DMBA-Dimethÿlbenzylamine intemal standard derivatized with (-) menthyl chloroformate S - Methamphetamine Calibration Curve

6 9 ugs in 20ul injection [SI Seleeiline Calibration Curve Data

Standard # Concentration S-Selegihe MC* DPVIBA** Ratio Mean &

ugs injecteci Peak Area Peak Area Standard Dev a

Statistical Data rz = 0.99464 m= 0.02070 b= - 0.07630

'SSelegiluie MC-ESelegdme derivatized with (-) menthyl chloroformate (selegiline weighted out)

**DMBA-Dirnethy lbenzylamine intemal standard derivatized rvith (-) menthyl chioroformate S Selegiline Calibration Curve

-O 4 8 12 16 20 24 28 32 36 40 uns in 20ul injection IR1 Selegiline Calibration Curve #1

- Standard # Concenhation R-Seleghe MC* 1 DMBA" ~a tioTG~& ugs injecteci Peak Area Peak Area Standard Dev 1

ta tistical Da ta r2 = 0.99343 m = 0.02329 b = - 0.15079

* R -Sele@ne MC- R-Selegiline derivatized with (-) rnenthyl chloroformate (selegilrne solid used)

** DMBA-Dimethylbenzylamine intemal standard derivatize with (-) menthyl chloroformate R Selegiline Calibration Curve #1

O 4 8 12 16 20 24 28 32 36 40 uas in 20 ul iniection IR1 Selegiline Calibration Curve #3

Concentration R-SelegLLine MC* DMBA" 1 Ratio ugs injected Peak Area Peak Area Standard Dev

Statistical Data rz = 0.99952 m = 0.03042 b = - 0.20560

*R-ÇelegJltie MC-R-Selegiluie derivatized with (-) menthyL chloroformate (selgiline in diloroform stock solution)

**DMBA-DLmethylbenzy Lamhe interna1 standard derivatized with (-) menthyl chloroformate R - Selegiline Calibration Curve #3

ugs in 20ul iniection Results of Tablet Assays Two separate lots of Selegiline hydrochloride tablets were assayed using

(-) menthyl chloroformate as the derivatizing agent and separated 5y the normal phase chromatographie conditions as described previously. Result of the analysis of ten Chinoin tablets (lot 06000289) show a mean label claim of 99.776 with a standard deviation of 4.4. Tnese results demonstrate that the method developed is accurate and applicable to the analysis of Çelegiline tablets. Analysis of another lot of tablets produced by Chiesi (lot 118) have a mu& lower mean label claim of 90.5% with a standard deviation of 12.5. men obtained these tablets had already reached their expiry date and therefore lower resulh are not totally unexpected. Additional analysis of these tablets did not show an improvement in the data, rather the results were lower with a mean label daim of 71.0 '/O and a standard deviation of 6.0 (n = 4). Calibration curves using a , a -dimethylbenzyIamine as the interna1 standard were used to calculate al1 tablet data. Chinoin Tablet Data

Lot # 0600289

PA - Peak Area

Sta tistical Data Mean Label daim = 99.7% Standard deviation + 4.4 Chiesi lumex Tablets First Batch Lot #Il8

- - 103.5 1 Tab 2 150.0 1.92702 4.62951 )0.41625 )0.41471 24.28 23.00 105.6 1 1 1.W78 4.56337 0.41317 [ 1 F

I 100.6 Tab 1A 146.9 154397 5.05733 0.30529 0.30161 19.42 23.55 82.5 1 1-56861 0.29799 ?. ?. 5.26389 1 r 1 102.0 Tab 3 149.0 1.47894 334767 03437 0.38203 33.87 2-82 100.2 1 1.53053 4.00476 0.37968 I I 101.4 Tab 1A 143.0 1.38092 4.69831 ' 0.29392 0.296971 19.22 Z. 18-42 1 1.34201 4.47311 0.30002

100.2 Tab 4 149.8 1.47-185 4.133% 10.35677 0.35639 21.79 2230 97.6 1.43625 4.03435 0.35601 1 1 102.9 Tab 1C 149.7 2.28086 7.87387 0.28967 0.28960 18.90 22.91 82.6 ' 1 2.23991 7.73639 0.28953

104.2 Tab 2B 148.8 1.17677 6.2035 0.2S970 0.140821 14.70 23.34 62.5 1 1.21501 6.33015 0.19194

101.0 Tab 5 152.5 1.96728 5.90527 0.33315 0.33644 20.92 22.08 94.7 1 1 2.01206 5.92277 10.33972 -- --! -

PA - Peak Area

StatisticaI Data Mean label daim = 90.5% Standard deviation + 12.5 Chiesi ïumex Tablets Second Batch Lot #il8

J Aliquot in1 Tab Tablet / Sel DMBN Ratio Ave ugs ( ug's '10 label Std via1 (mg) 1 # wt (mg) ( (PA) (PA) (PA) (PA) Assayed Expected daim # ' i ! 109.0 16 151.0 1.40533 5.46350 0.25622 0.3749 15.22 24.02 63.2 3 1.45126 5.63052 0.25775 1 103.2 7 151.4 12.15474 7.03992 0.30607 0.30312 16.70 22-72 73.3 3 1 2.14397 7.14283 0.30016 I 102.4 8 151.9 12.76408 8.49438 0.325-M 0.32378 17.40 22-47 77.4 3 2.75529 8.55248 0.32216 ii 202.6 9 149.9 1.60356 6.11123 0.26240 0.27983 15.96 22.S2 69.9 3 / 1.57083 5.2û457 0.2975

PA - Peak Area

Statistical Data Mean Iabel daim = 71.0 Standard deviation + 6.0 Discussion While there are several derivatizing reagents available for the chiral separation of primas. and secondary amines, there are few that are suitable for tertiary amines- (-) Menthyl chloroformate has been demonstrated to be a suitable derivatizing agent for the chiral separation of (5) Selegiline hydrochloride. Ushg the favorable solubility of Selegiline in chloroform a successful method (99.7 % label clairn) has been developed for a chiral HPLC assay of Selegiline hydrochloride tablets.

As previously mentioned the reaction of Selegiline with (-) rnenthyl chloroformate could result in the formation of three possible carbarnates due to the deavage of either the propargyl group, the methyl group or a combination of both. Studies clearly show that the reaction of (-) menthyl chloroformate with Selegiline produce the same carbamate as the reaction of methamphetamine with

(-) menthyl chloroformate. Therefore, the reaction of Selegiline with (-) menthyl chloroformate results in the dealkylation of the propargyl group. These results are consistent with results previously reported by Kapnang et al where the preferred cleavage order for a series of related tertiary amines is N-benzvl > N- allyl> N-ethyl > N-methyl ' (Kapnang, H. and Charles, G., 1983) Although the main objective of this thesis was to develop a chiral assay for Selegiline hydrochloride and Selegiline hydrochloride tablets, the chiral derivatization and assay procedure developed using (-) rnenthyl chloroformate can ako be used for the separation of racemic amphetamine, racemic methamphetamine and racemic desmethyl selegiline. Another benefit of the reaction of (-) menthyl diloroforrnate with Selegiline hydrochloride is that it consistently affords only one product. h al1 the reactions performed, the

' This cleavage order is reported for both ethyl and vinyl diloroformates

118 propargyl group was consistently cleaved. Finally, the separation is performed on a silica colum which is more rugged and less expensive than a chiral colunin* The main disadvantage of the derivatization procedure developed is the lengthy reaction time ( 60 minutes) needed for the reaction of (-) menthyl chloroformate with Selegiline hydrochloride. Although amphetamine, methamphetamine and desmethyl selegiline are formed using shorter reaction times, Seleglute needs both the extended time and high temperature. The retention time of the intemal standard a,cc-dimethylbenzylamine at approximately 25 minutes results in a chromatographie run time of thirty minutes. Studies could be done to identify another suitable amine as an interna1 standard that would elute prior to the Selegiline carbamate to reduce the run tirne of the chromatogram. The low yields of the reaction are also a concern. As previously mentioned the yields of the reaction of (-) menthyl chloroformate with Seleghe are substantially lower than the reaction of (-) menthyl diloroformate with either methamphetamine or desmethyl selegrluie. Trying to increase the yields by extending the reaction time from one hour to two and three hours resulted in an increase in yield of Selegrline carbamate. However it was also noticed that the dichloroethane had evaporated. Therefore, the increased yields could have been due to a more concentrated solution. The yields may be increased if the reaction could be camed out in a sample via1 whi& could withstand the heat and not allow for evaporation . Another way to increase the yield would be to use a more concentrated solution of (-) menthyl chloroformate. Perhaps a greater excess of (-) menthyl diloroformate reagent would increase the reaction rate and the overall yield. In addition to the development of a chiral HPLC assay of Selegiline hydrochloride, the reactions of methamphetamine, desmethyl selegiline and Selegiline with two additional achiral chloroformates, ethyl and 9- fluorenylmethyl chloroformate (FMOC) have also been evaluated. Studies have shown that the reaction of Selegiline with both ethyl chloroformate and FMOC resuit in formation of both desmethyl selegiline and methamphetamine carbamates. The reactions of the chloroformates studied with methamphetamine, desmethyl selegiline and Selegiline appear to be affected by the size of the groups attached to the nitrogen, the temperature of the reaction and the polarity of the derivatizing solvent. Reactions of al1 three chloroformates with methamphetamine showed much higher yields than those obtained with similar reactions with Selegiline. These results are consistent with those of Kometani et al (Kometani, T., Shunsaku, S. and Mitsuhashi, K., 1976) who reported that the reactivity of an amine to ethyl chloroformate decreased with an increase in the size of the group around the nitrogen atom. Even in the reaction of FMOC with Selegiline, the peak corresponding to the methamphetamine carbamate is larger than the peak corresponding to the desmethyl selegiline carbamate. Studies of al1 three chloroformates with methamphetamine, desmethyl selegiline and Selegiline showed much higher yields when the reactions were carried out in dichloroethane as compared to those employing benzene as the derivatizing solvent. Although the exact mechanism of the reaction is not fully understood, the reaction is believed to proceed by way of a quaternary intemediate followed by nucleophilic attack by chloride on one of the nitrogen substituents (Cooley, J. H. and Evain, E. J., 1989). The increased yields seen in dichloroethane may be due to the increased stability of the quaternary ammonium intermediate in a more polar solvent. Studies in benzene showed that the reaction of (-) menthyl chloroformate with amphetamine and methamphetamine can form the resulting carbamates at 600 C, whereas a temperature of 1000 C is needed to form Selegiline menthyl carbamate. Similarly, when reacting Selegiline with FMOC in benzene only a very small single product (correspondhg to the methamphetamine carbamate) was formed after two hours of heating, while the yields of the resulting carbarnates of methamphetamine ,amphetamine and desmethyl selegiline were higher after only one hou at 1000 C. Once again the reactivity may be related to the size of the substituents on the nitrogen atom as suggested by Kometani et al which would require harsher conditions for more complicated amines, such as Selegiline (Kometani, T., Shunsaku,S. and Mitsuhashi, K., 1976) . As expected due to the highly fluorescent nature of the compound, the reaction of FMOC with methamphetamine, desmethyl selegiline and Selegiline resulted in a more sensitive assay than the reactions with either ethyl chloroformate or (-) menthyl chloroformate. Although the results of the above experiments do provide further information pertaining to the reactions of chloroformates with primary, secondary and tertiary amines, it would be interesting to investigate the reactions of the above mentioned chiorofornates with tertiary amines bearing different substituents on the nitrogen. As previously mentioned Kapnang et al report a preferred cleavage order of N-benzyl > N-allyl > N-ethyl > N-methyl using vinyl and ethyl chloroformate. However the order rnay be different when derivatizing with either (-) menthyl chloroformate or FMOC. Ln addition the reaction conditions should be investigated, specifically the reactions of ethyl chloroformate and FMOC with Selegiline. The reaction after one hour in dichloroethane at 1000 C resulted in the formation of two carbarnates. However if the reaction was heated for a shorter period, at a lower temperature or at a reduced temperature for a shorter period of time, perhaps only one product would be formed. If one product could be formed then the reaction of Selegiline with the chiral (+) -1-(9-fluorenyl) ethyl chloroformate (FLEC) should be evaluated. Although 1chose to use a chiral derivatking agent followed by separation on a convention adùral column the discussions presented throughout this thesis demonstrate there are many alternative methods that could employed. Specifically the use of 2-naphthyl chloroformate followed by separation on either a cellulose or cyclodextrin chiral column may also result in a chiral separation. The introduction of the 2-naphthyl group would allow for increased sensitivity as well as introduce the needed naphthyl group for separation on either of these chiral colurnns. 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Robinson, J. B., Stereoselectivity and isoenzyme selectivity of monoamine oxidase inhibitors Biochern. Phamzacol., 34 (1985) 4105-4108. Salonen, J. S., Determination of the amine metabolites of Selegiline in biological fluids by capillary gas diromatographv J. Chromatogr.-,527(1990) 163-168.

Simon, K., Podanyi, B., Ecsery, 2. and Torok, Z., Absolute configuration and conformational analpis of (-)-(R)-Deprenyl and its homologues 1. Chem. Soc. Perkin Trtrns. 11, (1986) 111-1 15.

Simon, K., Bocskei, Z. and Torok, Z., X-ray diffraction study of Selegiline and related compounds Acta. Pham. Hung., 62 (1992) 225-230.

Simons, S. S. and Johnson, D.F., The structure of the fluorescent adduct formed in the reaction of O-phthalaldehydeand thiols with amines 1. Am. Chem. Soc., 98 (1976) 7098-7099.

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Tipton, K. F., Fowler, C.J. and Houslay, M. D., Monoamine oxidase: Basic and clinicalfrontiers, Excerpta Medica, Amsterdam, 1982, pp. 87.

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Wainer, 1. W., Drug Stereochem ist y, Analyt ical Methods and Pharmacology, Second Edn, Marcel Dekker, New York, 1993, pp. 65-106,139-182.

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Witte, D. K., de Zeeuw, R-A. and Drenth, B. F. H., Chiral derivatization of Promethazine with (-)menthylchloroformate for enantiomeric separation by RP- HPLC 1. High Resolut. Chrornntogr.: Chromatogr. Commun., 13 (1990) 569-571. Pre~arationof a- Phenylacetoacetonitrile -

hto a 2 liter round bottomed flask fitted with a separatory funne1 and reflux condenser, each protected with a CaC12 drying tube, was placed absolute ethanol (700 ml) and clean sodium metal (70g, 3 moles) added in srnail pieces at such a rate as to conhol the refuing of the solution. To the warm solution of sodium ethoxide was then added slowly with stirring a solution of benzyl cyanide (234g ;2 moles) in dry ethyl acetate (264g : 293 mis ;3 moles). The mixture was refluxed on a steam bath for two hours and then allowed to stand at room temperature ovemight. The mixture was diluted with water (2 L) and stirred to dissolve the solid. Crushed ice (IL) was added, the mixture extracted with ether (3 x 500 ml) and the ether extracts discarded. The separated aqueous phase was freed of any residual ether by bubbling air through the solution during 30 minutes and the solution was then acidified with a solution of glacial acetic acid (150m.l) in water (400ml). The precipitate was filtered, washed with water and the crude product (a- phenylacetoacetonitrile) used without hrther drying or purification for the preparation of benzyl methyl ketone. Pre~arationof Benzyl Meth 1 Ketone cJulian, 1938 #59>

Concentrated sulfuric acid (350 ml) was added to a 3 liter round bottorned flask and cooled to -100 C. The cmde, moist a- phenylacetoacetonitrile was added slowly to the cooled sulfuric acid with shaking and maintaining the reaction mixture temperature below 200 C.

When all the a-phenylacetoacetonitrile was added, the flask was warmed on a steam bath until the solution was complete and the then the flask was cooled to 00 C. Water (1750 ml) was added rapidly and the flask heated on a steam bath for 48 hours during which time the ketone layer separated (ketone layer contains much unreac ted a-phenylacetoacetonitrile , which is difficult to decarboxylate without adequate stirdtg of the reaction mixture). The ketone layer was separated and the acidic aqueous phase extracted with ether (500 ml). The combined oil and ether extract was dried over sodium sulfate, filtered and the solvent evaporated. [The residue tends to deposit crystals of a-phenylacetoacetonitrile which were filtered out and recycled through the decarboxylation reaction]. The residue was distilled the fraction b.p. 100-105° C at 12 mm being collected. The product was redistilled at atmospheric pressure, the material b.p. 214-2160 C being collected (literature b.p. 2160 C). KOH

hto a round bottomed flask was placed methylamine hydrochloride

(12.58 g , 0.1863 moles), potassium hydroxide (6.7 g, 0.1 194 moles) and benzylrnethyl ketone (5 g, 0.0373 moles) dissolved in methanol (50ml). The mixture waç stirred for one hour at room temperature. Sodium cyanoborohydride (1.5 g, 0.0227 moles) was added and the mixture stirred for twenty-four hours. The reaction mixture was evaporated under reduced pressure , water (30 ml) added, the solution acidified with hydrodoric acid, extracted with diloroform (3 x 30ml) and the chloroform extracts discarded. The aqueous solution was made alkaline with ammonium hydroxide and extracted with chloroform (3 x 30ml). The chloroform extracts were collected, dried (Molecular Sieve; Type 4A) filtered and evaporated under reduced pressure. The residue was distilled using a bulb distillation apparatus, the fraction b.p. 1000 C at 10 mm being collected. i.r. (liquid film) 3320 cm-' (NH);1600 un-' (aromatic); 1345 cm-', 1370 cm-' (C-N); 700 cm-1 ,750 cm-' (mono substituted benzene) (f)Methamphetamine hydrochloride (recrystallized from acetone) m.p. 134.5 C [Lit m.p. 135-137 Cl In a round bottom flask, methanol (25 ml), propargylamine hydrochloride (5.5 g , 0.06 moles), potassium hydroxide (2.5 g , 0.0445 moles) and benzyl methyl ketone (2 g, 0.015 moles) were added and auowed to stir at room temperature for one hour. Sodium cyanoborohydride ( 0.6 g, 0.0095 moles) was added and the mixture stirred for twenty-four hours. The mixture was evaporated under reduced pressure and water (30 ml) added. The solution was acidified with hydrochloric acid and extracted with ether ( 3 x 30 ml). The extracts were discarded and the aqueous solution made alkaline with ammonium hydroxide and extracted with chloroform (3 x 30 ml). The chloroform extracts were collected and dried (Moleculas Sieve; Type 4A), filtered and evaporated under reduced pressure. The residue was fractionally distilled, the fraction b.p. 48-500 C and 0.1 mm being collected. Yield 1.3 g i.r. (liquid film) 3300 cm- (c eC-H), 1600 an-'(aromatic); 700 cm-1 ,753 cm-1 (mono substituted benzene) To a solution of N-methylpropargylamine hydrochioride (4.8 g, 0.045 moles) in anhydrous methanol (12 ml) was added potassium hydroxide (1.683 g. 0.03 moles). After dissolving the potassium hydroxide, benzyl rnethyl ketone (3 g, 0.022 moles) was added and the mixture stirred for one hour at room temperature. Sodium cyanoborohydride (0.44 g, 0.007moles) was added and the mixture stirred for twenty-four hours. The solution was evaporated to dryness under reduced pressure. Water (60 mls)was added and the solution acidified with hydrochloric acid. The mixture was extracied with ether (3 x 30 ml), and the extracts discarded. The aqueous solution was made alkaline with ammonium hydroxide and extracted with chloroform (3 x 30 ml). The extracts were dried (Molecular Sieve,Type M) filtered and evaporated. The residue was fractionally distilled (bulb distillation), the fraction b.p. 58-600 C at O.lmm being collected. Yield 0.6 g i.r. (liquid film) 3300 cm- l (C C- H), 2100 cm- 1 (C C), 1600 cm- 1,1595 cm- 1 (aromatic); 705 cm-' ,745 cm-' (mono substituted benzene) Preoaration of the Menthyl carbarnate of (fl Metharn~hetarnine [Diastereorneric Methodl

A CH, CH3

Into a round bottomed flask was placed potassium carbonate (1.5 g), (k) methamphetamine (1 g, 0.0067 moles) in benzene (10 ml) and (-) menthyl chloroformate (3 ml: 3.06g: 0.01399 moles). The flask was stoppered and the reaction mixture stirred at room temperature for 4 days. Methanolic potassium hydroxide (25 ml, saturated solution) was added, the reaction mixture stirred ( 2 hours) and water (30 ml) added. The benzene layer was separated, washed with water and evaporated under reduced pressure. The residual liquid was placed in a sealed container. The infra-red spectum (liquid film) showed absorption peaks indicative of a carbamate, 1750 cm-' (C=O), 1260 cm-' (C-O). Pre~arationof the Menthyl carbamate of (f) Desmethvl Seleeiline

Into a round bottomed fiask was placed potassium carbonate (1.5 g), (+) methamphetamine (1 g, 0.00578 moles) in benzene (10 ml) and (-) menthyl chloroformate (2.5 ml: 2.55 g: 0.01166 moles). The flask was stoppered and the reaction mixture stirred at room temperature for 4 days. Methanolic potassium hydroxide (25 ml, saturated solution) was added, the reaction mixture stirred ( 2 hours) and water (30 ml) added. The benzene layer was separated, washed with water and evaporated under reduced pressure. The residual liquid was placed in a sealed container. The mfra-red spectum (liquid film) showed absorption peaks indicative of a carbamate and an acetylenic group. i.r. (liquid film), 3300 cm-' (CmCH), 1745 un-' (GO), and 1260 cm-1 ( C-O). Pre~arationof N-Acetvl-2-Phenvlisooro~vlamin~ (slight modification of the method of Ritter et al

(0.2 mole; 23.64g ; 26ml) and acetonitrile (0.3 mole;

12.2g ;15.61ml) were mixed and added to a solution of p-toluene sulphonic acid monohydrate (0.2 mole; 38g) dissolved in glacial acetic acid (100ml). The mixture was allowed to stand at room temperature with stirring for 72 hours and then poured onto cmshed ice (400g). The mixture was made alkaline with ammonia and extracted with ether (3 x 100ml), the extracts dried over potassium carbonate, filtered and the solvent evaporated. The residue was fractionally distilled and the following fraction collected.

i) b.p. 113 -118 at 0.4 mm 4.88g ii) b-p. 125 -131 at 0.4 mm 2.82g iii) b-p. 131 -140 at 0.4 mm Only fractions i) and ii) showed infrared spectra characteristic of amides. Preparation of 2-Amino-2-ohenvl~ro~ane( a-a-Dimethvlbenzvlamine) N-Acetyl-2-phenylisopropy1amine(7.6g) was dissolved in 95% ethanol (IOml) and added to a solution to alcoholic KOH (50rnl; 20%) and the solution reflwed during 96 hours. Water (2001111) was added and the solution steam distiiled, the distillate being collected in aqueous hydrochloric acid (volume of distillate approxima tely 3OOml). The distillate was evapora ted to dryness under reduced pressure, the residue dissolved in the minimum amount of water, made alkaline with ammonia and extracted with ether

(3 x 50ml). The extracts were dried (molecular sieve), filtered and the solvent evaporated. The residue was distilled (Kugelrohr), the material b.p. 750 C at 8mrn being collected. Yield 85 mgm. Pre~arationof N-Formyl-2-~henvliso~ropvlamine (Modification of Ritter method Into a 500ml three necked round bottomed flask fitted with a stirrer, thermometer, dropping funnel and reflux condenser comected to a trap containing 20% sodium hydroxide solution was added glacial acetic acid (125 mls). The material was cooled in an ice bath to 20 C and potassium cyanide (0.5 mole; 32.6g) added slowly while maintainhg the reaction mixture below 200 C. From the separatory funnel was added a previously cooled mixture of concentrated sulhric acid (68ml ; 1.25 mole) in glacial acetic acid (62.5m1). The addition was made slowly with stirring to keep the mixture below 200 C. The ice bath was removed and a-methylstyrene (0.5 mole; 59g; 65ml) added during 20 minutes while the temperature slowly rose to approximately 450 C. The mixture, which was quite viscous, was stirred at room temperature overnight. Air was bubbled through the mixture until free of hydrogen cyanide and the reaction mixture then poured into ice-water (750 g), the mixture neutralized with sodium carbonate and extracted with ether (3 x

100ml). The extracts were dried over sodium sulfate, filtered, the solvent evaporated and the residue distiiled. The material had a b.p. of 100-1100 C at 0.25mm and was a colorless viscous liquid. The infra-red spectrum showed absorption peaks indicative of an amide and was almost completely superimposable upon a spectrum of N- acetyl-2-phenylisopropylamine. Yield 31g Pre~arationof a-a-Dimethvlbenzvlamine N-Fomyl-2-phenylisopropylarnine (30g) was dissolved in absolute ethanol (150ml), potassium hydroxide pellets (30g) added and the solution refluxed during 40 hours. The mixture was diluted with water (400ml) and steam distilled until approximately 750ml of distillate had been collected. The acidified distillate was evaporated under reduced pressure and the solid residue dissolved in water (30 ml), the solution made alkaline with ammonia

and extracted with ether (3 x 50 ml). The extracts were dried (molecular sieve), filtered and evaporated to leave an oily residue which was distilled, the fraction b.p. 67-790 C at 2.5mrn being collected. Further distillations of the material showed b-p. 77-800 C at 8.5 mm. Yield 6.lg (57%based on formyl derivative consumed. [Lit b.p. 196-1970 C at 980 C at llmrn; 1000 C at 22mm, Dictionary of Organic Compounds, Heilbron and Brunbury] [N.B. Unchanged amide (17g) was recovered by ether extraction of the steam distillation residues] IMAW LVALUATION TEST TARGET (QA-3)

kg 12 II- = 1111pL 12 - L. ,,A IlIll&

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