An HPLC Ultraviolet Method Using Low Sample Volume and Protein Precipitation for the Measurement of Retinol in Human Serum Suitable for Laboratories in Low- and Middle-Income Countries. (1st July 2019)

• Record Type: Journal Article
• Authors: Chaudhary-Webb, Madhulika; Schleicher, Rosemary L; Erhardt, Juergen G; Pendergrast, Elizabeth C; Pfeiffer, Christine M
• Is Part Of: Journal of applied laboratory medicine. Volume 4:Number 1(2019)
• Page Start: 101
• Page End: 107
• Publication Date: 2019-07-01
• Subjects: Clinical chemistry -- Periodicals; Diagnosis, Laboratory -- Periodicals; 616.0756
• DOI: 10.1373/jalm.2018.027508
• Languages: English
• ISSNs: 2576-9456

Abstract

Abstract: Background: Assessing vitamin A status in populations remains a high public health priority for low- and middle-income countries. However, analytical difficulties with serum retinol measurements persist in international laboratories. Nearly all participants in a Centers for Disease Control and Prevention external quality assessment program use HPLC to measure serum retinol, but round-to-round results failing to meet acceptable criteria suggest the need to provide a straightforward stable HPLC ultraviolet (UV) method that can be adopted by these laboratories to improve performance. We present a protein precipitation HPLC-UV method that measures serum retinol below the deficiency cutoff value (<0.7 μmol/L or 20 μg/dL) that is suitable for low- and middle-income countries and uses commercially available materials. Methods: Serum (25 μL) added to retinyl acetate was precipitated with acetonitrile (125 μL) to extract retinol. Solvent-based calibration solutions required no extraction. Calibration used either single-point (50 μg/dL) or multipoint solutions (0.52–100 μg/dL). C18 column (4.6 × 100 mm) and acetonitrile with 0.1% triethylamine/water (83/17, v/v) as isocratic mobile phase (1.1 mL/min), achieved baseline separation (7 minutes). Results: With only 25 μL of serum, the limit of detection was 0.52 μg/dL. Single- and multipoint calibration generated equivalent results. Over several years, between-run imprecision was ≤7.1% in multiple quality-control materials. Overall mean (CV) method bias for NIST-certified reference materials (e-series) was −0.2% (5.8%). Maximally, 180 samples were processed within 24 h. Conclusions: This method was robust and stable over years and accurately measured serum retinol with low-volume samples. Thus, it may be of interest to low- and middle-income countries and to pediatric and finger stick applications.