Analysis of hair after contamination with blood containing cocaine and blood containing benzoylecgonine

In post-mortem work, blood is a potential source of external contamination of hair. The present study was carried out to investigate the amount of drug absorbed into hair which has been contaminated with blood containing either cocaine or BE. Solutions were prepared containing 0.05, 0.1, 0.2, 0.5 and 3.0 μg/mL of either cocaine or BE in human blood. Samples of approximately 3.2 g of drug-free hair were contaminated by soaking in the blood solutions for 5 min. They were then removed and left at room temperature. Approximately 0.5 g of hair was collected from each of the blood soaked hair samples at 6 h, 1, 2, 4 and 7 days after contamination. As each hair sample was collected it was shampoo-washed to prevent further drug absorption. Hair samples were analysed in triplicate using a fully validated method described previously. EME and cocaethylene were also measured in order to find out if cocaine or BE was breaking down to these compounds. Both cocaine and BE were absorbed into hair in significant concentrations when the concentration in the blood was 0.5 μg/mL or greater; cocaine was more readily absorbed than BE. Cocaine broke down to EME (<LOQ) at 0.5 μg/mL and to EME (>LOQ) and BE (<LOQ) at 3.0 μg/mL. When the blood concentration of cocaine was 0.5 μg/mL or less, there was no evidence of it breaking down to form BE. From the samples soaked in blood containing BE, there was no evidence of the BE breaking down. The absorption of drug into hair did not increase as the contamination period increased from 6 h to 7 days.     

吸毒者头发中海洛因、6-单乙酰吗啡、吗啡、可待因和乙酰可待因的分析及评价

目的确定海洛因吸毒者头发中海洛因、6-单乙酰吗啡（6-MAM）、吗啡（MOR）、可待因（COD）和乙酰可待因（AC）的浓度并考察其与国际毛发分析协会（Society of Hair Testing,SOHT）建议标准的适用性。方法 50个头发样品经冷冻研磨处理后采用液相色谱-串联质谱方法分析。结果所有样品中均检出6-MAM,并且浓度高于SOHT所建议的0.2 ng/mg,其中海洛因、6-MAM、MOR、COD和AC的浓度与其它研究报道无明显差异。头发中6-MAM：MOR的比率在0.15~36.27范围。结论由于各实验室间毛发样品前处理方法不同,而且存在吸食毒品中成分、剂量、吸食方式、代谢、头发颜色等诸多个体差异,本研究认为采用头发中6-MAM：MOR的比率大于1.3标准难以应用于鉴定海洛因吸毒。     

Concentrations of unconjugated morphine, codeine and 6-acetylmorphine in urine specimens from suspected drugged drivers

Concentrations of unconjugated morphine, codeine and 6-acetylmorphine (6-AM), the specific metabolite of heroin, were determined in urine specimens from 339 individuals apprehended for driving under the influence of drugs (DUID) in Sweden. After an initial screening analysis by immunoassay for 5-classes of abused drugs (opiates, cannabinoids, amphetamine analogs, cocaine metabolite and benzodiazepines), all positive specimens were verified by more specific methods. Opiates and other illicit drugs were analyzed by isotope-dilution gas chromatography-mass spectrometry (GC-MS). The limits of quantitation for morphine, codeine and 6-AM in urine were 20 ng/mL. Calibration plots included an upper concentration limit of 1000 ng/mL for each opiate. We identified the heroin metabolite 6-AM in 212 urine specimens (62%) at concentrations ranging from 20 ng/mL to > 1000 ng/mL. The concentration of 6-AM exceeded 1000 ng/mL in 79 cases (37%) and 31 cases (15%) were between 20 and 99 ng/mL. When 6-AM was present in urine the concentration of morphine was above 1000 ng/mL in 196 cases (92%). The concentrations of codeine in these same urine specimens were more evenly distributed with 35% being above 1000 ng/mL and 21% below 100 ng/mL. These results give a clear picture of the concentrations of unconjugated morphine, codeine and 6-acetylmorphine that can be expected in opiate-positive urine specimens from individuals apprehended for DUID after taking heroin.     

Distribution of 6-monoacetylmorphine and morphine in head and pubic hair from heroin-related deaths

There is limited published data to aid interpretation of analytical findings from hair analysis. The aim of the study was to collate 6-monoacetylmorphine (6-AM) and morphine concentrations in head and pubic hair from heroin users and to propose reference ranges and relate these to the amount of heroin used. The ratio of morphine-to-6-AM was also investigated. A total of 82 head hair samples divided into 173 segments of various lengths and 15 pubic hair samples were collected at postmortem from heroin users. The hair was analysed using a previously published method. A statistical evaluation demonstrated that in head hair, the lower, middle and upper concentration ranges of 6-AM were 0.1–0.9, 0.9–12.5 and 12.5–154.1 ng/mg and those of morphine were 0.1–0.8, 0.8–6.0 and 6.0–36.3 ng/mg. In pubic hair, the lower, middle and upper concentration ranges of 6-AM were 0.2–0.5, 0.5–2.3 and 2.3–18.2 ng/mg and those of morphine were 0.2–0.4, 0.4–2.4 and 2.4–13.3 ng/mg. The morphine-to-6-AM ratio showed a large variation. The ratio tended to decrease from proximal to distal segments. The statistical results suggest low, middle and high concentration ranges which we propose can be used for estimating the amount of heroin consumed into corresponding low or occasional, regular or habitual and heavy or excessive drug use. The ratio of morphine-to-6-AM showed great variation and did not support the proposal that a ratio less than 0.77 is needed to prove ingestion of heroin.     

The history of heroin abuse by assaying 6-monoacetylmorphine and morphine in human hair

The purpose of this study is to conclude the history of heroin abuse by assaying 6-monoacetylmorphine (MAM) and morphine (MOR) in human hair. The hair of heroin abuse was labeled and segmented, then washed and cut into fragments. After hydrolyses and extraction, 6-MAM and MOR in human hair were determined by GC/MS-SIM with selected ion monitoring. Results of the segmented hair were analyzed. It provided useful information about the history of heroin abuse (hair growth rate 1-1.5 cm/mon).     

High prevalence of 6-acetylmorphine in morphine-positive oral fluid specimens

Identification of 6-acetylmorphine, a specific metabolite of heroin, is considered to be definitive evidence of heroin use. Although 6-acetylmorphine has been identified in oral fluid following controlled heroin administration, no prevalence data is available for oral fluid specimens collected in the workplace. We evaluated the prevalence of positive test results for 6-acetylmorphine in 77,218 oral fluid specimens collected over a 10-month period (January-October 2001) from private workplace testing programs. Specimens were analyzed by Intercept immunoassay (cutoff concentration=30 ng/ml) and confirmed by GC-MS-MS (cutoff concentrations=30 ng/ml for morphine and codeine, and 3 ng/ml for 6-acetylmorphine). Only morphine-positive oral fluid specimens were tested by GC-MS-MS for 6-acetylmorphine. A total of 48 confirmed positive morphine results were identified. An additional 107 specimens were confirmed for codeine only. Of the 48 morphine-positive specimens, 32 (66.7%) specimens were positive for 6-acetylmorphine. Mean concentrations (+/-S.E.M.) of morphine, 6-acetylmorphine and codeine in the 32 specimens were 755+/-201, 416+/-168 and 196+/-36 ng/ml, respectively. Concentrations of 6-acetylmorphine in oral fluid ranged from 3 to 4095 ng/ml. The mean ratio (+/-S.E.M.) of 6-acetylmorphine/morphine was 0.33+/-0.06. It is suggested that, based on controlled dose studies of heroin administration, ratios >1 of 6-acetylmorphine/morphine in oral fluid are consistent with heroin use within the last hour before specimen collection. The confirmation of 6-acetylmorphine in 66.7% of morphine-positive oral fluid specimens indicates that oral fluid testing for opioids may offer advantages over urine in workplace drug testing programs and in testing drugged drivers for recent heroin use.     

The detection of acetylcodeine and 6-acetylmorphine in opiate positive urines

Acetylcodeine (AC), an impurity of illicit heroin synthesis, was investigated as a urinary biomarker for detection of illicit heroin use. One hundred criminal justice urine specimens that had been confirmed positive by GC/MS for morphine at concentrations >5000 ng/ml were analyzed for AC, 6-acetylmorphine (6AM), codeine, norcodeine and morphine. The GC/MS analysis was performed by solid phase extraction and derivatization with propionic anhydride. Total codeine and morphine concentrations were determined by acid hydrolysis and liquid/liquid extraction. AC was detected in 37 samples at concentrations ranging from 2 to 290 ng/ml (median, 11 ng/ml). 6AM was also present in these samples at concentrations ranging from 49 to 12 600 ng/ml (median, 740 ng/ml). Of the 63 specimens negative for AC, 36 were positive for 6AM at concentrations ranging from 12 to 4600 ng/ml (median, 124 ng/ml). When detected, the AC concentrations were an average of 2.2% (0.25 to 10.2%) of the 6AM concentrations. There was a positive relationship between AC concentrations and 6AM concentrations (r=0.878). Due to its very low concentration in urine, AC was found to be a much less reliable biomarker for illicit heroin use than 6AM in workplace or criminal justice urine screening programs. However, AC detection could play an important role in determining if addicts in heroin maintenance programs are supplementing their supervised diacetylmorphine doses with illicit heroin.     

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.     

The detection of group-specific antigens in hair after its contact with blood and ejaculate]

The aim of this study was the detection of AB0 antigens extrinsic for a person in his (her) hair. The hair was kept for 1-7 days in whole or diluted blood (96 experiments) and semen (11 experiments). Only the group-specific antigens, intrinsic for the subjects whose hair was examined, were detectable in the hair after this exposure.     

Determination of morphine in postmortem rabbit bone marrow and comparison with blood morphine concentrations

The aim of this study is to predict how long after time of death a buried body could be analyzed for opiates in soft tissues and to show the accessibility and suitability of bone marrow as a useful toxicological specimen from buried bodies. Morphine solutions were injected in nine albino rabbits. Doses ranged from 0.3 to 1.1 mg/kg with 0.1 mg/kg increments. One hour after the injections, the rabbits were sacrificed. Blood, urine and bone marrow samples were collected for analysis. After the whole bodies were buried, femur bone marrow specimens were collected on the seventh and fourteenth days. CEDIA was used to monitor morphine contents of the collected samples. All experimental cases showed that the increase in the given morphine doses correlated with the increase in blood and bone marrow morphine concentrations. High morphine concentrations were detected in urine samples, but there was no correlation between the urine and blood or urine and bone marrow morphine concentrations. Statistically meaningful increases in bone marrow morphine concentrations were found parallel to increase of blood morphine concentrations. Seventh and fourteenth day postmortem morphine concentrations also followed this correlation. Morphine concentrations in bone marrow at 7 and 14 day postmortem decreased consistently when compared with bone marrow morphine concentrations collected immediately after death. We conclude that in sudden death when other specimens are unavailable due to degradation, bone marrow can be a most useful specimen. Further experimental research in this area is required to validate bone marrow as an alternative tissue.     

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.     

Removing and identifying drug contamination in the analysis of human hair

The procedure used in this laboratory for removing and identifying contamination of hair specimens with drugs is demonstrated by its application to hair contaminated by various experimental models. The models include soaking; coating with drug followed by sweat conditions for 6 h; and soaking in a very high concentration of cocaine followed by storage and multiple shampoo treatments. A multi-part wash procedure along with a wash criterion is applied to all samples containing drug above the cutoff. The failure of the wash criterion is a signal that the sample may be positive due to contamination rather than use, and in the absence of other over-riding evidence, the sample would be considered to be negative for drug use. This Wash Criterion has also been tested with hair from subjects demonstrated to be drug users by one or more drug-positive urines; in these studies, all hair samples from demonstrated users passed the Wash Criterion test.     

Codeine and morphine blood concentrations increase during blood loss

During extensive blood loss, a plasma volume refill will take place by transfer of extravascular fluid into the circulation. Drugs present in this fluid may follow and cause a rise or a drop in blood drug concentration, depending on their levels and accessibility in the restoration fluid. This study explored the possible changes of codeine, and its metabolite morphine, in whole blood during a standardized exsanguination in the rat. Three doses containing 5 mg codeine were given orally. In eight rats, blood loss was accomplished by slowly withdrawing 0.8 mL blood at 10 min intervals during 70 min. In control rats, blood was withdrawn only at 0 and 70 min. At 70 min, the final/initial codeine and morphine concentration ratios were 0.70 +/- 0.38 and 0.88 +/- 0.47, respectively, in controls, but increased to 1.28 +/- 0.44 (p = 0.014) and 1.41 +/- 0.34 (p = 0.021), respectively, in exsanguinated rats. It is concluded that blood loss can affect blood drug concentrations.     

The measurement and interpretation of morphine in blood

A method for the determination of morphine is post-mortem blood by HPLC with electrochemical detection is described. Blood morphine levels in post-mortem cases are reported and the importance of these in causing death is discussed. These post-mortem levels are compared further with morphine levels in the blood of patients receiving morphine as an analgesic.     

Cause of death in heroin users with low blood morphine concentration

The blood morphine concentrations in cases of heroin-associated fatalities can vary considerably. Currently, a free-morphine concentration of > or = 100 ng/ml in blood is generally considered as potentially fatal. Moreover, it is a common observation that fatal cases of heroin-intoxication with blood morphine concentrations lower than 100 ng/ml occur. This poses the question of how the fatal cases with low blood morphine concentrations can be explained. In the study described here, 62 cases of morphine only intoxications were examined. The fatal cases were divided into two groups according to the free morphine concentrations measured in the blood of the heart (group I: free morphine concentration < 100 ng/ml, n = 21 cases; group II: free morphine concentration > or = 100 ng/ml, n = 41 cases). The two groups were compared as to circumstances of death, as well as to autopsy findings and histopathologic alterations. Overall, infections of the respiratory tract occurred significantly more often in group I (lower morphine concentrations) than in group II. In a second step, the group I cases were analyzed individually to get detailed information on the cause of death. In 19 of the 21 cases the authors could find a plausible explanation for death in combination with low free morphine concentrations in the blood.     

Effect of murine retroviral infection on hair and serum levels of cocaine and morphine.

LP-BM5 retrovirally infected female C57BL/6J mice were administered cocaine, morphine or both by daily intraperitoneal injection for 9 weeks. Drug concentrations were measured by radioimmunoassay in serum and in hair extracts. Hair samples obtained from all drug-treated mice were positive for the drug injected, while none of the saline-treated mice had detectable drug levels in hair or serum. The average morphine concentration obtained from non-infected mice was 11 ng/mg hair whereas the amount found in the LP-BM5-infected mice was significantly higher (20 ng/mg hair). Mice injected with both morphine and cocaine were given 50% of the regular dose of each drug and drug levels in the hair of these animals were approximately half that of mice injected with the full dose of the single drug. Non-infected mice treated with both drugs had a mean value of 7 ng morphine/mg hair and 374 ng cocaine/mg hair while retrovirus-infected mice had significantly higher concentrations, 10 ng morphine/mg hair and 1160 ng cocaine/mg hair (P less than 0.001). Serum concentrations of cocaine and morphine were significantly higher (P less than 0.01) in the retrovirus-infected animals from 40 min to 1.5 h. The increased concentrations of cocaine and morphine in serum during retrovirus infection are accompanied by a significant increase in the amount of drug incorporated into the hair matrix. This change indicates that retroviral infection may influence the disposition of these drugs in the systemic circulation.     

人体血液中吗啡的定性定量分析

目的　建立人体血液中吗啡的定性定量分析方法。方法　经过丙酸酐衍生化法 ,利用气质联用和气相色谱氮磷检测器对吗啡丙酸酐衍生物进行定性定量分析。结果　确定了人体血液中吗啡的气质联用和气相色谱氮磷检测器定性定量分析的色谱条件、吗啡气相色谱氮磷检测器定量分析的线性范围及吗啡最小检出量0 1ng。结论　该方法定性定量结果准确、可靠 ,定量线性范围可满足实际办案需要 ,为吸食鸦片类毒品人群血液中吗啡的定性定量分析提供了分析方法和依据     

Differentiation between drug use and environmental contamination when testing for drugs in hair

Personality disorders are associated with criminality and antisocial and borderline personalities as strong predictors of violence. Nevertheless antisocial patients show more instrumental violence, while borderline patients more emotional violence. We surveilled medical records of a personality disorder facility, searching data of aggression and crimes against property among 11 patients with antisocial personality disorder and 19 borderline personality disorder. We found that there are differences regarding engagement in violence and lawbreaking according to the personality disorder: antisocial patients statistically engage more in crimes against property than the borderline patients, and more in this kind of crime than in aggression, whilst borderline patients show a tendency to engage more in episodes of aggression and physical violence than antisocial patients, and less in crimes against property. We conclude that the distinct personality leads to a distinct pattern of crimes and violence: antisocial patients are cold and get more involved in crimes requiring more detailed planning, whilst borderline patients are impulsive and engage in explosive episodes of physical violence. Further studies on the association among personality disorder, behavior pattern and violence type may be useful for both treatment and criminal profiling.     

Differentiation between drug use and environmental contamination when testing for drugs in hair

The differentiation between systemic exposure and external contamination for certain drug groups has been frequently referred to as one of the limitations of in drug testing in hair. When hair samples are used, three steps are usually employed in order to minimise the possibility of external contamination causing a misinterpretation. The first consists of decontaminating hair samples by washing the hair before analysis, the second is the detection of the relevant metabolites in the hair samples and the third is the use of cut-off levels. Difficulty in the interpretation arises when metabolites are not detected either due to external contamination of the hair or low doses of the drugs used. A wash protocol needs to be practical and ideally remove any drug deposited on the external portion of the hair.