M E T H O D S OF A N A L Y S I S AND Q U A L I T Y C O N T R O L

D E T E R M I N A T I O N O F T H E ~ - G L Y C O L I C G R O U P

IN C O M P O N E N T S O F N U C L E I C A C I D S

V. STOICHIOMETRY OF THE OXIDATION REACTION OF RIBONUCLEOSIDES

AND NUCLECBIDE 5 ' -PHOSPHATES BY PERIODATE AS A FUNCTION

OF THE DURATION OF OXIDATION AND THE ptt VALUE

A . I . B u s e v a n d V. Y a . Z a k h a r a n s UDC577.113.4

The oxidation of the eis - a - glyeolic group in r ibonucleosides (I) and nucleoside 5' -phosphates (II) was found to be promis ing for the i r speetrophotometr ie [1], eo lor imetr ie [2], and t i t r imet r ie [3-5] determinat ions and the prepara t ion of der ivat ives of I and II, "dialdehydes" with biological act ivi ty [6-11]. The oxidation of I and II by periodate proceeds according to the equation:

O OHm(POoH)nil

He OH

where R is a heterocyel ie base, n =0 to 3. It is known that I and II react with periodate in a mola r rat io of 1 : 1 [12], and that this react ion is highly specific In dilute aqueous solutions at a pH value close to neutral, and that when NaIO 4 is used in a 3-5-fold excess , the oxidation proceeds quantitatively In nearly 30 min at room t e m - pera ture , o r in 1 h at 0~ [13]. Some authors c a r r y out the oxidation react ion for up to 3 [12] and 24 h at 20~ [111.

No sys temat ic studies on the s to ichiometry of this react ion as a function of the pit value o r the duration of oxidation a re descr ibed in the l i terature , and the re are no data on the appearance of an excess consumption of the oxidizing agent (the periodate). Since the co r rec tness of methods of analysis is dependent on the s tol- eh iometry of this reaction, the a im of this work was its study in o rde r to select the optimal conditions for the oxidation of I and II during their determination: For this purpose, we used mainly two types of oxidation, c o r - responding to the method of prepara t ion of the oxidizing agent, ioe., a solution of KIO~ in 0ol N sulfuric acid, where the oxidation proceeds at a pH of about 1.0-2.0, and a solution of NaIO 4 in water, where the oxidation proceeds at a p H o f a b o u t 4 . 0 - 7 . 0 , anda t i t r imet r ie var iant of the amplification method [4], with an accuracy within • 0~176 We limited ourse lves to the study of the oxidation react ion in the acid medium, since the "di- a ldehydes" I and II formed in the alkaline medium can readily undergo fur ther t rans format ions [13].

The resul ts a re listed in Table 1, which shows that the oxidation react ion of I and II by a 2-6-fold excess of periodate is more specific at pit of about 2.0 by using KIO 4 solution in dilute sulfuric acid, and is complete a f t e r 1-3 mira Increase in the duration of oxidation to 3-5 h does not lead to an excess consumption of the oxidizing agent, except in the case of inosine 5 ' - d i - and tr iphosphates and guanosine 5 ' - t r iphosphate . The presence of such a considerable l inear section on the curve of the dependence of the oxidizing agent consump- tion on the duration of oxidation does not require an accura te observat ion of the t ime of oxidation, which s impli- fies its per formance .

With an increase in pH to neutral values, a tendency to an increased consumption of the oxidation agent is observed. The most stable compounds towards overoxidation are I and their 5 ' -monophosphates , and the leas t stable are 5 ' - d i - and t r iphosphates of uridine and inosine. The oxidation of lnosine 5 ' - d i - and tr iphosphates by a NaIO 4 solution in water and of guanosine 5 ' - t r iphosphate by a KIO 4 solution in 0.1 N sulfuric acid proceeds without a pronounced l inear sect ion on the curve of the dependence of the consumption of the oxidizing agent on the duration of oxidation. During prolonged oxidation of I and II by NaIO 4 solution in water at a pit of about 4.0-7.0, the excess consumption of the oxidizing agent influences the resul ts of the determinat ions, which are too high. In the determinat ion of I and II, they must be oxidized by a solution of KIO 4 in 0.1 Nsulfur ie acid at a pH of about 2.0 for 10-15 min.

M. V. I~monosov Moscow State University. Transla ted f rom Khimiko-Farmatsevt ieheski i Zhurnal, VoL 12, No. 5, pp. 137-140, May, 1978. Original ar t ic le submitted September 6, 1977.

0091-150X/78/1205-0679S07.50 �9 1979 Plenum Publishing Corporat ion 679

TABLE 1. Amount of Periodate (n in moles) Reacting with i Mole of Ribonucleosides and Nucleoside 5'-Phosphates as a Function of pll Value and Duration of Oxidation at Room Temperature (18-20~

Concentration of com- pounds in reaction I [medium, M /

IPeriodate II {~-~ ~] ~ . o , ,

I Compound

Uridine

Uri .dine 5'-monoph_osphate acaa cisooium salt

Oridine 5'-diphosphate acid disodi~rn salt

Ufidiae 5'-triphosphate acid ui~odium salt

5-Ethyluridinc

5 - Bromouridine

Cytidine

Cytidinc P-monophos- phat e acid

Cytidine 5'-triphosphate

acid trisodium salt

Adenosine

Adenosine 5'-monophos- phate acid

Adenosine 5'-diphosphate

acid trisodium salt Adenosine 5'-triphos -

phate disodium salt tetrahydrate

Inosine

Inosine 5'-monophos- phate disodium s a l t

Inosine 5'-di~hosphate oisooi m salt

Inosine 5'-triphosphate disodium salt

Guanosine 5'-mono- phosphate disodium salt

Guanosine 5'-tfiphos- phate disodium salt

0,0125 0,0125

0,0125 0,0125

0,0125 0,0125

0,0125 0,0125 0.0125 0,0125 0,0125 0,0125 0,0125 0,0125

0,0125 0,0125

0,0125 0,0125 0,0125 0,0125

0,0125 0,0125

0,0125 0,0125

0,0100 0,0125 0,0125 0,0100 0,0100 0,0100 0,0125 0,0125

0,0125 0,0125

0,0125 0,0125

0,0125 0,0125

0,0125 0,0125

0,0125 0,0125

...... 6;665 2 , 5

0,005 2,5

0,005 2,5 0,005 2,5

0,0025 5 0,002 6,25

0,002 6,25 0,002 6,25 0,005 2,5 0,005 2.5 0,005 2~5 0,0025 0,005 2,5 0,005 2,5

0,005 2,5 0,005 2,5

0,025 5 0.002 6,25 0,005 2,5 0.005 2,5

0,005 2,5 0,005 2,5

0,005 2,5 0,005 2,5

0,004 2,5 0,005 2,5 0,005 2,5 0,004 2,5 0,004 2,5 0,004 2,5 0,005 2,5 0,005 2,5

0,005 2,5 0,005 2,5

0,005 2,5 0,005 2,5

0,O03 4,2 0,003 4,2

0,005 2,5 0,005 2,5

I ,OO5 2,5 0,005 2,5

* A d d i t i o n o f d i l u t e s u l f u r i c a c i d . ~ A d d i t i o n o f p h o s p h a t e b u f f e r s o l u t i o n s .

pH Duration of toxidation when n= 1_.000 ~ 0.002

cq 0

1,6 5,2

1,7 7,0

1,7 6,7 1,6 4,9 1,6

6,6

1,7 5,1

1,7 6,6 1,7 5,8

1,8 7,5

1,7 6,9

0 , 8 ' 1,7 3,6 5,9 6,8 7,9 1,7 6,7

t,7 6,9

1,7 6,3

1,7 3,5

1,7 7.0

1,7 3,1

I min_ 5 h 30 se~-3 h

30 see-5 h 30 sec-1 h

30 sec-5 h 30 sec-lO rain

30 see-5 h 30 sec-3 rain l rain--5 h l min--5 h 1 rain--5 h 2 min --4 h 30 sec --5 h

rain--5 h

l rain--5 h 30 sec=2 h

2 min-5 h 1 rain-3 h 1 min--5 h 1 rain--5 h

30 sec-5 h l min -4 h

2 min--5 h I min--I h

23 min--5 h m i a - 5 h

I min--3 h 30 see-5 h 3 rain--10 min 3 min--I h 2 min--5 h 2 min--3 h

I rain--3 h 30 sec=3 h

5 m i n - I h 20 rain--40 min

5 min--I h 5 rain--30 min

2 min--5 h 30 sec-2 h

2 min-lO min 3 min--3 h

1,002 1,005

1,000 1,044

1,006 1,053

1,008 1,060 1,003 1,003 1,00l 1,008 1,000 1,007

1,001 l,OlO

1,016 1,083 1,003 1.,000

1,004 1,040

1,006 1,033

1,008 1,002 1,005 1,003 1,029 1.034 1.001 1,020

1,010 1,216

1,017 1,074

1,014

1,004 1,031

1,014 1,010

Compounds I and I[ are rapidly oxidized by periodate apparently because D-ribose is present in them in the furanose form only, and the ~-glycolic group in the cis-form only, and in their solutions no equilibria are established between the optical isomers. In [14] a kinetic method for the identification and determination of ribose, maanose, fructose, sorbose, xytose, galactose, lactose, and saccharose is proposed by means of a perchlorate selective electrode, which is sensitive towards perlodate. Before carrying out the analysis, stand- ard solutions of carbohydrate and the sample solutions must be kept 2 days for an equilibrium between the optical isomers to be established. In the studies on the oxidation reaction of arabinose, xylose, ribose [15], glucose [16], and arabltol, xylitol, and ribitol [17] by a NaIO 4 solution, it was found that in several cases, when phosphate and bicarbonate buffer solutions were used, there was an excess consumption of the oxidizing agent, as we in fact found during the oxidation of the disodium salt of adenosine 5'-triphosphate in phosphate buffer solutions. It was also found [16] that the phosphate and bicarbonate buffer solutions considerably influence

the formal redox potential of the IO4-/IO 3- system.

6 8 0

EXPE RIMENTA L

Compounds I and II, the reagents and their preparation, and methods of determination are described in [4]. A 0.025 M (0.05 N) NaIO 4 solution was prepared by dissolving a weighed portion of a purified preparation in distilled water. The solution was then stored in a flask made of dark glass. A 10 ml portion of the periodate solution was added to 10 ml of an aqueous solution usually containing 0.1 mg-equiv, of I or II, and the mixture was lef~ to stand for the required time at room temperature, in a place protected from direct sunlight. The readings were taken at the following times: 30 see, 1, 2, 3, 5, 10, and 30 rain, 1, 2, 3, 4, 5, and 24 h. The pH was controlled by a pH-340 pH-meter.

L I T E R A T U R E C I T E D

1. J .S . Dixon and D. Lipkin, AnaLyt. Chem., 26, 1092-1093 (1954). 2. A. L Busev and V. Ya. Zakharans, Khim. Farm. Zh., No. 10, 135-140 (1977). 3. V. Ya. Zakharaos and U. Ya. Mikstais, Izv. Akad. Nauk I.~tv. SSR, Set. Khim., No. 1, 55-60 (1977). 4. A . I . Busev, V. Ya. Zakharans, and U. Ya. Mikstais, Khim. Farm. Zh., No. 3, 128-132 {1977). 5. A . I . Busev, V. Ya. Zakharans, and U. Ya. Mikstais, Khlm. Farm. Zh., No.4, 132-135 {1977). 6. M. Slavik, Ann. N. Y. Acad. Sci., 25.~5,266-268 (1975). 7. R .L . Cysyk, Cancer Chemother. Rep., 59, Pa~t I, 685-687 {1975). 8. J . G . Cory and C. B. George, Biochem. Biophys. Res. Commun., 52, 496-503 (1973). 9. J . G . Cory, M. M. Mansell, C. B. George, et aL, Arch. Biochem., 160,495-503 (1974).

10. W. Dvoach, H. Fletcher III, F. J. Gregory, et al., Cancer Res., 26, 2386-2389 (1966). 11o A.S. Jones, A. F. Markham, and It. To Walker, J. Chem. Soc., Perkin Trans. I, No. 14, 1567-1570 (1976). 12o Go Schmidt, Meth. EazymoL, 12, Part B (Nucleic Acids), 230-235 (1968). 13. N.K. Kochetkov, ~. I. Budovskii, E. D~ Sverdlov, et aL, Organic Chemistry of Nucleic Acids [in Russian],

Moscow (1970), p. 582. 14. Co E. Efstathiou and T. P. Hadjiioannou, Analyt. Chlm. Acta, 89, 55-63 (1977). 15. Z. Du~i~, Aeta Pharm. Jugosl., 25, 35-41 (1975). 16. Z. Du~i~ and A. Berka, Acta Pharm. Jugosl~ 25, 43-49 (1975). 17. Z. Du~iS, Arkh. Farm. (Beograd), 25,233-240 (1975).

I D E N T I F I C A T I O N OF A M I N A Z I N E , D I P R A Z I N E ,

AND P R ( ) t ~ A Z I N E IN S O L U T I O N S AND E V A L U A T I O N

OF T H E I R S T A B I L I T Y ( E X C H A N G E OF E X P E R I E N C E )

N. G. S e l e z e n e v , G. D. S o l o v ' e v a , E. A. T y r i n a , a n d V. M. I v a n o v a

UDC 615.214.22:547.869, 2].07

Phenothiazine derivatives have been identified by various methods - color reactions with oxidizing rea- gents, precipitation reactions, melting points, UV and IR spectra, and others [1-4]. Although many of these reactions are highly sensitive for the authentic compounds, in medicinals they are not sufficiently specific for the determination of the active substances in the presence of their decomposition products [5 I.

In our opinion, it is necessary to combine specific reactions with other raethods, for instance chromatog- raphy, in order to evaluate the authenticity and stability of aminaziae, dipraziae, and propaziae solutions. This combination of methods is especially advisable for the quantitative determination of the active substances ia the presence of their degradation products [6].

EXPERIMENTAL

Grade M paper and descending chromatography were used with the following solvent systems [1]: neutral, 1-butanol-ethanol-water (5 : 2 : 2); acid, 1-butanol-acetic ac id-wate r (4 : 1 : 5); and basic, 1-butanol- 1 N NHr (88:12). For development, UVlight (254 am), iodine vapor, and 1% chloramine solution were used.

L P. Par lor Ryazan Medical Institute, Moscow Industrial Chemical-Pharmaceutical Association, "Moskhimfarmpreparaty." Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 12, No. 5, pp. 140- 142, May, 1978.

0091-150X/78/1205-0681~07.50 �9 1979 Plenum Publishing Corporation 6~]