Practical experiences in application of hair fatty acid ethyl esters and ethyl glucuronide for detection of chronic alcohol abuse in forensic cases.

This article presents results from 1872 hair samples, which were analyzed for fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG). The results were evaluated in the context of self-reported drinking behavior, the use of hair cosmetics, the gender of the sample donors and hair sample length. For comparison, CDT and GGT in serum were available in 477 and 454 cases, respectively. A number of alcohol abstainers or low moderate drinkers and excessive drinkers were selected for assessment of cut-offs for FAEEs in the proximal 6cm hair segments and for EtG in the proximal 3cm hair segments. Cut-off values were assessed by ROC analysis. It was found that the cut-offs of 1.0ng/mg FAEE and 30pg/mg EtG presently used for excessive drinking lead to a low portion of false positives (4% and 3% respectively) but to a higher portion of false negatives (23% and 25% respectively). Comparison of the mean and medium concentrations in samples without any reported hair cosmetics (N=1079) and in samples with reported use of hair spray (N=79) showed an increase by the factor of about two for FAEE but no significant difference for EtG. Mean values of EtG were decreased by 80% in bleached samples (N=164) and by 63% in dyed samples (N=96). There was no significant effect of bleaching and dyeing on FAEE. Hair gel and hair wax, oil or grease showed no significant effect on both FAEE and EtG. With respect to gender and investigated hair length ambiguous results were obtained because of major differences in the compared subpopulations of male with higher alcohol consumption and mainly shorter hair, and less drinking female with longer hair. For excessive drinkers FAEEs in the 0-6cm hair segment and EtG in the 0-3cm segment decreased with increasing time of reported abstinence before sample collection. These drinkers attain the level of teetotalers only after more than 10 months of abstinence. In comparison to scalp hair, FAEEs recovered from armpit hair and leg hair were lower and from chest hair were higher. EtG in armpit hair was lower and in leg hair higher than in scalp hair. It is concluded that the combined use of FAEE and EtG essentially increases the accuracy of interpretation since both markers complement each other by a different sensitivity to sources of error.

Matrix and modifier effects in the supercritical fluid extraction of cocaine and benzoylecgonine from human hair.

The supercritical fluid extraction (SFE) behavior of cocaine and its major metabolite benzoylecgonine (BZE) was investigated and found to be highly dependent upon the chemical nature of the matrix and the manner in which the target drug analytes are incorporated into or on the matrix. The recovery of cocaine from Teflon wool, filter paper, drug-fortified hair, and drug user hair was studied using a variety of CO2/modifier mixtures. Incorporation of a triethylamine (TEA)/water modifier mixture provided dramatic improvements in the recovery of cocaine from interactive matrixes. The results suggest that the SF extractability of cocaine is not limited by analyte solubility; rather, desorption of cocaine from hair binding sites is a rate-limiting step in the SFE process. A displacement SFE mechanism is hypothesized in which TEA (as the triethylammonium cation) competes with cocaine for negatively charged hair binding sites. The dependence of extractability on hair/drug binding interactions allows the differentiation of cocaine present at different discrete sites in hair based on differences in SFE behavior. These findings suggest the potential for distinguishing exogenous (i.e., environmental) from endogenous (i.e., physiological) sources of drugs in hair. In contrast to the results observed for cocaine, SFE recoveries of BZE were poor from all matrixes and under all conditions studied. Its increased polarity, the presence of an additional binding site, and the possibility of multiple charged states suggest that poor BZE recoveries may be due to both poor analyte solubility and failure to desorb the analyte from hair binding sites under the conditions employed.

Studies of reproductive competence in male Dirofilaria immitis treated with milbemycin oxime.

Normal adult Dirofilaria immitis from a microfilaremic donor dog and D. immitis from donors rendered microfilaria (MF) negative by seven consecutive monthly doses of milbemycin oxime (500 micrograms/kg) were transplanted into three previously uninfected and untreated dogs. Two dogs received reciprocal combinations of treated and untreated D. immitis and the third received untreated adults of both sexes. A fourth dog served as an infected, milbemycin treated, non-transplanted control. Eleven weeks after pairing treated female with untreated male worms, a low-level microfilaremia developed in the recipient. Two of the three treated female worms recovered from this dog were non-fertile, and the third contained a small number of elongate and coiled embryos but no mature intrauterine (stretched) microfilariae (MFF). The dog receiving treated male and untreated female worms became microfilaremic after two weeks. Microfilaremia peaked at 37,000/ml 16 weeks after transplantation and declined over the next 20 weeks to 7,200/ml. Untreated females paired with treated males either became non-fertile or exhibited low numbers of developing embryos and MFF scattered throughout their reproductive tracts. Pairing of untreated male and female worms produced a mcirofilaremia during the second post-operative week, which plateaued around 15,000 MFF per ml. Females recovered after this pairing contained a normal pattern of embryonic development, including stretched MFF. There were no significant differences in the percentage composition or absolute numbers of developing and mature sperm in the reproductive tracts of treated and untreated male worms. However, the resumption of MF production in one milbemycin treated female worm after pairing with normal males and failure of treated males to sustain MF production in untreated female worms suggest that milbemycin oxime impairs the sexual competence of male D. immitis. This may explain the ability of this drug to bring about long term suppression of microfilaremia without immediate adulticidal activity.

Determination of fentanyl in hair: the case of the crooked criminalist.

When a State Crime Laboratory Director found that fentanyl patches were missing from a case submission stored in his evidence vault, he performed an investigation of those members of his staff who had access to the materials. As part of this investigation, two staff members were forced to submit to hair testing for fentanyl and other opiates and opioids. This unusual testing protocol was used to identify a senior Criminalist as a chronic abuser of fentanyl, and led to the rapid resolution of the case. To the best of our knowledge, this is the first report of the forensic analysis of hair for drugs of abuse in which the technology was used to identify an individual as a chronic fentanyl abuser.

The determination of cocaine in hair: a review.

The explosion of literature related to the analysis of hair for cocaine and its products is reviewed. In the commonly accepted applications of hair testing for cocaine, those related to criminal or civil investigations and pharmacotoxicologic studies occupy most of the relevant published work. This review uses detailed, 'binary' (yes/no) tables to demonstrate trends in the literature, and allows researchers and caseworkers quick access to the literature most important for answering a variety of questions.

Poppy seed ingestion as a contributing factor to opiate-positive urinalysis results: the Pacific perspective.

The possible contribution of poppy seed foods to positive opiate urinalysis results, especially from foods available in the Pacific Rim area, has recently become an issue for the U.S. Army Forensic Toxicology Drug Testing Laboratory in Hawaii. To assess the likelihood of this possible contribution, seven different poppy seed food products were consumed by male and female volunteers, and urine specimens were collected at time increments up to either 24 or 72 h. Specimens were evaluated for opiates using Roche Abuscreen radioimmunoassay (RIA), and all RIA positive specimens were analyzed for morphine and codeine using gas chromatography/mass spectrometry (GC-MS). Poppy seed cake, bagels, muffins, and rolls did not contain sufficient quantities of poppy seeds to give rise to opiate positive specimens by U.S. Department of Defense (DOD) GC-MS cutoff levels (morphine = 4000 ng/mL, codeine = 2000 ng/mL), although a number of specimens were positive by National Institute on Drug Abuse (NIDA) cutoff levels (morphine and codeine = 300 ng/mL). However, ingestion of poppy seed streusel or Danish pastry led to confirmed morphine and codeine positive specimens, irrespective of the use of DOD or NIDA confirmation cutoff values. In addition, significant amounts of codeine were observed in a number of these specimens. These findings argue against the unqualified application of previously published quantitative guidelines for eliminating poppy seed ingestion as a possible cause for a positive opiate urinalysis result.

Determination of deoxynivalenol (DON, vomitoxin) in wheat by high-performance liquid chromatography with photolysis and electrochemical detection (HPLC-hv-EC).

Deoxynivalenol (DON, vomitoxin) is a naturally occurring toxic fungal metabolite found in grains such as wheat, corn, rye, and barley. Current methods of analysis for DON and related trichothecene mycotoxins involve gas chromatography with electron capture detection, thin-layer chromatography, and high-performance liquid chromatography with ultraviolet detection. Improved sensitivity and selectivity for DON, as well as for the related compounds nivalenol and fusarenon-X, are made possible by the use of high-performance liquid chromatography with online, postcolumn photolysis and oxidative amperometric detection (HPLC-hv-EC). Conditions are optimized with respect to residence time in the photolytic reactor, applied potentials, mobile phase pH, and chromatographic parameters for the separation of DON, nivalenol, and fusarenon-X, and the technique is found to be linear over a range of 10 ppb-2 ppm with minimum detection limits on the order of 1-2 ng on-column (10-20 ppb). Since these compounds are not electroactive in the absence of photolysis, an additional mode of specificity is realized in addition to the chromatographic retention time and the dual-electrode response ratio. Some market samples of wheat naturally contaminated with DON are analyzed by this method, and the results are in agreement with those obtained by HPLC-UV and TLC analyses of the same samples.

Precolumn derivatization for improved detection in liquid chromatography-photolysis-electrochemistry.

Precolumn, homogeneous chemical derivatization with Sanger's reagent (2,4-dinitrofluorobenzene) is utilized to improve the chromatographic and detection properties of amino alcohols and amino acids. The 2,4-dinitrophenyl derivatives are separated using reversed-phase liquid chromatography and are detected using the hybrid photolysis-electrochemical (hv-EC) detector in tandem with UV absorbance detection. Following optimization of reaction, chromatographic, and detection variables, the derivatization-detection approach provides limits of detection in the low parts-per-billion range, with a linearity of roughly three orders of magnitude. Selectivity is based on retention times as well as dual electrode response ratios and a "lamp on/off" responsiveness criterion unique to the hv-EC detector. The method is applied to the determination of serine in beer.

An improved method for the rapid screening of illicit cocaine preparations using high performance liquid chromatography with electrochemical detection.

Electrochemical detection in HPLC offers a very sensitive and selective alternative to UV detection for a number of classes of compounds. While there have been no reports of the use of electrochemical detection for the determination of cocaine in illicit preparations or biological fluids, it is now possible to use on-line, continuous post-column photolytic derivatization to generate electroactive species from cocaine which may then be detected downstream using a conventional electrochemical detector operated at oxidative working electrode potentials. In this manuscript the construction, optimization and characterization of system parameters are discussed, and the sensitive and highly selective nature of this novel detection method is demonstrated to be uniquely suited to the rapid identification and quantitation of cocaine in simulated illicit preparations.

Analysis for penicillins and cefoperazone by HPLC-photolysis-electrochemical detection (HPLC-hv-EC).

Continuous, post-column, on-line, real-time photolytic derivatization or degradation can now be used following HPLC separation of various penicillin derivatives prior to conventional thin-layer, amperometric electrochemical detection using oxidative working potentials. Beta-lactam derivatives are separated by conventional reversed-phase HPLC, and each separated penicillin is then photolytically degraded to form, it is presumed, stable anionic species, which are then conveyed to the on-line electrochemical detector for qualitative and quantitative determinations. These methods of trace drug analysis have been applied to four separate penicillins or prodrugs, as well as one typical cephalosporin, viz. cefoperazone. Analytical parameters of the analysis have been determined, including dual electrode response ratios, sensitivity, minimum detection limits, linearity of calibration plots and the range of linear calibration. Finally, the analysis of cefoperazone-spiked saline solutions for i.v. administration has been performed in a single-blind study, as well as the determination of bacampicillin HCl in formulations obtained from a drug manufacturer in the United States. The overall method of analysis for these drugs has been demonstrated as being reproducible, accurate, and precise for at least five beta-lactam analogs. It is suggested that other beta-lactams will be amenable to these newer methods of analysis in a wide variety of sample matrices, including solid or liquid formulations, aqueous infusion solutions, and biological media, such as blood and urine.

Photolytic derivatization for improved LCEC determinations of pharmaceuticals in biological fluids.

Although electrochemical (EC) methods have been demonstrated to be sensitive and selective, wide use of EC detection in high performance liquid chromatographic (HPLC) assays in forensic and clinical toxicology laboratories has not been forthcoming. This fact is due to the general difficulty involved with the use of reductive EC detection methods, as well as to the lack of EC response in either the oxidative or reductive mode, for a number of classes of drugs having substantial clinical and forensic importance. The use of an on-line, post-column, continuous photolytic derivatization step, followed by conventional oxidative amperometric detection, alleviates many of these problems. In this report, the use of HPLC-photolysis-EC (HPLC-h nu-EC) for the trace determination of a number of controlled substances in biological fluids is presented. Following system optimization, the determination of phenobarbital, cocaine, methylphenidate, and several 1,4-benzodiazepines (and metabolites) is linear over three orders of magnitude. In addition, HPLC-h nu-EC offers a sensitive approach for these compounds, in that limits of detection (LODs) are all below 1 microgram/ml, ranging from 1 ng/ml to 750 ng/ml. The validity of this newer method is demonstrated in collaborative studies involving the trace determinations of phenobarbital in human serum, and chlordiazepoxide and its major metabolite, norchlordiazepoxide, in human urine. Finally, the authors' view of the role of HPLC-h nu-EC in the clinical and forensic toxicology laboratory is presented.