Jenny Moshin (Presenter)
SCIEX
Authorship: Adrian Taylor (1), Xiang He (2), Michael Jarvis (1) and Jenny Moshin (2)
(1) SCIEX, Concord, Canada. (2) SCIEX, Redwood City, USA
| Short Abstract Quadrupole Time-of-Flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. It affords accurate mass and mass resolution capabilities combined with sensitivity as well as the capability to perform simultaneous highly specific targeted quantification and non-targeted screening and the ability to perform retrospective data mining. Here we describe a new QTOF system with intuitive new software for easy adoption of accurate mass technology to forensic testing. In this paper we demonstrate that the new hardware and software combined allow the highest level of confidence for compound identification and quantification from blood and urine samples. |
Long Abstract
Introduction: (For research use only, not for use in diagnostic procedures).
Quadrupole Time-of-Flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. The technology overcomes many challenges faced when using traditional techniques and more significantly captures all information about the sample in one injection to allow for retrospectively mining the data. Using the accurate mass and mass resolution information from both TOF-MS and TOF-MS/MS acquired data allows for simultaneous highly specific targeted quantification and non-targeted screening. Here we describe a new bench-top QTOF system with revolutionary N-optic designed flight path and new, intuitive software for easy adoption of accurate mass technology to forensic testing. We demonstrate that the new hardware and software combined allow the highest level of confidence for compound identification and quantification from blood or urine samples in one seamless workflow.
Methods:
Blank human urine was spiked with more than 50 common drugs found in forensics setting at different concentration levels. For urine analysis, these samples, along with the un-spiked blank urine sample were diluted 10-fold in 10% methanol and centrifuged. The clear supernatants were transferred to the autosampler vials and 10 µL was injected. The blood samples were processed using a protein crash procedure involving drying down and reconstituting the resulting supernatant in 20% methanol and centrifuging prior to injecting onto the LC column. HPLC separation was performed using the SCIEX ExionLC™ AC system on a reverse-phase column (dimension: 50 × 2.1) at 30 degree centigrade. A 6.5 minute LC gradient was used. Data was collected on a SCIEX X500R QTOF mass spectrometer with SCIEX OS software: 1) TOF-MS survey scan with Information Dependent Acquisition (IDA)-triggering of up to 16 product ion scans or 2) SWATH® acquisition. For SWATH® acquisition, the precursor isolation window width was varied for each MS/MS experiment. An electrospray ionization source was used consisting of a Turbo V™ source containing a twin spray probe using the following parameters: 2500 V spray voltage, 35 psi curtain gas, 600 degree centigrade source temperature, 60 psi nebulizing and desolvation gas. Data processing was also performed in SCIEX OS software with simultaneous identification and quantification being accomplished in single software.
Preliminary Results:
Screening
1. Multiple confidence criteria including mass accuracy, retention time, isotope, and most importantly, library matching, were used.
2. The availability of MS/MS information for identification was required in this study through library searching the acquired spectra against a SCIEX high resolution forensic library containing MS/MS spectra of 1700 compounds. Excellent true positive rates were accomplished for both acquisition modes (89% with IDA and 98 % with SWATH® acquisition). This result is consistent with the expectation that the IDA-MS/MS data acquisition has the risk of occasionally missing acquiring MS/MS.
3. The acquisition of MS/MS data in the screening approach allowed the confident identification of structural isomers that have similar retention times through unique fragment ions and their ratios. One example is the ability to distinguish 3,4 Dimethylmethcathinone from 4-Ethylmethcathinone which have retention times of 4.41 and 4.61 respectively. Due to 3,4 Dimethylmethcathinone having a unique fragment ion at m/z 159.10425 and 4-Ethylmethcathinone having one at m/z 145.0886 allows determination of whether one or both of these compounds are present.
4. Extra settings such as threshold of ratio (vs control), intensity, and weighted combined score were used to ensure maximum true positive and minimum false identification rate.
Simultaneous Quantification
1. Linearity on the new QTOF system for the majority of compounds tested was four orders linear dynamic range covering 0.1 to 1000 ng/mL.
2. SWATH® acquisition ensures that all data, including MS/MS, is acquired at all times. Due to the fast scanning speed (up to 100 Hz at 35K resolution) in MS/MS mode, a fast cycle time (0.5 sec) was used for the 6.5-min LC method that allowed for one TOF-MS scan and 16 MS/MS scans that covered 120 to 500 m/z range. Further, variable-SWATH® acquisition-window approach was used to improve the MS/MS selectivity resulting in sensitive and unambiguous quantification using fragment ions. The MS/MS mode therefore provided better limit of detection amid higher specificity over the IDA approach where quantification is restricted to use of the precursor ion.
3. As well as identifying the compound based on accurate mass, retention time, isotope pattern and library searching, at the same time, confirmation and quantification was performed on the SWATH acquired data. Confirmation was based on unique fragment ions and their ratios as well as library searching and simultaneously quantification was performed on both precursor and fragment ions for determining the analyte concentration in the unknown.
Conclusion:
A sensitive and selective workflow was developed for toxicological drug screening in a forensic setting using the new X500R QTOF system. Both the IDA-MS/MS and the novel MS/MSAll with SWATH® acquisition were used for the screening task and both acquisition approaches yielded excellent performance. Further, with SWATH® acquisition which acquired all MS/MS data at all times in a looped fashion, consistent number of data points across the LC peak were acquired, and the resulted MS/MS data can not only be used for screening but also for quantification when lower detection limit was desired with complex matrix content in the samples. The newly designed software that accompanies the new bench-top QTOF instrument allows for simultaneous compound identification and quantification in one step all in one place. SCIEX OS software allows the easy adoption of accurate mass technology to routine forensic workflows.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | SCIEX |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |