Probing Fast Enantio-Recognition of Drugs with Multiple Chiral Centers by Electrospray-Tandem Mass Spectrometry and Its Mechanism

Round 1

Reviewer 1 Report

1. The authors claimed that the proposed method for separating chiral drugs is more efficient than HPLC. However, there is no comparison results with other methods and only provide the discussion on the proposed method. I suggest the authors to strengthen this in the discussion part.

2. What is the reason and motivation for using metal iron complexes as the selector to separate chiral drugs? Besides, how to remove the metal iron complexes after chiral drug separation and what is the drug purity after chiral separation? Please address these issues in the content.

3. The proposed method is not the only used to measure the properties of the drug complex such as bonding strength and stability mentioned in the results, which can also be measured by other analytical methods such as HPLC and NMR. Thus, what is the difference between the proposed method and other generally used methods? Please address this issue in the discussion part?

Author Response

Response to Reviewer 1 Comments

1. The authors claimed that the proposed method for separating chiral drugs is more efficient than HPLC. However, there are no comparison results with other methods and only provide the discussion on the proposed method. I suggest the authors to strengthen this in the discussion part.

Response 1:

Thank you very much for your patient reviewing work and helpful suggestions！

We did this work before. The entecavir enantiomers were discriminated by using tandem MS of metal coordination trimeric complexes in our lab. We compared the MS method with the HPLC method in the USP convention, which requires up to 11 minutes in gradient elution, but the MS method requires no more than 2 minutes [1]. Zhu et al used chiral column for separating ezetimibe stereoisomers and chromatographic time needs more than 20 min [2].

We have revised the description in the manuscript and the relevant change has been supplemented in the manuscript and has been marked in red from line 115 to 119.

 

2. What is the reason and motivation for using metal iron complexes as the selector to separate chiral drugs? Besides, how to remove the metal iron complexes after chiral drug separation, and what is the drug purity after chiral separation? Please address these issues in the content.

Response 2:

Thanks a lot for your professional suggestion!

Several methods have been used for chiral drug analysis successfully, including high-performance liquid chromatography (HPLC), capillary electrophoresis, and nuclear magnetic resonance. However, there are still some limitations to these techniques despite their significant contributions to stereoselective analysis of chiral drugs. HPLC requires chiral columns or chiral derivatization; the isolation efficiency of capillary electrophoresis is significantly influenced by the electrolyte solution; nuclear magnetic resonance necessitates a high concentration of samples and chiral shift reagent, etc. Therefore, it is important to develop a more efficient and simple method for chiral analysis. MS has also been gradually applied to the distinction of enantiomers in the chiral molecules in recent years. As a fast and simple high-throughput analysis method, MS can compensate for the low versatility of chiral columns and the complicated sample preparation process in liquid chromatography. It is clear that MS possesses advantages such as rapid analysis, high sensitivity, and reduced sample consumption. 

β-CD has been used as a chiral ligand to analyze chiral drugs based on its polysaccharide structure [3]. However, when only β-CD is used, the action of efficiency is often relatively low, and it cannot meet the needs of practical application [4]. Derivatization is a common method to majorize β-CD and enhance the interactions between β-CD derivatives and target drugs. The binding of metal ions to β-CD can better exert the role of β-CD as a chiral receptor and broaden the application of β-CD in chiral drug analysis.

In this research, the collision-induced dissociation (CID) combination with the metal-bound trimeric complex is one of the purely MS-based methods for chiral analysis. The metal iron complexes were not removed after chiral drug “separation” because this pure MS-based approach can achieve chiral discrimination in mixtures without separation.

3. The proposed method is not the only used to measure the properties of the drug complex such as bonding strength and stability mentioned in the results, which can also be measured by other analytical methods such as HPLC and NMR. Thus, what is the difference between the proposed method and other generally used methods? Please address this issue in the discussion part?

Response 3:

Thanks a lot for your professional suggestions and your careful work.

Compared with HPLC and NMR, MS shows a direct and accurate result. HPLC requires chiral columns or chiral derivatization, and nuclear magnetic resonance necessitates a high concentration of samples and chiral shift reagent, etc. MS does not need complicated sample preparation and expensive column but needs to find suitable ligands. 

We have revised the description in the manuscript and the relevant change has been supplemented in the manuscript and has been marked in red from line 512 to 521.

References

• Wang Y, Wang L, Chen X, et al. Chiral detection of entecavir stereoisomeric impurities through coordination with R-besivance and Zn^IIusing mass spectrometry. J Mass Spectrom. 2018;53(3):247-256. doi:10.1002/jms.4060
• Zhu B, Yao Y, Zhao Y, Sun T, Li Q. Study on the HPLC-based separation of some ezetimibe stereoisomers and the underlying stereorecognition process. Chirality. 2018;30(5):642-651.
• Ren X, Luo Q, Zhou D, et al. Thermoresponsive chiral stationary phase functionalized with the copolymer of β-cyclodextrin and N-isopropylacrylamide for high performance liquid chromatography. J Chromatogr A. 2020;1618:460904. doi:10.1016/j.chroma.2020.460904
• Agathokleous EA, Stavrou IJ, Kapnissi-Christodoulou C. Comparison of cyclofructan-, cyclodextrin-, and polysaccharide-based chiral stationary phases for the separation of pharmaceuticals. Anal Bioanal Chem. 2022;414(3):1323-1333. doi:10.1007/s00216-021-03754-1

Reviewer 2 Report

As attached.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

 General comment:

Authors did an interesting and comprehensive study on enantiomers recognition, but some pivotal improvement can be done before it can be accepted or publication. The manuscript also requires editing of English language, grammar, and style. Please find below the specific comments.

 

Specific comments:

1. Abstract: Please provide a research background that forms the fundamental of the present study at the beginning of the abstract. Please also provide a conclusion of the present study in the abstract.

Response 1:

Thanks a lot for your professional suggestion! Here is the revised Abstract.

The chiral drug is a very complex substance since individual enantiomer may differ in pharmacological and toxic effects, making it necessary to analyze enantiomers separately. In this study, we investigated the chiral differentiation of two ezetimibe enantiomers (i.e. SRS-EZM and RSR-EZM) and their mechanisms in complex with β-cyclodextrins (CDs) and metal ions as the auxiliary ligands. For this purpose, two complementary approaches have been employed: electrospray-tandem mass spectrometry (ESI-MS/MS) with Collision Induced Dissociation (CID) and molecular modeling methods including density functional theory (DFT) calculations and molecular dynamics (MD) simulations. The results showed a good agreement between experimental and theoretical data. It was demonstrated that SRS-EZM can be easily distinguished from RSR-EZM by applying CID in ESI-MS/MS. SRS-EZM is likely to form a more stable complex with β-CD and metal ions, and thus the [SRS-EZM]-Cu-[β-CD] cluster is more energetically difficult to lose the SRS-EZM molecule compared with RSR-EZM. Such difference may be attributed to the interactions between the drug molecule and the metal ion, as well as the cavity shape changes of the β-CDs upon complexation with molecular guests. Therefore, enantiomer in chiral drug can be recognized as ternary complexes of metal-analyte-β-CD by ESI-MS/MS with CID.

2. Introduction: Authors must provide more statements to justify the method selected; MS, the use β-CD.

Response 2:

Thanks a lot for your professional suggestions and your careful work.

We have provided more statements in Introduction and here is the detailed description.

Several methods have been used for chiral drug analysis in recent years, including high-performance liquid chromatography (HPLC), capillary electrophoresis, and nuclear magnetic resonance. However, there are still some limitations to these techniques despite their significant contributions to stereoselective analysis of chiral drugs. HPLC requires chiral columns or chiral derivatization; the isolation efficiency of capillary electrophoresis is significantly influenced by the electrolyte solution; nuclear magnetic resonance necessitates a high concentration of samples and chiral shift reagent, etc. Therefore, it is important to develop a more efficient and simple method for chiral analysis. MS has also been gradually applied to the distinction enantiomers in chiral molecule in recent years. As a fast and simple high-throughput analysis method, MS can compensate for the low versatility of chiral columns and the complicated sample preparation process in liquid chromatography. It is clear that MS possesses advantages such as rapid analysis, high sensitivity, and reduced sample consumption. 

β-CD has been used as chiral ligand to analyze chiral drugs based on its polysaccharide structure. However, when only β-CD is used, the action of efficiency is often relatively low, and it cannot meet the needs of practical application. Derivatization is a common method to majorize β-CD and enhance the interactions between β-CD derivatives and target drugs. The binding of metal ions to β-CD can better exert the role of β-CD as a chiral receptor and broaden the application of β-CD in chiral drug analysis.

In this research, the collision-induced dissociation (CID) combination with metal-bound trimeric complex is one of the purely MS-based methods for chiral analysis. This direct MS-based approach can achieve chiral discrimination in mixtures.

3. Line 30: Please check the font size used.

Response 3:

Thanks a lot for your professional suggestions and your careful work.

We have already carefully proofread and ensured the font size is appropriate.

 

4. Line 37: have been applied to… should be changed to has been applied for…

Response 4:

We have changed “various methods have been applied to…” into “various methods have been applied for…” in line 41.

5. Line 39: supercritical fluid chromatography 38 (SFC), thin-layer chromatography (TLC) and capillary electrophoresis (CE).

Response 5:

We have changed the relevant content in line 43.

6. Line 41: Authors have mentioned previously that HPLC, GC, SFC, TLC and CE all require too much time and needing plenty of sample. Is it the same with MS, which also requires a lot of time for analysis, and needs plenty of sample and perhaps rather expensive. This contradicts to the previous statement about other methods that have been used before. Please clarify.

Response 6:

Thanks a lot for your professional suggestion!

Mass spectrometry (MS)-hyphenated techniques such as LC-MS, GC-MS, and CE-MS all require much time and plenty of samples, which is the same with HPLC, GC, SFC, TLC, and CE because MS is used as a detector. In this study, the samples are directly injected into the MS system without hyphenated to other methods.

7. Line 47: Collision-induced dissociation should be changed to collision-induced dissociation.

Response 7:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 51 and marked in red.

8. Line 56-57: Please check for any grammatical errors.

Response 8:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript in line 60-61 and marked in red.

9. Line 58: Please remove etc. in the sentence.

Response 9:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript in line 63 and marked in red.

10. Line 61: What do authors mean by dedicated applications? Please provide examples, instead.

Response 10:

Thanks a lot for your professional suggestions and your careful work.

The relevant content in line 66 has been revised as “…exhibit wide range of utilities at reasonable prices” for clarity.

11. Line 65-69: Provide references to support this statement as authors said that many reported that it has low enantioselective binding and efficiency.

Response 11:

Thanks a lot for your professional suggestions and your careful work.

In the revised manuscript, we provide some references.

【1】Chatziefthimiou, S.; Inclán, M.; Giastas, P.; Papakyriakou, A.; Yannakopoulou, K.; Mavridis, I.; Molecular recognition of N-acetyltryptophan enantiomers by β-cyclodextrin. Beilstein J Org Chem. 2017, 13, 1572-1582.

【2】Asahara, C.; Iwamoto, T.; Akashi, M.; Shigemitsu, H.; Kida, T. Effective Guest Inclusion by a 6-O-Modified β-Cyclodextrin Dimer in Organic Solvents. Chempluschem. 2018, 83(9), 868-873. 

12. Line 76-81: Provide reference/s to support the statement here.

Response 12:

Thanks a lot for your professional suggestions and your careful work.

In revised manuscript line 84, we provide more references.

[3] Ma, Q.; Cong, W.; Liu, Y.; Geng, Z.; Lin, Y.; Wang, Z. Experimental and computational study on the enantioseparation of four chiral fluoroquinolones by capillary electrophoresis with sulfated-β-cyclodextrin as chiral selector. Chirality. 2021, 33(9), 549-557. 

13. Line 97: and to find…

Response 13:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 103 and marked in red.

14. Materials and methods: Please provide the full term for all the abbreviations mentioned; CuSO4, ZnCl2, MgCl2 …..

Response 14:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 132 and marked in red.

15. 2.2 Solution preparation: Please provide the initial volume of methanol used for dissolution prior to dilution. Define small amount of DMSO. Methanol should be written as MeOH? This section needs to be re-written in passive sentences and in past tense, accordingly. Please be consistent with the way the experimental units are written in the text. How were the solution mixed? Did it require stirring using a stirrer, and a specific duration?

Response 15:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 139 and marked in red.

16. Line 126: Are these solutions mixed, or prepared separately? Please clarify and rephrase the sentence, accordingly.

Response 16:

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All above solutions were mixed well separately and stored at 4℃ before use.

The relevant content has been revised in the manuscript line 150 and marked in red.

17. Line 147-148: The experiments were applied on Agilient Q-ToF 6550 later to ensure its accuracy. Please describe more on this statement.

Response 17:

After get Q-Trap MS data, we use the same MS parameter to verify the accuracy in Agilient Q-ToF 6550 to ensure the reproducibility of our method.

18. Line 187: spectrogram should be changed to chromatogram.

Response 18:

We think spectrogram here is appropriate because our method is a pure MS method.

19. Line 196: Cu²⁺ shows the best distinguishing effect.

Response 19:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 219 and marked in red.

20. Line 196-197: How did authors determine this? Any references to support this?

Response 20:

Thanks a lot for your professional suggestions.

According to the theory of collision-induced dissociation (CID), at the same collision energy, an unstable compound tends to produce more fragment ions.

In this study, on the basis of fragment ion peaks, we can evaluate the distinction between SRS- EZM and RSR- EZM. In our study, Pb²⁺ and Hg²⁺ have no difference in MS/MS spectrogram and no corresponding [Pb(EZM)₂(β-CD)-H]²⁺ and [Hg(EZM)₂(β-CD)-H]²⁺ ternary complexes found. So Pb²⁺ and Hg²⁺ have no benefits with β-CD for increasing the distinguishing effect on EZM enantiomers.

21. Line 202-204: Please provide reference to support this.

Response 21:

Compare with the fragment ion peaks we can evaluate the distinction between SRS- EZM and RSR- EZM. [Cu(EZM)₂(β-CD)-H]²⁺ has the largest difference in fragment ion peak. So Cu²⁺ shows the best distinguishing effect.

22. Figure 2: Please provide more description/summary of the findings in the figure caption.

Response 22:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 229 and marked in red.

23. Figure 3: Please provide figure with higher resolution. Please also provide more description/summary of the findings in the figure caption.

Response 23:

Thanks a lot for your professional suggestions and your careful work.

The figure has been changed and the relevant content has been revised in the manuscript in 244 and marked in red.

24. Line 234: which indicating should be changed to which indicates.

Response 24:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 257 and marked in red.

25. Line 236: Please check the sentence structure and amend accordingly.

Response 25:

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The relevant content has been revised in the manuscript line 260 and marked in red.

26. Line 240-244: Which figure are authors referring to? Please cite the figure accordingly in the text.

Response 26:

Thanks a lot for your professional suggestions and your careful work.

Figure 3 has been cited accordingly in the manuscript line 266 and marked in red.

27. Line 246: Please check whether differentiating is the correct term to use here.

Response 27:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 270 and marked in red.

28. 3.4.1. the [CuII(β-CD)]2+ complex should be changed to 3.4.1. The [CuII(β-CD)]2+ complex.

Response 28:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 273 and marked in red.

29. Line 262: What do authors mean by more regularly? Please explain more.

Response 29:

The authors intended to express that when the copper ion is initially placed close to the edge of the rim, as shown in Figs 4C and D, the β-CD stays in a more “natural” state, or namely, the structure presents a relatively “regular” circle.

The relevant content has been revised in the manuscript line 287 and marked in red.

30. Line 262-266: Please check for any grammatical errors, and amend accordingly.

Response 30:

Thanks a lot for your professional suggestions and your careful work.

According to your suggestion, we read the relevant content carefully and corrected some mistakes in line 287-291.

 

31. Line 268: Please check whether energy-unfavourable is the correct term used in this context.

Response 31:

Thanks for your professional suggestion.

We think energy-unfavourable here is appropriate, it means an unstable state with higher energy, and here is a reference.

[4] Yi, Z.; Yuan, Z.; Aggregation of a hepatitis C virus replicase module induced by ablation of p97/VCP. J Gen Virol. 2017, 98(7), 1667-1678.

32. Line 269-270: To satisfy the β-CD conformation requirement….

Response 32:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 293 and marked in red.

33. Line 272: In a word, the copper ion plays as a must in this complex… Please rephrase this.

Response 33:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 296 and marked in red.

34. Line 275-276: Authors mentioned in previous studies…. Hence, please provide more references to support this, not just one reference (reference 17).

Response 34:

Thanks a lot for your professional suggestions and your careful work.

In the revised manuscript, we provide some references.

[5] Wang, Y.; Wang, L.; Chen, X.; Sun, C.; Zhu, Y.; Kang, Y.; Zeng, S. Chiral detection of entecavir stereoisomeric impurities through coordination with R-besivance and ZnII using mass spectrometry. J Mass Spectrom. 2018, 53(3), 247-256.

[6] Li, R.; Niu, Y.; Zhao, N.; Yu, B.; Mao, C.; Xu, F. Series of new β-cyclodextrin-cored starlike carriers for gene delivery. ACS Appl Mater Interfaces. 2014, 6(6), 3969-3978.

35. Line 278: Please change could insert to… to could insert into…

Response 35:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 301 and marked in red.

36. Line 284: It is also reported for many times… Please provide references to support this.

Response 36:

Thanks a lot for your professional suggestions and your careful work.

“It is also reported for many times…” has been changed into “It is also reported …”. The relevant citations have been supplemented in the manuscript and marked in red.

【7】Thi, T.; Nauwelaerts, K.; Froeyen, M.; Baudemprez, L.; Van, M.; Augustijns, P.; Annaert, P.; Martens, J.; Van, H.; Van, G. Comparison of the complexation between methylprednisolone and different cyclodextrins in solution by 1H-NMR and molecular modeling studies. Journal of pharmaceutical sciences, 2010, 99(9), 3863–3873

【8】Madan, J.; Gundala, R.; Baruah, B.; Nagaraju, M.; Yates, C.; Turner, T.; Rangari, V.; Hamelberg, D.; Reid, D.; Aneja, R. Cyclodextrin complexes of reduced bromonoscapine in guar gum microspheres enhance colonic drug delivery. Mol Pharm. 2014, 11(12), 4339-4349.

37. Figure 4C is not cited in the text.

Response 37:

Thanks a lot for your professional suggestions and your careful work.

Figure 4C has been cited accordingly in the manuscript line 286 and marked in red.

38. Line 298-313: O1, O2…should be written in subscript as shown in Figure 5.

Response 38:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 326 and marked in red.

39. Please cite Figure 5A and 5B in the text accordingly.

Response 39:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 321-323 and marked in red.

40. Line 324-326: Please check the sentence and rephrase accordingly.

Response 40:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 346 and marked in red.

41. Line 329-331: Furthermore, the △R of the β-CD/Cu complex be-329 come smaller in the existence of copper ion, indicating that the Cu makes the entrance 330 become more round. Please provide more explanation/description on this.

Response 41:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 350 and marked in red.

42. Line 364: The rest of the Cu-O bond lengths.

Response 42:

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The relevant content has been revised in the manuscript line 386 and marked in red.

43. Line 369-379: Please check for any grammatical errors in the sentences, and amend accordingly.

Response 43:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 391 and marked in red.

44. Line 373: Thus, it is probable that… should be changed to Thus, it is possible that….

Response 44:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 395 and marked in red.

45. Line 378: contributes a lot… A lot seems vague, perhaps authors can replace this with contributes significantly.

Response 45:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 400 and marked in red.

46. Line 382-383: Is it supposed to be hydroxyl group? Please check for any grammatical errors in this sentence.

Response 46:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 405 and marked in red.

47. Line 394: insert in to… to be changed to insert into.

Response 47:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 416 and marked in red.

48. Line 395: the angel between… should be changed into the angle between…

Response 48:

Thank you for your suggestion. We apologize for our poor writing. According to your suggestion, the relevant content has been revised in the manuscript line 418 and marked in red.

49. Figure 6: No energy is shown in the figure. Please provide figure with higher resolution. What does the arrows in the figure refer to?

Response 49:

The energy differences are discussed in the text. As the absolute values of the internal energy of the substances cannot be determined, the calculated absolute values have no physical meanings. They are not listed in the figures to prevent misunderstanding. The black arrows in Figure 6E refer to the change of the binding pose of the drug from SRS to RSR.

A higher resolution Figure has been provided.

50. Line 418: is weaker compared to coordination…

Response 50:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 442 and marked in red.

51. Line 427: However, compared to the complex….

Response 51:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 452 and marked in red.

52. Line 434-436: Please check for any grammatical errors in these sentences, and rephrase accordingly.

Response 52:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 461 and marked in red.

53. Figure 7: No energy is shown in the figure. Please provide figure with higher resolution.

Response 53:

The energy differences are discussed in the text. As the absolute values of the internal energy of the substances cannot be determined, the calculated absolute values have no physical meanings. They are not listed in the figures to prevent misunderstanding.

A higher resolution figure has been provided.

54. Please describe more on the need of using different running time as presented in Figure 8.

Response 54:  

We did not use the different running time for the SRS and RSR systems. For both systems, 1 ns (namely 1000 ps) MD simulations were performed, as stated previously in section 2.3.2. As shown in Figure 8, different dynamic behaviors can be observed within 1 ns simulation duration: the RSR molecule run away from the CD quickly, while SRS can stay with it during the simulation. The snapshots at representative timepoints are presented. For the RSR case, the drug has drifted far enough away from the CD at 600 ps, so there is no need to show the snapshot at 1000 ps.

55. Please provide more description/ discussion for Figure 9.

Response 55:

The relevant content has been revised in the manuscript line 511 and marked in red.

56. Figure 9: Please provide a figure with better resolution. Scales for axes must be standardized, if possible.

Response 56:

The figure with better resolution has been provided. The distribution of the distances is normalized in the figure.

57. Line 491: it is shown….

Response 57:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 530 and marked in red.

58. Line 496: and forms a strong coordination…

Response 58:

Thanks a lot for your professional suggestions and your careful work.

The relevant content has been revised in the manuscript line 536 and marked in red.

59. More references should be provided to support the basis and findings of the present manuscript.

Response 59:

Thanks a lot for your professional suggestions and your careful work and we understand your concerns. In the revised manuscript, we have added more references.

Reviewer 3 Report

The manuscript entitled " Probing Fast Enantio-recognition of Drugs with Multiple Chiral Centers by Electrospray-Tandem Mass Spectrometry and its Mechanism " by wang et al. has been reviewed carefully for publication. The authors have proposed a new chiral separation/detection method to characterize the enantiomers. A manuscript can be considered for publication after addressing the below comments.

 

1. In 3.1 ESI-MS analysis of section 3. Result and discussion (line#179-183), it is advisable to mention the respective m/z readouts (i.e., 1007.114 or 342.9134, etc.) for the parent ion and/or fragmented ion mentioned for all the clusters mentioned. This helps the reader to better understand the results.
2. In 3.2, lines #202-204, Could you please elaborate on your conclusion to justify your findings and conclusion? It would be great if you can explain and compare each of the metal ions and how you come up with the conclusion " that loss of electron capability and the size of the radius are important factors that affect the discrimination function of metal ions as ligands.
3. About figure 2 A, B, C, and D, each one has two mass spectra assigned with it, however, there is no description for each of them. As an example, in Figure 2A, what do the different graphs represent? Also, the highlighted m/z [ 392.1456, 801.7286, 804.2257, 783.7531…] are not properly discussed in the result and discussion session.
4. In 3.3.1, Line #215-216, It says the corresponding abundance fragment ion of SRS-EZM is only 40% (Ref. fig-3). Could you please justify if it is exactly 40% or an average of 40% as the Mass Spectra of figure 3 (CE-16) shows the %intensity for 392.1449 m/z around 50% which is contradicting the write-up in lines #215-216? Also, I believe it is not advisable to make any conclusion about the intensity of fragment ion (392.1449 m/z) for the CE=16 where the parent ion (1007.8 m/z) is not abundantly fragmented. The parent ion should be fragmented by at least 90% to have a consistent and reproducible fragmentation pattern.
5. Also, the statement in lines #217-218 (When collision energy was set at 18V, this fragment ion can be completely decomposed. From the energy aspects, the binding of [Cu(SRS-EZM)2(β-CD)-H]2+ is stronger than [Cu(RSR-EZM)2(β-CD)-H]2+)] is contradicting with the respective mass spectrum referred in the Fig 3B where the fragment ion 392.1449 m/z is distinctly present. It is highly recommended to shows the m/z values in pure intensity (i.e. 3e4) instead of % intensity (i.e 90%). It is very difficult to interpret Fig3 A & 3B where all the fragment ion shows 90% intensity. It is hard to say that fragment ion is 392.1449 increasing or decreasing with the change of CE. I advise you to correct it.
6. In conclusion lines #487-488 (mainly the losses of EZM). Please mention the loss of EZM from the ternary complex. Please re-write the sentence for better clarity.
7. Could you please also include the limitation or disadvantages of this proposed new method over the other traditional analytical methods for chiral separation? Also, have you tried this new method for other drugs or compounds other than EZM? Does it have any specific pre-requirements for the application of this newly proposed method for chiral separation? Including this information will increase the interest of the other analytical scientists to see its feasibility and significance for their science.

 

Author Response

Response to Reviewer 3 Comments

The manuscript entitled " Probing Fast Enantio-recognition of Drugs with Multiple Chiral Centers by Electrospray-Tandem Mass Spectrometry and its Mechanism " by wang et al. has been reviewed carefully for publication. The authors have proposed a new chiral separation/detection method to characterize the enantiomers. A manuscript can be considered for publication after addressing the below comments.

1. In 3.1 ESI-MS analysis of section 3. Result and discussion (line#179-183), it is advisable to mention the respective m/z readouts (i.e., 1007.114 or 342.9134, etc.) for the parent ion and/or fragmented ion mentioned for all the clusters mentioned. This helps the reader to better understand the results.

Response 1:

Thanks a lot for your professional suggestions and your careful work.

The MS/MS spectrogram of [M^II(EZM)₂(β-CD)]²⁺ ternary complexes.  (M=Cu, Co, Zn, Mg, A. [Cu(EZM)₂(β-CD)]²⁺：m/z =1007.7987, B. [Co(EZM)₂(β-CD)]²⁺：m/z =1005.8000, C. [Zn(EZM)₂(β-CD)]²⁺：m/z =1008.2984, D. [Mg(EZM)₂(β-CD)]²⁺：m/z =988.3264).

In 3.1, we cite an instance to show a different form of possible parent ions and the specific results. The specific formula weight was shown in the following sections. For better understanding, we added respective m/z readouts to each chemical formula.

2. In 3.2, lines #202-204, Could you please elaborate on your conclusion to justify your findings and conclusion? It would be great if you can explain and compare each of the metal ions and how you come up with the conclusion " that loss of electron capability and the size of the radius are important factors that affect the discrimination function of metal ions as ligands.

Response 2：

Thank you very much for your careful work.

Different metal ions have different complexing actions. at the same collision energy, the abundance of the same fragment ion is different. So, by comparing the abundance of fragment ions, we can find out which metal ion is the best fit.

In our subsequent research, we present a possible conjecture, which is related to the loss of electron capability and the size of the radius. In order to keep rigorous, we have changed the description in the article.

3. About figure 2 A, B, C, and D, each one has two mass spectra assigned with it, however, there is no description for each of them. As an example, in Figure 2A, what do the different graphs represent? Also, the highlighted m/z [ 392.1456, 801.7286, 804.2257, 783.7531…] are not properly discussed in the result and discussion session.

Response 3:

Thank you for pointing out the shortcomings of our manuscript.

For the questions you point out, we sincerely apologize that we could not present in clearly and it led to ambiguity.

The revised descriptions in line 209-219 are as follows:

In addition, Zn²⁺, Mg²⁺ and Co²⁺ have shown a similar distinguishing effect to Cu²⁺. They all show one abundance difference of fragment ion in the corresponding [M^II(EZM)(β-CD)-H]²⁺ (M=Cu²⁺, Zn²⁺, Mg²⁺, Co²⁺) ternary complexes (SRS- EZM and RSR- EZM) MS/MS spectrogram (Fig. 2).

By comparing the abundance of this special fragment ion, we can discriminate SRS- EZM and RSR- EZM.

Among that, Cu²⁺ corresponds to fragment ion peak [EZM-H₂O+H]⁺ (m/z =392.14) and Zn²⁺, Mg²⁺, Pb²⁺ correspond to fragment ion peak [MII (EZM)₂(β-CD)-H]²⁺ (m/z =801.72, 804.22, 783.75). By comparison of the difference of fragment ion ratio of abundance between corresponding SRS-EZM and RSR-EZM ternary complexes, Cu²⁺ shows the best distinguishing effect.

4. In 3.3.1, Line #215-216, It says the corresponding abundance fragment ion of SRS-EZM is only 40% (Ref. fig-3). Could you please justify if it is exactly 40% or an average of 40% as the Mass Spectra of figure 3 (CE-16) shows the %intensity for 392.1449 m/z around 50% which is contradicting the write-up in lines #215-216? Also, I believe it is not advisable to make any conclusion about the intensity of fragment ion (392.1449 m/z) for the CE=16 where the parent ion (1007.8 m/z) is not abundantly fragmented. The parent ion should be fragmented by at least 90% to have a consistent and reproducible fragmentation pattern.

Response 4:

Thanks a lot for your professional comments!

Same as response 3, here we mainly want to represent a difference between SRS-EZM and RSR-EZM at the same collision energy.

As a result, the abundance shows a disparity. We have changed the ordinate in Fig. 3 and added Table 1 to show these differences.

The relevant change has been supplemented in the manuscript and has been marked in red from line 246 to 247.

Table 1. The difference in the fragment ions abundance of [Cu(EZM)₂(β-CD)]²⁺ ternary complexes at the different collision energy

	[Cu(RSR-EZM)2(β-CD)]2+	[Cu(SRS-EZM)2(β-CD)]2+	Difference value
CE10	12.4%	8.2%	4.2%
CE12	21.3%	13.1%	8.2%
CE14	43.2%	23.6%	19.6%
CE16	97.3%	46.7%	50.6%
CE18	99.4%	99.2%	0.2%
CE20	99.7%	99.6%	0.1%

5. Also, the statement in lines #217-218 (When collision energy was set at 18V, this fragment ion can be completely decomposed. From the energy aspects, the binding of [Cu(SRS-EZM)₂(β-CD)-H]²⁺ is stronger than [Cu(RSR-EZM)₂(β-CD)-H]²⁺)] is contradicting with the respective mass spectrum referred in the Fig 3B where the fragment ion 392.1449 m/z is distinctly present. It is highly recommended to shows the m/z values in pure intensity (i.e. 3e4) instead of % intensity (i.e 90%). It is very difficult to interpret Fig3 A & 3B where all the fragment ion shows 90% intensity. It is hard to say that fragment ion is 392.1449 increasing or decreasing with the change of CE. I advise you to correct it.

Response 5:

Thanks a lot for your professional suggestions!

According to the theory of collision-induced dissociation (CID), at the same collision energy, an unstable compound tends to produce more fragment ions.
As shown in Fig. 3 and Table1, when collision energy was less than 16V, diagnostic fragment ions did not completely dissociate from the parent ion. However, when collision energy was over 16V, the parent ion was completely fragmentation. So, we try to test which collision energy is the best to distinguish between SRS-EZM and RSR-EZM.

By comparing different collision energy and fragment ions, 16V was found to be the optimal collision energy to distinguish SRS-EZM from RSR-EZM. At 16V collision energy, more fragment ions were produced from RSR-EZM than SRS-EZM, showing a significant difference.

6. In conclusion lines #487-488 (mainly the losses of EZM). Please mention the loss of EZM from the ternary complex. Please re-write the sentence for better clarity.

Response 6:

Thanks a lot for your professional suggestions!

We have revised our sentence as follows:

In conclusion, electrospray-tandem mass spectrometry and molecular modeling were used in our study to probe the chiral analysis mechanism of RSR- and SRS-EZM. Collision-induced dissociation of the ternary complexes produces stable charge reduced intermediates, mainly the losses of the EZM from the ternary complexes.（line 524-527）

7. Could you please also include the limitation or disadvantages of this proposed new method over the other traditional analytical methods for chiral separation? Also, have you tried this new method for other drugs or compounds other than EZM? Does it have any specific pre-requirements for the application of this newly proposed method for chiral separation? Including this information will increase the interest of the other analytical scientists to see its feasibility and significance for their science.

Respose 7:

Thanks a lot for your professional suggestions!

To deeply understand the role of the drug enantiomers in the ternary complex and the application scope of our method, we studied the recognition of captopril and ibuprofen enantiomers by MS using Cu²⁺ and β-CD as ligand but failed. Captopril, β-CD, and metal ions cannot form a stable ternary complex because the sulfhydryl group of captopril is oxidated easily in the presence of metal ions. Ibuprofen, β-CD, and metal ions cannot form a ternary complex but can be recognized by D-glucose and metal ion complex, possibly due to the large opening size of β-CD relative to the ibuprofen molecule. Therefore, there are some limitations for MS with β-CD and metal ions complex method for recognition of enantiomers, such as suitable ligand, enantiomer`s stability, and molecular size. Majorizing this analytical method and increasing the universality is our future goal.

We have revised the description in the manuscript and the relevant change has been supplemented in the manuscript and has been marked in red from line 512 to 521.