Abstract

A square wave voltammetric method using a carbon fiber microelectrode (CFME) in 0.1 M phosphate-buffered saline (PBS) was developed and fully validated for the quantitative determination of fenitrothion (FNT) residues in soil, according to EURACHEM/CITAC guidelines and ISO/IEC 17025 requirements. FNT exhibited an irreversible diffusion-controlled cathodic peak at −1.02 V vs. saturated calomel electrode (SCE). Method validation supported by statistical analyses, including ANOVA, was employed. Key parameters such as pH, pulse amplitude, frequency, and step increments influencing the cathodic peak were optimized. Among the tested interferents, only 3-methyl-4-nitrophenol (MNP) significantly affected FNT detection, while no matrix effects were observed. The calibration curve was assessed using linear regression, which showed that the optimal line of best fit minimizes the sum of squared residuals. The calibration curve was linear in the range of 10 - 200 μg·L⁻¹ with R² = 0.9997. Recovery rates varied from 89.12% to 94.76%, with relative standard deviations (RSDs) between 1.19% and 1.47%. The method precision was confirmed with within-day and between-day RSDs of 1.404% and 1.579%, respectively. Good reproducibility was observed through comparative analysis by gas chromatography with a Nitrogen Phosphorus Detector (GC-NPD). LOD and LOQ were estimated and were close to 6.56 μg·kg⁻¹ and 19.87 μg·kg⁻¹, respectively. The FNT stock solution remained stable for at least three months at 7˚C. Measurement uncertainty was estimated as U = 0.04399 × C using the GUM approach. This validated electrochemical method offers a simple, accurate, and reliable approach suitable for environmental monitoring of FNT residues in soil.

Keywords

Fenitrothion, Carbon Fiber Microelectrode, Method Validation, Soil, ANOVA

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1. Introduction

Organophosphorus pesticides (OPs) are among the most widely used pesticides in agriculture for insect control because of their high toxicity to insects and limited persistence in the environment [1]. Fenitrothion (FNT) (O, O-dimethyl O-4-nitro-m-tolyl phosphorothionate) (Figure 1) is a contact insecticide and selective acaricide belonging to the organophosphate family [2].

Figure 1. Structure of fenitrothion [1].

In soil, it can evolve through biotic and abiotic processes, producing metabolites, of which the most stable and toxic one is 3-methyl-4-nitro phenyl (MNP) [3]. As an organophosphate pesticide, the principal function of FNT is to inhibit the activity of acetylcholinesterase [4] [5]. Consequently, the presence of pesticide residues and metabolites in food, water, and soil currently represents a major issue in environmental chemistry [6].

Owing to its potential toxicity to non-target organisms and the persistence of its fate in the environment, monitoring FNT residues in soil is crucial for environmental and food safety [7].

Therefore, the rapid and sensitive detection of FNT residues is essential for ensuring environmental protection and public health.

Conventional methods for the analysis of FNT residues in environmental matrices include chromatographic methods combined with specific detectors and spectroscopic methods [8]-[11]. These methods have high sensitivity and are suitable for laboratory detection but require expensive equipment and time-consuming sample pretreatment [12].

Electrochemical detection technology has the advantages of high sensitivity, accuracy, portability, simple operation, fast analysis, and low cost; thus, it has gradually become a research hotspot in the field of pesticide detection [12] [13].

Although numerous electroanalytical methods for FNT in various matrices have been published [6] [12] [14], most report only linearity, accuracy, reproducibility, and selectivity/specificity, omitting ruggedness testing, formal uncertainty estimation, matrix effects, and the stability of stock solution evaluation required by ISO/IEC 17025. Furthermore, no statistical test was used to demonstrate the validity of the evaluated parameters. This incomplete validation limits their adoption for routine regulatory monitoring.

For example,

• Xinsheng Liu et al., 2023 [12] developed a non-enzymatic electrochemical sensor based on nitrogen-doped mesoporous carbon@hydroxyl functionalized ionic liquid composites modified electrode for the detection of fenitrothion in food. The performance of the method was validated using key parameters such as linearity, accuracy, reproducibility, stability of the sensor, selectivity/specificity, and limits of detection and quantification. However, critical parameters like matrix effects, ruggedness of the method, stability of stock solution over time, and measurement uncertainty, required by ISO/IEC 17025, were not evaluated. Moreover, no statistical test was used to demonstrate the validity of the studied parameters;

• Mir Reza Majidi et al., 2009 [14] validated a quantitative electrochemical method for the determination of Fenitrothion in river water and commercial formulations by adsorptive stripping voltammetry with a carbon ceramic electrode. Despite linearity, repeatability, selectivity, accuracy, and reproducibility in gas chromatography, limits of detection and quantification were evaluated to demonstrate the fit for purpose; key parameters such as ruggedness, stability of stock solution over time, and measurement uncertainty, required by ISO/IEC 17025, were not assessed. Moreover, no statistical test was used to confirm the validity of the evaluated parameters;

• Tefera M. et al., 2015 [6] validated an electrochemical method for the quantitative analysis of FNT residues in soil using a sensor based on a Multi-wall Carbon Nanotubes Modified Glassy Carbon Electrode. The fitness for purpose of the method was evaluated by means of key parameters such as linearity, repeatability, reproducibility, accuracy, selectivity, and limits of detection and quantification.

Despite these parameters demonstrating the fit for purpose of the method, critical parameters like ruggedness, measurement uncertainty, and stability of stock solution over time, required by ISO/IEC 17025, were not evaluated. Furthermore, statistical tests were not used to justify the validity of the evaluated parameters.

Carbon fiber microelectrodes (CFMEs), owing to their small size and resistance to fouling, have demonstrated superior sensitivity and reduced matrix effects for organophosphates in complex environmental samples [15]-[18].

However, no fully validated CFME-based method for FNT in soil has been reported. Without such validation, laboratories cannot adopt CFME methods for regulatory compliance under ISO/IEC 17025, forcing reliance on more expensive and time-consuming chromatographic techniques.

According to the ISO 17025 (2017) requirements, “The laboratory shall validate non-standard methods, laboratory-developed methods, and standard methods used outside their intended scope or otherwise modified. The validation shall be as extensive as necessary to meet the needs of the given application or field of application” [19].

The objective of this study was to develop and validate an electrochemical method using a carbon fiber microelectrode for the quantitative analysis of FNT residues in soil using a single-laboratory approach in accordance with the requirements of ISO/IEC 17025 and EURACHEM/CITAC recommendations [20].

2. Experimental

2.1. Chemicals and Standards

Carbon fibers (diameter: 12 μm, length: 5 mm) were purchased from Cytec Engineered Materials (West Paterson, NJ, USA) and used for CFME elaboration. Standards of FNT (97%), MNP (98%), potassium ferrocyanide (99%), potassium mono- and dihydrogen phosphate (99%), sulfuric acid (98%), and sodium hydroxide (98%) were purchased from Sigma-Aldrich. Absolute ethanol (99.9%) and acetonitrile (99.9%) were purchased from AnalaR NORMAPUR. All solutions were prepared using deionized water (pH 6.5, conductivity < 0.1 μS/cm, and DOC < 0.1 mg/L).

2.2. Apparatus

A helical steel auger with 99% screw-on tools was employed for neutral soil sampling. A RETSCH brand analytical sieve with a 75 µm mesh, certified according to ISO 3310/1 standards, was utilized for sieving neutral soil samples. An OHAUS brand analytical balance with a precision of 10⁻⁴ g was employed to weigh the samples. For the separation of liquid and solid phases following extraction, a CU-5000 IEC brand centrifuge was used. Extracts were concentrated using a BüCHI brand R-200 and B-490 rotary evaporator.

A Rotating Tubular Divider TT-RSD200 brand was used for sampling replicates of blank soil and spiked soil samples.

An Agilent Technologies chromatograph equipped with an Agilent J&W HP-5 column (30 m × 0.32 mm × 0.25 µm) and a Nitrogen Phosphorus Detector was utilized for reproducibility assessment. GC-NPD conditions:

Injection volume 1 µL, splitless mode at 150˚C (held for 1 min), ramped at 10˚C/min to 260˚C; temperature of the detector 310˚C, carrier gas He at 1.5 mL/min. FNT retention time: 13.348 min.

A PalmSens Instrument PC233 Potentiostat (Netherlands), controlled using PalmSensVs1.60 software, was used to carry out voltammetric measurements.

PSLITE 1.7.3 software was used to control data management.

2.3. Analysis

Stock solution (1 g·L⁻¹) of FNT was prepared by dissolving the equivalent mass of FNT in absolute ethanol. Working solutions of FNT at 10 mg/L were then prepared by suitable dilution. 0.1 M PBS (pH 7.2) was used as the supporting electrolyte.

Electrochemical experiments were performed using square wave voltammetry (SWV), which offers higher sensitivity and faster scan rates than differential pulse voltammetry, making it suitable for trace-level FNT detection in complex matrices [15].

Measurements were performed using a PalmSens 3 type potentiostat (Netherlands) connected to a personal computer using Ivium PC and PSLite software. A three-electrode configuration was employed, consisting of a naked carbon fiber microelectrode as the working electrode (CFME) (diameter Φ = 12 μm), a saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the auxiliary electrode. The carbon electrode surface was renewed using a homemade electrochemical treatment. Indeed, the carbon fiber microelectrode was first pretreated electrochemically in a mixture of sulfuric acid H₂SO₄ (0.5 M)/ethanol (50/50 w/w) followed by a treatment in 0.1 M Phosphate Buffered Saline (PBS) pH = 7.2 using the following conditions: potential scanning rate: 100 mVs⁻¹ in the potential range −1.8 to +1 V for 20 cycles. Quality control of the CFME cleanliness was performed using visual and electrochemical tests. Electrochemical experiments were conducted in a 50 mL glass voltammetric cell at room temperature.

The appropriate solutions were transferred into the electrochemical cell. The analytical procedure for SWV was then optimized by systematically studying the experimental parameters that affect the responses, such as the pulse potential frequency related to the total pulse duration, amplitude of the pulse, and height of the potential step.

Scanning was performed from −1.8 to +1 V vs. SCE, with a step potential of 10 mV, an amplitude of 60 mV, and a pulse potential frequency of 60 Hz.

Before each experiment, the solutions were deaerated by bubbling nitrogen, and the electrochemical cell was maintained under a nitrogen atmosphere throughout the experiments.

For example, to record the SWV voltammograms of FNT, 50 mL of the supporting electrolyte was introduced into the electrochemical cell. Next, the supporting electrolyte was bubbled with nitrogen and stirred simultaneously for ten (10) minutes. The SWV voltammogram of the blank, represented by PBS, was then recorded ten seconds after stopping the stirring and bubbling of the solution.

Analytical curves were obtained for the pure electrolyte using the standard addition method.

This was carried out in 0.1 M PBS at pH 7.2 and at varying concentrations of FNT.

LOD and LOQ were calculated using formula (1):

$\text{LOD}=3.3\times S_0\text{and}\text{LOQ}=10\times S_0$ (Figure 2) (1)

where S₀ is the standard deviation.

Recovery (%) was calculated using formula (2):

$\text{Recovery rate}\left(\text{\%}\right)=\left(C_{\text{soil}}\left(\text{μg}\cdot {\text{kg}}^{-1}\right)\times 100\right)/\left(C_{\text{theor}}\left(\text{μg}\cdot {\text{kg}}^{-1}\right)\right)$ (2)

FNT residue levels in spiked soil were assessed using formula (3) and (4):

$C_{\text{soil}}\left(\text{μg}\cdot {\text{kg}}^{-1}\right)=C_{\text{cell}}\left(\text{μg}\cdot {\text{L}}^{-1}\right)\times \left(V_{\text{solvent}}\left(\text{L}\right)\right)/\left(m_{\text{soil}}\left(\text{kg}\right)\right)$ (3)

• C_soil: concentration of FNT residues in soil;

• C_cell (µg·L⁻¹): concentration of residues in the cell, evaluated using the calibration curve;

• C_theor: theoretical value expected from spiked soil;

• m_soil: mass of soil used for analysis.

$C_{\text{theor}}\left(\text{μg}\cdot {\text{kg}}^{-1}\right)=\left(C_{\text{stand}}\left(\text{μg}\cdot {\text{L}}^{-1}\right)\times V_{\text{stand}}\left(\text{L}\right)\right)/\left(m_{\text{soil}}\left(\text{kg}\right)\right)$ (4)

V_stand: volume of FNT standard solution used to spike soil.

2.4. Extraction and Clean-Up of FNT Residues

A 20 g soil sample with a predetermined moisture content was placed in a 250 mL Erlenmeyer flask. Acetonitrile was selected based on its high extraction efficiency for organophosphates in soil [8]. The mixture was stirred for one hour (optimized in preliminary tests to maximize recovery) and centrifuged at 500 rpm for 15 minutes to separate the solid and liquid phases. At the end of centrifugation, two phases were separated: the solid phase consisting of the soil and a liquid phase or aliquot, represented by the extracts. The aliquot was collected, filtered, and evaporated to dryness using a rotary evaporator at a temperature of approximately 50˚C. The dried residues were then solubilized with a minimum amount of absolute ethanol and transferred to a 50 mL flask. The flask was then filled with phosphate buffer at pH 7.2. The solution thus obtained was then transferred into the electrochemical cell for analysis.

3. Results and Discussion

3.1. Electrochemical Behavior of FNT

The electrochemical behavior of FNT was studied using square wave voltammetry, and the obtained signal is shown in Figure 3.

Figure 3 shows that no peak was observed in the SWV voltammogram of the PBS. However, in the presence of FNT (100 μg·L⁻¹), a well-resolved peak is observed at −1.02 V versus SCE. This peak is characteristic of the reduction of the nitro group (-NO₂) of FNT to a hydroxylamine group (-NHOH) according to the electrochemical equation below [13].

Figure 2. Electrochemical reduction of nitro group to hydroxylamine group [13].

Figure 3. Electrochemical behavior of PBS and FNT 100 µg·L⁻¹; pH = 7.2; frequency = 10 Hz; amplitude = 100 mV; increments = 20 mV; reference electrode: ECS.

3.2. Method Validation

The validation of the reported electrochemical method for the determination of fenitrothion (FNT) in soil was conducted comprehensively in accordance with EURACHEM/CITAC guidelines [20] and ISO/IEC 17025 [19] requirements, ensuring its fitness for purpose for quantitative analysis. The method underwent rigorous evaluation for key performance parameters, including ruggedness, specificity/selectivity, matrix effects, linearity, accuracy, precision, limits of detection (LOD) and quantification (LOQ), and measurement uncertainty.

According to IUPAC recommendations relating to harmonized guidelines for single-laboratory validation of methods of analysis [21], the key parameters of a method must be validated according to relevant criteria. Thus, this method will be validated when the following are met for the method to be adequate for its intended use:

• The calibration function must describe an optimal line of best fit, minimizing the sum of squared residuals.

• Precision < 5% RSD, at a 95% confidence interval.

• No detectable bias at 95% confidence interval.

• Limit of detection < 10 μg∙kg⁻¹, with RSD < 10% at the 95% confidence interval.

• Accuracy ranges from 80% to 120%, with RSD < 10% at the 95% confidence interval.

• No significant interference with the matrix.

• Any potential interferences identified and, if needed, compensated for.

• Critical reagents used for method validation must be stable during the period covering the tests.

• Measurement uncertainty < 10%, at a 95% confidence interval.

3.2.1. Ruggedness Test

Ruggedness evaluation was carried out using the Plackett-Burman experimental design (Table S1, Table 1, and Table 2). The standard deviation (S) of the results for spiked soil samples containing FNT at 60 μg∙kg⁻¹ was previously estimated under repeatability conditions as 1.07 μg∙kg⁻¹ based on eight determinations. The critical difference (${\Delta }_{\text{crit}}$ ) is calculated according to the following formula (5):

${\Delta }_{\text{crit}}=\frac{t_{\text{crit}}\times s}{\sqrt{2}}$ (5)

${\Delta }_{\text{crit}}=\left(2.262\times 1.07\right)/\sqrt{2}=1.711$

The results showed that factors such as pH, pulse potential frequency, pulse amplitude, and increment height significantly influenced the analytical response. These critical factors were subsequently optimized. The optimal values were: pH = 7.2; pulse potential frequency of 10 Hz; pulse amplitude of 100 mV; and an increment of 20 mV (Figure 4).

Table 1. Parameters evaluated in the ruggedness test.

Table 2. Evaluation of results from a ruggedness study of the analytical process.

Figure 4. Optimal values of pH, amplitude, frequency, and increments.

3.2.2. Specificity/Selectivity Assessment

FNT is detected based on the reduction potential of its nitro group. To ensure the specificity of the method, the voltammogram of FNT 100 µg∙L⁻¹ was recorded in the presence of electroactive compounds that could potentially interfere, such as profenofos, p-nitrophenol, p-aminophenol, 3-methyl-4-nitrophenol (MNP), dimethoate, $S{O}_4^{2-}$ , $N{O}_3^{-}$ , Na⁺, K⁺ ions. The results in Table 3 show that, apart from MNP, none of the studied interferents has a significant effect on the detection of FNT.

MNP, a major FNT metabolite in soil, strongly interferes with FNT detection, as both compounds exhibit similar reduction potentials. This limits the method’s selectivity in aged or heavily contaminated soils where MNP may coexist with FNT. Future work should explore chromatographic pre-separation or chemometric deconvolution to resolve this interference.

The interference of these two molecules has already been reported in many previous works and could be explained by the fact that the reduction potential of MNP is very close to the reduction potential of FNT [22] [23].

Table 3. Effect of interferents on the detection of FNT.

3.2.3. Matrix Effects

The matrix effect was evaluated based on the contribution of the residual current from the matrix to the faradaic current. Matrix effect affects the intercept and the slope of a calibration function and then sample quantification by increasing or decreasing the signal attributed to the measurand. To overcome matrix effect, standard addition has been used for calibration, and the value of the intercept has been evaluated to be equal to zero (Table 5).

However, a formal matrix-matched calibration study (comparing slopes in solvent vs. soil extract) was not performed; future work should confirm the absence of signal suppression or enhancement.

3.2.4. Assessment of the Calibration Function

Linear regression was used to assess the expected relationship between the analytical response and the concentration of FNT. The assays were carried out by the standard addition method, adding increasing volumes of the standard stock solution of FNT. A fresh stock solution of FNT was prepared each day, and all assays were performed under controlled light conditions to prevent photodegradation.

To confirm the reliability of the linear regression model, three sets of independent quality control samples at 15 µg∙L⁻¹, 30 µg∙L⁻¹, 50 µg∙L⁻¹, 90 µg∙L⁻¹, and 150 µg∙L⁻¹ were prepared by spiking from the standard stock solution of FNT. Control was carried out over a three-day period to improve precision and provide additional checks on the calibration solution.

In the absence of an independent standard solution, the same FNT standard solution was used to prepare the quality control solutions.

The plot of peak current versus the respective concentration of FNT was found to be linear in the working range (10 - 200 µg∙L⁻¹) and was represented by the linear equation: $I_p=0.0022\times C\left(\text{μg}\cdot {\text{L}}^{-1}\right)+0.0065$ with R² = 0.9997 (Figure 5). The statistical analysis of the data, based on the assumption that there is no relationship between variables at a 95% confidence interval, showed that the slope coefficient is significantly different from zero (Tables S2-S4, Table 4 and Table 5).

Furthermore, data analysis showed that the optimal line of best fit minimizes the sum of squared residuals, indicating that at least one predictor variable has a non-zero coefficient. That assumes linear regression is a useful model.

Moreover, the statistical analysis of quality control data showed that there is no significant difference between variances of QC data collected over three-day periods (Table S5 and Table 6).

Figure 5. Square wave voltammetry of different concentrations of FNT in PBS 0.01 M, pH = 7.2, frequency = 10 Hz, amplitude = 100 mV, increments = 20 mV, FNT concentration range: 10 - 200 µg∙L⁻¹.

Table 4. ANOVA calculations for least-squares linear regression.

Table 5. Statistical analysis of linear regression data.

Table 6. ANOVA table for Levene’s test for QC data.

3.2.5. Assessment of Method Accuracy

The bias of the method was evaluated using independently spiked blank soil samples and expressed as the relative recovery rate of the spiking. Specifically, five concentration levels—30 μg∙kg⁻¹, 60 μg∙kg⁻¹, 90 μg∙kg⁻¹, 120 μg∙kg⁻¹, and 150 μg∙kg⁻¹—were prepared from 500 g of blank soil.

To do this, an appropriate volume of the FNT standard solution was added to 100 g of blank soil to obtain the concentration corresponding to each level. Subsequently, each of the 100 g of fortified soil corresponding to each level was used to prepare three replicates using a Rotating Tubular Divider TT-RSD200.

All 15 independent spiked samples were subjected to the extraction procedure in the presence of five soil blank samples, as described in subsection 2.4 on “Extraction and Clean-up of FNT Residues”.

The aliquots were analyzed, and C_soil (μg∙kg⁻¹)—corresponding to the difference between the mean concentration of the spiked sample and the unspiked sample—was then calculated. Recovery (%) was finally calculated against matrix-matched standards using formula (2) above.

Experimental data and results of statistical analysis of the data are shown in Table S6, Table S7, and Table 7.

The results showed that there is no bias inherent in the method.

Recovery rates of 89.12% - 94.76% (Table 7) suggest that the extraction and dilution procedure minimizes matrix effects for the tested soil. However, validation was performed on a single neutral soil type; applicability to acidic, alkaline, or high-organic-matter soils requires further investigation.

Table 7. Statistical assessment of method accuracy.

3.2.6. Assessment of Method Precision

The precision of the method was evaluated by means of a nested design. Four batches of spiked blank soil samples were used to prepare four groups of eight independent samples. Samples were prepared from soil samples spiked to 60 μg·kg⁻¹ by adding an appropriate volume of FNT standard solution. Each group of samples was analyzed under repeatability conditions.

Three series of eight samples were analyzed over three different days using three different carbon fiber microelectrodes.

The fourth series of eight samples was used for reproducibility assessment, carried out by GC-NPD analysis using an Agilent J&W HP-5 column (30 m × 0.32 mm × 0.25 µm). The FNT peak was found at 13.348 min.

The results of the paired t-test showed that there is no significant difference between both analytical methods (Table S9), indicating that the method offers good reproducibility.

Statistical analyses of data (Table S8) are given in Table 8, Table S10, and Table S11.

Table 8. Statistical analysis of precision data.

3.2.7. Assessment of Limit of Detection (LOD) and Limit of Quantification (LOQ)

Due to the lack of a measurable signal obtained from soil blank samples, test samples with concentrations of analyte were prepared by spiking soil blank samples with a small volume of the FNT stock solution.

So, ten (10) independent measurements of the test samples were prepared and analyzed under repeatability conditions according to the whole measurement procedure, including any sample preparation steps. The standard deviation (S₀) was then calculated. No baseline correction was carried out on the test results.

The statistical analysis of the results is summarized in Table 9.

The LOQ was then validated by analyzing three spiked samples at the quantification limit, yielding recoveries of 91.43 to 93.87 μg∙kg⁻¹.

Table 9. Experimental data for LOD and LOQ assessments.

3.2.8. Assessment of the Stability of the FNT Stock Standard Solution

The stability of the standard stock solution of FNT (10 mg∙L⁻¹) was evaluated by measuring the current of a fresh 100 μg∙L⁻¹ quality control sample prepared from the standard stock solution of 100 mg/L FNT using the standard addition method. The stock solution was conserved at 7˚C for three months. Measurements were carried out once a week over a period of three months.

The stability of FNT in ethanolic stock solution was confirmed over three months at 7˚C (Table 10). However, FNT stability in spiked soil samples during storage was not assessed. To ensure accurate results, soil samples should be analyzed promptly after collection, or a stability study should be conducted to establish acceptable storage conditions.

Statistical analysis of the data in Table 10, Tables S12-S14 confirms the homogeneity of the variances of the data collected over three-month periods.

Table 10. Statistical analysis of data for stock solution stability assessment.

3.2.9. Measurement Uncertainty

Measurement uncertainty was estimated using the GUM approach [24].

Measurement uncertainty was estimated by integrating contributions from weighing, volumetric measurements, calibration, and reagent purity, resulting in an expanded uncertainty of U = 0.04399 × C_soil (Table S15).

The uncertainties associated with each component were combined accordingly:

$\frac{u\left(x\right)}{C_{\text{soil}}\left(\frac{\mu \text{g}}{\text{kg}}\right)}=\sqrt{\begin{array}{l}{0.00185}^2+{0.00087}^2+{0.00023}^2+{0.00003}^2+{0.00069}^2+{0.00001}^2\\ +{0.00012}^2+{0.00058}^2+{0.00255}^2+{0.00019}^2+{0.01732}^2\\ +{0.01155}^2+{0.00577}^2+{0.00058}^2+{0.00058}^2+{0.00219}^2\end{array}}$

$\frac{u\left(x\right)}{C_{\text{soil}}\left(\frac{\mu \text{g}}{\text{kg}}\right)}=\sqrt{0.00048}=0.02199$

$u\left(x\right)=0.02199\times C_{\text{soil}}\left(\frac{\mu \text{g}}{\text{kg}}\right)$

The expanded uncertainty $U\left(x\right)$ is obtained by applying a coverage factor of 2, based on a 95% confidence interval.

$U\left(x\right)=0.02199\times 2\times C_{\text{soil}}\left(\mu \text{g}/\text{kg}\right)$

$U\left(x\right)=0.04399\times C_{\text{soil}}\left(\mu \text{g}/\text{kg}\right)$

The results of the uncertainty assessment, which include both random and systematic errors, show that $\frac{U\left(x\right)\times 100}{C_{\text{soil}}\left(\frac{\mu \text{g}}{\text{kg}}\right)}=4.399\text{\%}$ . This value, being less than

10%, suggests that the true value lies within a smaller range. This suggests that the validated method is accurate and precise.

4. Conclusions

The electrochemical reduction of FNT on carbon fiber microelectrodes was studied, and the optimal conditions were used for the rapid quantitative analysis of FNT in soil samples.

The procedure for the extraction and cleanup of FNT residues from soil samples has the main advantages of being simple and quick. Carbon fiber microelectrodes are stable, reliable tools that are easy to design.

The proposed CFME method achieves within-day and between-day RSDs of 1.404% and 1.579%, respectively, and offers good reproducibility with GC-NPD.

Statistical tests were used to successfully demonstrate that the method can be validated according to ISO/IEC requirements and EURACHEM/CITAC guidelines.

Acknowledgements

The authors gratefully acknowledge Laboratoire National de Santé Publique for carrying out GC-NPD analysis.

Author Contributions

Boukaré KABORÉ: Writing—original draft, Software, Investigation, Formal analysis.

Amidou TALL: Writing—review & editing, Methodology, Formal analysis.

Bibata OUEDRAOGO: Writing—review & editing, Formal analysis.

Yibor Fabrice Roland BAKO: Writing—review & editing, Methodology, Formal analysis.

Issa TAPSOBA: Writing—review & editing, Supervision, Methodology, Formal analysis.

Appendix

Table S1. Results from a ruggedness study of the analytical process.

Table S2. Calibration curve data.

Table S3. ANOVA table for Levene’s test for linear regression.

Table S4. ANOVA calculation of data for calibration curve.

Table S5. Quality control data.

Table S6. Experimental data of accuracy assessment.

Table S7. ANOVA table for Levene’s test for accuracy.

Table S8. Intermediate precision data.

Table S9. Statistical analysis of GC-NPD and SWV data.

Table S10. ANOVA table for precision assessment with 3 groups of data each containing 8 replicates.

Table S11. Levene’s test for precision assessment with 3 groups of data each containing 8 replicates.

Table S12. data of stock solution stability assessment.

Table S13. ANOVA table for FNT stock solution stability assessment.

Table S14. Levene’s test for stock solution stability assessment.

Table S15. Sources of uncertainties and standard uncertainty estimate.

Conflicts of Interest

The authors declare no conflicts of interest.

References