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The synthesis of Pipecuronium bromide together with the investigation of its impurity profile required the solution of a number of analytical and structural problems. NMR spectroscopical methods received a major role in the structure elucidation of the synthesized compounds. The quantitative assessment of contaminations was also realized by 1H NMR spectroscopy. Out of the large amount of undertaken investigations, here we provide the structural characterization of the contaminating regiosimers of an important intermediate (5 alpha-androst-2-en-17-on) formed in the course of the synthesis, together with the NMR signal assignments of the end-product (Pipecuronium bromide) as measured in D2O and a mixture of CDCl3/DMSO-d6. The structure of a minor contamination of the end-product is also given.

As a consequence of the presence of two ester groups in its molecule the main decomposition route of pipecuronium bromide (active ingredient of Arduan injection) is hydrolysis. In addition to this, autoxidation can also take place. In order to obtain a stable dosage form liophylized ampoules containing mannitol were prepared. Mannitol is not only a filling material but its role is also to decrease the dielectric constant of the solvent water thus increasing the stability of the drug substance before and during the liophylization procedure and after the dissolution of the content of the ampoules for administration. In order to protect the drug molecule against oxidation inert gas atmosphere was used during the production of the ampoules. The preparation is compatible with basic infusion solutions.

The following methods are described for the analytical investigation of the intermediates of the synthesis of pipecuronium bromide (Arduan) (for the numbering of the intermediates and their impurities see Figure 1.). 1. Gas chromatographic methods (capillary GC using fused silica capillaries Ultra-2 and Silar 10C WCOT) for the impurity profiling of intermediates I, II, IV and V including the identification and spectroscopic characterization of their impurities; 2. TLC methods for the similar characterisation of the further intermediates (III, VI, VII and VIII); 3. Gas chromatographic assay methods for IV and V using fused silica capillary technique and internal standards; 4. Potentiometric titration methods for the determination of VII and VIII using 0.1 M hydrochloric acid as the titrant.

Pipecuronium bromide molecule contains two slightly basic nitrogen atoms in its two piperazine rings. Their pK values are quite similar to each other in consequence of their almost equivalent surroundings. Although the potentiometric titration used mostly for determination of pK values of organic compounds can be performed in this case too, but the polarimetric titration applied by us is more advantageous. Change of pH considerably influences the values of optical rotation; consequently, the curve obtained in this way is more utilizable than that of potentiometric titration. Protonation of two nitrogen atoms one after the other changes the optical rotation of the molecule in opposite direction. Consequently, a minimum can be seen on the curve of optical rotation versus pH value. The exact pK values were determined on the basis of the estimated parameters of a curve established corresponding to an equation and fitted to the measuring points. The sequence of deprotonation of nitrogen atoms could only be ascertained on the basis of the curves of polarimetric titration of compounds containing one piperazine ring; on the basis of the pK values and of the changing direction of optical rotation.

The mass spectral behaviour of the bis-quaternary salt pipecuronium bromide and some related Cn+ A-n type ammonium salts (n = 1, 2, 3) has been studied and compared. Under EI-MS conditions the evaporation of the samples preceded by in situ dealkylation led to formation of alkyl bromide and amine bases. It has been demonstrated that this technique, completed with quantitation of the relative amount of the decomposition products is applicable to get structure information about the basis part as well as the number and positions of the quaternary centres and the substituents at these centres. The FAB mass spectra of these mono-, bis- and this quaternary ammonium salts were found to be very interesting and provide direct structural information about the salt. They exhibit primary Cn+ intact cations and (Cn+ A-n + 1)+ single charged cluster ions and also fragments characteristic of partial structures at the quaternary centres.