49
Figure 3 Effect of flow rate on the elution profile obtained for benzoic acid on a polymeric SPE media.
samples where the flow rate has been intentionally altered to simulate this effect. It can be seen from the experiments performed that with the higher flow rate the analyte results in a greater level of breakthrough for the loading stage and that the amount of analyte that is eluted in the initial 100% methanol step is reduced, both of which have an effect on the effective recovery of the analyte.
This phenomenon is caused by the difference in time taken for the pressure driven flow compared to the time required for diffusion into the pores. Diffusion into the pore structure is required to initially capture the analyte of interest since this is where the majority of the surface area resides, thus at higher flow rates the compound simply does not get time to diffuse into the pore structure, and so analyte breakthrough is higher. During the elution part of the process the eluent is required to diffuse into the process to allow the analyte molecule to elute from the SPE media. If sufficient time is not given for this process to occur then the analyte molecule remains within the pore structure during that elution step. Robust assay development will take this effect into consideration, however the use of generic methodologies means that this is not always considered.
Chromatography
A chromatography column is designed to be used for multiple samples, and it is generally assumed for sample analysis that the chromatographic performance does not vary outside of specified performance criteria during the assay. However, it is evident that when using biological extracts that changes to the column are occuring, since the back pressure and chromatographic performance can alter throughout a batch of samples. The changes in back pressure and chromatographic performance are indicators that the surface of the column is changing and that interstitial space and/or frit porosity is being affected by matrix component build-up. Figure 4 demonstrates the effect of running a series of peptides, GSTAENAEYLR (GST), GSHQISLDNPYDQQDFFPK (GSH) and RPAGSVQNPVYHNQPLNPAPSR (RPAG) over a 6 hour period and the chromatographic deterioration that is observed. The chromatography was performed using a binary gradient from 10 - 40% of 0.025% tri-fluoroacetic acid (TFA) aq. and acetonitrile with 0.025% TFA over 10 minutes on a C18 based column. It can be seen that the peak shape deteriorates for all three components (GSH, RPAG and GST) and that there is a shift in the peak retention for one of the compounds as the stationary phase is modified.
In itself the deterioration of the stationary phase due to build-up of
Figure 4 The effect on chromatographic performance of running a column for 6 hours for 3 peptides.
matrix components is detrimental, however at least in the previous example there is an obvious effect that can be seen, and so it would be possible to troubleshoot the assay with a degree of confidence in the data. A different scenario exists however when considering components that are being injected onto the chromatographic system and are not being detected, such as non-ionisable compounds, or compounds with low ionisation efficiencies under the source parameter settings. For most bioanalytical assays this is the majority of the extracted sample, with phospholipids being a good example of compounds that are not routinely detected but which can have a potential effect on the mass spectrometry. Since the elution of these components of the extracted sample are not monitored, the chromatography will not be optimised, which can result in matrix component not eluting during a single chromatographic run.
Figure 5
Normalised detector response for phospholipids in ten water injections subsequent to an injection from a protein precipitated extract. Phospholipid m/z transistion labeled.
Figure 5 demonstrates this effect for a protein precipitated sample. Five phospholipid components are monitored, 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, 1-stearoyl- 2-hydroxy-sn-glycero-3-phosphocholine, glycerophosphocholine lipid, 1-hexadecanoyl-2-(9Z, 12Z–octadecadienoyl)-sn-glycero- 3-phosphocholine and 1-(9Z, 12Z–octadecadienoyl)-2-(5Z, 8Z, 11Z, 14Z–eicosatetraenoyl)-sn-glycero-3-phosphocholines. All of these compounds have the same phosphocholine daughter group which has a characteristic mass of 184.3, with the parent masses being; 496.4, 524.4, 704.4, 758.4 and 806.4 respectively. The chromatography has been described earlier in association with the data obtained for Figure 1. It can be clearly seen that the
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56
