Statement of Work (SOW).docx

Statement of Work (SOW)

The vendor will receive 470 milk samples from external partner as follows:

Samples collected at 8-12 weeks post-partum (T1).

The vendor will analyze these samples for human milk oligosaccharide (HMO) composition. A known amount of internal standard will be added at beginning of sample preparation to enable absolute HMO quantification. Lipids, proteins and salts will be removed through solid-phase Sep-Pak C18 and porous graphite carbon (PGC) microcolumns. The reducing end of the isolated oligosaccharides will be labelled with 2-aminobenzamide (2AB). The reaction will be facilitated in a thermocycler to guarantee constant and reliable conditions. Unreacted 2AB will be removed from 2AB-labeled oligosaccharides through solid-phase Sep-Pak silica microcolumns. 2AB-labeled oligosaccharides from different samples will be placed into the HPLC autosampler and chromatographically separated over an amide column and detected by an online-coupled fluorescence detector. The system allows for absolute quantification of the following 19 most abundant and structurally distinct HMO (constitute > 95% total human milk HMO):

2’-fucosyllactose (2’FL), 3-fucosyllactose (3FL), 3’-sialyllactose (3’SL), 6’-sialyllactose (6’SL), difucosyllactose (DFLac), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-Nfucopentaose (LNFP) I, LNFPII, LNFPIII, sialyl-LNT (LST) b, LSTc, difucosyl-LNT (DFLNT), disialyl-LNT (DSLNT), lacto-N-hexaose (LNH), fucosyl-LNH (FLNH), difucosyl-LNH (DFLNH), fucosyl-disialyl-LNH (FDSLNH), disialyl-LNH (DSLNH).

The vendor will provide an excel data sheet that lists the absolute concentrations (in nmol/mL and ug/mL) as well as relative concentrations (in % total) of the 19 HMOs in the provided samples. The data will also include information on Secretor status, HMO diversity and evenness, HMObound sialic acid and HMO-bound fucose.

Timeline:

Analysis of 470 milk samples should take place within approximately nine (9) months after the time of shipment.