English Title: Development of a reference measurement procedure for delta-9-tetrahydrocannabinol (THC) in serum using isotopic dilution mass spectrometry and vertification of THC-Glucuronide (THCglu) as a marker in urine to indicate THC in serum

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Abstract

In der vorliegenden Arbeit wurde eine Primärmethode für die quantitative Bestimmung von delta-9-Tetrahydrocannabinol (THC) in Serum entwickelt und validiert. Als Methode kam die Gaschromatographie-Massenspektrometrie (GC/MS) unter Anwendung der Isotopenverdünnungsanalyse zum Einsatz. Der Hintergrund ist der im Dezember 2004 in Deutschland eingeführte Grenzwert im §24a des Straßenverkehrsgesetzes (StVG) von 1 ng/mL THC in Serum. Diese Konzentration lässt es als möglich erscheinen, dass eine Beeinflussung der Fahrtüchtigkeit durch Cannabis vorliegen kann. Dementsprechend war das Ziel der Arbeit, zu untersuchen wie genau THC in diesem Bereich messbar ist. Mit Hilfe eines gut charakterisierten THC-Referenzmaterials wurden erstmals eine Rückführbarkeit der Methode sowie eine genaue Betrachtung der Messunsicherheit ermöglicht. Die Unsicherheitsbetrachtung der Messmethode wurde mittels der Top-down -Methode bestimmt, die mit dem Guide to the Expression of Uncertainty in Measurement (GUM) konform ist. Die erweiterte relative Messunsicherheit lag für die zwei untersuchten Konzentrationsniveaus (1 ng/mL und 2,4 ng/mL THC in Serum) unterhalb von 2 %, entsprechend einem Konfidenzintervall von 95 %. Dabei zeigten die Schwankungen der GC/MS-Messung den größten Einfluss auf das Unsicherheitsbudget, gefolgt von der Reinheitsbestimmung des THC-Referenzmaterials. Anhand der Untersuchungen wurde gezeigt, dass die THC-Konzentration im Bereich des bestehenden Grenzwertes mittels Primärmethode mit sehr geringer Messunsicherheit bestimmt werden kann. Die Primärmethode bildet somit die Grundlage für ein nationales Normal bereitgestellt, welches eine Rückführbarkeit für THC-Messungen mit hoher Genauigkeit ermöglicht. Die Primärmethode kann künftig zur Bestimmung von THC-Konzentrationen von Kontrollseren, Kalibrier- und Standardlösungen eingesetzt werden, welche in Routinelaboratorien Anwendung finden. Im praktischen Probengut wurde untersucht, ob THC-Glucuronid (THCglu) als direkter Nebenmetabolit des THCs in Urin einen brauchbaren Marker für die Anwesenheit von mindestens 1 ng/mL THC in Serum darstellt. In der Praxis eingesetzte immunologische Urintests basieren auf 11-nor-9-Carboxy-THC (CTHC) und dessen Glucuronid, den Hauptmetaboliten des THCs in Urin. Aufgrund der komplexen Bildungs- und Ausscheidungsverhältnisse von CTHC besteht jedoch nur ein begrenzter Zusammenhang zu der THC Konzentration in Serum. Für die Untersuchung wurden 211 Urinproben potentieller Cannabiskonsumenten auf THCglu und CTHC getestet. In einem anschließenden Vergleich mittels ROC-Analyse zeigte sich, dass beide Metaboliten des THCs etwa gleichwertig als diagnostische Marker in Urin geeignet sind und THCglu keine Verbesserung gegenüber CTHC aufweist. Für die THCglu-Messungen wurde daher ein neues Verfahren basierend auf Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC/MS/MS) an einem Triple Quadrupol-Massenspektrometer entwickelt und validiert. Die Studie ergab, dass THCglu kein besserer Marker für THC in Serum ist als CTHC.

Translation of abstract (English)

The present work on the "development of a reference measurement procedure for delta-9-tetrahydrocannabinol (THC) in serum using isotopic dilution mass spectrometry and vertification of THC-Glucuronide (THCglu) as a marker in urine to indicate THC in serum" was composed of a methodical part and a practice-related study. In the methodical part, the objective was the development and validation of a reference measuring method to determine delta-9-tetrahydrocannabinol (THC) by means of an isotopic dilution mass spectrometric (IDMS) method. THC is the psychoactive main component of cannabis. The background here is the threshold value of 1 ng/mL for THC in serum - introduced in Germany in December 2004 in Section 24a of the Straßenverkehrsgesetz (StVG) (Road Traffic Act). This concentration makes it appear possible that a person's fitness to drive can be influenced by cannabis. Within the scope of the work, investigation was made as to how accurately THC can be measured in this range of concentration. The IDMS method is regarded as a primary ratio method and is thus suitable for the development of a primary method. With the aid of this reference method, a national "standard" has been provided in order to enable traceability and comparability for THC measurements both at national and also at international level. First of all, a reference material for THC had to be created. To do this, very pure THC was purchased and subsequently characterised. The developed method was investigated within the scope of the validation as to linearity, analytical threshold values, accuracy and precision, and subsequently the total uncertainty of the THC determination was calculated. The uncertainty analysis of the GC/MS method was determined by means of the "top-down" method which complies with the Guide to the Expression of Uncertainty in Measurement (GUM). Thereby, the measurement uncertainty was investigated for two THC concentrations in serum (1 ng/mL and 2.4 ng/mL). The lower concentration level corresponds to the concentration of the existing threshold value in Germany. For both THC concentrations, an optimum and/or minimum uncertainty of less than 2 % was calculated. The spread of the GC/MS measurement indicated the greatest influence on the uncertainty budget, followed by the purity analysis. The providing of a national "standard" for THC has thus been successfully concluded. The next step will be the external quality control in the form of an international round robin test. The primary reference method could in future be used for the determination of the THC concentrations of control sera, calibration or standard solutions which find application in routine laboratories. Furthermore, within the scope of the work, a practice-related study for determining THCglu concentrations in 212 urine samples of potential cannabis consumers was carried out. THCglu represents an auxiliary metabolite of THC, which is present in urine following the corresponding consumption. The background of this investigation was to test whether THCglu would turn out to be a possible marker in urine for THC in serum. This possibility has already been presented in a laboratory study. The already described GC/MS methods for determining THCglu are based on an indirect determination of the analyte by means of a preceding cleavage to THC, which, however, in the case of an incomplete cleavage can lead to erroneous results. For this reason, a new process based on liquid chromatography, coupled with tandem-mass spectrometry (LC/MS/MS) on a triple-quadrupole mass spectrometer was developed. The production of an isotope-marked internal standard THCglu-D3 was realised enzymatically by means of UGT enzymes. The method was validated according to the directives for rare analytes. A correlation between the determined THCglu contents (related to creatinine) in urine and the THC contents in serum would mean that THCglu could be used as a marker for fresh cannabis consumption. In the comparison, however, it was not possible to determine a recognisable correlation between THCglu and THC. As opposed to the laboratory investigations with reproducible conditions, the tested samples originated from police stop-and-search operations and, as a result, comparable preconditions such as form of consumption, consumption type, as well as the time of the last consumption were not given. The study was unable to verify THCglu as general marker for THC in serum. Nevertheless, the investigation of THCglu could contain other important information. Further studies under reproducible conditions could lead to new findings on enzymatic generation as well as the decomposition of THCglu, whereby the newly developed LC/MS/MS method could be applied.