Semi-automated in vivo solid-phase microextraction sampling and the diffusion-based interface calibration model to determine the pharmacokinetics of methoxyfenoterol and fenoterol in rats.

In vivo solid-phase microextraction (SPME) can be used to sample the circulating blood of animals without the need to withdraw a representative blood sample. In this study, in vivo SPME in combination with liquid-chromatography tandem mass spectrometry (LC-MS/MS) was used to determine the pharmacokinetics of two drug analytes, R,R-fenoterol and R,R-methoxyfenoterol, administered as 5 mg kg(-1) i.v. bolus doses to groups of 5 rats. This research illustrates, for the first time, the feasibility of the diffusion-based calibration interface model for in vivo SPME studies. To provide a constant sampling rate as required for the diffusion-based interface model, partial automation of the SPME sampling of the analytes from the circulating blood was accomplished using an automated blood sampling system. The use of the blood sampling system allowed automation of all SPME sampling steps in vivo, except for the insertion and removal of the SPME probe from the sampling interface. The results from in vivo SPME were compared to the conventional method based on blood withdrawal and sample clean up by plasma protein precipitation. Both whole blood and plasma concentrations were determined by the conventional method. The concentrations of methoxyfenoterol and fenoterol obtained by SPME generally concur with the whole blood concentrations determined by the conventional method indicating the utility of the proposed method. The proposed diffusion-based interface model has several advantages over other kinetic calibration models for in vivo SPME sampling including (i) it does not require the addition of a standard into the sample matrix during in vivo studies, (ii) it is simple and rapid and eliminates the need to pre-load appropriate standard onto the SPME extraction phase and (iii) the calibration constant for SPME can be calculated based on the diffusion coefficient, extraction time, fiber length and radius, and size of the boundary layer. In the current study, the experimental calibration constants of 338.9±30 mm(-3) and 298.5±25 mm(-3) are in excellent agreement with the theoretical calibration constants of 307.9 mm(-3) and 316.0 mm(-3) for fenoterol and methoxyfenoterol respectively.

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.

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.