Evaluation of three state-of-the-art metabolite prediction software packages (Meteor, MetaSite, and StarDrop) through independent and synergistic use.

The aim of this study was to evaluate three different metabolite prediction software packages (Meteor, MetaSite, and StarDrop) with respect to their ability to predict loci of metabolism and suggest relative proportions of metabolites. A chemically diverse test set of 22 compounds, for which in vivo human mass balance studies and metabolic schemes were available, was used as basis for the evaluation. Each software package was provided with structures of the parent compounds, and predicted metabolites were compared with experimentally determined human metabolites. The evaluation consisted of two parts. First, different settings within each software package were investigated and the software was evaluated using those settings determined to give the best prediction. Second, the three different packages were combined using the optimized settings to see whether a synergistic effect concerning the overall metabolism prediction could be established. The performance of the software was scored for both sensitivity and precision, taking into account the capabilities/limitations of the particular software. Varying results were obtained for the individual packages. Meteor showed a general tendency toward overprediction, and this led to a relatively low precision (∼35%) but high sensitivity (∼70%). MetaSite and StarDrop both exhibited a sensitivity and precision of ∼50%. By combining predictions obtained with the different packages, we found that increased precision can be obtained. We conclude that the state-of-the-art individual metabolite prediction software has many advantageous features but needs refinement to obtain acceptable prediction profiles. Synergistic use of different software packages could prove useful.

Quantitative determination of glycopyrrolate in human plasma by liquid chromatography-electrospray ionization mass spectrometry: the use of a volatile ion-pairing agent during both liquid-liquid extraction and liquid chromatography.

The work presented here deals with the development of a quantitative tool for the determination of the quaternary ammonium anticholinergic glycopyrrolate in human plasma samples. Mepenzolate was used as an internal standard. The plasma samples were subjected to a suitable sample clean-up consisting of a simple and relatively fast, two step liquid-liquid ion-pair extraction procedure. The chromatography, using the same volatile ion-pair reagent heptafluorobutyric acid (HFBA), takes only 10 min. Relative standard deviation of retention times was never above 2.26% (n=36). The method was fully validated based on the US FDA Bioanalytical Method Validation Guidance for Industry. As such, a quantitative ESI-LC-MS(/MS) (TOF mass spectrometry) method was optimized for the absolute quantification of glycopyrrolate in human plasma in a concentration range from 0.101 to 101 ng/mL using a quadratic calibration function (R(2)=0.9995), y=-2.21 x 10(-4) (+/-3.93 x 10(-5))xx(2)+5.85 x 10(-2) (+/-5.27 x 10(-3))xx+4.08 x 10(-3) (+/-4.82 x 10(-4)). For the three QC concentrations (QC(1) 0.252, QC(2) 2.52, and QC(3) 25.2ng/mL) and the LLOQ (0.101 ng/mL), total precision was under 20% (18.0% (n=6) at the LLOQ) and maximum accuracy was 112% (88.9% for the LLOQ, n=6). Absolute matrix effect (maximum 133%+/-9.59, n=3), absolute recovery (better than 41.8%+/-2.22, n=3), relative (inter-subject) matrix effect (maximum 10.9%+/-1.45, n=4) and process efficiency (better than 45.2%+/-5.74, n=3) too were assessed at the 3 QC concentrations.

Stability study of the designer drugs "MDA, MDMA and MDEA" in water, serum, whole blood, and urine under various storage temperatures.

A controlled study was undertaken to determine the stability of the designer drugs MDA, MDMA and MDEA in pooled serum, whole blood, water and urine samples over a period of 21 weeks. The concentrations of the individual designer drugs in the various matrices were monitored over time, in the dark at various temperatures (-20, 4 or 20 degrees C), for a low (+/- 6 ng/ml for water, serum and whole blood and +/- 150 ng/ml for urine) and a high concentration level (+/- 550 ng/ml for water, serum and whole blood and +/- 2500 ng/ml for urine). Compound concentrations were measured using a validated HPLC assay with fluorescence detection. Our study demonstrated no significant loss of the designer drugs in water and urine at any of the investigated temperatures for 21 weeks. The same results were observed in serum for up to 17 weeks, and up to 5 weeks in whole blood. After that time, the compounds could no longer be analyzed due to matrix degradation, especially in the low concentration samples that were stored at room temperature. This study demonstrates that the designer drugs, MDA, MDMA and MDEA are stable when stored at -20 degrees C for 21 weeks, even in haemolysed whole blood.

Segmental analysis for cocaine and metabolites by HPLC in hair of suspected drug overdose cases.

Hair samples of eight postmortem cases were analyzed in segments of 1 to 3 cm for cocaine, benzoylecgonine and cocaethylene. Samples were prepared for analysis by digestion in 0.1 M HCl and subsequent extraction with mixed-mode solid-phase extraction columns. Measurement was made by reversed-phase, narrow-bore HPLC and fluorescence detection using two laboratory-made internal standards. The concentrations were in the region of 0.29-316 ng/mg of hair for cocaine, 0.43-141 ng/mg of hair for benzoylecgonine and 0.93-1.83 ng/mg of hair for cocaethylene. All eight investigated cases had cocaine-positive segments. In six of the cases, all segments were positive, suggesting regular cocaine use and two showed in-between negative segments indicating an interruption or a change of the abuse intensity. The results showed a second, remarkable observation, i.e. enormous concentration differences (factor >150) for both cocaine and benzoylecgonine between the different subjects. Furthermore, interindividual cocaine/benzoylecgonine ratios ranged from 0.02 to 8.43. We believe these observations could in part be attributed to both some of the still existing limitations in the analytical approach(es), especially the mandatory hair washing steps, and in our still too limited knowledge of the hair incorporation processes. Nevertheless, in some cases, segmental analysis proved to be an important tool to distinguish, together with postmortem examination, deadly chronic abuse from single acute drug overdosage.

Quantitative determination of amphetamine and alpha-phenylethylamine enantiomers in judicial samples using capillary gas chromatography.

alpha-Phenylethylamine was recently reported by us in various samples seized from the illicit drug circuit. At first, alpha-phenylethylamine was identified in powders that generally contained amphetamine and caffeine. Then, a couple, who were known drug users, was found dead in their apartment. Urine samples from both victims contained large amounts of amphetamine and alpha-phenylethylamine. All of the positive samples were re-examined with gas chromatography to determine the chirality of the detected drug or drugs. The homochiral derivatizing reagent N-trifluoroacetyl-L-prolyl chloride was used to convert the drug enantiomers into their corresponding diastereomeric derivatives. These derivatives were separated on an achiral stationary phase and detected using either flame ionization detection (FID) or on-line Fourier-transform infrared spectrometry (FTIR) for quantitative or qualitative purposes, respectively. Excellent separation was realized between all diastereomeric derivatives, and interference of excess, nonvolatile derivatizing reagent was reduced to a minimum. All powder samples consisted of racemic mixtures for alpha-phenylethylamine and for amphetamine. The urine samples also contained both enantiomers of alpha-phenyl ethylamine and amphetamine, albeit in varying proportions. These findings again substantiate the synthetic origin of alpha-phenylethylamine, attributing its presence in the urine of both victims to intentional or accidental intake. The disproportionate isomeric composition found in the urine samples confirms previous reports of a stereoselective metabolism for amphetamine enantiomers and suggests a similar pharmacokinetic profile for alpha-phenylethylamine.

Identification of alpha-phenylethylamine in judicial samples.

alpha-Phenylethylamine was recently identified in samples from several judicial cases using chromatographic (high-performance liquid chromatography-diode-array detection, gas chromatography-mass spectrometry, and gas chromatography-Fourier transform infrared detection) and spectrometric (nuclear magnetic resonance) techniques. In the first case, 1 kg of a white powder was found in a basement laboratory. It contained caffeine and more than 15% alpha-phenylethylamine. In the second case, two white powders were seized from a female. One powder consisted of pure amphetamine, and the other was a mixture of caffeine, amphetamine, and alpha-phenylethylamine. Four months later, a couple, who were known drug users, were found dead in their apartment. Urine samples of both victims contained large amounts of amphetamine together with alpha-phenylethylamine. Recently, 0.13 kg of a white powder and 0.30 kg of an orange powder were seized during a law enforcement operation. Both powders were mixtures of caffeine, amphetamine, and alpha-phenylethylamine. The data presented demonstrate the recent and unrelated repetitive occurrence of alpha-phenylethylamine in the circuit of illicit drugs.

Determination of chloral hydrate and metabolites in a fatal intoxication.

A young woman (32 years old) was found dead in her house. Screening of postmortem blood with enzyme multiplied immunoassay (EMIT) detected benzodiazepines, salicylic acid derivatives, and caffeine. These compounds were present in nontoxic concentrations as confirmed by thin-layer chromatography and high-performance liquid chromatography. The Fujiwara-Ross reaction on blood revealed the presence of chlorinated hydrocarbons in high concentrations. An optimized gas chromatographic method with electron capture detection allowed the identification and quantitation of chloral hydrate and both its metabolites, 2,2,2-trichloroethanol and trichloroacetic acid, in the available postmortem samples. The tissue concentrations indicated that chloral hydrate ingestion could be identified as the cause of this fatality.

Computer-aided screening for hallucinogenic and stimulant amphetamines with gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR).

An expert system applied as a screening test for amphetamine analogues found in recreational-drug exhibits (tablets or powders) is described. The knowledge base defining the reference Fourier transform infrared spectroscopic (FTIR) spectral patterns has been built according to criteria encompassing toxicological, pharmacological, and neurochemical aspects. The class identity of a compound is determined within seconds using soft independent modeling of class analogy (SIMCA). The predictive value of the system, as assessed at a testing accuracy of 95%, is expressed by a total correct classification rate of 93.93% and by a 96.30% rate of true-positive amphetamines. The specificity and the selectivity of the screening test, evaluated by testing 159 toxicologically relevant compounds, are discussed, emphasizing the chemical and physical factors affecting these parameters. Medicinal amphetamines giving cross-reactions with traditional screening techniques produce a negative result. The specificity of the system characterizes the expert system as a highly sensitive, selective, fast, and user-friendly screening test that screens for amphetamines with prediction accuracy adequate for investigations in analytical toxicology.

Quantitative gas chromatographic analysis of 3-cyano-3,3-diphenylpropionic acid, the acidic metabolite of bezitramide (Burgodin), in urine.

A sensitive gas chromatographic procedure with nitrogen-phosphorus detection was developed for the quantitative determination of 3-cyano-3,3-diphenylpropionic acid, the acidic metabolite of the narcotic analgesic bezitramide (Burgodin). Gas chromatography with mass spectrometric detection was used for confirmation. An internal standard, 5-cyano-5,5-diphenylvaleric acid, was synthesized and purified in our laboratory. The compound was extracted from 5 mL of hydrolyzed urine with n-hexane-ethyl acetate (85:15, v/v). Derivatization of the extract with an ethereal diazomethane solution was performed immediately before chromatographic analysis. Under the described conditions, the quantitation limit for 3-cyano-3, 3-diphenylpropionic acid in urine was 10 ng/mL. The extraction recovery was 82%. The calibration graph was linear over the concentration range from 10 to 500 ng/mL; at a 100-ng/mL concentration, within-day and day-to-day percent coefficients of variation of 1.9 and 3.7%, respectively, were obtained. Urine samples from 16 persons suspected of using bezitramide were analyzed, and they revealed metabolite concentrations that ranged from 10.5 to 88 ng/mL.

Tissue distribution of amphetamine isomers in a fatal overdose.

A young man (22 years old) died of a cardiorespiratory arrest a few hours following admission to the emergency department of a hospital. He was found lying seriously ill in the parking lot of a dance club. Screening of postmortem blood and urine with enzyme multiplied immunoassay (EMIT) detected only amphetamines, caffeine, and cotinine. Further screening of blood, urine, and stomach contents with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) was negative for all three matrices. Specific conditions for amphetamines were used for the gas chromatographic (GC) screening (GC-mass spectrometric [MS] and GC-nitrogen-phosphorus detection). This resulted in the preliminary identification of amphetamine in both blood and urine. Confirmation of the presence of amphetamine in all available postmortem specimens was provided by mass and infrared spectral data (GC-MS and GC-Fourier transform infrared spectrometry) after derivatization. Quantitative results and differentiation between the enantiomers of amphetamine were obtained after chiral derivatization. The calculated concentrations disclosed amphetamine ingestion as the cause of this fatality.

A fatal case of serotonin syndrome after combined moclobemide-citalopram intoxication.

We present a case involving a fatality due to the combined ingestion of two different types of antidepressants. A 41-year-old Caucasian male, with a history of depression and suicide attempts, was found deceased at home. Multiple containers of medication, the MAO-inhibitor moclobemide (Aurorix), the SSRI citalopram (Cipramil), and the benzodiazepine lormetazepam (Noctamid) as active substance, as well as a bottle of whiskey were present at the scene. The autopsy findings were unremarkable, but systematic toxicological analysis (EMIT, radioimmunoassay, high-performance liquid chromatography-diode-array detection [HPLC-DAD], gas chromatography-nitrogen-phosphorus detection, and gas chromatography-mass spectrometry) revealed the following: ethanol (0.23 g/L blood, 0.67 g/L urine), lormetazepam (1.65 microg/mL urine), cotinine (0.63 microg/mL blood, 5.08 microg/mL urine), caffeine (1.20 microg/mL urine), moclobemide (and metabolites), and citalopram (and metabolite). There upon, we developed a new liquid chromatographic separation with optimized DAD, preceded by an automated solid-phase extraction, for the quantitation of the previously mentioned antidepressive drugs. The results obtained for blood and urine, respectively, were as follows: Ro 12-5637 (moclobemide N'-oxide) not detected and 424 microg/mL; Ro 12-8095 (3-keto-moclobemide) 2.26 microg/mL and 49.7 microg/mL; moclobemide 5.62 microg/mL and 204 microg/mL; desmethylcitalopram 0.42 microg/mL and 1.22 microg/mL; and citalopram 4.47 microg/mL and 19.7 microg/mL. The cause of death was attributed to the synergistic toxicity of moclobemide and citalopram, both antidepressants, which, by intentional or accidental combined ingestion, can produce a potentially lethal hyperserotoninergic state. Based on the history of the case and pharmacology of the drugs involved, the forensic pathologists ruled that the cause of death was multiple drug intoxication, resulting in a fatal "serotonin syndrome," and that the manner of death was suicide.

Liquid chromatographic determination of cocaine, benzoylecgonine, and cocaethylene in whole blood and serum samples with diode-array detection.

An extraction with Bond Elut Certify solid-phase extraction (SPE) columns is developed for the isolation of cocaine, benzoylecgonine, and cocaethylene from whole blood and serum followed by reversed-phase liquid chromatography with diode-array detection. Two internal standards (2'-methylbenzoylecgonine and 2'-methylcocaine) with close structural resemblance to benzoylecgonine (a carboxylic acid) and to the two esters, cocaine and cocaethylene, are used in the analytical procedure. A thorough evaluation of this SPE and a comparison with different liquid-liquid extractions clearly show the superiority of the SPE. A linear response (correlation coefficient greater than 0.998) over a broad concentration range (0.025-5.0 micrograms/mL) is obtained. The sensitivity, specificity, precision (coefficients of variation less than 4.9% for within-day reproducibility and less than 5.3% for total reproducibility), and accuracy of the method are excellent for each analyte. Forensic blood samples from people suspected of cocaine abuse are analyzed and show the usefulness of the method, even for degraded postmortem samples.

Metabolic profiling of urinary organic acids by single and multicolumn capillary gas chromatography.

High-resolution gas chromatography (HRGC) and gas chromatography/mass spectrometry (GC/MS) are the techniques of choice to determine the retention indices of more than 200 organic acids as their trimethylsilyl (TMS) or oxime-trimethylsilyl derivatives. Several types of apolar and semipolar fused-silica capillary columns (OV-1, SE-52, and OV-1701), used to analyze and separate organic acids isolated from urine samples, are evaluated.

Solid-phase extraction technique for gas-chromatographic profiling of acylcarnitines.

We present a simple, new clean-up method for the gas-chromatographic profiling analysis of acylcarnitines. The use of a solid-phase, cation-exchange extraction combined with gas-chromatographic separation, based on the derivatization into acyloxylactones by Lowes and Rose (Analyst 1990;115:511-6), allows a selective and sensitive screening for acylcarnitines in urine. As such, a quantitative approach was developed for differential evaluation of acylcarnitines for detection of inborn errors of metabolism; the evaluation is both fast and routinely applicable in any biochemical laboratory. We validate the analysis method for acylcarnitines of various chain-lengths and present examples of its application to urine samples from diseased patients. We give special attention to the medium-chain acylcarnitines because of their association with medium-chain acylCoA dehydrogenase deficiency. Finally, the quantitative nature of the analysis allows evaluation of the acylcarnitine excretion over time.

Gas chromatographic/mass spectrometric identification of 3-hydroxydicarboxylic acids in urine.

Organic acid profiles were routinely analysed for two patients suffering from a massive 3-hydroxydicarboxyluria and five patients suffering from ketoaciduria. The identification of the urinary, saturated and unsaturated 3-hydroxydicarboxylic acids as their trimethylsilyl derivatives was performed with electron impact and positive chemical ionization gas chromatography/mass spectrometry. Additionally, their methylene units have been determined on SE-52 and OV-1701 types of stationary phases.

Potential of high-performance liquid chromatography with photodiode array detection in forensic toxicology.

The potentials and limitations of high-performance liquid chromatography-photodiode array detection are highlighted in respect to its use in the analysis of different biological matrices followed by the identification of unknowns. The logical analytical approach used in clinical and forensic toxicology, vital for the identification of one or more toxic substances as a cause of intoxication, is largely based on both simple and fast "general unknown screening" methods which cover most relevant drugs and potentially hazardous chemicals. In this field of systematic toxicological analysis, a literature overview shows that HPLC can play a substantial role. Both column packing material and eluent composition have their impact on intra- and interlaboratory reproducibility. In view of the sometimes different retention characteristics of various HPLC columns, several possibilities are addressed to enhance the discriminating power of primary retention parameters. The advantages of photodiode array detection as compared to UV detection have been of paramount importance to the success of HPLC in toxicological analysis. Dedicated libraries with spectral information and searching software are powerful tools in the process of identification of an unknown substance. In the present paper, these aspects are also verified in a number of real cases, i.e., trazodone and dothiepin, azide, chloroquine and cocaine, in which we illustrate from our own experience the potentials of HPLC-photodiode array detection in systematic toxicological analysis.

Quantitative colorimetric determination of urinary p-aminophenol with an automated analyzer.

We developed an automated colorimetric method for the quantitative determination of p-aminophenol with a Cobas Mira analyzer. The procedure can be used for the biological monitoring of human exposure to aniline. An absorbed aniline dose is extensively oxidized to p-aminophenol, which is excreted in urine mainly as glucurono- and sulfo- conjugates. After enzymatic hydrolysis, we reacted the free compound with resorcinol in the presence of manganese ions to form an indophenol dye, which is measured at 550 nm. Excellent accuracy (102.8%, 103.9%, and 96.8% at 2.5, 50, and 90 mg/L, respectively) and precision (7.7%, 2.1%, and 0.8% CV for within-run and 11.1%, 4.7%, and 4.6% for total reproducibility at 2.5, 50, and 90 mg/L, respectively) were achieved over a linear concentration range of 2.0 to 100 mg/L. The detection limit was 0.9 mg/L and no significant interference (except for o-aminophenol) was found for several investigated drugs and related compounds. The proposed method was used for a stability study and to analyze several samples from an occupational health screen.

Analysis of cocaine, benzoylecgonine, and cocaethylene in urine by HPLC with diode array detection.

A solid phase extraction method was developed for the isolation of cocaine, benzoylecgonine, and cocaethylene from urine followed by high-performance liquid chromatography/diode array detection. The application of a new solid hybrid phase extraction technology produced much cleaner extracts than conventional extraction procedures and made the selective extraction of substances with different polarities possible. Two internal standards with great structural resemblance to benzoylecgonine (a carboxylic acid) and to the two esters, cocaine and cocaethylene, respectively, were synthesized. A linear response over a broad concentration range was obtained. The sensitivity, specificity, and accuracy were satisfactory for each analyte. Hydrolysis of cocaine and cocaethylene to benzoylecgonine during extraction and analysis was less than 0.5%. The method described can be used to corroborate cocaine use, to establish cocaine overdoses, and to study pharmacological effects of cocaine and its metabolites.

Liquid chromatography-mass spectrometry in forensic toxicology.

Liquid chromatography-mass spectrometry has evolved from a topic of mainly research interest into a routinely usable tool in various application fields. With the advent of new ionization approaches, especially atmospheric pressure, the technique has established itself firmly in many areas of research. Although many applications prove that LC-MS is a valuable complementary analytical tool to GC-MS and has the potential to largely extend the application field of mass spectrometry to hitherto "MS-phobic" molecules, we must recognize that the use of LC-MS in forensic toxicology remains relatively rare. This rarity is all the more surprising because forensic toxicologists find themselves often confronted with the daunting task of actually searching for evidence materials on a scientific basis without any indication of the direction in which to search. Through the years, mass spectrometry, mainly in the GC-MS form, has gained a leading role in the way such quandaries are tackled. The advent of robust, bioanalytically compatible combinations of liquid chromatographic separation with mass spectrometric detection really opens new perspectives in terms of mass spectrometric identification of difficult molecules (e.g., polar metabolites) or biopolymers with toxicological relevance, high throughput, and versatility. Of course, analytical toxicologists are generally mass spectrometry users rather than mass spectrometrists, and this difference certainly explains the slow start of LC-MS in this field. Nevertheless, some valuable applications have been published, and it seems that the introduction of the more universal atmospheric pressure ionization interfaces really has boosted interests. This review presents an overview of what has been realized in forensic toxicological LC-MS. After a short introduction into LC-MS interfacing operational characteristics (or limitations), it covers applications that range from illicit drugs to often abused prescription medicines and some natural poisons. As such, we hope it can act as an appetizer to those involved in forensic toxicology but still hesitating to invest in LC-MS.

Is vitreous humour useful for the interpretation of 3,4-methylenedioxymethamphetamine (MDMA) blood levels? Experimental approach with rabbits.

As drug instability and redistribution are factors known to affect the interpretation of post-mortem blood levels, we questioned whether post-mortem vitreous humour concentrations could be useful as predictors for the MDMA load at the time of death. In a first series of in vivo experiments using rabbits, 3,4-methylenedioxymethamphetamine (MDMA) concentrations in plasma, blood and vitreous humour were studied as a function of time after intravenous (i.v.) administration of MDMA. Equilibration between the vascular compartment and vitreous humour was attained about 1 h after i.v. MDMA administration. In a second series of experiments, the post-mortem stability of MDMA in vitreous humour in relation to ambient temperature was investigated. Post-mortem MDMA concentrations in vitreous humour were closer to the ante-mortem blood levels when compared to cardiac blood samples. These preliminary investigations in the rabbit model indicate that measurements of vitreous humour concentrations could also be of interest for predicting the blood concentration at the time of death in humans.