bis-Polarographic analysis of psychotropic drugs

Bioelectrochemistry and Bioenergetics, 10 (1983) 25-36 A section of J. Elecrroanal. Chem., and constituting Vol. 155 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

25

Review article 492 bis-POLAROGRAPHIC

ANALYSIS

OF PSYCHOTROPIC

DRUGS

H. OELSCHLAGER Insiitut fiir Pharmazeutische Chemie D -&XI0 Frankfurt am Main (F.R G.)

der Johann

Wolfgang

Goethe - Universitijt,

Georg - Voigt - Str.

14,

(Revised manuscript received September 8th 1982)

SUMMARY The electrochemical quantitative analyses of drugs (and of their metabolites) in vivo have as a starting-point their behavior as pure substance in vitro. In this paper the polarographic behavior of psychotropic drugs is investigated and it is found that the drugs investigated (if necessary after appropriate functionalixation) are polarographically active and this behaviour can therefore be taken as a starting point for the further development of suitable analytical methods in vivo. The following drugs were investigated: benzodiaxepines (bromazepam, chlordiazepoxide, clonaaepam, diazepam, dipotassium chlorazepate, flunitrazepam; flurazepam, loraaepam, medaxepam, midaaolam, nitrazepam, oxazepam, pinazepam, prazepam, temazepam, triaaolam, clobaaam), meprobamate, phenothiazine- thioxantene- and butyrophenone-derivatives, sulpirid, reserpine, imipramine, thioridazine, amitriptyline, imipramine, opipramol, doxepine, chlorpromazine, levopromaaine, perarine, prothipendyl, chlorprothixene, triperidol, methamphetamine.

INTRODUCTION

The treatment of emotional and mental disorders by drug therapy was non-existent until 1952, when chlorpromazine as the first psychotropic drug was introduced into medical use. The number of patients suffering from neurosis, psychosis and psychopathic disorders is large and constantly increasing in all countries of the world, perhaps due to the modem way of life. Nearly all fundamental psychotropic agents have been developed in the period 1952-60. Today three important groups are differentiated: (1) minor tranquillizers (Fig. 1); (2) major tranquillizers (Fig. 2); (3) antidepressants (Fig. 3). Carbaminic acid esters (e.g. meprobamate), l&benzodiazepines and 1,5-benzodi-pines, as the main compounds, form the first group. Derivatives of phenothiazincs and butyrophenones and their analogues, and sulpirid and the alkaloid reserpine are the drugs of the second group, and non-planar tricyclic molecules are helpful as antidepressants. Figure 4 shows the steric structure of imipramine with its very rigid tricyclic moiety. The most commonly used antidepressants lack a reducible 0302-4598/83/$03.00

0 1983 Elsevier Sequoia S.A.

26 v3

Meprohmate

8

NH2 H2N- -0-H&-H&-C-CH,-O-CCti2-~~2-~~3

1,4-Benzodiazepines

1,5-Benzodiazepines

(Clobazam@)

Fig. 1. Structures of important minor tranquillizers.

Thioxanthenes

Phenothiazines

Ri R2

F Sulpirid

Butyrophenones

CH,O

Reserpine Fig. 2. Structures of important major tranquillizers.

27

k

I2

lmiprdmine

Q(J)

Opipramol

Qr--JJ

R

R

Amitriptyline

Doxepine

Fig. 3. Structures of important antidepressants.

group, being therefore electroinactive under polarographic aspects. Benzodiazepines, phenothiazines, butyrophenones and sulpirid are the leading drugs in psychiatric therapy. They enable the decrease of compulsory restraints on patients in the clinics and a remarkable shortening of hospital stay. Approximately 80% of the patients treated with these medicaments may return to their families and professions, and are not confined permanently to psychiatric institutions. C,,H24N,.HCI

IMIPRAMINE

Fig. 4. Structures of imipramine.

PROBLEMS

OF ANALYSIS

Most of the above-mentioned psychotropic drugs are used in small doses. In the case of some modem 1,Cbenzodiazepines 1 mg per tablet is the normal dose.

28

Therefore the content uniformity test according to USP XX is required, and specific analytical methods are needed for the determination of a single dose content without interference by excipients. This analytical problem may be demonstrated by the formulation of flunitrazepam tablets with a weight of 200 mg each and a content of 2 mg active principle only. Small amounts of 1,Cbenzodiazepines in formulations (tablets, dragtes, ampoules, suppositories, etc.) can be analysed by various methods: spectrophotometry, g.l.c., densitometry, voltammetry and recently by h.p.1.c. Using these methods, in most cases extraction of the active principle is necessary before quantitative measurement. The most universally employed separation technique is solvent extraction. In extraction from plasma it has been shown that non-polar solvents such as heptane coextract cause much less interference than some polar solvents, e.g. ethyl acetate. The formation of relatively stable emulsions, sometimes observed, can be avoided by use of slow agitating apparatus, e.g. the Linson mixer (Linson Instruments, AB Lars Ljungenberg h Co., Stockholm Sweden). In some cases the pH of the solution to be extracted also has a remarkable influence on the rejection of interferences. Among other separation techniques the use of t.1.c. and of macroreticular resins are very popular. The choice of the method of determination after extraction depends primarily on the physical and chemical properties of the molecules. Drugs of sufficient stability associated with poor polarity are vaporizable and can therefore be detected by g.1.c. Derivation by means of silylating agents often facilitates vaporization. If a drug is not vaporizable without degradation h.p.1.c. offers considerable advantages. The disadvantage of this method lies in the fact that numerous and complex parameters such as the partition coefficient and pH influence the result. In many cases no foregoing separation procedure is needed if voltammetric methods, especially differential pulse polarography (d.p.p.), are used for assay, because many of the excipients do not disturb the electrode process. This fact is noticeable, for some of them possess a distinct surface activity, but the resultant shifting of the half-wave potential (+) and the decrease of the faradaic current, or of peak height respectively, moves in a small range. For example, polyethylene glycol 6000 shifts U,,z of the first step of the d.c. polarographic wave of chlordiazepoxide only 30 mV to a more negative potential and lowers the diffusion current of both first steps by about 11%. On the other hand, magnesium stearate has no influence on the peak height [l]. Polarographic assays of psychotropic drugs in blood, plasma or urine need a selective clean-up in the presence of metabolites of the drug and coadministered drugs. In some cases omission of clean-up is demonstrated. For example, the three most important metabolites of medazepam in blood are N-desmethylmedazepam, diazepam and N-desmethyldiazepam. The peak heights of these four compounds, recorded by linear-sweep polarography, show considerable differences in the dependences on pH. This is a basis for analysis of medazepam and its metabolites in blood PI* The procedure is as follows: after coagulation of the blood the serum (1 cm3) is

29

made alkaline and extracted with petroleum ether at about 40-6O’C. After centrifuging of this extract the supematant is removed and the extraction of the aqueous phase is repeated. The combined extracts are evaporated in a water bath at 37’V in nitrogen atmosphere. The residue is subjected to cathode-ray polarographic analysis after dissolving in a B&ton-Robinson buffer, pH 3.5. (containing 10% dimethylformamide). Sample preparation is minimal only in toxicological investigations owing to the large amount of the administered drug. ESTIMATION

OF MINOR

TRANQUILLIZERS

Research work concerning I,4-benzodiazepines

Starting with chlordiazepoxide in 1961 the electrochemical properties of more than 15 1,Cbenzodiazepines (Table 1) have been investigated in our laboratory. In addition, we have elucidated the electrode processes. Exact knowledge of the electrode process is the basis of each correct voltammetric method because this information influences the accuracy of the analysis [3]. This influence depends on the predictable correlation of wave-height in d.c. polarography with n and D in the IlkoviE equation. Elucidation of the electrode process involves not only electroanalytical measurements but also in some cases difficult syntheses of supposed intermediates and final products of the electrochemical process. These compounds will be investigated with respect to their electrochemical behaviour which should correspond with the assumption of the overall electrode process. A very convincing experiment for the validity of the supposed electroanalytical reaction is the reduction of the depolarizer in a macroscale at controlled potential with subsequent isolation of the product and determination of its structure by routine methods of spectroscopy. The electroactive group of all 1,Cbenzodiazepines investigated is the -N=C< double bond, which is reduced at the d.m.e. under consumption of 2 e- and 2 H+ [4]. If the molecule. contains, additionally, further reducible groups, e.g. a nitro group, the electrode process becomes more complicated. Typical samples are the tranquillizers chlordiazepoxide, nitrazepam and oxazepam. Often, mixed-mechanism

TABLE 1 1,4-Benzodiazepines

investigated

Bromazepam Chlordiazepoxide ClOIMiZep@ll

Diazepam Dipotassium chlorazepate Flunitrazepam Flurazepam Lorazepam

Medaaepam Midazol~ Nitrazepam O==@m Pinaaepam prazepam Temazepam TriazOl~

+6e+6H+

-CH3NH2

Cl / b\

+2e +2H+ Shp

=fi-

;C=N-(48)

-

+2e-

,NHCH3

N=C, Step

:H-NH

)CH-NH

+2H+

,CH2

+2s+ZH+ -CH3NH2

Cd’5

Fig. 5. Polarography

of chlordiazepoxide

-

‘gH5

(CDO).

[including electrochemical (= e) and chemical (= c)] reactions are involved in the overall reduction process. Figure 5 shows that chlordiazepoxide is reduced at the d.m.e. in three steps under consumption of 6 e- and 6 H+ with subsequent ring contraction to a 3,4-dihydro-quinazoline derivative accompanied by elimination of methylamine. Also, in d.p.p. three separated peaks appear, the peak heights of each being linearly correlated to the concentration of the depolarizer [l] as depicted in Fig. 6. The reduction process of nitrazepam at the d.m.e. depends on the pH of the supporting electrolyte used [5]. In an acidic buffer two waves of the same height appear and 8 e- and 8 H+ are consumed totally; however, in an alkaline buffer

240-

Fig. 6. Differential

pulse polarogram of CD0

(A) and its intermediates

B and C.

31

Ni truepam NH- Co LHZ CH-N/H t 6”5

Fig.7. Polarographyof nitrazepam.

consumption of 6 e- and 6 H+ in two waves of different heights occurs owing to the fact that the intermediate hydroxylamino-derivative is reducible at a more negative potential only in the presence of protons. Therefore, in alkaline buffers, after reduction of the nitro group with consumption of 4 e- and 4 H+, only the reduction of the -N=C( double bond does occur (2 e-) as depicted in Fig. 7. Many difficulties had to be overcome in elucidation of the electrochemical behaviour of oxazepam at the d.m.e.. An e.c.e. mechanism explains the total consumption of 4 e- and 4 H+ as well as the elimination of water, with the subsequent formation of a ketimine-aldimine equilibrium as intermediate after the uptake of the first 2 e- and 2 H+ [6], as indicated in Fig. 8. The new l,Sbenzodiazepines, e.g. clobazam, are electroinactive according to the double lactam structure. Estimation of meprobamate The meprobamate molecule which possesses no electroactive group can be assayed by means of oscillopolarography in alkaline solution owing to a cut-in of

I

-H,O

+ ze-+2Hf Cd5

Fig. 8. Reductionmechanism(e.c.e.)of

Aidiminr

2 Z

C&

oxazepam

in

acidicbuffers.

32

CHrCH2-CH2-N(CH&

CH~CfH~CHTN(CH&

Chlorpromazine

Fig. 9. Polarography of chlorpromazine.

capacitive origin [7]. However, this determination is not very sensitive and could be influenced by all surface-active compounds present in the solution. Further, the electrode process of those electrochemical analyses is not correlated with a well-defined reaction specifically for the compounds to be assayed. ESTIMATION

OF MAJOR TRANQUILLIZERS

Research work concerning phenothiazines The major tranquillizers, e.g. phenothiazines and their analogues, lack a reducible electroactive group. They are oxidizable at a gold microelectrode, but this behaviour does not serve as the basis of a voltammetric assay. We have found that chlorpromazine and related compounds are oxidizable at the sulphur atom quantitatively and selectively using diluted nitric acid (2-3%) containing a small amount of sodium nitrite which increases the oxidation potential considerably (Fig. 9). The resulting sulphoxide is reducible at the d.m.e. under consumption of 2 e- and 2H+ in acidic buffers [S]. In more than 20 formulations such as tablets, drag&es, drops, ampoules, suppositories, etc. the content of the active principle (levomepromazine, perazine, prothipendyl, thioridazine) could be assayed with convenient standard deviations [9]. The phenothiazines investigated are given in Figure 10. No serious disturbance by the excipients was observed. In a smaIl number of cases difficulties arose. For example, a commercial thioridazine suspension contains Mg-Al-silicate as stabilizer. We observed that the sulphoxide formed will be

33

Levomepromazine [Z Diashreomeric

I-

Sulfowides]

Thioridazine

2 Diastereometic SulFoxides (5) as Racemates 1 srel

1

ste1 Tablets (25mg)

f 2.28 %

Dragees

Tablets (100 mg>

+ 0,77 %

Drops

2

1,03%

Drops

It 1,55%

Liquid

k

3,li

Amp&es

2 0,70%

Perazine

(extr.)

% Cextr.1

;rcl mg) k 2,17 %

Drops

+ (04%

Ampules

2 1,73%

Fig. 10. Phenothities

2,13x

Prothipendyl

Srel Tablets(100

?

(extr.)

Dragees

k 4,57

%

Drops

+

1,74%

Liquid

f

1,08%(extr.I

suppos.

If 1,53x

(extr.1

(c%tr.)

investigated.

adsorbed at the silicate quantitatively. Therefore, extraction of this sulphoxide was necessary before measurement. Further, we recognized that the unknown oxidation product of the preservative sorbic acid is polarographically active. During the

(7$)2

t

N(W)2

02N-CH2-(CH&-N(CH$2

Fig. 11. Assay of chlorprothixene formulations oia the degradation products.

oxidation process with diluted nitric acid an unexpected reaction occurs with neuroleptic chlorprothixene [lo]. After addition of nitric acid to the -C=Cdouble bond [ 10,l l] the side-chain of the molecule will be transformed into an aliphatic nitro-derivative cleaved from the ring structure. In the absence of the formed tricyclic ketone which is electroactive, a four-electron reduction wave can be observed which is suitable for determination, as depicted in Fig. 11. Research work concerning butyrophenones Volke et al. [ 1l] have shown that the keto group of the widely used butyrophenones, which are the most important neuroleptics, is reducible at the d.m.e. in acidic as well as in alkaline buffers which contain 50% dimethylformamide. The mechanism of the

F

F

/ Ayt&

7 *

Mercury Dimcr

;$er Mechanism

:

0~ + em-

/f Radical \

Fig. 12. Polarography of triperidol.

Compound

35 fH3 /\ cl- -

CHE-_PH-NH

Picrylfluoride CH,CN

CH3

CH5H5N

-458 mV DCP

DPP

Fig. 13. Functionalization of the electroinactive drug methamphetamine.

reaction is complicated because of the behaviour of the radical formed after the uptake of the first electron. Under certain conditions an uptake of nearly 2 e- does occur (Fig. 12). Sulpirid, as we have just found, is determinable after selective functionalization to its N-oxide with 3-chloroperbenzoic acid. The N-oxide is reducible in acidic buffers under consumption of 2 e- and H+. Reserpine can be estimated by reduction at the d.m.e. in aprotic solvents as well as by oxidation at a Pt electrode [12]. In the first case the reduction product is unknown while the oxidation product formed could be identified as its N-oxide. ESTIMATION OF ANTIDEPRESSANTS

The frequently used tricyclic antidepressants cannot yet be determined by voltammetric methods owing to their electroinactivity; attempts at functionalization have failed. In some cases catalytic waves caused by the advanced reduction of the protonated molecule are helpful, as observed with carbamazepine. Volke et al. [ 131have reported on the anodic oxidation of amitriptyline and other tricyclics at the platinum and gold rotating disc electrodes in acetonitrile, using tetrabutylammoniurn perchlorate as supporting electrolyte. A linear calibration was obtained over a concentration range of 2.5 to 15 X low4 M, as well as in biological fluids. In many cases no extraction procedure of the active principle is necessary because the excipients or the constituents of the serum do not disturb the electrode process. In this respect voltammetric analysis is not an alternative to spectroscopic methods but a superior method. Because voltammetric analysis requires a reducible or oxidizable group in the compound to be determined, it is of great importance that new functionalization reactions become more widely known. Such a new reaction occurring quantitatively is the use of picryl fluoride. Recently, Oelschlager and

36

hItiller [ 141were able to demonstrate the utility of picryl fluoride for the functionalization of the electroinactive methamphetamine, as shown in Fig. 13. A problem to be solved in the near future is the development of special electrodes and direct-measurement techniques for the estimation of spots on t.1.c. plates. In this way a highly specific method for quantitative determination of compounds, separated by t.l.c., would be helpful in comparison with densitometry as a second independent measurement. ACKNOWLEDGEMENTS

The experimental work was supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. The author thanks Prof. Dr. J. Volke of the J. Heyrovsky Institute of Physical Chemistry and Electrochemistry, Czechoslovak Academy of Sciences at Prague, for stimulating discussions over a long period. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14

H. OelschHger et al., Fresenius Z. Anal. Chem., 282 (1976) 123. H. Gelschlgger and V. Geppert, Arch. Pharm., 309 (1976) 1000. H. Oelschlttger, Lecture at the University of Regensburg, ref. Dtsch. Apoth. Ztg., 120 (1980) 1973. H. Gelschlager, Arch. Pharm., 296 (1963) 396. H. OelschHger et al., Arzneim. Forsch., 16 (1966) 82; H. Gelschl8ger et al., Arch. Pharm., 302 (1969) 241. H. Gelschlgger et al., Arch. Pharm., 302 (1969) 946; 303 (1970) 364; J. Electroanal. Chem., 25 (1970) 307. I. Hynie et al., Gas. Lek. &k., 103 (1964) 412. H. Oelschltlger and K. Bunge, Arch. Pharm., 307 (1974) 410. H. Oelschlager and R. Spohn, Doctoral Thesis, R. Spohn, University of Frankfurt/Main. 1980. H. Oelschlager and R. Spohn, Arch. Pharm., 314 (1981) 355. J. Volke et al., Pharmarie, 26 (1971) 399. A. Woodson and D. Smith, Anal. Chem., 42 (1970) 242; M.J. Allen and V.J. Powell, J. Elektrochem. Sot., 105 (1958) 541. J. Volke et al., J. Electroanal. Chem., 60 (1975) 239. H. Oelschlager and M. Muller, Pharmazie, 36 (1981) 807.