tims&#45;tof ms&#47;ms method development and calibration of mobility and mass for fentanyl analog screening

Harvesting the Orthogonality of LC and TIMS&amp;#45;TOF MS&amp;#47;MS to Screen Fentanyl Analogs

Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States1,2. The Center for Disease Control and Prevention (CDC) reported that the number of synthetic opioid-related overdose deaths from 2013 through 2021 was over 258,000. In 2021 alone, over 68,000 overdose deaths could be attributed to synthetic opioids, totaling 82% of all overdose-related deaths in the nation3. Since 2013, hundreds of fentanyl analogs have been identified, with varying potency4. With the emergence of illicitly manufactured fentanyl analogs, the schedule II synthetic opioid itself remains the most popular synthetic opioid available in the United States3. According to the Center for Forensic Science Research and Education (CFSRE), the top reported fentanyl analog in 2022 was fluorofentanyl, with additional non-fentanyl related synthetic opioids now being introduced into the volatile drug market supply at a rapid rate5.
Due to the overwhelming volume of fentanyl and fentanyl-related analogs circulating in the drug market, the DEA has implemented a fentanyl signature profiling program entitled Operation Death Dragon, in order to track the methodology used to synthesize these compounds with hopes of linking drug seizures back to their origin6. In 2018, 94% of the drug seizures were identified as being synthesized by the Janssen method, while the remaining 6% were synthesized with the Siegfried method6. The primary difference between the two methods is the presence of the fentanyl analog, benzyl fentanyl, detected as an impurity in the Janssen method, while the presence of despropionyl fentanyl (4-ANPP), a metabolite/precursor of fentanyl, is an impurity detected when synthesizing with the Siegfried method7.
The use of gas and liquid chromatography in tandem with mass spectrometry (GC-MS and LC-MS, respectively) for the targeted screening and quantification of synthetic opioids is regularly implemented in toxicology laboratories. GC-MS has been considered the gold standard for the detection of drugs of abuse in biological specimens. Access to publicly available mass spectral libraries8 and instrumentation marketed as plug and play systems9 are some reasons why GC-MS has remained an integral part in laboratories for both comprehensive screening as well as targeted quantification9,10. However, current GC-MS quantification methods in the literature tend to have a limited scope of analytes11 and quickly become outdated and not applicable to current casework. More importantly, the limits of detection and quantification are not comparable to LC-MS methods (&#60; 1 ng/mL)12, therefore increasing the potential for false-negative results. One such comparison between GC-MS and LC-MS which looked at postmortem specimens, noted that out of 134 positively identified cases of carfentanil, one of the most potent synthetic opioids to date, 104 of those cases screened negative for carfentanil utilizing GC-MS13. In drug analysis laboratories, GC-MS is more frequently used and amendable for synthetic opioids due to the high concentration of the analyzed samples. Yet, GC-MS is still utilized in conjunction with additional techniques such as Fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) for the confirmation of these compounds14. The application of GC-MS to the analysis of biological specimens in forensic toxicology requires sample preparation methods that include the extraction of these compounds using either liquid-liquid extraction (LLE) or solid-phase extraction (SPE)15. LLE can be conducted with a variety of solvents, however in a high-production lab, LLE may not be cost-efficient or timely. LLE consumes large quantities of solvents as well as sample volume, while the alternative, SPE, can be automated and requires minimum sample volume16. A recent GC-MS study reported the separation of 20 different isomeric fentanyl analogs using three separate GC thermal programs17. While baseline separation among isomers was successful, the applicability of this method to relevant forensic casework and workflows is limited.
LC-MS has gained popularity in forensic testing, especially due to the limitations of GC-MS when it comes to non-volatile and heat-sensitive compounds18,19. LC-MS screening utilizing triple quadrupoles (QQQ) and ion trap instrumentation have been successful in detecting synthetic opioids at low concentrations (&#60;1 ng/mL)12,20,21,22,23,24. Typically, these LC-MS methods are used as secondary confirmation methods to complement immunoassay and/or GC-MS results. In 2017, Shoff et al. at the Miami-Dade County medical examiner department (MDME) toxicology laboratory developed a comprehensive screening method for 44 opioid-related compounds using ultra high pressure liquid chromatography (UHPLC)-ion trap-MSn 13. Similar to targeted MRM methods, this ion trap method utilized a scheduled precursor list (SPL) containing retention times, precursor target ions, as well as primary daughter ions for MS3 spectral fragmentation when possible. What separates this screening method from methods developed on triple quadrupoles and linear quadrupole ion traps is the additional detail provided in the spectral data. What this screening method cannot provide is quantitative analysis, which is rarely developed on ion trap instrumentation, as well as the identification of unknowns13. LC-QQQ methods for synthetic opioids can simultaneously screen and quantify a predefined target list. The use of multiple reaction monitoring (MRM) transitions for the identification and quantification of compounds is a trusted data acquisition technique and is the most common technique utilized in the literature for the detection of synthetic opioids12,20.&#160;Reported linear ranges for the quantification of an array of synthetic opioids span from 0.01-100 ng/mL, with more potent synthetic opioids, such as carfentanil, detected at the sub-ng/mL range11,24,25,26,27.
The separation of isomeric synthetic opioids has been addressed in LC-MS methods. One such method separated 174 isomeric fentanyl analogs within a 16 min runtime using a biphenyl column28. In addition, fluctuations in LC-dependent parameters such as column efficiency, mobile phase pH, and pressure changes create retention time shifts that must be accounted for in targeted assays with consistent monitoring and larger collection windows (&#62;0.4 min), which may result in potential overlap of these once resolved isomers. Additional chromatographic techniques for the separation of isomers have been explored, including the use of 2-dimensional liquid chromatography (2D-LC)29; and while this technique does have the ability to provide orthogonal separation of compounds, the disadvantages outweigh the advantages including excessive run-times, cost, difficulty in method development, and usefulness compared to alternative separations30.
High resolution mass spectrometry (HRMS) is becoming increasingly reliable for the identification of synthetic opioids. Several instrumentation companies have marketed targeted methods developed for the purposes of identifying a broad range of synthetic opioids in complex biological matrices19,28,31. These methods, unlike previously discussed methods, can store analytical data for retroactive analysis. Thus, specimens that were previously analyzed with undetermined results can be revisited later for discovery of newly identified compounds. What separates HRMS from QQQ is accurate mass identification. While both MS techniques can accurately quantitate at low concentrations, HRMS has been proven to be more effective for initial screening and discovery of unknown compounds32,33,34. HRMS, specifically with time-of-flight (TOF) MS analyzers, has been at the forefront of NPS discovery, allowing forensic testing laboratories to deliver time-sensitive data on new compounds to both law enforcement and the scientific community to scale the spread of some of these compounds while increasing awareness and education33. The use of TOF for the detection of drugs of abuse has become extremely comprehensive, with methods containing an upwards of 600 compounds separated within a 10 min chromatographic program. Previous studies have been reported the advantages of trapped ion mobility spectroscopy (TIMS) coupled to TOF for detection and separation of isomeric opioids35.
Taking advantage of the orthogonality between liquid chromatography, trapped ion mobility spectrometry and mass spectrometry, the presented method provides a broad characterization of fentanyl analogs based on the retention time, isotopic pattern, mobility, and fragmentation pattern.

Author Spotlight: An Efficient Methodology to Confidently Differentiate and Characterize Fentanyl Analogs 

Fentanyl Analog Screening using LC-TIMS-TOF MS/MS

The scope of our research is to develop a method to discriminate and characterize isomeric analogs. This research aims to investigate if a method can be developed to confidently differentiate a large quantity, especially structural isomers. This protocol incorporates four modes of characterization that have previously not been coupled.Using four orthogonal methods of identification for the analogs should improve the ability to differentiate isomers that are difficult to differentiate with current standard procedures. Fentanyl is a big societal concern in the opioid epidemic. New analogs created in illicit labs are being found every day, and having a methodology that quickly and confidentially helps determine each analogs that are in a sample could improve the efficiency in drug law enforcement.

TIMS&#45;TOF MS&#47;MS Method Development and Calibration of Mobility and Mass for Fentanyl Analog Screening

To begin, TIMS-TOF MS/MS Method development, open the TIMS control application. Under MS settings, set the scan beginning and ending values to 50 mass to charge ratio and 1, 800 mass to charge ratio, respectively. Select the ion polarity mode as positive and the scan mode as parallel, accumulation serial fragmentation or passive.Next, move to the tabs under source and set the end plate offset to 500 volts, capillary to 4, 500 volts, nebulizer to 3.0 bar, dry gas to 10.0 liters per minute and dry temp to 200 degrees celsius. In the syringe pump settings, assign the syringe as Hamilton one milliliter. Enable the option active and set the flow rate to 80.0 microliters per hour.In the MS/MS tab, under precursor ions, set the number of passive ramps to eight, minimum charge to one and maximum charge to five. In scheduling settings, enable the precursor repetitions and under active exclusion, check the box to enable and set the release to 0.40 minutes after. Do not adjust the collision energy settings and isolation with settings.Then click to enable the TIMS stepping box. To perform calibration for both mass to charge ratio and mobility domains, under the mass to charge ratio calibration settings, select one of the preloaded tuning mix profiles in the reference list box. To calibrate, load the syringe for the TOF with a tuning mix solution.In the calibration mode, on the right side of the window, select between linear, quadratic, and enhanced quadratic to achieve the best score<and click calibrate to get as close to 100%as possible. Next, calibrate for mobility following the steps demonstrated previously for calibrating mass to charge ratio. Once calibrated, save the MS method by selecting the method tab on the top bar.In the dropdown menu, select save as to generate a new MS method file.

timstof-msms-method-development-calibration-mobility-mass-for

Research

JoVE Journal

Chemistry

HPLC Method Development for Screening of Fentanyl Analogs

Begin by preparing the mobile phases for high-performance liquid chromatography, or HPLC. For mobile phase A, or MPA, use five millimolar ammonium formate in water with 0.05%formic acid. For mobile phase B, or MPB, use 0.05%formic acid in a one-to-one methanol acetonitrile mixture.To begin the HPLC method development, open the liquid chromatography or LC software and create a new protocol. Then, click the Edit Method button to edit the binary gradient. Under the Binary Gradient tab, set the timestamps and concentrations of MPA and MPB.Set the stop time to 18 minutes, the flow rate to 0.400 milliliters per minute, and the minimum and maximum pressure limits to zero and 6, 000 PSI respectively. To initialize the HPLC, insert the LC column and column guards in the HPLC column section. Carefully observe the minimum and maximum column pressures.

Data Independent Acquisition &#40;dia&#41; Parallel Accumulation-Serial Fragmentation Method for Screening of Fentanyl Analogs

Once a TIMS TOF MS/MS data set has been collected in the data dependent acquisition, or DDA, passive mode, run the data-independent acquisition, or DIA, on the ion mobility software application. To do so, first load the previously saved DDA method. Change the mass spectrometry to DIA passive fragmentation.Leave all other settings the same. Under the MS/MS tab, select edit windows, then use the open analysis button located at the top of the DIA pop-up window and load the previously saved DDA data set. When a heat map appears at the bottom left, displaying a polygon of windows running diagonally across the graph, click and drag to resize the polygon so that it fits the data in the heat map.Set the number of mobility windows in the per mass width and the overlap. Click calculate windows on the bottom right of the pop-up to see the windows displayed with the new settings. Once suitable, click on apply.

Processing HPLC Ion Mobility TOF Data of Fentanyl Analog Screening

To process the HPLC ion mobility TOF data of fentanyl analog screening, begin by checking the calibration in the data analysis software. To do so, right-click on the file name under the analysis box and select properties. When the window, labeled File Name_Analysis Properties appears, select calibration status.From the drop-down box, select instrument calibration for the mass spectrometer and confirm that the error is no greater than 1 ppm. Then, select initial mobility calibration and confirm that the error is no greater than 1%To prepare a chromatogram in the data analysis software, right-click on chromatogram under the file name and select edit chromatogram. In type, click the drop-down box and select extracted ion chromatogram.Under filter, select all MS, and for scan mode, select all. If using masses to extract an ion, insert the theoretical mass-to-charge ratio of the molecule of interest, set the polarity to positive mode, and add the ion to the list. If using a formula to extract an ion, insert the formula for the molecule as well as the ion forms of interest for the chromatogram.For the mass spectrum, make sure the chromatogram is selected and right-click at the baseline of one edge of the compound peak and drag to the other edge of the peak. To generate a mobilogram, right-click on the left tab titled mobilogram and select edit mobilogram. When the window titled edit mobilogram traces appears, begin editing the chromatogram.In the retention time input, add the retention time range of the peak of interest. Once the parameters are selected, click add, followed by okay at the top right. To generate the compound spectra, at the bottom of the spectrum view window, sequentially select profile MS and fragment MS.At spectrum view, right-click and select copy compound spectra.To process the data, click on find, then select compounds manually, chromatogram, or mobilogram. Left-click and drag to highlight the desired peak to yield important information on the molecule of interest. Screening of the four isomers in a particular fentanyl analog screening kit revealed that the methoxyacetyl fentanyl and fentanyl carbamate separated well in the LC and mobility domains from the paramethoxyacetyl fentanyl and B-hydroxyl fentanyl.However, a clear separation of the latter two was achieved in their fragmentation patterns. Screening of three isomers in another kit showed that alpha-methyl-thiofentanyl and trans-3-methyl-thiofentanyl separated in the LC domain, but not in the mobility domain. However, trans-3-methyl-thiofentanyl and cis-3-methyl-thiofentanyl separated in the mobility domain and not in the LC domain.This data indicated the complementarity of the orthogonal LC and mobility domains in improving the identification of the isometric species.