SRM 1950 Metabolites in Human Plasma was prepared by Bioreclamation, Inc. (Hicksville, NY) according to specifications provided by NIST. The plasma pool was collected from 100 donors, with an equal number of men and women and from donors between 40 and 50 years of age. Donors were selected to be representative of the ethnic distribution of the U.S. population, based upon data from the U.S. Census in 2000. Donors were asked to fast overnight and to abstain from taking medications for 72 h prior to blood donation. A rapid glucose test was used to exclude individuals who did not comply with the fasting requirement. Individuals affected by overt disease or disorders and those having a body mass index (BMI) outside the 95th percentile were excluded.

Plasma was prepared from whole blood that was placed on ice immediately after collection, and lithium heparin was used as the anticoagulant. Each sample was centrifuged at 4 °C at 8000 × g for 25 min. Samples were processed and frozen within 60 min from the time of collection. Units of plasma were thawed once and blended under nitrogen. The resulting plasma pool was dispensed in 1 mL aliquots into vials, and the vials were then stored at −80 °C prior to shipment to NIST. Approximately 20 000 vials of SRM 1950 were delivered to NIST, and a unit of SRM 1950 consists of 5 vials.

Each unit of human plasma used in the preparation of SRM 1950 was screened by Bioreclamation and found to be nonreactive for hepatitis B surface antigen (HBsAg), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and human immunodeficiency virus 1 antigen (HIV-1Ag) by Food and Drug Administration (FDA)-licensed tests. However, because no test method can guarantee that these infectious agents are absent, appropriate safety precautions should be taken when handling this or any other potentially infectious human plasma or blood specimens.³⁹

Brief descriptions of the methods used in the value assignment of SRM 1950 are provided in the Certificate of Analysis⁴⁰ and are also included in the Supporting Information. Method details are also available online (http://srm1950.nist.gov). Some of the methods developed as part of the certification of SRM 1950 have been described in related publications.⁴¹⁻⁴³ Certification measurements were performed at NIST and at the Centers for Disease Control and Prevention (CDC, Atlanta, GA). The methods used at NIST had, in most cases, been used previously for value assignment of other SRMs. The methods used by CDC had previously been validated for use in the National Health and Nutrition Examination Survey (NHANES). Analyte quantification was based upon isotope-dilution mass spectrometry (ID-MS) whenever possible. For analytes such as the carotenoids where ID-MS methods were not available, liquid chromatographic methods with UV/visible detection (LC-UV) or fluorescence detection (LC-FL) were employed at NIST and CDC. For some of the fatty acids and carotenoids, value assignment measurements were performed using a single method at the CDC.

Traditional clinical markers such as glucose, cholesterol, and triglycerides can be useful as surrogate markers for classifying or stratifying samples in metabolomics research.^48,49 Glucose is one of the most abundant metabolites in serum or plasma, and glucose metabolism is also known to be altered in a number of disease states, including diabetes.⁸ Although electrolytes (calcium, magnesium, potassium, sodium) are not generally considered in metabolomics investigations, electrolyte imbalances are associated with a number of diseases. They are also some of the more abundant species in human serum or plasma.¹⁶ Steroid hormone profiles have been studied in relation to endocrine disorders and as potential cancer biomarkers.⁷

Table 1 summarizes the concentrations of the majority of the clinical markers that were value assigned in SRM 1950. For some analytes, mass concentrations presented in the table were calculated from mass fractions using the measured density of SRM 1950 (1.02086 g/mL). In general, each of these analytes was determined at NIST by a single method, as indicated in the table. Many of these methods are recognized as reference measurement procedures by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). The GC-MS procedures for the clinical analytes involved derivatization of the analyte of interest after isolation from the plasma matrix. Labeled internal standards were added at the beginning of the sample preparation process and allowed to equilibrate with the sample for a predetermined period of time. A similar approach was utilized for the LC-MS and LC-MS/MS analyses, but derivatization was not required in these methods. Samples for ICP-MS analysis were subjected to acid digestion, and an appropriate isotope spike was employed for quantification. For the GC-MS, LC-MS, and LC-MS/MS analyses, the relative expanded uncertainties associated with these measurements are generally in the range of 1−3%, and relative expanded uncertainties <1% are common for the ICP-MS methods. This method performance is reflected in the expanded uncertainties shown with each certified or reference value in Table 1.

Amino acids are the building blocks of proteins and also serve as important metabolic intermediates. Measurement of amino acids in biological fluids is routinely used in the diagnosis of metabolic disorders.⁵⁰ Thus, it is not surprising that metabolomics studies have frequently detected perturbations in amino acid concentrations that appear to be linked with particular disease states.⁵¹⁻⁵³ Research has also indicated that amino acid metabolism is altered in diabetes, and certain branched chain amino acids may have utility as early indicators of diabetes risk.^8,54 In some cases, changes in specific amino acid concentrations appear to have merit in classifying patients by disease subtype, as was reported for chronic obstructive pulmonary disease (COPD).⁵⁵

Amino acids were determined in SRM 1950 through a combination of data from four independent methods based upon liquid chromatography−tandem mass spectrometry (LC-MS/MS), gas chromatography−time-of-flight mass spectrometry (GC-TOF-MS), and two-dimensional GC coupled to time-of-flight mass spectrometry (GCxGC-TOF-MS).⁴¹ For the GC-TOF-MS analyses, either propyl chloroformate (PCF) or N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) was used as the derivatizing agent. Only MTBSTFA was used as the derivatizing agent for the GCxGC-TOF-MS measurements. Isotopically labeled internal standards were employed for each of the amino acids and were added at the beginning of the sample analysis. In general, good agreement was observed among the results from the four methods. Details on the results from the individual methods are presented elsewhere.⁴¹ Coefficients of variation (CVs) for the LC-MS/MS method were typically in the range of 1−3%, reflecting the excellent precision commonly associated with ID-MS methods. The observed CVs for the GC-TOF-MS and GCxGC-TOF-MS analyses were generally higher (≈5−10%), perhaps because of the additional sample preparation steps that were required for these methods.

The concentrations of 18 amino acids were assigned based upon a combination of the GC-MS and LC-MS/MS methods, and the certified and reference values are summarized in Table 2. Arginine and ornithine could not be determined in SRM 1950 by the GC-MS methods because of thermal instability of the arginine derivative, leading to potential conversion to ornithine. Histidine could not be determined by GC-TOF-MS because of a large interference from glucose, but adding a second dimension to the separation (GCxGC) removed the interference. Hence, employing multiple independent methods reduced the likelihood of undetected measurement bias and provided greater confidence in the value assignment of amino acids in SRM 1950. The results obtained in this work were also comparable to a related literature report on analysis of SRM 1950 by NMR⁵⁶ and to information on amino acid concentrations found in the Human Metabolome Database.¹⁷ SRM 1950 is the first serum or plasma-based NIST SRM with values assigned for amino acids.

Dysregulation of fatty acid metabolism has been implicated in a number of diseases, including diabetes⁸ and cancer.⁵² Some drug therapies have been shown to cause widespread changes in lipid profiles,⁵⁷ and differences in fatty acid concentrations have also been observed between groups having disparate levels of physical fitness.⁵⁸ Fatty acids were value assigned in SRM 1950 through a combination of GC methods with either flame ionization (FID) or mass spectrometric detection. A total of 26 fatty acids were determined in SRM 1950, including saturated, monounsaturated, and polyunsaturated fatty acids. The results are summarized in Table 3. Of these fatty acids, 14 were measured by a single method, either at NIST or at CDC. The two NIST methods (GC-FID and GC-MS) employed different sample preparation schemes and different chromatographic columns. The CDC method (GC-MS) represented a third approach and was independent from the NIST methodologies.

Figure 1 shows a comparison of results from the three methods for selected fatty acids. The certified or reference values derived from these results are also shown in the figure. In general, there was good agreement among the methods used, even for fatty acids present in SRM 1950 at low concentrations. Oleic acid, palmitic acid, and linoleic acid were the most abundant fatty acids measured in SRM 1950, and these results are consistent with U.S. population data from NHANES.⁵⁹ Omega-3 fatty acids are of interest because of their potential cardioprotective effects,⁴⁹ and several omega-3 fatty acids were measured in SRM 1950, as shown in Table 3. SRM 1950 represents the first serum or plasma-based NIST SRM with values assigned for fatty acids.

Numerous studies have attempted to discern the relationship between diet and the risk of disease. Nutrients such as carotenoids and vitamin D may reduce the risk of cancer or other diseases in certain populations.^60,61 However, identifying individuals more likely to respond to dietary interventions remains an elusive goal.⁶² Metabolomics studies may provide answers to some of the seemingly contradictory results that have been reported in nutrition research.⁶³

Vitamin A, vitamin E, and carotenoids were determined in SRM 1950 using a combination of LC-UV methods, as shown in Table 4. The NIST methods utilized two different types of chromatographic columns, and additional method details are provided in the Supporting Information. Whenever possible, the values in Table 4 represent a combination of the results obtained by NIST and CDC. In some cases, however, assigned values for the carotenoids were based solely upon measurements performed at CDC. Metabolites arising from two water-soluble vitamins (folate and vitamin B₆) and one additional fat-soluble vitamin (vitamin D) were also measured in SRM 1950, and these results are included in Table 4. The certified and reference values for these analytes were based primarily upon ID-MS methods, but an LC-FL method was included for analysis of pyridoxal 5′-phosphate, a vitamin B₆ metabolite.