An advanced automation platform coupled with mass spectrometry for investigating in vitro human skin permeation of UV filters and excipients in sunscreen products

Rationale

Systemic absorption of UV-filtering chemicals following topical application of sunscreens may present a safety concern. The Food and Drug Administration (FDA) had recommended an in vitro skin permeation test (IVPT) to evaluate the potential of this safety risk for the evaluation of sunscreens prior to clinical studies. Therefore, a sensitive and robust bioanalytical method(s) were required for IVPT studies of different topical sunscreen products.

Methods

An analytical procedure to quantitate sunscreen UV-filtering components and excipients in IVPT samples including avobenzone, octocrylene, oxybenzone, ecamsule, methylparaben and propylparaben was developed employing a RapidFire 360 robotic sample delivery system coupled with a triple quadrupole mass spectrometer. The analytical procedure was developed and validated according to the requirements of the FDA Bioanalytical Method Validation Guidance for Industry (2018).

Results

The analytical method provided a turnaround time of 12 seconds per sample and was determined to be accurate, precise, specific, and linear over the corresponding analytical ranges. The validated method was successfully applied for two IVPT studies for evaluating the skin permeation potential of UV-filtering chemicals and assisting with the selection of the sunscreen products for the clinical study conducted by the FDA.

Conclusions

This work highlights the first analytical procedure that has applied a non-chromatographic-MS/MS automation platform to an in vitro biopharmaceutics study. The analytical platform simultaneously quantitated four UV filters and two excipients in complex media to evaluate their permeation in IVPT studies. The sample throughput and analytical performance of advanced automation platforms indicate their analytical procedure has the potential to significantly advance the efficiency of IVPT studies to evaluate permeation of a wide variety of UV chemical filters and excipients for topical OTC sunscreen products.

     

Optimized switch-over between CHNS abundance and CNS isotope ratio analyses by elemental analyzer-isotope ratio mass spectrometry: Application to six geological reference materials

Rationale

Elemental abundances and isotopic ratios of carbon, nitrogen, sulfur and hydrogen have become important tools for reconstructing the evolution of Earth and life over geologic timescales, requiring accurate and precise analytical methods with high sample throughput. However, these measurements may require separate instruments for each task, such as an elemental analyzer (EA) with a thermal conductivity detector (TCD) for elemental abundances and an EA interfaced with a mass spectrometer for isotopic ratios.

Methods

To improve sample throughput and laboratory up-time, we developed a switch that allows converting an EA IsoLink™ system from a standalone mode using only a TCD to a mode for isotope ratio mass spectrometry (IRMS) within minutes. This permits accurate measurements of elemental abundances and isotopic ratios with high throughput and lower cost. We validated this method with six shale standards from the US Geological Survey (USGS) and compared our abundance data with those from another laboratory.

Results

Our results show that (a) abundance data agree well between the different laboratories and setups; (b) reproducible isotopic data can be obtained before and after the switch-over from EA standalone mode; and (c) the USGS rock standards cover a wide range in CHNS abundances and CNS isotopes, making them ideal reference materials for future geochemical studies.

Conclusions

This ideal analytical setup has the advantage that abundance measurements can be performed to determine optimal sample amounts for later isotopic analyses, ensuring higher data quality. Our setup eliminates the need for a separate EA while freeing up the mass spectrometer for other tasks during abundance measurements.

     

Method of micro-sampling human dentine collagen for stable isotope analysis

Rationale

Sampling of dentine for stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios in the direction of tooth growth allows the study of temporal changes to the diet and physiological stress of an individual during tooth formation. Current methods of sampling permanent teeth using 1 mm increments provide temporal resolution of 6–9 months at best depending on the tooth chosen. Although this gives sufficient sample sizes for reliable analysis by mass spectrometry, sectioning the dentine across the incremental structures results in a rolling average of the isotope ratios. A novel method of incremental dentine collagen sampling has been developed to decrease the collagen increment size to 0.35 mm along the incremental structures, thus reducing averaging and improving the temporal resolution of short-term changes within the δ¹³C and δ¹⁵N values.

Methods

This study presents data for a MicroMill-assisted sampling method that allows for sampling at 0.35 mm width × 1 mm depth increments following the incremental growth pattern of dentine. A NewWave MicroMill was used to sample the demineralised dentine section of modern donated human third molars from Sudan and compared to data from the same teeth using the 1 mm incremental sectioning method 2 established by Beaumont et al.

Results

The δ¹³C and δ¹⁵N isotopic data showed an increased temporal resolution, with each increment providing data for 2–4 months of dentine formation.

Conclusions

The data show the potential of this method for studying dietary reconstruction, nutritional stress, and physiological change with greater temporal resolution potentially to seasonal level and with less attenuation of the δ¹³C and δ¹⁵N values than was previously possible from human dentine.

     

New protocol for compound‐specific radiocarbon analysis of archaeological bones

Rationale

For radiocarbon results to be accurate, samples must be free of contaminating carbon. Sample pre‐treatment using a high‐performance liquid chromatography (HPLC) approach has been developed at the Oxford Radiocarbon Accelerator Unit (ORAU) as an alternative to conventional methods for dating heavily contaminated bones. This approach isolates hydroxyproline from bone collagen, enabling a purified bone‐specific fraction to then be radiocarbon dated by accelerator mass spectrometry (AMS).

Methods

Using semi‐preparative chromatography and non‐carbon‐based eluents, this technique enables the separation of underivatised amino acids liberated by hydrolysis of extracted bone collagen. A particular focus has been the isolation of hydroxyproline for single‐compound AMS dating since this amino acid is one of the main contributors to the total amount of carbon in mammalian collagen. Our previous approach, involving a carbon‐free aqueous mobile phase, required a two‐step separation using two different chromatographic columns.

Results

This paper reports significant improvements that have been recently made to the method to enable faster semi‐preparative separation of hydroxyproline from bone collagen, making the method more suitable for routine radiocarbon dating of contaminated and/or poorly preserved bone samples by AMS. All steps of the procedure, from the collagen extraction to the correction of the AMS data, are described.

Conclusions

The modifications to the hardware and to the method itself have reduced significantly the time required for the preparation of each sample. This makes it easier for other radiocarbon facilities to implement and use this approach as a routine method for preparing contaminated bone samples.

     

Peptidomic analysis of endogenous plasma peptides from patients with pancreatic neuroendocrine tumours

Rationale

Diagnosis of pancreatic neuroendocrine tumours requires the study of patient plasma with multiple immunoassays, using multiple aliquots of plasma. The application of mass spectrometry based techniques could reduce the cost and amount of plasma required for diagnosis.

Methods

Plasma samples from two patients with pancreatic neuroendocrine tumours were extracted using an established acetonitrile‐based plasma peptide enrichment strategy. The circulating peptidome was characterised using nano and high flow rate liquid chromatography/mass spectrometry (LC/MS) analyses. To assess the diagnostic potential of the analytical approach, a large sample batch (68 plasmas) from control subjects, and aliquots from subjects harbouring two different types of pancreatic neuroendocrine tumour (insulinoma and glucagonoma), were analysed using a 10‐min LC/MS peptide screen.

Results

The untargeted plasma peptidomics approach identified peptides derived from the glucagon prohormone, chromogranin A, chromogranin B and other peptide hormones and proteins related to control of peptide secretion. The glucagon prohormone derived peptides that were detected were compared against putative peptides that were identified using multiple antibody pairs against glucagon peptides. Comparison of the plasma samples for relative levels of selected peptides showed clear separation between the glucagonoma and the insulinoma and control samples.

Conclusions

The combination of the organic solvent extraction methodology with high flow rate analysis could potentially be used to aid diagnosis and monitor treatment of patients with functioning pancreatic neuroendocrine tumours. However, significant validation will be required before this approach can be clinically applied.

     

A novel laser desorption/ionization method using through hole porous alumina membranes

Rationale

A novel matrix‐free laser desorption/ionization method based on porous alumina membranes was developed. The porous alumina membranes have a two‐dimensional (2D) ordered structure consisting of closely aligned straight through holes of sub‐micron in diameter that are amenable to mass production by industrial fabrication processes.

Methods

Considering a balance between the ion generating efficiency and the mechanical strength of the membranes, the typical values for the hole diameter, open aperture ratio and membrane thickness were set to 200 nm, 50% and 5 μm, respectively. The membranes were coated with platinum on a single side that was exposed to the laser. Evaluation experiments were conducted on the feasibility of this membrane structure for an ionization method using a single peptide and mixed peptides and polyethylene glycol samples and a commercial matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometer in the positive ion mode.

Results

Results showed a softness of ionization and no sweet spot nature. The capillary action of the through holes with very high aspect ratio enables several loading protocols including sample impregnation from the surface opposite to the laser exposure side.

Conclusions

The feasibility study indicates that the through hole porous alumina membranes have several advantages in terms of usefulness over the conventional surface‐assisted laser desorption ionization (SALDI) methods. The proposed novel ionization method is termed Desorption Ionization Using Through Hole Alumina Membrane (DIUTHAME).

     

Absolute isotope ratios defining isotope scales used in isotope ratio mass spectrometers and optical isotope instruments

Rationale

The isotope delta is calculated from the isotope ratio of a sample and the absolute isotope ratio of the zero reference point defining each stable isotope international scale (R_std). Therefore, R_std requires accurate determination. However, the literature contains a large number of R_std values, and selection of different R_std may lead to inconsistency in reporting and recalculating stable isotope results.

Methods

We reviewed R_std used in the proprietary software provided by the manufacturers of stable isotope instruments commonly employed for analyses of stable HCNOS compositions. We compared the R_std values and assessed the potential implications of using different R_std and the normalization versus tank working gas standard for consistency in reporting stable isotope results.

Results

Different R_std values are used by different manufacturers of stable isotope analytical instruments. For R(²H/¹H)_VSMOW two different but very similar values are used, 0.00015575 and 0.00015576; for R(¹³C/¹²C)_VPDB three different values are used, 0.0111802, 0.0112372 and 0.01118028; and for R(¹⁵N/¹⁴N)_Air-N2 two values, 0.0036782 and 0.0036765, are used. All manufacturers are using the same value for R(¹⁸O/¹⁶O)_VSMOW, 0.00200520, but three different values for R(¹⁸O/¹⁶O)_VPDB, 0.002067200, 0.00208835 and 0.002088349. For R(³⁴S/³²S)_VCDT four different R_std are used, 0.0441509, 0.0441626, 0.044162589 and 0.0441520597.

Conclusions

The use of different R_std values may lead to differences in the isotope delta values obtained if the normalization versus working standard gas is applied. For the range of R_std used in proprietary software, the potential differences are lowest for oxygen (< 0.002 ‰) and nitrogen (< 0.001 ‰), and highest for carbon (0.107 to 0.112 ‰) and sulfur (0.023 ‰). Evaluation of the existing R_std values and recommendations for the best estimates are highly desirable to ensure worldwide consistency in stable isotope data reporting.

     

Position‐specific 13C/12C analysis of amino acid carboxyl groups – automated flow‐injection analysis based on reaction with ninhydrin

Rationale

The fundamental level of stable isotopic knowledge lies at specific atomic positions within molecules but existing methods of analysis require lengthy off‐line preparation to reveal this information. An automated position‐specific isotope analysis (PSIA) method is presented to determine the stable carbon isotopic compositions of the carboxyl groups of amino acids (δ¹³C_CARBOXYL values). This automation makes PSIA measurements easier and routine.

Methods

An existing high‐performance liquid chromatography (HPLC) gas handling interface/stable isotope ratio mass spectrometry system was modified by the addition of a post‐column derivatisation unit between the HPLC system and the interface. The post‐column reaction was optimised to yield CO₂ from the carboxyl groups of amino acids by reaction with ninhydrin.

Results

The methodology described produced δ¹³C_CARBOXYL values with typical standard deviations below ±0.1 ‰ and consistent differences (Δ¹³C_CARBOXYL values) between amino acids over a 1‐year period. First estimates are presented for the δ¹³C_CARBOXYL values of a number of internationally available amino acid reference materials.

Conclusions

The PSIA methodology described provides a further dimension to the stable isotopic characterisation of amino acids at a more detailed level than the bulk or averaged whole‐molecule level. When combined with on‐line chromatographic separation or off‐line fraction collection of protein hydrolysates the technique will offer an automated and routine way to study position‐specific carboxyl carbon isotope information for amino acids, enabling more refined isotopic studies of carbon uptake and metabolism.

     

High‐precision determination of lithium and magnesium isotopes utilising single column separation and multi‐collector inductively coupled plasma mass spectrometry

Rationale

Li and Mg isotopes are increasingly used as a combined tool within the geosciences. However, established methods require separate sample purification protocols utilising several column separation procedures. This study presents a single‐step cation‐exchange method for quantitative separation of trace levels of Li and Mg from multiple sample matrices.

Methods

The column method utilises the macro‐porous AGMP‐50 resin and a high‐aspect ratio column, allowing quantitative separation of Li and Mg from natural waters, sediments, rocks and carbonate matrices following the same elution protocol. High‐precision isotope determination was conducted by multi‐collector inductively coupled plasma mass spectrometry (MC‐ICPMS) on the Thermo Scientific™ NEPTUNE Plus™ fitted with 10¹³ Ω amplifiers which allow accurate and precise measurements at ion beams ≤0.51 V.

Results

Sub‐nanogram Li samples (0.3–0.5 ng) were regularly separated (yielding Mg masses of 1–70 μg) using the presented column method. The total sample consumption during isotopic analysis is <0.5 ng Li and <115 ng Mg with long‐term external 2σ precisions of ±0.39‰ for δ⁷Li and ±0.07‰ for δ²⁶Mg. The results for geological reference standards and seawater analysed by our method are in excellent agreement with published values despite the order of magnitude lower sample consumption.

Conclusions

The possibility of eluting small sample masses and the low analytical sample consumption make this method ideal for samples of limited mass or low Li concentration, such as foraminifera, mineral separates or dilute river waters.

     

A laser desorption ionization/matrix‐assisted laser desorption ionization target system applicable for three distinct types of instruments (LinTOF/curved field RTOF,LinTOF/RTOF and QqRTOF) with different performance characteristics from three vendors

Rationale

We have developed a target system which enables the use of only one target (i.e. target preparation set) for three different laser desorption ionization (LDI)/matrix‐assisted laser desorption ionization (MALDI) mass spectrometric instruments. The focus was on analysing small biomolecules with LDI for future use of the system for the study of meteorite samples (carbonaceous chondrites) using devices with different mass spectrometric performance characteristics.

Methods

Three compounds were selected due to their potential presence in meteoritic chondrites: tryptophan, 2‐deoxy‐d ‐ribose and triphenylene. They were prepared (with and without MALDI matrix, i.e. MALDI and LDI) and analysed with three different mass spectrometers (LinTOF/curved field RTOF, LinTOF/RTOF and QqRTOF). The ion sources of two of the instruments were run at high vacuum, and one at intermediate pressure. Two devices used a laser wavelength of 355 nm and one a wavelength of 337 nm.

Results

The developed target system operated smoothly with all devices. Tryptophan, 2‐deoxy‐d ‐ribose and triphenylene showed similar desorption/ionization behaviour for all instruments using the LDI mode. Interestingly, protonated tryptophan could be observed only with the LinTOF/curved field RTOF device in LDI and MALDI mode, while sodiated molecules were observed with all three instruments (in both ion modes). Deprotonated tryptophan was almost completely obscured by matrix ions in the MALDI mode whereas LDI yielded abundant deprotonated molecules.

Conclusions

The presented target system allowed successful analyses of the three compounds using instruments from different vendors with only one preparation showing different analyser performance characteristics. The elemental composition with the QqRTOF analyser and the high‐energy 20 keV collision‐induced dissociation fragmentation will be important in identifying unknown compounds in chondrites.

     

A simple and reliable method reducing sulfate to sulfide for multiple sulfur isotope analysis

Rationale

Precise analysis of four sulfur isotopes of sulfate in geological and environmental samples provides the means to extract unique information in wide geological contexts. Reduction of sulfate to sulfide is the first step to access such information. The conventional reduction method suffers from a cumbersome distillation system, long reaction time and large volume of the reducing solution. We present a new and simple method enabling the process of multiple samples at one time with a much reduced volume of reducing solution.

Methods

One mL of reducing solution made of HI and NaH₂PO₂ was added to a septum glass tube with dry sulfate. The tube was heated at 124°C and the produced H₂S was purged with inert gas (He or N₂) through gas‐washing tubes and then collected by NaOH solution. The collected H₂S was converted into Ag₂S by adding AgNO₃ solution and the co‐precipitated Ag₂O was removed by adding a few drops of concentrated HNO₃.

Results

Within 2–3 h, a 100% yield was observed for samples with 0.2–2.5 μmol Na₂SO₄. The reduction rate was much slower for BaSO₄ and a complete reduction was not observed. International sulfur reference materials, NBS‐127, SO‐5 and SO‐6, were processed with this method, and the measured against accepted δ³⁴S values yielded a linear regression line which had a slope of 0.99 ± 0.01 and a R² value of 0.998.

Conclusions

The new methodology is easy to handle and allows us to process multiple samples at a time. It has also demonstrated good reproducibility in terms of H₂S yield and for further isotope analysis. It is thus a good alternative to the conventional manual method, especially when processing samples with limited amount of sulfate available.

     

Analysis of ketone‐based neurosteroids by reactive low‐temperature plasma mass spectrometry

Rationale

Neurosteroids are important signalling molecules that modulate neuronal activity. Their low concentrations and low volatility make neurosteroid detection and quantification by ambient mass spectrometry challenging. Here we develop a reactive low‐temperature plasma mass spectrometry (LTP‐MS) method and demonstrate its potential for fast screening and quantification of neurosteroids in mouse brain.

Methods

Ketone‐based neurosteroids were analysed with the LTP‐MS method. The plasma of the LTP was heated in order to improve the desorption efficiency of low‐volatility neurosteroids. Methylamine with a concentration of 500 ppbv was employed as the reactive reagent. Neurosteroids in mouse brain tissue extracts were detected in 70 s with mass errors less than ±3 ppm due to coupling of the ion source with a high‐performance mass spectrometer.

Results

Reaction between neurosteroids and methylamine, seeded into the LTP gas stream, resulted in the formation of protonated methylamine–neurosteroid adducts with 5‐ to 100‐fold abundances, compared to [M + H]⁺ ions detected in non‐reactive LTP‐MS. The lowest detectable concentrations of neurosteroid standards were in the range of ng/mL. Concentrations of neurosteroids in male and female mouse brain extracts as determined with reactive LTP‐MS were on the level of ng/g, comparable to results obtained with high‐performance liquid chromatography–tandem mass spectrometry.

Conclusions

The developed reactive LTP‐MS is capable of providing sensitive identification and quantification of ketone‐based neurosteroids in mouse brain extracts with minimal sample treatment, and showcases the potential of reactive LTP‐MS as a tool for fast screening of neurosteroid levels in brain.

     

Intra‐ and inter‐tooth variation in strontium isotope ratios from prehistoric seals by laser ablation multi‐collector inductively coupled plasma mass spectrometry

Rationale

Strontium isotope ratios (⁸⁷Sr/⁸⁶Sr) in modern‐day marine environments are considered to be homogeneous (~0.7092). However, in the Baltic Sea, the Sr ratios are controlled by mixing seawater and continental drainage from major rivers discharging into the Baltic. This pilot study explores if variations in Sr can be detected in marine mammals from archaeological sites in the Baltic Sea.

Methods

⁸⁷Sr/⁸⁶Sr ratios were measured in tooth enamel from three seal species by laser ablation multi‐collector inductively coupled plasma mass spectrometry (LA‐MC‐ICP‐MS). The method enables micro‐sampling of solid materials. This is the first time that the method has been applied to marine samples from archaeological collections.

Results

The analyses showed inter‐tooth ⁸⁷Sr/⁸⁶Sr variation suggesting that different ratios can be detected in different regions of the Baltic Sea. Furthermore, the intra‐tooth variation suggests possible different geographic origin or seasonal movement of seals within different regions in the Baltic Sea through their lifetime.

Conclusions

The method was successfully applied to archaeological marine samples showing that: (1) the ⁸⁷Sr/⁸⁶Sr ratio in marine environments is not uniform, (2) ⁸⁷Sr/⁸⁶Sr differences might reflect differences in ecology and life history of different seal species, and (3) archaeological mobility studies based on ⁸⁷Sr/⁸⁶Sr ratios in humans should therefore be evaluated together with diet reconstruction.

     

Correlative mass spectrometry imaging,applying time‐of‐flight secondary ion mass spectrometry and atmospheric pressure matrix‐assisted laser desorption/ionization to a single tissue section

Rationale

Mass spectrometry imaging (MSI) is a powerful tool for mapping the surface of a sample. Time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) and atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) offer complementary capabilities. Here, we present a workflow to apply both techniques to a single tissue section and combine the resulting data for the example of human colon cancer tissue.

Methods

Following cryo‐sectioning, images were acquired using the high spatial resolution (1 μm pixel size) provided by TOF‐SIMS. The same section was then coated with a para‐nitroaniline matrix and images were acquired using AP‐MALDI coupled to an Orbitrap mass spectrometer, offering high mass resolution, high mass accuracy and tandem mass spectrometry (MS/MS) capabilities. Datasets provided by both mass spectrometers were converted into the open and vendor‐independent imzML file format and processed with the open‐source software MSiReader.

Results

The TOF‐SIMS and AP‐MALDI mass spectra show strong signals of fatty acids, cholesterol, phosphatidylcholine and sphingomyelin. We showed a high correlation between the fatty acid ions detected with TOF‐SIMS in negative ion mode and the phosphatidylcholine ions detected with AP‐MALDI in positive ion mode using a similar setting for visualization. Histological staining on the same section allowed the identification of the anatomical structures and their correlation with the ion images.

Conclusions

This multimodal approach using two MSI platforms shows an excellent complementarity for the localization and identification of lipids. The spatial resolution of both systems is at or close to cellular dimensions, and thus spatial correlation can only be obtained if the same tissue section is analyzed sequentially. Data processing based on imzML allows a real correlation of the imaging datasets provided by these two technologies and opens the way for a more complete molecular view of the anatomical structures of biological tissues.

     

Triboelectric nanogenerator (TENG) mass spectrometry of falsified antimalarials

Rationale

An epidemic of low‐quality medicines continues to endanger patients worldwide. Detection of such ‘medicines’ requires low cost, ambient ionization sources coupled to fieldable mass spectrometers for optimum sensitivity and specificity. With the use of triboelectric nanogenerators (TENGs), the charge required to produce gas‐phase ions for mass analysis can be obtained without the need for high‐voltage electrical circuitry, simplifying and lowering the cost of next‐generation mass spectrometry instruments.

Methods

A sliding freestanding (SF) TENG was coupled to a toothpick electrospray setup for the purposes of testing if falsified medicines could be fingerprinted by this approach. Extracts from both genuine and falsified medicines were deposited on the toothpick and the SF TENG actuated to generate electrical charges, resulting in gas‐phase ions for both active pharmaceutical ingredients and excipients.

Results

Our previous work had shown that direct analysis in real time (DART) ambient mass spectrometry can identify the components of multiple classes of falsified antimalarial medicines. Experiments performed in this study show that a simple extraction into methanol along with the use of a SF TENG‐powered toothpick electrospray can provide similar detection capabilities, but with much simpler and rugged instrumentation, and without the need for compressed gases or high‐voltage ion source power supplies.

Conclusions

TENG toothpick MS allows for rapid analyte ion detection in a safe and low‐cost manner, providing robust sampling and ionization capabilities.

     

Effects of the source gap on transmission efficiency of a quadrupole mass spectrometer

Rationale

Recent trends towards miniature and portable quadrupole mass spectrometry (QMS) entail challenges in instrumental sensitivity, which is influenced by 3D fringe field effects on ion transmission in the Quadrupole Mass Filter (QMF). The relationship of these effects with the gap from the ion source to the QMF entrance (source gap) is significant and little explored. We examine transmission characteristics experimentally and use the results to test the predictive accuracy of a recently developed 3D QMF simulation model. The model is then applied to directly investigate optimal transmission m/z ranges across multiple source gaps.

Methods

A portable single filter quadrupole mass spectrometer is used to analyse transmission characteristics across a range of common gases. We use an experimental approach originally proposed by Ehlert, enhanced with a novel method for absolute calibration of the transmission curve. Custom QMF simulation software employs the boundary element method (BEM) to compute accurate 3D electric fields. This is used to study the effects of the source gap on transmission efficiency.

Results

Experimental findings confirm a centrally peaked transmission curve; simulations correctly predict the optimal transmission location (in m/z) and percentage, and extend the experimental trend. We compare several methods for determining fringe field length, demonstrating how the size of the physical source gap influences both the length and the intensity of the fringe field at the QMF entrance. A complex relationship with ion transmission is revealed in which different source gaps promote optimal transmission at differing m/z ranges.

Conclusions

The presented results map the relationship between the source gap and transmission efficiency for the given instrument, using a simulation method transferrable to other setups. This is of importance to miniature and portable quadrupole mass spectrometers design for specific applications, for the first time enabling the source gap to be tailored for optimal transmission in the desired mass range.

     

The stable oxygen isotope ratio of resin extractable phosphate derived from fresh cattle faeces

Rationale

Phosphorus losses from agriculture pose an environmental threat to watercourses. A new approach using the stable oxygen isotope ratio of oxygen in phosphate (δ¹⁸O_PO4 value) may help elucidate some phosphorus sources and cycling. Accurately determined and isotopically distinct source values are essential for this process. The δ¹⁸O_PO4 values of animal wastes have, up to now, received little attention.

Methods

Phosphate (PO₄) was extracted from cattle faeces using anion resins and the contribution of microbial PO₄ was assessed. The δ¹⁸O_PO4 value of the extracted PO₄ was measured by precipitating silver phosphate and subsequent analysis on a thermal conversion elemental analyser at 1400°C, with the resultant carbon monoxide being mixed with a helium carrier gas passed through a gas chromatography (GC) column into a mass spectrometer. Faecal water oxygen isotope ratios (δ¹⁸O_H2O values) were determined on a dual‐inlet mass spectrometer through a process of headspace carbon dioxide equilibration with water samples.

Results

Microbiological results indicated that much of the extracted PO₄ was not derived directly from the gut fauna lysed during the extraction of PO₄ from the faeces. Assuming that the faecal δ¹⁸O_H2O values represented cattle body water, the predicted pyrophosphatase equilibrium δ¹⁸O_PO4 (Eδ¹⁸O_PO4) values ranged between +17.9 and +19.9‰, while using groundwater δ¹⁸O_H2O values gave a range of +13.1 to +14.0‰. The faecal δ¹⁸O_PO4 values ranged between +13.2 and +15.3‰.

Conclusions

The fresh faecal δ¹⁸O_PO4 values were equivalent to those reported elsewhere for agricultural animal slurry. However, they were different from the Eδ¹⁸O_PO4 value calculated from the faecal δ¹⁸O_H2O value. Our results indicate that slurry PO₄ is, in the main, derived from animal faeces although an explanation for the observed value range could not be determined.

     

Application of Bayesian analysis to the doubly labelled water method for total energy expenditure in humans

Rationale

The doubly labelled water (DLW) method is the reference method for the estimation of free‐living total energy expenditure (TEE). In this method, where both ²H and ¹⁸O are employed, different approaches have been adopted to deal with the non‐conformity observed regarding the distribution space for the labels being non‐coincident with total body water. However, the method adopted can have a significant effect on the estimated TEE.

Methods

We proposed a Bayesian reasoning approach to modify an assumed prior distribution for the space ratio using experimental data to derive the TEE. A Bayesian hierarchical approach was also investigated. The dataset was obtained from 59 adults (37 women) who underwent a DLW experiment during which the ²H and ¹⁸O enrichments were measured using isotope ratio mass spectrometry (IRMS).

Results

TEE was estimated at 9925 (9106‐11236) [median and interquartile range], 9646 (9167–10540), and 9,638 (9220–10340) kJ·day⁻¹ for women and at 13961 (12851–15347), 13353 (12651–15088) and 13211 (12653–14238) kJ·day⁻¹ for men, using normalized non‐Bayesian, independent Bayesian and hierarchical Bayesian approaches, respectively. A comparison of hierarchical Bayesian with normalized non‐Bayesian methods indicated a marked difference in behaviour between genders. The median difference was −287 kJ·day⁻¹ for women, and −750 kJ·day⁻¹ for men. In men there is an appreciable compression of the TEE distribution obtained from the hierarchical model compared with the normalized non‐Bayesian methods (range of TEE 11234–15431 kJ·day⁻¹ vs 10786–18221 kJ·day⁻¹). An analogous, yet smaller, compression is seen in women (7081–12287 kJ·day⁻¹ vs 6989–13775 kJ·day⁻¹).

Conclusions

The Bayesian analysis is an appealing method to estimate TEE during DLW experiments. The principal advantages over those obtained using the classical least‐squares method is the generation of potentially more useful estimates of TEE, and improved handling of outliers and missing data scenarios, particularly if a hierarchical model is used.

     

Quantification of 31 illicit and medicinal drugs and metabolites in whole blood by fully automated solid-phase extraction and ultra-performance liquid chromatography–tandem mass spectrometry

An efficient method for analyzing illegal and medicinal drugs in whole blood using fully automated sample preparation and short ultra-high-performance liquid chromatography–tandem mass spectrometry (MS/MS) run time is presented. A selection of 31 drugs, including amphetamines, cocaine, opioids, and benzodiazepines, was used. In order to increase the efficiency of routine analysis, a robotic system based on automated liquid handling and capable of handling all unit operation for sample preparation was built on a Freedom Evo 200 platform with several add-ons from Tecan and third-party vendors. Solid-phase extraction was performed using Strata X-C plates. Extraction time for 96 samples was less than 3 h. Chromatography was performed using an ACQUITY UPLC system (Waters Corporation, Milford, USA). Analytes were separated on a 100 mm?×?2.1 mm, 1.7 μm Acquity UPLC CSH C₁₈ column using a 6.5 min 0.1 % ammonia (25 %) in water/0.1 % ammonia (25 %) in methanol gradient and quantified by MS/MS (Waters Quattro Premier XE) in multiple-reaction monitoring mode. Full validation, including linearity, precision and trueness, matrix effect, ion suppression/enhancement of co-eluting analytes, recovery, and specificity, was performed. The method was employed successfully in the laboratory and used for routine analysis of forensic material. In combination with tetrahydrocannabinol analysis, the method covered 96 % of cases involving driving under the influence of drugs. The manual labor involved in preparing blood samples, solvents, etc., was reduced to a half an hour per batch. The automated sample preparation setup also minimized human exposure to hazardous materials, provided highly improved ergonomics, and eliminated manual pipetting.

Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell

We report on the combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS), and the latest-generation Orbitrap Eclipse Tribrid mass spectrometer for greatly improved single-cell proteome profiling. FAIMS effectively filtered out singly charged ions for more effective MS analysis of multiply charged peptides, resulting in an average of 1056 protein groups identified from single HeLa cells without MS1-level feature matching. This is 2.3 times more identifications than without FAIMS and a far greater level of proteome coverage for single mammalian cells than has been previously reported for a label-free study. Differential analysis of single microdissected motor neurons and interneurons from human spinal tissue indicated a similar level of proteome coverage, and the two subpopulations of cells were readily differentiated based on single-cell label-free quantification.

The combination of nanodroplet sample preparation, ultra-low-flow nanoLC, high-field asymmetric ion mobility spectrometry (FAIMS) and latest-generation mass spectrometry instrumentation provides dramatically improved single-cell proteome profiling.