21


Morphine, Hydromorphone, Norcodeine and Norhydrocodone, with the optimised LC condition. Figure 3 show example extracted ion chromatograms for 80 out of the 664 compounds using the optimised LC condition.


Retention time is a critical element for accurate identifi cation of each forensic analyte using this screening method. Therefore, retention time reproducibility tests were conducted for each compound to evaluate the robustness of the LC condition in this method. These tests indicate that the retention times generated from the optimised LC conditions are consistent and reproducible. Retention times measured on three separated analytical columns all have % coeffi cient of variation (CVs) of less than 5% for each of the 664 compounds.


The retention time determined by the optimised LC condition combined with high- resolution mass spectrometry (HRMS) and HR-MS/MS information, enable more accurate compound identifi cation than regular triple quad workfl ows. For example, the Noroxycodone (Figure 4A) and Oxymorphone (Figure 4B) have the exact same precursor ion and very similar MS/MS spectra. However, these two compounds were fully resolved by using the LC condition in this method. The retention time is 3.05 min for Noroxycodone and 2.10 min for Oxymorphone. Therefore, it is easier and more accurate to distinguish these two compounds by using retention time combined with MS and MS/MS information.


Figure 3. Extracted ion chromatograms (XICs) for multiple analytes (80 out of 600) show optimal peak separation.


samples, 10.0 µL of stock standard solutions were spiked into 90.0 µL of human whole blood matrix. The samples were extracted by using a protein precipitation procedure. Basically, 900 µL of Methanol: MeCN (50:50, v/v) were added into the above mixture and vortexed for 1 min then follow by 3 min sonication and another 1 min vortex. Then the samples were centrifuged for 5 min at 8,000 rpm. The supernatant was transferred out and completely dried down under nitrogen gas. The residues were reconstituted with 500 µL methanol: water (20:80, v/v).


LC separation


Analytes (10 µL sample injection volume) were chromatographically separated using a Phenomenex Kinetex®


2.6 µm phenyl-hexyl (50 x 4.6 mm) column. 10 mM ammonium formate in water was used as mobile phase A and 0.05% formic acid in methanol was employed as mobile phase B. The mobile phases were replaced every 2 days. A linear gradient (600 µL/min) from 10% B to 98% B in 7.0 min followed by 1.5 min of 98% B and 1.0 min of 10% B was employed.


Processing method settings


To identify compounds in the analysed samples, a targeted screening approach was employed using SCIEX OS software version 2.0. Samples were evaluated against a list of parameters containing the names, molecular formulas and retention times (RTs) for all targeted compounds. Appropriate integration parameters were defi ned for each component. For example, the compound, hydromorphone, was defi ned as the peak at 2.35 min (Figure 1) with a 30 second half time window. An MS/MS library [2] was used for MS/MS library matching.


The confi dence criteria used for screening were mass error, RT error, isotope ratio difference, and library score. A traffi c light system where different colours were assigned to different performance levels provided a way to assess the quality of the match. For example, in the case of mass error, green represented mass errors less than 5 ppm; orange, mass errors between 5 and 10 ppm; and red, mass errors larger than 10 ppm. A representative search result is shown in Figure 2.


Results and Discussion


For forensic analysis, it is imperative to be able to quickly test whole blood and urine samples with straightforward sample preparation to deliver clear accurate data, quickly. To this end, a single-injection method for screening 664 of the most up-to-date forensic compounds has been developed.


The method, denoted vMethod, allows for screening of 664 compounds in HR-MS/MS mode on a QTOF System.


To identify compounds in the analysed samples, a targeted screening approach was employed. Samples were evaluated against a list of parameters containing the names, molecular formulae and retention times for all targeted compounds. Appropriate integration parameters were defi ned for each component. For example, the compound hydromorphone was defi ned as the peak at 2.35 min (Figure 1) with a 30 second half time window. An MS/MS library was used for MS/MS library matching.


The confi dence criteria used for screening were mass error, retention time error, isotope ratio difference, and library score. A traffi c light system where different colours were assigned to different performance levels provided a way to assess the quality of the match. For example, in the case of mass error, green represented mass errors below 5 ppm; orange, mass errors between 5 and 10 ppm; and red, mass errors larger than 10 ppm A representative search result is shown in Figure 2.


The injection volume for the vMethod is 10 µl and sample matrices that can be analysed include whole blood and urine. The sample preparation protocol details all the steps required for the clean-up of both blood and urine samples and in just 10 minutes, detailed, verifi ed LC separation conditions are achieved with retention times.


Figure 1 shows an example of full chromatographic separation for 4 isomers, including


In addition, because the data was acquired in a non-targeted approach, the processing method designed here for screening targeted compounds can be quickly adjusted and used for unknown compound identifi cation using non-targeted data processing. Users can retrospectively analyse previously acquired MS and MS/MS data sets to screen for new compounds without having to re-inject samples, allowing data sets to be re-processed when newly identifi ed forensic targets are discovered. For retrospective data analysis, a new process method was built for 10 compounds including 5 initial compounds and 5 new compounds by using search parameters that included the compound’s name, formula and retention times. The updated processing method was then used to re-analyse data sets for the new compound.


Figure 4. Representative XIC HR-MS and HR-MSMS spectra for Noroxycodone and Oxymorphone


The vMethod gives data that provide both structural information and retention times to enhance identifi cation accuracy, especially for structurally similar isomers. The sample preparation procedures for urine and whole blood samples and library-search settings recommended in the method can help automate and confi dently establish the identifi cation of unknowns in an effi cient, all-in-one workfl ow.


Overall, the ability to identify structurally similar isomers was largely enhanced using the vMethod. In addition, because the data were acquired in a non-targeted approach the processing method designed here for screening targeted compounds can be quickly adjusted and used for unknown compound identifi cation using a non-targeted data processing. Users can retrospectively analyse previously acquired MS and MS/MS data sets to screen for new compounds without having to re-inject samples, allowing for data sets to be re-processed when newly identifi ed forensic targets are discovered.


Conclusions


This broad-based drug screening method offers a good balance between chromatographic separation and analysis time. The mass acquisition is designed to acquire analytes of interest in a semi-non-targeted fashion, allowing for high specifi city in tracing fragment ions back to precursor ions. In addition, it has the potential for more wide-ranging retrospective data analysis for determination of analytes not within the current scope of data processing.


References 1. The United Nations Offi ce on Drugs and Crime 2014 World Drug Report http://www.unodc.org/wdr2014/ 2. SCIEX Forensics High Resolution MS/MS Spectral Library 2.1, part number: 5059566


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