Significant enhancement of 11-Hydroxy-THC detection by formation of picolinic acid esters and application of liquid chromatography/multi stage mass spectrometry (LC-MS3): Application to hair and oral fluid analysis
Thieme D.,Institute of Doping Analysis |
Sachs U.,Albert Ludwigs University of Freiburg |
Sachs H.,Forensic Toxicological Center |
Moore C.,Immunalysis Corporation
Drug Testing and Analysis | Year: 2015
Formation of picolinic acid esters of hydroxylated drugs or their biotransformation products is a promising tool to improve their mass spectrometric ionization efficiency, alter their fragmentation behaviour and enhance sensitivity and specificity of their detection. The procedure was optimized and tested for the detection of cannabinoids, which proved to be most challenging when dealing with alternative specimens, for example hair and oral fluid. In particular, the detection of the THC metabolites hydroxyl-THC and carboxy-THC requires ultimate sensitivity because of their poor incorporation into hair or saliva. Both biotransformation products are widely accepted as incorporation markers to distinguish drug consumption from passive contamination. The derivatization procedure was carried out by adding a mixture of picolinic acid, 4-(dimethylamino)pyridine and 2-methyl-6-nitrobenzoic anhydride in tetrahydrofuran/triethylamine to the dry extraction residues. Resulting derivatives were found to be very stable and could be reconstituted in aqueous or organic buffers and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). Owing to the complex consecutive fragmentation patterns, the application of multistage MS3 proved to be extremely useful for a sensitive identification of doubly picolinated hydroxy-THC in complex matrices. The detection limits - estimated by comparison of corresponding signal-to-noise ratios - increased by a factor of 100 following picolination. All other species examined, like cannabinol, THC, cannabidiol, and carboxy-THC, could also be derivatized exhibiting only moderate sensitivity improvements. The assay was systematically tested using hair samples and exemplarily applied to oral fluid. Concentrations of OH-THC identified in THC-positive hair samples ranged from 0.02 to 0.29pg/mg. © 2014 John Wiley & Sons, Ltd.
Huge V.,Ludwig Maximilians University of Munich |
Lauchart M.,Ludwig Maximilians University of Munich |
Schelling G.,Ludwig Maximilians University of Munich |
Beyer A.,Ludwig Maximilians University of Munich |
And 2 more authors.
European Journal of Pain | Year: 2010
Background: NMDA receptors are involved in the development and maintenance of neuropathic pain. We evaluated the efficacy and safety of intranasal (S)-ketamine, one of the most potent clinically available NMDA receptor antagonists. Methods: Sixteen patients with neuropathic pain of various origins were randomized into two treatment groups: (S)-ketamine 0.2 mg/kg (group 1); (S)-ketamine 0.4 mg/kg (group 2). Plasma concentrations of (S)-ketamine and (S)-norketamine were measured over 6 h by High Performance Liquid Chromatography combined with mass spectrometry. Quantitative sensory testing (QST) was conducted before, during and after treatment. Side effects and amount of pain reduction were recorded. Results: Intranasal (S)-ketamine administration lead to peak plasma concentrations of 27.7 ± 5.9 ng/ml at 10 ± 6.3 min (group 1) and 34.3 ± 22.2 ng/ml at 13.8 ± 4.8 min after application (group 2). Maximal plasma concentrations of (S)-norketamine were 18.3 ± 14.9 ng/ml at 81 ± 59 min (group 1) and 34.3 ± 5.5 ng/ml at 75 ± 40 min (group 2). Pain scores decreased significantly in both groups with minimal pain at 60 min after drug administration (70 ± 10% and 61 ± 13% of initial pain in groups 1 and 2). The time course of pain decrease was significantly correlated with plasma concentrations of (S)-ketamine and (S)-norketamine (partial correlations: (S)-norketamine: -0.90 and -0.86; (S)-ketamine: -0.72 and -0.71 for group 1 and group 2, respectively). Higher dosing elicited significantly more side effects. Intranasal (S)-ketamine had no significant impact on thermal or mechanical detection and pain thresholds in normal or symptomatic skin areas. Conclusions: Intranasal administration of low dose (S)-ketamine rapidly induces adequate plasma concentrations of (S)-ketamine and subsequently of its metabolite (S)-norketamine. The time course of analgesia correlated with plasma concentrations. © 2009 European Federation of International Association for the Study of Pain Chapters.
Proof of cannabis administration by sensitive detection of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in hair using selective methylation and application of liquid chromatography- tandem and multistage mass spectrometry
Thieme D.,Institute of Doping Analysis |
Sachs H.,Forensic Toxicological Center |
Uhl M.,Bavarian State Criminal Police Office
Drug Testing and Analysis | Year: 2014
The identification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair represents an exceptional forensic analytical challenge due to low target concentrations in a complex matrix. Several dedicated techniques [gas chromatography - negative chemical ionization- tandem mass spectrometry (GC-NCI-MS/MS) or GC-GC-MS couplings] were specifically introduced into forensic toxicology aiming to a selective and sensitive identification of THCCOOH in hair. The combination of liquid-chromatography (LC) and MS/MS gained an outstanding relevance in forensic toxicology (including the detection of cannabinoids). However, its application to hair matrix is characterized by a lack of specificity which is due to the unspecific decarboxylation as most abundant fragmentation reaction. Therefore, various chemical modifications of the carboxyl and/or phenolic hydroxyl groups were examined to improve the selectivity. The selective methylation of the 9-carboxyl-group proved to be the most efficient derivatization procedure. Hair extracts were redissolved in acetonitrile and after addition of few milligrams of solid sodium carbonate derivatized with 25μL methyl iodide. The resulting THC-9-carboxymethylester was separated by conventional reverse phase LC and selectively detected using negative electrospray ionization by recording the fragmentation reactions 357→325 and 357→297. Resulting limits of quantification were below 100fg/mg. A further significant improvement was achieved by application of the multistage MS3 fragmentation 357→325→297. To verify the validity of this procedure, a systematic quantitative comparison of THCCOOH concentrations in hair with data from a well established GC-NCI-MS/MS technique was performed. Both techniques proved to be in good accordance (R2 =0.647, p=<0.001) and equally suitable for hair testing of THCCOOH. © 2013 John Wiley & Sons, Ltd.
Thevis M.,German Sport University Cologne |
Schanzer W.,German Sport University Cologne |
Geyer H.,German Sport University Cologne |
Thieme D.,Zoo Leipzig GmbH |
And 7 more authors.
British Journal of Sports Medicine | Year: 2013
The administration of musk extract, that is, ingredients obtained by extraction of the liquid secreted from the preputial gland or resulting grains of the male musk deer (eg, Moschus moschiferus), has been recommended in Traditional Chinese Medicine (TCM) applications and was listed in the Japanese pharmacopoeia for various indications requiring cardiovascular stimulation, antiinflammatory medication or androgenic hormone therapy. Numerous steroidal components including cholesterol, 5a-androstane-3,17-dione, 5β-androstane- 3,17-dione, androsterone, etiocholanolone, epiandrosterone, 3β-hydroxy- androst-5-en-17-one, androst-4-ene-3,17-dione and the corresponding urea adduct 3a-ureido-androst-4-en-17-one were characterised as natural ingredients of musk over several decades, implicating an issue concerning doping controls if used for the treatment of elite athletes. In the present study, the impact of musk extract administration on sports drug testing results of five females competing in an international sporting event is reported. In the course of routine doping controls, adverse analytical findings concerning the athletes' steroid profile, corroborated by isotope-ratio mass spectrometry (IRMS) data, were obtained. The athletes' medical advisors admitted the prescription of TCM-based musk pod preparations and provided musk pod samples for comparison purposes to clarify the antidoping rule violation. Steroid profiles, IRMS results, literature data and a musk sample obtained from a living musk deer of a local zoo conclusively demonstrated the use of musk pod extracts in all cases which, however, represented a doping offence as prohibited anabolic-androgenic steroids were administered.
Thieme D.,Institute of Doping Analysis
Bioanalysis | Year: 2012
Alternative specimens (e.g., hair and saliva) are well established in forensic toxicology and provide significant benefits as noninvasive, inexpensive alternatives to blood with access to improved long-term retrospection. Based on these experiences, the question of potential applications and limitations of alternative specimens in doping control arose. Compounds prohibited at all times (e.g., clenbuterol, β 2 agonists, estrogen-receptor modulators) may be successfully tested and clearly interpreted in alternative specimens. In contrast, prohibition of certain compounds in sport are limited to time ranges (e.g., stimulants are only prohibited in-competition), dosages or administration routes (e.g., systemic uptake of glucocorticosteroids). This cannot be properly differentiated by semiquantitative tests (e.g., hair analyses), but may be distinguished in saliva. Similarly, proof of external administration of endogenous steroids (e.g., testosterone) only seems to be achievable by quantitative analysis of saliva. Moreover, the retrospective monitoring of the relevance of social drugs or upcoming (unapproved) substances represents promising applications of hair tests in doping control. © 2012 Future Science Ltd.