Stereospecific Response of E/Z-isomers of N-Nitrososarcosine in LC-ESI-MS/MS.

The carcinogenic compound N-nitrososarcosine (NSAR) is found in foods and tobacco products, and its quantification is of great interest. Although the presence of two stereoisomers, E- and Z-NSAR, is well-known, individual investigation of the isomers has not been reported so far. The present study by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) reveals that (i) the mass spectrometric responses of the isomers differ by a factor of approximately two and (ii) the isomer ratio is unstable in freshly prepared standard solutions. As a consequence, NSAR concentrations determined by LC-ESI-MS/MS are biased if those facts are not taken into account. The method described here overcomes the difficulty of stereospecific response by adjusting the isomer ratio and was applied to 100 tobacco products and fully validated for moist and dry snuff reference materials showing expanded measurement uncertainties of ~20% and limits of quantification of ~20 ng/g.

The impact of perioperative atelectasis on antibiotic penetration into lung tissue: an in vivo microdialysis study.

OBJECTIVE: Postoperative pneumonia is a potentially devastating complication associated with high mortality in intensive care unit (ICU)-patients. One of the major predisposing factors is the perioperative occurrence of atelectatic formations in non-dependent lung areas. Perioperative ventilation/perfusion mismatch due to atelectasis may influence antibiotic distribution to lung tissue, hence increasing the risk of postoperative pneumonia. We evaluated whether differences in ventilation/perfusion mismatch can influence antibiotic distribution into lung tissue by means of in vivo microdialysis, comparing patients undergoing coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) (atelectasis model), with patients operated with the off-pump coronary artery bypass grafting (OPCAB)-technique. PATIENTS AND METHODS: We compared five patients operated with CPB (CPB-group) and five patients undergoing CABG with OPCAB-technique (OPCAB-group). Levofloxacin (500 mg) was administered intravenously, after surgery, in the ICU. Time versus concentration profiles of levofloxacin in lung tissue and plasma were measured at regular time-intervals. RESULTS: In the OPCAB-group, the median of the maximum concentration of levofloxacin in lung tissue (4.1 microg ml(-1) +/- 7, range 3.7-11.8 microg ml(-1)) was significantly higher compared with the CPB-group (2.5 microg ml(-1) +/- 0.3, range 2.0-2.9 microg ml(-1)) (P = 0.046). Median levofloxacin tissue/plasma area under the concentration curve (AUC) ratio in lung tissue was 0.3 +/- 0.2 (range 0.1-0.7) in the CPB-group versus 0.7 +/- 1.6 (range 0.4-0.8) in the OPCAB-group (P = 0.015). CONCLUSIONS: Data indicate that postoperative interstitial antibiotic concentration is influenced by perioperative atelectasis formation. Our findings suggest the re-evaluation of clinical dosing schemas of antibiotic therapy in a variety of diseases associated with atelectasis formation.

Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry.

Mycotoxins are toxic secondary metabolites produced by filamentous fungi that are growing on agricultural commodities. Their frequent presence in food and their severe toxic, carcinogenic and estrogenic properties have been recognised as potential threat to human health. A reliable risk assessment of mycotoxin contamination for humans and animals relies basically on their unambiguous identification and accurate quantification in food and feedstuff. While most screening methods for mycotoxins are based on immunoassays, unambiguous analyte confirmation can be easily achieved with mass spectrometric methods, like gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/mass spectrometry (LC/MS). Due to the introduction of atmospheric pressure ionisation (API) techniques in the late 80s, LC/MS has become a routine technique also in food analysis, overcoming the traditional drawbacks of GC/MS regarding volatility and thermal stability. During the last few years, this technical and instrumental progress had also an increasing impact on the expanding field of mycotoxin analysis. The aim of the present review is to give an overview on the application of LC-(API)MS in the analysis of frequently occurring and highly toxic mycotoxins, such as trichothecenes, ochratoxins, zearalenone, fumonisins, aflatoxins, enniatins, moniliformin and several other mycotoxins. This includes also the investigation of some of their metabolites and degradation products. Suitable sample pre-treatment procedures, their applicability for high sample through-put and their influence on matrix effects will be discussed. The review covers literature published until July 2006.

Characterization of stationary phases for gas chromatography by 29Si nuclear magnetic resonance spectroscopy III. Carborane-siloxane copolymers.

Five bis(dimethylsilyl)-m-carborane-siloxane polymers with methyl, phenyl, and 2-cyanoethyl ligands were characterized by (1)H, (11)B, (13)C, and (29)Si nuclear magnetic resonance (NMR) spectroscopy. All relevant chemical shifts are reported, whereas signal assignment was confirmed by 2D NMR spectroscopy. The chemical composition of the polymers was calculated from the (1)H and (29)Si NMR spectra. Only (29)Si NMR spectroscopy was able to quantify the methoxy end group, from which the average molecular weights were calculated. The copolymer Dexsil 300 turned out to have a regular microstructure, whilst the terpolymers Dexsil 400 and Dexsil 410 have only partly regular sequences. (11)B NMR spectroscopy confirmed the m-carborane structure and revealed some low molecular weight impurities.

Determination of fosfomycin in pus by capillary zone electrophoresis.

A method is described for the determination of fosfomycin in pus by capillary zone electrophoresis with reversed electroosmotic flow, and indirect UV absorbance detection. Sample pre-treatment is limited to removal of proteins and cell debris by adding the double volume of methanol, followed by vortexing for few seconds, and centrifugation at 15,000 x g for 2 min. The supernatant is directly injected into the instrument. Fosfomycin is separated from sample constituents with a background electrolyte at pH 7.25 (25 mM benzoate buffer with 0.5 mM hexadecyltrimethylammonium bromide added, adjusted to pH with tris(hydroxymethyl)-aminomethane (TRIS)). Separation is carried out in a capillary with 50 microm I.D., 64.5 cm total length, 56.0 cm to the detector, at 25 degrees C with -25 kV voltage applied. Due to the low absorbance of the analyte, indirect UV detection was performed at 254 nm using a bubble cell capillary. Sample was injected by pressure (450 mbar s). Repeatability for fosfomycin in spiked pus (from 8 or 10 consecutive injections of three different series at concentrations of 100 microg/mL of the antibiotic) was between 2.4 and 8.2% relative standard deviation (RSD). Accuracy (expressed as recovery of fosfomycin determined by three independent analysis at 10, 100 and 300 microg/mL fosfomycin added to plain pus) was between 75 and 102%. Intermediate reproducibility (n = 9 at three different days) was between 2 and 12% RSD. Limit of detection and limit of quantitation were 4.5 and 15 microg/mL, respectively. The concentration of fosfomycin in pus of patients treated with the antibiotic ranged up to 240 microg/mL. The concentration of other anionic pus constituents identified beside chloride (acetate, succinate, lactate, phosphate) ranged between 20 and 7800 microg/mL.

Tetramethyl-p,p'-sildiphenylene ether-dimethyl, diphenylsiloxane copolymers as stationary phases in gas chromatography.

A 33% tetramethyl-p,p'-sildiphenylene ether (SDPE)-67% dimethylsiloxane copolymer was prepared and characterized by 1H and 29Si NMR spectroscopy. The random copolymer was coated on fused-silica capillary columns and used as stationary phase in GC. Highly deactivated capillary columns with high separation efficiency and a working range from -10 to 400 degrees C were obtained. For comparison, a commercially available capillary column with an SDPE-diphenyl, dimethylsiloxane terpolymer was tested. The selectivity of SDPE phases is best described by the assumption that an SDPE unit is equivalent to two dimethylsiloxy and one diphenylsiloxy group. Both capillary columns exhibited low column bleed in combination with seriously increased elution temperatures.

Different responses of E:Z-isomers of pesticides in liquid chromatography-electrospray ionization-mass spectrometry.

The mass spectrometric behavior of four pairs of stereoisomers was investigated by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The E- and Z-isomers of the pesticides chlorfenvinphos, dimethomorph, mevinphos and phosphamidon-each with one double bond-were chosen for this study. The MS response of the individual isomers was investigated by infusing the isomers individually into the MS or after the separation of isomer mixtures via high-performance liquid chromatography (HPLC). In the case of dimethomorph, the same MS response was found for the two isomers. In contrast to that, the individual isomers of chlorfenvinphos, mevinphos and phosphamidon showed different MS response both in the single ion monitoring (SIM) mode in single quadrupole MS and multiple reaction monitoring (MRM) mode in tandem MS. The MS response of the isomers partly depends on (1) the declustering potential of the precursor ion in the SIM mode, (2) the selected transition and (3) the collision energy in the MRM mode. Consequently, quantification by summation of the peak areas of the isomers is inaccurate due to over- or underestimating of one of the stereoisomers. Accurate quantitative results can only be achieved when the compound-specific MS parameters are separately determined for each isomer. This can be done by using pure isomers or by the determination of the MS parameters after HPLC separation and the measurement of the actual isomer ratio with an independent technique.

Method development for the determination of 52 pesticides in tobacco by liquid chromatography-tandem mass spectrometry.

A method using reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry was developed for the determination of 52 pesticides in tobacco. The influence of mobile phase additives was investigated to improve sensitivity and accuracy of the method and to reduce matrix effects. The tobacco extracts were purified via a Chem Elut partition cartridge by consecutive elution with pentane followed by dichloromethane. The two fractions were further purified by Florisil solid-phase extraction with acetone or diethyl ether elution. An additional dispersive solid-phase extraction step with primary-secondary amine led to decreased recoveries of several pesticides due to degradation or binding to the sorbent. The method was validated for the tobacco types Burley, Oriental and Virginia. The recovery rates of almost all pesticides ranged between 70 and 120%. The limits of quantification were below or near the 10 ng/g level. Few but significant differences between the tobacco types could be found regarding recovery and sensitivity.

Method development for the determination of selected pesticides on tobacco by high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry.

A method was developed for the quantitative determination of alachlor, benalaxyl, clomazone, diflubenzuron, dimethomorph, diphenamid, ethofumesate, metalaxyl, methoprene, metobromuron and piperonyl butoxide on tobacco. The pesticides were extracted with water and methanol from five different types of tobacco. The extracts were purified by partition on an extraction cartridge containing diatomaceous earth. The purified extracts were analysed by reversed-phase high-performance liquid chromatography connected to an atmospheric pressure ionisation-electrospray-triple quadrupole mass spectrometer operating in the positive ion mode. Two different transitions and their relative intensities were monitored for unambiguous identification. All pesticides presented overall recovery rates between 35% and 110%. The trueness is near 100% and the interday precision is below 15%. The limits of quantifications are equal or below the guidance residue levels proposed by the Agrochemical Advisory Committee of CORESTA, an association of organisations having scientific research relative to tobacco.

In vivo microdialysis to measure antibiotic penetration into soft tissue during cardiac surgery.

BACKGROUND: Wound infections remain an important problem after cardiac surgery despite antimicrobial prophylaxis, causing increased mortality, morbidity, and costs. Penetration properties of antibiotics are altered by extracorporeal circulation, fluid resuscitation, surgery, and postoperative treatment measures. So far, interstitial antibiotic concentration has not been measured continuously during surgery. It remains uncertain whether the concentration of the prophylactic antibiotic is sufficient in interstitial tissue. Therefore, we measured interstitial concentrations of cefazolin in vivo during cardiac surgery. METHODS: Seven patients undergoing aortic valve replacement were studied in this prospective, observational, pharmacokinetic study. Cefazolin, 4 g, was administered before skin incision and additionally 2 g during skin closure. Microdialysis, an in vivo approach, was used to measure unbound interstitial drug concentrations. RESULTS: Cefazolin plasma concentration rose to a peak of 443 microg/mL (range, 169 to 802 microg/mL) within 20 minutes (range, 20 to 40 minutes). The maximum of interstitial concentration of cefazolin was observed within 60 minutes after antibiotic administration. Cefazolin tissue levels exceeded minimum inhibitory concentration values for most potential wound pathogens for more than 600 minutes after infusion. The maximum drug concentration of cefazolin in subcutaneous interstitial fluid was 22.6% of maximum plasma levels, comparable with 19.4% in muscular tissue. CONCLUSIONS: Cefazolin, administered in the high dose used at our institution, is effective for prevention against infection with the most prevalent pathogens during and immediately after cardiac surgery. Additionally, our data show that it is important to reevaluate clinical dosing schemas by means of direct in vivo measurements.

Determination of the minimum allowable operating temperature of stationary phases in capillary columns by inverse gas chromatography.

A method was developed to determine the minimum allowable operating temperature (MiAOT) of wall coated open tubular capillary columns. Two polyethylene glycol and fourteen polysiloxanes phases with different side groups (methyl, phenyl, cyanopropyl, trifluoropropyl, n-octyl) and backbone stiffening units (tetramethyl-p-silphenylene, tetramethyl-p,p'-sildiphenylene ether, carborane) were investigated by inverse gas chromatography. A sigmoidal profile of temperature versus column efficiency was found for almost all phases, as the column efficiency increased with temperature. The MiAOT was defined as that temperature where the column efficiency is half of its original value at elevated temperatures. It was found that the MiAOT of a stationary phase is approx. 60 K higher than its glass transition temperature.

Preparation and characterization of fused-silica capillary columns coated with m-carborane-siloxane copolymers for gas chromatography.

The carborane-siloxane copolymers Dexsil 300, a 34.5% bis(dimethylsilyl)-m-carborane-65.5% dimethylsiloxane copolymer, and Dexsil 400, a 24.9% bis(dimethylsilyl)-m-carborane-50.8% dimethyl, 24.3% methylphenylsiloxane copolymer, were coated on fused silica capillary columns and their gas chromatographic properties were evaluated. Their selectivity was evaluated using both Rohrschneider-McReynolds constants and triacylglycerol indices. The bis(dimethylsilyl)-m-carborane unit turned out to be equivalent to two dimethylsiloxy units and one half of a diphenylsiloxy unit. The m-carborane unit was found to cause a 15-25 K shift in the elution temperature between 120 and 360 degrees C. The working range was from 20 and 0 degrees C to 380 degrees C for Dexsil 300 and Dexsil 400, respectively. The column bleeding levels at 380 degrees C were below 20 and 15 pA for Dexsil 300 and Dexsil 400, respectively.

Increase of microcirculatory blood flow enhances penetration of ciprofloxacin into soft tissue.

The present study addressed the effect of microcirculatory blood flow on the ability of ciprofloxacin to penetrate soft tissues. Twelve healthy male volunteers were enrolled in an analyst-blinded, clinical pharmacokinetic study. A single intravenous dose of 200 mg of ciprofloxacin was administered over a period of approximately 20 min. The concentrations of ciprofloxacin were measured in plasma and in the warmed and contralateral nonwarmed lower extremities. The microdialysis technique was used for the assessment of unbound ciprofloxacin concentrations in subcutaneous adipose tissue. Microcirculatory blood flow was measured by use of laser Doppler flowmetry. Warming of the extremity resulted in an increase of microcirculatory blood flow by approximately three- to fourfold compared to that at the baseline (P < 0.05) in subcutaneous adipose tissue. The ratio of the maximum concentration (C(max)) of ciprofloxacin for the warmed thigh to the C(max) for the nonwarmed thigh was 2.10 +/- 0.90 (mean +/- standard deviation; P < 0.05). A combined in vivo pharmacokinetic (PK)-in vitro pharmacodynamic (PD) simulation based on tissue concentration data indicated that killing of Pseudomonas aeruginosa (ATCC 27853 and two clinical isolates) was more effective by about 2 log(10) CFU/ml under the warmed conditions than under the nonwarmed conditions (P < 0.05). The improvement of microcirculatory blood flow due to the warming of the extremity was paralleled by an increased ability of ciprofloxacin to penetrate soft tissue. Subsequent PK-PD simulations based on tissue PK data indicated that this increase in tissue penetration was linked to an improved antimicrobial effect at the target site.

Pharmacokinetics and pharmacodynamics of cefpirome in subcutaneous adipose tissue of septic patients.

The objective of the present study was to evaluate whether cefpirome, a member of the latest class of broad-spectrum cephalosporins, sufficiently penetrates subcutaneous adipose tissue in septic patients. After the administration of the drug at 2 g, tissue cefpirome concentrations in septic patients (n = 11) and healthy controls (n = 7) were determined over a period of 4 h by means of microdialysis. To assess the antibacterial effect of cefpirome at the target site, the measured pharmacokinetic profiles were simulated in vitro with select strains of Staphylococcus aureus and Pseudomonas aeruginosa. The tissue penetration of cefpirome was significantly impaired in septic patients compared with that in healthy subjects. For subcutaneous adipose tissue, the area under the concentration-versus-time curve values from 0 to 240 min were 13.11 +/- 5.20 g . min/liter in healthy subjects and 6.90 +/- 2.56 g . min/liter in septic patients (P < 0.05). Effective bacterial growth inhibition was observed in all in vitro simulations. This was attributed to the significantly prolonged half-life in tissue (P < 0.05), which kept the tissue cefpirome levels above the MICs for relevant pathogens for extended periods in the septic group. By consideration of a dosing interval of 8 h, the values for the time above MIC (T > MIC) in tissue were greater than 60% for pathogens for which the MIC was

In vivo measurement of levofloxacin penetration into lung tissue after cardiac surgery.

Nosocomial pneumonia is a severe complication after cardiac surgery (CS). Levofloxacin, a fluoroquinolone, qualifies for the therapy of postoperative pneumonia. However, penetration properties of levofloxacin into the lung tissue could be substantially affected by CS: atelectasis, low cardiac output after CS, high volume loads, and inflammatory capillary leak potentially influence drug distribution. The aim of our study was to gain information on interstitial antibiotic concentrations in lung tissue in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass. Therefore, six patients undergoing elective CS participated in this prospective study. A dose of 500 mg of levofloxacin was administered intravenously in addition to standard antibiotic prophylaxis immediately after the end of surgery. Time versus concentration profiles of levofloxacin in the interstitial lung tissue and plasma were determined. A microdialysis technique was used for lung interstitial concentration measurements. The microdialysis procedure was well tolerated in all patients and no adverse events were observed. The median area under the concentration curve (AUC) of levofloxacin in interstitial lung fluid was 18.6 microg.h/ml (range, 10.1 to 33.6). The median AUC for tissue (AUC(tissue)) of unbound levofloxacin/AUC(total) in plasma was 0.6 (range, 0.4 to 0.9). The median unbound AUC(tissue)/MIC was 2.4 (range, 1.3 to 4.2) for Pseudomonas aeruginosa. Our study demonstrated the feasibility and safety of microdialysis in human lung tissue in vivo after CS. The unbound AUC/MIC ratio revealed that levofloxacin used in the described manner was borderline sufficient for the treatment of nosocomial pneumonia caused by Klebsiella pneumoniae and insufficient for the treatment of pneumonia caused by Pseudomonas aeruginosa, because the breakpoint of 30 to 40 for AUC/MIC could not be reached by the conventionally used dosage schema in our post-CS setting. Penetration was lower than in previous reports.

Antibiotic abscess penetration: fosfomycin levels measured in pus and simulated concentration-time profiles.

The present study was performed to evaluate the ability of fosfomycin, a broad-spectrum antibiotic, to penetrate into abscess fluid. Twelve patients scheduled for surgical or computer tomography-guided abscess drainage received a single intravenous dose of 8 g of fosfomycin. The fosfomycin concentrations in plasma over time and in pus upon drainage were determined. A pharmacokinetic model was developed to estimate the concentration-time profile of fosfomycin in pus. Individual fosfomycin concentrations in abscess fluid at drainage varied substantially, ranging from below the limit of detection up to 168 mg/liter. The fosfomycin concentrations in pus of the study population correlated neither with plasma levels nor with the individual ratios of abscess surface area to volume. This finding was attributed to highly variable abscess permeability. The average concentration in pus was calculated to be 182 +/- 64 mg/liter at steady state, exceeding the MIC(50/90)s of several bacterial species which are commonly involved in abscess formation, such as streptococci, staphylococci, and Escherichia coli. Hereby, the exceptionally long mean half-life of fosfomycin of 32 +/- 39 h in abscess fluid may favor its antimicrobial effect because fosfomycin exerts time-dependent killing. After an initial loading dose of 10 to 12 g, fosfomycin should be administered at doses of 8 g three times per day to reach sufficient concentrations in abscess fluid and plasma. Applying this dosing regimen, fosfomycin levels in abscess fluid are expected to be effective after multiple doses in most patients.

Capillary electrophoresis analysis of fosfomycin in biological fluids for clinical pharmacokinetic studies.

A feasible capillary zone electrophoresis (CZE) method with indirect UV and contactless conductivity detection was developed for the determination of fosfomycin, an antibiotic, in human plasma and microdialysis samples. Samples were collected from test persons during a clinical trial. The background electrolytes used consisted of 25 mM benzoic acid and 0.5 mM hexadecyltrimethylammonium bromide, adjusted with tris(hydroxymethyl)aminomethane solution to pH 6.95 for plasma, and to pH 8.05 for microdialysis samples. CZE separations of the anionic analyte were carried out with reversed electroosmotic flow directed towards the anode. The limit of detection was between 0.6 and 2 microg/mL, depending on the matrix and the detection method. No sample preparation was needed for microdialysis samples; for plasma samples, proteins were precipitated with methanol (1+2, v+v), and the supernatant was analyzed. The yield determined with spiked samples was about 100%, the reproducibility of the entire method, expressed by the RSD% of three independent determinations of fosfomycin in triplicate after spiking Ringer's solutions and plasma samples, respectively, was better than 8%. The method is thus well-suited for clinical studies for the determination of the antibiotic in biological fluids.

Approaches for the coating of capillary columns with highly phenylated stationary phases for high-temperature GC.

Two highly phenylated tetramethyl-p-silphenylene-diphenylsiloxane copolymers were coated on fused silica capillary columns and used as stationary phases in GC. The copolymers offered new insights into the coating process and column preparation due to their physicochemical properties. The fused silica capillary surface had to be pretreated in various ways to achieve a homogeneous film and a well deactivated surface: etching with ammonium bifluoride; leaching with sodium hydroxide and hydrochloric acid; silylation with tetraphenyldimethyldisilazane and triphenylsilylamine. Droplet formation was observed on tetraphenyldimethyldisilazane silylated surfaces leading to capillary columns with low separation efficiency. The topology of inhomogeneous films was investigated by light microscopy, scanning electron microscopy, and Auger electron spectroscopy. It became apparent that the stationary phase did not form droplets but islands, which are connected by a wetting layer according to the Stranski-Krastanov growth mode. Both copolymers are potential stationary phases for high-temperature GC with promising properties. They offer a higher overall polarity than 75% phenyl, 25% methyl-polysiloxanes in combination with increased thermal stability and reduced bleed levels.