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PubMed2026

A liquid handling platform for standardized quantification of cell-free enzymatic activity encoded by antimicrobial resistance genes.

Antimicrobial resistance (AMR) is a growing global threat to human health, and rapid methods for characterizing emerging antimicrobial resistance genes (ARGs) are needed. Here, we develop a semi-automated workflow using cell-free gene expression systems to measure the activity of two ARGs encoded on plasmid DNA that produce rifampicin-inactivating and gentamicin-inactivating enzymes. We validated the use of a small benchtop Myra liquid handling system compared to manual pipetting, with no statistical differences observed. After optimizing the pre-incubation time of ARGs and dispensing protocol, expression of aac(3)-IIa increased the half-maximal inhibition concentration (IC50) of gentamicin by over 150-fold, whilst arr-3 increased the IC50 of rifampicin by ~20-fold compared to controls. This methodology for rapid, semi-automated ARG characterization offers a strategy to combat AMR by assessing novel ARGs identified through genomic surveillance or profiling activity of new or derivative antibiotics.

aminoglycoside resistance↗
PubMed2004

Spatial alternation deficits following developmental exposure to Aroclor 1254 and/or methylmercury in rats.

Polychlorinated biphenyls (PCBs) and methylmercury (MeHg) are ubiquitous environmental contaminants that alter cognitive function in both humans and animals. Because PCBs and MeHg often occur together in the environment, it is important to understand whether these two contaminants have the potential to interact, causing additive or greater than additive effects. The current study examined the combined effects of gestational and lactational exposure to Aroclor 1254 (A1254), a commercial PCB mixture, and MeHg on a series of spatial alternation tasks including cued spatial alternation (CA), non-cued spatial alternation (NCA), and delayed spatial alternation (DSA) in rats using standard two-lever operant testing chambers. Pregnant Long-Evans rats received either 6 mg/kg A1254 pipetted onto a Keebler Vanilla Wafer cookie (PCB-only group), 0.5 ppm. MeHg dissolved in the drinking water (MeHg-only group), 6 mg/kg A1254 + 0.5 ppm. MeHg (PCB + MeHg group), or corn oil vehicle and normal tap water (control group) beginning 28 days prior to mating and continuing through postnatal day 16. One male and one female from each litter began testing on spatial alternation at approximately 110 days of age. Animals were reinforced for pressing the lever opposite that pressed on the previous trial. In general, animals exposed to A1254 and/or MeHg were impaired relative to control rats on the NCA and DSA tasks. Significant reductions in NCA performance were observed in the MeHg-only and PCB + MeHg groups, while significant reductions in DSA performance were observed in the PCB-only and MeHg-only groups. The PCB + MeHg group showed a similar magnitude reduction in performance on DSA, but this difference was not statistically significant due to increased variability in that group. The reductions in DSA performance were observed across most of the delays, indicating that memory impairments were not likely the cause of the deficit. Instead, the DSA deficits following exposure to A1254 and/or MeHg are indicative of either an associative or attentional impairment. The results from the current study indicate that combined exposure to PCBs and MeHg does not exacerbate the PCB- or MeHg-induced impairments on spatial alternation tasks.

Animals↗
PubMed2005

Auditory deficits in rats exposed to an environmental PCB mixture during development.

Previous studies have indicated that developmental exposure to polychlorinated biphenyls (PCBs) may result in hearing impairment in rats. The cochlea is the suggested site of action, based upon one study demonstrating a loss of outer hair cells on the basilar membrane, and another demonstrating deficits in distortion product otoacoustic emissions (DPOAEs). The current study was conducted to assess the possible ototoxic effects of a unique PCB mixture formulated to model the congener profile of PCBs found in fish consumed by a human population in northeastern Wisconsin. Female Long-Evans rats were dosed orally with the PCB mixture beginning 28 days prior to breeding and continuing until the pups were weaned. Dams were fed one-half of a cookie onto which was pipetted 0, 1, 3, or 6 mg/kg of the PCB mixture dissolved in a corn oil vehicle. On postnatal day (PND) 21, pups were weaned, and one male and one female from each litter were randomly selected for auditory assessment. DPOAEs were measured to assess cochlear function, and auditory brainstem responses (ABRs) were measured to determine effects on central nervous system auditory pathways. DPOAE amplitudes were decreased, and DPOAE and ABR thresholds were elevated across a range of frequencies in PCB-exposed rats. These results support and extend previous reports of auditory impairment in PCB-exposed rats. Developmental exposure to PCBs may also result in subtle auditory impairments in humans, and if so, this may contribute to some of the cognitive deficits that have been observed in epidemiological studies.

Administration, Oral↗
PubMed1998

Behavior of transgenic mouse spermatozoa with galline protamine.

General morphology, physical and chemical stability of nuclei, and postfertilization behavior of spermatozoa from transgenic mice [TgN (Prml Gal) 223 Bri] containing nuclear avian protamine (galline) were compared to those in the spermatozoa of wild-type (Wild) mice. Galline to protamine I ratios in spermatozoal nuclei of transgenic mice, strains 3175 (T75) and 3177 (T77), were 1.94 and 5.62, respectively. Live T75 and T77 spermatozoa were indistinguishable in their gross morphology from Wild spermatozoa. However, unlike Wild and T75 spermatozoa, T77 spermatozoa were vulnerable to mechanical handling, as about 40% of heads and tails were separated after gentle pipetting in suspension. Motility of T77 spermatozoa was markedly inferior to that of T75 and Wild. Chromatin heterogeneity and instability of transgenic spermatozoal nuclei were evident by transmission electron microscopy, staining reaction to Giemsa, and, as apparent by both light microscopy and flow cytometry, reaction to SDS detergent. Wild and T75 spermatozoa fertilized 90% and 60% of zona-intact oocytes in vitro, respectively. T77 spermatozoa completely failed to fertilize and bound to zona surfaces very weakly, and none of them inserted their heads into the zona. Although inefficiently, T77 spermatozoa could fertilize zona-free oocytes in vitro, indicating some ability to undergo capacitation and spontaneous acrosome reaction in vitro. After microsurgical injection into oocytes, the rate of nuclear decondensation was the greatest in rooster spermatozoa, followed by T77, T75, and Wild spermatozoa.

Animals↗
PubMed2000

Development of in vivo-matured porcine oocytes following intracytoplasmic sperm injection.

The objective of this study was to assess the development of porcine ova fertilized by intracytoplasmic sperm injection (ICSI). Allyl trenbolone (Regumate) was used to synchronize estrus in 13 postpuberal gilts. Gilts were superovulated with pregnant mare serum gonadotropin and hCG. Ova were aspirated from 5- to 8-mm follicles at 36 h after hCG. Cumulus cells were removed by blunt dissection and pipetting in Beltsville embryo culture medium (BECM) supplemented with 0.1% hyaluronidase. Sperm were washed and resuspended in BECM + 8% polyvinylpyrrolidone. Ova (n = 237) that exhibited a polar body were centrifuged at 15 000 x g for 6 min and injected with a single spermatozoon. One hundred fifty-four ova were cultured in NCSU-23 medium in a 5% CO(2) in air environment for 168 h. Ova were fixed in acetic acid/ethanol and stained with 1% orcein. Sixty-nine ICSI ova were cultured for 24 h and transferred (mean = 23) to three recipients. Eighty-one ova (69%) that survived ICSI cleaved within 48 h. Thirty-eight percent (31/81) of these ova became blastocysts (mean +/- SEM = 24.7 +/- 1.1 cells). One recipient gave birth to three pigs. These results demonstrate that porcine embryos derived from ICSI can develop into live pigs.

Animals↗
PubMed2000

A quick anti-Xa-activity-based whole blood coagulation assay for monitoring unfractionated heparin during cardiopulmonary bypass: a pilot investigation.

UNLABELLED: We developed a quick and easy method to perform anti-Xa-activity-based whole blood assay and assessed its reliability for online monitoring of unfractionated heparins (UFHs) during cardiopulmonary bypass. Seventy-five microliters of a mixture of 1:3 large- and small-range Heptest reagent were transferred into blank cartridges of the ACT II device. The plastic flags for clot detection and stirring the sample and reagent were inserted and overlaid with 75 microL of Recalmix for recalcification. One-hundred fifty microliters of citrated whole blood were added and measurements performed. In vitro, the linearity of the test over a range of 1-8 IU/mL UFH, as well as the influence of variations in hematocrit (60%, 30%, and 20%), plasma coagulation factors (50%, 30%, and 20%) and platelets (100, 50, and 20 x 10(3)/microL) on the test results were assessed. In vivo measurements performed during cardiopulmonary bypass were compared with the chromogenic assay. The test revealed linearity to concentrations of 6 IU/mL of UFH and was not significantly influenced by the variations in the in vitro set-up despite a prolongation in samples with a hematocrit of 60%. In vivo, the correlation to the chromogenic test was R: = 0.90. The ACT II anti-Xa-UFH assay performed in whole blood was reliable when used over a wide range of conditions that could be encountered clinically. Although the test is useful for point-of-care monitoring, the necessity of individual calibrations and pipetting in the operation room requires further automation before its use in clinical practice. IMPLICATIONS: The ACT II anti-Xa-unfractionated heparin assay allows for reliable monitoring of large concentrations of UFH over a wide range of hematocrit, platelet, and coagulation factor levels. Further evaluation of this point-of-care device is indicated.

Adult↗
PubMed1991

Drug contamination from opening glass ampules.

Recently there have been several reports of postoperative sepsis due to the intravenous injection of contaminated solutions of propofol (Diprivan). The mechanism by which this contamination occurred has not been identified. This study sought to determine whether bacterial contamination of the contents of glass ampules can be decreased by swabbing the neck of the vial with alcohol prior to opening. Glass ampules of 1% propofol and 1% lidocaine were swabbed with a solution of Staphylococcus epidermidis. Half of these ampules were subsequently wiped with alcohol pads prior to being opened. An aliquot from each ampule was pipetted into a nutrient broth and allowed to incubate overnight at 37 degrees C. These solutions were plated on agar, incubated for 24 h, and then examined for bacterial growth. Three of the eight lidocaine ampules and six of the eight propofol ampules not cleaned with alcohol demonstrated evidence of bacterial contamination. The contents of all ampules that had been wiped with alcohol prior to being opened remained sterile (P less than 0.001 vs. non-alcohol-treated group for propofol ampules and P = 0.20 vs. non-alcohol-treated group for lidocaine ampules). These data suggest that bacterial contamination of propofol and lidocaine may occur as a result of opening glass ampules. Wiping the outside of the ampule with alcohol immediately prior to opening may decrease this risk.

Alcohols↗
PubMed2004

Surfactants attenuate gas embolism-induced thrombin production.

BACKGROUND: There are no pharmacologic strategies to prevent embolism bubble-induced blood clot formation. The authors conducted experiments to measure thrombin production in sheared whole blood in the presence and absence of bubbles and three surface-active compounds. METHODS: Blood samples were obtained from six volunteers seven times. The thrombin-specific substrate Boc-VPR-MCA was added to citrated blood diluted with HEPES-buffered saline. Experimental groups were as follows: sparging (air microbubble embolization) with surfactant present; sparging alone; surfactant alone; and neither surfactant nor sparging. The surfactants were Dow Corning Antifoam 1510US, Perftoran, and Pluronic F-127. Blood was sheared by a cone-plate viscometer at 100 and 500 s-1 for 5, 10, and 20 min at 37 degrees C, pipetted into excess stop buffer, and evaluated fluorimetrically. Mean values of fluorescence intensity +/- SDs for each group were compared using ANOVA. Differences were considered significant at P < 0.05 using the Bonferroni correction. RESULTS: For fixed shear rate, thrombin production increased 2.3- to 5.7-fold (P < 0.05) as shear duration lengthened. For fixed shear duration, thrombin production increased 1.9- to 3.9-fold (P < 0.05) with increasing shear rate. For fixed shear rate and duration, sparging increased thrombin production 2.1- to 3.7-fold (P < 0.05). Surfactant addition without sparging did not change thrombin production (P > 0.05). Surfactants attenuated thrombin production in sparged samples 31.8-70.9% (P < 0.05). CONCLUSIONS: Thrombin production is shear rate and duration-dependent. Sparging increases thrombin production. Surfactants added before sparging attenuate thrombin production. Surfactants may have a clinical application to attenuate gas embolism-induced clotting.

Blood Platelets↗
PubMed1998

Diversity of single potassium channels in isolated snail neurons.

Comparison of K+ channels in mollusk and mammalian neurons has been made to elucidate their fundamental properties. Using patch clamp cell-attached configuration, K+ channels in isolated snail neurons were separated into three subtypes: with big (BKC), medium (MKC) and small (SKC) unitary conductances. BKC and MKC were activated at -30 mV and SKC at more negative potentials. BKC and MKC proved sensitive to TEA, whereas SKC were sensitive to 4-AP. Cd2+ in the pipet decreased unitary conductance of BKC by 55% and of MKC by about 31%. Bath application of 5-HT selectively suppressed MKC. It is suggested that BKC can be referred to large conductance Ca(2+)-dependent K+ currents (KCa), MKC to intermediate conductance KCa and SKC channels comply with the characteristics of A current of mammals. These data show that KCa and A currents may be the most general types of currents generated by K+ channels.

Animals↗
PubMed1995

Near-patient measurements of methemoglobin, oxygen saturation, and total hemoglobin: evaluation of a new instrument for adult and neonatal intensive care.

OBJECTIVES: a) To evaluate the performance of a compact, new instrument that uses disposable cuvettes to measure total hemoglobin concentration, oxygen content, and the relative concentrations of oxy- and methemoglobin in 50-microL blood samples; b) to determine whether the instrument can be used for near-patient assessment of methemoglobinemia; and c) to ascertain whether problems commonly encountered in neonatal blood samples affect the instrument's performance. DESIGN: Prospective study, in which the test instrument was compared with a standard method. Samples of whole blood with and without bilirubin, fetal hemoglobin, and hemolysis were analyzed on the new (test) instrument and on a widely used cooximeter (OSM3 hemoximeter, Radiometer; reference instrument). SETTING: In vitro analyses of blood samples in clinical and university laboratories. MEASUREMENTS AND MAIN RESULTS: There was a close linear correlation between the methomoglobin measurements of the test instrument and those measurements of the reference instrument (slope = 0.989; r2 = .989). The average difference in mean assay values between the reference instrument and the test instrument was -0.59%, i.e., < 1% methemoglobin. Repeated measurements indicated the precision was 0.5% methemoglobin. Complete hemolysis of the sample reduced the methemoglobin reading by only 0.40%. Adding bilirubin (10 to 11 mg/dL [171 to 188.1 mumol/L]), increased the methemoglobin reading by 0.23%, increased the oxyhemoglobin reading by 0.45%, and increased total hemoglobin by 0.21 g/dL. Fetal hemoglobin also had minimal effects on the readings. CONCLUSIONS: The test instrument is fast and easy to operate. No sample preparation or pipetting is required. To operate the instrument, the user simply connects a syringe containing the blood sample to one of the disposable cuvettes, injects 50 microL of blood into the cuvette, and inserts the cuvette into the instrument. The test instrument automatically detects the presence of the cuvette, analyzes the sample, and displays the results in < 10 secs. The findings in this study indicate that the test instrument has sufficient accuracy for near-patient testing in intensive care units. The errors introduced by hemolysis, fetal hemoglobin, and bilirubin were too small to be of clinical importance. Thus, the test instrument is essentially unaffected by complications commonly encountered in neonatal blood. The capacity of the test instrument to measure methemoglobin makes it particularly useful if inhaled nitric oxide therapy becomes a standard clinical practice.

Adult↗
PubMed2000

Effect of gutta-percha solvents on surface microhardness of IRM fillings.

Eighty identical samples of IRM were prepared and embedded in acrylic resin. The samples were polished, rinsed, and divided into experimental groups and controls, 10 specimens each. Each experimental group was treated for 1 or 5 min with one of the following gutta-percha solvents: chloroform, xylene, or halothane. Sterile saline was used as a control. Treatment consisted of pipetting each solvent onto the external IRM surface. The specimens were then sealed in plastic assay tubes for the corresponding exposure periods. At each time period, surface microhardness of the samples was measured using a mini-load hardness tester with a 500 g load for 20 s. It was found that xylene and halothane caused significant reduction in surface microhardness of IRM after 5 min (p < 0.01). Chloroform caused significant reduction in surface microhardness of IRM after 1 min of treatment (p < 0.01). Chloroform caused total dissolution after 5 min of treatment. Saline caused only minor and nonsignificant changes in surface microhardness of IRM.

Acrylic Resins↗
PubMed2000

Resistance of a novel root canal sealer to bacterial ingress in vitro.

A dentin-bonding root canal sealer (ZUT) has been developed, consisting of an experimental glass ionomer cement (KT-308) and an antimicrobial silver-containing zeolite (0.2% by weight). This in vitro study evaluated the ability of ZUT used with or without gutta-percha, to resist bacterial ingress of Enterococcus faecalis over a period of 90 days. Canals of 80 single-rooted teeth were prepared with apical patency and filled as follows (n = 10): KT-308 alone; KT-308 with a single gutta-percha cone (SCGP); ZUT alone; ZUT with SCGP; AH26 alone; AH26 with SCGP; positive control-no root canal filling; and negative control-no root canal filling, with the apices of this group sealed with C&B Metabond cement. Teeth were coated with nail polish except for the apical 2 mm, and each tooth was sealed in a 4-ml glass vial, with an 18-gauge needle inserted through the vial cover and bonded into the pulp chamber with C&B Metabond cement. After sterilization with 2.5 Mrad gamma-radiation, Brain Heart Infusion broth with phenol red was injected into each vial. An inoculum of E. faecalis was pipetted through the needle into the pulp chamber every 5 days, and the broth was monitored daily for color change and turbidity. When change occurred, the broth was cultured for growth of E. faecalis. Kaplan-Meier survival analysis and the log-rank test revealed no significant differences among the three sealers used. The presence of gutta-percha, however, significantly improved resistance to bacterial ingress through obturated root canals (X, p < 0.05). Under the conditions of this study, the hypothesized advantage of ZUT (0.2% zeolite) was not demonstrated.

Anti-Infective Agents, Local↗
PubMed2003

In vitro cytotoxicity of medicated and nonmedicated gutta-percha points in cultures of gingival fibroblasts.

This investigation was designed to test the cellular toxicity of two medicated (Roeko activ point and Roeko Calcium Hydroxide) and four nonmedicated brands of gutta-percha (GP) points (Antaeos, DeTrey White, Roeko color, and Roeko Top color). The test points were transferred into a culture medium including the GP-point material with a concentration of 6 mg/ml, and eluates were obtained after 72 h. Five milliliters of each eluate were pipetted onto fibroblast cultures, incubated, and subsequently stained. Mitotic rates, cell densities, and the distribution of normal cells, pathologically altered and dead cells were determined and correlated with control cell cultures. Roeko activ point (containing chlorhexidine) resulted in the highest number of dead cells. The difference was statistically significant in comparison with all other materials. Concerning all parameters mentioned, the cytotoxicity of the points containing calcium hydroxide (Roeko Calcium Hydroxide) was not significantly different from all other points tested, with the exception of those containing chlorhexidine. All tested gutta-percha materials caused cytotoxic reactions in varying extents. Taking into consideration the limitations of an in vitro experiment, points containing calcium hydroxide and nonmedicated points seem to be the most recommendable products for clinical use.

Anti-Infective Agents, Local↗
PubMed2003

The effect of passive ultrasonic activation of 2% chlorhexidine or 5.25% sodium hypochlorite irrigant on residual antimicrobial activity in root canals.

Ninety-four single-canal roots were prepared using the step-down technique. Forty-two canals were irrigated with 2% chlorhexidine, 42 canals with 5.25% sodium hypochlorite (NaOCl), and 10 control canals with phosphate-buffered saline (PBS). The chlorhexidine and NaOCl groups were each then equally divided into a final irrigation group and a 1-min passive ultrasonic irrigation group. Canals were enlarged with a Parapost drill. The apical 3-5 mm was covered with nail polish. Canals were rinsed with PBS, dried, refilled with PBS, and stored. At 6 h, 20 microl of fluid was pipetted from each canal and placed into wells on agar plates, which were inoculated with Streptococcus sanguinis. The plates were incubated, and zones of inhibition were measured. Sampling was repeated at 24, 48, 72, 96, 120, 144, and 168 h. Residual antimicrobial activity with 2% chlorhexidine was statistically significantly superior to 5.25% NaOCl with irrigation alone and with final passive ultrasonic activation (p < 0.001). Chlorhexidine experimental groups demonstrated residual antimicrobial activity for as long as 168 h.

Anti-Infective Agents, Local↗
PubMed2002

Comparison of steatocrit and fat absorption in persons with cystic fibrosis.

BACKGROUND: Exocrine pancreatic insufficiency is a common problem in persons with cystic fibrosis causing malabsorption and poor growth. The 72-hour fecal fat study is the best qualitative measure of fat malabsorption used in clinical practice. This test has several drawbacks, which include cost and logistics. The steatocrit has been proposed as a rapid method to assess fat malabsorption. OBJECTIVES: The objective of this study was to determine whether the steatocrit is an accurate estimation of fat malabsorption in persons with cystic fibrosis. METHODS: Forty-nine stool samples for steatocrit processing were obtained from 72-hour fecal fat collections performed on twenty-seven persons with cystic fibrosis. Stools were weighed and homogenized with sand and water. The emulsified specimen was pipetted into heparinized capillary tubes and centrifuged for 15 minutes. The length of the fatty layer was compared to the length of the solid layer plus the fatty layer in each tube to determine the steatocrit value. Four steatocrit mixtures were evaluated: (1) standard steatocrit, (2) dilute steatocrit, (3) acid steatocrit, (4) dilute acid steatocrit. Steatocrit values were compared with the coefficient of fat absorption determined from the 72-hour fecal fat collection. RESULTS: The correlation coefficient of the steatocrit and the coefficient of fat absorption ranged from -.045 to -.491. Only the coefficient of fat absorption and the acid steatocrit had a significant correlation ( = 0.033). CONCLUSION: The steatocrit is an imprecise measure of fat absorption and not clinically useful in estimating steatorrhea.

Adolescent↗
PubMed2005

A comparison of techniques for analysing 90Y-Zevalin thin-layer chromatography plates.

OBJECTIVES: To simulate 90Y-Zevalin thin-layer chromatograms representing a range of radiochemical purities, to compare the radiochemical purities obtained with five techniques used to quantify 90Y on the plates and to measure the reproducibility of the five techniques at the minimum acceptable radiochemical purity of 95%. METHODS: Yttrium-90 solutions were pipetted onto the origin and solvent front lines of thin-layer chromatography (TLC) plates to simulate radiochemical purities of 90%, 92%, 94%, 95%, 96%, 98% and 100%. Each plate was analysed using three TLC scanners (Bioscan AR2000, Bioscan Mini-scan and an instrument constructed in-house) and two cut-and-count techniques: one using a sodium iodide well detector and the other a liquid scintillation counter. The reproducibility of each technique was measured by analysing the 95% plate 10 times. RESULTS: The radiochemical purities measured by the five techniques agreed well. The means of the seven results obtained with each agreed within 0.7%. The reproducibility of each technique was excellent. The coefficient of variation for 10 measurements was < or =0.3%. The signal to background ratios were satisfactory, ranging from 24 to 2.1 x 10(5). CONCLUSION: Each technique is suitable for analysing 90Y-Zevalin TLC plates.

Antibodies, Monoclonal↗
PubMed1981

Adaptation of EMIT procedures for maximum cost effectiveness to two different centrifugal analyzer systems.

We have adapted commercial immunoassay (EMIT) procedures for dilantin, phenobarbital, theophylline, carbamazepine, tobramycin, disopyramide, quinidine, procainamide, N-acetyl procainamide, and mysoline to two different centrifugal analyzers (Centrifichem and Multistat) using maximum dilutions of reagents to reduce reagent costs to approximately one-tenth of that incurred using the manufacturer's protocol. This savings due to the dilutions of reagents was primarily because of the ability to prolong the reaction time to achieve acceptably large absorbance changes over the absorbance measurement period. Correlation coefficients between values for drugs analyzed by these modified EMIT protocols and values obtained by other methods ranged from 0.927 to 0.994. Within-run precision and recovery values for these drugs were also entirely acceptable. We found that premature mixing of reagents in the rotors of the Multistat system and variable delivery of reagent in the Centrifichem 400 system were both caused by the surfactant in the EMIT buffer. The former problem is being resolved by the manufacturer; the latter problem was resolved by siliconizing the reagent pipet tips.

Centrifugation↗
PubMed1994

Determination of piroximone in human plasma by high-performance liquid chromatography.

A rapid and selective high-performance liquid chromatographic (HPLC) method using solid-phase extraction (SPE) for measuring piroximone in plasma samples has been developed. Human plasma and internal standard were pipetted onto a Bond Elut C18 extraction cartridge conditioned with methanol and water. Impurities and proteins were washed out with water. Piroximone and internal standard were eluted with methanol. After evaporation, the residue was dissolved in the mobile phase and the aliquot injected into the HPLC system. Piroximone and its related compounds were separated on a reversed phase C18 HPLC column maintained at 50 degrees C using a mobile phase consisting of phosphate buffer and methanol. Piroximone-N-oxide, piroximone, and internal standard were detected spectrophotometrically at 340 nm. The extraction recovery for piroximone was 94%. The within- and between-run coefficients of variation were < 3% in the concentration range of 320-5,000 ng/ml and < 17.5% at lower concentrations, e.g., 20 ng/ml. The limit of detection was 10 ng/ml.

Calibration↗