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Voltammetric determination of N-nitrosamines in the hypoglycemic agent tolazamide Pier Luigi Buldini 1, Vittorio Rossetti2, and Alessandro Toponi 1 1 CNR-LAMEL, Laboratorio Analisi Chimica dci Matcriali, Via dell'Idraulico 17/2, 1-40127 Bologna, Italy 2 FRANCIS SpA (Group SANOFI), Via Origgio 23, 1-21042 Caronno Pertusella, Italy

Voltammetrische Bestimmung von N-Nitrosaminen in dem hypoglykiimischen Mittel Tolazamid Summary. Exposure to N-nitrosamines has been indicted as one cause of cancer and so their presence has to be controlled. Voltammetry has been used for the detection of N-nitrosamines and has the advantage that it can be applied to both volatile and non-volatile compounds.

The proposed method consists in dissolving 50 mg of tolazamide drug in 1 M potassium hydroxide and, after deaeration, the solution is directly submitted to differential pulse polarography. The response at about - 1 . 5 0 V (vs. Ag/AgC1 reference electrode) is proportional to the N- nitrosamine content from 0.5 ppm to more than 1,000 ppm into the tolazamide sample, with a relative standard de- viation of + 3 - 4 % at 1 ppm. The sensitivity of the pro- posed method is essentially limited by a sharp peak due to N-[(hexahydro- 1H-azepin)carbonyl]-4-methylbenzen- sulphonamide (usually present as impurity in tolazamide), that occurs at about -1 .23 V (vs. Ag/AgC1 reference electrode).

N-nitrosamines have been indicted as one cause of cancer and have additional mutagenic, embryopathic and ter- atogenic actions. They can be formed in vivo by the action of nitrosating agents on secondary or tertiary amines. As nitrosamine investigations continue, the substances which may contain nitrosamine levels that are suspected to cause abnormal cellular transformations, are increasing. Among drugs, tolazamide is requested to be certified for the N- nitrosamine content (essentially due to 1-nitroso-lH-hexa- hydroazepine) because its synthesis is based on the nitrosa- tion of 1H-hexahydroazepine followed by its reduction to l-amino-lH-hexahydroazepine which reacts with 4-me- thylbenzensulphonylurethan giving N - {[(hexahydro - IH - azepin - 1 - yl) - amino]carbonyl} - 4 - methylbenzensulphon- anaide (tolazamide) [1] as reported in Fig. 1.

The polarographic behaviour of some N-nitrosamine has been investigated [2-7] and pulse polarography has been found to be a very sensitive technique for N-nitrosamine determination even if its rather poor selectivity makes a specific determination of one type in the presence of other ones impossible without a prior separation step. However, polarography has several advantages: it can be applied to both volatile and non-volatile compounds, it provides a rapid analysis with simple and inexpensive apparatus and it does not require sample preparation.

Offprint requests to: P. L. Buldini

c%-~}-s0z~2 . cxcoocz, 5 . c.3-@so2,.-cooc2%

%-~-S%NM-C00C2% + "2N-N~ ~ %-{~-S%~"-C0NH-N~

Fig. 1. Tolazamide synthesis

In general, at the mercury electrode, N-nitrosamines ex- hibit an irreversible, pH-dependent, four-electron reduction in acid solution to the corresponding hydrazine and irrevers- ible, pH-independent, two-electron reduction in basic solu- tion to nitrous oxide and the precursor amine.

The proposed method consists in dissolving 50 mg of tolazamide in 1 M potassium hydroxide and, after deaera- tion, the solution is directly submitted for differential pulse polarography. The response at about - 1.50 V (vs. Ag/AgC1 reference electrode) is proportional to the N-nitrosamine content from 0.5 ppm to more than 1,000 ppm in the tolazamide sample.

Preliminary studies

Effect of various electrolytes

The voltammetric behaviour of 1-nitroso-lH-hexahy- droazepine and N-{[(hexahydro-lH-azepin-l-yl)-amino]- carbonyl}-4-methylbenzensulphonamide does not seem to have been studied. A problem in the selection of the supporting electrolyte is the solubility of the tolazamide sample: while N-nitrosamines better reduce in acidic solutions, tolazamide dissolves in appreciable amounts only at pH-values over nine; when under, tolazamide solubiliza- tion is less than 0 .02-0.04% and so the resulting solution is too much diluted to permit sensitive determinations. The pH of the supporting electrolyte was proved to be the leading element in tolazamide solubilization.

The reduction of l-nitroso-lH-hexahydroazepine and N-{[(hexahydro-lH-azepin-l-yl)_amino]carbonyl}_4_methyl_ benzensulphonamide was examined in various supporting electrolytes.

At low pH-values (from 1 to 2), 0.01 M H2804, 0.5 M NHzSO3H, 0.01 M HOC6H3(COOH)SO3Na and the Britton & Robinson buffer at pH 2 were tested. The 1- nitroso-lH-hexahydroazepine peak was not well separated from the background in these supporting electrolytes and on the other hand, the N-{[(hexahydro-lH-azepin-l-yl)- amino]carbonyl}-4-methylbenzensulphonamide solubility is less than 0.01 -0 .02%; the only exception is the sodium salt

Fresenius Z Anal Chem (1987) 328:265-267 �9 Springer-Verlag 1987

Original papers

U.SET= -1.250 V

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Fig. 2. Typical polarogram ofa tolazamide drug sample. Curve 1 48.25 mg tolazamide crude sample; Curves 2, 3 and 4 100 ng ml- 1 standard additions

of 2-hydroxy-5-sulphobenzoic acid where the l-nitroso-lH- hexahydroazepine evidences a not analytically suitable peak with Up - 0.88 V (vs. Ag/AgC1 reference electrode).

The intermediate pI-I-range from 5 to 6, the Britton & Robinson buffer at pH 5 and some quaternary ammoni- um salts [(C2Hs)4NPF6, (CzHs)4NC104., (C4Hg)4NBr, (CzHs)4NBr, (C4H9)4NI] were examined. Tolazamide solubility keeps at about 0 .02-0.03% and 1-nitroso-lH- hexahydroazepine evidences a peak whose Up varies from - 1 . 4 5 V in 0.1 M (C2Hs)gNBr to - 1 . 5 1 V in 0.025M (C4H9)4NI. In addition, when examining the polarographic behaviour of the intermediate and impurities that may be present in the tolazamide drug, it is possible to see that in these supporting electrolytes the N-[(hexahydro-lH- azepin)carbonyl]-4-methylbenzensulphonamide peak over- laps the l-nitroso-lH-hexahydroazepine one so blocking in practice the N-nitrosamine determination. This potential impurity is present in the tolazamide drug due to the impossibility to obtain the 1-amino-lH-hexahydroazepine free of its starting material (1H-hexahydroazepine), see Fig. 1. 1H-hexahydroazepine also reacts with 4-methyl- benzensulphonylurethan to give N-[(hexahydro-lH-azepin)- carbonyl]-4-methylbenzensulphonamide.

Only in the Britton & Robinson buffer at pH 5, the 1-nitroso-lH-hexahydroazepine gives one peak with Up - 1.20 V that should permit the N-nitrosamine determina- tion, but the tolazamide solubility is even too low to permit a sensitive determination.

The addition of acetone for increasing the tolazamide solubility in the acid solutions was not successful, causing a rapid worsening of the polarographic background.

At basic pH, the Britton & Robinson buffers between 9 and 11, the ammonia buffers at p H 9 - 1 0 , 0 .1M (CH3)4NOH and 1 M KOH were examined. Tolazamide solubility was over 0 . 5 - 1 % and does not give any problem. Solubilities over 1% were not investigated because the presence of N-[(hexahydro-lH-azepin)carbonyl]-4-methyl- benzensulphonamide as impurity gives a polarographic re- sponse sufficiently near to the N-nitrosamine peak and large quantities of this impurity cause interference at the low N- nitrosamine contents.

In the basic supporting electrolytes l-nitroso-lH-hexa- hydroazepine evidences a peak whose Up is from -1 .47 to - 1.50 V (vs. Ag/AgC1 reference electrode), respectively, with diffusion currents that are similar to each other. The best results were obtained in 1 M KOH where the derivative peak is regular, well formed and very reproducible (see

266

T a b l e 1. Voltammetric characteristics of intermediates and im- purities occurring in the tolazamide preparation

Intermediate/impurity Up (vs. Ag/AgC1 reference electrode)

l H-Hexahydroazepine 1-Amino- 1 H-hexahydroazepine 4-Methylbenzensulphonamide 4-Methylbenzensulphonylurethan N-[(Hexahydro- 1 H-azepin)carbonyl]-

4-methylbenzensulphonamide

- 0.99 V -0.42 V - 0.92 v - 0.74 v; - 0.89 V

- 1 .23 V

Fig. 2); more diluted potassium hydroxide solutions cause worse polarographic background and more concentrated ones cause interference due to the shift of the impurity N- [(hexahydro - 1 H - azepin)carbonyl] - 4- methylbenzensul- phonamide peak to more negative potentials.

Voltammetric character&ties of intermediates and impurities of tolazamide preparation

In Table 1 are reported the polarographic characteristics in I M KOH of the intermediates and impurities that may occur in the tolazamide preparation.

The sufficiently near polarographic response of N- [(hexahydro-lH-azepin)carbonyl]-4-methylbenzensulphon- amide prevents the use of the method at extremely low N-nitrosamine concentrations. On the other hand, it is practically impossible to obtain tolazamide completely free from this impurity.

Deaeration of the supporting electrolyte

The l-nitroso-lH-hexahydroazepine response in 1 M KOH was not influenced by prolonged deaeration steps (at least to I h) with presaturated pure nitrogen, both in the presence of tolazamide and not.

E x p e r i m e n t a l

Reagents. Erbatron electronic grade potassium hydrox- ide and C. Erba RS polarographic grade reagents were used. Normal precautions for trace analysis were taken throughout the paper.

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Table 2. Polarographic conditions

Drop size Electrode type Measuring mode Pulse amplitude Start voltage End voltage

0.40 mm z ca. M.M.E. operated as D.M.E. Differential pulse polarography 100 mV -- 1.250 V -- 1.700 V

Purge time Replications Standard additions No. of standard additions Vessel volume

Verification voltage Voltage step Sweep rate Time step for measurements Peak width range (min/max) Tolerance for verification voltage Peak asymmetry (max)

240 s 3 (medium value obtained over 3 replications) 50 ~tl of a 200 ixg/1 standard solution 3 10 ml

- 1.500 V 6 mV 10 mV/s 600 ms 21/246 mV + 56 mV 9%

Solutions containing N-nitrosamines are disposed of properly using a well established procedure [8].

All organic materials used were synthetised and purified to more than 99.7% in the Francis SpA Laboratories.

Standard l-nitroso-lH-hexahydroazepine was prepared by reacting 1H-hexahydroazepine with nitrite ion under acidic conditions, then dried and distilled twice. Its purity was checked by G.L.C., capillary column and flame ioniza- tion detector, which showed it to be greater than 99.9% ; IR and UV spectra confirmed this result. Working standard at 200 ~tg 1- ~ was prepared by weighing a suitable quantity of standard l-nitroso-lH-hexahydroazepine in 0.01 M KOH. Less concentrated standards were prepared by diluting the 200 gg 1- ~ one with 0.01 M KOH.

Apparatus. The instruments used for the voltammetric mea- surements were a Metrohm (Herisau, Switzerland) 646 VA Processor equipped with 647 VA Stand, 675 VA Sample Changer and 665 Dosimat automatic addition burettes. The working electrode was a multi-mode electrode (MME), the reference electrode was an Ag/AgC1/c (KC1) = 3 M and the auxiliary electrode was a platinum tip about 2 x 65 mm long.

In Table 2 the operations listing of the 646 VA Processor is reported. Basic instrumental conditions are: pulse height 100 mV, drop time 600 ms, scan rate 10 mV s -~, analysis time (deaeration step and two replications included) 7 min.

A 10 ml cell is normally used. Solutions are deaerated with presaturated pure nitrogen for 4 min before analysis and thermostatted at 25.0 +_ 0.1~

Procedure. Dissolve about 50 mg of tolazamide in 10 ml of 1 M KOH directly in the polarographic cell. The listing that is summarized in Table 2 records the polarogram from - 1.25 to - 1.70 V, evaluates the peak by using only the rear half-peak in order to minimize the potential interference due to the N-[(hexahydro-lH-azepin)carbonyl]-4-methylben- zensulphonamide impurity at - 1.50 V (with a tolerance of + 55 mV) and compares it with three standard automatic additions of 50 gl of a 20.0 gg 1-1 1-nitroso-lH-hexahydro- azepine standard solution.

The whole procedure is automatically replicated two times in order to reduce the relative error by considering the mean of the three values obtained.

R e s u l t s a n d d i s c u s s i o n

To test the proposed method, several lots oftolazamide were examined. Some results are reported in Table 3.

When the prescribed conditions are used, the proposed method permits determinations of N-nitrosamines im-

Table 3. Determination of N-nitrosamines in tolazamide samples

Lot Crude (ppm) Sr (%) Purified (ppm)

Q/06/05 1.8 2.7 less than 0.5 Q/06/04 1.3 4.4 less than 0.5 Q/06/02 3.2 t.5 less than 0.5

purities as low as about 500 ng g-1 and a relative standard deviation of about + 3 - 4 % at 1 ppm.

Figure 2 shows a typical polarogram of a tolazamide sample where the 1-nitroso-lH-hexahydroazepine peak can be seen accompanied by the sharp peak due to the N-[(hexa- hydro - 1 H-azepin)carbonyl] - 4- methylbenzensulphonamide impurity present into the tolazamide sample. This sharp peak is close enough to interfere when determining low N-nitrosamine contents.

The poor selectivity of the polarographic technique in marking the different types of N-nitrosamines is not a prob- lem for drug characterization because only the total N-nitrosamine content is requested.

The sensitivity limit is on the contrary an important fac- tor because N-nitrosamine tolerance becomes smaller and smaller, so limiting the practical usefulness of the modern polarographic techniques also.

It is interesting to evaluate the polarographic technique as detector after a separation/enrichment step via high per- formance liquid chromatography in order to separate 1- nitroso-lH-hexahydroazepine from the tolazamide matrix.

At this moment problems are related to the choice of an eluent that may be a successful supporting electrolyte too and experiments are made for assuring the complete lack of missing (or forming) N-nitrosamines in the separation/ enrichment step.

R e f e r e n c e s

1. Wright JB, U.S.P. 3,063,903 Nov 13, 62 Upjohn 2. Franklin Smith W, Watkins P, Burmicz JS, Hanley HO (1975)

Anal Chim Acta 78 : 81 3. Heath DF, Tarvis JAE (1955) Analyst 80:613 4. Lydersen DL, Nagy K (1967) Fresenius Z Anal Chem 230:277 5. Chang SK, Harrington GW (1975) Anal Chem 47:1857 6. Hasebe K, Osteryoung JG (1975) Anal Chem 47:2412 7. Walters CL, Johnson EM, Ray N (1970)'Analyst 95:485 8. Castegnaro M, Egan H, O'Neill IK, Bartsch H, Fishbein L 0982)

In: Laboratory decontamination and destruction of nitrosamines in laboratory wastes. I.A.R.C. Sci Publ 43 "42

Received September 5, 1986; revised December l, 1986

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