Analytical Measurements: How Do You Know Your Results Are Right

24 Analytical Measurements: How Do You Know Your Results Are Right? WILLIAM HORWITZ

Downloaded by CORNELL UNIV on August 5, 2016 | http://pubs.acs.org Publication Date: August 10, 1981 | doi: 10.1021/bk-1981-0160.ch024

Bureau of Foods, Food and Drug Administration, HFF-101, 200 C Street, SW, Washington, DC 20204

The scientist these days has a new partner--the auditor. He is not a financial auditor, but rather an examiner of knowledge. He is a verifier of accounts, as the dictionary puts i t . In this case, he intends to verify that the public's trust in science is well founded. The presence of the science auditor is the result of revelations that some laboratories were submitting false or faulty data to government agencies as the basis for obtaining permission to expose the public and the environment to potentially hazardous materials, such as pesticides, food and color additives, and more recently, "toxic substances." In granting permission to use toxic chemicals to control agricultural pests, to construct protective food-contacting polymers, and to fabricate foods and articles useful to consumers, Congress required its public servants to assure themselves that no harm would occur to the ultimate users of the products. Congress did not require that the tests to assure absence of harm be performed by the presumably neutral government; on the contrary, they accepted the common portrait of a scientist as the altruistic individual whose main desire was to satisfy his thirst for knowledge. As a practical matter, however, we have discovered that there is a long road between the laboratory data and a regulatory petition that leads through the office of the laboratory manager, the vice president in charge of research and development, the chief legal counsel, and apparently more often than not, the director of public relations. It appears that we have placed so much emphasis on c e r t a i n t y that we are uncomfortable with u n c e r t a i n t y . In handling data, we tend to avoid and hide the u n c e r t a i n t i e s i n our obsession to produce " c l e a n " data. But c l e a n data are more a matter o f judgment than o f a c t u a l i t y . Raw data are f r e q u e n t l y d i s o r d e r l y i n the sense that they a r e f u l l of perturbations r e s u l t i n g from the many outside i n f l u e n c e s on the p a r t i c u l a r property we a r e measuring. The value which we obtain a t any given moment i s equivalent t o a s e r i e s o f one-frame s t i l l p i c t u r e s from a continuously running movie f i l m . As a r e s u l t o f t h i s discontinuous sampling o f a 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.


412

THE

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

continuous event, we o f t e n get the z i g - z a g patterns of p r o p e r t i e s with time, which abound i n the t o x i c o l o g i c a l j o u r n a l s , complete with the standard e r r o r s extending from each point, which o f t e n do not even have the decency of o v e r l a p p i n g each other. But we must always remember that unless we have v a r i a b i l i t y i n our measurements, we have no idea of the u n c e r t a i n t i e s i n our system. Raw data used to be a very simple concept: they were the numbers a c t u a l l y i n d i c a t e d by a measuring device r e g a r d l e s s of t h e i r being obtained by summing up the weights on a balance, read from the s c a l e on a buret, determined on an instrument d i a l , or a c t u a l l y measured on a recorder c h a r t . The a n a l y s t had f u l l c o n t r o l and r e s p o n s i b i l i t y over the production of the data at every step. He prepared h i s own reagents, c a l i b r a t e d h i s weights and volumetric glassware, and standardized the output o f h i s instruments. As e f f i c i e n c y experts and cost accountants penetrated l a b o r a t o r y management, some of these t e c h n i c a l r e s p o n s i b i l i t i e s were delegated to l e s s c o s t l y sources: Prepared reagents are purchased from a laboratory supply house or prepared by a c e n t r a l l o c a l u n i t ; glassware i s washed, s t o r e d , and d i s t r i b u t e d by a s p e c i a l i z e d o r g a n i z a t i o n ; r e s p o n s i b i l i t y f o r c a l i b r a t i o n i s assigned to the manufacturer of the equipment; and proper f u n c t i o n i n g of i n s t r u ments i s assumed to be b u i l t - i n by the instrument designers and computer o p e r a t o r s . T h i s s h i f t i n f u n c t i o n s i s not n e c e s s a r i l y bad. I t d i d r e l i e v e the a n a l y t i c a l chemist of numerous minor, but important, chores which were d i s t r a c t i o n s from higher l e v e l r e s p o n s i b i l i t i e s . But when these f u n c t i o n s were placed elsewhere, proper management r e q u i r e d that the performance o f these p r o f e s s i o n a l r e s p o n s i b i l i t i e s be a p p r o p r i a t e l y monitored to ensure s u i t a b l e o p e r a t i o n . Thus, the production of data s h i f t e d from a s t r a i g h t l i n e f u n c t i o n , e n t i r e l y under the d i r e c t s u p e r v i s i o n o f the p r o f e s s i o n a l s c i e n t i s t , to a maze-type o p e r a t i o n c h a r a c t e r i z e d by the i n t e r m i n g l i n g of the c r i t i c a l paths of a "PERT" c h a r t , managed by a l a b o r a t o r y d i r e c t o r . The demands f o r e f f i c i e n c y , coupled with the f a c t that many of our modern measurements cannot be obtained i n any other way than by mechanically or e l e c t r o n i c a l l y c o n t r o l l e d automatons, r e s u l t i n machines which measure the samples, execute the manipulations, determine the response, perform the c a l c u l a t i o n s , and present the f i n a l answer i n whatever form or u n i t s are d e s i r e d . The f i n a l value may be copied from a d i a l , recorded on tape, drawn on a c h a r t , or not presented at a l l , to be stored i n a computer f o r c o o r d i n a t i o n with past and f u t u r e values, presenting the e n t i r e sequence as the r e s u l t o f the experiment. These f i n a l r e s u l t s from machines are raw data j u s t as much as the d i r e c t measurements were. Whether or not the f i n a l r e s u l t s emanate d i r e c t l y from our manual observations or from our automated instruments i s r e a l l y not asking the r i g h t q u e s t i o n . The proper question we should c o n s t a n t l y be asking i s : "Are these data r i g h t ? " The o p e r a t i o n a l question i s : "How do we know that these data are r i g h t ? "



24.

HORWITZ

Analytical

Measurements

413

I intend to d i s c u s s that question i n t h i s paper. It i s a subject which i s r a r e l y d e a l t with i n the s c i e n t i f i c l i t e r a t u r e because our j o u r n a l s a r e not s e t up t o handle t h i s type o f d i s cussion. We p o l i t e l y assume that any measurement a s c i e n t i s t makes i s c o r r e c t . In our peer review system, the reviewer assumes that the data reported a r e c o r r e c t unless he f i n d s an i n t e r n a l i n c o n s i s t e n c y which the i n v e s t i g a t o r f a i l e d t o d e t e c t . T h i s i s the f i r s t l i n e o f defense i n any i n v e s t i g a t i o n — c o n s i s t e n c y . Very o f t e n i t i s the only l i n e o f defense because new i n f o r m a t i o n i s being developed f o r which there i s no e x t e r n a l guide. I f there a r e any guideposts, most l i k e l y they are s h i f t i n g guideposts because many i n v e s t i g a t i o n s determine how t h i n g s change with time. The s i t u a t i o n i s much l i k e being s e t down i n a dense f o r e s t and being t o l d t o f i n d your way out, with your only d i r e c t i o n i n d i c a t o r an o c c a s i o n a l glimpse o f the sun, whose p o s i t i o n s h i f t s with time. My purpose i s to provide a few general guideposts which may be h e l p f u l i n determining the r e l i a b i l i t y o f chemical and p h y s i c a l data. Most o f the problems and concepts discussed here have been developed as a r e s u l t o f the n e c e s s i t y f o r the review o f data produced from the chemical a n a l y s i s o f samples examined f o r comp l i a n c e with the Federal Food, Drug, and Cosmetic Act o r data submitted t o the Food and Drug A d m i n i s t r a t i o n (FDA) i n support o f a request f o r approval o f a r e g u l a t e d product. I cannot be o f much help i n e v a l u a t i n g b i o l o g i c a l data but you might f i n d the recent p u b l i c a t i o n on q u a l i t y assurance from the American P u b l i c Health A s s o c i a t i o n (1) u s e f u l i n t h i s r e s p e c t . I t c o n t a i n s chapters d e s c r i b i n g concepts which have been developed to assure the v a l i d i t y o f the product from l a b o r a t o r i e s i n v o l v e d i n such f i e l d s as anatomic pathology, c l i n i c a l chemistry, c l i n i c a l microbiology, c l i n i c a l t o x i c o l o g y , c y t o l o g y , hematology, immunology, and v i r o l ogy. However, you do not need any reference to know that something i s wrong when the computer p r i n t o u t shows the r e s u l t s o f examinat i o n o f the uterus o f a male r a t , the t e s t e s o f a female mouse, and d i e t consumption o f 9999.9 pounds by a 250 pound r a t ! Such information appeared i n the raw data supporting a recent submiss i o n t o FDA. Consistency The primary guidepost i n a l l data c o l l e c t i o n a c t i v i t i e s i s c o n s i s t e n c y . A s e r i e s o f measurements w i l l always f a l l i n t o one o f three c a t e g o r i e s : They w i l l go up; they w i l l go down; or they w i l l remain constant. T h i s i s not as t r i v i a l an observation as i t may seem. I mean to point out that measurements u s u a l l y f o l l o w a p a t t e r n and experiments a r e u s u a l l y designed t o determine that pattern. I f the measurements seem t o go up and down without a p a t t e r n , that i n i t s e l f i s a p a t t e r n . You a r e observing random v a r i a b i l i t y , which must be f a c t o r e d out t o d i s c o v e r the u n d e r l y i n g t r e n d . In f a c t , the reviewer should r e a l l y begin t o worry about the q u a l i t y o f the observations when there i s no reasonable



414

THE

PESTICIDE

CHEMIST

A N D M O D E R N

TOXICOLOGY

v a r i a b i l i t y component. Less than usual v a r i a b i l i t y suggests that some averaging has been going on. You can average out q u i t e a few w i l d r e s u l t s i f they a r e i n opposite d i r e c t i o n s and get a f a i r l y decent mean. The s t a t i s t i c i a n s heard o f averaging a long time ago and named i t " r e g r e s s i o n to the mean" f o r the a b i l i t y to hide poor data by t a k i n g enough o f i t . The data should a l s o be c o n s i s t e n t with corresponding i n f o r mation that may e x i s t i n the l i t e r a t u r e or from l a b o r a t o r y e x p e r i ence; i f not, an explanation i s c a l l e d f o r . The whole should equal the sum o f i t s parts and amounts o f products should be c h e m i c a l l y equivalent to the amounts o f r e a c t a n t s . Experiments should be designed to i n c o r p o r a t e as many s e l f - c h e c k i n g f e a t u r e s as poss i b l e , as f o r example, accounting f o r a l l components. However, i f one o f the f i g u r e s i s obtained by d i f f e r e n c e , the s e l f - c h e c k i n g feature i s l o s t . There i s a l s o a negative aspect to c o n s i s t e n c y . Data which are too c o n s i s t e n t are a l s o suspect. V a r i a b i l i t y patterns are u s u a l l y q u i t e r e p r o d u c i b l e from experiment to experiment. Less than usual v a r i a b i l i t y does not always mean b e t t e r and more c a r e f u l experimentation. To an a u d i t o r , i t may suggest the a p p l i c a t i o n o f mental telepathy or what, i n my student days, was known as graphite chemistry. V a r i a b i l i t y o f Measurements Beyond such simple concepts as consistency o f the data and i t s a d d i t i v e p r o p e r t i e s , we must understand the concept o f measurement i n a n a l y t i c a l chemistry because many o f the measurements that the t o x i c o l o g i s t makes are chemical i n nature. T h i s he has had to do i n s e l f defense because r a r e l y has he had a chemist at h i s beck and c a l l . U n t i l the l a s t decade or so, the chemist l a r g e l y ignored the area o f the a n a l y t i c a l chemistry o f r e s i d u e s and m e t a b o l i t e s . T h i s i s no longer the case. A n a l y t i c a l chemists i n the short space o f a few decades have given us some marvelous t o o l s i n the form o f the powerful r e s o l u t i o n s o f chromatography, the superb s e n s i b i l i t y o f v a r i o u s kinds o f spectroscopy and polarography, and the e x q u i s i t e s p e c i f i c i t y o f mass spectrometry. But d e s p i t e t h e i r power, we must always question the r e l i a b i l i t y of the information they are g i v i n g us. There are many causes or sources o f v a r i a b i l i t y . Some a r e very general and occur i n p r a c t i c a l l y a l l chemical measurements. Others are s p e c i f i c to the i n d i v i d u a l methods and thus are d i f f i c u l t to handle i n a general way. Therefore, we w i l l concentrate on the general aspects which must be considered i n a l l a n a l y t i c a l o p e r a t i o n s . One o f the most important i s sampling and handling o f the samples and a second i s what to do with the f i n a l a n a l y t i c a l r e s u l t s . These p o i n t s a r e not u s u a l l y covered i n most textbooks s i n c e they a r e r e a l l y o u t s i d e o f the a n a l y t i c a l o p e r a t i o n s . Sampling and handling o f the sample i s the beginning o f the sequence. The f i n a l d i s p o s i t i o n o f the a n a l y t i c a l r e s u l t s — h o w do


24.

H O R W i T Z

Analytical

415

Measurements

you i n t e r p r e t the d a t a — i s the end o f the sequence. Each o f these s u b j e c t s could support an extensive l e c t u r e on i t s own. A l l we can do i s t o point out now that both aspects a r e important and t h e i r neglect can lead t o j u s t as much t r o u b l e as poor a n a l y t i c a l work. Sampling Stated simply, the job o f the a n a l y t i c a l chemist i s to report what i s i n the container that he i s given. What the a n a l y s t t e l l s you only a p p l i e s t o what he works on. I f the t o x i c o l o g i s t g i v e s the chemist only h a l f a l i v e r , i t w i l l be the t o x i c o l o g i s t s job t o e x t r a p o l a t e t o the whole l i v e r , not the chemist's. The chemist should not take the r e s p o n s i b i l i t y f o r e x t r a p o l a t i n g the r e s u l t s of analyses t o the whole organ, complete t i s s u e , e n t i r e animal, o r to a l l animals. The designer o f the experiment should have taken i n t o c o n s i d e r a t i o n the purpose o f the work and b u i l t i n t o the m a t e r i a l sampled the a b i l i t y to e x t r a p o l a t e t o the d e s i r e d l e v e l of complexity. Therefore, one o f the f i r s t things that should be looked a t i s the design o f the experiment, t o ensure that the proper m a t e r i a l was s e l e c t e d f o r a n a l y s i s . Not only must the proper m a t e r i a l be s e l e c t e d f o r a n a l y s i s but i t must be handled properly to avoid contamination and a l t e r a t i o n . P l a s t i c i z e r s f r e q u e n t l y appear i n analyses from contact o f the sample with p l a s t i c c o n t a i n e r s or p r o t e c t i v e f i l m s . M e t a l l i c contaminants appear from contact with metal instruments, metal and p l a s t i c f o i l s and l i n e r s , s p a t u l a s , and g r i n d e r s . Adventitious compounds can appear from the most unexpected p l a c e s . Paper, f o r example, may c o n t a i n numerous c o a t i n g a d d i t i v e s ; f a t t y a c i d s and t h e i r d e r i v a t i v e s appear as coatings on p l a s t i c f i l m s and aluminum f o i l ; s i l i c o n e s a r e used to coat g l a s s . Therefore, i f samples are i n contact with common p r o t e c t i v e f i l m s and c o n t a i n e r s , they could pick up something which may i n t e r f e r e with your t r a c e analyses. I t i s good a n a l y t i c a l p r a c t i c e t o supply t o the chemist, p o r t i o n s o f a l l m a t e r i a l s which the samples may have contacted. These m a t e r i a l s would be examined as p o t e n t i a l sources o f unident i f i e d m a t e r i a l s appearing i n recordings or p r i n t o u t s . Conducting blanks through the e n t i r e procedure i s an absolute n e c e s s i t y i n t r a c e a n a l y s i s to account f o r minute amounts o f the analyte and i n t e r f e r e n c e s i n the reagents, absorbents, s o l v e n t s , water, and other m a t e r i a l s which contact the sample and i t s deri v a t i v e s during the a n a l y s i s . M a t e r i a l s which a r e o r d i n a r i l y considered i n e r t i n most chemical operations (e.g., s o l v e n t s , f i l t e r paper, d r y i n g agents such as sodium s u l f a t e ) may c o n t r i b u t e r e l a t i v e l y l a r g e q u a n t i t i e s o f i n t e r f e r i n g m a t e r i a l s as we go lower i n the c o n c e n t r a t i o n s c a l e . The preparation, sampling, and a n a l y s i s o f animal feeds deserve s p e c i a l a t t e n t i o n . The p r a c t i c a l i t i e s o f d i s t r i b u t i n g uniformly p a r t s per thousand, parts per m i l l i o n , and even parts per b i l l i o n o f a t e s t m a t e r i a l i n t o a heterogeneous feed mixture probably r e q u i r e the t a l e n t s o f a chemical engineer. We have


1



416

THE

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

described overcoming the d i f f i c u l t i e s i n the p r e p a r a t i o n o f an a n a l y t i c a l sample involved i n a f e e d i n g study f o r trace quant i t i e s o f metals ( 2 ) . S c a l i n g up t h i s mixing procedure a hundred or a thousand f o l d undoubtedly r e q u i r e s c o n s i d e r a b l e e x p e r i mentation and o p e r a t i o n a l c o n t r o l . A summary o f the feed mixing procedure f o r the l a r g e s c a l e t o x i c o l o g i c a l study conducted a t the National Center f o r T o x i c o l o g i c a l Research i s given by O i l e r et a l . (3). We can summarize the importance o f sampling by p o i n t i n g out that i n the case of the a n a l y s i s of peanuts f o r the mold metabolite a f l a t o x i n , at the parts per b i l l i o n l e v e l , 90? o f the t o t a l v a r i a b i l i t y i s derived from sampling the commodity and preparing the l a b o r a t o r y sample, and only 10? i s d e r i v e d from the a n a l y t i c a l operations. Based on the v a l i d a t i n g c o l l a b o r a t i v e study, the i n t e r l a b o r a t o r y c o e f f i c i e n t of v a r i a t i o n (CV) o f the method of a n a l y s i s alone i n t h i s case i s about 30? a t the 10 ppb l e v e l . The R e l i a b i l i t y of A n a l y t i c a l Methods The r o l e o f a n a l y t i c a l methods i n modern t o x i c o l o g y and i t s importance i n " r i s k assessment" can be summarized by a quotation from a recent report to the Environmental P r o t e c t i o n Agency (EPA) on pentachlorophenol (PCP) contaminants ( 4 ) : "A key problem to overcome i n order to make an adequate e v a l u a t i o n of the r e l a t i v e hazard o f PCP and i t s contaminants i s the l a c k of ready a v a i l a b i l i t y o f s u i t a b l y s e n s i t i v e and s p e c i f i c a n a l y t i c a l methods. Although progress has been made i n developing approp r i a t e a n a l y t i c a l c a p a b i l i t y , r o u t i n e a n a l y s i s has been hampered by the u n a v a i l a b i l i t y of s u i t a b l e a n a l y t i c a l standards f o r some of the isomers. In f a c t , the a v a i l a b i l i t y of a p p r o p r i a t e l y s p e c i f i c a n a l y t i c a l methods may be the r a t e l i m i t i n g f a c t o r i n a s s e s s i n g the hazard of d i o x i n s and r e l a t e d chemicals. Thus, when there are s e v e r a l isomers with widely d i f f e r i n g t o x i c i t i e s , as i n the case with hexachlorodibenzo-£-dioxins, analyses o f the isomers as a group only permit assessment o f hazard based upon the most t o x i c isomer. T h i s approach may, indeed, lead to overestimates of hazard, but, i n the absence of more d e f i n i t i v e analyses of s p e c i f i c t o x i c chemical species, i t i s necessary to t r e a t contamination data on a t o x i c o l o g i c a l l y worst-case b a s i s . " A n a l y t i c a l methods have two types o f c h a r a c t e r i s t i c s — s c i e n t i f i c and p r a c t i c a l . The s c i e n t i f i c c h a r a c t e r i s t i c s determine the r e l i a b i l i t y o f the a n a l y t i c a l data; the p r a c t i c a l c h a r a c t e r i s t i c s determine the u t i l i t y of the method. The s c i e n t i f i c a t t r i b u t e s o f a method i n c l u d e such things as accuracy, p r e c i s i o n , s p e c i f i c i t y , and l i m i t of r e l i a b l e measurement; the p r a c t i c a l a t t r i b u t e s


24.

H O R W i T Z

Analytical

Measurements

417

i n c l u d e c o s t o f performance, time r e q u i r e d , and l e v e l o f t r a i n i n g needed. For research purposes, the p r a c t i c a l aspects are o f secondary c o n s i d e r a t i o n ; f o r r e g u l a t o r y operations o f compliance and s u r v e i l l a n c e , p r a c t i c a l i t y i s o f great importance. Little enforcement i s p o s s i b l e u s i n g a method which turns out one a n a l y t i c a l value per day!


Specificity The fundamental property of a l l a n a l y t i c a l methods i s s p e c i f i c i t y — t h e t e s t s which a r e being a p p l i e d must measure what they purport to measure. For example, many t e s t s which measure c h l o r ide a l s o measure bromide and i o d i d e ; t h e r e f o r e such t e s t s are f o r h a l i d e s . They are u s e f u l f o r c h l o r i d e determinations because o f the absence o f the other h a l i d e s i n many m a t e r i a l s . C o l l o q u i a l l y we speak o f methods f o r c h l o r i d e s , but s t r i c t l y speaking such t e s t s are f o r h a l i d e s . When we d i d not have anything b e t t e r , we measured the organochlorine p e s t i c i d e residues by e x t r a c t i n g the p e s t i c i d e with an organic s o l v e n t and determining t o t a l c h l o r i d e (really total halide). I n i t i a l l y , we c a l l e d i t DDT, but as more r e l a t e d p e s t i c i d e s were introduced, i t had to be c a l l e d organoc h l o r i n e p e s t i c i d e s , then organochlorine compounds, and now we would have t o c a l l i t organic s o l v e n t - s o l u b l e organohalide mater i a l . We now know that a l o t o f what we assumed was DDT or r e l a t e d organochlorine p e s t i c i d e s , even by the e a r l y gas chromatographic methods, were i n a l l p r o b a b i l i t y PCBs ( p o l y c h l o r i n a t e d b i p h e n y l s ) . Schechter (5) had warned us about t h i s point many years ago with h i s example o f the "pre-DDT e r a " s o i l sample that had been kept i n a sealed c o n t a i n e r and had never been exposed to organochlorine pesticides. The gas chromatogram o f the m u l t i r e s i d u e method e x h i b i t e d a s e r i e s o f peaks, a number o f which had r e t e n t i o n times a t or c l o s e t o those of known p e s t i c i d e s . Schechter concludes, "Data reported without a p p l i c a t i o n o f s u i t a b l e confirmatory t e c h niques may not only be worthless, but what i s worse, i n c o r r e c t information may be s e r i o u s l y misleading and may be u n r e c t i f i a b l e . " We now have much b e t t e r t o o l s f o r a s s e s s i n g s p e c i f i c i t y than we had a t the beginning o f the p e s t i c i d e age. Gas and t h i n l a y e r chromatography can u s u a l l y detect the presence o f mixtures. They do not work so w e l l the other w a y — p r o v i n g the i d e n t i t y o f a pure compound. For t h i s you have to apply the instruments which work on the whole molecule, or a p p r e c i a b l e or c r i t i c a l f r a c t i o n s o f the molecule, such as i n f r a r e d spectroscopy, nuclear magnetic r e s o nance, or best o f a l l , mass spectrometry. But there are always footnotes or r e s e r v a t i o n s to the best o f techniques. In t h i s case, f o r unequivocal i d e n t i f i c a t i o n , apply the techniques only to pure samples; only a small amount i s needed, but i t must be pure! The r e q u i r e d s p e c i f i c i t y w i l l depend upon the purpose o f the a n a l y t i c a l r e s u l t s . The main need f o r s p e c i f i c i d e n t i f i c a t i o n o f a n a l y t e s l i e s with the t o x i c o l o g i s t s . They i n d i c a t e that many s i m i l a r compounds have s i g n i f i c a n t l y d i f f e r e n t t o x i c i t i e s . Some



418

THE

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

examples include the four c l o s e l y r e l a t e d a f l a t o x i n s (B]_, B2, G]_, G2) whose r e l a t i v e acute t o x i c i t i e s i n the d u c k l i n g cover a range of 10 to 1 (6). In the family of polynuclear hydrocarbons, some are reported as carcinogenic and some are not ( 7 ) . The most recent and complex example i s that o f the p o l y c h l o r i n a t e d dibenzo-£d i o x i n s (CDDs), present as contaminants i n 2,4,5-trichlorophenoxyacetic a c i d (2,4,5-T) and r e l a t e d e s t e r h e r b i c i d e s . There are 75 p o s s i b l e isomers of CDDs, from monochlorinated to o c t a c h l o r inated; there are 22 p o s s i b l e isomers of the t e t r a c h l o r o compound. The f u l l y c h l o r i n a t e d o c t a c h l o r i n a t e d compound i s r e l a t i v e l y i n e r t b i o l o g i c a l l y but the symmetrical 2,3,7,8-tetrachlorodibenzo-£d i o x i n (2378TCDD) has been c h a r a c t e r i z e d as the most potent small molecule t o x i n known ( 8 ) . As yet, the t o x i c o l o g i s t s have not been able to set a l i m i t of t o x i c o l o g i c a l i n s i g n i f i c a n c e as a target f o r method development f o r t h i s compound. They have merely i n d i c a t e d that the chemist should go as low as he can, c e r t a i n l y i n t o the parts per t r i l l i o n (ppt) r e g i o n . The c o n f l i c t i n g requirements of measuring i n the low ppt region, and at the same time being sure that the most t o x i c o l o g i c a l l y potent isomer i s the one which i s being measured, presents an i n t e r e s t i n g dilemma: To obtain s p e c i f i c i t y f o r 2378TCDD r e q u i r e s an extensive cleanup from DDE and PCBs, using s e v e r a l adsorption columns and high pressure l i q u i d chromatographic steps, and s e l e c t i v e c a p i l l a r y gas chromatography ( f o r isomer separation). With a p e r f e c t cleanup, any detector, from a mass spectrometer to an e l e c t r o n capture gas chromatographic detector, may be used to v e r i f y the presence of the t e t r a c h l o r o compound. S p e c i f i c i t y a l s o may be obtained through the use of a l e s s rigorous cleanup by r e l y i n g upon a very expensive high r e s o l u t i o n mass spectrometer to measure the exact peak l o c a t i o n s of the t e t r a c h l o r o compounds to 10 parts i n a m i l l i o n . At both extremes, as fewer ions (due to the compound of i n t e r e s t ) are monitored or as the cleanup i s shortened, a lower l e v e l of d e t e c t a b i l i t y i s achieved, but always at the expense of s p e c i f i c i t y . Furthermore, as the procedure becomes longer, l o s s e s become greater, accuracy and p r e c i s i o n d e t e r i o r a t e , and the operation becomes l e s s practical . Other problems, not n e c e s s a r i l y a f f e c t i n g d i f f e r e n t procedures to the same extent, include lengthy cleanups and gas chromatographic separations, u n a v a i l a b i l i t y of isomeric TCDD standards, and i m p u r i t i e s i n i s o t o p i c i n t e r n a l standards. The choice of d i f f e r e n t s i g n a l - t o - n o i s e r a t i o s by d i f f e r e n t l a b o r a t o r i e s a f f e c t s the d e t e c t i o n and measurement l i m i t s . Another aspect of the a n a l y s i s f o r TCDDs i s that the purpose of the work determines the degree of s p e c i f i c i t y that must be b u i l t i n t o an a n a l y t i c a l procedure. I f you are a regulatory agency s c i e n t i s t who must s u s t a i n the burden of proof against p o t e n t i a l questions from s k e p t i c a l s c i e n t i s t s and even more s k e p t i c a l lawyers, you w i l l include every p o s s i b l e point of a s s i s t a n c e , even s a c r i f i c i n g a low l i m i t of determination. I f you are embarked on


24.

H O R W i T Z

Analytical

Measurements

419


a s u r v e i l l a n c e program, to determine the extent of TCDD contamination i n the environment or i n the food supply, or a metabolism study, you need only s a t i s f y the s c i e n t i f i c questioning of your a s s o c i a t e s and s u p e r v i s o r s . I f you are engaged i n research, f o l l o w i n g a s p e c i f i c p r o t o c o l , where there e x i s t s c o l l a t e r a l information on presence and absence of the test m a t e r i a l and a dose-response curve to f a l l back on f o r the t e s t of consistency, a minimum amount of c h a r a c t e r i z a t i o n i s s u f f i c i e n t . In p r a c t i c e , then, a c h i e v i n g absolute s p e c i f i c i t y i s o f t e n not p o s s i b l e and sometimes not necessary. Absolute s p e c i f i c i t y i n trace a n a l y s i s can u s u a l l y be achieved only at the expense o f other a t t r i b u t e s of the procedure. Accuracy The accuracy of an a n a l y t i c a l r e s u l t i s measured by the d i f ference between the measured value and the true or assigned value. In most residue or contaminant work, we do not know the true value of the c o n s t i t u e n t we are measuring. We t h e r e f o r e have to f a l l back on the a r t i f i c i a l s i t u a t i o n of using the method of a d d i t i o n s to approximate the o r i g i n a l content of our a n a l y t e , or the f a r more d i f f i c u l t task of e s t i m a t i n g the true value by more d e f i n i t i v e methods. But we always seem to be thwarted i n our e f f o r t s to o b t a i n reasonable values f o r our a n a l y t e s . I w i l l use as an example a case which you would think would be a r e l a t i v e l y s t r a i g h t f o r w a r d a n a l y t i c a l p r o b l e m — t h e determinat i o n of the s t a b l e inorganic element chromium, which has an important r o l e i n the metabolism of carbohydrates. Figure 1 and Table 1 show the various published values f o r the chromium c o n c e n t r a t i o n i n human blood or plasma s i n c e 1948 as reported by Mertz i n 1975 (9), supplemented by some l a t e r values. I have drawn what appears to be a rough trend l i n e of values g e n e r a l l y decreasing s i n c e the e a r l y 1960s, which required the use of four c y c l e l o g paper. There i s general agreement now that the a c t u a l chromium content of blood i s c l o s e r to 1 ppb than to 1 ppm, yet every one of the almost two dozen c o n t r i b u t o r s to Figure 1, u s i n g s i x d i f f e r e n t types of methods, was s u f f i c i e n t l y convinced o f the soundness of h i s work to provide a r e f e r e e d paper o f f e r i n g h i s " t r u e " value as developed by the most modern, s e n s i t i v e , and r e l i a b l e procedure and instrumentation a v a i l a b l e at the time of presentation. T r y i n g to d i s c o v e r a pattern among the methods does not seem to lead anywhere. The spectrophotometric ( c o l o r i m e t r i c ) methods used i n i t i a l l y , which u s u a l l y have numerous steps, seem to give high, but not the highest, v a l u e s . Emission spectrometric methods appear to c l u s t e r i n the middle of the s c a l e . Atomic a b s o r p t i o n methods, some of which have extensive p r e l i m i n a r y cleanup steps, have a downward trend, p a r t i c u l a r l y a f t e r the i n t r o d u c t i o n of the g r a p h i t e furnace. Two of the most recent values were obtained by neutron a c t i v a t i o n with chemical separation i n one case (0.16 ppb)


THE

420


TABLE 1.

PESTICIDE

CHEMIST

A N DM O D E R N

TOXICOLOGY

REPORTED CHROMIUM CONCENTRATIONS IN BLOOD

Reference

Year

Method

Concentration ug/L (ppb)

Grushko, Ya. M. (Biokhimiya 13, 124-126; (CA 42, 8302i)

1948

ES

35

Urone, P. F. & Anders, H. K. (Anal. Chem. 22, 1317-1321)

1950

Sp

50

M o n a c e l l i , R., e t a l . ( C l i n . Chim. Acta 1, 577-582)

1956

ES

180

M i l l e r , D. 0. & Yoe, J . H. ( C l i n . Chim. Acta 4, 378-383)

1959

Sp

30

Paixao, L. M. & Yoe, J . H. ( C l i n . Chim. Acta 4, 507-514)

1959

ES

24

Herring, W. B., e t a l . (Am. J . C l i n . Nutr. 8, 846-854)

I960

ES

27

Volod'ko, L. V. & P r i s t u p a , Ch. V. ( V e s t s i Akad. Navuk B. SSR, No. 1, 107-109; ÇA 57, 11702a)

1962

ES

200

Schroeder, Η. Α., e t a l . ( J . Chronic D i s . 15, 941-964)

1962

Sp

390

Wolstenholme, W. A. (Nature 203, 1284-1285)

1964

SSMS

Glinsmann, W. H., et a l . (Science 152, 1243-1245)

1966

AA

27

Feldman, F. J . , et a l . (Anal. Chim. Acta 38, 489-497)

1967

AA

29

Niedermeier, W. & Griggs, J . H. ( J . Chronic D i s . 23, 527-535)

1971

ES

28

Hambridge, Κ. M. (Anal. Chem. 43, 103-107)

1971

ES

13


1000

24.

HORWiTZ

TABLE 1.

Analytical

Measurements

421

(continued)

Reference

Year


Cary, Ε. E. & Allaway, W. H. 1971 ( J . A g r i c . Food Chem. 19, 1159-1161)

Method

Concentration ug/L (ppb)

AA

10

Wolff, W. R., e t a l . (Anal. Chem. 44, 616-618)

1972 GC/MS

Davidson, I.W.F. & S e c r e s t , W.L. (Anal. Chem. 44, 1808-1812)

1972

AA

5.1

Davidson, I.W.F. & Burt, R.L. (Am. J . Obstet. Gynecol. 116, 601-608)

1973

AA

4.7

Pekarek, R. S., e t a l . (Anal. Biochem. 59, 283-292)

1974

AA

1.6

L i , R. T. & Hercules, D. M. (Anal. Chem. 46, 916-920)

1974

Chim

Versieck, J . et a l . ( C l i n . Chem. 24, 303-308)

1978

NA

Ward, Ν. I . , e t a l . (Anal. Chim. Acta 110, 9-19)

1979

NA AA

ES Sp SSMS AA GC MS Chim NA

= = = = = = = =

Emission spectroscopy Spectrophotometric (diphenylcarbazide) Spark source mass spectrometry Atomic a b s o r p t i o n Gas chromatography Mass spectrometry Chemiluminescence Neutron a c t i v a t i o n


146

0.16

20 20

422

THE


1000

PESTICIDE

CHEMIST

A N D M O D E R N

TOXICOLOGY

ι

100

r.*

o°

\

\ο \ \

\°

10

χ \ \ \

1.0

0.1 '

1950

1960

1970

1980

YEAR Figure 1.

Reported chromium concentration in blood as a function of date of publication



24.

H O R W i T Z

Analytical

Measurements

423

and by standard a d d i t i o n s i n the other (20 ppb), which d i f f e r by two orders o f magnitude. I t i s i n t e r e s t i n g that many o f these papers provide l i n e a r c a l i b r a t i o n curves and recovery o f added chromium which approximate 100%. E x c e l l e n t r e c o v e r i e s were reported d e s p i t e s e v e r a l breaches i n good a n a l y t i c a l p r a c t i c e , such as working a t concent r a t i o n s c o n s i d e r a b l y above the l e v e l o f i n t e r e s t , o p e r a t i n g with a performance blank that produces a background response that i s an a p p r e c i a b l e f r a c t i o n o f the measurement response, and the use o f samples suspected o f being contaminated. Standard Reference M a t e r i a l s (SRM) o f the National Bureau o f Standards (NBS) c e r t i f i e d f o r chromium were not a v a i l a b l e u n t i l the middle 1970s. The o r i g i n a l m a t e r i a l s , orchard leaves and spinach, c o n t a i n s e v e r a l parts per m i l l i o n o f chromium, w e l l o u t s i d e our range o f i n t e r e s t . A brewer's yeast c e r t i f i e d s p e c i f i c a l l y f o r chromium content a t 2.12*0.05 ug/g (SRM 1569) a l s o became a v a i l a b l e i n 1976. Bovine l i v e r SRM c e r t i f i e d a t 0.088 ug chromium/g i s now a l s o a v a i l a b l e . Some o f the l i s t e d procedures c l a i m high and even unique s p e c i f i c i t y , low l e v e l o f d e t e c t a b i l i t y , and extreme r a p i d i t y . Often these claims are made with no mention o f the r e l a t i v e magnitude o f the accompanying blank and with no evidence o f apprec i a t i o n o f the problem o f contamination. Frequently comparisons were made among methods but no mention i s made o f the s t a r t i n g p o i n t — w h e t h e r i t was the o r i g i n a l matrix or a common, prepared s o l u t i o n . Since i n almost a l l cases where concurrent methods were used, the v a r i o u s methods gave s i m i l a r r e s u l t s , i t may be assumed that they shared a common b a s i s f o r contamination, i f i t e x i s t e d . Thus we see that although s i x b a s i c a l l y d i f f e r e n t methods have been used f o r the determination o f chromium i n a common, presumably s t a b l e and f a i r l y constant b i o l o g i c a l s u b s t r a t e , blood, we do not know i t s chromium content. We cannot assume that the lowest value i s the most c o r r e c t s i n c e there may have been l o s s e s ; we can be q u i t e c o n f i d e n t that the high values were s u b j e c t to some contamination. Yet every method was v a l i d a t e d by s p i k i n g with known amounts o f chromium, and even with l a b e l e d 51cr i n some cases, with "excellent results." In many cases, blanks were mentioned as accounted f o r . I f we cannot decide on the c o n c e n t r a t i o n o f an i n o r g a n i c element a t t r a c e l e v e l s i n blood, how can we do b e t t e r with more complex and l e s s s t a b l e organic molecules i n t h i s and other t i s s u e s ? The chromium example i s not unique. We have s e v e r a l other i n t e r e s t i n g examples i n the area o f t r a c e a n a l y s i s o f b i o l o g i c a l m a t e r i a l s . Most o f them are from t r a c e element a n a l y s i s s i n c e t h i s s p e c i a l t y has been an a c t i v e area o f methods research f o r a t l e a s t h a l f a century, and there a r e a v a i l a b l e a number o f SRMs from the NBS f o r use as reference p o i n t s . The remarkable i n f l u e n c e o f methods o f a n a l y s i s on estimates of a r s e n i c intake i s shown by an e v a l u a t i o n o f the data given by J e l i n e k and Corneliussen (10) summarizing the a r s e n i c content o f FDA's " t o t a l d i e t " composites during the r e p o r t i n g periods o f 1967


THE


424

PESTICIDE

CHEMIST

A N D

M O D E R N

TOXICOLOGY

through 1975, supplemented by l a t e r , as yet unpublished, values through 1978. The average c a l c u l a t e d annual d a i l y intake o f a r s e n i c (as AS2O3) i s shown i n Figure 2. Substantial discont i n u i t i e s occur between 1970 and 1971 and between 1975 and 1976. In 1970 the program was c o n s o l i d a t e d i n a s i n g l e l a b o r a t o r y and the molybdenum blue method of a n a l y s i s was replaced by the s i l v e r d i e t h y l d i t h i o c a r b a m a t e procedure, with a r e s u l t i n g lowering of the blank and operation at a lower l i m i t of r e l i a b l e measurement. Thus much of the apparent decrease i n the a r s e n i c content of the d i e t (as AS2O3) from an average of 80 ug/day during the 1967-1970 period to 15 ug/day during 1971-1974 may be an a n a l y t i c a l a r t i f a c t that does not at a l l r e f l e c t a d r a s t i c decrease i n the a r s e n i c intake during t h i s p e r i o d . The 1975-1976 d i s c o n t i n u i t y c o i n c i d e s with a f u r t h e r method change from the s i l v e r d i e t h y l - d i t h i o c a r b a m a t e c o l o r i m e t r i c procedure to the hydride-atomic a b s o r p t i o n procedure. T h i s change brought the t o t a l d i e t values back up to those o r i g i n a l l y given by the molybdenum blue method. This i n t e r p r e t a t i o n i s r e i n f o r c e d by the f a c t that an i d e n t i c a l a r t i f a c t i s noted i n the Canadian t o t a l d i e t program, but a year e a r l i e r . The l e v e l of a r s e n i c (as As) found during the f i r s t quarter o f 1969, using a modified G u i t z e i t method, would c o n t r i b u t e to the d i e t not more than 95 ug/day. Subsequently the method was changed to the s i l v e r d i e t h y l d i t h i o c a r b a m a t e procedure. The maximum l e v e l s or a r s e n i c (as As) i n the t o t a l d i e t s dropped to not more than 30 ug/person/day i n 1970, 30 i n 1971, and 35 i n 1972-1973 (11). The Canadian program was discontinued before any f u r t h e r method change was introduced. Precision P r e c i s i o n i s the estimate of v a r i a b i l i t y of measurements. I t i s o f t e n confused with, or used interchangeably (and i n c o r r e c t l y ) with, accuracy. Accuracy r e f l e c t s systematic e r r o r ; p r e c i s i o n r e f l e c t s random e r r o r . The concept i s r e a l l y more complex s i n c e the systematic e r r o r term a l s o i s subject to random v a r i a b i l i t y , but f o r our purpose we can t r e a t the two a t t r i b u t e s o f a n a l y t i c a l methods as separate c h a r a c t e r i s t i c s . P r e c i s i o n i s a term which must be handled with care because there are many d i f f e r e n t p r e c i s i o n s . Any time there i s a source o f v a r i a b i l i t y , there i s a p r e c i s i o n a s s o c i a t e d with i t . I t i s u s u a l l y expressed as a standard d e v i a t i o n at a c e r t a i n l e v e l of analyte. I t can be a s s o c i a t e d with sampling as a random v a r i a b i l i t y w i t h i n a s i n g l e m a t e r i a l or as an among samples random v a r i a b i l i t y of a number of r e l a t e d m a t e r i a l s . The most common a n a l y t i c a l p r e c i s i o n terms are r e p e a t a b i l i t y , which i s the term a s s o c i a t e d with a s i n g l e operator ( w i t h i n - l a b o r a t o r y ) and r e p r o d u c i b i l i t y , which i s the term a s s o c i a t e d with d i f f e r e n t operat o r s i n d i f f e r e n t l a b o r a t o r i e s (between-laboratory). For research work, r e p e a t a b i l i t y i s most o f t e n reported; f o r r e g u l a t o r y work, the v a r i a b i l i t y between l a b o r a t o r i e s i s the most important. The



24.

H O R W i T Z

Analytical

Measurements

425

150 r

120

5

90

Û

Analytical Measurements: How Do You Know Your Results Are Right

Recommend Documents