Metabolomics Test Materials for Quality Control: A Study of a Urine Materials Suite.

There is a lack of experimental reference materials and standards for metabolomics measurements, such as urine, plasma, and other human fluid samples. Reasons include difficulties with supply, distribution, and dissemination of information about the materials. Additionally, there is a long lead time because reference materials need their compositions to be fully characterized with uncertainty, a labor-intensive process for material containing thousands of relevant compounds. Furthermore, data analysis can be hampered by different methods using different software by different vendors. In this work, we propose an alternative implementation of reference materials. Instead of characterizing biological materials based on their composition, we propose using untargeted metabolomic data such as nuclear magnetic resonance (NMR) or gas and liquid chromatography-mass spectrometry (GC-MS and LC-MS) profiles. The profiles are then distributed with the material accompanying the certificate, so that researchers can compare their own metabolomic measurements with the reference profiles. To demonstrate this approach, we conducted an interlaboratory study (ILS) in which seven National Institute of Standards and Technology (NIST) urine Standard Reference Material®s (SRM®s) were distributed to participants, who then returned the metabolomic data to us. We then implemented chemometric methods to analyze the data together to estimate the uncertainties in the current measurement techniques. The participants identified similar patterns in the profiles that distinguished the seven samples. Even when the number of spectral features is substantially different between platforms, a collective analysis still shows significant overlap that allows reliable comparison between participants. Our results show that a urine suite such as that used in this ILS could be employed for testing and harmonization among different platforms. A limited quantity of test materials will be made available for researchers who are willing to repeat the protocols presented here and contribute their data.

Standard Reference Material (SRM) 2378 fatty acids in frozen human serum. Certification of a clinical SRM based on endogenous supplementation of polyunsaturated fatty acids.

Dietary fatty acids can be both beneficial and detrimental to human health depending on the degree and type of saturation. Healthcare providers and research scientists monitor the fatty acid content of human plasma and serum as an indicator of health status and diet. In addition, both the Centers for Disease Control & Prevention (CDC) and the National Institutes of Health - Office of Dietary Supplements are interested in circulating fatty acids (FAs) because they may be predictive of coronary heart disease. The National Institute of Standards and Technology (NIST) provides a wide variety of reference materials (RMs) and Standard Reference Materials® (SRM®s) including blood, serum, plasma, and urine with values assigned for analytes of clinical interest. NIST SRM 2378 Fatty Acids in Frozen Human Serum was introduced in 2015 to help validate methods used for the analysis of FAs in serum, and consists of three different pools of serum acquired from (1) healthy donors who had taken fish oil dietary supplements (at least 1000 mg per day) for at least one month (level 1 material), (2) healthy donors who had taken flaxseed oil dietary supplements (at least 1000 mg per day) for at least one month (level 2 material), and (3) healthy donors eating "normal" diets who had not taken dietary supplements containing fish or plant oils (level 3 material). The use of dietary supplements by donors provided SRMs with natural endogenous ranges of FAs at concentrations observed in human populations. Results from analyses using two methods at NIST, including one involving a novel microwave-assisted acid hydrolysis procedure, and one at the CDC are presented here. These results and their respective uncertainties were combined to yield certified values with expanded uncertainties for 12 FAs and reference values with expanded uncertainties for an additional 18 FAs.

Development of urine standard reference materials for metabolites of organic chemicals including polycyclic aromatic hydrocarbons, phthalates, phenols, parabens, and volatile organic compounds.

Two new Standard Reference Materials (SRMs), SRM 3672 Organic Contaminants in Smokers' Urine (Frozen) and SRM 3673 Organic Contaminants in Non-Smokers' Urine (Frozen), have been developed in support of studies for assessment of human exposure to select organic environmental contaminants. Collaborations among three organizations resulted in certified values for 11 hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and reference values for 11 phthalate metabolites, 8 environmental phenols and parabens, and 24 volatile organic compound (VOC) metabolites. Reference values are also available for creatinine and the free forms of caffeine, theobromine, ibuprofen, nicotine, cotinine, and 3-hydroxycotinine. These are the first urine Certified Reference Materials characterized for metabolites of organic environmental contaminants. Noteworthy, the mass fractions of the environmental organic contaminants in the two SRMs are within the ranges reported in population survey studies such as the National Health and Nutrition Examination Survey (NHANES) and the Canadian Health Measures Survey (CHMS). These SRMs will be useful as quality control samples for ensuring compatibility of results among population survey studies and will fill a void to assess the accuracy of analytical methods used in studies monitoring human exposure to these organic environmental contaminants.

Application of inkjet printing technology to produce test materials of 1,3,5-trinitro-1,3,5 triazcyclohexane for trace explosive analysis.

The feasibility of the use of piezoelectric drop-on-demand inkjet printing to prepare test materials for trace explosive analysis is demonstrated. RDX (1,3,5-trinitro-1,3,5 triazcyclohexane) was formulated into inkjet printable solutions and jetted onto substrates suitable for calibration of the ion mobility spectrometry (IMS) instruments currently deployed worldwide for contraband screening. Gravimetric analysis, gas chromatography/mass spectrometry (GC/MS), and ultraviolet-visible (UV-vis) absorption spectroscopy were used to verify inkjet printer solution concentrations and the quantity of explosive dispensed onto test materials. Reproducibility of the inkjet printing process for mass deposition of the explosive RDX (1,3,5-trinitro-1,3,5 triazcyclohexane) was determined to be better than 2% for a single day of printing and better than 3% day-to-day.

Characterization of composition C4 explosives using time-of-flight secondary ion mass spectrometry and X-ray photoelectron spectroscopy.

The application of surface analytical techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) is explored as a means of differentiating between composition C4 plastic explosives (C-4). Three different C-4 samples including U.S. military grade C-4, commercial C-4 (also from the United States), and C-4 from England (PE-4) were obtained and analyzed using both ToF-SIMS and XPS. ToF-SIMS was able to successfully discriminate between different C-4 samples with the aid of principal component analysis, a multivariate statistical analysis approach often used to reduce the dimensionality of complex data. ToF-SIMS imaging was also used to obtain information about the spatial distribution of the various additives contained within the samples. The results indicated that the samples could potentially be characterized by their 2-D chemical and morphological structure, which varied from sample to sample. XPS analysis also showed significant variation between samples, with changes in the atomic concentrations, as well as changes in the shapes of the high-resolution C 1s and O 1s spectra. These results clearly demonstrate the feasibility of utilizing both ToF-SIMS and XPS as tools for the direct characterization and differentiation of C-4 samples for forensic applications.

Fabrication of polymer microsphere particle standards containing trace explosives using an oil/water emulsion solvent extraction piezoelectric printing process.

We present a methodology for fabricating polymer microspheres using inkjet printing of a biodegradable polymer containing either high explosives or high explosive simulant. Poly(DL-lactide/glycolide) 85:15 (PLGA) microsphere production is based on an oil/water emulsion solvent extraction process. The inkjet printing process allows for precise control of the microsphere diameter and the chemical composition. The microspheres can be used as calibrants or verification standards for explosives trace detection instruments. Gas chromatography/mass spectrometry analysis demonstrated that the composition of the microspheres was consistent with predicted concentrations based on the amount of analyte incorporated into the polymer solution and the inkjet operating parameters. We have demonstrated that the microspheres can be fabricated with a mass fraction of 70% of an analyte compound.

Determination of cyanide in blood by isotope-dilution gas chromatography-mass spectrometry.

BACKGROUND: Cyanide (CN) is a lethal toxin. Quantification in blood is necessary to indicate exposure from many sources, including food, combustion byproducts, and terrorist activity. We describe an automated procedure based on isotope-dilution gas chromatography-mass spectrometry (ID GC/MS) for the accurate and rapid determination of CN in whole blood. METHODS: A known amount of isotopically labeled potassium cyanide (K13C15N) was added to 0.5 g of whole blood in a headspace vial. Hydrogen cyanide was generated through the addition of phosphoric acid, and after a 5-min incubation, 0.5 mL of the headspace was injected into the GC/MS at an oven temperature of -15 degrees C. The peak areas from the sample, 1H12C14N+, at m/z 27, and the internal standard, 1H13C15N+, at m/z 29, were measured, and the CN concentration was quantified by ID. The analysis time was 15 min for a single injection. RESULTS: We demonstrated method accuracy by measuring the CN content of unfrozen whole blood samples fortified with a known amount of CN. Intermediate precision was demonstrated by periodic analyses over a 14-month span. Relative expanded uncertainties based on a 95% level of confidence with a coverage factor of 2 at CN concentrations of 0.06, 0.6, and 1.5 microg/g were 8.3%, 5.4%, and 5.3%, respectively. The mean deviation from the known value for all concentrations was <4%. CONCLUSION: The automated ID GC/MS method can accurately and rapidly quantify nanogram per gram to microgram per gram concentrations of CN in blood.

Characterization of surface organic components of human hair by on-line supercritical fluid extraction-gas chromatography/mass spectrometry: a feasibility study and comparison with human identification using mitochondrial DNA sequences.

This paper discusses results of a supercritical fluid extraction-gas chromatography/mass spectrometry (SFE-GC/MS) study of small samples ( 100 microg to 1 mg) of human scalp hair. The method offers a number of benefits including greater sensitivity than liquid extraction methods because the entire extractable mass is transferred to the analytical system, compared with only a few percent from a conventional liquid extraction/injection. The project's goals were to determine if SFE-GC/MS analyses of the surface-extractable components of an individual's hair yield consistent chemical profiles and to investigate if the profiles are sufficiently different to distinguish them from those of other individuals. In addition, the mtDNA sequences from ten of the same individuals used in the SFE-GC/MS study from four family units were determined, and, while the families were distinguishable, the maternal relations yielded identical sequences. In tandem, SFE-GC/MS and mtDNA techniques may provide valuable complementary data from forensic hair samples.

Evaluation of extraction techniques for the forensic analysis of human scalp hair using gas chromatography/mass spectrometry (GC/MS).

Preliminary research using on-line supercritical fluid extraction/gas chromatography-mass spectrometry (SFE/GC-MS) has shown that the natural and artificial surface components of human scalp hair are reproducible and differentiable. Therefore, these components may be useful for individualization or determining demographic characteristics or both. However, it is not known how the efficiency and selectivity of on-line SFE/GC-MS compares to other extraction methods. In this study, ultrasound, Soxhlet, and pressurized-fluid extraction were used to extract 1 mg to 1.3 g portions of a composite hair sample taken from an Asian male between the ages of 10 and 18. Percent extractables ranged from 0.9% to 5.6%, depending on the solvent used, and tended to increase with solvent polarity. Chemical analysis using GC/MS showed that the extracts contained large proportions of free fatty acids, squalene, cholesterol, and various wax esters. Finally, comparisons to SFE/GC-MS showed that this method possesses adequate efficiency, no observable differences in selectivity, and greater potential for miniaturization.

Radiocarbon as a tool to apportion the sources of polycyclic aromatic hydrocarbons and black carbon in environmental samples.

To determine the relative inputs of polycyclic aromatic hydrocarbons (PAHs) and black carbon (BC) in environmental samples from the combustion of fossil fuels and biomass, we have developed two independent analytical methods for determining the 14C abundance of PAHs and BC. The 5730 yr half-life of 14C makes it an ideal tracer for identifying combustion products derived from fossil fuels (14C-free) versus those stemming from modern biomass (contemporary 14C). The 14C abundance of PAHs in several environmental Standard Reference Materials was measured by accelerator mass spectrometry after extraction and then purification by high-performance liquid chromatography and preparative capillary gas chromatography. This method yields pure compounds that allow for a high degree of confidence in the 14C results. The PAHs data were then used to compare and evaluate results from an operationally defined thermal oxidation method used to isolate a BC fraction. The 14C compositions of PAHs and BC were very similar and suggest that the thermal oxidation method employed for isolating BC is robust and free from interferences by non-BC components. In addition, these data indicate that both the PAHs and the BC species derive mostly from fossil fuels and/or their combustion products.