| Objective:Combined the separation of high performance liquid chromatography (HPLC) and the sensitivity of tandem mass spectrometry (MS/MS), the aim of this study was to develop a novel,simple, feasibility and high-throughput approach for determination of retinol, retinyl acetate, retinyl palmitate and P-carotene in dried blood spots (DBS), and provided the foundation for detection of neonatal Vitamin A Deficiency with capillary blood.Methods:We investigated the molecular ions, fragments ions, declustering potential, entrance potential, collision energy, collision cell entrance potential, collision cell exit potential of retinol, retinyl acetate, retinyl palmitate and P-carotene to gain the best parameters of tandem mass spectrometry. Then we explored the solid phase, mobile phase, column temperature in detail to achieve the appropriate conditions of HPLC. Moreover, the optimal pretreatment methods were determined by peak areas in different extraction methods.Under the optimized conditions of tandem mass spectrometry and liquid chromatography, HPLC-MS/MS methods for retinol, retinyl acetate, retinyl palmitate and β-carotene in DBS were established. Linear range, precision, detection limits and accuracy were researched one by one. The relativity of retinol concentration between in DBS with this method and that in serum with HPLC was assessed.Results:The parameters of tandem mass spectrometry for retinol, retinyl acetate, retinyl palmitate and p-carotene were optimized. Retinol and its precursors were well separated on a C-8 column using a 95:5 (v/v) methanol/dichloromethane mixture at a flow rate of 0.2 ml/min. A volume of 100μl deionized water,200μl of a methanol and 300μl chloroform added to three DBS of diameter 3.2mm was found to be suitable for protein precipitation and retinoids extraction.The matrix effects of retinol, retinyl acetate, retinyl palmitate and β-carotene is 98.0%,96.7%,90.0% and 82.1%, respectively. Retinol and its precursors in calibration curves showed good linearity in the concentration range covered in the curves, with the correlation coefficients bigger than 0.99. The limits of detection were 16.2ng/ml,5.3 ng/ml,6.3 ng/ml,53.75 ng/ml, the limits of quantitation were 65 ng/ml,21.3 rig/ml,25.0 ng/ml, 53.75 ng/ml, the recoveries of samples were 85.5%,84.5%,83.5% and 73.5%, respectively, and within-and between-day coefficients of variance (CVs) 4.30%-7.35% and 4.85%-9.40%. The mean concentrations of retinol, retinyl acetate, retinyl palmitate and β-carotene in 50 children aged from 6 months to 70 months archived DBS were 0.48±α0.18μg/ml,0.115±0.06 μg/ml,0.02±0.01μg/ml,0.19±0.11μg/ml. It was showed correlation with the correlation coefficient 0.81 between the concentration of retinol in DBS measured by this HPLC-MS/MS method and that in serum from HPLC method.Conclusion:In the present study, a novel HPLC-MS/MS method to measure retinol, retinyl acetate, retinyl palmitate and β-carotene in DBS was established successfully. It was proved to be of high sensitivity, feasibility and high-throughput and would be useful for the evaluation of the retinol status in DBS and provides useful information for the treatment and prevention of vitamin A deficiency. |
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