Quantitative in vivo microsampling for pharmacokinetic studies based on an integrated solid-phase microextraction system

An integrated microsampling approach based on solid-phase microextraction (SPME) was developed to provide a complete solution to highly efficient and accurate pharmacokinetic studies. The microsampling system included SPME probes that are made of poly(ethylene glycol) (PEG) and C18-bonded silica, a fast and efficient sampling strategy with accurate kinetic calibration, and a high-throughput desorption device based on a modified 96-well plate. The sampling system greatly improved the quantitative capability of SPME in two ways. First, the use of the C18-bonded silica/PEG fibers minimized the competition effect from analogues of the target analytes in a complicated sample matrix such as blood or plasma samples, which is a common problem associated with solid coating SPME fibers for quantitative analysis. Moreover, the C18-bonded silica/PEG fibers provide high sensitivity and a large dynamic range that covers the possible sample concentration range during diazepam administration and elimination. Second, the kinetic calibration method offers more accurate quantitation than the calibration curve method for in vivo SPME, because it compensates for convection and matrix effects during sampling. Therefore, it is especially suitable as a fast sampling technique for pre-equilibrium SPME. Furthermore, with the high-throughput desorption device, the integrated system offers compactness and high efficiency. Its feasibility for in vivo sampling was demonstrated by monitoring diazepam pharmacokinetics and validated by conventional chemical assays and equilibrium SPME. In addition, we propose a simple method to determine the apparent distribution constant between an SPME fiber and a blood matix (Kfs) and the distribution constant between an SPME fiber and a pure PBS buffer sample matrix (Kfb). As a result, both total and free concentrations of the drug and its metabolites can be detected simultaneously. Accordingly, the binding constants to the blood matrix can be obtained, which are of special significance for clinical diagnosis and drug discovery.     

Determination of parabens in pharmaceutical formulations by solid-phase microextraction-ion mobility spectrometry

Solid-phase microextraction (SPME) coupled with ion mobility spectrometry (IMS) was used for the detection and quantitation of 4-hydroxybenzoate preservatives, methylparaben, ethylparaben, propylparaben, and butylparaben, in commercial pharmaceutical products. For the first time, SPME-IMS is described for the simultaneous detection, separation, and quantitation of multiple analytes in complex matrixes. The parabens are extracted from the samples using SPME, and the analytes on the fiber are heated by the IMS desorber unit and vaporized into the drift tube. The four preservatives differing only by a methyl group were separated in less than 18 ms. The analytical procedure was optimized for fiber coating selection, extraction time, sample pH, sample volume, ionic strength, and IMS conditions. Separation characteristics such as resolution, theoretical plates, and drift times of the parabens were also evaluated based on the direct interfacing of SPME to IMS. The conditions were tested using six over-the-counter topical products containing various combinations of preservatives. Analysis of the samples by SPME-IMS using benzyl paraben as an internal standard yields good comparison to an HPLC method, thereby reinforcing the applicability of this technique as a method for routine analysis. Limits of detection were 10 ng/mL for methylparaben and ethylparaben and 5 ng/mL for propylparaben and butylparaben. Good linearity range and reproducibility of less than 8% were obtained.     

Time-weighted average sampling with solid-phase microextraction device: implications for enhanced personal exposure monitoring to airborne pollutants

The solid-phase microextraction (SPME) device is used as a time-weighted average (TWA) sampler for gas-phase analytes by retracting the coated fiber a known distance into its needle housing during the sampling period. Unlike in conventional spot sampling with SPME, the TWA sampling approach does not allow the analytes to reach equilibrium with the fiber coating, but rather they diffuse through the opening in the needle to the location of the sorbent. The amount of analytes accumulated over time gives the measurement of the average concentration to which the device was exposed to. Depending on the sorbent used as the sink, TWA sampling for various analytes is possible with times ranging from 15 min to at least 16 h. Both the poly(dimethylsiloxane) (PDMS) and poly(dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber coating phases were tested, with the latter employing on-fiber derivatization for reactive carbonyl compounds, e.g., formaldehyde. Described herein are the theoretical and practical considerations for using the SPME device as a TWA sampler.     

Kinetics and the on-site application of standards in a solid-phase microextraction fiber

The kinetics of the desorption of analytes from a SPME fiber into an agitated sample matrix was studied, and a theoretical model was proposed to describe the dynamic desorption process, based on the steady-state diffusion of analytes in the extraction phase and in the boundary layer. It was found that the desorption of analytes from a SPME fiber into an agitated sampling matrix is isotropic to the absorption of the analytes onto the SPME fiber from the sample matrix under the same agitation conditions, and this allows for the calibration of absorption using desorption. The calibration was accomplished by exposing a SPME fiber, preloaded with a standard, to an agitated sample matrix, during which desorption of the standard and absorption of analytes occurred simultaneously. When the standard was the isotopically labeled analogue of the target analyte, the information from the desorption process, i.e., time constant a, could be directly used for estimating the concentration of the target analyte. When the standard varied from the target analyte, the mass-transfer coefficient of the analyte could be extrapolated from that of the standard. These predictions agree well with experimental results. This approach facilitates the full integration of sampling, sample preparation, and sample introduction, especially for on-site or in vivo investigations, where the addition of standards to the sample matrix, or control of the velocity of the sample matrix, is very difficult.     

Thin-film microextraction

The properties of a thin sheet of poly(dimethylsiloxane) (PDMS) membrane as an extraction phase were examined and compared to solid-phase microextraction (SPME) PDMS-coated fiber for application to semivolatile analytes in direct and headspace modes. This new PDMS extraction approach showed much higher extraction rates because of the larger surface area to extraction-phase volume ratio of the thin film. Unlike the coated rod formats of SPME using thick coatings, the high extraction rate of the membrane SPME technique allows larger amounts of analytes to be extracted within a short period of time. Therefore, higher extraction efficiency and sensitivity can be achieved without sacrificing analysis time. In direct membrane SPME extraction, a linear relationship was found between the initial rate of extraction and the surface area of the extraction phase. However, for headspace extraction, the rates were somewhat lower because of the resistance to analyte transport at the sample matrix/headspace barrier. It was found that the effect of this barrier could be reduced by increasing either agitation, temperature, or surface area of the sample matrix/headspace interface. A method for the determination of PAHs in spiked lake water samples was developed based on the membrane PDMS extraction coupled with GC/MS. A linearity of 0.9960 and detection limits in the low-ppt level were found. The reproducibility was found to vary from 2.8% to 10.7%.     

Anodized aluminum wire as a solid-phase microextraction fiber

The efficiency of anodized aluminum wire was investigated as a new fiber for solid-phase microextraction (SPME). Aluminum wires were anodized by direct current in a solution of sulfuric acid at room temperature and were conditioned at 300 degrees C for 30 min. These fibers were used for the extraction of some aliphatic alcohols, BTEX, and petroleum products from gaseous samples. The extracted analytes were transferred to a GC injector using an (inhouse-designed) SPME syringe that also allowed for an easy change of SPME fibers. The results obtained prove the ability of anodized aluminum wire as a new fiber for sampling of organic compounds from gaseous samples. This behavior is due most probably to the porous layer of aluminum oxide, which is formed on the metal surfaces. In this work, the optimum conditions for the preparation and conditioning of fibers and the extraction of analytes from gaseous samples were obtained. In the optimum conditions, one fiber was used in several equal analyses and the relative standard deviations were below 5% (n = 5). However, fiber-to-fiber reproducibility was 8% (n = 5). This fiber is firm, inexpensive, and durable and can be prepared simply.     

Evaluation of solid-phase microextraction for time-weighted average sampling of volatile sulfur compounds at ppb concentrations

The potential of solid-phase microextraction (SPME) for time-weighted average (TWA) sampling of volatile sulfur compounds in air at ppb concentrations was investigated. The target compounds (hydrogen sulfide, methanethiol (MeSH), ethanethiol (EtSH), dimethyl sulfide (Me2S), and dimethyl disulfide (Me2S2)) were extracted using SPME with a Carboxen-poly(dimethylsiloxane) fiber coating, and diffusion was controlled by keeping the fiber retracted within the needle of the sampling device. The effects of several important experimental variables (air velocity, direction of air flow, analyte concentration, humidity, temperature, extraction time) were studied. The uptake by the fiber was not affected by the direction of the air flow or the air velocity. The effects of concentration, humidity, temperature, and extraction time were examined in experiments with a central composite face design. The results showed that all or most of the investigated parameters had a significant impact on the uptake rates of H2S, MeSH, EtSH, and Me2S, which invalidated time-weighted average sampling of these compounds by SPME under the tested conditions. Moreover, reverse diffusion of H2S, MeSH, and EtSH occurred at 40% relative humidity. For Me2S2, the uptake rate had a variation of only 8% within the whole experimental domain, and the experimental value derived for the uptake rate was consistent with the theoretical value. This result was confirmed by comparative analyses of industrial samples by the standard addition method. Therefore, SPME appears to be a suitable technique for TWA sampling of Me2S2 using the Carboxen-poly(dimethylsiloxane) fiber coating. Finally, in an investigation of potential losses during storage of the fiber, no significant losses of the target compounds were detected after 3 days at -80 degrees C.     

Diffusion-based calibration for SPME analysis of aqueous samples

When an SPME fiber is exposed for a short period of time to a flowing fluid sample, the amount of extracted analyte depends on its diffusion coefficient in the matrix medium, and it can be correlated to its concentration using a simple mathematical model. This work discusses the extension of this approach, already validated for gaseous samples and SPME fibers coated with strong adsorbent coatings, to the diffusion-based quantification of analytes present in aqueous samples. Dilute aqueous solutions of aromatic hydrocarbons were used as model samples and vials were modified to use conventional magnetic agitation with controlled tangential flow of the test solution around the fiber. It was demonstrated that, with proper selection of the stirring speed and sampling time, the same diffusion-based quantitative model used for gas samples could be employed. Under optimal conditions, the concentrations of the evaluated aromatic hydrocarbons were estimated with relative standard deviations between 0.8 and 3.6% and without deviation from the expected values within this precision range. Considering the extraction times involved, between 30 and 60 s, the approach here presented is the fastest possible technique for direct extraction of analytes from liquid samples.     

Electrochemically controlled solid-phase microextraction based on conductive polypyrrole films

Solid-phase microextraction (SPME) fiber coatings based on conductive polypyrrole films were prepared for the electrochemical extraction and desorption of ionic analytes. Simple preparation of each of the PPY extraction coatings on a platinum wire was possible with a constant potential method, but more importantly, cycling of the film between oxidation and reduction potentials facilitated the extraction and desorption of ionic analytes. The analytes were desorbed into a sample aliquot of water and were determined by flow injection analysis using a mass spectrometer. The fiber coatings and the developed electrochemical SPME method were found to be stable and reproducible (RSD < 5%; N = 5) and could be extended to several cations and anions, confirming the versatility of the approach. Preconcentration of the analyte on the fiber was also possible by repeating the processes to increase the amount of analyte extracted.     

Time-weighted average passive sampling with a solid-phase microextraction device

A modified Solid-Phase Microextraction (SPME) device has been used as a passive sampler to determine the time-weighted average (TWA) concentration of volatile organic compounds (VOCs) in air. Unlike conventional sampling with SPME, in which the fiber is extended outside its needle housing, during TWA passive sampling, the fiber is retracted a known distance into its needle housing. The SPME passive sampler collects the VOCs by the mechanism of molecular diffusion and sorption on to a coated fiber as collection medium. This process has been shown to be described by Fick's first law of diffusion, whereby determination of the amounts of analytes accumulated over time enable measurement of the TWA concentration to which the sampler was exposed. A series of fibers, 100-microm poly(dimethylsiloxane), 65-microm poly(dimethylsiloxane)/divinylbenzene, and 75-microm Carboxen/poly(dimethylsiloxane), were tested for their "zero sink", face velocity, and response time behavior. Of the fibers tested, that coated with 75-microm Carboxen/poly(dimethylsiloxane) was found to be an excellent passive sampler for VOCs. TWA passive sampling with a SPME device was shown to be almost independent of face velocity and to be more tolerant of high and low analyte concentrations and long and short sampling times, because of the ease with which the diffusion path length could be changed. It was found that environmental conditions, e.g., temperature, pressure, relative humidity, and ozone, have little or no effect on sampling. The 75-microm Carboxen/poly(dimethylsiloxane) fiber can retain VOCs for up to two weeks without significant loss. When the SPME device was tested in the field and the results were compared with those from National Institute of Occupational Health and Safety method 1501, good agreement was obtained.     

Design and validation of portable SPME devices for rapid field air sampling and diffusion-based calibration

The use of SPME fibers coated with porous polymer solid phases for quantitative purposes is limited due to effects such as interanalyte displacement and competitive adsorption. For air analysis, these problems can be averted by employing short exposure times to air samples flowing around the fiber. In these conditions, a simple mathematical model allows quantification without the need of calibration curves. This work describes two portable dynamic air sampling (PDAS) devices designed for application of this approach to nonequilibrium SPME sampling and determination of airborne volatile organic compounds (VOCs). The use of a PDAS device resulted in greater adsorbed VOC mass compared to the conventional SPME extraction in static air for qualitative screening of live plant aromas and contaminants in indoor air. For all studied air samples, an increase in the number of detected compounds and sensitivity was also observed. Quantification of aromatic VOCs in indoor air was also carried out using this approach and the PDAS/SPME device. Measured VOC concentrations were in low parts-per-billion by volume range using only 30-s SPME fiber exposure and were comparable to those obtained with a standard NIOSH method 1501. The use of PDAS/SPME devices reduced the total air sampling and analysis time by several orders of magnitude compared to the NIOSH 1501 method.     

Sampling and analysis of airborne particulate matter and aerosols using in-needle trap and SPME fiber devices

A needle trap device (NTD) and commercial poly(dimethylsiloxane) (PDMS) 7-microm film thickness solid-phase microextraction (SPME) fibers were used for the sampling and analysis of aerosols and airborne particulate matter (PM) from an inhaler-administered drug, spray insect repellant, and tailpipe diesel exhaust. The NTD consisted of a 0.53-mm o.d. stainless steel needle having 5 mm of quartz wool packing section near the needle tip. Samples were collected by drawing air across the NTD with a Luertip syringe or via direct exposure of the SPME fiber. The mass loading of PM was varied by adjusting the volume of air pulled through the NTD or by varying the sampling time for the SPME fiber. The air volumes ranged from 0.1 to 50 mL, and sampling times varied from 10 s to 16 min. Particulates were either trapped on the needle packing or sorbed onto the SPME fiber. The devices were introduced to a chromatograph/mass spectrometer (GC/MS) injector for 5 min desorption. In the case of the NTD, 10 microL of clean air was delivered by a gas-tight syringe to aid the introduction of desorbed analytes. The compounds sorbed onto particles extracted by the SPME fiber or trapped in the needle device were desorbed in the injector and no carry-over was observed. Both devices performed well in extracting airborne polycyclic aromatic hydrocarbons (PAHs) in diesel exhaust, triamcinolone acetonide in a dose of asthma drug and DEET in a dose of insect repellant spray. Results suggest that the NTDs and PDMS 7-microm fibers can be used for airborne particulate sampling and analysis, providing a simple, fast, reusable, and cost-effective screening tool. The advantage of the SPME fiber is the open-bed geometry allowing spectroscopic investigations of particulates; for example, with Raman microspectroscopy.     

In-tube moleculary imprinted polymer solid-phase microextraction for the selective determination of propranolol

A molecularly imprinted polymer (MIP) material was synthesized for use as an in-tube solid-phase microextraction (SPME) adsorbent. The inherent selectivity and chemical and physical robustness of the MIP material was demonstrated as an effective stationary-phase material for in-tube SPME. An automated and on-line MIP SPME extraction method was developed for propranolol determination in biological fluids. This simplified the sample preparation process and the chromatographic separation of several beta-blocker compounds. The method developed for propranolol showed improved selectivity in comparison to alternative in-tube stationary-phase materials, overcoming the limitations of existing SPME coating materials. Preconcentration of the sample by the MIP adsorbent increased the sensitivity, yielding a limit of detection of 0.32 microg/mL by UV detection. Excellent method reproducibility (RSD < 5.0%) and column reusability (> 500 injections) were observed over a fairly wide linear dynamic range (0.5-100 microg/mL) in serum samples. To our knowledge, this is the first report on the automated application of a MIP material for in-tube SPME. The method was inexpensive, simple to set up, and simplified the choice of SPME adsorbent for in-tube extraction. The approach can potentially be extended to other MIPs for the determination of a wide range of chemically significant analytes.     

Determining chemical activity of (semi)volatile compounds by headspace solid-phase microextraction

This research introduces a new analytical methodology for measuring chemical activity of nonpolar (semi)volatile organic compounds in different sample matrices using automated solid-phase microextraction (SPME). The chemical activity of an analyte is known to determine its equilibrium concentration in the SPME fiber coating. On this basis, SPME was utilized for the analytical determination of chemical activity, fugacity, and freely dissolved concentration using these steps: (1) a sample is brought into a vial, (2) the SPME fiber is introduced into the headspace and equilibrated with the sample, (3) the SPME fiber is injected into the GC for thermal desorption and analysis, and (4) the method is calibrated by SPME above partitioning standards in methanol. Model substances were BTEX, naphthalene, and alkanes, which were measured in a variety of sample types: liquid polydimethylsiloxane (PDMS), wood, soil, and nonaqueous phase liquid (NAPL). Variable sample types (i.e., matrices) had no influence on sampling kinetics because diffusion through the headspace was rate limiting for the overall sampling process. Sampling time was 30 min, and relative standard deviations were generally below 5% for homogeneous solutions and somewhat higher for soil and NAPL. This type of activity measurement is fast, reliable, almost solvent free, and applicable for mixed-media sampling.     

Nonequilibrium solid-phase microextraction for determination of the freely dissolved concentration of hydrophobic organic compounds: matrix effects and limitations

Solid-phase microextraction (SPME) has recently been applied to measure the freely dissolved concentration, as opposed to the total concentration, of hydrophobic substances in aqueous solutions. This requires that only the freely dissolved analytes contribute to the concentration in the SPME fiber coating. However, for nonequilibrium SPME the sorbed analytes that diffuse into the unstirred water layer (UWL) adjacent to the SPME fiber can desorb from the matrix and contribute to the flux into the fiber. These processes were described as a model. Experimentally, an equilibrated and disconnected headspace was used as a reference for the freely dissolved concentration. The expected contribution of desorbed analytes to the uptake flux was measured for PCB no. 52 in a protein-rich solution, while it was not measured in a matrix containing artificial soil. The latter was possibly due to slow desorption of the analyte from the artificial soil. On the basis of the present study, a contribution of desorbed analytes to the uptake flux is expected only if(1) the rate-limiting step of the uptake process is diffusion through the UWL, (2) the concentration of the sorbed analyte is high, and (3) desorption from the matrix is fast.     

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.     

Application of solid-phase microextraction in the determination of diazepam binding to human serum albumin

In this paper, protein-drug interactions were studied by solid-phase microextraction (SPME) using diazepam binding to human serum albumin as a model system. Since drug compounds are normally polar and nonvolatile by nature, direct SPME is used in this work. The SPME extraction is an equilibrium process among the concentrations of the analyte partitioned onto the SPME fiber, free and bound drug in the solution. A calibration curve was first constructed by employing the amount of the analytes partitioned on the fiber versus the free analyte concentration in the solution in the absence of protein. In method I, the extraction was performed in the protein solution with known diazepam concentration. In method II, diazepam was first loaded onto the fiber by extracting in solution with known diazepam concentration. This fiber was subsequently transferred into the protein solution for desorption. The amount of the analyte left on the fiber was analyzed after the system reached equilibrium. The free drug concentration was then obtained from the calibration curve for both methods. The Scatchard plot was finally employed to obtain the number of binding sites and the equilibrium binding constants. Since only a very small amount of the protein solution is required (150 microL for each extraction), method II is very useful for circumstances where the protein amount is very limited. The direct measurement method proposed in this paper does not need a GC response factor, which significantly decreases the experimental error. The only measurement needed is the area count change (ratio) of the fiber injections before and after the protein was introduced into the solution. The difference between the direct measurement method for method I and method II is discussed. The result illustrated that the SPME direct measurement method provided both theoretical accuracy and simplicity in such applications.     

Calibration of a commercial solid-phase microextraction device for measuring headspace concentrations of organic volatiles

Solid-phase microextraction (SPME) is a versatile new technique for collecting headspace volatiles prior to GC analysis. The commercial availability of uniform SPME fibers makes routine, practical quantitation of headspace concentrations possible, but straightforward information for relating GC peak areas from SPME analyses to headspace concentrations has not been available. The calibration factors (amount absorbed by the fiber divided by headspace concentration) were determined for 71 compounds using SPME fibers with a 100 μm poly(dimethylsiloxane) coating. The compounds ranged from 1 to 16 carbons in size and included a variety of functional groups. Calibration factors varied widely, being 7000 times higher for tetradecane than for acetaldehyde. Most compounds with a Kovats retention index of <1300 on a nonpolar GC column (DB-1) equilibrated with the fiber in 30 min or less. A regression model is presented for predicting the calibration factor from GC retention index, temperature, and analyte functional class. The calibration factor increased with retention index but decreased with increasing sampling temperature. For a given retention index, polar compounds such as amines and alcohols were absorbed by the fibers in greater amounts than were hydrocarbons. Henry's law constants determined using SPME were in general agreement with literature values, which supported the accuracy of the measured calibration factors. An unexpected concentration dependence of calibration factors was noted, especially for nitrogen-containing and hydroxy compounds; calibration factors were relatively higher (the SPME fiber was more sensitive) at the lower analyte concentrations.     

Solid-phase microextraction field sampler

To facilitate the use of solid-phase microextraction (SPME) for field sampling, a new field sampler was designed and tested. The sampler was versatile and user-friendly. The SPME fiber can be positioned precisely inside the needle for time-weighted average sampling or exposed completely outside the needle for grab sampling. The needle is protected within a shield at all times, hereby eliminating the risk of operator injury and fiber damage. A replaceable Teflon cap is used to seal the needle to preserve sample integrity. Factors that affect the preservation of sample integrity (sorbent efficiency, temperature, sealing materials) were studied. The use of a highly efficient sorbent for the fiber is recommended as the first choice for the preservation of sample integrity. Teflon was a good material for sealing the fiber needle, had little memory effect, and could be used repeatedly. To address adsorption of high boiling point compounds on fiber needles, several kinds of deactivated needles were evaluated. RSC-2 fiber needles were the more effective. A preliminary field sampling investigation demonstrated the validity of the new SPME device for field applications.