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Product ion spectra obtained with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS) were applied to the identification of seized drug samples from atmospheric pressure matrix-assisted laser desorption/ionization product ion spectra (AP-MALDI-MS/MS spectra). Data acquisition was performed in the information-dependent acquisition (IDA) mode, and the substance identification was based on a spectral library previously created with LC-ESI/MS/MS using protonated molecules as precursor ions. A total of 39 seized drug samples were analyzed with both AP-MALDI and LC-ESI techniques using the same triple-quadrupole instrument (AB Sciex 4000QTRAP). The study shows that ESI-MS/MS spectra can be directly utilized in AP-MALDI-MS/MS measurements as the average fit and purity score percentages with AP-MALDI were 90% and 85%, respectively, being similar to or even better than those obtained with the reference LC/ESI-MS/MS method. This fact enables the possibility to use large ESI spectral libraries, not only to ESI analyses but also to analyses with other ionization techniques which produce protonated molecules as the base peak. The data obtained shows that spectral library search works also for analytical techniques which produce multi-component mass spectra, such as AP-MALDI, unless isobaric compounds are encountered. The spectral library search was successfully applied to rapid identification of confiscated drugs by AP-MALDI-IDA-MS/MS. Copyright © 2012 John Wiley & Sons, Ltd.

A method based on in-source collision-induced dissociation (ISCID) liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) and reverse target database search was developed and evaluated for drug screening and confirmation in analytical toxicology context. An established LC-TOFMS screening method, in which identification relies solely on protonated molecule accurate mass measurement, isotopic pattern fit, and retention time (RT), was completed to include 1-3 qualifier ions for each analyte in the database. The qualifier ions for 431 compounds were selected from the experimental ISCID spectra, and their molecular formulae were assigned by applying SmartFormula3D and MSFragmenter software. Three qualifier ions were assigned for 64.5%, two or three for 81.4%, one for 14.8%, and none for 3.7% of the compounds studied. Comparison between ISCID LC-TOFMS and LC-TOFMS with 25 authentic autopsy urine samples showed an improved confidence level with the ISCID method, as isomeric interferences were excluded in most cases. However, some false negative (FN) results were obtained at low concentration levels close to the reporting criteria. The cut-off concentration of the ISCID method was 10-100 ng/mL with 80% of the 49 representative compounds tested, and the level was approximately two times higher than that obtained by LC ion trap MS. The presented method enables simultaneous screening and confirmation whenever at least one qualifier ion is available, as applying an accurate mass precursor ion and one product ion surpasses the standard of four identification points that is required by the current EU protocol.

Besides its particular importance as a widely used therapeutic agent, insulin (and its synthetic derivatives) has been suspected, purported, and proven to be a lethal weapon in numerous cases of attempted or successful homicide and suicide. In addition to blood and urine as common matrices for clinical diagnosis and post-mortem analysis, vitreous humour has gained considerable attention in autopsy and follow-up investigations due to its ability to provide valuable information on cause and time of death. However, post-mortem insulin analyses using such specimens have been rare due to the limited penetration of peptide hormones into the vitreous body, and immunoassays were exclusively employed in those studies. In the present communication, the determination of insulin(s) from vitreous humour by means of immunopurification combined with ultrahigh performance liquid chromatography--high resolution/high accuracy (tandem) mass spectrometry is reported. Exploiting the constantly increasing sensitivity and robustness of modern mass-spectrometry-based instruments, the option to identify insulin in post-mortem vitreous samples is demonstrated with a specimen collected from a non-diabetic victim who died from an insulin overdose. This communication represents the first successful mass-spectrometry-based analysis of post-mortem material related to an insulin poisoning case.

A microfabricated re-usable multi-inlet fluidic connector chip in PDMS (polydimethylsiloxane) is introduced. A connector takes up ca. 1 mm 2 area and inlet density is limited principally by capillary tubing dimensions. Anisotropically etched silicon mold, with patterns matching those of the microfluidic chip, and dummy fibers have been used in PDMS casting. The connector chip material is self-sealing PDMS, which eliminates the need for gluing. This feature enables tubing to be attached and removed repeatedly, as long as the PDMS surfaces in contact with the microfluidic chip remain clean. Both imide-coated glass capillaries and polymeric PEEK (polyetheretherketone) tubes have been successfully connected to a microfluidic nebulizer chip which has one liquid and two gas ports. The connections were measured to withstand overpressures up to 220 and 20 kPa with and without additional compression, respectively. The fact that the connector chip is fabricated independently of the microfluidic chip improves significantly assembly yield of the fluidic system, because the microfluidic chip is not subject to gluing or other assembly operations. We tested the PDMS chip successfully with the nebulizer chip in mass spectrometry application. Even though room temperature performance of the connector chip was excellent, at elevated temperatures (above 50 °C) significant background signal originating from PDMS can be seen in mass spectra. This limits the usability of PDMS parts in sensitive mass spectrometry applications.

We performed a systematic study using a set of compounds with different proton affinities (PAs) on the ionization in atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS). The compounds studied included various aromatic molecules of different sizes. The PAs of these compounds were calculated using ab initio and hybrid density functional theory calculations at the B3LYP/6-31G(d) level of theory. We observed that only compounds with relatively high PAs above a threshold value of 920-950 kJ/mol were efficiently ionized as protonated molecules under AP-DIOS conditions and produced very clean mass spectra.

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.

A novel, microfabricated heated nebulizer chip for atmospheric pressure photoionization-mass spectrometry (APPI-MS) is presented. The chip consists of fluidic and gas inlets, a mixer, and a nozzle etched onto silicon wafer that is anodically bonded to a Pyrex glass wafer, on which an aluminum heater is sputtered. A krypton discharge lamp is used as the source for 10-eV photons to initiate the photoionization process. Dopant, delivered as part of the sample solution, is used to achieve efficient ionization. The use of the microfabricated heated nebulizer with APPI in the analysis of four analytes is demonstrated, and the spectra are compared to those obtained with a conventional APPI source. Ionization in positive and negative ion modes was successfully achieved and the spectra were mainly similar to those obtained with conventional APPI, indicating that the ionization in microfabricated and conventional APPI sources takes place by the same mechanisms. The flow rates with conventional APPI are approximately 100 muL/min, whereas the microchip heated nebulizer allows the use of flow rates 0.05-5 muL/min, thus being compatible with microfluidic separation systems or micro- and nano-LC. A stable signal was demonstrated throughout a 5-h measurement, which proved the excellent stability of the micro-APPI. The same heated nebulizer chip can be used for weeks.

This study presents coupling of a poly(dimethylsiloxane) (PDMS) micro-chip with electrospray ionization-mass spectrometry (ESI-MS). Stable electrospray is generated directly from a PDMS micro-channel without pressure assistance. Hydrophobic PDMS aids the formation of a small Taylor cone in the ESI process and facilitates straightforward and low-cost batch production of the ESI-MS chips. PDMS chips were replicated with masters fabricated from SU-8 negative photoresist. A novel coating, an amorphous diamond-like carbon-poly(dimethylsiloxane) hybrid, deposited on the masters by the filtered pulsed plasma arc discharge technique, improved significantly the lifetime of the masters in PDMS replications. PDMS chip fabrication conditions were observed to affect the amount of background peaks in the MS spectra. With an optimized fabrication process (PDMS curing agent/silicone elastomer base ratio of 1/8 (w/w), curing at 70 degree C for 48 h) low background spectra were recorded for the analytes. The performance of PDMS devices was examined in the ESI-MS analysis of some pharmaceutical compounds and amino acids.

We have studied the matrix effect within direct analysis of benzodiazepines and opioids from urine with desorption electrospray ionization-mass spectrometry (DESI-MS) and desorption atmospheric pressure photoionization-mass spectrometry (DAPPI-MS). The urine matrix was found to affect the ionization mechanism of the opioids in DAPPI-MS favoring proton transfer over charge exchange reaction. The sensitivity for the drugs in solvent matrix was at the same level with DESI-MS and DAPPI-MS (LODs 0.05–6 μg mL−1) but the decrease in sensitivity due to the urine matrix was higher with DESI (typically 20–160-fold) than with DAPPI (typically 2–15-fold) indicating better matrix tolerance of DAPPI over DESI. Also in MS/MS mode, DAPPI provided better sensitivity than DESI for the drugs in urine. The feasibility of DAPPI-MS/MS was then studied in screening the same drugs from five authentic, forensic post mortem urine samples. A reference measurement with gas chromatography-mass spectrometry (GC–MS) (including pretreatment) revealed 16 findings from the samples, whereas with DAPPI-MS/MS after sample pretreatment, 15 findings were made. Sample pretreatment was found necessary, since only eight findings were made from the same samples untreated.

An atmospheric pressure chemical ionization (APCI) microchip is presented for combining a gas chromatograph (GC) to a mass spectrometer (MS). The chip includes capillary insertion channel, stopper, vaporizer channel, nozzle and nebulizer gas inlet fabricated on the silicon wafer, and a platinum heater sputtered on a glass wafer. These two wafers are joined by anodic bonding creating a two-dimensional version of an APCI microchip. The sample from GC is directed via heated transfer line capillary to the vaporizer channel of the APCI chip. The etched nozzle forms narrow sample plume, which is ionized by an external corona discharge needle, and the ions are analyzed by a mass spectrometer. The GC-microchip APCI-MS combination provides an efficient method for qualitative and quantitative analysis. The spectra produced by microchip APCI show intensive protonated molecule and some fragmentation products as in classical chemical ionization for structure elucidation. In quantitative analysis the GC-microchip APCI-MS showed good linearity (r(2) = 0.9989) and repeatability (relative standard deviation 4.4%). The limits of detection with signal-to-noise ratio of three were between 0.5 and 2 micromol/L with MS mode using selected ion monitoring and 0.05 micromol/L with MS/MS using multiple reaction monitoring.

A miniaturized nebulizer chip for vaporization of liquid samples for mass spectrometry has been designed, fabricated, and characterized for fluidic and thermal performance. Silicon/glass chip has a liquid sample channel placed centrally between symmetric nebulizer gas channels. The liquid sample is nebulized and vaporised by an integrated platinum heater. The vaporized sample exits through an etched nozzle, and is ionized by an external corona needle. The ions are analysed by a mass spectrometer. The chip has been fabricated in both anisotropically wet etched and DRIE versions in silicon, with an anodically bonded Pyrex glass cover plate. Three different fluidic inlet designs are presented, with both through-wafer and edge insert versions. The shape of the erupting gas jet has been visualized by infrared thermography by using a low-diffusivity imaging screen and high heat capacity helium as a test gas. Dimensions of the jet's thermal footprint on the screen show that the jet is very narrow and confined, and this is confirmed in mass spectrometry results. This confined jet supplies the sample to the ionization region near corona tip, enabling efficient use of very small sample amounts and submicroliter flows.1591

A novel microchip heated nebulizer for atmospheric pressure chemical ionization mass spectrometry is presented. Anisotropic wet etching is used to fabricate the flow channels, inlet, and nozzle on a silicon wafer. An integrated heater of aluminum is sputtered on a glass wafer. The two wafers are jointed by anodic bonding, creating a two-dimensional version of an APCI source with a sample channel in the middle and gas channels symmetrically on both sides. The ionization is initiated with an external corona-discharge needle positioned 2 mm in front of the microchip heated nebulizer. The microchip APCI source provides flow rates down to 50 nL/min, stable long-term analysis with chip lifetime of weeks, good quantitative repeatability (RSD10%) and linearity (r(2)0.995) with linear dynamic rage of at least 4 orders of magnitude, and cost-efficient manufacturing. The limit of detection (LOD) for acridine measured with microchip APCI at flow rate of 6.2 muL/min was 5 nM, corresponding to a mass flow of 0.52 fmol/s. The LOD with commercial macro-APCI at a flow rate of 1 mL/min for acridine was the same, 5 nM, corresponding to a significantly worse mass flow sensitivity (83 fmol/s) than measured with microchip APCI. The advantages of microchip APCI makes it a very attractive new microfluidic detector.

This study focuses on porous silicon (pSi) fabrication methods and properties for desorption ionization on silicon mass spectrometry (DIOS-MS). PSi was prepared using electrochemical etching of n-type silicon in HF–ethanol solution. Porous areas were defined by a double-sided illumination arrangement: front-side porous areas were masked by a stencil mask, eliminating the need for standard photolithography, and backside illumination was used for the backside ohmic contact. Backside illumination improved the uniformity of the porosified areas. Porosification conditions, surface derivatizations and storage conditions were explored to optimize pSi area, pore size and pore depth. Chemical derivatization of the pSi surfaces improved the DIOS-MS performance providing better ionization efficiency and signal stability with lower laser energy. Droplet spreading and drying patterns on pSi were also examined. Pore sizes of 50–200 nm were found to be optimal for droplet evaporation and pore filling with the sample liquid, as measured by DIOS efficiency. With DIOS, significantly better detection sensitivity was obtained (e.g. 150 fmol for midazolam) than with desorption ionization from a standard MALDI steel plate without matrix addition (30 pmol for midazolam). Also the noise that disturbs the detection of low-molecular weight compounds at m/z < 500 with MALDI could be clearly reduced with DIOS. Low background MS spectra and good detection sensitivity at the 100–150 fmol level for pharmaceutical compounds were achieved with DIOS-MS.

The feasibility of atmospheric pressure desorption/ionization on silicon mass spectrometry (AP-DIOS-MS) for drug analysis was investigated. It was observed that only compounds with relative high proton affinity are efficiently ionized under AP-DIOS conditions. The limits of detection (LODs) achieved in MS mode with midazolam, propranolol, and angiotensin II were 80 fmol, 20 pmol, and 1 pmol, respectively. In MS/MS mode the LODs for midazolam and propranolol were 10 fmol and 5 pmol, respectively. The good linearity (r2 > 0.991), linear dynamic range of 3 orders of magnitude, and reasonable repeatability showed that the method is suitable for quantitative analysis. Copyright © 2003 John Wiley & Sons, Ltd.

This study presents a new design for chip-based electrospray ionization mass spectrometry (ESI-MS), in which ESI is generated by direct spraying from a polydimethylsiloxane (PDMS) micro-channel electrokinetically without pressure assistance. PDMS devices were fabricated using SU-8-masters, which were coated with amorphous PDMS-diamond hybrid to increase their life-time. The presented technique allows rapid and low-cost batch-production of the ESI-MS chips.

This study presents a novel porous silicon (pSi) fabrication method for the matrix-free desorption/ionization on silicon mass spectrometry (DIOS-MS). The fabrication conditions and surface treatments of pSi on DIOS-MS performance are explored. Utilization of pSi sample plates in the analysis of low molecular weight pharmaceutical compounds is shown.