Sources of milk samples used in validation experiments
Pooled samples of liquid milk from US lactating women (n = 5) with a relatively wide range of DHA levels (0.05 to 0.42 % DHA) that had been submitted for routine FA analysis were de-identified and used for method development. After the initial analysis, samples were stored at -80 °C until stability and validity experiments were conducted.
A second set of samples was collected in Malawi, Africa (n = 50) as part of a randomized trial of a lipid-based nutritive supplement (which did not include DHA or iTFA) for pregnant women . The trial was performed according to Good Clinical Practice guidelines and the ethical standards of the Helsinki Declaration. The protocol was approved by the College of Medicine Research and Ethics Committee, University of Malawi and the Ethics Committee of Pirkanmaa Hospital District, Finland. This trial was registered at clinicaltrials.gov with the identifier NCT01239693.
Breast milk samples from Malawi were collected at participants’ homes by following milk expression instructions that were given by female sample and data collectors. The mothers expressed the milk into clean plastic cups, which was immediately mixed thoroughly with a clean plastic spoon. Ten milliliters of milk was collected from this mixture into a clean falcon tube (the rest was given back for the child) and placed on ice for at most one hour. This was then taken to a satellite laboratory and aliquoted into five, 2 ml cryovials. These were stored temporarily at the satellite laboratory at -20 °C for no more than 2 days after which they were taken to a central sample archive laboratory and placed at -80 °C for long-term storage. The samples were shipped to the US on dry ice in accordance with International Air Transport Association sample shipment instructions.
Preparation of dried milk spots (DMS)
All DMS samples were prepared in the laboratory by placing two drops (≈ 50 uL) of thawed milk on absorbent paper (Ahlstrom 226, PerkinElmer, Greenville, SC) pretreated with OxyStop®, a proprietary antioxidant cocktail which delays oxidation of FA in dried whole blood .
A hole punch from the DMS card or 12.5 uL of thawed liquid milk were combined (1:40 parts) with the methylating mixture [boron trifluoride in methanol (14 %), toluene and methanol (35/30/35 v/v)], shaken and heated at 100 °C for 45 min. After cooling, 40 parts of both hexane and distilled water were added. After briefly vortexing the samples were spun to separate layers, and an aliquot of the hexane (upper) layer which contained the FA methyl esters was taken for analysis by gas chromatography (Shimadzu 2010; SP2560, 100-m column) as described previously . Data are expressed as a percent of total identified FA; a total of 26 FA between C10:0 – C22:6n-3 were identified. With each batch of DMS samples analyzed, two controls (one high and one low in DHA) were run. A 3-point standard curve was run at the beginning of each batch from which DHA levels of the unknowns were determined. Pure standards had been used to confirm the identities of DHA and iTFA, and additional confirmation has been made using gas chromatography-mass spectroscopy in our method development. The CVs for DHA were 6.0 % and iTFA 3.8 %.
Validation of liquid vs. dried milk spot fatty acid profile
Fifty-five samples of milk from Malawian and US mothers were used to compare the FA composition of liquid vs. DMS. To make DMS samples, 1iquid milk samples were thawed and one drop of milk was placed on the collection cards. Then the DMS samples were stored at room temperature in the dark for one week before analysis (to simulate the approximate time from collection to analysis in the field). The liquid samples analyzed immediately after thawing.
Stability of fatty acid profile in dried milk spots: varying time and temperature
The five pooled milk samples from US mothers were spotted onto pretreated cards. Several DMS cards from each pool were stored in the dark in reclosable plastic bags under each of the following conditions: room temperature (23 °C), refrigeration (4 °C), standard freezer (-20 °C), and research freezer (-80 °C). At Days 4, 7, 14, 21, and 28, DMS from the first three storage temperatures were analyzed for FA composition; the Day 0 sample was analyzed immediately after spotting and served as a reference. Additionally, DMS stored at -20 °C and -80 °C were tested at months 3, 6, 9, and 12, and then every 6 months for up to 3 years.
Pooled milk samples from five US mothers were placed on DMS cards and sent by express mail at ambient temperature to an affiliated lab in Seoul, South Korea (OmegaQuant Asia). Identical DMS samples were saved at OmegaQuant Analytics in Sioux Falls, SD. Sample analysis was coordinated to be done on the same day (about 1 week after shipping). Identical methods were used in both laboratories.
All statistics and graphs were analyzed and created with MiniTab (Student version, State College, PA, USA) and GraphPad Prism 6 (GraphPad software, San Diego, CA, USA). The primary FA of interest were DHA (C22:6n-3) and iTFA (C18:1 t plus C18:2 t). If necessary, the FA were log-transformed to meet normality requirements. Spearman correlations, linear regression, and paired t-tests were used to compare the liquid and DMS samples. Correlations between liquid and dry milk samples were calculated across the full range of DHA values, and for only those samples with DHA < 1.0 %. The latter is the range in which the vast majority of milk DHA levels lies worldwide . For the stability and inter-lab validation studies, an acceptable range of 15 % of the FA baseline or reference value was determined, as per FDA guidelines . If the FA value remained within the acceptable range over time and in different temperature conditions, it was considered stable. In addition, the entire FA profile was assessed for validity and stability and presented in the Additional files.