Cotton Dust - American Chemical Society

19 Histamine in Cotton Plant and Dust Determined by H i g h Performance Liquid Chromatography

Downloaded by CORNELL UNIV on September 2, 2016 | http://pubs.acs.org Publication Date: July 1, 1982 | doi: 10.1021/bk-1982-0189.ch019

J. H. WALL, L. L. MULLER, and R. J. BERNI U.S. Department of Agriculture, Agricultural Research Service Southern Regional Research Center, New Orleans, LA 70179 Histamine is q u a n t i f i e d i n cotton dust and frost-killed cotton p l a n t s . D i r e c t determination of histamine i n aqueous e x t r a c t s is achieved by an ionpairing separation mechanism on a CN bonded phase column and using H SO as both buffer and the counter-ion in a water: acetonitrile mobile phase. Fluorescent detection o f histamine is performed by a post-column derivatization with o-phthaldehyde. Histamine was found primarily associated with the < 20 µm fraction o f cotton dust and highly concentrated i n the leaves o f the plant. Inhalation o f histamine can cause a b r o n c h o c o n s t r i c t i o n , confusing the diagnosis o f byssinosis. E a r l i e r workers concluded that histamine's concentration i n cotton dust was not great enough t o elicit t h i s reaction. T h e i r conclusions were based on data generated from biological analytical testing. These instrumental r e s u l t s agree with their findings. 2

4

S c i e n t i f i c knowledge c o n c e r n i n g h i s t a m i n e has d e v e l o p e d i n s t a g e s s i n c e i t was f i r s t s y n t h e s i z e d i n 1 9 0 7 ( 1 ) . I t was i s o l a t e d from an e r g o t p r e p a r a t i o n by B a r g e r and D a l e i n 1 9 1 0 ( 2 J and i t s d r a m a t i c p h a r m a c o l o g i c a l e f f e c t s d e m o n s t r a t e d . Dale then e s t a b l i s h e d t h a t h i s t a m i n e was a normal c o n s t i t u t e n t o f mammalian t i s s u e and w i d e l y d i s t r i b u t e d t h r o u g h o u t t h e human body ( 3 ) Next was t h e o b s e r v a t i o n t h a t h i s t a m i n e i s l i b e r a t e d i n t i s s u e s by an a n t i g e n - a n t i b o d y r e a c t i o n and i s r e s p o n s i b l e f o r a l a r g e s h a r e o f t h e symptoms o f a n a p h y l a x i s and t h e a l l e r g i c s t a t e . H i s t a m i n e p l a y s an i m p o r t a n t r o l e i n p h y s i o l o g i c a l f u n c t i o n s o f a n i m a l s and man; f o r example, h i s t a m i n e l e v e l s change i n c e r t a i n pathological c o n d i t i o n s . Histamine l e v e l s o r i t s formation are a f f e c t e d a l s o by v a r i o u s s t r e s s s i t u a t i o n s , such as e x p o s u r e t o c h e m i c a l compounds, e n d o t o x i n s , h y p e r - o r h y p o t h e r m i c c o n d i t i o n s . This chapter not subject to U . S . copyright. Published 1982 American Chemical Society.

Montalvo; Cotton Dust ACS Symposium Series; American Chemical Society: Washington, DC, 1982.


302

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DUST

H i s t a m i n e i s a potent v a s o d i l a t o r and a s t i m u l a t o r o f smooth muscle c o n t r a c t i o n . Histamine also occurs n a t u r a l l y in plant t i s s u e s . It a r i s e s from t h e d e c o m p o s i t i o n o f h i s t i d i n e , but i t s f u n c t i o n has not been e l u c i d a t e d . H i s t a m i n e l e v e l s i n some p l a n t s are s u r p r i s i n g l y h i g h - 1 , 3 4 0 yg/g i n the b l o s s o m s o f the s p i n a c h p l a n t ( 4 J . I t i s the e x p o s u r e o f man and animal t o t h i s b o t a n i c a l h i s t a m i n e w i t h a p o s s i b l e p h y s i o l o g i c a l a c t i o n t h a t makes h i s t a m i n e o f a g r i c u l t u r a l i m p o r t a n c e . The i n h a l a t i o n o f c o t t o n d u s t , f o r i n s t a n c e , has been r e l a t e d t o b y s s i n o s i s , a r e s p i r a t o r y d i s e a s e i n v o l v i n g a lung d y s f u n c t i o n . Histamine, a n a t u r a l l y o c c u r r i n g c o n s t i t u e n t o f the cotton p l a n t , i s , t h e r e f o r e , p r e s e n t i n c o t t o n d u s t . In 1 9 3 2 M a i t l a n d and c o w o r k e r s ( 5 ) i d e n t i f i e d a h i s t a m i n e - l i k e s u b s t a n c e i n t o t a l c o t t o n d u s t c o l l e c t e d by a h i g h - v o l u m e f i l t e r . They found the l e v e l s ranged from 4 t o 2 0 yg/g u s i n g b i o l o g i c a l methods f o r h i s t a m i n e measurements. They a l s o found i n d i c a t i o n s t h a t t h e h i s t a m i n e was a s s o c i a t e d w i t h the f i n e r p a r t i c l e s which would be i n h a l e d i n t o the l o w e r a i r w a y s . MacDonald ( 6 ) c o n t i n u e d t h i s work and i n 1 9 3 4 c o n c l u d e d t h a t the h i s t a m i n e - l i k e compound was a c o n s t a n t c o n s t i t u e n t o f c o t t o n d u s t , but i t s l e v e l v a r i e d c o n s i d e r a b l y i n samples measured and was a l m o s t e n t i r e l y a s s o c i a t e d w i t h the ,6.) have been r e p o r t e d as low a s 4 yg/g. T h i s i s a p p r o a c h i n g t h e lower edge o f of uv s e n s i t i v i t y when t h i s amount i s d i s s o l v e d i n t h e e x t r a c t i o n s o l v e n t . T h i r d , most p l a n t e x t r a c t s have many compounds t h a t absorb i n t h i s r e g i o n making r e s o l u t i o n o f t h e h i s t a m i n e impossible. F l u o r e s c e n c e d e t e c t i o n was s e l e c t e d t o i n c r e a s e s e n s i t i v i t y and s e l e c t i v i t y . H i s t a m i n e has no n a t u r a l f l u o r e s c e n c e and a post-column d e r i v a t i z a t i o n w i t h OPT was found t o be f a c i l e . The OPT r e a c t i o n w i t h h i s t a m i n e o r any p r i m a r y amine w i l l o n l y o c c u r i n an a l k a l i n e medium. The d e r i v a t i z a t i o n r e a g e n t , pumped i n t o t h e system a f t e r t h e m i x t u r e has been s e p a r a t e d on t h e column, must be s t r o n g l y b a s i c t o n e u t r a l i z e t h e a c i d i n t h e m o b i l e phase. The s t r u c t u r e o f t h e OPT adduct has been found t o be dependent upon t h e pH a t which t h e r e a c t i o n i s c a r r i e d out as w e l l a s t h e s o l v e n t system ( 1 5 ) . The r e s p o n s e o f t h e f l u o r e s c e n c e g e n e r a t e d by t h e p o s t column d e r i v a t i z a t i o n o f h i s t a m i n e w i t h OPT as a f u n c t i o n o f t h e peak h e i g h t i s shown i n F i g u r e 1. The c u r v e i s l i n e a r between 0.01 and 0.1 yg o f h i s t a m i n e , c o r r e l a t i o n c o e f f i c i e n t = .9986. The volume o f i n j e c t i o n s were a d j u s t e d so t h e c o n c e n t r a t i o n o f h i s t a m i n e would f a l l w i t h i n t h e l i n e a r range o f t h i s c u r v e . HPLC S e p a r a t i o n o f H i s t a m i n e . T h e r e a r e t h r e e p o s s i b i l i t i e s f o r s e p a r a t i o n o f h i s t a m i n e b y HPLC; normal phase on s i l i c a , r e v e r s e d phase on a bonded s i l i c a column, o r an i o n - e x c h a n g e s e p a r a t i o n . P e r i n i (16) d e m o n s t r a t e d t h a t a c a t i o n - e x c h a n g e s e p a r a t i o n i s p o s s i b l e , but t h e a n a l y s i s time was l e n g t h y f o r e x a m i n a t i o n o f h i s t a m i n e i n animal b i o f l u i d s (70 m i n ) . H i s t a m i n e i s f r e e l y s o l u b l e i n water, i s s l i g h t l y s o l u b l e i n hot c h l o r o f o r m , and i s i n s o l u b l e i n l e s s p o l a r s o l v e n t s , making a



Figure 1.

Histamine response, post-column derivatization with OPT.


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normal phase s e p a r a t i o n on s i l i c a u n l i k e l y . A r e v e r s e d phase s e p a r a t i o n i s most p l a u s i b l e and w a t e r m o b i l e phases would s i m p l i f y the c l e a n - u p p r o c e d u r e f o r aqueous e x t r a c t s b e f o r e anaylsis. Examination o f h i s t a m i n e ' s s t r u c t u r e ( F i g u r e 2) r e v e a l s i t s m u l t i p l e i o n i z a t i o n p o s s i b i l i t i e s . The a p p r o a c h t o s e p a r a t i n g i o n i z a b l e compounds i n w a t e r m o b i l e phases i s t o o p e r a t e w i t h the compound c o m p l e t e l y i n the i o n i c form, c o m p l e t e l y i n the molecul a r f o r m , o r a t some f i x e d d e g r e e o f i o n i z a t i o n . I o n i z a t i o n i s c o n t r o l l e d t h r o u g h b u f f e r i n g the m o b i l e phase. G e n e r a l l y , phosphate b u f f e r s a r e used t o a c h i e v e the d e s i r e d d e g r e e o f i o n i z a t i o n . However, p h o s p h a t e s can c a u s e d e t e r i o r a t i o n o f the s t a i n l e s s s t e e l i n the i n s t r u m e n t upon p r o l o n g e d u s e . F o r c i n g the h i s t a m i n e c o m p l e t e l y i n t o the i o n i c form by the a d d i t i o n o f a s t r o n g m i n e r a l a c i d , H2SO4, t o the m o b i l e phase was the a l t e r n a t e approach s e l e c t e d . The pH o f a b u f f e r e d m o b i l e phase i s s i g n i f i c a n t not o n l y b e c a u s e r e t e n t i o n o f i o n i z a b l e compounds i s dependent upon pH but because bonded phase columns a r e made v i a an a c i d c a t a l y z e d r e a c t i o n . M a n u f a c t u r e r s ' recommendations a r e t o a v o i d any system w i t h a pH lower than 2 o r a c c e p t the r i s k o f r e v e r s i n g the bondi n g r e a c t i o n . However, the e f f e c t i v e pH e x p e r i e n c e d by a column in a w a t e r i o r g a n i c m i x t u r e i s d i f f i c u l t t o p r e d i c t . We have o p e r a t e d a t a pH o f 1.5 i n t h i s l a b o r a t o r y w i t h no adverse e f f e c t s t o the column, but e x e r c i s e the s i m p l e p r e c a u t i o n o f not a l l o w i n g the a c i d i c m o b i l e phase t o remain on the column o v e r n i g h t o r s t a t i c f o r l o n g p e r i o d s o f time (1 h r ) . We have p a r t i c u l a r l y n o t i c e d t h a t the CN column i s s t a b l e a t low pH's. The u p p e r l i m i t o f a c i d s t r e n g t h t o a v o i d column damage i s about 0.1% by volume ( 1 7 ) . The pKa o f the i m i d a z o l e r i n g i s near 6 (16) s o h i s t a m i n e would o n l y e x i s t a s an ion i n the a c i d i c (pH = 2-3) m o b i l e phase. One would p r e d i c t no r e t e n t i o n on a bonded phase column under t h i s c o n d i t i o n ; however, i t does o c c u r . F i g u r e 3 i s the s i m p l e s t way t o a c c o u n t f o r t h i s r e t e n t i o n . Here, the m i n e r a l a c i d a c t s as the c o u n t e r - i o n , as w e l l as the b u f f e r . A l l o f the h i s t a m i n e in the m o b i l e phase i s i n the i o n i c form and i s i n e q u i l i b r i u m w i t h the i o n - p a i r which i s o n l y s o l u b l e i n the s t a t i o n a r y phase c h e m i c a l l y bonded t o s i l i c a . H i s t a m i n e o n l y e l u t e s i n the i o n i c form and i s then d e r i v a t i z e d f o r d e t e c t i o n . A sharp peak i n the chromatogram w i t h good shape and no change i n r e t e n t i o n time w i t h v a r i a t i o n i n sample c o n c e n t r a t i o n i n d i c a t e s a w o r k i n g system. However, i f the p a i r e d i o n has some s o l u b i l i t y i n the m o b i l e phase, peak t a i l i n g o c c u r s . T h i s i o n - p a i r system was s t i l l h y d r o p h y l i c i n n a t u r e and w i t h a C3 o r C13 column peak t a i l i n g was e x t r e m e l y s e v e r e ( F i g u r e 4). G e n e r a l l y , a d j u s t m e n t s i n the w a t e r o r g a n i c r a t i o s w i l l n o t improve t h i s c o n d i t i o n . I t can be c o r r e c t e d by i n c r e a s i n g the c o n c e n t r a t i o n o f the c o u n t e r - i o n i n the m o b i l e phase, but, i n t h i s c a s e , adding more c o u n t e r - i o n would i n c r e a s e the a c i d


W A L L ET A L .

Histamine

in Cotton Plant and Dust


19.

Figure 3.

Ion-pairing retention mechanism.


307

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308

DUST

s t r e n g t h . The o n l y o t h e r p o s s i b i l i t y was t o change the p o l a r i t y o f t h e bonded phase; t h e r e f o r e , a CN column w i t h e q u i v a l e n t p o l a r i t y as s i l i c a i t s e l f was chosen and t a i l i n g e l i m i n a t e d as demonstrated by t h e peak shapes i n F i g u r e 4. U s i n g the CN column and an e l u e n t o f 25:75 a c e t o n i t r i l e : w a t e r w i t h 0.035% s u l f u r i c a c i d ( v : v : v ) , good peak shapes were o b t a i n e d w i t h no change i n r e t e n t i o n time w i t h v a r i a t i o n i n sample c o n c e n t r a t i o n . F i g u r e 5 r e p r e s e n t s a t y p i c a l chromatogram w i t h h i s t a m i n e e l u t i n g as the l a s t m a j o r peak. The f l o w r a t e was 6 ml/min w i t h t a t 0.5 m i n u t e s . H i s t a m i n e has a k' o f 4.4. Some o f the i n i t i a l peaks are f l u o r e s c e n t compounds as w e l l as some o t h e r amines. UV d e t e c t i o n a t v a r i o u s w a v e l e n g t h s showed no compounds e l u t i n g p a s t 6 min. UV d e t e c t i o n was u n s u i t a b l e f o r h i s t a m i n e due t o i n t e r f e r i n g compounds a b s o r b i n g from 200 t o 240 nm.


0

H i s t a m i n e E x t r a c t i o n . Some s e c o n d a r y p l a n t m e t a b o l i t e s a r e v e r y d i f f i c u l t t o e x t r a c t from t h e i r n a t u r a l m a t r i x and r e q u i r e l e n g t h l y s o x h l e t e x t r a c t i o n s . Complete h i s t a m i n e e x t r a c t i o n was r e l a t i v e l y s i m p l e . The HPLC s e p a r a t i o n was used t o d e s i g n a t e c h nique t o c o n f i r m a complete e x t r a c t i o n . The h i s t a m i n e peaks from t h r e e s e r i a l e x t r a c t i o n s on the same 3 grams o f c o t t o n p l a n t l e a v e s a r e shown i n F i g u r e 6. Post-column f l u o r e s c e n c e d e t e c t i o n o f t h e f o u r t h e x t r a c t i o n showed o n l y the s l i g h t e s t response even a t the h i g h e s t d e t e c t o r a m p l i t u d e . Three e x t r a c t i o n s accounted f o r from 95% t o 99% o f the h i s t a m i n e c o n t e n t . The p l a n t r e s i d u e s from t h e s e e x t r a c t i o n s y i e l d e d no a d d i t i o n a l h i s t a m i n e a f t e r s t a n d i n g a t ambient c o n d i t i o n s f o r two weeks. H i s t a m i n e C o n t e n t . V a l u e s f o r h i s t a m i n e i n c o t t o n d u s t and some r e l a t e d m a t e r i a l a r e g i v e n i n T a b l e I and the 14.7 yg/g f o r TABLE I H i s t a m i n e Content Sample Bracts Gin T r a s h T o t a l Dust < 20 urn Dust

uq/q 56.3 23.2 14.7 108.9

t o t a l d u s t agrees w i t h e a r l i e r work (.5,8). T h i s d u s t c o n t a i n e d about a 10% _< 20 ym f r a c t i o n , so 108.9 yg/g i s a c c u r a t e s i n c e h i s t a m i n e i s a s s o c i a t e d w i t h the f i n e r p a r t i c l e s . The b r a c t samples examined c o n t a i n e d o n l y about h a l f as much h i s t a m i n e as the f i n e d u s t which i s s u r p r i s i n g because i t has been e s t a b l i s h e d t h a t b r a c t and l e a v e s a r e t h e p r i n c i p l e c o n t r i b u t o r s t o the _< 20 ym f r a c t i o n o f c o t t o n d u s t ( 1 8 ) . Data shown i n T a b l e I I e x p l a i n s t h i s anomaly. The l e a v e s o f t h e c o t t o n p l a n t have 3 t o 4 times


19.

WALL E T AL.

Histamine


309

CN


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5

F L O W : 6 ml/ n l i

Figure 4.

Histamine with OPT derivatization. .035% H S0 (v); 6 mL/min. 2

H 0/CH CN(75:25); 2

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Figure 5. Histamine in cotton plant leaves, peak caused by histamine is indicated by the arrow. Key: column, radial-PAK CN; eluent, CH CN/H O(25:75);.035% HzSOrfv); detection, fluor., post-column with OPT; flow, 6 mL/min. 3

2


310

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DUST

1st 30 m L

2nd 30 m L


3rd 30 m L

Figure 6.

Water extraction efficiency. Histamine from 3g cotton plant leaves.

as much h i s t a m i n e as t h e b r a c t s . Such d i f f e r e n c e might r e s u l t from t h e f a c t t h a t t h e b r a c t i s a m o d i f i e d l e a f and p r o v i d e s p r o t e c t i o n f o r t h e young b o l l and perhaps a l i m i t e d p h o t o s y n t h e s i s f u n c t i o n . These r e s u l t s show t h a t h i s t a m i n e i s d i s t r i b u t e d TABLE I I H i s t a m i n e D i s t r i b u t i o n i n t h e Cotton Sample Bracts Leaves Stems Roots Fiber

P l a n t No. 1 yg/g 48.4 191.7 50.4 12.8 0

Plant

P l a n t No. 2 y g / g 74.2 229.5 33.0 42.5 0

t h r o u g h o u t t h e c o t t o n p l a n t , except f o r t h e f i b e r , w i t h a high c o n c e n t r a t i o n i n t h e l e a v e s . T h e r e s h o u l d be more b r a c t s than l e a v e s c o n t r i b u t i n g t o t h e f i n e d u s t so around 100 yg/g i n t h e £ 20 ym f r a c t i o n i s r e a s o n a b l e .


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WALL ETAL.

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311

Conclusions The HPLC method p r e s e n t e d i s based upon t h e s e p a r a t i o n of h i s t a m i n e i t s e l f and n o t a d e r i v a t i v e o f unknown s t r u c t u r e . An i o n - p a i r model i s proposed t o c o n t r o l r e t e n t i o n o f h i s t a m i n e on bonded phase columns. T h i s method and model s h o u l d be a p p l i c a b l e to a wide range o f b i o s y s t e m s , as w e l l as o t h e r aqueous p l a n t e x t r a c t s . Our r e s u l t s d e t e r m i n e d by HPLC a n a l y s i s a g r e e c l o s e l y w i t h t h o s e o b t a i n e d p r e v i o u s l y u s i n g b i o l o g i c a l methods b u t p r o v i d e g e a t e r r e p r o d u c i b i l i t y and s i m p l i c i t y .


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LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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Cotton Dust - American Chemical Society

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