Jochen Beyer

Detection and Quantification of New Designer Drugs in Human Blood: Part 1 - Synthetic Cannabinoids

Synthetic cannabinoids sprayed on herbal mixtures have been abused as a new designer drug all over the world since 2004. In 2008, the first compounds, CP 47,497 and JWH-018, were identified as active ingredients in these mixtures. Most of the compounds have been synthesized for research purposes and are potent CB1 and/or CB2 receptor agonists. To investigate the presence of synthetic cannabinoids in blood samples, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed using only 100 µL of blood. After the addition of 0.2 mL of trizma buffer, the blood was extracted using liquid-liquid extraction with 1 mL of 1-chlorobutane containing 10% of isopropanol for 5 min on a shaker at 1,500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N₂. The residue was reconstituted in methanol and injected into a Shimadzu 8030 LC-MS-MS system to separate and detect 25 synthetic cannabinoids. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory from 0.5-100 ng/mL, and from 5.0-500 ng/mL. for HU-210, CP 47,497 and the CP 47,497 C-8 homolog, respectively. The extraction efficiencies ranged from 30-101% and the matrix effects from 67-112%. Accuracy data were within the acceptance interval of ±15% (±20% at the lower limit of quantification) of the nominal values for all drugs.

Identification and quantification of 30 antipsychotics in blood using LC-MS/MS

Over the last decade, the prescription rates of antipsychotic (AP) drugs have increased worldwide. Studies have shown that the risk of sudden cardiac death is threefold higher among patients treated with APs. To investigate the presence of APs in postmortem cases, a liquid chromatography (LC)-MS/MS method was developed using only 0.1 ml of blood sample with 10 microl of internal standard (IS) (haloperidol-d(4), 1 microg/ml). After the addition of 0.2 ml of Trizma buffer, the blood sample was extracted using liquid-liquid extraction (LLE) with 1 ml of 1-chlorobutane for 5 min on a shaker at 1500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N(2). The residue was reconstituted in 0.05 ml acetonitrile containing 0.1% formic acid, vortexed for 30 s and an additional 0.45 ml of 50 mmol/l ammonium formate pH 3.5 was added and the sample vortexed; 0.1 ml of the final extract was injected into a Shimadzu Prominence HPLC system, with detection of drugs achieved using an Applied Biosystems 3200 Q-TRAP LC-MS/MS system equipped with a Turbo V ion source [electron spray ionization (ESI), multiple reaction monitoring (MRM) mode]. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory for all drugs, except olanzapine, from subtherapeutic to toxic concentrations. The lower limits of quantifications (LLOQs) corresponded to the lowest concentrations used for the calibration curves. With the exception of the lowest concentrations of bromperidol, buspirone and perphenazine, accuracy data were within the acceptance interval of +/- 15% (+/- 20% at LLOQ) of the nominal values for all drugs. The method has been proven to be useful for the routine analysis of APs in postmortem blood samples.

Analysis of toxic alkaloids in body samples

Many plants contain toxic alkaloids which may be dangerous to humans. Despite the large number of poisonous plants, cases of fatal plant poisonings are relatively rare. The frequencies of poisonings and the plants involved are often regionally specific. Plant poisonings can be aggregated into three categories: unintended ingestions, intended ingestions, and poisoning due to abuse of plant material. Unintended ingestions often occur in children or from a mix-up of plants and mushrooms in adults. Intended ingestions are common in homicides and suicides. Increasingly common is the abuse of plants for hallucinogenic reasons. Toxicological analysis of such alkaloids may help in diagnosis of poisoning or abuse cases. This review describes the toxic alkaloids aconitine, atropine, coniine, colchicine, cytisine, dimethyltryptamine, harmine, harmaline, ibogaine, kawain, mescaline, scopolamine, and taxine, which are often involved in fatal and non-fatal poisonings. The paper summarizes the symptoms of the intoxications and reviews the methods of detection of their toxic constituents in biological fluids.

The prevalence of drugs in injured drivers

In mid 2009 Victoria introduced compulsory drug testing of blood taken from all injured drivers taken to hospital. Δ(9)-Tetrahydrocannabinol (THC), methylamphetamine (MA) and 3,4-methylenedioxy-methylamphetamine (MDMA) are prohibited and if drivers are positive to any amount an automatic penalty is enforced. Laboratory screens were conducted on preserved blood using ELISA testing for cannabis metabolite and methylamphetamines and a fully validated LC-MS/MS method for 105 drugs including THC, amphetamines, opioids, benzodiazepines, antidepressants and antipsychotics and a number of other psychoactive substances using a minimum of two transitions per drug. Conventional GC-testing for ethanol was used to screen and quantify the presence of alcohol. 1714 drivers were tested and showed alcohol in 29% (≥ 0.01 g/100mL) and drugs in 35%. The positive rate for the three drugs prohibited by legislation was 12.5%. The prevalence of THC, MA and MDMA was 9.8%, 3.1%, and 0.8%, respectively. The range of THC concentrations in blood was 2-42 ng/mL (median 7) of which 70% had a concentration of 10 ng/mL or higher. The range of concentrations for MA and MDMA was 0.02-0.4 and 0.03-0.3mg/L (median for both drugs was 0.05 mg/L). Drugs of any type were detected in 35% of cases. The other drugs were largely prescribed drugs such as the antidepressants (9.3%) and benzodiazepines (8.9%). Neither 6-acetylmorphine nor cocaine (or benzoylecgonine) was detected in these cases.

The incidence of drugs of impairment in oral fluid from random roadside testing

Oral fluid (OF) has become a popular specimen to test for presence of drugs, particularly in regards to road safety. In Victoria, OF specimens from drivers have been used to test for the presence of methylamphetamine (MA) and Δ(9)-tetrahydrocannabinol (THC) since 2003 and 3,4-methylenedioxy-N-methylamphetamine (MDMA) since 2006. LC-MS/MS has been used to test the most recent 853 submitted OF specimens from Victoria Police for 31 drugs of abuse including those listed in the Australian Standard AS4760-2006. At least one proscribed drug was detected in 96% of drivers, of which MA was the most common (77%), followed by THC (42%), MDMA (17%) and the combination of all three (3.9%). Opioids were detected in 14% of drivers of which 4.8% were positive for 6-acetylmorphine and 3.3% for methadone. The incidence of the opioids tramadol (1.2%) and oxycodone (1.1%) were relatively low. Cocaine (8.0%) was as commonly detected as benzodiazepines (8.0%), and was almost always found in combination with MA (7.9%). Samples positive to benzodiazepines were largely due to diazepam (3.5%) and alprazolam (3.4%), with only 0.2% of drivers combining the two. Ketamine was also detected in 1.5% of cases. While the incidences of the proscribed drugs itself are concerning, it is clear that many drivers are also using other drugs capable of causing impairment.

Comparison of extraction efficiencies and LC–MS–MS matrix effects using LLE and SPE methods for 19 antipsychotics in human blood

Antipsychotic drugs are frequently associated with sudden death investigations. Detection of these drugs is necessary to establish their use and possible contribution to the death. LC–MS(MS) methods are common; however accurate and precise quantification is assured by using validated methods. This study compared extraction efficiency and matrix effects using common liquid–liquid and solid-phase extraction procedures in both ante-mortem and post-mortem specimen using LC–MS–MS. Extraction efficiencies and matrix effects were determined in five different blank blood specimens of each blood type. The samples were extracted using a number of different liquid–liquid extraction methods and compared with a standard mixed-mode solid-phase extraction method. Matrix effects were determined using a post-extraction addition approach—the blank blood specimens were extracted as described above and the extracts were reconstituted in mobile phase containing a known amount of analytes. The extraction comparison of ante-mortem and post-mortem blood showed considerable differences, in particular the extraction efficiency was quite different between ante-mortem and post-mortem blood. Quantitative methods used for determination of antipsychotic drugs in post-mortem blood should establish that there are no differences in extraction efficiency and matrix effects, particularly if using ante-mortem blood as calibrator.

The time-dependant post-mortem redistribution of antipsychotic drugs

The post mortem redistribution of ten commonly prescribed antipsychotic drugs (APs) was investigated. Femoral blood was collected from 273 cases at admission to mortuary (AD) and at post-mortem (PM). The PM samples were collected at various times up to nine days after admission and the sample pairs analysed using LC-MS/MS. The drugs included in this study were 9OH-risperidone (paliperidone), amisulpride, chlorpromazine, clozapine, haloperidol, olanzapine, promethazine, quetiapine, risperidone, and zuclopenthixol. Haloperidol, quetiapine and risperidone showed minimal changes between AD and PM specimens, whereas the majority of drugs showed significant changes between the sample pairs collected at different time points post mortem (p<0.01) in addition to an average concentration change greater than the uncertainty of measurement of the applied method. Average increases in blood concentrations after admission to the mortuary ranged up to 112% (chlorpromazine and olanzapine) but also decreases up to -43% (9OH-risperidone) were seen. There were large standard deviations between sample pairs and substantial day-to-day unpredictable changes that highlight the difficulty in the interpretation of drug concentrations post-mortem. Based on the presented data, we recommend that specimens for toxicological analysis should to be taken as soon as possible after admission of a deceased person to the mortuary in order to minimise the effects of the PM interval on the drug concentration in blood.

Screening procedure for detection of diuretics and uricosurics and/or their metabolites in human urine using gas chromatography-mass spectrometry after extractive methylation.

A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of diuretics, uricosurics, and/or their metabolites in human urine after extractive methylation. Phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by GC and identified by MS in the full-scan mode. The possible presence of the following drugs and/or their metabolites could be indicated using mass chromatography with the given ions: m/z 267, 352, 353, 355, 386, and 392 for thiazide diuretics bemetizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, hydrochlorothiazide, metolazone, polythiazide, and for canrenoic acid and spironolactone; m/z 77, 81, 181, 261, 270, 295, 406, and 438 for loop diuretics bumetanide, ethacrynic acid, furosemide, piretanide, torasemide, as well as the uricosurics benzbromarone, probenecid, and sulfinpyrazone; m/z 84, 85, 111, 112, 135, 161, 249, 253, 289, and 363 for the other diuretics acetazolamide, carzenide, chlorthalidone, clopamide, diclofenamide, etozoline, indapamide, mefruside, tienilic acid, and xipamide. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with reference spectra. This method allowed the detection of the abovementioned drugs and/or their metabolites in human urine samples, except torasemide. The limits of detection ranged from 0.001 to 5 mg/L in the full-scan mode. Recoveries of selected diuretics and uricosurics, representing the different chemical classes, ranged from 46% to 99% with coefficients of variation of less than 21%. After ingestion of the lowest therapeutic doses, furosemide was detectable in urine samples for 67 hours, hydrochlorothiazide for 48 hours, and spironolactone for 52 hours (via its target analyte canrenone). The procedure described here is part of a systematic toxicological analysis procedure for acidic drugs and poisons.

The analysis of antipsychotic drugs in human matrices using LC-MS(/MS)

Antipsychotic drugs (APs) are prescribed for a wide range of psychotic illnesses. With more than 35 APs currently available worldwide, this drug class has rapidly gained importance in both clinical and forensic settings. On account of their chemical properties, many APs are present in human specimens at very low concentrations, which complicate their detection using standard gas chromatography-mass spectrometry (GC-MS) procedures that often cannot provide the required sensitivity. Recent advances in liquid chromatography-(tandem) mass spectrometry LC-MS(/MS) technology have enabled accurate detection and quantification of these compounds in various human specimens, indicated by the increasing number of published methods. Method validation has been a particular focus of analytical chemistry in recent times. Recommendations set by several guidance documents are now widely accepted by the toxicology community, as reflected by the guidelines drafted by leading toxicological societies. This review provides a critical review of single-stage and tandem LC-MS procedures for the detection and quantification of APs, with a particular emphasis on appropriate method validation. The quality of published methods is inconsistent throughout the literature. While the majority of authors incorporate some validation experiments in their respective method development, a large number of published methods lack essential components of method validation, which are considered mandatory according to the guidelines. If adapting a method for the detection of APs for use in a laboratory, analysts should ensure successful validation experiments for appropriateness and completeness have been conducted, and perform additional experiments when indicated.

Alcohol congener analysis and the source of alcohol: a review

For many decades traditional alcohol congener analysis has provided the concentrations of fermentation by-product congeners found in blood, to ascertain if the claims of an individual regarding the alcoholic beverage(s) they have consumed were feasible, assisting in cases where after-drinking is involved. However, this technique does not provide information on the exact alcoholic beverage(s) consumed. More recently, ingredient biomarker congeners specific to certain alcoholic beverages have been detected in blood, making it possible to identify the particular alcoholic beverage consumed and therefore the source of alcohol (albeit only for a limited number of beverages). This novel approach may reduce current limitations that exist with traditional methods of detecting fermentation by-product congeners, which restrict the use of alcohol congener analysis internationally and for other medico-legal scenarios. This review examines the forensic application of alcohol congener analysis in determining the source of alcohol and other techniques.