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 1*1 of Canada Bibliothèque nationale du Canada

Acquisitions and Acquisitions et 8ibliographic Services services bibliographiques

395 Wellington Street 395. rue Wellington Ottawa ON K1A O N 4 ûttawaON KIAOiU4 Canada -da

Your itk votre reterenm

Our iUe Notre reterence

The author has granted a non- L'auteur a accordé une Licence non exclusive licence ailowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sen reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/^, de

reproduction sur papier ou sur format électronique.

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.

T h e 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

S tereochemical Techniques ............................................................................... ....... 10 Chromatograp hy ............................................................................................................ 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 Characteris tics 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

S tudies 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 C u ~ 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 G r a ~ h s

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

[S 1 Methamp hetamine Calibration Graph ............... .... ..............=...................... 107 .......................................... [SI Selegiline Calibration Curve Data ................... ... 108

. [SI Selegiline Calibration Curve Graph ................... ........................................... 109

.................... [RI Selegiline Calibration C u ~ 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 ................................................... -1 15

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

Chiesi Jumex Tablets O Second Batch O Lot# 118 .................................................... 117

List of Figures

Figure 1 O 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-dihydroxy phenyl) 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-dinucleo tide

(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(derived from

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

but differ in the spatial arrangement of the atoms.

the same structural formula

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

dias tereomers I

-

enantiomers

diastereomers

<

enan tio mers

I dias tereomers

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 m u t 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 2 R-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-workers in 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 (mobiIe phase: liquid)

Liquid-bondcd phase

Liquid-solid, or adsorp- tion

Ion exchange Sizc exclusion

Gas chromatography (GC) Gas-liquid (mobile phase: gas)

Gas-bonded phase

Supercritical-fluid chroma- tography (SFC) (mobile phase: supercritical fluid)

Liquid adsorbcd on a solid

Organic spccics bonded to a solid surfacc

Ioncxchange rcsin Liquid in interstices of a polymeric solid

Liquid adsorbed on a solid

Organic specics bondcd to a solid surface

Organic species bondtd to a solid surface

Partition bctwecn immis- cible liquids

Partition bctween liquid and bunded surface

Adsorption

Ion exchange Partitionlsicving

Partition betwcen gas and liquid

Partition ktwecn liquid and bonded surface

Adsorption

Partition betwttn super- critical fluid and bondcd 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/liquidW refers 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

compounds are generally

(Meyer, V. R., 1994).

by normal phase chromatography. Typically polar

best separated by normal phase chromatography

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 a t 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~orted bv the mobile hase to the detector * L J L

and the response is recorded using either an integrator or strip

signals that are recorded are Gaussian shaped peaks. Each peak

recorder. The

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. ï h i s 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 c m

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 col umn 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- heig ht 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.

h o t h e r 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 A 2 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

quanti ties;

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 c m separate 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. ï h i s 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 ~ e o r y , 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). ï h e 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 ~referred substrate 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.

Type I I -cellulose

Cyclobond 1, 11 ,111

Mixed Mode CSP

WE, Wl1, W M

AGP OVM H S A BSA

TVpe I

I I

1 I

11 1

I + III

IV

v Proteins

Interaction hydrogen bonding, dipnle slacking, iZ - K

Combina tion of h y drogen hmding, dipolc stacking, A - n fitting in cavity part of all the solutc rnust f i t ir the cavlty, a h hydrogen bonding and Ir-lt

hydrogen bond ing dipole stacking Ir- lt and cavi ty inclusion complex with transition metnl (M), chiral moleculc (A A) (L) and enantiomer o f racemic solute hydrogen bonding and hydrophobie interactions bctwcen protein and çolute. Alsri electrostatic

salute a lkyl carbinols, aryl substitutcd liydan toins lactanis, sulfoxides Contain phenyl t;roups, amides, iniides, keloncwj, esters

phcnytoin and mctab»lites, mpod crmtaining rnultiplc pheny 1 groups such as alcohols, amide, ester, ketone aromatic amines, alcohols, multiple phcnyl group aimpounds

a-amino acids mino and dicarboxylic acids containing a-OH

cationic and anioni mmpaunh, ephedrine, a, B- amino acids 1-1SA/BSA-anionic and neutral mmpounds. Necd aromatic and polai moicty

chiral selector (R)-N-(3,5 Dinitro benzoyl) phenyl glycine (S)-N-(3,5 DNBPG)

Microcrystalline cëllulose triacetate

Cellulose triaceta te Cellulose Mbenzoatc B-cyclodex trin y- cyclodextrin a - cyclodex trin Poly (triphenylmet hyl methacrylate) Chiral 18-crown-6-ethcr

Type 1- naphthylethyl carbamate Type 111- cyclodextrin

(WE) N-carboxymethyl(lR, 2s)-diphenylamino ethanol (W 11)- L-Praline (WM) - L-tert-Leucinc

AGP-ai-Acid protein OVM - Ovomucoid HSA-tiuman serurn albumin BSA- Bovine serum albumin

- Derivatizing :onvert alcohols, amines 07 :arboxylic acids to arornatic amide, urea or carliamate

h h o l s to csters, espccially para-nitrobcnzoic esters if Ot- near chiral ccnter, benzyl amides (or benzyl esters

lntroduce phcnyl groups. Chiral aliphatic amines hav w n converted to benazmide derivalives clicarl>«xylic acids have lieen converted to benzyl esters or amides Type 1 -Non aromalic amines and alcohols must be convertec Io dinitrophenyl derivative

derivatizd amino acids including N-acetyl, amide, N carbamoy 1, N-carbobenzoxy

Por HSA/DSA -most amino acids need ptecalurnn derivalizalion wilh N-acety benzenesulphonyl, DANSY L Often used in achiral/chiral systerns

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 s i k a 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 c m take 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~arations us 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 u I acid 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

the two diastereomeric

a chiral mobile phase

separation is due to the differences in the structure of

complexes. Ligand exchange chromatography with

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 h c t i o n 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 h c t i o n 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- (tnflurome thy1)pheny lacetic acid (Mosher's acid)

i C-C-OH

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

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

giucop yranosy l iso thiocyana te

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 ( o d y 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-basic naphthyl 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, C R . , 1985).

Among the chiral isothiocyanates employed as derivatizing agents,

2,3,4,6-tetra-0-acetyi-E-D-glucopyranosl isothiocyanate (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 (+) Amphetam

Figure 13

ine

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-glucopyranosyl isothiocyanate (GITC)

Figure 14

48

from illici t synthesis; s imilarly , the presence of solel y (R)-(-)-methamp he 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

Diphenyip yraline

T e r t i q Amines derivatized with 2-Naphthyl chloroformate

Figure 16

Other chloroformates commercially available a re ( - menthyl

chloroformate and the strongly fluorescent compounds 9-fluorenylmethyl

chloroformate (FMOC) and (+)-1-(9-F1uorenyl)ethyl Chloroformate (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, n o 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 ).

Thus,

formed will

Figure 18

if the propargyl group is cleaved from Selegiline, the product

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

methamp hetamine-

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 h t s 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 111 represents 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^ M e h o I Water

Method Develo~rnent Usine MethanoWater / Diethvl ether

MethanoüH,-O/Diethyl ether

Method DeveIo~ment Using MethanoVWater/TKF

* *Zohau CoIumn ***Flow rate in d m i n

(-) Menthyl carbamate derivative of (f) Methamphetamine RG = 1.12 COI-: ~ l t e x C-8 (150 x 4.6-1, Mobile 70/30 m e t h L i / ~ ~ 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~hetamine and 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 m l h i n , Detector set at 254 nm, 20 pl rnanual injection

Figure 21

(-) Menthyl carbarnate derivatives of (k) Methamphetamine Effect of 2% isopropanol ( P A ) 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

amphetamine were

set, two calibration curves of R-methamphetamine and R-

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 T a i h g

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 1 Jml/ 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 1 Sml/ 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 1 Sml/ 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 9 6 4 (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

a, a- Dimethylp heny lethy lamine

Aniline

[RI Arnp hetamine

[SI - a-Methylbenzylamine

a, a- Dimethy lbenzylamine

[RI - a-Methyl benzylamine

Derivatized Methamrhetamine

[RI Methamphetamine

[SI Me thamp he taamine

Retention T h e '

6.18 minutes

6.22 minutes

24.00 minutes

26.92 minutes

34.78 minutes

39.43 minutes

25.42 rninu tes

26.46 minutes

--

The retention tirnes are based on a mobile phase composition of 96:4 (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 e h , 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 ~Moroformate deriva 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 500~1 of (-) 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 b o t - 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 100~1 of standard solution to 200~1 of 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.

-0.0050 Aboorbancm

O.OQOQ O-OQSO I l ,

0.OIOQ 1 1 I 1 t 1

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

** T h information 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, i t 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 . 6 m ) 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

(+_)Desmethyl selegiline 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

(f) Selegiline Derivatized with Ethyl Chloroformate Derivatization in Dichioroethane, Separation on Silica Column Co lum: 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

Abeorbanca - a m s a a. 3 0 0 ~ a. 0050 o.0100

0.00 l 1 1 I i 1 1 1 1 1 I l l I I I

1 J

4 --5 -. - !:Il 1.5'1

10.00 -

Figure 33

*

1o.00 - ,

10.00 - i - 31.96 t

* f '

39.99 1 I I i i l K 8 1 1 " 1 i 1 1 -0.0050 0.0000 0.0050 0.0100

(+) Methamp hetamine deriva tized 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 1 Sml/ 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.itaso 0.0000 o.oa!o O.QldO

L 1 1 I I I 1 1 1 1

1 I fi t I I I 1

1 . 1 1 1 1 I I I I 1 ' I I I I

1

-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 . h l / 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

E x ~ e r h e n ta1

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 ~III cartridge [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 m n

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 thamphe tamine 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 p m 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), 5 0 0 ~ 1 of 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~aration of 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~hetamine Calibration Curve

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

Standard # Concentration

pgs înjected

R-bWMC* Peak Area

-DMBA** Peak Area

Ratio Mean &

Standard Dev 1

R - Methamphetamine Calibration Curve

6 9

ugs in 20ul injection

[SI- Metham~hetamine - Calibration Curve Data

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

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

1 Standard #

** DMBA-Dimethÿlbenzylamine intemal standard derivatized with (-) menthyl chloroformate

Ratio Mean &

Standard Dev Concentration ugs injecteci

S - W I C * Peak Area

DMBA" Peak Area

S - Methamphetamine Calibration Curve

6 9

ugs in 20ul injection

[SI Seleeiline Calibration Curve Data

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

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

**DMBA-Dirneth y lbenzylamine intemal standard derivatized rvith (-) menthyl chioroformate

Ratio DPVIBA** Peak Area

Standard # Mean &

Standard Dev a

Concentration ugs injecteci

S-Selegihe MC* Peak Area

S Selegiline Calibration Curve

-O 4 8 12 16 20 24 28 32 36 40

uns in 20ul injection

IR1 Selegiline Calibration Curve #1

ta tis tical Da ta r2 = 0.99343 m = 0.02329 b = - 0.15079

Standard #

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

** DMBA-Dimethylbenzylamine intemal standard derivatize with (-) menthyl chloroformate

Concenha tion ugs injecteci

R-Seleghe MC* 1 DMBA" ~a t i o Peak Area

-

T G ~ & Standard Dev Peak Area

1

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" ugs injected Peak Area Peak Area

1 Ratio 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. m e n

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

PA - Peak Area

!

StatisticaI Data

Mean label daim = 90.5%

Standard deviation + 12.5

103.5 1 Tab 2 150.0

146.9

F

100.6

?.

Tab 1A

1.92702 1.W78

154397

1

24.28

19.42 1 -56861

- -

4.62951 )0.41625 )0.41471 23.00

23.55

102.0

4.56337

5.05733 5.26389

101.4

0.29799

1

105.6 1 1

0.38203

0.41317 [

334767 Tab 3 100.2 33.87

82.5 0.30529

0 3 4 3 7

Tab 1A

1

I

2 - 8 2

1

I

1 0.30161

4.00476 149.0

0.37968

r 1.47894 1.53053

100.2

I 1

Tab 4

0.296971 19.22 Z . 18-42

149.8

143.0

0.35639 1.47-185 1.43625

1.38092 1.34201

0.28960 102.9

104.2

2.28086 2.23991

1.17677 1.21501

4.69831 ' 0.29392

97.6 21.79 4.133% 10.35677

Tab 1C

Tab 2B

4.47311

1 2230

1 82.6 ' 1

4.03435

7.87387 7.73639

6.2035

149.7

148.8

18.90

101.0

0.30002

0.35601

0.28967 0.28953

0.2S970 6.33015

5.90527

22.91

0.140821 14.70

152.5 Tab 5

0.19194 23.34 62.5

1.96728 2.01206

94.7

-- - 5.92277 10.33972

1

1 1

22.08 20.92 0.33315 0.33644

Chiesi ïumex Tablets Second Batch

Lot #il8

PA - Peak Area

Statistical Data

Mean Iabel daim = 71.0

Standard deviation + 6.0

Aliquot in1 Tab via1 (mg) 1 #

i

109.0 16

103.2 7

Ave (PA)

0.3749

0.30312

Tablet / Sel wt (mg) ( (PA)

!

DMBN Ratio

151.0

102.4

(PA)

17.40

15.96

1.40533 1.45126

0.30016

(PA)

22-47

22.S2

ugs ( ug's

0.30607

77.4

202.6

1

0.325-M 0.32216

J

Assayed

15.22

16.70

3

151.4 12.15474 7.14283

8.49438 8.55248

ii

8 0.32378

'10 label daim Expected

24.02

22-72

5.46350 5.63052

7.03992 1 2.14397 I

151.9 12.76408 2.75529

Std # '

0.25622 0.25775

9 69.9 3

63.2

73.3

0.27983 149.9 6.11123 5.2û457

3

3

1.60356 0.26240 0.2975 / 1.57083

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 c o l u m 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 S e l e g h e 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 h o u 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 1 chose 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. As with the FMOC derivatization, excess reagent

would have to be removed either by extraction or by reaction with a suitable

amine.

The work presented here illustrates the development of a chiral HPLC

separation for Selegihe hydrochloride. Although alternative methods for the

chiral separation of Selegiline hydrochloride may be possible, derivatization with

(-) menthyl chloroformate followed by separation on a conventional silica

column has been shown to be fairly simple and applicable to the analysis of

Çelegiline tablets. The information obtained regarding the reactions of primary,

secondary and tertiary amines with (-) menthyl chloroformate, ethyl

chloroformate and 9-fluorenylmethyl chloroformate could be valuable to both

the synthetic and analytical chemist.

References

Ahuja, S., Chiral Sepmations by Iiqzl id chronintogrnphy, Amencan Chemical Society,

Washington, KI 1991, pp. 43-100.

Menmark, S. G., Chrornatographic Enantiosepnrntion: Methods and Applicntiorzs, John Wiley & Sons, Toronto, 1988, pp. 15-16,27- 41,90-140.

Barksdale, J. M. and Clark., C.R., Liquid chrornatographic determination of the enantiomeric composition of amphetamine and related dmgs by diastereomenc derivatization \. Cliromntogr. Sci., 23 (1985) 176-180.

Beaulieu, N., Cyr, T.D., Graham, S. J. and Lovering, E. G., Liquid diromatographic method for Selegiline Hydrochloride and related compounds in raw matenals and tablets 1. Assoc. Off. Anal. Chem, 74 (1991) 453-455.

Birkmayer, W., Riederer, P.. Arnbrozi, L. and Youdim, M. B. H., Implications of combined treatment with Madopar and 1-deprenyl in Parkinson's disease Lmicet, II (1977) 43943-

Buck, R. H. and Knunmen, K., Resolution of amino acid enantiomers by high performance iiquid chromatography using automated pre-colurnn derivatization with a chiral reagent [. Chromntogr., 315 (1984) 279-285.

Buck, R. H. and Krummen, K., High-performance liquid chrornatographic determination of enantiomeric acids and alcohoIs after derivatization with a

chiral reagent 1. Chromntogr.. 387 (1987) 255-265.

Carey, F. A., Organic Chemistnj, McGraw-Hill Book Company, New York, 1987, p p 252-286.

Chafetz, L., Desai, M.P. and Sukonik, L., Trace decomposition of Selegiline. Use of worse-case kinetics for a stable drug 1. Phnrm. Sci., 83 (1994) 1250-1252.

Cooley, J. H. and Evain., E. J., Amine dealkylations with acyl dilorides Synthesis, 1 (1989) 1-7.

Cotzias, G. C., Deuelopments In Trentment For Parkinson's Disease , hledcom Press, 1971, pp. 25.

Cçerhati, T. and Forgacs, E., Retention of some monoamine oxidase inhibitory drugs on a Bcyclodextrin polymer-coated silica column 1. Chrornatogr. A,, 660 (1994) 313-318.

Dalgliesh, C. E., The optical resolution of arcmatic amino acids on paper

diromatograms 1. Chem. Soc., (1952) 3490-394.

Debowski, J., Sybilska, D. and Jurczak, J., The resolution of some chiral

compounds in reversed-phase high-performance liquid chromatography by

means of B-cydodextrin inclusion complexes Chrornatographin, 16 (1982) 198-200.

Deyl, Z., Hyanek, J. and Horakova, M., Profihg of amino acids in body fluids

and tissues by means of liquid diromatography [. Chromatogr., 379 (1986) 177-

250.

Doyle, T. D., Adams, W. A., Fry, F. S. Jr. and Wainer, LW., The application of HPLC chiral stationary phases to stereochernical problems of phamtaceutical interest: a general method for the resolution of enantiomenc amines as B- naphthyl-carbarnate derivatives \. Liqirid Chrornatogr., 9 (1986) 455471.

Ecsery, Z., Kosa, I., Knoll, J. and Somfai, E., Neth. Pat. App. 6,605,956 Chem. Abs.,

67 (1967) 21611~.

Einarsson, S., Selective determination of secondas. amino acids using precolumn derivatization with 9-fluorenylmethyl chloroformate and reversed-phase high- performance liquid chrornatography 1. Chromatogr., 348 (1985) 213-220.

Einarsson, S., Josefsson, B., Moller, P. and Sanchez, D., Separation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-b(9- Fluoreny1)ethyl chloroformate and reversed-phase liquid chromatography And.

Chem., 59 (1987) 1191-1195.

Eliel, E. L., Stereochemisty of Carbon Compouncis, McGraw-Hill, New York, 1962.

Forgacs, E., Use of principal cornponent analysis for the evaluation of the retention behavior of monoamine oxidase infubitory drugs on B-cyclodextrin

column 1. Phnrm. Biomed. Anal., 13 (1995) 525-532.

Fowler. J. S., 2-Methyl-3-butyn-2-01 as an acetylenic precursor in the Mannich reaction. A new synthesis of suicide inactivators of monoamine oxidase 1. Org. Chem, 42 (1977) 2637-2639.

Fowler, C. J., Callingham, B. A., Mantle, T.J. and Tipton, K.F., Monoamine

oxidase A and 8: A usefd concept? Biochemical Phnnnacology, 27 (1978) 97.

Fuller, R. W., Inhibition of monoamine oxidase by N-(phenoxyethyl) cydopropylamine. Correlation of inhibition with Hammett constants and

partition coefficients [. Med. Chem., 11 (1968) 397-398.

Gerlach, M., Riederer, P. and Youdim, M. B. H., The molecular pharmacologv of L-Deprenyl Eilr. J. Phannacol., 226 (1992) 97-108.

Gil-Av, E., Feibush, B. and Charles-Sigler, R., Separation of enantiomers by gas

liquid chromatography with an optically active stationary phase Tetrahedron Letf.,

(1 966) 1009-1015.

Girault-Vexlearschi, G., Influence de la ramifications des chaines hydrocarbonees sur la baside des amines III, Etude de l'equilibre d'ionisation des amines B d l .

Soc. Chim. Fr., 23 (1956) 589-606.

Greenstein, J. P. and Winitz, M., n e Chemistry ofilmino Acids, Wiiey, New York,

1961, pp. 1738.

Gubitz, G., Wintersteiger, R. and Hartinger, A., Fluorescence derivatization of

tertiary amines with 2-naphthyl chloroformate Chromatugr., 218 (1981) 51-56.

Hasegawa. M. and Matsubara, I., Gas chromatographie determination of optical

purities of amino acids using N-Trifluoracetyl menthyl esters Anal. Biochem., 63 (1975) 308-320.

Hutt, A. J. and Caldwell, J., The importance of Stereochemistry in the clinical

pharmacokinetics of the 2-aryl propionic acid non-steroidal anti-uillammatory

drugs Clin. Pharmacokinet., 9 (19%) 371-373.

Johnson, J. P., Some observations upon a new inhibitor of monoamine oxidase in

brain tissue Biochem. Phannacol., 17 (1968) 1285.

Johnson, D. M., Rerter, A., Collins, M. and Thompson, G.E., Enantiomenc purity of Naproxen by liquid chromatographie analysis of its diastereomeric octyl esters

J. Pham. Sci., 68 (1979) 112-111.

Julian, P. L., Oliver, J.J., Kimball, R.H., Pike, A.B. and Jefferson, GD., Methyl

benzyl ketone Org. Syn., 18 (1938) 66-69.

Julian, P. L. and Oliver, J.J., a-Phenylacetoacetonitrile (Acetobenzyl cyanide) Org. Syn., 18 (1938) 66-69.

Kamerling, J. P., Duran, M., Gerwig, G.J., Ketting, D., Bruinvis, L., VLiegenthart,

J.F.G. and Wadman, S.K., Determination of the absolute configuration of some

biologically important 2-hydroxydicarboxylic acids by capillary gas-liquid chromatography \. Chromatogr., 222 (1981) 276-283.

Kapnang, H. and Charles, G., Reaction des chloroformates sur les amines tertaires: cornpetition entre N-demethylation, desamination, N-debenzylation de N-dcsallylation Tetraliedron Lett., 24 (1983) 3233-3236.

Kinemuchi, H., Fowler, C.J. and Tipton, K.F., Snbstrate specz$ïcicities of the two f o m s of monoamine oxidase, Academic Press, London, 1984, pp. 253.

Knoll, J., Vizi, E.S. and Somogyi, G., Phenyiisopropyhnethylpropinylamine (E- 250), a monoamine oxidase inhibitor antagonizing effects of tyramine. Arzneim-

Forsch., 17 (1968) 1285-1297.

Knoll, J. and Magyar, K., Monoamine Oxiriase-New Vistas : Advances in

Biochemical Psycholpharmacology, Vol 5, Raven Press, New York, 1972, pp. 393

Knoll, J., The possible mechankm of action of (-)deprenyl in Parkinson's disease.

J. Neurnl TTansrn., 57 (1978) 33-38.

Knoll, J., Deprenyl (Selegiline): the history of its development and

pharmacological action Acta Neirrol. Sciznd. Suppl. , 95 (1983) 57-80.

Kometani, T., Shunsaku, S. and Mitsuhashi, K., On the cleavage of tertiary

amines with ethyl chloroformate Chem. Pham. Biil l . , 24 (1976) 342-349.

Konig, W. A. and Benecke, L, Gas chromatographic separation of chiral 2- hydroxy acids and Zalkyl substituted carboxylic acids J. Chromatogr., 195 (1980)

292-296.

Krimen, L. 1. and Cota, D.J., The Ritter Reaction Organic Renctions, 17 (1969) 213-

325.

La Croix, R., Pianezzola, E., Benedetti, and Strolin Benedetti, M., Sensitive high- performance liquid chrornatographic method for the determination of the three

main metabolites of Selegiline (L-deprenyl) in human plasma 1. Chromatugr. B, 656 (1994) 251-258.

LePage, L., Linder, W., Davies, G., Seitz, D. and Karger, B., Resolution of the

optical isomers of dansvl amino acids by reversed phase Liquid chromatography

with optically active metal chelate additives Annl. Chem., 51 (1979) 433-435.

Liu, J. H. and Ku,W. W., Detemination of enantiomeric N-Trifluoroacetyl-L-

prolyl diloride amphetamine derivatives by capillary gas chromatographylmass

spectrometry with chiral and achiral stationa. phases Annl. Cliem., 53 (1981)

21 80-2184.

Lough, W. J., Chiral Liqnid Chrornntography, Blackie and Son Ltd., Glasgow, 1989,

pp. 39-80.

Makino, Y., Ohta, S. and Hirobe, M., Enantiomeric separation of amphetamine by

high performance liquid chromatogcaphy using chiral crown ether-coated

reversed-phase packing: application to forensic analysis Forensic Sci. M., 78

(2996) 65-70.

Martin, A. J. P. and Synge, R. L. M., A new form of chromatogram employing

two liquid phases. 1. A theory of chromatography 2. Application to the micro-

determination of the higher monoamino-acids in proteins Biochem [., 35 (1941)

1358-1368.

Meyer, V. R., Prncticnl High-Prrfonnnnce Litpiri Chromatogrnphy, Second Edn, John

Wiley & Sons, Chichester, 1994, pp. 17-32.123-157-

Miller, K. R., Gal, J. and Ames, M. M., High Performance liquid diromatographic

resolution of enantiomers of 1-phenyl-2-aminopropanes (amphetamines) with

four dural reagents 1. Chromatogr., 307 (1984) 335-342.

Nagai, T. and Kamiyama, S., Assav of the optical isomers of methamphetamine

and amphetamine in rat urine using high-performance Liquid chromatography

with chiral cellulose-based columns 1. Chromatogr., 525 (1990) 203-209.

Noggle, F. T. J., DeRuiter, J. and Clark, C.R., Liquid chromatographie determination of the enantiomeric composition of methamphetamine prepared

from ephedrine and pseudoephedrine And. Cliem., 58 (1986) 1643-1648.

OLofson, R. A., Sdinur, R.C., Bunes, L. and Pepe, J.P., Selective N-dealkylation of

tertiary amines with vinyl chloroformate: An improved synthesis of naloxone Tetrnhedron Lett., (1977) 1567-1570.

Pasteur, L., The Fortncilrtions of Stereo Chernistry: Memoirs by Pasteur, Van 't Hoff, Le Bel and Wislicenw, Amencan Book Company, New York, 1901, pp. 1-33.

Pasutto, F. M., Mirror Images: The analysis of pharmaceutical enantiomers 1. Clin. Pharmacol., 32 (1992) 927-924.

Perrin, D. D., Physical Chernical Properties of Dnigs, Marcel Dekker, New York.

1980, pp. 1-48.

Pettersson, C. and Schill, G., Separation of enantiomeric amines by Ion- pair chrornatography 1. Chromatogr., 204 (1981) 179-183.

Pettersson, C. and No, K., Chiral resolution of carboxyiic and sulphonic acids by ion pair chromatography 1. Chromntogr., 282 (1983) 671-684.

Pettersson, C. and Josefsson, M., Chiral separation of amino alcohols by ion-pair chromatography Chromntopphin, 21 (1986) 321-326.

Pettersson, C., Liquid diromatographic separation of enantiomers using chiral

additives in the mobile phase Trends A d . Chem., 7 (1988) 209-217.

Pharmacopeial Convention, U. S., Sekgdine Hydrochloride- In process revision Phnmacopeial Forum, 20 (1994) 714.1-7150.

Pharmeuropa, Alternative method proposed for the defection of IS) Selegdine, 1996, pp. 24.

Pirkle, W. H., Pochapsky, T. C., Mahler, G. S., Corey, D. E., Reno, D. S. and

Alessi, D. M., Useful and easily prepared &al stationary phases for the direct

chrornatographic separation of the enantiomers of a variety of derivatized amines, arnino acids, alcohols and related compounds 1. Org. Chem., 51 (1986)

4991-5000.

Ramey, O. B., SLereochemist y, Heyden & Son Ltd., Philadelphia, 1981, pp. 69-97.

Ritter, R. J. and Minieri, P.P., A new reaction of nitriles 1; Amides from alkenes

and mononitdes J. Amer. Chem. Soc., 70 (1948) 4045-4048.

Ritter, J. J. and Kalish, J., a,a - Dirnethyl-P-phenylamine (Phenethylamine a, a -dimethyl) Org. Syn., 44 (1964) 44.

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) 11 1-1 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-phthalaldehyde and thiols with amines 1. Am. Chem. Soc., 98

(1976) 7098-7099.

Skidmore, M. W ., Hondbook of Derimtives for Chrornntography, Second Edn, John Wiley & Sons, Chichester, 1993, pp. 215-249.

Skoog, D. A. and Leary, J. J., Principles of lnstnimental Annlysis, Fourth Edition

Edn, Saunders CoLIege Publishing, Philadelphia, 1992, pp. 579-680.

Tipton, K. F., Fowler, C.J. and Houslay, M. D., Monoamine oxidase: Basic and

clinicalfrontiers, Excerpta Medica, Amsterdam, 1982, pp. 87.

Ullrich, K. J., Rurnrich, G., Neiteler, K., and Fritzsch, G., Contraluminal transport

of organic cation in the proximal tubule of the rat kidney Pflugers Arch., 420 (1992) 29-38.

Wainer, 1. W., Drug S tereochem ist y, Analyt ical Methods and Pharmacology, Second

Edn, Marcel Dekker, New York, 1993, pp. 65-106,139-182.

Wainer, 1. W., High Performance Liqitid Chrornatogtaphy: Fundarnerztal Pnnciples nnd

Pracf ice, John Wiley & Som, Toronto, 1994.

Witte, D. K., de Zeeuw, R-A. and Drenth, B. F. H., Chiral derivatization of

Promethazine with (-)menthyl chloroformate for enantiomeric separation by RP- HPLC 1. High Resolut. Chrornntogr.: Chromatogr. Commun., 13 (1990) 569-571.

Pre~aration of a- Phenylacetoacetonitrile - <Julian, 1938 #58>

h t o 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 hr ther drying or purification for the

preparation of benzyl methyl ketone.

Pre~aration of 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 slow ly 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-phenylace toacetonitrile , 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

h t o 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 subs tituted 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 ( f l 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~aration of 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~aration of N-Acetvl-2-Phenvlisooro~vlamin~

(slight modification of the method of Ritter et al <Ritter, 1948 #6b)

(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~aration of N-Formyl-2-~henvliso~ropvlamine

(Modification of Ritter method <Ritter, 1964 #62>

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~aration of 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)

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

APPLIEO - IMAGE. lnc 1653 East Main Street - -. - - Rochester. NY 14609 USA -- -- - - Phone: i l 6/482-O3ûû -- -- - - FU: i l 6/288-5989

O 1993. Applied Image. Inc.. Ail Rights Resewed