Advances in testing for sample manipulation in clinical and forensic toxicology - Part A: urine samples

In many countries, adherence testing is used to monitor consumption behavior or to prove abstinence. Urine and hair are most commonly used, although other biological fluids are available. Positive test results are usually associated with serious legal or economic consequences. Therefore, various sample manipulation and adulteration strategies are used to circumvent such a positive result. In these critical review articles on sample adulteration of urine (part A) and hair samples (part B) in the context of clinical and forensic toxicology, recent trends and strategies to improve sample adulteration and manipulation testing published in the past 10 years are described and discussed. Typical manipulation and adulteration strategies include undercutting the limits of detection/cut-off by dilution, substitution, and adulteration. New or alternative strategies for detecting sample manipulation attempts can be generally divided into improved detection of established urine validity markers and direct and indirect techniques or approaches to screening for new adulteration markers. In this part A of the review article, we focused on urine samples, where the focus in recent years has been on new (in)direct substitution markers, particularly for synthetic (fake) urine. Despite various and promising advances in detecting manipulation, it remains a challenge in clinical and forensic toxicology, and simple, reliable, specific, and objective markers/techniques are still lacking, for example, for synthetic urine

Advances in testing for sample manipulation in clinical and forensic toxicology—part B: hair samples

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology

Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated.We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level

Entwicklung des ersten Metaboliten-basierten LC-MSⁿ Medikamenten Screening Verfahrens für Urin

In the presented dissertation, the development of the first metabolite-based LC-MS screening approach is described. It consisted of establishing a simple and fast sample workup, fast and sufficient LC separation, MS settings for recording reproducible spectra, and a suitable screening concept. Using these methods, MS2 and MS3 spectra of parent drugs were recorded as well as those of their metabolites after having identified them in urine samples of rats and humans. By using the described methods, the new reference library (over 1,000 parent drugs, 2,700 metabolites, 100 biomolecules), and a sophisticated software tool, a new routine screening approach was established. This LC-MS screening approach is nowerdays an important part of the systematic toxicological analysis and showed excellent robustness and screening results in thousands of authentic patient samples. According to this, this LC-MS approach complements the established GC-MS approach. In addition, recent research projects are partly based on the developed workup, separation and/or detection methods. Finally, this procedure and the reference library could successfully be transferred to another apparatus type, which raised the hope of an instrument independent LC-MS reference library.Im Rahmen dieser Dissertation wurde die Entwicklung eines Metaboliten-basierten LC-MS Screenings beschrieben. Dazu wurden eine einfache und schnelle Probenvorbereitung, eine schnelle und effektive LC-Trennung, MS Settings zur Aufnahme reproduzierbarer Spektren und schließlich ein geeigneten Screening-Konzept entwickelt. Damit wurden MS2 and MS3 Spektren von Muttersubstanzen und ihren Metaboliten aufgenommen, die zuvor in Ratten- oder Patientenurinen identifiziert worden waren. Mit Hilfe dieser Methoden, der neuen Referenzbibliothek (über 1000 Muttersubstanzen, 2700 Metaboliten, 100 Biomoleküle) und einer speziellen Auswertesoftware konnte ein neues Routine-Screeningverfahren etabliert werden. Das entwickelte LC-MS System ist heutzutage ein wichtiger Bestandteil der systematischen toxikologischen Analyse. Bei mehreren tausend Patientenproben zeigte es sehr hohe Robustheit und sehr gute Screening-Ergebnisse. Zusammenfassend lässt sich festhalten, dass dieses LC-MS-Verfahren das etablierte GC-MS-Verfahren ideal ergänzt. Zusätzlich stellten die entwickelten Aufarbeitungs-, Trennungs- und/oder Detektionsmethoden eine wichtige Grundlage für andere aktuelle Forschungsprojekte des Arbeistkreises dar. Schließlich konnte gezeigt werden, dass dieses Verfahren und die zugrunde liegende Referenzbibliothek auf einen anderen Gerätetyp erfolgreich übertragen werden kann. Dies lässt Hoffnung auf eine langersehnte geräteunabhänige LC-MS Referenzbibliothek zu