Evaluation of multiple reaction monitoring cubed performed by a quadrupole-linear ion trap mass spectrometer for quantitative determination of 6-sulfatoxymelatonin in urine

•Evaluation of LC-MRM3 for quantitation of 6-Sulfatoxymelatonin in urine.•Strong degradation of MRM3 signal for 6-Sulfatoxymelatonin in water solutions.•MRM3 signal degradation disappears for 6-Sulfatoxymelatonin at analysis in urine.•6-Sulfatoxymelatonin and its deuterated analog behave very differently at MRM3.•MRM3 parameters for quantitation of 6-Sulfatoxymelatonin in urine were optimized. Liquid chromatography (LC) - mass spectrometry quantitative analysis of substances in biological samples is usually performed in the multiple reaction monitoring (MRM) variant. In complex biological matrices, strong interferences can be observed when using the LC-MRM method. Interference levels can be significantly reduced by using LC - multiple reaction monitoring cubed (MRM3). 6-sulfatoxymelatonin (6-SM) is a metabolite of melatonin, an important regulator of many biological processes. The quantitative analysis of 6-SM in urine allows monitoring of the melatonin level in the blood. The aim of the present work was to evaluate the LC-MRM3 method for the quantitative determination of 6-SM in urine. We found that for 6-SM in aqueous solutions, under some parameters of the MRM3 experiment, the effect of degradation of the MRM3 signal is observed. When analyzing 6-SM in urine, this signal degradation effect was significantly reduced. We have shown that optimization of such parameters of the MRM3 method as the linear ion trap fill time, the number of scans to sum, and the range of triple-stage scan allows obtaining the LC-MRM3 method, which is comparable to the LC-MRM in sensitivity and significantly exceeds it in selectivity.