Cavity Enhanced Immunoassay Measurements in Microtiter Plates using BBCEAS

We report on the first detailed use of broadband cavity enhanced absorption spectroscopy (BBCEAS) as a detection system for immunoassay. A vertical R ≥ 0.99 optical cavity was integrated with a motorised XY stage, which functioned as a receptacle for 96 well microtiter plates. The custom built cavity enhanced microplate reader was used to make measurements on a commercially available osteocalcin sandwich ELISA kit. A 30 fold increase in path length was obtained with a minimum detectable change in the absorption coefficient, αmin(t), of 5.3 × 10-5 cm-1 Hz-1/2. This corresponded to a 39 fold increase in the sensitivity of measurement when directly compared to measurements in a conventional microplate reader. Separate measurements of a standard STREP-HRP colorimetric reaction in microtiter plates of differing optical quality produced an increase in sensitivity of up to 115 fold compared to a conventional microplate reader. The sensitivity of the developed setup compared favorably with previous liquid-phase cavity enhanced studies and approaches the sensitivity of typical fluorometric ELISAs. It could benefit any biochemical test which uses single pass absorption as a detection method, through either the label free detection of biologically important molecules at lower concentrations or the reduction in the amount of expensive biochemicals needed for a particular test, leading to cheaper tests

Postmortem tissue distribution of morphine and its metabolites in a series of heroin related deaths

The abuse of heroin (diamorphine) and heroin deaths are growing around the world. The interpretation of the toxicological results from suspected heroin deaths is notoriously difficult especially in cases where there may be limited samples. In order to help forensic practitioners with heroin interpretation we determined the concentration of morphine (M), morphine‐3‐glucuronide (M3G) and morphine‐6‐glucuronide (M6G) in blood (femoral and cardiac), brain (thalamus), liver (deep right lobe), bone marrow (sternum), skeletal muscle (psoas) and vitreous humor in 44 heroin related deaths. The presence of 6‐monoacetylmorphine (6‐MAM) in any of the postmortem samples was used as confirmation of heroin use. Quantitation was carried out using a validated LC‐MS/MS method with solid phase extraction. We also determined the presence of papaverine, noscapine and codeine in the samples, substances often found in illicit heroin and that may help determine illicit heroin use. The results of this study show that vitreous is the best sample to detect 6‐MAM (100% of cases), and thus heroin use. The results of the M, M3G and M6G quantitation in this study allow a degree of interpretation when samples are limited. However in some cases it may not be possible to determine heroin/morphine use as in 4 cases in muscle (3 cases in bone marrow) no morphine, morphine‐3‐glucuronide or morphine‐6‐glucuronide was detected, even though they were detected in other case samples. As always postmortem cases of suspected morphine/heroin intoxication should be interpreted with care and with as much case knowledge as possible

Phenazepam is currently being misused in the UK

Phenazepam is a benzodiazepine not currently controlled in the United Kingdom, mainland Europe, or the United States. Developed in the 1970s for the treatment of epilepsy, alcohol withdrawal syndrome, insomnia, and anxiety,1 2 it is currently prescribed only in the former Soviet Bloc. However, recent reports from Sweden, Finland, and the US describe its illicit use.3 In the UK concern over the safety of phenazepam was raised in 2010, when three people in the East Midlands and six people in Scotland were admitted to hospital after phenazepam overdoses.4 5 These cases and increased seizures of the drug by police led the Scottish Government to issue warnings about phenazepam. Having been alerted about its presence, we began screening necropsy blood samples for phenazepam in our forensic toxicology laboratory in Dundee from the end of January 2011. To date, we have identified nine cases in which postmortem blood samples contained phenazepam. There was a history of drug misuse in all cases, and they occurred in men and women aged 31 to 45. Death was from the adverse effects of opiates in seven cases and from non-drug related causes in two. This many cases suggests that the use of phenazepam by drug misusers in the UK is on the rise. Phenazepam can be obtained legally on the internet so it could become more widely used as substitute for controlled benzodiazepines or designer drugs such as mephedrone. Doctors should be aware of both the availability and illicit use of phenazepam in the UK

Nefopam hydrochloride:A fatal overdose

Nefopam is a non-opiate analgesic commonly used for the treatment of moderate to severe pain. A case of a 37-year-old male who was found dead in the morning is presented. An autopsy was performed and femoral venous blood, heart blood, urine, and vitreous humor were submitted for toxicological analysis. A general drug screen detected the presence of nefopam, caffeine, nicotine, citalopram, gabapentin, amitriptyline, diazepam and paracetamol in cardiac blood. Nefopam was quantitated by high-performance liquid chromatography with diode-array detection. Nefopam was found at the following concentrations: 13.6 mg/L in unpreserved femoral blood; 14.7 mg/L in preserved (fluoride–oxalate) femoral blood; 21.2 mg/L in unpreserved cardiac blood and 4.5 mg/L in preserved vitreous. Citalopram was present at a concentration of 0.7 mg/L (femoral blood) and 0.9 mg/L (cardiac blood). Ethanol analyzed by headspace gas chromatography (GC–FID) was detected in preserved (fluoride–oxalate) vitreous (14 mg/100 mL) and preserved (fluoride–oxalate) urine 50 mg/100 mL. Death was attributed to atherosclerotic coronary artery disease and therapeutic drug toxicit

Deaths associated with new designer drug 5-IT

5-IT or 5-API is the common name for a newly emerging designer drug.1 It is a positional isomer of the tryptamine drug α-methyltryptamine and has the chemical structure (1H-indol-5-yl)propan-2-amine. Currently not controlled in Europe, it is covered, however, by the federal analogue acts in the USA2 and Australia.3 Very little is known about the acute or chronic effects of 5-IT. An oral dose of 20 milligrams is said to produce long lived stimulant effects, including increased heart rate, anorexia, diuresis, and slight hyperthermia for about 12 hours.4 Recently we identified 5-IT in postmortem blood samples of two young adults. The substance was found in combination with other drugs in one case. In the other, 5-APB/6-APB (a designer drug similar in some respects to the chemical structure of 3,4-methylenedioxyamphetamine (MDA)) was also found. The National Board of Forensic Medicine in Sweden has recently identified the drug in 14 deaths and 5-IT was said to be the direct cause of death in two of these cases.5 5-IT is inexpensive, easily available online as a so called research chemical and therefore has the potential for becoming a replacement for other recently banned designer drugs. The medical community should be aware of both the availability and use of 5-IT within the UK. The drug needs to be banned, but again regulatory control in the UK will be chasing designer drug innovation

Phenazepam:The drug that came in from the cold

Phenazepam [7-bromo-5-(2-chlorophenyl)-1, 3-dihydro-2H-1,4-benzodiazepine-2-one], sometimes called fenazepam (Fig. 1A), is part of the 1,4-benzodiazepine group of benzodiazepines along with drugs such as diazepam, nordazepam, oxazepam and temazepam. It is however one of very few of the 1,4-benzodiazepine group to contain a bromine atom, along with the active form of gidazepam, another soviet-developed benzodiazepine5 and bromazepam. As with other benzodiazepines, it is used clinically for its anxiolytic, anticonvulsant, muscle-relaxing and sedating properties,6 and is available as 0.5-mg and 1-mg tablets,6 injectable solutions (0.1%, 0.3%) or transdermal patches (Phenopercuten).7 Clinically the dose of phenazepam that is given depends on what condition is being treated but does not exceed 10 mg day−1, and is usually not more than 5 mg day−1. Illicitly, phenazepam has been reported being sold as a powder, tablets, in the USA, spiked in lysergic acid diethylamide (LSD) mimic blotters8 and 9 and in New Zealand phenazepam has been found in a synthetic cannabinoid mix called ‘Kronic’.10 Reported recreational doses of phenazepam are around 2–10 mg, but sometimes more.