Rifamycin XXIII. The Polarographic Behavior of Rifamycin B, Rifamycin 0, Rifamycin S, and Rifamycin SV GlAN GUALBERTO GALLO, LUlGl CHIESA, and PIER0 SENSl Research laboratories of lepefif S.p.A.,
Milan, lfaly
b The polarographic behavior of rifamycin 8, rifamycin 0, rifamycin S, and rifamycin SV i s described. The proportionality between concentration and diffusion current, the analysis of the reduction or oxidation curves, and the dependence of the half-wave potential on pH have been studied, and the number of electrons involved in the electrode processes has been determined. The polarographic method can be used for the quantitative determination of the four antibiotics studied. Rifamycin S and rifamycin SV are the oxidized and reduced form of a reversible oxidation-reduction system.
R
B is a neiy antibiotic discovered by Sensi et al. (6). (The name rifamycin has been adopted instead of rifomycin as in the foregoing papers in order to avoid a confusing similarity with the commercial name of other antibiotic drugs.) Although its strueture is not yet known, the chemical and physical properties have been described ( 5 ) . Rifamycin 0 is the oxidation product of the parent antibiotic (2) and gives by hydrolysis rifamycin S, which by reduction is transformed to rifamycin SV (3, 7'). I n this paper, the polarographic behavior of rifamycin E, 0, S, and SV is described. IFAMYCIN
EXPERIMENTAL
Reagents. Rifamycin B, 0, and S, as free acids, and rifamycin SV, as a potassium salt, were prepared in our laboratories and tested before use by physical and chemical methods (1-5).
Table 1.
Compounds Rifamycin B Rifamycin 0
(S.C.E.)
POTENTIAL iN VOLT
Figure 1. sec.-1/2
Polarogram of rifamycin B--m2/3f1/e = 1.60 mg.2/3 a. b.
1 M triethanolamine-hydrochloric acid, pH 7.5 2.2 mmoles per liter of rifamycin B in buffer
Methanol and buffer components were determined polarographically pure. Apparatus. An LKB Model 3266 B polarograph, coupled with a 10-mv. Leeds & Northrup recorder, was employed. The potential was measured us. a low resistance saturated calomel electrode. Contact with the solution was made via a salt bridge of saturated aqueous KCI. All p H measurements
were made with a Metrohm E 148 C p H meter. The viscosities were determined with a Hoppler viscosimeter; the densities were determined with a pycnometer. The magnetic stirring device and the fritted-glass diaphragm cell were constructed according to Stokes (9). Procedures. T h e current-voltage curves were obtained a t 20" =t0.1' C.,
Polarographic Characteristics of Rifamycin B and Rifamycin 0 at
Supporting Solution IM Triethanolaminehydrochloric acid buffer, pH 7.5 75% methanol and 25% 1M sodium acetateacetic acid buffer, pH
Slope, i id - i
-
m2/8t1/8
Em us. S.C.E., Volt
1.60
-1.25
Cm2/*t1'6 1.34
2.24
-0.20
2.03
id
us.
20" C.
Stokes-Einstein D
x
10-6
Potential cm.2 sec.-l 0.105
3.05
n 1.36
0.120
2.35
2.3
Porous Diaphragm Cell
D x
10-6,
cm.* sec.-l 5.14
n 1.04
...
...
5.5
VOL. 34, NO. 3, MARCH 1 9 6 2
423
PH
40 I
60
rically, and the L) values were calculated from the equation:
99
10
a
e
m 6-
--
-I
2
u-here K is the cell constant, t the diffusion time, VI and V 2 the conipartment volumes, and CI and CZ the final concentrations in the two compartments. To determine K , the cells were calibrated by allowing 0.1X KCl to diffuse into water a t 20" C. until 25% had passed through the diaphragm. The diffusion constant of KCl was calsq. cm. per culated as 1.658 X second a t 20" C. from the value 1.867 x 10-5 sq. cm. per second at 25" C.
, __
/
-
-
~ -
___
__
~
I
L--
/'
__--
-
-
P O T E N T I A L IN V O L T ( S C E )
Figure 2. Polarograms of rifamycin B in 50% methanol and acetate-acetic acid-sodium hydroxide, at various p H values (curves start from -0.8
volt
VI.
50% 1M sodium
S.C.E., 200 mv./abs.)
(9). RESULTS A N D DISCUSSION
-
8
Rifamycin B. Rifamycin I3 gives a reduction wave of El = - 1.25 volts us. S.C.E. in aqueous lM triethanol- 6 1 -amine-hydrochloric acid a t pH = 7.5, as shown in Figure 1 and reported r in Table I. The diffusion current is U " linearly proportional t o concentration 5 4 between 0 and 5 miiioles per liter, z and in solutions of this concentration, z , t h equantitative determination of 3 rifamycin B is polarographically feasu 2 ible. From the slope and the D and n values reported in Table I, the reduction wave can be considered due to a n electrode process irreversible and in0 volving one electron. The JTalue of 1.36 electrons obtained from the D *02 0 -0.2 -04 -06 -0 8 -1 0 -12 - 1.4 value calculated can be considered POTENTIAL IN VOLT ( 5 C E 1 fairly good if the approximation of Figure 3. Polarogram of rifamycin O-rnz/3t1/6 = 2.24 mg.*j3 sec.-1/2 the Stokes-Einstein equation is taken into account. a. 75% methanol and 25% 1M sodium acetate-acetic acid, pH 5.5 The u-ave of rifamycin B n a s studied b. 0.86 mmole p e r liter of rifamycin 0 in buffer a t different pH values, in 50% methanol and 50% 1Al sodium acetate-acetic acid-sodium hydroxide ; this buffer a n d t h e oxygen was removed from volts for rifamycin B, -0.5 volt for n a s adopted, despite its poor buffering rifamycin 0, and -0.3 volt for rifathe solutions with pure nitrogen. No poner a t the higher pH yalues, to mycin S and SV. maxima suppressors were used. The The values of diffusion coefficients avoid the complexities, n hich the use of resistance of all solutions was less for the four rifamycins, dissolved in t h a n 1000 ohms (as determined rvith different buffers might involve. A s their polarographic solvent system, a 50 c.p.s. Wheatstone bridge) so the shown in Figure 2 , the principal wave is a t 20" + 0.1" C. were calculated from I R drop correction was negligible in observed in the interval 4 to 9, a second the well known Stokes-Einstein equacomputing Elizvalues. T h e dropping n-ave of Ell2 = -1.5 ~ o l t sin the inmercury electrode characteristics, retion and measured with the porous terval 5 to 7 , and a third wave of ported in Tables I a n d 11, were dediaphragm cell method (9). I n this = - 1.64 volts a t pH values greater termined for the capillaries immersed method, the average diffusion time than 8. I n the plot of El against p H was 120 hours. The concentrations of in the supporting solutions a t a presfor the principal Bave, a slope 0.072 sure of 35 cm. of mercury, the drop the solutions in the compartments ~ o l tper pH unit was obtained, conwere determined spectrophotomettimes having been measured a t -1.5 ---
- -
I--
Y CL
CL
I-
Y
CL
Polarographic Characteristics of Rifamycin S-Rifamycin SV System a t
Table II.
20' C. Poroiis .~ -. -
Slope i Ella
Com-
pounds Rifamycin S Rifamycin SV
424
0
Supporting Solution 507, Methanol and
50y0 1 X sodium acetate-acetic acid buf-
fer, pH
=
6.0
ANALYTICAL CHEMISTRY
us. V l 2 )3t"6
2.26
S.C.E., Volt
Zd
C?n2/3 t1'6
+0.03
2.06
+0.03
1.51
- (id)a -i
Stokes-Einstein x 10-6
D
(idh
Diaphragm Cell
D
x 10-6
us.
Potential 0.035
71
1.7
1.82
2.04
2.63
1.71
...
. . .
, . .
...
Figure 4. Polarograms of rifamycin 0 in 75% methanol and ous pH values
25y0 1 M sodium acetate-acetic
[curves start from t0.2 volt
firniing the irreversibility of the clectrode process. Rifamycin 0. Rifamycin 0 gives trio reduction wives of El,* = -0.20 volt and -1.15 volts us. S.C.E. in 755% methanol and 25% 1 M sodium acetate-acetic acid at p H = 4.3 (the apparent pH value of t h e solution vas 5 5 ) , as shown in Figure 3 a n d reported in Table I. T h e diffusion current of t h e first wave is linearly proportional to concentration betneeii 0 a n d 1.5 mnioles per liter, (at higher concentrations the wave exhibits a niaxiniuni) and is suitable for a quantitative determination. From the slope and the D and n values re-
VI.
S.C.E., 200 mv./abs.)
ported in Table I, the first reduction wave can be considered due to a n electrode process irreversible and involving two electrons. The measuring of D with the porous diaphragm method was unsuccessful because we could not determine the concentration of rifamycin 0 in the two cell compartments as n e observed polarographically and spectrophotonietrically that rifamycin 0 underwent almost entirely the transformation to rifamycin S (B) during the period of diffusion. The polarographic naves of rifamycin 0 were studied in the pH interval 4 to 9 in solutions similar to that described aboie, as reported in Figure 4. The first nave is constant in the studied interval, while the second nave disappears a t p H values greater than 8; in the interval 8 to 9, a nely small reduction wave of E112 = 0.08 volt is detectable. I n the plot of Eliz against pH, a slope of 0.010 volt per pH unit for the first wave and O.Oi0 volt per pH unit for the second wave were obtained, both d u e s confirming the irreversibility of the elcxtrode process. Rifamycin S and Rifamycin SV. Rifamycin S gives a reduction wave of El,? = +0.03 volt vs. S.C.E. in 507, methanol and 507, l M sodium acetate-acetic acid a t p H = 5.4 (the apparent pH value of t h e solution was 6.0); rifamycin SV,dissolved in t h e same supporting solution, gives a n oxidation wave of t h e same Eliz. T h e polarogram of a mixture of t h e two rifamycins has the shape of a composite cathodic-anbdic wave, corresponding to a reversible process. The rifamycins can then be considered, respectively, the oxidized and the reduced form of a IeT-ersible oxidation-reduction system n-ith a potential, polarographically measured, of +0.03 volt us. S.C.E. The polarographic waves are reported in Figures 5 and 6 and described in Table 11. The diffusion current is linearly proportional to concentration between 0 and 5 mmoles per liter for rifamycin 8 and betneen 0 and 4 mnioles per liter
+
Figure 5. Polarograms of rifamycin S and rifamycin ! j V - ~ n * ' ~ f ' / ~= 2.26 mg*?!3set.-'
2
a. 50% methanol and 50% 1M sodium acetate-acetic acid, p H 6.0 b. 1 .E5 mmoles p e r liter of rifamycin S in buffer c. 2.33 mmoles p e r liter o f rifamycin SV in buffer
acid-sodium hydroxide, a t vari-
for rifamycin ST'; both can then be polarographically determined. The fact that the diffusion current constant of the oxidized form (rifamycin S, I = 2.06) is distinctly greater than that of the reduced form (rifamycin SV,I = 1.51) has already been observed for various oxidation-reduction systems (8). This is believed due to the difference between the diffusion coefficients of the two forms. From the slope and the D and n values reported in Table 11, the oxidation-reduction system can be considered reversible and involving two electrons in the electrode process. The determination of D for rifamycin SV was attempted with the porous diaphragm method but unsucccwfully ; in fact, rifamycin SV underwent, during the diffusion period, the transforniation to rifamycin S ( 3 ) ,checked p o l a ~ o -
---
2-y) Y
+02
0 P O T E N T I A L IN
-
_ _
-02
-
6
VOLT ( 5 C E I
VOL. 34, NO. 3, MARCH 1962
425
graphically and spectrophotometrically. The dependence of the composite cathodic-anodic wave on the p H was studied in the interval 4 to 8 in solutions similar to that described above. The shape of the curve remains the same, while the half-wave potential changes, giving a slope of 0.059 volt per pH unit, confirming a reversible process.
LITERATURE CITED
(1) Gallo, G. G., Sensi, P., Radaelli, P., Farmaco, E d . pract. 15, 283 (1960).
(2) Sensi, P., Ballotta, R., Greco, A. M., Farmaco, E d . sci. 15, 228 (1960). (3) Sensi, P., Ballotta, R., Greco, A. M., Gallo, G. G., Zbid., 16, 165 (1961). (4) Sensi, P., Coronelli, C., Nicolaus, B. J. R., J. Chromatog. 5 , 519 (1961). (5) Sensi, P., Greco, A. M., Ballotta, R., Antabaotzcs Ann. 1959-60, 262.
(6) Sensi, P., Margalith, P., Timbal, hl T. Farmaco, E d . sci. 14, 146 (1959). (7) iensi, P., Timbal, M. T., Maffii, G , Ezperientia 16, 412 (1960). (8) Smith, L. I., Kolthoff, I. M., Wawzonek, S., Ruoff, P. M., J. Am. Chem. SOC. 63, 'iois (194i). (9) Stokes, R. H., Zbid., 72,763 (1950). '
RECEIVEDfor review July 24, 1961. Accepted December 26, 1961.
Rapid Spectrophotometric Method for the Determination of Itaconic, Citric, Aconitic, and Fumaric Acids CLARK G. HARTFORD' Miles Chemical Co., Division o f Miles laboratories, Elkhart, ind.
b A rapid, simple spectrophotometric method is presented for the quantitative determination of microgram quantities of itaconic, citric, frans-aconitic, cis-aconitic, and fumaric acids using the Furth and Herrmann reaction. The basic color reaction for citric acid has been extended to the other four acids and to certain combinations of these. However, the method does not distinguish between the aconitic acids and citric acids nor between fumaric and itaconic acids except in a qualitative manner. Absorption vs. wavelength curves illustrate the type and degree of interference between the acids. Beer's law is obeyed except in the case of citric acid and a t higher concentrations, aconitic acid.
F
are available in the literature for the quantitative determination of itaconic and acoiiitic acids. Bromination of the unsaturate is w e d in the majority of cases (4, 7 , Q), while oxidation with permanganate (8) is used in one. The accuracy of bromination has been questioned ( 8 ) , and this author has encountered difficulty in the determination of the end point of the permanganate titration of itaconic and aconitic acid. These methods are not applicable to mixtures of these acids in microgram quantities. Several methods (1, 10, 12-14) for the estimation of citric acid depend on the Furth and Herrmann reaction (6), in which color is formed in the presence of pyridine and acetic anhydride. The method of Marier and Boulet ( I O ) has been used extensively in our laboratories with excellent results. I n using EW METHODS
Present addresa, Bowaters Southern Paper Corp., Calhoun, Tenn. 1
426
ANALYTICAL CHEMISTRY
this method, fumaric, itaconic, transaconitic, &-aconitic, and isocitric are the only substances found which interfere in the microgram range. Godin (6) used this color reaction for the quantitative determination of fumaric, aconitic, itaconic, and citric acids, after preliminary separation by paper chromatography; Diffen (3) used i t in a spot test. The present inveitigation had as its goal the developmcJnt of a rapid niicro quantitative procedure for determining itaconic, trans-aconitic, citric, and fumaric acids separately and in mixtures using the Furth and Herrmann reaction. DEVELOPMENT OF METHOD
UJe of the idcntical conditions given by hlarier and I3oult.t (10) for citric acid gives the mauimum sensitivity and accuracy in the dotrrmination of itaconic, tram-aconitic, cis-aconitic, and fumaric acids. The absorptioii characteristics of the color drvelopcd nith citric and trans-aconitic acids wrre almost identical (Figure 1, a and c), although the greater absorptivity obtained n ith tmns-aconitic acid permits increased sensitivity in its determination. The aconitic acids can be distinguishcd qualitatively from citric by this method. Citric acid, in fact, becomes aconitic acid with the removal of the -OH and -H, and formation of a double bond. Since the double bond is present in aconitic acid, color was developed immediately upon addition of acetic anhydride. Color took several minutes to develop in dilute citric acid solution, although in more concentrated solutions, color develops inimediatcly but
darkens 1% ith time. Cis-aconitic acid has a curve similar to that for transaconitic acid (Figure 1, a and b ) except for the pronounced hump at 407 m p . This hump is absent or only slightly visible in trans-aconitic acid depending on the sample source. The absorption curve of the colored material from itaconic acid (Figure 2. a) has t n o m a s i m a - o n e at 385 m p and a second smaller one a t 407 mg. At 420 mp, the absorption is equivalent to about 1670 of that a t 385 mp, but a t 435 mp there is absorption equivalent to only 1% of that a t 385 mp, Citric and aconitic acid measured at 435 mp gave almost the same value as at 420 mp. Absorbance a t 385 mp for citric and aconitic acid is approximately equivalent to 65T0 and 6 i 7 0 , respectively. of the values read a t 435 m p for a 100-pg. sample. Fumaric acid gave an absorption curve (Figure 2, b) similar in many respects to that of itaconic acid having two peaks a t the same wavelength, 385 mp and 407 mp, but with a shoulder a t 368 mp and a somewhat broader base. Fumaric acid a t 385 mp has only about 4% of the absorbance as itaconic acid a t 385 nip on an equal molar basis. Thc similarity of itaconic and fumaric acid curves was a t first believed due to an itaconic impurity in the fumaric acid. Since itaconic acid is much more soluble than fumaric acid, a sample was recrystallized from n ater. However, this gave a curve idcntical to that of the starting fumaric acid sample. RECOMMENDED METHOD
Marier and Boulet's method ( I O ) , with only slight modification, is recommended.
