# Rapid Residue Analysis of Phthalanilic Acid in Cereals and Oilseed Rape by High-Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry

Authors: Aijuan Zhang; Li Zhou; Miao Yu; Yizhi Feng; Yanli Bian; Jinju Pan; Bojun Zuo; Lin Liang
Journal: Journal of Chemistry
 (2022)

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## Abstract

Phthalanilic acid is widely used in wheat and oilseed rape fields in China, but there is no analysis method to detect its residues in wheat and oilseed rape substrates. To accurately evaluate the health and environmental impact of phthalanilic acid residues on wheat gains and rapeseeds, it is urgent to establish a reliable analytical method for its detection. In this study, a rapid and sensitive method was developed based on QuEChERS and LC-MS/MS to detect phthalanilic acid in cereals (wheat, rice, corn, and millet), wheat straw, and rapeseeds. Optimization of the detection conditions showed that the mobile phase of acetonitrile-0.1% formic acid solution had the best chromatographic resolution and sensitivity. The average recovery rates of phthalanilic acid in cereals, wheat straw, and rapeseeds ranged from 88% to 113% with relative standard deviations (RSDs) of 1%–6%. There were good linear relationships with the correlation coefficients (r) higher than 0.9990 at concentrations of 0.01–5 mg L−1. The limits of quantification (LOQ) for phthalanilic acid in cereals, wheat straw, and rapeseeds were 0.01 mg kg−1, 0.05 mg kg−1, and 0.02 mg kg−1, respectively. These results show that the method established in this study is convenient and reliable for routine monitoring of phthalanilic acid in cereals, wheat straw, and rapeseeds.


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## Body

## 1. Introductions

Phthalanilic acid (Figure1) is a recently discovered plant growth regulator with robust internal absorption and two-way conductivity [1]. It quickly seeps into the plant system through foliar spraying, improving stress resistance in flowers and fruits, thus enhancing quality and yield [2]. Phthalanilic acid has been extensively applied in agricultural production in recent years due to its protective effects on fruits and vegetables [3–6]. However, several countries, including China, CAC, the United States, Australia, South Korea, and Japan have not formulated corresponding limit standards and residue limit standards for using phthalanilic acid in agricultural products. It is noteworthy that unreasonable use of plant growth regulators in actual production may lead to residues in food and the environment, endanger food safety, and potentially affect human health. Studies involving the use of rats as experimental animal models have shown that phthalanilic acid has a damaging effect on the immune system. Furthermore, oral exposure of 300 mgkg−1 phthalanilic acid for 28 consecutive days has been shown to cause various degrees of oxidative damage to the spleen and thymus of mice [7, 8].Figure 1
The chemical structure and secondary mass spectrum of phthalanilic acid.At present, most studies on phthalanilic acid have mainly focused on toxicity [6, 7], action mechanism [9–11], and synthesis [12]. There are also a few studies on the methods for analyzing the levels of phthalanilic acid residues [13–16]. Zhao et al. used modified QuEChERS and UPLC-MS/MS methods to determine the levels of phthalanilic acid residue in soil, bean, fruits, and vegetables and found that the LOQ of phthalanilic acid in all matrices was 0.01 mg kg−1 [13, 14]. Wei et al. established a HPLC-based method for analyzing phthalanilic acid contents in rat blood and tissue samples to examine the distribution and metabolism of phthalanilic acid in rat tissues. The results showed that phthalanilic acid distributed quickly and widely in rats, with relatively high selectivity to the kidney [15, 16]. Li et al. studied the dissipation and final residue of phthalanilic acid in apple and found that the half-lives of phthalanilic acid in apple were 3.7∼5.8 days, and the final residues were all lower than the LOQ (0.01 mg kg−1) [2].Although the analytical method of phthalanilic acid has been established in fruits and vegetables, no country or organization has formulated the maximum residue limit of phthalanilic acid in agricultural products to date. Therefore, there is a need to establish the analytical method of phthalanilic acid in agricultural products, as this will be crucial in evaluating its environmental and health risks. The QuEChERS method was first proposed by Anastassiades and Lehotay in 2003 [17]. The method can select suitable extraction solvents and purification fillers according to the characteristics of samples and target compounds. It has the advantages of rapidity, simplicity, and safety. The QuEChERS method has been widely used to analyze pesticide residues in food [18, 19] and environmental samples [20]. Moreover, the QuEChERS method combined with LC-MS/MS has been used to determine the residues of phthalanilic acid in vegetables [13] and fruits [2]; however, there is no report on the application of the method for the analysis of pesticide residues in cereals and oilseed rape. Compared with fruits and vegetables, cereals and rape substrates are more complex and require more stringent purification conditions. In this study, we optimized the QuEChERS method and the detection conditions of chromatography and mass spectrometry to improve the detection of phthalanilic acid residues in cereals, wheat straw, and rapeseeds. The findings of this study provide a technical support for the systematic evaluation of phthalanilic acid content in agricultural products.


## 2. Materials and Methods

### 2.1. Chemicals and Reagents

Phthalanilic acid analytical standard (purity 99.5%) was obtained from Chem Service Inc. (Pennsylvania, USA). Sodium chloride (analytical grade) and formic acid (HPLC-grade) were purchased from Beijing Chemical Reagents Company (Beijing, China). Acetonitrile (HPLC-grade) was obtained from Thermo Fisher Scientific (Waltham, USA). The sorbents octadecyl silica (C18), graphitized carbon black (GCB), and primary secondary amine (PSA) were purchased from Agela Technologies, Ltd. (Tianjin, China). Organic filter (0.22μm) was obtained from MedJen (Tianjin) Technology Co., Ltd. (Tianjin, China). Ultrapure water was obtained from Exceed-Cd-08 Labpure water system (Chengdu, China).


### 2.2. Instrumentation

HPLC was conducted using the Agilent 1290II-6460 high-performance liquid chromatography mass spectrometer (Santa Clara, CA, USA) equipped with Electrospray Ion Source (ESI). The Poroshell 120 EC-C18 (100 mm × 4.6 mm, 2.7μm) column was used to separate the target at a run temperature of 30°C. The isocratic mobile phase consisted of water acidified with 0.1% formic acid and acetonitrile (30 : 70, v/v), and the flow rate was 0.5 mL min−1. The injection volume was 5 µL, and the temperature of the injection chamber was 15°C. The analyses were performed in an electronegative mode with a 4.0 kV capillary voltage, 300°C dry gas temperature, 15 psi nebulizer, and 11 Lmin−1 dry gas flow rate. Typical chromatograms are shown in Figure 2.Figure 2
LC-MS/MS chromatograms of (a) acetonitrile solvent, (b) blank rapeseed sample, (c) phthalanilic acid standard solution (0.01 mg L−1), (d) spiked rapeseeds sample (0.02 mg kg−1), (e) blank wheat grains sample, (f) spiked wheat grains sample (0.01 mg kg−1), (g) blank wheat straw sample, (h) spiked wheat straw sample (0.05 mg kg−1), (i) blank corn sample, (j) spiked corn sample (0.01 mg kg−1), (k) blank rice sample, (l) spiked rice sample (0.01 mg kg−1), (m) blank millet sample, and (n) spiked millet sample (0.01 mg kg−1).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)


### 2.3. Standard Solutions

Stock solution of phthalanilic acid (1000 mg L−1) was diluted with pure acetonitrile and stored at −18°C until use. Working standard solutions (5, 1, 0.5, 0.1, 0.05, and 0.01 mg L−1) of phthalanilic acid were prepared with acetonitrile and blank sample extracts from the stock solutions by serial dilution. The series of working standard solutions were prepared afresh and used within 24 hours.


### 2.4. Sample Preparation

Exactly 10.0 g of homogenized cereals (2.0 g of wheat straw and 5.0 g of rapeseeds) was weighed into a 50 mL plastic centrifuged tube, and 10 mL water was added and left to stand at room temperature. After 10 min, 10 mL of 1% formic acid acetonitrile was added and the tube was shaken for 5 min. Subsequently, sodium chloride (4 g) was added to the tube and the mixture was vortexed again for 5 min. The tube was then centrifuged for 5 min (RCF 2077 g). Then, 2 mL of the supernatant was purified by adding 100 mg C18 and 30 mg PSA for cereals and rapeseeds and 100 mg C18 and 30 mg GCB for wheat straw. The cleaned extracts were then centrifuged for 1 min (RCF 2077 g), and the upper layer solvent was filtered through 0.22µm nylon filters.


### 2.5. Method Validation

Recovery experiments of phthalanilic acid were conducted using cereals, wheat straw, and rapeseed samples. The standard solution was spiked with samples at concentrations of 0.01, 0.1, and 1 mg kg−1 for cereals, 0.05, 0.5, and 5 mg kg−1 for wheat straw, and 0.02, 0.2, and 2 mg kg−1 for rapeseeds. Each recovery trial was repeated five times.In this study, the matrix effect was evaluated by the following formula [18, 21].(1)ME=the matrix standard curvethe solvent standard curve×100%.ME of 90∼110% indicates that the matrix has no obvious effect; ME < 90% indicates a weakening effect; and ME > 110% indicates a matrix-enhancing effect.


## 2.1. Chemicals and Reagents

Phthalanilic acid analytical standard (purity 99.5%) was obtained from Chem Service Inc. (Pennsylvania, USA). Sodium chloride (analytical grade) and formic acid (HPLC-grade) were purchased from Beijing Chemical Reagents Company (Beijing, China). Acetonitrile (HPLC-grade) was obtained from Thermo Fisher Scientific (Waltham, USA). The sorbents octadecyl silica (C18), graphitized carbon black (GCB), and primary secondary amine (PSA) were purchased from Agela Technologies, Ltd. (Tianjin, China). Organic filter (0.22μm) was obtained from MedJen (Tianjin) Technology Co., Ltd. (Tianjin, China). Ultrapure water was obtained from Exceed-Cd-08 Labpure water system (Chengdu, China).


## 2.2. Instrumentation

HPLC was conducted using the Agilent 1290II-6460 high-performance liquid chromatography mass spectrometer (Santa Clara, CA, USA) equipped with Electrospray Ion Source (ESI). The Poroshell 120 EC-C18 (100 mm × 4.6 mm, 2.7μm) column was used to separate the target at a run temperature of 30°C. The isocratic mobile phase consisted of water acidified with 0.1% formic acid and acetonitrile (30 : 70, v/v), and the flow rate was 0.5 mL min−1. The injection volume was 5 µL, and the temperature of the injection chamber was 15°C. The analyses were performed in an electronegative mode with a 4.0 kV capillary voltage, 300°C dry gas temperature, 15 psi nebulizer, and 11 Lmin−1 dry gas flow rate. Typical chromatograms are shown in Figure 2.Figure 2
LC-MS/MS chromatograms of (a) acetonitrile solvent, (b) blank rapeseed sample, (c) phthalanilic acid standard solution (0.01 mg L−1), (d) spiked rapeseeds sample (0.02 mg kg−1), (e) blank wheat grains sample, (f) spiked wheat grains sample (0.01 mg kg−1), (g) blank wheat straw sample, (h) spiked wheat straw sample (0.05 mg kg−1), (i) blank corn sample, (j) spiked corn sample (0.01 mg kg−1), (k) blank rice sample, (l) spiked rice sample (0.01 mg kg−1), (m) blank millet sample, and (n) spiked millet sample (0.01 mg kg−1).
(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)


## 2.3. Standard Solutions

Stock solution of phthalanilic acid (1000 mg L−1) was diluted with pure acetonitrile and stored at −18°C until use. Working standard solutions (5, 1, 0.5, 0.1, 0.05, and 0.01 mg L−1) of phthalanilic acid were prepared with acetonitrile and blank sample extracts from the stock solutions by serial dilution. The series of working standard solutions were prepared afresh and used within 24 hours.


## 2.4. Sample Preparation

Exactly 10.0 g of homogenized cereals (2.0 g of wheat straw and 5.0 g of rapeseeds) was weighed into a 50 mL plastic centrifuged tube, and 10 mL water was added and left to stand at room temperature. After 10 min, 10 mL of 1% formic acid acetonitrile was added and the tube was shaken for 5 min. Subsequently, sodium chloride (4 g) was added to the tube and the mixture was vortexed again for 5 min. The tube was then centrifuged for 5 min (RCF 2077 g). Then, 2 mL of the supernatant was purified by adding 100 mg C18 and 30 mg PSA for cereals and rapeseeds and 100 mg C18 and 30 mg GCB for wheat straw. The cleaned extracts were then centrifuged for 1 min (RCF 2077 g), and the upper layer solvent was filtered through 0.22µm nylon filters.


## 2.5. Method Validation

Recovery experiments of phthalanilic acid were conducted using cereals, wheat straw, and rapeseed samples. The standard solution was spiked with samples at concentrations of 0.01, 0.1, and 1 mg kg−1 for cereals, 0.05, 0.5, and 5 mg kg−1 for wheat straw, and 0.02, 0.2, and 2 mg kg−1 for rapeseeds. Each recovery trial was repeated five times.In this study, the matrix effect was evaluated by the following formula [18, 21].(1)ME=the matrix standard curvethe solvent standard curve×100%.ME of 90∼110% indicates that the matrix has no obvious effect; ME < 90% indicates a weakening effect; and ME > 110% indicates a matrix-enhancing effect.


## 3. Results and Discussion

### 3.1. Optimization of Detection Conditions

The appropriate mass spectrometry and liquid chromatography conditions were determined for optimum sensitivity and separation. Phthalanilic acid standard solution (1 mg L−1) was injected into the mass spectrometer using the direct injection method. The target was scanned with an electrospray power supply in positive and negative ion scanning modes. The results showed that phthalanilic acid has a good ionization effect in the positive ion scanning mode and obtained the characteristic ion peak (M + H). Thus, the mass spectrometry parameters including collision voltage and dwell time were further optimized to determine the most suitable monitoring ions. The mass spectrum acquisition parameters of phthalanilic acid used in this study have been reported previously (Table 1). However, we made a few modifications. Specifically, the mass spectrometry acquisition mode used in this study adopted a negative ion mode, and the corresponding parent ions, qualitative and quantitative fragments, were also different [2, 13]. Considering the complex matrix in cereals, straw, and rapeseed in this study, the positive ion collection mode with high response under the condition of the instrument was preferred in this study. The elution effects of methanol water system and acetonitrile water system as mobile phases were investigated. The results showed that the system pressure of methanol water system was significantly higher than that of acetonitrile water system, and the peak shape of the target was wide. Further optimization of the chromatographic conditions revealed that adding trace formic acid improves the peak type and ionizes the test substance, thus enhancing the response of the test compound. The use of acetonitrile-0.1% formic acid water system as the mobile phase obtained better peak type without impurity peak interference. Li et al. [2] also used different proportions of acetonitrile and formic acid water as the mobile phase and achieved good separation. Therefore, it was selected as the mobile phase, and the proportion and flow rate of the mobile phase was adjusted to control the retention time of phthalanilic acid.Table 1
Summary of detection conditions for phthalanilic acid.
MatrixChromatographic columnMobile phasePurifying agentParent ion (m/z)Product ions (m/z)Collision energy (eV)PolarityReferenceWheat gains, wheat straw, and rapeseedsPoroshell 120 EC-C18 (100 mm × 4.6 mm, 2.7μm)Acetonitrile/0.1% formic acid aqueous solutionC18 + PSA,24294 (quantitation)3Positive—C18 + GCB1499AppleC18Acetonitrile/0.1% formic acid aqueous solutionPSA240.1196 (quantitation)3NegativeLi et al(100 mm × 2.1 mm, 3.5µm)11815Soybean, fruits, and vegetablesHSS T3 column (100 mm × 2.1 mm, 1.8μm)Methanol/waterGCB, florisil240118 (quantitation)16NegativeZhao et al.19614


### 3.2. Optimization of Extraction and Purification Conditions

The QuEChERS pretreatment method is fast, simple, and inexpensive and is widely used in pesticide residue analysis. However, this method is only suitable for the pretreatment of fruits and vegetables with high water content [17]. Samples with low water content such as wheat grains and rapeseeds require the addition of a certain proportion of ultrapure water in the extraction process [18, 22]. Also, since phthalanilic acid contains a carboxyl group, 0.1% formic acid is added to the extraction solvent to enhance extraction efficiency for better recovery [23]. The addition of water increases the recovery rate, which in turn increases the levels of organic acids, pigments, fats, and other impurities in the extract [24, 25]. In this study, the sample clean-up process involved three typical clean-up agents, C18, PSA, and GCB. Notably, C18 can remove nonpolar and medium-polar compounds from polar samples; PSA mainly removes organic acids, pigments, sugars, and fats in nonpolar samples; and GCB is a weakly polar or nonpolar adsorbent that can remove impurities such as hydrophobic compounds like chlorophyll and carotenoids [23, 26]. The adsorption of phthalanilic acid and matrix standard solutions with different dosages of three purifiers was evaluated. The results showed that PSA and GCB were adsorbed to certain levels with increased adsorbent dose, except C18 (Figure 3). The adsorption of PSA and GCB on phthalanilic acid in acetonitrile was stronger than that of other substrates. This may be due to the adsorption of other impurities in the matrix by PSA and GCB, thus reducing phthalanilic acid adsorption. Of note, the main components of cereals and straw are starch, protein, and pigment; rapeseed matrix is complex and rich in fat, protein, and some other lipophilic compounds. Thus, a purifier may not achieve the ideal purification effect. Therefore, the purification effects of the three purification agents mixed at different ratios were further compared. C18 (100 mg) was mixed with different doses of PSA and GCB to compare the recoveries at different ratios because C18 had no adsorption on phthalanilic acid. The results (Figure 4) showed that the purification recovery rates of C18 in combination with different doses of PSA (except 50 mg PSA) and GCB met all the requirements of the European Union guidelines (SANTE/11813/2017), requiring an average recovery rate of 70%–120% and a relative standard deviation of less than 20% [27] (European Commission, 2017).Figure 3
The recoveries of phthalanilic acid clean-up by different adsorbents in acetonitrile, cereals, wheat straw, and rapeseed matrix blank solutions.Figure 4
The recoveries of phthalanilic acid in six substrates by different adsorbents combination at the level of 0.1 mg kg−1 addition.


### 3.3. Method Validation

#### 3.3.1. Linearity and LOQ

The regression coefficients for phthalanilic acid in the matrices were all >0.9990. The parameters of the linear regression equation and correlation coefficient are shown in Table2. The LOQ of the target compound in cereals, wheat straw, and rapeseeds was determined at the minimum additive recovery concentration, and the limit of quantification was 0.01 mg kg−1, 0.05 mg kg−1, and 0.02 mg kg−1, respectively.Table 2
Performance characteristics of the method for phthalanilic acid in cereals, wheat straw, and rapeseeds.
CropsSpiked level(mg·kg−1)Average recovery(%) (n = 5)RSD(%)MatrixAcetonitrileLOQ(mg·kg−1)Regression equationrRegressionequationrWheat grains0.011036Y = 15020 X + 1115r = 0.9992Y = 20487 X + 1638r = 0.99900.010.19811923Wheat straw0.051133Y = 6214 X + 98r = 0.99900.050.510015964Rapeseeds0.02884Y = 10170 X + 249r = 0.9999Y = 20271 X + 1594r = 0.99900.020.29122933Corn0.01935Y = 45569 X + 2023r = 0.99970.010.19541953Rice0.01901Y = 41500 X + 1399r = 0.9997Y = 48582 X + 10592r = 0.99590.010.1107211032Millet0.01933Y = 37143 X + 2097r = 0.99940.010.110611941


#### 3.3.2. Accuracy and Precision

The accuracy and precision of this method were measured by additive recovery and relative standard deviation. The recovery test for phthalanilic acid was performed at three levels in three blank matrices, and each level was repeated five times (Table2). According to the extraction and purification method, the recovery rate of phthalanilic acid was between 88% and 113%, while the RSD was between 1% and 6%. These results show that the accuracy and reproducibility of this method meet the requirements of pesticide residue analysis [27].


#### 3.3.3. Matrix Effect

In this study, phthalanilic acid had good recovery rates in the different matrices under the optimized combination of different contents of purifiers. The matrix effect was measured and calculated to reduce the damage of impurities to the instrument [28–30]. The results of the matrix effect of phthalanilic acid in different matrices and purifiers are shown in Figure 5. The matrix effect of phthalanilic acid in the different matrices ranged from 20% to 93%. The matrix effect was lowest in cereals and rapeseed matrices when the purifiers were C18 (100 mg) and PSA (30 mg). Meanwhile, the matrix effect of the straw was lowest when the purifiers were C18 (100 mg) and GCB (30 mg). To reduce the influence of the matrix effect on the instrument during the detection process (under the condition of ensuring enhanced recovery rate), a purifying agent with little influence on the matrix effect was selected as the extraction method. After comprehensive comparison of recovery and matrix effect, C18 (100 mg) and PSA (30 mg) were selected as purifiers for cereals and rapeseeds, while C18 (100 mg) and GCB (30 mg) were selected for wheat straw.Figure 5
The matrix effects of phthalanilic acid in six substrates by different adsorbents combination at the level of 0.1 mg kg−1 addition.


### 3.4. Practical Application

A total of 24 wheat grains samples, 24 straw samples, and 20 rapeseeds samples from the field were tested to verify the reliability of the method. The residues of phthalanilic acid in wheat grains and rapeseeds were lower than the LOQ. Phthalanilic acid residues were detected in four wheat straw samples, and the residues ranged from 0.056 to 0.52 mg/kg (Table3).Table 3
The terminal residues of phthalanilic acid in wheat grains, wheat straw, and rapeseeds.
CropsDose (mg·kg−1)Spray timesInterval to harvest (d)Residue (mg·kg−1)∗HR (mg·kg−1)Wheat grains200230–50<0.01 (12)0.01Wheat straw<0.05 (8), 0.056, 0.078, 0.17, 0.520.52Rapeseeds266.6227∼75<0.02 (10)0.02Note. ∗Average of 2 samples; HR, highest residue.


## 3.1. Optimization of Detection Conditions

The appropriate mass spectrometry and liquid chromatography conditions were determined for optimum sensitivity and separation. Phthalanilic acid standard solution (1 mg L−1) was injected into the mass spectrometer using the direct injection method. The target was scanned with an electrospray power supply in positive and negative ion scanning modes. The results showed that phthalanilic acid has a good ionization effect in the positive ion scanning mode and obtained the characteristic ion peak (M + H). Thus, the mass spectrometry parameters including collision voltage and dwell time were further optimized to determine the most suitable monitoring ions. The mass spectrum acquisition parameters of phthalanilic acid used in this study have been reported previously (Table 1). However, we made a few modifications. Specifically, the mass spectrometry acquisition mode used in this study adopted a negative ion mode, and the corresponding parent ions, qualitative and quantitative fragments, were also different [2, 13]. Considering the complex matrix in cereals, straw, and rapeseed in this study, the positive ion collection mode with high response under the condition of the instrument was preferred in this study. The elution effects of methanol water system and acetonitrile water system as mobile phases were investigated. The results showed that the system pressure of methanol water system was significantly higher than that of acetonitrile water system, and the peak shape of the target was wide. Further optimization of the chromatographic conditions revealed that adding trace formic acid improves the peak type and ionizes the test substance, thus enhancing the response of the test compound. The use of acetonitrile-0.1% formic acid water system as the mobile phase obtained better peak type without impurity peak interference. Li et al. [2] also used different proportions of acetonitrile and formic acid water as the mobile phase and achieved good separation. Therefore, it was selected as the mobile phase, and the proportion and flow rate of the mobile phase was adjusted to control the retention time of phthalanilic acid.Table 1
Summary of detection conditions for phthalanilic acid.
MatrixChromatographic columnMobile phasePurifying agentParent ion (m/z)Product ions (m/z)Collision energy (eV)PolarityReferenceWheat gains, wheat straw, and rapeseedsPoroshell 120 EC-C18 (100 mm × 4.6 mm, 2.7μm)Acetonitrile/0.1% formic acid aqueous solutionC18 + PSA,24294 (quantitation)3Positive—C18 + GCB1499AppleC18Acetonitrile/0.1% formic acid aqueous solutionPSA240.1196 (quantitation)3NegativeLi et al(100 mm × 2.1 mm, 3.5µm)11815Soybean, fruits, and vegetablesHSS T3 column (100 mm × 2.1 mm, 1.8μm)Methanol/waterGCB, florisil240118 (quantitation)16NegativeZhao et al.19614


## 3.2. Optimization of Extraction and Purification Conditions

The QuEChERS pretreatment method is fast, simple, and inexpensive and is widely used in pesticide residue analysis. However, this method is only suitable for the pretreatment of fruits and vegetables with high water content [17]. Samples with low water content such as wheat grains and rapeseeds require the addition of a certain proportion of ultrapure water in the extraction process [18, 22]. Also, since phthalanilic acid contains a carboxyl group, 0.1% formic acid is added to the extraction solvent to enhance extraction efficiency for better recovery [23]. The addition of water increases the recovery rate, which in turn increases the levels of organic acids, pigments, fats, and other impurities in the extract [24, 25]. In this study, the sample clean-up process involved three typical clean-up agents, C18, PSA, and GCB. Notably, C18 can remove nonpolar and medium-polar compounds from polar samples; PSA mainly removes organic acids, pigments, sugars, and fats in nonpolar samples; and GCB is a weakly polar or nonpolar adsorbent that can remove impurities such as hydrophobic compounds like chlorophyll and carotenoids [23, 26]. The adsorption of phthalanilic acid and matrix standard solutions with different dosages of three purifiers was evaluated. The results showed that PSA and GCB were adsorbed to certain levels with increased adsorbent dose, except C18 (Figure 3). The adsorption of PSA and GCB on phthalanilic acid in acetonitrile was stronger than that of other substrates. This may be due to the adsorption of other impurities in the matrix by PSA and GCB, thus reducing phthalanilic acid adsorption. Of note, the main components of cereals and straw are starch, protein, and pigment; rapeseed matrix is complex and rich in fat, protein, and some other lipophilic compounds. Thus, a purifier may not achieve the ideal purification effect. Therefore, the purification effects of the three purification agents mixed at different ratios were further compared. C18 (100 mg) was mixed with different doses of PSA and GCB to compare the recoveries at different ratios because C18 had no adsorption on phthalanilic acid. The results (Figure 4) showed that the purification recovery rates of C18 in combination with different doses of PSA (except 50 mg PSA) and GCB met all the requirements of the European Union guidelines (SANTE/11813/2017), requiring an average recovery rate of 70%–120% and a relative standard deviation of less than 20% [27] (European Commission, 2017).Figure 3
The recoveries of phthalanilic acid clean-up by different adsorbents in acetonitrile, cereals, wheat straw, and rapeseed matrix blank solutions.Figure 4
The recoveries of phthalanilic acid in six substrates by different adsorbents combination at the level of 0.1 mg kg−1 addition.


## 3.3. Method Validation

### 3.3.1. Linearity and LOQ

The regression coefficients for phthalanilic acid in the matrices were all >0.9990. The parameters of the linear regression equation and correlation coefficient are shown in Table2. The LOQ of the target compound in cereals, wheat straw, and rapeseeds was determined at the minimum additive recovery concentration, and the limit of quantification was 0.01 mg kg−1, 0.05 mg kg−1, and 0.02 mg kg−1, respectively.Table 2
Performance characteristics of the method for phthalanilic acid in cereals, wheat straw, and rapeseeds.
CropsSpiked level(mg·kg−1)Average recovery(%) (n = 5)RSD(%)MatrixAcetonitrileLOQ(mg·kg−1)Regression equationrRegressionequationrWheat grains0.011036Y = 15020 X + 1115r = 0.9992Y = 20487 X + 1638r = 0.99900.010.19811923Wheat straw0.051133Y = 6214 X + 98r = 0.99900.050.510015964Rapeseeds0.02884Y = 10170 X + 249r = 0.9999Y = 20271 X + 1594r = 0.99900.020.29122933Corn0.01935Y = 45569 X + 2023r = 0.99970.010.19541953Rice0.01901Y = 41500 X + 1399r = 0.9997Y = 48582 X + 10592r = 0.99590.010.1107211032Millet0.01933Y = 37143 X + 2097r = 0.99940.010.110611941


### 3.3.2. Accuracy and Precision

The accuracy and precision of this method were measured by additive recovery and relative standard deviation. The recovery test for phthalanilic acid was performed at three levels in three blank matrices, and each level was repeated five times (Table2). According to the extraction and purification method, the recovery rate of phthalanilic acid was between 88% and 113%, while the RSD was between 1% and 6%. These results show that the accuracy and reproducibility of this method meet the requirements of pesticide residue analysis [27].


### 3.3.3. Matrix Effect

In this study, phthalanilic acid had good recovery rates in the different matrices under the optimized combination of different contents of purifiers. The matrix effect was measured and calculated to reduce the damage of impurities to the instrument [28–30]. The results of the matrix effect of phthalanilic acid in different matrices and purifiers are shown in Figure 5. The matrix effect of phthalanilic acid in the different matrices ranged from 20% to 93%. The matrix effect was lowest in cereals and rapeseed matrices when the purifiers were C18 (100 mg) and PSA (30 mg). Meanwhile, the matrix effect of the straw was lowest when the purifiers were C18 (100 mg) and GCB (30 mg). To reduce the influence of the matrix effect on the instrument during the detection process (under the condition of ensuring enhanced recovery rate), a purifying agent with little influence on the matrix effect was selected as the extraction method. After comprehensive comparison of recovery and matrix effect, C18 (100 mg) and PSA (30 mg) were selected as purifiers for cereals and rapeseeds, while C18 (100 mg) and GCB (30 mg) were selected for wheat straw.Figure 5
The matrix effects of phthalanilic acid in six substrates by different adsorbents combination at the level of 0.1 mg kg−1 addition.


## 3.3.1. Linearity and LOQ

The regression coefficients for phthalanilic acid in the matrices were all >0.9990. The parameters of the linear regression equation and correlation coefficient are shown in Table2. The LOQ of the target compound in cereals, wheat straw, and rapeseeds was determined at the minimum additive recovery concentration, and the limit of quantification was 0.01 mg kg−1, 0.05 mg kg−1, and 0.02 mg kg−1, respectively.Table 2
Performance characteristics of the method for phthalanilic acid in cereals, wheat straw, and rapeseeds.
CropsSpiked level(mg·kg−1)Average recovery(%) (n = 5)RSD(%)MatrixAcetonitrileLOQ(mg·kg−1)Regression equationrRegressionequationrWheat grains0.011036Y = 15020 X + 1115r = 0.9992Y = 20487 X + 1638r = 0.99900.010.19811923Wheat straw0.051133Y = 6214 X + 98r = 0.99900.050.510015964Rapeseeds0.02884Y = 10170 X + 249r = 0.9999Y = 20271 X + 1594r = 0.99900.020.29122933Corn0.01935Y = 45569 X + 2023r = 0.99970.010.19541953Rice0.01901Y = 41500 X + 1399r = 0.9997Y = 48582 X + 10592r = 0.99590.010.1107211032Millet0.01933Y = 37143 X + 2097r = 0.99940.010.110611941


## 3.3.2. Accuracy and Precision

The accuracy and precision of this method were measured by additive recovery and relative standard deviation. The recovery test for phthalanilic acid was performed at three levels in three blank matrices, and each level was repeated five times (Table2). According to the extraction and purification method, the recovery rate of phthalanilic acid was between 88% and 113%, while the RSD was between 1% and 6%. These results show that the accuracy and reproducibility of this method meet the requirements of pesticide residue analysis [27].


## 3.3.3. Matrix Effect

In this study, phthalanilic acid had good recovery rates in the different matrices under the optimized combination of different contents of purifiers. The matrix effect was measured and calculated to reduce the damage of impurities to the instrument [28–30]. The results of the matrix effect of phthalanilic acid in different matrices and purifiers are shown in Figure 5. The matrix effect of phthalanilic acid in the different matrices ranged from 20% to 93%. The matrix effect was lowest in cereals and rapeseed matrices when the purifiers were C18 (100 mg) and PSA (30 mg). Meanwhile, the matrix effect of the straw was lowest when the purifiers were C18 (100 mg) and GCB (30 mg). To reduce the influence of the matrix effect on the instrument during the detection process (under the condition of ensuring enhanced recovery rate), a purifying agent with little influence on the matrix effect was selected as the extraction method. After comprehensive comparison of recovery and matrix effect, C18 (100 mg) and PSA (30 mg) were selected as purifiers for cereals and rapeseeds, while C18 (100 mg) and GCB (30 mg) were selected for wheat straw.Figure 5
The matrix effects of phthalanilic acid in six substrates by different adsorbents combination at the level of 0.1 mg kg−1 addition.


## 3.4. Practical Application

A total of 24 wheat grains samples, 24 straw samples, and 20 rapeseeds samples from the field were tested to verify the reliability of the method. The residues of phthalanilic acid in wheat grains and rapeseeds were lower than the LOQ. Phthalanilic acid residues were detected in four wheat straw samples, and the residues ranged from 0.056 to 0.52 mg/kg (Table3).Table 3
The terminal residues of phthalanilic acid in wheat grains, wheat straw, and rapeseeds.
CropsDose (mg·kg−1)Spray timesInterval to harvest (d)Residue (mg·kg−1)∗HR (mg·kg−1)Wheat grains200230–50<0.01 (12)0.01Wheat straw<0.05 (8), 0.056, 0.078, 0.17, 0.520.52Rapeseeds266.6227∼75<0.02 (10)0.02Note. ∗Average of 2 samples; HR, highest residue.


## 4. Conclusions

In this study, an analytical method for determining phthalanilic acid residues in different matrices of cereals, wheat straw, and rapeseeds was established. The sample pretreatment operation is simple, and the performance indicators such as the linear range, LOQ, average addition recovery, and RSD of the method meet the requirements of pesticide residue analysis. Thus, the method is suitable for rapid and accurate identification and quantification of phthalanilic acid residues.



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*Source: 5684797-2022-07-30.xml*