Qualitative method validation and uncertainty evaluation via the binary output: I – Validation guidelines and theoretical foundations

Qualitative methods have an important place in forensic toxicology, filling central needs in, amongst others, screening and analyses linked to per se legislation. Nevertheless, bioanalytical method validation guidelines either do not discuss this type of method, or describe method validation procedures ill adapted to qualitative methods. The output of qualitative methods are typically categorical, binary results such as “presence”/“absence” or “above cut-off”/“below cut-off”. Since the goal of any method validation is to demonstrate fitness for use under production conditions, guidelines should evaluate performance by relying on the discrete results, instead of the continuous measurements obtained (e.g. peak height, area ratio). We have developed a tentative validation guideline for decision point qualitative methods by modeling measurements and derived binary results behaviour, based on the literature and experimental results. This preliminary guideline was applied to an LC-MS/MS method for 40 analytes, each with a defined cut-off concentration. The standard deviation of measurements at cut-off ( ) was estimated based on 10 spiked samples. Analytes were binned according to their %RSD (8.00%, 16.5%, 25.0%). Validation parameters calculated from the analysis of 30 samples spiked at and (false negative rate, false positive rate, selectivity rate, sensitivity rate and reliability rate) showed a surprisingly high failure rate. Overall, 13 out of the 40 analytes were not considered validated. Subsequent examination found that this was attributable to an appreciable shift in the standard deviation of the area ratio between different batches of samples analyzed. Keeping this behaviour in mind when setting the validation concentrations, the developed guideline can be used to validate qualitative decision point methods, relying on binary results for performance evaluation and taking into account measurement uncertainty. An application of this method validation scheme is presented in the accompanying paper (II – Application to a multi-analyte LC-MS/MS method for oral fluid).