Md. Nazmul Alam

Aptamer-functionalized solid phase microextraction-liquid chromatography/tandem mass spectrometry for selective enrichment and determination of thrombin.

In this publication, a novel solid phase microextraction (SPME) coating functionalized with a DNA aptamer for selective enrichment of a low abundance protein from diluted human plasma is described. This approach is based on the covalent immobilization of an aptamer ligand on electrospun microfibers made with the hydrophilic polymer poly(acrylonitrile-co-maleic acid) (PANCMA) on stainless steel rods. A plasma protein, human α-thrombin, was employed as a model protein for selective extraction by the developed Apt-SPME probe, and the detection was carried out with liquid chromatography/tandem mass spectrometry (LC-MS/MS). The SPME probe exhibited highly selective capture, good binding capacity, high stability and good repeatability for the extraction of thrombin. The protein selective probe was employed for direct extraction of thrombin from 20-fold diluted human plasma samples without any other purification. The Apt-SPME method coupled with LC-MS/MS provided a good linear dynamic range of 0.5-50 nM in diluted human plasma with a good correlation coefficient (R(2)=0.9923), and the detection limit of the proposed method was found to be 0.30 nM. Finally, the Apt-SPME coupled with LC-MS/MS method was successfully utilized for the determination of thrombin in clinical human plasma samples. One shortcoming of the method is its reduced efficiency in undiluted human plasma compared to the standard solution. Nevertheless, this new aptamer affinity-based SPME probe opens up the possibility of selective enrichment of a given targeted protein from complex sample either in vivo or ex vivo.

Review of geometries and coating materials in solid phase microextraction: Opportunities, limitations, and future perspectives

The development of new support and geometries of solid phase microextraction (SPME), including metal fiber assemblies, coated-tip, and thin film microextraction (TFME) (i.e. self-supported, fabric and blade supported), as well as their effects on diffusion and extraction rate of analytes were discussed in the current review. Application of main techniques widely used for preparation of a variety of coating materials of SPME, including sol-gel technique, electrochemical and electrospinning methods as well as the available commercial coatings, were presented. Advantages and limitations of each technique from several aspects, such as range of application, biocompatibility, availability in different geometrical configurations, method of preparation, incorporation of various materials to tune the coating properties, and thermal and physical stability, were also investigated. Future perspectives of each technique to improve the efficiency and stability of the coatings were also summarized. Some interesting materials including ionic liquids (ILs), metal organic frameworks (MOFs) and particle loaded coatings were briefly presented.

Advances in Solid Phase Microextraction and Perspective on Future Directions

Solid phase microextraction (SPME) is a versatile, non-exhaustive sample preparation tool that has been demonstrated to be well-suited for facile and effective analysis of a broad range of compounds in a plethora of studies. A growing number of reports describing diverse SPME workflows for novel investigations in a variety of fields, such as flavor and fragrance investigations, environmental studies, and diverse bioanalytical applications, among others, corroborate the applicability of this microextraction tool in the analytical sciences

Numerical modeling of solid-phase microextraction: binding matrix effect on equilibrium time.

Solid-phase microextraction (SPME) is a well-known sampling and sample preparation technique used for a wide variety of analytical applications. As there are various complex processes taking place at the time of extraction that influence the parameters of optimum extraction, a mathematical model and computational simulation describing the SPME process is required for experimentalists to understand and implement the technique without performing multiple costly and time-consuming experiments in the laboratory. In this study, a mechanistic mathematical model for the processes occurring in SPME extraction of analyte(s) from an aqueous sample medium is presented. The proposed mechanistic model was validated with previously reported experimental data from three different sources. Several key factors that affect the extraction kinetics, such as sample agitation, fiber coating thickness, and presence of a binding matrix component, are discussed. More interestingly, for the first time, shorter or longer equilibrium times in the presence of a binding matrix component were explained with the help of an asymptotic analysis. Parameters that contribute to the variation of the equilibrium times are discussed, with the assumption that one binding matrix component is present in a static sample. Numerical simulation results show that the proposed model captures the phenomena occurring in SPME, leading to a clearer understanding of this process. Therefore, the currently presented model can be used to identify optimum experimental parameters without the need to perform a large number of experiments in the laboratory.

Insights into the Effect of the PDMS-Layer on the Kinetics and Thermodynamics of Analyte Sorption onto the Matrix-Compatible Solid Phase Microextraction Coating

The currently presented research investigated the performance of matrix compatible PDMS-overcoated fibers (PDMS-DVB/PDMS) as compared to unmodified PDMS/DVB coatings using aqueous samples and employing a wide range of analyte polarities, molecular weights, and functionalities. In the first part of the work, a kinetic approach was taken to investigate the effect of the PDMS outer layer on the uptake rate of analytes during the mass transfer process. In short, the results can be simplified into two models: (1) the rate-limiting step is the diffusion through the coating and (2) the rate-limiting step is the diffusion through the aqueous diffusional boundary layer. For polar compounds, according to the theoretical discussion, the rate-limiting step is the diffusion through the coating; therefore, the outer PDMS layer influences the uptake rate into the matrix compatible coatings. On the other hand, for nonpolar compounds, the rate-limiting step of the uptake process is diffusion through the aqueous diffusional boundary layer; as such, the overcoated PDMS does not affect uptake rate into the matrix-compatible coatings as compared to DVB/PDMS fibers. From a thermodynamic point of view, the calculated fiber constants further corroborate the hypothesis that the additional PDMS layer does not impair the extraction phase capacity.

Numerical Simulation and Experimental Validation of Calibrant-Loaded Extraction Phase Standardization Approach

We present the kinetics of calibrant release and analyte uptake between the sample and calibrant-loaded extraction phase, CL-EP, with a finite-element analysis (FEA) using COMSOL Multiphysics software package. Effect of finite and infinite sample volume conditions, as well as various sample environment parameters such as fluid flow velocity, temperature, and presence of a binding matrix component were investigated in detail with the model in relation to the performance of the calibration. The simulation results supported by experimental data demonstrate the suitability of the CL-EP method for analysis of samples with variation of the sample environment parameters. The calibrant-loaded approach can provide both total and free concentrations from a single experiment based on whether the partition coefficient (Kes) value being used is measured in a matrix-matched sample or in a matrix-free sample, respectively. Total concentrations can also be obtained by utilizing CL-EP in combination with external matrix-matched calibrations, which can be employed to automate the sampling process and provide corrections for variations in sample preparation, matrix effects, and detection processes. This approach is also suitable for very small volumes of sample, where addition of an internal standard in the sample is either troublesome or can change the sample characteristics.

Development of a Microfluidic Open Interface with Flow Isolated Desorption Volume for the Direct Coupling of SPME Devices to Mass Spectrometry

Technologies that efficiently integrate the sampling and sample preparation steps with direct introduction to mass spectrometry (MS), providing simple and sensitive analytical workflows as well as capabilities for automation, can generate a great impact in a vast variety of fields, such as in clinical, environmental, and food-science applications. In this study, a novel approach that facilitates direct coupling of Bio-SPME devices to MS using a microfluidic design is presented. This technology, named microfluidic open interface (MOI), which operates under the concept of flow-isolated desorption volume, consists of an open-to-ambient desorption chamber (V ≤ 7 μL) connected to an ionization source. Subsequently, compounds of interest are transported to the ionization source by means of the self-aspiration process intrinsic of these interfaces. Thus, any ionization technology that provides a reliable and constant suction, such as electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), or inductively coupled plasma ionization (ICP), can be hyphenated to MOI. Using this setup, the desorption chamber is used to release target compounds from the coating, while the isolation of the flow enables the ionization source to be continuously fed with solvent, all without the necessity of employment of additional valves. As a proof of concept, the design was applied to an ESI-MS/MS system for experimental validation. Furthermore, numerical simulations were undertaken to provide a detailed understanding of the fluid flow pattern inside the interface, then used to optimize the system for better efficiency. The analytical workflow of the developed Bio-SPME-MOI-MS setup consists of the direct immersion of SPME fibers into the matrix to extract/enrich analytes of interest within a short period of time, followed by a rinsing step with water to remove potentially adhering proteins, salts, and/or other interfering compounds. Next, the fiber is inserted into the MOI for desorption of compounds of interest. Finally, the volume contained in the chamber is drained and moved toward the electrospray needle for ionization and direct introduction to MS. Aiming to validate the technology, the fast determination of selected immunosuppressive drugs (e.g., tacrolimus, cyclosporine, sirolimus, and everolimus) from 100 μL of whole blood was assessed. Limits of quantitation in the subppb range were obtained for all studied compounds. Good linearity (r2 ≥ 0.99) and excellent precision, with (8%) and without (14%) internal standard correction, were attained.

Effect of Transport Parameters and Device Geometry on Extraction Kinetics and Efficiency in Direct Immersion Solid-phase Microextraction

An alternative strategy to increase mass transfer entails geometry optimization of the extraction systems including design of solid-phase microextraction (SPME) probes. In this work, a computational model was employed to elucidate practical aspects such as efficiency and kinetics of extraction by employing several new geometries. Extraction of a model analyte at static conditions with the configurations, such as thin-film, fiber, coated tip, and nanoparticles, was numerically simulated to obtain an in-depth understanding of the advantages and limitations of each geometry in microextraction and exhaustive modes. The attained results associated with the equilibration time dependency on shape were in good agreement with previously reported experimental observations. They demonstrate that the mass-transfer is highly dependent on the size and shape of the coatings and increases with a decrease in size of the devices particularly rapidly below 10 μm caused by radial diffusion effect. Nevertheless, extractions performed using octadecyl-functionalized magnetic nanoparticles demonstrated that higher enrichment factors are achievable with the use of a fewer number of particles in comparison to factors achieved via exhaustive extraction, where a larger number of particles must be employed, confirming theoretical predictions. The conclusions reached are valid for any extraction method. The results obtained herein are very useful toward the design and optimization of future extraction technologies and approaches.

Development and validation of eco-friendly strategies based on thin film microextraction for water analysis

The aim of the current study is the establishment of Green Analytical Chemistry strategies for water analysis by elimination/reduction of hazardous chemicals, energy consumption, and waste generation throughout the entire analytical workflow. The first approach introduced in this manuscript consists of addition of water to a sampling vessel that contains a thin film microextraction (TFME) device, followed by removal of the device after equilibration, and subsequent quantification of the extracted components by thermal desorption GC/MS. In this approach, namely the in-bottle TFME approach, analyte-loss associated errors that stem from analyte adherence to glass surfaces and/or degradation are avoided as extraction occurs in situ, while analytes are isolated from a complex matrix that contains degradation agents (bacteria, oxidizing or reducing species, etc.). This procedure also circumvents the splitting of original samples into sub-samples. The second approach involves the use of portable TFME devices that facilitate on-site extraction of compounds, therefore eliminating the use of collection vessels, a factor known to potentially introduce errors into analysis. The herein described procedure involves attachment of the TFME device to drill accessories, analyte extraction via direct immersion into sampled site, and subsequent on-site instrumental analysis, which is carried out with the use of a portable GC/MS containing an appropriate thermal desorption interface, or alternatively, by transferring the membrane to the laboratory for bench-top GC/MS analysis. To facilitate a better understanding of the processes governing the developed approaches, modeling by COMSOL Multiphysics® software was performed. The findings of this study were applied for further method optimization, and the optimized developed methods were then applied for on-site surface water analyses. Finally, the greenness of the developed methods was evaluated with use of the eco-scale assessment, with obtained scores compared to that of the US EPA 8270 method.

Effect of Binding Components in Complex Sample Matrices on Recovery in Direct Immersion Solid-Phase Microextraction: Friends or Foe?

The development of matrix compatible coatings for solid-phase microextraction (SPME) has enabled direct extraction of analytes from complex sample matrices. The direct immersion (DI) mode of SPME when utilized in conjunction with such extraction phases facilitates extraction of a wide range of analytes from complex matrices without the incurrence of fouling or coating saturation. In this work, mathematical models and computational simulations were employed to investigate the effect of binding components present in complex samples on the recovery of small molecules varying in logP for extractions carried out using the direct immersion approach. The presented findings corroborate that the studied approach indeed enables the extraction of both polar and nonpolar analytes from complex matrices, provided a suitable sorbent is employed. Further results indicated that, in certain cases, the kinetics of extraction of a given analyte in its free form might be dependent on the desorption kinetics of their bound form from matrix components, which might lower total recoveries of analytes with high affinity for the matrix. However, the binding of analytes to matrix components also enables SPME to extract a balanced quantity of different logP analytes, facilitated by multiphase equilibria, with a single extraction device.