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PubMedDate not supplied

Reversed-phase high-performance liquid chromatography determination of selected phenolic acids in propolis concentrates in terms of standardization for drug manufacturing purposes.

A reversed-phase high-performance liquid chromatography method with gradient elution was developed for the determination of the caffeic, p-coumaric, and ferulic acids in propolis concentrates. Solid-phase extraction on an RP18 column was applied for preliminary purification, and chromatographic separation was performed on 100 RP18e Lichrospher column of particle size 5 microm. The mobile phase was obtained by mixing in appropriate ratios 0.03 mM NaH2PO4, acidified with H3PO4 up to pH = 3.0, with acetonitrile to obtain a gradient in the elution process. Spectrophotometric detection was conducted at 320 nm. Under the established conditions, the method featured high sensitivity, good precision, and comparability of results, as proven by method validation and statistical analysis of the obtained results. The limits of detection were 0.315, 0.325, and 0.695 microg/mL for caffeic, p-coumaric, and ferulic acids, respectively. The corresponding recovery values were 98.14, 101.05, and 99.42% and the linearity ranges from 1.31 to 99.18 microg/mL, 1.52 to 119.16 microg/mL, and 2.42 to 184.14 microg/mL. The precision of the method was expresed by relative standard deviation values that did not exceed 3%. It was also shown that the propolis concentrates under examination had similar antibacterial activity against Staphylococcus aureus ranging from 119.8 to 124.3 microg/mL, contrary to model mixtures that showed no antibacterial activity.

Anti-Bacterial Agents↗
PubMed2006

Environmental impact of toxic metals and metalloids from the Muñón Cimero mercury-mining area (Asturias, Spain).

This paper presents the results of the sampling surveys carried out in order to evaluate the environmental problems associated to La Soterraña, an abandoned Hg mine in Asturias, north of Spain. In particular, this paper overviews the impact of mining and metallurgical activities on terrestrial and aquatic environments. The wastes generated during the mining activity (ore extraction and processing) and later accumulated on the ground, contain great amount of sulphides, becoming potentially acid-generating. Consequently, the mobility of heavy metals and other ecotoxic elements is enhanced. Wastes are generally located close to watercourses and on very steep hillsides, where they are exposed to oxidative weathering, posing a significant risk due to the release of ecotoxic elements to the environment. Background levels were determined at sites, which had not been directly affected by mercury mining. A multielemental geochemical study of mining wastes, soils, stream sediments, water and air samples collected in the area of influence of the old mining and metallurgical works was carried out. Total Hg and As concentrations in soils reach values up to 502 and 19,940 mg kg(-1), respectively, 500 and 2000 times higher than the local background levels. The effects of mining seem to be intense both in waters and stream sediments, as well as in the local atmosphere, whose Hg content is 10 times higher than the background level in the area. Therefore, the target carcinogenic risk was exceeded for As and Hg in La Soterraña site.

Air Pollutants, Occupational↗
PubMed1996

beta-agonists in animal feed. III: Optimization of the clean-up and the end-determination step.

This study represents the second part of an interlaboratory study intended to develop an official modular Community confirmatory method for the detection of beta-agonists in animal feed. Homogeneous pools of primary extracts were prepared by means of an extraction module based on the conclusions of a previous part of this work. The primary extracts were further processed by four laboratories each using a different clean-up scheme. The final extracts thus obtained were cross-distributed between the same laboratories and measured either by GCMS or HPLC. Two laboratories (B and D) applied separate clean-up schemes for clenbuterol and salbutamol. All clean-up schemes for clenbuterol were found to be compatible with all end-determination steps. In contrast, for salbutamol clean-up method D was found not to be compatible with the end-determination steps applied by laboratories B and C. The results of this study have clearly demonstrated that the clean-up methods for both clenbuterol and salbutamol applied by laboratory B yielded superior recoveries with an acceptable standard deviation. Therefore, in conclusion to this study, the participating laboratories recommend the clean-up schemes applied by laboratory B to serve as part of the official Community confirmatory method.

Adrenergic beta-Agonists↗
PubMed2004

Automated segmentation of lumbar vertebrae in digital videofluoroscopic images.

Low back pain is a significant problem in the industrialized world. Diagnosis of the underlying causes can be extremely difficult. Since mechanical factors often play an important role, it can be helpful to study the motion of the spine. Digital videofluoroscopy has been developed for this study and it can provide image sequences with many frames, but which often suffer due to noise, exacerbated by the very low radiation dosage. Thus, determining vertebra position within the image sequence presents a considerable challenge. There have been many studies on vertebral image extraction, but problems of repeatability, occlusion and out-of-plane motion persist. In this paper, we show how the Hough transform (HT) can be used to solve these problems. Here, Fourier descriptors were used to describe the vertebral body shape. This description was incorporated within our HT algorithm from which we can obtain affine transform parameters, i.e., scale, rotation and center position. The method has been applied to images of a calibration model and to images from two sequences of moving human lumbar spines. The results show promise and potential for object extraction from poor quality images and that models of spinal movement can indeed be derived for clinical application.

Algorithms↗
PubMed2006

Process-based reactive transport modeling of a permeable reactive barrier for the treatment of mine drainage.

Reactive transport modeling of a permeable reactive barrier for the treatment of mine drainage was used to integrate a comprehensive data set including pore water chemistry and solid phase data from several sampling events over a >3-year time period. The simulations consider the reduction of sulfate by the organic carbon-based treatment material and the removal of sulfate and iron by precipitation of reduced mineral phases including iron monosulfides and siderite. Additional parameters constraining the model include dissolved H2S, alkalinity and pH, as well as a suite of solid phase S-fractions identified by extractions. Influences of spatial heterogeneity necessitated the use of a 2-dimensional modeling approach. Simulating observed seasonal fluctuations and long-term changes in barrier reactivity required the use of temperature dependent rate coefficients and a multimodal Monod-type rate expression accounting for the variable reactivity of different organic carbon fractions. Simulated dissolved concentrations of SO4, Fe, H2S, alkalinity and pH, as well as solid phase accumulations of reduced sulfur phases generally compare well to observed trends over 23 months. Spatial variations, seasonal fluctuations and the time-dependent decline in reactivity were also captured. The modeling results generally confirm, and further strengthen, the existing conceptual model for the site. Overall sulfate reduction and S-accumulation rates are constrained with confidence within a factor of 1.5.

Hydrogen-Ion Concentration↗
PubMed2005

Gill and liver histopathological changes in yellow perch (Perca flavescens) and goldfish (Carassius auratus) exposed to oil sands process-affected water.

The extraction of bitumen from the Athabasca oil sands (Alberta, Canada) produces significant volumes of process-affected water containing elevated levels of naphthenic acids (NAs), ions, and polycyclic aromatic hydrocarbons (PAHs). The sublethal response of aquatic organisms exposed to oil sands constituents in experimental aquatic environments that represent possible reclamation options has been studied. In this study, the effects of process-affected waters on gill and liver tissues in yellow perch (Perca flavescens) and caged goldfish (Carassius auratus) held in several reclamation ponds at Syncrude's Mildred Lake site have been assessed. Following a 3-week exposure, significant gill (epithelial cell necrosis, mucous cell proliferation) and liver (hepatocellular degeneration, inflammatory cell infiltration) histopathological changes were noted in fish held in waters containing high levels of oil sands process-affected water. In addition, measurements of gill dimensions (gill morphometrical indices) proved sensitive and provided evidence of a physiological disturbance (gas exchange) with exposure to oil sands materials. Due to the complexity of oil sands process-affected water, the cause of the alterations could not be attributed to specific oil sands constituents. However, the histopathological parameters were strong indicators of exposure to oil sands process-affected water and morphometrical data were sensitive indicators of pathological response, which can be used to identify the interactive effects of ionic content, NAs, and PAHs in future laboratory studies.

Animals↗
PubMed2005

Protamylasse, a residual compound of industrial starch production, provides a suitable medium for large-scale cyanophycin production.

Protamylasse is a residual compound occurring during the industrial production of starch from potatoes. It contains a variety of nutrients and all necessary minerals and could be used as a carbon, nitrogen, and energy source for the growth of bacteria and also for cyanophycin (CGP) biosynthesis. Media containing protamylasse as the sole compound diluted only in water were therefore examined for their suitability in CGP production. Among various bacterial strains investigated in this study, a recombinant strain of Escherichia coli DH1 harboring plasmid pMa/c5-914::cphA6803, which carries the cyanophycin synthetase structural gene (cphA) from Synechocystis sp. strain PCC6803, was found to be most suitable. Various cultivation conditions for high CGP contents were first optimized in shake flask cultures. The optimized conditions were then successfully applied to 30- and 500-liter fermentation scales in stirred tank reactors. A maximum CGP content of 28% (wt/wt) CGP per cell dry matter was obtained in 6% (vol/vol) protamylasse medium at an initial pH of 7.0 within a cultivation period of only 24 h. The CGP contents obtained with this recombinant strain employing protamylasse medium were higher than those obtained with the same strain cultivated in mineral salts medium or in expensive commercial complex media such as Luria-Bertani or Terrific broth. It was shown that most amino acids present in the protamylasse medium were almost completely utilized by the cells during cultivation. Exceptions were alanine, tryptophan, tyrosine, and most interestingly, arginine. Furthermore, CGP was easily isolated from protamylasse-grown cells by applying the acid extraction method. The CGP exhibited a molecular mass of about 26 to 30 kDa and was composed of 50% (mol/mol) aspartate, 46% (mol/mol) arginine, and 4% (mol/mol) lysine. The use of cheap residual protamylasse could contribute in establishing an economically and also ecologically feasible process for the biotechnological production of CGP.

Amino Acids↗
PubMed2001

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants.

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.

Animals↗
PubMed1992

[Immunologic reaction and ventilatory function in hemp-processing workers].

The relationship between immunological status and respiratory function was studied in 42 hemp workers and in 49 control workers. The highest prevalence of positive skin tests was recorded for a mixture of hemp and flax (64%), followed by flax (48%), hemp on a combining machine (41%), hemp on a carding machine (38%), hemp on spinning and weaving machines (33%), and hemp on a softening machine (20%). The prevalence of positive skin tests in control workers was smaller and varied from 21 to 5%. Increased IgE was found in 35.7% of the hemp workers and in 5.0% of the controls (P less than 0.05). Hemp workers with positive skin tests had a significantly higher prevalence of chronic respiratory symptoms than those with negative skin tests. Acute ventilatory capacity reductions did not differ in respect to the immunological status. An extract of hemp dust caused a constriction of an isolated guinea pig tracheal smooth muscle in vitro. Our data suggest that hemp dust as well as producing an immunological effect may exert an irritant effect on the respiratory system in exposed textile workers.

Adult↗
PubMedDate not supplied

Mass transfer of terpenes through a silicone rubber membrane in a liquid-liquid contacting system.

Terpenoids are important compounds for the fragrance industry, and recently, biocatalytic methods have been developed to produce them from cheap monoterpenes, such as alpha-pinene oxide. The biotransformation of alpha-pinene oxide using resting cells of Pseudomonas fluorescens NCIMB 11671 produces isonovalal (cis-2-methyl-5-isopropylhexa-2,5-dienal), which is a fragrance. However, this biotransformation has technical problems including the following: alpha-pinene oxide undergoes autoxidation in water and light; it is hydrophobic and relatively toxic to the biocatalyst; and it suffers from product inhibition. Therefore, removal of isonovalal as it is formed should reduce its toxicity and increase volumetric productivity and production yield. Aqueous-organic two-phase (AOTP) systems can be used in the biotransformation of hydrophobic substrates and can protect biocatalysts from toxic substrates and products. However, the formation of stable emulsions makes further downstream processing and continuous operation difficult. One solution to these problems is to use a solid-phase membrane between the aqueous and organic phases in a Membrane Bioreactor for Biotransformation (MBB). Since there are no data in the literature on the behavior of terpenes and terpenoids with solid-phase membranes, or their mass transfer behavior, the objective of this work was to measure these parameters for a wide range of compounds so as to be able to design an MBB, and to gain a greater understanding of their behavior in these types of systems. Organic/aqueous (P (org) (aq)) and membrane/aqueous (P (mem) (aq)) partition coefficients were measured first, and subsequently used to quantify the overall mass transfer coefficients (k(ov)). The overall mass transfer coefficient (k(ov)) of alpha-pinene oxide through the membrane was found to be 2.5 x 10(-5) m x s(-1) using thicknesses of both 250 and 1,000 microm. Extraction kinetics were successfully described using a resistance-in-series model and were controlled by the aqueous boundary layer and/or membrane resistance (k(mem)), while organic film resistance played an insignificant role. Aqueous film resistance (k(aq)) was found to be the limiting step for mono- and diterpenes, and the effect of the hydrodynamics on k(ov) was successfully predicted using a Wilson plot. However, the extraction kinetics of larger terpenes, such as steroids, were influenced solely by the k(mem), suggesting that membrane diffusivity also depended on the size of the permeating molecule. Finally, the influence of other terpene byproducts on the flux of alpha-pinene oxide was investigated and found to decrease the flux into the organic phase by up to 10%.

Aldehydes↗
PubMed1976

[The quality of meat and lard of pigs fed waste fat derived from perchlorethylene extraction].

Throughout the fattening period, six pigs of the Landrace breed were fed waste fat from perchlorethylene extraction added to the feed ration at a 5-per-cent rate. The waste fat from the rendering plant contained 0.45% residual perchlorethylene. Two groups, each containing six animals of the same breed, one group being fed a ration without any fat supplement and the other being fed a ration containing 5% of its volume as rendering-plant fat obtained by pressing, were examined at the same time during the trial. The basic composition of meat was not affected by the feeding of waste fat from rendering plants. The lard of pigs fed a supplement of rendering-plant fat with perchlorethylene oxidated somewhat more quickly during storage than the lard of control pigs. The sensory quality of meat stock, boiled and roast meat, lard, and scraps was not affected by the feeding of rendering-plant fat; the taste and flavour of the products were normal. No residues of perchlorethylene were detected either organoleptically or instrumentally in the meat and lard of pigs.

Animal Feed↗
PubMed2004

Integrated biological treatment of recalcitrant effluents from pulp mills.

This work aimed at determining the degree of depuration of a recalcitrant effluent (weak black liquor, WBL) achieved in a series treatment consisting of a first stage methanogenic fluidised bed reactor followed by a second stage aerobic, upflow reactor packed with "biocubes" of Trametes versicolor immobilised onto small cubes of holm oak wood. The mesophilic, lab scale methanogenic fluidised bed reactor contained a microbial consortium immobilised onto granular activated carbon 500 microm average size. The process removed decreasing amounts of organic matter at decreasing hydraulic retention times (HRT), eventually reaching an average of 50% at 0.5 day HRT. Colour and ligninoid removals also decreased with decreasing HRT. Although the methanogenic fluidised bed reactor provided an effective treatment for the degradable organic matter, important concentrations of recalcitrant organic matter and colour still remained in the anaerobic effluent. This anaerobic effluent was fed to the aerobic packed bed reactor. Two HRT were tested in this unit, namely 5 and 2.5 days. The reactor averaged an organic matter removal in the range of 32% COD basis, during an experimental run of 95 days. Colour and ligninoid contents were removed in high percentages (69% and 54%, respectively). There was no significant difference in reactor performance at 5- and 2.5-day HRT. There was a positive correlation between pollutant removal efficiencies and Laccase activity in crude extracts of the reactor liquor. No supplemental soluble carbohydrate was required to sustain the fungus activity and the consistent reactor performance. Overall, the two-stage treatment achieved approximately a 78% removal of the original organic matter of the WBL (COD basis) and ca. 75% of colour and ligninoid contents.

Bacteria, Anaerobic↗
PubMed2002

Impact of a commercial peat moss operation on water quality and biota in a small tributary of the Richibucto River, Kent County, New Brunswick, Canada.

The St-Charles Plain (Kent County, New Brunswick, Canada) commercial peat moss operation has been ongoing since 1983. To process the peat, a dry extraction method is used that requires extensive drainage of the peat bog. The water is directed toward sedimentation ponds, where it drains into a small brook, which feeds into a river affected by tidal salt water. Water discharge from the bog contains large amounts of peat particles that deposit in the surrounding watershed. As a result, the pH of the freshwater sites that receive the drainage water from the commercial operation, is fairly acidic (pH 3.9-4.7). Water samples from or near the peat moss operation have a higher concentration of total phosphorous and total organic carbon. The peat particles contain relatively high levels of total mercury, as reflected by analysis of peat sediments. However, the water samples contained low levels of dissolved mercury. Indigenous samples of biota-namely, sand shrimps (Crangon septemspinosa) and mummichogs (Fundulus heteroclitus)-did not contain mercury levels higher in the impacted sites than in the reference sites. Introduced blue mussels (Mytilus edulis) did not accumulate significant amounts of mercury during a 62-day exposure in the study area. Overall, the data suggest that although relatively large amounts of mercury-containing peat particles are discharged into the ecosystem, bioaccumulation of mercury in the biota does not occur.

Animals↗
PubMed2006

Fermentative production of superoxide dismutase with Kluyveromyces marxianus.

This work sought to develop a fermentative process for the microbial production of superoxide dismutase (SOD), to overcome extraction from animal tissues. Twenty-eight wild-type yeast strains were screened for SOD productivity. Kluyveromyces marxianus L3 showed the highest SOD activity (62 U mg(-1)) and was used for process development. Oxidative stress conditions and parameters affecting oxygen transfer rate were exploited to improve production. The effects of dilution rate (0.067 vs 0.2 h(-1)), aeration pressure (0.3 vs 1.2 bar) and H(2)O(2) (0 vs 50 mM) were studied during chemostat experiments. Low dilution rate, high pressure and H(2)O(2) resulted in an increase in CuZn-SOD up to 475 U mg(-1). When a regulation of oxygen saturation was applied during batch cultures, CuZn-SOD was progressively higher at 60, 80 and 90% dissolved oxygen tension (DOT) (250, 330 and 630 U mg(-1), respectively). Furthermore, the highest growth rate and biomass yield were achieved at 90% DOT, this being therefore the best DOT condition for high overall productivity. Growth and productivity on different carbon sources were compared. Specific activity was higher on glycerol than on lactose or glucose (496, 454 and 341 U mg(-1), respectively). The highest biomass yield was achieved on lactose. It may be therefore the best substrate for SOD production.

Biotechnology↗
PubMed2006

Production of triacetylhydroxytyrosol from olive mill waste waters for use as stabilized bioantioxidant.

A hydroxytyrosol triacetyl derivative was very efficiently produced as a highly pure stabilized antioxidant compound by a short treatment of olive mill waste water (OMWW) organic extracts, rich in hydroxytyrosol, with an acetylating mixture composed of HClO4-SiO2 and Ac2O (Chakborti and Gulhane reaction), in mild and safe conditions. A successive single step of middle pressure liquid chromatography (MPLC) purification of the reaction product was performed, with an overall yield of 35.6%. (This process, including both the Chakborti and Gulhane reaction and the MPLC purification, is protected by an international patent under PCT/IT2005/000781.) The o-diphenol triacetyl derivative was also produced by direct reaction of hydroxytyrosol, previously purified by MPLC, with HClO4-SiO2 and Ac2O, with an overall yield of 29.5%. A further procedure for the production of the hydroxytyrosol triacetyl derivative was consistent with the direct treatment of raw OMWW with the acetylating agent and a single step of MPLC purification, with an overall yield of 27.6%. The purified natural triacetylhydroxytyrosol confirmed the same strong protective effects against the oxidative stress in human cells as the corresponding synthetic compound, likely because of the biochemical activation of the acetyl derivative into the active parent hydroxytyrosol by esterases. We therefore propose the utilization of OMWW for recovering hydroxytyrosol as a natural antioxidant in a chemically stabilized form, with a good yield, which can be potentially used as a nontoxic functional component in nutritional, pharmaceutical, and cosmetic preparations.

Antioxidants↗
PubMed2001

Utilization of brewer's yeast cells for the production of food-grade yeast extract. Part 1: Effects of different enzymatic treatments on solid and protein recovery and flavor characteristics.

Yeast extract was produced from brewer's yeast of a beer factory by combined enzymatic treatments using endoprotease, exoprotease, 5'-phosphodiesterase, and adenosine monophosphate (AMP)-deaminase. Effects of enzyme combination, enzyme dosages and treatment sequence on the recovery of solid and protein, flavor and compositional characteristics were investigated. Exoprotease dosage strongly affected the recovery of protein and degree of hydrolysis (DH) and sensory characteristics. When the yeast cells were treated using optimal combination of endoprotease and exoprotease (0.6% Protamex and 0.6% Flavourzyme), high solid recovery (48.3-53.1%) and the best flavor profile were obtained. Among various treatment sequences using multiple enzymes, treatment with protease followed by nuclease resulted in the highest 5'-guanosine monophosphate (5'-GMP) content. The optimal concentrations of both 5'-phosphodiesterase and AMP-deaminase were found to be 0.03%. After treatments using optimal combination of enzyme, enzyme dosages and treatment sequence for four enzymes, a high solid yield of 55.1% and 5'-nucleotides content of 3.67% were obtained.

AMP Deaminase↗
PubMed2001

Chlorothalonil and its 4-hydroxy derivative in simple quartz sand soils: a comparison of sorption processes.

Quartz sandy soils from Simcoe, Ontario, Canada and North Carolina had sorption properties for chlorothalonil that were nearly the same. For labile surface sorption kinetics, the Simcoe soil gave a pseudo first-order rate constant of kS1 = (7.4 +/- 0.7) x 10(-2) days-1. At equilibrium, the labile surface sorption capacity theta c of Simcoe soil for chlorothalonil was 23.8 x 10(-6) (mol/g). The sorption properties of the 4-hydroxy derivative of chlorothalonil were different in two important respects. They were larger by an order of magnitude, and they were substantially different for the two soils. Sorption by the Simcoe soil was too fast for kinetics measurements by the on-line HPLC micro extraction method, but for the North Carolina soil kS1 = (1.15 +/- 0.01) days-1 was recorded. For the Simcoe and North Carolina soils, respectively, theta c > 200 (mumol/g) and theta c approximately 113 (mumol/g). Two conclusions can be drawn. First, the replacement of the Cl by OH on the 4 position of chlorothalonil makes the sorption effects much greater. Second, the stronger interactions are associated with a greater sensitivity to small differences in the chemical compositions of the soils. Subtle soil properties causing significant effects might include small amounts and physical structures of organic matter and metal oxides. This implies that, for predictive computer models, mechanism parameters will have to be correlated in two dimensions: chemical structure, and the composition and amounts of chemical materials in soils.

Adsorption↗
PubMed2001

Selected reaction monitoring LC-MS determination of idoxifene and its pyrrolidinone metabolite in human plasma using robotic high-throughput, sequential sample injection.

The generation of large numbers of samples during early drug discovery has increased the demand for rapid and selective methods of analysis. Liquid chromatography-tandem mass spectrometry (LC-MS-MS), because of its sensitivity, selectivity, and robustness, has emerged as a powerful tool in the pharmaceutical industry for many analytical needs. This work presents a high-throughput selected reaction monitoring LC-MS bioanalytical method for the determination of idoxifene, a selective estrogen receptor modulator, and its pyrrolidinone metabolite in clinical human plasma samples. The described method uses short, small-bore columns, high flow rates, and elevated HPLC column temperatures to perform LC separations of idoxifene and its metabolite within 10 s/sample. Sequential injections were accomplished with a 215/889 multiple probe liquid handler (Gilson, Inc.), which aspirates eight samples simultaneously and performs its rinse cycle parallel to sample injection, resulting in minimum lag time between injections. This high-throughput method was applied to the determination of idoxifene and its metabolite in clinical human plasma samples. Sample preparation employed liquid/liquid extraction in the 96-well format. Method validation included determination of intra- and interassay accuracy and precision values, recovery studies, autosampler stability, and freeze-thaw stability. The LOQ obtained was 10 ng/mL for idoxifene and 30 ng/mL for the metabolite. Using idoxifene-d5 as an internal standard, idoxifene showed acceptable accuracy and precision values at QC level 1 (QC1, 15 ng/mL), level 2 (QC2, 100 ng/mL), and level 3 (QC3, 180 ng/mL) (85.0% accuracy +/- 12.0% precision, 95.1 +/- 4.9%, and 90.3 +/- 4.7%, respectively). The pyrrolidinone metabolite also showed acceptable accuracy and precision values (using no internal standard for quantitation) at QC1 (60 ng/mL), QC2 (100 ng/mL), and QC3 (180 ng/mL) (104.9 +/- 14.4%, 91.1 +/- 13.0%, and 90.8 +/- 12.2%, respectively). The validated method was applied to the analysis of 613 human clinical plasma samples. An average run time of 23 s/sample (approximately 37 min/ 96-well plate or over 3,700 sample/day) was achieved. The successful validation presented indicates that rapid methods of analysis can efficiently and reliably contribute to the fast sample turnaround required for high sample number generating processes.

Chromatography, Liquid↗