Dear Reviewers and Editor,

Thank you for reviewing our manuscript titled: “Analysis, Identification and Confirmation of Synthetic Opioids using Chloroformate Chemistry: Retrospective Detection of Fentanyl and Acetylfentanyl in Urine and Plasma Samples by EI-GC-MS and HR-LC-MS”.

We have addressed the first reviewer’s comments and these are highlighted in the “revised manuscript” and present in the “manuscript” new versions of the document. The first reviewers had three comments. The first two were straightforward (i.e., replace discussion by conclusion and a line feed in Line 606). The last one was a suggestion to simplify titles for the figures. We have attempted to reduce the text and insert informative titles for each figure as suggested and per the journal’s request. We hope these changes are sufficient enough so as to not impact the overall description of the figure by the captions below it. Please let us know if we would need to modify further. Again, these changes are highlighted in the “revised manuscript” and present in the “manuscript” new versions of the document.

To the Editor, we have formatted the manuscript per guidelines set by the journal. All these changes can be easily followed by the highlighted “revised manuscript” new version of the document. In addition, we removed the Disclaimer and Auspices statements from the Acknowledgements section. The new, revised funding statement can be found in the "Response to Reviewers" letter.

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Submitted filename: Response_to_Reviewers.pdf

Dear Reviewers and Editor,

Thank you for reviewing our manuscript titled: “Analysis, Identification and Confirmation of Synthetic Opioids using Chloroformate Chemistry: Retrospective Detection of Fentanyl and Acetylfentanyl in Urine and Plasma Samples by EI-GC-MS and HR-LC-MS”.

We have addressed the first reviewer’s comments and these are highlighted in the “revised manuscript” and present in the “manuscript” new versions of the document. The second reviewer did not have any specific comments on the manuscript. The first reviewer had three comments. The first two were straightforward (i.e., replace discussion by conclusion and a line feed in Line 606). The last one was a suggestion to simplify titles for the figures. We have attempted to reduce the text and insert informative titles for each figure as suggested and per the journal’s request. Below is an itemized list these changes in the revised version:

1. The word discussion was changed to conclusion in the main text.

2. Line 606 was denoted as a line feed. We have deleted this.

3. Titles of the Figures have been formatted and simplified as much as possible while retaining their clear, descriptive nature. The changes made are itemized below and the edits are highlighted in yellow. A tracked changes version of the Figure file has been also uploaded in the overall review of the manuscript for your convenience.

4. Fig 1. Synthetic opioids belonging to the fentanyl family. (a) Chemical structures of fentanyl, acetylfentanyl and their main metabolites, the potencies of each opioid relative to morphine are given in brackets; (b) commonly employed antidotes against fentanyl poisoning along with their circulation half-lives.

5. Fig 2. Chemical modification of fentanyls. (a) Reaction of fentanyl with 2,2,2-trichloroethoxycarbonyl chloride (Troc-Cl) to yield two unique, abiotic products that bear structural features of the original opioid (grayed boxes); (b) overall protocol to aid in the confirmation of fentanyl and norfentanyl in a biological sample by EI-GC-MS and targeted HR-LC-MS.

6. Fig 3. Extraction and reaction of fentanyl with Troc-Cl when spiked in plasma at two concentrations (10 and 20 ng/mL). (a) GC chromatogram of extracted plasma sample; (b) extracted ion (m/z = 245) chromatogram for fentanyl (rt = 32.8 min.) for low concentration spike; (c) extracted ion (m/z = 245) chromatogram for fentanyl for high concentration spike; (d) GC chromatogram of the reaction between extracted plasma sample and Troc-Cl; (e) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the low concentration sample showing no discernible peak while (f) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the high concentration sample shows a clear peak at rt = 33.03 min.

7. Fig 4. Mass spectral data associated with the reaction between fentanyl-spiked plasma matrices with Troc-Cl. Mass spectra for (a) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (20 ng/mL) with Troc-Cl, note that the spectrum is complex and it is the result of an additional Troc-containing interference (Troc-norfentanyl peaks indicated in red); (b) Expansion of the molecular ion peak region for Troc-norfentanyl where the peaks arising from our product are highlighted in red and those from the Troc-containing interference are in gray; mass spectra for (c) extracted fentanyl when spiked at 10 ng/mL and (d) extracted fentanyl when spiked at 20 ng/mL; (e) mass spectrum for 2-phenylethyl chloride for the lowest fentanyl concentration spike and (f) for the highest fentanyl concentration.

8. Fig 5. Extraction and reaction of fentanyl with Troc-Cl when spiked in urine. (a) GC chromatogram of extracted urine sample; (b) extracted ion (m/z = 245) chromatogram for fentanyl (rt = 32.8 min.) for low concentration urine spike (5 ng/mL); (c) extracted ion (m/z = 245) chromatogram for fentanyl for high concentration urine spike (10 ng/mL); (d) GC chromatogram of the reaction between extracted urine sample and Troc-Cl; (e) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the low concentration urine sample (rt = 33.03 min.); (f) extracted ion (m/z = 149) chromatogram for Troc-norfentanyl for the high concentration spiked urine sample.

9. Fig 6. Mass spectral data associated with the reaction between fentanyl-spiked urine matrices with Troc-Cl. Mass spectra for (a) extracted fentanyl when spiked at 5 ng/mL, (b) extracted fentanyl when spiked at 10 ng/mL, (c) Troc-norfentanyl product arising from the treatment of fentanyl-spiked urine extract (10 ng/mL) with Troc-Cl, (d) and 2-phenylethyl chloride, the second confirmatory by-product from the reaction; mass spectra. Mass spectra for Troc-norfentanyl and 2-phenylethyl chloride contain interfering signals from other matrix components, but in both cases the base peaks can be observed (m/z = 149 and m/z = 91 respectively).

10. Fig 7. Extraction and reaction of acetylfentanyl with Troc-Cl when spiked in plasma. (a) GC chromatogram of extracted plasma sample; (b) extracted ion (m/z = 231) chromatogram for acetylfentanyl (rt = 32.3 min.) for low concentration plasma spike (50 ng/mL); (c) extracted ion (m/z = 231) chromatogram for fentanyl for high concentration plasma spike (200 ng/mL); (d) GC chromatogram of the reaction between extracted plasma sample and Troc-Cl; (e) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the low concentration plasma sample (rt = 32.6 min.); (f) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the high concentration spiked plasma sample.

11. Fig 8. Mass spectral data associated with the reaction between acetylfentanyl-spiked plasma matrices with Troc-Cl. Mass spectra for (a) extracted fentanyl when spiked at 50 ng/mL, (b) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (50 ng/mL) with Troc-Cl, (c) and 2-phenylethyl chloride, the second confirmatory by-product from the reaction; mass spectra for (d) extracted fentanyl when spiked at 100 ng/mL, (e) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (100 ng/mL) with Troc-Cl, (f) and 2-phenylethyl chloride for the highest concentration spike.

12. Fig 9. Extraction and reaction of acetylfentanyl with Troc-Cl when spiked in urine. (a) GC chromatogram of extracted urine sample; (b) extracted ion (m/z = 231) chromatogram for acetylfentanyl (rt = 32.3 min.) for low concentration urine spike (20 ng/mL); (c) extracted ion (m/z = 231) chromatogram for fentanyl for high concentration urine spike (100 ng/mL); (d) GC chromatogram of the reaction between extracted urine sample and Troc-Cl; (e) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the low concentration urine sample (rt = 32.6 min.); (f) extracted ion (m/z = 135) chromatogram for Troc-norfentanyl for the high concentration spiked urine sample.

13. Fig 10. Mass spectral data associated with the reaction between acetylfentanyl-spiked urine matrices with Troc-Cl. Mass spectra for (a) extracted fentanyl when spiked at 20 ng/mL, (b) Troc-norfentanyl product arising from the treatment of fentanyl-spiked urine extract (20 ng/mL) with Troc-Cl, (c) and 2-phenylethyl chloride, the second confirmatory by-product from the reaction; mass spectra for (d) extracted fentanyl when spiked at 100 ng/mL, (e) Troc-norfentanyl product arising from the treatment of fentanyl-spiked plasma extract (100 ng/mL) with Troc-Cl, (f) and 2-phenylethyl chloride for the highest concentration spike.

14. Fig 11. Analysis by HR-LC-MS of extraction and reaction of fentanyl with Troc-Cl when spiked in plasma. (a) Total ion chromatogram (TIC) of extracted plasma sample; (b) extracted ion ([M+H+] = 337.4865 � 5 ppm) for fentanyl (rt = 13.85 min.) for low concentration plasma sample (10 ng/mL); (c) extracted ion ([M+H+] = 337.4865 � 5 ppm) for high concentration in plasma sample (20 ng/mL); (d) Total ion chromatogram (TIC) of reaction between extracted plasma sample with Troc-Cl; (e) extracted ion ([M+H+] = 407.7160 � 5 ppm) for Troc-norfentanyl for low concentration spiked plasma sample; (f) extracted ion ([M+H+] = 407.7160 � 5 ppm) for Troc-norfentanyl for the high concentration spiked plasma sample showing its appearance at rt = 15.91 min.

15. Fig 12. Analysis by HR-LC-MS of extraction and reaction of fentanyl with Troc-Cl when spiked in urine. (a) Ion chromatogram of extracted urine sample; (b) extracted ion ([M+H+] = 337.4865 � 5 ppm) for fentanyl (rt = 13.85 min.) for low concentration urine sample (5 ng/mL); (c) extracted ion ([M+H+] = 337.4865 � 5 ppm) for high concentration in urine sample (10 ng/mL); (d) Ion chromatogram of reaction between extracted urine sample with Troc-Cl; (e) extracted ion ([M+H+] = 407.7160 � 5 ppm) for Troc-norfentanyl for low concentration spiked urine sample; (f) extracted ion ([M+H+] = 407.7160 � 5 ppm) for Troc-norfentanyl for the high concentration spiked urine sample showing its appearance at rt = 15.91 min.

16. Fig 13. Analysis by HR-LC-MS of extraction and reaction of acetylfentanyl with Troc-Cl when spiked in plasma. (a) Total ion chromatogram (TIC) of extracted plasma sample; (b) extracted ion ([M+H]+ = 323.4595 � 5 ppm) for acetylfentanyl (rt = 12.46 min.) for low concentration plasma sample (50 ng/mL); (c) extracted ion ([M+H]+ = 323.4595 � 5 ppm) for acetylfentanyl for high concentration plasma sample (200 ng/mL); (d) Total ion chromatogram (TIC) of the reaction of extracted urine sample with Troc-Cl; (e) extracted ion ([M+H+] = 393.6890 � 5 ppm) for Troc-norfentanyl for low concentration spiked plasma sample; (f) extracted ion ([M+H+] = 393.6890 � 5 ppm) for Troc-norfentanyl for the high concentration spiked plasma sample showing its appearance at rt = 15.42 min.

17. Fig 14. Analysis by HR-LC-MS of extraction and reaction of acetylfentanyl with Troc-Cl when spiked in urine. (a) Total ion chromatogram (TIC) of extracted urine sample; (b) extracted ion ([M+H]+ = 323.4595 � 5 ppm) for acetylfentanyl (rt = 12.46 min.) for low concentration urine sample (20 ng/mL); (c) extracted ion ([M+H]+ = 323.4595 � 5 ppm) for acetylfentanyl for high concentration urine sample (100 ng/mL); (d) Total ion chromatogram (TIC) of the reaction of extracted urine sample with Troc-Cl; (e) extracted ion ([M+H+] = 393.6890 � 5 ppm) for Troc-norfentanyl for low concentration spiked urine sample; (f) extracted ion ([M+H+] = 393.6890 � 5 ppm)for Troc-norfentanyl for the high concentration spiked urine sample showing its appearance at rt = 15.42 min.

We hope these changes are sufficient enough so as to not impact the overall description of the figure by the captions below it. Please let us know if we would need to modify further. Again, these changes are highlighted in the “revised manuscript” and present in the “manuscript” new versions of the document.

To the Editor, we have formatted the manuscript per guidelines set by the journal. All these changes can be easily followed by the highlighted “revised manuscript” new version of the document. Below is an itemized list of all the changes in the revised version.

1. Line 29: Abstract title changed to font size = 18.

2. Line 57: Introduction title changed to font size = 18.

3. Line 157: Materials and methods title changed to font size = 18.

4. Line 159: Chemicals and reagents title changed to font size = 16.

5. Line 173: EI-GC-MS Analysis Method title changed to font size = 16.

6. Line 191: HR-LC-MS Analysis Method title changed to font size = 16.

7. Line 209: Sample preparation and extraction title changed to font size = 16.

8. Line 231: Results and Discussion title changed to font size = 18.

9. Line 577: Conclusion title (changed from originally Discussion) changed to font size = 18.

10. Line 607: Acknowledgements title changed to font size = 18 and now reads: “The authors would like to thank Dr. Carolyn J. Koester and Mr. Armando Alcaraz for engaging discussions regarding GC-MS/LC-MS analysis and their critical reading of the manuscript.”

11. Lines 613-614: The disclaimer and auspices statements where funding source is mentioned have been removed and added to the bottom of this rebuttal letter.

12. All references annotations within the main text of the manuscript have been changed from parentheses ( ) to brackets [ ].

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14. All References within the main text of the manuscript have been formatted accordingly to fit the journal’s guidelines (including the addition of DOI info). Below is an itemized list of all the references changed and the changes are highlighted.

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Lines 1848-1849: 34. Cunningham SM, Haikal NA, Kraner JC. Fatal Intoxication with Acetyl Fentanyl. J. Forensic Sci. 2016;61(Suppl 1):S276-S280. DOI: 10.1111/1556-4029.12953

Lines 1851-1853: 35. Yonemitsu K, Sasao A, Mishima S, Ohtsu Y, Nishitani Y. A Fatal Poisoning Case by Intravenous Injection of "Bath Salts" Containing Acetyl Fentanyl and 4-Methoxy PV8. Forensic Sci. Int. 2016;267:e6-e9. DOI: 10.1016/j.forsciint.2016.08.025

Lines 1855-1858: 36. Wnuk SF, Valdez CA, Khan J, Moutinho P, Robins MJ, Yang X, et al. Doubly homologated dihalovinyl and acetylene analogues of adenosine: Synthesis, interaction with S-adenosyl-L-homocysteine hydrolase, and antiviral and cytostatic effects. J. Med. Chem. 2000;43(6):1180-1186. DOI: 10.1021/jm990486y

Lines 1860-1863: 37. Wnuk SF, Ro B-O, Valdez CA, Lewandowska E, Valdez NX, Sacasa PR, et al. Sugar-modified conjugated diene analogues of adenosine and uridine: Synthesis, interaction with S-adenosyl-L-homocysteine hydrolase, and antiviral and cytostatic effects. J. Med. Chem. 2002;45:2651-2658. DOI: 10.1021/jm020064f

Lines 2100-2103: 38. Showalter BM, Reynolds MM, Valdez CA, Saavedra JE, Davies KM, Klose JR, et al. Diazeniumdiolate ions as leaving groups in anomeric displacement reactions: a protection-deprotection strategy for ionic diazeniumdiolates. J. Am. Chem. Soc. 2005;127(41):14188-14189. DOI: 10.1021/ja054510a

Lines 2105-2108: 39. Valdez CA, Saavedra JE, Showalter BM, Davies KM, Wilde TC, Citro ML, et al. Hydrolytic reactivity trends among potential prodrugs of the O2-glycosylated diazeniumdiolate family. Targeting nitric oxide to macrophages for antileishmanial activity. J. Med. Chem. 2008;51(13):3961-3970. DOI: 10.1021/jm8000482

Lines 2110-2114: 40. Valdez CA, Leif RN, Hok S, Vu AK, Salazar EP, Alcaraz A. Methylation protocol for the retrospective detection of isopropyl-, pinacolyl- and cyclohexylmethylphosphonic acids, indicative markers for the nerve agents sarin, soman and cyclosarin, at low levels in soils using EI-GC-MS. Sci. Total Environ. 2019;683:175-184. DOI: 10.1016/j.scitotenv.2019.05.205

Lines 2116-2119: 41. Valdez CA, Leif RN, Alcaraz A. Effective methylation and identification of phosphonic acids relevant to chemical warfare agents mediated by trimethyloxonium tetrafluoroborate for their qualitative detection by gas chromatography-mass spectrometry. Anal. Chim. Acta, 2016;933:134-143. DOI: 10.1016/j.aca.2016.05.034

Lines 2350-2354: 42. Valdez CA, Marchioretto MK, Leif RN, Hok, S. Efficient derivatization of methylphosphonic and aminoethylsulfonic acids related to nerve agents simultaneously in soils using trimethyloxonium tetrafluoroborate for their enhanced, qualitative detection and identification by EI-GC–MS and GC–FPD. Forensic Sci. Int. 2018;288:159-168. DOI: 10.1016/j.forsciint.2018.04.041

Lines 2356-2359: 43. Valdez CA, Leif RN, Hok, S. Carbene-based difluoromethylation of bisphenols: Application to the instantaneous tagging of bisphenol A in spiked soil for its detection and identification by electron ionization gas chromatography-mass spectrometry. Sci. Rep. 2019;9:17360. DOI: 10.1038/s41598-019-53735-9

Lines 2361-2363: 44. Foerster EH, Mason MF. Preliminary studies on the use of n-butylchloride as an extractant in a drug screening procedure. J. Forensic Sci. 1974;19(1):155-162. PMID: 4853734

Lines 2365-2367: 45. Strano-Rossi S, Alvarez I, Tabernero MJ, Cabarcos P, Fernández P, Bermejo, AM. Determination of Fentanyl, Metabolite and Analogs in Urine by GC/MS. J. Applied Toxicol. 2011;31(7):649–654. DOI: 10.1002/jat.1613

Lines 2542-2543: 46. Gardner MA, Sampsel S, Jenkins WW, Owens JE. Analysis of Fentanyl in Urine by DLLME-GC-MS. J. Anal. Toxicol. 2015;39(2):118-125. DOI: 10.1093/jat/bku136

Lines 2545-2548: 47. Strano-Rossi S, Bermejo AM, de la Torre X, Botrè F. Fast GC-MS Method for the Simultaneous Screening of THCCOOH, Cocaine, Opiates and Analogues Including Buprenorphine and Fentanyl, and Their Metabolites in Urine. Anal. Bioanal. Chem. 2011;399(4):1623-1630. DOI: 10.1007/s00216-010-4471-4

Lines 2550-2552: 48. Sisco E, Burns A, Moorthy AS. Development and evaluation of a synthetic opioid targeted gas chromatography mass spectrometry (GC-MS) method. J. Forensic Sci. 2021;66:2369-2380. DOI: 10.1111/1556-4029.14877

Lines 2554-2556: 49. Moore A, Foss J, Juhascik M, Botch-Jones S, Kero F. Rapid Screening of opioids in seized street drugs using ambient ionization high resolution time-of-flight mass spectrometry. Forensic Chem. 2019;13:100149. DOI: 10.1016/j.forc.2019.100149

Lines 2558-2560: 50. Weldon ST, Perry DF, Cork RC, Gandolfi AJ. Detection of Picogram Levels of Sufentanil by Capillary Gas Chromatography. Anesthesiology. 1985;63:684-687. DOI: 10.1097/00000542-198512000-00021

Lines 2769-2773: 51. Van Nimmen NFJ, Poels KLC, Veulemans HAF. Highly Sensitive Gas Chromatographic-Mass Spectrometric Screening Method for the Determination of Picogram Levels of Fentanyl, Sufentanil and Alfentanil and Their Major Metabolites in Urine of Opioid Exposed Workers. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2004;804(2):375-387. DOI: 10.1016/j.jchromb.2004.01.044

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Lines 2779-2781: 53. UNODC. Recommended Methods for the Identification and Analysis of Fentanyl and Its Analogues in Biological Specimens. United Nations Office on Drugs and Crime, Vienna, 2017. DOI: 10.18356/aca7aca5-en.

Lines 2783-2785: 54. Anderson DT, Muto JJ. Duragesic Transdermal Patch: postmortem Tissue Distribution of Fentanyl in 25 Cases. J. Anal. Toxicol. 2000;24:627-634. DOI: 10.1093/jat/24.7.627

Lines 2787-2789: 55. Ogilvie L, Stanley C, Lewis L, Boyd M, Lozier M. Notes from the Field: Acetyl Fentanyl Overdose fatalities-Rhode Island, March-May 2013. Morbidity and Mortality Weekly Report, 2013;62:703-704.

Lines 2960-2962: 56. McIntyre IM, Trochta A, Gary RD, Malamatos M, Lucas JR. An Acute Acetyl Fentanyl Fatality: A Case Report with Postmortem Concentrations. J. Anal. Toxicol. 2015;39(6):490-494. DOI: 10.1093/jat/bkv043

Lines 2964-2965: 57. Fort C, Curtis B, Nichols C, Niblo, C. Acetyl Fentanyl Toxicity: Two Case Reports. J. Anal. Toxicol. 2016;40(9):754-757. DOI: 10.1093/jat/bkw068

Lines 2967-2969: 58. Katselou M, Papoutsis I, Nikolaou P, Spiliopoulou C, Athanaselis S. Old Opioids, New Concerns: The Case of Acetylfentanyl. Forensic Toxicol. 2016;34:201-212. DOI: 10.1007/s11419-016-0310-4

In addition, we removed the Disclaimer and Auspices statements from the Acknowledgements section. The new, revised funding statements are below.

“Disclaimer: This document (LLNL-JRNL-839586) was prepared as an account of work sponsored by an agency of the United States government. Neither the United States government nor Lawrence Livermore National Security, LLC, nor any of their employees makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States government or Lawrence Livermore National Security, LLC. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States government or Lawrence Livermore National Security, LLC, and shall not be used for advertising or product endorsement purposes.

Auspices statement: This work was performed under the auspices of the U. S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. The was funded fully by a Mid-Career Research Grant awarded by the Lawrence Livermore National Laboratory (PLS-21-FS-036) to C. A. V.”

Please let us know if there are other edits/corrections that need to be amended. We might have missed some and we apologize in advance for this.

Best regards,

Carlos Valdez and co-authors

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