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Polymorphonuclear cells (neutrophils) are the first cells that arrive at sites of infections. According to the current dogma, they are involved in eliminating bacteria, after which they die through apoptosis. We now demonstrate that enhanced IgA-induced phagocytosis of bacteria or beads by neutrophils led to increased cell death. Nuclear changes and positivity for the general cell death marker 7-aminoactinomycin D were observed, but the absence of annexin V membrane staining supported that neutrophils did not die via apoptosis, in contrast to neutrophils that had not phagocytosed bacteria. Moreover, increased release of neutrophil extracellular traps (NETs) was observed, which was most likely due to augmented production of reactive oxygen species after uptake of IgA-opsonized particles. Blocking the IgA Fc receptor FcαRI abrogated phagocytosis and NET formation. Thus, FcαRI triggering on neutrophils resulted in a rapid form of cell death that is referred to as NETosis, as it is accompanied by the release of NETs. As such, IgA may play a prominent role in mucosal inflammatory responses, where it is the most prominent Ab, because it enhanced both phagocytosis of bacteria and formation of NETs, which are effective mechanisms that neutrophils employ to eliminate pathogens.

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the endogenous CYP 3A4/5 marker 4β-hydroxycholesterol in human K(2)-EDTA plasma. It is based on alkaline hydrolysis to convert esterified to free 4β-hydroxycholesterol, followed by analyte extraction from plasma by hexane and purification of the hexane extract by normal-phase solid-phase extraction. The analyte is chromatographically separated from endogenous isobaric plasma oxysterols and excess cholesterol by a 16-min reversed-phase gradient on a C18 column; detection is performed by atmospheric pressure photoionization tandem mass spectrometry in the positive ion mode, using toluene as a dopant. Using 400μl of plasma, 4β-hydroxycholesterol can be quantified in the concentration range 10.0-250nM. Validation results show that the method is sufficiently selective towards endogenous plasma sterols and capable of quantifying the analyte with good precision and accuracy. The analyte is sufficiently stable in all relevant matrices and solvents; the addition of the anti-oxidant butylated hydroxytoluene to prevent in vitro formation of 4β-hydroxycholesterol from cholesterol during storage or analysis is not necessary, provided that long-term frozen storage of plasma occurs at -70°C., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Cytarabine is an anti-tumor drug that is currently under investigation for treatment in combination with other anticancer drugs for the chemotherapy of leukemia. The quantitative determination of cytarabine in plasma is challenging due to the required sensitivity, its in vitro instability and the presence of an isobaric endogenous compound, cytidine. Owing to the polarity of cytarabine, conventional chromatography cannot provide adequate separation of the analyte and the interfering compounds. A few analytical methods have been reported for the determination of cytarabine in plasma, but their sensitivity was not sufficient since most of these methods apply spectrophotometric detection., Results: In this article, an LC-MS/MS method is described for the determination of cytarabine in human plasma down to the sub ng/ml level. To prevent conversion of cytarabine by cytidine deaminase, whole blood samples were stabilized with tetrahydrouridine directly after the collection of whole blood at the clinical site. Cation-exchange SPE was employed to extract cytarabine from 50 µl human plasma. UHPLC on high strength silica T3 column (100 × 2.1 mm × 1.8 µm) was applied to achieve adequate separation of cytarabine from cytidine within a reasonable run time of 5 min. A triple quad mass spectrometer equipped with an ESI source was used for detection., Conclusion: The method was linear over the concentration ranges of 0.500-500 ng/ml. The intra- and inter-day relative standard deviation (precision) as well as the bias (accuracy) were well below 15%. In the presence of tetrahydrouridine, cytarabine was sufficiently stable under all relevant conditions. The method was successfully applied to support a clinical pharmacokinetic study with a low dose of cytarabine.

Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C(18) stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS-MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.

Ion suppression effects were observed during the determination of clenbuterol in urine with solid-phase extraction/multiple-stage ion-trap mass spectrometry (SPE/MS(3)), despite the use of atmospheric pressure chemical ionisation. During SPE, a polymeric stationary phase (polydivinylbenzene) was applied. Post-cartridge infusion of analyte to the SPE eluate after the extraction of blank urine was performed to obtain a profile of the suppression. Single and multiple-stage MS were performed to provide insight in the suppressing compounds. The ion suppression was mainly ascribed to two m/z values, but still no identification of the compounds was achieved from the multiple-stage MS data. No ionisable and non-ionisable complexes and/or precipitation of clenbuterol with matrix compounds were observed. A concentration dependence of the percentage of suppression was observed. Up to 70% of the signal was suppressed upon post-cartridge infusion of 0.22 microg/mL (at 5 microL/min) clenbuterol into the eluate, and this decreased to about 4% at infusion of 22 microg/mL clenbuterol. Molecularly imprinted polymers were used to enhance the selectivity of the extraction. Although matrix components were still present after extraction, no interference of these compounds with the analyte was observed. However, the bleeding of the imprint from the polymer (brombuterol) caused significant ion suppression., (Copyright 2002 John Wiley & Sons, Ltd.)

To determine sub-ppb levels of drugs in biological samples, selective, sensitive and rapid analytical techniques are required. This work shows the possibilities for high-throughput analysis of solid-phase microextraction (SPME) directly coupled to an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionisation source. As no chromatographic separation is performed, the SPME procedure is the time-limiting step. Direct immersion SPME under non-equilibrium conditions permits the determination of lidocaine in urine within 10 min. After a 5 min sorption time with a 100 microm polydimethylsiloxane-coated fibre, the extraction yield of lidocaine from urine is about 7%. When applying 4 min desorption, using a mixture of ammonium acetate buffer (pH 4.5) and acetonitrile (85 + 15 v/v), about 10% of the analyte is retained on the fibre. An extra cleaning step of the fibre is therefore used to prevent carry-over. By use of tandem MS, no matrix interference is observed. The detection limit for lidocaine is about 0.4 ng ml(-1) and the intraday and interday reproducibility are within 14% over a concentration range of 2-45 ng ml(1).

Solid-phase extraction-pipette tips (SPE-PTs) were used for micro solid-phase extraction of lidocaine and diazepam from plasma. Off-line extraction was followed by on-line desorption. On-line desorption was carried out by direct coupling of the SPE-PTs with the liner of the programmed-temperature vaporiser. This coupling only required shortening of the liner by maximally 16 mm, cutting the SPE-PT, and equipping the remaining part with two O-rings. Due to the heating of the injector the SPE-PTs were heated as well, which resulted in a significant amount of impurities. Pre-heating and pre-washing was performed prior to the extraction to reduce the impurity level. The internal coupling device was applied successfully for the analysis of plasma samples with gas chromatography (GC) and mass-selective detection. Detection limits of 0.75 ng/ml and 2.5 ng/ml were obtained for lidocaine and diazepam, respectively, using 200 microl plasma. Recoveries for both compounds were about 80%. Although it is possible, the internal coupling device was not developed to be used as such. The main goal of this coupling was to show the feasibility of the integration of SPE-PTs with GC and to realize an important step to new automated SPE-GC systems.

Solid-phase extraction (SPE) was coupled to ion-trap mass spectrometry to determine clenbuterol in urine. For SPE a cartridge exchanger was used and, after extraction, the eluate was directly introduced into the mass spectrometer. For two types of cartridges, i.e. C18 and polydivinylbenzene (PDVB), the total SPE procedure (including injection of 1 mL urine, washing, and desorption) has been optimised. The total analysis, including SPE, elution, and detection, took 8.5 min with PDVB cartridges, while an analysis time of 11.5 min was obtained with C18 cartridges. A considerable amount of matrix was present after extraction of urine over C18 cartridges, resulting in significant ion suppression. With PDVB cartridges, the matrix was less prominent, and less ion suppression was observed. For single MS, a detection limit (LOD) of about 25 ng/mL was found with PDVB cartridges. With C18 cartridges an LOD of only about 50 ng/mL could be obtained. Applying tandem mass spectrometry (MS/MS) did not lead to an improved LOD due to an interfering compound. However, a considerable improvement in the LOD was obtained with MS3. The selectivity and sensitivity were increased by the combination of efficient fragmentation of clenbuterol and reduction of the noise. Detection limits of 2 and 0.5 ng/mL were obtained with C18 and PDVB cartridges, respectively. The ion suppression was 4 to 45% (concentration range: 250 to 1.0 ng/mL) after extraction of urine using PDVB cartridges, and up to 70% ion suppression was observed using C18 cartridges. With MS4, no further improvement in selectivity and sensitivity was achieved, due to inefficient fragmentation of clenbuterol and no further reduction of noise.

Solid-phase extraction-pipette tips were used for micro solid-phase extraction of lidocaine and diazepam. Off-line desorption was done after in-vial collection for reference purposes, whereas with on-line desorption the eluate was directly introduced in the gas chromatograph. With both methods the total eluate (100 microl) was introduced into the GC system, which was equipped with a programmed-temperature vaporiser (PTV) for large volume injection. For on-line desorption a laboratory-made coupling device was developed to connect the pipette tips with the injector of the PTV. The coupling device was applied successfully since no leakage occurred at the connection of the coupling device and the pipette tip. No significant differences in recovery of lidocaine and diazepam and in presence of impurities were observed between chromatograms obtained with either off-line or on-line desorption. Preliminary experiments with standard solutions showed recoveries of about 75% for a concentration level of 1 microg/ml. The system seems particularly suitable for high-throughput analysis.

The use of a programmed temperature vaporiser (PTV) with a packed liner was evaluated for the injection of large volumes (up to 100 microl) of plasma extracts in a gas chromatograph. Solvent purity, which is essential when large volumes are injected into the GC system, was determined. Special attention was paid to the purity of the solvents used for the solid-phase extraction (SPE) procedure. For this SPE method, ethyl acetate was used as the extraction and reconstitution solvent, and thus the purity of the ethyl acetate was critical, especially when a non-selective GC detector was applied. The liquid capacity and inertness of different packed liners were investigated. The liner packed with ATAS "A" (modified Chromosorb-based material with special treatment) was found to be the most suitable for the analysis of the tested drugs. Good linearity in response for variations in volume and concentration was observed. A comparison was made between the applicability of flame ionisation detection (FID) and mass-selective detection (MSD). When 50-microl volumes of plasma extracts were injected with the PTV, the detection limits for secobarbital, lidocaine, phenobarbital and diazepam were about 50-times lower than when 1-microl volumes were injected. The detection limits of the tested compounds in plasma for injection of 50-100 microl plasma extract are 5-10 ng/ml for GC-FID whereas plasma concentrations of 250 pg/ml can be detected using the selected ion monitoring (SIM) mode of a MSD. For non-selective GC-FID, the background from a 50-microl injection was substantially larger than with 1-microl injection as a result of co-injected plasma matrix components and solvent impurities. These background effects were less with GC-MSD in the total ion current mode and virtually absent with GC-MSD in the SIM mode.

The potential of micellar electrokinetic chromatography (MEKC) for the profiling of cocaine samples is described. An MEKC system containing sodium dodecyl sulfate (SDS) and methanol was optimized using a test mixture of cocaine, its common impurities (benzoylecgonine, norcocaine, tropacocaine, and trans-cinnamoylcocaine), and several degradation products. The effect of pH, percentage modifier, and concentration surfactant on the separation has been investigated. The optimal separation buffer for cocaine samples consisted of 75 mM SDS, 17.5% methanol, and 25 mM borate (pH 8.3) and was well suited to separate components of diverse polarity in one run. Various cocaine seizures have been analyzed with the MEKC system and their signatures were compared. The electrokinetic chromatograms obtained were characteristic, and differences and similarities among the samples could easily be observed. Several impurities were identified in the samples by means of migration times and comparison of recorded and library UV spectra. The composition of the samples was determined semiquantitatively using relative corrected peak areas.