Analytical Methods for Nitrosamines - ACS Publications - American

23 Analytical Methods for Nitrosamines PHILLIP

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Downloaded by COLUMBIA UNIV on March 3, 2015 | http://pubs.acs.org Publication Date: December 9, 1981 | doi: 10.1021/bk-1981-0174.ch023

Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68105

Reliable analytical methods are available for determination of many volatile nitrosamines at concentrations of 0.1 to 10 ppb in a variety of environmental and biological samples. Most methods employ d i s t i l l a t i o n , extraction, an optional cleanup step, concentration, and final separation by gas chromatography (GC). Use of the highly specific Thermal Energy Analyzer (TEA) as a GC detector affords simplification of sample handling and cleanup without sacrifice of selectivity or sensitivity. Mass spectrometry (MS) is usually employed to confirm the identity of nitrosamines. U t i l i z a t i o n of the mass spectrometer's capability to provide quantitative data affords additional confirmatory evidence and quantitative confirmation should be a required criterion of environmental sample analysis. Artifactual formation of nitrosamines continues to be a problem, especially at low levels (0.1 to 1 ppb), and precautions must be taken, such as addition of sulfamic acid or other nitrosation inhibitors. The efficacy of measures for prevention of artifactual nitrosamine formation should be evaluated in each type of sample examined. Reliable methods are a v a i l a b l e f o r determination of n i t r o s a m i n e s , e s p e c i a l l y v o l a t i l e n i t r o s a m i n e s , i n a v a r i e t y of foods, environmental samples, commercial products, blood and animal t i s s u e s . Reviews of these methods are a v a i l a b l e ( 1 , 2) and d e s c r i p t i o n s of some s t a t e - o f - t h e - a r t procedures are i n c l u d e d i n papers on nitrosamine occurrence i n t h i s volume. This paper i s not intended t o be a comprehensive review of h i s t o r i c a l developments or of the many v a r i a t i o n s of procedures

0097-6156/81 /0174-0331 $ 0 5 . 0 0 / 0 © 1 9 8 1 American Chemical Society In N-Nitroso Compounds; Scanlan, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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c u r r e n t l y i n use, but r a t h e r w i l l focus upon some of the major remaining problems. Examples of these problems and some s o l u t i o n s to them are taken from a n a l y t i c a l s t u d i e s i n progress i n our l a b o r a t o r y and from the recent l i t e r a t u r e . The continued existence of r e a l problems i n nitrosamine a n a l y s i s i s i l l u s t r a t e d by the recent report (3) of up to 23 ppb of N - n i t r o s o dimethylamine (NDMA) and 28 ppb of N-nitrosodiethylamine (NDEA) i n commercial vegetable o i l s , while l e v e l s less than 1 ppb were found when s p e c i a l precautions were taken to avoid a r t i f a c t s ( 4 ) . Major emphasis i n s t u d i e s of N-nitroso compounds i n foods has been p l a c e d upon v o l a t i l e nitrosamines, i n part because these compounds are r e l a t i v e l y easy to i s o l a t e from complex matrices by v i r t u e of t h e i r v o l a t i l i t y . Procedures u t i l i z i n g atmospheric pressure or vacuum d i s t i l l a t i o n have been used by most i n v e s t i g a t o r s , with v a r i a t i o n s of the method of Fine e_t a l . (5) being among the most popular. This procedure employs vacuum d i s t i l l a t i o n of a mineral o i l suspension of the sample with o p t i o n a l a d d i t i o n of water to improve nitrosamine recovery from low moisture content samples (6). The usual approach to prevent i o n of nitrosamine formation during a n a l y s i s i n v o l v e s adding sulfamic a c i d or ascorbate to destroy r e s i d u a l n i t r i t e at an e a r l y stage of sample p r e p a r a t i o n . Aqueous d i s t i l l a t e s are e x t r a c t e d , u s u a l l y with d i c h l o r o methane (DCM), concentrated to small volumes, g e n e r a l l y i n a Kuderna-Danish evaporator, and examined by gas chromatography (GC) using a s p e c i f i c d e t e c t i o n system. A d d i t i o n a l chromatographic cleanup may be r e q u i r e d , depending on the complexity of the sample and s p e c i f i c i t y of the chromatographic d e t e c t o r . The subnanogram s e n s i t i v i t y and s p e c i f i c i t y of the Thermal Energy Analyzer (TEA) (2) make a d d i t i o n a l p u r i f i c a t i o n of concentrates unnecessary f o r most samples. The TEA has become the d e t e c t o r of choice i n l a b o r a t o r i e s performing large numbers of nitrosamine analyses because of the minimal sample preparat i o n r e q u i r e d p r i o r t o GC-TEA determination and the r e l a t i v e freedom from f a l s e p o s i t i v e r e s u l t s experienced i n i t s use i n many l a b o r a t o r i e s . The TEA responds to some compounds other than those c o n t a i n i n g the N-nitroso group, but methods f o r t e n t a t i v e l y confirming the i d e n t i t y of TEA-positive chromatographic peaks have been d e s c r i b e d . E l i m i n a t i o n of TEA response by p h o t o l y t i c decomposition of nitrosamines by u l t r a v i o l e t r a d i a t i o n (366 nm) i s e f f e c t i v e i n p r o v i d i n g t e n t a t i v e confirmat i o n and i n i d e n t i f y i n g response from compounds other than nitrosamines ( 8 ) . Methods f o r c l a s s i f y i n g TEA response based on u l t r a v i o l e t i r r a d i a t i o n and chemical procedures appear to e l i m i n a t e most ambiguities other than p o s s i b l e i n t e r f e r e n c e from C - n i t r o s o compounds ( 9 ) . Confirmation of i d e n t i t y of nitrosamines v i a an independent d e t e c t i o n system i s d e s i r a b l e since a higher l e v e l of confidence i s achieved i f a d i f f e r e n t p h y s i c a l property or s t r u c t u r a l c h a r a c t e r i s t i c i s measured. Mass spectrometry (MS) has been used

In N-Nitroso Compounds; Scanlan, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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for t h i s purpose by many i n v e s t i g a t o r s . S e n s i t i v i t y of low r e s o l u t i o n MS was s u f f i c i e n t t o confirm the i d e n t i t y of NDMA at the 4 ppb l e v e l i n 400 g samples of smoked marine f i s h (10). At least 50 ng NDMA was r e q u i r e d for i n j e c t i o n i n t o the GCMS system to produce a mass spectrum of s u f f i c i e n t i n t e n s i t y f o r unambiguous c o n f i r m a t i o n . Use of high r e s o l u t i o n MS (Μ/ΔΜ 2:10,000) f o r determination of v o l a t i l e nitrosamines was described by T e l l i n g et a l . ( 11 ). L i m i t s of 1 ppb were achieved by measuring the NO. peaks at m/z 30 from nitrosamines u t i l i z i n g high r e s o l v i n g power to achieve s p e c i f i c i t y rather than chromato graphic cleanup of sample concentrates. Developments based on t h i s approach provided r e s u l t s comparable to those obtained by GC-TEA by measuring the molecular ions of NDMA and N - n i t r o s o p y r r o l i d i n e (NPYR) at m/z 74.0480 and m/z 100.0637, r e s p e c t i v e l y (JL2). These authors reported d e t e c t i o n l i m i t s f o r NDMA i n foods of 1 yg/kg by high and low r e s o l u t i o n MS and 0.02 yg/kg by GC-TEA: 250 g samples were used. When a high performance mass spectrometer was employed, a d e t e c t i o n l i m i t of 0.3 pg, corresponding t o 0.001 ppb, was achieved (13). L i q u i d - l i q u i d p a r t i t i o n i s used to e x t r a c t v o l a t i l e nitrosamines from aqueous d i s t i l l a t e s and as the i n i t i a l i s o l a t i o n step i n determination of n o n - v o l a t i l e nitrosamines. Since vacuum d i s t i l l a t i o n i s o f t e n the most time-consuming and cumbersome step i n the a n a l y t i c a l procedure, d i r e c t e x t r a c t i o n i s an a t t r a c t i v e a l t e r n a t i v e when sample p h y s i c a l c h a r a c t e r i s t i c s and composition are a p p r o p r i a t e . Column e x t r a c t i o n (14) i s a t t r a c t i v e f o r t h i s a p p l i c a t i o n , since it permits the use of l a r g e r volumes of e x t r a c t i o n solvent than i s f e a s i b l e i n conventional batch e x t r a c t i o n apparatus. Columns c o n t a i n i n g k i e s e l g h u r (15) were used f o r DCM e x t r a c t i o n of aqueous d i s t i l l a t e s from beer, and c e l l u l o s e c a r t r i d g e s f o r d i r e c t e x t r a c t i o n of rumen f l u i d with DCM (JU6). Column-extraction permits use of sequential solvents f o r sample cleanup, avoids s u b j e c t i n g l a b i l e samples t o high temperatures r e q u i r e d i n d i s t i l l a t i o n , and i s r e a d i l y adaptable t o simultaneous processing of m u l t i p l e samples. E x t r a c t i o n and chromatographic s e p a r a t i o n may be combined by appropriate choice of column packing and solvents (17). I t i s important t o remember that most published d e t e r minations of nitrosamines i n foods and beverages were obtained using d i s t i l l a t i o n and the s u b s t i t u t i o n of any new i n i t i a l i s o l a t i o n method r e q u i r e s c a r e f u l and thorough e v a l u a t i o n . Determination of nitrosamines i n a i r samples has r e v e a l e d s i g n i f i c a n t exposure of some groups of people i n occupational s e t t i n g s . A v a r i e t y of approaches f o r i s o l a t i n g nitrosamines from a i r have been used, but s o r p t i o n on a s o l i d has emerged as a convenient and r e l i a b l e method. The sorbents employed should be chemically i n e r t while possessing adequate c a p a c i t y , and should permit easy e l u t i o n . Thermal (18, 19) and solvent e l u t i o n (20, 21) have been used. A r t i f a c t u a l formation of nitrosamines during sampling or desorption can be a problem i n atmospheres



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c o n t a i n i n g amines and oxides of n i t r o g e n . A nitrosamine sampling system which e l i m i n a t e s in s i t u n i t r o s a t i o n has been described (20). An i n t e r n a l standard i s d e s i r a b l e i n any q u a n t i t a t i v e trace environmental a n a l y s i s . The i d e a l i n t e r n a l standard should behave i n a manner i d e n t i c a l to that of the analyte i n a l l the procedures followed f o r i s o l a t i o n , p u r i f i c a t i o n , and determination without producing i n t e r f e r e n c e . This i s a d i f f i c u l t requirement t o meet f o r nitrosamines, e s p e c i a l l y f o r NDMA. Because of the s p e c i a l r e g u l a t o r y p o s i t i o n occupied by foods and beverages, a great deal of a t t e n t i o n has been given to development and a p p l i c a t i o n of a n a l y t i c a l procedures for them. Improved procedures have r e s u l t e d i n q u a n t i t a t i o n and c o n f i r mation l e v e l s i n the range of 1 to 10 ppb with sample amounts of 10 to 250 g. Detection l i m i t s f o r foods are i n the 0.1 to 1 ppb range. Detection l i m i t s of 0.1 to 1 ppm appear adequate f o r p e s t i c i d e formulations (17, 22), while s e n s i t i v i t y of 0.01, 0.005, and 0.2 ppb were reported f o r N-nitrosodi-n-propylamine i n water, s o i l , and crops, r e s p e c t i v e l y (23). Detection l i m i t s of 0.2 to 1 ppm f o r N-nitrosodiethanolamine (NDELA) i n cosmetic products and i n g r e d i e n t s (24) and higher l i m i t s i n metal-working f l u i d s (25) are probably too high, i n view of recent evidence that NDELA i s a more potent carcinogen than p r e v i o u s l y b e l i e v e d (26, 2_7). In g e n e r a l , d e t e c t i o n l i m i t s have been set by the c a p a b i l i t i e s of a v a i l a b l e a n a l y t i c a l methods r a t h e r than by c o n s i d e r a t i o n of t o x i c o l o g i c a l potency. Detection l i m i t s f o r most a n a l y t i c a l procedures are determined by response of the d e t e c t i o n system, p o t e n t i a l i n t e r f e r e n c e s , and by the maximum sample quantity which can be conveniently processed i n the a n a l y t i c a l l a b o r a t o r y . In the s p e c i a l case of nitrosamines, precursor amines and n i t r o s a t i n g agents are u s u a l l y present i n the sample and a r t i f a c t u a l formation of nitrosamines during sample handling i s a problem which must be recognized and c o n t r o l l e d by the a n a l y s t . M i n i mizing the number of steps and amount of sample handling (28) and removing at l e a s t one of the precursors p r i o r to a n a l y s i s are the most common approaches to minimizing a r t i f a c t formation. Even with these p r e c a u t i o n s , p r a c t i c a l d e t e c t i o n l i m i t s may be determined by l e v e l s of nitrosamines produced a r t i f a c t u a l l y or present as contaminants i n s o l v e n t s , reagents, and apparatus. Nitrosamine contamination has been reported i n d e i o n i z e d water (29, 30), amines (314), blood c o l l e c t i o n tubes with rubber stoppers (32), and l a b o r a t o r y glassware washing detergents (33 ). Examination of procedural c o n t r o l (blank) samples w i l l u s u a l l y detect contamination, but a r t i f a c t u a l formation of nitrosamines i s a more d i f f i c u l t problem t o detect and prevent.


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Materials. A l l s o l v e n t s were " D i s t i l l e d i n G l a s s " f r o m B u r d i c k and J a c k s o n (Muskegon, M I ) . A l l o t h e r r e a g e n t s were ACS g r a d e , e x c e p t where o t h e r w i s e n o t e d . C e l i t e 560 ( J o h n s M a n v i l l e , Lompoc, CA) was s c r e e n e d t o remove p a r t i c l e s s m a l l e r t h a n 60 mesh and u s e d w i t h o u t f u r t h e r t r e a t m e n t . N i t r o s a m i n e s t a n d a r d s were o b t a i n e d f r o m Thermo E l e c t r o n C o r p . (Waltham, MA) and d i l u t e d t o a p p r o p r i a t e c o n c e n t r a t i o n s w i t h DCM. N - N i t r o s o b i s ( 2 - h y d r o x y p r o p y l ) a m i n e (BHP) a n d NDELA were p r e p a r e d by t h e E p p l e y I n s t i t u t e C h e m i c a l S e r v i c e s U n i t . D i s t r i b u t i o n R a t i o s . A i r - w a t e r , e t h e r - w a t e r and DCM-water d i s t r i b u t i o n r a t i o s were measured as d e s c r i b e d p r e v i o u s l y (34) u s i n g GC-TEA. Column E x t r a c t i o n . Aqueous s a m p l e s and d i s t i l l a t e s were added t o g l a s s c h r o m a t o g r a p h i c tubes or p l a s t i c s y r i n g e b a r r e l s c o n t a i n i n g 0.5 g C e l i t e 560 p e r g o f s a m p l e . A f t e r 20 t o 30 m i n o f e q u i l i b r a t i o n , t h e c o l u m n s were e l u t e d w i t h 100 ml o f DCM [ f o r NDMA, NPYR and N - n i t r o s o m o r p h o l i n e (NM0R) o r e t h y l a c e t a t e ( f o r NDELA and B H P ) ] . R e s i d u a l s o l v e n t was removed f r o m t h e c o l u m n s by a p p l y i n g n i t r o g e n p r e s s u r e . E x t r a c t s were d r i e d w i t h Na4SO4 and c o n c e n t r a t e d t o 1 ml i n a K u d e r n a - D a n i s h apparatus (NDMA, NPYR, and NM0R) i n a 50°C w a t e r b a t h o r i n a r o t a r y e v a p o r a t o r f o r NDELA and BHP, u s i n g a 30 C w a t e r b a t h . T r i i s o p r o p a n o l a m i n e ( E a s t m a n l o t B8X) was p r e p a r e d as a 1 M s o l u t i o n w i t h 0.01 M s u l f a m i c a c i d a d d e d . C o n c e n t r a t e d s u l f u r i c a c i d was a d d e d t o a d j u s t t o pH 1 t o 2. One p o r t i o n was exposed t o u l t r a v i o l e t r a d i a t i o n f r o m a " B l a k - R a y " B-100A lamp ( U l t r a v i o l e t P r o d u c t s , I n c . , San G a b r i e l , CA) f o r 4 h r . a t a d i s t a n c e o f a p p r o x i m a t e l y 30 cm. The s o l u t i o n was s t i r r e d d u r i n g i r r a d i a t i o n . I r r a d i a t e d and u n t r e a t e d s o l u t i o n s were s a t u r a t e d w i t h ammonium s u l f a t e and 10-ml s a m p l e s were t r a n s f e r r e d t o c o l u m n s c o n t a i n i n g 15 g C e l i t e 560 and e q u i l i b r a t e d f o r 30 m i n . Columns were e l u t e d w i t h e t h y l a c e t a t e , and t h e e x t r a c t s were d r i e d and c o n c e n t r a t e d . The c o n c e n t r a t e was t r a n s f e r r e d t o a 5-ml R e a c t i - v i a l ( P i e r c e C h e m i c a l Co., R o c k f o r d , I L ) and volume was r e d u c e d t o a p p r o x i m a t e l y 0.1 ml i n a s t r e a m o f d r y n i t r o g e n . A f t e r a d d i t i o n o f 0.5 ml _ t - b u t y l d i m e t h y l c h l o r o s i l a n e / i m i d a z o l e r e a g e n t ( A p p l i e d S c i e n c e , S t a t e C o l l e g e , P A ) , t h e v i a l was c a p p e d and h e l d a t 60 C f o r 30 min i n a h e a t i n g b l o c k and t h e n c o o l e d t o room t e m p e r a t u r e . Hexane (0.5 m l ) and e i t h e r 0.5 ml w a t e r o r 0.5 ml 10% NaOH s o l u t i o n were a d d e d . A f t e r s h a k i n g and s e p a r a t i o n o f p h a s e s , t h e h e x a n e l a y e r was r e m o v e d and t r a n s f e r r e d t o a s e c o n d R e a c t i - v i a l . The r e a c t i o n m i x t u r e was e x t r a c t e d t w i c e more w i t h hexane and t h e e x t r a c t s were c o m b i n e d and volume r e d u c e d t o 1 ml i n a s t r e a m o f d r y n i t r o g e n . P o r t i o n s w e r e t a k e n f o r GC-TEA o r GCMS a n a l y s i s . The same d e r i v a t i z a t i o n p r o c e d u r e was u s e d t o d e t e r m i n e NDELA i n m e t a l w o r k i n g f l u i d s


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and cosmetic p r e p a r a t i o n s . In some cases, it was necessary to add a p o r t i o n of C e l i t e t o these samples p r i o r to t r a n s f e r r i n g them t o the e x t r a c t i o n columns. Beer Samples. The beer samples were examined as part of the American S o c i e t y of Brewing Chemists (ASBC) and 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 Chemists (AOAC) c o l l a b o r a t i v e s t u d i e s of NDMA i n beer. D u p l i c a t e samples were analyzed by the column e x t r a c t i o n procedure and the ASBC d i s t i l l a t i o n procedure (35). The AOAC procedure (36) was s i m i l a r , except that a l a r g e r sample (50 vs. 25 g) was examined and sulfamic a c i d was added t o minimize a r t i f a c t u a l formation of nitrosamines. Both methods u t i l i z e N-nitrosodipropylamine (NDPA) as an i n t e r n a l standard. In the AOAC procedure, the beer was t r e a t e d with d i l u t e HC1 and sulfamic a c i d , and the added a c i d was then n e u t r a l i z e d by a d d i t i o n of d i l u t e a l k a l i . V o l a t i l e nitrosamines were c o l l e c t e d by atmospheric pressure d i s t i l l a t i o n . The d i s t i l l a t e was made a l k a l i n e and e x t r a c t e d with DCM. The e x t r a c t was d r i e d and concentrated t o 1.0 ml and an a l i q u o t was analyzed by GC-TEA. We used these concentrates, without f u r t h e r cleanup, f o r e v a l u a t i n g the GCMS high r e s o l u t i o n s e l e c t e d ion monitoring procedure. D i r e c t GC I n j e c t i o n of Amines. Morpholine, 2,6-dimethylmorpholine, or p y r r o l i d i n e i n methanol (2.6 t o 2.8 g/ml) s o l u t i o n s were i n j e c t e d d i r e c t l y i n t o the GC i n l e t at 210 C. P o r t i o n s of these s o l u t i o n s were exposed to u l t r a v i o l e t r a d i a t i o n (366 nm) f o r 16 hours p r i o r t o i n j e c t i o n . GC-TEA A n a l y s i s . A Bendix model 2200 GC and Thermo E l e c t r o n model 502 TEA were used. The GC i n j e c t o r temperature was 210 C. The TEA p y r o l y s i s furnace was operated at 450 C and the c o l d trap was held at -150 C i n isopentane s l u s h . Oxygen flow t o the ozonator was 20 cc/min and i n d i c a t e d pressure was 1.5 t o r r at a helium flow r a t e of 20 cc/min. TEA output was processed by a d i g i t a l i n t e g r a t o r (Spectra Physics System I ) . For the AOAC beer samples, a 2 m χ 2 mm g l a s s column packed with 8.5% Carbowax 20 M + 0.85% NaOH on 100/120 mesh Chromosorb G was used at 130 C and a helium flow r a t e of 20 cc/min. Retention times of NDMA and NDPA were 4.5 and 12.2 min, r e s p e c t i v e l y . For the ASBC c o l l a b o r a t i v e study, a 1 m χ 2 mm g l a s s column c o n t a i n i n g 6% Carbowax 20 M-TPA on 100/120 mesh Chromosorb G was operated at 90 C with 20 cc/min helium flow r a t e . Retention times were 3.6 and 11.3 min f o r NDMA and NDPA, r e s p e c t i v e l y . For determination of nitrosamines i n amines, a 2 m χ 2 mm, 10% Carbowax 20 M-TPA on 100/120 mesh Chromosorb G column was operated at 190 C with a c a r r i e r gas flow r a t e of 20 cc/min. Retention times were : NPYR, 6.6 min; NMOR, 7.4 min. For a n a l y s i s of BHP and NDELA TBDMS d e r i v a t i v e s , a 1 m χ 2 mm n i c k e l a l l o y column packed with 1% Carbowax 20 M-TPA on 100/120 mesh Chromosorb G was operated at 150 C and a c a r r i e r


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gas f l o w r a t e o f 20 m l / m i n . R e t e n t i o n t i m e s o f BHP-TBDMS NDELA-TBDMS were 8.1 and 10.0 m i n , r e s p e c t i v e l y .


337

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GCMS A n a l y s i s . The GCMS s y s t e m u t i l i z e d a B e n d i x m o d e l 2200 c h r o m a t o g r a p h c o n n e c t e d t o an Α Ε Ι MS902 mass s p e c t r o m e t e r , e q u i p p e d w i t h a h i g h s p e e d pumping s y s t e m ( 9 - S y s t e m s , M o r r i s t o w n , NJ) f o r t h e i o n s o u r c e . No c a r r i e r gas s e p a r a t o r was u s e d . F o r d e t e r m i n a t i o n o f n i t r o s a m i n e s and TBDMS d e r i v a t i v e s o f h y d r o x y - n i t r o s a m i n e s , c o l u m n s and o p e r a t i n g c o n d i t i o n s were i d e n t i c a l t o t h o s e f o r GC-TEA a n a l y s e s : F o r most work, t h e He f l o w r a t e was 15 c c / m i n and t h e c o l u m n e f f l u e n t was s p l i t 1:1 b e t w e e n a f l a m e i o n i z a t i o n d e t e c t o r and t h e mass s p e c t r o m e t e r . The s t a i n l e s s s t e e l s p l i t t e r , s o l v e n t v e n t v a l v e ( C a r l e Instruments, I c F u l l e r t o n , C A ) , and a s s o c i a t e d p l u m b i n g were m a i n t a i n e d a t 190 -200 C i n a B e n d i x v a l v e o v e n w h i c h a l s o c o n t a i n e d a h i g h vacuum i s o l a t i o n v a l v e f o r t h e mass s p e c t r o m e t e r and an a d j u s t a b l e h e a t e d g l a s s e n t r y t u b e (R.H. A l l e n Co., B o u l d e r , CO). The mass s p e c t r o m e t e r was b y p a s s e d f o r the time r e q u i r e d t o e l u t e the s o l v e n t . n

Q

The mass s p e c t r o m e t e r r e s o l v i n g power was s e t a t 10,000 (107o v a l l e y ) . A c c e l e r a t i n g v o l t a g e was 8 k v and e l e c t r o n c u r r e n t was 450 pa. E l e c t r o n e n e r g y was a d j u s t e d t o m i n i m i z e helium i o n i z a t i o n . I n d i c a t e d p o t e n t i a l was 30v. E l e c t r o n m u l t i p l i e r v o l t a g e was 3 k v and t h e p r e a m p l i f i e r r e s i s t o r was 10 ohms. A n a r r o w mass r a n g e , a p p r o x i m a t e l y 500 ppm, was s c a n n e d , u s i n g e i t h e r t h e p e a k m a t c h i n g s y s t e m o f t h e MS902 o r a m i c r o p r o c e s s o r - c o n t r o l l e d s y s t e m b u i l t i n o u r l a b o r a t o r y . The latter a p p l i e d a l i n e a r v o l t a g e sweep t o t h e e l e c t r o s t a t i c a n a l y z e r p l a t e s and, v i a the r e f e r e n c e v o l t a g e c o n t r o l , t o the a c c e l e r a t i n g v o l t a g e . S i m i l a r p e r f o r m a n c e was o b t a i n e d w i t h b o t h s y s t e m s , b u t t h e m i c r o p r o c e s s o r p r o v i d e d more f l e x i b i l i t y . P e r f l u o r o a l k a n e - 2 2 5 (PGR, G a i n e s v i l l e , F L ) was a d m i t t e d through a g l a s s i n l e t s y s t e m t o p r o v i d e r e f e r e n c e p e a k s . A n a l y t i c a l and r e f e r e n c e p e a k s f o r t h e n i t r o s a m i n e s s t u d i e d a r e shown i n T a b l e I . Sample and r e f e r e n c e p e a k s were s c a n n e d a l t e r n a t e l y a t a r e p e t i t i o n r a t e o f a p p r o x i m a t e l y 1 s e c and were m o n i t o r e d on an o s c i l l o s c o p e . When t h e n i t r o s a m i n e peak a p p e a r e d , t h e o s c i l l o g r a p h i c r e c o r d e r c h a r t d r i v e was e n g a g e d and r e m a i n e d on u n t i l t h e p e a k d i s a p p e a r e d . N i t r o s a m i n e q u a n t i t i e s were e s t i m a t e d by c o m p a r i n g t h e sum o f sample p e a k h e i g h t s m e a s u r e d f r o m t h e c h a r t ( u s u a l l y 10 t o 20 v a l u e s ) w i t h v a l u e s d e r i v e d f r o m i n j e c t i o n o f standard solutions. Results

and

Discussion

I n t e r n a l Standards. A compound s e l e c t e d as an i n t e r n a l s t a n d a r d i d e a l l y s h o u l d b e h a v e i n a manner i d e n t i c a l t o t h a t o f the a n a l y t e i n a l l s e p a r a t i o n steps i n the a n a l y t i c a l process and s h o u l d be m e a s u r e d by t h e same f i n a l d e t e r m i n a t i o n m e t h o d . D i s t i l l a t i o n f r o m a q u e o u s s y s t e m s and s o l v e n t p a r t i t i o n a r e t h e



338

N-NITROSO COMPOUNDS

Table I . Mass S p e c t r a l Peaks Employed f o r I d e n t i f i c a t i o n of Nitrosamines Relative Intensity

Reference Peak

Nitrosamine

Composition

NDMA

C H N 0

74.0480

1

100

69.9986

NMEA

C H N 0

88.0637

1

70

80.9952

NPYR

C H N 0

100.0637

1

90

99.9936

C

4 10 2°

99.9936

C

6 14 2°

C

12 29 2°3 2

C

14 33 2°3 2

2

3

8

4

NDEA NDPA NDELA-TBDMS BHP-TBDMS

2

6

8

2

2

2

m/z

N

102.0793

1

H

100

N

130.1106

1

H

20

123.9936

N

S i

305.1716

2

H

50

304.9825

N

S i

333.2029

2

H

50

331.9871

MÎ-tBu


23.

ISSENBERG

Analytical

Methods for Nitrosamines

339

most widely used separation methods i n determination of v o l a t i l e nitrosamines. Table I I presents air-water and solvent-water d i s t r i b u t i o n r a t i o s measured f o r ΝDMA and two nitrosamines which have been employed as i n t e r n a l standards f o r NOMA determination. Table I I . D i s t r i b u t i o n Ratios of Some V o l a t i l e Nitrosamine


Air-Water Ν DMA NMEA NDPA

7.1 12 25

Dis{: r i b u t i o n Ratio (x 1CT ) DCM-Water }

3.8 15 140

Nitrosamines

Ether-Water 0.28 0.99 22

It i s c l e a r that n e i t h e r NMEA nor NDPA i s appropriate f o r an i n t e r n a l standard i n NDMA determination i f c r i t e r i a are i n t e r p r e t e d s t r i c t l y , but both compounds have been used f o r t h i s purpose. A d d i t i o n of a nitrosamine, not normally present i n the sample, i s h e l p f u l i n d e t e c t i n g any gross e r r o r s i n the procedure, but the a d d i t i o n should not be considered t o be i n t e r n a l s t a n d a r d i z a t i o n . U t i l i z a t i o n of NMEA or NDPA to i n d i c a t e recovery of NDMA can lead to s i g n i f i c a n t e r r o r s . In most reports of the a p p l i c a t i o n of these " i n t e r n a l standards", recovery of a l l nitrosamines was c l o s e to 100%. Under these c o n d i t i o n s , any added compound would appear t o be a good i n t e r n a l standard, but none i s necessary. NDMA i s a p a r t i c u l a r l y d i f f i c u l t compound f o r use of i n t e r n a l s t a n d a r d i z a t i o n because of i t s anomalous d i s t r i b u t i o n behavior. I£ mass spectrometry i s employed f o r q u a n t i t a t i v e determination, H- or N-labeled NDMA could be added as i n t e r n a l standard. Because the l a b e l e d m a t e r i a l would c o e l u t e from GC columns with the unlabeled NDMA, t h i s approach i s unworkable when GC-TEA i s employed or when high r e s o l u t i o n MS s e l e c t e d ion monitoring i s used with the equipment described above. R a d i o i s o t o p e - l a b e l e d nitrosamines have proven valuable i n development of a n a l y t i c a l methods and for demonstrating e f f i c i e n c y of recovery of nitrosamines from tobacco products and smoke (37-39). The very high s p e c i f i c a c t i v i t y r e q u i r e d f o r low p a r t - p e r - b i l l i o n determinations has discouraged most a n a l y s t s from using t h i s approach. Unless a radiochromatographic detector with adequate s e n s i t i v i t y i s a v a i l a b l e , samples must be counted independently of the f i n a l chromatographic determination, and one of the advantages of i n t e r n a l s t a n d a r d i z a t i o n , c o r r e c t i o n for v a r i a t i o n i n volume i n j e c t e d , i s l o s t . Column E x t r a c t i o n . Results obtained by the standard ASBC d i s t i l l a t i o n procedure and by C e l i t e column e x t r a c t i o n are compared i n Table I I I . The ASBC method included


340

iV-NITROSO COMPOUNDS

Table I I I . NDMA Concentrations Beer, Using ASBC Method and C e l i t e Column E x t r a c t i o n

χ


NDMA

NDMA Found ( g / l ) ASBC Method C e l i t e Column E x t r a c t i o n u

Added

2.08

1.3

2.0

2.55

1.4

2.0

5.67

4.2

4.0

6.02

4.9

5.1

10.42

7.8

8.0

0

ND (

Analytical Methods for Nitrosamines - ACS Publications - American

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