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The aim of refining 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.
Figure 1 – LC Gradient conditions
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.
Figure 2 Extracted ion chromatograms of multiple drugs including NPS
this, the NPS may increase potency and alter the side effects of the designer drug. Once the NPS has been modified, it becomes difficult if not impossible to analyse.
In this study 85 analytes were selected which would challenge chromatographic separation and test the instrument to detect and screen the compounds in an ultra-fast time frame. Although in a real-life case it would be highly unlikely that any sample would contain this many forensic drugs.
Experimental
In a recent application note ‘Ultra-Fast Forensic Toxicological Screening and Quantitation under 3 Minutes using Sciex X500R QTOF System and Sciex OS 1.0 Software’ [4] novel psychoactive substances were addressed in the study of urine samples. Twenty samples were diluted in 10% methanol and centrifuged to reduce the possibility of matrix interference. The clear supernatants were transferred to autosampler vials which were spiked with 85 compounds including opioids and synthetic cannabinoids. The urine samples contained a calibrator set
spiked with compounds described in Table 1. There are 7 levels of calibrators: 40%, 80%, 100%, 200%, 300%, 500% and 1000% which are against the ng/mL value of each compound in table one, for example for the compound of 6-MAM, a metabolite of heroin, the calibration range was 4ng/ mL to 100ng/mL. Using such a calibration range enable low level detection, sub cut off level through to a high level of concentration at 100ppb. There were also 20 urine samples with unknown number of compounds. The injection volume was 5 µL with a dilution factor for this sample set was a x4, again this reduces matrix interferences and the need for instrument maintenance.
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 sufficient 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.
In this study the LC was coupled with the Sciex X500R QTOF system, a high- resolution quadrupole time-of-flight (QTOF) mass spectrometer. The X500R is suited for screening applications because the data generated from these systems provides structural information for every possible analyte. Typically, scanning across the full mass range provides the fine spectral details of the precursor ions of the analytes. After coupling to a quadrupole mass filter, 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
data cycle, the instrument begins by acquiring the TOF-MS information first, and then sequentially follows that by acquiring the MS/MS information of all precursor ions across a specified mass range in pre-divided Q1 mass isolation windows. SWATH® acquisition records MS/MS information of everything all the
with. In this technique’s
Figure 3 Principle of the analytical technique.
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