Following common protocol for the extraction of absorbed residues [2 (link)], each ceramic sample was first ‘cleaned’ by removing the outer layer of the ceramic surface before collecting approximately 2 g of ceramic powder by drilling into the pot. An acidified methanol extraction (AE) method was applied to all samples following common procedure [32 (link)]. In brief, a mixture of methanol (4 ml) and sulphuric acid (800 µl) was added to 1 g of ceramic powder alongside an internal standard (10 µl of C34:0) before heating at 70°C for 4 h. After centrifuging, the supernatant was extracted and transferred to a clean labelled hatch tube. The lipid extract was separated from the acid by adding 2 ml of n-hexane, removing the supernatant and passing through a filter pipette to neutralize the sample with potassium carbonate (K2CO3). This process was repeated three times. The samples were dried under a gentle stream of nitrogen, and then dissolved in n-hexane and transferred to an auto-sampling vial with a micro insert. A second internal standard (10 µl of C36:0) was added before analysis by GC techniques.
A solvent extraction (SE) was undertaken on the majority of samples to extract and identify tri-, di- and mono-acylglycerols and/or wax esters, which do not remain intact after AE. Briefly, a mixture of dichloromethane-methanol (DCM 2 : 1, v/v) was added to the remaining 1 g of ceramic sample. The samples were sonicated for 15 min and then centrifuged. The supernatant was transferred to a clean labelled hatch tube. These steps were performed three times. The solution was then dried to completion under a gentle stream of nitrogen.
The ceramic powder remaining after SE was then submitted to acidified butanol extraction (ABE) [15 (link),16 (link)]. Here, 2 ml of n-hexane and 1 ml of boron trifluoride solution (BF3) in butanol were added to the sample. After heating for 2 h at 80°C, the samples were centrifuged. The supernatant was then transferred to a clean labelled hatch tube and neutralized using a saturated solution of sodium carbonate. To ensure no more sample was trapped in the ceramic powder, 2 ml of DCM was added to the sample, vortexed, centrifuged and the supernatant transferred to the same extract. After centrifugation, the organic phase was transferred to a new clean labelled hatch tube. The aqueous phase was extracted twice more with DCM. The combined organic phases were washed twice with water, and dried under nitrogen. The dried extracts from SE and ABE were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethyl-chlorosilane and heated for 1 h at 70°C. n-hexane was added to redissolve the extract before transferring to an auto-sampling vial which contained 10 µl of the second internal standard C36:0 for GC analysis.
A solvent extraction (SE) was undertaken on the majority of samples to extract and identify tri-, di- and mono-acylglycerols and/or wax esters, which do not remain intact after AE. Briefly, a mixture of dichloromethane-methanol (DCM 2 : 1, v/v) was added to the remaining 1 g of ceramic sample. The samples were sonicated for 15 min and then centrifuged. The supernatant was transferred to a clean labelled hatch tube. These steps were performed three times. The solution was then dried to completion under a gentle stream of nitrogen.
The ceramic powder remaining after SE was then submitted to acidified butanol extraction (ABE) [15 (link),16 (link)]. Here, 2 ml of n-hexane and 1 ml of boron trifluoride solution (BF3) in butanol were added to the sample. After heating for 2 h at 80°C, the samples were centrifuged. The supernatant was then transferred to a clean labelled hatch tube and neutralized using a saturated solution of sodium carbonate. To ensure no more sample was trapped in the ceramic powder, 2 ml of DCM was added to the sample, vortexed, centrifuged and the supernatant transferred to the same extract. After centrifugation, the organic phase was transferred to a new clean labelled hatch tube. The aqueous phase was extracted twice more with DCM. The combined organic phases were washed twice with water, and dried under nitrogen. The dried extracts from SE and ABE were derivatized by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethyl-chlorosilane and heated for 1 h at 70°C. n-hexane was added to redissolve the extract before transferring to an auto-sampling vial which contained 10 µl of the second internal standard C36:0 for GC analysis.
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