The author would like to acknowledge the initial support of Dr Cesar Ramirez during initial method developments.

1. Sample preparation
<ol>
	<li>Store the fentanyl analog screening kit at -20 &#176;C once they have been received. Resuspend each sample to a final concentration of 400 &#181;g/mL by the addition of 500 &#181;L of pure LC/MS grade methanol.</li>
	<li>Mix using a plate mixer or vortexer at 400 rpm for 1 h or vortex at medium speed for 15 min. After resuspension store the vials at -20 &#176;C.</li>
	<li>Dilute each standard to a final concentration of 1 ng/mL using pure LC/MS grade methanol.</li>
	<li>Group the standards into groups, such that each group does not contain any isomeric standards. See Supplementary Table 1 for the breakdown of the 14 groups of fentanyl standards.</li>
</ol>
2. HPLC mobile phases preparation
<ol>
	<li>Prepare mobile phase A (MPA) using 5 mM/L ammonium formate (NH4HCO2) in H2O with 0.05% formic acid (85%).
	<ol>
		<li>Weigh out 0.078 g of NH4HCO2, pour into a 250 mL volumetric flask and fill with water to about 2/3rd. Swirl to mix well.</li>
		<li>Once fully dissolved, add 0.125 mL of formic acid (85%). Fill with water to the fill line (250 mL) and mix.</li>
	</ol>
	</li>
	<li>Prepare mobile phase B (MPB) using 0.05% formic acid in a 1:1 methanol acetonitrile mixture.
	<ol>
		<li>Pour 125 mL of acetonitrile into a volumetric flask and add methanol until it reaches the fill line.</li>
		<li>Swirl to mix well. Add 0.125 mL of 0.05% formic acid to the flask and mix to homogenize.</li>
	</ol>
	</li>
</ol>
3. HPLC method development
<ol>
	<li>Open the LC software to the home screen. In the sample table, located at the center of the window, create a new protocol.
	<ol>
		<li>Annotate the vial column with the sample location. Annotate the sample ID column using the format YYYYMMDD_NAME (representative descriptor). Annotate the volume column with the desired the injection volume (15 &#181;L).</li>
	</ol>
	</li>
	<li>Annotate the data path column with the location where the data will be saved to.</li>
	<li>HPLC method input
	<ol>
		<li>Select New from the bar in the sample table located in the middle of the screen. Under the separation method, select the Drop-down Arrow and click New Method in the pop-up window.</li>
		<li>A new window will pop up labeled separation method, here edit the acquisition time or the instrument control framework (ICF) system method.</li>
		<li>Double click the Edit Method button for the ICF system to open up the popup to edit the binary gradient, as seen in Figure 1.</li>
		<li>Under the binary gradient tab, a visual representation of the gradient is on the left and the breakdown of the gradient is on the right, set the time stamps, the flow rate and the MPA and MPB concentrations.</li>
		<li>Ensure the stop time is set to 18 min, the flow rate to 0.400 mL/min, and pressure limit min and max at 0 psi and 6000 psi.</li>
		<li>Set the time stamp concentrations as following: 
		At 0.00 min, set B concentration to 20.0. 
		At 1.50 min, set B concentration to 25.0. 
		At 3.00 min, set B concentration to 27.0. 
		At 6.00 min, set B concentration to 27.0. 
		At 6.50 min, set B concentration to 30.0. 
		At 7.00 min, set B concentration to 95.0. 
		At 16.00 min, set B concentration to 95.0. 
		At 16.50 min, set B concentration to 20.0.</li>
	</ol>
	</li>
</ol>
4. Initialization of the HPLC
<ol>
	<li>In the HPLC column section, insert the LC column (monolithic C18 HPLC column 100 mm x 4.6 mm) and column guards (guard column 5 mm x 4.6 mm). Pay attention to the minimum and maximum column pressure.</li>
	<li>Run a sample blank (same buffer solution) to test for any leaks and create a baseline. Look for leaks. Leaks could be observed by column pressure fluctuations. If there are leaks determine where they might be coming from and tighten the connections.</li>
	<li>Run a sample blank (same buffer solution) using the desired LC program to precondition the column.</li>
</ol>
5. timsTOF MS/MS method development
<ol>
	<li>Open the timsControl application. On the left side of the window are the MS and TIMS Settings.
	<ol>
		<li>Under MS settings set the scan beginning and ending to 50 m/z and 1800 m/z, respectively. Select positive mode for the ion polarity and select parallel accumulation-serial fragmentation for the scan mode.</li>
		<li>Under TIMS Settings set the Mode to custom, 1/K0 start to 0.40 Vs/cm2, 1/K0 end to 1.85 Vs/cm2, ramp time set to 150.0 ms, and MS averaging set to 1.</li>
	</ol>
	</li>
	<li>In the lower selection of tabs under Source perform the following changes in the two setting boxes.
	<ol>
		<li>In the source, set the end plate offset to 500 V, capillary to 4500 V, nebulizer to 3.0 bar, dry gas to 10.0 L/min, and dry temp to 200 &#176;C.</li>
		<li>In the syringe pump settings, ensure the syringe is Hamilton 1 mL, active is enabled, and the flow rate set to 80.0 &#181;L/h.</li>
	</ol>
	</li>
	<li>In the Tune tab perform the following changes in the setting tabs for General, Processing, and TIMS.
	<ol>
		<li>Under general settings tab, setup the following sections: transfer, collision cell, quadrupole, focus pre TOF, and detection.
		<ol>
			<li>Under the transfer settings, set the deflection 1 delta to 70.0 V, funnel 1 RF to 341.0 Vpp, CID energy to 0.0 eV, funnel 2 RF to 300.0 Vpp, multipole RF to 300.0 Vpp.</li>
			<li>Under the collision cell settings, ensure that the collision energy is set to 6.0 eV and the collision RF to 1200.0 Vpp.</li>
			<li>Under the quadrupole settings, ensure ion energy is set to 6.0 eV and low mass to 250.00 m/z.</li>
			<li>Under the focus pre TOF settings, set transfer time to 75.0 &#181;s and pre-pulse storage to 5.0 &#181;s.</li>
		</ol>
		</li>
		<li>Under the processing settings tab, set up the following sections: mass spectra peak detection and mobility peak detection.
		<ol>
			<li>In the mass spectra peak settings, unselect sum of intensities (area) and set the absolute threshold to 667.</li>
		</ol>
		</li>
		<li>Under the TIMS tab, set up the following sections: offsets, ion change control, and advanced parameters.
		<ol>
			<li>Under the offsets setting set &#916;t1 to -20.0 V, &#916;t2 to -100.0 V, &#916;t3 to 40.0 V, &#916;t4 to 80.0 V, &#916;t5 to 0.0 V, &#916;t6 to 120.0, and collision cell in to 250.0 V.</li>
			<li>Under the ion charge control settings, click the box to enable and set the target intensity to 5.00 M.</li>
			<li>Under the advanced parameters, enable the lock accumulation to mobility range.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>In the MS/MS tab set the Scan mode to parallel accumulation-serial fragmentation and set up the following sections: precursor ions, scheduling, active exclusion, collision energy settings, isolation width settings, and TIMS stepping.
	<ol>
		<li>Under precursor ions, set the number of parallel accumulation-serial fragmentation ramps to 8, charge minimum to 1, and charge maximum to 5.</li>
		<li>Under scheduling settings, enable the precursor repetitions. Under active exclusion, check the box to enable and set the release to 0.40 min after.</li>
		<li>Do not adjust the collision energy settings and isolation width settings. Click the TIMS Stepping Box to enable.</li>
	</ol>
	</li>
</ol>
6. Mobility and mass calibration
<ol>
	<li>Perform calibration for both m/z and mobility domains. Under the m/z calibration settings, select one of the preloaded tuning mix profiles in the reference list box that appear in the drop-down menu.</li>
	<li>To calibrate, load the syringe for the TOF with a tuning mix solution. On the right side of the window, use the settings titled calibration mode for the various calibration types to be set to achieve the highest score.</li>
	<li>Ensure that the score is as close to 100% as possible. Switch between linear, quadradic and enhanced quadratic to achieve the best score.</li>
	<li>Calibrate for mobility following steps 6.1-6.3 used for calibrating m/z.</li>
	<li>Once calibrated, save the MS method by selecting the Method tab on the top bar. In the drop-down menu, select Save As to generate a new MS method file.</li>
</ol>
7. Creating data independent acquisition (dia) parallel accumulation-serial fragmentation method
<ol>
	<li>Once a data set has been collected in data dependent acquisition (dda) parallel accumulation-serial fragmentation, run the experiments in dia. Open the ion mobility software application and load the dda method saved in step 6.5.</li>
	<li>Leave all the settings the same expect for the MS setting, change this from parallel accumulation-serial fragmentation to dia- parallel accumulation-serial fragmentation.</li>
	<li>On the bottom of the panel, select the MSMS tab and then click on Window Editor to open the dia window popup shown in Figure 2.</li>
	<li>Load the previously saved dda data set (.m file) using the open analysis button located at the top of the popup window.</li>
	<li>A heat map appears at the bottom left displaying windows with 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 (corners are selectable by double clicking on the lateral lines a point will appear that can be dragged and resized).</li>
	<li>To the right of the windows are the window settings. Set the mass width (50 is go to) and set the horizontal overlap, both in Da.</li>
	<li>Set the number of mobility windows in per mass width and the vertical overlap.</li>
	<li>Click Calculate Windows on the bottom right of the popup to see the windows displayed with the new settings. Once suitable, click Apply dia-PASEF Windows to Method. This will close the popup bringing it back to the home screen.</li>
</ol>
8. HPLC ion mobility TOF data processing
<ol>
	<li>Open the data analysis software. At the top left corner click on the File tab and select Open from the drop down. In the new files window select the files of choice and click Open.</li>
	<li>Check the calibration. Right click on the file name under the analysis box and select Properties. A window labeled file name_analysis properties will appear. Select Calibration Status.</li>
	<li>From the drop-down box, select Instrument Calibration for the Mass Spectrometer. Confirm that the error is no greater than 1 &#181;g/mL. Select Initial Mobility Calibration and confirm that the error is no greater than 1 &#181;g/mL.</li>
	<li>Prepare a chromatogram in the data analysis software as described below.
	<ol>
		<li>Right click on Chromatogram under the file name, select Edit Chromatogram. In type, click the drop-down box and select Extracted Ion Chromatogram.</li>
		<li>Under filter, select All MS and with scan mode select All. This is to view the peaks for the molecule of interest rather than only its fragments.</li>
		<li>Below this, look for the two filter options for molecule selection: masses or formula highlighted in blue.</li>
		<li>If using masses to extract an ion, insert the theoretical m/z of the molecule of interest. For this case select (mass for representative result) m/z.</li>
		<li>If using formula to extract an ion, insert the formula for the molecule as well as the ion forms of interest for the chromatogram. In this case, select the protonated ion, [M+H]+.</li>
		<li>Set the polarity to positive mode.</li>
	</ol>
	</li>
	<li>Mass spectrum
	<ol>
		<li>Right click at the baseline of the peak of the compound and drag to the other edge of the peak. This will create a mass spectrum of fragments within that retention time.</li>
	</ol>
	</li>
	<li>Generation of Mobilogram
	<ol>
		<li>Right click on the left tab titled mobilogram and select Edit Mobilogram. A window titled edit mobilogram traces will appear. Follow steps 8.4.2-8.4.7 for editing the chromatogram.</li>
		<li>In the retention time input, add the retention time range of the peak of interest.</li>
		<li>Once the parameters are selected, click Add followed by Ok at the top right of the edit mobilogram traces window.</li>
		<li>After a short amount of time, the software will process the desired selections and output a chromatogram. Repeat steps 8.6.1-8.6.3 for all ions in the mixture.</li>
		<li>Generating the compound spectra
		<ol>
			<li>At the bottom of the spectrum view window, select Profile MS and Fragment MS. This will allow to include ions from full scan and PASEF.</li>
			<li>At spectrum view, right click and select Copy Compound Spectra. With the spectrum data that appears at the right, find more information on the compound such as resolution resolving power, intensity, and signal to noise ratio (S/N).</li>
		</ol>
		</li>
		<li>Data processing
		<ol>
			<li>To process the data, manually integrate the chromatogram and mobility peaks to yield important information on the molecule of interest.</li>
			<li>Right click Find and select Integrate only chromatogram or mobilogram. Left click and drag to highlight the desired peak. The information will be displayed which includes retention time, area, S/N, and mobility.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Fentanyl+and+18+Novel+Fentanyl+Analogs+and+Metabolites+by+LC-MS-MS,+and+report+of+Fatalities+Associated+with+Methoxyacetylfentanyl+and+Cyclopropylfentanyl.">Analysis of Fentanyl and 18 Novel Fentanyl Analogs and Metabolites by LC-MS-MS, and report of Fatalities Associated with Methoxyacetylfentanyl and Cyclopropylfentanyl.</a> Journal of Analytical Toxicology. 42 (9), 592-604 (2018).</li><li>Lurie, I. S., Berrier, A. L., Casale, J. F., Iio, R., Bozenko, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+illicit+fentanyl+using+UHPLC-MS/MS.">Profiling of illicit fentanyl using UHPLC-MS/MS.</a> Forensic Science International. 220 (1-3), 191-196 (2012).</li><li>Krajewski, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+fentanyl+analog+screening+kit+toward+the+identification+of+emerging+synthetic+opioids+in+human+plasma+and+urine+by+LC-QTOF.">Application of the fentanyl analog screening kit toward the identification of emerging synthetic opioids in human plasma and urine by LC-QTOF.</a> Toxicology Letters. 320, 87-94 (2020).</li><li>Pirok, B. W. J., Stoll, D. R., Schoenmakers, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Developments+in+Two-Dimensional+Liquid+Chromatography:+Fundamental+Improvements+for+Practical+Applications.">Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications.</a> Analytical Chemistry. 91 (1), 240-263 (2018).</li><li>Keller, T., Keller, A., Tutsch-Bauer, E., Monticelli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+ion+mobility+spectrometry+in+cases+of+forensic+interest.">Application of ion mobility spectrometry in cases of forensic interest.</a> Forensic Science International. 161 (2-3), 130-140 (2006).</li><li>Klingberg, J., Keen, B., Cawley, A., Pasin, D., Fu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+high-resolution+mass+spectrometric+analyses+of+new+psychoactive+substances.">Developments in high-resolution mass spectrometric analyses of new psychoactive substances.</a> Archives of Toxicology. 96 (4), 949-967 (2022).</li><li>Fleming, S. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+U-47700,+a+Novel+Synthetic+Opioid,+in+Human+Urine+by+LC-MS-MS+and+LC-QToF.">Analysis of U-47700, a Novel Synthetic Opioid, in Human Urine by LC-MS-MS and LC-QToF.</a> Journal of Analytical Toxicology. 41 (3), 173-180 (2017).</li><li>Guale, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+LC-TOF-MS+screening+for+drugs,+metabolites,+and+collateral+compounds+in+forensic+toxicology+specimens.">Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.</a> Journal of Analytical Toxicology. 37 (1), 17-24 (2013).</li><li>Palmquist, K. B., Swortwood, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data-independent+screening+method+for+14+fentanyl+analogs+in+whole+blood+and+oral+fluid+using+LC-QTOF-MS.">Data-independent screening method for 14 fentanyl analogs in whole blood and oral fluid using LC-QTOF-MS.</a> Forensic Science International. 297, 189-197 (2019).</li><li>Adams, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+isomeric+opioids+in+urine+using+LC-TIMS-TOF+MS.">Analysis of isomeric opioids in urine using LC-TIMS-TOF MS.</a> Talanta. 183, 177-183 (2018).</li></ol>

Matthew Willetts and Melvin A. Park are employees of Bruker Daltonics Inc, the manufacturer of the timsTOF Pro2 commercial instrument. All the other authors declare no conflicts of interest.

The analytical separation of biological samples containing high isomeric content can be analytically challenging. In this paper, the method described aims to characterize 29 isomer sets, for a total of 185 analogs from a 250 opioid standard kit. During the test group preparation, it is important to ensure that there are no two analogs with m/z that cannot be experimentally distinguished. The data described here uses standards not from a human-based matrix, if this method is to be used in a field experiment; additional trials using the matrix of interest are necessary to ensure the method applicability. The matrix might cause the analytes to ionize differently than they do in a matrix free experiment making it imperative to determine what prevalent ion forms will be observed. In the analysis, the identification is based on the isotopic patterns of the most abundant forms (e.g., [M+H]+1 and [M+Na]+1).
The choice of LC program is critical in cases where the isomers are not separated in the mobility domain. Decreasing the slope of the gradient can increase the specificity of the method by increasing the difference between the chromatographic peaks.
During data analysis it is important that nonrelevant signals are ignored when determining the retention time and mobility. The advantages of HRMS allow for narrow m/z windows (typical mass resolution of ~50,000) with additional mobility filtering, thus reducing the chemical noise.
The method described here provides a reference library for the screening for fentanyl analogs from an unknown sample. The reported RT, K, isotopic and MS fragmentation pattern are used as qualifiers during the data processing and unknown assignment. When compared to traditional MRM LC-QQQ analysis, while similar LOD are achievable13,19,27, the unique advantage is to screen for alternative opioids not included in the database generated using the FID kit. Each new opioid will require the analysis using a synthetic standard for its final validation.

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.

<table><tbody><tr><td>Ammonium formate for HPLC</td><td>Fluka</td><td>17843-50G</td><td></td></tr><tr><td>Eppendorf&amp;nbsp;Snap-Cap Microcentrifuge Safe-Lock Tubes</td><td>Fisher</td><td>05-402-25</td><td></td></tr><tr><td>ESI-L Low Concentration Tuning mix</td><td>Agilent</td><td>G1969-85000</td><td></td></tr><tr><td>Fentanyl Analog Screening (FAS) Kit&amp;nbsp;</td><td>Cayman Chemical</td><td>9003237, 9003286, 9003380, 9003381</td><td>kit of 250 snthetic opioids, 210 fentanyl analogs, broken up into one kit and emergent panel versions 1-4</td></tr><tr><td>Formic acid Optima LC/MS</td><td>Fisher</td><td>A117-50</td><td></td></tr><tr><td>Onyx guard column (5 x 4.6 mm)&amp;nbsp;</td><td>Phenomenex</td><td>CHO-7649</td><td>guard column for C18 columns</td></tr><tr><td>Onyx monolithic C18 HPLC column (100 x 4.6 mm)</td><td>Phenomenex</td><td>CHO-7643</td><td>reverse phase C18 LC column</td></tr><tr><td>Optima grade acetonitrile&amp;nbsp;</td><td>Fisher</td><td>A996-4</td><td></td></tr><tr><td>Optima grade methanol</td><td>Fisher</td><td>A454-4</td><td></td></tr><tr><td>Optima grade water</td><td>Fisher</td><td>W7-4</td><td></td></tr><tr><td>Pipette</td><td>Fisher</td><td>05-719-510</td><td>kit of 1-10 &amp;micro;L, 10-100 &amp;micro;L, and 100-1000 &amp;micro;L pipette</td></tr><tr><td>Pipette tips 10&amp;micro;L</td><td>Fisher</td><td>94060100</td><td></td></tr><tr><td>Pipette tips 1000&amp;micro;L</td><td>Fisher</td><td>94056710</td><td></td></tr><tr><td>Pipette tips 200&amp;micro;L</td><td>Fisher</td><td>94060310</td><td></td></tr><tr><td>Plate mixer</td><td>IKA&amp;nbsp;</td><td>MS 3 D S1</td><td>IKA MS 3 digtital</td></tr><tr><td>Prominence LC-20 CE ultrafast liquid chromatograph</td><td>Shimadzu, Japan</td><td></td><td>equiped with DGU-20A5, LC-20AD, SIL-20AC, CTO-20A, SPD-M20A, CBM-20A, SPD-20A</td></tr><tr><td>timsTOF Pro</td><td>Bruker Daltonics Inc., Billerica, MA</td><td></td><td>timsTOF instrument with PASEF&amp;nbsp;</td></tr></tbody></table>

fentanyl analog screening using lc-tims-tof ms/ms

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States. Most recent cases of fentanyl-related overdose are linked to illicitly manufactured fentanyl and its associated extreme potency. In the present work, we describe a high-throughput analytical protocol for the screening of fentanyl analogs. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry allow for the separation and assignment of hundreds of fentanyl analogs from a single sample in a single scan. The described approach takes advantage of the recent development of data-dependent acquisition and data-independent acquisition using parallel accumulation in the mobility trap followed by sequential fragmentation using collision-induced dissociation. The fentanyl analogs are confidently assigned based on their retention time, mobility, and MS fragmentation pattern.

The fentanyl analog screening kit of 250 analogs standards were divided into 14 groups: 12 groups of 17 analogs and 2 groups of 16 analogs, to avoid m/z interferences. Each analog is further characterized by their m/z, retention time (RT), mobility (K), and MS/MS fragmentation pattern.
Examples of isomeric separations are shown in Figure 3 and Figure 4 for the C22H28N2O2 and C21H28N2OS chemical formula, respectively. 
The fentanyl analog screening kit contains four isomers with the C22H28N2O2 chemical formula (see Figure 3). Inspection of Figure 4 shows the [M+H]+ extracted ion chromatogram, mobilograms, isotopic pattern and MS/MS fragmentation pattern. The methoxyacetyl fentanyl and fentanyl carbamate separate well in the LC and mobility domains from the para-methoxy acetyl fentanyl and B-hydroxyl fentanyl. However, a clear separation between the para-methoxy acetyl fentanyl and B-hydroxyl fentanyl is achieved by their fragmentation patterns.
The fentanyl analog screening kit contains three isomers with the C21H28N2OS chemical formula. Inspection of Figure 4 shows the [M+H]+ extracted ion chromatogram, mobilograms, isotopic pattern and MS/MS fragmentation pattern. The a-methyl thiofentanyl and (&#177;)-trans-3-methyl thiofentanyl separate in the LC domain but not in the mobility domain. Moreover, (&#177;)-trans-3-methyl thiofentanyl and (&#177;)-cis-3-methyl thiofentanyl separate in the mobility domain and not in the LC domain. This shows the complementarity of the LC and mobility separation improving identification of the isomeric species through the orthogonality of domains. Figure 4D summarizes this result provided by LC and mobility.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65562/65562fig01.jpg" /> 
Figure 1: Gradient ramp in the software. ICF system method dialog gradients time points applied during separation dependent on the percentage of B concentration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65562/65562fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65562/65562fig02.jpg" /> 
Figure 2: dia-PASEF window settings. Under dia-PASEF MS methods the MS/MS settings are unique. The graph on the left shows the window polygons adjusted from the data recorded in the dda-PASEF method. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65562/65562fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65562/65562fig03.jpg" /> 
Figure 3: Typical extracted ion chromatogram, extracted mobilogram, and amplified MS projection for isomeric opioids. (A) Structures of methoxyacetyl fentanyl, para-methoxy acetyl fentanyl, &#946;-hydroxy fentanyl, fentanyl carbamate and the respective (B) typical retention time, (C) typical mobility, (D) isomeric signal, (E) major fragments and (F) representation of the fragmentation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65562/65562fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65562/65562fig04.jpg" /> 
Figure 4: Typical extracted ion chromatograms and extracted mobilograms for isomeric opioids. (A) Structures of &#945;-methyl thiofentanyl, (&#177;)-trans-3-methyl thiofentanyl, and (&#177;)-cis-3-methyl thiofentanyl. For the respective isomers (B) typical retention time, (C) typical mobility, (D) orthogonality between the retention time and mobility. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65562/65562fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Mixture groups. This table outlines which opioids are in each of the 14 mixtures created for this procedure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65562/Supplementary table 1_RE.xlsx" target="_blank">Please click here to download this File.</a>

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

A method for the screening of fentanyl analogs based on their retention time, mobility, and mass spectrometry fragmentation pattern.

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

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its ...

fentanyl-analog-screening-using-lc-tims-tof-msms

Nanyang Technological University

Research

Without Section

JoVE Journal

Chemistry

2.0K Views.  Florida International University. De reikwijdte van ons onderzoek is het ontwikkelen van een methode om isomere analogen te onderscheiden en te karakteriseren. Dit onderzoek heeft tot doel te onderzoeken of er een methode kan worden ontwikkeld om met vertrouwen een grote grootheid, met name structurele isomeren, te differentiëren. Dit protocol omvat vier karakteriseringswijzen die nog niet eerder zijn gekoppeld.Het gebruik van vier orthogonale identificatiemethoden voor de analogen zo...

Video: Auteur in de schijnwerpers: een efficiënte methodologie om fentanyl-analogen met vertrouwen te differentiëren en te karakteriseren 

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Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the local to federal levels. The continuing evolution of PFAS chemistries presents a challenge to the environmental monitoring, where ongoing development of targeted methods necessarily lags the discovery of new chemical compounds. There is a need, therefore, to have forward-looking methodologies that can detect emerging and unexpected compounds, monitor these species over time, and resolve details of their chemical structure to enable future work in human health. To this end, non-targeted analysis by high-resolution mass spectrometry offers a broad base detection approach that can be combined with almost any sample preparation scheme and provides significant capabilities for compound identification after detection. Herein, we describe a solid-phase extraction (SPE) based sample concentration method tuned for shorter chain and more hydrophilic PFAS chemistries, such as per fluorinated ether acids and sulfonates, and describe analysis of samples prepared in this fashion in both targeted and non-targeted modes. Targeted methods provide superior quantification when reference standards are available but are intrinsically limited to expected compounds when performing analysis. In contrast, a non-targeted approach can identify the presence of unexpected compounds and provide some information about their chemical structure. Information about chemical features can be used to correlate compounds across sample locations and track abundance and occurrence over time.

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Environment

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Natural Product Discovery with LC-MS/MS Diagnostic Fragmentation Filtering: Application for Microcystin Analysis

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural features, many compounds within the same class undergo similar MS/MS fragmentation and have several identical product ions and/or neutral losses. The purpose of diagnostic fragmentation filtering (DFF) is to efficiently detect all compounds of a given class in a complex extract by screening non-targeted LC-MS/MS datasets for MS/MS spectra that contain class specific product ions and/or neutral losses. This method is based on a DFF module implemented within the open-source MZmine platform that requires sample extracts be analyzed by data-dependent acquisition on a high-resolution mass spectrometer such as quadrupole Orbitrap or quadrupole time-of-flight mass analyzers. The main limitation of this approach is the analyst must first define which product ions and/or neutral losses are specific for the targeted class of natural products. DFF allows for the subsequent discovery of all related natural products within a complex sample, including new compounds. In this work, we demonstrate the effectiveness of DFF by screening extracts of Microcystis aeruginosa, a prominent harmful algal bloom causing cyanobacteria, for the production of microcystins.

Diagnostic fragmentation filtering, implemented into MZmine, is an elegant, post-acquisition approach to screen LC-MS/MS datasets for entire classes of both known and unknown natural products. This tool searches MS/MS spectra for product ions and/or neutral losses that the analyst has defined as being diagnostic for the entire class of compounds.

Natural products are often biosynthesized as mixtures of structurally similar compounds, rather than a single compound. Due to their common structural ...

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

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

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