ESI response

Analytical methods for the detection of residues of semicarbazide use derivatisation with 2-nitrobenzaldehyde and LC-MS detection. Figure 18 shows the positive ESI response for a 1 ppm solution of semicarbazide after derivatisation and concentration. The main peak 2 at 16 min shows the expected 209 (M+H)+ ion of the 2-nitrobenzaldehyde derivative of semicarbazide together with its sodium adduct ion at m/z 231 (Figure 19). 

Amad, M.H. Cech, N.B. Jackson, G.S. Enke, C.G. Importance of Gas-Phase Proton Affinities in Determining the ESI Response for Analytes and Solvents. J. Mass Spectrom. 2000, 35, 784-789. 

FIGURE 6 Effects of soivents and additives on ESI response ofTyr-Gly-Gly-Phe-Leu (Leucine Enkephaiin) (courtesy ofThermo Finnigan). 

It is of primary importance to understand how the chemical-physical properties of a given compound can influence its response in ESI-MS. In fact, ESI response can vary significantly among different analytes, depending not only on their concentration, but also on their properties [29-33],... 

Other studies demonstrated that ESI response can be highly suppressed if the real samples do not undergo a pretreatment step, indicating the importance of this procedure in limiting matrix effects [36],... 

As envisioned previously, ESI response is concentration-dependent. A linear response versus concentration is up to the maximum concentration of about 10 M. When analyte concentration exceeds this limit, the ESI response levels off. This is because ESI intensity is proportional to the surface concentration of an ion. At about lO M, the droplet surface is completely saturated and higher concentration will not increase the total number of surface charges available for ion formation [25]. This will impact the high concentration end of quantitation using LC/ESl-MS. For the low concentration end, the detection limit depends on the sensitivity of LC/MS system, including efficient ion transfer/detection and removal of chemical noise in the system. 

In order to optimize the LC/MS/MS system, the authors investigated the effects of methanol content, ammonium acetate concentration, and the percentage of acetic/formic acids in the mobile phase on the ESI response (m/z 744 to 495 transition) (Figure 7-19). They found that the best ESI response was obtained at -80% (v/v) of methanol (Figure 7-20A).They also discovered that the ammonium acetate (5 mM) could be applied as a buffer in the mobile phase to achieve better reproducible separation between ET-743 and the internal standard. It is well known that the addition of acetic acid or formic acid in the mobile phase can suppresse the ionization of residual silanols on silica-based reversed-phase columns for LC/MS analysis (Figure 7-19C). In positive ion mode, however, the acids can form an ion pair with the MH+... 

M.H. Amad, N.B. Cech, G.S. Jackson, C.G. Enke, Importance of gas-phase proton affinities in determining the ESI response for analytes and solvents, J. Mass Spectrom., 35 (2000) 784. 

N.B. Cech, C.G. Enke, Relating ESI response to non-polar character of small peptides. Anal. Chem., 72 (2000) 2717. 

R. Bonfiglio, R.C. King, T.V. Olah, K. Merckle, The effects of sample preparation methods on the variability of ESI response for model drug compounds. Rapid Commun. Mass Spectrom., 13 (1999) 1175. 

The most widely applied LC method for the separation of peptides and proteins is reversed-phase LC (RPLC). A typical mobile-phase composition is an acetonitrile-water gradient with a fixed concentration of TFA (typically 0.05-0.5%). TEA acts as an ion-pairing agent enhancing the retention of peptides and proteins, but also masks secondary interactions with the silica-based stationary phase. TFA is a volatile additive, but due to its ion-pairing properties and effect on the surface tension, it may significantly suppress the ESI response in positive-ion mode. 

It is widely recognized that sensitivity in either ESI or APCI is improved as the percentage of organic modifier is increased, due to the improved facility for desolvation. Adequate reverse-phase retention (/< = 1 to 5) is therefore important when LC-MS is performed. Temesi and Law studied the effect of LC mobile-phase composition on ESI response for a series of 35 compounds [18]. In their investigation, the signal response for positive-ion ESI was on average 10-20% higher in MeOH than ACN. 

Hop et al. [56,59] demonstrated that ESI response factors could be normalized using nanoelectrospray. In this report, 25 compounds from 6 structurally distinct classes were tested using silicon chip-based nanoelectrospray devices (NanoMate) with <300 nL/min flow rate. The MS responses between these 25 compounds were relatively consistent (within 2.2-fold). This can be compared to standard LC-MS data which would have a 21-fold difference in response therefore, it was a big improvement. 

Currently, it is generally believed that the IDM model applies to ions with significant surface activity (i.e., hydrophobic molecules) and the CRM model applies largely to hydrophilic species. Thus, proteins and metal cations may follow CRM, whereas peptide and fatty acids follow IDM. As a consequence, the ESI response depends on the nature of the analyte. Enke has put forward an... 

The development of quantitative MS/MS methods is hard, not only due to the lack of conunercial analytical standards but also because other difficulties must be considered, such as the low general solubility of PFASs in common LC solvents and their tendency to form micelles, the adhesion of the PFASs to labware and instruments, the difficulty of separating polymeric PFASs in conventional LC columns, and the ability of PFASs to form aggregate and adducts that influence ESI responses [38]. 

Figure 5.32 Typical ESI calibration curve generated for a concentration range covering six ordars of magnitude. The analyte was the surface active trimethyldecylammonium cation, dissolved in 50 50 water methanol containing 0.5 % acetic acid. If response (observed signal intensity) is directly proportional to concentration, such a log-log plot should have a slope of 1.0, and this is indeed observed over the concentration range 10 -10 mol.L . Positive deviations from linearity at the low end are attributed to chemical background interference, and negative deviations at the high end to saturation in ESI response. Reproduced from Cech and Enke, Mass Spectrom. Revs. 20, 362 (2001), with permission of John Wiley Sons, Ltd.

The first calibration is performed on a racemic sample, to determine the correction factor q accounting for differences in ESI response between compounds differing by a methyl group. A second calibration is carried out with a sample of alcohol or amine of known enantiomeric excess, which allows the determination of the selectivity factor s (the ratio kfast/ sW = f/ s) through the following equation ... 

---