A mysterious autosampler problem is solved.
We recently experienced some mysterious and sporadic liquid chromatography (LC) results using an ultrahigh-pressure LC (UHPLC) system. This incident took us a week of intensive investigation involving many hypotheses and numerous blind alleys until we found the root cause. We believe this issue has not been previously reported and could easily be missed by many LC users (1–4). Here is our story.
The ProblemOur problems began when we performed potency and impurity assays of two newly developed clinical drug products (capsules and tablets). We followed a validated 42-min procedure that had been used successfully for two years and modified it into a faster and equivalent 15-min UHPLC method (5). The sample preparation procedure in the modified method uses 20 mM ammonium formate, pH 3.7, as the extraction solvent. We used an Agilent model 1290 UHPLC (Santa Clara, California, USA) with 3-μL injections and performed many area-percent analyses without any issues. However, this time the potency assay failed system suitability tests because the response factors for duplicate standard (calibrator) preparations differed by ~20%. Curiously, peak area repeatability from the same vial was excellent (<0.3% RSD), indicating that the autosampler was not malfunctioning. Duplicate standard preparations were then made by two analysts. Highly variable results and high responses for the calibrator were observed, which led to artificially low potency values of the drug products. We decided to launch a more systematic investigation.
We started our investigation by interviewing the primary analyst and checking all the calculations for the method. We noted higher than expected responses from some calibrator vials but not others — even though all calibrator solutions (~0.5 mg/mL) should have provided nearly identical responses. It seemed quite inconceivable that we obtained responses from two duplicate calibrator preparations that varied as much as 20–30%.