tives by combining the features of both. Instead it was considerably poorer than the parent compound. For one currently using GBHA as a colorimetric reagent for calcium, the 5-methyl derivative would be a good substitute if reagent stability has been a problem. This reagent needs a more alkaline medium for color development, but this should limit interference. The 4-methyl derivative would be of value to one needing greater sensitivity than the parent cornpound offers; here too, a more basic medium is desirable. There was little difference between the methyl derivatives and the parent compound toward interferences when the tests were run under the same conditions of alkalinity. The parent compound decomposed in basic solution under nitrogen to glyoxal and the amine used in its preparation. This colorless solution, on exposure to air, rapidly developed the same colors observed when the parent compound was allowed to decompose in basic solution. The decomposition colors
were due to the air oxidation of the amine in basic solution. Glyoxal undergoes the benzilic acid rearrangement to the salt of glycolic acid in basic solution (6). The formation of a complex with the reagent limits the amount of reagent in solution, but some of the reagent is always present to decompose, because the complex is in' equilibrium with its components. For this reason, the complexes decompose too. For a really satisfactory reagent for calcium of this type, some means must be found to prevent this decomposition. If the carbon-nitrogen double bond could he incorporated in a ring, the problem of decomposition would be solved. Such a compound might be 8,8'dihydroxy-2,2'-biquinoline. LITERATURE CITED
(1) Ashkinazi, Ya., Rabinovich, M., Zh. Prikl. Khim. 7. 939 119341: C d . 29. 2521 (193.5). (2) Bayer, Ernst, Chem. Ber. 90, 2325 (1957).
(3) Bent, H., French, C., J . Am. Chem. SOC.63. 568 11941). (4) Diehl; H.; Lindstrom, F., A N A L . CHEM.31,414 (1959). ( 5 ) Diepolder, E., Chem. Her. 42, 2917 (1909). ( 6 ) Evans, W.,Adkins, H., J . A m . Chem. SOC.41, 1411 (1919). ( 5 ) Holler, .4.,Huggett, C., Rathman, F., Zbad.. 72. 2034 119.50). (8) Kerr, J.)Ana&st85,867 (1960). (9) hlueller, C., Pelton, W., J . i i m . Chem. SOC.71, 1504 (1949). (10) Yeunhoeffer, O., Kolbel, H., Chem. Rer. 68, 260 (1935). ( 1 1) Proskouriakoff, A,, Titherington, R., J . A m . Chem. SOC.52,3982 (1930). (12) Schultz, G., Chem. Rer. 40, 4322 (1907). (13) VrschlabskG, M., OkBE, A., Collection Czech. Chem. Commun. 27, 246 (1962). (14) Williams, K. T., Wilson, J. R., ANAL.CHEY.33, 244 (1961).
RECEIVED for reviex February 11, 1964. Accepted March 19, 1964 Taken in part from the M S thesis of Carl W Milligan S w t h Carolina Academy of Sciences Xleeting, Columbia, A4pril 1963, and Southeastern Regional LIeeting, ACS, Charlotte, November 1963 Work supported by the Xational Science Foundation, research grant S o G19699
Spectrophotometric Determination of Streptozotocin ARLINGTON A. FORIST Biochemical Research Division, The Upjohn Co., Kalamazoo, Mich.
b Need for a rapid procedure for the determination of streptozotocin has led to the development of a method involving acid cleavage of the N-nitrosomethylamide group to yield nitrous acid, diazotization of sulfanilic acid with the resulting nitrous acid, and coupling of the diazonium salt with N( 1 -naphthyl)ethylenediamine dihydrochloride to produce an azo compound with maximum absorption at 550 mp. Analysis of standard samples has given a mean recovery of lOOyo with a standard deviation of Recovery of added streptozotocin from filtered fermentation beer has been 97.8 2 3.070(mean 2 standard deviation) and the results obtained by the chemical procedure have agreed with those found by microbiological assay.
l.Oyo.
S
treptozotocin is a broad spertrum antibiotic of unknown btructure (9). An empirical formula of CliH2?S5012 has been suggested and the molecule contains an N-nitrosomet hylamide group (I) ( 4 ) . Chemical, physical, and 0 YO
II
I CH?
R-C--S-
I biological propertie. of streptozotocin have bren tiemibed (3-5, 8, 9 ) , and an 1338
ANALYTICAL CHEMISTRY
eltenqive study of the stability of thii: antibiotic in solution has al\o been reported ( 2 ) . ?1Iicrobiological methods for the determination of qtreptozotocin by a disk-plate a.say against Proteus uidgarzs and for the arialjsis of blood plasma utilizing Proteus rettgeri have been described ( 8 ) . Garrett ( 2 ) employed polarography as an analytical tool in itudying the qolution Ytaldity of this antibiotic. Seed for a rapid procedure for the determination of -treptoxotocin as bulk drug and in fermentation beers led to the development of the present qwtrophotometric method based on a quantitative adaptation of the spot teht of Feiql and S e t o (1) for the detection of ,Y-nitroso compounds. EXPERIMENTAL
Reagents. .hetic acid, 3Oy0 (v./v.). Hydrochloric acid, 6 s . Acetate buffer, pH 4.0, approximately 0.1.U in acetic acid and 0.0251 in sodium acetate. Sulfanilic acid, 1% in 3Oy0 acetic acid. Color reagent, 0.5% sulfanilic acid and 0.1% S ( 1 - naphthy1)ethylenediamine dihydrochloride (KED) in 30% acetic acid. A 100 i 1-mg. portion of S E D is added to 50 nil. of 1% sulfanilic acid in 30y0acetic acid and the resulting solution is diluted to 100 ml. with 307, acetic acid. This reagent was prepared fresh daily as it discolors on standing.
Standard, stock solution. In the present work a stock solution containing about 10 mg. of streptozotocin in 25 nil. of acetate buffer was employed. This solution is st'able for a t least a week under refrigeration. Standard, working solution. One milliliter of the stock solution is diluted to 25 ml. with the acetate buffer (concentration about 15 pg, per ml.). This solution is stable for a t least 4 days under refrigeration. Apparatus. Glass-stoppered test tubes. These were prepared bv sealing t,he ends of T 19/38 outside ground joints and fitting them with -$ 19/38 stoppers. Constant temperature water bat,h, 60" C . Spectrophotomet,er. A Beckman Model B spectrophotometer with 1-cm. cells was employed for absorbance measurement's. Procedure. A sample solution is prepared which contains from 3 to 30 pg. of streptozotocin per ml. of the acetate buffer. For solid preparations, about 20 mg. of sample are dissolved in the acetate buffer and diluted to 25 ml. One milliliter of the resulting solution is then diluted to 50 ml. with buffer. For fikered beers, a 1 to 10 dilution with the buffer is made. One milliliter of the sample solution is transferred to a glass-stoppered test tube, followed by 5 ml. of the color reagent and 1 nil. of 6 S HC1. The resulting solution is mised and the stoppered tube placed in the 60' C.
Table I. Color Development from Streptozotocin as a Fiinction of Time at
60" C. Reaction time, rninu tes .i
10 20 30 40 50
60 or
A d 0.280 0 337 0 365 0 377 0 392 0 391 0 399
14.88 pg. per ml.
Table II. Determination of Standard Samples of Streptozotocin
Taken, pg./ml.
8.93 11 90 14 88 20 83 29 76
Found, pg./ml.
9.07 8.91 11.98 11 91 14.79 14 79 21 09 20 89 29 57 29 69
Recovery,
5%
101 6 99.8 100.7 100.1 99.4 99.4 101.2 loo 3 99.4 99 8
Alean and standard deyiiation 100 0 1 1 . 0
bath for 45 minutes The sample is then removed, cooled to room temperature, and absorbanca a t 550 mp is determined us. a reagent blank similarly prepared. Two 1-ml. aliquot.; of the working solution of the standard are carried through the procedure in parallel with the unknown. The streptozotocin content of the unknown is then calculated from the response obtained with the qtandard. Beer's law is followed over the recommended concentration range (absorbance per p g . per ml. of sample is approximately 0.026). RESULTS A N D C W X J S S l O N
The procedure piwented for the determination of streptozotocin is based on a reaction of the S-nitroso group. The method is a quantitative adaptation of a spot test for N-nitroso compounds described by Frigl and S e t o ( I ) with a substitution of ;V(l-naphthy1)ethylenediarnine dihydrocmhloride for l-naphthylamine as the c o u i h g reagent. A similar reagent rnixture has been reported l y Saltzman ( 7 ) for the determination of nitrogen dioxide in the atmospherr. The reactions involved in the prchent procedure are acid cleavage of the .Y-nitrosn group to yield nit,rous acid, diazotization of ::ulfanilic acid with the resulting nitrou-: ;acid, and coupling of the diazonium salt with 'Y(1nai)hth!-l)c,thylrnrtlia nine dihydro-
chloride to produce an azo compound with an absorption maximum at 550 mcr Development of the azo compound as a function of reaction time a t 60" C. is shown in Table I. Under the conditions used, masimum color development is reached within 40 minutes and remains constant for a t least an additional 20 minutes. A 45-minute reaction period has been employed for routine measurements. A%ftercolor derelopment, absorbance a t 550 mp is unchanged over a 3-hour period a t room temperature wit'h exposure to usual laboratory lighting. After 7 2 hours, absorbance decreases by only 3.594 indicating no problem of instability of the color, thereby permitting considerable flexibility of operat,ion. -%bsorbance a t 550 mp follows Beer's law over the range 3 to 30 pg. of streptozotocin per ml. of sample. I n routine use, standards have been run in parallel with unknown samples as described above. However, escellent day-to-day reproducibility indicates that it should be possible to operate with a standard curve. Application of the method to a series of standard solutions has given a mean recovery and standard deviation of 100.0 + 1.0% (Table 11). -4nalysis of a filtered beer containing added streptozotocin has given a mean recovery and standard deviation of 97.8 i 3.0cT, over the range 30 to 120 p g . per ml. of beer (Table 111). Chemical ay of the beer agreed with the assay and no correction for background a t 550 mp caused by the beer was necessary. The present procedure is applicable to the determination of streptozotocin in filtered fermentation beers as w~11as in simpler systems. The method i q rapid, accurate, and precise, and well suited t,o the analysis of large numbers of samples. It has been possible to est,pnd this procedure, with minor modifications, to the determination of streptozotocin in blood and other tissues (10) and to studies of the solution stability of this antibiotic ( 2 ) . The method also has been adapted to the assay of fermentation beers with t,he Technicon AutoAnalyzer by G. C. Prescott (Fermentation Research and Development, The lipjohn Co.) ( 6 ) . A brief examination of the stability of streptozotocin in the acetate buffer a t pH 4.0 was made. Other studies have indicated optimum stability of the antibiotic a t this pH ( 2 , 4 ) . Rewlts in Table IV show that the stnck solutinn is stable in terms of this assay fnr at least a week under refrigeration and the working solution for at least 4 days. At room temperature, the working solution s h o w an apparent loss of ,5% in one day and about 13% in 5 days. dctnally, deromposition of streptozotocin at rnom temperature probably is f
Table 111. Recovery of Streptozotocin Added to a Filtered Beer"
Added, pg. ml. 0 29 8 59.5
89.3 119 0
Found, pg./ml. 167 3 167 3 194 6 197 3 225.7 225.7 2.52.9 254.1 286 8 286 4
Recovered, pg./ml.
Recovery,
7c
. . 27 3 30 0 58.4 58.4 85.6 86.8 119 5 119 1
91 6 100 7 98.2 98.2 95.9 97.2 100 4 100 1
Mean and standard deviation 97.8 f 3 0 a
Bioassay, 167 pg./ml.
Table IV. Stability of Streptozotocin in Acetate Buffer, p H 4
Stor- Temage pera- Remaintime, ture, ing, % days "C.
Solution Stock, 372 fig./ml. Working, 14 88 a./ml. Working, 14 88 pg. /ml.
1 5 7 4
4 4 4 4
1 5
25 25
100 8 100 5 100 5
99 2
94 5 85 7"
4 Color developed raDidly at room temperature before addition of 6.V HCI; probably free nitrite present.
greater than this aq there is an indication of cleavage t o nitrite under these conditions. Sitrite is a positive interference in this method. Standard solutions should be stored in the refrigerator. ACKNOWLEDGMENT
The author is indebted to J. H. Ford and associates for the microbiological assay data. LITERATURE CITED
(1) Feigl, F., Neto, C. C., . h A I , . CHEM 28, 1311-12 (1956). ( 2 ) Garrett, E. R., J . d m . Phnrm. Assoc., Sci. Ed. 49, 767-77 (19601. (3) Hanka, L. J., Sokolski. T. T., Antibid. .4nn. 1959/60, n D . 2h,5-61. (4) Herr, R. R., Ehle, T. E., Rerpy, 31.E., Jahnke, H. K., l h i d . , pp, 236-40. ( 5 ) Lewis, C., Barhiers, A . R., Ibid., pp. 247-54. ( 6 ) Prescott, G. C., the Upjohn Co., unpuhlished data, 1959. CHEM.26, (7) Saltzman, R. E., -4~~1,. 1949-.i5 (1954). (8) Sokolski, W. T., l'avra. J . J., H i . n k ~ , L. J., Antibiot. ilnn. 1959/60, nn. 241-6. 191 , , T-avra. J. J.. IIeRoer. C.. IIietz. A , . Hnnka, 'Id. J., Pokolski, IT. T., Ibii.1 pp. 230-5. (IO) Wallach. r>. P., The Upjohn Co., unpublished data, 1 9 3 . RECEIVEDfor review January 16, 1964. Accepted )larch 30, 1964. VOL. 36, NO. 7, JUNE 1964
1339
