UG Laboratorium voor Toxicologie/Ref4U
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.
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.
Katleen van Uytfanghe