Toxicological detection of ethylenediamine and piperazine antihistamines and their metabolites in urine by computerized gas chromatography-mass spectrometry

Summary A gas chromatographic-mass spectrometric procedure for the detection of the following ethylenediamine and piperazine antihistamines and their metabolites in urine after acid hydrolysis, extraction and acetylation is described: Adeptolon, antazoline, bamipine, buclizine, chlorcyclizine, chloropyramine, cinnarizine, clemizole, cyclizine, etodroxizine, histapyrrodine, hydroxyzine, meclozine, mepyramine, oxatomide and tripelenamine. The acetylated extract was analysed by computerized gas chromatography-mass spectrometry. Using ion chromatography with the selective ions m/z 58, 72, 85, 125, 165, 183, 198 and 201 the possible presence of ethylenediamine or piperazine antihistamines or their metabolites was indicated. The identity of positive signals in the reconstructed ion chromatograms was then confirmed by a visual or computerized comparison of the stored full mass spectra with the reference spectra. The ion chromatograms, reference mass spectra and gas chromatographic retention indices (OV-101) are documented. The procedure presented is integrated in a general screening procedure (general unknown analysis) for several groups of drugs.

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Toxikologischer Nachweis von Ethylendiamin- und Piperazin-Antihistaminica und ihren Metaboliten im Urin mittels computerunterstützter Gas-Chromatographie-Massenspektrometrie

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Part of these results was reported at the Symposium Klinisch-Toxikologische Analytik of the Austrian and German Societies of Clinical Chemistry, Salzburg, Austria, September 14–16, 1987 [20]     

Mass spectrometry of the trimethylsilyl derivatives of medicinal barbiturates

The electron ionization mass spectra of the trimethylsilyl derivatives of a number of medicinal barbiturates have been examined. The trimethylsilyl derivatives are useful in the identification of barbiturates by gas liquid chromatography/mass spectrometry (g.l.c.-m.s.). Mass spectra of these derivatives are characterized by abundant high mass ions which permit the ready distinction of one barbiturate from another. The prominent and characteristic [M - 15] ion is an effective monitor of molecular weight, while other unique fragmentations and/or varying patterns of relative abundance serve to differentiate isomers.     

固相萃取/离子阱气相色谱质谱联用对血液中多种安眠药物的同时测定

建立了气相色谱-质谱联用测定血液中多种安眠药物的分析方法,通过固相萃取提取并富集血液样品中的9种常见巴比妥类、吩噻嗪类和苯二氮杂类安眠药物,采用离子阱二级质谱检测其含量。通过优化萃取溶液pH值及气相色谱-质谱联用分析条件,对9种安眠药物进行了定量分析。采用离子阱串联质谱(MS/MS)技术消除基底干扰,给出被测物结构信息。9种安眠药物的检出限为0.04~0.10 mg/L,回收率为78%~93%。方法用于人心血中安眠药物的检测,检出安定0.6 mg/L。该方法高效、简单、灵敏度高,可用于血液中巴比妥类、吩噻嗪类和苯二氮杂类安眠药物的同时测定。     

Capillary gas chromatographic separation of urinary organic acids. Retention indices of 101 urinary acids on a 5% phenylmethyl silicone capillary column

Gas chromatographic retention indices (methylene units) are reported for 101 urinary organic acids as their trimethylsilyl and oximated trimethylsilyl derivatives on a 5% phenylmethyl silicone fused silica capillary column. Using anion exchange chromatography, organic acids were extracted from urines of five healthy individuals, seven patients with neuroblastoma, and nine patients with inherited organic acidurias. Separation of the various acids was achieved by capillary gas chromatography and identification was done by mass spectrometry using a computerized library search program. All identifications were confirmed by visual comparison with reference mass spectra. Standard deviations of the retention indices for all acids were less than 0.035 methylene units and for 46 acids less than 0.01 methylene units. Three chromatograms of urine from individuals with neuroblastoma, phenylketonuria, and propionic acidemia and one from a healthy individual are shown.     

Screening procedure for detecting butyrophenone and bisfluorophenyl neuroleptics in urine using a computerized gas chromatographic-mass spectrometric technique

A method for the identification of butyrophenone and bisfluorophenyl neuroleptics and their predominant basic metabolites in urine after acid hydrolysis is described. The acetylated extract is analysed by computerized gas chromatography-mass spectrometry. An on-line computer allows rapid detection using mass fragmentography with the masses m/e 112, 123, 134, 148, 169, 257, 321 and 189, 191, 223, 233, 235, 245, 287, 297. The identity of positive signals in the reconstructed mass fragmentograms is established by a comparison of the entire mass spectra with those of standards. The mass fragmentograms and the underlying mass spectra are documented.     

Development of a multi-target screening analysis for 301 drugs using a QTrap liquid chromatography/tandem mass spectrometry system and automated library searching

A new multi-target screening (MTS) procedure for drugs in blood and urine for toxicological analysis has been developed using a hybrid triple-quadrupole linear ion trap mass spectrometer (QTrap) for the fast detection and identification of 301 forensically important drugs, e.g. tranquilizers (benzodiazepines), hypnotics, drugs of abuse (opiates, cocaine, amphetamines, cannabinoids), antidepressants, neuroleptics, and some cardiac drugs, in one single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Samples were extracted either with liquid-liquid extraction or solid-phase extraction. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information-dependent acquisition (IDA) experiment. Finally, drug identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The advantage of this newly developed method is the possibility to detect and identify 301 drugs in one single LC/MS/MS run.     

Scanning thin-layer chromatography-liquid secondary ion mass spectrometry and its application for investigation of drug metabolites

Thin-layer chromatography-mass spectrometry including both direct and scanning thin-layer chromatography-liquid secondary ion mass spectrometry, has been used to obtain good separation and good mass spectra of non-volatile and thermally unstable mixtures. The methods are very simple, useful and easy to perform, and they can overcome the problems associated with indirect and direct coupling of liquid chromatography and mass spectrometry. Scanning thin-layer chromatography-liquid secondary ion mass spectrometry can be carried out like gas chromatography-mass spectrometry, and gives good quality chromatograms and mass spectra. These methods were used to analyse drug metabolites and mixtures of other organic compounds.     

Systematic toxicological analysis of drugs and their metabolites by gas chromatography-mass spectrometry.

Gas chromatographic-mass spectrometric (GC-MS) procedures for the systematic toxicological analysis of several categories of drugs relevant to clinical toxicology, forensic toxicology and doping control are reviewed. Papers from 1981 to 1991 are taken into consideration. They describe the detection of acute or chronic intoxication and the detection of drug abuse. Screening procedures are included for the following categories: barbiturates and other sedative-hypnotics, anticonvulsants, benzodiazepines, antidepressants, phenothiazine and butyrophenone neuroleptics, central stimulants (amphetamines, cocaine), hallucinogens (LSD, phencyclidine, tetrahydrocannabinol), opioid (narcotic) and other potent analgesics, non-opioid analgesics, antihistamines (histamine H1-receptor blockers), antiparkinsonian drugs, beta-blockers (beta-adrenoceptor blockers), antiarrhythmics (class I and IV), diuretics, laxatives and their metabolites. Methods for confirmation of results obtained by screening procedures using immunoassay or chromatographic techniques are also included. GC-MS procedures for the simultaneous detection of several categories of drugs, the so-called "general unknown analysis", are reviewed. The toxicological question to be answered and the consequence for the choice of an adequate method, the sample preparation and the chromatography itself are discussed. The basic information about the biosample assayed, work-up, GC column, mass spectral detection mode, reference data and sensitivity of each procedure are summarized in tables, arranged according to the category of drug. Examples of typical GC-MS applications are presented. Fragment ions that are suitable for mass spectral screening for particular categories of drugs and for general unknown are tabulated.     

Accurate mass filtering of ion chromatograms for metabolite identification using a unit mass resolution liquid chromatography/mass spectrometry system

Acceleration of liquid chromatography/mass spectrometric (LC/MS) analysis for metabolite identification critically relies on effective data processing since the rate of data acquisition is much faster than the rate of data mining. The rapid and accurate identification of metabolite peaks from complex LC/MS data is a key component to speeding up the process. Current approaches routinely use selected ion chromatograms that can suffer severely from matrix effects. This paper describes a new method to automatically extract and filter metabolite-related information from LC/MS data obtained at unit mass resolution in the presence of complex biological matrices. This approach is illustrated by LC/MS analysis of the metabolites of verapamil from a rat microsome incubation spiked with biological matrix (bile). MS data were acquired in profile mode on a unit mass resolution triple-quadrupole instrument, externally calibrated using a unique procedure that corrects for both mass axis and mass spectral peak shape to facilitate metabolite identification with high mass accuracy. Through the double-filtering effects of accurate mass and isotope profile, conventional extracted ion chromatograms corresponding to the parent drug (verapamil at m/z 455), demethylated verapamil (m/z 441), and dealkylated verapamil (m/z 291), that contained substantial false-positive peaks, were simplified into chromatograms that are substantially free from matrix interferences. These filtered chromatograms approach what would have been obtained by using a radioactivity detector to detect radio-labeled metabolites of interest.     

Identification of phase I and phase II metabolites of benfluron and dimefluron in rat urine using high-performance liquid chromatography/tandem mass spectrometry

Biotransformation products of two potential antineoplastic agents, benfluron and dimefluron, are characterized using our integrated approach based on the combination of high-performance liquid chromatography (HPLC) separation of phase I and phase II metabolites followed by photodiode-array UV detection and electrospray ionization tandem mass spectrometry (MS/MS). High mass accuracy measurement allows confirmation of an elemental composition and metabolic reactions according to exact mass defects. The combination of different HPLC/MS/MS scans, such as reconstructed ion current chromatograms, constant neutral loss chromatograms or exact mass filtration, helps the unambiguous detection of low abundance metabolites. The arene oxidation, N-oxidation, N-demethylation, O-demethylation, carbonyl reduction, glucuronidation and sulfation are typical mechanisms of the metabolite formation. The interpretation of their tandem mass spectra enables the distinction of demethylation position (N- vs. O-) as well as to differentiate N-oxidation from arene oxidation for both phase I and phase II metabolites. Two metabolic pathways are rather unusual for rat samples, i.e., glucosylation and double glucuronidation. The formation of metabolites that lead to a significant change in the chromophoric system of studied compounds, such as the reduction of carbonyl group in 7H-benzo[c]fluorene-7-one chromophore, is reflected in their UV spectra, which provides valuable complementary information to MS/MS data.     

Screening procedure for the detection of antiparkinsonian drugs and their metabolites in urine using a computerized gas chromatographic-mass spectrometric technique

Summary A method for the identification of antiparkinsonian drugs and their metabolites in urine after acid hydrolysis is described. The acetylated extract is analysed by computerized gas chromatography-mass spectrometry. An on-line computer allows for the rapid detection using mass chromatography with the masses m/z 86, 98, 136, 150, 165, 196, 197 and 208. The identity of positive signals in the reconstructed mass chromatogram is established by a comparison of the stored entire mass spectra with those of standards. The mass chromatogram, the underlying mass spectra and the gas chromatographic retention indices (OV 101) are documented.

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Screening-Verfahren zum Nachweis von Parkinson-Mitteln und ihren Metaboliten im Urin mit Hilfe einer Computerunterstützten GC-MS-Technik

Zusammenfassung Eine Methode zum Nachweis von Parkinson-Mitteln und ihren Metaboliten im Urin nach saurer Hydrolyse wird beschrieben. Der acetylierte Extrakt wird mit der Kopplung Gas-Chromatographie-Massenspektrometrie-Datenverarbeitung analysiert. Ein on-line-Computer erlaubt eine rasche Auswertung durch die Verwendung der Massenchromatographie mit den Massen m/z 86, 98, 136, 150, 165, 196, 197 und 208. Die Verbindungen, die im rekonstruierten Massenchromatogramm angezeigt werden, können durch Vergleich der zugrunde liegenden Massenspektren mit Referenzspektren identifiziert werden. Das Massenchromatogramm, die zugrunde liegenden Massenspektren und die gaschromatographischen Retentionsindices (OV-101) werden gezeigt.

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Dedicated to Prof. Dr. Dr. h. c. mult. Egon Stahl on the occasion of his 60th birthday     

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP® LC-MS/MS system and library searching

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a “scheduled” survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP^® hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization–MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography–mass spectrometry, and liquid chromatography–diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).     

Scope and limitations of a general unknown screening by gas chromatography—mass spectrometry in acute poisoning

The gold standard for the diagnosis of acute poisoning is toxicological analysis. Because information on the incorporated toxic substance provided by the patient or his relatives is known from experience to be unreliable in about 40% of all intoxications, a screening procedure that covers most relevant drugs and toxicants is required rather than an analytical procedure optimized for the identification of a single class of substances. The special task for a general unknown screening procedure is to identify a toxic substance among endogenous or food-derived substances as well as environmental toxicants in a biological matrix on an emergency basis. Because the unknown toxic substance may vary considerably in its physicochemical properties and its concentration range, a universally applicable screening procedure is required. Although gas chromatography—mass spectrometry has been used for three decades, it still offers many unique advantages in terms of sensitivity, specificity, reliability, and coverage of a large number of toxic substances. Because the procedure has to be kept as simple and as short as possible, compromises have to be made with respect to extraction, derivatization, and mass-spectral techniques. The specimen of choice for a general unknown screening is—if available—urine. The standard mode of ionization is electron impact. The identification of unknown substances is highly challenging because, in our experience, previously unknown metabolites may be detected rather frequently in acute poisoning. Although an automated mass spectra library search considerably facilitates the identification process, expert knowledge on the identification of substances not included in the library as well as knowledge in clinical toxicology and metabolism is indispensable.     

Massenspektrometrischer Nachweis von Arzneimittelmetaboliten (Barbiturate, Noludar, Pyramidon) im Rahmen der forensischen Analyse

Zusammenfassung Die massenspektrographische Identifizierung von aus biologischem Material isolierten Metabolitensubstanzen verschiedener Arzneimittel (Barbiturate, Noludar, Pyramidon) wird an Hand der einzelnen Massenspektren und ihrer daraus abgeleiteten Zerfallsschemata besprochen. In Zukunft wird die Massenspektrographie, vor allem kombiniert mit der Gas-Chromatographie, sicherlich mehr als bisher mit dazu beitragen, viele, häufig sehr differenzierte Probleme des Arzneimittelmetabolismus zu klären.

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Mass-spectrometric detection of drug · metabolites (barbiturates, noludar, pyramidon) in forensic analysis

The mass-spectrometric identification of the metabolites isolated from biological material is discussed by means of the single mass spectra and of fragmentation patterns derived from them. In future, mass spectrography, mainly in combination with gas chromatography, wil certainly more than hitherto contribute to the elucidation of many difficult problems of drug metabolism.

     

Generic dealkylation: a tool for increasing the hit‐rate of metabolite rationalization,and automatic customization of mass defect filters

The use of exact mass liquid chromatography/mass spectrometry (LC/MS) for drug metabolism studies has increased significantly in recent years. Firstly, exact mass measurements facilitate identification of standard biotransformations through the use of narrow window extracted ion chromatograms, which are typically highly selective relative to signals from matrix or dosing components. Secondly, novel metabolites can be characterized via elemental formula calculations and high‐resolution product ion spectra. Furthermore, biological background ions can be removed by the use of mass defect filters (MDFs) which filter out ions based on the decimal component of their m/z value. Here, we describe an approach which we term ‘generic dealkylation’ that in association with other data interpretation tools adds significant value to the assignment process. Generic dealkylation uses a simple strategy to identify those bonds which have the potential to be cleaved by metabolism. In combination with standard phase 1 and phase 2 biotransformations, this allows creation of a chemically intelligent MDF which balances the need to remove matrix background with the requirement of avoiding filtering true metabolites. Secondly, generic dealkylation increases the hit‐rate at which non‐trivial (i.e. not covered by simple phase 1 oxidations or direct phase 2 conjugations) metabolites can be directly rationalized. The value of the generic dealkylation approach is illustrated by its application to determination of in vitro metabolic routes for two commercial drugs, nefazodone and indinavir. Copyright © 2009 John Wiley & Sons, Ltd.     

In silico methods for predicting metabolism and mass fragmentation applied to quetiapine in liquid chromatography/time‐of‐flight mass spectrometry urine drug screening

Current in silico tools were evaluated for their ability to predict metabolism and mass spectral fragmentation in the context of analytical toxicology practice. A metabolite prediction program (Lhasa Meteor), a metabolite detection program (Bruker MetaboliteDetect), and a fragmentation prediction program (ACD/MS Fragmenter) were used to assign phase I metabolites of the antipsychotic drug quetiapine in the liquid chromatography/time‐of‐flight mass spectrometry (LC/TOFMS) accurate mass data from ten autopsy urine samples. In the literature, the main metabolic routes of quetiapine have been reported to be sulfoxidation, oxidation to the corresponding carboxylic acid, N‐ and O‐dealkylation and hydroxylation. Of the 14 metabolites predicted by Meteor, eight were detected by LC/TOFMS in the urine samples with use of MetaboliteDetect software and manual inspection. An additional five hydroxy derivatives were detected, but not predicted by Meteor. The fragment structures provided by ACD/MS Fragmenter software confirmed the identification of the metabolites. Mean mass accuracy and isotopic pattern match (SigmaFit) values for the fragments were 2.40 ppm (0.62 mDa) and 0.010, respectively. ACD/MS Fragmenter, in particular, allowed metabolites with identical molecular formulae to be differentiated without a need to access the respective reference standards or reference spectra. This was well exemplified with the hydroxy/sulfoxy metabolites of quetiapine and their N‐ and O‐dealkylated forms. The procedure resulted in assigning 13 quetiapine metabolites in urine. The present approach is instrumental in developing an extensive database containing exact monoisotopic masses and verified retention times of drugs and their urinary metabolites for LC/TOFMS drug screening. Copyright © 2009 John Wiley & Sons, Ltd.     

Hyphenated mass spectrometric techniques-indispensable tools in clinical and forensic toxicology and in doping control

Hyphenated mass spectrometric techniques, particularly gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), are indispensable tools in clinical and forensic toxicology and in doping control owing to their high sensitivity and specificity. They are used for screening, library-assisted identification and quantification of drugs, poisons and their metabolites, prerequisites for competent expertise in these fields. In addition, they allow the study of metabolism of new drugs or poisons as a basis for developing screening procedures in biological matrices, most notably in urine, or toxicological risk assessment. Concepts and procedures using GC/MS and LC/MS techniques in the areas of analytical toxicology and the role of mass spectral libraries are presented and discussed in this feature article. Finally, perspectives of their future position are discussed.     

气相色谱/质谱结合化学计量学解析方法用于植物精油成分分析

使用气相色谱/质谱(GC-MS)法对缬草精油和含有缬草精油的混配精油的化学成分进行了分析.采用直观推导式演进特征投影法(HELP)对二维数据中的色谱重叠峰进行了解析,得到两种精油的各个物质的纯色谱和纯质谱,同时利用交互移动窗口因子分析法(AMWFA)直接比较两种精油中的共有组分,提取出共有组分的纯质谱,通过与HELP法解析出的质谱进行比较,发现AMWFA法比HELP法能更快速鉴别出混配精油中的缬草精油.     

Purification and quantitative analysis of urinary prostanoids in human and in rat by packed and capillary gas chromatography-negative-ion chemical-ionization mass spectrometry

We describe a method for the quantitative analysis of prostaglandin (PG) E2 and the major urinary metabolites PGI2 and thromboxane (Tx) A2 in human and in rat by combined gas chromatography and negative-ion chemical-ionization mass spectrometry. The procedure is based on the sequential use of small columns with distinct properties combined with a thin-layer chromatography step, for the extraction and the purification of urinary prostaglandins. The compounds are then analysed as their pentafluorobenzyl ester-O-methyloxime-trimethylsilyl ether derivatives, using either packed or capillary columns. Deuterated analogues are used as internal standards. The method was established by using tritiated prostaglandins covering the extremes of polarity in order to optimize the recovery of prostanoids as well as the quality of the chromatograms and spectra. The overall recovery was 24%. Standard curves were obtained by the same procedure and found to be reproducible, with a maximal day-to-day variation of +/- 5%. The relatively simple approach required for the sequential extraction and purification of prostaglandins on small columns of distinct properties, combined with the highly specific and highly sensitive method of detection, places this procedure among the most reliable method for measuring urinary prostanoids in both humans and animals. In addition, the procedure is faster than classical approaches and necessitates smaller amounts of samples and solvents.     

Distinction between melanins derived from different precursors using pyrolysis/gas chromatography/mass spectrometry and the NIST mass spectral search algorithm

A method was required to objectively determine whether various melanins were synthesised from different precursors, and whether synthesis from the same precursor was reproducible. Melanins have a complex, heterogeneous, polymeric structure, making them difficult to characterise and compare. Pyrolysis chromatography may be useful for such large molecules that are not amenable to analysis by other methods, but the resulting chromatograms are usually complex and difficult to compare. Techniques used to objectively differentiate between such chromatograms often employ statistical methods that are difficult to use and interpret without specialised knowledge. Melanins were analysed by pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS). Software was developed to automate the conversion of the resulting total ion current (TIC) chromatograms to pseudo-mass spectra (PMS), consisting of a row vector of chromatographic peak areas (analogous to ion abundances) ordered along a retention time axis (analogous to m/z ratio). The National Institute of Standards and Technology (NIST, USA) mass spectral search program, which is used widely as an objective measure of the degree of similarity between mass spectra, was then used to generate match factors for comparisons of the generated PMS. Match factors between melanins synthesised from the same precursors were not significantly different, while match factors between melanins synthesised from different precursors were significantly smaller. The reproducibility of the pyrolysis technique was reasonable, with the majority of relative standard deviation (RSD) values of match factors from melanins synthesised from the same precursor, being below 10% (n=5 for each melanin type). While the method does not allow the unequivocal identification of individual melanin types in isolation, it can be used to compare melanins from different sources and objectively estimate the degree of similarity between them on the basis of significant differences between their pyrograms.