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.     

An express immunological method for detection of human seminal plasma.

Monoclonal antibodies (Mabs) against human seminal plasma (HSP) were produced and during screening procedures dissociation constants of the antigen/antibody complexes were determined. Mab 1E5 was selected for further studies because of its high reactivity in an enzyme-linked immunoassay (ELISA) and high affinity for its corresponding antigen. The specificity of Mab 1E5 was checked in absorption ELISA with human organ extracts and some biological secretions. It was established that the 1E5-corresponding epitope was a thermostable peptide moiety which could be detected in HSP, only. This monoclonal antibody was used for the development of an express method for detection of human semen. The assay was applied for screening of 57 cases of suspected rape. A complete correlation was found between the results obtained by the proposed test and by routine microscopic methods. The newly designed immunoassay is reliable, it is easily performed and it is less time-consuming.     

Drug screening in urine--common pitfalls in measurement and interpretation

Nowadays immunoassays are used for the rapid screening of drugs world-wide. Despite the fact that they are a highly valuable tool for the test of legal and illicit drugs, there is a non-negligible risk of false positive and false negative findings, and many pitfalls must be taken into account when using these tests in an uncritical manner and without valid confirmation procedures. In accordance with the committee for "problems of threshold values for medical and addictive drugs" set up by German scientific societies (in particular GTFCh, DGRM and DGVM), immunoassays are recommended as a useful tool for screening. But confirmation analysis with conclusive methods (GC/MS or LC/MS) is indispensable for valid substance identification, discrimination between active and inactive metabolites, detection of escort substances and accurate determination of concentrations in body fluids.     

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC–MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-μg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010–2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC–MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 μg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.     

Evaluation of two immunoassay procedures for drug testing in hair samples

A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively.     

新精神活性物质检验鉴定面临的挑战和对策

新精神活性物质更新快,种类繁多,缺乏国际条约的监管,列入联合国管控目录困难,加之一些国家和国际社会对大麻等毒品管控降低,给我国在新精神活性物质类毒品的监管带来困难.具有成瘾性和社会危害性的新精神活性物质才能成为列管对象,毒物分析技术人员在列管程序中扮演极为重要的角色.我国对新精神活性物质的监管法规规定了相关列管程序,毒...     

The detection of cocaine in hair specimens using micro-plate enzyme immunoassay.

The analysis of hair for drugs of abuse is becoming increasingly popular and is under consideration by the Division of Health and Human Services as a possible alternative or adjunct to urinalysis in workplace programs. The detection of cocaine in human hair using a commercially available micro-plate enzyme immunoassay is described for the first time. Sample size and incubation time were the major variables in the optimization of the method. In order to validate the procedure, the method was applied to 105 consecutive hair samples routinely received into our laboratory. The samples were simultaneously analyzed by the Micro-Plate immunoassay (EIA), as well as our current fluorescence polarization immunoassay (FPIA) procedure and gas chromatography-mass spectrometry (GC/MS). The sensitivity of the EIA and FPIA assays were 75% and 67.8% respectively; specificity 97.4% and 80.5% respectively; and efficiency 91.4 and 77.1% respectively. The Micro-Plate EIA was shown to be a valid alternative to other immunoassay screening methods for the detection of cocaine in hair by demonstrating increased sensitivity, specificity and efficiency over our current technique.     

Comparison of the MTP immunoassay with EMIT in blood screening for drugs

We compared the MTP immunoassay with EMIT for the screening of drugs of abuse (opiates, cannabinoids, cocaine metabolites and amphetamines) in whole blood samples. These blood samples were obtained from the German police, when driving under the influence of drugs of abuse was suspected. For screening with the MTP immunoassay 25 microliters of serum or blood (without any pretreatment) was pipetted into the wells of the microtiter plates and the procedure was followed as described. Prior to screening with a Cobas Mira and EMIT reagents, the samples were treated with acetone to precipitate serum proteins. The cutoff for all drugs of abuse was set at 10 ng per ml of serum or blood. In most cases there was a good agreement between the negative and positive results of the two screening assays. The agreement between the two assays in the detection of opiates and cocaine was 91% and 93%, respectively, and for cannabinoids and amphetamines approximately 80%. The MTP immunoassay was more sensitive than EMIT for the detection of cannabinoids--but at the same time the MTP immunoassay was less specific. Both screening assays have a sensitivity of 100% for the detection of opiates and cocaine, but the specificity of the EMIT--also for opiates--was substantially lower. The MTP immunoassay has in respect to amphetamines a very high sensitivity, whereas the sensitivity of EMIT for amphetamines is inacceptable due to losses during sample preparation. The specificity of MTP immunoassay for amphetamines is not optimal, because a relatively large amount of samples tested false-positive for amphetamines at the cutoff of 10 ng/ml. In summary the MTP immunoassay, although not automated, performs well in comparison with EMIT, especially if the sample preparation for EMIT testing ist considered.     

Comparison of results for quantitative determination of morphine by radioimmunoassay, enzyme immunoassay, and spectrofluorometry.

The quantitative results (accuracy and precision) for determination of opiates by radioimmunoassay (RIA), enzyme immunoassay (EMIT), and spectrofluorometry on split samples are compared. A variety of physiological samples were studied, including random urine from a methadone maintenance clinic and postmortem urine, blood, bile, brain, and lung tissue from heroin-induced or heroin-related deaths. The opiate concentrations detected by the two immunoassay methods were in good agreement with each other in the absence of interfering substances which are believed to react with the antimorphine antibodies. The immunoassay results were in agreement within the relative standard deviation with the fluorometry results in 55% of the urine samples and 80% of the blood samples. The immunological methods are superior to fluorometry for quantitation of morphine in urine samples due to quenching interferences in fluorometry from urine. They were comparable to fluorometry for quantitation of morphine in blood samples.     

The detection of cotinine in hydrolyzed meconium samples.

To date, the screening of meconium for the determination of tobacco exposure in newborns has proven difficult. It was hypothesized that cotinine forms reversible Schiff base bonds with free amino functions on proteins, therefore, hydrolysis of meconium would be necessary for the detection of 'free' cotinine. One-hundred-and-two (102) meconium samples received into our laboratory were extracted using a routine non-hydrolysis screening procedure for drugs of abuse. Separate aliquots of the specimens were hydrolyzed and re-extracted according to the same procedure. The results of the two methods were compared using a highly specific cotinine micro-plate enzyme immunoassay procedure (EIA). Of the non-hydrolyzed samples, 33% were positive for cotinine, while 79% of the hydrolyzed samples were cotinine-positive. Common drugs of abuse did not interfere with the analysis. Micro-plate EIA provides a rapid, simple and reliable screening method for the determination of cotinine in meconium following hydrolysis and extraction. In general, the meconium specimens received into our laboratory are from newborns considered to be at risk for post-natal problems due to suspected drug and/or alcohol abuse during pregnancy.     

Hair analysis by immunological methods from the beginning to 2000

Immunoassays for hair testing must satisfy three requirements: (1) They must have cross-reactivity with parent drug and lipophilic metabolites actually found in hair (2) they must not experience interference from the dissolved hair matrix and (3) they must be titered for cutoffs appropriate to the drug concentrations found in hair. Because the analytes found in hair after drug use are generally the parent drug or its lipophilic metabolites, immunoassays developed and intended for urine testing are not suitable for hair. Immunoassays whose antibodies are bound to a solid support, such as coated-tube radioimmunoassay or coated-plate ELISA tests, experience less matrix interference than those which use other means of separation of bound and free fractions. Homogenous assays are not suitable for hair testing because the hair matrix frequently interferes in the detection of the signal. Historically radioimmunoassays for drugs of abuse were first used for detecting drugs in hair. Currently ELISAs and coated-plate 96 well microplate EIAs are employed for screening hair digests or extracts for drugs. The optimum cutoffs for immunoassays for drugs in hair should be chosen based on the analyte concentration which produces the fewest false positive or false negative results when applied to tests of hair from known users and non-users of drugs. A hair immunoassay test at these cutoffs should have a sensitivity and specificity of better than 90%. The predictive value of the test will depend on the prevalence of drug use in the tested population. Cutoffs or decision thresholds for immunoassays used for screening for drugs should not be at the limit of detection of the assay because that produces a very large incidence of false positives. Because immunoassays are ligand-binding assays, they have a short range of linearity with low precision at both ends of the range. In the future, immunoassays will continue to be used for screening hair and other matrices for drugs of abuse because they provide rapid, inexpensive automated procedures for separating negative specimens from those which are suspected of containing drugs. For forensic purposes, all positive results must be confirmed by an independent analysis using a procedure based on a different property of the analyte. An immunoassay test should not be confirmed by a second immunoassay test but by a chromatographic test performed on a different dissolved or extracted aliquot of the original specimen.     

Determination of lysergic acid diethylamide in body fluids by high-performance liquid chromatography and fluorescence detection--a more sensitive method suitable for routine use

A new method for determination of lysergic acid diethylamide (LSD) in body fluids by high-performance liquid chromatography and fluorescence detection was developed based on previously published methods. The new method is suitable for confirmation of samples tested positive by immunoassay, avoiding loss of LSD by absorption to surfaces. The reduced loss of LSD results in improved sensitivity. This is achieved by adding ethylene glycol to the samples, which cover glass surfaces. This principle can similarly be used to improve analysis of other drugs. Body fluids for analysis included urine and whole blood. An internal standard was applied for quantification of LSD. The new method offers satisfying precision data and has a detection limit of less than 0.05 ng/nL.     

Significantly increased detection rate of drugs of abuse in urine following the introduction of new German driving licence re-granting guidelines

In this paper we present the first assessment of the new German driving licence re-granting medical and psychological assessment (MPA) guidelines by comparing over 3500 urine samples tested under the old MPA cut-offs to over 5000 samples tested under the new MPA cut-offs. Since the enzyme multiplied immunoassay technique (EMIT) technology used previously was not sensitive enough to screen for drugs at such low concentrations, as suggested by the new MPA guidelines, enzyme-linked immunosorbent assay (ELISA) screening kits were used to screen for the drugs of abuse at the new MPA cut-offs. The above comparison revealed significantly increased detection rates of drug use or exposure during the rehabilitation period as follows: 1.61, 2.33, 3.33, and 7 times higher for 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), morphine, benzoylecgonine and amphetamine respectively. The present MPA guidelines seem to be more effective to detect non-abstinence from drugs of abuse and hence to detecting drivers who do not yet fulfil the MPA requirements to regain their revoked driving licence.     

Benzodiazepine findings in blood and urine by gas chromatography and immunoassay

Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.     

STATEWIDE SPECIAL EDUCATION SURROGATE PARENT PROGRAMS: ENSURING QUALITY ADVOCACY TO ALL FOSTER CHILDREN WITH SPECIAL EDUCATION NEEDS

The Individuals with Disabilities Education Improvement Act (IDEIA) protects foster children's rights to have a special education decision maker. For foster children who do not have a natural or adoptive parent or a responsible adult in their life to take on this role, IDEIA requires that a special education surrogate parent be appointed by appropriate procedures. Under IDEIA, these procedures are delegated to the states. Each state must ensure that local education agencies (LEAs) delineate methods for recruiting and maintaining a pool of available special education surrogate parents. Due to differing state laws and LEA procedures, there are many discrepancies in the quality and availability of special education surrogate parents. To combat these problems, this Note proposes principles for administrative regulations establishing statewide special education surrogate parent programs by examining existing statewide programs. Administered through a state's Department of Education in collaboration with child welfare agencies, statewide special education surrogate parent programs guarantee well‐qualified decision makers who will advocate for all children eligible for special education services.     

The determination of drugs of abuse in whole blood by means of FPIA and EMIT-dau immunoassays--a comparative study

Six groups of common drugs of abuse (cannabinoids, benzoylecgonine, opiates, barbiturates, benzodiazepines and amphetamines) were determined in whole blood after acetone precipitation, using enzyme multiplied immunoassay (EMIT dau) and fluorescence polarisation immunoassay (FPIA--Abbott TDx and ADx) methods. Both methods, designed primarily for urine, allowed the determination of all above mentioned class of drugs but amphetamine. Only 1 ml of a pre- or postmortem blood sample was needed. The sensitivity of cannabinoids determination was higher by FPIA. The FPIA method gave more precise results, particularly in the case of autopsy blood. The method was applied for drug screening in autopsy and police blood samples. The results (both positive and negative) were in agreement with those obtained with chromatographic methods.     

Development of a specific fragmentation pattern-based quadrupole-Orbitrap™ mass spectrometry method to screen drugs in illicit products

Over the past decade, illicit drugs have been founded in marketed products, which pose a risk to public health. In particular, newly designed analogues synthesized by chemical modification of parent compounds to avoid detection by authorities are frequently detected worldwide. Although many analytical methods for determination of drugs have been reported, analytical methods using high-resolution mass spectrometry, which has the advantage of rapid screening and accurate identification of new substances, are necessary to control illicit drugs in marketed products. In this study, a rapid analytical method using an Orbitrap™ mass spectrometer for identification of illicit drugs in marketed products was developed. The 32 drugs were classified as benzodiazepine-, synthetic cannabinoid-, amphetamine- and benzylpiperazine-type drugs according to their chemical structures, and from their fragmentation patterns in tandem mass spectrometry spectra of an established method. The method validation gave a limit of detection of 0.06–5.30 ng/mL and a limit of quantification of 0.18–16.50 ng/mL, high linearity (R² > 0.994) and mean recoveries of spiked matrix-blank samples ranging from 83.7% to 117.1%. Approximately 71% of 21 samples collected over 3 years were found to individually contain one of four types of benzodiazepines or two different synthetic cannabinoids. In one case, levels as high as 827.2 mg/g were measured suggesting adulteration at high levels, which suggests that potential illicit products containing drugs should be regularly screened to protect public health.     

The general toxicology unknown. I. The systematic approach

The general toxicology unknown often presents challenges and interests to toxicologists. A systematic analytical approach to search for drugs or poisons is presented here. The preliminary screening analyses were as follows: alcohol by gas chromatography (GC), ethchlorvynol colorimetric analysis, enzyme multiplied immunoassay technique (EMIT), basic drug screening by GC, and neutral and weakly acidic drug screening by GC. Other additional analyses were performed depending on the special circumstance of each individual case and the results of these preliminary analyses. Positive findings were confirmed by computerized gas chromatography/mass spectrometry when practical. Quantitation was performed by GC whenever possible.     

Spatially offset Raman spectroscopy (SORS) for the analysis and detection of packaged pharmaceuticals and concealed drugs

Spatially offset Raman spectroscopy (SORS) is a powerful new technique for the non-invasive detection and identification of concealed substances and drugs. Here, we demonstrate the SORS technique in several scenarios that are relevant to customs screening, postal screening, drug detection and forensics applications. The examples include analysis of a multi-layered postal package to identify a concealed substance; identification of an antibiotic capsule inside its plastic blister pack; analysis of an envelope containing a powder; and identification of a drug dissolved in a clear solvent, contained in a non-transparent plastic bottle. As well as providing practical examples of SORS, the results highlight several considerations regarding the use of SORS in the field, including the advantages of different analysis geometries and the ability to tailor instrument parameters and optics to suit different types of packages and samples. We also discuss the features and benefits of SORS in relation to existing Raman techniques, including confocal microscopy, wide area illumination and the conventional backscattered Raman spectroscopy. The results will contribute to the recognition of SORS as a promising method for the rapid, chemically specific analysis and detection of drugs and pharmaceuticals.