Standard Operating Procedure for POP Sampling in Breast Milk

Standard Operating Procedure for
POP Sampling in Breast Milk
Page 7 of 20
5.6 Preparation of composite samples
The samples must be handled by qualified personnel, to guarantee their integrity. Individual milk
samples must be homogenized by heating them at 38 °C and shaking them for 10 minutes, after which
the laboratory must prepare individual samples and composite samples. The individual samples must
contain 25 ml of breast milk for the analysis of analytically simple POPs, as pesticide POPs and PCB
markers. As to the composite sample, 10 ml of each one of the 50 individual samples must be taken to
obtain a 500-ml composite sample. Of those 500 ml, 50 ml must be kept aside for the analysis of simple
POPs by the participant countries, and the rest, 450 ml, will be analyzed by the WHO Reference
Laboratory. The latter will analyze both analytically simple and complex POPs, such as dioxin-like PCBs,
PCDD and PCDF. The rest of the sample collected from individual donors, (15 ml), must be mixed to
obtain a 750-ml composite sample that will be sent to WHO for its Human Milk Global Bank. The Bank’s
reserve will be used in the future if new POPs are added to the Stockholm Convention, as well as for
other scientific purposes. Countries with adequate resources may opt to obtain more than one
composite sample from the 50 individual samples; for example, one composite sample obtained with 25
individual samples. However, not all the composite samples need to contain 500 ml. If the country
wishes to analyze other contaminants in aliquots of the composite samples, the protocol for sample
collection will need to be modified accordingly (either to include more donors or to increase the volume
of each milk sample). More information is available at the WHO Reference Laboratory.
5.7 Laboratory testing and analytical quality
All the composite samples will be tested at the Laboratory for Dioxins and Agrotoxic Substances
of the "Chemisches and Veterinäruntersuchungsamt Freiburg" (CVUA: Community Reference
Laboratory for Chemical Testing of Foods of Animal Origin), in Freiburg, Germany. This laboratory was
appointed as a WHO Reference Laboratory for the study, having met all the requirements established by
WHO at the Fourth Round for the Interlaboratory Calibration of PCDD, PCDF and PCB levels in human
milk.
The Reference Laboratory follows a rigorous quality control program to guarantee the accuracy
and reliability of the results obtained in the study. The laboratory runs this program daily in the routine
testing for PCDD, PCDF and PCB and POP agrotoxics, primarily on foodstuff samples.
The elements below are part of the quality program:
Testing of reagent blank samples;
Testing of fortified samples (refined vegetal oil);
Testing of reference samples (known concentrations);
The analytical procedures for the PCDD/PCDF and PCB will be performed in accordance with
the procedure described by Malisch and Leeuwen (2002) 1 . Following freeze-drying, the sample will be
extracted with ethanol/toluene (70:30, v:v) for 8 hours in a "Twisselmann" extractor. The raw extract,
primarily consisting of fat, will be purified with ether and fortified with PCDD/PCDF and PCB in-house
standards labeled with 13 C. The fat from the extract will be removed using gel permeation
chromatography (Bio Beads S-X3). After treating the extract in a multilayer column (silica, silica/sulfuric
acid), the PCDD/PCDF and PCB will be separated in a Florisil column. The PCDD/PCDF fraction will be
treated in an activated charcoal column and later analyzed in a High Resolution Gas Chromatographer
1 Malisch, R; Leeuwen FXR. (2002). Organohalogen Compounds 56, 317-320.