ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
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by the design of the system. The optimum scan time appears to be about 10 ms/level which requires some 60 ms/scan for the six levels. To keep this in perspective, it should be noted t h a t the most popular commercial vidicon system involves a 30-ms scan time for a single level. The most serious limitation of the present system in our view is the stray light figure. This probably can be improved with some modifications of the present system or other completely different designs. Nevertheless, the work demonstrates that it is possible to use the two-dimensional character of the silicon vidicon to aid the range vs. resolution tradeoff.
(3) Felkel, H. L., Jr.; Pardue, H. L. Anal. Chem. 1978, 50, 602. (4) Felkel, H. L., Jr.; Pardue, H. L. Clin. Chem. ( Winston-Salem, N.C.) 1978, 24, 602. (5) Harrison, G. R.; Lord, R. C.; Lofbourow, J. R. "Practical Spectroscopy"; Prentice Hall: New York, 1948; p 548. (6) Felkel, H. L., Jr.; Pardue, H. L. Anal. Chem. 1977, 49, 1112. (7) Hoffman, R. M.; Pardue, H. L. Anal. Chem. 1978, 50, 1458. ( 8 ) Slavin, W. Anal. Chem. 1963, 35, 561. (9) Grant, G. H.; Kachmer, J. F. In "Fundamentals of Clinical Chemistry"; Tietz, N. W., Ed.; W. B. Saunders Co.: Philadelphia,Pa., 1976; pp 299-304. (10) McDowell, A. E.; Pardue, Harry L. J . Pharm. Sci. 1978, 67, 822. (11) Clark, W. M. "Determination of ktydrogen Ions", 3rd 4.;William and Wilkins Co.: Baltimore, Md., 1928; p 84. (12) Barde, W. R. J . Am. Chem. SOC. 1924, 46, 581.
LITERATURE CITED
RECEIVED for review February 16, 1979. Accepted April 12, 1979. This work was supported by Grant No. CHE 75-1550
(1) Danielsson, A,; Lindblom, P. Appl. Spectrosc. 1976, 30, 151. (2) Wood, D. L.; Dargis, A. B.; Nash, D. L. Appl. Spectrosc 1975, 27, 310.
AD1 from the National Science Foundation.
Decomposition at 90 "C of the Cholinesterase Substrate Indoxyl Acetate Impregnated on Paper Supports Thaddeus J. Novak, Lester W. Daasch, and Joseph Epstein" Research Division, Chemical Systems Laboratory, Aberdeen Proving Ground, Maryland 2 10 10
Decomposition of cholinesterase substrate indoxyl acetate impregnated on paper supports and stored at 90 OC was found to be due to hydrolysis caused by water occluded or physically absorbed on cellulosic paper. While an initiating reaction between the indoxyl acetate and the cellulosic hydroxyl groups in the paper was not entirely ruled out as a secondary cause of the degradation, no evidence for participation of the hydroxyl groups was found. Significant improvement in the indoxyl acetate stability was realized by substituting a Teflon support or acetylated papers for the unacetylated paper support.
Procedures commonly used for the analysis (and detection) of organophosphorus ester pesticides and nerve gases make use of the fact that these esters rapidly and irreversibly inactivate esterase enzymes, and thus render them incapable of hydrolyzing their substrates. With proper choice of the esterase, substrate, and reaction conditions, the detection and estimation of concentrations of two nerve gases in water at the parts-per-trillion level have been made ( I ) . An important factor in the sensitivity of the analysis is the speed of inactivation, and therefore the source of the enzyme (and the substrate) is often determined by the class of compounds to be analyzed. One particularly useful adaptation of the enzymatic method is in detection of nerve gases in field water samples (2). 'The test reagents are impregnated on papers. A shortcoming of the test, however, is that a contact time of 20 min between water containing 0.01 mg/L of the nerve gas isopropylmethylphosphonofluoridate (Sarin) only partially inhibits a test paper impregnated with cholinesterase obtained from horse serum. For an unequivocal detection a t a particular level, the enzyme must be totally inhibited so t h a t there is no change in color due to the hydrolysis of a chromogenic substrate. The level set for the maximum permissible concentrations of Sarin in drinking water has been 0.02 mg/L (3) but is now con-
sidered to be too high by a factor of approximately 4 especially for tropical areas where the consumption of water exceeds 5 L per day. The detection nerve gases a t the proposed level of 0.005 mg/L using the test papers described in Ref. 2 would take an unacceptably long time. To decrease the required time for incubation (or increase the sensitivity), it has been decided ( 3 )to replace horse serum cholinesterase with eel cholinesterase, whose rate of inactivation by Sarin is approximately four times that of the horse serum cholinesterase ( I ) . The use of eel cholinesterase precipitated the requirement t h a t the previously used chromogenic substrate, 2,6-dichloroindophenylacetate, acceptable for horse serum cholinesterase (2), be replaced. Indoxyl acetate, an excellent substrate of eel cholinesterase ( I ) has been selected for this purpose. It is envisioned that both the substrate and the enzyme will be impregnated on papers. In the final development of this analytical technique, the acceptability of the technique will depend upon, at least in part, the functioning of the reagents on the paper, especially after storage under laboratory conditions (shelf-life) and also after storage a t elevated temperatures (up to 100 "C)for short periods of time. This paper presents our studies on the stability of indoxyl acetate on paper a t 90 "C. EXPERIMENTAL Chemicals, Solvents, and Materials. Except where anhydrous indoxyl acetate (Sigma Chemical Co.) was used, the indoxyl acetate (97%)was from Aldrich Chemical Co. Isatoic anhydride (recrystallizedgrade).was from Sherwin Williams, Inc. Isatin and anthranilic acid were obtained from Chem Services, Inc. Methanol (ACS grade) was from Fishe jcientific, Inc.; acetone (GC-Spectrophotometric grade) was from J. T. Baker Chemical Co.; and dichloromethane (distilled-in-glass grade) was from Burdick and Jackson Laboratories, Inc. Whatman papers no. 1, no. ,:I no. 3 MM were from Reeve Angel, Inc. S&S papers Type 2495, Type 2497, and Type 2498, were from Schleicher & Schuell, Inc. Teflon tape (John Crane
This article not subject to U.S. Copyright. Published 1979 by the American Chemical Society
1272
ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
m:
Cl-+OO
,I1
\I
0
'
I L
It
( 4 ) . Compound 111 melted at 262-3 "C [reported, 262-3 "C ( 5 ) and 261 "C (611. Mass spectra did not allow us to make a distinction between structures IVa and IVb. Structure IVa is preferred over structure IVb because TLC analysis of a mixture produced by adding 1 drop of aqueous 10% NaOH to a methanol solution (0.1 mL) containing indoxyl acetate (ca. 0.1 mg) and Compound 111 (ca. 0.1 mg) indicated that Compound IV was the major product, and it seems more likely that the ketone carbonyl rather than the amide carbonyl of I11 would have been involved in the condensation that gave IV. Compound V was found to have the same R, value (silica gel, dichloromethane) as an authentic sample of isatin and, after extraction from the gel, V gave the proper response in a selective spot test for isatin (7). Compounds VI and VI1 on TLC plates, displayed the same Rf values and gave the same yellow color when sprayed with Van Urks reagent (8)as authentic specimens. Acetic acid was detected by observing the m / e 60 peak of acetic acid in the vapors generated by storing indoxyl acetate (10 mg) in a closed 10-mL volumetric flask at 90 "C for 8 weeks and then reheating the flask at 90 "C while performing the analysis. The relative abundance of the degradation products was visually estimated from TLCs of the extracts of degraded indoxyl acetate impregnated Whatman no. 1 disks that had been stored at 90 "C for 8 weeks. The estimates were based on the sizes of the spots and the extent of fluorescence quenching produced by the spots when the TLCs were illuminated with 254-nm light. Quantitative TLC Analyses. Aliquots ( 8 - p L ) of standard sohtions of indoxyl acetate or of the sample extracts were applied to the TLC plates by means of 8-pL capillary pipets (Analtech, Inc.). Thin layer plates were eluted with dichloromethane until the solvent migrated a distance of 10 cm up the plate. The TLC plates were allowed to air dry for approximately 15 min before scanning. The measured parameters were fluorescence quenching. Quantitation was achieved by averaging the weights of the recorded peaks obtained from triplicate scans on each indoxyl acetate spot. W a t e r Analyses. Samples of the support materials analyzed were from previously unopened packages obtained from the suppliers and were all handled in an air-conditioned environment (50% relative humidity) for ca. 30 min before they were analyzed. The water content of the support materials was determined on a Du Pont Instruments Solids Moisture Analyzer Model 26-321A. The measurement is based on coulometry. In the procedure, moisture is desorbed from the sample in an oven and swept into a detector cell with dry nitrogen where it is adsorbed on a thin film of phosphorus pentoxide contained between two platinum electrodes. Quantitative electrolysis of the water then occurs. The amount of current required for the electrolysis corresponds to the amount of water electrolyzed. The accuracy of the measurements was *2%, Effects of Air on the Indoxyl Acetate Degradation at 90 "C. Two Whatman no. 1 filter paper disks (1.27-cm diameter) were wet with a solution (10 pL) containing indoxyl acetate (10 mg) in acetone (1 mL). After the disks were allowed to air dry in the dark, they were inserted into glass tubes (ca. 7.5 X 0.6 cm). The ends of one of the tubes were sealed in a Bunsen flame while the ends of the other tube were drawn into smaller openings. Argon was passed through the tube with the narrowed openings for 5 min to remove most of the air and the tube was then sealed in the Bunsen flame. After both samples were stored in an oven at 90 "C for 1 week, the disk in the tube in which most of the air had been displaced by argon became blue while the one in the other tube became violet. TLCs of methanol extracts from the disks irdicated that much less indirubin was produced in the blue disk t h a in the violet one and that the blue disk contained indigo. Spectral Data in Figure 2. Whatman no. 1 filter paper disks (1.27-cm diameter) were wet with solutions (10 pL) of indoxyl acetate (10 mg) in methanol (1 mL) that also contained 0, 50, or 100 p L of added water. After the disks were allowed to air dry in the dark, they were sealed in glass tubes and stored in an oven at 90 "C for 3 days. Afterward, the disks were removed from the tubes and the soluble materials were extracted into methanol (5 mL) by allowing the disks to soak for 5 min with periodic agitation. The spectra of the extracts were obtained in 1.00-cm quartz cells with methanol as the reference. The spectrum (curve A, Figure 2) of the extract of a disk that had not been subjected to storage
a,: H
J
I\
Figure 1. Structures of compounds discussed in the text
Thred-Tape) was obtained from Crane Packing Co., Morton Grove, Ill. Thin layer chromatography (TLC) was carried out on precoated glass plates (0.25-mm thick silica gel 60-F2, layer) obtained from E. M. Laboratories, Inc. Preparative chromatography was carried out on precoated glass plates Type PLQF obtained from Quantum Industries, Inc. Equipment and Instrumentation. A model 51325-1 (Gelman, Inc.) chromatography chamber was used for the elutions. Quantitation of indoxyl acetate on thin layer chromatograms was performed on a Kontes densitometer using the fluorescence quench mode. The output from the densitometer was recorded on a 10-inch stripchart recorder (Beckman, Inc.). Electronic absorption spectra were obtained on a Cary Model 14 spectrophotometer using 1.00-cm matched cells. Methanol was used in the reference cell. Mass spectra were obtained on a Perkin-Elmer-Hitachi Model RMU-6E at 70 eV. Water analyses were performed using a DuPont Instruments Solids Moisture Analyzer Model 26-321-A. Procedures. Isolation and Identification of Indoxyl Acetate Degradation Products Impregnated o n W h a t m a n No. I Filter Paper. Whatman no. 1filter papers impregnated with an acetone solution of indoxyl acetate (Compound I, Figure 1) (18 mg/mL) were allowed to air dry in the dark. Samples were then placed in covered glass dishes and stored in an oven at 90 "C for 8 weeks, and then were allowed to soak in methanol, acetone, or dichloromethane for up to 72 h to recover the degradation products. The extracts of store? samples were applied to preparative chromatography plates that were later eluted to the top of the plate twice using dichloromethane. The bands of the separated products were scraped from the plates and adsorbent from each band was packed into a disposable Pasteur pipet (Fisher Scientific, Inc.) into which a piece of glass wool had been inserted to prevent loss of the adsorbent. Compounds were removed from the adsorbent by passing dichloromethane through the columns and the solid products were recovered by allowing the dichloromethane to evaporate a t ambient temperature. Mass spectra of Compounds 11,111,and IV (Figure 1) isolated from the degradation mixture displayed molecular ions and fragmentation patterns consistent with the indicated structures. Compound I1 from the degradation mixture showed the same spectral characteristics and had the same R, value (silica gel, dichloromethane) as a sample of I1 prepared according to Russell
ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
LOU
300
400
500
WAVELkkGTH
600
700
,nw
Figure 2. Electronic absorption spectra of methanol extracts of disks containing indoxyl acetate on Whatman no. 1 filter paper before 90 " C storage (A) and after storage at 90 "C for 3 days with disks containing no added water (B), 10 fig of water (C), and 100 v g of water (D)
4YB 5 a
04
0 2 -
I
300
A piece of S&S Type 2497 chromatography paper (1.27 cm square) was suspended just above 0.1 mL of the indicator solution in a 10-mL test tube for the incubation. After the tube was stoppered, it was stored in an oven a t 90 "C and the indicator was checked periodically for a color change. It was found that the indicator remained green during the first 8 h of storage but that it had become red when it was observed again 16 h later. Wettability of Paper Supports. Two disks (1.27-cm diameter) of each support material, Whatman no. 1 filter paper and S&S Type 2497 chromatography paper, were impregnated with indoxyl acetate by wetting each disk with a solution (10 ILL) containing indoxyl acetate (10 mg) in methanol (1 mL). After the disks had been allowed to air dry in the dark, one disk made with each support material was wet by adding 1drop of methanolic NaOH while the other was wet by adding 1 drop of an aqueous NaOH solution. Blue color was produced immediately in each disk except the one made from the S&S chromatography paper which was wet with the aqueous NaOH solution. Even after standing 1 h, the aqueous NaOH solution failed to produce a blue color in the disk made with the S&S Type 2497 paper. Test for Acetylation of the Paper Support. Acetylation was not detected when a Whatman no. 1 filter paper disk (1.27 cm diameter) containing indoxyl acetate (100 pg) was stored at 90 "C for 8 weeks, washed with methanol to remove residual indoxyl acetate and degradation products, and tested for acetylation using hydroxylamine and ferric chloride spray reagents (9). Under the same conditions, S&S Type 2495 (21YC acetylated) chromatography paper gave an intense purple color. Effect of Various Support Materials on the Stability of Indoxyl Acetate at 90 "C. Support materials included in these tests were Whatman papers no. 1, no. l;, and no. 3MM; S&S papers Type 2495, Type 2497, and Type 2498; and Teflon Thred-Tape. Disks (1.27 cm) of each support were wet with a solution (10 vL) containing indoxyl acetate (10 mg) in acetone (1 mL). (A methanol solution was used with the S&SType 2495 and Type 2497 papers which are acetylated and are affected by acetone.) After allowing the disks to air dry in the dark, each disk was inserted between two pieces of the same material that was used to make the disk and this combination was inserted between two microscopes slides that were later clamped to prevent indoxyl acetate from volatilizing during storage. The disks were then stored in an oven at 90 "C for 2 weeks. A strong violet discoloration was produced with all of the stored samples except those made with the acetylated papers (S&S Type 2495 and Type 2497 papers) and the Teflon tape. The latter supports acquired only very slight discolorations.
RESULTS AND DISCUSSION
00 200
1273
400 'NAVELEUGTH
600
500 r w
700
,
Figure 3. Electronic absorption spectra of methanol extracts of disks containing indoxyl acetate on Schleicher & Schuell Type 2497 chromatography paper before (A) and after (B) storage at 90 " C for 14 days
above ambient temperature was run for comparison. Spectral Data in Figure 3. Two disks (1.27-cm diameter) of S&S Type 2497 chromatography paper were wet with a solution (10 pLj of indoxyl acetate (10 mg) in methanol (1mL). After the disks were allowed to dry in the dark, the spectrum of one of the disks (curve A, Figure 3) was obtained using the same procedure that was used to obtain the spectra in Figure 1. The other disk was sealed in a glass tube and stored in an oven at 90 "C for 2 weeks. The spectrum of the methanol extract of the stored disk (curve B, Figure 3) was obtained using the same procedure that was used to obtain curve A. Incubation of Acetylated Paper with Water. Indicator solution was prepared for this experiment by diluting 1 drop of Universal Indicator Solution (Eastman Organics, Catalogue no. A4953) with water to produce a light green solution. The diluted indicator was tested with aqueous acetic acid and it was found that the addition of 1 pL of an aqueous solution containing 5 pg of acetic acid caused 0.1 mL of the indicator solution to turn red.
Decomposition of the colorless indoxyl acetate (I), impregnated on Whatman no. 1filter papers, was visually evident after 15-min storage in the dark a t 90 "C. After storage for 2 months where densitometry on TLC indicated that approximately 75% of the original indoxyl acetate had decomposed, products were isolated by preparative chromatography and identified by spectroscopy, comparison of thin layer chromatography R, values with those of authentic specimens, and by the colors produced with visualization reagents. The compounds identified in order of their abundance were: I1 > 111 > IV > V 2 VI > VII. Acetic acid was also shown to be formed by mass spectrometry. Indirubin (11) is violet, 111 is yellow, and IV is purple. Other experiments, in which the quantity of air was restricted during the time of indoxyl acetate degradation produced some indigo (VIII). Also, it was noted in experiments carried out in melting point tubes a t the melting point of indoxyl acetate, that color changes originated a t the interface of the molten indoxyl acetate and the air. From these results and observations, it was speculated that the major product, the violet indirubin (II), is favored through the sequence of reactions given by Equations 1-4. I H,O (or free hydroxyl groups) IX (indoxyl) +CH,COOH (1)
+
-
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 8, JULY 1979
--
IX + O2 VI11 (indigo) + 2 H 2 0 VI11 + 302 2 V (isatin) + H 2 0 V
+ IX
-
I1 + HzO
(2)
(3) (4)
I n the presence of copious quantities of oxygen, indigo is not found since it is converted to isatin. Oxidation of indigo t o isatin is documented (5). T h e second highest abundant compound, viz. indolo(2,1-~)-quinazoline-6,12-dione (111) is thought to be formed via the oxidation of VI11 as follows:
VI11 + 0
2
-
I11
(5 )
In connection with Equation 5, the oxidation of indigo by air during sublimation to I11 was reported by Perkin in 1907 (6). It is speculated t h a t the purple IV is formed by condensation of I11 and I X (keto form), according to Equation 6. I11
+ IX
-
IV
+ H20
The origin of isotoic anhydride (VI), another compound which was found in traces is though to be by oxidation of isatin; anthranilic acid (VII), from isotoic anhydride. In support of this, Kolbe was able to form isotoic anhydride by oxidation of isatin with chromic acid in acetic acid, and hydrolysis of isotoic anhydride with mineral acids was shown to lead to anthranilic acid ( I O ) . The conditions under which the degradation of indoxyl acetate have been studied are considered sufficiently close to those described above (viz., acidic oxidations) to lead to the conclusion t h a t the compounds identified in this study were formed through the sequence of reactions listed above. T h e initiation of the degradation is depicted as a simple hydrolysis (or transesterification with the hydroxyl groups of cellulose) with subsequent condensation and oxidations producing water so as to sustain the hydrolytic reaction. As would be expected if the water was the initiator, the rate of hydrolysis of indoxyl acetate impregnated on paper was water concentration dependent (Figure 2), with 100 pg of water added to a disk containing 100 pg of indoxyl acetate (molar ratio H20:I,10:l) causing a complete hydrolysis of the indoxyl acetate after 3 days’ storage a t 90 “C and 10 pg of H20 (molar ratio l:l),causing an approximately 50% hydrolysis of the indoxyl acetate in the same time period. A 20% decomposition is also seen on “dry” paper, suggesting t h a t there is a significant quantity of water which remains on the “dry” paper. Analysis of the water content of disks of %’hatman no. 1 filter paper prepared in an air-conditioned room at 50% R H showed that they did in fact contain from 3.6 to 5.4% H20 removable by heating the disks to 90 “C. The latter fact also made clear that a disk weighing ca. 10 mg would be expected to contain 500 pg of water; dehydration to a point where the quantity of water would be low enough as to provide a “stable” substrate does not appear to be practical because of the high rate of readsorption of water. T h e moisture content of hydrophobic papers could be expected to be less than that of cellulosic papers, but the actual percentage of moisture was not known. We tested acetylated chromatographic paper (Schleicher & Schuell, Type 2497) as a support matrix for the indoxyl acetate. I t contained from 1.3 to 2.1% moisture, less than that of the cellulosic paper, but still an undesirable amount for use as an indoxyl acetate support since it is more than enough to hydrolyze all the indoxyl acetate that is in the substrate disks. Nevertheless, tests showed after storage of acetylated papers impregnated with indoxyl acetate for 14 days a t 90 “C (Figure 3), less than 5% of the indoxyl acetate had decomposed. Thus although both the acetylated and nonacetylated cellulose papers contained quantities of moisture in large excess over that needed for a stoichiometric hydrolytic reaction, indoxyl acetate
impregnated in acetylated cellulosic paper showed very little hydrolysis as compared with the ester impregnated on a nonacetylated cellulose paper. The mechanism of protection of the acetylated cellulosic papers was therefore investigated. Among the mechanisms thought possible were: (a) a preferential reaction of the acetyl groups of the cellulose with water (the cellulose acetate acts as a dehydrating agent), and (b) a protection which is accomplished by preventing moisture from contacting the indoxyl acetate by physical means. T h a t the acetylated cellulose filters could act as postulated in (a) appeared plausible both from a statistical and kinetic viewpoint. Comparing the numbers of acetyl groups available for reaction with water in the substrate and on the paper, there are about 200 times the number of acetyl groups attached to the cellulose as there are in the substrate (the disk of paper weighs ca. 22 mg; the quantity of indoxyl acetate on the disk is 0.1 mg; the paper is considered to be ca. 50% acetylated). With respect to the kinetic aspect, it was thought possible that the hydrolysis rate of a partially acetylated cellulose could be higher than that of indoxyl acetate because of an intramolecular catalytic assist of an a-hydroxyl group (11). However, incubation of water with papers of acetylated cellulose, for 8 h a t 90 “C failed to produce, in a closed test tube. sufficient acetic acid to affect a pH indicator solution, Le.. 5 wg of acetic acid were not produced. Twenty-four hours of incubation at this temperature produced a color change in the indicator solution. A 5-pg yield of acetic acid represents
