Mass Spectrometry

A newly discovered method is described for generating gas-phase ions from volatile and nonvolatile compounds. The method, matrix-assisted ionization (MAI), is both simple and sensitive, requiring only the vacuum inherent with all mass spectrometers and a suitable matrix, eliminating the need for lasers, electric fields, nebulizing gas, and even heaters to generate gas-phase ions. MAI is applicable for the direct analysis of drugs from biological fluids and tissue without prior purification. By placing matrix only on a specific surface area of interest and exposure to the vacuum of the mass spectrometer, ions are observed from compounds within the targeted surface area of tissue exposed to the matrix solution, thus allowing rapid and simple interrogation of “features of interest.” The limit of detection for drug standards is low attomoles and clean full mass range mass spectra are obtained from low femtomoles of the drug.

The purpose of this study was the development of various analytical MS methods to investigate the chemical composition of e-liquids used in electronic cigarettes and characterize their quality. Low-quality nicotine (the main active compound), glycerol, propylene glycol (solvents), or flavors could greatly increase the toxicity. The search of alkaloid contaminants of nicotine was performed by LC–MS-MS after a deep study of fragmentation pathways by high resolution ESI-MS. A fully validated method for quantitation of organic polar impurities such as cotinine, anabasine, myosmine, nornicotine, and N-nitroso-nornicotine and nicotine itself was developed using MS coupled to UHPLC. To evaluate organic volatile toxicants, headspace from e-cigarette refill liquids was sampled by SPME to perform GC–MS analysis. Finally, heavy metal residues as inorganic toxicants were determined by ICP-MS after simple dilution. A number of cases of contamination by metals (mainly arsenic) was detected.

We have developed a range of analytical workflows using mass spectrometry, in a regulated environment, to support pharmaceutical companies in the development and control of their monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs). High-resolution mass spectrometry is a powerful tool for the analysis of antibodies, but is not readily compatible with a number of chromatographic techniques using high-salt mobile phases. Herein, we present the development and use for marketed mAbs and ADCs of 2D LC–MS via an online desalting step. We demonstrate the importance of such a setup for the determination of drug:antibody ratio (DAR), and the analysis of molecularity, fragmentation, and charge variants (deamidation, oxidation), notably under stress conditions. We discuss the advantages of 2D LC–MS in a regulated environment.

A rapid, accurate, and precise method for the quantification of trypsin inhibitor activity was evaluated. The method utilizes alpha hydroxyl acid capped oligo-lysines [hydroxy acid (Lys)n] or alpha hydroxyl acid capped oligo-lysines-methionine [hydroxy acid (Lys-Met)] as substrates. Hydrolysis of the oligopeptides yields unique chemical residues that were readily quantified with electrospray–mass spectrometry (ESI-MS). Accuracy and precision of the approach compared favorably with that of the standard test method.

Our annual review of products introduced at Pittcon or during the previous year, broken down by the following categories: accessories, atomic spectroscopy, components, imaging, mass spectrometry, mid-IR, NIR, NMR, Raman, software, UV-vis, and X-ray.

Monoclonal antibodies (mAbs) have been increasingly used as biotherapeutic agents and a number of new mAbs are currently in the drug pipeline. Over the next five years the patent on at least nine major biotherapeutic monoclonal antibodies will expire, opening the door for development and marketing of generic forms known as Biosimilars. In this paper a review of the central role mass spectrometry coupled to liquid chromatography plays in characterizing these antibodies is presented. Contemporary top down and middle-up approaches using mass spectrometry and various novel separation techniques to measure the intact masses of mAbs and their subunits or domains are highlighted. Example data of an innovator mAb, Humira (adalimumab) are presented showing the identities and relative abundances of the isoforms associated with this mAb. Similarly the current state of classical peptide mapping using reversed-phase chromatography and tandem mass spectrometry with scan- dependent acquisition is briefly reviewed. Novel approaches that speed analysis and provide information on post translational modifications, glycosylation, and disulfide mapping are discussed. Example data of stressed and unstressed samples of adalimumab are also presented to demonstrate peptide mapping data and modifications to the antibody. Lastly, the current use of mass spectrometry in glycoprofiling of mAbs is reviewed. Example glycan data for adalimumab generated by a novel labeling scheme and sensitive to detection by both fluorescence and mass spectrometry will be presented.

Simultaneous quantification of metabolites in biological fluids, where they are present at different concentration levels, is usually a challenging analytical task. One of the steps that should be undertaken to increase analytical method efficiency is optimization of the electrospray ionization source (ESI), which can be especially helpful in increasing method sensitivity of agents with poor ionization characteristics. We present here a step-by-step ESI optimization strategy with the use of the design of experiments (DoE. This multivariate statistical approach allows effective evaluation of the effects of multiple factors and interactions among factors on a given response in a minimum number of experimental runs.

Naltrexone (Depade, Re Via, Trexan) is a potent narcotic antagonist structurally similar to oxymorphone and naloxone. It blocks the subjective effects of heroin and other opiates and is primarily used in the management of opioid dependence and alcohol dependence. While conjugated 6β-naltrexol is the major urinary metabolite in man, conjugated naltrexone and free 6β-naltrexol are also major urinary species. To assist with monitoring substance abuse patients who are prescribed naltrexone, a rapid, selective, and sensitive LC–MS-MS method was developed to analyze for both naltrexone and 6β-naltrexol post-enzymatic hydrolysis. The validation of this method and some representative patient data are discussed in this report.

The enhanced resolution of comprehensive two-dimensional gas chromatography (GCxGC) was combined with the increased resolving power, speed, and mass accuracy of the Pegasus® HRT's mass analyzer to confidently characterize molecules in light cycle oil (LCO) and vacuum gas oil (VGO). Optimized chromatographic and mass spectrometry parameters were implemented to improve data acquisition, processing, and heteroatomic speciation of these light to midlevel petroleum fractions. Software tools were utilized to process the data and facilitate robust compound identifications. GCxGC-HRT data was processed using comprehensive Peak Find and resulted in comprehensive characterization of molecules in LCO and VGO samples. Compound classes consisted of, but were not limited to alkanes, cycloalkanes, aromatics, benzothiophenes, and carbazoles. Selective processing of alkylbenzothiophenes and dibenzothiophenes was conducted by retrospectively processing data using rapid two-dimensional, accurate mass Target Analyte Finding (TAF).

This article provides useful tips for smooth validation of multi-analyte LC–MS-MS methods and summarizes important validation outcomes for 295 analytes, including more than 200 mycotoxins.

Imaging techniques using vibrational spectroscopy, mass spectrometry (MS), and atomic force microscopy have all been advancing and gaining momentum in recent years. There is great potential power in these imaging techniques, particularly in the biomedical field. Thomas Bocklitz of at the Friedrich-Schiller-University Jena is working to better harness the power of these techniques by combining them.

Nowadays, biotherapeutic proteins are available in different formats such as fusion proteins, monoclonal antibodies, or antibody-drug conjugates. The complexity of these molecules requires advanced and comprehensive characterization to guarantee their potency and safety. This work provides an overview of a methodology using an innovative capillary electrophoresis-tandem mass spectrometry coupling (CE-MS-MS) for the characterization of biologics primary structure. This method was applied to perform biosimilarity assessment between two mAbs, distinguishing minor differences like a sole amino acid substitution. Such a level of characterization is permitted by cumulating the specificities of both CE and high-resolution tandem MS using a sheathless interface, therefore renewing the interest for this type of coupling.

The quality and safety of ready-to-eat packaged foods-such as salads-is very difficult for consumers and suppliers to judge, and improving this situation is the focus of a Europe-wide research project. Part of the project is devoted to the development of better methods to detect and analyze the volatile organic compounds released from relevant food types, in an effort to identify biomarkers for quality and microbial contamination. This article examines one important food (melon) and shows how a method based on thermal desorption (TD) with gas chromatography-time-of-flight-mass spectrometry (GC–TOF-MS) can elucidate how key volatiles vary with time of storage and with the size of the melon pieces. The article highlights how such analytical information will be of value in efforts to improve the quality and safety of ready-to-eat foods.

There is often insufficient prevention to ensure safe swimming environments. Recreation water illness (RWI), most commonly in the form of digestional track illness as well as skin, ear, and respiratory infections, are often caused by water contamination from human waste. Stercobilin is a very stable and suitable chemical biomarker of human waste that has the potential to be used for waste monitoring in public swimming facilities. Using solid phase extraction (SPE) techniques paired with high-resolution mass spectrometry (HRMS), we have developed a robust method used for swimming pool water monitoring to create safer swimming environments.

Do the signal-to-noise ratios presented by instrument vendors accurately reflect improvements in mass spectrometers? We review factors influencing the validity of vendor SNR specifications, and argue that the statistical alternative of instrument detection limits is more consistent with regulatory guidelines and a more relevant indicator of instrument performance.

Beyond the long-established, optical standard techniques of photometry and immunoassay, LC-API-MS-MS has opened new horizons for clinical pathology. This is related to biomedical research and standardization as well as to routine diagnostic testing. It can be expected that the latter field will see important growth, including the introduction of automated MS-based analyzer systems. This will shift the application of mass spectrometric tests from a few specialized laboratories to many standard clinical laboratories with a lower level of analytical expertise.

We recently spoke to Gary Duncan and Wendy Russell of the Rowett Institute of Nutrition & Health in Aberdeen, Scotland, about the significance of phytochemical bioavailability to human health and the important role of liquid chromatography linked to tandem mass spectrometry (LC–MS-MS) in their research.

Worldwide trends in illicit drug use and production have shifted toward an increase in synthetic analogues and the emergence of new variations in their manufacture.

In most countries, herbal medicinal products (HMPs) are introduced into the market without proper scientific evaluation or enforced safety and toxicological studies.

This article describes the development of a new data-independent acquisition (DIA) workflow for protein quantification that uses a mass spectrometer that combines three types of mass analyzers to achieve lower limits of detection (LOD), higher sensitivity, more accurate quantitative results, wider dynamic range, and better reproducibility than existing high-resolution accurate-mass (HRAM) tandem mass spectrometry (MS-MS) DIA workflows.

A multiresidue method has been developed and validated for the analysis of methylxanthines (caffeine and its metabolites) and cotinine in human plasma.

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This interview with Steven J. Ray, an Associate Scientist in the Department of Chemistry at Indiana University in Bloomington, Indiana, discusses his work with a new form of mass spectrometry (MS) for analyzing complex samples.

Military jet fuel (JP-8) is very similar to commercial jet fuel (Jet A) except for the presence of three additives, fuel system icing inhibitor, corrosion inhibitor–lubricity improver (CI-LI), and antistatic additive, which are added to improve characteristics of JP-8.

Direct sample analysis coupled to high-resolution time-of-flight mass spectrometry (TOF-MS) may be effective at analyzing synthetic cathinones, especially for qualitative analysis, because it does not require potentially tedious sample preparation.

Canola largely contains unsaturated fatty acids, thus rendering it clear in most cases after extraction and refining.

Simple, sensitive, rapid, selective, and precise reversed-phase liquid chromatography (LC), electrospray ionization mass spectrometry (ESI-MS), and tandem MS (ESI-MS-MS) methods were developed and validated for the determination of 2-hydroxy-4-(methylthio)-butanoic acid (HMTBA) in bovine serum and sea water matrix. HMTBA is the ?-hydroxy analog of the sulfur-containing amino acid methionine and is extensively used as a methionine supplement in poultry and bovine feed.

Native mass spectrometry, the method by which noncovalent protein complexes are retained in the gas phase for intact mass analysis, is gaining interest as a method for intact protein characterization. The development of a modified orbital ion trap platform for high-resolution analyses has expanded the role of native mass spectrometry to address the challenges of intact protein characterization.

Ionization of small, large, volatile, and nonvolatile compounds with charge states nearly identical to electrospray ionization are produced from a solid matrix or solution with high sensitivity utilizing the vacuum inherent with any mass spectrometer. With the proper matrix, analytes can be analyzed from ambient conditions or by direct introduction into vacuum.

The identification of nontargeted species in environmental and commercial samples by mass spectrometry can be very difficult. In this article, authors from Eastman Chemical Company describe their systematic approach for the identification of nontargeted species using nominal and accurate mass data, searching both mass spectral and "spectra-less" databases.