BLT Stages

Excessive alcohol consumption is more and more common. Alcohol use can be detected in blood, urine and hair. Alcohol biomarkers are used for this purpose. In this study, the alcohol biomarker phosphatidylethanol (PEth) is used. This marker can be measured by microsampling. Routinely at the Gent University Volumetric Absorptive Microsampling (VAMS) are used, but Tasso devices or dried blood spots (DBS) can also be used. The Tasso devices are validated by comparison with the VAMS . of the use of conversion factor for Peth results obtained via Tasso devices will be validated, based on previously obtained concentrations of PEth with VAMS. The DBS will also be compared with VAMS, as well as different extraction techniques for both. A solid phase extraction protocol, making use of  Phospholipid Removal Plate (PLD), with which PEth is extracted from whole blood is also validated. A liquid chromatograph/mass spectrometer is used to determine the concentration of PEth and to determine how much PEth is or is not lost after whole blood extraction. The different extraction methods for VAMS and DBS give similar results, meaning that both extraction methods may be used. The validation of the Tasso devices causes some ambiguity. In one measurement there are corresponding results, in the other measurement there are not. These are issues that can be investigated further. The PLD certainly needs to be developed further. A lot of adjustments to the protocol were made, whereby the recovery was often very good, but the absolute signal was poor. This makes a lot of contradictory results, so there is still uncertainty as to whether or not the PLD can be integrated as a sample preparation step.

 

This bachelor project focusses on the implementation and validation of a cannabinoid screening method using high resolution mass spectrometry. The Laboratory for Toxicology from the University of Ghent performs measurements in blood, urine and other biological matrices for the detection of medication and drugs of abuse. In their portfolio is the confirmation of cannabis use by the screening of urine for cannabinoids. Cannabis is a highly abused illicit drug and poses e.g., a lot of risk in terms of driving. One application of this screening is upon driver’s license regranting, where the presence of cannabis must be excluded. The laboratory uses gas chromatography-mass spectrometry as a semi-quantitative confirmation of the presence of 11-nor-delta 9-carboxy-tetrahydrocannabinol (THC-COOH). Because of the intensive sample preparation and need for derivatization before GC/MS, the laboratory strives for a simpler, less intensive, but comparable method for the detection and confirmation of cannabinoids. The sample preparation of urine for the liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-MS-QqTOF) method contains less steps than the sample preparation for GC/MS. Moreover, LC-MS-QqTOF makes it possible to analyze less volatile components without the need for derivatization.

To implement this method in the routine laboratory an optimization of the sample preparation is required. The implementation also includes a validation of the limit of detection (LOD) and matrix effects. The validation of the LOD provides information about the sensitivity and specificity of the instrument. The matrix effects provide information about the interference of matrix components in the ionization process. At the end of the experiments a comparison between the LC-MS-QqTOF and GC-MS is made. This is carried out by comparing the concentration of THC-COOH in forensic samples obtained by the GC-MS and LC-MS-QqTOF.

For the determination of the LOD two blanc urines and solvent were used. The samples were spiked using a cannabinoid standard at a concentration level of 10, 15, 20 and 30 ng/ml. A blank of every sample was also included. This resulted in a limit of detection of 10 ng/ml for THC-COOH, 11-OH-THC and cannabinol. The LOD for cannabidiol was set at 15 ng/ml and 20 ng/ml for THC. Matrix effects were determined by spiking ten urines and repeatedly water as solvent at a concentration level of 50 ng/ml. The areas of the different components were evaluated in urine and water. The majority of the samples showed ion suppression (internal standard compensated) and enhancement between the accepted interval of 100 ± 15 %, with a variation below 15 %. When the matrix effects were less than 85 % or larger than 115 %, the CV was above 15 %. By reducing the variation, the matrix effects might be improved. When comparing the concentration of forensic samples obtained by the GC-MS and LS-MS-QqTOF a large difference is observed, with a minimal deviation of 30 %. This may be due to a calibration bias. It can also be caused by not adding the internal standard at the most optimal step during sample preparation for both calibrators and samples upon LC-MS-QqTOF analysis.

The results of this preliminary validation show that there is a great potential for this screening method for cannabinoids with the LC-MS-QqTOF. However, there is still need for further validation of the method in terms of accuracy, precision and robustness. 

 

High resolution mass spectrometry (HRMS) is becoming more prevalent as a screening tool in forensic toxicology. HRMS is being explored as an alternative technique, as the instruments collect untargeted, accurate mass data, which permit identification of a broad range of toxicological relevant compounds within a single analytical run.

The tendency to use scientific supported evidence in forensic cases is becoming more important, the use of different independent methods for systematic toxicological analysis can significantly increase the reliability of the evidence. Therefore, The Laboratory of Toxicology (UGent) initiated research to validate and implement a new screening method using HRMS, as published by Thoren et al. 

A number of factors that influence the quality and reliability of the results will be evaluated before implementation. The retention time, limit of detection and the matrix effects in urine and vitreous humor are validated for common forensic toxicological compounds, using a standard mix with 108 compounds.

The accuracy of the calibration is determined for the ‘TOF MS APCI solution’ method and the ‘TOF MS APCI solution – with product’ method. The first method is more accurate, the average bias is respectively 0.0005 ± 0.00015 Da and 0.0013 ± 0.00059 Da. Levey-Jenningsplots are set up for the system function check and the internal standard to evaluate the quality of the system. The spectral library is extended with 20 reference standards, the optimal method is to infuse the compounds into the mass spectrometer. The retention time is determined for 98 compounds (91%). The best sensitivity (72.5%) and specificity (99.2%) is obtained with a retention time width of 0.3 min and a mass width of 0.01 Da. The limit of detection and matrix effect is validated for 75 components (77%). Nevertheless, it is recommended to perform the matrix effects experiment under better conditions.

HRMS has potential to be used as an additional method for systematic toxicological analysis. The method can detect a wide range of compounds with sufficient sensitivity, both in urine and vitreous humor. However, further research into the possibilities and disadvantages of the screening method is necessary to improve the sensitivity of the method.

 

Abnormal thyroid function has important public health consequences. Disagreement between results from different free thyroxine (fT4) methods has been a problem that limits their clinical utility. Not necessarily for diagnosis and/or monitoring of patients, but e.g. for establishing common reference intervals and clinical decision limits, to develop evidence-based clinical practice guidelines and application of consistent standards of medical care.

To reach a solution, a reference measurement procedure (RMP) for fT4 was developed based on equilibrium dialysis isotope dilution-liquid chromatography-tandem mass spectrometry. The equilibrium dialysis (ED) part of the RMP has to adhere to strictly determined parameters. In this project a robustness study evaluates the performance of different ED materials to prove their suitability for analytical inter-laboratory studies.

A set of parallel tests was performed with the endorsed RMP against the introduction of new ED-materials (i.e. other cell designs or differences in membrane cut-off). The results show that the ratio between the volume of sample per area of exposed membrane has a big influence on the time needed to reach equilibrium. The best suited cells for the procedure – Dianorm Macro 1 S - were the ones with a bigger ratio. An increase in the molecular weight cut-off of the membrane (from 5 kDa to 10 kDa) was also proved possible.

Given the influence of the cell’s design on the dialysis’ time, a study to determine the time needed to reach equilibrium is advised for all different cell-types prior to their use in the RMP.

 

This bachelor thesis can be seen as a preparative step for developing and validating the eventual reference measurement procedure for rT3. This procedure will likely be published by the Ref4U of the UGent. This RMP for rT3 can aid to the study of positive involvement of T3 and rT3 measurements, in diagnosis and monitoring of patients with thyroid disease.. Especially for people who, after treatment, still have no relief of symptoms. The ratio of T3 and rT3 can also help to explain a wrong metabolism which TSH and FT4 can’t do. For now this procedure is in its early stages. The procedure consists of a protein precipitation whereafter the analyt is extracted using double liquid/liquid extraction etylacetate. The detection of the analyte is done using liquid chromatography – Tandem Massa spectrometrie. For the primary validation of the procedure the precision was determined by using the CLSI EP 5 manual. The imprecision calculated complies with the predetermined criteria, i.e. a total coefficient of variation below 5% and a within run coeffficient of variation below 2,5%. For the trueness of the procedure it can be said that for now only relatively high concentrations of rT3 can be measured with high reliability. More experiments are needed to find out what the reasons are and how to improve the method. In addition, the method needs optimization in order to make it robust enough to also measure other matrixes such as pools or samples based on depleted serum. 

 

The aim of this bachelor thesis is to integrate a method for a quantitative determination of GHB in a routine laboratory. The method, which is already developed and validated, has to be transferred to the GC-MS in the routine laboratory. After validation, the determination of GHB can be added to the scope of ISO 17025 accreditation. With this accreditation the Laboratory of Toxicology proves that it works with a good functioning management system and that the obtained results are reliable.

The original method is completely validated. Hence to integrate this method in the routine toxicological laboratory only the instrument specific characteristics have to be validated. Consequently the limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy and precision were tested. 

No tests were performed for the LOD and LOQ. The LOD was determinated on 1 µg/ml in the original methode. Because the Laboratory of Toxicology works with a cut-off level of 5 µg/mL no additional experiments were performed. Only the signal to noise ratios for the lowest calibrator and the calibrator closest to the cut-off level were verified in order to confirm that these meet the specifications for LoD and LoQ respectively. The results of the S/N ratio of the LOD and LOQ were both higher than the criteria of respectively three for the LOD and five for the LOQ.

A six-point calibration curve was tested for five days, and its linearity was evaluated. Finaly, we used weighted lineair regression (1/x2) to calculate the results. Note, at each concentration level, the response was corrected for the response of the blanc.

To test the accuracy and precision three quality control (QC) samples were made, independent from the calibration curve. They were tested for five days in a row together with the calibration curve. Quality specifications were: a bias within ± 15% (within ± 20% for the lowest calibrator) and a preciosion, expressed as CV, less than 15% (or 20% for the lowest calibrator).

The results for two of the three QC samples are between the criteria for accuracy, only for the lowest sample, the accuracy is borderline. More measurements are needed to increase the confidence in the result. Also for the precision, although the results are below the specifications, from a statistical point of view more measurements are needed to confirm these observations. Finally, results calculated with the calibration curve were compared with results calculated via 1-point calibration. The rationale was that, the 1-point calibration curve would be the routine labortories first screening test. If a results is higher than the cut-off level, it would be quantitated with using a calibration curve. We could prove that results calcualted in either way are comparable, and the conclusions made for accuracy and precision are similar.