Biocompatible Solid-phase Microextraction Research Articles

High throughput quantification of prohibited substances in plasma using thin film solid phase microextraction

Simple, fast and efficient sample preparation approaches that allow high-throughput isolation of various compounds from complex matrices are highly desired in bioanalysis. Particularly sought are methods that can, without sacrificing time, easily remove matrix interferences capable of inducing ionization suppression/enhancement, or causing detrimental effects in instrumental performance. In this work, an automated high-throughput sample preparation method using thin film solid phase microextraction (SPME) for the analysis of multiple prohibited substances in plasma is proposed. A biocompatible SPME extraction phase made of hydrophilic–lipophilic balance particles immobilized with polyacrylonitrile (PAN) demonstrated satisfactory extraction capabilities for 25 compounds of a wide range of polarities (logP from −2 to 6.8). Due to the well-known biocompatible characteristics of PAN-based SPME coatings, minimum sample handling was required. Experimental conditions for pre-conditioning, extraction, wash and desorption were carefully optimized for the proposed method. By taking full advantage of the 96 thin film handling capability of the automated system, a preparation time of approximately 1.5min per sample can be achieved. Satisfactory results in terms of absolute matrix effects were found for the majority of the studied analytes, given that 24 out of 25 compounds exhibited values in the range of 100 and 120%. The method was validated in terms of linearity (R2>0.99), inter and intra-day accuracy (85–130%) and precision (<20%) and limits of quantitation (0.25–10ngmL−1 for most compounds).

Analysis of Human Saliva Metabolome by Direct Immersion Solid-Phase Microextraction LC and Benchtop Orbitrap MS

Saliva samples collected from one 58 year old male and one 35 year old female during 7 days of fasting were analyzed by direct immersion of both C18 and mixed-mode biocompatible solid-phase microextraction fibers, in combination with a LC-MS method using a benchtop orbitrap instrument in both positive and negative ionization modes, in order to evaluate the difference in terms of metabolite coverage. The mixed-mode coating provided better results, with the simultaneous extraction of a higher number of both hydrophilic and hydrophobic metabolites. The ability of detected features to distinguish differences between the individuals and changes in saliva metabolome induced by diet was also demonstrated. Saliva may be useful for medical diagnostics as it is non-invasive. The use of biocompatible solid-phase microextraction fibers can play an important role as an alternative sample preparation method for untargeted LC-MS metabolomics studies on human saliva because it provides simultaneous extraction of metabolites with a wide range of polarity, thus allowing the detection of changes in metabolic pathways with unsupervised statistical analyses.

In vivo solid-phase microextraction for monitoring intravenous concentrations of drugs and metabolites

This protocol for in vivo solid-phase microextraction (SPME) can be used to monitor and quantify intravenous concentrations of drugs and metabolites without the need to withdraw a blood sample for analysis. The SPME probe is inserted directly into a peripheral vein of a living animal through a standard medical catheter, and extraction occurs typically over 2-5 min. After extraction, the analytes are removed from the sorbent and analyzed by, for example, liquid chromatography-tandem mass spectrometry. It has been validated in comparison with conventional blood analysis, and we describe here the in vitro experiments typically conducted during method development. The new-generation biocompatible SPME probes are designed specifically for extraction of semi-volatiles and nonvolatiles directly from aqueous samples and can be steam sterilized. Sorbents are coated on fine-gauge surgical steel wire (200-μm diameter), which is more rugged and biocompatible than conventional fibers (100-μm fused silica fiber). They incorporate a binding agent that resists fouling by the biological matrix and does not cause an immune response in the experimental animal. The sorbents used (coating thickness of ∼50 μm) are selected for their affinity for the types of small molecules of interest. The procedure is illustrated by the analysis of benzodiazepines with polypyrrole-coated wires inserted into peripheral blood vessels of beagles, although it can be adapted for use in smaller animals. The in vivo sampling can require as little as 1 min, in which case the entire procedure from sampling to instrumental analysis can take as little as 30 min.

Coupling of solid‐phase microextraction continuous bed (monolithic) capillaries with capillary zone electrophoresis for direct analysis of drugs in biological fluids

Hyperlink robust biocompatible solid-phase microextraction (SPME) devices were prepared using continuous bed (monolithic) restricted-access media (RAM) as the SPME capillary insert. The RAM-based SPME approach was able to simultaneously separate proteins from a biological sample, while directly extracting the active components of caffeine, paracetamol and acetylsalicylic acid from the drug NeoCitramonum. The devices were interfaced with a CZE system and fully automated analysis for sample preconcentration, desorption, separation and quantification of analytes was evaluated. Comparative study of in-line coupled SPME-CZE using RAM and RP capillary inserts was carried out. Using an SPME (RAM) insert, the calculated caffeine, paracetamol and acetylsalicylic acid LODs in a bovine plasma sample were 0.3, 0.8 and 1.9 ng/mL, respectively.

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.

Approaches for coupling solid-phase microextraction to nanospray

Biocompatible solid-phase microextraction (SPME) devices were prepared using two restricted access materials (RAM) as the SPME coating. The restricted access materials were immobilized on steel and platinum wires. The selective coating eliminated most of the matrix interference, which allowed the coupling to mass spectrometry without further purification. The SPME devices were interfaced to mass spectrometry by electronanospray. Several experimental set-ups are described and discussed herein. For the in situ extraction of peptides from the tryptic digests, trypsin was immobilized both on steel wires and on the inside wall of a vial. The devices were incubated together with the RAM–SPME devices and a protein (casein) solution. After the protein digestion, the resulting peptides were analyzed by SPME/nanospray. The vial approach provided the best results; up to eight peptides could be identified which corresponds to a sequence coverage of 58%. The limit of detection of SPME/nanospray for the extraction of peptides from an aqueous solution was about 50 fmol/mL. The results demonstrate that the direct coupling of SPME to nanospray can reduce analysis time and is an attractive alternative to conventional approaches like Zip–Tip purification.

Monitoring of drugs and metabolites in whole blood by restricted-access solid-phase microextraction coupled to liquid chromatography–mass spectrometry

Robust biocompatible solid-phase microextraction (SPME) devices were prepared using various alkyldiol-silica (ADS) restricted-access materials (RAM) as the SPME coating. The ADS-SPME approach was able to simultaneously fractionate the protein component from a biological sample, while directly extracting diazepam and the major metabolites N-desmethyldiazepam, oxazepam and temazepam, and overcame the present disadvantages of direct sampling in biological matrices by SPME. The devices were interfaced with an LC–MS system and an isocratic mobile phase was used to desorb, separate, and quantify the analytes. The calculated diazepam, nordiazepam, temazepam, and oxazepam detection limits were 20, 20, 30, and 35 ng/ml in heparinized blood, respectively. The method was confirmed to be linear over the range of 50–1000 ng/ml with an average linear coefficient ( R 2) value of 0.996. The injection repeatability and intra-assay precision of the method were evaluated over ten injections at concentrations of 50, 200, and 500 ng/ml, resulting in a R.S.D. of ca. 10%. The robustness of the ADS-SPME device was evaluated for future use in in vivo studies, providing many direct extractions and subsequent determination of benzodiazepines in blood. For the extraction of the peptides angiotensin I, II, and III from blood, a novel restricted access material with cation exchange properties was evaluated. The ion-exchange diol silica improved the extraction efficiency of peptides relative to the conventional ADS material with reversed phase extraction centers.

Bio-compatible in-tube solid-phase microextraction capillary for the direct extraction and high-performance liquid chromatographic determination of drugs in human serum

A restricted access material (RAM), alkyl-diol-silica (ADS), was used to prepare a highly bio-compatible solid-phase microextraction (SPME) capillary for the automated and direct in-tube extraction of several benzodiazepines from human serum. The bifunctionality of the ADS extraction phase prevented fouling of the capillary by protein adsorption while simultaneously trapping the analytes in the hydrophobic porous interior. This the first report of a restricted access material utilized as an extraction phase for in-tube SPME. The approach simplified the required apparatus in comparison to existing RAM column switching procedures, and more importantly eliminated the excessive use of extraction solvents. The biocompatibility of the ADS material also overcame the existing problems with in-tube SPME that requires an ultrafiltration or other deproteinization step prior to handling biological samples, therefore further minimizing the sample preparation requirements. The calculated oxazepam, temazepam, nordazepam and diazepam detection limits were 26, 29, 22 and 24 ng/ml in serum, respectively. The method was linear over the range of 50–50 000 ng/ml with an average linear coefficient ( R 2) value of 0.9998. The injection repeatability and intra-assay precision of the method were evaluated with five injections of a 10-μg/ml serum sample (spiked with all compounds), resulting in an average RSD<7%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.

Direct Determination of Benzodiazepines in Biological Fluids by Restricted-Access Solid-Phase Microextraction

A biocompatible solid-phase microextraction (SPME) fiber was prepared using an alkyl-diol-silica (ADS) restricted-access material as the SPME coating. The ADS-SPME fiber was able to simultaneously fractionate the protein component from a biological sample, while directly extracting several benzodiazepines, overcoming the present disadvantages of direct sampling in biological matrixes by SPME. The fiber was interfaced with an HPLC-UV system, and an isocratic mobile phase was used to desorb, separate, and quantify the extracted compounds. The calculated clonazepam, oxazepam, temazepam, nordazepam, and diazepam detection limits were 600, 750, 333, 100, and 46 ng/mL in urine, respectively. The method was confirmed to be linear over the range of 500-50000 ng/mL with an average linear coefficient (R2) value of 0.9918. The injection repeatability and intraassay precision of the method were evaluated over 10 injections, resulting in a RSD of approximately 6%. The ADS-SPME fiber was robust and simple to use, providing many direct extractions and subsequent determination of benzodiazepines in biological fluids.