The poppy seed defense: a novel solution.

A major toxicological challenge is distinguishing whether morphine in urine, in the absence of 6-monoacetylmorphine (6-MAM), originates from 'street' heroin use or poppy seed ingestion. Manufacturing byproducts from the synthesis of illicit heroin include those that originate from the reaction of acetic anhydride with the alkaloid impurity, thebaine, which undergoes skeletal rearrangement, resulting in compounds with a 2-(N-methylacetamido)ethyl side-chain. The hypothesis that the tertiary amide in this side-chain is resistant to endogenous hydrolysis was supported from in-vitro experiments; a glucuronide metabolite (designated 'ATM4G') was identified that may be used as a marker of 'street' heroin administration. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for this metabolite was then performed on selected urine specimens from 22 known heroin users, these being negative on routine testing for 6-MAM by gas chromatography-mass spectrometry (GC-MS), using the generally applied reporting threshold of 10 ng/mL, but positive for the presence of morphine. Peaks corresponding to the retention time for the metabolite marker were clearly observed for 16 of the 22 samples, with variations of the ratios of its three dependent ions being within ± 30% of that produced in vitro. Conversely, 6-MAM was detected in only 3 samples, but at concentrations <1 ng/mL. Such a high frequency for the presence of the metabolite marker in urine, in the absence of 6-MAM, is noteworthy and suggests that detection of this metabolite may offer an important advance in forensic toxicology, allowing the development of a new and more definitive test for heroin abuse and thus a potential solution to the so-called 'poppy seed defense'.

Aminoindanes--the next wave of 'legal highs'?

Due to its closed ring system, 2-aminoindane is a conformationally rigid analogue of amphetamine. Internet websites offering synthetic compounds as 'research chemicals' have recently been advertising 5,6-methylenedioxy-2-aminoindane (MDAI), 5, 6-methylenedioxy-N-methyl-2-aminoindane (MDMAI), 5-iodo-2-aminoindane (5-IAI), and 5-methoxy-6-methyl-2-aminoindane (MMAI). The chemistry, pharmacology, and toxicological aspects of this new class of psychoactive substances are reviewed, as these could become the next wave of 'legal highs'.

Marine biofouling on fish farms and its remediation.

The fish farming industry suffers significantly from the effects of biofouling. The fouling of cages and netting, which is costly to remove, is detrimental to fish health and yield and can cause equipment failure. With rapid expansion of the aquaculture industry, coupled with the tightening of legislation on the use of antifouling biocides, the problems of fish farm biofouling are increasing. The nature of the biological communities that develop on fish farm equipment and the antifouling practices that can be employed to reduce it are described here. Particular emphasis is placed on antifouling legislature and the future needs of the industry. The biological communities that develop on fish cages and netting are distinctive, in comparison to those that foul ships. Temperate species of particular importance, because of their cosmopolitan distribution and opportunistic nature, include the blue mussel Mytilus edulis and the ascidian Ciona intestinalis. Antifouling practices include predominantly the use of copper-based antifoulant coatings, in combination with practical fish husbandry and site management practices. The antifouling solutions presently available are not ideal, and it is widely accepted that there is an urgent need for research into combatant technologies. Such alternatives include the adoption of "foul-release" technologies and "biological control" through the use of polyculture systems. However, none of these have, as yet, been proven satisfactory. In view of current legislative trends and the possible future "phasing out" of available antifouling materials, there is a need to find alternative strategies.

Drug absorption may be delayed after stroke: results of the paracetamol absorption test.

BACKGROUND: Autonomic changes are frequent after stroke but it is not known whether gastric emptying is altered. We have investigated this using the paracetamol absorption test. METHODS: 12 acute stroke patients and 13 healthy controls of similar age received 1 g oral paracetamol tablets. We studied all patients within 24 h of the stroke and 5 days later. Standard pharmacokinetic measurements were derived from the plasma paracetamol-time curve. RESULTS: In acute stroke patients, mean plasma T(max) was delayed compared with that in controls (96.3 vs 46.2 min, P=0.015). The C(max) of paracetamol was also lower (16.1 vs 23.9 mg l(-1), P=0.028). The area under the curve of paracetamol did not differ significantly in acute stroke patients and controls. CONCLUSIONS: Gastric emptying appears to be delayed in acute stroke patients, and this may result in delayed pharmacological action of orally administered drugs.

Simple high-performance liquid chromatographic method to monitor vigabatrin, and preliminary review of concentrations determined in epileptic patients.

A simple and rapid high-performance liquid chromatographic method has been developed for the determination of vigabatrin concentrations in plasma or serum. The assay uses only 100 microL of specimen and has been found to be linear over a concentration range of 1 to 50 mg/L. The limit of detection has been determined as 1 mg/L, and the between-batch coefficient of variation for the two internal quality controls routinely analysed (n = 33) has been found to be less than 5%. There was no evidence of interferences in the assay from other commonly prescribed anti-epileptic drugs. This method has been applied to routine clinical specimens to determine the concentration of vigabatrin in 47 patient specimens over a 12-month period. It was found that only 63% of the male group and 53% of the female group were within the proposed target concentration of 5 to 35 mg/L. In addition, it was found that 26% of the male group and 36% of the female group were found to have concentrations below 5 mg/L, which may indicate lack of compliance and/or lack of therapeutic efficacy of treatment.

Application and validation of a urinary methadone metabolite (EDDP) immunoassay to monitor methadone compliance.

A total of 1381 urine specimens were screened using a Microgenics CEDIA urinary primary methadone metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EDDP) immunoassay (cut-off calibrator concentration of 100 microg/L) in combination with a Dade Behring EMIT urinary methadone immunoassay (cut-off calibrator concentration of 300 microg/L). Of these, 642 (46%) were found to be positive using the EDDP assay but only 541 (39%) were found to be positive by the methadone assay. Out of the 108 specimens which were EDDP-positive but negative by the methadone assay, 47 (7%) could not be confirmed as positive using the routine in-house method of gas chromatography incorporating nitrogen-specific detection. Of these 47 results, 38 were re-screened using a more sensitive gas chromatography-mass spectrometry (GC-MS) technique, which demonstrated the presence of EDDP in every case. There was insufficient specimen to analyse the remaining nine samples. There were seven specimens which gave negative results by the EDDP and GC-MS assays but which had methadone concentrations ranging from 4000 to 37,500 microg/L. These were therefore presumed to be 'spiked' with methadone, i.e. to have had methadone added to the specimen to yield positive screening results and simulate compliance. It is concluded that the Microgenics CEDIA EDDP assay is a sensitive and reliable technique to determine the compliance of subjects prescribed methadone for opiate detoxification and maintenance.

Estimating antemortem drug concentrations from postmortem blood samples: the influence of postmortem redistribution.

AIMS: To compare blood drug concentrations during life with postmortem drug concentrations measured from a peripheral site and a central site. METHODS: Coroner's cases from October 1990 to July 1997 were reviewed. Six cases had data on both antemortem and postmortem blood drug concentrations. The postmortem to antemortem ratio was compared with the postmortem central to peripheral ratio, using cardiac blood as a central site and femoral blood as a peripheral site. RESULTS: Drugs that have a high postmortem central to peripheral ratio; that is, drugs that exhibit considerable postmortem redistribution, also have high postmortem to antemortem ratios. CONCLUSIONS: A large degree of error can arise from attempting to estimate antemortem drug concentrations and the ingested dose from postmortem measurements. The chosen site and technique for postmortem blood sampling can greatly influence the concentration of drug measured.

Using amphetamine isomer ratios to determine the compliance of amphetamine abusers prescribed dexedrine.

The amphetamine isomer ratios (l-amphetamine/d-amphetamine) in 373 urine specimens submitted for analysis over a two-year period have been determined using a chiral derivatizing agent in conjunction with a gas chromatograph fitted with a nitrogen-specific detector. All of the specimens were collected from known or suspected amphetamine abusers, some of which were prescribed dexedrine for maintenance and detoxification. The mean (+/- 1 standard deviation [SD]) l/d-amphetamine isomer ratio for 147 specimens from compliant subjects prescribed dexedrine was 15.0% (+/- 4.9%). The mean (+/- 1 SD) l/d-amphetamine isomer ratio from 165 subjects abusing illicit amphetamine was 98.5 (+/- 27.5%). The calculation of l/d-amphetamine isomer ratios in urine has been found to be a rapid method for determining the compliance of subjects prescribed dexedrine and is therefore a useful technique for the continued management of amphetamine abusers. In addition, 17 specimens of illicit amphetamine powder (assumed to be a racemic mixture) were submitted to the laboratory for analysis. Using a combination of gas chromatography with and without chiral derivatization, the powders were found to have a mean l/d-amphetamine isomer ratio of 89.2% (range 72.2% to 98.3%) and mean purity (w/w) of 21.5% (range 3.4% to 71.0%) relative to pure dl-amphetamine substance.

A pilot study to determine the usefulness of the urinary excretion of methadone and its primary metabolite (EDDP) as potential markers of compliance in methadone detoxification programs.

Fourteen subjects (selected on the basis of compliance with the methadone-maintenance program prescribed by the consultant psychiatrist in charge of their treatment) undergoing opiate detoxification by methadone-replacement therapy were studied to determine if a relationship exists between the dose of methadone prescribed and the urinary excretion of methadone and/or its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). After the derivation of this relationship, it was hoped that the urinary concentrations of methadone and/or EDDP could be used as a noninvasive technique to monitor the methadone compliance of 56 drug abusers. Despite statistically significant correlations (p<0.001) between methadone dose and urine concentrations of methadone and EDDP, the large variation in concentrations measured in the urine of drug abusers negated the possibility of any clear-cut relationship being confirmed. However, it may be possible to use excretion data to monitor individual compliance but only through long-term monitoring of individual subjects to establish their own intraindividual variation in excretion patterns.

Screening for drugs of abuse (II): Cannabinoids, lysergic acid diethylamide, buprenorphine, methadone, barbiturates, benzodiazepines and other drugs.

Requirements for the provision of an efficient and reliable service for drugs of abuse screening in urine have been summarized in Part I of this review. The requirements included rapid turn-around times, good communications between requesting clinicians and the laboratory, and participation in quality assessment schemes. In addition, the need for checking/confirmation of positive results obtained for preliminary screening methods was stressed. This aspect of the service has assumed even greater importance with widespread use of dip-stick technology and the increasing number of reasons for which drug screening is performed. Many of these additional uses of drug screening have possible serious legal implications, for example, screening school pupils, professional footballers, parents involved in child custody cases, persons applying for renewal of a driving licence after disqualification for a drug-related offence, doctors seeking re-registration after removal for drug abuse, and checking for compliance with terms of probation orders; as well as pre-employment screening and work-place testing. In many cases these requests will be received from a general practitioner or drug clinic with no indication of the reason for which testing has been requested. This also raises the serious problems of a chain of custody, provision of two samples, stability of samples, and secure and lengthy storage of samples in the laboratory-samples may be requested by legal authorities several months after the initial testing. The need for confirmation of positive results is now widely accepted but it may be equally important to confirm unexpected negative results. Failure to detect the presence of maintenance drugs may lead to the patient being discharged from a drug treatment clinic and, if attendance at the clinic is one of the terms of continued employment, to dismissal. It seems likely that increasing abuse of drugs and the efforts of regulatory authorities to control this, will lead to the manufacture of more designer drugs. Production of substituted phenethylamines was facilitated by the drug makers' cook book, 'PIHKAL' (Phenethylamines I Have Known And Loved) by Dr Alexander Shulgin and Ann Shulgin, and production of substituted tryptamines is promised in their next book, TIHKAL. Looking to the future, laboratories will need to ensure that they can detect and quantitate an ever-increasing number of drugs and related substances. The question of confidence in results of drugs of abuse testing raised in 1993 by Watson has assumed even greater importance as a result of attention focused on the OJ Simpson trial in Los Angeles. Toxicological investigations are likely to be challenged more frequently in the future. Even if analyses have been performed by GC-MS, there is a need to establish the level of match between the spectrum of the unknown substance and a library spectrum which is considered acceptable for legal purposes. It will also be essential to ensure that computer libraries contain spectra for all substances likely to be encountered in drugs of abuse screening.

The measurement of morphine in the hair of heroin abusers.

Several techniques have been described for the determination of morphine in hair as a method of monitoring past heroin use. However, although some of the techniques [notably radioimmunoassay (RIA)] may appear relatively simple to perform, any results obtained must be interpreted with caution. In this study, hair specimens from four known heroin abusers were sectionally analysed by a specific RIA for morphine. Prior to analysis, all hair sections were cleaned to remove any possible surface contamination. Five different hair digestion procedures were evaluated to determine the most effective method that could be used to liberate morphine from hair. The greatest analytical recovery was obtained by incubation with 1.0 M sodium hydroxide for 18 h at 55 degrees C, neutralization with 1.0 M hydrochloric acid, and pH adjustment with 0.1 M phosphate buffer (pH 7.0). The morphine concentrations detected in the hair specimens ranged from 0.5 to 13.2 ng/mg of hair. It was also found that the use of shorter length segments (e.g. 1 cm length) gave a clearer, more detailed picture of the historic pattern of heroin use in the four subjects studied.

The effect of glutaraldehyde adulteration of urine specimens on syva EMIT II drugs-of-abuse assays.

The effect of glutaraldehyde (the active component of "UrinAid") on Syva EMIT II drugs-of-abuse screening assays was studied. It was found that, dependent on the assay involved, concentrations of between 0.75 and 2.00% (v/v) of glutaraldehyde in urine could give rise to false-negative screening results. A simple method for identifying urine specimens that have been adulterated with glutaraldehyde, based on final absorbance rate readings (dA/min), is proposed.

Elemental mercury-induced skin granuloma: a case report and review of the literature.

BACKGROUND: Injection of elemental mercury is rare and only some 72 cases have been reported in the literature over the period 1923-1995. Direct subcutaneous injection or extravasation of mercury injected into blood vessels can produce local granulomata and abscesses. Unless intravascular mercury injection has occurred, clinical signs of mercury toxicity are usually absent though four cases of systemic toxicity have been reported following isolated subcutaneous injection without evidence of elemental mercury dissemination. CASE REPORT: We report a further case of subcutaneous injection by gunshot of elemental mercury, with subsequent granuloma formation, in a 19-year old man who was admitted with an eight month history of a tender enlarging mass in his left antecubital fossa, while on active military service. Surgical removal of mercury from a presumed mercury-tipped bullet was undertaken but was incomplete and the patient declined further operative intervention as he remained asymptomatic. Chelation therapy was not instituted. Serum and urine mercury concentrations were measured for six years after presentation. CONCLUSIONS: We recommend that cases of subcutaneous metallic mercury injection should be managed by complete surgical excision of the granuloma under X ray control and serial monitoring of blood and urine mercury concentrations.

Routine therapeutic monitoring of lamotrigine in epileptic patients using a simple and rapid high performance liquid chromatographic technique.

We have developed a simple and rapid high performance liquid chromatographic technique to determine lamotrigine concentrations in epileptic patients and validated it using external quality control material. The method has been used to monitor the lamotrigine concentration in 70 specimens from 61 patients. Only 50% of the specimens had concentrations within the proposed target range of 1 mg/L to 4 mg/L, and there was no relationship between lamotrigine concentration and age, sex, other anti-epileptic treatments, or dose, although this could in part be explained by concomitant anti-epileptic therapy. We suggest that lamotrigine should be monitored therapeutically in order to assess its efficacy and audit its use as an anti-epileptic treatment, especially with the introduction of this relatively new drug as monotherapy.

An investigation into the extent of possible dilution of specimens received for urinary drugs of abuse screening.

Recent American and Swedish studies have shown an increase in "false" negative results when analysing dilute urine specimens for drugs of abuse. In the light of these studies, it was decided to perform a pilot study to determine the extent of possible specimen adulteration and dilution in a random batch of 50 urine specimens presented to this laboratory, using creatinine, osmolality, pH and relative density. It was found that 20% of the specimens were outside the pH range associated with the optimum working of Syva EMIT Drugs of Abuse in Urine (DAU) immunoassay screening techniques, and that if the National Institute on Drug Abuse (NIDA) recognized dilution cut-off of 1.8 mmol/L for urine creatinine concentration is applied, 84% of the specimens surveyed here would need to be repeated to ensure accurate results. Because of these findings, it is recommended that routine creatinine and pH estimations should be performed on all specimens submitted for urinary drugs of abuse screening, or at least when unexpectedly negative results are obtained.

Screening for drugs of abuse. I: Opiates, amphetamines and cocaine.

(1) In order to provide an efficient and reliable service for drugs of abuse screening in urine, the laboratory should analyse 20-30 samples per week, and the staff should include a scientist with special expertise in the subject. (2) Turnaround times should be between 2-3 days of sample collection. To achieve this aim it may be necessary to make special arrangements for the delivery of samples to the laboratory. Results should preferably be transmitted by electronic mail or facsimile with the necessary precautions for security and confidentiality: hardcopy reports may also be required. (3) Good communications between the requesting clinician and the laboratory are essential. An advisory service should be provided by the laboratory and clinicians should be encouraged to discuss requests and results with laboratory staff. It is important that the laboratory inform doctors of the range of substances detected and the sensitivity and specificity of laboratory assays. (4) Assays should be performed according to the manufacturer's protocols, or by modified methods that have been rigorously validated. Quality control samples should be included in each analytical run and participation in an external quality assessment scheme, e.g. UKNEQAS, is essential to provide independent confirmation and confidence that results compare with those from other laboratories. Other requirements include adequate training and supervision of staff, and careful recording of samples and results. (5) Drugs to be tested will depend on the drug 'scene' in the area but should include those drugs regularly prescribed for maintenance therapy (e.g. methadone, dihydrocodeine, benzodiazepines), and drugs frequently misused (e.g. heroin, buprenorphine, amphetamines, cocaine). (6) Positive results obtained by preliminary screening methods e.g. EMIT, should be confirmed by another analytical technique, e.g. TLC, GC or GC-MS. If there are potentially serious or legal implications, and in employment and preemployment testing, confirmation of positive results is mandatory. In some cases, e.g. checking for methadone or benzodiazepine compliance, it may be considered unnecessary to confirm positive results although possible spiking of samples cannot be excluded without checking for the presence of metabolites by a chromatographic procedure.

A preliminary evaluation of five rapid detection kits for on site drugs of abuse screening.

There are now several rapid drugs of abuse testing kits that have been designed for near-patient testing and on-site clinic screening for drug abuse. These kits are sold on the basis of their cost and rapid generation of accurate results. Five such kits have been evaluated by comparison with recognized and established methodologies. All of the kits evaluated were found to lack both sensitivity and specificity. An unacceptable proportion of false negative and false positive results were observed for most kits. This raises the question of their usefulness in the near-patient and clinic situation.