Rapid identification of ergot derivatives by 1H-NMR spectroscopy

Journal

Pergamon

Rapid identification spectroscopy ALAN

of Pharmaceutical

& Biomedical

Analysis. Vol. 12, No. 1, pp. 27-40, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0731-7085/94 $6.00 + 0.00

of ergot derivatives by ‘H-NMR

F. CASY*

School of Pharmacy and Pharmacology,

University of Bath, Claverton Down, Bath BA2 7AY, UK

Abstract: The 400 MHz ‘H-NMR spectra of some therapeutically important ergot derivatives (three bases, four protonated bases and four dihydroergoline salts) are analysed in terms of the low field chemical shift region (above 5 ppm), common resonances of rings C and D (below 5 ppm) and C-8 substituent features. Attention is drawn to data of specific analytical value, and a scheme for the rapid identification of members of this group of ergots proposed. Features which provide evidence of the solute conformation of ring D, and isomerization to less active C-8 epimers are also emphasized. Keywords:

Ergots

(ergolines);

dihydroergolines;

‘H-NMR.

Introduction ‘H-NMR Spectroscopy is a valuable technique for the identification of individual members of a closely related group of compounds with similar electronic, vibrational and other properties. Examples of the application of NMR in this regard are differentiation of the penicillin, cephalosporin and tetracycline families of antibiotics [l-3]. Ergot (ergoline) derivatives, both natural and synthetic variants, likewise form a group which presents problems of specific identification. This paper presents a review of reports on the NMR features of the group and extends it to include most derivatives in therapeutic use [4]. Data of specific analytical value are highlighted; in addition features that provide evidence of stereochemistry (notably the conformation of ring D) and isomerization to less active C-S epimers are emphasized. The described analyses concern first common features of the ergoline nucleus 1 (low field: aromatic, vinylic and acidic protons above 5 ppm: alicyclic ring protons), and secondly those of the C-8 substituent (peptidebased moiety or simple function as in ergometrine I and methysergide 2) which primarily carry specificity (Structure 1 and 2). Literature reports on ergolines refer exclusively to free bases [5-81 whose ‘H-NMR spectra are generally well resolved, as confirmed in the further examples of this paper. *Correspondence

to 5 Piplar

Ground,

Structures

1

R=%-ICH@Ie)CIQOH

2

R=NHCI3(33Y3$OH with H-N’ rsplaced by M-N’

1 and 2

Data on protonated bases, the usual therapeutic form of ergots, make up the bulk of the present results. With the exception of certain C-S function and aromatic signals, broad resonances often of poor resolution were encountered. These features of salt spectra limited signal assignment in many cases but, as will be shown, did not preclude the designation of diagnostic points. The broad nature of resonances due to alicyclic ring protons of ergoline salts may be a consequence of conformational equilibria set up as a result of proton exchange at 6-N and/or changes in conformation about the C-8 amido bond (less probable: single COR features were observed, mostly sharply resolved). Spectral assignments shown in Tables l-3 were aided by previous analyses of lysergic acid diethylamide (220 MHz) [6] and ergotamine of (270 MHz) [7] made by consideration chemical shifts and coupling constants

Bradford-on-Avon

BA15

27

lXF, UK.

in DMSO-d,

Ergocryptine

in DMSO-d,

in CDC&NaOD

(3~) in CDC13

(3b) in CDCll

work tartrate

Ergocristine

Present

(3a) in CDC13 270 MHz [7)

bases”

(Y.21)d

8.05 brs’

(9.18)”

8.03 brs’

not seen

8.14 d (
I-NH

of some ergoline

Ergotamine

characteristics

and solvent

Spectral

Compound

Table 1 ‘H-NMR

(10.74)d

9.82 s

(10.74)d

9.7 s

not seen

9.04 s

20-NH

6.95-7.21

7.24 dd (7.5,<1) 7.19 t (7-8) 7.15 dd (-7,l)

7.02-7.35

m

m”

mh

mb

mb

7.13-7.57

7.1-7.5

7-7.5

12,13,14-H

Low field region

5 ppm)

within 6.9-7.3

6.94 brs’

within 7-7.4

6.94 brs’

6.94 d (-2)

6.91 dd (1.8.0.5)

2-H

(above

6.22 brs 6.45 m’

6.39 dd (6.--l)

6.3 brs 6.5 brd’

6.4 brd (5.8)

6.37 dd (-632)

6.34 dd (5.5,2.0)

Y-H

7.45 brs

(1.5)

7.38 d

within 7-7.4

within 7.46-7.57

not seen

6.91 s

l5’-OH

NMR SPECTROSCOPY

OF ERGOT DERIVATIVES

i

a

29

30

ALAN

F. CASY

NMR SPECTROSCOPY

OF ERGOT

DERIVATIVES

supported by spin-decoupling experiments. In this paper spectra were run at 400 MHz and analyses assisted by 2D ‘H-‘H correlation (COSY) plots. Salts were examined as solutions in DMSO-d6 and, when solubility allowed, D20. The former solvent allowed the detection of N-H and O-H resonances, both D20-exchangeable.

Materials and Methods ‘H-NMR Spectra were obtained on a JEOL GX 400 spectrometer. Samples of approximately 10 mg were dissolved in the appropriate solvent (0.5 ml) and examined without degassing at the ambient probe temperature (20°C) and employing the standard conditions of 32K data points with digital resolution of 0.18 Hz per point [9]. The homonuclear ‘H-‘H chemical shift correlated 2D plots were obtained using the standard COSY-45 pulse sequence [lo]. Solvents employed were CDC13 and CDsOD for bases, and DMSO-de, CD30D and DzO for salts. TMS served as reference in all cases except that of DzO when DSS was used. Samples of bromocriptine mesylate, dihydroergotamine mesylate, ergotamine methysergide maleate and ergotartrate, metrine maleate were supplied by Sandoz Pharmaceuticals, and pergolide mesylate by Lilly Research Laboratories. The Home Office Forensic Science Service provided samples of dihydroergocornine and dihydroergocristine mesylates. Ergocryptine and ergocristine bases were purchased from Sigma.

Results and Discussion

Free base ergolines The ‘H-NMR features ergots, ergotamine 3a, ergocristine 3b (Structure

of three C-8 peptidic ergocryptine 3c and 3) are given in Table

31 1. It was possible to identify l-H(NH) and 2-H by a COSY cross-peak in spectra of ergocryptine (3~) and ergocristine (3b). The vinylic 9-H signal (broad resonance 6.34-6.4 ppm in CDC13, 6.22-6.3 ppm in DMSO-de) was accompanied by a minor signal to low field (near 6.5 ppm) in DMSO-d6 spectra, evidence that this solvent induces epimerization at C-8. Most aliphatic ring proton signals were resolved in all three base spectra and their multiplicities and “J coupling magnitudes were consistent with a pseudo-axial C-8 substituent half-chair conformation (4) (Structure 4) for ring D stabilized by an intramolecular hydrogen bond [7]. The coupling interactions of 5l3H and So-H include both allylic and homoallylic contributions [6, 71, and in consequence both resonances are characteristically broad (Fig. 1).

Structure 4 Base conformation.

C-8 Substituent features The 5’-H resonance was well defined in all spectra as a narrow triplet near 4.6 ppm, as were those of the benzyl methylene protons (AB of ABX system) of ergotamine (3a) and ergocristine (3b) (dd near 3.4 and 3.25 ppm). Methyl features distinguished ergotamine 3a (2’-Me, s near 1.5 ppm), ergocristine 3b (2’CHMe,, 2d near 1 ppm) and ergocryptine 3c (2’-CHMe, and 5’-CH,CHMe2, 4d near 1 ppm). The presence of the C-S epimer of ergocryptine was apparent from the minor 5’H resonance upfield of the major signal in the spectrum run in DMSO-d6.

Protonated ergolines

(a) (b) (c) (d) (e)

ergotamine ergocristine ergocryptine bromocriptine erogcornine

Structure

3

R,

R2

R?

H H H Br H

Me CHMez CHMe, CHMe, CHMe,

CHzPh CHzPh CH,CHMe> CH,CHMe2 CHMez

Ergometrine 1 and methysergide 2 maleates were the simplest cases examined since they do not carry peptidic units at C-S (Table 2). Only a single NH resonance (20-NH) was present in the spectrum of methysergide run in DMSO-d6 - the l-NH signal was replaced by an NMe singlet near 3.8 ppm additional to that of 6NMe. Aryl signal integrals distinguished ergotamine 3a tartrate (9, includes 5’-CH,Ph) and bromocriptine 3d mesylate (3, 2-H re-

in CD,OD

Bromocriptine

Ergotamine

in CD,OD

in DMSO-d,

in DMSO-d,

(3d) mesylate

(3a) tartrate

in DMSO-d,

-

7.15-7.26

7.06-7.27

9.5 s

11.8 s -

7.05-7.35

rnp

m

mh

7.01 d(4.5) 7.17-7.3 m

9.4 s

-

7.16-7.21 m 7.34 dd(6, 2)

X.14 d(7.5)’

10.75 brs’

-

-

(2) maleate

m

7.13 brs 7.23 m 7.39 dd(6, 2.5)

7.11-7.18 7.29 d(7)

12,13,14-H

Low field region .__

-

8.26 d(7)”

10.97 brs’

Methysergide

in DMSO-d,

bases)”

20-NH

salts (protonated

l-NH

of some ergoline

-

(1) maleate

-__

characteristics

and solvent .--__

Spectral

in DzO

Ergometrine

Compound

Table 2 ‘H-NMR 5 ppm)

-

-

near 7’

within 7.01-7.3

within 7.16-7.21

within 7.13-7.23

within 7.11-7.2’

2-H

(above

ionq

6.56 brs

6.42 brs

6.57 brs 6.72 brs’

6.62 brs

3.24 s mesylate

3.37 s mesylate

4.24 s tartrate

6.24 s maleate

6.05 s maleate

6.55 brs

6.05 s maleate

Counter

6.21 s maleate

-~~

.~_

6.48 brs

6.52 brs

9-H ___

___

within 3.68-3.9 within 3.75 m

within 3.84 m

3.41 brt (-10)

3.55 t(12)

within 3.75 m

in D,O

Methysergide in DMSO-d,

in CD,OD

Bromocriptine in DMSO-d,

Ergotamine in DMSO-d,

in CDaOD

(3d) mesylate

(3a) tartrate

(2) maleate

3.75 t(ll-12)

\

\

within 3.6-3.8

Ergometrine in DMSO-d,

(1) maleate

3.5 mr

and solvent

4B-H

Compound

4~H

Table 2 Continued

m

m

3.24 brs

4.1 brm

3.1 m’

3.12 s 3.8 s ( I-NMe)

3.08 s 3.8 s (I-NMe)

3.11 brs

3.06 brs

3.17 brs

_~

m

m

m

m

l

3.6 brdd (-12,3)’

3.5 dd(12,-5)c

within 3.15 m’

within 3.75 mc

within 3.68-3.9

within 3.6-3.8

2.87 t(12.5)

2.8 t(-12)’

2.58 t(--I1)‘.k

2.94 t(- 12)’

2.83 t(12)

2.88 brt (- 12)

2.85 t(-1oy

of rings C and D (below 5 ppm) .____~ _ 7a-H 7B-H 6-NMe

resonances

4.08 brm

l

within 3.84 m

within 3.15 m

within 3.68-3.9

within 3.6-3.8

5B-H

Common

4.37 brsr

4.4 brs”

3.6 (?) mc+

4.lY brs”

4.25 brs’

4.01 br+”

4.25 brs’+

801-H

-

in

1.0 t’ 0.82 d(6.7)‘ I. 17 d(6.7)’

3.6 m

1.51.1.68 m(X-line)

cross-peak

4.55 t(6-7)

4.36 t(-7)’

13’.CH,CHMe, I .6-iXm’

I.8 m

4.51 t(5-6)

-

5’.H

m’

4.01 m (overlaps Xa-H)

2O-NHClJMe 3.84 m’

Other ,ignals

3.5 m’

3.1 m

1.95 m’

\

2.33 m’

I .9 mc

3.85 t(6-7)’

3.55-3.75

CHzOH 4.83 rnT

I .9S m(2’)‘ 2.24 m(5’)’

2’-CHMe, + 5’.CH?CHMe, _ I A53.3 m‘

3.X4 m

3.75 t(7.5)’

111-H

-

1.9 m’

IV-H

2O-NHCHEt 3.69 m

1.75 m

9’.H

-

-

X,-H

C-8 Subhtituent features

I3’-CH,Ph 3.1 m 3.2 dd(12.5,6)

by COSY

2WNHCHEtCH,OH 3.5 m A.

2WNHCHCH,Me I .38,1.62 m(8:line)

3.55 dd(l I .3,6.4) 3.64 dd(ll.5,4.8)

2@NHCHMeCH,OH 3.4 m(8-line)c-e

CH, signals

“Footnotes of Table 1 apply. “Absent after DzO exchange; linked to CH2 of C-8 substituent ?he integral of 3 is relative to that of the 9-H resonance (1). 4Mesylate and tartrate resonances fall below 5 ppm. ‘3.55 t(-10) after D,O. ‘Unresolved. ‘Overlaps with the 13’-CHZ signal. “8a-H and CHMe signals overlap.

CD?OD

2’.CHMe, + YCH,CtiMe’ 0.79 d(6.4)0.92 d(6.8j 0.95 d(7.3) I .O8 d(6.8)

Bromocriptine mesylate in DMSOpd,

(3d)

2’-Me 1.5s I .49 5’

t(overlap)

0.97JI.99

Ergotamine (3a) tartrate in DMSO-de

in CD,OD

20.NHCHCH,Me -0.87 t(7.3)

(2)

Methysergide maleate in DMSO-d,

20.NHCHMe I .09 d(6.7y

Me signals

1.16 d(6.7)

DMSO-d,,

(1)

and

in DzO

in

maleate

Ergometrine

Compound solvent

Table 2 Continued

NMR SPECTROSCOPY

OF ERGOT

Figure 1 Part of the 400 MHz’H-NMR

spectrum

35

DERIVATIVES

of ergocryptine

base 3c in CDCI,.

placed by Br) from other members of this group. Although resolution of ring C-D proton signals of salts was inferior to that of the Table 1 bases, it proved possible to assign many individual resonances by the aid of COSY plots and other means. The 7@-H signals (mostly 2.8-2.9 ppm) were generally well resolved; their appearance revealed a strong coupling (-12 Hz) to 8~H indicative of preference for a pseudo-equatorial 8-R half chair conformation of ring D (5) (Structure 5). The SWH resonances near 4 ppm were characteristically broad (the 4P-H signal was obscured by overlap with that of 5P-H). 5

Structure 5 Ring D conformation

shift scale is ppm from TMS.

maleate. In the peptidic examples 3a and 3d, characterized by 5’-H triplet signals near 4.5 ppm, 2’ (Me for 3a, CHMe* for 3d) and 5’ (CH2Ph for 3a, CH2CHMe2 for 3d) spectral features were all resolved. Signals due to ring G (pyrrolidino protons) of 3a and 3d, absent from spectra of the non-peptidic examples, were assignable to various multiplet resonances (8’, 11’ near 3 and 3.8, respectively, 9’, 10’ 1.9-2.33 ppm). Dihydroergoline protonated bases The absence of a vinylic proton resonance near 6.5 ppm in spectra of the examples given in Table 3 immediately revealed them as 9,10dihydroergoline derivatives 6 (Structure 6). Integrals of the aryl proton band differentiated pergolide 7 (Structure 7) and dihydrocornine

R

C--

for salts.

C-8 Substituent features COSY plots clearly delineated the C-8 features of ergometrine 1 maleate (the 8-line CHCH,OH signal was specially well resolved in the D20 spectrum), and methysergide

The chemical

I

6 Structures 6 and 7 For structure 6: (a)-(e)

as for 3.

10.9 s

(6b) mesylate

(6e) mesylate

dihydroergocristine in DMSO-d,

Dihydroergocornine in DMSO-d, 10.9 s

-

11.94 brs

11.04 brs

in Da0

(6a) mesylate

(7) mesylate

l-NW

of some dihvdroergoline

in DMSO-d,

characteristics

and solvent

Soectral

Dihydroergotamine in DMSO-d,

Pergoiide

Compound

Table 3 ‘H-NMR salts’

9.25 s

9.3 s

-

9.6 brs

20-NH

(above

5 ppm)

lpr

brs lpr tf-7) 2pr t(-7) 2pr d(7.9) 1 pr d(7.9) 2 pr

H-12.14: 6.83 d(7). 7.22 d(7.9) H-13: 7.09 t(7-8) _~_~~

6.86 d(7) Ipr 7.06-7.28 m 8pr

6.96 7.04 7.09 7.17 7.23

6.54 d(7.2)

H-12 or 14: 6.9 d(7) 7. 13-7.3h

H-12,14: 7.0 d(7). 7.36 d(7.9) H-13: 7.23 t(7.9)

12,13.14-H

Low field region

..-.

7.16 brs

within 7.06-7.28

6.96-1.23

within

within 7.13-7.3

7.25 brs

2-H - .._____.

7.16

10.0 brs

-

Y.95 brs 6.42 s

9.9 brs

~__. Other acidic-H

and solvent

Dihydroergocornine in DMSO-d,

(6e)

Dihydroergocristine (6b) mesylate in DMSO-d,

in DzO

Dihydroergotamine (6a) mesylate in DMSO-d6

Pergolide (7) mesylate in DMSO-d,

Compound

Table 3 Continued

5

\

b

5

(3.7)d brm

(3.72)d dd (lOJ.5)

5

5

I

\

(3.7)d brm

s

G;h;

5P-H

3.3-3.5

(3.69)d brd(l1)

4P-H

within 2pr m 2.9-3.1

4a-H

3.0 brs

3.0 brs

(2.74)d s

3.03 s

(6.Pr”)”

6-Me

Common

’

(3.5)d m’

(3.3-3.6)“ m”

(3.58)d brd(-11)

(3.58)d brd(14)

70(-H

resonances

\

’

’

2.8-3.5’

within 2pr m 2.9-3.1

7@-H

\

base of 2.4 Me signal

8&-H

l

\

\

l

2.85 brd( 14)

9a-H

of rings C and D (below 5 ppm)

-1.6 brq(-12)

1.61 brq(-12.5)

within m 1.75-2.2

1.62 brq(-11)

1.4 brq(-14)

9P-H

’

’

*

’

within 4pr m 3.3-3.5

lOa-H

2.43 s

2.34 s

(2.73)d s

2.33 s

2.41 s

Me of mesylate

2’-Me 1.53s

2’-CH&? O.Y2,1.05 d(6.7)

(6b)

(6e)

Dihydroergocristine in DMSO-d,

Dihydroergocornine in DMSO-d,

4.56 t(4.5)

13’.CH,Ph 3.05?@14,7) 3.15 dd(I4,4)

a 4pr m 3.3-3.5

4.36 d(3)

4.56 t(6)

4.52 t(5.5)

5’.H

3.05 dd(14.6) 3.26 dd(14.7)

13’~CH?Ph 3.07 dd(-14,5)” 3.18 dd(-14.7)

X-CHC&$ 2.58 dd(13,7) 2.69 dd(13,5)

CH2 signals

“Footnotes of Tables I and 2 apply. u 6-NPr”: Me I .03 t(7); MeCH, I .7-l .85 m; NmL within “The higher field doublet ismasked by other resonances.

2’-CHm: + 5’.CH&? I .OO d(6.7) I .05 d(7.0) 1. IO d(7.0) 1. I6 d(6.7)

1.52 s

in DzO

(6a)

Dihydroergotamine in DMSO-d,

Me signals

S-& 2.4 s

and solvent

Pergolide (7) mesylate in DMSO-d,

Compound

Table 3 Continued

’

’

3.35-3.48

3.2-3.5

8’-H

m

C-S Substituent

m

within I .8-2.2

within 1.7-2.1

1.8-2.0

1.7-I .8.

within

within 1.7-1.8, I .8-2.0

-

9’-H

features

m

m

m

m

within I .8-2.2

within 1.7-2.1

within Y’-H signals

within O’-H signals

-

;O’-H

m

m

3.82 dd(9.6)

3.8 t(-7)

3.72 dd(lO,5.5)

t(-7)

3.75

I II-H

signals

2’-CHMeZ 2.22 m(6.7) 5’.CHMe? 2.48%

2’-CHMe? 2.1 m(6.7)

-

Other

NMR SPECTROSCOPY

OF ERGOT

6e (both 4) from dihydroergotamine 6a and dihydrocristine 6b (both 9). The spectrum of the non-peptidic example pergolide 7 was best resolved in regard to ring C-D proton signals. Entry to its analysis was gained by assignment of C&I&S (a typical 8-line signal) which led via COSY cross-peaks to &x-H and other resonances. The 9P-H signal near 1.4 ppm was unique in displaying three large couplings (producing an apparent quartet) consistent with a pseudo-eq-S-CH,SMe ring D chair conformation 8 (Structure 8) for this dihydroergoline derivative (in dihydroergolines with bulky 6-N substituents a twist-boat conformation of ring D is favoured [8]). The low field position of the signal due to its geminal partner (9~H) may be attributed to aromatic deshielding. Ring C-D assignments are in accord with those made for festuclavine base (6-NMe, SMe analogue of 7) once allowance for Nprotonation effects are made [5]. Proton signals below 4 ppm were poorly resolved in all peptidic dihydroergoline spectra due to extensive overlap of ring C-D and pyrrolidino (ring G) resonances. However, all spectra revealed an apparent quartet near 1.5 ppm of separations -12 Hz assigned to 9P-H.

a Structure

39

DERIVATIVES

8

C-8 Features The presence of a peptidic C-S substituent in derivatives 6a, b and e was revealed by observation of a .5’-H resonance near 4.5 ppm in their spectra; signal multiplicities (t for 6a and 6b, d for 6e) served to pin-point dihydroergocornine (5’-CHMeJ. The S-line signal diagnostic of 5’-C&Ph was clearly resolved in spectra of dihydroergotamine 6a and dihydroergocristine 6b, as were 2’ and 5’ methyl features (2’-Me for 6a, 2’-CHMe2 for 6b, 2’and 5’-CHMe2 for 6e). COSY cross-peaks allowed assignment of other features of the C-8 substituent. Proposed diagnostic scheme Once there is reason to suspect an analyte to be an ergot, e.g. by its positive response in the coloration reaction with p-dimethylaminobenzaldehyde [ll, 121, examination of the pattern

of the low field region of its ‘H-NMR spectrum run in DMSO-dh will confirm the presence of an ergoline skeleton (moving from low to high field: two 1 pr singlets, multiplet of integral 3 or more, 1 pr singlet). Absence of the highest field 1 pr singlet of this group near 6.5 ppm (due to the 9-H vinylic proton) reveals a dihydroergoline derivative. Only three low field features of this kind are seen in the spectrum of methysergide 2 (l-NH + l-NMe). C-8 Substituent evidence. The next point to check is the presence or absence of a 1 pr resonance near 4.5 ppm (usually a narrow triplet). Such signals are due to 5’-H of a peptide-based C-8 substituent and take the form of triplet in spectra of 3/6 a-d and doublets in those of the ergocornines 3e and 6e. The count of high field Me doublet signals near 1 ppm differentiates ergocryptine 3c and bromocriptine 3d (both 4) from ergocristine 3b (2) and ergotamine 3a (nil). Identities of the last two examples are confirmed by additional C-8 substituent signals (8-line 5’-C&Ph resonance within 3.0-3.5 ppm for 3a and 3b, 2’-Me singlet near 1.5 ppm for 3a). Dihydroanalogues 6a-d may be differentiated similarly from their spectra. Careful integration of the aromatic signal near 7.0 ppm serves to distinguish ergocryptine (integral 4) from its 2bromo congener 3d (integral 3). C-8 Spectral features of the non-peptidic derivatives readily identify ergometrine 1 (Me doublet near 1.1 ppm, 8-line C&OH signal near 3.5 ppm) and methysergide 2 (Me triplet near 0.9 ppm, CEMe resolved 1 pr multiplets near 1.4 and 1.6 ppm). The dihydro-derivative pergolide 7 is best characterized by its 6NCH,CH,Me (m and t near 1.8 and 1.0, -_ respectively) and S-Me (s 2.4 ppm) resonances.

Acknowledgements - Thanks are due to Sandqz Pharmaceuticals (Dr W.S. Phillips), Lilly Research Laboratories (T.L. Jeatran) and the Home Office Forensic Science Service (Dr D. Osselton) for generous provision of the ergots detailed under “Materials and Methods”, and Mews R. Hartell and D. Wood for recording the spectra.

References [l] S.K. Branch, A.F. Casy and E.M.A. Ominde, J. Pharm. Biomed. Anal. 5, 73-103 (1987). [2] S.K. Branch and A.F. Casy, Prog. Med. Chem. 26, 355-436 (1989). [3] A.F. Casy and A. Yasin, J. Pharm. Biomed. Anal. 3, 281-292 (1983). [4] Martindale: The Extra Pharmacopoeia, 29 edn, pp.

40

[5]

[6] [7] [8]

ALAN

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F. CASY

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