Simple Analytical Strategy for Screening Three Synthetic Cathinones (α-PVT, α-PVP, and MDPV) in Oral Fluids

Round 1

Reviewer 1 Report

In the literature, the most used technique for the analysis of cathinones in the salivary matrix is ​​LC-MS / MS, having a high sensitivity and requiring a minimum pre-treatment of the salivary sample.   The authors propose GC / MS as an alternative analytical technique, notoriously less performing than LC-MS / MS and which usually requires a sample extraction process. However, in this paper there are innovative solutions such as the use of the bar adsorptive microextraction (BAµE) which allow to limit the performance gap between the two techniques and to contain the sample pre-treatment procedure.   The optimization of the BAµE undoubtedly remains the strong point of this study, thanks to the large number of tests carried out on a truly important number of sorbent coatings.    Unfortunately for the problems listed below, The presented method suffers from some problems attributable to the chromatographic technique used.   The use of gas chromatography without the use of derivatizers, allows you to save time in the analytical process, but causes serious thermo-degradation problems, which is why the authors were able to search for only 3 of the 6 analytes provided.   The use of derivatizers implies minimal treatment times, compared to the result that can be obtained. Probably, testing more derivatizing reagents, trying to optimize reaction times, would have been a way to increase the very small analysis panel and give more importance to the work.   These problems, combined with the poor linearity of the MDPV curve, have led to the method being configured as a screening test rather than as a quantitative method.   Line 44 - It is not clear what the quoted percentages refer to: to the new cathinones discovered, to those most used or to those seized. The period to which they refer is not specified either. TO   Line 84 - Perhaps it is appropriate to point out all six analytes that it was initially proposed to include in the method, clarifying that it was only possible to insert three of them.   Line 92 - Explain why seized material was used instead of certified standards.   Line 160 - The values ​​used to optimize the parameters in paragraph 2.2 are reported, but then the parameters used in the method are reported in the Results. If this structure is maintained, it is advisable to create a link that connects paragraph 2.2 to that of the Results.   Line 166 - Also report in the text the sorbent coatings tested and reported only in Figure 1.   Line 233 - The number of levels making up the calibration curve is not reported; the LOQ is not included in the calibration curve, for what reason?   Line 331 - Paragraph 3.3 presents the concentration ranges found in the real samples for MDPV and α-PVP, which are respectively 20.5-831.7 and 81.9-935.0 μg / L. In the previous paragraphs, an LOD of 30 μg / L and an LOQ of 90 μg / L were reported. How then was it possible to find a concentration lower than the LOQ and LOD?   Line 322 - repetition error, the preposition "as" is present twice;   In the introduction it is recommended to add the following works for completeness:

Graziano S, Anzillotti L, Mannocchi G, Pichini S, Busardò FP. Screening methods for rapid determination of new psychoactive substances (NPS) in conventional and non-conventional biological matrices. J Pharm Biomed Anal. 2019 Jan 30;163:170-179. doi: 10.1016/j.jpba.2018.10.011.

Zaami S, Giorgetti R, Pichini S, Pantano F, Marinelli E, Busardò FP. Synthetic cathinones related fatalities: an update. Eur Rev Med Pharmacol Sci. 2018 Jan;22(1):268-274. doi: 10.26355/eurrev_201801_14129.

La Maida N, Di Trana A, Giorgetti R, Tagliabracci A, Busardò FP, Huestis MA. A Review of Synthetic Cathinone-Related Fatalities From 2017 to 2020. Ther Drug Monit. 2021 Feb 1;43(1):52-68. doi: 10.1097/FTD.0000000000000808.

 

Di Trana A, Mannocchi G, Pirani F, La Maida N, Gottardi M, Pichini S, Busardò FP. A Comprehensive HPLC–MS-MS Screening Method for 77 New Psychoactive Substances, 24 Classic Drugs and 18 Related Metabolites in Blood, Urine and Oral Fluid. J Anal Toxicol 2020 Aug 20; bkaa103.doi: 10.1093/jat/bkaa103.

Author Response

We greatly appreciate the comments and suggestions of reviewer #1 and made the necessary changes and adaptation throughout the manuscript, in particular by adding the suggested references in the introduction section. We also acknowledge the limitations of the proposed work, especially regarding the thermal degradation of the selected compounds, resulting in poor linearity and comprehensiveness. We highlighted this fact in the manuscript However, the proposed methodology is an alternative analytical cycle to monitor the selected compounds in oral fluid, particularly when LC-MS/MS is not available. Moreover, we chose to apply a more direct approach to screening these compounds in the oral fluids, which required minimal steps in the sample preparation stage. Nevertheless, the suggestion of reviewer #1 will certainly be considered in future works.

Line 44 – We agree and corrected to the following: “By 2020, several hundred of NPS have been detected and around 830 are now being monitored by the European Monitoring Centre for Drug and Drug Addition (EMCDDA). Synthetic cathinones (SC) are the second largest class of NPS being monitored by the EU Early Warning System by (19 %), after synthetic cannabinoids (24 %) [2]”.

Line 84 – We agree and corrected to the following: “The present contribution aimed to evaluate the performance of BAµE prior to GC-MS operating in selected-ion monitoring acquisition mode (SIM), to screen levels of six SCs including α-PVP, α-PVT, MDPV, MDPBP, methylone and MDPPP in oral fluids.”

Line 92 – We used seized materials because when we developed the methodology, MDPPP and α-PVT were recently detected NPS and were unavailable as certified standards.

Line 160 - We agree with the reviewer and added a clear link between the two sections.      

Line 166 – We agree with the reviewer and added the requested information.

Line 233 – We added the number of levels in the calibration curve. We did not include the LOQ in the calibration curve because the imprecision at that level was too high (RSD > 15 %). For this reason, we decided to start the calibration at 0.25 mg/L.

Line 331 – The presented LOD and LOQ of 30 and 90 μg/L, respectively are only for the instrumental analysis. When we took into consideration the enrichment process from the oral fluid samples though BAμE-LD, the established LOD was 100 μg/L. For this reason, we believe that the proposed analytical method can be used to detected most samples containing the target compounds.

Line 322 – We corrected it to: “…could still be used as an alternative methodology to…”.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments to the authors

In this paper, the authors study the applicability of a BAµE-µLD/GC-MS method for the determination of three synthetic cathinones (α-PVP, α-PVT, and MDPV). In fact, their initial intention was to determine 6 cathinones. However, because of thermal degradation during injection, some of these compounds were discarded. So, I do not see how the method can improve other methodologies previously developed. For instance, reference 7 reported a method for the determination of 12 synthetic cathinones, including the ones evaluated in the present study, in oral fluid using SPE and UHPLC-MS/MS. Indeed, they obtained better performances that in the present work. Thus, I cannot see the advantages of using GC-MS for this analysis, since LC instruments are also common instrumentation in analytical labs. Nevertheless, perhaps I am wrong, what I have understood after reading the manuscript is that what they really want to demonstrate is the applicability of the sample treatment (BAµE-µLD) technique for this kind of compounds. If this is the case, I do not see why they chose GC-MS as determination technique knowing in advance that these compounds suffer thermal degradation. Anyway, I think that this manuscript can be published in this journal, but I encourage the authors to highlight the advantages of BAµE-µLD over other sample treatment techniques and try to better describe the advantages of the entire method (BAµE-µLD/GC-MS).

In addition, some specific comments that I think will help you to improve your manuscripts are as follows:

• Line 66: I do not understand why the amount of a drug determine in this kind of matrix represents the free fraction. Could you explain it to me?
• Concentration should be written using the format: μg L^-1.
• Standards and materials section
  • Provide the supplier for all the reagent and phases for the BAµE you have used. Even if you cite references 17 and 19, in the present manuscript a minimum description is needed
  • I do not see the relevance of the RMN information for the objective of the manuscript. Delete this part.
• BAµE-µLD assay section
  • BAµE abbreviation has been previously introduced but µLD not. Please define it.
  • Lines 165-171: In this section final condition should be stated. Optimized parameters should be discussed in the Results section
• Oral fluid samples section
  • Line 186: The first two parameters. Which parameters?
  • Line 187: to which volume of oral fluid sample were the 100 µL of working standard spiked?
  • The working standard was spiked before diluting the oral fluid with water, wasn’t it?
  • Line 191: fragmentograms? do you mean total ion chromatogram?
• Instrumental set-up section
  • Instruments used need to be specified
  • Although some experimental conditions coincide with previous studies, a minimum of information is required. For instance, what about MS parameters?
• Results section
  • Line 235: Why did you not test linearity from the LOQ?
  • Line 268: was the BAµE-µLD optimized using the standard solutions in water or did you use oral fluid matrix?
  • Comment here the studied conditions (see comment above)
  • Line 286: did you checked extended time?
  • Section 3.3: have you studied the reproducibility when samples in oral fluid were analyzed?
  • Section 3.3: have you checked matrix effect?
  • Section 3.3: did you prepare calibration standards in oral fluid?
• Revise the manuscript for grammar or spelling errors. See here some examples:
  • Line 58: have been increasing increased
  • Line 68, 80: once?
  • Line 77: alternatives
  • Line 149: ware were
  • Line 148 and others: ºC should be changed by ^oC
  • Line 230: obtaing obtained
  • Line 278 and 282: supplementary
  • Line 300: as followed follows
• Figures:
  • Figure 1: specify other conditions
  • Figure 2: Fragmentogram Total ion chromatogram
• Supplementary material: check figure numbering

Author Response

We greatly appreciate the comments and suggestions of reviewer #2 and made the necessary changes and adaptation throughout the manuscript, in particular by highlighting the advantages of BAµE-µLD over other sample treatment techniques and to better describe the advantages of the entire method (BAµE-µLD/GC-MS).

We understand the concerns described by the reviewer, and although thermal degradation occurred and SPE with UHPLC-MS/MS presented better results than the proposed approach, we believe that our methodology has significant advantages over those ones. First, LC-MS/MS are not as widespread as GC-MS systems, which makes the later a more comprehensive and common technique. Secondly, SPE does not take into consideration the contemporary Green Analytical Chemistry Principles. And third, we explicitly said that the methodology can only be used as a screening technique, which can be combined with a quantitative approach. In sum, the proposed analytical cycle is a greener and easier to apply than the current ones.

• Line 66 – The free fraction of a drug represents the unconjugated form of it. In this case, no additional hydrolysis processes are required for analysis.
• We agree with the μg L^-1 format and made the necessary corrections.
• Standards and materials section
  • We added some information regarding the suppliers for reagents and phases used
  • We also put the RMN information as supplemental material.
• BAµE-µLD assaysection
  • µLD is now defined in the introduction
  • Lines 165-171 were changed to explicitly show the optimized parameters.

• Oral fluid samples section
• Line 186: The parameters were recovery and sensitivity. This was added to the manuscript
• Line 187: We spiked 500 µL of oral fluid sample with the 100 µL of working standard. This is now more explicit in the text.
• The oral fluid was spiked with the working solution before diluting with water
• Line 191: You are correct. It refers to the total ion chromatogram operating in the SIM mode. This was corrected.
• Instrumental set-up section
• We did not specify the instruments used because they are exactly the same as described in the references, including MS conditions (except m/z ions monitored in SIM mode).
• Results section
• Line 235: We did not include the LOQ in the calibration curve because the imprecision at that level was too high (RSD > 15 %). For this reason, we decided to start the calibration at 0.25 mg/L.
• Line 268: The BAµE-µLD optimization assays were performed in ultrapure water, as already described in in the methods section.
• The studied conditions were added to the manuscript
• Line 286: We performed these assays for 16h to compare the selectivity of the coating material when equilibrium is achieved.
• Section 3.3: We have studied the reproducibility when oral fluid samples were used. This was depicted as the RDS of recovery assays.
• Section 3.3: We did not check ion suppression or increment due to matrix effects, since we did not use a quantitative approach.
• Section 3.3: We prepared calibration standards in solvent that were used for spiking the oral fluid samples
• Revise the manuscript for grammar or spelling errors. See here some examples:
• Line 58: Corrected.
• Line 68, 80: Corrected throughout the manuscript.
• Line 77: We changed to “suitable methodologies”
• Line 149: Corrected
• Line 148 and others: ºC was changed to ^oC throughout the manuscript.
• Line 230: Corrected
• Line 278 and 282: Corrected
• Line 300: Corrected
• Figures:
• Figure 1: More information was added
• Figure 2: Correcte
• Supplementary material: Figure numbering was checked

Author Response File: Author Response.docx

Reviewer 3 Report

In this work, the authors have proposed an extraction method for the screening of synthetic cathinones in oral fluids based on a bar adsorptive microextraction followed by microliquid desorption. The studied drugs were analysed by GC-MS.The analytical method was developed by studying  several experimental parameters influencing the separation and extraction method.

In this study the extraction method is not entirely new, as the same system has been used for the extraction of benzodiazepines and ketamine and metabolites. Considering that this analytical method has been presented as a possible screening of cathinones, due to the easy thermal degradation of most of these compounds, in my opinion GC-MS is not the most compatible separation technique. There are in literature several articles in which the separation of cathinones has been optimized by HPLC. Since the extraction method is easy, fast and efficient, it is also important to choose a separation method compatible with the analytes studied in order to carry out a real screening. The advantages of GC reported by the authors are undoubtedly undiscussed but it is always necessary to consider the performance of the analytical method towards the compounds under study. Otherwise, such this method cannot be used as an analytical routine tool. In my opinion, the manuscript cannot be published in this specific journal.

Other minor comments

• The description of the NMR characterization of the pure compounds can be put in the supplemental material.
• The extraction parameters have to be deeply discussed.
• To show the efficiency of the extraction method it is important to make a comparison with the other cathinone extraction methods reported in the literature.

Author Response

We greatly appreciate the comments and suggestions of reviewer #3 and made the necessary changes and adaptation throughout the manuscript, in particular by highlighting the advantages of BAµE-µLD over other sample treatment techniques and better discussion of the extraction parameters.

We understand the concerns described by the reviewer, and although thermal degradation occurred and HPLC or LC-MS presented better results than the proposed approach, we believe that our methodology has significant advantages over those ones. First, LC-MS/MS are not as widespread as GC-MS systems, which makes the later a more comprehensive and common technique. Secondly, most applications do not take into consideration the contemporary Green Analytical Chemistry Principles. And third, we explicitly said that the methodology can only be used as a screening technique, which can be combined with a quantitative approach. In sum, the proposed analytical cycle is a greener and easier to apply than the current ones.

We also put the NMR characterization in supplementary material and the extraction parameters were extensively discussed. We already provided a comparison with other extraction methods.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

"As was stated before, although the developed methodology cannot be applied for quantitative purposes, it can still be used to detect, confirm, as well as for semi quantification of the three SCs in oral fluids, since these compounds were detected in this type of sample matrix in the ranges proposed by this approach [10]; MDPV and α-PVP were detected in the range of 20.5-831.7 and 81.9-935.0 µg L-1 , respectively". 

This sentence must be corrected because in my opinion, Authors can not semi-quantificate or quantificate under the LOQ of their methodology. Furthermore LOD is 100 µg L-1 in oral fluid thus the concentration value of a real sample can not be under LOD and LOQ. 

Please reconsider semi-quantitative data because is not accetable results out of your methodology limits. 

Author Response

We greatly appreciate the comments and suggestions of reviewer #1 and made the necessary changes and adaptation throughout the manuscript. We especially acknowledge that the proposed analytical approach is only suitable for qualitative analysis of the three synthetic cathinones. Please check the highlighted parts of the manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have corrected most of my suggestions, however, from my point of view they have failed in highlighting the advantages of the proposed method over other established methodologies. In fact, as main advantages, they mention the availability of GC-MS instrumentation in laboratories and the fact that their procedure follows the Green Analytical Chemistry Principles. Then, having in mind that there are other methods with better performances, they present the BAµE-µLD/GC-MS as a screening and semi-quantitative technique. Nevertheless, according to them, samples can be analyzed on site using fast detection kits, then results have to be confirmed using more advanced techniques. For such a purpose, do you think that the GC-MS method will be the best option instead of LC-MS?

Author Response

We greatly appreciate the comments and suggestions of reviewer #2 and made the necessary changes and adaptation throughout the manuscript. We especially highlighted that although most oral samples from substance abuse individuals can be analysed on site using fast detection kits, this cannot be easily performed for recently introduced NPS on the market, since they have distinct physico-chemical characteristics from classical drugs. For this reason, presumptive positive samples must be later confirmed using more advanced techniques such as LC-MS and GC-MS.

We also acknowledge that for these type of compounds, LC-MS presents analytical advantages over GC-MS instrumentation. However, the latter can be a suitable alternative, whenever LC-MS is not available. In our case, we demonstrate that the miniaturized sample preparation technique, in combination with GC-MS, can be used alternatively to LC-MS, to confirm the presence of the target compounds. Please check the highlighted parts of the manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript can be published 

Author Response

We greatly appreciate the positive feedback from reviewer #3.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

The authors responded fairly to reviewers' questions. The article is acceptable for publications.

Author Response

We greatly appreciate the positive feedback from reviewer #1.

Author Response File: Author Response.docx

Reviewer 2 Report

see attached file

Comments for author File: Comments.docx

Author Response

We greatly appreciate the positive feedback from reviewer #2. Furthermore, we corrected each sentence, phrase, or word pointed out by the reviewer. Nevertheless, we made additional minor English corrections throughput the manuscript. Please check the highlighted parts of the document.

Author Response File: Author Response.docx