ANALYTICAL
Method
BIOCHEMISTRY
88,612-618
for Detection
DIANE Department
of Human
(1978)
of 2-Azahypoxanthine and Rat Urine’
in Human
D. BEAL AND GEORGE T. BRYAN
Oncology, University of Wisconsin Madison. Wisconsin 53706
Center
for
Health
Sciences,
Received October 20. 1977: accepted March 20, 1978 A new, simple, reproducible, and quantitative method for the detection of 2azahypoxanthine (2-AH) in urine is described. The method is based on removal of interfering substances from urine by cation- and anion-exchange chromatography. Quantitative determination of 2-AH eluted from the anion-exchange resin involves its conversion to 5-diazoimidazole-4-carboxylic acid and subsequent coupling of this compound withN-(I-naphthyl)ethylenediamine. The resultant dye product has an absorption maximum at 505 nm in 2.4 N HCI, which is linearly related to the original 2-AH concentration in the range of O-8 pg/ml. The only substance found to interfere and not be removed or destroyed is p-acetamidophenyl glucuronide, an excretion product of phenacetin- and acetaminophen-containing drugs.
Investigation of the in vitro degradation of the anti-neoplastic agent, 5(3,3-dimethyl- I-triazeno)imidazole-4-carboxamide (DTIC), demonstrated that the drug decomposed to dimethylamine and 5-diazoimidazole-4carboxamide (diazo-ICA) in dilute acid solutions, especially in the presence of ultraviolet light (1). The diazo-ICA thus formed spontaneously cyclized to 2-azahypoxanthine (imidazo[4,5-d]-u-triazin-4(3H)-one, 2-AH) (Fig. 2) (2), an analog of hypoxanthine in which carbon atom 2 is replaced by a nitrogen (3). In studying the metabolism and mechanism of action of DTIC in viva, it became necessary to determine if 2-AH was excreted after drug administration. To accomplish this, a highly specific and quantitative analytical method for urinary 2-AH was required. No previously described method for determination of 2-AH or other 2-azapurines was found, necessitating the development of an entirely new analytical procedure for the determination of 2-AH. The present paper reports a method for the detection of 2-AH in human and rat urine. It involves separation of 2-AH from interfering urinary components by ion-exchange chromatography and its quantitative determination by formation of an azo dye product. Tests used to identify the dye product are also provided. I Presented in part at the 1976 Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, New Orleans, Louisiana, August 16, 1976. 0003-2697/78/0882-0612$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
612
DETECTION
OF 2-AZAHYPOXANTHINE
IN URINE
613
METHODS
Materials. 5-Aminoimidazole-4-carboxamide (AIC) hydrochloride was purchased from Bachem, Inc. (Marine Del Rey, Calif.); 4-nitroimidazole from Aldrich Chemical Co., Inc. (Milwaukee, Wis); and imidazole4methanol picrate and N-( I-naphthyl)ethylenediamine (NEDA) dihydrochloride from Eastman Kodak Co. (Rochester, N. Y.). AG 50W(H+) (12% cross-linkage, 200-400 mesh) and AG l(Cl-) (8% cross-linkage, 200-400 mesh) resins, obtained from Bio-Rad Laboratories (Richmond, Calif.), were made into 1:l slurries with water before use. SpragueDawley rats were purchased from Sprague-Dawley Co. (Madison, Wis.). Other chemicals and supplies were obtained from commercial sources. Zen-exchange chromatography ofurine. Twenty-four-hour urine collections, preserved under lo- 15 ml of toluene, were obtained from consenting laboratory workers and hospitalized cancer patients. Specimens were kept refrigerated at 4°C until assayed. A 10% aliquot of a 24-hr urine was adjusted to pH 1.5-2.0 with 6 N HCl and then, if necessary, was filtered through Whatman No. 50 filter paper. The acidified sample was passed through an AG 50W(H+) column (1.6 cm o.d. x 4 cm) by gravity. The column was washed with 50 ml of 0.01 N HCl. The effluent and wash were combined and the pH was adjusted to 8.9-9.1 with 6 N NaOH. The resultant sample was passed through an AG l(C1F) column (1.2 cm o.d. x 6 cm) under 0.5 psi of pressure. The column was washed with 20 ml of distilled water and the combined effluent and wash were discarded. 2AH was eluted from the column with 100 ml ofO.O1 N HCl under pressure. Twenty-four-hour urines collected from male Sprague-Dawley rats were preserved under 2 ml of toluene and assayed within 48 hr. The volume was adjusted to 50 ml with 0.01 N HCl (pH-2.0), and 25 ml of the sample was fractioned as described above. Recovery of 2-AH was determined from duplicate samples of urine with 2-AH added prior to column treatment. Spectrophotometric quantitation of 2-AH in urine. A 5.0-ml aliquot of the 0.01 N HCl eluate from the AG l(Cll) column, 0.2 ml of 2% NaNO,, and 2.5 ml of concentrated HCl were dispensed into a lo-ml volumetric flask and mixed. The flask was then covered with a glass sphere 2 cm in diameter and was placed in a 90°C water bath for 3.5 hr. After this time, the flask was removed and allowed to cool for 30 min before 1.0 ml of 5 N NaOH and 0.1 ml of 10% ammonium sulfamate were added and the sample was mixed. Two-tenths milliliter of 1% NEDA was then added, the volume was adjusted to 10.0 ml with distilled water (final HCl concentration, 2.4 N), and the final solution was thoroughly mixed. After 30 min, the optical absorption of the sample was compared with a blank of 2.4 N HCI at 505 nm in a Bausch and Lomb Spectronic 20 spectrophoto-
614
BEAL
AND
BRYAN
meter. Quantitative comparisons were made with a 2-AH standard curve prepared in a similar fashion. Preparation of azo dye derivatives. Imidazole-Cmethanol picrate was changed to the sulfate form as described by Totter and Darby (4), using H&SO, (96%) instead of HCl (37%). 2-AH was synthesized from diazoICA according to Shealy et al. (1,2); 5aminoimidazole (AI), from 5nitroimidazole according to Rabinowitz (5), as modified by Cusack et al. (6); and 5-aminoimidazole-4-carboxylic acid (AICA), from imidazole-C methanol sulfate according to Gireva and Dobychina (7). Because of their instability, AI and AICA were not isolated but were reacted immediately after formation with NaNO, and NEDA as follows: 50 ml of HCl (37%) and 10.0 mmol of NaNO, were added to 3.0 mmol of the given imidazole in 50 ml of water (AI) or in 50 ml of phosphate buffer (pH 8.5) (AICA). The reaction mixture was placed on a magnetic stirrer for l-2 min before 10 mmol of ammonium sulfamate was added, and the mixture was again stirred for l-2 min. Upon addition of 4.0 mmol of NEDA, the dye product formed and began to precipitate out of solution within a few minutes. The reaction mixture was stirred for 1 hr and filtered, and the precipitate were dried over CaCl, in a vacuum desiccator and stored at -20°C. The dye product formed from AIC was synthesized by the above method, except that 3.0 mmol of AIC in 100 ml of 6.0 N HCl was used as the starting material. The dye product from 2-AH and another from AIC were also synthesized as described above, except that 3.0 mmol of 2-AH and AIC, each in 100 ml of 6.0 N HCl, were used, and the reaction mixtures were heated at 90°C for 6 hr after adding NaNO, and were cooled for 2 hr before adding ammonium sulfamate and NEDA. Identijkution of dye product formed from 2-AH and NEDA. Identification of the dye product from 2-AH and NEDA was accomplished by comparing several of its physical properties with those of known dye products. Physical properties were determined on the following instruments: melting points, block-type melting point apparatus; visible and uv spectra, Beckman DB-GT spectrophotometer; and infrared spectra, Beckman AccuLab 4. Dye products, dissolved in EtOH, were chromatographed on Silica Gel G in isopropanol: H,O: cont. NH, (80: 15:5) and in n-propanol: cont. NH3 (6:4) and on aluminum oxide (AI,O,) glass plates in pyridine: H,O (1: 1). All chromatograms were developed in the ascending manner and were visualized directly. RESULTS
AND DISCUSSION
Acid hydrolysis of 2-AH and diazotization of the resultant product were completed within 3.5 hr under the conditions specified. The diazo intermediate proved to be very stable, remaining unchanged for 6 months
DETECTION
OF Z-AZAHYPOXANTHINE
615
IN URINE
FIG. 1. Linear relationship between 2-AH concentration (micrograms per milliliter) and absorbance at 505 nm after formation of azo dye as described under Methods (18 samples per point, bars represent *SD). Linear regression line: y = 0.0894x + 0.004.
when kept in 6 N HCl at 4°C. Coupling of the intermediate occurred at room temperature and demonstrated a linear response between the original 2-AH concentration (O-8 pg/ml) and optical absorbance at 505 nm in the presence of excess NEDA (Fig. 1). Maximum color development occurred within 30 min and was stable for a minimum of 30 min. Recovery of various quantities of 2-AH added to duplicate human or rat urine samples is shown in Table 1. Ion-exchange chromatography TABLE OF 2-AH
RECOVERY
SAMPLES
AFTER
PRIOR
ADDITION
TO ISOLATION
1
OF INCREASING
AMOUNTS
BY ION-EXCHANGE
Percentage 2-AH
OF 2-AH
TO URINE
CHROMATOGRAPHY
recovered”
Added (PB)
Human
100 200 400 600 1000 2000 5000
95.0 91.5 93.7 93.0 92.6
Overall U Mean k SD. “n = 8. “n = 48. d n = 56.
recovery
urine * + -r2 +
5.7” 2.1 0.9 2.7 1.9
97.4 + 1.3 93.9
2 2.1’
Rat urine 100.3 92.5 94.2 95.0 93.4 95.8 94.1
” 2 t + + 2 f
4.8” 1.5 1.6 1.2 1.0 1.3 1.3
95.1 t 2.6d
616
BEAL
AND
BRYAN
proved to be quantitative with overall recoveries of 93.9 2 2.1% (mean + SD) from human urine and 95.1 ? 2.6% from rat urine. All normally occurring urinary components which interfered with the calorimetric assay were removed by the ion-exchange resins or were destroyed during the acid hydrolysis procedure. The only substance found to interfere was p-acetamidophenyl glucuronide, an excretion product of phenacetinand acetaminophen-containing drugs. The presence of this compound can be eliminated by prescribing various other analgesic drugs. Since the reaction pathway for calorimetric determination of 2-AH had not been described previously, it was of interest to identify the reaction intermediate and final dye product. Ultraviolet absorption studies revealed that 2-AH decomposed to imidazole during acid hydrolysis, indicating that the dye product contained an intact imidazole moiety. The reaction intermediate was diazotizable, implying the presence of a 5-amino group. Unless the compound underwent a rearrangement, the only other position expected to be substituted was position 4, the most likely substituent being a-H, -COOH, or -CONH, moiety. Synthesis ofdye products containing the above moieties enabled comparison of their physical properties with those of the dye product from 2-AH. None of the dye products melted below 300°C but they became charred [AI, 162°C; AIC, 190°C; AICA, 2-AH, and AIC (heated), -204”Cl and gave off yellow- to red-colored fumes [AI, 180- 190°C; AIC, 190°C; AICA, 2-AH, and AIC (heated), 250-26o”C]. As shown in Table 2, absorption maxima in the visible region varied with the solvent used. Spectra from the 2-AH dye product most closely resembled those from the AICA and AIC (heated) dye products. Movement of the 2-AH dye product on tic again mimicked that of the AICA and AIC (heated) dye products (Table 3). Infrared spectra were identical for the 2-AH, AICA, and AIC (heated) dye products. Comparison of these physical properties revealed that the reaction intermediate was 5-diazoimidazole-4-carboxylic acid and TABLE COMPARISON
OF VISIBLE
ABSORPTION
2
MAXIMA
Absorption
OF AXOIMIDAZOLE
maxima
DYE
PRODUCTS
(nm)
Dye product formed from
2.4 N HCI
0.1 N HCI
HZ0
MeOH
2-AH AICA AIC (heated) AIC AI
500 500 500 510 521
518 520 516 520 489
501 501 501 500 464
502 501 501 496 473
0.1 N NaOH 475 475 475 498 455
DETECTION
OF 2-AZAHYPOXANTHINE TABLE
COMPARISON
OF
R,
VALUES
617
IN URINE
3
OF AZOIMIDAZOLE
DYE PRODUCTS
R, Values Dye product formed from
Pyridine:H,O ( 1: l)/A1,O,”
2-AH AICA AIC (heated) AIC AI a Solvent
n-Propanobconc. NH, (6:4)/Silica Gel G”
0.032 0.032 0.032 0.363 0.435 systemklc
Isopropanol:H,O:conc. (80: 15:5)/Silica
0.476 0.476 0.476 0.621 0.694
NH, Gel G”
0.255 0.257 0.253 0.410 0.492
plate.
that the resultant dye product was 5[4-([(Z-aminoethyl)amino]naphthyl)azolimidazole-4-carboxylic acid (Fig. 2). Although 5-diazoimidazole-4carboxylic acid was stable in solution, the dye product underwent decarboxylation in solution and in solid form even when stored at -20°C. 2-AH was not found in normal rat or human urine. Detection of a diazotizable compound in the 0.01 N HCI eluate using the present method does not definitively prove the presence of urinary 2-AH, since all false
NEDA e
‘$?
\
NHCH2CH2NH2
8\ 5-
imidazole-4-carboxylic
FIG.
2. Reaction
/
[(4-[(Z-Aminoethyl)omino]naphthyl~ozo]-
pathway
acid
for calorimetric
determination
of 2-AH.
618
BEAL
AND BRYAN
positive sources have not necessarily been determined. Such proof requires use of characterization tests as described under Methods or mass spectrometry as described by Lower et al. (8). The present method offers a simple, reproducible, and quantitative means of detecting 2-AH in urine. For our purposes, it will allow us to determine if this compound, and therefore its parent compound diazoICA, are formed in vivo after DTIC administration. This report also represents the disclosure of a new type of reaction for 2-azapurines and the synthesis of a stable diazo compound. ACKNOWLEDGMENTS This work was supported in part by U. S. Public Health Service Grants CA 13290, CA 14520, and CA 20432 from the National Cancer Institute, USPHS. The technical assistance of Ms. K. K. Whitnable and Ms. E. Torres is gratefully acknowledged. Special thanks is given to Ms. F. Baier for help in preparation of the manuscript.
REFERENCES 1. Shealy, Y. F., Krauth, C. A., and Montgomery, J. A. (1962) J. Org. Chem. 27, 2150-2154. 2. Shealy, Y. F., Struck, R. F., Holum, L. B., and Montgomery, J. A. (1961) J. Org. Chem.
26, 2396-2401.
3. Wolley, D. W., and Shaw, E. (1951)J. Biol. Chem. 189, 401-410. 4. Totter, J. R., and Darby, W. J. (1955) in Organic Syntheses, Collective Vol. 3, Vols. 20-29, (Homing, E. C., ed.), p. 461, Wiley, New York. 5. Rabinowitz, J. C. (1956) J. Biol. Chem. 218, 175-187. 6. Cusack, N. J., Shaw, G., and Litchfield, G. J. (1971) J. Chem. Sot. C., 1501-1507. 7. Gireva, R. N., and Dobychina, N. S. (1959) Izv. Tomsk. Politekh. Inst. 102, 108-113 [Chem. Abstr. (1963) 59, 1622dl. 8. Lower, G. M., Jr., Lanphear, S. P., Johnson, B. M., and Bryan, G. T. (1977) .I. Toxicol. Environ. Health 2, 1095- 1107.
Method
BIOCHEMISTRY
88,612-618
for Detection
DIANE Department
of Human
(1978)
of 2-Azahypoxanthine and Rat Urine’
in Human
D. BEAL AND GEORGE T. BRYAN
Oncology, University of Wisconsin Madison. Wisconsin 53706
Center
for
Health
Sciences,
Received October 20. 1977: accepted March 20, 1978 A new, simple, reproducible, and quantitative method for the detection of 2azahypoxanthine (2-AH) in urine is described. The method is based on removal of interfering substances from urine by cation- and anion-exchange chromatography. Quantitative determination of 2-AH eluted from the anion-exchange resin involves its conversion to 5-diazoimidazole-4-carboxylic acid and subsequent coupling of this compound withN-(I-naphthyl)ethylenediamine. The resultant dye product has an absorption maximum at 505 nm in 2.4 N HCI, which is linearly related to the original 2-AH concentration in the range of O-8 pg/ml. The only substance found to interfere and not be removed or destroyed is p-acetamidophenyl glucuronide, an excretion product of phenacetin- and acetaminophen-containing drugs.
Investigation of the in vitro degradation of the anti-neoplastic agent, 5(3,3-dimethyl- I-triazeno)imidazole-4-carboxamide (DTIC), demonstrated that the drug decomposed to dimethylamine and 5-diazoimidazole-4carboxamide (diazo-ICA) in dilute acid solutions, especially in the presence of ultraviolet light (1). The diazo-ICA thus formed spontaneously cyclized to 2-azahypoxanthine (imidazo[4,5-d]-u-triazin-4(3H)-one, 2-AH) (Fig. 2) (2), an analog of hypoxanthine in which carbon atom 2 is replaced by a nitrogen (3). In studying the metabolism and mechanism of action of DTIC in viva, it became necessary to determine if 2-AH was excreted after drug administration. To accomplish this, a highly specific and quantitative analytical method for urinary 2-AH was required. No previously described method for determination of 2-AH or other 2-azapurines was found, necessitating the development of an entirely new analytical procedure for the determination of 2-AH. The present paper reports a method for the detection of 2-AH in human and rat urine. It involves separation of 2-AH from interfering urinary components by ion-exchange chromatography and its quantitative determination by formation of an azo dye product. Tests used to identify the dye product are also provided. I Presented in part at the 1976 Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, New Orleans, Louisiana, August 16, 1976. 0003-2697/78/0882-0612$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
612
DETECTION
OF 2-AZAHYPOXANTHINE
IN URINE
613
METHODS
Materials. 5-Aminoimidazole-4-carboxamide (AIC) hydrochloride was purchased from Bachem, Inc. (Marine Del Rey, Calif.); 4-nitroimidazole from Aldrich Chemical Co., Inc. (Milwaukee, Wis); and imidazole4methanol picrate and N-( I-naphthyl)ethylenediamine (NEDA) dihydrochloride from Eastman Kodak Co. (Rochester, N. Y.). AG 50W(H+) (12% cross-linkage, 200-400 mesh) and AG l(Cl-) (8% cross-linkage, 200-400 mesh) resins, obtained from Bio-Rad Laboratories (Richmond, Calif.), were made into 1:l slurries with water before use. SpragueDawley rats were purchased from Sprague-Dawley Co. (Madison, Wis.). Other chemicals and supplies were obtained from commercial sources. Zen-exchange chromatography ofurine. Twenty-four-hour urine collections, preserved under lo- 15 ml of toluene, were obtained from consenting laboratory workers and hospitalized cancer patients. Specimens were kept refrigerated at 4°C until assayed. A 10% aliquot of a 24-hr urine was adjusted to pH 1.5-2.0 with 6 N HCl and then, if necessary, was filtered through Whatman No. 50 filter paper. The acidified sample was passed through an AG 50W(H+) column (1.6 cm o.d. x 4 cm) by gravity. The column was washed with 50 ml of 0.01 N HCl. The effluent and wash were combined and the pH was adjusted to 8.9-9.1 with 6 N NaOH. The resultant sample was passed through an AG l(C1F) column (1.2 cm o.d. x 6 cm) under 0.5 psi of pressure. The column was washed with 20 ml of distilled water and the combined effluent and wash were discarded. 2AH was eluted from the column with 100 ml ofO.O1 N HCl under pressure. Twenty-four-hour urines collected from male Sprague-Dawley rats were preserved under 2 ml of toluene and assayed within 48 hr. The volume was adjusted to 50 ml with 0.01 N HCl (pH-2.0), and 25 ml of the sample was fractioned as described above. Recovery of 2-AH was determined from duplicate samples of urine with 2-AH added prior to column treatment. Spectrophotometric quantitation of 2-AH in urine. A 5.0-ml aliquot of the 0.01 N HCl eluate from the AG l(Cll) column, 0.2 ml of 2% NaNO,, and 2.5 ml of concentrated HCl were dispensed into a lo-ml volumetric flask and mixed. The flask was then covered with a glass sphere 2 cm in diameter and was placed in a 90°C water bath for 3.5 hr. After this time, the flask was removed and allowed to cool for 30 min before 1.0 ml of 5 N NaOH and 0.1 ml of 10% ammonium sulfamate were added and the sample was mixed. Two-tenths milliliter of 1% NEDA was then added, the volume was adjusted to 10.0 ml with distilled water (final HCl concentration, 2.4 N), and the final solution was thoroughly mixed. After 30 min, the optical absorption of the sample was compared with a blank of 2.4 N HCI at 505 nm in a Bausch and Lomb Spectronic 20 spectrophoto-
614
BEAL
AND
BRYAN
meter. Quantitative comparisons were made with a 2-AH standard curve prepared in a similar fashion. Preparation of azo dye derivatives. Imidazole-Cmethanol picrate was changed to the sulfate form as described by Totter and Darby (4), using H&SO, (96%) instead of HCl (37%). 2-AH was synthesized from diazoICA according to Shealy et al. (1,2); 5aminoimidazole (AI), from 5nitroimidazole according to Rabinowitz (5), as modified by Cusack et al. (6); and 5-aminoimidazole-4-carboxylic acid (AICA), from imidazole-C methanol sulfate according to Gireva and Dobychina (7). Because of their instability, AI and AICA were not isolated but were reacted immediately after formation with NaNO, and NEDA as follows: 50 ml of HCl (37%) and 10.0 mmol of NaNO, were added to 3.0 mmol of the given imidazole in 50 ml of water (AI) or in 50 ml of phosphate buffer (pH 8.5) (AICA). The reaction mixture was placed on a magnetic stirrer for l-2 min before 10 mmol of ammonium sulfamate was added, and the mixture was again stirred for l-2 min. Upon addition of 4.0 mmol of NEDA, the dye product formed and began to precipitate out of solution within a few minutes. The reaction mixture was stirred for 1 hr and filtered, and the precipitate were dried over CaCl, in a vacuum desiccator and stored at -20°C. The dye product formed from AIC was synthesized by the above method, except that 3.0 mmol of AIC in 100 ml of 6.0 N HCl was used as the starting material. The dye product from 2-AH and another from AIC were also synthesized as described above, except that 3.0 mmol of 2-AH and AIC, each in 100 ml of 6.0 N HCl, were used, and the reaction mixtures were heated at 90°C for 6 hr after adding NaNO, and were cooled for 2 hr before adding ammonium sulfamate and NEDA. Identijkution of dye product formed from 2-AH and NEDA. Identification of the dye product from 2-AH and NEDA was accomplished by comparing several of its physical properties with those of known dye products. Physical properties were determined on the following instruments: melting points, block-type melting point apparatus; visible and uv spectra, Beckman DB-GT spectrophotometer; and infrared spectra, Beckman AccuLab 4. Dye products, dissolved in EtOH, were chromatographed on Silica Gel G in isopropanol: H,O: cont. NH, (80: 15:5) and in n-propanol: cont. NH3 (6:4) and on aluminum oxide (AI,O,) glass plates in pyridine: H,O (1: 1). All chromatograms were developed in the ascending manner and were visualized directly. RESULTS
AND DISCUSSION
Acid hydrolysis of 2-AH and diazotization of the resultant product were completed within 3.5 hr under the conditions specified. The diazo intermediate proved to be very stable, remaining unchanged for 6 months
DETECTION
OF Z-AZAHYPOXANTHINE
615
IN URINE
FIG. 1. Linear relationship between 2-AH concentration (micrograms per milliliter) and absorbance at 505 nm after formation of azo dye as described under Methods (18 samples per point, bars represent *SD). Linear regression line: y = 0.0894x + 0.004.
when kept in 6 N HCl at 4°C. Coupling of the intermediate occurred at room temperature and demonstrated a linear response between the original 2-AH concentration (O-8 pg/ml) and optical absorbance at 505 nm in the presence of excess NEDA (Fig. 1). Maximum color development occurred within 30 min and was stable for a minimum of 30 min. Recovery of various quantities of 2-AH added to duplicate human or rat urine samples is shown in Table 1. Ion-exchange chromatography TABLE OF 2-AH
RECOVERY
SAMPLES
AFTER
PRIOR
ADDITION
TO ISOLATION
1
OF INCREASING
AMOUNTS
BY ION-EXCHANGE
Percentage 2-AH
OF 2-AH
TO URINE
CHROMATOGRAPHY
recovered”
Added (PB)
Human
100 200 400 600 1000 2000 5000
95.0 91.5 93.7 93.0 92.6
Overall U Mean k SD. “n = 8. “n = 48. d n = 56.
recovery
urine * + -r2 +
5.7” 2.1 0.9 2.7 1.9
97.4 + 1.3 93.9
2 2.1’
Rat urine 100.3 92.5 94.2 95.0 93.4 95.8 94.1
” 2 t + + 2 f
4.8” 1.5 1.6 1.2 1.0 1.3 1.3
95.1 t 2.6d
616
BEAL
AND
BRYAN
proved to be quantitative with overall recoveries of 93.9 2 2.1% (mean + SD) from human urine and 95.1 ? 2.6% from rat urine. All normally occurring urinary components which interfered with the calorimetric assay were removed by the ion-exchange resins or were destroyed during the acid hydrolysis procedure. The only substance found to interfere was p-acetamidophenyl glucuronide, an excretion product of phenacetinand acetaminophen-containing drugs. The presence of this compound can be eliminated by prescribing various other analgesic drugs. Since the reaction pathway for calorimetric determination of 2-AH had not been described previously, it was of interest to identify the reaction intermediate and final dye product. Ultraviolet absorption studies revealed that 2-AH decomposed to imidazole during acid hydrolysis, indicating that the dye product contained an intact imidazole moiety. The reaction intermediate was diazotizable, implying the presence of a 5-amino group. Unless the compound underwent a rearrangement, the only other position expected to be substituted was position 4, the most likely substituent being a-H, -COOH, or -CONH, moiety. Synthesis ofdye products containing the above moieties enabled comparison of their physical properties with those of the dye product from 2-AH. None of the dye products melted below 300°C but they became charred [AI, 162°C; AIC, 190°C; AICA, 2-AH, and AIC (heated), -204”Cl and gave off yellow- to red-colored fumes [AI, 180- 190°C; AIC, 190°C; AICA, 2-AH, and AIC (heated), 250-26o”C]. As shown in Table 2, absorption maxima in the visible region varied with the solvent used. Spectra from the 2-AH dye product most closely resembled those from the AICA and AIC (heated) dye products. Movement of the 2-AH dye product on tic again mimicked that of the AICA and AIC (heated) dye products (Table 3). Infrared spectra were identical for the 2-AH, AICA, and AIC (heated) dye products. Comparison of these physical properties revealed that the reaction intermediate was 5-diazoimidazole-4-carboxylic acid and TABLE COMPARISON
OF VISIBLE
ABSORPTION
2
MAXIMA
Absorption
OF AXOIMIDAZOLE
maxima
DYE
PRODUCTS
(nm)
Dye product formed from
2.4 N HCI
0.1 N HCI
HZ0
MeOH
2-AH AICA AIC (heated) AIC AI
500 500 500 510 521
518 520 516 520 489
501 501 501 500 464
502 501 501 496 473
0.1 N NaOH 475 475 475 498 455
DETECTION
OF 2-AZAHYPOXANTHINE TABLE
COMPARISON
OF
R,
VALUES
617
IN URINE
3
OF AZOIMIDAZOLE
DYE PRODUCTS
R, Values Dye product formed from
Pyridine:H,O ( 1: l)/A1,O,”
2-AH AICA AIC (heated) AIC AI a Solvent
n-Propanobconc. NH, (6:4)/Silica Gel G”
0.032 0.032 0.032 0.363 0.435 systemklc
Isopropanol:H,O:conc. (80: 15:5)/Silica
0.476 0.476 0.476 0.621 0.694
NH, Gel G”
0.255 0.257 0.253 0.410 0.492
plate.
that the resultant dye product was 5[4-([(Z-aminoethyl)amino]naphthyl)azolimidazole-4-carboxylic acid (Fig. 2). Although 5-diazoimidazole-4carboxylic acid was stable in solution, the dye product underwent decarboxylation in solution and in solid form even when stored at -20°C. 2-AH was not found in normal rat or human urine. Detection of a diazotizable compound in the 0.01 N HCI eluate using the present method does not definitively prove the presence of urinary 2-AH, since all false
NEDA e
‘$?
\
NHCH2CH2NH2
8\ 5-
imidazole-4-carboxylic
FIG.
2. Reaction
/
[(4-[(Z-Aminoethyl)omino]naphthyl~ozo]-
pathway
acid
for calorimetric
determination
of 2-AH.
618
BEAL
AND BRYAN
positive sources have not necessarily been determined. Such proof requires use of characterization tests as described under Methods or mass spectrometry as described by Lower et al. (8). The present method offers a simple, reproducible, and quantitative means of detecting 2-AH in urine. For our purposes, it will allow us to determine if this compound, and therefore its parent compound diazoICA, are formed in vivo after DTIC administration. This report also represents the disclosure of a new type of reaction for 2-azapurines and the synthesis of a stable diazo compound. ACKNOWLEDGMENTS This work was supported in part by U. S. Public Health Service Grants CA 13290, CA 14520, and CA 20432 from the National Cancer Institute, USPHS. The technical assistance of Ms. K. K. Whitnable and Ms. E. Torres is gratefully acknowledged. Special thanks is given to Ms. F. Baier for help in preparation of the manuscript.
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