Method Study And Application Of Determination Of Vitamin D In Serum Based On Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS)

Objective:The level of endogenous vitamin D metabolites was low.In order to improve the sensitivity and accuracy of the detection of vitamin D metabolites in human serum,liquid-liquid extraction,derivatization and LC-MS/MS technology and internal standard method were used to establish a simple,rapid,sensitive,reproducible,high recovery and high accuracy detection method,which was applied to the research of vitamin D metabolites related diseases.The tested vitamin D metabolites included 25hydroxyvitamin D₂（25OHD₂）,25 hydroxyvitamin D₃（25OHD₃）,1,25-hydroxyvitaminD₂[1,25-dihydroxyvitaminD₂,1,25（OH）₂D₂],1,25-hydroxyvitamin D3[1,25-dihydroxyvitamin D₃,1,25（OH）₂D₃].Method:Firstly,the mass spectrum parameters and conditions of the target analyte were optimized by using the standard sample.Then LC-MS/MS technology was used to optimize the pretreatment steps of serum samples.The specific steps include:the selection of deproteinizing solvent,the selection of type and times of liquid-liquid extraction solvent,the selection of sample redissolving solvent and its proportion,the selection of molar amount of derivatization reagent,the selection of derivatization time,etc.In this study,the positive ion electrospray ionization mode was used for multiple reaction monitoring.Standard curve combined with deuterium isotope internal standard method was used for accurate quantification.A method for simultaneous determination of 25（OH）D₂,25（OH）D₃,1,25（OH）₂D₂and 1,25（OH）₂D₃in serum was established.The linear range,limit of detection,limit of quantitation,matrix effect,recovery,intra-day and inter-day precision of the method were also investigated.Results:In this study,four kinds of vitamin D metabolites and internal standard 25（OH）D₃-d₆were detected by mass spectrometry and LC-MS/MS.The optimized MRM parameters were obtained,including the parent ion/daughter ion pair,DP,CE,CEP,EP and CXP values of each substance.The optimum pretreatment conditions were methanol as deproteinization solvent,methyl tert-butyl ether（MTBE）as extraction solvent,and the extraction times were two.Methanol-water（9:1,V/V,0.1%FA）was used as the redissolving solvent.The molar ratio of the derivatized substance to the tested substance was 9000,and the derivatization time was 10 min.After derivatization with PTAD,the detection sensitivity of the four vitamin D metabolites was increased by 10-66 times.Under the optimized conditions,four kinds of vitamin D metabolites in serum,25（OH）D₂,25（OH）D₃,1,25（OH）₂D₂and 1,25（OH）₂D₃can be detected simultaneously by LC-MS/MS,and the analysis time is 12 min.The linear ranges of 25（OH）D₂and 25（OH）D₃were 1-100 ng?m L^-1,the detection limits were 0.3ng?m L^-1and the quantitation limits were 1.0 ng?m L^-1.The linear ranges of1,25（OH）₂D₂and 1,25（OH）₂D₃were 5-100 ng?m L^-1,the detection limits were 1.5ng?m L^-1and the quantitation limits were 5.0 ng?m L^-1.The recoveries at low,medium,and high concentrations were 76.12%-124.11%,96.84%-125.24%,72.03%-103.65%respectively,and the coefficient of variation（CV）was 0.91%-13.95%.The intra-day precision was less than 11.46%,the coefficient of variation was 2.92%-11.46%,the inter-day precision was less than 6.82%,the coefficient of variation was0.20%-6.82%.Conclusions:In this study,a method for the determination of vitamin D metabolites 25（OH）D₂,25（OH）D₃,1,25（OH）₂D₂and 1,25（OH）₂D₃was established based on liquid-liquid extraction,derivatization pretreatment and LC-MS/MS.The results showed that the pretreatment process was simple and the derivatization reaction conditions were mild.The sensitivity of mass spectrometry was greatly improved after derivatization.At the same time,the method showed a wide linear range,good repeatability,intra-day precision and inter-day precision,as well as good recoveries at low,medium,and high concentrations.The internal standard method was used for accurate quantification.Finally,the method was applied to the analysis of vitamin D metabolites in healthy people,diabetes,and hyperlipidemia patients.