Pesticide Analytical Methodology - American Chemical Society

6

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 8, 2016 | http://pubs.acs.org Publication Date: October 30, 1980 | doi: 10.1021/bk-1980-0136.ch006

A Critical Comparison of Pre-Column and Post -ColumnFluorogenic Labeling for the HPLC Analysis of Pesticide Residues Η. A. MOYE Pesticide Research Laboratory, Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611 P. A. ST. JOHN American Instrument Company, Silver Spring, MD 20910 The concept of "fluorogenic labeling" had its beginnings in the early 1950's when dyes containing a naphthalene nucleus were adsorbed to proteins through weak molecular attractions (1). In an effort to better understand the effects of proteins on naph thalene dye fluorescence Hartly and Massey (2) in 1956 reacted 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride) with various amino acids. Other authors have detected dansylated amino acids by thin-layer chromatography (3-8). Dansyl chloride reacts with phenols as well and was used by Frei, Lawrence, Hope and Cassidy to derivatize hydrolyzed N-methyl carbamate pesticides prior to HPLC analysis on silica columns with fluorometric detect ion (9). Early work by Chen demonstrated that dansyl derivatives of amino acids suffered from severe quenching in protic solvents (10). More recently Froehlich and Murphy (11) have shown two to three fold enchancement in fluorescent intensity of dansylated amino acids over pure water by employing 30% dimethyl sulfoxide as solvent. Primary and secondary amines have been quantitated by thin layer chromatography after derivatization with 7-chloro-4-nitro -benzo-2-oxa-1,3-diazole (NBDCl, 12). The reagent has a l s o been used f o r a n a l y s i s o f reduced nitrosamines employing a d s o r p t i v e mode HPLC (13). We have reported on the use o f 9-fluorenylmethyl c h l o r o formate (FM0CC1) as a pre-column f l u o r o g e n i c l a b e l i n g reagent f o r primary and secondary amines, and found i t to be p a r t i c u l a r l y s u i t a b l e f o r aqueous based HPLC systems such as i o n exchange where both dansyl c h l o r i d e and NBDC1 d e r i v a t i v e s e x t e n s i v e l y l o s e quan tum e f f i c i e n c i e s with concurrent l o s s e s i n l i m i t s o f d e t e c t i o n (14). As w e l l , we have a l s o reported on a post-column f l u o r o g e n i c l a b e l i n g HPLC arrangement f o r the a n a l y s i s of N-methylcarbamate p e s t i c i d e s employing the primary amine s p e c i f i c o - p h t h a l i c d i carboxaldehyde-mercaptoethanol (ΟΡΑ-MERC) reagent (15). T h i s sys tem was e x t e n s i v e l y studied by Krause (16), and found to be sen s i t i v e , s e l e c t i v e , r e p r o d u c i b l e and s t a b l e . By s u b s t i t u t i n g an o x i d a t i v e calcium h y p o c h l o r i t e reagent f o r the h y d r o l y t i c sodium

0-8412-05 81 -7/80/47-136-089$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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hydroxide reagent i n t h i s system we have discovered that glyphosate h e r b i c i d e (n-phosphonomethylglycine) can be r e a d i l y cleaved to produce a primary amine which r a p i d l y r e a c t s with the OPA-MERC reagent to produce a fluorophore (15). We chose the FM0CC1 reagent and the OPA-MERC system to study the r e l a t i v e merits of pre-column and post-column f l u o r o g e n i c l a b e l i n g when u t i l i z e d i n the development of HPLC based r e s i d u e procedures f o r glyphosate h e r b i c i d e (GLYPH) and i t s major metab o l i t e , aminomethylphosphonic a c i d (AMPA). Some of the c o n c l u s i o n s presented here are based upon experimental data while others a r i s e from conceptual examination of the two approaches. Additionally, some of the c o n c l u s i o n s recorded here are not d e f i n i t i v e but r e s u l t from a l i m i t e d number of observations and are included i n order to a s s i s t the reader i n understanding why c e r t a i n choices were made during the development of the procedures. P a r a l l e l , and i n f r e q u e n t l y d u p l i c a t i v e , e f f o r t s of the type described here were judged by the authors to be j u s t i f i a b l e i n l i g h t of the recent worldwide i n t e r e s t i n the h e r b i c i d e glyphosate i n a d d i t i o n to the f a c t that the only p r e v i o u s l y published r e s i d u e procedure f o r the h e r b i c i d e (17) has g e n e r a l l y become to be r e garded as lengthy, cumbersome and subject to low r e c o v e r i e s . Apparatus

and Reagents

Pre-column L a b e l i n g (FM0CC1). A f l u o r o m e t r i c HPLC was con s t r u c t e d from two Waters A s s o c i a t e s model 6000A pumps, a Waters A s s o c i a t e s model 660 solvent programmer, a Rheodyne model 7010 sample i n j e c t i o n v a l v e equipped with a 20 y l sample loop and an American Instrument Co. Aminco-Bowman spectrophotofluorometer model 4-8202, equipped with a model B16-63019 flow through c e l l . Chromatograms were recorded on a Sargent model MR s t r i p chart recorder. A Corning CS #0-54 c u t o f f f i l t e r (290 nm) was placed before the p h o t o m u l t i p l i e r tube (IP28) to reduce the s c a t t e r e d l i g h t from the e x c i t a t i o n monochromator. E x c i t a t i o n was at 270 nm and emission at 315 nm; a 150 W. xenon arc lamp was used as a source. S l i t program was set at 3,3,3,3,3,5. Separations were achieved on Waters A s s o c i a t e s μ Carbohydrate (4 mm χ 30 cm) or μ NH£ (4 mm χ 30 cm) columns operated i n the anion exchange mode. I s o c r a t i c o p e r a t i o n , unless otherwise s p e c i f i e d , was conducted at 1.0 ml/min with pH 4 phosphate b u f f e r (0.1 M) c o n t a i n i n g 25% aceton i t r i l e by volume. Various solvent programs were a l s o attempted. Acetone was p e s t i c i d e grade; a l l other reagents were reagent grade. Cation exchange sample cleanup was performed on a 2.4 cm χ 50 cm g l a s s column having an i n t e g r a l 500 ml r e s e r v o i r and a r e movable stopcock. E x a c t l y 190 g of hydrogen form Dowex 50W -X8, 100-200 mesh, e q u i l i b r a t e d with 1 l i t e r of 0.1N HC1, was used to pack each column and d i s c a r d e d a f t e r use. Water e x t r a c t s of crops were concentrated on a Bilchi Rotovapor model R under minimum pressue from a water a s p i r a t o r ;

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

6.

MOYE

A N D ST. J O H N

Pre- and Post-Column

Fluorogenic


condensor coolant was kept a t 5°C by a c i r c u l a t i n g water bath.

Labeling

91

refrigerated

Post-Column L a b e l i n g (OPA-MERC). An arrangement ( F i g . 1) s i m i l a r to that p r e v i o u s l y reported f o r the determination of N-methylcarbamate p e s t i c i d e s (15) was constructed from a Waters A s s o c i a t e s model 6000 pump, a Rheodyne 7010 i n j e c t o r with a 20 μΐ loop, a 4 mm χ 25 cm s t a i n l e s s s t e e l column packed with 13.5 μ Aminex A-27 (Biorad L a b o r a t o r i e s ) , two M i l t o n Roy Model 196-0066001 reagent pumps, an American Instrument Co. Fluoromonitor equip ped with ΟΡΑ f i l t e r s (360 and 455 nm) or a G i l s o n Spectra/glo fluorometer s i m i l a r l y equipped. Chromatograms were recorded on a V a r i a n model A20 s t r i p chart recorder (100 mv). Oxidant (calcium h y p o c h l o r i t e ) was pumped and mixed with the HPLC column eluent v i a model CJ3031 Kel-F "T"s (Laboratory Data C o n t r o l ) and 1.6 mm O.D. χ 0.5 mm I.D. T e f l o n tubing. A 10.6 m delay c o i l of s i m i l a r tubing (0.29 ml volume) was used to provide a delay time of about 1 min. 40 sec. before e n t e r i n g another Kel-F "T" i n t o which the OPA-MERC reagent was pumped ; a 0.6 m l e n g t h o f tubing c a r r i e d the mixture to the fluorometer. The ΟΡΑ reagent was made up as pre v i o u s l y described (15). E x a c t l y 1 g was d i s s o l v e d i n 10 ml of dioxane and d i l u t e d to 1 l i t e r with pH 10, 0.125 M borate b u f f e r to which 1 ml of MERC was added. Thermostatting of the columns was accomplised a t 62°C with a small o i l f i l l e d bath (model PY1, Bench Scale Equipment Co.). Mobile phase f o r the a n a l y t i c a l HPLC i o n exchange column was 0.1 M H3PO4 at 1.0 ml/min. Calcium h y p o c h l o r i t e was prepared by d i s s o l v i n g 12 mg of HTH ( O l i n Co.) and 11.6 g NaCl i n 1 l i t e r of 0.1 M K H P 0 and a d j u s t i n g the pH to 9.0 with 10 M KOH. 2

4

Experimental Pre-column L a b e l i n g (FM0CC1). FM0CC1 r e a c t s v i a an Sn2 mech anism with the amino n i t r o g e n of both primary and secondary amines producing a carbamate having a f l u o r e n y l group as the fluorophore (14). K i n e t i c s of the r e a c t i o n with the primary amine AMPA were impossible to measure by HPLC, however, that of GLYPH showed a r a p i d r e a c t i o n ( F i g . 2 ) . The m i c r o - s c a l e d e r i v a t i z a t i o n s were performed by p l a c i n g 0.1 ml of 10~ -10~ M GLYPH or AMPA along with 0.9 ml of 0.025 M pH 9 sodium borate, 0.9 ml of acetone and 0.1 ml of 10"" M s o l u t i o n of FM0CC1 i n acetone, i n t o a T e f l o n capped 16 mm χ 125 mm c u l t u r e tube. The s o l u t i o n s were incubated at 23°C f o r 20 min. without shaking or s t i r r i n g a f t e r which 3 ml p o r t i o n s of e t h y l ether were used to wash away excess reagent. Appropriate d i l u t i o n s were made with water before i n j e c t i o n i n t o the l i q u i d chromatograph. A t y p i c a l chromatogram using the μ Carbohydrate column i s shown i n F i g . 3. When 10" M GLYPH was d e r i v a t i z e d and compared to an a u t h e n t i c standard, as p r e v i o u s l y described (14), 109% conversion was c a l c u l a t e d , which could be explained by a somewhat impure a u t h e n t i c standard. 7

3

2

5


PESTICIDE

A

PUMP

MOBILE


PHASE

35FT DELAY COIL

AMINEX A27 COLUMN χ 62*C

A Ca(CIO)

ΟΡΑ

2

SOLUTION

pH 9

Figure 7.

FLUOROMETER

PUMP

PUMP

SAMPLE INJECTION VALVE

METHODOLOGY

HoPQa

0.1 M

A

ANALYTICAL

Schematic of post-column fluorogenic OPA-MERC

WASTE

HPLC arrangement

40'

5-

oJ

° 0

I

5

I

Î0

I

15

I

20

1

25

1

30

35

MINUTES Figure 2. Kinetics curve of GLYPH-FMOCCl reaction as monitored by HPLC (5 X 10~ M glyphosate, 5 χ 10 M FMOCCl in 1:1 acetone :0.025U sodium borate, pH 9.0, 23°C) 5

4


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M O Y E

AND

ST.

J O H N

Pre-

and

Post-Column

Fluorogenic

Labeling

93

Crop E x t r a c t i o n and Cleanup (FM0CC1). The e x t r a c t i o n p r o cedure found i n the P e s t i c i d e A n a l y t i c a l Manual (17) was u t i l i z e d throughout. I n t h i s procedure 100 g of chopped c r o p , 100 ml of chloroform and 200 ml of water were added to a 1 quart Mason j a r and blended a t medium speed f o r 15 min. The j a r was r i n s e d w i t h 2 χ 20 ml of Η 0 and the combined contents and r i n s e s d i s t r i b u t e d e q u a l l y between three 60 mm χ 120 mm polypropylene c e n t r i f u g e b o t t l e s . A f t e r c e n t r i f u g a t i o n a t 10,000 rpm f o r 20 min. the aqueous l a y e r s were combined and r o t a r y evaporated to 50 ml a t which time the pH was a d j u s t e d to 1.0 w i t h concentrated HC1. A l l of the sample (50 ml + 10 ml wash) was p l a c e d a t the top of the 50W - X8 column and e l u t e d a t 3.5 ml/min w i t h 0.1 N HC1. GLYPH appeared i n the 280 to 400 ml f r a c t i o n and AMPA appeared i n the 580 to 800 ml f r a c t i o n . These f r a c t i o n s were r o t a r y evapo r a t e d s e p a r a t e l y to approximately 4 ml which were then t r a n s f e r r e d to a 5 ml v o l u m e t r i c and made to volume w i t h a 1 ml r i n s e of the flask.


2

Crop D e r i v a t i z a t i o n (FM0CC1). D e r i v a t i z a t i o n of crop f r a c t i o n s c o l l e c t e d from the 50W - X8 column was performed w i t h a p r o p o r t i o n a t e l y l a r g e r amount of FM0CC1 to accommodate reagent scavenging by c o - e x t r a c t i v e s t h a t were not f u l l y i s o l a t e d by the column. I n a d d i t i o n , K2CO3 (150 mg) had to be added to the ex tremely a c i d i c (pH 0) c o n c e n t r a t e so that the borate b u f f e r was not consumed. E x a c t l y 1 ml of e i t h e r the GLYPH or AMPA f r a c t i o n was p l a c e d i n a T e f l o n capped c u l t u r e tube and approximately 150 mg of K2CO3 was added w i t h shaking to b r i n g the pH to 11. Along w i t h 4 ml of H2O 5 ml of 0.1 M FM0CC1 i n acetone was added to the tube which was capped and reacted a t 23°C f o r 20 min. The r e a c t i o n mix was washed 3 times w i t h 5 ml of e t h y l e t h e r , d i l u t e d to 10 ml w i t h H2O and i n j e c t e d onto the HPLC. Comparisons were made to standards i n 0.1 M HC1 which were s i m i l a r l y d e r i v a t i z e d . Crop E x t r a c t i o n and Cleanup (OPA-MERC). GLYPH and AMPA were e x t r a c t e d and cleaned up p r i o r to post-column f l u o r o g e n i c l a b e l i n g HPLC d e t e r m i n a t i o n i n e x a c t l y the same manner as f o r the FM0CC1 procedure w i t h the e x c e p t i o n that the concentrated h i g h l y a c i d i c f r a c t i o n s from the 50W - X8 columns were a d j u s t e d w i t h 10 M KOH to pH 3-8. T h i s was necessary i n order to prevent adverse s h i f t i n g of the HPLC mobile phase pH and subsequent s h i f t s i n r e t e n t i o n and d e t e r i o r a t i o n of peak shape. R e s u l t s and D i s c u s s i o n HPLC S e p a r a t i o n s . The columns chosen f o r the FM0CC1 and OPAMERC experiments were a r e s u l t of s e v e r a l c o n s i d e r a t i o n s and ob s e r v a t i o n s . Although a l l four columns used operated i n the anion exchange mode o n l y the s i l i c a p a r t i c l e columns (μ Carbohydrate and μΝΗ2) were s u c c e s s f u l i n chromatographing GLYPH and AMPA, o s t e n s i b l y due to the i n t e r a c t i o n of the f l u o r e n y l moiety of the


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2ng GLYFMOC

9

12

15

MINUTES

Analytical Letters

Figure 3. Typical chromatogram of AMPA and GLYPH derivatives (μ carbo hydrate column, 25% acetonitrile:0.1M KH PO pH 4; 1 mL/min flow, 20 /xL injection (là) 2

it

Figure 4. Chromatogram and elution pattern, as determined by collecting 1-mL fractions following injection of radiolabeled A MP A and GLYPH, showing enhanced retention of GLYPH-FMOC (μ carbohydrate column, 25% acetonitrile/0.025M KH PO pH 4; 1 mL/min flow) t

it



6.

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A N D ST. J O H N


Fluorogenic

Labeling

95

d e r i v a t i v e w i t h the p o l y s t y r e n e - d i v i n y l b e n z e n e polymer forming the p l a s t i c bead. Even 0.1 M H3PO4 was u n s u c c e s s f u l i n e l u t i n g e i t h e r d e r i v a t i v e . These columns, however, i n c o n t r a s t to the p l a s t i c bead type (HA-X10 and Aminex A-27) could be r e a d i l y programmed by both pH and i o n i c s t r e n g t h . The quartenary ammonium p l a s t i c bead type column was necessary i n the OPA-MERC postcolumn f l u o r o g e n i c l a b e l i n g arrangement, however, due to c o n t i n ued column bleed from the s i l i c a p a r t i c l e columns which caused extremely high background f l u o r e s c e n c e . In sharp c o n t r a s t to the e a s i l y programmable s i l i c a p a r t i c l e columns the p l a s t i c bead column could not be programmed a t a l l and took s e v e r a l hours to e q u i l i b r a t e when even small changes were made i n i o n i c strength or pH. E f f i c i e n c i e s f o r the two types of columns were both somewhat low a t about 1500 t h e o r e t i c a l p l a t e s f o r the p l a s t i c bead column and only about 1000 f o r the s i l i c a p a r t i c l e columns ; the l a t t e r were more prone to d e t e r i o r a t i o n from crop c o - e x t r a c t i v e s , dropping to only about 600 a f t e r months of use, while the p l a s t i c bead column was not measurably a f f e c t e d . While i t i s g e n e r a l l y recognized that pre-column d e r i v a t i z a t i o n f r e q u e n t l y leads t o improved r e s o l u t i o n i n a multicomponent mixture our observations w i t h the FM0CC1 d e r i v a t i v e s of GLYPH and AMPA i n d i c a t e that i t can be sometimes d e t r i m e n t a l , as seen i n F i g . 4 where ^ C l a b e l e d GLYPH and AMPA were chromatographed und e r i v a t i z e d ; 1 ml f r a c t i o n s were c o l l e c t e d and counted i n a s c i n t i l l a t i o n spectrometer. When GLYPH and AMPA were d e r i v a t i z e d and chromatographed a s h i f t i n GLYPH r e t e n t i o n i s observed, from 14 min. to 20.5 min.; the AMPA r e t e n t i o n remained unchanged a t 7.5 min. Since, of s i x amino a c i d s , a l l e l u t e d before AMPA and presented no i n t e r f e r e n c e s the increased r e t e n t i o n o f GLYPH appears to only increase a n a l y s i s time, a d e f i n i t e detriment. Post Column D e r i v a t i z a t i o n (OPA-MERC) O p t i m i z a t i o n . GLYPH response was measured as a f u n c t i o n o f the OPA-MERC reagent flow and Ca(C10)2 reagent flow. Flows were v a r i e d from 0.2 to 0.6 ml/min; while each was being v a r i e d the other was held a t 0.3 ml/min, which was observed to be the optimum flow f o r both of them when column mobile phase (0.1 M H3PO4) was h e l d a t 1.0 ml/min. The C a ( C 1 0 ) cleavage r e a c t i o n of GLYPH was not s t u d i e d ext e n s i v e l y . By exposing GLYPH t o Ca(C10)2 a t pH 2 and then p r e column d e r i v a t i z i n g with FM0CC1 followed by chromatography on the yNH column i t was apparent that g l y c i n e was one r e a c t i o n product that was produced which could then r e a c t w i t h OPA-MERC. By lengthening the delay c o i l from 5 f t to 35 f t , g i v i n g an i n c r e a s e i n r e a c t i o n time from 14 sec. to 1 min. 38 sec. , an increase i n GLYPH peak area by a f a c t o r o f 2 was r e a l i z e d . Even though i t was apparent that GLYPH was not being completely converted to a primary amine the s e n s i t i v i t y was adequate f o r r e s i d u e s t u d i e s and r e p r o d u c i b i l i t y was good (see Crop Recoveries s e c t i o n ) . 2

2



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ANALYTICAL

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Column Cleanup S t u d i e s . One of the primary goals of t h i s study was to achieve a s i g n i f i c a n t r e d u c t i o n i n time r e q u i r e d f o r sample e x t r a c t i o n and cleanup, as w e l l as to e l i m i n a t e one, or p r e f e r a b l y both, of the d e r i v a t i z a t i o n steps used i n the ΡAM method. Many and v a r i e d attempts were made at s i n g l e column cleanup of f r u i t s and v e g e t a b l e s , i n c l u d i n g c a t i o n exchange, anion exchange, a d s o r p t i o n , r e v e r s e phase and molecular s i z e . Of these, c a t i o n exchange on a 50 χ 2.4 cm column w i t h 190 g of 100-200 mesh 50W - X8 appeared to be the most s a t i s f a c t o r y . R e t e n t i o n of g l y phosate was s t r o n g l y dependent upon eluent pH; maximum r e t e n t i o n was observed at 0.05 - 0.1 M HC1 and was the key to the s i n g l e column cleanup s i n c e i t allowed f o r maximum s e p a r a t i o n of GLYPH from sugars, e t c . , which were not r e t a i n e d by the column. With 0.1 M HC1 as eluent AMPA began coming o f f the column at 580 ml, w e l l separated from crop i n t e r f e r e n c e s . GLYPH began e l u t i n g a t 280 ml; i t s f r a c t i o n contained a l l of the s i g n i f i c a n t crop peaks which were apparent on the HPLC chromatogram but which d i d not i n t e r f e r e w i t h the GLYPH peak s i n c e they were e a r l y e l u t e r s . The e l u t i o n p a t t e r n s are i l l u s t r a t e d i n F i g . 5. Crop R e c o v e r i e s , Pre-Column D e r i v a t i z a t i o n (FM0CC1). As i l l u s t r a t e d i n F i g . 6 i t was p o s s i b l e to recover GLYPH a t 0.1 ppm e s s e n t i a l l y q u a n t i t a t i v e l y from cantaloupe u s i n g the FM0CC1 p r e column d e r i v a t i z a t i o n procedure. However, upon changing HPLC chromatographic c o n d i t i o n s f o r AMPA by l o w e r i n g the mobile phase i o n i c s t r e n g t h there appeared to be s e v e r a l i n t e r f e r e n c e s which were unresovable from AMPA-FMOC. Since such i n t e r f e r e n c e s seemed insurmountable and i t was necessary to q u a n t i t a t e AMPA, no r e c o v e r i e s were attempted f o r GLYPH a l o n e . Crop R e c o v e r i e s , Post-Column D e r i v a t i z a t i o n (OPA-MERC). The good to e x c e l l e n t r e c o v e r i e s r e a l i z e d f o r a l l crops s t u d i e d r e s u l t e d from the e f f i c i e n c i e s of both the c a t i o n exchange cleanup and anion exchange a n a l y t i c a l columns, the care taken i n the r o t a r y e v a p o r a t i o n of the samples ( 5 °C condenser temp., maximum a s p i r a t i o n and 40°C water b a t h ) , and the s e l e c t i v i t y of the OPAMERC reagent. As seen i n Table I , r e c o v e r i e s a t 0.1 ppm f o r AMPA ranged from 61 to 82% and f o r GLYPH from 70 to 96%. T y p i c a l chromatograms are i l l u s t r a t e d i n F i g s . 7 and 8. The o v e r a l l r e p r o d u c i b i l i t y of the post-column f l u o r o g e n i c l a b e l i n g HPLC as w e l l as the proposed procedure f o r AMPA and GLYPH i s i l l u s t r a t e d by the r e p l i c a t e r e c o v e r i e s from cucumber which were measured over a two day p e r i o d (Table I I ) :



MOYE

AND

Figure 5.

ST.

J O H N

Pre-

and Post-Column

Fluorogenic

Labeling

Elution pattern of GLYPH (-Ο-) and AMPA (-φ-) from 50 cm 2.4 cm 50W — X8 column as a function of eluent pH


X


PESTICIDE

STANDARD

li O

Figure (μΝΗ pH 4) (μΝΗ 2

2

Li 3

ANALYTICAL

METHODOLOGY

C A N T E L O U P E CHECK

ι 6

ι ι 9 0 MINUTES

1 3

1 6

1 9

6. Chromatogram showing (top) recovery of GLYPH from cantaloupe column, 1 mL/min flow, 20 μ-L injection, 25% acetonitrile/O.lM KH PO , and (bottom) cantaloupe coextractives interfering with AMPA derivative column, 1 mL/min flow, 20 /JL injection, 25% acetonitrile/0.025M KH PO pH 4) È

2

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k

6.

MOYE

A N D

ST.


J O H N

Table I .


C r

Crop Recoveries

S i n g l e recovery C a l c u l a t e d from C a l c u l a t e d from Average of f i v e

Labeling

99

a t 0.1 ppm

AMPA

°P

Cantaloupe Cranberries Jalapeno peppers Pumpkin ^ Cucumber a. b. c. d.

Fluorogenic

GLYPH 92% 76 70 90 96

68% 61 65 68 82

sample; average o f d u p l i c a t e i n j e c t i o n s . peak h e i g h t s . peak areas. r e p l i c a t e samples.

Table I I . R e p r o d u c i b i l i t y o f Recoveries a t at 0.1 ppm from Cucumber a

Sample No.

AMPA

GLYPH

81% 83 81 83 84

108% 89 96 97 92

= = a. b. c.

82.4% 1.3

χ = 96.4% s = 7.2

Average o f d u p l i c a t e i n j e c t i o n s . C a l c u l a t e d from peak h e i g h t s . C a l c u l a t e d from peak areas.

Standard d e v i a t i o n s f o r AMPA r e c o v e r i e s were l e s s than 2%, e x c e l l e n t f o r r e p l i c a t e r e c o v e r i e s w h i l e the percentage remained high (82.4%). While GLYPH r e c o v e r i e s have always been higher than AMPA f o r a l l crops s t u d i e d the v a r i a b i l i t y has a l s o been higher o s t e n s i b l y due to the incompleteness of the Ca(C10)2 cleavage r e a c t i o n . S t i l l , a standard d e v i a t i o n of l e s s than 8% appears acceptable. A time and cost a n a l y s i s f o r the determination of two r e s i d u e



100

PESTICIDE

ANALYTICAL

CHECK

Figure 7. Chromatograms of (left) untreated (check) cucumber and (right) AMPA-fortifled cucumber at 0.1 ppm (Aminex A-27 column, 0.1M H PO , 1 mL/min flow, 20 pL injection, atten 2X) s

h

METHODOLOGY

0.1 ppm

15.0

Figure 8. Chromatograms of (left) untreated (check) cucumber and (right) GLYPH-fortified cucumber at 0.1 ppm (Aminex A-27 column, 0.1M H,PO , 1 mL/min flow, 20 pL injection, atten 10X) h


6.

M O Y E

A N D ST. J O H N


Fluorogenic

Labeling

101

samples i n c l u d i n g a three p o i n t a n a l y t i c a l curve f o r AMPA and GLYPH i s shown i n Table I I I :

Table I I I . Time and Cost A n a l y s i s f o r AMPA and GLYPH Residue Determination (OPA-MERC) a


Step 1. 2. 3. 4. 5. 6.

7.

a. b. c.

Extraction Centrifugation Rotary evaporation (50 ml) Column prep. (50W - X8) Column e l u t i o n Rotary evaporation (5 ml) HPLC Totals

Hours

% of 8 hour day

$/sample

0.5 0.5 1.0

6 6

0.22

13 b

2.7 1.3 2.0° 8.0

—

34 16 25 100

— —

12.62 — —

0.10 12.94

Two samples Columns prepared previous day. Includes 3 p o i n t a n a l y t i c a l curve.

Conclusions For the a p p l i c a t i o n d e s c r i b e d here a post-column f l u o r o g e n i c l a b e l i n g approach was devised which was demonstrated to be s u f f i c i e n t l y s e n s i t i v e (0.1 ppm), r e p r o d u c i b l e , economical and r e l a t i v e l y r a p i d compared to the e x i s t i n g ΡAM procedure. By nor m a l i z i n g on an anion exchange HPLC s e p a r a t i o n two f l u o r o g e n i c reagents were chosen that had demonstrated high quantum y i e l d s and thus were capable of producing high s e n s i t i v i t i e s f o r s t a n dards . I t was necessary to choose a pre-column f l u o r o g e n i c l a b e l i n g reagent (FM0CC1) which d e r i v a t i z e d both primary (AMPA) and secondary amines (GLYPH). T h i s reagent a l s o d e r i v a t i z e s a l c o h o l s under the c o n d i t i o n s that were used and consequently could be expected to be scavanged by crop c o e x t r a c t i v e s while producing p o s s i b l e i n t e r f e r e n c e s ; t h i s was indeed observed f o r the cantaloupe AMPA f r a c t i o n . Conversely, s i n c e GLYPH i s cleaved by Ca(C10) to produce a primary amine which r e a c t s with OPA-MERC, a primary amine s p e c i f i c reagent, fewer p o t e n t i a l i n t e r f e r e n c e s ought to be expected, as was observed. Amino a c i d s , as w e l l as s e v e r a l n a t u r a l l y o c c u r r i n g phosphonic and s u l f o n i c a c i d s d i d not i n t e r f e r e with the post-column f l u o r o g e n i c l a b e l i n g determination o f the e a r l y e l u t i n g AMPA. A peak e l u t i n g a f t e r AMPA, which d i d not i n t e r f e r e a t the 0.1 ppm l e v e l and occurred i n s e v e r a l crops, was not i d e n t i f i e d . 2



102

PESTICIDE

ANALYTICAL

METHODOLOGY

It was observed that the quartenary ammonium p l a s t i c bead column packings were rugged and l e s s prone to being fouled by crop c o e x t r a c t i v e s than were the chemically bonded s i l i c a p a r t i c l e columns. A f t e r over a year of use they r e t a i n e d t h e i r ef f i c i e n c y and s e n s i t i v i t y . T h e i r o p e r a t i o n with a c i d i c buffers (pH 1-4) was p e r f e c t l y compatible with the need to perform the Ca(C10)2 cleavage of GLYPH under a c i d i c c o n d i t i o n s . Fortunately, the b u f f e r i n g a c t i o n of the ΟΡΑ-MERC reagent was adequate to s h i f t the pH to 10, an optimum for the r i n g formation to o c c u r . The procedure described here takes f u l l advantage of the water s o l u b i l i t y of the two a n a l y t e s , t h e i r a n i o n i c and c a t i o n i c behavior, the speed of HPLC separations and i t s s u i t a b i l i t y for the a n a l y s i s of n o n - v o l a t i l e compounds.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Weber, G.; Laurance, D. J. R. Biochem. J., 1954, 51, xxxi. Hartley, B. S.; Massey, V. Biochim. Biophys. Acta, 1956, 21, 58. Gros, C.; Labousse, B. Europ. J. Biochem., 1969, 7, 463. Schmer, G.; Kreil, G. J. Chromatog., 1967, 28, 458. Seiler, N.; Weichmann, J. Experientia, 1964, 20, 559. Deyl, Z.; Rosmus, J. J. Chromatog., 1965, 20, 514. Morse, D.; Horecker, B. L. Anal. Biochem., 1966, 14, 429. Mesrob, B.; Holeysovsky, V. J. Chromatog., 1966, 21, 135. Frei, R. W.; Lawrence, J. F.; Hope, J.; Cassidy, R. M. J. Chromatogr. Sci., 1974, 12, 40. Chen, R. F. Arch. Biochem. Biophys., 1967, 120, 609. Froehlich, P. M.; Murphy, L. D. Anal. Chem., 1977, 49, 1606. Lawrence, J. F.; Frei, R. W. Anal. Chem., 1972, 44, 2046. Klimisch, H -J.; Ambrosius, D. J. Chromatog., 1976, 121, 93. Moye, H. A.; Boning, A. J. Anal. Lett., 1979, 12(B1), 25. Moye, H. A.; Scherer, S. J.; St. John, P. A. Anal. Lett., 1977, 10(13), 1049. Krause, R. T. J. Chromatogr. Sci., 1978, 16, 281. Pesticide Analytical Manual, Food and Drug Administration, Washington, D. C., Pest. Reg. Sec. 180.364.

RECEIVED

February 7, 1980.


Pesticide Analytical Methodology - American Chemical Society

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