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Vitamin D-binding protein (VDBP), a serum transport protein for 25-hydroxyvitamin D [25(OH)D], has three common proteoforms which have co-localized amino acid variations and glycosylation. A monoclonal immunoassay was found to differentially detect VDBP proteoforms and methods using liquid chromatography-tandem mass spectrometry (LC-MS/MS) might be able to overcome this limitation. Previously developed multiple reaction monitoring LC-MS/MS methods for total VDBP quantification represent an opportunity to probe the potential effects of proteoforms on proteolysis, instrument response and quantification accuracy. VDBP was purified from homozygous human donors and quantified using proteolysis or acid hydrolysis and LC-MS/MS. An interlaboratory comparison was performed using pooled human plasma [Standard Reference Material® 1950 (SRM 1950) Metabolites in Frozen Human Plasma] and analyses with different LC-MS/MS methods in two laboratories. Several shared peptides from purified proteoforms were found to give reproducible concentrations [≤2.7% coefficient of variation (CV)] and linear instrument responses (R2≥0.9971) when added to human serum. Total VDBP concentrations from proteolysis or amino acid analysis (AAA) of purified proteoforms had ≤1.92% CV. SRM 1950, containing multiple proteoforms, quantified in two laboratories resulted in total VDBP concentrations with 7.05% CV. VDBP proteoforms were not found to cause bias during quantification by LC-MS/MS, thus demonstrating that a family of proteins can be accurately quantified using shared peptides. A reference value was assigned for total VDBP in SRM 1950, which may be used to standardize methods and improve the accuracy of VDBP quantification in research and clinical samples. [ABSTRACT FROM AUTHOR]

A roundtable to discuss monitoring of serum 25-hydroxyvitamin D [25(OH)D] in the NHANES was held in late July 2009. Topics included the following: 1) options for dealing with assay fluctuations in serum 25(OH)D in the NHANES conducted between 1988 and 2006; 2) approaches for transitioning between the RIA used in the NHANES between 1988 and 2006 to the liquid chromatography tandem MS (LC-MS/MS) measurement procedure to be used in NHANES 2007 and later; 3) approaches for integrating the recently available standard reference material for vitamin D in human serum (SRM 972) from the National Institute of Standards and Technology (NIST) into the NHANES; 4) questions regarding whether the C-3 epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3] should be measured in NHANES 2007 and later; and 5) identification of research and educational needs. The roundtable experts agreed that the NHANES data needed to be adjusted to control for assay fluctuations and offered several options for addressing this issue. The experts suggested that the LC-MS/MS measurement procedure developed by NIST could serve as a higher order reference measurement procedure. They noted the need for a commutability study for the recently released NIST SRM 972 across a range of measurement procedures. They suggested that federal agencies and professional organizations work with manufacturers to improve the quality and comparability of measurement procedures across all laboratories. The experts noted the preliminary nature of the evidence of the 3-epi-25(OH)D3 but felt that it should be measured in 2007 NHANES and later. J. Nutr. 140: 2030S-2045S, 2010. doi: 10.3945/jn.110.121483.

Vitamin D exists in two major forms, vitamin [D.sub.3] and vitamin [D.sub.2]. Vitamin D helps the body absorb calcium and promote optimal bone health. Both forms of vitamin D are metabolized to 25-hydroxyvitamin D in the body, and the levels of 25-hydroxyvitamin [D.sub.3] [25(OH)[D.sub.3]] and 25-hydroxyvitamin [D.sub.2] [25(OH)[D.sub.2]] in serum are considered the best indicators of vitamin D status. A candidate reference measurement procedure for serum 25(OH)[D.sub.3] and 25(OH)[D.sub.2] has been developed and critically evaluated. The deuterated compounds 25(OH)[D.sub.3]-[D.sub.3] and 25(OH)[D.sub.2]-[D.sub.3] are used as internal standards for 25(OH)[D.sub.3] and 25(OH)[D.sub.2], respectively. The 25(OH)[D.sub.3] and 25(OH)[D.sub.2] and their respective labeled internal standards are simultaneously extracted from serum using liquid liquid extraction prior to reversed-phase liquid chromatography--tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a cyano (CN) column for both 25(OH)[D.sub.3] and 25(OH)[D.sub.2]. Atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) were used for LC-MS/MS. The accuracy of the method was evaluated by recovery studies of measuring 25-hydroxyvitamin D [25(OH)D] in spiked samples with known 25(OH)D levels. The recoveries of the added 25(OH)[D.sub.3] and 25(OH)[D.sub.2] ranged from 99.0% to 101.0%. The absolute recoveries with this method were 97% and 92% for 25(OH)[D.sub.3] and 25(OH)[D.sub.2], respectively. Excellent precision was obtained with between-set coefficients of variation (CVs) of 0.2-0.6% for 25(OH)D levels > 1 ng/g and within 2% for the level of < 1 ng/g. Chromatographic separation of 25(OH)[D.sub.3] and 25(OH)[D.sub.2] from their respective isomers 3-epi25(OH)[D.sub.3] and 3-epi-25(OH)[D.sub.2] was achieved. The limit of detection at a signal-to-noise ratio of ~3 was 40 pg of 25(OH)D on column (or ~0.15 ng/g as expressed as a concentration). This candidate reference measurement procedure for serum 25(OH)[D.sub.3] and 25(OH)[D.sub.2] demonstrates good accuracy and precision and low susceptibility to interferences. It can be used to provide an accuracy base to which clinical methods for 25(OH)[D.sub.3] and 25(OH)[D.sub.2] can be compared and that will serve as a standard of higher order for measurement traceability. 10.1021/ac9026862

A suite of five ephedra-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for ephedrine alkaloids, synephrine, caffeine, and selected toxic trace elements. The materials represent a variety of natural, extracted, and processed sample matrixes that provide different analytical challenges. The constituents have been determined by multiple independent methods with measurements performed by NIST and by three collaborating laboratories. The methods utilized different sample extraction and cleanup steps in addition to different instrumental analytical techniques and approaches to quantification. In addition, food--matrix proximates were determined by National Food Processor Association laboratories for one of the ephedra-containing SRMs. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials.

A new technique is demonstrated for the simultaneous concentration and high-resolution separation of chiral compounds. With temperature gradient focusing, a combination of a temperature gradient, an applied electric field, and a buffer with a temperature-dependent ionic strength is used to cause analytes to move to equilibrium, zero-velocity points along a microchannel or capillary. Different analytes are thus separated spatially and concentrated in a manner that resembles isoelectric focusing but that is applicable to a greater variety of analytes including small chiral drug molecules. Chiral separations are accomplished by the addition of a chiral selector, which causes the different enantiomers of an analyte to focus at different positions along a microchannel or capillary. This new technique is demonstrated to provide high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile reversal of peak order (desirable for analysis of trace enantiomeric impurities), and high resolving power (comparable to capillary electrophoresis) in combination with greater than 1000-fold concentration enhancement enabling improved detection limits. In addition, chiral temperature gradient focusing allows for real-time monitoring of the interaction of chiral analyte molecules with chiral selectors that could potentially be applied to the study of other molecular interactions. Finally, unlike CE, which requires long channels or capillaries for high-resolution separations, separations of equivalent resolution can be performed with TGF in very short microchannels (mm); thus, TGF is inherently much more suited to miniaturization and integration into lab-on-a-chip-devices.