Influence of Sampling Site and Fluid Flow on the Accuracy of Total Body Clearance Calculation.

Studies have showed that by assuming arteriovenous drug concentrations are homogenous after intravenous injection, the determination of total body clearance based on venous drug concentrations is often inaccurate. This study considers the use of a fluidic pharmacokinetic profile generator where 28 different profile types were generated corresponding to a physiological model with varying sampling sites, administration locations, and fluid flow rates. Clearance was calculated using established equations, commercial software, and recently proposed models. The results show large differences in clearance values calculated with published equations and commercial software relative to the actual value of clearance. Alterations in sampling site, administration location, and fluid flow rates each influence the extent of calculation errors. The data show that a significant drug concentration gradient exists within the central circulatory system. The results show that the best way to address this issue would be to inject the drug at a peripheral location to allow for sufficient mixing and then sample from a large vein. Extrapolating for missing data can also lead to large errors in clearance calculation; this can be addressed by collecting more samples early after IV bolus administration or by collecting data during steady state conditions for an IV infusion.

In vivo monitoring of rat brain endocannabinoids using solid-phase microextraction.

Aim: Solid-phase microextraction is proposed to measure concentrations of anandamide and 2-arachidonoyl glycerol in live rat brains in response to stress. Materials & methods: Solid-phase microextraction fibers were prepared from steel with 1.5 mm extraction coating. 24 male rats were divided into groups based on brain region, stria terminalis or posterior hypothalamus and loud noise or control groups. The fibers were desorbed in acetonitrile-water (75:25) and analyzed by ultraperformance LC-MS/MS. The linear range of the method was 0.05-50 ng/ml and the in vivo concentrations were found to be between 0.3 and 40 ng/ml. Conclusion: The new approach was successfully used to determine the concentrations of anandamide and 2-arachidonoyl glycerol in vivo and could be used in the future to measure other endogenous compounds.

Transdermal sampling of vitamin D3 and 25-hydroxyvitamin D3.

AIM: Transdermal analysis is proposed for vitamin D3 and its hydroxylated metabolite to overcome problems associated with blood analysis. METHODS: Vitamin D3 was extracted directly from skin with solid patches and liquid phases. Deuterium-labeled vitamin D3 was added to the extraction solutions to compensate for variability and accurately determine the rate of transdermal transfer. Of the different extraction solvents tested, 50:50 octanol:isopropanol showed the best results, with an accuracy of 115% and reproducibility better than 30%. CONCLUSION: The research shows that transdermal route can be used for analysis of vitamin D3 in porcine skin. When microneedles are used, accurate measurements were obtained in 1 h. With intact skin, the highest accuracy was obtained when extraction was done for 2 h.

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 5­lipoxygenase-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.

Evaluation of in vivo solid phase microextraction for minimally invasive analysis of nonvolatile phytochemicals in Amazonian plants.

INTRODUCTION: Although solid phase microextraction (SPME) has been used extensively for fingerprinting volatile compounds emitted by plants, there are very few such reports for direct insertion SPME. In this research, direct contact of SPME probes with the interstitial fluid of plants was investigated as a method for phytochemical analysis. OBJECTIVE: Medicinal plants from the Amazon have been the source of numerous drugs used in western medicine. However, a large number of species used in traditional medicine have not been characterized chemically, partly due to the difficulty of field work. In this project, the phytochemical composition of plants from several genera was fingerprinted by combining convenient field sampling by solid phase microextraction (SPME) with laboratory analysis by LC-MS. The new method was compared with classical sampling followed by liquid extraction (LE). METHODOLOGY: SPME probes were prepared by coating stainless steel wires with a mixture of polyacrylonitrile and either RP-amide or HS-F5 silica particles. Sampling was performed by inserting the microextraction probes into various tissues of living plants in their natural environment. After in vivo extraction, the probes were sealed under vacuum and refrigerated until analyzed. The probes were desorbed in mobile phase and analyzed on a Waters Acquity UPLC with triple quadrupole mass spectrometer in positive ion mode. RESULTS: Twenty Amazonian plant species were sampled and unique metabolomic fingerprints were obtained. In addition, quantitative analysis was performed for previously identified compounds in three species. Comparison of the fingerprints obtained by in vivo SPME with those obtained by LE showed that 27% of the chromatographic features were unique to SPME, 57% were unique to LE, and 16% were common to both methods. CONCLUSION: In vivo SPME caused minimal damage to the plants, was much faster than traditional liquid extraction, and provided unique fingerprints for all investigated plants. SPME revealed unique chromatographic features, undetected by traditional extraction, although it produced only half as many peaks as ethanol extraction.

Development of supported liquid-phase microextraction probes for in vivo PK studies.

BACKGROUND: A new sample preparation method termed supported liquid-phase microextraction is proposed. With this technique, the extraction phase is a liquid immobilized inside the pores of a membrane coated on a solid support. METHODOLOGY: Supported liquid-phase microextraction probes were prepared by coating wires with porous polyacrylonitrile followed by saturation with 1-octanol. The probes were introduced inside hypodermic needles and used for in vivo extraction of oxybutynin from the blood and tissues of rabbits. The linear range of the method was from 0.5 to 500 ng/ml. CONCLUSION: The proposed method was successfully applied to monitor the PK profile of oxybutynin. The drug followed a two-compartment model, with a volume of distribution of 14 l/kg and a half-life of 76 min.

Investigating transdermal delivery of vitamin D3.

Transdermal delivery of therapeutic amounts of vitamin D3 is proposed to overcome its variable oral bioavailability, especially for people who suffer from fat malabsorption. The main challenge for this delivery route is to overcome the barrier properties of skin, especially for very lipophilic compounds such as vitamin D3. In this study, the effect of different penetration enhancers, such as oleic acid, dodecylamine, ethanol, oleic acid in propylene glycol, isopropyl myristate, octyldodecanol, and oleyl alcohol in propylene glycol were evaluated in vitro for their effectiveness in delivering vitamin D3 through polyamide filter, polydimethylsiloxane membrane, and porcine skin. A diffusion cell was used to study the transdermal permeability of vitamin D3. Ointment formulations of vitamin D3 were prepared containing the most widely used penetration enhancers, oleic acid, and dodecylamine. The ointment containing oleic acid as chemical penetration enhancer did not improve delivery compared to control. On the other hand, the formulation containing dodecylamine as a penetration enhancer did improve the transdermal delivery of vitamin D3. However, statistical significance and an amount high enough for nutritional supplementation purposes were reached only when the skin was pretreated with 50% ethanol. In these conditions, the ointment delivered an amount of 760-ng vitamin D3 per cm(2) of skin. The research shows promise that transdermal delivery could be an effective administration route for vitamin D3 when ethanol and dodecylamine are used as penetration enhancers.

Normalized vitamin D metabolite concentrations are better correlated to pharmacological effects than measured concentrations.

BACKGROUND: Vitamin D deficiency has been associated with a multitude of diseases, ranging from fractures to cancer. Nearly 99% of vitamin D metabolites are bound to proteins, altering the relationship between concentration and activity. METHODS & RESULTS: Normalized concentrations were calculated and validated using published data regarding the correlation of 25-hydroxyvitamin D with bone mineral density. In addition, healthy and kidney disease subjects were recruited for preliminary investigations. Use of the normalizing equations resulted in statistically significant improvements in the relationship between vitamin D metabolites and several markers of health status. CONCLUSION: Normalized concentrations are similar to clinically reported values and are easier to interpret than free or bioavailable concentrations, since their values match the range of measured total concentrations.

Calculation of normalized drug concentrations in the presence of altered plasma protein binding.

BACKGROUND AND OBJECTIVE: In 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. METHODS: When 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. RESULTS: The 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. CONCLUSIONS: A 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.

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.

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.

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.

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.

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.

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.

Blood sampling without blood draws for in vivo pharmacokinetic studies in rats.

PURPOSE: Pharmacokinetic (PK) studies in rats typically involve removal of serial blood samples following dosing. This research illustrates the development of a fast in vivo microextraction technique that has the potential to partly replace current sampling techniques based on blood draws, especially in the case of small animals. METHODS: The proposed sampling procedure is based on solid phase microextraction (SPME), an approach that has continued to revolutionize sampling and sample preparation ever since its discovery a decade ago. In vivo microextraction is faster than conventional methods, interferes minimally with the investigated system, minimizes errors associated with sample preparation and limits exposure of lab personnel to hazardous biological samples. RESULTS: Here we show the successful application of fast microextraction during a full PK study with diazepam in rats. The results were found to correlate very well with a standard analytical method. Three calibration strategies--external, standard on the fiber, and double extraction--were employed to correlate the amount extracted with the in vivo concentration. CONCLUSIONS: Our results demonstrate the unique advantages of this technique and highlight its utility as a valuable new tool for fast bioanalysis in support of in vivo sampling and PK studies, particularly during the early drug discovery process. This approach can be used not only for drugs, but also for other exogenous or endogenous compounds.

Biocompatible solid-phase microextraction coatings based on polyacrylonitrile and solid-phase extraction phases.

The applications of solid-phase microextraction (SPME) are continuously expanding, and one of the most interesting current aspects consists of applying SPME for fast analysis of biological fluids. The goal of this study is to develop biocompatible SPME coatings that can be utilized for in vivo and in vitro extractions, in direct contact with a biological matrix such as blood or tissue. The biocompatibility of the proposed new coatings is confirmed by X-ray photoelectron spectroscopy, and their performance is tested by developing an SPME/HPLC method for analysis of verapamil, loperamide, diazepam, nordiazepam, and warfarin in buffer solutions and in human plasma. The coatings prove to be biocompatible by not adsorbing proteins and are successfully applied for fast drug analysis and assay of drug plasma protein binding.