Synthesis and characterization of a molecularly imprinted polymer and its application as SPE enrichment sorbent for determination of trace methimazole in pig samples using HPLC-UV

A novel molecularly imprinted polymer that could be applied as enrichment sorbent was prepared using methimazole (MMZ) as the template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker. Though evaluated by static, kinetic and competitive adsorption tests, the polymer exhibited high adsorption capacity, fast kinetics and good selective ability. A method for determination of trace MMZ was developed using this polymer as enrichment sorbent coupled with high performance liquid chromatography focusing on complex biological matrices. Under the optimum experimental conditions, the MMZ standard is linear within the concentration range studied, that is, from 0.5 μg L⁻¹ to 150 μg L⁻¹ (r² = 0.9941). Lower limits of detection (LOD, at S/N = 3) and quantification (LOQ, at S/N = 10) in pig samples were 0.63 μg kg⁻¹ and 2.10 μg kg⁻¹ for kidney, 0.51 μg kg⁻¹ and 1.70 μg kg⁻¹ for liver, 0.56 μg kg⁻¹ and 1.86 μg kg⁻¹ for muscle, respectively. Recoveries and relative standard deviation (RSD, n = 9) values for precision in the developed method were from 71.14% to 88.41% and from 2.53% to 6.18%.     

Microextraction in packed sorbent for analysis of antidepressants in human plasma by liquid chromatography and spectrophotometric detection

A sensitive and reproducible method by microextraction packed sorbent and liquid chromatography with UV detection (MEPS/LC–UV) is described for the determination of new generation antidepressants (sertraline, mirtazapine, fluoxetine, citalopram and paroxetine) in human plasma samples. The MEPS variables, such as sample volume, pH, number of extraction cycles (draw–eject), and desorption conditions (solvent and solvent volume of elution) influenced the MEPS/LC efficiency significantly. Important factors in the optimization of MEPS efficiency, as well as washing steps and carryover effect are discussed. The analyses were carried out using small sample volumes (400 μL), and in a short time period (3 min for the entire sample preparation step). The MEPS/LC–UV method was shown to be linear at concentrations ranging from the limit of quantification (LOQ) to 1000 ng mL⁻¹. The LOQ values ranged from 10 to 25 ng mL⁻¹. The inter-day precision of the method presented coefficient of the variation ranging from 1.3% to 8.7%. On the basis of analytical validation, it is shown that the MEPS/LC–UV methodology is adequate for antidepressant analysis, from therapeutic to toxic levels. In order to evaluate the proposed method for clinical use, the MEPS/LC–UV method was applied to analysis of plasma samples from elderly depressed patients.     

Efficiencies and costs of larval growth in different food environments (Asteroidea: Asterina miniata)

The ocean is a nutritionally heterogeneous environment. For feeding larval forms, food variability has significant consequences for growth and later recruitment success. In this study, the physiological and biochemical responses to a range of different food concentrations (unfed, 4, 20, and 40 algal cells μl^− 1) were examined in larvae of the asteroid, Asterina miniata. Measurements of growth, protein synthesis rates, and the energetic cost of protein synthesis were made. Under conditions of rapid growth, protein comprised a larger percent (66%) of a larva's organic biomass compared to similar-aged, slower-growing larvae (26%). Larvae fed at the highest food concentration tested (40 algal cells μl^− 1) had a protein depositional efficiency of 80% (± 16%), a value 3-fold higher than larvae fed 20 algal cells μl^− 1 (28% ± 11%). Also, faster-growing larvae required 3-fold less energy per unit mass of protein growth. Larvae fed 40 algal cells μl^− 1 deposited protein at a respiratory cost of 65 ± 11 pmol O₂ h^− 1 (μg protein)^− 1; larvae fed 20 algal cells μl^− 1 had a cost of 192 ± 47 pmol O₂ h^− 1 (μg protein)^− 1. While there were differences in the cost to deposit protein (i.e., protein growth, the balance of synthesis and degradation), there were no differences in the energetic cost of protein synthesis for all food concentrations tested. The energetic cost of protein synthesis was fixed at 13.8 (± 0.92) Joules (mg protein synthesized)^− 1 and was independent of developmental stage, growth rates, and large changes (58-fold) in protein synthesis rates. A major conclusion from this study is that larvae grown in high-food environments not only grew faster, but did so for considerably less energy. Defining the complex relationships of food availability and metabolic efficiency will provide more accurate predictions of larval growth under variable food conditions in the ocean.     

Denitrifying sulfide removal and carbon methanogenesis in a mesophilic, methanogenic culture

The inhibitory effects of 90-189 mg l⁻¹ of sulfide and 25-75 mg-N l⁻¹ of nitrate on methanogenesis were investigated in a mixed methanogenic culture using butyrate as carbon source. In the initial phase of 90 mg l⁻¹ S²⁻ test, autotrophic denitrification of nitrate occurred with sulfide as the electron donor. Then the sulfate-reducing strains converted the produced sulfur back to sulfide via heterotrophic oxidation pathway. Methanogenesis was not markedly inhibited when 90 mg l⁻¹ of sulfide was dosed alone. When 25-75 mg-N l⁻¹ of nitrate was presented, initiation of methanogenesis was seriously delayed. Nitrogen oxides (NO_x), the intermediates for nitrate reduction via denitrification pathway, inhibited methanogenesis. The 90 mg l⁻¹ of sulfide favored heterotrophic dissimilatory nitrate reduction to ammonia (DNRA) pathway for nitrate reduction. Possible ways of maximizing methane production from an organic carbon-rich wastewater with high levels of sulfide and nitrate were discussed.     

A chemiluminescence method to detect malondialdehyde in plasma and urine

A highly sensitive and convenient method to detect malondialdehyde (MDA) in specimens of plasma and urine was developed using high-performance liquid chromatography combining chemiluminescence detection. MDA was separated through a reverse-phase C18 column at a flow rate of 0.8 ml min⁻¹. It was then detected by a chemiluminescence detector. Variables that affected chemiluminescence reaction, including mobile phase, flow rate, chemiluminescence reagent, parameters of the photomultiplier tube, and temperature, were studied. This assay was linear from 0.50 to 50 μmol L⁻¹, with limits of detection and quantification of 0.08 and 0.30 μmol L⁻¹, respectively. The recoveries were in the range of 92.2 to 98.5%, and the intra- and interday reproducibilities were obtained with relative standard deviations of less than 4% and less than 6%, respectively. MDA in plasma and urine was stable for 12 h at 4 °C. MDA levels were 2.74, 3.19, and 3.20 μmol L⁻¹ for plasma and 1.25, 2.04, and 1.58 μmol L⁻¹ for urine, respectively. The proposed method is simple, rapid, and sensitive with low cost. Taken together, this method can be suitable for detecting MDA in tissue and feed specimens.     

Selectivity and grazing impact of microzooplankton on phytoplankton in two subtropical semi-enclosed bays with different chlorophyll concentrations

Microzooplankton grazing rates were compared between two sites (S1 and S2) in the coastal seas of eastern Hong Kong with similar physio-chemical parameters, but different chlorophyll concentrations. During the period from March 2007 to January 2008, six sets of dilution experiments, combined with high performance liquid chromatography and phytoplankton size fractionation (< 200 μm, < 20 μm and < 5 μm), were carried out to study the microzooplankton grazing rate on phytoplankton of different taxonomic groups and sizes. Although total chlorophyll a concentrations were much higher in S1 (4.98-18.42 μg l^− 1) than in S2 (0.29-1.68 μg l^− 1), size composition of phytoplankton was relatively similar between the two sites. Measured as chlorophyll a, phytoplankton growth rates (− 0.84-1.91 d^− 1 in S1; 0.03-2.85 d^− 1 in S2) and microzooplankton grazing rates (0.00-2.26 d^− 1 in S1; 0.00-1.49 d^− 1 in S2) for all three size fractions were similar between the two bays. Phytoplankton growth rates and microzooplankton grazing rates measured as other pigments for phytoplankton of different size fractions did not show strong variations. Microzooplankton grazing impact, expressed as the ratio of microzooplankton grazing rate to phytoplankton growth rate, was generally higher in S1 than in S2, although the difference was not statistically significant. High microzooplankton grazing impact on alloxanthin (1.00-45.85) suggested strong selection toward cryptophytes. Our results provided no evidence for size selective grazing on phytoplankton by microzooplankton.     

Hollow fiber-based liquid phase microextraction with factorial design optimization and gas chromatography–tandem mass spectrometry for determination of cannabinoids in human hair

A new method, based on hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography–tandem mass spectrometry (GC–MSMS), was developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in samples of human hair. Since hair is a solid matrix, the samples were subjected to alkaline digestion using NaOH. The aqueous solutions obtained were extracted using a 6 cm polypropylene fiber (600 μm i.d., 200 μm wall thickness, 0.2 μm pore size) for each extraction. A 2⁵⁻¹ fractional factorial design for screening, and a central composite design for optimization of significant variables, was applied during development of the extraction method. The variables evaluated were the type of extraction solvent, pH, stirring speed, extraction time, and acceptor phase volume. The optimized conditions for the proposed extraction procedure were 10 mg of hair sample; 20 μL of butyl acetate; aqueous (pH 14) donor phase containing 6.8% NaCl; 600 rpm stirring speed; 20 min extraction time. A linear response was obtained in the ranges 1–500 pg mg⁻¹ (CBD and CBN) and 20–500 pg mg⁻¹ (THC), with regression coefficients >0.99. Precision, determined as the relative standard deviation, was 3.3–8.9% (intra-day) and 4.4–13.7% (inter-day). Absolute recoveries varied in the ranges 4.4–4.8% (CBD), 7.6–8.9% (THC) and 7.7–8.2% (CBN). Limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were 0.5–15 pg mg⁻¹ and 1–20 pg mg⁻¹, respectively. The method was successfully used to determine CBD, THC and CBN in hair samples from patients in a drug dependency rehabilitation center. Concentrations varied in the ranges 1–18 pg mg⁻¹ (CBD), 20–232 pg mg⁻¹ (THC) and 9–107 pg mg⁻¹ (CBN), confirming the suitability of the method for monitoring studies.     

Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations

Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l⁻¹, 3 g yeast extract l⁻¹ and 5 g peptone l⁻¹ gave the maximum ethanol production efficiency with concentration, productivity and yield of 120.68 ± 0.54 g l⁻¹, 2.01 ± 0.01 g l⁻¹ h⁻¹ and 0.51 ± 0.00 g g⁻¹, respectively. When sugarcane molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34 ± 0.78 g l⁻¹, 1.52 ± 0.01 g l⁻¹ h⁻¹ and 0.45 ± 0.01 g g⁻¹, respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol concentration and yield for approximately 14% when compared to those of the unsupplemented juices.     

Two-stage anaerobic digestion of biodegradable municipal solid waste using a rotating drum mesh filter bioreactor and anaerobic filter

A rotating drum mesh filter bioreactor (RDMFBR) with a 100 μm mesh coupled to an anaerobic filter was used for the anaerobic digestion of biodegradable municipal solid waste (BMW). Duplicate systems were operated for 72 days at an organic loading rate (OLR) of 7.5 gVS l⁻¹ d⁻¹. Early in the experiment most of the methane was produced in the 2nd stage. This situation gradually reversed as methanogenesis became established in the 1st stage digester, which eventually produced 86–87% of the total system methane. The total methane production was 0.2 l g⁻¹ VS_added with 60–62% volatile solids destruction. No fouling was experienced during the experiment at a transmembrane flux rate of 3.5 l m⁻² h⁻¹. The system proved to be robust and stably adjusted to a shock loading increase to 15 gVS l⁻¹ d⁻¹, although this reduced the overall methane production to 0.15 l g⁻¹ VS_added.     

A liquid chromatography method for quantifying caffeine dissolution from pharmaceutical formulations into colloidal,fat-rich media

A simple and rapid high-performance liquid-chromatography method is presented that permits quantification of caffeine in colloidal fat emulsions proposed as new ‘biorelevant’ dissolution media (Intralipid™ and various milks). Using a mobile phase of 0.1 M sodium acetate (pH 4.0) and acetonitrile (89.5:10.5, v/v) at 1 ml min⁻¹, the drug and internal standard (7-β-hydroxyethyltheophylline) were eluted within 8 min. Caffeine extraction was undertaken by protein precipitation in ice-cold 12% (w/v) trichloroacetic acid and centrifugation at 10,000 rpm for 15 min. This simple extraction method generated caffeine recovery values (corrected for % fat content) of 75.4 ± 1.4–100.6 ± 5.5%. The limit of detection was within the range 0.25–0.4 μg ml⁻¹ and linearity was demonstrated in each medium up to 125 μg ml⁻¹. Precision was <11.5% RSD and intra- and inter-day accuracy was 93.4–109.3%. The validated method was applied to in vitro USP dissolution tests in milk which compared the kinetics of caffeine release from (i) extended release matrices containing hydroxypropyl methylcellulose (HPMC) and (ii) an immediate release commercial analgesic tablet. Good reproducibility was obtained in both extended and immediate release dissolution tests. The method provides high-throughput quantification of this common drug in fat emulsions used as biorelevant dissolution media.     

Growth, reproductive and photosynthetic responses to copper in the yellow-horned poppy, Glaucium flavum Crantz.

Glaucium flavum Crantz. is found in an anthropized coastal grassland at the joint estuary of the Tinto and Odiel rivers (SW Spain), growing under the influence of high levels of copper contamination derived from nearby petrochemical industries, with no obvious adverse affects on the performance of the plant. In addition, this species exhibits a series of ecological characteristics which may render it appropriate for use in the phytoremediation of contaminated areas. Nonetheless, the response of G. flavum to elevated copper concentrations has not been studied. A greenhouse experiment was conducted to investigate the effects of a range of Cu concentrations (0 to 47 mmol l⁻¹) on the growth, reproduction and photosynthetic performance of G. flavum, by measuring relative growth rate, fruit and seed production, chlorophyll fluorescence parameters, gas exchange and photosynthetic pigment concentrations. We also determined total copper, nitrogen, phosphorous, sulphur, calcium and magnesium concentrations. G. flavum survived with concentrations of up to 730 mg Cu kg⁻¹ DW in the leaves, when treated with 30 mmol Cu l⁻¹ (2000 mg l⁻¹). Quantum efficiency of PSII, net photosynthesis rate, as well as leaf Ca and Mg concentrations were all negatively affected by Cu concentrations greater than 9 mmol l⁻¹ in the nutrient solution. Our results indicate that the reduction in photosynthetic performance may be attributed to the adverse effect of excess Cu on the photosynthetic apparatus of the plant, both directly, via a decrease in pigment concentrations, and indirectly, via interference of Cu with Ca ions of PSII. Growth and seed production were only slightly affected by leaf tissue concentrations as high as 230 mg Cu kg⁻¹ dry mass, which suggests that this species could play an important role in phytoremediation of Cu-contaminated soils.     

Synergistic production of L-arabinose from arabinan by the combined use of thermostable endo- and exo-arabinanases from Caldicellulosiruptor saccharolyticus

The optimum conditions for the production of l-arabinose from debranched arabinan were determined to be pH 6.5, 75 °C, 20 g l⁻¹ debranched arabinan, 42 U ml⁻¹ endo-1,5-α-l-arabinanase, and 14 U ml⁻¹ α-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus and the conditions for sugar beet arabinan were pH 6.0, 75 °C, 20 g l⁻¹ sugar beet arabinan, 3 U ml⁻¹ endo-1,5-α-l-arabinanase, and 24 U ml⁻¹ α-l-arabinofuranosidase. Under the optimum conditions, 16 g l⁻¹l-arabinose was obtained from 20 g l⁻¹ debranched arabinan or sugar beet arabinan after 120 min, with a hydrolysis yield of 80% and a productivity of 8 g l⁻¹ h⁻¹. This is the first reported trial for the production of l-arabinose from the hemicellulose arabinan by the combined use of endo- and exo-arabinanases.     

Lead stress in seedlings of Avicennia marina, a common mangrove species in South China, with and without cotyledons

The effects of lead (Pb; 0-1000 mg L⁻¹) stress on the growth and biochemical responses of seedlings of Avicennia marina were examined, with and without cotyledons. After 50 days exposure to Pb, the growth of A. marina was not affected at low concentrations (0-50 mg L⁻¹ Pb). Roots tolerated to high Pb concentrations, with a significant reduction in biomass only at 1000 mg L⁻¹ Pb. In leaves and stems, 500 mg L⁻¹ Pb already caused a significant decline in biomass (0.6-fold). Accumulation of Pb occurred mainly in roots, with some accumulation in cotyledons but very little in leaves. Pb concentrations in both roots and cotyledons were proportional to the Pb levels in the substrate (y = 25.945x − 4281, r² = 0.67, P = 0.001 for roots, and y = 0.249x + 45.636, r² = 0.879, P < 0.001 for leaves). In treatments with 500 and 1000 mg L⁻¹ Pb, nitrogen concentrations in cotyledons were higher, while the carbon to nitrogen ratios were significantly lower than in the control without Pb. The Pb levels had significant positive effects on sugar content, MDA concentration and POD activity in both roots and leaves, while the removal of cotyledons significantly decreased the POD activity and MDA content in roots A. marina seedlings according to the two-way multivariate analysis of variance test. The sugar content in the cotyledon of Pb-treated seedlings was significantly lower than that in the control (without Pb), suggesting that more carbohydrate reserves (e.g., sugar) stored in cotyledons had been mobilized to leaves and even roots under Pb treatment.     

Primary production,respiration and calcification of the temperate free-living coralline alga Lithothamnion corallioides

Calcification and primary production responses to irradiance in the temperate coralline alga Lithothamnion corallioides were measured in summer 2004 and winter 2005 in the Bay of Brest. Coralline algae were incubated in dark and clear bottles exposed to different irradiances. Net primary production reached 1.5 μmol C g⁻¹ dry wt h⁻¹ in August and was twice as high as in January–February. Dark respiration showed significant seasonal variations, being three-fold higher in summer. Maximum calcification varied from 0.6 μmol g⁻¹ dry wt h⁻¹ in summer 2004 to 0.4 μmol g⁻¹ dry wt h⁻¹ in winter 2005. According to P–E curves and the daily course of irradiance, estimated daily net production and calcification reached 131 μg C g⁻¹ dry wt and 970 μg CaCO₃ g⁻¹ dry wt in summer 2004, and 36 μg C g⁻¹ dry wt and 336 μg CaCO₃ g⁻¹ dry wt in winter 2005. The net primary production of natural L. corallioides populations in shallow waters was estimated at 10–600 g C m⁻² y⁻¹, depending on depth and algal biomass. The mean annual calcification of L. corallioides populations varied from 300 to 3000 g CaCO₃ m⁻². These results are similar to those reported for tropical coralline algae in terms of carbon and carbonate productivity. Therefore, L. corallioides can be considered as a key element of carbon and carbonate cycles in the shallow coastal waters where they live.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.     

Spermatozoa input concentrations and RNA isolation methods on RNA yield and quality in bull (Bos taurus)

Sperm RNA can be used to understand the past spermatogenic process, future successful fertilization, and embryo development. To study the sperm RNA composition and function, isolation of good quality RNA with sufficient quantity is essential. The objective of this study was to assess the influence of sperm input concentrations and RNA isolation methods on RNA yield and quality in bull sperm. The fresh semen samples from bulls (n = 6) were snap-frozen in liquid nitrogen and stored at −80 °C. The sperm RNA was isolated using membrane-based methods combined with TRIzol (RNeasy + TRIzol and PureLink + TRIzol) and conventional methods (TRIzol, Double TRIzol, and RNAzol RT). Based on fluorometric quantification, combined methods resulted in significantly (P < 0.05) higher total RNA yields (800–900 ng/30–40 × 10⁶) as compared with other methods and yielded 20 to 30 fg of RNA/spermatozoon. The quality of RNA isolated by membrane-based methods was superior to that isolated by conventional methods. The sperm RNA was observed to be intact as well as fragmented (50–2000 bp). The study revealed that the membrane-based methods with a cocktail of lysis solution and an optimal input concentration of 30 to 40 million sperm were optimal for maximum recovery of RNA from bull spermatozoa.     

Biodegradation of phenol using Corynebacterium sp. DJ1 aerobic granules

The single-culture Corynebacterium sp. DJ1 aerobic granules were cultivated and were utilized to degrade high-strength phenolic wastewater. These granules can degrade phenol at sufficient high rate without severe inhibitory effects up to phenol concentration of 2000 mg l⁻¹. Furthermore, the kinetic characteristic noted for these granules yields a zero-order phenol degradation behavior with 500–1500 mg l⁻¹ phenol, which facilitates reactor design and scale up. With added acetate to promote cell growth, this single-culture aerobic granular system yields the highest phenol degradation rate reported in granular literature.     

Quantum dot-based assay for Cu quantification in bacterial cell culture

A simple and sensitive method for quantification of nanomolar copper with a detection limit of 1.2 × 10⁻¹⁰ M and a linear range from 10⁻⁹ to 10⁻⁸ M is reported. For the most useful analytical concentration of quantum dots, 1160 μg/ml, a 1/K_sv value of 11 μM Cu²⁺ was determined. The method is based on the interaction of Cu²⁺ with glutathione-capped CdTe quantum dots (CdTe–GSH QDs) synthesized by a simple and economic biomimetic method. Green CdTe–GSH QDs displayed the best performance in copper quantification when QDs of different sizes/colors were tested. Cu²⁺ quantification is highly selective given that no significant interference of QDs with 19 ions was observed. No significant effects on Cu²⁺ quantification were determined when different reaction matrices such as distilled water, tap water, and different bacterial growth media were tested. The method was used to determine copper uptake kinetics on Escherichia coli cultures. QD-based quantification of copper on bacterial supernatants was compared with atomic absorption spectroscopy as a means of confirming the accuracy of the reported method. The mechanism of Cu²⁺-mediated QD fluorescence quenching was associated with nanoparticle decomposition.