Importance of Sample Preparation Diluent for Reliability of HPLC Method

Ludmila Mironchik, Global R&D, Teva Pharmaceutical Industries, Ltd., Netanya 4250483, Israel
Zina Zupnik, Global R&d, Teva Pharmaceutical Industries, Ltd., Netanya 4250483, Israel
Larissa Kelmansky, Global R&d, Teva Pharmaceutical Industries, Ltd., Netanya 4250483, Israel
Gregory Verba, Global R&d, Teva Pharmaceutical Industries, Ltd., Netanya 4250483, Israel
Sofia Vals, Global R&d, Teva Pharmaceutical Industries, Ltd., Netanya 4250483, Israel
Vladimir Ioffe, Global R&d, Teva Pharmaceutical Industries, Ltd., Netanya 4250483, Israel

HPLC‐UV method for assay and determination of impurities for Laquinimod capsules was developed using a volatile buffer, ammonium acetate, in mobile phase and sample diluent, to allow identification of unknown impurities and degradation products using LC‐MS equipment. Later, the principles of this method were adopted for the method for Laquinimod tablets. However, instead of volatile ammonium acetate buffer, a phosphate buffer having the same pH was taken for preparation of mobile phase and sample diluent. The reasons for this substitution were:

• Phosphate buffer has much lower absorption in UV at low wavelengths, which allows to achieve much smoother baseline and enhance signal‐to‐noise ratio thus improving method sensitivity
• Since capsules and tablets, having the same strength, are prepared from the same granulate, new unknown impurities or degradation products were not expected. Therefore, there is no need to adjust the HPLC method to MS detector.

However, during method validation, we faced an unexpected problem: standard of one of the known impurities was found to be unstable in solution. This observation was made during accuracy test, for solutions of drug product spiked with the standard of this impurity. Such instability was attributed to interaction with phosphate buffer, since no stability issues have ever been experienced while using ammonium acetate buffer. Therefore, for sample preparation, especially when analyzing impurities / degradation products, we used an alternative diluent, having the same aqueous/organic ratio as the mobile phase, but not containing phosphate buffer.

Results: This diluent, not containing phosphate buffer, is fully compatible with the mobile phase, ensured stability of all the known components (standards of API and known impurities) and thus guarantied reliability of results of analyses.

Conclusions: Possible interactions between components of the analyte and components of the diluent / mobile phase should be taken into account in development of HPLC methods.