Quantitative Determination of Diaminocarboxylic Acids and Related Compounds by Rapid Paper Electrophoresis MARY A. DORAN Eastern Research laboratory, The Dow Chemical Co., Frarningharn, Mass.
b A study of intermediate products formed during the preparation of EDTA required the development of rapid methods for the simultaneous determination of aminocarboxylic acids and related compounds. Cupric complexes of EDTA, (ethylenedinitril0)triacetic acid, N,N-ethylenediglycine, N,N’-ethylenediglycine, and nitrilotriacetic acid were separated by low voltage paper electrophoresis. The resulting bands were developed with sodium diethyldithiocarbamate, extracted with ethyl alcohol, and measured spectrophotometrically. Precision is &2.5% of amount present or k0.025 actual %. A similar procedure involving paper electrophoresis and ninhydrin staining resolved and evaluated the following quantitatively: N,N-ethylenediglycine, N,N’-ethylenediglycine, N-(2-aminoethyl)glycine, 2oxo-1 -piperazineacetic acid, 2-oxopiperazine, iminodiacetic acid, glycine, and ethylenediamine. Precision is *5% of amount present or h0.03 actual %.
A
of the products formed during the carboxymethylation of ethylenediamine (EDA) (compound abbreviations are given in Table I) , commercially important as a production process for EDTA, required the development of procedures for the detection and quantitative determination of a wide variety of related compounds in the presence of each other. The postulated reaction of formaldehyde and sodium cyanide or of glycolonitrile with EDA to form EDTA is outlined below. N INVESTIGATIOK
g:
HZNCH~CH~SHZ 0
II
+
-
4 NsCN 4 4 HOCHzCN 4 NaOH
8
(HzNCCH*)zNCHzCHzN(CHz NH&
HCHO
ANALYTICAL CHEMISTRY
(NECCHZ)~NCH~CHZN( CHIC~N)~ 0
8
I/ +( N ~ ~ C C H ~ ) ~ N C H Z C H Z N0Na)z ( C H Z (1)
I n addition to the reactions indicated, mono-, di,- and trisubstitution of EDA can take place. Mono-, di-, and trisubstituted nitriles, amides, and acids of ammonia are also possible reaction intermediates. Although the present 1752
study is limited essentially to acid forms of the intermediates predicted, it is expected that the methods described here will be adapted successfully for the remaining compounds. LeBlanc (‘7) and LeBlanc and Daniel (3) have developed a polarographic method for the determination of SEDDA, U-EDDA, and NTA in the presence of EDTA. The cyclization in acid medium of S-EDDA and U-EDDA to their respective ketopiperazines , S-KP and U-KP, was observed. The polarographic method, however, was unsuitable for general use here because of interferences arising from the multicomponent system being studied. An ideal analytical procedure for this system should be simple, rapid, free of interference, and micro or semimicro in nature. Ninhydrin (ll2,3-triketohydrindene) staining in conjunction with paper chromatography (8)or paper electrophoresis (9) has been used widely for the identification and determination of amino acids. Xinhydrin also reacts with compounds containing -NH2 and - N H groups (6). Accordingly, micropaper electrophoresis (PEP) was tried, and effective conditions were developed to resolve components of the reaction mixtures, nine of which were ninhydrin positive. These are summarized in Table I. The presence of multiple carboxylic groups in the more highly substituted compounds favored the possibility of strong complex formation with metal ions. The Cu(I1) chelates of 5 compounds were separated by paper electrophoresis. Sodium diethyldithiocarbam-
ate was an effective detecting agent for the complexed copper. Analytical procedures based on electrophoretic separation, treatment with developing agent, and extraction of colored bands with subsequent spec-
trophotometric measurements are described in detail. EXPERIMENTAL
Standards. ED.4, E D T A , KTA, I M D A , and G L were available in pure form. 2-KP was prepared by the method of Aspinall (1). EDMA-dihydrochloride, S-KP, and S-EDDA were recovered from carboxymethylation reaction mixtures. A solution, prepared from material containing 90% UEDDA, was standardized by potentiometric titration and by the paper electrophoretic copper diethyldithiocarbamate (CuDTC) method described in this report. ED3A, U-KP, and 3-KP have not been isolated. Paper Electrophoresis. Sample solutions (pH 8 to 10) representing different degrees of substitution in E D A were diluted to 0.2M with respect to concentration of E D A and/or substituted products. Strongly alkaline solutions (pH > 10) were adjusted to p H 10.0 with hydrochloric acid before dilution. Diluted samples showed no change after storage in the refrigerator for 1 week. The Cu(I1) complex solution mas prepared in the following manner. An aliquot of undiluted sample solution was adjusted to p H 7.0 with 5M HCl. Cupric chloride solution, l M , was added until the p H reading showed no appreciable decrease upon continued dropwise addition of the reagent. Readjustment to pH 7.0 was made Kith 1M potassium hydroxide. The solution was diluted quantitatively t o 0.2M concentration and filtered to remove insoluble cupric hydroxide formed in the presence of excess Cu(I1) ion. Standard solutions of Cu(I1) EDTA and Cu(I1) STA, prepared in the described manner, have remained stable a t room temperature for a month. Electrophoretic separations were carried out in a Spinco Model RD-2 electrophoresis cell with a Duostat regulated power supply, using Schleicher and Schuel 2043 A mgl. filter paper. KOmodification of the general procedure for the operation of the instrument was necessary (2). Diluted test solutions 4 t o 10 pl,, were transferred by micropipet t o the sample striper and applied t o the paper strip. For ninhydrin positive components, separations were made by electrophoresis in 1 M acetic acid at a constant current of 8 ma. for
45 minutes. The voltage changed during this time from 400 to 330 volts. Shorter runs, 20 to 30 minutes, were used in the analyses of samples containing EDA. Buffer solution, p H 7.2, used for the resolution of copper complex bands, was prepared by mixing 170 ml. of 0.2.zI disodium phosphate with 3 nil. of I&' citric acid and diluting to 2 liters. Electrophoresis proceeded for 1 to 2 hours a t a constant current of 18 ma. The voltage reading was 430 volts a t the beginning of the run, 330 volts after 1 hour, and 300 volts after 2 hours. .4t the end of the time interval desired, the rack holding the strips was extended to a horizontal position and transferred to a drying oven at 75" C. for 10 minutes. Ninhydrin Color Development. The dried strips were passed through a solution containing 0.5 gram of ninhydrin in 100 ml. of 95y0 acetone for 2 seconds, drained, and dried at 75' C. for 10 minutes. Figure 1 shows t h e migration Dattern for a remesentative mixture 'of IMD-4, ED3A, SE D D A , U-EDDX, glycine, S-KP, E D M A , 2-KP. and E D A . The yellow colored bands were cut out and placed in test tubes. The color was extracted by adding 5.0 ml. of glacial acetic acid and immersing the tubes in a boiling water bath for 1 minute. Absorbances were read within 30 minutes in a Beckman 1Model D U spectrophotometer at 430 mp. The purple bands were eluted with 5.0 ml. of 7574 ethyl alcohol containing 4 X mmole of cupric chloride per liter (6). Extraction a t room temperature was complete in 45 minutes. Photometric measurements were made within 30 minutes a t 510 mp. Blanks for both determinations were obtained by suitable extraction of a n unstained portion of the paper strip. S-EDDA was determined in band No. 2 (Figure 1A) by extracting the purple band with glacial acetic acid. When the solution was heated ( 5 minutes a t 100" C,), the yellow color of S-KP, indicating the cyclization of S-EDDX was produced. At the same time, U-EDDA was converted to the colorless U-KP. Two paper strips in each analytical run xere reserved for purposes of standardization, leaving six strips for three duplicate runs of sample solutions. As ninhydrin color development mas not reproducible in repeated runs, i t was necessary to include known amounts of standard solutions with each analysis. Linearity of absorbance us. concentration was established for 1 to 8 X lO-5.Tf solutions of all standards. The molar absorptivities lvere in the range of 4000 (EDMA) to 10,000 (S-KP). Ordinarily, 2 aliquots, 4 and 8 p1. of 0 1M solutions of S-KP, EDMA, and glycine were sufficient for the construction of standard curves for the calculation of component concentrations. For samples containing significant amounts of EDA, a standard curve for this compound was required. Recoveries of E D M A and S-KP were determined by adding known quantities of each to standard solutions of carboxymethylation mixtures, Re-
~
Table I.
~~~
Key to Abbreviations of Compounds Determined by Paper Electrophoretic Methods
Abbreviation
EDTA
Name and Formula (Ethylenedinitri1o)tetraacetic acid HOOCCHz\ /CHzCOOH SCHZCHzN HOOCCH~/
Reaction with Ninhydrin
Negative
\CH~COOH
(Ethylenedinitri1o)triacetic acid
HOOCCHz, ED3A
/CHzCOOH NCH2CHZS HOOCCH,/ H'
Purple
2-0xo-1,4-piperazinediaceticacid Ha Hz
/e-c
3-IiP
HOOCCHZ--9
\c-c
HA
U-EDDA
U-KP
/ 'S-CH~COOH
I'
0
S,X-Ethylenediglycine /CHzCOOH HZXCH~CH~X \CH~COOH 3-Oxo-1-piperazineacetic acid Hz Hz HN
/c-c \c-c/ 11
0
Xegative
\S-CHzCOOH
Purple
Segative
H?
N ,Ai'-Ethylenediglycine H 00CC H n
/CHzCOOH
SEDDA
Purple 2-Oxo-1-piperazineacetic acid
Hz
,c-c S-KP
Hz
HX
's-CH~COOH \C-C/
H2
Yellow
I
0
X-(2-Aminoethyl )glycine
EDM.4
/CHzCOOH HzXCHzCHzN \H 2-Oxopiperazine Hz Hz
Purple
,c-c
2-KP
HN
\c-c/ H2
GL
13ID.A
\SH
Y ?I lo w
11
0
Glycine HtNCH2COOH Iminodiacetic acid /CHzCOOH HS \CH~COOH Nitrilotriacetic acid /CHzCOOH
Purple
Purple
NT-4
~~-CH~COOH
Xegative
EDA
\CH&OOH Ethylenediamine HsNCHzCH2NHZ
Blue
VOL. 33, NO. 12, NOVEMBER 1961
1753
A
E
.
Figure 1 Electrophoretic separation in 1M acetic acid at 8 ma., color development with ninhydrin; solid bands, purple; shaded bands, yellow Running A. 4 5 minufes 1. I M D A a n d E D 3 A 2. S-EDDAand U-EDDA
3.
GL
4. 5. 6. 7.
S-KP EDMA 2-KP EDA
Time 8. 720 minutes l a . IMDA l b . ED3A
20. 2b.
S-EDDA U-EDDA
sults of this experiment are given in Table 11. The precision of the method is *5% of the amount present or k0.03 actual %, whichever is the greater. Copper Diethyldithiocarbamate Reaction (CuDTC) (4, 11). The dried
strips with resolved copper complex bands were passed through a 0.2% aqueous solution of sodium diethyl-
II. Determination of EDMA and S-KP in Mixtures (Composition of mixtures with respect to other components: EDTA 0.057M, ED3A 0.002M, S E D D A 0.005M, NT.4 0.003M) Moles/Liter .~
Table
dithiocarbamate (DTC) for 2 seconds, drained, and dried at 75' C. for 10 minutes. The electrophoretic separation of Cu(1I)EDTA. Cu(1I)NTA. Cu(II)S-EDDA; and Cu(II)U-EDDA is shown in Figure 2. Selected bands were cut out and transferred to test tubes containing 5.0 or 10.0 ml. of absolute alcohol. The tubes were immersed in a 60" C. water bath for 30 seconds and alloi\ed to stand for 5 minutes a t rooin temperature for complrte extraction of the brown color. For each series, a single section from a n unstained portion of the electrogram served as a blank. Absorbances were read a t 432 m p . A series of standard solutions of Cu(IIjST.4 and Cu(IIjEDT.4 and cupric chloride was analyzed. A linear plot of absorbances us. copper content x i s obtdined for the range 1 to 8 X l O - ~ X copper. The molar absorptivityl computed from the graph, was (1.23 =t 0.01) X lo4 for all three compounds and was used for the calculation of copper concentration of eluted bands. Stability of color was tested by allowing a solution to stand for 2 hours a t room temperature and then heating a t the boiling temperature for 15 minutes. The observed decrease a t 0.352 absorbance was 0.002. Variation of concentration (0.2 to 2.0%) and age (freshly prepared to 3 days old) of D T C reagent caused =t2% deviation from the standard calibration curve. Recoveries of Cu(1I)EDTA and Cu(1I)NTA were checked by adding known quantities of these two materials to previously analyzed solutions and analyzing them. The results of this study are in Table 111. The precision of the method is =t2.597, of the amount present or dz0.025 actual %, whichever is the greater. The limit of detection is 0.05 mole 70. U-KP and 3-KP are ninhydrin inactive and do not complex Cu(I1) ions and therefore they cannot be determined directly by either the ninhydrin or CuDTC method. They may be converted to the corresponding diaminocarboxylic acids by adjusting the test solution to pH > 12 and boiling. The differencc between the concentrations of U-EDDA and ED3;1 before and after basic treatment is a measure of these ketopiperazines initially present in the solution.
Error,
-4dded 0,0252
0.0504 0.0756
Found EDhf.4 0.0262 0 0266 0.0497
0.0769 0.0757
0.0630
0.bssl
0.0530
0.0532 0.0260 0.0257
7% $3.9 6 -1.4 +1.7 +5 I
+o. 1
+4.9
S-KP
0.0265
... 0.0794
1754
$0.3 -1.9 -3.0
...
0.0797
0.0805
.,.
+0.4
+1.4
ANALYTICAL CHEMISTRY
A
E
Figure 2. Electrophoretic separation of Cu(ll) chelates in pH 7.2 buffer solution, 18 ma., color development with sodium diethyldithiocarbamate Running Time A. 1 hour B. 2 hours a. Cu(II)EDTA b. Cu(II)NTA c. Cu(II)ED3A d. CU(II)U-EDDA e. Cu(II)S-EDDA
can be increased to give quantitative results (6). High salt concentration in the sample resulted in slow irregular migration and poor resolution of components. Careful adjustment of highly alkaline samples to a p H of 10.0 only was necessary to avoid this effect. Attempts to desalt the solution by the addition of ethyl alcohol or by treatment with ion exchange resin were not effective. The former process caused some precipitation of products while the latter produced partial conversion of open chain acids to the corresponding osopiperazines. The copper diethyldithiocarbamate (CuDTC) method of analysis is dependent on the amount of copper complesed and therefore should be applicable to
RESULTS A N D DISCUSSION
The nature of the reaction of ninhydrin with the aminocarboxylic acids and the osopiperazines, 2-KP and S-KP, has not been studied extensively. However, visible spectra of eluates of all purple bands are the same and correspond to the colored product (Ruhemann's purple) (IO) obtained with standard amino acids, glycine, lysine, and alanine. Spectra of glacial acetic extracts of the yellow bands produced by 2-KP and S-KP are identical. Purple alcoholic solutions tend to be unstable. By adding Cu(I1) to the eluting solution, the reproducibility
Table 111. Determination of Copper by Diethyldithiocarbamate Method
Moles per Liter Total Found Cu(I1) EDTA 0 0894 0248 0 1143 0 0249 0496 0 1388 0 0494 Cu(I1) NT.4 0 0244 0681 0 0945 0 0701 0852 0 1096 0 0852
Added
0 0
0 0
Error,
3' %
Control 4-0 4 -0 4 Control 9
+;
any ligand which forms a stable complex with Cu(I1) ions in known molar ratio a t p H 7.0, provided that this complex remains essentially undissociated during the electrophoretic run. For example, EDTA, ED3A, NTA, SEDDA, and U-EDDA migrate in the form of the complex and show a characteristic copper band for each compound. Under identical conditions the compounds EDA, EDMA, glycine, and I M D A produce bands of both the copper chelate and the uncomplexed ligand. Interference in analyses by such materials is not significant. Satisfactory results have been obtained in the analysis of a reaction mixture in whirh glycine, IMDA, and E D A each comprised 5%, EDMA 25% of total products. The main application of these paper electrophoretic methods is the determination of the distribution of products in carboxymethylation reaction mixtures. Electrophoresis with ninhydrin color development has bern applied in equilibrium studies of the diamino carboxylic acids which form cyclic imides according to the equation
where RI
= H or CHICOOH RZ = H or CHzCOOH
procedures are well suited for analysis of biological fluids (blood, serum, urine, gastric juice, etc.) for trace amounts of
It has also been helpful as a screening test during the separation of E D M A from a commercial carboxymethylation reaction mixture. Both methods have been combined in establishing the identity of ED3A, reported as a n impurity in EDTA. but not isolated. The location of the ninhydrin purple band with respect to the bands of S-EDDA, U-EDDA, and EDMA (Figure 1B) as well as the formation of a stable Cu(I1) chelate with a characteristic position in the migration pattern (Figure 2B) is indicative of the compound ED3A. Cyclization to 3-KP is indicated by the absence of the ninhydrin band in acid solutions. The reappearance of the ninhydrin band after basic treatment proves the rerersibility of the cyclization reaction (Equation 2). The methods have potential usefulness in other fields. As E D T A has been widely used in agricultural and biological studies, a method for the detection and determination of E D T A and its degradation products should find many applications. Electrophoretic
EDTA. LITERATURE CITED
(1) Aspinall, S. R., J. Am. Chem. SOC. 62, 1202 (1940). (2) Beckman SPINCO Division, Beckman
Instruments, Inc., Fullerton, Calif., Paper Electrophoresis Instruction Manual, Model R. (3) Daniel, R. L., LeBlanc, R. B., ANAL. CHEM.31, 1221 (1959). (4) Feigl, F., “Spot Tests in Inorganic Analvsis.” Vol. I. 5th ed..,. D. 433. Elsevier, “Amsterdam,’1958. (5) Feigl, F., “Spot Tests,” Vol. 11, 4th ed., p. 208, Elsevier, Amsterdam, 1954 (6) Giri, K. V., Radhakrishnan, A. N., Vaidvnathan, C. S., Xature 170. 1025 (1952). ( 7 ) LeBlanc, R. B., Aiv-4~.CHEM.31, 1840 (1959). (8) Lederer, E., Lederer, >I “Chroma., tography,” p. 204, Elsevier, Amsterdam, 1955. (9) Lederer, M., “Introduction to Paper Electrophoresis and Related Methods,,’ p. 88, Elsevier, Amsterdam, 1957. (10) McCaldin, D. J., Chem. Reus. 60, 39 (1960). (11) Welcher, F. J., “The Analytical Uses of Ethylene diamine t e traace tic Acid,” p. 291, Van Nostrand, New York, 1958. RECEIVEDfor review March 3, 1961. Accepted June 9, 1961. Detroit Anachem Conference, Detroit, Mich., October 1961.
Ion Exchange Method for Determination of 1-1sonicotinyl-2-acetylhydrazide (Acetylated Isoniazid) in Biological Fluids ALFRED HELLER, JOHN E. KASIK, LEON CLARK, and LLOYD J. ROTH Deparfment o f Pharmacology, The University of Chicago, Chicago 37, 111.
b A direct, rapid method for the determination of l -isonicotinyl-2-acetylhydrazide (acetylated isoniazid) utilizes ion exchange column chromatography. It permits determination of acetylated isoniazid in the presence of isoniazid, isonicotinic acid, and other metabolites, and has been adapted for analysis in biological fluids. p Aminosalicylic. acid does not interfere. The analysis may be carried out b y either radioisotopic or colorimetric procedures.
I
S O N I C O T I K Y L H Y D R A Z I D E(isoniazid,
isonicotinic acid hydrazide) is a n important agent in the therapy of tuberw:!osis and its metabolism is of considerable theoretical and practical
interest (8, 11). The major nietabolite of isoniazid in man is the acetylated derivative, 1 - isonicotinyl 2 - acetylhydrazide (hereafter refcrred to as acetylated isoniazid), although the amount of drug acetylated varies widely from individual to individual (6). Since this derivative has considerably less bacteriostatic activity than isoniazid (S), i t has been postulated that the amount of isoniazid acetylated may be a factor in the success or failure of therapy with this drug. In addition, the suggestion has been made that inhibition of this acetylation by p-aminosalicylic acid (PAS) may account in part for the value of combined therapy with these trvo agents. Hughes has described a method for quantitative determination of acetylated
-
isoniazid using an extraction procedure followed by colorimetric determination (6). Belles and Littleman determined the acetylated derivative by an ion exchange procrdure ( I ) , but their method has subsequently been found to include a considerable error due to isonicotinic acid ( 2 ) . The report prescnted hcre describes a direct method for the quantitative separation and determination of acctylated isoniazid by ion exchange chromatography. MATERIALS
Don-ex l-XS, analytical grade, 100to 200-mesh, a strongly anionic ion exchange resin (J. T. Baker Chemical Co., Phillipsburg, K. J.) was prepared in the pyruvate form by the following VOL, 33, NO. 12, NOVEMBER 1961
0
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