The effect of sodium fluoride preservative and storage temperature on the stability of 6-acetylmorphine in horse blood, sheep vitreous and deer muscle

This study examined the in vitro stability of 6-acetylmorphine (6AM) in horse blood, sheep vitreous humour (VH) and homogenised deer muscle stored under different storage conditions. The stability of 6AM in horse blood is of interest because many toxicological laboratories utilise this matrix for the preparation of blood calibration and check standards and the latter are typically stored during routine use. Data on the storage stability of 6AM in human VH is extremely limited and no data has been reported in muscle. In the absence of human samples, 6AM stability was demonstrated in sheep vitreous and deer muscle. Blood and VH were stored with and without NaF at room temperature (RT), 4 and -18°C for 84 days. Muscle tissue homogenates were prepared in water with and without NaF and also in phosphate buffer (pH 6.0) containing NaF. Homogenates were stored for 31 days at RT, 4 and -18°C. Morphine and 6AM were extracted using SPE and quantified by GC-ion trap-MS/MS. In the absence of NaF, 6AM could not be detected after 7 and 14 days in blood stored at RT and 4°C, respectively. Although at -18°C 6AM was stable for 7 days (12% loss), only 54% was detected by day 84. The addition of NaF to horse blood increased 6AM stability substantially at every temperature. Further, the rate of degradation was found to be significantly slower in blood preserved with 2% NaF compared with 1% NaF (p=.05). 6AM was stable for the study period in preserved blood (1 and 2% NaF) stored at -18°C. For laboratories utilising horse blood in the preparation of standards, preservation with 1% NaF (minimum) and storage at -18°C is recommended. The addition of NaF to VH was essential for 6AM stability. Irrespective of temperature substantial losses (≥ 42%) were observed in unpreserved sheep VH by day 7. In preserved VH the concentration declined by only 22% on day 7 following storage at RT and no loss observed in VH stored at 4 and -18°C at the same time. In muscle, 6AM was stable for 7 days in preserved samples stored at RT and in all samples stored at 4°C and below. The addition of NaF increased the stability of 6AM substantially in muscle. The increased stability of 6AM in VH and muscle preserved with fluoride was attributed to inhibition of bacterial action and the subsequent reduction in the rate of putrefaction of these tissues.     

Ethanol stability in unpreserved refrigerated antemortem blood

Ethanol stability in preserved antemortem blood has been widely studied since it is a common practice in cases involving suspected impaired driving to collect antemortem blood in evacuated blood tubes containing sodium fluoride. In some situations, antemortem blood is submitted to a forensic laboratory for ethanol analysis in evacuated blood tubes that contain only an anticoagulant. There has been limited research on ethanol stability in antemortem blood stored without a preservative. On two occasions, antemortem blood was collected from five ethanol-free individuals into 6-ml Vacutainer® tubes containing only 10.8 mg potassium EDTA. The blood tubes were spiked with ethanol to approximately either 0.08 or 0.15 g/dl. Dual-FID headspace gas chromatography was used to analyze 58 blood tubes, 29 from each session, for ethanol 1 day after sample collection and again after 1 year of refrigerated storage (~4°C). Statistically significant decreases in ethanol were detected at the 0.05 level of significance. Mean decreases in ethanol after 1 year of storage for the 0.08 and 0.15 g/dl samples were 0.013 and 0.010 g/dl, respectively. The mean ethanol decrease across all tubes was 0.012 g/dl. The range of decreases for the 58 blood tubes was 0.003–0.018 g/dl. The mean ethanol decreases measured in this unpreserved antemortem blood are comparable in magnitude to those previously observed in antemortem blood containing sodium fluoride after 1 year of refrigerated storage. Ethanol did not increase in the antemortem blood samples despite the absence of sodium fluoride.     

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4°C and -18°C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4°C and -18°C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18°C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4°C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4°C and had declined by 81% following storage at -18°C. At 4°C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18°C was essentially stable for the study period whereas at 4°C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4°C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18°C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH.     

Quantification of Morphine,Codeine, and Thebaine in Home‐Brewed Poppy Seed Tea by LC‐MS/MS

Recently, medical examiners reported two cases of a 21‐year‐old male and 24‐year‐old male with high amounts of morphine in their blood at autopsy. It was suspected that the decedents ingested lethal amounts of morphine from home‐brewed poppy seed tea. No studies to date have investigated opium alkaloid content extracted from poppy seeds by home‐brewing methods. Various poppy seed products were purchased from online sources and extracted with four home‐brewing methods representative of recipes found on drug user forums. Morphine, codeine, and thebaine were quantified in the tea extracts by liquid chromatography‐tandem mass spectrometry using a validated analytical method. Morphine, codeine, and thebaine concentrations from seeds were <1–2788 mg/kg, <1–247.6 mg/kg, and <1–124 mg/kg, respectively. Alkaloid yield varied between extractions, but regardless of extraction conditions, lethal amounts of morphine can be rinsed from poppy seed coats by home‐brewing methods.     

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid

The purpose of this study was to evaluate the efficiency of the Cozart^® RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart^® RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart^® RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography–mass spectrometry (GC–MS). The samples were stored frozen at −20 °C until analysis by GC–MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC–MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.     

血液样品中防腐剂对碳氧血红蛋白稳定性的影响

目的研究临床上常用8种试剂对血液样品中碳氧血红蛋白(HbCO)稳定性的影响。方法将血液样品分为高、低两个HbCO浓度组,选用临床常用的甲醛、氟化钠、乙二胺四乙酸二钠、亚硝酸钠、草酸钾、肝素钠、柠檬酸钾及氟化钠与草酸钾混合物(1:3)8种试剂,按常用浓度分别添加到血样品中,并于添加后0h、2h、8h、24h、3d、7d用紫外可见光分光光度法检测其中HbCO饱和度,用统计学方法进行结果分析。结果本实验选用的8种试剂只有甲醛和亚硝酸钠对HbCO的稳定性影响较为显著,而其余6种对HbCO稳定性的影响无统计学意义。结论在检验疑似CO中毒并经甲醛或亚硝酸钠防腐的检材时应慎重,以免导致错误的鉴定结论。     

Bacterial Degradation of Risperidone and Paliperidone in Decomposing Blood

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high‐performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole‐cleaved degradation products for both drugs were identified as 2‐hydroxybenzoyl‐risperidone and 2‐hydroxybenzoyl‐paliperidone utilizing liquid chromatography quadrupole‐time‐of‐flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.     

Elevated morphine concentrations determined during infant death investigations: artifacts of withdrawal of care

Abstract:  Three cases are reported of elevated postmortem blood morphine concentrations (189–3036 ng/mL) that were observed during the course of death investigations involving three children ranging in age from 1 week to 2 years, all of whom underwent withdrawal of life support. In all three cases, the presence of opiates in postmortem blood was indicated by immunoassay (ELISA) and quantitative confirmatory analysis of free morphine concentrations in postmortem blood was performed by solid-phase extraction followed by gas chromatography/mass spectrometry (GC/MS) in the selected ion monitoring mode. While the practice of withdrawing life support from terminally ill patients, with the accompanying administration of narcotics/analgesics has been reported in the medical literature, it has not been adequately described in the forensic literature. The implications of this practice on the forensic toxicological interpretation of morphine findings are discussed. To our knowledge, this is the first report of postmortem morphine concentrations arising directly from administration in conjunction with withdrawal of care in pediatric patients.     

Studies on the stability and detection of cocaine, benzoylecgonine, and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in whole blood using Abuscreen radioimmunoassay

A study was undertaken to assess the stability and the radioimmunoassay (RIA) detection of cocaine, benzoylecgonine (BZE), and 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in whole blood while stored in 4 different kinds of blood collection tubes for up to 30 days at refrigeration and room temperatures. At various intervals, the tubes were sampled and analyzed using Abuscreen RIA. Also, semi-quantitative data derived from RIA analysis of forensic blood specimens were compared with quantitative data acquired using gas chromatography (GC) or GC/mass spectrometry (GC/MS) on the same specimens. RIA and chromatographic studies revealed that BZE and THC-COOH were stable in blood under all conditions studied. Cocaine, however, was found not to be stable in blood, especially when stored at room temperatures. Despite cocaine's instability in blood, RIA was able to detect the presence of cocaine and its breakdown products in blood under all conditions studied.     

The Pharmacokinetics of Morphine and Codeine in Human Plasma and Urine after Oral Administration of Qiangli Pipa Syrup

Papaveris pericarpium, a natural source of morphine and codeine, is the principal active component in many antitussive traditional Chinese medicines. We herein report the first PK study of papaveris pericarpium in human plasma and urine following oral administration of single (15, 30, 60 mL) and multiple dose (15 mL) of Qiangli Pipa Syrup (MOR 0.1 mg/mL, COD 0.028 mg/mL) by monitoring morphine and codeine using a HPLC‐MS/MS method. Their T_max and t_1/2 values are independent of dosages, while the AUC_0?t linearly increased with higher dosages, indicating linear PK characteristics. AUC_0?t increased obviously after multiple doses, indicating possible risk of accumulative toxicity. Urine studies suggested risks of positive opiate drug tests with a cutoff of 300 ng/mL, which lasted 6–14 h at different doses. These results provide important information for clinical safety, efficacy and rational drug use of Qiangli Pipa Syrup and also guide the related judicial expertise of its administration.     

Morphine and 6-acetylmorphine concentrations in blood, brain, spinal cord, bone marrow and bone after lethal acute or chronic diacetylmorphine administration to mice

The aim of this study was to evaluate postmortem incorporation of opiates in bone and bone marrow after diacetylmorphine (heroin) administration to mice. Mice were given acute (lethal dose of 300 mg/kg) or chronic (10 and 20 mg/kg/24 h for 20 days) intraperitoneal administration of diacetylmorphine. The two metabolites of diacetylmorphine, 6-acetylmorphine (6-AM) and morphine, were extracted from whole blood, brain, spinal cord, bone marrow and bone (after hydrolysis) using a liquid/liquid method. Quantification was performed by gas chromatography-mass spectrometry (GC/MS). Results showed that after acute administration, opiates were present in all studied tissues. Morphine concentrations appeared to be higher than those of 6-AM in blood (52.4 microg/mL versus 27.7 microg/mL, n=12), bone marrow (87.8 ng/mg versus 8.9 ng/mg, n=6) and bone (0.85 ng/mg versus 0.43 ng/mg, n=6), but 6-AM concentrations were higher than those of morphine in brain (14.0 ng/mg versus 7.4 ng/mg, n=12) and spinal cord (27.8 ng/mg versus 20.8 ng/mg, n=12). No correlation was found for both compounds between blood concentrations and either brain, spinal cord, bone or bone marrow concentrations while a significant one was found between brain and spinal cord concentrations either for morphine (r=0.89, n=12, p<0.001) or 6-AM (r=0.93, n=12, p<0.001), the concentration being higher in spinal cord than in brain. When bones were stored for 2 months, only 6-AM remained in bone marrow but not in bone. After chronic administration, mice being sacrificed by cervical dislocation 24 h after the last injection, no opiate was detected in any studied tissues. Further studies are required, in particular in human bones, but these results seem to show that 6-AM could be detect in bone marrow several weeks after the death and could be an alternative tissue for forensic toxicologist to detect a fatal diacetylmorphine overdose, even if no correlation between blood and bone marrow was observed. On the other hand, neither bone tissue nor bone marrow will allow the confirmation of a chronic diacetylmorphine use.     

Gamma-hydroxybutyric acid stability and formation in blood and urine

Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples.     

Multiple homicides as a result of chloroform poisoning: case report and experimental study.

Two cases (involving five murder victims) of multiple homicide by inhalational chloroform intoxication are reported. In the discussion of the findings the valence of toxicological analyses is underlined with regard to the possibility of forcible external suffocation due to occlusion of the respiratory orifices by means of a chloroform-soaked soft covering. In addition storage experiments were performed at +4, +20 and -20 degrees C with cadaver blood mixed with chloroform. The optimal solution for avoiding volatile losses was stored in glass tubes with ground glass stoppers. In cases of unclear death in which involvement of volatile substances is suspected it is, therefore, advisable to preserve an additional blood sample at -20 degrees C in glass tubes that are only opened for the analysis of volatile substances.     

An investigation into the use of a portable cyanoacrylate fuming system (SUPERfume®) and aluminum powder for the development of latent fingermarks

Abstract: Few techniques offer “in situ” methods of friction ridge skin mark development. “In situ” development reduces mark transportation, degradation, and often cost. The effectiveness of cyanoacrylate fuming using the SUPERfume^® and dusting with aluminum powder for latent fingermark development on several nonporous surfaces, stored in various temperature environments for time periods up to 52 weeks, was investigated. Five thousand and four hundred latent fingermarks were deposited under controlled conditions and graded. The results suggested that cyanoacrylate fuming (SUPERfume^®, Foster and Freeman, U.K.) was more effective at developing latent fingermarks on textured and smooth plastic surfaces and for marks stored in temperatures of 37°C, whereas aluminum powder was more effective on glass, enameled metal paint, and varnished wood, and for storage temperatures below 20°C. There were no significant benefits to using either technique for marks older than 24 h, but it was possible to develop fingermarks following 52 weeks of storage using both techniques.     

The Stability of 4-Chloromethcathinone in Blood and Vitreous Humor

We present results of our study on the stability of 4-chloromethcathinone (4-CMC) in authentic postmortem peripheral blood and vitreous humor samples. The stability of 4-CMC was determined in postmortem blood samples (for a period of 90 days) and vitreous humor (30 days) at three different temperatures: −15°C, +4°C, and + 23°C. The analyses were carried out using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). In both materials, the lowest 4-CMC stability was demonstrated at room temperature. The blood samples stored in a freezer (−15°C) showed stability for the entire study period (90 days), while in the case of the vitreous humor sample stored at the same temperature the concentration of the substance decreased by 53% after 30 days. The study carried out in authentic postmortem blood and vitreous humor samples confirms the previous reports of 4-CMC instability in biological material. Authors suggest that the biological material should be stored frozen until analyses are carried out as soon as possible after collection of the material.     

Refining Stable Oxygen and Hydrogen Isoscapes for the Identification of Human Remains in Mississippi,

Isoscape refinement is an essential component for accurately predicting region‐of‐origin in forensic investigations involving isotope analysis of unidentified human remains. Stable oxygen (δ¹⁸O) and hydrogen (δ²H) isotopes were measured from 57 tap water samples collected across Mississippi to model refined isoscapes for the state. A tap water conversion equation, δ¹⁸O_tw=1.64 δ¹⁸Op?31.35, was developed for the southeastern USA to test the prediction accuracy of the δ¹⁸O_tw isoscape using individuals with known residential histories. A local Mississippi resident (USAFA‐134) was assigned with 90% probability to the correct region‐of‐origin reported by the participant. Assignments for Georgia residents (USAFA‐118 and USAFA‐205) had variable results, predicting USAFA‐118 from Mississippi and USAFA‐205 as a nonlocal resident. Stable isotope values often overlap geographically and a multi‐isotope approach should be used when narrowing region(s)‐of‐origin(s). This study demonstrates the utility of refining isoscapes and the importance of tissue calibration in prediction assignments of human remains.     

Evaluation of a method for simultaneous quantification of codeine, ethylmorphine and morphine in blood.

Codeine, ethylmorphine and morphine are the most commonly detected opiates in forensic blood samples in Norway. A method for the simultaneous quantification of these opiates utilizing solid phase extraction and gas chromatography-mass spectrometry has been evaluated. The detection limits were 0.026 mumol/l for codeine, 0.025 mumol/l for ethylmorphine and 0.032 mumol/l for morphine (corresponding to 7.8, 7.8 and 9.1 micrograms/l, respectively). The analytical variations at concentrations of 1.0 mumol/l codeine, 1.0 mumol/l ethylmorphine and 0.5 mumol/l morphine were less than 5%.     

The Stability of Collected Human Scent Under Various Environmental Conditions*,†

Abstract: Human scent evidence collected from objects at a crime scene is used for scent discrimination with specially trained canines. Storage of the scent evidence is usually required yet no optimized storage protocol has been determined. Storage containers including glass, polyethylene, and aluminized pouches were evaluated to determine the optimal medium for storing human scent evidence of which glass was determined to be the optimal storage matrix. Hand odor samples were collected on three different sorbent materials, sealed in glass vials and subjected to different storage environments including room temperature, ?80°C conditions, dark storage, and UVA/UVB light exposure over a 7‐week period. Volatile organic compounds (VOCs) in the headspace of the samples were extracted and identified using solid‐phase micro‐extraction–gas chromatography/mass spectrometry (SPME–GC/MS). Three‐dimensional covariance mapping showed that glass containers subjected to minimal UVA/UVB light exposure provide the most stable environment for stored human scent samples.     

Comparative Toxicology of Intentional and Accidental Heroin Overdose*

Abstract: The demographic and toxicological characteristics of deliberate (SUI, n = 50) and accidental (ACC, n = 927) fatal heroin overdose cases were examined. SUI cases were more likely to be female, had lower body mass indices, were more likely to be enrolled in treatment and less likely to have hepatic pathology. The median blood morphine concentration of SUI cases was significantly higher than that of ACC cases (0.70 vs. 0.40 mg/L, p < 0.001). Blood morphine concentrations of >1 mg/L were seen among 38.0% of SUI cases compared to 13.9% of ACC cases. Being a member of the SUI group remained a significant independent predictor of higher morphine concentrations after controlling for the effects of potential confounders (p < 0.001), other significant predictors being the absence of alcohol (p < 0.001), the presence of methadone (p < 0.05), and the presence of cocaine (p < 0.05). The current data are consistent with the view that suicide forms a small, but distinct, category of heroin overdose cases, rather than overdose being a parasuicidal phenomenon per se.