Oral History Interview, Cook, J William

HISTORY OF THE U. S. FOOD AND DRUG ADMINISTRATION

Interview between: J. William Cook Retired Director, Division of

Pesticide Chemistry and Toxicology F .~., and .. Fred L. Lofsvold, FDA - James Harvey Young, Emory University - Falls Church, Virginia

June 18, 1980 INTRODUCTION

This is a transcription of a taped interview, one of a series conducted by Robert G. Porter and Fred L. Lofsvold, retired employees of the U. S. Food and Drug Administration. The interviews were held with retired F.D.A. employees whose recollections may serve to enrich the written record. It is hoped that these narratives of things past will serve as source material for present and future researchers; that the stories of important accomplishments, interesting events, and distinguished leaders will find a place in training and orientation of new employees, and may be useful to enhance the morale of the organization; and finally, that they will be of value to Dr. James Harvey Young in the writing of the history of the Food and Drug Administration. The tapes and transcriptions will become a part of the collection of the National Library of Medicine and copies of the transcriptions will be placed in the Library of Emory University. DEPARTMENT OF HEALTH. EDUCATION. AND WELFARE PUBLIC HEALTH SERVICE FOOD AND DRUG ADMINISTRATION

TAPE INDEX SHEET CASSETTE NuMBER(.S) 1 and 2 GENERAL TOPIC OF INTERVIEW: History of the Food & Drug Administration DATE: 6/18/80 PLACE: Falls Church, Virginia LENGTH: 100 minutes INTERVIEWEE INTERVIEWER *

NAME: J. WlCLlAMOOK NAME: ~r~dL. ~nf~~~ld. .

ADDRESS: ADDRESS: U.S. Food & Drug Administration Denver, Colorado FDA SERVICE DATES: FROM 1939 TO 1972 RETIRED? Yes TITLE: Director, Division of Pesticide Chemistry and Toxicolpqy (If retired, title of last FDA position) CASSETT SIDE EST.TIME PAGE SUBJECT NO. 1 NO./ ON TAPE I NO. Introduction. History of organo-phosphorus pesticides. Development of fly bioassay method for organic pesticides. "Mills Procedure for analysis by paper chromatography" developed. FDA Pesticide Manual. PCB problem - background and analytical solution. Pesticide methods development through AOAC Collaboration. Analysis for multiple pesticide residues by FDA Manual methods.

FDA approval of new pesticides and tolerances for residues. Cook develops spot tests for organo-phosphorus pesticides. Examples of use of spot tests.

2 A 10 Discovery of the thermionic detector for gas- (First 1 1 iquid chromatograph. minute of 18 Another example of use of spot tests. SideAis 23 Cook's participation in international blank) committees.

Cook receives HEW award: Commissioner Larrick's and Commissioner Edwards' interest in research. End of recording * Also James Harvey Young, Professor of History, Emory University, Atlamta, GA.

- This recording is being made with J. William Cook, a retired scientist from the Food and Drug Administration at his resi- dence in . The recording is part of the FDA oral history project. The date is June 18, 1980.

A1 so present is James Harvey Young, Professor of History,

Emory University. My name is Fred Lofsvold.

FL: Mr. Cook, would you briefly describe your career with the Food and Drug Administration?

JWC: I started with the Food and Drug in September of 1939, reporting to the Seattle District. About two years later, I was transferred to a sublaboratory of Seattle District in

Portland, Oregon. During those two periods, I did general laboratory work, including quite a bit of pesticide analysis for lead, arsenic and fluorine. But Mr. Harvey asked me to transfer to San Francisco to do some general research on foods. I moved to San Francisco in 1943 and was there 9 years. Then I was transferred to Washington, D.C. into the Division of Food. I had developed some expertise in the use of enzymes looking for contaminants in foods while in San

Francisco. Enzymatic techniques were useful in measuring the organo-phosphorus pesticide chemicals so I began to ap- ply them to FDA's problems and that got me into pesticide work, beginning about 1952. I continued mainly in the field of pesticides until I retired in 1972. JHY: Would you say just a word about your education before you became employed by FDA?

JWC: OK, I received my Bachelors and Masters from Oregon

State with a major in Chemistry and a minor in Nutrition. I was interested in the biological aspects of chemistry. My

first job out of Oregon State was on the Agricultural Exper-

iment Station staff of Washington State College, now Wash-

ington State University. I was in the School of Agricul- ture, Biochemistry Department and did cooperative work with the Department of Poultry on poultry nutrition. From there

I went to the Food and Drug Administration.

JHY: Can you remember the first moment that you encountered the idea of an organo-pesticide? In contrast with the metal lic ones?

JWC: No, I really can't remember when I first knew about the general subject of organo-pesticides. I remember being fascinated by the plant growth hormones, a subject which guided people into herbicides. That was about 1936-37.

I guess it was when I first came to D.C. in 1952 and was attempting to use the enzymes as means of determining food contaminants, that I became fascinated with the organo- phosphorus compounds. There was some secret work being done in the Division of Food at the same time on the war nerve gases.. The organo-phosphorus pesticides are related

to the so-cal led war nerve gases, the main difference being

that the pesticides usually are solids, or liquids rather

than gases at room temperature.

JHY: I got you off the track there, but since there was a

bridge in history of kinds of things used for pesticides, I

was just curious about where you saw them first, the new

ones coming?

JWC: For years I had read the current scientific journals

and was always attracted by articles that concerned com-

pounds with biological activity. In those days companies that were developing data on new pesticide chemicals brought data on chemistry and toxicology to FDA scientists in order to inform us and to get our reactions to their procedures and data. (This was before such data were required to be submitted by the Miller Amendment to FDA Law in 1954.) When there were meetings concerning organo-phosphorus compounds I was asked to attend. I quickly became interested and intri- gued with both the chemistry and the biological effects of these compounds. Organic phosphorus compounds were developed in Germany during World War 11. Schrader who worked for Farbenfabriken Bayer at Leverkusen, Germany, was working on them as possible insecticides. The Germans recognized the fantastic toxicity of these compounds and did develop them as war nerve gases. It wasn't until after the wiar, when some U.S. people went into Germany that we got acquainted with these compounds and brought some of them to the U.S.

I thought I would give you a little history of the development of the general chemical analysis for pesticides that we in Food and Drug developed through continued inten- sive research and organization. The first sort of general method was developed by Dr. Ed Laug in the Division of

Toxicology. He used the fruitfly as a bioassay organism. He extracted plants, did some clean-up or purification of the extracts, and then exposed groups of flies to the extracts.

An estimate of the pesticide in the plant extract was made by comparing the number of flies killed by the extracts, to the number of flies killed by the standards. This simple procedure was useful, but it was soon found to have a number of limitations. Dr. A1 Klein in the Division of Food was working closely with Dr. Laug and he found that some samples of food had naturally occurring compounds present which were extracted and had a tendency to either give a false positive or a false negative result. He did considerable work in attempting to clean up the extracts to eliminate the false results and samples for improving the certainty for quantities and identity. Later, Mr. Paul Mills joined the Divi- sion of Food and he too began to do work to improve the technique. The fly bioassay was developed primarily for a screening technique, i.e., a technique of looking for any pesticide that may be present in a sample of unknown history. But, of course, it was useful for those samples in which the pesticide was known and a quantitative result was - needed. The pesticides in use at that time were primarily the chloro-organics, DDT, chlordane, benzene hexachloride, etc. Mills was interested in improving the screening techniques because most samples FDA analyzed were of unknawn pesticide spray history and many new chemicals were being developed and put on to the market. He used paper chromatography as a means to clean up or purify the extracts from the plants so that there would be less uncertainty, i .e., fewer false positivies and negatives in the fly bioassay. As he worked with the paper chromatography and was able to detect amd measure the chemicals on the paper, he found that estimate better quantitative results on the paper @ he could than was possible to achieve with fly bioassay and also was able to identify the chemical which was usually not possible by fly bioassay. He did continue doing both the fly bioassay and the paper chromatography for a while, but finally just stopped using the fly bioassay. Also he found that the paper chromatography did not give a complete clean up. Again, he got some false positives and some fa1 se negatives unless the sample was fairly well purified before it was put on to the paper. He started using column chromatography to further purify the extracts. Owen Winkler, in the Division of Food, had been using columns to do clean up for some arsenic and lead work. Mills got some of his clues from Mr.

Winkler. The clean up or purification procedures developed

by Mr. Mills to give really good results with the paper

chromatography were found to be very useful when gas

chromatography was developed.

In 1959, Mr. Mills published an article in which he

pulled together the various steps in pesticide analysis

identifying the type of detectors for gas chromatography,

the clean up or purification procedures, etc. That is the

basis for a screening or multi-detection system now utilized

by Food and Drug Adminstration and by many other labora-

tories throughout the world. The procedure is generally

known as the Mills procedure; however it has been expanded

and developed by a number of other people over the past 20

years to the point where 100, or more pesticides, including

chlorinated compounds, carbamates and organo-phosphorus

compounds, can be run through the scheme to obtain qualitative and quantitative data with a high degree of certain-

ty. The procedure is useful for a large variety of food

products. A number of adaptations have been added so that,

if there is some uncertainty, an identifying procedure can be run on certain portions of the extracts to give a second

analysis to give a higher degree of certainty of the identi-

ty. This publication was in the Journal of the Assoaiation

of Official Agricultural Chemists, Vol. 42, page 734, 1959.

The sequel to the Mills Procedure, that is, the greatly ela-

borated procedure, is a loose-leaf publication of the Food and Drug Administration called Volume I Pesticide Analyti- cal Methods.

JHY: This is a complex manual, constantly updated, and, so far as we know, until very recently, still being updated and definitely employed for analyses of pesticide residues by FDA chemists. Now in this complex book of procedures which could be applied, are there incidents in which applications had an important regulatory influence, or in some other way became important to society that you could c ite for us?

JWC: Yes, one very striking example is that , by use of this technique, it is possible to separate the PCBs from the pesticide chemicals. The PCBs, (polychlorinated biphenyl compounds), which have become controversies in many places, originally showed up on gas chromatograms as interfering peaks. A separate clean-up procedure or column was devised to separate the PCBs from DDTs, etc., so that more accurate analysis of DDT could be made. But, likewise, even though PCB is not a pesticide, this procedure permitted us to develop a very good procedure for the PCBs, in foods, in packaging materials and in plastic food wraps, etc. That is an outgrowth of this analytical procedure.

JHY: It was sort of a serendipitous side result that initi- ally was kind of a problem to you, but later on proved to be of regulatory benefit. JWC: Right. This separation of the PCBs from the pesticide

chemicals proved to be a real boon to us when the National

Cash Register Company's no-carbon copying paper found its

way into the manufacturing of food packages and then

migrated into food.

JHY: That is to say the scraps of paper were mixed with

other paper.

JWC: Right. And re1at ivel y large quantities of PCBs were

in the cardboard containers.

I JHY: Is that because the PCBs were in the mechanism by which the carbon transfer was made in these papers?

JWC: Right. The dyes that produced the color on the second

and third copies were dissolved in PCBs and those were en-

capsulated into minute capsules and were in a layer on the

paper. When pressure was put on paper by the pen or type-

writer, those capsules broke and allowed the dye to dye the

second paper. All the scraps would have heavy layers of these chemicals and those scraps and the waste paper cast

out by the industries and government, as scrap, were incor-

porated into the material from which most cardboard is

made. FL: Then the PCBs would migrate from the packing, the

cardboard, to the food that was packed in that kind of a

package?

JWC: That's correct. But, besides that, the PCBs got into

other elements of the environment, such as the streams, and

thereby contaminated fish and other food products. There

were many other sources of PCBs, besides the copying paper,

which contributed to PCBs in the environment. This analyti-

cal procedure was easily adapted to PCB analysis in a

va r iety of food and other products.

JHY : You were more involved personally in developinsg the

methods, rather than in employing the methods with r~egardto

food products, which had been seized as suspect and needed

to be tested. This was done somewhat more in the fimeld. So you were on the research side of improving the techniques by

which analyses could be made to find the residues.

JWC: I did not do laboratory work on any phase of the Mills Procedure. In 1959 I became Chief of the Pesticide Branch.

My job there was to supervise and encourage the continued

improvement of the procedure, as well as supervise the peti-

tion review for tolerances. In 1963 I became Deputy

Director of the Division of which Pesticide Branch was a

part. As Deputy I continued to monitor the pesticide work. Our division was involved with improving this technique, but we also did a good deal of testing to be sure that it would 0 work and we did work with the field districts a good deal in performing these analyses. It so happens that the problem

of PCBs in cardboard packages developed at the time when we

first produced this clean up procedure for eliminating the

PCBs from the DDTs. And so our laboratory did get deeply

involved in the analysis. We even helped industry labora-

tories, had meetings of industry people and field la~boratory

people to help them proceed with the analysis for th'em-

selves.

JHY: Was this also true in connection with pesticide resi-

dues, did you meet with industry in an effort to put your

joint talents together in such a way as to reduce or elim-

inate problems in connection with residues getting into the

food supply?

JWC: Oh, yes in many ways. For instance, the procedure as

it is in the manual now has been the result of a fantastic

amount of research work, most of that research work has been

oriented toward the theme of the Association of Official

Analytical Chemists over the years. And there are many

references in the manual to the collaborative work done in

the AOAC system. And that system involves state and federal

Food and Drug scientists, as well as industry, municipal

health and food officials. Many times the samp'les for

collaborative studies were sent to a number of Food and Drug District laboratories and may have gone to a number of industry state, municipal, and other governmental laboratories, including Canadian Food and Drug, to determine the results that they obtained as against those obtained by the author or developer of the various aspects of the procedure.

The results of these collaborative studies were then presented to the Association at their yearly fall meeting, and are heard by all interested parties. Any interested party is welcome to attend the meeting and has opportunity to comment. The results are then reviewed by Association committees and accepted or rejected as official or unofficial methods and ultimately incorporated into the AOAC manual.

JHY: One of the striking things about the manual is that it provides procedures for analyzing samples that may have multiple pesticides in the sample. Now why, from the state of the marketplace, was it necessary to develop such complex procedures? Does this mean that apples or lettuce or some other product in this early age of the new pesticides were being sprinkled with many pesticides so that you had samples that might have 4 or 5 and therefore needed the new complex procedures?

JWC: The procedures in this manual are really relatively simple. Many, many of the pages you see are data which show the chemist the supporting data for each page, 'for example, some tables show the X recovery of maybe 100 chemicals through a certain step of the procedure, etc. There are many facets to the answer to that question. Generally speaking, the same lettuce sample, for instance would not

necessarily have a variety of chemicals on it, but there were a number of chemicals that were registered for use on lettuce, or apples. Therefore, we wanted a procedure to analyze lettuce that would identify and measure any possible chemical or combination of chemicals that may be present on samples of unknown spray history, without going through a multitude of individual analyses. You indicated that this procedure is complex. Basically, to the chemist, this is much less complex than running the procedures for a number of different chemicals on lettuce. With this multi-residue procedure, it is necessary only to make one extract, one clean-up or purification, one pass through the gas chromatograph, and any of a large number of chlorinated and many organophosphorus pesticides that may be present would be identified and measured. Such a lettuce sample can be analyzed for all these pesticides in a matter of a new hours, in contrast to many days by individual methods.

JHY: Now most samples would only have one or two pesticides on them?

JWC: That's right. So again this is sort of a fortuitous outgrowth of attempting to make a good simple analysis. With DDT for instance, it used to be a relatively difficult to be certain that you were analyzing for and measuring DDT. In an attempt to make that analysis and adding these new techniques of chromatographies and broad spectrum detectors, when we determined DDT, we automatically determined the others, so we just took advantage of that.

JWC: For instance, when the first petitions for aldrin, dieldrin and endrin came, the method's sensitivity was so poor in relation to the toxicity, that we decided not to set a tolerance for endrin because the method would not determine the amount that we considered to be toxic. Aldrin and dieldrin were marginal methods with respect to toxicity. And those chemical methods were very complex and very non- sensitive. The new procedure that is in this manual is so simple and so accurate, so sensitive to dieldrin and aldrin both that they can be determined if only one thousandth of one millionth of a gram was present, aldrin responds so beautifully in this multiresidue procedure that aldrin was used as a reference standard to be sure that the gas chroma- tagraph was working properly and that the techniques are working properly. So it was just a fortuitous benefit to all the problems of analysis to work and develop this kind of technique.

JHY: That really makes it much more clear to me. Thank you. Now, let me throw in a judgmental question, you were watching the new pesticides come along and developing tests to detect them. And also the Food and Drug Administration had the responsibility at this period of deciding whether or

not new ones should go on the marketplace. As you, as a scientist deeply involved in this look back upon your experience when these new ones were coming on the market so fast, what is your impression about the part of FDA's responsibility toward admitting them? Do you feel that FDA was properly cautious about admitting them to the marketplace or too cautious or too lenient?

JWC: A number of the pesticide chemicals, primarily the chloro-organics, were in use before the 1954 Miller Amend- ment to FDC Act. This amendment mandated that FDA set tolerances based on chemical, toxicity and residue data submitted to FDA by the manufacturer. Before the Miller

Amendment, Food and Drug had to prove in court that the food, that that particular sample of food was poisonous and did not have responsibility or authority of approving or disapproving of a chemical before it was in use.

JHY: It had to set tolerances after the things were on the market?

JWC: The only official tolerances set before t'he Miller

Amendment was after long drawn out hearings held in 1950. At those hearings, the proponents did not have to have any toxicity or other data except to show that the chemical had been used. In 6 months of formal hearings, 20,000 pages of test imony were recorded. From this, Food and Drug did set some tolerances based on the use data and FDA's and Public

Health's knowledge of toxicity.

JHY: Even though there night be a toxicity question still somewhat open.

JWC: Right, when the Miller Amendment was enacted, t provided that toxicity data had to be provided to the D vision of Pharmacology so they could determine a safe level and a method had to be available to adequately analyze for the tolerance level in food before the tolerance could be established. Of course, some of the tolerances established through the Miller Amendment were before we had really good methods. So there was a degree of uncertainty in the methods of analysis for some of these compounds. For instance,

DDT--the analysis for it was based on the total chlorine analysis even though there is some chlorine normally present in food products. For example, 0.2 part per million DDT might be calculated from the chlorine present in an untreated sample. And then you might calculate 0.4 parts per million based on a sample that had been treated with DDT.

The chemist subtracted the two tenths from the 'four tenths and said that the DDT present was two tenths. But the calculation was made on the assumption that the chlDrine did in fact come from DDT. However, this was not a certainty.

It could have been other problems. Now in the case of the aldrin and dieldrin that I referred to before, again it was basically a total chlorine procedure. With a blank of 0.1 ppm calculated as aldrin and a sample of 0.1 ppm and the proposed tolerance in the range of .l,there was uncertainty. Even though the Miller Amendment mandated method submission to FDA, we felt that we had to have more efficient analytical procedures to do adequate surveys of food samples in commerce. The mu1 ti-residue procedure previous1y described was the answer to many of the methods problems we faced in setting and enforcing the tolerances. And the toxicity -- there is never enough data to be absolutely sure of anything, so judgements had to be made on all data.

Sometimes tolerances were not established because of lack of proof of safety. Sometimes we went back and rescinded tolerances or lowered some tolerances because of new data.

JHY: So you did feel a sense of urgency because you knew that things were going on the market and that there was scientific uncertainty. It was part of your task to develop the scientific certainty which would clarify some of these doubtful matters. JWC: We assumed it to be our task. Certainly the law didn't say that, but we assumed it to be our task to continue to develop methods to make it easier for us to know or easier for us to make surveys to find the end product of having set a tolerance and to pass judgment on extension of the tolerances to other products. When I discuss the organo-phosphorus history, I'll bring in some details on that. As I said previously, when I worked in San Francisco,

I was attempting to use enzyme systems to help identify food contaminants. I was transferred to D.C. in 1952 to continue that kind of work. There wasn't anybody else doing similar work. Mr. Vorhes, who was then the Director for the Divi- sion of Food, suggested that I look around and find some aspect of the Division work that might be enhanced with the use of the enzyme systems. I went to Nutrition and to

Antibiotics and people in the Division of Food, various aspects of foods, freezing and so forth--and the pesticide people and I, of course, was studying the literature a good deal. It seemed to me that one of the best uses of the enzyme work at that tine would be the organo-phosphorus compounds, because these compounds are toxic by virtue of the fact that they are inhibitors of the cholinesterase enzymes. The cholinesterase enzymes hydrolyze to a compound called acetylcholine. Acetylcholine is involved in the transmission of nerve impulses. Therefore muscle activity is based on acetylcholine being formed and hydrolyzed quickly. When those enzymes are inhibited, the person becomes rigid or has tremors. I attended meetings with industry as industry brought in data to tell Food and Drug about the chemicals that they were anticipating putting into use, especially when organo-phosphorus compounds were discussed. It was then that I learned about the earlier work done on the organo-phosphorus compounds. When I started looking into the literature to see how people were doing the research on the esterase systems and in inhibition of esterases, I found that most of them had relatively com- plicated (for those days) pieces of equipment . For me that was impossible, because Food and Drug was pretty poor then.

Our total budget was about five million dollars and that's for the whole nation. There was no laboratory equipment really, even beakers and pipettes available for me. I had to figure out some less expensive way to do the work. I had been fascinated with what is called spot tests and that is the use of qualitative reagents that reveal small am)ounts of chemicals spread out like ink in a blotter. For instance, in

San Franciso, I devised a test for urea by having the enzyme urease put in a piece of paper along with a dye that would change with acid-base. So if you put a spot or a chunk of flour or some wheat grains or something like that that had rat urine on them, they would turn the paper a different color in spots where there was some urine. It's simple, cheap and quite accurate for identification of urea, a component of urine from mammals. FL: Incidentally, we still use that in our cases that we bring on insanitation.

JWC: Do you really?

FL: Yes.

JWC: I wondered about that the other day. So I thought I might as well start to work with a similar technique for the organo-phosphorus compounds, if it were at all possible.

However, I didn't think right at first to use the enzyme spot test. So I started by doing chromatography on the 0-P coupounds. I got a suggestion from Joe Levine, a chemist in the Division of Pharmaceutical Chemistry, that the sulfur in most 0-P pesticides might be sensitive to bromine. So I used that reaction as a spot test. After I developed the chromatogram, I sprayed the whole paper with a bromine containing compound called N-Brom-succinimide. Then I superimposed that with a dye chemical (fluorescein) spray that was sensitive to bromine. Wherever the sulfur in the organo-phosphorus compound used up the bromine, it was not available then to change the color of the fluorescein. The brominated fluorescein is pink and the non-brominated is yellow. So I'd have yellow spots where there was organo- phosphorus compounds. Most of the organo-phosphorus compounds that were used as pesticides had sulfur to stabilize them. I found that to be a very useful test and almost immediately I discovered some startling reactions of the organo-phosphorus compounds. First I spotted Systox, chromatographed it, and sprayed my two reagents. Beautiful, just like that. In less than an hour I had a beautiful analysis showing the two isomeric compounds which comprise technical Systox. So I thought well I'd better test some of the other organo-phosphorus compounds to see if they're equally sensitive. So I took an 8" square piece of paper and spotted 10 different 0-P pesticides in microgram quantities.

JWC: First, I spotted Systox on the paper. Then I washed the small spotting pipette and then spotted parathion. I repeated the wash and spotting of eight other 0-P products.

When I chromatographed and sprayed the spot test reagents, I found that Systox wasn't Systox any more. I got different spots than I did when I spotted it alone on the chronato- gram. I immediately thought, well gee whiz, the only difference between now and the other time was it took me maybe 15 or 20 minutes to spot the chemicals on the chromatogram, as against previously it was just a matter of seconds. So I took three different pieces of paper, and spotted them with Systox. I left one under the fluorescent lamp that I was working under. I stuck one in the drawer, and I took one over to the window and let the sun shine on it. And the one where even the fluorescent light was on it changed. The one in the drawer didn't change. And the one in the window changed. So that showed me that spread out in a thin layer on the paper Systox is almost instantaneously changed from the technical product of 2 isomers to two other compounds that are more soluble in water. It probably was the explanation of why Systox is a systemic insecticide.

Because, even though it is basically an oil soluble technical product, it immediately changed to more nearly water soluble compounds. And the combination of the water and oil solubility permits it to go into the plant and translocate.

There were changes in the other 0-P pesticides also.

Knowing that some of these technical products were good in vitro inhibitors of cholinesterase, and some of them were not good in vitro inhibitors of cholinesterase, I thought I would devise another test in which I did superim- pose both spot tests, the cholinesterase inhibition and the bromine-fluorescein test. Much to my surprise, I found that the bromination technique that I used for the first spot test converted the non-cholinesterase in vitro inhibitors, tors, to in vitro inhibitors. This technique gave me a tool to determine or visualize some of the general chemical char- acteristics of these compounds. From this line of work I developed many useful clues to look for when petitions came in for new 0-P compounds. I was able then to accept or reject confidently the data that were submitted in the petitions. I almost felt that at times I knew more about the compounds than the companies that made them.

JHY: This distinction that was new to science that you had discovered, was interesting analytical ly, but could you hypothesize things from it about relative degrees of toxicity or toxicity in different parts of the body, or a ything of that kind? Did it have implications of that sort?

JWC: I gave considerable consideration to the possib lity that some degree of projection of toxicity to humans from animal studies could be made. For instance, I postulated that it might be possible to determine the in vitro inhibitory effect of a series of organo-phosphorus cornpoun~ds on a number of different enzyme systems in experimental animals and some in human, then knowing the toxic effect of these compounds on the experiental animals make a calculated projection of the toxicity to man.

We ran one experiment with this in mind, but did not follow it up. In that experiment we measured the in v itro inhibitory effect of (1) parathion (2) malathion and (3) methyl parathion on 8 different enzyme sources. Those eight were: 1. Rat red blood cells

2. Rat whole blood

3. Rat plasma 4. Rat brain

5. Dog plasma

6. Dog red blood cell s

7. Human red blood cells

8. Human plasma It was interesting to find that "... the inhibitory properties of the three pesticides are most similar in their effect on rat brain; the greatest difference is less than a factor of 3." "...the greatest dissimilarity (is) in their effects on rat plasma; parathion is 10 times more effective than methyl parathion and about 3000 times more effective than malathion".

This work was possible only because of the conversion of the technical product to in vitro inhibitors by the use of the bromine oxidation previously noted. This experiment was only the first step toward the postulated purpose of comparative toxicities to man.l Unfortunately if was not pursued.

The techiques were aids in pursuing (to a more complete finish, I might add) some other phenomena that were significant to us, such as, the low toxicity of parathion to cows in contrast to the extreme toxicity of parathion to dogs.2

Another was some work where we showed the reason

Jane McCaulley and J. W. Cook, JAOAC 42, 197-200, (1959)

J. W. Cook Ag. and Food Chem., 5, 859-863 (1957) why malathion has such a low toxicity to mammals in contrast to its being a good pesticide3, and also an exploration of the high degree of potentiation of toxicity when malathian and EPN were fed to animals simultaneously4

JHY: But it is a clue, a warning flag, in a sense.

JWC: These spot tests and chromatographic pictures and the I above described experiments helped a good deal in the evaluation of petitions, because, by this time, any petition on organo-phosphorus compounds would come to me to eva1uate.

FL: These petitions were applications from compan market a product?

JWC: I say petitions--before the Miller Amendment, the people used to bring in data on methods and data on toxicity and ask us to review it. Not really say yes or no, but they did bring it in to review it because they knew they had to ultimately deal with Food and Drug. In reviewing the petitions and keeping in mind the many experiments from our lab I could help resolve some questions that arose in my mind. But, when the Miller Amendment was passed, then, of course, we had to get full petitions including methods of

J. W. Cook, Jane R. Blake, George Yip and Martin Williams, JAOAC 41, 399-411 (1958)

J. W. Cook, Jane R. Blake and Martin W. Williams, JAOAC 40, 664 (1957) and subsequent papers. analysis and toxicity data. Again these chromatograms helped me a good deal in judging whether the type of method of analysis, the determinative step of the analysis or sometimes even the extraction procedures were adequate to be able to extract these compounds of different solubilities that were produced by the light effect and oxidations to produce the terminal, effective residue which might not be the same as the product sprayed, such as in the care of

Systox. I had a number of experiences in which I had to turn down petitions on the basis that I thought that the method was not really testing what the sponsors thoulght they were testing. Sometimes they would agree and sometimes they felt I was wrong. I was fortunate in being able to take them out in the laboratory and demonstrate why I tho~ught their data were wrong. And on the basis of those demon- strations, some of them went back and took another look at their chemical. In a couple of instances, they had brought in cholinesterase inhibition methods of analysis for their compound and I knew from my work that the pure compound was not a cholinesterase inhibitor unless it was converted somehow. In some instances, one spectacular one, I questioned that their method of analysis was right on the basis of the color compound they were producing. I showed them in the lab why I felt that their method was wrong. They went back to their lab and did not question me. They ultimately developed a very good method of analysis.

They also found out some things about their compound that they did not know previously. They found that the terminal residue was different than they thought it was and would, in fact, not yield the color compound they had used as the basis of the original method.

Another interesting toxicological problem was fairly easily solved with the aid of the simple spot tests I had developed. Dr. Frawley, in the Division of Toxicology, discovered that there was marked potentiation of the toxicities of the two 0-P compounds, EPN and malathion when they were fed simultaneously. Potentiation is a conditio~nin which two or more chemicals administered simultaneously produce more biological effect than the sum of the effects of the individals. Frawley reported that an effective in vivo dose of EPN depressed plasma cholinesterase more than red cell cholinesterase; on the other hand, an effective dose of malathion depressed the same cholinesterases in the reverse order. Simultaneous adrninstrationn of EPN and malathion depressed the two enzymes similar to malathion.

So it appeared that EPN acted to make malathion more toxic.

A simple in vitro experiment using rat liver homogenates showed the liver rapidly converted malathion to another compound whereas if EPN was added before malathion, then the malathion was not changed. This simple in vitro experiment was easily accompl ished by use of the simple spot tests. These and other experiments helped us considerably in evaluating the toxicity data. When the potentiation of these two compounds was first found, we had a concern that there may be some mysterious aspect of potentiation which would lead into the need for extreme amounts of work to be able to evaluate the safety of other 0-P compounds.

JHY: How did this help out, then? It gave you a method of checking each possible potentiation.

JMC: Well, as far as malathion and EPN were concerned, potentiation wasn't a mystery any more. For instance, I tested the combination, the in vitro combination of a number of organo-phosphorus compounds with malathion and ca8me to the conclusion that -- the data showed that in vitro, parathion was a very potent potentiator of malathion. In other words, it inhibited the destruction of the malathion as did

EPN. On the other hand, parathion (which is itself much more toxic than EPN) would kill the animal before it had any opportunity to inhibit the enzyme that hydrolized malathion.

There was very little possibility that there was any greater hazard from the two than from the one alone. I did this with other compounds too and came to a judgment that we could do tests like this that would give us a little better feeling in making interpretation of combinations. JHY: Did potentiation really raise a nightmare vision that, if enough of these got out, that there might be kind of a wholesale disaster?

JWC: Well, I suppose that could have gone through people's minds. We did, in fact, stop processing petitions right when we first found the potentiation. For some time we asked the petitioners of each of the 0-P compounds that they do potentiation work between their compound and other compounds that were already in tolerance. That, of course, amounted to a tremendous amount of work on their part. They did that, but this was finally stopped. That is we didn't ask them to do any more of the combination potentiation.

Partly because we felt that the new data had not shoswn any potentiation of other combinations and nothing refut'ed the in vitro experiments that we had done.

JHY: Fine.

JWC: The best analytical procedure for the organo-chl orine compounds had been developed quite extensively with gas chromatography as the determinative step. That is it had gotten away almost completely from paper chromatography to gas chromatography. It so happened that a detector called the electron capture detector was extremely sensitive to many chlorinated organic compounds, including many of the chlorinated pesticides. There are different sensitivities for different compounds, but there is extreme sensitivity for some of the very toxic organo-chlorines and therefore made an ideal combination for analysis. Only one or two of the organo-phosphorus compounds were responsive to the electron capture detector because they had chlorine a1 so. lady in my laboratory had done gas chromatography at NIH before coming to FDA. Her name is Laura Giuffrida. She said she would like to try doing gas chromatography of organo-phosphorus compounds. I almost attempted to dis- courage her, because we had no detection system; but we d not have a gas chromatograph available either. So she went to NIH, where she had worked previously, and made use of their gas chromatograph which had a flame ionization detector on it. That is the gases that came off of the column would go through a flame and be ionized and change a current to give a response that could be recorded on a . graph. This (flame ionization) is sensitive to any organic compound so it does not discriminate an 0-P or 0-C compound from sugar. Once she decided to clean that detector. So she took it apart and cleaned it and put it back together.

When she did that and put an organo-phosphorus compownd through, she got roughly 20 thousand times increase in response over what she had been obta ning. That is, let's say, a compound that had 10 carbons nd one phosphorus would be 20,000 times more responsive than one that was 10 carbons with no phosphorus. However, this h gh response was limited to the first few samples put through the dector. Since she had worked with a pH meter and found how sensitive that detector was to perspiration and salt off the fingers, she thought maybe the fact that she handled that detector as she put it back in the instrument might be contributing somehow to the high response. So she coated the electrode with a little salt and found that she then got this 20,000 times response which persisted. This detector is used throughout the world and is known as a, "thermionic dector".

This development opened up a new world of analysis to us, because there had been no good way of measuring 0-P compounds. So it now occurred to us that, with this detector available, it could be possible to incorporate it into the

Mills procedure. We found that it was relatively simple to modify the sol vents and the clean-up procedures to include both the 0-P and 0-C chemicals, then we added a second detector to the gas chromatograph. That is, the effluent from the column would go through the chlorine detector first, which is non-destructive, and pass on into the thermionic detector and thereby give readings for both groups of compounds in the same analytical procedure. Thus, some of the 0-P compounds, but not all of them, were included into the whole analytical scheme. We thought that this was a significant enough discovery to merit an award.

We were able to convince the Department to give her an award. I was told that it was the largest financial award ever given by HEW. It was $1500. JHY: To anybody in FDA, you mean?

JWC: To anybody in the Department. That was my understandi ng. Now, going back to the spot test technique it was useful in studying other chemicals relating to petitions. One example is as follows: Parathion is extremely toxic to dogs, for instance, one part per million will produce a depression of blood cholinesterase. On the other hand, large quantities fed to a cow will not affect their cholinesterase or permit any parathion to pass into the milk. So I judged that something had to be happening to the parathion before it got to the bloodstream of the cow because, generally speaking, parathion when fed at toxic levels will go from the bloodstream into other mammals' milk and into the meat. The Department of Agriculture had a cow at Beltsville that had a opening into the rumen, so we fed the cow parathion and removed samples from the rumen of this cow.

JHY: Was that Dr. Beaumont?

JWC: Yes, I think so, that's right. By the time I got the samples into the laboratory there was no longer any parathion present. But again a spot appeared migrating to a different position. Some literature indicated to me the possibility that there might be a chemical reduction of parathion, so I checked and found that the nitro group of parathion had been reduced to an amino group. That compound 0 is much less toxic and therefore it gave me better assurance in approving of a tolerance for parathion on plants being

fed to cows, because it would not be transmitted to the

milk.

JHY: The reduction would result from some chemical in the

cow that was not in the dog?

JWC: That's right. The cow has a very large rumen which

digests the food before the food reaches the true stomach.

The rumen is sort of a fermentation factory; it's filled

with organisms of various kinds which act upon the food.

Basically the cow lives on organisms and the organisms live

on the plants that the cow eats. Dogs have a much simpler

digestive system.

JHY: And it wasn't necessary for you to figure out what the

chemical was that did this?

JWC: It was an enzyme system, a reductive enzyme. I pub-

lished this research in Agriculture and Food Chemistry,

volume 5, number 11, page 859, November, 1957.

In July of '64 through December of '69 the Bureau of

Food published or republished the selective publications of

the Bureau of Science research, in quite large volumes, each

of which runs in the neighborhood of 600 pages. JHY: And that was for 6 months.

JWC: That's right.

FLL: And these were articles that had been published in scientific journals?

JWC : Right. Only selected ones, not all of them. Reprints from the Journal of Microbiology, for instance, Journal of -Nutrition, Journal of Food Science, and so forth.

FLL: And a good percentage of those covered the kind of work we've been talking about and method development and research, and pest icide analysis?

JWC: That's right

In 1963 I was sent to Rome to attend a meeting of the

Food and Agriculture Organization's Pesticide Committee.

There I was asked to be on a working committee on pesticide residues. We met for a one or two week period each year, and I continued on that committee for about eight years. It was very very fascinating. There were members from about 10 different countries on the committee. We attempted to set up tolerances that we thought could be considered international to1erances which would he1 p the Food and' Agriculture

Organization he1 p their underdeveloped countries in providing themselves with food. In other words, it got into how they could do their agriculture and still make use of pesticides, and keep them within tolerance levels that other nations, at least, considered to be safe.

I was also asked to become a member of the Pesticide

Section of the International Union of Pure and Applied Chem- istry, which also met someplace in Europe each year. In this section we attempted to either provide methods of analysis to interested organizations, including Food and

Agriculture Organizations or World Health Organization or to suggest to people throughout the world methods that should be developed or expanded for the use of those two organizations. We also provided methods of analysis to anmother group called The Codex Alimentarius. These were to be incorporated into a compendium of monographs on food products in international trade, so that the standards on those monographs would be acceptable to all nations and so that residue levels or contents or composition did not become an inhibitory actor in international trade. A lot of work was involved in both committees because, generally speaking, we in Food and Drug had more data available for both these committee meet ngs than did some of the other people from other countries for they would have only that which they found in the literature, and maybe not too much of that; whereas we would have petition after petition. So it turned out to be a big challenge, but it was a lot of fun. FLL: Were you the only United States representative on

those committees?

JWC: On the FA0 one, yes, for most of the time. On the IUPAC committee I was the only U.S. member; there were some

other U.S. associate members. Companies could send people,

generally by invitation, who were able to provide data that

would be useful to the Committee. For instance, American

Cyanamid would send one or two people, or Dow Chemical would

send people, depending on the chemicals of t eirs that we

were going to be considering.

The FA0 Committee was giving considerat on to all the

data that we could find on the amount of res due that might

be contributed to food products from various rates of appl i-

cation, time intervals, and type of product, and weather

-. -,, -,, conditions, and t deciding whether the residue was the

parent compound or some metabolic product. We used the term

terminal residue, which may be a metabolite or some other

compound produced by the effect of light on the parent

pesticide. We tried to evaluate the methods of analysis by

which those data were acquired. We developed monographs to

incorporate our studies and recommendations. We also

wanted to have a method of analysis that would be useful for

the various countries to use in judging the products re-

ceived by their own countries. Of course, I always at-

tempted to promote the multi-detection methods that we had developed in FDA, primarily because we and other people had put in so much effort to be relatively certain th t each step in the method had been tested to show its va ue; whereas in many instances the data were developed by an unknown method or impure chemicals or problems wh ch raised questions on the validity of the data.

JHY: You mean another nation?

JWC: I mean other laboratories in the U.S. and in other nations. Well, even in our own country, a lot of the data that you find in the literature are pretty difficult to interpret. Of course, this is exactly the kind of thing that we had to do in evaluating petitions for all of the compounds for which we did set tolerances. A petition would consist sometimes of from one or two experiments, to maybe 100 different experiments in a variety of publications, plus data that were acquired by the company's laboratories in their own experimental work, or work that they had contracted out to some university, which may or may not be published. Then from evaluating each one of those individual experiments, one has to make a decision as to the validity of the results of each experiment. Then an overall evaluation has to be made of all of the experiments. And sometimes when you get dozens of experiments you find many of them say yes and many of them say no. So then you have to pass some kind of judgment on which ones you can accept as being valid, in relation to establishing a tolerance, and which ones you feel you must reject. If the methods of analysis seem to be adequately supported, you are inclined more to depend on those data than on data from some method that is not so well known or adequately supported. So in the petition work, essentially we were doing a fair amount of "paper" research without leading to some more laboratory work but to a decision on, in the case of the petitions, yes or no whether a tolerance can be established. I used some of the same data to support the FA0 and IUPAC recommendations. Generally speaking, I tried to promote the use of our multi-residue methods for adoption and to recommend people for their next work. But it is very difficult to talk people into this because each seemed to want to do it the way they're used to, or they want to take credit for the method that they have developed. No other group has ever put the effort, the combined effort, to study in relatively infinite detail essentially all of the steps and other aspects of a multi-residue analysis, as we have on this

Mil1s procedure.

JHY: It isn't that the results of your complex multip1 e approach are inaccessible, because this volume here on the table before us, "Pesticide Analytical Manual" Volume I, which we have referred to before, would be readily available to any laboratory in any nation which wished to take it and employ it. JWC: That's right. Some people have some valid arguments against it though, for instance, the method uses acetonitrile for extraction. There is not a pure form available in some nations so they must buy it from the U.S. So some prefer to use a different solvent. If a different solvent

is used, then you may have quite a different procedure to follow to clean up or purify the extract beyond that step.

So I can understand why some people would be reluctant to use our multi-residue method. But it's still a good approach.

In Cortina, Italy at a IUPAC meeting I had an interesting and rewarding experience at an informal meeting of friends. Dr. Batora from Czechoslovakia, stated that he wanted to thank me because he became interested in pesticide chemistry from reading my papers on malathion and potentiation. He pointed out that he did the first work on pesticides in his country, and represented Czechoslovakia at this international meeting. Then Dr. Pekka Koivistoinen, from

Finland, who had been very active in this field for a long time, said he was pleased and interested that Dr. Batora would say that because he himself became interested in pesticides from the same series of papers of mine.

JHY: Have. you cited this article on the tape?

JWC: I didn't give the reference. The reference is the

Journal of AOAC, May, 1955, pg. 399. The title is "Malathionase Activation and Inhibition". and others that follow.

JHY: Well that certainly is a tribute to have stimulated two men of this kind who eventually became your peers in an international venture of this sort. Mr. Cook has just

brought a printed award which was given to him which has his photograph at the top and his title, Acting Director - Divi- sion of Pesticide, Chemistry, and Toxicology, Bureau of Foods, Pesticides and Product Safety - Food and Drug Administration. This accolade for "sustained high quality of performance and exceptional supervisory ability in chemical research on the nature and measurement of intermediate and terminal residues of pesticides in food." Now, I've just read the citation and will you please explain the award?

JWC: Well, the award consists of a distingui shed service certificate from the U.S. Department of Health, Education and Welfare. Also, I was awarded a departmental gold metal about the size of an old silver dollar and is entitled

"Medal of Award for Distinguished Service" with my name engraved

JHY: Th is is from the Department of HEW? JWC: Right. It was presented in a ceremony, a relatively

large departmental ceremony.

FLL: That is the highest award that the Department issues,

I believe. What is the date on the certificate?

JWC: 1970. April 10, 1970.

JHY: And you said that before the Department would accept a

candidate for this award, that candidate had to have

received an award from the agency?

JWC: We1 1, yes, the FDA Award of Merit.

FLL: Which is the highest award that the Agency issues.

Bill, do you have any stories about any of the Commissioners you served under that would sort of illuminate how they operated?

WJC: With respect to the laboratory operations, yes, I can

comment that Mr. Larrick used to have meetings over in the

South Agriculture Building where our laboratories were located and is away from his own office. Periodically, he would have these meetings and ask the Division Directors to

supply chemists or other scientists to report on their work

in the laboratory. This, of course, helped him understand what our problems were and what we were doing, but it also gave the individuals in the laboratory the feeling that he cared; that he was interested in what was going on in the organization. Some of the other commissioners had no meetings of similar nature. Dr. Edwards did ask that there be presentations, but he was not as interested in the details, but wanted more broad reviews of a subject from the scientists, rather than their specific research. For instance, I was asked to give a report on mercury. We were doinlg some work on mercury, but most of my report was from literature.

This did not give the laboratory people the feeling that Dr.

Edwards was interested in the scientists themselves or in their work or accomplishments.

FLL: Were those presented by operating researchers, or presented by branch and division directors?

JWC: In the case of Mr. Larrick's meetings, they were presented by the person doing the research, yes. Also the room was large enough that other scientists could attend these meetings. Not so with Dr. Edwards' meetings.

JHY: And this was in the early '60s when you were at that building?

JWC: Yes, well it could have been even the late '50s and the early '60s, right. FLL: But the Edwards presentations, were they by the

individual researchers?

JWC: Not that I recall. In some instances there may have been. Like in some of the aflatoxins and other mycotoxins,

some of the people doing some of the laboratory work probably gave some of the talks, I'm not absolutely sure of that.

(END OF THE RECORDING) L

Reprinled from ANALYTICAL CUEMISTRY ANNUAL RNIEWS, Vcl. 37, Pow 130R. April I965

Pesticide Residues 1. William Cook and Sidney Williamc t of Food Chemistry, Food and Drug Administrotion, U. S. Department of Health, Education, and Welfore, dington, D. C.

ETHODOLOQY for residue analysis may become part of our environment. recognition that, for these determinative M has advanced rapidly during The chemist cannot know which of the steps to he useful and dependable, the current review period, irom ~ovem- hundreds of possible pesticide chemical great care must be exercised to avoid ber 1962 through October la. No- residues to look for in samples of air, interferences, false responses, and in- table progress h& been made in the de water, soil, plants, human and animal correct inter~retations of chromato- velopment and refinement of methods tissues, prepared foods, etc. There is an grams. of analysis by which any or all of a urgent need for general arocedures that Lykken (134) emohasiees the point large number of pesticide residue chemi- can identify andmeasurea large number that if a aukpie is &t representat&e of cals can be detected and measured in of chemicals at one time. Thev must LC the lot of material &om which it is ob- one general operation. This is of highly sensitive and accurate, since it is tained, results of malysis will not be L particular significance because great essential that all monitoring of our en- valid or useful. IIe presents valuable interest has developed~ntside the vironment he at a level considerably information for anyone involved in scientific community as well as within- below any "tolerance" or otherwise residue work and discusses factors which in the possible presence of pesticide critical level, so that trends can be more must be considered in designing experi- chemicals in all parts of our environ- readily recognized and Bssessed for men&, such as proper sampling, comment, including man himself. Only significance. Upward or downward positing, quartering, storage, and ship by the use of improved methodology trends in any portion of our environment of samples. will it be wssible to accom~lishthe task ment will be recognizable only when the Greater importance has been mi& of detecting, identifying, ahd measuring methodology becomes sufficiently sensi- to efficient extraction of the pesticide the manv".wssihle residual nesticide tive and accumt&so that analyses in the residue from the mmple. The use of chemicals. It is only after the presence frwtion-of-a-partrper-million or even mixtures of solvents such as hexsne and or absence of these chemicals in any part part-per-billion range become routinely isopropanol to achieve a single con- of our environment has been proved depndable (Fischbach, H. Pub. 1082, tinuous phase with the aqueous medium unequivpcauy that the medical man, Xational Research Council... 11. 55.. Nov. of food products has been studied the lawyer, the lawmaker, the adminis- 29,1962). (14, 101, I@, 147, 176, 192). Some traton in government and in industry, The multiple detection procedure of investigato~xdehydrated the tissues by and other ;ntemted groups can &&s Mills (146) for chlorinated pesticides is using anhydrous sodium sulfate, which he significance of such residues. still. being extended and modified. The improved the extraction of the residue There are 300 to 400 chemicals determinative steps most useful for with an organic solvent (10, 50, '141, registered for use on food products these general procedures are still the 186). *alone, and a few hundred more are fom of chromatography-gas liquid, The use of solvents which dissolve the registered for other uses whereby they thin Layer, and paper. There is growing pesticidal chemicals while maintaining

130 R . ANALYllCAl CHEMBTRY 1 miscibility with the aqueous medium ol to those unfamiliar with the sub Burke and Holswade (46) present the tissues, such as acetone (93,1187, ject. similar data for micmcoulometnc gss 186) and acetonitrile (14,64, l67), is With the search for ever-increasing chromatography. Relention ti- rela- reported. Such solvents give generally sensitivity and speed of analyak has tive to aldrin are lidted for 87 chlorinated higher values than those obtained by the come the resliaation that these desirable use of solvents that are immiscible with goals encourage the production of tissue media, such as hexane alone. mettda in which unreragnid side ef- lists the amwat of each pesticide Care. must be exercised not to pass fects, minor interferences, slight judgment on the efficiency of extraction amounts of contaminants, and any deflection may he found very uaodd. merely on the basis of high recoveries of lack of care in the use of equipment or Recovery data indicate that responses the chemical added during the analytical interpretation of responses can produce are linear when the peaticide is present procedure. Good recoveries of the greatly misleading and inaccurate re- above a definite minimum quantity. chemical added may be achieved when sults. The need for pmper cleanup of armple the degree of extraction of the actual Loveloek (IS), in a general discus- extract hefore inieetion ie m~h.si.ed. weathered residue is poor. sion of electron absorption detectors, and oonditiona 'and p ~ 10; u ~ Gas chromatography culumna cur- points out that with complex mixtures most ediective use are deecribed. The rently available are sensitive to small (such as are usually present in residue general level of eensitivity obtainable is amounts of certain impurities. A num- analysis) these detedors may give " . . . giveneaO.01 p.p.m. ber of papers emphasize the neceeaity of not only inaccmte hut even totslly Shuman and Collie (177) dencrib good cleanup before the extraeta can be false results!' Causes of varioua fah the preparation of a gna ehrnm&gmphy chromatographed to yield unambiguous mponses, both positive and negative, column; they alsD mphesirs the need results (23, 27, 44, 46, 69, 1.90, 1%)). are diarmssed and a puIse-ssmpling for proper conditioning. They remm- These columns must be carefully and technique which minimiwa the emrs is mend a &foot, &mm. i.d. column packed thoroughly "conditioned" (&, & 65, described. Barney, Stanley, and Cook with lvo Dnw Corning 200 (12,500 177) before they can be used routinely (I@, working with Systox, ,have shown osntistokes) silicon fluid on Andtmm to obtain good qualitative and quanti- that in a pwrly designed detector, ABS. Other workers (&,&Ihve .bo tative results. electron eapture and ionization may found this type of column superior for Barry and Hundley (17)have edited a take place at the same time and that pesticide work. "Pesticide Analytical Manual" com- pulse mode of detector operation will not De Fauhert-Maunder, E!gan, snd prising a compilation of methods and eliminate interferencesfrom ionisstion. Roburn (65)give details for prepuing a other information weiul to a residue Burke and Giutirida (44) point out columu. Good columos, columns &h analyst. Although developed as a guide the need for adequate cleanup before a decompose pesticides, comtruotion, for chemists in the laboratories of the aample extraat k injeoted into the elee- Linearity of respnnaen aod &&ling of Food and Drug Administration, it has tron capture gas chromatograph. They detectam. edi& of nte of inisotiop been distributed to many others as well. show that injection of poorly cleaned It presents information on sampling, extracts may contaminate a column extraction, cleanup, and determinative and result in weak or spurious responses. a gl& injection liner ia procedures; techniques for preparing Siee solventa used must he "pure," Beckman and Bevenue ($4) and conditioning columns; and lists of mlitillatiun is frequently requid. the e5ect of oolumn relative retention times and detectable The usr of plastic containera for aolventa on recovery of chlorinated hydm- quantities of many pesticide8 for dif- is diicuuraged, sinoe extractahlea in the carbons. Workii with &fa& by ferent chromatographic procedures and plastiw may cause response of the elm '/,-inch columns snd a micmmulomstric detectors. tron capture detector: In some eases detector, they checked mlnmos made Although gas chromatography with these spurious ediecta are so strong that of coppr, 8tain1~steal, aluminum, d various detecton is now the most mpomdue to pesticide residues may quarts. Copper tuhiigave the pooreat popular technique in residue analyak, he completely maeked. The need for recaveries, quarta the bed. Alumhum other procedures have not been proper preconditioning of the gas and stginless steel were sstisfwtory. neglected. Thin layer chmmatography, chmmatography column is slso thor- It has been apparent for mme time with its gwater speed of development ouahly discussed. Unless ..prouerly . con- that complete reliance on the retention and increased resolution and sensitivity, ditioned, the column mayca use deg- time for identifieatinn of a 00mpoyld has to some extent replaced paper radation of some pesticides. Not only may yield erroneaus &. Flobimn chromatography. Colorimetric, ultra- may the degradation result in lass of the and Riebardsbn (170) empkined the. violet, and infrared procedures also are pesticide hut also the degradation need for caution in interpreting the IS. used. products may cause mpnnse8 at the sulta of gss chrnmtqg.phy of pht retention times of some other common and animal extracts, both ss to htiW GAS CHROMATOGRAPHY-GENERAL pesticides for which they may he mis- and ouantitv. when onlv me mlumn is The grestest advances during this taken. Quipment and operating used.' -The; describsd four ditlemnt period have been made in gas chroma- parameters described nermit detection columns and tabulated the reanlutinn of tography. Because of the extreme bf chlorinated pestitid&, such as hepts pairs of pesticides on thea vnriuw sensitivity of the electron capture de- chlor ewxide, st levels of 0.01 to 0.001 columns. tector, it haa been used by many p.p.m.- Relntive retention ties are Gonldeo, Gondwin, aod hvies (88, workers. Dimick and Hartmann (64) listed for 65 pesticides. 89) were alao concerned about impmvins have publiihed a general description of Bonelli, Hartmann, and Dimick (56) the oertaiat~ a~tifi=tion.TIW electron capture gas chromatography as describe two columns wed with electron found that a column packed witb a used in pesticide analysis. They diiw capture gas chromatogrsphy. Pesti- 2.5% silicon oil and 0.25T0 EpiLotc 1001 the principle of electron capture and cidw which cannnt be resolved on one on CeJite gave good mlution. With che geometry and operating parameters column may he mlvd on the other. column temperature of 163O and gs of the detector. Although only one Operating parametars, sensitivity data, flow of 100 ml. nitmgen per minute, specific instrument is described and and retention time for a number of obteined complete sep.ntion of at some of the steps in the outlined pro- pesticides, including chlorinated, 11 pesticides in 30 minutea. They .bo cedure have since been improved, this organophosphom, and nrganosulfn~~, propwed the use of simult.nents* report doe provide a good introduction are given. hmatogaphy wing five pnlbl

VOL 37, NO. 5, AUB 1965 columns leading to one electron capture The results ohtained from the two grams of original sample was injected. detector. The stationary nhases of the chromatogram can be used to char- Construction and operating conditions columns differ so that 3 to-5 peaks may acterize the pesticide. of the detector are described, and re- be obtained for each pesticide. They Gutenmann and Lisk (97, 99) pre- tention time lists for 23 organo- called this a "spectrochromatogram" pared hrominated derivatives which had phosphate compounds are given. d stated that the pattern is char- strong electron capturing ability and Burchfield, Rhoades, and Wheeler cteristic of the specific pesticide. They chromatographed these as a means of (42) report the development of a micro- also described the use of a halogen- obtaining increased sensitivity. They coulometric detection system which 1s .-sensitive cell of the type used in de- worked with diphenyl, Guthion, MCP, specific for phosphorus. The effluent tecting refrigerator leaks. The response and MCPB as pure compounds, and from the usual gas chromatographic to individual chlorinated pesticides of also used this technique to determine column is passed through a quartz tube this cell differs from that of the electron residues of CIPC, monuron, diuron, and heated to 950' C., with hydrog,en as capture cell. By connecting this de- linuron in fruits and vegetables. Re the carrier gas. Organic compounds are tector in series with the electron capture wveries from crops of 76 to 116% were reduced to hydrocarbons, watere PHa, detector and recording responses from obtained at levels of 0.05 to 1.2 p.p.m. Ha, and HC1. The latter thre, comr both detectors, identification of in- when only one pesticide was present at pounds precipitate silver ion nd so register on a microcoulometric t tration dividual compounds is made more a time. 1. reliable. A word of caution may be in order cell. Insertion of a short si ma gel Programmed temperature gas about using this technique in any pro- column removes HC1; substitution of chromatography is ah coming into cedure which does not include extensive AllOI for silica gel removes both HCI use as a means of improving resolution cleanup of residue before gas chroma- and HS and permits measurement of and separation, speedidg up runs, and tography. Valuable information may PHI with absolute spec:ficity. Response chromatographing mixtures containing he obtained when only one pesticide is of the cell to PH*, Ha, and HC1 is in both very fast and very slowly elut- added and when the untreated crop is therstioof2 "2:l. When amodel C-100 microcoulometer at maximum sensi- inn- comwunds. Burke (45).. . used nro- available m that chromatogram of grammed temperature with a micro- sample and control can be compared. tivity is used, 0.1 pg. of P gives a peak coulometric detector: be tabulated However, if the technique is used on area of 5 square inohes. Cleaned-up relative retention times for 22 com- crops of unknown spray history with no extracts from crops examined do not pounds. Other workers have ahused control cro~available for comparison, interfere with the reduction or detection this technique (.20, 56,118). chro~natc,~rsm.iwould probahlycontain steps. The technique of preparation of a so many unihtified and unidentifiablt. peaks -that accurate interpretation CHLORINATED RESTICIDES- derivative of a pesticide before in- GENERAL PROCEDURES jection into the gas chromatograph has would be impoasihle. been continued. For some time, it has Bache, Lisk, and Laos (19) prepared More attention has been given to the been the practice to convert 2,4-D and nitro derivatives of MCP, MCPB, and development of multiple detection pro- other chlorinated phenoxy acid NAA in order to increase the response of cedures for the chlorinated pesticides herbicides to their methyl esters be- these herbicides on the electron capture than for any other class of pesticides. cause the free acids will not pass through nas chromatonraoh. They used this This is only natural, since these com- the common gas chromatographic col- technique to- determine * MCP, and pounds are widely used and many are so umns. Derivatives are now being used MCPB in timothy and ueas and NAA in persistent that traces of some com- for other reasons. Klein and Watts apples, and repoited fiding residues of wunds. such as DDT, are being found (1.20)found that Perthane, o,pf-DDT, MCP on snap bean plants treated with ;host 'everywhere. 'Moreover, these and p,p'-DDD have similar retention MCPB. comwunds have been found to be more times and are difficult to resolve on The above discussion haa been con- amenable to this type of analytical many gaa chromatographic oolum~; cerned primarily with gas chroma- method. however, olefins of these three com- tograph detectors for halogenated com- Mills, Onley, and Gaither (146)have pounds prepared by refluxing cleaned- pounds. One of the most exciting and combined and modified previously re up sample extracts with 2% NaOH promising developments of the past ported methods to provide a rapid, in ethanol were separated on a bfwt year was the appearance of two dis- simple procedure for extracting and gas chromatographic column of Celite similar detector systems, each of which cleaning up residues from nonfatty 545 with a 2.5% coating of SF-% and is rewrted to be hinhlv-- swcific. for foods. Used with gas chromatography, 2,2 - diethyl - 1,3 - propandioliso- phosphorus - containing compounds. thin layer chromat~graphy, or paper phthalate polyester (1:l). Klein and Giuffrida (86) modified a conventional chromatography, the procedure d Watts ohtained recoveries ranging from flame ion&a&on detector by fusing a determine 21 chlorinated pesticides. 84 to 105% from samples of leafy sodium sslt onto the electrode. The Good recoveries were ohtained of 5 vegetables containing residues added at result was a detector 600 times as pesticides added to 11 products at levels of 1 to 10 p.p.m. One striking responsive to a compound containing 10 level8 from 0.02 to 0.2 p.p.m. benefit from the use of the olefins is that carbons and 1 phosphorus atom as was Taylor, Rea, and Kkhy (186) ex- the Perthane olefin gives an electron the conventional flame detector. Re- tracted chlorinated ~eaticideresidues capture response about ten times grater sponse to compounds containing six f&&snimal tiiue by blending the tissue than Perthane. chlorine atoms w88 twenty times 8s with acetone and anhydrous h?%%h. Beckman and Berkenhotter (24)) great, while the response to compounds The pesticides wete -transferred to used derivatives to increase the reli- containing neither CI nor P was the same hexane and injected into a gas chroma- ability of identification of pesticide as that of the conventional flame tograph. Recoveries for lindane, endrin, residues. They separated the individual ionisation detector. When the ex- dieldrin, p,pl-DDE, and heptsohlor - -. traction ~dureof Mills, Onley. epoxide ranged from 75 to 99% at 2.5 wit{ a them& Gnductivity deteotor and ~aither(146) wae 4,diasinon; to 10 p.p.m. levels. and then dechlorinated the individual mnnel, psrsthion, ethion, and Trithion, Several pmcedmhave been repotted - fractions with sodium and liquid am- when added to bmcoli at levels of 0.05 for extracting chlorinated pesticide a- residues from water. Kah and Way- monk After thdt thev chroma- and 0.1 p.p.m., were wily detected. tographed the dechlorinatk portions There was no interference from crop man (115) describe a continuous ex- again and ohtained chromatogrsm~. materials even when the equivalent of 5 twtor wing duxing petroleum ether. The water sample was passed through the determinative step in which the Florid mlumn procedure and then de- the extractor at a rate of 0.5 to 1.0 samples were ext,racted&th acetone and termined by mi&ocoulometric gas chm- liter per hour. Nonpolar compounds the residues nartitioned into Skellv- matogra~hy. Fish oil sam~learecld were extracted by the petroleum ether, solve B, a portion of which was injech additIo&l treatment of thd 15% &te concentrated, and determined by elec- into the gas chromatoaraph without from the Florisil column to eliminate tron capture gas chromatography. ~additional&anup. ~hei;sed techni- interferenw. Saponification and MgO- Some of the intermediates in the nmnu- cal grade solvents without purification Celite column cleanup were ueed prior facture of aldrin and endrin were deter- andkported recoveries raning from 60 to injectioninto the gas chromatograph. mined at levels as low as 0.3 p.p.b. to 112% for 11 pesticides, with sensi- McCully and McKinley (186) used a by using a 135-liter sample. Infrared tivities from 0.04 to 0.001 p.p.m. (The freezing technique to separate chlori- spectra can be run on extracts after writers believe that it is well to caution nated pesticide residues from fata and cleanup on alumina columns. readers once more against using such oh. The fat or oil was dissolved in a Breidenbach d al. (38) describe abbreviated procedw unless the spray bengene-acetone mixture (I + 19) and equipment and procedures for collecting history of the crop is known and un- the fat precipitated by d i to large volume samples of water by carbon treated samples of the same product are -70° C. The solution was filtered adsorption as well as analysis of discrete available for comparison determina- through a charooal-wwd Eellulose ool- bottled samples of water. The prooe- tion.) umn and concentrated for injection into dure used hy the Public Health Service- The determination of chlorinated the electron capture gas chromstograph. Water Pollution Surveillance System for pesticide residues in fatty foods hss A special apparatus for we in thia pro- analysis of carbon-chloroform extract presented a special problem, since the cedure was dwribed in a smnd pub. by thin layer chromatography, elec- pesticides are fat- .and oil-ealuble and lication (196). Working with organo- tron capture, and micnxoulometric gas sepmtion is difficult. The analysis of phosphate compounds, hby and chromatography, and infrared is rn milk pdnts an added challenge in Lsws (67) reported that freeaing out of ported, but no data on the efficiency of that, for many pdures, the fat must waxlike sutmtances from scetone solu- such a system are presented. first be separated from the milk. tion removed some imppities but also Schwarts el ol. (176) have used elec- Onley (161) has reported a rapid removed pesticides. tron capture gas chromatography for method for milk which combines the Ott and Gunther (163) used forced determining "Polystream," a mixture of usual two step into one. Instead of volatiliration to separate pesticide chlorinated benzenes, in c1a.m~ and 6mt separstii the fat from the milk residues from butter fat by use of a oysters. and then extracting the pesticide from newly designed device. The fat wss Minyard and Jackson (147) analyzed the fat, the milk is blended with a heated to about 1W0 C. and volatiles 101 samples of commercial animal feeds, mixture of acetonitrile, ethyl ether, were carried 'to a aled trap by s using electron capture gas chroma- dioxme, and acetone (3:l:l:l) and stream of nitrugen. Determination wss tography. They extracted the samples anhydrous dumsulfate. After filter- made by microcoulometric g~ chronm with an isopropanolSkellysolve B mixing, water is added and the residues are tography; the entire analysis reqnired ture (1 + 3) and used a Florisil column tmferred to petroleum ether. From about one hour. Sensitivity was re- cleanup. They state that they were this point, a modilication of the Mills ported at about 0.5 p.p.m. for some of able to detect less than 1p.p.b. of most procedure is followed. By electron the more common chlorinated peaticidea. of the chlorinated pesticides. capture gas chmmatogmphy, sat* However, DDT broke down to form Baets (14) reported using No~it-A factory recoveries were obtained for 19 some DDEsnd DDD. for cleaning up sample extracts. The pesticides at levels ranging from 0.005 De Faubert-Maunder, Egsn, snd sample was extracted by blending with toO.1 p.p.m. Rohurn (6t) compared dimethylform- acetonitrile or mixed solvents and the Henderson (108) reported a cohbo- amide and dimethvlarlfoxide for ex- residuea were partitioned into petroleum rative atudy involving two samples of traoting residues f& h-e solutions ether. An aliquot was evaporated to milk and 22 laboratories. Advantages of fsts, and reported that dimethyl- drynm, taken up in benzene, ahaken and disadvantages of various methods formamide gave better recovariee. with NoritrA, and filtered. The filtrate were discussed, snd results by paper Thev dacribed ~dwforSnavrinn was reported to be suitable for injection chromatography and microcoulometric ssm&~ of fat,'mik, butter, andese&T into the microcoulometric gas chroma- and electron capture gas chroma- Mo& (161) used a column containing tograph but it could not be used for togmphy were compsred. 100 gram8 of steadsrdLed Fbrisil 40 electron capture gas chromatography Several me- have been reported clean up as much as 2 gmma of fat. because of excessive interference. The for separating fat from dairy pmducts F'eaticidea were eluted from the column method is reported to have given satis prior to pesticide analysis. Langlois, with %methylene ohbride in peh- factory recoveries of 11 chlorinsted Stemp, and Liska (131) used the con- leum ether in a form adequate for pesticides from carrots, collards, okra, ventional Babcock test procedure and spotting the entire sample for psper and peas at 0.1 to 1.0 p.p.m. levels. reported that although endrin was ap- chromatography. Recoveries of lindane and BHC, how- parently destroyed, DDT, DDE, Langbis, Stemp, and Lisks (1W) ever, were as low as 53%. lindane, heptsehlor, heptsehlor epoxide, extraoted pesticide reaidw from dairy Moats (162) reported a onestep and dieldrin weie recovered satis pmducta by grinding the -plea with cleanup prncedure using a column of factarily. Lampert (1%) used a de- Florid that was psrtidly'de~tiMted Nuchar C lmlite 545 (1 + 2). tergent solution and a Babcook cream by the addition of 5% water to the With paper chromatography as the test bottle for separat'hg the fat from sdsorbent. The mixture was added to determinative step, sensitivity was milk. the top of a Florisil column end the believed to be about 0.03 to 0.1 p.p.m. A number of di5erent ways have been pesticides were eluted witb WOmethyl- McKinley, Coffin, and McCully.ilS7) s# for separation or cleanup of ene chloride in petrolemu ether. The have reviewed cleanup pmcedw for pesticide residues fmm fata and oils. elute wan evaporated and the ddw both chlorinated and organophosphate Eidelman (67)used dimethylsulfoxide taken up in he for injection into the pesticide residues. They point out the to extract the reeiduea from acetone and electron captnm gm chmmaw. advantages and limitations of the petroleum ether solutions of fat. After The pdure tooL 30 to 90 minutea various methods, and list 41 references. water was added and the residues were and recoveries were reportad M being Gutenmann and Lisk (9.9) have used partitioned into petroleum ether, they coneistently better than 90%. The electron capture gas chromatography ss were further cleaned up by the Mills sensitivity was stated to be 0.05 p.p.m.

VOL 37, NO. 5, AMl I 965 1- 1 for DDT and endrin and 0.1 ppm. ges chromatography. They also report reaction is sensitive to about 10 ME.- for some of the other chlorinated the use of thin layer chromatography on aldrin. pesticides. The same procedure was uncleaned plant extracts as a rapid Bache (11) used thin laver chroma- also adapted for analyzing egg yolk and screening method for DDT and DDE in tography ' & determine 'amiben in ultry tissue (181, 182). The fact plants. tomatoes. The samole was treated with at in this procedure different volumes Paper chromatography continues to sodium hydroxide; the hydrolyzed f eluting solvent are used for different receive attention. Mitchell (148) re- amiben was extracted and, after transfer @ pesticides presents a difficulty. For ports the minimum detectable quan- to acetone, spotted on silica gel plates. example, to remove endrin, 650 ml. of tities of 22 chlorinated pesticides, using Bache, Gutenmann, and Lisk (12) used eluting solvent and 90 minutm of the ~~~~~-phenox~e

solvent systems for each of 62 pesticides, With gas chromatography as the ex- rewrted at levels of 0.04 to 0.12 . p.p.m. ~ including chlorinated compounds, or- amining medium, dieldrin so exposed on a whole milk basis. ganophosphates, and carbamates, and showed one derivative; endrin showed Blinn and Gunther (81) present the they discuss chromogenic sprays, choice one main product and several minor results of a collahontive study of two of solvent system, and the use of thin ones; aldrin showed dieldrin and a small versions of a colorimetric procedure for layer chromatography a a cleanup pro- amount of another derivative; p,p'- DDT in milk and butter fat. They pre cedure. DDE, three main ~roductsand several fer the version which includes an oxida- Kawashiro and Hosogai (114) have minor ones; p,pl-TDE, a small amount tion step in the cleanup. reported a new spray reagent for de- of a dehydrochlorinated product; and Klein, Watts, and Damico (121) tecting chlorinated pesticides on silica p,pl-DDT, a small amount of DDE. used the conversion of DDT to DDE gel thin layer chromatography plates. The a,8, 7, and 8 isomers of BHC did as confirmation of identity in the The plates are sprayed with 0.5% not show any reaction products. analysis of butter and oils for DDT. o-tolidine or o-dianisidine and then ir- They used the Mills procedure for ex- radiated with ultraviolet at 2536 A. traction and cleanup and determined The pesticides appear as green spots DDT by electron capture gas chroma- CHLORINATED PESTICIDES-- tography. A second aliquot was con- against a white background. Smounts SPECIFIC PROCEDURES of 0.5 to 1pg. are detectable for many verted to DDE by treatment with of the pesticides. This reagent does not sodium hydroxide, end again chroma- appear to be as sensitive as the con- Friestad (77) reports a spctro- tographed. ventional AgNOs. photometric method for aldrin which re- Hardin and Sarten (101) compared Morley and Chiha (153) have used quires prior cleanup of the sample. The five nrocedures for extracting DDT thin layer chromatography as a cleanup aldrin is reacted with nitrosyl chloride, from field-treated collards. hey found procedure for gas chromatography. then heated in acid to form dihydro- that blending first with isopro~ylal- Samples were spotted and developed on chlorketo aldrin. This compound reacts cohol and then with hexane &ve better each half of a plate. One half of the in alcoholic solution with mdinitro- recoveries than did tumbling or grinding plate then was covered with aluminum benzene and potassium hydroxide to with hexane. foil and the other half sprayed and ex- give a red-violet color, which is ex- Espadas and Loaeza (70) used aniline posed to ultraviolet light to locate the tracted with chloroform and its ab- in place of alcoholic sodium methylate spots. Similar areas on the covered half sorbance read at 525 In*. Dieldrin and or alcoholic potassium hydroxide in the then were scraped off and extracted for endrin inkrfwe with the mmy. This color development after nitration of

134 R . ANALYWAL CHEMISTRY DDT. They state that a more stable column and injected into the gas chm esterify any free 2,4-D. The methcd color was obtained and the method was matograph. The sensitivity of the was sensitive to 0.01 p.p.m. simplified. method is reported as 0.01 p.p.m. Both paper and thin layer chroms- Beckman and Bevenue (88) deter- Recoveries of 90 to 100% were obtained W~bv were used bv Abbott d d. mined Dilsn residues in peam by using at levels of 0.05 to 1.0 p.pm. (1jtd ditermine MCPA, MCPB, 2,4-D, microcoulometric gas chromatography Burke and Mills (46)used a modifies- 2,4DB, and 2,4,5-T in soil and water after cleanup with Nuchar. DDT, if tion of the Mills procedure to determine The thin layer procedure present, was removed by elution from a Thiodan and Tedion waidues. DDT dinweb and DNOC fmm the above Florisil column. was removed by elution from the herbicides and detected their pm&. Albert (6) has suggested a mdflca- Florid columns with 6% ethyl ether in Other chromatographic procedure for tion which saves about two hours in the petroleum ether. Thiodan and Tedion determining some of these herbicides in determination of endrin by the Mills then were eluted with 30% ethyl ether in various crop are described by Bewue, procedure. He reports that the 15% petroleum ether. Determiuation was Zweig, and Nash (66)for 2,4D in dry eluate can cleaned up and fat elimi- made by micmulometric gas chroms- crop and wahuta; by Yip (198) for nated by passing the eluate through a tography. Extrsda of broccoli required 2,4D in wheat; by Gutenmann and potsssium hydroxide-Celite column. additional cleanup on a column of Lkk (94) for 2,4-D and 2,4DB in This replaces the lengthy saponification sodium sulfate, attapulgua clay, Celite forage; by (1CO) for MCP in mil; and

step. By electron capture gas chroma- 545, and Nuchar 190 N. Dieldrin aud by. (98)~ forailvex in water. tography, the recoveries from cam&, endrin, if present, gave overl?pping ~aoudand Luh (69) deernib a onions, collards, and broccoli at the with the column used m the colorimetric method for determining 0.05 p.p.m. level ranged from 100 to work. 2,4,5-T in cauhed apricots at a level 2 106%. EDITOR'SNWE. This pw A colorimetric method for the deter- about 1 p.p.m. After cleanup on cedure may work equally well for mination of Thiodau in vegetables and activated Wc aluminum oxide, the dieldrin. beef fati8 dmcribed by Maitlen, Walker, extracted midue waa reacted with Gordon, Haines, and Martin (87) and Weatlske (140. Vegetables were chromdropic acid and sulfuric acid. determined Kelthane in milk colori- extraated by tumbling with an n- The resulting color. was read at 565 metrically with a sensitivity of 0.01 heme-isop&pnnol mixture (2 + 1). mr The authors atate that about 40% p.p.m. based on whole milk or about Sugar beet extracte required cleanup by of the 2,4,6T was converted to a ann- 0.25 D.D.m. on the fat basis. Thev ex- shaking with a charcoal-maguesium bind fo¬ extrsct.ble with ether. tracLd the fat from the milkUand oxide mixture. Beef fat was ground CosLley, Campbell, and McFarren hydrolyzed the Kelthane with tetnr- with anhydmus sodium ~ulfateand ex- (68) ud 8 aomeahai aimikr color m methylammonium hydroxide. The re- tracted with n-pentme. Cleanup in- action to determine 2,PD and its leased chloroform was determined by cluded acetonitrile partitioning and the butoxyethanol ester in fish and ahd&h the Fujiwara reaction. The procedure use of a Florisil column. The actual at a eeneitivity reported to be 0.1 eliminated the need for a speeial steam determination nas carried out in a p.p.m. The blended amp lea were system. single ti& tube; an. aliquot was evap treated with sodium hydroxide to Ackermauu. Carbone. and Kuchar orated, methannlic sodium hvdroxide- hydrolyze the ester to 2,4-D wbich, (4) modified 'an earlier' spectrophote pyrid&e magent was added; and the after aeidi6cation, was extracted with metric method for the determination of developed color was read at 520 20. benzene. A FloM column was uaed pentachloronitrobe11~enein soil and Of 4.5 pesticides teeted, only captan, for clamp, after which the cobr was crop. They used a direct extraction chlordane, heptachlor, and ovex caused developed with ohrornotmpic acid and from food products with ethanol and any interference. A somewhat Bimikr read at 570 mr. Chchroma~y partition into petroleum ether to elimi- pmcsdure had been used earlier by was used to verify the identity of the nate pigments. The method was aid Butler, Maitlen, and Fahey (47) to 2,4D. to be sensitive to about 3rrg. determine Thiodm in atnwbemes and Kirkland and Pew (118) used tem- The A.O.A.C. method for sulfone was alfalfa. peratwe-progrd micmeoulometzic studied by Shuman (176). He found A madification of the colorimetric gas chromatography for the detelmina- that hexane as the stripping solvent method for toxaphene has been reported tion of the herbicides Trysben (2,8,6 gave better recovery from fieldsprayed by Nikolov and Donev (160). They triehlorobemic acid) and %bar @dy- peaches, whereas benzene worked better stste that the sensitivity of the deter- chlorinated, beoroic ad). Tha for apples. He checked the remahder of mination was incdabout tenfold by herbicidm were extracted fmm samplm the fruit by blending with an iso- preliminary treatment with nitric acid of sorghum, wheat, harley, pineapple, propanol-benzene mixture and found before devdopment of the color. and 6ng.u cane by blending with methyl that the stripping had removed over ethyl ketone. The extncted mid- 90% of the midues. were clesned up and converted to the Two gas chromatographic columns methyl aeters before they mre hum- are described bv Carev (48) for use in A number of me have appeared tographed. Setisfactory anslysee rere the determiasti& of 2,3,5,6tetrachloro- for the determination of the chloro- &ed out at 0.04 p.p.m. nitrosnieole in vegetables and grains by phenoxy acscids and their eaters in food Getsendaner (84)-reports a me* electron capture gas-chromatography. products. The free acids are generally for the determination of dalspon m By using a cleanup column of aetivated converted to their methyl esters for nanberries, baaanss, and corn cobs. niguesia and diatomaceous earth, as determination by p chromatogrsphy. The detarmiuation was made by eke- little as 0.02 p.p.m. can be detected This k necessary buaethe free acids tmn capture @In chromatograpqy uain( without interference from croos. will not through the common sili- a column consisting of 3.85% diethyh Beck- and Bevenue (h)describe con oil g& chromat&aphic column. glycol sdipate polyester and phosphoric a method for the determination of tetra- Bevenue, Zweig, and Nash (m de- said on Qss Chmm 8 or Chmmamrb chlorothiophene and 1,2dibronur scribe a method for determining 2,4-D W.A.W. These columns permitted the 3-chloropropane in brueeels s~routaand and ita estern in wtstocS, using micro- nucceaaful ahromstognphii of dsbpon walnut me&, using electron capture gas coulometric gia chroma&aPhy. as the free acid so long as the acidity of chromatography. The sample is Cleanup on a Florid column nas found the column (KPO4) was rhsint.ined. blended with petroleum ether, and the nemwm to remow unidentified inter- Recoverim at levels of 0.26 to 5 p.p.m. extract is p& through a Florisil ferences; and diammethani was used to ranged from 80 to 100%. ORGANOPHOSPHATES- concentrated, and injected into the gas many of the compounds were not re- GENERAL PROCEDURES chromatograph. Two columns are covered. The development of multiple de- described, and relative retention times Zadroaiusks (t@ determined para- tection schemes for the organophosphate and sensitivities for 19 organophosphate thion. methvl oarathion. malathion, and ticides has proved more difficult than compounds are reported. diasiLon i"n ' strawlbekies, cabbage, r the chlorinated. Extraction and Anyone usina conventional electron soinach. eto.. at levels of 0.5 to 2 p.D.m. eanup procedures, to be useful, must capture gas chromatography for deter- dy a psper ihromatographic ~rocidure. rnbe capable of handling compounds of mining organophosphate residues should After hromination, fldorescein was used widely differing polarities, since the remember that thielectron capture de- to detect the spots on the chmma- parent pesticides and their metabolites tector is much more responsive to tograms. range from oil-soluble to water-soluble. halogenated compounds. Chlorinated Three color reagents (metanil yellow, Coffin and Savary (54) report a pro- pesticide residues, if present even in yellow RFS, and methyl orange) for eedure which includes acetonitrile ex- trace amounts. can be mistaken for detectiug thiophosphsltes on paper chro- traction, elution from polyethylene- significant amounts of organophosphates matogrh are reported by Dutt and coated bnina with slightli acidified with similar retention times. Seow (65). Metanil yellow was found 40% acetonitrile. partitioning into Gutenmanu and Lisk (96) took ad- to be the beat of the three when tested chloroform, and fina elution -of the vantaae of thk hi& electron capture on parathion, malathion, diaainon, and organophosphates from Magnesol with repoise tb halcge&ted compoun& in a dimethoate. The limit of detection was successive portions of chloroform, ace- method they used to determine ethion lto2rg. tone, and methanol. The pesticides and malathion in solutions. They re Get5 and Friedman (83) studied were separated by paper chroma- ported that organophosphates which cholinesterase inhibition methods of tography and the spots were located by contained methoxy or ethoxy groups detectiug organophosphates on paper one of several means. The portions of reacted with HI (Zeisel alkoxyl reac- chromatograms. They developed two the paper chromatogram containing the tion) to form methyl and ethyl iodides. procedures. In one, a direct method, spots were cut out and phosphorus was These were injected into the gas chroma- the developed chromatomam itself was determined after digestion in a Sch& tograph. It is pointed out that alcohols, sprayed first with e&yme-indicator niger flask. Recoveries of 80 to 107% ethers, and esters must be removed, solution and then with the substrate. were obtained for 41 organophosphate since they also undergo the Zeisel re In the other, or indirect, procedure, standards and for 25 organophosphates action. This procedure, of course, does after a second sheet of paper had been added to lettuce at levels of 0.4 to 1.5 not identify the wmpound being meas- sprayed with the enzyme indicator solu- p.p.m. Eight compounds tested were ured other than as one containing a tion, it was placed in firm contact with not determined by this procedure. methoxy or ethoxy group. the developed chromatogram and in- As mentioned earlier, a big step for- Croshy and Laws (57) used gas cuhated for 15 minutes. Then the ward in the methodology for organo- chromatography as an additional second sheet was treated with substrate phosphate residues has been the de- cleanup step in preparing extracts for and the spots were developed on it. velopment of gas chromatographic de infrared determination. The entire McKinlev and Joh8l (1%) described ctors highly specific and sensitive for effluent from the gas chromatograph the use of-liver es,@me inhibition for hosphorus-containing compounds (&, was caught by passing it through detection of ornanoohosphate spots on 8186). It is anticipated that as they b methylene chloride. After evaporation paper chromat&ra&. he substrate come more readily available them de- of the methylene chloride, the residue was 1-naphthy! aeetste and the color teetors will find widespread use in the was dissolved in carbon &sulfide and the reagent was amne fast hlue RR. develooment of comdete oroeedures infrared spectrum was determined over About 30 organophosphate pesticides that will be adequate fbr the hetermina- the 5- to 15-micron ranee. us in^ a cavitv and metabolites, as well as carharyl, tionof both the parent pesticide chemi- cell with beam eonden& andscale ex- were studied. Most of the compouoda cal and the significant metabolic pander. Good spectra were obtained were detectable at levels between 0.01 products. Meanwhile earlier detecton with as little as 1 rg. residue. Re- and 0.50 fig.; some required as much as are being used. Nelson (167) msde use coveries of 50 to 80% were obtained 3a. of microcoulometric gas chroma- from fruit and vegetables at 0.2 to 4.0 Thin layer chromalography has also to~~rauhvwith the sulfur cell for deter- p.p.m. levels, and 40 to 50% at the been used in the determination of mFniig "lo thiophosphate pesticides in lower limit of 0.1 p.p.m. Data are re- organophosphates. Uchiyama and Okui fruits and veeetables- at levels r& ported for 15 organophosphate com- (190) list R, values for 14 compounds from 0.15 to 1.5 p.p.m. Samples we; pounds. chromatographed on silica gel plates extracted hv hlendine with aceto- Frehse (74) has written a most ex- using a heme-acetone (4 + I) mixture nitrile, and the residues were partitioned tensive review on the use of infrared in as developing solvent. Bunyan (41) into wtroleum ether. No additional wticide residue analysis. Although it adapted both the bmmophenol hlue- cleanup was used. It was pointed out covers all clssses of wticides, the silver nitrate reagent for thiophosphates that the study was made only on parent greatest emphasis is on organdphos- and the cholinesterase inhibition method wmpounds. Many of these formed ohate.. Amonn the subjects discussed for use on thin layer chromatography. toxic metabolites which contained no are extraction ind cleaiup procedures The bromophenol hhe-eilver nitrate nulfur or were water-soluhle and so (where it is pointed out that thorough reagent was found to be more sensitive would not be detected by this procedukl cleanup is indispensable), cells, solvents, on silica gel plates (<0.1 to 0.6 rg.) &an. Hammond. and Thornson (66). analysis of solid substances, special than on alumina (about 0.5 rg.). The v , . ,, using electron capture gas chroma- equipment, and the infrared char- cholineaterase inhibition technique tography, obtained reeoveriea of 73 to acteristics of organophosphate pati- would not work directly on the thin 91% for a number of the parent organo- cidei. layer plates and required the use of phoaphatea from lettuce, oniona, apples, MacRse and McKinley (159) used a sprayed paper placed in contact with etc. The eamples were blended with an Soka-Floe and activated charooal col- the plate, similar to the method de- thy1 methyl ketone-heme mixture umn to clean up midues prior to paper acribed above (83). Again, silica gel a3 + 2) and the extract was washed with chromatography. Two systems are plates worked better than alumina. sodium sulfate solution, passed through described which can be used to identify Several procedures based upon the anhydrous sodium sulfate and then 13 parent organophosphates. How- molybdenum hlue method for deter- through an alumina or magneaia column, ever, when added to crop extracts, mination of phosphorus after extraction, cleanup, and oxidation to inorganic hydrogen sulfide, which was swept into tography to determine Fenitm- phosphate have been reported. Blimn a receiver containing dnc acetate and thion ldimethyl(5methyl-Pnitmphenyl (88)used a Sch6niger combustion flask reacted with Npdimethyl-pplienyl- phospliorothiobate)l, pirathion, -Chloi- to determine dimethoate in a number of enedismine hydrochloride to form thion, and paraoxon in cocor beam. agricultural products, with a dtivity methylene blue, the absorbance of Cox (66, 66) studied the mbrimetrie. of 0.1 p.p.m. Brewerton (39) used which wasread at 670 mu. procedure for Guthion in whiah perchloric acid digestion. Isseva and Giang and Schecbter~(86)determined pesticide is hydmlyd to anthranili a Enoshevskaya (109) used a mixture of dimethoate m milk and various cmps by acid, diswtid, and coupled N-(1-m~hthv1)ethvlenedismine nitric and sulfuric acids and potassium B colorimetric pmdure, which me& with .&. permsfga~~teas the digeting and ured both the parent compound and its dihydmehloride. He rep& a cobb oxidizmg agent in determining a number oxygen dog. The compounds were mtive skrdy in which Guthion wss of the organophosphates. hydmlywd with akdi to thioglycolic added to various fruits and vegeteblm These last three procedures, of corn, acid, which was mxtd with dium at lev& of 0.3 to 1.56 p.pm. Re- do not identify the pesticide but simply phosphd&tuneptete, and the ab- coveriee rsnged from 58 to 187%. measure the total phosphorus. How- sorbence we8 measured at 720 w. Miles (144) deamibw a new and npid ever, when comhii with paper or Em and Frepr (88) used paper colorimetric metbod 601 Guthion, Ethyl thin layer chmmstogmphy, the method chmmstogmphy to detumine dimeth- Guthion, and their oxygen It become more specific. The cleaned-up oate in milk. The dimethoata was isbssedonthediremtcouplineoftbe sample extracts may be chmma- extracted fmm milk with an ethyl pesticide with N-(1-naphthyl)ethylena tographed on paper or thin layer and ether-hexane mixture. After transfer to dismine dihydmchbrids in the p- the identity of the residue determined hexme, the extract was cleaned UP on a of acetic and hydmhb~acids to pm- by the R, bf the spot. The spots then n0&icolumn and then Bpottedfor dma purple eolution with abaorptinn may be cut out from the paDer chmma- pauer chromatognu,hy.--- After develop- mpdmumat568mr. Tbssamplawm t&am or soraped off the thin layer m&t the paper ass sprayed with extrscted by blending or tumbling with plate and total phosphorus determined 2,GdihmmpN-abmp-quinonde. chlomf0rmand~~uprith.n toobtain a quantitative value. Ruelene Dimkthoata showed up ss a red spot. Attaclqy-&elite mirtrue. The orygen in milk (1%) and phosphsmidon in Won, Guthion, Systox, Trithion, analogs wm epamtd from the prtmt vegetables and fruits (8)have been de- snd malsthion did not mot. compouadn on a Bbriril column. Re- termined in such a msnner by paper Cem (@) reporta a colorimetric coverim fmm fruits and vellet.bka chromatomauhv. .Thin laver ehmmk method, bred on the Fujiwara reac- ranged from 78 ta S7%. tography'LA bken used ai&larlY in the tion, for the dekmimtion of Diptern Ftehae, Niessen, and Ti& (76, 78) deternunation of dimethoate (180). (trichlodon) in foods. report an infnrebmetlmd for fenthion A rather novel approach' for aemi- Mibui sC d. (I@) deacribe a colori- (Lebsycid) in beet laavea, httma, csb ouantitative determination of 0~0-metric method for DDVF' based on an hagq apples, and ch* as rdl ss bhcmphateg was taken by B&X, orangsrad complax tbst is formed be- olives snd olive oil. After &m&m Doml, aud Tho- (40). It is baaed twean DDVF' and acetone in the and cbup, the duems oxitlined upon the fact that etersses fmm varioua plsll~~caof dcoholia potaaaium hy- with ptueium pm!mgamta. bovine organs sepamte into five to seven dmxide. Absorption at 370 w follows donebsnd at 7.55 mtcmaswss4f zones when extiacts of the or- are the Beer-Lambed law. They report quantitation and the speEtrum from 7 submitted to agar-gel electrophoresis on that the metbod is ahapplicable to to 11 micronsfor~EPtion.Amim* micrmeope slides. Total or partial Dipterex and Dibmm, and the pm plumphorn detmmbation may .bo be disappearance of one or more of the dm@for Dibmm isdescribed (1.50)). run on the oleaned-op naldne. zone occurred when organophosphates Bun and Johnson (188) have de- Bstes and Rnwlspds (18. 10) have were added to the extracts prior to veloped.~By biopsay procedure which electrophoresis. Inhibition pattern for can determine as little as 0.1 p.p.m. 14 compounds and procedures for the DDVP in the pleeence of many other analysis of samples of unknown Wry ilwecticides. are described. With kidney extracts, Archer d d. (9) report a nompecific the sensitivity is reported as 0.05 p.p.m. cholinestaaae inhibition procedure for for mme of the pesticides. the detanninstim of ethion in olives. Theyusedpemeth acid tomidire the ORCANOPHOSPHAlES- ethion huee the olehic compounds SIEClflC PROCEDURES present in olivw interfered with the Van Middelem, Waites, and Win usual bmmine treatment. The mew, (191) used both electron capture gas with modified bup,worked well on chromatography and the dinitrochlom a numhw of fruibend vegetsblen. benzene colorimetric method to deter- Grabsm and Orwall (BO) describe a Thin they believe to be due to the fo* mine dimethoate in snap beans and pmaQve in which the ethion in hy- tion of free fatty soid inthe bran dnrinr: found reaults by the two procedures in dmlyd with ethandic sodium hy- stow. Rn~Bnds(17s) &minstd good agreement. Only the parent com- dmxide and the diethyl phosphom interference in the determinbh of pound was actually measured, since the dithioic soid formed in determined md.tbion in pimento by usin# pob- oxygen analog was not recovered by the speetmphotometsidy a. its complex ethy-ted d& 4 .aid- prooedurea. copper sdt abeorbing at 418 w. To dedaluminsch.aupodumm. A modification of the colorimetric make the prooedure specific for ethion, Fisober and UBlidh (7s) Ie.prt method for dhninon wae used by Enos Delnav is eliminated by a merauric infrsred method for the determip.tion and Frear (69)to determine dimethoate chloride treatment and other phosphate of malathion in kohlrabi, lettuco, and in fruits and forage. The eample was @aides sre eliminated by a dilute cauiieower. The clbl&lp exbab extracted with a mlvent lvambg with eodium hydmude wd. The method rere blved in carbon disulMs nature of sample) and, after ciiup by is rewrted ss a~~limbleto a number of the sbmrption hbbdat 9.82 mkmm mlvent extraction, the dimethoata was fn& and v&bles. used to determine the mdntbion. s extracted from heme with hyb Damn. Do-. and Thsin ((11) Conaidemhle work h. bem duw on bromic acid. Acid hydrolyaie pmducod used &mn Gptire gas chroms- the detmmbtion of parathion and

VOL 37, NO. 5, NBl 1965 1t7R related compounds. Van Middelem, hydrobred to release formaldehyde (140) describes a m& method for Waites, and Wilson (199) studied vari- which is thed rescted with chrome Andel in meat in which the mrmel is also ous extraction and cleanup procedures tropic acid. Resnent an* cron hlsnks hvdrolvsed and the trichloronhenol for parathion in leafy vegetables. They are-required, ind beauthora point out s&&'lled, but, ip place of Giga found blending with a mixture of kc- weaihle interference from formaldehyde colorimetric determination, the tri- ropy1 alcohol and benzene to be the h the air or fmm phosgene in ihe chlomphenol is determined directly by referred method of extraction. chloroform. ultraviolet spectroscopy at 315 mN. eStraight tumbling with benzene gave To determine phorate, Blinn (99) Thii method, however, is not applicable very low recoveries. They also describe d thin layer chromatograph with to samples containing less thin 1 a chmmatographic cleanup column infrared or the colorimetric cXmm* p.p.m. which is superior to shaking the raw tropic acid method. The residue was Adarns, Anderson, and MeDougall extract with a decoloriringmixture. oxidised by peracetic acid to the oxygen (6) report a paper chromatographic George (89) reports a micro method analog sdfone. By using thin layer method for determining Systox (deme- for the determination of parathion and chromatom~hvthe residue was sem- ton) and its toxic metabolites. An such similar compounds as methyl rated fm& &ential interfering &ti- ethanol solution of the extracts is Darathion. binsnacrvl. EPN. and cides and wsitivelv identified. A cleaned UD on a column of acid-washed huthion. ' This iethod'is based'on the palladium ckoride c-hmogenic &put alumina and then is chromatographed Averell-Norria colorimetric nmedure did not interfere with the colorimetric on siliconetreated naper. The pawr is but is said to.& ten timea more sensi- or infrared determination of the eluted sprayed with pota&&m perm&&nate tive. In the determination, Guthion swts. Excellent infrared spectra were and treated with potagsium hydroxide. was firat hydmlyaed to break the nitm- obtgined with as little as 7 &. by using Systox and its most importent metah- gen ring. Karatbane and Chlorthion ultramicmwt99sium bromide pellets olites form 0,Odiethylphosphom- interfere in the method but unby- and beem-condenser. Blinn (90j later thioic acid which is then detected by drolysed Guthion gives only a slight compared the ability of 12 oxidants to spraying with 2,6dibmmo-N-ohlorc-p color. convert phorate to its oxygen analog quinoneimine. This procedure is said to Coffi and McKinley (65) report both sulfone. He report6 that m-chlomper- have a sensitivity of 0.3 p.p.m. and to a colorimetric and a paper chroma- bensoic acid worked best. He also sug- distinguish residues of Systox and its tographic method for parathion, methyl gests the use of silica gel thin layer metabolites in the presence of other parathion, EPN, and their oxohs. To plates bn!Tered at pH 6 to prevent orarno~hos~hate- nestiiides. determine the total pnitrophenol, the hydrolytic decomposition of the organo- ~ro&nkL (I&) ddaacribes a method cleaned-UD extract is treated with phosphateesters. for detecting Svstox in air. The method hydrogen' peroxide and potassium hy- Although Archer el al. (10) ahused is based on chcability nf the thiol isomer droxide and the ~nitmnhenate is veracetic acid to oxidire nhorate, the to extinguish the Bumcence of -in. measured colorimet~callyat 400 w. bhorate was determined sf& oxidation Geldmacher-MaUnckrodt and Weigel To determine individual comwunds, the hv cholinestelgse inhibition. Potatoes (81) studied the ration of the hy- cleaned-up extracts are cbrdma- w&e snlyred by extracting with chlom drolysis products of $ystox and Meta- graphed on paper, the developed form and anhydrous sodium muate Systox with heavy metals. They sug- hmmatogram is treated with bmmine without further cleanup. Sugar beet gest the we of copper and cobalt solu- eand potassium hydroxide, and the in- leaf extracts were cleaned up on a tions as spray resgends after separation dividual spots of pnitrophenate are sodium carbonatecelite 545-charcoal of the compounds by thin layer chroma- eluted and read at 4M)u.Recoveries column, and cottonseed extracts on a tography. of !34 to 101% are reported from lettuce, Florisil column. A spectrophotofluarometrie method strawberries, and apples at levels from Cholinestelgse inhibition was used by for Zmophos and its oxygen analog 0.4 to 1.3 p.p.m. Aa little as 1 fig. of Blumen (JJ) to determine Phoedrin in is described by Kiigemagi and Terriere each compound was readily detected on fruits and v&tahles. A modification of (116). After extraction and cleanup, the the paper chromatogram, and ammatic the Dmcedure in which unhvdrolvsed. . residue is hydmlysed and washed with aminea did not interfere. acet;lcholine is mnverted to hy- strong alkali. It is then activated at Kubitova (In) described a method dronamic acid and rescted with ferric 315 m* and the Buoaescence measured for parathion and pnitrophenol in chloride to form a red complex was at 375 w. The method was tried on a animal tissue. The eamnle wea blended studied collaboratively. Recoveries number of fruits and vegetables and is with acetone, the residue tderred to fmm apples,. cabbage, and tomatoes said to have a sensitivity of 0.05 p.p.m. chloroform, and pnitrophenol extracted ranged from 70 to 117% at levels of with a sodium carbonate solution. In 0.164and 0.328 p.p.m. the determination of total parathion and Claborn and Ivey (61) report a pnitmphenol, a seoond- sample was colorimetric method for determining A general infrsred method for the extracted and parathion was hydrolyd Nemacide (VC-13) and mnnel in animal determination of N-methyl carhamates to pnitrophenol. The pnitrophenol tiseue. After extraction and cleanup. in plants has been dmribed by Niessen was determined as an indophenol blue the peaticideaare hydrolysed and the r& and Frehee (168). gamnles were ex- after reduction with titanium trichloride sultinn ehlomohenols are steamdintilled tracted by blending with'acetone. In- and reaction with o-cml. and reacted 'with 4aminoaotipyrine. terferinp: nhtmaterial was .nrecioitated . Gajan (79) developed a polsrogrsphic The reaultim color is extracted into a with an-aBmmonium cbloride-phosphoric method for the determination of para- nitrometban&Pyridinemixture and read acid coagulating solution and, after thion. It was tested on green beans, at 490 w. 8ensitivity is estimated to additional cleanup on alumins, the in- apples, tcmatoes, broccoli, spinach, and be 0.05 p.p.m. Teasley (186) uda frared SpeCtNm fl'Olll 2.95 to 2.83 mi- bruwls spmuta and wan able to detect different version of the colorimetric crons was reoorded. The ahrotion at as little asO.l n.n.m. nanthion. Methvl procedure to determine Nemacide 2.88 micmidue to the N-H stktching thranilate iid pdi&phenol did not [O,(Miethyl 0-(2,4dichlomphenyl)- vibration was used for auantitgtion. As terfere. phoephomthioate] in fnik and vege- little as 0.2 p.p.m. df the pesticides - Several methods have been proposed tables. The method was rrubjected to could be determined. for the determination of ~horate. collaborative study, and although three After the infrared determination, the Waldmn el 02. (196) used an iIhProved collaboratom obtained fair results, two earbon diiulfide solution was used for colorimetric method in which phorate is others were unable to do so. Magat thin Layer chromatagmphy on alumina G, which served to determine the use of a new color reagent in determining used for fodder. A dilute sodium hy- identity of the pesticide. R, values are Zcctran in oeachesaud cottod. The droxide extract of the sample was listed for seven compounds. Zectran w& extracted from the sample acidified and extracted with pitmleum An infrared method was used by and hydrolyzed to yield 4dimethpl- ether. The solvent was evaporated and Ferguson e2 d.(71) to determine CIPC amino-3,&xylenol, which then ww re- the residue was dblved in 5 ml. of in white potatoes. After extraction and acted with luteoarsenotungstic wid. ethanol and treated with 0.5 ml. of cleanup the residue was dissolved in Abwrbance was measured at 700 w. propanol and 2 drop of 10% apueonm carbon disulfideand the infrared speetra The lntmarsenotungstic acid is said to potgssinm cyanide to produce an orange0 were obtained of the solution in 0.5- he highly specific for Mimethylamino- color, which was memured. The mm. cells. Peaks at 1110 and 1210 3,5-xylenol. method is said to be good for residues as em.-' were used for calculation. Monu- Callm 168) modified the standard low 860.1 p.p.m. ron and diuron did not interfere and the priced& 'fo; dithiocarhamates. The three compounds could he distinguished residue was deoomposed directly on the FUNGICIDES hv their infrared s~ectra. The method crop and the evolved carbon hllide Gunther. Blinn. and Barklev-.. (9.9) &used on samples which contained he- was collected and reacted with a solu- describe 8,procedure for determining tween 2 and 15D.D.m. CIPC. To detel- tion of cupric acetate and diethanol- biohenvl and o-ohenylohenol- -- in aitrus mine CIPC in miik and urine, Gard and amine in ethanol. Absorbance was fkit. -~hesample was blended with Ferguson (80) used modifications of measured at 435 w. Cullen points out water and the residues were ieolated by other methods. The CIPC was hy- that the dithimbamatea decompose steam diiillation into cyclohexan~. drolyzed; the 3-chloroaniline was di very quiekly when in a slurry of a crop After senamtion. the o-~henylphenol tilled, diazotized, and coupled with or in contact with slightly polar sol- was c~"~~ed&th phitmb-luene N-(1-naphthy1)-ethylene diamine dihy- vents. Samples should either be ans- diaronium fluobomte and determined drochloride. In order to obtain con- lyzed immediately after harvest or ~lorimetricallyat 540 mr. Biphenyl sistent low blanks, it was necessary to from for storage. The method was was determined diitly by maswe add formali to the urine and to age it tested on ferham, siram, maneb, sineb, ment of its absorbanceat 248 mr. for48 hours prior to analysis. thiram, and metinun. Souci and Maier-Haarlaender (179) Hardon, Bmink, and Van der Pol used a similar procedure for biphenyl (102) made use of similar diazotiaation DNTW COMPOUNDS but modified.the stesm &tillation ap and coupling to determine dichloran From a study of methods for deter- paratus. Rajzman (169) mports (2,&lichloro-4-nitroanilinef,a fungicide. mining the dinitro compounds, Bogga method for biphenyl in citnu, fruit bseed Although not a carhamate, it is listed (84) concludes that the paper chroma- upon the blue color given by hiibewl here since it can, if pmsent, interfere in tographic procedure is still the heat with sulfuric acid and trace of formd- the determination of some of the ear- general method. He Ma Rj values fo~ dehvde and ferric iron. The abnorbpnae hamstes. six compounds for both the aqueous wm"messured at 610 w. There is no Johnson (110) conducted a collabora- and nouaqucou~~ m . interference from mhenyl~henol: it tive study of the colorimetric method for Potter (168) determined Dinoeeb in does give a pink co& but-this d&ap carbaryl. After minor modifications potatoea by measuring its absorbance in pesrs during treatment with duri were made to improve the method, an ethyl methyl ketone at 379 mp after acid. a additional collaborative study was NU extraction and cleanup. Voml and Deehm (194) used (111) on samples of apples and lettuce. Abbott and Thornson (8, 3) used a ete& distillation t~ sepamte o-phmyl- Recoveries averaged 87.8%. wedgdayer type of plate chroma- phenol from citrus fruit. The o-phwl- Chiba and Morlev (60) introduced a togmphy as cleanup in the determina- ihenol waa then mted with 2,6 rapid thin layer chro~&tographic screen- tion of Dinwb in a number of fruits dibromoquinone-chlomimide and ah- in=-. orocedure for carbawl without any and vegetables. The plates were coated sorbance was measured at 619 mr. prior cleanup. The sample was ex- with a layer of silica gel-kieselguhr, To determine diphenybmine in tracted by blending with methylene which varied in thickness from 2 mm.at apples, Gutenmsnn and Lkk (96) chloride &d anhyd& sodium sulfate, one edge to 0.1 mm. on the opposite wed eiectron capture gas chmms- eva~orated, dissolved in petroleum edge. The sample extract was applied togmphy. The residue was extncted ether, and 'spotted on silica gel plates. as a streak near the thick edge and the and brominated to form what was After development, the plates were plate was developed. The yellow believed to he a hexabromo derivative of sprayed with methanolie sodium hy- Diwb band was then scraped off; diphewlamine, which was then injected droxide and the hydmlyzed I-naphthol the pticide was eluted with a solvent into the ge. chmmatogaph. %lvents was coupled by spraying with a solution and determined by infrared or gas were redistilled and contsot with mh- of pnitmhemne diaaonium fluoborste. chromatography or colonmetrically by her, which might contain dipbenyl- The authors note that with suitable the method of Potter (168) dmribed amine, was avoided. cleanup much lower amounts of ear- above. Andmn and Adam (7) report a haryl can he detected. Kilgore and Cheng (116) note that colorimetric method for the det&ina- Bracha (57) used a different Karathane diilved in N,Ndimethyl- tion of Dexon @dimethyhmho- diazonium salt in the determination of formamide gives a strong yellow color benlenedisw sodium &Ifonate) in aom, 0 - isopropoxyphenyl - N - methyl- without the addition of alkali. They cottod, and several other orope. carhamate. For the determination of used this phenomenon as a besis for the The aample was boded with 1% residues on various surfaces, he coupled determination of Karathane in fruit. mdium snlfite and tbe Dexon wm the hydrolysed insecticide 'with dik A hewe extract of the samole was isolated by dialysis. The Dem was tized 3-nitroaniline4~ulfonie acid cleaned up, either on a Fbrisil odlumn or than reac&d with ~euoreinoland sodium and measured the absorbance at 490 by wsshing with concentrated sulfuric hydroxide and inndiated with Wt mr The developed color was very acid, and evaporsted. The residue was fkm two projection spotlighb -to stable in water. This method hss been diilved in N.Ndimethylformsmide pdnce a yellow color red at adapted for the determination of car- and absorbance .read at 444 mr. A mr haryl, Isolan, Pyrolan, Dimetilan, and sensitivity of about 0.05 p.p.m. was Paanrela (164) eondnated a adlaho

Hercules AC-5727 (m-isopropylphenyl-~ ~~. ~ attained. tive studv of the colorimetric method N-n~ethylcarhamate). Heiniuch and Pmr (106) report a [Steller d d., .I.Agr. Food Cb. 4 Marquardt and Luee (142) report the method for dini-resol in plants 480 (IBW)] for dodine in fruit at livela of 0.1 to 10.4p.p.m. Recoveries ranged acetic acid was determined from the by Martin and Schwartsman (143). from 64 to 119%, with most values fall- abtbance at 360 mp. Reooveries from apples, cabbage, ing between 80 and 110%. Young, Shimahukw, and Aono (199) spinach, and mustard gmm at 1.4 to Kleinman (lB) conducted a collab determined naphthslenescetic acid- in 2.4 p.p.m. levels rsnaed from 84 to ative study of the colorimetric pineapples hy i& ultraviolet absorharm lzo%. thod for glyodin in pears and peaches. after eliminating interfcrenees by oxida- Munday (166) conducted a collabora- veries averaged about 88% for tion with wkiumne-k. tive study in which the A.O.A.C. mwpears and 88% for 'peaches. Two ~etun&aand ~&insonil66)based method for piperonyl butoxide was collaborators, however, reported dif- their method for simaxi~rein olant tiasue teated on a number of procegsed grain ficulties with peaches. on the ultraviolet abeorbahce of hy- pmducts. It was found that plant Nieasen, Frehse, and Tiets (269) droxysimazine. After extraction and extractives gave an abnormal brown developed a quantitative procedure for cleanup, the simmine was treated with color and interfered in the determina- Funailon (Bayer 32394) midues on sulfwic acid and hydrolyzed to hy- tion. apples, using a microtitrstion in a two- dmxysimasine. The absorbance then Hoffman and Gordon (107, 108) phase chloroform-water system. Apples was measured at 225,240, and 255 mr. studied the A.O.A.C. colorimetric meth- were stripped with chl&oform, waxes Benfield and Chilwell (N)have ods for arsenic and found that the were removed by precipitation from proposed a method for determining the arsine-molybdenum blue method gave cold methanol solution, and the ex- a-triasines in soil and in crops by gas slightly better reproducibility thsn did tract was cleaned up on an alumina chromatography of the cleaned-up ex- the silver diethyldithiocarbamate pm- column. The Fungilon then was tract. They used a 4foot column cedure, although both were suitable for titrated with Aeml OT (dioctyl- packed with 0.1% ethylene glycol determining arsenic in foods. They re- sodium-sulfosuccinste), with methylene adipate polyester on glaes heads. An port that antimony does not interfere blue as indicator. At the end point, unusual feature of their method was the with the arsine-mdybdenum blue color intensity was equal in the two addition of a second related triaaine to method and that its interference with layers. Glyodin and dodine are re- the sample ea an internal standard be- the silver dithiocarbamste method can ported to interfere. fore extraction. Final determination be orevented bv addillp more stannous involved only the ratio between the chketo the ieneratif;g mixture. MISCEUANEOUS PESTICIDES amounts of the two component6 present. Methods for cvanide have been re- Blinn and Gunther ($8) developed a viewed by Bark end Higson (16) who Analytical methods have heen re- procedure for diitinguiishing between compare and evaluate the various pro- ported for a number of herbicides and residues of Aramite and OW-9 in food- oedupx. Jones and Schwartaman (118) other growth regulstors in addition to stuffs. The two scaricides have similar rewrt a rapid method for detcrminillp: those diiscuesal above. structures, and OW-9 responds to the mercury ii wheat ccmtaining tmd HEH (&hydmxyethylhydraaine), usual colorimetric pmcedure for seed kernels. The trmted kernels were also known as “Odors," is used to Aramite. To dkthpkh between them, picked out visudy ender ultraviolet duce flowering in pineapples. Thomas Blinn and Gunther used gas and thin light and burned in a SchSniger com- d Ackermann (187) have developed a layer chromatography as well a% gas bustion hk. Mercury was deter- lorimetric method for its dete& chromatography of their parent car- mined with dithiroqe. Good agree- 4ion. After extraction with water and binols after hydrolysis. ment with the officialA.O.A.C. method removal of interfering color pigments Tieta d d. (188) made use of a red is claimed. An snslpis can be com- with ion exchange resins, the HEH is nickel chelate mmnlex formed with am- pleted in about two hours. reacted with cinnsmaldehyde to produce monia to determine Eradex (2,Squinox- Pickard and Martip (167) describe a yellow color which is read at 420 alioedithiol cyclic trithiocarbonate) in a method for detennlninn mercurv in mp. fruit. ~be0rb;encewas measured at530 soil. The sample is digested with-sul- Fletcher and Zalik (75) developed a mr Havens, Adams, and Anderson furic and nitric acids and selenium. and method for Sindoleacetic acid in which (108) used a similar reaction to deter- the mercury is ditiMed from bd.iling a methanolic extract of the plant mate- mine Mo188tgn (&methyl-2,3-quinox- sulfuric acid with hpdrogen chloride rial was chromatomaphed on wper and alinedithiol cyclic carbonate) in apples gae. After treatment with EDTA and part of the chm&~mwk sprayed and pears. They read the abeorbsnce dim thiosulfate, the mercury is with a chmmoaenic mntto locate the at540mp. determined with dithimne. indoleacetic &id. The corresponding Sinclsir, Lindgren, and Forbee (178) Phillips, Bowman, and Schultheis R, region from the wpmyed lues was determined ethylene chlorobmmide, (186) have developxi a screening pm- eluted with methanol and the ultra- using the pmcednw of Sinclair d d. cedure, the main purwse of which is to violet speotrum wss determined. Ab for ethylene dihmmide [J.Em. Entmol. single out samnles tbt mav contain mrbance at 280 mr wea uaed for quan- 55, 236 (1962)l. This procedure con- ov&toleranee &dues. ~i&~,or- titative determination. sisted of &am distillation, alkaline asoic chlorine, and aaetylcholinesterase Lane (It$) conducted a oollsborative hydmlyaia, and determination of the inhihition detkrminatiok were run on study of the colorimetric method for bromide. It was pointed out that since the same extract. Comparison of vari- maleic hydwide in potatoes. Re- ethvlene chlomhmmide undernoes de#- ous ratios provided the his for char- coveries were satisfactory. radktion in pmducte, inor&ic h& acterisation and estimation of most Zweig el al. (m1) developed a method mides should slao be determined. insecticides that inhibited cholinesteraae for the determination of naphthalene- Kimummd Miller (1'7) modified the or that contained chlorine. acetic acid in olives, using gna chroma- colorimetric method to determine Polumgraphy has come into wider tography as part of the cleanup pm metsldehydein plant material. Emulsi- use in residue dys$ work. Davidek cedure. The olives were blended with fication pmblems were m1ved by ass- and Janicek (60) arid Kosmatyi and chloroform and hyhhloric wid and ing the extract through a Florex &no. Shlyapak (123) uaed polupolerogrsphy to extract wss passed through alumina Objectionable interlering colors were determine DDT, and Gajan (79) made silica gel columns. The &due eliminated by evaporat& the chlom- use of the technique b determine pan- en wea methyluted with dimethane form extract to dryne~n. thion. Nangniot and Dardene (166) aF p$s and injected into the chroma- After minor modifioations, the ultra- dwribe t bpolangraphic methods tograph. Fractions were collected. The violet method tor nicotine in fruite and for determining oaptan and lolpet eluate was nitrsted and naphthalene- veptables WM studied oollsbontively (Phaltan) on pknte. Veksler and Tsukervanik (193) liit (1964). (54) Coffm, D. E., Savary, G., M.,47, a number of defoliants which can be (13) Bache, C. A,, Lisk, D. J.,,Loos, 875 (1964). M. A., J. Assoc. Ofi. Agr. Chwls 47, (55) Cox, W. S., Ibid., 46, 229 (1963). determined quantitatively by polarog- 348 (1964). (56) Ibid., 47, 280 (1964). raphy. They state that there is a (14) Baetz, R. A,, Ibid., p. 322. (57) Ckosby, N. T., Laws, E. Q., Andual, relationship between the polarogrsphic (15) Bark, L. S., Himon, H. G., Anolyst 89,319 (1964). behavior of the compounds and their 88, 751 (1963). (58) Cullen, T. E., ANAL.CB&Y. 3 (16) Barney, J. E., 11, Stanley, C. W., 221 (1964). aotivities as defoliants. Cook C. E., ANAL. CUEU. 35, 2206 (59) Daoud, N. H., Luh, B. S., FmfM84 Morris and Haenni (164) determined (1\ - -OR<^ - -,. Ind. 7, 33 (1962); C.A. m, 1641% the infrared spectra (2 to 35 microns) (17) Barry H. C., Hundle ,J G , Pesti- (1964). of 24 pesticides, using potassium bromide c~de~naiytical Manual 6.k he t of (60) Davidek, J., Janicek, G., Z. And. disks. They discuss the relation of Health, Education, and Welfare, %hod Ch.1106.2) 194. 431 (1963); C.A. 59. 4494 and Drug Admin.), Vol. 1 (1963). %-""",. absorotion hand to structure and note (18) Batas, A. N., Rowlands, D. G., (61) Dawson, J. A,, Donegsn, L., Thin, the maxima of analytical significance. Analyst 89,286 (1964). E. M., Analyst 89,495 (1964 Guillemin (91) seoarated the isomers (19) Ibid., p. 288. (62) De FaubertMaunder, bk J., Egan, .. . (20) Beckman, H. F., Berkenhotter, P.. H., Godly, E. W., Hammond, E. W., of BHC by gas chromatography, using ANAL.C~Y.35,242 (1963). Roburn, J., Thornson, J., Ib*I., p. 168. a 3 m. X 6 mm. stainless steel column (21) Beckman, H., Bevenue, A., J. Agr. (63) De Faubert-Maunder, M J., Egan, packed with 40- to 60-mesh glass beads Food Ch.11,479 (1983). H., Roburn, J Zbid., p. 157. coated with 0.25% polypropylene glycol (22) Ibid., 12,183(1964). (64) Dimick, 2. P., Hart-n, H., (23) Ibid., p. 245. Re& Rev., Vol. 4 Academic Press, Nix 1025. (24) Beckman, H., Bevenue, A,, J. Chre Inc., New York, 1963. In spite of advances in instrumenb mdag. 10 233(1963). (65) Dutt, M. C., Seow, P. H., J. Agr. tion, bioassay methods continue to have (25) ent tie id, C. A,, Chilwell, E. D., Food Ch.11 467 (1963). their uses. Sum el ol. (184) discuss Anolyst 89, 475 (1964). (66) Egan, H., $ammond, E. W., Thom- (26) Bevenue, A,, Zweig, G., Nash, N. L., eon, J., Andys689,175 (1964). factors that may affect results and sng- J. Assoe. O& Agr. Chisb 45, 990 (67) Rdedelmsn, M., J. Asaoc. O&. Agr. gest precautions to he taken in bic- IIW',, Chi& 46,182 (1963). assavs. Weinmann (197) describes (68) Enos, H. F., Jr., Frear D. E. H., metiods of purifying the ekraots and J. Agr. Food Chem. 10 447 (1962). (69) Enos, H. F., Jr., hear, D. E. E., evaluatme results. Funderburk and M.,12,175 (1964). ~awrencC(78)determined Diquat and (70) Espadas, R. S., Lomza, J. 0.. Paraquat by measuring their bleaching (Conusion Nacl. Erradicaeion Palu- effect on duckweed (Lanaminor L.). dismo), CNEP Bd. 4, No. 2, BB (1960); 191. C.A. 58, 10673f (1963). as little as 0.0005 p.p.m. Diquat or (38IM., p. 204 (71) Ferguson, C. E., Jr., Gard, L. N., 0.00075 p.p.m. Paraquat can be de- (33) Blumen, bid.,47,272 (1964). R.H.,DIeex,K.W.,J.Agr. tected. (34) Bog@ H. M., Zbid., p. 346. %%cm. 11,428 (1963). Two methods make use of newer (35) ~onedi,E. J., Hartmano, H., Dim- (72) Fischer, W., Uhlich, U. NodrF. ick, K. P., J. Agr. Food Chern. 12, 333 DaU. P,zmmaeh* (k"'~ad~) techniques to accomplish the familiar (1964). 13,28 (1961); C.A. 59,804%(1963). determination of organic chlorides in (36) Bosin, W. A,, ANAL.CHEU. 35. 833 fat. Krzeminski and Landmann (136) (1963). 903 (1963). used sodium in liquid ammonia to (37) Brachs, P., J. Agr. Food Chem. 12, 461 (1964). "Baycr" 16, 182 (1963-64). release the chloride from pesticide resi- (38) Breidenbach, A. W., Liehtenber (75) Frehae, H., Niemen, H., Tietc, H., dues and then determined the chloride J. J., Henke, C. F., smith D. J., Eichef Zbid., IS, 148 (1962-63). potentiometrically. Schmitt and Zweig herger, Jr., J. W., ~tieili,H., U. S. (76) Ibid., p. 160. (174) used neutron activation to deter- D t. of H&, Educutia, and Welfare, (77) Friastad. H. 0..ANAL.Cnm. 35. mine total organic chloride in butter ~%icHdlh Swviec Pub. No. 1241 ' 1'012 (1963j. llW)~\ ---.,. (78) Funderburk, Jr., H. H., Lawrence, fat. The sensitivity is reported to be (3?!l&werton, H. V., N.Z. J. gEi. 6,418 J. M., Nature 199, 1011 (1963). 10 p.p.h. total organic chloride and ,..7W,. (791 Gaian. R. J.. J. Assw. Oflic.- Aw. the time of analysis to be less than 1 (40) Bruaux, P., Dormal, S., Thomas, G., ' dhkd46,216'(1963). hour per sample, which can be reduced Ann. Bid. Clin. (Paris) 22, 375 (80) Gard, L. W., Ferguson, Jr.. C. E., (1964); C.A. 61.6303a (1964). J. Agr. FoodChcnh 11,234(1963). considerably if many specimens are (41) Bunyan, P. J. Analyal89,615 (1964). (81) Geldmacher-Mallinehmdt, M., processed simultaneously. (42) Burohfield d P Rhoades, J. W., Weigel, U., Arch. Td.20, 114 Whealer, R. j., DivGion of Agricultural (1963); C.A. 59, 10708b (1963). BIBLIOGRAPHY and Food Chemistry of American (82) George, D. A., J. Aaaoc. O&. Agr. Chemical Society, 148th Meeting, ACS, Chisb46,960 (1963). (1) Abbott, D. C., Egan, H., Hammond, Chicago, IU., September 1964. (83) Getz, M. E., Friedman, S. J., Zbid, E. W., Thomsan, J., Analyst 89, 480 (43) Burke, J., J. Asaoc. Ofi. Ap. Chew ',. 7M.".. (1964). isla 46,19841963). (84) Getzendwer, M. E. Ibid., p. 269. (2) Abbott, D. C., Thomson, J., Ibid., p. (44) Burke, J., Giuffrida, L., Ibid., 47,326 (85) Gian P. A., ~ehechter, M. S., RlR~ (1964). J. Agr. Food hem. 11.63 (1963). (3jAbbott, D. C., Thomson, J., Chem. (45) Burke, J., Holswade, W., Ibid., p. (86)Chisfa Giuffrids.47,293 L.. (1964).J. Asaoc. O&. Agr. Ind. (Lads) 12,481 (1964). Rd5 (4) Ackermann, H. J., Carbone, L. J., ($Burke, J., Mills, P. A., Bid., 46, 177 (87) Gordon, C. F., Hainee, L. D., Kuchar, E. J., J. Agr. Food Ch.11, (1%). Martin, J. J.,J. Agr. Food Chem. 11.84 297 (1963). (47) Butler, L. I., Maitlen J. C., Fahey, (N'w. (5) Adams, J. M., Anderson, C. A,, J. E., J. Agr. Food 6hem. 10, 479 (88) Goulden, R., Goodwin, E. S., Davies, MeDougall, D., Ibid., p. 178. (1962). L., Andyal88.941(1963). (6) Albert, R. A., J. Assoc. Ofi. Agr. (48) Carey, W. F., J. Aaaoc. Ofi. Agr. (89)Ibid., p. 951. Chemists 47,659 (1964). Chisfa46,876 (1963). (7) Anderson, C. A., Adams, J. M., J. (49) Cerne, V., Nohrunp7(1)6C4(1963); (90) Graham, J. R., Omoll, E. F., J. Agr. Agr. Food Chem. 11, 474 (1963). C.A. 59,6895~(1963). Food Chem. ll,67 (1963). (8) Anliker, R., Menzer, R. E., Ibid., p. (50) Chiha, M., Modey, H. V., J. Assoc. (91) Guillemin, C. L., And. Chim. Ada 291. O&. Agr. Chwb47, 667 (1964). 27,213 (1962); C.A. 58,8898 (1963). (9) Archer, T.E., Winterlin, W. L., Zweig, (51) Claborn, H. V., Ivey, M. C., Ibid., p. (92) Gunther, F. A., Blmn, R. C., Barthy, G., Beckman, H. F., Ibid., p. 471. 871. J. H., Analyst 88.36 (1963). (10) Archer, T. E., Zweig, G., Winterlin, (52) Coakley, J. E., Campbell, J. E., (93) Gutenmann, W. H., Lisk, D. W. L., Francis, E. K., Ibid., p. 58. McFarren, E. F., J. Agr. Food Chem. 12, J. Agr. Food Chem. 11.301 (1983). (11) Bache, C. A., J. Assoc. O&. Agr. 262 (1964). (94)M.,p. 304. a Chemisk 47,355 (1964). (53) Coffin, D. E., McKinley, W. P., J. (95) Ibid., p. 468. (12) Bache, C. A., Gutenmann, W. H., Aasoc. Ofi. Agr. Chrmisb 46, 223 (96)Ibid., p. 470. Lisk, D. J., J. Agr. Food Chem. 12, 185 (1963). (97)Ibid., 12, 46 (1964).

VOL 37. NO. 5, AP111 1965 . 141 R (98) Gutenmann, W. H., Lik, D. J., rin r-Verlag, Berlin-Gottingen- (171) Rabum, J., Ibid., 38, 1555(1963). J. Am. Water Wmh Assoc. 56, 189 2.e~de$ber (1963). (172) Rowlands, D. G., Andyst 89, 498 (1964). (135) MCCU% K A McKinley, W. P., (1964). (99) Gutenmann, W. H., Lkk, D. J., J. Aaaoc. &.hi;. Chmiab 47, 652 (173) Schafer, M. L., Buach, K. A,, J. Aaaoc. O5c. Aar. Chmnda %, 859 IIW\ Cam bell, J. E., J. hiry Sci. 4, 1025 (166% (174) Schmitt, R. A,, Zweig, G., J. Agr. Fwd Chem. 10, 481 (1962). (175) Schwartz, N., Gaffney, H. E., Schmutzer, M. S., Stetano, F. D., J. Assoc. Ofi. Agr. Chemiats %, 893 (1963). (176) Shuman, H.,Ibid. p 195. (177) Shuman, H., ~dbe,J. R., Ibid., (1964). p. 992. (104) Heinisoh, E., Neubert, P., J:Prakt. (178) Sinclair, W. B., Lindgren, D. L., Chem. 22, 267 (1963); C.A. 60, 7385f Forbes, R., J. Econ. Elnt. 57,346 (1964). (179) Souc~, S. W., Maier-Haarlaender, G.. Z. Lebenan.-UnlerswA-Forach.119, 217 (1963); C.A. 59,6688e (1963). (180) Steller, W. A,, Cumy, A. N., J. Assoc. Ofi. Agr. Chemiats 47, 645 (1964). (181) Stemp, A. R., Liska, B. J., Lsnglois, B. E., Stadelman, W. J., PouUry Sci. 43,273 (1964). (182) See ref. 130. (183) Sun, Y. P., Johnson, E. R., J. Aam. O&. Agr. Chemists %, 524 (1963). (184) Sun, Y. P., Jo son E R Pan- kaskie, J. E., Earles. *., 's&; J. Y. T., Ibid., p. 530. (185) Taylor, A., Res, R.E.,Kirby, D. R., Andyat 89,497 (1964). (188) Teasley, J. I., J. Asam. Ofi. Agr. Chemists47,287 (1960. (187) Thomas, M. P., hckermann, H. J., J. A .Fwd Chem. la, 432 (1964). (188) Flet& H.,Oamap, M. F., Frebee, H.,Niessen, H.,P mchulc-Nachr. BW. "Bayer," 15, 166 (1%). (154) Morris, Jr., W. W., Haenni, E. O., (189) Tmtaenko, M. A,, Nme v ObbP Irn. 46,964 (1963). Sad.-Khina. Analiza (Raboty p~Prom.- (155) hunday, W. H.,Ibid., p. 244. Srmil. Khim.) p. 175 (1962); C.A. 59, (156) Nangniot, P., Dardene, G., Bull. 449 (1963) Inal. Am. Sf&. Rceh. Uanbbuz 31 (lW)%chiya& M., Okui, S., Shokuhin (I), 110 (1963); C.A. 60. 126038 Eia ' ku Zasahi 3, 277 (1962); C.A. (1964 5s,%9a (1963). (157) dhn, R. C., J. Assm. O&. AGT. (191) Van Middelem, C. H., Waited, R. Chrmiab 47,289 (1964). E., J. Agr. Fwd Chem. 12.178 (1,964). (158) Nieasen, H., Frehp, H.; P* (192) Van Middelem, C. H., Waited, R. schuiz-Nachr. "Baycr, 16, 205 (1983- E., Wihn, J. W..Ib ,11,56(1963). 64). (193) %ksler, E. A., %ukervsnlk, I. P., (159) Niessen, H.,$ehse, H.,Tietz, H., Uzbekak. Khim. Zh. 6, No. 4, 55 (1962); Ibid., 15,172 (1962-63). C.A. 58,3834~(1963 (1W) Nikolov, N. I., Donev, L., Zh. (194) Vogel, J., De&mea J., Ma. Add. Khim. 18, 532 (1963); C.A. WfeLebem. H 54,'330 (1963); 59,4494g (1963). C.A. 60 i1289f (I&$. (161) Onley, J. H. J. Aaam. O& Agr. (195) wafdmn, A. C., Pasarela, N. R., Cihanisb 47,317 (1964). Woolford, M. H.,%re, J. H.,J. Agr. (162) Onley, J. H., Milk, P. A,, Ibid., Fwd Chem. 11, 241 (1963). 45,9B (1962). (196) Walker, K. C., ernan, M., J. Assoc. (163) 0% D. I,Guntber, F. A., J. Agr. ON.Apr. Chemwk 250 (1963). Fwd Ch.12,239 (1964). (197) Weinmann, W.3. Lcbenm.-Ut*cr- (164) Peearela, N. R., J. Aaaoc. Olfic. d.-Fmach. 119, 334 (1963); C.A. A .Chumwfa47,300(1964). S9,6896b(1963). (1657~etunova, A. A,,Martinson, E. E., (198) Yip, G., J. Aaroc. Olfic. Agr. C h Firid. Ebt. 10, 729 (1963); C.X. 60, wls 47,343 (1964). 1964). (199) Young, A. Y., Shimahukuro, S., (AB%ps, W. F., Bowman, M. C., Schultheir R. J.. J. Am. Fowl Clbnr Aono, L., J. Agr. Fbal Chem. 11, 132 (1964). (1963). (131) hglois, B. E., Stem A. R., lo, 486(1&). ' Liska, B. J., J. Dai~Sci. 46, k4 (1963). (167) PicLard, J. A,,Martin, J. T., J. Sci. (203) Zad~~imka,J., R-iki Panalu+ (132) Leshy, J. S., Taylor, T., Andyd Fwd Agrl4, 706 (1963). wcqo ZaWu H*. 15. 5 (1964); C.A. 88,882 (1963). (168) Potter, J. A,,Andgat 88,661 (1963). 61,6262b (1964). (1% Iaaeyk, J. E., ANAL.CUM. 35. (160) Ra'zman, A.:Ibid., p. 117. (201) Zweig, G., Gutriick, D. L., Gulli, ",."-I. (170) Robinson, J., Richardson, A., Chem R., Archer, T. E., Kartmann, H. T.. 1) Lykken, L., Ruiduc Rcv. 3, '19. Ind. ~Lmdrm)35,1480 (1963). J. Agr. Foad Chnn. 13,59 (1964). Reprinted from ANALYllCAL CHEMISTRY ANNUAL REVIEWS, Vol. 39, Page 142R, April 1967

westicide Residues Sidney Williams and J. Williom Cook, Division of Food Chemistry, Food and Drug Administration, U. S. DeNrtmenf of Health, Education, ond Welfare, Washington, D. C.

EsEAnCH IN THE FIELD of method- response for each of these; thus the tion by all workers iq the field. Because R ology for residue analysis has re- possible misinterpretation is more ob- of these problems a@d because the au- ceived much attention during the two- vious. This problem has been given thors believe that @e use of chemical year review period from November 1964 a great deal of study and considerable nomenclature excln$ively would make through October 1966. In preparing progress has been made as indicated in the review difficult to follow, ve have this review, no attempt has been made numerous places throughout the review. arbitrarily adopted a mixed system. to include all articles dealing with resi- Closely associated with the field of In Frear's "Pestidide Index" (Ill), due analyses but, rather, the authors methodology, particularly with the probably the most comprehensive list- have tried 'MI select those which, in mnltidetection methods, is that of the ing of pesticides, cofnmon names, trade their opinion, will be most useful tothe "nature of the terminal residues." A and chemical namesare listed and cross- worker in this field. pesticide chemical may be altered by its indexed. We have tried to use the Much of the literature refers to the environment after application to yield name mhich we believe will be most "multidetection" methods in which terminal residues sufficiently close to n~eaningfulto the reader and, where many chemicals can be determined the parent chemical to appear as a possible, to use a @me which may be during one analysis. The possible in- response to the detection system of a found in Frear's Index. Common clusion of so many compounds in one multidetection method. Developments names which appear in the United analysis is a tremendous step forward in this field should be closely watched States Food and Drug Administration but it doe8 increme the ~roblemof and taken into consideration as aids in tolerance regulations have been used interpreting the responses properly so the interpretation of responses in many where possible. aommon names of that one ha? adeauate sssurance of the of the methodsnow available. other compounds have heen used when- ident,ity of the compounds reported. There is no single uniform system of ever they exist and pre considered to be This problem is basically no different nomenclature used in the literature for well known. For those compounds best from interpteting the response of the pesticide chemicals, so for many pesti- known by a single trade name we have methods such as a color reaction cides there is no single generally rec- used the trade name in capitalized the "wst &mid" or "specific" ognized name. Both the coined patent, form. In those instances where the lethods; for example the "specific" names and the common names vary common or trade name may be con- colorimetric method for oarathion also with the country and worker; thus fusing or is not well known, we have responds to paraoxon, methyl para- there may he a. number of names to tried to include the chemical name as thion, EPN, Chlorthion, etc. The describe the same compound. On the well. Thus the teader should, mith GLC methods generally give separate other hand, some names have rewgni- the help of Frear's Index, be able to

142 R ANALYTICAL CHEMISTRY identify aln~ost every wmpound re- are also divided into sections dealing measuring the intensity of selected farred to in this paper. with pollution, toxicity and toxi- r. atomic lies and molecular bands, the lhe literature on pesticide residues cological factors, analysis, etc. system can be made quantitative and has maintained its rapid growth in the This two-year period has ah seen hlghly specific for the halogens, phos- two years since the previous biannual the beginnings of attempts to automate phorus, and sulfur. Bache and Lis review (76). Gunther (133) has con- pesticide residue analysis. Gunther el (13) used this emission spectrome tinued his excellent series of "Residue d. (136) designed a system for the detector to analyze a number of fda.r. Reviews" and 15volumes have now been determination of total chlorine. They for such organophosphorus pesticides published. These volumes contain arti- wmbined an automatic combustion ap as diazinon, dimethoate, disulfoton cles on mauy aspects of pesticide paratus with a continuous flow chloride @iSyaton), ethion, parathion, and residues written by experts in the spe- ion detector. It was capable of han- ronnel by*measuring the intcnsity of the cific fields. Volume 1 of the Food and dling as much as 2-gram equivalents of 2535.65 A. line. Rewveries of 72- Drug Administration's "Pesticide An& plant extractives and had a useful range 115% were obtained at levels ranging lytieal Manual" (191, probably the of 0.01 to 500 ppm. The burning cycle from 0.03 to 0.60 ppm. most widely used manual of methods for of 7 minutes was automatic and the Brody and Chey (60) developed a pesticide residue analysis, has under- chloride ion measured and recorded. flame photometric detector for deter- gone continuous revision and updating. Ott and Gunther (833) rewrted an mining sulfur- and phosphor~ntain- The methods in this volume have been automated colorim&io ~hosphorus ing wmpounds. The flame emission studied in FDA laboratories and are determination which had a sensitivity spectra were generated in a hydmga- known to be useful combinations of well below 0.1 irg phosphorus per air flame and narrow bandpasa inter- extraction, cleanup, and determinative milliliter of ha1 solution. Samples ference filters used for isolation of the steps which yield quite satisfactory were cleaned up and burned in a phosphorus and sulfur emission at 526 qualitative and quantitative results for Schoniger flask and the solution was m+ and 394 m+, respectively. The many wmpounds. transferred to an AuloAnalyzer which sensitivity was said to he 0.25 ng for The U. S. Public Health Service has carried out the analysis and recorded the malathion or parathion and in the sub- published a 2-volume "Guide to the result. Several procedures were re- microgram range for sulfur-contsining Analysis of Pesticide Residues" (66) ported for the automatic determination compounds. which includes methods for the analysis of cholinesteraee inhibition usine the Conlson (77) introduced an electro- of water and soil as well as foods. AutoAnalyzer. Voss (E83) descrged a lytic wnductivity detector for gas However, this is primarily a compilation urocedure in which acetvlthiocholine chromatography. The effluent from of methods recommended by a variety was used as the substratk. The lib- the GLC eolumn wss passed through a of laboratories and which have not erated thiocholine acted on 5,5-dithiohis- combustion tube where the compounds necessarily been used or tested by the Znitrohenzoic acid producing a wlor were oxidized to COI, Sod301 and U. S. Public Health Service. which was measured at 4%3 mp. Ott HC1. The gases were paased into a Several new periodicals of interest and Gunther (%?I, ,839) used acetyl- stream of deionized water and into an have appeared in the period covered by choline as the substrate and measured electrolytic wnductivity cell where th this review. The "Bulletin of Environ- the transmittance at 555 m+ as atrected wnductivity was measured by a simp mental Contamination and Toxicology" by change in wlor of phenol red. Whes.t&ane bridge. The detector was (53) is a bimonthly journal designed to la All of the above procedurea required 10,000 times as sensitive for halogen provide a rapid publication in fields prior extraction and cleanup before the or sulfur comoounds as for wbon or including pesticide residue methodology. sample solution could be placed in the nitrogen. The first issue was dated January- AWoAnalyzer. The first fully auto- Coulson (78) later modified this February 1966. mated anslysis was reported by Gunther system to de&i&ne nitrogen-wntaining The U. S. Department of Agriculture and Ott (157) for the determination of compounds. The effluent from the began the publication of "Pesticides biphenyl in citrus fruit rind. The GLC, instead of being oxidized, was Documentation Bulletin" (E@) on sample was automatically homogenized reduced with hydrogen in the presence March 10, 1965. This biweekly is a in water and the biphenyl steam dis- of a catalyst to change any nitrogen to ". . . wmputer produced permuted title tilled. Oils and waxes were removed NH,. Any acids formed were removed index in three parts: Keyword Index, with H80, and a cyclohexane solution with Sr(0H). and the NHI was passed Bibliography, and Author Index!' This of the biphenyl was passed through the through to the electrolytic wnductivity is excellent as a general reference for cell of a rewrdiog W spectropho- detector. The appratus was used for those interested in many aspects of tometer with readings taken at 246 me the determination of such nitrogen- pesticides but is of little value to the and recorded. The useful range was wntei wmpounds as simasine, residue analytical chemist as a specific said to he from 1 to 150 ppm on a parathion, amitrole, eta. reference because only those words whole fruit basis with a reproducibility Martin (198) renorted a method for which appear in the title of an article of about 3%. the deten&nation'of nitrogen-cantain- will appear as entries. There is no ine eom~oundswhich also reduced the grouping of subjects such as "methods GAS CHROMATOGRAPHY efiuent -from the GLC column with of analysis" or "chemical methods of Another area which received con- hydrogen to produce NHs. The NHa analysis" nulees those words appear in siderable attention was detectors for gas was then passed into a titration cell the titles of the original article. chromatography. As the need for de where it was automatically titxated More recent and potentially mast teetors capable of selective specificity with wulometridy generated hydro- valuable to the pesticide residue chemist for wmpounds wnteming halogens, gen ion. However, with some com- is the "Health Aspects of Pesticides- phosphorus, sulfur, and nitrogen became pounds, some of the nitrogen atoms Literature Bulletin" (146). This is an greater, several new approaches were were converted to elemental nitrogen experimental monthly publication of taken. McCormack, Tong, and Crmke which would not he wnverted to NHI the Office of Pesticides of the U. S. (@4) developed a detector based on Guthion, for example, gave only 3 Public Health Service. The first issue, selective monitoring of the emission recovery. Although the method dated September 1966, contained 117 spectra of the eluted organic compound. developed for petroleum industry use, abstracts including 26 of recent articles Using argon as the carrier gas, the it was said to be applicable to peaticite* dealing with analysis. In addition to spectra are excited in the plasma of a analysis. author and subject indexes, the conten$ 2450-Mc electrodeless discharge. By A number of papers were puhli&d on the thermionic detector, all of which and HCI (by ~1~03could be removed, CHLORINATED PESTICIDES gave strong support to the validity of thus giving the systems high specificity. the detector. Beckman and Gauer The electron capture detector con- General Procedudes. Chlorinated (26)rcviewed the literature and develop tinues in wide use but only a few papers pesticides continue to be the most ent of the sodium thermionic detec- suggested modifications. Yauger et al. widely used group and it is natural (287) reported the use of Ni63 as the that methodology for these com- eandr. operationThey described of a detector the constructionbased on radioactivity source, the big advantage pounds received a great deal of att'en- Giuffrida's design. Hartmann (145) being that such a detector can be tion. Beynon and Ellgar (36) prepared reported a thermionic detector for operated at temperatures up to 300' C. an excellent review of work published phosphorus in which cesium bromide Abbott and de Fanbert Maunder (3) up to May 1965. They list 324 was used as the alkali metal source. described a simple electron capture references and cover all aspects of The cesium bromide plus a suitable detector that could be constructed from residue analysis from thc collection and filler mas pressed under high pressure to a standard 75-ohm co-axial cable plug storage of samples through extraction form a ceramic-like pellet which was and a strip of tritiated copper foil at a and cleanup to the numerous means of then shaped to serve as the tip of the total cost of material of less than 510. quantitation and identification. burner. Coahran (71) described a Gas chromatography has become so Mumma and coworkers (218) in- modified detector in which a ceramic accepted in pesticide residue analysis vestigated the effectiveness of a com- tube filled with granular anhydrous that its use in procedures is now taken monly used extraction procedure in Na2S04was placed around the jet. for granted much like the analytical removing pesticide residues which had Giuffrida and Ives (123) used dual balance or a spectrophotometer. How- been picked up by growing crops. detectors in an investigation of cleanup ever, several papers have appeared Using crops grown in soil containing procedures for organophosphorus pesti- which treat gas chromatography as a dieldrin, they found that the videly cide residues. The effluent from the general topic. Guddnowica (125) com- used hesanc-isopronanol (2: 1) extrac- GLC column was passed through a piled a vast amount of data on the tion procedure rerpoved only about stream splitter and into two detectors. use of electron capture gas chroma- 640/0 of the dieldrin present. When this The response of a regular flame ioni~a- tography in pesticide residue analysis. was followed by a 1Zhourextraction tion detector was indicative of the He listed the R, and sensitivities for a with a 1:l mixture of chloroform and amount of vlant extractives uresent large number of the pesticides on a methanol complete extraction of the and thus oi the cleanup e&iency. variety of columns. Burke and Hol- dieldrin was obtained. The remonse of the thermionic detector swade (68) tested 17 liquid phases in In the past tvo years, several col- showed' the amount of pesticides re- the search for a GLC column which laborative studies were nlade of widely covered. would elute the common pesticides in a used analytical pmcedures. Johnson Giuffrida, Ives, and Rostwick (124) different order than the widely used (167) reported on a study of the Mills' described and explained the operating DC-200 column. They recommended procedure involvingi the determination parameters for electron capture and the a column prepared by mixing equal of heptachlor epoxide and dieldrin in hermionic detectors. Specific details portions of Gas Chrom Q coated with evaporated milk bnd in butterfat. ere given on how to adjust each 15% QF-l and Gas Chrom Q coated The results for 20 I&oratories shoved a edetector and GLC system for most with 10% DC-200. They listed reten- standard deviation of 10.039 ppm for suitable operation for residue analyses. tion times and response data on the heptachlor epoxide at the 0.29-ppm This paper should be required reading for column for over 85 pesticide chemicals level and a standard deviation of residue analysis using gas chroma- using both the electron capture and the +0.052 ppm for dieldrin at the tography. microcoulometric detector. 0.2Sppm level. Several collaborative Karmen (173) described a stacked Berck (29) listed retention times, studies of the Mils, Onley, Gaither &me ionization detector for phos- both absolute and relative to n-pentane, procedure were alsm reported. Krause phorus and chlorine. He indicated for 34 fumigants on a column packed (187) studied the recovery of aldrin, that the detector worked because phos- with 10% SE30 on Diatoport-S. DDE, and methoxychlor from potatoes. phorus and halides increased the vapor Kanazawa (171) evaluated and com- Gaul (115) investig@ted the recovery of pressure of the alkali metal and thus pared columns with two liquid phases, lindane, heptachlor, and TDE from made more of it available for ionization. 5% Dow Silicone 11 and 2% polyethyl- endive and cauliflqwer, and Davidson Abel, Lanneau, and Stevens (5) re- ene glycol, for the separation of chlorin- (87) reported on the determination of norted a modified stacked flame detector, ated and phosphorus pesticides and BHC, p,pl-DDT aqd endrin in apricots clai~nin~a controlled specificity for herbicides. Linear ranges, sensitivities, and strawberries. Each study demon- phosphates and halides in the order of and separation efficiencies are reported. strated the validity of the procedure. 100,000-200,000 to 1 over other organic: Gaul (114) compared five methods of d large number of articles describe species. measurine GLC Deaks and discussed the general procedures lor the determination Burchfield el al. (54)discussed various problems with -toxaphene, chlordane, of chlorinated pestitide residues. Many types of GLC detectms, pointing out and bHC. She suggested ways of meas- of these are modifications of previously the advantages of each. Burchfield uring the peak areaswhen the pesticides reported methods. Gunther and Bark- and Wheeler (57) described the use of were separate and in mixture. It was ley (134) modified ;a microcoulometeric the microcoulometric detector in both also pointedout that in determining BHC gas chromatograp! so that, when de- the oxidative and reductive modes. the analyst should bear in mind that sired, the GLC cmlumn could be by- Burchfield el al. (56) also reported the the electron capture response to the passed with the s$mple going directly use of the microcoulometric detector @-isomer is about 50Yo of the response to the combustion furnace. This per- for the determination of phosphorus, to the other isomers. mitted essy detetmination of "total sulfur, and chlorine. The effluent from Giu5rida (121) described a GLC sys- chlorides." Adva tages of the arrange- he GLC was carried through a reducing tem for the collection of fractions for ment include a m $e accurate measure- en with H*, forming pH,, H$S, md infrared analysis. The fractions were ment of toxaphebe since the entire ak CI. All three products mere meas- collected individually directly on KBr residue registered as one peak. ured by a microcoulometric titration and then pressed into micro disks. Robertson and Tyo (246) determined cell with silver electrodes. 13y insere About 10 mg of KBr was used and good chlorinated pesticides in oysters using a ing subtraction columns before the spectra were obtained with as little as continuous perforated basket centrifuge cell, KC1 (by silica gel) or both HzS 3 pg of pesticide. for extraction of sample rvith acetonitrile. After partitioning of the resi- tuzzi (2%) reported a rapid procedure partitioning into petroleum ether, the dues into petroleum ether, the deter- for the analysis of fish, meat, and fat extracts were further cleaned up on an mination was made by electron capture by electron capture GLC. The method activated Florisil wlumn and deter- GLC. Recoveries for heptachlor, hep combined the use of a mixture of ace- mined by electron capture GLC. tachlor epoxide, DDE, and DDT tone, methyl Cellosolve, and formamide bert (876) used a wlumn of ranged from 97 to 115% at the 0.16 to extract the pesticide residues with magnesium oxide, and Celite 545 ppm level. Kadis and Jonasson (170) the use of calcium stearate to wagulate replace the Florisil column in used a modification of the method of and hold fatty constituents. Recover- atdvsis of oils bvelectron canture GLC. Langlois et al. [Milk and Food Technol. ies ranged from 7&108% at levels of Sah; (249) de&nnined alhin, hepta- 27,202 (19M)I to determine chlorinated 0.003-1.0 ppm. Kotula and Moats chlor, endrin, and dieldrin in wheat pesticides in blood. The sample was (183) used TLC to analyze eggs or using electron capture GLC. The ground with Florisil, transferred to a poultry fat samples in less than 2 hours. ground samples were extracted with Florisil column, and eluted with 30% Extraction was with ethyl ether with acetonitrile in a Soxhlet; residues were methylene chloride in petroleum ether. cleanup on a esrbon-Celite 545 wlumn. partitioned into petroleum ether and After evaporation and solution in As an alternative, fat could be dissolved cleaned up on a magnesia-Celite hexane, analysis was by electron capture in petroleum ether and cleaned up on a wlumn. GLC. Jain and coworkers (161) used Florisil column. In each case, suction Several procedures have been re- a simplified procedure to determine 23 ~vasused to speed up the elution from ported for the determination of chlorin- pesticides including chlorinated, organo- the column. l&ht chlorinated com- ated pesticide residues in water. phosphorus, aud a nitro compound in pounds were determined with a sensi- Lamar and coworkers (190) extracted blood. The sample was extracted with tivity of about 0.1 ppm. Sawyer (254) large (up to 4 liters) samples of water an acetone-ether mixture (1: I), evap- used acetone to extract chlorinated with hexane and used electron capture orated, taken up in hexane, and pesticides from eggs. After partitioning GLC for the determinative step. Smith injected into an electron capture GLC. into petroleum ether, the residues were and Eichelberger (260) described a thin There was no interference from the cleaned up on a Florisil wlumn for layer chromatographic cleanup of the blood but the sensitivity was limited by determination by micmeoulometric or carbon chloroform extract (of water) the size of sample that could be chro- electron capture GLC. In addition to resulting in a solution suitable for matographed (equivalent to 1 mg being fast, it was claimed that this electron capture GLC. Lerenard and blood). procedure eliminated interferences some- Simon (194)used an automatic liqnid- Radomski and Fiserova-Bergerova times found in other procedures. Cum- liquid extractor which they found ($46) described the determination of mings and wworkem (83) combined capable of extracting 8CkS@& of lindane chlorinated pesticides in tissues using features of the method of Stemp el d. and dieldrin from water at wncentra- electron capture GLC. They blended [Poullry Sn'. 43, 273 (1964)l with tions of 1ppb. Sanderson and Ceresia the sample with petroleum ether, added those of Mills el al. [JAOAC 46, 186 (263) reported on a continuous liquid- anhydrous Na,SO,, made to volume (1963)j for the analpis of eggs. The liquid extraction apparatus. With a with petroleum ether, and injected an sample was ground with Florisil and sample flow rate of 1 literfiour, r aliquot into the GLC without any anhydrous NasSO, and the mixture wveries of about 90% at the 1-ppb le cleanup. Sensitivities were reported transferred to the top of a Florisil were obtained. Tensley and Cox (87'1 in the range from 0.001 to 0.06 ppm. column. The pesticides were eluted wmpared extraction produres for* Hamence and co~vorkers(14.8) analyzed in two fractions and wncentrated for removing endri and DDT from soils. animal tissue by extracting with acetone, analysis by electron capture GLC. They reported that the Immerex extra- partitioning the residues into petro- The sensitivity was reported as 0.001 tion method was the best. The proce- leum ether, extracting with acetonitrile, ppm, and recoveries for lindane, hepta- dure involved a 16-hour extraction with and again partitioning into petroleum chlor epoxide, DDT, dieldrin, and en- whexsnmtone (9 + 1) in an Im- ether. Final cleanup was on an alu- &in ranged from 78 to 109%. merex tester, an apparatus designed for mina column. Determination mas by Moats (dlL\ used TLC to determine tbe analysis of bituminous paving electron capture GLC. To co~~firm chlorinated pesticides in dairy products mixture which uses an extraction basket identity and separate compounds with with a sensitivitv of about 0.125 nom on for the sample container. similar retention times, aliquots were a fat basis. ~kmpand ~iski'(266) Samuel (262) reported a screening reacted with HUr, alwholic KOH, and reported a simplified and shortened procedure for chlorinated and thio- chlorine, and gas chromatography wss procedure for the analysis of milk. phosphorus pesticides in daii products, repeated. Data are given for 12 com- A 10-ml samole of milk was mixed with fruits, vegetables, and animal tissue. pounds. deactivated horisil, slurried with 20% After sample extraction, a wmbiiation Stanley and LeFavoure (263) used a CHL% in ~etroleumether. and decanted of 1 or more of 3 cleanup procedures perchloric-acetic acid mixture to digest through s'column of deactivated Flori- prepared the sample for final analpis by samples of animal tissues. The fat and sil. The eluate was evaporated; the electron capture or microcoulometric pesticides were extracted with n-hexane residue was taken up in hexane and in- GLC. Rewverim of 7&1CQ% were and cleaned up on a sulfuric acid-Celite jected into an electron capture GLC. reported at levels of 0.05-2 ppm. Water column before determination by eleetron Recoveries were over90% atlevels of 0.1 soluble ~r~anothiophnsphoms wm- capture GLC. Aldrin, dieldrin, and to 10 ppm whole milk basis. It was pounds do not wme through the endrin are destroyed by the procedure. stated that 4MO samples wuld be procedure. Parker el al. ($38) combined portions cleaned up by one technician in a day. Considerable use has been mgde of of previously reported methods for the Giutrrida d d. (122) described a thin layer chromatography in the determination of chlorinated pesticide procedure for milk, fats, and oils. analysis for chlorinated pesticides. Ma- residues in animal and human tissues. .Milk was extracted with acetone and theme and Bathalter (240) described a Frozen samples were blended with Dry the residues were partitioned into pe- deanup procedure makinguse of 8- X 8- Ice to a powder and extracted with troleum ether. Fats and oils were inch plates with channels 10 mm wide hexane. Acetonitrile extraction and a dissolved in petroleum ether. The 2 mm deep which were filled with AlzO column containing Florisil, Celite, 8th- samples were transferred to a wlumn G coating. Sample extracte were spot- pulgus clay, and charcoal were used for of deactivated Florid, and after removal ted on individual chl~noels and the* cleanup before determination by elec- of solvent pesticides were eluted with plates developed twice with two dif- tron capture GLC, Onley and Ber- acetonitrile containing 10% H20. After ferent solvents. This separated the pesticide residues from the plant ex- the steam bath without loss of pesticide. the first 4 major peaks of chlordane were tracts and after elution from the Moats and Kotula (315) speeded up the small or had disappeared, nrhile the lmt scra~edoff adsorbent. the residues elution from cleanup columns by using 3 peaks were not chapged significantly. in suitable fork for electron suction. They reported that elution Gajan and Link (IlSJused oscilloscopic pture GLC. rates of 250 ml/min from Florid polarography for the determination of Kovacs (186) used 3'/p X 4-inch columns and 100 ml/min from carbon- DDT. They reported that with an for TLC and renorted that Celite columns gave good recoveries electrolyte of 0.1M tetramethyl am- as many as 26 chlorinated pesticides without adversely affecting the cleanup. monium bromide in 50% aqueous could be resolved in 5-10 minutes and Mumma and Kantner (917) made use acetone-ethanol, only DDT and those identified. The lower limit of detection of the mass spectrometer for more analogs such as methosychlor contain- for many of the commo~~lvused chlorin- positive identification of pesticides. ing the trichloroethane group gave re- ated &;ticides was 0.C65 pg. Crab- They determined the mass spectra of sponses in the -0.3 to -1.7-volt range. tree (79) used microscope slides coated several chlorinated pesticides and found The regular wave showed a peak indi- with Alsos and developed in hexane in that each gave easily recognizable cating the total o,p'- and p,pi-DDT 3'/1 minutes for rapid confirmation of molecular ion petlkr and characteristic whereas the derivative showed two identity. Beckman and Winterlin ($7) ion fragments. Their procedure was peaks whose ratios was equal to the described what they called "thin-strip to collect the GLC neaks in medicine ratio of the two isomers. thin layer c!hromatography." They droppers containing GLC column pack- Several papers dealt with the deter- used a tool to scrape mated 8 X ins material. The ~esticidewas washed mination of DDT. Dingle (90) modi- 8inch plates in such a manner that 02,concentrated, snd injected into the fied Davidow's sulffuric acid cleanup individual TLC strips or channels 4 mm mass spectrometer. The procedure has for fat [JBOBC 33, 130 (1950)) and wide were formed. As many as 20 been run on dieldrin, DDT, and DDE obtained solutions suitable for PC, channels could be used on one plate. from wheat and alfalfa. The sensitivity GLC, IR, or colorimatric determination. The advantages claimed were that, since was 0.1 ppm, and 0.1 pg has given a Recoveries rvere said to be better than the spots could not spread, sensitivity good mass spectrum. Payne and Cox 98%. Stempkovskaya and Vekshtein was increased and that it was essier to ($39) used infrared for the identification ($66) reported a modification of the remove separated spots for GLC and of chlorinated pesticide residues in Schecter-Haller technique using KNOa IR. Engst el nl. (99) used silica acid sludge, soils, industrial effluents, and or NH,NOI with *SOn in place of gel plates and reported the detection fish and other aquatic fauna. Column fuming HNOI. Thay also described a of 6 chlorinated pesticides with a sensi- and thin laver chrornatoeraohv."were stable artificial color standard consisting tivity of 0.05 pg. Abbott and co- used for the cleanup and separation of of a solution of CuCl*, crystal violet, workers (4) studied the effect of tem- the individual pesticides. Minyard and and K2Cr107 ~hichwas said to cor- perature on R, values. R, values for Jackson ($13) attempted to make respond to the color produced by 100 16 chlorinated pesticides in 16 solvent/ identification bv electron capture GLC pg of DDT. Crosby and Archer (80)

adsorbent svsterm were~ niven. Ball-~~ - ~-~ more certain through the use of flnsh determined the DDT group in milk, chmiter an2 Toelg (16) investigated heater inserts packed with various blood, and tissue as their dehydrohalo- orescence indicators for TLC. salts to modify the pesticides. A genated compounds after treatment C'wenty-four substances were studied. number of salts were investigated, and with KOII. Estraqtion mas with pen- Fluorescence or quenching of spots at the authors suggested the possible use tane and deternlidation by electron levelu of 0.02-5 pg were noted with six of several modiiers in parallel ahead of a capture GLC. Tha first foot of the reagents. Adamovic (7), investigating single column and detector. Sequential GLC column was packed with calcium spray reagents for TLC, reported that injection of an extract into the various carbide to remove $races of water and the chlorinated uesticides under ultra- modifiers would produce normal and ethanol. Beckman and coworkers ($41, violet light reacted with aromatic modified chromatograms which were after showing that the l~esticideswere amines to form characteristicallv colored characteristic of the pesticide. Lee present only in the yolk of eggs, dcter- spots even without zinc chkde or and coworkers (193) enwuntered a mined DDT and DOE by extracting the iodine. A total of 18 aromatic amines contaminant which had the same R, yolks with acetone. The estract was were tested with the 6 most promising on a silicone elastomer/Celite GLC evaporated; the residue was taken up in showing sensitivities down to 0.5 pg. column as aldrim, and on a column of hesane and cleaned up on a Florisil There have been several papers on Apiezon L had the same Rf as P-BHC. column befare determination by electron chlorinated pesticides which are of By means of infrared they identified the capture GLC. Rscoveries at 0.05- general interest to the residue chemist. eontaminant as dibutyl phthalate which 1.0-ppm levels averaged 94%. The Gunther, Hylin, and Spenger (135) they thought came from plastic con- time required for aqalysis was less than investigated the nature of the organic tainers and plastic-based paints used in 1 hour per sample. Schnntner and chlorine interferences in the total the laboratory. Schnitzerling (Z56)used gradient elution halogen methods for organic chlorine Specific hocedures. Shuman and from a cooled, water-jacketed, silicic pesticides. Using CP tracer, they Cieri ($57) reported a method for acid column to separate DDT and its have tentativelv identified the inter- determining residues of chlorbenside metabolites into indkvidual components. ferences as quat&ary chloride salts of including its sulfoxide and sulfone Compounds separated included o,p1-and lecithins. Burke el al. (59) studied the oxidation nroducta. Samules were ex- p,pi-DDT, DDE, p,p'-DDD, Kelthane, losses of pesticides in virious methods tracted according to the 'Mills, Onley, p,p'-dichlorobenzo~~~enune,and p,p'- of concentrating solutions down to Gaither orocedure and all forms of DDA. volumes of 0.5 ml or less. They found chlorbenside residue converted to the Hansen (143) reported that the that large losses occurred when the sulfone by oxidation with chromic- colorimetric meth~d of Jones and solutions were evaporated by a stream acetic acid solution. After cleanup Riddick [ANAL.CHIEM.23, 349 (1951)) of air. Losses increased as the residual on an A120s column, determination for the determinatimn of Dilan could be lume approached drynoss and the was made by electron capture GLC. made about 10 times more sensitive by rcentage losses were greater when Thrush (375) compared the electron decreasing the total volume of the final smaller amount8 of pesticides were e capture gas chromatogram of a chlor- solution while keeuine. the same ratios present. They found that by using a dane standard with that of a weathered of reagents. miero Snyder column, solutions eould he chlordane residue found on squash. Harrison (ILL), . .. ~vasable to determine rapidly concentrated to 0.14.3 ml on He noted that in the weathered residue, endrin in wildlife in the presence of large amounts (100-500-fold) of DDE herr and Watts (369) developed a rapid n~olybdenum blue reaction after wet and dieldrin by making a preliminary cleanup procedure for organophosphate digestion or Schaniger hkcombustion. separation on TLC before using GLC. pesticides which also shows promise of RI's are listed for 20 compounds. Although recoveries were slihtly better being useful as a general cleanup methud Thin layer rhromatography was used on silica gel plates, alumina gave better for many other types of wmlwunds. by a number of workers. separation of endrin from dieldrin. The procedure, called sweep codistill* (185) separated thinphosphorusKOw*co Engel et al. (97) determined heptachlor tion, makes use of a heated short glass pounds on Ah0& plates and located and heptachlor epoxide in alfalfa hay column packed with glass wool. An the pesticides with a tetrabromophenul- by blending the sample with water and ethyl acetate extract of the sample was phthlein-AgNOa spray. Rl's were ethanol and then extracting with hexane. injected into the tube, and pesticides given for 19 compounds and 11 of thw They used the procedures of Samuel and solvent were swept through into a were detectable at the 0.05-pg level. (252) for cleanup. Ott et d. ($56) receiver by a stream of Nz. Repeated Barney (18) investigated previously used thin layer chromatography and injectiuns of small amounts of ethyl reported chromogenic reagents for or- oscillop~larograph~to determine p,pf- acetate resulted in nearly complete ganophosphorus compounds and devel- Kelthane and p,p'-dichlorohenzophe- recovery of the pesticides while crop oped 2 new tests. The developed plates none. They suggested that in analyz- material remained in the tube. The were sprayed with HI solution, heated, ing crop extracts, TLC be used for effluent was clean enough for gas chro- sprayed with ammonium persulfate cleanup before polarographing the sam- matography using the thermionic detec- solution. heated amin. and s~raved ple. Mestres and Chave (m9) deter- tor. Recoveries from five crops forti- with a-onium moiybdate follokedhy mined linhne in flour by extracting fied with a mixture of earbophenothion huflered benzidine solution. The Dro- with acetonitrile, cleaning the extract (Trithion), diazinon, malathion, para- cedure determined dl compounds tested on a NaSO, and Florid column, and thion, and methyl parathion ranged except for one ~hos~honium- - comwund. then using electron capture GLC. from 89 to 101%, and only 20 minutes omitting the ammonium perklfate Recovery of 0.25 ng of lindane was 95 i was required to clean up each sample. resulted in a test which detected orpano- 3 To determine toxaphene in milk, Getz and Watts (118) reported a phosphates but only some of the fat, blood, and hay, Archer and Crosby colorimetric procedure which had a organophosphonic acids. The lower (12) used KOH to dehydrohalogenate sensitivity of 2 pg of yganophosphorus limit of detection was less than 1 fig. the toxaphene before injection into the compound. A cleaned up residue was Watts ($84) adapted the p-nitrobenzyl electron capture GLC. Advantages heated with +@nitrobenzyl) pyridine pyridine reagent (118) for use as a claimed for the procedure were very and eyclohexylamine for 2 minutes, chromogenic spray in paper and thm rapid and effective cleanup, a higher diluted, and the absorbance was read at layer chromatography. Twenty organo- and more compact peak which eluted 520 mp. The procedure worked for all phosphorus pesticides tested gave dis- before the DDT group, and a two-fold 24 organophosphorus pesticides tested. tinct and persistent spots. The test increase in sensitivity. Recoveries Getz (117) described a procedure in was sensitive to about 0.5 for both ranged from 74-95Yo at 0.1 and 0.5- which the organophosphorus compounds the thio and nonthio organophosphorus ppm levels. Faucheux (108) investi- were quantitatively converted to ortho- pesticides. Klienko (180) used All0 gated the use of diphenylamineZnCI, phosphates hv ammonium persulfate plates and 3 chromogenic sprays fo as a chromogenic reagent for toxaphene, and &en measured as molybd&mm blue detecting organophosphorus residues.e DDT, and chlordane on alumina TLC with an absorbance at 660 nu. Residues Zadrozinska (891) used silica gel and plates. Characteristic colors were ob- were sepmated on paper chromato- talc adsorbents with 16 mobile pbases. tained from these pesticides. Five pg grams, the spots were cut out, and the El-Rdai and Hopkins (96) described each of toxaphene and chlordane could determination was run directly with the the use of plates coated with cellulose be detected in mixture. DDT and piece of paper. Sensitivity was 0.1 powder containing 10% C&04 as binder, TDE could be estimated semiquantita- pg P, equivalent to about 1 pg pesticide. for the separation of organophosphorus tively when all forms xvere present. Irudayasamy and Natarajan (159) used pesticides and their oxons. Two sol- Color reactions of 34 pesticides at the paper chromatography for the deter- vent systems were used, each including 20-pg level were reported. mination of thiophosphorus pesticides. an immobile salvent phase. Four spray After development, the dried chromate reagents were used including one based ORGANOPHOSPHORUS PESTICIDES gram was exposed to bromine vapors, on choliesterase inhibition directly on air dried, and sprayed with a solution the plate which was seasitive to 0.001- General Procedures. Storherr and of Congo red. The pesticides appeared 2 ng of the various pesticides. The coworkers (268) reported a procedure as blue spots on a red background and authors discussod the choice of systems they used for the determination of five were stable for 10 days if protected from for specific separations and listed RI organophosphorus pesticides (malathion, light. The test was sensitive to 0.5 pg values. Melchiorri et al. (m) used parathion, methyl parathion, diazinon, of pesticide. Zadrosinska ($90) used Silica Gel GF 254 plates for the separa- and carbophenothion) in a number of paper. ~ chromatoaraphy- - in the deter- tion and identiJication of 13 organo- vegetables and fruits. The sample was mi~lntiot~of organopb,hosphorus pesticides phosphorus pesticides in vegetable oils. blended with acetonitrile and filtered. in various food c~o~s.He extracted the Various solvent systems are described. The extract was concentrated under sample with carbbn tetrachloride and Shm6 (250) studied the chromatog- vacuum to remove the acetonitrile and after separation by paper chromatography of 10 organophosphorus com- was then extracted with ethyl acetate. raphy used enzymatic and fluorescein pounds on silica gel using 16 solvent After cleanup on a column of carbon and methods for making the spots visible. systems and reported Rl values. He Celite, the pesticides were determined Bates (W)also used paper chromatog- used two chromogenic reagents, one by GLC using the thermionic detector. raphy but extracted the food samples (Bh, FeCla and sulfosalicylic acid) had Other aliquots were exanlined by the with acetone, eooled the extracts to a sensitivity of ahout 5 irg end the other colorimetric p-nitrobenzyl pyridine -80°C. and filtered them to remove fats (palladium chloride) a sensitivity of method (118), a total -.phosphorus prow- and waxes and used a MgO column for about 2 pg. Stanley (26.2) used 3 dure (fly), and by paper chromatog- further cleanup. The pesticides were 1-inch microscope slides coated wia raphy. Watts and Stmherr (285) separated and idenaed by 2-dimen- silica gel-G. He listed R, values for 31 described a rapid extraction procedure sional paper chromatography. For organophosphorus compounds in 6 sol- in which the crop sample was extracted quantitiation, the spots were cut out and vent systems and described 7 spray by blending with ethyl acetate. Stor- phosphorus was determined by the reagents. cides. In addition to giving the spec- of 0.1 ppm. The two compounds could tra, rearrangement and fragmentation be separated from each other and from patterns are discussed. other insecticides through procedures Specific Procedures. Ijlinn and that are described. Paaarela (41) used a colorimetric pro- The insecticide, diethyl-l-(2,bdi- cedure to determine Abate, (0,0, chloropheny1)-2-chldrovinyl phosphate, 0',0' - tetrsmethyl - 0,O' - thiodi - p - or compound 4072, is known in England able to separate any combination of 19 phenylene phosphorothioate) in water, as chlorfeuvinphos or by the trade organophosphorus pesticides by the mud, and oysters. After extraction name Birlane. Clhorn and Ivey (70) use of three GLC columns, although no and cleanup, the Abate was hvdro- described a procedure for its deter- one column gave complete separation lyzed to 4,4'-thiodiphenol and reacted mination in milk and tissue in which of all 19 compounds. Hrivnak and with 4-aminoantipwine.. under oxidizing compound 4072 is hydrolyzed to 2,2',4'- Pastorek (15:) reported the successful conditions. The color formed was trichloroacetophenone and determined separation of I1 0,O-dialkyl-O-(4- extracted into butanol and read at 510 as such by electron capture GLC. nitropheny1)thiophosphates describing I. Recoveries from water at 0.02% Beynon and coworkers (54) reported the columns and operating conditions 0.045 ppm levels ranged from 79 to the analysis for compound 4072 in soil used. Nelson (223) used microcoulo- 8870 and from mud and oysters at 0.1- and crops. After extraction of the metric GLC for the determination of 16 0.66 ppm levels, 60 to 82%. sample and cleanup On a Florisil column, thiophosphates in 25 c.rops at levels as Katague and Anderson (174) used the insect,icidewas determined either by low as 0.1 ppm. Samples were ex- electron capture GLC for the deter- cholinesterase inhiution or by electron tracted by the Mills, Onley, Gaither mination of Bay 37289 (0-ethyl-O- capture GLC. Cmpound 4072 con- procedure [JAOAC 46, 181 (1963)l 2,4,5 - trichlorophenyl - ethylphos- sists of 6Yo cis isomer and 90% trans. and the residues partitioned into petro- phonothioate), its oxygen analog, and When these isomers were gas chromato- leum ether before the gas chromatog- Z,4,5-trichlorophenol, in a number of graphed as the intact compounds, they raphy. Recoveries of over 70% were crops including alfalfa, beans, carrots, had different retention times. Sensi- obtained for all compounds except and potatoes. After extraction of the tivity of the two determinative proce- Guthion (16%), demeton (46%), and sample with acetone/benzene, the 2.4.5- dures wasabout equd, 0.01 ppm. Robin- dimethoate (0%). Later modific~tions trichlorophenol wss removedwith 0.1~ son et al. ($47) determined compound (224) increased the recovery of di- NaOH for separate determination. Bay 4072 in sheep fat, limr, and other tissues. methoate and Guthion to 70-98Yo. 37289 and its oxygen analog were the; The parent compmund and its me- Cook and coworkers (74) studied the hydrolyzed to 2,4,5-trichloropheno!, tabolite, trichlorqcetophenone, mere electron capture response of 7 organo- acetylated, and injected into the GLC. separated from chlorinated pesticides phosphorus pesticides in an attempt to The sensitivity of the method was and from each other on a column of un- correlate structure to response. They about 0.1 ppm with recoveries ranging activated Florisil. Each was then deter- found that in general the electron from 75 to 104%. mined by electron capture GLC. The ffinity changed in the manner: Several procedures have been reported sensitivity for compound 4072 was for the determination of Bidrin (di- 0.003 ppm and for the trichloroaceto- 0 S methvl ~hos~hateof 3-hvdroxv-N.N- phenone 0.001 ppm. Bazzi and Fab- II

water salted out and the acetonitrile~~~ The method, which is specific for anisidine to give a yellow color with extract evaporated. The residue was methyl vinyl phosphates will thus also maximum absorbance at 375 mp. subiected to distillation under a vacuum detect Phosdrin and phosphamidon. Boone (45) used microcoulometric of &out 0.5 micron with the pesticide Murphey et d. (219) described a GLC to determine DDVP and naled residues being collected on a cold finger ~rocedurein which Bidrin was hvdro- (Dibrom) in apples, cabbages, and cooled with liquid nitrogen. The resi- iyzed with NaOH and the resilting carrots. A silicic acid column was used due~were rinsed off, adjusted to volume, dimethvlamine distilled and determined for cleanup. Buachler et al. (51) and injected into a GLC. For identifi- colorim~trica~l~as dimethyl dithiocar- modified and improved the resorcinol cation, the peaks of interest were col- banlate following addition of Cui2 and method for the detqrmination of DDVP. lected and their infrared spectra ob- CSz. Recoveries from alfalfa, lettuce, El-Refsi and Giuffrida (95) used tained. Hermann (151) used frustrated orange peel, string beam, etc. at levels GLC with the thermionic detector multiple internal reflection (FMIR) of 0.2-10 ppm ranged from 80 to 108%. to determine DDYP and trichlorfon infrared for the identification of trace The color reaction is specific for di- (Dipteres) in water and in formulations. amounts of organophosphorus pesticides alkylamines. Thus, N,N-dimethylcar- They also studied the hydrolysis rates eluted from column, paper, and thin bamates such as Dimetilan isolan, and of each pesticide and the rate of con- layer chromatograms. Pvrolan would interfere and could be version of trichlorfon to DDVP. An- Nangniot (221) determined 22 phos- determined by this reaction. Lau (192) derson and coworkers (10) reported a graphic~horicacid polarography. ester pesticides byOperating oscillo- used cholinesterase inhibition for the method for the &termination of tri- a determination of both Bidrin and the chlorfon and its metabolites, chloral conditions for each are listed. closely related Azodrin (dimethyl phos- hydrate, and trichloroethanol, in plant Damico (55) determined the mass phate of 3-hydrosy-N-methyl-cis-cro- and animal tissue using electron capture spectra of 23 organophosphorus pesti- tonamide) in crops with a sensitivity GLC. After extraction and cleanup,

148 R . ANALYTICAL CHEMISTRY trichlorfon and chloral hydrate were methoate gave a straight line. Re- Szyszko (296) used oscillographic injected into the GLC with trichlorfon coveries from fruits and vegetables at polarography for the detennination of breaking dovn and both compounds 0.1-0.5 ppm levels ranged from 80 Guthion. A most characteristic curve registering as chloral. To determine to 108%. George eI al. (116) described wm obtained using a pH 4.0 acetate the trichlorethanol, a separate aliquot a colorimetric method for dimethoate in bufferas electrolyte. was acetylated to form trichloroethyl- plants and milk. After extraction and Bowman and Beroza (46) reported acetate and then cbromatographed. cleanup, the residue was treated with two procedures for the determination Mustafa et at. (220) used a wlorimetric methanolic NaOH and 1-chloro-2,4di- of Imidan [O,O-dimethyl-S-phthalimi- procedure for determinimg trichlorfon. nitrobenzene to form a wlor which was domethyl phosphorodithioate] in milk The sample was spotted on a filter paper read at 505 mp. Although the oxygen and wrn plants. After extraction and impregnated with 3,5-dintrobenzoic acid analog would react, it did not come cleanup, electron capture GLC wna used and heated at 'iOoC. for 2 minutes. through the cleanup. The authors for the determination step using a Trichlorfon gave a bhe spot, which was tested 33 insecticides, 3 herbicides, and wlumn which was preconditioned by measured in e densitometer with a 550 1fungicide and found that they did not injection of Imidan just prior to use. mp filter. The reaction, based on the interfere with the analysis. Smart A colorimetric method, based on the cleavage of the P -C bond and reaction (259) compared three colorimetric pro- hydrolytic cleavage to liberate fod- of the phosphite estors with the 3,Sdi- cedures for the determination of di- dehyde which was then reacted with nitrobeuzoic scid, distinguished between methoate in fruits and vegetables, and chromohpic acid, was also described trichlorfon arid DDVP, which did not reported that the procedure of Chilwell although it was not so good as the GLC react. Szyszko (898)reported an oscil- and Beecham worked best. Bache and procedure. lopolarographic method for trichlorfon Li (15) reported the use of GLC with Gutenmann and coworkers (1%) in which l'mdane and DDT did not the emission spectra detector (.%?Oh)for abo used electron capture GLC for the interfere. However, maneb and zineb the determination of dimethoate and determination of Imidan. They re- did interfere. Szyszko (297) also re- phorate in soil. ported that Imidan, its oxygen analog, porkd an oscillopolarographic method Blinn and Boyd (10) reported a folpet, and phthalic acid dl had the for demeton-S-methyl in foods where colorimetric as well as a thin layer proce- same retention time. It was therefore nmneb and zineb did not interfere. dure for the detenninalion of the 61thio- beKeved that aU broke down to phthalic Giang and Schechter (119) described a lane insecticides, 2diethoxyphosphimo- anhydride on GLC. method for the determination of de- thioylimino-1,3-ditbiolane,and its oxy- Gudzinowicz (1%) described some of meton and its metabolites in fruits and gen analog. After extraction and clean- the GLC properties of fenthion (0,O- vegetables. After extraction and cleanup, the insecticides wuld be determined dimethyl-0 - 14- (methy1thio)-m-tolyll- up, the parent compounds and me- on TLC plates made with equal parts phosphorothioate) also known as Lebay- tabolites were all converted to the sul- of silica gel-G and silica gel-HF. rid. He used both electron capture and fanes by oxidation with m-chloroperhen- Under ultraviolet lit, the compounds flame ionization detectors and reported zoic acid. After additional cleanup on appeared as dark areas on a fluorescent that as little as22 ng was easily detected. a cellulose column, the residue was dis- background. In the wlorimetric proce- Koivistoinen et d. (Be) studied solved in CSa and read in a 5-mm in- dure, the cleaned up residue was procedures for the extraction of frared cavity cell using 5x scale treated first with acid and then with thion from fruits using a wlorimetric expansion. The absorption at 7.55 alkali to form thiooyanate which was procedure for the determinative step. microns was used for calculation. converted to cyanogen bromide and They reported that for samples ana- Recoveries at 0.6 ppm levels ranged reacted with bensidine in pyridine to lyzed 2-3 days after pesticide appliea- from 76 to 102%. form an intense red solution with an tion, tumbling the unmacerated fruit Gilmore and Cortes (120) used dual absorption maximum at 530 mp. with benzene gave the highest values. band TLC as cleanup for the determins- Adams and Anderson (8)reported a However, for samples with longer tiou of diazinon. By means of a spectrophotofluorometric procedure for periods between application and anal- divider in the applicator, the plates were the determination of Guthion [0,0- ysis, procedures which &led for blend- coated with a mixture of Darw G60 and dimethyl - S - 4 - 0x0 - 1,2,3 - benzo- ing of sample with pohr or mixed Solka-Floc on the lower 4 cm and with triaziu-3(4H)-ylmethyl phosphorodi- solvents gave higher values. Mestres silica gel H on the remaining 16 cm. thioate] in milk and meat. After ex- and Chave (210) described a pmeedure The crude extract along with a standard traction and cleanup by liquid-liquid for the determination of malathion in mere applied to the charcoal-cellulose partitioning and the use of an alumina flour which involved extraction with band, and after development, the sample column, the pesticide was hydrolyzed to acetonitrile and petroleum ether and spots, located by comparison with the anthranilic acid, and divided into 2 Florisil wlumn cleanup. Determina- standard R,'s, mere removed for analy- equal aliquots; standard hydrolyzed Gu- tion was by GLC using paired them- sis. Fifty grams of spinach was puri- thion wes added to one. The fluores- ionic and flame ionization detectors. fied by the above procedure and re- cence of both solutions was measured at Sensitivity was reported as 0.1 ppm. coveries as followed by S35 labeled 400 mp using an activation wavelength A number of workers reported pro- diazinou, averaged 957Z0. Abbott and of 340 mp. The oxygen dog was cedures for the determination of para coworkers (2) reported the use of measured as well as the parent wm- thion. Lodi (195) used electron capture multiband chromatoplates for the TLC pound. Sensitivity of the procedure GLC for its determination in wine and determination of dimethoate. They was reported as 0.005 ppm in milk, biological materials. With wine, a prepared plates having 3 bands of 0.02 ppm in tissue, and 0.03 ppm in fat. preliy cleanup by paper or thin different adsorbents and spotted cleaned Anderson and coworkers (11) used a layer chromatography was needed. up sample extracts. Development somewhat similar procedu;e 'for the Ott el d. (284) described a rapid thin separated the dimethoate from the re- determination of the anthelmintic. N- laver orocedure havinn a sensitivitv of maining plant nlateriah and most hydroxynaphthalimide diethyl ihos- other organophosphorus compounds. phate. Based on the procedure of P. The dimethoate spots were made A. Giang [J. Agt. Food Ch.9, 42 visible by spraying with Brilliant green (1961)l for the sulfur analog, Bayer Szyszko (295) used oscillopolarography and exposure to bromine vapor. The 22,408, the fluoresmce ,was measured in which zineb did not interfere but square root of the spot areas plotted at 480 nyl using an activation wave- web did. Beckman and coworkers against the log. of amount of di- length of 372 ma. (25)analyzed for parathion in cole crops, using a Florisil column to remove crop potato extract. Adsorbents, solvent the official AOAC method to determine interferences and chlorinated pesticides systems, and spray reagents were dis- carbaryl in bees, using a Florisil column before the final determination by either cussed. In a later publication, Henkel for additional cleanup. electron capture GLC or the Avrrill- (150) reported on 3 thin layer chromato- Gajan el al. (118) keported an oscillo- orris colorimetric method. graphic systems and 5 chromogenic graphic polarographic procedure Moye and Winefordner (216) re- color development systems for a number whereby carbaryl could be determined in aported a rapid method for the deter- of N-methyl and N,N-dimethyl car- the presence of a-naphthol. Using a minat,ion of p-nitrophenol in urine, using hamates. Limits of detections for these modified cleanup, recoveries of car- phosphorimetry. The method could carbamates ranged from 0.05 to 0.15 haryl from fortified crops averaged determine as little as 0.01 pg in 5 ml of pg. Hylin (158) used thin layer chro- 95% at levels from 0.2 to 10.0 ppm. urine in 40 minutes with an average matography to determine the dithio- Among a number d pesticides tested, recovery of 88%. Skuric (258) de- carbamates on leaves. R, values were only o-phenylphenol interfered. Engst scribed a fluorometric method for the given for ziram, t,hiram, zineb, maueb, and coworkers (10) formed nitro determination of methyl paraoxon based and others. Sensitivity mas approxi- derivatives of carbaryl by treatment on the oxidation of indole in the mately 2.5 pg. with HNOI. These derivatives were presence of methyl paraoxon. Zielinski and Fishbein (293) presented then determined quantitatively by both Winnett and Katz (286) described a data on the GLC behavior of N-sub- d.c. and pulsed pdlarography with a - colorimetric procedure for phorate (Thi- stituted carbamates on 3 different sensitivity of 0.5 and 0.005 ppm of car- met) in vegetables in which the cleaned columns while Fishbein and Zielinski haryl, respectively. up phomte residue was hydrolyzed with (107) described the GLC behavior of Finocchiaro and Benson (105) used I HBr and the released HzS determined as the trimcthylsilyl derivatives of a thin layer chromatography for the P methylene blue. Claborn and Ivey number of carhamates and urea. determination of c&rbaryl. After the (69) determined ronnel in milk and in Damico and Benson (86) developed and samples were spotFd and the phtes animal tissues, using electron rapture tabulated the mas spectra of 14 car- developed, the carbaryl was hydro- GLC after cleanup on a Florisil column. bamate pesticides. The significant lyzed by spraying with KOH and then As little as 0.001 ppm could be deter- fragmentation ions were noted and ions coupled with p-nitfobenzenediazonium mined in milk and 0.005 ppm in tissues. were postulated for 8 N-methylcar- fluoborate to produce blue spots. The Sulfotepp was measured by oscillo- bamate rearrangements. Chen and procedure was sensitive to about 0.05 polarography by Szyszko ($99). Renson (63) reported the infrared spec- ppm and distingui$hed carbaryl from Sumithion [0,0 - dimethyl - O - (3 - tra of 32 carbamate pesticides and wnaphthol, which had a lower RI. methyl - 4 - nitrophenyl)phosphorothio- model compounds. The characteristic Dmgle (91) determined carbaryl in ate] has been determined colorimetri- absorption frequencies and associated cattle dipping solutions by simple cally by alkaline hydrolysis to sodium structures were tabulated in a summary dilution with eths~loland measuring 3-methyl4-nitrophenolatewith the ab- and presented in a correlation chart. the absorbance at a80 m*. Correction sorbance at 400 mp being measured for Broderick et al. (49) reported that for cl-naphthol was based on its absorb- uantitation. ICovac and Sohler (184) methyl anthranilate, because of its ance at 324 mp. sed this procedure following extraction absorption bands at 2.86 and 2.95 Johnson and Btansbury reported 0 and cleanup on .41,0a thin layer plates microns, interfered in the infrared similar colorimetric procedures for the to determine Sumithion in fruits and determination of methylcarhamates. determination of Temik, 2-methyl- vegetables at levels as low as 0.1 ppm, They described a method for removing 2(methylthio) pmpionaldehyde O- while Franz and Kovac (110) reported a methyl anthranilate in the analysis of (methylcarbamoyl) oxime (165) and similar determination in milk. Oi and Concord grapes for carbamate residues. for Tranid, 3-eso.chloro-6-endocyano- Umeda (827) used infrared for the Specific Procedures. Johnson and 2 - norbornanone - O - (methylcar- simultaneous determination of Sumi- Stansbury (162) reviewed the various bamoy1)-oxime (166). The oxime t thion and methyl parathion in spinach methods for determination of carbaryl carhamates were hydrolyzed with NaOH P and lettuce at about 1 ppm. The in a variety of products and in water, to form the oxime which was then absorption peak at 10.3 microns was and described extraction and cleanup hydrolyzed with HCI to release hydrox- used to measure the Sumitbion and the procedures. ylamine. The hydtosylamine was oxi- pal; at 10.8 microns for methyl par* Gutenmann and Lisk (139) used dized with iodine to nitrous acid which thion. Coahran (78) reported the use electron capture GLC for the determi- diazotized sulfanilic acid. The latter of GLC with the thermionic detector for nation of carbaryl in various crops. was coupled with l-naphthylamine to the determination of Zinophos (0,O- After extraction and cleanup, the form a color which was read at 530 mp. diethyl-0-2-pyrazinyl phosphorothio- carbaryl was hydrolyzed to a-naphthol, The sensitivity of these methods was ate) in soil following overnight estrac- which was then brominated with I and xported to be abowt 0.03 ppm. Niessen tion in a Soxhlet. 13rz in glacial acetic acid. The hro- and Frehse ($85) described a colori- minated residue was taken up in benzene metric procedure for the determination CARBAMATES and injected into the GLC, which actu- of Bayer 30,007 (Baygon, Unden) ally determined brominated l-naphthyl (o-isopropoxyphenyl methylcarhamate) General Procedure. Eberle and acetate. Van Middelem and coworkers in leafy vegetable& After extraction Gunther (94) conducted an extensive (278) reported a somewhat similar and cleanup, the eesticide was saponi- investigation of 5 carbamates-car- technique in which the a-naphthol fied, neutralized, and treated with baryl, Dimetilau, isolan, Pyrolan and formed by hydrolysis w;ls brominated triethanolamine, anninoantipyrinc, and Zectran. They studied the effect of with bromine and the electron capture K3Fe(CN), to form a color read at 490 natural sunlight and ultraviolet light GLC determination made of brominated w. on these compounds and presented n-naphthol. Results were reported for useful basic information concerning their levels as low as 0.1 ppm. Benson and nalytical behavior with GLC, TLC, Finocchiaro ($8) modified the official oscillographic polarography, and fluo- AOAC colorimetric method for carbaryl Enzyme inhibition coutinues to be a aresrence spectrometry. Henkel (149) [Johnson, D. P., JAOAC 47,283 (1964) ] useful tool in pesticide residue work. described the TLC behavior of herbicidal to eliminate the need for special equip- Its lack of specificity, objectionable as carhamates and presented methods for ment and to shorten the timeof analysis. it may be in many uses, actually en- their determinatiou in soil, water, and Johnson and Stansbury (164) modified hanres its value as a screening tool,

150 R . ANALYTICAL CHEMISTRY

* /' since dangerous amounts of inhibitor HERBICIDES Recoveries ranged from $9 to 93% at may be detected no matter of what 0.00024.4 pprn levels. Crmby and nature. I3eynon and Stoydin (37) Faust and Hunter (104) have reviewer1 Ilowers (81) reported a method for the reported such a rapid screening test for the chemical methods for the detection determination of 2,4D in milk and other cholinesterase inhibition making use of of aquatic herbicides includmg diquat, high protein samples where the 2,4 agar-agar plates. As little as 0.001 paraquat, and the phenoxy alkyl acids. may he hound to the sample. The pg of DDVP and other inhibitors could They discussed various cleanup and refluxed the sample with NaOH anrn be detected. determinative procedures. Henkel methanol to release the 2,4-D which was Ortloff and Franz (830) conducted (148) reported on the TLC behavior of methylated for electron capture GLC the test for detection of organo- the phenoxyalkyl acid herbicides. Ad- determination. Yip (288) used pm- phosphorus pesticides on TLC plates, sorbents and pretreatment, liquid grammed temperature microwulometric using either 2-azobenzene-1-naphbhyl- phases, R,'s and reagents for detection GLC to determining a number of the acetate (yielding white spots on a were discussed. Homgai and Kawsshio chlorinated herbicides in oils. Rewv- red background) or indoxyl acetate (156) separated 16 herbicides in mixtures eries ranged from 87 to 113% at 0.02- (white spots on blue) as substrate. by TLC, using various nonpolar and 0.08 ppnl levels. Yip and Ney (%9) Ackermann (6) used silica gel TLC polar solvents. Johnson (168) described deterrnioed free 2,4-D and its atera in plates for the semiquantitative estima- a colorimetric method for the determi- milk and forage. After extraction, tion of organophosphorus and carbamate nation of N-1-naphthylphthalamic acid cleanup, and methylation, determina- pesticides. Beam and Hankmson (83) in cabbage, asparagus, and alfalfameal. tion was made by both miorocoulo- reported s, procedure for the determina- The sample was heated with zinc and metric GLC and paper chromatography. tion of organophosphorus compounds NaOH and the released 1-naphthylamine Flansggn d al. (108) reported a paw and carbxryl in milk based on cholin- steam distilled. After cleanup, the 1- chromatographic procedure for dalapon, esterase inhibition, naphthylamine was wupled with diazo- using AgIi08 and phenoxyethanol as Several workers described the auto- tized sulfanilie acid and the absorbance chromagenic reagent. Smith and CCF mation of cholinesterase inhibition read at 535 m+ workers ($61) described a method for determinations using the Technicon Olney and wworkers (88) used a dicamba & ndk and a number of crops, AutoAnalyzer. Among these are Voss modified vrocedure for the electron using electron capture GLC after meth- (883) and Ott and Gunther (831) capture Gkdetermination of amiben ylation. Meulemans and Upton (211) whose proeedurcs required prior extrac- in vewtables. The eamvle was dkested determined dichlobenil and its metab- tion and cleanup. In a later publica- withalkali to release a&ben from any olite 2,6-dichlorohenzoio acid in fruits, tion, Ott and Gunther (838) used the complexes. After extraction and clean- soil, water, and fish. The two were spots scraped off a TLC plate as input up, it was methylated and further separated and determined by electron sample for the AutoAnalyzer. cleaned up on a Florisil wlumn before capture GLC after cleanup. The di- Guilbault and Kramer (188) re- being injected into the GLC. Sensitiv- chlohenil was chromatographed as such ported 2 new fluorogenic substrates, ity of 0.01 ppm was reported. but the metabolite was first methylated. resorufin butyrate and indoxyl acetate. Hilton and Uyehara (162) modified Beynon and coworkers (36) reported an Both are uoufluorescent compounds the colorimetric procedure of Storherr electron capture GLC method for which are hydrolyzed by cholinesterase and Burke [JAOAC 44, 196 (1961)l to determination of dichlobenil and Chlor- to highly fluorescent materials. As determine amitrole in sugar cane. Re- thimid (2,6-dichlorothiobenzamide)*in little as 0.0003 units/ml of horse serum coveries ranged from 71 to 125% at crops, soils, and water. Several extra* cholinesterase could be determined. levels of 0.0025 to 0.5 ppm. Pease tion and cleanup procedures and 3 GLC However, in addition to cholinesterase, (840) used temperature programmed, columns are described. Rewveriee such enzymes ils acylase, acid phos- microcoulometric GLC for the determi- ranged from 80 to 100D/o at levels of phatase, and chymotrypsin also hydro- nation of bromacil in tissue, plants, and 0.03-5.0 ppm. Boyack et al. (48) wed lyzed the substrates to varying degrees. soil. Recoveries averaged 98% at levels GLC with a &me ionization detector to Bauman et d. (81) reported an imrno- of 0.04 to 5.6 ppm. determine diphenamid in vegetables and bilized enzyme system which could be A number of methods have been peanuts, with a sensitivity of 0.05 ppm. used for continuous monitoring of reported for the determination of 2,4-D Endhardt and McIiinlev (98) substrate concentration and thus for and other chlorophenoxy alkyl acid studied the polarographic behsvior of the detection of cholinesterase inhibi- herbicides. Hagin and Liswtt (141) diuuat. Using previouslg vublished ex- tors. A urethane foam pad was Jercribwl R procPdure for rhc determine+ trhtion and &anup prb&dures, they impregnated with starch-immobilized tion oi2.4-1) and 2,11)Rill forw1.111ant- were able to determine diquat polaro- cholinesterase and a solution of the which made use of quick free& and graphically at levels as low as 0.01 ppm substrate, butyrylthiocholine, passed blanchii of the plant material before with recoveries of 8P97%. through it. Any inhibition acting on extraction. Determination was by elec- Calderbank and Yuen (61) described the enzyme reduced the hydrolysis to tron capture GLC after esterification an improved ultraviolet method for easily oxidized thiocholiue iodide. This with diammethane. diquat in potatoes. After extraction caused a change in current flowing Meagher ($05) reported a procedure and cleanup on a cation exchange ool- through 2 platinum electrodes placed for 2,4D and 2,4,5T in citrus. The umn, the diquat was reduced to a free on opposite sides of the pad and thus peel was extracted by blending with hot radical with sodium dithionite and its signaled the presence of an inhibitor. acetone, and the bound, the free acid, absorbance read at 379 nut. Earlier, Guilbault and Kramer (131) used a and the ester forms were separated, and they had reported a sidm method for similar immobilized enzyme pad in a each was hydrolyzed to the free acid. paraquat (60). Katz (176) reported continuous fluorometric system for The free acids were esteriIied with Z both colorimetric and TLC procedures determining anticholinesterase com- butoxyethanol saturated with HC1 gas for five substituted urea herbicides in pounds in air and water. The sub- and cleaned up on a Florisil column water. After extraction with chlom strates, the acetyl and butyl esters of before determination by electron cap form, diuron, monuron, linuron, ne 1- and Znaphthol, which do not ture GLC. Chromatographing the com- uron, and fenuron were hydrolyzed,9 fluoresce, were continuously passed pounds as their butoxyetbyl esters had diazotized, and coupled with N-(1- through the pad and were hydrolyzed the advantage that the long retention naphthy1)ethylenediamine dihydrochlo- to the fluorescent phenols. Upon inhi- times separated the peaks from inter- ride to form magenta dyes which were bition, the fluorescence dropped. ferenees present near the solvent front. extracted with ?I-butanol and read at

VOL. 39, NO. 5, APRIL 1967 151 11 555 mr. TLC mith ninhydrin spray which was steam distilled, diizotized 5 ppm in citrus fruit and 5 mg/wrapper. reagent wm used for identificaittion of the and coupled with N-(1-naphthy1)ethyl- McCarthy and Winkfordner ($02) com- specific herbicide. enediamine The absorbance, memured hind a TLC cleanup with phosphori- Gutennmann and Lisk (I/@) ued elec- at 530 mp, prmitkd detection as low as metric determinatidn for biphenyl in ron capture GLC to determine DNOC, 0.05 1q,rn. oranges. For the phosphorimnetry they Several workers have reported meth- used an exitation wwelength of 275 mp milk,NOSBP,apple*,iosynil, and grains. and hromoxynil They noted in 6 ods for the determination of the 8- and emission of 470 mp. Vogel and that ' reacting the phenolic pesticides triazines. Mattson et al. ($01) described Deshusses (281) reported a GLC proce- with diazomethane to form the methyl a procedure for the determination of dure for biphenyl in citrus fruit and ethers eliminated trailing on the GLC. both chloro and methylmercaptyl s-tri- mappers. The biphenyl was distilled Roggs (42) also reported the superior azines, using miaocoulometric GLC and absorbed in cyclohexane, which was chromatographic behavior of the methyl with the chlorine and sulfur cells, respec- injected into a GLC with an ionization ethers of the dimitrophenols. Bache tively. A sensitivity of 0.05 ppm was detector. Sensitivity was reported as and Lisk (14) reported a similar GLC attainable for most crops and recoveries 0.5 ppm. Viel (879) reported a colori- procedure for ioxynil hut used the emis- ranged from 75 to 107%. Abhott and metric method for the determination of sion spectrometric detector of Mc- wworkers (1) used thin layer chroma- captan and folpet im grapes and straw- Cormack, Tong, and Cooke ($04) to tagraphy to determine 8 s-triazines in berries. After extraction and cleanup, measure the atomic iodine line at 2062 soil and water. Using silica gel G as the the dried residue was treated with A. Ford and coworkers (109) described adsorbent the developed chromatograms pyridine and then with KOH and the a colorimetric procedure for the deter- were sprayed with an 0.570 solution of absorbance read at 435 mp. Fishbein mination of norea (Herhan) in vege- Brilliant green andexposed to Brafumes. et al. (106) used thin layer chromatog- tables, grains, and oil seeds with a The triazines appeared as deep green raphy on silica gel f$r the determination sensitivity of 0.1 ppm. The herbicide spots on an off white background and of captan and Captap; (2-mercaptohenzo- was hydrolyzed by caustic to dimethyl were immediately marked in outline. thiazole). As chtomogenic reagents, amine and the primary bicyelic amine For quantitation, the square root of the they used resorcinol in glacial acetic acid which were both steam distilled. After area of the spots plotted against the log for captan and cuptic chloride-hydros- reaction with I-fluoro-2,4-dinitroben- of weight trihe gave a straight line. ylamine for Captes. Cheng and Kilgore zene, the complex with the bicyclic Manner (197) also used TLC to detect 8 (64) described an electron capture GLC amine was separated out on an alumina s-triazines on silica gel GF254. Ninety- method for the determination of Botran column and the absorbane in alkaline one mobile solventBYstem and Rj's for (2,6-dichloro-4-nitroaniline) in stored dimethylformamide read at 443 mp. each are listed. Plates were examined fruits. A sensitivity of 0.01 ppm was Koivistoinen and Karinpiili (181) re- under ultraviolet light (254 mk) with the attained by tumbling the macerated ported a modified procedure for IPC s-triazines appearing as dark brown sample mith benzene, drying the benzene and CIPC on fruits and vegetables. spots on a yellowish green, auorescing with Na2S04, filtming, and injecting Samples were extracted by tumbling background. The spots could be eluted into the GLC. %gel and Deshusses with benzene and the herbicides hydro- for additional determinations. Radke (880) reported a ipolarographic proce- yaed. Theamines were steam distilled, el al. (344) evaluated the pyridinealkali dure for 2,6dlchloro4-nitroaniline ediazotized, and coupled with N-(1-naph- colorimetric method for the determina- which had an accuracy of +3Yoat levels thy1)ethylenediamhle; the absorbance tion of atrazine. They showed that the of 2-7 ppm. was read at 555 rup. Recoveries from color intensity increased with acidity Hoffman and mworkers (154) re- spinach, cabbage, tomatoes, and straw- of the system and that 20' * 2' C was a berries ranged from 86 to 113% at suitable temoerature for color develop- 0.5-200 ppm levels. ment. Chiba and Morley (66) reported in tobacco. For the colorimetric deter- Pease (842) described a gas chroma- a microcoulometric GLC method for tri- mination, the residqes were extracted by tographic method for the determination chloroacetic acid in wheat sensitive to blending with benmne, cleaned up on a of the herbicide 3-cyclohexyl-5,6-tri- 0.1 ppm. Compounds suchas Kelthane, Florisil column, evqporated, dissolved in methyleneurncil in sugar beets and soil. which could break down to give CHCls absolute alcohol, tlriethylamine added, Using the Rame ioniaation detector, crop interf~red. and absorbance read at 640 mp. In the blanks ran as high as 0.04 ppm. Merkle TLC method, the developed plates were et al. (207) used electron capture GLC FUNGICIDES sprayed with diethjilamine and the spots

romuared~~~ with standards. Miller (2121 after methylation to determine picloram . ~~ (4-amino-3,5,6-trichloropicolinicacid) in Gunther and Ott (137) described a investigated 4 col&imetric methods foi. soil. They noted that the acidity of fully automated procedure for the deter- dichlone and combined uarts of 2 for the extracting solvent (acidified acetone) mination of biphenyl in citrus fruit rind. collaborative studp. T& sample was was very important. It had to be acid The sample was automatically homog- strio~ed.. with benzene and cleaned uu on enough to convert the picloram to the enized and steam distilled; waxes and aFlorisil column, a@dcolor wasdeveloped free acid but not soacid as to convert the oils were removed from distillate and the with dimethvlamine for reading at 495 amino group to a quaternary salt. biphenyl in cyclohexane was fed through mp. Ten collabodators analyzid sam- Kerr and Olney (176) determined a cell of a recording ultraviolet spectro- ples containing 0154.0 ppm dichlone trifluralin in vegetables by electron photometer. Chioffi (67) used TLC on and obtained recoveries with an overall capture GLC and prometryne by hy- silica gel to determine biphenyl and range of 78-112%. Sensitivity of the drolysis to hydroxypropasine which was o-phenylphenol in lemons. Norman and method was 0.25 measuredspectrophotometrically. Dres- coworkers (926) used TLC for cleanup Thornton and 'Tm,nderson ($73) used cher (92) described 2 methods for deter- in the determination of biphenyl in citrus electron capture GLC for the determina- mining pyrazon. In one procedure fruit and wrappers. Sample extracts tion of Chemagra 2635, a mixture of which can be used for detection on paper were smtted on Eastman silica gel 1.2.4-trichloro-3.5-tlinitrobeneene and r thin layer chromatograms, the pyra- chromagrams and, after development, 1:2;3-trichloro-4;6-dinitrobenzene. The zon was diaaotized, losing its chlorine the spots were located under ultraviolet sensitivity of the method vas 0.1 pum eatom, and was then coupled with 2- light: The spots were then cut out, and recoGeries frmm cucumbers, pow naphthol to form a dye. In the second extracted with ethanol, and the absorb- toes, spinach, cottbnseed, etc. were over procedure, pyrazon was treated with ance of the biphenyl was measured at 85%. Lyalikov and Solonar (196) de- NaOH-methanol to split off dine 248 m. Sensitivity was reported as scribed the polarographic determination of hexachlorobutadiene and stated that interferences. They found that the flu- determining mercury in rice following other chlororwnic comoounds did not dinestion with nitric and nerchloric interfere. acids. An excellent and thorough study Cullen and Stanovick (88) used elec- trati& over the range of 0.2-50 fig. of the dithizone method for mercury in tron capture GLC for the'determination They later reported (132) an ultra-sensi- foods was recently reported (169). of korax, 1-chloro-2-uitropropane, in tive specific qualitative test for cyanide, Each step in the procedure was eval- vegetables. The sample was blended using pnitrohenzaldehyde and o-dinitro- uated and the resulting method studied with benzene and methanol and after benzene to form a highly colored blue collaboratively. Rewveries at 0.1 ppm washing and drying the benzene solution complex by which as little as 3 nano- were exdent and the sensitivity was was injected into the GLC. Recoveries grams total cyanide could he detected. thought to he 0.05 pprn (dried sample). averaged 80-102% at 0.W54.1 pprn Steller et al. ($64) described a colori- Neutron activation has also been used levels. Voloshchenko and Klisenko metric method for cyanimide on cotton- for the determination of mercury. IGm (888) described a colorimetric method seeds. The seeds were extracted by and Silverman (177) used it for the for the determination of Mylone, (3,5- tumbling with water followed by cleanup analvsis of wheat and tobacco, makinga dimethyl - 1,3,5,2H - tetrahydrothiidi- with activated charwal. The cyan- che&cal separation after irra&ition I& azine-2-thione). The compound was imide was then reacted with a solution fore measuriw activity of '"Hz. Torni- hydrolyzed with acid to release CS1 of trisodium pentacyanoammine ferroate zawa and co&rkers (277) used neutron which was reacted with diethylamine to give a red wlor which was read at activation to determine mercury in rice. and cnpric acetate to form copper dithio- 530 u. The sensitivity of the method Again, a chemical separation was made carbamate. The sensitivity of the was 0.03 pprn and rewveries at levels after irradiation hut these workers meas- method was reported as 0.02 ppm and of 0.0H.20 pprn averaged about 85%. ured "Wg. recoveries from vegetables ranged from Cottonseed has been analyzed for Hearth et al. (147) reported an oscillo- 93 to 120%. Cotta-Ramusino and Stac- DEE" (S,S,S-tributyl phosphorotrithic- polarographic method for the determi- chini (76) reported a spectrofluoro- ate), using gas chromatography afkr nation of Morestan (&methyl-2,3quin- metric method for the determination of Florisil wlumn cleanup. Thomas and oxalinedithiol cyclic carbonate) in o-phenylphenol on citrus fruit. The ex- Harris (278) wdthe microcoulometric orange rind. The hexane stripping solu- tract was diluted with 0,lN NaOH and detector while Thornton and Anderson tion was concentrabd and cleaned up on the fluorescence measured at 425 mp, (874) used electron capture detection in silica gel TLC plates. The spots were using an excitation wavelength of 325 mp. their oroeedure. Bielorai and Alumot located by their fluorescence under ultra- (38) reported a procedure for the deter- violet light, scraped off, and eluted with MISCELLANEOUS PESTICIDES mination of ethylene dibromide in foods ethanol for the polarographic determina- and feeds, using"G~~with aflame ionize tion. Martin and Schwartzman (199) Kroeller (188) used the colorimetric tion detector. Benaene was added to reported that the ultraviolet spectro- method for arsenic in food based on the the sample and distilled. The distilled photometric method for niwtine, at reaction of AsHa with silver diethyldi- benzene was dried and then injected into times, wuld not distinguish between thiomrbamate after wet digestion and the GLC. Results by this method were crop interference from mushd greens preliminary sepnration by distillation in good agreement with the chemical and nicotine; tbey described a TLC PIC- from HCI. Kirchmann and Roderhourg titrimetric method at 15-1500 pprn cedure which did make the diitinction. (179) used radioactivation for the deter- levels. Kimura and Miller (178) re- Narahu and coworkers ($88) used a mination of arsenic in plant matter. ported a thin layer chromatographic the gss chromatograph with a thed After irradiation the arsenic was sepa- procedure for the determination of gib- wnductivity detector to determine rated by wet ashing and precipita- berellic acid in rhubarb having a sensi- pentachlorophenol in soy sauce. They tion as As& before measurement of AP. tivity of 3 ppb. The gibberellic acid chromatographed the PCPas the phenol, The limit of detection was 2 X spots were located on the acidified silica using dehydroacetic acid as an internal gram. Banderis (17) reported a colori- gel plate by their fluorescence under standard. Cheng and Kilgore (66) in metric method for the determination of ultraviolet light. Zieliaski and Fishbein determining pentachlorophenol and its chlorates in plants and soil. It was (892) reported that they wuld gas sodium salt in fruits, first methylated based on the reaction of chlorates with chromatoeranh maleic hvdrazide after these wmpounds with dimomethane HCI to release chlorine. The chlorine reacting itinLpyridine with hexamethyl- before using electron capture GLC for was reacted with o-tolidine to form a disilazane in the presence of trimethyl- the determinative sten. Akisada (9) de- yellow color which was read at 448 mp. chlorosilane. Hoffman et al. (153) dis- scribed a wlorimetricmethod for Gnta- Several methods have been reported cussed ~ossibleinterferences in the color- chloroohenol and tetrachloronhenol in for the determination of cyanide. imetric method for maleic hydrazide urine and in air. The phe;lols were Kroeller (189) used a specially designed and described a Norit-A clmu~to distilled off from the acidified urine still to distill cyanide from foods under eliminate interferences. Lane i191) while the air was passed through an nitrogen. The distilled HCN was con- conducted a collaborative study of the absorbing solution containing a borate verted to cyanobromide which reacted colorimetric method for malei; hydra- buffer at pK 7.13. They were then with pyridine-benzidine to form a red side [J. R. Lane. JAOAC 46, 211 reacted with 4-aminoantipyrine and color which was measured. Guilbault (1963j1. Five ~ilaboratorsobhed ILFe(CN)6and the colors extracted into and Kramer (189) reported a fluoro- average recoveries of 7M2% from sam- xylene. The absorbance was measured metric method in which the cvanide waa ples of cranberries, potatoes, onions, etc. at 4i0 mrr for trtrachlomphenol and at reacted with quinone monoxime benzene forti6ed at 1.3- to 85-ppm levels. 570 inp for pcnuichlorophenol. Zielin~ki sulfonate ester in dimethvlsulfoxide to A cold vapor atomic absorption ap and Fi3hheul (P.9 I) ass chromatonranhed give a green fluorescence. With an paratus was designed by Schachter (255) piperonyl bu&d; and a nngb& of excitation wavelength of 440 mfi and to measure submicrogram quantities of 3,4methylenedioxyphenyl derivatives, emission of 500 mG as little as 0.5 pg of mercury in the vapor phase at room tem- both as the compounds themselves and cyanide was easily detected and no other perature. Using thisspparatus, Pappas as the methyl apd trimethylsilyl deriva- ions were found to interfere. These and Rosenberg developed procedures for tives of these wmpounds. Mestres and authors (130) investigated this reaction the determination of mercury in wheat Barrios (%IS) used gas chromatography and those of various other quinone (836) and in fish and eggs (237) at levels to determine propylene oxide and pro- derivatives with cyanide, studying the as low as 0.01 ppm. Epps (101) used pyleue glycol in fruit. By means of a effect of substituents, solvents, pH, and the colorimetric dithizone method for system in which 1-20 mg samples were

VOL. 39, NO. 5, APRIL 1967 . 153 R introduced directly into the injection classes and the 50 chemicals most con,- he obtained from as little as 1 fig of chamber, they demonstrated that pro- monly found, there were 1.35 X 10j6 pesticide. The prgcedure, which has pylene oxide was rapidly absorbed by possible different wmbinations that the been used to idenqify pesticides sopa- prunes in which it was hydrolyzed to residue chemist could encounter. rated by GLC, consi&cd of incorporating propylene glycol. In exploring methods for the determi- the sample into 4 mg of KBr in a micro- Delfel (88) described the use of HI as a nation of organo-metallic fungicides on pellet formed in a %mm diameter hole 0color reagent for the detection of rote- crops, Gudzinowics and Luciano (187) in folded aluminum foil. It was pointed none on paper chromatagrams. Rote- showed that atomic absorption could be out that it vas essential to compare none gave a characteristic blue color used to determine zinc, manganese, and sample spectra witb standard spectra with the reagent while elliptone gave a iron. However, the amounts of these obtained in the same manner since weak pink or violet color. None of the other metals found in untreated plants were so bands are missing at mirrogram levels. materials present in crude extracts of high that their measurement did not Derris ellipliea or Tephrosia vogelii gave seem a promising means of detecting LITERATURE CITED any color with the reagent. Delfel (89) fungicide residues. (1) Ahbott, D. C., Blmting, J. A,, Thom- also studied the TLC behavior of rote- Beroza and Bowman (SO) introduced son, J., Analyst 90, 356 (1965). none and related compounds and de- the concept of p-values-based on the (2) Ibid., 91, 94 (1966). scribed a number of solvent systems and distribution ratios between 2 immiscible (3) Ahbott, U. C., db Fauhert Maunder, chromogenic agents to give desired sepa- solvents-as the basis for identification M. J., Chem. Ind. ( ondon) 1965, p. 82. (4) Ahbott, U. C., &an, II., Thomson, rations. Johnson and Stansbury (163) of pesticide residues and other com- J., J.Chromatog. 16,481 (1964). reported a colorimetric method for the pounds. In its simplest form, a solution (5) Abel, K.. Lanaeau, K., Stevens, defoliant, sodium cis-3-cl~loroacrylate of the residue was analyzed by GLC and R. K., J. Assac. Ofie. Anal. Chemists (Prep), in cottonseeds. The sample was then after shaking with an equal volume 49.. 1022- -- ilQRRl~~ ...., (6) Ackermann, H., Nahrung 10, 273 acidified and the free acid extracted by of immiscible solvent it was again ana- (1966); Cn. 65,9Q57a(1966). blending with 1-butanol. After cleanup, lyzed by GLC. The ratio of the 2nd (7) Adamuvlc, V. M., J. Chromatog. 23, it was reacted with pyridine and NaOH result to the first is the p-value. The 274 1lQlXj to produce a colored solution which was authors refined the procedure by the use passed through an alumina coluum and of a 5-plate Craig counter current dis- then read at 395 mp. Tosaphene, chlor- tribution apparatus (31); they listed dane, DDT, and TDE did not interfere. p-values for 131 pesticides in 6 binary Christian and coworkers (68) described systems (45); they designed an appa- a polarographic method for selenium in ratus for rapid extraction (33) and a bioloeical materials while Cummines et device as well as an equation for obtain- al. (84)used a colorimetric proce'dure ing p-values using nonequilihrated sol- measuring the absorbance of a complex vents (46). They (33) also studied the of selenium with 3,3'-diaminobenzidine. extraction of added pesticides from milk Pease ($41) determined sulfamates in with hexane-diethyl ether with and apples and pears by removing all the ~vithoutprior mixing of sample witb sulfates and then reducing the sulfa- ethanol. They found that without eth- mates to HIS which was reacted with anol, the extractionefficiencies paralleled p-dimethylaminoaniline to form methyl- the polarities as judged by p-values. (1966). ene blue. Bowmall and Beroza (47) Farrow et al. (108) reported a cleanup (17) Banderis, A., J. Sci. Food Agr. 16, reported a gas chromtographic proce- procedure for both chlorinated and 558 (1965). (18) Barney 11, J. E., J. Chromatog. 20, dure for the determination of tepa, organophosphate pesticides based on 334 (1965). apholate, hempa, and several other vacuum sublimation. The dried sample (19)Barry, 11. C., Hundloy, J. G., chemosterilant.3. Using the flame pho- extract was subjected to vacuum sub- Johnson, L. Y., "Pestmde Analytical tometric detector of Brody and Chaney limation at 85°C. for 15 minutes and Manual," U. S. Dept. of Health, Edu- cation, and Welfare, Food and Drug (50) they could detect as little as 0.1 ng the r~esticideresidu~ were collected on Admin., Vol. 1, 1963. of the sterilants. Bullard (58) used a cold finger cooled with Dry Ice-ace- (20) Bates, J. A. R., Analyst 90, 453 11 4R.5) GLC rvith flame ionization detector to tone. The residues were rinsed from the \ -.. --,. determine tetramine (tetramethylene- cold finger, made to volume, and in- (21) Baumnn, E. K., Gaadson, L. H., Guilhault, G. G., K amer, D. N., ANAL. disulfotetramine), a systemic toxicant jected into the electron capture GLC. CHEM.37,1378 (10&). used to keep animals "from feeding on The procedure was tested on 35 pesti- (22) Bami, B., Fnbbdini, R., Alti Cav. seed and young seedlings. Recoveries cides in spinach and recoveries for 25 Quaua. 3", 96 (19611); C.A. 63, 35358 from a variety of foliages consistently of these exceeded 80Yo. Most of the (1965). (23) Beam, J. E., Elankinson, D. J., averaged above 80'%. Billy and cc- others were recovered in the 60+wo J. Dairv Sei. 47, 1297 (1964). n,orkers (39) reported a spectrophoto- range except for a fev low values from (24) Beckman, H., Bdvenoe, A,, Carroll, metric procedure for the determination vaxy plant extractives. K., Erro, F., J. dssoe. Ofic. Anal. of the lampricide, 3-trifluoromethyl4- Rvbakov (8U) reviewed the use of Chemists 49,996 (1966). .., (25) Beekmsn, H., Btvenue, A., Ganer, nitrophenol (TFM),in water and in fish polarography and discussed methods for W. O.,Erro, F., Ibid, 48,1169 (1965). tissue. After deanup by liquid-liquid the analvsis of nesticides containinr (26) Beckman, H., Gauer, W. O., Bull. partitioning and ion exchange column sulfur, phosphor&, chlorine, and nitro- Enuiron. Contam. l'otieol. 1, 149 (1966). the determination mas made by measur- gen. (27) Bockman, H., Winterlin, W., Ibid., y.n 7R ing the absorbance at 395 mp. Coffin (73) reviewed the nse of paper (28) Bensan, W. R., Finocchisro, J. M., chromatography in pesticide residue J. Assoc. ORLc. Am. Chemists 48. 676 MISCELLANY~.---~.. analysis, discussing its advantages and disadvantages as well as various detec- Duggan (93) described the procedures tion systems. sed by the Food and Drug Administra- Salo and Salminen ($51) tabulated to validate multiple residue methods TLC data for 29 common pesticides on the varieties of foods. To illustrate under a number of solvent systems. (1065). the magnitude of the problem, he Chen (63) deseribed a micro technique (32) Benms, M., Bowman, M. C., ANAL. pointed out that with the 12 major food for infrared by which good spectra could CEIEM.38, 837 (1966).

154 R . ANALYTICAL CHEMISTRY

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'(S981) ZSL '8P SlVulay3 U~V'3gO '.)OSSV .f "3 'U 'IIOSpN (SZZ) (262) Stanley, C. W., J. Chromalag. 16, (287) Yauger, Jr., W. L., Addison, L. M., 467 (1964). Stevens, R. K., J. Assoc. Ofie. Awl. (263) Stanley, R. L., LcFavo.ure, H. T., Chemi-ls 49,1053 (1966). J. Assoc. OJie. Agr. Chemtsts 48, 666 (288) Yip, G., J. Assac. O&. Agr. (1965). Chemists 47, lllG (1964). (264) Steller, W. A,, Frederick, I. B., (289) Yip, G., Ney, Jr., R. E., W Morgan, P. W., J. Ayr. Fowl Chem. 13, 14,167(1966). 329 (1965). (290) Zadrozinska, J., Roaiki Panstwe (265) Sternp, A. R., Liska, B. J., J. Dairy @a we00 Z&u Hig. 16, 53 (1965); ScC 48,985 (1965). C.A. 62.168760 f 1965). (266) Sternpkovskaya, .L. A., Vekshtein, (291) I&& 39"7; 64,26&h(l%6). M. A,, F'opr. Pitanw 24, 17 (1965); p. C.A: C.A. 64, 8870h (1666). (292) Zielinski, Jr., W. L., Fishbein, L., (267) Stevens, R. D., Yan Middelem, J. Chromntoo. 20.140 f 19651. C. H.. J. Am. Food Chem. 14. 149 (293) ~i&nic,&:, w.'L., kiahbein, L., J. Gas Chtmnotoo. 3.333 (1965). (294) Zietinski, J;, W. L.; Fishbein, L., ANAL.CEEM.38.41 f 1966). (295) Sz szkq 6;&ik Pamhmocgo ~akduHig., 16,445 (1965); C.A. 64, 11760ef1966).. . (296) Szyszko, E., Fann. Polska 21, 81 (1965); C.A. 63,7562h (1965). (297) Ibid., p. 483; C.A. 64, 2860h 1~---.,~1966). (298) Ibid., 22, 161 (1966); C..4. 65, 127735 (1966). (299) Ibid., p. 249; C.A. 65, 12773~ (1966).

vor. 39, NO. 5, APW 1967 157 R