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>...

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

<ol>
	<li>Gladden, R. M., Martinez, P., Seth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fentanyl+Law+Enforcement+Submissions+and+Increases+in+Synthetic+Opioid-Involved+Overdose+Deaths+-+27+States,+2013-2014.">Fentanyl Law Enforcement Submissions and Increases in Synthetic Opioid-Involved Overdose Deaths - 27 States, 2013-2014.</a> Morbidity and Mortality Weekly Report. 65 (33), 837-843 (2016).</li><li>Rudd, R. A., Seth, P., David, F., Scholl, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increases+in+Drug+and+Opioid-Involved+Overdose+Deaths+-+United+States,+2010-2015.">Increases in Drug and Opioid-Involved Overdose Deaths - United States, 2010-2015.</a> Morbidity and Mortality Weekly Report. 65, 1445-1452 (2016).</li><li>Department of Justice Drug Enforcement Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+of+Abuse:+A+DEA+Resource+Guide.">Drugs of Abuse: A DEA Resource Guide.</a> Department of Justice Drug Enforcement Administration. , (2022).</li><li>Vardanyan, R. S., Hruby, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fentanyl-related+compounds+and+derivatives:+current+status+and+future+prospects+for+pharmaceutical+applications.">Fentanyl-related compounds and derivatives: current status and future prospects for pharmaceutical applications.</a> Future medicinal chemistry. 6 (4), 385-412 (2014).</li><li>Center for Forensic Science Research and Education (CFSRE). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q4+2022+NPS+Opioids+Trend+Report.">Q4 2022 NPS Opioids Trend Report.</a> Center for Forensic Science Research and Education (CFSRE). , (2022).</li><li>Drug Enforcement Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fentanyl+SIgnature+Profiling+Program+Report.">Fentanyl SIgnature Profiling Program Report.</a> Drug Enforcement Administration. , (2019).</li><li>Drug Enforcement Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+a+Chemical+Precursor+Used+in+the+Illicit+Manufacture+of+Fentanyl+as+a+List+I+Chemical.">Control of a Chemical Precursor Used in the Illicit Manufacture of Fentanyl as a List I Chemical.</a> Drug Enforcement Administration. , (2007).</li><li>Valdez, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+Chromatography-Mass+Spectrometry+Analysis+of+Synthetic+Opioids+Belonging+to+the+Fentanyl+Class:+A+Review.">Gas Chromatography-Mass Spectrometry Analysis of Synthetic Opioids Belonging to the Fentanyl Class: A Review.</a> Critical Reviews in Analytical Chemistry. 52 (8), 1938-1968 (2022).</li><li>Churley, M., Cuadra-Rodriguez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+Note:+Screen+More+Drugs+with+the+Agilent+GC/MS+Toxicology+Analyzer+with+a+High+Efficiency+Source.">Application Note: Screen More Drugs with the Agilent GC/MS Toxicology Analyzer with a High Efficiency Source.</a> Agilent Technologies Incorporation. , (2017).</li><li>Gerace, E., Salomone, A., Vincenti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+Approaches+in+Fatal+Intoxication+Cases+Involving+New+Synthetic+Opioids.">Analytical Approaches in Fatal Intoxication Cases Involving New Synthetic Opioids.</a> Curr Pharm Biotechnol. 19 (2), 113-123 (2018).</li><li>McIntyre, I. M., Trochta, A., Gary, R. D., Malamatos, M., Lucas, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Acute+Acetyl+Fentanyl+Fatality:+A+Case+Report+With+Postmortem+Concentrations.">An Acute Acetyl Fentanyl Fatality: A Case Report With Postmortem Concentrations.</a> Journal of Analytical Toxicology. 39 (6), 490-494 (2015).</li><li>Marchei, E., 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=New+synthetic+opioids+in+biological+and+non-biological+matrices:+A+review+of+current+analytical+methods.">New synthetic opioids in biological and non-biological matrices: A review of current analytical methods.</a> Trends in Analytical Chemistry. 102, 1-15 (2018).</li><li>Shoff, E. N., Zaney, M. E., Kahl, J. H., Hime, G. W., Boland, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+Identification+of+Fentanyl+Analogs+and+Other+Opioids+in+Postmortem+Cases+by+UHPLC-Ion+Trap-MSn.">Qualitative Identification of Fentanyl Analogs and Other Opioids in Postmortem Cases by UHPLC-Ion Trap-MSn.</a> Journal of Analytical Toxicology. 41 (6), 484-492 (2017).</li><li>Drug Enforcement Administration. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+Working+Group+for+the+Analysis+of+Seized+Drugs+(SWGDRUG)+Recommendations.">Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) Recommendations.</a> Drug Enforcement Administration. , (2022).</li><li>Peters, F. T., 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=A+systematic+comparison+of+four+different+workup+procedures+for+systematic+toxicological+analysis+of+urine+samples+using+gas+chromatography-mass+spectrometry.">A systematic comparison of four different workup procedures for systematic toxicological analysis of urine samples using gas chromatography-mass spectrometry.</a> Analytical and Bioanalytical Chemistry. 393 (2), 735-745 (2009).</li><li>Remane, D., Meyer, M. R., Peters, F. T., Wissenbach, D. K., Maurer, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+simple+procedure+for+liquid-liquid+extraction+of+136+analytes+from+different+drug+classes+for+development+of+a+liquid+chromatographic-tandem+mass+spectrometric+quantification+method+in+human+blood+plasma.">Fast and simple procedure for liquid-liquid extraction of 136 analytes from different drug classes for development of a liquid chromatographic-tandem mass spectrometric quantification method in human blood plasma.</a> Analytical and Bioanalytical Chemistry. 397 (6), 2303-2314 (2010).</li><li>Cayman Chemical. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supplemental+Material:+Differentiation+of+Isobaric+and+Isomeric+Fentanyl+Analogs+by+Gas+Chromatography-Mass+Spectrometry+(GC-MS).">Supplemental Material: Differentiation of Isobaric and Isomeric Fentanyl Analogs by Gas Chromatography-Mass Spectrometry (GC-MS).</a> Cayman Chemical. , (2018).</li><li>Armenian, P., Vo, K. T., Barr-Walker, J., Lynch, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fentanyl,+fentanyl+analogs+and+novel+synthetic+opioids:+A+comprehensive+review.">Fentanyl, fentanyl analogs and novel synthetic opioids: A comprehensive review.</a> Neuropharmacology. 134, 121-132 (2018).</li><li>Zhang, Y., 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=Development+and+application+of+a+High-Resolution+mass+spectrometry+method+for+the+detection+of+fentanyl+analogs+in+urine+and+serum.">Development and application of a High-Resolution mass spectrometry method for the detection of fentanyl analogs in urine and serum.</a> Journal of Mass Spectrometry and Advances in the Clinical Lab. 26, 1-6 (2022).</li><li>Concheiro, M., Chesser, R., Pardi, J., Cooper, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+Toxicology+of+New+Synthetic+Opioids.">Postmortem Toxicology of New Synthetic Opioids.</a> Frontiers in Pharmacology. 9, 1210 (2018).</li><li>Mohr, A. L., 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+Novel+Synthetic+Opioids+U-47700,+U-50488+and+Furanyl+Fentanyl+by+LC-MS/MS+in+Postmortem+Casework.">Analysis of Novel Synthetic Opioids U-47700, U-50488 and Furanyl Fentanyl by LC-MS/MS in Postmortem Casework.</a> Journal of Analytical Toxicology. 40 (9), 709-717 (2016).</li><li>Moody, M. T., Diaz, S., Shah, P., Papsun, D., Logan, B. 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+analogs+and+novel+synthetic+opioids+in+blood,+serum/plasma,+and+urine+in+forensic+casework.">Analysis of fentanyl analogs and novel synthetic opioids in blood, serum/plasma, and urine in forensic casework.</a> Drug Testing and Analysis. 10 (9), 1358-1367 (2018).</li><li>Seither, J., Reidy, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confirmation+of+Carfentanil,+U-47700+and+Other+Synthetic+Opioids+in+a+Human+Performance+Case+by+LC-MS-MS.">Confirmation of Carfentanil, U-47700 and Other Synthetic Opioids in a Human Performance Case by LC-MS-MS.</a> Journal of Analytical Toxicology. 41 (6), 493-497 (2017).</li><li>Strayer, K. E., Antonides, H. M., Juhascik, M. P., Daniulaityte, R., Sizemore, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC-MS/MS-Based+Method+for+the+Multiplex+Detection+of+24+Fentanyl+Analogues+and+Metabolites+in+Whole+Blood+at+Sub+ng+mL(-1)+Concentrations.">LC-MS/MS-Based Method for the Multiplex Detection of 24 Fentanyl Analogues and Metabolites in Whole Blood at Sub ng mL(-1) Concentrations.</a> ACS Omega. 3 (-1), 514-523 (2018).</li><li>Kahl, J. H., Gonyea, J., Humphrey, S. M., Hime, G. W., Boland, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+of+Fentanyl+and+Six+Fentanyl+Analogs+in+Postmortem+Specimens+by+UHPLC-MS-MS.">Quantitative Analysis of Fentanyl and Six Fentanyl Analogs in Postmortem Specimens by UHPLC-MS-MS.</a> Journal of Analytical Toxicology. 42 (8), 570-580 (2018).</li><li>Fogarty, M. F., Papsun, D. M., Logan, B. 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. 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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 tr...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium formate for HPLC</td>
			<td>Fluka</td>
			<td>17843-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf&#160;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&#160;</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)&#160;</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&#160;</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 &#181;L, 10-100 &#181;L, and 100-1000 &#181;L pipette</td>
		</tr>
		<tr>
			<td>Pipette tips 10&#181;L</td>
			<td>Fisher</td>
			<td>94060100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips 1000&#181;L</td>
			<td>Fisher</td>
			<td>94056710</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips 200&#181;L</td>
			<td>Fisher</td>
			<td>94060310</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate mixer</td>
			<td>IKA&#160;</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&#160;</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.

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.

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...

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

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