The Levels Of B-vitamins In Human Milk And The Effect Factors

Background and ObjectivesHuman milk has been recommended as the optimal food for infants and met almost all the nutrient requirements for infants aged 0 to 6 months. The B-vitamins from human milk are the only source for exclusively breastfed infants. The contents of B-vitamins in human are closely associated with infants’ health.There has been growing interests in B-vitamins levels of human milk in the recent years. The effect factors mainly included the lactation stages, maternal dietary intake, socioeconomic status, rural and urban areas and so on. The analysis methods mainly included the microbiological, chemical, radioactive methods, enzyme immunoassays methods and high performance liquid chromatography (HPLC). The reference firstly used the ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze the 5 free forms of B-vitamins in human milk. However, these methods have some disadvantages, such as non-standard collecting human milk samples, small samples size. Moreover, each method could be used for only one individual vitamin with labor-intensive and time-consuming pretreatments and a larger volume of human milk was required for these determinations. Additionally, flavin adenine dinucleotide was not usually considered.It is necessary to develope the rapid, sensitive, efficient method to analyze the more B-vitamins in human milk for larger samples. It helps people to understand the B-vitmains in human milk and effect factors, guide the maternal dietary intake, improve the infants’ physical status and provide the data for making the adequate intakes of B-vitamins for infants.Subjects and methodsThrough selecting the mass spectrometer parameter, analysis column, mobile phase and pretreatment method, we developled the UPLC-MS/MS method to analyze the levels of B-vitamins in human milk. The performance for analysis was evaluated by linearity, sensitivity, accuracy, precision, recoveries, matrix effects and stability.The human milk samples (1778) were randomly selected from the sample bank of the National high Technique R & D Programm (863 Projects). We analyzed these samples by UPLC-MS/MS and described the data according to different lactation stages,20 survey plots,11 provinces,6 districts, South and North areas, rural and urban areas, coastal and inland areas.To analyze the B-vitamins in different maternal education, economic level, delivery way, physical index and dietary intake, independent sample t-test and analysis of variance (ANOVA) was used. The correlations of two factors were analyzed and effect factors were screen out using the linear regression equation.ResultsThe relative recovery ranged from 80.1 to 120.2% and the accuracy was determined to be 98.3 to 108.0%. Intra-day and inter-day variation were 3.4-19.9% and 5.9-18.1%, respectively. This method has been successfully applied to the simultaneous quantification of thiamin, riboflavin, pyridoxal, pyridoxine, pyridoxamine, nicotinamide, nicotinic acid, flavin adenine dinucleotide (FAD), biotin, and pantothenic acid in human milk.The median concentrations of free form of 6 B-vitamins in colostrums, transitional milk and mature milk were respectively as follows:thiamin 5.0 μg/L,6.7μg/L and 27.2 μg/L; riboflavin 29.3 μg/L,41.5 μg/L and 32.2 μg/L; niacin 464.2 μg/L,675.1 μg/L and 635.7 μg/L; vitamin B-6　4.7 μg/L,18.8 μg/L and 70.3 μg/L; FAD 808.7 μg/L,1174.6 μg/L and 1023.9 μg/L; pantothenic acid 1770.9 μg/L,2628.3 μg/L and 2119.4 μg/L. At present, the contents of six B-vitamins (thiamin, riboflavin, niacin, vitamin B-6, FAD and pantothenic acid) were all lowest in colostrum. The levels of thiamin and vitamin B-6 increased and the levels of riboflavin, niacin, FAD and pantothenic acid first increased and then decreased as lactation stage increased. The contents of thiamin, riboflavin and niacin were significantly lower in rural areas than in urban areas and the contents of vitamin B-6 were significantly higher in rural areas than in urban areas. There were no differences of contents of FAD in rural and urban areas. The contents of pantothenic acid were significantly higher in rural areas than in urban areas in postpartum 0-30 days and results were similar between rural and urban areas after postpartum 30 days. The contents of thiamin, riboflavin and niacin were significantly higher in coastal areas than in inland areas and the contents of vitamin B-6, FAD and pantothenic acid were significantly lower in coastal areas than in inland areas.The effect factors mainly included lactation stages, maternal dietary intake, district, rural and urban areas, coastal and inland areas, maternal occupation, maternal education and delivery ways. The province factors affected the levels of 10 B-vitamins in human milk. The lactation stages affected the levels of thiamin, riboflavin, pyridoxal, pyridoxine, pyridoxamine, nicotinamide, nicotinic acid, biotin, and pantothenic acid in human milk. The maternal dietary intake affected the levels of thiamin, pyridoxal, pyridoxine, pyridoxamine, nicotinamide, nicotinic acid, FAD, biotin, and pantothenic acid in human milk. The maternal education affected the levels of riboflavin, pantothenic acid, pyridoxamine and biotin in human milk. The maternal occupation affected the levels of riboflavin and pantothenic acid in human milk. The coastal and inland areas affected the levels of thiamin, pyridoxal, pyridoxine, pyridoxamine and FAD in human milk. The rural and urban areas affected the levels of nicotinamide, nicotinic acid, pyridoxal, pyridoxine and FAD in human milk. The delivery ways affected the levels of pyridoxamine and pantothenic acid in human milk.ConclusionsThis study developed the rapid, sensitive, efficient UPLC-MS/MS method for simultaneous analysis of ten B-vitamins in human milk. We analyzed the 1778 human milk samples by UPLC-MS/MS method and firstly reported the levels of B-vitamins in human milk systematically according to different lactation stages,20 survey plots,11 provinces,6 districts, South and North areas, rural and urban areas, coastal and inland areas. This paper showed that the levels of B-vitamins in human milk were controlled by actation stages, maternal dietary intake, district, rural and urban areas, coastal and inland areas, maternal occupation, maternal education and delivery ways.