The reliability of immunoassay for determining the presence of opiates in the forensic setting

Urine immunoassays are commonly used as a rapid screen for drugs of abuse in emergency room, hospital, clinic, and forensic settings. The authors were concerned whether or not a negative screen of the urine for opiates was of significance and indicative that analysis of blood for opiates was not necessary. Specifically, we wished to determine whether a negative test for opiates by immunoassay absolutely rules out an acute overdose, and if not, what percentage of cases with negative results have opiates in the blood. A retrospective analysis was performed using the toxicology results for cases ruled an acute narcotic overdose at the Bexar County Medical Examiner's Office between 1998 and 2003. One hundred eighty-three cases met the criteria for the study. A false-negative rate of approximately 15% was found using an immunoassay as compared with blood analysis for narcotics. The authors feel that while this rate may be acceptable in a clinical setting, it is unacceptable in a forensic setting.     

ELISA-based determination of opiates in purulent transformation of cadaveric material

Abuse of drugs has been a topical issue up to the present-day. Opiates like morphine and heroin are still prevalent. The routine methods of determination of drugs in blood made within the forensic medical expertise are time-consuming and samples must be specially prepared for them. ELISA, when used for the determination of opiates in blood and urine, significantly cuts the test time. According to our research results, the diagnosis of opiates by ELISA is advisable at the preliminary stage of the forensic expertise of cadaver; the method is informative, which is of extra importance in cases of purulent transformation of cadaveric material.     

刺激性毒物西阿尔（CR）的检验

目的简要介绍刺激性毒物西阿尔的物理、化学性质及检验方法;方法使用直接提取法对常见检材进行提取,使用TLC、GC/MS方法对其进行定性分析,以西阿尔在不同展开系统下的Rf值和特征质谱图作为定性依据;结果可快速准确的对西阿尔进行定性分析;结论该方法简便,易行,准确,可靠,可用于案件检验。     

Immunochemical methods in detection of opiates in tissues and organs

Conditions of isolation of opiates from tissues and analysis of the resultant extracts by immunochemical methods are discussed. The main characteristics of OPIATES U kits (Pharmatech) for polarization fluorescent immunoanalysis of cadaveric material (liver) are determined. Immunochemical methods for forensic chemical analysis for opiates are experimentally compared.     

Performance of immunoassays in screening for opiates,cannabinoids and amphetamines in post-mortem blood

Several immunoassay methods for screening of abused drugs in whole blood were evaluated in post-mortem forensic toxicology. Blood samples known to be positive or negative for opiates, cannabinoids or amphetamines by gas chromatography-mass spectrometry (GC-MS) were analysed by EMIT II Plus and EMIT d.a.u., Syva RapidTest and Triage 8 after acetone precipitation. In these experiments, the EMIT immunoassay method was modified by using the Dade Behring VIVA analyser to detect substances more sensitively. Low concentrations of abused drugs were detected in blood samples. The sensitivities of the modified EMIT method for opiates, cannabinoids and amphetamines were 100, 86 and 98%, respectively, whereas the values were below 86% with the other methods. The specificities of all immunoassay methods for opiates and cannabinoids were 83% or above but 51-85% for amphetamines. Sample rejection occurred in a few cases with the EMIT amphetamine assays. The modified EMIT immunoassay system presented here seems to be useful for screening of drugs of abuse in post-mortem blood samples, especially when urine is not available.     

Diagnostic implication of blood and urine morphine content in alcohol intoxication

A total of 198 cases of acute parenteral poisoning with opiates are characterized. The range of concentrations of opiates metabolites in the blood and urine, main causes of death due to opiate poisoning in alcohol intoxication are analysed. Opiates toxicity was assessed with the logit-regression method and dose-effect curves valid for analysis of relationships between probability of death and opiate metabolites concentration in blood and urine. Correlation between probability of death and detection of morphine and ethanol in biological media of the victims is considered. Concentrations of morphine in blood and urine definitely indicating opiates poisoning in alcohol intoxication as a cause of death are determined.     

Validation of thin layer chromatography with AccuTOF-DART™ detection for forensic drug analysis

Abstract: Thin layer chromatography (TLC) is a technique that is commonly employed in the forensic drug analysis of pharmaceutical preparations. Detection is typically accomplished using various visualization spray reagents. Conventional gas chromatography–mass spectrometry (GC‐MS) analysis is typically performed to confirm the TLC results. Depending on the drugs tested and the instrument conditions required, this confirmation can take up to an hour to complete. Direct analysis in real time (DART?) is an ionization source, coupled to an accurate‐mass time‐of‐flight mass spectrometer that has the capability to ionize materials under ambient conditions. To streamline analysis, the combination of TLC with DART? detection is proposed to screen and subsequently identify drug compounds, all from the same TLC plate. DART? confirmations of TLC analyses take <10 min to complete and compare favorably to GC‐MS in sensitivity and selectivity. This study validates the use of TLC‐DART in the forensic identification of the components of several pharmaceutical preparations.     

A comparative analysis of Cannabis material

Extracts of 100 plant-like or resinous materials were analyzed for CBD, CBC, delta 9-THC, and CBN by GC using two different column packings and by GC-MS. Our independent identification of these cannabinoids confirmed those of other forensic science analysts who used microscopic examination, the Duquenois-Levine color test, and TLC for their analyses of the same samples. The identifications of cannabinoids by forensic acience analysts using TLC were corroborated by GC-MS analysis of hexane extracts of appropriate chromatogram spots.     

Detection of cannabinoids in urine

Described in the paper are results of comparative examination of urine by the methods of immunochromatographic express analysis (ICA), fluorescence-polarization immune analysis (FIA) and of thin-layer chromatography (TLC) made for the purpose of detecting consumers of hemp products. A high specificity and a good sensitivity of the methods were demonstrated, which is a basis for using them as preliminary tests in detecting cannaboids and their metabolites in urine. The methods were evaluated quantitively and qualitatively versus the etalon method of chromato-mass-spectrometry. A possibility was pointed out to apply the methods of ICA and TLC as system analysis in the determination of 11-nordelta-9-tetrahydrocannabinol-9-carbonic acid in urine of marijuana users.     

The hair analysis proficiency testing program of the French Society of Analytical Toxicology

In an effort to improve laboratories performing hair analysis in forensic cases, the French Society of Analytical Toxicology (S.F.T.A.) has implemented a proficiency testing program since 1992. Actually about 10 laboratories are participating. Each survey is dedicated to one analyte or one pharmacological class: opiates (6-monoacetylmorphine, morphine and codeine), cocaine and benzoylecgonine, tetrahydrocannabinol, buprenorphine and norbuprenorphine, beta-blockers (metoprolol, atenolol), beta 2-agonists (salbutamol, clenbuterol). Animal hair was tested for clenbuterol. Prior to sending, hair samples were reduced to a powdered form, well mixed to ensure homogeneity, and then tested by GC/MS or HPLC/MS. Results confirm those obtained in a preliminary study on opiates and cocaine analysis in hair: a common analytical procedure has to be used by all the participants, including hydrolysis of hair. It is essential to work on authentic drug-positive hair samples and not on spiked samples. Participation at this program is free of charge and considered as an educational tool. Comparison of the results with those of other laboratories in Europe and USA shows that the analytical methods used during this program are in accordance with all the other procedures.     

Hair analysis of opiates in mothers and newborns for evaluating opiate exposure during pregnancy

The increasing interest in toxicological hair analysis as a marker of human exposure to xenobiotics such as illicit substances or therapeutic drugs, has been made feasible by the extension of mass spectrometry, a highly sensitive method of detection. A newborn exposed to drugs in utero can suffer from a varying degree of withdrawal syndrome, a few days after birth. If of opiate origin, the withdrawal syndrome can be treated with morphine, among other therapeutics, but it is not easy to diagnose because of atypical symptoms presented by neonates and especially when maternal drug addiction has not been revealed. To assess and measure toxicological factors linked with the appearance and the severity of this syndrome, maternal and neonatal matrices such as urine, meconium and hair were collected during a protocol approved by the ethical committee. Opiates in particular were measured with GC-MS and potential combined dependences (cannabis, cocaine, amphetamine, LSD and benzodiazepines) and/or substitutive therapeutics (methadone or buprenorphine) were also assessed in 17 mother/neonate couples. Gestational opiate exposure profiles were drawn up and linked with the observed withdrawal syndromes. A withdrawal syndrome seems to appear more frequently after foetal exposure to an association of opiates/substitutive molecules (8 out of 10 withdrawal syndromes observed in this study), although the impact of cocaine and benzodiazepines must also be taken into account. The results obtained in neonatal hair make it possible to affirm foetal drug exposure and are in accordance, for the majority, with the appearance of a neonatal withdrawal syndrome (NWS). Neonatal hair analysis could contribute to assess in utero exposure to opiates, particularly when results in urine and meconium are negative or when these matrices are not available.     

The analysis of the methylenedioxy derivatives of amphetamine]

Methods for analysis of narcotics belonging to amphetamine methylene dioxy derivatives (MDD) are reviewed. The characteristics of these agents, their metabolism, and methods used for their detection and identification (TLC, GC, HPLC, GC/MS) are described. Methods for their extraction from biological objects (human urine and hair) are described. Efficacy of MDMA and MDEA from the urine by different extractants is assessed. The data demonstrate different potentialities for detection and identification of amphetamine MDD, including those in biological specimens (human urine and hairs), by numerous chromatographic methods.     

阿片类成瘾者血清、尿中吗啡TLCS分析

建立阿片类成瘾者血清、尿液中吗啡的薄层色谱扫描 (TCLS)定量检测方法。样品经酸、碱性水解后调至pH9,氯仿 /异丙醇 ( 9∶1)萃取及GDX 40 3柱固相萃取 ,在紫外区可见光区薄层扫描。测得 3种萃取方法吗啡回收率分别为 75 3 %± 4 9% ,80 9%± 3 2 %和 79 4%± 3 5 % ,血清、尿中吗啡最低检出浓度分别为 0 1μg·ml-1,0 0 5 μg·ml-1(信噪比≥ 3 )。本法可用于阿片类药物成瘾者或中毒者血、尿中吗啡的检测。     

Evaluation of Tamm‐Horsfall Protein and Uroplakin III for Forensic Identification of Urine

Abstract: In this study, Tamm‐Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium‐specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT‐PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.     

Comparative analysis of illicit heroin samples

The comparative analysis of street heroin samples is still an object of scientific discussion. A combination of a wide range of analytical techniques is necessary to obtain a valid amount of information about the sample composition, and it is impossible to define an unique analytical approach. We suggest a complete analytical sequence based on analysis of volatile compounds, opiates, diluents, adulterants and metals, by head space gas chromatography (HS/GC), gas chromatography mass spectrometry (GC/MS), thin layer chromatography (TLC), high pressure liquid chromatography (HPLC) and atomic absorption (AA) using a sample amount as low as 50-100 mg. The outlined procedure can be successfully applied to routine work, thus obtaining suitable information about a sample's chemical composition. This helps to attribute or exclude common sources of separate specimens. Results obtained on 33 street heroin samples confiscated in the metropolitan area of Rome are listed.     

A quantitative densitometric determination of heroin and cocaine samples by high-performance thin-layer chromatography

The traditional thin-layer chromatography (TLC) is a sensitive technique frequently used in chemical analysis for semiquantitative determinations. In the last years a high-performance thin-layer chromatography system (HPTLC) has been developed. It assures quantitative determinations with direct densitometric measurements of chromatographic spots in situ on the plate. Here, we wish to report a method for the drug determination in street samples, that employs a CAMAG TLC/HPTLC Scanner Photodensitometer and a CAMAG LINOMAT III for sample application. Samples of illicit drug traffic (50 samples of street heroin and 30 samples of street cocaine) are tested and the gas-liquid chromatography (GLC) and HPTLC determinations are compared. The HPTLC method has proven to be sensitive, precise and accurate for quantitative determinations of drugs in street samples. Furthermore this method may be useful in forensic chemistry to show a "fingerprint" of each sample.     

Dependence of acute opiate poisoning lethal outcome on the gender, age, and duration of addiction

The article presents toxicological characteristics of 198 cases of acute parenteral poisoning with morphine and heroin in the range of concentrations of their metabolites in the blood and urine which occur in practice. Risk of death is quantified in the range of possible morphine concentrations in the blood in acute opiates intoxication with reference to gender and age. Assessment of the criteria of quantitative opiates toxicity was made according to the method of logit regression and dose-effect curve suitable for analysis of correlations between probability of death and blood concentrations of opiates metabolites. Conditional lethal doses in respect to gender and age are calculated.     

The determination of lysergide (LSD) in urine by high-performance liquid chromatography-isotope dilution mass spectrometry (IDMS)

The use of isotope dilution mass spectrometry (IDMS) has been investigated for the forensic confirmation of lysergic acid diethylamide (LSD) in urine by LC-MS. The advantages of using a deuterated analog of LSD as an internal standard over methysergide are discussed. This study includes a comparison of the electrospray mass spectra of LSD, LSD-d3 and methysergide, and discusses the choice of suitable ions for use in selected ion monitoring (SIM) mode. An IDMS method is presented for the LC-MS confirmation of LSD in urine, with a limit of quantification (LOQ) of 0.5 ng/mL, reflecting the forensic requirement at this laboratory. Under some circumstances the LOQ can be improved to 0.1 ng/mL. This method is linear in the range tested (up to 10 ng/mL LSD in urine) and has been validated in terms of accuracy and precision.     

An Investigation of Normal Urine with a Creatinine Concentration Under the Cutoff of 20 mg/dL for Specimen Validity Testing in a Toxicology Laboratory,

In clinical and forensic toxicology laboratories, one commonly used method for urine specimen validity testing is creatinine concentration. In this study, workplace guidelines are examined to determine their relevance to forensic and clinical toxicology samples. Specifically, it investigates the occurrence of urine creatinine concentrations under 20 mg/dL and notes potential issues with factors influencing creatinine concentration by utilizing a simple, novel method consisting of cation‐paring high‐pressure liquid chromatography in tandem with ultraviolet detection to determine the creatinine concentration in 3019 donors. Of the 4227 sample population in this study, 209 (4.94%) were below the cutoff value of 20 mg/dL for dilute urine. Because there are many factors that can influence the urinary creatinine concentration, samples that have creatinine under the 20 mg/dL cutoff do not always implicate sample adulteration.     

Screening for drugs in forensic blood samples using EMIT urine assays

A screening method for the detection of drugs in haemolysed whole blood has been evaluated. Methanolic extracts of 300 forensic blood samples known to be positive or negative for drugs were analysed with EMIT d.a.u. assay kits for amphetamine, cannabinoids, opiates and benzodiazepines (the latter to analyse for diazepam and the main metabolite N-desmethyldiazepam). There were very few false positive results, except for the amphetamine assay in postmortem blood samples, where 9% were false positive. For amphetamine and cannabinoids a few false negatives were found, these were from samples with very low drug concentrations. No false negatives were found for opiates and diazepam. The present modification of the EMIT d.a.u. method seems to be a good method for screening of drugs in forensic blood samples, except for amphetamine in postmortem samples. The method is simple and requires only 0.5 ml blood.