High Performance Mass Spectrometry - American Chemical Society

6 Organic Trace Analysis Using Direct Probe Sample Introduction and High Resolution Mass Spectrometry WILLIAM F. HADDON Western Regional Research Center, U.S. Department of Agriculture, Albany, CA 94710

Many interesting problems in organic trace analysis are inaccessible to combined gas chromatographymass spectrometry (GC/MS). For example, a recent treatise on the analysis of organic pollutants in water points out that between 80 and 90 percent of the extractable organic compounds in polluted water f a i l , even after derivitization, to pass through a gas chromatographic column (1). One approach for introducing less volatile compounds into the mass spectrometer in a highly purified state is the use of a liquid chromatograph coupled directly to the mass spectrometer (2,3,4). However, as an alternative to utilizing chromatographic separation prior to analysis, we can consider the mass spectrometer itself as an analytical instrument which is capable in itself of performing highly efficient separations, based, for example, on mass or energy differences of generated ions or on selective ionization, as well as precise identification and quantitation, based on reference mass spectra obtained separately on known compounds. This use of a mass spectrometer for combined separation-identification functions might be called "mass spectrometer-mass spectrometer" (MS/MS) analysis, with the direct probe serving as the vehicle for sample introduction. A number of laboratories have reported results using this approach, and the methods used to achieve selectivity in complex mixtures have included field ionization (5), chemical ionization (6,7), negative chemical ionization (8,9), collisional activation (10), mass-analyzed ion kinetic energy (11), and high resolution electron ionization (EI) (12-15) techniques. This report describes the use of the high resolution EI method in which particular ions are monitored as a function of temperature as the components of interest volatilize ©0-8412-0422-5/78/47-070-097$10.00/0

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HIGH PERFORMANCE MASS SPECTROMETRY

from t h e d i r e c t i n t r o d u c t i o n p r o b e . The term h i g h r e s o l u t i o n s e l e c t e d i o n m o n i t o r i n g (SIM) i s used t o d e s c r i b e t h i s type o f experiment ( 1 6 ) , and the i o n abundance p r o f i l e r e c o r d e d as a f u n c t i o n o f t e m p e r a t u r e i s c a l l e d an e x a c t mass fragmentogram. In many o r g a n i c t r a c e a n a l y s i s p r o b l e m s , the p r e p a r a t i o n o f h i g h l y p u r i f i e d samples amenable t o low r e s o l u t i o n mass s p e c t r a l i d e n t i f i c a t i o n r e q u i r e s ex tensive e f f o r t . Employing i n c r e a s e d mass r e s o l u t i o n t o a c h i e v e h i g h e r s e l e c t i v i t y can reduce the e x t e n t o f sample p u r i f i c a t i o n s i g n i f i c a n t l y i n many c a s e s , and r e c e n t examples o f GC-high r e s o l u t i o n SIM f o r b o t h b i o f l u i d a n a l y s i s (17,18) and e n v i r o n m e n t a l m o n i t o r i n g (19) a r e i l l u s t r a t i v e . In many l a b o r a t o r i e s concerned w i t h a n a l y z i n g p a r t i c u l a r components o f complex m i x t u r e s , the s a v i n g s i n t h e c o s t o f sample p r e p a r a t i o n made p o s s i b l e by the a v a i l a b i l i t y o f h i g h r e s o l u t i o n SIM c a p a b i l i t y may j u s t i f y the h i g h e r i n i t i a l c o s t o f a s u i t a b l e h i g h r e s o l u t i o n mass s p e c t r o m e t e r . Furthermore, s m a l l r a d i u s ( 6 - i n c h o r l e s s ) m a g n e t i c s e c t o r mass s p e c t r o meters h a v i n g b o t h the f a s t scan r a t e s r e q u i r e d by GC/MS a p p l i c a t i o n s and the c a p a b i l i t y f o r h i g h r e s o l u t i o n (10-20,000) o p e r a t i o n w i t h good s e n s i t i v i t y a r e becoming a v a i l a b l e c o m m e r c i a l l y . On a t l e a s t one s m a l l r a d i u s i n s t r u m e n t o f t h i s t y p e , image c u r v a t u r e c o r r e c t o r s have been employed t o enhance the h i g h r e s o l u t i o n p e r f o r m a n c e , and f u r t h e r improvements i n r e s o l u t i o n and s e n s i t i v i t y f o r these s m a l l r a d i u s i n s t r u ments w i l l u n d o u b t e d l y be f o r t h c o m i n g . Two examples d e s c r i b e d below from the f i e l d s o f c l i n i c a l and f o o d c h e m i s t r y i l l u s t r a t e the u t i l i t y o f t h e h i g h r e s o l u t i o n d i r e c t probe method f o r t r a c e analysis. In a d d i t i o n , p e s t i c i d e s i n human t i s s u e have been measured a t h i g h r e s o l u t i o n on a p h o t o - p l a t e equipped mass s p e c t r o m e t e r (Γ5), and s i m i l a r h i g h r e s o l u t i o n methods have been employed t o measure p e s t i c i d e s i n f o o d samples (9) and b i o f l u i d s (8) u s i n g n e g a t i v e c h e m i c a l i o n i z a t i o n , and t o q u a n t i t a t e drug m e t a b o l i t e s r a p i d l y i n b l o o d plasma (7.) u s i n g c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y . These papers c o n t a i n e x c e l l e n t d e s c r i p t i o n s o f the p r a c t i c a l problems o f p r e p a r i n g samples and q u a n t i t a t i n g the r e s u l t s . Experimental H i g h r e s o l u t i o n SIM o p e r a t i o n can be a c h i e v e d on a m a g n e t i c s e c t o r mass s p e c t r o m e t e r by i n c o r p o r a t i n g a s w i t c h e d v o l t a g e d i v i d e r c i r c u i t , or by employing c o m p u t e r - d r i v e n power s u p p l i e s .

6. HADDON

99

Organic Trace Analysis

The o v e r - a l l e l e c t r o n i c s t a b i l i t y n e c e s s a r y f o r q u a n t i t a t i v e work depends on t h e p r e c i s i o n r e q u i r e d and on t h e mass r e s o l u t i o n s e l e c t e d f o r t h e a n a l y s i s . These s t a b i l i t y r e q u i r e m e n t s c a n be d e r i v e d from t h e G a u s s i a n c u r v e o f F i g u r e 1, w h i c h r e p r e s e n t s a mass s p e c t r a l peak. The 10 p e r c e n t v a l l e y d e f i n i t i o n o f mass s p e c t r o m e t e r r e s o l u t i o n c o r r e s p o n d s t o t h e o r d i n a t e v a l u e a t a d i s t a n c e 2.44 σ from t h e c e n t e r o f t h e peak (5 p e r c e n t o f maximum h e i g h t f o r a s i n g l e p e a k ) , where σ i s t h e s t a n d a r d d e v i a t i o n o f t h e c u r v e . The o v e r - a l l i n s t r u m e n t s t a b i l i t y , S, i n p a r t s p e r m i l l i o n (ppm) f o r a p a r t i c u l a r a n a l y s i s i s 6

S = (a /2.44)(10 /R) U) where σ i s the number o f s t a n d a r d d e v i a t i o n s t o t h e o r d i n a t e a t t h e r e q u i r e d p e r c e n t a c c u r a c y , and R i s t h e mass r e s o l u t i o n (Μ/ΔΜ, 10 p e r c e n t v a l l e y d e f i n i t i o n ) x

T a b l e I . Mass S p e c t r o m e t e r S t a b i l i t y i n P a r t s p e r M i l l i o n Necessary f o r Q u a n t i t a t i v e High R e s o l u t i o n S e l e c t e d Ion M o n i t o r i n g . Resolution

Desired Q u a n t i t a t i v e Accuracy

(Percent):

99

95

90

80

16.

45.

61.

89.

9.8

27.

36.

53.

10,000

4.9

13.

18.

26.

15,000

3.3

9.0

12.

18.

20,000

2.4

6.7

9.2

2.7

3.6

3,000 5,000

50,000

.98

C a l c u l a t e d from Eq. 1. 'Μ/ΔΜ based on 10 p e r c e n t v a l l e y

13. 5.

definition.

T a b l e I l i s t s t y p i c a l c a l c u l a t e d v a l u e s o f S. These v a l u e s show t h a t q u a n t i t a t i v e p r e c i s i o n o f 90 p e r c e n t o r g r e a t e r s h o u l d be e a s i l y a c h i e v e d on most commercial mass s p e c t r o m e t e r s w h i c h have been d e s i g n e d f o r a c c u r a t e mass measurement a p p l i c a t i o n s . When o n l y one o r two peaks need t o be m o n i t o r e d , t h e c i r c u i t r y o f an e x i s t i n g peak matcher c a n be modi f i e d t o m o n i t o r b o t h peaks by s w i t c h i n g between peak t o p s . (2,19) On one w i d e l y used commercial h i g h r e s o l u t i o n i n s t r u m e n t , t h i s i s done by d i s c o n n e c t i n g t h e peak matcher scan c o i l s ( 2 ) . A l t e r n a t i v e l y , r e p e t i t i v e

100


s c a n s o v e r narrow mass ranges can be u t i l i z e d , but a t reduced s e n s i t i v i t y . An advantage o f t h i s l a t t e r dynamic method o f r e c o r d i n g d a t a i s t h a t the peak p r o f i l e information i s continuously a v a i l a b l e during the e x p e r i m e n t . An i n c r e a s e beyond two c h a n n e l s can be a c h i e v e d w i t h o u t a computer system by i n c o r p o r a t i n g a s w i t c h a b l e v o l t a g e d i v i d e r c i r c u i t (13) (20) ( 2 1 ) . I t i s imp o r t a n t f o r a c i r c u i t o f t h i s type to employ samplea n d - h o l d a m p l i f i e r s f o r each c h a n n e l t o p r o v i d e cont i n u o u s o u t p u t s i g n a l s s u i t a b l e f o r d r i v i n g an o s c i l l o g r a p h i c or m u l t i p l e pen r e c o r d e r . Computer-Controlled Power S u p p l i e s f o r H i g h R e s o l u t i o n SIM. A d e d i c a t e d computer can p e r f o r m b o t h d a t a c o l l e c t i o n and i n s t r u m e n t c o n t r o l f u n c t i o n s f o r h i g h r e s o l u t i o n SIM when used i n c o n j u n c t i o n w i t h a programmable power s u p p l y f o r the mass s p e c t r o m e t e r a c c e l e r a t i n g and e l e c t r i c s e c t o r v o l t a g e s . The computer p r o v i d e s some a d d i t i o n a l b e n e f i t s over a h a r d - w i r e d system: The number o f mass c h a n n e l s can be l a r g e and can be changed d u r i n g the a n a l y s i s ; the f r a c t i o n o f t i m e a t a p a r t i c u l a r mass v a l u e can be v a r i e d a c c o r d i n g t o the e x p e c t e d abundance o f the peak; and the s y s t e m , when s u i t a b l y programmed, can sweep s h o r t mass r e g i o n s t o p r o v i d e peak p r o f i l e i n f o r m a t i o n w h i c h can be used b o t h t o a s c e r t a i n the degree o f i n t e r f e r e n c e from i s o b a r i c i o n s near the e l e m e n t a l c o m p o s i t i o n o f i n t e r e s t , and t o make a u t o m a t i c p e r i o d i c a d j u s t m e n t s o f the a c c e l e r a t i n g v o l t a g e t o compensate f o r d r i f t i n voltages. F i g u r e 2 shows the programmed power s u p p l y c i r c u i t r y u s e d i n our l a b o r a t o r y f o r h i g h r e s o l u t i o n SIM on a CEC 21-110A d o u b l e - f o c u s s i n g mass s p e c t r o m e t e r . Many o f the e l e c t r o n i c and programming d e t a i l s , i n c l u d i n g c h o i c e o f s u i t a b l e power s u p p l y u n i t s , a r e from the Washington U n i v e r s i t y system o f Holmes f221 f o r low r e s o l u t i o n SIM. In the USDA system a s i n g l e programmer (D/A c o n v e r t e r ) w i t h a 1 - v o l t o u t p u t d r i v e s a p a i r o f power s u p p l i e s i n s e r i e s , one w i t h a v o l t a g e g a i n o f 100 (KEPCO OPS-2000, Kepco, I n c . , F l u s h i n g , N.Y.) f o l l o w e d by a second w i t h a g a i n of -1 (KEPCO NTC2000). These power s u p p l i e s d i r e c t l y r e p l a c e the e l e c t r i c s e c t o r s u p p l y f o r m e r l y used w i t h the i n s t r u ment. The 21-110 r e q u i r e s p l u s and minus 375 v o l t s f o r n o m i n a l 8 kv. o p e r a t i o n . We d e r i v e a f i x e d p o r t i o n o f t h i s v o l t a g e from the i n t e r n a l r e f e r e n c e s u p p l y o f the OPS-2000 t h r o u g h R and R , and a v a r i a b l e p o r t i o n from the D/A c o n v e r t e r (DATEL DAC-169, 16 b i t p r e c i s i o n , D a t e l Systems, I n c . , Canton, Ma.) t h r o u g h R i . The 2

3

6. HADDON

101


KEPCO

NTC-2000

100 (EA+E.)

0 to +1000V

INV (IN) E (0 to +1V or 0 to +10V) A

SPC-16 D/A

CONVERTER INTERFACE 16-BIT LATCH

LOGIC SIGNALS

DATEL DAC-169 16 BIT D/A CONVERTER

-100 EA +EI] 0 to -1000V

Rl

NULL

WNr-

KEPCO OPS-2000

10KÛ

R2

10KÛ

+6.2V REF

Figure 2. Programmed power supply interface for CEC 21-110 mass spectrometer

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s e t t i n g o f R d e t e r m i n e s the f r a c t i o n o f f i x e d v o l t a g e a c c o r d i n g to the r e q u i r e m e n t s o f mass range and p r e c i s i o n . For a t e n p e r c e n t mass r a n g e , each d i g i t a l s t e p o f the programmed v o l t a g e c o r r e s p o n d s t o a 1.5 ppm change i n mass. The r e l a t i o n s h i p between mass and v o l t a g e o u t p u t f o r programmed a c c e l e r a t i n g v o l t a g e o p e r a t i o n i s 3

where m i s the mass i n f o c u s a t the c o l l e c t o r w i t h the D/A c o n v e r t e r a t 0 v o l t s , or d i g i t a l b i t s e t t i n g , r e q u i r e d t o ' ? o c u s mass m. ~Eq. 2 i s d e r i v e d from the mass s p e c t r o m e t e r e q u a t i o n by w r i t i n g a c c e l e r a t i n g v o l t a g e as a sum o f the a c c e l e r a t i n g v o l t a g e a t m p l u s the added v o l t a g e from the D/A c o n v e r t e r and a p p l i e s to b o t h s i n g l e and d o u b l e focussing sector instruments. F i g u r e 3 shows t h a t a l i n e a r r e l a t i o n s h i p i s o b t a i n e d f o r Eq. 2 f o r the 21110 mass s p e c t r o m e t e r m o d i f i e d as i n F i g u r e 2 over the mass range 293-331. V a l u e s of D/A c o n v e r t e r v o l t a g e w h i c h f o c u s p a r t i c u l a r e x a c t masses can be d e t e r m i n e d by l o c a t i n g i n t e r n a l r e f e r e n c e p e a k s , t y p i c a l l y d e r i v e d from p e r f l u o r o k e r o s e n e (PFK), and u t i l i z i n g them t o d e r i v e the s l o p e and i n t e r c e p t by l e a s t squares f i t to Eq. 2. A l t e r n a t i v e l y , the s p e c t r o m e t e r can be s e t to the c e n t e r o f a known peak, w h i c h g i v e s m directly, and k can be c a l c u l a t e d by m a n u a l l y l o c a t i n g one or more a d d i t i o n a l r e f e r e n c e peaks as V j j / a i s v a r i e d . The computer can be programmed to a d j u s t the app l i e d a c c e l e r a t i n g v o l t a g e d u r i n g an e x p e r i m e n t t o compensate f o r i n s t a b i l i t i e s i n the mass s p e c t r o m e t e r by sweeping a s i n g l e r e f e r e n c e peak p e r i o d i c a l l y , and c h a n g i n g the v o l t a g e a t each m/e a c o n s t a n t amount t o c o r r e c t f o r s h i f t s i n peak c e n t r o i d , a l t h o u g h t h e r e a r e s m a l l e r r o r s i n t h i s p r o c e d u r e because mass and v o l t a g e a r e not l i n e a r l y r e l a t e d (Eq. 2 ) . Feedback c o n t r o l o f t h i s type a l l o w s the use o f l e s s e x p e n s i v e power supp l i e s , w h i c h o f t e n have e x c e l l e n t s h o r t term s t a b i l i t y but p o o r e r l o n g term or t e m p e r a t u r e s t a b i l i t y (2_2) . M i c r o p r o c e s s o r - b a s e d SIM modules are becoming a v a i l a b l e , and one c o m m e r c i a l system s u i t a b l e f o r h i g h r e s o l u t i o n measurement i n c l u d e s a peak s t a b i l i z i n g c a p a b i l i t y and the f a c i l i t y f o r m o n i t o r i n g up to 8 m/ev a l u e s ( 2 3 ) . As w i t h a l l SIM c i r c u i t s l i m i t e d t o peak s w i t c h i n g , the one drawback r e l a t i v e to a computerbased system i s t h a t peak p r o f i l e i n f o r m a t i o n i s not e a s i l y o b t a i n e d . A s o l u t i o n to t h i s p r o b l e m i s to devote t h r e e c h a n n e l s o f the peak s e l e c t o r t o the same peak, one a t the c e n t e r , and two a t the 10 p e r c e n t 0

0

0

6. HADDON


103

3.6 3.4 -

y/ 293 299 / #

#

3.3 -

0

/

0.2

0.4

0.6

# 3

0.8

°°

1.0

VD/A, VOLTS

Figure 3. Mass-voltage relationship for programmed accelerating voltage operation of 21110 mass spectrometer. M = m/e value focused at collector. V = voltage output of D/A converter. D/A

104


i n t e n s i t y p o i n t s on the h i g h and low mass s i d e o f the peak. Three e x a c t mass fragmentograms c o i n c i d e n t i n t i m e ( t e m p e r a t u r e ) and h a v i n g 1:10:1 i n t e n s i t y r a t i o s c o n f i r m the absence o f i n t e r f e r i n g i s o b a r i c i o n s d u r i n g the a n a l y s i s ( 2 4 ) . Applications Aflatoxin Analysis. I n our l a b o r a t o r y we have measured s m a l l amounts o f a f l a t o x i n s i n foods and f e e d s , and i n the b i o f l u i d s o f e x p e r i m e n t a l a n i m a l s (25-27). A f l a t o x i n s are metab o l i t e s o f the mold A s p e r g i l l u s F l a v u s , and one o f the metabolites, a f l a t o x i n B i , i s of p a r t i c u l a r i n t e r e s t because i t i s a n a t u r a l l y o c c u r r i n g c a r c i n o g e n w h i c h i s sometimes p r e s e n t a t up to s e v e r a l p a r t s - p e r - b i l l i o n (ppb) i n some a g r i c u l t u r a l p r o d u c t s (28-30). The s t r u c t u r e s and p a r t i a l EI mass s p e c t r a o f s i x a f l a t o x i n s are shown i n F i g u r e 4, and the mass s p e c t r a o f about 50 a d d i t i o n a l m y c o t o x i n s a r e a v a i l a b l e e l s e w h e r e (3JL). P u r i f i c a t i o n and subsequent t r a n s f e r o f a f l a t o x i n s i n t o the mass s p e c t r o m e t e r i s d i f f i c u l t because the compounds a r e u n s t a b l e when h i g h l y p u r i f i e d , bond s t r o n g l y t o g l a s s s u r f a c e s , and cannot be s e p a r a t e d by gas chromatography. These f a c t o r s r e s t r i c t the use o f low r e s o l u t i o n mass s p e c t r a l a n a l y s i s f o r s t r u c t u r a l l y s p e c i f i c v e r i f i c a t i o n or q u a n t i t a t i o n of a f l a t o x i n s . The use o f h i g h r e s o l u t i o n SIM on p a r t i a l l y p u r i f i e d a f l a t o x i n c o n t a i n i n g m i x t u r e s i n t r o d u c e d v i a the d i r e c t probe s i g n i f i c a n t l y l o w e r s the l i m i t s o f d e t e c t i o n f o r mass s p e c t r a l a n a l y s i s o f t h e s e compounds, and has the a d d i t i o n a l advantage t h a t p r i o r i s o l a t i o n p r o c e d u r e s can be s i m p l i f i e d (12) . By u s i n g h i g h r e s o l u t i o n SIM we have d e t e c t e d a f l a t o x i n s B i and Mi i n e x p e r i m e n t a l m i l k samples a t 19 and 140 ppb, r e s p e c t i v e l y . An a n a l y t i c a l e x t r a c t i o n scheme f o r o b t a i n i n g p a r t i a l l y p u r i f i e d a f l a t o x i n s from f r e e z e - d r i e d m i l k and s u i t a b l e f o r use i n c o n j u n c t i o n w i t h h i g h r e s o l u t i o n mass s p e c t r a l a n a l y s i s i s shown i n F i g u r e 5. E x t r a c t A c o n t a i n s a l l o f the a f l a t o x i n s . In e x t r a c t s Β and C, w h i c h are more h i g h l y p u r i f i e d , a f l a t o x i n s B i and Mi a r e p a r t i a l l y s e p a r a t e d because of d i f f e r e n c e s i n p a r t i t i o n c o e f f i c i e n t s i n the e x t r a c t i o n s o l v e n t s (32). H i g h r e s o l u t i o n SIM d a t a f o r e x t r a c t s A and C a r e shown i n F i g u r e s 6 and 7. These d a t a o f i n t e n s i t y vvs. mass r e p r e s e n t scans o v e r 0.3 amu f o r each a p p r o p r i a t e i n t e g r a l m/e a t mass r e s o l u t i o n o f 7,000. The m i d d l e s c a n f o r e x t r a c t A ( F i g . 6 ) , w h i c h was r e c o r d e d a t the t e m p e r a t u r e o f maximum r a t e o f v o l a t i l i z a t i o n o f the a f l a t o x i n s , shows the Mi m o l e c u l a r i o n a t m/e 328, the

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M-16 i o n o f Mi and m o l e c u l a r i o n o f B i (same e l e m e n t a l c o m p o s i t i o n , C i H i 0 ) a t m/e 312, and the M-29 i o n o f Mi a t m/e 299. The abundance r a t i o [M] ·/[M-29] agrees w i t h i n e x p e r i m e n t a l e r r o r w i t h t h e r a t i o o f t h e s e peaks i n t h e r e f e r e n c e low r e s o l u t i o n mass spectrum o f a f l a t o x i n Mi shown i n F i g u r e 4 and c o n f i r m s t h e absence o f i n t e r f e r e n c e a t a r e s o l u t i o n o f 7000 f o r this particular extract. Figure 7 gives equivalent d a t a f o r e x t r a c t C o b t a i n e d from t h e same sample o f f r e e z e - d r i e d m i l k . The a d d i t i o n a l peak a t m/e 314 i s from 12 ng a f l a t o x i n B , added as an i n t e r n a l s t a n d a r d . The abundance r a t i o s between a f l a t o x i n i o n s a t m/e 312 and 328 i n t h e s e scans r e f l e c t t h e d e c r e a s e d amount o f a f l a t o x i n B f o r e x t r a c t C w h i c h r e s u l t e d from s o l v e n t p a r t i t i o n i n g during extraction. Thus f o r e x t r a c t A ( F i g . 6) t h e i n t e n s i t y r a t i o o f C i H i 0 t o C i H i 0 i s 0.32, compared t o 0.06 f o r pure Mi (see F i g . 4 e ) , but f o r e x t r a c t C ( F i g . 7) t h e r a t i o i s 0.092, w h i c h i n d i c a t e s l e s s a f l a t o x i n B , as e x p e c t e d . The methodo l o g y t o q u a n t i t a t e t h e compounds i s c u r r e n t l y b e i n g developed. A n o t a b l e f e a t u r e o f t h e d i r e c t probe method f o r a f l a t o x i n a n a l y s i s i s an enhancement o f s e n s i t i v i t y w h i c h o c c u r s when p a r t i a l l y p u r i f i e d samples o f a f l a t o x i n a r e r u n on t h e mass s p e c t r o m e t e r , r e l a t i v e t o measurements on h i g h l y p u r i f i e d samples. Apparently, t h i s i s because b o n d i n g t o t h e s u r f a c e o f t h e sample c o n t a i n e r s i s s u b s t a n t i a l l y reduced i n a complex mixt u r e f o r t h e s e compounds. The d a t a o f F i g u r e 8 i l l u s t r a t e t h i s enhancement o f s e n s i t i v i t y f o r a f l a t o x i n B i added t o an e x t r a c t o f p o o l e d human u r i n e s . The c u r v e s o f F i g . 8 a r e e x a c t mass fragmentograms, r e c o r d e d d u r i n g r a p i d t e m p e r a t u r e programming o f t h e d i r e c t probe from about 50°C t o 250°C, w i t h the mass s p e c t r o m e t e r tuned t o the m o l e c u l a r i o n o f B , m/e 312.0635, a t 7000 r e s o l u t i o n . A c o m p a r i s o n o f t h e response o b t a i n e d i n c u r v e A o f F i g . 8 f o r 1.3 ng p u r e B i w i t h t h a t o f c u r v e D, o b t a i n e d from 0.03 ng B i added t o the u r i n e e x t r a c t , i l l u s t r a t e t h e 1 0 0 - f o l d enhancement o f s e n s i t i v i t y f o r d e t e c t i n g a f l a t o x i n s B i i n a complex m i x t u r e . The s e n s i t i v i t y enhancement i s somewhat g r e a t e r f o r a f l a t o x i n M i , f o r w h i c h n e g l i g i b l e response i s o b t a i n e d f o r 30 ng o f p u r i f i e d compound, compared t o a s i g n a l t o background r a t i o o f b e t t e r than 100 f o r 13 ng i n t r o d u c e d i n a m i x t u r e , as shown i n F i g s . 6 and 7. Q u a n t i t a t i v e H i g h R e s o l u t i o n SIM f o r A n a l y z i n g P u r i n e s i n B l o o d and T i s s u e . Q u a n t i t a t i v e d i r e c t probe measurements a t t h e 2100 ppm l e v e l f o r f i v e p u r i n e components o f human b l o o d 7

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Figure 5. Analytical extraction of aflatoxins B and M from freeze-dried milk t

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Figure 6. High resolution SIM scans of intensity in millivolts (mV) vs. mass for 13 ng aflatoxin M and 1.7 ng aflatoxin B in 100fig dried milk extract A. Direct introduction probe temperature increases from A to C. t

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Figure 7. High resolution SIM scans of intensity in millivolts (mV) vs. mass for extract C. 12.4 ng aflatoxin B (mass 314) added as internal standard. Direct probe temperature increases from A to B. 2

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111

and t i s s u e have been r e p o r t e d by Snedden and P a r k e r u s i n g h i g h r e s o l u t i o n SIM (13) ( 3 3 ) . The h i g h r e s o l u t i o n mass s p e c t r a l a n a l y s i s f o r t h e p u r i n e s p r o v i d e d q u a n t i t a t i v e measurement o f h y p o x a n t h i n e ( I ) , x a n t h i n e ( I I ) , u r i c a c i d ( I I I ) , a l l o p u r i n o l (IV) and p u r i n o l (V) (see F i g u r e 9 ) , w h i c h a r e i m p o r t a n t i n t h e s t u d y and t r e a t m e n t o f gout. The use o f h i g h r e s o l u t i o n mass s p e c t r o m e t r y made p o s s i b l e a c c u r a t e a n a l y s i s a t t h e s e l e v e l s w i t h o u t p r e - p u r i f i c a t i o n o f t h e samples. More r e c e n t l y t h e same group has used h i g h r e s o l u t i o n mass s p e c t r o m e t r y t o q u a n t i t a t e o e s t r o g e n and p r o g e s t e r o n e i n human o v a r i a n t i s s u e a t 10-50 ppm, a g a i n u s i n g p r o c e d u r e s t h a t r e q u i r e no c h e m i c a l s e p a r a t i o n o r d e r i v i t i z a t i o n p r i o r t o mass s p e c t r a l a n a l y s i s ( 1 4 ) . To i n s u r e t h a t t h e i r method i s v a l i d , t h e a u t h o r s r e c o r d e d a h i g h r e s o l u t i o n spectrum o f t h e m i x t u r e t o be a n a l y z e d a t t h e chosen r e s o l u t i o n , i n t h i s case 20,000, and compared t h e abundances o f c h a r a c t e r i s t i c mass peaks i n t h e e x p e r i m e n t a l sample w i t h those i n t h e h i g h r e s o l u t i o n r e f e r e n c e spectrum o f t h e pure compound. F i g u r e 10 i l l u s t r a t e s t h e e x c e l l e n t agreement o b t a i n e d a t f i v e o f t h e s i x masses examined f o r I I I i n human muscle t i s s u e . S i m i l a r experiments w i t h the o t h e r p u r i n e s o f F i g u r e 9 y i e l d e d a s e t o f masses a p p r o p r i a t e f o r a n a l y z i n g I-V t o g e t h e r . I n t e g r a t i n g t h e s e l e c t e d i o n p r o f i l e s ( e x a c t mass fragmentograms) a t f i v e masses as a f u n c t i o n o f temp e r a t u r e gave a s e t o f r e l a t i v e peak a r e a s . These peak a r e a s were r e l a t e d t o t h e r e l a t i v e amounts o f I-V u s i n g a s e t o f f i v e l i n e a r s i m u l t a n e o u s e q u a t i o n s and r e sponse c o e f f i c i e n t s d e v e l o p e d from runs on p u r i f i e d compounds, a c c o r d i n g t o e s t a b l i s h e d p r o c e d u r e s o f q u a n t i t a t i v e mass s p e c t r o m e t r y ( 3 4 ) . The i n t r o d u c t i o n o f a c c u r a t e l y weighed (mg amounts) samples f a c i l i t a t e d the c a l c u l a t i o n o f a b s o l u t e c o n c e n t r a t i o n s . An e v a l u a t i o n o f t h e s e n s i t i v i t y and measurement e r r o r f o r t h e a n a l y s i s r e v e a l s t h a t d i f f e r e n t methods o f sample p r e p a r a t i o n g i v e d i f f e r e n t q u a n t i t a t i v e p r e cision. F i g . 11 shows t h a t t h e b e s t r e s u l t s were o b t a i n e d u s i n g powdered samples o f d e s s i c a t e d b l o o d and muscle t i s s u e . D e p o s i t i n g t h e samples on t h e d i r e c t probe s u r f a c e ( g o l d ) by e v a p o r a t i o n o f an aqueous s o l u t i o n gave s i g n i f i c a n t l y lower p r e c i s i o n f o r a g i v e n sample amount, a p p a r e n t l y because o f i n t e r a c t i o n s between t h e sample and t h e s u r f a c e o f t h e probe f o r t h e more p o l a r compounds. When a l e s s p o l a r s u b s t a n c e , c a f f e i n e , was a n a l y z e d , t h e r e was no dependence o f p r e c i s i o n on t h e method o f p r e p a r i n g t h e sample, as shown i n F i g . 11.

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Figure 8. Exact mass fragmentogram for C H 0 (aflatoxin Bi molecular ion, m/e 312.0635): A, 1.65 ng B from reference solution; B, blank, extract B; C, urine extract plus 0.13 ng B ; D, urine extract plus 0.030 ng B . î7

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Discussion The mass s p e c t r o m e t e r i s w e l l s u i t e d t o t r a c e a n a l y s i s o f the t y p e d i s c u s s e d h e r e f o r two r e a s o n s : The p r o c e s s o f i o n p r o d u c t i o n by the mass s p e c t r o m e t e r i s g e n e r a l l y l i n e a r o v e r many o r d e r s o f magnitude o f sample c o n c e n t r a t i o n ; and the s e l e c t i v i t y o f the method can be e x t r e m e l y g r e a t when e i t h e r h i g h mass r e s o l u t i o n , a s e l e c t i v e i o n i z a t i o n method, or a c o m b i n a t i o n of t h e s e a r e employed t o e f f e c t s e p a r a t i o n between d i f f e r e n t components o f m i x t u r e s . The wide dynamic range n e c e s s a r y f o r q u a n t i t a t i o n i s w e l l accomodated by electron multiplier detection. Photoplate detection w i l l be g e n e r a l l y l e s s s u i t a b l e f o r t r a c e a n a l y s i s problems o f t h i s n a t u r e because of extensive blackening of the p h o t o g r a p h i c p l a t e near i n t e n s e l i n e s , and nonl i n e a r i t y of response. The a f l a t o x i n s a r e f a v o r a b l y a n a l y z e d by h i g h r e s o l u t i o n SIM i n complex m i x t u r e s i n p a r t because o f t h e i r low r a t i o o f hydrogen t o c a r b o n , w h i c h l e a d s t o an e x a c t mass v a l u e below the masses o f o t h e r sub stances, p r i m a r i l y l i p i d s , t y p i c a l l y present i n food e x t r a c t s . Many o t h e r e n v i r o n m e n t a l p o l l u t a n t s i n c l u d i n g h a l o g e n a t e d p e s t i c i d e s and many o f the common m y c o t o x i n s (35.) have comparable o r g r e a t e r mass d e f e c t s and s h o u l d be amenable t o a n a l y s i s by t h i s method. We have f r e q u e n t l y o b s e r v e d peaks below the e x a c t mass p o s i t i o n s o f the a f l a t o x i n s , as shown f o r the scans a t m/e 299 and 312 o f F i g u r e 6. These peaks may a r i s e from h a l o g e n a t e d p e s t i c i d e c o n t a m i n a n t s . High reso l u t i o n SIM may be a v a l u a b l e complement t o GC/MS and t o n e g a t i v e c h e m i c a l i o n i z a t i o n t e c h n i q u e s (8_,_9) f o r such compounds. Surface e f f e c t s . I n t e r a c t i o n s w i t h the s u r f a c e o f cont a i n e r s used t o i n t r o d u c e samples v i a the d i r e c t probe can a f f e c t d e t e c t i o n l i m i t s a d v e r s e l y , and an under s t a n d i n g o f s u r f a c e e f f e c t s appears to be i m p o r t a n t f o r f u l l y u t i l i z i n g the method i n t r a c e a n a l y s i s . In a d e t a i l e d s t u d y o f s u r f a c e i n t e r a c t i o n s f o r s m a l l pep t i d e s , Friedman (36-38) d e s c r i b e s the r a t e o f v o l a t i l i z a t i o n (dn/dt) o f a pure compound from a s u r f a c e by the e q u a t i o n %

H

= N Ae" o

E / R T

(3)

where N i s the number o f sample m o l e c u l e s i n i t i a l l y on the s u r f a c e , A i s a f r e q u e n c y f a c t o r , and the exponen t i a l term e ~ ' g i v e s the f r a c t i o n o f m o l e c u l e s a t t e m p e r a t u r e Τ w i t h s u f f i c i e n t energy t o overcome s u r face bonding. Ε i s the a c t i v a t i o n energy f o r removing 0

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a m o l e c u l e from the s u r f a c e . A h i g h r a t e o f v o l a t i l i z a t i o n i s r e a l i z e d by i n c r e a s i n g N , f o r example by d i s p e r s i n g the sample by e v a p o r a t i o n from s o l u t i o n and by r e d u c i n g E, by u s i n g an i n e r t d i r e c t probe s u r f a c e . The enhanced s e n s i t i v i t y f o r a f l a t o x i n s i n mixt u r e s t h a t we o b s e r v e i s c o n s i s t e n t w i t h Friedman's model f o r s u r f a c e v o l a t i l i z a t i o n . Apparently other compounds c o d e p o s i t e d from s o l u t i o n a l o n g w i t h the a f l a t o x i n s e i t h e r occupy a c t i v e s i t e s on the sample tubes p r e f e r e n t i a l l y , o r s h i e l d the a f l a t o x i n s from them, r e s u l t i n g i n more e f f i c i e n t v o l a t i l i z a t i o n a r i s i n g from a r e d u c t i o n i n E. The c u r v e s o f measurement e r r o r f o r the p u r i n e s ( F i g . 1 1 ) , w h i c h i n d i c a t e lower p r e c i s i o n f o r more p o l a r compounds, a r e s u g g e s t i v e o f s u r f a c e i n t e r a c t i o n s ^ but the i n t e r p r e t a t i o n i s l e s s o b v i o u s , because d i s p e r s i n g the compounds over the g o l d s u r f a c e o f the sample probe a c t u a l l y l o w e r e d the p r e c i s i o n o f the a n a l y s i s . The p r e s e n c e o f h i g h concent r a t i o n s of i n o r g a n i c s a l t s probably accounts f o r t h i s e f f e c t f o r the p u r i n e s . I n Friedman's s t u d y o f s m a l l peptide v o l a t i l i z a t i o n , traces of i n o r g a n i c s a l t s l o w e r e d the r a t e o f v o l a t i l i z a t i o n s u b s t a n t i a l l y a t a given temperature (38). The s u r f a c e e f f e c t p r o b l e m i s an i m p o r t a n t a r e a f o r f u r t h e r study. I t has been s u g g e s t e d r e c e n t l y t h a t a r e d u c t i o n i n s u r f a c e b o n d i n g e x p l a i n s the s p e c t r a o b t a i n e d from " n o n - v o l a t i l e " compounds a b s o r b e d on a c t i v a t e d (surface-prepared) tungsten emitter wires u s i n g f i e l d d e s o r p t i o n mass s p e c t r o m e t r y (39,40.). M i n i m i z i n g s u r f a c e i n t e r a c t i o n s appears p r o m i s i n g as a g e n e r a l method t o enhance s e n s i t i v i t y f o r mass s p e c t r a l a n a l y s i s of l e s s v o l a t i l e substances. Of c o u r s e , i t i s i m p o r t a n t t o r e c o g n i z e t h a t the c h e m i c a l i n s t a b i l i t y o f a p a r t i c u l a r s u b s t a n c e may be s i g n i f i c a n t l y d e c r e a s e d r e l a t i v e t o i t s s t a b i l i t y i n a s o l u t i o n or s o l i d sample m a t r i x ; f o r example, a f l a t o x i n s d i s p e r s e d on a s u r f a c e show g r e a t l y enhanced r e a c t i v i t y to UV l i g h t . Ion Source C o n t a m i n a t i o n . C o n t a m i n a t i o n o f the i o n s o u r c e , w h i c h may o c c u r r a p i d l y i n some c a s e s , can a p p r e c i a b l y r e d u c e the s e n s i t i v i t y o f the method. T h i s f a c t o r must be weighed w i t h the advantages o f u s i n g the d i r e c t probe when a l t e r n a t i v e GC/MS p r o c e d u r e s a r e a p p l i c a b l e . The r a p i d i t y o f s o u r c e c o n t a m i n a t i o n v a r i e s w i d e l y i n the few examples r e p o r t e d . Snedden and P a r k e r e x p e r i e n c e d l e s s t h a n 20 per c e n t d e c l i n e i n s e n s i t i v i t y i n s i x months o f p u r i n e a n a l y s i s ( 1 3 J . C o n v e r s e l y , Dougherty (8.) found t h a t s e n s i t i v i t y f o r p o l y c h l o r i n a t e d b i p h e n y l (PCB) d e t e c t i o n was s e v e r e l y r e d u c e d a f t e r r u n n i n g t e n samples o f u n p u r i f i e d human u r i n e e x t r a c t . M i n i m a l s o l v e n t e x t r a c t i o n and column 0

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chromatography p r i o r t o d i r e c t probe a n a l y s i s s o l v e d t h i s p r o b l e m , as i t has i n our l a b o r a t o r y f o r a f l a t o x i n a n a l y s i s . Other approaches t o r e d u c i n g i o n source c o n t a m i n a t i o n may be a p p l i c a b l e i n some c a s e s . F o r example, i t s h o u l d be p o s s i b l e t o m e c h a n i c a l l y v e n t t h e v o l a t i l i z e d sample away from t h e i o n i z a t i o n chamber u n t i l t h e temperature o f v o l a t i l i z a t i o n f o r t h e compounds o f i n t e r e s t i s reached (see F i g . 8 ) , t h e r e b y reducing the q u a n t i t y o f m a t e r i a l which enters the i o n source. Chemical B i n d i n g E f f e c t s . The c h e m i c a l s t a t e o f t h e sample c a n a f f e c t i t s v o l a t i l i t y . A comparison o f the q u a n t i t a t i v e mass s p e c t r a l measurements f o r u r i c a c i d i n b l o o d plasma w i t h t h e c o n c e n t r a t i o n v a l u e s o b t a i n e d by e n z y m a t i c a n a l y s i s s u g g e s t s t h a t t h e mass s p e c t r o meter d e t e c t s o n l y t h e u r i c a c i d w h i c h i s bound l o o s e l y t o b l o o d p r o t e i n s , and n o t t h e u r i c a c i d i n s o l u t i o n , w h i c h may e x i s t p r i m a r i l y as t h e sodium s a l t . These d a t a , w h i c h must be c o n s i d e r e d p r e l i m i n a r y , suggest t h a t mass s p e c t r a l a n a l y s i s , when p e r f o r m e d w i t h o u t p r i o r sample t r e a t m e n t , may i n d i c a t e i n d i r e c t l y t h e e x t e n t o f c h e m i c a l b i n d i n g f o r s m a l l m o l e c u l e s i n some cases. Conclusions High r e s o l u t i o n s e l e c t e d i o n m o n i t o r i n g measurements on samples i n t r o d u c e d on t h e d i r e c t probe c a n p r o v i d e a c c u r a t e and h i g h l y s p e c i f i c q u a l i t a t i v e and q u a n t i t a t i v e i n f o r m a t i o n about compounds p r e s e n t i n t r a c e amounts. T h i s m i x t u r e a n a l y s i s method i s app l i c a b l e t o some compounds w h i c h cannot be s e p a r a t e d by gas chromatography because o f low v o l a t i l i t y o r chemical instability. The h i g h e s t s e n s i t i v i t i e s a r e obt a i n e d f o r s u b s t a n c e s h a v i n g l a r g e mass d e f e c t s and t h i s embraces a number o f c l a s s e s o f compounds, i n c l u d i n g h a l o g e n a t e d p e s t i c i d e s and m y c o t o x i n s , w h i c h a r e o f p a r t i c u l a r i n t e r e s t i n e n v i r o n m e n t a l and f o o d c h e m i s t r y . M a g n e t i c s e c t o r mass s p e c t r o m e t e r s h a v i n g s u f f i c i e n t e l e c t r o n i c s t a b i l i t y f o r a c c u r a t e mass measurement c a n be m o d i f i e d f o r a c c u r a t e q u a n t i t a t i v e h i g h r e s o l u t i o n s e l e c t e d i o n m o n i t o r i n g measurements.

References 1. Keith, L.H., in Identification and Analysis of Organic Pollutants in Water, L.H. Keith, ed. (Ann Arbor Science Publishers, Ann Arbor) 1976, p. iii. 2. McFadden, W.H., Schwartz, H.L. and Evans, S., J. Chromatogr. (1976) 122, 389.

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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Arpino, P.J., Dawkins, B . J . , and McLafferty, F.W., J. Chromatogr. Sci. (1974) 12, 574. Carroll, D.I., Dzidic, I., Stillwell, R.N., Haegele, K.D. and Horning, E.C., Anal. Chem. (1975) 47, 2369. Sphon, J.Α., Dreifuss, P.A. and Schulten, H.R., J. Assoc. Official Anal. Chemists, (1977) 60, 73. Hunt, D.F. and Ryan III, J.F., Anal. Chem., (1972) 44, 1306. Weinkam, R.J., Rowland, M. and Meffin, P . J . , Biomed. Mass Spectrom. (1977) 4, 42. Dougherty, R.C. and Piotrowska, K., Proc. Nat'l. Acad. Sci. USA, (1976) 73, 1777. Dougherty, R.C. and Piotrowska, K., J. Assoc. Official Anal. Chemists, (1976) 59, 1023. Levson, K. and Schulten, H.R., Biomed. Mass Spec trom. (1976) 3, 137. Soltero-Rigau, E. Kruger, K.L. and Cooks, R.G., Anal. Chem. (1977) 49, 435. Haddon, W.F., Masri, M.S., Randall, V.G., Elsken, R.H., and Meneghelli, B . J . , J. Assoc. Official Anal. Chemists (1977) 60, 107. Snedden, W. and Parker, R.B., Anal. Chem. (1971) 43, 1651. Snedden, W. and Parker, R.B., Biomed. Mass Spec trom. (1976) 3, 275. Hutzinger, O., Jamieson, W.D., and Safe, S., Nature (London) (1974) 252, 698. Fenselau, C., Anal. Chem. (1977) 49, 563A. Millington, D.S., Buoy, M.E., Brooks, G., Harper, M.E. and Griffiths, Κ., Biomed. Mass Spectrom. (1975) 2, 219. Millington, D.S., J. Steroid Biochem. (1975) 6, 239. Evans, K.P., Mathias, Α., Mellor, Ν., Silvester, R. and Williams, A.E., Anal. Chem. (1975) 47, 821. Gallegos, E . J . , Anal. Chem. (1975) 47, 1150. Hammar, C.G., and Hessling, R., Anal. Chem. (1971) 43, 298. Holmes, W.F., Holland, W.H., Shore, B.L., Bier, D.M. and Sherman, W.R., Anal. Chem. (1973) 45, 2063. Vacuum Generators, Ltd., Altrincham, Cheshire, England. Millington, D.S., personal communication. Haddon, W.F., Wiley, M. and Waiss, A.C., Anal. Chem. (1971) 43, 268. Masri, M.S., J. Am. Oil Chem. Soc. (1970) 47, 61. Masri, M.S., Haddon, W.F., Lundin, R.E., and Hseih, D.P.H., J. Agric. Food Chem. (1974) 22, 512.

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28. Wilson, B.J. and Hayes, A.W. in Toxicants Oc curring in Foods, National Academy of Sciences, Washington, D.C., pp. 372-423 (1973). 29. Heildelberger, C., Annu. Rev. Biochem. (1975) 44, 79. 30. Ong, T.M., Mutat. Res. (1975) 32, 35. 31. Aflatoxin Mass Spectral Data Bank, Pohland, A.E. and Sphon, J.Α., ed., U.S. Food and Drug Adm., Bureau of Foods, Division of Chem. and Phys., Washington, D.C. 32. Masri, M.S., Page, J.R., and Garcia, V.G., J . Assoc. Official Anal. Chemists (1969) 52, 641. 33. Parker, R.B., Snedden, W., and Watts, R.W.E., Biochem. J . (1969) 115, 103. 34. Kiser, Introduction to Mass Spectrometry and Its Applications, Prentice-Hall, Inc., Englewood Cliffs (1965), p. 216. 35. Kingston, D.G.I., J. Assoc. Official Anal. Chemists (1976) 59, 1016. 36. Beuhler, R . J . , Flanigan, E., Greene, L . J . and Friedman, L . , Biochem. and Biophys. Res. Commun. (1972) 46, 1082. 37. Beuhler, R.J., Flanigan, E., Greene, L . J . and Friedman, L . , Biochem. (1974) 13, 5061. 38. Beuhler, R.J., Flanigan, Ε., Greene, L . J . and Friedman, L . , J . Am. Chem. Soc. (1974) 96, 3990. 39. Soltman, B., Sweeley, C.C. and Holland, J.F., Anal. Chem. (1977) 49, 1164. 40. Hunt, D.F., Shabanowitz, J . and Botz, F.K., Anal. Chem. (1977) 49, 1160. RECEIVED December 30, 1977

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