Molecular imaging of humain hair with MeV-SIMS: A case study of cocaine detection and distribution in the hair of a cocaine user

Luka Jeromel; Nina Ogrinc; Zdravko Siketić; Primož Vavpetič; Zdravko Rupnik; Klemen Bučar; Boštjan Jenčič; Mitja Kelemen; Matjaž Vencelj; Katarina Vogel-Mikuš; Janez Kovač; Ron M. A. Heeren; Bryn Flinders; Eva Cuypers; Žiga Barba; Primož Pelicon

doi:10.1371/journal.pone.0263338

Peer Review History

Original SubmissionSeptember 27, 2021
7 Dec 2021 Decision Letter - Joseph Banoub, Editor PONE-D-21-29597Molecular imaging of humain hair with MeV-SIMS: a case study of cocaine detection and distribution in the hair of a cocaine userPLOS ONE Dear Dr. Barba, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. 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Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Joseph Banoub, Ph,D., D. Sc. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following financial disclosure: Work at JSI was supported by the Slovenian research agency grants No. P1-0112, I0-0005, J7-9398 and N1-0090. Additionally, resources and within the EU H2020 project No. 824096 “RADIATE ” and IAEA CRP projects F11019 “Development of Molecular Concentration Mapping Techniques Using MeV Focused Ion Beams" and F11021 ”Enhancing Nuclear Analytical Techniques to Meet the Needs of Forensic Science”. N.O. acknowledges funding by the European Union, European Social Fund, and the support from FP7 European Union Marie Curie IAPP Program, BRAINPATH. A traveling grant for a long stay abroad of E.C was awarded by Fonds Wetenschappelijk Onderzoek (FWO). Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: Work at JSI was supported by the Slovenian research agency grants No. P1-0112, 231 I0-0005, J7-9398 and N1-0090. Additionally, resources and within the EU H2020 project 232 No. 824096 “RADIATE” and IAEA CRP projects F11019 “Development of Molecular 233 Concentration Mapping Techniques Using MeV Focused Ion Beams” and F11021 234 ”Enhancing Nuclear Analytical Techniques to Meet the Needs of Forensic Science”. N.O. 235 acknowledges funding by the European Union, European Social Fund, and the support 236 from FP7 European Union Marie Curie IAPP Program, BRAINPATH. A traveling 237 grant for a long stay abroad of E.C was awarded by Fonds Wetenschappelijk Onderzoek 238 (FWO). Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: Work at JSI was supported by the Slovenian research agency grants No. P1-0112, I0-0005, J7-9398 and N1-0090. Additionally, resources and within the EU H2020 project No. 824096 “RADIATE ” and IAEA CRP projects F11019 “Development of Molecular Concentration Mapping Techniques Using MeV Focused Ion Beams" and F11021 ”Enhancing Nuclear Analytical Techniques to Meet the Needs of Forensic Science”. N.O. acknowledges funding by the European Union, European Social Fund, and the support from FP7 European Union Marie Curie IAPP Program, BRAINPATH. A traveling grant for a long stay abroad of E.C was awarded by Fonds Wetenschappelijk Onderzoek (FWO). Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. Additional Editor Comments: I have added the second referee comments below. Please try to answer the comments of referee #1 when you resubmit this manuscript. Referee #2 comments The paper by Jeromel,L. et al looks at distribution of cocaine in human hair, using TOF-SIMS. The paper is well written and edited, and is recommended for publications. However the authors may clarify a few points listed below in the manuscript. These are suggestions only. Somewhere in the introduction, the question regarding the standards such as cholesterol or the specific amino acids should be clarified. Why were these specific standards used, considering cholesterol is hydrophobic compared to the amino acids. Do these ions have any specific desorption rates compared to cocaine? Was free cocaine in solution form studied? I am not sure how changes occur in “post mortem” state of the hair cells? Kindly clarify. In the last figure the zones of high concentration (red) of cocaine at specific spots of the hair is due to especial location of the hair due to any structures of the hair? Why are the signals stronger from these specific areas? Kindly describe the figure in more details. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In the manuscript “Molecular imaging of humain hair with MeV-SIMS: a case study of cocaine detection and distribution in the hair of a cocaine user”, Jeroma et al. test a methodology for examining cocaine distribution within human hair. For this reviewer, it took a little time to discern whether the authors were presenting a research article or a methodology article. Regardless, the examination of cocaine in hair is far from novel, and there are a number of methodologies for this purpose, including mass spectrometry based; the authors did provide a sufficient introduction to cover some of these methodologies. Specific comments are as follows: 1. The authors need to examine the same hair more than once, to show the variability of the method. 2. More than one hair sample should be examined, to ensure comparable spectra can be obtained from cocaine users. 3. Please provide the ms/ms spectra for m/z 304, to verify to a reader that the ion is indeed cocaine. 4. Can the cocaine be quantified (rather than providing relative intensity information) using this method? This would be quite important. 5. Some modest edits to language (including within the title) are needed. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. Attachments Attachment Submitted filename: KN PLOS ONE Review Joe.docx https://doi.org/10.1371/journal.pone.0263338.r001
Revision 1
13 Jan 2022 Author Response List of changes made based on the remarks of the Reviewer 1: Technical questions Reviewer #1 “The authors need to examine the same hair more than once, to show the variability of the method.” We have analyzed the sample more than once, and the measurements have been done over a long period of time (several months). Results remained the same, however the ion yield of molecules, such as cocaine, has been reduced when the samples aged due to the oxidation of the samples. We revised the text accordingly so it is now clear that the measurements were performed multiple times “More than one hair sample should be examined, to ensure comparable spectra can be obtained from cocaine users.” Due to the strict ethical clearance policy, we only obtained one sample. However, multiple samples of the same subject were analyzed within the work of Flinders (see ref 31), and later one of these samples was forwarded to Jožef Stefan Institute. The manuscript was changed accordingly. “Please provide the ms/ms spectra for m/z 304, to verify to a reader that the ion is indeed cocaine.” Samples of the same subject were analyzed with MALDI MS/MS mass spectrometry imaging, where the exact mass of cocaine is measurable. See ref 31. After MeV-SIMS analysis, the same set of samples was also measured by DAPNe analysis, where Orbi trap mass spectrometer also confirmed the presence of cocaine molecular peak. The text was changed accordingly. “Can the cocaine be quantified (rather than providing relative intensity information) using this method? This would be quite important.” Quantification with MeV-SIMS, as well as common SIMS, is not possible due to the high impact the surrounding matrix on the ionization probability. A standardization of secondary ion yield of the molecule in relation to the concentration, given the constant matrix (e.g. collagen in human hair) would be possible, however even such approach would lead to questionable results due to uneven composition of biological tissues. Our scope remained only on providing relative intensity information. “Some modest edits to language (including within the title) are needed.” We have revised the text again. Technical questions Reviewer #2 “Somewhere in the introduction, the question regarding the standards such as cholesterol or the specific amino acids should be clarified. Why were these specific standards used, considering cholesterol is hydrophobic compared to the amino acids. Do these ions have any specific desorption rates compared to cocaine? Was free cocaine in solution form studied?” Amino-acids, such as arginine, leucine, phenylalanine, etc. as well as cholesterol are, to our knowledge, commonly used in SIMS community as a reference/calibration standards. Therefore, we could draw some explicit comparisons between sputtering rate of MeV and keV ions, not only from the keV-SIMS measurements done at JSI, but also worldwide, and the results encouraged us to develop the method further. The masses of such amino acids and cholesterol are also within the same domain as the mass of cocaine, so such standards provided us the information about expected molecular yields from human hair. “I am not sure how changes occur in “post mortem” state of the hair cells? Kindly clarify.” We measured the samples several time over a significant period of time (several months), so we gained some insight into aging of the samples. The localization of cocaine and other hair-specific molecules remained the same, at least on the above cellular size level. However, the intensity of the signal was rapidly diminishing, due to deposition of oxidation layers on the sample. “In the last figure the zones of high concentration (red) of cocaine at specific spots of the hair is due to especial location of the hair due to any structures of the hair? Why are the signals stronger from these specific areas? Kindly describe the figure in more details.” The concentration distribution of cocaine is highly unique compared to distribution of other hair – specific ions. Latter are distributed very homogeneously, which indicates, that there are no significant structural impacts on the secondary ion yield. Indeed, this can greatly influence the results of surface sensitive methods, therefore we were very observant on the intensities of the total ion yield, as well as some other peaks. Among those, the cocaine peak is the only one showing uneven distribution. As it can be seen in the last figure, the gap between the intensiy peaks is speculated to be due to the detection of each individual dose of cocaine that the subject has taken. Through this conclusion, we speculate, that the second dose was taken 12 hours after the first one, and in both cases the cocaine was depositing on the hair for approx. 3 hours. Editorial changes Technical -Page 5, line 150, sample preparation, technical: “A set of hair samples previouisly analysed using the MALDI TOF-MS technique was used to examine the evolution of the cocaine signal by MeV-SIMS along the hair longitudinal cross-section. All of the samples were collected post-mortem and were used previously by Cuypers et al. [29] and Flinders et al. [31].” has been changed to: “A set of hair samples, which were previously analyzed by means of MALDI TOF-MS technique, was used to examine the evolution of the cocaine signal by MeV-SIMS along the hair longitudinal cross-section. All of the samples were collected post-mortem and were used previously by Cuypers et al. [29] and Flinders et al. [31]. Within these works, more sets of samples from the same subject were analyzed, and provided similar results within the sets” -Page 5, line 178, Results and discussion, technical: “The spectra obtained from the surface of the longitudinally cut hair samples are shown in Fig. 5. “, has been changed to: “The spectra from one of the measurements of the longitudinally cut hair samples are shown in Fig. 5.” -Page 5, line 181, Results and discussion, technical: After “The molecular peak of cocaine at m/z of 304 was detected only in the hair 181 sample of the drug user.” we added: “The exact mass of cocaine at m/z=304.1540 has before been measured by MALDI MS/MS on another set of samples (see ref 31)” Language - Page 1, abstract, language: “During the growth of human hair, several biomedical substances present in the human body are incorporated into the hair” has been changed to: “Human hair absorbs numerous biomolecules from the body during its growth” -Page 1, abstract, language: “.It could be either assessed by chemical extraction and analysis of hair bundels, or by direct single hair fibre analysis by mass spectroscopy imaging (MSI).” has been changed to: “It could be either assessed by chemical extraction and further analysis of hair bundels, or by direct single hair fibre analysis with mass spectroscopy imaging (MSI).” -Page 1, abstract, language: “In this work, we analyzed the cocaine distribution in individual hair samples using MeV-SIMS.” has been changed to: “Within this work, we analyzed the cocaine distribution in individual hair samples using MeV-SIMS.” -Page 1, line 1, Introduction, language: “The presence of illegal chemical substances in human body is usually monitored by bulk chemical analysis or spectroscopic methods of biological specimens [1–5] such as urine, saliva, sweat, and hair” has been changed to: “The presence of illegal chemical substances in human body is usually monitored by bulk chemical analysis, or by spectroscopic methods of biological specimens [1–5], such as urine, saliva, sweat, and hair” -Page 2, line 9, Introduction, language: “A number of pharmaceutical substances, as well as illegal substances were detected in hair [9, 10] with a variety of analytical techniques.” has been changed to: “Various pharmaceutical substances, as well as illegal substances were detected in hair [9, 10] with numerous analytical techniques.” -Page 2, line 26, Introduction, language: “Typical cocaine concentrations found in hair samples after its administration range from 0.5 to 216 ng/mg [23].” has been changed to: “Typical cocaine concentration values in hair samples after its administration have been reported from 0.5 to 216 ng/mg [23].” -Page 2, line 42, Introduction, language: “Longitudinal sectioning of hair, which reveals its interior, allows the probing primary ion beam with energies of a few keV used in Secondary Ion Mass Spectroscopy (SIMS) to access the hair matrix and enables the detection of chemicals embedded in the hair.” has been changed to: “Longitudinal sectioning of the hair, which reveals its interior, allows the primary ion beam, used for Secondary Ion Mass Spectroscopy (SIMS), to access the hair matrix, thus enabling the detection of chemicals embedded in the hair.” -Page 2, line 52, Introduction, language: “In a classic SIMS set-up, absolute yields range from 10−4 to 10−3 per single impinging particle. To increase the absolute molecular yields and the associated chemical sensitivity, cluster beams are introduced in SIMS, such as Au+ n , SF+ 5 , C+ 60, Bi+ 3 and Ar+ (n) clusters with n= 55 60–3000 [32, 33] and, more recently, the use of water clusters in SIMS improves both aspects even further [34, 35].” has been changed to: “In a classic SIMS set-up, the absolute yields range from 10−4 to 10−3 per single impinging particle. To increase absolute molecular yields and the associated chemical sensitivity, cluster beams were introduced in SIMS, such as Au+ n , SF+ 5 , C+ 60, Bi+ 3 and Ar+ (n) clusters with n= 55 60–3000 [32, 33]. More recently, the use of water clusters in SIMS has been demonstrated to improve both aspects even further [34, 35].” -Page 3, line 76, Experimental, language: “Time-Of-Flight (TOF) mass spectrometer for MeV-SIMS is installed at the high-energy focused-ion-beam facility of the Jožef Stefan Institute (JSI) (Fig.1).” has been changed to: “Time-Of-Flight (TOF) mass spectrometer for purpose of MeV-SIMS analysis has been implemented at the high-energy focused-ion-beam facility of the Jožef Stefan Institute (JSI) (Fig.1).” -Page 5, line 173, Measurements, language: “For MeV-SIMS, the sample holder is tilted for 55 degrees in order to be positioned perpendicularly to the axis of the TOF spectrometer.” has been changed to: “For MeV-SIMS, the sample holder is tilted 55 degrees with regards to the primary beam axis in order to be positioned perpendicularly to the axis of the TOF spectrometer.” -Page 6, line 190, Results and discussion, language: “This result indicates the method’s high sensitivity for cocaine metabolically incorporated in the interior hair structure and its insensitivity to eventual external contamination of the hair.” has been changed to: “This result indicates high sensitivity for cocaine metabolically incorporated in the interior hair structure, which is not affected by external contamination of the hair.” -Page 6, line 212, Conclusion, language: “In the reported case of cocaine detection in human hair, we observe pronounced longitudinal spikes of the cocaine levels.” has been changed to: “In the reported case of cocaine detection in human hair, we observe pronounced longitudinal spikes of cocaine’s relative concentration.” Attachments Attachment Submitted filename: Letter_to_Editor_manuscript_Jeromel_et_al.docx https://doi.org/10.1371/journal.pone.0263338.r002
18 Jan 2022 Decision Letter - Joseph Banoub, Editor Molecular imaging of human hair with MeV-SIMS: a case study of cocaine detection and distribution in the hair of a cocaine user PONE-D-21-29597R1 Dear Dr. Barba, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Joseph Banoub, Ph,D., D. Sc., FCIC, FRCS Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0263338.r003
Formally Accepted
17 Mar 2022 Acceptance Letter - Joseph Banoub, Editor PONE-D-21-29597R1 Molecular imaging of humain hair with MeV-SIMS: a case study of cocaine detection and distribution in the hair of a cocaine user Dear Dr. Barba: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Joseph Banoub Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0263338.r004

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