Methods of preparing and analyzing breast milk
View Patent ↗The present disclosure provides methods of preparing breast milk for analysis analyzing breast milk, determining reference ranges for analytes in breast milk and testing breast milk for a panel of analytes. Aspects of the present disclosure provide dietary and lifestyle recommendations for the lactating mother-based on measured concentration of analytes. Retesting is disclosed to measure the influence of maternal diet on breast milk composition.
1. A method for analyzing breast milk for total fatty acid concentrations comprising the steps of:
a) collecting a human breast milk sample from a lactating individual;
b) freezing the breast milk sample at a temperature between −70 degrees C. to −80 degrees C.;
c) thawing the breast milk sample above 20 degrees C.;
d) homogenizing the breast milk sample by sonicating to disrupt cell structures in the breast milk sample;
e) performing liquid liquid extraction by combining the breast milk sample with chloroform, methanol, and water in a volume ratio of approximately 1:2:0.8 in a separating vessel to form an upper layer and a lower layer;
f) separating the lower layer containing chloroform and lipids;
g) dissolving the lower layer in a nonpolar solvent;
h) adding boron trichloride in methanol to form an extract mixture;
i) heating the extract mixture at about 60 degrees C. for approximately ten minutes;
j) cooling the extract mixture with water and hexane and mixing;
k) extracting an upper organic layer to provide a sample extract; and
l) performing gas chromatography-mass spectrometry analysis on the sample extract to provide measured total fatty acid concentrations.
2. The method of claim 1 wherein the step of collecting a human breast milk sample comprises collection of breast milk over a 24 hour period.
3. The method of claim 1 wherein the nonpolar solvent of step g) comprises hexane.
4. The method of claim 3 wherein the concentration of boron trichloride in methanol is 12% weight/weight.