2.4. Chemical Analysis and Quality Control

Non-fasting venous blood was drawn from each woman at the time of inclusion; 14 mL divided into two separate sterile BD vacutainer^® plastic tubes, one containing EDTA and the other containing heparin as anticoagulant. EDTA is the preferred anticoagulant for heavy metal analyzes, while the full blood in the heparin tube was intended for the OCP and PCB analyses. Each tube was gently inversed 5−6 times before placed in a cooling box at temperature of four degree Celsius (≤4.0 °C). Within 12 h, the blood in the EDTA tube was centrifuged at 2000 rpm for 15 min. The plasma was then transferred to a 12 mL (16 × 100 mm) cultured glass tube with cap. The whole blood sample from the heparin tube was transferred to a 12 mL cultured glass tube. All samples were stored in a freezer with temperature at minus 20 degree Celsius (−20 °C) until they were referred to the Pasteur Institute. The freezer was securely placed in a locked room at the Trauma Care Foundation head office in Battambang, Cambodia.

The plasma samples were transported to Pasteur Institute in Nha Trang, Vietnam, by TSP Express Cambodia Co. Ltd. on 28 October 2016. The whole blood samples, frozen and stored in cooling box, were taken to Pasteur Institute by car from Battambang by the research assistant.

The samples were analyzed at Nha Trang Pasteur Institute, Vietnam, for 18 PCBs and 19 organochlorine pesticides as well as the most relevant toxic and essential metals.

The chemical analysis was performed according to the international quality control system QA/QC established by the Centre de Toxicologie du Quebec (https://www.inspq.qc.ca/CTQ). Plasma samples were extracted by liquid–liquid extraction with ethanol, de-ionized water saturated with ammonium sulfate, and hexane. The OCs were separated from the lipids by a Florisil column manually packed with 3.0 g of 0.5% deactivated Florisil and 2 g of granulated sodium sulfate on top of the columns. The OCs were eluted with 11 mL hexane: dichloromethane (3:1 v/v). The collected fraction was evaporated to 0.5 mL by Centrivap Concentrator. The sample was evaporated to 200 μL before transfer to a gas chromatography (GC)-vial with an insert capillary, then reduced further to 20 μL, and octa-chloronaphthalene (OCN) was added as a recovery standard.

The gas chromatography (GC) was performed using a Shimadzu GC 2010 fitted with a Shimadzu AOC 20 S auto sampler (Injector: AOC 20 I) connected to a Shimadzu MS QP2010 plus spectrometer. A 30 m SLB−5 MS column (0.25 mm i.d. and 0.25 μm film thickness; Supelco) was used. The instrument was operated in the selected ion monitoring (SIM) mode, employing positive electron-impact ionization (EI+) as the source for PCB and OCPs.

Next, 13 C-labeled PCB 77 was used as an internal standard for PCB 28/31, PCB 33, PCB 52, PCB 74, PCB 95, PCB 101, and PCB 99. Similarly, 13 C-labeled PCB 118 was used as an internal standard for PCB 149, PCB 118, PCB 153, PCB 105, PCB 138, and PCB 158, as well as an internal standard for the rest of the PCBs. Finally, 13 C-labeled HCB was used as an internal standard for HCB, 13 C-labeled β –HCH for HCH, 13 C-labeled p,p’-DDT for DDT, and 13 C- labeled p,p’-DDE for the rest pesticides. The different compounds were identified from their SIM masses and retention times. For each analyte, the ratio of two masses was monitored. Peaks with differences in isotopic ratio greater than 20% compared to the quantification standard were rejected and not quantified. The samples were analyzed for 22 PCBs and 19 pesticides. For every 10 samples, a blank was analyzed to assess laboratory-derived (i.e., inadvertent) sample contamination. The method detection limit (LOD) was calculated as: LOD = Mean of blank concentration + 3 × SD of blank concentration.