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

The abuse of heroin (diamorphine) and heroin-related deaths are increasing around the world. The interpretation of the toxicological results from suspected heroin-related deaths is notoriously difficult, especially in cases where there may be limited samples. 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 liquid chromatography-tandem mass spectrometry (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 four cases in muscle (three cases in bone marrow) no morphine, M3G, or M6G were 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.

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 motorized 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 redistribution of the heroin metabolites morphine and morphine-3-glucuronide in rabbits over 24 h.

The interpretation of postmortem drug levels is complicated by changes in drug blood levels in the postmortem period, a phenomena known as postmortem drug redistribution. We investigated the postmortem redistribution of the heroin metabolites morphine and morphine-3-glucuronide in a rabbit model. Heroin (1 mg/kg) was injected into anesthetised rabbit; after 1 h, an auricular vein blood sample was taken and the rabbit was euthanised. Following death rabbits were placed in a supine position at room temperature and divided into three groups namely (1) immediate autopsy, (2) autopsy after 30 minutes and (3) autopsy 24 h after death. Various samples which included femoral blood, cardiac blood, lung, liver, kidney, vitreous humour, subcutaneous and abdominal fat, liver, bone marrow and skeletal muscle were taken. The samples were analysed with a validated LC-MS/MS method. It was observed that within minutes there was a significant increase in free morphine postmortem femoral blood concentration compared to the antemortem sample (0.01 ± 0.01 to 0.05 ± 0.02 mg/L).Various other changes in free morphine and metabolite concentrations were observed during the course of the experiment in various tissues. Principal component analysis was used to investigate possible correlations between free morphine in the various samples. Some correlations were observed but gave poor predictions (>20 % error) when back calculating. The results suggest that rabbits are a good model for further studies of postmortem redistribution but that further study and understanding of the phenomena is required before accurate predictions of the blood concentration at the time of death are possible.

Analysis of phenazepam and 3-hydroxyphenazepam in post-mortem fluids and tissues.

Phenazepam is a benzodiazepine that is predominantly used clinically in the former Soviet states but is being abused throughout the wider world. This study reports the tissue distribution and concentration of both phenazepam and 3-hydroxyphenazepam in 29 cases quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a variety of post-mortem fluids (subclavian blood, femoral blood, cardiac blood, urine, vitreous humour) and tissues (thalamus, liver and psoas muscle). In 27 cases, the cause of death was not directly related to phenazepam (preserved (fluoride/oxalate) femoral blood phenazepam concentrations 0.007 mg/L to 0.360 mg/L (median 0.097 mg/L). In two cases, phenazepam was either a contributing factor to, or the certified cause of death (preserved (fluoride/oxalate) femoral blood 0.97 mg/L and 1.64 mg/L). The analysis of phenazepam and 3-hydroxyphenazepam in this study suggests that they are unlikely to be subject to large post-mortem redistribution and that there is no direct correlation between tissues/fluid and femoral blood concentrations. Preliminary investigations of phenazepam stability comparing femoral blood phenazepam concentrations in paired preserved (2.5% fluoride/oxalate) and unpreserved blood show that unpreserved samples show on average a 14% lower concentration of phenazepam and we recommend that phenazepam quantitation is carried out using preserved samples wherever possible.

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 toxicity.

Four fatalities involving 5-IT.

The new designer drug 5-(2-aminopropyl)indole (5-IT) is an indole derivative with stimulant properties. Its synthesis was first described by Albert Hofmann and Franz Troxler in 1962. We report four deaths associated with 5-IT and a validated high-performance liquid chromatography method for quantitation of the drug. In all four deaths, an autopsy was performed, and femoral venous blood, heart blood, urine and vitreous humor were submitted for toxicological analysis. The blood specimens were subjected to comprehensive testing that included alcohol analysis by headspace gas chromatography (GC-FID), acidic/neutral, basic drug and opiates screening by liquid chromatography-mass spectrometry (LC-MS-MS), and acidic/neutral, basic and acidic drugs screening by HPLC. In Case 1, a 25-year-old male, 3,4-methylenedioxymethamphetamine (MDMA; <0.08 mg/L) and 5-IT (preserved femoral blood 1.2 mg/L; unpreserved femoral blood 0.8 mg/L; cardiac blood 1.2 mg/L; vitreous 0.8 mg/L and urine >10 mg/L) were detected, and death was attributed to the toxic effects of 5-IT. In Case 2, a 25-year-old female, 3,4-methylenedioxy-N-methylcathinone (methylone, not quantitated), 6-(2-aminopropyl)benzofuran (6-APB; femoral blood <0.08 mg/L) and 5-IT (preserved femoral blood 1.0 mg/L; unpreserved femoral blood 0.9 mg/L; cardiac blood 2.6 mg/L; vitreous 1.4 mg/L and urine >10 mg/L) were detected, and death was attributed to the toxic 'cocktail effects' of the drugs. In Case 3, a 22-year-old male with a history of epilepsy, 5-IT (0.5 mg/L femoral blood) and 6-APB (0.2 mg/L femoral blood) were detected, and death was attributed to the toxic effects of the drugs, with the role of epilepsy being indeterminate. In Case 4, a 25-year-old female, 5-IT (0.4 mg/L femoral blood), amphetamine (0.4 mg/L femoral blood), MDMA (1.5 mg/L femoral blood), 4-methyl-N-ethylcathione, 3,4-methylenedioxyamphetamine HCl (MDA), benzylpiperazine and 6-APB were detected, and death was attributed to the 'cocktail effect' of the drugs.

Stability of 3,4-methylenedioxymethampetamine (MDMA), 4-methylmethcathinone (mephedrone) and 3-trifluromethylphenylpiperazine (3-TFMPP) in formalin solution.

Occasionally, the only postmortem samples available for analysis are contaminated with formaldehyde, either due to embalming prior to sampling or because analysis is carried out only when formalin-fixed tissues retained for histological study are available. Formaldehyde reacts with several drugs of forensic interest that contain either a primary or a secondary amine group to form their N-methyl derivatives. We investigated the stability of 3,4-methylenedioxymethampetamine (MDMA), 4-methylmethcathinone (mephedrone) and 3-trifluromethylphenylpiperazine (3-TFMPP) in formalin solutions using three different formaldehyde concentrations (5, 10 and 20%) and three different pHs (3.0, 7.0 and 9.5). Analysis was performed using high-performance liquid chromatography with diode array detection to determine the percentage degradation of each drug over time, up to 60 days. MDMA, mephedrone and 3-TFMPP are unstable in formalin solutions, with the degradation rate increasing with increasing pH. After 28 days in 20% formalin, pH 9.5, there remained 57% of the initial 3-TFMPP concentration, 11% of the initial MDMA concentration and 4% of the initial mephedrone concentration. Forensic toxicologists should be aware that, when analyzing for these drugs in an embalmed body or in tissues stored in formalin solutions, the methylated form of the secondary amine-containing drug could be a more useful analyte than the parent drug.

Discrimination of Sri Lankan black teas using fluorescence spectroscopy and linear discriminant analysis.

BACKGROUND: The quality of teas is currently graded using trained tea tasters, whose evaluation can sometimes be subjective. In this study the simple fluorescence-based technique of total luminescence spectroscopy (TLS) in conjunction with data classification using principal component analysis (PCA) was applied to discriminate between teas from 11 different Sri Lankan plantations. Solvent extraction of the tea samples was followed by TLS to record excitation-emission matrices in the excitation range 250-590 nm and emission range 300-700 nm. RESULTS: The application of PCA and linear discriminant analysis (LDA) allowed the successful classification of all 11 teas using only the first two principal components. LDA demonstrated how the technique was able to discriminate between all teas correctly with 100% classification. CONCLUSION: Further development of this work could lead to a simple device that could be used by tea manufacturers instead of or alongside trained tea tasters to grade teas.

Meptazinol and ethanol: a fatal intoxication.

Meptazinol (Meptid(®)) is an analgesic drug that is used to treat mild to moderate pain including postoperative pain, obstetrical pain, and the pain of renal colic. This case reports a death due to the combined effects of meptazinol and alcohol in a man with significant heart disease and alcoholic liver disease. A 57-year-old male was found unresponsive in his bed at home with empty blister packets of meptazinol around him. A general drug screen detected the presence of meptazinol, and caffeine and metabolites, in cardiac blood. Analysis, both quantitative (HPLC-DAD) and qualitative (HPLC-DAD, LC-MS), of meptazinol was carried out. Meptazinol was found at the following concentrations: 15.5 mg/L in unpreserved femoral blood; 18.6 mg/L in preserved (fluoride-oxalate) femoral blood; 52.1 mg/L in unpreserved cardiac blood; 16.8 mg/L in preserved vitreous; 61.7 mg/L in unpreserved urine; and 9.8 g/L in stomach contents. Ethanol, analyzed by headspace GC-FID, was present in preserved (fluoride-oxalate) femoral venous blood, urine, and vitreous at concentrations of 232 mg/100 mL, 297 mg/100 mL, and 192 mg/100 mL, respectively. Death was attributed to meptazinol and ethanol toxicity, with atherosclerotic coronary artery disease as a contributing factor.

Liquid-phase broadband cavity enhanced absorption spectroscopy (BBCEAS) studies in a 20 cm cell.

Sensitive liquid-phase measurements have been made in a 20 cm cell using broadband cavity enhanced absorption spectroscopy (BBCEAS). The cavity was formed by two high reflectivity mirrors which were in direct contact with the liquid-phase analytes. Careful choice of solvent was required to minimise the effect of background solvent absorptions. Measurements were made on the broad absorber Sudan black, dissolved in acetonitrile, using a white LED light source and R > or = 0.99 cavity mirrors, leading to a cavity enhancement factor (CEF) of 82 at 584 nm. The sensitivity as measured by the minimum detectable change in the absorption coefficient (alpha(min)) was 3.4 x 10(-7) cm(-1). Further measurements were made on the strong absorber methylene blue dissolved in acetonitrile at 655 nm. A white LED was used with the R > or = 0.99 cavity mirrors, leading to a CEF of 78 and alpha(min) = 4.4 x 10(-7) cm(-1). The use of a more intense red LED also allowed measurements with higher reflectivity R > or = 0.999 cavity mirrors, leading to a CEF of 429 and alpha(min) = 2.8 x 10(-7) cm(-1). The sensitivity was limited by dark noise from the detector but nevertheless appears to represent the most sensitive liquid-phase absorption measurement to date.

Broadband cavity enhanced absorption spectroscopy as a detector for HPLC.

The first demonstration of a cavity enhanced absorption spectroscopy (CEAS) based technique, applied to HPLC detection is reported. Broadband cavity enhanced absorption spectroscopy (BBCEAS) has been used for detection in a HPLC system (HPLC-BBCEAS). Measurements were made on the dyes rhodamine 6G and rhodamine B between 450 and 600 nm. The sensitivity of the measurements as determined by the minimum detectable change in the absorption coefficient, alpha(min), were 2.9 x 10(-5) cm(-1) at 527 nm and 1.9 x 10(-5) cm(-1) at 556 nm, the peak absorption wavelengths of rhodamine 6G and rhodamine B, respectively. The limits of detection (LOD) for the two dyes were 426 and 271 pM, respectively. The LOD of the HPLC-BBCEAS setup was found to be between 54 and 77 times lower than with a Perkin-Elmer HPLC (series 200) comprising a 200EP photodiode array detector. The sensitivity of the developed setup also compared favorably with the previous single wavelength HPLC-CRDS studies while using a considerably lower cost experimental setup and simpler experimental methodology. The use of BBCEAS detection also allowed the discrimination following an isocratic HPLC separation of the nearly co-eluting dyes rhodamine 6G and rhodamine B.

Liquid-phase broadband cavity-enhanced absorption spectroscopy measurements in a 2 mm cuvette.

A novel implementation of broadband cavity enhanced absorption spectroscopy (BBCEAS) has been used to perform sensitive visible wavelength measurements on liquid-phase solutions in a 2 mm cuvette placed at normal incidence to the cavity mirrors. The overall experimental methodology was simple, low cost, and similar to conventional ultraviolet-visible absorption spectroscopy. The cavity was formed by two concave high reflectivity mirrors. Three mirror sets with nominal reflectivities (R) of R = 0.99, 0.9945, and 0.999 were used. The light source consisted of a high intensity red, green, blue, or white LED. The detector was a compact charge-coupled device (CCD) spectrograph. Measurements were made on the representative analytes, Ho(3+), and the dyes brilliant blue-R, sudan black, and coumarin 334 in appropriate solvents. Cavity enhancement factors (CEF) of up to 104 passes for the high reflectivity mirrors were obtained. The number of passes was limited by relatively high scattering and absorption losses in the cavity, of approximately 1 x 10(-2) per pass. Measurements over a wide wavelength range (420-670 nm) were also obtained in a single experiment with the white LED and the R = 0.99 mirror set for Ho(3+) and sudan black. The sensitivity of the experimental setup could be determined by calculating the minimum detectable change in the absorption coefficient alpha(min). The values ranged from 5.1 x 10(-5) to 1.2 x 10(-3) cm(-1). The limit of detection (LOD) for the strong absorber brilliant blue-R was 620 pM. A linear dynamic range of measurements of concentration over about two orders of magnitude was demonstrated. The overall sensitivity of the experimental setup compared very favorably with previous generally more experimentally complex and expensive liquid-phase cavity studies. Possible improvements to the technique and its applicability as an analytical tool are discussed.