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High-Resolution, Accurate-Mass Forensic Toxicology Screening in Blood Samples Using a Q Exactive Mass Spectrometer

Authors: Isabelle Morel1, Sylvie Lepage1, Benedicte Duretz2, Angela C. De Pietro3

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1Forensic Toxicology Laboratory, Rennes Hospital, Rennes, France; 2Thermo Fisher Scientific, Les Ulis, France; 3Nova Analtica, So Paulo, SP, BR


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Forensic scientists and forensic toxicologists need to identify an unlimited number of compounds in complex matrixes with the capability of retrospective data analysis for quick and confident analysis. The major challenge is to separate the analytes of interest from the matrix and accurately identify them. In this work a bench-top quadrupole-Orbitrap™ ultra-high resolution mass spectrometer routinely capable of better than 5 ppm mass accuracy and 140,000 FWHM resolution was evaluated with Thermo Scientific™ ExactFinder™ 2.0 data processing software, for forensic toxicology screening in blood samples. A comparison with Targeted Screening on a Triple Quadrupole MS using the SRM (Selected Reaction Monitoring) mode and also UPLC/Diode Array Detection (DAD) was done.
Blood samples were spiked with 20 μl of an internal standard solution (Flurazepam at 1 mg/L) and extracted with TOXI-TUBES™ A (Agilent Tec.). UHPLC separation was performed on a high-pressure gradient pump with a 30 min gradient using Hypersil™ GOLD PFP column 150 x 2.1 mm 3 μm. Mobile phases were 10 mM Ammonium formate and 0.1% Formic acid in water (A) and 0.1% Formic acid in Acetonitrile (B). Compounds were detected on a mass spectrometer equipped with an Orbitrap mass analyzer. A Heated Electrospray Source Ionization (HESI) probe was used as an ion source. The instrument was operating in alternating positive and negative full scan mode. Each Full Scan was followed by 8 HR-MS2 scans in positive mode and 3 HR-MS2 scans in negative mode. Precursor selection was done in the data-dependent operation mode where the most intense ion of the previous scan was selected for fragmentation. Resolution was set to 70,000 FWHM for each full scan mode and 17,500 FWHM for MS2 scan acquisition. MS2 spectra were acquired with a Normalized Collision Energy (NCE) of 70. For comparison, data have been acquired on a UPLC instrument equipped with a DAD detector using a 15 min LC gradient and a library with 612 molecules. On a Triple Quadrupole MS system, six different targeted LC-MS/MS methods have been used to acquire data in SRM mode. This method includes 97 molecules. Identification of the analytes is performed using the exact mass of the precursor, the retention time, the isotopic distribution and the fragment exact masses.

Thirty-nine samples were analyzed and compared using the 3 different technologies. We have been able to identify 143 compounds with the HRAM approach, 121 with the 6 targeted forensic screening methods on the triple quadrupole MS and 69 compounds by DAD. Fewer analytes have been identified using DAD despite the 612 analytes in the library. Sensitivity is the main concern with this technique. Moreover, DAD may provide some false results. For ex.: estazolam has been identified in DAD but not confirmed by the MS technologies; alprazolam was not detected with DAD but was confirmed by the other 2 approaches.
The Triple Quadrupole approach gives good results in terms of positive hits identified. However, the identification is confirmed using six different SRM methods i.e. the same sample has to be injected several times. Moreover these six methods contain only 97 analytes.

The HRAM LC-MS/MS approach identified the largest number of compounds for forensic toxicology with the 650 analytes library. This HRAM method also allows for retrospective data analysis. Data collected by this approach show benefits of high-resolution screening over both the triple quadrupole approach and DAD detection.