Nanospray ionization

Figure 14.5 Modified-ESI source for the direct infusion of undiluted ILs. A stainless steel wire is placed in the spray, leading to the optimal vaporization of the IL. Additionally, an orthogonal ESI source is used. Only a part of the IL ions is transferred into the MS, thus minimizing pollution of the source. (Modified from Dyson, R J. et al.. Direct probe electrospray (and nanospray) ionization mass spectrometry of neat ionic liquids. Chem. Commun., 2204, 2004. Reproduced by permission of the Royal Society of Chemistry.)...

Dyson, P. J. et al.. Direct probe electrospray (and nanospray) ionization mass spectrometry of neat ionic liquids. Chem. Commun., 2204, 2004. 

NSI Nanospray ionization (NSI) is a low-flow (10-500-nL/min) ESI technique with many advantages over conventional-flow ESI ( 200 xL/min) for the analysis of drugs, metabolites, peptides, proteins, and other macromolecules. Advantages of NSI over ESI include decreased sample consumption and increased sensitivity (Wilm and Mann, 1996 Corkery et al., 2005). NSI can be used for LC-MS or direct-infusion MS analysis of molecules (Wickremsinhe et al., 2006 Ramanathan et al., 2007c). 

Ramanathan, R., Zhong, R., Blumenkrantz, N., Chowdhury, S. K., and Alton, K. B. (2007c). Response normalized liquid chromatography nanospray ionization mass spectrometry. J. Am. Soc. Mass. Spectrom. 18 1891-1899. 

Ramanathan, R. et al., Early assessment of metabolites in clinical studies Potential applications of NanoSpray ionization mass spectrometry, in 10th Annual Symposium on Chemical and Pharmaceutical Structure Analysis, Langhome, PA, 2007. 

Nanospray ionization is a variation of regular ESI in that the typical flow rate is reduced to between 30 and 200 nL/min. Because of the very low flow rate at which samples are consumed, this technique is rapidly being integrated in many analytical applications including proteomics, metabolite characterization, and pharmaceutical analysis. An approach of combining fraction collection with automated chip-based NSI MS was recently introduced for metabolite identification (Hop, 2006 Staack et al., 2005). LC effluent was collected into a 96-well plate and the fraetions of interest were infused using an automated chip-based nanospray system for structure elucidation. 

TABLE 5.2 Experimental conditions for electrospray, micospray and nanospray ionization... 

Waters Corporation Synapt. The samples are solutions of biological material, and the ion sources are uniformly EIS, nanospray ionization, or matrix-assisted laser desorption and ionization as described in Chapters 4,9,15, and 18. Studies with the Synapt traveling wave instrument have revealed details of biomolecular ions in the gas phase that are not available by MS alone or by other methods. The full meaning of such studies and relevance for in vivo biomolecular activity is currently under discussion and debate " nonetheless, IM-MS for explorations of biomolecules certainly has affected the visibility of mobility as a measurement method and the level of technology that has been advanced through pharmaceutical and medical concerns. 

Two factors favor the use of nanoelectrospray ionization for coupling microfluidic devices to mass spectrometers. The first is the similarity between the conventional puUed-glass capillary tips and the nanospray nozzles developed for microdevices discussed above the second springs from the linear geometry of microfluidic channels. Thus, nanospray ionization techniques are probably the most likely to be used for the construction of robust interfaces between... 

The most common instrumentation for the analysis of biomarkers includes microbore and capillary reversed-phase chromatography coupled to a triplestage quadrupole (TSQ) mass spectrometer or ion trap, with an atmospheric pressure ionization source such as electrospray ionization (ESI), nanospray ionization (NSI), or atmospheric pressure chemical ionization (APCI). Ion trap mass spectrometers provide higher sensitivity in full-scan mode, which is useful for product ion identification of a metabolite however, TSQs are used most often due to improved sensitivity for quantification in multiple reaction... 

Another example where this problem can arise involves drug discovery requiring bioanalytical quantitation of drug metabolites in plasma when reference standards are not available. Two innovative approaches to this fairly common problem were proposed recently. The first of these approaches (Valaskovic 2006) exploits very low flow rate nanospray ionization that exhibited a distinct trend towards equimolar responses of similar compounds (in this case a set of well known drugs) when the flow rate was reduced from 25 to less than lOnL.min (see also Section 5.3.5b). A more uniform response between... 

Figure 11.35 Diagram illustrating a method for providing absolute quantitation of metabolites in incurred serum samples when no analytical standard is available. The method exploits the trend towards equimolar responses for a drug candidate and its metabolite at low flowrates ( 10 nL.min ) that permits measurement of the concentration ratio of metabolite to parent compound via the observed ratio of signals for the two using nanospray ionization (1 nano/Ip,nano)- However, use of a fast nonselective extraction procedure like protein precipitation for the LC-ESl-MS/MS assays is not suitable for the nanospray experiment since the extracts are too complex, so a more selective procedure, liquid-liquid extraction (LLE) in this case, must be used to prepare these extracts. The connection between the two procedures is achieved by analyzing the LLE extracts by both nanospray and the LC-ESI-MS method used for the assays of the incurred samples, yielding an LC-ESI-MS peak area ratio (A] lc/ms/- p,lc/ms) comparison with (iM.mmo/Ip.nano) that is interpreted as the concentration ratio. This comparison yields a calibration factor k ji = (lM,nano/Ip,nano)/(AM.LC/Ms/Ap Lc/Ms) that enables quantitation of the metabolite relative to that of the parent compound subsequently, absolute quantitation of the latter by conventional LC-MS using an analytical standard to prepare a calibration curve permits absolute quantitation of the metabolite also. Only a limited number of nanospray analyses (one in the example shown) is required to derive a k j value applicable to a complete set of study samples. Reproduced from Valaskovic, Rapid Commun. Mass Spectrom. 20, 1087 (2006), with permission of John Wiley Sons Ltd.

The flow rates are 70 pl/min (for Xbridge column) and 0.5 pi/ min (nanospray tips) with the gradient remaining same for both columns. Eluted peptides are suitable for either electrospray or nanospray ionization, respectively, for transferring peptides from... 

ESI is used at low flow rates (<50 pi min ), even down to the nl min level. In the latter case, this is referred to as nanospray. The ionization rate increases with reduced flow, which is the reason for using narrow-bore columns with nanospray ionization. 

The second trend to watch is miniaturization. This is advantageous not only because sample amount is often limited but also because performance may be improved, and running costs can be reduced (e.g., by using less chemicals). Small size also means that more equipment can be put into the (often-limited) laboratory space. The main limitation of miniaturization is sensitivity. In this respect MS, due to its sensitivity, is also invaluable. Luckily an important mass spectrometric technique, nanospray ionization, is ideally suited for coupling to nano-HPLC [34] (requires nl/min flow rates). This reduces sample requirement, and also facilitates coupling MS with electrophoretic techniques. 

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