A Look At Column Reproducibility: Analysis Of Endogenous Steroid Hormones In Serum

By Kenneth D. Berthelette, Jennifer M. Nguyen, Chelsea Plummer, Kim Haynes, Jonathan E. Turner, and Jonathan P. Danaceau

Having high quality products, particularly liquid chromatography (LC) stationary phases and columns, ensures that the data generated for an assay is accurate and reproducible over time. The quality of LC stationary phases can be measured by batch-to-batch reproducibility testing for a given assay to ensure that each manufactured lot of material performs similarly. Most batch-to-batch tests are performed using three batches of material made within a certain time period, such as those within a method validation kit. While these materials can work well, their use may only provide a snapshot of the overall quality of the product. To get the full picture, historical batches of material should be tested, as this provides a better scope of overall product quality. 

The ACQUITY HSS T3 stationary phase is a commonly used C₁₈ bonded phase using fully porous silica particles. This phase is designed to retain polar analytes better than a traditional C₁₈ without compromising the separation of hydrophobic compounds. This phase has been used in many applications and publications for separations ranging from small molecule pharmaceuticals in dosage form to bioanalytical applications.^4-6 To demonstrate the quality of the HSS T3 material, three columns were packed with different batches of material. These materials were made five years apart ranging from 2016 to 2021. All three columns were then used to separate endogenous steroid hormones in human serum to assess the reproducibility of the data across all columns.