RESUMO
RATIONALE: For the characterization of the chemical composition of complex matrices such as tobacco smoke, containing more than 6000 constituents, several analytical approaches have to be combined to increase compound coverage across the chemical space. Furthermore, the identification of unknown molecules requiring the implementation of additional confirmatory tools in the absence of reference standards, such as tandem mass spectrometry spectra comparisons and in silico prediction of mass spectra, is a major bottleneck. METHODS: We applied a combination of four chromatographic/ionization techniques (reversed-phase (RP) - heated electrospray ionization (HESI) in both positive (+) and negative (-) modes, RP - atmospheric pressure chemical ionization (APCI) in positive mode, and hydrophilic interaction liquid chromatography (HILIC) - HESI positive) using a Thermo Q Exactive™ liquid chromatography/high-resolution accurate mass spectrometry (LC/HRAM-MS) platform for the analysis of 3R4F-derived smoke. Compound identification was performed by using mass spectral libraries and in silico predicted fragments from multiple integrated databases. RESULTS: A total of 331 compounds with semi-quantitative estimates ≥100 ng per cigarette were identified, which were distributed within the known chemical space of tobacco smoke. The integration of multiple LC/HRAM-MS-based chromatographic/ionization approaches combined with complementary compound identification strategies was key for maximizing the number of amenable compounds and for strengthening the level of identification confidence. A total of 50 novel compounds were identified as being present in tobacco smoke. In the absence of reference MS2 spectra, in silico MS2 spectra prediction gave a good indication for compound class and was used as an additional confirmatory tool for our integrated non-targeted screening (NTS) approach. CONCLUSIONS: This study presents a powerful chemical characterization approach that has been successfully applied for the identification of novel compounds in cigarette smoke. We believe that this innovative approach has general applicability and a huge potential benefit for the analysis of any complex matrices.
RESUMO
BACKGROUND: Owing to the lack of an internationally recognized tacrolimus reference material and reference method, current LC-MS and immunoassay test methods used to monitor tacrolimus concentrations in whole blood are not standardized. The aim of this study was to assess the need for tacrolimus assay standardization. METHODS: We sent a blinded 40-member whole-blood tacrolimus proficiency panel (0-30 µg/L) to 22 clinical laboratories in 14 countries to be tested by the following assays: Abbott ARCHITECT (n = 17), LC-MS (n = 9), and Siemens Dade Dimension (n = 5). Selected LC-MS laboratories (n = 4) also received a common calibrator set. We compared test results to a validated LC-MS method. Four samples from the proficiency panel were assigned reference values by using exact-matching isotope-dilution mass spectrometr at LGC. RESULTS: The range of CVs observed with the tacrolimus proficiency panel was as follows: LC-MS 11.4%-18.7%, ARCHITECT 3.9%-9.5%, and Siemens Dade 5.0%-48.1%. The range of historical within-site QC CVs obtained with the use of 3 control concentrations were as follows: LC-MS low 3.8%-10.7%, medium 2.0%-9.3%, high 2.3%-9.0%; ARCHITECT low 2.5%-9.5%, medium 2.5%-8.6%, high 2.9%-18.6%; and Siemens/Dade Dimension low 8.7%-23.0%, medium 7.6%-13.2%, high 4.4%-10.4%. Assay bias observed between the 4 LC-MS sites was not corrected by implementation of a common calibrator set. CONCLUSIONS: Tacrolimus assay standardization will be necessary to compare patient results between clinical laboratories. Improved assay accuracy is required to provide optimized drug dosing and consistent care across transplant centers globally.
