Dietmar Hein

Automation of solid-phase microextraction in high-throughput format and applications to drug analysis.

The automation of solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was accomplished using a 96 multiwell plate format, a SPME multifiber device, two orbital shakers, and a three-arm robotic system. Extensive optimization of the proposed setup was performed including coating selection, optimization of the fiber coating procedure, confirmation of uniform agitation in all wells, and the selection of the optimal calibration method. The system allows the use of pre-equilibrium extraction times with no deterioration in method precision due to reproducible timing of extraction and desorption steps and reproducible positioning of all fibers within the wells. The applicability of the system for the extraction of several common drugs is demonstrated. The optimized multifiber SPME-LC-MS/MS was subsequently fully validated for the high-throughput analysis of diazepam, lorazepam, nordiazepam, and oxazepam in human whole blood. The proposed method allowed the automated sample preparation of 96 samples in 100 min, which represents the highest throughput of any SPME technique to date, while achieving excellent accuracy (87-113%), precision (<or=20% RSD), and sensitivity (limit of quantitation 4 ng/mL). Automated SPME provides unique advantages over automated solid-phase extraction (SPE) including lower cost, the ability to quantitatively determine free and total drug concentrations in a single biofluid sample, and the ability to directly process whole blood samples with absolutely no sample pretreatment required.

Multibed Needle Trap Devices for on Site Sampling and Preconcentration of Volatile Breath Biomarkers

To facilitate their use in trace gas analysis, the adsorption capacity of needle trap devices (NTDs) was increased by combining three adsorbent materials and increasing total adsorbent amount. The use of 22 gauge needles, application of internally expanding desorptive flow technique without cryofocusation and a new on site alveolar sampling method for NTDs provided sensitivity in the parts per trillion range of VOC concentrations without loosing precision or linearity. LODs were 0.4 ng/L for isoprene, 0.5 ng/L for dimethyl sulphide, 0.9 ng/L for 2-butenal, 1.0 ng/L for hexane, 1.2 ng/L for pentane, 2.3 ng/L for hexanal, 5.3 ng/L for pentanal, and 8.3 ng/L for acetone. R of calibration curves were consistently >0.98. Loss of volatile aldehydes during storage for 7 days was less than 10%. Needle trap devices packed with more than one adsorbent material represent a promising alternative to SPE and SPME for analysis of volatile organic compounds in the low parts per billion/parts per trillion range. Crucial problems of clinical breath analysis concerning sensitivity of analytical methods, limited stability, and decomposition of breath compounds during sampling and storage could be solved.

Needle trap micro-extraction for VOC analysis: effects of packing materials and desorption parameters.

Combining advantages of SPE and SPME needle trap devices (NTD) represent promising new tools for a robust and reproducible sample preparation. This study was intended to investigate the effect of different packing materials on efficacy and reproducibility of VOC analysis by means of needle trap micro extraction (NTME). NTDs with a side hole design and containing different combinations of PDMS, DVB and Carbopack X and Carboxen 1000 and NTDs containing a single layer organic polymer of methacrylic acid and ethylene glycol dimethacrylate were investigated with respect to reproducibility, LODs and LOQs, carry over and storage. NTDs were loaded with VOC standard gas mixtures containing saturated and unsaturated hydrocarbons, oxygenated and aromatic compounds. Volatile substances were thermally desorbed from the NTDs using fast expansive flow technique and separated, identified and quantified by means of GC-MS. Optimal desorption temperatures between 200 and 290°C could be identified for the different types of NTDs with respect to desorption efficiency and variation. Carry over was below 6% for polymer packed needles and up to 67% in PDMS/Carboxen 1000 NTDs. Intra and inter needle variation was best for polymer NTDs and consistently below 9% for this type of NTD. LODs and LOQs were in the range of some ng/L. Sensitivity of the method could be improved by increasing sample volume. NTDs packed with a copolymer of methacrylic acid and ethylene glycol dimethacrylate were universally applicable for sample preparation in VOC analysis. If aromatic compounds were to be determined DVB/Carboxen 1000 and DVB/Carbopack X/Carboxen 1000 devices could be considered as an alternative. PDMS/Carbopack X/Carboxen 1000 NTDs may represent a good alternative for the analysis of hydrocarbons and aldehydes. NTME represents a powerful tool for different application areas, from environmental monitoring to breath analysis.

Sample preparation with solid phase microextraction and exhaustive extraction approaches: Comparison for challenging cases.

In chemical analysis, sample preparation is frequently considered the bottleneck of the entire analytical method. The success of the final method strongly depends on understanding the entire process of analysis of a particular type of analyte in a sample, namely: the physicochemical properties of the analytes (solubility, volatility, polarity etc.), the environmental conditions, and the matrix components of the sample. Various sample preparation strategies have been developed based on exhaustive or non-exhaustive extraction of analytes from matrices. Undoubtedly, amongst all sample preparation approaches, liquid extraction, including liquid-liquid (LLE) and solid phase extraction (SPE), are the most well-known, widely used, and commonly accepted methods by many international organizations and accredited laboratories. Both methods are well documented and there are many well defined procedures, which make them, at first sight, the methods of choice. However, many challenging tasks, such as complex matrix applications, on-site and in vivo applications, and determination of matrix-bound and free concentrations of analytes, are not easily attainable with these classical approaches for sample preparation. In the last two decades, the introduction of solid phase microextraction (SPME) has brought significant progress in the sample preparation area by facilitating on-site and in vivo applications, time weighted average (TWA) and instantaneous concentration determinations. Recently introduced matrix compatible coatings for SPME facilitate direct extraction from complex matrices and fill the gap in direct sampling from challenging matrices. Following introduction of SPME, numerous other microextraction approaches evolved to address limitations of the above mentioned techniques. There is not a single method that can be considered as a universal solution for sample preparation. This review aims to show the main advantages and limitations of the above mentioned sample preparation approaches and the applicability and capability of each technique for challenging cases such as complex matrices, on-site applications and automation.

Investigation of the Effect of the Extraction Phase Geometry on the Performance of Automated Solid-Phase Microextraction

A new configuration of C(18) thin film extraction phase designed for high sample throughput has been developed and applied to the analysis of benzodiazepines in spiked urine samples using high performance liquid chromatography coupled with tandem mass spectrometry. The high throughput analysis was achieved with the use of a robotic autosampler which enabled parallel analyte extraction in a 96-well plate format. Factors affecting data reproducibility, extraction kinetics, sample throughput, and reliability of the system were investigated and optimized. The intrawell reproducibility was 4.5-7.3%, while interwell reproducibility was 7.0-11% in urine and PBS samples. The limits of detection and quantitation were 0.05-0.15 ng/mL and 0.2-2.0 ng/mL for all analytes, respectively. By comparison with optimized automated multifiber SPME relying on rod geometry, the C(18) thin films showed higher extraction rates (approximate 2-fold increase) and hence higher sample throughput because of the improved configuration and more effective agitation/mass transfer. In addition, this new configuration provided an extraction phase with greater surface area to volume ratio and greater extraction phase volume, which resulted in approximately 2-fold increase in the extraction capacity for diazepam compared with the extractions with automated multifiber SPME rod geometry. The results of this investigation demonstrated the advantages of using thin films to improve extraction kinetics and sensitivity of automated SPME methods for high performance liquid chromatography.

Development and Application of a Needle Trap Device for Time-Weighted Average Diffusive Sampling

A simple, cost-effective analysis combining solventless extraction, thermal desorption, and determination of volatile organic compounds (VOCs) was developed and validated. A needle trap device (NTD) packed with the sorbent Carboxen1000 was used as a time-weighted average (TWA) diffusive sampler to collect target compounds by molecular diffusion and adsorption to the packed sorbent. This process can be described with derivations of Ficks first law of diffusion, which expresses the relation between the TWA concentrations to which the passive sampler is exposed and the mass of analytes adsorbed to the packed sorbent in the sampler. The effects of experimental factors such as temperature, pressure, humidity, and face velocity were taken into account in applying diffusive sampling under nonideal conditions. This study demonstrates that NTD is effective for air analysis of benzene, toluene, ethylbenzene, and o-xylene (BTEX), due to the good adsorption/desorption quality of Carboxen 1000 and to the special geometric shape of the needle with a small cross section avoiding the need for calibration. Storage tests showed good storage stability for BTEX. Verification of the theoretical model showed good agreement between theoretical and experimental sampling rates. Method validation done against NIOSH method 1501, SPME, and NTD active sampling revealed good agreement between those methods. Automated NTD sample introduction to a gas chromatograph facilitates the use of this technology for industrial hygiene applications.

Protocol for the development of automated high-throughput SPME-GC methods for the analysis of volatile and semivolatile constituents in wine samples.

Ever since the invention of gas chromatography (GC), numerous efforts within the chromatographic community have been directed toward the development of fast GC methods. However, the developments in high-speed GC technologies have simultaneously created demand for the availability of compatible detection and sample preparation methods, so that the speed of the overall analytical process is increased. Solid phase micro extraction (SPME) is a sample preparation technique developed to address the need for rapid sample preparation. Therefore, the objective of this protocol is to outline recent developments in SPME technology that can be applied toward high-throughput automated qualitative and quantitative analyses of volatile and semivolatile compounds in wine samples. The use of this protocol facilitates routine high-throughput determinations of 200–500 analytes of different physicochemical properties with SPME step requiring only 10–15 min per sample.

Solid Phase Microextraction Devices Prepared on Plastic Support as Potential Single-Use Samplers for Bioanalytical Applications

This study presents new thin-film solid phase microextraction (SPME) devices prepared on plastic as potential single-use samplers for bioanalysis. Polybutylene terephthalate (PBT) was selected as a support due to its well-known chemical resistance, low cost, and suitability as a material for different medical grade components. The herein proposed samplers were prepared by applying a hydrophilic-lipophilic balanced (HLB)-polyacrylonitrile (PAN) coating on rounded and flat PBT pieces previously sanded with regular sandpaper. SPME devices prepared on PBT were evaluated in terms of robustness, chemical stability, and possible interferences upon exposure to different solvents and matrixes. Rewarding results were found when these samplers were employed for the quantitative analysis of multiple doping substances in common biological matrixes such as urine, plasma, and whole blood. Finally, the proposed thin-film SPME devices made on a PBT were evaluated by conducting multiple extractions from whole blood and plasma using the Concept 96 system. Results showed that more than 20 extractions from plasma and whole blood can be performed without observed decreases in coating performance or peeling of the extraction phase from the plastic surface. These findings demonstrate the robustness of PAN-based coatings applied on such polymeric substrate and open up the possibility of introducing new alternatives and cost-effective materials as support to manufacture SPME biocompatible devices for a wide range of applications, particularly in the clinical field.

Thin-Film Microextraction Coupled with Mass Spectrometry and Liquid Chromatography–Mass Spectrometry

Thin-film microextraction (TFME) is a format of solid-phase microextraction (SPME) technique which offers improvement of sensitivity without sacrificing time through the increase of available surface area and extractive phase volume. This technique offers significant advantages which make it attractive for many analytical/bioanalytical applications. This review discusses the fundamental principle of TFME and its benefits versus the rod fiber geometry of SPME, and demonstrates the agreements of the experimental data for the available TFME systems with the theoretical concept. The current configurations, coating chemistries, coating preparation methods, and applications for TFME system are reported.