Analysis of erlotinib and its metabolites in rat tissue sections by MALDI quadrupole time-of-flight mass spectrometry

A qualitative and quantitative analysis of erlotinib (RO0508231) and its metabolites was carried out on rat tissue sections from liver, spleen and muscle. Following oral administration at a dose of 5 mg/kg, samples were analyzed by matrix-assisted laser desorption ionization (MALDI) with mass spectrometry (MS) using an orthogonal quadrupole time-of-flight instrument. The parent compound was detected in all tissues analyzed. The metabolites following drug O-dealkylation could also be detected in liver sections. Sinapinic acid (SA) matrix combined with the dried-droplet method resulted in better conditions for our analysis on tissues. Drug quantitation was investigated by the standard addition method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on the tissue extracts. The presence of the parent compound and of its O-demethylated metabolites was confirmed in all tissue types and their absolute amounts calculated. In liver the intact drug was found to be 3.76 ng/mg tissue, while in spleen and muscle 6- and 30-fold lower values, respectively, were estimated. These results were compared with drug quantitation obtained by whole-body autoradiography, which was found to be similar. The potential for direct quantitation on tissue sections in the presence of an internal standard was also investigated using MALDI-MS. The use of alpha-cyano-4-hydroxycinnamic acid (CHCA) as the matrix resulted in better linearity for the calibration curves obtained with reference solutions of the drug when compared to SA, but on tissue samples no reliable quantitative analysis was possible owing to the large variability in the signal response. MS imaging experiments using MALDI in MS/MS mode allowed visualizing the distribution of the parent compound in liver and spleen tissues. By calculating the ratio between the total ion intensities of MS images for liver and spleen sections, a value of 6 : 1 was found, which is in good agreement with the quantitative data obtained by LC-MS/MS analysis.     

Multidimensional Mass Spectrometry of Synthetic Polymers and Advanced Materials

Multidimensional mass spectrometry interfaces a suitable ionization technique and mass analysis (MS) with fragmentation by tandem mass spectrometry (MS²) and an orthogonal online separation method. Separation choices include liquid chromatography (LC) and ion‐mobility spectrometry (IMS), in which separation takes place pre‐ionization in the solution state or post‐ionization in the gas phase, respectively. The MS step provides elemental composition information, while MS² exploits differences in the bond stabilities of a polymer, yielding connectivity and sequence information. LC conditions can be tuned to separate by polarity, end‐group functionality, or hydrodynamic volume, whereas IMS adds selectivity by macromolecular shape and architecture. This Minireview discusses how selected combinations of the MS, MS², LC, and IMS dimensions can be applied, together with the appropriate ionization method, to determine the constituents, structures, end groups, sequences, and architectures of a wide variety of homo‐ and copolymeric materials, including multicomponent blends, supramolecular assemblies, novel hybrid materials, and large cross‐linked or nonionizable polymers.     

Noninvasive determination of human cortisol and dehydroepiandrosterone sulfate using liquid chromatography-tandem mass spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for measurements of steroids in human saliva has garnered increased interest in the area of clinical psychoneuroendocrinological research. However, performance characteristics of LC-MS/MS methods for the analysis of steroids in saliva are limited. Human saliva samples were collected via passive drool. Cortisol and dehydroepiandrosterone sulfate (DHEA-S) in the samples were extracted together, resolved on a C18-A column, and analyzed using tandem mass spectrometry. The LC-MS/MS method had limits of quantitation of 0.03 and 0.06 ng/mL for DHEA-S and cortisol, respectively. Method evaluations showed coefficient variation (%CV) of inter-assay ranging 4.6–17.9% for DHEA-S and cortisol, recoveries of 102.4–109.5% for DHEA-S and 94.6–98.3% for cortisol, and assay linearity with R² = 0.9964 for DHEA-S (1.0–25.0 ng/mL) and R² = 0.997 (1.0–25.0 ng/mL) for cortisol. No cross contamination among samples was observed. Human saliva showed 20% and 18% ion enhancement effect for DHEA-S and cortisol assay, respectively. No interference by ten common steroids was detected. Regression analysis of method comparisons with laboratory-developed test (LDT) method revealed R² = 0.9688 (LC-MS/MS = 0.9665 LDT-LC-MS/MS − 0.7355) for cortisol, and R² = 0.9039 (LC-MS/MS = 1.0173 LDT-LC-MS/MS + 3.6797) for DHEA-S. Reference ranges for young adults were determined to be 0.3–5.9 ng/mL for females and 0.1–5.6 ng/mL for males for salivary cortisol, and 0.6–7.4 ng/mL for females and 0.6–10.1 ng/mL for males for salivary DHEA-S. An LC-MS/MS method for quantifying cortisol and DHEA-S in human saliva was developed and validated for clinical and psychoneuroendocrinological research that require noninvasive means of measuring these hormones.

     

Quantitative MALDI-MS<Superscript><Emphasis Type="Italic">n</Emphasis></Superscript> analysis of cocaine in the autopsied brain of a human cocaine user employing a wide isolation window and internal standards

Detection of drugs in tissue typically requires extensive sample preparation in which the tissue is first homogenized, followed by drug extraction, before the extracts are finally analyzed by LC/MS. Directly analyzing drugs in intact tissue would eliminate any complications introduced by sample pretreatment. A matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS ⁿ ) method as been developed for the quantification of cocaine present in postmortem brain tissue of a chronic human cocaine user. It is shown that tandem mass spectrometry (MS² and MS³) increase selectivity, which is critical for differentiating analyte ions from background ions such as matrix clusters and endogenous compounds found in brain tissue. It is also shown that the use of internal standards corrects for signal variability during quantitative MALDI, which can be caused by inhomogeneous crystal formation, inconsistent sample preparation, and laser shot-to-shot variability. The MALDI-MS ⁿ method developed allows for a single MS³ experiment that uses a wide isolation window to isolate both analyte and internal standard target ions. This method is shown to provide improved precision [∼10–20 times reduction in percent relative standard deviation (%RSD)] for quantitative analysis compared to using two alternating MS³ experiments that separately isolate the target analyte and internal standard ions.     

Validated hydrophilic interaction LC-MS/MS method for determination of 2-pyrrolidinone residue: applied to a depletion study of 2-pyrrolidinone in swine liver

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of 2-pyrrolidinone in swine liver was developed and validated. After the fortification of 2-pyrrolidinone-d₆ as the internal standard, 2-pyrrolidinone in swine liver was extracted by acetonitrile, and the supernatant was led through a C18 + WAX mixed-mode solid phase extraction (SPE) cartridge. Furthermore, the eluate was adjusted to pH 5.0 and then led through a strong cationic exchange SPE cartridge. 2-Pyrrolidinone and 2-pyrrolidinone-d₆ were concentrated and eluted by acetonitrile containing 2% ammonium hydroxide. The final eluate was acidified and then injected for hydrophilic interaction LC-MS/MS analysis. Mass spectrometry detection was carried using positive turbo-ion spray ionization mode. The multiple reaction monitoring transitions were 86 → 69 for 2-pyrrolidinone and 92 → 75 for 2-pyrrolidinone-d₆. The C18 + WAX mixed-mode SPE cleanup greatly prevented the rapid contamination of mass spectrometer. The further SCX SPE cleanup thoroughly eliminated the absolute matrix effect. Solvent calibration standards could be readily used for quantitative analysis of 2-pyrrolidinone with excellent precision and accuracy. Endogenous levels of 2-pyrrolidinone in some blank matrices was readily determined. Full recoveries were readily achieved by the optimize extraction protocol, and thus the role of 2-pyrrolidinone-d₆ was to just compensate the variation of the injections. The detection limit was 5 ng g⁻¹ swine liver. The validated method was applied to a depletion study of 2-pyrrolidinone in swine liver following intramuscular administration of a drug 2-pyrrolidinone formulation. The matrix effect from tissue samples usually represented a technical challenge for LC-MS/MS analysis, and a very small molecule such as 2-pyrrolidinone also represented a technical barrier for LC-MS/MS analysis. However, the extraction protocol developed in the present study reached the best outcome: zero matrix effect and full recovery.     

Direct profiling of myelinated and demyelinated regions in mouse brain by imaging mass spectrometry

One of the newly developed imaging mass spectrometry (IMS) technologies utilizes matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to map proteins in thin tissue sections. In this study, we evaluated the power of MALDI IMS as we developed it in our (Bruker) MALDI TOF (Reflex IV) and TOF-TOF (Ultraflex II) systems to study myelin patterns in the mouse central nervous system under normal and pathological conditions. MALDI IMS was applied to assess myelin basic protein (MBP) isoform-specific profiles in different regions throughout the mouse brain. The distribution of ions of m/z 14,144 and 18,447 displayed a striking resemblance with white matter histology and were identified as MBP isoform 8 and 5, respectively. In addition, we demonstrated a significant reduction of the MBP-8 peak intensity upon MALDI IMS analysis of focal ethidium bromide-induced demyelinated brain areas. Our MS images were validated by immunohistochemistry using MBP antibodies. This study underscores the potential of MALDI IMS to study the contribution of MBP to demyelinating diseases.     

Sterically hindered phenols in negative ion mobility spectrometry–mass spectrometry

Negative corona discharge atmospheric pressure chemical ionization (APCI) was used to investigate phenols with varying numbers of tert‐butyl groups using ion mobility spectrometry–mass spectrometry (IMS‐MS). The main characteristic ion observed for all the phenolic compounds was the deprotonated molecule [M–H]⁻. 2‐tert‐Butylphenol showed one main mobility peak in the mass‐selected mobility spectrum of the [M–H]⁻ ion measured under nitrogen atmosphere. When air was used as a nebulizer gas an oxygen addition ion was seen in the mass spectrum and, interestingly, this new species [M–H+O]⁻ had a shorter drift time than the lighter [M–H]⁻ ion. Other phenolic compounds primarily produced two IMS peaks in the mass‐selected mobility spectra measured using the [M–H]⁻ ion. It was also observed that two isomeric compounds, 2,4‐di‐tert‐butylphenol and 2,6‐di‐tert‐butylphenol, could be separated with IMS. In addition, mobilities of various characteristic ions of 2,4,6‐trinitrotoluene were measured, since this compound was previously used as a mobility standard. The possibility of using phenolic compounds as mobility standards is also discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Ion mobility spectrometry – Mass spectrometry coupling for synthetic polymers

This review covers applications of ion mobility spectrometry (IMS) hyphenated to mass spectrometry (MS) in the field of synthetic polymers. MS has become an essential technique in polymer science, but increasingly complex samples produced to provide desirable macroscopic properties of high‐performance materials often require separation of species prior to their mass analysis. Similar to liquid chromatography, the IMS dimension introduces shape selectivity but enables separation at a much faster rate (milliseconds vs minutes). As a post‐ionization technique, IMS can be hyphenated to MS to perform a double separation dimension of gas‐phase ions, first as a function on their mobility (determined by their charge state and collision cross section, CCS), then as a function of their m/z ratio. Implemented with a variety of ionization techniques, such coupling permits the spectral complexity to be reduced, to enhance the dynamic range of detection, or to achieve separation of isobaric ions prior to their activation in MS/MS experiments. Coupling IMS to MS also provides valuable information regarding the 3D structure of polymer ions in the gas phase and regarding how to address the question of how charges are distributed within the structure. Moreover, the ability of IMS to separate multiply charged species generated by electrospray ionization yields typical IMS‐MS 2D maps that permit the conformational dynamics of synthetic polymer chains to be described as a function of their length.     

Effective monitoring for ractopamine residues in samples of animal origin by SPR biosensor and mass spectrometry

Ractopamine (RCT) is a member of the β-2-agonist (β-agonist) family. It is licensed for use as an animal growth promoter in more than 20 countries worldwide, including the United States and Canada, but is either not licensed or prohibited by over 150 others, including those within the European Union. The issue of the use of RCT in livestock bound for human consumption has risen to prominence recently following the decision by The People's Republic of China to ban the import of pork from a number of processing plants after finding traces of RCT in shipments from the U.S.A.In order to monitor for the illegal use of such compounds within Europe, there is a requirement to have a robust and reliable testing scheme capable of the detection of low concentrations of RCT. In the present study an optical biosensor screening assay was developed. The developed assay was compared with a liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) confirmatory procedure. These methods were used to study the ability to detect RCT in pigs following treatment. Both testing procedures were capable of detecting low μg kg⁻¹ concentrations of the drug in urine and liver. Liver was found to be a less suitable sample matrix, with RCT residue levels being undetectable after 5 days withdrawal of the drug. Urine samples however still contained detectable RCT residues several weeks after withdrawal. The correlation (as measured by r²) between the biosensor and LC-MS/MS methods was 0.99 and 0.97 for urine and liver samples, respectively.It is concluded that testing regimes based on RCT analysis in liver are less likely to detect illegal administration of the drug than those based on urine analysis. Urine samples provide an excellent matrix for the detection of RCT residues for an extended period post withdrawal.     

A review of recent, unconventional applications of ion mobility spectrometry (IMS)

The applications of ion mobility spectrometry (IMS) have grown exponentially beyond its uses for explosive, illicit drug and chemical warfare agent monitoring in recent years. Instrumental developments including new drift tube materials and ionization sources have enabled the manufacturing of more sophisticated and affordable IMS equipment for the advantageous analysis of samples with no pretreatment. The most recent applications of IMS include quality control and cleaning validation procedures in the pharmaceutical industry; determinations of contaminants in food samples; clinical analyses of biological fluids; environmental analyses of contaminants in gaseous, liquid and solid samples; and (bio)process quality control monitoring. Coupling IMS with MSⁿ has enabled the analysis of very complex samples and the extraction of knowledge unavailable from isolated MS measurements, especially in the polymer and petroleomic industries.     

Tetraalkylammonium halides as chemical standards for positive electrospray ionization with ion mobility spectrometry/mass spectrometry

Chemical standards for positive ion mode electrospray ionization ion mobility spectrometry/mass spectrometry (ESI(+)-IMS/MS) are suggested. The low clustering tendency of tetraalkylammonium halides makes them ideal chemical standards for ESI(+)-IMS/MS. A homologous series of these compounds forms a useful external standard for instrument testing and resolution calibration of an IMS instrument. Selected homologues or a mixture of tetraalkylammonium halides can be used as mobility standards in the analytical runs. Absolute and relative reduced mobilities were calculated for C2--C8, C10 and C12 tetraalkylammonium halides. Absolute reduced mobilities in nitrogen were 1.88, 1.56, 1.33, 1.15, 1.02, 0.92, 0.84, 0.73, and 0.67 cm2 V(-1) s(-1), for C2--C8, C10 and C12 tetraalkylammonium halides, respectively. Relative reduced mobilities (relative to 2,6-di-tert-butylpyridine) for the same compounds were 1.20, 1.00, 0.855, 0.743, 0.658, 0.59, 0.54, 0.47, and 0.43, respectively.     

Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the visualization of molecular distributions within tissue sections. While providing excellent molecular specificity and spatial information, absolute quantification by MALDI IMS remains challenging. Especially in the low molecular weight region of the spectrum, analysis is complicated by matrix interferences and ionization suppression. Though tandem mass spectrometry (MS/MS) can be used to ensure chemical specificity and improve sensitivity by eliminating chemical noise, typical MALDI MS/MS modalities only scan for a single MS/MS event per laser shot. Herein, we describe TOF/TOF instrumentation that enables multiple fragmentation events to be performed in a single laser shot, allowing the intensity of the analyte to be referenced to the intensity of the internal standard in each laser shot while maintaining the benefits of MS/MS. This approach is illustrated by the quantitative analyses of rifampicin (RIF), an antibiotic used to treat tuberculosis, in pooled human plasma using rifapentine (RPT) as an internal standard. The results show greater than 4-fold improvements in relative standard deviation as well as improved coefficients of determination (R²) and accuracy (>93% quality controls, <9% relative errors). This technology is used as an imaging modality to measure absolute RIF concentrations in liver tissue from an animal dosed in vivo. Each microspot in the quantitative image measures the local RIF concentration in the tissue section, providing absolute pixel-to-pixel quantification from different tissue microenvironments. The average concentration determined by IMS is in agreement with the concentration determined by HPLC-MS/MS, showing a percent difference of 10.6%.

Open image in new window

Graphical Abstract ?

     

Electrospray Ionization Mass Spectrometry: A Powerful Platform for Noble‐Metal Nanocluster Analysis

Electrospray ionization mass spectrometry (ESI‐MS) is an analytical technique that measures the mass of a sample through “soft” ionization. Recent years have witnessed a rapid growth of its application in noble‐metal nanocluster (NC) analysis. ESI‐MS is able to provide the mass of a noble‐metal NC analyte for the analysis of their composition (n, m, q values in a general formula [M_nL_m]^q), which is crucial in understanding their properties. This review attempts to present various developed techniques for the determination of the composition of noble metal NCs by ESI‐MS. Additionally, advanced applications that use ESI‐MS to further understand the reaction mechanism, complexation behavior, and structure of noble metal NCs are introduced. From the comprehensive applications of ESI‐MS on noble‐metal NCs, more possibilities in nanochemistry can be opened up by this powerful technique.     

IMSPeptider: A computational peptide collision cross‐section area calculator based on a novel molecular dynamics simulation protocol

Introduction of ion mobility mass spectrometry (IMS/MS) into the proteomic workflow provides an orthogonal separation to the widely used LC‐MS platforms. IMS also provides structural information that could facilitate peptide identification. However, the lack of tools capable of predictive power in a high‐throughput fashion makes peptide global profiling quite challenging. To target this issue, a computational workflow was developed based on biophysical principles to predict the collision cross‐section area (CCS) of peptides as measured from IMS/MS experiments. Hosted on a web server, it allows the user to input a primary sequence (query) and retrieve information on peptide structure, sequence, and corresponding CCS. The current version is designed to identify peptide sequences up to 23 residues in length, in its higher charge state, based on a match of the molecule m/z and CCS. The protocol was validated against a 128‐sequences‐dataset and CCS predicted within 2.8% average error. © 2013 Wiley Periodicals, Inc.     

Pharmaceutical metabolite profiling using quadrupole/ion mobility spectrometry/time‐of‐flight mass spectrometry

The use of hybrid quadrupole ion mobility spectrometry time‐of‐flight mass spectrometry (Q/IMS/TOFMS) in the metabolite profiling of leflunomide (LEF) and acetaminophen (APAP) is presented. The IMS drift times (T_d) of the drugs and their metabolites were determined in the IMS/TOFMS experiments and correlated with their exact monoisotopic masses and other in silico generated structural properties, such as connolly molecular area (CMA), connolly solvent‐excluded volume (CSEV), principal moments of inertia along the X, Y and Z Cartesian coordinates (MI‐X, MI‐Y and MI‐Z), inverse mobility and collision cross‐section (CCS). The correlation of T_d with these parameters is presented and discussed. IMS/TOF tandem mass spectrometry experiments (MS² and MS³) were successfully performed on the N‐acetyl‐p‐benzoquinoneimine glutathione (NAPQI‐GSH) adduct derived from the in vitro microsomal metabolism of APAP. As comparison, similar experiments were also performed using hybrid triple quadrupole linear ion trap mass spectrometry (QTRAPMS) and quadrupole time‐of‐flight mass spectrometry (QTOFMS). The abilities to resolve the product ions of the metabolite within the drift tube and fragment the ion mobility resolved product ions in the transfer travelling wave‐enabled stacked ring ion guide (TWIG) demonstrated the potential applicability of the Q/IMS/TOFMS technique in pharmaceutical metabolite profiling. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of trifluoroacetic acid and other short-chain perfluorinated acids (C2-C4) in precipitation by liquid chromatography-tandem mass spectrometry: comparison to patterns of long-chain perfluorinated acids (C5-C18)

This paper provides analytical chemical information on selected new molecular entities (NMEs) which are drugs that have recently been approved by the FDA. These are the antiretroviral drugs, atazanavir, indinavir and emtricitabine, the antibacterial gemifloxacin, rosuvastatine which is a cholesterol-lowing drug, the anti-cancer drug gefitinib and aprepitant for neurological disorders. Electrospray ionisation-quadrupole ion trap mass spectrometry (ESI-MSⁿ) was employed to generate tandem mass spectrometric (MS²) data of the drugs studied and structural assignments of product ions were supported by quadrupole time-of-flight mass spectrometry (QToF-MS/MS). These fragmentation studies were then utilised in the development and validation of a specific and sensitive liquid chromatographic method (LC–ESI-MS²) to identify and determine these drugs at therapeutic concentration levels in serum after a single protein precipitation procedure with acetonitrile. In addition, this method was compared to the application of gas liquid chromatography-flame ionisation detection (GLC-FID) and differential pulse polarography (DPP) for the analysis of these NMEs in serum.     

Suitability of a fully 13C isotope labeled internal standard for the determination of the mycotoxin deoxynivalenol by LC-MS/MS without clean up

Very often, the accuracy of quantitative analytical methods for the determination of mycotoxins by liquid chromatography (LC)-mass spectrometry (MS) and LC-MS/MS is limited by matrix effects during the ionization process in the MS source. Stable isotope labeled standards are best suited to correct for matrix effects and to improve both the trueness and the precision of analytical methods employing LC-MS and LC-MS/MS. This paper describes the successful use of fully ¹³C isotope labeled deoxynivalenol [(¹³C₁₅)DON] as an internal standard (IS) for the accurate determination of DON in maize and wheat by LC electrospray ionization MS/MS. To show the full potential of (¹³C₁₅)DON as IS, maize and wheat extracts were analyzed without further cleanup. Subsequent to calibration for the LC-MS end determination, DON was quantified in matrix reference materials (wheat and maize). Without consideration of the IS, apparent recoveries of DON were 29±6% (n=7) for wheat and 37±5% (n=7) for maize. However, the determination of DON in the reference materials yielded 95±3% (wheat) and 99±3% (maize) when (¹³C₁₅)DON was used as an IS for data evaluation.     

Sites of metabolic substitution: investigating metabolite structures utilising ion mobility and molecular modelling

Drug metabolism is an integral part of the drug development and drug discovery process. It is required to validate the toxicity of metabolites in support of safety testing and in particular provide information on the potential to form pharmacologically active or toxic metabolites. The current methodologies of choice for metabolite structural elucidation are liquid chromatography/tandem mass spectrometry (LC/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. There are, in certain cases, examples of metabolites whose sites of metabolism cannot be unequivocally identified by MS/MS alone. Utilising commercially available molecular dynamics packages and known quantum chemistry basis sets, an ensemble of lowest energy structures were generated for a group of aromatic hydroxylated metabolites of the model compound ondansetron. Theoretical collision cross–sections were calculated for each structure. Travelling‐wave ion mobility (IMS) measurements were also performed on the compounds, thus enabling experimentally derived collision cross‐sections to be calculated. A comparison of the theoretical and experimentally derived collision cross‐sections were utilised for the accurate assignment of isomeric drug metabolites. The UPLC/IMS‐MS method, described herein, demonstrates the ability to measure reproducibly by ion mobility, metabolite structural isomers, which differ in collision cross‐section, both theoretical and experimentally derived, by less than 1 Ų. This application has the potential to supplement and/or complement current methods of metabolite structural characterisation. Copyright © 2010 John Wiley & Sons, Ltd.     

高效液相色谱-串联质谱在兽药残留分析中的应用

高效液相色谱-串联质谱以其特有的快速高效分离和定性、定量准确等优势,在兽药残留检测领域应用广泛。本文系统地介地绍了高效液相色谱-串联质谱(HPLC-MS/MS)在兽药残留分析中的研究进展,并对该领域今后的发展趋势进行了展望。引用文献66篇。