Use of Standard Reference Material 2242 (Relative Intensity Correction Standard for Raman Spectroscopy) for microarray scanner qualification.

As a critical component of any microarray experiment, scanner performance has the potential to contribute variability and bias, the magnitude of which is usually not quantified. Using Standard Reference Material (SRM) 2,242, which is certified for Raman spectral correction, for monitoring the microarray fluorescence at the two most commonly used wavelengths, our team at the National Institute of Standards and Technology (NIST) has developed a method to establish scanner performance, qualifying signal measurement in microarray experiments. SRM 2,242 exhibits the necessary photostability at the excitation wavelengths of 635 nm and 532 nm, which allows scanner signal stability monitoring, although it is not certified for use in this capacity. In the current study, instrument response was tracked day to day, confirming that changes observed in experimental arrays scanned are not due to changes in the scanner response. Signal intensity and signal-to-noise ratio (S/N) were tracked over time on three different scanners, indicating the utility of the SRM for scanner qualification.

Microarray Scanner Performance Over a Five-Week Period as Measured With Cy5 and Cy3 Serial Dilution Slides.

To investigate scanner performance and guide development of an instrument qualification method, slides with replicates of successive dilutions of cyanine 5 (Cy5) and cyanine 3 (Cy3) dyes (referred to as dye slides) were scanned. The successive dilutions form a dose-response curve from which performance can be assessed. The effects of a variety of factors, including the number of scans and slide storage conditions, on scanner performance over a five-week period were investigated and tracked with time series charts of dye signal intensity, signal-to-noise (S/N), signal background, slope, and limit of detection (LOD). Scanner drift was tracked with a known stable reference material, Standard Reference Material (SRM) 2242. The greatest effect on the figures of merit was the dye, with the Cy5 dye showing signs of degradation after one week of scanning independent of all other factors while the Cy3 dye remained relatively stable. Use of the charts to track scanner performance over time holds promise for development of a method for microarray scanner performance qualification. Although not a prescription for performance qualification, this introductory study provides sufficient information regarding the use of dye slides to enable the user to institute a preliminary test method.

Quantitative determination of disaccharide content in digested unfragmented heparin and low molecular weight heparin by direct-infusion electrospray mass spectrometry.

Heparins and low molecular weight heparins (LMWHs) are heterogeneous glycosaminoglycans derived from natural sources that are prescribed as anticoagulants. In this work, a direct-infusion electrospray ionization mass spectrometry (ESI-MS) method was applied to the quantitative analysis of known disaccharides in various native heparins and LMWHs after digestion with heparinase enzymes. Disaccharide deltaUA2S-->GlcNS6S was found to compose the majority of all samples analyzed (81-88%). The values were significantly higher than those reported by previously published methods. The disaccharide isomer pair deltaUA-->GlcNS6S/deltaUA2S-->GlcNS was also detected in all samples at lower levels (11-19%). While digestion with heparinases I and II revealed a limited number of disaccharides, the addition of heparinase III to digests led to the detection of disaccharide deltaUA2S-->GlcNAc6S in native porcine heparin. This result indicated the importance of utilizing all three heparinases to gain maximum information when analyzing heparin and LMWH digests. This method displayed good between-day (4-6%) and between-digest (1-2%) reproducibility in separate experiments. To determine if the digestion matrix was suppressing the signal of low-abundance disaccharides, several disaccharides were exogenously added at low levels (1-10 pmol/mg) to a quenched digest reaction. Analysis revealed that low level disaccharides were detectable in this matrix above the limits of detection (0.1-0.2 pmol/mg) and quantitation (0.2-0.7 pmol/mg). While this method was unable to distinguish between disaccharide isomers, it utilized simple mass spectrometry instrumentation to provide useful quantitative data for characterizing preparations of native heparin and LMWH, which could be used to compare various marketed preparations of these popular anticoagulants.

Determination of caffeine and caffeine-related metabolites in ephedra-containing standard reference materials using liquid chromatography with absorbance detection and tandem mass spectrometry.

The concentrations of caffeine and caffeine-related compounds in 2 ephedra-containing reference materials have been determined by 3 independent methods with measurements performed by the National Institute of Standards and Technology (NIST) and a collaborating laboratory. Results from the 3 methods were used for value assignment of caffeine, theobromine, and theophylline in these Standard Reference Materials (SRMs). The methods used at NIST to determine the concentration levels of caffeine, theobromine, and theophylline in SRM 3243 Ephedra-Containing Solid Oral Dosage Form and SRM 3244 Ephedra-Containing Protein Powder used reversed-phase liquid chromatography with absorbance detection and tandem mass spectrometry. These reference materials are part of the first suite in a series of NIST SRMs that provide concentration values for multiple components in dietary supplements. These SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and similar materials.

Preparation and characterization of a suite of ephedra-containing standard reference materials.

The National Institute of Standards and Technology, the U.S. Food and Drug Administration, Center for Drug Evaluation and Research and Center for Food Safety and Applied Nutrition, and the National Institutes of Health, Office of Dietary Supplements, are collaborating to produce a series of Standard Reference Materials (SRMs) for dietary supplements. A suite of ephedra materials is the first in the series, and this paper describes the acquisition, preparation, and value assignment of these materials: SRMs 3240 Ephedra sinica Stapf Aerial Parts, 3241 E. sinica Stapf Native Extract, 3242 E. sinica Stapf Commercial Extract, 3243 Ephedra-Containing Solid Oral Dosage Form, and 3244 Ephedra-Containing Protein Powder. Values are assigned for ephedrine alkaloids and toxic elements in all 5 materials. Values are assigned for other analytes (e.g., caffeine, nutrient elements, proximates, etc.) in some of the materials, as appropriate. Materials in this suite of SRMs are intended for use as primary control materials when values are assigned to in-house (secondary) control materials and for validation of analytical methods for the measurement of alkaloids, toxic elements, and, in the case of SRM 3244, nutrients in similar materials.

Comparison by LC-MS and MALDI-MS of prostate-specific antigen from five commercial sources with certified reference material 613.

OBJECTIVE: Prostate-specific antigen (PSA) samples from five commercial sources were compared with PSA from the European Commission Community Bureau of Reference, known as CRM 613, using liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). DESIGN AND METHODS: The effects of storage at various temperatures, multiple freeze-thaw cycles and lyophilization vs. solution storage were investigated. RESULTS: : CRM 613 and the five commercially available samples varied in their heterogeneity and in the amount of PSA with a molecular mass of 28,430 kDa. Few changes were observed among any of the samples during the stability study or study of the effect of lyophilization and freeze-thaw cycles. CONCLUSIONS: Use of many of the commercial PSA sources as authentic PSA standards for calibrants for in vitro diagnostic tests could lead to significantly biased PSA measurements. Although all the PSA samples appear to be relatively stable for the time periods and conditions studied, some of the commercially available samples contain limited amounts of PSA and included other unidentified components.

Development of a new standard reference material: SRM 1955 (homocysteine and folate in human serum).

Total homocysteine (tHCY) and folate are interrelated biomarkers for arteriosclerosis and coronary heart disease. Although many different methods for both tHCY and folate are clinically available, the intermethod and interlaboratory results are often poor, resulting in the need for a matrix reference material and reference methods. The National Institute of Standards and Technology (NIST) has developed isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/ tandem mass spectrometry (LC/MS/MS) methods for determination of tHCY and several folate forms including 5-methyltetrahydrofolic acid (5MT) and folic acid (FA). Additionally, a method for simultaneous measurement of tHCY, 5MT, and FA has been developed and validated. In collaboration with the Centers for Disease Control and Prevention (CDC), mass spectrometric methods and methods used in clinical laboratories have been applied to characterize a new Standard Reference Material (SRM), SRM 1955, "Homocysteine and Folate in Human Serum," containing low, medium, and high levels of tHCY and 5MT. Additionally, FA, 5-formyltetrahydrofolic acid (5FT), vitamin B12, and total folate values are provided. Use of the new SRM should improve clinical measurements and will permit traceability to internationally recognized certified reference materials, as described by European Directive 98/79/EC on in vitro diagnostic medical devices.

Simultaneous quantification of homocysteine and folate in human serum or plasma using liquid chromatography/tandem mass spectrometry.

A unified extraction and quantification procedure based on stable isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the simultaneous determination of total homocysteine and folate (5-methyltetrahydrofolic acid and folic acid) levels in human serum and plasma. This is the first report documenting the simultaneous extraction and quantification of these structurally dissimilar analytes. Analytes are quantitatively isolated from samples (500 microL) prior to LC/MS/MS analysis using a two-step stabilization process combined with C18 solid-phase extraction. The method exhibits excellent linearity over 4 orders of magnitude for each analyte. Measurement repeatability (RSD, N = 2) ranged from 0.3% to 3% for all analytes over 1 day of analysis. Total method variability (RSD, N = 6) ranged from 0.7% to 10% for all analytes over three independent days of analysis. The accuracy and practical applicability of the method were demonstrated by applying the method to the quantitative determination of each analyte in a new NIST serum Standard Reference Material (NIST SRM 1955 Homocysteine and Folate in Frozen Human Serum) and in a small subset of normal donor plasma samples.

Capillary electrophoresis for the investigation of prostate-specific antigen heterogeneity.

Prostate-specific antigen (PSA) is a single-chain glycoprotein that is used as a biomarker for prostate-related diseases. PSA has one known posttranslational modification, a sialylated diantennary N-linked oligosaccharide attached to the asparagine residue N45. In this study capillary electrophoresis (CE) was employed to separate the isoforms of seven commercially available free PSA samples, two of which were specialized: enzymatically active PSA and noncomplexing PSA. The free PSA samples examined migrated as four to nine distinct, highly resolved peaks, indicating the presence of several isoforms differing in their oligosaccharide compositions. Overall, the use of CE provides a rapid, reproducible method for separation of PSA into its individual isoforms.

Determination of ephedrine alkaloids in dietary supplement standard reference materials.

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.

Development and evaluation of an isotope dilution LC/MS method for the determination of total homocysteine in human plasma.

Elevated plasma homocysteine has been identified as a strong and independent risk factor for cardiovascular diseases, and recently, it has been associated with the development of dementia in older adults. Selected ion-monitoring isotope-dilution LC/MS (electrospray) has been developed and evaluated as a reference method for the accurate determination of total homocysteine in human plasma. Homocysteine is quantitatively isolated from plasma via the use of anion-exchange resins and then detected and quantified in stabilized plasma extracts with selected ion-monitoring LC/MS. This method is shown to be highly comparable to LC/MS/MS determinations in terms of its analytical accuracy and precision, yet this alternative measurement approach does not necessitate the enhanced instrumentation or added expense required of tandem MS/MS determinations. LC/MS detection of homocysteine was linear (standard error of the estimate for the regression line was 0.0323) over 3 orders of magnitude, and the calculated limits of detection and quantification were 0.06 micromol/L (0.12 ng on column) and 0.6 micromol/L (1.2 ng on column), respectively. Independent calibration curves showed excellent linearity (r2 > or = 0.996) between 0 and 25 micromol/L homocysteine over a 3-day period. The accuracy and precision of total homocysteine measurements for patient samples and quality control pools using LC/MS were compared to total homocysteine measurements using LC/MS/MS, GC/MS, FPIA, and LC-FD. LC/MS performed well in relation to the other homocysteine methods in terms of its capability to accurately quantify plasma homocysteine over the normal range (5-15 micromol/L).

Comparison of isotope dilution mass spectrometry methods for the determination of total homocysteine in plasma and serum.

Two independent methods have been critically evaluated and applied to the measurement of total homocysteine in serum and plasma: solid-phase anion extraction (SPAE) gas chromatography/mass spectrometry (GC/MS) and protein precipitation liquid chromatography/tandem mass spectrometry (LC/MS/MS). In addition, analysis of samples prepared by SPAE was accomplished by liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS. These methods have been used to determine total homocysteine levels in several existing serum-based Standard Reference Materials (SRMs) from the National Institute of Standards and Technology and in patient plasma samples provided by the Centers for Disease Control and Prevention. The precision of the homocysteine measurements in serum and plasma was critically evaluated, and method comparisons were carried out using Bland-Altman plots and bias analysis. On the basis of the excellent precision and close agreement of the mass spectrometric (MS) methods, the MS-based methods will be used for certification of a serum-based SRM for homocysteine and folates.

Structural characterization and detection of kale flavonoids by electrospray ionization mass spectrometry.

Sensitive and precise analytical methods are needed for flavonols, a subclass of flavonoids that has strong antioxidant activity. We report an improved method for identifying the predominant flavonols, quercetin and kaempferol, by collisionally activated dissociation (CAD) and quantifying them by high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) in the selected ion monitoring mode. Practical applications of the method were demonstrated using several kale and biological samples. Two commercial kale samples were found to have 77 or 244 ppm quercetin and 235 or 347 ppm kaempferol (ppm = microg of quercetin/g of kale or microg of kaempferol/g of kale by fresh weight, 5-15% relative standard deviation). Blanching was found to reduce the flavonols to approximately 60% of the levels found in the unblanched kale. Isotopically labeled kale (cultivar Vates) grown in a greenhouse under an atmosphere of (13)CO(2) was found to have much lower flavonol levels. UV-A and UV-B supplementation during kale growth in the greenhouse was found to enhance both quercetin and kaempferol levels in Vates kale. The UV-B-supplemented kale not only produced more flavonols but the quercetin-to-kaempferol ratio was also higher than the UV-A-supplemented or the nonsupplemented kale. Recovery of flavonols from kale was approximately 60% based on spike and recovery trials with rutin, a glycoside of quercetin. Recovery of flavonols from biological samples spiked with rutin ranged from 96% for urine to 70% for plasma. Compared to UV detection, ESI-MS in the deprotonation mode provided lower detection limits, and both higher sensitivity and selectivity, in addition to structural characterization of the kale flavonols by CAD.