24,25-dihydroxyvitamin D3 in serum: sample purification with Sep-Pak C-18 cartridges and liquid chromatography before protein-binding assay.

1983 ◽  
Vol 29 (10) ◽  
pp. 1806-1807 ◽  
Author(s):  
M L Traba ◽  
M Babé ◽  
C de la Piedra ◽  
A Marín
Keyword(s):  
Liquid Chromatography ◽  
Protein Binding ◽  
Serum Sample ◽  
High Performance ◽  
Binding Assay ◽  
Reversed Phase ◽  
Specific Method ◽  
Dihydroxyvitamin D3 ◽  
Protein Binding Assay ◽  
24,25 Dihydroxyvitamin D3

Abstract We describe a precise, specific method for measuring 24,25-dihydroxyvitamin D3 in human serum. A 2-mL serum sample is extracted with acetonitrile and passed through a Sep-Pak C-18 cartridge. The sample is further purified by "high-performance" liquid chromatography under isocratic conditions on a normal-phase column (Radial-Pak silica-gel cartridge), then subjected to a protein-binding assay. The mean concentration of 24,25-dihydroxyvitamin D3 in serum from 22 normal adults (measured during the spring) was 2.9 micrograms/L (SD 1.9, range 6.3-0.42 microgram/L). The intra-assay CV was 7.7%, the interassay CV 11.2%. Purification of the sample with Sep-Pak C-18 and liquid chromatography on normal plus reversed-phase columns leads to a mean value of 3.4 micrograms/L (SD 1.6 micrograms/L, n = 12), not significantly different from results with our method.

Issues of methodology, standardization and metabolite recognition for 25-hydroxyvitamin D when comparing the DiaSorin radioimmunoassay and the Nichols Advantage automated chemiluminescence protein-binding assay in hip fracture cases

2003 ◽  
Vol 40 (5) ◽  
pp. 546-551 ◽  
Author(s):  
Paul Glendenning ◽  
Jane M Noble ◽  
Mario Taranto ◽  
Alexander A Musk ◽  
Marjory McGuiness ◽  
...  
Keyword(s):  
Vitamin D ◽  
Hip Fracture ◽  
Protein Binding ◽  
High Performance ◽  
Binding Assay ◽  
Negative Bias ◽  
Radioactive Label ◽  
Hydroxyvitamin D ◽  
Protein Binding Assay ◽  
25 Hydroxyvitamin D

Background: Deficiency of vitamin D is commonly associated with hip fracture and treatment with vitamin D reduces hip fracture rates. Consequently, the demand for assays to measure 25-hydroxyvitamin D (25-OHD) has increased. The Nichols Advantage chemiluminescence protein-binding assay (CLPBA) for 25-OHD is a first-generation automated immunoassay with decreased turnaround time, reduced manual handling and non-radioactive label. Methods: We compared the CLPBA to the DiaSorin radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) for the measurement of 25-OHD using 161 samples from hip fracture patients and samples before and after institution of ergocalciferol (vitamin D2) therapy. Results: A negative bias for the CLPBA at concentrations below 30 nmol/L and a positive bias at 25-OHD values above 30 nmol/L compared with the RIA resulted in diagnostic discordance for one in three samples when using 30 and 50 nmol/L as decision limits. HPLC analysis confirmed the presence of a negative bias for the CLPBA at low values. Both immunoassays under-estimate 25-hydroxyvitamin D2. Conclusions: The discordance between 25-OHD values may be due to differences in standardization of each assay relative to HPLC. Our results emphasize the need for assay-specific clinical decision limits.

Simultaneous determination of uric acid and creatinine in plasma by reversed-phase liquid chromatography.

1985 ◽  
Vol 31 (1) ◽  
pp. 109-112 ◽  
Author(s):  
A Zhiri ◽  
O Houot ◽  
M Wellman-Bednawska ◽  
G Siest
Keyword(s):  
Uric Acid ◽  
Liquid Chromatography ◽  
High Performance ◽  
Ammonium Acetate ◽  
Limit Of Detection ◽  
Reversed Phase ◽  
Specific Method ◽  
Reversed Phase Liquid Chromatography ◽  
Phase Liquid

Abstract Because numerous substances, endogenous compounds as well as xenobiotics, interfere with determination of uric acid and creatinine, we have devised a more nearly specific method by which we can simultaneously determine uric acid and creatinine in plasma by "high-performance" liquid chromatography. We used a mobile phase of ammonium acetate (30 mmol/L) and methanol (156 mmol/L) at pH 7.0 and a flow rate of 1 mL/min. We used a C18 reversed-phase column, and measured absorbance at 235 nm, the wavelength corresponding to the absorption maximum of uric acid and creatinine. Uric acid and creatinine could be determined in less than 5 min by directly injecting plasma diluted fivefold; use of a precolumn eliminated the need for deproteinization. We evaluated the precision, recovery, linearity, specificity, and limit of detection of the method and we checked for analytical interference by 37 currently used drugs. We compared results with those obtained by routine and reference methods.

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