Surface‐activated chemical ionization and cation exchange chromatography for the analysis of enterotoxin A

Surface‐activated chemical ionization (SACI) has been widely used in recent years for the analysis of different compounds (e.g. peptides, street drugs, amino acids). The main benefits of this technology are its high sensitivity and its effectiveness under different chromatographic conditions [i.e. ion exchange chromatography and reversed‐phase (RP) chromatography]. Here we used SACI in conjunction with quadrupole time‐of‐flight mass spectrometry to analyze enterotoxin A, which is produced by Staphylococcus aureus, in milk matrix using both RP and ion exchange chromatographies. SACI had increased sensitivity as compared with electrospray ionization. Moreover, the higher quantitation efficiency of this technique, mainly in terms of limit of detection (0.01 ng/ml), limit of quantitation (0.05 ng/ml), linearity range (0.05–50 ng/ml), matrix effect, accuracy (intraday and interday accuracy errors were 9.2% and 10.3%, respectively) and precision (intraday and interday precision errors were 5.3% and 12.8%, respectively), is shown and discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Surface-activated chemical ionization ion trap mass spectrometry in the analysis of drugs in dilute urine samples. Part II: analysis of morphine and other street drugs

The new ionization method, called surface-activated chemical ionization (SACI), was employed for the analysis of fives drugs (morphine, codeine, 6-monoacetylmorphine (6-MAM), benzoylecgonine and cocaine) by ion trap mass spectrometry. The results so obtained have been compared with those achieved by using atmospheric pressure chemical ionization (APCI), no-discharge-APCI and electrospray ionization (ESI) clearly showing that SACI is the most sensible one mainly due to the high ionization efficiency and the lower chemical noise. The performance of SACI in terms of sensitivity and linearity was compared with the sensitivity and linearity obtained using APCI, no-discharge-APCI and ESI, showing that the new SACI approach gives rise to the best results. Then, SACI was used to analyze morphine, codeine, 6-MAM, benzoylecgonine and cocaine in urine samples. After the optimization of the instrumental parameters for a mixture of the standard compounds, eight urine samples were analyzed. They were strongly diluted (1 : 20 and 1 : 100) in order to prevent the chromatographic column damage due to the matrix composition. Furthermore, the diluted urine samples were directly analyzed, without pretreatment, through LC-MS and LC-MS/MS, and the obtained results are reported.     

A novel interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

A variable flow "peak trapping" liquid chromatography (LC) interface has been developed for the coupling of nanoscale LC to electrospray ionization mass spectrometry (ESI-MS). The presented peak trapping LC interface allows for the extended analysis time of co-eluting compounds and has been employed for the identification of proteins via tandem mass spectrometry (MS/MS). The variable flow process can be controlled either manually or in a completely automated manner where the mass spectrometer status determines the status of the variable flow interface. When the mass spectrometer operates in MS survey mode, the interface is operated in a so-called "high-flow" mode. Alternatively, the interface is operated in a "low-flow" mode during MS/MS analysis. In the "high-flow" mode of the variable flow process the column flow rate is typically around 200 nL/min, whereas in the "low-flow" mode the column effluent is introduced into the source of the mass spectrometer at 25 nL/min. In addition to the flow reduction during MS/MS analysis, the gradient is paused to preserve the peptide separation on the analytical nanoscale LC column. The performance of the variable flow nanoscale LC/MS/MS interface is demonstrated by the automated analysis of standard peptide mixtures and protein digests utilizing variable flow, data-dependent scanning MS/MS techniques, and automated database searching.     

Surface-activated chemical ionization in the analysis of arginine in plasma samples

Alterations of arginine plasma levels are involved in several disorders of amino acid metabolism such as hurtnup, argininosuccinic aciduria, histidinemia, citrullinuria, and cystinuria. In this work a new liquid ionization source, surface-activated chemical ionization (SACI), has been used to analyze arginine in human and rat plasma samples. Arginine was extracted and diluted ten times through protein precipitation. The diluted arginine samples were then analyzed using an ion-exchange chromatographic column coupled with the SACI source and an ion trap analyzer using MS(3) monitoring in order to increase the sensitivity and specificity of the analysis. The multiple-point standard additions method was used to quantify the arginine. This method was employed to eliminate the matrix effect that affects all liquid ionization sources (APCI, ESI, SACI, etc.), and also does not require use of an internal standard. High-quality results in terms of sensitivity, limit of detection, lower limit of quantitation, linearity and reproducibility, are demonstrated.     

Surface-activated chemical ionization ion trap mass spectrometry in the analysis of amphetamines in diluted urine samples

A new ionization method, named surface-activated chemical ionization (SACI), was employed for the analysis of five amphetamines (3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDE), amphetamine and methamphetamine) by ion trap mass spectrometry. The results so obtained have been compared with those achieved by using atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) using the same instrument, clearly showing that SACI is the most sensitive of the three. The limit of detection and linearity range for SACI were compared with those obtained using APCI and ESI, showing that the new SACI approach provides the best results for both criteria. SACI was used to analyze MDA, MDMA MDE, amphetamine and methamphetamine in four urine samples, and the quantitation results are compared with those achieved using ESI.     

Surface‐activated chemical ionization time‐of‐flight mass spectrometry and labeling‐free approach: two powerful tools for the analysis of complex plant functional proteome profiles

Surface‐activated chemical ionization (SACI) has been widely used in recent years to analyze a range of different compounds (e.g., peptides, street drugs, amino acids). The main benefits of this technology are its high sensitivity and its effectiveness under different chromatographic conditions. Here, we used SACI in conjunction with a highly selective quadrupole time‐of‐flight mass analyzer to characterize a complex proteome pattern after separation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). The data obtained were compared with those obtained using the micro‐electrospray ionization (ESI) approach, which showed that using SACI strongly increased the number of detectable proteins. The higher sensitivity is mainly due to the ability of SACI to selectively produce singly charged species of high intensity under full‐scan conditions and doubly charged species for tandem mass spectrometric (MS/MS) peptide characterization by simply changing the ionization conditions during data acquisition. Copyright © 2010 John Wiley & Sons, Ltd.     

Surface-activated no-discharge atmospheric pressure chemical ionization

A new ionization method named surface-activated chemical ionization (SACI) has been realized. In this invention a commercially available atmospheric pressure chemical ionization (APCI) chamber, employed without any corona discharge (no-discharge APCI), has been modified with the insertion of a gold surface, leading to a significant improvement in the ionization efficiency. The ionization of the sample takes place by both gas-phase and surface-activated processes. This new ionization source is able to generate ions with high molecular mass and low charge states, leading to improved sensitivity and reduced noise. The new device has been tested in the analysis of some peptides. A comparison between the performance with and without the presence of the surface, and the optimization of the operating conditions (nebulizing gas flow, sample solution flow, pH of solution, and surface area), are reported and discussed.     

Analysis of underivatized amino acid mixtures using high performance liquid chromatography/dual oscillating nebulizer atmospheric pressure microwave induced plasma ionization-mass spectrometry

A dual oscillating capillary nebulizer (OCN) in conjunction with an atmospheric pressure microwave induced plasma ionization (AP-MIPI) source was applied to the analysis of underivatized amino acid mixtures. It was found that, compared to the single OCN, the dual OCN enhanced the sensitivity of detection several fold. Enhanced sensitivity was compound dependent. For small molecules, such as amino acids, it was 2-5 times more sensitive, while for larger molecules such as peptides it was more than an order of magnitude. The increase in sensitivity was attributed to the enhanced nebulization of the new torch. By using water/ acetonitrile containing 0.1% nonafluoropentanoic acid as the high performance liquid chromatography (HPLC) mobile phase and a C18 column, all common amino acids were separated and detected. A comparison between the results obtained using microwave induced plasma, atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) at flow rates compatible with micro LC (10-100 microL/min) showed a higher sensitivity of detection with the AP-MIPI technique for the analysis of underivatized amino acids.     

The use of chemical derivatization to enhance liquid chromatography/tandem mass spectrometric determination of 1-hydroxypyrene, a biomarker for polycyclic aromatic hydrocarbons in human urine

This article presents an analytical approach that used chemical derivatization to enhance mass spectrometric (MS) response in electrospray ionization (ESI) mode of 1-hydroxypyrene (1-OHP), a commonly used biomarker to monitor human exposure to polycyclic aromatic hydrocarbons (PAHs). The enhancement successfully enabled the desired detection of 50 pg/mL in human urine. The introduction of an MS-friendly dansyl group to 1-OHP enhanced both ionization efficiency in the ESI source and collision-activated dissociation (CAD) in the collision cell. The response increase was estimated to be at least 200-fold, and enabled the reduction of sample size to only 100 microL. The selective MS detection also facilitated a fast (run time 3 min) liquid chromatography (LC) method which successfully resolved the analyte and interferences. The sample processing procedure included enzymatic hydrolysis of glucuronide and sulfate conjugates, liquid-liquid extraction, derivatization with dansyl chloride and a final liquid-liquid extraction to generate clean extracts for LC/MS/MS analysis. This approach has been validated as sensitive, linear (50-1000 pg/mL), accurate and precise for the quantitation of 1-OHP in human urine. This is the first report of using chemical derivatization to enhance MS/MS detection with fast chromatography in the determination of 1-OHP in human urine.     

Compensation voltage shifting in high-field asymmetric waveform ion mobility spectrometry-mass spectrometry

The separation and ion focusing properties of High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) depend on desolvated ions entering the device, leading to a compound-specific, reproducible compensation voltage (CV) for each ion. This study shows that the conditions identified for stable spray and satisfactory ion desolvation in normal electrospray ionization mass spectrometry (ESI-MS) operation might significantly differ from those required for FAIMS-MS. In a typical setup with high-flow electrospray conditions, ions could be incompletely desolvated, resulting in the formation of unidentified clusters with differing behavior in a FAIMS environment. This causes compound-specific shifts of as much as 10 V in CV values when the mobile phase composition and/or flow rate are varied. The shifts diminish and finally disappear when the flow rate of methanol, used as mobile phase, is reduced to 40 microL/min and that of acetonitrile to 20 microL/min. The reproducibility of the observed CV was determined by scanning the CV while infusing a five-component mixture into a 400 microL/min flow of methanol or 50:50 acetonitrile/water. The relative standard deviation (RSD) for these multiple scans ranged from 0.7% to 6%. Therefore, under a constant set of experimental parameters, the CV does not shift appreciably. These observations have an impact on method development strategies. High flow rates can be used with the FAIMS device, since the CV values are reproducible, but it is likely that clusters are forming. Therefore, CV scans should be performed under conditions which mimic the chromatographic elution or flow injection analysis conditions, including matrix composition, to minimize errors in CV determination. An alternative approach is to determine the liquid flow rate at which the CV becomes compound-specific and to split the mobile phase stream accordingly. These experimental results may be specific to the setup used for this study and may not be directly applicable to other instrument FAIMS devices.     

A simplified device for protein identification by microcapillary gradient liquid chromatography-tandem mass spectrometry

A simplified device and procedure have been developed for microcapillary gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS). This procedure has proved useful in identifying low level quantities of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel bands. Microelectrospray needles are packed with reversed-phase resin and function both as a high performance liquid chromatography (HPLC) column and a nanospray mass spectrometer tip when interfaced between an HPLC and ion trap mass spectrometer. Variable submicroliter flow rates are generated by flow splitting between the microelectrospray capillary and an HPLC system. A manual injector is used to inject a protein digest mixture that binds to the column and is then washed at a high flow rate (2 microL/min post split). Gradient elution of bound peptides was initiated by the injection of a filled loop of 70% v/v methanol (5 microL) concomitant with a reduction of flow rate (0.1 microL/min post split). This forms a diffusion-dependent gradient of variable length (typically 15-30 min in length) depending upon the final flow rate. Chromatographic separations of a standard solution digest demonstrate that this diffusion-dependent gradient provides reasonable separations such that multiple peptide identifications by MS/MS can be obtained. Application of this methodology to the analysis of several in-gel-digested gel-separated proteins is presented to demonstrate its utility.     

Electrospray ionization/atmospheric pressure photoionization multimode source for low-flow liquid chromatography/mass spectrometric analysis

Analysis of several polar and non-polar compounds is performed with a newly developed dual electrospray ionization/atmospheric pressure photoionization (ESI/APPI) or ESPI source. Several variables are considered in the source, such as ESI probe heater temperature, solvent flow, dopant effects, repeller plate voltage, source geometry and photon energy (Kr vs. Ar lamp). Direct photoionization resulting in a molecular radical cation [M](*+) dominates at high temperatures (>400 degrees C) and low flow rates (<200 microL/min). Indirect photo-induced chemical ionization (PCI) involving solvent molecules becomes important at lower temperatures and higher solvent flow rates. Indirect PCI is enhanced using an Ar lamp, which yields comparable [M+H](+) signal but poorer [M](*+) signal than the Kr lamp at lower temperatures and higher flow rates. This is in support of our recent finding that the Ar lamp results in a solvent-dependent enhancement of analyte molecules via PCI. Analysis of 12 compounds in methanol under low-flow conditions (10 microL/min) demonstrates that the dual ESPI source performs favorably for most compounds versus the standard ESCI source, and significantly better than ESCI for the analysis of unstable drugs, like flurbiprofen. Several factors contributing to the benefits of the ESPI source are the shared optimal geometry for ESI and APPI sources and soft ionization of APPI versus APCI.     

Surface-activated chemical ionization ion trap mass spectrometry for the analysis of cocaine and benzoylecgonine in hair after extraction and sample dilution

Surface-activated chemical ionization (SACI) was employed for the analysis of cocaine and its metabolite, benzoylecgonine, extracted from hair. Following decontamination and acid hydrolysis procedures on the hair sample, the sample solution was diluted (1:10) and directly analyzed by liquid chromatography/surface-activated chemical ionization multiple collisional stage single reaction monitoring mass spectrometry (LC/SACI-MS(3)-SRM) without solid-phase extraction (SPE) pre-purification and concentration procedures. To increase the selectivity of the method, MS(3) was chosen instead of the less selective MS/MS. This data was compared with that achieved using gas chromatography/mass spectrometry (GC/MS), the reference method used by the Italian Government Institute of Health protocol. The limits of detection (LODs) were 0.003 ng/(mg hair) for cocaine and 0.02 ng/(mg hair) for benzoylecgonine and the limits of quantitation (LOQs) were 0.01 ng/(mg hair) for cocaine and 0.04 ng/(mg hair) for benzoylecgonine. The squared correlation coefficient (R(2)) of the calibration curve was 0.9887-0.9980 for cocaine and 0.9987-0.9997 for benzoylecgonine. The percent accuracy error was 2-5% for both cocaine and benzoylecgonine using the LC/SACI-MS(3)-SRM approach, whereas it was higher for benzoylecgonine (20-25%) using the LC/SACI-MS/MS-SRM approach compared with the GC/MS data due to hair matrix contamination. In both cases, high precision was achieved (1-3% precision error), which confirmed the stability of the developed methods.     

Reducing glycerophosphocholine lipid matrix interference effects in biological fluid assays by using high-turbulence liquid chromatography

Matrix interferences can severely affect quantitative assays of biological samples when electrospray ionization (ESI) is employed with liquid chromatography/tandem mass spectrometry (LC/MS/MS). A major source of matrix interferences for plasma sample analyses is the presence of glycerophosphocholine (GPCho) lipids. The efficiency of online high-turbulence liquid chromatography (HTLC) extraction for eliminating these lipids is evaluated and the interfering effects of endogenous lipids on human plasma assays are measured for pharmaceutical compounds having a wide variety of chemical properties. It is found that GPCho lipids, represented by 16:0, 18:1 and 18:0 LPC (lysophosphatidylcholine) and 16:0-18:2 PC, cause variations for hydrophobic compound analyses even when optimal online HTLC extraction conditions are employed. The efficiency for lipid removal depends on the organic content of the transfer solvent, but turbulent flow loading has no significant effect.     

Liquid chromatography/mass spectrometry in anabolic steroid analysis--optimization and comparison of three ionization techniques: electrospray ionization,atmospheric pressure chemical ionization and atmospheric pressure photoionization

The applicability of liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the detection of the free anabolic steroid fraction in human urine was examined. Electrospray ionization (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization methods were optimized regarding eluent composition, ion source parameters and fragmentation. The methods were compared with respect to specificity and detection limit. Although all methods proved suitable, LC/ESI-MS/MS with a methanol-water gradient including 5 mM ammonium acetate and 0.01% acetic acid was found best for the purpose. Multiple reaction monitoring allowed the determination of steroids in urine at low nanogram per milliliter levels. LC/MS/MS exhibited high sensitivity and specificity for the detection of free steroids and may be a suitable technique for screening for the abuse of anabolic steroids in sports.     

Temperature‐programmed capillary high‐performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry for analysis of fatty acid methyl esters

A new capillary high‐performance liquid chromatography method with atmospheric pressure chemical ionization mass spectrometry was developed for the analysis of fatty acid methyl esters and long‐chain alcohols. The chromatographic separation was achieved using a Zorbax SB‐C18 HPLC column (0.3 × 150 mm, 3.5 μm) with a mobile phase composed of acetonitrile and formic acid and delivered isocratically at a flow rate of 10 μL/min. The column temperature was programmed simply, using a common column oven. Good reproducibility of the temperature profile and retention times were achieved. The temperature programming during the isocratic high‐performance liquid chromatography run had a similar effect as a solvent gradient; it reduced retention times of later eluting analytes and improved their detection limits. Two atmospheric pressure chemical ionization sources of the mass spectrometry detector were compared: an enclosed conventional ion source and an in‐house made ion source with a glass microchip nebulizer. The enclosed source provided better detectability of saturated fatty acid methyl esters and made it possible to determine the double bond positions using acetonitrile‐related adducts, while the open chip‐based source provided better analytical figures of merit for unsaturated fatty acid methyl esters. Temperature‐programmed capillary high‐performance liquid chromatography is a promising method for analyzing neutral lipids in lipidomics and other applications.     

Characterization of copolymers by high performance liquid chromatography

Size exclusion chromatography (SEC) is capable of evaluating the molecular weight distribution (MWD) of a sample. Information about the chemical composition distribution can be gained by gradient high performance liquid chromatography (gradient HPLC), where a poor starting eluent is, in the course of the separation, substituted by another one of increasing elution strength. Both normal-phase and reversed-phase systems can be employed. The combination of SEC and gradient HPLC enables chromatographic cross-fractionation to be performed efficiently.     

Ultra-fast mass fingerprinting by high-affinity capture of peptides and proteins on derivatized poly(glycidyl methacrylate/divinylbenzene) for the analysis of serum and cell lysates

The development of support materials in mass fingerprinting is an important task required for diagnostic markers in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The material-based approach, which we introduced as material-enhanced laser desorption/ionization (MELDI), focuses not only on different functionalities, but also emphasizes the morphology, i.e. porosity or particle size of the carrier material. As a result, it provides a quick and sensitive platform for effective binding of peptides and proteins out of different biofluids, e.g. serum, spinal fluid, urine or cell lysates, and to subsequently analyze them with MALDI-TOF MS. This approach includes a built-in desalting step for serum protein profiling and is sensitive enough to detect proteins and peptides down to 100 fmol/microL. Here we co-polymerized glycidyl methacrylate (GMA) with divinylbenzene (DVB) using thermal polymerization to yield a GMA/DVB polymer for further modifications. Different affinities have been created, such as immobilized metal ion affinity (IDA-Cu2+), reversed-phase (RP) and anion-exchanger (AX) chromatography. The diverse derivatizations and the dispersity of the particles created by different chemical synthetic approaches were confirmed by characteristic infrared (IR) peaks. The polymerization carried out by non-stirring yielded an average pore radius of 6.1 microm (macro-pores) that enhanced the binding capacity enormously by offering enlarged surface areas. Moreover, atomic absorption spectrometry (AAS) provided the metal content loaded on iminodiacetic acid (IDA) in the case of poly(GMA/DVB)-IDA-Cu2+. To summarize, the optimized MELDI approach is sensitive in its performance, extremely fast and can be adapted to robotic systems for routine analysis, allowing sample preparation in less than 5 min in contrast to the conventional surface-enhanced laser desorption/ionization (SELDI) methods.     

Investigation of chromatographic peak broadening in supercritical fluid chromatography/atmospheric pressure chemical ionization mass spectrometry

Multimode ionization source allows for switching between different ionization techniques, for example, electrospray and atmospheric pressure chemical ionization, within a single analysis. Supercritical fluid chromatography can handle a wide polarity range of substances from hydrophilic to lipophilic in a single run and can undoubtedly benefit from versatility of this ion source. Nevertheless, we observed a significant chromatographic peak broadening effect in atmospheric pressure chemical ionization mode during supercritical fluid chromatography‐mass spectrometry analysis of volatile flavor compounds with a dual ion source named ESCi (Waters). Surprisingly, this effect was not related to the separation process but was triggered solely by the ion source conditions. Neither of photodiode array detector, electrospray mode nor a dedicated atmospheric pressure chemical ionization source suffered from such a phenomenon. Chromatographic peak profiles of ten test substances obtained with the dual ion source were compared with photodiode array detector data as a reference. The broadening effect was more pronounced for volatile compounds with low polarity. Dependence of peak broadening on the ion source settings was systematically investigated. Tuning of desolvation gas flow and its temperature dramatically reduced peak distortion and increased detection sensitivity.     

Investigation on the role of pneumatic aspects in electrospray, desorption electrospray surface ionization and surface activated chemical ionization

This review reports the results of some studies carried out by us on the role of pneumatic aspects in electrospray and desorption electrospray surface ionization, with the aim to propose some relevant aspects of the mechanisms involved in these ionization methods. Electrospray ion sources, with the exception of the nano- electrospray source, operate with the concurrent action of a strong electrical field and a supplementary coaxial gas flow. The electrical field is responsible for electrospraying of the analyte solution but the use of a coaxial gas flow leads to a significant increase of the analyte signal and allows the use of higher solution flows. However, by employing capillary voltages much lower than those necessary to activate the electrospray phenomenon, analyte ions are still observed and this indicates that different mechanisms must be operative for ion production. Under these conditions, ion generation could take place from the neutral pneumatically sprayed droplet by field-induced droplet ionization. Also in the case of desorption electrospray ionization (DESI), and without any voltage on the spraying capillary as well as on the surface of interest, ions of analytes present on the surface become detectable and this shows that desorption/ionization of analytes occurs by neutral droplets impinging the surface. Consequently, the pneumatic effect of the impinging droplets plays a relevant role, and for these reasons the method has been called pneumatic assisted desorption (PAD). Some analogies existing between PAD and surface activated chemical ionization (SACI), based on the insertion of a metallic surface inside an atmospheric pressure chemical ionization source operating without corona discharge, are discussed.