| The demand of infant for vitamins by breast milk is difficult to meet and vitamin deficiency can cause many diseases,so it is quite essential to add a variety of vitamin nutritional supplements in infant formula.Vitamins can be divided into water-soluble vitamins(such as vitamin C,B group)and fat-soluble vitamins(such as vitamin A,D,E,K).There are many methods to detect the content of vitamins in infant formula,such as microbial method,spectrometric method,chromatography technology and so on.Due to the need to detect a wide variety of vitamins,whose sample pretreatment and detection methods cannot be compatible with each other,the whole detection processes are always laborious and timeconsuming,and does not comply with the concept of green environmental protection of modern analysis and determination.Based on the physicochemical properties of vitamins and related researches,it had been established to determination of 14 kinds of water-soluble vitamins and 6 kinds fat-soluble vitamins by ultra-performance liquid chromatography-tandem mass spectrometry(UPLCMS/MS)and supercritical fluid chromatography-tandem mass spectrometry(SFC-MS/MS),which can not only greatly reduce detection workload,shorten the analysis time and improve the analysis efficiency,but also reduce the use of organic reagents to a great extent,which was convenient and environmental-friendly.The system suitability of these methods was investigated respectively by detecting quality control standard infant formulas.The results showed that these methods are sensitive,accurate and applicable for simultaneous determination and analysis of vitamins in infant formula.The main research contents and results were as follows:1.A rapid and sensitive UPLC-MS/MS method was developed for the simultaneous quantitation of 14 water-soluble vitamins in infant formula.The pretreatment procedure and detection condition were optimized.Internal standard method was adopted to calculate the contents.Single sample analysis time was only 6 min under MRM mode.The results showed that 14 kinds of vitamins exhibited a good linearity(R2>0.9961)with the LOQ(limit of quantitative)of 10 ?g/L for vitamin C,50 ?g/L for Taurine,lower than 1.0 ?g/L for other compounds.The detection results of two kinds of quality control standard are within limits with the intra-and inter-day precisions of 0.60%~8.78% and 0.80%~23.29%.Compared to the results of National Standard method,the determination deviations were less than 15%.Thus,a simple and rapid determination method of water-soluble vitamins in infant formula was established taking advantage of the high efficiency and high sensitivity of UPLC-MS/MS.2.A rapid and sensitive UPLC-MS/MS method was developed for simultaneous determination of the fat-soluble vitamins A,D2,D3,?-tocopherol in infant formula.With the isotope internal standards to correct operating errors and matrix effects,samples were hydrolyzed by KOH solution and extracted by n-hexane,and then separated on UPLC HSS T3 column.The instrumental analysis time of a single sample was 9min under MRM mode.Linearity in the range of 20~200 ?g/L were good(R2>0.9960).The recoveries of vitamin A,D2,D3,?-tocopherol were 97.54%,107.5%,88.06% and 101.0% respectively,the LOQs were 2,10,1.2 and 0.2 ?g/L;intra-and inter-day precisions were less than 10%.Thus,this method was simple,effective,accurate,and applicable for the determination of fat-soluble vitamins A,D2,D3,?-tocopherol in infant formula.3.The SFC-MS/MS method was developed for simultaneous determination of fatsoluble vitamins by the optimized saponification procedure as above,but after being blowing to all dry,the sample was redissolved by n-heptane: isopropanol 8:2 solution,of which 2 ?L was injected to the SFC-MS/MS system.Vitamin D2,D3,?-tocopherol,K1 and K2 showed good linearity(R2?0.9975)in the range of 0.1~1.0,0.05~0.5,0.5~5.0,0.5~5.0 and 0.5~5.0 mg/L respectively.Operating errors and matrix effects were corrected by the corresponding internal standards,the recoveries of vitamin D2,D3,?-tocopherol,K1 and K2 in quality control standard were improved from 89.61%,95.57%,104.0%,78.77% and 70.05% to 100.9%,98.67%,106.4%,105.8% and 96.85%,the intra-day precisions were less than 8.71% and the inter-day precisions were less than 12.54% with LOQs(limit of quantitation)of 80,100,50,5 and 20 ?g/L respectively,which can meet the quantitation requirements of fat-soluble vitamins in infant formula.4.Combined with the SFC-MS/MS detection technology,the enzymatic hydrolysis pretreatment method was established to focus on the determination of vitamin K1 and K2 in infant formula.The enzymatic hydrolysis method was used to solve the problem of vitamin K alkali intolerance in saponification method,and to improve the recovery of vitamin K.The recoveries of vitamin K1 and K2 in quality control standard sample were 109.5% and 99.46%,the intra-day precisions were less than 10.57%,the inter-day precisions were less than 13.02%,which can meet the determination requirements of fat-soluble vitamin in infant formula.Due to the result of matrix effect of VK1-2H7 was 98.18%,the accuracy of the result would not be affected calculated by the external standard method with the absence of isotope internal standard,while the expenses of this work would be greatly reduced.5.Based on the chemical properties and ionization mechanisms of target compounds,there was a further discussion on the influence factors of MS/MS response.The optimum peak shape of nicotinamide was occurred with pH of 5.5,and the break peak and leading peak were occurred when pH decreased from 5.5 to 2.The response of vitamin B1 increased from pH 5 to pH 2 and significantly increased when the concentration of ammonium acetate solution changed from 0 to 10 mmol/L.The fat-soluble vitamins showed higher sensitivity with ESI source rather than APCI source with UPLC-MS/MS analysis,but the case was contrary with SFC-MS/MS analysis.These studies provided the theoretical basis for the improvement of accuracy and sensitivity of determination of vitamins by chromatography tandem mass sepectrometry technology. |
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