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.

Production of compound A and carbon monoxide in circle systems: an in vitro comparison of two carbon dioxide absorbents.

Two new generation carbon dioxide absorbents, DrägerSorb Free and Amsorb Plus, were studied in vitro for formation of compound A or carbon monoxide, during minimal gas flow (500 ml x min(-1)) with sevoflurane or desflurane. Compound A was assessed by gas chromatography/mass spectrometry and carbon monoxide with continuous infrared spectrometry. Fresh and dehydrated absorbents were studied. Mean (SD) time till exhaustion (inspiratory carbon dioxide concentration >or= 1 kPa) with fresh absorbents was longer with DrägerSorb Free (1233 (55) min) than with Amsorb Plus (1025 (55) min; p < 0.01). For both absorbents, values of compound A were < 1 ppm and therefore below clinically significant levels, but were up to 0.25 ppm higher with DrägerSorb Free than with Amsorb Plus. Using dehydrated absorbents, values of compound A were about 50% lower than with fresh absorbents and were identical for DrägerSorb Free and Amsorb Plus. With dehydrated absorbents, no detectable carbon monoxide was found with desflurane.

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.

Only carbon dioxide absorbents free of both NaOH and KOH do not generate compound A during in vitro closed-system sevoflurane: evaluation of five absorbents.

BACKGROUND: Insufficient data exist on the production of compound A during closed-system sevoflurane administration with newer carbon dioxide absorbents. METHODS: A modified PhysioFlex apparatus (Dräger, Lübeck, Germany) was connected to an artificial test lung (inflow at the top of the bellow approximately/= 160 ml/min CO2; outflow at the Y piece of the lung model approximately/= 200 ml/min, simulating oxygen consumption). Ventilation was set to obtain an end-tidal carbon dioxide partial pressure of approximately 40 mmHg. Various fresh carbon dioxide absorbents were used: Sodasorb (n = 6), Sofnolime (n = 6), and potassium hydroxide (KOH)-free Sodasorb (n = 7), Amsorb (n = 7), and lithium hydroxide (n = 7). After baseline analysis, liquid sevoflurane was injected into the circuit by syringe pump to obtain 2.1% end-tidal concentration for 240 min. At baseline and at regular intervals thereafter, end-tidal carbon dioxide partial pressure, end-tidal sevoflurane concentration, and canister inflow (T degrees(in)) and canister outflow (T degrees(out)) temperatures were measured. To measure compound Ainsp concentration in the inspired gas of the breathing circuit, 2-ml gas samples were taken and analyzed by capillary gas chromatography plus mass spectrometry. RESULTS: The median (minimum-maximum) highest compound Ainsp concentrations over the entire period were, in decreasing order: 38.3 (28.4-44.2)* (Sofnolime), 30.1 (23.9-43.7) (KOH-free Sodasorb), 23.3 (20.0-29.2) (Sodasorb), 1.6 (1.3-2.1)* (lithium hydroxide), and 1.3 (1.1-1.8)* (Amsorb) parts per million (*P < 0.01 vs. Sodasorb). After reaching their peak concentration, a decrease for Sofnolime, KOH-free Sodasorb, and Sodasorb until 240 min was found. The median (minimum-maximum) highest values for T degrees(out) were 39 (38-40), 40 (39-42), 41 (40-42), 46 (44-48)*, and 39 (38-41) degrees C (*P < 0.01 vs. Sodasorb), respectively. CONCLUSIONS: With KOH-free (but sodium hydroxide [NaOH]-containing) soda limes even higher compound A concentrations are recorded than with standard Sodasorb. Only by eliminating KOH as well as NaOH from the absorbent (Amsorb and lithium hydroxide) is no compound A produced.

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.

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 determination of vapor-phase compound A in sevoflurane anesthesia using gas chromatography-mass spectrometry.

BACKGROUND: During low-flow or closed-circuit anesthesia with the fluorinated inhalation anesthetic sevoflurane, compound A, an olefinic degradation product with known nephrotoxicity in rats, is generated on contact with alkaline CO(2) adsorbents. To evaluate compound A formation and thus potential sevoflurane toxicity, a reliable and reproducible assay for quantitative vapor-phase compound A determination was developed. METHODS: Compound A concentrations were measured by fully automated capillary gas chromatography-mass spectrometry with cryofocusing. Calibrators of compound A in the vapor phase were prepared from liquid volumetric dilutions of stock solutions of compound A and sevoflurane in ethyl acetate. 1,1,1-Trifluoro-2-iodoethane was chosen as an internal standard. The resulting quantitative method was fully validated. RESULTS: A linear response over a clinically useful concentration interval (0.3-75 microL/L) was obtained. Specificity, sensitivity, and accuracy conformed with current analytical requirements. The CVs were 4.1-10%, the limit of detection was 0.1 microL/L, and the limit of quantification was 0.3 microL/L. Analytical recoveries were 100.6% +/- 10.1%, 102.5% +/- 7.3%, and 99.0% +/- 4.1% at 0.5, 10, and 75 microL/L, respectively. The method described was used to determine compound A concentrations during simulated closed-circuit conditions. Some of the resulting data are included, illustrating the practical applicability of the proposed analytical approach. CONCLUSIONS: A simple, fully automated, and reliable quantitative analytical method for determination of compound A in air was developed. A solution was established for sampling, calibration, and chromatographic separation of volatiles in an area complicated by limited availability of sample volume and low concentrations of the analyte.

Determination of the designer drugs 3, 4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxyamphetamine with HPLC and fluorescence detection in whole blood, serum, vitreous humor, and urine.

BACKGROUND: The popular designer drugs 3, 4-methylenedioxymethamphetamine (MDMA) and 3, 4-methylenedioxyethylamphetamine (MDEA) can be determined in serum, whole blood, and urine, but also in vitreous humor. The latter matrix is interesting when dealing with decomposed bodies in a toxicological setting. METHODS: After extraction, chromatographic separation was achieved on a narrow-bore C(18) column by gradient elution with fluorometric detection; results were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The method was linear over the range of 2-1000 microg/L for whole blood, serum, and vitreous humor, and 0.1-5 mg/L for urine. Extraction recoveries were >70%, imprecision (CV) was 2.5-19%, and analytical recoveries were 95.5-104.4%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.8 and 2 microg/L, respectively, for whole blood, serum, and vitreous humor, and 2.5 microg/L and 0.1 mg/L, respectively, for urine. Excellent correlations between the quantitative LC-fluorescence and LC-MS/MS results were obtained. We found the following concentrations in a thanatochemical distribution study in rabbits: in serum, 5.3-685 microg/L for MDMA and from the LOQ to 14.5 microg/L for 3, 4-methylenedioxyamphetamine (MDA); in whole blood, 19.7-710 microg/L for MDMA and from the LOQ to 17.8 microg/L for MDA; in vitreous humor, 12.1-97.8 microg/L for MDMA and from the LOQ to 3.86 microg/L for MDA. In routine toxicological urine samples, concentrations ranged from LOQ to 14.62 mg/L for MDA, from LOQ to 157 mg/L for MDMA, and from LOQ to 32.54 mg/L for MDEA. CONCLUSIONS: The HPLC method described is sensitive, specific, and suitable for the determination of MDMA, MDEA, and MDA in whole blood, serum, vitreous humor, and urine.

In vitro compound A formation in a computer-controlled closed-circuit anesthetic apparatus. Comparison with a classical valve circuit.

BACKGROUND: Few data exist on compound A during sevoflurane anesthesia when using closed-circuit conditions and sodalime with modern computer-controlled liquid injection. METHODS: A PhysioFlex apparatus (Dräger, Lübeck, Germany) was connected to an artificial test lung (inflow approximately 160 ml/min carbon dioxide, outflow approximately 200 ml/min, simulating oxygen consumption). Ventilation was set to obtain an end-tidal carbon dioxide partial pressure (Petco2) approximately 40 mmHg. Canister inflow (T degrees in) and outflow (T degrees out) temperatures were measured. Fresh sodalime and charcoal were used. After baseline analysis, sevoflurane concentration was set at 2.1% end-tidal for 120 min. At baseline and at regular intervals thereafter, Petco2, end-tidal sevoflurane, T degrees in, and T degrees out were measured. For inspiratory and expiratory compound A determination, samples of 2-ml gas were taken. These data were compared with those of a classical valve-containing closed-circuit machine. Ten runs were performed in each set-up. RESULTS: Inspired compound A concentrations increased from undetectable to peak at 6.0 (SD 1.3) and 14.3 (SD 2.5) ppm (P < 0.05), and maximal temperature in the upper outflow part of the absorbent canister was 24.3 degrees C (SD 3.6) and 39.8 degrees C (SD 1.2) (P < 0.05) in the PhysioFlex and valve circuit machines, respectively. Differences between the two machines in compound A concentrations and absorbent canister temperature at the inflow and outflow regions were significantly different (P < 0.05) at all times after 5 min. CONCLUSION: Compound A concentrations in the high-flow (70 l/min), closed-circuit PhysioFlex machine were significantly lower than in conventional, valve-based machines during closed-circuit conditions. Lower absorbent temperatures, resulting from the high flow, appear to account for the lower compound A formation.

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.

Evaluation of automated single mass spectrometry to tandem mass spectrometry function switching for comprehensive drug profiling analysis using a quadrupole time-of-flight mass spectrometer.

A liquid chromatographic mass spectrometric strategy for systematic toxicological analysis (STA) is presented using the automatic 'on-the-fly' single mass spectrometry mode to tandem mass spectrometry mode (MS to MS/MS) switching abilities of a quadrupole time-of-flight (Q-TOF) instrument. During the chromatographic run, the quadrupole is initially set to transmit all masses until (an) ion(s) reaches a certain set threshold. Thereupon, the quadrupole automatically switches to the MS/MS mode, selecting the ion(s), which are subsequently fragmented in the high-efficiency hexapole collision cell, thus generating product ions that are further mass analyzed by the TOF. By limiting the TOF spectral accumulation time in the MS/MS mode to a statistically acceptable minimum, the quadrupole almost instantly switches back to the MS mode. Qualitative information, comprising the complementary MS ([M + H](+) ion mass) and MS/MS (informative product ion profile) data, as well as quantitative information obtained by integration of the MS extracted ion chromatogram(s), can be obtained in one single acquisition. Optimization of the automatic switching parameters, such as threshold, TOF spectral accumulation time, detection window and collision energy, was carried out by injection of a mix of 17 common drugs which were not necessarily baseline separated in the chromatographic system used. Indeed, the complete separation of the drugs is not deemed necessary since up to 8 different ions can 'simultaneously' be selected for MS/MS if they reach the preset criteria. In addition, the quantitative performance of the method was defined. In a second phase, the developed method was field-tested. To that end, the resulting data from extracts of urine samples were compared with and found to be in close concordance with those obtained by a standard toxicological analysis. This innovative approach clearly holds the potential for a substantial advance in the introduction of LC/MS in STA.

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.

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.

Narrow-bore HPLC in combination with fluorescence and electrospray mass spectrometric detection for the analysis of cocaine and metabolites in human hair.

A simple, but sensitive and specific, high-performance liquid chromatographic assay for cocaine, cocaethylene, and benzoylecgonine is described. Using direct fluorometric detection, the procedure is particularly interesting for the routine analysis of human hair samples. In the sample preparation part, the hair samples are cut and washed and two internal standards with close structural resemblance to benzoylecgonine and cocaine as well as to cocaethylene are added. Subsequently, the hair samples are homogenized, hydrolyzed overnight in a 0.1 M HCl solution at 56 degrees C, and extracted on IST Confirm HCX solid-phase extraction columns. Chromatographic separation is achieved on a narrow-bore Hypersil BDS C18 column (125 x 2.1 mm, 3 microns) by gradient elution with an ammonium acetate buffer-methanol/acetonitrile mixture. For the fluorometric detection, excitation and emission wavelengths of 242 and 315 nm, respectively, are used. This analysis protocol affords a method of high sensitivity and specificity which has been fully evaluated and validated. The data presented show good accuracy and linearity with excellent reproducibility and recovery. Because unequivocal identity confirmation is mandatory in forensic applications, an extension of the analysis protocol was accomplished toward mass spectrometric detection. We succeeded in a simple methodological transfer from LC/FL to LC/ESI-MS/MS, thus providing two complementary approaches after a single, common sample-processing step. Hair samples from 29 fatalities, all known drug users and suspected victims from a drug overdose, were analyzed in this way. Of the investigated samples, 12 were positive and the concentrations found range from 0.98 to 938 ng/mg of hair for cocaine and from 1.45 to 388 ng/mg of hair for benzoylecgonine. Traces of cocaethylene were also found in two of the hair samples. The results obtained with LC/ESI-MS/MS were in close agreement with those obtained with LC/FL, positively confirming the isolates' identity and structure by means of the resulting MS/MS spectra.

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.

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.

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.

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.