Chemical Derivatization Techniques in Pesticide ... - ACS Publications

12 Chemical Derivatization Techniques in Pesticide Analysis Advances and Applications W. P. COCHRANE Downloaded by RUTGERS UNIV on February 25, 2017 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch012

Laboratory Services Division, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C5 In common with other analysts, the pesticide analyst has two major problems facing him during the analysis of a sample, namely, 1. The IDENTITY of the pesticide present 2. The AMOUNT of pesticide or its residue in the sample. In both these areas, chemical derivatisation has traditionally played a role and with the advent of gas chromatography an even more important role. The reasons for preparing a derivative suitable for GC analysis are many and varied and have been discussed thoroughly in a number of books and reviews (1-6). For convenience they are summarised in Table I . As can be seen, two different types of chemical derivatisation techniques are mentioned under Item 4 of Criteria, There is the chemical derivatisation of a pesticide as a pre-requisite of the method of analysis, e.g. esterification of the chlorophenoxy acids, as well as derivatisation as a method for confirmation of identity. The former must meet a l l the requirements associated with a practical, viable analytical procedure while for the latter the emphasis is on speed, ease of operation and reproducibility. Apart from the above reasons d e r i v a t i s a t i o n i s a l s o r e q u i r e d i n the analyses of many o f the newer p e s t i c i d e s which have one o r more f u n c t i o n a l groups which need p r o t e c t i o n i n order to f a c i l i t ate GC work. Also the i n c r e a s e d use o f high pressure l i q u i d chromatography (HPLC) n e c e s s i t a t e s p r e - or post-column d e r i v a t i s a t i o n since the choice o f HPLC detectors i s l i m i t e d compared with GC. The increased use o f d e r i v a t i s a t i o n r e a c t i o n s i s evident from the gradual increase i n the number o f p u b l i c a t i o n s d e a l i n g w i t h the subject. Figure 1 shows the y e a r l y v a r i a t i o n i n numbers o f publ i c a t i o n s d e a l i n g with d e r i v a t i s a t i o n i n p e s t i c i d e a n a l y s i s over the p e r i o d 1963 to 1978. While the i n t e r e s t i n d e r i v a t i s a t i o n techniques i n organophosphorus i n s e c t i c i d e a n a l y s i s has remained f a i r l y constant and a low l e v e l of a c t i v i t y , the OC i n s e c t i c i d e s underwent an i n c r e a s e d p e r i o d o f a t t e n t i o n from 1968-1972 which has s t a b l i z e d over the l a s t few years. I t i s i n the i n s e c t i c i d a l carbamate and h e r b i c i d e areas that an o v e r a l l steady increase i n the use o f d e r i v a t i s a t i o n r e a c t i o n s f o r q u a n t i t a t i v e and con0-8412-05 81 -7/80/47-136-231 $05.00/0 © 1980 American Chemical Society Harvey et al.; Pesticide Analytical Methodology ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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A.

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METHODOLOGY

Advantages Gained by Chemical D e r i v a t i s a t i o n and Some Reaction C r i t e r i a

Advantages: 1. 2. 3. 4. 5. 6. 7.

Β.

ANALYTICAL

Improved e x t r a c t a b i l i t y during the clean-up procedures. Change i n v o l a t i l i t y c h a r a c t e r i s t i c s . Increase i n thermal s t a b i l i t y of compounds. Improve chromatographic or s e p a r a t i o n behaviour. Increase i n s e n s i t i v i t y of d e t e c t i o n . Impart s e l e c t i v i t y . A i d i n c o n f i r m a t i o n of i d e n t i t y .

Criteria : 1. 2. 3. 4. 5. 6.

D e r i v a t i v e must be formed r a p i d l y . P r e p a r a t i o n should r e q u i r e minimum of manipulation. D e r i v a t i v e should be r e l a t i v e l y s t a b l e . Reaction must be q u a n t i t a t i v e and/or r e p r o d u c i b l e . Short (acceptable) GC r e t e n t i o n time f r e e from back ground or reagent i n t e r f e r e n c e s . Good s e n s i t i v i t y .

firmatory a n a l y s i s has o c c u r r e d . T h i s has been augmented i n r e cent years by i n t e r e s t i n the a n a l y s i s of f u n g i c i d e s , noteably e t h y l e n e t h i o u r e a and the e t h y l e n e b i s d i t h i o c a r b a m a t e s , as evidenced by the trend o f the o v e r a l l t o t a l curve. Since a number of reviews (4,5,7,8) cover the l i t e r a t u r e p r i o r to 1975, t h i s r e view w i l l h i g h l i g h t the more recent advances i n d e r i v a t i s a t i o n procedures or techniques as i l l u s t r a t e d w i t h a p p l i c a t i o n s from the 4 main groups o f p e s t i c i d e s , namely, h e r b i c i d e s , OCs, OPs and f u n gicides . Herbicides E s t e r i f i c a t i o n of the chlorophenoxy a c i d s to t h e i r r e s p e c t i v e methyl e s t e r s by reagents such as diazomethane, BF^/methanol or a c i d methanol mixtures have been f o r l o n g the standard procedure f o r the analyses of these compounds. Recently a number of workers have been i n v e s t i g a t i n g the use of new and more EC s e n s i t i v e e s t e r i f i c a t i o n reagents f o r general use. This has come about since the EC d e t e c t i o n of the methyl e s t e r s of MCPA or MCPB, which contain only one c h l o r i n e each, are very poor. A l s o the methyl e s t e r of MCPA has a very short r e t e n t i o n c l o s e to the s o l v e n t f r o n t when analysed i n c o n j u n c t i o n with 2,4-D, 2,4,5-T fenoprop, e t c . The a c i d s and the v a r i o u s e s t e r s i n v e s t i g a t e d are shown i n Table I I . In t r a n s e s t e r i f i c a t i o n work by Y i p (9) the 2 - c h l o r o e t h y l e s t e r was d i s c a r d e d s i n c e the background p a t t e r n of the d e r i v a t i v e gave too many peaks. However, Woodham et^ al. (10) used f

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

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Table I I :


Compound

2,4-D 2,4-DB 2,4,-DP 2,4,5-T 2,4,5-TB 2,4,5-TP MCPA MCPB MCPP 2,3,6-TBA Picloram Dicamba Fenac References

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Techniques

D e r i v a t i v e s o f the Chiorophenoxyalky1 A c i d s and Some Other A c i d i c H e r b i c i d e s

2,2,2-trichloro e t h y l

2-chloro ethyl

2,2,2-trifluoro ethyl

X X X X

X

X

X

X

X X X

ΈΈΈ-

X X X X X X X X X X X

X X 9-14

9-14

14

13, 15

1/Pentafluorobenzyl a BCl^/2-chloroethanol reagent to determine residues o f 2,4-D i n s o i l , sediment and water samples while Gutenman and L i s k (11) used BF^/2-chloroethanol f o r e s t e r i f i c a t i o n o f MCPA i n s o i l e x t r a c t s . Woodham eit a l (10) found that although the 2 - c h l o r o e t h y l e s t e r had the d e s i r e d s e n s i t i v i t y , the r e a c t i o n produced l e s s i n t e r f e r e n c e and gave a l o n g e r r e t e n t i o n time, i t was s t i l l s u b j e c t to i n t e r ferences from c e r t a i n s o i l types ( F i g . 2A). A f t e r d i e t h y l e t h e r e x t r a c t i o n a clean-up step was added employing an a l k a l i n e wash t o remove i n t e r f e r i n g substances. The lower l i m i t o f s e n s i t i v i t y f o r t h i s method i n s o i l s i s approximately 0.01 ppm f o r 2,4-D ( F i g . 2B). More r e c e n t l y (12, 13) B C l ^ : 2-chloroethanol was used t o determine chlorophenoxy a c i d r e s i d u e s i n n a t u r a l waters. I t was found, as would be expected, that t h i s reagent produced l i t t l e or no product f o r 2,3,6-TBA and dicamba due to the s t e r i c hinderance o f σ-Cl atoms. The p r a c t i c a l l i m i t s o f d e t e c t i o n ranged from a lower l i m i t 0.01 ug/L f o r fenoprop t o a h i g h o f 2-5 ug/L f o r MCPB. Y i p (9) showed that t r i f l u o r o e t h a n o l i n c r e a s e d the ECD response o f 2,4,5-T and fenoprop but n o t MCPA o r 2,4-D. More r e c e n t l y , Mierzwa and Witek (14) found that a f t e r e s t e r i f i c a t i o n of 2,4-D and MCPA with 20% 2 , 2 , 2 - t r i c h l o r o e t h a n o l i n t r i f l u o r o a c e t i c an hydride i n the presence o f I^SO, a lower l i m i t o f 0.096 ppb 2,4-D and 0.06 ppb MCPA i n 1 l i t r e samples o f water was o b t a i n e d . As i n most of the d e r i v a t i s a t i o n r e a c t i o n s , removal o f excess


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1974

YEAR

Figure 1. Variation in number of publications dealing with chemical derivatization techniques for pesticides from 1962-1978 ((+) total (including fungicides); (A) herbicides; (\J) OCs (including Mirex, PCB's, etc.); (O) DP's)

RE T E N T I O N

T I M E (mm )

Journal of Agricultural and Food Chemistry

Figure 2. Chromatographic tracing of (Α) α 2,4-Ό fortified soil sample (2-chloroethyl ester) without alkali treatment and (B) samples with alkali pre-wash: (a) a blank soil sample; (b) soil sample fortified with 0.013 ppm 2,4-Ό acid; and (c) soil sample fortified with 0.667 ppm 2,4-Ό acid (\0)



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reagents i s mandatory p r i o r to ECD a n a l y s i s . I t was found that the s e n s i t i v i t y o f the e s t e r s i n c r e a s e d from the 2 - c h l o r o e t h y l to t r i f l u o r o e t h y l to the t r i c h l o r o e t h y l d e r i v a t i v e . With MCPA the t r i c h l o r o e t h y l e s t e r was 100 times more s e n s i t i v e than the 2 - c h l o r o e t h y l e s t e r , while with 2,4-D the i n c r e a s e was o n l y a f a c t o r o f 10. S i m i l a r l y , Chau and co-workers (15) have shown that the PFB e s t e r s are more EC s e n s i t i v e than the corresponding 2 - c h l o r o e t h y l e s t e r s . For example, the PFB e s t e r o f MCPA i s 2.5 times g r e a t e r than that obtained with the 2 - c h l o r o e t h y l e s t e r and 1000 times greater than MCPA methyl e s t e r . Normally, the e s t e r i f i c a t i o n r e a c t i o n with p e n t a f l u o r o b e n z y l bromide (PFBBr) i s much simpler t o perform s i n c e i t takes p l a c e i n acetone which i s e a s i l y evaporated a f t e r the r e a c t i o n . However, the PFBs tend t o have longer r e t e n t i o n times and t h i s reagent r e a c t s under b a s i c c o n d i t i o n s with both phenols and c a r b o x y l i c a c i d s . I t has been reported that an 0V-101/0V-210 column separated 9 PFB e s t e r s , o n l y the MCPA and 2,4-DP over-lapped,while a 5% DC-200 or 3% DC-11 column gave comp l e t e s e p a r a t i o n o f 10 PFB e s t e r s . O v e r a l l , i t would appear that the s e n s i t i v i t i e s o f the PFB and t r i c h l o r o e t h y l e s t e r s are e q u i v a l e n t , with the f i n a l choice depending upon background i n t e r ference from the sample and s u i t a b l e column s e p a r a t i o n c h a r a c t e r i stics . Organochlorine I n s e c t i c i d e s and Related Compounds Since w e l l - e s t a b l i s h e d chemical d e r i v a t i s a t i o n techniques a l ready e x i s t f o r the m a j o r i t y o f the organochlorine i n s e c t i c i d e s (5, T) i t i s not s u r p r i s i n g t h a t recent a c t i v i t y i n t h i s area has centred round compounds such as Kepone, Mirex, HCB, the PCBs, e t c . which c o - i n t e r f e r e i n both the i d e n t i f i c a t i o n and q u a n t i t a t i o n o f pesticide residues. Kepone (chlordecone) can be converted back t o Mirex by p e r c h l o r i n a t i o n ( F i g . 3) u s i n g a 4:1 r a t i o o f phosphorus p e n t a c h l o r i d e to aluminium c h l o r i d e i n carbon t e t r a c h l o r i d e a t 145 C f o r 3 h r s . i n a c l o s e d tube (16). To e l i m i n a t e any Mirex that may have been o r i g i n a l l y p r e s e n t , the s e p a r a t i o n o f Kepone was performed on a m i c r o - F l o r i s i l column p r i o r t o d e r i v a t i s a t i o n . S i m i l a r l y , a microF l o r i s i l column was u t i l i z e d a f t e r r e a c t i o n to remove e a r l y e l u t i n g peaks from the gas chromatograms, and e s p e c i a l l y when the t o t a l amount of Kepone present was l e s s than 25 ng. T h i s d e r i v a t i s a t i o n technique was found s e n s i t i v e t o ppb l e v e l s o f Kepone i n environmental and b i o l o g i c a l samples. For example, Figure 4 shows an o y s t e r e x t r a c t before and a f t e r d e r i v a t i s a t i o n (16) . The Kepone l e v e l averaged 0.07 ppm (upper chromatogram) i n t h i s p a r t i c u l a r sample which was run on a 4% SE-30/6% 0V-210 column. The r e a c t i o n was found t o be q u a n t i t a t i v e and 8 o f the more common 0C p e s t i c i d e s were found to disappear on r e a c t i o n or give der i v a t i v e s with GC r e t e n t i o n d i f f e r e n t than Mirex. Mirex has been a s s o c i a t e d with the c o n t r o l o f the imported f i r e ant i n the south-eastern USA, where i t was subsequently


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PEAKS


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ANALYTICAL

Journal of Agricultural and Food Chemistry

Figure 4.

Electron-capture gas chromatograms of an oyster extract before and after derivatization ((PCl /AlCl ), 0.66 mg injected (16)) 5

3



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found i n non-target organisms i n c l u d i n g man (17). S i m i l a r l y , Mirex and/or photomirex (8-monohydromirex) have been i d e n t i f i e d as major contaminants i n f i s h from Lake Ontario (18), cormorant eggs (19) and Canadian Human milk (20). Mirex and photomirex are p a r t i c u l a r l y d i f f i c u l t to determine i n the presence o f l a r g e q u a n t i t i t e s o f PCB without p r i o r s e p a r a t i o n since t h e i r r e t e n t i o n times are s i m i l a r to that of major heptachlorobiphenyls on most GC columns. Lane e t a l (21) s t u d i e d the p h o t o l y t i c and o * - i r r a d i a t i o n of duck egg t i s s u e s c o n t a i n i n g n a t u r a l l y o c c u r i n g Mirex. Although 7 d e r i v a t i v e s were obtained on p h o t o l y s i s and 8 from ϊ - i r r a d i a t i o n the major product from both r e a c t i o n s was i d e n t i f i e d as photomirex ( F i g . 3 ) . In t h i s instance UV p h o t o l y s i s was c a r r i e d out at 254 nm on the a c t u a l egg homogenate and caused a 36% de crease a f t e r 48 h r s . whereas f-irradiation r e s u l t e d i n a 64% l o s s of Mirex. I r r a d i a t i o n , t h e r e f o r e , could be used as a c o n f i r m a t o r y t e s t so long as the r e s u l t i n g chromatograms do not become too complex f o r i n t e r p r e t a t i o n . As p o s t u l a t e d i n a previous r e view (5), Mirex can a l s o be confirmed by r e d u c t i o n o f the gem-dichloro methylene group with C r C l - (22) . In p r a c t i s e , three major peaks are obtained, the r a t i o s of which were found to vary w i t h the CrCl^/acetone r a t i o and temperature o f r e a c t i o n . None of these products appear to be photomirex. Reaction was c a r r i e d out overnight at 55-60°C or room temperature f o r 32 h r s . or l o n g e r . While photomirex could be completely reacted to give a major p r o duct with a d i f f e r e n t r e t e n t i o n time than Mirex, Kepone only r e acted p a r t i a l l y . Lewis et_ al^ (23) employed a d i f f e r e n t approach i n that M i r e x - c o n t a i n i n g human t i s s u e e x t r a c t s were subjected to a diethylamine a s s i s t e d p h o t o l y s i s a t wavelength> 280 nm to s e l e c t i v e l y e l i m i n a t e PCB i n t e r f e r e n c e s i n PCB/Mirex mixtures. A 100 min. p h o t o l y s i s of an A r o c h l o r 1260/Mirex mixture i n 10 ml hexane c o n t a i n i n g diethylamine r e s u l t e d i n complete e l i m i n a t i o n of the i n t e r f e r i n g h e p t a c h l o r o b i p h e n y l component and only a 0-5% l o s s of Mirex. However, some i n t e r f e r e n c e s appear t o remain i n the photomirex region of the chromatograms. A l s o i t has been pointed out by Mes and co-workers (20) t h a t although Mirex can be i d e n t i f i e d by GC, u s i n g diethylamine a s s i s t e d p h o t o l y s i s to l i m i t PCB i n t e r f e r e n c e , q u a n t i t a t i o n at low l e v e l s (0.01-0.1 ppm) r e main questionable s i n c e the workers found poor agreement between t h e i r GC and mass spectrometry data. Other i n d i r e c t approaches to the PCB/Mirex problem have been the p e r c h l o r i n a t i o n of the PCBs to decachlorobiphenyl with Sb CI,.-containing reagents (24) and n i t r a t i o n of the PCBs w i t h a 1:1 mixture o f cone. H^SO^/ fuming HNO^ (25) . Shown i n Figure 5 i s a chromatogram o f an ex t r a c t of a h e r r i n g g u l l egg from Lake Ontario a f t e r clean-up on a 1% d e a c t i v a t e d F l o r o s i l column; note that Mirex i s only partially r e s o l v e d from a h e p t a c h l o r o b i p h e n y l peak. The lower t r a c i n g i s the same e x t r a c t a f t e r a 30 min. n i t r a t i o n at 70 C. Mirex and photomirex are the only major c o n s t i t u e n t s to s u r v i v e n i t r a t i o n . HCB, ^-nonachlor and small amounts o f mono- and d i hydromirex compounds c o n s t i t u t e most of the minor peaks remaining


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RETENTION TIME (MINS.)

ηίΖ ·Ί , ° 88 from Lake Ontario after cleanup on a t lor ml column; (B) same extract after nitration (represents about twice as much sample as A) Conditions: & χ 4 mm glass column with 1% SP-2100 on Supelcoport 100/120 mesh; flow rate 40 mL/min Argon/methane; oven 190°C; Ni detector. (

}

f

a

h e r r i n g

g u l 1

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i n the chromatogram. Also c i s - and trans-chlordane can be q u a n t i t a t e d a f t e r n i t r a t i o n (25) . HCB i s recovered i n low y i e l d s (*Ί0%) p r i m a r i l y due to v o l a t i l i s a t i o n r a t h e r than r e a c t i o n with the a c i d mixture. S i m i l a r l y , l i n d a n e r e c o v e r i e s are e r r a t i c . A f t e r a c o l l a b o r a t i v e study i n v o l v i n g sediment, carp, e e l and g u l l egg samples, t h i s n i t r a t i o n technique was found r e l i a b l e f o r Mirex and photomirex a t l e v e l s ^ 1 0 ppb i n the presence o f 1,000-fold greater l e v e l s of PCB (26). Hexachlorobenzene (HCB) has a t t r a c t e d i n c r e a s e d a t t e n t i o n due to i t s common occurence i n human b l o o d , milk and adipose t i s s u e , w i l d l i f e samples, wheat and l a k e water. Various approaches ( F i g . 6) to the chemical c o n f i r m a t i o n o f HCB have i n cluded the p r e p a r a t i o n of a pentachlorophenyl p r o p y l e t h e r when t r e a t e d with KOH i n 1-propanol (27) . The d e r i v a t i v e had a l o n g e r GC r e t e n t i o n time than HCB. Another two step approach by H o l d r i n e t (28) i n v o l v e d r e a c t i o n of HCB w i t h 0.2N KOH i n e t h y l e n e g l y c o l under r e f l u x to produce pentachlorophenol which i s then e s t e r i f i e d with diazomethane to give the PCP methyl ether d e r i v a t i v e . I t was o r i g i n a l l y demonstrated by Baker (29) that HCB i s converted to monoethoxypentachlorobenzene when r e f l u x e d w i t h sodium e t h o x i d e . More r e c e n t l y , i t has been shown (30) that ex tended treatment ( i . e . g r e a t e r than 6 hrs.) w i t h NaOEt w i l l f u r t h e r convert the monoethoxy d e r i v a t i v e to diethoxy compounds. Since the p u r i f i e d diethoxy d e r i v a t i v e obtained from a ρrep-scale r e a c t i o n gave 2 major chromatographic peaks, i t i s suggested a mixture of isomers i s produced. S i m i l a r l y , C r i s t and co-workers (31)confirmed HCB i n adipose t i s s u e by the p r e p a r a t i o n of s e v e r a l d e r i v a t i v e s from v a r i o u s a l c o h o l s . I t was found that r e a c t i o n with a K0H/2p r o p a n o l / p y r i d i n e mixture f o r 10 min. at 100 C produced mono-isopropoxy pentachlorobenzene while a f t e r 30 min. b i s - i s o p r o p o x y t e t r a chlorobenzene was formed. The d i - s u b s t i t u t e d d e r i v a t i v e was l e s s subject to f u r t h e r s u b s t i t u t i o n than the mono-derivative and r e p r o d u c i b i l i t y of the r e a c t i o n was more e a s i l y c o n t r o l l e d such that a lower l e v e l of 5 ppb HCB could be confirmed i n f a t t y samples. The f l e x i b i l i t y of t h i s r e a c t i o n was shown by the use of other a l c o h o l s (EtOH, 1-PrOH, 1-BuOH) and the c o n f i r m a t i o n of 0.3 ppm HCB i n r a t adipose t i s s u e c o n t a i n i n g 160 ppm A r o c h l o r 1016. Since d e r i v a t i s a t i o n to the di-isopropoxy d e r i v a t i v e was not f e a s i b l e due to i n t e r f e r e n c e from an A r c c h l o r peak, con f i r m a t i o n based on the mono-substituted d e r i v a t i v e was p o s s i b l e . Organophosphorus and Carbamate I n s e c t i c i d e s Most OP i n s e c t i c i d e s may be determined d i r e c t l y by GC u s i n g the p h o s p h o r u s - s e l e c t i v e flame photometric detector (FPD) which helps to minimize clean-up. However, i t must be emphasised that the FPD i s o n l y a " s e l e c t i v e d e t e c t o r f o r phosphorus (at 540 nm) or sulphur (at 394 nm) and not an "element s p e c i f i c d e t e c t o r " . This i s i l l u s t r a t e d i n F i g . 7 which shows the 4 o x i d a t i o n products 1 1


PESTICIDE A N A L Y T I C A L M E T H O D O L O G Y


240

R= Ethyl, 1 - P r o p y l . 2 - P r o p y l or tert.-Butyl

Figure 6.

Reactions used for the confirmation and/or quantitation of hexachlorobenzene (HCB)

Figure 7. Characteristics of methidathion and its sodium hypochlorite oxidation products on 1% DEGs at 200°C with flame photometric detection (P-mode)



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obtained from the macro-scale r e a c t i o n of n e u t r a l i s e d sodium hypo c h l o r i t e w i t h methidathion (Supracide ) (32). G e n e r a l l y , o x i d a t i o n of the P-S groups i n such i n s e c t i c i d e s as malathion, d i a zinon, p a r a t h i o n , f e n i t r o t h i o n , e t h i o n , e t c . y i e l d t h e i r r e s p e c t i v e oxons. A s i n g l e oxon d e r i v a t i v e peak i s the r e s u l t of h y p o c h l o r i t e o x i d a t i o n of methidathion a t the ng l e v e l but 4 p r o ducts were observed at the macro-scale. Although t h i s chromato gram was obtained u s i n g the Ρ-mode o f the FPD only one peak, namely methidaoxon,peak 2, contains phosphorus. The other 3 p r o ducts c o n s i s t of 2 t h i a d i a z o l i n o n e r i n g s l i n k e d by methylene, s u l f i d e or d i s u l f i d e groupings r e s p e c t i v e l y . A l s o a 10:1 r e s ponse r a t i o was obtained when e q u i v a l e n t amounts of the sulphide and d i s u l p h i d e were recorded on the S- and Ρ-channels,respectively, of the FPD. O x i d a t i o n of phorate w i t h NaOCl or ^ °2 phorate oxon s u l f o x i d e and not phorate oxon. Recently the q u a n t i t a t i v e o x i d a t i o n of oxydemeton methyl with KMhO^ to i t s corresponding s u l f o n e has been used f o r i t s determination i n a v a r i e t y o f p l a n t and animal t i s s u e s down to a l i m i t of 0.01 ppm (33). An a l t e r n a t i v e approach to the a n a l y s i s of oxydemeton metyl, which contains a d i a l k y l s u b s t i t u t e d s u l f o x i d e moiety, i s v i a t r i f l u o r a c e t y l a t i o n ( F i g . 8) (34) . Reaction of oxydemeton methyl with t r i f l u o r a c e t i c anhydride at 100° f o r 15 min. r e s u l t s i n a mono-trifluoroacetoxy d e r i v a t i v e which thermally degrades oncolumn to give 2 peaks, the c i s - and trans-isomers of dehydrooxydemeton-methyl. Other compounds w i t h a s u l f o x i d e moiety that can give mono-or di-TFA d e r i v a t i v e s s u i t a b l e f o r confirmatory purposes are d a s a n i t , mesurol s u l f o x i d e , nemocur s u l f o x i d e , a l d i c a r b s u l f o x i d e , counter s u l f o x i d e and oxycarboxin. Due to the poor chromatographic c h a r a c t e r i s t i c s o f s u l f o x i d e s , a n a l y s i s was p r e v i o u s l y performed f o l l o w i n g o x i d a t i o n to t h e i r r e s p e c t i v e s u l fones. Now with mesurol, a l l 3 o x i d a t i o n products plus phenols can be analysed simultaneously. Of course, t r i f l u o r a c e t y l a t i o n and methylation of NH-containing OPs and carbamates are p o s s i b l e but these procedures have been e x t e n s i v e l y covered i n previous reviews (5, T) . 2

r

e

s

u

l

t

s

i

n

However, two other areas are o f i n t e r e s t ( F i g . 9) namely, a) d e r i v a t i s a t i o n of phenols or amines to phosphorus-containing compounds and b) on-column methylation. In the former, phosphory l a t i o n of 7 a l c o h o l s and 12 phenols with d i e t h y l chlorophosphate i n the presence o f t r i e t h y l a m i n e at 60-70 C i n benzene has been reported (35). A l i p h a t i c a l c o h o l s , such as methanol, butanol or isoamyl a l c o h o l r e a c t i n s t a n t a n e o u s l y while phenol and eresoIs r e q u i r e ca 1-1 1/2 h r s . and x y l e n o l s 3-4 h r s . f o r best r e s u l t s . The r e s u l t i n g a l k y l or a r y l d i e t h y l phosphates were s e l e c t i v e l y detected at the 10-25 ng l e v e l with the P-mode FPD. Similarly, Jacob et_ a l (36) used dimethyl t h i o p h o s p h i n i c c h l o r i d e i n the presence of excess t r i e t h y l a m i n e to convert primary a l i p h a t i c , aromatic and h e t e r o c y c l i c amines to the corresponding N-dimethylt h i o p h o s p h i n i c amides. The excess reagent was e a s i l y removed by t r e a t i n g the r e a c t i o n mixture w i t h methanol-sodium hydrogen c a r -


242

PESTICIDE

CH O 3

ANALYTICAL METHODOLOGY

Ο

s

A p ,° O.ÏCF3 CH (/ S.CH .CH.S.CH .CH

CH

CH 0" S.CH .CH .S.CH .CH 3

N

2

2

2

V

3

3

2

3

ΔΙ

OXYDEMETON METHYL CH3O

CH 0 2


2

JQ N

S.CH=CH.S.CH CH 2

3

0.5 f sd

u Journal of Agricultural and Food Chemistry

Figure 8. Reaction of oxydemeton methyl with trifluoroacetic anhydride (TFAA) and the GC characteristics of the two products (cis- and trans-dehydro-oxydemeton methyl) on 3% OV-17 at 190°C (34)

PHOSPHORYLATION

α)

Ο R-OH + CI-P-IOCjH,). 1

5

1

3

/

b§-70*C

Ο R-0-P(OC,H_L + HCI 2 52

R = alkyl or aryl.

b)

S R'-NH + CI-P(OCH ) 2

2

3

J

1

2

—

3

-,

-20fo + 20°C

S R-NH-P-(OCH ) + HCI 3

J

2

l

R = primary aliphatic, aromatic or heterocyclic amines

Figure 9.

Phosphorylation reactions of alcohols and amines


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bonate and the d e r i v a t i v e s were w e l l separated on a SE-30 o r OV-17 c a p i l l a r y column. Although a lower l i m i t of d e t e c t i o n of 0.5 pg f o r N - d i m e t h y l t h i o p h o s p h i n y l a n i l i n e was obtained with a rubidium sulphate AFID, an FPD d e t e c t o r c o u l d a l s o be used. T h i s d e r i v a t i s a t i o n technique was a p p l i e d to twenty aromatic o r h e t e r o c y c l i c amines as w e l l as 6 amino a c i d e s t e r s . However, i t should be p o i n t e d out that dimethylthiophosphinic c h l o r i d e r e a c t s and i s as s e n s i t i v e , i f not more so, to moisture problems than TFAA. Since the r e s u l t i n g product c o n t a i n s Ρ i t too gives a FPD/AFID response. The general subject of on-column p y r o l y t i c methylation has been r e c e n t l y reviewed (37) and, i n p a r t i c u l a r , t h i s technique has been the subject of a number of papers d e a l i n g w i t h the i d e n t i f i c a t i o n and q u a n t i t a t i o n of many organophosphorus p e s t i c i d e s and r e l a t e d d i a l k y l phosphorothioates (38-AO) as w e l l as carbamate p e s t i c i d e s (41-43^. GLC on-column t r a n s e s t e r i f i c a t i o n of OP i n s e c t i c i d e s at 260 C u s i n g trime thylphenylammonium hydroxide i n methanol, r e s u l t s i n s h o r t - c h a i n t r i a l k y l phosphates which can be q u a n t i t a t e d using the P-mode FPD ( F i g . 10). The exact s t r u c t u r e of the t r i a l k y l phosphate formed i s dependent upon the s t r u c t u r e of the o r i g i n a l OP i n s e c t i c i d e . Parathion i s t r a n s e s t e r i f i e d to give diethylmethylthiophosphate (DEMTP) while azinphos methyl y i e l d s t r i m e t h y l dithiophosphate (TMDTP). The minimum detectable l i m i t f o r malathion was 400 pg. The technique was a p p l i e d to the determination of chlorphoxim at l e v e l s o f 10 ppb i n f i s h and 0.1 ppb i n water. I t has been suggested that methanolic TMPAH q u a n t i t a t i o n of d i a l k y l phosphorothioates and phosphorodithioates could provide a convenient and s a f e a l t e r n a t i v e to the use o f diazomethane where methylation i s the f i n a l step i n OP residue a n a l y s i s (40). S i m i l a r l y , on-column methylation has been a p p l i e d to carbamate p e s t i c i d e s c o n t a i n i n g an a c t i v e N-H group. Wien and Tanaka (41) showed t h a t N - a r y l carbamates are methylated on-column with t r i me thy 1 a n i l i n i urn hydroxide-methanol to give the i n t a c t N-methyl and N - a r y l d e r i v a t i v e s . On the other hand N-methyl, 0 - a r y l c a r bamates such as c a r b a r y l or carbofuran y i e l d e d o n l y the methyl ethers o f t h e i r r e s p e c t i v e phenols. T h i s work has now been ex tended to s u l f u r - c o n t a i n i n g carbamates such as methomyl, methiocarb, a l d i c a r b , e t c . (42-43). Here the oxime h y d r o l y s i s products of these carbamates are chromâtοgraphed as the 0-methyl oximes. T h i s r e a c t i o n has been a p p l i e d to the a n a l y s i s o f oxamyl (lower l e v e l 0.5 ug i n 50 g sample) i n s o i l u s i n g the S-mode FPD. However, i t must be p o i n t e d out that to date, the a p p l i c a t i o n of the phosphorylation and on-column methylation techniques have had only l i m i t e d a p p l i c a t i o n . Phenols and

Fungicides

Pentachlorophenol (PCP) i s widely used as a p e s t i c i d e i n a g r i c u l t u r e and as a wood p r e s e r v a t i v e i n i n d u s t r i a l and domestic products. PCP together with other chlorophenols which a r i s e as


PESTICIDE

244

ON-COLUMN

ο)

ANALYTICAL

METHYL AT ION

S NQ Q>-OP-(OCH ) 3

2

2

T M

6

^

c

H

-

N0 Q OCH CH30P(OCH ) >

2

3 +

3

S b) ( C H 0 ) . p . S - Ç H . C 0 E t CH C0 Et 2


CHC0 Et CHC0 Et

2

2

2

TMPT

Methyl Parathion

3

METHODOLOGY

2

2

S (CH 0) -PSCH, 3

2

2

TMDTP

Malathion

c) ^ ^ - N - C - O C H - M e

+

™ £ •

σ

MeO NCOCHMe

Propham

d)

Me NC-C=NOCNHMe SCH-, 2

Oxamyl

Figure 10.

HN

Examples

TMAH

M e N C - C = NOMe 2

SCH

3

Oxamyl methoxime

of on-column methylation reactions of organophosphorus and carbamate pesticides

NH II

S

Figure

11.

Acylation reactions of ethylenethiourea (ETU) with dichloroacetic anhydride (DC A A ) and dichloroacetyl chloride (DCACl)



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metabolites of the OP and carbamate i n s e c t i c i d e s , and phenoxyacids h e r b i c i d e s , have been v a r i o u s l y determined i n t a c t using FID d e t e c t i o n or,more commonly> as t h e i r e t h e r s , e s t e r s or s i l y l der i v a t i v e s (7) i n environmental, a g r i c u l t u r a l and mammalian samples. The more commonly used d e r i v a t i v e i s the methyl e t h e r , using d i a zomethane, a f t e r e x t r a c t i o n and/or an acid/base p a r t i t i o n i n g c l e a n up s t e p . However, r e c e n t l y Edgerton and Moseman (44) compared previous methods f o r the residue a n a l y s i s of PCP i n urine and found an Alumina clean-up column f o l l o w i n g d e r i v a t i s a t i o n was necessary f o r l e v e l s below 30 ppb. A l s o a 1 h r . c l o s e d system a c i d h y d r o l y s i s step using HC1 gave as much as a 1 7 - f o l d higher PCP l e v e l than d i d other methods, i n d i c a t i n g PCP forms conjugates i n u r i n e . The h y d r o l y s i s , methylation, column clean-up p r o cedure allowed l e v e l s of 1 ppb PCP to be detected i n u r i n e . Also a lower l e v e l of 0.01 ppm PCP i n fish,shrimp and o y s t e r s has been obtained a f t e r e t h y l a t i o n with diazoethane followed by a F l o r i s i l clean-up (45) . When the e t h y l a t i o n procedure was used on seawater e x t r a c t s i n t e r f e r e n c e s were observed at concentrations lower than 0.01 ppb u s i n g ECD-GLC. Formation of the amyl d e r i v a t i v e using diazopentane, i n c r e a s e d the GC r e t e n t i o n time s u f f i c i e n t l y to separate PCP from e a r l y e l u t i n g peaks. Concentrations of PCP as low as 2 ppt c o u l d be detected on sea water. I n t e r e s t i n g l y , the same workers a l s o u t i l i z e d HPLC with UV d e t e c t i o n of the f r e e phenol with clean-up to o b t a i n d e t e c t i o n l i m i t s o f 5 ppm i n t i s s u e s and 2 ppb i n sea water. The l i m i t of PCP acetate i s about 1-2.5 pg w i t h a Ni-ECD (46, 47) which gave a 5 ppb l i m i t of d e t e c t a b i l i t y f o r PCP i n adipose t i s s u e (47). In the l a s t few years a number o f r e p o r t s have appeared on an e x t r a c t i v e p e n t a f l u o r o b e n z y l a t i o n procedure f o r phenols and c a r b o x y l i c a c i d s to enhance t h e i r e l e c t r o n - c a p t u r i n g p r o p e r t i e s . Here the e x t r a c t i o n and d e r i v a t i z a t i o n of the phenol or a c i d i s accomplished i n one step by the p a r t i t i o n of the phenol (or acid) anion from the aqueous phase as an i o n - p a i r with a quaternary ammonium i o n i n t o an organic phase which contains the d e r i v a t i z a t i o n reagent (48-51). This technique has been a p p l i e d to the e x t r a c t i v e p e n t a f l u o b e n z y l a t i o n of 2,4-D, and MCPA i n water w i t h a d e t e c t i o n l i m i t of 1-3 ug/L (52). A l s o , the p r i n c i p l e has been a p p l i e d to the e x t r a c t i v e d e r i v a t i z a t i o n of ethylene t h i o u r e a (ETU) i n water. Even though ETU can be GC chromâtοgraphed i n t a c t on Versamid 900 and Carbowax 20 M packed columns (52, 53) or various c a p i l l a r y columns (54), by f a r the most popular approach has been the a l k y l a t i o n of the t h i o c a r b o x y l group and i n some i n s t a n c e s , double d e r i v a t i z a t i o n i n v o l v i n g the NH-group ( 7 ) . D e r i v a t i z a t i o n has r e s u l t e d i n improved GC c h a r a c t e r i s t i c s and i n c r e a s e d s e n s i t i v i t y using EC d e t e c t i o n . However, these procedures i n v a r i a b l y require lengthy r e a c t i o n times at r e f l u x temperatures to achieve acceptable y i e l d s . A l s o , under these c o n d i t i o n s any parent e t h y l enebisdithiocarbamate f u n g i c i d e residue that i s present i s con v e r t e d to ETU to a c e r t a i n extent. Figure 11 shows the r e a c t i o n sequence f o r the room temperature e x t r a c t i v e N - a c y l a t i o n of ETU



246

PESTICIDE

ι 0

1 2

Retention

1 4 Time

ANALYTICAL

METHODOLOGY

1 6 (min.)

Figure 12. (A) Standard of dichloroacetyl-ETU (4.0 ng); (B) blank apple juice extract; (C) apple juice extract spiked with 1 ppm ETU after extractive derivatiza tion with DCA A. Conditions: 6' X 2 mm i.d. glass column with 3% OV-330 on 80-100 mesh Chromosorb 750; flow rate, 30 mL/min helium; oven, 200°C; NP alkali flame ionization detector.



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from water u s i n g d i c h l o r o a c e t i c anhydride i n C ^ C l ^ . To e f f e c t e f f i c i e n t e x t r a c t i o n , a c e t o n i t r i l e was used (as phase t r a n s f e r agent) i n a 1:10 r a t i o w i t h the water. Reaction i s complete i n 3 min. Although S - a l k y l a t i o n c o u l d be p o s t u l a t e d as a p o s s i b l e r e a c t i o n pathway, t h i s was discounted a f t e r the d i - a c y l a t e d d e r i v a t i v e was prepared from the mono-product u s i n g d i c h l o r o a c e t y l c h l o r i d e . NMR and GC/MS a n a l y s i s showed that e l i m i n a t i o n of HC1 together w i t h r i n g - c l o s u r e occurs on GC i n j e c t i o n to give a sharp, s i n g l e peak on 0V-17 o r 0V-330 columns. O r i g i n a l l y , t h i s e x t r a c t i v e a c y l a t i o n procedure was used as a r a p i d s c r e e n i n g procedure f o r the presence o f ETU i n water samples a t the 0.ΟΙ Ο.05 ppm l e v e l (56) and has now been extended t o f r u i t and vegetable j u i c e s . Figure 12 shows the chromatographic c h a r a c t e r i s t i c s o f the ETU d e r i v a t i v e on an 0V-330 column and d e t e c t i o n u s i n g NP heated-bead d e t e c t o r . A l s o i l l u s t r a t e d i s the before and a f t e r chromatograms o f a spiked apple j u i c e sample a t the 1 ppm l e v e l . In c o n c l u s i o n , i t i s obvious that chemical d e r i v a t i z a t i o n continues and w i l l continue t o p l a y an important r o l e i n both q u a l i t a t i v e and q u a n t i t a t i v e a n a l y s i s o f p e s t i c i d e s , t h e i r r e s i dues and m e t a b o l i t e s . One o f the major advantages being that d e r i v a t i z a t i o n gives an improvement i n s e l e c t i v i t y as a r e s u l t of the formation o f a c h a r a c t e r i s t i c d e r i v a t i v e which responds s e l e c t i v e l y t o c e r t a i n GC and HPLC d e t e c t o r s .

LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

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COCHRANE

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RECEIVED February 7,

1980.


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