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Analysis of drugs and proteins in biological matrices, using different types of sample preparations and mass spectrometry
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes bioanalysis of small and large molecules in biological matrices and includes screening of illegal drugs in urine with high resolution mass spectrometer, bioanalysis of MTH1 inhibitors in mice plasma and quantification of proteins in plasma and cell lysates.

Screening of illegal drugs was based on high resolving power mass spectrometer (QTOF) and the results were compared to immunoassays. For the study, the nine most commonly abused drugs were selected for screening of a large number of authentic urine samples. Evaluation of the screening results showed that the QTOF generated a low rate of false positive and false negative results and can be used as an alternative or a complementary to immunoassays.

In another study, a bioanalytical method for the two new anticancer targets TH588 and TH287 was developed and validated. The compounds inhibit the MTH1 protein that is required for cancer cell survival. To study the pharmacokinetic properties of the substances, a bioanalytical method was developed using a fast sample preparation followed by LC-MS/MS analysis. Validation showed that the method was linear, accurate and precise in the studied concentration range. Stability tests showed that the substances were stable in mice plasma and under different storage conditions. The study also addresses other important factors such as solubility, chromatography and fragmentation mechanisms.

Two other studies focused on quantification of specific proteins in biological specimens; plasma and bacterial lysates. The matrices are rich in endogenous proteins making quantitative analysis of target proteins challenging. Therefore a new strategy is proposed that combines known procedures in a way it has not been used before. The methods are based on quantification with isotopically labelled peptides added after proteolytic digestion of the sample with immobilized thermolysin or pepsin. The sample digest was analysed on LC-MS/MS and LC/LC-MS/MS systems.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholm Universtiy , 2014. , 59 p.
Keyword [en]
Bioanalysis, drugs, proteins, liquid chromatography, two-dimensional liquid chromatography, mass spectrometry
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
URN: urn:nbn:se:su:diva-107689ISBN: 978-91-7447-984-3 (print)OAI: oai:DiVA.org:su-107689DiVA: diva2:749474
Public defence
2014-11-07, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 13:00 (Swedish)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 2: Manuscript.

Available from: 2014-10-16 Created: 2014-09-24 Last updated: 2014-10-23Bibliographically approved
List of papers
1. Development and validation of method for TH588 and TH287, potent MTH1 inhibitors and new anti-cancer agents, for pharmacokinetic studies in mice plasma
Open this publication in new window or tab >>Development and validation of method for TH588 and TH287, potent MTH1 inhibitors and new anti-cancer agents, for pharmacokinetic studies in mice plasma
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-107705 (URN)
Available from: 2014-09-24 Created: 2014-09-24 Last updated: 2014-09-25
2. A bioanalytical method for quantification of thioredoxins in Bacillus anthracis by digestion with immobilized pepsin and LC-MS/MS and on-line LC/LC-MS/MS
Open this publication in new window or tab >>A bioanalytical method for quantification of thioredoxins in Bacillus anthracis by digestion with immobilized pepsin and LC-MS/MS and on-line LC/LC-MS/MS
(English)Manuscript (preprint) (Other academic)
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-107723 (URN)
Available from: 2014-09-25 Created: 2014-09-25 Last updated: 2014-09-25
3. Digestion of Enolase and Carbonic Anhydrase as Model Proteins for Therapeutic Proteins in Blood Plasma with Immobilized Thermolysin and Quantification of Some of the Peptides by LC/LC-MS/MS
Open this publication in new window or tab >>Digestion of Enolase and Carbonic Anhydrase as Model Proteins for Therapeutic Proteins in Blood Plasma with Immobilized Thermolysin and Quantification of Some of the Peptides by LC/LC-MS/MS
2014 (English)In: Chromatographia, ISSN 0009-5893, E-ISSN 1612-1112, Vol. 77, no 1-2, 59-74 p.Article in journal (Refereed) Published
Abstract [en]

There is a need for fast method development in the early drug discovery phase of therapeutic proteins. Thermolysin has not been used for quantification of proteins in blood plasma earlier. It is a thermostable protease which permits the use of high temperatures for fast hydrolysis of proteins. Model proteins were digested with immobilized thermolysin on agarose gel. Protein-specific peptides were selected for quantitation and quantified based on stable isotope dilution. Protein digests of blood plasma were cleaned and separated using an automated LC/LC-MS/MS system. Essential digestion parameters that influence thermolysin hydrolytic activity were optimized for high peptide yield. The validated methods were selective, linear, precise and accurate with a limit of detection of 2 nM for both proteins. The proposed strategy for method development could be valuable for quantification of proteins in blood plasma samples. The study underscores and discusses important features of the enzymatic digestion and chromatographic separation.

Keyword
On-line two-dimensional liquid chromatography, Mass spectrometry, Hydrolysis with thermolysin, Enzyme immobilization, Quantification of proteins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-100656 (URN)10.1007/s10337-013-2562-z (DOI)000329230500007 ()
Note

AuthorCount:4;

Available from: 2014-02-12 Created: 2014-02-10 Last updated: 2017-12-06Bibliographically approved
4. Evaluation of a direct high-capacity target screening approach for urine drug testing using liquid chromatography-time-of-flight mass spectrometry
Open this publication in new window or tab >>Evaluation of a direct high-capacity target screening approach for urine drug testing using liquid chromatography-time-of-flight mass spectrometry
Show others...
2012 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, no 909, 6-13 p.Article in journal (Refereed) Published
Abstract [en]

In this study a rapid liquid chromatography-time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8 mu m particles. Total analysis time was 4 min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100 ng/mL for amphetamines and opiates, and 5 ng/mL for buprenorphines, with lower limits of quantification were 2.8-41 ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97-0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography-time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC-MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (+/- 20 mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography-time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays.

Keyword
Screening, Drugs of abuse, Urine, Time-of-flight mass spectrometry, Liquid chromatography
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-83938 (URN)10.1016/j.jchromb.2012.10.006 (DOI)000311764400002 ()
Available from: 2012-12-17 Created: 2012-12-17 Last updated: 2017-12-06Bibliographically approved

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