Chapter 14 Imidazole Alkaloids

421

Chapter 14 IMIDAZOLE ALKALOIDS

14.1. HPLC systems ..................................................... References.

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421 422

14.1. HPLC SYSTEMS Pilocarpine i s widely used in ophtalmology in eye-drops.However, in solution i t may undergo epimerization t o isopilocarpine, and even hydrolysis t o pilocarpic acid. In b o t h cases loss of pharmacological activity i s the result. Therefore. the analysis of ophtalmic solutions for pilocarpine and i t s decomposition products have been the subject of several investigations. The f i r s t methods described for the analysis of pilocarpine used a cation-exchange resin in combination with a basic DeGraw e t a 1 . l and Urbanyi e t al.3 used a TRIS buffer, 2 b u t i t was found t o degradecolumn performance . A sodium phosphate buffer - t r i e d instead of the TRIS buffer - caused problems as to the reproducibility of the separation 2 . Khalil 4 used an octadecyl and a cyanopropylsilane column in s e r i e s in order t o analyze pilocarpine in the presence of preservatives in ophtalmic preparations. Tetrahydrofuran - borate buffer (pH 9.2)(3:7) was used as mobile phase. Noordam e t a1 .5’9 reported the separation of pilocarpine. and the decomposition products mentioned above, on an octadecyl column with a mobile phase consisting of a mixture of water and methanol (97:3) containing 5%potassium hydrogen phosphate (pH 2.5)(Fig.14.1). I t was found t h a t increasing the s a l t concentration and lowering the pH lead t o improved s e l e c t i v i t y and peak shape. Kennedy and McNamara” found that replacing the octadecyl type of column in the method of Noordam e t a1.5’9 with a phenyl type of stationary phase reduced the analysis time, whilst improving peak shapes and resolution of pilocarpine and i t s degradation products. As mobile phase, 5%potassium dihydrogen phosphate (pH 2.5) in water was found t o be the most suitable. Kneckze6 applied ion-pair HPLC t o analyze ophtalmic solutions containing pilocarpine, physostigmine and rubreserine (see Chapter 8, Table 8.7, Fig.8.7). To improve the sensitivity of the pilocarpine analysis - enabling determinations in biological fluids - Mitra e t a1.’ developed a derivatization technique by means of which pilocarpine was quaternarized with the aid of e-nitrobenzylbromide (0.25 mg/ml - 24 hrs a t 4OoC). The quaternary derivatives were analyzed on an octadecyl column using 0.001 M sodium octanesulfonate in methanol - water ( 4 : l ) as mobile phase. the derivatization technique described was also applicable t o other amines. Straight-phase HPLC was used by Dunn e t a l . l O , whereby the alkaloids were separated on s i l i c a gel with hexane - 2% amnonia in propanol (7:3). To avoid interference of UV absorbing n i t r a t e , samples were passed through an ion-exchange column prior t o HPLC. Bundgaard and tiansen” separated pilocarpine and i t s degradation products on a s i l i c a gel column using methanol - 2 M phosphoric acid - water (3:5:92) containing 3% sodium s u l f a t e as mobile phase (Fig.14.2). The poor separation of pilocarpine and isopilocarpine a t a column temperature of 20-25’ C was improved by increasing the temperature t o 40’ C.

References p. 4 2 2

422

Detection o f p i l o c a r p i n e i s most s e n s i t i v e a t i t s UV maximum o f 215 nm. However. d e t e c t i o n a t 220 nm improves the s t a b i l i t y o f the baseline, whereas only a minor decrease - 5% o f the

-

peak h e i g h t o f p i l o c a r p i n e i s observed as compared w i t h 215.11m~~.The d e t e c t i o n l i m i t a t 215 nm i s about 0.04 pg, the d e t e c t i o n l i m i t obtained f o r the r e f r a c t i v e index method i s about 9 6 pg . REFERENCES

1 J . I . DeGraw, J.S. Engstrom and E. W i l l i s , J. P h a r m . Sci., 64 (1975) 1700. 2 J.D. Weber, J. ASSOC. Off. Anal. C h e m . , 59 (1976) 1409. 3 T. Urbanyi, A. Piedmont, E. W i l l i s and G. Manning, J . P h a r m . s c i . , 65 (1976) 257. 4 S. K.W. K h a l i l , J. P h a r m . Sci., 66 (1977) 1625. 5 A. Noordam, K. Waliszewski, C. Olieman, L. Maat and L. Beyerman, J . C h r o m a t o g r . , 153

(1978) 271. 6 M. Kneczke, J . C h r o m a t o g r . . 198 (1980) 529. 7 A.K. M i t r a , B.L. Baustian and T.J. Mikkelson. J. P h a r m . Sci., 69 (1980) 257. 8 J.J. O'Donnell. R. Sandman and M.V. Drake, J . P h a r m . Sci., 69 (1980) 1096. 9 A. Noordam, L. Maat and H.C. Beyerman, J . P h a r m . S c i . , 70 (1981) 96. 10 D.L. Dunn, B.S. S c o t t and E.D. Dorsey, J . P h a r m . Sci.. 70 (1981) 446. 11 J.M. Kennedy and P.E. McNamara, J . C h r o m a t o g r . , 212 (1981) 331. 12 H. Bundgaard and S.H. Hansen. rnt. J . P h a r m . , 10 (1982) 281. 13 M.V. Drake, J.J. O'Donnell and R.P. Sandman, J . P h a r m . S c i . , 71 (1982) 358.

3

2

min

20

10

0

o

1

8

1'2

min

Fig. 14.1. Separation o f p i l o c a r p i n e and degradation products5 Column Lichrosorb RP18 10 um (300x4 m I D ) , mobile phase 5% potassium dihydrogen phosphate i n water - methanol (97:3)(pH 2.5), f l o w r a t e 1.5 ml/min, d e t e c t i o n w i t h d i f f e r e n t i a l r e fractometer. Peaks: 1, pilocarpine; 2, i s o p i l o c a r p i n e ; 3, p i l o c a r p i c acid; 4, i s o p i l o c a r p i c acid. Fig. 14.2. Separation o f p i l o c a r p i n e and degradation products" Column Lichrosorb Si60 5 um (250~4.6 mn ID), mobile phase methanol - 2 M phosphoric a c i d water (3:5:92) containing 3% o f anhydrous sodium s u l f a t e , f l o w r a t e 1.2 ml/min. d e t e c t i o n UV 214 nm. Peaks: 1, pilocarpine; 2, isopilocarpine; 3, p i l o c a r p i c acid; 4. i s o p i l o c a r p i c acid; 5. n i t r a t e ; 6, unknown decomposition product.

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P2 t 3

TABLE 14.1 HPLC ANALYSIS PILOCARPINE AN0 RELATED ALKALOIDS

P

P

M

ALKALOIDS*

OTHER COMPOUNDS

AIMS

STATIONARY PHASE

COLUMN D I M . LxIO(mm)

1

0.1M Na-phosphate b u f f e r ( p H 9.0) i n 5% isoprOH

2

100x6

0.2M T r i s buffer-isoprOH(95:5)(pH 9 )

3

300x4 300x4

THF-borate b u f f e r ( p H 9 . 2 ) ( 3 : 7 )

Analysis synthesized p i 1

P i 1, i s o p i l ,pi l a c

A n a l y s i s ophtalmic s o l u t i o n s Aminex-7 7-11 um

Pi1,isopil

A n a l y s i s i n pharmaceuticals

Aminex-7 7-11 urn

A n a l y s i s i n pharmaceuticals

uBondapak C18 and WBondapak CN i n series

A n a l y s i s i n pharmaceuticals ( F i g . 14.1)

LichrosorbRP18 l O ~ m 3 0 0 x 4 o r N u c l e o s i l C18 150x4

A n a l y s i s i n pharmaceuticals (Table 7.7, Fig.7.7)

UBondapak C18

P i 1 .benzal k o n i um

Hydroxypropylmethylcellulose

P i 1, isopi 1 ,pi l a c .

is o p i 1ac

P i 1 .physostigmi ne. r u b r e s e r i ne

-

Pi1,isopi Pi1,isopi isopilac

,pilac,

Salicylate,phenethyl alcohol, methyl paraben

100x6 65x5.5

4 5% KH2P04 i n H20-MeOH(97:3)(pH 2.5) 59 9

3 0 0 ~ 3 . 9 MeOH-0.005M aq. h e p t a n e s u l f o n i c a c i d (pH 3.6)(2:3)

A n a l y s i s i n b i o l o g i c a l f l u i d s UBondapak C18 3 0 0 ~ 3 . 9 MeOH-H O ( 4 : l ) c o n t a i n i n g 0.001M Na-ocby d e r i v a t i z a t i o n p i 1 tanesubonate A n a l y s i s i n pharmaceuticals L i c h r o s o r b RP18 lOpm250x4.6 5% KH2P04 i n H20-MeOH(97:3)(pH 2.5)

wn

P i 1. i s o p i

A n a l y s i s i n pharmaceuticals

Si60 Hibar 5

Pi1,isopi ,pilac, isopilac

A n a l y s i s i n pharmaceuticals

UBondapak Phenyl

P i 1 , i s o p i l ,pi l a c , isopilac

A n a l y s i s p i 1 degradation p r o d u c t s i n b a s i c aqueous solutions(Fig.14.2)

L i c h r o s o r b Si60 5um 2 5 0 ~ 4 . 6 MeOH-2M H PO -H 0(3:5:92) 3% Na2S043

*

REF.

0.2M T r i s b u f f e r ( p H 9.2) i n 5% isoprOH

Pi1,isopil

Aminex A-7

MOBILE PHASE

2 5 0 ~ 4 . 6 Hexane-2% NH40H i n isoprOH(7:3) 3 0 0 ~ 3 . 9 5% KH2P04 i n H20(pH2.5)

6 7 8,13 10 11

containing

12

p i l = p i l o c a r p i n e , i s o p i l = i s o p i l o c a r p i n e , p i l a c = p i l o c a r p i c acid, i s o p i l a c = i s o p i l o c a r p i c a c i d

P

N w