A complex method requiring determination of assay, purity and impurities in a highly potent API (HPAPI) for a phase I-III project required development, evaluation and validation using ultra-performance liquid chromatography (UPLC). The two specified process generated impurities, each with different solubility profiles, needed to be resolved and quantified. The specified impurities were also treated as highly potent materials requiring special handling through containment procedures such as powered air purifying respirators, gowning and degowning strategies.
The situation
The challenge was to determine the suitable diluent for the solution preparations, as whilst the HPAPI and one of the specified impurities was soluble in concentrated inorganic acid, the other specified impurity was only soluble in a fluorinated organic acid. The stock solutions required preparation in their respective solvents, whilst a common diluent needed to be established for further development to obtain suitable chromatography. The highly potent nature of the materials did not prevent Sterling from successfully overcoming any of the method development challenges.
A 50 mM ammonium phosphate buffer, methanol (gradient) was used as the mobile phase. The diluent solution tried to test the chromatography, as well as the retention time marker, standard and sensitivity, was a mixture of dilute inorganic acid and methanol. From the experiments, it was observed that split peaks were reported in the chromatograms attributed to the diluent, indicating it was not suitable for the method. Therefore, the next step was to try a different diluent. Dilute inorganic acid (0.3M HCL) was used as a diluent for all the solution preparations to observe the peaks obtained during chromatography. Using concentrated acids for preparation of the stock solutions may have hampered their stability, and therefore, a set percentage of concentrated acid sufficient to dissolve the compound was used. The solutions were then diluted to final volume with the diluted acids.
The switch to the 0.3 M HCL diluent resolved the challenge presented by the peaks splitting. The method parameters and instrument conditions used during development. The method enabled us to perform the chromatography required to demonstrate that the method was sensitive, peaks were resolved and that the impurities in the unspiked HPAPI were well below the specification limit. This meant that the method was deemed suitable for further evaluation.
Further work performed
The method was then evaluated for system precision, linearity, accuracy and precision, specificity, limit of detection (LOD) and limit of quantification (LOQ) with the evaluation meeting the acceptance criteria for said parameters. For accuracy and precision, the impurities were spiked at different percentages of the nominal concentration of the API to obtain the data, which was well within the acceptance criteria.
Validation work was performed in line with ICH guidelines for a late phase assay and impurities method. The method was validated for linearity, accuracy and precision, LOD, LOQ, specificity, stability, robustness and intermediate precision. Late phase analytical methods require additional parameters to be tested, such as robustness and intermediate precision as a part of the validation, so these were incorporated in the validation protocol.
The validation work met all acceptance criteria parameters, proving that the method developed was sensitive, selective and robust for its intended purpose. The percentage of individually specified and total impurities in the HPAPI was also well below the specification criteria provided by the customer, demonstrating our ability to work with HPAPI materials, and the expertise of our analytical scientists in developing complex phase appropriate analytical methods.
