Pharmacokinetics of tranexamic acid in patients undergoing cardiac surgery with use of cardiopulmonary bypass.

We conducted a study to assess pharmacokinetics of high-dose tranexamic acid for 24 h after administration of the drug in patients undergoing cardiac surgery with cardiopulmonary bypass. High-dose tranexamic acid involved a bolus of 30 mg.kg(-1) infused over 15 min followed by a 16 mg.kg(-1) .h(-1) infusion until chest closure with a 2 mg.kg(-1) load within the pump prime. Tranexamic acid followed first-order kinetics best described using a two-compartment model, with a total body clearance that approximated the glomerular filtration rate. Mean plasma tranexamic acid concentrations during the intra-operative period and in the first 6 postoperative hours were consistently higher than the suggested threshold to achieve 100% inhibition and 80% inhibition of tissue plasminogen activator. With recent studies implicating high-dose tranexamic acid as a possible aetiology of postoperative seizures following cardiac surgery, the minimum effective yet safe dose of tranexamic acid in high-risk cardiac surgery needs to be refined.

Quantitative liquid chromatographic and tandem mass spectrometric determination of vitamin D3 in human serum with derivatization: a comparison of in-tube LLE, 96-well plate LLE and in-tip SPME.

Sensitive and selective methods based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection were developed for the determination of vitamin D(3) in human serum. Derivatization of vitamin D(3) and its stable isotope labeled internal standard provided highly sensitive quantification and selective detection from endogenous compounds. Samples were prepared using the in-tube liquid-liquid extraction (LLE), 96-well plate LLE, and in-tip solid phase micro-extraction (SPME) in 96-well format. In all methods, the MS/MS detection was performed using Applied Biosystems-Sciex API 3000 tandem mass spectrometers interfaced with a heated nebulizer probe and operated in the positive ionization mode. Both tube and plate LLE methods achieved a lower limit of quantitation (LLOQ) of 0.5 ng/mL when 1.0 and 0.4 mL of human serum was processed, respectively, and were validated in the concentration range of 0.5-25 ng/mL; while for the in-tip SPME method, LLOQ was 5 ng/mL with only 0.1 mL of human serum required. Comparisons were made among three different methods, including precision and accuracy, sample throughput, recovery and matrix effects.

Analysis of ergosterol for the detection of mold in soils by automated on-fiber derivatization headspace extraction-SPME-GC/MS.

The estimate of ergosterol ([5,7,22-ergostatrien-3beta-ol] has been used by many to relate its concentration to the amount of mold in soils. This new method using on-fiber derivatization-solid phase microextraction-GC/MS method for the analysis of ergosterol presents a quick and straightforward method where low detection limits (1.5 ppb) and good limit of quantitation range (3 ppb to 90 ppm) can be achieved with careful control of analytical parameters. After saponification of real soil samples, sampling without extensive workup can be performed and analysis by a standard addition method can be utilized to deduce the original sample concentration of ergosterol. Peak area extraction analysis by MS SIM on selected characteristic fragment ions gives results with RSD < or = 2.2%.

Automation of in-tip solid-phase microextraction in 96-well format for the determination of a model drug compound in human plasma by liquid chromatography with tandem mass spectrometric detection.

Studies using in-tip solid phase microextraction (in-tip SPME) in a 96-well plate format are conducted to investigate the feasibility of SPME automation. The sample preparation process, including extraction and desorption, was fully automated and coupled with currently commercially available automated liquid handling systems. Several process parameters including extraction time and speed, and desorption time were investigated. An LC-MS/MS method has been developed and validated to determine the levels of a drug compound (MK-0533) in human plasma that demonstrates the suitability of this new approach. The developed method has a lower limit of quantitation (LLOQ) of 5 ng/mL when 0.25 mL of human plasma is processed and is validated in the concentration range of 5-2, 000 ng/mL. The successful application of the assay in clinical sample analysis indicates that in-tip SPME can be easily automated and has great potential to be used for high throughput quantitative determination of drugs in pharmaceutical industry.

Automated high-throughput method using solid-phase microextraction-liquid chromatography-tandem mass spectrometry for the determination of ochratoxin A in human urine.

A new automated, high-throughput method for the determination of ochratoxin A (OTA) in human urine samples has been optimized and validated using solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry (SPME-LC-MS/MS). High-throughput was achieved by simultaneous preparation of up to 96 samples using multi-fiber SPME device and multi-well plates. A carbon-tape coating was chosen for the first time as the best extracting phase for this contaminant. The proposed method required only minimal sample pre-treatment to adjust sample pH to 3.0 using a dilution (1:1) with 0.5M phosphate-buffered saline. A simple gradient guaranteed a good chromatographic separation from matrix interferences in only 8min. Relative recovery (%), precision and linearity validation results met Food and Drug Administration acceptance criteria at three concentration levels (1, 10, and 50ng/mL), indicating excellent performance of the proposed method. Limits of detection and quantitation were 0.3 and 0.7ng/mL in urine, respectively. OTA determination in urine is a good marker for human exposure to this mycotoxin. It is also less invasive than blood analysis. This method is fully automated and the SPME technique is simpler, less time-consuming and cheaper compared with most widely adopted clean-up procedures for OTA extraction from urine.

Comparison of solid-phase microextraction and liquid-liquid extraction in 96-well format for the determination of a drug compound in human plasma by liquid chromatography with tandem mass spectrometric detection.

Two sensitive and selective methods based on solid phase microextraction (SPME) and liquid-liquid extraction (LLE) in 96-well format, in combination with high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection have been developed to determine a model drug compound in human plasma. Both assays were performed on an Applied Biosystems-Sciex API 4000 tandem mass spectrometer interfaced with a turbo ion-spray probe and operated in the negative ionization mode. A lower limit of quantitation (LLOQ) of 1 ng/mL achieved when 0.25 mL of human plasma was processed. In both methods, a stable isotope labeled internal standard was utilized. The methods were validated in the concentration range of 1-500 ng/mL. The intraday precision (%C.V.) of the method using LLE was 0.8% at LLOQ, and was equal to or lower than 3.3% at all other concentrations, while the intraday precision (%C.V.) of the method using SPME was 6.9% at LLOQ, and was equal to or lower than 5.7% at all other concentrations. Based on the direct comparison of the two methods and their successful applications in clinical sample analysis, it may be concluded that SPME may be considered and used as an alternative approach for quantitative determination of drugs in pharmacokinetic studies.

Development of immunoaffinity solid phase microextraction probes for analysis of sub ng/mL concentrations of 7-aminoflunitrazepam in urine.

We report on the development of solid phase microextraction probes for drug analysis, prepared with antibodies specific for benzodiazepines covalently immobilized to the surface. In the technique, immobilized antibody probes are exposed to a sample containing the drug for 30 min. Extracted drugs are subsequently desorbed from the probes in 500 microL of methanolic desorption solution, which is dried, reconstituted in a small volume of injection solution and analysed by LC-MS/MS. The antibodies were characterized both before and after immobilization, to facilitate the rational selection of antibodies for such analyses. Polyclonal and monoclonal antibodies were compared as was the impact of affinity purification of the polyclonal antibody to isolate the drug-specific fraction. The probes were evaluated for utility in analyzing 7-aminoflunitrazepam at sub ng/mL concentrations in urine, which is expected to be found several days after a single oral dose of 2 mg of flunitrazepam. Such analyses are required in monitoring for abuse of this drug, both in terms of 'club drug' use and in cases of drug-facilitated sexual assault. In these cases drug concentrations in blood and urine are much lower than in chronic abuse cases and are difficult to analyse by conventional methods. The method developed has a limit of detection of 0.02 ng/mL, with accuracy ranging from 1% to 27% and precision (% R.S.D.) ranging from 2% to 10% between the lower and upper limits of quantitation for the analysis of 7-aminoflunitrazepam in urine. The dynamic range of the method is from 0.02 ng/mL, which is limited by the instrument sensitivity, to 0.5 ng/mL, which is approaching the capacity of the probes. This would allow for quantitative analysis of samples at concentrations below that measurable by many other methods for general benzodiazepines analysis from urine, and a highly selective screen for samples at higher concentrations. The method has similar limits of detection to the most sensitive literature methods specifically designed for such analysis but with the advantage of significantly simplified sample preparation. This simplification makes the technique more amenable for use by both professionals and non-professionals.

Polydimethylsiloxane rod extraction, a novel technique for the determination of organic micropollutants in water samples by thermal desorption-capillary gas chromatography-mass spectrometry.

A novel, simple and inexpensive approach to absorptive extraction of organic compounds from environmental samples is presented. It consists of a polydimethylsiloxane rod used as an extraction media, enriched with analytes during shaking, then thermally desorbed and analyzed by GC-MS. Its performance was illustrated and evaluated for the enrichment of sub- to ng/l of selected chlorinated compounds (chlorobenzenes and polychlorinated biphenyls) in water samples. The new approach was compared to the stir bar sorptive extraction performance. A natural ground water sample from Bitterfeld, Germany, was also extracted using both methods, showing good agreement. The proposed approach presented good linearity, high sensitivity, good blank levels and recoveries comparable to stir bars, together with advantages such as simplicity, lower cost and higher feasibility.

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 (R2) 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.

Verapamil drug metabolism studies by automated in-tube solid phase microextraction.

Verapamil is a common calcium antagonist described with antianginal, antihypertensive and antiarrythmic properties. The metabolites of verapamil have also shown pharmacological properties and therefore sample preparation and analysis techniques capable of metabolic screening for verapamil are important. In-tube SPME is a relatively new method integrating sample extraction, concentration and introduction into one single step without the use of organic solvents. The capability of in-tube SPME in bioanalysis has been reviewed but there has been no application described in the field of drug metabolism. Since automation and interfacing of in-tube SPME coupled to liquid chromatography-mass spectrometry (LC-MS) is possible, we confirm in this study that it is a powerful method to monitor the main metabolites of verapamil in various biological matrices like plasma, urine and cell culture media. Further, we show that it could also be used in routine pharmacokinetics measurements. An in-tube SPME LC-MS method was developed to extract and analyze the metabolic profile of verapamil from biological matrices. The detection limit for verapamil, gallopamil, norverapamil and PR22 were 52, 53, 65 and 83 ng/ml (UV detection) and 5, 6, 6 and 8 ng/ml (MS detection), respectively. The precision of the method was calculated in various biological matrices and the average % R.S.D. (N=5) for verapamil, gallopamil, norverapamil and PR22 was 3.9, 3.7, 3.8 and 4.3% (MS detection), respectively. The linear dynamic range was determined to be 100-800 ng/ml (UV detection) with a total sample preparation and analysis time of 34 min.

Miniaturization of capillary isoelectric focusing.

Miniaturization of whole-column imaging capillary isoelectric focusing (CIEF) is discussed. A 1.2 cm capillary was used as a separation column for CIEF. The experimental results for the analysis of two pI markers and the protein myoglobin showed that good CIEF separation results could be obtained. Secondly, a light-emitting diode (LED) was used as the light source for the whole-column absorbance imaging detection. The focusing of both the pI markers and myoglobin were observed with the LED light source. The whole-column imaging CIEF instrument was simplified and miniaturized by the use of the LED. Further developments are also discussed.

Direct LC analysis of five benzodiazepines in human urine and plasma using an ADS restricted access extraction column.

An alkyl-diol-silica (ADS) precolumn was used for the direct and on-line extraction of several benzodiazepines from serum and urine. The protein component of the biological sample was flushed through the ADS column, while simultaneously extracting the benzodiazepine compounds in the pores of the ADS stationary phase. The role of hydrophobic interactions in the extraction mechanism was confirmed. Column switching was employed to elute the extracted analytes from the ADS column into a high-performance liquid chromatography reverse-phase C18 column for the isocratic separation and UV detection of the benzodiazepines. Sample preconcentration via large volume injections was possible, improving the limits of detection. The calculated clonazepam, oxazepam, temazepam, nordazepam and diazepam detection limits were 38.8, 24.2, 31.7, 31.3, 45.0 ng/ml in serum, respectively, and 48.4, 24.5, 31.7, 33.1, 52.9 ng/ml for urine, respectively. The method was linear over the range of 50-10000 ng/ml in both matrices with an average linear coefficient (R(2)) value of 0.9918. The injection repeatability and intra-assay precision of the method were evaluated over ten injections, resulting in a percent relative standard deviation <5%. The ADS extraction column was robust, providing many direct injections of biological fluids for the extraction and subsequent determination of benzodiazepines.

Solid phase microextraction.

Solid Phase Microextraction (SPME) uses a small volume of sorbent dispersed typically on the surface of small fibres, to isolate and concentrate analytes from sample matrix. After contact with sample, analytes are absorbed or adsorbed by the fibre phase (depending on the nature of the coating) until an equilibrium is reached in the system. The amount of an analyte extracted by the coating at equilibrium is determined by the magnitude of the partition coefficient of the analyte between the sample matrix and the coating material. After the extraction step, the fibres are transferred, with the help of a syringe-like handling device, to analytical instrument, for separation and quantitation of target analytes. This technique integrates sampling, extraction and sample introduction and is a simple way of facilitating on-site monitoring. Applications of this technique include environmental monitoring, industrial hygiene, process monitoring, clinical, forensic, food, flavour, fragrance and drug analyses, in laboratory and on-site analysis.

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.

Biological sample analysis with immunoaffinity solid-phase microextraction.

A theophylline antiserum was covalently immobilized on the surface of a fused silica fiber, modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde, and used as a selective and sensitive extraction medium for the immunoaffinity solid-phase microextraction (SPME) determination of theophylline in serum samples. The specificity of the immunoaffinity SPME fiber was first investigated using a fixed concentration of [3H]theophylline together with various amounts of interference, possessing no cross-reactivity with the theophylline antibody. No significant non-specific binding was observed. The reproducibility of the fiber preparation and the immunoaffinity SPME analysis was also investigated, resulting in a relative standard deviation of 6.1% for five analyses of the same fiber. The antigen-antibody binding isotherm was obtained by analyzing theophylline standards of various concentrations (0.1-5 ng mL(-1)) until saturation values were reached. Initial binding of theophylline was linear with a r2 = 0.968. The cross-reactivity of the theophylline immunoaffinity SPME fiber for the structural analog caffeine was investigated by adding various amounts of caffeine in the presence of theophylline at a saturation concentration and produced a low cross-reactivity value of 0.1%. Finally. spiked serum samples (10 and 50 ng mL(-1)) were successfully analyzed with an excellent correlation with the standard binding isotherm, thus confirming the performance of the immunoaffinity SPME coating for improved bioanalysis.

Sampling and Raman confocal microspectroscopic analysis of airborne particulate matter using poly(dimethylsiloxane) solid-phase microextraction fibers.

Commercial poly(dimethylsiloxane) (PDMS) 7-microm solid-phase microextraction (SPME) fibers were used for sampling and Raman spectroscopic analysis of a tailpipe diesel exhaust, candle smoke, cigarette smoke, and asbestos dust. Samples were collected via direct exposure of the SPME fiber to contaminated air. The mass loading for SPME fibers was varied by changing the sampling time. Results indicate that PDMS-coated fibers provide a simple, fast, reusable, and cost-effective air sampling tool for airborne particulates. The PDMS coating was stable; Raman bands of the PDMS coating were observed exactly at the same wavenumber positions before and after air sampling. Raman spectroscopic analysis resulted in identification of several characteristic bands allowing chemical speciation of particulates. The advantage of the SPME fiber is the open bed geometry allowing for application of various spectroscopic methods of particulate analysis. This paper describes the first-ever combined application of SPME technology with Raman confocal microspectroscopy for sampling and analysis of airborne particulates. Advantages of the combination of solid-phase microextraction and Raman microspectroscopy for airborne particulate analysis are discussed. Challenges associated with combined SPME sampling and Raman analysis of single particles are also described.

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.

Field sampling and determination of formaldehyde in indoor air with solid-phase microextraction and on-fiber derivatization.

A new sampling and analysis method for formaldehyde in indoor air was tested in several indoor air surveys. The method was based on the use of solid-phase microextraction (SPME) poly(dimethylsiloxane)/divinylbenzene,65-microm fiber and gas chromatography. Indoor air surveys included grab and time-weighted average (TWA) sampling and were completed at six locations using (a) the SPME method employing on-fiber formaldehyde derivatization with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride and (b)the conventional National Institute for Occupational Safety and Health (NIOSH) 2451 method. Sampling time for SPME fiber ranged from 10 min for grab sampling to 8 h for TWA sampling. Sampling locations included a residential house, a rental apartment, an office building, and industrial workplaces. The air concentrations measured by SPME ranged from 10 to 380 ppbv and correlated well with those estimated by the NIOSH method. Results also indicated thatin some cases the formaldehyde concentrations measured in residential air could be much higher than those allowed in occupational settings. The SPME method proved to be accurate, fast, sensitive, and cost-efficient in field sampling applications. This research should be of interest to research, industrial, and regulatory agencies as well as to the general public concerned with indoor air quality.

Air sampling and analysis of volatile organic compounds with solid phase microextraction.

Solid phase microextraction (SPME) presents many advantages over conventional analytical methods by combining sampling, preconcentration, and direct transfer of the analytes into a standard gas chromatograph (GC). Since its commercial introduction in the early 1990s, SPME has been successfully applied to the sampling and analysis of environmental samples. This paper presents an overview of the current methods for air sampling and analysis with SPME using both grab and time-weighted average (TWA) modes. Methods include total volatile organic compounds (TVOCs), formaldehyde, and several target volatile organic compounds (VOCs). Field sampling data obtained with these methods in indoor air were validated with conventional methods based on sorbent tubes. The advantages and challenges associated with SPME for air sampling are also discussed. SPME is accurate, fast, sensitive, versatile, and cost-efficient, and could serve as a powerful alternative to conventional methods used by the research, industrial, regulatory, and academic communities.

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.