Bernhard X. Mayer

Impaired target site penetration of beta-lactams may account for therapeutic failure in patients with septic shock.

ObjectiveCurrent guidelines for adjusting antimicrobial therapy regimens commonly are based on drug concentrations measured in plasma. In septic patients, however, the interstitial space of soft tissues in addition to the central compartment represents the target site of infection. We thus hypothesized that one explanation for therapeutic failure during antibiotic treatment might be the inability to achieve effective antimicrobial concentrations in the interstitial space fluid of soft tissues. This is corroborated by the fact that piperacillin, a frequently administered &bgr;-lactam antibiotic, often fails to be effective despite documented susceptibility of the causative pathogen in vitro. DesignProspective comparative study of two groups. SettingThe intensive care unit and research ward of an university hospital. SubjectsSix patients with septic shock and a control group of six gender- and age-matched healthy volunteers. InterventionsTo measure piperacillin penetration into the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue, we employed microdialysis after a single intravenous administration of 4.0 g of piperacillin to patients and healthy volunteers. Piperacillin concentrations were assayed by using reversed-phase high-pressure liquid chromatography. Measurements and Main Results In septic shock patients, interstitial piperacillin concentrations in skeletal muscle and subcutaneous adipose tissue were five- to ten-fold lower than corresponding free plasma concentrations (p < .03). Mean piperacillin concentrations in subcutaneous adipose tissue never exceeded 11 &mgr;g/mL, which is below the minimal inhibitory concentration for a range of relevant pathogens in patients with septic shock. ConclusionThe results of the present study demonstrate that in septic shock patients, piperacillin concentrations in the interstitial space may be subinhibitory, even though effective concentrations are attained in plasma. The lack of success of antimicrobial therapy in these patients thus might be attributable to inadequate target site penetration of antibiotics.

How to increase precision in capillary electrophoresis.

This review surveys approaches on how to improve precision in capillary zone electrophoresis and micellar electrokinetic chromatography. Many different techniques have been employed successfully to improve instrument precision and to facilitate method transfer between instruments and laboratories. Operational parameters as well as theories will be discussed in detail.

Surgery and intensive care procedures affect the target site distribution of piperacillin.

Objective Therapeutic failure of antibiotic therapy has been ascribed to pharmacokinetic alterations in compromised patient populations. The present study, therefore, aimed at examining the influences of cardiac surgery and intensive care procedures on the postoperative target site distribution of piperacillin. For this purpose, the penetration of piperacillin to the interstitial space fluid, the relevant target site for most bacterial infections, was compared between patients after aortic valve replacement and healthy volunteers. Design Comparative study in two study populations. Setting The intensive care unit and research ward of a university hospital. Patients The study population included six otherwise healthy patients scheduled to undergo aortic valve replacement and a control group of six healthy male volunteers. Interventions After the administration of a single iv infusion of 4.0 g piperacillin, free piperacillin concentrations were measured in the interstitium of skeletal muscle and subcutaneous tissue by in vivo microdialysis and in venous serum. Piperacillin concentrations were assayed with reversed phase high-performance liquid chromatography. Measurements and Main Results Interstitial piperacillin concentrations in muscle and subcutaneous adipose tissue were significantly lower in patients compared with volunteers with the area under the curve for the interstitium/area under the curve for serum concentration ratios ranging from 0.25 to 0.27 and from 0.43 to 1.22 in patients and volunteers, respectively (p < .05 between groups). The terminal elimination half-life was markedly prolonged in patients, leading to a concomitant increase in t > minimal inhibitory concentration (MIC) values, the relevant surrogate for therapeutic success of therapy with &bgr;-lactam antibiotics, for strains with MIC50 <4 &mgr;g/mL. For strains with MIC50 >20 &mgr;l/mL, however, inadequate target site concentrations were attained in the patient population. Conclusions During the postoperative and intensive care periods, target site concentrations of piperacillin are markedly altered and decreased. This may also be true for other antibiotic agents and may have clinical implications in that current dosing guidelines may result in inadequate target site concentrations for high-MIC strains. Conceivably, this could lead to therapeutic failure in some patients.

Simultaneous determination of levofloxacin and ciprofloxacin in microdialysates and plasma by high-performance liquid chromatography

The fluoroquinolones levofloxacin and ciprofloxacin were simultaneously determined in microdialysis and plasma samples by reversed-phase high-performance liquid chromatography (HPLC) and fluorescence detection. After a simple sample preparation step the analytes were separated in the isocratic mode within 12 min. The calibration curve for levofloxacin was linear from 0.0156 to 5 gm l −1 and 0.02–12.5 gm l −1 in microdialysates and plasma samples, respectively. The limits of quantification for levofloxacin and ciprofloxacin were 0.0156 and 0.1 gm l −1 in microdialysis samples, and 0.02 and 0.1 gm l −1 in plasma samples, respectively. Some experimental details concerning microdialysate dilution and plasma protein precipitation were described to ensure accurate quantitation. The assay can be used as a routine method to analyse microdialysis and plasma samples from clinical studies.

Plasma and tissue pharmacokinetics of cefpirome in patients with sepsis

Objective Broad initial antibiotic treatment is crucial for patients experiencing septic shock or severe sepsis. Fourth-generation &bgr;-lactam antibiotics, such as cefpirome, are frequently favored in these conditions because of their low toxicity and wide antimicrobial coverage. From recent data, however, there is circumstantial evidence that one reason for the high mortality rate of patients with sepsis might be an impaired penetration of antimicrobial agents from the central compartment to the infectious focus. Thus, the present study aimed at describing penetration properties of cefpirome to the target site of many bacterial infections, which is the extracellular space fluid of soft tissues. Design Prospective comparative study of two groups. Setting An intensive care unit and research ward in a university hospital. Subjects The study population included 12 patients with septic shock or severe sepsis and a control group of six overall age-matched healthy volunteers. Interventions To measure cefpirome penetration into the interstitial space fluid of skeletal muscle, we employed microdialysis after single intravenous administration of 2.0 g of cefpirome to patients and healthy volunteers. Measurements and Main Results Maximum concentration and area under the concentration vs. time values in interstitium were significantly lower in patients compared with the control group (p < .004). Cefpirome area under the concentration time values for plasma were 16.0 ± 1.1 mg·min/mL (mean ± sem) and 18.8 ± 1.1 mg·min/mL in patients and healthy volunteers, respectively (p = .075, not significant). In both study groups, mean cefpirome concentrations in interstitium and plasma exceeded 28 &mgr;g/mL throughout the observation period of 240 mins and covered completely minimal inhibitory concentration values for a range of clinically relevant pathogens. Conclusion Cefpirome concentrations reached in tissue interstitium and plasma exceeded minimal inhibitory concentrations of most clinically relevant pathogens in patients with sepsis. Thus, cefpirome exhibits a tissue pharmacokinetic profile, which seems to be particularly valuable for the empirical therapy of patients with sepsis.

Target site concentrations after continuous infusion and bolus injection of cefpirome to healthy volunteers

Recent data indicate a higher level of effectivity of β‐lactam antibiotics if serum concentrations are kept above the minimal inhibitory concentration (MIC) of the pathogen. This concept would favor continuous infusion over bolus dosing. However, it is usually not the serum concentration but the free interstitial concentration in the target tissue that determines antibiotic activity. We therefore set out to measure effective drug concentrations in the interstitial space of muscle and subcutaneous adipose tissue and to compare trough levels and times above the MIC after bolus versus continuous infusion of cefpirome.

Micellar electrokinetic chromatography for the analysis of cefpirome in microdialysis and plasma samples obtained in vivo from human volunteers

Pharmacokinetics of drugs in the human interstitial space fluid can be monitored by means of microdialysis. However, the small‐volume microdialysis samples containing low drug concentrations require a sensitive analytical method. In the present study, micellar electrokinetic chromatography (MEKC) is described for the quantification of cefpirome in human microdialysis and plasma samples. Sample preparation of human plasma samples by ultracentrifugation was suitable for comparison of plasma and microdialysate concentrations. Limits of quantification were 2 μg/mL and 0.3 μg/mL for plasma and microdialysate samples, respectively. The limit of detection (LOD) was estimated at 0.2 μg/mL for the plasma and microdialysate samples. In conclusion, MEKC is a reliable and reproducible technique for measuring cefpirome concentrations in microdialysates as well as centrifuged plasma samples.

Investigation of the triacylglycerol composition of iceman's mummified tissue by high-temperature gas chromatography

The pattern of intact triacylglycerols of a skin sample from the 5300-year-old Iceman mummy (nicknamed Otzi) was resolved on a diphenyl-dimethylpolysiloxane stationary phase by high-temperature gas chromatography. Adipocere from a 64-year-old glacier mummy as well as recent human subcutaneous fat served as a comparison in this study. Qualitatively, the results for mummy samples were similar with well-preserved saturated, but decomposed unsaturated, triacylglycerols, the latter being predominant in subcutaneous fat. Excellent preservation of triacylglycerols with odd carbon numbers and branched acyl chains was observed. The results presented here shed new light on the process of mummification.

Synthesis of fluorine-18-labeled ciprofloxacin for PET studies in humans

Ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-quinoline-3-carboxylic acid), a widely-prescribed antibiotic, was labeled with fluorine-18 with the aim to perform positron emission tomography studies in humans for pharmacokinetic measurements. Due to a lack of chemical activation of ciprofloxacin for a direct nucleophilic exchange reaction a novel two-step synthetic approach, which employed an activated 6-fluoro-7-chloro substituted precursor molecule, was developed. The radiosynthesis yielded, starting from 52.5 +/- 11.3 GBq of [(18)F]fluoride, 1.3 +/- 0.6 GBq (n = 13) [(18)F]ciprofloxacin ready for intravenous administration in about 130 min synthesis time. A series of analytical tests was performed in order to prove the identity of the radiolabeled compound and its suitability for human applications.

Comparison of UV and tandem mass spectrometric detection for the high-performance liquid chromatographic determination of diclofenac in microdialysis samples

High-performance liquid chromatography (HPLC) was used to analyze microdialysis samples obtained in vivo from human subcutaneous adipose tissue after topical application of the nonsteroidal anti-inflammatory drug diclofenac. For the reliable determination of diclofenac two different detection principles were applied in two different laboratories. One HPLC method utilized UV-detection at 280 nm, the other one used selected reaction monitoring mass spectrometry (MS). The HPLC-UV and -MS methods offered low limits of quantification of 10 and 1 ng/ml and an accuracy between 94.0-126.7 and 89.3-110.9%, respectively. However, a comparison showed that the HPLC-UV method failed to determine diclofenac in biological matrices, as both false negative and positive values were found. HPLC-MS is clearly superior to HPLC-UV due to a much more selective detection, increased sensitivity and shorter run times.