A Simultaneous Extraction/Derivatization Strategy for Quantitation of Vitamin D in Dried Blood Spots Using LC–MS/MS: Application to Biomarker Study in Subjects Tested for SARS-CoV-2

Round 1

Reviewer 1 Report

The manuscript by Chhonker et al. examined a simultaneous extraction/derivatization strategy for quantitation of Vitamin D in dried blood spots using LC-MS/MS. They found that our developed LC−MS/MS method may be used for quantification of Vitamin D and its metabolites in DBS and may be applied to investigations of the emerging role of these compounds in various physiological processes.

The major conclusions of this research are justified by the results. The methodology seems to be correct in most experiments and the results of this work may be worth publishing. However, the study requires improvement in some aspects. Please consider the following points:

Minor revision:

1. In figure 1, the figures were not very clear, please replace them.

2. In line 330, the authors described that the absolute mean recoveries are shown in Table 6, but where is table 6?

3. Did the authors compare this method with the published methods?

4. Did the authors use the clinical samples to compare the accuracy of this method and other published methods? How many clinical samples were used?

Author Response

The manuscript by Chhonker et al. examined a simultaneous extraction/derivatization strategy for quantitation of Vitamin D in dried blood spots using LC-MS/MS. They found that our developed LC−MS/MS method may be used for quantification of Vitamin D and its metabolites in DBS and may be applied to investigations of the emerging role of these compounds in various physiological processes.

 

The major conclusions of this research are justified by the results. The methodology seems to be correct in most experiments and the results of this work may be worth publishing. However, the study requires improvement in some aspects. Please consider the following points:

 

Minor revision:

 

1. In figure 1, the figures were not very clear, please replace them.

Reply: It has been replaced by the high-quality image

2. In line 330, the authors described that the absolute mean recoveries are shown in Table 6, but where is table 6?

Reply: It is typological error and has been corrected to table 3.

3. Did the authors compare this method with the published methods?

Reply: Provided in the introduction and tabular form of comparison of previously reported LC-MS/MS methods related to the quantitation of vitamin D in dried blood spots or human serum included in supplementary material table S2.

4. Did the authors use the clinical samples to compare the accuracy of this method and other published methods? How many clinical samples were used?

Reply: 909 clinical samples were used in our analysis.  In section 3.8 we have included the conc. range of all analytes found in the clinical samples, which were within the calibration range and also compared with other published methods. We will briefly present the statistical significance and clinical relevance of this data to our future manuscript. In this one, we have highlighted the methodology part.

 

Reviewer 2 Report

Reviewer comments:

In the manuscript (IJMS-2210678) the authors have developed and validated according to FDA guidelines an efficient and robust LC-MS/MS method for the simultaneous quantitation of vitamin D2 and vitamin D3  and their 25-hydroxy metabolites in human blood. The advantage of applied LC-MS/MS method for the determination of vitamin D is its better specificity, sensitivity and accuracy for vitamin D assay compared to immunoassay method. Beside this, this method used DBS as the sample matrix which used only 40 ul of patient’s blood ensuring the micro sampling technique. 

The manuscript can be very interesting for readers, because it offers the new method of vitamin D quantification which is very sensitive with LLOD of 0.78 ng/mL. This is very important because in real biological samples the concentrations of individual vitamin D metabolites are common below the quantitation level obtained for other developed methods.

In this form, the manuscript needs several minor corrections before the final decision. Please, take into account below some of the comments and suggestions for the improvement of your manuscript quality. In the pdf file of the manuscript the text for the correction has been marked. 

Title page

Lines 6-11 and 16-21

Please, replace bold text with normal. Correct font size in E-mail.

Line 29 and in the other part of manuscript

According to the WHO, abbreviation for the coronavirus disease 2019 is COVID-19.

Line 32

Replace Formic Acid with formic acid

Line 33

Replace ml with mL. Delete s in techniques.

Line 37 

In a sentence, ng/mL can be omitted after the first three numbers.

Lines 39, 99

Replace V with v in Vitamin D.

Lines 46-49 and in the rest of the manuscript.

Individual forms of vitamin D like D2, D3 should be written as D2 and D3.

Line 49

Please, add s to the form.

Line 51-57

Please replace the text with:

The first biotransformation reaction occurs in the liver where vitamins D2 and D3 are converted by the hydroxylation reaction into corresponding 25-hydroxy metabolites (25(OH)D3 or  25(OH)D2)). The reaction is  catalysed by two isoforms of microsomal cytochrome p450 (CYP27A1 and CYP2R1). In the next step, in the renal proximal tubule of the kidney, corresponding 25-hydroxy metabolites of vitamins D2 and D3 are converted into 1,25-dihydroxy or 24,25-dihydroxy metabolites by the action of microsomal enzymes CYP27B1 or CYP24A1, respectively . 

Line 57

Please, insert possible dihydroxy between  two metabolites. Add of vitamin D after metabolites.

Line 77

Please, replace participants tested for Covid-19 and negative Covid-19 with patients positive or negative on COVID-19

Line 89

Add its after and

Line 91 

Replace l with L.

Line 94

Delete full stop before references.

Line 96

Please, replace the quantitation of vitamin D and primary metabolites with the simultaneous quantitation of two major forms of vitamin D (D2 and D3) and their 25-hydroxy metabolites in DBS.

Material and methods

Line 104 and in the rest of manuscript

Please replace Vit D2 or Vit D3 with vitamin D2 or vitamin D3.

Line 106

Please, insert (MeOH) after Methanol.

Line 119

Insert space after 664.

Lines 120-122 and in other part of text

Please, replace serum with plasma. For this experiment human blood with anticoagulant was probably used, so after that you can only get plasma and different blood cells. 

Line 122

Please, insert concentration of buffer.

Line 126

Delete m in gm

Line 142 

Insert its after and.

Line 143 and line 277

Insert m after µ, replace 2.1* 100mm, with 2.1x 100 mm,

Lines 145 and 146

MeOH and formic acid instead of methanol and Formic Acid. 

Line 147

Insert B after %.

Table 1

Please, correct the table.

Each analyte in one row.

Vit D2 and D3 replace with vitamin D2 and Vitamin D3. Use subscript for the number of vitamins. Retention Time replace with Retention time (min), MRM replace with MRM (precursor/fragment ions)

Insert * 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) below the table

Lines 158-160

Please delete this sentence, the same sentence is in line 104-106.

Line 160-162

Please, replace ….. each analyte with Orginal stock solutions of vitamin D3, vitamin D2, 25(OH)D2 and 25(OH)D3,were mixed…. 

Line 162

Insert curves after calibration

Line 169

Please replace title with

Preparation of artificial blood spiked and DBS samples 

Line 170

New title Preparation of blood and DBS cards

Line 173 

Replace RBC with red blood cells (RBC). Delete - in vivo.

Line 175

Insert (sample) or the vitamin D free artificial blood (blank) between blood and on.

Line 181, 186

Insert space

Line 185

Insert After that, solution of  before ZnSO4

Line 198

Replace Fa with formic acid

Line 201

Delete ) after % and insert after water.

Line 235

Formatting 

Line 248

Replace hr with hours.

Line 250

Replace D with D2 and D3

Line 251

Please, insert statistical analysis.

Results and discussion

Line 255

Delete  internal standards and parentheses.

Line 258 

Replace signal-to noise ratio with S/N.

Line 263

Delete (scheduled multiple reaction monitoring).

Line 262

Did you think the next:

To compare the value of obtained data collected in conventional MRM with sMRM mode, data were further analysed  and it found that sMRM mode was superior to the conventional MRM mode, because MRM transition was monitored only during short retention time window maximizing the dwell times.

Lines 269 and 270

Inser (TableS1) after 367.4. Insert (Table 1) after Da.

Line 285

Please, use room temperature or RT in the manuscript . Please look at lines 187 188 and other parts of the manuscript where the room temperature is mentioned. 

Line 288

Please, explain why there are differences between obtained RT of analytes in the text (Line 289), Table 1 and Figure 1. Image 1 should be in a higher resolution. Supplementary data add chromatograms of IS, as well as at least several clinical samples which contain different levels of vitamins D and their metabolites.

Line 299

Insert a after 1.

Line 301

Replace signal-to-noise ratio with S/N.

Lines 304-305

Suggestion of title for Figure 1.

Representative MRM ion-overlay chromatograms of vitamin D2 and D3 and their 25-hydroxy metabolites extracted from DBS prepared from vitamin D free artificial blood (a) blank, and  with standard spiked at LQC (b) and MQC level (c).  (d) Clinical Sample(#4200007901B2) extracted from DBS.

Table 2

Correct name of analyte. Put levels in the second row, add y after da.

Line317

Replace D and with D2 and vitamin D3 and their  

Line 319 

Space and mL.

Line 330

Replace Table 6 with Table 3.

Line 333

Insert in the title of table recovery obtained for pre- or post extraction spike, Which of them?

Suggested title for the table 3

 

The extraction recovery (%) of 25(OH)D2, 25(OH)D3, vitamin D2 and vitamin D3 after pre- or post extraction spike of human DBS. Values are given as a mean value±SD of three experiments.

Table 3

In table 3 replace (Mean ± SD, n#3) with (%), delete % in front of Extraction. Check which data are related to vitamin D2 and vitamin D3.

Line 357

Delete space in ice-chilled

Lines 357-395

Please correct marked text according to the previous comments.

Line 384

Insert space before DBS.

Line 394

Insert (2020) after et al.

References

Please, check the font style.

Supplementary data

Please correct Supplementary Table 1 and Titles of figure 1 and figure 2.

 

Comments for author File: Comments.pdf

Author Response

In the manuscript (IJMS-2210678) the authors have developed and validated according to FDA guidelines an efficient and robust LC-MS/MS method for the simultaneous quantitation of vitamin D2 and vitamin D3  and their 25-hydroxy metabolites in human blood. The advantage of applied LC-MS/MS method for the determination of vitamin D is its better specificity, sensitivity and accuracy for vitamin D assay compared to immunoassay method. Beside this, this method used DBS as the sample matrix which used only 40 ul of patient’s blood ensuring the micro sampling technique. 

The manuscript can be very interesting for readers, because it offers the new method of vitamin D quantification which is very sensitive with LLOD of 0.78 ng/mL. This is very important because in real biological samples the concentrations of individual vitamin D metabolites are common below the quantitation level obtained for other developed methods.

In this form, the manuscript needs several minor corrections before the final decision. Please, take into account below some of the comments and suggestions for the improvement of your manuscript quality. In the pdf file of the manuscript the text for the correction has been marked. 

Title page

Lines 6-11 and 16-21

Please, replace bold text with normal. Correct font size in E-mail.

Reply: Corrected

Line 29 and in the other part of manuscript

According to the WHO, abbreviation for the coronavirus disease 2019 is COVID-19.

Reply: Corrected

Line 32

Replace Formic Acid with formic acid

Reply: Corrected

Line 33

Replace ml with mL. Delete s in techniques.

Reply: Corrected

Line 37 

In a sentence, ng/mL can be omitted after the first three numbers.

Reply: Corrected

Lines 39, 99

Replace V with v in Vitamin D.

Reply: Corrected

Lines 46-49 and in the rest of the manuscript.

Individual forms of vitamin D like D2, D3 should be written as D2 and D3.

Reply: In the literature vitamin D2 and D3 had been reported without a subscript. We would like to follow this pattern.

Line 49

Please, add s to the form.

Reply: Corrected

Line 51-57

Please replace the text with:

The first biotransformation reaction occurs in the liver where vitamins D2 and D3 are converted by the hydroxylation reaction into corresponding 25-hydroxy metabolites (25(OH)D3 or  25(OH)D2)). The reaction is  catalysed by two isoforms of microsomal cytochrome p450 (CYP27A1 and CYP2R1). In the next step, in the renal proximal tubule of the kidney, corresponding 25-hydroxy metabolites of vitamins D2 and D3 are converted into 1,25-dihydroxy or 24,25-dihydroxy metabolites by the action of microsomal enzymes CYP27B1 or CYP24A1, respectively. 

Reply: Thank you for your suggestion. Correction has been done with track changed.

Line 57

Please, insert possible dihydroxy between two metabolites. Add of vitamin D after metabolites.

Reply: Corrected

Line 77

Please, replace participants tested for Covid-19 and negative Covid-19 with patients positive or negative on COVID-19

Reply: Corrected

Line 89

Add its after and

Reply: Corrected

Line 91 

Replace l with L.

Reply: Corrected

Line 94

Delete full stop before references.

Reply: Corrected

Line 96

Please, replace the quantitation of vitamin D and primary metabolites with the simultaneous quantitation of two major forms of vitamin D (D2 and D3) and their 25-hydroxy metabolites in DBS.

Reply: Corrected

Material and methods

Line 104 and in the rest of manuscript

Please replace Vit D2 or Vit D3 with vitamin D2 or vitamin D3.

Reply: Corrected

Line 106

Please, insert (MeOH) after Methanol.

Reply: Corrected

Line 119

Insert space after 664.

Reply: Corrected

Lines 120-122 and in other part of text

Please, replace serum with plasma. For this experiment human blood with anticoagulant was probably used, so after that you can only get plasma and different blood cells. 

Reply: Corrected. Thank you for the clarification.

Line 122

Please, insert concentration of buffer.

Reply: Inserted

Line 126

Delete m in gm

Reply: Corrected

Line 142 

Insert its after and.

Reply: Corrected

Line 143 and line 277

Insert m after µ, replace 2.1* 100mm, with 2.1x 100 mm,

Reply: Corrected

Lines 145 and 146

MeOH and formic acid instead of methanol and Formic Acid. 

Reply: Corrected

Line 147

Insert B after %.

Reply: Corrected

Table 1

Please, correct the table.

Each analyte in one row.

Vit D2 and D3 replace with vitamin D2 and Vitamin D3. Use subscript for the number of vitamins. Retention Time replace with Retention time (min), MRM replace with MRM (precursor/fragment ions)

Insert * 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) below the table

Reply: Corrected.

Lines 158-160

Please delete this sentence, the same sentence is in line 104-106.

Reply: Deleted

Line 160-162

Please, replace ….. each analyte with Original stock solutions of vitamin D3, vitamin D2, 25(OH)D2 and 25(OH)D3,were mixed…. 

Reply: Corrected

Line 162

Insert curves after calibration

Reply: Corrected

Line 169

Please replace title with

Preparation of artificial blood spiked and DBS samples 

Reply: Corrected

Line 170

New title Preparation of blood and DBS cards

Reply: Corrected

Line 173 

Replace RBC with red blood cells (RBC). Delete - in vivo.

Reply: Corrected

Line 175

Insert (sample) or the vitamin D free artificial blood (blank) between blood and on.

Reply: Corrected

Line 181, 186

Insert space

Reply: Corrected

Line 185

Insert After that, solution of  before ZnSO4

Reply: Corrected

Line 198

Replace Fa with formic acid

Reply: Corrected

Line 201

Delete ) after % and insert after water.

Reply: Corrected

Line 235

Formatting 

Reply: Corrected the formatting.

Line 248

Replace hr with hours.

Reply: Corrected

Line 250

Replace D with D2 and D3

Reply: Corrected

Line 251

Please, insert statistical analysis.

Reply: Included in the text.

Results and discussion

Line 255

Delete  internal standards and parentheses.

Reply: Corrected

Line 258 

Replace signal-to noise ratio with S/N.

Reply: Corrected

Line 263

Delete (scheduled multiple reaction monitoring).

Reply: Corrected

Line 262

Did you think the next:

To compare the value of obtained data collected in conventional MRM with sMRM mode, data were further analysed  and it found that sMRM mode was superior to the conventional MRM mode, because MRM transition was monitored only during short retention time window maximizing the dwell times.

Reply: Thank you for the writing suggestion.

Lines 269 and 270

Inser (TableS1) after 367.4. Insert (Table 1) after Da.

Reply: Corrected

Line 285

Please, use room temperature or RT in the manuscript. Please look at lines 187 188 and other parts of the manuscript where the room temperature is mentioned. 

Reply: Corrected all parts of the manuscript.

Line 288

Please, explain why there are differences between obtained RT of analytes in the text (Line 289), Table 1 and Figure 1. Image 1 should be in a higher resolution. Supplementary data add chromatograms of IS, as well as at least several clinical samples which contain different levels of vitamins D and their metabolites.

Reply: Thank you for the suggestion. RT has been corrected in the text. In figure 1, chromatogram of one clinical sample has already been included.

Line 299

Insert a after 1.

Reply: Corrected

Line 301

Replace signal-to-noise ratio with S/N.

Reply: Corrected

Lines 304-305

Suggestion of title for Figure 1.

Representative MRM ion-overlay chromatograms of vitamin D2 and D3 and their 25-hydroxy metabolites extracted from DBS prepared from vitamin D free artificial blood (a) blank, and  with standard spiked at LQC (b) and MQC level (c).  (d) Clinical Sample(#4200007901B2) extracted from DBS.

Reply: Corrected

Table 2

Correct name of analyte. Put levels in the second row, add y after da.

Reply: Corrected

Line317

Replace D and with D2 and vitamin D3 and their  

Reply: Corrected

Line 319 

Space and mL.

Reply: Corrected

Line 330

Replace Table 6 with Table 3.

Reply: Corrected

Line 333

Insert in the title of table recovery obtained for pre- or post extraction spike, Which of them?

Reply: In the methods part we have the extraction recovery calculation method. It is the peak area ratio of the pre-extraction spiked analyte to post-extraction spiked analyte.

Suggested title for the table 3

 

The extraction recovery (%) of 25(OH)D2, 25(OH)D3, vitamin D2 and vitamin D3 after pre- or post extraction spike of human DBS. Values are given as a mean value±SD of three experiments.

Reply: Corrected

Table 3

In table 3 replace (Mean ± SD, n#3) with (%), delete % in front of Extraction. Check which data are related to vitamin D2 and vitamin D3.

Reply: Corrected

Line 357

Delete space in ice-chilled

Reply: Corrected

Lines 357-395

Please correct marked text according to the previous comments.

Reply: Corrected

Line 384

Insert space before DBS.

Reply: Corrected

Line 394

Insert (2020) after et al.

Reply: Corrected

References

Please, check the font style.

Supplementary data

Please correct Supplementary Table 1 and Titles of figure 1 and figure 2.

 

 

Round 2

Reviewer 1 Report

no