- Email: [email protected]
Direct current and differential pulse polarography of penicillamine
J. Electroanal. Chem., 95 (1979) 201--210 © Elsevier Sequoia S.A., Lausanne - - P r i n t e d in The Netherlands
201
DIRECT CURRENT A N D DIFFERENTIAL PULSE POLAROGRAPHY OF PENICILLAMINE
MOHAMMED JEMAL ~ and ADELBERT M. KNEVEL
Department of Medicinal Chemistry and Pharmacognosy, School o f Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Ind. 47907 (U.S.A.) (Received 17th May 1977; in revised form 26th May 1978)
ABSTRACT The direct current and differential pulse polarographic behavior of the sulfhydryl group of penicillamine in 0.056 M citrate buffer of pH 6.45 was investigated. Depending on the concentration of penicillamine, one, two, or three waves were obtained. In most of the concentration regions investigated (1 × 10--5--1 × 10 - 3 M), a linear relationship was found to exist between concentration and the d.c. wave height. In the case of d.p. polarography, a linear relationship was obtained below the 5 x 10 - 4 M level. A study of the effects of mercury pressure on d.c. wave heights, the effects of Triton X-100 on both d.c. and d.p. polarograms, and the analysis o f electrocapillary curves indicates that penicillamine exhibits an adsorption wave. Log plot analysis indicates that the electrode reaction product is RSHg. The calculated d.p. peak current was found to be lower than the experimental current, and the diffusion coefficient of penicUlamine in 0.056 M citrate buffer of pH 6.45 was found to be 7.8 × 10 - 6 cm 2 s- 1 . INTRODUCTION
One of the problems in the use of penicillin as a therapeutic agent is penicillin allergy. Of the various degradation products which can be formed in reconstituted penicillin solutions during recommended periods of storage, penicillamine has been shown to be an antigenic determinant in the penicillin allergic reaction [1--3]. On the other hand, penicillamine is used in therapy as a metal complexing agent and is official in the United States Pharmacopeia XIX
(U.S.P.). There is no report in the literature on the systematic study of the polarographic behavior of peniciUamine. However, Page reported the presence of a catalytic wave in a penicillin preparation inactivated with 0.1 M sodium hydroxide, hydrolyzed in a warm 0.1 M hydrochloric acid and then dissolved in Brdicka ammoniacal cobalt buffer solution [4,5]. The catalytic wave was attributed to the sulfhydryl group o f penicillamine obtained from penicillin. A m e t h o d for analysis of penicillin was developed based on the amperometric titration of the sulfhydryl group of penicillamine which was obtained by hydrolysis of penicillin [6]. The U.S.P. m e t h o d for the assay of penicillamine is based on the titration with mercuric acetate using diphenylcarbazone as the endpoint indicator. Present address: The Squibb Institute for Medical Research, N e w Brunswick, N.J. 0 8 9 0 3 , U.S.A.
202
The purpose of this study is to investigate the direct current (d.c.) and differential pulse (d.p.) polarographic behavior of penicillamine based on the sulfhydryl group. Since parenteral penicillin preparations are usually marketed with citrate buffer so that reconstituted penicillin solutions have a pH of a b o u t 6.5, a 0.056 M citrate buffer of pH 6.45 was used as the supporting electrolyte. EXPERIMENTAL
Apparatus The polarographic instrument used in this research was the PAR Model 174A polarographic analyzer (Princeton Applied Research Corporation, Princeton, N.J.). The polarographic analyzer was used in conjunction with PAR Model 174/70 drop timer. The polarographic cell used was PAR Model 9301 cell bott o m fitted with PAR Model 9300 cell top. All polarographic data were obtained with a three-electrode system. The working electrode was dropping mercury, the counter electrode was a platinum wire, and the reference electrode was a saturated calomel electrode Model 3-712 (Coleman Instruments, Oak Brook, Ill.). The mercury capillary had the following characteristics: in 0.056 M citrate buffer of pH 6.45 at open circuit, the mercury flow rate was 2.57 mg s -1 and the natural drop time was 3.0 s at a mercury reservoir height of 78.6 cm (uncorrected for back pressure). Except when the effect of mercury pressure on wave height was studied, all data were obtained with mercury height of 78.6 cm. Natural drop time was used for all d.c. polarograms and automatic drop time of 2 s was used for all d.p. polarograms. A scan rate of 2 mV s -1 and a pulse amplitude of 25 mV were employed. The low-pass filter was set at 3.0 for d.c. polarography b u t at off for d.p. polarography. Deoxygenation was performed with pre-purified nitrogen. Nitrogen was passed through a two-way stopcock so that it could be directed either into the solution or above the solution as needed. An omnigraphic Model 2000 XY recorder was used (Houston Instrument, Bellaire, Texas).
Materials DL-penicillamine was obtained commercially and was labelled 99 + % (Aldrich Chemical Company, Inc., Milwaukee, Wisc.). Thin-layer chromatography exhibited two spots, one for penicfllamine and the other for penicillamine disulfide (precoated silica gel plates, EM Laboratories, Elmsford, N.Y.; n-butanol-acetic acid-water: 66--17--17; RF values of 0.35 for penicillamine and 0.10 for penicillamine disulfide). Since polarography o f freshly prepared penicillamine solutions did n o t show any penicillamine disulfide, the penicillamine disulfide spot seen on thin-layer chromatography plates was attributed to the oxidation o f penicillamine during the chromatographic development. {Note that penicillamine gives anodic waves as discussed below whereas penicillamine disulfide gives only.cathodic waves at potentials more negative than --1.0 V, depending on pH.) Scintillation grade Triton X-100 was used as obtained commercially (Eastman Kodak Company, Fair Lawn, N.J.).
203
Method
All polarographic experiments were conducted at 22 + 1 ° C. Citrate buffer, 0.056 M at pH 6.45 was prepared with 16.0 g 1-1 of trisodium citrate dihydrate and 0.51 g 1-1 of citric acid m o n o h y d r a t e . Penicillamine stock solutions were prepared in double distilled water. Their concentrations decreased upon keeping and were checked before use by means of d.p. polarography. Penicillamine solution o f the order of 5 × 10 -3 M deteriorated at the rate of 0.5% per hour at 22 + 1 ° C. A 0.6% w/v or 0.1% Triton X-100 stock solution was prepared in double distilled water. RESULTS AND DISCUSSION
A preliminary study of a 1 X 10 -4 M penicillamine solution in buffers of different pH values was conducted to determine the polarographic behavior of this compound. A single, well defined anodic wave of penicillamine was obtained for each buffer investigated. The half-wave potential shifted to a more negative valiJe when the pH was increased as shown in Fig. 1. In the low pH region, the half-wave potential changed linearly with pH whereas in the high pH region the rate o f change with pH was smaller. The slope of the linear portion of the plot is 0.062 V pH -1, which is in good agreement with the theoretically expected value of 0.059 V at 22°C [7]. Typical d.c. and d.p. polarograms o f penicillamine in citrate buffer of pH 6.45 are presented in Fig. 2, where a 5.0 × 10-4 M penicillamine solution
.---4
0.E
04 > --IN bJ I 0.2
I 4
I 8
pH
I 12
-0.5 -0.3
-0.1 "1"0.1 -0.7 - 0 . 5 - 0 . 3 -0.1 "1"0.1
E vs SCE, Volts
Fig. 1. Half-wave potential of 1.0 × 10 --4 M penicillamine versus pH. Buffers used for data in the plot: McIlvaine buffer (2.2--7.8), S~brenson's borate buffer (7.8--9.2), and S~renson's glycine buffer (8.6--12.8). Fig. 2. Typical d.c. and d.p. polarograms o f penicillamine in citrate buffer of pH 6.45. Concentrations of penicillamine: 5.0 X 10 --5 M for (a) and (c); 5.0 X 10 --4 M for (b) and (d). Positive scanning from left to right was done from initial potential of --0.70 V to about +0.2 V. The point of start of scan indicates the zero current axis. The polarographic analyzer was set to display anodic current as if it were cathodic current i.e. positive current.
~204
exhibits three waves whereas a 5.0 X 10 -5 M solution exhibits only one wave. The third wave, which occurred just before the discharge of mercury, started to appear at and above 6.0 X 10 -5 M on d.p. polarograms but it was n o t discernible on d.c. polarograms at such low concentrations. From the fact that the third wave starts to appear at concentrations n o t high enough for the second wave, which occurs at a less positive potential, to appear and from the results of the nature of the first and second waves (see below), it appears t h a t the third wave is n o t of faradaic origin. The d.c. curve of this wave is poorly defined even at high concentrations (Fig. 2d) and is difficult to analyze. The data presented in this paper involve only the first and the second waves. C o n c e n t r a t i o n e f f e c t s o n d.c. p o l a r o g r a p h y
The second wave started to appear at about 1.40 × 10-4 M and then the wave height increased with concentration. Once the second wave appeared, the height o f the first wave (0.53 pA) was independent of concentration. The effects of concentration on the d.c. polarograms of penicillamine are summarized in Table 1, and show that the relationship between concentration and current is roughly linear (i.e. current/concentration is roughly constant) for concentrations of penicillamine which do not give the second wave. For concentrations which give the second wave, linear relationship between concentration and total wave height (i.e. wave height measured at the plateau of the second wave from the residual current) is obtained for concentrations above about 4.0 X 10 -4 M. Penicillamine solutions of concentrations below 4.0 X 10-4 M (and above 1.40 X 10-4 M) gave higher values of current/concentration, because the second wave did n o t form a well defined plateau due to poor resolution from the third wave and hence accurate measurement of wave height was n o t possible. Thus, except for this narrow concentration region where it is difficult to measure wave height, there is a linear relationship between concentra-
TABLE 1 Effects o f concentration o n d.c. p o l a r o g r a p h y o f p e n i c i l l a m i n e (PA) Concentrations o f 1.45 X 10 --4 M and above gave the second wave in a d d i t i o n t o t h e first wave. In t h e s e cases, the wave height was t h e s u m o f t h e h e i g h t s o f t h e first a n d s e c o n d waves; i.e., the height as m e a s u r e d at the p l a t e a u o f the second wave from the extrapolated line o f t h e residual c u r r e n t curve 104 [ P A ] / m o l 1-1
Wave h e i g h t /pA
(Wave h e i g h t / concentration)
E1/2/V (SCE) the first wave
/pA/mM -1 0.110 0.578 0.963 1.45 1.93 3.85 6.74 8.67 11.0
0.044 0.216 0.355 0.816 0.924 1.59 2.63 3.31 4.10
4.00 3.74 3.69 5.63 4.79 4.13 3.90 3.82 3.73
--0.39 --0.42 -0.43 --0.43 -0.44 -0.46 -0.48 -0.49 --0.50
for
205 tion and current when wave height is measured (from the residual current) at the plateau of the second wave for solutions which give the second wave. The half-wave potential of the second wave was nearly independent of concentration. However, the half-wave potential of the first wave shifted to a more negative value when concentration was increased (Table 1 and Fig. 2).
Concentration effects on d.p. polarography As expected from the d.c. polarograms, the d.p. polarograms of penicillamine exhibited the second peak at about 1.4 × 10 -4 M. For penicillamine solutions which give the second peak, the relationship between concentration and the sum of the heights of the two peaks was non-linear and irregular. Therefore, the analysis of the first peak only was pursued. The effects of concentration on the first peak are summarized in Table 2. The peak height increased steadily with concentration until 6.74 × 10-4 M and then started to decrease slightly. It was interesting to note that the height of the first peak kept increasing with concentration even after the second peak appeared (note t h a t in the case of d.c. the first wave remained independent of concentration once the second wave appeared). As can be seen from Table 2, there is a linear relationship between concentration and peak height in the low concentration region. The potential of the second peak was nearly independent of concentration. On the other hand, the potential of the first peak shifted to a more negative value as concentration was increased. Since the current/concentration value for d.p. polarography is a b o u t 10 times higher than that for d.c. polarography, the d.p. technique is more useful for the analysis o f small amounts of penicillamine. The useful analytical region, in which there is a direct proportionality between peak height and concentration, is below the 5 X 10-4 M level. The general behavior of penicillamine d.p. polarography versus concentration is similar to the d.p. polarographic behavior of other sulfhydryl compounds [8--10]. TABLE 2 Analysis of the first d.p. polarographic peak of different concentrations of penicillamine (PA) At 1.93 X 10--4 M and above, the polarograms exhibited the second peak in addition to the first peak. In these cases the data listed belong to the first peak only 104 [PA ]/mol 1-1
0.0250 0.110 0.550 0.963 1.93 4.82 6.74 8.67 11.0
Peak height
(Peak height/
Peak potential
/~A
concentration) /~A/mM-1
V(SCE)
0.113 0.495 2.48 4.36 8.89 20.60 21.83 21.01 19.65
45.2 45.0 45.1 45.3 46.1 42.7 32.4 24.2 17.9
--0.38 -0.40 -0.42 -0.42 -0.44 -0.46 -0.48 -0.49 -0.50
206
Effects o f mercury pressure on d.c. waves The height of the first wave of a peniciUamine solution which gives only a single wave changed in direct proportion to h"°C1/2 This indicated that the first OFt * wave of penicillamine was diffusion controlled as long as the second wave did n o t appear. The height of the first wave of a penicillamine solution which gives the second wave varied in direct relation to hco,r. This indicated that the limiting height (i.e. that height of the first wave which is independent of concentration) is adsorption (or film formation) controlled. The results of the analysis of the total wave height (i.e. the sum of the heights of the first and second waves) in 1/2 relation to mercury pressure show that the total wave height varied as hcorr indicating that it is diffusion controlled.
Diffusion coefficient o f penicillamine The Ilkovic equation was used to determine the diffusion coefficient of penicillamine in a 1.0 × 10 -3 M solution in 0.056 M citrate buffer o f pH 6.45. The current measured at the plateau of the second wave was used (3.73 #A). Drop time of 2.60 s (see Fig. 3) and mercury flow rate of 2.57 mg s -1 were employed. The diffusion coefficient thus calculated was 7.8 X 10 -~ cm 2 s-1.
Drop time curves A comparison of the drop time curves of citrate buffer o f pH 6.45 and 1.0 × 10 -3 M penicillamine solution in citrate buffer of pH 6.45 is presented in Fig. 3. The presence of penicillamine causes depression of drop times at potentials more positive than --0.4 V and this indicates that there is adsorption (or film formation) in this potential region. Since - 0 . 4 V lies at the plateau of the first wave of a 1.0 X 10 -~ M penicillamine solution, this means t h a t there is no depression of drop times in the potential region where penicillamine alone, i.e. w i t h o u t the electrode reaction product, is found in the vicinity of the electrode. In the potential region between - 0 . 4 V and +0.02 V, t h e potential at which the plateau of the second wave begins, both penicillamine
3°E
w
3.2
~ z.8
e
a
2.4
2:
I -o.,
I -oz
E/V
vs.
I -t.3
-,
I
SCE
Fig. 3. Drop time curves. (a) Citrate buffer of pH 6.45, (b) 1.0 X 10-3 M penicillamine in citrate buffer of pH 6.45.
207
and the product are found. It is possible that either one or both cause the depression of drop times in this potential region. The fact that depression of drop times continues b e y o n d +0.02 V, where the concentration of penicillamine at the electrode is virtually zero, indicates that the product is adsorbed on the mercury electrode. Thus the shapes of the drop time curves indicate the presence of adsorption confirming the conclusion made from the study of the effect o f mercury pressure on wave heights.
Effects of Triton X-IO0 The effects of Triton X-100 on the first d.c. wave of a penicillamine solution which does n o t give the second wave are summarized in Table 3. The wave height was only slightly affected by the presence o f Triton X-100. The halfwave potential shifted to a more positive value with increase in Triton X-100 concentration until 0.008% and then remained practically the same with a further increase in Triton X-100 concentration. In the case of a solution giving two waves (4.32 × 10 -4 M), the height of the first wave increased from a value of 0.53 pA to 0.75 pA when 0.003% Triton X-100 was added and then remained independent of a further increase in Triton X-100. The half-wave potential o f the first wave shifted from --0.46 V to --0.36 V in the presence of 0.008% Triton X-100 and then did n o t change with increase in Triton X-100. The total wave height measured at the plateau of the second wave was n o t affected by the presence of Triton X-100 and also the half-wave potential of the second wave did n o t change at all. The effects of Triton X-100 on the first d.p. peak o f a penicillamine solution which does n o t give the second peak are summarized in Table 4. Both the peak height and peak potential were affected. The height and potential of the first peak for a solution giving the second peak were also affected by the presence of Triton X-100. It was generally found t h a t the peak potential of the first peak was shifted by about 0.10 V by presence of a sufficient a m o u n t of Triton X-100 (about 0.008%), but the ratio between peak height in the presence of a given a m o u n t o f Triton X-100 to that in the absence of Triton X-100 changed with the concentration of penicillamine. Neither the height nor the potential of the second peak was affected by Triton X-100. The fact t h a t the first wave of peniciUamine is affected by the presence of Triton X-100 whereas the second wave is n o t indicates that the two waves are of a different nature. TABLE 3 Effects o f T r i t o n X-100 o n the single d.c. wave o f 4.32 X 10 - 5 M penicillamine solution T r i t o n X-100/% W/V
Wave h e i g h t / p A
E1/2/V (SCE)
0 0.003 0.008 0.012 0.020
0.166 0.176 0.160 0.148 0.154
--0.42 --0.34 --0.32 --0.31 --0.32
208 TABLE 4 Effects of Triton X-100 on the single d.p. peak of 4.32 X 10 - 5 M penicillamine solution Triton X-100/% W/V
Peak height/pA
Peak potential/V (SCE)
0 0.003 0.008 0.012 0.020
1.95 2.26 1.24 0.84 1.32
--0.42 --0.35 --0.32 -0.30 -0.32
The shift of the first wave to a more positive potential by Triton X-100 can be explained by theories on adsorption in polarography [11--15]. If the first wave is a facilitated wave due to the adsorption of penicillamine or its electrode reaction product, then the coverage of the dropping mercury electrode by Triton X-100 would prevent adsorption by penicillamine or its reaction product; hence a more positive potential would be required for the electrode reaction to take place. If the second wave is an inhibited wave due to film formation by penicillamine electrode reaction product, then one could imagine that coverage of the dropping mercury electrode by Triton X-100 would cause the same kind of inhibition as the electrode reaction product, and, hence, the first wave would move to a more positive potential. The change of d.p. peak height, especially the increase of it, in the presence of Triton X-100 is difficult to explain. There are a few examples in the literature where surfactants either decreased [16--18] or increased [18] d.p. peak heights but the causes were n o t well investigated.
Log plot analysis To determine the nature of the electrode reaction of penicillamine, 10g plot analysis of the first wave for a solution which does n o t give the second wave was undertaken. As shown in Fig. 4, a plot of E versus log[(id--i)/i] gives a
0.6 ~)
~6 -o.~
g
-I.E .-J
-3,0
~.
I 0.38 - E'/V
I 0.44 v s . SCE
I 0.50
I
Fig. 4. Log plot analysis of 8.65 × 10 - 5 M penicillamine in citrate buffer of pH 6.45. (a) log(id--i)/i VS. E, (b) log(id---i)2/i VS. E, (c) log(/d--/) vs. E.
209
straight line whereas plots of E versus log[(id--i)2/i] and E versus log(/d--/) give curved lines. These results indicate that the electrode reaction p r o d u c t is mercurous mercaptide of the type RSHg [7,19]. The slope of the straight line is 0.06 V, which agrees well with the theoretical value of 0.059 V for a reversible one-electron reaction. The potential at which the straight line passes through zero is --0.42 V, which agrees well with the half-wave potential obtained directly from the polarogram. Thus the electrode reaction of penicillamine is: RSH + H g ~ RSHg + H ÷ + e
where RSH is HS
s CH C I
CHs
H J
C
COOH (penicillamine)
J
NH2
Experimental and calculated peak height In the concentration region where there is proportionality between concentration and peak height, peak height/concentration is equal to 45 pA mM -1 (Table 2). In the low and high concentration regions where d.c. wave height shows proportionality with concentration, wave height/concentration is equal to a b o u t 3.8 #A mM -1 (Table 1). Theoretical peak height/concentration can be calculated from experimental wave height/concentration using the equation [16,20] : ip
•
3r
a -- 1
where ip is the peak current, id is the d.c. diffusion current, r is the drop time, t is the time between pulse application and current measurement; o -exp(~ AEnF/RT), where n is the number of electrons transferred per molecule, F is the faraday constant, R is the molar gas constant, T is absolute temperature, and Z ~ is the pulse amplitude. In the polarographic analyzer PAR Model 174A, t is equal to 48 ms. The peak current/concentration value calculated using the above equation is 3.84 #A mM -1 . In the polarographic analyzer PAR Model 174A, the differential pulse m o d e has a ten-fold gain in the o u t p u t n o t present in the other modes. The peak currents reported in this paper are the inflated values as read from the instrument and, therefore, must be divided by ten to obtain the true values. Thus the experimental peak current/concentration is really 4.5 pA mM -1, which is still larger than the value calculated from the theoretical equation. While an experimentally determined peak current could be lower than theoretical peak current as calculated above due to instrumental artifact and irreversibility [ 16], it is n o t obvious w h y the experimental peak current is higher. The use of d.p. polarography for the simultaneous analysis of penicillamine and benzylpenicillenic acid, another sulfhydryl c o m p o u n d implicated in penicillin allergy and found in parenteral penicillin preparations, will be reported in the future.
210 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
M . A . S c h w a r t z , J. P h a r m . Sci., 5 8 ( 1 9 6 9 ) 6 4 3 . M. J e m a l , S . L . H e m a n d A . M . K n e v e l , J . P h a r m . Sci., 6 7 ( 1 9 7 8 ) 3 0 2 . G.A. Caron, Immunology, 6 (1963) 81. J.E. Page, Analyst, 73 (1948) 214. J . E . P a g e in t h e p a p e r o f E. L e s t e r - S m l t h , A n a l y s t , 7 3 ( 1 9 4 8 ) 1 9 7 . J . G r a f n e t t e r o v a , Clin. C h i m . A c t a , 11 ( 1 9 6 5 ) 1 2 8 . W. S t r i c k s a n d I.M. K o l t h o f f , J . A m e r . C h e m . S o c . , 7 4 ( 1 9 5 2 ) 4 6 4 6 . D . R . C a n t e r f o r d a n d A . S . B u c h a n a n , J. E l e c t r o a n a l . C h e m . , 4 4 ( 1 9 7 3 ) 2 9 1 . D.R. Canterford, J. Electroanal. Chem., 52 (1974) 144. F. P e t e r a n d R . R o s s e t , A n a l . C h i m . A c t a , 7 9 ( 1 9 7 5 ) 4 7 . R . B r d i e k a , Z. E l e k t r o c h e m . , 4 8 ( 1 9 4 2 ) 2 7 8 . O . H . Miiller, T r a n s . E l e c t r o c h e m . S o c . , 8 7 ( 1 9 4 5 ) 4 4 1 . R.E. Benesch and R. Benesch, J. Phys. Chem., 56 (1952) 648. L. J u l i e n a n d M . L . B e r n a r d , R e v . C h i m . Min., 5 ( 1 9 6 8 ) 5 2 1 . D . R . C a n t e r f o r d a n d A . S . B u c h a n a n , J. E l e c t r o a n a l . C h e m . , 4 5 ( 1 9 7 3 ) 1 9 3 . D.J. Myers and J. Osteryoung, Anal. Chem., 45 (1973) 267. K. Hasebe and J. Osteryoung, Anal. Chem., 47 (1975) 2412. E. J a c o b s e n a n d H . L i n d s e t h , A n a l . C h i m . A e t a , 8 6 ( 1 9 7 6 ) 1 2 3 . I.M. K o l t h o f f a n d C. B a r n u m , J . A m a r . C h e m . S o c . , 6 2 ( 1 9 4 0 ) 3 0 6 1 . E.P. P a r r y a n d R . A . O s t e r y o u n g , A n a l . C h e m . , 3 7 ( 1 9 6 5 ) 1 6 3 4 .
201
DIRECT CURRENT A N D DIFFERENTIAL PULSE POLAROGRAPHY OF PENICILLAMINE
MOHAMMED JEMAL ~ and ADELBERT M. KNEVEL
Department of Medicinal Chemistry and Pharmacognosy, School o f Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Ind. 47907 (U.S.A.) (Received 17th May 1977; in revised form 26th May 1978)
ABSTRACT The direct current and differential pulse polarographic behavior of the sulfhydryl group of penicillamine in 0.056 M citrate buffer of pH 6.45 was investigated. Depending on the concentration of penicillamine, one, two, or three waves were obtained. In most of the concentration regions investigated (1 × 10--5--1 × 10 - 3 M), a linear relationship was found to exist between concentration and the d.c. wave height. In the case of d.p. polarography, a linear relationship was obtained below the 5 x 10 - 4 M level. A study of the effects of mercury pressure on d.c. wave heights, the effects of Triton X-100 on both d.c. and d.p. polarograms, and the analysis o f electrocapillary curves indicates that penicillamine exhibits an adsorption wave. Log plot analysis indicates that the electrode reaction product is RSHg. The calculated d.p. peak current was found to be lower than the experimental current, and the diffusion coefficient of penicUlamine in 0.056 M citrate buffer of pH 6.45 was found to be 7.8 × 10 - 6 cm 2 s- 1 . INTRODUCTION
One of the problems in the use of penicillin as a therapeutic agent is penicillin allergy. Of the various degradation products which can be formed in reconstituted penicillin solutions during recommended periods of storage, penicillamine has been shown to be an antigenic determinant in the penicillin allergic reaction [1--3]. On the other hand, penicillamine is used in therapy as a metal complexing agent and is official in the United States Pharmacopeia XIX
(U.S.P.). There is no report in the literature on the systematic study of the polarographic behavior of peniciUamine. However, Page reported the presence of a catalytic wave in a penicillin preparation inactivated with 0.1 M sodium hydroxide, hydrolyzed in a warm 0.1 M hydrochloric acid and then dissolved in Brdicka ammoniacal cobalt buffer solution [4,5]. The catalytic wave was attributed to the sulfhydryl group o f penicillamine obtained from penicillin. A m e t h o d for analysis of penicillin was developed based on the amperometric titration of the sulfhydryl group of penicillamine which was obtained by hydrolysis of penicillin [6]. The U.S.P. m e t h o d for the assay of penicillamine is based on the titration with mercuric acetate using diphenylcarbazone as the endpoint indicator. Present address: The Squibb Institute for Medical Research, N e w Brunswick, N.J. 0 8 9 0 3 , U.S.A.
202
The purpose of this study is to investigate the direct current (d.c.) and differential pulse (d.p.) polarographic behavior of penicillamine based on the sulfhydryl group. Since parenteral penicillin preparations are usually marketed with citrate buffer so that reconstituted penicillin solutions have a pH of a b o u t 6.5, a 0.056 M citrate buffer of pH 6.45 was used as the supporting electrolyte. EXPERIMENTAL
Apparatus The polarographic instrument used in this research was the PAR Model 174A polarographic analyzer (Princeton Applied Research Corporation, Princeton, N.J.). The polarographic analyzer was used in conjunction with PAR Model 174/70 drop timer. The polarographic cell used was PAR Model 9301 cell bott o m fitted with PAR Model 9300 cell top. All polarographic data were obtained with a three-electrode system. The working electrode was dropping mercury, the counter electrode was a platinum wire, and the reference electrode was a saturated calomel electrode Model 3-712 (Coleman Instruments, Oak Brook, Ill.). The mercury capillary had the following characteristics: in 0.056 M citrate buffer of pH 6.45 at open circuit, the mercury flow rate was 2.57 mg s -1 and the natural drop time was 3.0 s at a mercury reservoir height of 78.6 cm (uncorrected for back pressure). Except when the effect of mercury pressure on wave height was studied, all data were obtained with mercury height of 78.6 cm. Natural drop time was used for all d.c. polarograms and automatic drop time of 2 s was used for all d.p. polarograms. A scan rate of 2 mV s -1 and a pulse amplitude of 25 mV were employed. The low-pass filter was set at 3.0 for d.c. polarography b u t at off for d.p. polarography. Deoxygenation was performed with pre-purified nitrogen. Nitrogen was passed through a two-way stopcock so that it could be directed either into the solution or above the solution as needed. An omnigraphic Model 2000 XY recorder was used (Houston Instrument, Bellaire, Texas).
Materials DL-penicillamine was obtained commercially and was labelled 99 + % (Aldrich Chemical Company, Inc., Milwaukee, Wisc.). Thin-layer chromatography exhibited two spots, one for penicfllamine and the other for penicillamine disulfide (precoated silica gel plates, EM Laboratories, Elmsford, N.Y.; n-butanol-acetic acid-water: 66--17--17; RF values of 0.35 for penicillamine and 0.10 for penicillamine disulfide). Since polarography o f freshly prepared penicillamine solutions did n o t show any penicillamine disulfide, the penicillamine disulfide spot seen on thin-layer chromatography plates was attributed to the oxidation o f penicillamine during the chromatographic development. {Note that penicillamine gives anodic waves as discussed below whereas penicillamine disulfide gives only.cathodic waves at potentials more negative than --1.0 V, depending on pH.) Scintillation grade Triton X-100 was used as obtained commercially (Eastman Kodak Company, Fair Lawn, N.J.).
203
Method
All polarographic experiments were conducted at 22 + 1 ° C. Citrate buffer, 0.056 M at pH 6.45 was prepared with 16.0 g 1-1 of trisodium citrate dihydrate and 0.51 g 1-1 of citric acid m o n o h y d r a t e . Penicillamine stock solutions were prepared in double distilled water. Their concentrations decreased upon keeping and were checked before use by means of d.p. polarography. Penicillamine solution o f the order of 5 × 10 -3 M deteriorated at the rate of 0.5% per hour at 22 + 1 ° C. A 0.6% w/v or 0.1% Triton X-100 stock solution was prepared in double distilled water. RESULTS AND DISCUSSION
A preliminary study of a 1 X 10 -4 M penicillamine solution in buffers of different pH values was conducted to determine the polarographic behavior of this compound. A single, well defined anodic wave of penicillamine was obtained for each buffer investigated. The half-wave potential shifted to a more negative valiJe when the pH was increased as shown in Fig. 1. In the low pH region, the half-wave potential changed linearly with pH whereas in the high pH region the rate o f change with pH was smaller. The slope of the linear portion of the plot is 0.062 V pH -1, which is in good agreement with the theoretically expected value of 0.059 V at 22°C [7]. Typical d.c. and d.p. polarograms o f penicillamine in citrate buffer of pH 6.45 are presented in Fig. 2, where a 5.0 × 10-4 M penicillamine solution
.---4
0.E
04 > --IN bJ I 0.2
I 4
I 8
pH
I 12
-0.5 -0.3
-0.1 "1"0.1 -0.7 - 0 . 5 - 0 . 3 -0.1 "1"0.1
E vs SCE, Volts
Fig. 1. Half-wave potential of 1.0 × 10 --4 M penicillamine versus pH. Buffers used for data in the plot: McIlvaine buffer (2.2--7.8), S~brenson's borate buffer (7.8--9.2), and S~renson's glycine buffer (8.6--12.8). Fig. 2. Typical d.c. and d.p. polarograms o f penicillamine in citrate buffer of pH 6.45. Concentrations of penicillamine: 5.0 X 10 --5 M for (a) and (c); 5.0 X 10 --4 M for (b) and (d). Positive scanning from left to right was done from initial potential of --0.70 V to about +0.2 V. The point of start of scan indicates the zero current axis. The polarographic analyzer was set to display anodic current as if it were cathodic current i.e. positive current.
~204
exhibits three waves whereas a 5.0 X 10 -5 M solution exhibits only one wave. The third wave, which occurred just before the discharge of mercury, started to appear at and above 6.0 X 10 -5 M on d.p. polarograms but it was n o t discernible on d.c. polarograms at such low concentrations. From the fact that the third wave starts to appear at concentrations n o t high enough for the second wave, which occurs at a less positive potential, to appear and from the results of the nature of the first and second waves (see below), it appears t h a t the third wave is n o t of faradaic origin. The d.c. curve of this wave is poorly defined even at high concentrations (Fig. 2d) and is difficult to analyze. The data presented in this paper involve only the first and the second waves. C o n c e n t r a t i o n e f f e c t s o n d.c. p o l a r o g r a p h y
The second wave started to appear at about 1.40 × 10-4 M and then the wave height increased with concentration. Once the second wave appeared, the height o f the first wave (0.53 pA) was independent of concentration. The effects of concentration on the d.c. polarograms of penicillamine are summarized in Table 1, and show that the relationship between concentration and current is roughly linear (i.e. current/concentration is roughly constant) for concentrations of penicillamine which do not give the second wave. For concentrations which give the second wave, linear relationship between concentration and total wave height (i.e. wave height measured at the plateau of the second wave from the residual current) is obtained for concentrations above about 4.0 X 10 -4 M. Penicillamine solutions of concentrations below 4.0 X 10-4 M (and above 1.40 X 10-4 M) gave higher values of current/concentration, because the second wave did n o t form a well defined plateau due to poor resolution from the third wave and hence accurate measurement of wave height was n o t possible. Thus, except for this narrow concentration region where it is difficult to measure wave height, there is a linear relationship between concentra-
TABLE 1 Effects o f concentration o n d.c. p o l a r o g r a p h y o f p e n i c i l l a m i n e (PA) Concentrations o f 1.45 X 10 --4 M and above gave the second wave in a d d i t i o n t o t h e first wave. In t h e s e cases, the wave height was t h e s u m o f t h e h e i g h t s o f t h e first a n d s e c o n d waves; i.e., the height as m e a s u r e d at the p l a t e a u o f the second wave from the extrapolated line o f t h e residual c u r r e n t curve 104 [ P A ] / m o l 1-1
Wave h e i g h t /pA
(Wave h e i g h t / concentration)
E1/2/V (SCE) the first wave
/pA/mM -1 0.110 0.578 0.963 1.45 1.93 3.85 6.74 8.67 11.0
0.044 0.216 0.355 0.816 0.924 1.59 2.63 3.31 4.10
4.00 3.74 3.69 5.63 4.79 4.13 3.90 3.82 3.73
--0.39 --0.42 -0.43 --0.43 -0.44 -0.46 -0.48 -0.49 --0.50
for
205 tion and current when wave height is measured (from the residual current) at the plateau of the second wave for solutions which give the second wave. The half-wave potential of the second wave was nearly independent of concentration. However, the half-wave potential of the first wave shifted to a more negative value when concentration was increased (Table 1 and Fig. 2).
Concentration effects on d.p. polarography As expected from the d.c. polarograms, the d.p. polarograms of penicillamine exhibited the second peak at about 1.4 × 10 -4 M. For penicillamine solutions which give the second peak, the relationship between concentration and the sum of the heights of the two peaks was non-linear and irregular. Therefore, the analysis of the first peak only was pursued. The effects of concentration on the first peak are summarized in Table 2. The peak height increased steadily with concentration until 6.74 × 10-4 M and then started to decrease slightly. It was interesting to note that the height of the first peak kept increasing with concentration even after the second peak appeared (note t h a t in the case of d.c. the first wave remained independent of concentration once the second wave appeared). As can be seen from Table 2, there is a linear relationship between concentration and peak height in the low concentration region. The potential of the second peak was nearly independent of concentration. On the other hand, the potential of the first peak shifted to a more negative value as concentration was increased. Since the current/concentration value for d.p. polarography is a b o u t 10 times higher than that for d.c. polarography, the d.p. technique is more useful for the analysis o f small amounts of penicillamine. The useful analytical region, in which there is a direct proportionality between peak height and concentration, is below the 5 X 10-4 M level. The general behavior of penicillamine d.p. polarography versus concentration is similar to the d.p. polarographic behavior of other sulfhydryl compounds [8--10]. TABLE 2 Analysis of the first d.p. polarographic peak of different concentrations of penicillamine (PA) At 1.93 X 10--4 M and above, the polarograms exhibited the second peak in addition to the first peak. In these cases the data listed belong to the first peak only 104 [PA ]/mol 1-1
0.0250 0.110 0.550 0.963 1.93 4.82 6.74 8.67 11.0
Peak height
(Peak height/
Peak potential
/~A
concentration) /~A/mM-1
V(SCE)
0.113 0.495 2.48 4.36 8.89 20.60 21.83 21.01 19.65
45.2 45.0 45.1 45.3 46.1 42.7 32.4 24.2 17.9
--0.38 -0.40 -0.42 -0.42 -0.44 -0.46 -0.48 -0.49 -0.50
206
Effects o f mercury pressure on d.c. waves The height of the first wave of a peniciUamine solution which gives only a single wave changed in direct proportion to h"°C1/2 This indicated that the first OFt * wave of penicillamine was diffusion controlled as long as the second wave did n o t appear. The height of the first wave of a penicillamine solution which gives the second wave varied in direct relation to hco,r. This indicated that the limiting height (i.e. that height of the first wave which is independent of concentration) is adsorption (or film formation) controlled. The results of the analysis of the total wave height (i.e. the sum of the heights of the first and second waves) in 1/2 relation to mercury pressure show that the total wave height varied as hcorr indicating that it is diffusion controlled.
Diffusion coefficient o f penicillamine The Ilkovic equation was used to determine the diffusion coefficient of penicillamine in a 1.0 × 10 -3 M solution in 0.056 M citrate buffer o f pH 6.45. The current measured at the plateau of the second wave was used (3.73 #A). Drop time of 2.60 s (see Fig. 3) and mercury flow rate of 2.57 mg s -1 were employed. The diffusion coefficient thus calculated was 7.8 X 10 -~ cm 2 s-1.
Drop time curves A comparison of the drop time curves of citrate buffer o f pH 6.45 and 1.0 × 10 -3 M penicillamine solution in citrate buffer of pH 6.45 is presented in Fig. 3. The presence of penicillamine causes depression of drop times at potentials more positive than --0.4 V and this indicates that there is adsorption (or film formation) in this potential region. Since - 0 . 4 V lies at the plateau of the first wave of a 1.0 X 10 -~ M penicillamine solution, this means t h a t there is no depression of drop times in the potential region where penicillamine alone, i.e. w i t h o u t the electrode reaction product, is found in the vicinity of the electrode. In the potential region between - 0 . 4 V and +0.02 V, t h e potential at which the plateau of the second wave begins, both penicillamine
3°E
w
3.2
~ z.8
e
a
2.4
2:
I -o.,
I -oz
E/V
vs.
I -t.3
-,
I
SCE
Fig. 3. Drop time curves. (a) Citrate buffer of pH 6.45, (b) 1.0 X 10-3 M penicillamine in citrate buffer of pH 6.45.
207
and the product are found. It is possible that either one or both cause the depression of drop times in this potential region. The fact that depression of drop times continues b e y o n d +0.02 V, where the concentration of penicillamine at the electrode is virtually zero, indicates that the product is adsorbed on the mercury electrode. Thus the shapes of the drop time curves indicate the presence of adsorption confirming the conclusion made from the study of the effect o f mercury pressure on wave heights.
Effects of Triton X-IO0 The effects of Triton X-100 on the first d.c. wave of a penicillamine solution which does n o t give the second wave are summarized in Table 3. The wave height was only slightly affected by the presence o f Triton X-100. The halfwave potential shifted to a more positive value with increase in Triton X-100 concentration until 0.008% and then remained practically the same with a further increase in Triton X-100 concentration. In the case of a solution giving two waves (4.32 × 10 -4 M), the height of the first wave increased from a value of 0.53 pA to 0.75 pA when 0.003% Triton X-100 was added and then remained independent of a further increase in Triton X-100. The half-wave potential o f the first wave shifted from --0.46 V to --0.36 V in the presence of 0.008% Triton X-100 and then did n o t change with increase in Triton X-100. The total wave height measured at the plateau of the second wave was n o t affected by the presence of Triton X-100 and also the half-wave potential of the second wave did n o t change at all. The effects of Triton X-100 on the first d.p. peak o f a penicillamine solution which does n o t give the second peak are summarized in Table 4. Both the peak height and peak potential were affected. The height and potential of the first peak for a solution giving the second peak were also affected by the presence of Triton X-100. It was generally found t h a t the peak potential of the first peak was shifted by about 0.10 V by presence of a sufficient a m o u n t of Triton X-100 (about 0.008%), but the ratio between peak height in the presence of a given a m o u n t o f Triton X-100 to that in the absence of Triton X-100 changed with the concentration of penicillamine. Neither the height nor the potential of the second peak was affected by Triton X-100. The fact t h a t the first wave of peniciUamine is affected by the presence of Triton X-100 whereas the second wave is n o t indicates that the two waves are of a different nature. TABLE 3 Effects o f T r i t o n X-100 o n the single d.c. wave o f 4.32 X 10 - 5 M penicillamine solution T r i t o n X-100/% W/V
Wave h e i g h t / p A
E1/2/V (SCE)
0 0.003 0.008 0.012 0.020
0.166 0.176 0.160 0.148 0.154
--0.42 --0.34 --0.32 --0.31 --0.32
208 TABLE 4 Effects of Triton X-100 on the single d.p. peak of 4.32 X 10 - 5 M penicillamine solution Triton X-100/% W/V
Peak height/pA
Peak potential/V (SCE)
0 0.003 0.008 0.012 0.020
1.95 2.26 1.24 0.84 1.32
--0.42 --0.35 --0.32 -0.30 -0.32
The shift of the first wave to a more positive potential by Triton X-100 can be explained by theories on adsorption in polarography [11--15]. If the first wave is a facilitated wave due to the adsorption of penicillamine or its electrode reaction product, then the coverage of the dropping mercury electrode by Triton X-100 would prevent adsorption by penicillamine or its reaction product; hence a more positive potential would be required for the electrode reaction to take place. If the second wave is an inhibited wave due to film formation by penicillamine electrode reaction product, then one could imagine that coverage of the dropping mercury electrode by Triton X-100 would cause the same kind of inhibition as the electrode reaction product, and, hence, the first wave would move to a more positive potential. The change of d.p. peak height, especially the increase of it, in the presence of Triton X-100 is difficult to explain. There are a few examples in the literature where surfactants either decreased [16--18] or increased [18] d.p. peak heights but the causes were n o t well investigated.
Log plot analysis To determine the nature of the electrode reaction of penicillamine, 10g plot analysis of the first wave for a solution which does n o t give the second wave was undertaken. As shown in Fig. 4, a plot of E versus log[(id--i)/i] gives a
0.6 ~)
~6 -o.~
g
-I.E .-J
-3,0
~.
I 0.38 - E'/V
I 0.44 v s . SCE
I 0.50
I
Fig. 4. Log plot analysis of 8.65 × 10 - 5 M penicillamine in citrate buffer of pH 6.45. (a) log(id--i)/i VS. E, (b) log(id---i)2/i VS. E, (c) log(/d--/) vs. E.
209
straight line whereas plots of E versus log[(id--i)2/i] and E versus log(/d--/) give curved lines. These results indicate that the electrode reaction p r o d u c t is mercurous mercaptide of the type RSHg [7,19]. The slope of the straight line is 0.06 V, which agrees well with the theoretical value of 0.059 V for a reversible one-electron reaction. The potential at which the straight line passes through zero is --0.42 V, which agrees well with the half-wave potential obtained directly from the polarogram. Thus the electrode reaction of penicillamine is: RSH + H g ~ RSHg + H ÷ + e
where RSH is HS
s CH C I
CHs
H J
C
COOH (penicillamine)
J
NH2
Experimental and calculated peak height In the concentration region where there is proportionality between concentration and peak height, peak height/concentration is equal to 45 pA mM -1 (Table 2). In the low and high concentration regions where d.c. wave height shows proportionality with concentration, wave height/concentration is equal to a b o u t 3.8 #A mM -1 (Table 1). Theoretical peak height/concentration can be calculated from experimental wave height/concentration using the equation [16,20] : ip
•
3r
a -- 1
where ip is the peak current, id is the d.c. diffusion current, r is the drop time, t is the time between pulse application and current measurement; o -exp(~ AEnF/RT), where n is the number of electrons transferred per molecule, F is the faraday constant, R is the molar gas constant, T is absolute temperature, and Z ~ is the pulse amplitude. In the polarographic analyzer PAR Model 174A, t is equal to 48 ms. The peak current/concentration value calculated using the above equation is 3.84 #A mM -1 . In the polarographic analyzer PAR Model 174A, the differential pulse m o d e has a ten-fold gain in the o u t p u t n o t present in the other modes. The peak currents reported in this paper are the inflated values as read from the instrument and, therefore, must be divided by ten to obtain the true values. Thus the experimental peak current/concentration is really 4.5 pA mM -1, which is still larger than the value calculated from the theoretical equation. While an experimentally determined peak current could be lower than theoretical peak current as calculated above due to instrumental artifact and irreversibility [ 16], it is n o t obvious w h y the experimental peak current is higher. The use of d.p. polarography for the simultaneous analysis of penicillamine and benzylpenicillenic acid, another sulfhydryl c o m p o u n d implicated in penicillin allergy and found in parenteral penicillin preparations, will be reported in the future.
210 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
M . A . S c h w a r t z , J. P h a r m . Sci., 5 8 ( 1 9 6 9 ) 6 4 3 . M. J e m a l , S . L . H e m a n d A . M . K n e v e l , J . P h a r m . Sci., 6 7 ( 1 9 7 8 ) 3 0 2 . G.A. Caron, Immunology, 6 (1963) 81. J.E. Page, Analyst, 73 (1948) 214. J . E . P a g e in t h e p a p e r o f E. L e s t e r - S m l t h , A n a l y s t , 7 3 ( 1 9 4 8 ) 1 9 7 . J . G r a f n e t t e r o v a , Clin. C h i m . A c t a , 11 ( 1 9 6 5 ) 1 2 8 . W. S t r i c k s a n d I.M. K o l t h o f f , J . A m e r . C h e m . S o c . , 7 4 ( 1 9 5 2 ) 4 6 4 6 . D . R . C a n t e r f o r d a n d A . S . B u c h a n a n , J. E l e c t r o a n a l . C h e m . , 4 4 ( 1 9 7 3 ) 2 9 1 . D.R. Canterford, J. Electroanal. Chem., 52 (1974) 144. F. P e t e r a n d R . R o s s e t , A n a l . C h i m . A c t a , 7 9 ( 1 9 7 5 ) 4 7 . R . B r d i e k a , Z. E l e k t r o c h e m . , 4 8 ( 1 9 4 2 ) 2 7 8 . O . H . Miiller, T r a n s . E l e c t r o c h e m . S o c . , 8 7 ( 1 9 4 5 ) 4 4 1 . R.E. Benesch and R. Benesch, J. Phys. Chem., 56 (1952) 648. L. J u l i e n a n d M . L . B e r n a r d , R e v . C h i m . Min., 5 ( 1 9 6 8 ) 5 2 1 . D . R . C a n t e r f o r d a n d A . S . B u c h a n a n , J. E l e c t r o a n a l . C h e m . , 4 5 ( 1 9 7 3 ) 1 9 3 . D.J. Myers and J. Osteryoung, Anal. Chem., 45 (1973) 267. K. Hasebe and J. Osteryoung, Anal. Chem., 47 (1975) 2412. E. J a c o b s e n a n d H . L i n d s e t h , A n a l . C h i m . A e t a , 8 6 ( 1 9 7 6 ) 1 2 3 . I.M. K o l t h o f f a n d C. B a r n u m , J . A m a r . C h e m . S o c . , 6 2 ( 1 9 4 0 ) 3 0 6 1 . E.P. P a r r y a n d R . A . O s t e r y o u n g , A n a l . C h e m . , 3 7 ( 1 9 6 5 ) 1 6 3 4 .