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An HPLC-UV Method to Assess Human Plasma 25(OH)D 3 .

Authors :
Tijerina A
Garza A
López A
Cavazos N
Romo A
Heya MS
Bouzas C
Tur JA
Salas R
Source :
Nutrients [Nutrients] 2024 Jul 18; Vol. 16 (14). Date of Electronic Publication: 2024 Jul 18.
Publication Year :
2024

Abstract

The aim of this study was to validate an HPLC-UV method to assess vitamin D status by determining the linearity and precision of the 25-hydroxyvitamin D <subscript>3</subscript> (25(OH)D <subscript>3</subscript> ) calibration curve, the limits of detection, quantitation and robustness of the method, and its accuracy. A second stock solution of 25(OH)D <subscript>3</subscript> was prepared (500 ng/mL), and working dilutions (5, 10, 20, 30, 40, and 50 ng/mL) were prepared for a calibration curve. The HPLC equipment had a UV-Vis diode-array detector and utilized an Acclaim <superscript>TM</superscript> 120 C18 column (5 µm, 4.6 × 250 mm) with a flow rate of 1.2 mL/min, a column temperature of 30 °C, and the standards and samples were maintained at 4 °C, with an injection volume of 100 µL. Detection of 25(OH)D <subscript>3</subscript> was determined at 265 nm, with a retention time of 4.0 min. The validation was conducted according to the FDA Validation of Analytical Procedures: Guidance for Industry. Vitamin D was extracted from plasma samples using acetonitrile (ACN)-0.1% formic acid (2:1 v / v ), and the percentage of recovery was calculated. The proposed method conditions gave excellent linearity (R <superscript>2</superscript> = 0.9989) and the linearity coefficient was R <superscript>2</superscript> > 0.99 for 25(OH)D <subscript>3</subscript> . The detection and quantification limits were 1.1703 ng/mL and 3.5462 ng/mL, respectively. Decreasing or increasing the reading temperature by 1 °C decreased the response units (AU) of vitamin D, 25(OH)D <subscript>3</subscript> . When the current flow rate decreased by 0.2 mL/min (1.0 mL/min), the retention time increased to 4.913 min, whereas an increase of 0.2 mL/min of the proposed flow rate (1.4 mL/min) decreased the retention time to 3.500 min. The percentage of recovery varied from 92.2% to 97.1%. The proposed method to quantify a vitamin D metabolite (25(OH)D <subscript>3</subscript> ) in human plasma samples was reliable and validated.

Details

Language :
English
ISSN :
2072-6643
Volume :
16
Issue :
14
Database :
MEDLINE
Journal :
Nutrients
Publication Type :
Academic Journal
Accession number :
39064747
Full Text :
https://doi.org/10.3390/nu16142304