New Algorithms using All Ions MS/MS for the Identification of Isobaric Drugs in Blood Samples Using LC/MS/MS High Resolution Mass Spectrometry Martin Josefsson1; Bernhard Wuest2; Markus Roman1; 1National Board of Forensic Medicine Linköping, Sweden; 2Agilent Technologies GmbH, Waldbronn, Germany

In recent years accurate mass TOF and Q-TOF mass spectrometers have become more standard in forensic toxicology laboratories. The need to analyze unknown drugs and their metabolites requires sophisticated software tools to handle accurate mass data. We developed a novel algorithm which enables unprecedented accuracy in identification of drugs and their metabolites. The identification of isomeric compounds without the need of a standard is particularly important as many metabolite standards are not commercially available. The use of a co-elution score makes it possible to find fragments without a priori knowledge of the fragmentation.

In this study, post-mortem blood samples were analyzed to evaluate the ability of the All Ions MS/MS algorithm to correctly identify isobars and isomers. By combining MS and MS/MS experiments with All Ions MS/MS, users gain increased analytical speed and accuracy, significantly improved identification of compounds of interest, and increased productivity in forensic toxicology laboratories. Laboratories that analyze panels of drugs can import All Ions MS/MS results directly into their MassHunter Quantitative Analysis software for more productive identification and quantitation in a single software.

Introduction

Acquisition PCDL FindbyFormula on MS low channel

EICs for fragments on MS high channel

Correlate qualified fragments with target

Qual

F .d data file

S Targets

Find

m/z, ion species

Extract

Qualify and confirm fragments

Export

Align EICs of fragments with parent EIC

Targets fragment ions

Figure 1: All Ions MS/MS process

Sample Preparation Blood samples were fortified at 0.1 μg/g with a 30 compound test mixture in replicates (n=3) based on the protocol in a recently published paper 1. LC Conditions MS Conditions

Experimental

Agilent UHPLC 1290 System

Column Acquity UPLC

Mobile phase A= 10mM ammonium formate + 0.05% formic acid in water B= 0.05% formic acid in acetonitrile

Flow rate 0.5 mL /min

Gradient program Time (min) B (%)

0 – 0.6 1

0.7 5

8 50

10 – 11 95

Stop time 12

Agilent 6540 Q-TOF Mass Spectrometer Ion source AJS ESI Polarity Positive/Negative Ion spray voltage 3500 V (Pos) /-3000 V (Neg) Dry gas temperature 300 Dry gas 6 L/min (N2) Nebulizer pressure 35 psi (N2) Sheath Gas Flow 10 L/min (N2) Sheath Gas Temp 375°C MS parameters (MS mode) Scan range 50 – 1000 amu Scan rate 1.5 spectra/sec All Ions MS/MS mode Three segments at 0 eV, 20 eV, and 40 eV Scan range 50 – 1000 amu Scan rate 3 spectra/s Automatic internal reference mass correction was applied using purine (m/z 121.0509) and HP921 (m/z 922.0098

A B C

Figure 6: Batch at a glance MassHunter Qualitative Software view with (A) the quantitative ion, (B) the qualitative ions, and (C) the MS accurate mass and pattern identification

Conclusions Drugs of interest in forensic toxicological investigations have been used to demonstrate the analytical utility of the All Ions MS/MS software capability in MassHunter. The All Ions MS/MS method was used to screen for the presence of targeted compounds. These compounds were identified first using the accurate mass and isotopic pattern of the precursor ion, and then confirmed using the associated fragment ions. Easier method setup and more sophisticated data processing results were observed with increased confidence in target compound identifications. Overall, All Ions MS/MS enables a more productive analysis. Moreover, the data can be reanalyzed to look for additional compounds without the need to re-inject samples. A special software view summarizes important compound information in a single window which makes data review much faster and easier. From there, a quant method can be built with MS and MS/MS information to further improve the productivity of the analysis and to enable quantitation and identification in single software package. The comparison of All Ions MS/MS results to those of an MS method proved that the sensitivity is comparable. References

1: Roman, Markus et al. Liquid chromatography/ time-of-flight mass spectrometry analysis of postmortem blood samples for the targeted toxicological screening. Anal.Bioanal Chem 2013, DOI 10.1007/s00216-013-6798-0

B

C

For Forensic Use