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LC-MS/MS is a powerful analytical tool in forensic toxicology testing that can support a variety of testing regimes such as screening, confirmation and quantitative workflows. More specifically, analysis of NPS using LC-MS/MS provides many advantages, including the ability to reliably detect new drugs and their metabolites from a variety of biological matrices.

In this blog, we are going to discuss the benefits of accurate mass spectrometry for non-targeted NPS analysis and how those benefits can provide additional levels of confirmation. Forensic toxicologists are often concerned about screening for and identifying unknown substances that have recently surfaced on the recreational drug market. The speed and sensitivity of accurate mass spectrometers can be leveraged to perform MS scanning experiments to search for unknown molecular ions while also performing selective MS/MS scanning functions. This provides comprehensive compound fragmentation on all the analytes present in the sample. Because these fragments are acquired in high resolution, the detected NPS can be accurately identified through spectra library database searching.

Accurate mass spectrometers, such as quadrupole time-of-flight (QTOF) systems, are flexible platforms that can support both quantitative and qualitative analysis. Below are some of the most common acquisition methods that can be leveraged for both targeted and non-targeted workflows.

Targeted workflows:

• High-resolution multiple reaction monitoring (MRM^HR): as its name implies, MRM^HR is a high-resolution version of the traditional MRM assay. This targeted approach enables acquisition of high-resolution fragments, which intrinsically reduces matrix background and provides more specificity. As a result, MRM^HR is commonly used for NPS analysis to provide sensitive quantitation of low-concentration drugs and metabolites in complex biological matrices with a high degree of accuracy and precision. The acquisition of selective high-resolution accurate mass MS/MS information using the MRM^HR workflow can also be combined with spectral library searching and ion ratio for identification purposes, proving added levels of confirmation.

Check out this tech note to learn how the MRM^HR on the SCIEX X500R QTOF system was used for the quantitation and identification of low levels of NPS in human urine samples: Read now

Non-targeted workflows:

Acquisition methods such as data-dependent acquisition (DDA) and SWATH data-independent acquisition (DIA) can be leveraged to acquire high-resolution spectra from single sample sets in a routine testing laboratory environment.

• Data-dependent acquisition (DDA): DDA is an acquisition strategy that automatically selects candidate precursor ions for further MS/MS analysis. If any ions acquired in the survey (TOF MS) scan meet the DDA criteria set by the user, the instrument triggers dependent MS/MS spectra for each of the qualifying candidates. With DDA, identification of analytes using the full scan MS/MS spectrum can be quite precise and specific given that the mass spectrometer is fragmenting only one analyte at a time. The acquired, dependent MS/MS information (full scan MS/MS spectrum) can be used to confirm identification of analytes using spectra library database searching.
• SWATH data-independent acquisition (DIA): unlike DDA, SWATH DIA fragments every detectable precursor eluting from the LC column and entering the mass spectrometer, not just a subset. For each cycle, the instrument focuses on a narrow mass window of precursors and acquires MS/MS data from all precursors detected within that window. This mass window is then stepped across the entire mass range, systematically collecting MS/MS data from every mass and from all detected precursors. This results in a truly comprehensive MS/MS data set that includes all detectable compounds extracted from biological samples, creating a digital record of the sample. The resulting fragments can also be used to confirm identification of the substance present in the samples using spectral library matching. One of the advantages of this DIA method is that it provides the ability to perform retrospective analysis on previously acquired data sets to screen for the presence of new substances without having to re-inject samples.

Learn more about the benefits of DDA and SWATH DIA for non-targeted workflows.

And check out these technical notes that compare the two workflows for the screening and quantitation of NPS in biological matrices:

Fast Forensic Toxicological Screening and Quantitation in Under 3 Minutes 

Expanding NPS screening capabilities in the forensic toxicology laboratory

In summary, QTOF systems are the mass spectrometers of choice for NPS screening because of their ability to acquire high-resolution accurate mass precursor and fragment data to improve confidence in NPS detection, providing added flexibility for non-targeted workflows. The use of accurate mass spectrometry for NPS analysis reduces the effects of complex matrices and background interferences, which results in higher data quality, lower LOQs and fewer false positives. Overall, the ability to acquire full MS/MS fragmentation spectra of all the analytes present in the samples enhances compound identification through spectral library matching. In addition, previously acquired data sets can be retrospectively analyzed to look for the presence of newly identified NPS should new questions about a sample arise.

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