A non-routine HPLC approach, direct large-volume-injection, was demonstrated for the analysis of drug products at strengths as low as 0.01 mg per dose unit. When combined with UV detection, it enabled a direct injection of 1.8 mL of drug-product-sample into a single analytical column and determination of impurities of the drug products at a concentration as low as 2 ng/mL, or 0.1% of active drug in full compliance with the ICH guideline. Through an on-column focusing approach such a large volume of sample was first concentrated on-line, and then chromatographed. The impurities were successfully identified and quantified using matching placebo as controls. The limit of detection and limit of quantitation were found to be 0.4 and 1.3 ng/mL, respectively. Accuracy (recovery) and precision (repeatability) were 97.7-109.2% and 3.4-8.6%, respectively.

In facing the challenges of analyzing low-dose-drug-products, a series of analytical control strategies were established. Through a series of examples, the utilization of these control strategies, including environmental contamination, chromatographic background, drug recovery, column disturbance, peak shape, retention variation and data processing, is thoroughly discussed in this paper. On-column focusing was achieved through a series of optimizations, including a factorial experiment design.