Phase-resolved detection in ion-mobility spectrometry

Throughput has been improved in an ion-mobility spectrometer by means of continuous ion-beam modulation and phase-resolved detection. Conventional ion-mobility spectrometry (IMS) utilizes brief packets of ions for separation in the gas phase. In contrast, this preliminary report outlines a new detection method based on phase resolution and involves monitoring the ion signal continuously, which raises the duty cycle of the instrument from approximately 1% (conventional IMS) to 50%. The greater duty cycle is shown to improve signal-to-noise ratios (S/N). However, the higher S/N levels did not translate into higher sensitivity values when calibration plots were constructed. It is demonstrated that even analytes with small differences in drift times can be modulated at frequencies that maximize the measured phase differences between ions. Phase-resolved signals can be processed with a Fourier transform and chemometric data reduction to simplify identification of the analyte from its phase spectra.     

Mechanical ion gate for electrospray-ionization ion-mobility spectrometry

A novel ion gate for electrospray-ionization atmospheric-pressure ion-mobility spectrometry (ESI-IMS) has been constructed and evaluated. The ion gate consisted of a chopper wheel with two windows—one for periodic ion passage from the ESI source into the drift region and the other for timing and synchronization purposes. The instrument contained a 45.0 cm long drift tube comprising 78 stainless steel rings (0.12 cm thick, 4.90 cm o.d., 2.55 cm i.d.). The rings were connected together in series with 3.34-MΩ resistors. The interface plate and the back plate were also connected with the first and the last rings, respectively, of the drift tube with 3.34-MΩ resistors. A potential of −20.0 kV was applied to the back plate and the interface plate was grounded. The drift tube was maintained at an electric field strength of ∼400 V cm⁻¹. An aperture grid was attached to the last ring in front of a Faraday plate detector, center-to-center. Several sample solutions were electrosprayed at +5.0 kV with +500 V applied to the ion gate. Baseline separations of selected benzodiazepines, antidepressants, and antibiotics were observed with moderate experimental resolution of ∼70.     

Combined ion-mobility and mass-spectrometry investigations of metallothionein complexes using a tandem mass spectrometer with a segmented second quadrupole

Rabbit metallothionein (MT) 2A complexes with Cd(II), Zn(II), Ag(I), Cu(I), Hg(II), arsenite, monomethylarsonous acid (MMA), and dimethylarsinous acid (DMA) have been examined using ion-mobility measurements and mass spectrometry in a triple-quadrupole mass spectrometer equipped with a segmented second quadrupole that doubled as an ion-mobility cell [Guo, Y.; Wang, J.; Javahery, G.; Thomson, B. A.; Siu, K. W. M. An Ion-Mobility Spectrometer with Radial Collisional Focusing. Anal. Chem.2005, 77, 266-275]. The metal ions confer conformational rigidity on the MT complexes, which counteracts Coulombic repulsion among protons added as a result of electrospray. Triply and quadruply protonated Cd(7)MT2A have smaller cross-sections than the Cd(7)MT2A structure deduced from published NMR data. For the 6+ ions, the As(6)MT2A complex has a cross-section of 790 A(2); the MMA(10)MT2A complex, 920 A(2); and the DMA(20)MT2A complex, 1220 A(2). This increase in cross-section of the As(III) species, from As(3+) to MMA to DMA, is interpreted as a consequence of decreasing multiple coordination and increasing number of methyl groups.     

Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-of-flight mass spectrometry

This paper presents an improved analytical method for glycosylation structural characterizations of a monoclonal antibody (mAb) using a newly developed quadrupole ion-mobility time-of-flight (ESI-Q-IM-TOF) mass spectrometer. Using this method, high-resolution mass spectra were acquired to produce the overall glycosylation profile of the mAb. Additionally, the light and heavy chains from the reduced antibody were separated in the gas phase by the ion mobility functionality of the instrument, allowing accurate mass measurement of each subunit. Furthermore, the glycan sequences, as well as the glycosylation site, were determined by a two-step sequential fragmentation process using the unique dual-collision-cell design of the instrument, thus providing detailed characterizations of the glycan structures.     

Comparison of negative and positive modes of ion-mobility increment spectrometry in the detection of heroin vapors

Some analytical characteristics of ion-mobility increment spectrometry (IMIS) in the negative and positive modes were compared in detecting diacetylmorphine vapors. It was shown experimentally that the sensitivity of IMIS in the negative mode to diacetylmorphine depends on the concentration of the water vapors in the drift gas: the lower the concentration, the higher the sensitivity. Data for cocaine are presented for comparison. Sampling diacetylmorphine and cocaine in the ionization chamber of the ion-mobility increment spectrometer was performed using a chromatograph with a multicapillary gas-chromatographic column.     

A segmented ring,cylindrical ion trap source for time-of-flight mass spectrometry

An ion trap source has been designed for use with time-of-flight (TOF) mass analysis. Two thin diaphragms make up a segmented ring electrode; the end cap electrodes are planar wire mesh. The potential field produced by the rf voltage applied between the ring and end cap electrodes resembles that of the cylindrical ion trap. The trapped ion population for ions created by electron impact exhibits linear growth against a first-order loss that has a time constant of about 50 µs; no ion loss occurs when the electron beam is off. The observed value of q _z at low-mass cutoff for rf ion storage is ?0.84. Pulsed extraction of all ions is accomplished by switching the trap electrodes from rf to voltages required to provide a linear dc extraction field. The TOF flight path includes a wide energy range reflectron. Better than unit mass resolution is achieved through m/z 500 without collisional ion cooling. With an extraction rate of 1 kHz and a recording rate of 4 spectra per second, a linear working curve is obtained between 36 pg and 18 ng of chlorobenzene delivered chromatographically. The system has demonstrated the potential to achieve a very high sample utilization efficiency at high spectral generation rates.     

Flow injection system for atomic absorption spectrometry

A manifold is described which permits the analysis of less than 1 ml of sample solution. Water- and air-compensation methods for the introduction of the carrier stream into the nebulizer are effective in obtaining sensitive and reproducible measurements of magnesium, but air-compensation gives higher peak responses.     

Flow injection calibration methods for atomic absorption spectrometry

The use of an atomic absorption spectrometer as a detector in flow injection analysis is briefly reviewed. A new simplified model is described for the dispersion effects observed with such systems; the model is based on considering the dispersion to be due to a single hypothetical mixing chamber located immediately prior to the measurement stage. The utility of this approach is demonstrated for two methods of calibration commonly used in atomic absorption spectrometry, and it is shown that flow injection sample and standard handling techniques are comparable to manipulation with volumetric apparatus. The flow injection method has a number of advantages for the analogue of the standard addition method. The use of an exponential concentration gradient is proposed as a novel method of calibration using a single concentrated standard. Results are presented for the determination of chromium in standard steels.     

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab

Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.     

Statistical design of experiments as a tool in mass spectrometry

This Tutorial is an introduction to statistical design of experiments (DOE) with focus on demonstration of how DOE can be useful to the mass spectrometrist. In contrast with the commonly used one factor at a time approach, DOE methods address the issue of interaction of variables and are generally more efficient. The complex problem of optimizing data-dependent acquisition parameters in a bottom-up proteomics LC-MS/MS analysis is used as an example of the power of the technique. Using DOE, a new data-dependent method was developed that improved the quantity of confidently identified peptides from rat serum.     

Flow modulation comprehensive two-dimensional gas chromatography-mass spectrometry with a supersonic molecular beam

A new approach of flow modulation comprehensive two-dimensional gas chromatography-mass spectrometry (GC x GC-MS) with supersonic molecular beam (SMB) and a quadrupole mass analyzer is presented. Flow modulation uniquely enables GC x GC-MS to be achieved even with the limited scan speed of quadrupole MS, and its 20 ml/min column flow rate is handled, splitless, by the SMB interface. Flow modulation GC x GC-SMB-MS shares all the major benefits of GC x GC and combines them with GC-MS including: (a) increased GC separation capability; (b) improved sensitivity via narrower GC peaks; (c) improved sensitivity through reduced matrix interference and chemical noise; (d) polarity and functional group sample information via the order of elution from the second polar column. In addition, GC x GC-SMB-MS is uniquely characterized by the features of GC-MS with SMB of enhanced and trustworthy molecular ion plus isotope abundance analysis (IAA) for improved sample identification and fast fly-through ion source response time. The combination of flow modulation GC x GC with GC-MS with SMB (supersonic GC-MS) was explored with complex matrices such as diesel fuel analysis and pesticide analysis in agricultural products.     

Effect of additives in a carrier gas on the behavior of ions in an ion-mobility increment spectrometer

The effect of additives (ethanol, acetic acid, water, chloroform, and acetone) in air used as the carrier gas on the changes in the spectra of the H⁺(C₆H₅NH₂) ion was studied for an ion-mobility increment spectrometer (IMIS) operating in tandem with a mass spectrometer (MS). It was shown that the presence of additives could improve both the resolution of the instrument and the sensitivity of the method. The resolving power was increased fourfold using air saturated with an ethanol vapor, while the sensitivity was increased 11-fold upon adding a mixture of ethanol and acetic acid to the carrier gas. The dependences on the concentration of the additives indicate that the formation of the clusters is the reason for these effects. The presence of an ethanol vapor in the carrier gas almost completely eliminates the effect of the present water vapor; therefore, air saturated with ethanol vapors can be used in the IMIS-MS instruments without drying.     

Improved design for high resolution electrospray ionization ion mobility spectrometry

An improved design for high resolution electrospray ionization ion mobility spectrometry (ESI-IMS) was developed by making some salient modifications to the IMS cell and its performance was investigated. To enhance desolvation of electrospray droplets at high sample flow rates in this new design, volume of the desolvation region was decreased by reducing its diameter and the entrance position of the desolvation gas was shifted to the end of the desolvation region (near the ion gate). In addition, the ESI source (both needle and counter electrode) was positioned outside of the heating oven of the IMS. This modification made it possible to use the instrument at higher temperatures, and preventing needle clogging in the electrospray process. The ion mobility spectra of different chemical compounds were obtained. The resolving power and resolution of the instrument were increased by about 15-30% relative to previous design. In this work, the baseline separation of the two adjacent ion peaks of morphine and those of codeine was achieved for the first time with resolutions of 1.5 and 1.3, respectively. These four ion peaks were well separated from each other using carbon dioxide (CO₂) rather than nitrogen as the drift gas. Finally, the analytical parameters obtained for ethion, metalaxyl, and tributylamine indicated the high performance of the instrument for quantitative analysis.     

Retention studies of acrylamide for the design of a robust liquid chromatography-tandem mass spectrometry method for food analysis

A wide range of solid phases for SPE (solid-phase extraction) (n=14) and HPLC (n=9) were compared regarding the chromatographic retention of acrylamide. For SPE, a hydroxylated polystyrene-divinylbenzene copolymer phase (ENV+) gave the strongest retention. Twenty millilitre of water per gram solid phase could be passed with less than 5% loss of acrylamide from the column, thus enabling significant enrichment of food extracts. Other polymer phases gave varying degrees of retention, while silica bonded phases gave low retention. For HPLC, columns were evaluated both in reversed-phase and aqueous normal-phase (hydrophilic interaction chromatography) modes. The best retention was obtained with a phase comprising porous graphitic carbon (Hypercarb), giving a k-value of 4 with water as the mobile phase. Based on these investigations, a method for analysis of acrylamide in food using liquid chromatography-tandem mass spectrometry was designed to meet the demands of a collaborative validation trial. A comparative investigation of solid phases has not been published earlier. Thus, the paper should provide a base for new method developments regarding clean-up, enrichment and chromatography of acrylamide. In addition, the detailed standard operating procedure (SOP) method, as used in a collaborative validation trial, is provided as an electronic supplement (www.elsevier.com).     

Novel design for drift tubes in ion mobility spectrometry for optimised resolution of peak clusters

In the past decade, Ion Mobility Spectrometry has established a very strong foot hold in medical and biological applications due to its numerous advantages including sensitivity, ruggedness and reproducibility. During the analysis of complex samples such as human breath, it is very probable that two or more analytes form peak clusters due to similar drift times and pre-separation times, thus hindering the identification of the analytes. Furthermore, such overlapping of signal makes quantification very difficult or even impossible. Resolving these peak clusters is important to enable proper identification and quantification of analytes detected for diagnosis. Hence, we designed a drift tube with variable length for investigating the influence of varying drift lengths and electric field on resolution. Peak cluster formations usually seen between acetone and the reactant ion peak, between the dimer peaks of 2-Heptanone and 4-Heptanone have been resolved with the new drift tube after optimisation. These novel drift tubes could easily negate the peak clusters often encountered when complex medical and biological samples are measured with the ion mobility spectrometer. Furthermore, the fact that these drift tubes can be altered in length thereby providing a wide range of electric fields (from 50 to 3300 V.cm⁻¹), opens up new research options in ion motions in an electric field.     

Flow injection micelle-mediated methodology for determination of lead by electrothermal atomic absorption spectrometry

A simple and sensitive flow injection micelle-mediated method was developed for the determination of trace Pb by electrothermal atomic absorption spectrometric (ET-AAS). The formation of the analyte-entrapped surfactant micelles by online merging of the analyte solution with O,O-diethyldithiophosphate solution and Triton X-114 solution sequentially, the adsorption of the resultant analyte-entrapped surfactant micelles onto a microcolumn packed with silica gel. The adsorbed analyte was then eluted with methanol and determined by ET-AAS. With consumption of 6.0 mL sample solution, a detection limit of 0.016 μg L⁻¹, and a concentration factor of 21.6 were obtained at a sample throughput of 20 h⁻¹. The relative standard deviation was 5.1% (c = 2.0 ng mL⁻¹, n = 7). The method has been successfully applied to the analysis of trace Pb in water sample with recoveries ranging from 94.0 to 99.0%. The certified reference material was also analyzed for validation, and the determined value obtained was in good agreement with the certified value.