Analysis of alkyl-anthraquinone derivatives from hydrogen peroxide industrial process, using LC × LC-HRMS with shifting gradients†

The characterization of industrial working solutions containing numerous structurally related compounds and isomers requires the use of two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS). The separation of alkyl-anthraquinone derivatives produced during hydrogen peroxide production was achieved by coupling a biphenyl and a C18 in the first and second dimensions, combined with the use of continuous shifting gradients in the second dimension (²D). The use of shifting gradients offers a significant advantage over regular gradients, with a 20% increase in occupancy and better separation of isomers eluted within the same modulation. Additionally, MS spectra were improved by reducing ion suppression in the source. The analysis of the industrial solutions produced a list of 226 peaks with 75 different molar masses, thereby confirming the presence of many isomers. The excellent repeatability of the retention times (within ±0.28 s in the second dimension) enabled the method to be used to study the differences between production batches, which were divided into two groups according to their measured hydrogen peroxide yield. Using multivariate statistical tools on the area of each peak, a total of 191 variables, including 11 clusters of insufficiently resolved isomers, were studied by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). A simple Wilcoxon test performed on the most pertinent variables was able to confirm that 28 peaks could be used to monitor the industrial process. This work highlighted the efficacy of combining a very powerful separation technique with the implementation of two simple statistical tests to rapidly identity a set of discriminant markers in a highly complex industrial mixture.