Mass Spectrometry

Using examples from our analysis of L-carnitine and acyl-L-carnitines, we give specific guidance for the use of mass spectrometry in quantitative analysis, as applied to clinical research and clinical pharmacology. We focus on quantitative accuracy and analytical selectivity as keys to successful implementation of mass spectrometric methods in clinical applications

Accurate evaluation of chemical modifications such as asparagine deamidation and aspartic acid isomerization is an essential component of comprehensive characterization of therapeutic monoclonal antibodies (mAbs). When located in the complementarity determining regions (CDRs), these modifications can cause a loss of function, impacting product efficacy and safety, resulting in the designation of the modification as a critical quality attribute. However, artifactual modifications can be introduced by analytical procedures, and distinguishing modifications as either critical quality attributes or method-induced artifacts is an important objective for product development. Conventional peptide mapping coupled with ultrahigh-resolution mass spectrometry offers advanced capabilities for definitive characterization of protein therapeutics. However, experimental conditions such as digestion time and pH can influence the observed level of chemical modifications, usually leading to over-estimation. In this work, a new peptide mapping method was developed specifically for mAb characterization that employs optimal enzyme pH for robustness, but short digestion times and time-course elements to minimize and monitor deamidation/isomerization, respectively, allowing a more accurate assessment of potential CDR sequence liabilities.

Chronic kidney disease or kidney complication resulting from another systematic disorder can impact the organ’s blood filtering capability resulting in the passage of blood-born proteins through the kidneys and into urine.  Clinical analyses for blood proteins in urine are performed to assess proper kidney function or to monitor a diagnosed disorder.  Serum albumin is a common target in these clinical assays and detection of elevated SA levels in urine is termed Albuminuria. Because of normal variability in urine content and volume multiple measurements are often made in comparison to creatitine levels within the same urine sample and reported as a ratio (ACR).  Demonstrated here is a novel means for quantifying albumin and creatinine directly from the same urine sample using MALDI-TOF mass spectrometry.  Standard addition of albumin and deuterated creatinine (d3) into control urine produced a linear and quantitative response (R2 = 0.99 and 0.98) and is used to quantify both analytes across their clinically relevant ranges. This MS-based method represents a simple, fast, attractive alternative to currently clinical methods.

This article investigates the use of gas chromatography–time-of-flight mass spectrometry (GC–TOF MS) to fragrance-profile three essential oils (ginger, wintergreen and rosemary). As well as considering the compositional differences between the oils, we will examine the use of peak deconvolution to identify closely-eluting compounds, and explore the use of soft electron ionization, assisted by comparison of ion ratios, to discriminate between isomeric monoterpenes that are difficult to identify at conventional 70 eV ionization energies due to their very similar mass spectra.

Compounds that are added as fragrances to personal care products (PCPs) can also be allergens or skin irritants for some consumers. Knowing whether these compounds are present in a product is important for both consumers with known allergies and for manufacturers in order to be compliant with various regulations related to allergens. Here, a GC-TOFMS method was developed to screen for and quantify regulated allergens in approximately 5 minutes. This method utilized a short and narrow chromatographic column along with mathematical deconvolution of the TOFMS data to separate the target allergens from each other in the standards and from matrix interference in samples. Calibration equations were compiled for standards from 1 ppb to 1 ppm (on-column) with excellent linearity and correlation coefficients. These were applied to various commercially-available perfume and cologne samples to determine quantitative information for the targeted allergens. The full-mass range data acquisition also provided for non-targeted characterization and comparisons to better understand the aroma profile of each sample. The reported method reduced analysis time for allergen screening while simultaneously increasing the acquired information about the PCP samples.

The power of nontargeted metabolite profiling is illustrated in a study focused on the determination of molecular markers in malting barley that are predictive of desirable malting quality for brewing applications. The metabolite extraction, detection, and analysis methods are highthroughput and reproducible, and therefore, this approach represents a practical addition to the plant breeder’s molecular toolbox.

Water samples were obtained from the Tar River and a local water treatment plant in eastern North Carolina in spring 2013 and fall 2015 to monitor the presence of a panel of pharmaceutical and personal care products (PPCPs). Samples were extracted by solid phase extraction (SPE) or liquid-liquid extraction and analyzed for parent PPCPs and their metabolites by liquid chromatography-time of flight mass spectrometry (HPLC-TOFMS) and gas chromatography-mass spectrometry (GC-MS). Both extraction and detection methods were compared by their recoveries and detection limits for each compound. Many parent PPCPs and their metabolites were detected including: carbamazepine, iminostillbene, oxcarbazepine, epiandrosterone, loratadine, β-estradiol, triclosan, and others. Liquid-liquid extraction was found to give overall superior recoveries. Furthermore, HPLC-TOFMS gave lower detection limits than GC-MS. Library searching of additional peaks identified further compounds with biological activity. Additionally, the effectiveness of the treatment plant on the removal of the compounds of interest is discussed.

An LC-MS method has been developed for simultaneous quantification of buprenorphine and its three metabolites, namely norbuprenorphine, buprenorphine glucuronide, and norbuprenorphine glucuronide Chromatographic separation was achieved on a C18 column with a gradient of acetonitrile and ammonium acetate buffer (25 mM, pH 6.6). The method run time was 7.5 min. Quantification was performed by selected ion monitoring of [M+H]+ ions of norbuprenorphine glucuronide (590), norbuprenorphine (414), buprenorphine glucuronide (644) and buprenorphine (468). Naloxone (328) (328ng/mL) was used as an internal standard. The samples were processed by protein precipitation and extraction recovery was ≥95% with minimal observed matrix effects (

In this study we report on the use of a field-portable GC-MS with rapid sampling techniques such as solid-phase micro extraction, purge-and-trap, thermal desorption, and heated headspace to provide a fast response for in-field-SVOCs analyses for a wide variety of environmental-type samples including potable waters, tea, plants and road gravel. We will show that this field-portable approach can provide the required sensitivity, selectivity for the effective analysis of SVOCs with very high boiling points such as polycyclic aromatic hydrocarbon (PAHs), pesticides, phenolic compounds and phthalate esters in a number of different field-based samples, in less than 10 minutes.

Moxidectin formulations help to reduce hair loss and irritation due to parasite worms in animals. So Estimation of Moxidectin in hair is important to evaluate therapeutic levels, distribution & accumulation, however estimation is also useful to evaluate harm to birds when they eat animal hair. Hence Moxidectin estimation is required for pharmacokinetic as well as environmental exposure study. Objective of the present work is to develop a rapid, selective method for the estimation of Moxidectin in Cattle Hair by LC-MS/MS. Oxcarbazepine used as a internal standard. Moxidectin extracted from cattle hair by liquid-liquid extraction using Sorenson’s Buffer as digestion solvent for incubation & methyl tert-butyl ether as an extraction solvent. Detection was performed over the range 0.026 to 1.000 ng/mG using MRM in positive polarity at unit resolution under turbo ion spray whereas separation was achieved on Kinetex 100 x 4.6 mm, 5u EVO C18 100A column with Methanol : 10mM Amonium formate pumped as gradient flow with 4.50min run time. Q1 is 640.45 whereas Q3 is sum of 528.50 and 498.50. Validation parameters shown reliable results. Method is applied for the estimation of Moxidectin in cattle Hair.

We present a brief review of this year's ASMS conference, which took place June 5–9 in San Antonio, Texas.

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.