Detection of drugs using XAD-2 resin. II:Analysis of liver in medical examiner's cases

Liver tends to concentrate drugs in quantities generally higher than those found in blood or other body compartments. This fact as well as the general availability of liver in postmortem cases makes it an important specimen for comprehensive toxicologic investigation. A scheme for the analysis of liver for drugs with tissue hydrolysis, XAD-2 resin extraction, and TLC has been developed and the parameters affecting recovery have been studied. The hydrolysis of liver specimens at various pH conditions resulted in an improved recovery for morphine by using pH 2 (2N hydrochloric acid). Recoveries of barbiturates, codeine, and meperidine were essentially the same at pH 2 and pH 3. A considerable loss (22 to 55%) was observed for four drugs (pentobarbital, morphine, codeine, and meperidine) as a result of drug binding to the tissue pellets during the process of centrifuging the liver homogenates. This method is recommended as a comprehensive screening procedure for drugs in liver tissue. For quantitative purposes, however, it is necessary to determine a correction factor for all the losses occurring at the various steps of the procedure. This procedure compared favorably with other procedures for liver analysis reported in literature.     

Detection of drugs using XAD-2 resin. I:Choice of resin, chromatographic conditions, and recovery studies

Amberlite XAD-2, a nonionic polystyrene divinylbenzene resin, was first used for the analysis of drugs in urine and a number of reports have described the development at optimal conditions for extraction, including type of resin columns, pH conditions, and eluting solvents. XAD-4 and XAD-7 resins were compared to the similarly structured XAD-2 resin and no significant advantage over the XAD-2 resin for drug screening was observed. A quantity of 5 to 6 g of resin was found to have sufficient capacity for the extraction of 200 ml of pentobarbital solution (1 mg/100ml). A column flow rate of approximately 15 ml/min (gravitational flow) was sufficient for analysis and slower rates were not more efficient. A mixture of ethyl acetate and 1,2-dichloroethane (3:2) was found to give best overall recovery (66 to 94%) of drugs, the resulting extracts being reasonably free of interfering substances. A pH value of 8.5 is recommended as optimum for comprehensive analysis of acidic and basic drugs. Recovery studies were conducted on spiked samples to determine drug losses occuring during various steps in the XAD-2 extraction procedure for four acidic (amobarbital, secobarbital, pentobarbital, and phenobarbital) and four basic (morphine, codeine, meperidine, and methadone) drugs. A relatively small amount (0 to 5%) of the drugs was not adsorbed by the resin and amounts varying from 6 to 40% failed to be desorbed by the eluting solvent. Additional losses occurred during the removal and analysis of TLC spots. Recovery of drugs from aqueous solutions analyzed with the XAD-2 resin were compared to recoveries reported in the literature with other XAD-2 resin methods for the extraction of drugs from urine. Recovery of phenobarbital, morphine, and codeine improved by 4 to 23% while recoveries of amobarbital, pentobarbital, secobarbital, methadone, and meperidine were 4 to 28% less efficient when compared to literature data.     

The differential elution of drugs from XAD-2 resin.

Biological fluids and tissue extracts prepared according to a previously published method were passed through a column of Amberlite XAD-2 resin for removal of drugs. The differential elution of the adsorbed drugs from the resin was performed by sequential elution of the drugs in four steps. The column was first washed with 30 ml of 0.05M sodium acetate buffer, pH 4.55. Drugs exhibiting acidic or neutral characteristics were then eluted from the column with 100 ml of chloroform in 20-ml aliquots. The column was then washed with 30 ml of 0.1M potassium carbonate, which was discarded. Drugs exhibiting basic characteristics were then eluted from the column with 100 ml of chloroform:isopropanol (3:1) in 20-ml aliquots. Sensitivity of drug detection with this method by thin-layer chromatography was 0.5 mug/ml in a 10-ml sample for nearly all drugs tested.     

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%.     

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.     

A rapid screening procedure for drugs and poisons in gastric contents by direct injection-HPLC analysis

A high performance liquid chromatographic method for toxicological drug screening of gastric content has been developed. The samples were diluted (1:3-1:30) in 0.01 N hydrochloric acid and injected into a reverse phase column for separation by gradient elution. Mobile phase consisted of solvent A (acetonitrile/water 90:10, 0.01 M sodium dodecylsulphate, 0.5% v/v glacial acetic acid) and solvent B (water/acetonitrile 90:10, 0.01 M sodium dodecylsulphate, 0.5% v/v glacial acetic acid); the gradient was programmed from 20 to 80% A in 30 min. The flow was kept constant at 1.5 ml/min. Two home-made internal standards, butyrylsalicylic acid and diacetyltubocurarine with retention times of 5.6 and 21.4 min, respectively, were used. Drugs are identified by matching their relative retention times and UV spectra (200-400 nm) with those contained in a home made library of more than 340 reference compounds (9 analgesics, 22 antidepressants, 30 antihistamines, 14 antihypertensives, 21 antirheumatics, 15 beta-blockers, 9 bronchodilators, 10 Ca antagonists, 14 diuretics, 26 neuroleptics, 25 tranquilizers, and other significant xenobiotic compounds). The fluorometric (FLD) emission spectrum (280-700 nm; excitation wavelength, 230 nm) was used as a further identification. At 50mg/l analyte concentrations, the injection of gastric content after dilution (1:3) produced S/N ratios in the range 8-140. The method is simple, rapid, rather inexpensive and proved to be a useful means of investigation if used in combination with GC-MS screening in blood. On the other hand, the system suffers from a relatively limited sensitivity for compounds with a low UV absorption and from interferences due to the presence in the matrix of some highly UV- and FL-responsive compounds (e.g. tryptophan).     

A semiautomated radioimmunoassay for mass screening of drugs of abuse.

A rapid, semiautomated radioimmunoassay system for detection of morphine, barbiturates, and amphetamines is described. The assays are applicable to large drug abuse screening programs. The heart of the system is the automatic pipetting station which can accomplish 600 pipetting operations per hour. The method uses 15 to 30 mul or urine for the morphine and barbiturate assay and 100 mul for the amphetamine and combined morphine/barbiturate assays. A number of other drugs were tested for interference with the assays and the results are discussed.     

A single-column procedure on Bond Elut Certify for systematic toxicological analysis of drugs in plasma and urine.

A single-column solid-phase extraction procedure was developed for the screening of acidic, neutral, and basic drugs from plasma. The recoveries of all 25 tested drugs exceeded 82%. After the plasma had been diluted with phosphate buffer (pH 6.0), the drugs were extracted using a single Bond Elut Certify column. The acidic and most of the neutral drugs were eluted by acetone/chloroform (1:1) and the basic drugs were eluted by 2% ammoniated ethyl acetate. Some neutral drugs appeared in both fractions. The two fractions were collected separately and evaporated until approximately 100 microL of solvent remained in the tube. Both fractions were analyzed separately on a gas chromatograph equipped with a wide-bore capillary column and a flame ionization detector. The procedure could also be used for urine samples.     

A routine screening method for the major metabolite of methyl phenidate in the urine of greyhounds

A qualitative urine screening procedure for the determination of the major urinary metabolite of methyl phenidate, α-phenyl-2-piperidine acetic acid (ritalinic acid), has been devised.The method involves a salting-out extraction, conversion of the metabolite to the parent drug using methanol-hydrogen chloride, and simultaneous two-column gaschromatographic detection. Final confirmation of the presence of the drug is by two-system thin-layer chromatography. The method is suitable for the detection of illicitly administered methyl phenidate in greyhounds.     

The evaluation of five electrophoretic phenotyping systems for routine screening of bloodstains

The performance of the polymorphic marker systems group-specific component (GC), phosphoglucomutase-1 (PGM), alpha-2-HS-glycoprotein (A2HS), haptoglobin (Hp), and erythrocyte acid phosphatase (EAP) was evaluated on control bloodstains. The major factors considered were: sensitivity of the test system; stability of the marker; laboratory economics of each test; and distinguishing power (Dp) of the system. GC was considered to be the most suitable marker for routine screening because of its high stability and Dp, and the sensitivity of the immunoblotting detection method. PGM and A2HS were the next most valuable markers followed by Hp. EAP could only be considered useful when large amounts of relatively fresh bloodstain were available.     

Juvenile arrest rates for burglary: A routine activities approach

Juveniles comprise a substantial portion of the offenders arrested for burglary in the United States each year. Using a Hierarchical Multivariate Linear Model, the current research examines juvenile burglars, by gender, utilizing a routine activities approach. This analysis was performed using data on the thirty five largest cities in Texas, between 1990 and 2004. Increased incidents of juvenile arrests for burglary, in both genders, occurred where there were high levels of poverty and low levels of female headed households. Juvenile males appeared to be arrested more for burglary in areas where there were high levels of unemployment and non-white individuals, while juvenile females were arrested for burglary in places where there were higher numbers of males between the ages of 19 and 24 years. Results suggest that the current measures of routine activities theory better explains variation in juvenile male arrests for burglary.     

Victimization risks and routine activities: A theoretical examination using a gender-specific and domain-specific model

This paper examines female and male victimization risks in general and in three domains: home, work, and leisure/public. In doing so, the analysis is based on a popular victimization model: the routine activities/lifestyle theory of victimization. There are several critiques of the routine activities/lifestyle theory research at present. Most tests of this theory use a sample of victims that does not distinguish between specific populations. Further, research on victimization risks needs domainspecific models of victimization because lifestyle can encompass a large variety of behaviors in several different settings, all of which do not have the same risk of victimization (Lynch, 1987). Analyses using data from the National Crime Survey’s Victim Risk Supplement (1983) indicate the importance of analyzing specific populations and domains in any evaluation of routine activities/lifestyle victimization theory because the determinants of victimization are not identical between men and women or between the domains of home, work, and leisure/public.     

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.     

Application of a simple extraction procedure using aqueous ammonia to the analysis of basic drugs in blood by gas chromatography

A simple and reliable previously reported extraction procedure has been extended to the analysis of a wide range of basic and neutral drugs in postmortem blood and other tissues. The method employs 200 microliters of blood treated with water and ammonia and extracted with diethyl ether. The concentrated extract is generally sufficiently clean for direct analysis by gas chromatography and high pressure liquid chromatography. Recovery, precision and linearity data are presented for selected drugs.     

Drugs in motorists traveling Swedish roads: on-the-road-detection of intoxicated drivers and screening for drugs in these offenders

This paper deals with the police officer's or police doctor's ability to find drivers under the influence of drugs. We have also studied whether the protocol on the driver's previous histories of drug intake is useful for directing the chemist in his analytical approach to revealing intoxicants in the suspects' body fluids. A comprehensive procedure for screening traffic-hazardous drugs in the urine was found necessary and is described. By using this method, we have studied the incidence of drunken drivers with detectable medicinal or illicit agents. The results demonstrate that 91% of those drivers found by the officer or doctor of the police to be on intoxicants other than ethanol, carried some kind of traffic-hazardous drug in their body fluids, and that the doctor was a better judge than the police in identifying these offenders. By using a series of chemical methods for drug screening, we found that every third driver suspected of drunken driving due to ethanol, but not to other intoxicants, held some kind of a traffic-hazardous drug substance in his urine; benzodiazepines and cannabinoids were the most common findings. The data imply that 34% of these suspects revealed their intakes of traffic-dangerous intoxicants. We conclude that the judgements of both the officer and doctor of the police are needed for an efficacious detection of drivers under the influence of drugs. Moreover, the results infer that the chemist has to screen for intoxicants to reveal these in a suspect driver. We also conclude that drugs, particularly the benzodiazepines or cannabinoids, may be commonly encountered in drunken drivers, suspected of being inebriated by ethanol but no other toxicants.     

A proposal for cadaver organ procurement: routine removal with right of informed refusal

In order to alleviate the shortage of vital organs for transplant, we propose a system of routine removal of cadaver organs with an option of informed refusal by family. Unless an individual registered an objection during his or her lifetime, or unless the family objected to the procedure, clinicians would be permitted routinely to salvage vital organs for transplant. Our proposal charts a middle path between the current ineffective policy based on "encouraged voluntarism" and "presumed consent" policies that promise effectiveness at the cost of violating traditional ethical and legal principles. In this article, we analyze the failure of the current regime, articulate a policy of routine removal subject to the right of informed refusal, and defend this policy against several possible objections.