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
1755
Table
I.
Recovery of C14-Acetylated Isoniazid from Water Solution, Urine, and Blood Plasma by Isotopic Analysis
Fluid Water
Added 1. C14-acetylated isoniazid 2. C I4-acetylated isoniazid C14 isoniazid
C14isonicotinic acid
3 . CI4 iuoniazid
100
% Acetylated Isoniazid Found 93.6 i 1.1 93.9 i 0 8
100
0 . 7 & 0.0
pg./Ml. 100 100 100
C14 isonicotinic acid 100 Urine 1. C14-acetylated isoniazid 100 95.6 & 0 . 5 2. C14-acetylated isoniazid 100 100.2 & 0 . 2 C14isoniazid 100 CI4isonicotinic acid 100 3. CI4isoniazid 0.9 f 0 . 2 100 C14 isonicotinic acid 100 Blood plasma 1. C "-acetylated isoniazid 10 96.6 f 1.5" 2. CI4-acetylated isoniazid 10 98.0 f 0 . 0 C14isoniazid 10 10 C14 isonicotinic acid 3. C14 isoniazid 10 0.7 f 0.4 C14 isonicotinic acid 10 4. (214-acetylated isoniazid 1 .o 95.3 f 1.6b 1.o 5 . 04-acetylated isoniazid 96.6 f 1.1 CI4isoniazid 1.0 C14 isonicotinic acid 1.o 6. C14 isoniazid 1.0 0.9 f 0.1 1.o C14 isonicotinic acid % Recovery for added acetylated isoniazid from blood plasma by colorimetric analysis. 98.5 f 0.6. I, 100.6 f 3.4. Values reported f standard errors. Each value represents a minimum of three determinations.
in R, and configuration to the radioactive band found on autoradiography. Nonlabeled isoniazid chromatographed side by side on the same filter paper strips was identical in color and R, value to the isotopically labeled compound. Specific activity of the labeled isoniazid was 6.7 X IO7 d.p.m. per mg. Acetylated isoniazid with a C14 label in the carboxyl position was prepared from the C14 isoniazid b y the method used for the nonisotopic synthesis (la). Isonicotinic acid labeled with C14 was obtained as a precursor in the synthesis of C1* isoniazid. Both C14 acetylated isoniazid and C14 isonicotinic acid were shown to be radiochemically pure in the manner used for labeled isoniazid. Specific activity of CILacetylated isoniazid was 3.8 X 106 d.p.m. per mg. and for CI4 isonicotinic acid was 1.15 X 108 d.p.m. per mg. APPARATUS
The chromatographic columns used for ion exchange analysis were made from 10-mm. 0.d. borosilicate glass tubing 50 em. in length, fitted at the lower end with a microburet tip and a t the upper with a 24/40 female borosilicate glass joint to accommodate a reservoir. Radioisotopic analysis was performed using 7-sq. cm. aluminum planchettes in a Nuclear Chicago Model 161-4 scaler with a Model C-110 automatic sample changer. Colorimetric determinations were carried out in a Beckman DU spectrophotometer. PROCEDURE
procedure. The resin was placed in 5 volumes of 1.OA: HC1 and stirred for 8 to 12 hours, followed by batch washing with distilled water until the supernate was p H 6.5 as tested with pHydrion paper. Five volumes of 1 . 0 s S a O H were added to the resin and stirred for 8 to 12 hours, followed by batch washing with distilled water to p H 6.5. The resin was then placed on the pyruvate cycle by addition of 10% (v./v.) pyruvic acid (Matheson, Coleman and Bell, Norwood, Ohio) and stirred for 4 hours. The resin was allowed to settle and the supernate decanted. A fresh 10% solution of pyruvic acid was added and stirred for 12 hours, after which it was washed with distilled water until the p H of the supernatant wash water was 6 . 5 . Xonisotopic isoniazid and isonicotinic acid (Sutritional Biochemical Corp., Cleveland, Ohio) were recrystallized from boiling 95% ethyl alcohol [isoniazid, m.p. l i l - 2 " C. (uncorr.): isonicotinic acid. m.p. 320" C . iuncorr.)]. Acetylated iqoniazid, m.p. 164-5" C. (uncorr.), v a s synthesized by the method of Yale et al. (121. Elemental analysis: C 54.18%. 13 5.44%, K 23.547, (theoretical, C %.73To7,, H 5.03'%, S 23.46%). Infrared spectrophotometric analysis of the acetylated isoniazid revealed the addition of a carboxyl stretching vibration a t 1865 cm.-l when comparcd with isoniazid. 1756
ANALYTICAL CHEMISTRY
Isoniazid labeled with C14 in the carboxyl position was synthesized in our laboratory by a modification of the method of Murray and Langham (9). I n the original synthetic scheme the esterification of isonicotinic acid is accomplished by treating the acid with thionyl chloride to form isonicotinyl chloride, which is in turn treated with alcohol. While this procedure in our hands resulted in good chemical yields, i t also gave rise to-isotopically labeled contaminants which could not be removed by standard purification methods. Although the esterification procedure of LaForge ( 7 ) gave consistently lower yields, i t resulted in a product free of contaminants of this type. The product thus obtained had the same melting point and ultraviolet and infrared spectrum as authentic nonisotopic isoniazid. Radiochemical purity was established chromatographically on Whatman S o . 1 filter strip utilizing the following three descending systems: butyl alcoholwater 1 to 1; butyl alcohol-waterethyl alcohol-acetic acid 45 : 34 :20: 1; isopropyl alcohol-water 85: 15. Treatment of the paper strips with cyanogen bromide gas followed by spraying with benzidine dissolved in acetone and water (4) revealed a single yellow spot identical
The determination of acetylated isoniazid is based on the ability of Dowex I charged on the pyruvate cycle to remove isoniazid and its nonacetylated metabolites from biological fluids. Resin (17-ml. wet volume) is placed in each column and washed with 50 to 100 ml. of deionized water prior to use. Resin is prevented from passing through the column by placing a small amount of washed glass wool a t the lower end of the column prior to filling. Son-protein-containing solutions, including urine, may be analyzed directly without prior treatment in volumes up to 5 ml. Protein-containing solutions such as blood plasma are pretreated with 0.5 ml. of 30% (w./v.) trichloroacetic acid per 5 nil. of plasma, followed by centrifugation. Two milliliters or less of the supernate is transferred to the column and allowed to run onto the resin. (Larger volumes of trichloroacetic acid containing supernate may cause leakage of metabolites from the column.) The column is washed n i t h distilled water and EO ml. of effluent is collected a t a flow rate of 7 t o 10 drops per minute. (Variations from this flow rate lead to nonquantitative recoveries of acetylated isoniazid.) Isotopic Procedure. The acetylated isoniazid which passes through the column may be determined by isotopic techniques if labeled drug has been used.
Colorimetric Procedure. The column effluent may be analyzed colorimetrically by the procedure of Nielish (IO) as modified by Bellcs (1). In our laboratory acetylated isoniazid is converted to isonicotinic acid by acid hydrolysis with 2 ml. of concentrated HC1 in an autoclave at 15 pounds for 45 minutes; the sample is dried under vacuum a t 50" C. and the colored complex formed by the addition of Chloramine T and potassium cyanide in a barbital buffer is measured in a Beckman DU spectrophotometer a t 600 mp. In the case of blood plasma containing more than 1 pg. per ml. of acetylated isoniazid, 5 ml. of column effluent is carried through the colorimetric procedure. For samples with 1 pg. per ml. or less, 10 ml. is required. Standard solutions were prepared containing C14-acetylated isoniazid alone and in combination with CL4isoniazid and isonicotinic acid, these three substances comprising the major fraction of isoniazid and metabolites found in human urine and plasma (6). The radioisotopkally labeled compoundswere diluted with carrier, so that the specific activity was approximately equal for each metabolite. Urine samples containing 100 pg. per ml. of each C14labeled metabolite and plasma samples containing 10 and 1.0 pg. per ml. were analyzed for acetylated isoniazid. The results are shown in Table I. C14-labeled isoniazid was administered to both human volunteers and male Sprague Dawley rats. Urine samples collected from these subjects were analyzed for acetylated isoniazid. The column effluent thus obtained was lyophilized, redissolved in a small amount of distilled water, and subjected to chromatographic analysis on Whatman No. 1 filter paper strips with development by butyl alcohol-water (1 to 1) or isopropyl alcohol-v-ater (85 to 15). Urine samples before column analysis and solutions containing isoniazid, isonicotinic acid, and acetylated isoniazid were run simultaneously with the colunin effluent. Autoradiograms were made of these strips, which were subsequently developed colorimetrically.
Four male Sprague Dawley rats were injected with C14 isoniazid and blood plasma from these animals was analyzed for acetylated isoniazid after column treatment by both isotopic and colorimetric procedures as a comparison of the two methods. We have routinely determined free or unaltered isoniazid on aliquots of blood plasma or urine by the fluorometric procedure of Peters (11), in addition to the column analysis, and thereby obtained values for both free and acetylated isoniazid in biological fluids.
only one band identical in R, and color reaction to acetylated isoniazid, indicating that this and only this derivative had passed through the column. Comparison of isotopic and colorimetric analysis of the column effluent for acetylated isoniazid in rat blood plasma after administration of C1* isoniazid gave values which agreed within 4%. ACKNOWLEDGMENT
The authors thank Robert C. Hinman, Jr., for technical assistance. LITERATURE CITED
RESULTS AND DISCUSSION
In Table I are given the recovery values for C14-acetylated isoniazid from standard solutions of this compound either alone or in combination with C14 isoniazid and isonicotinic acid in water solution or added to urine or plasma. Such recoveries are not affected by the presence of p-aminosalicylic acid, a drug frequently used in conjunction with isoniazid. Since there are several metabolites of isoniazid such as the hydrazones and isonicotinuric acid, and others as yet unidentified metabolites which were not included in the mixtures prepared and analyzed, urine from rats and human volunteers administered C14 isoniazid was also subjected to analysis and the effluent chromatographed as described above. These studies showed that the column effluent of urine samples contained only acetylated isoniazid as determined by autoradiography or color development of the paper strips. There was only one iadioactive band and development with cyanogen bromide and benzidine showed the deep purple color of acetylated isoniazid and the same R , value as a known sample of this derivative run simultaneously. The untreated urine contained six labeled compounds, while the column effluent on chromatographic analysis revealed
(1) Belles, Q. C., Littleman, 11. L., Am. Rev. Respiratory Diseases 81, 364 (1960). (2) Belles, Q. C., Littleman, &I L.,.
personal communication.
(3) Bernstein, J., Lott, W. A., Steinberg, B. A., Yale, H. L., Am. Rev. Tuberc. 65, 357 (1952). (47 Cuthbertson, W.F. J., Ireland, D. M., Wolf, W.,Biochem. J . 55, 669 (1953). (5) Hughes, H. B., J . Pharmacol. Ezptl. Therd . 109, 444 (1953). (6) €3ug es, H. B., Biehl, J. P., Jones,
1
A. P., Schmidt, L. H., Am. Rev. Tuberc.
70, 266 (1954). (7) LaForge, F. B., J . Am. Chem. SOC. 50, 2477 (1928). (8) Mitchell, R. S., Bell, J. C., S e z v Engl. J . Med. 157,1066 (1957). (9) Murray, Arthur 111, Langham, IV. H., J . Am Chem. SOC.74, 6289 (1952). (10) Nielish, IT., Chem. Ztg. 81, 32 (1958). (11) Peters, J . B., A m . Rev. Respiratory Diseases 81, 485 (1960). (12) Yale, H. L., Losee, Kathryn, Mar-
tins, J., Hoking, Mary, Perry, F. M., Bernstein, J., J . Am. Chem. SOC.75, 1933 (1953).
RECEIVEDfor review March 31, 1961. Accepted August 22, 1961. Work su ported in part by grants in aid from t f e National Tuberculosis Association, The Tuberculosis Institute of Chicago and Cook County, Contract No. AT(11-1)45 between the University of Chicago and the Atomic Energy Commission, and the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago.
Conductivity Method for Determination of Urea WEI-TSUNG CHIN and WYBE KROONTJE Agronomy Department, Virginia Agricultural Experiment Station, Virginia Polytechnic Institute, Blacksburg, Va.
b
An accurate and rapid method has been developed for the determination of urea based on the difference in electrical conductivity of urea and ammonium carbonate produced from urea by urease in solution. The principle and adaptation of this method are discussed. Examples of calculation and comparison with an established method are given.
T
general methods used for the determination of urea in a solution can be classified into four categories: urease-Kjeldahl or nesslerization method (8, 9 ) , hypobromite manometric method (6), p-dimethylaminobenzaldehyde or diacetyl spectrophotometry (1, IO), and chromatographic method (6). The accuracy and adaptability of these methods are usually HE
limited by the presence of colored or interfering substances, complexity in procedure, and narrow testing range of urea concentration. The conductivity method eliminates some of these limitations. EXPERIMENTAL
Apparatus and Reagents. tivity bridge and cell. VOL. 33,
NO. 12,
NOVEMBER 1961
Conduc1757
