Optimized evolution of a toxicology laboratory from immunoassay to the SCIEX X500R QTOF System
Oscar G. Cabrices1, Dean Fritch2, Melanie Stauffer2, Nadine Koenig2, Derrick Shollenberger2, Jennifer Gilman2, and Adrian M. Taylor3
1SCIEX, USA; 2Health Network Laboratories, USA; 3SCIEX, Canada
The rapid growth of novel psychoactive substances (NPS), like synthetic cannabinoids, bath salts and novel opioid analogs has required the NSC-ADID to updated its list of target analytes for impaired driving and motor vehicle fatality forensic testing. GC-MS and immunoassay are common approaches but there is growing understanding of the need for LC-MS testing, for Tier II compounds. A comprehensive drug screening workflow for the analysis of forensic DUID blood samples has been successfully developed using the SCIEX X500R QTOF System based on the new NSC-ADID recommendations. Using substantially less blood sample, it is demonstrated that the LC-MS assay can efficiently detect up to 60 key forensic compounds in a single assay.
Over the past decade, the National Safety Council’s Alcohol, Drugs and Impairment Division (NSC-ADID) started an initiative to standardize forensic toxicology laboratory testing for cases involving driving under the influence of drugs (DUID).Â
Target forensic compounds of interest were divided into two tiers: Tier I drugs include the most frequently encountered drugs found in DUID casework, and those which could be screened and confirmed with commercially available immunoassay and GC/MS instrumentation. Tier II analytes were those that had limited occurrence or required more advanced instrumentation such as LC/MS-MS, which is typically not readily available in every forensic laboratory.
More recently, the NSC-ADID made further changes on the list of target analytes for impaired driving and motor vehicle fatality forensic testing, due to recent advances in analytical technology and rapidly growing of novel psychoactive substances (NPS), like synthetic cannabinoids, bath salts and novel opioid analogs1.Â
In this technical note, a comprehensive drug screening workflow for the analysis of forensic DUID blood samples is described. The methodology was developed using a simplified sample preparation approach in combination with the SCIEX X500R QTOF System following the new NSC-ADID recommendations for forensic testing in DUID and motor vehicle fatality cases.
Sample preparation: Control whole blood samples were spiked with a stock standard solution mixture containing all the different drugs for initial method development. A detailed list of the forensic compounds targeted, including accurate mass information and limits of detection (LOD) used for this screening are detailed on Supplement Information of this technical document. Forensic DUID case samples and controls were extracted for LC/MS screening using the following protocol.2
Liquid Chromatography: HPLC separation was performed at 30 °C on a Phenomenex Kinetex Phenyl-Hexyl column (50 × 2.1 mm, 2.6µm) on the SCIEX ExionLCâ„¢ AC System using the following conditions: Mobile Phase A: 10 mM Ammonium Acetate in H2O:ACN (90:10). Mobile Phase B: 10 mM Ammonium Acetate in ACN:H2O (90:10) plus 0.1% Formic Acid. LC separation conditions are detailed below: Â
Mass spectrometry and data analysis: MS and MS/MS data were collected using the SCIEX X500R QTOF System. For all positive ionizable compounds: Information Dependent Acquisition (IDA) was used. On the other hand, for the negative ionizable target compounds: MRMHR workflow with the Apply TOF start/stop mass feature was used. Both screening strategies began with a TOF MS experiment. Detailed acquisition parameters are displayed in Figure 4.
Targeted data processing was performed using SCIEX OS Software for positive analyte identification based on previously determined criteria. Four main confidence criteria were used including mass error, retention time, isotope ratio difference, and library score. Subsequently, a combined score was computed based on these four confidence categories with custom weightings.
The vMethod Application for 664 forensic compounds3 was initially used to obtain retention times and MS/MS spectra quality to build a data analysis processing method for the 60 target forensic compounds of interest.
Two different acquisition strategies were utilized to streamline the screening workflow. For all positive ionizable compounds IDA-MS/MS was chosen as the acquisition mode, as it enabled the easy collection of precursor ions, and multiple dependent MS/MS scans on several of the most abundant precursor/candidate ions. Subsequently resulting in IDA-MS/MS spectra free of interfering species leading to accurate MS/MS library spectral matching.
Figure 5 displays XIC chromatograms showing the detection of all target compounds analyzed with both positive and negative electrospray ionization modes in control blood samples spiked at the LODs, based on the latest NSC-ADID recommendations.1
Throughout the method development process, it was important to obtain high combined scores for all compounds based on the four main confidence criteria defined in the processing method. Additional qualification criteria were implemented by setting an analyte concentration threshold based on the LODs to minimize false positives and/or false negative hits. Figure 6 shows the successful detection of 6-MAM and Fentanyl at their corresponding LODs, with mass errors less than 2ppm and MS/MS scores over 90%.Â
Throughout the method development process, it was important to obtain high combined scores for all compounds based on the four main confidence criteria defined in the processing method. Additional qualification criteria were implemented by setting an analyte concentration threshold based on the LODs to minimize false positives and/or false negative hits. Figure 6 shows the successful detection of 6-MAM and Fentanyl at their corresponding LODs, with mass errors less than 2ppm and MS/MS scores over 90%.Â
Table 1 shows the average (n=9) combined scores obtained for all 55 target compounds, in control blood samples spiked at the LOD analyzed over the course of 3 days. Inter-day reproducibility resulted in %RSDs ranging between 1-10% for the target analytes.
It was found that THC-COOH had sufficient S/N ratios (> 200) and mass error less than 1 ppm at the LOD (10 ng/mL) for positive identification. However, low-abundance MS/MS spectra were obtained at that concentration level, subsequently resulting in an average combined score of 70%. Further optimization on the sample extraction protocol is recommended to enhance THC-COOH sensitivity and MS/MS fragmentation.
One of the principal goals of developing this comprehensive analysis workflow was to successfully migrate the current immunoassay approach to the SCIEX X500R QTOF System. The current immunoassay sample preparation and analysis workflow utilizes 1mL of forensic blood sample and 2mL of acetonitrile for extraction, whereas with the QTOF MS strategy the laboratory was able to reduce the sample size to 100 µL while still meeting the NSC-ADID recommended cutoffs.
The ability of meeting these cutoffs with minimal sample is ideal, as often forensic case samples are limited in volume. Additionally, it eliminates the laboratory’s need for using multiple reagent kits (9 kits currently utilized) as the QTOF MS approach provides the enhanced selectivity and sensitivity to streamline the detection of Tier I and Tier II compounds.
As part of the implementation plan, 30 forensic DUID case samples were screened with both immunoassay and QTOF MS for results comparison.
Table 2 shows all compounds detected in the 30 forensic DUID samples examined with both immunoassay analyzer and the SCIEX X500R QTOF system. Compounds highlighted in green were specifically detected using QTOF MS but missed or classified as a single compound class (e.g., OPI for Opiates and metabolites) by the immunoassay approach.
Figures 1 and 7 show the detailed analysis of two different DUID samples in the study. In reference to the sample displayed on Figure 7, the immunoassay analyzer detected THC-COOH exclusively.Â
However, when analyzed with the SCIEX X500R QTOF System, the same compound was identified but also three compounds of interest, which were not tested by immunoassay were detected:
It is important to highlight that norfentanyl was considered a positive hit although obtaining a combined score of 53.9%. Analyte review based on the acceptance criteria like retention time, mass error on the TOF-MS scan, concentration threshold (> 1 ng/mL) as well as parent drug metabolism pathway knowledge, were supporting evidence of compound presence in the forensic DUID sample.
A comprehensive drug screening workflow for the analysis of forensic DUID blood samples has been successfully developed using the SCIEX X500R QTOF System based on the new NSC-ADID recommendations.
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