Solid-phase extraction of tramadol from plasma and urine samples using a novel water-compatible molecularly imprinted polymer

In this study, a novel method is described for the determination of tramadol in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and tramadol as template molecule. The novel imprinted polymer was used as a solid-phase extraction (SPE) sorbent for the extraction of tramadol from human plasma and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for the MIP cartridges were studied. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of tramadol. The limit of detection (LOD) and limit of quantification (LOQ) for tramadol in urine samples were 1.2 and 3.5 μg L⁻¹, respectively. These limits for tramadol in plasma samples were 3.0 and 8.5 μg L⁻¹, respectively. The recoveries for plasma and urine samples were higher than 91%.     

Water column anammox and denitrification in a temperate permanently stratified lake (Lake Rassnitzer,Germany)

We studied microbial N₂ production via anammox and denitrification in the anoxic water column of a restored mining pit lake in Germany over an annual cycle. We obtained high-resolution hydrochemical profiles using a continuous pumping sampler. Lake Rassnitzer is permanently stratified at ca. 29 m depth, entraining anoxic water below a saline density gradient. Mixed-layer nitrate concentrations averaged ca. 200 μmol L⁻¹, but decreased to zero in the anoxic bottom waters. In contrast, ammonium was <5 μmol L⁻¹ in the mixed layer but increased in the anoxic waters to ca. 600 μmol L⁻¹ near the sediments. In January and October, ¹⁵N tracer measurements detected anammox activity (maximum 504 nmol N₂ L⁻¹ d⁻¹ in ¹⁵NH₄⁺-amended incubations), but no denitrification. In contrast, in May, N₂ production was dominated by denitrification (maximum 74 nmol N₂ L⁻¹ d⁻¹). Anammox activity in May was significantly lower than in October, as characterized by anammox rates (maximum 6 vs. 16 nmol N₂ L⁻¹ d⁻¹ in incubations with ¹⁵NO₃⁻), as well as relative and absolute anammox bacterial cell abundances (0.56% vs. 0.98% of all bacteria, and 2.7×10⁴ vs. 5.2×10⁴ anammox cells mL⁻¹, respectively) (quantified by catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) with anammox bacteria-specific probes). Anammox bacterial diversity was investigated with anammox bacteria-specific 16S rRNA gene clone libraries. The majority of anammox bacterial sequences were related to the widespread Candidatus Scalindua sorokinii/brodae cluster. However, we also found sequences related to Candidatus S. wagneri and Candidatus Brocadia fulgida, which suggests a high anammox bacterial diversity in this lake comparable with estuarine sediments.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

Task-specific ionic liquid-assisted extraction and separation of astaxanthin from shrimp waste

Astaxanthin, as an outstanding antioxidant reagent, was successfully extracted from shrimp waste by the ionic liquids based ultrasonic-assisted extraction. Seven kinds of imidazolium ionic liquids with different cations and anions were investigated in this work and one task-specific ionic liquid in ethanol with 0.50 mol L⁻¹ was selected as the solvent. At the optimized ultrasonic extraction conditions, the extraction amount of astaxanthin increased 98% (92.7 μg g⁻¹) compared to the conventional method (46.7 μg g⁻¹). Furthermore, the extracted solution was isolated through the solid-phase extraction with a molecularly imprinted polymer sorbent. After loading the samples on molecularly imprinted polymer cartridge, the different washing and elution solvents, such as water, methanol, n-hexane, acetone and dichloromethane, were evaluated, and finally, astaxanthin was separated from the shrimp waste extract.     

Nitrogen nutrition of Canna indica: Effects of ammonium versus nitrate on growth, biomass allocation, photosynthesis, nitrate reductase activity and N uptake rates

The effects of inorganic nitrogen (N) source (NH₄⁺, NO₃⁻ or both) on growth, biomass allocation, photosynthesis, N uptake rate, nitrate reductase activity and mineral composition of Canna indica were studied in hydroponic culture. The relative growth rates (0.05-0.06 g g⁻¹ d⁻¹), biomass allocation and plant morphology of C. indica were indifferent to N nutrition. However, NH₄⁺ fed plants had higher concentrations of N in the tissues, lower concentrations of mineral cations and higher contents of chlorophylls in the leaves compared to NO₃⁻ fed plants suggesting a slight advantage of NH₄⁺ nutrition. The NO₃⁻ fed plants had lower light-saturated rates of photosynthesis (22.5 μmol m⁻² s⁻¹) than NH₄⁺ and NH₄⁺/NO₃⁻ fed plants (24.4-25.6 μmol m⁻² s⁻¹) when expressed per unit leaf area, but similar rates when expressed on a chlorophyll basis. Maximum uptake rates (V_max) of NO₃⁻ did not differ between treatments (24-35 μmol N g⁻¹ root DW h⁻¹), but V_max for NH₄⁺ was highest in NH₄⁺ fed plants (81 μmol N g⁻¹ root DW h⁻¹), intermediate in the NH₄NO₃ fed plants (52 μmol N g⁻¹ root DW h⁻¹), and lowest in the NO₃⁻ fed plants (28 μmol N g⁻¹ root DW h⁻¹). Nitrate reductase activity (NRA) was highest in leaves and was induced by NO₃⁻ in the culture solutions corresponding to the pattern seen in fast growing terrestrial species. Plants fed with only NO₃⁻ had high NRA (22 and 8 μmol NO₂⁻ g⁻¹ DW h⁻¹ in leaves and roots, respectively) whereas NRA in NH₄⁺ fed plants was close to zero. Plants supplied with both forms of N had intermediate NRA suggesting that C. indica takes up and assimilate NO₃⁻ in the presence of NH₄⁺. Our results show that C. indica is relatively indifferent to inorganic N source, which together with its high growth rate contributes to explain the occurrence of this species in flooded wetland soils as well as on terrestrial soils. Furthermore, it is concluded that C. indica is suitable for use in different types of constructed wetlands.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

The antioxidant effect of the mesoionic compound SYD-1 in mitochondria

The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75 μmol mg⁻¹ protein) inhibited the lipoperoxidation induced by Fe³⁺/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0 μmol mg⁻¹ protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α′-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25–1.0 μmol mg⁻¹ protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9–19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25–1.0 μmol mg⁻¹ protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca²⁺/phosphate by approximately 30% (1.0 μmol mg⁻¹ protein). When Ca²⁺/H₂O₂ were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0 μmol mg⁻¹ of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.     

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%.     

A high-throughput screening (HTS) immunochemical method for the analysis of stanozolol metabolites in cattle urine samples

A high-throughput immunosorbent solid-phase extraction (HTS-IS-SPE) procedure coupled to enzyme-linked immunosorbent assay (ELISA) has been established for the analysis of stanozolol (St) and its main metabolite in cattle, 16β-hydroxy-stanozolol (16βOH-St), in cow urine samples. The chemical structure of the immunizing hapten 2′H-androst-2-eno[3,2-c]-pyrazol-17-hemiglutarate 5 (hapten A) has been designed to accomplish simultaneous detection of St and 16βOH-St. The antibodies obtained have been used to establish a microplate ELISA method able to detect these metabolites with IC₅₀ values of 0.57 μg L⁻¹ and 1.46 μg L⁻¹, respectively in PBST. Immunosorbents prepared by covalently attaching the antibodies to Sepharose, efficiently removed the matrix interferences caused by the cattle urine samples. Moreover, St and 16βOH-St were efficiently extracted from urine samples as demonstrated by LC–MS/MS analysis. The immunosorbents are filled on small mini-columns arranges on a 96-SPE-setup compatible with the microplate based ELISA methods. Samples and standards can be run in parallel which increment considerably the speed of the screening method. The recovery values of the whole HTS-IS-SPE-ELISA procedure has found to be 112 ± 10% and St can be detected in hydrolyzed urine samples with LOD of 1.26 ± 0.46 μg L⁻¹ using just 1 mL of sample. As proof-of-concept the urinary excretion profile of St treated animals has been investigated by analyzing individual sampling points. Results from pooled urine samples have also been compared with the results obtained by GC–MS analysis demonstrating the StIR equiv. measured with the HTS-IS-SPE-ELISA protocol are in accordance with the St and 16βOH-St levels found with the chromatographic method. The analytical procedure is rapid, effective and the detectability achieved is below the MPRL (minimum performance required levels) recommended by CRL (Community Reference Laboratory) to the European Community.     

Substrate recognition and hydrolysis by a fungal xyloglucan-specific family 12 hydrolase

Biochemical studies to elucidate the structural basis for xyloglucan specificity among GH12 xyloglucanases are lacking. Accordingly, the substrate specificity of a GH12 xyloglucanase from Aspergillus niger (AnXEG12A) was investigated using pea xyloglucan and 12 xylogluco-oligosaccharides, and data were compared to a structural model of the enzyme. The specific activity of AnXEG12A with pea xyloglucan was 113 μmol min⁻¹ mg⁻¹, and apparent k_cat and K_m values were 49 s⁻¹ and 0.54 mg mL⁻¹, respectively. These values are similar to previously published results using xyloglucan from tamarind seed, and suggest that substrate fucosylation does not affect the specific activity of this enzyme. AnXEG12A preferred xylogluco-oligosaccharides containing more than six glucose units, and with xylose substitution at the −3 and +1 subsites. The specific activities of AnXEG12A on 100 μM XXXGXXXG and 100 μM XLLGXLLG were 60 ± 4 and 72 ± 9 μmol min⁻¹ mg⁻¹, respectively. AnXEG12A did not hydrolyze XXXXXXXG, consistent with other data that demonstrate the requirement for an unbranched glucose residue for hydrolysis by this enzyme.     

Effects of the methane-inhibitors nitrate,nitroethane, lauric acid,Lauricidin and the Hawaiian marine algae Chaetoceros on ruminal fermentation in vitro

The effects of several methane-inhibitors on rumen fermentation were compared during three 24 h consecutive batch cultures of ruminal microbes in the presence of nonlimiting amounts of hydrogen. After the initial incubation series, methane production was reduced greater than 92% from that of non-treated controls (25.8 ± 8.1 μmol ml⁻¹ incubation fluid) in cultures treated with nitroethane, sodium laurate, Lauricidin^® or a finely-ground product of the marine algae, Chaetoceros (added at 1, 5, 5 and 10 mg ml⁻¹, respectively) but not in cultures treated with sodium nitrate (1 mg m1⁻¹). Methane production during two successive incubations was reduced greater than 98% from controls (22.5 ± 3.2 and 23.5 ± 7.9 μmol ml⁻¹, respectively) by all treatments. Reductions in amounts of volatile fatty acids and ammonia produced and amounts of hexose fermented, when observed, were most severe in sodium laurate-treated cultures. These results demonstrate that all tested compounds inhibited ruminal methane production in our in vitro system but their effects on fermentation differed.     

Relationship of rapid light curves of variable fluorescence to photoacclimation and non-photochemical quenching in a benthic diatom

The response of rapid light–response curves (RLCs) of variable fluorescence to changes in short- and long-term photoacclimation status was studied in an estuarine benthic diatom. The diatom Nitzschia palea was grown under low- (LL, 20 μmol m⁻² s⁻¹) and high-light (HL, 400 μmol m⁻² s⁻¹) conditions, with the purpose of characterising the effects of long-term photoacclimation on (i) steady-state light–response curves (LC) of relative electron transport rate, rETR, (ii) the response of RLCs to changes in ambient irradiance (E, the irradiance to which the sample is acclimated to immediately before the RLCs), (iii) the relationship of RLCs to LC parameters and non-photochemical quenching (NPQ). Photoacclimation to LL and HL conditions induced distinct light–response patterns of rETR and NPQ. Higher growth light resulted in rETR vs. E curves with lower initial slopes (α, 0.591 μmol⁻¹ m² s vs. 0.661 μmol⁻¹ m² s, for HL and LL, respectively) and markedly higher maximum rates (rETR_m, 95.9 vs. 29.3), reached under higher E levels (higher light-saturation coefficient, E_k: 162.4 μmol m⁻² s⁻¹ vs. 44.3 μmol m⁻² s⁻¹). Acclimation to HL induced bi-phasic NPQ vs. E curves, with minimum values reached under low E levels (15–25 μmol m⁻² s⁻¹) and not on dark-acclimated samples. The response of RLCs to changes in ambient irradiance varied with the long-term photoacclimation status of the samples. The initial slope, α_RLC, decreased monotonically with E in LL cultures, from 0.68 to 0.25 μmol⁻¹ m² s, while varied bi-phasically in HL-acclimated samples. Typically, α_RLC of HL cultures increased under low E, reaching a maximum of 0.61 μmol⁻¹ m² s under 25–55 μmol m⁻² s⁻¹, and decreased gradually under higher E levels to 0.25 μmol⁻¹ m² s. RLC maximum rETR, rETR_m,RLC, and saturation coefficient E_k,RLC, increased with E following a saturation-like pattern, with the HL cultures presenting markedly higher values for all the E range (maximum rETR_m,RLC values were 108.6 and 33.4 for HL and LL cultures, respectively). An inverse relationship was consistently found between α_RLC and NPQ, both on LL and HL cultures, causing strong correlations (P < 0.001 in all cases) between NPQ and the high light-induced decrease of α_RLC, Δα_RLC. RLCs were confirmed to also provide information on the long-term photoacclimation status, as significant correlations (P < 0.001 both for HL and LL cultures) were verified between E_k and an index based on RLC parameters, Ê_k, both for LL and HL cultures. These results reinforce the usefulness of RLCs as a tool for inferring on the short- and long-term photoacclimation status of samples with different long-term light histories, through the estimation of LC parameters and the monitoring of NPQ levels.     

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.     

Horizontal or vertical photobioreactors? How to improve microalgae photosynthetic efficiency

The productivity of a vertical outdoor photobioreactor was quantitatively assessed and compared to a horizontal reactor. Daily light cycles in southern Spain were simulated and applied to grow the microalgae Chlorella sorokiniana in a flat panel photobioreactor.The maximal irradiance around noon differs from 400 μmol photons m⁻² s⁻¹ in the vertical position to 1800 μmol photons m⁻² s⁻¹ in the horizontal position. The highest volumetric productivity was achieved in the simulated horizontal position, 4 g kg culture⁻¹ d⁻¹. The highest photosynthetic efficiency was found for the vertical simulation, 1.3 g of biomass produced per mol of PAR photons supplied, which compares favorably to the horizontal position (0.85 g mol⁻¹) and to the theoretical maximal yield (1.8 g mol⁻¹). These results prove that productivity per unit of ground area could be greatly enhanced by placing the photobioreactors vertically.     

Hypoxia and sulphide influence gamete production in Ulva sp.

Gamete production after exposure to hypoxia or sulphide was studied in the marine macroalga Ulva sp. collected in the Sacca di Goro, Italy. Experiments were carried out on discs (12 mm diameter) of thalli cultured in artificial sea water in laboratory at 20 ± 1 °C, 152 μmol m⁻² s⁻¹, 16 h photoperiod and 30‰ salinity. Dehydration of thallus was used as inducer of gametogenesis and growth and gamete release during recovery after 10, 20, 30 or 40 min dehydration (20 ± 1 °C, 25% humidity) were analysed. Unlike non-dehydrated thalli the dehydrated ones produced gametes. Thallus discs, non-dehydrated or subjected to 30 min dehydration, were exposed to hypoxia (1.78–4.02 μmol O₂ L⁻¹) or sulphide (1 mM) for 3, 5, or 7 days at 20 °C in the dark. Non-dehydrated and dehydrated thalli maintained in normoxic conditions in the dark were the controls. Gamete density was checked by counting at the end of the incubation period and during the subsequent 7 days of recovery under 16 h photoperiod in normoxic conditions. Non-dehydrated thalli maintained in normoxic conditions in the dark released gametes when returned to light suggesting that dark constitutes a stimulus to gamete production. The presence of gametes at the end of 3 days incubation of dehydrated thalli in normoxia demonstrated that gametogenesis can occur even in the dark. However, gametes were not present at the end of incubation in hypoxic and sulphidic conditions. Actually, during hypoxic incubation oxygen consumption in D-thalli was very low, only 0.117 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ compared to 5.93 × 10⁻³ μmol O₂ mg⁻¹ h⁻¹ in normoxia, denoting a reduction of the metabolic rate that could not sustain gametogenesis. During recovery after incubation in normoxic, hypoxic or sulphidic conditions densities of gametes from dehydrated thalli showed significant differences and resulted after hypoxia > after normoxia > after sulphide. Differences in non-dehydrated thalli were not significant. Dehydrated thalli, still green at the end of the incubation period, underwent blanching in the course of recovery in parallel to gamete production, while non-dehydrated thalli maintained their green colour even after exposure to sulphide. Our findings suggest that macroalga Ulva sp. can survive exposure to darkness, severe hypoxia and high sulphide levels and can maintain gamete production even when the exposure to these stress conditions is joined to dehydration.     

Biomass production and nitrogen and phosphorus removal by the green alga Neochloris oleoabundans in simulated wastewater and secondary municipal wastewater effluent

Biomass productivity of 350 mg DCW L⁻¹ day⁻¹ with a final biomass concentration of 3.15 g DCW L⁻¹ was obtained with Neochloris oleoabundans grown in artificial wastewater at sodium nitrate and phosphate concentrations of 140 and 47 mg L⁻¹, respectively, with undetectable levels of residual N and P in effluents. In secondary municipal wastewater effluents enriched with 70 mg N L⁻¹, the alga achieved a final biomass concentration of 2.1 g DCW L⁻¹ and a biomass productivity of 233.3 mg DCW L⁻¹ day⁻¹. While N removal was very sensitive to N:P ratio, P removal was independent of N:P ratio in the tested range. These results indicate that N. oleoabundans could potentially be employed for combined biofuel production and wastewater treatment.     

Voltammetric determination of cefixime in pharmaceuticals and biological fluids

Electroreduction and adsorption of cefixime was studied in phosphate buffer by cyclic voltammetry (CV), differential pulse cathodic adsorptive stripping voltammetry (DPCAdSV), and square-wave cathodic adsorptive stripping voltammetry (SWCAdSV) at hanging mercury drop electrode (HMDE). These fully validated sensitive and reproducible cathodic adsorptive stripping voltammetric procedures were applied for the trace determination of the bulk drug in pharmaceutical formulations and in human urine. The optimal experimental parameters were as follows: accumulation potential = −0.1 V (vs. Ag/AgCl, 3 M KCl), accumulation time = 50 s, frequency = 140 Hz, pulse amplitude = 0.07 V, and scan increment = 10 mV in phosphate buffer (pH 2.6). The first peak current showed a linear dependence with the drug concentration over the range of 50 ng ml⁻¹ to 25.6 μg ml⁻¹. The achieved limit of detection and limit of quantitation were 3.99 and 13.3 ng ml⁻¹ by SWCAdSV and 7.98 and 26.6 ng ml⁻¹ by DPCAdSV, respectively. The procedure was applied to assay the drug in tablets. Applicability was also tested in urine samples. Peak current was linear with the drug concentration in the range of 1 to 60 μg ml⁻¹ of the urine, and minimum detectability was found to be 12.6 ng ml⁻¹ by SWCAdSV and 58.4 ng ml⁻¹ by DPCAdSV.     

Quantification of carnosine-related peptides by microchip electrophoresis with chemiluminescence detection

A microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of carnosine-related peptides, including carnosine, homocarnosine, and anserine, in biological samples. A simple integrated MCE-CL system was built to perform the assays. The highly sensitive CL detection was achieved by means of the CL reaction between hydrogen peroxide and N-(4-aminobutyl)-N-ethylisoluminol-tagged peptides in the presence of adenine as a CL enhancer and Co²⁺ as a catalyst. Experimental conditions for analyte labeling, MCE separation, and CL detection were studied. MCE separation of the above-mentioned three peptides took less than 120 s. Detection limits (signal/noise ratio [S/N] = 3) of 3.0 × 10⁻⁸, 2.8 × 10⁻⁸, and 3.4 × 10⁻⁸ M were obtained for carnosine, anserine, and homocarnosine, respectively. The current MCE-CL method was applied for the determination of carnosine, anserine, and homocarnosine in human cerebrospinal fluid (CSF) and canine plasma. Homocarnosine was detected at the micromolar (μM) level in the CSF samples analyzed, whereas the levels of carnosine and anserine in these samples were below the detection limit of the assay. Interestingly, both carnosine and anserine were detected in the canine plasma samples, whereas homocarnosine was not.