17
These transitions were selected from the characteristic fragmentation behaviour observed from full-scan CID mass spectra from these target compounds (Figure 4). The infusion ion current signal area ratios between amlodipine and its D4 internal standard were plotted against the concentration of the standards. Typically the data acquisition for each sample was for 30 seconds and a 20 second portion of the ion current from the analyte and the internal standard, for each sample, were calculated as a ratio and plotted against the concentration for each standard and QC sample. The TriVersa NanoMate spray voltage was 1.65 kV with a head pressure of 0.3 psi on the sample extract contained within the pipette tip.
Discussion
Infusion nanoESI analysis of amlodipine in fortified dried blood spots.
Figure 2. Photograph of DBS card holder showing the four PEEK ferrules which form a boundary region when the cover plate is closed tightly onto a DBS card.
Figure 4 shows the full-scan collision-induced dissociation (CID) mass spectrum for D4 amlodipine (Figure 4a) and amlodipine (Figure 4b). These data were obtained via infusion nanoESI analysis of analytical standards of each compound using an AB SCIEX 4000 QTRAP mass spectrometer operated in the positive ion mode. In these infusion experiments, unlike LC/MS analyses, the analyte ion current signal is constant and allows signal averaging which can be beneficial when very low levels of target analytes are present.
The individual steps involved in using the TriVersa NanoMate platform for liquid extraction-surface sampling and subsequent infusion nanoESI-MS have been recently described in the literature [11]. In general, this includes an initial step where the robotic arm picks up a conductive pipette tip and moves the tip to the extraction solvent reservoir in order to pick up a 1-10 microliter aliquot of the extraction/spray solvent. Then, the pipette tip is positioned above the surface to form a micro liquid droplet maintained between the pipette tip and the sample surface. A sequence of dispense-aspirate steps affects an extraction of the sample surface whereupon the pipette tip is moved by the robot to the inlet of the microfabricated ESI chip and infusion nanoESI mass spectral acquisition is achieved employing either positive or negative ion detection.
Essential to the above approach of liquid extraction surface analysis is the formation of the micro liquid droplet maintained between the pipette tip and the sample surface such as a dried blood spot (DBS).
Figure 3. Photograph of a DBS card containing applied spots of whole blood. In this version stainless steel ferrules are employed to form the boundary region on the DBS card when the cover is clamped closed.
Mass spectrometry experimental conditions
An AB Sciex 4000 QTRAP tandem triple quadrupole mass spectrometer (Concord, Ontario,Canada L4K 4V8) was equipped with a TriVersa NanoMate (Advion BioSciences, Inc, Ithaca, NY 14850), which had been upgraded to LESA capabilities. The upgrade involved software changes that allow the conventional disposable pipette tip to access a special solvent reservoir affixed at one end of the platform. The DBS card holder described above had the same footprint as an ANSI 96-well plate so it could be placed directly into the sample holder of the platform stage of the system. The X,Y,Z coordinates for the four ferrule regions of the card holder were programmed into the TriVersa NanoMate to allow sequential sampling access to each dried blood spot sample. The 4000 QTRAP mass spectrometer was operated in positive ion nanoelectrospray SRM mode (approximately 200 nL/min infusion flow rate and the DBS sample typically extracted in 7 microliters of solvent). The transitions monitored included m/z 409.2 > m/z 237.9 for amlodipine and m/z 413.3 > m/z 238.2 for D4 amlodipine, respectively, with a dwell time of 50 msec. for each transition and employing nitrogen as the collision gas.
If the sample surface is highly absorptive, the liquid disperses away from the pipette tip and cannot be retrieved for subsequent infusion mass spectrometry. The purpose of the DBS sample card holder described in this report (Figure 2) is to create a defined region formed by the perimeter of the tapered stainless steel or PEEK ferrules to allow confinement and recovery of the extract.
Introduction of the extraction/spray solvent into the DBS card holder reservoir (Figure 5a) produces a small pool of solvent which is intended to extract sample of interest contained on the surface (Figure 5b).
The diameter of the DBS surface extraction region in this work was 3mm and defined by the perimeter of the ferrules located in the hinged cover of the DBS card holder.
Figure 5a). CAD drawing showing a pipette tip inserted into a PEEK ferrule of the DBS card holder to extract the first dried spot sample.
b). Cross section CAD drawing showing the pipette tip inserted into the PEEK ferrule to a location just above the surface of the DBS spot while dispensing the extraction solvent to the confined region within the perimeter of the PEEK ferrule.
Figure 4. Collision-induced dissociation mass spectra for tetradeuterated amolipine (upper panel) and amlodipine (lower panel).
Figure 6. Calibration curve for the infusion SRM MS analysis of fortified whole blood samples using the device shown in Figure 5AB ranging from10 ng/mL to 10,000 ng/mL.
INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2012
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