Methadone and illegal drugs in hair from children with parents in maintenance treatment or suspected for drug abuse in a German community.

BACKGROUND: Children living in homes with drug-addicted parents are in a steady danger of poisoning and may suffer from neglect, maltreatment, and lagging behind in development. Hair analysis could be a suitable way to examine this endangering exposure to drugs. METHODS: Hair samples from 149 children (aged 1-14 years) living with parents substituted by methadone and/or suspected for abuse of illegal drugs, and from 124 of the parents in a German community were investigated by liquid chromatography-hybrid quadrupole time-of flight mass spectrometry and by headspace solid phase microextraction gas chromatography-mass spectrometry for methadone, heroin, cocaine, amphetamines, ecstasy, cannabinoids and benzodiazepines and their metabolites or degradation products (32 compounds). RESULTS: From the children's hair, only in 35 samples, no drugs were detected. Cannabinoids were found in 56 samples, in 20 of them as the only drug. In the remaining 95 samples, methadone was identified 35 times with additional use of illegal drugs in 28 cases. Drug use in the children's environment was obvious for heroin in 44 cases, cocaine in 73 cases, amphetamine or ecstasy in 6 cases, and diazepam in 8 cases. The concentrations varied from limit of quantification to 2.16 ng/mg of methadone, 11.1 ng/mg of 6-acetylmorphine, 17.8 ng/mg of cocaine, 3.29 ng/mg of amphetamine, and 0.72 ng/mg of Δ-tetrahydrocannabinol. In general, hair from younger children contained higher concentrations than from their elder siblings. Systemic incorporation of methadone, cocaine, or cannabinoids appeared likely from detection of the nonhydrolytic metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in 11 cases, norcocaine in 16 cases, and 11-nor-9-carboxy-Δ-tetrahydrocannabinol in 9 cases. Within the families, hair samples of children and parents provided often the same drug pattern. External deposition from smoke and by contact with contaminated surfaces or parent's hands and systemic deposition after passive smoking, administration, or oral intake by hand-to-mouth transfer were discussed as alternative incorporation mechanisms into hair. CONCLUSIONS: Altogether, investigation of children's hair proved to be a useful way to detect endangering drug use in their environment and lead to a more thorough inspection and measures to improve their situation in many of the cases.

Hair as a novel noninvasive method to study the impact of pregnancy on CYP2D6 metabolizing capacity.

A novel method is described to investigate changes in the drug metabolizing capacity of CYP2D6 during pregnancy using hair analysis.

Formation of the N-methylpyridinium derivative to improve the detection of buprenorphine by liquid chromatography-mass spectrometry.

The legally defensible identification of the narcotic, analgesic buprenorphine, in biological specimen requires considerable sensitivity due to its low therapeutic dosages and corresponding target concentrations. Application of liquid chromatography-electrospray ionisation-mass spectrometry, which became the default method for buprenorphine detection, is impeded by the disadvantageous fragmentation of the stable precursor ion producing unspecific product ions of comparatively low abundance. A chemical modification to form the N-methylpyridinium ether derivative of buprenorphine is presented to improve the selectivity and sensitivity of its detection by liquid chromatography-mass spectrometry (LC-MS). The reaction of buprenorphine with 2-fluoro-1-methyl-pyridinium-p-toluene-sulfonate and triethylamine as catalyst was accomplished in acetonitrile at an ambient temperature yielding a chemically stable derivative. Fragmentation of the permanently charged precursor ion (m/z = 559) leads to the formation of diagnostic and abundant fragments (e.g. m/z = 443 and 450) representing all parts of the molecule. The application of the technique to the identification of buprenorphine in hair samples demonstrates a high specificity, availability of sufficient qualifier ions and a significant (approximately 8-fold) improvement of detection limits with respect to comparable experiments based on underivatised buprenorphine.

Determination of hydroxy metabolites of cocaine in hair samples for proof of consumption.

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC-positive samples for these compounds was performed by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS). Once identified, mass transitions for o-, p- and m-isomers of hydroxy COC as well as p- and m-isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC-positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p-OH-BE, m-OH-BE as well as p-OH-NCOC and m-OH-NCOC have been identified for the first time in COC-positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC-positive hair samples.

Determination of hydroxy metabolites of cocaine from hair samples and comparison with street cocaine samples.

Drugs which are commonly smoked or sniffed (e.g. cocaine), can contaminate hair through smoke or dust; therefore testing for metabolites, especially hydroxy metabolites, is highly recommended. The presence of hydroxy metabolites in street-cocaine (COC) has been discussed. To check if detection of hydroxy metabolites definitely proves ingestion, the presence of these metabolites in street COC samples has to be checked. It is expected that the more hydrophilic hydroxy metabolites of COC are incorporated into the hair-matrix to a lesser extent. For this study 576 COC positive hair samples (≥0.1ng COC/mg hair) were analysed by LC-MS/MS for benzoylecgonine (BE), norcocaine (NC), cocaethylene (CE), ortho-, meta- and para-hydroxy COC (o-, m-, p-OH-COC), meta- and para-hydroxy BE (m-, p-OH-BE), and meta- and para-hydroxy NC (m-, p-OH-NC). The results were compared with the respective metabolite/COC concentration ratios in 146 street COC samples, confiscated by the Bavarian police. Peak areas were used to estimate BE/COC, NC/COC, CE/COC and hydroxy metabolites/COC. Similar metabolic ratios were found for o-OH-COC in 88% of the samples, but for p-OH-COC and m-OH-COC only in 5.1% and 6.8%, respectively. Notably, p- and m-OH-BE as well as p- and m-OH-NC could not be identified from seized samples. We propose that area ratios exceeding the ratios of street COC more than twice or identification of OH-BE and OH-NC enable to differentiate COC consumption from contamination. Using these criteria, consumption of the drug could be proven in 92% of COC positive samples. As detection of meta- and para-hydroxy metabolites using the above mentioned criteria is a reliable tool to distinguish between ingestion and external contamination, it is recommended to implement their measurement into daily routine work.

Examination of a long-term clozapine administration by high resolution segmental hair analysis.

The long-term administration of clozapine could be verified by fine segmentation and analysis of single hairs of one person to examine the history of a multiple poisoning case. Segments of 1-2.5mm length were extracted by ultrasonification in 30 microl of the mobile phase (mixture of methanol+water, 50+50). By application of isocratic liquid chromatography and using narrow bore columns (Synergy Polar-RP, Phenomenex), an acceleration and miniaturization of the HPLC-MS-MS assay could be achieved. Total amounts of clozapine down to 30 fg (on column) and its desmethyl metabolite could be analysed in multiple reaction monitoring mode. According to typical sample amounts of approximately 16 microg, relevant hair concentrations higher than 1 pg/mg were detected. Significant and reproducible concentration profiles along the hair fibres revealed characteristic administration cycles. The administration time course - in particular the time of its termination - could be verified with a precision of a few days. The accuracy and reproducibility of the concentration profile was proven based on multiple investigations of single hairs. An individual hair growth rate of 0.55 mm/day was determined with a relative standard deviation of 8% by comparison of concentration profiles in hairs collected after a time span of 165 days.

Post-mortem review of fentanyl-related overdose deaths among identified drug users in Southern Bavaria, Germany, 2005-2014.

BACKGROUND: Two decades ago, there were only single case reports on deaths in Europe following the consumption of illicitly manufactured fentanyl by problem drug users. Today, lethal fentanyl intoxication is now no longer a rarity. Since 2005, a rapid increase of lethal fentanyl-related intoxications in the drug scene has been observed at the Institute of Legal Medicine, Ludwig Maximilians University, Munich. We hypothesized that this rise is the result of the launch of fentanyl matrix patches in Germany in 2004, their broad acceptance, their diversion from the regulated supply chain, and incautious prescription by medical care providers. METHODS: Post-mortem toxicological reports were reviewed for lethal fentanyl-related intoxications between 2004 und 2014. Blood and tissue samples were tested by GC/MS or LC-MS/MS. The results of police investigations, autopsy reports, and the database of the Institute of Legal Medicine, LMU, were analysed to identify problem drug users and to detect the source of fentanyl as well as the routes of administration. RESULTS: Between 2005 and 2014, 242 overdose victims with post-mortem toxicological detection of fentanyl were found. In the majority of cases, fentanyl matrix patches were the source of fentanyl. CONCLUSION: The onset of fentanyl-related deaths coincided with the launch of transdermal fentanyl matrix patches in Germany in 2004. Several approaches, such as providing drug users with information on the possible risks of fentanyl consumption, education of medical caregivers, and also monitoring of the prescription of fentanyl patches, are required to reduce the number of fentanyl-related deaths in drug addicts.

Liquid chromatography-quadrupole-time-of-flight mass spectrometry screening procedure for urine samples in forensic casework compared to gas chromatography-mass spectrometry.

This work represents the development, validation, and application of a liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) screening method for the detection of pharmaceutical substances and illicit drugs (acidic, basic, and neutral organic drugs) in urine samples. Time-of-flight mass spectrometry was performed using an LC-Triple TOF 5600 system with electrospray ionization operated in both positive and negative mode, respectively. The limits of detection (LODs), determined for 34 substances, were < 10 ng/mL for 91% of the compounds. The limits of quantitation (LOQs) were < 20 ng/mL for 91% of the substances. The identification of the compounds was based on exact mass (< ± 5 ppm), retention time (<2%) if available, isotopic pattern fit (<10%) and library hit (>70%). These four parameters served as identification criteria and are discussed according to their role in identifying compounds even without reference substances. In routine casework, two in-house XIC (extracted ion chromatogram) lists, consisting of 456 protonated and 26 deprotonated compounds were used and retention times for 365 compounds were available. Compared to the results found with the established gas chromatography-mass spectrometry (GC-MS) procedure, the findings with the LC-QTOF-MS screening method showed a good comparability. Results that were not detected by LC-QTOF-MS because of a missing entry in the targeted XIC list could retrospectively be confirmed by simply entering the elemental formula of the relevant substance into the software and reprocessing the sample. LC-QTOF-MS offers an attractive technique for the fast and specific identification of illicit drugs and toxic compounds in urine samples. Copyright © 2016 John Wiley & Sons, Ltd.

Two fatalities associated with synthetic opioids: AH-7921 and MT-45.

In this study, two fatalities associated with the synthetic opioids AH-7921 and MT-45 are reported. Within the last few years, both compounds have emerged on the recreational drug market and are sold as "research chemicals" on the internet. In the first case, a 22-year-old woman was found dead in the bedroom of her apartment by two of her friends. A plastic bag labeled "AH-7921" was found in the apartment and the two friends stated that the deceased had consumed AH-7921 prior to her death. The woman was a known drug addict. In the second case, a 24-year-old man was found dead in his room by his mother. The deceased was sitting on a chair in front of his desk slumped over. Several bags of white powder labeled "MT-45", "Methoxmetamine" and "Methoxphenidine" were found in his room. Toxicological analyses of femoral blood, heart blood, liver, pericardial fluid, urine, vitreous humor and stomach content of the deceased were performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Time-of-flight mass spectrometry was carried out on an LC-Triple TOF 5600 system (AB Sciex) with electrospray ionization operated in positive mode. In the first case, additional hair analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-QTOF-MS. In both cases, the relevant synthetic opioid could be detected in all analyzed samples. The concentration of AH-7921 was determined to be 450µg/L in femoral blood. MT-45 was present at a concentration of 2900µg/L in femoral blood. Besides methoxmetamine which could qualitatively be detected in femoral blood, urine and stomach content no methoxphenidine was found. In summary, deaths of the young individuals could be, by exclusion of other causes of death, attributed to the consumption of an overdose of AH-7921 and MT-45, respectively.

Mitragynine concentrations in two fatalities.

Two cases of fatalities are reported of which the recreational use of Mitragyna speciosa ("kratom") could be confirmed. One of these cases presents with one of the highest postmortem mitragynine concentrations published to date. Our results show that even extremely high mitragynine blood concentrations following the consumption of kratom do not necessarily have to be the direct cause of death in such fatalities as a result of an acute overdose. The two cases are compared with regard to the differences in mitragynine concentrations detected and the role of mitragynine in the death of the subjects. Irrespective of the big differences in mitragynine concentrations in the postmortem blood samples, mitragynine was not the primary cause of death in either of the two cases reported here. Additionally, by rough estimation, a significant difference in ratio of mitragynine to its diastereomers in the blood and urine samples between the two cases could be seen.

Significant enhancement of 11-Hydroxy-THC detection by formation of picolinic acid esters and application of liquid chromatography/multi stage mass spectrometry (LC-MS(3) ): Application to hair and oral fluid analysis.

Formation of picolinic acid esters of hydroxylated drugs or their biotransformation products is a promising tool to improve their mass spectrometric ionization efficiency, alter their fragmentation behaviour and enhance sensitivity and specificity of their detection. The procedure was optimized and tested for the detection of cannabinoids, which proved to be most challenging when dealing with alternative specimens, for example hair and oral fluid. In particular, the detection of the THC metabolites hydroxyl-THC and carboxy-THC requires ultimate sensitivity because of their poor incorporation into hair or saliva. Both biotransformation products are widely accepted as incorporation markers to distinguish drug consumption from passive contamination. The derivatization procedure was carried out by adding a mixture of picolinic acid, 4-(dimethylamino)pyridine and 2-methyl-6-nitrobenzoic anhydride in tetrahydrofuran/triethylamine to the dry extraction residues. Resulting derivatives were found to be very stable and could be reconstituted in aqueous or organic buffers and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). Owing to the complex consecutive fragmentation patterns, the application of multistage MS3 proved to be extremely useful for a sensitive identification of doubly picolinated hydroxy-THC in complex matrices. The detection limits - estimated by comparison of corresponding signal-to-noise ratios - increased by a factor of 100 following picolination. All other species examined, like cannabinol, THC, cannabidiol, and carboxy-THC, could also be derivatized exhibiting only moderate sensitivity improvements. The assay was systematically tested using hair samples and exemplarily applied to oral fluid. Concentrations of OH-THC identified in THC-positive hair samples ranged from 0.02 to 0.29pg/mg.

Cannabinoids in hair: strategy to prove marijuana/hashish consumption.

Delta9-Tetrahydrocannabinol (THC) and 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THCA) are equally used to indicate consumption of cannabis (hashish and marijuana). Publications of the early 90's demonstrate the possibilities of determining THC, cannabinol (CBN), and cannabidiol (CBD). All these substances are present in cannabis smoke and can be incorporated into the hair only by contamination. Generally, washing procedures should prevent false positive results, but finally it cannot be excluded that traces of THC may be found in hair after mere passive cannabis smoke exposure. Three authentic cases illustrate the problems originating in the exclusive determination of THC/CBN. The first example is the case of a couple living together in an apartment. Both persons' hair samples had been taken and gave positive results for THC and CBN. The male subject admitted smoking cannabis several times per day, but the female mate denied any consumption. Examination of the hair for THCA showed a high level (>6.6 pg/mg) in the sample of the male person and negative results (LOQ 0.1 pg/mg) in the sample of his mate. The second case hair is of a self admitted cannabis user's hair and was tested first by an immunoassay and GC/MS with a negative result. Nevertheless, the THCA concentration quantified in his sample was 2.7 pg/mg hair. The third hair sample is of a 2-year-old child that was tested positive for cannabis by using an immunochemical test. No THC and CBN were detectable by GC/MS, however, trace amounts of THCA using GC/MS/MS. A comparative study of hair samples (screening for cannabinoids using ELISA test, THC determination by GC/MS, THCA by GC/MS/MS) showed that only 26 segments of 66 were positive for both THC and THCA. Thirteen were negative for THC and positive for THCA, and six were positive for THC but negative for THCA. The cases were selected by an ELISA test or re-examined when the blood/urine results or the statement of the accused did not match with a THC outcome. The most appropriate strategy to prove cannabis consumption is immunochemical initial test followed by a GC/MS/MS confirmation of THCA.

Proficiency test for the analysis of hair for drugs of abuse, organized by the Society of Hair Testing.

Eighteen laboratories interested in the analysis of human hair for drugs of abuse participated in a proficiency test (PT) organized by the Society of Hair Testing (SoHT) in 2001. Samples sent to the participants included one drug-free hair sample and two samples from drug users, sent in the form of short segments previously checked for homogeneity by three reference laboratories. Participants were requested to analyze the samples following the standard procedure used routinely in their laboratories.The compounds present in the samples included opiates, cocaine and metabolite, cannabinoids and amphetamines. All the laboratories analyzed opiates, cocaine and benzoylecgonine (BE); only 10 analyzed amphetamines, and 9 cannabinoids. Various methods were used to extract drugs from the hair-enzyme treatment, acidic, basic and methanol extractions. All the laboratories employed GC-MS, with the exception of two which used GC-MS/MS and LC-MS/MS, respectively. Six laboratories performed initial screening tests by RIA, ELISA or EMIT. Results show that the laboratories performed well qualitatively, since they successfully identified all the analytes that they tested, with the exception of eight false results. However, the scatter of quantitative results was high.

The neuropathology of cocaine abuse.

Cocaine abuse represents a worldwide significant forensic issue as it is becoming widely recognized as one of the most dangerous illicit drugs in common use today. Besides cardiovascular complications, psychiatric and neurologic symptoms are the most common manifestations of cocaine toxicity. The latter include seizures, movement disorders and cerebrovascular complications. In chronic cocaine abusers morphological, physiological, and neurochemical abnormalities have been demonstrated by using neuroradiological techniques such as computed tomography, magnetic resonance imaging, positron emission tomography or single photon emission computed tomography. The spectrum of neuropathologic changes encountered in the brains of cocaine abusers is broad, but the major findings consist of ischemic and hemorrhagic stroke, subarachnoid and intracerebral hemorrhages and cerebral ischemia. Especially persons with underlying arteriovenous malformation or aneurysm are at risk for such events. Except for a few instances of vasculitis, the etiology of cocaine-related cerebrovascular accidents is still unclear. Besides pharmacologically-induced vasospasm, impaired hemostasis and platelet function and decreased cerebral blood flow have been proposed. At the cellular level, abnormalities in the expression of transcription factors and changes of brain neurotransmitter systems have been reported.

Proof of cannabis administration by sensitive detection of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid in hair using selective methylation and application of liquid chromatography- tandem and multistage mass spectrometry.

The identification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair represents an exceptional forensic analytical challenge due to low target concentrations in a complex matrix. Several dedicated techniques [gas chromatography-negative chemical ionization-tandem mass spectrometry (GC-NCI-MS/MS) or GC-GC-MS couplings] were specifically introduced into forensic toxicology aiming to a selective and sensitive identification of THCCOOH in hair. The combination of liquid-chromatography (LC) and MS/MS gained an outstanding relevance in forensic toxicology (including the detection of cannabinoids). However, its application to hair matrix is characterized by a lack of specificity which is due to the unspecific decarboxylation as most abundant fragmentation reaction. Therefore, various chemical modifications of the carboxyl and/or phenolic hydroxyl groups were examined to improve the selectivity. The selective methylation of the 9-carboxyl-group proved to be the most efficient derivatization procedure. Hair extracts were redissolved in acetonitrile and after addition of few milligrams of solid sodium carbonate derivatized with 25 µL methyl iodide. The resulting THC-9-carboxymethylester was separated by conventional reverse phase LC and selectively detected using negative electrospray ionization by recording the fragmentation reactions 357➔325 and 357➔297. Resulting limits of quantification were below 100 fg/mg. A further significant improvement was achieved by application of the multistage MS3 fragmentation 357➔325➔297. To verify the validity of this procedure, a systematic quantitative comparison of THCCOOH concentrations in hair with data from a well established GC-NCI-MS/MS technique was performed. Both techniques proved to be in good accordance (R(2)=0.647, p = <0.001) and equally suitable for hair testing of THCCOOH.

Hair Analysis for Determination of Isoniazid Concentrations and Acetylator Phenotype during Antituberculous Treatment.

Background. Analysis of isoniazid (INH) uptake has been based on measurement of plasma concentrations providing a short-term and potentially biased view. Objectives. To establish hair analysis as a tool to measure long-term uptake of INH and to assess whether acetylator phenotype in hair reflects N-acetyltransferase-2 (NAT2) genotype. Design and Methods. INH and acetyl-INH concentrations in hair were determined in patients on INH treatment for M. tuberculosis infection using high pressure liquid chromatography/mass spectrometry. Acetyl-INH/INH ratios were correlated with NAT-2 genotype. Results. Hair concentrations of INH, determined in 40 patients, were not dependent on ethnic group or body mass index and were significantly higher in male compared to female patients (median (range) 2.37 ng/mg (0.76-4.9) versus 1.11 ng/mg (0.02-7.20) (P = 0.02). Acetyl-INH/INH ratios were a median of 15.2% (14.5 to 31.7) in homozygous rapid acetylator NAT-2 genotype and 37.3% (1.73 to 51.2) in the heterozygous rapid acetylator NAT-2 genotype and both significantly higher than in the slow acetylator NAT-2 genotype with 5.8% (0.53 to 14.4) (P < 0.05). Conclusions. Results of hair analysis for INH showed lower concentrations in females. Acetyl-INH/INH ratios were significantly lower in patients with slow acetylator versus rapid acetylator genotypes.

Acute demyelination in a person with amphetamine abuse.

We report the case of a 31-year-old woman, admitted to the hospital for chest pain, dying a few days later from septic multiorgan failure, and showing at autopsy foci of acute demyelination in the occipital lobe. Gas chromatography/mass spectrometry analysis revealed the presence of amphetamine in the demyelinated area, which might be considered as the pathogenic agent, since other causes for demyelination could be excluded. This case represents the first report showing a demyelinating process due to a street drug.

Changes in antidepressant metabolism in pregnancy evidenced by metabolic ratios in hair: a novel approach.

BACKGROUND: Depression and other psychiatric illnesses are common during pregnancy and are often treated with antidepressants. Physiological changes of pregnancy may alter the pharmacokinetics of medications and ultimately affect the dose required to maintain effective therapy. Human hair offers a safe, non-invasive way to monitor long term systemic exposures to medications. OBJECTIVE: To determine whether the ratio of hair antidepressant: major metabolite differed when early and late pregnancy was compared to the postpartum period. METHODS: Segmental analyses using liquid chromatography-mass spectrometry-mass spectrometry were performed on hair samples. The mean concentration of parent compound and metabolite was found for each trimester and the postpartum period. RESULTS: Twelve women provided hair samples of which nine samples were long enough to analyze the first and third trimesters along with the postpartum period. Citalopram, venlafaxine, fluoxetine and sertraline were the antidepressants studied. In the citalopram group, a statistically significant difference existed between the citalopram:norcitalopram ratio when the first trimester was compared to the postpartum period (0.89+/-0.26 versus 1.4+/-0.24 respectively, p=0.022). A statistically significant difference also existed between the third trimester and the postpartum period for the citalopram group (0.9+/-0.14 and 1.4+/-0.24 respectively, p=0.048). No other statistically significant differences were found. CONCLUSION: It is important that variations in drug metabolism during pregnancy be considered as these changes may necessitate a dosage adjustment to ensure that therapeutic failure does not occur during pregnancy.

Formation of the N-methylpyridinium ether derivative of propofol to improve sensitivity, specificity and reproducibility of its detection in blood by liquid chromatography-mass spectrometry.

In spite of comparatively high therapeutic concentrations in blood, the quantitation of propofol may cause analytical challenges due to its pharmacokinetic (e.g. short half life, high distribution volume) and physicochemical (significant volatility) particularities. Moreover, a considerable and concentration dependent protein binding and a substance association to erythrocytes result in an irreproducible distribution between whole blood and serum. A possible redistribution in stored samples needs to be compensated during sample preparation or included into the result interpretation. The new analytical approach is based on a formation of N-methylpyridinium derivatives of propofol and corresponding internal standards and permits a significant ( approximately 300 fold) increase of detection limits in LC-MS/MS. Derivatization is achieved by a direct conversion of the acetonitrile supernatant of a protein precipitation with 2-fluoro-1-methyl-pyridinium-p-toluene-sulfonate using triethylamine as catalyst. The derivative exhibits a high solvent stability and provides--in contrast to the unchanged parent compound--a sufficient number of diagnostic qualifier fragments to fulfil common identification criteria. By using 2-tert-butyl-6-methylphenol as internal standard (instead of the commonly applied thymol), a better compensation of matrix effects could be achieved. Owing to its high robustness, appropriate quantitation limits (LLOQ approximately 13 ng/mL), minimum sample amount and preparation effort, the assay could efficiently be applied for quantitative propofol analyses in pharmacokinetic studies with high sampling rates.

Ethylglucuronide as a potential marker for alcohol-induced elevation of urinary testosterone/epitestosterone ratios.

The potential influence of alcohol consumption on endogenous steroids has already been described in the literature. In those studies the ethanol level after ingestion was monitored using its concentration in blood, urine or saliva. Corresponding methods are not commonly used in anti-doping laboratories. Ethylglucuronide (EtG), which can be easily detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), appears to be a more suitable parameter for this purpose. It is slowly excreted into the urine and indicates alcohol intake for a much longer period than blood or urinary alcohol and it is therefore routinely used for legal purposes as an alcohol consumption marker. In pharmacokinetic studies that aimed to establish calculation models after ethanol intake, the formation of EtG was observed to coincide with elevated urinary testosterone/epitestosterone (T/E) ratios. Similarly, large amounts of EtG were correlated with abnormal steroid profiles found in routine doping samples. In this pilot study, several cases with significantly elevated T/E ratios were associated with urinary EtG concentrations higher than 50 microg/mL. These findings confirmed recent intake of ethanol in considerable amounts and suggest a connection to changes in specific steroid profile parameters. Owing to the ease with which procedures to determine EtG can be carried out, and the potential for such procedures to be introduced into screening schemes, the inclusion of this marker in the final evaluation of suspicious outliers in T/E ratio longitudinal studies would seem to be very useful.