Uncertainty estimation strategies for quantitative non-targeted analysis.

Non-targeted analysis (NTA) methods are widely used for chemical discovery but seldom employed for quantitation due to a lack of robust methods to estimate chemical concentrations with confidence limits. Herein, we present and evaluate new statistical methods for quantitative NTA (qNTA) using high-resolution mass spectrometry (HRMS) data from EPA's Non-Targeted Analysis Collaborative Trial (ENTACT). Experimental intensities of ENTACT analytes were observed at multiple concentrations using a semi-automated NTA workflow. Chemical concentrations and corresponding confidence limits were first estimated using traditional calibration curves. Two qNTA estimation methods were then implemented using experimental response factor (RF) data (where RF = intensity/concentration). The bounded response factor method used a non-parametric bootstrap procedure to estimate select quantiles of training set RF distributions. Quantile estimates then were applied to test set HRMS intensities to inversely estimate concentrations with confidence limits. The ionization efficiency estimation method restricted the distribution of likely RFs for each analyte using ionization efficiency predictions. Given the intended future use for chemical risk characterization, predicted upper confidence limits (protective values) were compared to known chemical concentrations. Using traditional calibration curves, 95% of upper confidence limits were within ~tenfold of the true concentrations. The error increased to ~60-fold (ESI+) and ~120-fold (ESI-) for the ionization efficiency estimation method and to ~150-fold (ESI+) and ~130-fold (ESI-) for the bounded response factor method. This work demonstrates successful implementation of confidence limit estimation strategies to support qNTA studies and marks a crucial step towards translating NTA data in a risk-based context.