Abstracts of Recent Papers from the staff of the Regional Laboratory for Toxicology

Last modified: February 17, 2003

Abstracts have now been placed into a separate file for speed of downloading the titles of papers.

Methods: A literature search was conducted regarding the use and evaluation of near-patient testing devices for drugs-of-abuse screening. The results of this research, together our own practical evaluations of such devices, have been collated into this review.

Results: It is proposed that although near-patient testing devices may be useful in remote areas or where rapid action needs to be taken, it should be remembered that they provide only initial screening data and may yield false-positive or flase-negative results. Such devices need to be used with caution because a rapid but unconfirmed result may lead to misdiagnosis and inappropriate treatment for those who have a drug problem. It should be noted that a single result, which may be inaccurate, could lead to the cessation of treatment and a failure to provide care for those in greatest need. In addition, false-positive results may also have medico-legal implications, especially with the initiation of the drug testing and treatment orders.

Conclusions: Near-patient testing devices for drugs of abuse could be an expensive and potentially inaccurate means to monitor patient treatment and drug abuse status. 

© 2002 American Association for Clinical Chemistry

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Analysis of 4-methylthioamphetamine (4-MTA) in clinical specimens

S. P. Elliott

 Abstract

 There has been much publicity, particularly in Europe, regarding a new phenylethylamine based compound called 4-methylthioamphetamine (4-MTA), also known as para-methylthioamphetamine (p-MTA), MTA or "Flatliner". Chemically, 4-MTA is an amphetamine derivative and is a non-neurotoxic potent serotonin-releasing agent and reversible inhibitor of rat monoamine oxidase-A. Its effects, therefore, appear different to amphetamine.

Analysis of various plasma and urine specimens in three clinical cases implicating "Ecstasy" ingestion, revealed the presence of 4-MTA. Presumed metabolites were also detected, with one compound identified as being 4-MTA sulphoxide. The concentrations of 4-MTA measured in the plasma ranged from 0.131 mg/L to 0.760 mg/L. In one patient, the 4-MTA concentration was determined in a series of plasma samples; this allowed a presumptive half-life of approximately 7 hours to be estimated for 4-MTA in plasma. This paper describes the first reported data regarding possible pharmacokinetics of 4-MTA in humans and presents the first reported non-fatal instances of 4-MTA intoxication in the U.K.

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Urinary Opiate Profiles – a Study of Heroin Users in the West Midlands, UK.

 C. Meadway, S. George 

 Abstract

The presence of the heroin metabolite 6-monoacetylmorphine (6-MAM) in urine is used to definitively identify recent heroin abuse. A rapid and sensitive GC- MS method for the simultaneous analysis of codeine, norcodeine, morphine, normorphine and 6-MAM in urine was developed and successfully applied to the analysis of 321 ‘heroin positive’ urine specimens from individual subjects (identified by the presence of 6-MAM), to provide quantitative urinary opiate excretion data for heroin abusers. The cohort analysed was composed of 238 males (age range 16 to 53 years) and 83 females (age range 16 to 50 years). The concentrations of free 6-MAM, morphine and codeine determined in these 321 specimens ranged between 103 µg/L – 246,312 µg/L, 129 µg/L – 193,600 µg/L and 103 µg/L – 519,000 µg/L respectively. Free norcodeine and normorphine concentrations were found to range between 143 µg/L – 50,200 µg/L and 205 µg/L – 149,700 µg/L respectively.

A statistically significant relationship was determined between subject age and 6-MAM concentration, possibly indicating opiate tolerance in these individuals. In addition, significant correlations were determined between the free concentrations of urinary opiates and their primary metabolites in spot urine specimens following heroin use. It is hoped that by studying the relationships between opiates and their metabolites in subjects where the route of administration has been established, that a model can be developed to follow the compliance if individuals prescribed diamorphine for opiate addiction problems. It is hoped that such a model could aid in both the initial diagnosis and the continued clinical management of opiate abusers.

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The effect of heat inactivation to minimise the risk of HIV infection on the results obtained for Therapeutic Drug Monitoring.

 S. George 

 Abstract

The effect of human immunodeficiency virus (HIV) inactivation by heat treatment on therapeutic drug monitoring results obtained for caffeine, carbamazepine, digoxin, ethosuximide, lamotrigine, phenobarbitone, phenytoin, theophylline and valproic acid was studied using temperatures of 56^OC and 60^OC and incubation times ranging from 0 to 120 minutes.

It was found that there were slight decreases (less than 5%) in the concentrations determined for caffeine, carbamazepine, ethosuximide, phenobarbitone, theophylline and slight increases (less than 5%) in phenytoin and valproic acid concentrations as the time of treatment increased. Lamotrigine concentrations were unaltered throughout the study. Digoxin showed the largest change in concentration with a maximal increase of 8% from 1.3 µg/L to 1.4 µg/L when heated at 56^OC for 30 minutes and 18% from 1.1 µg/L to 1.3 µg/L when heated at 60^OC for 30 minutes. However, none of these changes were statistically significant (p > 0.05) indicating that there was no overall clinical or statistical significant effect of heat treatment on the results obtained for therapeutic drug monitoring results for any drug studied.

The mechanisms for these alterations in concentration have not been elucidated; however, it is concluded that heat inactivation to negate exposure to HIV virus will not affect the therapeutic drug monitoring results when determined in biohazard specimens that have been heated prior to their analysis.

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Development of a high-performance liquid chromatography retention index scale for toxicological drug screening.

Simon P. Elliott and Keith A. Hale

Abstract

An efficient and practical analytical method for correcting HPLC retention data has been produced using an HPLC diode-array UV detector system. The system is based on retention indices (RI) and is to be used primarily for the identification of toxicologically relevant drugs involved in clinical and forensic toxicology. The RI correction method was chosen as it provided slightly greater degree of reproducibility than using relative retention time (RRT), particularly for acidic and neutral drugs. Development of the system involved the establishment of the optimal chromatographic conditions and extensive studies of the stability of the system. An acetonitrile gradient elution was used with RI values determined by interpolation from a series of specifically chosen basic and acidic/neutral marker drugs which eluted at regular intervals to produce a linear RI scale. It was found that two separate RI scales were required for basic and acidic/neutral drugs. The use of multiple drug markers as primary retention index standards had not been previously applied to HPLC general drug screening and comparison with a recently published database suggests that the system may also provide improved selectivity.

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Applications of a HPLC-DAD drug screening system based on retention indices and UV spectra.

Simon P. Elliott and Keith A. Hale

Abstract

An analytical database of over 250 toxicologically relevant drugs, using HPLC with diode array UV detection, has been extensively applied to both clinical and forensic toxicology. This general drug screening system, based on a mixed phase (OD/CN) column and gradient reverse-phase HPLC, can identify a wide range of basic, acidic and neutral drugs and metabolites, some of which are not amenable to GC and TLC. Compounds are identified using both retention index (RI) value (calculated by interpolation between a series of reference drug markers) and UV spectral data. It has been previously shown that this chromatographic system provides long-term reproducibility and has potentially useful selectivity differences compared to those based on ODS columns. Development has been undertaken to improve the speed and practicality of the system for emergency toxicology screening by increasing the rate of the elution gradient, while maintaining the applicability of the RI database. The database has also been found to be an important tool in determining optimum strategies for the adaptation of the system for quantitative analyses of drugs and metabolites under isocratic conditions.

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The measurement of morphine in the hair of heroin abusers

S George and R A Braithwaite

SUMMARY.

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 1.0M sodium hydroxide for 18h at 55°C, neutralization with 1.0M hydrochloric acid, and pH adjustment with 0.1M 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.

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Opiate concentrations following the ingestion of poppy seed products - evidence for 'the poppy seed defence'

C. F. E. Meadway

The Substance Abuse and Mental Health Services Administration¹ stipulated 300 ng/ml cut-off limit for opiate assays has been called into question due to positive results being obtained following the ingestion of poppy seed containing food products.

To establish the plausibility of ‘the poppy seed defence’ the concentrations of codeine, norcodeine, morphine, normorphine and thebaine (a potential marker for seed ingestion) in several varieties of seeds were quantified by GC-MS. The maximum morphine and codeine concentrations were found to be 33.2 and 13.7 m g/g seed respectively. The country of origin of the seed specimen and the preparation of the seeds before their use was found to influence the alkaloid concentration determined.

All urine specimens analysed following the consumption of bread rolls (mean 0.76 g seed per roll) by four subjects produced negative results in the EMIT opiate assay with the exception of one subject (body weight 63.0 kg) who consumed two poppy seed rolls. In this subject opiate positive screening results (Behring EMIT II opiate assay) were obtained for up to 4 - 6 h post ingestion with maximum urinary morphine and codeine concentrations of 832.0 ng/ml (@ 2 - 4 h post ingestion) and 47.9 ng/ml (@ 0 - 2 h post ingestion) respectively being achieved.

Following the ingestion of poppy seed cake (mean 4.69 g seed per slice) by four individuals opiate positive screening results were obtained for up to 12 - 24 h. In one subject (dose equivalent to 0.07 g poppy seed/kg body weight) maximum urinary morphine and codeine concentrations of 302.1 ng/ml (@ 0 - 2 h) and 83.8 ng/ml (@ 2 - 4 h) respectively were recorded.

These findings demonstrate the plausibility of the poppy seed defence. Great care should therefore be taken when interpreting the data produced from drugs of abuse screening for opiates.

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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

S. George

Abstract

Fourteen subjects (selected on the basis of compliance to their prescribed methadone maintenance program by the consultant psychiatrist in charge of their treatment) undergoing opiate detoxification by methadone replacement therapy were studied to determine whether or not 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). Following the derivation of this relationship, it was hoped that the urinary concentrations of methadone and/or EDDP could be used as a non-invasive technique to monitor the methadone compliance of 56 drug abusers.

Despite statistically significant correlation’s (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 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 intra-individual variation in excretion patterns.

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The Effect of Glutaraldehyde Adulteration of Urine Specimens on Syva EMIT II Drugs-of-Abuse Assays

S. George and R.A. Braithwaite

Abstract

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 readings (dA/min), is proposed.

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

S. George and R.A. Braithwaite

Abstract

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 of Drug Abuse (NIDA) recognised 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.

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

S. George and R.A. Braithwaite

Abstract

There are now several rapid drugs of abuse testing kits that have been designed for near-patient testing and on-site clinical 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 the of 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.

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Analysis of etorphine in post-mortem samples by HPLC with UV diode-array detection.

Forensic Sci. Intl., 101; 9-16

Elliott, S.P and Hale, K.A. (1999)

Abstract

Etorphine is a synthetic narcotic analgesic usually used in veterinary medicine. It possesses an analgesic potency up to 1000 times greater than morphine and is therefore used in low doses, primarily for tranquillising large animals. For veterinary use, etorphine is usually available in its commercial formulation as Immobilon^®, when in combination with acepromazine or methotrimeprazine. Due to the potency of etorphine, only very low doses are required to produce adverse or fatal effects. This paper describes a method for detecting and quantifying etorphine using HPLC with UV diode array detection (HPLC-DAD) and demonstrates the advantage of the technique for the detection of Immobilon^® at low doses. In a forensic case involving Immobilon^®, the etorphine concentrations measured in post mortem femoral vein and heart blood specimens were 14.5 mg/L and 23.5 mg/L, respectively. No etorphine was detected in the urine. To our knowledge this is the first time post mortem etorphine concentrations have been reported.

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A previously unidentified acepromazine metabolite in man: implications for the measurement of acepromazine in blood.

J. Anal. Tox., 23; 367-371

Elliott, S.P. and Hale, K.A. (1999)

Abstract

HPLC-DAD results obtained during the investigation of two cases involving acepromazine prompted us to study the stability of the drug in blood. It was found that acepromazine can undergo in vitro conversion by human red blood cells to 2-(1-hydroxyethyl)promazine, a product which has been reported as a minor urinary metabolite in horse urine but not previously identified in humans. Further, our analytical findings in the two cases examined suggest that 2-(1-hydroxyethyl)promazine may be the major unconjugated metabolite of acepromazine in humans. These findings have important implications for the analytical toxicology of acepromazine.

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Fatal poisoning with a new phenylethylamine: 4-methylthioamphetamine (4-MTA).

J. Analytical Toxicology, 24; 85-89

Elliott, S.P. (2000)

Abstract

There has been much publicity in the U.K. regarding a new phenylethylamine based compound called 4-methylthioamphetamine (4-MTA), also known as para-methylthioamphetamine (p-MTA), MTA or "Flatliner". Chemically, 4-MTA is an amphetamine derivative and is a non-neurotoxic potent serotonin-releasing agent and reversible inhibitor of rat monoamine oxidase-A.

Analysis of post-mortem blood and urine specimens in a case implicating MDMA, revealed the presence of 4-MTA, at a concentration of 4.6 mg/L in femoral blood and 87.2 mg/L in the urine. The concentration of 4-MTA in peri-mortem blood was measured at 4.2 mg/L. This is the first reported case of death involving 4-MTA in the U.K. and the first case known to only involve 4-MTA.

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Laboratory Screening for Drugs of Abuse.

S.George

Abstract

Drugs of abuse is a growing national problem, the true extent of which may never be determined. The laboratory screening for drugs of abuse is a complex procedure requiring extensive resource allocation in terms of both manpower and equipment. Screening techniques are used to confirm or refute clinical diagnoses, and thereby aid patient management. However, screening methods may only rule out the specimens that are negative for drugs thought to be abused. For several assays a more sensitive and specific technique needs to be applied to determine the exact identity of the drug found in the specimens submitted for analysis from the subject under scrutiny. It is the level of sophistication of this challenging analytical field that results in only specialist laboratories being able to perform this work in its entirety.

Introduction

1. The pattern and range of substances abused is constantly changing according to supply and demand. If heroin is in short supply then addicts may turn to injecting benzodiazepines such as temazepam until their supply is restored. What is sold as 'ecstasy' may be amphetamine, methylenedioxymethamphetamine (MDMA, ecstasy) or aquarium oxygenating tablets. There has also been a recent resurgence of LSD in the rave scene, but at lower concentrations than ere abused in the 1960's. In Glasgow the GP prescribing ban on buprenorphine (potent narcotic analgesic) has brought the incidence of positive screening results down from 60% to around 10% in addicts, but has led to an increase in the abuse of benzodiazepines.

2. Prescribed medication and some over-the- counter remedies may produce "false positive" results. For example, codeine taken for pain relief will give positive opiate screening results, and nasal decongestants may give rise to positive amphetamine screening results. These issues may only be resolved by the use of confirmatory screening techniques outlined below.

3. Some investigations may have important medico-legal implications. This is especially true if children are involved, for example the "Munchausen syndrome by proxy" cases where children are administered drugs by their parents as a cry for attention/help, or perhaps where children accidentally ingest drugs found in the home of drug abusers...............

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Measurement of selenium in clinical samples.

Annal Clinical Biochem., 36: 301-315.

T. M. T. Sheehan and D. J. Halls.¹

^{1 The Trace Element Unit, Department of Clinical Biochemistry, Glasgow Royal Infirmary University NHS Trust, Castle Street, Glasgow G4 0SF.}

Selenium (Se) is a metalloid element, of atomic weight 79, located in group VI of the periodic table.¹ Its biochemistry, toxicology and nutritional importance have been reviewed regularly and thoroughly over the last decade.^2-5 The element is essential for mammals, including humans, as a component of two enzymes, glutathione peroxidase and iodothyronine 5'-de-iodinase. In addition, a distinct selenium-containing protein, selenoprotein P, has been identified in plasma, but its function as yet remains unclear.⁶ Selenium deficiency has been investigated with respect to a considerable number of human illnesses,⁴ an issue raised recently in the UK in response to falling dietary intake.⁷ A prophylactic role for the element in cancer prevention is the subject of both intense debate and epidemiological study⁸ and there are suggestions that this action may be pharmacological rather than essential.^9,10 In animal studies it has been demonstrated that benign viruses can become virulent by passing through a selenium-deficient host.¹¹

Selenium, and its measurement in biological materials, may become clinically relevant in situations of excessive or insufficient intake. Except for isolated areas of China, where human selenosis has been endemic,¹² selenium poisoning in humans is uncommon, but may be serious nonetheless. Toxicity due to the ingestion of concentrated solutions¹³ and incorrectly formulated health supplements¹⁴ has been reported. Features of acute poisoning include vomiting and diarrhoea, ‘garlic-breath’, mucosal damage, metabolic acidosis and muscle spasm; however, these may also arise, in part, from the low pH and any non-selenium components of ingested solutions. Classical chronic selenosis manifests as gastro-intestinal disturbance, ‘garlic-breath’, loss of hair and nails, infection of the nail beds and skin lesions and, in extreme cases, neurological impairment. In these circumstances selenium analysis can confirm ingestion, important if diagnosis is difficult;¹⁵ otherwise, its value is limited as it is not necessarily a good indicator of the severity of poisoning or its prognosis, and the possibilities for active systemic removal of ingested selenium are almost non-existent.¹³ Similarly, the value of biological monitoring of occupational exposure to the element is confined to monitoring and documenting the extent of exposure.¹⁶

More relevant, as evidenced by current clinical practice, is the biochemical monitoring of selenium in patients deemed to be at risk of deficiency as a consequence of illness or therapy. The basis for this concern originates in the two classic Chinese manifestations of endemic selenium deficiency: Keshan syndrome,¹⁷ a cardiomyopathy prevalent amongst children and women, and Kashin-Beck disease,¹⁸ an osteoarthropathy afflicting children. Both of these conditions were unique, in that large human populations exhibited specific symptomology, fresh incidence of which was dramatically reduced by intensive dietary supplementation with selenium. Following awareness of these phenomena, occidental accounts of symptomatic iatrogenic selenium deficiency appeared. These reported cardiomyopathy (three fatal), myalgias, haematological abnormalities and discoloration of hair and nails and have been reviewed by Lockitch.⁴ All the patients had been on ‘selenium-free’ long-term parenteral nutrition, the fatalities occurring after 2, 6 and 8 years. Further studies indicated that, although symptomatic selenium ………………………………………….

This article was prepared under the auspices of the Analytical Investigation Standing Committee of the Association of Clinical Biochemists.

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The application of drugs of abuse screening on critically ill patients.

Care of the Critically Ill. 15: 180-185.

Robin A. Braithwaite. 1999

The abuse of drugs and other substances including alcohol has become an increasingly widespread problem in society1.In addition, the diversity of drugs being abused continues to increase each year, with new so-called 'designer drugs' appearing on the drug scene, sometimes without prior warning. The drugs that are commonly abused are shown in TABLE 1. The age at which people take drugs is becoming younger each year2,3, also the parents of many young children may be drug users and thus increase the risk of accidental poisoning4. As a consequence, the ingestion of drugs of abuse has become a more important factor to be considered in the diagnosis and management of acute medical emergencies, including the card of the critically ill5. The presence of drugs is an additional risk factor in those patients requiring anaesthesia or undergoing surgical intervention. A growing number of cases also have medico-legal aspects that may cause problems at a later stage when evidence is presented in court.

In many situations,; drug usage may be suspected from the history, or circumstances of the case. It is important to document. the patient's recent history carefully as this may sometimes give clues to the type of drug being used eg history of heroin use or collapse at a 'rave' party where .'ecstasy is frequently used. However, the history given by drug users can, sometime be, extremely unreliable. In addition the composition and purity of street,: drugs, can be highly , variable. The presentation of the patient and signs and symptoms of poisoning can be helpful in deciding the scope of any drug screening to be carried out. Evidence of intravenous usage may also suggest a history of drug use and potential risks regarding HIV and Hepatitis B and C viruses. There is also a frequent association between drug and alcohol abuse, sudden collapse, violence and trauma5.

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Positively negative - Drug Testing Uncovered

Drug Link November/December 1999

Claire Meadway, Shashi Parmar, Steve George. 1999

Understanding the laboratory approach to drugs testing answers many questions surrounding the results.

The rise in the drug using population is reflected in increased numbers of screening requests received by specialist laboratories. Advances in technology enable the 'labs' to considerably increase the repertoire of tests available, in line with the diversity of drugs being used. The laboratory aims to provide analytical information and interpretation, which aids in the initial assessment and longer term treatment of drug users. Tests are usually carried out on urine specimens, because high concentrations of drugs and their metabolites are detectable following drug use. Urine tests are non-invasive and large volume specimens are easily obtained.

Link to full article: (PosNeg.TIF - group 4 TIFF file, 310KB, 3 pages)

To save (download) the TIFF file, right click on the above link and choose the option "Save Target As..."

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Using Amphetamine Isomer ratios to Determine the Compliance of Amphetamine Abusers Prescribed Dexedrine.

J. Analytical Toxicology, 24; 223-227

S George, RA Braithwaite

ABSTRACT

The amphetamine isomer ratios (l-amphetamine/d-amphetamine) in 373 urine specimens submitted for analysis over a three year period have been determined using a chiral derivatising 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 (+ 1sd) l/d-amphetamine isomer ratio for 147 specimens from compliant subjects prescribed dexedrine was 15.0% (+ 4.9%). The mean (+ 1sd) 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, a total of 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 derivatisation, 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 weight/weight of 21.5% (range 3.4% to 71.0%) relative to pure dl-amphetamine substance.

Introduction

Amphetamine was first commercially available in 1932 as a nasal mucosa dilator and was used extensively during World War II to combat fatigue (1). However, it is the euphoric and stimulant properties that have resulted in amphetamine becoming Britain's most commonly abused stimulant, second only to cannabis in the overall league table of illicit drug use (2). The number of persons found guilty, cautioned, or dealt with for drug offences relating to amphetamine in the U.K. increased from 3532 in 1991 to 10,364 in 1995, with the number of seizures rising from 6821 to 15,443 over the same period. The quantity of amphetamine seized also increased from 421 kg in 1991 to 819 kg in 1995 (3).

Amphetamine is one of the most potent sympathomimetic amines in stimulating the central nervous system with dose-related psychic effects (4). It can be abused by oral, nasal, or intravenous routes. The development of tolerance, combined with the desire for the feeling of power, hyperactivity, and euphoria, can lead a heavy intravenous user to injecting per day. It should be noted that abused street amphetamine, also known as whizz, sulphate, or speed, is of relatively low purity, typically around 5% (1,2).

Illicit amphetamine is produced as a racemate composed of two isomers, the d-isomer and the i-isomer, in a ratio of 1:1. Dexedrine, the d-isomer of amphetamine, has 34 times the central stimulant activity of the i-isomer and is used as an effective substitute and harm-reduction mechanism for subjects undergoing amphetamine stabilisation and withdrawal-treatment programs. The i-isomer has been found to be slightly more potent in its cardiovascular effects, leading to increased blood pressure and arrhythmias at high doses (4,5).

The renal elimination of amphetamine, a weak base, is pH dependent with an elimination half-life ranging between 7 and 34 h. Normally, around 30% of the dose is excreted unchanged in the urine, but this may increase to around 60-70% under acidic conditions (elimination half-life of 8 to 10.5 h) or decrease to between land 7% (elimination half-life of 16 to3l h) in alkaline urine specimens (1,6).

The urinary elimination of amphetamine has been found to be stereospecific with the elimination half-life of the d-isomer (average of 13 h) being slightly shorter than the i-isomer (average of 17 h) under alkaline conditions (1,7). Under acidic conditions, the clearance through renal excretion is more rapid, so the difference in isomer half-life is minimized (1). All of these drug-clearance issues will impact the detection of amphetamine in the urine of drug abusers and the subsequent interpretation of analytical findings.................................

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

J Clin Pathol 2000;53:282-285

D S Cook, R A Braithwaite, K A Hale

Abstract

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 redo; that is, drugs that exhibit considerable postmortem redistribution, also have high postmortem to antemortem ratios.

Keywords: postmortem blood sampling; drug concentrations, toxicological analysis.

Introduction

The processes of postmortem redistribution result in the migration of drugs between blood and tissues. The rate and extent of this movement varies according to several factors, including the nature of the drug and the time interval between death and postmortem specimen collection. Within the torso, the major organs constitute potential drug pools, and the gastrointestinal tract might contain considerable quantities of unabsorbed drug, and thus central blood is subject to redistribution from these local organs. peripheral blood, such as femoral blood) is subject to redistribution influences only from local tissue~musde and fat. In general, redistribution into central vessels is greater than redistribution into peripheral vessels. The difference between the two sites is known as the central to peripheral (C/P) ratio. For these reasons, the blood specimen of choice for toxicological analysis after death is a femoral venous sample, ideally collected from a ligated vessel,^7-10 although inevitably there will be situations in which such sample collection is not possible.

Often, pathologists or toxicologists are quested to estimate the   amount of drug present at the time of death, or the number of tablets consurned. This assumes that the drug concentration found at postmortem examination is a reliable estimate of that present at the time of death. There is a lack of evidence that such an errtrapolation is possible; in only a few cases reported in the literature are antemortem blood concentrations available for comparison with values from a variety of sites at postmortem examination.^{14, 17-19} Such comparisons have not been carried out enensively because antmortem samples are often not available for analysis. In 1978, Vorpahl and Coe¹⁷ collected data pertaining to antemortern and postmortem blood digoxin concentrations. The chosen postmortem blood sampling site was the left ventricle. Their results showed that in all 27 cases, postmortem cardiac blood concentrations were significantly higher than antemortern blood concentrations. Blood from the femoral vein was only collected in 11 cases; in these cases, the average postmortem to antemortem (PM/AM) ratio was 1.4, and in nine of the 11 cases the postmortem concentration was higher than the antemortem value.

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

Ann. Clin. Biochem. 37: 350-354.

S. George, S. Parmar, C.Meadway, R A Braithwaite,

ABSTRACT

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 (cutoff calibrator concentration of 100 ng/mL) in combination with a Dade Behring EMIT urinary methadone immunoassay (cutoff calibrator concentration of 300 ng/mL). Of these 642 (46%) were found to be positive using the EDDP assay but of these only 541 (39%) were found to be positive by the methadone assay.

Out of the 642 EDDP positive specimens, 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 9 samples. In addition, the EDDP and GC-MS assays produced negative screening results for 7 specimens that had methadone concentrations ranging from 4,000 to 37,500 ng/mL and these were therefore presumed to be “spiked” with methadone, i.e. 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.

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

Ann. Clin. Biochem. 37: 338-343.

S. George, L. Gill, R.A. Braithwaite

ABSTRACT

A simple and rapid high performance liquid chromatographic method has been developed for the determination of vigabatrin in plasma or serum. The assay uses only 100 µL of specimen and has been found to be linear over the concentration range of 1 to 50 mg/L. The limit of detection has been determined to be 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 twelve month period. It was found that only 63% of the male group and 53% of the female group were within the proposed target range 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.

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The Toxicology of Selenium

Bulletin of the International Association of Forensic Toxicologists. Vol. XXVIII (3) 1998

T. M. T. Sheehan

ABSTRACT

Selenium (Se) is a metalloid, of atomic weight 79, located in group VI of the periodic table. Two elemental allotropes occur, a red colloidal, amorphous form and black, crystalline selenium. Other oxidation states are -2 (selenide), +4 (selenite) and +6 (selenate). In addition, it can substitute for sulphur to produce selenocysteine and selenomethionine. Geographical distribution of selenium is widespread, but geological deposition varies markedly to produce regions of very low and very high selenium content; bioavailability is favoured by alkaline soils. The element is used in the electronics industry, in the manufacture of paints, pigments and glass, and in shampoos. Selenium is an essential trace element for mammals, including humans, forming an integral part of the molecule of two enzymes: glutathione peroxidase and iodothyronine 5’­deiodinase. Recommended daily intake (U.S.A.) is 13-70µg depending on age. Selenium status is usually assessed by assay of the element in plasma or whole blood, 'normal' values varying with geological selenium content, and increasing with age until circa 17 yrs old. This laboratory's adult plasma reference range is 70-130µg/1, twice that for New Zealand, a low-selenium area and half the values seen in selenium -replete areas of the U.S.A. Whole blood [Se] is 25-50% higher than that of plasma. Urinary excretion is usually well below 50µg/24 hours. Analysis of selenium in body fluids is usually by molecular fluorescence or atomic absorption spectroscopy using either hydride generation or direct electrothermal atomisation.

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

Ann. Clin. Biochem. 34: 460-510

D. Simpson, R.A. Braithwaite, D.R. Jarvie, M.J. Stewart, S. Walker, I.W. Watson, B. Widdop.

ABSTRACT

Screening urine samples for drugs of abuse is important for the confirmation of drug misuse and for the clinical management of drug misusers. Laboratories providing this service receive a steadily increasing number of samples, and the reasons for requesting drug screens may vary considerably. Many such requests have potential legal implications: for example, suspected poisoning of children, cases relating to child custody, screening prison inmates, pre-employment screening, and drug testing in the work place. In addition, laboratories may become involved in screening for a wide range of prescribed and abused drugs in persons suspected of driving under the influence of drugs.1,2

Drug maintenance therapy for harm minimization is now widely used in general practice as well as in drug treatment centres. Drugs prescribed are usually methadone, dihydrocodeine and benzodiazepines although other drugs, including buprenorphine, pentazocine, amphetamine, desipramine and fluoxetine, have been used. An important aspect of screening for maintenance drugs is the need to eliminate or at least minimize the chance of false negative tests for patients prescribed these drugs. Unexpected negative results in this situation should be confirmed to prevent possible serious implications.

Patterns of drug abuse continue to change: substances now being abused include the anaesthetic ketamine, a-methylfentanyl ('China white'), N-methyl-l- (3,4-methylene-dioxyphenyl)-2-butanamine (MBDB), and y-hydroxybutyrate (GHB, 'liquid X', 'liquid E'), which is, used as a diet supplement and as a sedative in alcohol withdrawal regimes.

Hospital laboratories currently receive only a relatively small number of requests for the measurement of anabolic steroids although an increasing number of parents, general practitioners and psychiatrists are concerned about the health of young people abusing steroids. Laboratories testing for sporting bodies must be accredited by the International Olympic Committee (IOC). It is likely, however, that there will be an increasing need for laboratories to act as reference centres for clinical and non-I0C samples.

Urine drug screening for Road Traffic Act offences is becoming increasingly important in the USA and in Europe. A survey' has shown that 35% of the drugs and driving cases in London and the surrounding area involved drug abuse; benzodiazepines and narcotic drugs were detected in 38% and 13% of the cases, respectively. In the USA screening of urine samples revealed that 59% of reckless drivers who were not intoxicated with alcohol tested positive for cocaine, marijuana, or both. The European Community has issued a directive relating to the classification of drugs according to their effect on the ability to drive, and member countries should have implemented regulations necessary to comply with this directive by July 1996.' With this aim in view, information in package inserts will enable doctors to advise patients about a drug's effect on the ability to drive. Drug screening for driving offences could provide an additional source of revenue for hospital laboratories.

The development of new analytical procedures and improvement of existing techniques are essential aspects of drugs of abuse screening. Syva EMIT (Enzyme Multiplied Immunoassay Technique) methods (Behring Diagnostics UK Ltd, Milton Keynes, UK) have been adapted for use on a number of analysers, with modifications to make them more cost-effective such as dilution of the manufacturer's reagents, 4 or the use of in-house reagents.5 It is important that rigorous validation of modified methods, including assessment of accuracy, precision and specificity, is carried out before routine use. Validation of modified methods is now mandatory to satisfy Control of Substances Hazardous to Health (COSHH) regulations and for laboratory accreditation, and is also necessary for protection against litigation. New commercial kits based on antibody-coated microplate enzyme immunoassay (EIA) and the Auto-Lyte EIA for use on a range of analysers, are marketed by Cozart Bioscience Ltd (Abingdon, UK). A method based on cloned enzyme donor immunoassay (CEDIA) has been introduced by Boehringer (Boehringer Mannheim, Mannheim, Germany). The increasing number of methods suitable for near-patient testing include the ONTRAK and ONTRAK TESTCUP assays (Roche Diagnostics, Welwyn Garden City, UK), EZ-SCREEN test (EDITEK Inc, Burlington, NC, USA), Triage 7 and 8 cassettes (E Merck, Darmstadt, Germany), and Bionike A/Q` One Step immunochemical tests (Bionike, South San Francisco, CA, USA) available in test card and test strip forms (see later section on Nearer patient/on-site testing). Application of chiral analysis to drugs of abuse assays has been reported. This form of analysis has been used, for example, to differentiate between the steroisomers of amphetamine and methamphetamine, which may be present as a result of, prescribed medication, over-the-counter preparations or illicit drugs. Dexamphetamine is widely prescribed as a harm reduction measure while street amphetamine is composed of the racemic form. 9

Routine screening for drugs of abuse is almost exclusively performed on urine samples although other matrices, including plasma, hair, saliva and sweat, may be used. Measurement of plasma concentrations of prescribed drugs can be helpful in the management of drug misusers. For example, measuring methadone concentrations has been reported to be of value in confirming that individuals complaining of withdrawal symptoms do indeed have abnormally low drug concentrations in plasma.10 Measurement of drugs in hair is used in the USA and a service is provided in the UK by Tricho-Tech Ltd, Cardiff. There are, however, some problems associated with the use of this matrix including passive contamination of hair, removal of bound drugs by washing, and a lack of knowledge of the way in which drugs pass into hair and the stability of drugs in hair." Hair testing will probably be a valuable tool for the future but at present it is laborious and expensive.

Sweat analysis is another non-invasive pro­cedure, which offers many of the advantages of hair analysis. Sweat, collected from sweat patches which can be worn for several days to several months at a time, has been used to test for heroin, cocaine and metabolites using gas chromatography-mass spectrometry (GC­MS).12,11 Cone et al. 12 suggest that the sweat patch could be a useful monitoring device for surveillance of individuals in treatment and probation programmes.

A previous review, Screening for Drugs of Abuse (1),11 provided background information on analytical methods for the detection of amphetamines, opiates and cocaine in urine. This review deals with cannabinoids, lysergic acid diethylamide (LSD), buprenorphine, methadone, barbiturates, benzodiazepines, and other drugs (fentanyl and its derivatives, ketamine, pentazocine, cyclizine and dipipa­none, propoxyphene and phencyclidine).

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Antidepressants. In: analytical toxicology for clinical, forensic and pharmaceutical chemists.

Volume 5. Brandenberger H and Maes R.A.A., Walter de Gruyter, Berlin - New York. pp. 481-494.

R.A. Braithwaite.

Introduction

The first tricyclic antidepressant to be introduced was imipramine (Tofranil) by Geigy in 1957. Following its success, the synthesis of a large number of chemically related compounds soon yielded many other effective antidepressants, the most well known of which was amitriptyline. The use of this group of drugs expanded rapidly during the 1960's and 1970's and became agents of choice in the treatment of depressive disorders. However, reports of toxicity, particularly in overdosage, soon appeared in the medical literature (1). At the present time tricyclic antidepressants, because of their proven efficacy and low cost, are widely prescribed, but remain one of the commonest causes of acute self-poisoning world-wide, and are responsible for a large number of overdose fatalities (2-6). Approximately 10-20% of all patients who present to hospital with self-poisoning may have taken a tricyclic antidepressant, also a high proportion of these patients require intensive care (4). Tricyclic anti­depressants are also associated with a relatively high incidence of adverse-effects in normal therapeutic usage (7).

A number of "second generation" heterocyclic antidepressants were developed during the 1970's such as trazodone, mianserin, maprotiline and amoxapine (8). Although structurally unrelated to the tricyclic group of antidepressants, some of these drugs show a similar pharmacological action and toxicity profile (8). In the last decade a "third generation" of specific serotonin re-uptake inhibitor antide­pressants (SSRI's) has been introduced, the most well known member of this group being fluoxetine (Prozac) (9). This new group of antidepressants appears to be cli­nically effective, with a greatly reduced toxicity in overdose (10). There is therefore an important requirement for the availability of specific and sensitive analytical techniques for the qualitative and quantitative measurement of antidepressant drugs and their active metabolites in body fluids and tissues. The main clinical applications for the measurement of these drugs is shown in table 1.

Table 1. Main Clinical Applications for the Measurement of Tricyclic and Newer Antidepressants

1. Emergency toxicology

2. Therapeutic drug monitoring

3. Investigation of adverse drug reactions

4. Forensic toxicology

5. Clinical trials of antidepressant action

6. Pharmacokinetic studies

 Pharmacokinetics

 Chemical structures of the tricyclic and some newer antidepressants

The tricyclic heterocyclic and newer selective serotonin reuptake inhibitor (SSRI) antidepressants form a chemically very heterogeneous group of compounds. The three ring so-called "tricyclic" dibenzodiazepine structure of imipramine and some congeners (e.g. clomipramine, lofepramine) and the dibenzoeycloheptadiene struc­ture of amitriptyline are shown in figure 1. Minor modification of the central ring structure is found with dothiepin and doxepin, or outer aromatic ring in the case of clomipramine. The three ring system incorporating a central seven membered ring is the common feature of all of the drugs in this group. All these drugs, apart from lofepramine, have short aliphatic tertiary or secondary amino side-chains. The N-desmethyl metabolites of amitriptyline and imipramine (nortriptyline and desi­pramine respectively) are pharmacologically active, and were also developed as ef­fective antidepressants in their own right. There is therefore a need to have analytical techniques that are able to measure tertiary amine parent drugs and their active N-desmethyl metabolites. The most important "second generation" heterocyclic antidepressants and the four major "third generation" SSRI's are shown in figure 2.

 Pharmacokinetics and metabolism

The pharmacokinetics and metabolism of the tricyclic antidepressants have been intensively studied over many years, particularly in the case of amitriptyline and imipramine and their respective metabolites, nortriptyline and desipramine (11). These compounds are generally well absorbed from the gastrointestinal tract, and because of their high lipid solubility, drug concentrations found in whole blood and plasma are much lower than in tissues. …………………………………….

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The Presence of Gamma-Hydroxybutyric Acid (GHB) in Postmortem Biological Fluids

J. Analytical Toxicology, 25; 152 - Letter to the Editor (March 2001)

S P Elliott

In recent years,  the popular drug of abuse gamma-hydroxybutyric acid (GHB) has caused a great deal of public and scientific controversy, one aspect of which is the apparent presence of GHB in biological specimens. Previously published articles have suggested that endogenous GHB can be present at toxicologically significant concentrations in postmortem blood (up to 433mg/L), but not in postmortem urine samples (1,2). It was therefore believed that the presence of GHB in a urine sample may indicate GHB ingestion. LeBeau et al. (3) also reported GHB (at concentrations lower than 15 mg/L) in citrate-buffered, antemortem blood. The interpretation of GHB concentrations found in either antemortem or postmortem specimens have therefore become difficult.

In order to obtain further evidence of the prevalence of endogenous GHB, a preliminary study involving post-mortem blood and urine specimens from 13 GHB unrelated fatalities (including instances of drowning and hanging) were analysed for the presence of GHB by both gas chromatography-mass spectrometry (GC-MS) and gas chromatography flame ionization detection. Detection involved identification of GHB-diTMS and gamma-butyrolactone. Table 1 shows, the concentrations of GHB measured in the specimens by GC-MS. Specimens analysed were less than six months old, stored in unpreserved containers, and frozen at -20^oC prior to analysis. In addition, GHB was detected but not quantitated in vitreous humor obtained in case 13. The results show that GHB is present in almost all postmortem samples analyzed (limit of detection, 1 mgL), and in many cases, at concentrations that would indicate GHB ingestion and potential intoxication. In particular, based on the small number of samples analyzed, there does not appear to be any obvious relation between GHB concentration and the blood/urine ratio. In five years of CHB analysis by the Laboratory, only one confirmed fatal case of GHB intoxication has been recorded where GHB was detected at 356 mg/L in the postmortem blood (significant supporting evidence found at the scene) (4).

Hence, these findings further complicate potential interpretation of suspected GHB-related fatalities as it does not appear to be possible to infer GHB ingestion if detection is confirmed in both postmortem blood and urine samples, as previously believed.

References

1. E.L. Fieler, D.E. Coleman, and R.C. Baselt. GHB concentrations in pre- and postmortem blood and urine. Clin. Chem. 44: 692 (1998).

2. R.C. Stephens, D.E. Colernan, and R.C. Baselt. in vivo stability of endogenous gamma-hydroxybutytate in postmortem blood. J. Forensic Sci. 44(1): 231 (1999).

3. M.A. LeBeau, M.A. Montgomery, R.A. Jufer and M.L. Miller. Elevated GHB in citrate-buffered blood. J. AnaL Toxicol. 24: 383-384 (2000).

4. K.A. Hale. Regional Laboratory for Toxicology, Birmingham, U.K. Personal comunication, 1995.

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