Determination of sulfonic acid degradates of chloroacetanilide and chloroacetamide herbicides in groundwater by LC/MS/MS

An analytical method is presented which permits qualitative and quantitative analysis of sulfonic acid degradates of three chloroacetanilide herbicides (acetochlor, alachlor, and metolachlor) and one chloroacetamide herbicide (dimethenamid) in groundwater at trace levels with determination by LC/MS/MS. The analytes were isolated from groundwater by solid-phase extraction (SPE). The final samples were analyzed by reversed-phase HPLC with MS/MS detection utilizing a pneumatically assisted, and heat-assisted electrospray interface (TurboIonSpray). Unique precursor/production pairs were obtained in the MS/MS mode which permitted conclusive identification of each analyte, even when the analytes coeluted. Quantification was performed by generation of an external calibration curve. Excellent linearity was obtained over a calibration range from 0.25 to 10 ng injected on-column, with all linear correlation coefficients exceeding 0.999. Method performance for this analytical procedure was validated by analyzing groundwater samples fortified at levels of 0.1, 1, and 50 ppb. The average recovery at each fortification level for each analyte exceeded 89%. Excellent method precision was demonstrated with percent relative standard deviations of less than 10% for all analytes at all fortification levels.     

Simultaneous determination of diphenhydramine, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine using liquid chromatography/electrospray tandem mass spectrometry

Our studies on drug disposition in chronically instrumented pregnant sheep involve simultaneous administration of the antihistamine diphenhydramine (DPHM), its deuterated analogue ([2H10]DPHM) and their metabolites to the mother or the fetus via various routes. Such studies require sensitive and selective mass spectrometric methods for quantitation of these labeled and unlabeled compounds in order to assess comparative maternal and fetal drug metabolism. The objective of this study was to develop and validate a liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantitation of DPHM, its N-oxide metabolite and their deuterium-labeled analogues in ovine plasma and urine. Samples spiked with the analytes and the internal standard, orphenadrine, were processed using liquid-liquid extraction. The extract was chromatographed on a propylamino LC column and MS/MS detection was performed in the positive ion electrospray mode using multiple reaction monitoring. The linear concentration ranges of the calibration curves for the N-oxides and the parent amines were 0.4-100.0 and 0.2-250.0 ng ml-1, respectively. In validation tests, the assay exhibited acceptable variability (< or = 15% at analyte concentrations below 2.0 ng ml-1 and < 10% at all other concentrations) and bias (< 15% at all concentrations), and the analytes were stable under a variety of sample handling conditions. Using this method, the labeled and unlabeled N-oxide metabolite was identified in fetal plasma after DPHM and [2H10]DPHM administration. This method will be used further to examine the comparative metabolism of diphenhydramine to its N-oxide metabolite in the mother and the fetus.     

Confirmation and quantitation of selected sulfonylurea, imidazolinone, and sulfonamide herbicides in surface water using electrospray LC/MS

A multianalyte method has been developed for the confirmation and quantitation of 16 selected sulfonylurea, imidazolinone, and sulfonamide herbicides in surface water. Samples are acidified, the analytes are extracted using RP-102 extraction cartridges, and the extracts are cleaned-up using an anion-exchange cartridge (SAX) stacked on top of an alumina cartridge. The final extracts are evaporated to dryness, redissolved in 10:90 or 20:80 acetonitrile/water, and analyzed using electrospray LC/MS. Confirmation criteria were defined so that identification of the analytes could be made with a reasonable degree of scientific certainty. Confirmation required that (1) LC/MS retention times of the analytes be within 1% of the retention times of the standards, (2) the molecular ion and two characteristic fragment ions per analyte be present, and (3) ion abundance ratios of the fragment ions relative to the molecular ion be within 20% of the ion ratios obtained for the standards. Each of the analytes in all of the samples used to validate this method met these confirmation criteria. Quantitation of the analytes at 0.1 and 1.0 ppb was demonstrated, with average recoveries between 70% and 114% and relative standard deviations that were less than 13%. The limit of quantitation (LOQ) for this method is 0.1 ppb (ng/mL) for all analytes. Six water types were used for the validation of this method.     

Low picogram determination of Ro 48-6791 and its major metabolite, Ro 48-6792, in plasma with column-switching microbore high-performance liquid chromatography coupled to ion spray tandem mass spectrometry

A coupled liquid chromatography/tandem mass spectrometry assay was developed for simultaneous determination of Ro 48-6791 and its secondary amine metabolite in human plasma samples with a quantification limit for both compounds of 1 pg/mL using a 1 mL plasma aliquot. The method exploits the enhanced mass sensitivity of a microbore (300 microns i.d.) reversed-phase capillary column coupled to an ion spray probe combined with tandem mass spectrometry. A straightforward column-switching system was utilized to focus the analytes onto a microbore trapping column following solid-phase extraction of a 50 microL plasma sample extract from liquid/liquid extraction. Backflushing of the retained analytes from the trapping column onto the microbore capillary column provided the requisite high peak concentration for high sensitivity. The inter-assay precision and accuracy for Ro 48-6791 and its metabolite, at 10 pg/mL, were found to be 3.4%, and 105%, and 9.1%, and 99.9%, respectively. The calibration curves were linear over the range 1 to 1000 pg/mL. The method proved to be sufficiently rugged for analysis of samples.     

Antileishmanial chalcones: statistical design, synthesis, and three-dimensional quantitative structure-activity relationship analysis

A large number of substituted chalcones have been synthesized and tested for antileishmanial and lymphocyte-suppressing activities. A subset of the chalcones was designed by using statistical methods. 3D-QSAR analyses using 67 (antileishmanial activity) and 63 (lymphocyte-suppressing activity) of the compounds for the training sets and 9 compounds as an external validation set were performed by using the GRID/GOLPE methodology. The Smart Region Definition procedure with subsequent region selection as implemented in GOLPE reduced the number of variables to approximately 1300 yielding 3D-QSAR models of high quality (lymphocyte-suppressing model, R2 = 0. 90, Q2 = 0.80; antileishmanial model, R2 = 0.73, Q2 = 0.63). The coefficient plots indicate that steric interactions between the chalcones and the target are of major importance for the potencies of the compounds. A comparison of the coefficient plots for the antileishmanial effect and the lymphocyte-suppressing activity discloses significant differences which should make it possible to design chalcones having a high antileishmanial activity without suppressing the proliferation of lymphocytes.     

Multiresidue method for analysis of pesticides in liquid whole milk

A multiresidue method to analyze liquid whole milk for 59 compounds was developed. The method involves a single extraction and cleanup strategy for many classes of compounds--organophosphorus compounds (OPs), organochlorine compounds (OCs), N-methylcarbamates (MCs), etc. Initial extraction is performed with ethanol-ethyl acetate as solvent and sodium sulfate as drying agent. A portion of the extract is concentrated to an oily consistency, and target analytes are partitioned into acetonitrile. Further cleanup is achieved by sequential solid-phase extractions--octadecyl (C18)-bonded silica cartridges followed by aminopropyl (NH2)-bonded silica cartridges. The solvent is exchanged to acetone for analysis by gas chromatography (GC) with electrolytic conductivity, flame photometric (FPD) or mass spectrometric (MS) detection and to methanol for analysis by liquid chromatography with postcolumn derivatization. Typical limits of detection (LODs; peak-to-peak signal-to-noise ratio > or = 3) were 0.3 ppb for OPs, 0.9 ppb for OCs and MCs, and 9 ppb for mass-selective detection. From the level of quantitation (LOQ = 3.33 x LOD) to 10 x LOQ, linear instrument response was observed for all detectors except the FPD which required a second-order calibration curve. Average recoveries for spikes at the LOQ ranged from 69 to 127% with standard deviations of about 10%. Similar accuracy and precision were observed for fortifications at 5 x LOQ and 10 x LOQ. The method was used to analyze 20 milk samples from various liquid milk processing plants. Incurred residues were confirmed by high-resolution GC/MS and GC/MS/MS.     

Gas-liquid chromatographic determination of T-2 toxin and diacetoxyscirpenol in corn and mixed feeds

A gas-liquid chromatographic method has been developed for the simultaneous determination of the T-2 toxin and diacetoxyscirpenol (DAS) in corn and mixed feeds. The analytes are extracted with aqueous methanol. Ammonium sulfate is used to denature and precipitate proteinaceous compounds and to salt out low polar compounds. The analytes are selectively concentrated into chloroform, which is washed with aqueous potassium hydroxide to remove acidic compounds. Residual interferences are removed by silica gel column chromatography. Heptafluorobutrylimidazole (HFBI) is added to form esters of the analytes. The HFB-esters are separated on an SE-30 glass column and measured witha 63Ni electron capture detector, using methoxychlor as an internal standard. The method has been applied to corn, livestock feeds, and pet food. The limit of detection is 100 ng T-2 toxin and 25 ng DAS/G. Average recoveries of 105 and 97% and coefficients of variation of 17 and 10% were obtained for T-2 toxin and DAS, respectively.     

Correction for non-ideal tracer pharmacokinetic disposition by disposition decomposition analysis (DDA)

PURPOSE: Pharmacokinetic (PK) studies assume that the tracer's PK is equivalent to the parent compound. This assumption is often violated. The aim of this work is to present a method enabling the ideal tracer PK, i.e. the PK of the parent compound, to be predicted from the non-ideal tracer. METHODS: The procedure uses a disposition decomposition-recomposition (DDR) that assumes that the labeling mainly changes the elimination kinetics while the distribution kinetics is not significantly affected. In the DDR procedure an elimination rate constant correction factor (kCOR) is determined from a simultaneously fitting to plasma concentration data resulting from an i.v. injection of both the tracer and the parent compound. The correction factor is subsequently used to predict the ideal tracer PK behavior from the disposition function (i.v. bolus response) of the non ideal tracer. RESULTS: The DDR method when applied to plasma level data of erythropoietin (r-HuEPO) and its iodinated tracer (125I-r-HuEPO) from a high (4000U/kg) and a low (400U/kg) dosing of r-HuEPO in newborn lambs (n=13) resulted in excellent agreements in the elimination rate corrected dispositions in all cases (r=0.995, SD=0.0095). The correction factor did not show a dose dependence (p > 0.05). The correction factors were all larger than 1 (kCOR=1.94, SD=0.519) consistent with a reduction in the EPO elimination by the iodination labeling. CONCLUSIONS: The DDR tracer correction methodology produces a better differentiation of the PK of endogenously produced compounds by correcting for the non-ideal PK behavior of chemically produced tracers.     

The kinetics of bioaccumulation of zinc, copper, lead and cadmium by oysters (Crassostrea iredalei and C. belcheri) under tropical field conditions

Cyanobacterial neurotoxins have been implicated in animal deaths resulting from drinking contaminated water. Anatoxin-a (AN) and homoanatoxin-a (HMAN) have previously been analysed using high-performance liquid chromatography (HPLC) with UV detection, but this procedure is insufficiently sensitive and is subject to interferences. A sensitive fluorimetric (FL) method for determining AN was recently developed using derivatisation with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and this has been applied to the simultaneous determination of AN, HMAN and their epoxy and dihydro degradation products. Microscale syntheses were used to prepare the dihydro and epoxy derivatives from AN and HMAN. These compounds were produced in high yields, as confirmed by electrospray MS and HPLC-FL of their benzoxadiazole derivatives. All six NBD derivatives were readily separated using isocratic reversed-phase HPLC. The recoveries of these compounds from spiked water samples, using weak cation-exchange (WCX) solid-phase extraction (SPE), were 83.2-84.9% at concentrations of 10 micrograms/l. The R.S.D. values were 1.7-3.9% (n = 8) and the limits of detection were better than 10 ng/l for all six compounds, illustrating the high sensitivity of the method. This methodology was successfully applied to the analysis toxin degradation products in natural samples. Dihydroanatoxin-a (0.8 mg/g) was isolated from a benthic Oscillatoria bloom from Caragh Lake, Ireland, and was found to contain two isomers but their ratio was different from that found in the synthetic material.     

Radioimmunoassay of 8-arginine-vasopressin (antidiuretic hormone) in human plasma

A radioimmunoassay for 8-arginine-vasopressin (AVP) measurement in human plasma has been developed and evaluated, using a commercial preparation of an antibody of AVP. Detection limit of the assay was 0.4 pg. A simple acetone extraction procedure gave a recovery of 65% of added [125I]AVP. The overall sensitivity in the assay was 1.0 pg/ml when 2 ml plasma samples were extracted. The antigenic sites of the employed antibody seemed to be a combination of amino acid residues in the tripeptide tail and the pentapeptide ring. This can explain that the antibody was almost completely insensitive to chemically or enzymatically degraded AVP. The inter-assay coefficient of variation for the control plasma pools averaged 17%. A good correlation to plasma osmolalities above 290 has been found. AVP level in recumbent subjects (n = 8) with plasma osmolalities in the normal range was 2.8 +/- 1.0 pg/ml (mean +/- SD) and in ambulatory subjects (n = 10) on ad lib. water intake 4.5 +/- 1.9 pg/ml (mean /+- SD).     

AMT catalepsy and hypokinesia: interaction with morphine and cocaine

The first successful analysis of iodine compounds in serum and urine by mass fragmentography using GC-MS combined system was performed. The equipment used was Shimadzu LKB 9000 GC-MS (MID-PM). The TMSi derivatives of the compounds were analyzed by GC-MS system equipped with a 3 ft X 3 mm column packed with 1% OV-1 and the temperature was programmed from 200 degrees to 320 degrees C at 10 degrees C/min. The mass specturm showed molecular ions at m/e 523, 649, 741, 867 and 993 which correspond to the TMSi derivatives of MIT, DIT, T2, T3 and T4 respectively. The base peak at m/e 218 was applied to establish the precise quantitative evaluation of T4 and related compounds by mass fragmentography. The following results were obtained: 1) The minimum detectable limits of the compounds injected into the column were ca. 10 pg for MIT, DIT, 20pg for T2, 50pg for T3, and 500 pg for T4 respectively. 2) The sensitivity was of the order of ng or pg which enables quantitation with 1 ml of human serum and urine sample. 3) West's method was very convenient for extracting the compounds from biological fluids, and procedure can be carried out easily in a short time. The recovery rates were ca. 16.0% for MIT, 26.6% for DIT, 61.5% for T2, 71.6% for T3, and 85.1% for T4 respectively. 4) The ability to simultaneously analyze various iodoaminoacids is sure to be effectively utilized in studies to elucidate the relative importance of these hormones and their metabolic changes in various physioloigcal and pathological states of human beings.     

Analysis of estrone, estradiol and estriol in serum and urine by mass fragmentography using GC-MS (author's transl)

The first successful analysis of estrogens in serum and urine was performed by mass fragmentography using a GC-MS combined system. The equipment used was Shimadzu LKB 9000 GC-MS (MID-PM). The TMSi derivative of the compounds were analyzed using the GC-MS system equipped with a 3 ft x 3mm column packed with 1.5% OV-1 and the temperature was programed from 200 degrees to 260 degrees C at 10 degrees C/min increments. The mass spectrum showed molecular ions at m/e 342, 416 and 504 which correspond to the TMSi derivatives of Estrone (E-1), Estradiol (E-2) and Estriol (E-3) respectively. Molecular ion peak of each compound was applied to establish the precise quantitative evaluation of E-1, E-2 and E-3. 1) The minimum detectable limits of the compounds injected into the column were ca. 2 pg for E-1, E-2 and 5 pg for E-3 respectively. 2) The sensitivity was of the order of ng or pg which enables quantitation with 2 ml of human serum and urine samples obtained from normally ovulating women. 3) This method was very convenient for extracting the compounds from biological fluids, and the procedure can be carried out easily in a short time. 4) Mass fragmentography makes possible the simultaneous measurements of E-1, E-2 and E-3 in samples obtained from pregnant women.     

Estrogen-mediated neuroprotection after experimental stroke in male rats

BACKGROUND AND PURPOSE: We have previously shown that 17beta-estradiol reduces infarction volume in female rats. The present study determined whether single injection or chronic implantation of estrogen confers neuroprotection in male animals with middle cerebral artery occlusion (MCAO) and whether there is an interaction with endogenous testosterone. METHODS: Male Wistar rats were treated with 2 hours of reversible MCAO. In protocol 1, acute versus chronic estrogen administration was examined in groups receiving the following: Premarin (USP) 1 mg/kg IV, immediately before MCAO (Acute, n=13, plasma estradiol=171+/-51 pg/mL); 7 days of 25 microg (E25, n=10, 10+/-3 pg/mL) or 100 microg 17beta-estradiol (E100, n=12, 69+/-20 pg/mL) by subcutaneous implant; or saline (SAL, n=21, 3+/-1 pg/mL). Laser-Doppler flowmetry was used to monitor the ipsilateral parietal cortex throughout the ischemic period and early reperfusion. At 22 hours of reperfusion, infarction volume was determined by 0 2,3,5-triphenyltetrazolium chloride staining and image analysis. In protocol 2, rats were castrated to deplete endogenous testosterone and then treated with estradiol implants: castration only (CAST, n= 13, estradiol=5+/-2 pg/mL), sham-operated (SHAM, n= 10, 4+/-2 pg/mL), estradiol implant 25 microg (CAST+E25, n=16, 7+/-2 pg/mL) or 100 microg (CAST+E100, n=14, 77+/-14 pg/mL). RESULTS: Cortical infarct volumes were reduced in all estrogen-treated groups: Acute (21+/-4% of ipsilateral cortex), E25 (12+/-5%), and E100 (12+/-3%) relative to SAL (38+/-5%). Caudate infarction was similarly decreased: Acute (39+/-7% of ipsilateral striatum), E25 (25+/-7%), and E100 (34+/-6%) relative to SAL (63+/-4%). Castration did not alter ischemic outcome; cortical and caudate infarction (percentage of respective ipsilateral regions) were 37+/-5% and 59+/-5% in CAST and 39+/-7% and 57+/-5% in SHAM, respectively. Estrogen replacement reduced infarction volume in castrated animals in cortex (19+/-4% in CAST+E25 and 12+/-4% in CAST+E100) and in caudate (42+/-6% in CAST+25 and 20+/-7% in CAST + 100). Laser-Doppler flowmetry results during ischemia and reperfusion was not different among groups. CONCLUSIONS: Both acute and chronic 17beta-estradiol treatments protect male brain in experimental stroke. Testosterone availability does not alter estradiol-mediated tissue salvage after MCAO.     

Reversed-phase high-performance liquid chromatographic determination of enoxacin and 4-oxo-enoxacin in human plasma and prostatic tissue. Application to a pharmacokinetic study

A simple high-performance liquid chromatographic method has been developed for the simultaneous determination of enoxacin and 4-oxo-enoxacin in plasma and prostatic tissue. The work-up procedure involves a liquid-liquid extraction step followed by isocratic chromatography on a reversed-phase analytical column, with ultraviolet absorbance detection (lambda = 340 nm). Using a mobile phase of 20.9% (v/v) acetonitrile buffer (pH 2.1), adequate retention time and separation among the analytes has been obtained using tetrabutylammonium hydroxide included in the eluent. Retention times are 5.2 min for enoxacin, 6.8 min for pefloxacin and 12 min for 4-oxo-enoxacin. For plasma and prostatic tissue, the precision of the assay was below 9%. The percent recovery from the nominal values for accuracy ranged from 94 to 108%. The limits of quantitation were 20 ng/ml for plasma and 50 ng/g for tissue (precision < 18%). The detection limits were 10 ng/ml and 25 ng/g, respectively. The calibration curves were linear from 20 to 1000 ng/ml for plasma and from 50 to 2500 ng/g for tissue. In plasma, the extraction recoveries averaged 52% for enoxacin and 63% for 4-oxo-enoxacin. In prostatic tissue, they were 57 and 76% for the two analytes, respectively. This method has been employed for the determination of enoxacin and 4-oxo-enoxacin in plasma and prostatic tissue samples from patients following repeated oral administration of enoxacin (400 mg twice a day for four days).     

Association between mother-infant salivary contacts and caries resistance in children: a cohort study

We selected 327 7-month-old infants and divided them into two groups based on the frequency of salivary close contacts between mother and infant. Five to seven years later, all first-born children (N = 55) whose dental development had been followed regularly, were examined for dental caries and prevalence of salivary mutans streptococci (MS) and lactobacilli. The children with frequent maternal close contacts (F group, N = 21) had significantly less MS in saliva than the children with rare close contacts (R group, N = 34, P = 0.02). Only 19% of the children in F group compared with 56% in R group had experienced caries in their primary molars and/or canines (P < 0.01). A significantly greater proportion of the children in F group (57%) than in R group (27%, P < 0.05) had a high intake of sugar-containing foods and drinks in a 2-day dietary history. The F and R groups did not differ significantly with respect to other children's caries risk factors, or in age, sex, stage of dental development, dental treatment, or the social aspects studied. There were no significant differences between F and R groups in maternal caries experience, salivary MS or lactobacillus counts, or in maternal background factors (age, breast feeding, or education). Frequent transfer of maternal saliva to the mouth of the baby before tooth eruption was negatively associated with oral infection by MS and to caries in the primary dentition, possibly due to protective immune mechanisms.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells

Two-dimensional echocardiographic (2-D) planimetry and the Doppler pressure half-time (PHT) method have been used to estimate mitral valve area (MVA) in patients with mitral stenosis (MS). Recently, the proximal isovelocity surface area (PISA) method has been shown to be accurate for calculating MVA. The purpose of this study was to compare the PISA method with previous methods. Thirty patients with MS were studied; 17 had pure MS, 4 combined mild MR, 6 combined mild AR, and 3 combined MR and AR. Color Doppler flow mapping was performed at an aliasing (blue-red interface) velocity of 14 cm/sec using the zero-baseline shift. MVA was calculated as 2 x 3.14 x R2 x 14 x (theta/180) / PFV, where R is the distance from aliasing to orifice, 14 is the aliasing velocity, theta is the internal angle of the mitral valve, and PFV is the peak flow velocity at the mitral orifice. MVA was also calculated using the 2-D and PHT methods, and compared with the PISA method. MVA calculated using the PISA method correlated well with the 2-D (r=0.90, p < 0.01, SEE = 0.18 cm2) and PHT methods (r=0.82, p < 0.01, SEE = 0.24 cm2). Compared with the 2-D method, the standard error of the estimate of the PISA method was - 0.14+/-0.18 cm2 and the percent error was -10.4+/-18.9%. Compared with the PHT method, the standard error of the estimate of the PISA method was + 0.01+/-0.24 cm2 and the percent error was +3.4+/-34.6%. MVA calculated using the PISA method correlated well with the 2-D and PHT methods in patients with pure MS or with MS combined mild regurgitation. The PISA method may be useful for calculating MVA as an alternative method.     

Column switching in capillary liquid chromatography-tandem mass spectrometry for the quantitation of pg/ml concentrations of the free basic drug tolterodine and its active 5-hydroxymethyl metabolite in microliter volumes of plasma

A capillary column switching system was developed for the determination of low, unbound concentrations of the basic drug tolterodine and its active 5-hydroxymethyl (5-HM) metabolite in human plasma. Free concentrations of tolterodine and 5-HM at pM and nM (pg/ml and ng/ml) levels were obtained by ultrafiltration of 40-400 microliters plasma at 37 degrees C. The free fraction (%) was independent of the plasma concentrations of the analytes. Detection of the analytes was performed by sheathless electrospray tandem mass spectrometry in the multiple-reaction monitoring mode. The selectivity of the mass spectrometric detection and the additional clean-up on the pre-column allowed direct injection of the ultrafiltrated plasma samples. Tolterodine and 5-HM were pre-concentrated on a reversed-phase capillary pre-column (1 cm x 200 microns) and subsequently backflushed onto the separation column (25 cm x 200 microns). The stability of the chromatographic system was good; a large number of ultrafiltrated plasma samples could be injected and the relative standard deviation of the retention times was typically < or = 1% (within-day). The accuracy was between 86 and 105% and the precision was between 1 and 7% without the use of an internal standard. Linear calibration curves were obtained between 100 pM and 100 nM.     

Mycoplasmas in wild turkeys living in association with domestic fowl

One hundred and nineteen Merriam's wild turkeys (Meleagris gallopavo merriami) and 31 domestic chickens coexisting on a ranch in west-central Colorado (USA) were surveyed for mycoplasmosis by serologic and cultural methods. Although no clinical signs were apparent in any wild turkeys tested, 51 (43%) had positive rapid plate agglutination (RPA) reactions for M. gallisepticum (MG) and/or M. synoviae (MS); 37% of 56 adults and 48% of 63 subadults were classified as positive reactors to MG and/or MS. No turkeys tested in 1992 (n = 61) and 17 (29%) of 58 turkeys tested in 1993 were RPA-positive for M. meleagridis (MM). Hemagglutination inhibition (HI) test results were negative for MG, MS and MM as were most enzyme-linked immunosorbent assay (ELISA) test reactions (MG = 99%, MS = 93%, MM = 87%). Immunoblotting showed mild to moderate reactivity to MG proteins in 49% of 41 samples tested. Most chickens were strongly positive for MS by RPA (81%), HI (58%) and ELISA (87%); 48% also were positive for MG by RPA but all were MG-negative by HI and ELISA. No pathogenic mycoplasmas were isolated from either group of birds. Mycoplasma gallopavonis was commonly identified from the wild turkeys, and M. gallinaceum was isolated from both the chickens and wild turkeys. In a transmission study conducted in 1994, disease-free domestic turkeys failed to seroconvert when co-housed with wild turkeys from this population that were RPA-positive for MG. Collectively, the results of this study were inconclusive regarding the status of pathogenic mycoplasmas within this wild turkey population.     

Pharmacokinetic/pharmacodynamic evaluation of systemic effects of flunisolide after inhalation

The pharmacokinetics and pharmacodynamics of flunisolide were studied in healthy volunteers after inhalation. In the morning on the day the study began, volunteers inhaled 0.5 mg of flunisolide with and without oral administration of charcoal, or 1 mg, 2 mg, and 3 mg of flunisolide with concomitant administration of charcoal. A placebo group was used to assess the endogenous cortisol, granulocyte, and lymphocyte baseline levels. Flunisolide plasma levels were determined by high-performance liquid chromatography using a tandem mass spectrometer as detector (HPLC/MS/MS). Cortisol plasma levels and differential white blood cell counts were obtained over 12 hours. An integrated pharmacokinetic/pharmacodynamic (PK/PD) model was applied to link the flunisolide plasma concentrations with the effects on lymphocytes, granulocytes, and cortisol. Maximum concentration levels of 3 to 9 ng/mL of flunisolide were observed after 0.2 to 0.3 hours for all of the investigated doses. The terminal half-life ranged from 1.3 to 1.7 hours. There was no statistical difference between treatments in the presence or absence of orally administered charcoal. The pharmacokinetic/pharmacodynamic (PK/PD) models satisfactorily described the time-courses of the effects on granulocytes, lymphocytes, and cortisol suppression. The resulting E50-values (concentrations to induce 50% of the maximum effect) concurred with the reported values of in vitro receptor binding affinities. The duration of the systemic effects were short because of the short half-life of the drug. Cumulative cortisol suppression increased with dose administration and ranged from 20% to 36%. The PK/PD simulations resulted in a smaller degree of cortisol suppression for the drug administered at 10 PM. The cumulative change from baseline was slightly smaller for the effects on granulocytes and lymphocytes than those on cortisol. This information promotes the comparison with other inhaled glucocorticoids.