# Development and Validation of an HPLC-UV Method for the Determination Bis(2-ethylhexyl) Phthalate Ester in Alcoholic Beverages

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## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

#### 2.1. Chemical and Reagents

#### 2.2. Instrumentation

#### 2.3. Procedure

^{2}coefficient > 0.999000 should be taken as a graph. All the above calculations and diagrams are conducted with the Chemstation software. Next is the analysis of the sample; the solution we prepared in part A is injected into the liquid chromatograph with a syringe through the instrument’s inlet. The result given by the liquid chromatograph is expressed in mg/L with one decimal place.

#### 2.4. Method Validation

#### 2.4.1. Specificity

- In a real sample of Ouzo, 4 independent spikes of DEHP were made at concentrations of 0.3, 0.7, 1.1, and 1.5 mg/L and measured following the entire analytical procedure. Additionally, unspiked “ouzo” was measured. The spike was conducted by adding a small volume of a dense standard to avoid dilution of the sample.
- In pure solvent ethyl alcohol 40% v/v, 4 independent spikes of DEHP were made at concentrations of 0.3, 0.7, 1.1, and 1.5 mg/L and were measured following the entire analytical procedure. The pure solvent without spike was also measured.

#### 2.4.2. Linearity

#### 2.4.3. Limit of Detection (LOD) and Limit of Quantification (LOQ)

#### 2.4.4. Accuracy

#### 2.4.5. Precision

#### 2.4.6. Range

#### 2.4.7. Ruggedness

#### 2.5. Assessment of Uncertainty

_{bias,N}: the relative standard uncertainty by NORDTEST of the systematic errors (method precision). The recovery data were used to calculate it. u

_{(Rw)}: the relative standard uncertainty from intra-laboratory reproducibility. For its calculation, the data of the control sample were used [13,14].

## 3. Results and Discussion

#### 3.1. Specificity

#### 3.1.1. Elution Time and Separation Capacity of DEHP

#### 3.1.2. Ouzo Matrix Effect Check

#### 3.2. Linearity

#### 3.2.1. Statistical Data Checks

#### 3.2.2. Simple Linear Regression

#### 3.2.3. Linearity Checks (Linear Range)

#### 3.3. Limit of Detection (LOD) and Limit of Quantification (LOQ)

#### 3.4. Accuracy

_{0}= 0.62 mg/L DEHP.

_{i}(y-axis), are presented in terms of the corresponding known concentrations of the samples, μ

_{i}(x-axis). Additionally, the regression line is plotted using the least squares method. Finally, with the help of Excel we obtain the regression analysis table, a part of which is shown in Figure 8.

- For the slope b = 0.9875: H
_{0}: b = 1 vs. H_{A}: b ≠ 1, to ascertain whether the value of b is statistically equal to monad, the 95% confidence interval of b should contain monad. From the regression analysis table, Figure 8 (below), CI 95% = (0.863; 1.112). Since CI includes monad we accept the null hypothesis, H_{0}: b = 1. Therefore, we conclude that there is no “proportional” systematic error. - For the intercept a = 0.0315: H
_{0}: a = 0 vs. H_{A}: a ≠ 0, to ascertain whether the value of a is statistically equal to zero, the 95% confidence interval of a should contain zero. From the regression analysis table, Figure 8 (below), CI 95% = (−0.167; 0.230). Since CI includes zero, we accept the null hypothesis, H0: a = 0. Therefore, we conclude that there is no “standard” systematic error [9].

#### 3.5. Precision

- Repeatability:
- −
- Standard deviation$:{\mathrm{s}}_{\mathrm{r}}=\sqrt{{\mathrm{s}}_{\mathrm{error}}{}^{2}}=\sqrt{0.00061}=0.02470\mathrm{mg}/\mathrm{L}$.
- −
- Relative standard deviation, from equation ${\mathrm{RSD}}_{\mathrm{r}}=\frac{{\mathrm{s}}_{\mathrm{r}}}{\overline{\mathrm{x}}}100$ = 2.3%, as $\overline{\mathrm{x}}$ = 1.07 mg/L the average of the measurements in Table 7.
- −
- Limit of repeatability, from equation $\mathrm{r}=2.8\times {\mathrm{s}}_{\mathrm{r}}$: r
_{s}= 0.069 mg/L, for two repeated measurements under repeatability conditions and 95% significance level.

- Intermediate precision:
- −
- Standard deviation:${\mathrm{s}}_{\mathrm{I}}=\sqrt{{\mathrm{s}}_{\mathrm{day}}{}^{2}+{\mathrm{s}}_{\mathrm{error}}{}^{2}}=\sqrt{0.00057+0.00061}=0.0344$ mg/L.
- −
- Relative standard deviation, from equation ${\mathrm{RSD}}_{\mathrm{I}}=\frac{{\mathrm{s}}_{\mathrm{I}}}{\overline{\mathrm{x}}}100$ = 3.2%, as $\overline{\mathrm{x}}$ = 1.07 mg/L the average of the measurements in Table 7.
- −

_{r}and RSD

_{I}values satisfy the acceptable values predicted based on the concentration level of DEHP$\left(\overline{\mathrm{x}}=1.07\frac{\mathrm{mg}}{\mathrm{L}}\right)$ [16]. Additionally, it is worth noting that there does not seem to be any variation in the measurements between the different analysis days. This is proven statistically, for a significance level of 95%, by the p-value = 0.082 > 0.05, which is shown in Table 6.

ANOVA: DEHP mg/L versus Day |
---|

Factor Type Levels Values |

day random 8 1; 2; 3; 4; 5; 6; 7; 8 |

Analysis of Variance for DEHP mg/L |

Source DF SS MS F P |

day 7 0.0122750 0.0017536 2.86 0.082 |

Error 8 0.0049000 0.0006125 |

Total 15 0.0171750 |

S = 0.0247487 R-Sq = 71.47 R-Sq(adj) = 46.51% |

Expected Mean |

Square for Each |

Term (using |

Variance Error unrestricted |

Source component term model) |

1 day 0.00057 2 (2) + 2 (1) |

2 Error 0.00061 (2) |

#### 3.6. Range

#### 3.7. Ruggedness

**Table 7.**Experimental design of five-parameter two-level factors, to evaluate the ruggedness of method (according to Taguchi experimental design, seven-parameter two-level factors).

Experiment | Factors | mg/L DEHP | |||||||
---|---|---|---|---|---|---|---|---|---|

A | B | C | D | E | F | G | |||

1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1.07 | 1.03 |

2 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | 1.00 | 1.02 |

3 | 1 | 2 | 2 | 1 | 1 | 2 | 2 | 1.03 | 1.03 |

4 | 1 | 2 | 2 | 2 | 2 | 1 | 1 | 1.00 | 1.05 |

5 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1.07 | 1.04 |

6 | 2 | 1 | 2 | 2 | 1 | 2 | 1 | 1.04 | 1.06 |

7 | 2 | 2 | 1 | 1 | 2 | 2 | 1 | 1.05 | 1.07 |

8 | 2 | 2 | 1 | 2 | 1 | 1 | 2 | 1.09 | 1.03 |

#### 3.8. Assessment of Uncertainty

#### 3.8.1. Calculation u_{bias,N}

_{bias,N}, is calculated from Equation (4) [14]:

_{conc}: A certified DEHP reference standard was used for spike, for which the certificate gave purity = 99.90%, and uncertainty a = ±0.05% for a significance level of 95%. In these cases, assuming a normal distribution, the standard uncertainty is given by Equation (5):

_{vol}: A volumetric flask (class A) V

_{bottle}= 100 mL was used for the spike of the sample, which according to the manufacturer has a tolerance of a = ±0.08 mL. The standard uncertainty due to the tolerance, assuming a quadratic distribution, is:

^{−4}°C

^{−1}[22]. Therefore, the change in volume of liquid contained in the bottle is:

^{−4}) = 0.26 mL

_{pipette}= 0.050 mL were transferred, which according to the manufacturer have a tolerance of a = ±0.001 mL. Thus, the standard uncertainty of the pipette volume, assuming a quadratic distribution, is:

_{vol}is:

#### 3.8.2. Calculation u(R_{w})

_{w}), was calculated using the data collected from the control sample measurements (a total of 36 measurements over one year on the same control sample). From these data, $\overline{\mathrm{x}}$ = 0.9258 mg/L DEHP and s = 0.017948 mg/L DEHP. Therefore, the relative standard uncertainty from the intra-laboratory reproductivity, u(R

_{w}), will be equal to the relative standard deviation, expressed as a % [14]:

_{w}) = 1.9386144%

#### 3.8.3. Calculation of Combined Relative Uncertainty by NORDTEST

_{bias,N}and u(R

_{w}), calculated above, Equation (1) gives the uncertainty according to NORDTEST [14]:

_{c}= 7.9% ή 0.079

## 4. Conclusions

## Author Contributions

## Funding

## Institutional Review Board Statement

## Informed Consent Statement

## Data Availability Statement

## Conflicts of Interest

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**Figure 1.**Chromatogram, HPLC, of a DEHP standard solution of concentration 0.7 mg/L, to check the specificity of the method.

**Figure 2.**Chromatogram, HPLC, pure ethyl alcohol solvent 40%vol, to check the specificity of the method.

**Figure 5.**Calibration curve for DEHP determination method. The diagram shows: the 95% confidence and prediction intervals and the coefficient of determination, r

^{2}.

**Table 1.**Factors and levels studied during the evaluation of the ruggedness of the DEHP determination method in Ouzo.

Factor | Levels | ||
---|---|---|---|

Regular Value | Deliberate Change | ||

1 | 2 | ||

A | Concentration of methanol solution in acetonitrile | 1.00% | 0.99% |

Β | Gradient of mobile phase solvents: A: Ultrapure water B: Acetonitrile with 1% methanol | −3 min A: 37.5%, B: 62.5% −10 min A: 0%, B: 100% −20 min A: 0%, B: 100% −22 min A: 37.5%, B: 62.5% | −3 min A: 36.5%, B: 63.5% −10 min A: 1%, B: 99% −20 min A: 1%, B: 99% −22 min A: 36.5%, B: 63.5% |

C | Mobile phase flow rate | 1.00 mL/min | 0.98 mL/min |

D | Column oven temperature | 25.0 °C | 25.5 °C |

E | Wave length | 225 nm | 226 nm |

Experiment | Parameter | ||||||
---|---|---|---|---|---|---|---|

A | B | C | D | E | F | G | |

1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |

2 | 1 | 1 | 1 | 2 | 2 | 2 | 2 |

3 | 1 | 2 | 2 | 1 | 1 | 2 | 2 |

4 | 1 | 2 | 2 | 2 | 2 | 1 | 1 |

5 | 2 | 1 | 2 | 1 | 2 | 1 | 2 |

6 | 2 | 1 | 2 | 2 | 1 | 2 | 1 |

7 | 2 | 2 | 1 | 1 | 2 | 2 | 1 |

8 | 2 | 2 | 1 | 2 | 1 | 1 | 2 |

A’ part | Concentration Level | Duplicate Per Level | DEHP Concentration, mg/L | Area |

1 | 1 | 0.3 | 34.65 | |

1 | 2 | 0.3 | 41.72 | |

1 | 3 | 0.3 | 39.98 | |

2 | 1 | 0.7 | 96.47 | |

2 | 2 | 0.7 | 99.12 | |

2 | 3 | 0.7 | 95.86 | |

3 | 1 | 1.1 | 148.80 | |

3 | 2 | 1.1 | 153.69 | |

3 | 3 | 1.1 | 150.97 | |

4 | 1 | 1.5 | 205.14 | |

4 | 2 | 1.5 | 207.12 | |

4 | 3 | 1.5 | 203.34 | |

B’ part | SUMMARY | |||

DEHP Concentration, mg/L | Mean Value Area | Standard Deviation, s | RSD,% | |

0.3 | 38.78 | 3.684 | 9.498 | |

0.7 | 97.15 | 1.733 | 1.784 | |

1.1 | 151.15 | 2.450 | 1.621 | |

1.5 | 205.20 | 1.891 | 0.921 |

X_{i,sp} | Known Concentration, μ _{i} (μ_{i} = x_{i}, sp + x_{0}, x_{0} = 0.62) | Method under Validation, x_{i} |
---|---|---|

0.3 | 0.92 | 0.94 |

0.7 | 1.32 | 1.35 |

1.1 | 1.75 | 1.70 |

1.5 | 2.12 | 2.14 |

Day: | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|

DEHP mg/L: | 1.11 | 1.05 | 1.09 | 1.06 | 1.04 | 1.01 | 1.07 | 1.09 | 1.05 | 1.03 | 1.10 | 1.06 | 1.10 | 1.14 | 1.04 | 1.06 |

Analysis of Variance |
---|

Source DF Adj SS Adj MS F-Value p-Value |

A 1 0.003025 0.003025 5.48 0.041 |

B 1 0.000025 0.000025 0.05 0.836 |

C 1 0.000100 0.000100 0.18 0.680 |

D 1 0.000625 0.000625 1.13 0.313 |

E 1 0.000400 0.000400 0.72 0.415 |

Error 10 0.005525 0.000553 |

Lack-of-Fit 2 0.000625 0.000313 0.51 0.619 |

Pure Error 8 0.004900 0.000613 |

Total 15 0.009700 |

**Table 9.**DEHP recovery measurements in Ouzo, in the context of internal quality control of the accuracy of the method.

Measurement | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |

recovery Ri% | 93 | 94 | 93 | 91 | 95 | 91 | 92 | 97 | 95 | 92 | 91 | 91 |

Measurement | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | |

recovery Ri% | 93 | 94 | 91 | 90 | 94 | 91 | 92 | 92 | 93 | 94 | 91 |

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**MDPI and ACS Style**

Gemenetzis, E.G.; Alygizakis, N.A.
Development and Validation of an HPLC-UV Method for the Determination Bis(2-ethylhexyl) Phthalate Ester in Alcoholic Beverages. *Appl. Sci.* **2023**, *13*, 3194.
https://doi.org/10.3390/app13053194

**AMA Style**

Gemenetzis EG, Alygizakis NA.
Development and Validation of an HPLC-UV Method for the Determination Bis(2-ethylhexyl) Phthalate Ester in Alcoholic Beverages. *Applied Sciences*. 2023; 13(5):3194.
https://doi.org/10.3390/app13053194

**Chicago/Turabian Style**

Gemenetzis, Evangelos G., and Nikiforos A. Alygizakis.
2023. "Development and Validation of an HPLC-UV Method for the Determination Bis(2-ethylhexyl) Phthalate Ester in Alcoholic Beverages" *Applied Sciences* 13, no. 5: 3194.
https://doi.org/10.3390/app13053194