Identifying reliable ions for the statistical differentiation of structurally similar fentanyl analogs

Definitive identification of fentanyl analogs based on mass spectral comparison is challenging given the high degree of structural and, hence, spectral similarity. To address this, a statistical method was previously developed in which two electron-ionization (EI) mass spectra are compared using the unequal variance t-test. Normalized intensities of corresponding ions are compared, testing the null hypothesis (H₀) that the difference in intensity is equal to zero. If H₀ is accepted at all m/z values, the two spectra are statistically equivalent at the specified confidence level. If H₀ is not accepted at any m/z value, then there is a significant difference in intensity at that m/z value between the two spectra. In this work, the statistical comparison method is applied to distinguish EI spectra of valeryl fentanyl, isovaleryl fentanyl, and pivaloyl fentanyl. Spectra of the three analogs were collected over a 9-month period and at different concentrations. At the 99.9% confidence level, the spectra of corresponding isomers were statistically associated. Spectra of different isomers were statistically distinct, and ions responsible for discrimination were identified in each comparison. To account for inherent instrument variations, discriminating ions for each pairwise comparison were ranked based on the magnitude of the calculated t-statistic (t_calc) value. For a given comparison, ions with higher t_calc values are those with the greatest difference in intensity between the two spectra and, therefore, are considered more reliable for discrimination. Using these methods, objective discrimination among the spectra was achieved and ions considered most reliable for discrimination of these isomers were identified.