1. All Ions MS/MS
High Productivity Forensic Screening with More Confident Identification
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September 22, 2018

2. Screening vs. Targeted Analysis
Topics
Screening vs. Targeted Analysis
What is All Ions MS/MS?
Drug Screening with All Ions MS/MS
The demand for greater analytical productivity combined with legally defensible results for the screening of forensic samples have placed substantial demands on traditional analytical techniques. This presentation describes the use of a new technique, “All Ions MS/MS”, to rapidly screen and identify drugs of forensic toxicological interest.
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September 22, 2018

3. Non-Target Screening vs. Targeted Quantitation
LC/QQQ
Benefits:
Most Sensitive Technique
Good Quantitative Results
Drawbacks:
Limited number of analytes
Non-target compounds undetected
LC/TOF or LC/Q-TOF
Analysis Options:
MS only screening
MS/MS with accurate mass library for confident identification
Benefits:
Detect, Quantify, and Identify hundreds or thousands of compounds in a single injection
Reanalyze old sample data any time without reinjection upon discovery of new compounds
Targeted quantitation using a triple quadrupole LC/MS system is tried and true method for getting the best possible sensitivity and quantitative accuracy for a targeted list of compounds. However, a targeted list limits the total number of compounds which can be analyzed in a particular run. Non-target screening is a technique which is done on TOF or Q-TOF LC/MS. One screening technique uses MS only (without fragmentation) and matching precursor ions to the accurate mass formulae in an accurate mass database. In order to take confidence in identification to the next level, the precursors can be fragmented in order to search an accurate mass library with accurate mass MS/MS spectra.
The benefits of untargeted screening include the ability to detect, identify, and quantify compounds in one injection. In addition, it is possible to re-process data at any time if a new compound is discovered and added to the database. This means samples can be re-analyzed without pulling them out of the freezer, re-prepping them, and reinjecting.
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September 22, 2018

4. Non-Target Forensic Screening The Value of the Agilent Solution
Robust hardware
Sample Preparation Products
HPLC and UHPLC Columns
Automated software workflows
Comprehensive Accurate Mass Libraries
LC/MS Application Kits
First-class service and application support
Agilent truly provides a one-vendor solution from sample preparation, separation, MS analysis, data processing, and reporting. Agilent’s highly-regarded service and support keep you running with maximum efficiency. Our LC/MS Application Kits provide databases, libraries, methods, columns, standards, and on-site support in order to get your analysis up and running with maximum efficiency.
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September 22, 2018

5. Agilent Forensic Toxicology PCDL
Database with 9,000+ Compounds
Designer Drugs
Hallucinogens
Stimulants
Opioids
Benzodiazepines
Hypnotics
Neuroleptics
Barbituates
Antidepressants
Antiepileptics
Library spectra for 3,000+ Compounds
Accurate Mass Correction
Molecular Structure
Quality Controlled
CAS Number/PubChem Link
Agilent’s Forensic Toxicology Personal Compound Database and Library (PCDL) is the largest and most comprehensive commercial library on the market. In addition to the fact that it contains over 9,000 compounds in the database, it also has undergone an extensive quality control process to verify that every entry is high quality and that the peaks are corrected to the theoretical accurate mass (instead of the experimental values which can vary from one instrument to another).
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September 22, 2018

6. Screening vs. Targeted Analysis
Topics
Screening vs. Targeted Analysis
What is All Ions MS/MS?
Drug Screening with All Ions MS/MS
The demand for greater analytical productivity combined with legally defensible results for the screening of forensic samples have placed substantial demands on traditional analytical techniques. This presentation describes the use of a new technique, “All Ions MS/MS”, to rapidly screen and identify drugs of forensic toxicological interest.
Confidentiality Label
September 22, 2018

7. All Ions MS/MS-What is it?
Step 1: Fragmentation without Isolation on TOF or Q-TOF
First Scan: Low fragmentation energy to analyze precursors
Second Scan: High fragmentation energy to analyze fragment ions
Low energy spectrum
High energy spectrum
Shows Precursor Ions
All Ions MS/MS is a new acquisition mode and data processing tool which enables fragmentation without isolation on Agilent TOF and Q-TOF LC/MS systems. The acquisition mode alternates scans between high and low energy fragmentation modes. All ions introduced into the mass spectrometer are first analyzed with a low fragmentation energy (precursors), and then a high fragmentation energy to give fragment ion information which can be used for identification and confirmation.
Shows Fragment Ions
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September 22, 2018

8. All Ions MS/MS-What is it?
Step 2: Software Extracts, Correlates, and Confirms
Find by Formula uses library to extract precursors from low energy MS channel
All Ions MS/MS uses library to extract fragments from high energy channel, gives coelution score
Software automatically matches precursors with fragment ions: Coelution Plot with Coelution Score
Agilent’s MassHunter Qualitative Analysis software then uses a unique Find by Formula algorithm to extract precursor ions in the acquired data using Agilent’s accurate mass databases, with Accurate mass formula information for thousands of forensic compounds of interest. If a precursor match is found, and the isotopic pattern matches the database, the All Ions MS/MS software automatically extracts fragment ions for that compound which are present in our accurate mass MS/MS libraries. The extracted precursor and fragment ion peaks are plotted to compare their coelution, and are assigned a coelution score in order to qualify with certainty that a particular fragment corresponds to a particular precursor ion.
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September 22, 2018

9. All Ions MS/MS: What is the Value?
More Confident Identification
Simple Method Set Up
High Productivity Software Workflow
Reanalyze Old Samples Without Reinjection to Find New Compounds
Forensic Toxicology PCDL and Application Kit with 9, Forensic Compounds of Interest
All Ions MS/MS provides more confident MS/MS identification, without the complicated method setup required for most targeted MS/MS analyses. Since every ion is analyzed and fragmented, there is no need to set up MRM transitions, or set analytical thresholds. The automated software workflow makes data processing simple and fast, making it compatible with high productivity environments. In the event that new drugs of abuse are discovered at a later date, data files can be re-interrogated without re-injecting samples. In addition, Agilent provides LC/MS Application Kits for Forensic Toxicology, which include accurate mass databases and libraries, methods, LC columns, standards, and on site support. These Agilent-developed solutions make method setup and implementation faster and simple for new LC/MS users.
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September 22, 2018

10. Screening vs. Targeted Analysis
Topics
Screening vs. Targeted Analysis
What is All Ions MS/MS?
Drug Screening with All Ions MS/MS
The demand for greater analytical productivity combined with legally defensible results for the screening of forensic samples have placed substantial demands on traditional analytical techniques. This presentation describes the use of a new technique, “All Ions MS/MS”, to rapidly screen and identify drugs of forensic toxicological interest.
Confidentiality Label
September 22, 2018

11. Application of All Ions MS/MS Untargeted Drug Screening in Postmortem Blood
Purpose of Application:
Evaluate Efficiency of All Ions MS/MS Workflow
Unambiguously Identify Coeluting Analytes at Different Concentrations
The analytical method uses a high resolution TOF or Q-TOF mass spectrometer to quickly and easily analyze samples, determine whether target compounds are present and at what concentration Blood samples fortified with a 30 compound test mixture at a concentration of 0.1 µg/g in replicates (n=3) were analyzed using an LC/Q-TOF instrument with “All Ions MS/MS” software processing tools in order to validate the effectiveness of the technique for rapid generation of accurate results, despite the presence of similar and isomeric compounds at different concentrations. In addition, two post-mortem blood samples were analyzed to test the capability of “All Ions MS/MS” for the identification of isobars and isomers. Combining MS and MS/MS experiments and using All Ions MS/MS increases analytical speed and accuracy, significantly improves unambiguous identification of compounds of interest, and increases productivity in forensic toxicology laboratories. In addition, laboratories that analyze panels of drugs can import All Ions MS/MS results directly into their MassHunter Quantitative Analysis software for more productive compound identification and quantification.
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September 22, 2018

12. Experimental Untargeted Drug Screening in Postmortem Blood
Preparation of post-mortem blood samples
Liquid/liquid extraction with 90:10 acetonitrile:ethanol
250 mg blood µL extraction solvent
2 µL of extract injected
Run time: 12 minutes
Two Analyses:
30-compound test standard
Blood sample spiked at 0.1 µg/g
Authentic post-mortem blood samples were prepared using liquid/liquid extraction with acetonitrile/ethanol 90/10 (250 mg blood µL extraction solvent. An aliquot of 2 µL of this extract were injected.
UHPLC column
Acquity UPLC, HSS T3, 2.1mm x 150mm, 1.8 µm
Mobile phase
A:10mM Ammonium formate % Formic acid in Water
B: 0.05% Formic acid in Acetonitrile
Gradient program
Min % B 1%
% %
% % % %
Stop time 12.0
Flow rate
0.5 mL/min
In order to compare results from MS and All Ions MS/MS experiments, a 30-compound test standard and a blood sample spiked at 0.1 µg/g were used.
Ion Source Parameters:
Gas Temp: oC Gas Flow: 6 L/min Nebulizer: 35 PSI Sheath Gas Temp: 375 oC Sheath Gas Flow: 10 L/min
MS Parameter (MS-Mode):
Scan Range: 50 – 1000 amu Scan Rate: 1.5 spectra/sec
All Ions MS/MS Mode:
3 Scan Segments at 0 eV, 20 eV and 40 eV Scan Range: 50 – 1000 amu Scan Rate: 3 spectra/sec
All Modes:
Automatic internal reference mass correction using Purine (m/z ) and HP 921(m/z )
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September 22, 2018

13. Evaluation of All Ions MS/MS Automated Workflow Untargeted Drug Screening in Postmortem Blood
Step 1: Find by Formula Extracts Precursors using PCDL database
The data were analyzed using the Find by Formula (FbF) algorithm MassHunter Qualitative Analysis rev. B Find by Formula evaluates a chemical formula and determines if a compound with that formula is present in the high resolution mass spec data. This approach uses a modified version of the Find by Formula algorithm which supports the All Ions MS/MS technique. Mass peaks in the low energy channel are first searched against the Forensics and Toxicology Personal Compound Database and Library (PCDL) rev. B (aka Broecker, Herre & Pragst MS/MS library, version 4.1) for compounds which have the same m/z values. From this a search a set of putative identifications is automatically compiled.
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September 22, 2018

14. Evaluation of All Ions MS/MS Automated Workflow Untargeted Drug Screening in Postmortem Blood
Step 2: Extract Corresponding Fragment Ions
Fragments from PCDL
For this compiled list of compounds, the fragment ions in the MS/MS spectra from the PCDL were used to extract those ions in the high energy channel in order to evaluate their correlation with corresponding precursor ions.
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September 22, 2018

15. Overlaid Precursor and Fragment Ion Chromatograms
Evaluation of All Ions MS/MS Automated Workflow Untargeted Drug Screening in Postmortem Blood
Step 3: Precursor and Fragment Correlation/Coelution
Overlaid Precursor and Fragment Ion Chromatograms
Coelution Plot
Both the precursors and fragment ions are extracted as ion chromatograms and evaluated using a co-elution score. The co-elution score is derived from a technique which is similar to the Peak Purity method used in UV chromatography (Sievert, Hans-Jürgen P.; Drouen, Anton C.J.H., 1993). The software calculates a number that accounts for factors including abundance, peak shape (symmetry), peak width, and retention time. The scores can be plotted and made available to the user for inspection in a Co-elution Plot.
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September 22, 2018

16. Coeluting with ~50x Concentration Difference
Differentiation and Identification of Isomers Untargeted Drug Screening in Postmortem Blood
Alprazolam
Coeluting with ~50x Concentration Difference
Methadone
From the data analysis it is immediately apparent that alprazolam and methadone co-elute. In addition, the concentration of methadone is much greater than alprazolam. The concentration of the compounds in this example are both at therapeutic levels, but Methadone is present at about 50x higher concentration.
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September 22, 2018

17. Similar precursor m/z values and overlapping isotopic clusters
Differentiation and Identification of Isomers Untargeted Drug Screening in Postmortem Blood
Similar precursor m/z values and overlapping isotopic clusters
Since methadone and alprazolam have similar precursor ion values (m/z and ) it could be difficult to distinctly identify either compound using traditional LC/MS methods. Using another high resolution accurate mass technique, for example, targeted MS/MS or data-dependent MS/MS would have produced a mixed spectrum. However, an examination of the co-elution scores and plots automatically generated by the All Ions MS/MS software clearly identified the compounds in the sample using both fragment ions from the PCDL and information for their isotopic clusters.
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September 22, 2018

18. Compound Confirmation-Methadone Untargeted Drug Screening in Postmortem Blood
Red outline overlays theoretical isotopic pattern and abundance with acquired precursor data for methadone
Methadone confirmed with seven qualified fragments
Each of the fragments is flagged as either “Qualified” or rejected (with a reason). The isotopic cluster is displayed with a red overlay indicating the theoretical abundance and spacing. As can be seen there is excellent agreement for both methadone (above) and alprazolam (next slide) despite the large difference in their concentrations. The methadone precursor match to Agilent’s accurate mass database (top), and was confirmed with seven qualified fragments (bottom).
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September 22, 2018

19. Compound Confirmation-Alprazolam Untargeted Drug Screening in Postmortem Blood
Red outline overlays theoretical isotopic pattern and abundance with acquired precursor data for alprazolam Alprazolam confirmed with two qualified fragments, despite low concentration
Alprazolam was extracted and confirmed with a good precursor isotopic match (top) and two qualified fragments (bottom) despite a significantly lower concentration with a coeluting analyte.
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September 22, 2018

20. High Productivity: Compound Details View
Compound List
Compound ID Results
Overlaid Chromatograms
Low Energy (MS) Spectrum
Results of the analysis can be reviewed within two modules of the Qualitative Analysis software: Navigator View, which allows the viewing of multiple data files chosen from a Data Navigator window, and the new Compound Details View (shown here), which shows Identification Results, Co-elution plots, MS and fragment spectra for one compound at a time. Users are able to manually review and change compound selections in the Compound Details View by quickly browsing through them in the Compound List.
High Energy Fragment Spectrum
Co-elution Plot
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September 22, 2018

21. Conclusions All Ions MS/MS Increases Confidence in ID
Simple Method Setup
Automated data processing
Accurate Mass MS/MS Library Extraction
Coelution Plot with Coelution Score
Reanalyze data any time, without reinjection of samples
Review data quickly, increasing productivity
Drugs of interest in forensic and toxicological investigations have been used to demonstrate the analytical utility of a new All Ions MS/MS software capability in MassHunter. We rapidly generated a data processing method for high resolution accurate mass data from a Q-TOF mass spectrometer. The “All Ions MS/MS” method was used to screen for the presence of target compounds. These compounds were identified first using the accurate mass and isotopic pattern of the precursor ion, and then confirmed using the co-elution of the associated fragment ions. The All Ions MS/MS approach enables simpler method setup, more sophisticated data processing and results in more confident in target compound identifications. Overall, this technique leads to a more productive analysis and the ability to re-interrogate data to look for additional compounds without re-injecting samples. The Compound Details View summarizes important compound information in a single window, which means that large batches of sample results can be reviewed quickly for even greater productivity.
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September 22, 2018