Development and application of a statistical approach to establish equivalence of unabbreviated mass spectra.

摘要

In many regulatory applications, identification is based on mass spectral comparisons of a compound to a reference standard or library; however, no confidence level associated with the match is determined. Described herein is a means for determining statistical equivalence to the mass spectral identification of an unknown compound. A statistical model was developed to predict standard deviations, which were used in an unequal variance t-test to compare spectra at every m/z ratio over the entire scan range. If determined to be statistically indistinguishable at every m/z ratio, the random-match probability (RMP) was calculated, assessing the probability that the characteristic fragmentation pattern of the mass spectra would occur by random chance alone.;Due to the challenge of differentiating similar mass spectra, the method was initially developed using alkane and alkylbenzene standards of varying concentrations. Using the developed method, replicate spectra were successfully associated at the 99.9% confidence level, with RMP values ranging from 10 -29 to 10-50. Despite the similarity in fragmentation patterns, spectra were distinguished from others in the homologous series. Moreover, the alkane spectra were appropriately associated to, and discriminated from, normal and branched alkanes in a standard reference library at the 99.9% confidence level.;The statistical method was further investigated using salvinorin A, the hallucinogenic compound in the plant Salvia divinorum. Spectra of salvinorin A were statistically associated to those of salvinorin A standards, with RMP values ranging from 10-126 to 10-134, and were distinguished from spectra of salvinorins B, C, and D at the 99.9% confidence level. Statistical association of salvinorin A spectra from eight different geographical locations was possible at 90.0 to 99.9% confidence levels, with RMP values ranging from 10-37 to 10-126 , while discrimination was possible at the 99.9% confidence level for salvinorins B and C and 99.0 to 99.9% for salvinorin D. In addition, 441 different Salvia species and varieties were screened for salvinorin A using the developed method. Mass spectra of compounds with similar retention times were statistically discriminated from salvinorin A at the 99.9% confidence level.;Lastly, mass spectra of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), phentermine, and psilocin case samples (n = 36) were collected by an accredited forensic laboratory using their routine procedures. Using the developed method, these spectra were statistically associated to corresponding reference standards at the 99.9% confidence level, with RMP values ranging from 10-37 to 10-41. The spectra of the case samples were discriminated from other reference standards at the 99.0% or 99.9% confidence level. Moreover, the case samples were appropriately associated to, and discriminated from, spectra in a standard reference library at the 99.0% or 99.9% confidence level.;Therefore, a method was developed for assigning statistical significance to the comparison of mass spectra that is simple and rapid. This method may be useful for industrial quality control as well as for many regulatory applications, such as identification of environmental pollutants, food and drug contaminants, and controlled substances.