Robin A. Braithwaite

Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry

Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 microL of the 250 microL extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabolite following a small oral dose of ketamine (50 mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospective detection of administration. The detection limit for ketamine and norketamine was 0.03 ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respectively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection.

The prevalence of drugs and alcohol found in road traffic fatalities: a comparative study of victims

Researchers have studied the involvement of drugs and alcohol in fatal road traffic incidents, but with particular emphasis on the possible impairment of the driver. This paper describes a comparative study of drug and alcohol findings in various victim groups (drivers of cars, vans or lorries, car passengers, motorcyclists, motorcycle passengers, cyclists and pedestrians) between 2000 and 2006. Post-mortem blood and urine specimens submitted were analysed by immunoassay, GC-NPD, GC-FID, GC-MS and HPLC-DAD. The results of 1047 cases indicated 54% of all victims were positive for drugs and/or alcohol, with the highest percentage of positive findings occurring in pedestrians (63%). Males between the ages of 17-24 were most likely to be involved in a road traffic accident, whether being in control of a vehicle (driver) or involved indirectly (car passenger, pedestrian, motorcycle passenger). A wide range of drugs were detected (e.g., drugs of abuse, anti-convulsants, anti-histamines, anti-inflammatories, anti-psychotics, cardiac drugs and over-the-counter products), but alcohol and cannabinoids were the most frequent substances across the victim groups. When detected, alcohol was commonly above the legal driving limit in blood and urine (>63% in those in control and >60% not in control). Overall, the presence of drugs and/or alcohol was of similar frequency in those victims in control (55% of driver, 48% of motorcyclists, 33% of cyclists) and not in control of a vehicle (52% of car passengers, 63% of pedestrians). This degree of frequency strongly implicates the involvement of drugs and alcohol in road traffic incidents and infers an effect on driving ability and individual impairment.

Use of human microsomes and deuterated substrates: an alternative approach for the identification of novel metabolites of ketamine by mass spectrometry.

In vitro biosynthesis using pooled human liver microsomes was applied to help identify in vivo metabolites of ketamine by liquid chromatography (LC)-tandem mass spectrometry. Microsomal synthesis produced dehydronorketamine, seven structural isomers of hydroxynorketamine, and at least five structural isomers of hydroxyketamine. To aid identification, stable isotopes of the metabolites were also produced from tetra-deuterated isotopes of ketamine or norketamine as substrates. Five metabolites (three hydroxynorketamine and two hydroxyketamine isomers) gave chromatographically resolved components with product ion spectra indicating the presence of a phenolic group, with phenolic metabolites being further substantiated by selective liquid-liquid extraction after adjustments to the pH. Two glucuronide conjugates of hydroxynorketamine were also identified. Analysis by LC-coupled ion cyclotron resonance mass spectrometry gave unique masses in accordance with the predicted elemental composition. The metabolites, including the phenols, were subsequently confirmed to be present in urine of subjects after oral ketamine administration, as facilitated by the addition of deuterated metabolites generated from the in vitro biosynthesis. To our knowledge, phenolic metabolites of ketamine, including an intact glucuronide conjugate, are here reported for the first time. The use of biologically synthesized deuterated material as an internal chromatographic and mass spectrometric marker is a viable approach to aid in the identification of metabolites. Metabolites that have particular diagnostic value can be selected as candidates for chemical synthesis of standards.

Detection of ketamine and its metabolites in human hair using an integrated nanoflow liquid chromatography column and electrospray emitter fritted with a single porous 10 μm bead.

Targeting metabolites incorporated into hair following drug administration is useful for evidential purposes as this approach can aid in differentiating between administration and passive exposure. Greater analytical sensitivity is required than for targeting the parent drug alone. A 20 μm i.d. fused silica capillary column with an integrated electrospray emitter fritted with a single porous 10 μm polymeric bead has been successfully fabricated to facilitate this purpose. The sensitivity gains through the use of these integrated single fritted columns coupled to a nanoelectrospray source (nanoflow-LC nanoESI) over conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) columns was explored by their application to the detection of ketamine and its phase I metabolites in human hair. Hair was collected from 4 volunteers following the administration of a small oral dose of ketamine (50 mg) and subsequently analysed by the capillary-LC nanoESI approach. The drug and its metabolites were extracted from hair using solid phase extraction following a methanolic wash, pulverisation with a ball mill and acid digestion. From a 50 μL extract, 1 μL was injected and the method provided a limit of detection estimated to be 5 fg on column for ketamine and norketamine and 10 fg for dehydronorketamine. The single porous frit minimises extra column band broadening and offers an alternative fritting approach which reduces the blocking of the electrospray emitter, in comparison with other approaches such as sintering and polymerisation.

Metabolites of lorazepam: Relevance of past findings to present day use of LC‐MS/MS in analytical toxicology

The advent of liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the sensitivity it confers, permits the analysis of both phase I and II drug metabolites that in the past would have been difficult to target using other techniques. These metabolites may have relevance to current analytical toxicology employing LC-MS/MS, and lorazepam was chosen as a model drug for investigation, as only the parent compound has been targeted for screening purposes. Following lorazepam administration (2 mg, p.o.) to 6 volunteers, metabolites were identified in urine by electrospray ionization LC-MS/MS, aided by the use of deuterated analogues generated by microsomal incubation for use as internal chromatographic and mass spectrometric markers. Metabolites present were lorazepam glucuronide, a quinazolinone, a quinazoline carboxylic acid, and two hydroxylorazepam isomers, one of which is novel, having the hydroxyl group located on the fused chlorobenzene ring. The quinazolinone, and particularly the quinazoline carboxylic acid metabolite, provided longer detection windows than lorazepam in urine extracts not subjected to enzymatic hydrolysis, a finding that is highly relevant to toxicology laboratories that omit hydrolysis in order to rapidly reduce the time spent on gas chromatography-mass spectrometry (GC-MS) analysis. With hydrolysis, the longest windows of detection were achieved by monitoring lorazepam, supporting the targeting of the aglycone with free drug for those incorporating hydrolysis in their analytical toxicology procedures.

Chapter 11 – Aminoindane Analogues

One of the less well known compound classes of the so-called novel psychoactive substances are represented by analogues and derivatives that are based on the 2-aminoindane nucleus, which due to its structure, is a conformationally rigid analogue of amphetamine. Published animal and in vitro studies of the pharmacological profile of various aminoindanes indicate an involvement of serotonin release and re-uptake. In addition, drug discrimination studies carried out in rats pointed towards some pharmacological properties that are also shared with 3,4-methylenedioxymethamphetamine (MDMA). At first glance, one might be tempted to consider whether such substances would display empathogenic or entactogenic effects similar to MDMA and/or whether drug users might consider these substances as potential MDMA-replacements, particularly those on the lookout for substances that are not under control in most countries. At present there is very limited information about the acute and chronic human toxicity of various aminoindane derivatives, with the extent of current availability and trends in recreational use of this class of drugs being uncertain. However, from a clinical and forensic toxicology perspective, it is important to be able to identify the possible presence of such compounds and their potential metabolites in biological fluids and tissues, also to follow any trend in their use and need for control measures to be introduced. This chapter will provide an account of selected aminoindanes regarding their chemistry, pharmacology and toxicology and concludes that, so far, aminoindanes appear to play a minor role.

Signal enhancement of glucuronide conjugates in LC‐MS/MS by derivatization with the phosphonium propylamine cation tris(trimethoxyphenyl) phosphonium propylamine, for forensic purposes

Although chemical derivatization for signal enhancement in drug testing is most often associated with gas chromatography, it also has the potential to improve the detection of analytes poorly ionized by atmospheric pressure ionization techniques, such as electrospray ionization used in liquid chromatography-mass spectrometry. A number of acidic compounds, namely drug glucuronides (e.g. conjugates of temazepam, oxazepam, lorazepam, morphine, testosterone, epitestosterone, 5-α-dihydrotestosterone, androsterone, p-nitrophenol, and paracetamol) were successfully derivatized with tris(trimethoxyphenyl) phosphoniumpropylamine to introduce a quaternary cation functionality to the analytes. Benzodiazepine glucuronides were more specifically investigated, and following positive mode electrospray ionization mass spectrometry, average improvements to peak areas as a result of derivatization were 67-, 6-, and 7- fold for temazepam, oxazepam, and lorazepam glucuronides. Average improvements to the signal-to-noise ratios for temazepam, oxazepam, and lorazepam glucuronides were 1336-, 371- and 217-fold, respectively. The values obtained for the derivatized conjugate were also typically higher than those for the underivatized parent drug. Urine containing benzodiazepine glucuronides was also successfully derivatized. The data indicates potential for the use of charge derivatization to improve the detection of molecules with acidic functionalities by liquid chromatography-mass spectrometry (LC-MS) techniques in certain scenarios.