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Using dendron chemistry, we developed stability-enhanced carboxylate surface-modified (negatively charged dendron) AuNPs (Au-NCD). Since the carboxylate surface of Au-NCD is optimal for complexation with cisplatin (Pt) moieties, we further synthesized Pt-loaded Au-NCD (Au-NCD/Pt) to serve as potential therapeutic anticancer agents. The size distribution, zeta potential, and surface plasmon resonance of both Au-NCDs and Au-NCD/Pt were characterized via dynamic light scattering, scanning transmission electron microscopy, and ultraviolet–visible spectrophotometry. Surface chemistry, Pt uptake, and Pt release were evaluated using inductively coupled plasma-mass spectrometry and X-ray photoelectron spectroscopy. Colloidal stability in physiological media over a wide pH range (1–13) and shelf-life stability (up to 6 months) were also assessed. Finally, the cytotoxicity of both Au-NCD and Au-NCD/Pt to Chinese hamster ovary cells (CHO K1; as a normal cell line) and to human lung epithelial cells (A549; as a cancer cell line) was evaluated. The results of these physicochemical and functional cytotoxicity studies with Au-NCD/Pt demonstrated that the particles exhibited superlative colloidal stability, cisplatin uptake, and in vitro anticancer activity despite low amounts of Pt release from the conjugate. [ABSTRACT FROM AUTHOR]

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 [micro]L) prior to LC/ MS/MS analysis using a two-step stabilization process combined with [C.sub.18] 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.

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.

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 [micro]mol/L (0.12 ng on column) and 0.6 [micro]mol/L (1.2 ng on column), respectively. Independent calibration curves showed excellent linearity ([r.sup.2] [greater than or equal to] 0.996) between 0 and 25 [micro]mol/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 [micro]mol/L).

A method has been developed for the direct microscale determination of 12 catechins in green and black tea infusions. The method is based on liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS). Standard catechin mixtures and tea infusions were analyzed by LC/APCI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identifies of eight major catechins and caffeine in tea were established based on LC retention times and simultaneously recorded mass spectra. In addition, monitoring of the catechin-specific retro Diels--Alder fragment ion at m/z 139 throughout the chromatogram provided a unique fingerprint for catechin content in the samples that led to the identification of four minor chemically modified catechin derivatives in the infusions. This report is the first to describe the comprehensive determination of all 12 reported catechins in a single analysis. The utility of LC/APCI-MS for providing routine separation and identification of catechins at femtomole to low-picomole levels without extraction or sample pretreatment, and its potential as a standard analytical tool for the determination of polyphenols in natural products and biological fluids, are discussed.

For environmental studies assessing uptake of orally ingested engineered nanoparticles (ENPs), a key step in ensuring accurate quantification of ingested ENPs is efficient separation of the organism from ENPs that are either nonspecifically adsorbed to the organism and/or suspended in the dispersion following exposure. Here, we measure the uptake of 30 and 60 nm gold nanoparticles (AuNPs) by the nematode, Caenorhabditis elegans, using a sucrose density gradient centrifugation protocol to remove noningested AuNPs. Both conventional inductively coupled plasma mass spectrometry (ICP-MS) and single particle (sp)ICP-MS are utilized to measure the total mass and size distribution, respectively, of ingested AuNPs. Scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS) imaging confirmed that traditional nematode washing procedures were ineffective at removing excess suspended and/or adsorbed AuNPs after exposure. Water rinsing procedures had AuNP removal efficiencies ranging from 57 to 97% and 22 to 83%, while the sucrose density gradient procedure had removal efficiencies of 100 and 93 to 98%, respectively, for the 30 and 60 nm AuNP exposure conditions. Quantification of total Au uptake was performed following acidic digestion of nonexposed and Au-exposed nematodes, whereas an alkaline digestion procedure was optimized for the liberation of ingested AuNPs for spICP-MS characterization. Size distributions and particle number concentrations were determined for AuNPs ingested by nematodes with corresponding confirmation of nematode uptake via high-pressure freezing/freeze substitution resin preparation and large-area SEM imaging. Methods for the separation and in vivo quantification of ENPs in multicellular organisms will facilitate robust studies of ENP uptake, biotransformation, and hazard assessment in the environment.

Quantum dots (QDs) are semiconductor nanocrystals exhibiting unique optical properties that can be exploited for many practical applications ranging from photovoltaics to biomedical imaging and drug delivery. A significant number of studies have alluded to the cytotoxic potential of these materials, implicating Cd-leaching as the causal factor. Here, we investigated the role of heavy metals in biological responses and the potential of CdSe-induced genotoxicity. Our results indicate that, while negatively charged QDs are relatively noncytotoxic compared to positively charged QDs, the same does not hold true for their genotoxic potential. Keeping QD core composition and size constant, 3 nm CdSe QD cores were functionalized with mercaptopropionic acid (MPA) or cysteamine (CYST), resulting in negatively or positively charged surfaces, respectively. CYST-QDs were found to induce significant cytotoxicity accompanied by DNA strand breakage. However, MPA-QDs, even in the absence of cytotoxicity and reactive oxygen species formation, also induced a high number of DNA strand breaks. QD-induced DNA damage was confirmed by identifying the presence of p53 binding protein 1 (53BP1) in the nuclei of exposed cells and subsequent diminishment of p53 from cytoplasmic cellular extracts. Further, high-throughput real-time PCR analyses revealed upregulation of DNA damage and response genes and several proinflammatory cytokine genes. Most importantly, transcriptome sequencing revealed upregulation of the metallothionein family of genes in cells exposed to MPA-QDs but not CYST-QDs. These data indicate that cytotoxic assays must be supplemented with genotoxic analyses to better understand cellular responses and the full impact of nanoparticle exposure when making recommendations with regard to risk assessment.

The predominant adrenergic protoalkaloid found in the peel and fruit of bitter orange, Citrus aurantium, is synephrine. Synephrine is reputed to have thermogenic properties and is used as a dietary supplement to enhance energy and promote weight loss. However, there exists some concern that the consumption of dietary supplements containing synephrine or similar protoalkaloids may contribute to adverse cardiovascular events. This study developed and validated a positive-ion mode liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of the major (synephrine) and minor (tyramine, N-methyltyramine, octopamine, and hordenine) adrenergic protoalkaloids in a suite of National Institute of Standards and Technology (NIST) bitter orange Standard Reference Materials (SRMs): SRM 3258 Bitter Orange Fruit, SRM 3259 Bitter Orange Extract, and SRM 3260 Bitter Orange Solid Oral Dosage Form. The limit of quantitation (LOQ) for all protoalkaloids is approximately 1 pg on-column, except for octopamine (20 pg on-column). Additionally, the method has a linear dynamic range of > or =3 orders of magnitude for all of the protoalkaloids. Individual, as well as "total", protoalkaloid levels (milligrams per kilogram) in the NIST SRMs were determined and compared to the levels measured by an independent liquid chromatography/fluorescence detection (LC/FD) method. Satisfactory concordance between the LC/MS/MS and LC/FD protoalkaloid measurements was demonstrated. LC/MS/MS analysis of the protoalkaloids in the SRMs resulted in mean measurement imprecision levels of < or =10% coefficient of variation (% CV).

Two independently developed liquid chromatography (LC) methods for the quantitative determination of biotin in multivitamin/multielement tablets (NIST Standard Reference Material 3280 (SRM 3280)) are described. The methods use distinctly different tablet extraction solvents (methanol vs 1.5% aqueous formic acid) and analyte detection principles (mass spectrometry (MS) versus evaporative light-scattering detection (ELSD)) to ensure quantitative reliability. The use of different extraction and detection procedures allows cross-validation of the methods and enhances confidence in the final quantitative results. Both methods yield highly comparable results for the mean level of biotin (LC/MS = 26.5 mg/kg +/- 0.3 mg/kg (n = 12); LC/ELSD = 24.7 mg/kg +/- 1.7 mg/kg (n = 12)) in SRM 3280, yet the methods differ considerably in their analytical characteristics. The isotope-dilution LC/MS method exhibits excellent linearity from 0.02 ng to 77 ng biotin on-column with a method limit of detection (LOD) and limit of quantification (LOQ) of 0.02 ng (S/N > 3) and 0.06 ng (S/N > 10) biotin on-column, respectively. The LC/ELSD method exhibits good linearity from 155 ng to 9900 ng biotin on-column with a method LOD and LOQ of 155 ng (S/N > 3) and 310 ng (S/N > 10) biotin on-column, respectively. Method performance data indicates that the LC/MS method is analytically superior to the LC/ELSD method; however, either method in combination with SRM 3280 should provide quality assurance, accuracy, and traceability for biotin levels in multivitamin/multielement dietary supplements.

Catechins are polyphenolic plant compounds (flavonoids) that may offer significant health benefits to humans. These benefits stem largely from their anticarcinogenic, antioxidant, and antimutagenic properties. Recent epidemiological studies suggest that the consumption of flavonoid-containing foods is associated with reduced risk of cardiovascular disease. Chocolate is a natural cocoa bean-based product that reportedly contains high levels of monomeric, oligomeric, and polymeric catechins. We have applied solid-liquid extraction and liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometry to the identification and determination of the predominant monomeric catechins, (+)-catechin and (-)-epicatechin, in a baking chocolate Standard Reference Material (NIST Standard Reference Material 2384). (+)-Catechin and (-)-epicatechin are detected and quantified in chocolate extracts on the basis of selected-ion monitoring of their protonated [M + H](+) molecular ions. Tryptophan methyl ester is used as an internal standard. The developed method has the capacity to accurately quantify as little as 0.1 microg/mL (0.01 mg of catechin/g of chocolate) of either catechin in chocolate extracts, and the method has additionally been used to certify (+)-catechin and (-)-epicatechin levels in the baking chocolate Standard Reference Material. This is the first reported use of liquid chromatography/mass spectrometry for the quantitative determination of monomeric catechins in chocolate and the only report certifying monomeric catechin levels in a food-based Standard Reference Material.

Standard Reference Material (SRM) Baking Chocolate was recently issued, and the process used for value assignment of nutrient concentrations is reported herein. SRM 2384 is intended for use as a primary control material for assigning values to in-house control materials and for validation of analytical methods for the measurement of fatty acids, proximates, vitamins, and elements in chocolate and similar high-fat matrices. The Certificate of Analysis for SRM 2384 provides assigned values for concentrations of fatty acids, proximates, vitamins, elements, and total dietary fiber, for which product labeling is required by the Nutrition Labeling and Education Act of 1990, as well as for catechins, caffeine, theobromine, and theophylline. These assigned values were based on measurements by NIST and/or collaborating laboratories.