The sampling campaign protocol was approved by the Regional Committee for Medical and Health Research Ethics in Norway (2013/1269).
The participants were contacted around two weeks before the date of the first visit, and PP pre-cleaned containers of different sizes were distributed to them, together with instructions on how to weigh and collect some of the samples. They were also requested to complete and return the following forms and questionnaires: a) Consent form to be signed prior to the collection of the samples, where the
participants agreed to the collection and use of these samples; b) Food diary (FD), containing detailed information about the individual food sample content (ingredient by ingredient), amount, packaging materials, cooking methods and utensils, etc. used for every meal collected during the two consecutive days of food sample collection period; c) Food frequency questionnaire (FFQ), containing generic information about the average consumption and frequency of a wide range of food items (n = 225) of the participants during the last whole year (Brantsæter et al., 2008); and d) Indoor environment questionnaire (IEQ), which collected information about house and other frequent indoor environments, lifestyle, habits, daily used products as well as basic personal information.
Finally, three appointments were scheduled with every participant. These comprised: a) two visits in two consecutive days to their homes to collect all the samples but blood; and b) one extra appointment with a research nurse at NIPH to collect blood samples.
3.2.1 Dust Samples
Participants were told when contacted to not discard their vacuum cleaner bags until the sampling appointment. During the second visit, the bags were collected from the vacuum cleaner by the researchers, wrapped in aluminium foil and kept in a plastic bucket at room temperature. In case the participants needed to replace the bag before the appointment, they had to wrap it themselves in aluminium foil and keep it at room temperature until its collection.
The bags were cut with scissors and tweezers and the dust was sieved with a 500 µm mesh sieve. 2 g of sieved dust were placed in 30 mL pre-cleaned PP containers and kept at 4 °C until they were shipped to Birmingham.
For blanks, six empty pre-cleaned 30 mL PP containers were sent to Birmingham.
Once there, 2 g of clean sodium sulphate were added to each bottle, shaken for 2 minutes and stored together with the dust samples. The purpose of these blanks was the identification of potential cross-contamination from the PP bottles, as well as from the storage places.
3.2.2 Food Samples
Food samples were divided in two groups: Solid and liquid.
The food sampling protocol was established following duplicate diet method (Shim, Oh and Kim, 2014) during a period of two consecutive days. In it, a duplicate portion of all food consumed by the participant over a specific period of time is weighed (by the use of a kitchen scale measuring ± 0.1 g), recorded in a food diary, and mixed. In this study, participants received instructions on how to collect the food samples, and four bottles were given to them: Two for solid food samples (relating to day 1 and day 2 of the sampling), and another two for the liquid food samples (also relating to day 1 and day 2 of the sampling); as well as two food diaries to complete relating to day 1 and day 2. Samples and food diaries were collected during the second visit to be homogenised within the following 24 hours.
All the solid food samples were weighed and homogenised in a food processor (Robot-coupe Blixer 3) for 2 minutes. Then, 100 g were weighed, placed in 250 mL pre-cleaned PP containers and stored at - 20 °C until they were shipped to Birmingham. All the liquid food samples were homogenised by shaking for 1 minute.
Then, 100 g were weighed and stored following the same procedure as solid food samples.
For solid food blanks, six pre-cleaned 2 L PP bottles were each filled with 100 g of diatomaceous earth, shaken for 1 minute, emptied into the food processor, homogenised for 2 minutes and placed in 250 mL pre-cleaned PP containers. The purpose of these blanks was the identification of potential cross-contamination from the food processor, as well as from the storage places.
For liquid food blanks, six empty cleaned 2 L PP bottles and six empty PP pre-cleaned 125 mL containers were sent to Birmingham. Once there, 100 mL deionised water were added to each 2 L PP bottle, shaken for 1 minute and placed in the 250 mL PP containers. The purpose of these blanks was the identification of potential cross-contamination from the PP bottles, as well as from the storage places.
3.2.3 Blood Samples
Blood samples were collected by a research nurse at the NIPH and placed in 10 mL plastic Vacutainer® serum tubes. The samples were left to coagulate for 1 hour and centrifuged at 2,500 rpm for 15 minutes. The supernatant serum fractions were transferred into 2 mL PP cryogenic vials and stored at - 80 °C until they were
shipped to Birmingham. Once there, the samples were kept at -20 °C until their analysis.
For blanks, six empty pre-cleaned 2 mL tubes were sent to Birmingham. Once there, 2 mL of calf serum were added to each tube, shaken for 2 minutes and stored together with the blood samples at - 20 °C. The purpose of these blanks was the identification of potential cross-contamination from tubes, as well as from the storage places.
3.2.4 Sample Storage
All the material used during the sampling campaign (including PP bottles and containers) was rinsed with methanol, dried at room temperature and kept closed or wrapped in aluminium foil until its use.
Dust samples were shipped to Birmingham at room temperature and stored at 4°°C until their analysis. Food samples were sent to Birmingham in expanded polystyrene containers filled with dry ice. Once in Birmingham, liquid food samples were placed in the freezer at – 20 °C until their analysis, while the solid food samples were weighed, freeze-dried, weighed again and homogenised with a coffee blender prior to storage at – 20 °C in 125 mL pre-cleaned glass containers until their analysis. Serum samples were sent to Birmingham in expanded polystyrene containers filled with dry ice. They were stored there at – 20 °C until their analysis.
All blanks were stored together with the corresponding samples.
All samples were removed from the fridge (4 °C) or freezer (- 20 °C) and allowed to reach room temperature before commencing sample treatment. Any remaining sample was replaced in the fridge or freezer until the end of the project.
The concentrated extracts to be injected into the high performance liquid chromatography coupled to tandem (triple quadrupole) mass spectrometer (HPLC-MS/MS(QqQ)) system were kept in low volume amber glass screw neck vials (200 µL) with screw neck cap and PTFE/silicone septum, purchased from Waters® Corp.
These vials were stored at – 20 °C before the analysis. Once the samples were analysed, the screw cap was replaced and the vials were stored back at – 20 °C until the end of the project.