Challenges in the identification and quantification of an unknown impurity in chenodeoxycholic acid drug substance

In 2018 the Amsterdam University Medical Centre decided to prepare chenodeoxycholic acid (CDCA) capsules (also known as pharmacy compounding) for patients with the genetic metabolic disease cerebrotendinous xanthomatosis (CTX) when the product with a marketing authorization was commercially unavailable for patients. However, after reanalysis, unknown impurities were identified in the CDCA active pharmaceutical ingredient (API) using thin-layer chromatography from the European Pharmacopoeia (Ph.Eur.) monograph. Therefore, the API did not comply with the Ph.Eur. specifications for related substances and as a result, pharmacy compounding was halted and an investigation was initiated to identify and quantify the unknown impurities. Meanwhile, a second CDCA API was sourced from another manufacturer. However, this API also appeared to contain an unknown impurity. This impurity could be identified as a dimer of CDCA using reversed phase liquid chromatography mass spectrometry. Since the Ph.Eur. at the time did not describe a suitable analytical method for the quantification of this new impurity, a high pressure liquid chromatography with differential refractometer (HPLC-RI) method was developed to quantify the dimer. Subsequently, in 2019, a new draft version of the CDCA Ph.Eur. monograph was published, including the dimer as a new impurity together with a HPLC-RI method for its identification and quantification. The CDCA-dimer is classified as non-toxic and permitted in the CDCA API up to a maximum of 0.5 %. Because the API complied with the updated Ph.Eur. specifications, pharmacy compounding of CDCA capsules could be resumed.