**4. Conclusions**

HILIC is a popular chromatographic method for the analysis of hydrophilic compounds. The separation mechanism in HILIC is more complicated in regards to reversed phase HPLC due to the various kinds of interactions that rule the retention. Despite the increased number of publications in the last years, the separation mechanism in HILIC is still under investigation and many analysts are puzzled over the use of this chromatographic method [32,33]. Thus, understanding the influence of operational parameters in HILIC is crucial for an effective method development. The chemical nature of bisphosphonates, both polar and ionic, makes them perfect candidates for HILIC. In this work the chromatographic behavior of two nitrogen-containing bisphosponates, namely risedronate and zoledronate, and one non-nitrogen-containing bisphosphonate, namely tiludronate, has been thoroughly investigated under zwitterionic HILIC conditions. The results indicate that apart from partition, which is the dominant separation mechanism in HILIC, electrostatic repulsions play an important role to the retention of bisphosphonates. Peak tailing of the two nitrogen-containing bisphosphonates was improved by the addition of sodium pyrophosphate to the mobile phase. A zwitterionic hydrophilic interaction liquid chromatography method coupled to diode-array detection was developed, validated and applied to the quantitation of risedronate in pharmaceutical formulations. The proposed method is stability-indicating since it allows accurate and precise quantitation of risedronate in tablets without any interference from excipients or degradation products, and it is

applicable to routine quality control of risedronate in tablets. A run time of less than 6 min ensures rapid quantitation.

**Author Contributions:** I.P., E.T., C.P. and A.V. participated in designing the study. E.T., E.K. and I.P. conducted the study. Data collection and analysis was performed by I.P., E.T., and A.V. The manuscript was written by I.P., C.P. and A.V.

**Funding:** This research received no external funding.

**Acknowledgments:** We would like to acknowledge George Chorafakis for proofreading the manuscript.

**Conflicts of Interest:** The authors have no conflict of interest.
