| Human milk is considered as the most ideal natural food for infants and it could meet nutritional needs of the growth and development for infants. As it contains high-quality protein, fat, a variety of minerals, vitamins and other nutrients, it efficiently promotes the infant’s growth of intelligence, strengthen the nervous system, while improving infant immunity. It is recommended as the best food for infant feeding by the World Health Organiz ation(WHO). It contains 3%-5% of lipids, which are one of the most important compounds in human milk that provides energy and nutrition for infants. Lipids consist of triglycerides, phospholipid, sphingolipids and steroids, etc. Though steroids do not occupy much in the components of lipids, they are of great importance in the process of growth and development of infants.The main components of steroids are cholesterol and steroid hormones, in which cholesterol is essential for growth and maturity of infants’ organs and, in particular, for growth of brain and nervous system and steroid hormones could adjust in vivo metabolism of infants and the development and promotion of gender consciousness. More focus was made on the problems whether the intake of hormones could cause Child Precocious Puberty(CPP) and degradation of male characteristics as studies on hormones are deepening. Currently, most of Chinese families having infants could not achieve breast-feeding because of various reasons and thus there are demands for infant formula milk powder. The difference between components of infant formula milk powder and those of standard human milk determines whether needs for growth and development of infants could be met. Therefore, it is especially important to n ormalize the analysis of components of human milk and infant formula milk powder in order to make it closer to human milk. Lipidomics is a systematic comprehensive analysis for all the lipids within the organisms, to determine the structure and function, understanding of lipid and revealling the close contact between the lipid metabolism and cell, tissue, organ, and even the physiological and pathological processes. Steroidomic is a branch of the lipidomic, which is a comprehensive analysis of steroid substances in the lipid. In this study, a total of 37 human milk including colostrum period(1 ~ 5 d), transitional milk period(6 ~ 20 d) and mature milk period(after 21 d) from 15 lactating women were as samples to make absolute quantification analysis and s tudy the dynamic change of components during lactation period.(1) An effective method was proposed for the determination the content of cholesterol in human milk and the dynamic change trend during lactation period by gas chromatography –mass spectrometry(GC-MS). After saponifying the analyte by KOH-ethanol solution, it was extracted by the mixed solution of ethyl ether and petroleum ether, dehydrated by anhydrous Na2SO4 and concentrated by nitrogen blowing. The analyte was separated and quantitatively analyzed using GC-MS on the Agilent DB-5MS capillary column(30 m×0.25 mm, 0.25 μm). Use Agilent gas chromatograph workstation and Agilent mass spectrometer workstation to analyz e the chromatogram and mass spectrometry. The results showed that this method is highly sensitive and accurate for the qualitative and quantitative analysis of cholesterol in human milk. The SPSS 17.0 software was used to analyze the difference of cholesterol coontent in lactation period, and the difference of cholesterol content in different lactation period was significant( P < 0.05). From the experimental results, the cholesterol concentration in colostrum was 202.81±3.72 mg/L. With the extending lactation period the concentration of cholesterol was decreased rapidly and the average contents of the human milk in transition and mature period were 171.19±3.24 mg/L and 123.95±3.12 mg/L. The recovery was reached at 94% and RSD was below 4%, LOD was 0.15 mg/L and LOQ was 0.52 mg/L.(2) An effective method was established with an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) for determination of 9 steroid hormones in Chinese human milk from northeastern part of China. Human milk s amples were treated with Glucose aldehyde glycosides enzyme and aryl sulfatase for enzymatic hydrolysis, extracted by acetonitrile, purified by solid-phase extraction column and concentrated, separated by an Acquity UPLC XDB C18 chromatographic column(100 mm×2.1 mm, 1.7 μm) and detected by reaction monitoring for multiple-reaction monitoring(MRM) scan pattern. The experimental data were obtained by using AB analyst software for mass spectrometer to analyze the experimental results obtained by chromatography and mass spectrometry. The results showed that the calibration curve with a good linear correlation coefficient(R2) that was above 0.99 in the range of 0.1-20 ng/m L. The results also showed that this method has good sensitivity and accuracy. The relative standard deviation fell between 1.89% and 4.02% and the method of the LOD was in the range of 0.10~0.25 ng/m L, and LOQ was in the range of 0.33-0.86 ng/m L. The SPSS 17.0 software was applied to analyze difference of steroid hormones contents at differenr stages of lactation. The contents of estradiol and testosterone in human milk at three stages of lactation had significant statistical differences(P < 0.05), while the contents of estrone, estriol, progesterone, dehydroepiandrosterone and androstenedione in colostrum period had significant statistical differences with another two stages of lactation(P < 0.05). There were significant differences in the content of cortisone in transitional milk and mature milk(P < 0.05). In addition to cortisone, androsterone and dehydroepiandrosterone, the other 6 kinds of steroid hormones contents decreased with the extension of lactation period. The content of cortisone in human milk decreased with the extension of lactation period before increasing, in contrast to androsterone. Dehydroepiandrosterone was detected initially in colostrum milk period but not the other two periods.The study applies steroidomics method combining with chromatography–mass spectrometry instruments to make qualitative and quantitative analysis for steroids in human milk including cholesterol and several kinds of important steroid hormones, and make deep understanding of the composition of steroid in human milk and the trend of dynamic change for contents. It has important meaning for the lipid base of infant formula milk powder to become closer to the components of lipids in human milk and for developing humanized infant formula milk powder and improving infants’ health. It could also provide the theoretical foundation for deepening the study of human milk lipidomics. |
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