20 Analytical Aspects: An Introduction WILLIAM HORWITZ
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Bureau of Foods, Food and Drug Administration, HFF-101, 200 C Street SW, Washington, DC 20204
We frequently hear statements regarding the fantastic advances in analytical chemistry over the last several decades. These statements tell us how the analytical chemist has increased his ability to detect and measure many chemicals from parts per thousand to parts per million, then to parts per billion, and now even parts per trillion. Such statements are finding their way into the morning newspaper, complete with 3 and 4 significant figures. Rarely, however, do we see a discussion as to whether or not these figures are correct. Even i f they are correct, what is their reliability or its converse, its uncertainty. Even among scientists, a report from a laboratory showing the presence of several parts per trillion of a toxic chemical is accepted without question. What is worse, the accompanying uncertainties of analytical measurements are not recognized by many analytical chemists. The first two lectures in this session on the Analytical Chemist and Modern Toxicology will introduce some of the marvels which have been accomplished with the aid of analytical chemistry. Our f i r s t two speakers are from the National Center f o r T o x i c o l o g i c a l Research o f the Food and Drug A d m i n i s t r a t i o n a t J e f f e r s o n , Arkansas. The National Center f o r T o x i c o l o g i c a l Research has been one o f the f i r s t i n s t i t u t i o n s that has been u s i n g good l a b o r a t o r y p r a c t i c e s both i n t o x i c o l o g y and i n a n a l y t i c a l chemistry. The o r i g i n a l concept o f t h i s i n s t i t u t i o n was to handle what was termed a "mega-mouse" study. Sampling s t a t i s t i c s t e l l us that the p r o b a b i l i t y o f determining a very low incidence o f cancer i n animals, say a t l e v e l s o f 0.1? or 1$, r e q u i r e s tens o f thousands i f not hundreds o f thousands o f t e s t animals to be sure to d i f f e r e n t i a t e between a tenth o f a percent i n d u c t i o n and the background. The sheer l o g i s t i c s o f such an o p e r a t i o n soon scaled down the o r i g i n a l v e r s i o n to a 25,000 mouse v e r s i o n on a known This chapter not subject to U.S. copyright. Published 1981 American Chemical Society Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
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standard carcinogen as a model substance i n a t e s t run. The r e s u l t s o f t h i s f i r s t g i g a n t i c experiment were r e c e n t l y published i n the J o u r n a l o f Environmental Pathology and T o x i c o l o g y . In conducting t h i s study, numerous problems and s i d e i s s u e s have been handled i n s e t t i n g up, maintaining, and i n t e r p r e t i n g the experiment. But one o f the main t o o l s was a n a l y t i c a l chemistry, maintaining the i n t e g r i t y and p u r i t y o f the d i e t , water, and a i r without i n t e r r u p t i o n f o r more than a two-year period, a s s u r i n g the presence of the a n t i c i p a t e d amount of the t e s t substance as w e l l as i t s p u r i t y and i n t e g r i t y , p r o v i d i n g the chemical i n f o r m a t i o n required to determine the t o x i c i t y p r o f i l e such as metabolism and pharmokinetics, and f i n a l l y i n t e r p r e t i n g the r e s u l t s of a l l of the analyses. Along the way, a d d i t i o n a l p e r i p h e r a l but important chemical operations were necessary to guarantee the s a f e t y o f the personnel and animals from contamination, and the d i s p o s a l o f a l l exposed and contaminated experimental m a t e r i a l s . Many new a n a l y t i c a l methods had to be developed and v a l i d a t e d to assure t h e i r a p p l i c a b i l i t y to the problem at hand. In a d d i t i o n , s p e c i f i c a t i o n s had to be developed to p r o t e c t the experimental animals and the c o n t r o l s from exposure to m a t e r i a l s which might perturb the responses sought. In order to i n t e r p r e t the r e s u l t s from chemistry and t o x i cology programs, i t i s necessary to apply s t a t i s t i c s . Dr. Tiede w i l l point out the major s t a t i s t i c a l t o o l s required i n t h i s a r e a . An important t h i n g to remember i n s t a t i s t i c s i s that to measure small e f f e c t s or small q u a n t i t i e s you need l a r g e samples. A l s o , i f you wish to be more c o n f i d e n t of your r e s u l t s , you need a l a r g e r sample. Only i f you wish to be sloppy and not be very c o n f i d e n t , can you get along with a small number o f samples. This applies whether you are measuring t o x i c o l o g i c a l e f f e c t s or p h y s i c a l amounts of substances i n micrograms, nanograms, or picograms. Another important f a c t to remember i s that i t i s impossible to design, conduct, or i n t e r p r e t any work i n these areas without a working knowledge of s t a t i s t i c s . At the very l e a s t the s c i e n t i s t must become an amateur s t a t i s t i c i a n i n order to t e l l the s t a t i s t i c i a n what i s wanted and to understand the answer that i s provided. In my l e c t u r e I am warning you that the a n a l y t i c a l chemist i s nowhere near as good as he t h i n k s he i s or that he makes i t appear that he i s . For those who do not wish to be confused by v a r i a b i l i t y , the a n a l y t i c a l chemist w i l l g i v e you a number. But the v a r i a b i l i t y i s s t i l l there. To p r o p e r l y i n t e r p r e t chemical values i n terms of b i o l o g i c a l phenomena, the a n a l y t i c a l v a r i a b i l i t y must be removed to assure that the f i n a l r e s u l t s are t r u l y of t o x i c o l o g i c a l s i g n i f i c a n c e and not merely the a n a l y t i c a l e r r o r of the chemist. The t o x i c o l o g i s t must be p a r t i c u l a r l y wary when the chemist operates near the l i m i t s of measurement. I t appears
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
20.
H O R W i T Z
Analytical
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Introduction
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that f a l s e p o s i t i v e s and f a l s e negatives are inherent i n the data when a method i s pushed beyond i t s c a p a b i l i t i e s . The t o x i c o l o g i s t and chemist, not the s t a t i s t i c i a n , must stay c l e a r o f that precipice. F i n a l l y , Dr. McKinney, from h i s vast experience i n environmental chemistry w i l l point out some very p r a c t i c a l matters, such as how t o handle the sample to protect i t from things which w i l l mimic the compound sought. To emphase t h i s point even f u r t h e r , much o f the t r a c e element analyses i n b i o l o g i c a l m a t e r i a l s i n l i t e r a t u r e today i s i n v a l i d because the i n v e s t i g a t o r was unaware that metal t o o l s have a s u f f i c i e n t s o l u b i l i t y or f r a g i l i t y t o impart s i g n i f i c a n t amounts o f trace elements i n t o a b i o l o g i c a l sample from mere contact. The i n t r i g u i n g advertisements o f instrument manufacturers suggest that they have the true s a l v a t i o n f o r a n a l y t i c a l problems. Very o f t e n the instrument manufacturer w i l l apply h i s equipment t o the i d e a l s i t u a t i o n o f a pure compound i n a pure s o l v e n t . The r e s u l t s a r e s t r i c t l y true, but may be g r o s s l y misleading i f a p p l i e d t o a b i o l o g i c a l matrix. The same a p p l i e s t o recovery s t u d i e s even i n b i o l o g i c a l matrices. The n a t i v e compound may be t i g h t l y bound by r e a c t i o n or by absorption and may be l o s t t o your determination, although a d d i t i o n s above t h i s point are recovered s a t i s f a c t o r i l y . Under other c o n d i t i o n s , these m a t e r i a l s which do not respond t o a normal a n a l y s i s a r e released t o give an unant i c i p a t e d e f f e c t . Furthermore, never f o r g e t blanks and c o n t r o l s . Any i r r e g u l a r i t y i n t h e i r values r e q u i r e s f u r t h e r i n v e s t i g a t i o n . Blanks are a l s o c r i t i c a l f o r proper s t a t i s t i c a l i n t e r p r e t a t i o n o f c a l i b r a t i o n f u n c t i o n s , r e c o v e r i e s , and l i m i t s o f measurement. Only by keeping i n mind a l l o f these various f a c t o r s which tend t o subvert or mislead the i n v e s t i g a t o r , can the e x q u i s i t e r e s u l t s described by Dr. McKinney, be obtained.
R E C E I V E D March 18,
1981.
Bandal et al.; The Pesticide Chemist and Modern Toxicology ACS Symposium Series; American Chemical Society: Washington, DC, 1981.
