Analytical Investigation of UDCA Peak Purity by UPLC/MS/MS
Challenge: The technological objective of this project was to investigate the peak purity of Ursodeoxycholic Acid drug substance and drug product with forced degradation by employing Ultra Performance Liquid Chromatography/Mass Spectrometry (UPLC/MS) techniques. Ursodeoxycholic acid (UDCA) is one of the secondary bile acids, which are metabolic by-products of intestinal bacteria. Essentially, Diteba was committed to investigate the degradation pathways and impurity profiles of Ursodeoxycholic Acid drug product by assessing the Ursodeoxycholic Acid peak purity by using a UPLC coupled with Mass Spectrometry (MS).
Solution: A method was previously developed to determine related substances of Ursodeoxycholic Acid by using UPLC technology coupled with an Evaporative Light Scattering (ELS) detector. As the Ursodeoxycholic Acid and its degradation products do not absorb UV, MS technology was employed to investigate the Ursodeoxycholic Acid peak purity.
UPLC MS/MS Analysis of Ursodeoxycholic Acid API, drug product and placebo samples were carried out to assess the purity of Ursodeoxycholic Acid peak. This was performed to show the Ursodeoxycholic Acid peak generated as a result of forced degradation (acid, base, heat, light and peroxide) were free from any coeluting related substance.
The Total Ion Chromatograms (TIC) and full MS scans of degraded Ursodeoxycholic Acid peaks are similar to that of the reference standard. The peaks with 525.5 and 553.5 Da for some of the samples are attributed to a different batch of solvent used in the analysis and are not uncommon in this type of analysis. 
It is also important to note that any likely related substance coeluting with Ursodeoxycholic Acid would have a smaller m/z than 391.4 (peak). Thus it could be concluded that the related substances method for Ursodeoxycholic Acid was stability-indicating with respect to the specificity of the analytical method.
Background subtraction eliminated many peaks attributed to solvent cluster ions. However, there was still some background noise that remained. In order to determine whether these peaks came from the sample, an acceptance percentage based on the base peak was calculated by dividing the intensity of base peak in each sample by the highest intensity peak found in the diluent. The full MS scan revealed a combined spectrum across the Ursodeoxycholic Acid peak.
In order to maintain compatibility with the ELS results, the chromatographic conditions were modified based on the method provided by the sponsor as a standard to couple with MS. The MS parameters were optimized on the Ursodeoxycholic Acid peak to provide maximum and minimum fragmentation.
Result: With background subtraction, full MS scan spectra of blank (diluent), Ursodeoxycholic Acid reference standard and samples were collected and analyzed by individually comparing the TIC's of samples to diluent and reference standard. Suspect impurity responses, if found, were then investigated by comparing the extracted ion chromatograms of suspect responses to the Ursodiol reference standard.