Abstract
Breast milk contains human milk oligosaccharides (HMOs), including sialyllactose (SL). SL is composed of sialic acid and lactose, and is divided into 3′-SL and 6′-SL according to the binding position. SL has immunoprotective effects against bacteria and viruses, and acts as a probiotic in the gastrointestinal tract. In this study, we developed a bioanalytical method for simultaneous analysis of 3′-SL and 6′-SL in liver and kidney tissues of Yucatan minipigs using liquid chromatography–tandem mass spectrometry (LC-MS/MS) under conditions optimized in our previous study. LC-MS/MS was performed using a hydrophilic interaction liquid chromatography (HILIC) column (50 mm × 2.1 mm, 3 μm) with a mobile phase consisting of 10 mM ammonium acetate in water (pH 4.5) and acetonitrile with gradient elution at a flow rate of 0.3 mL/min. A surrogate matrix method using water was applied for analysis of endogenous SL. The developed method was validated with regard to selectivity, linearity, precision, accuracy, the matrix effect, recovery, parallelism, dilution integrity, carryover, and stability according to the US Food and Drug Administration guidelines. We performed a tissue distribution study of minipigs, and analyzed liver and kidney tissues using the developed method to determine the tissue distribution of 3′-SL and 6′-SL. The tissue concentrations of 3′-SL and 6′-SL were readily measurable, suggesting that the method would be useful for evaluating the tissue distributions of these compounds in minipigs.
Highlights
The major sialyloligosaccharides in breast milk are 30 - and 60 -sialyllactose (30 -SL and 60 -SL, respectively), disialyllacto-N-tetraose (DSLNT), and sialyllacto-N-tetraose (SLNT). 30 -SL and 60 -SL are endogenous compounds within the body, and the level of 30 -SL is maintained during lactation, but 60 -SL level decreases gradually in breast milk over time [1]
We developed an analytical method in minipig tissues using simplified sample preparation steps, as well as a shortened preparation time compared to previous studies
The methanol extract was evaporated with a SpeedVac concentrator and redissolved with reconstitution solution
Summary
The major sialyloligosaccharides in breast milk are 30 - and 60 -sialyllactose (30 -SL and 60 -SL, respectively), disialyllacto-N-tetraose (DSLNT), and sialyllacto-N-tetraose (SLNT). 30 -SL and 60 -SL are endogenous compounds within the body, and the level of 30 -SL is maintained during lactation, but 60 -SL level decreases gradually in breast milk over time [1]. 30 -SL and 60 -SL are endogenous compounds within the body, and the level of 30 -SL is maintained during lactation, but 60 -SL level decreases gradually in breast milk over time [1]. Previous studies analyzed human milk oligosaccharides (HMOs) in milk and biological matrices using various methods, including high-performance liquid chromatography–ultraviolet detection. Molecules 2020, 25, 5721; doi:10.3390/molecules25235721 www.mdpi.com/journal/molecules (HPLC-UV) [9], high-performance anion-exchange chromatography with pulsed amperometric detection [10,11], capillary electrophoresis (CE) [12], matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [13,14,15], MALDI-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) [16], electrospray ionization mass spectrometry (ESI-MS) [17], and microfluidic chip–time-of-flight mass spectrometry (HPLC-Chip/TOF MS) [18,19].
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