Category: Formulation and Quality
Purpose: In the frame of making up a generic drug product (DP), it was developed a suitable and specific method for the identification of the disaccharide counter-ion of the DP by using the HPLC technique. The identification purpose consists in the comparison of both DP and reference standard chromatographic profiles.
During the method development, the chromatographic profile of the disaccharide reference standard showed the presence of a shoulder next to the main peak and an additional peak, while they were not observed in both API and DP solutions.
By performing some investigational experiments, it has been demonstrated that the shoulder next to the main peak was due to different conformations that disaccharide could have in the solution of CaSO4 1.2 mM used for standard preparation and as mobile phase, while the additional peak was due to the formation of its lactone occurred during the solubilization of the disaccharide powder.
Methods: The chromatographic analysis of the counter-ion was performed by using an High Performance Liquid Chromatography (HPLC) system with RI detector.
During the method development, the separation was performed by an isocratic elution with CaSO4 1.2 mM. After the investigational activities, the mobile phase was changed in H2SO4 0.450 mM.
The column used was HYPERREZ XP CARBOHYD 100 x 7.7 mm, 8 µm (flow rate 0.5 mL/min, injection volume 5 µL, run time 15 min), with a temperature of 60 °C. The autosampler was set at 5°C.
During the investigational activities the samples were prepared by adding the equimolar ratio of Ca(OH)2 and after a reaction of 30 minutes, it was stopped by adding mobile phase (H2SO4 0.450 mM) to the sample solutions. Additionally, the NMR analysis was performed by using Nuclear Magnetic Resonance (NMR) BRUKER working at 600 Hz equipped with cryoprobe.
The interest in performing a further investigation was related to the fact that the shoulder and additional peak were only detected in the disaccharide standard material.
To this end, some experiments were required to understand the root-cause of the shoulder and extra peak genesis. In details, the strategy was the following: in a primary stage it was investigated on the potential identification/characterization of the species responsible for both the shoulder and additional peak genesis. In a second stage, the method was optimized to avoid their formations.
Results: The investigation started with the HPLC analysis of both the API, DP and disaccharide standard material dissolved in CaSO4 1.2 mM: only the standard material freshly made showed the presence of the shoulder next to the main peak, while it was not observed if injected after 24 hours. On the other hand, both the two standard solutions showed the presence of the additional peak.
The disaccharide standard material dissolved in CaSO4 1.2 mM was characterized by NMR analysis (1H monodimensional, 2D 1H-1H TCOSY, 1H DOSY): data showed that the disaccharide in CaSO4 1.2 mM exists in two conformations. Additionally, the NMR experiments together with HPLC results confirm that the samples ageing influences the stabilization of one of the two conformations, not observing the shoulder. The NMR analysis also showed the presence of the related lactone into the solution. This product is formed directly during the dissolution of the disaccharide standard powder in CaSO4 1.2 mM, as reported in the relevant scientific literature.
The NMR output suggested to change the mobile phase, considering the probable influences of the pKa of the entity, thus a solution of H2SO4 0.450 mM (pH ~ 3.1) was made. The usage of this kind of solution promotes the formation of the lactone (~ 28 % peak area in CaSO4 1.2 mM, ~41% peak area in H2SO4 0.450 mM).
Starting from a bibliographic research, a basic pH could prevent the lactone formation. From that, a quantity of Ca(OH)2 (pH ~11) was added and a subsequent reaction for 30 minutes takes place. Finally, the samples were diluted at the final concentration of 1.0 mg/mL with H2SO4 0.450 mM.
The salification of this entity avoids the detection of the lactone peak.
Conclusion: The data obtained showed that the shoulder was due to different conformations that disaccharide counter-ion could have in solution by using CaSO4 1.2 mM for both the separation and standard preparation. The extra peak was due to the formation of its lactone during the powder solubilization.
The shoulder formation was solved by using H2SO4 0.450 mM for both the separation and standard preparation, while the extra peak was solved by adding Ca(OH)2 at equimolar ratio in a reaction of 30 minutes.
Stefano Lanciotti– Ferentino, Lazio, Italy