Chronic treatment with zinc and antidepressants induces enhancement of presynaptic/extracellular zinc concentration in the rat prefrontal cortex

Zinc exhibits antidepressant-like activity in preclinical tests/models. Moreover, zinc homeostasis is implicated in the pathophysiology of affective disorders. The aim of the present study was to examine the effect of chronic zinc, citalopram and imipramine intraperitoneal administration on the presynaptic and extracellular zinc concentration in the rat prefrontal cortex and hippocampus. We used two methods: zinc–selenium histochemistry (which images the pool of presynaptic-vesicle zinc) and anodic stripping voltammetry (ASV) for zinc determination in microdialysate (which assays the extracellular zinc concentration). We report that chronic (14×) zinc (hydroaspartate, 10 and 65 mg/kg) and citalopram (20 mg/kg) administration increased the pool of presynaptic zinc (by 34, 50 and 37%, respectively) in the rat prefrontal cortex. The 21% increase induced by imipramine (20 mg/kg) was marginally significant. Likewise, zinc (hydroaspartate, 65 mg/kg), citalopram and imipramine increased the extracellular zinc (although with a different pattern: time point, area under the curve and/or basal level) in this brain region. Furthermore, zinc induced an increase in presynaptic (by 65%) and extracellular zinc (by 90%) in the hippocampus, while both citalopram and imipramine did not. These results indicate that all of the treatments increase presynaptic/extracellular zinc concentrations in the rat prefrontal cortex, which may then contribute to their antidepressant mechanisms. Alterations induced by zinc (but not antidepressants) administration in the hippocampus may be related to specific zinc mechanisms. All the data (previous and present) on the effect of antidepressant treatments on the presynaptic/extracellular zinc concentrations suggest the involvement of this biometal presynaptic/synaptic homeostasis in the antidepressant mechanism(s).     

<Superscript>1</Superscript>H NMR-based metabolic profiling and target analysis: a combined approach for the quality control of <Emphasis Type="Italic">Thymus vulgaris</Emphasis>

The characterization of T. vulgaris plant material for quality control purposes was performed by NMR-based methods. Direct extraction of 141 T. vulgaris samples with DMSO-d ₆ enabled the obtainment of crude extracts with a representative composition in terms of both volatile and non-volatile constituents. The acquisition of 600 MHz ¹H NMR spectra resulted in a dataset which was analyzed by a combination of metabolic profiling and target analysis approaches. Preliminary analysis of the ¹H NMR spectra was performed by principal component analysis, which revealed sample discrimination on a chemotype basis (thymol, carvacrol and linalool chemotypes). Further minor discriminative constituents were identified as p-cymene, γ-terpinene, rosmarinic acid, and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl. Metabolite identification was accomplished by 1D and 2D NMR techniques and supported by spiking experiments. Fast dereplication of constituents not available as reference compounds was performed by HPLC–SPE–NMR experiments. A targeted approach based on qHNMR was validated for quantification of the identified secondary metabolites. Validation was performed in terms of precision (intra-day RSD ≤ 4.51%, inter-day RSD ≤ 4.18%), repeatability (RSD ≤ 2.30%), accuracy (recovery rates within 93.4 and 103.4%), linearity (correlation coefficients ≥ 0.9990), robustness, and stability. The amount of the dominant monoterpene in thymol, carvacrol, and linalool chemotypes was respectively found to be within 0.4–2.6, 0.7–2.3, and 1.1–3.6% (w/w). Variable amounts of the precursors p-cymene and γ-terpinene were found in thymol and carvacrol chemotypes. The highest amount of rosmarinic acid and 3,4,3′,4′-tetrahydroxy-5,5′-diisopropyl-2,2′-dimethylbiphenyl in the analyzed samples was respectively 4.6 and 0.4% (w/w). Since quantification is performed on a weight basis, the essential oil content can be estimated based on the sum of the quantified monoterpenes. The NMR-based analysis of T. vulgaris represents a more comprehensive approach in comparison to traditional chromatographic methods such as GC and LC, respectively employed for the analysis of volatile and non-volatile constituents. Further advantages lie in the simple sample preparation, rapidity and reproducibility of the NMR analysis.     

Aflatoxin B<Subscript>1</Subscript> Contamination of Traditionally Processed Peanuts Butter for Human Consumption in Sudan

A survey was carried out to detect the presence of aflatoxin B₁ in 60 duplicated samples (120 samples) of peanuts butter purchased from the local markets and other traditionally prepared and distributed by the street sellers in Khartoum state, Sudan. AflaTest-P affinity column was used to extract the toxin from the samples, and the concentration was measured by calibrated Vicam fluorometer. Aflatoxin B₁ was detected at variable levels in 100% of the screened samples. Traditionally prepared samples showed the highest incidence of aflatoxin B₁ which is above the internationally regulated tolerance levels (5–20 ppb). The means and the ranges of the aflatoxin B₁ recovered were as follows: 63.9 ppb (29–128 ppb), 54.5 ppb (21–131 ppb) and 101 ppb (17–170 ppb) for samples collected from Khartoum, Khartoum North and Omdurman areas, respectively. Samples from retail stores presented relatively low aflatoxin B₁ incidences 14.5 ppb (1–57 ppb), but only 30% of the samples revealed aflatoxin level below 10 ppb. Laboratory segregated and carefully prepared butter from good grade nuts showed the lowest levels of this toxin (3.3 ppb; 2–6 ppb). The results showed that peanuts butter prepared by the street sellers and distributed by the retail stores are evidently hazardous to human health. There is therefore urgent need for strong form of quality control measures and public awareness. The use of excellent grade peanuts and care during processing and storage are priority.     

Competitive binding of Fe<Superscript>3+</Superscript>, Cr<Superscript>3+</Superscript>, and Ni<Superscript>2+</Superscript> to transferrin

Competitive binding of Fe³⁺, Cr³⁺, and Ni²⁺ to transferrin (Tf) was investigated at various physiological iron to Tf concentration ratios. Loading percentages for these metal ions are based on a two M ⁿ⁺ to one Tf (i.e., 100% loading) stoichiometry and were determined using a particle beam/hollow cathode–optical emission spectroscopy (PB/HC-OES) method. Serum iron concentrations typically found in normal, iron-deficient, iron-deficient from chronic disease, iron-deficient from inflammation, and iron-overload conditions were used to determine the effects of iron concentration on iron loading into Tf. The PB/HC-OES method allows the monitoring of metal ions in competition with Fe³⁺ for Tf binding. Iron-overload concentrations impeded the ability of chromium (15.0 μM) or nickel (10.3 μM) to load completely into Tf. Low Fe³⁺ uptake by Tf under iron-deficient or chronic disease iron concentrations limited Ni²⁺ loading into Tf. Competitive binding kinetic studies were performed with Fe³⁺, Cr³⁺, and Ni²⁺ to determine percentages of metal ion uptake into Tf as a function of time. The initial rates of Fe³⁺ loading increased in the presence of nickel or chromium, with maximal Fe³⁺ loading into Tf in all cases reaching approximately 24%. Addition of Cr³⁺ to 50% preloaded Fe³⁺–Tf showed that excess chromium (15.0 μM) displaced roughly 13% of Fe³⁺ from Tf, resulting in 7.6 ± 1.3% Cr³⁺ loading of Tf. The PB/HC-OES method provides the ability to monitor multiple metal ions competing for Tf binding and will help to understand metal competition for Tf binding.     

Fluctuation in recoverable nickel and zinc resistant copiotrophic bacteria explained by the varying zinc ion content of Torsa River in different months

Heavy metal content analysis of River Torsa in India did not indicate any alarming level of toxicity for human consumption but revealed variation at the ppb level in different months. The variation in recoverable nickel and zinc resistant copiotrophic (or eutrophic) bacterial counts was explained by the variation of the zinc content (34.0–691.3 ppb) of the river water in different sampling months. Growth studies conducted with some purified nickel and/or zinc resistant strains revealed that pre-exposure of the cells to ppb levels of Zn²⁺, comparable to the indigenous zinc ion concentration of the river, could induce the nickel or zinc resistance. A minimum concentration of 5–10 μM Zn²⁺ (325–650 ppb) was found effective in inducing the Nickel resistance of the isolates. Zinc resistance of the isolates was tested by pre-exposing the cells to 4 μM Zn²⁺ (260 ppb). The lag phase was reduced by 6–8 h in all the cases. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicated that some of the Torsa River isolates, having inducible nickel and zinc resistance, are members of the genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Serratia and Moraxella.     

T-2 and HT-2 toxin analysis in cereals and cereal products following IAC cleanup and determination via GC-ECD after derivatization

The authors present a new and sensitive method for the determination of T-2- und HT-2 Toxin in cereals and cereal products in the low ppb level. A representative part of the cereal sample is extracted with a mixture of methanol-water (90:10) and the extract is cleaned on the commercially available immunoaffinity column T-2test™ (IAC), eluted with methanol, derivatized by pentafluorpropionic anhydride (PFPA) and measured on a GC-ECD. The method has been successfully validated on wheat, rye and oats. The recovery rates with wheat and rye endowed on a level of 50 ppb and with 85 ppb naturally contaminated oats were 71–115% with a coefficient of variation of 5.7–19.5%. The detection limits of the method with a signal to noise level of 3:1 were 1.5–2.3 μg/kg for HT-2 and 1.1–1.7 μg/kg for T-2 toxin. Financial support: Federal Ministry of Food and Agriculture (part of the project 05HS 001 — Improvement and validation of type A trichothecene (T-2 toxin and HT-2 toxin) analysis and occurrence of these mycotoxins in food marketed in Germany)     

Method validation for the determination of ochratoxin A in green and soluble coffee by immunoaffinity column cleanup and liquid chromatography

A method was validated for the determination of ochratoxin A (OTA) in soluble and green coffee. Performance parameters evaluated included selectivity, accuracy, intermediate precision, linearity, limit of detection, limit of quantitation, and ruggedness. The method was found to be selective for OTA in both matrices tested. Recovery rates from soluble coffee samples ranged from 73.5 to 91.2%, and from green coffee samples from 68.7 to 84.5%. The intermediate precision (RSDr) was between 9.1 and 9.4% for soluble coffee and between 14.3 and 15.5% for green coffee analysis. The linearity of the standard calibration curve (r²) was <0.999 for OTA levels of 1.0–20.0 μg/kg in coffee samples. The limit of detection was determined to be 0.01 ng of OTA on column, while the limit of quantitation was found to be 0.03 ng on column. The limit of quantitation is equivalent to 0.6 μg/kg in soluble coffee samples and 0.3 μg/kg in green coffee samples. The results of the ruggedness trial showed two factors are critical for soluble coffee analysis: the extraction method, and the flow rate of the mobile phase. For green coffee analysis two critical factors detected were the extraction method and the storage temperature of the immunoaffinity column. Five samples of soluble coffee and 42 of green coffee were analysed using the validated method. All soluble coffee samples contained OTA at levels that ranged from 8.4 to 13.9 μg/kg. Six of the 42 green coffee samples analysed (14.3%) contained OTA at levels ranging from 0.9 to 19.4 μg/kg. The validated method can be used to monitor OTA levels in Colombian coffee for export or for local consumption.     

4D prediction of protein <Superscript>1</Superscript>H chemical shifts

A 4D approach for protein ¹H chemical shift prediction was explored. The 4th dimension is the molecular flexibility, mapped using molecular dynamics simulations. The chemical shifts were predicted with a principal component model based on atom coordinates from a database of 40 protein structures. When compared to the corresponding non-dynamic (3D) model, the 4th dimension improved prediction by 6–7%. The prediction method achieved RMS errors of 0.29 and 0.50 ppm for Hα and HN shifts, respectively. However, for individual proteins the RMS errors were 0.17–0.34 and 0.34–0.65 ppm for the Hα and HN shifts, respectively. X-ray structures gave better predictions than the corresponding NMR structures, indicating that chemical shifts contain invaluable information about local structures. The ¹H chemical shift prediction tool 4DSPOT is available from .     

Application of Anodic Stripping Voltammetry for Zinc, Copper, and Cadmium Quantification in the Aqueous Humor: Implications of Pseudoexfoliation Syndrome

Anodic stripping voltammetric (ASV) procedure, using mercury film electrode, was optimized and applied to determine the concentrations of zinc, cadmium, and copper in the aqueous humor. Concentration levels as low as 1 ppb of the test metals was possible to be detected using short electrolysis times (120 s) and microquantities of aqueous humor (up to 35 μL). As a first application of the voltammetric analysis of trace metals in the aqueous humor, the role of the three selected trace elements in the pseudoexfoliation (PEX) syndrome was examined. Samples from aqueous humor were collected during cataract extraction from patients with and without PEX. The zinc and copper concentration levels in the aqueous humor did not show statistically significant difference in the study and control group. Cadmium was detected in a small number of samples, without however statistical differences between the two groups. ASV proved to be a highly precise and sensitive tool for the quantification of heavy metal ions in aqueous humor. Further studies may lead to useful conclusions for the role of zinc, copper, or cadmium in PEX syndrome.     

Sensitive and rapid HPLC quantification of tenofovir from hyaluronic acid-based nanomedicine

The purpose of this study was to develop and validate a rapid, sensitive, and specific reversed-phase high-performance liquid chromatography method for the quantitative determination of native tenofovir (TNF) for various applications. Different analytical performance parameters such as linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), and robustness were determined according to International Conference on Harmonization (ICH) guidelines. A Bridge™ C18 column (150 × 4.6 mm, 5 μm) was used as stationary phase. The retention time of TNF was 1.54 ± 0.03 min (n = 6). The assay was linear over the concentration range of 0.1–10 μg/mL. The proposed method was sensitive with LOD and LOQ values equal to 50 and 100 ng/mL, respectively. The method was accurate with percent mean recovery from 95.41% to 102.90% and precise as percent RSD (relative standard deviation) values for intra-day, and inter-day precision were less than 2%. This method was utilized for the estimation of molar absorptivity of TNF at 259 nm (ε ₂₅₉ = 12,518 L/mol/cm), calculated from linear regression analysis. The method was applied for determination of percentage of encapsulation efficiency ( 22.93 ± 0.04%), drug loading (12.25 ± 1.03%), in vitro drug release profile in the presence of enzyme (43% release in the first 3 h) and purification analysis of hyaluronic acid-based nanomedicine.     

Chiral separation of aliphatic primary amino alcohols as o‐phthaldialdehyde/mercaptoethanol derivatives on polysaccharide‐based chiral stationary phases

A sensitive chiral high performance liquid chromatography (HPLC) method for the determination of aliphatic primary amino alcohol isomers with o‐phthaldialdehyde/mercaptoethanol precolumn derivatization has been developed and validated. Seven chiral columns were tested in a reversed phase mode. Excellent enantioseparation with the resolution more than 2.0 was achieved on Chiralcel OJ‐3R. The effect of various chromatographic conditions including column temperature, acetonitrile content in the mobile phase, buffer pH, buffer concentration, and buffer type in the mobile phase on the retention and the selectivity was investigated. The final mobile phase consisted of binary mixture of 20mM ammonium formate solution with acetonitrile (75:25; v/v). The analyses were performed at mobile phase flow rate of 1.0 mL/min and the column temperature of 40°C. The fluorescence detection was performed at excitation wavelength of 345 nm and emission wavelength of 450 nm. The developed method was fully validated in terms of linearity, sensitivity, accuracy, precision, intermediate precision, and selectivity according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines using internal normalization procedure. The proposed chiral method was proved to be highly sensitive, simple, and rapid and was successfully applied to the determination of D‐Valinol content in commercially available samples of L‐Valinol.     

Spectrofluorimetric Method for the Determination of Doxepin Hydrochloride in Commercial Dosage Forms

A novel spectrofluorimetric method has been developed for the determination of doxepin hydrochloride in commercial dosage forms. The method is based on the fluorescent ion pair complex formation of the drug with eosin Y in the presence of sodium acetate–acetic acid buffer solution of pH 4.52 which is extractable in dichloromethane. The extracted complex showed fluorescence intensity at λ_em = 567 nm after excitation at 464 nm. The calibration curve was linear over the working range of 0.1–0.8 μg ml⁻¹. Under the optimized experimental conditions, present method is validated as per International Conference on Harmonization guidelines. The limit of detection for the developed method is 2.95 ng ml⁻¹. The method has been successfully applied to the determination of doxepin hydrochloride in commercial dosage forms. The results are compared with the reference spectrofluorimetric method.     

Resonance Scattering Spectral Detection of Trace Pb<Superscript>2+</Superscript> Using Aptamer-Modified AuPd Nanoalloy as Probe

The resonance scattering spectral probe for Pb²⁺ was obtained using aptamer-modified AuPd Nanoalloy. In the pH 7.0 Na₂HPO₄–NaH₂PO₄ buffer solution, the aptamer interacted with AuPd nanoalloy particles to form stable aptamer-AuPd nanoalloy probe for Pb²⁺ that is stable in high concentration of salt. The probe combined with Pb²⁺ ions to form a G-quadruplex and to release AuPd nanoalloy particles that aggregate to form big particles which led the resonance scattering (RS) intensity enhancing. The reaction solution was filtered by 0.15 μm membrane to obtain the filtration containing aptamer-AuPd nanoalloy probe that has strong catalytic effect on the electrodeless nickel particle plating reaction between Ni(II) and PO₂³⁻ that exhibited a strong RS peak at 508 nm. The RS intensity at 508 nm decreased when the Pb²⁺ concentration increased. The decreased intensity (ΔI _508nm) is linear to the concentration of 0.08–42 nM Pb²⁺, with regress equation of DI_508nm = 16.3 c + 1.5 \Delta {I_{{5}0{\rm{8nm}}}} = {16}.{3}\,c + {1}.{5} , correlation coefficient of 0.9965, and detection limit of 0.04 nM Pb²⁺. The RS assay was applied to the analysis of Pb²⁺ in wastewater, with satisfactory results.     

Thermally induced denaturation and aggregation of BLG-A: effect of the Cu<Superscript>2+</Superscript> and Zn<Superscript>2+</Superscript> metal ions

There is growing evidence that metal ions can accelerate the aggregation process of several proteins. This process, associated with several neuro-degenerative diseases, has been reported also for non-pathological proteins. In the present work, the effects of copper and zinc ions on the denaturation and aggregation processes of β-lactoglobulin A (BLG-A) are investigated by differential scanning calorimetry (DSC), fluorescence, electron paramagnetic resonance (EPR) and optical density. The DSC profiles reveal that the thermal behaviour of BLG-A is a complex process, strongly dependent on the protein concentration. For concentrations ≤0.13 mM, the thermogram shows an endothermic peak at 84.3°C, corresponding to denaturation; for concentrations >0.13 mM an exothermic peak also appears, above 90°C, related to the aggregation of the denaturated BLG-A molecules. The thioflavin T fluorescence indicates that the thermally induced aggregates show fibrillar features. The presence of either equimolar Cu²⁺ or Zn²⁺ ions in the protein solution has different effects. In particular, copper binds to the protein in the native state, as evidenced by EPR experiments, and destabilizes BLG-A by decreasing the denaturation temperature by about 10°C, whereas zinc ions probably perturb the partially denaturated state of the protein. The kinetics of BLG-A aggregation shows that both metal ions abolish the lag phase before the aggregation starts. Moreover, the rate of the process is 4.6-fold higher in the presence of copper, whereas the effect of zinc is negligible. The increase of the aggregation rate, induced by copper, may be due to a site-specific binding of the metal ion on the protein.     

Determination of selenium in duplicate diets of residents of Pinhel,Portugal, by neutron activation

A rapid cyclic instrumental neutron activation analysis (CINAA) method has been used to determine the selenium content of 27 duplicate diet samples from each of the 27 districts surrounding Pinhel, Portugal. The accuracy and precision of the CINAA method have been evaluated by analyzing certified reference materials and observed to be within ±5–10% for samples containing at least 40 ppb of selenium. The detection limit has been found to vary between 26–42 ppb selenium depending on the sample composition. The average daily dietary intake has been calculated as 37 μg of selenium per day.     

Statistical analysis of structural characteristics of protein Ca<Superscript>2+</Superscript>-binding sites

To better understand the biological significance of Ca²⁺, we report a comprehensive statistical analysis of calcium-binding proteins from the Protein Data Bank to identify structural parameters associated with EF-hand and non-EF-hand Ca²⁺-binding sites. Comparatively, non-EF-hand sites utilize lower coordination numbers (6 ± 2 vs. 7 ± 1), fewer protein ligands (4 ± 2 vs. 6 ± 1), and more water ligands (2 ± 2 vs. 1 ± 0) than EF-hand sites. The orders of ligand preference for non-EF-hand and EF-hand sites, respectively, were H₂O (33.1%) > side-chain Asp (24.5%) > main-chain carbonyl (23.9%) > side-chain Glu (10.4%), and side-chain Asp (29.7%) > side-chain Glu (26.6%) > main-chain carbonyl (21.4%) > H₂O (13.3%). Less formal negative charge was observed in the non-EF-hand than in the EF-hand binding sites (1 ± 1 vs. 3 ± 1). Additionally, over 20% of non-EF-hand sites had formal charge values of zero due to increased utilization of water and carbonyl oxygen ligands. Moreover, the EF-hand sites presented a narrower range of ligand distances and bond angles than non-EF-hand sites, possibly owing to the highly conserved helix–loop–helix motif. Significant differences between ligand types (carbonyl, side chain, bidentate) demonstrated that angles associated with each type must be classified separately, and the EF-hand side-chain Ca–O–C angles exhibited an unusual bimodal quality consistent with an Asp distribution that differed from the Gaussian model observed for non-EF-hand proteins. The results of this survey more accurately describe differences between EF-hand and non-EF-hand proteins and provide new parameters for the prediction and design of different classes of Ca²⁺-binding proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Michael Kirberger, Xue Wang, and Hai Deng contributed equally to this article.     

Fluorescent Protein-Based Optical Biosensor for Copper Ion Quantitation

In the present study, spectroscopic determinations of copper ions using chimeric metal-binding green fluorescent protein (His6GFP) as an active indicator have been explored. Supplementation of copper ions to the GFP solution led to a remarkable decrease of fluorescent intensity corresponding to metal concentrations. For circumstances, rapid declining of fluorescence up to 60% was detected in the presence of 500 μM copper. This is in contrast to those observed in the case of zinc and calcium ions, in which approximately 10–20% of fluorescence was affected. Recovery of its original fluorescence up to 80% was mediated by the addition of ethylenediamine tetraacetic acid. More importantly, in the presence of metal ions, the emission wavelength maximum remains unchanged while reduction of the optical density of the absorption spectrum has been observed. This indicates that the chromophore’s ground state was possibly affected by the static quenching process. Results from circular dichroism measurements revealed that the overall patterns of circular dichroism spectra after exposure to copper ions were not significantly different from that of the control, where the majority of sharp positive band around 195–196 nm in combination with a broad negative deflection around 215–216 nm was obtained. Taken together, it can be presumed that copper ions exerted their static quenching on the fluorescence rather than structural or conformational alteration. However, notification has to be made that some peptide rearrangements may also occur in the presence of metal ions. Further studies were conducted to investigate the feasibility of using the His6GFP as a sensing unit for copper ions. The His6GFP was encapsulated in Sol-gel and immobilized onto the optical fiber connected with a fluorescence detecting device. The Sol-gel was doped into the metal solution where the quenching of fluorescence could be monitored in real time. The sensing unit provided a high sensitivity of detection in the range of 0.5 μM to 50 mM with high selectivity for copper ions. All these findings open up a high potential to apply the fluorescent protein-based bioanalytical tool for copper determination in the future.     

Deuterium isotope effects on <Superscript>15</Superscript>N backbone chemical shifts in proteins

Quantum mechanical calculations are presented that predict that one-bond deuterium isotope effects on the ¹⁵N chemical shift of backbone amides of proteins, ¹Δ¹⁵N(D), are sensitive to backbone conformation and hydrogen bonding. A quantitative empirical model for ¹Δ¹⁵N(D) including the backbone dihedral angles, Φ and Ψ, and the hydrogen bonding geometry is presented for glycine and amino acid residues with aliphatic side chains. The effect of hydrogen bonding is rationalized in part as an electric-field effect on the first derivative of the nuclear shielding with respect to N–H bond length. Another contributing factor is the effect of increased anharmonicity of the N–H stretching vibrational state upon hydrogen bonding, which results in an altered N–H/N–D equilibrium bond length ratio. The N–H stretching anharmonicity contribution falls off with the cosine of the N–H···O bond angle. For residues with uncharged side chains a very good prediction of isotope effects can be made. Thus, for proteins with known secondary structures, ¹Δ¹⁵N(D) can provide insights into hydrogen bonding geometries. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Determination of melamine and cyanuric acid in human urine by a liquid chromatography tandem mass spectrometry

Melamine was found to be the etiological factor for the urinary stones epidemic in infants and young children in China in 2008. Urine level of melamine and its analog cyanuric acid may be useful markers for the evaluation of toxic effects. Liquid chromatography tandem mass spectrometry methods for the individual determination of melamine and cyanuric acid in human urine are described. Using isotope labeled internal standards during liquid–liquid extraction, the method was fully validated by verifying specificity, linearity, LLOQ, intra- and inter-assay precision and accuracy, matrix effect, recovery and stability. Calibration curves with good linearity (r = 0.9999) over the concentration range from 10 to 5000 ng/ml, intra-assay precision <10% and inter-assay precision <15%, accuracy between 93.0 and 111.6% were obtained with multiple reaction monitoring mode for melamine and cyanuric acid in human urine. The methods were successfully applied to the analysis of urine samples collected from 86 infants and 110 adults.     

Liquid seaweed extracts identified using <Superscript>1</Superscript>H NMR profiles

Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The increasingly regulated international trade in such products requires that they be standardized and defined to a degree not previously required. We examined commercially available extracts using quantitative ¹H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products examined. In addition to recognizing extracts made from different seaweeds, analysis of the ¹H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different manufacturers. This result established that the process used to make an extract is an important variable in defining its composition. A comparison of the ¹H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble extracts when all data are pooled. Quantitative analysis using ¹H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at least an order of magnitude (2.5–33 mg/mL; R ² > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard. Issued as NRCC no. 42,652.