Katherine E. Sharpless

C30 stationary phases for the analysis of food by liquid chromatography.

The introduction of a polymeric C30 liquid chromatographic column by Sander et al. [Anal. Chem., 66 (1994) 1667] designed for the separation of carotenoid isomers, has led to the development of improved analytical methods for these compounds. Subsequent commercial availability of polymerically bonded C30 columns has facilitated these advances, and applications to a wide variety of separation problems with biological samples have been described. This report provides a comprehensive review of applications of polymeric C30 columns, utilized in the determination of carotenoids, retinoids, and other nutrients and related compounds in complex, natural-matrix samples.

Development and Certification of a Standard Reference Material for Vitamin D Metabolites in Human Serum

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Healths Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.

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.

Liquid chromatographic determination of carotenoids in human serum using an engineered C30 and a C18 stationary phase

A C30 stationary phase was specifically engineered for carotenoid separations, and carotenoid measurements using this column are compared with those obtained using a somewhat more conventional C18 column. Both methods were used to contribute measurements for the certification of carotenoids in Standard Reference Material 968b, Fat-Soluble Vitamins and Cholesterol in Human Serum. Analytes were extracted from the serum into hexane. Measurements on the C18 column were made using a gradient of acetonitrile, methanol, and ethyl acetate, which is described in detail elsewhere. Measurements on the C30 column were made using a gradient of water, methanol, and methyl tert.-butyl ether.

Certification of standard reference materials containing bitter orange

A suite of three dietary supplement standard reference materials (SRMs) containing bitter orange has been developed, and the levels of five alkaloids and caffeine have been measured by multiple analytical methods. Synephrine, octopamine, tyramine, N-methyltyramine, hordenine, total alkaloids, and caffeine were determined by as many as six analytical methods, with measurements performed at the National Institute of Standards and Technology and at two collaborating laboratories. The methods offer substantial independence, with two types of extractions, two separation methods, and four detection methods. Excellent agreement was obtained among the measurements, with data reproducibility for most methods and analytes better than 5% relative standard deviation. The bitter-orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods.

Isotope Dilution Liquid Chromatography - Mass Spectrometry Methods for Fat- and Water-Soluble Vitamins in Nutritional Formulations

Vitamins are essential to human health, and dietary supplements containing vitamins are widely used by individuals hoping to ensure they have adequate intake of these important nutrients. Measurement of vitamins in nutritional formulations is necessary to monitor regulatory compliance and in studies examining the nutrient intake of specific populations. Liquid chromatographic methods, primarily with UV absorbance detection, are well established for both fat- and water-soluble measurements, but they do have limitations for certain analytes and may suffer from a lack of specificity in complex matrices. Liquid chromatography-mass spectrometry (LC-MS) provides both sensitivity and specificity for the determination of vitamins in these matrices, and simultaneous analysis of multiple vitamins in a single analysis is often possible. In this work, LC-MS methods were developed for both fat- and water-soluble vitamins and applied to the measurement of these analytes in two NIST Standard Reference Materials. When possible, stable isotope labeled internal standards were employed for quantification.

Development of liquid chromatographic methods for the determination of phytosterols in Standard Reference Materials containing saw palmetto.

Liquid chromatographic (LC) methods using atmospheric pressure chemical ionization/mass spectrometric (APCI-MS) detection were developed for the separation and analysis of the phytosterols campesterol, cycloartenol, lupenone, lupeol, beta-sitosterol, and stigmasterol. Brassicasterol and cholesterol were also included for investigation as internal standards. The methods were used to identify and quantify the phytosterols in each of two Serenoa repens (saw palmetto) Standard Reference Materials (SRMs) developed by the National Institute of Standards and Technology (NIST). Values obtained by LC-MS were compared to those obtained using the more traditional approach of gas chromatography with flame ionization detection. This is the first reported use of LC-MS to determine phytosterols in saw palmetto dietary supplement materials.

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.

The stability of retinol, α-tocopherol, trans-lycopene, and trans-β-carotene in liquid-frozen and lyophilized serum

Abstract The concentrations of retinol, α-tocopherol, and trans -β-carotene in lyophilized serum stored at −25°C and −80°C have been monitored for 10 years. There was no evidence of degradation of any of these compounds over the 10-year period. Retinol, α-tocopherol, and trans -β-carotene were less stable at −25°C in liquid-frozen serum than they were in lyophilized serum. At −80°C, trans -β-carotene levels were stable for up to 3 years of storage in liquid-frozen serum. Both retinol and α-tocopherol appeared stable in liquid-frozen serum for at least 5 years at −80°C. The effect of repeated freeze/thaw cycles on retinol, α-tocopherol, trans -lycopene, and trans -β-carotene in liquid-frozen and reconstituted lyophilized serum both stored at −20°C was also studied. Retinol, α-tocopherol, trans -lycopene, and trans -β-carotene in reconstituted lyophilized serum stored at −20°C were stable for at least 3 days with minimal (

Development of saw palmetto (Serenoa repens) fruit and extract standard reference materials

As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed two standard reference materials (SRMs) representing different forms of saw palmetto (Serenoa repens), SRM 3250 Serenoa repens fruit and SRM 3251 Serenoa repens extract. Both of these SRMs have been characterized for their fatty acid and phytosterol content. The fatty acid concentration values are based on results from gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS) analysis while the sterol concentration values are based on results from GC-FID and liquid chromatography with mass spectrometry analysis. In addition, SRM 3250 has been characterized for lead content, and SRM 3251 has been characterized for the content of β-carotene and tocopherols. SRM 3250 (fruit) has certified concentration values for three phytosterols, 14 fatty acids as triglycerides, and lead along with reference concentration values for four fatty acids as triglycerides and 16 free fatty acids. SRM 3251 (extract) has certified concentration values for three phytosterols, 17 fatty acids as triglycerides, β-carotene, and γ-tocopherol along with reference concentration values for three fatty acids as triglycerides, 17 fatty acids as free fatty acids, β-carotene isomers, and δ-tocopherol and information values for two phytosterols. These SRMs will complement other reference materials currently available with concentrations for similar analytes and are part of a series of SRMs being developed for dietary supplements.