AOAC Final Action Methods in 2018

Table 2014.08D. ( continued )

No. of laboratories No. of replicates

Mean concn, µg/kg

Mean recovery, %

PAH

s r , µg/kg s R , µg/kg RSD r , % RSD R , % HorRat

9 9 9 8 9 9 8 9 8 9

18 18 18 16 18 18 16 18 16 18

20.0 71.0

80.1 88.7 84.9 86.2 83.2 80.3 81.6 85.1 84.3 81.7

1.0 5.3 7.3 6.0 3.0 6.1 9.5 2.2 2.2 8.0

2.2 9.4

4.8 7.5 6.9 3.1 7.2 7.6 4.7 6.4 3.5 4.9

10.7 13.2 13.9 15.4 13.2 13.3 11.0

0.37 0.55 0.62 0.75 0.51 0.57 0.54 0.37 0.35 0.48

Naph

106.2 193.9

14.7 29.8

Phe

41.6 80.3

5.5

10.7 22.5

203.9

Pyr

34.0 63.2

3.3 5.3

9.8 8.4

163.4

16.6

10.2

( b ) Elute with 10 mL hexane–dichloromethane (3 + 1, v/v), collecting 0.5 mL elution fractions in 20 evaporation tubes or flasks. Add 0.5 mL isooctane to each elution fraction and evaporate down to 0.5 mL using the optimized evaporation conditions. Analyze each fraction by GC/MS. ( c ) Determine PAH elution profile by plotting analyte response (peak area or height) in a given fraction normalized to the sum of analyte responses in all tested fractions vs the elution volume. See Figure 2014.08B for an example of a PAH elution profile. It is recommended to add an additional 0.5 mL on top of the determined elution fraction (corresponding to 100% recovery) as a safety margin ensuring good analyte recoveries in routine practice. This would result in the optimum elution volume of 7 mL for the silica cartridge tested in Figure 2014.08B . ( d ) To check the effectiveness of fat removal, dissolve 100 mg pure fish oil (or any suitable fat) in 1 mL hexane and apply it onto the silica gel cartridge, which was conditioned with 6 mL hexane–dichloromethane (3 + 1, v/v) and 4 mL hexane. Elute with the optimum elution volume of hexane–dichloromethane (3 + 1, v/v), which was determined in the previous step (e.g., 7 mL for the example in Figure 2014.08B ). Collect this fraction in an evaporation tube or flask, which empty weight (after heating in an oven to remove moisture) was recorded to four decimal places

( 10 ) Condition a silica SPE column (1 g silica gel with approximately 0.2 g of muffled anhydrous sodium sulfate on the top) with 6 mL hexane–dichloromethane (3 + 1, v/v) and 4 mL hexane. ( 11 ) Apply the extract in hexane onto the silica SPE cartridge. ( 12 ) Elute with hexane–dichloromethane (3 + 1, v/v) using volume determined for the given silica gel SPE cartridges from the elution profiles of target analytes and fat, which are dependent on the silica deactivation, see Note ( 4 ) below. Collect the eluent. ( 13 ) Add 0.5 mL isooctane (and 1–2 mL ethyl acetate) to the eluent as a keeper and gently evaporate down to 0.5 mL to remove hexane and dichloromethane from the final extract. ( 14 ) Transfer the final extract into an autosampler vial for the GC/MS analysis. Notes : ( 1 ) The fat capacity of the 1 g silica gel SPE column is approximately 0.1 g. If the 5 mL ethyl acetate extract aliquot contains more than 0.1 g fat, it is necessary to use a smaller aliquot volume to avoid sample breakthrough during the cleanup step. ( 2 ) Ethyl acetate should not be present in the extract applied to the silica cartridge because it can affect the extract polarity, thus potentially retention of fat and analytes on the silica gel. The coextracted fat and 50 µL isooctane act as keepers during the first evaporation step (step 8 ), thus the evaporation should be conducted gently until there is no significant change in the volume, i.e., until only the isooctane and coextracted fat are left in the evaporation tube or flask. ( 3 ) Addition of 1–2 mL ethyl acetate to the eluent in step  13 is recommended for a better control of the evaporation process and higher absolute recoveries of volatile PAHs. ( 4 ) The deactivation and storage of silica gel SPE cartridges can vary, potentially resulting in different amounts of water in the silica, thus its potentially different retention characteristics. Therefore, it is important to test the elution profiles of PAHs and fat and determine the optimum volume of the elution solvent to ensure adequate analyte recoveries and fat cleanup. The following procedure is recommended: ( a ) Prepare a PAH solution in hexane by combining 50 µL of the Working PAH Solution A and 1 mL hexane in a vial. Mix well and apply onto a silica SPE column (1 g silica gel with approximately 0.2 g of muffled anhydrous sodium sulfate on the top), which was conditioned with 6 mL hexane–dichloromethane (3 + 1, v/v) and 4 mL hexane.

10 g of homogenized sample - Add 13 C -PAH mixture, vortex, equilibrate (15 min)

Extraction: - Add 5 mL (or 10 mL) water and 10 mL EtOAc, shake (1 min) - Add 4 g anh. MgSO4 and 2 g NaCl, shake (1 min), centrifuge - Evaporate 5 mL aliquot of extract, reconstitute in 1 mL hexane

Silica-SPE clean-up: - Condition 1g silica with 6 mL hexane:DCM (3:1, v/v ) and 4 mL hexane - Apply sample - Elute with 10 mL of hexane:DCM (3:1, v/v )

GC -MS(/MS) analysis

Figure 2014.08A. Flow chart of the method for determination of PAHs in seafood using GC/MS. Figu 2014.08A. Flow chart of the method for determination of PAHs in seafood using GC/MS.

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