Florin Marcel Musteata

Automated solid-phase microextraction and thin-film microextraction for high-throughput analysis of biological fluids and ligand-receptor binding studies.

This protocol describes how to perform automated solid-phase microextraction (SPME) and thin-film microextraction (TFME) in a 96-well plate format for high-throughput analysis of drugs, metabolites and any other analytes of interest in biological fluids using liquid chromatography–electrospray tandem mass spectrometry. Sample preparation time required is typically 1 min per sample; hence, the throughput achievable with automated SPME/TFME is comparable with automated 96-well liquid–liquid extraction and solid-phase extraction methods, but greater than most online solid-phase extraction methods. The technique is applicable to complex samples such as whole blood without additional pretreatment. The amount of analyte extracted by SPME/TFME is proportional to the free (unbound) concentration of the analyte; hence, SPME/TFME can be used to determine both total and free concentrations of analytes from a single biofluid sample and to perform automated ligand–receptor binding studies in order to determine binding affinity and/or overall extent of ligand binding to a complex biofluid.

In vivo solid-phase microextraction for single rodent pharmacokinetics studies of carbamazepine and carbamazepine-10,11-epoxide in mice

The use of solid-phase microextraction (SPME) for in vivo sampling of drugs and metabolites in the bloodstream of freely moving animals eliminates the need for blood withdrawal in order to generate pharmacokinetics (PK) profiles in support of pharmaceutical drug discovery studies. In this study, SPME was applied for in vivo sampling in mice for the first time and enables the use of a single animal to construct the entire PK profile. In vivo SPME sampling procedure used commercial prototype single-use in vivo SPME probes with a biocompatible extractive coating and a polyurethane sampling interface designed to facilitate repeated sampling from the same animal. Pre-equilibrium in vivo SPME sampling, kinetic on-fibre standardization calibration and liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) were used to determine unbound and total circulating concentrations of carbamazepine (CBZ) and its active metabolite carbamazepine-10,11-epoxide (CBZEP) in mice (n=7) after 2mg/kg intravenous dosing. The method was linear in the range of 1-2000ng/mL CBZ in whole blood with acceptable accuracy (93-97%) and precision (<17% RSD). The single dose PK results obtained using in vivo SPME sampling compare well to results obtained by serial automated blood sampling as well as by the more conventional method of terminal blood collection from multiple animals/time point. In vivo SPME offers the advantages of serial and repeated sampling from the same animal, speed, improved sample clean-up, decreased animal use and the ability to obtain both free and total drug concentrations from the same experiment.

Naturally occurring hypothermia is more advantageous than fever in severe forms of lipopolysaccharide- and Escherichia coli-induced systemic inflammation.

The natural switch from fever to hypothermia observed in the most severe cases of systemic inflammation is a phenomenon that continues to puzzle clinicians and scientists. The present study was the first to evaluate in direct experiments how the development of hypothermia vs. fever during severe forms of systemic inflammation impacts the pathophysiology of this malady and mortality rates in rats. Following administration of bacterial lipopolysaccharide (LPS; 5 or 18 mg/kg) or of a clinical Escherichia coli isolate (5 × 10(9) or 1 × 10(10) CFU/kg), hypothermia developed in rats exposed to a mildly cool environment, but not in rats exposed to a warm environment; only fever was revealed in the warm environment. Development of hypothermia instead of fever suppressed endotoxemia in E. coli-infected rats, but not in LPS-injected rats. The infiltration of the lungs by neutrophils was similarly suppressed in E. coli-infected rats of the hypothermic group. These potentially beneficial effects came with costs, as hypothermia increased bacterial burden in the liver. Furthermore, the hypotensive responses to LPS or E. coli were exaggerated in rats of the hypothermic group. This exaggeration, however, occurred independently of changes in inflammatory cytokines and prostaglandins. Despite possible costs, development of hypothermia lessened abdominal organ dysfunction and reduced overall mortality rates in both the E. coli and LPS models. By demonstrating that naturally occurring hypothermia is more advantageous than fever in severe forms of aseptic (LPS-induced) or septic (E. coli-induced) systemic inflammation, this study provides new grounds for the management of this deadly condition.

Monitoring free drug concentrations: challenges

Measurement of drug concentrations in biological samples is of utmost importance in many research areas. The information about the amount of drug in a biological sample can be given as either total concentration, which ignores the interaction of the drug with the sample matrix, or as free concentration, which shows the portion of molecules able to diffuse through membranes and exert biological activity. Although the historical trend has been towards determining total concentrations, measurement of free concentrations is becoming more important since it correlates better with pharmacological and toxicological effects. This review will discuss the most popular experimental approaches for monitoring free drug concentrations, based on the type of sample to be investigated and the kind of information to be collected. It is shown that the current challenges in measuring free concentrations are: convenience, accuracy, precision, wide applicability, availability of accurate and precise reference methods, ruggedness, and standardized sample conditions.

Analytical methods used in conjunction with solid-phase microextraction: a review of recent bioanalytical applications

Integration of sampling and sample preparation with various analytical instruments is a highly desirable feature for any analytical method. This is most conveniently achieved by using microextraction techniques or various microdevices. Among these techniques, solid-phase microextraction (SPME) is particularly remarkable due to its simplicity and effectiveness. This review discusses the most recent applications of SPME in bioanalysis, grouped according to the analytical instrument that SPME is coupled to. It is shown that one of the most important aspects of such analytical methods is the ability of SPME to perform direct and selective extraction of analytes from complex biological samples. By far, the most popular method continues to be SPME coupled to GC. Nevertheless, the last 2 years have witnessed significant advances in other areas, such as successful automation of SPME coupled to liquid chromatography and the development of new coatings suitable for direct extraction from biological samples. Furthermore, a few bioanalytical applications based on direct coupling of SPME to MS, ion mobility spectrometry, CE and analytical chemiluminescence have been reported.

Pharmacokinetic applications of microdevices and microsampling techniques.

Pharmacokinetic studies require information regarding drug concentration at numerous time points during the process of absorption, distribution, metabolism and excretion. In order to obtain reproducible and good-quality data, the sampling method is as important as the bioanalytical method. A further difficulty in performing pharmacokinetic studies is related to the limited amount of sample that can be collected in some cases. Since analytical methods should interfere as little as possible with the investigated organism, microsampling techniques are a natural choice for pharmacokinetic studies. Accordingly, microdevices and microsampling approaches have been used increasingly in recent years for a wide variety of analytical applications, including analysis of drugs in biological samples. Such techniques not only reduce the amount of reagents needed for analysis, but are also faster and less disrupting. This review provides a brief overview of contemporary microsampling techniques: collection of small sample aliquots, ultrafiltration, microdialysis, solid-phase microextraction, biosensors and microfluidics. It is concluded that recent developments in microsampling and microdevices promise to streamline pharmacokinetic studies and bring bedside monitoring of therapeutic drugs into clinical practice.

Overview of extraction methods for analysis of vitamin D and its metabolites in biological samples.

In the last decade the scientific and medical community was confronted with a renewed interest in vitamin D and its metabolites, interest prompted by new discoveries regarding the association between members of the vitamin D family and a great number of physiological functions and pathological states. An impressive number of research projects have helped clear the path towards a better understanding of the functions of vitamin D and have resulted in the development of numerous methods of analysis. This review focuses on the various extraction methods used for analysis of vitamin D in research or clinical settings. Two main extractive methods are usually employed: liquid-liquid extraction and solid-phase extraction. Some methods use no extraction step and direct analysis is performed at the cost of significantly increased matrix interference. On the other hand, other methods use combined extraction techniques, and even additional derivatization steps in order to increase the sensitivity and accuracy of the analysis. The method of choice ultimately depends on the research question and the purpose of the study.

Calculation of Normalized Drug Concentrations in the Presence of Altered Plasma Protein Binding

Background and ObjectiveIn many clinical situations, measurement of the total drug concentration does not provide the needed information concerning the fraction of unbound drug in plasma, which is available for pharmacodynamic action. To address this, a ‘normalized concentration’ can be calculated on the basis of the observed total drug concentration and the serum protein level. Up to now, this method has only been applied to phenytoin. Several equations for calculating normalized concentrations of phenytoin have been published, many leading to different results. Regrettably, all of the equations in the current literature are based on an outdated model of drug binding to human serum albumin and are based on the fraction of unbound drug, which is known to depend on both protein and drug concentrations. In response to the relatively new scientific evidence about drug binding to human plasma proteins, the objective of the present study is to develop a general method for calculating normalized drug concentrations in the presence of altered plasma protein binding.MethodsWhen several drug molecules can be bound by a protein molecule, multiple equilibria are established; these equilibria may be formulated in terms of a stoichiometric analysis or a site-oriented analysis. Both models are currently encountered in the scientific literature, sometimes without clear identification of which model is used. The present study presents the basic equations for both models and shows how the normalized concentration can be calculated on the basis of the measured drug concentration, the protein level and the binding constants.ResultsThe normalized concentration can be calculated for any drug, using the same simple equation regardless of the binding model and the number of binding proteins. Explicit solutions are presented for particular cases of clinical importance. The new model is validated by comparison with the Winter-Tozer equation for calculating the normalized phenytoin concentration and is found to be equivalent for concentrations close to therapeutic concentrations. In the case of phenytoin, the main advantage of the new equation is that it also works outside the linear binding range.ConclusionsA new comprehensive method for calculating normalized drug concentrations is developed, allowing drug concentrations to be interpreted correctly in cases of altered drug-protein binding. The calculations are based on binding constants and are applicable to any protein level and drug concentration, without being limited to linear binding of drugs to proteins. The new model is expected to become important in pharmacokinetic-pharmacodynamic modelling, allometric scaling and population pharmacokinetics because it provides the ability to accurately take into account physiological and pathological changes in protein binding. As a direct clinical application, the equations can be used to calculate normalized drug concentrations in patients with abnormal protein levels, such as the elderly, trauma patients and paediatric patients.

Determination of free and deconjugated testosterone and epitestosterone in urine using SPME and LC–MS/MS

BACKGROUND A thin sheet of polydimethylsilosane membrane was used as an extraction phase for solid-phase microextraction. Compared with fiber or rod solid-phase microextraction geometries, the thin film exhibited much higher extraction capacity without sacrificing extraction time due to its higher area-to-volume ratio. The analytical method involved direct extraction of unconjugated testosterone (T) and epitestosterone (ET) followed by separation on a C18 column and detection by selected reaction monitoring in positive ionization mode. RESULTS The limit of detection was 1 ng/l for both T and ET. After method validation, free (unconjugated) T and ET were extracted and quantified in real samples. Since T and ET are extensively metabolized, the proposed method was also applied to extract the steroids after enzymatic deconjugation of urinary-excreted steroid glucuronides. CONCLUSION The proposed method allows quantification of both conjugated and unconjugated steroids, and revealed that there was a change in the ratio of T to ET after enzymatic deconjugation, indicating different rates of metabolism.

Lipoxin A4, a 5‐lipoxygenase pathway metabolite, modulates immune response during acute respiratory tularemia

Respiratory infection with Francisella tularensis (Ft) is characterized by a muted, acute host response, followed by sepsis‐like syndrome that results in death. Infection with Ft establishes a principally anti‐inflammatory environment that subverts host‐cell death programs to facilitate pathogen replication. Although the role of cytokines has been explored extensively, the role of eicosanoids in tularemia pathogenesis is not fully understood. Given that lipoxin A4 (LXA4) has anti‐inflammatory properties, we investigated whether this lipid mediator affects host responses manifested early during infection. The addition of exogenous LXA4 inhibits PGE2 release by Ft‐infected murine monocytes in vitro and diminishes apoptotic cell death. Tularemia pathogenesis was characterized in 5‐lipoxygenase‐deficient (Alox5−/−) mice that are incapable of generating LXA4. Increased release of proinflammatory cytokines and chemokines, as well as increased apoptosis, was observed in Alox5−/− mice as compared with their wild‐type counterparts. Alox5−/− mice also exhibited elevated recruitment of neutrophils during the early phase of infection and increased resistance to lethal challenge. Conversely, administration of exogenous LXA4 to Alox5−/− mice made them more susceptible to infection thus mimicking wild‐type animals. Taken together, our results suggest that 5‐LO activity is a critical regulator of immunopathology observed during the acute phase of respiratory tularemia, regulating bacterial burden and neutrophil recruitment and production of proinflammatory modulators and increasing morbidity and mortality. These studies identify a detrimental role for the 5‐LO–derived lipid mediator LXA4 in Ft‐induced immunopathology. Targeting this pathway may have therapeutic benefit as an adjunct to treatment with antibiotics and conventional antimicrobial peptides, which often have limited efficacy against intracellular bacteria.