472  Liu et al.: Journal of AOAC International Vol. 98, No. 2, 2015 RESIDUES AND TRACE ELEMENTS

Determination of Multiresidues of Three Acid Herbicides in Tobacco by Liquid Chromatography/Tandem Mass Spectrometry Shanshan Liu, Zhaoyang Bian,1 Fei Yang, Zhonghao Li, Ziyan Fan, Hongfei Zhang, Ying Wang, Yange Zhang, and Gangling Tang China National Tobacco Quality Supervision and Test Center, Zhengzhou, 450001, People’s Republic of China

A method to determine residues of the three acid herbicides, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, and 3,6-dichloro-2methoxybenzoic acid (dicamba), in tobacco using LC/MS/MS is presented. Because these herbicide residues in tobacco might exist in different forms (free acid, salt, and ester), tobacco samples were first pretreated by alkaline hydrolysis and then the pH was adjusted in order to convert the residues completely to their free acid forms. Dichloromethane extraction and dispersive SPE using C18 sorbent were carried out before LC/MS/MS analysis, and quantification was performed using the internal standard method. Linearity was good for all 2 analytes (R ≥ 0.999) in the concentration range of 0.02 to 0.5 mg/kg. LODs were below 0.05 mg/kg. Recoveries ranged from 80.4 to 93.5%, and RSD was below 10%. This simple, efficient, and sensitive method can be applied to the determination of residues of the three acid herbicides in tobacco.

A

s the most used broadleaf weed herbicides and the second most used selective herbicides in the world, acid herbicides are widely applied in the production of crops, fruits, tea, cotton, and tobacco. Although typical acid herbicides, such as 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4-dichlorophenoxyacetic acid (2,4-D), and 3,6-dichloro2-methoxybenzoic acid (dicamba; Figure 1), are considered to have low toxicity, they have been proven to be endocrine disruptors. After being taken in, these acid herbicides might cause soft tissue sarcoma in human body; they also show placenta toxicity in animals. These compounds are systemic herbicides that could be readily absorbed by leaves and roots and transported to areas of metabolic activity (1, 2). In the guidance residue levels (GRLs) for 118 agrochemicals released by the Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA) Agrochemical Advisory Committee in 2008, GRLs of 2,4,5-T, 2,4-D, and dicamba are 0.05, 0.20, and 0.20 mg/kg, respectively (3). GC, GC/MS, and LC/MS/MS (4–6) have been proposed for the determination of acid herbicide residues. The analytes need to be derivatized before GC analysis. The derivatization will affect the accuracy of the determination and the reagents used Received May 12, 2014. Accepted by AK September 24, 2014. 1   Corresponding author’s e-mail: [email protected] DOI: 10.5740/jaoacint.14-089

are normally toxic. LC/MS/MS does not require a derivatization step and can provide lower LODs and better selectivity in the multiple reaction monitoring (MRM) mode. Yang et al (7). reported a determination of chlorinated phenoxyacid herbicides in tobacco using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction. It is a general method but not suitable for the determination of the esters or salts. Acid herbicides might be manufactured in different forms such as esters and salts, i.e., 2,4-D that is manufactured and sold as butyl 2,4-dichlorophenoxyacetate and sodium-2,4dichlorophenoxyacetate (1). Because the acid herbicide residues in tobacco might exist in different forms (free acid, salt, and ester), direct extraction of samples without proper pretreatment using only acidic solvent as described in the literature will probably lead to a decrease in recovery (8). Zhang et al. (9) reported a determination of phenoxyacid, sulfonylurea, triazine, and other selected herbicides by mixed-mode SPE coupled with LC/MS/MS in which the pretreatment process was complicated and time consuming. In this study, an LC/MS/MS method was developed for the determination of residues of three acid herbicides in tobacco. Pretreatment of tobacco samples was carried out in order to completely convert the residues into their free acid forms. Experimental Apparatus An LC/MS/MS system (API 4000, AB Sciex, Framingham, MA); refrigerated table top centrifuge (3-30K, Sigma, St. Louis, MO); vortex mixer (Talboys Troemner, Thorofare, NJ); ultrasonic cleaner (KQ-700DB, Kunshan Ultrasonic Instruments Co., Kunsha, China); and digital balance (TEG612-L, Sartorius, Goettingen, Germany) were used. Chemicals and Standards Methanol (pesticide residue grade; DUKSAN, Gyeonggi-do, South Korea); CH2Cl2 (pesticide residue grade, J.T. Baker, Phillipsburg, NJ); toluene (pesticide residue grade, J.T. Baker); acetonitrile (pesticide residue grade, DUKSAN); acetic acid (HPLC grade, TEDIA, Fairfield, OH); C18 sorbent (Agilent Technologies, Santa Clara, CA); Florisil (TEDIA); anhydrous MgSO4 (Tianjin Kemiou Chemical Reagent Co., Tianin City, China); NaOH (Tianjin Chemical Reagent Factory Tianjin Chemical Reagent Factory); and concentrated H2SO4 (Luoyang Haohua Chemical Reagent Co., China) were used. 2,4,5-T, 2,4-D, and dicamba and their methyl ester derivatives

Liu et al.: Journal of AOAC International Vol. 98, No. 2, 2015  473 Table  1.  MRM parameters for 2,4,5-T, 2,4-D, dicamba, and the IS Analytes 2,4,5-T Figure  1.  The structures of 2,4,5-T, 2,4-D, and dicamba.

were purchased from Dr. Ehrenstorfer GmbH (Augsburg Germany); 2,4,5-T-D4, 2,4-D-D3, and dicamba-D3 were purchased from CDN Co. (Pointe-Claire, Canada). Standard Solutions Individual stock solution of the herbicides at a concentration of 1.0 mg/mL was prepared in methanol. The mixed working standard solution, which was also used as a spiking solution for validation experiments, was prepared by diluting the stock solutions in CH2Cl2 at 1.0 µg/mL of each herbicide. The obtained solutions were all stored in a refrigerator at –18°C. The matrix-matched calibration solutions were used to establish the calibration curves. Calibration solutions were prepared by diluting the working standard solution with the extract of blank control samples to obtain a concentration of each herbicide between 2 and 50 ng/mL. Preparation of Spiked Samples As reported (7), we used organically grown samples that were previously determined to be free of pesticide residues as blank control matrix in the recovery experiments and for the preparation of matrix-matched calibration solutions. The tobacco samples and spiked samples were prepared as reported by Yang et al. (7). Sample Pretreatment A 1.0 g sample was weighed into a 50 mL centrifuge tube and spiked with 200 µL mixed internal standard (IS) solution containing 1.0 µg/mL 2,4,5-T-D4, 2,4-D-D3, and dicamba-D3. Then, 10 mL of 0.5 M NaOH solution was added into the centrifuge tube, and the sample was soaked for 30 min to be sufficiently hydrated. After that, the mixture was vortexed for 2 min at 2000 rpm, and 0.5 mL concentrated H2SO4 (9.2 M) was added to adjust the pH to below 1.5. Afterward, 10 mL CH2Cl2 was added, and the mixture was vortexed again at 2000 rpm for 2 min and then left to stand. A 1 mL volume of subnatant was transferred into a 1.5 mL centrifuge tube containing 50 mg MgSO4 and 10 mg C18 sorbent. The mixture was vortexed at 2000 rpm for 2 min and centrifuged at 10 000 rpm for 2 min. Then, 200 μL of supernatant was transferred and diluted to 1 mL with CH2Cl2. The sample was then filtered with a 0.22 µm filter (Anpel Scientific Instrument Co., Ltd, Shanghai, China) prior to LC/MS/MS analysis. LC/MS/MS Analysis LC analysis was performed with a Waters Atlantis dC18 column (3 µm, 2.1 × 150 mm). Methanol containing 0.1% acetic acid was used as the mobile phase (flow rate 0.2 mL/min). The

2,4-D Dicamba 2,4,5-T-D4 2,4-D-D3 Dicamba-D3

Parent ion, m/z

Product ion, m/z

DP, V

CE, V

255.0

197.0

–30

–15

253.0

195.0

–30

–15

221.1

162.8

–40

–15

219.1

160.8

–40

–15

219.1

175.1

–55

–7.5

221.1

177.0

–55

–7.5

258.9

198.8

–40

–17

256.7

196.5

–40

–17

223.7

165.8

–34

–17

221.8

164.2

–34

–17

223.8

179.8

–26

–7

221.9

177.9

–26

–7

injection volume was 5 μL and the column temperature 25°C. Total analysis time was 5 min. Electrospray ionization MS (ESI) was performed in negative ion mode with ion spray potential of –4500 V. The nebulizer gas (air) pressure was 10 psi and the ion source temperature 450°C. Analytes and IS were measured for quantitative analysis in the MRM mode. Declustering potential (DP) and collision energy (CE) for each parent–product ion pair are listed in Table 1. Results and Discussion Sample Pretreatment Optimization The pKa of the carboxylic groups in 2,4,5-T, 2,4-D, and dicamba are 2.85, 2.73, and 1.97, respectively. With current pretreatment methods reported in the literature, only an acidic solvent is used to extract the analytes, which usually makes the determined concentration lower than the actual value. In this study, we focused on the optimization of the pretreatment process in order to determine acid herbicides existing in different forms. Therefore, NaOH was used to convert all acid herbicides in tobacco into a salt, and the pH of solution was then adjusted to below 1.5. As a result, all the acid herbicides were converted into their acid form and could be readily extracted for the following purification and analysis. Optimization of Tobacco Moisture Adjustment and Selection of NaOH Solution Concentration Due to the complicated chemical composition of tobacco, direct extraction with organic solvent will lead to a significant matrix effect that can be ameliorated by adjusting the moisture content of dry tobacco prior to extraction. Because NaOH solution needs to be applied for hydrolyzing the acid herbicide residues in salt and ester forms, tobacco samples were pretreated before analysis with an NaOH aqueous solution of optimized concentration in order to achieve both the moisture adjustment and the hydrolysis in one step. Normally, moisture adjustment would simply be carried out by soaking tobacco for a certain time. In order to make sure that the hydrolysis was completely accomplished, the influence of

474  Liu et al.: Journal of AOAC International Vol. 98, No. 2, 2015 Table  2.  The effect of extraction agents on the recoveries of 2,4,5-T, 2,4-D, and dicamba in different volume ratio Dichloromethane

2,4,5-T

2,4-D

Dicamba

Toluene

Acetonitrile

Volume ratio

Extraction yield, %

Extraction yield calculated with IS, %

Extraction yield, %

Extraction yield calculated with IS, %

Extraction yield, %

Extraction yield calculated with IS, %

1

45.6

100.0

48.2

106.6

73.6

106.0

2

76.7

98.7

73.6

112.3

83.7

105.8

3

87.2

95.0

88.8

100.7

83.8

99.9

4

105.3

104.1

90.5

101.1

88.6

99.4

5

108.4

106.5

102.0

114.0

87.4

98.8

1

98.5

100.5

47.0

100.3

68.1

97.0

2

95.4

93.2

65.6

79.1

74.2

96.7

3

109.3

98.4

85.2

99.2

96.8

103.7

4

95.2

95.3

107.9

43.9

97.0

96.1

5

99.7

109.7

110.6

144.8

91.7

89.8

1

81.6

101.5

74.9

95.2

54.2

101.7

2

84.6

95.4

74.0

91.4

71.4

93.2

3

96.0

105.1

86.1

82.1

77.2

117.0

4

99.4

95.6

138.0

502.1

63.7

81.8

5

107.8

99.4

141.9

76.8

76.9

91.9

different pretreatment methods on recovery was investigated. Tobacco samples spiked with standards were first soaked in 10 mL of 0.5 M NaOH solution for 30 min and then vortexed for 1, 2, 5, and 10 min; ultrasonicated for 10, 20 30, and 60 min; or continued to be soaked for 15, 30, 60, and 90 min. The results showed no significant differences, but the method with vortex step was chosen for its higher efficiency. NaOH solutions with various concentrations (0.25, 0.5, 0.75, and 1.0 M) were used to evaluate the effect of NaOH concentration on recovery. No significant difference was observed, and the moderate concentration (0.5 M) was thus chosen in the following study. Extraction Solvent Selection and Extraction Ratio Optimization In order to get good analytical results, acid herbicides need to be extracted from an acidic water phase by an organic solvent. Most of the water soluble impurities will stay in the water phase, and the extract can be purified. Based on the polarities and solubilities of acid herbicides, CH2Cl2 (hydrophobic), toluene (hydrophobic), and acetonitrile (hydrophilic) were used as a solvent to extract standards from the spiked mixture of 10 mL 0.5 M NaOH solution and 0.5 mL 9.2 M H2SO4 solution. Different volume ratios (extraction solvent/aqueous solution) are listed in Table 2. Phase separation was found for all three extraction solvents due to their hydrophobicity or salt effect. Samples were taken from the organic phase. The extraction yields were calculated and are shown in Table 2. It was found that the extraction yield increased with the volume of organic solvent added. However, the concentration of target compound was diluted while the volume ratio was increasing, which reduced the sensitivity of the analysis. The extraction efficiency of acetonitrile was low because of its high solubility in water. Isotope marker was added as an IS to calculate the extraction yield (Table 2). The

results suggested that with the IS calibration, good extraction yield would be obtained no matter how much extraction agent was used. Therefore, 10 mL extraction agent was enough considering extraction yield, recovery, and sensitivity. The color of the extract with acetonitrile was darker than those with the other two extraction solvents. This was due to the high polarity of acetonitrile, which could extract more substances from the tobacco. The extraction efficiency of toluene was close to that of CH2Cl2. However, toluene affected the peak pattern and caused severe peak broadening and splitting. A clean spectrum and good peak pattern could be achieved using CH2Cl2. Therefore, CH2Cl2 was selected as the extraction solvent. The compatibility of the extraction solvent and LC mobile phase was also investigated. The CH2Cl2 in the extract was removed by a nitrogen flush. Then the samples were dissolved in methanol, and analyzed by LC/MS/MS, and compared with the sample in dichloromethane. The results suggested that replacing CH2Cl2 with methanol would not affect the LC separation. So the sample pretreatment could be simplified, and a nitrogen flush and solvent change were not necessary. Sorbent Selection and Optimization Primary-secondary amine (PSA), C18, graphitized carbon black (GCB), and Florisil are commonly used sorbents for purification of extracts. PSA sorbent, which is frequently used in the QuEChERS pretreatment method (10–13), is the base sorbent used for cleanup as it can remove many matrix substances such as pigments, fatty acids, and organic acids. The effects of various sorbents on sorption efficiency and recovery were studied, and the results indicated that although acid herbicides could be sorbed onto PSA, C18, and GCB, reducing the recovery, C18 sorbent exhibited the lowest affinity to acid herbicides and excellent performance in extract purification. The influences of different amounts of C18 sorbent (10, 15,

Liu et al.: Journal of AOAC International Vol. 98, No. 2, 2015  475 Table  3.  The effect of the matrix on the detection of 2,4,5-T, 2,4-D, and dicamba Detector response Standard solution Matrix-matched

Solvent

Ratio

2,4,5-T

46100

50100

0.92

2,4-D

51700

66200

0.78

Dicamba

19800

22000

0.90

50, 75, and 100 mg/mL extract) were investigated. The sample was filtered and analyzed by LC/MS/MS. After purification by C18 sorbent, the color of the extract became lighter, the number of peaks of impurities was reduced, and the sensitivity of detection increased. The results revealed that 10 mg of C18 sorbent for 1  mL extract was enough for sample purification. There was no significant improvement in the cleanup effect when more sorbent was added. Matrix Effect Many components extracted with the analytes during the extraction process can affect the quality of the analytical results, which is known as matrix effect and is normally unavoidable. Since the tobacco composition is very complicated, a severe matrix effect would considerably influence the qualitative and quantitative analysis. However, the matrix effect can be reduced by using matrix-matched standard solutions, diluting the extract, adding an IS, and optimizing the purification method. The matrix effect on the detector response for 2,4,5-T, 2,4-D, and dicamba was investigated by comparison of the response differences between matrix-matched standard solutions and pure solvent standard solutions. The matrix-matched standard solutions (containing 0.2 mg/kg of each acid herbicide standard in blank tobacco matrix solution) were prepared as reported earlier (12, 13). As shown in Table 3, a matrix effect occurred for all three acid herbicides, but 2,4-D was affected most. As a result, matrix-matched standard solutions were used to minimize the matrix effect. Also, isotope markers of the three acid herbicides were added as ISs, and C18 sorbent was used to purify samples to reduce the matrix effect on the analytical results. As pointed out in the literature, diluting the extract would Table  4.  The effect of dilution ratio on the 2,4,5-T response differences between matrix-matched standard solutions and pure solvent standard solutions Relative intensity Dilution ratio of the matrix Concentration of 2,4,5-T, mg/kg

Solvent standard solution

5

2.5

5/3

0.10

1.00

0.84

0.72

0.67

0.25

1.00

0.95

0.77

0.64

0.50

1.00

0.85

0.75

0.69

1.00

1.00

0.85

0.71

0.55

2.50

1.00

0.91

0.71

0.60

Figure  2.  TIC chromatogram of 2,4-D, 2,4,5-T, and dicamba under the optimized condition.

effectively reduce the matrix effect (11). The effect of dilution ratio (5/3, 2.5, and 5 times) on the 2,4,5-T response differences between matrix-matched standard solutions and pure solvent standard solutions was studied. The results (Table 4) indicated that the matrix effect was reduced while increasing the dilution ratio. However, the concentration of target compound would be too low to be detected if the dilution ratio was too high. The dilution ratio of 5 was chosen based on the results of this study and the literature (13). Optimization of LC/MS/MS Parameters Methanol and acetonitrile are usually used as mobile phase solvents in LC/MS/MS. It was found that adding 0.1% acetic acid in the mobile phase improved the peak patterns and the ionization of the target compounds in the ESI source. In consideration of the compatibility of the mobile phase and extraction solvent, methanol with 0.1% acetic acid was used as the mobile phase in LC/MS/MS. The MS optimization of the target compounds was performed by flow injection analysis of their individual standard solutions at a concentration of 0.1 mg/L in methanol. The precursor ion and the product ions were chosen in this process, as well as the optimum DP and entrance potential for the precursor ion and the CEs for the product ions. In the selected reaction monitoring (SRM) process, the most abundant product ions (SRM1) were chosen for quantification and the second ones in abundance (SRM2) for identification. The quantitative and qualitative ion pairs of the target compounds and their main parameters of DP and CE of are summarized in Table 1. The HPLC/MS/MS total ion current (TIC) chromatogram of 2,4-D, 2,4,5-T, and dicamba under the optimized conditions is shown in Figure 2. Linearity and LOQ The linearity was investigated with matrix-matched standard calibration solutions at five different levels under the optimized conditions. The linear range was between 5 and 100 ng/mL. The linear relation and R2 values are summarized in Table 5. The linearity of the response for the studied compounds was very good, with all R2 values higher than 0.999. LODs and

476  Liu et al.: Journal of AOAC International Vol. 98, No. 2, 2015 Table  5.  The retention times, linearity, regression coefficients, LOD, and LOQ of 2,4,5-T, 2,4-D, and dicamba Retention time, min

Dependent linear equation

R2

LOD, mg/kg

LOQ, mg/kg

0.003

0.010

2,4,5-T

3.17

y = 0.6539x + 0.0002 0.9998

2,4-D

2.77

y = 0.9498x + 0.0446 0.9991

0.003

0.010

Dicamba

4.16

y = 0.2907x + 0.0146 0.9990

0.005

0.017

a

 y = Intensity; x = Concentration ratio of analyte to its IS.

the pH was adjusted in order to convert the residues completely to their free acid forms. CH2Cl2 extraction and dispersive SPE using C18 sorbent were carried out before LC/MS/MS analysis. Dilution of the samples before LC/MS/MS analysis decreases the matrix effects, and quantification was performed using the IS method. This simple, efficient, and sensitive method can be applied in the residue determination of the three acid herbicides in tobacco. References

Table  6.  Recoveries and RSDs of 2,4,5-T, 2,4-D, and dicamba in tobacco Recovery, %

RSD, %

2,4,5-T

87.7–93.5

5.17

2,4-D

81.5–89.6

6.58

Dicamba

80.4–87.3

7.03

LOQs, defined respectively as three and 10 times the maximum baseline height of the blank, are also summarized in Table 5. Recoveries and Precision To study the recovery of method, tobacco samples were spiked with standards at concentrations of 0.02, 0.05, and 0.10 mg/kg for 2,4,5-T because its GRL is 0.05 mg/kg, and at 0.05, 0.20, and 0.25 mg/kg for 2,4-D and dicamba because their GRL is 0.2 mg/kg. Five replicate samples were prepared for each concentration level and the precision of this method was determined by calculating the RSD of replicate measurements. The recovery and RSD values listed in Table 6 exhibit the high accuracy and good precision, respectively, of the extraction method. Conclusions The developed method allows qualitative and quantitative determination of residues of three acid herbicides, 2,4,5-T, 2.4-D, and dicamba, in tobacco using LC/MS/MS. Tobacco samples were first pretreated by alkaline hydrolysis, and then

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tandem mass spectrometry.

A method to determine residues of the three acid herbicides, 2,4,5-trichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, and 3,6-dichloro-2-met...
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