Introduction:
Human milk oligosaccharides (HMO) are an important component in the development of healthy infants and are the focus of intense analytical and synthetic research. Previous studies have shown that HMOs play a role in bolstering the health of nursing infants by acting as a food source for beneficial gut microbes and by acting as decoy receptors for intestinal pathogens. Due to the complexities of the matrix, sample preparation for HMO analysis is both time consuming and highly prone to variation. In this study, we developed an automated sample preparation in the 96-well plate format that achieves rapid-throughput while cutting down on variation and eliminating cross contamination.

Methods:
Human milk samples were homogenized and then the liquid handler (Opentrons OT-2)aliquoted a specified amount into each well and performed a Folch extraction and then an ethanol precipitation. The samples were subjected to reduction using NaBH4 and then cleaned up using PGC SPE. The samples were injected into an Agilent 1260 series HPLC coupled to an Agilent 6530 HPLC-QTOF-MS. Compounds were separated on a PGC column over 40-minutes which enables detection of approximately 125 human milk oligosaccharides.

Preliminary Data:
When automating a process, there are two key challenges: controlling cross contamination and ensuring reproducibility. To demonstrate that our method was free of cross contamination, we prepared method blanks next to our highest concentration standard and then quantitated the amount of 3’FL that was present in the method blanks using the external calibration curve method. Our highest standard contains 0.573 mg/mL of 3’FL, and the adjacent method blanks were found to contain on average 2.17x10^-5 mg/mL with a standard deviation of 1.68x10^-5 mg/mL (N=3). Our LLOQ was determined to be 1.66x10^-4 mg/mL (from N=7 blanks), hence the liquid handler did not introduce any measurable cross contamination.
To demonstrate reproducibility, seven samplings (N=7) of the same milk sample were prepared by the liquid handler and we looked at the responses of the 20 most abundant compounds in human milk. Looking at the CV for these responses, we found that the maximum CV was 14.19% for LNnH, and the minimum was 2.29% for 2'FL. The mean CV was 6.74% with a SD of 3.05%, which suggests a high level of reproducibility in our method.

Novel aspect:
Automated procedure on an open-source liquid handler for analyzing large batch HMO samples at higher reproducibility and speed.

Aaron Stacy

Appearances