Jörg Teske

Scheduled multiple reaction monitoring algorithm as a way to analyse new designer drugs combined with synthetic cannabinoids in human serum with liquid chromatography–tandem mass spectrometry

Here, we describe the development and application of a liquid chromatography-tandem mass spectrometry method with positive electrospray ionisation and scheduled multiple reaction monitoring algorithm (s-MRM) to analyse synthetic cannabinoids (SC) combined with new designer drugs (NDD) in human serum. A Luna 5μm C18 (2) 100A, 150mm×2mm analytical column and a mobile phase consisted of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v) - both with 10mM ammonium acetate and 0.1% acetic acid (pH=3.2), were used for the separation. A binary flow pumping mode with a total flow rate of 0.400mL/min was used. A single sample extraction with 1-chlorobutane for both substance groups was performed. Acceptable linearity in the validated calibration ranges of 0.05-1ng/mL for SC and 1-50ng/mL for NDD was achieved. The limit of detection was not greater than 0.02/0.40ng/mL and the limit of quantification not greater than 0.05/0.50ng/mL for SC/NDD respectively. The presented study revealed that this method is a very effective way for sensitive SC and NDD identification in human serum and has useful application in hospitals, therapy centres and forensic psychiatric centres. S-MRM ensures a method upgrade with a smaller loss of sensitivity, precision and accuracy in comparison to traditional MRM methods. Also addition of new SC and NDD can be performed in the future.

Analysis of synthetic cannabinoids in abstinence control: Long drug detection windows in serum and implications for practitioners

Controlled studies on the elimination kinetics and the long-term detection of synthetic cannabinoids in humans are lackingand will most probably not become available in the near futurebecause of the unfavorable risk profiles of these substances. Forthis reason, we aim to share our observations on this topic afterhaving analyzed more than 4200 serum samples for syntheticcannabinoids until late 2012. The samples were received frompatients of forensic psychiatric clinics, rehabilitation clinics orwithdrawal treatment centers located in two different regionsof Germany (federal states of Baden-Wurttemberg and Bavariaas well as Lower Saxony). Due to a high prevalence of syntheticcannabinoid use, most of the institutions sent follow-up samplesfrom patients who tested positive by LC-MS/MS analysis.A particularly striking observation was made in a number ofcases in which the widely used synthetic cannabinoids JWH-081,JWH-122 or JWH-210 were detectable for up to 102 days followingself-reported cessation of drug use, leading to estimated terminalelimination half-lives of up to 41 days. Consistent with the claimofabstinenceduringthisperiod,acontinuousdecreaseofserumconcentration levels was noted. This observation raised thepossibility that synthetic cannabinoids may be extensively dis-tributed into deeper compartments (e.g. fat tissue), hence lead-ing to an extended window of detection due to redistributionof these substances into the bloodstream, especially followingheavy consumption. Moreover, our preliminary data suggestthat this effect is even more pronounced than in the case ofΔ

Cardiac glycosides from Yellow Oleander (Thevetia peruviana) seeds

Thevetia cardiac glycosides can lead to intoxication, thus they are important indicators for forensic and pharmacologic surveys. Six thevetia cardiac glycosides, including two with unknown structures, were isolated from the seeds of the Yellow Oleander (Thevetia peruviana (Pers.) K. Shum., Apocynaceae). LC-ESI⁺-MS(/MS) analysis under high-resolution conditions used as a qualitative survey of the primary glycosides did not lead to fragmentation of the aglycones. Acid hydrolysis of the polar and non-volatile thevetia glycosides under severe conditions yielded the aglycones of the thevetia glycosides and made them amenable to GC-MS analysis. Comparison of mass spectral fragmentation patterns of the aglycones, as well as high-resolution mass spectrometric and NMR data of four of the primary thevetia glycosides including the two unknowns, revealed the structures of the complete set of six thevetia glycosides. The identified compounds are termed thevetin C and acetylthevetin C and differ by an 18,20-oxido-20,22-dihydro functionality from thevetin B and acetylthevetin B, respectively. The absence of an unsaturated lactone ring renders the glycosides cardio-inactive. The procedures developed in this study and the sets of analytical data obtained will be useful for screening and structure assessment of other, particularly polar, cardiac glycosides.

Small molecule adduct formation with the components of the mobile phase as a way to analyse valproic acid in human serum with liquid chromatography-tandem mass spectrometry

A valproic acid (VPA) LC-MS/MS analytical method using analyte adduct formation was developed and validated in human serum. The fragmentation of the sodium acetate adduct (mass transition: 225/143) and acetic acid adduct (mass transition: 203/143) were used as the target and qualifier mass transition, respectively. A Luna 5 μm C18 (2) 100 A, 150 mm×2 mm analytical column and a mobile phase consisting of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v), both with 10mM ammonium acetate and 0.1% acetic acid (pH=3.2) were used. A binary flow pumping mode with a total flow rate of 0.4 mL/min was applied. Protein precipitation with 1 mL of the mobile phase B was used as sample preparation. The calculated limit of detection/quantification was 0.45/1.0 μg/mL and the inter-/intra-day precision was <6%. The application of a deuterated internal standard resulted in a good adduct formation reproducibility. The strategy applied made the VPA LC-MS/MS analysis in human serum on the basis of two mass transitions possible. Therefore, it is an interesting alternative for the VPA pseudo multiple reaction monitoring methods (mass transition 143/143) and a proof that the developed strategy is also useful for the analysis of compounds which do not produce any stable ion fragments detectable by tandem mass spectrometry.

Postmortem concentration distribution in fatal cases involving the synthetic opioid U-47700

Dear Editor, U-47700 is a synthetic drug from the group of new psychoactive substances, used illicitly by recreational opioid users. Animal models revealed that U-47700 is around 7.5 times more potent in binding to opioid receptors than morphine. Its high potency might lead to numerous lethal intoxications, like reported recently after U-47700 consumption [1–4]. Especially, a combined application of U-47700 with other opioids has to be classified as dangerous [5]. Like already pointed out, there is insufficient information about U-47700 concentration in biological samples [6]. Therefore, with this letter, we wanted to present our data concerning U-47700 concentration distribution in postmortem specimens collected after two lethal intoxications. In fatalities investigated, both decedents were found dead on a bed. Because U-47700 was characterized as a selective and strong opioid receptor agonist, generally leaving uncharacteristic findings, the autopsies performed could not reveal the cause of death. Since cerebral/lung oedema and a full urinary bladder were found, a possible intoxication was discussed. The postmortem specimens collected by forensic pathologists underwent blood alcohol analysis, broad toxicological examinations with general unknown analyses, and appropriate confirmations of targeted drugs. No relevant concentrations of other substances than U-47700 could be detected (Table 1). Appropriate quantifications in postmortem specimens, performed on the basis of liquidliquid extraction with 1-chlorobutane, standard addition (with spikes added at three levels), fentanyl-D5 applied as internal standard, and liquid chromatography-tandem mass spectrometry, revealed U-47700 concentrations as presented in Table 1. As expected, femoral blood concentrations were lower than the urine/heart blood concentrations and can be defined as high when compared with toxic morphine levels. They can also be classified as lower than the concentration discussed by Elliott et al. [1] but higher than the data presented by Mclntyre et al./Mohr et al. [2, 3]. Regarding all the circumstances, the cause of death could be explained by the U-47700 consumption in both cases. It is well known that the interpretation of postmortem drug concentration is a big challenge [7]. Scarcity of information on reference concentrations in postmortem matrices makes the interpretation even more problematic. Since the analyses in cases discussed were performed in a variety of postmortem specimens, the concentration distribution data provided can be * Marek Dziadosz analytiker@chemist.com; Dziadosz.Marek@mh-hannover.de

Letter to the Editor—Consumption of Levamisole in Cocaine Preparations

Sir, The relative long cocaine distribution chain is responsible for cocaine dilution with different cheaper components used as adulterants such as hydroxyzine, lidocaine, procaine, benzocaine, caffeine, boric acid, phenacetin, and levamisole (1). This psychoactive substance modification leads to a weight increase and in consequence to higher profits. Among these adulterants, levamisole gained a lot of attention in recent years, as this drug was reported as especially dangerous to cocaine consumers due to its numerous side effects on human health (agranulocytosis, ANCA-associated vasculitis, retiform purpura, and others) (2,3). Levamisole as an imidazothiazole derivative used to be applied as a veterinary anthelminthic medication but also to treat pediatric nephritic syndrome and rheumatoid arthritis (2). The reason for combining cocaine with levamisole is still unknown. It is believed that levamisole is added directly during cocaine manufacture to potentiate its effects. The prevalence of levamisole-adultered cocaine was investigated recently in North America and was reported to be as high as 88% (4). In this letter, we wanted to report the results of our study concerning the investigation of the levamisole prevalence in cocaine-positive blood samples analyzed in our laboratory and collected during police traffic controls. For these purposes, the LC-MS/MS analytical method used in the routine for the analysis of cocaine and its metabolites was modified to make a qualitative levamisole analysis possible simultaneously. Samples, which contained at least 1 ng/mL benzoylecgonine (cocaine metabolite), were defined as cocaine-positive samples and used in the data evaluation for the purpose of the levamisole prevalence determination. The analysis of 133 cocaine-positive samples in the stretch of 3 months (July–September 2014) revealed that 111 were also positive for levamisole, which equals in a levamisole prevalence of 83%. The LC-MS/MS analytical method applied for the qualitative analysis of levamisole based on the detection of two mass transitions (multiple reaction monitoring mode: 204.9/178.1 for the target and 204.9/123.0 for the qualifier) and was characterized by a limit of detection of 0.14 ng/mL. The presented study revealed that levamisole is present in the majority of cocaine consumed in Lower Saxony. The obtained prevalence was comparable with the data presented by Lynch et al. for cocaine samples in North America. Therefore, this drug has to be considered not only by clinicians in cocaine users with unexpected symptoms such as otherwise unexplained agranulocytosis (5) but also by forensic pathologists in their expertise (2). As the prevalence of levamisole seems to be quite high, and furthermore, different adverse effects of this drug are well known and documented, the application of analytical methods which detect not only cocaine and its metabolites but additionally levamisole can be very useful. Additionally, it has to be considered that the knowledge about adulterants in all illicit drugs and their effect on human health is very important and has a big impact on the correct data interpretation in the forensic/ clinical expertise.

Method validation of a survey of thevetia cardiac glycosides in serum samples

A sensitive and specific liquid chromatography tandem mass spectrometry (HPLC-ESI(+)-MS/MS) procedure was developed and validated for the identification and quantification of thevetin B and further cardiac glycosides in human serum. The seeds of Yellow Oleander (Thevetia peruviana) contain cardiac glycosides that can cause serious intoxication. A mixture of six thevetia glycosides was extracted from these seeds and characterized. Thevetin B, isolated and efficiently purified from that mixture, is the main component and can be used as evidence. Solid phase extraction (SPE) proved to be an effective sample preparation method. Digoxin-d3 was used as the internal standard. Although ion suppression occurs, the limit of detection (LOD) is 0.27 ng/ml serum for thevetin B. Recovery is higher than 94%, and accuracy and precision were proficient. Method refinement was carried out with regard to developing a general screening method for cardiac glycosides. The assay is linear over the range of 0.5-8 ng/ml serum. Finally, the method was applied to a case of thevetia seed ingestion.

Imatinib quantification in human serum with LC-MS3 as an effective way of protein kinase inhibitor analysis in biological matrices.

Abstract Background: As imatinib gained a lot of attention in the field of medicine, appropriate methods are needed for drug analysis. LC-MS/MS combined with complex sample preparation and column enrichment is usually the method of choice when high sensitivity is necessary. The application of LC-MS3 in imatinib quantification has not been discussed in the literature. Methods: An LC-MS3 imatinib quantification method was developed and validated in human serum. The sample preparation was based on the liquid-liquid extraction of 50 μL human serum. Chromatographic separation was performed using a Luna 5 μm C18 (2) 100 A, 150 mm×2 mm column and the elution was done using a mobile phase consisting of A (H2O/methanol=95/5, v/v) and B (H2O/methanol=3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid. Results: The conditions applied resulted in a limit of detection/quantification value of 0.14/0.45 ng/mL reached without a sophisticated sample preparation technique or enrichment column application. It could be demonstrated that MS3 detection is a very effective way of sensitive imatinib quantification. Further, it could be stated that the strategy presented can be very useful for a sensitive analysis of other protein kinase inhibitors, because their molecule structure is appropriate for MS3 detection. Conclusions: The presented analytical strategy is an effective way of protein kinase inhibitor analysis in human serum.

Monitoring of levamisole concentration in serum of traffic participants after cocaine consumption

This study highlights the problem of levamisole‐adulterated cocaine in context of active traffic participation. For the purposes of levamisole concentration monitoring in human serum, an analytical method based on LC‐MS/MS and solid‐phase extraction was applied. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm column and a mobile phase consisting of A (H2O/methanol = 95/5, v/v) and B (H2O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and with 0.1% acetic acid (pH = 3.2), were used. The validation experiments demonstrated that the method applied was appropriate for levamisole quantification in human serum. For 23% of levamisole‐positive samples, the concentrations exceeded 20 ng/mL. Therefore, the interaction of this drug with cocaine has to be considered as important for active traffic participation. As a consequence, monitoring of levamisole concentration in human serum is recommended, as long as it is used as cocaine adulterant.

Letter to the editor-the problem of caffeine consumption in the bodybuilding scene

Sir, Caffeine is an alkaloid which can be found in coffee, tea leafs, or kola nuts. It is responsible for a relative mild stimulation of the central nervous system and is the most frequently consumed substance with centrally stimulating effects (1). Therefore, nowadays it is not possible to imagine the life without caffeine. The newest trend among caffeine consumers who want to provide a higher stimulating effect is beverages sold as energy drinks and loaded with this substance together with other additives such as sugar and herbal components (2). Symptoms of caffeine consumption are dose-dependant, and in higher doses, even intoxications can be expected. Regular caffeine use can also lead to addiction with a withdrawal syndrome after discontinuation of consumption (3). Although numerous toxic effects have been described, lethal caffeine intoxications are rare and therefore not reported in the literature frequently (4–6). This substance is also a popular ingredient of different legal highs which main components responsible for the desired effect are analyzed with complex analytical methods (7). Caffeine is detected in the majority of biological samples due to dietary exposure to this substance. However, this drug gained also attention in the bodybuilding scene and is widely used as supplement. The bodybuilders can find a lot of information in the internet, which suggests using caffeine powder/ pills instead of coffee (8). It is stated that in this form, a better dosage control can be achieved. However, a big problem is the caffeine tolerance, which occurs after multiple caffeine use and diminishes its value for exercise performance. This fact is a reason why higher caffeine doses would be needed to achieve the expected stimulating effect. Bodybuilders are also advised to consume caffeine pills/powder early in the morning, about 1 h before the workout (to achieve the highest stimulating effect). This strategy enables also the elimination of caffeine during the day, so no interference with the sleep should occur. The problem of caffeine abuse in the bodybuilding scene was described by FitzSimmons et al. In this documented case, a caffeine intoxication with a caffeine serum concentration of 12.3 mg/L was reported (9). However, after three days on the ward, the patient remained seizure-free and had normal renal function. Caffeine toxic levels are considered to be 15 mg/L and higher, and lethal concentrations are expected to be above 100 mg/L (4). In this context, we wanted to mention the following case. A 25-year-old was found dead on the floor of his office. The last time he was seen alive by his friends was the day before death. Vomit was found on the body and in the surrounding area. No preexisting illness was known. However, a year before, he passed a psychiatric examination in consequence of his aggressive behavior. The only auxiliary information provided by the police was the fact that the decedent was an active bodybuilder and consumer of different drugs which he used to enhance his performance. The fact that he was mentally fragile was also known. The autopsy did not show any signs of violence or pathological change of the organs. On the basis of this information and investigations performed by the police, toxicological analyses were strongly recommended by the forensic pathologists. After the postmortem materials were screened for different drugs on the basis of cloned enzyme donor immunoassay (CEDIA—urine), gas chromatography–mass spectrometry (GC-MS —urine), and high-performance liquid chromatography with diode array detection (HPLC-DAD—heart blood), very high signals for caffeine were obtained. Other relevant substances could not be detected. The appropriate quantification was applied with HPLC-DAD (calibration range: 10–300 mg/L; limit of detection/ quantification: 0.12/0.43 mg/L). The HPLC-DAD analyses performed revealed a caffeine concentration of 255 mg/L in femoral blood and of 214 mg/L in heart blood. A documented series of 14 fatalities after caffeine overdosage revealed a blood concentration range of 79–344 mg/ L (10). As caffeine concentrations in human blood greater than 100 mg/L are considered to be lethal (4), the quantified caffeine values could explain the cause of death. The achieved results are consistent with the case history, because the deceased was an active bodybuilder and caffeine powder consumer. Therefore, the death was caused by accidental caffeine intoxication on the basis of bodybuilding performance enhancement. A competing cause of death could not be found. The popularity of caffeine consumed in form of coffee, pills/ powder, or other beverages is responsible for the fact that this drug is present in most forensic samples, when a general unknown analysis is performed. However, caffeine as a drug with toxic effect (in higher doses) on human health is often underestimated. It has to be considered that caffeine concentrations above 15 mg/L can cause toxic symptoms (5,6). As caffeine taken in excess can cause tremor, tachycardia, vomiting, convulsions, cardiac arrhythmias, coma, and death, it should be always considered in the forensic expertise. In fatalities in the bodybuilding scene this drug is very important, because overconsumption of different substances is quite common to enhance the bodybuilding performance (like demonstrated in this letter). This overconsumption may cause different toxic effects or even be lethal, when higher concentrations are reached.