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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
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March 11 2019
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Source: J Appl Lab Med. 4(1):101-107
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Details:
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Alternative Title:J Appl Lab Med
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Personal Author:
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Description: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.
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Pubmed ID:31639712
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Pubmed Central ID:PMC6945745
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Volume:4
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Issue:1
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