Screening and Quantification of Pain and AntidepressantDrugs in Human Urine by Liquid Chromatography-High Resolution Mass Spectrometry

摘要

This method was developed and validated to screen and quantify patient samples in a production environment. Standards and QCs were prepared as single solutions with all analytes present. In order to meet method acceptance, the qualitative and quantitative criteria for each analyte must be met. In this way, the method was designed to have a sufficient number of scans in full-scan mode in order to quantify the parent mass, as well as have a minimum of one MS~2scan to evaluate the accurate mass of fragments and the library search parameters. Having this in mind, two methodologies that collect targeted MS~2 scans were assessed during method development: data dependent MS~2 acquisition (ddMS~2), and data independent acquisition (DIA). During this evaluation, DIA proved to be superior to ddMS~2 in consistently collecting at least one MS~2scan for all analytes at the LLOQ level. The resolution value was also tested during method development. Resolution is a critical parameter given that the method does not include a sample clean-up step. A resolution of 70,000 was chosen as the minimum value that did not result in parent mass or isotope interferences. In order to overcome the problems with accuracy and matrix interference for some of the compounds, the deuteratedand/or C-13 form of each of the analytes was used as an internal standard. Additionally, the hydrolysis step was optimized for time, temperature and IMCSzyme concentration. The optimal conditions that yielded consistent hydrolysis values ≥80% for all the commercially available glucuronide standards were incubation at 65°C for one hour, with 5000 Fishman units/mL of enzyme concentration in the master mix solution. It is important to note that codeine 6-glucuronide, and morphine 6-glucuronide analytes are not effectively cleaved by the majority of the β-glucuronidaseenzymes available in the market. In this method, codeine 6-glucuronide was the analyte that took longer to hydrolyze, but was consistently cleaved by IMCSzyme above 80% at one hour with the conditions specified above. In addition to the parameters shown in Table 1 and Table 2, the internal standard-normalized matrix factor for nine independent matrix sources was also evaluated, and found to be ≤ 14.0%RSD for all the analytes. Assessments of specificity, carryover, and impact from blood contamination (5% v/v) were also performed, and found to be acceptable. The results presented here show the successful validation of the method for the screening and quantification of 47 drugs and metabolites in human urine using the Q-Exactive Orbitrap.