Lorna T. Sniegoski

Development of a Standard Reference Material for Metabolomics Research

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Interlaboratory comparison studies on the analysis of hair for drugs of abuse

Eleven laboratories interested in the analysis of human hair for drugs of abuse participated in a study to determine how well drugs could be detected and quantified in hair. For the two exercises completed to date, substances to be determined were limited to cocaine, benzoylecgonine, and morphine. Samples sent to the participating laboratories included hair from drug users, drug-free hair, and hair into which drugs had been soaked. For the first exercise, the hair samples were sent as powders; for the second, they were in the form of short segments. Results from these studies have shown that the laboratories, with a few exceptions, have performed very well qualitatively. However, scatter in quantitative results was high. Various approaches were used to liberate drugs from the hair, with the most commonly used, acid extractions and enzyme digestions, producing similar results. Laboratories using GC/MS generally performed well and reported no false positives. In contrast, one laboratory analyzing hair directly using MS/MS without extractions produced three of the four false positives and the worst quantitative results.

Preparation and value assignment of Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum.

Standard Reference Material 968c Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for retinal, delta-, gamma-, and alpha-tocopherol, trans- and total beta-carotene, and cholesterol in human serum. Values are also reported for 16 additional compounds including lutein, zeaxanthin, alpha- and beta-cryptoxanthin, lycopene, alpha-carotene, retinyl palmitate, and 25-hydroxyvitamin D. The certified values for the fat-soluble vitamins and carotenoids in SRM 968c were based on the agreement of results from the means of at least two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the medians from an interlaboratory comparison study among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol in the SRM are the means of results obtained using the NIST definitive method, gas chromatography-isotope dilution mass spectrometry.

Preparation and value assignment of standard reference material 968e fat-soluble vitamins, carotenoids, and cholesterol in human serum

Standard Reference Material 968e Fat-Soluble Vitamins, Carotenoids, and Cholesterol in Human Serum provides certified values for total retinol, γ- and α-tocopherol, total lutein, total zeaxanthin, total β-cryptoxanthin, total β-carotene, 25-hydroxyvitamin D3, and cholesterol. Reference and information values are also reported for nine additional compounds including total α-cryptoxanthin, trans- and total lycopene, total α-carotene, trans-β-carotene, and coenzyme Q10. The certified values for the fat-soluble vitamins and carotenoids in SRM 968e were based on the agreement of results from the means of two liquid chromatographic methods used at the National Institute of Standards and Technology (NIST) and from the median of results of an interlaboratory comparison exercise among institutions that participate in the NIST Micronutrients Measurement Quality Assurance Program. The assigned values for cholesterol and 25-hydroxyvitamin D3 in the SRM are the means of results obtained using the NIST reference method based upon gas chromatography-isotope dilution mass spectrometry and liquid chromatography-isotope dilution tandem mass spectrometry, respectively. SRM 968e is currently one of two available health-related NIST reference materials with concentration values assigned for selected fat-soluble vitamins, carotenoids, and cholesterol in human serum matrix. This SRM is used extensively by laboratories worldwide primarily to validate methods for determining these analytes in human serum and plasma and for assigning values to in-house control materials. The value assignment of the analytes in this SRM will help support measurement accuracy and traceability for laboratories performing health-related measurements in the clinical and nutritional communities.

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Abstract We describe a manual fluorometric method for the quantitation in protein solutions of total free amino compounds, expressed as norleucine. A trichloroacetic acid deproteinization step is employed, and o-phthalaldehyde, buffered with phosphate at pH 9,2, is used as the fluorogenic reagent. The method is linear, reproducible, and rapid. Recoveries of amino acids added to serum are quantitative. Sensitivity is in the picomole range. Results on unselected patient sera are diseussed.

Demonstrating the comparability of certified reference materials.

Certified reference materials (CRMs) enable the meaningful comparison of measurement results over time and place. When CRMs are used to calibrate or verify the performance of a measurement system, results produced by that system can be related through the CRM to well-defined, stable, and globally accessible reference(s). Properly done, this directly establishes the metrological traceability of the results. However, achieving the meaningful comparison of results from measurement systems calibrated and/or verified with different CRMs requires that the different materials truly deliver the same measurand, that is, are “the same” within stated uncertainty except for differences in the level of the analyte of interest. We here detail experimental and data analysis techniques for establishing and demonstrating the comparability of materials. We focus on (1) establishing a uniform interpretation of the common forms of CRM uncertainty statements, (2) estimating consistent measurement system response uncertainties from sometimes inconsistent experimental designs, (3) using “errors-in-variables” analysis methods to evaluate comparability studies and novel graphical tools for communicating results of the evaluation to reviewing authorities and potential CRM customers, and (4) augmenting established comparability studies with new materials using measurements provided by the certifying institution. These experimental and data analytic tools were developed in support of the Joint Committee for Traceability in Laboratory Medicine’s efforts to enhance the reliability of clinical laboratory measurements and are illustrated with potassium and cholesterol measurands of clinical relevance; however, these tools can be applied to any group of materials that deliver the same nominal measurand with stated value and uncertainty.

NIST reference materials to support accuracy in drug testing

Substance abuse is a major problem worldwide. There is considerable emphasis placed upon testing individuals for evidence of use of controlled substances. Because the consequences of a positive test can be quite severe, laboratories conducting such tests must rigorously follow a carefully designed quality assurance program. Such a QA program should include use of reference materials to assure that the methods used to detect and quantify drugs are providing accurate results. The National Institute of Standards and Technology (NIST) supports accuracy in drugs of abuse testing by providing Standard Reference Materials (SRMs) with certified concentrations of drugs of abuse in urine- and hair-based reference materials. NIST, working in collaboration with the College of American Pathologists (CAP), has developed urine-based SRMs for marijuana metabolite, cocaine metabolite, morphine and codeine, and morphine glucuronide and CAP Reference Materials for amphetamines and phencyclidine. Certification measurements performed at NIST involve two independent methods for each analyte, one of which always uses GC/MS with the other usually being an LC method with either MS or UV detection. Work has recently been completed on a seven component drug in urine SRM. In addition NIST conducts research in the analysis of hair for drugs of abuse. To assist laboratories testing hair for that purpose, NIST has developed two drugs in hair reference materials.

Certification of drugs of abuse in a human serum standard reference material: SRM 1959

A new standard reference material (SRM) for drugs of abuse in human serum (SRM 1959) has been developed. This SRM is intended to be used as a control material for laboratories performing analysis of drugs of abuse in blood to evaluate the accuracy of their methods. SRM 1959 is a frozen human serum material fortified with seven compounds for which analyses are performed to determine evidence of illegal drug use: benzoylecgonine (BZE), methadone (METH), methamphetamine (MAMP), morphine (MOR), nordiazepam (NOR), phencyclidine (PCP), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-9-COOH). Two independent methods involving isotope dilution (ID)-gas chromatography/mass spectrometry (GC/MS) and ID-liquid chromatography/mass spectrometry (LC/MS) were used for the value assignment. For THC-9-COOH, an additional measurement using LC/tandem mass spectrometry (LC/MS/MS) was also included. All methods used isotopically labeled compounds as internal standards and solid-phase extractions to isolate the analytes from the serum. The GC/MS methods used different clean-up procedures from those used for the LC/MS-based methods. Repeatability with within-set coefficients of variation (CVs) ranged from 0.5% to 4.3% for the GC/MS methods and from 0.2% to 1.2% for the LC/MS-based methods. Intermediate precision with between-set CVs for all the methods ranged from 0.1% to 1.1%. Agreement between the GC/MS and LC/MS methods ranged from 0.8% to 8.8%. The results from the methods were combined to obtain the certified concentrations and their expanded uncertainties.

Validation of New Instrumentation for Isotope Dilution Mass Spectrometric Determination of Organic Serum Analytes

A major activity in the 20 year collaboration between the Analytical Chemistry Division at NIST and the College of American Pathologists (CAP) has been the development of highly accurate and precise “definitive” methods for important clinical analytes in human serum. Definitive methods for organic analytes use isotope dilution/gas chromatography/mass spectrometry and require a mass spectrometer capable of making highly precise measurements of the ratio between the ion intensities of a characteristic ion from the analyte of interest and its stable-isotope-labeled analog. Recently, the mass spectrometer used for 20 years for definitive method development and measurements was replaced with a modern instrument capable of automated operation, with accompanying gains in convenience and sample throughput. Switching to the new instrument required modifications of measurement protocols, acceptance criteria, and ratio calculations with background corrections to go along with automated instrument operation. Results demonstrated that the two instruments gave comparable results for measurements of both urea and cholesterol in samples from various serum-based Standard Reference Materials [SRMs] and College of American Pathologists materials.

CCQM-K11: The determination of glucose in serum

A Key Comparison on the determination of glucose in human serum organized by the Consultative Committee on Amount of Substance (CCQM) was carried out in 2001. To address the measurement traceability needs of the clinical chemistry community, the CCQM is undertaking Key Comparisons to document the capabilities of national metrology institutes (NMIs) that provide measurement services in this area. This Key Comparison, along with two others recently completed, CCQM-K6 and CCQM-K12, determination of total cholesterol and creatinine, respectively, in serum, will provide a basis for evaluating the capabilities of NMIs for the determination of well-defined, organic substances of similar molecular weights and concentrations in human serum. Participants in CCQM-K11 included: National Institute of Standards and Technology (NIST) [USA], Coordinating Laboratory; Korea Research Institute of Standards and Science (KRISS) [S Korea]; and Physikalisch-Technische Bundesanstalt (PTB) [Germany]. A frozen human serum material was analysed by each of the participants using ID-GC/MS-based methods. Agreement of results among the participants was good, with a relative range of <1%, yielding a Key Comparison Reference Value of 0.7762 ? 0.0088 mg/g. These results were in accordance with the good agreement found for two serum materials in a pilot study (CCQM-P8) involving the same participants. In conclusion, three NMIs have demonstrated their ability to make reference measurements of glucose in serum using isotope dilution-based methods that, when executed properly, provide measurements that are precise, accurate and SI traceable. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the Mutual Recognition Arrangement (MRA).