Ultimate 3000 variable wavelength detector

Reversed-phase (RP) high-performance liquid chromatography (HPLC) analysis was carried out using an UltiMate 3000 RS UHPLC pump, UltiMate 3000 autosampler, UltiMate 3000 Variable Wavelength Detector (UV detection at λ = 220 nm (Dionex, Germering, Germany)), and radio-detector (Gabi Star, Raytest; Straubenhardt, Germany). We used Jupiter 5 μm C18 300 Å 150 × 4.6 mm (Phenomenex Ltd. Aschaffenburg, Germany) as the column, with acetonitrile (ACN)/H₂O/0.1% trifluoroacetic acid (TFA) as the mobile phase. Gradient A: 0.0–3.0 min 10% ACN, 3.0–16.0 min 10–60% ACN, 16.0–18.0 min 60% ACN, 18.0–18.1 min 60–10% ACN, 18.1–22.0 min 10% ACN; flow rate of 1 mL/min.

Five-milliliter blood samples were collected by brachial venipuncture into amber tubes with gel, centrifuged, and stored in dry ice at a temperature of −20 °C for transportation to IMIP’s translational research laboratory. Serum retinol measurement (a dependent variable) was analyzed using high-performance liquid chromatography (HPLC), as recommended by the WHO [9 ,19 ], according to the technique established by Furr et al. [20 ]. The analysis was performed using a Dionex UltiMate 3000 HPLC system (Sunnyvale, CA, USA) with an Acclaim C18 column (5 µm × 4.6 mm × 250 mm) coupled with a C18 Nucleosil guard column, and an injection volume of 20 µL. Detection was performed using a UV detector (Dionex UltiMate 3000 Variable Wavelength Detector) at 325 nm. Retinyl acetate obtained from Sigma was used as a standard, peak retention was 3.6 min, and the mobile phase flow was 1.5 mL/min. The cut-off points used for the classification of retinol levels were: deficient: <0.35 μmo/L; low: ≥0.35 and <0.70 μmol/L; acceptable: ≥0.70 and <1.05 μmol/L; and adequate ≥1.05 μmol/L [14 ]. VAD was defined as retinol levels <0.70 μmol/L, as recommended by the WHO [9 ,19 ]; therefore, the cumulative prevalence of vitamin A levels classified as deficient or low (serum retinol levels <0.70 μmol/L) was adopted as the endpoint for the univariate and multivariate analyses.

Reversed-phase high-performance liquid chromatography analysis was performed with the following instrumentation: UltiMate 3000 RS UHPLC pump, UltiMate 3000 autosampler, UltiMate 3000 column compartment (25 °C oven temperature), UltiMate 3000 Variable Wavelength Detector (Dionex, Germering, Germany; UV detection at λ = 220 nm) a radio detector (GabiStar, Raytest; Straubenhardt, Germany), Jupiter 5 μm C₁₈ 300 Å 150 × 4.6 mm (Phenomenex Ltd. Aschaffenburg, Germany) column with acetonitrile (ACN)/H₂O/0.1% trifluoroacetic acid (TFA) as mobile phase; flow rate of 1 mL/min; gradient: 0.0–1.0 min 10% ACN, 1.0–12.0 min 10–60% ACN, 13.0–15.0 min 60–80% ACN, 15.0–16.0 min 80–10% ACN, 16.0–20.0 min 10% ACN.