High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]⁺ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]^- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS ⁿ analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.     

Characterization of a distributed plasma ionization source (DPIS) for ion mobility spectrometry and mass spectrometry

A recently developed atmospheric pressure ionization source, a distributed plasma ionization source (DPIS), was characterized and compared to commonly used atmospheric pressure ionization sources with both mass spectrometry (MS) and ion mobility spectrometry (IMS). The source consisted of two electrodes of different sizes separated by a thin dielectric. Application of a high RF voltage across the electrodes generated plasma in air yielding both positive and negative ions. These reactant ions subsequently ionized the analyte vapors. The reactant ions generated were similar to those created in a conventional point-to-plane corona discharge ion source. The positive reactant ions generated by the source were mass identified as being solvated protons of general formula (H₂O)_nH⁺ with (H₂O)₂H⁺ as the most abundant reactant ion. The negative reactant ions produced were mass identified primarily as CO₃⁻, NO₃⁻, NO₂⁻, O₃⁻ and O₂⁻ of various relative intensities. The predominant ion and relative ion ratios varied depending upon source construction and supporting gas flow rates. A few compounds including drugs, explosives and amines were selected to evaluate the new ionization source. The source was operated continuously for 3 months and although surface deterioration was observed visually, the source continued to produce ions at a rate similar that of the initial conditions.     

Characterization of Ni(II) complexes of Schiff bases of amino acids and (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide using ion trap and QqTOF electrospray ionization tandem mass spectrometry

This work demonstrates the application of electrospray ionization mass spectrometry (ESI-MS) using two different mass analyzers, ion trap and hybrid quadrupole time-of-flight (QqTOF) mass analyzer, for the structural characterization of Ni(II) complexes of Schiff bases of (S)-N-(2-benzoylphenyl)-1-benzylpyrrolidine-2-carboxamide with different amino acids. ESI enables the determination of molecular weight on the basis of rather simple positive-ion ESI mass spectra containing only protonated molecules and adducts with sodium or potassium ions. Fragmentation patterns are characterized by tandem mass spectrometric experiments, where both tandem mass analyzers provide complementary information. QqTOF data are used for the determination of elemental composition of individual ions due to mass accuracies always better than 3 ppm with the external calibration, while multistage tandem mass spectra obtained by the ion trap are suitable for studying the fragmentation paths. The novel aspect of our approach is the combination of mass accuracies and relative abundances of all isotopic peaks in isotopic clusters providing more powerful data for the structural characterization of organometallic compounds containing polyisotopic elements. The benefit of relative and absolute mean mass accuracies is demonstrated on the example of studied Ni(II) complexes. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Effect of electrospray ionization conditions on low-energy tandem mass spectra of peptides

The effect of electrospray ionization (ESI) conditions on low-energy tandem mass spectra of peptides in the relative molecular mass range 400–1200 was examined. For singly charged peptide ions the source skimmer potential (which determines the degree of acceleration of the ions through the intermediate pressure region in the source) can strongly influence the extent of fragmentation observed in tandem mass spectra, especially at low collision energies. For each peptide there is an optimum skimmer potential which represents a balance between generating ions with sufficient internal energy for subsequent tandem mass spectrometric experiments and inducing the onset of other processes such as source fragmentation. The fragmentation which can be achieved in tandem mass spectra with high skimmer potentials differs from ESI source fragmentation for the same peptides. We have found that fragmentation in ESI mass spectra depends both on skimmer potential and on solvent pH, presumably because the latter determines the proportion of doubly charged species generated from a given peptide. Low-energy tandem mass spectra of peptides following ESI are equally as sensitive to peptide structure and the type of adduct studied (e.g. [M + H]⁺ vs. [M + NH₄]⁺) as tandem mass spectra obtained following older ionization methods such as fast atom bombardment.     

Identification of new phase II metabolites of xanthohumol in rat in vivo biotransformation of hop extracts using high-performance liquid chromatography electrospray ionization tandem mass spectrometry

Polyphenolic compounds occurring in hop extracts and their phases I and II metabolites formed during in vivo rat biotransformation have been analyzed using HPLC/MS/MS with electrospray ionization (ESI). Two main groups of polyphenolics are present in the hops, i.e., xanthohumol related compounds and so called α- and β-bitter acids (humulones and lupulones). In our study, hybrid quadrupole-time-of-flight (QqTOF) analyzer is used for the identification of both natural phenolics and their metabolites due to the possibility of accurate mass measurements in full scan and tandem mass spectra supported by MSⁿ data obtained with the ion trap analyzer. Both ESI polarity modes are used for the determination of molecular weights based on [M+H]⁺ and [M−H]⁻ ions in the full scan spectra and the structural information in subsequent tandem mass spectra. The emphasis is given on the elemental composition determination of individual metabolites based on accurate masses typically better than 5 ppm even with the external calibration. Advanced software tools are used for the metabolite identification using the comparison of the blank chromatogram with the real incubation sample together with the software prediction and detection of possible metabolites. Chromatograms of rat incubations are also compared with chromatograms of pure rat feed, rat feed enriched with hop extracts and the placebo experiment. More than ten compounds originating from the hops are identified in rat feces, two of them belong to phase I metabolites and five compounds are phase II metabolites.     

L‐Valine assisted distinction between the stereo‐isomers of D‐hexoses by positive ion ESI tandem mass spectrometry

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry (ESI‐ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 amino acids which are basic acidic amino acids and amides. The ions of amino acid–hexose complexes were further investigated by tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave rich fragmentation, such as the complex ions of isoleucine, phenylalanie, tyrosine, and valine. We found that hexoses could be complexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine–hexose complexes coordinated by potassium ion, the MS² spectra of the ion at m/z 453 yielded unambiguous differentiation. And the fragmentation ions are sensitive to the stereochemical differences at the carbon‐4 of hexoses in the complexes, as proved by the MS². Copyright © 2010 John Wiley & Sons, Ltd.     

环钯化二茂铁亚胺-膦配合物的电喷雾离子阱质谱研究

采用电喷雾离子阱质谱法(ESI-MS)对10种环钯化二茂铁亚胺-膦配合物的质谱特征进行了研究, 获得了其结构碎片信息, 对其质谱裂解途径进行了解析. 结果表明, 在正离子检测方式下可以得到强的准分子离子峰[M-Cl]+簇, 它们的(+) ESI-MSn(n=1~3)质谱主要产生碳-膦键断裂的碎片, 同时也能观察到Pd—P或Pd—C键的断裂, 这些特征为此类化合物及其结构类似物的结构推断提供了依据.     

Determination of polymer additives by liquid chromatography coupled with mass spectrometry. A comparison of atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI)

A method for the determination of polymer additives like antioxidants, UV absorbers and processing stabilizers using liquid chromatography (LC) coupled with atmospheric pressure photoionization mass spectrometry (APPI-MS) is presented. Ion source parameters were optimized regarding temperatures, gas flow rates, and voltages applied. Detection limits were determined using APPI with or without dopant and were compared with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Differences between APPI, ESI and APCI are pointed out and the effect of the dopant toluene and acetone is discussed. The optimized method yielded detection limits between 0.001 mg L⁻¹ and 0.022 mg L⁻¹ for 15 different analytes. Linear calibration plots could be obtained for all solutes over a wide concentration range showing satisfying repeatability with standard deviations of peak areas between 3.4% and 7.6%. The results indicate that the developed method can be regarded as suitable for the quantitative determination of polymer additives even at low concentration levels.     

新型环钯化二茂铁亚胺-膦配合物的质谱特征

采用电喷雾离子阱质谱法(ESI-MS)对两种环钯化二茂铁亚胺-膦配合物的质谱特征进行了研究,获得了它们的结构碎片信息,对其质谱裂解途径进行了解析.结果表明:在正离子检测方式下可以得到强的准分子离子峰[M-C l]+和[M-I]+簇,它们的(+)ESI-MSn(n=1~4)质谱主要产生碳-膦键断裂的碎片,同时也能观察到钯-磷键的断裂.这些特征为此类化合物及其结构类似物的结构推断提供了依据.     

Direct desorption/ionization of analytes by microwave plasma torch for ambient mass spectrometric analysis

Ambient ionization is the new revolution in mass spectrometry (MS). A microwave plasma produced by a microwave plasma torch (MPT) at atmospheric pressure was directly used for ambient mass spectrometric analysis. H₃O⁺ and NH₄⁺ and their water clusters from the background are formed and create protonated molecules and ammoniated molecules of the analytes. In the full‐scan mass spectra, both the quasi‐molecular ions of the analytes and their characteristic ionic fragments are obtained and provide evidence of the analyte. The successful detection of active compounds in both medicine and garlic proves that MPT has the efficient desorption/ionization capability to analyze solid samples. The obtained decay curve of nicotine in exhaled breath indicates that MPT‐MS is a useful tool for monitoring gas samples in real time. These results showed that the MPT, with the advantages of stable plasma, minimal optimization, easy, solvent‐free operation, and no pretreatment, is another potential technique for ambient MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Charge remote fragmentation of fatty acids cationized with alkaline earth metal ions

Fatty acids can be collisionally activated as [M ? H + Cat]⁺, where Cat is an alkaline earth metal, by using tandem mass spectrometry. High-energy collisional activation induces charge remote fragmentation to give structural information. In the full scan mass spectra molecular ions are easily identified, particularly when barium is used as a cationizing agent; ions are shifted to a higher mass, lower chemical noise region of the mass spectrum. Moreover, the isotopic pattern of barium is characteristic, and the high mass defect of barium allows an easy separation of the cationized analyte from any remaining interfering ions (chemical noise), provided medium mass-resolving power is available. An additional advantage is that most of the ion current is localized in [M ? H + Cat]⁺ species. Structural analysis of fatty acids can be performed when the sample size is as low as 1 ng.     

通过确定初始电离位点预测吲哚类生物碱质谱的特征裂解规律

确定初始电离位点是EI源质谱解析中一个至关重要的环节. 本文提出了一种确定初始电离位点的新方法. 该法通过计算和分析分子离子自旋密度, 进而比较从中性分子到分子离子的电荷变化和键长变化, 从而确定初始电离位点. 对简单吲哚类生物碱质谱的特征裂解机理进行了预测, 结果与标准物质的质谱吻合. 在此基础上, 与传统的根据基团电离能确定电离位点的方法进行了比较. 结果表明, 该法优于电离能方法. 此法不仅可用于预测以α-裂解为主导的吲哚生物碱和其它含氮化合物的质谱裂解规律, 还为揭示其它小分子化合物以及气相多肽离子的裂解机理奠定了基础.     

全氯代多环芳烃的大气压化学电离质谱

８种全氯代多环芳烃的大气压化学电离质谱（ＡＰＣＩ－ＭＳ）测试结果表明,在ＡＰＣＩ离原中,所有全氯代多环芳烃均能形成质量较样品分子量少１９的负离子,该类负离子是样品分子离子解离一个氯原子后再结合一个氧原子的产物。当样品含有一个五元环时,仍能观察到样品的分子离子峰。ＡＰＣＩ－ＭＳ是一种全氯代多环芳烃或其它弱极性有机物的理想质谱分析方法。     

Chemical Ionization—A Mass-Spectrometric Analytical Procedure of Rapidly Increasing Importance

The mode of ionization of a molecule has a strong influence on its behavior in the mass spectrometer and thus on the information that can be obtained from its mass spectrum. In chemical ionization a reagent gas, e.g. methane, is first ionized by electron impact. The ions formed in ion-molecule reactions, in particular [CH₅]⁺, [C₂H₅]⁺, and [C₃H₅]⁺, then react “chemically” with the substrate M in fast acid/base type reactions to form ions of the type [MH]⁺, [M(C₂H₅)]⁺, etc., which subsequently fragment to various extents. Alternatively, chemical ionization can be effected by charge exchange, in that ions of a reagent gas, e.g. [He]^+?, react with the substrate M to form molecular ions [M]^+·. Chemical ionization can thus be conducted in a more or less mild fashion and the extent of the fragmentation can be controlled over a very wide range.     

Mass spectra of doubly charged ions

The mass spectra of biological molecules, whose molecular mass exceeds 10 kDa, invariably contain multiply charged ions. For example, a survey scan of a small protein will produce singly, doubly and triply protonated molecules, the intensity of the doubly charged species often being greater than that of the singly charged entity. Although the spectra resulting from doubly charged peptides have not previously been studied, collisional activation of such doubly charged species may result in significant additional information pertaining to molecular structure. The techniques employed to study ions originating from multiply charged species were linked scanning of constant B/E and tandem mass spectrometry, namely low collision energy spectra acquired on a BEQQ hybrid instrument. The methodology was applied to model compounds whose tandem mass spectrometry characteristics are well known, e.g. gramicidin S and angiotensin I. The results for the product ions of the [M + 2H]²⁺ species of the models were obtained which highlight the methodology required for high-mass materials.     

Application of atmospheric pressure ionization HPLC-MS-MS for the analysis of natural products

Summary The development of techniques utilizing atmospheric pressure ionization, namely atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI), has pioneered the coupling of liquid chromatography (HPLC) with mass spectrometry in recent years. Both ESI and APCI generate ions from polar and labile biomaterials with remarkable ease and efficiency. In particular, the use of HPLC with tandem mass spectrometry (MS-MS) opens further dimensions in the field of bioorganic analysis. Thus, HPLC-MS-MS provides the tools for direct elucidation of the structure and variety of polar natural compounds in complex matrices. In order to develop efficient and straightforward strategies for the analysis of polar natural products, the potential and the limitations of these hyphenated analytical techniques are discussed using heterocyclic aromatic amines, fumonisins, acylated glycoconjugates and regioisomeric fatty acid hydroperoxides as examples. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

2,2-二羟甲基丙二酸二乙酯的化学电离质谱分析

化学电离(chem ical ionization,CI)是由Munson和Field[1]于1966年提出的一种质谱电离技术,其原理实际上是分子-离子反应[2,3]。在化学电离条件下,常常可以得到丰度很强的准分子离子峰,没有或很少碎片离子,能准确提供相对分子质量信息,但缺乏结构信息。我们在实验中发现,采用适     

Characterization of prenylflavonoids and hop bitter acids in various classes of Czech beers and hop extracts using high-performance liquid chromatography–mass spectrometry

Hops contain a wide range of polyphenolic compounds with antioxidant properties divided in various chemical classes. These compounds are detected in hop extracts and also in beer as its main product. Based on the careful optimization of column type, column packing, mobile phase composition and gradient steepness, two high-performance liquid chromatography–mass spectrometry (HPLC/MS) methods have been developed. The first method using Purospher Star RP-8e column and the gradient of aqueous acetonitrile containing 0.3% formic acid is optimized for the separation of low polar polyphenolic compounds, while the second one with Zorbax SB-CN column is used for more polar hops and beer components. In this work, more compounds are detected in comparison to previous reports. In total, 49 low polar and 37 polar compounds are detected in studied samples and their molecular weights are determined based on atmospheric pressure chemical ionization (APCI) mass spectra. Some compounds are identified based on the interpretation of their full scan and tandem APCI mass spectra, retention behavior and UV spectra, while the full structure elucidation of other species still requires further research. The quantitation of xanthohumol related prenylflavonoids and bitter acids is done with two detection techniques (APCI-MS and UV detection) providing comparable results. Both compound classes (i.e., prenylflavonoids and bitter acids) are separated and quantitated in a single HPLC run, where numerous other polyphenolics are detected as well.     

Validation of a qualitative screening method for pesticides in fruits and vegetables by gas chromatography quadrupole-time of flight mass spectrometry with atmospheric pressure chemical ionization

A wide-scope screening method was developed for the detection of pesticides in fruit and vegetables. The method was based on gas chromatography coupled to a hybrid quadrupole time-of-flight mass spectrometer with an atmospheric pressure chemical ionization source (GC-(APCI)QTOF MS). A non-target acquisition was performed through two alternating scan events: one at low collision energy and another at a higher collision energy ramp (MS^E). In this way, both protonated molecule and/or molecular ion together with fragment ions were obtained in a single run. Validation was performed according to SANCO/12571/2013 by analysing 20 samples (10 different commodities in duplicate), fortified with a test set of 132 pesticides at 0.01, 0.05 and 0.20 mg kg⁻¹. For screening, the detection was based on one diagnostic ion (in most cases the protonated molecule). Overall, at the 0.01 mg kg⁻¹ level, 89% of the 2620 fortifications made were detected. The screening detection limit for individual pesticides was 0.01 mg kg⁻¹ for 77% of the pesticides investigated. The possibilities for identification according to the SANCO criteria, requiring two ions with a mass accuracy ≤±5 ppm and an ion-ratio deviation ≤±30%, were investigated. At the 0.01 mg kg⁻¹ level, identification was possible for 70% of the pesticides detected during screening. This increased to 87% and 93% at the 0.05 and 0.20 mg kg⁻¹ level, respectively. Insufficient sensitivity for the second ion was the main reason for the inability to identify detected pesticides, followed by deviations in mass accuracy and ion ratios.     

Compositional elucidation of heavy petroleum base oil by GC × GC‐EI/PI/CI/FI‐TOFMS

Comprehensive two‐dimensional gas chromatography (GC × GC) coupled to time‐of‐flight mass spectrometry is a powerful separation tool for complex petroleum product analysis. However, the most commonly used electron ionization (EI) technique often makes the identification of the majority of hydrocarbons impossible due to the exhaustive fragmentation and lack of molecular ion preservation, prompting the need of soft‐ionization energies. In this study, three different soft‐ionization techniques including photo ionization (PI), chemical ionization (CI), and field ionization (FI) were compared against EI to elucidate their relative capabilities to reveal different base oil hydrocarbon classes. Compared with EI (70 eV), PI (10.8 eV) retained significant molecular ion (M^+·) information for a large number of isomeric species including branched‐alkanes and saturated monocyclic hydrocarbons along with unique fragmentation patterns. However, for bicyclic/polycyclic naphthenic and aromatic compounds, EI played upper hand by retaining molecular as well as fragment ions to identify the species, whereas PI exhibited mainly molecular ion signals. On the other hand, CI revealed selectivity towards different base oil groups, particularly for steranes, sulfur‐containing thiophenes, and esters, yielding protonated molecular ions (M + H)⁺ for unsaturated and hydride abstracted ions (M‐H⁺) for saturated hydrocarbons. FI, as expected, generated intact molecular ions (M^+·) irrespective to the base oil chemical classes. It allowed elemental composition by TOFMS with a mass resolving power up to 8000 (FWHM) and a mass accuracy of 1 mDa, leading to the calculation of heteroatomic content, double bond equivalency, and carbon number of the compounds. The qualitative and quantitative results presented herein offer a unique perspective into the detailed comparison of different ionization techniques corresponding to several hydrocarbon classes.