Comparing novel small-angle x-ray scattering approaches for absolute size and number concentration measurements of spherical SiO2particles to established methods.

Biomedical analytical applications, as well as the industrial production of high-quality nano- and sub-micrometre particles, require accurate methods to quantify the absolute number concentration of particles. In this context, small-angle x-ray scattering (SAXS) is a powerful tool to determine the particle size and concentration traceable to the Système international d'unités (SI). Therefore, absolute measurements of the scattering cross-section must be performed, which require precise knowledge of all experimental parameters, such as the electron density of solvent and particles, whereas the latter is often unknown. Within the present study, novel SAXS-based approaches to determine the size distribution, density and number concentrations of sub-micron spherical silica particles with narrow size distributions and mean diameters between 160 nm and 430 nm are presented. For the first-time traceable density and number concentration measurements of silica particles are presented and current challenges in SAXS measurements such as beam-smearing, poorly known electron densities and moderately polydisperse samples are addressed. In addition, and for comparison purpose, atomic force microscopy has been used for traceable measurements of the size distribution and single particle inductively coupled plasma mass spectrometry with the dynamic mass flow approach for the accurate quantification of the number concentrations of silica particles. The possibilities and limitations of the current approaches are critically discussed in this study.

Determination of the Nanoscale Silica Mass Fraction by AF4/ICP-MS with Isotope Dilution Analysis Using 29Si-Enriched Silica Nanoparticles.

A methodology based on the use of asymmetrical flow field-flow fractionation (AF4) coupled to ICP-MS with size fraction-targeted isotope dilution analysis (IDA) has been developed, validated, and applied for the first time to determine the mass fraction of nanoscale silica (SiO2). For this purpose, 29Si-enriched SiO2 nanoparticles, to be used as an IDA spike/internal standard, were synthesized and characterized in-house. Double IDA was used to quantify an aqueous suspension of Stöber silica particles of similar characteristics to those of the 29SiO2 nanoparticle (NP) spike using a representative test material of natural Si isotopic composition as the calibrant. For fumed SiO2 NP in a highly complex food matrix, a methodology based on single IDA with AF4/ICP-MS using the same 29SiO2 NP spike was developed and validated. Relative expanded measurement uncertainties (k = 2) of 4% (double IDA) and 8% (single IDA) were achieved for nanoscale silica mass fractions of 5143 and 107 mg kg-1 in water suspension and food matrix, respectively. To assess the accuracy of AF4/ICP-IDMS for the characterization of SiO2 NP in a food matrix, standard addition measurements on samples spiked with Aerosil AF200, also in-house characterized for Si mass fraction, were undertaken, with an average recovery of 95.6 ± 4.1% (RSD, n = 3) obtained. The particle-specific IDA data obtained for both SiO2 NP-containing samples were also compared with that of post-AF4 channel external calibration using inorganic Si standards. The mass fractions obtained by IDA agreed well with those obtained by external calibration within their associated measurement uncertainties.

Traceable characterization of hollow organosilica beads as potential reference materials for extracellular vesicle measurements with optical techniques.

The concentration of cell-type specific extracellular vesicles (EVs) is a promising biomarker for various diseases. However, concentrations of EVs measured by optical techniques such as flow cytometry (FCM) or particle tracking analysis (PTA)  in clinical practice are incomparable. To allow reliable and comparable concentration measurements suitable reference materials (RMs) and SI-traceable (SI-International system of units) methods are required. Hollow organosilica beads (HOBs) are promising RM candidates for concentration measurements of EVs based on light scattering, as the shape, low refractive index, and number concentration of HOBs are comparable to EVs of the respective size range that can be detected with current optical instrumentation. Here, we present traceable methods for measuring the particle size distribution of four HOB types in the size range between 200 and 500 nm by small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM), as well as the number concentration by single-particle inductively coupled plasma mass spectrometry (spICP-MS). Based on the size and shape results, traceable reference values were obtained to additionally determine the refractive index of the shell of the HOB samples by FCM. Furthermore, the estimated refractive indexes of the HOBs plausibly agree with the refractive indexes of EVs of corresponding size. Due to their narrow size distribution and their similar shape, and low refractive index, all HOB samples studied are suitable RM candidates for calibration of the measured sample volume by optical methods within the photon wavelength range used, and thus for calibration of number concentration measurements of EVs in the size range indicated. This was confirmed as the number concentration values obtained by PTA and two independent flow cytometric measurements agreed with the concentration reference values obtained by two independent spICP-MS measurements within the calculated uncertainty limits.

Selenium speciation studies in cancer patients to evaluate the responses of biomarkers of selenium status to different selenium compounds.

This work presents the first systematic comparison of selenium (Se) speciation in plasma from cancer patients treated orally with three Se compounds (sodium selenite, SS; L-selenomethionine, SeMet; or Se-methylselenocysteine, MSC) at 400 µg/day for 28 days. The primary goal was to investigate how these chemical forms of Se affect the plasma Se distribution, aiming to identify the most effective Se compound for optimal selenoprotein expression. This was achieved using methodology based on HPLC-ICP-MS after sample preparation/fractionation approaches. Measurements of total Se in plasma samples collected before and after 4 weeks of treatment showed that median total Se levels increased significantly from 89.6 to 126.4 µg kg-1 Se (p < 0.001), particularly when SeMet was administered (190.4 µg kg-1 Se). Speciation studies showed that the most critical differences between treated and baseline samples were seen for selenoprotein P (SELENOP) and selenoalbumin after administration with MSC (p = 5.8 × 10-4) and SeMet (p = 6.8 × 10-5), respectively. Notably, selenosugar-1 was detected in all low-molecular-weight plasma fractions following treatment, particularly with MSC. Two different chromatographic approaches and spiking experiments demonstrated that about 45% of that increase in SELENOP levels (to ~ 8.8 mg L-1) with SeMet is likely due to the non-specific incorporation of SeMet into the SELENOP affinity fraction. To the authors' knowledge, this has not been reported to date. Therefore, SELENOP is probably part of both the regulated (55%) and non-regulated (45%) Se pools after SeMet administration, whereas SS and MSC mainly contribute to the regulated one.

Isotope Dilution Analysis for Particle Mass Determination Using Single-Particle Inductively Coupled Plasma Time-of-Flight Mass Spectrometry: Application to Size Determination of Silver Nanoparticles.

This paper describes methodology based on the application of isotope dilution (ID) in single-particle inductively coupled plasma time-of-flight mass spectrometry (spICP-ToFMS) mode for the mass determination (and sizing) of silver nanoparticles (AgNPs). For this purpose, and considering that the analytical signal in spICP-MS shows a transient nature, an isotope dilution equation used for online work was adapted and used for the mass determination of individual NPs. The method proposed measures NP isotope ratios in a particle-to-particle approach, which allows for the characterization of NP mass (and size) distributions and not only the mean size of the distribution. For the best results to be obtained, our method development (undertaken through the analysis of the reference material NIST RM 8017) included the optimization of the working conditions for the best precision and accuracy in isotope ratios of individual NPs, which had been only reported to date with multicollector instruments. It is shown that the precision of the measurement of these ratios is limited by the magnitude of the signals obtained for each NP in the mass analyzer (counting statistics). However, the uncertainty obtained for the sizing of NPs in this approach can be improved by careful method optimization, where the most important parameters are shown to be the selection of the spike isotopic composition and concentration. Although only AgNPs were targeted in this study, the method presented, with the corresponding adaptations, could be applied to NPs of any other composition that include an element with different naturally available isotopes.

Synthesis, characterisation and multi-modal intracellular mapping of cisplatin nano-conjugates.

The synthesis of nanocarriers for the delivery of the antitumor drug cisplatin is reported. Multimodal-imaging consisting of surface enhanced Raman scattering and laser ablation inductively coupled plasma time of flight mass spectrometry was used to visualise the intracellular uptake of both the nanocarrier and drug.

Nano-bio interactions of upconversion nanoparticles at subcellular level: biodistribution and cytotoxicity.

Background: Modern medicine requires intensive research to find new diagnostic and therapeutic solutions. Recently, upconverting nanoparticles (UCNPs) doped with lanthanide ions have attracted significant attention. Methods: The efficient internalization of UCNPs by cells was confirmed, and their precise cellular localization was determined by electron microscopy and confocal studies. Results: UCNPs colocalized only with specific organelles, such as early endosomes, late endosomes and lysosomes. Furthermore, experiments with chemical inhibitors confirmed the involvement of endocytosis in UCNPs internalization and helped select several mechanisms involved in internalization. Exposure to selected UCNPs concentrations did not show significant cytotoxicity, induction of oxidative stress or ultrastructural changes in cells. Conclusion: This study suggests that UCNPs offer new diagnostic options for biomedical infrared imaging.

Metrological Protocols for Reaching Reliable and SI-Traceable Size Results for Multi-Modal and Complexly Shaped Reference Nanoparticles.

The study described in this paper was conducted in the framework of the European nPSize project (EMPIR program) with the main objective of proposing new reference certified nanomaterials for the market in order to improve the reliability and traceability of nanoparticle size measurements. For this purpose, bimodal populations as well as complexly shaped nanoparticles (bipyramids, cubes, and rods) were synthesized. An inter-laboratory comparison was organized for comparing the size measurements of the selected nanoparticle samples performed with electron microscopy (TEM, SEM, and TSEM), scanning probe microscopy (AFM), or small-angle X-ray scattering (SAXS). The results demonstrate good consistency of the measured size by the different techniques in cases where special care was taken for sample preparation, instrument calibration, and the clear definition of the measurand. For each characterization method, the calibration process is described and a semi-quantitative table grouping the main error sources is proposed for estimating the uncertainties associated with the measurements. Regarding microscopy-based techniques applied to complexly shaped nanoparticles, data dispersion can be observed when the size measurements are affected by the orientation of the nanoparticles on the substrate. For the most complex materials, hybrid approaches combining several complementary techniques were tested, with the outcome being that the reliability of the size results was improved.

New Model for Quantifying the Nanoparticle Concentration Using SERS Supported by Multimodal Mass Spectrometry.

Surface-enhanced Raman scattering (SERS) is widely explored for the elucidation of underlying mechanisms behind biological processes. However, the capability of absolute quantitation of the number of nanoparticles from the SERS response remains a challenge. Here, we show for the first time the development of a new 2D quantitation model to allow calibration of the SERS response against the absolute concentration of SERS nanotags, as characterized by single particle inductively coupled plasma mass spectrometry (spICP-MS). A novel printing approach was adopted to prepare gelatin-based calibration standards containing the SERS nanotags, which consisted of gold nanoparticles and the Raman reporter 1,2-bis(4-pyridyl)ethylene. spICP-MS was used to characterize the Au mass concentration and particle number concentration of the SERS nanotags. Results from laser ablation inductively coupled plasma time-of-flight mass spectrometry imaging at a spatial resolution of 5 µm demonstrated a homogeneous distribution of the nanotags (between-line relative standard deviation < 14%) and a linear response of 197Au with increasing nanotag concentration (R2 = 0.99634) in the printed gelatin standards. The calibration standards were analyzed by SERS mapping, and different data processing approaches were evaluated. The reported calibration model was based on an "active-area" approach, classifying the pixels mapped as "active" or "inactive" and calibrating the SERS response against the total Au concentration and the particle number concentration, as characterized by spICP-MS. This novel calibration model demonstrates the potential for quantitative SERS imaging, with the capability of correlating the nanoparticle concentration to biological responses to further understand the underlying mechanisms of disease models.

Accumulation of molybdenum in major organs following repeated oral administration of bis-choline tetrathiomolybdate in the Sprague Dawley rat.

Molybdenum is an essential dietary trace element required for several critical enzyme systems. High intake is associated with toxicity in ruminants and animal studies. The proposed therapeutic use of molybdenum-based drugs poses a potential risk for accumulation through chronic administration of therapeutic doses of this element. The current experiment was designed to study the effect of daily dosing of a molybdenum compound, bis-choline tetrathiomolybdate (TTM), in Sprague Dawley rats using laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-ToF-MS) and two dosing levels of TTM for up to 3 months. To investigate if molybdenum accumulation was associated with tissue toxicity, histopathology, haematology and clinical biochemistry markers of toxicity were incorporated into the study design. There were no behavioural signs of toxicity to the rats, and no clinical or anatomic pathology was associated with treatment. The current data did show a progressive accumulation of molybdenum within the adrenal gland, kidneys, liver, spleen, brain and testes. Although this was not associated with tissue toxicity within the 3-month study design, greater exposure over a longer period of time has the potential for producing adverse pathophysiological cellular function. Tissue toxicity, as a result of local excessive accumulation of molybdenum over time, has clear implications for the therapeutic use of molybdenum in humans and demands sensitive monitoring of tissue molybdenum levels to avoid toxicity. The current study highlights the shortcomings of conventional biomonitoring approaches to detect molybdenum accumulation with the goal of avoiding molybdenum-associated toxicity.

Synthesis of Porous Hollow Organosilica Particles with Tunable Shell Thickness.

Porous hollow silica particles possess promising applications in many fields, ranging from drug delivery to catalysis. From the synthesis perspective, the most challenging parameters are the monodispersity of the size distribution and the thickness and porosity of the shell of the particles. This paper demonstrates a facile two-pot approach to prepare monodisperse porous-hollow silica particles with uniform spherical shape and well-tuned shell thickness. In this method, a series of porous-hollow inorganic and organic-inorganic core-shell silica particles were synthesized via hydrolysis and condensation of 1,2-bis(triethoxysilyl) ethane (BTEE) and tetraethyl orthosilicate (TEOS) in the presence of hexadecyltrimethylammonium bromide (CTAB) as a structure-directing agent on solid silica spheres as core templates. Finally, the core templates were removed via hydrothermal treatment under alkaline conditions. Transmission electron microscopy (TEM) was used to characterize the particles' morphology and size distribution, while the changes in the chemical composition during synthesis were followed by Fourier-transform infrared spectroscopy. Single-particle inductively coupled plasma mass spectrometry (spICP-MS) was applied to assess the monodispersity of the hollow particles prepared with different reaction parameters. We found that the presence of BTEE is key to obtaining a well-defined shell structure, and the increase in the concentration of the precursor and the surfactant increases the thickness of the shell. TEM and spICP-MS measurements revealed that fused particles are also formed under suboptimal reaction parameters, causing the broadening of the size distribution, which can be preceded by using appropriate concentrations of BTEE, CTAB, and ammonia.

A fit-for-purpose copper speciation method for the determination of exchangeable copper relevant to Wilson's disease.

Exchangeable copper (CuEXC), mainly comprised copper (Cu) bound to albumin, has been proposed as a specific marker of Cu overload in Wilson's disease (WD). To the author's knowledge, there are no methods capable of determining reliably CuEXC to meet the requirements and challenges faced by a clinical trial. The present work describes a novel speciation strategy for the determination of the main Cu-species in human serum by anion-exchange high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). A label-free protein quantification approach was conducted where the concentration of Cu associated to the protein fraction was based on its relative peak area distribution and the total Cu concentration in the sample. Such a methodology was characterized in terms of selectivity, sensitivity, precision, and robustness. Due to the lack of speciated Cu-reference materials, protein recovery was assessed by comparison with that of species-specific (SS) isotope dilution (ID). For this, a double SS HPLC-ICP-IDMS method for Cu-albumin was developed and presented here for the first time. Three human sera (two frozen LGC8211 and ERM®-DA250a, and the lyophilised Seronorm™ Human) were analyzed using both the relative and ID quantification methods. The validated relative approach, with relative expanded uncertainties (k = 2) between 5.7 and 10.1% for Cu-albumin concentrations ranging from 112 to 455 µg kg-1 Cu, was found to be able to discriminate between healthy and WD populations in terms of Cu-albumin content. Also, using such methodology, underestimation of CuEXC by the classical EDTA/ultrafiltration method was demonstrated. The methodology developed in this work will be invaluable for quality control assessment and WD drug monitoring. This work describes a Cu-protein quantification approach for the determination of exchangeable Cu relevant to Wilson's Disease.

Guidance for characterization of in-house reference materials for light element stable isotope analysis.

RATIONALE: Preparation of in-house reference materials (RMs) is an important aspect of light element stable isotope analysis. While some relevant information is available, there is as yet no clear set of guidelines available covering all aspects of in-house production and characterization of RMs. METHODS: To address this need, the experience of production of certified reference materials under accreditation to ISO 17034:2016 and ISO/IEC 17025:2017 has been distilled into guidance for production of in-house RMs that are fit-for-purpose. RESULTS: The guidance provided covers five areas: (i) planning; (ii) material considerations including preparation, packaging, and storage; (iii) measurements and assessments; (iv) value and uncertainty assignment; and (v) monitoring and use. CONCLUSIONS: In-house RMs prepared by following this guidance can be used to provide traceability to measurement results when used for normalization or for quality control and/or assurance purposes.

Assessment of changes in autophagic vesicles in human immune cell lines exposed to nano particles.

BACKGROUND: Safe and rational development of nanomaterials for clinical translation requires the assessment of potential biocompatibility. Autophagy, a critical homeostatic pathway intrinsically linked to cellular health and inflammation, has been shown to be affected by nanomaterials. It is, therefore, important to be able to assess possible interactions of nanomaterials with autophagic processes. RESULTS: CEM (T cell), Raji (B lymphocyte), and THP-1 (human monocyte) cell lines were subject to treatment with rapamycin and chloroquine, known to affect the autophagic process, in order to evaluate cell line-specific responses. Flow cytometric quantification of a fluorescent autophagic vacuole stain showed that maximum observable effects (105%, 446%, and 149% of negative controls) were achieved at different exposure durations (8, 6, and 24 h for CEM, Raji, and THP-1, respectively). THP-1 was subsequently utilised as a model to assess the autophagic impact of a small library of nanomaterials. Association was observed between hydrodynamic size and autophagic impact (r2 = 0.11, p = 0.004). An ELISA for p62 confirmed the greatest impact by 10 nm silver nanoparticles, abolishing p62, with 50 nm silica and 180 nm polystyrene also lowering p62 to a significant degree (50%, 74%, and 55%, respectively, p < 0.05). CONCLUSIONS: This data further supports the potential for a variety of nanomaterials to interfere with autophagic processes which, in turn, may result in altered cellular function and viability. The association of particle size with impact on autophagy now warrants further investigation.

On-column internal standardisation as an alternative calibration strategy for speciation analysis: feasibility demonstration through analysis of inorganic As in rice.

Species-specific isotope dilution mass spectrometry (SS-IDMS) has been the calibration method of choice for high accuracy speciation analysis because it can correct for detector sensitivity drifts, matrix effects, and analyte loss during sample preparation and analysis. However, in many cases SS-IDMS calibration is either not applicable (e.g. for monoisotopic elements) or not feasible (e.g. limited by the cost and availability of like-for-like isotopically enriched species). The work presented here demonstrates the potential of a novel on-column species-specific internal calibration approach, which is based on the chromatographic injection of the same species of the analyte as the internal standard (IS), after the sample injection. It can compensate for on-column analyte losses and signal drift and can be applied with any detector capable of recording time-resolved data, provided that enough species resolution can be achieved. The feasibility of this novel calibration strategy for accurate quantitative elemental speciation in complex matrices is demonstrated here through the analysis of inorganic arsenic in rice. An expanded uncertainty (k = 2) of <10% was obtained for a mass fraction range of 60 to 300 µg kg-1 inorganic-As (i-As) in dry rice products. The method is currently used for the certification of i-As in baby food matrices to support Commission Regulation (EU) 2015/1006 in regard to the maximum levels of i-As in foodstuffs.

AF4-UV-ICP-MS for detection and quantification of silver nanoparticles in seafood after enzymatic hydrolysis.

A method based on asymmetric flow field-flow fractionation (AF4) coupled to ultraviolet-visible (UV-vis) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) has been developed for silver nanoparticles (Ag NPs) detection and quantification in bivalve molluscs. Samples were pre-treated using a conventional enzymatic (pancreatin and lipase) hydrolysis procedure (37 °C, 12 h). AF4 was performed using a regenerated cellulose (RC) membrane (10 kDa, 350 µm spacer) and aqueous 5 mM Tris-HCl pH = 7.4 as carrier. AF4 separation was achieved with a program that included a focusing step with tip and focus flows of 0.20 and 3.0 mL min-1, respectively, and an injection time of 4.0 min. Elution of different size fractions was performed using a cross flow of 3.0 mL min-1 for 15 min, followed by linear cross flow decrease for 7.5 min, and a washing step for 9.4 min with no cross flow. Several bivalve molluscs (clams, oysters and variegated scallops) were analysed for total Ag content (ICP-MS after microwave assisted acid digestion), and for Ag NPs by the method presented here. Results show that Ag NPs are detected at the same elution time than proteins (UV monitoring at 280 and 405 nm), which suggests a certain interaction occurred between Ag NPs with proteins in the enzymatic extracts. AF4-UV-ICP-MS fractograms also suggest different Ag NPs size distributions for selected samples. Membrane recoveries, determined by peak area comparison of fractograms with and without application of cross flow, were within the 49-121% range. Confirmation of the presence Ag NPs in the investigated enzymatic extracts was demonstrated by SEM after an oxidative pre-treatment based on hydrogen peroxide and microwave irradiation.

Demethylation of Methylmercury in Bird, Fish, and Earthworm.

Toxicity of methylmercury (MeHg) to wildlife and humans results from its binding to cysteine residues of proteins, forming MeHg-cysteinate (MeHgCys) complexes that hinder biological functions. MeHgCys complexes can be detoxified in vivo, yet how this occurs is unknown. We report that MeHgCys complexes are transformed into selenocysteinate [Hg(Sec)4] complexes in multiple animals from two phyla (a waterbird, freshwater fish, and earthworms) sampled in different geographical areas and contaminated by different Hg sources. In addition, high energy-resolution X-ray absorption spectroscopy (HR-XANES) and chromatography-inductively coupled plasma mass spectrometry of the waterbird liver support the binding of Hg(Sec)4 to selenoprotein P and biomineralization of Hg(Sec)4 to chemically inert nanoparticulate mercury selenide (HgSe). The results provide a foundation for understanding mercury detoxification in higher organisms and suggest that the identified MeHgCys to Hg(Sec)4 demethylation pathway is common in nature.

Systematic study of the selenium fractionation in human plasma from a cancer prevention trial using HPLC hyphenated to ICP-MS and ESI-MS/MS.

This work represents the first systematic speciation study of selenium (Se) in plasma from subjects participating in a pilot study for a cancer prevention trial (PRECISE). This involved supplementation of elderly British and Danish individuals with selenised yeast for 6 months and 5 years, respectively, at 100, 200, and 300 µg Se/day or placebo. Speciation data was obtained for male plasma using HPLC-ICP-MS and HPLC-ESI-MS/MS. With the proposed strategy, approximately 1.5 mL of plasma was needed to determine total Se concentration and the fractionation of Se in high molecular weight (HMW) and low molecular weight (LMW) pools, and for quantification and identification of small Se species. For the first time, Se-methyl-selenocysteine (MSC) and methyl-2-acetamido-2deoxy1-seleno-ß-D-galactopyranoside (Selenosugar-1) were structurally confirmed in plasma after supplementation with selenised yeast within the studied range. Determination of selenomethionine (SeMet) incorporated non-specifically into albumin (SeALB) was achieved by HPLC-ICP-MS after hydrolysis. By subtracting this SeMet concentration from the total Se in the HMW pool, the concentration of Se incorporated into selenoproteins was calculated. Results from the speciation analysis of the free Se metabolite fraction (5% of total plasma Se) suggest a significant increase in the percentage of Se (as SeMet plus Selenosugar-1) of up to 80% of the total Se in the LMW fraction after 6 months of supplementation. The Se distribution in the HMW fraction reflects a significant increase in SeALB with Se depletion from selenoproteins, which occurs most significantly at doses of over 100 µg Se/day after 5 years. The results of this work will inform future trial design. Graphical abstract.

Systematic comparison of post-column isotope dilution using LC-CO-IRMS with qNMR for amino acid purity determination.

Determination of the purity of a substance traceable to the International System of Units (SI) is important for the production of reference materials affording traceability in quantitative measurements. Post-column isotope dilution using liquid chromatography-chemical oxidation-isotope ratio mass spectrometry (ID-LC-CO-IRMS) has previously been suggested as a means to determine the purity of organic compounds; however, the lack of an uncertainty budget has prevented assessment of the utility this approach until now. In this work, the previously published ID-LC-CO-IRMS methods have not only been improved by direct gravimetric determination of the mass flow of 13C-labelled spike but also a comprehensive uncertainty budget has been established. This enabled direct comparison of the well-characterised ID-LC-CO-IRMS method to quantitative nuclear magnetic resonance spectroscopy (qNMR) for purity determination using valine as the model compound. The ID-LC-CO-IRMS and qNMR methods provided results that were in agreement within the associated measurement uncertainty for the purity of a sample of valine of (97.1 ± 4.7)% and (99.64 ± 0.20)%, respectively (expanded uncertainties, k = 2). The magnitude of the measurement uncertainty for ID-LC-CO-IRMS determination of valine purity precludes the use of this method for determination of purity by direct analysis of the main component in the majority of situations; however, a mass balance approach is expected to result in significantly improved measurement uncertainty.