Isobaric Interferences Research Articles

Derivatization with 2-hydrazino-1-methylpyridine enhances sensitivity of analysis of 5α-dihydrotestosterone in human plasma by liquid chromatography tandem mass spectrometry

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of DHT by LC-MS/MS. Derivatization of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of DHT, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies.

Structural analysis of a compound despite the presence of an isobaric interference by using in-source Collision Induced Dissociation and tandem mass spectrometry.

The presence of an isobaric contaminant can drastically affect MS and MS/MS patterns leading to erroneous structural and quantitative analysis, which is a real challenge in mass spectrometry. Herein, we demonstrate that MS and MS/MS structural analysis of a compound can be successfully performed despite the presence of an isobaric interference with as low as few millidaltons mass difference by using pseudo-MS3 . To this end, in-source collisional excitation (in-source CID) and the Survival Yield (SY) technique (energy-resolved collision induced dissociation MS/MS) were performed on two different source geometries: a Z-spray and an orthogonal spray (with a transfer capillary) ionization sources on two different mass spectrometers. By using soft ionization conditions, the SY curve for the mixture is a linear combination of the SY curves from the pure compounds demonstrating the presence of two components in the mixture. In the case of harsher ionization conditions, the SY curve of the mixture perfectly overlaps the SY curve from the pure analyte. This observation demonstrates the isobaric interference has been completely removed by in-source CID fragmentation, independently of the source design, leaving then the analyte precursor ions only. Therefore, by measuring the MS spectrum in harsh ionization conditions and according to SY criterium, the compound of interest can be made free from isobaric interference paving the way for, for example, unequivocal HPLC-MS as well as HPLC-MS/MS structural and quantitative analysis despite the presence of a co-eluting isobaric interference.

Determination of low-level 135Cs and 135Cs/137Cs atomic ratios in large volume of seawater by chemical separation coupled with triple-quadrupole inductively coupled plasma mass spectrometry measurement for its oceanographic applications

Radioisotopes of cesium are powerful tracer for oceanographic studies. In this work, a novel method was developed for determination of ultra-low level 135Cs and 137Cs in seawater using triple-quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS). Cesium was pre-concentrated from up to 45 L seawater samples using ammonium molybdophosphate (AMP) adsorption, following a selective leaching of cesium using Sr(OH)2. The cesium was further purified from interfering elements using AMP-PAN and cation-exchange chromatography. Sr(OH)2 leaching was found to be an effective approach for selective exchange of cesium from the AMP sorbent without dissolution, which avoids the problem of separation of huge amount of NH4+ and MoO42− in the following steps. The decontamination factors for barium and rubidium with the developed method were more than 4 × 107 and 800, respectively. The separated 135Cs and 137Cs were measured using ICP-MS/MS by employing N2O as reaction gas to further elimination of isobaric (i.e. 135Ba and 137Ba) and polyatomic ions interferences. A detection limit of 1.5 × 10−16 g L−1 for 135Cs in seawater was achieved. The concentrations of 135Cs in seawater from Baltic Sea, Danish straits and Roskilde Fjord were determined using the developed method to identify the sources of 135Cs, the water masses exchange in this region was investigated using 135Cs and 137Cs.

On-tissue chemical derivatization in mass spectrometry imaging.

Mass spectrometry imaging (MSI) combines molecular and spatial information in a valuable tool for a wide range of applications. Matrix‐assisted laser desorption/ionization (MALDI) is at the forefront of MSI ionization due to its wide availability and increasing improvement in spatial resolution and analysis speed. However, ionization suppression, low concentrations, and endogenous and methodological interferences cause visualization problems for certain molecules. Chemical derivatization (CD) has proven a viable solution to these issues when applied in mass spectrometry platforms. Chemical tagging of target analytes with larger, precharged moieties aids ionization efficiency and removes analytes from areas of potential isobaric interferences. Here, we address the application of CD on tissue samples for MSI analysis, termed on‐tissue chemical derivatization (OTCD). MALDI MSI will remain the focus platform due to its popularity, however, alternative ionization techniques such as liquid extraction surface analysis and desorption electrospray ionization will also be recognized. OTCD reagent selection, application, and optimization methods will be discussed in detail. MSI with OTCD is a powerful tool to study the spatial distribution of poorly ionizable molecules within tissues. Most importantly, the use of OTCD−MSI facilitates the analysis of previously inaccessible biologically relevant molecules through the adaptation of existing CD methods. Though further experimental optimization steps are necessary, the benefits of this technique are extensive.

Techniques for measuring carbon and oxygen isotope compositions of atmospheric CO2 via isotope ratio mass spectrometry.

Measuring the stable isotope compositions of atmospheric CO2 is common in earth and atmospheric sciences, and various analytical methods have been developed utilizing continuous-flow (CF) or dual-inlet (DI) isotope ratio mass spectrometry (IRMS). Air is typically collected via passive, manual, or automated collection methods and the volume of the air sample ranges from 10 to 300 mL for CF-IRMS to >1 L for DI-IRMS to yield a measurable amount of atmospheric CO2 gas. It has been determined that the integrity of vials and flasks for air sample storage can be compromised after 3 days of air collection for δ13 C values and within 10 hours for δ18 O values. Air samples must be purified after collection to remove constituents of air, such as Ar, O2 , N2 , N2 O, and water vapor, to avoid isobaric interferences during mass spectrometric measurement. Purification is generally undertaken by utilizing commercial or custom-made preconcentration devices, the blanking method for CF-IRMS, or an offline/online cryogenic separation using a vacuum line for DI-IRMS. Ambient N2 O is a component of air that may affect analytical results and thus must either be corrected for or be removed using a gas chromatographic column. In some cases, water is removed during air collection by using a common chemical desiccant, magnesium perchlorate (Mg(ClO4 )2 ), or by a dry ice/alcohol mixture (-78°C). Lastly, a linearity issue for IRMS due to the low amount of purified CO2 from a typical ambient air sample must be considered. In general, analytical precisions of 0.02-0.21‰ and 0.04-0.34‰ for CF-IRMS and 0.01-0.02‰ and 0.01-0.02‰ for DI-IRMS are expected for δ13 C and δ18 O measurements, respectively.

Complementing Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging with Chromatography Data for Improved Assignment of Isobaric and Isomeric Phospholipids Utilizing Trapped Ion Mobility-Mass Spectrometry.

Lipids, such for example the multifaceted category of glycerophospholipids (GP), play a major role in many biological processes. High-resolution mass spectrometry is able to identify these highly diverse lipid species in combination with fragmentation experiments (MS/MS) on the basis of the accurate m/z and fragmentation pattern. However, for the differentiation of isomeric lipids or isobaric interferences, more elaborate separation methods are required. Especially for imaging techniques, such as matrix-assisted laser desorption/ionization (MALDI)-MS imaging, the identification is often exclusively based on the accurate m/z. Fragmentation via MS/MS increases the confidence in lipid annotation in imaging approaches. However, this is sometimes not feasible due to insufficient sensitivity and significantly prolonged analysis time. The use of a separation dimension such as trapped ion mobility spectrometry (TIMS) after ionization strengthens the confidence of the identification based on the collision cross section (CCS). Since CCS libraries are limited, a tissue-specific database was initially generated using hydrophilic interaction liquid chromatography-TIMS-MS. Using this database, the identification of isomeric lipid classes as well as isobaric interferences in a lipid class was performed using a mouse spleen sample in a workflow described in this study. Besides a CCS-based identification as an additional identification criterion for GP in general, the focus was on the distinction of the isomeric GP classes phosphatidylglycerol and bis(monoacylglycero)phosphate, as well as the differentiation of possible isobaric interferences based on the formation of adducts by MALDI-TIMS-MS imaging on a molecular level.

Investigation and Resolution of Interference in the LC-QTOF-MS Detection of 4-MePPP.

An interference resulting in the false-positive detection of the synthetic cathinone 4-MePPP in urine was suspected following the recent addition of 4-MePPP spectral data to an LC-QTOF-MS drug library. Although positive detection criteria were achieved, it was noted that all urine samples suspected of containing 4-MePPP also concurrently contained high levels of tramadol and its associated metabolites. Using QTOF-MS software elucidation tools, candidate compounds for the suspected interference were proposed. To provide further confidence in the identity of the interference, in silico fragmentation tools were used to match product ions generated in the analysis with product ions predicted from the theoretical fragmentation of candidate compounds. The ability of the suspected interference to subsequently produce the required product ions for spectral library identification of 4-MePPP was also tested. This information was used to provide a high preliminary confidence in the compound identity prior to purchase and subsequent confirmation with certified reference material. A co-eluting isobaric interference was identified and confirmed as an in-source fragment of the tramadol metabolite, N,N-bisdesmethyltramadol. Proposed resolutions for this interference are also described and subsequently validated by retrospective interrogation of previous cases of suspected interference.

Chemical Derivatization-Aided High Resolution Mass Spectrometry for Shotgun Lipidome Analysis.

Chemical derivatization coupled with nano-electrospray ionization (nESI) and ultra-high resolution accurate mass spectrometry (UHRAMS) is an established approach to overcome isobaric and isomeric mass interference limitations, and improve the analytical performance, of direct-infusion (i.e., "shotgun") lipidome analysis strategies for "sum composition" level identification and quantification of individual lipid species from within complex mixtures. Here, we describe a protocol for sequential functional group selective derivatization of aminophospholipids and O-alk-1'-enyl (i.e., plasmalogen) lipids, that when integrated into a shotgun lipidomics workflow involving deuterium-labeled internal lipid standard addition, monophasic lipid extraction, and nESI-UHRAMS analysis, enables the routine identification and quantification of >500 individual lipid species at the "sum composition" level, across four lipid categories and from >30 lipid classes and subclasses.

A high throughput Rb-Sr dating method using solution tandem ICP-MS/MS (87Sr/86Sr) and standard addition calibration ICP-MS (Rb/Sr)

Routine generation of large Rb-Sr age datasets has been limited by the time-consuming requirement of chromatographic isolation of Sr from Rb to avoid the isobaric interference of 87Rb+ on 87Sr+. Recently, direct measurement of interference-free 87Sr/86Sr has become possible with the advent of tandem quadrupole inductively coupled plasma mass spectrometry (ICP-MS/MS). Here, we describe a method that uses ICP-MS/MS to measure 87Sr/86Sr combined with a standard addition calibration for precise determination of Rb/Sr, allowing efficient generation of Rb-Sr ages. The method is demonstrated on a suite of ~50 to 90 Myrs (million years) old celadonite samples that are dated with age uncertainties typically of ±2 to 5 Myrs provided the sample Rb/Sr is >2. A method to separate the phyllosilicate fraction from the bulk sample, because it commonly has a higher Rb/Sr more suitable for dating, using differential settling is also described.•Differential settling allows high Rb/Sr phyllosilicates to be separated from other phases•ICP-MS/MS allows 87Sr/86Sr measurement without chromatographic isolation of Sr+ from Rb+•Single-spike standard addition ICP-MS was used for Rb/Sr measurement

Resolving isobaric interference towards the determination of 137Cs and 90Sr using laser-ionization mass spectrometry

Resolving isobaric interference in situ using laser-ionization mass spectrometry (LIMS).

In situ Fe isotopic analyses of fourteen reference materials using a synthetic Cr standard for mass bias and isobaric interference corrections by femtosecond LA-MC-ICP-MS

A new method for the direct determination of Fe isotopic compositions in silicate rock samples by fs LA-MC-ICP-MS.

Precise δ88/86Sr determination on a MC-ICP-MS by an improved method combining Zr-empirical external normalization isobaric interference correction and 84Sr–87Sr double spike

Zr-EEN isobaric interference correction coupled with the 84Sr–87Sr double spike method allows highly accurate and precise δ88/86Sr determination on a MC-ICP-MS.

Multi-class pesticide analysis in cannabis oil using coated blade spray and solid-phase microextraction with liquid chromatography coupled to mass spectrometry

The application of solid-phase microextraction (SPME) coupled to mass spectrometry to provide a high-throughput and cost-effective solution to multi-residue analysis of pesticides in cannabis oil samples has not been extensively explored. In this work, the method development steps for the extraction of an initial target list of 74 pesticides from cannabis oil via SPME for analysis with both LC-MS/MS and coated blade spray (CBS) are presented. The exploration of a washing step to remove adhered oil whilst minimally desorbing extracted analytes along with the implementation of central composited design investigation to examine compound extraction kinetics in the non-polar matrix yielded a workflow that was validated via both instrumental techniques. Of the initial target list, 37 pesticides were found to be suitable for screening or quantitation via CBS with performance validated via LC-MS/MS. The majority of compounds were found to meet the EU SANTE guidelines for analysis (i.e. linearity, precision, accuracy) whilst reaching limits of quantitation below or at Health Canada minimum regulatory limits (majority at 10 ng/mL). Examination of factors contributing to poor quantitation of pesticides via CBS are shared and explored, such as contributing isobaric interference sourced from plant byproducts and carrier oil, and comparison of signal-to-noise values achieved in cannabis oil when compared to the cannabis-free medium-chain triglyceride oil used as a carrier oil to stress the importance of matrix-match method development.

Simultaneous determination of platinum group elements and rhenium mass fractions in road dust samples using isotope dilution inductively coupled plasma-tandem mass spectrometry after cation exchange separation

Measurement of Platinum Group Element (PGE: Ru, Rh, Pd, Os, Ir, and Pt) and Rhenium (Re) in environmental samples is a difficult task due to their ultra-trace level concentrations, and these metals suffer from severe isobaric and polyatomic interference. These kinds of issues have been solved for environmental samples having simple matrices such as tunnel dust and moss. However, a scope of improvement still exists in challenging sample matrices such as road dust, soil, and rocks. In this study, a method that enables measurement of PGE and Re mass fractions in road dust and rock samples is proposed. The method involves matrix removal using a cation-exchange column followed by measurement using inductively coupled plasma tandem mass spectrometry (ICP-MS/MS, also known as ICP- QQQ-MS) with 10% NH3 in He as reactive gas in Collision/Reaction Cell. This study emphasizes that the usage of a simple cation-exchange column is an important step over the framework developed by Suoranta et al., 2012, which was developed to measure PGE and Re fractions in environmental samples having a simpler matrix such as moss samples. The method combines high pressure (130 bars) and high temperature (220 °C) digestion procedure in combination with isotope dilution (ID) as a calibration strategy, PGE and Re separation using cation exchange resin, and their measurements using ICP-MS/MS. The method was validated with road dust (BCR-723) and ultramafic komatiite (OKUM) having a complex matrix. The measured PGE and Re mass fractions of the two-measured reference material show excellent agreement with their certified values and have intermediate precisions ranging from 0.4–7.7% RSD. We conclude that the combination of single-stage cation-exchange chromatography with ID-ICP-MS/MS measurements enables measurement results with uncertainties fit for purpose for PGE and Re mass fractions in environmental samples with a complex matrix.

Billion-year exposure ages in Gale crater (Mars) indicate Mount Sharp formed before the Amazonian period

The erosion rates and mechanisms operating on Mount Sharp in Gale crater, Mars were assessed via experiments performed by the SAM instrument to determine the cosmogenic noble gas contents of Murray mudstone formation samples Mojave 2 and Quela. Previous measurements of samples from the Aeolis Palus depression between Mount Sharp and the north rim of Gale crater indicate that scarp retreat-generated surfaces formed within the last 100 Ma. In contrast, Mojave 2 yielded exposure ages of 1,320±240 (3He), 910±420 (21Ne), and 310±60 Ma (36Ar). Quela gave a 3He age of 1,460±200 Ma; 21Ne and 36Ar from this sample could not be quantified due to isobaric interferences. The discordant and young 36Ar exposure age in Mojave 2 is likely the result of interaction with water which dissolved the chlorine-bearing host phases of this nuclide. The most probable exposure scenario is that both Mojave 2 and Quela have been at the surface for the most recent ∼1 Ga after the overlying few meters of rock were removed in a geologically rapid exhumation episode. Based on local geomorphology, scarp retreat is the most likely mechanism for the exposure at these two sites. The exposure ages measured throughout Curiosity's traverse indicate that the net removal of rock has proceeded more recently on Aeolis Palus than on the lower slopes of Mount Sharp. The implied differential erosion rate is insufficient to explain how Mount Sharp formed, even over billions of years. Instead, given that the surfaces on Mount Sharp have existed for >1 Ga, the mountain must have formed early, likely during the Hesperian. This study provides direct quantitative support for inferences based on crater counts that Mount Sharp had eroded to close to its current form before onset of the Amazonian.

Isotope Dilution-Total Evaporation-Thermal Ionization Mass Spectrometric Direct Determination of Radioactive Strontium-90 in Microdrop Samples.

Thermal ionization mass spectrometry (TIMS) was used to directly quantify an ultratrace of radioactive 90Sr in microliter droplet samples. No chemical separation was required in removing isobaric interferences on M = 90 such as 90Zr and organic molecules in the mass spectrum because the difference in evaporation and ionization (emission) temperature among organic molecules, Zr and Sr, allows us to control the emission manner and significantly suppress the isobaric interferences. Direct quantification was achieved by improving the intercalibration of Faraday cups and ion counting in an isotope dilution (ID) method. Furthermore, the use of a total evaporation method (TE) enhanced the detection efficiency by the complete collection of the 90Sr ion beam from the samples and minimized the complexity of the fractionation effect in the isotope ratio calculation. In this study, 1 fg of 90Sr (equal to activity of 5 millibecquerel (mBq)) in a very low-volume sample with 108 times greater isobaric interference from 90Zr was successfully measured using the proposed ID-TE-TIMS method. The limit of detection was 0.029 fg (equal to 0.15 mBq) without any preconcentration. To demonstrate the wide usability of this method, low-volume samples of tears, eyelashes, saliva, environmental standards, and water samples (i.e., seawater and ground water) were analyzed within 1 h. The relationship of the measured values between this ID-TE-TIMS method and a radiometric analysis was shown to have good linearity.

Strontium isotope analysis of apatite via SIMS

We report the development of a SIMS analytical method for in situ Sr isotope analysis of the mineral apatite at an analytical scale of 15 μm or less. Experiments were conducted on a series of reference apatites of widely differing Sr contents at three different mass resolution settings: low (3000), moderate (4200) and very high (16000), using a large-geometry CAMECA 1280 ion microprobe retrofitted with a HyperionTM oxygen plasma ion source. Peak-stripping algorithms tailored to the different mass-resolution settings were developed to correct for significant interferences on the target strontium isotope species in the mass spectra, namely Ca dimers, CaPO, Rb and SrH, and for instrumental mass fractionation. The most consistently accurate and precise 87Sr/86Sr ratios were achieved at the 3000 mass resolution setting with appropriate energy filtering and use of a cold trap. On average, corrected 87Sr/86Sr ratios by this method were elevated by ~0.8‰ above equivalent TIMS and solution ICPMS data, with consistent behaviour across the range of Sr concentrations. Individual spot precision of ± 0.001 to 0.004 (2SE) was achieved, depending on Sr content. The key advantages of SIMS analysis compared to existing LA-MC-ICP-MS methods are the high spatial resolution, low sample consumption, and the high mass resolution that allows for the elimination of problematic isobaric interferences. Our approach is particularly useful for the analysis of small or valuable targets, such as mineral inclusions or meteoric samples, and for apatite with high REE contents. However, the much larger volume of material consumed during LA-MC-ICP-MS analysis allows for substantially better precision than the SIMS method presented here. The highly variable trace element composition of natural apatites, involving both cationic and anionic substitutions, necessitates careful characterisation of prospective targets prior to any in situ analysis, in addition to matching to appropriate reference materials.

Simple, Rapid Lipidomic Analysis of Triacylglycerols in Bovine Milk by Infusion-Electrospray Mass Spectrometry.

Bovine milk is a complex mixture of lipids, proteins, carbohydrates, and other factors of which lipids comprise 3-5% of the total mass. Rapid analysis and characterization of the triacylglycerols (TAG) that comprise about 95% of the total lipid is daunting given the numerous TAG species. In the attached methods paper, we demonstrate an improved method for identifying and quantifying TAG species by infusion-based "shotgun" lipidomics. Because of the broad range of TAG species in milk, a single internal standard was insufficient for the analysis and required sectioning the spectrum into three portions based upon mass range to provide accurate quantitation of TAG species. Isobaric phospholipid interferences were removed using a simple dispersive solid-phase extraction step. Using this method, > 100 TAG species were quantitated by acyl carbon number and desaturation level in a sample of commercially purchased bovine milk.

Mass spectral database for TOF-SIMS of stable isotopes of Sr and Zr

Nuclear accidents (e.g., at the Chernobyl and Fukushima Daiichi nuclear power plants) release considerable amounts of radioactive pollutants into the environment. Radioactive particles in dust and soil can range from a few micrometers to several micrometers in size. Detection of these micrometer-sized particles requires a fast and effective analytical method. Because time-of-flight secondary ion mass spectrometry (TOF-SIMS) exhibits high spatial resolution and parallel detection capability, it is suitable for analyzing micrometer-sized radioactive particles. However, due to isobaric interferences, TOF-SIMS cannot effectively distinguish between isobaric elements such as 90Sr and 90Zr. Resonant ionization sputtered neutral mass spectrometry (R-SNMS) eliminates isobaric interferences and enables the measurement of isotope ratios of isotopes approaching the detection limit. However, R-SNMS cannot be performed independently because its results are based on the TOF-SIMS spectra. This study provides a database of reference spectra for Sr and Zr stable isotopes, which can act as important reference for further analysis of radioactive particles in environmental samples.

Simultaneous Compound-Specific Analysis of δ33S and δ34S in Organic Compounds by GC-MC-ICPMS Using Medium- and Low-Mass-Resolution Modes.

Compound-specific isotope analysis of sulfur (δ34S-CSIA) in organic compounds was established in the last decade employing gas chromatography connected to multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS). However, δ33S-CSIA has not yet been reported so far. In this study, we present a method for the simultaneous determination of δ33S and δ34S in organic compounds by GC-MC-ICPMS applying medium- and also low-mass-resolution modes. The method was validated using the international isotope reference materials IAEA-S-1, IAEA-S-2, and IAEA-S-3. Overall analytical uncertainty including normalization and reproducibility for δ33S and δ34S was usually better than ±0.2 mUr (σ) for analytes containing at least 100 pmol of S. Further, it is demonstrated that, despite small isobaric interferences, results obtained at low mass resolution are indistinguishable from medium mass resolution offering the benefit of increased sensitivity and versatility of this method. Additionally, the method was applied for the δ33S and δ34S isotope analysis of industrially produced organic compounds to investigate potential mass-independent fractionation (MIF). The relation between δ34S and δ33S in these compounds followed a mass-dependent fractionation trend (MDF; Δ33S ≤ ±0.2 mUr). Degradation of dimethyl disulfide by direct photolysis caused a small but significant MIF (Δ33S = 0.55 ± 0.04 mUr, n = 3), demonstrating sufficient sensitivity of the method for these types of studies.