Competitive Protein Binding Assay of Naproxen by Human Serum Albumin Functionalized Silicon Dioxide Nanoparticles

We have developed a new competitive protein binding assay (CPBA) based on human serum albumin functionalized silicon dioxide nanoparticles (nano-SiO2-HSA) that can be used for naproxen determination in urine. Compared with a conventional multi-well reaction plate, nano-SiO2 with a high surface-area-to-volume ratio could be introduced as a stationary phase, markedly improving the analytical performance. Nano-SiO2-HSA and horseradish peroxidase-labeled-naproxen (HRP-naproxen) were prepared for the present CPBA method. In this study, a direct competitive binding to nano-SiO2-HSAwas performed between the free naproxen in the sample and HRP-naproxen. Thus, the catalytic color reactions were investigated on an HRP/3,3′5,5′-tetramethylbenzidine (TMB)/H2O2 system by the HRP-naproxen/nano-SiO2-HSA composite for quantitative measurement via an ultraviolet spectrophotometer. A series of validation experiments indicated that our proposed methods can be applied satisfactorily to the determination of naproxen in urine samples. As a proof of principle, the newly developed nano-CPBA method for the quantification of naproxen in urine can be expected to have the advantages of low costs, fast speed, high accuracy, and relatively simple instrument requirements. Our method could be capable of expanding the analytical applications of nanomaterials and of determining other small-molecule compounds from various biological samples.

Determination of Serum 25-hydroxy Cholecalciferol Using High-Performance Liquid Chromatography: A Reliable Tool for Assessment of Vitamin D Status

This study was undertaken to design and set up a rather simple, reliable, and less expensive high-performance liquid chromatography (HPLC)-based method to assay 25(OH)D as a diagnostic tool for vitamin D assessment. Serum proteins were precipitated using ethanol and, after 10 minutes incubation at room temperature, methanol:isopropanol. The extraction was performed using hexane followed by evaporation under nitrogen flow. The sediment was then reconstituted in methanol and passed through a polypropylene filter. To run the chromatographic analysis, 20 μL of the filtrate was injected to the column. Peaks of 25(OH)D2 and 25(OH)D3 were both detected using a UV detector set at 265 nm. With a flow rate of 1.2 mL/minute, peaks of D3 and D2 vitamers were detected around 9.5 and 10.7 minutes, respectively. The intra- and inter-assay variations were 8.1% and 12.6%, respectively, and the recovery percent was found to be 100 ± 5%. To compare the procedure with conventional methods, 90 serum samples from subjects (48 females and 42 males) aged 40.5 ± 13.9 yrs, were analyzed for 25(OH)D using HPLC, competitive protein-binding assay (CPBA), and radioimmunoassay (RIA). Generally, CPBA and RIA assays both showed over-estimation of serum 25(OH)D, compared to HPLC. Though all three methods correlated significantly with each other, with the strongest between HPLC and RIA (r = 0.87, p < 0.001), both RIA and CPBA were found unreliable in detection of some deficient samples.

HPLC Method for 25-Hydroxyvitamin D Measurement: Comparison with Contemporary Assays

Background: The concentration of 25-hydroxyvitamin D [25(OH)D] in serum has been designated the functional indicator of vitamin D (VitD) nutritional status. Unfortunately, variability among 25(OH)D assays limits clinician ability to monitor VitD status, supplementation, and toxicity. Methods: We developed an HPLC method that selectively measures 25-hydroxyvitamin D2 [25(OH)D2] and D3 [25(OH)D3] and compared this assay with a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method, a competitive protein-binding assay (CPBA) on the Nichols Advantage™ platform, and an RIA from Diasorin. Results: For the new HPLC assay, between-run CVs were 2.6%–4.9% for 25(OH)D3 and 3.2%–13% for 25(OH)D2; recoveries were 95%–102%; and the assay was linear from 5 μg/L to at least 200 μg/L. Comparison data were as follows: for HPLC vs LC-MS/MS, y = 1.01x − 4.82 μg/L (Sy|x = 4.93 μg/L; r = 0.996) for 25(OH)D3, and y = 0.902x − 0.566 μg/L (Sy|x = 2.56 μg/L; r = 0.9965 for 25(OH)D2; for HPLC vs Diasorin RIA, y = 0.709x − 5.86 μg/L (Sy|x = 7.35 μg/L; r = 0.7509); and for HPLC vs Nichols Advantage CPBA, y = 1.00x − 3.60 μg/L (Sy|x = 32.7 μg/L; r = 0.6823). Conclusions: The new HPLC method is reliable, robust, and has advantages compared with the Nichols Advantage CPBA and the Diasorin RIA. The Nichols Advantage CPBA overestimated or underestimated 25(OH)D concentrations predicated on the prevailing metabolite present in patients’ sera.