Determination of perfluorinated alkyl acid concentrations in biological standard reference materials

Standard reference materials (SRMs) are homogeneous, well-characterized materials used to validate measurements and improve the quality of analytical data. The National Institute of Standards and Technology (NIST) has a wide range of SRMs that have mass fraction values assigned for legacy pollutants. These SRMs can also serve as test materials for method development, method validation, and measurement for contaminants of emerging concern. Because inter-laboratory comparison studies have revealed substantial variability of measurements of perfluoroalkyl acids (PFAAs), future analytical measurements will benefit from determination of consensus values for PFAAs in SRMs to provide a means to demonstrate method-specific performance. To that end, NIST, in collaboration with other groups, has been measuring concentrations of PFAAs in a variety of SRMs. Here we report levels of PFAAs and perfluorooctane sulfonamide (PFOSA) determined in four biological SRMs: fish tissue (SRM 1946 Lake Superior Fish Tissue, SRM 1947 Lake Michigan Fish Tissue), bovine liver (SRM 1577c), and mussel tissue (SRM 2974a). We also report concentrations for three in-house quality-control materials: beluga whale liver, pygmy sperm whale liver, and white-sided dolphin liver. Measurements in SRMs show an array of PFAAs, with perfluorooctane sulfonate (PFOS) being the most frequently detected. Reference and information values are reported for PFAAs measured in these biological SRMs.

Milk and serum standard reference materials for monitoring organic contaminants in human samples

Four new Standard Reference Materials (SRMs) have been developed to assist in the quality assurance of chemical contaminant measurements required for human biomonitoring studies, SRM 1953 Organic Contaminants in Non-Fortified Human Milk, SRM 1954 Organic Contaminants in Fortified Human Milk, SRM 1957 Organic Contaminants in Non-Fortified Human Serum, and SRM 1958 Organic Contaminants in Fortified Human Serum. These materials were developed as part of a collaboration between the National Institute of Standards and Technology (NIST) and the Centers for Disease Control and Prevention (CDC) with both agencies contributing data used in the certification of mass fraction values for a wide range of organic contaminants including polychlorinated biphenyl (PCB) congeners, chlorinated pesticides, polybrominated diphenyl ether (PBDE) congeners, and polychlorinated dibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners. The certified mass fractions of the organic contaminants in unfortified samples, SRM 1953 and SRM 1957, ranged from 12 ng/kg to 2200 ng/kg with the exception of 4,4′-DDE in SRM 1953 at 7400 ng/kg with expanded uncertainties generally <14 %. This agreement suggests that there were no significant biases existing among the multiple methods used for analysis.

Comparison of targeted peptide quantification assays for reductive dehalogenases by selective reaction monitoring (SRM) and precursor reaction monitoring (PRM)

Targeted absolute protein quantification yields valuable information about physiological adaptation of organisms and is thereby of high interest. Especially for this purpose, two proteomic mass spectrometry-based techniques namely selective reaction monitoring (SRM) and precursor reaction monitoring (PRM) are commonly applied. The objective of this study was to establish an optimal quantification assay for proteins with the focus on those involved in housekeeping functions and putative reductive dehalogenase proteins from the strictly anaerobic bacterium Dehalococcoides mccartyi strain CBDB1. This microbe is small and slow-growing; hence, it provides little biomass for comprehensive proteomic analysis. We therefore compared SRM and PRM techniques. Eleven peptides were successfully quantified by both methods. In addition, six peptides were solely quantified by SRM and four by PRM, respectively. Peptides were spiked into a background of Escherichia coli lysate and the majority of peptides were quantifiable down to 500 amol absolute on column by both methods. Peptide quantification in CBDB1 lysate resulted in the detection of 15 peptides using SRM and 14 peptides with the PRM assay. Resulting quantification of five dehalogenases revealed copy numbers of <10 to 115 protein molecules per cell indicating clear differences in abundance of RdhA proteins during growth on hexachlorobenzene. Our results indicated that both methods show comparable sensitivity and that the combination of the mass spectrometry assays resulted in higher peptide coverage and thus more reliable protein quantification.

A simple QuEChERS-like extraction approach for molecular chemical characterization of organic aerosols: application to nitrated and oxygenated PAH derivatives (NPAH and OPAH) quantified by GC–NICIMS

An extraction procedure based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) approach has been developed and used for analysis of particle-bound nitrated and oxygenated PAH derivatives (NPAH and OPAH, respectively). Several analytical conditions, for example GC injection temperature and MS detection settings, were optimized. This analytical procedure enabled simultaneous GC–NICIMS quantification of 32 NPAH and 32 OPAH (or other oxygenated compounds), including typical components of secondary organic aerosol (SOA) formed by photooxidation of PAH (e.g. 2-formyl-trans-cinnamaldehyde and 6H-dibenzo[b,d]pyran-6-one). The QuEChERS-like approach was optimized, including the nature of the extraction solvent, the sorbent used for clean-up, and extraction time. The final extraction procedure was based on brief mechanical agitation (vortex mixing for 1.5 min), with 7 mL acetonitrile as solvent. Because dispersive solid-phase extraction (d-SPE) did not provide satisfactory results, SPE using SiO₂ was selected for sample purification. Identical results were obtained when the QuEChERS-like and traditional pressurised solvent extraction (PLE) procedures were compared for analysis of fortified ambient air particle samples. The procedure was validated by analysis of two aerosol standard reference materials (NIST SRM 1649b (urban dust) and SRM 2787 (fine particulate matter, <10 μm)). For numerous NPAH and OPAH, this is the first report of their quantification in both SRMs. Compared with other extraction methods, including PLE, the QuEChERS-like procedure resulted in increased productivity and reduced extraction cost. This paper shows that QuEChERS-like extraction procedures can be suitably adapted for molecular chemical characterization of aerosol samples and could be extended to other categories of compound.

Study of the performance of three LC-MS/MS platforms for analysis of perfluorinated compounds

The analytical suitabilities of three different liquid chromatography–tandem mass spectrometry (LC-MS/MS) systems, (1) triple quadrupole (QqQ), (2) conventional 3D ion trap (IT), and (3) quadrupole–linear IT (QqLIT), to determine trace levels of perfluorinated compounds (PFCs) in fish and shellfish were compared. Sample preparation was performed using alkaline extraction and solid-phase-extraction cleanup. This evaluation was focused on both quantitative (sensitivity, precision, and accuracy) and qualitative (identification capabilities) aspects. In the three instruments, the former facet was evaluated using selected reaction monitoring (SRM), which is the standard mode for quantitative LC-MS/MS analysis. Accuracy was similar in the three systems, with recoveries always over 70 %. Precision was better for the QqLIT and QqQ systems (7–15%) than for the IT system (10–17%). The QqLIT (working in SRM mode) and QqQ systems offered a linear dynamic range of at least 3 orders of magnitude, whereas that of the IT system was 2 orders of magnitude. The QqLIT system achieved at least 20-fold higher sensitivity than the QqQ system, and this was at least tenfold higher sensitivity than for the IT system. In the IT system, identification was based on sensitive full mass range acquisition and MS ⁿ fragmentation and in the QqLIT system, it was based on the use of an information-dependent-acquisition scan function, which allows the combination of an SRM or MS full scan acting as the survey scan and an enhanced product ion scan followed by MS³ as the dependent scan in the same analysis. Three instruments were applied to monitor the content in fish and shellfish (anchovies, swordfish, tuna, mussels, and oysters) obtained from Valencia and Barcelona markets (Spain). The eight target PFCs were detected at mean concentrations in the range from 10 ng ?kg^-1 (perfluoro-7-methyloctanoic acid and perfluoro-1-decanesulfonate) to 4,200 ng ?kg^-1 (perfluoropentanoic acid). Furthermore, perfluoroheptanoic and perfluoroundecanoic acids (not covered as target analytes) were identified in some samples.

Controlled Ion Ejection from an External Trap for Extended m/z Range in FT-ICR Mass Spectrometry

An auxiliary rf waveform of the same amplitude and phase applied to all the rods of an ion accumulation multipole creates an m/z-dependent axial pseudo potential. Controlled decrease of the auxiliary rf amplitude releases ions from the accumulation multipole sequentially from high to low m/z. The slope of the auxiliary rf voltage ramp is adjusted so that ions of different m/z reach the center of the ICR cell at the same time point, which mitigates the typical time dispersion observed in external source FT-ICR and extends the observable mass range for a single data acquisition by 2- to 3-fold. For complex mixture analysis, twice the number of elemental compositions are assigned when the auxiliary rf ejection is applied compared with the standard gated trapping.

Three-dimensional molecular reconstruction of rat heart with mass spectrometry imaging

Cardiovascular diseases are the world’s number one cause of death, accounting for 17.1 million deaths a year. New high-resolution molecular and structural imaging strategies are needed to understand underlying pathophysiological mechanism. The aim of our study is (1) to provide a molecular basis of the heart animal model through the local identification of biomolecules by mass spectrometry imaging (MSI) (three-dimensional (3D) molecular reconstruction), (2) to perform a cross-species validation of secondary ion mass spectrometry (SIMS)-based cardiovascular molecular imaging, and (3) to demonstrate potential clinical relevance by the application of this innovative methodology to human heart specimens. We investigated a MSI approach using SIMS on the major areas of a rat and mouse heart: the pericardium, the myocardium, the endocardium, valves, and the great vessels. While several structures of the heart can be observed in individual two-dimensional sections analyzed by metal-assisted SIMS imaging, a full view of these structures in the total heart volume can be achieved only through the construction of the 3D heart model. The images of 3D reconstruction of the rat heart show a highly complementary localization between Na⁺, K⁺, and two ions at m/z 145 and 667. Principal component analysis of the MSI data clearly identified different morphology of the heart by their distinct correlated molecular signatures. The results reported here represent the first 3D molecular reconstruction of rat heart by SIMS imaging.

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Quantitative determination of memantine in human plasma by GC using negative ion chemical ionization MS detection after derivatization with a new reagent, o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride

A method is presented for the quantitative determination of memantine in plasma by use of the derivatization reagent o-(pentafluorobenzyloxycarbonyl)-2,3,4,5-tetrafluorobenzoyl chloride. Memantine can be quantitatively analyzed down to 49?pg per mL of plasma using a 250?μL sample and negative ion chemical ionisation mass spectrometry (GC-NICI-MS). Plasma samples were made alkaline with carbonate buffer and extracted with n-hexane. The extracts were treated with reagent solution for 20?min, concentrated, and submitted to GC-NICI-MS. The method is rapid because extraction and derivatization occur in one single step. Amantadine is used as an internal standard. The utility and robustness of the assay is demonstrated by giving data on specificity, linearity, accuracy and precision, benchtop stability, freeze-thaw stability, autosampler stability, aliquot analysis, and prospective analytical batch size accuracy.

Compressive Mass Analysis on Quadrupole Ion Trap Systems

Conventionally, quadrupole ion trap mass spectrometers eject ions of different mass-to-charge ratio (m/z) in a sequential fashion by performing a scan of the rf trapping voltage amplitude. Due to the inherent sparsity of most mass spectra, the detector measures no signal for much of the scan time. By exploiting this sparsity property, we propose a new compressive and multiplexed mass analysis approach—multi Resonant Frequency Excitation (mRFE) ejection. This new approach divides the mass spectrum into several mass subranges and detects all the subrange spectra in parallel for increased mass analysis speed. Mathematical estimation of standard mass spectrum is demonstrated while statistical classification on the parallel measurements remains viable because of the sparse nature of the mass spectra. This method can reduce mass analysis time by a factor of 3–6 and increase system duty cycle by 2×. The combination of reduced analysis time and accurate compound classification is demonstrated in a commercial quadrupole ion trap (QIT) system.

Soft Ionization of Saturated Hydrocarbons, Alcohols and Nonpolar Compounds by Negative-Ion Direct Analysis in Real-Time Mass Spectrometry

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O₂]￣^?. No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

Simultaneous determination of water-soluble vitamins in SRM 1849 Infant/Adult Nutritional Formula powder by liquid chromatography–isotope dilution mass spectrometry

Assessing dietary intake of vitamins from all sources, including foods, dietary supplements, and fortified foods, would be aided considerably by having analytical methodologies that are capable of simultaneous determination of several vitamins. Vitamins naturally present in foods may occur in different chemical forms, with levels ranging over several orders of magnitude. Vitamins in dietary supplements and fortified foods, however, are typically added in a single chemical form, and matrix issues are usually not as complex. These sources should thus be relatively amenable to approaches that aim for simultaneous determination of multiple vitamins. Our recent work has focused on development of liquid chromatography (LC)–UV/fluorescence and LC–tandem mass spectrometry methods for the simultaneous determination of water-soluble vitamins (thiamine, niacin, pyridoxine, pantothenic acid, folic acid, biotin, and riboflavin) in dietary supplement tablets and fortified foods, such as formula powders and breakfast cereals. As part of the validation of our methods and collaboration in characterization of a new NIST SRM 1849 Infant/Adult Nutritional Formula powder, we report data on SRM 1849 using isotope dilution mass spectrometric methods. Use of available NIST Standard Reference Materials® as test matrices in our method development and validation gives a benchmark for future application of these methods. We compare three chromatographic approaches and provide data on stability of vitamin standard solutions for LC-based multiple vitamin determinations.

Boundaries of Mass Resolution in Native Mass Spectrometry

Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.

Solid phase extraction of Cd(II), Pb(II), Zn(II) and Ni(II) from food samples using multiwalled carbon nanotubes impregnated with 4-(2-thiazolylazo)resorcinol

Multiwalled carbon nanotubes were impregnated with 4-(2-thiazolylazo)resorcinol and used for the separation and preconcentration of Cd(II), Pb(II), Zn(II) and Ni(II) ions from food samples. The analytes were quantitatively recovered at pH 7.0 and eluted with 3?mol?L^?1 acetic acid. The effects of pH value, flow rate, eluent type and sample volume on the recoveries, and the effects of alkali, earth alkali and transition metals on the retention of the analytes were studied. The method was validated using the standard certified reference materials SRM 1570A (spinach leaves) and IAEA 336 (lichen), and the results were found to be compatible with the certified values of reference materials. The new enrichment procedure was applied to the determination of these ions in various food samples.

Establishing Drug Resistance in Microorganisms by Mass Spectrometry

A rapid method to determine drug resistance in bacteria based on mass spectrometry is presented. In it, a mass spectrum of an intact microorganism grown in drug-containing stable isotope-labeled media is compared with a mass spectrum of the intact microorganism grown in non-labeled media without the drug present. Drug resistance is determined by predicting characteristic mass shifts of one or more microorganism biomarkers using bioinformatics algorithms. Observing such characteristic mass shifts indicates that the microorganism is viable even in the presence of the drug, thus incorporating the isotopic label into characteristic biomarker molecules. The performance of the method is illustrated on the example of intact E. coli, grown in control (unlabeled) and ¹³C-labeled media, and analyzed by MALDI TOF MS. Algorithms for data analysis are presented as well.

Improved MALDI-TOF Microbial Mass Spectrometry Imaging by Application of a Dispersed Solid Matrix

The key step in high quality microbial matrix-assisted laser desorption/ionization mass spectrometry imaging (microbial MALDI MSI) is the fabrication of a homogeneous matrix coating showing a fine-grained morphology. This application note addresses a novel method to apply solid MALDI matrices onto microbial cultures grown on thin agar media. A suspension of a mixture of 2,5-DHB and α-CHCA is sprayed onto the agar sample surface to form highly homogeneous matrix coatings. As a result, the signal intensities of metabolites secreted by the fungus Aspergillus fumigatus were found to be clearly enhanced.

Gas-Phase Arylmethyl Transfer and Cyclodeamination of Argentinated N-Arylmethyl-Pyridin-2-Ylmethanimine

In collisional activation of argentinated N-arylmethyl-pyridin-2-ylmethanimine, a neutral molecule of AgNH₂ is eliminated, carrying one hydrogen from the methylene and the other one from the ortho position (relative to the ipso carbon) of the aryl ring. Taking argentinated N-benzyl-pyridin-2-ylmethanimine for example, the proposition that the AgNH₂ loss results from intramolecular arylmethyl transfer combined with cyclodeamination is rationalized by deuterium labeling experiments, blocking experiments, and theoretical calculations. The structure of the final product ion from loss of AgNH₂ was confirmed further by multistage mass spectrometry.

Elimination of Benzene from Protonated N-Benzylindoline: Benzyl Cation/Proton Transfer or Direct Proton Transfer?

Collision-induced dissociation (CID) of protonated N-benzylindoline and its derivatives was investigated by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Elimination of benzene was observed besides hydride transfer and electron transfer reactions. D-labeling experiments and accurate mass determinations of the product ions confirm that the external proton is retained in the fragment ion, and the elimination reaction was proposed to be initiated by benzyl cation transfer rather than proton transfer. Benzyl cation transfer from the nitrogen atom to one of the sp²-hybridized carbon atoms in the indoline core is the key step, and subsequent proton transfer reaction leads to the elimination of benzene. Density functional theory (DFT)-based calculations were performed and the computational results also support the benzyl cation/proton transfer mechanism.

Measurement of mercury species in human blood using triple spike isotope dilution with SPME-GC-ICP-DRC-MS

The measurement of different mercury compounds in human blood can provide valuable information about the type of mercury exposure. To this end, our laboratory developed a biomonitoring method for the quantification of inorganic (iHg), methyl (MeHg), and ethyl (EtHg) mercury in whole blood using a triple-spike isotope dilution (TSID) quantification method employing capillary gas chromatography (GC) and inductively coupled dynamic reaction cell mass spectrometry (ICP-DRC-MS). We used a robotic CombiPAL® sample handling station featuring twin fiber-based solid-phase microextraction (SPME) injector heads. The use of two SPME fibers significantly reduces sample analysis cycle times making this method very suitable for high sample throughput, which is a requirement for large public health biomonitoring studies. Our sample preparation procedure involved solubilization of blood samples with tetramethylammonium hydroxide (TMAH) followed by the derivatization with sodium tetra(n-propyl)borate (NaBPr₄) to promote volatility of mercury species. We thoroughly investigated mercury species stability in the blood matrix during the course of sample treatment and analysis. The method accuracy for quantifying iHg, MeHg, and EtHg was validated using NIST standard reference materials (SRM 955c level 3) and the Centre de Toxicologie du Québec (CTQ) proficiency testing (PT) samples. The limit of detection (LOD) for iHg, MeHg, and EtHg in human blood was determined to be 0.27, 0.12, and 0.16 μg/L, respectively.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC–MS with in-source fragmentation

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography–mass spectrometry (LC–MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.