Liquid Chromatography-mass Spectrometry Instrument Research Articles

Targeted Quantification of Amino Acids by Dansylation.

Quantification of amino acids in biological samples is a critical tool for studying metabolism. Although many methods for amino acid analysis exist, important considerations include ease of sample preparation, dynamic range, reproducibility, instrument availability, and throughput. Here, we present a simple, rapid, and robust method for the analysis of amino acids by chemical derivatization and liquid chromatography-mass spectrometry (LC-MS). We provide a detailed protocol for the analysis of 20 proteinogenic amino acids in biological samples which will enable straightforward implementation on modern LC-MS instruments.

Seasonal variations and potential sources of biomass burning tracers in particulate matter in Nanjing aerosols during 2017-2018.

Biomass burning (BB) is an important source of atmospheric particulate matter and can adversely affect air quality, visibility, human health, and climate change. To study the characteristics and potential source regions of BB tracers in PM2.5, a liquid chromatography-mass spectrometry instrument (HPLC-MS/MS) is applied in this study to develop and validate a method to determine organic tracers of BB in 397 aerosol samples. The total mean concentrations of 17 tracers measured in 2017 and 2018 were 333.32ngm-3 and 243.45ngm-3, respectively. Among them, the concentration of levoglucosan was the highest among all the tracers, with 325.63ngm-3 in 2017 and 237.47ngm-3 in 2018. The BB tracers showed obvious seasonal variations characteristics, most of which were abundant in winter. However, the concentrations of 3,4-dimethoxyacetic acid and sinapinic acid were higher in summer and spring than that in the other seasons. There were obvious differences in the Potential Source Contribution Factor (PSCF) model results of the BB's potential source area annually and in different seasons. The results of the potential source analysis showed that Beijing-Tianjin-Hebei had a great impact on the Nanjing air quality in 2017. Finally, five source factors for BB were identified based on the Positive Matrix Factor (PMF) model, and these were cellulose, hardwood, softwood, grass, and secondary formed. During the 2 years, cellulose was the largest contributor to biomass burning. Owing to the different fire conditions each year, the contribution of the five factors to the BB tracers was also different. For example, the contribution of softwood to the BB tracers was greater in 2018 (8.4%) than in 2017 (5.2%), while the contributions of hardwood and cellulose did not change significantly.

Possibilities of Liquid Chromatography Mass Spectrometry (LC-MS)-Based Metabolomics and Lipidomics in the Authentication of Meat Products: A Mini Review.

The liquid chromatography mass spectrometry (LC-MS)-based metabolomic and lipidomic methodology has great sensitivity and can describe the fingerprint of metabolites and lipids in pork and beef. This approach is commonly used to identify and characterize small molecules such as metabolites and lipids, in meat products with high accuracy. Since the metabolites and lipids can be used as markers for many properties of a food, they can provide further evidence of the foods authenticity claim. Chromatography coupled to mass spectrometry is used to separate lipids and metabolites from meat samples. The research data usually is compared to lipid and metabolite databases and evaluated using multivariate statistics. LC-MS instruments directly connected to the metabolite and lipid databases software can be used to assess the authenticity of meat products. LC-MS has good selectivity and sensitivity for metabolomic and lipidomic analysis. This review highlighted the combination of metabolomics and lipidomics can be used as a reference for analyzing authentication meat products.

A Modular and Expandable Ecosystem for Metabolomics Data Annotation in R.

Liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics experiments have become increasingly popular because of the wide range of metabolites that can be analyzed and the possibility to measure novel compounds. LC-MS instrumentation and analysis conditions can differ substantially among laboratories and experiments, thus resulting in non-standardized datasets demanding customized annotation workflows. We present an ecosystem of R packages, centered around the MetaboCoreUtils, MetaboAnnotation and CompoundDb packages that together provide a modular infrastructure for the annotation of untargeted metabolomics data. Initial annotation can be performed based on MS1 properties such as m/z and retention times, followed by an MS2-based annotation in which experimental fragment spectra are compared against a reference library. Such reference databases can be created and managed with the CompoundDb package. The ecosystem supports data from a variety of formats, including, but not limited to, MSP, MGF, mzML, mzXML, netCDF as well as MassBank text files and SQL databases. Through its highly customizable functionality, the presented infrastructure allows to build reproducible annotation workflows tailored for and adapted to most untargeted LC-MS-based datasets. All core functionality, which supports base R data types, is exported, also facilitating its re-use in other R packages. Finally, all packages are thoroughly unit-tested and documented and are available on GitHub and through Bioconductor.

Functional surface modification using tailored ultrashort-pulse laser light for applications in Liquid Chromatography Mass Spectrometry instruments

Ultrashort-pulse lasers allow to deposit extremely large peak intensities into a material, albeit with an exquisite precision. Thus, they have become a prevalent tool in many processes that require micro-precision material modification. One such applications is the micro-structuring of functional surfaces in Liquid Chromatography Mass Spectrometry instruments. Laser light can be tailored to induce permanent changes to the surface properties of materials not easily achieved by other means, for example to change the local chemistry, the local crystal structure and the local morphology. Such methods are expected to affect the behaviour of electrodes in a Liquid Chromatography Mass Spectrometry instrument. Structuring the incident laser light enables tailored laser-material interactions to be controlled with very high spatial resolutions, leading to extremely accurate control of the local surface properties relative to the bulk and other regions on the surfaces.

Multi-Omic Single-Shot Technology for Integrated Proteome and Lipidome Analysis.

Mass spectrometry (MS) serves as the centerpiece technology for proteome, lipidome, and metabolome analysis. To gain a better understanding of the multifaceted networks of myriad regulatory layers in complex organisms, integration of different multiomic layers is increasingly performed, including joint extraction methods of diverse biomolecular classes and comprehensive data analyses of different omics. Despite the versatility of MS systems, fractured methodology drives nearly all MS laboratories to specialize in analysis of a single ome at the exclusion of the others. Although liquid chromatography-mass spectrometry (LC-MS) analysis is similar for different biomolecular classes, the integration on the instrument level is lagging behind. The recent advancements in high flow proteomics enable us to take a first step towards integration of protein and lipid analysis. Here, we describe a technology to achieve broad and deep coverage of multiple molecular classes simultaneously through multi-omic single-shot technology (MOST), requiring only one column, one LC-MS instrument, and a simplified workflow. MOST achieved great robustness and reproducibility. Its application to a Saccharomyces cerevisiae study consisting of 20 conditions revealed 2842 protein groups and 325 lipids and potential molecular relationships.

Undergraduate forensic biochemistry laboratory experiment to detect doping in animal hair using LCMS

Doping using performance enhancing banned substances is a serious problem in almost every sport competition. Not surprisingly, the detection of these contra banned drugs is an area of active and continuous improvement and innovation by bioanalytical chemists. Additionally, most students working out in the gym and taking part in various sports need to be made aware of the doping and the health problems associated with it. Science or STEM students, in particular chemistry students, must not only be made aware of these issues, but also be taught that chemistry (and science) can provide solutions to such real-life issues. To this end, a newly developed forensic laboratory experiment is described that guides students to learn liquid chromatography mass spectrometry instrumentation (LC-MS) to detect four common doping drugs cortisol, dexamethasone, methyl prednisolone and flumethasone in camel hair samples. In addition, the project is also designed to reinforce the importance of hair analysis as an additional sample matrix, complementary to saliva, blood and urine tests, in doping applications. In addition to learning various aspects of sample preparation, extraction, and LC-MS principles, students will also learn how to validate this method according to Food and Drugs Administration guidelines for intra and inter day precision and accuracy, recovery, stability and linearity. This “applied forensic science” experiment was successfully implemented in a biochemistry undergraduate research course to enhance students' learning of doping issues as well as important bio-analytical and forensic biochemistry concepts. Student survey confirmed that this laboratory experiment was successful in achieving the objectives of raising awareness of doping control in students and illustrating the usefulness of chemistry in solving real-life problems. This experiment can be easily adopted in an advanced biochemistry laboratory course and taught as an inquiry-guided exercise. Such hands-on and engaging experiments should be part of undergraduate curriculum to foster deeper interest and innovation in STEM subjects to better prepare the next-generation workforce in science and technology.

Subnanogram proteomics: Impact of LC column selection, MS instrumentation and data analysis strategy on proteome coverage for trace samples

One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5ng to 50ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading to a ∼3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ∼95% for 0.5ng samples and by ∼42% for 2ng samples. Using the best combination of the above variables, we were able to identify >3000 proteins from 10ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. The present ultrasensitive LC–MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.

Signatures for Mass Spectrometry Data Quality

Ensuring data quality and proper instrument functionality is a prerequisite for scientific investigation. Manual quality assurance is time-consuming and subjective. Metrics for describing liquid chromatography mass spectrometry (LC–MS) data have been developed; however, the wide variety of LC–MS instruments and configurations precludes applying a simple cutoff. Using 1150 manually classified quality control (QC) data sets, we trained logistic regression classification models to predict whether a data set is in or out of control. Model parameters were optimized by minimizing a loss function that accounts for the trade-off between false positive and false negative errors. The classifier models detected bad data sets with high sensitivity while maintaining high specificity. Moreover, the composite classifier was dramatically more specific than single metrics. Finally, we evaluated the performance of the classifier on a separate validation set where it performed comparably to the results for the testing/training data sets. By presenting the methods and software used to create the classifier, other groups can create a classifier for their specific QC regimen, which is highly variable lab-to-lab. In total, this manuscript presents 3400 LC–MS data sets for the same QC sample (whole cell lysate of Shewanella oneidensis), deposited to the ProteomeXchange with identifiers PXD000320–PXD000324.

Contribution of various types of liquid chromatography–mass spectrometry instruments to band broadening in fast analysis

When performing fast LC with 50mm narrow-bore columns packed with small particles, the LC instrumentation can give rise to non-negligible band broadening. In the present study, the loss in chromatographic efficiency attributed to nine different mass spectrometers of various brands, ionization source geometries and types of analyzers was assessed. In their standard configurations, the extra-column variance of these UHPLC–MS systems was estimated to vary from 20 to >100μL2. However, it was demonstrated that these differences arise exclusively from the chromatographic system (i.e., injector, tubing, valves, heater) and from the tubing employed to interface the UHPLC instrument with the MS device. By minimizing the tubing used for each UHPLC system, the extra-column variance was reduced to approximately 17–19μL2 at 600μL/min, for all types of configurations. To achieve optimal chromatographic performance, it is therefore of prime importance to optimize the UHPLC configuration prior to conducting MS. The tubing located between the UHPLC system and the ionization source entrance was found to be particularly critical, as it contributes to band broadening even in the gradient mode. Using an optimized UHPLC–MS configuration, the loss in efficiency with a 50×2.1mm I.D. column was negligible for k>7. However, the efficiency loss with 1mm I.D. columns remained non-negligible for all current instrumentation, even for solutes with a value of k>20. Indeed, for a mixture of isobaric substrates and metabolites analyzed in gradient mode, the peak widths decreased by approximately 50% between a standard and optimized UHPLC–MS configuration, considering a 50×2.1mm, 1.7μm column. The peak broadening was changed by 230% on a 50×1mm, 1.7μm stationary phase, for the same system configurations.

Liquid Chromatography–Mass Spectrometry Calibration Transfer and Metabolomics Data Fusion

Metabolic profiling is routinely performed on multiple analytical platforms to increase the coverage of detected metabolites, and it is often necessary to distribute biological and clinical samples from a study between instruments of the same type to share the workload between different laboratories. The ability to combine metabolomics data arising from different sources is therefore of great interest, particularly for large-scale or long-term studies, where samples must be analyzed in separate blocks. This is not a trivial task, however, due to differing data structures, temporal variability, and instrumental drift. In this study, we employed blood serum and plasma samples collected from 29 subjects diagnosed with small cell lung cancer and analyzed each sample on two liquid chromatography-mass spectrometry (LC-MS) platforms. We describe a method for mapping retention times and matching metabolite features between platforms and approaches for fusing data acquired from both instruments. Calibration transfer models were developed and shown to be successful at mapping the response of one LC-MS instrument to another (Procrustes dissimilarity = 0.04; Mantel correlation = 0.95), allowing us to merge the data from different samples analyzed on different instruments. Data fusion was assessed in a clinical context by comparing the correlation of each metabolite with subject survival time in both the original and fused data sets: a simple autoscaling procedure (Pearson's R = 0.99) was found to improve upon a calibration transfer method based on partial least-squares regression (R = 0.94).

An introduction to liquid chromatography–mass spectrometry instrumentation applied in plant metabolomic analyses

Over the past decade the application of non-targeted high-throughput metabolomic analysis within the plant sciences has gained ever increasing interest and has truly established itself as a valuable tool for plant functional genomics and studies of plant biochemical composition. Whilst proton nuclear magnetic resonance ((1)H-NMR) spectroscopy is particularly appropriate for the analysis of bulk metabolites and gas chromatography mass spectrometry (GC-MS) to the analysis of volatile organic compounds (VOC's) and derivatised primary metabolites, liquid chromatography (LC)-MS is highly applicable to the analysis of a wide range of semi-polar compounds including many secondary metabolites of interest to plant researchers and nutritionists. In view of the recent developments in the separation sciences, leading to the advent of ultra high performance liquid chromatography (UHPLC) and MS based technology showing the ever improving resolution of metabolite species and precision of mass measurements (sub-ppm accuracy now being achievable), this review sets out to introduce the background and update the reader upon LC, high performance (HP)LC and UHPLC, as well as the large range of MS instruments that are being applied in current plant metabolomic studies. As well as covering the theory behind modern day LC-MS, the review also discusses the most relevant metabolomics applications for the wide range of MS instruments that are currently being applied to LC.

Recent developments in the application of liquid chromatography–tandem mass spectrometry for the determination of organic residues and contaminants

Since the introduction of atmospheric pressure ionisation (API) liquid chromatography–mass spectrometry (LC-MS) for routine analysis in the early 1990s, large improvements in terms of sensitivity and robustness of the MS instruments have been achieved and new mass analysers such as the linear ion traps and a Fourier transformation (FT) ion trap MS (Orbitrap) have been designed for LC-MS/MS analysis of small molecules [1]. Improvements of LC-MS instrumentation, the establishment of maximum levels for e.g. pesticides [2, 3], mycotoxins [4] and pharmaceuticals [5] and the definition of strict analytical quality assurance criteria (e.g. [6, 7]) have greatly contributed to the increasing popularity of LC-MS/MS in recent years. Most applications of LC-MS/MS to the analysis of residues and contaminants (e.g. in food and environmental samples) focus on the accurate quantification or semi-quantitative screening of target compounds and the confirmation of their identity. Recent publications clearly indicate a trend towards multi-target methods covering up to more than 100 compounds in a single chromatographic run as well as towards screening of (mostly highly polar) transformation products of known residues and contaminants with the major goal to study their metabolisation or degradation during numerous processes. This article presents a short introduction to the variety of mass analyser configurations and recent developments of high-performance liquid chromatography (HPLC) instrumentation. Additionally, three key topics of LC-MS/MS—matrix effects, multi-analyte methods and confirmatory analysis— are briefly discussed.

Confirmation of Peanut Protein Using Peptide Markers in Dark Chocolate Using Liquid Chromatography−Tandem Mass Spectrometry (LC-MS/MS)

Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.

Determination of Acepromazine, Ketamine, Medetomidine, and Xylazine in Serum: Multi-Residue Screening by Liquid Chromatography-Mass Spectrometry

A large variety of drugs are administered to large and small animals by veterinary clinicians for sedation, anesthesia, muscle relaxation, and analgesia. The present paper reports a simple and rapid multi-residue detection and quantitation method for four chemically different drugs: medetomidine, xylazine, ketamine, and acepromazine. Chromatographic separation was carried out on a liquid chromatography-mass spectrometry instrument with a C18-reversed-phase column. Fragmentation patterns were determined with atmospheric pressure chemical ionization mass spectrometry set to operate in a positive selective ion monitoring mode. The method was determined to be linear over the range of concentrations tested (2.0-100.0 ng/mL). Accuracy, precision, and specificity were evaluated and the method was determined to be applicable to detection of medetomidine, xylazine, ketamine, and acepromazine in serum samples of multiple animal species (canine, equine, and bovine). Matrix limits of quantitation were determined to be 5.0 ng/mL for all four analytes, and recoveries ranged between 82.0 and 118%, with a 3.0-18.3% relative standard deviation.

A liquid chromatography-mass spectrometry assay for analyzing sulfonamide antibacterials in cattle and fish muscle tissues.

A simple and rapid method able to determine residues of 12 sulfonamide (SAs) antibacterials in cattle and trout muscle tissues is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography-mass spectrometry (LC-MS). The LC-MS instrumentation was equipped with an electrospray source and a single quadrupole. After 0.8 g of a flesh sample containing the analytes is deposited on sand (crystobalite), this material is packed into an extraction cell. SAs are extracted by flowing 4 mL of water through the cell heated at 80 degrees C. A 0.5-mL aliquot of the bovine tissue extract is then directly injected into the LC column, while the fish tissue extract is filtered prior to LC-MS analysis. MS data acquisition was performed in the positive-ion mode and monitoring at least three ions for each target compound. Confirmatory ions were produced by the in-source collision-induced dissociation process. At the tolerance levels issued by the EU and U.S. Food and Drug Administration, i.e., 100 ppb, recovery of the analytes in bovine and trout muscle tissues was 75-98% with RSDs ranging between 1 and 8%. Estimated limits of quantification (S/N = 10) were 6-15 ppb for SAs in bovine muscle tissue and 3-13 ppb in trout fillet. When trying to reduce the analysis time by using a short chromatographic run time, severe ion signal suppression was experienced for the early-eluted SAs. This effect was traced to competition effects by polar endogenous coextractives, maybe proteinaceous species, which are eluted in the first part of the chromatographic run. This unwelcome effect was removed by simply adopting more selective chromatographic conditions.

A General Unknown Screening Procedure for Drugs and Toxic Compounds in Serum using Liquid Chromatography-Electrospray-Single Quadrupole Mass Spectrometry

A complete general unknown screening procedure was developed using liquid chromatography-mass spectrometry (LC-MS), a coupling that can increase the range of compounds amenable to MS. Sample preparation was by solid-phase extraction on a mixed-mode support in parallel with serum deproteination in order to recover the most hydrophilic compounds. Chromatography employed a reversed-phase narrow-bore column (150 x 1-mm i.d.) and a 50-min gradient elution at low flow-rate (50 microL/min), compatible with the electrospray source used without splitting nor heating. The single quadrupole LC-MS instrument used was operated in the 100 to 1100 mu mass range in both the positive and negative modes, with two different, alternated collision-induced dissociation voltages in the source, in order to obtain the molecular or pseudo-molecular ions as well as fragments for the compounds analyzed. The addition of spectra obtained at low and high fragmentation voltages gave reconstructed spectra for each polarity, representing library entries. Finally, a program was created in order to detect the peaks of interest in the chromatographic noise using a very efficient signal processing algorithm, compute their relative retention time with respect to the internal standard (glafenine), draw their reconstructed spectra, search them in the libraries, and edit a report.

Validation of liquid chromatography—mass spectrometry methods: interlaboratory studies

Developments over the last 15 years have led to widespread acceptance and commercialization of two liquid chromatography—mass spectrometry (LC-MS)interfaces: thermospray (TSP) and particle beam (PB). Very few LC-MS methods have been developed and formally adopted, however, by the U.S. Environmental Protection Agency (EPA). This article addresses the difficulties encountered with current LC-MS instrumentation in validating the methods and discusses some of the results of three interlaboratory EPA studies, including a thermospray-LC-MS (TSP-LC-MS) study for chlorinated herbicides, a TSP-LC-MS study for carbamate pesticides, and a particle beam-LC-MS (PB-LC-MS) study for benzidines. Differences in features among the various LC-MS interfaces made it impractical to impose strict guidelines to the participants in conducting these analyses. Consequently, difficulties were encountered in analyzing the data; nonetheless, interesting and useful data, as outlined in this article, were obtained.

Corona discharge ionization liquid chromatography—mass spectrometry interface for target compound analyses

The design and construction of a dedicated liquid chromatography—mass spectrometry instrument for detecting and assaying trace-level target compounds are described. It employs a microbore high-performance liquid chromatograph interfaced directly to a point-to-point high voltage corona discharge ionization source with a quadrupole mass analyser. Applications are described using reversed-phase chromatography for the detection of route-specific impurities in formulated medicinal preparations at the 5 ppb level for patent protection, and for the detection of biogenic amines in blood plasma without derivatization.