Electrospray interface small molecules

Plastic microdevices for high-throughput screening with MS detection were also prepared for detection of aflatoxins and barbiturates. These devices incorporated concentration techniques interfaced with electrospray ionization MS (ESI-MS) through capillaries [2], The microfluidic device for aflatoxin detection employed an affinity dialysis technique, in which a poly (vinylidene fluoride) (PVDF) membrane was incorporated in the microchip between two channels. Small molecules were dialyzed from the aflatoxin/antibody complexes, which were then analyzed by MS. A similar device was used for concentrating barbiturate/antibody complexes using an affinity ultrafiltration technique. A barbiturate solution was mixed with antibodies and then flowed into the device, where uncomplexed barbiturates were removed by filtration. The antibody complex was then dissociated and electrokinetically mobilized for MS analysis. In each case, the affinity preconcentration improved the sensitivity by at least one to two orders of magnitude over previously reported detection limits. 

Capillary electrokinetic chromatography (CEKC) with ESI-MS requires either the use of additives that do not significantly impact the ESI process or a method for their removal prior to the electrospray. Although this problem has not yet been completely solved, recent reports have suggested that considered choices of surfactant type and reduction of electro-osmotic flow (EOF) and surfactant in the capillary can decrease problems. Because most analytes that benefit from the CEKC mode of operation can be effectively addressed by the interface of other separations methods with MS, more emphasis has until now been placed upon interfacing with other CE modes. For small-molecule CE analysis, in which micellar and inclusion complex systems are commonly used, atmospheric pressure chemical ionization (APCI) may provide a useful alternative to ESI, as it is not as greatly affected by involatile salts and additives. 

Commercially, Agilent Technologies produces chips for both direct infusion into a mass spectrometer and for HPLC-MS applications. The chips accommodate nanoflow rates with an electrospray ionisation source, are about the size of a credit card and are rensable. The infusion chip is for collecting direct MS or tandem MS data. The protein HPLC chip has both a sample enrichment and CIS separation column on the chip, as well as the connections and spray nozzles for electrospray. There are also a small molecule chip and a glycan chip. The chip being used is placed in a Chip Cube MS interface which positions the sprayer tip perpendicular to the MS inlet (Figure 10.7). 

Atmospheric pressure ionization (API). The need to analyze polar componnds and the necessity to interface LC with MS led to the development of techniqnes where the ionization occurs at atmospheric pressure outside the vacuum chamber, and the resulting ions are transferred directly into the mass analyzer. Electrospray ionization (ESI) is the most successful of the API methods because of the range of molecular masses to which it can be applied, from small molecules to proteins. Other API methods include atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo-ionization (APPI), and also the recently developed surface ionization methods such as desorption electrospray ionization (DESI) and direct analysis in real time (DART) (see below and Sections 2.2.2 and 2.2.3). 

Nowadays, interfacing separation methods such as high-performance hquid chromatography (HPLC) or CE [54] to MS is already a routine technique. MS methods based on so-called soft ionization techniques, which include fast atom bombardment, laser desorption and electrospray ionization (ESI), have allowed the analysis of biological macromolecules that in the past could have been analyzed only by extensive cleavage and derivatization. Of these methods, the two most preferred for biomolecules are ESI [55] and matrix-assisted laser desorption/ionization(MALDI) [56,57], for which time-of-flight (TOE) and ion trap mass analyzers are the most frequentiy used mass analysis methods. However, these methods are equally suitable for small molecules, such as metabolites or the products of organochemical reactions. 

The observation of multiple charging of proteins in electrospray ionization attracted much attention all major instrument manufacturers introduced an API system to be used in combination with an electrospray interface. At the same time, it was found that electrospray ionization is not only suitable for the mass analysis of large molecules, but is also a very efficient ionization technique of small polar or ionic molecules, e.g. drugs and their metabolites. In the past few years, hundreds of dedicated API-MS systems equipped with electrospray and APCI interfaces have found their way into many different laboratories, especially within pharmaceutical companies and biochemistry/biotechnology laboratories. 

Thermospray and, more recently, electrospray ionization have found wide application as an interface technology between HPLC instruments and mass spectrometers. They represent powerful techniques for the analysis of complex lipids directly from solutions (Henion and Lee, 1990 Murphy, 1993). In most instances, the total HPLC eluant can be sent directly into the heated thermospray ion source. Here, the combination of heat and eluant velocity creates a plume of small-diameter particles suspended in a vapor (nebulization). A strong electric charge forms on the surface of the liquid particles and as the droplets evaporate the increase in charge ionizes analyte molecules that are discharged directly from the droplet into the gas phase. From here, they may enter the mass spectrometer directly. 

Following Dole s [4] work, John Fenn introduced somedecisive improvements that allowed a mass spectrometer to be interfaced to an electrospray source [5, 6] and clearly demonstrated that ESI-MS could be used very effectively for the analysis of small ions and molecules [5] as well as peptides and proteins with a molecular mass extending into the megadalton range [6]. This work had a big impact and started the ESI-MS revolution that is continuing to this day. 

ESI was first proposed by Malcolm Dole in 1968 who noticed that the Coulombic fission cascade would eventually lead to sufficiently small drops which contained a single solute molecule that retained some of the drop charge such that a fully desolvated gas phase ion would ultimately be left once all the solvent evaporates, this mechanism being known as the charge residue model (CRM). These efforts were, however, largely unsuccessful practically due to the use of an ion-drift spectrometer to which the electrospray was interfaced. It was John Fenn, then at Yale University, who later developed a practical method for electrospray ionization mass spectrometry (ESI-MS) that allowed the identification and structure analysis of biomacromolecules of virtually unlimited molecular weights to an accuracy of 0.01% by averaging... 

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