Screening postmortem blood and tissues for nine classes [correction of cases] of drugs of abuse using automated microplate immunoassay

A commercially available enzyme-linked immunosorbant assay (ELISA) was evaluated as a screening procedure for the detection of nine classes of abused drugs in postmortem blood and tissue specimens. Specifically, postmortem blood, fluid and/or tissue homogenates were screened for amphetamine (AMP), methamphetamine (MET), barbiturates (BARB), benzodiazepines (BZD), cannabinoids (CNB), cocaine (benzoylecgonine; BE), morphine-specific (MOR), opiates (class; OPI), phencyclidine (PCP) and lysergic acid diethylamide (LSD) by ELISA and by coated tube radioimmunoassay (CTR) (BARB, BE, OPI, PCP, LSD) or double-antibody radioimmunoassay (DAR) (AMP/METH, BZD, CNB). Specimens that screened 'positive' by any method were confirmed and quantitated by gas chromatography/mass spectrometry (GC/MS). The only assay that appeared to perform less optimally than RIA was the MOR assay (five false negatives). However, this assay is very specific for free morphine while the GC/MS confirmation method provided a total morphine value. The OPI assay was more sensitive, producing fewer false negatives, and is recommended for broad class opiate screening. EIA is an adequate alternative to RIA for screening postmortem specimens, including blood and tissue, for nine major classes of drugs.     

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.     

Interpretation of postmortem digoxin levels: evaluating a "corrective factor" for postmortem blood digoxin concentration

Interpretation of postmortem serum digoxin levels is made difficult above all by a possible prefinal or postmortem rise in digoxin concentrations in the blood. To compensate for this postmortem increase, Eriksson et al. (1984) divided the level of postmortem digoxin in femoral venous blood by a factor of 1.5; in the opinion of these authors, postmortem digoxin levels still exceeding "therapeutic levels" after division by 1.5 are an index of digoxin overdose. The diagnostic value of this "correction factor" was investigated. In 56 cases with documented digoxin medication, samples of postmortem femoral venous blood were taken and the level of digoxin determined. In none of the cases had there been a clinical diagnosis of digoxin intoxication. Fifty percent of the measured values were above "therapeutic levels" (0.7 ng/ml to 2.2 ng/ml). Following division by 1.5, 20% of the cases still showed levels exceeding 2.2 ng/ml; the highest "corrected" value was 4.44 ng/ml. Taking into account the length of time between final dosage and death, individual differences in sensitivity to digitalis glycoside, and the complexity of ante- and postmortem dispersion processes, we concluded for the cases we studied that an (undetected) digoxin overdose was not even likely in those cases whose postmortem values after division by 1.5 lie above "therapeutic levels". The "correction factor" proposed by Eriksson et al. (1984) is only of limited diagnostic value; at best the "corrected" values can give an approximate indication of the corresponding antemortem serum digoxin concentrations. In particular, "corrected" values only a little above "therapeutic levels" could not confirm suspicion of an overdose with sufficient certainty.     

Screening analysis for medicinal drugs and drugs of abuse in whole blood using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS)-Toxicological findings in cases of alleged sexual assault

An ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) method for simultaneous screening of 46 medicinal drugs and drugs of abuse in whole blood was developed and validated. The method includes most of the commonly used and abused drugs such as amphetamines, cocaine, benzodiazepines, and opioids. Chromatographic separation of the targeted drugs was achieved using a Waters ACQUITY UPLC coupled to a Waters Micromass LCT Premier XE time-of-flight mass spectrometer. The total chromatographic run time was 13.5min injection to injection. The estimated method LOQ is in the range of 0.06-27ng/g, which is below the therapeutic levels for each of the drugs analyzed but LSD. The extraction recovery ranged from 6% to 197% with median value 95% and mean value 82%. Matrix effect ranged from 81% suppression to 29% enhancement of the signals compared to signals obtained in the absence of biological matrix. The method was tested on 55 authentic forensic toxicology samples confirming the same positive results as found using the routine analytical procedures as well as some additional compounds. Recently there has been considerable attention paid to drug-facilitated sexual assault and the toxicological findings in these cases. As part of a pilot study to investigate the prevalence of medicinal drugs, drugs of abuse, and alcohol in victims of alleged sexual assault, biological specimens were obtained from 167 victims being examined at the Sexual Assault Center in Aarhus, Denmark. The obtained blood samples were analyzed using the novel screening method supported by additional analyses for e.g. THC and alcohol. 124 victims reported they have been drinking alcohol prior to the assault (74%). Alcohol analyses revealed 59 positive findings (48%). 35 of the cases were found positive for one or more drugs excluding alcohol (21%). 20 of the victims reported they have been subject to a drug-facilitated sexual assault (12%). For the victims suspecting drug-facilitated sexual assault, the toxicological analyses revealed four positive for alcohol and nine victims were positive for one or more drugs, with six of the victims found positive for benzodiazepines or other drugs with sedative effects. It was notable that victims tested positive for medicinal drugs and drugs of abuse as well as victims of alleged drug-facilitated sexual assault in average underwent medical examination later than the whole study population.     

Stability of drugs in stored postmortem femoral blood and vitreous humor

The stability of 46 drugs in postmortem femoral blood stored for one year at -20 degrees C was investigated. The drugs included benzodiazepines, antidepressants, analgetics and hypnotics. For seven drugs we found a significant change in the concentration between the first and second analysis. Five substances; ethanol, desmethylmianserin, 7-amino-nitrazepam, THC and zopiclone showed a decrease in the concentration whereas the concentrations of two substances; ketobemidone and thioridazine increased. However, the changes observed were not of such an order that it would affect the interpretation in normal forensic casework. We also investigated the possible influence of potassium fluoride on the concentrations of the 46 drugs in vitreous humor after storage for one year. For two substances, ethanol and zopiclone, there were significantly lower concentrations in the samples without potassium fluoride. Furthermore, we also studied the correlation between the concentrations in femoral blood and vitreous humor. For 23 substances there was a significant difference between the concentrations in the vitreous humor and femoral blood. Significant correlations between the concentrations in these two specimens were found for 23 substances, indicating that vitreous humor can be an alternative specimen when blood samples are not available, provided that such correlation exists for the particular substance. Statistical analysis also revealed a correlation between the degree of protein binding of the different drugs and percentage of vitreous/femoral blood concentrations.     

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.     

Optimization and validation of CEDIA drugs of abuse immunoassay tests in serum on Hitachi 912

Due to sensitive limits of detection of chromatographic methods and low limit values regarding the screening of drugs under the terms of impairment in safe driving (§ 24a StVG, Street Traffic Law in Germany), preliminary immunoassay (IA) tests should be able to detect also low concentrations of legal and illegal drugs in serum in forensic cases. False-negatives should be avoided, the rate of false-positive samples should be low due to cost and time. An optimization of IA cutoff values and a validation of the assay is required for each laboratory. In a retrospective study results for serum samples containing amphetamine, methylenedioxy derivatives, cannabinoids, benzodiazepines, cocaine (metabolites), methadone and opiates obtained with CEDIA drugs of abuse reagents on a Hitachi 912 autoanalyzer were compared with quantitative results of chromatographic methods (gas or liquid chromatography coupled with mass spectrometry (GC/MS or LC/MS)). Firstly sensitivity, specificity, positive and negative predictive values and overall misclassification rates were evaluated by contingency tables and compared to ROC-analyses and Youden-Indices. Secondly ideal cutoffs were statistically calculated on the basis of sensitivity and specificity as decisive statistical criteria with focus on a high sensitivity (low rates of false-negatives), i.e. using the Youden-Index. Immunoassay (IA) and confirmatory results were available for 3014 blood samples. Sensitivity was 90% or more for nearly all analytes: amphetamines (IA cutoff 9.5 ng/ml), methylenedioxy derivatives (IA cutoff 5.5 ng/ml), cannabinoids (IA cutoff 14.5 ng/ml), benzodiazepines (IA cutoff >0 ng/ml). Test of opiates showed a sensitivity of 86% for a IA cutoff value of >0 ng/ml. Values for specificity ranged between 33% (methadone, IA cutoff 10 ng/ml) and 90% (cocaine, IA cutoff 20 ng/ml). Lower cutoff values as recommended by ROC analyses were chosen for most tests to decrease the rate of false-negatives. Analyses enabled the definition of cutoff values with good values for sensitivity. Small rates of false-positives can be accepted in forensic cases.     

Isolation of drugs from blood and tissues with XAD-2 bags

Nylon bags containing 2-g portions of Amberlite XAD-2 resin were used for systematic analysis of drugs in biosamples. The procedure requires 10 or less grams of material, two XAD-2 bags, and enables rapid and economical isolation of most common drugs. The method was demonstrated on autopsy blood spiked with 19 of the most common drugs, and was routinely used in cases of fatal and non-fatal poisoning. The eluates were clean and suitable for direct gas chromatographic and ultraviolet spectrophotometric analysis.The procedure used appeared more convenient than XAD-2 column extraction procedures. Classic solvent extraction methods were usually less efficient.     

Screening and quantification of antipsychotic drugs in human brain tissue by liquid chromatography-tandem mass spectrometry: application to postmortem diagnostics of forensic interest

A quantitative LC-MS/MS method has been developed for the simultaneous determination of 17 antipsychotic drugs in human postmortem brain tissue. Sample preparation was performed using Hybrid Solid Phase Extraction-Precipitation technology for the removal of endogenous protein and phospholipid interferences. The chromatographic separation was performed for 16 min on a C8 column, which used a gradient elution of formate ammonium and acetonitrile, and a flow rate gradient. Triple quadrupole mass spectrometry was employed to generate tandem mass spectrometric (MS/MS) data of the target analytes to select the ion m/z signals. Quantitation of the analytes was performed by operating in the dynamic multiple reaction monitoring (dMRM) mode using an electrospray ionization interface. Calibration curves prepared in the spiked brain tissue were linear in the range 20-8000 ng/g (r(2)>0.993) for all drugs (except olanzapine). Within- and between-day coefficients of variation were lower than 25% for all drugs at the LOQ. The LOQ in the matrix ranged between 2 ng/g and 80 ng/g. The method was successfully applied to the unequivocal identification and accurate quantification of antipsychotic drugs in human postmortem brain tissues: therefore, this method can be used in forensic investigations.     

Evaluation of a photodiode array/HPLC-based system for the detection and quantitation of basic drugs in postmortem blood.

A systematic analytical approach has been developed for liquid chromatography determination of a number of basic drugs in postmortem blood. Using selective extraction, that is, back extraction into 0.2N sulfuric acid and 6N hydrochloric acid after the initial extraction with toluene under basic conditions (from 2 mL of blood), basic and weakly basic drugs, such as propranolol and diazepam, can be simultaneously quantitated and identified with a high degree of confidence. A microcomputer-based photodiode array detector was used to evaluate peak purity and facilitate peak identification. An automatic library search was performed at the end of each analysis using the system software. The method was validated for within-day and between-day precision for ten basic drugs at two concentrations. The coefficient of variation for the between-day precision was less than 8.7%. Accuracy of the assay was tested at four concentrations using linear regression analysis. The coefficients of determination (r2) for all ten drugs were greater than 0.99, and their slopes were close to unity. The chromatographic conditions developed are suitable for the screening of several basic, acidic, amphoteric, and neutral drugs. Retention data and ultraviolet spectral data for 119 drugs on two reversed-phase columns, using acidic mobile phases, are also presented.     

Immunofluorescence detection of drugs in postmortem tissues: a new technique with potential for assessment of drug influence in cause of death

This report describes a new technique, immunofluorescence, for the detection and possible characterization of drug content in postmortem tissues. By using antisera generated against a drug-protein conjugate, the stabilization of tissue-sequestered drug is accomplished by incubation of fresh frozen sections of tissue with dilute solutions of rabbit anti-drug antibodies. Secondary incubation with a fluorescence-labeled anti-rabbit immunoglobulin labels these points of sequestration. Tissue sections so stained are examined by fluorescence microscopy. In studies with rats given graded doses of morphine sulfate, there were discernible differences in tissue binding of morphine in brain sections from animals treated "therapeutically," fatally, and chronically. Extension of these studies to human autopsy material is anticipated and potential problems are discussed. This technique offers the forensic toxicologist the potential for evaluating the drug content of tissues in situ.     

An evaluation of fused silica capillary columns for the screening of basic drugs in postmortem blood: qualitative and quantitative analysis

Fused silica capillary columns (Durabond) have been evaluated for the screening of more than 100 basic drugs in postmortem blood samples. The combination of these columns, nitrogen-phosphorus detectors, and SKF-525A (internal standard) allows for the simultaneous screening and quantitation of several basic drugs such as amphetamines, amitriptyline, and codeine. Approximately 2000 blood samples have been analyzed by this procedure. The use of capillary columns results in excellent baseline stability and this, together with an autosampler and data system, enables unattended overnight operation. "Double peaking" associated with splitless injection can be a problem as can sensitivity for some of the polar drugs; however, with the extraction procedure described and the equipment used, the screening of blood for basic drugs is improved when compared with packed column technology.     

Guidelines for postmortem protocol for ocular investigation of sudden unexplained infant death and suspected physical child abuse

Postmortem examination is a cornerstone in identifying the cause of unexplained sudden death in children. Even in cases of suspected or known abuse, an autopsy may help characterize the nature of the abuse, which is particularly important in the forensic autopsy of children in the first 3 to 4 years of life when inflicted neurotrauma is most common. Forensic examinations are vital in cases that might otherwise be diagnosed as sudden infant death syndrome. The ocular autopsy in particular may demonstrate findings that were not appreciated on antemortem clinical examination. This protocol for postmortem examination of the eyes and orbits was developed to promote more consistent documentation of findings, improved clinical and forensic decision making, and more replicable and coherent research outcomes.     

Screening for the presence of drugs in serum and urine using different separation modes of capillary electrophoresis

Capillary electrophoresis (CE) is a modern separation technique that has some distinct advantages for toxicological analysis, such as a high efficiency, fast analysis, flexibility, and complementary separation mechanisms to chromatographic methods. CE can be applied in various modes, which each have a different separation mechanism or selectivity. The most common mode is capillary zone electrophoresis (CZE), in which charged analytes migrate in a buffer under the influence of an electric field. In micellar electrokinetic chromatography (MEKC), micelles are added to the buffer which interact with the analytes. MEKC can also be used for the separation of neutral compounds. In non-aqueous CE (NACE), the aqueous buffer is replaced by a background of electrolytes in organic solvents. A sample that needs to be screened can easily be analyzed subsequently by these CE modes using the same instrumentation.The aim of the study was to develop procedures for the analysis of basic and acidic drugs in serum and urine using CZE, MEKC, and NACE. A test mixture that consisted of six basic and six acidic compounds was used to study the separation behavior of five CE methods. The results showed that three methods (based on CZE, MEKC, and NACE) were suitable for the analysis of basic compounds and three methods (based on CZE and MEKC) for the analysis of acidic compounds.For the extraction of analytes from serum and urine, a solid-phase extraction (SPE) and a liquid-liquid extraction (LLE) method were compared. Both SPE and LLE methods provided clean extracts after extraction of the basic compounds from serum and urine. The extracts of acidic compounds contained more matrix interferences, especially for urine. The SPE method had some advantages compared to LLE, as it lead to cleaner extracts and higher peaks, and as it elutes basic and acidic compounds in one fraction.The potentials and pitfalls of the various methods for screening purposes in analytical toxicology are discussed.     

A micromethod for the isolation of drugs from blood using amberlite XAD-2

The extraction of drugs from small blood samples (1 ml or less) for subsequent quantitative determination is described. Isolation was carried out by adsorption of the drugs to Amberlite XAD-2 resin utilizing a batch procedure that enabled the simultaneous extraction of up to 200 samples in approx. 5 hours. A new desorption technique yielded extracts of high purity that could be used directly for gas chromatographic or radioimmunological determinations, even if hemolyzed or putrid blood was to be examined. The following 26 substances were quantitated after addition to postmortem blood speciments at concentrations of 1-10 microgram/ml: tilidine, diphenhydramine, dibenzepine, imipramine, chlorpromazine, amphetamine, pentazocine, phenacetin, methaqualone, meprobamate, parathion, diazepam, digoxin, beta-methyldigoxin, carbromal, glutethimide, amobarbital, pentobarbital, cyclobarbital, phenobarbital, diphenylhydantoin, carbutamide, tolbutamide, glycodiazin, tolazamide and chlorpropamide. Thereby recoveries of 60-100% could be achieved. The reproducibility of the procedure was satisfactory as demonstrated by coefficients of variation of 3.7-8%.     

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.