*2.5. Enzymatic Method Determination of Acetic Acid Content*

The method reported in the kit instruction (Enzytec acetic acid) was followed. The Enzytec fluid Acid combination Standard (ID-No 5460, 3 × 3 mL) was used to calibrate the automated photometric systems.

### *2.6. Spectrophotometric Method Validation Parameters*

The linearity, precision, sensitivity, and measurement uncertainty were determined.

In the linearity assessment, a matrix-match calibration curve was plotted at 0, 0.06, 0.13, 0.25, and 0.50 g/L for acetic acid.

The method precision was tested, performing ten analyses of the same sample

$$\text{Reprodability} = \frac{\text{Standard deviation of analyzed samples}}{\text{Standard deviation of reference samples}} \tag{1}$$

Normality, by the Shapiro–Wilk test [13], and the presence of anomalous data, by the Huber test [14], were evaluated to define the method's precision.

The method sensitivity was evaluated by LLOQ (limit of quantification: LOQ = 10σS) and LLOD (limit of detection: LOD = 3.3σS) determinations, where σ is the relative standard deviation and S is the slope of the standard curve.

The method accuracy was determined, performing ten analyses with both methods (Community [4] and enzymatic), determining the residual distribution by the Saphiro–Wilk test, and controlling for anomalous data by *t*-tests.

Type A and B uncertainties were measured following the EURACHEM/CITAC guide [15]. Type A was estimated from 10 repeated readings of the same sample.

$$\text{U Type A} = \sqrt{\frac{\text{variance}}{\text{Degrees of freedom}}} \tag{2}$$

Type B was determined with a metrology approach.

U(t) is the uncertainty associated with 20 mL pipette use U(t). It was obtained considering a certificate of calibration (0.016 mL) and repeatability (0.00050 mL).

U(p) is the uncertainty associated with 10 mL pipette use. It was obtained considering a certificate of calibration (0.096 mL) and repeatability (0.00020 mL).

U(ct) is the uncertainty associated with the calibration curve. It was obtained for the standard, which was measured at three concentrations in triplicate.

$$\text{U(ct)}\,\text{S}\frac{\text{x/y}}{b}\*\sqrt{1/\text{n}+1/\text{m}}\tag{3}$$

S = standard deviation of the residual

n = points used for the calibration line

m = readings taken for each sample

U(mr) is the uncertainty associated with a standard preparation.

U(bt) is the uncertainty associated with balances. It was determined considering a certificate of calibration (0.00060 g), repeatability (0.000029 g), and stability (0.000032 g).

U(m) is the uncertainty associated with the use of a 100 mL flask. It was obtained considering a certificate of calibration (0.01 mL) and repeatability (0.00030 mL).

*Foods* **2020**, *9*, 761

U(k) is the uncertainty associated with the use of a 250 mL flask. It was obtained considering a certificate of calibration (0.025 mL) and repeatability (0.00040 mL).

2

Compound uncertainty: <sup>q</sup> (*U Type A*) <sup>2</sup> + (*U Type B*) The accuracy was tested by Student's *t* test:

$$\text{Accuracy} = \frac{\left| \overline{\mathbf{X}}\_{\text{Commutality}} - \overline{\mathbf{X}} \right|}{\sqrt{\mathbf{S}\_{\text{r}}^{2} + \mathbf{U}\_{\text{Commutity}}^{2}}} \le t\_{\text{PV}} \tag{4}$$

X = Community method value Xsy/<sup>x</sup> = medium repeatability values S 2 <sup>r</sup> = standard deviation<sup>2</sup> U 2 CRM <sup>=</sup> reference material uncertainty<sup>2</sup> Uncertainty (*t*pν) = k × σr.

#### *2.7. Statistical Analysis*

The data were analyzed using the software Statistica Version 7.0 (StatSoft, Hamburg, Germany). The normality of the data was verified by applying the Shapiro–Wilk *W*-test. A *p*-value of >0.05 indicates a normal distribution. The non-parametric Huber test determined the outliers.

#### **3. Results**

#### *3.1. Method Linearity*

The method linearity, by regression coefficient determination (Figure S1), and residuals distribution, by ANOVA tests (Figure S2), were evaluated. R<sup>2</sup> = 0.99; the residuals were normally distributed.

#### *3.2. Method Sensitivity*

The method detection limit was tested by repeated analyses of blank samples. The LLOD and LLOQ were derived from the regression curve. The quantitation limit (LLOD) of an individual analytical procedure is the lowest amount of acetic acid in a sample that can be quantified with suitable precision and accuracy. The lower limit of detection was 0.0063 g/L. The detection limits (LLOQ) were determined as the concentration giving a peak height three times the background noise. The Lower Limit of Quantitation was 0.0253 g/mL. The LLOQ dilution factor was used to determine the lower end of the measuring range. It was obtained by dividing the (read concentration × 10) / (weight × rate) (0.098 g/100 mL). The last point of the calibration curve line was the upper end of the measuring range (0.40 g/100 mL).

#### *3.3. Measurement Uncertainty*

Type A uncertainty due to method repeatability was 0.005 (Table S1). Type B uncertainties—due to method repeatability and associated with the standard preparation, the calibration curve, the balances, the flasks (100 mL and 250 mL), and the pipettes (10 mL and 20 mL)—were less than 10% of the results (Table S2).
