Online restricted access media with liquid chromatography and tandem mass spectrometry for the direct analysis of small molecules in biological fluids represents an interesting alternative to time-demanding traditional sample preparation techniques. In this study, important considerations concerning the development of a restricted access media with liquid chromatography and tandem mass spectrometry method for the analysis of dansylated estrogens in biological matrix are presented. Parameters influencing peak tailing and trapping efficiency were evaluated. The key factors included the ion strength of the mobile phase, a loading flow rate of the sample onto the trap column, and selection of a proper stationary phase of the trap column for a given set of analytes. These parameters have proven to be essential for minimizing any unwanted chromatographic peak tailing. The bulk derivatization of the analytes in the biological fluids and its relationship to the observed matrix effects was evaluated as well.
5 Figures and Tables
Figure 1. Diagram of valve positions in the instrumental set-up for analysis of estrogens by trap-and-elute LC–MS/MS.
Table 1. LC time program of the gradient analysis including valve positions
Figure 4. The effect of the different stationary phases of trap columns (C18, C8, C4) upon peak asymmetry and sensitivity of the detection. Panels B, D, and F show the analyte bands eluted from the RAM without the analytical column present in the system.
Figure 5. Evaluation of matrix effects in (A) unstripped CSF, (B) PBS-BSA, and (C) lipid-free PBS-BSA.
Figure 6. Comparison of peak areas of E1*, E2*, E2*, and E3* when BSA was added before derivatization, before derivatization with triple the amount of DNS, and after derivatization.
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