Chapter 9 Ergot Alkaloids

357

Chapter 9 ERGOT ALKALOIDS

9.1. Reversed-phase HPLC ...............................................................

9.2. I o n - p a i r HPLC.....................................................................

............................................................. ......................................................................... .............................................................................

9.3. Straight-phase HPLC.. 9.4. Detection References

357 362 362 366 368

The e r g o t a l k a l o i d s can be d i v i d e d i n t o two main groups: the l y s e r g i c a c i d a l k a l o i d s and the c l a v i n e a l k a l o i d s . The a n a l y t i c a l problems concerning the l y s e r g i c a c i d a l k a l o i d s can be summarized as f o l l o w s :

1. Analysis o f LSD and r e l a t e d compounds.

2. Separation o f ergometrine and the ergotamine and the ergotoxine groups o f a l k a l o i d s . 3. Separation o f the i n d i v i d u a l a l k a l o i d s w i t h i n each o f the groups mentioned.

4. Separation o f t h e C-8 stereoisomers ( - i n e / i n i n e ,

normal/iso-).

5. Separation o f t h e dihydroderivatives of the ergotoxine group o f a l k a l o i d s and ergotamine. For each o f the separations mentioned, HPLC has been used. The analysis o f LSO i s sometimes described i n connection w i t h t h e analysis o f drugs o f Reviews on HPLC analysis o f LSD have been given16’49959. L u r i e and Weber32’49 described a semipreparative HPLC separation o f LSD t o enable f u r t h e r i d e n t i f i c a t i o n o f i t by means of d i f f e r e n t spectral data. T w i t c h e t t e t a1.28 reported the analysis o f LSD i n body f l u i d s by means o f a combination o f HPLC, f l u o r i m e t r y and radioimnuno-assay. The separation o f the n a t u r a l l y occurring e r g o t a l k a l o i d s derived from l y s e r g i c a c i d i n groups has been i n v e s t i g a t e d i n a long series o f studies

19,25,34,35,36,41.43.50,52,57,58,60,61

and so has the separation o f the various components w i t h i n each group, p a r t i c u l a r l y w i t h i n the ergotoxine group 25.34s36,41,43’51’60161.The C-8 stereoisomers can u s u a l l y be separated w i t h o u t d i f f i c u l t y 10.14,17 9 19~25~26,34,36,41~43947,51~52960~61~ t h e separation of t h e d i hydrod e r i v a t i v e s of the ergotoxine group of a l k a l o i d s has a l s o been achieved6g11*19g25~33r34g41s60~

61, S t a b i l i t y studies o f ergotamine17, ergometrine31 and d i h y d r ~ e r g o t a m i n ehave ~ ~ been p e r f o r med using HPLC f o r the separation o f the a l k a l o i d and i t s degradation products. Schauwecker e t a1.22 developed a method f o r the t r a c e enrichment o f ppb q u a n t i t i e s o f e r g o t a l k a l o i d s i n urine. al.

HPLC analysis o f c l a v i n e a l k a l o i d s has been c a r r i e d o u t by Wurst e t a1.29 and Eckers e t 57,58

9.1.

REVERSED-PHASE HPLC Reversed-phase HPLC has been widely used f o r the analysis o f both LSD and e r g o t a l k a l o i d s .

The f i r s t reversed-phase separation o f e r g o t a l k a l o i d s was reported by Jane and Wheals3 i n connection w i t h the analysis o f LSD. P e l l i c u l a r beads w i t h chemically bonded octadecyl groups were used i n combination w i t h methanol

-

0.1% aqueous amnonium carbonate (3:2) as mobile phase.

However, the mobile phase proposed by V i v i l e c c h i a e t a1.6 f o r the separation o f the dihydroergotoxine a1 kaloids on octadecyl columns ( a c e t o n i t r i l e

Relerrncu p. 3138

-

aqueous amnonium carbonate) has been

368 TABLE 9.1 CAPACITY FACTORS ( k ' ) AN0 SEPARATION FACTORS FOR ERGOT ALKALOIDS ON REVERSED-PHASE PACKINGS" '34 Co1umn:Lichrosorb RP2 (5 p m ) , RP8 (10 pin) o r RP18 (10 pm)(250x2 o r 4 mn 10). mobile phase a c e t o n i t r i l e - 0.01M aqueous amonium carbonate (2:3), d e t e c t i o n UV 320 o r 280 nm. A1 k a l o i ds

Lichrosorb RP2 (I

Lysergic a c i d Ergometri ne ma1eate Ergometrini ne Ergotamine t a r t r a t e

0.16

Lichrosorb RP8 Lichrosorb RP18 k' (I k (I 0.20

0.10

:::: :::: :::::

2.11 3.21 1.46 1.37 1.10

9*87 12.91 14.50

1.53 1.12

1.47

7.15 9.11 9.82

1.27

1*54 2'27 5.34

Ergocrypti ne Ergocornine

67.8 *24

Ergocri s t i n e Ergotami n i ne Ergocorninine Ergocrypti nine Dihydroergotamine t a r t r a t e Oihydroergocornine methanesulfonate Oihydroergocryptine methanesulfonate O i hydroergocristine methanesul fonate

8.45

1.47 2.35

1.08

1.08

''11

16:66

9.43

1.75

1.44

::;;

OS8'

20.50

::::!

2.27 3.95 1.63 1.57 1.10

29.6

1.86

6":;

~~:~~ 1.10 o:;i

7.94 9.7

1.58

1.50 1.20

TABLE 9.2 SEPARATION OF ERGOTAMINE AN0 ITS DECOMPOSITION R e l a t i v e capacity f a c t o r s (k;=k;/kh) o f decomposition product [ ( r e l a t i v e t o ergotamine E) Column pBondapak C18 (300x4 m ID), mobile phase a c e t o n i t r i l e - 0.01M aqueous ammonium carbonate, l i n e a r gradient i n 15 min from 3:37 t o 1:1, f l o w r a t e 8.0 ml/min, d e t e c t i o n UV 320 nm. Alkaloid

kb

Rel. standard d e v i a t i o n [%I 0.3 0.4 0.3 1.0

Ergotaminine 1.18 Aci -ergotami ne 0.77 0.98 Aci-ergotaminine Lysergic a c i d amide 0.45 I s o l y s e r g i c a c i d amide 0.61 Lysergic a c i d 0.08 Isolysergic acid 0.18

0.8 1.2 1.5

TABLE 9.3 SEPERATION OF SOME ERGOT Column Lichrosorb RP18. 10 pm (250x4 mn ID), mobile phase 0.1% ammonium acetate i n aceton i t r i l e - water (35:65). f l o w r a t e 3.5 ml/min, d e t e c t i o n UV 254 nm. A1 kal oids

Retention time(min)

Ergometrine Ergometri n i ne Papaverine(interna1 standard) Ergosi ne Ergotami ne Ergocorni ne Ergocrypti ne Ergocri s t i n e Ergotami n i ne Ergocorninine Ergocryptinine Ergocristinine

1.6 2.4 4.0 6.2 6.6 10.0 11.4 16.2 27.0 42.0 48.0 56.0

369

TABLE 9.4

SEPARATION

OF ERGOTOXINES

___________

AND OIHYDROERGOTOXINES~~ _______~

~~~

~

A1 k a l o i d

~

k‘

Ergocornine O i h y d r o e r g o c o r n i ne a-Ergocryptine D i h y d r o - a - e r g o c r y p t i ne 8 - E r g o c r y p t i ne Dihydro-B-ergocryptine

4.22 4.38 5.00 5.44 5.78 5.91

Column L i c h r o s o r b RP18 10 pm ( 2 5 0 ~ 4 . 6 mm I D ) , m o b i l e phase t e t r a h y d r o f u r a n ammonium a c e t a t e ( 2 : 3 ) , f l o w r a t e 1.5 ml/min, d e t e c t i o n UV 322 nm (Fig.9.5).

-

0 . 0 1 M aqueous

found t o have w i d e r a p p l i c a t i o n s f o r t h e s e p a r a t i o n o f e r g o t a l k a l o i d s . The s o l v e n t was t e s t e d by D o l i n a r ”

and Szepesy e t a1 .25934 i n c o m b i n a t i o n w i t h d i f f e r e n t reversed-phase packings.

O o l i n a r found t h a t t h e p a c k i n g w i t h c h e m i c a l l y bonded o c t y l groups was t h e most s u i t a b l e s t a t i o n a r y phase f o r t h e s e p a r a t i o n o f e r g o t a l k a l o i d s w i t h a wide range o f p o l a r i t i e s (Fig.9.1). The s e p a r a t i o n s o b t a i n e d on an o c t a d e c y l column were s i m i l a r ( F i g . 9 . 2 ) . column (RP2) and a s p h e r i c a l o c t a d e c y l column ( S p h e r i s o r b ODS),

On a d i m e t h y l s i l y l

t h e less p o l a r a l k a l o i d s were

n o t as w e l l s e p a r a t e d as on t h e above-mentioned columns; however, t h e more p o l a r a l k a l o i d s were b e t t e r separated ( F i 9 . 9 . 3 ) . Szepesy e t a 1 . 2 5 * 3 4 i n v e s t i g a t e d t h e e f f e c t o f t h e c h a i n l e n g t h o f t h e c h e m i c a l l y bonded a l k y l groups on t h e s e p a r a t i o n o f t h e a l k a l o i d s . B e s t r e s u l t s f o r t h e e r g o t o x i n e and d i h y d r o e r g o t o x i n e groups of a l k a l o i d s were o b t a i n e d w i t h t h e o c t a d e c y l s t a t i o n a r y phase. An i n c r e a s e o f c h a i n l e n g t h o f t h e alkanes caused an i n c r e a s e o f t h e c a p a c i t y f a c t o r s o f t h e a l k a l o i d s and, t o some e x t e n t , a l s o an i n c r e a s e i n t h e s e l e c t i v i t y ( T a b l e 9 . 1 ) . The f o u r a l k a l o i d s o f t h e e r g o t o x i n e group, as w e l l as t h r e e d i h y d r o e r g o t o x i n e a l k a l o i d s , c o u l d be separated. S i m i l a r sys tems have a1 s o been used i n o t h e r inves t i g a t i o n s l 7 * 18’21’22937’39950. F o r t h e q u a n t i t a t i v e a n a l y s i s o f ergotamine and i t s decomposition p r o d u c t s ( T a b l e 9.2). Bethke e t a1 , I 7 f o u n d t h a t a l i n e a r g r a d i e n t was necessary when a c e t o n i t r i l e

-

aqueous amno-

nium carbonate was used as m o b i l e phase. A s t e p w i s e g r a d i e n t as used b y E r n i and F r e i 1 8 c o u l d be used, b u t some problems arose w i t h t h e q u a n t i t a t i o n o f t h e chromatogram. However. t h e s t e p wise gradient procedure o f f e r e d p o s s i b i l i t i e s f o r the analysis o f very low concentrations o f d i h y d r o e r g o t o x i n e a l k a l o i d s (ppb r a n g e ) . I n j e c t i o n volumes o f up t o s e v e r a l hundred m i l l i l i t r e s c o u l d be a p p l i e d : t h e a l k a l o i d s were adsorbed on t h e t o p o f t h e column and t h e y were subseq u e n t l y e l u t e d by means o f t h e s t e p w i s e W e h r l i e t a l .24 i n v e s t i g a t e d t h e i n f l u e n c e o f o r g a n i c bases ( p r i m a r y , secondary. t e r t i a r y and q u a t e r n a r y amines) on t h e s t a b i l i t y o f c h e m i c a l l y bonded reversed-phase m a t e r i a l s . I t was found t h a t w i t h sodium h y d r o x i d e and q u a t e r n a r y amines, t h e s i l i c a t e s t r u c t u r e o f t h e p a c k i n g was r a p i d l y a t t a c k e d , making columns u s e l e s s w i t h i n 1-3 days. Decomposition o f such m a t e r i a l s i s m a i n l y due t o d i s s o l u t i o n o f t h e s i l i c a g e l . However, when w o r k i n g w i t h h i g h pH values, p r i m a r y , secondary and t e r t i a r y a l k y l a m i n e s can be used w i t h o u t a n o t i c e a b l e decrease i n t h e q u a l i t y o f t h e column. T r i e t h y l a r n i n e was found t o be a good choice. I n c r e a s e i n t h e w a t e r content o f the eluents tested ( a c e t o n i t r i l e

-

water

-

base) gave an i n c r e a s e i n t h e d i s s o l u -

t i o n o f t h e s i l i c a g e l . F o r t h e a n a l y s i s of e r g o t a l k a l o i d s . a c e t o n i t r i l e was p r e f e r r e d above methanol because o f a b e t t e r s e l e c t i v i t y , a h i g h e r p l a t e number, and a l o w e r p r e s s u r e drop. The v a r i o u s amines t e s t e d were compared f o r t h e i r i n f l u e n c e on t h e r e t e n t i o n b e h a v i o u r o f t h e

Referencea p. 368

360 TABLE 9.5 SEPARATION OF ERGOT ALKALOIDS AND LSDZ8 S1: column Spherisorb 5-ODS ( 1 0 0 ~ 4 . 6mn ID), mobile phase methanol - 0.025M disodium hydrogen phosphate (65:35)(pH 8). f l o w r a t e 1.0 ml/min. S2: column Spherisorb S5W (150~4.6 mn ID), mobile phase methanol - aqueous ammonium n i t r a t e (3:2), f l o w r a t e 1 ml/min. Detection UV 280 nm o r fluorescence ( e x c i t a t i o n 320 nm, emission 400 nm).

s1

A1 k a l o i d

s1

s2

l.OO(4.9ml) 2.04 0.38 0.23 0.61 0.53 1.15 1.68 2-0xo-LSD 0.65 D-Lysergic a c i d monoethylamide 0.52 D i hydroergocorni ne 2.60 D i hydroergocri s t i n e 3.68 D i hydroergocryptine 2.63

D-LSD Iso-LSD 0-Lysergami de D-Lysergi c a c i d Lysergol Lumi-LSD

1.00 (3.4ml) 1.28 2.76 0.65 0.76 0.85 0.59

0.81

0.74 0.76 0.71

D i hydroergotami ne Ergocorni ne Ergocri s t i n e Ergocrypti ne Ergocrypti nine Ergometri ne Ergometri nine Ergosi ne Ergosinine Ergotamine Ergothioneine Methysergi de Methylergometrine

s2

0.81

2.48 1.39 2.31 1.86 2.08 0.26 0.47 1.22 1.22 1.57 >6 0.65 0.39

0.79

0.81

0.78 0.72 0.79 0.79 0.78 0.75 0.81 >2.35 0.85 0.76

~~

ergot alkaloids. Hartmann e t a1 .33 found t h a t some reversed-phase columns showed i n s u f f i c i e n t s t a b i l i t y against a mobile phase such as a c e t o n i t r i l e

-

aqueous ammonium carbonate. However, by r e -

p l a c i n g amnonium carbonate by a 0.33 M phosphate b u f f e r (pH 7.5) s i m i l a r r e s u l t s could be obtained for the separation o f the dihydroergotoxine a l k a l o i d s , and the problem concerning the i n s t a b i l i t y o f the s t a t i o n a r y phase was avoided. S i m i l a r r e s u l t s were a l s o obtained w i t h triethylamine. A mobile phase c o n s i s t i n g o f water

-

acetonitrile

-

t r i e t h y l a m i n e (32:8:1)

and an octadecyl column gave a complete r e s o l u t i o n o f a l l f o u r dihydroergotoxine a l k a l o i d s (Fig.9.4).

A s i m i l a r method was applied by A l i and Strittmatter3’.

-

(35:65) containing 0.1% ammonium acetate and tetrahydrofuran

Acetonitrile

-

water

0.01 M amnonium acetate (3:2)

have been used i n combination w i t h octadecyl columns t o separate, respectively. various e r g o t a l k a l o i d s (Table 9.3)36 and some ergotoxine-type a l k a l o i d s (Fig.9.5. Table 9.4) 60 . To avoid decomposition o f reversed-phase co\umn m a t e r i a l by b a s i c solvents. Sondack31 preferred acetonitrile

-

acetic acid

- water

(30:1:79)

i n combination w i t h an octadecyl s t a t i o -

nary phase t o separate ergometrine and i t s decomposition products. T w i t c h e t t and co-workers (12,27,28.46)

found t h a t a mobile phase c o n s i s t i n g o f methanol

- 0.025

M aqueous disodium

hydrogen phosphate (65:35)(pH 8) i n combination w i t h an octadecyl column was b e t t e r s u i t e d f o r the analysis o f LSD than a normal-phase (Table 9.5). I n connection w i t h the development o f a post-column reactor, G f e l l e r e t a1.42 used a s t a t i o n a r y phase w i t h chemically bonded d i o l groups, which allowed the use o f e x c l u s i v e l y aqueous mobile phases (0.01 M, pH 3, phosphate b u f f e r ) . A s e r i e s o f a l k a l o i d s was separated, v i z . d i hydroergotamine and bromocryptine. Wurst e t a1 .29’43 reported the separation o f clavines and l y s e r g i c a c i d d e r i v a t i v e s on a s t a t i o n a r y phase containing alkylamine groups i n combination w i t h n e u t r a l organic solvents (Tables 9.6 and 9.7). For the clavines, the best separation was obtained w i t h d i e t h y l e t h e r

- ethanol

(84:16);

-

under i s o c r a t i c conditions

-

a g r a d i e n t e l u t i o n was, however, found

t o g i v e b e t t e r r e s u l t s . For the a l k a l o i d s derived from l y s e r g i c acid, d i e t h y l e t h e r

-

ethanol

361 TABLE 9.6 RELATIVE RETENTIONS

OF CLAVINE ALKALOIDS AND SIMPLE DERIVATIVES OF LYSERGIC ACID2’

Column Micropak NH , 10 pm (250x2 mn ID). f l o w r a t e 1 ml/min. d e t e c t i o n UV 225 and 240 nm. Mobile phase S1: d?ethyl e t h e r ethanol (84:16) S2: d i e t h y l e t h e r - ethanol (80:20) 53: d i e t h y l e t h e r isopropanol (70:30) 54: d i e t h y l e t h e r - isopropanol (60:40) S5: chloroform - isopropanol (9O:lO) S6: chloroform isopropanol (80:20)

-

-

A1 kal o i d Paspacl a v i ne I s o s e t o c l a v ine Lysergene Setoclavi ne I s o l y s e r g i c a c i d amide Lysergi ne Agroclavi ne Pyroclavine Festucl a v i ne Penniclavine Lysergic a c i d amide Pal ic l a v i n e Elymoclavine Lysergol Chanocl a v i ne r e t e n t i o n volume agroc l a v i n e (ml)

s1

s2

s3

54

55

S6

0.11 0.25 0.38 0.55 0.83 0.88 1.00 1.45 2.08 2.80 2.90 3.12 3.22 3.50

0.11 0.26 0.39 0.61

8.00

1.49 1.98 2.65 2.51 2.25 2.90 2.99 6.03

0.11 0.27 0.46 0.67 0.69 1.20 1.00 1.80 2.37 2.97 2.29 1.66 3.17 3.48 8.56

0.14 0.26 0.45 0.67 0.64 1.14 1.00 1.63 2.01 2.72 1.85 1.26 2.72 2.83 6.33

0.12 0.70 0.58 0.98 0.58 1.24 1.00 1.65 1.46 5.05 4.00 3.85 4.48 5.80 9.83

0.12 0.70 0.63 1.05 0.55 1.30 1.00 1.71 1.47 3.85 2.95 2.99 3.05 4.30 6.03

4.67

3.00

4.90

3.50

2.67

1.90

0.80

0.87 1.00

TABLE 9.7 RELATIVE RETENTIONS OF ERGOT ALKALOIDS43 Column Micropak NH , 10 pm (250x2 mn ID)(A) and Lichrosorb NH2. 10 r a t e 1 ml/min, d e t g c t i o n UV 310 nm. Mobile phase S1: d i e t h y l ether - ethanol (93:7) column B S2: d i e t h y l e t h e r ethanol (88:12) column A S3: d i e t h y l ether - ethanol (84:16) column A 54: d i e t h y l ether isopropanol (60:40) column A S5: chloroform isopropanol (9O:lO) column A

-

A1 k a l o i d Ergocryptine Ergocryptini ne Ergocorni ne Ergocorni nine Ergocristine Ergocri s t i n i n e Ergostine Ergostinine Ergosine Ergos in i ne Ergotami ne Ergotaminine Ergometrine Ergometri n i ne Lysergic a c i d amide I s o l y s e r g i c a c i d amide 8-Hydroxy-ergotamine Agroclavine r e t e n t i o n volume agroc l a v i n e (ml)

Rsferenca p. 368

-

s1

s2

53

54

55

2.82 1.59 3.06 2.00 3.76 2.35 7.38 3.15 8.47 3.09 12.44 4.56 12.82 3.82

0.50

0.47 0.21 0.56 0.30 0.78 0.38 1.30 0.57 1.21 0.46 1.94 0.88 4.16 1.00

0.31 0.14 0.32 0.18 0.45 0.26 0.82 0.37 0.71 0.28 1.08 0.52 2.55 0.57 1.85 0.64

0.13 0.03 0.12 0.05 0.15 0.04 0.33 0.05 0.47 0.07 0.53 0.07 4.93 0.98 4.00 0.58

-

0.25 0.64 0.34 0.89 0.45 1.85 0.75 1.83 0.63 2.25 0.94 5.12 1.05

2.90

8.88 1.00

-

0.83

1.00

1.00

1.00

1.00

1.10

6.00

4.67

3.50

2.67

pm

(250x2 mn IO)(B). f l o w

362

(88:12) o r (93:7) gave very good separation. However, i n this case a gradient e l u t i o n was a l s o preferred. The components of the ergotoxine group were best separated w i t h diethyl e t h e r ethanol (97.5:2.5). Harzer62 used a column switching technique t o confirm the i d e n t i t y of LSO. An octyl type of stationary phase and a straight-phase s i l i c a gel column were used i n combination with the solvent methanol - 0.3% potassium dihydrogen phosphate i n water (pH 3 ) ( 1 : 1 ) .

9.2. ION-PAIR HPLC Fluorimetric ion-pair chromatography has been applied f o r ergotamine, ergotaminine and dihydroergotamine i n connection w i t h the analysis of pharmaceutical preparations containing tropane a l k a l ~ i d s ~ ~ (Chapter ’ ~ ~ ’ ~4 )~. ’ ~ ~ Lurie described ion-pair chromatography of LSD. As pairing-ion heptanesulfonic acid (0.005 M ) ” or methanesulfonic acid (0.005 M)32’49 was used in a mobile phase of water - methanol a c e t i c acid (59:40:l)(pH 3.5) and a microparticulate octadecyl material as s t a t i o nary phase. For semipreparative work, the concentration of the pairing-ion was increased t o 0.04 M i n order t o reduce t a i l i n g and t o eliminate peak s p l i t t i n g ( see a l s o Chapter 7 ) 54-56 . Post column derivatization i n connection w i t h ion-pair chromatography was used by Lawrence e t al.48 f o r , e.g., ergotamine (Chapter 4 ) . Ali and Strittmatter3’ studied the separation of some dihydroergot alkaloids on reversed-phase columns (octadecyl and octyl) w i t h a c e t o n i t r i l e - water mixtures containing s a l t s ( c i t r a t e , acetate or bromide) as mobile phase. Best r e s u l t s were obtained with a mobile phase of pH 7 i n combination w i t h an octyl column. Under such conditions the dihydroergotoxine alkaloids and dihydroergotamine could be separated with three d i f f e r e n t salt-containing solvents (Table 9. 8 ) . In acidic media the separations were inadequate. The a- and B-isomers of dihydroergocrypt i n e could only be separated a t a h i g h pH (12.3) w i t h a c e t o n i t r i l e - methanol - diethylamine (375:65:21) as mobile phase, i . e . a solvent system s i m i l a r t o t h a t mentioned by Hartmann e t

.

a l . 33 A normal-phase ion-pair chromatographic method f o r the separation of ergot alkaloids has been reported by Szepesi e t ale6’. The behaviour of some alkaloids upon changes i n the d i - ( 2 -ethylhexyl)phosphoric acid concentration i s shown i n Table 9.9. The increase of the concent r a t i o n of the counter-ion showed a nearly l i n e a r relationship with the increase of the capac i t y factor of the ergot alkaloids. Due t o the strong interaction of 10-camphorsulfonic acid and the ergot alkaloids, systems w i t h t h i s counter-ion were unsuitable f o r ergot alkaloids (Table 8 . 9 ) . Under basic conditions t h e alkaloids could a l s o be separated using t h i s s o r t of mobile phase (Table 9.10). 3.3. STRAIGHT-PHASE HPLC Wittwer and Kluckhohn’ analyzed LSD and a s e r i e s of ergot alkaloids on s i l i c a gel with a c e t o n i t r i l e - diisopropyl e t h e r a s mobile phase. A s i m i l a r system was used by Perchalski et a1.7 f o r the determination of ergotamine i n plasma. Heacock e t a1.‘ performed some preliminary investigations on the analysis of ergot alkaloids on p e l l i c u l a r s i l i c a gel packings. By means o f chloroform - methanol - ethyl a c e t a t e a c e t i c acid (60:20:50:3), reasonable r e s u l t s were achieved. Water-deactivated columns in combination with chloroform - methanol were a l s o used w i t h good r e s u l t s .

-

363

TABLE 9.8 CAPACITY FACTORS AN0 SEPARATION FACTORS PHASE COLUtIN39

(a)

FOR SOME ERGOT ALKALOIDS ON A REVERSED

Column, Lichrosorb RP8. 7 rn (250x4.6), mobile phases, S 1 a c e t o n i t r i l e - water - t r i ethanolamine - c i t r i c a c i d (45 m l t 60 m l t 0.4 m l + 0.166 g), pH = 7.1; S2 a c e t o n i t r i l e water - triethanolamine - sodium acetate (45 m l t 60 m l t 1 m l t 0.3 g), pH = 7.1, adjusted by a d d i t i o n o f two drops a c e t i c acid; S3 a c e t o n i t r i l e - water tetradecyl t r i m e t h y l amnonium bromide (45 m l t 60 m l t 0.1 g ) , pH = 7.1, adjusted by a d d i t i o n o f two drops triethanolamine; S4 a c e t o n i t r i l e water - amnonium carbonate (45 m l t 60 ml t 0.04 9). pH = 8.3; 5 5 a c e t o n i t r i l e water diethylamine (375:625:21), pH = 12.3.

-

-

ALKALOIDS

MOBILE PHASE

s1 d i hydroergotamine d i hydroergocornine dihydro-a-ergocryptine d i hydroergocristine d i hydro-8-ergocrypti ne

-

k' 2.68 3.45 4.92

52

1.29 1.43 1.12

5.50

s3

k'

a

3.29 4.00 5.71 6.24

54

k'

a

1.21 1.43 1.09

2.08 2.65 3.84 4.22

k'

a

1.27 1.45 1.10

55

k'

a

1.30 1.75 2.15 3.05

1.35 1.23 1.42

a

4.66 5.72 8.21 9.26

1.23 1.44 1.13

TABLE 9.9 DEPENDENCE OF CAPACITY RATIOS MEASURED FOR SOME ERGOT ALKALOIDS ON DI-(2-ETHYLHEXYL) PHOSPHORIC ACDI ( OHP) CONCENTRATION^^ Conditions: $ondapak CN column ( 3 0 0 ~ 3 . 9mm I.D.); d e t e c t i o n a t 280 nm. Compound

1 cm3/min;

Eluent composition (%) Hexane 65 65 Chloroform 20 20 A c e t o n i t r i l e 15 15 DHP ( t f ) .

8-Ergocryptinine a-Ergocrypti n i ne Ergocorni n i ne Ergocri s t i n i n e 6-Ergocryptine a-Ergocrypti ne Ergocornine Ergocri s t i ne Ergotamine Ergotaminine Ergometrine O i hydro-6-ergocrypti ne D i hydro-a-ergocrypti ne Dihydroergocornine D i hydroergocristine D i hydroergotami ne

e l u e n t flow-rate,

I

-

65 20 15 0.0005 0.001 0.005

0.90 0.89 0.90 0.89 0.90 0.89 1.07 1.07 1.07 1.18 1.07 1.18 1.28 1.36 1.41 1.50 2.72 2.36 1.48 1.39 12.0 12.0 2.79 1.75 2.79 1.75 3.28 2.00 3.55 2.21 4.17 3.71

65 20 15

65 20 15

65 20 15 0.01 0.025

60 70 23 17 17 13 0.005 . . 0.005

1.17 4.71 1.17 4.71 1.41 6.46 1.41 7.21 1.69 1.96 1.69 1.96 1.93 2.21 2.10 2.21 3.62 2.93 5.55 15.9 15.2 1.59 1.41 1.59 1.41 1.85 1.71 1.97 1.71 3.28 2.64

10.4 10.4 12.9 14.3 2.38 2.38 2.77 2.77 3.54 25.9

3.70 3.70 4.63 5.22 2.41 2.41 2.78 2.78 4.11 16.8

4.15 4.15 4.48 5.52 1.48 1.48 1.67 1.67 2.37 12.0

9.67 9.67 12.7 14.6 3.37 3.37 3.89 4.15 5.35 28.3

1.69 1.69 2.00

2.00 2.00 2.26 2.26 3.37

1.15 1.15 1.30

2.70 2.70 3.10 3.10 4.63

2.00

3.00

1.30

1.96

-

TABLE 9.10 INFLUENCE OF DHP CONCENTRATION I N THE PRESENCE OF DEA I N THE ELUENT ON THE CAPACITY RATIOS ( k ' ) AN0 SELECTIVITY FACTORS ( r i j ) OBTAINED FOR NATIVE AND HYDROGENATED ERGOT PEPTIDE ALKALOIDS~~ Conditions as i n Table 9.9 Compound DEA (H) DHP (H)

Eluent, hexane-isopropanol

zXy3 10k'

Hydrocortisone Predni sol one 6-Ergocryptinine a-Ergocryptinine Ergocornin i ne Ergocri s t i nine 6-Ergocryptine

1.87 2.10 2.29

:;1.22

a-Ergocryptine Ergocornine Ergocristine

1'26

Ergotarninine Ergotarnine

4.81 4*35

1.11

Dihydroergocornine Dihydroergocristine Dihydroergotarnine

2.48 3.06 4.06

k'

'ij

1.85 2.10

22.94 *29 3.58 2.06 '.06 2.42 3.06

Dihydro-6-ergocryptine 2.10 D i hydro-a-ergocrypti ne 2.10

7.5~10-4 10-3

10-3 rij

l.oo

1.17

o.lo

:::!

2*40

$:1;

3.95 2.74

5::;

4.48

:;I: ;:!; X;; 4.52

(80:20)

k'

7.5~10-4 1.5~10-3 1j

1.87 2.01

1.00 1.25 1.32

o.lo

;:;

43.47 3;:

1.18 1'39

1.20

l.oo

1.11 '03*

5.88

i::: ;:;

2.94 4.31 4.81

k'

r.

'ij

1.93 2.10

1.43

:46.07 3

1.05

2:;

1.07

l.o

1.47

1.62

o.lo

1.10 1.33

3.53

4.53

$i;

;2:

7.5~10-4 4x10-3

k'

'i j

k'

1.10

1.90 2.13 10.4 11.5

'050

4.27 4*87 4.87 7.20

1'48

1.98

&gg

2.77

1.87 2.12

1.10

z:

1.35

6.15 8.60 4.40

1.72

:i!2

1.15

3.26 3.58

7.80

;i!:

7.5~10-4 2x10-3

::;: 7.51

1.43

i::: 6.03

i:

1.47

'ij

;;::

:::; A: 3.07 3.70

1.48

7.13

365

TABLE 9.11 CAPACITY FACTORS ( k ' ) OF ERGOT ALKALOIDS ON SILICA GEL PACKINGS WITH DIFFERENT Column Lichrosorb Si60, 10 um (250x2 n ID), mobile phase S 1 n-hexane - chloroform - ethanol (40:40:10), 52 chloroform - methanol (95:5), S3 chloroform-etlianol (95:5), d e t e c t i o n UV 280 o r 320 nm ~~

~~~

s1

s2

S3

17.41 10.00 6.27 1.90 1.90 1.90 1.40

-

-

A1 k a l o i d Ergometrine maleate Ergometrinine Ergotamine t a r t r a t e Ergocornine Ergocrypti ne Ergocristine Ergotami nine

1.80 0.50 0.50 0.50 0.28

0.60 0.60 0.60 0.37

~

~~

Alkaloid

s1

s2

53

Ergocorninine Ergocryptinine Ergocri s t i n i ne Dihydroergocorninea Dihydroergocryptinea Dihydroergocristinea

0.93 0.93 0.93

0.25 0.25 0.25 1.75 1.65 1.50

0.13 0.13 0.13

-

-

a As methanesulfonate s a l t TABLE 9.12 CAPACITY FACTORS ( k ' ) AND SEPARATION FACTORS (a) FOR ERGOT ALKALOIDS ON SILICA GEL PACKINGS WITH DIFFERENT ELUENTS51 Column Lichrosorb Si60, 5 um (250x4.6), mobile phase S 1 hexane - chloroform - a c e t o n i t r i l e (60:25:15), 52 idem i n r a t i o (56:22:22), 53 idem i n r a t i o (55:20:25), S4 hexane - chloroform - a c e t o n i t r i l e - methanol (55:20:25:3), f l o w r a t e 100 ml/h, d e t e c t i o n UV 320 nm. Alkaloid 6-Ergocrypti n i ne a-Ergocryptinine Eraocristinine Ergocorninine Ergotami n i ne 6-Ergocrypti ne a-Erqocryptine Ergoirisitine Ergocornine Ergometri n i ne

s1

k'

k'

a

2.04 2.54 3.24 7.48 9.28 10.80

1.00 1.08 1.32 1.40 2.78 3.29 3.67 4.78 4.40

1'24 1.28 2.31

1'24 1.16

Ergotami ne Ergometrine

52 a

i.:!

I."'

'"' 4.67

20.54

k' 0.95 1.00 1.22 2.75 3.09 3.54

s3

k'

a

"05 1.22 2.25

"" 1.15

0.77 0.77 0.77 0.77 1.01 1.21 1.21 1.21 1.21 2.11

:g;

s4

a

i::

::;;

2.32

TABLE 9.13 CAPACITY FACTORS OF SOME ERGOT ALKALOIDS ON A POROUS POLYSTYRENE STATIONARY PHASE41 Column H i t a c h i Gel no 3011-0, 5-7 m ( 5 0 0 ~ 4 . 6 nun ID). mobile phase S 1 n-hexane t r i e t h y l a m i n e (70:30:0.5), 52 cyclohexane - ethanol - t r i e t h y l a m i n e (7n:30:0.5), - chloroform - t r i e t h y l a m i n e (5:95:0.5), d e t e c t i o n UV 280 nm. Alkaloids

k' i n

E rgornet r ine a-Ergocrypti ne Ergocornine Ergocristine a-Ergocryptinine Ergocorni n i ne Ergocristinine Ergotami n i ne Oihydro-aergocryptine Oihydroergocornine Dihydroergocristine

Referenew p. 366

S1

s2

8.68 1.47 1.85 3.07 1.78 2.45 4.31 5.57 1.13 1.48 2.38

5.15 0.73 0.88 1.31 0.89 1.15 1.82 2.41 0.57 0.71 1.00

'

s3 26 0.76 0.84

1.11

2.14 2.86 3.23 3.41 1.53 1.96 2.37

-

ethanol S3 _?-hexane

366

Jane 9 separated a wide range of drugs of abuse on microparticulate s i l i c a gel w i t h polar mobile phases containing ammonium n i t r a t e solutions. LSD could be analyzed w i t h methanol 0.2 M ammonium n i t r a t e solution (3:2)(Fig.9.6). The system has been successfully applied t o the analysis of LSD13’15’16’28’45’46, but f o r the analysis of ergot alkaloids the above men-

tioned reversed-phase system gave b e t t e r resul ts28’46 (Table 9.5). Szepesy e t a l . 25’34’35 found microparticulate s i l i c a gel t o be very useful f o r the separation of the alkaloids present in the ergometrine, ergotamine. ergotoxine and dihydroergotoxine groups. The stereoisomers could be separated on s i l i c a gel (Table 9.11). To obtain a good separation of the various alkaloids within each group, reversed-phase chromatography was found to be best (Table 9.1). Later51, a straight-phase system was developed f o r the separation o f the ergotoxine alkaloids (Table 9.12. Figs. 9.7 and 9.8). By means of a solvent system consisting o f hexane - chloroform - a c e t o n i t r i l e - methanol (50:20:25:3), a rapid group separation could be achieved (Fig.9.9). Aigner e t al.14 investigated the separation of some drugs, e.g. ergotamine and ergotaminine, on s i l v e r iodide impregnated s i l i c a g e l . Multi-component drugs could be separated with chloroform - diethylamine as mobile phase on a 1.09% s i l v e r iodide impregnated s i l i c a gel column. Quercia e t a1 .I1 used a microparticulate aluminium oxide column to separate the dihydroderivatives of some ergot alkaloids and pentane - methanol (98:2) o r (97:3) as mobile phase. Yoshida e t al.41 isolated ergotoxine and ergotoxinine from ergot by means of a s i l i c a gel column and cyclohexane - acetone ( 1 : l ) as mobile phase. The ergotoxinine group o f alkaloids was separated on a porous polystyrene, modified by hydroxymethyl groups by using _I-hexane ethanol - triethylamine (70:30:0.5) as mobile phase. The ergotoxine group alkaloids and their dihydro derivatives could be separated in t h i s system (Table 9.13). For the LC-MS analysis of clavine alkaloids in ergot fermentation broth, Eckers e t a1.57*58 separated the alkaloids on s i l i c a gel with the mobile phase dichloromethane - methanol - concentrated ammonia (95: 5:O. 1) (Fig .9.10). 9.4. DETECTION The intense fluorescence of LSD provides the basis f o r a very s e n s i t i v e and s e l e c t i v e detection o f t h i s compound - 10 pg. The excitation wavelength used i s about 325 nm13*15g16’ 21’28’53 and the emission i s measured a t 389 nm53, 400 nm4128 o r 420 nm13’15916’21. Prolonged i r r a d i a t i o n - 5 min - of LSD trapped in a scanning fluorimetric detector, r e s u l t s i n the conversion i n t o a non-fluorescent lumi-derivative. The disappearance of the fluorescence upon UV-irradiation has been used t o distinguish LSD from other fluorescent compounds t h a t do not exhibit t h i s b e h a v i o ~ r ~ ~ The ’ ~ ~same ’ ~ ~principle . has been used f o r the i d e n t i f i c a t i o n o f ergot alkaloids by Scholten and Frei44. A photochemical reaction detector was designed i n which the eluted ergot alkaloids were i r r a d i a t e d f o r 20 sec with 327 nm UV-light - and the emission was measured a t 410 nm. Dihydroderivatives were i r r a d i a t e d a t 280 nm and the fluorescence measured a t 340 nm. Under such conditions a 90-99% decrease o f the fluorescence was found f o r the 17 alkaloids investigated. The influence of the solvent on the quenching of the fluorescence was studied by Heacock e t al.‘. From Table 9.14 i t i s c l e a r t h a t only chloroform causes considerable quenching. The fluorescence maxima of some ergot alkaloids a r e l i s t e d in Table 9.15‘.

367

TABLE 9.14 INFLUENCE OF SOLVENT ON FLUORESCENCE INTENSITY OF E R G O T A M I N E ( ~ . ~ X ~ O - ~ M ) ~

hex= 350 nm

Solvent

Re1a t i ve i n t e n s i t y ( % )

100

100% Ethanol 50% D i i s o p r o p y l e t h e r a 50% Cyclohef;anea 50% Acetong 50% Hexane 50% Benzenea 50% Chloroforma

130 113 96 91 109 13

a 50% m i x t u r e s w i t h a b s o l u t e e t h a n o l TABLE 9.15 EMISSION MAXIMA AND RELATIVE FLUORESCENCE INTENSITIES FOR SOME ERGOT ALKALOIOS I N ACETONE SOLUT ION^.

xPy=

350 nm, c o n c e n t r a t i o n = 100 ppm.

A1 k a l o i d

,Amau

emission

Relative intensity(%)

~~

Ergotami ne Ergotami n i n e E r g o c r i s t i ne E r g o c r y p t i ne E r g o c r y p t i n i ne Ergocornine Ergocorninine Ergosi ne b L y s e r g i c a c i d amide I s o l y s e r g i c a c i d amide 0-Lysergic a c i d Ergotrate Isosetoclavifle Elymoclavine Agrocl a v i n e 0-Lysergic acid diethylamide

400 397 400 403 39 7 402 398 398 393 393 392 403 393 394 392 397

40.2

100.0

21.0 22.1 62.2 18.3 83.8 56.1 1.1 61.5 32.8 28.9 82.2 0.3 0.5 62.2

b C o n c e n t r a t i o n = 20 ppm, due t o low s o l u b i l i t y o f compound i n acetone. Fluorescence d e t e c t i o n has found w i d e a p p l i c a t i o n i n t h e a n a l y s i s o f e r g o t a l k a l ~ i d s ~ ’ ’ ~ ’

36*50. S c o t t and Lawrence”

found t h a t f o r t h e f l u o r i m e t r i c d e t e c t o r used i n t h e i r i n v e s t i -

g a t i o n s , t h e optimum wavelength f o r e x c i t a t i o n was 235 nm. Baker e t a1.4 compared a f l u o r i m e t r i c d e t e c t o r and d e t e c t o r s w i t h v a r i a b l e wavelength (334 nm) and s i n g l e wavelength (254 nm) f o r t h e a n a l y s i s o f i l l i c i t LSO samples. The f l u o r i m e t r i c and t h e v a r i a b l e wavelength d e t e c t i o n a l l o w e d a n a l y s i s w i t h o u t sample p r e t r e a t m e n t , whereas s e v e r a l peaks i n t e r f e r e d w i t h LSO by d e t e c t i o n a t 254 nm. For a s p e c i f i c d e t e c t i o n o f t h e e r g o t o x i n e a l k a l o i d s , Szepesy e t a1.25 used a wavelength o f 320 nm, f o r t h e i r d i h y d r o d e r i v a t i v e s 280 nm. Bethke e t a1.17 a p p l i e d s i m u l t a n e o u s d e t e c t i o n a t 280 and 320 nm f o r ergotamine and i t s decomposition p r o d u c t s . D e t e c t i o n a t 241 nm 52

has a l s o been employed

.

S i n c e t h e l u m i a l k a l o i d s have o n l y v e r y s m a l l a b s o r p t i o n a t 320 nm, t h e y do n o t i n t e r f e r e w i t h t h e d e t e c t i o n o f t h e e r g o t a l k a l o i d s w i t h a 9,lO-double

References p. 368

bond. The d i h y d r o and t h e l u m i

368 a l k a l o i d s can be detected a t 280 nm. the lumi a l k a l o i d s w i t h an increase o f s e n s i t i v i t y by a f a c t o r o f 1000 when compared w i t h the 320 nm d e t e c t i o n l i m i t . Szepesy e t al.25 used t h e wavelength 320 nm and 280 nm f o r t h e d e t e c t i o n o f the ergotoxine and the dihydroergotoxine alkaloids, r e s p e c t i v e l y . For q u a n t i t a t i v e determinations o f e r g o t a l k a l o i d s , Wurst e l a l , 2g’43 made use o f several wavelengths

-

310. 282, 254. 240 and 225 nm. I n t h a t way, p a r t i a l l y resolved a l k a l o i d s could

be q u a n t i f i e d . White45 developed an electrochemical d e t e c t i o n method f o r t h e HPLC, a l s o s u i t a b l e f o r the analysis o f LSD and some phenothiazine d e r i v a t i v e s . Santi e t a1.10s26 as w e l l as G f e l l e r e t a1 .30’38 studied t h e s e l e c t i v e d e t e c t i o n o f ergotamine as p i c r a t e i o n - p a i r i n mu1 ti-component preparations. This method i s discussed i n Chapter 4. Frei4’

has reviewed r e a c t i o n l i q u i d chro-

matography, i n c l u d i n g the method mentioned above.

A post column f l u o r e s c e n t i o n - p a i r i n g technique has been developed f o r e r g ~ t a m i n e ~The ~. major advantage o f t h i s technique i s an improvement o f the s e l e c t i v i t y , as no i n t e r f e r i n g peaks due t o non-ion-pairing compounds are observed i n u r i n e analyses (see Chapter 4). Lawrence e t a1.48 described a s i m i l a r technique, b u t they used an organic e l u e n t i n com42

b i n a t i o n w i t h a s i l i c a gel column, instead o f an aqueous e l u e n t as used by G f e l l e r e t a l . (see Chapter 4).

Eckers e t a1.57*58 reported the analysis o f c l a v i n e a l k a l o i d s i n e r g o t fermentation b r o t h by means o f LC-MS (Fig.9.10). than CI-MS;

I n general. EI-MS was found t o g i v e more i n f o r m a t i v e r e s u l t s

however, the a l k a l o i d s p a l l i c l a v i n e decomposed during EI-MS, n e c e s s i t a t i n g the

l a t t e r method f o r the analysis o f t h a t a l k a l o i d . Various conditions f o r the mass spectrometry were studied. REFERENCES

1 J.D. Wittwer and J.H. Kluckhohn, J . C h r o m a t o g r . sci., 11 (1973) 1. 2 R.A. Heacock. K.R. L a n g i l l e , J.D. MacNeil and R.W. Frei. J . C h r o m a t o g r . . 77 (1973) 425. 3 I.Jane and 8.6. Wheals, J. Chromatogr., 84 (1973) 181. 4 O.R. Baker, R.C. Williams and J.C. Steichen, J . C h r o m a t o g r . sci.. 12 (1974) 499. 5 M.L. Chan, C. Whetsell and J.O. McChesney, J . C h r o m a t o g r . S c i . , 12 (1974) 512. 6 R.V. Vivilecchia, R.L. Cotter. R.J. Limpert, N.Z. Thimot and J.N. L i t t l e , J. Chromatogr., 99 (1974) 407. 7 R.J. Perchalski, J.O. Winefordner and B.J. Wilder, A n a l . C h e m . , 47 (1975) 1993. 8 P.J. Twitchett. Chem. B r . , 11 (1975) 443. 9 I . Jane. J . C h r o m a t o g r . , 111 (1975) 227. 10 W. Santi, J.M. Huen and R.W. Frei. J . C h r o m a t o g r . , 115 (1975) 423. 11 V. Quercia, L. Turchetta, V. Cuozzo and I.D o n a t e l l i , ~ 0 1 1 .C b i m . Farm., 115 (1976) 810. 12 P.J. Twitchett, A.E.P. Gorvin, A.C. Moffat, P.L. Williams and A.T. Sullivan, i n H i g b - p r e s s u r e L i q u i d C h r o m a t o g r a p h y i n C l i n i c a l C h e m i s t r y , E d i t o r P.F. Dixon, A c a d e m i c P r e s s , L o n d o n , 13 14 15 16 17 18 19 20 21 22 23 24

.

1976. = 0.210 ----

6:B. Wheals, I b i d e m , p 211 R. Aigner, H. S p i t z y and R.W. F r e i , J . C h r o m a t o g r . s c i . , 14 (1976) 381. J. C h r i s t i e , M.W. White and J.M. Wiles, J . C h r o m a t o q r . , r 129 119761 496. 6.6. Wheals, J . C h r o m a t o g r . , 122 (1976) 85. 1 H. Bethke, 6. Delz and K. S t i c k , J . C h r o m a t o g r . . 123 11976) 193. F. Erni and R.W. F r e i , J . C h r o m a t o g r . , 125 (1976) 269. J. Oolinar, C h r o m a t o g r a p h i d , 10 (1977) 364. i Lurie, J. Assoc. off. Anal. C h e m . , 60 (1977) 10h5. E. Johnson, A. Abu-Shumays and S.R. Abbottt, J . C h r o m a t o g r . , 134 (1977) 107. P. Schauwecker, R.W. F r e i and F. Erni. J. C h r o m a t o g r . , 136 (1977) 63. J.R. Anderson, G.L. Blackman and I . H . Pitman, A u s t r . J . Pharm. S c i . , 7 (1978) 73. A. Wehrli, J.C. Hildenbrand, H.P. K e l l e r , R. Stamplfi and R.W. F r e i , J. Chromatogr., 149 (1978) 199.

.

I.

,

369

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

L. Szepesy, I . Feher, G. Szepesi and M. Gazdag. J . C h r o m a t o g r . , 149 (1978) 271. J.M. Huen, R.W. F r e i , W. Santi and J.P. Thevenin, J . C h r o m a t o g r . , 149 (1978) 359. P.J. Twitchett, P.L. Williams and A.C. Moffat, J . C h r o m a t o g r . , 149 (1978) 683. P.J. Twitchett. S.M. Fletcher, A.T. S u l l i v a n and A.C. Moffat. J . C h r o m a t o g r . , 150 (1978) 73. M. Wurst, M. F l i e g e r and Z. Rehacek. J . C h r o m a t o g r . . 150 (1978) 477. J.C. G f e l l e r , J. Huen and J.P. Thevenin, J . C h r o m a t o g r . . 166 (1978) 133. D.L. Sondack, J . C h r o m a t o g r . , 166 (1978) 615. I.S. L u r i e and J.M. Weber, J . L i q . C h r o m a t o q r . , 1 (1978) 587. W. Hartmann, M. Rodiger, W. Ableidinger and H. Bethke, J . Pharm. S c i . , 67 (1978) 98. L. Szepesy, I . Feher, G. Szepesi and M. Gazdag, Magy. Kem. ~ o l y ,84 (1978) 375. P. Horvath, G. Szepesi and A. Kassai, P l a n t a Med.. 33 (1978) 407. P. Hatinguais, 0. Beziat, P. Negol and R. Tarroux, Trav. SOC. Pharm. M o n t p e l l i e r , 38 (1978)

329.

G. Megges, A r c h . K r i m i n o l . , 164 (1979) 25. J.C. G f e l l e r , J.M. Huen and J.P. Thevenin, C h r o m a t o g r a p h i a , 12 (1979) 368. S.L. A l i and T. S t r i t t m a t t e r , I n t . J . P h a r m . , 4 (1979) 111. R.W. F r e i , J . C h r o m a t o g r . , 165 (1979) 75. A. Yoshida, S. Yarnazaki and T. Sakai, J . C h r o m a t o g r . , 170 (1979) 399. J.C. G f e l l e r . G. Frey, J.M. Huen and J.P. Thevenin, J . C h r o m a t o g r . , 172 (1979) 141. M. Wurst, M. F l i e g e r and 2 . Rehacek, J . C h r o m a t o q r . , 174 (1979) 401. A.H.M.T. Scholten and R.W. F r e i , J . C h r o m a t o q r . , 176 (1979) 349. M.W. White, J . C h r o m a t o q r . . 178 (1979) 229. R.E. Ardrey and A.C. Moffat. J. F o r e n s i c Sci. Soc., 19 (1979) 253. F. Luccioni, A. B a r l a t i e r and 0. Lena, P h a r m . d c t a Helv., 54 (1979) 69. J.F. Lawrence, U.A.T. Brinkman and R.W. F r e i , J . C h r o m a t o g r . , 185 (1979) 473. I.S. Lurie, I n t e r n a t i o n a l Laboratory. (1980) 61. P.M. Scott and G.A. Lawrence, J . d g r i c . Food Chem., 28 (1980) 1258. G. Szepesi, M. Gazdag and L. Terdy, J. C h r o m a t o g r . , 191 (1980) 101. R. Fankel and I . Slad, z. d n a l . Chem., 303 (1980) 208. P.O. Edlund, J. C h r o m a t o g r . , 226 (1981) 107. I.S. L u r i e and S.M. Demchuk. J . L i o . C h r o m a t o -u r . . . 4 (1981) 337. I.S. L u r i e and S.M. Demchuk; J . L i g . C h r o m a t o g r . , 4 (1981j 357. I.S. Lurie, J . L i q . C h r o m a t o g r . , 4 (1981) 399. C. Eckers, D.E. Games, D.N.B. Mallen and B.P. Swann, A n a l . P r o c . , 8 (1982) 133. C. Eckers, O.E. Games, D.N.B. Mallen and 8.P. Swann, B i o m e d . M a s s S p e c t r o m . , 9 (1982)

162.

59 T.A. Gough and P.8. Baker, J . C h r o m a t o q r . S c i . , 20 (1982) 289. 60 8. Herlnyi and S. Gorog. J . C h r o m a t o g r . , 238 (1982) 250. 61 G. Szepesi, M. Gazdag and R. Ivancsics, J . C h r o m a t o g r . , 241 (1982) 153. 62 K. Harzer, J . C h r o m a t o q r . . 249 (1982) 205.

.u

370

11

101

1 I

I

I

1

1

I

1

0

L

8

12

16

20

min

Fig. 9.1. Separation o f some ergot a l k a l o i d s 1 9 Column Lichrosorb RP8 5 pm ( 1 2 5 ~ 4 . 2 mn 10). mobile phase a c e t o n i t r i l e - 0.02% aqueous ammonium carbonate ( 2 : 3 ) , f l o w r a t e 100 ml/h, d e t e c t i o n UV 254 nm. Peaks: 1, l y s e r g i c acid; 2, i s o l y s e r g i c acid; 3, l y s e r g i c a c i d amide and ergometrine; 4 , i s o l y s e r g i c a c i d amide; 5, e r gometrinine; 6, ergosine; 7, ergotamine; 8, ergocornine; 9, ergocryptine ( a and 8 ) ; 10, ergoc r i s t i n e ; 11, ergosinine; 12, ergotaminine; 13, ergocorninine; 14, ergocryptinine; 15, ergoc r i s t i n i n e ; x, unknown. (reproduced w i t h permission from r e f . 19, by the courtesy o f F r i e d r . Vieweg & Sohn, Wiesbaden) 19 F i g . 9.2. Separation o f some e r g o t a l k a l o i d s Column Lichrosorb RP18 10um ( 1 2 5 ~ 4 . 2 mm I D ) , mobile phase a c e t o n i t r i l e - 0.02% aqueous ammonium carbonate (42:58), f l o w r a t e 100 ml/h, d e t e c t i o n UV 254 nm. Peak numbering as i n Fig. 9.1. (reproduced w i t h permission from r e f . 19, by the courtesy o f F r i e d r . Vieweg E Sohn, W i esbaden)

11 5

13

1

0

1

L

I

8

12

I

16

I

min

F i g . 9.3. Seperation o f some ergot a l k a l o i d s 1 9 ( 1 2 5 ~ 4 . 2 mm I D ) , mobile phase Column Spherisorb ODS 5 a c e t o n i t r i l e - 0.02% aqueous ammonium carbonate (42:58), f l o w r a t e 100 ml/h. d e t e c t i o n U V 254 nm. Peak numbering 1-8 as i n Fig. 9.1.. peak 9, ergosinine; 10, ergocryptine; 11, e r g o c r i s t i n e , ergocorninine and ergotaminine; 12, e r gocryptinine; 13, e r g o c r i s t i n i n e ; x, unknown. (reproduced w i t h permission from r e f . 19, by the courtesy o f F r i e d r . Vieweg & Sohn. Wiesbaden)

371

I

0

I

5

10

15

min

50

min

40

30

20

10

0

Fig. 9.4. Separation of dihydroergotoxine alkaloids33 Column Lichrosorb RP18 5 pin (150x3 mm ID), mobile phase water - a c e t o n i t r i l e - triethylamine (32:8:1). flow r a t e 1.0 ml/min, detection UV 280 nm. Peaks: 1, dihydroergocornine; 2 , dihydro- a-ergocryptine; 3, dihydroergocristine; 4, dihydro-B-ergocryptine. (reproduced with permission from ref. 33, by the courtesy o f Journal Pharmaceutical Sciences) Fig. 9.5. Analysis of ergocornine and ergocryptine i n fermentation liqour 60 Column Lichrosorb RP18 10 pm ( 2 5 0 ~ 4 . 6mn ID), mobile phase tetrahydrofuran - 0.01 M aqueous ammonium acetate (2:3), flow r a t e 1.5 ml/min, detection UV 322 nm. Peaks: 1, ergocornine; 2. a-ergocryptine;3,6-ergocrypti ne; 4, ergocorni nine; 5 , a-ergocrypti nine; 6 , 6-ergocrypti ni ne; 7 , ergometrine; 8 , ergometrinine.

i

li

'"1

I

_A_ L

O

Fig. 9.6. HPLC analysis of LSD and some related compounds9 Column P a r t i s i l 6 p m ( 2 5 0 ~ 4 . 6mm ID), mobile phase methanol - 0.2 M aqueous ammonium n i t r a t e ( 3 : 2 ) , flow r a t e 1 ml/min, detection UV 320 nm. Peaks: 1, lysergic a c i d ; 2 , lysergamide; 3 , LSD; 4, isoLSD. Fig. 9.7. Separation of ergotoxine alkaloids 51 Column Lichrosorb Si60 5 pm ( 2 5 0 ~ 4 . 6mm I D ) , mobile phase hexane - chloroform - a c e t o n i t r i l e (56:22:22), flow r a t e 100 ml/min, detection UV 320 nm. Peaks: 1, ~ e r g o c r y p t i n i n e ; 2 , cr-ergocryptinine; 3, ergocorninine; 4, B-ergocryptine; 5, a-ergocryptine; 6 , ergocornine; x, unknown.

References p. 368

312

3

I

2

51 F i g . 9.8. S e p a r a t i o n o f f o u r isomers o f e r g o c r i s t i n e Column L i c h r o s o r b S i 6 0 5 pm ( 2 5 0 ~ 4 . 6mn I D ) , m o b i l e phase hexane - c h l o r o f o r m - a c e t o n i t r i l e (56:22:22), f l o w r a t e 100 ml/h. d e t e c t i o n UV 320 nm. Peaks: 1, e r g o c r i s t i n i n e ; 2, a c i - e r g o c r i s t i n i n e ; 3, e r g o c r i s t i n e ; 4, a c i - e r g o c r i s t i n e ; x, unknown. F i g . 9.9. S e p a r a t i o n o f e r g o t a l k a l o i d s 5 1

Column L i c h r o s o r b S i 6 0 5 pm ( 2 5 0 ~ 4 . 6 mn ID), m o b i l e phase hexane - c h l o r o f o m - a c e t o n i t r i l e methanol (55:20:25:3), f l o w r a t e 100 ml/h, d e t e c t i o n UV 320 nm. Peaks: 1, e r g o c r i s t i n i n e , e r g o c o r n i n i n e , a- and 8 - e r g o c r y p t i n i n e ; 2, e r g o t a m i n i n e ; 3, e r g o c r i s t i n e , ergocornine,a- and 8 - e r g o c r y p t i n e ; 4, e r g o m e t r i n i n e ; 5, ergotamine; 6, e r g o m e t r i n e ; x, unknown.

.O

10

20

30

LO

5’0

60 min

F i g . 9.10. Reconstructed t o t a l i o n c u r r e n t t r a c e o b t a i n e d by E I LC/MS of e r g o t Column S p h e r i s o r b 5W s i l i c a g e l (250x5 mn I D ) , m o b i l e phase d i c h l o r o m e t h a n e - methanol c o n c e n t r a t e d ammonia (95:5:0.1). f l o w r a t e 1 ml/min. Peaks: 1, s o l v e n t f r o n t ; 2, a g r o c l a v i n e ; 3, s e t o c l a v i n e ; 4 , f e s t u c l a v i n e ; 5, p a l l i c l a v i n e o r isomer; 6, p a l l i c l a v i n e o r isomer; 7 , N-noragrocl a v i n e ; 8, elymoclavine; 9, penni c l a v i n e ; 10, isochanoclavine; 11, n o r c h a n o c l a v i n e s I and 11; 12. c h a n o c l a v i n e I ; 13, c h a n o c l a v i n e 11. (reproduced w i t h p e r m i s s i o n o f John W i l e y & Sons, L t d . )

TABLE 9.16 HPLC ANALYSIS ERGOT ALKALOIDS I N AN0 FROM FUNGI ALKALOIDS *

AIMS

Lysac, Ergta,Ergtaine,Ergct,Ergctine diHErgct,Ergcp,Ergcpine, diHErgcp,Ergco,Ergcoine, di HErgco ,Ergm,Ergmine Aocl ,Chcl ,Elcl ,Fcl ,Pcl ,Pycl, Scl,isoScl,paspaclavine, pal iclavine,Lysam, isolysam, Lysgol ,Lysg,lysergen Ergta ,Ergtaine,Ergct,Ergcti ne

Analysis plant extracts,fermented Lichrosorb Si60,lO wn products and pharmaceutical preparations (Table 9.1 and 9.11) Lichrosorb RP2,RP8 and RP18 Analysis clavines and simple Micropak NH2,lO fl 1 ysergi c acid derivatives (Table 9.6)

COLUMN D I M . MOBILE PHASE LxID mm

STATIONARY PHASE

Analysis in crude Ergot Ergta ,Ergtaine,Ergs ,Ergsine, Analysis in scl erotia ,cul ture Ergct,Ergctine,Ergcp,Ergcplne, media and pharmaceutical proErgco,Ergcoine,Ergm,Ergmine ducts (Table 9.3) papaverine Analysis in crude Ergot and pharErgtaine,Ergct,Ergctine, di HErgct ,a-Ergcp, a-Ergcpi ne, maceuti cal preparations diHErgcp,Ergco,Ergcoine, (Table 9.13) diHErgco,Ergm Agcl ,Lysam,isolysam Analysis in fermentation media Ergta,Ergtaine,B-OH-Ergta, and pharmaceutical preparations Ergs,Ergsine,Ergst,Ergstine, (Table 9.7) Ergct ,Ergctine,Ergcp, Ergcpi ne, Ergco,Ergcoine,Ergm,Ergmine Ergta ,Ergtaine ,Ergs ,Ergsine, Analysis ergot a1 kaloids in flour Ergct ,Ergcti ne,Ergcp ,Ergcpine, Ergco .Ergcoine,Ergm, Ergmine Ergta,Ergtaine,Ergct,ErgctAnalysis ergotoxine alkaloids (Table 9.12 and Fig.9.7.9.8 ine,u-, 8-Ergcp,a-,B-Ergcpine, Ergco,Ergcoine,Ergm,Ergmine and 9.9)

Micropak Si-10 Li chrosorb RP18,lO un

Ergta,Ergtaine,Ergct,Ergcti-

Silica gel C8, 5 1 ~ n

ne ,a-Ergcp ,8-Ergcp ,Ercni ne, Ergco ,Ergcoi ne

Analysis in sclerotium material

*For abbreviations see footnote Table 9.19

Hitachi Gel no 3301-0 5-7 un Lichrosorb NH2,10 fl or Micropak NH2, 10 pm

250x2 250x2 250x2

250x2 250x4

REF.

n-hexane-CHC13-EtOH(4:4:1) -CHC13-MeOH(95:5) CHCl2-EtOH(95 :5) CHCl3-isoprOH(9: 1) ,(8: 2) Et20-isoprOH(7:3). (6:4) EtzO-EtOH(84:16) .(8:2) also gradient elution CHCl3-EtOH( 95 :5) 0.1% (NH4)OAc in ACN-H20 (35:65)

29 35

36 500~4.6 n-hexane-EtOH-TrEA( 70:30 :0.5) n-hexane-CHC13-TrEA(5:95:0.5) cycl ohexane-EtOH-TrEA( 70: 30:O. 5) 41 CHC13;isoprOH(9:l) 250x2 EtpO-1 soprOH( 6:4) EtzO-EtOH(84: 16) (88:12), (93: 7) also gradient elution 43 250~4.6 ACN-O.02M aq. (NH )2CO3(43:57), (35 :65), (48 :72) 50 250~4.6 Hexane-CHC1 -ACN(56:22:22), (60:25:15),?55:20:25) Hexane-CHC13-ACN-MeOH 51 (55:20:25 :3) 250x4 ACN-H20( 55:45) containing 0.04% (NH4)CO? 52 3

Lichrosorb RP8, 5

rrn

Lichrosorb Si60,5 ~n

w 4 w

Agcl,norAgcl,Chcl I and 11, Analysis i n e r g o t f e r m e n t a t i o n b r o t h w i t h LC-MS (Fig.9.10) isoChcl 1,norChvl I and 11, Elcl,Fcl,Pcl,Pycl,Scl ,isoScl, norisoScl .palliclavine

Spherisorb 561

250x5

00s-type 5 um

250x5

NH2-tyne,

5 ,jn

not given

CH2C12-MeOH-conc. NH40H (95:5:0.1) MeOH-HpO-conc.NH OH (60:40:0.!) isooctane-CH2C12-MeOH( 5:4: 1 )

Ergm,Ergmi ne ,Ergco,Ergcoine, a-Ergco, 6-Ergcoine, E-Ergcp, a-Ergcnine,diHErgco ,di H- aErgcn,diH- &Ergco

Separation ( F i g .9.5 ,Table 9.4)

L i c h r o s o r b RP18, 10 un

250~4.6 THF-O.01M aq NH40Ac(2:3)

15 E r c o t a l k a l o i d s

Separation w i t h s t r a i n h t phase i o n - p a i r HPLC (Tables 9.9, 9.10)

rSondapak CN

3 0 0 ~ 3 . 9 Hexane-CHC1 ACN-di-(2-ethylhexyl)phosp&ric a c i d i n various r a t i o s

57.58

60 61

TABLE 9.17 HPLC ANALYSIS ERGOT ALKALOIDS I N PHARMACEUTICAL PREPARATIONS ALKALOIDS*

OTHER COnPOUNDS

AIMS

STATIONARY PHASE

Agcl ,Elcl Pcl .isoScl ,LSD, Lysg,isoLysg,Ergta, Ergtaine,Ergs, Ergs ine, E r g c t , E r g c t i n e ,Ergcp, Ergcpine,Ergco, Ergcoi ne ,Ergm

Preliminary investigation o f s e p a r a t i o n w i t h HPLC

diHErgct,diHErgcp, diHErgco

Separation on small p a r t i c l e UBondapak C18 s i z e column packings Separation as i o n - p a i r s S p h e r o s i l XOB,5-10 rn loaded w i t h 0.03M p i c r i c a c i d and b u f f e r DH 5 S i l i c a g e l 100, 5 fl loaded w i t h 0.06M p i c r i c a c i d and b u f f e r PH 5 A n a l y s i s i n pharmaceutical Micropsk A1-5 preparations

Ergta .atroDine, scopolamine

diHergta,diHErgct, diHergcp,diHErgco

Corasil

COLUMN DIM. LxID(mn)

MOBILE PHASE

1 0 0 ~ 2 . 4 CHCl3-MeOH-EtOAc-AcOH(60: CHCl3-MeOH( 100:4)

REF. 20: 50: 3 )

2

*For a b b r e v i a t i o n s see f o o t n o t e Table 9.19

300x4

ACN-O.01M aq.(NH4)2C03(2:3)

6

CHC13 s a t . w i t h 0.05M p i c r i c 1 0 0 ~ 2 . 8 a c i d i n pH 5 b u f f e r CHC13.sat.with 0.06M p i c r i c 1 0 0 ~ 2 . 8 a c i d i n pH 5 b u f f e r 250x2 Pentane-MeOH(98:2) ,( 97:3)

10

11

P

; I

B

Ergta,Ergtaine atropine,scopo1 ami ne ,caffeine

Butalbital , phenobarbital

Separation on silver impregnated silica gel

P 0 m m

Ergta ,Ergtaine, aciErgta ,aciErgtaine,Lysam,isoLysam,Lysac, isoLysac.lumiErgta Ergta .Ergtaine aci Ergta ,aci Ergtaine Lysam,isoLysam, Lysac,isoLysac Ergta,Ergtaine,Ergs, Ergsine,Ergct,Ergctine,diHErgct, a-, B-Ergcp,Ergcpine.diHErgcp, Ergco,Ergcoi ne, Ergm,Ergmine, Lysam,isoLysam, Lysac,isoLysac Ergct,a-. kErgcp, Ergco

Qua1 ity control ergotamine preparations (Table 9.2)

14

17 Separation witn stepwise gradient system

Nucleosil C18 5 m

150x3

Separation with reversed phase HPLC (Fig.9.1,9.2, 9.3)

Spherisorb ODS,5 un

125~4.2

Lichrosorb RP2,5 un Lichrosorb RP8.5 im Lichrosorb RP18,5 im

125~4.2 ACN-O.O2%aq (NH4)2C03( 15:85) 125~4.2 ACN-O.O2%aq(NH4)2C03(2:3) 125~4.2 ACN-0.02%aq( NH4)2C03( 2: 3), (38:62)

Influence of organic bases Lichrosorb RP18,5 run on the stability reversed phase stationary phases Lichrosorb Si60,lOim Analysis pharmaceutical preparations (Table 9.1 and 9.10)

Ergta,Ergtaine, Ergct ,Ergctine, diHErgct, Ergcp ,Ergcpine, di HErgcp Ergco,Ergcoine, diHErgco, Ergm,Ergmine,Lysac Ergta,Ergtaine, butalbita1,pheno- Separation with ion-pair diHErgta,tropane barbita1,barbitchromatography alkaloids,caffeine a1,pizotifene Ergm,Ergmi ne, Lysac

CHCl3-DEA( 99.99 :O .01) Lichrosorb Si100,5 un imp.with 1.09% AgI not given gradient A CHC13-hexane(l:l) B CHC13-MeOH-DEA(90:10:0.5) linear gradient from 16 to 92% B i n A (1.5-2.5 min) ACN-O.OlM(NH4)2C03(2:1),(1:1) uBondapak C18 or 300x4 300~4.6 Nucleosil C18,lO un

Analysis ergometrine preparations

ACN-O.OlM(NH )2CO3 stepwise graiient : 8% ,15%,30%, 40%,50% and 60%

18

19

250~4.6 0.05M TrEA in ACN-H20(1:1)

24 250x2

n-hexane-CHC1 EtOH (4:4 : 1 ) iTHCl3-MeOH(95?;) CHC13-EtDH(95:5)

Lichrosorb RP2,RP8, RP18

250x2

ACN-O.01M aq. (NH4)2C03( 2: 3 )

Lichrosorb Si100,5 pin loaded with 0.06M picric acid and buffer pH 6 UBondapak C18

150x3

CHC13 sat.with O.O€M picric acid and buffer pH 6

25,34

300x4

ACN-AcOH-H20(20: 1:79)

26.30, 38 31 0 4

cn

Separation dihydroergot o x i n e i n pharmaceutical p r e p a r a t i o n s (Fig.9.4) A n a l y s i s i n pharmaceutical p r e p a r a t i o n s (Table 9.8)

diHErgct,diHa-Ergcp, d i Hs-Ergcp .diHErgco diHErgta,diHErgct, d i Ha-Ergcp, d i Hg-Ergcp,diHErgco

L i c h r o s o r b RP18,5 p

150x3

L i c h r o s o r b RP18,lO pm L i chrosorb RP8,7 rm

250x4 ACN-HzO-DEA(375:625:21) 2 5 0 ~ 4 . 6 ACN-H20( 45:60 )+0.049( NH4)eC03 ACN-H 0 TrEA(45:60:0.4)+citric i 6 6 9 ) .pH 7.1 acid

HzO-ACN-TrEA(32:8:1) H20-MeOH-TrEA( 25 :3.6: 1)

33

(6.

ACN-H20-TrEA(45:60:1)+0.3g

sodium acetate,pH 7.1 ACN-H20(45:60)+0. l g t e t r a d e c y l t r i r n e t h y l a m o n i um bromide pH 7.1 Ergtaine,Ergct, E r g c t i ne, d i Ergct, a-Ergcp ,a-Ergc p i ne,di Ha- Ergcp Ergco ,Ergcoine, diHErgco,Ergm diHErgta,bromocryptfne.atropine, emetine,ephedrine

Analysis i n pharmaceutical p r e p a r a t i o n s (Table 9.13)

H i t a c h i Gel no 3011-0 5-7 ldn

cyclohexane-EtOH-TrEA(70:30:0.5) 41

P i n d o l o l ,guanfacin,ketotifen, p i z o t i f e n ,clemastine

Post column d e r i v a t i z a t i o n L i c h r o s o r b DIOL.10 m using the f l u o r i m e t r i c L i c h r o s o r b RP8.10 um i o n - p a i r technique

wn

Ergta ,Ergtaine, a c i E r g t a ,aciErgtaine,Ergct,Ergcp, Ergco

D e t e c t i o n w i t h a photochemical r e a c t i o n d e t e c t o r

L i c h r o s o r b RP18,5

diHErgta

S t a b i l i t y studies

uBondapak C18

Detection i o n w i t h postcolumn d e r i v a t i z a t i o n

L i c h r o s o r b Si60.5 vm

Ergta,atropine

39

5 0 0 ~ 4 . 6~-hexane-EtOH-TrEA(70:30:0.5) n-hexane-CHC1 3-TrEA( 5:95: 0.5)

250x4 0.1M phosphate b u f f e r (pH 3 ) 1 0 0 ~ 4 . 6 MeOH-0.02M aq. phosphate b u f f e r (PH 3)(3:2)

42

1 2 0 ~ 4 . 6ACN-O.01M NaHC03(42:58), (38:62),pH 2.2 o r 8.5 44

Hydroxyatrazine

300x4 60x3

ACN-H20(2:3)

47

0.1M b u t y r i c a c i d i n CHC13MeOH( 9: 1 )

48

?

TABLE 9.18

E

HPLC ANALYSIS LSD AND RELATED COMPOUNDS I N DRUG SEIZURES AND AS PURE COMPOUNDS

0

ALKALOIDS

$ P P

*

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

COLUMN D I M LxlD(mn)

MOBILE PHASE

Sil-X

C o r a s i l I1

500~2.3 610~2.3

ACN-( isopr)20 ( 4 :6) ACN-(isopr)pO (25:75)

m

LSD,i soLSD,Lysac, Lysam, isolysam

STP,strychnine, phencycl i d i ne

Ergta,Ergtaine,diHErgta,

Ergct,Ergctine,diHErgct Ergcp ,Ergm ,MeErgm, MeMeErgm

A n a l y s i s i11i c i t preparations

REF.

, 1

LSO ,isoLSD,Lysac, Lysam,Lysgol .Ergta, Ergs ,Ergsi ne,Ergct, Ergctine,diHErgct. Ergcp.Ergcpine. diHErgcp, Ergco ,di HErgco , Ergm, Ergmine. MeErgm,MeMeErgm

I d e n t i f i c a t i o n LSO i n i l l i c i t p r e p a r a t i o n s by HPLC and GLC

C o r a s i l C18

1200x2.2

MeOH-0.1% aq. (NH4)2C03 (3:2)

L SD

Comparison o f photometric d e t e c t o r s f o r HPLC Identification street drugs

Zorbax S i l

250~2.1

CH2C1 2-MeOH-AcOH( 70:30:0.1)

C o r a s i l II,37-5Om

500~2.3

Separation drugs o f abuse (Fig.9.6)

Partisil 6

un

250~4.6

MeOH-0.2 M NHqN03(3:2)

A n a l y s i s i l l i c i t samples

P a r t i s i l 5 um

250~4.9

MeOH-O.3% aq. ( NH4)2C03 (3:2)

300x4

0.005M h e p t a n e s u l f o n i c a c i d i n MeOH-AcOH-H20 (40:1:59), pH 3.5

LSO,codeine,heroin, methadon,cocaine, strychnine,mescaline,quinine

3.8

Barbiturates,amphetami nes , various other drugs o f abuse

LSO ,isoLSD,Lysac, Lysam LSD LSD,isoLSD,Lysac, Lysam,Ergta,Ergm

Phencyclidine, N-methylpropylamide,various o t h e r drugs o f abuse

I o n - p a i r chromatography f o r pbondapak C18 t h e s e p a r a t i o n o f t h e drugs o f abuse

*For a b b r e v i a t i o n s see f o o t n o t e Table 9.19

4 Cyclohexane-cyclohexylamine (98.8:O .2 ) g r a d i e n t e l u t i o n (1inear) A to B A s k e l l y B-95%EtOH-dioxanecyc 1oh exy 1ami ne (99. I :50:25 :13) B idem (686:100:200:14) 5

9 16

20

LSD

Fluorescence d e t e c t i o n f o r HPLC-analysis LSD

Micropak MCH-10

250~2.2

ACN-O.1M (1:l)

LSD

Photochemical d e t e c t i o n f o r i d e n t i f i c a t i o n LSD

Spherisorb DDS

100~4.6

MeOH-aq. 0.025M Na2HP04 (65:35) pH 8

LSD,isoLSD

Semipreparative HPLC f o r uBondapak C18 the i d e n t i f i c a t i o n o f drugs o f f o r e n s i c i n t e r e s t P a r t i s i l - 1 0 ODS

LSD,lysergic a c i d methylpropylamide LSD

Benzocaine Phenothiazine type drugs

LSD,isoLSD,Lysac, Lysam,Lysgol , Ergta,diHErgta, Ergs ,Ergsine, Ergct,diHErgct, Ergcpine,diHErgcp, Ergco,diHErgco, Ergm,MeErgm,

A n a l y s i s i l l i c i t preparations

300~4.4 250~9.4

Bondapak C18

aq.(NH4)2C03

21 27,E

0.005M methanesulfonic a c i d i n MeOH-AcOH-H20(40:1:59), pH 3.5 0.04M methanesulfonic a c i d i n MeOH-AcOH-H20(40:1:59), 32, pH 3.5 49

300x4

ACN-H 0-1% aq.(NH4)2C03 (4OO:E72: 28)

37

Electrochemical d e t e c t o r

S i l i c a C S y l o i d 74,

2DOx4.6

MeOH- pH 10.2 NH4N03 b u f f e r (9:l) 45

Analysis i l l i c i t prepar a t i o n s w i t h TLC, HPLC and MS (Table 9.5)

Sherisorb 5 ODS

100~4.6

Spherisorb S5W

15Dx4.6

MeOH-0.025M Na2HP04(65: 35) PH 8 MeOH-O.2M NH4N03

7m

AK

HPLC ANALYSIS ERGOT ALKALOIDS I N BIOLOGICAL MATERIAL ALKALOIDS*

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

E r g t a i ne

Reserpine

Determination i n plasma

S i l i c a g e l 10

LSD,isoLSD

Detection i n b i o l o g i c a l f l u i d s , i n combination w i t h TLC

Partisi

diHErgct ,diHErgcp, diHErgco

Trace enrichment technique

Nucleos I C18, 5 un

Determination i n i n t e s t i n a l homogenate

Partisi

Ergta

Quinine

UII

6un

10/25 ODS

COLUMN D I M LxID(mn)

MOBILE PHASE

REF.

250~2.2

( i s o p r ) 0-ACN-MeOH (69.5: 36:O. 5 )

250~4.6

MeOH-0 .2M aq (11:9)

100x3

ACN-O.1M (2:3)

250~4.6

ACN-1% a c e t a t e b u f f e r pH 6.5 (55:45)

. NH4N03

aq.(NH4)2C03

7

15,13

22 23

P

f

I

P 0

m m

0-LSD,isoLSD,Lysac, Lysam,Lysgol , lumiLS0,Z-oxoLSD, Lysac monoethylamide,Ergta, diHErgta ,Ergs, E r g s i ne,Ergct, diHErgct,Ergcp, Ergcpine,diHErgcp, Ergco ,diHErgco, Ergm,Ergmi ne, MeErgm,MeMeErgm. Ergothioneine diHErgta,bromoc r y p t ine ,m e t ine , hyoscyamine, ephedr ine

A n a l y s i s LSO i n body f l u i d s Spherisorb 5 ODS (Table 9.5) Spherisorb S5W

100~4.6

D e t e c t i o n w i t h p o s t - c o l umn L i c h r o s o r b RP8 f l u o r i m e t r i c i o n - p a i r technique Lichrosorb Oiol

100x4 .6

28 Various drugs

5 um

E r g t a ,Ergtaine, MeErgm ,MeMeErgm, Ergct

A n a l y s i s i n plasma

H y p e r s i l OOS,

LSO

O e t e c t i o n i n serum and urine

L i c h r o s o r b RP8,7w w i t h column s w i t c h to L i c h r o s o r b S i 60, 5 Irn

*Abbreviations used i n Tables 9.16 Agcl Ccl Chcl E lc l Fcl Fucl Mcl Pcl Pycl Scl Lysam

150~4.6

MeOH-0.025M NazHP04(65:35) PH 8 MeOH-0.2M NHqN03(3:2)

-

250~4.6

MeOH-0.02M phosphate b u f f e r (PH 3)(3:2) 0.1M phosphate b u f f e r (PH 3 ) 42

250~4.6

ACN-O.OlM (1:l)

250x4

A MeOH-0.3% KH2PO4 (pH=3)

125x4

(NH4)2C03( 3:7), 53

(1:l)

B MeOH-1% (NH4)2C03(3:2)

s o l v e n t A f o r column s w i t c h system 62

9.19:

Agrocl a v i ne Costaclavine Chanocl a v i n e Elymoclavi ne F e s t u c l a v i ne Fumiclavine Molliclavine Penni c l a v i n e Pyrroclavine Setoclavine L y s e r g i c a c i d amide ( e r g i n e )

Lysac LSD LYsg Lysgol Ergm Ergmi ne Ergta E r g t a i ne MeErgm MeMeErgm Ergst

Lysergic a c i d 6-Lysergide Lysergine Lysergol Ergometrine (ergobasine o r ergonovine) Ergometri n i ne Ergotamine Ergotami n i n e Methylergometrine 1-Methylmethylergometrine (methysergide) E r g o s t i ne

Ergs t i n e Ergs Ergs ine Ergtox Ergct Ergctine Ergcp Ergcpi ne Ergco Ergcoine d i H-

E r g o s t i n i ne Ergosi ne Ergosi n i n e E r g o t o x i ne( ErgcttErgcptErgco) E r g o c r i s t i ne Ergocri s t i n i n e E r g o c r y p t i ne Ergocrypti nine Ergocornine Ergocorni n i ne dihydro-

w -a W