Simultaneous determination of Vitamin A, 25-hydroxyl vitamin D3 α-tocopherol in small biological fluids by liquid chromatography-tandem mass spectrometry

A sensitive method for the simultaneous analysis of vitamin A (VA), 25-hydroxyl vitamin D-3 (25-OH VD3) and alpha-tocopherol (VE) in children plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Sample preparation chose the solid phase extraction. 100 mu L of plasma was mixed with 300 mu L ethanol contained 4 mu L isotope-labelled analytes. After a series operation, the supernatant was applied to the solid phase extraction (SPE) plate (HLB mu Elution plate). The eluate was evaporated, and reconstituted in 100 mu L methanol. And then, 6 mu L reconstituted sample was injected into LC-MS/MS. Quantitative analysis was carried out by multiple reaction monitoring (MRM) with a positive mode electrospray (ESi +). Separations of VA, 25-OH VD3 and VE were performed on an Acquity UPLC reversed-phase Phenyl-Hexyl analytical column (CSH, 2.1 x 50 nun, 1.7 mu m). Gradient elution was used at a flow rate of 0.4 mL/min with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid, 5 mM ammonium formate in acetonitrile. The total time of analysis was 10 min. The method had a limit of quantification (LOQ) of 10.03, 1.20, and 0.04 ng/mL for VA, 25-OH VD3 and VE in methanol, respectively. The linear calibration curves were fitted over the range of 0.14-14.32 mu g/mL, 1.80-180.29 ng/mL, and 6.03-602.99 ng/mL for VA, 25-OH VD3 and VE in methanol. The correlation coefficients were greater than 0.998 for all analytes. The recoveries for all analytes were between 80 and 120% with the inter- and intra-day precisions (presented as relative standard deviation, RSD%) less than 10.0%. Analysis of VA, 25-OH VD3 and VE in recurrent respiratory tract infection children plasma and anemic infants' fingertip blood was then carried out using this method and statistical analysis of the data with statistic package for social science 20.0 (SPSS 20.0). Using this method, multiple fat-soluble vitamins could be detected at the same time. Solid phase extraction was used to simplify sample pretreatment. mu Elution plate used here could reduce the sample volume, only 100 mu L sample was used in this method, and 6 mu L reconstituted sample was injected into LC-MS/MS. This makes the method appropriate for larger sample pretreatment, and suitable for children, especially infants and newborns' sample detection, in whom the circulation blood was low.