25 Table 1. Compound list/Cuttoff concentrations
Figure 2. Extracted ion chromatograms of multiple drugs including NPS
Typically, scanning across the full mass range provides the fi ne spectral details of the precursor ions of the analytes. After coupling to a quadrupole mass fi lter, such a mass spectrometer (Quadrupole-TOF or QTOF) can provide the full-scan information of not only the precursors ions, but also all the product ions in very high resolving power. Modern QTOF systems provide the capability of switching between MS and MS/MS scans instantly, enabling structural information to be obtained very quickly.
The analytical experiment to acquire the data was MS/MSAll with. In this technique’s data cycle, the instrument begins by acquiring the TOF-MS information fi rst, and then sequentially follows that by acquiring the MS/MS information of all precursor ions across a specifi ed mass range in pre-divided Q1 mass isolation windows. SWATH®
acquisition records MS/MS information of everything
With any drug screen, the time it takes to get a result is critical and in this paper the development of a robust LC confi guration and gradient was key to getting accurate and confi dent data. The HPLC separation was performed with a Phenomenex Synergi Hydro-RP column (20 × 2 mm, 2.5 µm). Mobile phase A was 10 mM ammonium formate in water and mobile phase B was 0.1% (v/v) formic acid in methanol. The image (Figure 1) details the gradient conditions that were employed in this study.
Prior to the development of this method a 2-minute LC method [5] had been created using a Phenomenex Kinetex Phenyl-Hexyl column 50 × 2.1mm, 2.6µm 00B-4495-E0, however to improve the retention of the polar species the Hydro RP column was selected and the LC runtime was extended by 0.5 minute. Ensuring suffi cient retention of the very polar species was important to allow the diversion of the salt-containing eluates in the beginning of the gradient to waste. The aim of refi ning the LC conditions was to evenly distribute all the eluting analytes throughout the duration of the data acquisition window to reduce the number of co-eluting analytes thus minimising ion suppression and matrix effects.
To preserve the column lifetime an organic wash was performed at the end of the gradient run followed by aqueous re-equilibration so retention time reproducibility was maintained. Within the 2.5 min LC runtime several groups of isomers could be resolved, in Figure 2 Morphine at 0.47 mins, hydromorphone at 0.98 mins, norcodeine at 1.08 mins were shown in Inset A, oxymorphone at 0.46 min and noroxycodone at 1.14 min were shown in Inset B, and amitriptyline at 1.54 min and EDDP at 1.65 min were shown in Inset C.
In this study the LC was coupled with the Sciex X500R QTOF system, a high-resolution quadrupole time-of-fl ight (QTOF) mass spectrometer. The X500R is suited for screening applications because the data generated from these systems provides structural information for every possible analyte.
all the time, and it signifi cantly improves the MS/MS data quality by allowing sequentially programed Q1 isolations therefore more selective MS/MS data collection compared to other MS/MSAll techniques. Figure 3 shows the principal behind this acquisition experiment and Figure 4 details the settings for this acquisition on the X500R QTOF system.
Figure 3. Principle of the analytical technique.
Analysis of the data at spiking level 1 shows clear resolution and separation with confi dent identifi cation. Figure 5 shows the extracted ion chromatograms of selected forensic analytes in urine at spiking level 1.
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