Determination of muramic acid in organic dust by gas chromatography-mass spectrometry

A method is described for the quantitation of muramic acid, a marker of bacterial peptidoglycan, in organic dust. House dust samples were hydrolysed in hydrochloric acid and then extracted with hexane to remove hydrophobic compounds. The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography-mass spectrometry. The muramic acid derivative gave two peaks upon injection into the gas chromatograph-mass spectrometer. Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5–1500 ng of muramic acid. In a house dust sample, 40 ng of muramic acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed). The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust.     

Ergosterol Content in Various Fungal Species and Biocontaminated Building Materials

This paper reports the ergosterol content for microbial cultures of six filamentous fungi, three yeast species, and one actinomycete and the ergosterol levels in 40 samples of building materials (wood chip, gypsum board, and glass wool) contaminated by microorganisms. The samples were hydrolyzed in alkaline methanol, and sterols were silylated and analyzed by gas chromatography-mass spectrometry. The average ergosterol content varied widely among the fungal species over the range of 2.6 to 42 μg/ml of dry mass or 0.00011 to 17 pg/spore or cell. Ergosterol could not be detected in the actinomycete culture. The results for both the fungal cultures and building material samples supported the idea that the ergosterol content reflects the concentration of filamentous fungi but it underestimates the occurrence of yeast cells. The ergosterol content in building material samples ranged from 0.017 to 68 μg/g of dry mass of material. A good agreement between the ergosterol concentration and viable fungal concentrations was detected in the wood chip (r > 0.66, P ≤ 0.009) and gypsum board samples (r > 0.48, P ≤ 0.059), whereas no relationship between these factors was observed in the glass wool samples. For the pooled data of the building materials, the ergosterol content correlated significantly with the viable fungal levels (r > 0.63, P < 0.0001). In conclusion, the ergosterol concentration could be a suitable marker for estimation of fungal concentrations in contaminated building materials with certain reservations, including the underestimation of yeast concentrations.     

Ochratoxin A in airborne dust and fungal conidia

Farm workers are often exposed to high concentrations of airborne organic dust and fungal conidia, especially when working with plant materials. The purpose of this investigation was to study the possibility of exposure to the mycotoxin ochratoxin A (OTA) through inhalation of organic dust and conidia. Dust and aerosol samples were collected from three local cowsheds. Aerosol samples for determination of total conidia and dust concentrations were collected by stationary sampling on polycarbonate filters. Total dust was analysed by gravimetry, and conidia were counted using scanning electron microscopy. A method was developed for extraction and determination of OTA in small samples of settled dust. OTA was extracted with a mixture of methanol, chloroform, HCI, and water, purified on immunoaffinity column, and analysed by ion-pair HPLC with fluorescence detection. Recovery of OTA from spiked dust samples (0.9–1.0 μg/kg) was 74% (quantitation limit 0.150 μg/kg). OTA was found in 6 out of 14 settled dust samples (0.2–70 μg/kg). The total concentration of airborne conidia ranged from < 1.1 × 10⁴ to 3.9 × 15⁵ per m³, and the airborne dust concentration ranged from 0.08 to 0.21 mg/m³. Conidia collected from cultures of Penicillium verrucosum and Aspergillus ochraceus contained 0.4–0.7 and 0.02–0.06 pg OTA per conidium, respectively. Testing of conidial extracts from these fungi in a Bacillus subtilis bioassay indicated the presence of toxic compounds in addition to OTA. The results show that airborne dust and fungal conidia can be sources of OTA. Peak exposures to airborne OTA may be significant, e.g., in agricultural environments. This revised version was published online in August 2006 with corrections to the Cover Date.     

Quantification of ergosterol and 3-hydroxy fatty acids in settled house dust by gas chromatography-mass spectrometry: comparison with fungal culture and determination of endotoxin by a Limulus amebocyte lysate assay.

Ergosterol and 3-hydroxy fatty acids, chemical markers for fungal biomass and the endotoxin of gram-negative bacteria, respectively, may be useful in studies of health effects of organic dusts, including domestic house dust. This paper reports a method for the combined determination of ergosterol and 3-hydroxy fatty acids in a single dust sample and a comparison of these chemical biomarkers determined by gas chromatography-mass spectrometry with results from fungal culture and Limulus assay. Analyses of replicate house dust samples resulted in correlations of 0.91 (ergosterol in six replicates; P < 0.01) and 0.94 (3-hydroxy fatty acids in nine replicates; P < 0.001). The amounts of ergosterol (range, 2 to 16.5 ng/mg of dust) correlated with those of total culturable fungi (range, 6 to 1,400 CFU/mg of dust) in 17 samples, (r = 0.65; P < 0.005). The amounts of endotoxin (range, 11 to 243 endotoxin units/mg of dust) measured with a modified chromogenic Limulus assay correlated with those of lipopolysaccharide (LPS) determined from 3-hydroxy fatty acid analysis of 15 samples. The correlation coefficient depended on the chain lengths of 3-hydroxy acids used to compute the LPS content. The correlation was high (r = 0.88 +/- 0.01; P < 0.001) when fatty acid chains of 10 to 14 carbon atoms were included; the correlation was much lower when hydroxy acids of 16- or 18-carbon chains were included. In conclusion, the results of the described extraction and analysis procedure for ergosterol and 3-hydroxy fatty acids are reproducible, and the results can be correlated with fungal culture and endotoxin activity of organic dust samples.     

Total and Free Ergosterol in Mycelia of Saltmarsh Ascomycetes with Access to Whole Leaves or Aqueous Extracts of Leaves

Three species of saltmarsh ascomycetes were grown in the presence of all of the constituents of their natural substrate (leaves of cordgrass) or were presented only with aqueous extracts of the leaves. These two growth-condition treatments had no significant effect on total ergosterol content of the fungal mycelia, contrary to an earlier hypothesis that availability of plant lipids would lower fungal ergosterol contents. Mycelial content of free ergosterol was about twice as variable as that for total (free plus esterified) ergosterol. Total ergosterol (data pooled for all species) was strongly correlated to organic mycelial mass (r² = 0.43, P < 0.00001, and slope = 4.59 μg of ergosterol mg of organic mass^-1).     

Bioactive extracts of Carum copticum L. enhances efficacy of ciprofloxacin against MDR enteric bacteria

The widespread occurrence of extended spectrum β-lactamases (ESβLs) producing enteric bacteria and their co-resistance with flouroquinolones has impaired the current antimicrobial therapy. This has prompted the search for new alternatives through synergistic approaches with herbal extracts. In this study Carum copticum (seeds) was extracted first in methanol and then subsequently extracted in different organic solvents. MIC of plant extracts, ciprofloxacin and thymol was determined by broth micro-dilution method using TTC. Synergism between plant extracts and ciprofloxacin was assayed by the checkerboard method. Chemical constituents of active extracts were analyzed by GC-MS. Methanolic, hexane and ether extract of Carum copticum exhibited significant antibacterial activity with MIC values ranged from 0.25 mg/ml to 2.0 mg/ml. Synergy analysis between Carum copticum extracts and ciprofloxacin combinations revealed FIC index in the range of 0.093–0.25. About 81% ciprofloxacin resistant ESβL producing enteric bacteria were re-sensitized in the presence of 15.6–250 μg/ml of methanolic extract of Carum copticum. Moreover, ciprofloxacin showed 8 to 64 folds reduction in MIC in presence of 250 and 500 μg/ml of hexane extract. Whereas, 4–32 folds reduction in MIC of ciprofloxacin was achieved in the presence of 31.25 and 62.5 μg/ml of ether extract, indicating synergistic enhancement of drug activity. The chemical analysis of hexane and ether extracts by GC-MS revealed the common occurrence of one or more phenolic hydroxyl at different locations on benzene ring. This study demonstrated the potential use of herbal extract of Carum copticum in combination therapy against ESβL producing bacteria.     

Sensitive and simple determination of mannitol in human brain tissues by gas chromatography–mass spectrometry

A simple, reliable and sensitive gas chromatographic–mass spectrometric method was devised to determine the level of mannitol in various human brain tissues obtained at autopsy. Mannitol was extracted with 10% trichloroacetic acid solution which effectively precipitated brain tissues. The supernatant was washed with tert.-butyl methyl ether to remove other organic compounds and to neutralize the aqueous solution. Mannitol was then derivatized with 1-butaneboronic acid and subjected to GC–MS. Erythritol was used as an internal standard. For quantitation, selected ion monitoring with m/z 127 and 253 for mannitol and m/z 127 for internal standard were used. Calibration curves were linear in concentration range from 0.2 to 20 μg/0.1 g and correlation coefficients exceeded 0.99. The lower detection limit of mannitol in distilled water was 1 ng/0.1 g. Mannitol was detected in control brain tissues, as a biological compound, at a level of 50 ng/0.1 g. The precision of this method was examined with use of two different concentrations, 2 and 20 μg/0.1 g, and the relative standard deviation ranged from 0.8 to 8.3%. We used this method to determine mannitol in brain tissues from an autopsied individual who had been clinically diagnosed as being brain dead. Cardiac arrest occurred 4 days later.     

Dominance of mixed ether/ester,intact polar membrane lipids in five species of the order Rubrobacterales: Another group of bacteria not obeying the “lipid divide”

The composition of the core lipids and intact polar lipids (IPLs) of five Rubrobacter species was examined. Methylated (ω-4) fatty acids (FAs) characterized the core lipids of Rubrobacter radiotolerans, R. xylanophilus and R. bracarensis. In contrast, R. calidifluminis and R. naiadicus lacked ω-4 methyl FAs but instead contained abundant (i.e., 34–41 % of the core lipids) ω-cyclohexyl FAs not reported before in the order Rubrobacterales. Their genomes contained an almost complete operon encoding proteins enabling production of cyclohexane carboxylic acid CoA thioester, which acts as a building block for ω-cyclohexyl FAs in other bacteria. Hence, the most plausible explanation for the biosynthesis of these cyclic FAs in R. calidifluminis and R. naiadicus is a recent acquisition of this operon. All strains contained 1-O-alkyl glycerol ether lipids in abundance (up to 46 % of the core lipids), in line with the dominance (>90 %) of mixed ether/ester IPLs with a variety of polar headgroups. The IPL head group distribution of R. calidifluminis and R. naiadicus differed, e.g. they lacked a novel IPL tentatively assigned as phosphothreoninol. The genomes of all five Rubrobacter species contained a putative operon encoding the synthesis of the 1-O-alkyl glycerol phosphate, the presumed building block of mixed ether/ester IPLs, which shows some resemblance with an operon enabling ether lipid production in various other aerobic bacteria but requires more study. The uncommon dominance of mixed ether/ester IPLs in Rubrobacter species exemplifies our recent growing awareness that the lipid divide between archaea and bacteria/eukaryotes is not as clear cut as previously thought.     

Fluorescent/ultraviolet absorbing ester derivative formation and analysis of eicosanoids by high-pressure liquid chromatography

A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B₂ chromatographs as two species and coelutes with PGF_2α. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF_1α, 6-keto-PGE₁, 13,14-dihydro-6,15-diketo-PGF_1α). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF_1α extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF_1α quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.     

Production of biomass, carotenoid and other lipid metabolites by several red yeast strains cultivated on waste glycerol from biofuel production – a comparative screening study

This study was focused on a comparison of growth and production properties of seven red yeast strains of the genus Rhodotorula, Sporobolomyces and Cystofilobasidium cultivated on glycerol substrate. Production of enriched yeast biomas and specific yeast metabolites (carotenoids, ergosterol, lipids) was evaluated on medium with glucose, pure technical glycerol and/or waste glycerol from biofuel production (40 g/L) and mixture of glycerol and glucose (1:3, 1:1, 3:1; C/N ratio 57 in all cultivations). All tested strains were able to utilize glycerol as the only carbon source. Production of biomass on waste glycerol was in most strains higher than in control as well as in medium with pure technical glycerol and reached 15.97–21.76 g/L. Production of carotenoids and ergosterol was better in glucose medium than in medium with glycerol only. Nevertheless, using glycerol medium with addition of glucose, higher yields of total carotenoids, beta-carotene and ergosterol were obtained than in control. The highest yields of total pigments were reached by Sporobolomyces roseus (3.60 mg/g cell dry weight (CDW); glycerol:glucose 1:3), Sporobolomyces salmonicolor (2.85 mg/g CDW; glycerol:glucose 1:3) and Rhodotorula glutinis (2.80 mg/g CDW; glycerol:glucose 3:1) In glucose medium, most tested strains except Cystofilobasidium capitatum (22.6 %) produced neutral lipids in the range of 11–15 %. Production of triacylglycerols in all strains was in 10–30 % better in glycerol medium, in which Rhodotorula aurantiaca and Sporobolomyces shibatanus also reached intracellular triacylglycerol concentrations up to 20 % of biomass. This study has shown that oleaginous red yeasts could have great potential for converting crude glycerol to valuable lipids and carotenoids in respect of efficient bioresources utilization.     

Optimization of polysaccharide and ergosterol production from Agaricus brasiliensis by fermentation process

Statistically based experimental designs were applied for the fermentation optimization of polysaccharide and ergosterol production from Agaricus brasiliensis in a stirred-fermenter. Culture temperature, agitation speed and initial pH were identified to have significant effects on polysaccharide and ergosterol production by a Plackett–Burman design. These three significant factors were subsequently optimized using steepest ascent method and Box–Behnken design. The validity of the optimum conditions was verified by separate experiments in which the polysaccharide and ergosterol yields were increased by 57% and 43%, respectively, as compared to those under unoptimized fermentation conditions.     

Uranium quantification in semen by inductively coupled plasma mass spectrometry

In this study we report uranium analysis for human semen samples. Uranium quantification was performed by inductively coupled plasma mass spectrometry. No additives, such as chymotrypsin or bovine serum albumin, were used for semen liquefaction, as they showed significant uranium content. For method validation we spiked 2 g aliquots of pooled control semen at three different levels of uranium: low at 5 pg/g, medium at 50 pg/g, and high at 1000 pg/g. The detection limit was determined to be 0.8 pg/g uranium in human semen. The data reproduced within 1.4–7% RSD and spike recoveries were 97–100%. The uranium level of the unspiked, pooled control semen was 2.9 pg/g of semen (n = 10). In addition six semen samples from a cohort of Veterans exposed to depleted uranium (DU) in the 1991 Gulf War were analyzed with no knowledge of their exposure history. Uranium levels in the Veterans’ semen samples ranged from undetectable (<0.8 pg/g) to 3350 pg/g. This wide concentration range for uranium in semen is consistent with known differences in current DU body burdens in these individuals, some of whom have retained embedded DU fragments.     

Synergistic antifungal activity of statin–azole associations as witnessed by Saccharomyces cerevisiae- and Candida utilis-bioassays and ergosterol quantification

BackgroundFrequent opportunist fungal infections and the resistance to available antifungal drugs promoted the development of new alternatives for treatment, like antifungal drug combinations.AimsThis work aimed to detect the antifungal synergism between statins and azoles by means of an agar-well diffusion bioassay with Saccharomyces cerevisiae ATCC 32051 and Candida utilis Pr_1–2 as test strains.MethodsSynergistic antifungal effects were tested by simultaneously adding a sub inhibitory concentration (SIC) of statin (atorvastatin, lovastatin, pravastatin, rosuvastatin or simvastatin) plus a minimal inhibitory concentration (MIC) of azole (clotrimazole, fluconazole, itraconazole, ketoconazole or miconazole) to yeast-embedded YNB agar plates, and a positive result corresponded to a yeast growth inhibition halo higher than that produced by the MIC of the azole alone. Yeast cell ergosterol quantification by RP-HPLC was used to confirm statin–azole synergism, and ergosterol rescue bioassays were performed for evaluating statin-induced ergosterol synthesis blockage.ResultsGrowth inhibition was significantly increased when clotrimazole, fluconazole, itraconazole, ketoconazole and miconazole were combined with atorvastatin, lovastatin, rosuvastatin and simvastatin. Highest growth inhibition increments were observed on S. cerevisiae (77.5%) and C. utilis (43.2%) with a SIC of simvastatin plus a MIC of miconazole, i.e. 4 + 2.4 μg/ml or 20 + 4.8 μg/ml, respectively. Pravastatin showed almost no significant effects (0–7.6% inhibition increase). Highest interaction ratios between antifungal agents corresponded to simvastatin–miconazole combinations and were indicative of synergism. Synergism was also confirmed by the increased reduction in cellular ergosterol levels (S. cerevisiae, 40% and C. utilis, 22%). Statin-induced ergosterol synthesis blockage was corroborated by means of ergosterol rescue bioassays, pravastatin being the most easily abolished inhibition whilst rosuvastatin being the most ergosterol-refractory.ConclusionsSelected statin–azole combinations might be viable alternatives for the therapeutic management of mycosis at lower administration doses or with a higher efficiency.     

Measurement of ergosterol,diaminopimelic acid and glucosamine in soil: evaluation as indicators of microbial biomass

Methods were developed for the measurement of ergosterol, diaminopimelic acid (DAP) and glucosamine in soil as possible indicators of, respectively, fungal, bacterial and total microbial biomass. Ergosterol, obtained by saponification of methanol extracts of soil, was measured by high pressure liquid chromatography with ultra-violet detection. DAP and glucosamine in acid hydrolysates of soil were separated and assayed by quantitative paper chromatography. Physical losses in extraction (generally < 15%) were quantified using ¹⁴C-labelled compounds. Amount (with coefficients of variation) in grassland and arable soils were 0.99–2.06 μg ergosterol (2–16%), 17–163 μg DAP (10–36%) and 505–2109 μg glucosamine (6–23%) per g soil. Evaluation of the DAP and glucosamine figures on the basis of known soil biomass data indicated that these compounds were largely associated with non-living organic matter. In contrast, the ergosterol measured was of the order expected from the fungal biomass present, and this substance may therefore provide a valuable biomass indicator.     

Microdetermination of the 6,15-diketo-13,14-dihydroprostaglandin F1alpha in human plasma using gas chromatography/selected ion monitoring with [18O]6,15-diketo-13,14-dihydro-prostaglandin F1alpha as an internal standard

We devised a simple and effective purification method for the microdetermination of 6,15-diketo-13,14-dihydro-prostaglandin F1alpha (DK), a metabolite of prostacyclin (PGI2). [18O]DK was synthesized from the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O] water to obtain an internal standard for the gas chromatography/selected ion monitoring (GC/SIM) of DK. The methyl ester-methoxime-tert-butyldimethylsilyl ether derivative was prepared, then gas chromatography/selected ion monitoring was carried out by monitoring the ion at m/z 613.4 for DK and at m/z 617.4 for an internal standard. A good linear response over the range of 10 pg to 10 ng was demonstrated. We detected DK to a level of about 297.8 pg/ml in human plasma. This method can be used to determine DK in biological samples.     

Synthesis and antibacterial studies of binaphthyl-based tripeptoids. Part 2

A compact synthesis of 15 new binaphthyl-based dicationic tripeptoids and one biphenyl based dicationic tripeptoid is described. Fourteen of these tripeptoids resulted from variation of the C-2′ ether substituent of the binaphthyl unit. An O-iso-butyl ether binaphthyl derivative was found to be the most active against Staphylococcus aureus (MIC 1.95 μg/mL). The biphenyl analogue also showed good activity against S. aureus (MIC 1.95 μg/mL). These compounds, however, were less active against four vancomycin-resistant strains of enterococci (VRE) than some of our previously developed compounds that had an O-iso-pentyl ether substituent on the binaphthyl unit and a C-2 l-Leu moiety.     

Mesoionic compounds with antifungal activity against Fusarium verticillioides

Background

Fungi contaminate the food of humans and animals, are a risk to health, and can cause financial losses. In this work, the antifungal activities of 16 mesoionic compounds (MI 1–16) were evaluated against mycotoxigenic fungi, including Aspergillus spp., Fusarium verticillioides and Penicillium citrinum. Furthermore, the decreased ergosterol in the total lipid content of Fusarium verticillioides was investigated.

Results

F. verticillioides was the most sensitive fungus to the mesoionic compounds. Among the evaluated compounds, MI-11 and MI-16 presented higher antifungal effects against F. verticillioides, with MIC values of 7.8 μg/ml, and MI-2 and MI-3 followed, with MICs of 15.6 μg/ml. The most active compounds were those with heterocyclic ring phenyl groups substituted by electron donor moieties (MI-11 and MI-16). Among some compounds with higher activity (MI-2, MI-11 and MI-16), decreased ergosterol content in the total lipid fraction of F. verticillioides was demonstrated. MI-2 reduced the ergosterol content approximately 40% and 80% at concentrations of 7.8 μg/ml and 15.6 μg/ml, respectively, and MI-11 and MI-16 decreased the content by 30% and 50%, respectively, when at a concentration of 7.8 μg/ml.

Conclusion

These findings indicate that mesoionic compounds have significant antifungal activity against F. verticillioides.     

Fungi and fungal products in some Canadian houses

Building related illness prompted a study in the winter of 1986 to identify and quantify and fungal products present in c. 50 Canadian homes. Of these, 70% had been reputedly associated with health problems. Building parameters, i.e. air change rate and the internal moisture levels, were measured, and the fungi present were characterized and quantified along with their metabolites. Air and dust samples were analyzed and the fungal biomass in the dust was measured by a procedure which involved determination of ergosterol by a gas chromatograph/mass spectrometer system. Some 42 fungal species were identified in air, samples of which were further analyzed for fungal volatiles. Penicillium was the most common genus in both air and dust, together with Cladosporium and Alternaria. The potentially hazardous fungus Aspergillus fumigatus was found in only two houses, and Strachybotry atra in only one. New criteria are suggested to define the acceptable standards for indoor fungal levels in air during winter.     

High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis

Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine. In addition, small amounts of epinephrine and norepinephrine were detected. Logarithmic-phase strain NT-1 cells contained 249±44 pg dopamine/10⁶ cells compared to 477±42 pg/10⁶ cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells. The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells. There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells.     

Development of sensitive high-performance liquid chromatography with fluorescence detection using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride as a labeling reagent for determination of bisphenol A in plasma samples

A sensitive HPLC method for determination of bisphenol A (BPA) in plasma samples using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a fluorescence labeling reagent was developed. The fluorescence labeling reaction was completed within 10 min at room temperature. DIB-Cl reacts with the phenolic hydroxyl group of BPA in the presence of triethylamine (TEA). The DIB-Cl derivative of BPA (DIB-BPA) was separated within 30 min with an ODS column using acetonitrile–water (90:10, v/v) as the isocratic eluent. Calibration graphs were linear over the range of 1.0–100 ng/ml (r=0.999). The detection limit of DIB-BPA was 0.05 ng/ml (2.5 pg) at a signal-to-noise ratio of 3. The relative standard deviations (RSDs) of the method for between-run were 1.0–5.0%. The analytical recoveries of known amounts (1.0 and 100 ng/ml) of BPA-spiked rabbit plasma were around 95%.