.,Inalyti'ca Chimic.a ,,lt'ta. 78 (1975) 383--389 i~ Elsevier Scientific Publishing Comp,'my.'Amsterdam - Printed in The Netherlands

A RAPID METHOD BILLION AMOUNTS

383

FOR THE DETERMINATION OF SUB-PART PER OF N-NITROSO COMPOUNDS IN F O O D S T U F F S

D. H. FINE. D. P. R O U N B E H L E R und P. E. O E T T I N G E R

Thermo Elet'tpolt Corl,ortttitm. 85 Fir.~t Avenue. Wtdtham. Mtt,~.sat'h,svtts 02154 ( U . S . A . ) (Received Ist F e b r u a r y 1975)

Since M a g e e and B a r n e s t discovered that d i m e t h y l n i t r o s a m i n e caused m a l i g n a n t t u m o r s in the rat, there has been m u c h c o n c e r n a b o u t the possible w i d e s p r e a d o c c u r r e n c e of N - n i t r o s o c o m p o u n d s in the e n v i r o n m e n t 2. M a n y Nn i t r o s o c o m p o u n d s are earcinogenic3; trace levels o f d i m e t h y l n i t r o s a m i n e a n d N - n i t r o s o p y r r o l i d i n e have been f o u n d in cured foods such as fried bacon "~.-~ a n d s m o k e d fish -~-('. Analytical m e t h o d s , sensitive at the ltg k g - t level in the original foodstuff. have been d e v e l o p e d for the f o u r t e e n m o s t volatile n i t r o s a m i n e s 4-7. T h e p r o c e d u r e s are c o m p l e x a n d t i m e - c o n s u m i n g , requiring digestion, extraction, a n d steam or v a c u u m distillation followed by extensive clean-up. T o achieve sensitivity at the ltg k g - t level in the original foodstuff, c o n c e n t r a t i o n of the extract by at least a factor of 1000 is essenti~d. Even then, the nitrogen-specific C o u l s o n electrolytic c o n d u c t i v i t y a n d / o r the alkali flame ionization gas c h r o m a t o g r a p h (g.c.) d e t e c t o r s w h i c h are gener~dly used 8 are n o t specific for N - n i t r o s o c o m p o u n d s , a n d c o n f i r m a t i o n by gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y (g.c.-m.s.) is m a n d a t o r y . Redistilled a n d repurified s o l v e n t s ;rod reagents are needed, a n d e l a b o r a t e p r e c a u t i o n s are required to o b t a i n reliable results. T h e time required p e r analysis is long, and the cost per analysis is high. T o a c h i e v e Itg k g - ~ sensit.ivity in the original material, the existing clean-up p r o c e d u r e s h a v e b e e n designed to elimimlte "'interfering" c o m p o u n d s . T h u s a negative result o n a p a r t i c u l a r foodstuff indicates that n o n e o f the fourteen N - n i t r o s o c o m p o u n d s tested are present, b u t d o e s not imply the presence o r a b s e n c e of o t h e r N - n i t r o s o ¢ o m p o t t n d s a m e n a b l e to g.c. With the a d v e n t of the g.c.-thermal energy a n a l y z e r '~ (t.e.a.) interf~lce ~0 w h i c h is specified for N - n i t r o s o c o m p o u n d s it is n o w possible to identify and q u a n t i f y N - n i t r o s o c o m p o u n d s a m e n a b l e to g.c. This p a p e r r e p o r t s a new analytical a p p r o a c h w h i c h requires only two or three s i m p l e steps: v a c u u m distillation from mineral oil (time required 50 min), e x t r a c t i o n with d i c h l o r o m e t h a n e ( 10 r a i n ) a n d , d e p e n d i n g o n the sensitivity required, c o n c e n t r a t i o n o n a K u d e r n a D a n i s h e v a p o r a t o r (20 rain). An analysis therefore takes less t h a n 90 rain, w i t h sensitivity r o u t i n e l y at the 0.05-0.01 ltg kg - t c o n c e n t r a t i o n level. M o r e o v e r , because t h e c l e a n - u p has been k e p t to a taainimum c o m m e n s u r a t e w i t h g.c. c o l u m n c o m p a t i b i l i t y , the m e t h o d facilitates q u a n t i t a t i o n of all N - n i t r o s o c o m p o u n d s anaenable to g.c. Since the gas c h r o m a t o g r a p h - t h e r m a l energy a n a l y z e r (g.c.-t.e.a.) is tt u n i q u e in its selectivity to c o m p o u n d s c o n t a i n i n g e x t r e m e l y heat-labile n i t r o s o g r o u p s , a rapid analysis p r o c e d u r e has been d e v e l o p e d .

,384

D . H . F I N E . D. P. R O U N B E H L E R . P. E. O E T T I N G E R

T h e foodstuff to be ~lnalyzed is g r o u n d up and mixed with alkali. C r u d e mineral oil is a d d e d : the c o n t e n t s are then h e a t e d gently tinder v a c u u m a n d the distillate is frozen out in a cold trap. The N - n i t r o s o c o m p o u n d s dissolve in the mineral oil as it p e n e t r a t e s into the c o m p l e x food matrices, so e l i m i n a t i n g the need for digestion o r fine g r i n d i n g of the foodstuff. At the e n d of the e x t r a c t i o n step the food m a t r i x is still visible, even t h o u g h the N - n i t r o s o c o m p o u n d s have been r e m o v e d . By k e e p i n g the oil u n d e r redtlced presstlre, all the c o m p o u n d s which tire soluble in tin o i l - w a t e r e m u l s i o n a n d are m o r e volatile than the oil are distilled as t h e y are extracted. In a d d i t i o n to N - n i t r o s o c o m p o u n d s , o r g a n i c alnd inorganic nitrates ~~ give al response o n g.c.-t.e.a. C o m b i n e d extraction a n d v a c u u m distillation from w a r m mineral oil effectively o v e r c o m e s this obstacle: inorganic nitrates a n d nitrites d o not have sufficiently high v a p o r pressures to be distilled tinder these mild conditions, a n d o r g a n i c nitrites d e c o m p o s e d u r i n g heating. A l t h o u g h the c r u d e distillate cain be i n t r o d u c e d directly into the g.c. w i t h o u t further clean-up, d i c h l o r o m e t h a n e extraction is simple a n d e x t e n d s the life o f the g.c. column. EXPERIMENTAL R etl.qetl l x

Because g.e.--t.e.a, is so selective, pure an,'dytical reagents are not necessary; commerci,'ll g r a d e s o d i u m h y d r o x i d e , h y d r o c h l o r i c acid, a n d m i n e r a l oil are suitable. If the d i c h l o r o m e t h a n e e x t r a c t is to be c o n c e n t r a t e d on a K u d c r n a D a n i s h e v a p o r a t o r , redistilled d i c l a l o r o m e t h a n e should be used. ,4 l~paraltc,¢

A specially designed single c o l u m n , t e m p e r a t u r e - p r o g r a m m ~ l b l e , gas c h r o m a t o g r a p h , e q u i p p e d with ;l t.e.a, d e t e c t o r ~ was used. T h e t,e.a, tr~p was kept at -- 159':~C. T h e gas c h r o m a t o g r a p h i c c o l u m n was p r e p a r e d fi'om stainless steel tube (10 ft x~- in. o.d.) p a c k e d with 157.1, (w/w) F F A P {'free fatty acid phase) o n c h r o m o sorb W (acid washed. D M C S treated. 80-100 mesh). T h e c o l u m n was t e m p e r a t u r e p r o g r a m m e d at 5°C rain-~ from 140 to 210':'C. P l ' f j u e f h l l ' £ .a

A b o u t 2{) g of the food sample, cut into thin slices or minced, was weighed into a 500-ml r o u n d - b o t t o m e d flask, to which was added 4, ml of 0.1 M sodium hydroxide and 20 ml of crude mineral oil. The contents were heated slowly "tinder v,'lcuum. T h e distillate was collected in a glass finger at - - 1 9 2 ' C . W h e n the flask t e m p e r a t u r e reached I10"C (after c,. 40 min). the distillate was r e m o v e d a n d the c o n t e n t s o f the t h r e e cold fingers were c o m b i n e d . T h e i n t e r - c o n n e c t i n g tubing was rinsed with water, the w,'lshings plus distillate were transferred to a 125-ml s e p a r a t i n g funnel, 4 ml o f 0.1 M h y d r o c h l o r i c acid was atdded, a n d the washings were e x t r a c t e d w i t h d i e h l o r o m e t h a n e (3 × 5 mi). T h e c o m b i n e d e x t r a c t s cain be ; m a l y s e d by g.e.-t.e.a. (sensitivity 5 ;tg kg-~ of original foodstuff), but if incre,'~sed sensitivity is required, the d i c h l o r o m e t h a n e extract is concentr,'lted on a 3-stage K u d e r n a D,'lnish evapor,'ltor; c o n c e n t r a t i o n to 0.2 ml gives ,'¢ sensitivity of 0.05 ,,g kg -~. If g r e a t e r sensitiviiy is required, m o r e t h a n 20 g of sample is taken,

N-NITROSO COMPOUNDS

IN F O O D S T U F F S

385

N - N i t r o s o stamlm'ds

T w o st,'tndard n i t r o s a m i n e Calibration s t a n d a r d s p r o v i d e d by the lntern,'ttional A g e n c y for Research in C a n c e r ( I A R C ) o f t h e W o r l d Health O r g a n i z a t i o n , Lyon, Frtnace. a n d by the US F o o d a n d D r u g A d m i n i s t r a t i o n ( F D A ) . Waslaington, D.C. w e r e used. T h e I A R C s t a n d a r d c o n t a i n e d 23.9 Itg ¢,~f d i m e t h y l n i t r o s a m i n e ( D M N ) p e r ml, 25.3 Itg o f d i e t h y l n i t r o s a m i n e ( D E N ) pet" ml. 29.0 Itg of dibutylnitrosanaine ( D B N ) p e r ml a n d 30.0 ltg o f N - n i t r o s o p y r r o l i d i n e ( P Y R N ) per ml. The F D A s t a n d a r d cont',fined 0.5 ltg r n l - ~ o f each o f ON, IN. metlaylethyhaitrostm~ine ( D P r N ) . ethylbutyhaitros,'~mine, p r o l g y l b u t y l n i t r o s a m i n e , methyhua~yhaitros,'uaaine, I,)BN, nit r o s o p i p e r i d i n e , P Y R N . n i t r o s o m o r p h o l i n e trod dianaylnitros,'maine: a g.c.---t.e.a. c l a r o m a t o g r a n a o f this st,'tndttrd m i x t u r e is s h o w n in Fig. 1. RIz-SULTS A N D D I S C U S S I O N

T h e I A R C s t a n d a r d s o l u t i o n w:Ls ; t d d e d to c a n n e d t u n a t'ish a n d c a n n e d c o r n e d beef :tt the fi Itg k g - t concent,'t~tion level. "The chl-omt~togr:ma of the c a n n e d t u n a fish. after recovery, is s h o w n in Fig. 2(a) a n d tht~t of the c t m n e d c o r n e d beef. in Fig. 3. R e c o v e r i e s were 71-75"i, for D M N , 90"~, Ibr D E N . 95-98'",i for D B N . a n d I00'!.~i for P Y R N . T h e relatively low r e c o v e r y o f the m o s t volatile c o m p o u n d . D M N , wtm a t t r i b u t e d to losses d u r i n g v a c u u m distillation. Figt, re 2 (b) is the c h r o m a t o g r a m fi'om c a n n e d t u n a fish. showing 0.20 ltg k g - t O1" D M N a n d 0.46 Hg k g - t o1' P Y R N . identified from their retention times. F i g u r e 4 is the clarom,'ttogram fi'onl a soytt btzan oil s:maple used to deep-fry c o d f i s h a n d french fi'ies. T h e o n l y g.c. p e a k o b s e r v e d was that o f D P r N . w h i c h

'--

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I Il

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g

Time

( rain )

I=ig. I. C h r o , n : ~ t o g r a n l (g.c....-l.e.a.) gt'~nc~nl.l'~.llJt~n o r 0.5 lILt ii11- i

Ibr

a slantlard

i11ixtl.ll'e o f

14 v o l a l . i l u

,litro.~a,uinu.~.

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at

~t

O£N

1

1

I

I

I PYRN

11'

'

OMN

OBN

I

1 8

4

I 12 Time

16 (rain)

I 24

20

I 2@

Fig. 2a. C h r o m a t o g r a m of canned tunis fish extract following recovery of it mixture of DMN. DEN, DBN, a n d PYRN. each at 5 ltg k g - I . Fig. 2b. C h r o m a t o g r a m of a canned tuna fish extract, showing the presence of D M N (0.2/,~g k g - ~) and P Y R N (0.45 #g k g - l ) .

DMN DEN PYRN

DBN

I ,4

8

l I

12 16 Time (rain)

20

24

I 28

Fig, 3. Chromatogram from a canned beef cxtr:tct following recovery of a mixture of D M N . DBN, and PYRN, each at 2 pg k g - ' .

DEN.

387

N - N I T R O S O C O M P O U N D S IN F O O D S T U F F S

was a d d e d as a n internal s t a n d a r d at the 0.5 #g k g - 1 level. R e c o v e r y o f the D P r N was 100~,,. T h e soya b e a n oil was s h o w n not to contain N - n i t r o s o c o m p o u n d s a m e n a b l e to o u r g.c. conditions, d o w n to the 0. I ltg k g - 1 level. F i g u r e 3 shows t h a t the c o r n e d beef e x t r a c t gave several smaller p e a k s in a d d i t i o n to the D M N , D E N , D B N , a n d P Y R N w h i c h were a d d e d intentionally. N i t r o s o p i p e r i d i n e (0.2/ag k g - t ) was identified f r o m its retention time but the o t h e r g.c. p e a k s did not m a t c h the retention times of the s t a n d a r d n i t r o s a m i n e s available. T h e conc e n t r a t i o n of the unknovcn p e a k s w a s ca. O. 1-0.6 ltg k g - 1 ( e s t i m a t e d from the k n o w n mole response a n d a s s u m i n g a m o l e c u h t r weight of 100). Figure 5 shows the c h r o m a t o g r a m o f a fried b a c o n extract; D M N (5.9 ltg kg - t ) and P Y R N (52 ltg kg - I ) were identified from their retention times. D P r N was a d d e d as an internal s t a n d a r d at the 0.5 Fig k g - 1 level. At a h i g h e r gain, t.e.a, s h o w e d the recovery o f D P r N to be 100~,,. T h e r e are five unidentified g.c. I

1

I

4

I

I

I

E,

I

12

+

"I""~

16

Time ( rain}

I

""s' ~ 'F'` ~"

20

i

I 24

1

I

28

Fig. 4. C h r o m a t o g r a m from soya bean oil used to fry codfish and french fries. Dipropylnitrosamine (0.5 irE, k g - t) was added as all internal standard.

~YRN

~k

DMN Time

(rain)

Fig. 5. C h r o m a t o g r a m of a fried b a c o n extract, showing D M N (5.9 Hg k g - t ) and PYRN (52 gig k g - t ) D i p r o p y l n i t r o s a m i n e was added as an internal standard (0.5 #g k g - t ) : the recovery was 95%.

388

D. H, F I N E . IZ), P, R O U N B E H L E R .

P. I-'. O E T T I N G E R

peaks in Fig. 5: four o f these ",trc p r o b a b l y N - n i t r o s o c o m p o u n d s at a p p r o x i m a t e l y 1 ~tg kg -~. T h e g.c. peak i m m e d i a t e l y after that of D M N does n o t have the s y m m e t r i c a l p e a k s h a p e characteristic of a N - n i t r o s o c o m p o u n d : a l t h o u g h it und o u b t e d l y c o n t a i n s a labile n i t r o s o group, it is p r o b a b l y not a n N - n i t r o s o compound. T h e c b r o m a t o g r , ' u n s p r e s e n t e d d o not s h o w any evidence o f c o n a p o u n d s interfering with the ;tnalysis. In c o n t r a s t to the p r o c e d u r e s of existing techniques, clean-up has been k e p t to an a b s o l u t e m i n i m u m . T h e only restraint is the necessity to o b t a i n a r e p r e s e n t a t i v e extract which c,'m be injected into a gas chromatograph. Identification by g.c.-t.e.a, d e p e n d s u p o n final identification o f the nitrosyl radical, a n d can be c o m p a r e d directly with the use o f g.c.-m.s, for N O + ion mass (29.997) identification. T h e selectivity o f t.e.a, to nitrosyl radicals is at least as g o o d •'is can be achieved by h i g h - r e s o l u t i o n m.s.. for there is virtually no o t h e r o r g a n i c c o m p o u n d w h i c h can p,'iss t h r o u g h ,'t t r a p at - 159'C, a n d then react with o z o n e to give the c h a r a c t e r i s t i c e m i s s i o n in the near infi','t-red. A l t h o u g h t h e ion s o u r c e o f the m a s s s p e c t r o m e t e r is non-selective, the t.e.,'l, catalytic pyrolyzer subjects the c o m p o u n d s to a ~/ery rnild t h e r m a l shock, sufficient o n l y to break the N - N O b o n d . T h e selectivity o f g.c.-l.e.a, to N - n i t r o s o c o m p o u n d s is greater t h a n any o t h e r ,'m,'dytic,'d technique. T h i s is e x p l a i n e d by the fact that a c o m p o u n d giving a response on g.c.-t.e.a, m u s t pass six successive screening steps us follows: (1) the c o n l p o u n d m u s t n o t d e c o m p o s e in the g.c. injection port ( o r g a n i c nitrites a n d Nnitroso a m i d e s d e c o m p o s e instantly, a n d the nitrosyl radical is eluted t o g e t h e r with the solvent fi'ont); (2) the c o m p o u n d m u s t have sufficient vapor p r e s s u r e to elute fi'om the g.c. c o l u m n ; (3) the c o m p o u n d must be eluted from the g.c. a c c o r d i n g to the c o l u m n characteristics; (4) the c o m p o u n d m u s t d e c o m p o s e u n d e r the mild catalytic pyrolysis c o n d i t i o n s o f the t.e.a, pyrolyzer: ( 5 ) t h e c o m p o u n d s m u s t survive a cold t r a p at - - 1 5 9 " C (virtually all organic c o m p o u n d s are t r a p p e d at this t e m p e r a t u r e ) " (6) the c o m p o u n d m u s t react with o z o n e to give a characteristic emission in the near infrared. C o l l a b o r a t i v e ',m,'flyses with o t h e r laboratories are currently u n d e r way, a n d a t t e m p t s to identify t h e " u n k n o w n " g.c.-t.e.a, peaks found in m e a t a n d fried b a c o n are in progress. We t h a n k Larry Keefer ( N a t i o n a l C a n c e r Institute, Bethesda. M a r y h m d ) a n d l-'red Huffm,'m, D a v i d Lieb a n d F i r o o z Rufeh ( T h e r m o E!ectron C o r p o r a t i o n ) for m a n y helpful discussions. We are i n d e b t e d to F r a n k C a m p a g n a for technical assistance. Dr. E. W a l k e r ( I A R C , Lyon, France) supplied the c o l l a b o r a t i v e nitros a m i n e m i x t u r e : Total Fazio ( F D A , W a s h i n g t o n ) supplied the m i x t u r e of 14 nitrosamines. T h i s w o r k w a s s u p p o r t e d by C o n t r a c t N O 1 C P 4 5 6 2 3 with the N a t i o n a l C a n c e r Institute. U. S. D e p a r t m e n t of Flealth. E d u c a t i o n a n d Welfare. SUMMARY

A new ;malytical c l e a n - u p t e c h n i q u e , designed specifically for tile analysis of N - n i t r o s o c o m p o u n d s with a t h e r m a l energy analyzer a n d a gas c h r o m a t o g r a p h , is described. O n l y t w o steps are r e q u i r e d : v a c u u m distillation f r o m mineral oil

N-NITROSO

COMPOUNDS

IN F ( ) O D S T U F F S

389

f o l l o w e d by e x t r a c t i o n with d i c h l o r o m e t h a n e . T h e sensitivity for N - n i t r o s o cornp o u n d s in foodstuffs is at the 5 ltg k g - ~ level, but if a d i c h l o r o m e t h a n e extract is c o n c e n t r a t e d on a K u d e r n a D a n i s h e w l p o r a t o r , a 100-fold increase is attainable. T h e m e t h o d can be used for a n y f o o d s t u f f or b i o l o g i c a l material. ;tnd t he q u a n t i t a t i o n o f a n y N - n i t r o s o c o m p o u n d s a m e n a b l e to g a s c h r o m a t o g r a p h y is p o s s i b l e . REFERENCFS

I 2 3 4 5 6 7 8 t) 11) II

P. N. M a g e e and J. M. Barnes, Brit. ,!. ('~owel'. I0 (1956) 114. W, Lijinsk.v and S. S. Epstein. N a t u r e ( L o n , h m ) , 225 (1970) 21. P. N, M:lg¢~a. Food C'osmet. "l'oxic'ol,. 9 ( 1971 ) 207. N. P. Sen. B. D o n a l d s o n . J. R. J y e n g a r a n d T. Panalaks. N a t u r e (Lon,hm). 241 (1973) 473. N . T . C r o s b y . J. K. F o r u m a n . J. F, Palframata. and R. Sawyer. Ntttto'e ( l . o n d o n ) . 238 (1972) 342. T. l:azio. J. N. Dalllit:o. J. W. l-h~ward. R. I-I, W h i t e a n d J. O. Waits. ,I..,ItH'. I,'ood Chem.. 19(1971)251. J. M. E s s i g m a n n and P. Issenbt:rg. d. Food St'i.. 37 (1972) 684. J. F. l~alfi'am:ln. J. M a c r l a b and N. T. Crosby. ,I. C'lo'omtltO~.lr.. 76 (1973)307. I). !1. Fine. F. Rttfeh ;tnd D. Lieb. Ntttto'e ( L o n d o n ) . 247 ( 1 9 7 4 ) 3 0 9 . I). H. F i n e and D. P. R o u n b e h l u r . . I . Cht'tnHtttoqr.. 109 (1975) 271. 1). H. Finu. I). Lieb a n d D, P. Rotmbehle.r. Ant//. Chem.. 47 { 1975) I1~,%