Detection of 7-Dehydrocholesterol and Vitamin D3 Derivatives in Honey
, Venkatram Atigadda 1, Pawel Brzeminski 1,3, Adrian Fabisiak 1,3, Edith K. Y. Tang 4, Robert C. Tuckey 4 and Andrzej T. Slominski 1,2,*Round 1
Reviewer 1 Report
The manuscript entitled “Detection of 7-dehydrocholesterol and Vitamin D3 derivatives in honey” investigated the presence of vitamin D3 and its hydroxy derivatives in honey, thus implicating honeybees in the production of secosteroids. This manuscript is suitable to be published in Molecules, after revisions.
All abbreviations should be described when used for the first time.
Why the authors opted to use dichloromethane to extract the target analytes? Why not other solvent?
The concentration should be expressed in the same units, sometimes appears ng/g other µg/g or only ng. The units of limit of quantification is ng? The authors should provide more information related to method validation.
In figures, retention time should be RT, as well as through the manuscript.
According to section 4.1, several standards are synthetized by the authors. The authors confirm the structure of these standards, before LC-MS analysis? Please add this information to the manuscript.
Section 4.2. honey (70 mL) extracted with 175 mL dichloromethane…. then the authors written honey (100g) is extracted with 3 x 150 mL dichloromethane….Why this different process? Is too confuse.
Please add a conclusion section with main achievements.
Author Response
The manuscript entitled “Detection of 7-dehydrocholesterol and Vitamin D3 derivatives in honey” investigated the presence of vitamin D3 and its hydroxy derivatives in honey, thus implicating honeybees in the production of secosteroids. This manuscript is suitable to be published in Molecules, after revisions.
Reply
We thank the reviewer for the time and effort to review the paper
All abbreviations should be described when used for the first time.
Reply
We now describe all abbreviations when used for the first time. The changes are highlighted in yellow.
Why the authors opted to use dichloromethane to extract the target analytes? Why not other solvent?
Reply
Dichloromethane was chosen because it is an easy to use two-phase system that efficiently extracts secosteroids into the organic phase. It is widely used in the extraction of secosteroids from aqueous phases. The appropriate references where dichloromethane has been used are included in the revised version of the manuscript. In addition, methylene chloride is a non-polar solvent and most lipophilic solutes have very good solubility in methylene chloride. Due to the lipophilic nature of vitamin D analogs, we rationalized that the expected compounds to have good solubility in methylene chloride. In addition, due to its low boiling point, methylene chloride can be evaporated at room temperature under a slight vacuum.
The concentration should be expressed in the same units, sometimes appears ng/g other µg/g or only ng. The units of limit of quantification is ng? The authors should provide more information related to method validation.
Reply
We used ng/g only for presenting concentration of 20(OH)D3 in honey where as ng is used for describing exact amount of 20(OH)D3. We did not use µg/g as a unit in the manuscript. The standard curve is in Fig. S2, while LOQ in Fig. S3, so the information related to method validation is shown.
In figures, retention time should be RT, as well as through the manuscript.
Reply
We have corrected this. However, the abbreviation (RT) was explained in appropriate sections of the manuscript.
According to section 4.1, several standards are synthetized by the authors. The authors confirm the structure of these standards, before LC-MS analysis? Please add this information to the manuscript.
Reply
Yes, structures were confirmed by NMR and the data on this has been published. We therefore added the sentence below to Section 4.1:
“The identities of all synthesized secosteroidal standards were determined by mass spectrometry and NMR at the time of synthesis [24, 26, 62-69].”
Section 4.2. honey (70 mL) extracted with 175 mL dichloromethane…. then the authors written honey (100g) is extracted with 3 x 150 mL dichloromethane….Why this different process? Is too confuse.
Reply
These two different extractions were performed by two different authors contributing to the manuscript and they chose the two different units (mL and g) for the amounts of honey measured. After the first extraction method was used, we realized that the second extraction was more efficient. It is well established that repeating liquid-liquid extraction three times will recover the maximum possible amount of solute. Therefore, to improve the method we changed to the second procedure and extracted the aqueous layer three times with methylene chloride, and the 20(OH)D3 quantification was performed using this extraction method. It is therefore necessary to mention both extraction methods in the manuscript.
Please add a conclusion section with main achievements.
Reply
The conclusion section has been identified per reviewer request
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The aim of the paper was to investigate the presence of vitamin D3 and its hydroxy derivatives in honey collected during summer months in the Birmingham (honey extract from the local source) and honey commercially available from Walmart (honey extract from the commercial source) by LC-MS method. The manuscript is well constructed and the idea of the work seems to be very interesting. The results are clearly presented, and the conclusions are consistent with the results obtained. In my opinion, the submission requires minor edition and improvement at some points, main of which are listed below.
Minor issues:
L139-140: Please close the gap in the text.
L220: Please describe the type of honey, that is, from which potentially flowering plants bees have produced the tested honey.
L235-238, 244-252: The gradient profile for LC-MS assay should be given using percentages of solvent B. For example: “The elution profile was as follows: 0-15 min, 40-100% B (v/v); 15-30 min, 100% B; ….”. What was the time for equilibration?
Additionally, please shorten Figure titles. At this point, it is not necessary to re-describe the equipment used or the conditions of analysis.
The Authors should also add the Table with all identified compounds, containing names of the components, retention times, and MS data, etc.
Author Response
The aim of the paper was to investigate the presence of vitamin D3 and its hydroxy derivatives in honey collected during summer months in the Birmingham (honey extract from the local source) and honey commercially available from Walmart (honey extract from the commercial source) by LC-MS method. The manuscript is well constructed and the idea of the work seems to be very interesting. The results are clearly presented, and the conclusions are consistent with the results obtained. In my opinion, the submission requires minor edition and improvement at some points, main of which are listed below.
Reply
We thank the reviewer for the time and effort to review the paper
Minor issues:
L139-140: Please close the gap in the text.
Reply
This has been done
L220: Please describe the type of honey, that is, from which potentially flowering plants bees have produced the tested honey.
Reply
This information we have available on the honey in provided in the materials and methods
“Honey (70 mL) collected during summer months in the Birmingham, Alabama area was extracted with methylene chloride (175 mL).”
“In addition, commercially available unprocessed honey from Nature Nate’s Corporate (McKinney, TX) was purchased from Walmart”
To make these point clear for the reader we added following sentence to the Results section:
“Honey collected during summer in the Birmingham, AL area, or purchased commercially (see section 4.2)”
L235-238, 244-252: The gradient profile for LC-MS assay should be given using percentages of solvent B. For example: “The elution profile was as follows: 0-15 min, 40-100% B (v/v); 15-30 min, 100% B; ….”. What was the time for equilibration?
Reply
We changed all gradient profile following reviewer’s suggestions and all injections were performed after equilibration in which a delta column pressure reached around 10 psi. See below for the modified text.
“The pre-purification was performed using conditions as follows: 0-15 min, 40–100% acetonitrile in water (v/v) at a flow rate of 0.5 mL/min; 15-45 min, 100% acetonitrile at a flow rate of 0.5 mL/min; 45-65 min, 100% acetonitrile at a flow rate of 1.5 mL/min…….
……..as follows: 0-3 min, 20- 60% methanol or acetonitrile (v/v); 3-3.2 min, 60-97% methanol or acetonitrile in water (v/v); 3.2-4.8 min, 97% methanol or acetonitrile in water (v/v) at a flow rate of 0.3 mL/min. For the ACQUITY UPLC BEH C18 column, elution was performed with a gradient of methanol in water (all containing 0.1% formic acid) as follows: 0-2 min, 40% methanol in water (v/v); 2-3 min, 40-85% methanol in water; 3-6 min, 97% methanol in water (v/v) at a flow rate of 0.3 mL/min. For the Atlantis C18 column, elution was with a gradient of methanol in water (containing 0.1% formic acid for LC-MS) as follows: 0-20 min, 85-100% methanol in water; 20-30 min, 100% methanol in water (v/v) at a flow rate of 0.5 mL/mL. For the Pursuit 200Å PFP column, elution was performed with a gradient of methanol (containing 0.1% formic acid) as follows: 0-20 min, 40-100% methanol in water (v/v); 20-30 min, 100% methanol in water (v/v) at a flow rate of 0.5 ml/mL. All injections were performed after re-equilibration in the initial solvent until the delta column pressure reached around 10 psi”
Additionally, please shorten Figure titles. At this point, it is not necessary to re-describe the equipment used or the conditions of analysis.
Reply
We shortened the Figure titles. Columns and mobile phase information remain, because all analyses were performed using different columns and mobile phases. This information is therefore necessary to identify the exact procedure.
The Authors should also add the Table with all identified compounds, containing names of the components, retention times, and MS data, etc.
Reply
We added Table 1 with the requested information on page 12, and quoted the Table with the following sentence (last paragraph of Results):
"Table 1 shows a summary of the information on the metabolites detected in honey, including the LC system used, RT and detected masses."
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors reported the presence of 7DHC, D3 and L3, and of species corresponding to 20(OH)-7DHC, 20(OH)D3, 1,20(OH)2D3, 25(OH)D3 and 1,25(OH)2D3 in honey. The manuscript is very interesting and is well written in good English and in an adequate format for Molecules. The paper is suitable for publication.
Author Response
The authors reported the presence of 7DHC, D3 and L3, and of species corresponding to 20(OH)-7DHC, 20(OH)D3, 1,20(OH)2D3, 25(OH)D3 and 1,25(OH)2D3 in honey. The manuscript is very interesting and is well written in good English and in an adequate format for Molecules. The paper is suitable for publication.
Reply
We thank the reviewer for the time and effort to review the paper and for positive evaluation of its content.
Author Response File: Author Response.pdf
