Saliva testing after single and chronic administration of dihydrocodeine

In the present study, concentrations of dihydrocodeine and its metabolites in saliva and serum were compared after single low-dose and chronic high-dosage administration of the drug. In the first investigation, blood and saliva were collected periodically from six subjects after oral administration of 60 mg dihydrocodeine. In the second study, 20 subjects on oral dihydrocodeine maintenance provided single samples of blood and saliva simultaneously. Serum protein binding of salivary analytes and their recovery from the adsorbing material of the collection device as well as pH values of saliva samples were determined. The fluids were analyzed for dihydrocodeine and the major metabolites by high-performance liquid chromatography. In the single dose study dihydrocodeine was the only analyte found in saliva for up to 12–24 h post-dose. The half-life of dihydrocodeine in saliva was about twice that found in blood. The ratios of saliva/ serum concentrations ranged from 1.2 to 17.0. After chronic high-dosage use, dihydrocodeine was the main salivary analyte and N-nordihydrocodeine was present in a few samples. Saliva/serum concentration ratios of dihydrocodeine were strongly dependent on the pH value of saliva and, to a lesser extent, on serum-protein binding. The saliva/ serum ratios were more similar after chronic administration. The data suggest a passive diffusion process as the underlying mechanism for the transport of dihydrocodeine into saliva. After both single and chronic use, the presence of the drug in saliva can be used as evidence of recent substance administration. Received: 24 February 1999 / Accepted: 9 September 1999     

Effect of the shampoo Ultra Clean on drug concentrations in human hair

The influence of the special shampoo Ultra Clean (Zydot Unlimited, Tulsa, Oklahoma) on the results of hair analyses was investigated. Hair samples from persons (n = 14) with a known history of drug abuse were collected at autopsy. The hair samples were divided into separate strands which were analyzed both after washing with Ultra Clean and without treatment. Hair analyses were performed by methanol extraction under sonication, purification by solid phase extraction and GC/MS in SIM mode according to routine procedures for tetrahydrocannabinol (THC), cocaine, amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDE), heroin, 6-monoacetylmorphine (6-MAM), morphine, codeine, dihydrocodeine and methadone. All drugs originally present in the hair fibers were still detected after a single application of Ultra Clean. However, a slight decrease in drug concentrations could mostly be observed e.g. cocaine (n = 10) -5%, 6-MAM (n = 12) -9%, morphine (n = 12) -26%, THC (n = 4) -36%. The findings clearly demonstrated that drug substances had not been sufficiently removed from human hair by a single Ultra Clean treatment to drop their concentrations below the limit of detection of the analytical method applied.     

Radioimmunoassay of hair for determining opiate-abuse histories

Heroin and morphine metabolites can be detected in hair with the use of commercially available radioimmunoassay reagents and with minor sample preparation. Hair samples obtained from morphine-treated mice and heroin users contained nanogram levels of the drug per milligram of hair (single human hair). The results of the hair analyses for all subjects admitting the use of heroin were positive, whereas the results of only 30% of thin-layer chromatographic urinanalyses of these same subjects were positive. In addition, differences in drug concentration for sections of hair near the scalp and near the distal end correlated with the length of time the drug had been used. These results exemplify the potential advantages of the use of hair analysis over urine and serum analyses in terms of accessibility, sample stability, and long-term retention of information.     

Vergiftungs- und Todesfälle durch Substitutionsmittel im Umfeld von substituierten Drogenabhängigen

A total of seven cases of accidental ingestion of methadone or dihydrocodeine by four children and three adults are reported of which four were fatal. In each case, someone in the environment was taking methadone or dihydrocodeine as a substitute drug for heroin addiction who obviously did not realize the dangers of methadone for non-addicts. Possible preventive measures are the usage of child-proof containers with adequate labels for take-home medications. Furthermore substituted addicts have to be thoroughly indoctrinated concerning the toxicity and hazards of methadone.     

Judicial applications of hair testing for addicts in Crete: sectional hair analysis of heavy heroin abusers

Laboratory examination of hair from drug users has been employed for the last 5 years in Crete, in addition to the psychiatric or other forensic clinical examinations necessary for the confirmation of a person's use of drugs. The present study reports results of total and sectional head, axillary and pubic hair analysis of imprisoned abusers under interrogation and awaiting presentation at the Crime Court. Morphine levels in total head hair samples 1, 2 and 4 months after preliminary imprisonment ranged from 1.2 to 38.2, 0.5 to 22.5 and 0.1 to 4.9 ng/mg of hair respectively, while the maximum morphine levels amongst all studied groups of those arrested were found in the sections of axillary and pubic hair. Notably high morphine levels in head, axillary and pubic hair sections (13.7, 8.4 and 18.1) ng/mg respectively) were measured, even in abusers who had been imprisoned between 2 and 4 months. Hair tests may considerably assist to evaluate the systematic present and past abuse of heroin and other drugs. Consequently, it may be used as valuable expertise evidence during questioning and in court.     

A specific immunoassay for the determination of morphine and its glucuronides in human blood

The development of specific antisera for immunochemical determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide is described. Morphine was N-demethylated to normorphine and N-alkylated to give N-aminopropyl-normorphine as hapten for antisera against morphine. As haptens for antisera against morphine-3-glucuronide and morphine-6-glucuronide, N-aminopropyl-nor-morphine was glucuronidated in position 3 or 6 respectively. Each of these three haptens were coupled to BSA employing the glutaraldehyde method to obtain three different immunogens. Immunisation of rabbits with these conjugates gave anti-morphine, anti-morphine-3-glucuronide and anti-morphine-6-glucuronide antisera, which were tested in a competitive, heterogeneous radioimmunoassay. Tracers for this radioimmunoassay procedure were synthesised by substitution of morphine and morphine-6-glucuronide in position 2 with ¹²⁵I and indirect iodination of the morphine-3-glucuronide hapten according to the method of Bolton and Hunter. The resulting antisera show very specific reactions with morphine, morphine-3-glucuronide and morphine-6-glucuronide. Cross reactivities of each antiserum with structurally related opiates and opioides are very low. The cross reactivities of the anti-morphine antiserum against morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine were less than 0.3%, the anti-morphine-3-glucuronide antiserum against morphine, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine less than 0.1% and the anti-morphine-6-glucuronide antiserum against morphine, morphine-3-glucuronide, codeine or dihydrocodeine less than 0.1%, against codeine-6-glucuronide less than 2.3%. The determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood samples (limit of detection = 3, 1, 0.5 ng/g) of nine cases of fatal heroin overdose with this radioimmunoassay method and the comparison with a GC/MS method is described. Received: 22 December 1997 / Received in revised form: 23 March 1998     

Ultrastructural alterations and environmental exposure influence the opiate concentrations in hair of drug addicts

Hair samples were taken at autopsy from the head of 1 male and 1 female subject both known as drug abusers. Some of the strands were bleached by in-vitro cosmetic treatment. The bleached hair as well as the original hair samples were partly exposed to water or soil prior to further investigations and drug monitoring. The exposure times were 4 weeks or 6 months for water and 6 months for soil. The hair fibers were examined by transmission electron microscope (TEM) and by scanning electron microscope (SEM) investigations. The electron microscope studies confirmed that all experimental conditions had produced morphological alterations in the hair fibers. After exposure to water or to soil for 6 months as well as after storage of the clipped bleached hair in tap water at room temperature for 4 weeks, drug monitoring of formerly positive hair samples gave negative results. After storage of natural hair in soil or in water for 4 weeks the opiate levels had dramatically decreased. The samples were screened by fluorescence polarization immunoassay after enzymatic digestion. The results were confirmed by GC/MS.     

Investigation of illicit heroin seized in Malaysia: physical characteristics and chemical profiling

The high number of illicit heroin cases in Malaysia has led to the local forensic laboratory profiling of the physical and chemical characteristics of 311 street samples submitted as heroin cases in 2010. Visual examination and physical characteristics of the heroin substance and plastic packages are described. The preferred ranges of the weight of substances per package and the dimensions of the plastic package/receptacle are defined. Analysis of eight major components (three adulterants and five opium alkaloids) is also detailed. It was found that most samples contained less than 10% heroin base and more than 80% caffeine. Physical and chemical data together provide a quick overview of the samples seized within the country. Principal component analysis of the five major opium alkaloids present in the seized samples suggested that there were at least two sources responsible for the samples analyzed.     

Differentiation of heroin and cocaine using dual-energy CT—an experimental study

Objective

To evaluate if heroin and cocaine can be distinguished using dual-energy CT.

Materials and methods

Twenty samples of heroin and cocaine at different concentrations and standardized compression (SC) were scanned in dual-energy mode on a newest generation Dual Energy 64-row MDCT scanner. CT number, spectral graphs, and dual-energy index (DEI) were evaluated. Results were prospectively tested on six original samples from a body packer. Wilcoxon’s test was used for statistical evaluation.

Results

Values are given as median and range. Under SC, the CT number of cocaine samples (?29.87 Hounsfield unit (HU) [?125.85; 16.16 HU]) was higher than the CT number of heroin samples (?184.37 HU [?199.81; ?159.25 HU]; p?<?0.01). Slope of spectral curves for cocaine was ?2.36 HU/keV [?7.15; ?0.67 HU/keV], and for heroin, 1.75 HU/keV [1.28; 2.5 HU/keV] (p?<?0.01). DEI was 0.0352 [0.0081; 0.0528] for cocaine and significantly higher than for heroin samples (?0.0127 [?0.0097; ?0.0159]; p?<?0.001). While CT number was inconclusive, all six original packs were correctly classified after evaluation of the spectral curve and DEI. In contrast to the CT number, slope of the spectral curve and DEI were independent of concentration and compression.

Conclusion

The slope of the spectral curve and the DEI from dual-energy CT data can be used to distinguish heroin and cocaine in vitro; these results are independent of compression and concentration in the measured range.     

In situ labeling of DNA reveals interindividual variation in nuclear DNA breakdown in hair and may be useful to predict success of forensic genotyping of hair

Hair fibers are formed by keratinocytes of the hair follicle in a process that involves the breakdown of the nucleus including DNA. Accordingly, DNA can be isolated with high yield from the hair bulb which contains living keratinocytes, whereas it is difficult to prepare from the distal portions of hair fibers and from shed hair. Nevertheless, forensic investigations are successful in a fraction of shed hair samples found at crime scenes. Here, we report that interindividual differences in the completeness of DNA removal from hair corneocytes are major determinants of DNA content and success rates of forensic investigations of hair. Distal hair samples were permeabilized with ammonia and incubated with the DNA-specific dye Hoechst 33258 to label DNA in situ. Residual nuclear DNA was visualized under the fluorescence microscope. Hair from some donors did not contain any stainable nuclei, whereas hair of other donors contained a variable number of DNA-positive nuclear remnants. The number of DNA-containing nuclear remnants per millimeter of hair correlated with the amount of DNA that could be extracted and amplified by quantitative PCR. When individual hairs were investigated, only hairs in which DNA could be labeled in situ gave positive results in short tandem repeat typing. This study reveals that the completeness of DNA degradation during cornification of the hair is a polymorphic trait. Furthermore, our results suggest that in situ labeling of DNA in hair may be useful for predicting the probability of success of forensic analysis of nuclear DNA in shed hair.     

Toxicological detection of pholcodine and its metabolites in urine and hair using radio immunoassay,fluorescence polarisation immunoassay,enzyme immunoassay and gas chromatography-mass spectrometry

Summary Pholcodine (3-O-(2-morpholinoethyl)-morphine) is used in many countries as an antitussive without analgesic or addictive properties. It is of forensic relevance that pholcodine interferes with opiate immunoassays. In this paper a gas chromatographic-mass spectrometric (GC-MS) procedure for the precise and sensitive detection of pholcodine and its metabolites in urine and hair, after acid hydrolysis, extraction and acetylation, is presented. Furthermore, detection of pholcodine using radio immunoassay (RIA), fluorescence polarisation immunoassay (FPIA) and enzyme immunoassay (EIA) for opiates is described. Using GC-MS, unmodified pholcodine could be detected in urine samples 4–7 weeks after ingestion of a single therapeutic dose of 50 mg of pholcodine, the desmorpholinohydroxy metabolite for 1–2 weeks and the other metabolites (nor-, nordesmorpholinohydroxy-, hydroxy-, oxo- and noroxo-pholcodine) only during the first few hours. Morphine could also be detected in urine samples for the first few days. It was however mainly formed artificially during acid hydrolysis and only in trace amounts by metabolism. All the immunoassays tested gave positive results in urine samples during the first week taking the cut-off values recommended by the manufacturer into consideration. If values between the cut-off and the detection limit were taken into consideration, RIA and FPIA gave positive results for 2–4 weeks and EIA up to 2 weeks. Pholcodine could also be detected by RIA and GC-MS in samples of head hair clipped 10 weeks after ingestion of 50 mg and in daily shaved samples of beard hair over a period of three weeks after ingestion of three doses of 60 mg. It can be concluded that the widely used immunoassays for opiates show positive results in urine and hair samples for a long time after ingestion of the non-opioid pholcodine and that these results can be confirmed by the GC-MS procedure described in this paper.     

Changes in place of origin of heroin seized in Denmark from 1981 to 1986

Summary Hundred thirty-eight samples of heroin weighing more than 0.1g seized between 1981 and 1986 were characterized according to their contents of opium alkaloids, adulterants, and diluents together with their form and color. The chemical fingerprint was used to establish a change in the heroin during the period. As compared to the first few years covered by the survey, a predominant number of the samples at the end of the period were in the base form and contained the opium alkaloids papaverine and noscapine. In particular, the concentration of noscapine as related to the heroin content of each sample had increased considerably, indicating Pakistan or Iran as being the places of origin of most of the heroin seized in Denmark at the end of the period.     

Concentrations of Δ9-tetrahydrocannabinol,cocaine and 6-monoacetylmorphine in hair of drug abusers

Hair samples taken from 850 individuals with presumed drug abuse were tested simultaneously forΔ⁹tetrahydrocannabinol (THC), cocaine, heroin, the primary heroin metabolite 6-monoacetylmorphine (6-MAM) and morphine. The drugs were extracted with methanol under sonication. Compared to other extraction procedures this solvent extraction technique provides high extraction yields and less experimental effort. The analyses were carried out using gas chromatography - mass spectrometry (GCMS) in selected ion monitoring (SIM) mode. This procedure allows the simultaneous detection of amphetamine, methylenedioxyamphetamine (MDA), methylenedioxymetbamphetamine (MDMA) and methylenedioxyethylamphetamine (MDE). THC was found in 104 (12.2%), cocaine in 230 (27%) and 6-MAM in 141 (16.6%) samples. In addition to 6-MAM, morphine was detected in 87 (10.2%) and heroin in 38 samples (4.5%). The concentrations found were in a range 0.009-16.7 ng/mg for THC, 0.037-129.68 ng/mg for cocaine, 0.028-79.82 ng/mg for 6-MAM, 0.045-53.14 ng/mg for heroin and 0.011-7.800 ng/mg for morphine. The statistical distribution of the drug concentrations compared with the self-reported consumption behaviour of the users may possibly lead to a better understanding of the relationship between drug dosage and corresponding concentrations in hair.     

Buprenorphine and norbuprenorphine findings in hair during constant maintenance dosage

It is still a matter of debate whether a positive correlation between the dose and the amount of drug in the hair exists. Drugs such as buprenorphine (BUP) used under controlled conditions present an opportunity to prove a possible relationship. Due to discrepant findings of BUP/norbuprenorphine (NBUP) ratios in hair, in vitro degradation of both analytes in diluted acid was also investigated. The levels of BUP and NBUP in proximal hair sections from 18 subjects participating in a maintenance program were determined by liquid chromatography/tandem mass spectrometry following incubation with methanol and subsequent liquid/liquid extraction. BUP and NBUP were incubated in diluted hydrochloric acid at 60°C for up to 24 h. The alleged rearrangement products were simultaneously monitored. All hair samples tested positive for BUP (lower limit of detection–0.238 ng/mg hair) and NBUP (0.043–0.961 ng/mg hair). The concentration of NBUP in hair was consistently higher than that of BUP except for a single specimen. Degradation of BUP and NBUP was dependent on time; hydrolysis of NBUP occurred faster than that of BUP. The concentration of BUP and NBUP will be underestimated if analytes are recovered by acidic procedures. NBUP should be monitored in hair samples besides BUP for the sum of both BUP and NBUP may provide an estimate of BUP exposure following long-term administration of the drug.     

The possibilities of hair analysis in the determination of involuntary doping in sports

Although not yet fully recognised by international sporting committees, hair analysis in doping control may be a useful adjunct to drug testing of urine. It may permit access to retrospective information and the identification of banned substances, especially when exogenous abuse has to be distinguished from other forms of involuntary exposure to identical substances. Negative hair results coupled with positive urine samples may be used to draw conclusions of involuntary doping in sports whenever athletes claim not to have ingested any drug, identical substances are present in their environment or are normal constituents of food and beverages served to them immediately before the competition. Two cases are well described in the literature in which hair analyses were fundamental in documenting positive doping after urinalysis. In Brazil, 2 cases of athletes testing positive for banned substances caught our attention because of the possibility of involuntary doping; hair analysis, if performed, may have helped to clarify the results of the urinalysis. Despite the fact that it cannot be used for routine control and overrule positive urinalysis, hair analysis can detect long term exposure as well as those substances which are not excreted in urine. In the current International Olympic Committee (IOC) code, hair analysis is not yet considered useful even in special cases of doping control.     

Chemometric procedures for analyzing trace organic impurities present in street doses of heroin via a constant weight approach

This paper seeks to evaluate whether the chemical profiling data obtained with a constant weight approach can be used in the classification of highly cut heroin samples. A constant weight of a seized substance instead of the conventional weight equivalent to 15 mg heroin base was used to profile the manufacturing impurities. The study attempts to optimize four clustering tools using 12 impurity peaks extracted from the heroin samples (<2.3% purity) analyzed at 650 mg sample weight to find the most ideal statistical techniques for sample classification. The effectiveness of four clustering tools, namely the principal component analysis (PCA), hierarchical cluster analysis (HCA), K-means clustering (KMC) and discriminant analysis (DA) was assessed using 25 heroin samples derived from five known batches. HCA and DA proved promising in clustering the related samples. Finally, only the HCA was then employed to evaluate the general relationships between 46 unknown heroin samples.     

Unusual patterns of I-131 contamination

Whole body imaging with radioiodine can detect functioning metastases, which can often be effectively treated with appropriate amounts of radioiodine. Non-physiologic I-131 uptake detected on images is usually interpreted as suggesting functioning thyroid metastases. However, extra-thyroidal I-131 accumulation does not always imply thyroid cancer metastases and has been reported on many occasions, including various non-thyroidal neoplasms, and contamination by body secretions. In order to avoid unnecessary therapeutic interventions it is extremely important to properly distinguish false-positive sites of I-131 localization. Three patients with unusual radioiodine contamination patterns, either presented for the first time or rarely presented in the existing literature, were reported. Reported cases consist of contamination in hair (due to styling hair with sputum), contamination in neck (due to drooling during sleep) and, contaminated chewing gum. False positive contamination sources were clarified by careful examination of patients and further images when necessary.     

Statistical evaluation of morphological data of Japanese head hair and the screening of evidential hair samples by cluster analysis

Intra-individual and inter-individual variations in Japanese head hair morphology were reevaluated and the screening of evidential hair samples using a statistical method was investigated. A quantification of the morphological data was used as a system in which morphological features obtained by macroscopic and microscopic observations can be objectively processed in the form of numerical information. For confirming the availability of morphological features for hair comparison, the inter-individual comparison of ten male Japanese was investigated by discrimination analysis using 18 variables including five original numerical morphological features (variables) and 13 variables obtained from six morphological features by quantification. The majority of the comparisons showed a high level of discrimination between two individuals. For morphologically narrowing down the number of evidential hair samples collected from a crime scene, a screening method was investigated by cluster analysis using 18 variables. In an experimental model of evidential hair samples derived from 20 individuals, hair samples from 13 individuals were successfully discriminated using the cluster analysis described in the present investigation.     

Vitreous humor as an alternative sample to blood for the supercritical fluid extraction of morphine and 6-monoacetylmorphine

The use of vitreous humor as an alternative sample to blood was investigated for the detection of heroin abuse by quantifying levels of morphine and 6-monoacetylmorphine (6-MAM) in post-mortem samples. The levels achieved in each of the two toxicological specimens were compared on a case-to-case basis to determine if a correlation existed. A total of 20 positive morphine cases were examined. In general, the levels of morphine in blood were higher than in the corresponding vitreous humor samples, with some correlation existing. 6-MAM was found in 15 blood samples and 17 vitreous humor samples. Although no correlation was found between the levels of 6-MAM in blood and vitreous humor, the latter may still be used for verification of heroin abuse.     

Mumie ohne Geheimnisse – Identifizierung, Eingrenzung der Liegezeit und Nachweis von Morphin

In 2010 the mummified corpse of a 25-year-old male drug user was found in his apartment after a 2-year postmortem interval and had died just after being released from an 11-month prison sentence. The initial postmortem examination revealed skeletization and advanced stage mummification. The autopsy revealed surprisingly well preserved thigh muscles in addition to disintegration of the parenchymatous organs and nearly complete desiccation. The comprehensive toxicological investigations provided evidence of morphine (0.41 ng/mg in hair, 0.05 µg/g in muscle, 1.95 µg/g in liver, 0.54 µg/g in kidneys, 70 ng/g in bone and 7.57 ng/g in bone marrow), 6-acetylmorphine (0.15 ng/mg in hair) and codeine (0.05 ng/mg in hair and 0.01 µg/g in muscle) due to heroin consumption. The concentrations measured in the postmortem specimens indicated lethal heroin intoxication and the distribution ratio of the concentrations contributed to clarifying the cause of death. The state of preservation of the body, the scope of investigations and the results make the case just as newsworthy as the rarity of comparable cases.