Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies

The study of the metabolic fate of drugs is an essential and important part of the drug development process. The analysis of metabolites is a challenging task and several different analytical methods have been used in these studies. However, after the introduction of the atmospheric pressure ionization (API) technique, electrospray and atmospheric pressure chemical ionization, liquid chromatography/mass spectrometry (LC/MS) has become an important and widely used method in the analysis of metabolites owing to its superior specificity, sensitivity and efficiency. In this paper the feasibility of LC/API-MS techniques in the identification, structure characterization and quantitation of drug metabolites is reviewed. Sample preparation, LC techniques, isotope labeling, suitability of different MS techniques, such as tandem mass spectrometry, and high-resolution MS in drug metabolite analysis, are summarized and discussed. Automation of data acquisition and interpretation, special techniques and possible future trends are also the topics of the review.     

Electron ionization mass spectral fragmentation of deoxynivalenol and related tricothecenes

Careful analysis of the electron impact (EI) mass spectral data obtained for the trimethylsilyl (TMS) ethers of known trichothecene mycotoxins of the deoxynivalenol group permitted the construction of a database useful for the identification of these mycotoxins directly from a gas chromatography/mass spectrometry (GC/MS) run. Structures of the ions at m/z 103, 117, 147 and 191 were elucidated by high-resolution mass spectrometry (HRMS) and a fragmentation scheme was suggested. The relative abundances of these ions in the mass spectra of the trichothecenes allowed a fast structural diagnosis during analysis of biological matrices. A new mycotoxin of this group, 3-acetylnivalenol, was tentatively identified by using MS data interpretation only.     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Applications of solid-phase microextraction in food analysis

Food analysis is important for the evaluation of the nutritional value and quality of fresh and processed products, and for monitoring food additives and other toxic contaminants. Sample preparation, such as extraction, concentration and isolation of analytes, greatly influences the reliable and accurate analysis of food. Solid-phase microextraction (SPME) is a new sample preparation technique using a fused-silica fiber that is coated on the outside with an appropriate stationary phase. Analyte in the sample is directly extracted to the fiber coating. The SPME technique can be used routinely in combination with gas chromatography (GC), GC–mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC) or LC–MS. Furthermore, another SPME technique known as in-tube SPME has also been developed for combination with LC or LC–MS using an open tubular fused-silica capillary column as an SPME device instead of SPME fiber. These methods using SPME techniques save preparation time, solvent purchase and disposal costs, and can improve the detection limits. This review summarizes the SPME techniques for coupling with various analytical instruments and the applications of these techniques to food analysis.     

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.     

Application of gas and liquid chromatography coupled to time‐of‐flight mass spectrometry in pesticides: Multiresidue analysis

Analysis of pesticide residues in water and food matrices is an active research area closely related to food safety and environmental issues. In this aspect mass spectrometry (MS) coupled to gas chromatography (GC) and liquid chromatography (LC) has been increasingly used in the analysis of pesticide residues in water and food. The increasing interest in application of high‐resolution mass spectrometry with time‐of‐flight (TOF) and hybrid triple quadrupole TOF in pesticide analysis is due to its capability of performing both targeted and nontargeted analysis. This article discusses an overview of the application of GC‐TOF‐MS and LC‐TOF‐MS in water and food matrices.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

Analysis of hydroxylated polybrominated diphenyl ether metabolites by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

Hydroxylated polybrominated diphenyl ether (OH‐PBDEs) metabolites have the potential to cause endocrine disruption as well as other health effects. Currently, gas chromatography/mass spectrometry (GC/MS) after derivatization is used for the analysis of OH‐PBDEs. However, there is a need for the direct analysis of OH‐PBDEs at relatively low concentrations in environmental and biological samples. Liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI‐MS/MS) was evaluated for the analysis of nine OH‐PBDEs, ranging from tri‐ to hexabrominated. Separation of the nine isomeric metabolites was achieved with reversed‐phase liquid chromatography, followed by detection by APCI‐MS in negative mode. Notably, a significant decrease in ionization was observed in 6‐hydroxyl‐substituted PBDE metabolites in the presence of an ortho‐substituted bromine, relative to the other hydroxylated metabolites. This is probably due to the formation of dioxins in the source as a result of the high‐temperature conditions, which prevented ionization by hydrogen abstraction. The MS/MS experiments also provided evidence of the neutral losses of HBr and Br₂, indicating the possible use of neutral loss scanning and selected reaction monitoring (SRM) for the screening of brominated metabolites in samples. The applicability of LC/APCI‐MS/MS was demonstrated for the analysis of metabolites of BDEs 47 and 99 formed in human liver microsomes. The LC/APCI‐MS/MS method was able to detect metabolites that had previously been identified by GC/MS following derivatization. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of two oxy-pyrimidine metabolites of diazinon in urine by gas chromatography/mass selective detection and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry

An analytical method was developed for the determination in urine of 2 metabolites of diazinon: 6-methyl-2-(1-methylethyl)-4(1H)-pyrimidinone (G-27550) and 2-(1-hydroxy-1-methylethyl)-6-methyl-4(1H)-pyrimidinone (GS-31144). Two of the urine sample preparation procedures presented rely on gas chromatography/mass selective detection (GC/MSD) in the selected ion monitoring mode for determination of G-27550. For fast sample preparation and a limit of quantitation (LOQ) of 1.0 ppb, urine samples were purified by using ENV+ solid-phase extraction (SPE) columns. For analyte confirmation at an LOQ of 0.50 ppb, classical liquid/liquid partitioning was used before further purification in a silica SPE column. An SPE sample preparation procedure and liquid chromatography/electrospray ionization/mass spectrometry/mass spectrometry (LC/ESI/MS/MS) were used for both G-27550 and GS-31144. The limit of detection was 0.01 ng for G-27550 with GC/MSD, and 0.016 ng when LC/ESI/MS/MS was used for both G-27550 and GS-31144. The LOQ was 0.50 ppb for G-27550 when GC/MSD and the partitioning/SPE sample preparation procedure were used, and 1.0 ppb for the SPE only sample preparation procedure. The LOQ was 1.0 ppb for both analytes when LC/ESI/MS/MS was used.     

Metabonomics investigation of human urine after ingestion of green tea with gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry and (1)H NMR spectroscopy

A method using gas chromatography/mass spectrometry (GC/MS), liquid chromatography/mass spectrometry (LC/MS) and (1)H NMR with pattern recognition tools such as principle components analysis (PCA) was used to study the human urinary metabolic profiles after the intake of green tea. From the normalized peak areas obtained from GC/MS and LC/MS and peak heights from (1)H NMR, statistical analyses were used in the identification of potential biomarkers. Metabolic profiling by GC/MS provided a different set of quantitative signatures of metabolites that can be used to characterize the molecular changes in human urine samples. A comparison of normalized metabonomics data for selected metabolites in human urine samples in the presence of potential overlapping peaks after tea ingestion from LC/MS and (1)H NMR showed the reliability of the current approach and method of normalization. The close agreements of LC/MS with (1)H NMR data showed that the effects of ion suppression in LC/MS for early eluting metabolites were not significant. Concurrently, the specificity of detecting the stated metabolites by (1)H NMR and LC/MS was demonstrated. Our data showed that a number of metabolites involved in glucose metabolism, citric acid cycle and amino acid metabolism were affected immediately after the intake of green tea. The proposed approach provided a more comprehensive picture of the metabolic changes after intake of green tea in human urine. The multiple analytical approach together with pattern recognition tools is a useful platform to study metabolic profiles after ingestion of botanicals and medicinal plants.     

Mass spectrometric analysis of cyclofenil and its metabolites in human urine

Using gas chromatography/electron impact-mass spectrometry (GC/EI-MS) and high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), the structures of cyclofenil metabolites in human urine have been assigned. The hydroxyl metabolites liberated from the glucuronide conjugates after acid hydrolysis were characterized as the trimethylsilyl (O-TMS) derivatives using GC/MS. The conjugate glucuronide forms were detected without hydrolysis by HPLC/MS. Cyclofenil was not observed in urine. Tentative structures for the two metabolites are proposed.     

Isotope abundance analysis methods and software for improved sample identification with supersonic gas chromatography/mass spectrometry

We present newly developed isotope abundance analysis (IAA) methods and software which are used to derive elemental formula information from experimental mass spectral data of molecular ion isotopomeric abundances. The software, using a novel method, can also be used to automatically confirm or reject NIST library search results, thereby significantly improving the confidence level in sample identifications. In the case of IAA confirmation of the NIST library results, sample identification is unambiguous, since the confirmation is achieved by two independent sets of data and analytical methods. In the case of a rejection, such as when the molecule is not included in the library's databases, the IAA software independently provides a list of elemental formulae with declining order of matching to the isotopomeric experimental data, in a similar way to accurate mass measurements with costly instruments. IAA is ideally applicable to gas chromatography/mass spectrometry (GC/MS) (and liquid chromatography/electron ionization mass spectrometry (LC/EI-MS)) with a supersonic molecular beam (SMB) since it requires a trustworthy and highly abundant true molecular ion that is unique to the SMB-MS systems, plus the absence of self chemical ionization and vacuum background noise, again unique features of GC/SMB-MS. The various features of the IAA methods and software are described, their performance is demonstrated with the analysis of experimental GC-SMB-MS data and the IAA concept is compared with accurate mass alternatives. The combination of IAA and GC/SMB-MS is believed to be superior to accurate mass GC/MS in view of the general availability of trustworthy molecular ions for an extended range of compounds.     

Purity assessment of commercially available crystalline deoxynivalenol

Deoxynivalenol (DON) obtained from 2 commercial sources was characterized, and its purity was determined. The structural identity of DON was confirmed by 1H and 13C-nuclear magnetic resonance (NMR) spectroscopy, gas chromatography with mass spectrometric (GC/MS) detection, and infrared/attenuated total reflectance (IR/ATR) spectroscopy. NMR spectra showed shifts that varied from previously published data. However, we established a complete, unambiguous assignment for all signals. Chromatograms obtained by GC/MS were almost identical for both investigated samples and confirmed the structure of DON. Likewise, IR/ATR spectra verified the identity of DON. The degree of purity was determined by liquid chromatography (LC) with a variable wavelength detector, LC/MS/MS, GC with electron-capture detection (GC-ECD), and ultraviolet (UV) spectrophotometry. The purity check using LC showed a single peak in both chromatograms. With LC/MS/MS measurements, we could detect small amounts of impurities in the crystalline DON from both sources. In data obtained by GC-ECD, no differences in purity were observed. The UV measurements showed an absorption maximum at 217 nm. The mean epsilon(m) of the extinction coefficients was calculated as 6727 (L/cm/mol) for DON (Sigma) and 6825 (L/cm/mol) for DON (Biopure). Finally, the purity of DON from the 2 commercial sources was calculated as >96 and >98%, respectively. Although the DON produced by both providers can be considered sufficiently pure for routine analysis of trichothecenes in food and feed, this work again demonstrated that the impurity of the solid mycotoxin constitutes the greatest contribution to the overall uncertainty of a mycotoxin calibrant.     

Use of liquid chromatography/time-of-flight mass spectrometry and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids

The process of metabolite identification is essential to the drug discovery and development process; this is usually achieved by liquid chromatography/tandem mass spectrometry (LC/MS/MS) or a combination of liquid chromatography/mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR) spectroscopy. Metabolite identification is, however, a time-consuming process requiring an experienced skilled scientist. Multivariate statistical analysis has been used in the field of metabonomics to elucidate differences in endogenous biological profiling due to a toxic effect or a disease state. In this paper we show how a combination of liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and multivariate statistical analysis can be used to detect drug metabolites in a biological fluid with no prior knowledge of the compound administered.     

LC–MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography–mass spectrometry (GC–MS). LC–MS/MS also offers higher flexibility than immunoassays because LC–MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC–MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC–MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC–MS/MS will become a key instrument in routine clinical laboratories.     

LC-MS/MS-based multibiomarker approaches for the assessment of human exposure to mycotoxins

Mycotoxins are toxic fungal secondary metabolites that frequently contaminate food and feed worldwide, and hence represent a major hazard for food and feed safety. To estimate human exposure arising from contaminated food, so-called biomarker approaches have been developed as a complementary biomonitoring tool besides traditional food analysis. The first methods based on radioimmunoassays and enzyme-linked immunosorbent assays as well as on liquid chromatography were developed in the late 1980s and early 1990s for the carcinogenic aflatoxins and in the last two decades further tailor-made methods for some major mycotoxins have been published. Since 2010, there has been a clear trend towards the development and application of multianalyte methods based on liquid chromatography–electrospray ionization tandem mass spectrometry for assessment of mycotoxin exposure made possible by the increased sensitivity and selectivity of modern mass spectrometry instrumentation and sophisticated sample cleanup approaches. With use of these advanced methods, traces of mycotoxins and relevant breakdown and conjugation products can be quantified simultaneously in human urine as so-called biomarkers and can be used to precisely describe the real exposure, toxicokinetics, and bioavailability of the toxins present. In this article, a short overview and comparison of published multibiomarker methods focusing on the determination of mycotoxins and relevant excretion products in human urine is presented. Special attention is paid to the main challenges when analyzing these toxic food contaminants in urine, i.e., very low analyte concentrations, appropriate sample preparation, matrix effects, and a lack of authentic, NMR-confirmed calibrants and reference materials. Finally, the progress in human exposure assessment studies facilitated by these analytical methods is described and an outlook on probable developments and possibilities is presented.

Analytical separation and detection methods for flavonoids

Flavonoids receive considerable attention in the literature, specifically because of their biological and physiological importance. This review focuses on separation and detection methods for flavonoids and their application to plants, food, drinks and biological fluids. The topics that will be discussed are sample treatment, column liquid chromatography (LC), but also methods such as gas chromatography (GC), capillary electrophoresis (CE) and thin-layer chromatography (TLC), various detection methods and structural characterization. Because of the increasing interest in structure elucidation of flavonoids, special attention will be devoted to the use of tandem-mass spectrometric (MS/MS) techniques for the characterization of several important sub-classes, and to the potential of combined diode-array UV (DAD UV), tandem-MS and nuclear magnetic resonance (NMR) detection for unambiguous identification. Emphasis will be on recent developments and trends.     

Multi-mycotoxin analysis of animal feed and animal-derived food using LC–MS/MS system with timed and highly selective reaction monitoring

Mycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC–MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food.

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Graphical abstract Multi-mycotoxin analysis of animal feed and animal-derived food using LC-MS/MS

     

Development and in-house validation of a robust and sensitive solid-phase extraction liquid chromatography/tandem mass spectrometry method for the quantitative determination of aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 μg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.     

Mass spectrometry applied to the analysis of estrogens in the environment

Environmental analytical chemistry has recently changed focus from analysis of non-polar, persistent contaminants (e.g. polychlorinated biphenyls and dioxins) to more polar and labile compounds that interfere with biological processes. For example, natural and synthetic estrogens and their metabolites have been detected in sewage treatment plant effluents at nanogram/liter concentrations that are similar to those at which both total sex reversal and intersex (containing both testes and ova) is induced in fish exposed to these compounds in laboratory experiments. The development of techniques for the analysis of natural and synthetic estrogens in biological fluids (i.e. serum and urine) has been a priority in the biomedical field. However, the recent recognition that estrogen hormones are contaminants in the environment that may contribute to endocrine disruption has focused attention on the need for highly sensitive and specific techniques that are applicable for trace analysis in complex environmental matrices. Three optimized mass spectrometric protocols have been developed for the determination and quantitation of steroid hormones in environmental matrices using gas chromatography/tandem mass spectrometry (GC/MS/MS), liquid chromatography/mass spectrometry selected ion monitoring, (LC/MS - SIM) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The advantages and disadvantages of each method are presented.