Extraction of vitamin D

The serum extraction method was a modification of a previously described method⁹. To the 500 µL serum sample a mixture of 200 µL of Acetonitrile: Methanol (70:30) was added. The tube was briefly vortexed and then sonicated for 30 min (Labman Scientic Instruments, Chennai, India). After sonication, 1 mL hexane was added followed by brief vortexing and centrifugation at 10,000 rpm for 10 min. 900 µL of hexane was carefully transferred to a fresh tube without disturbing the phase separation. The hexane was evaporated to dryness using nitrogen concentrator (KeMi scientific, Pune India). The residue was reconstituted in 200 µL of methanol followed by vortexing to ensure uniform mixing.

DBS extraction method was a modification of a previously described method¹⁰. A circular disc of 3 mm diameter (corresponding to 3.2 µL of whole blood) was punched from a spot on the DBS card using a semi-automated puncher (Horizon Speciality, USA). For the assay, 8 discs from each participant were used (corresponding to 25.6 µL of whole blood). The punch was cleaned with 70% ethanol prior to and after taking sample from each study participant. The discs were placed into 2 mL Eppendorf tubes along with 250 µL of water. Each tube was vortexed for 30 s and then sonicated for 1 h using a sonicator (Labman Scientific Instruments, Chennai, India). 250 µL of methanol was added to each tube and the tubes were sonicated for another 30 min. Subsequently, 500 µL of hexane was added to each tube followed by centrifugation at 10,000 rpm for 10 min. Approximately 400 µL of hexane was carefully aspirated in a fresh tube without disturbing the phase separation and evaporated to dryness using nitrogen concentrator (Kemi Scientific, Pune India). The residue was reconstituted in 25 µL of methanol followed by vortexing to ensure uniform mixing.

The LC–MS system used was Shimadzu 8045 triple quadrupole mass spectrometer (MS) with the electrospray ionization (ESI) source in positive ion polarity mode fitted with Nexera X2 LC-30A UHPLC system. Data were collected in the multiple reaction monitoring (MRM) mode monitored for various vitamin D components in the extract using the following MS parameters: oven temperature 30 ∘C, interface temperature 298 ∘C, desolvation line temperature 196 ∘C, nebulizing gas flow 3.0 L/min, heating gas flow 10.0L/min, drying gas flow 18.0 L/min, IG vacuum 1.7e − 003 Pa, PG vacuum 6.9e + 001 Pa, CID Gas 230 KPa. Each vitamin D3 species and internal standard was identified and quantified using three MRM transitions reported previously, that for the various species were: (i) 25(OH)D3 = 401.2 → 383.3, 401.2 → 365.4, 401.2 → 159.1,; (ii) vitamin D3 = 385.2 → 259.2, 385.2 → 241.0, 385.2 → 107.3; and (iii) the internal standard [²H₆]-25(OH)D3 = 407.5 → 389.5, 407.5 → 371.0, 407.5 → 107.0⁹. Isolation and separation of analytes by chromatography was achieved by using a Shimadzu –Premier C18 150 mm × 2.1 × 3 µm column without derivatization. A typical LC–MS consisted of 10 min, with mobile phase A = water + 0.1% formic acid, and B = methanol + 0.1% formic acid, with a flow rate of 400 μL/min, and had the following gradient sequence: (i) equilibration with solvent B at 90% from 0.0 to 1.4 min, (ii) linear increase in solvent B from 90 to 100% from 1.5 to 4 min, and (iii) equilibration and washing with solvent B at 90% from 4.1 to 10 min. MS acquisition was stopped after 5 min of the LC gradient, and the remaining eluent was directed to waste collection system.