ANALYST. J U N E 1984. VOL. 109 759

Electroanalytical Studies of Certain Nitro and Benzodiazepine Drugs at Rotating Disc Electrodes

Edmund Bishop and Waqar Hussein* University of Exeter, Chemistry Department, Stocker Road, Exeter, EX4 4QD, UK

~~

Nitro groups have proved to be diversely reducible, best at gold electrodes in alkaline media, the immunosuppressive azathioprine by one or two steps to the hydroxylamine, the antiamoebic metronidazole by two steps to the amine, and the hypnotic nitrazepam by a single step to the amine. Neither the 1,2-carbon-nitrogen double bond, nor the N-oxide group in diazepines, was active at solid electrodes. Rapid voltammetric determination of the nitro-compounds at gold electrodes was effective and gave a mean relative standard deviation of 1 %. Electrode kinetic parameters have been determined for the nitro compounds. Cyclic voltammetry of azathioprine was uninformative.

Keywords: Pharmaceutical nitro compounds and benzodiazepines; rotating disc electrode voltammetry; cyclic voltammetry; electrode kinetic parameters; reaction mechanisms

Nitro compounds are not particularly common among phar- maceuticals, but examples are to be found in many classes of drug, and nitration is often used as a preliminary step in polarographic determination. The examples studied are drawn from widely prescribed drugs of different classes; all have been reduced at mercury electrodes, but no investiga- tions at solid electrodes have been reported. Nitrazepam is a benzodiazepine, and benzodiazepines are reducible at mer- cury electrodes through the carbon-nitrogen double bond, but no examination at solid electrodes or by anodic oxidation has appeared in the literature.

Azathioprine is the prime drug in tissue survival, organ transplants and autoimmune disorder. It is metabolised to 6-mercaptopurine, itself commonly used as an antineoplastic agent, especially in acute leukaemia in children. It has been determined by polarographic reduction,1.2 but the reaction is undefined. Other methods include spectrophotometry,3.4 fluorimetry,jT6 titrimetry,7 TLC,2.3,8 HPLC9-'7 and ion- exchange chromatography. 18 Metronidazole is effective against protozoal and anaerobic bacterial infection; it is active in both intestinal and extra-intestinal amoebiasis, but is not cardiotoxic, and is the drug of choice in severe amoebiasis. It and related compounds have been determined at the dropping-mercury electrode,19-21 but the number of electrons and the reaction mechanism remain undefined. Other methods of determination include spectrophotometry,22-24 GLC,25-27 HPLC2S31 and TLC.32-34 Nitrazepam is a note- worthy night-time hypnotic, with a wider safety margin in overdosage, and less depressant than other hypnotics (although dependence may occur after prolonged use, this is less pronounced than with barbiturates to which nitrazepam is preferred). Considerable interest has been displayed in the polarographic reduction of nitrazepam and other benzodiaze- pine^.^'-^^ Reduction of the carbon-nitrogen double bond at the 1,2-position in the diazepine ring at mercury consumes two electrons, and two- or four-electron reduction of the nitro group is reported. A search for. the first reaction at solid electrodes was, therefore, apposite.

Experimental The rotating electrode assembly and its associated electronics, electrode activation, voltammetric and coulometric proce- dures, other apparatus, glassware and solution manipulation and deoxygenation have been described.44 The samples, supplied by the manufacturers listed in Table 1, were of Drug

* Present address: Department of Pharmaceutics, Faculty of Pharmacy, University of Karachi, Karachi-32, Pakistan.

Standard grade: chlordiazepoxide, supplied as the hydro- chloride, was converted into sulphate as previously des- ~ r i b e d . ~ ~ Alkaline solutions of nitro compounds were pre- pared by dissolution in 5 ml of ethanol and dilution to volume with an appropriate sodium carbonate solution to give the required concentrations of drug and base. The standard scan speed was 5 mV s-1.

Results and Discussion None of the compounds listed in Table 1 showed anodic activity at either gold or platinum electrodes within the solvent potential range in media ranging from 0.1 mol 1-1 sulphuric acid to 0.1 mol 1-1 sodium carbonate. Nor did any of the diazepines show any cathodic activity assignable to the carbon-nitrogen double bond in the diazepine ring at either gold or platinum electrodes over a similar range of conditions. The N-oxide group in chlordiazepoxide is also inactive at solid electrodes.

Voltammetry of the Nitro Compounds

In 0.1 mol 1-1 sodium carbonate, azathioprine gave a single cathodic wave at a platinum electrode but, as shown in Fig. l(a), the wave is ill-defined and progressively merges with the solvent wave as the rotation speed increases. At a gold electrode the background wave is favourably moved to more negative potentials, so revealing reasonably well formed single waves at higher concentrations, Fig. l ( b ) , while at lower concentrations, Fig. l(c), a second wave appears at lower rotation speeds, but merges with the solvent wave as the rotation speed is increased. That the electrode process is not affected by adsorption is demonstrated by the curves, B, obtained by repeating the scan at 50 Hz without intervening activation of the electrode, and C for 10 Hz. There is no loss in wave height, but the second wave disappears at lower concentrations. The limiting current versus square root of frequency plots for the first wave have reasonable intercepts and suffer a change of slope at higher rotation speeds. The separate limiting current versus concentration plots for the two ranges of 0-10-3 and 1-10 x 10-3 mol 1-1 show zero intercepts and excellent linearity, but the slopes are smaller for the higher concentration range. Azathioprine in alkaline solution therefore obeys the Levich relationship at gold electrodes. No useful voltammograms were obtained in acidic media.

Metronidazole did not give a cathodic wave at a platinum electrode in acidic media, but did so at a gold electrode, Fig. 2(a). Although Levich behaviour was displayed at low

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760 ANALYST, JUNE 1984, VOL. 109

Table 1. Compounds examined

Proprietary name

Imuran

C.A. number Generic name Structure Batch number Manufacturer

446-86-6 Azathioprine A N 0 40583 Wellcome Foundation

0 2 N PCH3

CH3 I

OsN

443-48- 1 Metronidazole LN 6 May and Baker Flagyl Ltd.

146-22-5 Nitrazepam Mogadon 0768936 Roche Products 0 H //

02N q- -N

58-25-3 Chlordiazepoxide NHCH3 0808303 Roche Products hydrochloride

CI q 2 * H c l L

CsH5 0

Librium

439-14-5 Diazepam 0905376 Roche Products YH3 0

Valium

-N

84649-1 Lorazepam 652 D Wyeth Acivan Laboratories

604-75-1 Oxazepam Serenid-D Serenid Forte

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ANALYST, JUNE 1984, VOL. 109 76 1

0.4 Q E 2 0.8 .

1.2

1.6

E N vs. S.C.E.

Fig. 1. Voltammograms of various concentrations of azathioprine in 0.1 mol 1-1 sodium carbonate. (a ) 2 x 10-3 rnol 1 - 1 at platinum.; (6) 5 X mol 1 - I at gold; and ( c ) 1 x 10-? mol I - ' at gold electrodes. Curve B, rescan at 50 Hz without intervening reactivation of electrode; C , the same at 10 Hz; and 1-5 denote rotation speeds of 10, 20, 30, 40 and SO Hz. Electrode area, 0.503 cm2; scan speed. 5 mV s l ; temperature, 25 "C; and solutions purged for 6 h with oxygen-free nitrogen

0

0.8

a 1.6 E . u -

2.4

3.2

0.4 0 - 0.4

0

0.5

1 .o

1.5

2.0 -0.8 -0.4 -0.6 -0.8 -1.0

E N vs. S.C.E.

0

1.6

3.2

4.8

6.4

.o

Fig. 2. ( b ) and (c) 2 x 10-3 mol 1 - 1 in 0.1 mol 1 1 sodium carbonate at ( b ) platinum and (c) gold electrodes. Conditions as in Fig. 1

Voltammograms of various concentrations of metronidazole. (a ) 5 X lop3 mol 1 - ' in 0.1 mol 1-' sulphuric acid at gold;

concentrations and rotation speeds, the crossing of the voltammograms did not encourage further investigation. However, the isospotential point, at which current and potential become independent of rotation speed, is of interest as an example of the alpha-kink predicted t h e ~ r e t i c a l l y ~ ~ but not hitherto observed experimentally. In alkaline media, cathodic waves were produced at a platinum electrode, Fig. 2(6), and moved to more negative potentials with increasing rotation speed or concentration, eventually merging with the solvent wave. The Levich plots were non-linear, and there is a high and unexplained standing current at pre-wave potentials; the system is, therefore, not useful for quantitative purposes. Gold electrodes in 0.1 moll- ' sodium carbonate afforded the best results. Fig. 2(c) shows the waves produced by 2 x 10-3 rnol 1-1 metronidazole. Potentials move to more negative values as the rotation speed or concentration increases, the second wave moving more rapidly than the first. Both Levich plots are good for each wave, and the best results came from the summation limiting currents of the two waves.

Nitrazepam gave no reduction wave in acidic media at platinum electrodes. A single wave appeared at gold elec- trodes, Fig. 3(a) , well defined at lower rotation speeds, but with movement to more negative potentials at higher frequen- cies and concentrations and intersection as with metronidaz- ole, but without forming an alpha-kink. Conditional obe- dience to the Levich relationship was shown if the limiting currents were measured at a fixed potential. In 0.1 rnol I - '

sodium carbonate solution, defective voltammograms were produced at platinum electrodes, Fig. 3(b), with high initial currents, again as with metronidazole. Limiting currents did not show Levich dependence. A single good wave was produced at gold electrodes, Fig. 3(c) , with limiting currents in excellent accord with the Levich relationship. A yellow precipitate appeared and increased in amount on progressive scanning.

No previous work at solid electrodes has been located, and most of that at mercury electrodes has been qualitative or empirical. Determination of an-values35 is not helpful; two waves for diazepoxides38 have been found each to be two-electron, the first reduction of the N-oxide and the second reduction of the carbon-nitrogen double bond. Two waves reported for nitrazepam39 were assigned to a four-electron reduction of the nitro group to the hydroxylamine followed by the two-electron reduction of the double bond. Other polarographic work on nitro compounds includes a report46 of a single wave in more concentrated solutions splitting into two waves in more dilute solution, the first of four and the second of two electrons, as for nitrazepam at mercury,3~ and perhaps azathioprine in this work, but not for nitrazepam at solid electrodes. The first wave has been claimed47 to be more useful in polarographic determination of nitro compounds, which is the case for azathioprine here. A two-step reaction has been postulated21 for metronidazole, the first four- electron reduction to the hydroxylamine and the second two-electron reduction to the amine, in pH 9 buffer solution.

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762 ANALYST, JUNE 1984. VOL. 109

0

0.5

1 .o

6 5 1.5 - 2.0

2.5

3.0 0 -0.2 -0.4 -0.6 -0.8 -1.0

0

0.5

1 .o

1.5

2.0 -0.5 -0.7 -0.9 -1.

EIV vs. S.C.E.

0

2.0

4.0

6.0

8.0

-0.4 -0.8 -1.2 -1.6 -2.0

Fig. 3. Voltammograms of various concentrations of nitrazepam. (a ) 2 X 10W rnol 1-1 in 0.1 mol 1-1 sulphuric acid at gold; ( b ) 2 x 10-3 rnol 1-1 in 0.1 mol 1-1 sodium carbonate at platinum; and (c) 5 X 10V3 mol 1-1 in 0.1 rnol 1 1 sodium carbonate at gold electrodes. Conditions as in Fig. 1

-2.0 4 E .

-1.0

0

2.0

4.0 Q E 1 -u

6.0

8.0

I I

+0.4 0 -0.4 -0.8

-1.0

0

2.0

4.0

6.0

8.C

I

+0.4 0 -0.4 -0.8 EiV vs. S.C.E.

Fig. 4. without intervening activation of the electrode

Cyclic voltammograms of 5 x 10-3 rnol 1-1 azathioprine in 0.1 mol 1-1 sodium carbonate at a stationary gold electrode. B, Recycle

Analytical Validity

Analyses of calibration results for the two ranges of azathio- prine, the two waves of metronidazole and the two media for nitrazepam at gold electrodes are given in Table 2. The calibrations are satisfactory despite the large intercepts for metronidazole. Platinum electrodes are unsuitable. Further, to test the voltammetric method, sets of four solutions of the Drug Standards were prepared and the limiting currents measured at each of five rotation speeds at activated gold electrodes, the concentrations calculated from slope and intercept, and the results examined statistically. Each set of measurements was completed in 10 min including activation of the electrode. The results, involving propagation of error from five calibrations, are shown in Table 3. The mean relative standard deviation of the 24 results is just over 1%. The rapid voltammetric method is, therefore, satisfactory.

Electrode Kinetics

The half-wave potentials, mass and charge-transfer rate constants and charge-transfer coefficients, a, have been determined over a range of conditions, and tabulations of values are available from the authors. The values have been calculated by pattern theory48 and are referenced to the half-wave potentials because conditional potentials are not accessible. The half-wave potentials are approximately -0.7 V versus S.C.E. for azathioprine and the first wave of metronidazole, about -1.1 V versus S.C.E. for the second wave of metronidazole and -0.85 V versus S.C.E. for nitrazepam in sodium carbonate and -0.35 V versus S.C.E. in sulphuric acid. The half-wave potentials become more nega- tive on increasing the mass-transfer rate, by increasing rotation speed or concentration. The mass-transfer rate constants fall in the range 1-2 x 10-6 1 cm-2 s-1, increasing

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ANALYST, J U N E 1984, VOL. 109 763

Table 2. Example calibrations by R D E voltammetry. Electrode. gold; area. 0.503 cm2; temperature, 25 "C; and n = 4

Azathioprin in 0.1 mol 1-1 sodium carbonate solution Range 0-10 x 1 0 k 4 mol I - I . .

Range 0-10 x 10-3 moll-- I

Metronidazole in 0.1 mol 1 I sodium carbonate solution. Range 0-10 x

First wave . . . . . . . . mol 1-1

Secondwave . . . . . .

Nitrazepam In 0.1 mol 1 - 1 sodium carbonate solution. Range 0-10 x 10-3 moll-' . . . . . . . .

In 0.1 mol I- ' sulphuric acid. Range 0-10 x 10-3 moll- 1 . .

. .

. .

. .

. .

. .

Nominal Slope/ Intercept/ t'requency/Hz mA 1 mmol-1 mA

10 20 30 40 50 10 20 30 40 50

10 20 30 40 50 10 20 30 40 50

10 20 30 40 50

10 20 30 40 50

0.261 15 0.349 66 0.420 00 0. SO7 35 0.580 48 0.223 22 0.278 29 0.328 35 0.374 15 0.414 22

0.269 62 0.369 52 0.4.53 54 0.510 53 0.569 74 0.236 63 0.270 96 0.308 08 0.306 73 0.335 19

0.351 67 0.508 33 0.646 33 0.740 41 0.831 12

0.270 00 0.374 15 0.450 95 0.520 95 0.571 51

-0.002 77 0.000 46 0.000 00 0.002 41 -0.000 05 0.009 35 0.025 71 0.01679 0.01 1 56 0.008 06

0.299 23 0.300 48 0.337 89 0.452 63 0.348 68 0.679 08 1.13692 1.426 15 1.838 46 2.085 38

-0.005 00 -0.018 33 0.017 96 0.011 02 0.040 31

0.000 00 0.001 56

-0.000 95 -0.00095 -0.021 22

Correlation coefficient

0.999 68 0.999 98 1 .000 00 0.999 74 0.999 99 0.999 84 0.999 68 0.999 63 0.999 91 1.000 00

0.999 98 0.999 99 0.999 77 0.999 92 0.999 98 0.999 87 0.999 99 0.999 96 0.999 97 0.999 88

0.999 99 1 .000 00 0.999 96 0.999 99 1 .000 00

1 .000 00 0.999 99 0.999 96 0.999 97 0.999 96

S.d. of S.d. of slope/ residualsimA mA 1 mmol-1

0.001 94 0.000 59 0.000 00 0.003 48 0.000 58 0.012 27 0.020 00 0.026 90 0.014 87 0.000 72

0.005 55 0.005 57 0.029 50 0.011 47 0.005 73 0.010 71 0.004 16 0.008 32 0.006 93 0.015 25

0.002 89 0.002 89 0.017 38 0.011 43 0.003 57

0.000 00 0.004 59 0.01 1 55 0.011 55 0.01429

0.003 30 0.000 98 0.000 00 0.005 73 0.000 95 0.002 02 0.003 50 0.004 44 0.002 45 0.000 13

0.000 94 0.000 95 0.004 87 0.003 22 0.001 61 0.001 87 0.000 71 0.001 41 0.001 18 0.002 59

0.000 68 0.000 68 0.002 87 0.002 00 0.000 62

0.000 00 0.000 76 0.001 90 0.001 90 0.002 50

R.s.d. of slope, Yo

1.26 0.28 0.00 1.13 0.16 0.91 1.26 1.35 0.66 0.03

0.35 0.26 1.07 0.63 0.28 0.79 0.26 0.46 0.38 0.77

0.19 0.13 0.44 0.27 0.08

0.00 0.20 0.42 0.37 0.44

Table 3. Precision of determination by R D E voltammetry. Electrode, gold; area, 0.503 cm2; temperature, 25 "C; and medium, 0.1 moll-' sodium carbonate solution except as indicated, Each result arises from measurement of the limiting current at each of five rotation speeds

Azathioprine Metronidazole Nitrazepam

Low range High range R.s.d. , O/o R.s.d., '/a

Concentration/ Concentration/ Concentration/ 1st 2nd Concentration/ In In mmo11-l R.s.d., '/o mmoll - 1 R.s.d., O/O mmoll- wave wave mmol l-1 Na2C03 H2S04 0.200 042 2.50 2.000 42 3.44 2.000 52 0.17 0.75 1.999 93 0.67 1.16 0.500 105 0.19 5.001 05 1.32 5.001 30 1.89 0.23 4.999 82 0.96 0.99 0.800 168 0.74 8.001 68 0.60 8.002 08 2.82 0.74 7.999 70 0.52 0.82 1 .000 21 0.31 10.002 10 2.22 10.002 60 0.45 1.02 9.999 63 0.23 0.12

with increasing rotation speed but independent of concentra- tion. The charge-transfer rate constants increase with increas- ing rotation speed, but are little affected by concentration changes, and are about 3 x 10-6 1 cm-2 s-1 for azathioprine and the first wave of metronidazole. The second step for the latter is about twice as fast as the first. Nitrazepam shows the fastest reaction; the rate constant k = 6.3 x 10-6 1 cm-2 s-1 in sodium carbonate and about 10% slower in sulphuric acid. The a-values lie between 0.2 and 0.4 and are little affected by mass-transport rates.

Cyclic Voltammetry of Azathioprine

At all scan rates in the potential span +0.4 to -1.0 V versus S.C.E. a single cathodic peak appeared in the first cycle only,

Fig. 4, and no anodic peak indicative of re-oxidation could be observed. Further, cycling without reactivation of the elec- trode, scan B, showed complete suppression of the cathodic peak; the reaction product is, therefore, strongly adsorbed on the electrode surface. The absence of an anodic peak accords with observations at mercury electrodes.39 Variation of peak potential (Ep) and current (Zp) with scan rate is shown in Table 4. The variation of peak current with square root of scan rate is non-linear and not interpretable.

Coulometry and Reaction Mechanisms

Determination of the number of electrons involved in the reactions was made by passage of a series of determinate charges at constant current and measurement of the resultant

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764 ANALYST, JUNE 1984, VOL. 109

Table 4. Cyclic voltammetry of 5 x 10-3 mol 1-1 azathioprine in 0.1 mol 1-1 sodium carbonate solution at a gold electrode

Scan rate/ E, vs. mV s- 1 S. C. E./V I&mA

200 -0.585 0.75 300 -0.675 0.85 400 -0.730 0.95 600 -0.825 1.145 780 -0.900 1.120

1000 -0.960 1.08

decrease in the limiting current. Azathioprine gave good results of 25.0-25.6% decrease per electron equivalent, indicating a four-electron reduction. Metronidazole gave a wide scatter of results indicating 5.56-8.33 electrons, but a sufficient concentration of results permitted a decision of six electrons. This behaviour may explain the lack of a literature value. Nitrazepam gave low values in the range 14.75-15.0% loss in wave height per electron equivalent, suggesting that the number of electrons is six but that there may be a further reaction proceeding to a small extent. A six-electron reaction in acidic and basic media has been reported.49.50

Because none of the other diazepines showed reducibility at solid electrodes, it may be concluded that nitrazepam shares with metronidazole a six-electron mechanism of reduction of the nitro group, via nitroso and hydroxylamine intermediates, to the amine. There is no evidence of a second wave for nitrazepam, in contrast to the distinct separate waves at mercury electrodes,3Y and so no halt at the hydroxylamine. Metronidazole does give two waves, and from the slopes of the calibrations it would appear that at lower concentrations the first step is a four-electron reduction to the hydroxylamine, followed by a two-electron step to the amine. The reduction of azathioprine follows a single four-electron pathway to the hydroxylamine in more concentrated solution and this splits into two two-electron steps in more dilute solution.

We thank May and Baker Ltd.. Roche Products, The Wellcome Foundation and Wyeth Laboratories for the gift of the Drug Standard materials listed in Table 1 and the Royal Society for the SEL transfer standard DVM. W. H. thanks the Government of Pakistan for the award of a Scholarship, and the University of Karachi for the grant of leave of absence.

1.

2.

3. 4. 5. 6. 7. 8.

9.

10. 11.

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Paper A3143 7 Received December 7th, I983 Accepted January 10th) I984

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