Postcolumn infusion

Shou, W. Z. Naidong, W. Postcolumn infusion study of the dosing vehicle effect in the liquid chromatography/tandem mass spectrometric analysis of discovery pharmacokinetic samples. Rapid Commun Mass Spectrom 2003, 17, 589-597. 

Alternatively, matrix effects can be evaluated using postcolumn infusion methods described in detail in this chapter and elsewhere (King et al., 2000 Weng and Halls, 2002 Mei, 2005). 

As shown in Fig. 1.7, the method for evaluating ion suppression/enhancement encountered during a bioanalytical assay involves injection of a processed blank matrix sample on the column with continuous postcolumn infusion of a mixture of an analyte and an internal standard into the LC stream. The analyte and the internal standard are monitored (MRM or SRM scan) throughout the entire LC ran time while the matrix components are eluting from the column. Data from a matrix effect experiment obtained using the postcolumn addition method are given in Fig. 1.8. 

Figure 1.7. Postcolumn infusion method to evaluate matrix effect originally described by Bonfiglio et al. (1999) and King et al. (2000). (Reprinted with permission from Bakhtiar and Majumdar, 2007.)...

Tolonen et al. [167] described a simple and efficient method for determination of labile protons in drug metabolites using postcolumn infusion of deuterium oxide in LC/MS experiments with ESI and TOF-MS. The number of exchangeable protons in analytes hydroxyl, amine, thiol, and carboxylic acid protons can easily be determined by comparing the increase in m/z values after H/D-exchange occurring on line between... 

Fig. 6.5 Diagram of system set-up for postcolumn infusion test for matrix effect. The analyte in the mobile phase was infused by a syringe pump at about 10 pL/min. The blank matrix extract or the test control (mobile phase or water blank extract) was injected into the analytical column. The effluent from the analytical chromatographic column was mixed...

Fig. 6 Profile of postcolumn infusion of carvedilol with an injection of control plasma extract (lot 3, the problematic lot) overlaid with the LC-MS/MS chromatograms of carvedilol and its deuterated internal standard (D5-carvediol) to demonstrate a significant difference in ion suppression (-25 %) due to even a very small difference in retention time (0.02 min) between carvediolol-S (1.93 min) and its deuterated internal standard (1.91 min). Reproduced from ref. [35] with permission from Elsevier...

To avoid the expensive use of deuterated solvents for H/D exchange experiments, Tolonen et al. [21] have described the postcolumn infusion of D2O to facilitate the LC-MS detection and identification of labile protons in a column eluant. Whilst acknowledging the potential limitations with respect to a reduced level of exchange, and hence sensitivity, compared to the use of deuterated mobile-phase solvents, they optimized the column effluent flow rate (via a splitting connector) with the infused D2O flow rate to enable the very useful determination of up to four labile protons. The method was exemplified by the differentiation of hydroxylated metabolites of the alkaloidal drugs imipramine and omeprazole (Figure 13.5) from the N-oxide and sulfone metabolites, respectively [21]. This was a differentiation that could not be achieved by high-resolution mass measurements. 

Figure 7-14 Postcolumn infusion system. Mobile phase or specimen extracts are injected into the HPLC system,The analyte being evaluated is continuously infused, postcoiumn, and is mixed with the column effluent through a tee before entering the electrospray interface.

FIGURE 11.6 Comparison of infusion chromatograms generated with (A) APCI, and (B) ESI. The matrix in this case was dog plasma prepared by protein precipitation. Analysis was performed by isocratic LC-MS/MS with a 2.1 x 50-mm Zorbax SB-C18 column at a flowrate of 0.5 mL/min (APCI) or 0.25 mL/min (ESI). Postcolumn infusion of urapidil was adjusted to achieve similar analyte delivery to each source. Detection of urapidil occurred by SRM. (Reprinted from King et al. [19], with permission from Elsevier Science, Inc.)... 

Fig. 3 Schematic representation of continuous infusion of an internal standard and postcolumn (postelution) mixing with LC eluent prior to being directed to a mass spectrometer (MS) for detection...

A second protocol involves postcolumn continuous infusion of compound into the MS detector.The instrumental setup includes a syringe pump connected via a tee to the column effluent (Figure 7-14). Because the compound being tested is introduced into the mass detector at a constant rate, a constant electrospray ionization response should... 

The frequency of ion suppression or other deleterious effects can be evaluated by different experimental methods. The most common method is the comparison of the instrument response for an injected sample prepared in the mobile phase to the same concentration added to a preextracted blank sample matrix. The second method involves postcolumn continuous infusion of the compound into the MS instrument In the third method, standard line slope comparison is also used to evaluate ion suppres-sion. FuU-scan mass spectra can also be a useful method to study what impurities (in the blank of extracted sample matrix or in the LC mobile phase) are responsible for ion suppression and to try to remove these compounds. 

Figure 5.19 Effect of HPLC injections on the APCI signal from the analyte infused post-column at a constant rate the isocratic mobile phase was 80 % acetonitrile and 20 % aqueous ammonium acetate (1 % w/v), and the analyte solution that was infused postcolumn was 1 p.g.mL in the mobile phase, (a) Infusion of parent compound with no injection on the HPLC (mobile phase only) (b) enhancement effect of injection of water on the signal from infused analyte (c) suppression effect of injection of extract of a blank plasma sample on the signal from the infused analyte (see Section 5.3.5a). The diagonal line is drawn to connect the injection times in the different chromatograms. Reproduced from Sangster (2004), Rapid Commun. Mass Spectrom. 18, 1361, with permission of John Wiley Sons, Ltd.

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