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Turbulent flow online extraction coupled to LC-MS. Application for identification and quantification of drugs and their metabolites


Müller, Daniel Michael. Turbulent flow online extraction coupled to LC-MS. Application for identification and quantification of drugs and their metabolites. 2012, ETH Zürich, Chemie Biologie Pharmazie .

Abstract

In recent years, automation of processes became more and more important in LC-MS analysis in clinical laboratories. There are two important reasons for this development: (a), optimization of the processes, and (b), improvement of patient safety because of the reduced risk of human errors.
In LC-MS analysis of biological samples, sample preparation is usually the most time-consuming step, even though it is much less complicated than for GC-MS. Therefore, automation for LC-MS processes should be started by automation of the extraction.
For the most efficient automation, an online coupling of the extraction process to the LC-MS system should be able. Turbulent flow chromatography, a recently developed online extraction technique, fulfilled this prerequisite. Compared to online-coupled solid phase extraction, a technique that is used more frequently in therapeutic drug monitoring (TDM) and the clinical and forensic toxicological field, the system needs less maintenance and is less prone to mechanical failures, which makes it very attractive. The first aim of this project was the development and validation of a method using online extraction with turbulent flow chromatography for the TDM of two frequently used immunosuppressants, everolimus and sirolimus. With the automation, the throughput of the method was increased significantly compared to an existing method. On the contrary, the complexity of the method was decreased, increasing also the total robustness of the method.
As second main part of the project, an automated toxicological screening system using online extraction with turbulent flow chromatography was developed and thoroughly validated, including method comparisons using real patient samples. The screening system was adequate for the toxiclogical screening of urine, serum and heparinized plasma samples, circumventing extensive manual sample preparation steps due to the online extraction. Using only 600 μL of urine or 100 μL of heparinized plasma or serum, the system was able to detect more than 700 substances, including metabolites. For a specific identification, MS2 and MS3 spectra as well as the retention time were taken as identification criteria. In serum and heparinized plasma, about 80 % of the substances, for which a therapeutic range could be found in the literature, could be identified with a limit of identification below the lower limit of the therapeutic range. The turn-around time until the reporting of the results was about 2 hours for urine samples and 45 min for serum or heparinized plasma samples after arrival of the samples in the laboratory. This was substantially faster than for the previously used GC-MS method.
To answer the question, which matrix should be selected for toxicological screening purposes, a patient sample comparison between urine and heparinized plasma samples was performed. In the sample pairs having different compounds identified in the two matrices, it could clearly be demonstrated that plasma better reflected the current exposure to drugs whereas urine has a longer detection window and therefore allowed the detection of compounds being no more present in the systemic circulation.
The metabolism of new designer drugs entering the black market in fast pace is often unknown. However, knowledge of the metabolites is important in order to make the designer drugs detectable in toxicological screening approaches, or to study the contributions of the metabolites to the pharmacological or toxic effects of the parent compound. Therefore, an automated online metabolism method using human liver microsomes was developed. It was compared to an established offline approach and shown to give very comparable findings. Furthermore, as an example, the metabolism of 11 cathinones, a new type of designer drugs, was studied. In conclusion, two analytical methods, one for TDM of everolimus and sirolimus, and the other for a fully automated toxicological screening procedure for over 700 compounds, have been developed and fully validated. Both methods proved to be robust, increased the throughput of samples and optimized the respective workflows. Heparinized plasma should be used as matrix for a toxicological screening in acute situations, whereas urine is superior in the detection of compounds being no more present in the systemic circulation. The online metabolism method allowed an easy approach to study the metabolism of drugs or drugs of abuse using human liver microsomes.

Abstract

In recent years, automation of processes became more and more important in LC-MS analysis in clinical laboratories. There are two important reasons for this development: (a), optimization of the processes, and (b), improvement of patient safety because of the reduced risk of human errors.
In LC-MS analysis of biological samples, sample preparation is usually the most time-consuming step, even though it is much less complicated than for GC-MS. Therefore, automation for LC-MS processes should be started by automation of the extraction.
For the most efficient automation, an online coupling of the extraction process to the LC-MS system should be able. Turbulent flow chromatography, a recently developed online extraction technique, fulfilled this prerequisite. Compared to online-coupled solid phase extraction, a technique that is used more frequently in therapeutic drug monitoring (TDM) and the clinical and forensic toxicological field, the system needs less maintenance and is less prone to mechanical failures, which makes it very attractive. The first aim of this project was the development and validation of a method using online extraction with turbulent flow chromatography for the TDM of two frequently used immunosuppressants, everolimus and sirolimus. With the automation, the throughput of the method was increased significantly compared to an existing method. On the contrary, the complexity of the method was decreased, increasing also the total robustness of the method.
As second main part of the project, an automated toxicological screening system using online extraction with turbulent flow chromatography was developed and thoroughly validated, including method comparisons using real patient samples. The screening system was adequate for the toxiclogical screening of urine, serum and heparinized plasma samples, circumventing extensive manual sample preparation steps due to the online extraction. Using only 600 μL of urine or 100 μL of heparinized plasma or serum, the system was able to detect more than 700 substances, including metabolites. For a specific identification, MS2 and MS3 spectra as well as the retention time were taken as identification criteria. In serum and heparinized plasma, about 80 % of the substances, for which a therapeutic range could be found in the literature, could be identified with a limit of identification below the lower limit of the therapeutic range. The turn-around time until the reporting of the results was about 2 hours for urine samples and 45 min for serum or heparinized plasma samples after arrival of the samples in the laboratory. This was substantially faster than for the previously used GC-MS method.
To answer the question, which matrix should be selected for toxicological screening purposes, a patient sample comparison between urine and heparinized plasma samples was performed. In the sample pairs having different compounds identified in the two matrices, it could clearly be demonstrated that plasma better reflected the current exposure to drugs whereas urine has a longer detection window and therefore allowed the detection of compounds being no more present in the systemic circulation.
The metabolism of new designer drugs entering the black market in fast pace is often unknown. However, knowledge of the metabolites is important in order to make the designer drugs detectable in toxicological screening approaches, or to study the contributions of the metabolites to the pharmacological or toxic effects of the parent compound. Therefore, an automated online metabolism method using human liver microsomes was developed. It was compared to an established offline approach and shown to give very comparable findings. Furthermore, as an example, the metabolism of 11 cathinones, a new type of designer drugs, was studied. In conclusion, two analytical methods, one for TDM of everolimus and sirolimus, and the other for a fully automated toxicological screening procedure for over 700 compounds, have been developed and fully validated. Both methods proved to be robust, increased the throughput of samples and optimized the respective workflows. Heparinized plasma should be used as matrix for a toxicological screening in acute situations, whereas urine is superior in the detection of compounds being no more present in the systemic circulation. The online metabolism method allowed an easy approach to study the metabolism of drugs or drugs of abuse using human liver microsomes.

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Item Type:Dissertation
Referees:von Eckardstein Arnold, Rentsch Savoca Katharina, Altmann Karl-Heinz, Zenobi Renato
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Clinical Chemistry
Dewey Decimal Classification:610 Medicine & health
540 Chemistry
Language:English
Date:2012
Deposited On:03 Apr 2012 10:00
Last Modified:05 Apr 2016 15:38
Number of Pages:227
Publisher DOI:https://doi.org/10.3929/ethz-a-007139663
Related URLs:http://opac.nebis.ch/F/?local_base=NEBIS&CON_LNG=GER&func=find-b&find_code=SYS&request=007139663

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