Food Analytical Methods

, Volume 11, Issue 5, pp 1303–1311 | Cite as

Simultaneous Determination of Aflatoxin B1, Bisphenol A, and 4-Nonylphenol in Peanut Oils by Liquid-Liquid Extraction Combined with Solid-Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

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Abstract

An analytical method based on liquid-liquid extraction combined with solid-phase extraction and isotope dilution-ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was well developed for simultaneous determination of aflatoxin B1 (AFB1), bisphenol A (BPA), and 4-nonylphenol (4-NP) in peanut oil. After adding isotope internal standards, the samples were firstly diluted by normal hexane and then extracted by acetonitrile and Carb/PSA solid-phase extraction cartridge in sequence to obtain the extracted solution. All the extracted solution was merged and was subsequently dried to near dryness by a mild nitrogen stream. Three target analytes were separated on a Phenomenex Luna C18 chromatographic column, quantified by an internal standard method and detected by ESI positive (ESI+) and negative (ESI) subsection acquisition modes under multi-reaction monitoring (MRM) conditions. Results demonstrated that the three target analytes exhibited excellent linearity in their corresponding concentration ranges of 0.1–100.0 μg/L with correlation coefficients all greater than 0.998. The corresponding method limits of quantitation (MLOQ, S/N = 10) of AFB1, BPA, and 4-NP were 0.2, 1.0, and 2.0 μg/kg, respectively. Moreover, the mean recoveries for negative samples spiked at three concentration levels were calculated between 87.7 and 105.1% with relative standard deviation (RSD, n = 6) ranging from 2.2 to 7.9% and the interday precision (n = 5) ranging from 5.0 to 8.7%. Finally, the method was successfully applied to analyze 52 peanut oil samples, and AFB1 and 4-NP were detected in 43 samples with the concentrations in the ranges of 0.5–69.4 and 9.3–77.8 μg/kg, respectively. None of BPA was detected in any samples.

Keywords

Peanut oil Aflatoxin B1 Bisphenol A 4-Nonylphenol Solid-phase extraction Liquid chromatography-tandem mass spectrometry (LC-MS/MS) 

Introduction

Aflatoxin B1 (AFB1), one of the most toxic mycotoxins, is mainly produced by Aspergillus flavus and Aspergillus parasiticus. It is considered as an unavoidable, naturally occurring contaminant of peanut and peanut oil (Zhang et al. 2014; Ji et al. 2016). Mounting evidence has indicated that AFB1 is implicated in the etiology of various human diseases and has strong genotoxic, carcinogenic, and immunotoxic effects on humans and animals (Yu et al. 2015a; Saad-Hussein et al. 2014; Sabet et al. 2017). It has been classified into I grade carcinogens by the International Agency for Research on Cancer (IARC) of the World Health Organization since 1993. Therefore, many countries have legislated on the maximum levels of AFB1 in foods to reduce its harm to humans (Sergeyeva et al. 2017). By this token, reliable and sensitive method should be established for AFB1 determination in food products.

Bisphenol A (BPA) and nonylphenol (NP) are considered synthetic endocrine-disrupting chemicals because they can alter immune functions, produce sexual dysfunction, or cause cancer at low concentrations (Salgueiro-Gonzalez et al. 2012; Kim et al. 2015). They were identified as the priority hazards by European Union and other countries due to their serious harm to humans. In fact, phenol pollutants in various food products are almost always paid great attention and their restrictions have been stringently legislated in a slew of countries including China (Xian et al. 2017). Commonly, BPA and NP are widely used in the production of plastic industry for better performance, whose products are often employed in the processing of peanut oil production. It is therefore unavoidable that a certain amount of BPA and NP migrate into oil products and further threaten human health. However, the control for BPA and NP in peanut oil is still at risk monitoring level domestic and overseas. It is of importance to further impose a limit on the requirement of BPA and NP. Considering these circumstances, sensitive methods established for determination of BPA and NP in peanut oil, are extremely required.

To date, the methods for AFB1 determination in plant oils have been well established (Bao et al. 2010; Fan et al. 2015). The method for BPA determination was mainly established in milk samples (Deceuninck et al. 2015), human urine (Provencher et al. 2014), canned foods (Cunha and Fernandes 2013), beverages (Shao et al. 2005), and other ready-made meals (Regueiro and Wenzl 2015), while that for NP determination was mostly in dairy products (Lv et al. 2014) and aqueous samples (Shih et al. 2015). However, only a few methods established for the simultaneous determination of BPA and NP in peanut oils have been reported with fairly low detection efficiency (Niu et al. 2011; Wu et al. 2016a) and these methods leave much to be desired. Commonly, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) is almost always employed for the determination of harmful substances in foodstuffs, which possesses fairly high sensitivity, qualitative and quantitative accuracy (Yu et al. 2015b; Rodriguez-Gomez et al. 2015; Wu et al. 2012; Dong et al. 2018; Dong et al. 2016; Luo et al. 2016a; Luo et al. 2016b). In addition, it deserves to be mentioned that it always takes much effort to extract the favorable compounds from oil products. It has been reported that gel permeation chromatography (GPC), liquid-liquid extraction, and solid-phase extraction are widely served for the pretreatment in oil matrix (Zgola-Grzeskowiak and Grzeskowiak 2013; Tran et al. 2013; Yang et al. 2015). However, GPC is gradually being phased out due to its weakness such as time-consuming, solvent-consuming, and environmentally unfriendly. Neither liquid-liquid extraction nor solid-phase extraction can completely extract the target analytes in the oil samples. They are inexhaustive to remove lipids so as to effectively reduce the interference of impurities in the UPLC-MS/MS analysis.

Therefore, in the present study, a two-step extraction method was employed to fully extract three target analytes. After complete extraction, a sensitive and rapid isotope dilution-UPLC-MS/MS method was developed and validated for the simultaneous determination of AFB1, BPA, and 4-NP (their structures are provided in Supplementary materials) in peanut oils. The established method was verified to possess high detection efficiency compared to the existing method for individual target analytes. The method was successfully applied for the determination of AFB1, BPA, and 4-NP in a total of 52 peanut oil products, confirming its suitability in the determination of general pollutants in plant oils.

Materials and Methods

Chemicals, Reagents, and Standards

Standards of AFB1, BPA, and 4-NP with purity ≥ 98% were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany), so was the isotope internal standard of 4-n-NP-d 4. Isotope internal standards of U-[13C17]-AFB1 in acetonitrile at a concentration of 0.5 μg/mL and BPA-d 4 with purity ≥ 98% were purchased from Shanghai Anpel Scientific Instrument Co. Ltd. (Shanghai, China).

Methanol, acetonitrile, normal hexane, and dichloromethane (UPLC grade) were all purchased from Merck Chemicals Co., Ltd. (Darmstadt, Germany). Laboratory-made ultrapure water (18.0 MΩ cm) was used in the whole experiment. Carb/N-propyl ethylenediamine (PSA) solid-phase extraction (500 mg, 6 mL, Supelclean Co., USA) was selected as adsorption cartridge. It is generally activated by 10 mL of methanol-dichloromethanemethanol (2/8, v/v) and identical volume of methanol, then stabilized by 6 mL of normal hexane before use.

Fifty-two peanut oil samples were used in this study, among which, 20 samples termed S1–S20 were collected from local supermarkets and 32 numbered S21–S52 were from individual workshops.

Instrumentation

An ACQUITYTM ultra-high performance liquid chromatography and Waters Xevo™ TQ tandem triple quadrupole mass spectrometer (UPLC-MS/MS, Waters Co., USA) were employed for sample analyses in this study. The samples were vortex mixed with a MS3 basic vortex mixer (IKA GmbH, Germany) and centrifuged by 5418 high-speed centrifuge (Eppendorf Corp., Germany). Turbo Vap LV concentrator (Biotage Co., USA) was used for the concentration of extracted solution. Milli-Q Gradient A10 system prepared for ultrapure water in the present work was purchased from Millipore Corp., Bedford, USA.

Stock and Standard Solutions

Appropriate amounts of three target analytes and three isotope internal standards were dissolved in methanol to respectively obtain the individual standard stock solutions. A mixed standard solution of AFB1 at a concentration of 0.2 mg/L and BPA and 4-NP at a concentration of 2 mg/L was prepared by progressively diluting the individual standard stock solution with methanol, and then stored at 4 °C. Analogously, a mixed isotope internal standard solution of U-[13C17]-AFB1 at a concentration of 0.1 mg/L and BPA-d 4 and 4-n-NP-d 4 at a concentration of 1 mg/L was obtained by diluting with methanol, and stored at − 20 °C. Working standard solutions (AFB1, 2 μg/L; BPA, 20 μg/L; 4-NP, 20 μg/L) were freshly prepared by diluting the mixed standard stock solution with methanol, among which all contained 2 μg/L of U-[13C17]-AFB1, 20 μg/L of BPA-d 4, and 20 μg/L of 4-n-NP-d 4.

Sample Preparation

Sample (1.00 g) (accuracy of 0.01 g) was weighed into a 10-mL glass centrifuge tube, and then, 20 μL of mixed isotope internal standard solution was added in. After vortex mixed for 1 min, 3 mL of normal hexane was added in and the sample was then vortex mixed for 30 s, followed by 5 mL of acetonitrile addition and 2 min vortex mixture. Subsequently, the sample solution was centrifuged at 3500 rpm for 5 min. The supernatant composed of normal hexane and oil phase was transferred into Carb/PSA solid-phase extraction column that have been activated and stabilized. The below solution extracted by acetonitrile was kept in the glass centrifuge tube for prepared standby.

The sample solution was collected which was out flown from Carb/PSA solid-phase extraction cartridge. The column was washed with 8 mL of normal hexane to remove triglycerides, then 4 ml of methanol-dichloromethane (2/8, v/v) was applied to elute AFB1, BPA, and 4-NP. The collected eluent coupled with acetonitrile extracted solution mentioned above was fully mixed and then dried to near dryness by a mild nitrogen stream in a 50 °C water bath. Methanol (2.0 mL) was added in and dispersed by vortex mixing. The obtained mixture was finally filtered through a PTFE filter membrane (0.22 μm) and analyzed by UPLC-MS/MS.

UPLC-MS/MS Conditions

UPLC Condition

Chromatographic separation was studied on Phenomenex Luna C18 (50 mm × 2.00 mm, 3 μm). The flow rate was set as 0.4 mL/min, and the column temperature was kept constant at 30 °C. The mobile phase consisted of ultrapure water (A) and methanol (B). The gradient elution program was performed as follows: 0.0–1.7 min, 50%–80% B; 1.7–2.0 min, 80%–100% B; 2.0–4.0 min, 100% B; 4.0–4.1 min, 100%–50% B; 4.1–6.0 min, 50% B. The injection volume was 5 μL.

MS/MS Condition

MS/MS detection was performed on a triple quadrupled mass spectrometer detector equipped with a jet stream electrospray ionization (ESI) source under multi-reaction monitoring (MRM) conditions. ESI positive (ESI+) and negative (ESI) subsection acquisition modes were used for quantification with a capillary voltage of 1.0 kV, wherein 0–1.4 min was in ESI+ mode and 1.4–4.0 min in ESI mode. The ion source temperature and desolvation temperature were respectively optimized at 150 and 400 °C. Additionally, the flow rates of the desolvation gas (nitrogen), cone gas (nitrogen), and collision gas (Ar of high purity) were set at 800 L/h, 50 L/h, and 0.15 mL/min, respectively. The specific MS parameters for the three TAs and the isotope internal standards, such as monitoring ion pair (m/z), cone voltage, and collision energy, are displayed in Table 1.
Table 1

The specific MS parameters for three target analytes and the isotope internal standards in MS/MS analysis

Compounds

Parent ion

Cone (V)

MRM 1a (m/z)

CE (eV)

MRM 2b (m/z)

CE (eV)

AFB1

[M+H]+

40

313.3 > 241.2

40

313.3 > 285.3

25

U-[13C17]-AFB1

[M+H]+

40

330.4 > 301.3

18

BPA

[M-H]

30

227.0 > 212.0

20

227.0 > 133.0

25

BPA-d 4

[M-H]

35

231.0 > 216.0

20

4-NP

[M-H]

35

219.1 > 133.0

30

219.1 > 147.0

30

4-n-NP-d 4

[M-H]

35

223.2 > 110.0

35

CE collision energy

aMRM 1, quantifier transition

bMRM 2, qualitative transition

Results and Discussion

Optimization of Mass Spectrometry Conditions

Generally, AFB1 could attain high response under ESI+ mode ionization and BPA and 4-NP were commonly detected under ESI mode ionization. In fact, it remains necessary to optimize the mass spectrometry parameters in the new established method because of the preferable parameters slightly varying from different instruments. In the corresponding ESI+ and ESI mode, methanol-water (1:1, v/v) was selected as mobile phase at the flow rate of 0.2 mL/min. The standards/internal standard solutions in the range of 0.5–1.0 mg/L were respectively injected into the electrospray ion source via the peristaltic pump at the flow rate of 5 μL/min. The qualitative and quantitative ions of each target compound were selected. AFB1 is a derivative of dihydrofuronaphthoate, containing a bisfuran ring and an oxazolidinone (also named coumarin). The parent ion m/z 313 was prone to neutral loss (-CO2) and comes into being the fragment ion of m/z 269 on the basis of lactone structure contained in oxygen mixed naphthalene of AFB1. The ester keton in AFB1 could result in ring cracking via α fracture and i fracture and the neutral loss (-CO) naturally happened. After the closed-loop reaction, the fragment ion of m/z 269 was thus obtained. Similarly, the ester keton in the fragment ion of m/z 269 was also prone to ring cracking and neutral loss (-CO). However, closed-loop reaction was suppressed due to steric hindrance and the fragment ion of m/z 241 was finally attained. As for 4-NP, daughter ions of m/z 161, 147, and 133 were obtained by the loss of alkyl groups (C4H10, C5H12, and C6H14) from the parent ion m/z 219. Daughter ions of m/z 212, 133, and 93 were, respectively, attained by the loss of CH3, C6H6O, and C9H11O in BPA. The highest and second highest fragment ions were selected as qualitative and qualitative ions for each target analyte. After the characteristic ion pairs selected, the collision energy, cone voltage, and the other parameters were optimized to obtain the best response of the analytes. The optimized parameters are presented in Table 1.

Optimization of Chromatography Conditions

It has reported that C18 column is commonly used for the separation of AFB1, BPA, and 4-NP. As for aqueous mobile phase, 0.1% formic acid is often adopted for AFB1 detection, while 0.05% ammonia was mostly employed for BPA and 4-NP detection, all of which could achieve favorable ionization efficiency. Pure methanol was used for the preparation of standards and sample solutions in this work, which did not match with the initial proportion of the mobile phase. Solvent effect would easily occur if the column with small diameter and minor stuff was used. Considering this circumstance, Phenomenex Luna C18 column (50 mm × 2.00 mm, 3 μm) was thus selected and excellent results were also obtained in later work.

In this paper, AFB1, BPA, and 4-NP were designed to be isolated in the same elution program. It was found that the response of BPA and 4-NP were severely inhibited when using 0.1% formic acid, while the response of AFB1 was seriously suppressed when adopting 0.05% ammonia. Water was therefore used as aqueous mobile phase. The chromatographic behavior of acetonitrile-water and methanol-water on the three TAs was investigated. Results showed that the responses of BPA and 4-NP separated with methanol-water were two times higher than those separated with acetonitrile-water. Methanol-water was thus selected as mobile phase for gradient optimization in the next work. Moreover, the gradient elution program was also optimized and described in the “Materials and Methods” section. Under this optimized gradient elution program, all the analytes have symmetrical sharp peaks and best response, and AFB1 and BPA are fully separated to adapt to the ESI+ and ESI subsection acquisition modes in mass spectrometry. In addition, three TAs could be well separated in 4 min and attain symmetrical peak shape. The extracted ion chromatograms of three target analytes standard solutions and three isotope internal standards are displayed in Fig. 1.
Fig. 1

Selected ion chromatograms of AFB1 (2 μg/L), BPA (20 μg/L), 4-NP (20 μg/L) and their corresponding isotope internal standards under the optimized instrumental conditions

Optimization of Pretreatment Conditions

The extraction and purification of trace lipid-soluble compounds in oil and fat has been a hot and difficult issue in detecting field, to which worldwide scholars continuously devote themselves. For example, a previous work reported the method for determination of BPA and alkylphenol in plant oil based on gel permeation chromatography purification treatment (Niu et al. 2011). However, this purification treatment is time-consuming, expensive, and solvent-consuming. C18 solid-phase extraction column was also employed by researchers for purifying BPA (Maragou et al. 2006). In addition, methanol-water solution was selected as extraction solvent and immunoaffinity extraction column was employed for purification to determine aflatoxin in oils and fats (Bao et al. 2013). These technologies mentioned above all possess particular features, which provide quite good references for this study.

Commonly, acetonitrile is insoluble in fat and has strong permeability and good versatility. Considering these characteristics, the effect of acetonitrile-oil liquid extraction method was investigated on the recovery experiment of negative peanut oil samples at first. Due to the large viscosity of peanut oils, they were diluted with 3 mL of normal hexane before liquid-liquid extraction treatment, which was beneficial for their flow ability and extraction efficiency. Afterwards, 5 mL of acetonitrile was added in and the samples were fully vortex extracted for 2 min. The acetonitrile extraction solution was collected for analysis. It was found that the recoveries of AFB1, BPA, and 4-NP were 93.2 ± 8.1, 60.7 ± 11.5, and 20%, respectively. Moreover, the recoveries of BPA and 4-NP had no significant improvement even repeated extraction with acetonitrile.

Under this circumstance, the effect of fractional extraction technique was further evaluated. Concretely, acetonitrile was selected as the first step extraction solvent and solid-phase extraction technology was employed to fully extract and purify the remaining TAs in normal hexane coupled with oil phase. Three different solid-phase extraction cartridges, including NH2 (500 mg/6 mL, Waters), Carb/NH2 (500 mg/6 mL, Supelclean), and Carb/PSA (500 mg/6 mL, Supelclean), were used to test the purification effect. In detail, 1.0 g of the negative peanut oil sample was extracted with acetonitrile and normal hexane layer was collected to add a certain amount of standard solution of BPA and 4-NP, resulting in 20 μg/kg of BPA and 4-NP contained in sample solution. Subsequently, the sample solution was respectively treated with the three solid phase extraction columns. Compared to mixed solution contained same pure solvent, the absolute recovery was calculated. Results presented in Fig. 2 suggested that the recoveries of the TAs treated with Carb/PSA reached the highest. It may be attributed to the fact that Carb/PSA cartridge is composed of identical amount of Carb and PSA, wherein Carb with large specific surface area could effectively absorb impurities such as steroids and PSA could absorb fatty acids, organic acids, polar pigments, and other impurities. Moreover, PSA could retain the target phenols through dipole-dipole interaction with -OH to form hydrogen bonds. The impurity leaching and target elution can be thus achieved with different polar solvents. In fact, Carb/NH2 has similar properties mentioned above. However, Carb/PAS exerts better purification effect due to the fact that PSA with two amino groups possesses stronger ion exchange capacity than NH2 column. Therefore, Carb/PAS cartridge was used for purification in further experiments.
Fig. 2

Recoveries of BPA and 4-NP treated with different solid phase extraction cartridges

On the basis of the adsorption properties of the Carb/PSA cartridge and the matrix composition of the peanut oil samples, the elution conditions were optimized according to the elution curve. Most triglycerides were removed by adding 8 mL of normal hexane at first. The elution effect of different cartridges of methanol-dichloromethane (2/8, v/v) on TAs were then investigated. Results indicated that BPA and 4-NP could be completely eluted by 4 mL of methanol-dichloromethane (2/8, v/v).

The above conditions were further validated in negative samples. The acetonitrile extract and the eluted solution purified by Carb/PAS cartridge were merged together, which was used for the UPLC-MS/MS analysis after concentration and re-dissolution. It was found that the three target analytes were fully extracted and purified, and favorable recoveries were also observed.

Method Validation

Selectivity

The selectivity of the developed method was evaluated by the analyses of three target analytes in 20 negative samples, which were pretreated and detected under the optimized conditions mentioned above. No interference peaks were observed in the three target analytes, revealing the favorable selectivity of this developed method.

Linearity Range, ILOQs, MLOQs, and ME

Under optimized instrumental conditions mentioned above, the analytical characteristics of this developed method, such as linearity range, linear equations, instrument limit of quantitation (ILOQ), and method limit of quantitation (MLOQ), were investigated to evaluate the efficiency of the method and the possibility of method application to real samples. A series of mass concentrations (0.1, 0.2, 1.0, 5.0, 10.0, and 50.0 μg/L of AFB1; 0.5, 1.0, 5.0, 20.0, 50.0, and 100.0 μg/L of BPA; 1.0, 2.0, 10.0, 20.0, 50.0, and 100.0 μg/L of 4-NP) of pure solvent standard working solutions and matrix calibration standard working solutions were prepared with the negative sample extraction solution and pure solvent, respectively, wherein all contained 2 μg/L of U-[13C17]-AFB1 and 20 μg/L of BPA-d 4 and 4-n-NP-d 4. The ratio of the peak area of each TA and its corresponding internal standard peak area was used as ordinate y. The corresponding mass concentration (μg/L) was used as abscissa x. Linear regression analysis was performed by plotting the ratios of the y value on the ordinate versus x value on the abscissa. Results shown in Table 2 indicated that all of the correlation coefficients were greater than 0.998, revealing the good linear relationship between the quantitative ion peak areas and analyte concentrations. The limit of detection (LOD) and limit of quantification (LOQ) of the established method refer to the triple signal-to-noise ratio (S/N = 3) and tenfold signal-to-noise ratio (S/N = 10), respectively (Dang et al. 2017; Wu et al. 2016c; Xian et al. 2016). Generally, the instrument LOD (ILOD) and instrument LOQ (ILOQ) were measured by the standard solution with the pure solvent, while the method LOD (MLOD) and method LOQ (MLOQ) were determined by calibration matrix solution (Xian et al. 2016; Zeng et al. 2016). As shown in Table 2, we could find that the ILOQs for AFB1, BPA, and 4-NP were 0.1, 0.5, and 1.0 μg/L, while MLOQs were 0.2, 1.0, and 2.0 μg/kg, indicating the high sensitivity of the established method.
Table 2

Linear range, linear equations, correlation coefficient (R 2), ILODs, MLOQs, and ME for three target analytes

Analytes

Linear range (μg/L)

Linear equation (solvent), R 2

Linear equation (matrix), R 2

ILOQ (μg/L)

MLOQ (μg/kg)

ME ± SD (n = 3)

AFB1

0.1~50.0

y = 0.5416x + 0.1081, 0.9995

y = 0.4932x + 0.0698, 0.9992

0.1

0.2

0.93 ± 0.05

BPA

0.5~100

y = 0.0536x + 0.0215, 0.9989

y = 0.0475x + 0.0196, 0.9981

0.5

1.0

1.05 ± 0.05

4-NP

1.0~100

y = 0.0616x + 0.0543, 0.9986

y = 0.0592x + 0.0527, 0.9982

1.0

2.0

0.89 ± 0.08

The matrix enhancement and matrix suppression effects often occur to influence the accurate quantification of the target analytes in the UPLC-MS/MS determination. The matrix effect (ME) of the developed method was also thus investigated. A series of matrix calibration solutions and standard solutions of same concentrations were prepared by using the extraction solution of 20 negative samples and pure solvent, respectively. The ME was identified by the slope value of the matrix calibration working curve versus that of the pure solvent standard working curve. It has been reported that ME > 1 and ME < 1 represent matrix enhancement and matrix suppression, respectively (Dong et al. 2015; Dong and Xiao 2017; Wu et al. 2016b). As displayed in Table 2, the ME of this developed method was observed in the range of 0.80–1.19, manifesting the ME of the target analytes could be ignored.

Recovery, Accuracy, and Precision

The methodological indicators, consisting of recovery, accuracy, and precision, were investigated by the addition of the negative sample recovery test (n = 6). Specifically, the mixed standard solutions at three different concentration levels (AFB1 with 1 × MLOQ, 10 × MLOQ, and 1 × limit of maximum residue (20 μg/kg); BPA and 4-NP with 1 × MLOQ, 2 × MLOQ, and 10 × MLOQ) were added into the negative samples. At the optimal experiment condition, each added level includes six parallel experiments and the recovery and intraday precision was calculated. Additionally, relative experiment was continuously conducted for 5 days to determine the interday precision (n = 5) at the middle added level. Results presented in Table 3 showed that the average recoveries of three added levels were in the range of 87.7–105.1%, and the intraday precision ranged from 2.2 to 7.9%. Moreover, the average recoveries of middle added level in the range of 89.7–95.3% and interday precision (n = 5) in the range of 5.0–8.7% were favorably observed. By this token, this developed method possesses excellent recovery, accuracy, and precision.
Table 3

Recovery, accuracy, and precision of three target analytes in different negative samples

Compound

Added (μg/kg)

Recovery (%, n = 6)

Intraday precision (%, n = 6)

Recovery (%, n = 5)

Interday precision (%, n = 5)

AFB1

0.2, 2.0, 20.0

101.8, 94.7, 97.9

5.7, 2.2, 3.1

95.3

5.0

BPA

1.0, 2.0, 10.0

105.1, 92.6, 89.7

7.9, 5.2, 4.4

92.0

8.7

4-NP

2.0, 4.0, 20.0

87.7, 93.9, 90.2

7.5, 5.1, 4.5

89.7

7.4

Method Applicability and Comparison

The method established in this work was adopted to determine a total of 52 peanut oil samples collected from local supermarket and individual workshops. Unfortunately, the obtained results indicated that AFB1 in the range of 0.5–69.4 μg/kg were detected in 43 samples, wherein 29 were collected from the individual workshops. In addition, the AFB1 concentrations detected in four samples collected from individual workshops were substandard and all exceeded the limit value of 20.0 μg/kg (Fig. 3). 4-NP was detected in 12 samples with a concentration range of 9.3–77.8 μg/kg, among of which seven were collected from individual workshops. Meantime, none of BPA was detected in any samples. The ion chromatograms of AFB1 and 4-NP are shown in Fig. 4.
Fig. 3

Detecting results of the three target analytes in peanut oil samples

Fig. 4

The ion chromatogram of AFB1 and 4-NP detected in typical samples

In order to further validate the applicability of the method established in this work, another two methods, respectively, recorded in GB 5009.22-2016 (GB 5009.22- 2016) and reported by Niu et al. (2011), were adopted to detect the three TAs in four typical samples and their detecting effects were compared. As presented in Supplementary materials, we found that two sample pretreatments and two instrumental detections were involved in the two selected methods, while only one pretreatment or instrumental method was contained in the developed method in relation to simultaneous determination of the three target analytes. Moreover, this developed method exerted more favorable detection efficiency, sensitivity, accuracy, recovery, and precision than reported methods, revealing its good applicability.

Conclusions

In this work, the samples were extracted by a two-step extraction method consisted of liquid-liquid extraction and solid-phase extraction. On the basis of this extraction method, a sensitive and rapid method, using isotope dilution method and UPLC-MS/MS with ESI+ and ESI modes, was developed and validated for the simultaneous determination of AFB1, BPA, and 4-NP in peanut oil. Preferable experimental conditions were obtained by optimizing the instrumental parameters and pretreatment extraction. According to methodological indicators, the results indicated that this developed method possesses good specificity, recoveries, accuracy, intraday precision, and interday precision, so does the detection efficiency. In addition, this method was verified to be suitable for simultaneous determination of AFB1, BPA, and 4-NP in peanut oil products. Results indicated that AFB1and 4-NP were commonly found in peanut oil products and these products collected from small worships were substandard, to which great and continuous attention should be paid.

Notes

Compliance with Ethical Standards

Conflict of Interest

Hongling Deng declares that he has no conflict of interest. Xinguo Su declares that he has no conflict of interest. Haibo Wang declares that she has no conflict of interest.

Ethical Approval

This article does not contain any studies with animals performed by any of the authors.

Informed Consent

Not applicable.

Supplementary material

12161_2017_1113_MOESM1_ESM.docx (26 kb)
ESM 1 (DOCX 26.3 kb)
12161_2017_1113_MOESM2_ESM.docx (16 kb)
ESM 2 (DOCX 16.0 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Guangdong Food and Drug Vocational CollegeGuangzhouChina

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