Quantification of 25-hydroxyvitamin D<sub>3</sub> in dried blood spots as compared to plasma among Indian adults

Ashwini V Shete; Jyoti Sawant; Rajani Bagul; Ujjwala Ghule; Sarang S. Saluke; Christopher R. Sudfeld

doi:10.12688/f1000research.149829.2

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Brief Report

Revised

Quantification of 25-hydroxyvitamin D₃ in dried blood spots as compared to plasma among Indian adults

[version 2; peer review: 1 approved, 1 approved with reservations]

Ashwini V Shete¹, Jyoti Sawant¹, Rajani Bagul¹, Ujjwala Ghule¹, Sarang S. Saluke², Christopher R. Sudfeld ³

Ashwini V Shete¹, Jyoti Sawant¹, [...] Rajani Bagul¹, Ujjwala Ghule¹, Sarang S. Saluke², Christopher R. Sudfeld ³

PUBLISHED 06 Jun 2025

Author details Author details

¹ ICMR National AIDS Research Institute, Pune, Maharashtra, India
² Bio-Analytical Technologies, Pune, Maharashtra, India
³ Global Health and Population, Harvard T.H. Chan School of Public Health, Boston, USA

Ashwini V Shete
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Jyoti Sawant
Roles: Investigation, Methodology, Validation, Writing – Review & Editing

Rajani Bagul
Roles: Investigation, Methodology, Validation, Writing – Review & Editing

Ujjwala Ghule
Roles: Investigation, Methodology, Validation, Writing – Review & Editing

Sarang S. Saluke
Roles: Formal Analysis, Investigation, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Christopher R. Sudfeld
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background

Vitamin D may play an important role in later-life physical and cognitive health. Vitamin D status is standardly assessed in serum and plasma; however, collection, transport, and storage costs make large epidemiologic studies challenging. We assessed the agreement of 25-hydroxyvitamin D₃ (25(OH)D₃) quantification from dried blood spots (DBS) as compared to standard plasma assessment among older Indian adults.

Methods

A total of 58 adults over 45 years of age who resided in Pune, India were enrolled in the study from July 2020 to June 2021. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess 25(OH)D₃ concentrations in paired plasma and DBS samples.

Results

Plasma and DBS 25(OH)D₃ concentrations were highly correlated (Pearson’s correlation = 0.976). The median 25(OH)D₃ concentration of the study population assessed by plasma was 14.6 ng/mL (Q1=12.0, Q3= 18.1) while the median concentration assessed in DBS was 12.8 ng/mL (Q1=11.0, Q3= 16.6). 25(OH)D₃ concentrations measured from DBS were on average 6% (95% CI: 2-13%) lower than concentrations assessed by plasma across the observed 25(OH)D₃ distribution.

Conclusions

We found good agreement between 25(OH)D₃ quantification between DBS and plasma and our findings indicate that DBS can be used in epidemiologic studies of vitamin D among Indian adults.

Keywords

Vitamin D, Calcifediol, Micronutrients, Dried Blood Spot Testing, Validation Study

Corresponding author: Christopher R. Sudfeld

Competing interests: No competing interests were disclosed.

Grant information: The research was funded by National Institute on Aging Grant Number (P30AG024409)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2025 Shete AV et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Shete AV, Sawant J, Bagul R et al. Quantification of 25-hydroxyvitamin D₃ in dried blood spots as compared to plasma among Indian adults [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Research 2025, 13:512 (https://doi.org/10.12688/f1000research.149829.2) First published: 20 May 2024, 13:512 (https://doi.org/10.12688/f1000research.149829.1) Latest published: 06 Jun 2025, 13:512 (https://doi.org/10.12688/f1000research.149829.2)

Revised Amendments from Version 1

A text update of the manuscript was made to address comments from the reviewers. In summary, we have made four updates: i) adjusted text in the introduction to provide information on sunlight and diet as contributors to vitamin D status, ii) clarified LC-MS/MS specifications, iii) added a formula to estimate plasma vitamin D3 from dried blood spot assessment and iv) added two limitations on 25(OH)D2 and intra-assay precision to the discussion section.

See the authors' detailed response to the review by Katarzyna Dzik

Introduction

Globally, it is estimated that one billion people are vitamin D deficient (25(OH) D <20 ng/mL), while almost 50% of the global population is estimated to be vitamin D insufficient (25(OH) D 20-30 ng/mL).¹ Sun exposure and dietary sources of vitamin D (e.g. fatty fish, milk, cheese, vitamin supplements) contribute to vitamin D status.² Older adults may be at greater risk of vitamin D deficiency due to decreased production of vitamin D in the skin, reductions in sun exposure and physical activity, decreased appetite, and changes in dietary habits.³ In India, studies have indicated that vitamin D deficiency is common among the general adult population; however, few studies have included population-based samples.⁴

Vitamin D status in humans is best indicated by 25-hydroxyvitamin D (25(OH)D), which is the total of the 25-hydroxy forms of 25-hydroxyvitamin D₂ (25(OH)D₂) and 25-hydroxyvitamin D₃ (25(OH)D₃).⁵ Serum or plasma quantification of 25(OH) D are the standard biological specimens used in the assessment of vitamin D status.⁶ However, in large epidemiologic studies, collection and storage of plasma or serum are often not feasible due to the cost and logistic requirements of refrigeration after blood collection and cryogenic storage, particularly in resource-limited settings. As a result, dried blood spot (DBS) collection is becoming more common in epidemiologic studies to overcome these barriers. Prior studies have found that measurement of 25(OH) D from DBS with liquid chromatography-tandem mass spectrometry (LC-MS/MS) can yield valid and reliable results.⁷^–¹³ However, older age may alter vitamin D binding protein levels and affinity and influence vitamin D level estimations in the elderly population.¹⁴

We conducted a study to evaluate the agreement of 25(OH)D₃ assessment in DBS as compared to plasma concentrations among Indian adults over 45 years of age. The results of our study are intended to inform the ability to use DBS to assess vitamin D status in large population-based studies of vitamin D status among older adult populations in India.

Methods

Study design

The study enrolled 58 adult participants over 45 years of age who resided in Pune, India. Participants were contacted for enrollment at ICMR-NARI Facility for Collaborative Clinical Research. The study enrolled participants from July 2020 to June 2021.

Sample collection

Each participant had 4 mL of blood collected with a lavender top vacutainer (ethylenediaminetetraacetic acid (EDTA) anticoagulant). 50 μl of whole blood was spotted on predefined circles of Whatman 903 filter paper using a calibrated pipette. The DBS were air-dried and then stored at -20°C by placing them individually into a zip lock bag along with desiccants and a humidity indicator card. The blood sample was also centrifuged at 1500 g for 15 minutes to isolate plasma. The plasma samples were stored at -80°C until testing.

Chemicals and reagents

The study utilized D6-25-hydroxyvitamin D3 (IS) and 25-hydroxyvitamin D3 (25(OH)D3) from Sigma Aldrich (St. Louis, USA, catalog numbers H-074-1ML and 17938-1MG, respectively), HPLC grade methanol from J. T Bakers (Phillipsburg, NJ, USA, catalog number 8402.2500), formic acid from Sigma Aldrich (St. Louis, USA, catalog number F0507-100ML), acetonitrile from J. T Bakers (Phillipsburg, NJ, USA, catalog number 02-002-180), ammonium formate from Loba Chemie (Mumbai, India, catalog number 01210), and zinc sulfate pentahydrate from Thomas Baker (Mumbai, India, catalog number 169722). A set of 5 control samples with known target values of 25(OH)D₃ were obtained from the DEQAS Vitamin D External Quality Assessment Scheme (London, UK, no catalog number).

Standard preparation and calibration

A stock solution of the standard, 25-hydroxycholecalciferol (25(OH)D₃), was prepared in ethanol at a concentration of 1 mg/mL. The standard was diluted to prepare liquid calibrators of concentrations of 5, 10, 20, 40, 60, 80, & 125 ng/mL and controls with the concentrations of 25, 50, and 100 ng/mL using 0.1%formic acid in methanol: water (80:20 v/v) as a diluent. DBS calibrators were prepared in artificial blood consisting of washed RBCs and phosphate buffer pH 6.5 (in water) mixed in 1:1 proportion. 50 μl of the spiked blood was spotted on Whatman filter paper number 903 to prepare standards of concentrations of 5, 10, 20, 40, 60,80, and 125 ng/mL. Controls were prepared similarly with concentrations of 25, 50, and 100 ng/mL.

LC-MS/MS quantification of 25(OH)D₃ in plasma

The procedure for measuring 25(OH)D₃ in plasma samples was as follows: 100 μL internal standard (50 ng in 100% methanol) was added to 100 μL of plasma samples and vortexed for 5 minutes. This was followed by the addition of 50 μL zinc sulfate pentahydrate (0.2 M) and 300 μL acetonitrile: methanol (70:30) with vortexing steps in between. The tubes were centrifuged for 10 minutes at 14000 rpm at 10°C. 200 μL supernatant was transferred to HPLC vials for analysis.

Sample preparation/extraction procedure for DBS

Two 6mm DBS punches were placed in Eppendorf tubes, to which 100 μL internal standard (50 ng in 100% methanol) was added and vortexed for 5 minutes. Thereafter 50 μL of zinc sulfate pentahydrate (0.2 M) and 300 μL acetonitrile: methanol (70:30) were added sequentially with vortexing steps after each addition. The tubes were centrifuged for 10 minutes at 14000rpm at 10°C and 200 μL supernatant was transferred to an HPLC vial for further analysis.

LC-MS/MS conditions

The system consisted of 4000 QTrap (A B Sciex) along with the Shimadzu LC 20AD LC System (Shimadzu Corporation, Singapore, no catalog number) with electrospray ionization (ESI) controlled by Analyst 1.4.2 software (SCIEX, MA, USA, https://sciex.com/products/software/analyst-software , no catalog number). Copyright license was obtained for the Analyst 1.4.2 software. The following HPLC conditions were used:

1. Column: Agilent Poroshell 120 EC-C18, (2.1×50 mm, 2.7-Micron particle size)
2. Column temperature: 40°C
3. Injection volume: 10 μl
4. The mobile phase: A: Methanol and B: 10 mM Ammonium Formate in HPLC grade Water
5. The mobile phase composition: A: 90% and B: 10%. Isocratic flow.
6. Flow rate: 0.4 mL/min
7. Sample temperature: 5°C

Validation of quantitation of 25(OH)D3 by LC-MS/MS method

The LC-MS/MS assay method was validated as per the guidelines for the bioanalytical method development and validation as well as CLSI C62 Liquid Chromatography-Mass Spectrometry Methods; Approved Guidelines.¹⁵^,¹⁶ The LOQ (limit of quantitation) was set at 5 times the concentration of the LOD (Lower limit of Detection). The LOD was determined by the lowest concentration and its peak-to-peak signal to noise ratio, which was required to be greater than 5. Linearity was determined by analyzing the standard graph for 25(OH)D₃. Accuracy was determined by comparing the expected versus observed values for the in-house prepared control samples. The control samples were assayed on 5 consecutive days with 6 replicates per day for calculating inter-assay and intra-assay precision, respectively. Recovery was determined by spiking known amounts (5 and 100 ng/mL) of 25(OH)D₃ in DBS samples, and spiked concentration areas were compared with spiked blank extracted samples. Processed sample stability was assessed after storing the control samples on an autosampler tray at 5°C or benchtop at ambient temperature for 24 hours. Carryover was determined by analyzing the blank sample after the highest standard was measured.

Statistical analysis

Linearity for plasma quantification and medians and interquartile ranges (IQRs) were presented for 25(OH)D₃ concentrations for paired plasma and DBS assessments. A Pearson’s correlation was calculated and a Passing and Bablock analysis was conducted to compare plasma and DBS measurements. A Bland-Altman analysis was also conducted to estimate the mean bias and limits of agreement. Analyses were conducted with SAS version 9.3 and MedCalc version 20.027.

Results

Plasma 25(OH)D₃ quantitation by LC-MS/MS method was validated first before proceeding to DBS validation. Retention time for 25(OH)D₃ was observed to be 1.14 minutes. Plasma standards were linear between 5.0 to 125.0 ng/mL with r² = 0.9988. Accuracy for the controls ranged from 95.02 to 101.3% and intra-assay precision varied from 5.0-6.4%. Vitamin D EQAS samples processed by LC-MS/MS method showed bias ranging from -3.32 to 3.26%.

We then performed LC-MS/MS method validation on DBS samples that were spiked with known 25(OH)D₃ concentrations in artificial blood. The comparative results of plasma and DBS sample validation are presented in Table 1. DBS 25(OH)D₃ standards showed good linearity over the working range from 5.0 to 125.0 ng/mL (r² = 0.9967). Calibration curves for plasma and DBS standards are plotted in Figure 1. The peak-to-peak signal-to-noise ratio for 1 ng/mL standard was ~7. So LOD for the method was 1 ng/mL and the LOQ was 5 ng/mL. Accuracy for the analyte ranged from 100.50% to 113.75%. Intra-assay CVs varied from 5.13% to 13.39% and inter-assay CVs were 6.92% to 13.77% when measured using the in-house prepared QC samples. Recovery of 25(OH)D₃ varied from 78.11-82.85% while the recovery of the internal standard was up to 90%. Retention time for 25(OH)D₃ was observed to be 1.14 minutes. 25(OH)D₃ was found to be stable after the QC samples were processed and left either on the autosampler tray at 5°C or benchtop at ambient temperature for 24 hours. No carryover was observed in the LCMS/MS analysis at a concentration of 100 ng/mL.

Table 1. Results of LC MS/MS method validation for 25(OH)D₃ quantitation using plasma and DBS standards/controls spiked with known 25(OH)D₃ concentrations.

Parameters	Plasma	Dried Blood Spot (DBS)
Linearity (R²)	0.9988	0.9967
Linearity range	5 to 125 ng/mL	5 to 125 ng/mL
Limit of detection	1 ng/mL	1 ng/mL
Limit of quantification	5 ng/mL	5 ng/mL

Concentrations	5 ng/mL	25 ng/mL	100 ng/mL	5 ng/mL	25 ng/mL	100 ng/mL
Accuracy (%)	95.02	97.588	101.29	100.50	113.75	103.17
Inter-assay precision (CV%)	0.21	3.96	9.51	13.39	9.05	5.13
Intra-assay precision (CV%)	6.42	5.01	6.06	6.92	8.82	13.77
Recovery (%)	-	-	-	82.84	-	78.11
Processed sample stability (%) on Autosampler	-	-	-	93.12	-	99.33
Processed sample stability (%) on Bench Top	-	-	-	100.01	-	102.30

Figure 1. Standard calibration curves for 25(OH)D₃ concentrations (ng/mL) (x-axis) and Analyte area/IS area ratio (y-axis) in plasma (a) and (b) dried blood spot (DBS) samples.

We then proceeded to compare human plasma and DBS samples from study participants. A total of 58 adults participated in the study of which 30 (51.7%) were male and 28 (48.3%) were female. The median age of study participants was 52 years (Q1 = 47, Q3 = 55). The median plasma 25(OH)D₃ concentration of the study population was 14.6 ng/mL (Q1 = 12.0, Q3 = 18.1, range = 8.0-33.6) while the median DBS 25(OH)D₃ concentration was 12.8 ng/mL (Q1 = 11.0, Q3 = 16.6, range = 7.7-31.9). Plasma and DBS 25(OH)D₃ concentrations were highly correlated with a Pearson’s correlation of 0.976 (p < 0.0001).

Figure 2 presents a plot of the plasma and DBS 25(OH)D₃ concentrations of study participants. The Passing and Bablock equation for the relationship between DBS and plasma concentrations in the study population was DBS 25(OH)D₃ concentration (ng/mL) = -0.23 (95% CI: -0.98 to 0.66) + 0.94 × plasma 25(OH)D₃ concentration (ng/mL) (95% CI: 0.87 to 0.98). The corresponding equation to estimate plasma 25(OH)D₃ is plasma 25(OH)D₃ (ng/mL) = 1.0638 * [DBS 25(OH)D₃ concentration (ng/mL) + 0.23]. There was no indication of non-linearity (p-value = 0.20). Therefore, 25(OH)D₃ concentrations as assessed by DBS were on average 6% (95% CI: 2-13%) lower than concentrations assessed in plasma across the observed 25(OH)D₃ distribution. Figure 3 presents the Bland-Altman plot. The mean bias ± standard error between plasma and DBS was 1.31 ± 0.16 ng/mL, and the limits of agreement were -1.14 to + 3.77 ng/mL.

Figure 2. 25(OH)D₃ concentrations (ng/mL) in plasma (x-axis) and in dried blood spots from human samples (DBS; y-axis).

Blue line indicates the Passing and Bablok regression equation with red dashed lines and shading indicating 95% confidence intervals. The identity line is presented as a green dashed line.

Figure 3. Bland-Altman plot for 25(OH)D₃ assessment in plasma and in dried blood spots.

Difference in concentrations between plasma and DBS (y-axis) plotted against mean of plasma and DBS measurements (x-axis).

Discussion

Our study indicated satisfactory performance characteristics for the assessment of 25(OH)D₃ in plasma and DBS samples using standards and spiked controls. Subsequently, among samples from Indian adults older than 45 years of age, we found a good agreement of measurement of 25(OH)D₃ from DBS and plasma samples. We found that 25(OH)D₃ concentrations in DBS samples were on average 6% (95% CI: 2-13%) lower than paired plasma samples. The difference was consistent across the observed range of 25(OH)D₃ in plasma from 8-32 ng/mL.

Our finding that 25(OH)D₃ quantification in dried blood spots is a valid alternative to standard plasma assessment is in line with prior validation studies in other settings and populations.⁷^–¹² Further, most prior studies have also found that 25(OH)D₃ assessed in DBS is systematically lower than serum or plasma concentrations.⁷^,⁹^,¹¹^,¹² A study conducted among adults 22-48 years in India found that mean 25(OH)D₃ concentrations were similar between DBS and serum, and there was no difference in the classification of vitamin D sufficiency and insufficiency.⁹

The study had several limitations. First, we did not quantify 25(OH)D₂ concentrations in the study population. Nevertheless, it is estimated that 25(OH)D₃ accounts for the majority of the total 25(OH) D concentrations among adults in India and there is no current large-scale fortification with ergocalciferol (vitamin D₂) in the country.⁹^,¹² Therefore, the assessment of 25(OH)D₃ may capture most of the variation in 25(OH) D concentrations in our study population. However, future studies should also evaluate dried blood spot assessment of 25(OH)D₂ in India to more accurately assess total 25(OH) D. We also saw some evidence of a moderately high intra-assay precision (CV%) for DBS at the 100 ng/mL level, and therefore, future studies may also examine higher ranges of 25(OH)D₃. Second, we did not adjust for the hematocrit fraction. However, a prior study suggests that adjustment for hematocrit fraction in the normal range had minimal effect on DBS 25(OH)D₃ quantification.¹³ Measurement of hematocrit in large population-based studies may not be efficient in terms of resources for the potentially small increase in precision.

Overall, we found good agreement between 25(OH)D₃ quantification by LC-MS/MS in DBS as compared to standard plasma assessment. DBS assessment of 25(OH)D₃ may help expand population-based vitamin D research in adult populations in India and other settings.

Ethics and consent

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the National AIDS Research Institute Ethics Committee (Ref No. NARI-EC/2017-03, Approval date: 18 August 2020), Indian Council of Medical Research (Ref No. 54/17/Indo-foreign/Ger/17-NCD-II, Approval date: 9 October 2017), and the Harvard T. H. Chan School of Public Health Institutional Review Board (Ref. No. IRB17-0778, 8 June 2017). All participants provided written informed consent.

Data availability statement

Figshare: Vitamin D Validation – India, Sudfeld, Christopher (2024). Vitamin D Validation - India. figshare. Dataset. https://doi.org/10.6084/m9.figshare.25665483.v3.¹⁷

This project contains the following underlying data:

• vitd4222024.csv

Data are available under the terms of Creative Commons Zero (CC0) license.

Software availability

The LC-MS/MS data was analyzed with SCIEX Analyst 1.4.2 software. A free version of software able to complete this analysis is available from Open MS (https://openms.de/).

Acknowledgments

We would like to thank the participants who made this study possible.

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Comments on this article Comments (0)

Version 2

VERSION 2 PUBLISHED 20 May 2024

Author details Author details

Jyoti Sawant
Roles: Investigation, Methodology, Validation, Writing – Review & Editing

Rajani Bagul
Roles: Investigation, Methodology, Validation, Writing – Review & Editing

Ujjwala Ghule
Roles: Investigation, Methodology, Validation, Writing – Review & Editing

Sarang S. Saluke
Roles: Formal Analysis, Investigation, Methodology, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Christopher R. Sudfeld
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The research was funded by National Institute on Aging Grant Number (P30AG024409)
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article Versions (2)

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Published: 06 Jun 2025, 13:512

https://doi.org/10.12688/f1000research.149829.2

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Published: 20 May 2024, 13:512

https://doi.org/10.12688/f1000research.149829.1

© 2025 Shete AV et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Shete AV, Sawant J, Bagul R et al. Quantification of 25-hydroxyvitamin D₃ in dried blood spots as compared to plasma among Indian adults [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Research 2025, 13:512 (https://doi.org/10.12688/f1000research.149829.2)

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Reviewer Report 30 Jun 2025

Katarzyna Dzik, University of Gdansk, Gdańsk, Poland

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https://doi.org/10.5256/f1000research.182852.r390095

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Reviewer Report 04 Sep 2024

Katarzyna Dzik, University of Gdansk, Gdańsk, Poland

Approved with Reservations

https://doi.org/10.5256/f1000research.164335.r312373

The manuscript entitled "Quantification of 25-hydroxyvitamin D₃ in dried...." is well written and shows a simple yet important set of data presented in acceptable form.
I have one major concern.
In table 1. results show that the accuracy of DBS and plasma is different for 5 and 25 ng/mL concentrations. Also, there is very high CV% for DBS at 5 ng/mL concentration- 13.39. This results might impact the concentration corresponding to vitamin D deficiencies, therefore might be at the great importance. Please address this data in the discussion.

Minor comments.
1. Please unify - over — in the description of LC-MS/MS conditions. It might be misleading and seems that the column temperature is minus 40℃.

2. In the introduction the sentence „Dietary sources of vitamin D (…) are not regularly consumed in most parts of the world and therefore exposure to sunlight is the primary source of vitamin D for most individuals globally.” seems to be very misleading. The Authors cite a paper by Ann Pretice, however in the original paper by Ann Pretice we can find a sentence „The relative contributions of sunshine exposure and dietary intake to vitamin D supply and the achievement of adequacy in an individual, therefore, depend on many factors.” It would be better to avoid this sentence, or write more information to avoid misleading.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: neurodegeneration, vitamin D, exercise,

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 16 Jun 2025

Christopher Sudfeld, Global Health and Population, Harvard T.H. Chan School of Public Health, Boston, USA

16 Jun 2025

Author Response

Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a ... Continue reading Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a potentially higher CV% at 100 ng/mL in the DBS - as a result, we have added this limitation for future studies to look at even higher concentrations. We have noted "We did see some evidence of a moderately high intra-assay precision (CV%) for DBS at the 100 ng/mL level and therefore future studies may also examine higher ranges of 25(OH)D₃."

Minor Comment #1 - We have used colon : rather than - to avoid confusion on the temperatures in particular.

Minor Comment #2 - We have clarified this sentence to be more general about vitamin D sources. "Sun exposure and dietary sources (e.g., fatty fish, milk, cheese, vitamin supplements) contribute to vitamin D status (2).
Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a potentially higher CV% at 100 ng/mL in the DBS - as a result, we have added this limitation for future studies to look at even higher concentrations. We have noted "We did see some evidence of a moderately high intra-assay precision (CV%) for DBS at the 100 ng/mL level and therefore future studies may also examine higher ranges of 25(OH)D₃."

Minor Comment #1 - We have used colon : rather than - to avoid confusion on the temperatures in particular.

Minor Comment #2 - We have clarified this sentence to be more general about vitamin D sources. "Sun exposure and dietary sources (e.g., fatty fish, milk, cheese, vitamin supplements) contribute to vitamin D status (2).
Competing Interests: No competing interests. Close
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Author Response 16 Jun 2025

Christopher Sudfeld, Global Health and Population, Harvard T.H. Chan School of Public Health, Boston, USA

16 Jun 2025

Author Response

Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a ... Continue reading Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a potentially higher CV% at 100 ng/mL in the DBS - as a result, we have added this limitation for future studies to look at even higher concentrations. We have noted "We did see some evidence of a moderately high intra-assay precision (CV%) for DBS at the 100 ng/mL level and therefore future studies may also examine higher ranges of 25(OH)D₃."

Minor Comment #1 - We have used colon : rather than - to avoid confusion on the temperatures in particular.

Minor Comment #2 - We have clarified this sentence to be more general about vitamin D sources. "Sun exposure and dietary sources (e.g., fatty fish, milk, cheese, vitamin supplements) contribute to vitamin D status (2).
Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a potentially higher CV% at 100 ng/mL in the DBS - as a result, we have added this limitation for future studies to look at even higher concentrations. We have noted "We did see some evidence of a moderately high intra-assay precision (CV%) for DBS at the 100 ng/mL level and therefore future studies may also examine higher ranges of 25(OH)D₃."

Minor Comment #1 - We have used colon : rather than - to avoid confusion on the temperatures in particular.

Minor Comment #2 - We have clarified this sentence to be more general about vitamin D sources. "Sun exposure and dietary sources (e.g., fatty fish, milk, cheese, vitamin supplements) contribute to vitamin D status (2).
Competing Interests: No competing interests. Close
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Reviewer Report 13 Aug 2024

Christopher T Sempos, National Institutes of Health (NIH), Bethesda, MD, USA; Vitamin D Standardization Program, Havre de Grace, MD, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.164335.r300657

Summary: Object is to develop a dried blood spot (DBS) assay for the measurement of plasma 25(OH)D3 concentration that is accurate and precise compared to the Vitamin D Standardization Program (VDSP) standardized LC-MS/MS assay. That is, results from the LC-MS/MS assay can be interpreted as the "True" 25(OH)D concentration.

Comments:
(1) P3, Chemicals and Reagents: The authors indicate that the that the LC-MS/MS produced results that were within -5% to +5% of the true 25(OH)D concentration of the DEQAS samples, As a result, the LC-MS/MS assay appears to be standardized according to criteria adopted the Vitamin D Standardization Program (VDSP) -- that is 25(OH)D3 measurements by the LC-MS/MS assay are the true concentration. If the authors reversed X and Y in Figure 2 the resulting regression equation could be used to standardize the DBS values to the true 25(OH)D concentration. I suggest that the make that change and then in the Discussion indicate the importance measuring the True 25(OH)D concentration for public health policy.

(2) What do the authors mean when they write that "...25(OH)D3 accounts for the majority of the total 25(OH)D among adults in India..."? Even small quantifiable amounts of 25(OH)D2 can make an enormous difference when estimating the prevalence of low/deficient total 25(OH)D levels.

From the National Indian Survey or other sources what are the 25(OH)D2 concentrations by age and sex. if this assay is to be used in the study of vitamin D in adults it needs to be established that 25(OH)D2 levels are close to nil in Indian adults.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. Shete A, Sawant J, Bagul R, Ghule U, et al.: Quantification of 25-hydroxyvitamin D3 in dried blood spots as compared to plasma among Indian adults. F1000Research. 2024; 13. Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Coordinator, Vitamin D Standardization Program

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VERSION 2 PUBLISHED 20 May 2024

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Christopher T Sempos, National Institutes of Health (NIH), Bethesda, USA; Vitamin D Standardization Program, Havre de Grace, USA
Katarzyna Dzik, University of Gdansk, Gdańsk, Poland

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30 Jun 2025 | for Version 2

Katarzyna Dzik, University of Gdansk, Gdańsk, Poland

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Approved

The manuscript is now acceptable for indexing.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

neurodegeneration, vitamin D, exercise,

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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04 Sep 2024 | for Version 1

Katarzyna Dzik, University of Gdansk, Gdańsk, Poland

8 Views Cite this report Responses(1)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

neurodegeneration, vitamin D, exercise,

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Author Response

16 Jun 2025

Christopher Sudfeld, Global Health and Population, Harvard T.H. Chan School of Public Health, Boston, USA

Thank you for the thoughtful review of the manuscript. We have addressed each of your points below

Major comment #1 - We agree there was some indication of a potentially higher CV% at 100 ng/mL in the DBS - as a result, we have added this limitation for future studies to look at even higher concentrations. We have noted "We did see some evidence of a moderately high intra-assay precision (CV%) for DBS at the 100 ng/mL level and therefore future studies may also examine higher ranges of 25(OH)D₃."

Minor Comment #1 - We have used colon : rather than - to avoid confusion on the temperatures in particular.

Minor Comment #2 - We have clarified this sentence to be more general about vitamin D sources. "Sun exposure and dietary sources (e.g., fatty fish, milk, cheese, vitamin supplements) contribute to vitamin D status (2).

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Competing Interests

No competing interests.

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Reviewer Report

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13 Aug 2024 | for Version 1

Christopher T Sempos, National Institutes of Health (NIH), Bethesda, MD, USA; Vitamin D Standardization Program, Havre de Grace, MD, USA

9 Views Cite this report Responses(0)

Approved With Reservations

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

References

1. Shete A, Sawant J, Bagul R, Ghule U, et al.: Quantification of 25-hydroxyvitamin D3 in dried blood spots as compared to plasma among Indian adults. F1000Research. 2024; 13. Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Coordinator, Vitamin D Standardization Program

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Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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[9] 9. Lote-Oke R, Pawar J, Kulkarni S, et al.: A LC-MS method for 25-hydroxy-vitamin D3 measurements from dried blood spots for an epidemiological survey in India. Sci. Rep. 2020; 10(1): 19873. PubMed Abstract | Publisher Full Text | Free Full Text

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[11] 11. Man PW, Heijboer AC, van der Meer IM , et al.: Agreement between measurement of 25-hydroxyvitamin D3 in dried blood spot samples and serum in a Chinese population in the Netherlands. J. Steroid Biochem. Mol. Biol. 2019; 195: 105472. PubMed Abstract | Publisher Full Text

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[13] 13. Newman MS, Brandon TR, Groves MN, et al.: A liquid chromatography/tandem mass spectrometry method for determination of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in dried blood spots: a potential adjunct to diabetes and cardiometabolic risk screening. J. Diabetes Sci. Technol. 2009; 3(1): 156–162. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Srikanth P, Chun RF, Hewison M, et al.: Associations of total and free 25OHD and 1,25(OH)2D with serum markers of inflammation in older men. Osteoporos. Int. 2016; 27(7): 2291–2300. PubMed Abstract | Publisher Full Text | Free Full Text

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Quantification of 25-hydroxyvitamin D3 in dried blood spots as compared to plasma among Indian adults

Abstract

Background

Methods

Results

Conclusions

Keywords

Revised Amendments from Version 1

Introduction

Methods

Study design

Sample collection

Chemicals and reagents

Standard preparation and calibration

LC-MS/MS quantification of 25(OH)D3 in plasma

Sample preparation/extraction procedure for DBS

LC-MS/MS conditions

Validation of quantitation of 25(OH)D3 by LC-MS/MS method

Statistical analysis

Results

Table 1. Results of LC MS/MS method validation for 25(OH)D3 quantitation using plasma and DBS standards/controls spiked with known 25(OH)D3 concentrations.

Figure 1. Standard calibration curves for 25(OH)D3 concentrations (ng/mL) (x-axis) and Analyte area/IS area ratio (y-axis) in plasma (a) and (b) dried blood spot (DBS) samples.

Figure 2. 25(OH)D3 concentrations (ng/mL) in plasma (x-axis) and in dried blood spots from human samples (DBS; y-axis).

Figure 3. Bland-Altman plot for 25(OH)D3 assessment in plasma and in dried blood spots.

Discussion

Ethics and consent

Data availability statement

Software availability

Acknowledgments

References

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Quantification of 25-hydroxyvitamin D₃ in dried blood spots as compared to plasma among Indian adults

LC-MS/MS quantification of 25(OH)D₃ in plasma

Table 1. Results of LC MS/MS method validation for 25(OH)D₃ quantitation using plasma and DBS standards/controls spiked with known 25(OH)D₃ concentrations.

Figure 1. Standard calibration curves for 25(OH)D₃ concentrations (ng/mL) (x-axis) and Analyte area/IS area ratio (y-axis) in plasma (a) and (b) dried blood spot (DBS) samples.

Figure 2. 25(OH)D₃ concentrations (ng/mL) in plasma (x-axis) and in dried blood spots from human samples (DBS; y-axis).

Figure 3. Bland-Altman plot for 25(OH)D₃ assessment in plasma and in dried blood spots.