*2.8. Validation Method*

NPs are frequently regarded as a single compound when their environmental occurrence, transit, removal, and toxicity are investigated. However, technical NPs comprise a mixture of over 100 isomers and congeners with different side chain lengths and branches [15]. The NP chromatogram presents more than 10 isomer peaks with various nonyl substituent branching configurations. Due to the characteristic of NPs being present in more than 100 isomers, some of the individual peaks can contain several isomers and are, therefore, not pure compounds. Furthermore, their compositions vary between manufacturers, and the concentration and exact type of each isomer are unknown [16]. The concentration and type for each isomer in standard branched NP chemicals used for the present investigation were also not identified.

Individual NPs are not commercially accessible, and the standard chemical is a mixture of isomers with alkyl groups with varying branches. Validation for the analysis and calibration curve, therefore, proceeded with 4-n-NP, a single linear compound whose concentration can be accurately determined, instead of branched NPs. The proposed analytical method was validated in terms of its linearity, accuracy, precision, and repeatability.

For high-fat matrices, the linearity of the calibration curve was determined at five points (*<sup>n</sup>* = 3), with concentrations of 4-n-NP ranging from 1.00 × <sup>10</sup><sup>2</sup> to 2.00 × 103 ng/mL at five points (1.00 × 102, 2.00 × 102, 5.00 × <sup>10</sup>2, 1.00 × 103, and 2.00 × 103 ng/mL), while for low-fat matrices, it ranged from 1.00 × 101 to 1.00 × 103 ng/mL at five points (1.00 × 101, 2.00 × 101, 1.00 × 102, 5.00 × <sup>10</sup>2, and 1.00 × <sup>10</sup><sup>3</sup> ng/mL). A calibration curve was obtained using the peak area and concentration ratio of 4-n-NP to 4-n-NP 13C6.

To determine the sensitivity of the method, the limit of detection (LOD) and limit of quantification (LOQ) were determined as follows:

$$\text{LOD} = 3.3 \sigma/\text{S LOQ} = 10 \sigma/\text{S}$$

where S is the standard curve slope and σ is the RSD of the y-intercept.

Accuracy and precision were calculated as a percentage of spiked 4-n-NP in each matrix, and then compared with the AOAC guideline standard value (AOAC, 2012). Internal standards as well as 4-n-NP at three different concentrations were added to each sample. Intraday and interday precision and accuracy were validated by determining analytes in three replicates at the nominal concentrations on three consecutive days. The nominal concentrations were 5.00 × <sup>10</sup>2, 1.00 × <sup>10</sup>3, and 2.00 × 103 ng/mL for the highfat matrices, and 1.00 × 102, 5.00 × 102, and 1.00 × 103 ng/mL for the low-fat matrices. Precision was calculated as the RSD by dividing the standard deviation by the mean, while measurement accuracy was determined by expressing the calculated concentration as a percentage of the nominal concentration.
