Cocktail approach for in vivo phenotyping of 5 major CYP450 isoenzymes: development of an effective sampling, extraction, and analytical procedure and pilot study with comparative genotyping.

In this study, the authors developed a phenotyping method for CYP1A2, 2C9, 2C19, 2D6, and 3A4 using a cocktail of 100 mg caffeine, 125 mg tolbutamide, 20 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. A simple sampling scheme was established collecting 3 blood samples at 0, 4, and 24 hours followed by solid-phase extraction and liquid chromatography/tandem mass spectrometry analysis. After addition of 8 deuterated internal standards and extraction, the analytes were separated using gradient elution with ammonium acetate and methanol. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple-reaction monitoring mode with positive electrospray ionization. The assay was validated according to international guidelines: limits of quantification (LOQs) were between 0.25 and 1.0 ng/mL for all analytes, except for paraxanthine and caffeine (20 ng/mL). Extraction efficiencies ranged between 77% and 103% and matrix effects between 23% and 95%; precision and accuracy data fulfilled accepted criteria. Calibration curves from LOQ to 1000 ng/mL were established for undiluted and 1:10 diluted plasma (r > 0.998). The method was tested in a pilot study with 14 volunteers. Additional genotyping of the probands generally demonstrated good accordance with the measured phenotyping indices but also disclosed certain contradictory results.

Quality evaluation of tandem mass spectral libraries.

Tandem mass spectral libraries are gaining more and more importance for the identification of unknowns in different fields of research, including metabolomics, forensics, toxicology, and environmental analysis. Particularly, the recent invention of reliable, robust, and transferable libraries has increased the general acceptance of these tools. Herein, we report on results obtained from thorough evaluation of the match reliabilities of two tandem mass spectral libraries: the MSforID library established by the Oberacher group in Innsbruck and the Weinmann library established by the Weinmann group in Freiburg. Three different experiments were performed: (1) Spectra of the libraries were searched against their corresponding library after excluding either this single compound-specific spectrum or all compound-specific spectra prior to searching; (2) the libraries were searched against each other using either library as reference set or sample set; (3) spectra acquired on different mass spectrometric instruments were matched to both libraries. Almost 13,000 tandem mass spectra were included in this study. The MSforID search algorithm was used for spectral matching. Statistical evaluation of the library search results revealed that principally both libraries enable the sensitive and specific identification of compounds. Due to higher mass accuracy of the QqTOF compared with the QTrap instrument, matches to the MSforID library were more reliable when comparing spectra with both libraries. Furthermore, only the MSforID library was shown to be efficiently transferable to different kinds of tandem mass spectrometers, including "tandem-in-time" instruments; this is due to the coverage of a large range of different collision energy settings-including the very low range-which is an outstanding characteristics of the MSforID library.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples.

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Development of an LC-MS/MS method for the quantitation of 55 compounds prescribed in combined cardiovascular therapy.

This paper reports an LC-MS/MS method with positive electrospray ionization for the screening of commonly prescribed cardiovascular drugs in human plasma, including compounds with antihypertensive (57), antidiabetic (12), hypolipemiant (5), anticoagulant (2) and platelet anti-aggregation (2) effects. Sample treatment consisted of a simple protein precipitation with MeOH/0.1 M ZnSO4 (4:1, v/v) solution after the addition of internal standard, followed by evaporation and reconstitution. Analytes separation was performed on a Polar-RP column (150 m x 2 mm, 4 µm) using a gradient elution of 15 min. The MS system was operated in MRM mode, monitoring one quantitation and one confirmation transition for each analyte. The recovery of the protein precipitation step ranged from 50 to 70% for most of the compounds, while some were considerably affected by matrix effects. Since several analytes fulfilled the linearity, accuracy and precision values required by the ICH guidelines, the method proved to be suitable for their quantitative analysis. The limits of quantitation varied from 0.38 to 9.1 µg/L and the limits of detection from 0.12 to 5.34 µg/L. The method showed to be suitable for the detection of plasma samples of patients under cardiovascular treatment with the studied drugs, and for 55 compounds reliable quantitative results could be obtained.

Monitoring of herbal mixtures potentially containing synthetic cannabinoids as psychoactive compounds.

Herbal mixtures like 'Spice' with potentially bioactive ingredients were available in many European countries since 2004 and are still widely used as a substitute for cannabis, although merchandized as 'herbal incense'. After gaining a high degree of popularity in 2008, big quantities of these drugs were sold. In December 2008, synthetic cannabinoids were identified in the mixtures which were not declared as ingredients: the C(8) homolog of the non-classical cannabinoid CP-47,497 (CP-47,497-C8) and a cannabimimetic aminoalkylindole called JWH-018. In February 2009, a few weeks after the German legislation put these compounds and further pharmacologically active homologs of CP-47,497 under control, another cannabinoid appeared in 'incense' products: the aminoalkylindole JWH-073. In this paper, the results of monitoring of commercially available 'incense' products from June 2008 to September 2009 are presented. In this period of time, more than 140 samples of herbal mixtures were analyzed for bioactive ingredients and synthetic cannabimimetic substances in particular. The results show that the composition of many products changed repeatedly over time as a reaction to prohibition and prosecution of resellers. Therefore neither the reseller nor the consumer of these mixtures can predict the actual content of the 'incense' products. As long as there is no possibility of generic definitions in the controlled substances legislation, further designer cannabinoids will appear on the market as soon as the next legal step has been taken. This is affirmed by the recent identification of the aminoalkylindoles JWH-250 and JWH-398. As further cannabinoids can be expected to occur in the near future, a continuous monitoring of these herbal mixtures is required. The identification of the synthetic opioid O-desmethyltramadol in a herbal mixture declared to contain 'kratom' proves that the concept of selling apparently natural products spiked with potentially dangerous synthetic chemicals/pharmaceuticals is a continuing trend on the market of 'legal highs'.

LC-MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum.

Since the late 1990s and early 2000s, derivatives of well-known designer drugs as well as new psychoactive compounds have been sold on the illicit drug market and have led to intoxications and fatalities. The LC-MS/MS screening method presented covers 31 new designer drugs as well as cathinone, methcathinone, phencyclidine, and ketamine which were included to complete the screening spectrum. All but the last two are modified molecular structures of amphetamine, tryptamine, or piperazine. Among the amphetamine derivatives are cathinone, methcathinone, 3,4-DMA, 2,5-DMA, DOB, DOET, DOM, ethylamphetamine, MDDMA, 4-MTA, PMA, PMMA, 3,4,5-TMA, TMA-6 and members of the 2C group: 2C-B, 2C-D, 2C-H, 2C-I, 2C-P, 2C-T-2, 2C-T-4, and 2C-T-7. AMT, DPT, DiPT, MiPT, DMT, and 5MeO-DMT are contained in the tryptamine group, BZP, MDBP, TFMPP, mCPP, and MeOPP in the piperazine group. Using an Applied Biosystems LC-MS/MS API 365 TurboIonSpray it is possible to identify all 35 substances. After addition of internal standards and mixed-mode solid-phase extraction the analytes are separated using a Synergi Polar RP column and gradient elution with 1 mM ammonium formate and methanol/0.1% formic acid as mobile phases A and B. Data acquisition is performed in MRM mode with positive electro spray ionization. The assay is selective for all tested substances. Limits of detection were determined by analyzing S/N-ratios and are between 1.0 and 5.0 ng/mL. Matrix effects lie between 65% and 118%, extraction efficiencies range from 72% to 90%.

ESI-MS/MS library of 1,253 compounds for application in forensic and clinical toxicology.

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions-in some cases solutions made of tablets-were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.

Determination of basic drugs of abuse in human serum by online extraction and LC-MS/MS.

A new quantitation method for the determination of drugs of abuse (opiates, amphetamine and derivatives, cocaine, methadone and metabolites) in serum by using online extraction coupled to liquid chromatography (LC)-mass spectrometry (MS)/MS has been developed. The online extraction is carried out using two extraction columns simultaneously and one analytical column. One extraction column is loaded, while the other one is eluted by a gradient. The elution gradient also separates the analytes in the analytical column. For the sample preparation, serum is spiked with a mixture of deuterated analogues of the drugs. After protein precipitation with methanol/zinc sulphate, centrifugation, evaporation and reconstitution, the sample is injected into the LC system. The quantitation is based on the analysis of two multiple reaction monitoring transitions per drug. The recovery of the protein precipitation step is over 80% for all analytes. Intra- and interday precision, as relative standard deviation, is lower than 6%, and in the case of accuracy, RE is lower than 15%. Only the most polar analytes showed matrix effects. The limits of quantitation for the analysed compounds vary between 0.5 and 2.8 ng/mL. The developed method was used to quantify basic drugs in samples "from driving under the influence of drugs" cases. The results were compared with those obtained by using solid-phase extraction-GC-MS.

Fatal and severe codeine intoxication in 3-year-old twins--interpretation of drug and metabolite concentrations.

This work presents two cases of codeine intoxication in 3-year-old monozygotic twin brothers while treated with a codeine slow-release formulation. One child had to be admitted to the hospital, whereas the other one died at home after aspiration of gastric content. The concentrations of codeine and major metabolites including morphine and corresponding glucuronide conjugates were measured by liquid chromatography-tandem mass spectrometry in serum, urine, cerebrospinal fluid, and brain tissue, respectively. A genetic polymorphism study was carried out in order to determine the ability of the children to metabolize codeine by O-demethylation. A pharmacokinetic calculation was also performed to estimate the administered dose of codeine in question. High concentrations of all substances were found in samples of both children. The pharmacokinetic estimate suggests an overdose of codeine, and the possible reasons for the high opiate concentrations are discussed. Furthermore, the postmortem distribution--during and after resuscitation--might play a major role in the interpretation of postmortem concentration levels.

Kinetics in serum and urinary excretion of ethyl sulfate and ethyl glucuronide after medium dose ethanol intake.

The direct ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), are of increasing importance for clinical and forensic applications, but there are only few studies on the kinetics of EtG in serum and none on EtS. In this study, 13 volunteers (social drinkers) drank ethanol in the form of white wine to reach a blood alcohol concentration of 0.51 +/- 0.17 g/kg, and blood and urine samples were analyzed for EtG and EtS simultaneously by chromatography-tandem mass spectrometry (LC-MS/MS). Mean peak serum EtG and EtS concentrations were 2.9 +/- 1.3 and 2.8 +/- 1.6 micromol/l, respectively, and were reached between 4.0 +/- 0.9 h after the start of drinking (3.0 +/- 0.5 h for EtS). The mean time differences between reaching maximum blood ethanol levels and serum metabolite levels were 2.3 +/- 0.9 h for EtG and 1.2 +/- 0.5 h for EtS. In the last blood samples collected (10-11 h after the start of drinking), 11 (of 13) volunteers were still positive for EtG in serum, whereas only 2 were positive for EtS. In the serum of one female person, no EtS was detectable at any time; however, it was excreted in the urine in (low) concentrations. Ethanol was detectable in the serum for up to 8.6 h after the start of drinking, whereas EtG and EtS were detectable up to more than 5.8 h (EtG) and 4.0 h (EtS), respectively. Mean peak urinary concentrations were 401 +/- 232 micromol/l for EtG and 266 +/- 153 micromol/l for EtS, and mean peak levels were reached 6.2 +/- 0.9 h (EtG) and 5.3 +/- 1.2 h (EtS) after the start of drinking. Maximum concentrations of EtG and EtS in serum showed a wide interindividual variation and could not be correlated to the maximum blood ethanol concentrations. Correlations (p < 0.001, Kendall's Tau b) were found when comparing pairs of parameters, but mostly involved areas under the curve (AUC) of metabolites or of ethanol; one correlation linked the peak concentrations of EtG and EtS in urine.

Validated quantitation of angiotensin II receptor antagonists (ARA-II) in human plasma by liquid-chromatography-tandem mass spectrometry using minimum sample clean-up and investigation of ion suppression.

For the quantitation of angiotensin II receptor antagonists (ARA-II) in human plasma, a method using liquid-chromatography (LC)-electrospray ionization tandem mass spectrometry (MS/MS) has been developed with respect to simple sample clean-up and investigation of ion suppression effects. For sample preparation, protein precipitation using zinc sulphate and methanol showed advantages in speed, recovery, and reproducibility over solid-phase extraction. A triple quadrupole mass spectrometer (Sciex API 365) with turbo ionspray source was used for detection of compounds with multireaction monitoring (MRM) of two transitions per compound. Suppression effects caused by endogenous matrix compounds were investigated by post-column infusion of analytes and LC analysis of precipitates of blank plasma samples and could be excluded. A validation was performed for the ARA-II drugs (valsartan, irbesartan, losartan and its active metabolite EXP 3174, eprosartan, candesartan, and telmisartan). The developed method showed good intra- and interday precision (<12% relative standard deviation) and accuracy (<11.5% bias) at different concentrations for all the studied compounds. The calculated lower limits of quantitation were between 7 and 13 ng/mL, and the compounds were stable during the analytical process. These rather expensive drugs against hypertension are prescribed with increasing numbers in Europe and the industrialized nations. Complications might arise from overdosage or metabolic disorders. However, drug monitoring is not usually performed. Because the therapeutic concentrations range from a few nanograms to hundreds of nanograms per milliliter for the different drugs, and they are not amenable to gas chromatography/MS analysis because of their high molecular weight and polarity, the LC-MS/MS method is the golden standard for therapeutic drug monitoring and for clinical and forensic toxicology of ARA-II drugs.

Hydrolysis and transesterification reactions of candesartan cilexetil observed during the solid phase extraction procedure.

Candesartan cilexetil is an angiotensin receptor antagonist widely used in the treatment of high blood pressure. This prodrug is metabolised into candesartan, which blocks the receptors AT1 for angiotensin II decreasing the blood pressure levels. During the development of a solid phase extraction procedure for the chromatographic determination of eight antihypertensive compounds, lack of linearity and reproducibility was observed only for candesartan cilexetil. Due to this fact, a stability study for this prodrug was performed. It showed that the lack of linearity and reproducibility was based on hydrolysis and transesterification processes which occurred during the drying step after elution with methanol into glass tubes. These phenomena could be reproduced artificially under basic conditions, which demonstrated the presence of basic residues in glass tubes. The study of this potential hydrolysis and transesterification reactions is very important to assure that labile drugs containing ester groups remain unaffected.

Electrospray-ionization MS/MS library of drugs as database for method development and drug identification.

An ESI MS/MS library of 800 compounds has been developed and a collection of data is now available for Analyst 1.4 and higher. Compounds include forensically important drugs, such as illegal drugs, some deuterated analogues, hypnotics, amphetamines, benzodiazepines, neuroleptics, antidepressants and many others. For setting up the library of product ion spectra, 20-200 ng of the compounds have been injected either by flow injection or via a short LC-column, the precursor ions were chosen from the Q1 scan spectra, and product ion spectra were generated by CID in the collision cell using three different collision energies (20, 35 and 50 eV). Three spectra of each compound have been collected and compound names, CAS numbers, formulas and molecular weights have been added in the database, which has been generated by the Analyst software. The library can be used for compound identification during general unknown screening analysis by combination of Q1 scan techniques and subsequent MS/MS analysis in a second analytical run. Quantitative procedures for multi drug analysis using Multiple Reaction Monitoring can be established by selection of product ions and suitable collision energies from the library. For publication of the spectra, PDF-files have been generated and can be viewed on-line as supplementary data or from the website in alphabetical order: (supplementary data, should be made available via ELSEVIER-WEBSITE or via ).

Ethyl sulphate: a direct ethanol metabolite reflecting recent alcohol consumption.

BACKGROUND: Ethyl sulphate (EtS), a direct ethanol metabolite, appears to offer potential as a biomarker for recent alcohol consumption. Although its window of assessment is similar to that of ethyl glucuronide (EtG), there are differences between the two markers in their pathways for formation and degradation. AIMS: (a) To assess the excretion of EtS compared to EtG and ethanol in drinking experiments with healthy volunteers, and (b) to elucidate the possibility of using the two metabolites for monitoring abstinence in substance use disorder patients during rehabilitation treatment. DESIGN, SETTING, PARTICIPANTS: (a) Nine drinking experiments were performed by six healthy volunteers (two females, four males), with a mean age of 34.1 years (20-62), average oral intake of 0.2 g/kg ethanol (0.1-0.61), and having 74 spot urine samples. (b) Thirty-six substance abuse patients (mean age 41.9 years, 20-59; 22 males, 14 females) in a rehabilitation programme after withdrawal, producing 98 urine samples. Ethyl glucuronide and ethyl sulphate were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) using d5-EtG and d5-EtS, respectively, as an internal standard. FINDINGS: (a) VOLUNTEERS: EtG and EtS were detectable for up to 36 hours and reached the limits of determination in urine at 20.6 hours and 21.2 hours (median), respectively, after ethanol intake. EtG-100 (standardized to a creatinine of 100 mg/dl) reached its maximum level at 2.8 hours and EtS-100 at 2.1 hours (median) after the beginning of the experiment. Of the ethanol ingested, 0.022% was excreted as EtS in one volunteer. Eight samples were positive for EtS only and six for EtG only. Spearman's rank correlation coefficients of 0.84 (P < 0.0001) between EtG and EtS and 0.87 (P < 0.0001) between EtG-100 and EtS-100 were found. (b) PATIENTS: of the 98 urine samples evaluated, 27 were positive for EtS and of these only 20 were also positive for EtG. Spearman's rank correlation coefficients of 0.84 (P < 0.0001) between EtG and EtS and 0.82 (P < 0.0001) between EtG-100 and EtS-100 were found. CONCLUSIONS: The data from patients and volunteers suggest that the direct ethanol metabolite ethyl sulphate has the potential to serve as a biomarker of recent ethanol intake. Because EtG and EtS are formed via different pathways they might be used conjointly, thereby increasing sensitivity.

Stability of ethyl glucuronide in urine, post-mortem tissue and blood samples.

The stability of ethyl glucuronide (EtG) under conditions of degradation was examined in urine samples of nine volunteers and in post-mortem tissue (liver, skeletal muscle) and blood taken from seven corpses at autopsies. Analysis was performed via LC-MS/MS. EtG concentrations in urine samples ranged from 2.5 to 296.5 mg/l. When stored at 4 degrees C in airtight test tubes, EtG concentrations remained relatively constant; when stored at room temperature (RT) for 5 weeks in ventilated vials, variations of EtG concentrations ranged from a 30% decrease to an 80% increase, with an average of 37.5% increase. Liver and skeletal muscle tissue of three corpses with positive blood alcohol concentrations (BAC; ranging from 0.106 to 0.183 g%) were stored for 4 weeks and analysed periodically. EtG concentrations decreased 27.7% on average in 4 weeks storage at RT but EtG was still detectable in all samples with initial EtG concentrations higher than 1 mug/g. Blood and liver samples of four corpses with negative BACs were stored at RT after addition of 0.1 g% ethanol, and no new formation of EtG was observed.

Forensic confirmatory analysis of ethyl sulfate--a new marker for alcohol consumption--by liquid-chromatography/electrospray ionization/tandem mass spectrometry.

Ethyl sulfate (EtS)--a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others--was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M - H]- m/z 125 and product ions m/z 97 [HSO4]- and m/z 80 [SO3]-. Pentadeuterated EtS (D5-EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M - H]- m/z 130, product ions m/z 98 [DSO4]- and m/z 80 [SO3]-). After addition of D5-EtS and D5-EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers' urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs.