separation of alkaloid isomers and stereoisomers …

SEPARATION OF ALKALOID ISOMERS AND STEREOISOMERS WITH

BETA-CYCLODEXTRIN BONDED PHASES

by

KAREN DENISE WARD, B.S.

A THESIS

IN

CHEMISTRY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

MASTER OF SCIENCE

August, 1987

ACKNOWLEDGMENTS

This manuscript Is gratefully

dedicated to those who made It all possible:

Dr. D. W. Armstrong, my parents and family,

Soon and the lab gang, and my Lord Jesus Christ.

And most especially, with love and

appreciation, this Is for Timothy Joseph Ward.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS 11

LIST OF TABLES iv

LIST OF FIGURES V

CHAPTER

I. INTRODUCTION 1

Nicotine and Its Metabolites I

Cyclodextrins in Separations 5

II. METHODS AND MATERIALS II

Methods 11

Materials 12

III. RESULTS AND DISCUSSION 13

Separation of Structural Isomers and Homologous 13 Compounds

Separation of Enantiomers 24

Separation of Metabolites 42

Separation of Derivatized Nicotinoids 52

IV. CONCLUSIONS 59

REFERENCES 61

ill

LIST OF TABLES

1. Structures and Chromatographic Data for Homologous 14 Series of Alkaloids Related to Nicotine

2. Structures and Chromatographic Data for Structural 20 Isomers Related to Nicotine

3. Chromatographic Data Obtained at pH = 7.1 for a 25 Series of Alkaloids Related to Nicotine

4 StructuresfortheOptically Active Alkaloids 27 Related to Nicotine

5. Resolution, Selectivity, and Retention Data for Various 30 Optically Active Alkaloids Related to Nicotine

6. Resolution, Selectivity, and Retention Data for 41 Nicotine and Analogues Separated with a p-Cyclodextrin Mobile Phase

7. Structures of Nicotine Metabolites and 43 Naturally Occurring Tobacco Alkaloids

8. Retention Data For Naturally Occurring Nicotine 45 Alkaloids and Metabolites

9. Retention, Resolution, and Separation Values 55 for Derivatized Enantiomers

IV

LIST OF FIGURES

1. The Structure of the Tobacco Alkaloid Nicotine 2 with the Ring Numbering System Shown

2. Structure of 3-Cyclodextrin (A) and Two of the 6 Glucopyranose Units that Illustrate Details of the a-(1,4) Glycosidic Linkage (B), and the Numbering System Employed to Describe the Ring System

3. A Schematic of Cyclodextrin Bonded to a Silica Gel 8 Support and Reversibly Forming an Inclusion Complex with a Chiral Molecule

4. A Chromatogram Showing the Separation (From 17 Left to Right) of Azetidine Nicotine, Nicotine, and N'-Methylanabasine

5. A Chromatogram Showing the Separation (From 22 Left to Right) of Nornicotine, Nicotine, Myosmlne, and p-Myosmine

6. Plot of Enantiomeric Resolution Versus Methanol ZZ Content of the Mobile Phase for N'-Benzylnornicotine

7. Plots Showing the Effect of Mobile Phase Composition 35 (i.e., % Acetonitrile) on the Enantiomeric Resolution of N'-(2,2-D1fluoroethyl)nornicotlne (•) and N'-Benzylnornicotine (jo)

8. Chromatogram Showing the Resolution of Racemic 39 N"-(2,2,2-Trifluoroethyl)nornicotine (Ro = 1.5 ) (A), N'-Benzylnornicotine (Re = 1.9) (B), andw-(2-Naphthyl-methyDnornicotine (Ro = 1.5) (C) on a 25 cm p-Cyclodextrin Column

9. Separation of Naturally Occurring Nicotine Alkaloids 47 and Metabolites

10. Separation of Nicotine Alkaloids and Metabolites 50 Using A 5% Acetonitrile, 95% Aqueous Triethyl-ammonium Acetate (1%) Mobile Phase

11. Structures of Menthyl- and Benzyl-Carbamate 53 Derivatives of Nicotinoids

12. Chromatogram of Compound 4 57

CHAPTER I

INTRODUCTION

Nicotine and It.q Metabolites

Nicotine is primarily consumed as inhaled cigarette smoke,

consequently scientists in the field of smoking research are Interested

in the quantitation of nicotine and its metabolites. The adverse effects

of cigarette smoking on health depend largely on the amount of "tar"

reaching the lungs and the amount of nicotine in the blood. The

"tar"-to-nicotine ratio is usually constant in each brand of cigarette (1),

therefore the determination of nicotine provides information on the

amount of "tar" reaching the lungs. This could provide much of the data

necessary for the study of the epidemiology of smoking (1). Recently,

other forms of nicotine such as nicotine-containing chewing gum,

"smokeless cigarettes," and smokeless tobacco have become popular.

Researchers have investigated the monitoring of the levels of nicotine

and its major metabolites, cotinine and nicotine-N*-oxide, in human

plasma as a means to determine nicotine-intake from these sources.

The separation and quantitation of nicotine and related alkaloids,

termed nicotinoids, is necessary in a number of other important areas.

These include studies involving the relationship between structure and

reactivity (2, 3) of nicotine analogues. The structure and numbering

system of nicotine is shown in Figure 1. Nicotine analogues can be

formed by making substitutions on the pyridine or pyrrolidine ring.

These analogues can then be used in animal studies to determine their

f!i f ! h ' sii i ii i-: I': P- i'

ifiMhii {•• ?•• !•; !=: i -

FiQure 1 The Structure of the Tobacco Alkaloid Nicotine ^ with the Ring Numbering System Shown.

pyridine Ring

Pyrrolidine Ring

4

activity with respect to nicotine. For example, 6-methylnicotine

exhibits as much physiological activity as nicotine, whereas the

2-methyl and 4-methyl isomers are nearly inactive (4, 5). However,

relatively few nicotine analogues have been synthesized and separated.

Other areas in which the separation and quantitation of nicotinoids are

important are alkylation reactions (3, 6), alkaloid metabolism (7), plant

breeding control (8), familial dermatitis, and anaphylactic reactions (9).

A relatively small number of nicotinoids (i.e., mainly nicotine,

cotinine, and some other metabolites) have been separated by

conventional chromatographic means. The earliest reported methods

involved thin layer chromatography and gas chromatography (10-16).

More recently, solvent extraction followed by gas chromatography/mass

spectrometry and high performance liquid chromatography (HPLC)

methods have been shown to be sensitive and efficient (1, 7-9, 17-25).

The HPLC-based separations utilized either reversed phase or ion

exchange packings. In most studies only two to four compounds needed

to be resolved. The most complex separation reported involved a mixture

of eight nicotine-related metabolites (7).

Very little separation data for structural isomers related to

nicotine or homologous compounds has been reported, possibly because

these types of analytes are known to be difficult to separate by

conventional reversed phase LC. The isolation of optically pure isomers

has also been a difficult problem (26, 27). For example, the most recent

methods for resolving nornicotine still involve making diastereomeric

derivatives, such asN'-(methoxycarbonyl)nornicotine, followed by

5

recrystallization or preparative LC (28). There are few reports on the

resolution (by any method) of any other derivatized nicotine analogue.

Cvclodextrins In Seoarations

Cyclodextrins, also known as cycloamylases, are chiral, toroidal

shaped molecules which are composed of glucose units which are bound

via a-(l,4) linkages. The structure of p-cyclodextrin, which contains

seven glucose units, is shown in Figure 2. The interior of the cavity,

which contains no hydroxyl groups, is relatively hydrophobic in

comparison to polar solvents like water (29-33). As a consequence,

cyclodextrins are able to complex a variety of water insoluble or slightly

soluble compounds. The entrance of the cyclodextrin cavity is lined with

the 2- and 3-hydroxyl groups. It is possible that an included solute could

hydrogen bond with the 2-hydroxyl groups, thus forming an extremely

stable inclusion complex.

As a result of the inclusion complex formation, stable

cyclodextrin bonded phases have been shown to be an effective liquid

chromatography (LC) stationary phase for the separation of a variety of

enantiomers (34-40). In order for chiral recognition to occur, there

should be a relatively "tight fit" between the hydrophobic species and the

cyclodextrin cavity (30, 31, 34, 35, 37, 40), p-Cyc1odextrin (3-CD) is

known to form tight inclusion complexes with many compounds that

contain two to four rings (29). A simplified model for Inclusion complex

formation is illustrated in Figure 3. Since nicotine alkaloids contain at

least two ring moieties, it is reasonable to assume that p-cyclodextrin

Figure 2. Structure of (3-Cyclodextr1n (A) and Two of the Glucopyranose Units that Illustrate Details of the a-(l,4) Glycosidic Linkage (B), and the Numbering System Employed to Describe the Ring System.

A.

B. ^ ^ ^ 0

4 CH2OII

8

ii

f

Figure 3. A Schematic of Cyclodextrin Bonded to a Silica Gel Support and Reversibly Forming an Inclusion Complex with a Chiral Molecule.

. w ^ I + ,t-Y rv-> 'bK:w

10

bonded phases might be able to resolve these enantiomers. In addition,

cyclodextrin-containing mobile phases have been used in conjunction

with achiral stationary phases to resolve some racemic mixtures

(41 -43). Therefore, both p-cyclodextrin bonded phases and

p-cyclodextrin-containing mobile phases may be utilized to achieve

enantiomeric resolution of a variety of optically active nicotinoids.

The effectiveness of the cyclodextrin bonded phases in HPLC Is not

merely restricted to the separation of enantiomers. Cyclodextrin bonded

phases have also been utilized in the separation of diastereomers and In

routine separations (30, 34, 36, 44-46). Unlike all other LC stationary

phases, cyclodextrin bonded phases can discriminate between very

similar compounds on the basis of their geometry or spatial orientation

(35), as a result of the inclusion complex formation. Solutes with

different shapes form inclusion complexes with cyclodextrin of different

stabilities (36). The most stable complex is usually retained longest

while the compound which forms the least stable complex is eluted first.

A number of tobacco alkaloids and nicotine metabolites were separated

using the p-cyclodextrin bonded phases. The effect of structure on

chiral and isomeric recognition was examined as well

CHAPTER II

METHODS AND MATERIALS

Methods

All separations were done at room temperature (21 T ) with a

Shimadzu LC-4A liquid chromatograph. The compounds were detected at

254 nm with a variable wavelength detector containing a 13 LL flow

cell. All samples were dissolved In acetonitrile or methanol (depending

on the mobile phase composition) prior to injection. Columns (25 x 0.46

cm) containing p-CD bonded to 5 iim silica were obtained from Advanced

Separation Technologies, Whippany, NJ. The void volume of the column

was determined by injecting neat methanol. The peak-trough

combination caused by the change in refractive index was used as the

marker for the void volume. Flow rates, solvent compositions and pHs

are given in the respective tables and figures.

The order of elution for the enantiomers was established from

consecutive injections of the individual pure enantiomer, as.well as

samples containing the racemic form spiked and unspiked with one

enantiomer. Precautions were taken to ensure that the peaks seen were

actually that of the enantiomers. The absence of peak overlap, which

could be caused by contaminants, was demonstrated by the constant

peak-height ratios obtained by detection at different wavelengths.

Presumably a contaminant would demonstrate noticeably different

absorption values from that of the enantiomers. In some cases, the

circular dlchrolsm spectra of eMiirug fractii®ins were takwi m ©nier t®

confirm the separations.

CI packed (5 u.m) mlcrocolurmis were maidle as prewiiawjBlljf irepoirlteeli

(47). The columns were 1 m x 250 iim i d A VaUc® iliniteTOil Msp

microlnjector was used to introduce 0:2 iiL of s®IiiJitfi«iiini limit® tdte s sttemi.

The chiral mobile phase consisted of 203: atpecnus aceitioiniijibrilll® sai)ijjralj[£(!tl

with p-CD. The microcolumn system pressmre was 230 attmni anflJ idtne ffflssw

rate was l.3M.Lym1n.

Materials

HPLC grade methanol, acetonitrile. trieitllTiyllanniiiiiie, amll waiter were

obtained from Fisher Scientific Company. Ttie mniolbijlle pitnases tueetdl were

mixtures of acetonitrile-buffer (0/100 to 100/0, v/v), aiMJ

methanol-buffer (0/100 to 100/0, v/v). Buffers were preparedl ll!>j

making a 1 % solution of triettnylamine in water aiml adding gflaciiaH aceltiic

acid until the desired pH was obtained.

The nicotlnold 2,3"-dipyridyl was obtained fnami SUgpa Onemniijoll

Company (St. Louis, MO). All other alkaloids were graciiousl^ izllaTfialeidl ^

Jeffrey Seeman (Philip Morris Company, Ridwnond, VM.

Free p-cyclodextrin was obtained from Advanced Separatiiiani

Technologies, Whippany, NJ. The p-cyclodextrin motoiilie plhiase was

prepared by saturating a 20% acetonitrile/80?5 water saliutlon wlltjt?i

P-cyclodextrin.

CHAPTER III

RESULTS AND DISCUSSION

Separation of Strtictural Isomers and Homologous Comnounds

The structure and separation data for ten related nicotinoids is

given in Table 1. The ability of the p-cyclodextrin bonded phase to

discriminate between structurally similar compounds is evident. Figure

4 shows the complete separation of the homologues: azetidine nicotine

(4-membered ring), nicotine (5-membered ring), and N'-methylanabasine

(6-membered ring) in fifteen minutes. The relative retention appears to

be directly controlled by the size of the saturated ring (Table 1). An

increase in retention is observed as the ring system attached to the

pyridine ring is increased in size. Presumably the larger ring size

produces a tighter inclusion complex which results in a longer retention

time.

The effect of derivatizing the N' position in the pyrrolidine system

is apparent from the retention behavior of nicotine, nornicotine, and

N'-benzylnornicotine (Table 1). The capacity factor nearly doubles in

going from a hydrogen atom to a methyl group, then almost triples when

the methyl group is replaced with a benzyl substituent. This may be due

to the increased ability of N'-benzylnornicotine to form a stronger

inclusion complex than nicotine or nornicotine, but the effects of

increased hydrophobicity for benzyl versus methyl or hydrogen must also

be considered. The effect of adding an ethyl side chain to the pyridine

system is shown in the retention behavior of nicotine and

13

Table 1. Structures and Chromatographic Data for Homologous Series of Alkaloids Related to Nicotine.

Compounds

•* CHj

Azetidine Nicotine

H*^ CHi

Nicotine^^

^MT CH,

N*-Methylanabasine

Nicotine

jyx. 6-Ethylmcot1ne

Or? Nornicotine

^ H * ^ CHJ

Nicotine

0"^^

k-a

0.513

0.706

0.981

0.406

0.775

0.241

0.451

1.240

at>

1.38

1.40

1.91

1.87

2.75

Rs^

2.53

2.68

480

3.15

7.67

Mobile Phase^

0/100^

5/95^

5/95^

N'-Benzylnornicotine

15

Table 1. Continued.

Compounds

^ ^ H

2-Phenylpyrrol1dlne

^ - ^ CHJ

1 -tiethyl-2-phenylpyrrol1d1ne

^ * ^ CHJ

1 -Methy1-2-phenylpyrrol1d1ne

^-^ CHJ

4-ison1cot1ne

k'a

0.653

0.792

0.875

1.300

ab

1.22

1.50

Rs^

3.00

2.37

Mobile Phase^

5/95^

0/100^

^Capacity factor, k" = (retention volume of solute-void volume)/vo1d volume.

^Selectivity factor, a = k'2/k'

^Resolution factor, Rg = 2(d1stance between peaks)/(sum of the bandwidths of the two peaks).

< Numbers refer to the percent volume of acetonitrile in aqueous triethylammonium acetate (1 %) buffered to pH 41. The flow rate was l.OmL/min.

16

Table 1. Continued.

^Two 25 cm p-cyclodextrin columns connected in series were used.

One 25 cm p-cyclodextrin column was used.

Figure 4. A Chromatogram Showing the Separation (From Left to Right) of Azetidine Nicotine, Nicotine, and N'-Methylanabasine. The Flow was l.OmL/min. Other Separation Conditions are Given In Table 1.

18

V I — \ — I -

4 8 12 16

RETEIfTION TIME, M I N

Id

19

6-ethylnicotine. The capacity factor increased from 0.41 to 0.78 upon

the addition of an ethyl group at the number 6 position of the pyridine

ring. This may be due to the fact that the ethyl side chain allows

6-ethylnicotine to fit "tighter" into the cyclodextrin cavity and therefore

be retained longer.

Three sets of structural isomers were examined. These include:

(a) nornicotine from 4-isonornicotine and 2-isonornicotine, (b) nicotine

from 2-isonicot1ne and 4-isonicotine, and (c) myosmlne from

p-mysomine. In all cases the Isomers differed only in the location of the

nitrogen heteroatom in the pyridine ring (Table 2). Figure 5 shows the

separation of nornicotine, nicotine, myosmlne, and p-myosmlne, which

are baseline resolved in twelve minutes. One of the more interesting

aspects of this data involves the retention of the 4- or p-isomer relative

to that of the naturally occurring Isomer (where the nitrogen heteroatom

is in the meta position). In all cases the 4- or p-isomer is retained

longer by the p-CD bonded phase (Table 2). Since all of the structural

isomers are the same size, the increased retention of the 4- or

p-isomers may be due to additional dipolar or electrostatic Interactions

with the cyclodextrin. It is well known that many complexed solutes can

hydrogen bond with the 2- and 3-hydroxy1s at the mouth of the

cyclodextrin cavity and that this interaction also can lead to chiral

recognition in cyclodextrin based enantiomeric separations (37, 40). In

this study, the increased retention of the 4- or p-isomers may be due to

the increased proximity of the pyridine nitrogen to the mouth of the

cyclodextrin cavity which allows better hydrogen bond formation.

Table Z Structures and CJTroinatoQrMzifiiiic QMa toar StiruiidtMraill ilssmnsrs Related to Nicotine.

Compounds k-a c ^

09 Nomkxitlne

4-isorarnicot1ne

2-lsonormcotine

2-lsonicot1ne

c^ Nicotine

4-lsonlGotine

Myosmlne

p-Myosmine

0.279

0.367

0.514

0.658

0.739

0,863

0.967

1.830

1J2 1I..3Q)

1.410 1.66

1.89

1.12 I

1.31 K83

3.96 5.95^

21

Table 2. Continued.

^Capacity factor, k' = (retention volume of solute-void volume)/void volume.

•^Selectivity factor, a = k'2/k"

^Resolution factor, Rs = 2(distance between peaks)/(sum of the bandwidths of the two peaks).

^Numbers refer to the percent volume of acetonitrile in aqueous triethylammonium acetate (1 %) buffered to pH 41. The flow rate was I.OmL/min.

®Two 25 cm p-cyclodextrin columns connected in series were used.

One 25 cm p-cyclodextrin column was used.

Figure 5. A Chromatogram Showing the Separation (From Left to Right) of Nornicotine, Nicotine, Myosmlne, and p-Myosmine. The Flow Rate was 1.0 mL/min. Other Separation Conditions are Given In Table 2.

23

4 6 8 10

RETENTION TIME, M I N 12 14

24

The effect of pH on retention and selectivity is shown in Table 3.

In all cases, retention increased with increasing pH. This is a

consequence of the alkaloids going from their protonated form (at pH 41)

to their unprotonated form at higher pHs. The more hydrophobic the

alkaloid, the greater was the increase in retention in going from pH = 41

to 7.1 (see N'-benzylnornicotine for example). Interestingly, there were

no changes in the retention order (i.e., selectivity) at different pHs. The

efficiency (peak widths) deteriorated significantly at the higher pHs for

these particular compounds as a result of the longer retention on the

column.

Separation of Enantiomers

Table 4 gives the structure of nicotine and nineteen chiral

analogues. The effect of structure on chiral recognition was examined by

using these systematically altered nicotine analogues. Note that the

structural changes fall Into three general classes. In one group of

compounds, the substituent from the pyrrolidine nitrogen was altered

with each substituent varying in size, electron withdrawing ability,

hydrogen bonding ability, or orientation. The second group of compounds

were analogous to those in the first group except that the pyrrolidine

ring was opened (compounds 12-15, Table 4). In the third group, changes

were restricted to the pyridine ring (compounds 16-20, Table 4).

Table 5 gives the separation data for ten racemic nicotinoids that

were resolved using a p-cyclodextrin bonded phase column. Note that

two different mobile phases were tested. The best separations were

25

Table 3. Chromatographic Data Obtained at pH = 7.1 for a Series of AlkaloidsRelated to Nicotine.

Compounds k* Mobile Phase Column^

Nornicotine 4-lsonorn1cot1ne 2-lsonornicotine

2-lsonicotine Nicotine 4-lsonicot1ne

Nicotine 6-Ethylnicot1ne

Nornicotine Nicotine N'-Benzylnornicotine

Azetidine nicotine Nicotine N'-Methylanabasine

Myosmlne p-Myosmine

2-Phenylpyrrolidine 1-Methyl-2-phenyl-

pyrrolidine

1-Methy1-2-phenyl-pyrrolidine

4-lsonicotine

1.13 1.31 1.81

0,87 1.53 1.79

3.05 5.24

1.42 3.06 9.79

2.24 3.01 3.61

3.28

3.35

1.12 1.16

1.12

1.52

5/95

10/90

5/95

5/95

5/95

5/95

20/80

20/80

II

II

II

26

Table 3. Continued

^Numbers refer to the percent volume of acetonitrile in aqueous triethylammonium acetate (1 %) buffered to pH 7.1. The flow rate was 1.0 mL/min.

• 11 = two 25-cm p-cyclodextrin columns.

27

Table 4 Structures for the Optically Active Alkaloids Related to Nicotine.

Compound Name Structure

1. Nicotine

CH]

2. l-Methyl-2-(2-pyrldyl)pyrrolid1ne H ^ N f CH]

N' J^^' 3. l-Methyl-2-(4-pyrldyl)pyrrol1d1ne ^^ CHJ

4. N'-(Ethyl)nomlcotlne

L-CHj

5. N*-(2,2-D1fluoroethyl)norn1cotine \ ^ , L^CFjH

6. N'-(2,2,2-Trlfluoroethyl)norn1cotine »* LCFJ

7. N'-Benzylnornicotine (f J ^ H ,_^

28 Table 4. Continued.

Compound Name Structure

*M

8. N'-(2-Methylbenzyl)norn1cot1ne

9. N'-(2-Naphthylmethyl)norn1cotine

10. N'-( 1 -NaphthylmethyDnornicotine

11. N'-Benzoylnornicotine

12. N-Benzyl-N,a-d1methyI-3-pyrid1nemethanamine

13. N,a-Dimethyl-N-(2-naphthylmethyl)- ^ < ^ s ^ 3-pyrid1nemethanam1ne ([ J ^ M

14. N,a-Dimethyl-N-(l-naphthylmethyl)- ^--^^j^..^ 3-pyr1dinemethanamine 5 ^jJ^?

N

15. N-Ethyl-N,a-dimethylphenylmethamine r ^ V ^ >

CH,


:ompound Name Structure

Cr9 16. l-Methyl-2-phenylpyrrolid1ne

CH3

17. l-Benzyl-2-phenylpyrrol1dine ^^555^.^"^

18. 6-Ethylnicotine | j ^

CH3

^f''^^—(

19. 6-Butyln1cotine

20. 5,6-Cyclohexenonlcotine

30

Table 5. Resolution, Selectivity, and Retention Data for Various Optically Active Alkaloids Related to Nicotine.

Mobile Flow Compound k' a Rg Phase Rate Column^

N'-(2,2-D1fluoroethyl)- 3.86 1.03 1.14 10/90 1,0 nornicotine 0.60 1.00 100/0 1.0

N'-(2,2,2-Trifluoroethyl)- 1.03 1.09 1.51 25/75 1.0 nornicotine 0.64 1.19 1.29 100/0 1.0

5,6-Cyclohexenonicotine 2.97 1.04 1.50 25/75 1.0 416 1.07 1.57 100/0 1.0

N'-Benzylnornicotine 2.82 1.18 2.36 30/70 1.0 2.23 1.14 1.47 100/0 1.0

N'-(2-Methylbenzyl)- 8.13 1,07 1.54 10/90 0.8 nornicotine 1.22 1.15 1.17 100/0 1.0

N-Benzyl-N,a-dimethyl- 2.05 1.03 0.80 20/80 0.5 3-pyridinemethanamine 2.53 1.00 100/0 0.5

N'-(2-Naphthylmethyl)- 5.35 1.07 2.02 30/70 0.8 nornicotine 3.37 1.02 1.12 100/0 0.8

N,a-D1methyl-N- 1.64 1.09 1.21 20/80 0.5 (2-naphthylmethyl)- 2.83 1.00 - — 100/0 0.8

nornicotine

31

Table 5. Continued.

Mobile Flow Compound k' a Rg Phase Rate Column^

N'-Benzoylnornicotine 475 1.05 1.32 5/95 0.5 406 1.00 100/0 1.0

1-Benzyl-2-phenyl- 6.70 1.02 0.30 10/90 0.5 pyrrolidine 448 1.00 100/0 1.0

The capacity factor corresponds to the first eluting enantiomer.

^The mobile phase consisted of acetonitrile/aqueous triethylammonium acetate (pH 7.1).

1 = one 25-cm p-cyclodextrin column, II = two 25-cm p-cyclodextrin columns connected in series.

32

obtained with hydro-organic mobile phases with acetonitrile as the

organic modifier. The aqueous portion of the mobile phase was buffered

at a pH of 7.1 with triethylammonium acetate. It was noted that

decreasing the pH of the mobile phase usually decreased enantiomeric

resolution. At pHs less than 5.0, no enantiomeric separations were

observed. Previous reports indicated that both retention and resolution

decreased on p-cyclodextrin columns with increasing organic modifier

concentration. This is shown for N'-benzylnornicotine In Figure 6 and for

N'-(2,2-difluoroethyl)nornicotine in Figure 7. When a solute elutes near

the void volume of the column, further increases in modifier

concentration have little effect on retention and selectivity. This

general type of behavior was observed for the nicotinoids with methanol

modifier (Figure 6). However, an unusual type of retention behavior was

observed for approximately half of the nicotinoids when acetonitrile was

used as the organic modifier. A retention minimum was reached between

60 and 80% (by volume) acetonitrile. Further Increases in acetonitrile

concentration (up to 100% organic modifier) caused a reversal in

retention behavior. Similar minima have been observed on achiral

reversed phase columns and were attributed either to residual silanol

groups or solubility limitations of the solute. However, in this study

enantiomeric resolution also increased at high acetonitrile concentration

(see N'-benzylnornicotine in Figure 7). As the bonded p-cyclodextrin is

the only chiral constituent of the column it should be responsible for the

observed behavior. This change in retention behavior (Figure 7) may

indicate a change in the mechanism of chiral recognition. Currently it is

Figure 6. Piot of Enaaitoenefic-Resolution-Versasi^tliaQOt' CoBteat of t h o ^ ^ f i e PhaseTor ^J'-Bsez tl— nemlcetinfe NQte4he-D1 f fereflce -i n -SBapftfaKl: LoeatiOR-of Th+s^Zpcve^Versus-the-AQato9©.ua?tot Uswig/vcefconttpfle^

3^

m

1

3-

2-

11-

w n

IID\

II II • 1 ir'

••

• " i r - "»••—

4tS) StiD

CP'

T i— r^—rr • n

aBoo 7300 aSD Srttdlhflfoll

<m

Figure 7. Plots Showing the Effect of Mobile Phase Composition (i.e., % Acetonitrile) on the Enantiomeric Resolution of N'-(2,2-D1fluoro-ethyDnornicotine (•) and N'-Benzylnornicotine (o). Note That There are Two Different Types of Behavior at High Modifier Concentration. The Minima in the Second Curve may Indicate a Change in the Mechanism for Chiral Recognition.

36

e

20 40 60 % Acetonitrile

80 1

100

37

not known If retention is due solely to interaction between the external

hydroxyls and the nicotlnold (as in a normal phase separation) or if some

kind of complexation is involved. Interestingly, the enantiomeric elution

order of N'-benzylnornicotine is identical In hydro-organic mobile phases

and 100% acetonitrile (i.e., the R-isomer elutes before the S-lsomer).

It Is evident from the data in Table 5 that hydrogen bonding,

steric, and pKa effects are important in p-cyclodextrin mediated chiral

recognition. For example, racemic N'-(2-naphthylmethyl)nornicotine Is

easily resolved on the p-cyclodextrin bonded phase. However, the

1-naphthy1methyl Isomer (Table 4) shows absolutely no chiral

recognition even though it Is well retained. An analogous phenomenon

was observed previously for 1- and 2-naphthyl and naphthylamide

derivatized amino acids (34, 35). Apparently the orientation of the

1-naphthy1 group in these compounds precludes chiral recognition by

p-cyclodextrin. The addition of an a-methyl group to the phenyl ring of

N'-benzylnornicotine decreases enantiomeric resolution (Table 5).

Interestingly, the presence of the pyrrolidine ring does not seem to be an

absolutely necessary structural feature for chiral recognition by

p-cyclodextrin. Essentially all racemic nicotine analogues that can be

resolved on the p-cyclodextrin bonded phase are also resolved when the

pyrrolidine ring is opened (Table 5).

Compounds 4 through 6 in Table 4 show the effect of electron

withdrawing groups on chiral recognition. Racemic N'-(ethyl)nornicotine

does not separate on a p-cyclodextrin column. Substituting fluorine for

hydrogen at the 2-ethyl position results in chiral recognition and

38

resolution (Table 5). The more fluorines (i.e., N'-(2,2,2-trif1uoro-

ethyDnornicotine) the better the separation. The presence of fluorine

allows additional hydrogen bonding to take place and alters the basicity

(pKa) of the pyrrolidine nitrogen.

Changes in the pyridine ring of the nicotinoids also affect chiral

recognition. For example, the most easily resolved racemate was

N'-benzylnornicotine (Table 5). Substituting a phenyl group for the

pyridine ring (i.e., 1-benzyl-2-phenylpyrrol1d1ne) very nearly eliminates

chiral recognition by the p-cyclodextrin bonded phase (Table 5). Addition

of alkyl groups to the pyridine ring of nicotine (compounds 18 and 19,

Table 4) did not seem to enhance the separation of the unresolved

nicotine analogues. However, the cyclic analogue of 6-butyln1cot1ne (i.e.,

racemic 5,6-cyc1ohexenon1cot1ne, Table 4) was baseline resolved (Table

5). A typical separation of three of these racemates is presented in

Figure 8.

Many of the nicotlnold enantiomers can be separated on achiral

columns with cyclodextrin-containing mobile phases. In this case the

cyclodextrin is both adsorbed on the stationary phase and is present as a

mobile phase carrier molecule. The relative retention of the

enantiomeric pairs is opposite to that found for the p-cyclodextrin

bonded phase. Table 6 lists several racemates that were resolved using

a p-cyclodextrin mobile phase as well as the relevant retention data.

The bonded chiral phase approach was generally thought to be superior

for solutes that could be resolved by both methods. This was because the

"chiral mobile phase additive approach" tended to produce broad peaks

Figure 8. Chromatogram Showing the Resolution of Racemic N'-(2,2,2-Tr1fluoroethyl)nornicotine (Re = 1.5) (A), N'-Benzylnornicotine (Re = 1.9) (B), and N'-(2-Naphthylmethyl)nornieotine (Re = 1.5) (C) on a 25 cm p-Cyclodextrin Column. This Particular Separation Used Gradient Elution from 10/90 (v/v) Acetonitrile/1% Aqueous Triethylammonium Acetate (pH = 7.1) to 70/30 in 20 Minutes. The Flow Rate was 1.0 mL/min and the Wavelength was 254 nm.

40

Retention Time (min.)

Table 6. Resolution, Selectivity, and Retention Data for Nicotine and Analogues Separated with a p-Cyclodextrin Mobile Phase.

Compound

Nicotine l-Methyl-2-(2-pyridyl)-

pyrrolidine 1-Methyl-2-(4-pyridyl)-

pyrrolidine 1-Methyl-2-phenyl-

pyrrolidine

5,6-Cyclohexenonlcotine N'-(2,2-Difluoroethyl)-

nornlcotine N'-(2,2,2-Tr1fluoroethyl)-

nornicotine N'-Benzylnornicotine N*-(2-Naphthylmethyl)-

nornicotine

k't)

6.56 7.33

5.63

5.75

5.98 3.22

453

6.08 5.93

a

1.12 1.13

1.09

1.07

1.07 1.08

1.11

1.08 1.08

Rs

1.7 1.4

1.2

1.5

1.2 0.6

1,2

1.5 1.4

^Mobile phase: p-cyclodextrin saturated solution in 20% acetonitrile/80% water, column length: Im. Flow rate: 1.3M.L/min.

• The capacity factor corresponds to the first eluting enantiomer.

42

(poor efficiency) and required longer columns and separation times. The

most significant thing about the p-cyclodextrin mobile phase technique

was that a few of the racemates (including nicotine) could be resolved by

this approach despite the lack of success on the p-cyclodextrin bonded

phase. Compounds 1 through 4 (Table 6) in particular gave good

resolution with p-cyclodextrin mobile phases while showing little or no

chiral recognition with p-cyclodextrin bonded phases. The reasons for

this are not entirely clear, but may be related to changes in the binding

ability of the bonded phase and to the multiple equilibria possible when

p-cyclodextrin is in free solution (48).

Separation of Metabolites

The names and structures of twelve naturally occurring

nicotinoids and metabolites are given in Table 7. The effects of pH and

organic modifier on selectivity and retention were examined. Table 8

lists the chromatographic conditions along with the retention data for

the nicotinoids. The separation which resulted in the most resolved

peaks was done at a pH of 5.5 with methanol as the organic modifier. The

chromatogram is reproduced in Figure 9, A. Out of the twelve

nicotinoids, two sets of compounds tended to coelute (i.e., compounds 2

and 12, and compounds 4 and 10). Chromatogram B in Figure 9 shows the

separation obtained at pH 5.5 with acetonitrile as the organic modifier.

The change in selectivity with the change in the organic modifier used is

evident by comparing chromatogram A with B. Note that compounds 4 and

10 coelute when methanol was used (Figure 9, A), but were easily

43

Table 7. Structures of Nicotine Metabolites and Naturally Occurring Tobacco Alkaloids.

Compound Structure

1. (S)-(-)-Anabas1ne

2. (R,S)-Anatablne

3. (S)-(-)-Cotln1ne

4 2,3'-Dlpyridyl

H

H ..;i K M^O

CHs

5. (S)-(-)-N-Methylanabas1ne

6. Myosmlne


Compound Structure

7. (S)-(-)-N1cotine N Cr9

CH3

8. (IR, 2S)-anti-Nicotine-N'-ox1de [I J ^ M '

9. (IS, 2S)-syn-N1cot1ne-N'-ox1de ^x^^Ji^i^

10.

11.

Nicotyrene

Norcotinine

O" • ^ N ^

K^ CH3

\^o H

12. (S)-(-)-Nornicot1ne "N cr?

45

Table 8. Retention Data For Naturally Occurring Nicotine Alkaloids and Metabolites.

Coi

1. 2. 3. 4 5.

6. 7, 8.

9.

10.

11. 12.

Tipound

(S)-(-)-Anabaslne (R,S)-Anatab1ne (S)-(-)-Cotinine 2,3'-Dipyridyl

k' with Acetonitrile

4 1 ^

0.20 0.15 0.78 1,87

(S)-(-)-N-Methy1anabas1ne0.37

Myosmlne (S)-(-)-N1cotine (IR, 2S)-ant1-N1cot1ne-N'-oxide (IS, 2S)-syn-N1cotine-N'-oxIde Nicotyrene

Norcotinine (S)-(-)-Nornicotine

0.60 0.28 0.71

0.60

1.41

0.37 0.15

Modifier

pH

5.5b

0.36 0.30 1.21 2.59 0.66

1.48 0,55 0.98

0.98

2.87

0.66 0.30

7.1C

0.80 0.80 1.15 2.46 1.87

1.60 1,53 0.64

0.64

2.94

0.56 0.80

k- with Methanol Modifier

pH

5.5^

0.36 0.22 1.43 3,97 0.79

2.03 0.65 1.08

0.98

3.97

0.83 0,22

^The mobile phase composition was 5/95 (v/v) acetonitrile/1% triethylammonium acetate (aq). The flow rate was 1.0 mL/min.

• The mobile phase composition was 5/95 (v/v) acetonitrile/1% triethylammonium acetate (aq). The flow rate was 1.0 mL/min.

46

able 8. Continued.

^The mobile phase composition was 5/95 (v/v) acetonitrile/1% riethylammonium acetate (aq). The flow rate was 1.0 mL/min.

The mobile phase composition was 5/95 (v/v) methanol/1% riethylammonium acetate (aq). The flow rate was 1.0 mL/min.

Figure 9. Separation of Naturally Occurring Nicotine Alkaloids and Metabolites. A. Chromatographic Conditions: 5% Methanol. 95% Triethylammonium Acetate (1%), pH = 5.5, Flow Rate = 1.0 mL/min, One 25 cm p-Cyclodextrin Column. B. Chromatographic Conditions: 5% Acetonitrile, 95% Triethylammonium Acetate (1%), pH = 5.5, Flow Plate = 1.0 mL/min, One 25 cm p-Cyclodextrin Column.

48

10 .-.'0


B.

10 15 .'0


49

resolved when acetonitrile was the organic modifier (Figure 9, B).

Conversely, though metabolites 8 and 9 coelute when the organic

modifier was acetonitrile (Figure 9, B), these peaks are partially

resolved when methanol was used (Figure 9, A).

Figure 10 shows the separations obtained at pH 4.1 (chromatogram

A) and pH 7.1 (chromatogram B). The selectivity changes with changing

pH, For example, when the pH was 4 1 , nicotlnold 10 eluted before

compound 4 (Figure 10, A), but when pH was 7.1, this order was reversed

(Figure 10, B). Also, compound 11 elutes in the midst of several peaks

when pH 4.1 was used (Figure 10, A), but compound 11 was the first

eluting peak when pH was 7.1 (Figure 10, B).

By examining the retention data in Table 8, It Is evident that three

distinct types of chromatographic behavior are present. The first type Is

exhibited by those nicotinoids whose retention increases with Increasing

pH. Compounds such as anabaslne, anatabine, N'-methylanabasine,

nicotine, and nornicotine fall into this catagory. A second type of

behavior is shown by compounds whose retention increases to a point,

and remains at that point. Nicotinoids exhibiting this behavior are

cotinine, 2,3'-d1pyridyl, myosmlne, nicotyrene, and norcotinine. The

third type of retention behavior is characterized by first increasing

retention with increasing pH, then as the pH continues to increase, the

retention decreases. This results in a retention maxima. The

metabolites ant1-n1cotine-N'-oxide and syn-nicotine-N'-oxide are

classified in this group.

Figure 10. Separation of Nicotine Alkaloids and Metabolites Using A 5% Acetonitrile, 95% Aqueous Triethylammonium Acetate (1%) Mobile Phase. A. pH = 4.1, Flow Rate = 1.0 mL/min, One 25 cm p-Cyclodextrin Column. B. pH = 7.1, Flow Rate = 1.0 mL/min, One 25 cm p-Cyclodextrin Column.

J7'' ' 2J2 1,16.9


B.

0 0 cr


52

The selectivity changes greatly with changes In pH and with the

organic modifier used. These selectivity changes can be exploited to

obtain the best separation if a sample contains only a few of these

alkaloids or metabolites.

Separation of Derivatized Nicotinoids

As mentioned previously, some of the nicotinoids showed no

enantiomeric resolution on the p-cyclodextrin bonded phase.

Presumably, these compounds were too small to interact sufficiently

with the cyclodextrin cavity to achieve chiral recognition. This was

reported previously in the case of amino acid separations (34). While the

amino acids showed no enantiomeric resolution on p-cyclodextrin bonded

phases, the dansyl-derivatized amino acids were easily resolved. Thus

by derivatizing the compound, resolution sometimes can be achieved.

A series of menthyl- and benzyl-carbamate derivatized nicotinoids

(Figure 11) were synthesized and chromatographed. The menthyl-

carbamate derivative of anabaslne, 2, exhibited excellent separation

with a resolution value of 10, as shown in Table 9. However, the

menthyl-carbamate derivative of nornicotine, 1, was not resolved,

despite its structural similarity. Even more surprising was the fact that

derivative 3, which entirely lacks the second ring structure, does exhibit

resolution. Apparently the second ring structure is not a prerequisite for

chiral recognition in this case.

Compound 4, which contains two chiral centers, exhibited four

distinct peaks at certain solvent ratios. The chromatogram is shown in

Figure 11. Structures of Menthyl- and Benzyl-Carbamate Derivatives of Nicotinoids.

CHJ

CH3 CH3

I

54

T>-o^

CH, £ " '

CH3' CH3

3

55

Table 9. Retention, Resolution, and Separation Values for Derivatized Enantiomers.

Compound Mobile Flow k' a Rg Phase^ Rate

(mL/min)

30/70 1.0 2.78

30/70 1.0 2.02, 5.10 2.53 10.0

20/80 1.0 466, 13.58 2.91 9.4

10/90 0.5 5.89, 6.15 1.04 0.6 1.56 3.7

9.24, 9.63 1.04 0.4

20/80 1.0 1.70, 2.09 1.18 0.8

^The mobile phase consisted of acetonitrile/trlethylammonium acetate (1%),pH = 7.1, (v/v).

56

Figure 12. These peaks might correspond to the RR, RS, SR, and SS

isomers; however, this has not been confirmed by other techniques.

Figure 12 Chrom^bctGrafrrafCjMnia^^ wer€E 30/70 AcstoTltrilfe^AqufisoMsTnei^ amfTranium-Acet tEE(T%D; pl^=^7:i, R&wv' Rati&*(J.5nUynTln-.

'%K.

• = + — 3 l(p 1 ^

^Hettn:Tij m'^ " t ir^;^' i-iTi H"0)

CHAPTER IV

CONCLUSIONS

p-Cyclodextrin bonded phases showed excellent ability to separate

structurally similar nicotinoids. The resolution of both homologous

series of compounds and structural Isomers was achieved. The retention

of the homologous compounds tended to increase as the size of the

homologue increased. In the case of the structural isomers, the

compounds with nitrogen in the para position were retained longer than

the meta isomers. This is the first reported separation of most of these

nicotine-related alkaloids.

p-Cyclodextrin complexation was used to resolve several racemic

alkaloids related to nicotine. The p-cyclodextrin bonded phases show

much greater efficiency than the technique using cyclodextrin as a chiral

mobile phase additive. Some of the alkaloids, such as N'-benzyl

nornicotine, showed retention and resolution minima on the

p-cyclodextrin bonded phases. These minima may indicate a change in

the mechanism of chiral recognition at the different mobile phase

compositions.

A mixture of tobacco alkaloids, and nicotine and Its metabolites

were chromatographed on the p-cyclodextrin bonded phase. This is the

first reported attempt to separate a complex mixture of nicotinoids. The

best separation was achieved with methanol as the organic modifier, and

ten of the twelve compounds were resolved. It was observed that

59

60

selectivity could be changed by adjusting the pH of the buffer, or by

using different organic modifiers.

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separation of alkaloid isomers and stereoisomers …

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