The Ochratoxins and Other Related Compounds

Mycotoxins and Other Fungal Related Food Problems Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 09/22/18. For personal use only.

12 The Ochratoxins and Other Related Compounds STANLEY NESHEIM Division of Chemistry and Physics, Food and Drug Administration, 200 "C" Street, S. W., Washington, D.C. 20204

The ochratoxins are toxic metabolites elaborated by several species of Aspergillus ochraceus as well as by Penicillium viridicatum. The discovery, chemistry, history, sources, oc­ currence, toxicology, and fate in foods and feeds of these metabolites are reviewed. Emphasized are the analytical methods for the detection and estimation of the ochratoxins with reference to their applicability in commodities such as grains, nuts, beer, and animal tissues. Similarities and dif­ ferences in sampling procedures, extraction methods and solvents, extract purification, toxin detection and estimation, and chemical and biological techniques of confirmation of identity are described. Other metabolites which interfere with current methods are listed. Suggestions to direct fur­ ther efforts in developing analytical methods of ochratoxins are proposed.

In a screening program to determine possible causes of certain animal diseases in South Africa, Scott showed that three of five strains of Aspergillus ochraceus Wilh isolated from local domestic legumes and cereal products were toxigenic (1). The toxic metabolites were later isolated and chemically characterized (2, 3, 4). The major toxic com­ pound was named ochratoxin A; several related compounds were also isolated and identified (Figure 1). More recently one or more of the ochratoxins have been isolated from various mold species of the Asper­ gillus and Penicillium genera (5-14). These molds are commonly found in soils and on food and feed commodities such as nuts, dried fish, grains, beans, peas, coffee, spices, alfalfa, and meats (9, 10, 12, 15-24). Ochra­ toxin A, the 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3-R-methyl isocou­ marin amide of L-β-phenylalanine (Figure 1), is the most toxic of the 276

12.

277

Ochratoxins

NESHEIM

ochratoxins ( 2 5 )

a n d is p r o d u c e d i n h i g h e s t y i e l d ( 2 6 ) .

s t r u c t u r e has b e e n c o n f i r m e d b y synthesis ( 2 7 , 2 8 ) . t o x i n B , its d e c h l o r o a n a l o g , a r e a c i d i c c o m p o u n d s a n d under ultraviolet light.

Its c h e m i c a l

Both it and ochra­ fluoresce

intensely

T h e y are r e l a t i v e l y h e a t stable, r e s i s t i n g a u t o -

claving i n oatmeal a n d cereal (29).

H o w e v e r , t h e y are l a r g e l y d e s t r o y e d

i n coffee b y h e a t i n g to 2 0 0 ° C f o r 5 m i n o r m o r e u n d e r s i m u l a t e d coffee roasting conditions (21).

B o t h ochratoxins A a n d Β h a v e b e e n f o u n d as

n a t u r a l c o n t a m i n a n t s of f o o d a n d feeds s u c h as w h e a t , b a r l e y , oats, coffee beans, c o r n , r y e , a n d d r i e d w h i t e beans ( 9 , 1 0 , 1 2 , 1 4 , 29-39). d e t e c t e d r a n g e d f r o m < 1 0 / * g / k g to 28 m g / k g .

T h e levels

T h e h i g h e r levels w e r e

f o u n d i n l o w e r g r a d e or m o l d y m a t e r i a l s . I n D e n m a r k , w h e r e

samples

w e r e t a k e n f r o m districts e x p e r i e n c i n g a h i g h i n c i d e n c e of s w i n e n e p h r o p ­ a t h y , 5 8 % of 33 samples w e r e c o n t a m i n a t e d , a n d a v e r a g e d 3 m g / k g .

In

o t h e r surveys there a n d elsewhere i n v o l v i n g n o n s e l e c t e d samples t h e i n c i ­ d e n c e a n d degree of c o n t a m i n a t i o n w a s l o w . T o c o n t r o l , r e d u c e o r e l i m i ­ nate o c h r a t o x i n c o n t a m i n a t i o n , factors w h i c h c a n m o d i f y m o l d ment (including moisture, temperature, aeration, time, a n d h a v e b e e n i n v e s t i g a t e d (21, 40, 41).

develop­ substrate)

T h e f a t e of o c h r a t o x i n i n the n o r m a l

use of c o n t a m i n a t e d p r o d u c t s has also b e e n s t u d i e d . A s m e n t i o n e d a b o v e , u n d e r s i m u l a t e d coffee r o a s t i n g c o n d i t i o n s t h e ochratoxins are l a r g e l y d e s t r o y e d (21),

b u t 3 0 % r e m a i n e d i n t a c t i n o a t m e a l o r r i c e cereals after

3 h r of a u t o c l a v i n g ( 2 9 ) .

O n m a l t i n g a n d b r e w i n g beer f r o m barley con­

t a m i n a t e d w i t h 1-5000 / x g / k g o c h r a t o x i n A the b e e r c o n t a i n e d f r o m /Ag/1.; this corresponds to a 2 - 7 % Several

investigators

transmission

studied

the

effects p r o d u c e d b y t h e ochratoxins.

6-20

(42).

toxicological

and

pathological

T h e L D o for ochratoxin A a d m i n ­ 5

i s t e r e d per os varies f r o m 2.1-4.67 m g / k g for the c h i c k , s w i n e , a n d t r o u t to 22 m g / k g for the f e m a l e r a t (12, 25, 30, 33, 43-57).

O c h r a t o x i n A has

b e e n r e p o r t e d as teratogenic to m i c e a n d rats ( 5 1 , 59, 5 9 )

a n d to t h e

c h i c k e n e m b r y o (60);

teratogenic

other investigators h o w e v e r r e p o r t n o

effects i n c h i c k e n e m b r y o s

(61,

62).

A dose of 200 /xg o c h r a t o x i n A / k g

b o d y w e i g h t has b e e n r e p o r t e d to p r o d u c e nephrosis i n p i g s (31,

Figure 1. Ochratoxin A: R =H,R = Cl; ochratoxin B: Ri = R2 = H; ochratoxin C: R = -CH -CH , R = Cl 1

2

1

2

S

2

52).

278

MYCOTOXINS

T h e d e p o s i t i o n of o c h r a t o x i n A i n the l i v e r , k i d n e y , fat, a n d m u s c l e tissues of s w i n e a n d the e x c r e t i o n of o c h r a t o x i n A i n c o w s ' m i l k h a v e b e e n r e ­ ported

(30,63,64).

T o h e l p detect, c o n t r o l , a n d e l i m i n a t e o c h r a t o x i n f r o m h u m a n a n d a n i m a l foods a n d feeds, sensitive a n a l y t i c a l m e t h o d s screening methods have been developed.

i n c l u d i n g some

S o m e of these h a v e b e e n u s e d

f o r t h e s u r v e i l l a n c e of v a r i o u s c o m m o d i t i e s a n d to detect the n a t u r a l o c c u r r e n c e of ochratoxins.

T w o of the o c h r a t o x i n m e t h o d s h a v e b e e n

tested i n i n t e r l a b o r a t o r y c o l l a b o r a t i v e studies (65, 66)

and were adopted

"official first a c t i o n ' for i n c l u s i o n i n O f f i c i a l M e t h o d s of A n a l y s i s " of t h e A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s (67).

S u c h a d o p t i o n means

t h a t the m e t h o d has b e e n v a l i d a t e d b y t e s t i n g i n s e v e r a l laboratories o n i d e n t i c a l sets of samples c o n t a i n i n g ochratoxins at u n k n o w n levels o c c u r ­ r i n g b o t h as a n a t u r a l c o n t a m i n a n t a n d as a n a d d e d c o n t a m i n a n t . A l t h o u g h s e v e r a l g o o d g e n e r a l r e v i e w s c o v e r i n g b r o a d areas of t h e subject ( 1,12,14,33,47, 72, 73)

68-71

) a n d r e v i e w s of some of the m e t h o d s

(25,

h a v e b e e n p r e p a r e d , n o i n - d e p t h r e v i e w of t h e m e t h o d o l o g y has

been made.

T h i s c h a p t e r attempts to s u m m a r i z e a l l the w o r k o n m e t h ­

o d o l o g y i n c l u d i n g m u c h t h a t has b e e n scattered as parts of reports i n o t h e r areas, fields, o r d i s c i p l i n e s . M o s t of the p u b l i s h e d o c h r a t o x i n m e t h o d s d e s c r i b e d b e l o w

apply

o n l y to t h e analysis of o c h r a t o x i n s A a n d B . A f e w m e t h o d s h a v e b e e n a p p l i e d to t h e analysis of the m e t h y l a n d e t h y l esters (2-4,15, 67, 74-78).

26, 43,

65,

A l t h o u g h the esters of o c h r a t o x i n A are as t o x i c as the p a r e n t

c o m p o u n d s (47),

t h e y h a v e b e e n f o u n d o n l y i n substrates m o l d e d u n d e r

l a b o r a t o r y c o n d i t i o n s a n d at m u c h l o w e r concentrations t h a n those ochratoxins A a n d Β

of

(4).

A U o c h r a t o x i n m e t h o d s consist of t w o or m o r e of the f o l l o w i n g b a s i c steps: lot s a m p l i n g , a n a l y t i c a l s a m p l e p r e p a r a t i o n , e x t r a c t i o n of the toxins, p u r i f i c a t i o n o r c o n c e n t r a t i o n , d e t e c t i o n , q u a n t i t a t i o n , a n d c o n f i r m a t i o n of i d e n t i t y of the t o x i n . T h e m e t h o d s differ i n c o m m o d i t y a p p l i c a b i l i t y , sen­ s i t i v i t y ( o r m i n i m u m d e t e c t i o n l e v e l ) , a c c u r a c y , p r e c i s i o n , a m o u n t of v a l i d a t i o n d a t a s u p p o r t i n g t h e m e t h o d , cost of m a t e r i a l s , e q u i p m e n t , t i m e , a n d labor. T h e c h o i c e of a m e t h o d is d i c t a t e d b y factors s u c h as t y p e a n d n u m ­ b e r of samples t o b e a n a l y z e d a n d i n f o r m a t i o n r e q u i r e d for the p a r t i c u l a r s t u d y . T h e a n a l y s t s h o u l d n o t b e l i m i t e d to a n y specific m e t h o d .

For a

s u r v e y , f o r e x a m p l e , h e c a n a n d s h o u l d use t h e s i m p l e s t r e l i a b l e m e t h o d a v a i l a b l e (i.e.,

a screening m e t h o d )

to e l i m i n a t e the m a n y

negative

samples a n d t h e n p e r h a p s a p p l y a n o t h e r , m o r e r i g o r o u s m e t h o d to the p o s i t i v e samples. O f course t h e s c r e e n i n g m e t h o d s h o u l d h a v e a s e n s i t i v ­ i t y a d e q u a t e to the needs of t h e s u r v e y . M e t h o d s are c o n t i n u a l l y i m p r o v e d as n e w t e c h n i q u e s a p p e a r a n d

Ochratoxins

279

are a p p l i e d to o l d p r o b l e m s .

T h e a n n u a l r e p o r t of t h e G e n e r a l R e f e r e e

12.

NESHEiM

o n M y c o t o x i n s o f t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s is p u b ­ l i s h e d e v e r y y e a r i n t h e M a r c h issue o f t h e Journal Official

Analytical

Chemists

ments i n m e t h o d o l o g y . s u c h as Ό c h r a t o x i n s ,

of the Association

of

a n d provides a continuing review of develop­

A s s o c i a t e Referees a r e assigned t o separate topics ,,

" A f l a t o x i n s i n Coffee," " M y c o t o x i n s i n G r a i n s , "

to n a m e a f e w , u n d e r t h e r e v i e w of t h e G e n e r a l Referee.

T h e Associate

Referees also r e p o r t progress i n t h e i r assignments b u t o n a less r e g u l a r basis.

Lot Sampling F o r m y c o t o x i n s i n g e n e r a l o r f o r ochratoxins specifically, t h e c o n t a m i ­ n a t i o n i n t h e c o m m o d i t y m a y b e i n s m a l l pockets o r i n a f e w o f m a n y u n i t s of t h e c o m m o d i t y .

A . ochraceus

is k n o w n to p r o d u c e as h i g h as

1.3-1.5 m g o c h r a t o x i n A / k g o n c h o p p e d c o r n , p o l i s h e d r i c e , o r w h e a t b r a n ( 2 9 ) a n d 3.9 g / k g o n s h r e d d e d w h e a t ( 7 9 ) . T h e s e h i g h levels c a n b e associated w i t h v e r y s m a l l portions o f a t o t a l lot. T h e r e f o r e , l a r g e samples m u s t b e t a k e n to increase t h e p r o b a b i l i t y of i n c l u d i n g c o n t a m i ­ n a t e d p o r t i o n s o f heterogeneous lots.

C o m m o n p r a c t i c e f o r aflatoxin

analysis is to t a k e a 25 k g s a m p l e f o r larger k e r n e l m a t e r i a l s s u c h as nuts a n d a five k g s a m p l e f o r c o m m o d i t i e s s u c h as s m a l l grains. T h e c o n s i d e r a ­ tions i n v o l v e d i n s a m p l i n g peanuts h a v e b e e n p u b l i s h e d

(80-82).

Analytical Sample Preparation T h e effectiveness of s o m e a p p r o a c h e s to r e d u c t i o n of t h e l o t s a m p l e to a n a n a l y t i c a l s a m p l e , 1 0 - 3 5 0 g, has b e e n s t u d i e d ( 8 3 , 84).

Generally

10 g is c o n s i d e r e d too s m a l l f o r a d e q u a t e a n a l y t i c a l s a m p l i n g ; c u r r e n t l y a 100-g s a m p l e is r e c o m m e n d e d .

F o r s m a l l samples (


100 m g f r o m

10-g samples of c o m m o d i t i e s s u c h as grains or nuts. T h i s m i x t u r e is t h e r e ­ f o r e f u r t h e r f r a c t i o n a t e d ( u s u a l l y b y T L C ) , a n d the toxins are d e t e r m i n e d b y v i s u a l o r i n s t r u m e n t a l c o m p a r i s o n o n t h i n l a y e r plates of the

fluores­

c e n c e intensities of the u n k n o w n spots w i t h spots of the T L C standards. T h e m a n y solvents a n d absorbents w h i c h h a v e b e e n u s e d for the T L C d e t e c t i o n a n d e s t i m a t i o n of ochratoxins a n d some t y p i c a l R

f

values are

l i s t e d i n T a b l e V . I t s h o u l d b e e m p h a s i z e d t h a t n e a r l y a l l the

methods

d e p e n d o n g o o d T L C r e s o l u t i o n of t h e toxins f r o m e a c h other a n d f r o m i n t e r f e r i n g m a t e r i a l s . T h e T L C R values are not v e r y r e p r o d u c i b l e , a n d t

u n k n o w n spots are best i d e n t i f i e d b y c h r o m a t o g r a p h y w i t h k n o w n o c h r a ­ t o x i n standards a d d e d to the extract either i n s o l u t i o n or o n the o r i g i n s p o t o n the T L C p l a t e . I d e n t i c a l R values of u n k n o w n a n d s t a n d a r d , e v e n t

i n s e v e r a l T L C systems, d o not p o s i t i v e l y i d e n t i f y t h e u n k n o w n .

Usually

f u r t h e r c o n f i r m a t i o n of i d e n t i t y m u s t b e m a d e . T h i s is discussed

below.

12.

285

Ochratoxins

NESHEIM

R

Table V .

V a l u e s ' of Ochratoxins for T h e i r Separation, Detection and Quantitation

t

Ochratoxins: Esters" of Solvent"

Α Thin Layer

Β

Chromatography

A (Silica

C e H - H O A c , 25:1 C H -HOAc,9:l

45

24

C H6-HOAc,8:2 C H -H0Ac,3:l C H -HOAc,4:l

50 69

35 45

65

C H - H O A c - H 0 90:10:1 C H - M e O H - H O A c , 18:1:1

32 65

50

100

C e H e - M e O H - H O A c , 12:2:1 C H e - M e O H - H O A c , 24:2:1

60 60

52 35

14

0

0

e

55 67

Β Gel) 30 46

e

e

e

e

6

6

e

6

c

2

e

C H -CCl -nBuOH-EtOAcformic acid, 35:25:20:19:1 C H - M e O H - E t O A c , 15:3:1 C H - E t O A c - f o r m i c acid, 70:30:1 C H - a c e t o n e - f o r m i c acid, 80:20:1 C H -BuOH-EtOAc-formic acid, 69:15:12:4 C He-BuOH-EtOAc-formic acid, 50:15:33:2 Toluene-EtOAc-formic acid, 5:4:1 e

6

4

100

4,27 1, 5 - 9 , 1 3 , 26, 3 7 , 7 5 , 79,90 111,112 78,86,110 27, 6 2 , 1 0 4 , 108,111 113 35-38,96 8,21,35,36, 6 7 , 7 4 , 76 77,96,117 1-3,86,111 9,10,85,87 100,115-117 15 14, 9 7 , 1 0 5 28

e

6

31 50

6

e

46

e

e

74

65

81

15

71

63

80

15

e

6

e

Toluene-EtOAc-formic acid, 6:3:1 EtOAc-t-PrOH-H 0,5:2:1 Et20-MeOH-H 0-formic acid, 95:4:1:1 CHCI -acetone,93:7 CHCls-EtOAc-formic acid, 60:40:1 CHCls-acetone-i-PrOH, 85:12.5:2.5 2

2

3

74

References

28

70

55-70 42 27

25

95

13,18,21,28, 3 5 , 3 6 , 75, 87, 9 0 , 9 1 , 94-96,99102, 111, 118 6,7,9,10,98, 116 97 10 91 119 15

286

MYCOTOXINS

Table V .

Continued Ochratoxins: Esters' of

Solvent*

Α

CHCl -acetone-formic acid, 80:20:1 C H C l - M i B K , 4:1 (Oxalic acid impregnated silica) D i - i s o p r o p y l ether ( O x a l i c acid impregnated silica) Hexane-acetone-HOAc, 18:2:1

A

Β

References

65

3

48

3

Thin Layer

Β

28,102 20

1,120

35

28 50

Chromatography

67,74,76

(Alumina)

CHCl -acetone,4:l

9

3

Paper i-PrOH-3iV (ΝΗ ) 00 ,3:1 Hexane (formamide i m p r e g n a t e d paper) 4

Silica

2

3

Gel Mini-Column

Chromatography 65 50 Chromatography

18

2,3,86 4

Detection

Toluene-EtOAc-formic acid, 5:4:1

4

89

° RfxlOO.

See a, Table I. • Methyl or ethyl esters of ochratoxins A and B . Fluorescent band detected on 4 mm X 7 cm silica gel column.

b

d

T h e m e t h o d of v i s u a l c o m p a r i s o n of fluorescence intensities suffers f r o m p o o r p r e c i s i o n ( > db 2 0 % ) ( 6 5 ) a n d d e p e n d s g r e a t l y o n t h e a n a ­ lyst's a b i l i t y a n d experience. T h e T L C d e n s i t o m e t r y is m o r e objective b u t suffers m o r e f r o m i n s t r u m e n t a l p r o b l e m s , interferences, a n d p o o r r e s o l u t i o n i n T L C . T h e types of T L C s c a n n i n g i n s t r u m e n t s , l i n e a r ranges a n d sensitivities o b t a i n e d w i t h t h e m are s h o w n i n T a b l e V I . A l s o l i s t e d i n T a b l e V I are o t h e r a n a l y t i c a l m e t h o d s . T h e s e are less sensitive o r m o r e c o m p l i c a t e d to use b u t h a v e b e e n u s e d i n s p e c i a l c i r c u m s t a n c e s as i n d i ­ c a t e d i n references l i s t e d i n t h e t a b l e .

Chemical Confirmation of Identity T h e presence of ochratoxins i n a s a m p l e is i n i t i a l l y b a s e d o n t h e i d e n t i t y of t h e R values of t h e u n k n o w n s w i t h those o f s t a n d a r d o c h r a ­ toxins. A d d i t i o n a l p r o o f is m o s t f r e q u e n t l y o b t a i n e d b y d e v e l o p i n g c h r o m a t o g r a m s i n several solvent systems. T h i s is c o n c l u s i v e e v i d e n c e o n l y i n a negative sense—i.e., t h a t t h e i n i t i a l suspect spots are n o t o c h r a ­ toxins. F o r p o s i t i v e p r o o f t h e toxins are separated a n d p u r i f i e d b y p r e t

12.

Table V I .

287

Ochratoxins

NESHEIM

Instrumental Methods for Quantitation of Ochratoxins (ng/spot) Linear Range

Instrument

Sensitivity

CV

h

References

TLC Fluorodensitometry P h o t o v o l t , m o d e l 530 10-100 7.1 15,26,91,101 Ozumor,SD-91,Asuka 0-5000 170 14,105 Kogyo Co., Tokyo H i t a c h i F l u o r o spec6 5 , 74 trometer, M P F - 2 A , T L C accessory 018-0057 Schoeffel, S D 3000 3-10 65,77 A m i n c o - B o w m a n spectro 0-10 0.5-1.0 3.6 5 , 4 3 , 8 5 , 1 1 4 , 121 photo fluorometer A m i n c o - B o w m a n spectro 0.25-5 0.25 52,117 p h o t o fluorometer (after exposing T L C spots t o N H ) F o u r different i n s t r u 6.4 32,42, 52,65,67, ments ( P h o t o v o l t , 74 Schoeffel, A m i n c o Bowman and Hitachi) Miscellaneous Techniques U V spectrophotometry sensitivity 15,18,26, 28,43-45, 0.5-5 /xg/ml 67,74, 85,98,103, 109,110,112,118, 121-123 U V spectrophotometric dialysis 121 and titration Phosphorescence a n d fluorescence 118 H y d r o l y s i s followed b y G L C 43,121 analysis for phenylalanine R a d i o c h e m i c a l methods 62,99,108,110 a

3

In acid medium : excitation maximum 310^340 nm, emission maximum 440-475 n m ; i n basic medium: excitation maximum 360-390 nm, emission maximum 427445 nm. br* m - χ £ · χ· standard deviation Χ 100 ° Coefficient of variation = average paratory T L C a n d identified b y one or m o r e of the p h y s i c a l c h e m i c a l or β

bioassay techniques ( T a b l e V I I ) . A l m o s t every instrument for measuring p h y s i c a l parameters has been a p p l i e d t o t h e i d e n t i f i c a t i o n o f o c h r a t o x i n s ( i r , u v , N M R , a n d mass spec­ trometry).

P h y s i c a l a n d c h e m i c a l p r o p e r t i e s s u c h as m e l t i n g p o i n t s ,

e l e m e n t a l a n a l y s i s , a n d c h e m i c a l d e r i v a t i v e f o r m a t i o n h a v e also u s e d . T h e b a s i c T L C sprays l i s t e d i n T a b l e V c h a n g e t h e

been

fluorescence

of

o c h r a t o x i n s A a n d Β f r o m g r e e n t o b l u e b y f o r m i n g a salt w i t h t h e phenolic functional group.

288

MYCOTOXINS

Table VII.

Chemical Confirmation of Identity of Ochratoxins

TLC Sprays

References

N E U O H or N H fumes 3

Triethylamine NaHC0 FeCl K O H (20%) A1C1 3

3

3

Repeated TLC with up to 10 different solvent systems Solubility in NaHCO solution Chemical derivatives M e t h y l esters a n d ethers s

5 - 7 , 1 0 , 1 3 , 3 5 , 36, 87, 9 0 , 9 1 , 93, 94,99-101,117,119 87 67,74 7,104,120 75 6,7 4 , 9 , 27, 2 8 , 3 5 , 3 6 , 8 6 , 8 7 , 90, 95 9, 9 0 , 9 4 , 3 5 - 3 8 2-4, 7,10, 26,31, 3 5 , 3 7 , 3 8 , 9 4 , 9 9 , 104 4, 26, 6 5 , 6 7 , 52, 74 15,100 15 15

E t h y l esters Acetates Trifluoroacetates T r i m e t h y l s i l y l ether Spectral methods V i s u a l c o m p a r i s o n u n d e r short a n d long u v light 76,94 Fluorescence spectrophotometry, 5 acid-base Ultraviolet spectrophotometry 2-4, 9,15,18,26,28,44, 98,100, 102,122 Infrared spectrophotometry 2-4,13,15,26-28,102 Optical rotatory dispersion and 2 - 4 , 1 3 , 2 7 , 28,104 polarimetry N u c l e a r m a g n e t i c resonance 2-4,13,26, 27,31, 69,102,104,124 M a s s spectrometry 2-4, 26-28, 69,104,125 M e l t i n g points, C , H , N analysis 2-4,13, 26,27,98,102,124 A T L C i d e n t i f i c a t i o n m a y b e m a d e b y p r e p a r i n g t h e esters or ethers of b o t h the u n k n o w n a n d s t a n d a r d o c h r a t o x i n s a n d c o m p a r i n g t h e T L C characteristics ( R / s ) of these d e r i v a t i v e s .

Biological Tests T a b l e V I I I lists s o m e of t h e m o r e sensitive tests w h i c h h a v e b e e n d e v i s e d to test o c h r a t o x i n f o r b i o l o g i c a l a c t i v i t y . T h e s e are f o r t h e m o s t p a r t q u a l i t a t i v e i n n a t u r e . I n c o n j u n c t i o n w i t h other p h y s i c a l a n d c h e m ­ i c a l d a t a some of t h e b i o l o g i c a l tests are o c c a s i o n a l l y u s e d for c o n f i r m i n g t h e i d e n t i t y of o c h r a t o x i n s .

Ochratoxin-Kelated Metabolites (Table IX) T h e f e w c h e m i c a l l y r e l a t e d d e r i v a t i v e s of t h e ochratoxins r e p o r t e d so f a r i n c l u d e , besides t h e e t h y l a n d m e t h y l esters of ochratoxins A a n d

12.

289

Ochratoxins

NESHEIM

Table VIII.

B i o l o g i c a l Tests f o r O c h r a t o x i n

Type Response

System

Sensitivity

C h i c k e n embryo

death

LD

Z e b r a fish l a r v a Brine shrimp

death death

20% mortality

Brine shrimp larvae

death

LC

0 . 0 1 - 17 μg/egg

5 0

5

1.7

0

/xg/ml

A Reference 25,60-62,70, 78, 126 129 70,130

1 /Ag/ml

megaterium g r o w t h inhibition growth Bacillus cereus mycoides inhibition growth Tetrahymena pyriinhibition formis H S M H e L a cell cytotoxicity T r a c h e a l organ death culture D a y old duckling death D a y old chick death Bacillus

131 LC l O r t / m l ; 17% m o r t a l i t y 0.2 /xg/disc 35, 3 6 , 1 2 7 , 1 fig/disc 128 127 1.5 j t t g / d i s c 5 0

1%200

ftg/ml

10/xg/ml LC

5

0

150

LD

1.7

ftg/ml

132 22, 6 8 , 9 7 133

1-4 1 3 5 - 1 6 6 / A g / c h i c k 107, 1 2 2 , 1 3 4

/Ag/duckling 5 0

B , 4-hydroxy-ochratoxin A a n d ochratoxin a and β ( F i g u r e 1).

Methods

f o r one o r m o r e of t h e m e t h y l a n d e t h y l esters h a v e b e e n p u b l i s h e d 65, 74-77).

T h e y are as t o x i c as the p a r e n t c o m p o u n d s ( 2 5 )

p r o d u c e d i n r a t h e r l o w y i e l d b y the m o l d ( 4 ) .

(4,

b u t are

Ochratoxin D reported b y

Scott is s y n o n y m o u s w i t h 4 - h y d r o x y o c h r a t o x i n A . O c h r a t o x i n a is d e r i v e d f r o m ochratoxin A b y r e m o v i n g the phenylalanine moiety.

S e v e r a l of t h e

other c o m p o u n d s s h o w n i n T a b l e I X are r e l a t e d to the o c h r a t o x i n s o n l y i n t h a t t h e y are p r o d u c e d b y the same m o l d s a n d h e n c e c a n i n t e r f e r e b o t h i n c h e m i c a l analyses, tests, a n d i n b i o l o g i c a l assays or t o x i c i t y studies. T h e d e r i v a t i v e s f o r the most p a r t are r e l a t i v e l y n o n t o x i c ( 2 5 ) .

Methods

of analysis are a v a i l a b l e for some of these substances b u t h a v e n o t b e e n r e f e r e n c e d b e c a u s e t h e y are b e y o n d the scope of this r e v i e w . Conclusion T h e m u l t i p l i c i t y of m e t h o d s i n use as o u t l i n e d here i n d i c a t e s the l a c k of a single m e t h o d a d e q u a t e f o r a l l tasks. M e t h o d o l o g y w i l l p r o b a b l y b e g r e a t l y i m p r o v e d i n the near f u t u r e w h e n t h e p r o m i s i n g t e c h n i q u e h i g h efficiency l i q u i d c h r o m a t o g r a p h y

of

(already successfully a p p l i e d to

t h e analysis of t h e m y c o t o x i n s , aflatoxins, p a t u l i n , a n d s t e r i g m a t o c y s t i n ) is a p p l i e d to o c h r a t o x i n s . I t is h o p e d t h a t t h e f u t u r e w i l l b r i n g r e l i a b l e , r a p i d , i n e x p e n s i v e , a n d e v e n a u t o m a t e d m e t h o d s to effectively a n d e c o ­ n o m i c a l l y protect the food a n d feed supply.

290

MYCOTOXINS

Table I X . Ochratoxins, Related Metabolites and Derivatives Produced by T w o Major Ochratoxin-Producing Fungi Compound Ochratoxin A and Β M e t h y l a n d e t h y l esters of ochratoxins A and Β 4 - H y d r o x y ochratoxin A Ochratoxin T and T O c h r a t o x i n a a n d β* A

a

c

a

L - A l a n i n e and L-leucine de­ r i v a t i v e s of o c h r a t o x i n a 4-Hydroxyaspergillic acid Aspocracin Brevianamide A and Β Citrinin Emodin 3- (1,2-Epoxy propyl) -5,6dihydro-5-hydroxy-6methyl-2-one Ergosterol Erythritol Griseofulvin M e l l e i n (ochracin) 4-Hydroxymellein Oxalic acid Penicillic acid

L-Prolyl-L-leucine anhydride L-Prolyl-L-valine anhydride Secalonic acid Viridicatin Viridicatol Viridicatum toxin Unidentified toxins

A. ochraceus

P. viridi­ catum

+ + + +

+

1,12,13,93,98,119 4

+ +

1 2 , 9 8 , 104 78 2 9 , 4 4 , 88, 9 9 , 1 0 4 , 107, 111, 1 1 2 , 1 1 3 , 117 136

+ + + + + + + + +

+ +

+ + +

+ + + +

+ + +

Reference

4,97 137,138 139 9,10,12,31,50,119 14 140

13,14 14 141 1, 22, 125 96,135 9,50 4, 5, 1 4 , 2 2 , 9 1 , 9 3 , 9 7 , 1 0 3 , 1 1 9 , 142, 143 1 1 14,145 146 147 141 11,16, 2 3 , 9 3 , 9 7 , 119,144,148-152

Ochratoxins A and C in which the phenylalanine moiety has been replaced with tyrosine. * Ochratoxins in which the phenylalanine moiety has been removed. β

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12.

NESHEIM

Ochratoxins

291

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292

MYCOTOXINS

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12.

NESHEIM

Ochratoxins

293

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294

MYCOTOXINS

108. Maebayashi, Y., Miyaki, Κ., Yamazaki, M., Chem. Pharm. Bull. (1972) 20, 2172. 109. Pitout, M. J., Toxicol. Appl. Pharmacol. (1968) 13, 299. 110. Wei, R., Strong, F. M., Smalley, Ε. B., Appl. Microbiol. (1971) 22, 276. 111. Nel, W., Purchase, I. F. H., J. S. Afr. Chem. Inst. (1968) 21, 87. 112. Pitout, M. J., Biochem. Pharmacol. (1969) 18, 485. 113. Steyn, P. S., Holzapfel, C. W., Ferreira, N . P., Phytochemistry (1970) 9, 1977. 114. Lemieszek, K., Abstracts of the 2nd International Union of Pure and Applied Chemistry Sponsored International Symposium Mycotoxins in Food. Pulaway, Poland (1974) July 23-25. 115. Chu, F. S., J. Ass. Offic. Anal. Chem. (1970) 53, 696. 116. Scott, P. M., Lawrence, J. W., van Walbeek, W., Appl. Microbiol. (1970) 20 839 117. Trenk, H.L., Chu, F. S., J. Ass. Offic. Anal. Chem. (1971) 54, 1307. 118. Neely, W. C., West, A. D., J. Ass. Offic. Anal. Chem. (1972) 55, 1305. 119. Ciegler, Α., Fennell, D. I., Sansing, G. Α., Detroy, R. W., Bennett, G. Α., Appl. Microbiol. (1973) 26, 271. 120. Steyn, P. S., J. Chromatogr. (1969) 45, 473. 121. Shu, F. S., Arch. Biochem. Biophys. (1971) 147, 359. 122. Chu, F. S., Noh, I., Chang, C. C., Life Sci. (1972) 11, Part I, 503. 123. Pitout, M . J., "Mycotoxins in Human Health," I. F. H . Purchase, Ed., p. 53, Macmillan, London, 1971. 124. Yamazaki, M., Maebayashi, Y., Miyaki, K., Tetrahedron Lett. (1971) 25, 2301. 125. Doster, R. C., Sinnhuber, R. O., Wales, J. H., Food Cosmet. Toxicol. (1972) 10, 85. 126. Choudhury, H., Carlson, C. W., Poultry Sci. (1973) 52, 1202. 127. Broce, D., Grodner, R. M., Killebrew, R. L., Bonner, F. L., J. Ass. Offic. Anal. Chem. (1970) 53, 616. 128. Clements, N. L., Abstracts of the American Association of Cereal Chemists —American Oil Chemists Society Joint Meeting, Washington, D.C. (1968). 129. Abedi, Ζ. H., Scott, P. M., J. Ass. Offic. Anal. Chem. (1969) 52, 963. 130. Brown, R. F., J. Amer. Oil Chem. Soc. (1969) 46, 119. 131. Harwig, J., Scott, P. M., Appl. Microbiol. (1971) 21, 1011. 132. Hayes, A. W., Melton, R., Smith, S. J., Bull. Environ. Contam. Toxicol. (1974) 11, 321. 133. Cardeilhac, P. T., Nair, K. P. C., Colwell, W. M., J. Ass. Offic. Anal. Chem. (1972) 55, 1120. 134. Peckham, J. C., Doupnik, B., Jr., Jones, Ο. H., Jr., Appl. Microbiol. (1971) 21, 492. 135. Cole, R. J., Moore, J. H., Davis, N . D., Kirksey, J. W., Diener, U . L., J. Agric. Food Chem. (1971) 19, 909. 136. Pawlowski, Ν. E., Doster, R. C., Lee, D. J., Nixon, J. E., Sinnhuber, R. O., "Abstracts of Papers," 167th National Meeting, ACS, March 31-April 5, 1974, Los Angeles, Calif. 137. Myokei, R., Sakurai, Α., Chang, C., Kodaira, Y., Takahashi, N., Tamura, S., Tetrahedron Lett. (1969). 138. Myokei, R., Sakurai, Α., Chang, C., Kodaira, Y., Takahashi, N., Tamura, S., Agric. Biol. Chem. (1969) 33, 1491. 139. Wilson, B. J., Yang, D. T. C., Harris, T. M., Appl. Microbiol. (1973) 26, 633. 140. Moore, J. H., Murray, T. P., Marks, M. E., J. Agric. Food Chem. (1974) 22, 697. 141. Hutchison, R. D., Steyn, P. S., van Rensburg, S. J., Toxicol. Appl. Pharmacol. (1973 ) 24, 507.

12.

NESHEIM

Ochratoxins

295

142. Udagawa, S., Ichinoe, M., Kurata, H., "Proceedings First U.S.-Japan Conference on Toxic Micro-Organisms," U.S. Department of Interior, Washington, D.C. (1970) pp. 174-187. 143. Wu, M. T., Ayres, J. C., Kohler, P. E., Appl. Microbiol. (1974) 27, 427. 144. Budiarso, I. T., Carlton, W. W., Tuite, J. F., Vet. Pathol. (1970) 7, 531. 145. Yamazaki, M., Maebayashi, Y., Miyaki, K., Chem. Pharm. Bull. (1971) 19, 199 146. Cunningham, K. G., Freeman, G. G., Biochem. J. (1953) 53, 328. 147. Luckner, M., Mohammed, Y. S., Tetrahedron Lett. (1964) 29, 1987. 148. Carlton, W. W., Tuite, J., Caldwell, R., J. Amer. Vet. Med. Ass. (1973) 163, 1295. 149. McCracken, M . D., Carlton, W. W., Tuite, J., Food Cosmet. Toxicol. (1974) 12, 79. 150. Ibid., 89. 151. Ibid., 99. 152. Zwicker, G. M., Carlton, W. W., Tuite, J., Food Cosmet. Toxicol. (1973) 11, 989. RECEIVED December 16, 1974.