Scan, data-dependent

Figure 12.16 Potential dependent SFG spectra (a) and the Stark tuning plot (b) from chemisorbed CO on Pt nanoparticles in a CO-saturated 0.1 M H2SO4 electrolyte. Each spectrum was acquired for 10 s (forward scan data only are shown). The potential was scanned from — 0.20 to 0.70 V (vs. Ag/AgCl) at 1 mV/s. Pt nanoparticles were of approximately 7 nm size, and were immobilized on an Au disk.

MS/MS Duty Cycle Typical MS/MS analysis is a serial process, relying on the selection of precursors (peptides) in MS mode, followed by high-energy fragmentation in MS/MS. This process is termed data dependent acquisition (DDA). The duty cycle for the completion of MS and MS/MS cycles (the time necessary for MS/MS spectrum acquisition) is of primary importance. When the separation performance is viewed from the mass spectrometry perspective, the peak capacity can be characterized by the number of MS/MS scans, yielding successful... 

Data-dependent acquisition ability has been developed and incorporated into most software packages [MetaboLynx, Xcalibur, and Analyst Information Dependent Acquisition (IDA)]. In data-dependent acquisition mode, a mass spectrometer decides on the fly whether to collect MS/MS or MSn data, remain in full scan MS mode, or conduct other survey scans based upon user-defined criteria. Product ion spectra of potential metabolites can be automatically acquired in a single LC/ MS run. However, false positives may be generated due to highly intense matrix ion signals that may inadvertently trigger MS/MS or MSn scan functions. 

The major advantage of this instrument is that qualitative and quantitative analysis can be performed in the same LC-MS run. As an example in a data-dependent experiment, the selected reaction monitoring mode can be used as a survey scan and the enhanced product ion mode (EPI) as a dependent scan. The consequence is that for each quantified analyte a confirmatory MS/MS spectmm can be obtained. 

When working with non-radiolabeled drugs the major challenge is to find metabolites in the biological matrices. Because the enzymes responsible for metabolism are quite well characterized metabolic changes can partially be predicted. For example hydroxylation of the parent drug is in many cases the principal metabolic pathway. From a mass spectrometric point of view it results in an increase of 16 units in the mass spectrum. In the full-scan mode an extracted ion current profile can be used to screen for potential metabolites. In a second step a product ion spectrum is recorded for structural interpretation. Ideally, one would like to obtain relative molecular mass information and the corresponding product ion spectrum in the same LC-MS run. This information can be obtained by data dependant acquisition (DDA), as illustrated in Fig. 1.39. 

In this case the survey scan was set as a full scan and the dependent scan as a product ion scan. The problem with data dependent acquisition is to determine the selection criteria. In most cases the system picks up the most abundant ion in the full scan spectrum. An inclusion list with masses of potential metabolites or exclusion list of known interferences significantly improves the procedure. In the example shown in Fig. 1.39, a procedure called dynamic background subtraction (DBS) was applied. This procedure considers chromatographic peak shapes and monitors not the most abundant signal in the spectrum but the largest increase of an ion in a spectrum. The advantage is that once a signal of a peak has... 

Fig. 1.39 LC-MS data dependent analysis of vinpocetin in rat urine using dynamic background substraction (DBS) on a triple quadrupole linear ion trap. (A) Full scan MS (survey scan) trace. (B) Enhanced product ion scan (dependent scan). The major peak at 3.9 min corresponds to apovinpocetin, the minor one at 2.9 min to the hydroxylation product of apovinpocetin (m/z 339).

Depending on the manufacturer, it may be possible to process the scan data for viewing and deriving bone metric analysis on the scan unit itself (see Note 11). In the event that a viewing or a bone analysis package is locally unavailable, several commercially available software packages are offered for download (see Note 12). 

Figure 5.1. Full-scan MS and data-dependent MS/MS spectra of verapamil generated from a microsomal incubation sample.

Table 5.2 shows a representative set of accurate mass measurements of 50 pharmaceutical compounds which were acquired using an eight-scan event experiment on the LTQ-FTICR (Josephs et al., 2004b). Mass spectra from all eight scan events were acquired within 7 s, the width (at the base) of a typical chromatographic peak, as shown in Fig. 5.3 for warfarin (MW 308). The first scan event is a full-scan positive-ion MS acquired in the ion trap. If the expected protonated molecule is detected above a preset threshold, a second scan event, an ion trap (IT) data-dependent MS/MS scan, is triggered. This is followed by a third scan event, where the protonated molecule is accurately measured in the FT, and a fourth scan event, a full-scan MS/MS in the FT. 

After all four positive scans are completed (typically within 3 s), the polarity is switched and the fifth scan event records a negative-ion full-scan MS. If the expected protonated molecule is not detected in the positive mode, the second, third and fourth scan events are skipped and the fifth (negative-ion-mode) scan event is triggered. Similar to the positive-ion mode, if the expected [M-H]- ion is detected in the full-scan MS, IT data-dependent MS/MS (sixth), FT accurate MS (seventh), and FT MS/MS (eighth) scan events are acquired. Clearly demonstrated here is the ability of the LTQ-FT to handle multiple experiments on a chromatographic time scale. One might question the need for such an elaborate data-dependent scheme when apparently all that is needed is an accurate mass determination followed by a data-dependent accurate mass MS/MS spectmm. Apart from the fact that using the... 

TABLE 5.2. Accurate Mass Measurements Gathered in an 8-scan Event Data Dependant LC-MS Experiment... 

Figure 5.3. Warfarin mass spectra from eight-scan event data-dependent experiment gathered on a chromatographic scale (7 s wide at peak base). Fragments detected with accuracy <2 ppm.

Triolo, A., Altamura, M., Dimoulas, T., Guidi, A., Lecci, A., and Tramontana, M. (2005). In vivo metabolite detection and identification in drug discovery via LC-MS/MS with data-dependent scanning and postacquisition data mining. J. Mass Spectrom. 40 1572-1582. 

Data-dependent software programs allow real-time decisions to be made during an analysis. This approach features a preestablished threshold for the detection of a peak during full-scan mass spectrometry and MS/MS scan modes. If a peak of interest is detected in real-time, then the mass spectrometer is switched from full-scan mode to another scan mode to obtain more information from the same analysis. For example, the system may be automatically switched to the production mode during the analysis of a chromatographic peak to obtain substructural information. Thus, more detailed information is obtained in fewer analyses. This powerful... 

The energy spectrum of constant angle, polychromatic coherent scatter may be transformed with the help of Eq. 3 into a diffraction profile depending on the momentum transfer, x, variable. The position of the active X-ray focus yields one spatial coordinate (y position) of the voxel irradiated for the 2-D scan data. This is complemented by the X voxel coordinate (= RP cos(0)) derived from the Z detector coordinate. The third spatial dimension of the scattering volume in the object under investigation in the Z direction is known from the position of the conveyor belt on which the object is translated and... 

SCAN gives a printout or summary of the quality of the data in a contig. The quality of the data depends on the number of times it has been sequenced and the particular uncertainty codes used in each gel reading. It divides the data into five categories and the quality codes are described below ... 

---