A Novel LC-MS/MS Method For Detecting Three Kinds Of B Vitamins In DBS And Its Application In Two Hospitals

Objective:Developed and optimized a method for the quantitative detection of VitB₁,VitB₂,and VitB₆ by using dried blood spots combined with LC-MS/MS,and simultaneously quantified and compared the nutritional status of VitB₁（Thiamin）,VitB₂（Riboflavin）and VitB₆（Pyridoxalphosphate,PLP）in 140 children（excluding nutrient deficiencies and suffering from blood system diseases）aged from 0 to 7 years in two hospitals in Beijing and Xining by a novel LC-MS/MS,in order to provide a realistic basis for children’s health improvement.Methods:A method for the quantitative analysis of vitamins B₁,B₂ and B₆ in dried blood spots by internal standard method using LC-MS/MS was established under the optimal mass spectrometry,chromatography,and extraction conditions.Linear range,and detection Limits,accuracy,precision,and consistency of the results of dried blood spots and homologous liquid whole blood.In addition,a random selection of 140 children aged 0 to 7 years who attended the outpatient clinics of the Capital institute of pediatrics and Women and children’s hospital of Qinghai in August 2016（excluding nutrient deficiencies and suffering from blood system diseases）.There were 72 children from Capital institute of pediatrics and 68 children from Women and children’s hospital of Qinghai.Children were divided according to age:42cases in infant group（0^&lt;1 year old）,60 cases in infant group（1³ years old）,preschool group（>3⁷ years old）.Forty microliters of peripheral blood from children was collected and deposited on blank paper to prepare dried blood spots.Concentrations of VitB₁,VitB₂and VitB₆ in dried blood spots were analyzed using the novel LC-MS/MS method.Results:VitB₁,VitB₂,VitB₆ linear range are 0.5¹00 ng/ml,0.2⁴0 ng/ml,0.5¹00 ng/ml,respectively.The limits detections were 0.5,0.2,0.5 ng/ml respectively.The accuracies were-5.99%⁰.97%,-5.15%¹.33%,-7.55%⁵.47%respectively.The recoveries of samples were 97.65%¹11.15%,98.37%¹03.3%,80.67%⁹0.50%respectively.And within-run coefficients of variance（CVs）3.22%⁸.26%,1.64%¹.30%,1.72%⁷.53%,between-run coefficients of variance（CVs）0.29%¹3.11%,0.19%⁸.49%,0.50%¹0.80%.The stability coefficients of VitB₁,VitB₂,and VitB₆ within 30 days are all less than 15%,it shows that the three kinds of B vitamins in the dried blood spots are stable at room temperature.The application of this method to detect the whole blood of 140 children in two hospitals in Beijing and Xining showed that the level of VitB₁（thiamin）in the whole blood of children in Beijing area hospital（7.59 ng/mL）was significantly higher than that of hospital children in Xining area（4.27 ng/mL）（t=4.89,P<0.05）.There was no difference in the levels of thiamin（2.86 ng/mL vs 2.88 ng/mL）and PLP（24.83 ng/mL vs 25.10 ng/mL）in children and whole blood of Beijing and Xining Hospital.Conclusion:A new method for simultaneous quantitative detection of VitB₁,VitB₂ and VitB₆ in whole blood was established using dry blood spots（DBS）combined with high performance liquid chromatography-tandem mass spectrometry（LC-MS/MS）.This method can accurately quantify VitB₁,VitB₂,and VitB₆ levels in children.Using this method to detect140 children,the results showed that the VitB₂（riboflavin）level of children from hospital in Beijing was better than that from hospital in Xining.Among the total children,the VitB₁（thiamin）and VitB₂（riboflavin）levels of infant were higher than baby and preschool children.