Pesticide Analytical Methodology - American Chemical Society

16 Reduction of Radioactive Metabolic Data Using

Downloaded by NORTH CAROLINA STATE UNIV on January 2, 2018 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch016

A D e s k - T o p Computer Network W. L. SECREST, W. C. FISCHER, D. P. RYSKIEWICH, J. E. CASSIDY, and G. J. MARCO Ciba-Geigy Corporation, Agricultural Division, P.O. Box 11422, Greensboro, NC 27409

In order to r e g i s t e r a chemical with the Environmental P r o t e c t i o n Agency (EPA) f o r use as a p e s t i c i d e , sound s c i e n t i f i c data must be presented and s t r i c t requirements be met. The average time r e q u i r e d to generate data which s a t i s f y these c r i t e r i a i s p r e s e n t l y estimated to be nine years from the o r i g i n a l s y n t h e s i s to the appearance of an a g r i c u l t u r a l chemical i n the marketplace. Extensive metabolism i n f o r m a t i o n i s necessary f o r complete understanding of t o x i c o l o g i c a l , environmental, and biochemical e f f e c t s of the chemical and to support the r e s i d u e data. A s i n g l e h e r b i c i d e , Dual®, f o r use on a s i n g l e crop, soybeans, r e q u i r e d 46 volumes of data submitted to the EPA f o r review. These volumes represented a stack of paper 10 f e e t tall. In order f o r the metabolism group to meet t h e i r t i g h t deadlines f o r our extensive c o n t r i b u t i o n to t h i s volume of i n f o r mation, we e x t e n s i v e l y use computers f o r c o l l e c t i o n , r e d u c t i o n , summarization, and i n t e r p r e t a t i o n of data. Three general questions about p e s t i c i d e s i n a g r i c u l t u r a l products and the environment must be answered: where i s i t and i t s m e t a b o l i t e s , how much of these are there and what are the m e t a b o l i t e s . The Metabolism S e c t i o n of the Biochemistry Department uses a v a r i e t y of a n a l y t i c a l techniques to answer these questions. Metabolism s t u d i e s are g e n e r a l l y done with r a d i o a c t i v e carbon ( ^ C ) , which provides a convenient method f o r d e t e c t i o n and q u a n t i t a t i o n of a r a d i o a c t i v e p e s t i c i d e . A p r e f e r r e d way to measure r a d i o a c t i v i t y i s with a l i q u i d s c i n t i l l a t i o n counter (LSC). From 1/2 to 22 minutes can be r e q u i r e d per sample v i a l . Between 300 and 600 v i a l s are counted on an average day. Outputs from the LSC must be c o r r e c t e d f o r background r a d i a t i o n and counting e f f i c i e n c y . The p a r t s - p e r - m i l l i o n (ppm) equivalent to the r a d i o a c t i v e chemical i n a b i o l o g i c a l , s o i l or water sample can then be determined by accounting f o r a l i q u o t s i z e , s p e c i f i c a c t i v i t y , and other c o r r e c t i o n f a c t o r s s p e c i f i c f o r the sample. P a r t i t i o n i n g c h a r a c t e r i s t i c s and chromatographic data can be t r e a t e d i n a l i k e manner. Although many 0-8412-0581-7/80/47-136-287$05.00/0 © 1980 American Chemical Society Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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modern LSC(s) o f f e r options which c a l c u l a t e dpm from raw counting data, these options o f f e r l i t t l e or no advantage i n t h i s a p p l i c a t i o n because of the many a d d i t i o n a l c a l c u l a t i o n s and approp r i a t e f o r m a t t i n g of r e s u l t s necessary. Based on past experience, the f o l l o w i n g times would be r e q u i r e d to reduce the data from 100 samples; more than four hours f o r hand c a l c u l a t i o n s , 3 hours f o r a simple programmable hand c a l c u l a t o r , 30 minutes f o r a time-share computer s e r v i c e , and l e s s than 2 minutes f o r d i r e c t l y i n t e r f a c e d minicomputers. Hardware The desk top computer network i s shown i n F i g u r e 1. The l i q u i d s c i n t i l l a t i o n counters which we r o u t i n e l y use to generate metabolism data i n c l u d e two Beckman LS-200 s e r i e s l i q u i d s c i n t i l l a t i o n counters and two Tracor Mark I I I l i q u i d s c i n t i l l a t i o n counters, however, other LSC makes and models may be i n t e r f a c e d . The Tracor counters are equipped with two standard communications output connectors. One i s used to d r i v e the p r i n t e r , the other i s a v a i l a b l e to the user. The Beckman counters use a unique t e l e t y p e d r i v e r but an i n t e r f a c e produced by W i l l i a m Palmer I n d u s t r i e s can s p l i t the s i g n a l and provide a standard communicat i o n output. Therefore, a l l data output from the l i q u i d s c i n t i l l a t i o n counters are a v a i l a b l e i n an e l e c t r i c a l standard format, RS-232C. Hardware i n t e r f a c i n g was t h e r e f o r e s i m p l i f i e d and data c o l l e c t i o n was a matter of software development. The data r e d u c t i o n hardware i s based on a Hewlett-Packard 9825A desk top computer. I t i s supported by one megabyte of f l e x i b l e d i s k storage, a p r i n t e r / p l o t t e r and the necessary i n t e r face equipment f o r o n - l i n e LSC data c o l l e c t i o n . R e l i a b i l i t y was a prime f a c t o r when the hardware was chosen. The LSC(s) and data system run v i r t u a l l y unattended, 24 hours a day, 365 days a year. Samples are t y p i c a l l y counted f o r two minutes each plus one minute f o r the e x t e r n a l standard. Therefore, data from the four counters are r e c e i v e d by the HP9825A at an average i n t e r v a l of 45 seconds. With such a demand on the system, computers r e q u i r e good s e r v i c e support, more so than other instruments. Since i n s t a l l a t i o n i n May, 1977, there have been l e s s than two work days of cumulative downtime. The system has s i n c e been upgraded to a desk top computer network because of the increased load on the i n i t i a l HP9825A system. An HP9835A has been added so that data r e d u c t i o n and a n a l y s i s w i l l not i n t e r f e r e with data c o l l e c t i o n . T h i s second desk top computer i s supported by one megabyte of f l e x i b l e d i s k memory, a 180 CPS p r i n t e r , and a four c o l o r X-Y p l o t t e r . An HP2647 graphics t e r m i n a l was added to provide a d d i t i o n a l remote access to the data bases being generated. The system lends i t s e l f to expansion and continued modernization.

Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


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1 MEG

4 LSC

FLEXIBLE

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Figure 1.

Desk-top computer network


289

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Software LSC Data C o l l e c t i o n . The LSC data are c o l l e c t e d by the HP9825A u s i n g what i s known as b u f f e r e d I/O ( i n p u t / o u t p u t ) . A p o r t i o n of the computer's memory i s set a s i d e f o r the data from each LSC. As the LSC(s) output data, i t i s s t o r e d d i r e c t l y i n the b u f f e r assigned to the p a r t i c u l a r counter without otherwise a f f e c t i n g the program which i s running. A f t e r a predetermined number of c h a r a c t e r s have been r e c e i v e d or a s p e c i f i c c h a r a c t e r has been r e c e i v e d , the computer jumps to a subroutine which s t o r e s the counter data. Each LSC i s a u t o m a t i c a l l y assigned a p r i o r i t y , and data output by two or more counters are handled simultaneously according to the p r i o r i t y . Data storage a c t u a l l y i n v o l v e s r e f o r m a t t i n g the i n f o r m a t i o n from each LSC, and s t o r i n g i t i n a p a r t i c u l a r sequence as comp a c t l y as p o s s i b l e . Each LSC manufacturer outputs data i n a d i f f e r e n t format and f r e q u e n t l y s e v e r a l d i f f e r e n t output formats are a v a i l a b l e on an i n d i v i d u a l counter. The computer i s programmed to convert these d i f f e r e n t outputs to a standard format f o r use i n l a t e r computations. Nonessential data i n each output l i n e are discarded to improve storage e f f i c i e n c y . A f t e r the data have been reformatted, the computer reads the corresponding l i n e of data from the f l e x i b l e d i s k . The o l d e s t data are d i s c a r d e d and r e p l a c e d with the new data. The e n t i r e l i n e i s then r e s t o r e d which completes the sequence. This procedure always r e t a i n s the l a s t two passes of data. With the completion of storage, the computer r e t u r n s to the main program and continues from where the i n t e r r u p t i o n occurred. The time l o s t during the i n t e r r u p t i o n i s t y p i c a l l y l e s s than two seconds. Because t h i s equipment runs c o n t i n u o u s l y , p r o v i s i o n s must be made f o r the i n e v i t a b l e power f a i l u r e s and o c c a s i o n a l d e v i a t i o n s from the expected data formats. The computer has a b u i l t - i n p r o v i s i o n f o r power f a i l u r e s . The software i s w r i t t e n so that the program which was running at the time the power went o f f i s reloaded and r e s t a r t e d . The only data which i s l o s t i s that which had been s t o r e d i n the b u f f e r but not s t o r e d on the f l e x i b l e d i s k a t the time of the power f a i l u r e . E r r o r s may a r i s e from a v a r i e t y of sources, i . e . , when the data c o l l e c t i o n program d e t e c t s that something has gone wrong. The software i s w r i t t e n to assume the worst p o s s i b l e circumstance. The e r r o r , the l i n e number i n which the e r r o r occurred, and the data are p r i n t e d on the i n t e r n a l s t r i p p r i n t e r . It i s a l s o assumed that the program might have been loaded i n c o r r e c t l y . Therefore, the computer i s d i r e c t e d to r e l o a d and r e s t a r t the program. T h i s f e a t u r e allows e r r o r s i n the software or hardware to be r a p i d l y traced and c o r r e c t e d . At the present time, the a d d i t i o n a l sample data r e q u i r e d f o r the c a l c u l a t i o n s are entered through the computer keyboard. These data i n c l u d e a l i q u o t s i z e s , moisture f a c t o r s , combustion e f f i c i e n c i e s , p a r t i t i o n i n g volumes, e t c . Prompts appear on the



16.

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computer d i s p l a y asking f o r each e n t r y . T h i s makes the system easy to use and new personnel are able to l e a r n the system q u i c k l y . F i g u r e 2 i s an example of these data e n t r i e s . The data may be checked and c o r r e c t e d i f necessary. The example shown i s t i s s u e combustion data from an animal balance study. The computer not only puts numerical data i n t o formulas and produces an answer, i t can make judgments on the data and produce more meaningful i n f o r m a t i o n . For example, a t o l e r a n c e of + 10% i s s e t f o r the two passes of LSC data. When t h i s t o l e r a n c e i s exceeded, the computer assumes the most recent data are the most accurate. The output i s noted so that the v i a l s can be l e f t i n the LSC f o r another pass. The computer not only c o r r e c t s f o r background r a d i a t i o n , sample quenching and instrument e f f i c i e n c y , i t c a l c u l a t e s the s t a t i s t i c s f o r LSC counting as presented by C u r r i e (1). The data outputs are, t h e r e f o r e , more meaningful, because d e t e c t i o n and q u a n t i t a t i o n l i m i t s are known. Output. The computer produces a hard copy output f o r each data s e t entered. T h i s output i n c l u d e s the "raw" counting data, l a b o r a t o r y personnel e n t r i e s , intermediate c a l c u l a t i o n s , and the f i n a l r e s u l t s . The format i s easy to use and allows the r e s u l t s to be hand c a l c u l a t e d to check the computations. The computer has a r e a l time c l o c k which dates each output and provides a space f o r the i n d i v i d u a l to s i g n . T h i s s a t i s f i e s the proposed EPA g u i d e l i n e s f o r "raw" data. An example of a hard copy output which may be obtained i s shown i n F i g u r e 3. In t h i s example, the entered data (Figure 2) i s used w i t h the LSC data to c a l c u l a t e the ppm and percent of C dose i n v a r i o u s animal t i s s u e s a f t e r combustion. Another hard copy output i s shown i n F i g u r e 4. In t h i s example, i o n exchange column chromatographic data are expressed i n histogram form. The upper histogram i s the C e l u t i o n p a t t e r n of metabolites i n a crop e x t r a c t and the lower histogram i s a H l a b e l e d standard which was added to the e x t r a c t f o r cochromatographic purposes. These formatted outputs are stored on a f l e x i b l e d i s k e x a c t l y the same as p r i n t e d . Later the data on the d i s k are stored by sample number and p r o j e c t . A l l raw and reduced data f o r a p r o j e c t are stored on a s i n g l e f l e x i b l e d i s k . A f t e r the data have been stored on a f l e x i b l e d i s k , i t i s a simple matter f o r the computer to make summaries or do trend a n a l y s e s . An example of one of these summaries i s shown i n F i g u r e 5. In t h i s example, the percent of C remaining i n the p e e l and f r u i t at v a r i o u s time periods a f t e r spraying apples with a C l a b e l e d compound, was analyzed by a curve f i t program. 1 U

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Expansion. As data p r o c e s s i n g equipment becomes more "bench" o r i e n t e d , data from other instruments can be input d i r e c t l y to the network. A Hewlett-Packard 1084B HPLC i s p r e s e n t l y i n t e r f a c e d a l l o w i n g reduced data from i t s processor


PESTICIDE

292


File

Name: w s l Number 7 8 9 10 11 12

ANALYTICAL

Description

Entry

Background F i r s t Sample

-30.0000 25.0000 18.0000 35.0000 0.96C0 43.6G00 Back F a t 0.2281 0.1898 82.0000 Omental F a t 0.1788 0.1564 215.0000 Leg M u s c l e 0.2165 0.1730 270.0000 Tenderloin 0.1546 0.1709 100.0000 Br e i n 0.1967 0.1518 47.0000 Heart 0.1839 0.1624 116.0000 0.2432 0.1780 183. 0000 Kidney 0.1924 0.1406 426.0000 Liver 0.2536 0.2192 2800.0000 338800000.0000 10.0000

Combustion E f f i c i e n c y Specific Activity Sample Ν a ire 1st A l i q u o t Weight 2nd A l i q u o t h e i g h t T o t a l Sample S i z e Sample Nane 1st A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sample S i z e Sample Name 1st A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sample S i z e Sample Name 1 s t A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sample S i z e Sample Nane 1st A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sample S i z e Sample Nane 1st A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sam:ple S i z e Sample Name 2nd A l i q u o t W e i g h t T o t a l Sample S i z e Sample Name 1st A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sample S i z e Sample Name 1st A l i q u o t Weight 2nd A l i q u o t W e i g h t T o t a l Sample S i z e D a i l y Dose (dpn) Number o f Days Dosed

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figure 2.

METHODOLOGY

Tissue combustion data entered for an animal balance study


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Pesticide Analytical Methodology - American Chemical Society

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