【摘要】：研究背景和目的人乳為嬰兒最佳食品,它能夠提供0-6月齡嬰兒幾乎所有營養(yǎng)成分。人乳中B族維生素是純母乳喂養(yǎng)嬰兒攝入的唯一來源。人乳中B族維生素的水平將直接關(guān)系嬰兒的健康。近年來越來越多的國內(nèi)外學(xué)者關(guān)注人乳中B族維生素的水平。目前的研究涉及到影響人乳中B族維生素水平的因素主要有哺乳階段、膳食因素、社會經(jīng)濟(jì)學(xué)因素、農(nóng)村和城市等。人乳中B族維生素的研究方法方面,主要有微生物法、化學(xué)法、放射分析法、免疫分析法、HPLC法,2012年有人首次應(yīng)用UPLC/MS-MS方法分析了人乳中5種游離B族維生素的水平。然而目前的研究仍存在一些不足,主要是乳樣采集缺乏標(biāo)準(zhǔn)化(如任意次乳樣或幾毫升乳樣)；樣本量較小,缺乏代表性；分析方法通常一次只能測定一種維生素、前處理復(fù)雜、分析周期長、檢測成本高；較少的研究測定了人乳中黃素腺嘌呤二核甘酸(FAD)的水平。因此開發(fā)一種高效、快速、準(zhǔn)確的方法,進(jìn)行大樣本人乳中B族維生素水平的分析,將有助于了解人乳中B族維生素的水平及其影響因素,指導(dǎo)乳母膳食,改善嬰兒健康狀況,為修訂嬰兒B族維生素適宜攝入量提供科學(xué)依據(jù)。研究對象和方法通過優(yōu)化質(zhì)譜參數(shù)、分析柱、流動相、前處理方法等,建立了·UPLC-MS/MS法分析人乳中B族維生素水平,用回收率、準(zhǔn)確性、精密度、檢出限、線性范圍、穩(wěn)定性、基質(zhì)效應(yīng)等因素進(jìn)行了方法學(xué)確認(rèn)。從中國母乳成分?jǐn)?shù)據(jù)庫樣品中抽樣,抽取1778份母乳樣品,采用UPLC-MS/MS法分析了1778份人乳樣品的B族維生素水平,按不同哺乳階段、20個調(diào)查點、全國11省、6大區(qū)域、南北地區(qū)、農(nóng)村和城市、沿海和內(nèi)陸進(jìn)行了描述和歸納。描述不同乳母學(xué)歷、經(jīng)濟(jì)水平、分娩方式、體格測量、膳食攝入指標(biāo)下的人乳樣品中的B族維生素水平,用兩獨立樣本的t檢驗和單因素方差分析檢驗不同指標(biāo)下人乳中B族維生素水平的差異；分析兩因素間的相關(guān)性,通過線性回歸方程篩選出人乳中的10種B族維生素水平各自的影響因素。研究結(jié)果UPLC-MS/MS方法前處理簡單,線性范圍擬合良好,準(zhǔn)確度為98.4-108.0%,日內(nèi)精密度和日間精密度分別為3.4-19.9%和5.9-18.1%、穩(wěn)定性好,達(dá)到分析要求,適合大批量人乳樣品多種B族維生素同時分析。初乳、過渡乳、成熟乳中B族維生素水平的中位數(shù)依次分別為：硫胺素(5.0μg/L、6.7μg/L、 27.2 μg/L),核黃素(29.3μg/L、41.5 μg/L、32.2μg/L),煙酸(464.2 μg/L、675.1 μg/L、635.7 μg/L)、維生素B6(4.7μg/L、18.8μg/L、70.3μg/L)、FAD (808.7 μg/L、1174.6μg/L、1023.9 μg/L)、泛酸(1770.9 μg/L、2628.3 μg/L、2119.4 μg/L)。目前分析的人乳中B族維生素水平在初乳階段水平均是最低的,硫胺素和維生素B6水平隨哺乳期增大而增大,核黃素、煙酸、FAD和泛酸水平隨哺乳期增大均呈現(xiàn)先增大后減小的變化趨勢。整個哺乳階段里,農(nóng)村乳母的硫胺素、核黃素和煙酸水平低于城市乳母,農(nóng)村乳母的維生素B6水平高于城市乳母,城市乳母的FAD水平與農(nóng)村乳母相近,農(nóng)村乳母的泛酸水平在哺乳期0-30天里高于城市乳母,30天以后二者差異逐漸變小。沿海地區(qū)乳母乳汁的硫胺素、核黃素和煙酸水平高于內(nèi)陸地區(qū)乳母,沿海地區(qū)乳母乳汁的FAD、泛酸和維生素B6水平低于內(nèi)陸地區(qū)乳母。影響人乳中B族維生素水平的因素主要有哺乳階段、膳食、地區(qū)、沿海和內(nèi)陸、農(nóng)村和城市、乳母學(xué)歷、乳母職業(yè)、分娩方式。省份地區(qū)因素影響人乳中10種B族維生素水平；哺乳階段影響人乳中除FAD以外的9種B族維生素水平,膳食因素影響人乳中除核黃素以外的9種B族維生素水平,乳母學(xué)歷影響人乳中核黃素、泛酸、吡哆胺、生物素水平；乳母職業(yè)影響人乳中核黃素、泛酸水平；沿海和內(nèi)陸地區(qū)影響人乳中硫胺素、吡哆醛、FAD、吡哆醇、吡哆胺水平；農(nóng)村和城市影響人乳中尼克酰胺、吡哆醛、FAD、尼克酸、吡哆醇水平；分娩方式影響人乳中泛酸和吡哆胺水平。結(jié)論本研究建立了高效、快速的UPLC-MS/MS法分析人乳中10種B族維生素水平,檢測方法具有良好的準(zhǔn)確性和精密度；分析了全國1778份人乳樣品,首次系統(tǒng)報道了全國20個調(diào)查點、11省份、6大區(qū)域、南北地區(qū)、農(nóng)村和城市、沿海和內(nèi)陸、不同哺乳階段的人乳中B族維生素水平；研究發(fā)現(xiàn)人乳中B族維生素的影響因素主要包括哺乳階段、膳食、地區(qū)、沿海和內(nèi)陸、農(nóng)村和城市、乳母學(xué)歷、乳母職業(yè)、分娩方式。
[Abstract]:The background and purpose of the study are the best food for infants, which can provide almost all of the nutritional ingredients for infants from 0 to 6 months of age. Vitamin B in human milk is the only source of pure breast-fed infants. The level of B vitamins in human milk will directly relate to the health of the infant. In recent years, more and more domestic and foreign scholars are concerned about the level of B vitamins in human milk. At present, the factors affecting the level of vitamin B in human milk mainly include the feeding stage, the dietary factor, the social economic factor, the rural and the city, and so on. In the aspect of the study of B vitamins in human milk, the level of five free B vitamins in human milk was analyzed by the method of micro-organism, chemical method, radioanalysis, immunoassay, HPLC, and the first time in 2012 by UPLC/ MS-MS. However, there are still some deficiencies in the present study, such as the lack of standardization in the collection of milk samples (such as any sub-sample or a few ml of milk samples), the small sample size and the lack of representativeness; the analytical method can only measure one kind of vitamin at a time, the pretreatment is complicated, the analysis period is long, and the detection cost is high; The level of fAD in human milk was determined by a few studies. therefore, a high-efficiency, fast and accurate method is developed, and the analysis of the vitamin level of the B-family in the human milk of the large sample is carried out, So as to provide a scientific basis for revising the appropriate intake of vitamin B vitamins. The method of UPLC-MS/ MS was established to analyze the level of B vitamins in human milk by optimizing the mass spectrum parameters, the analytical column, the mobile phase and the pretreatment method. The recovery, the accuracy, the precision, the detection limit, the linear range and the stability of the human milk were determined by the method of UPLC-MS/ MS. The matrix effect and other factors have been confirmed by the methodology. 1778 human milk samples were sampled from Chinese milk component database samples, and 1778 human milk samples were analyzed by UPLC-MS/ MS. The levels of vitamin B in 1778 human milk samples were analyzed by UPLC-MS/ MS. The coastal and inland areas have been described and summarized. The vitamin levels of B vitamins in human milk samples were tested by t-test and single-factor analysis of variance of two independent samples, and the differences in vitamin levels of B-group in human milk were tested by t-test and single-factor analysis of variance of two independent samples. The correlation between two factors was analyzed, and the influence factors of 10 B vitamins in human milk were selected by linear regression equation. The method of UPLC-MS/ MS was simple, the linear range fit well, the accuracy was 98.4-108.0%, the intra-day precision and the day precision were 3.4-19.9% and 5.9-18.1%, and the stability was good. The median of the vitamin levels in the colostrum, the transitional milk and the mature milk were: thiamine (5.0. mu.g/ L, 6.7. mu.g/ L, 27.2. mu.g/ L), riboflavin (29.3. mu.g/ L, 41.5. mu.g/ L, 32.2. mu.g/ L), nicotinic acid (464.2. mu.g/ L, 675.1. mu.g/ L, 635.7. mu.g/ L), vitamin B6 (4.7. mu.g/ L, 18.8. mu.g/ L, 70.3. mu.g/ L), FAD (808.7. mu.g/ L, 1174.6. mu.g/ L, 1023.9. mu.g/ L), pantothenic acid (1770.9. mu.g/ L, 2628.3. mu.g/ L, 2119.4. mu.g/ L). At present, the level of B vitamins in human milk is the lowest in the phase of the colostrum, and the levels of thiamine and vitamin B6 increase with the increase of the lactation period, and the levels of riboflavin, niacin, FAD and pantothenic acid increase with the increase of the lactation period. The level of thiamine, riboflavin and niacin in the rural lactating mother is lower than that of the city's milk mother in the whole lactation period, and the level of the vitamin B6 of the dairy mother in the rural area is higher than that of the urban milk mother, and the FAD level of the urban milk mother is similar to that of the rural dairy mother. The level of pantothenic acid in the rural lactating women is higher than that of the city's milk mother in the period 0-30 days of the lactation period, and the difference between them gradually becomes smaller after 30 days. The levels of thiamine, riboflavin and niacin in the breast milk of the coastal area are higher than that in the inland areas, and the levels of FAD, pantothenic acid and vitamin B6 in the milk in the coastal area are lower than that in the inland areas. The factors affecting the level of vitamin B in human milk mainly include the feeding stage, the diet, the area, the coastal and the inland, the rural and the city, the mother's degree, the mother's occupation and the mode of delivery. The factors of the province affect the level of 10 B vitamins in human milk, and the level of vitamin B vitamins except FAD in human milk is affected by the feeding stage, and the dietary factors affect the levels of vitamin B vitamins except for riboflavin in human milk, and the milk mother's degree affects the riboflavin and pantothenic acid in human milk. in that coastal and inland areas, the human milk, such as thiamine, calcitral, fad, trialdol, and alammine, are present in the coastal and inland areas; and in the rural and urban areas, the human milk, such as nicholamide, fad, fad, nicotinic acid, The levels of pantothenol in human milk were affected by delivery, and the level of pantothenic acid in human milk was affected by delivery. Conclusion The present study established an efficient and rapid UPLC-MS/ MS method for the analysis of 10 B-family vitamin levels in human milk. The detection method has good accuracy and precision.1778 human milk samples were analyzed. The first system reported 20 investigation points,11 provinces,6 large areas and north and south areas. The level of vitamin B in human milk in rural and urban, coastal and inland and different lactation stages is studied. The factors affecting the vitamin in human milk mainly include the feeding stage, the diet, the area, the coastal and the inland, the rural and the city, the mother's degree, the mother's occupation and the mode of delivery.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R155.5;O657.63

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