Scale up and Application of Biosurfactant from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in Enhanced Oil Recovery

There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y _p/x), biosurfactant on sucrose (Y _p/s), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g⁻¹, 0.18 g g⁻¹, and 0.03 g l⁻¹ h⁻¹, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y _x/s, Y _p/x, Y _p/s, and Y of 0.42 g g⁻¹, 0.595 g g⁻¹, 0.25 g g⁻¹, and 0.057 g l⁻¹ h⁻¹, respectively. The biosurfactant maximum production, 2.5 g l⁻¹, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K _L a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s⁻¹, respectively. Comparison of K _L a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K _L a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.     

Ethanol Production from Cashew Apple Bagasse: Improvement of Enzymatic Hydrolysis by Microwave-Assisted Alkali Pretreatment

In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L⁻¹ of NaOH (372 ± 12 and 355 ± 37 mg g_glucan⁻¹) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15–30 min) and microwave power (600–900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g_CAB-M⁻¹) increased glucose concentration to 15 g L⁻¹. The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L⁻¹ and 1.41 g L⁻¹ h⁻¹, respectively.     

Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis–Menten constant (K _M) of the enzyme and V _max of the reaction. The obtained K _M of the reaction was 14 ± 3 g L⁻¹ (41 μM). Furthermore, inhibition by adenosine 5′-monophosphate (AMP) was evaluated, and the K _M^App value was determined to be 12 ± 2 g L⁻¹ (35 μM) for 7.5 and 15 μM AMP, respectively, with V _max decreasing from 0.1 ± 0.03 to 0.05 ± 0.01 g L⁻¹ min⁻¹. Therefore, AMP exerted a non-competitive inhibition.     

Determination of butoxyacetic acid (biomarker of ethylene glycol monobutyl ether exposure) in human urine candidate reference material

Ethylene glycol monobutyl ether (EGBE), an industrial solvent, is absorbed by the body not only by inhalation but also by dermal absorption (liquid or vapour). EGBE is metabolized to butoxyacetic acid (BAA). Pooled freeze-dried urine candidate reference material (RM) was prepared from urine obtained from persons occupationally exposed to EGBE. This material has the advantage of containing butoxyacetic acid in both the free and conjugated (glutamine and glycine) forms, as found in native urine. In all GC method modifications used, acid hydrolysis was used to release BAA from its conjugated form. The amount of butoxyacetic acid in homogeneity and stability testing was measured by GC after derivatisation with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. Detection was by MS in EI mode, in the authors’ laboratory. For interlaboratory comparison of the reference material GC methods with MS, FID, and ECD were used. Different extraction solvents (dichloromethane–isopropanol 2:1, ethyl acetate, or dichloromethane) and derivatisation reagents (trimethylsilyldiazomethane, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide) were used. Using ANOVA (at the statistical level α = 0.05) no changes were found in the concentration of butoxyacetic acid during fifteen month isochronous stability testing, or in homogeneity testing. The uncertainty contributions were u _h = 8.8 mg L⁻¹ and u _s = 6.5 mg L⁻¹. The concentration of butoxyacetic acid in freeze-dried urine RM was evaluated from the results of eight laboratory data sets within an interlaboratory comparison by use of the interactive statistical software IPECA. The contribution to total uncertainty derived from interlaboratory comparison was u _i = 12.7 mg L⁻¹. The reference value (c = 273 ± 33 mg L⁻¹) is an unweighted arithmetic average of accepted results. The value is traceable to the pure butoxyacetic acid (98% w/w; Acros Organic #257760010) used as calibrant. The uncertainty given is combined expanded uncertainty derived from the results from interlaboratory comparison, and from homogeneity and stability tests (k = 2). The reference material will be used to verify method performance in the biological monitoring of occupational exposure to EGBE.     

Comparison of different sample treatments for the analysis of ochratoxin A in wine by capillary HPLC with laser-induced fluorescence detection

Ochratoxin A (OTA) is a mycotoxin naturally found in various foods, including wine. As OTA is considered as a possible human carcinogen, the maximum concentration for this compound has been established at 2 μg kg⁻¹ in wine by the EU (Directive (CE) No 1881/2006). Typically, immunoaffinity columns have been used for its extraction. However, simpler, more efficient and less contaminant extraction systems are demanding. In this work, dispersive liquid–liquid microextraction using ionic liquid as extractant solvent (IL-DLLME) and the QuEChERS procedure, have been evaluated and compared for extraction of OTA in wine samples. Laser-induced fluorescence (LIF, He–Cd Laser excitation at 325 nm) coupled with capillary HPLC has been used for the determination of OTA, using a sodium dodecyl sulfate micellar solution in the mobile phase to increase the fluorescence intensity. Matrix-matched calibration curves were established for both methods, obtaining LODs (3× S/N) of 5.2 ng·L⁻¹ and 85.7 ng·L⁻¹ for IL-DLLME and QuEChERS, respectively. Clean extracts were obtained for white, rose and red wines with both methods, with recoveries between 88.7–94.2% for IL-DLLME and between 82.6–86.2% for QuEChERS. The precision was evaluated in terms of repeatability (n = 9) and intermediate precision (n = 15), being ≤ 8.5% for IL-DLLME and ≤ 5.4% for QuEChERS.     

Production of Lipids Containing High Levels of Docosahexaenoic Acid by a Newly Isolated Microalga, <Emphasis Type="Italic">Aurantiochytrium</Emphasis> sp. KRS101

In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.     

Effect of organic load on the performance and methane production of an AnSBBR treating effluent from biodiesel production

Currently, there is an increasing demand for the production of biodiesel and, consequently, there will be an increasing need to treat wastewaters resulting from the production process of this biofuel. The main objective of this work was, therefore, to investigate the effect of applied volumetric organic load (AVOL) on the efficiency, stability, and methane production of an anaerobic sequencing batch biofilm reactor applied to the treatment of effluent from biodiesel production. As inert support, polyurethane foam cubes were used in the reactor and mixing was accomplished by recirculating the liquid phase. Increase in AVOL resulted in a drop in organic matter removal efficiency and increase in total volatile acids in the effluent. AVOLs of 1.5, 3.0, 4.5 and 6.0 g COD L⁻¹ day⁻¹ resulted in removal efficiencies of 92%, 81%, 67%, and 50%, for effluent filtered samples, and 91%, 80%, 63%, and 47%, for non-filtered samples, respectively, whereas total volatile acids concentrations in the effluent amounted to 42, 145, 386 and 729 mg HAc L⁻¹, respectively. Moreover, on increasing AVOL from 1.5 to 4.5 g COD L⁻¹ day⁻¹ methane production increased from 29.5 to 55.5 N mL CH₄ g COD⁻¹. However, this production dropped to 36.0 N mL CH₄ g COD⁻¹ when AVOL was increased to 6.0 g COD L⁻¹ day⁻¹, likely due to the higher concentration of volatile acids in the reactor. Despite the higher concentration of volatile acids at the highest AVOL, alkalinity supplementation to the influent, in the form of sodium bicarbonate, at a ratio of 0.5–1.3 g NaHCO₃ g COD_fed⁻¹, was sufficient to maintain the pH near neutral and guarantee process stability during reactor operation.     

Stannum film electrode for square wave voltammetric determination of trace copper(II)

An anodic stripping voltammetric procedure for the determination of Cu(II) at an in situ-plated stannum film electrode (SnFE) was described. The results indicated that the SnFE had an attractive electroanalytical performance, with two distinct voltammetric stripping signals for copper and stannum, and showed the superior advantage for the determination of copper compared with the bismuth film electrode. Several experimental parameters were optimized. The SnFE exhibited highly linear behavior in the concentration range from 1.0 to 100.0 μg L⁻¹ of Cu(II) (r = 0.994) with the detection limit of 0.61 μg L⁻¹ (S/N = 3), and the relative standard deviation for a solution containing 40.0 μg L⁻¹ Cu(II) was 2.2% (n = 8). The procedure has been successfully applied for the determination of Cu(II) in lake water sample.     

Synthesis of hydrophilic polyurethane particles in non-aqueous inverse miniemulsions

It is shown that it is possible to synthesize high molecular weight hydrophilic polyurethane particles by reacting either tolylene-2,4-diisocyanate or isophorone diisocyanate and oligoethylene glycol (M _n ∼200 g mol⁻¹) in non-aqueous inverse emulsions. This procedure offers the advantage that the formation of polyurea can be prevented in consequence of the absence of water in the emulsion. Apparent molecular weights of hydrophilic polyurethane as high as 19,000 g mol⁻¹ (M _n) were obtained.     

Retinol fluorescence in lecithin/<Emphasis Type="Italic">n</Emphasis>-butanol/water aggregates: a new improvement for its analysis in cosmetics without pretreatment

The possibilities of different media formed by lecithin/n-butanol (n-BuOH)/water ternary mixtures for the analysis of all-trans-retinol by fluorescence have been studied. Fluorescence intensity of retinol increases in the presence of different types of aggregates formed in these media. Analytical features are good, the detection limit and quantification limit have micrograms per liter levels, and the linear range and sensitivity are appropriate to determine retinol in cosmetic samples. The analysis of retinol in anti-wrinkle creams can be achieved directly without any pretreatment of the sample. The vesicles built up from a biocompatible surfactant (lecithin) in aqueous solution with a low amount of n-BuOH permit an appropriated media for a simple, rapid, and sensitive analytical method. This method has a linear range between 64.1 and 800 μg L⁻¹, a sensitivity of 202.3 L mg⁻¹, and a low detection and quantification limit at 19.2 and 64.1 μg L⁻¹, respectively.     

1,3,5,7-Tetramethyl-8-(<Emphasis Type="Italic">N</Emphasis>-hydroxysuccinimidyl butyric ester)difluoroboradiaza-<Emphasis Type="Italic">s</Emphasis>-indacene as a new fluorescent labeling reagent for HPLC determination of amino acid neurotransmitters in the cerebral cortex of mice

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L⁻¹ pH 7.8 boric acid buffer. The separation was performed on a C₁₈ column with methanol–water–buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L⁻¹ H₃Cit–0.10 mol L⁻¹ NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L⁻¹. The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.     

Analytical method for assessing potential dermal exposure to pesticides of a non-agricultural occupationally exposed population

To measure dermal exposure of a non-agricultural occupationally exposed population to pesticides, a new method has been developed for analysis of 13 pesticides from different classes (fungicides, herbicides, insecticides) on dermal patches. The method includes extraction of the patches and analysis of the pesticides by GC–MS and/or HPLC–fluorescence. Water-soluble pesticides (glyphosate and glufosinate) on patches were ultrasonically extracted twice with ultra-pure water for 10 min and analysed by HPLC–fluorescence after derivatisation with FMOC. Organic-soluble pesticides (bifenthrin, cyprodinil, difufenicanil, fludioxonil, oxadiazon, pyriproxyfen, clopyralid, 2,4-D, fluroxypyr, 2,4-MCPA, and triclopyr) were extracted ultrasonically twice for 10 min with 70:30 dichloromethane–acetonitrile and analysed by GC–MS directly or after derivatisation with N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide. Detection limits varied between 3 and 4 μg L⁻¹ for water-soluble pesticides and between 1 and 10 μg L⁻¹ for organic-soluble pesticides.     

Enhanced Production of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine by Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> Hemoglobin Using a Novel Expression System in <Emphasis Type="Italic">Corynebacterium crenatum</Emphasis>

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A₅₆₂⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.     

The Fed-Batch Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase (DERA) in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Exponential Feeding Strategy Control

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes a sequential aldol reaction useful in synthetic chemistry. In this work, the effect of a feeding strategy on the production of a thermophilic DERA was investigated in fed-batch cultures of recombinant Escherichia coli BL21 (pET303-DERA008). The predetermined specific growth rate (μ _set) was evaluated at 0.20, 0.15, and 0.10 h⁻¹, respectively. The DERA concentration and volumetric productivity were associated with μ _set. The cells synthesized the enzyme most efficiently at μ _set = 0.15 h⁻¹. The maximum enzyme concentration (5.12 g/L) and total volumetric productivity (0.256 g L⁻¹ h⁻¹) obtained were over 10 and five times higher than that from traditional batch cultures. Furthermore, the acetate concentration remained at a relatively low level, less than 0.4 g/L, under this condition which would not inhibit cell growth and target protein expression. Thus, a specific growth rate control strategy has been successfully applied to induce fed-batch cultures for the maximal production of the thermophilic 2-deoxyribose-5-phosphate aldolase.     

Production and analytical characterization of neopterin immunoreagents for biosensor developments

Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins. The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 μg L⁻¹, respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 μg L⁻¹. The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations ranging from 3.2 to 103 μg L⁻¹ could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5–16 μg L⁻¹ (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into multianalyte biochip detection systems.     

Enantioselective piezoelectric quartz crystal sensor for d-methamphetamine based on a molecularly imprinted polymer

A piezoelectric quartz crystal (PQC) sensor based on a molecularly imprinted polymer (MIP) has been developed for enantioselective and quantitative analysis of d-(+)-methamphetamine (d(+)-MA). The sensor was produced by bulk polymerization and the resulting MIP was then coated on the gold electrode of an AT-cut quartz crystal. Conditions such as volume of polymer coating, curing time, type of PQC, baseline solvent, pH, and buffer type were found to affect the sensor response and were therefore optimized. The PQC-MIP gave a stable response to different concentrations of d(+)-MA standard solutions (response time = 10 to 100 s) with good repeatability (RSD = 0.03 to 3.09%; n = 3), good reproducibility (RSD = 3.55%; n = 5), and good reversibility (RSD = 0.36%; n = 3). The linear range of the sensor covered five orders of magnitude of analyte concentration, ranging from 10⁻⁵ to 10⁻¹ μg mL⁻¹, and the limit of detection was calculated as 11.9 pg d(+)-MA mL⁻¹ . The sensor had a highly enantioselective response to d(+)-MA compared with its response to l(−)-MA, racemic MA, and phentermine. The developed sensor was validated by applying it to human urine samples from drug-free individuals spiked with standard d(+)-MA and from a confirmed MA user. Use of the standard addition method (SAM) and samples spiked with d(+)-MA at levels ranging from 1 × 10⁻³ to 1 × 10⁻² μg mL⁻¹ showed recovery was good (95.3 to 110.9%).     

Effect of Substrate Concentration on Dark Fermentation Hydrogen Production Using an Anaerobic Fluidized Bed Reactor

The effect of substrate (glucose) concentration on the stability and yield of a continuous fermentative process that produces hydrogen was studied. Four anaerobic fluidized bed reactors (AFBRs) were operated with a hydraulic retention time (HRT) from 1 to 8 h and an influent glucose concentration from 2 to 25 g L⁻¹. The reactors were inoculated with thermally pre-treated anaerobic sludge and operated at a temperature of 30 °C with an influent pH around 5.5 and an effluent pH of about 3.5. The AFBRs with a HRT of 2 h and a feed strength of 2, 4, and 10 g L⁻¹ showed satisfactory H₂ production performance, but the reactor fed with 25 g L⁻¹ of glucose did not. The highest hydrogen yield value was obtained in the reactor with a glucose concentration of 2 g L⁻¹ when it was operated at a HRT of 2 h. The maximum hydrogen production rate value was achieved in the reactor with a HRT of 1 h and a feed strength of 10 g L⁻¹. The AFBRs operated with glucose concentrations of 2 and 4 g L⁻¹ produced greater amounts of acetic and butyric acids, while AFBRs with higher glucose concentrations produced a greater amount of solvents.     

Synthesis,Crystal Structure and Raman Spectrum of Hydrazinium(+2) Fluoroarsenate(III) Fluoride,N<Subscript>2</Subscript>H<Subscript>6</Subscript>AsF<Subscript>4</Subscript>F

Summary.  Hydrazinium(+2) fluoroarsenate(III) fluoride was prepared by the reaction of hydrazinium(+2) fluoride and liquid arsenic trifluoride. N₂H₆AsF₄F is stable at 273 K, but decomposes slowly at room temperature. N₂H₆AsF₄F crystallizes in the orthorhombic space group Pnn2 with a = 774.0(2) pm, b = 1629.2(4) pm and c = 436.6(1) pm; V = 0.5506(3) nm³, Z = 4 and d _c  = 2.461 g cm⁻³. The structure consists of N₂H₆ ²⁺ cations, AsF₄ ⁻ anions, and F⁻ anions and is interconnected by a hydrogen bonding network. Distorted trigonal-bipyramidal AsF₄ ⁻ units are very weakly interconnected and form chains along the b axis. Bands in the Raman spectrum are assigned to the vibrations of N₂H₆ ⁺² cations and AsF₄ ⁻ anions. Corresponding author. E-mail: adolf.jesih@ijs.si Received April 18, 2002; accepted July 15, 2002     

On-line ionic imprinted polymer selective solid-phase extraction of nickel and lead from seawater and their determination by inductively coupled plasma-optical emission spectrometry

Nickel(II) and lead(II) ionic imprinted 8-hydroxyquinoline polymers were synthesized by a precipitation polymerization technique and were used as selective solid phase extraction supports for the determination of nickel and lead in seawater by flow injection solid phase extraction on-line inductively coupled plasma-optical emission spectrometry. An optimum loading flow rate of 2.25 mL min⁻¹ for 2 min and an elution flow rate of 2.25 mL min⁻¹ for 1 min gave an enrichment factor of 15 for nickel. However, a low dynamic capacity and/or rate for adsorption and desorption was found for lead ionic imprinted polymer and a flow rate of 3.00 mL min⁻¹ for 4-min loading and a flow rate of 2.25 mL min⁻¹ for 1-min elution gave a enrichment factor of 5. The limit of detection was 0.33 μg L⁻¹ for nickel and 1.88 μg L⁻¹ for lead, with a precision (n = 11) of 8% (2.37 μg Ni L⁻¹) for nickel and 11% (8.38 μg Pb L⁻¹) for lead. Accuracy was also assessed by analyzing SLEW-3 (estuarine water) and TM-24 (lake water) certified reference materials, and the values determined were in good agreement with the certified concentrations.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.