Comparison of Widely Targeted Metabolomics and Untargeted Metabolomics of Wild Ophiocordyceps sinensis

Round 1

Reviewer 1 Report

I don't think there is enough novelty/significance in this work and the English fluency is very poor and also enough references were not cited. Literature search by the author about this work is very poor. I reject this manuscript for publication.

Author Response

Reply to Reviewer #1

Dear Reviewer,

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits.

Comments: “I don't think there is enough novelty/significance in this work and the English fluency is very poor and also enough references were not cited. Literature search by the author about this work is very poor. I reject this manuscript for publication.”

Response：We appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. Your comments made us aware of the problem with the article. Therefore, fully consider your comments and make revisions in the process of rewriting the article. First of all, this paper was a comparative study on the metabolome of Ophiocordyceps sinensis, hoping that the comparison results of the two methods can provide methodological guidance for Ophiocordyceps sinensis. Secondly, the rewritten article has been revised by professional teachers and language editing companies, hoping to ensure your smooth reading. We also strengthened reference reading and citation, with 43 references cited in our study. Your comments are very important to the revision of the article, and we look forward to your comments.

We attach the modified documents and records.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments for author File: Comments.pdf

Author Response

Reply to Reviewer #2

Dear Reviewer,

Thank you very much for giving us an opportunity to revise our manuscript, and we also appreciate you very much for your positive and constructive comments and suggestions on our manuscript.

Comments: “This manuscript describes the explore metabolites present in stroma and sclerotia of Ophiocordyceps sinensis. Researchers have used both targeted and untargeted approaches to address the following:

• The difference between the metabolites between the stroma and sclerotia.
• Comparing the differences by using various basic Stastical analysis.
• Comparing general metabolite profile towards biosynthesis and pathway analysis.
• Comparing targeted Vs untargeted metabolomics approaches.

Here experimental design seems to be quite simple for both the omics analysis, but the conclusions and the data interpretation done by the authors are of highly importance that demonstrates advantages between targeted and untargeted methods. Here they have just done the profiling experiment and the data has been reported as just identified species without proper conclusions or interpretations on the identified metabolites.”

Thank you very much for your opinion. We have rewritten the article based on your comments, placing more emphasis on the methods, results, and initial discussion of the mechanism. In the remainder of this letter, we discuss each of your comments individually along with our corresponding responses. To facilitate this discussion, we first retype your comments in italic font and then present our responses to the comments.

Comment 1: Results/methods section should contain the details of obtaining the 778 metabolites, where did they get these transitions and how did they identify the metabolites.

Response 1: Thank you very much for your professional review, which was a great help in revising the article. The identification of metabolites is the key and difficult point of the metabolome. We have highlighted this work in the article, and the methods and results are presented in the article. Specifically, databases for metabolite identification were described in the “Methods”.

Comment 2,3: Results section needs to be re written, this section reads to me like methods section. Similar sentences are repeated in both of these sections. In the results section, it would be better to explain the obtained results in detail and with detailed explanations of its importance.

Response 2,3: Your comment reminds us to focus on the description of the results, which is crucial to the structure of the article. We have rewritten the Results section to describe the research results in detail. On the other hand, we also explain the obtained results in detail and with detailed explanations of its importance.

Comment 4: The detailed list of the both the targeted metabolites and untargeted metabolites masses need to be disclosed in the supplementary list. This will help the readers to replicate the methodology.

Response 4: Thank you for your careful observation to make the revision of the article more specific. We have added details of differential metabolites in the article, as shown in Table 1 and Table 2.

Table 1 Differential metabolites in samples of the widely targeted metabolomics

Number	Name	m/z	VIP	P-vlue	RT(min)
1	N-Acetyl-L-phenylalanine	206.10	3.910	0.0058	5.877
2	5-Methyltetrahydrofolic acid	458.20	3.479	0.0006	3.280
3	Riboflavine	377.10	2.906	0.0064	5.056
4	Tripdiolide	377.20	2.787	0.0110	5.136
5	N-Acetyl-L-leucine	174.10	2.721	0.0030	5.377
6	4-Hydroxy-3-methoxymandelate	181.10	2.431	0.0015	3.273
7	Bromocriptine	654.20	2.346	0.0351	12.035
8	Lumichrome	243.10	2.051	0.0034	6.697
9	N1-Methyl-2-pyridone-5-carboxamide	153.10	2.049	0.0033	2.628
10	Nicotinic acid adenine dinucleotide	664.10	2.023	0.0006	0.753
11	Tetrahymanol	443.40	1.987	0.0130	10.573
12	Citrostadienol	427.40	1.906	0.0025	12.285
13	11β-Hydroxyandrost-4-ene-3,17-dione	303.20	1.851	0.0231	6.838
14	Pyridoxine	170.10	1.811	0.0417	1.552
15	Trans-zeatin-riboside	352.00	1.717	0.0439	4.310
16	3-(Carboxymethylamino)propanoic acid	148.10	1.712	0.0276	0.672
17	L-Arginine	175.10	1.704	0.0022	0.600
18	2-Picolinic acid	124.00	1.701	0.0236	1.326
19	Melibiose	341.10	1.699	0.0011	4.546
20	Salsolinol	180.10	1.695	0.0113	2.266
21	L-Homoserine	120.10	1.680	0.0005	0.671
22	L-Threonine	120.10	1.673	0.0004	0.640
23	Gemcitabine	264.10	1.671	0.0290	0.745
24	L-Homoglutamic acid	162.10	1.665	0.0176	0.728
25	Hesperidin	611.20	1.661	0.0002	6.809
26	Flavin adenine dinucleotide (FAD)	786.20	1.658	0.0386	4.082
27	Isodiospyrin	375.10	1.648	0.0332	11.278
28	3-Aminoisobutanoic acid	104.10	1.620	0.0154	0.940
29	Cupressuflavone	539.10	1.608	0.0207	9.667
30	Aminomalonic acid	120.00	1.601	0.0004	0.655
31	L-Citrulline	176.10	1.599	0.0075	0.660
32	N,N-Dihydroxy-L-phenylalanine	198.10	1.595	0.0235	2.315
33	Docosahexaenoic acid	329.20	1.572	0.0204	12.603
34	5-Methyl-2-furaldehyde	111.00	1.566	0.0144	0.755
35	13(S)-HPOT	311.20	1.538	0.0104	11.465
36	5-Carboxyvanillic acid	213.00	1.522	0.0355	3.522
37	Isopulegol	155.10	1.483	0.0001	2.460
38	Serotonin	177.10	1.467	0.0280	2.612
39	Dipterocarpol	443.40	1.466	0.0038	12.052
40	1-Aminocyclopropanecarboxylic acid	102.00	1.460	0.0020	0.700
41	Betaine aldehyde	102.10	1.443	0.0495	0.753
42	Phenethylamine	122.10	1.441	0.0222	2.820
43	Thiamine	265.10	1.425	0.0386	0.701
44	7-Hydroxyflavone	239.10	1.418	0.0421	0.562
45	Cytosine	112.10	1.412	0.0285	0.630
46	6,8-Diprenylnaringenin	409.20	1.409	0.0017	12.621
47	D-Proline	116.10	1.366	0.0438	0.755
48	L-Ornithine	133.10	1.365	0.0129	0.550
49	5-(3-pyridyl)-2-hydroxytetrahydrofuran	166.10	1.357	0.0111	2.803
50	2-carboxybenzaldehyde	151.00	1.333	0.0374	3.072
51	L-Proline	116.10	1.309	0.0166	0.730
52	4-Hydroxybenzaldehyde	123.00	1.287	0.0313	1.905
53	6-Aminocaproic acid	132.10	1.282	0.0170	1.837
54	Sarsasapogenin	417.30	1.276	0.0167	12.084
55	L-Histidine	156.10	1.269	0.0245	0.590
56	Vanillic acid	169.00	1.251	0.0460	3.776
57	(+)-Pteryxin	387.10	1.236	0.0343	11.106
58	L-Asparagine	133.10	1.223	0.0400	0.640
59	p-Aminobenzoate	138.10	1.220	0.0273	0.736
60	Carnosol	329.20	1.202	0.0026	12.147
61	5-Aminolevulinate	132.10	1.201	0.0230	1.555
62	(S)-(-)-perillyl alcohol	135.10	1.200	0.0450	11.602
63	Ethyl 3,4,5-trimethoxybenzoate	241.10	1.170	0.0047	0.529
64	5-Aminovaleric acid	118.10	1.157	0.0452	1.108
65	Cardanol	303.30	1.148	0.0293	12.688
66	1H-Indole-2,3-dione	148.00	1.145	0.0467	2.794
67	Oxymorphone	302.10	1.133	0.0405	11.184
68	Ailanthone	377.20	1.121	0.0307	3.687
69	β-sitosterol	397.40	1.085	0.0096	12.930
70	5,6-DHET	339.30	1.080	0.0264	13.034
71	Anacrotine	352.20	1.024	0.0233	9.550

 

Table 2 Differential metabolites in samples of the untargeted metabolomics

Number	Name	m/z	VIP	P-vlue	RT(min)
1	Sancycline	414.14	2.349	0.0190	5.185
2	Vignatic acid B	519.29	2.284	0.0151	4.656
3	Glycyrin	382.14	2.191	0.0123	7.114
4	4'-O-Methylkanzonol W	350.12	2.134	0.0182	7.115
5	Pirbuterol	240.15	2.009	0.0362	0.713
6	Neotame	378.22	1.978	0.0126	4.295
7	Rubrophen	380.13	1.963	0.0124	7.810
8	5'-Dehydroadenosine	265.08	1.916	0.0058	3.025
9	Nï‰-hydroxy-L-arginine	190.11	1.846	0.0102	0.607
10	Geniposidic acid	374.12	1.840	0.0031	4.467
11	3-Phenylpropanoic acid	150.07	1.797	0.0457	4.055
12	L-Arginine	174.11	1.783	0.0210	0.625
13	Phthalic acid	166.03	1.716	0.0327	0.894
14	Methionylleucine	262.14	1.652	0.0132	3.654
15	Epalrestat	319.03	1.635	0.0157	0.613
16	N-Phenyl-beta-D-glucopyranosylamine	255.11	1.632	0.0160	2.719
17	Tes (buffer)	229.06	1.618	0.0427	0.648
18	4-Pyridoxic acid	183.05	1.592	0.0288	2.897
19	(2R)-2-î²-gdimboa	373.10	1.584	0.0148	1.071
20	Ethosuximide	141.08	1.571	0.0029	2.716
21	12-Azabenz[a]anthracene	229.09	1.569	0.0064	9.210
22	Vorinostat	264.15	1.551	0.0264	3.953
23	(Methylthio)pyrazine	126.03	1.540	0.0055	0.695
24	Nicotinamide adenine dinucleotide	663.11	1.537	0.0189	0.979
25	Acmimycin	346.17	1.512	0.0052	1.228
26	Pentothal	242.11	1.439	0.0071	2.868
27	Phenylethyl alcohol	122.07	1.438	0.0016	4.042
28	Coumarin	146.04	1.433	0.0033	1.208
29	Pentobarbital	226.13	1.425	0.0156	0.710
30	Doxercalciferol	412.33	1.417	0.0089	9.951
31	Vitamin D2	396.34	1.416	0.0418	9.957
32	2-Hydroxycinnamic acid	164.05	1.409	0.0019	1.206
33	5,10-Methenyltetrahydrofolate	456.16	1.409	0.0010	0.697
34	Bicine	163.08	1.407	0.0254	0.655
35	Delta-guanidinovaleric acid	159.10	1.399	0.0021	1.085
36	L-Alanyl-L-proline	186.10	1.376	0.0006	0.706
37	Noradrenaline	169.07	1.374	0.0006	0.706
38	5-Oxidanidyl-5-oxidanylidenenorvalylarginine	302.15	1.372	0.0086	0.704
39	Febrifugine	301.14	1.354	0.0369	4.430
40	L-Tyrosine	181.07	1.353	0.0006	1.040
41	Hydroxytorsemide	364.12	1.338	0.0008	0.665
42	L-dopa	197.07	1.324	0.0220	0.992
43	Aspirin	180.04	1.322	0.0213	0.991
44	Piracetam	142.07	1.318	0.0225	1.405
45	Boldenone undecylenate	452.33	1.310	0.0129	9.950
46	γ-L-glutamyl-L-tyrosine	310.12	1.303	0.0027	2.800
47	N-acetyl-L-tyrosine	223.08	1.297	0.0248	3.208
48	Val-ser	204.11	1.286	0.0006	0.707
49	Methohexital	262.13	1.284	0.0147	3.621
50	Succinyl proline	215.08	1.278	0.0174	0.991
51	Benzoic acid	122.04	1.275	0.0028	0.744
52	Piperonyl sulfoxide	324.18	1.272	0.0163	10.098
53	Reticuline	329.16	1.270	0.0141	0.652
54	Liafensine	366.19	1.258	0.0013	9.830
55	Dihydroxyphenylalanine	197.07	1.245	0.0202	0.884
56	Lumichrome	242.08	1.244	0.0138	5.674
57	D-Xylonic acid	166.05	1.243	0.0044	0.649
58	DL-Mevalonic acid	148.07	1.233	0.0073	0.693
59	Ethenodeoxyadenosine	275.10	1.231	0.0411	1.428
60	Pro-gln	243.12	1.224	0.0034	0.692
61	Mebutamate	232.14	1.222	0.0116	0.718
62	Pipemidic acid	303.13	1.221	0.0068	1.349
63	γ-Glutamyl-S-(1-propenyl) cysteine sulfoxide	306.09	1.221	0.0451	3.240
64	11-Nitro-1-undecene	199.16	1.209	0.0355	6.186
65	Asp-tyr	296.10	1.203	0.0015	2.456
66	7-Formyldehydrothalicsimidine	411.17	1.199	0.0163	0.680
67	N2-(1-Carboxyethyl)-2'-deoxyguanosine	339.12	1.191	0.0184	1.391
68	Deoxyadenosine	251.10	1.191	0.0005	3.118
69	Coumarone	118.04	1.190	0.0272	1.210
70	6-Hydroxy-5-methoxyindole glucuronide	339.10	1.172	0.0290	4.627
71	1-Linoleyl-sn-glycerol 3-phosphate	434.24	1.170	0.0394	9.579
72	Choline o-sulfate	183.06	1.160	0.0058	0.664
73	4-Hydroxybenzaldehyde	122.04	1.157	0.0381	1.216
74	L-Egothioneine	229.09	1.154	0.0274	0.699
75	1-Stearyl estercitric acid	444.31	1.148	0.0036	9.412
76	NS-102	261.07	1.144	0.0014	4.748
77	N-Ribosylhistidine	287.11	1.141	0.0038	0.660
78	N-Acetylmuramic acid	293.11	1.134	0.0177	1.363
79	4-Aminopyridine	94.05	1.134	0.0206	0.627
80	Porphobilinogen	226.10	1.117	0.0209	2.900
81	N-Phenylacetylglutamic acid	265.09	1.108	0.0029	2.371
82	Carglumic acid	190.06	1.100	0.0143	0.605
83	N-Methylethanolamine phosphate	155.04	1.093	0.0420	0.654
84	N-Acetyl-L-glutamic acid	189.06	1.091	0.0172	1.183
85	N2-(carboxymethyl)arginine	232.12	1.089	0.0172	0.690
86	Lodenosine	253.10	1.084	0.0351	9.214
87	Arecoline	155.09	1.070	0.0291	0.715
88	Diphenolic acid	286.12	1.067	0.0079	0.659
89	Gabapentin	171.13	1.052	0.0414	4.803
90	γ-Aminobutyric acid	103.06	1.052	0.0111	0.639
91	Phe-gln	293.14	1.043	0.0090	3.321
92	Bardoxolone methyl	505.32	1.029	0.0210	9.358
93	Indospicine	173.12	1.017	0.0464	1.433
94	3'-Deaminofusarochromanone	277.13	1.015	0.0431	2.437
95	2'-Deoxycytidine	227.09	1.014	0.0314	4.673
96	Troxipide	294.16	1.003	0.0140	2.704

 

 

Comment 5: It is not clear from the figures and the methods section, that how many replicates did they use to obtain the conclusions, from the PCA plots it seems to be 3 replicates, are they biological or technical replicates.

Response 5: We are very sorry for our negligence of describe. In fact, we used 3 biological replicates, and added to the Methods section of the manuscript.

Comment 6: The figures in the article is not clear, it needs to be redrawn by increasing the font.

Response 6: It is really true as Reviewer suggested that figures in the article is not clear. Therefore, we have made appropriate adjustments to the image.

Comment 7: Figure 2, column chart needs to be redrawn in another format, the stacked format is bit confusing and can’t read any text in the figure.

Response 7: We grateful your advice, as you said that the stacked format is bit confusing and can’t read any text in the figure. We redrawn the column chart and plotted the relative abundance of column chart the top 20 metabolites according to the VIP value to observe the difference between OSBSz and OSBSh.

Comment 8: There is a lack of explanation of the observed metabolites in respective stroma and sclerotia samples. I suggest researchers to explain the reasons behind these metabolites and why do we observe the similar and different in this stroma and sclerotia samples.

Response 8: Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. In the Results and Discussion section of the article, we have added descriptions of some metabolites. We found important metabolites responsible for the difference between OSBSz and OSBSh, and these metabolites play important roles in the growth of the stroma and sclerotia.

Comment 9: I would suggest to researchers to describe the mechanisms behind the common components that were found in these two methods.

Response 9: Special thanks to you for your good comments. Based on your suggestion we also explored he common components that were found in these two methods in the Discussion section and they have a particular role for OSBSz and OSBSh.

Comment 10: The article needs to rewritten focusing the broad profiling experiment. Some of the experimental details are repeated both in the methods section and also in the results section. Repetition of the same thing multiple times is not good.

Response 10: Thank you for taking the time to review our manuscript, as you said, there are many problems with this article and your suggestions are crucial to the revision of the manuscript. Therefore, in the process of rewriting the article, we consider your comments to enrich the article. In the discussion section of the article, there is also more emphasis on discussing the mechanism of difference.

We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript described performing both targeted and untargeted approach to profile the metabolome of Ophiocordyceps sinensis. Overall, the study design and result is OK. However, a significant writing quality improvement is required for this manuscript. I just listed some typos or grammar errors caught by me. The authors need to read the manuscript carefully and fix all the typos and grammar errors.

(1) Line 49 delete “analysis techniques”

(2)Line 53, change “different samples. Such as” to “different samples, such as”

(3)Line 65-66 change “to detected” to “to detect”

(4)Line 233 “liquid phase” should be “liquid chromatography”

(5)Line 233, 237 “mass spectrometry” should be “mass spectrometer”

(6)Line 245 insert spaces to “1minat” and “100mg”

(7)Line 259 “drained” should be “dried down”

Other concerns:

(1)Line 113, could authors’ column separate L or D-arginine? If not, the authors should delete L or D before arginine.

(2)Figure 2 and 4, the authors should provide high resolution figure.

Author Response

Reply to Reviewer #3

We are very grateful to your comments for the manuscript. According with your advice, we tried our best to amend the relevant part and made some changes in the manuscript. These changes will not influence the content and framework of the paper. All of your questions were answered below. 

Comments: “The manuscript described performing both targeted and untargeted approach to profile the metabolome of Ophiocordyceps sinensis. Overall, the study design and result is OK. However, a significant writing quality improvement is required for this manuscript. I just listed some typos or grammar errors caught by me. The authors need to read the manuscript carefully and fix all the typos and grammar errors.

(1) Line 49 delete “analysis techniques”

(2)Line 53, change “different samples. Such as” to “different samples, such as”

(3)Line 65-66 change “to detected” to “to detect”

(4)Line 233 “liquid phase” should be “liquid chromatography”

(5)Line 233, 237 “mass spectrometry” should be “mass spectrometer”

(6)Line 245 insert spaces to “1minat” and “100mg”

(7)Line 259 “drained” should be “dried down”

Other concerns:

(1)Line 113, could authors’ column separate L or D-arginine? If not, the authors should delete L or D before arginine.

(2)Figure 2 and 4, the authors should provide high resolution figure.”

Comment 1：(1) Line 49 delete “analysis techniques”

(2)Line 53, change “different samples. Such as” to “different samples, such as”

(3)Line 65-66 change “to detected” to “to detect”

(4)Line 233 “liquid phase” should be “liquid chromatography”

(5)Line 233, 237 “mass spectrometry” should be “mass spectrometer”

(6)Line 245 insert spaces to “1minat” and “100mg”

(7) Line 259 “drained” should be “dried down”

Response 1： Thank you for the detailed review. We have carefully and thoroughly proofread the manuscript to correct all the grammar and typos. We rewritten the article according to the review comments. LetPub for its linguistic assistance during the preparation of this manuscript.

Comment 2：Line 113, could authors’ column separate L or D-arginine? If not, the authors should delete L or D before arginine.

Response 2：Thank you for your valuable advice, in fact, there are differences between L-arginine and D-arginine. L-arginine is an essential amino acid for the human body, which cannot be synthesized by the human body and needs to be ingested from food. D-arginine is derived from L- Arginine is used as raw material, and it is obtained by racemization reaction.

Comment 3：Figure 2 and 4, the authors should provide high resolution figure.

Response 3： It is really true as Reviewer suggested that figures in the article is not clear. We have improved the resolution of the figure.

I wish this revision will be acceptable for publication in your journal.

Thank you for your consideration. I am looking forward to hearing from you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The manuscript can be published after minor revisions. See below:

The authors have improved the manuscript quite better from the previous version. I still would suggest the authors to revise the manuscript more thoroughly, especially the abstract part (writing needs to be improved), and make sure to revise all other sections including the conclusion part once or twice. 

Author Response

Reply to Reviewer #1

Dear Reviewer,

Thank you very much for giving us an opportunity to revise our manuscript, and we also appreciate you very much for your positive and constructive comments and suggestions on our manuscript.

Comments: “The manuscript can be published after minor revisions. The authors have improved the manuscript quite better from the previous version. I still would suggest the authors to revise the manuscript more thoroughly, especially the abstract part (writing needs to be improved), and make sure to revise all other sections including the conclusion part once or twice.”

Response：We appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. Therefore, we carefully revised the abstract, discussion and other sections. Thank you very much for your professional review and pointing out the direction for our manuscript. We sincerely hope that this manuscript can be published and look forward to your review.We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory.

Author Response File: Author Response.pdf

Reviewer 2 Report

Thanks for considering all those coments and taking time to revise the manuscript.  the paper reads much better now. is it possible to increase the font of x and y axis of figure 4.

Author Response

Dear Reviewer, 

Thank you very much for your time involved in reviewing the manuscript and your very encouraging comments on the merits.
Comments: “Thanks for considering all those coments and taking time to revise the manuscript. the paper reads much better now. is it possible to increase the font of x and y axis of figure 4. ”
Response: Thank you very much for your comments in the round 1 of comments, your comments are very professional and detailed. We made some changes to the abstract and discussion, and also adjusted Figure 4. Thanks again for your suggestion, your comments are very important to the revision of the article, and we look forward to your comments.

Author Response File: Author Response.pdf

Reviewer 3 Report

I'm glad to see the authors revised the manuscript carefully. I think the writing quality of the manuscript is OK now. In the previous round of review, I asked the authors if their LC column was able to separate L- or D- metabolites. The authors didn't understand my question. I know the L- or D- are different metabolites and have different physiological functions, but I’m talking about separation of them on a LC column. It is impossible to separate L- or D- metabolites on C18 column without derivatization. The authors should check the raw data carefully to confirm this.  The authors should remove L-/D- in text and heatmaps if this is true.

Author Response

Reply to Reviewer #3

Thank you very much for giving us an opportunity to revise our manuscript, and we also appreciate you very much for your positive and constructive comments and suggestions on our manuscript. Your professional advice is essential for the revision of the article.

Comment: “I'm glad to see the authors revised the manuscript carefully. I think the writing quality of the manuscript is OK now. In the previous round of review, I asked the authors if their LC column was able to separate L- or D- metabolites. The authors didn't understand my question. I know the L- or D- are different metabolites and have different physiological functions, but I’m talking about separation of them on a LC column. It is impossible to separate L- or D- metabolites on C18 column without derivatization. The authors should check the raw data carefully to confirm this.  The authors should remove L-/D- in text and heatmaps if this is true.”

Response：We are very sorry for misunderstanding your advice before, we take your advice very seriously, and consulted experts and teachers due to our lack of knowledge. As you said, it is impossible to separate L- or D- metabolites on C₁₈ column without derivatization, and we remove L-/D- in text and heatmaps. In addition, we focused on revising the abstract and discussion sections. Thanks again for your professional review.

 

Author Response File: Author Response.pdf